Biologically-Inspired Systems Volume 1
Series Editor Stanislav N. Gorb
For further volumes: http://www.springer.com/series/8430
Hermann Ehrlich
Biological Materials of Marine Origin Invertebrates
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Dr. Hermann Ehrlich Institute of Bioanalytical Chemistry Dresden University of Technology Bergstr. 66 01069 Dresden Germany
[email protected]
ISBN 978-90-481-9129-1 e-ISBN 978-90-481-9130-7 DOI 10.1007/978-90-481-9130-7 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2010933606 © Springer Science+Business Media B.V. 2010 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)
Preface
Biological substances appeared in marine environments at the dawn of evolution. At that moment, the first organisms acquired the ability to synthesize polymer chains which were the basis, in their turn, for the formation of the building blocks that fueled the so-called self-assembling process. They, in their turn, produced more complicated structures. The phenomenon of three main organic structural and scaffolding polymers (chitin, cellulose, and collagen) probably determined the further development and evolution of bioorganic structures and, of course, the organisms themselves. All the three biopolymers, notwithstanding their differences in chemical composition, have the common principles in their organization: nanofibrils with the diameter 1.5–2 nm, the ability to self-assemble, production of fibrillar and fiber-like structures with hierarchical organization from nano—up to macrolevels, the ability to perform both the role of scaffolds and the templates for biomineralization and formation of the rigid skeletal structures. Chitin and collagen in particular played the determining role in the formation of skeletal structure in marine invertebrate organisms. These two biopolymers possess all the qualities needed to refer to them simultaneously as biological materials and biomaterials, the latter thanks to their successful application in biomedicine. The fact that modern science finds chitin and collagen both in unicellular and in multicellular invertebrates in fossil and modern species confirms beyond a doubt the success of these biological materials in the evolution of biological species during millions of years. I realize that this success should be consolidated at genetic level and the detection of corresponding conserved genes must be the main priority. The abundance of silica as well as calcium and carbonate ions in the ancient marine environments on one hand, and the existence of chitin and collagen primary scaffolds in primitive biological form on the other hand led to the formation of unique biocomposites, possessing completely new qualities. The diversity of skeletal forms of marine invertebrates impresses man both with its exceptionality and strict conformity with mathematic and thermodynamic laws. Nature performs here the role of the first, and doubtless brilliant, engineer without using any equipment or computer support, which is inconceivable in the creation of any construction nowadays.
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Many engineering solutions that we observe in unicellar marine organisms can also be found in further developed organisms. I think that the chitin system which came into existence as a result of polymerization of N-acetylglucosamine into polyN-acetylglucosamine, already in ancient bacteria and fungi, is even older than the collagen system. Chitin is more resistant in extreme conditions: both to a wide pH range and to changes in temperature up to 300◦ C. But both polymers can be found in the cuticles of many inhabitants related to the hydrothermal vent fauna and both materials can also be found in marine organisms living in Arctic and Antarctic waters. How, and on the basis of which chemical laws, the skeletal formation, for example, in silica– chitin as well as silica–collagen-based deep-sea glass sponges at –1.5◦ C takes place is still a puzzle. If a rubber laying in a certain element of a spaceship is frozen, it cannot be launched. People perish and a very complicated heavy-weight construction is destroyed as happened in the tragedy of the Shuttle. But marine invertebrates, living under the thick ice in Antarctic, manage not only to exist but also to swim, run away and pursue, overtake, hold, gnaw, drill, suck, multiply, and live their life from an egg or capsule, to larva, and to the grown-up species. Everywhere and at every level biological materials, hard and sharp, elastic and gel-like, successfully perform their role with the only aim—to survive, a mission that has been achieved over billions of years. Even nowadays, when marine invertebrates are confronted with completely foreign heavy metal and ion pollutants, the fight for survival is undiminished and skeletal systems are built using nickel, strontium, or uranium. One can only learn from them and think it over! Thus, Extreme Biomimetics could now be proposed as the novel direction in biomaterials science. If biomaterials science can be referred to as one of the directions in materials science, the science of biological materials of marine origin does not exist at all, including the classification of these materials. In my opinion, the level of modern science nowadays allows the start of serious and systematic research into biological materials of marine origin because their formation and the principles of their organization are the bases of the evolution of biomaterials of the highest level, like the bones or teeth of human beings. It is not a coincidence that the collagen of a primitive sponge is homologous to that of a human being. All of us realize that it is not the interest in the peculiarities of the shell skeletal construction that comprises the driving force in the development of modern materials science, but elementary solutions for the human problems of toothache or osteoporosis. Let us add to the appearance of such a unique direction in science the field of Military Biomaterials. Huge sums of money are spent on solutions for these very often artificially created and painful problems of mankind. However, having invited them, our scientific community is confronted with the problem of a lack of basic knowledge pertaining to the peculiarities of biological material creation and is becoming aware of the necessity for detailed research of the shell, crawfish, or diatom. I was also faced with this problem, when in 2003 Professor Hartmut Worch from Max-Bergmann Center of Biomaterials in Dresden, as an outstanding engineer, asked me to work on a problem: the speedy creation of an artificial bone.
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However, being a biologist, I decided before starting work on this task, to look back at the sources of skeletal formation in living organisms. I found marine sponges to be good representatives for this process and thus I started my “dive” into the strange world of marine biological materials. My professor is a pensioner now, the best brains in the world are still working hard in the field of artificial bone creation. To tell you the truth, even if I live to become a pensioner, I am not sure that we will be able to find out the exact mechanism for the skeleton formation of even primitive marine sponge. I can mention the diversity of marine species, their habitat in the depths, and unfavorable, to human beings at least, climate zones as restricting factors. In spite of the great interest in the marine biological materials on the part of scientists dealing with the problems of biotechnology, bioorganic and bioanalytical chemistry, materials science, solid state physics, crystallography, mineralogy, bionics, and biomimetics, there is not a single scientific center in the world today that can claim as its focus this scientific research. In my book on biological materials of marine origin I have made an attempt at classifying them utilizing their great diversity of forms. The book consists of 8 parts, an introduction, 35 chapters, an epilogue, and an addendum including more than 2,000 references. Many of the photos have been published for the first time. I have also paid much attention to the historic factors, as it is my opinion that the names of the discoverers of unique biological structures should not be forgotten. I am fully aware of the fact, that due to interaction of many fields, I cannot satisfy the interest of the scientists in the above-mentioned fields of science, but I hope that all of them will acquire new knowledge. There are so many institutions and individuals to whom I am indebted for the gift or loan of material for study that to mention them all would add pages to this monograph. It may be sufficient to say that without their cooperation, this work could hardly have been attempted. I also thank Prof. Catherine Skinner, Prof. Edmund Bäeuerlein, Prof. Victor Smetacek, Prof. Dan Morse, Prof. George Mayer, Prof. Hartmut Worch, and Prof. Eike Brunner for their support and permanent interest in my research. I am grateful to Vasily V. Bazhenov, Denis V. Kurek, René Born, Sebastian Hunoldt, and Andre Ehrlich for their technical assistance. To Dr. Allison Stelling and Mrs. Tatiana Motschko, I am thankful for taking excellent care of manuscripts and proofs. To my parents, my wife, and my children, I am under deep obligation for their patience and support during hard times. Dresden, Germany
Hermann Ehrlich
Introduction
We probably know more about the Moon than we do about the bottom of the sea Ole Jorgen Lonne, Ph.D.
Abstract The first and generalized classification of biological materials of marine origin is proposed as follows: Biomineralized Structures and Biocomposites; Non-mineralized Structures; Macromolecular Biopolymers; Self-made Biological Materials. The biological, chemical, and materials diversity of the marine environment is immeasurable and therefore is an extraordinary resource for the discovery of new bioactive substances, drugs, toxins, pigments, enzymes, and bioluminescence-based markers; as well as biopolymers, bioadhesives, bioelastomers, and hierarchically structured biocomposites. Recent technological and methodological advances in structure elucidation, genomics, proteomics, organic synthesis, bioinspired materials chemistry, biological assays, and biomimetics have resulted in the isolation and clinical evaluation of various novel pharmacological preparations and biomaterials. These compounds range in structural class from simple linear peptides to complex biopolymers. Equally as diverse are the molecular modes of action by which these molecules impart their biological activity (Newman and Cragg 2004; Weiner 1997). Beyond their importance as a food source, the world’s seas have always been bountiful providers of special materials valued for human health and pleasure. Access to this resource historically has been hindered by the apparent hostility of the seawater environment to manufactured materials and engineering concepts of terra firma. In spite of the extraordinary potential of the marine environment for new biomaterials, the environmental risks and exploration costs have been prohibitive. In the past decade, new tools in biotechnology have been introduced that are producing extraordinary new products and assays based on the new understanding of genetic factors and their expression as complex biological molecules. Applying these tools to the marine environment provides opportunities to unlock similar micro-molecular vaults of marine biomedical products so that they can join other ix
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macro-biomaterials that have already been harvested from the sea for thousands of years (Weber 1993). Dramatic developments in understanding the fundamental underpinnings of life have provided exciting opportunities to make marine bioproducts. This achievement using marine biotechnology is an important part of the economy in the USA, Japan, China, Korea, Russia as well as in European Community (Attaway 1993; Powers 1995). According to MarineBiotech.org (www.marinebiotech.org), marine biotechnology, as the name implies, utilizes the rich biodiversity found in the world’s oceans for applications in biotechnology. Marine biotechnology has recently been embraced as a field of great potential by both molecular biologists and the biotechnology industry. The oceans cover nearly 70% of the earth’s surface and comprise 90–95% of the biosphere by volume of living organisms on earth and thus contain a
Fig. 1 We probably know more about the Moon than we do about the bottom of the sea (image from the IMAX film “Volcanoes of the Deep Sea,” courtesy Rutgers University and The Stephen Law Company)
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tremendous range of diverse biological resources and unique conditions. For example, the largely unexplored deep-sea hydrothermal vents (Fig. 1) represent a treasure trove of biodiversity, as do extreme ocean environments such as cold polar waters and the deep ocean floor characterized by intense pressure. Although deep ocean exploration is still in its infancy, many experts now believe that the deep sea harbors are some of the most diverse ecosystems on earth. This diversity holds tremendous potential for human benefit. More than 15,000 natural products have been discovered from marine microbes, algae, and invertebrates, and this number continues to grow. The uses of marine-derived compounds are varied, but the most exciting potential uses lie in the medical realm. More than 28 marine natural products are currently being tested in human clinical trials, with many more in various stages of preclinical development (Maxwell et al. 2005). Marine biotechnology focuses not only on the growing use of marine life in the food, cosmetic, and agricultural industries such as aquaculture, but also on little known forms of deep ocean life. While the goal of modern marine biotechnology is on biomedical applications of natural marine products, we also should consider how these organisms and molecules will be renewably collected from marine life or mined from the sea surface, the subsurface, and the seafloor. Selection of suitable materials and coatings for sea surface or underwater processing facilities will be critical to minimize environmental impact and to maximize process efficiency. Self-cleaning and drag-reducing materials also have a key role to play as assistive technologies in the seeding, harvesting, and development of natural marine products. Bioprospecting inspires businessmen to consider the value of marine conservation, because new cures and new materials help to put a price tag on the value of biodiversity research. Theoretically, nature has an “inspiration value” that justifies its protection.
1 Species Richness and Diversity of Marine Biomaterials An exciting “marine pipeline” of new drugs and biomaterials has emerged from intense efforts over the past decade to more effectively explore the rich biological, chemical, and materials diversity offered by marine life. The number of marine taxa, particularly the large complex forms, increased dramatically with the onset of the Cambrian explosion about 540 million years ago (Knoll 2001). Sepkoski’s classic work documented a steady increase in the number of taxa during the Phanerozoic, with the exception of five big events during which diversity suffered mass depletion (see for review Sala and Knowlton 2008). The events at the end of the Ordovician, Permian, and Cretaceous periods were due to only mass extinctions, whereas the loss in diversity in the late Devonian and at the end of the Triassic was a result of low origination as well as high extinction. However, this paradigm of monotonic increase broken only by mass extinction events has been recently questioned because of sampling artifacts associated with the fossil record and some authors suggest that during some geological periods taxonomic diversity might have remained stable (Bambach et al. 2004).
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Ecosystems have also changed over geological time with feedbacks that have changed earth’s physical properties (e.g., creation of the present atmosphere). Although the information on ecosystem diversity over geological times is not as good as that on taxonomic diversity, it is clear that the number of marine ecosystems and ways of making a living has increased since the primordial pre-Cambrian ocean. Examples include the marine Mesozoic revolution (MMR) that followed the end-Permian mass extinction. During the MMR, there was a proliferation of new plant and animal taxa associated with an increase in trophic diversity, from infaunal suspension and detritus feeders (animals that live in the sediment and filter the water or eat detritus on the bottom) to nektonic carnivores (animals that swim and eat invertebrates and fish in the water column). Understanding mass extinctions is of particular importance because some have argued that the impact of humans could potentially approach the scale of that caused by asteroids. We clearly have yet to approach the 98% species extinction level that occurred at the end of the Permian, but this should not be used to justify complacency, as threshold effects could result in rapid collapses with little warning. Extinction events associated with global warming are potentially very informative with respect to understanding how marine organisms might respond to a warmer world. Knowledge about changes in biodiversity in the past is essential to understanding potential scenarios of change in the future. Identifying the knowable unknowns will help us to identify research priorities and understand the limitations of management. Before humans began to significantly exploit the ocean, the only disturbances resetting the successional clock and causing sudden declines in biodiversity at all levels were environmental disturbances of the type outlined above. However, human activities are without doubt now the strongest driver of change in marine biodiversity at all levels of organization; hence, future trends will depend largely on humanrelated threats (Barnes 2002). Although marine species richness may only total 4% of global diversity, life began in the sea and much of the diversity in the deep branches of life’s tree is still primarily or exclusively marine (Briggs 1994). For example, 35 animal phyla are found in the sea, 14 of which are exclusively marine, whereas only 11 are terrestrial and only one exclusively so (Ormond et al. 1997). It is not truly known how many species inhabit the world’s oceans; however, it is becoming increasingly clear that the number of microbial species is many times larger than previously estimated, enough that marine species in total may approach 1–2 million.Our understanding of major changes in marine diversity over deep time is comparatively good, thanks to the excellent fossil record left by many marine organisms, although considerable sampling problems limit the potential for accurate, fine grained analyses. In contrast, our knowledge of marine diversity at present is poor compared to our knowledge for terrestrial organisms and an appreciation for the dramatic changes in marine ecosystems that have occurred in historic times is only just beginning to emerge. There are approximately 300,000 described marine species, which represent about 15% of all described species. There is no single listing of these species, but
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any such listing would be only an approximation owing to uncertainty from several sources. As a consequence, the total number of marine species is not known to even an order of magnitude, with estimates ranging from 178,000 species to more than 10 million species (Poore and Wilson 1993). The two biggest repositories of marine biodiversity are coral reefs (because of the high number of species per unit area) and the deep sea (because of its enormous area). Estimates for coral reefs range from 1 to 9 million species, but are very indirect, as they are based on a partial count of organisms in a large tropical aquarium or on extrapolations stemming from terrestrial diversity estimates. Estimates for the deep sea are calculated using actual field samples, but extrapolations to global estimates are highly controversial. The largest estimate (10 million benthic species) was based on an extrapolation of benthic macrofauna collected in 233 box cores (30 × 30 cm each) from 14 stations, although others suggested 5 million species as a more appropriate number (Grassle and Maciolek 1992; Gray 2001; O’Dor and Gallardo 2005; Pimm and Raven 2000). What is clear from these data is that we have a remarkably poor grasp of what lives in the ocean today, although ongoing programs such as the Census of Marine Life (Malakoff 2003) should yield greatly improved estimates in the not too distant future. However, intensive surveys of individual groups point to the enormous scale of the task ahead. Thus, marine biotechnology’s promising future reflects the tremendous biodiversity of the world’s oceans and seas. The promise of marine biotechnology also reflects many marine organisms’ need to adapt themselves to the extremes of temperature, pressure, and darkness that are found in the world’s seas. The demands of the marine environment have led these organisms to evolve unique structures, metabolic pathways, reproductive systems, and sensory and defense mechanisms. Many of these same properties have important potential applications in the human world. There are no doubts that diversity of biological materials of marine origin is almost equivalent to the marine biodiversity. However, in contrast to zoological classification of species, the corresponding classification of biological materials is not yet established. I make an attempt here to represent a very preliminary and generalized classification of biological materials of marine origin as follows: • Biomineralized Structures and Biocomposites (skeletal formations, macroand microscleres, spicules, spines, bristles, cell walls, cyst walls, loricae, etc.) • Non-mineralized Structures (bioelastomers like abductin, resilin, gorgonin, spongin; antipathin, bioadhesives like byssus and related DOPA-based polymers; biocements, and glues) • Macromolecular Biopolymers (marine polysaccharides of algal origin; chitin, collagen). • Self-made Biological Materials (tubular structures of marine invertebrates like some foraminifera or worms which are made due to co-agglutination of external mineral debris, sand grains, or other particles).
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Fig. 2 Diversity of biological materials from marine invertebrates: (a) sea urchin (calcareous spines and tests, echinochrom-like pigments); (b) holoturia (collagens, Cuvierian tubules as bioadhesives), (c) bivalvia molluscs (shell, mother pearl, nacre, byssus); (d) sea stars (mineralized structures, adhesives) (image courtesy A.V. Ratnikov)
In principle, each marine organism possesses both mineralized and nonmineralized structures (Fig. 2); however, there are numerous species which are lacking a mineral component. In these cases, some species use complex crosslinking-based biochemical reactions which lead to hardening (sclerotization) of organic matter; while another species developed unique constructs wherein mineralized and non-mineralized skeletal parts are distributed alternately (e.g., Isididae bamboo corals). All of the examples listed above have in common their tremendous biomimetic potential—the driving force for bioinspiration and development of novel materials as well as technologies. For example, some marine organisms are sessile and must employ sophisticated methods to compete for a place to anchor. Barnacles and mussels, which depend on their ability to attach to solid surfaces for survival, have developed bio-adhesives that stick to all kinds of wet surfaces (Deming 1999; Dickinson et al. 2009). Current research into the ways that marine organisms adhere to wet surfaces, or prevent other organisms from adhering to them, is yielding useful new technologies. These technologies include both adhesion inhibitors (e.g., antifouling coatings for ship hulls) and new types of adhesive such as medical “glues” for joining tissue or promoting cell attachment in tissue engineering applications (Dalsin et al. 2003; Cha et al. 2008; Hwang et al. 2007a, b; Kamino 2008). In spite of the sea’s vast potential as a source of new biotechnologies, this domain remains relatively unexplored and few marine biotechnology products and services have been commercialized to date. Indeed, the vast majority of marine
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organisms (primarily microorganisms) have yet to be identified. Even for known organisms, there is insufficient knowledge to permit their intelligent management and application.
References Attaway DH, Zaborsky OR (eds) (1993) Marine biotechnology. Pharmaceuticals and bioactive natural products, vol I. Plenum, New York Bambach RK, Knoll AH, Wang SC (2004) Origination, extinction, and mass depletions of marine diversity. Paleobiology 30:522–522 Barnes DKA (2002) Biodiversity – invasions by marine life on plastic debris. Nature 416:808 Briggs JC (1994) Species diversity: land and sea compared. Syst Biol 43:130–135 Cha HJ, Hwang DS, Lim S (2008) Development of bioadhesives from marine mussels. Biotechnol J 3(5):631–638 Dalsin JL, Hu BH, Lee BP, Messersmith PB (2003) Mussel adhesive protein mimetic polymers for the preparation of nonfouling surfaces. J Am Chem Soc 125:4253 Deming TJ (1999) Mussel byssus and biomolecular materials. Curr Opin Chem Biol 3:100–105 Dickinson GH, Vega IE, Wahl KJ et al (2009) Barnacle cement: a polymerization model based on evolutionary concepts. J Exp Biol 212:3499–3510 Grassle JF, Maciolek NJ (1992) Deep-sea species richness: regional and local diversity estimates from quantitative bottom samples. Am Nat 139:313–321 Gray JS (2001) Marine diversity: the paradigms in patterns of species richness examined. Sci Mar 65:41–46 Hwang DS, Gim Y, Yoo HJ, Cha HJ (2007a) Practical recombinant hybrid mussel bioadhesive fp-151. Biomaterials 28:3560–3567 Hwang DS, Sim SB, Cha HJ (2007b) Cell adhesion biomaterial based on mussel adhesive protein fused with RGD peptide. Biomaterials 28:4039–4045 Kamino K (2008) Underwater adhesive of marine organisms as the vital link between biological science and materials science. Mar Biotechnol (NY) 10(2):111–121 Knoll AH (2001) Life on a young planet: the first three billion years of evolution on earth. University Press, Princeton, NJ Malakoff D (2003) Scientists counting on census to reveal marine biodiversity. Science 302:773 Maxwell S, Ehrlich H, Speer L (2005) Medicines from the deep: the importance of protecting the high seas from bottom trawling. Natural resources defense council issue paper. Newman DJ, Cragg GM (2004) Marine natural products and related compounds in clinical and advanced preclinical trials. J Nat Prod 67:1216–1218 O’Dor R, Gallardo VA (2005) How to census marine life: ocean realm field projects. Sci Mar 69(Suppl 1):181–189 Ormond R, Gage J, Angel M (eds) (1997) Marine biodiversity: patterns and process. University Press, Cambridge, UK Pimm SL, Raven P (2000) Biodiversity – extinction by numbers. Nature 403:843–845 Poore GCB, Wilson GDF (1993) Marine species richness. Nature 361:597–598 Powers DA (1995) New frontiers in marine biotechnology: opportunities for the 21st century. In: Lundin CG, Zilinskas RA (eds) Marine biotechnology in the Asian Pacific region. The Word Bank and SIDA, Stockholm Sala E, Knowlton N (2008) Global marine biodiversity trends. In: Duffy JE (Topic ed), Cleveland CJ (ed) Encyclopedia of earth. National Council for Science and the Environment, Environmental Information Coalition, Washington, DC Weber P (1993) Abandoned seas: reversing the decline of the oceans. WorldWatch Paper No 116, Worldwatch Institute, Washington, DC Weiner RM (1997) Biopolymers from marine prokaryotes. Trends Biotechnol 15:390–394
Contents
Part I
Biomaterials
1 Biomaterials and Biological Materials, Common Definitions, History, and Classification . . . . . . . . 1.1 Definitions: Biomaterial and Biological Material 1.2 Brief History of Biomaterials . . . . . . . . . . . 1.3 Classification of Biomaterials . . . . . . . . . . 1.3.1 Metals and Alloys . . . . . . . . . . . . 1.3.2 Ceramics . . . . . . . . . . . . . . . . 1.3.3 Polymers . . . . . . . . . . . . . . . . 1.3.4 Composites . . . . . . . . . . . . . . . 1.4 Requirements of Biomaterials . . . . . . . . . . 1.5 The Future of Biomaterials . . . . . . . . . . . . 1.6 Conclusions . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . Part II
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Biominerals and Biomineralization
2 Biominerals . . . . . . . . . . . . . . . . . . . . 2.1 Biominerals of Marine Invertebrate Origin . 2.1.1 Calcium-Based Biominerals . . . 2.1.2 Magnesium-Based Biominerals . . 2.1.3 Barite-Based Biominerals . . . . . 2.1.4 Fe-Based Biominerals . . . . . . . 2.1.5 Vanadium (Biomineral?) . . . . . 2.1.6 Strontium-Based Biominerals . . . 2.1.7 Boron . . . . . . . . . . . . . . . 2.1.8 Titanium-Based Biominerals . . . 2.1.9 Copper-Based Biominerals . . . . 2.1.10 Zinc-Based Biominerals . . . . . . 2.1.11 Manganese Oxides . . . . . . . . 2.1.12 Germanium-Based Biominerals . . 2.1.13 Silica-Based Biominerals . . . . . 2.2 Conclusion . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .
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3 Biomineralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Biomineralization–Demineralization–Remineralization Phenomena in Nature . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Principles of Demineralization: Isolation of Organic Matrices 4.2 Structural Biopolymers as Common Templates for Biomineralization . . . . . . . . . . . . . . . . . . . . . . 4.2.1 Chitin . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2 Collagen . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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7 Paleodictyon Honeycomb Structure . . . . . . . . . . . . . . . . . . 7.1 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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5 Multiphase Biomineralization . . . . . . . . . . . . . . . 5.1 Silica–Aragonite–Chitin Biocomposites in Demosponges (Demospongiae: Porifera) . . . . . 5.2 Radula as Example of Multiphase Biomineralization 5.3 Silica–Chitin–Apatite Biocomposites of Brachiopoda 5.4 Copepoda Teeth as a Multiphase Biocomposite . . . 5.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . Part III
Biomineralized Structures and Biocomposites
6 Hierarchical Biological Materials . . . . . . . . . . . . . 6.1 Cellular Structures . . . . . . . . . . . . . . . . . . 6.2 Honeycomb Structures: From Nano- to Macroscale . 6.3 Siliceous Honeycomb Cellular Structures in Diatoms 6.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .
8 Peculiarities of the Structural Organization of the Glass Sponges’ (Hexactinellida) Skeletons . . . . . . . . . . . . . . 8.1 Glass Sponges (Hexactinellida) . . . . . . . . . . . . . 8.2 Demosponges (Demospongiae) . . . . . . . . . . . . . 8.3 Lithistid Sponges . . . . . . . . . . . . . . . . . . . . . 8.4 Cellular Structures in Glass Sponges . . . . . . . . . . . 8.5 Eiffel’s Design in Skeletal Frameworks of Glass Sponges 8.6 Spiculogenesis . . . . . . . . . . . . . . . . . . . . . . 8.7 The Role of the Organic Matrix in Biosilica Formation by Sponges . . . . . . . . . . . . . . . . . . . . . . . . 8.7.1 Silicatein-Based Silicification . . . . . . . . . . 8.7.2 Chitin- and Collagen-Based Silicification . . .
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8.8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
180 180
9 Phenomenon of Interspace Mineralization in the Bilayered Organic Matrix of Deep-Sea Bamboo Coral (Anthozoa: Gorgonacea: Isididae) . . . . . . . . . . . . . . . . . . . . . . . . . 9.1 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
187 193 193
10
Bamboo Corals as Living Bone Implants . . . . . . . . . . . . . . . 10.1 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
195 199 199
11
Sand Dollar Spines . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
201 206 207
12
Molluscs Spicules . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1 Spicules of Nudibranchia . . . . . . . . . . . . . . . . . . . . . 12.2 Spicules in Aplacophora . . . . . . . . . . . . . . . . . . . . . 12.3 Spicules in Polyplacophora (Chitons) . . . . . . . . . . . . . . 12.4 Onchidella Spicules . . . . . . . . . . . . . . . . . . . . . . . 12.4.1 Onchidella celtica: Silica-Containing Slug or Mystery? 12.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
211 212 219 225 228 230 237 237
Part IV 13
Non-mineralized Structures
Spongin . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.1 Spongin as a Halogenated Scleroprotein . . . . . . . 13.2 Spongin as a Collagenous Protein . . . . . . . . . . 13.3 Function of Spongins in Natural Environments . . . 13.4 Mechanical Properties of Spongin-Based Skeletons . 13.5 Spongin as a Three-Dimensional Scaffold for Tissue Engineering . . . . . . . . . . . . . . . . . . . . . . 13.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .
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245 246 248 251 252
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252 254 254
14
Gorgonin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.1 Introduction into the History and Chemistry of Gorgonin . . . 14.2 Mechanical Properties of Gorgonin-Based Skeletons . . . . . 14.3 Gorgonin-Based Skeletons and Paleoceanographic Dynamics 14.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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257 258 262 265 266 267
15
Antipathin . . . . . . . . . . . . . . . . . . . . . . . . . 15.1 Brief Introduction into Black Corals . . . . . . . . 15.2 Chemistry of Black Corals . . . . . . . . . . . . . 15.3 Material Properties of Antipathin-Based Skeletons
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271 271 273 275
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15.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
276 277
16
Rubber-Like Bioelastomers of Marine Origin 16.1 Hinge Ligament . . . . . . . . . . . . . 16.2 Chemistry of the Hinge Ligament . . . . 16.3 Structural Features of Hinge Ligaments . 16.4 Conclusion . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .
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279 279 281 284 286 286
17
Capsular Bioelastomers of Whelks . . . . . . . . . . . . . . . . . . 17.1 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
289 296 296
18
Byssus: From Inspiration to Development of Novel Biomaterials . 18.1 Byssus—An Ancient Marine Biological Material . . . . . . . 18.2 Why Molluscs Produce Different Kinds of Byssus . . . . . . 18.3 Chemistry of Byssus and Related Proteins . . . . . . . . . . . 18.3.1 M. edulis Adhesive Protein-2 (Mefp-2) . . . . . . . . 18.3.2 M. edulis Adhesive Protein-4 (Mefp-4) . . . . . . . . 18.4 Biomechanics and Materials Properties of Byssus . . . . . . . 18.5 Biocomposite-Based Byssus . . . . . . . . . . . . . . . . . . 18.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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299 301 303 305 307 308 309 312 313 314
19
Abductin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.1 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
319 322 322
20
Resilin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.1 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
323 325 326
21
Adhesion Systems in Echinodermata 21.1 Sea Urchins . . . . . . . . . . . 21.2 Sea Cucumbers . . . . . . . . . 21.3 Sea Stars . . . . . . . . . . . . 21.4 Conclusion . . . . . . . . . . . References . . . . . . . . . . . . . . .
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327 328 329 331 333 333
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Adhesive Gels from Marine Gastropods (Mollusca) 22.1 The Role of Mucus in Gastropod Gels . . . . . 22.2 Chemistry of Gastropod Gels . . . . . . . . . . 22.3 Possible Mechanism of Cross-Linking . . . . . 22.4 Conclusion . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . .
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335 336 338 338 339 340
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Barnacle Cements . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.1 Barnacles—Crustaceans That Mimic Molluscs . . . . . . . . . 23.2 “First-Kiss” Adhesion Behavior in Barnacles . . . . . . . . . .
341 341 343
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23.3 Barnacle Cements . . . . . . . . . . . . . . . . . . . . . . . . 23.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Part V
345 346 347
Suction-based Adhesion in Marine Invertebrates
24
Suctorian Protozoa . . 24.1 Suctorian Ciliates 24.2 Conclusion . . . References . . . . . . .
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351 351 356 356
25
Trichodina Sucker Disk . . . . . . . . . . . . . . . . . . . . . . . . 25.1 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
359 362 362
26
Giardia Suction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.1 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
365 368 368
27
Suction in Molluscs 27.1 Limpets . . . 27.2 Cephalopods 27.3 Conclusion . References . . . . .
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371 371 372 376 377
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Halogenated Biocomposites . . . . . 28.1 Polychaetes Jaws . . . . . . . . 28.2 Crustaceans Alternative Cuticles 28.3 Conclusion . . . . . . . . . . . References . . . . . . . . . . . . . . .
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379 382 386 388 388
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Chitin–Protein-Based Composites . . . . . . . . . . . . . . 29.1 The Highly Flexible Setae of Hairy Lobster K. hirsuta 29.2 S. crosnieri . . . . . . . . . . . . . . . . . . . . . . . 29.3 Structural Features of E. sinensis Setae . . . . . . . . 29.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .
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391 392 395 398 402 403
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Part VI 30
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Macromolecular Biopolymers
Chitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30.1 Two- and Three-Dimensional Chitinous Scaffolds of Poriferan Origin . . . . . . . . . . . . . . . . . . . . 30.2 Modern View on Toxicity, Immunology, Biodegradation, and Biocompatibility of Marine Chitin . . . . . . . . . . 30.2.1 Toxicity . . . . . . . . . . . . . . . . . . . . . 30.2.2 Immunology . . . . . . . . . . . . . . . . . . . 30.2.3 Biodegradability . . . . . . . . . . . . . . . . .
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30.2.4 Biocompatibility . 30.2.5 Wound Dressing . . 30.2.6 Tissue Engineering 30.3 Conclusion . . . . . . . . . References . . . . . . . . . . . . .
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418 420 422 422 423
Marine Collagens . . . . . . . 31.1 Poriferan Collagens . . . 31.2 Coelenterates Collagens 31.3 Molluscs Collagens . . . 31.4 Echinoderm Collagens . 31.5 Conclusion . . . . . . . References . . . . . . . . . . .
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427 428 430 434 435 438 438
32
Self-Made Biological Materials of Protozoans 32.1 Testate Amoeba . . . . . . . . . . . . . . 32.2 Gromiids . . . . . . . . . . . . . . . . . 32.3 Tintinnids . . . . . . . . . . . . . . . . . 32.4 Xenophyophores . . . . . . . . . . . . . 32.5 Conclusion . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .
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445 445 447 448 450 452 452
33
Foraminifera . . . . . . . . . . . . . . . . . . . . . . . . . 33.1 Foraminifera: Agglutination Versus Biomineralization 33.2 Silk-Based Shell of Stannophyllum zonarium . . . . . 33.3 Sponge-Imitating Giant Foraminifer . . . . . . . . . . 33.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .
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455 455 459 461 463 463
34
Polychaete Worms: From Tube Builders to Glueomics . . . . . . . 34.1 Larvae Metamorphosis and the Initial Phases of Tube Formation 34.2 The Chemistry of Tube Construction . . . . . . . . . . . . . . . 34.3 Features of the Pectinariid Tubes . . . . . . . . . . . . . . . . . 34.4 Biomimetic Potential of Polychaetes Bioadhesives . . . . . . . 34.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
465 468 471 474 476 479 480
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Part VII
Part VIII 35
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Self-Made Biological Materials
Extreme Biomimetics
Life in Extreme Environments: From Bacteria to Diatoms . . . . . 35.1 Eurythermal Marine Biota as Source for Development of Novel Biomaterials . . . . . . . . . . . . . . . . . . . . . . 35.2 Biosilicification in Geothermal and Hydrothermal Environments 35.3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
485 486 491 496 496
Contents
Epilogue . . . . . . . . . . . References . . . . . . . . Additional Sources Internet Resources
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499 502 503 503
Addendum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
505 522 549
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
565
Part I
Biomaterials
The union of biology with materials science and engineering represents one of the most exciting scientific prospects of our time. As currently few biologists know much about engineering and even fewer engineers know much about biology, the expectations of future advances seem unbounded. Robert O. Ritchie (2008)
Materials Science and Engineering is a young and vibrant discipline that has, since its inception in the 1950s, expanded into three directions: metals, polymers, and ceramics (and their mixtures, composites). Biological materials are being added to its interests, starting in the 1990s, and are indeed its new future (Meyers et al. 2008). Biomaterials represent a central theme in a majority of the problems encountered. These fields have evolved into a very interdisciplinary arena building on traditional engineering principles that bridge advances in the areas of materials science, life sciences, nanotechnology, and cell biology, to name a few (Wnek and Bowlin 2008). This trend of interdisciplinary research to solve the most challenging yet compelling medical problems has been embraced in the field and is leading to the betterment of human health. It is evident that the fields of biomaterials and biomedical engineering are continually changing due to the rapid creation and advancement in technology in more traditional areas as well as rapidly developing areas (e.g., tissue engineering). Indeed, the field of biomaterials has become one of the fastest growing areas in materials science, as bioengineering has become in engineering (Ritchie 2008).
References Meyers MA, Chen PY, Lin AYM et al (2008) Biological materials: structure and mechanical properties. Prog Mater Sci 53:1–206 Ritchie RO (2008) Editorial. J Mech Behav Biomed Mater 1(3):207 Wnek GE, Bowlin GL (2008) Encyclopedia of biomaterials and biomedical engineering, 2nd ed (four-volume set). Informa Healthcare. London, New York
Chapter 1
Biomaterials and Biological Materials, Common Definitions, History, and Classification
Abstract Biomaterial can be defined as any material used to make devices to replace a part or a function of the body in a safe, reliable, economic, and physiologically acceptable manner. Biological material is a material produced by a biological system. Most biological materials can be considered as composites. Composite materials are those that contain two or more distinct constituent materials or phases, on a microscopic or macroscopic size scale. The modern biomaterials science is defined and explained through the introduction of biotechnology and advances in the understanding of human tissue compatibility. Developing from bio-inert materials to biodegradable materials, biomaterials are widely used in medical devices, tissue replacement, and surface coating applications. In this chapter the history of biomaterials, their classification, requirements, state of the art, as well as a future are discussed.
1.1 Definitions: Biomaterial and Biological Material According to Williams (1999), biomaterials science is the study of the structure and properties of biomaterials, the mechanisms by which they interact with biological systems and their performance in clinical use. I agree with the explanation of biomaterial and biological material proposed by Park and Lakes in the third edition of their renowned book (Park and Lakes 2007). According to these authors, a biomaterial can be defined as any material used to make devices to replace a part or a function of the body in a safe, reliable, economic, and physiologically acceptable manner. A variety of devices and materials are used in the treatment of disease or injury. Commonplace examples include sutures, tooth fillings, needles, catheters, bone plates. A biomaterial is a synthetic material used to replace part of a living system or to function in intimate contact with living tissue. The Clemson University Advisory Board for Biomaterials has formally defined a biomaterial to be “a systemically and pharmacologically inert substance designed for implantation within or incorporation with living systems” (cited by Park and Lakes 2007).
H. Ehrlich, Biological Materials of Marine Origin, Biologically-Inspired Systems 1, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9130-7_1,
3
4
1
Biomaterials and Biological Materials
By contrast, a biological material is a material, such as bone, skin, or artery, produced by a biological system. The major difference between biological materials and biomaterials (implants) is viability. There are other equally important differences that distinguish living materials from artificial replacements. First, most biological materials are continuously bathed with body fluids. Exceptions are the specialized surface layers of skin, hair, nails, hooves, and the enamel of teeth. Second, most biological materials can be considered as composites (Park and Lakes 2007). Composite materials are those that contain two or more distinct constituent materials or phases, on a microscopic or macroscopic size scale. The term “composite” is usually reserved for those materials in which the distinct phases are separated on a scale larger than the atomic, and in which properties such as the elastic modulus are significantly altered in comparison with those of a homogenous material. Accordingly, fiberglass and other reinforced plastics as well as bone are viewed as composite materials, but alloys such as brass or metals such as steel with carbide particles are not. Natural composites often exhibit hierarchical structures in which particulate, porous, and fibrous structural features are seen on different length scales. Composite materials offer a variety of advantages in comparison with homogenous materials. However, in the context of biomaterials, it is important that each constituent of the composite be biocompatible and that the interface between constituents not be degraded by the body environment. Composites currently used in biomaterial applications include the following: dental filling composites; bone particle or carbon fiber reinforced methyl methacrylate bone cement and ultrahigh molecular weight polyethylene; and porous surface orthopedic implants (Park and Lakes 2007). A Biomedical material (Williams 1999) is material intended to interface with biological systems to evaluate, treat, augment or replace any tissue, organ, or function of the body. There are numerous papers in the modern literature where authors use terms like “nanomaterials” and “bionanomaterials.” According to Williams (2008, 2009), the term “nanomaterial” should not exist because it is senseless. The discussion about nanomaterial provides a hint of the analysis of a biomaterial that follows, since a prefix which is an indicator of scale cannot specify the integer that follows (in this case a material) unless that integer can be qualified by that scale. In other words, it is very clear what a nanometer is because nano means 10−9 and a meter is a measure of length. In the case of the term nanomaterial, the question arises, what is it about the material that is 10−9 ? Is it the dimension of a crystal within the material, or of a grain boundary, a domain, or a molecule, or is it a parameter of a surface feature of the sample, or perhaps of the resistivity or thermal conductivity of the material. “Clearly this is nonsense,—said Williams,—but one has to accept that nanomaterials are here to stay, with even some journal titles containing the word” (Williams 2009). There are both nanobiomaterials and nanostructured biomaterials, which should be differentiated from each other (Dorozhkin 2009). Nanobiomaterials refers to
1.2
Brief History of Biomaterials
5
individual molecular level biomaterials, such as single proteins, while nanostructured biomaterials refers to any biomaterials whose structure or morphology can be engineered to get features with nanometer-scale dimensions (Thomas et al. 2006). In this book, I use the term “biological materials.” However, some of them, like chitin and collagens from marine invertebrates as well as coral hydroxyapatite, have been described in the literature as biomaterials because of their applications in biomedicine and tissue engineering.
1.2 Brief History of Biomaterials Development of biomaterials science from historical point of view has been thoroughly described by Popp (1939), Weinberger (1948), Harkins and Koepp Baker (1948), Baden (1955), Sivakumar (1999), Ratner and Bryant (2004), Staiger et al. (2006), Park and Lakes (2007). Based on data reported in these works, I take the liberty to represent a brief history of biomaterials as follows. The Egyptians during the reign of Ramses II had specialists for the treatment of the teeth and the oral cavity. Palatal defects were treated at that time with laminated sheets of gold. The Edwin Smith Papyrus, which is about 2000 years old, contains accounts of fractures of the facial bones and several case reports, and Case 15 describing a traumatic perforation of the maxilla complicated by injury to the zygomatic arch. It is likely that some forms of obturators were used in Egypt as early as 2600 BC . Popp states that the ancient Egyptians also made artificial ears, noses, and eyes. Galen in the second century BC described clefts of the palate. Some of the earliest biomaterial applications were as far back as ancient Phoenicia where loose teeth were bound together with gold wires to tie artificial ones to neighboring teeth (Teoh 2004). Amatus Lusitanus is credited with having invented the obturator between 1511 and 1561. The first scientific description of congenital and acquired defects of the maxilla and their treatment was given by Pare in his Chirurgie in 1541. He specifically described defects of the palate with bone destruction caused by arquebus shots, stab wounds, or syphilitic gumma, describing also the accompanying speech deficiency and giving general principles of treatment. He used a flat, vaulted, metallic plate in gold or silver with a sponge attached to it. The sponge was introduced into the defect, where it expanded with readily absorbed nasal and oral secretions, thus holding the obturator base in position. Pare mentioned the speech improvement resulting from the use of the appliance. Hollerius in 1552, following Pare’s work, also advocated the use of sponges fixed on a gold plate. In 1565 Alexander Petronius described palatine obturators (De morbo Gallico). He used wax, tow, and sponges for the bulb section of the appliance. Further progress was made by Guillemeau, who described a technique for the construction of obturators around the year 1600. Pierre Fauchard, often called “the Father of Dentistry,” described five types of obturators in his classic work, Le Chirurgien-Dentiste. He was the first to discard sponges and advocate an obturator-bulb fixed to a denture base. He also described the retention of full upper dentures by means of atmospheric pressure, adhesion,
6
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Biomaterials and Biological Materials
and peripheral seal, methods which have application in obturator therapy. In 1756 Lorenz Heister further perfected Fauchard’s appliances. Iron wire was reported to have been used as early as 1775 for fracture fixation (Wnek and Bowlin 2008). Delabarre introduced the soft-hinged velum in 1820. Bourdet noticed the tendency of acquired palatal defects to close spontaneously (which might be only an observation of local recurrence of malignant disease). He designed obturators consisting of a plate of gold held by ligature wires to the abutment teeth. Until about 1820, obturators were primarily used for the treatment of acquired defects of the hard palate. Claude Martin used for the first time on 13 April 1887 an immediate prosthetic appliance in conjunction with surgery. The poor results obtained by his predecessors in the restoration of the resected mandible (partial and hemi-resection) prompted his work. According to Martin, immediate prosthesis consists of the replacement of the resected bone fragment by an appliance fixed in the soft tissues before closure of the wound. This idea is the first hint of the modern principles of immediate bone grafting, fixed Vitallium (cobalt–chromium–molybdenum alloys), or tantalum implants used for the restoration of bone loss in the mandible in today’s plastic and reconstructive surgery. With the advent of the Iron Age and Industrial Revolution, steel materials were used in the nineteenth century as bone plates and screws to fix fractures. Fixing fractures with screws allowed a stronger fixity than the earlier method of fixing with metallic wires. Steel made from nickel-plating steel and vanadium steel later replaced carbon steel materials as steel corrodes easily in the human body. However, these newer materials were not sufficiently corrosion resistant. It also became clear that they become toxic inside the human body. Historically speaking, until Dr. J. Lister’s aseptic surgical technique was developed in the 1860s, attempts to implant various metal devices such as wires and pins constructed of iron, gold, silver, platinum were largely unsuccessful due to infection after implantation. The aseptic technique in surgery has greatly reduced the incidence of infection. Many recent developments in implants have centered around repairing long bones and joints. In the early 1900s bone plates were successfully implemented to stabilize bone fractures and accelerate their healing. Lane of England designed a fracture plate in the early 1900s using steel. Sherman of Pittsburgh modified the Lane plate to reduce the stress concentration by eliminating sharp corners. He used vanadium alloy steel for its toughness and ductility. R (Co–Cr-based alloy) was found to be the most inert material Subsequently, Stellite for implantation by Zierold in 1924. Soon 18-8 (18 w/o Cr, 8 w/o Ni) and 18-8 s Mo (2–4 w/o Mo) stainless steels were introduced for their corrosion resistance, with 18-8 s Mo being especially resistant to corrosion in saline solution. Later, another R was introduced into medical practice. alloy (19 w/o Cr, 9 w/o Ni) named Vitallium The first use of magnesium was reported by Lambotte in 1907, who utilized a plate of pure magnesium with gold-plated steel nails to secure a fracture involving the bones of the lower leg (Lambotte 1932). The attempt failed as the pure magnesium metal corroded too rapidly in vivo, disintegrating only 8 days after surgery and producing a large amount of gas beneath the skin.
1.2
Brief History of Biomaterials
7
Albee and Morrison first studied calcium phosphate (CaP) compounds in 1920, injecting tricalcium phosphate (TCP) into animals to test its efficacy as a bone substitute. A noble metal, tantalum, was introduced in 1939, but its poor mechanical properties and difficulties in processing it from the ore made it unpopular in orthopedics, yet it found wide use in neurological and plastic surgery. Smith-Petersen in 1931 designed the first nail with protruding fins to prevent rotaR . tion of the femoral head. He used stainless steel but soon changed to Vitallium Thornton in 1937 attached a metal plate to the distal end of the Smith-Petersen nail and secured it with screws for better support. Later in 1939, Smith-Petersen used an artificial cup over the femoral head in order to create new surfaces to substiR R R , Bakelite , and Vitallium . tute for the diseased joints. He used glass, Pyrex The latter was found more biologically compatible, and 30–40% of patients gained usable joints. Similar mold arthroplastic surgeries were performed successfully by the Judet brothers of France, who used the first biomechanically designed prosthesis made of an acrylic (methyl methacrylate) polymer. The same type of acrylic polymer was also used for corneal replacement in the 1940s and 1950s due to its excellent properties of transparency and biocompatibility. Thus, according to Ratner and Bryant (2004), the modern era of medical implants might be traced back to an observation made by British ophthalmologist Harold Ridley in the late 1940s. While examining Spitfire fighter pilots who had shards of canopy plastic unintentionally implanted in their eyes from enemy machine gun fire, he noted that these shards seemed to heal without ongoing reaction. He concluded that the canopy plastic, poly(methyl methacrylate), might be appropriate for fashioning implant lenses for replacing cataractous natural lenses. His first implantation of such a lens was in 1949. His observation and innovation led to the development of modern intraocular lenses (IOLs) that are now being implanted in over 10 million human eyes each year and have revolutionized treatment for those with cataracts. At about the same time that Harold Ridley was innovating IOLs, Charnley was developing the hip implant, Vorhees invented the vascular graft, Kolff was revolutionizing kidney dialysis, and Hufnagel invented the ball and cage heart valve (Ratner and Bryant 2004). These pioneers, in an era before principles for medical materials were established, proved feasibility, saved lives, and evolved the foundations that we build on today. In 1952, Ray et al. developed hydroxyapatite (HA), a combination of various CaP compounds, testing healing of nonunions in rats and guinea pigs. However, commercial application of HAs did not occur until the 1970s (Weiss 2003). By the late 1960s engineers, chemists, and biologists, in collaboration with physicians, were formalizing design principles and synthetic strategies for biomaterials. In particular, the idea that the release of toxic leachables from biomaterials will adversely affect healing was formalized—this toxicology idea is implicit in today’s definition of biocompatibility. As developments took place in biology and materials science, biomaterials researchers were quick to incorporate these new ideas into biomaterials. By the time of the 1950s–1960s, blood vessel replacements were in clinical trials and artificial heart valves and hip joints were in development.
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Thus, till the polymer industry was developed in 1950s, the metallic materials were mainly used. The first quarter century, 1950–1975, of biomaterials development was dominated by the characteristics of the materials intended for prostheses and medical devices. Blood vessel implants were attempted with rigid tubes made of polyethylene, acrylic polymer, gold, silver, and aluminum, but these soon filled with clot. The major advancement in vascular implants was made by Voorhees, Jaretzta, and Blackmore in 1952, when they used a cloth prosthesis R N copolymer (polyvinylchloride and polyacrylonitrile) and later made of Vinyon R R R R , Dacron , Teflon , and Ivalon . Through the experimented with nylon, Orlon pores of the various cloths a pseudo- or neointima was formed by tissue ingrowths. This new lining was more compatible with blood than a solid synthetic surface, and it prevented further blood coagulation. Heart valve implantation was made possible only after the development of open-heart surgery in the mid-1950s. Starr and Edwards in 1960 made the first commercially available heart valve, consisting of a silicone rubber ball poppet in a metal strut. Concomitantly, artificial heart and heart assist devices have been developed. Important in the early days was the long-term integrity of the biomaterial as well as its non-toxic nature. Biological interactions that were considered included the non-toxic nature of the biomaterial as well as its normal inflammatory and wound healing responses when implanted. Many materials were described as being inert, but this was a confusing descriptor as it did not adequately and appropriately describe material changes following implantation or cell and tissue responses to the implanted biomaterial. It eventually became clear that materials could change without adversely affecting the function and interaction of the biomaterial, prosthesis, or medical device. Likewise, modulation of the inflammatory and wound healing responses could occur without altering the function of the biomaterial, prosthesis, or medical devices. From 1970 to 2000, biological interactions with biomaterials started to be more extensively investigated. The discovery by Hench and co-workers that a range of compositions of modified phosphosilicate glasses has the ability to form a stable chemical bond with living tissues (bone, ligament, and muscle) opened a completely new field in biomedicine (Hench et al. 1971). Since then, many artificial biomaterials based on, or inspired by, Hench’s glasses have been developed and successfully employed in clinical applications for repairing and replacing parts of the human body. This field is continuously expanding: new processing routes have extended the range of applications toward new and exciting directions in biomedicine (Hench and Polak 2002), many of which still rely on the original Hench’s base formulation, 45S5 Bioglass, which has now become the paradigm of bioactive materials. Advances in our knowledge of biological mechanisms, for example, the coagulation, thrombosis, and complement pathways, led to a better understanding of biological interactions with biomaterial surfaces. In the 1980s, the revolution in techniques for the study of cell and molecular biology led to their application to the investigation of interactions occurring at biomaterial interfaces. More recently, with the advent of the areas of tissue engineering and regenerative medicine, heavy emphasis has been placed on biological interactions with biomaterials. What is the state of the art today? Surprisingly, gold is still quite popular!
1.2
Brief History of Biomaterials
9
Recently, it was shown that implants of pure metallic gold release gold ions which do not spread in the body, but are taken up by cells near the implant (Larsen et al. 2008). It was hypothesized that metallic gold could reduce local neuron inflammation in a safe way. Bioliberation, or dissolucytosis, of gold ions from metallic gold surfaces requires the presence of disolycytes, i.e., macrophages, and the process is limited by their number and activity. Novel metal-based biomaterials were also developed during last decade. For example, bulk metallic glasses (BMGs) are a promising biomaterial due to their superior mechanical properties and corrosion and wear resistance over the metallic biomaterials used currently (Ashby and Greer 2006). The in vitro and in vivo results indicate that the BMGs are in general nontoxic to cells and compatible with cell growth and tissue function. Unique about BMGs is that chemistry, atomic structure, and surface topography (Kumar et al. 2009) can all be varied independently and the effect of the individual contribution on the biocompatibility was revealed in this work. The ability to precisely net-shape complex geometries combined in a single processing step, with patterning of the surface, will enable us to program desirable and predictable cellular response into a three-dimensional biomaterial (Schroers et al. 2009). The modern biomaterials science is defined and explained through the introduction of biotechnology and advances in the understanding of human tissue compatibility. Developing from bio-inert materials to biodegradable materials, biomaterials are widely used in medical devices, tissue replacement, and surface coating applications. Without doubts, the market situation is also one of the driving forces in recent times. Improved patient benefits form the most important factor stimulating market growth for biomaterials, where major segments are as usual ceramics, metals, polymers, and composites. Reconstructive surgery and orthobiologics are the dominant segments in orthopedic biomaterials today. Placement of endosseous implants has improved the quality of life for millions of people. It is estimated that over 500,000 total joint replacements, primarily hips and knees, and between 100,000 and 300,000 dental implants are used each year in the United States alone (Wnek and Bowlin 2008). Total joint arthroplasty relieves pain and restores mobility to people such as those afflicted with osteoarthritis, and dental implants provide psychological and aesthetic benefits in addition to improving masticatory function for edentulous patients. Modern biomaterials found applications not only in orthopedic, cardiovascular, gastrointestinal, wound care, urology, and plastic surgery, but in such directions as brain repair (Zhong and Bellamkonda 2008). As reported in Nature Reviews article (Orive et al. 2009), recently developed biomaterials can enable and augment the targeted delivery of drugs or therapeutic proteins to the brain, allow cell or tissue transplants to be effectively delivered to the brain, and help to rebuild damaged circuits. Similarly, biomaterials are being used to promote regeneration and to repair damaged neuronal pathways in combination with stem cell therapies. Many of these approaches are gaining momentum because nanotechnology allows greater control over material–cell interactions that induce specific developmental processes and cellular responses including differentiation, migration and outgrowth.
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1.3 Classification of Biomaterials The reader can find different kinds of classification proposed for biomaterials in the literature, especially in the books listed in the Table 1.1. Due to limited space, I include in this chapter only very common information about this topic.
Table 1.1 Books related to biomaterials Year
Title
Author(s)
Publisher
1948
An Introduction to the History of Dentistry with Medical and Dental Chronology and Bibliographic Data Cell Wall Mechanics of Wood Tracheids On Growth and Form, 2nd ed. Strength of Biological Materials
Weinberger BW
The C.V. Mosby Company, St. Louis, D.D.S., New York
Mark RE
Elden HR
Yale University Press, New Haven Cambridge University Press, Cambridge Williams and Wilkins (Company, Baltimore) Gordon and Breach scientific Publishers Prentice-Hall, Englewood Cliffs, NJ Wiley, New York
Fung YC, Perrone N, Anliker M
Prentice-Hall, Englewood Cliffs, NJ
Fraser RDB, MacRae TP, Rogers GE
Thomas, Springfield
Montagna W, Parakkal PF Preston RD
Academic Press, New York Chapman and Hall, London Pitman, London
1967 1968 1970
1970 1971 1971 1972
1972
1974 1974 1975 1975 1976
1976
1977
Physical Properties of plant and Animal Materials Organic Chemistry of Biological Compounds Biophysical Properties of the Skin Biomechanics: Its Foundation and Objectives Keratins: Their Composition, Structure, and Biosynthesis The Structure and Function of Skin, 3rd ed. The Physical Biology of Plant Cell Walls Structural Materials in Animals Biology of the Arthropod Cuticle Mechanical Design in Organism Wood Structure in Biological and Technological Research Chitin
Thompson DW Yamada H (Edited by Evans FG) Mohsenin NN Barker R
Brown CH Neville AC Wainwright SA, Biggs WD, Currey JD, Gosline JW Jeronimidis G. In: Baas P, Bolton AJ, Catling DM Muzzarelli RAA
Springer-Verlag, New York Princeton University Press, Princeton The University Press, Leiden Pergamon Press, UK, Oxford
1.3
Classification of Biomaterials
11 Table 1.1 (continued)
Year
Title
Author(s)
Publisher
1980
Guidelines for Physicochemical Characterization of Biomaterials. Devices and Technology Branch National Heart, Lung and Blood Institute Mechanical Properties of Biological Materials Introduction to Composite Materials Mechanical Properties of Bone
Baier RE
NIH Publication No. 80-2186
Vincent JFV, Currey JD
Cambridge University Press, Cambridge Technomic Pub. Co., Westport, CT American Society of Mechanical Engineers, New York The C.V. Mosby Company, St. Louis, MO Princeton University Press, Princeton Princeton University Press, Princeton Wiley, New York
1980 1980 1981
Tsai SW, Hahn HT Cowin SC
1983
Biomaterials in Reconstructive Surgery
Rubin LR
1984
Mechanical Adaptations of Bone The mechanical Adaptations of Bones Cellulose Chemistry and Its Applications Cellulose Chemistry and Its Applications Cellulose: Structure, Modification, and Hydrolysis Biomechanics: Motion, Flow, Stress, and Growth Handbook of Bioactive Ceramics, Volume II—Calcium Phosphate and Hydroxyapatite Ceramics Biomaterials: Novel Materials from Biological Sources Structural Biomaterials
Currey JD
1984 1985 1985 1986
1990 1990
1991
1991
Currey JD Nevell TP, Zeronian SH Nevell TP, Zeronian SH Young RA, Rowell RM
Fung YC Yamamuro T, Hench L, Wilson J
Springer-Verlag, New York CRC Press, Boca Raton
Byrom D
Macmillan
Vincent JFV
Princeton University Press, Princeton ButterworthHeinemann, Oxford Williams & Wilkins, Baltimore Marcel Dekker, New York
1992
Materials Selection in Mechanical Design
Ashby MF
1992
Allografts in Orthopedic Practice Biological Performance of Materials: Fundamentals of Biocompatibility
A. Czitrom and Gross A
1992
John Wiley and Sons, New York John Wiley and Sons, New York
Black J
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Table 1.1 (continued) Year
Title
Author(s)
Publisher
1992
Biomaterials—An Introduction, 2nd ed. Biological Performance of Materials, 2nd ed. Biomechanics: Mechanical Properties of Living Tissues, 2nd ed. Composite Materials for Implant Applications in the Human Body Composite Materials: Engineering and Science Applied Dental Materials
Park JB and Lakes RS
Plenum Press, New York Marcel & Dekker, New York Springer-Verlag, New York
1992 1993
1993
1994 1994 1994
1994
1994
1995
1995
1995
1996
1996
1997 1997
Implantation Biology: The Host Response and Biomedical Devices Hierarchical Structures in Biology as a Guide for New Materials Technology
Hierarchical Structures in Biology as a Guide for New Materials Technology Proteins at Interfaces II. Fundamentals and Applications Self-reinforced Bioabsorbable Polymeric Composites in Surgery Biomedical Applications of Synthetic Biodegradable Polymers Biomaterials Science: An Introduction to Materials in Medicine An Introduction to Composite Materials Biomechanics: Circulation, 2nd ed. Protein-Based Materials
Black J Fung YC
Jamison RD and Gilbertson LN Matthews FL and Rawlings RD McCabe JF Greco RS
National Materials Advisory Board, Commission on engineering and Technical systems, National research Council, NMAB- 464 Tirrell DA
Horbett TA, Brash JL
Rokkamen P, Törmälaö P
American Society of Testing and Materials, Philadelphia, USA Chapman & Hall, London Blackwell Science Publications, Oxford CRC Press, London
National Academy Press, Washington, DC
National Academy Press, Washington, DC American Chemical Society, Washington, DC Tampereen, Pikakapio, Tampere, Finland
Hollinger JO
CRC Press, London
Ratner BD, Hoffman AS, Schoen FJ, and Lemons JE Hull D and Clyne TW
Elsevier Science, New York
Fung YC McGrath KP, Kaplan DL
Cambridge University Press, Cambridge, UK Springer-Verlag, New York Birkhäuser, Boston
1.3
Classification of Biomaterials
13 Table 1.1 (continued)
Year
Title
Author(s)
Publisher
1998
The Chemistry, Biology, and Medical Applications of Hyaluronan and Its Derivatives Biomaterials in Surgery
Laurent TC
Portland Press, London
Walenkamp GHIM, Bakker FC Hill D
New York, Stuttgart
1998 1998
1999
1999 2000
2000 2001 2001 2001 2002
2002
Design Engineering of Biomaterials for Medical Devices Basic Transport Phenomena in Biomedical Engineering A Primer on Biomechanics The History of Metallic Biomaterials, Metallic Biomaterials, Fundamentals and Applications Bone Cements Structural Biological Materials Bone Biomechanics, 3rd ed. Chitin: Fulfilling a Biomaterials Promise Heterogeneous Materials: Microstructure and Macroscopic Properties Integrated Biomaterials Science
Fournier RL
Taylor & Francis, PA, Philadelphia
Lucas GL, Cooke FW, Friis EA Sumita M, Ikada Y, and Tateishi T
Springer, New York
Kühn K-D Elices M
Springer, Berlin Pergamon
Cowin SC (ed) Khor E
CRC Press, Boca Raton, FL Elsevier, Oxford
Torquato S
Springer, New York
Barbucci R
Kluwer Academic/Plenum, New York Narosa Publishing House, New Delhi, India John Wiley & Sons, New York
2002
Biomaterials
Bhat SV
2002
An Introduction to Tissue-Biomaterial Interactions Bones: Structure and Mechanics Calcium Phosphate Bone Cements: A Comprehensive Review
Dec KC, Puleo DA, and Bigirs R
2002 2003
2003
Failure in Biomaterials, in Comprehensive Structural Integrity series, vol. 9
John Wiley & Sons, New York
Currey JD Weiss DD
Teoh SH
ICP, Tokyo
Princeton University Press, Princeton Journal of Long-Term Effects of Medical Implants, 13(1)41−47 Elsevier, London, UK
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Biomaterials and Biological Materials
Table 1.1 (continued) Year
Title
Author(s)
Publisher
2004
Engineering Materials for Biomedical Applications
Teoh SH
2005
Biomaterials Science: An Introduction to Materials in Medicine Surfaces and Interfaces for Biomaterials Medical Textiles and Biomaterials for Healthcare An Introduction to Biomaterials Mechanics of Biological Tissue Cellular Transplants: From Lab to Clinic The Gecko’s Foot Biomedical Polymers
Ratner BD, Hoffman AS, Schoen FJ, Lemons JE
World Scientific Publishing Co. Pte. Ltd. Academic Press, New York
2005 2005
2006 2006 2007 2007 2007 2008 2008 2008
Cellular Response to Biomaterials Shape Memory Alloys for Biomedical Applications Orthopaedic Bone Cements
2008
Natural-Based Polymers for Biomedical Applications
2008
Bioceramics and Their Clinical Applications Dental Biomaterials: Imaging, Testing and Modelling Orthodontic Biomaterials
2008
2009 2009
Bulk Metallic Glasses for Biomedical Applications
2009
Mechanical Behaviour of Materials Biomaterials and Regenerative Medicine in Ophthalmology Bone Repair Biomaterials
2009
2009 2009
Biomaterials and Tissue Engineering in Urology
Vadgama P Anand SC, Miraftab M, Rajendran S, Kennedy JF Guelcher SA, Hollinger JO Holzapfel GA, Ogden RW Halberstadt C and Emerich DF Forbes P Jenkins M Di Silvio L Yoneyama T, and Miyazaki S Deb S Reis RL, Neves NM, Mano JF, Gomez ME, Marques AP, Azevedo HS Kokubo T Curtis RV and Watson TF Matasa CG and Chirita M Schroers J, Kumar G, Hodges TM, Chan S and Kyriakides TM Meyers M, Chawla C Chirila TV
Planell JA, Best SM, Lacroix D, Meroli A Denstedt J and Atala A
Woodhead Publishing Ltd. Woodhead Publishing Ltd. CRC Taylor & Francis Springer, New York Academic Press Fourth Estate, London Woodhead Publishing Ltd. Woodhead Publishing Ltd. Woodhead Publishing Ltd. Woodhead Publishing Ltd. Woodhead Publishing Ltd.
Woodhead Publishing Ltd. Woodhead Publishing Ltd. Technica-Info Kishinev JOM, 61, 21–29
Cambridge University Press Woodhead Publishing Ltd. Woodhead Publishing Ltd. Woodhead Publishing Ltd.
1.3
Classification of Biomaterials
15 Table 1.1 (continued)
Year
Title
Author(s)
Publisher
2009
Biomaterials for Treating Skin Loss Materials Science for Dentistry, 9th ed. Biomedical Composites
Orgill DP, Blanco C
Woodhead Publishing Ltd. Woodhead Publishing Ltd. Woodhead Publishing Ltd. Woodhead Publishing Ltd. Woodhead Publishing Ltd. Woodhead Publishing Ltd.
2009 2009 2010 2010 2010
2010
2010 2010
2010
Injectable Biomaterials: Science and Applications Biomaterials for Artificial Organs Bioactive Materials in Medicine: Design and Applications Surface Modification of Biomaterials: Methods, Analysis and Applications Biotextiles as Medical Implants Novel Biomedical Hydrogels: Biochemistry, Manufacture and Medical Implant Applications Regenerative Medicine and Biomaterials for the Repair of Connective Tissues
Darvell BV Ambrosio L Vernon B Lysaght M Zhao X, Courtney JM and Qian H Williams R
Woodhead Publishing Ltd.
King MV and Gupta BS
Woodhead Publishing Ltd. Woodhead Publishing Ltd.
Rimmer S
Archer C and Ralphs J
Woodhead Publishing Ltd.
Based on the nature of material these can be further classified into following manner: Metals and Alloys; Ceramics, Polymers, and Composites.
1.3.1 Metals and Alloys Metals were among the first orthopedic biomaterials and are commonly used to this day (see Section 1.2). Currently, most orthopedic implants are made from either stainless steel, titanium or one of its alloys, or a cobalt–chrome alloy, although tantalum and Nitinol metals have also been used.
1.3.2 Ceramics These kinds of biomaterials are well described and classified in Encyclopedia of Biomaterials and Biomedical Engineering (Wnek and Bowlin 2008).
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Biostable Ceramics. Aluminum oxide (alumina, ASTM F-603) and a zirconium oxide (zirconia) compound (ASTM F-1873) are the two most common biostable ceramics. Biostable ceramics neither resorb nor induce osteoblastic apposition on their surfaces within the body. Advantages are that both aluminum oxide and zirconium oxide are strong and stable (so there is no need to follow degradation products); while the disadvantages include having weak interface with bone or tissue, low shock resistance, high modulus, and the potential for catastrophic failures. Bioactive Ceramics. The most common examples are bioactive glasses (Bioglass) (see Hench (1998)), bioactive glass-ceramics (Ceravitals A-W Ceramic). Their main advantage is good bonding to tissue and bone; their disadvantage is that they are not as strong as biostable ceramics (Hench 1998; Hench and Andersson 1993; Hench and Paschall 1973; Hench and West 1996). Bioresorbable Ceramics. Various apatites and other calcium phosphate and carbonate-containing bioceramics like Biobase and Cerasorb. (More detailed reviewed by Dorozhkin 2009.) Ceramic Bone Cements.Ceramic bone cements are another active area of research and clinical use. Several different approaches have been taken in the development of a variety of ceramic-based bone cements. Examples of bone cements are discussed in detail in the reviews by Kühn (2000) and Weiss (2003).
1.3.3 Polymers Most of the biodegradable polymeric products on the market are made from only a few polymers, many of which were first used in sutures. The most common suture materials are the polylactic and glycolic acid polymers and copolymers, the trimethylene carbonate copolymers, and polydioxanone. The advantages of the biodegradable polymeric products include the following: they disappear, so longterm stress shielding is not a concern; there are no long-term device or materials problems; and no second operation is required for removal. The biodegradable polymeric products can be used for drug delivery.
1.3.4 Composites Composite materials can be generally defined as those materials having two or more distinct material phases. Porous materials may also be considered composite materials, with one phase composed of void or air spaces. Composite biomaterials for dentistry, for example, are mostly based on combinations of silane-coated inorganic filler particles with dimethacrylate resin. The filler particles used are either barium silicate glass, quartz, or zirconium silicate and are usually combined with 5–10% weight of 0.04 μm particles of colloids silica. A hydroxyapatite–polyethylene composite has been developed for use in orthopedic implants. The material knits together with bone, maintains good mechanical properties, and can be shaped or trimmed during surgery using a scalpel.
1.4
Requirements of Biomaterials
17
Interestingly, Wegst and Ashby (as reviewed by Anderson 2006) classify biological (natural) materials into four groups: Ceramics and ceramic composites: These are biological materials where the mineral component is prevalent, such as in shells, teeth, bones, diatoms, and spicules of sponges. Polymer and polymer composites: Examples of these are the hooves of mammals, ligaments and tendons, silk, and arthropod exoskeletons. Elastomers: These are characteristically biological materials that can undergo large stretches (or strains). The skin, muscle, blood vessels, soft tissues in body, and the individual cells fall under this category. Cellular materials: Typically are the light weight materials which are prevalent in feathers, beak interior, cancellous bone, and wood.
1.4 Requirements of Biomaterials The design or selection of a specific biomaterial depends on the relative importance of the various properties that are required for the intended medical application. Physical properties that are generally considered include hardness, tensile strength, modulus, and elongation; fatigue strength, which is determined by a material’s response to cyclic loads or strains; impact properties; resistance to abrasion and wear; long-term dimensional stability, which is described by a material’s viscoelastic properties; swelling in aqueous media; and permeability to gases, water, and small biomolecules. In addition to the mechanical, thermal, and surface properties of materials, other physical properties could be important in particular applications of biomaterials: electrical, optical, absorption of x-rays, acoustic, ultrasonic, density, porosity, and diffusion (Park and Lakes 2007). The success of a biomaterial in the human body depends on the controlled bulk properties (mechanical as well as a match of tissues at the site of implantation) and the surface properties on the micrometer and nanoscale (Fig. 1.1). The shape and bulk properties of biomaterials should mimic the tissues which they are meant to augment or replace. The surface chemistry and topography of the implant material determine how the host tissues interact with the implant. Therefore, the ability to fabricate complex shapes with a wide range of surface topographies is an important property of a biomaterial (Schroers et al. 2009). Depending on the application, differing requirements may arise. Sometimes these requirements can be completely opposite. In tissue engineering of the bone, for instance, the polymeric scaffold needs to be biodegradable so that as the cells generate their own extracellular matrices, the polymeric biomaterial will be completely replaced over time with the patient’s own tissue. In the case of mechanical heart valves, on the other hand, we need materials that are biostable, wear-resistant, and which do not degrade with time. Materials such as pyrolytic carbon leaflet and titanium housing are used because they can last at least 20 years or more (Teoh 2004).
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Fig. 1.1 The foreign body reaction as illustrated here is the normal reaction by higher organisms to an implanted synthetic material. A biomaterial implanted into the body, however, induces a different response, termed the foreign body reaction. A biomaterial elicits nonspecific protein adsorption immediately upon implantation. Many different proteins adsorb to the surface in a range of conformations from native to denatured. Nonspecific protein adsorption may be an instigator in the foreign body reaction. A number of different cells, such as monocytes, leukocytes, and platelets, adhere to these biomaterial surfaces and as a result may lead to upregulation of cytokines and subsequent proinflammatory processes. The end stage of the foreign body reaction involves the walling off of the device by an avascular, collagenous fibrous tissue that is typically 50–200 μm thick (adapted from Ratner and Bryant 2004)
According to Teoh (2004), the requirements of biomaterials can be generally grouped into four broad categories: 1. Biocompatibility: The material must not disturb or induce an un-welcoming response from the host, but rather promote harmony and good tissue-implant integration. An initial burst of inflammatory response is expected and is sometimes considered essential in the healing process. However, prolonged inflammation is not desirable as it may indicate tissue necrosis or incompatibility. 2. Sterilizability: The material must be able to undergo sterilization. Sterilization techniques include gamma, gas (ethylene oxide (ETO)), and steam autoclaving. Some polymers such as polyacetal will depolymerize and give off the toxic gas formaldehyde when subjected to high-energy radiation by gamma rays. These polymers are thus best sterilized by ETO. 3. Functionability: The functionability of a medical device depends on the ability of the material to be shaped to suit a particular function. The material must therefore
1.5
The Future of Biomaterials
19
be able to be shaped economically using engineering fabrication processes. The success of the coronary artery stent—which has been considered the most widely used medical device—can be attributed to the efficient fabrication process of stainless steel from heat treatment to cold working to improve its durability. 4. Manufacturability: It is often said that there are many candidate materials that are biocompatible. However, it is often the last step, the manufacturability of the material that hinders the actual production of the medical device. It is in this last step that engineers can contribute significantly.
1.5 The Future of Biomaterials The future of new biomedical materials is dependent upon the development of an enhanced knowledge base of molecular, cellular, and tissue interactions with materials. The general trend in biomaterials is to use and employ materials that play an active role in tissue regeneration rather than passive and inert materials. Therefore, understanding how a material interacts with the surrounding environments, including cells and tissue fluid, allows material design to be tailored so that implants can be constructed to promote a specific biological response, helping them better perform their function. This class of materials has been described as the “Third Generation” of biomaterials (Abou Neel et al. 2009). Anderson (2006) proposed two goals of Materials Scientists to study biomaterials: (a) The “materials” approach of connecting the (nano-, micro-, meso-) structure to the mechanical properties is different from the viewpoint of biologists and chemists, since it analyzes them as mechanical systems. This has yielded novel results and is helping to elucidate many aspects of the structure heretofore not understood. (b) The ultimate goal of synthesizing bioinspired structures is a novel approach within the design and manufacture. This approach has yielded some early successes such as Velcro (the well-known hook-loop attachment device) in which the material components were conventional and their performance was biomimicked. A new direction consists of starting at the atomic/molecular level (bottom-up approach) through self-assembly and to proceed up in the dimensional scale, incorporating the hierarchical complexity of biological materials. This approach is at the confluence of biology and nanotechnology and is already yielding new architectures that have potential applications in a number of areas, including quantum dots, photonic materials, drug delivery, tissue engineering, and genetically engineered biomaterials. Unfortunately, war crises became to be an additional driving force in development of novel biomaterials with specific features today. Thus, for example, the Pentagon recently funds major initiatives in biomaterials. The US Department
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of Defense has selected Princeton engineers to lead two new multi-institutional research initiatives, aimed at inventing materials that adapt themselves to changing loads and environments. The structural materials project will be led by Ilhan Aksay, professor of chemical engineering, and is to receive $7.5 million. The grants were among 69 recently announced by the Pentagon as part of its Multidisciplinary University Research Initiative (MURI) program. The goal of the materials science project, sponsored by the Army Research Office, is to develop adaptive materials that are able to repair and strengthen themselves when needed. A key to replicating such functions will be to develop porous materials, similar to the structure of bone, through which new material can flow to repair weak spots. The researchers plan to develop an embedded sensing system to monitor and locate which areas need strengthening. They also will investigate methods for directing and moving fluid within the material to where it is needed. Similar investigation has been carried out at Center for Military Biomaterials Research in New Jersey since 2004. Another research project, dedicated to development of a combination of bone cement and antibiotics, was supported by research grants from the US Army Medical Research Acquisition Activity (USAMRAA), Orthopedic Trauma Research Program, and the National Institutes of Health Public Health Service Awards. The study was published online on January, 2, 2009 in the Journal of Orthopedic Research. In this work, researches used bone cement infused with an antibiotic called colistin—one of the last-resort antibiotics for drug-resistant Acinetobacter baumannii—to treat mice infected with samples of the bacteria taken from soldiers wounded in Iraq and Afghanistan. After 19 days, only 29.2% of the mice still had detectable levels of A. baumannii. R P bone cement” must An antibiotic chosen for use with this “Surgical Simplex elute from the cement at high enough levels to provide antibacterial protection during the initial 72 h after implantation while remaining at safe, non-toxic levels in the serum and urine. To provide this effective release, the antibiotic must be able to withstand the heat generated by polymerization. The civil sector of the biomaterials market also seems to be optimistic. Biomaterials products had a market size of $25.5 billion in 2008, and the biomaterial device market size was $115.4 billion in the same year, and is expected to reach $252.7 billion in 2014. This massive revenue potential highlights the immense opportunity in the market. According to a new market research report, “Global biomaterials Market (2009–2014),” published by Markets and Markets (http://www.marketsandmarkets .com), the total global biomaterials market is expected to be worth US$58.1 billion by 2014, growing at a CAGR of 15.0% from 2009 to 2014. The US market is expected to account for nearly 42% of the total revenues. The biomaterials market today has already crossed $28 billion. While the orthopedic biomaterials market was the biggest segment in 2008 with $9.8 billion, the cardiovascular biomaterial market is estimated to be dominant segment in 2014 with an estimated $20.7 billion. The cardiovascular biomaterial market is expected to grow with a CAGR of 14.5% from 2009 to 2014, mainly due to increasing stress levels that have in turn increased the incidence of cardiac arrest.
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The US market is the largest geographical segment for biomaterials and is expected to be worth $22.8 billion by 2014 with a CAGR of 13.6% from 2009 to 2014. Europe is the second largest segment and is expected to reach $17.7 billion by 2014 with a CAGR of 14.6%, and the Asian market size is estimated to increase at the highest CAGR of 18.2% from the year 2009 to 2014. Improvement in fabrication technology and new product development at competitive prices will be the key to future market growth. The USA and Europe hold a major share of the global biomaterials market; while emerging economies such as China, India, Japan, Brazil, Russia, and Romania represent a high growth rate.
1.6 Conclusions Biomaterials are either modified natural or synthetic materials which find applications in a wide spectrum of medical and dental implant and prosthesis for repair, augmentation, or replacement of natural tissues. The past decade has witnessed the emergence of a new set of tools, combinatorial and high-throughput screening, in biomaterials development. Numerous articles as well as books cited in this chapter covers recent examples of high-throughput and combinatorial studies of biomaterials. Assembly of nanoscale materials and functional hierarchical structures is a big challenge now faced by nanotechnology. Learning from biology, using biopolymers as scaffolds to control the synthesis and organization of materials like living tissues provides the perfect solution to determine the progress in biomaterials science. In the following chapters, I want to illustrate the biomimetic potential and discuss features, advantages and imperfections of a broad variety of unique biological materials of marine origin from nanoto macroscale.
References Abou Neel EA, Pickup DM, Valappil SP et al (2009) Bioactive functional materials: a perspective on phosphate-based glasses. J Mater Chem 19:690–701 Albee F, Morrison H (1920) Studies in bone growth. Ann Surg 71:32–38 Anderson JM (2006) The future of biomedical materials. J Mater Sci Mater Med 17:1025–1028 Ashby MF, Greer AL (2006) Metallic glasses as structural materials. Scripta Materialia 54(3): 321–326 Baden E (1955) Prosthetic therapy of congenital and acquired clefts on the palate: an historical essay. J Hist Med Alld Sci X(3):290–301 Dorozhkin S (2009) Nanodimensional and nanocrystalline apatites and other calcium orthophosphates in biomedical engineering, biology and medicine. Materials 2:1975–2045 Harkins CS, Koepp Baker H (1948) Twenty years of cleft palate prosthesis. J Speech Hear Dis 13:23–30 Hench LL (1998) Bioceramics. J Am Ceram Soc 81:1705–1728 Hench LL, Andersson OH (1993) Bioactive glasses. In: Hench LL, Wilson J (eds) An introduction to bioceramics. of Singapore: World Scientific, Republic of Singapore Hench LL, Paschall HA (1973) Direct chemical bond of bioactive glass-ceramic materials to bone and muscle. J Biomed Mater Res Symp 4:25–42
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Hench LL, Polak JM (2002) Third-generation biomedical materials. Science 295:1014–1017 Hench LL, Splinter RJ, Allen WC et al (1971) Bonding mechanisms at the interface of ceramic prosthetic materials. J Biomed Mater Res 2:117–141 Hench LL, West JK (1996) Biological applications of bioactive glasses. Life Chem Rep 13: 187–241 Kühn K-D (2000) Bone cements. Springer, Berlin Kumar G, Tang HX, Schroers J (2009) Nanomoulding with amorphous metals. Nature 457(7231):868–872 Lambotte A (1932) L’utilisation du magnesium comme materiel perdu dans l’osteosynthèse. Bull Mém Soc Nat Chir 28:1325–1334 Larsen A, Kolind K, Pedersen DS et al (2008) Gold ions bio-released from metallic gold particles reduce inflammation and apoptosis and increase the regenerative responses in focal brain injury. Histochem Cell Biol 130:681–692 Orive G, Anitua E, Pedraz JL et al (2009) Biomaterials for promoting brain protection, repair and regeneration. Nat Rev Neurosci 10:682–692 Park JB, Lakes RS (2007) Biomaterials – an introduction, 3rd ed. Springer, New York, Berlin Popp H (1939) Zur Geschichte der Prosthesen. Med Welt 13:961–964 Ratner BD, Bryant SJ (2004) Biomaterials: where we have been and where we are going. Annu Rev Biomed Eng 6:41–75 Ray R, Degge J, Gloyd P et al (1952). Bone regeneration. J Bone Joint Surg Am 34A(3): 638–647 Schroers J, Kumar G, Hodges TM et al (2009) Bulk metallic glasses for biomedical applications. JOM 61:21–29 Sivakumar R (1999) On the relevance and requirements of biomaterials. Bull Mater Sci 22: 647–655 Staiger MP, Pietak AM, Huadmai J et al (2006) Magnesium and its alloys as orthopedic biomaterials: a review. Biomaterials 27:1728–1734 Teoh SH (2004) Engineering materials for biomedical applications. World Scientific, Singapore Thomas V, Dean DR, Vohra YK (2006) Nanostructured biomaterials for regenerative medicine. Curr Nanosci 2:155–177 Weinberger BW (1948) An introduction to the history of dentistry with medical and dental chronology and bibliographic data. St. Louis, The C.V. Mosby Company. D.D.S., New York Weiss DD (2003) Calcium phosphate bone cements: a comprehensive review. J Long-Term Eff Med Implants 13(1):41–47 Williams DF (1999) The Williams dictionary of biomaterials. University Press, Liverpool Williams DF (2008) The relationship between biomaterials and nanotechnology. Biomaterials 29:1737–1738 Williams DF (2009) On the nature of biomaterials. Biomaterials 30:5897–5909 Wnek GE, Bowlin GL (2008) Encyclopedia of biomaterials and biomedical engineering, 2nd ed (four-volume set). Informa Healthcare. London, New York Zhong Z, Bellamkonda R (2008) Biomaterials for the central nervous system. J R Soc Interface 5:957–975
Part II
Biominerals and Biomineralization
Chapter 2
Biominerals
Abstract Biominerals may be deposited within the organism, and within its immediate surroundings or environment, by the metabolism of the living creature. The physiological pathways, by which organisms precipitate minerals, and the forms and functions of the skeletons they fashion have been shaped by natural selection through geologic time. These metabolic routes and the skeletons they form have conserved expressions that continue throughout evolution. Many biomineralized tissues of marine invertebrate origin are composite materials, containing a biologically produced organic matrix and nano- or microscale amorphous or crystalline minerals. Calcium-, magnesium-, barite-, iron-, vanadium-, strontium-, boron-, titanium-, copper-, zinc-, manganese-, germanium-, and silica-based biominerals of marine invertebrate origin are discussed. Of the intriguing scientific topics that are receiving renewed attention today, the study of biomineral formation based on organic templates is one of the most fascinating. All biominerals are common minerals, easily accommodated in the usual definition of “mineral” but they may have distinct morphologies and have been found to make unique contributions to well-known life forms (Skinner 2005). By the 1930s, there were approximately 10 different minerals known to be present in living organisms. This changed when Heinz A. Lowenstam published a paper (Lowenstam 1962a) describing the presence of magnetite in chitons. Magnetite is a relatively hard iron oxide, previously thought by chemists to be formed only at very high pressures and temperatures. Lowenstam noticed that limestone outcroppings near the ocean shore were being undercut by the scrapping action of chitons, and he went on to show that the surface of the lateral radular tooth of the chiton was covered with magnetite. Since that discovery, numerous biominerals with a range of chemical compositions have been discovered. Most of them are listed in the book by Lowenstam and Weiner (1989). Lowenstam and Weiner describe 38 “common” minerals found in metazoans. The following cations have been discovered in biomineral formations: Ba, Ca, Cu, Fe, K, Mn, Mg, Na, Ni, Pb, Si, Sr, and Zn. In living organisms, they are usually found as hydroxides, oxides, and sulfates or as sulfides, carbonates, and phosphates. Recently, it was reported (Skinner 2005) that biominerals can be classified in the
H. Ehrlich, Biological Materials of Marine Origin, Biologically-Inspired Systems 1, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9130-7_2,
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same framework as minerals. Minerals are classified by composition based on the anionic constituents, and representatives in many of the 78 mineral classes listed in Dana’s New Mineralogy (Gaines et al. 1997) can be found in metazoans. Biominerals may be deposited within the organism, and within its immediate surroundings or environment, by the metabolism of the living creature (Skinner 2000). The physiological pathways, by which organisms precipitate minerals, and the forms and functions of the skeletons they fashion have been shaped by natural selection through geologic time. These metabolic routes and the skeletons they form have conserved expressions that continue throughout evolution (Knoll 2003). The number and varieties of biomineralizers recently reviewed by Ehrlich et al. (2008) can be best appreciated by the fact that approximately 128,000 species of molluscs, about 800 species of corals, more than 5,000 species of sponges (including 525 species of glass sponges), 700 species of calcareous green, red, and brown algae, more than 300 species of deep-sea benthic foraminifera, and 200,000 diatom species have been described (Mann and Droop 1996). Mineral materials, including calcium-based biominerals, are commonly produced throughout a wide range of phyla ranging from archaea, bacteria, and fungi, through lower and higher plants, to the Chordata. Among the higher phyla, most of the work has been on Cnidaria, Mollusca, Arthropoda, Echinodermata, and Chordata (Wilt 2005). Many forms of calcium carbonate (CaCO3 ) and calcium phosphates, with a range of compositions, are made by living organisms. Further, there are organisms whose mineralized tissues may be formed from one or several of the calcium carbonate polymorphs. The polymorph expression may change between the larval and adult forms (Skinner 2005). For example, in the ideal crystalline form of calcium carbonate, three different polymorphs may be exhibited: vaterite, aragonite, and calcite (Addadi et al. 2003). Calcium carbonate is also found in its amorphous form (amorphous calcium carbonate, ACC), as a monohydrocalcite (CaCO3 • H2 O), or as the hexahydrate, ikaite (CaCO3 • 6H2 O) (Gaines et al. 1997). The calcium phosphate phases found in hard tissues include the mineral known as brushite, calcium phosphate dihydrate (CaHPO4 • 2H2 O, DCPD), octacalcium phosphate (Ca8 H2 (PO4 )6 • 5H2 O, OCP), tricalcium phosphate (β-Ca3 (PO4 )2 , βTCP), hydroxylapatite (Ca5 (PO4 )3 OH, HAP), and fluorapatite (Ca5 (PO4 )3 F, FAP) (Fig. 2.1). All the compositions listed here are those accepted as the ideal with well-defined crystal structures; these minerals typically occur naturally with these compositions. The minerals contribute to the strength of tissues and skeletons, to eggshells, as well as to the hardness of claws and teeth (Cameron 1990). For example, the transformation of amorphous calcium phosphate to crystalline dahlite in the radular teeth of chitons (Mollusca) was reported by Lowenstam and Weiner (1989). Vertebrate and particularly human calcium-based biominerals can be divided into two types (Skinner 2000), which are listed below: 1. Essential calcium, which is a normal part of the expected physiology of human systems, such as the mineral matter found in bones (reviewed by Dorozhkin
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Fig. 2.1 SEM images of fluorapatite crystals (a). The surface of such crystal possesses characteristic features (b) (images courtesy: Paul Simon)
2007; Glimcher 2006; Skinner 2005) and teeth (reviewed by Fincham et al. 1999; Robinson et al. 1995; Simmer and Hu 2001). There are 208 bones in the skeleton and 32 teeth in the oral cavity of a normal adult (Skinner 2000). 2. Unexpected and undesired or pathologic mineral deposits of calcium, including the following: • • • • • • • • • • • •
pancreatic calculi (Jin et al. 2002) and stones (Multinger et al. 1983); renal stones (Kageyama et al. 2001); kidney stones (Khan et al. 2002; Ryall et al. 2000); urinary calculi (Suto and Wooley 1972), stones (Prien and Prien 1968; Rose 1977; Williams et al. 2006), and cystoliths (Saetre 1954); gallstones (Been et al. 1979); bladder stones (Chaudhri et al. 2007); rhinoliths (calculus present in the nasal cavity) (Celikkanat et al. 1997; Rasinger et al. 1985; Shaw 2007; Vink et al. 2002); tonsilloliths (oropharyngeal concretions) (Cerny and Bekarek 1990; Mesolella et al. 2004); vaginoliths—vaginal calculi (Cetinkursun et al. 2001; Malhotra et al. 2004; Malik et al. 2006); cardiolytes (Gilinskaya et al. 2003); cutaneous calculi (Moulik et al. 1974; Neild and Marsden 1985; Tezuka 1980); enteroliths (Lopez and Welch 1991; Pantongrag-Brown et al. 1996; Rudge 1992);
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• sialoliths—salivary submandibular (Burstein et al. 1979) and parotid gland stones (Slomiany et al. 1983; Thompson 1973); • ptyaliths—calculus in a salivary glands (Anneroth et al. 1975); • dental calculi (Rabinowitz et al. 1969). The formation of crystals in pathological mineralization follows the same principles as normal calcifications (Magalhaes et al. 2006). Biogenic calcium-based minerals have also been well documented within the plant kingdom (Franceschi and Horner 1980; Franceschi and Nakata 2005). The most common phytocrystals are formed from the calcium oxalate (Cox) hydrates, namely the calcium oxalate monohydrate and calcium oxalate dihydrate. Typically, Cox crystals appear intracellularly in specialized cells called crystal idioblasts. Extracellular deposits are a characteristic feature of numerous gymnosperm species and the ontogeny of extracellular deposits in coniferous gymnosperms indicates extracellular origin. However, within Plantae, the carbonate biomineralization of marine and freshwater algae is replaced by silica phytolith mineralization in the epidermis of some vascular plants, especially grasses, sedges, and the sphenoid genus Equisetum (Harrison 1996; Knoll 2003). Another kind of organic–mineral composite discovered in plants is known as cystoliths. These formations are heavily calcified wall ingrowths that occur in specialized cells called lithocysts in leaves, stems, and sometimes roots of species restricted to a few angiosperm families, notably Moraceae, Urticaceae, and Acanthaceae (Metcalfe and Chalk 1983). Lithocysts are usually localized in the upper and/or the lower epidermis and are associated with many of the photosynthetic cells in every plant species that has been investigated thus far, suggesting a relationship between CaCO3 deposition in cystoliths and photosynthesis (Okazaki et al. 1986). The cystolith is a spindle-shaped body composed of concentric layers of longitudinally orientated cellulose microfibrils and are associated with pectins and other cell wall polysaccharides. At maturity the cystoliths are heavily impregnated with calcium carbonate (Watt et al. 1987). Silica-based biominerals identified in chordates, including human and mammals, are represented in the Addendum (Table 1).
2.1 Biominerals of Marine Invertebrate Origin 2.1.1 Calcium-Based Biominerals Calcium-based marine invertebrate’s skeletons have been optimized by natural selection over millions of years to physically support and physiologically maintain diverse tissue types encompassing a variety of functions (Green et al. 2002). Because of the numerous excellent experimental papers, as well as books and reviews published during the last 120 years (Chave 1954; Dreschel 1896; Herdman
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1884; Leitmeir 1910; Lippmann 1973; Milliman 1974; Vinogradov 1933) on calcium carbonate and calcium phosphate biominerals of marine invertebrate origin [see, e.g., publications by H. Lowenstam, A. Veis, S. Weiner, L. Addadi, J. Aizenberg, F. Wilt listed in my previously published reviews (Ehrlich et al. 2008, 2009), as well as in this book], I will focus this work on other biominerals that have received less attention from the scientific community. In particular, I will seek to inform the reader about the very rare or unusual materials that form the basic material for skeletal scaffolds of protists, diatoms, and other marine animals. The quantitative data regarding inorganic constituents of marine invertebrates can be found in the fundamental works by Clark and Wheeler published in 1922, as well as by Vinogradov in 1954. The diversity of invertebrate’s biominerals is briefly represented below. 2.1.1.1 Calcium Oxalate (Weddellite) Cramer (1891) was the first to chemically demonstrate that the calcium oxalate dihydrate mineral weddellite was present as a skeletal component of marine algae Bornetella sphaerica. Flajs (1977a, b) was the first to show this mineral in the scanning electron microscope in Bornetella nitida (Dasycladales) and to prove that it is indeed part of the algae skeleton, and not a contaminant. The skeletal formations of Bornetella species also contain aragonite as well as Mg- and Sr-based carbonates (Berger et al. 1997). Weddellite has been identified as a microarchitectural component of the gizzard plates from the deepwater gastropod Scaphander cylindrellus (Lowenstam 1968). This was the first indication of a non-pathologic precipitation of this mineral by an animal species. A new occurrence of weddellite in sediments from the Weddell Sea, Antarctica, is also reported by Lowenstam, lending support to the earlier interpretation that the mineral is an authentic constituent of the sediments in this area. In addition to being found in Mollusca, weddellite and calcite (anhydrous calcium carbonate phase) were also found as components of concretions in the renal sac of the ascidian tunicate Molgula manhattensis (Saffo and Lowenstam 1978). The presence of weddellite along with urate in the concretions suggests a resemblance to human kidney stones, although, unlike the latter, the concretions in Molgula do not seem to be pathologic deposits. 2.1.1.2 Fluorite (CaF2 ) In both vertebrates and invertebrates, fluoride is largely accumulated in skeletal structures. There is little or no accumulation in soft, edible tissues, with the exception of fish skin (Wright and Davison 1975). Fluorine has long been reported as a component of calcareous marine sediments of planktonic origin and of invertebrate skeletons (Monniot et al. 1995). Vinogradov (1953) noted that all mollusc shells contain fluorine and further specified that marine species have a higher content of this element compared to freshwater ones. The presence of fluorine was reported in diverse calcified structures of marine
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invertebrates: holothurians (Lowenstam and Rossman 1975); mussels and crustaceans (Wright and Davison 1975); polychaetes (Vovelle et al. 1989); and ascidian spicules (Monniot et al. 1995). X-ray diffraction patterns show that the statoliths of marine mysid crustaceans are composed of fluorite, and this mineral is also a principal phase of the gizzard plates of some tectibranch gastropods (Lowenstam 1968). The occurrence of fluorite in crustacean mysid statoliths (Lowenstam and McConnell 1968) confirms the earlier interpretations based on insufficient documentation. Fixation of fluorine in hard tissues of marine invertebrates is extensive in the shelf seawaters and minor in the bathyal zone of the oceans. Mysid shrimps of the genus Schistornysis precipitate either fluorite (CaF2 ) or calcium carbonate. For example, in Schistornysis spiritus, 3.79 μg calcium and 3.60 μg fluoride are precipitated. This corresponds to 9.3 mg of CaF2 and 2,788 mg ambient water, respectively. Concentration factors with respect to statocyst water mass are 610 and 183.000, respectively. This high fluoride accumulation for the formation of statoliths alone contributes to a fluoride content of 4,500 ppm with respect to dry body weight (Wittman and Ariani 2008). 2.1.1.3 Calcium Sulfate-Based Biominerals Calcium sulfate dihydrate (CaSO4 ·• 2H2 O) statoliths in scyphozoan medusa (Aurelia sp.) were reported by Spangenberg and Beck in 1968. Later, Chapman (1985) showed that cubomedusan statoliths are composed of gypsum as well and also found that the statolith was lamellated—however, he regarded it as a cleavage in the gypsum—whereas statoliths of a number of scyphozoan medusa (Aurelia aurita, Cyanea capillata, Cyanea lamarckii—Semaeostomeae; Periphylla periphylla—Coronata; Rhizostoma octopus—Rhizostomeae) possess the unusual biomineral bassanite (calcium sulfate hemihydrate, CaSO4 ·• 1/2H2 O) (Becker et al. 2005; Tiemann et al. 2002).
2.1.2 Magnesium-Based Biominerals Calcite skeletons of marine algae (Böhm 1973) and animal organisms (Chave 1954) often contain high concentrations of magnesium carbonate. The percentage varies considerably among different taxonomic entities, although the magnesium content is positively correlated with water temperature (Milliman et al. 1971). This is most pronounced in the Corallinaceae, which have the highest magnesium concentration (up to 30% MgCO3 ) and a near linear correlation of concentration and temperature. Changes in the growth rate of these algae may effect changes in the magnesium content of the calcite through the interaction of factors such as the diffusion rate of magnesium through the cell wall and the construction of an organic matrix or a template (Kolesar 1978). Intriguingly, magnesium plays a role in geotaxis of some ctenophores or comb jellies (Aronova 1987). The gravitation receptor in the aboral organ is formed from four symmetrical groups of gravitational mechanoreceptor cells. Their apices face
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the aboral organ cavity that is isolated from seawater and is limited by a cupola from long, glued flagella emerging from peripheral epithelial cells. Thereby the autonomous medium comparable with endolymph of otolith organs is preserved above the receptor epithelium. In most animals, the main chemical element of the otolith apparatus is known to be calcium that forms with various anions compounds such as gypsum, apatite, calcite, aragonite, vaterite, and fluorite. Additionally anions such as Mg, Na, K, Fe, Sr, P, and Be (Vinnikov et al. 1981) are found as well. According to results from X-ray structural analysis, the highest percentage of electrolyte composition of the otolith apparatus of the comb jelly Beroe cucumis, including intracellular concretions and free otoconia, is in magnesium and calcium carbonates, where the highest percentage belongs to Mg (44.8%) (Aronova 2009). A significant part of the jellyfish otoconia consists of a dense organic material, in which crystallization of inorganic phases occurs. Magnesium is an important component of the skeletons of corals (Dodd 1967). Thus, ion micro-probe imaging of the aragonite skeleton of Pavona clavus, a massive reef-building coral, shows that the distribution of magnesium is strongly correlated with the fine-scale structure of the skeleton and corresponds to the layered organization of aragonite fibers surrounding the centers of calcification, which have up to 10 times higher magnesium concentration (Meibom et al. 2004). This indicates a strong biological control over the magnesium composition of all structural components within the skeleton. The authors suggested that magnesium may be used by the coral to actively control the growth of the different skeletal crystal components. Another form of magnesium-based biomineral which is distributed within marine organisms is brucite (Mg(OH)2 ). Biologically mediated formation of brucite implies the development of a microenvironment with high pH and low pCO2 (Noreen and Holmes 2006; Nothdurft et al. 2005). Only a few studies report the biomineralization of brucite, which occurs in red algae (Schmalz 1965) and in nudibranch spicule (Cattaneo-Vietti et al. 1995). In contrast to Onchidella sp. which contains siliceous spicules, Doridacean opisthobranchs have calcium carbonate spicules in their mantle, foot, gills, and rhinophores, which are generally fusiform or spheroid in shape. The fusiform spicules are mainly composed of calcite and brucite, with a small percentage of fluorite (CaF2 ) (Cattaneo-Vietti et al. 1995). Brucite has been identified in the skeletons of several genera of coral, and its existence provides information toward understanding the processes that take place within microenvironments of the carbonate skeleton, beneath the living surface of the coral. Brucite was found to be concentrated within green bands that occur in some Montastraea faveolata coral skeletons (Nothdurft et al. 2005). These green bands are thought to be associated with a high-pH environment created by endolithic algae. Moreover, formation of brucite was recently reported in diatoms (Tesson et al. 2008). The diatom Phaeodactylum tricornutum was shown to form microbialite in response to alkalinization of its growth medium. After 4 days of growth in an airbubbled culture, the pH of the medium reached 10 due to the photosynthetic activity of the microalgae, causing brucite crystals to appear around the cells. At the same time, ionization of dissolved silicates occurred and led to Si sorption on brucite
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crystals. The involvement of sulfated and carboxylated polysaccharides in the initial sorption of magnesium on diatom cells was interpreted by the authors as a biologically mediated process. It is suggested that carboxyl groups of the cell walls form sorption sites for Mg nucleation.
2.1.3 Barite-Based Biominerals Barite precipitates in the seawater column when surface-dwelling plankton is consumed by zooplankton, creating microenvironments where barite is supersaturated and precipitates. These planktons concentrate barium in their shells and in their organic matter, and thus their decay in the presence of sulfate creates the supersaturated conditions necessary for barite precipitation (Hubert et al. 1975; Puchelt 1972). Acantharia, surface-dwelling protists that make their shells out of strontium sulfate (celestite) (see below), may be particularly important in marine barite precipitation. Another possible source of barite in pelagic sediments is the Xenophyophorea, benthic protists that form barite exoskeletons, which may be agglutinated from falling barite crystals in the ocean biomineralizes (Hopwood et al. 1995; Schulze and Thierfelder 1905). The Xenophyophorea are the largest deep-sea protists, ranging in size from a few millimeters up to 25 cm (Tendal 1972). Their main diagnostic morphological features are the following: (i) an agglutinated test composed of foreign particles (“xenophyae”), (ii) a cytoplasm organized as a multinucleate plasmodium enclosed within a branching system of organic tubes (granellare), (iii) strings of stercomata (stercomare) closely associated with the granellare system, and (iv) numerous intracellular barium sulfate crystals (granellae) (Tendal 1972). X-ray diffraction, electron diffraction, and electron microscopy studies have been performed on barium sulfate crystals from three xenophyophore species (Aschemonella ramuliformis, Reticulammina labyrinthica, Galatheammina lamina) obtained at bathyal and abyssal depths in the northeastern Atlantic (Gooday and Nott 1982). Two populations of crystals were observed. The first were tablets, ∼2 μm in length and rhombic or hexagonal in outline. The second population consisted of much smaller particles (< 0.5 μm) of poor crystallinity. A comparison of the larger xenophyophore crystals with synthetically grown crystals indicated that the former probably grew at low supersaturation (S < 25) in solutions of low-to-moderate ionic strength (I < 1.0 M). Interestingly, diatom-mediated barite precipitation in microbial mats calcifying at a warm sulfur spring system in Northwestern Utah (USA) was recently reported (Bonny and Jones 2007). Diatoms proliferate in the warm (∼ 48◦ C), saline, bicarbonate, and sulfur-rich spring water and form thick microbial mats in association with cyanobacteria and sulfate-reducing bacteria. These mats are lithified by minerals precipitating from the spring water, and diatoms are trapped and preserved in situ in porous calcite deposits (tufas). Cells of cyanobacteria and sulfate-reducing bacteria are encrusted or impregnated with calcite as the microbial mats lithify. Diatom frustules, however, are commonly associated with microcrystalline barite:
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barite precipitates in haloes around diatom bundles, lines and fills diatom frustules, and even replaces diatom silica. Bioaccumulation of barium in diatom tissues and adsorption of barium to diatom extracellular polysaccharides mediate barite saturation in lithifying microbial mats and are directly responsible for precipitation of primary barite at the Stinking Hot Springs (Bonny and Jones 2007). The phenomenon described above suggests the presence of barite-containing frustules in marine diatoms living under corresponding barium-rich environmental conditions.
2.1.4 Fe-Based Biominerals Many invertebrates use crystals of metal salts to harden their cutting, rasping, and grinding equipment. The teeth of marine invertebrates, especially molluscs, possess different Fe-based biominerals (Webb et al. 1990). Best known among the groups that have mineralized teeth are the limpets (Gastropoda: Acmaeidae, Patellidae) and chitons (Polyplacophora). In chitons, the second lateral tooth is mineralized primarily by magnetite (FeO) (Lowenstam 1962a). However, ferrihydrite (5Fe2 O3 • 9H2 O), lepidocrocite (γ-FeOOH), maghemite (γ-Fe2 O3 ), goethite (α-FeOOH), and apatite (crystalline calcium phosphate) also occur (Kim et al. 1989; Kirschvink and Lowenstam 1979; Lowenstam 1967; Mizota and Maeda 1986; St Pierre et al. 1992; Shaw et al. 2008; Towe and Lowenstam 1967; Webb et al. 1989). In limpets, the radular teeth are only impregnated with goethite and opal (SiO2 • nH2 O) (Lowenstam 1962b; Mann et al. 1986; Sone et al. 2005). The presence of amorphous iron oxide and hydroxyapatite in the highly modified radular apparatus of Falcidens sp. (Caudofoveata) has also been reported (Cruz et al. 1998). Additionally, amorphous, hydrous, ferric phosphatic dermal granules were discovered in Molpadia (Holothuroidea: Echinodermata) (Lowenstam and Rossman 1975). Marine sponges (Porifera) are known to build their numerous skeletal structures of calcium carbonate or silica. However, the first occurrence of crystalline iron mineralization in the phylum Porifera and the first indication of hard tissue formation among the keratosa demosponges have also been reported (Towe and Rützler 1968). Thus, reddish-brown granules embedded in the spongin fibers of some keratose sponges consist of very fine crystallites of poorly organized lepidocrocite. It was confirmed that the small granules of iron are attached to the sponge skeletal fibers and are not from foreign materials. The iron content in spongin-based skeleton of some demosponges may contribute up to 10% of the skeleton ash (Towe and Rützler 1968). Lepidocrocite, calcite, and goethite (α-FeOOH) were also identified within spongin fibers of a common bath sponge Spongia officinalis by Vacelet et al. in 1988. Unfortunately, nothing is currently known about the biological significance of this biomineralization, which is unusual to find in sponges. However, the finding has an economic importance, as high amounts of iron are said to depreciate the value of commercial sponges and to affect the durability of the skeleton (Sella 1912). It was noted (Vacelet et al. 1988) that values of iron content are significantly higher in a strongly polluted habitat than in pure water.
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2.1.5 Vanadium (Biomineral?) Although the unusual phenomenon whereby some ascidians accumulate vanadium to levels more than 10 million times higher than those in seawater has attracted researchers in various fields, the physiological roles of vanadium remain to be explained (Brand et al. 1989; Ciereszko et al. 1963; Michibata et al. 2003). The following hypotheses were recently discussed (Ciancio et al. 2004; Odate and Pawlik 2007): (i) cellulose of the tunic might be produced by vanadocytes; (ii) vanadium-containing vanadocytes might reversibly trap oxygen under conditions of low oxygen tension, (iii) vanadium in ascidians acts to protect them against fouling or as an antimicrobial agent. However, most of the proposals put forward do not seem to be supported by sufficient evidence. Therefore, we have not yet obtained any clue to resolve the physiological roles of vanadium in ascidians. Attempts to characterize this phenomenon can be expected to promote more information about the unusual accumulation of vanadium by one class of marine organisms. Probably, studies on possible existence of vanadate–cellulose-based biominerals in ascidians could give us an answer.
2.1.6 Strontium-Based Biominerals Marine invertebrates are known to be involved in strontium cycle in the world ocean (Odum 1949). Radiolarian and molluscs (Odum 1951a) are the key players in this phenomenon (Odum 1951b). Acantharians are a chemically unique group of organisms that secrete skeletal (Bütschli 1906) and cystic structural forms composed of celestite (SrSO4 ) (Bernstein et al. 1987, 1992; Odum 1951a; Veizer 1978). They are documented as ubiquitous and abundant marine protozoans, frequently outnumbering their siliceous radiolarian and carbonate foraminiferan. Bernstein et al. (1987) reported comparative data on particulate strontium sulfate fluxes and strontium-to-chlorinity ratios to provide insights into the strontium cycle of the North Pacific. Free-drifting sediment traps were used to derive large particle fluxes between depths of 100 and 3,500 m in the eastern and western North Pacific Ocean. Flux data revealed substantial quantities of acantharian skeletons and cysts (both made of strontium sulfate) settling through the upper kilometers of the water column. The greatest fluxes of celestite were detected at 400 m. Minimal to nondetectable fluxes noted at and below 900 m provide evidence that at this juncture, the majority of acantharian specimens had dissolved, thereby contributing to the pool of dissolved strontium. These fluxes of particulate strontium and model calculations for fluxes of dissolved strontium indicate that acantharians play an important role in the ocean’s strontium budget. In concert with these observations, previous investigations that report Ba as a principal trace constituent in acantharian celestite (Bernstein et al. 1992; Rieder et al. 1982) lead to the hypothesis that acantharians are important to the biogeochemical cycles of both Ba and Sr (Bernstein et al. 1998). However, Sr together with a little Ba is found in the Remanella species too (Rieder et al. 1982).
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2.1.7 Boron Findings in several disciplines suggest that boron, an abundant element in the ocean, is involved in a range of aspects of marine life and it is considered an essential trace element (Harriss 1969). While all boron-containing and boron-binding low molecular weight metabolites are of prokaryotic origin, boron also plays an important role in stabilizing plant and algal cell walls by cross-linking carbohydrates (Black and Mitchell 1952). The molecular biology of boron transporters in different branches of the tree of life is beginning to emerge, but the current knowledge remains far from phylogenetically representative (Carrano et al. 2009). While a number of studies report that boron is essential for the growth and development of marine algae, the specific role(s) of the element remain(s) unclear. However, Lewin demonstrated the requirement of boron for the growth of marine pennate and centric diatoms (Lewin 1965), as cell division was significantly reduced at boron concentrations less than 0.5 mg L−1 (∼0.05 mM; i.e., ∼10% of natural seawater concentration) and ceased completely at lower concentrations (Lewin 1966a). The occurrence of boron–silica-based biocomposites in diatoms has not yet been reported. The role of boron within mollusc shells is also unknown; however, the constant relationship between seawater salinity and boron concentration suggests that boron can be used as a measure of salinity. It was suggested, on the basis of analyses of the boron content of shells belonging to the bivalved mollusc Mytilus edulis, that salinity could be reconstructed from biomineral records of boron (Roopnarine et al. 1998). Molluscan shells grow by incremental accretion and preserve within them geochemical records of their environments. Therefore, if boron concentration is being controlled by an external factor, the ontogenetic boron profiles of different shells, contemporaneous and sympatric, should be similar.
2.1.8 Titanium-Based Biominerals Diatoms are known to bioaccumulate trace levels of titanium (Riley and Roth 1971), and organisms collected from the marine environment can contain 0.01–0.13 wt% (1,254 ppm) Ti in silica (Martin and Knauer 1973). Recently, a two-stage bioreactor cultivation process was used to metabolically insert titanium into the patterned biosilica frustule of the diatom Pinnularia sp. by controlled feeding of soluble titanium and silicon to the silicon-starved cell culture suspension (Jeffryes et al. 2008). The addition of titanium to the diatom cells had no detrimental effects on the growth of the organism and preserved the nano- and microstructure of the frustule biosilica. Co-uptake of Ti and Si was required for maximum incorporation of titanium into the frustule biosilica. Titanium was preferentially deposited as a nanophase lining the base of each frustule pore. Numerous peptides showed high affinity to titanium dioxide in experiments in vitro (Dickerson et al. 2008); however there is no information about titanium– protein complexes isolated from biological objects. To my best knowledge, only one titanium-based skeletal structure observed in foraminifera in a natural environment
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has been described. This structure was reported in Bathysiphon argenteus, a species of foraminifera, which employs a titanium mineral in its test (Cole and Valentine 2006).
2.1.9 Copper-Based Biominerals Distribution of copper within organisms of marine invertebrates is, unfortunately, mostly determined due to heavy metal contamination in the sea (Bryan 1976). The regulation of the copper adsorption seems to be a complex phenomenon. For example, it was reported (Ozoh 1994) that under low temperature conditions (12◦ C), accumulation of copper by the ragworm, Hediste (Nereis) diversicolor, increased at a low salinity of 7.6% but was reduced at intermediate and high salinities of 15.25 and 30.5%, respectively. Copper contents of the worms at low salinity and temperature ranged from 68 to 185 μg/g. Under increasing temperature of 17 and 22◦ C, bioavailability of copper to the worms increased irrespective of salinity gradient. The copper contents of the worm ranged from 59 to 784 μg/g dry weight. It is established that copper is the major metal component of the glycerid polychaete jaws (Gibbs and Bryan 1980; Gibbs et al. 1981). The bloodworm, Glycera dibranchiata, has four black jaws that grab and bite its prey. The jaws are hollow, and the space is used to conduct venom into the prey. It was found that the jaw tip exhibits the ordered crystalline structure of the copper-based biomineral atacamite (Cu2 Cl(OH)3 ) (Lichtenegger et al. 2002). It was reported that the jaw system is more complex, containing zinc, iron, and unmineralized copper compounds as well (Lichtenegger et al. 2005). For example, X-ray absorption spectroscopy studies showed that a fraction of copper in the jaw is present in its oxidation state of Cu(I), which is in contrast to the mineral that exclusively contains Cu(II). X-ray fluorescence imaging also revealed traces of copper in the jaw base, which is devoid of mineral. Traces of iron were found as well but were spatially correlated with the copper mineral, suggesting a substitution of copper atoms by iron in the atacamite mineral. Zinc was evenly dispersed throughout the jaw matrix, quite analogous to zinc in Nereis jaw, a related worm species where unmineralized zinc serves to cross-link and harden the proteinaceous matrix. Overall, as reported recently (Moses et al. 2008), Glycera jaws are composed of ∼40 wt% protein, 40 wt% melanin, and <10 wt% metals and minerals, the latter being mostly in the form of unmineralized copper (Cu) with small amounts of mineralized atacamite fibers. The Cu in all forms is concentrated at the tip of the jaw, typically within ∼30 μm of the surface. Additionally, the jaw protein is very rich in glycine and histidine.
2.1.10 Zinc-Based Biominerals Zinc was detected as a major inorganic component in the jaws of Nereid worms by Bryan and Gibbs (1979). For example, analyses of the jaws of Nereis versicolor
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have shown zinc to be a major inorganic component, amounting to between 0.5 and 2.4% of the dry weight and accounting for up to 40% of the total body burden of zinc. In this species, zinc concentrations in the jaws were not related to those of the habitat sediments. Zinc concentrations in N. diversicolor increased during periods of rapid growth and may represent a physiological demand for this metal (Howard and Brown 1983). Analyses of the jaws of Nereis virens have shown that various metals are present in detectable quantities; in order of decreasing concentration these are [with ranges (μg/g dry weight) in brackets] as follows— zinc (10,130–18,360), sodium (1,150–2,690), magnesium (616–1,080), calcium (531–1,130), iron (444–1,180), manganese (510–952), potassium (270–960), and copper (4–80), as well as lead (8·7), cadmium (0·1), and silver (<0.1) (Bryan and Gibbs 1980). The most concentrated metal zinc is not uniformly distributed throughout the jaw, as the highest level occurs in the distal tip (45,740 μg/g) and falls to a much lower level in the basal part (1,790 μg/g). The contribution of some jaw metals to the total body burden varies according to the body size: in small worms, the jaw zinc accounts for over 40% of the total body burden, but in large worms, it accounts for 30% or less; jaw manganese contributes over 50% of the total body burden in small worms, dropping to about 20% with increasing size. For silver, cadmium, copper, iron, and lead the jaws generally contribute less than 1% to the total body burden. Recently, more attention is paid to investigations of microstructure and mechanical properties of zinc-containing worm jaws (Birkedal et al. 2005; Lichtenegger et al. 2003). It was shown that the local hardness and the stiffness of the jaws correlate with the local zinc concentration, pointing toward a structural role for zinc (Khan et al. 2006). This zinc is always found to be tightly correlated with chlorine, suggesting the presence of a zinc–chlorine compound. No crystalline inorganic phase was found, however, and results from X-ray absorption spectroscopy further exclude the presence of simple inorganic zinc–chlorine compounds in amorphous form. The correlation of local histidine levels in the protein matrix and zinc concentration leads authors to hypothesize a direct coordination of zinc and chlorine to the protein. Broomell et al. (2006) demonstrated directly, for the first time, that Zn plays a critical role in the mechanical properties of histidine-rich Nereis jaws. Using nanoindentation, they showed that removal of Zn by chelation decreases both hardness and modulus by over 65%. Moreover, reconstitution of Zn yields a substantial recovery of initial properties. Modulus and hardness of Zn-replete jaws exceed those attainable by current engineering polymers by a factor of >3. Zn-mediated histidine cross-links are proposed to account for this enhancement in mechanical properties. Zinc silicate is another zinc-based biomineral investigated in marine invertebrates, especially in the diatoms. Zinc incorporation into the siliceous cell wall (frustule) of the freshwater diatom Stephanodiscus hantzschii was studied for Zn2+ concentrations ranging from 25 pmol L−1 to 25 nmol L−1 (Jaccard et al. 2009). Concentrations of intracellular Zn were positively correlated with Zn in the frustule, suggesting that Zn in the frustule originated from intracellular pools. The processes leading to Zn incorporation into the cell wall were examined by determining the role(s) of Si and Mn via competition experiments. Results demonstrated that Zn
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competed with Mn for incorporation into the frustule. Zn/Si values for S. hantzschii were consistent with field data obtained from Lake Geneva (Switzerland). Ellwood and Hunter (2000) have investigated the incorporation of Zn and Fe into the frustule of the marine diatom Thalassiosira pseudonana. Zn levels in the cell wall generally increased over a wide range of free Zn2+ concentrations in culture media, leading the authors to suggest that Zn content in fossil frustules could be used to track past changes in oceanic concentrations of free Zn2+ .
2.1.11 Manganese Oxides Deep-sea minerals in polymetallic nodules, crusts, and hydrothermal vents are formed not only by mineralization but also by biologically driven processes involving marine microorganisms (Wang and Müller 2009a, b).Within the nodules, freeliving and biofilm-forming bacteria provide the matrix for manganese deposition (Glasby 2006), and in cobalt-rich crusts, coccolithophores represent the dominant organisms that act as bio-seeds for an initial manganese deposition. Manganese aggregates occur as both crystallized varieties and disordered fine-grained phases with significant ore grade and up to 50–60 vol% of X-ray amorphous components. X-ray amorphous nanosized Mn oxides in Fe–Mn nodules from the Pacific Ocean floor were recently examined from the standpoint of their biogenic origin (Lysyuk 2009). SEM examination showed abundant mineralized biofilms on the studied samples. The chemical composition of bacterial mass has been reported as follows (wt%): 28.34 MnO, 17.14 Fe2 O3 , 7.11 SiO2 , 2.41 CaO, 17.90 TiO2 , 1.74 Na2 O, 1.73 Al2 O3 , 1.30 MgO, 1.25 P2 O5 , 1.25 SO3 , 0.68 CoO, 0.54 CuO, 0.53 NiO, and 0.50 K2 O. The chemical composition of fossilized cyanobacterial mats within the interlayer space of nodules is reported as follows (wt%): 48.35 MnO, 6.23 Fe2 O3 , 8.76 MgO, 5.05 Al2 O3 , 4.45 SiO2 , 3.63 NiO, 2.30 Na2 O, 2.19 CuO, 1.31 CaO, and 0.68 K2 O and comprises direct evidence for participation of bacteria in Mn oxide formation. This phase consists of mineralized glycocalyx consisting of nanosized flakes of todorokite. Native metals (Cu, Fe, and Zn) as inclusions of 10–20 μm in size were identified in ferromanganese nodules as well. The formation of native metals can be explained by their crystallization under highly reducing conditions maintained by organic matter.
2.1.12 Germanium-Based Biominerals Germanium (Ge) is a scarce but not an extremely rare element in the Earth’s crust (about 1.6 ppm Ge crustal average). In plants, Ge can partially substitute for B in the case of boron deficiency, although deficiency symptoms are still seen with a lag period of approximately 1–3 weeks (Rosenberg 2008). In a biogeochemical respect, germanium and silicon react very similarly. Their molar ratio is typically in the order of 0.6 × 10−6 , with significant deviations only where germanium is complexed and transported, e.g., by humic-rich waters. Germanium is a very conservative element
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in biogeochemical terms. It hardly shows involvement in any biogeochemical reaction cycles and is mainly present in the form of complexes or hydroxo compounds of the tetravalent germanium. The only naturally occurring organogermanium compounds are mono- and dimethylgermanium, which are believed to be formed by microbiological activity in continental zones containing Ge-rich minerals, then are leached into rivers, and finally into the open sea (Rosenberg 2008). Siliceous organisms take up Ge (IV) as if it were a very heavy isotope of silicon (Lewin 1966b). Uptake studies have been performed with Ge radioisotopes, which indicate that at low Ge/Si ratios (0.01), most of the Ge is incorporated into the cell wall of the diatom (Azam et al. 1973). For the smaller portion that is incorporated into the different cell organelles, some Ge/Si fractionation was observed by Mehard et al. (1974). The diatoms Nitzschia alba, Navicula pelliculosa, Cylindrotheca fusiformis, and Cyclotella nana took up radioisotopically labeled germanic acid, 68 Ge(OH) , from their growth media and incorporated up to 80% of it into the 4 silica of their cell walls (Azam 1974). Silicification appeared to be required for germanium incorporation. Ge mimics the metabolic pathway of silicon at low concentrations and affects silicon transport and uptake by acting as a classical competitive inhibitor at high concentrations. Such behavior has been observed for all creatures that require silicon as an essential element or which undergo silicification. For example, Ge acid at concentration levels of 1–25 mg L−1 disrupts siliceous spicule formation in freshwater sponges (Simpson et al. 1983). The effect of germanium on the secretion of siliceous spicules by the freshwater sponge Spongilla lacustris was investigated by exposing germinating and hatching gemmules to varying concentrations of germanium (Ge) in the presence of silicon (Si) (Simpson 1979). Results were analyzed quantitatively and qualitatively and demonstrated that a [Ge]/[Si] (= molar ratio) of 1.0 completely inhibits silicon deposition. Intermediate ratios (0.5, 0.1, 0.01) which are permissive to spicule appearance result in fewer, shorter, and thinner spicules, in proportionately fewer microscleres and in short bulbous megascleres. The size of the bulb increases with increasing [Ge]/[Si], while the length of the bulbous megascleres decreases with increasing [Ge]/[Si]. Microscleres do not demonstrate these graded responses suggesting that they are secreted in an all or none manner. Swellings produced in pond water and bulbs produced in germanium appear to decrease in size with time, indicating a spreading of the accumulated silica. The effect of germanium on spicule secretion can be partially explained by its ability to uncouple the growth in length of the axial filament from the growth of the surrounding silicalemma. Under these conditions, excess silicalemma is produced in which silica accumulates as bulbs in short spicules. Continuous exposure to Ge is necessary to produce this altered morphology. It is conjectured that the bulbs may be retained due to an inhibition of spreading which in turn may be caused by the incorporation of germanium into the silica (Simpson 1979). Herewith, I take a liberty to suggest that phenomenon described above can be used in the future as a key method for the biotechnological development of germanium-based biomineralized structures using sponges, for which cultivation technologies have recently been well developed.
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2.1.13 Silica-Based Biominerals Silica-based biocomposites also occur widely in nature (Fig. 2.2). Cell membranes of microorganisms might function as seed crystals for Si precipitation, which is well known from biogeosystems with Si supersaturation, e.g., geothermal springs (Sommer et al. 2006). Due to their small size, bacteria as a group have the highest surface area-to-volume ratio of any group of living organisms and this, together with the presence of charged chemical groups on their cell surface, is responsible for the potent mineral-nucleating ability of these cells (Douglas 2005). Silicic acid which has been taken up from soil solution (actively or passively) is precipitated primarily as amorphous silica at cell walls, lumens, and in the intercellular voids of plants (Ma et al. 2006). Silica–cuticle double layers and silica–cellulose double layers were observed on the surface of leaves,
Fig. 2.2 SEM images of siliceous spines and scales of heliozoan Placocystis sp. Bar scales—250 nm (a), 300 nm (b, c) (images courtesy: Ludmila Gaponova)
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stem, and hulls (Yoshida 1965). Some plant cystoliths also contain silicon and are covered in a sheath of siliceous material (Watt et al. 1987). Phytogenic silica in the form of phytoliths is regarded as the major component of the biogenic silica pool in soils, followed by diatom skeletons and sponge spicules (Clarke 2003). Siliceous skeletons and spicules are also known to be present in different Protozoa, e.g., radiolarians (Hertwig 1879), silicoflagellates (Ehrenberg 1830), and sarcodines (Dujardin 1841). In animals, not only siliceous skeletons are limited to glass sponges and demosponges spicules (Uriz 2006), but also there are minor occurrences such as the opalized mandibular blades of boreal copepods (Sullivan et al. 1975) and micrometer-scale silica tablets formed intracellularly in the epidermis of some brachiopod larvae (Williams et al. 2001). Siliceous structures were also identified within ascidians (Monniot et al. 1995). For example, the Henze precipitate, a peculiar blue-green microparticulate obtained by lysis of the blood cells of the ascidian Phallusia mammillata (Protochordata), was investigated with atomic force microscopy (AFM), scanning electron microscopy (SEM), and X-ray microanalysis. The microparticulates measured 50–100 μm in diameter and appeared irregular in shape. X-ray analysis showed that the elements present in these same precipitates were mainly C, Si, Al, and O. The microparticulate composition appeared close to that of natural waxes or lacquers, embedding amorphous silicates and/or other Si–Al components. The unusual occurrence of Si in ascidian blood and its role still remain unclear to this day. The high silicon content of certain radular sites of the Patellacea (Mollusca, Gastropoda) has been shown by infrared absorption spectra to be fixed in the form of the mineral opal (Lowenstam 1971). The presence of opalized base plates only in the families Acmaeidae and Lepetidae appears to be of taxonomic significance. Minimum values of the volume of fixation and of the turnover rate of opal by the Patellacea are currently being calculated to assess the role of this previously neglected taxon in biological fixation of silica in the oceans. The significance of these organisms points to the much needed study of the silica transport system in tissue-grade Metazoa. Interestingly, some Mollusca species, e.g., Onchidella celtica, possess siliceous penial structures of different morphologies (Labbé 1933a, b, 1934a, b). Moreover, different kinds of silica-based spicular formations were identified within cuticle layers of these slugs (Labbé 1933a, b, 1934a, b) (see also Chapter 12). Experiments including the detailed analysis with respect to the identification of the nature and origin of silica-based formations reported previously in different representatives of these Silicodermates (Labbé 1933a, b, 1934a, b) are now in progress in our laboratory. Unique siliceous structures that occur in marine glass sponges (Fig. 2.3) are described and discussed below (see Chapter 8). Because of the wide diversity of silica in skeletal structures of different organisms and especially of marine invertebrates, I summarized numerous data regarding this topic in Table 1 (see Addendum).
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Fig. 2.3 Hyalonema sieboldii glass sponge individual anchoring spicule is high flexible and can be bent in the circle
2.2 Conclusion Thus, many biomineralized tissues of marine invertebrate origin are composite materials containing a biologically produced organic matrix and nano- or microscale amorphous or crystalline minerals, most of which are represented by calcium carbonate and silica. During the processes of biomineralization, the organic material acts variously as nucleator, cooperative modifier, and matrix or mold for the mineral ions. The resulting tissue has properties very different from those of the pure minerals themselves. The stiff mineral prevents the organic matrix (proteins, peptides, polysaccharides, lipids) from yielding, while the organic matrix prevents the mineral from cracking (Treccani et al. 2003). Why some marine organisms utilize silica or germanium rather than calcium carbonate or goethite as a structural material is unknown. Probably, it depends on external factors like temperature, pH, salinity, aerobic or anaerobic conditions, as well as on toxicity, or on supersaturation with respect to corresponding mineral phase, and/or on structural (fibrillar or globular), physico-chemical (charged or uncharged, acidic or neutral), as well as on interface (hydrophobic or hydrophilic) peculiarities of organic matrixes which also can differ during different stages of ontogenesis. Unfortunately, chemical pollution of the oceans can lead to appearance of novel, unknown, and “bizarre” forms of biominerals whose development is a kind of biological response of the organism to artificially determined supersaturation of the corresponding metallic ions. As example of this phenomenon, the reader can observe the unique electron microscope images of the crystalline iron-based mineral phase growing on nanofibers of marine sponge collagen (Fig. 2.4), which was isolated from the animal that was accidentally located in an environment highly contaminated with this metal. Intriguingly, however, materials science point of view is that this image stimulates ideas to develop novel iron–collagen-based biocomposites with undoubtedly interesting properties for technical applications. I endorse the suggestion recently made by Livingston et al. (2006) that even if biomineralized structures per se appeared independently in different lineages, a common set of genetic and developmental mechanisms, i.e., a biomineralization “toolkit,” may have been repeatedly employed. Biomineralization phenomena are probably one of the most widely studied topics in modern geochemistry, materials
References
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Fig. 2.4 SEM images: iron mineralization of collagen fibrils (adapted from Vacelet et al. 1988)
science, biomedicine, and biomimetics (Estroff 2008). The following chapters summarize the basic information available on common and alternative organic templates with respect to both calcification and silicification in vivo as well as in vitro and allow for the first time a broader consideration of the field of biological materials of marine origin.
References Addadi L, Raz S, Weiner S (2003) Taking advantage of disorder: amorphous calcium carbonate and its roles in biomineralization. Adv Mater 15:959–970 Anneroth G, Eneroth CM, Isacsson G (1975) Crystalline structure of salivary calculi. A microradiographic and microdiffractometric study. J Oral Pathol 4:266–272 Aronova MZ (1987) Sensory systems of Ctenophora. Doctorate Sci. Dissertation, Leningrad Aronova MZ (2009) Structural models of “simple” sense organs by the example of the first Metazoa. J Evol Biochem Physiol 45:179–196 Azam F (1974) Silicic acid uptake in diatoms studies with [68 Ge] germanic acid as tracer. Planta 121:205–212 Azam F, Hemmingsens BB, Volcani BE (1973) Germanium incorporation into the silica of diatom cell walls. Arch Mikrobiol 92:11–20 Becker A, Sötje I, Paulmann C et al (2005) Calcium sulfate hemihydrate is the inorganic mineral in statoliths of Scyphozoan medusae (Cnidaria). Dalton Trans 1:1545–1550 Been JM, Bills PM, Lewis D (1979) Microstructure of gallstones. Gastroenterology 76(3):548–555 Berger S, Kaever MJ, Reichekl B (1997) Influence of ecological conditions on magnesium and strontium content and ultrastructure of the calcareous skeleton of Bornetella sphaerica (Zarnadini), Dasycladales. Paläont Z 71(3/4):189–195
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Chapter 3
Biomineralization
Abstract In the process of biomineralization, organisms typically accumulate the precursors (e.g., ions of metals) required to synthesize biominerals from their respective environments. We have a broad diversity of inorganic precursors in natural marine environments, as well as organic scaffolds and specific templates that may be found in marine organisms. But, why some organisms utilize, for example, silica rather than calcium carbonate as a structural material is unknown. It is remarkable that an organic matrix also plays a key role as an interlinkage between a diagenic mineral (rocky substrate) and the biomineral (skeleton of organism) in marine invertebrates. The active role of the organic matrix in biomineralization is fundamental and represents a source of inspiration for future nanotechnologies with a bottom-up approach. Biominerals are produced by marine organisms for specific purposes including structural support, sensing (geotaxis), ion storage, and toxic waste removal. A process by which biological systems produce inorganic minerals in vivo is called biomineralization. In the process of biomineralization, organisms typically accumulate the precursors (e.g., ions of metals) required to synthesize biominerals from their respective environments (water, soil, food) (Currie et al. 2006). Organisms are able to transport these precursors from the environment in which they are found into the organism. Subsequently, the precursors may be stored, transported in vivo, and converted into biominerals. Traditionally, biomineralization processes are broadly classified as follows (Currie et al. 2006): • Processes in which minerals are synthesized for a specific biological function. Biomineralization in this class is strictly under biological (genetic) control (e.g., skeletons formation). • Processes in which minerals are formed without any apparent specific function (pathological biomineralization). These biominerals may be useful, detrimental, or benign to the organisms producing them (e.g., calculi).
H. Ehrlich, Biological Materials of Marine Origin, Biologically-Inspired Systems 1, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9130-7_3,
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Biomineral-containing materials are actually composites of biomacromolecules and inorganic salts (or oxides), and they have structural and mechanical properties that may be somewhat different to those of their individual components. Moreover, there are differences between classical nucleation theory of crystal formation and phenomena observed in biomineralization processes that occur in nature. According to classical nucleation theory (Nancollas 1982; Volmer 1939), the crystallization of inorganic minerals starts from their constituting ions, which, on the basis of their ionic complementarity, form small clusters in a stochastic process of dynamic growth and disintegration. These clusters become stable when a critical size is reached. At this point, the increasing surface energy, related to the growing surface area, is balanced by the reduction of bulk energy, which in turn is related to the formation of a crystal lattice. The resulting primary nanoparticles form the critical crystal nuclei that are the basis of further growth through the associated reduction of the Gibbs free energy of the system (Currie et al. 2006). However, in contrast to what is described by classical nucleation theory, calcium carbonate crystal formation, for example, has been shown to occur from a transient amorphous precursor phase, both in biological and in biomimetic systems. Thus, in 1997 it was demonstrated that sea urchin larvae form their calcitic spicules by first depositing a highly unstable mineral phase called amorphous calcium carbonate (Weiner et al. 2009). This strategy has since been shown to be used by animals from other phyla, and for both aragonite and calcite. Recent evidence shows that vertebrate bone mineral may also be formed via a precursor phase of amorphous calcium carbonate (Weiner 2006). This strategy thus appears to be widespread (Beniash et al. 2006; Politi et al. 2004, 2008). The challenge now is to understand the mechanisms by which these unstable phases are initially formed, how they are temporarily stabilized, and how they are then destabilized and transformed into a crystalline mature product (Weiner et al. 2009). Intriguingly, it was recently shown that CaCO3 nucleation is preceded by the formation of nanometer-sized pre-nucleation clusters, which is also not predicted by classical nucleation theory (Pouget et al. 2009).The nanoscopic pre-nucleation clusters, which have been visualized by these authors, are the smallest stable form of CaCO3 currently known. These clusters are likely the building blocks of the amorphous precursor particles observed in biomineralization. Based on the highly regulated biological environments, biomineralization can be classified as an intracellular, intercellular, or extracellular process (Subburaman et al. 2006). An understanding of how organisms select, localize, and concentrate elements is gained by investigating biologically controlled biomineralization processes. Such studies can yield information on how minerals are nucleated, spatially located, or segregated, as well as the internal microstructure and bulk morphology produced, and how inorganic/organic interfaces are controlled. It is also generally assumed that the organic matrix plays an important role in crystal growth, as well as contributing to the biomechanical properties of the mineralized tissue formed (Weiner and Traub 1984). In many mineralized tissues, the organic matrix forms two- or three-dimensional structures onto (or into) which the mineral crystallites grow. The two categories of matrix components have been called “framework” or
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Fig. 3.1 The foot-like structures of W. leuckarti glass sponge (a), of Isidella sp. octocoral (b), and of the black coral (Antipatharia) are destined for attachment of the organisms to hard substrates
“surface” constituents: one for the more hydrophobic, insoluble macromolecules and the other for the more acidic, soluble macromolecules, respectively (Weiner et al. 1983). I want to note the fundamental role of an organic matrix in the biomineralization processes of marine invertebrates with the examples shown in Fig. 3.1. The reader can see the foot (holdfast) skeletal fragments corresponding to the three different organisms: the glass sponge Walteria leuckarti, the octocoral Isidella sp., and the black coral Antipatharia sp. The principle of attachment to hard substrates using a foot-like structure is common for all of these organisms, but they drastically differ from each other in their mineral phases. Thus, the glass sponge skeleton is made of amorphous silica, while the mineralized parts of Isidella octocoral are of crystalline calcite. The black coral hard tissues are, however, represented by chitin–protein–polyphenolic composite material, which lacks any kind of mineral. Therefore, it seems absolutely correct to make the suggestion that an exact organic matrix is initially responsible for attachment between animal and substrate. The matrix forms a foot-like structure, which was clearly optimized for the survival of the organisms during evolution. There are also no doubts about the ancient character of this phenomenon, especially in the case of glass sponges, which have been found in Precambrian fossils (Reitner and Wörheide 2002). Furthermore, it is remarkable that an organic matrix also plays a key role as an interlinkage between a diagenic mineral (rocky substrate) and the biomineral (skeleton of organism). The active role of the organic matrix in biomineralization is fundamental and represents a source of inspiration for future nanotechnologies with a bottom-up
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approach (Lee and Choi 2007; Sanchez et al. 2005). The building of discrete or extended organic architectures in biomineralization often involves hierarchical processing in which the molecular-based organic assemblies are used to provide frameworks, as well as determining the organization of inorganic materials. The inorganic constituents are in turn exploited as pre-fabricated units in the production of higher order complex microstructures (Aizenberg et al. 2005; Fratzl 2007; Lakes 1993; Mann 1995; Meyers et al. 2006; Pouget et al. 2007). In biological systems, almost all calcified tissues contain a distinctive assemblage of acidic proteins, glycoproteins, or both, which are capable of influencing crystal growth of calcium-based mineral phases. One example of the mechanisms used is the intercalation of some of the acidic macromolecules into the crystal lattice (Berman et al. 1993). Control of crystal growth by acidic matrix macromolecules is an important process in the formation of many mineralized tissues (Addadi and Weiner 1985; Moradian-Oldak et al. 1992). Acidic and unusually acidic proteins are a feature shared by most—if not all—calcium carbonate-mineralizing phyla (Marin and Luquet 2007). Highly acidic macromolecules are postulated to be intermediates in tissue mineralization, because they can sequester many calcium ions and have been shown to occur in high concentrations at mineralizing foci in distantly related organisms (Marsh 1994). Negatively charged carboxylate groups also have the ability to bind calcium ions and induce the nucleation of the related biominerals in nature. It was hypothesized that the carboxyl group is, in many tissues, the organic– mineral interface (Gilbert et al. 2005). In this hypothesis, the carboxyl group is the molecular “glue” of choice for biominerals in skeletal structures that occur in organisms. However, acidic proteins and acidic polysaccharides are not involved in biosilicification phenomena. Thus, we have a broad diversity of inorganic precursors in natural marine environments, as well as organic scaffolds and specific templates that may be found in marine organisms. But why some organisms utilize, for example, silica rather than calcium carbonate as a structural material is unknown (Mann 1995). The following results reported by Rieder and co-workers in 1982 may help us to come nearer to an answer to this complex question. The authors comparatively investigated Acantharia, Remanella, and Loxodes protists of the order Karyorelictida. Acantharia is a subclass of essentially marine pelagic protozoans in the class Actinopodea and is characterized by skeletal rods made of strontium sulfate (celestite). Opinions differ as to whether Ca or Ba is present in the rods. Remanella is a karyorelictid ciliate, so called because they have macronuclei which do not divide, which is thought to be an ancestral trait. Remanella is commonly found in marine sediments or associated with detritus, especially in areas with little oxygen. Remanella have a hook-shaped anterior end and a line of so-called Müller’s bodies along the convex dorsal side of their bodies. In Acantharia and Remanella, the ratio of Sr:Ba is nearly the same, and it is comparable with the ratio found in seawater. However in Loxodes (Fenchel and Finlay 1984), which is closely related to Remanella, nothing but Ba was found. This difference might be explained as follows. Both ciliates have to incorporate a heavy mineral into the Müller’s bodies, which may function as a mechanoreceptor.
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Conclusion
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These organelles are specialized vacuoles (7–10 μm in diameter) containing a heavy load of BaSO4 (3–3.5 mm in diameter, density 4.5 g/cm3 )—the Müller body— which is fixed to a modified ciliary stalk (Fenchel and Finlay 1986). The proposed mechanism is analogous to graviperception in Metazoa. Gravity is perceived by the bending of a ciliary complex that changes the membrane potential, which in turn determines the activity of the cilia and thus the swimming activity of the cell (Hemmersbach and Häder 1999). It is easier for organisms to incorporate Ba in freshwater than in seawater, because the ratio Sr:Ba is 5:1, while in seawater this ratio is 400:l (Rieder et al. 1982). These examples show that the listed protozoa (different perhaps with the Acantharia) have a mechanism that discriminates very well against the lighter elements in the group IIa of the periodic system. The heavier elements will be incorporated in the same way as the main component. The amount of the heavier elements incorporated is limited by their content in the environment. There is a great difference between the ionic radii in this group of elements. The heavier the ions, the greater their radii. Thus there may be one basic mechanism of discrimination, one that changed little during evolution. This mechanism might use the different radii of the ions. If so, there should be related organisms that cannot distinguish between the elements as well as Remanella or Loxodes. Other species of the order Karyorelictida, like Geleia or Trachelocerca, change the ratio Ca:Sr:Ba found in the environment to a great extent, but they cannot discriminate as well against the lighter elements. In all protozoa tested, however, very good discrimination against elements of group IIa is found, with the exception of one Trachelocerca species which incorporates Mn. Why is Sr or Ba incorporated into these unicellular organisms? Why don’t they use Ca (except Prorodon), which they could obtain very easily? The authors suggested that Loxodes and Remanella use Ba and Sr because they are heavier than Ca, and the heavier mineral will be more advantageous in a mechanoreceptor. But why is Sr used in Acantharia while Ca, Sr, and Ba are used in Geleia and Trachelocerca? There was no explanation for the Trachelocerca species found in Sylt (Germany) that incorporates Ca, Sr, Ba, and perhaps a little Mn, while another species from northern Italy incorporates Ca, Sr, and Mn. Nor is it determined which chemical compound of Mn is present in the animal. Presumably there must be another incorporation mechanism, different from the ones that incorporate Ca, Sr, and/or Ba. It appears, however, that all of these protozoa could serve as good model organisms for investigating the mechanism of incorporation of Ca, Sr, and Ba (Rieder et al. 1982).
3.1 Conclusion A general mode of biomineralization that addresses several levels of biomineral formation has been suggested by Nancollas (1982), Lowenstam and Weiner (1989), Mann (1995, 2001), Simkiss and Wilbur (1989), Addadi and Weiner (1992), Addadi et al. (2002), and Cölfen and Mann (2003), and the best overall sources of
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information and discussions on this topic have been recently provided by Bäuerlein (2007), Meyers et al. (2008), Ehrlich et al. (2008, 2009), Weiner (2008), and Estroff (2008). Therefore, because of the very comprehensive information on biomineralization available (see also Table 2 in Addendum), I will take the liberty of discussing below only the most recent events regarding this subject matter, which were recently discovered in our laboratory. The name of the process we discovered is multi-phase biomineralization.
References Addadi L, Beniash E, Weiner S (2002) Assembly and mineralization processes in biomineralization: strategies for forming biological composite materials. In: Jones W, Rao CNR (eds) Supramolecular organization and materials design. University Press, Cambridge Addadi L, Weiner S (1985) Interactions between acidic proteins and crystals – stereochemical requirements in biomineralization. Proc Natl Acad Sci USA 82:4110–4114 Addadi L, Weiner S (1992) Control and design principles in biological mineralization. Angew Chem Int Ed Engl 31:153–169 Aizenberg J, Weaver JC, Thanawala MS et al (2005) Skeleton of Euplectella sp.: structural hierarchy from the nanoscale to the macroscale. Science 309:275–278 Bäuerlein E (2007) Handbook of biomineralization. Wiley-VCH, Weinheim, 440 pp Beniash E, Addadi L, Weiner S (2006) Cellular control over spicule formation in sea urchin embryos: a structural approach. J Struct Biol 125:50–52 Berman A, Hanson J, Leiserowitz L et al (1993) Biological control of crystal texture: a widespread strategy for adapting crystal properties to function. Science 259:776 Cölfen H, Mann S (2003) Higher-order organization by mesoscale self-assembly and transformation of hybrid nanostructures. Angew Chem Int Ed 42:2350–2365 Currie HA, Patwardhan SV, Perry CC et al (2006) Natural and artificial hybrid biomaterials. In: Kicckelbick G (ed) Hybrid materials: synthesis, characterisation and application. Wiley-VCH, Weinheim Ehrlich H, Koutsoukos PG, Demadis KD et al (2008) Principles of demineralization: modern strategies for the isolation of organic frameworks. Part I. Common definitions and history. Micron 39:1062–1091 Ehrlich H, Koutsoukos PG, Demadis KD et al (2009) Principles of demineralization: modern strategies for the isolation of organic frameworks. Part II. Decalcification. Micron 40: 169–193 Estroff LA (2008) Introduction: biomineralization. Chemical Rev 108:4329–4331 Fenchel T, Finlay BJ (1984) Geotaxis in the ciliated protozoon loxodes. J Exp Biol 110:17–23 Fenchel T, Finlay BJ (1986) The structure and function of Müller vesicles in loxodid ciliates. J Protozool 33:69–76 Fratzl P (2007) Biomimetic materials research: what can we really learn from nature’s structural materials? J R Soc Interf 4:637–642 Gilbert PUPA, Albrecht M, Frazer BH (2005) The organic–mineral interface in biominerals. Rev Mineral Geochem 59:157–185 Hemmersbach R, Häder D-P (1999) Graviresponses of certain ciliates and flagellates. FASEB J 13(Suppl):S69–S75 Lakes R (1993) Materials with structural hierarchy. Nature 361:511–515 Lee SW, Choi CS (2007) The correlation between organic matrices and biominerals (myostracal prism and folia) of the adult oyster shell, Crassostrea gigas. Micron 38:58–64 Lowenstam HA, Weiner S (1989) On biomineralization. University Press, Oxford, New York Mann S (1995) Biomineralization and biomimetic materials chemistry. J Mater Chem 5(7): 935–946
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Mann S (2001) Biomineralization: principles and concepts in bioinorganic materials chemistry. University Press, Oxford, New York Marin F, Luquet G (2007) Unusually acidic proteins in biomineralization. In: Bäuerlein E (ed) Handbook of biomineralization. Wiley-VCH, Weinheim Marsh M (1994) Polyanions and biomineralization. In: Allemand D, Cuif J-P (eds) Biomineralization 93, 7th international symposium. Part 1, no 7 fundamentals of biomineralization. Bulletin de 1’Institut océanographique de Monaco 14(1):121–128 Meyers MA, Chen P-Y, Lin AY-M et al (2008) Biological materials: structure and mechanical properties. Progr Mater Sci 53:1–206 Meyers MA, Lin AYM, Seki Y et al (2006) Structural biological composites: an overview. J Minerals 58:34–41 Moradian-Oldak J, Frolow F, Addadi L et al (1992) Interactions between acidic matrix macromolecules and calcium phosphate ester crystals: relevance to carbonate apatite formation in biomineralization. Proc R Soc Lond Ser B Biol Sci 247:4755 Nancollas GH (1982) Biological mineralization and demineralization. Springer, Heidelberg Politi Y, Arad T, Klein E et al (2004) Sea urchin spine calcite forms via a transient amorphous calcium carbonate phase. Science 306:1161–1164 Politi Y, Metzler RA, Abrecht M et al (2008) Transformation mechanism of amorphous calcium carbonate into calcite in the sea urchin larval spicule. PNAS 105:17362–17366 Pouget E, Dujardin E, Cavalier A et al (2007) Hierarchical architectures by synergy between dynamical template self-assembly and biomineralization. Nat Mater 6:434–439 Pouget EM, Bomans PHH, Goos JACM et al (2009) The initial stages of template-controlled CaCO3 formation revealed by Cryo-TEM. Science 323:1455–1458 Reitner J, Wörheide G (2002) Non-lithistid fossil Demospongiae – origins of their palaeobiodiversity and highlights in history of preservation. In: Hooper JNA, Van Soest RWM (eds) Systema porifera: a guide to the classification of sponges. Kluwer Academic/Plenum, New York Rieder N, Ott HA, Pfundstein P et al (1982) X-ray microanalysis of the mineral contents of some protozoa. J Protozool 29(1):15–18 Sanchez C, Arribat H, Giraud-Guille MM (2005) Biomimetism and bioinspiration as tools for the design of innovative materials and systems. Nat Mater 4:277–288 Simkiss K, Wilbur KM (1989) Biomineralization. Academic, San Diego Subburaman K, Pernodet N, Kwak SY et al (2006) Templated biomineralization on self-assembled protein fibers. Proc Natl Acad Sci USA 103:14673–14678 Volmer M (1939) Kinetik der phasenbildung. Steinkopff, Dresden Weiner S (2006) Transient precursor strategy in mineral formation of bone. Bone 39:431–433 Weiner S (2008) Biomineralization: a structural perspective. J Struct Biol 163:229–234 Weiner S, Mahamid J, Politi Y et al (2009) Overview of the amorphous precursor phase strategy in biomineralization. Front Mater Sci China 3:104–108 Weiner S, Traub W (1984) Macromolecules in mollusk shells and their functions in biomineralization. Phil Trans R Soc Lond Ser B 304:421–438 Weiner S, Traub W, Lowenstam HA (1983) Organic matrix in calcified exoskeletons. D. Reidel Publishing Co, Dordrecht, Holland
Chapter 4
Biomineralization–Demineralization– Remineralization Phenomena in Nature
Abstract Biomineralization, demineralization, and remineralization phenomena are three fundamental parts of the mineral–organic matrix circuit occurring in numerous specialized environments in nature. It is apparent that demineralization and biomineralization should be envisioned as the two sides of the same coin. Nature often uses these in admirable synergy to achieve a multitude of purposes. Interestingly, the agents, principles, and mechanisms of chemical dissolution that are found or take place in natural environments reported in this chapter seem to parallel the explanation of demineralization processes in vitro. Better understanding of the mechanisms leading to demineralization of biominerals in natural environments and better characterization of reaction conditions might allow us to more clearly identify specific characteristics useful for the development of new techniques based on gentle, biologically inspired demineralization with respect to isolation of organic templates. Additionally, from the author’s point of view, the organic template could be involved in the regulation of biomineralization phenomena directly, or via the numerous functionally active and structurally diverse molecules and macromolecules which may become attached to it. It is suggested that chitin and collagen are common and alternative scaffolds as well as templates in both calcification and silicification phenomena in Nature. Biological mineralization and demineralization play a vital part in our life and the environment around us (Liu and Lim 2003). And it is the removal of the mineral component that permits access to the organic matrix by extracellular organic compounds produced by biological systems. The possibility of this kind of attack, and cellular remodeling, is in many respects functionally similar to the chemical dissolution mechanisms of demineralization. Thus, demineralization is the process of removing minerals, in the form of mineral ions, from biominerals that takes place both in nature (physiological and pathological demineralization in organisms and bioerosion) and in laboratories, where the dissolution of mineral phases is determined by the practical goals or pure scientific interest relating to the isolation and investigation of organic matrix. To investigate the controlling mechanisms typically found in bioorganic materials and matrices, new techniques can be identified to mimic the regeneration of
H. Ehrlich, Biological Materials of Marine Origin, Biologically-Inspired Systems 1, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9130-7_4,
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these “hard” tissues, which ensures that the resulting bionanostructure and mechanical properties will be the same as or very similar to those of the natural tissue (Liu and Lim 2003). To understand the fundamental processes leading to demineralization, we must first focus on the phenomena that many natural systems have in common. At the very early stages of tissue organization and mineral nucleation are the most general needs, after which specific control of mineral processes including dissolution would allow differentiation into characteristics unique to each organism or organ. For example, in vivo bone remodeling and tooth caries share the same first step— dissolution of the mineral phase by the generation of low pH solutions (Collins et al. 2002); however the origin of these processes in these distinct structures is different. Physiological demineralization has been investigated in human and animal organisms, including marine invertebrates. Most attention has been focused on bone resorption as a necessary event during bone growth, tooth eruption, and fracture healing. Bioerosion is the second important example of demineralization occurring naturally with a history as long as that for biomineralization. It is known to be a major process driving the degradation of carbonate skeletal material and rocky limestone coasts in all marine and some freshwater environments (Wisshak et al. 2005). In concert with biologically mediated demineralization, physico-chemical dissolution and mechanical abrasion are rampant in these environments. Thus, most of the research into modern and ancient bioerosions has been conducted on the degradation of calcium carbonate substrates such as corals, shells, and limestones, resulting in the production of fine fractions of carbonate sediments. If a biomineralized structure are considered as a composite of organic matrix (protein, polysaccharide, lipid) and mineral, then three pathways of demineralization in the natural environment have been identified (Collins et al. 2002): (1) chemical deterioration of the organic phase; (2) chemical deterioration of the mineral phase; (3) biological (microorganisms, enzymes) attack of the composite. The first of these three pathways is relatively unusual in that it is extremely difficult to dissolve or alter the organic phase without first or simultaneously effecting the intimately associated mineral phase and will occur only in environments that are geochemically stable for the mineral component. However, because rates of biomolecular deterioration in the burial environment are slow, such biocomposite destruction could yield useful biomolecular information. In most environments, biocomposites are not in thermodynamic equilibrium with the soil solution and undergo total chemical deterioration. Dissolution of the mineral exposes the organic matrix to microbially determined deterioration (biodeterioration), and in most cases the initial phase of dissolution will be followed by microbial attachment (pathway 3). Biological attack also proceeds by initial demineralization; therefore paths 2 and 3 are functionally equivalent. However, in a biocomposite that follows path 3, the damage is more localized than in path 2, and regions equivalent to path 1 may therefore exist outside these zones of destruction (Collins et al. 2002).
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Another example of naturally occurring demineralization is the so-called phenomena of dark decalcification (Tentori and Allemand 2006). Kawaguti and Sakumoto (1948) noted “intake of Ca2+ ” in all scleractinian corals exposed to light and “output of Ca2+ ” in all corals exposed to dark. They argued that the skeleton formation was favored by the alkaline pH (8.84–9.15) of the incubation medium, which was assumed to be result of photosynthesis by zooxanthellae in the coral; correspondingly, the drop in pH (8.00–7.80) in the dark was the reason for the “resolution of the skeleton [sic]” (Kawaguti and Sakumoto 1948). Chisholm (2000) observed dark decalcification in coralline algae incubated at various depths and explained this as a result of previous light exposure or the acidification caused by cell respiration. It is also possible that the tissue recovery verified visually underwater was overestimated and that decalcification was due to tissue injury. Experiments on soft corals (Sarcophyton sp. and Sinularia sp.) indicated that these corals also decalcify in the dark (Tentori and Allemand 2006). These authors suggest that diurnal calcification–decalcification cycles probably control coral sclerite size and shape. Interestingly, the agents, principles, and mechanisms of chemical dissolution that are found or take place in natural environments reported above seem to parallel the explanation of demineralization processes in vitro. Better understanding of the mechanisms leading to demineralization of biominerals in natural environments and better characterization of reaction conditions might allow us to more clearly identify specific characteristics useful for the development of new techniques based on gentle, biologically inspired demineralization. Living forms appear to create specialized environments in concert with the biomineralized tissue formations and probably have been doing so since life first appeared (Skinner 2005). Biomineralization and demineralization phenomena are only two parts of the mineral–organic matrix circuit occurring in such specialized environments in nature (Fig. 4.1). The third part of this biochemical cycle is remineralization. Remineralization is the process of restoring the solid minerals—through the transfer of anions and cations—to a nucleation site where the lattices leading to mineral structures are generated. This process follows demineralization and is observed in vivo in a host of natural environments. A remineralization process in vitro has also been established.
Fig. 4.1 Schematic view of “biomineralization– demineralization– remineralization” cycle occurring in nature
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Remineralization also plays an important role in natural environments. The following experiments were recently described by Le Cadre et al. (2003). To study the pH effects, cultures with pH values in the range of normal marine (pH 7.9–8.3) were prepared using hydrochloric acid to lower the pH to 7. Foraminifera Ammonia beccarii was collected and introduced into these different environment cultures. Under neutral pH (7.0) conditions, pseudopodial emission was reduced or stopped. Then the animal’s external mineralized tissues, “the test” became opaque as a result of superficial alteration, which is the first stage of test decalcification. Decalcification progressively extended over the whole test, first destroying the mineralized areas of the last chambers, which contain less tissue, i.e., are thinner. After 15 days, only interlocular walls were preserved, giving the test a star-shaped characteristic of an advanced stage of decalcification. If a specimen was maintained in low pH conditions, the entire test was sometimes entirely destroyed and only the cytoplasm, covered with the inner organic layer, remained. On the other hand, if a specimen with a partially dissolved test was placed in a solution at normal pH, it was able to rebuild its test. Remineralization was somewhat different from the original calcification and was accompanied, in most cases, by morphological abnormalities (e.g., abnormal expansions, irregular chamber sizes, wall with concave form). These observations show that temporary acidification of the environment, causing partial decalcification of the test, is able to induce morphological abnormalities of foraminiferal tests during recalcification. This acidification may be caused by the anthropogenic impacts or a natural cause. In both cases, deformation of foraminiferal tests yields information on environmental characteristics of the area. Therefore the observations on foraminiferal tests and their use as bioindicators of pollution in coastal environments is now one of the areas under development in the discussion on climate change (Le Cadre et al. 2003). Similarly, effects of structural conformations were observed in experiments with octocorals. Isolated spicules of the gorgonian Leptogorgia virgulata were decalcified using 0.5 M EDTA solution (pH 7.5) and exposed to an artificial seawater solution to evaluate the ability of the spicules matrix to recalcify (Watabe et al. 1986). Recalcification of the decalcified spicule matrices occurred after 48 h in the artificial seawater solution containing NaCl, CaCl2 , KCl, MgSO4 , MgCl2 , and NaHCO3 . Decalcified matrices which retained the configuration of normal spicules assumed a form upon recalcification similar to, but not identical with, undecalcified spicules. Recalcification also occurred in the use of decalcified matrices that did not retain the form of normal spicules. Most of the recalcified matrices showed reduced calcium content when compared with normal spicules. The experiments demonstrated that completely decalcified spicule matrices can initiate recalcification and influence mineral form (Watabe et al. 1986). Thus, morphological abnormalities observed in different biomineral formations during and after remineralization might be a common phenomenon, because also in case studies using high-resolution transmission electron microscopy and relating to the growth of apatite crystals in the remineralized enamel, similar effects were obtained. The growth of newly formed crystals and the regrowth of pre-existing
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Principles of Demineralization: Isolation of Organic Matrices
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enamel crystals occurred extensively in remineralized enamel (Tohda et al. 1990). With advancing growth, the crystals came into contact and fused with each other, forming large crystals with hexagonal outlines. Various kinds of crystalline defects, including edge dislocation, low-angle grain boundary, and lattice shifting, were frequently detected between the fusing crystals. These observations confirm the previous suggestion that processes of de- and remineralization (shell, bone resorption, caries) must be regarded as abnormal biomineralization processes (Winterberg 1898; Krampitz and Graser 1988). A better understanding of the biomineralization–demineralization– remineralization mechanisms at the molecular level will result in more effective strategies for the development and establishment of novel methods, tools, and approaches for science, engineering, and medicine.
4.1 Principles of Demineralization: Isolation of Organic Matrices One of the most troublesome points when dealing with demineralization is making a distinction between naturally occurring and in vitro or laboratory or applied demineralization. One action known as “quick and deep” demineralization comes from a chemical point of view where very aggressive chemical reagents have been used, which led to harsh destruction of both mineral and organic phases and to corresponding artifacts. For example, HF-based silica dissolution procedure could drastically change the structure of glass sponge spicular proteins (Croce et al. 2004). As a consequence, when this is the dissolution technique, we can learn little about the real nature of the organic matrix of the biomineral or the actions of skeletal formation. The isolation of an organic component from any natural biomineralized material whether mineralized with calcium- or silica-containing compounds is indispensable but in our minds the most efficient and effective technique should be based not on fast dissolution of the inorganic component but instead on a slow, biomimetically inspired process that would spare the organic component in the biomineral-based naturally occurring composites and not result in artifacts. Thus, how the Nature does this job? The phenomenon of biological demineralization, including biologically induced decalcification, is widely distributed in nature. The calcibiocavicole activity (Carriker and Smith 1969) is a fact well investigated and described for different unicellular and metazoan organisms. That the nature of the environment may be a factor in the development of the penetrating habit is suggested by the fact that the great majority of calcibiocavitic species are marine (indeed, cariogenic microorganisms in oral cavities of vertebrates function in a “marine-like” environment). Grazing on surface biofilms by hard-toothed higher animals and invertebrates, as well as by the growth of chasmolitic and cryptoendolithic microbes, can result in significant physical and chemical erosion of biominerals (Garcia-Pichel 2006). Cavitation of biogenic calcareous substrata may provide bacteria, fungi, and algae with nutrients from the organic matrix which are not otherwise available (Carriker and Smith 1969; Smith 1969). Aerobic heterotrophic bacteria, fermenting, sulfide-oxidizing, and nitrifying
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bacteria can dissolve acid-labile minerals due to the production of acids as byproducts of metabolism (carbonic, organic, sulfuric, and nitric acids, respectively) (Ehrlich 1996). Also lichens and fungi in soils produce organic acids such as lactic, succinic, oxalic, citric, acetic, and α-keto acids (reviewed in Kalinowski et al. 2000). These dissolved acids and other organic exudates can affect pH in weathering solutions and thereby promote or inhibit etching. Organic ligands can complex cations in solution, inhibit precipitation, or lower the saturation index in solution and enhance dissolution indirectly. Simple and complex organic ligands are able to adsorb on the mineral surface and thus modify their dissolution rates via polarizing the metal–oxygen bonds or bridging several reactive surface centers together as described for the application to ligand-promoted or ligand-inhibited dissolution of carbonates (Jordan et al. 2007; Pokrovsky and Schott 2001), oxides (Pokrovsky et al. 2005), and silicates (Golubev et al. 2006; Golubev and Pokrovsky 2006). A large variety of organic and inorganic substrates such as wood, mollusc shells, bryozoan skeletons, crustacean carapaces, and corals have been found infested with boring marine foraminiferans (Venec-Peyre 1996; Wisshak and Rüggeberg 2006). They act as bioeroders of skeletal grains and contribute to the production of fine and very fine particles (Venec-Peyre 1987). The Foraminifera represent one of the most ecologically important groups of marine heterotrophic protists, the evolutionary history of which is well known for biomineralized lineages, and many of these are key indices in biostratigraphic, paleoceanographic, and paleoclimatic reconstructions (Pawlowski et al. 2003). Venec-Peyre (1987) suggests that the boring processes in the case of foraminifera are chemical in nature. Due to the unicellular nature of the borers, the relevant borings cannot result from the activity of differentiated organs (rasps or another adapted system) as with other borers. Regarding the perforated calcareous species, the protoplasm itself secretes calcium carbonate required for test elaboration. The cell probably acts by gradually and completely dissolving the substratum, resulting in the fragile-looking aspects of the cavity outline, and by the removal of ions. For agglutinated foraminiferan species, the process is somewhat different. The dissolution of the substrate seems to be incomplete and leads to the weakening of the carbonate framework into minute aggregates. The latter, as the various wastes on the surface of the substrate, are moved and accumulated near the foraminifera by pseudopodia. Secondarily, the aggregates are gathered on the organic lining and joined by a cement secreted by the cell. The purpose of such penetration may be to protect themselves against water turbulence and to provide material for test construction (Venec-Peyre 1987). Sponges (Porifera) are known as the first multi-cellular organisms in earth. Whereas many sponges are chemically or mechanically defended, some may have no such defenses and may have the competitive advantage of using a substrate that other organisms cannot use. One such strategy is to bore into the carbonate substrate that is out of reach for most predators, with the additional advantage of using a space unavailable to their competitors (Zundelevich et al. 2007). Thus, boring sponges, mostly from the family Clionidae, generally dominate the bioeroder community
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(Calcinai et al. 2000; Risk et al. 1995). These sponges inhabit cavities which they excavate in coral, the valves of living molluscs, dead shells, and calcareous rock. During penetration the substrate is gradually destroyed as the sponge hollows out an extensive system of cavities and tunnels. Preliminary studies revealed that these excavations are produced as small fragments of calcareous material are removed by a special type of amoebocyte which exhibits an etching activity (Cobb 1969). Cellular penetration occurs along the interface where these cells contact the substrate and is characterized by a unique pattern of cell–substrate relationships. Each active cell releases a substance which dissolves the substrate around its edge, forming a linear etching which corresponds in size and shape to the contours of the cell. Deeper etching occurs at the cell edge, moving gradually downward through the initial etching, and sinks into the substrate in a noose-like fashion. During this movement, the cell border is drawn down through the slit-like crevice cut by the cell edge, while the nucleus remains in position on the surface of the substrate within the original etched outline. Eventually, the undercutting action is completed and a small chip is freed from the substrate. Thus, penetration is achieved by the precise cellular release of a chemical agent which dissolves the calcareous substrate along restricted zones of contact between cell and substrate (Cobb 1969). Carbonic anhydrase and acid phosphatase are probably involved in this process (Pomponi 1980). Rates of bioerosion induced by sponges depend on several biotic and abiotic factors, including nutrient and food availability, temperature, physiological state of organism, density and type of substrate, and the presence of etching occurring at the cell edge (Schönberg 2002a, b; Zundelevich et al. 2007). Previously, Vacelet (1981) proposed that sponge zooxanthellae have an influence on the calcium carbonate solubility and a general stimulatory effect on the host metabolism. Recently, Schönberg (2006) showed that growth and erosion of the zooxanthellae Australian bioeroding sponge Cliona orientalis are enhanced in light. However, Zundelevich et al. (2007) reported that the measured bioerosion rate in the case of sponge Pione cf. vastifica was 2.3 g m–2 sponge day−1 , showing seasonal but not diurnal variations, suggesting that the zooxanthellae harboring the sponge have no effect on its boring rate. The “mud worm,” Polydora websteri, is a small polychaete borer which lives in the shells of oysters and other molluscs, and has long been considered a pest of bivalves (Lunz 1940). Its presence may stimulate the mollusc to secrete extra layers of shell around the worm’s burrow. In this manner, Polydora causes its host to divert energy to shell deposition, perhaps leaving its weakened host prey to other enemies and diseases. In terms of the boring processes, Blake and Evans (1973) summarize three mechanisms in Polydora: a chemical mechanism, where special glands secrete acid solutions to dissolve substrates; a mechanical mechanism, where the enlarged modified setae on the fifth setiger abrade the substrate; and a combined chemical and mechanical mechanism. Polydora websteri penetrate all layers of the oyster shell, including prismatic, calcite-ostracum, hypostracum, periostracum, and internal conchiolin layers of visible thickness (Haigler 1969).
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Worms induced to settle directly on test substrates at room temperature bored chalky deposits within 24 h, calcite-ostracum and hypostracum within 1 week, and conchiolin layers within a month. The author reported that the ability of adult worms and their larvae to bore hard substrates is determined by a chemical agent. This chemical is not conchiolinase, for Iceland spar is composed of pure calcite and lacks the conchiolin matrix which binds calcium carbonate crystals together in most oyster shell layers. The adult and larvae of P. websteri did produce acid in seawater agar medium with phenol red indicator, and pieces of Iceland spar introduced into the medium after the worms were removed were etched in a manner indistinguishable from etched areas in artificial blisters (Haigler 1969). Another Polydora species Polydora villosa is found only in living corals colonies, where the infection rates range from 15 to 100% (Liu and Hsieh 2000). The fine architecture on the inner surface of the U-shaped passages exhibits characteristics of abrasion made by P. villosa. The rough characteristics, such as abrasion pitting and etching seen in the inner surface of the U-shaped passages in P. villosa, are similar to those found in coral skeletal crystals that have been dissolved by hydrochloric acid, acetic acid, or EDTA (Williams and Margolis 1974). Therefore Liu and Hsieh (2000) suggested that this polychaeta secretes acids to erode the coral skeleton. During the boring period, P. villosa might curve its body, allowing minimum exposure to seawater, thus preventing the acid from being diluted. Carriker et al. (1967, 1974), Carriker (1961), and Carriker and Williams (1978) determined that the boring of holes in the shell of bivalve prey by predatory muricid and naticid gastropods, to obtain food, consists of two alternating phases: (i) chemical, in which an accessory boring organ (ABO) or demineralization gland secretes an uncharacterized substance that etches and weakens the shell at the site of penetration, and (ii) mechanical, during which the radula rasps off and swallows some of the weakened shell as minute flakes. However, it is generally believed that chemical boring has evolved as a specialization of mechanical boring (Morton and Scott 1988). Development of a microelectrode has enabled in 1967 the first continuous recording of the pH of the secretion of the normally functioning ABO of the shell-boring predatory snail Urosalpinx. The recording was made in an incomplete borehole in a glass-shell model. The minimum pH recorded was 3.8; hitherto the secretion had been considered neutral (Carriker et al. 1967). The ABO was first described in Dolium galea (Naticidae) by Troschel (1854). Schiemenz (1891) first suggested that this ABO secretes an acid. Ankel (1937), by placing freshly cut naticid ABOs against the shell, obtained shallow dissolution in a few hours and postulated the presence of a calcase. In 1978 Carriker and Williams hypothesized that a combination of HCl, chelating agents, and enzymes in a hypertonic mucoid secretion released by the ABO dissolves shell during hole boring. The similarity of patterns of dissolution etched by the ABO secretion and those produced artificially by HCl and EDTA as reported by Carriker (1978) supports the hypothesis that these chemicals, or chemicals similar to them, are constituents of the ABO secretion. Lactic and succinic acids and a chitinase-like enzyme were also suggested as possible agents in shell dissolution (Carriker and Williams 1978).
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The fine structure of shell etched by the secretion was contrasted in these experiments with normal shell and shell solubilized artificially. A synoptic series of scanning electron micrographs of representative regions of the normal shell of Mytilus edulis was prepared to serve as a standard for ultrastructural interpretations of the pattern of dissolution (Carriker 1978). It was suggested that preferential dissolution of shell matrix by the ABO secretion is functionally advantageous to boring gastropods because it increases the surface area of mineral crystals exposed to solubilization and facilitates removal of shell units from the surface of the borehole by the radula. Day (as reported in Carriker and Smith 1969) in a study of the shell-dissolving secretion of the snail, Argobuccinum, found that H2 SO4 is present and accounts for 67% of the CaCO3 dissolving activity of the secretion; the remaining solubilization of the shell is achieved by some other unidentified component which may be a chelating agent. Corals are phylogenetically basal metazoans, possess a simple anatomy, can be easily manipulated for physiological studies, and possess a majority of genes in common with vertebrates (Tambutté et al. 2007). They are therefore good models for biochemical, physiological, and evolutionary studies concerning either the transport of ions or the synthesis of organic matrix involved in biomineralization processes. Demineralization of coral skeletons using different techniques and chemical agents leads to isolation of the following biomacromolecules as reported in literature: chitin (Wainwright 1963), collagens (Franc et al. 1985; Kingsley et al. 1995), gorgonin (Ehrlich et al. 2006b; Noé and Dullo 2006; Sherwood et al. 2006), canthaxanthin (Cvejic et al. 2007), and a wide diversity of proteins (reviewed in Tambutté et al. 2007). Scleractinians (stony corals) are a group of calcified anthozoan corals, many of which populate shallow water tropical to subtropical reefs. Most of these corals calcify rapidly and their abundance on reefs is related to a symbiotic association with zooxanthellae. These one-celled algal symbiotes live in the endodermal tissues of their coral host and are thought to be responsible for promoting rapid calcification (Stanley 2003). Recently, Fine and Tchernov (2007) presented an experimental approach documenting how coral skeletons dissolve as a physiological response to increased atmospheric CO2 . Thirty coral fragments from the five coral colonies of the scleractinian Mediterranean species were subjected to pH values of 7.3–7.6 and 8.0–8.3 (ambient) for 12 months. The corals were maintained in an indoor flow-through system under ambient seawater temperatures and photoperiod. After 1 month in acidic conditions, morphological changes were seen, initially polyp elongation, followed by dissociation of the colony form and complete skeleton dissolution. Surprisingly, after 12 months, when transferred back to ambient pH conditions, the experimental soft-bodied polyps calcified and reformed colonies. It should be noted that the organic matrix of scleractinian corals can be divided into “soluble” and “insoluble” components (Weiner 1984) depending on the extraction method used, and consequently care must be taken when comparing and interpreting results.
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Moreover, in 1980, Johnston (1980) warned that a differentiation should be made between “skeletal organic matrix” and “skeletal organic material” and suggested that the latter involved the skeletal organic matrix plus all other contaminating components such as endoliths and trapped tissues. From that time, in order to avoid such contamination, the chemical treatment of corals was carefully performed on the powders of skeletons (usually created using sodium hydroxide and/or bleach) in order to obtain only the skeletal organic matrix. Typical procedure is described by Cuif et al. (1999) in their study on 24 species of corals collected in very various environments, ranging from cold and/or deep seas to tropical lagoons of Polynesian atolls. Coral samples were thoroughly cleaned with sodium hypochlorite, rinsed, and oven dried (40◦ C) overnight. Dried skeletal fragments were powdered. The resulting powder was calibrated to regulate the decalcification process made in very standardized conditions. To carry out the full sequence of mineralizing matrix characterizations, 3 g of the calibrated powder was dispersed in 25 ml of Milli-Q water (magnetic stirring), then decalcification was made by the addition of ultra-pure acetic acid under permanent control of the pH. After the complete dissolution of the mineral phase, a low-speed centrifugation allowed soluble and insoluble matrices to be separated. Desalting of soluble components was done on Sephadex G-25 gel-based, low-pressure standard system, allowing salt and organic compounds of lower molecular weights to be removed. To obtain reliable biochemical information, these two successive steps of preparation play a major role, allowing removal of most of the external contaminants, both insoluble (i.e., possible remains of fungal or algal cell membranes) and low-weight soluble compounds that could have been introduced by seawater flow through coral skeletons (Cuif et al. 1999). Similar decalcification method was described recently for scleractinian coral Stylophora pistillata (Puverel et al. 2005b). Microcolonies of this coral species were cleaned by removing soft tissues with 1 M NaOH at 70◦ C. The skeletons were then rinsed with ultrapure water, dried at 60◦ C overnight, and ground to a fine powder with a mortar and pestle. The powder was resuspended in ultrapure water and decalcified by adding ultrapure acetic acid up to pH 4.0 in the presence of protease inhibitors. After complete dissolution of aragonite, soluble and insoluble matrices were separated by centrifugation (10 min, 10,000×g). To desalt soluble components, the supernatant containing the soluble organic matrix was ultrafiltered and then lyophilized. Using antibodies raised against soluble organic matrix isolated from coral S. pistillata as described above, the authors presented direct evidence for the role of calicoblastic cells in organic matrix synthesis and secretion. The same decalcification method was used in comparative study on S. pistillata, known as branched robust coral, and Pavona cactus, a leafy complex coral (Puverel et al. 2005a). Soluble organic matrix of both coral species was shown to contain high amounts of potentially acidic amino acids and glycine. However, proportions of glycosaminoglycans and SDS-PAGE analysis of soluble organic matrix proteins were very different. Internal peptide sequences of two matrix proteins (one from each species) were obtained. One sequence of S. pistillata is unusual because it contains a long poly-aspartate domain, as described in proteins belonging to the calsequestrin family and in proteins from molluscan species.
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Recently, Rahman and Isa (2005) documented demineralization procedure for spicules of soft coral Lobophytum crassum (Rahman and Isa 2005). The colony of the corals was cut using sharp scissors into small pieces. The pieces were ground five to six times with a mixer machine and washed with tap water until the spicules were obtained. The collected spicules were stirred vigorously in 1 M NaOH for 2 h to remove the fleshy tissues and debris. The mechanically and chemically cleaned spicules were extensively washed in distilled water and decalcified in 0.5 M EDTA (pH 7.8) overnight. The decalcifying solution was centrifuged with 4000 rpm (15 min) to remove any insoluble material followed by filtration with filter paper. The authors reported that soluble organic matrix comprised 0.03% of the spicule weight. The SDS-PAGE analysis of the preparation showed four protein bands. The 67-kDa protein appears to be glycosylated. Moreover, the isolated organic matrix possesses carbonic anhydrase activity which functions in calcium carbonate crystal formations, indicating that organic matrix is not only a structural protein but also a catalyst. It is generally agreed that enzymes play a role in biomineralization– demineralization–remineralization phenomena. The principal enzymes of those mentioned in the literature are carbonic anhydrase, alkaline phosphatase (Chave 1984), phosphoprotein phosphatase (Kreitzman et al. 1969; Kreitzman and Fritz 1970), and vacuolar-type H+ -ATPase (Ziegler et al. 2004). Carbonic anhydrase speeds equilibrium reactions in the CO2 –H2 O system, whereas alkaline phosphatase decouples inorganic phosphorous from organophosphorus compounds. Both enzymes commonly occur at sites of carbonate and phosphate mineralization, and also at many non-calcifying sites and in non-calcifying organisms. Is it possible than enzyme determined desilicification also occurring in natural environments? Dissociation of solid silica in spicular formations of marine sponges was observed and visualized using SEM (Fig. 4.2). Besides the silica-anabolic enzymes, the silicateins, another enzyme termed silicase (silase), have been identified in the marine sponge Suberites domuncula. Silicase is able to depolymerize amorphous silica (Schröder et al. 2003). The expression of the gene encoding this silica-catabolic enzyme is strongly upregulated in response to higher concentrations of silicon, like the expression of silicatein (Krasko et al. 2000) and collagen (Krasko et al. 2000; Schröder et al. 2000). The silicase cDNA has been identified in primmorphs from S. domuncula, applying the technique of differential display of transcripts. The deduced polypeptide is closely related to the carbonic anhydrases. Most of the amino acids which are characteristic of the eukaryotic-type carbonic anhydrase signature are also present in the sponge silicase (Prado Figueroa et al. 2008). Carbonic anhydrases are a family of zinccontaining enzymes. The three conserved histidine residues which are characteristic of these enzymes are also found in the deduced sponge protein at aa181, aa183, and aa206. These histidine residues bind a zinc ion. It is assumed that the reaction mechanism of the sponge enzyme is analogous to that of other zinc-dependent enzymes involved in ester hydrolysis. The Zn2+ ion (= Lewis acid) interacts with water (= Lewis base). The hydroxide ion formed by splitting of the water molecule is bound to the zinc ion. This hydroxide ion then performs a nucleophilic attack at one
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Fig. 4.2 SEM image of the biologically mediated dissolution of silica layers observed on the surface of S. domuncula demosponge spicule (image courtesy: Carsten Eckert)
of the silicon atoms of the polymeric silicate. In the next step the zinc complex binds to silicon under cleavage of the Si–O bond in the polymeric silicate. The transiently formed zinc-bound silicate is then hydrolyzed by water, resulting in the release of the silicic acid product and regeneration of the zinc-bound hydroxide. Based on its ability to dissolve or to etch silica substrates, the silicase is of interest for a wide range of applications in nanobiotechnology. Isolation, purification as well as cloning of the gene encoding this enzyme are described in detail in the corresponding patent (Müller et al. 2007). Thus, biomimetic principles of demineralization are listed and discussed above. What is, however, with the man-made approach? Here are some examples. Wet chemical etching of silicate glasses in aqueous HF solutions is a subject which has been studied over many years. The first report originates from the discovery of HF by Scheele (1771). The specific property of HF-containing solutions to attack the glass is related to the presence of fluorine-containing species in solution: F− , HF, and HF2 . The dissolution mechanism, in particular the role of the various fluorine-containing species, has been reviewed by Spierings (1991, 1993). The HF2 ions are adsorbed on surface silanol groups, the HF molecules on vicinal silanol groups, and H+ ions on surface-bridging oxygens in siloxane units. Fluorinecontaining adsorption complexes have been observed at hydrated SiO2 surfaces in gaseous HF by infrared spectroscopy. These are transformed into surface groups such as =Si−F and =Si−O−SiF3 . The adsorption of HF and HF2 increases the electronic density on the bridging oxygen in the siloxane unit. This in turn makes these oxygens more basic, so more H+ ions are adsorbed, which leads to more siloxane bonds being broken per time unit, i.e., a kind of catalytic effect. The ratedetermining step is then the breakage of the siloxane bond by the combined action of the adsorbed species.
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Judge (1971) found that the etching rate depends on the concentration of HF molecules but does not depend on the concentration of the HF2 ion. This result showed that solutions with a pH of 7 and higher that contain essentially all fluorides in the deprotonated state exhibit essentially zero rate of SiO2 dissolution, which indicates that a HF2 or a F ion in solution is quite benign and much less reactive than HF molecule. Previous experimental studies showed that HF etching of SiO2 films was enhanced by the addition of water. Consequently, H2 O may play a direct role in the etching mechanism itself. Unlike many physical properties, no linear relations are observed between the composition of the glass and its dissolution rate. The dissolution rate of a multicomponent silicate glass is found to be largely determined by two factors: the degree of linkage or connectivity of the silicate network and the concentration of SiO2 in the glass. It is proposed that the dissolution of the glasses is preceded by the leaching of alkali and alkaline earth components present in the glass, followed by the subsequent dissolution of the leached layer. Probably fluorine species will diffuse into the leached layer to enhance the dissolution rate. Analysis of the activation energy data indicates that in some corrosive glasses, the leaching itself becomes rate determining. Similar to silicate glasses, silica-based skeletons of biological origin are also examples of multi-component silica because of the presence of organic compounds as well as K+ and Na+ . By virtue of their embedded, mineralized skeletons, sponges are of great interest to both evolutionary biologists and materials scientists. As the most basal metazoans, they are the key to understanding the evolution of both calcium- and silicon-based biomineralization. The manifestation of this mineralization, a skeleton of spicules embedded in the body of the sponge, is typically a complex arrangement of calcite or silica. For example, the skeletal spicules of glass sponges (Hexactinellida, Porifera) are valuable model systems for the investigation of structure–function relationships in biomaterials, with the ultimate goal of identifying design strategies for new synthetic materials. Demineralization of the siliceous spicules of sponges has already been described the nineteenth century (Schultze 1860) based on the use of hydrofluoric acid. Kölliker was the first to describe the use of HF solution for the demineralization of Hyalonema spicules (Kölliker 1864). In 1888, Sollas reported on sponge spicule desilicification methods based not only on HF but also on boiling solutions of KOH (Sollas 1888). A HF etching procedure developed for microscopic investigations of the structure of sponge siliceous spicules was described by Vosmajer and Wijsman (1905) and this is still used today. A similar HF-based desilicification method was used by Schmidt in 1926 for a comparative study of organic and inorganic substances within the spicules of Hyalonema and Monorhaphis species (Schmidt 1926). HF dissolution of silica was used more recently to visualize the sponges’ axial filaments but was satisfactory only for determining their gross morphology; unfortunately, it also had the drawback of partially masking the filament’s fine detail (Simpson et al. 1985). The removal of silica from the face of the block by soaking in HF circumvents some problems but results in the loss of the freed
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filaments which are no longer supported by a surrounding matrix to hold them in the block (Willenz 1983). Also, silicateins were isolated from siliceous spicules of Tethya aurantia by dissolving the silica in HF/NH4 F solutions (Cha et al. 1999). However, the fibrillar organic matrix was described for Euplectella sp. spicules that had been desilicated using HF gas rather than solution (Travis et al. 1967). Even nowadays, HF-based techniques are still in use (Weaver and Morse 2003). As pointed out early by Bütschli (1901), this rapid technique may produce artifacts (Croce et al. 2004; Kono et al. 1992; Schröder et al. 2006). “Collateral damages” due to the use of HF in biological objects are well known. Hydrofluoric acid cleaves disulfide bonds, e.g., in keratin (Schiettecatte et al. 2003), is a highly specific reagent for the cleavage of phosphate–oxygen bonds (Fuchs and Gilvarg 1978), dephosphorylates phospholipids (Shaw and Stead 1974), phosphoglycerides, and phosphoglycolipids (Reddy et al. 1976), removes polysaccharides from peptidoglycan polysaccharide complexes, and hydrolyzes oligosaccharides (Jürgens and Weckesser 1986). In addition, fluoride binds to calcium and magnesium in hard tissues, modified cell membranes, organelles, and enzymes (Gubbay and Fitzpatrick 1997). This problem becomes obvious, for example, during the analysis of the silaffins from diatoms as well as the silicateins from sponges; both peptides were shown to become dephosphorylated by HF treatment. Therefore, NH4 F had to be used which did not remove the phosphate groups. Another demineralization method (Conley 1998) which is based on the use of Na2 CO3 or 0.5 M NaOH with subsequent heating to 85◦ C is also inapplicable for the isolation of native proteinaceous matrices because it leads to the denaturation of proteins. In order to overcome this obstacle, novel, slow-etching methods, which use solutions of 2.5 M NaOH (or at least 1% sodium dodecyl sulfate, SDS, or 1% rhamnolipid biosurfactant) at 37◦ C and take 14 days, have been recently developed (Ehrlich et al. 2006a). The following mechanism can be put forth on the basis of the reaction of alkaline desilicification of organic silicon materials (Fig. 4.3). Hydroxyl ions primarily attack and subsequently break the stronger siloxane bonds (Si–O–Si) located on the surface of the silicone component.
Fig. 4.3 Principle schema of the alkali–silica reaction (left, crystalline silica; right, amorphous silica) (images courtesy: Denis Kurek)
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The negative charge that appears as a result of this bond breakage is balanced by positively charged ions of the alkali element: Si − O − Si + 2NaOH → 2Si − O − Na + H2 O During the reaction of alkaline desilicification, the hydroxide ions penetrate into the SiO2 particles, therefore weakening the structure of the latter. Such a pattern of disintegration of the lattice structure by alkaline hydroxide is virtually impossible in the case of highly crystallized silicates (quartz); however, and in contrast to amorphous silicates (opal A), this process proceeds very efficiently, due to the increased surface area and irregular open structure of the particles (Ferraris 1999). It is apparent that both decalcification and desilicification are intricately complex mechanisms that serve several purposes, from organisms’ survival to structural protection to calcium or silicon balance. It is apparent that demineralization and biomineralization should be envisioned as the two sides of the same coin. Nature often uses these in admirable synergy to achieve a multitude of purposes. Continued advances in instrument technology will make it possible to further untangle unresolved issues. There is a plethora of opportunities for innovative research and applied science based on gentle, biomimetic, demineralization approach.
4.2 Structural Biopolymers as Common Templates for Biomineralization Biological mineralization of tissues in living organisms relies on organic molecules that preferentially nucleate minerals and control their growth. This process is often referred to as “templating,” but this term has become generic, denoting various proposed mineral–organic interactions including both chemical and structural affinities (Subburaman et al. 2006). First of all, the definition of “template for mineralization” seems appropriate with respect to the organic component. It is known that organic biomolecules such as peptides, proteins, proteoglycans, lipids, and polysaccharides are involved in most, if not all, stages of biomineral formation, from transport to nucleation and growth through to structure stabilization. In the generation of such well-defined composite biomineralized structures, molecular recognition between the organic and inorganic species has been proposed as essential. The mineralizing specifics can arise from individual or combinations of interfacial or non-bonding interactions such as electrostatic interactions, hydrogen bonding, and the hydrophobic effect and may also include stereochemical effects (Patwardhan et al. 2007). According to a definition proposed by Mann (2001), “The organic matrix is a pre-formed insoluble macromolecular framework that is a key mediator of controlled biomineralization. The matrix subdivides the mineralization spaces, acts as a structural framework for mechanical support, and is interfacially active in nucleation.” Templating is described as one of the principles of biomineral architecture by Liu and Lim (2003) in their investigations on the structural synergy between biominerals and
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biosubstrates. These authors suggested that the nucleation of mineral materials will be best templated by substrates having an excellent structural correlation with the crystalline phase. In the process of nucleation, increases in the size of initial mineral precipitate should overcome a free energy barrier, so-called nucleation barrier, before it can become a stable growth center for the mineral phase in the system. It was shown, for example, that collagen fibers are much more effective in lowering the nucleation barrier for hydroxylapatite (HAP) than non-calcium phosphate particles and therefore serve as better templates. The structural synergy between the biominerals and collagen seems to be optimal in this regime (Liu and Lim 2003). One of the remarkable characteristics of many biominerals in mineralized tissues is the precise control of the mineral crystallographic orientation. The usual explanation for this control is templating, where the spacing and the orientation of the functional groups of the substrate somehow “match” the atomic arrangement of a precise crystal face (Harding and Duffy 2006). The templating explanation implicitly assumes that the control of orientation is due to the lowering of the energy of a specific organic–mineral interface. I accept the definition of a template of biomineralization proposed by De Yoreo and Vekilov (2003) in their milestone work entitled Principles of Nucleation and Crystal Growth: . . .there is a substantial body of evidence to suggest that proteins and other organic molecules serve as “templates,” providing preferential sites for nucleation and controlling the orientation of the resulting crystals.
Additionally, from my point of view the organic template could be involved in the regulation of biomineralization phenomena directly or via the numerous functionally active and structurally diverse molecules and macromolecules which may become attached to it. I suggest that chitin and collagen are common and alternative scaffolds as well as templates in both calcification and silicification phenomena in Nature.
4.2.1 Chitin There are two principal skeletal systems which support the cellular structure of animal tissues: those determined by chitinous and the other by collagenous frameworks (Rudall 1955). The collagenous skeleton consists principally of the unique fibrous protein collagen I together with varying quantities of non-collagenous proteins, while the chitinous system utilizes aminopolysaccharide chitin together with proteins which are not collagen-like. Both chitin and collagen are usually formed as extracellular secretions that are conspicuously fibrous at different hierarchical levels (nanofibrils–microfibrils–fibers) and these structures are often the sites for eventually heavy deposits of both amorphous and crystalline minerals. The chitinous system is mainly of ectodermal origin and forms characteristic exoskeletons. The collagenous system is almost entirely of mesodermal origin and thus internal within the body. The two systems therefore are normally of quite different cellular origin and could be considered as entirely independent systems.
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Phylogenetic results suggest that the chitin systems of fungi and animals are related (Wagner 1994) and that the chitin system is very ancient in contrast to collagen system. However, Celerin et al. reported in 1996 collagen in fungi which normally contain chitin. The explanation proposed is as follows. All true collagenous proteins currently known are found exclusively in the kingdom Animalia (Garrone 1978; Garrone et al. 1993). They have been used alone or in combination with other extracellular matrix (ECM) components to argue that animals are a monophyletic group. It has been suggested that the ECM is a primitive feature of multicellularity in animals (Garrone et al. 1993; Wainright et al. 1993). However, all of these authors acknowledge that the ECM did not arise de novo with the first multicellular animal. Thus, it is implied that components of the ECM evolved before the first Metazoan. This suggests that one of the major components of the animal ECM, namely collagen, is much more primitive than has been thought or documented previously and probably evolved prior to the divergence of fungi and animals. Strong affinities exist between the collagens and other ECM components, including fibronectin, glycosaminoglycans, and proteoglycans (Reddi 1984). Fungal fimbriae are surface appendages that were first described on the haploid cells of the smut fungus Microbotryum violaceum; they are long (1–20 g), narrow (7 nm), flexible structures that have been implicated in cellular functions such as mating and pathogenesis (Celerin et al. 1996). Since this initial description, numerous fungi from all five phyla have been shown to produce fimbriae on their extracellular surfaces. Based on these results, it is suggested that the proteinaceous subunits of fimbriae should be termed fungal collagens. The unexpected finding of collagen in the members of the Mycota suggests that it may have evolved from a common ancestor that existed before the divergence of fungi and animals. Further, it was hypothesized that native fungal fimbriae can function as a mammalian extracellular matrix component. They can act as a substratum which permits corresponding animal cells to adhere, spread, and proliferate in a manner similar to animal collagens. The chitin–protein system is the reverse of the collagenous system in connective tissue. The polysaccharide fibrous framework (chitin) is reinforced and modified by a protein matrix, while a protein fibrous framework (collagen) is reinforced and modified by a polysaccharide matrix (Rudall and Kenchington 1973). The chitinous system and the collagenous system can replace one another as is seen when comparing the cuticles of arthropods (insects) with those of nematodes and annelids (worms) (Rudall 1955). Evidence for the presence of chitin in lower chordates, like ascidians, and lancelets as well as in the epidermal cuticle of fishes (Wagner et al. 1993) implies that the vertebrates inherited their chitin synthesis capability from their ancestors. It might seem appropriate to explain the relationships between chitin and collagen production in terms of natural selection and thus look toward the total skeletal needs for proper function in the animal as being satisfied by either as a convenience based on other considerations. Structures selected through evolution often take on similar geometries, via similar principles. These architectures can now be obtained experimentally with chitin and collagen (Giraud-Guille 1988). The common morphologies and structural features of both have nanostructural units
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of 1.5 nm (triple helices of collagen) and 2 nm (crystallites of chitin) which are represented as nanofibrils. Chemical multi-functionality of both systems can explain why chitin and collagen synthesis capabilities could have been preserved during evolution in organisms in which the calcification or silicification could be based on structurally similar but chemically different macromolecules. Chitin is, after cellulose, the most abundant polysaccharide in nature. It is known to occur as a component of the cell wall in fungi and diatoms, and also in diverse structures of at least 19 animal phyla (Willmer 1990). Chitin as a rigid scaffold is well known in arthropod cuticle. Arthropods, which include the crustaceans (e.g., crabs, lobsters, and other isopods), insects (e.g., wasps, bees, ants, beetles), arachnids (e.g., spiders, scorpions, ticks, mites), centipedes, millipedes, and several lesser groups, account for approximately 80% of all known animal species (Al-Sawalmih 2007). The stiff chitin exoskeleton of arthropods may have been a decisive factor in the success of this phylum (Martin et al. 2007). It generally occurs associated with different kinds of proteins, other polysaccharides, minerals, usually calcium and magnesium carbonates, lipids, and pigments in different proportions depending on the organism. Interaction between chitin and other molecules and elements is often the key way to rigidification (Kamada et al. 1991) of the specific biological material. For example, a major component of the rigidification in fungal walls is the cross-linking between chitin and glucans (Wessels et al. 1990). Chitin is rapidly degraded by enzymatic hydrolysis on death of organisms. Although a labile molecule from enzymatic point of view, chitin is relatively resistant to decay when complexed with protein in invertebrate cuticles (Flannery et al. 2001). Hence, arthropod cuticle, where histidyl and catechol cross-linking often occurs, has been observed in the more recent fossil records. However, evidence exists for the survival of chitin in fossils over geological timescales (Stankiewicz et al. 1997). From a structural point of view chitin performs the same function among invertebrates as cellulose does in the plant kingdom. Chitin [poly(β-(1-4)-N-acetyl-D-glucosamine)], a natural polysaccharide, was first defined in 1823 (Odier 1823) (Fig. 4.4). Depending on its source, chitin occurs in two main allomorphs, namely the α- and β-forms (Blackwell 1973; Rudall and Kenchington 1973), which can be differentiated by infrared and solid-state NMR spectroscopy together with X-ray diffraction (Rinaudo 2006). A third allomorph γ-chitin has also been described (Rudall 1969; Rudall and Kenchington 1973), but from a detailed analysis, it is just a variant of the family (Atkins 1985). α-Chitin is by far the most abundant; it occurs in fungal and yeast cell walls, in krill, in lobster and crab tendons and shells, in shrimp shells, as well as in insect cuticle. It is also found in and presumably produced by various marine living organisms as reported in the review by Rinaudo (2006). Other occurrences mentioned include the harpoons of cone snails (Olivera et al. 1995), the oral grasping spine of Sagitta (Atkins et al. 1979; Chanzy 1998; Saito et al. 1995), and the filaments ejected by the seaweed Phaeocystis (Chrétiennot-Dinet et al. 1997). These more exotic α-chitins have proved particularly interesting for structural studies since, in comparison with the abundant arthropod chitin, some of them present remarkably high crystallinity
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Fig. 4.4 Structure of chitin
(Rudall 1976) and high purity as they are synthesized in vivo and do not contain pigment, protein, or calcite. In addition to the native chitin, pure α-chitin may be formed via recrystallization from solution (Helbert and Sugiyama 1998; Persson et al. 1990), in vitro biosynthesis (Bartnicki-Garcia et al. 1994; Ruiz-Herrera et al. 1975), or enzyme-based polymerization (Sakamoto et al. 2000). β-Chitin, the more rare form, is found in association with small proteins in squid pens (Rudall 1969; Rudall and Kenchington 1973) and in the tubes synthesized by pogonophoran and vestimentiferan worms (Blackwell et al. 1965; Gaill et al. 1992). It also occurs in aphrodite chaetae (Lotmar and Picken 1950) as well as in the lorica built by some seaweeds or protozoa (Herth et al. 1977, 1986). A particularly pure form of β-chitin is found in the spines excreted by the diatom Thalassiosira fluviatilis (Dweltz et al. 1968; Herth et al. 1986; Revol and Chanzy 1986). As of today, it has not been possible to obtain β-chitin either from solution or by in vitro biosynthesis. β-Chitin is less crystalline than α-chitin because of its parallel structure of the chains. β-Chitin as metastable polymorph can transform into stable α-chitin because it possesses the unique property of intercalating small molecules within its lattice (Saito et al. 2000). Crystallographic and electron microscopic studies show a close association of chitin with protein and suggest a quantitative relationship between these components but not covalent bonding (Attwood and Zola 1967). Chitin is insoluble in general solvents due to its specific structure, which is based on the hydrogen bonding among acetamide groups, hydroxyl groups, and carbonyl groups (Khor and Lim 2003). The chitin molecule consists of N-acetylD -(+)-glucosamine (GlcNAc) residues, including the acetamide group at the C-2 position of glucosamine, the secondary hydroxyl group at C-3, and the primary hydroxyl group at C-6 positions (Jayakumar and Tamura 2008). Chitin oligomers of more than 10 monomers are sparingly soluble in water and spontaneously assemble into fiber (Kaufmann et al. 2007).
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Hierarchical organization is an essential characteristic of numerous biological materials based on chitin. In all cases, the first level of the organization of these materials is the chitin chain along the c-axis where hydrogen ions are laterally spaced by 0.475 nm with a monomer length of 1.032 nm. The second level is nanofibrils of about 2–3 nm in diameter, each containing 19 chains and are about 300 nm long. The number of chitin chains in the nanofibril is probably close to a minimum for stability; hence, the chitin nanofibrils present an optimum surface area for interfacial interactions within corresponding structural formations (e.g., cuticles) (Vincent 2002). The lateral nanofibrillar dimensions can range from 2.5 to 25 nm, depending upon the organism (Goodrich and Winter 2007). The third level is microfibrils. For example, chitin microfibrils are a major component of the walls of stipe cells in fungi. They occur as shallow helices, which may be right or left handed (Kamada et al. 1991). The fourth level is “fiber” of more than 1 μm in diameter (Al-Sawalmih 2007). There is diversity of the occurrence of chitin aggregation within one organism. For example, in shell-free eolid nudibranchs (Mollusca), chitin appears in three different organs: in the radula teeth, in cuticles of the head alimentary tract, and as intracellular granules in the epidermal cells of the skin and the gut epithelium, known as the spindles (Martin et al. 2007). In contrast, chitin armor in arthropods is a granular chitin, which while protecting the animal does not interfere with the suppleness and flexibility of this tissue. In response to nematocysts fired by tentacles of hydroid prey (Cnidaria), the epidermal cells of these molluscs release masses of chitin microgranules, which then form aggregates with the nematocyst tubules, having the effect of insulating the animal from the deleterious action of the Cnidaria tentacles. The chitin in different invertebrates could be regarded as the substrate which binds other macromolecules that in turn induce nucleation of the mineral phase (Poulicek et al. 1986). This is well described for mollusc shells and specifically for nacre where chitin has been suggested as an important component of the molluscan organic matrix (Mann 1988). The operculum of Gastropoda molluscs, the permanent plate which covers the aperture when the animals withdraw into their shell, is a metapodial production, corneous or calcified. Poulicek (1983) reported that the chitin content of the operculi varies according to the degree of calcification: high calcification corresponds to a high amount of chitin in the matrix in relation to other organic molecular components, and vice versa. In the operculi of 114 different species of gastropod examined by this author, chitin was present only in those which are calcified and was completely lacking in non-calcified corneous ones. Chitin contributes the framework at the base of the biological hierarchy for calcification (Weiss and Schönitzer 2006): an alignment between the orientation of chitin fibers and the crystallographic axes of the mineral phase was observed in brachiopod and mollusc shells as well as in vitro (Falini et al. 2001, 2002). The current model (Levi-Kalisman et al. 2001) of mollusc shell nacre formation is as follows: the matrix consists of sheets of β-chitin that are surrounded by a silk-like protein gel. Small proteins are either attached to the chitin or distributed within the silklike protein gel. A mixture of chitin–protein pre-fills the space to be mineralized, and the chitin is the ordered structure that ultimately dictates the orientation of
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the mature crystals (Addadi et al. 2006). Inhibition of chitin secretion in molluscs causes disintegration of the several different calcareous layers present in these species (Machado et al. 1991) and this can take place with distinctly different mineral species. For example, the α-chitin–aragonite association in the nacreous layer of mollusc Anodonta cygnea (Machado et al. 1991) is different from the composition of the shell of Lingula unguis (Brachiopoda) which contains HAP and β-chitin. Iijima and co-workers (1990) showed that in this latter mineralized tissue the crystallographic axes of the c-axis of the apatite are parallel to the fiber axis of β-chitin. Furthermore, a close relationship of unit cell dimensions of chitin and apatite indicates that the structure of the organic matrix may play a role in assisting the deposition and orientation of the apatite crystals. Mineralization phenomena within crustacean cuticle have been widely investigated. The three or four upper layers of this cuticle are mineralized, with calcium carbonate precipitated into the twisted lamellar structure of the chitin–protein cholesteric matrix (Bouligand 1972). Three-dimensional chitinous matrix is present in exoskeletons of numerous crustaceans. For example, the exoskeleton of the American lobster Homarus americanus is a nanocomposite consisting of chitin– protein fibers, reinforced with amorphous calcium carbonate (ACC) and a small amount of crystalline calcite (Al-Sawalmih et al. 2008). These researchers showed for the first time using X-ray microdiffraction that the crystalline calcium carbonate fraction is associated with the chitin–protein fibers and that the calcite crystallites have a fibrous morphology with the crystallographic c-axis oriented perpendicular to the cuticle surface, again supporting a relationship between calcite and the organic chitin–protein fibers. Recently, Heredia et al. (2006) have reported an amorphous form of α-chitin in the brown shrimp Penaeus aztecus shell coupling with calcitic spherulites. There are several examples in the literature where chitin was investigated both in vivo and in vitro as the sole template for mineralization or in association with other molecules (see also Table 4.1). Wainwright (1963) reported that in corals Pocillopora damicornis the major organic constituent of the skeleton was chitin in an unusually pure state. The skeleton of P. damicornis differs from all calcified structures previously described in its lack of associated fibrous protein or mucopolysaccharides. This Hawaiian reef coral was found to contain at least 99.9% by weight of the mineral aragonite, present as submicroscopic crystals in spherulitic arrangements. The organic component of the skeleton contains 0.01–0.1% of the total weight and has three microscopic constituents: (1) non-chitinous filaments of lime-boring algae, (2) a dispersed network of fibers 1 μm in diameter, and (3) a transparent, milky, regionally birefringent matrix of chitin. A spongework of chitin fibrils, average diameter 20 μm, was inferred to be randomly oriented in the plane of the skeletogenic epithelium perpendicular to the direction of growth of the long axes of the aragonite crystals. The skeletal development can be traced from the initial mineral deposit by the larva after its attachment, through the formation of the larval skeleton and growth into a fully formed, branching colony. The chitin fibrils conform to the contour of the skeletogenic epithelium and later deposition of aragonite crystals accounts for the
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Table 4.1 Chitin as universal scaffold and template for biomineralization Calcification
Silicification
In vivo Mg–calcite and aragonite Hall et al. (2002) in Bryozoa skeletons Calcified hydrozoan Miglietta (2006) skeletons Pocillopora coral skeleton Wainwright (1963) Chitin–calcite–chitin layers in Ostracoda valves Calcite, vaterite, and amorphous α-chitin in crustacean Pandalus borealis Amorphous calcium carbonateintercalated between chitinous lamella in crayfish C. quadricarinatus HAP and β-chitin in Lingula (Brachiopoda) shell
In vitro Yeast cell wall calcification: OCP Growth of calcite on crustacean α-chitin Aragonite, vaterite, and calcite on β-chitin scaffolds Ca phosphates on β-chitin scaffolds Ca phosphates on α-chitin
Glass sponge skeletons: Farrea occa Aphrocallistes vastus (α-chitin) Glass sponge spicules: Euplectella aurantiacum (α-chitin)
Ehrlich et al. (2007a)
Mikkelsen et al. (1997)
Rossella fibulata (α-chitin)
Ehrlich et al. (2008b)
Shechter et al. (2008)
Silica–chitin–goethite in limpet teeth Silica–chitin–apatite
Sone et al. (2007) Williams et al. (2001)
Kesling (1956)
Ehrlich and Worch (2007a)
Iijima and Moriwaki Silica teeth within chitinous Beklemishev (1990) mandible blade in calanoid (1954); Miller copepods (Crustacea) et al. (1990) Chitinous scaffold in diatom Brunner et al. cell wall (2009) Wang et al. (2008)
Silica–β-chitin composites
Ogasawara et al. (2000) Manoli et al. (1997) Silica–α-chitin composites Ehrlich et al. (2008b) Falini et al. (2002) Glycidoxypropyltrimeth Miyazaki et al. oxysilane–chitin composite (2007) Falini et al. (2001) Wan et al. (1998); Ge et al. (2004); Geçer et al. (2008)
formation of all skeletal elements of Pocillopora. It was suggested that (1) the amide group of the chitin molecule is responsible for the ability of certain organic substrates to be calcified (thus protein is not a necessary component of such substrates); (2) zooxanthellae in Pocillopora contribute a product via photosynthesis of the monomer of the chitin matrix to the coral; and (3) chitin synthesis thus depends on the activity of zooxanthellae and the rate of chitin synthesis controls the rate of skeletogenesis in Pocillopora (Wainwright 1963).
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Recently, Miglietta (2006) described Hydractinia antonii sp. nov., a new, partially calcified hydractiniid (Cnidaria: Hydrozoa: Hydractiniidae). This species found in offshore waters of the Aleutian Islands (Alaska) was dredged from the bottom at 139–145 m depth. This is the third known species of extant calcified Hydractiniidae that shows a unique colony structure. The base is heavily calcified and the distal parts are ramified and have a solid chitinous structure. Branches are all in one plane and the colony reaches up to 18 cm in height. Kesling (1956) described calcified chitin-based structures in Ostracoda as follows: Each valve contains two calcareous parts. The distal part is the outer lamella; it is the only part seen when the carapace is closed. The proximal part, a narrow flange just inside the free edge, is the duplicature, so-called because it duplicates tile chitin-calcite-chitin structure of the outer lamella. The two parts are joined together by a thin layer of chitin, the adhesive strip.
Mobilization of calcium in crustaceans during the molt cycle from the cuticle to transient calcium deposits is widespread. The dynamics of calcium transport to transient calcium deposits called gastroliths and to the cuticle over the course of the molt cycle was recently studied in the crayfish Cherax quadricarinatus (Shechter et al. 2008). In this species, calcium was deposited in the gastroliths during premolt and transported back to the cuticle during post-molt and was shown by digital X-ray radiographic analysis. The predominant mineral in the crayfish is amorphous calcium carbonate embedded in an organic matrix composed mainly of chitin. Scanning electron micrographs of the cuticle during premolt showed that the endocuticle and parts of the exocuticle were the source of most of the labile calcium, while the epicuticle did not undergo degradation and remained mineralized throughout the molt cycle. The gastroliths are constructed of concentric layers of amorphous calcium carbonate intercalated between chitinous lamella. Chitin was also found to be a substrate favoring the deposition of calcite crystals from stable supersaturated solutions at pH 8.50 and at 25◦ C in in vitro experiments. The mineralization, studied by Manoli et al. (1997) at constant solution supersaturation, made for relatively large amounts of overgrowth to be formed and identified exclusively as calcite. The induction periods varied markedly with level of supersaturation. The conditions of the experiments were such that the supersaturated solutions remained stable for periods up to 2 days, their stability verified by the constant pH and calcium concentration. It was shown that a low degree of supersaturation is a better representation of the physiological environment, where the free calcium concentration is low due to complexation of the element with molecules in biological fluids. When the solutions were seeded with chitin, the calcium carbonate overgrowth followed without the addition of any intermediate molecule. Other in vitro experiments reported by Sugawara et al. (2006) had a CaCO3 precipitation from a supersaturated solution of calcium carbonate with a chitin substrate in the presence of CAP-1 (calcification-associated peptide) for 20 h. The chitin matrix for the crystallization was prepared by spin coating the polysaccharide solution onto glass substrates. SEM observation revealed that microfibrils of
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about 10 nm diameter were formed and an X-ray diffraction pattern of this matrix confirmed it as α-chitin. In the absence of CAP-1, rhombohedral calcite crystals were formed on the chitin matrix. Composites of β-chitin with calcium carbonate polymorphs were prepared by precipitation of the mineral into a chitin scaffold using a double diffusion system (Falini et al. 2002). The β-chitin was obtained from the pen of the Loligo sp. squid. The three main polymorphs of calcium carbonate, namely aragonite, calcite, and vaterite, were observed. It was suggested that their location within the matrix was a function of the polymorph. The supersaturation inside the compartmentalized space in the chitin governs the location and crystal polymorph (Falini and Fermani 2004). Moreover, oriented precipitation of apatite crystals in/by β-chitin was observed in an in vitro experiment without any protein (Trautz and Bachra 1963). It is now clear that the role of chitin in calcification can be in the pure form as well as in the form of a chitin–protein complex (Machado et al. 1991). However, the role of chitin in biosilicification is still not completely understood. The presence of chitin as structural component of different Protozoa species was also reported (Herth 1980; Herth et al. 1977; Mulisch and Hausmann 1989), however never with respect to a silica–chitin composite material. We hypothesized (Ehrlich et al. 2007a) that silica–chitin biocomposites might be identified in Protozoa as well as in choanoflagellate-like protists as ancestral organisms which are architecturally close to sponge larvae (Maldonado 2004). Steenkamp et al. (2006) suggested that colonial non-mineralized choanoflagellate-like protists gave rise to the first animals (i.e., sponges), while chitinous thecate choanoflagellate-like protists gave rise to the first fungi. On the other hand, results published by Carr et al. (2008) unambiguously show that choanoflagellates are an ancient monophyletic group. Thus, there is no evidence that Metazoa are derived from a choanoflagellate ancestor or that any division of choanoflagellates has an exclusive relationship at the genetic level with Metazoa. β-Chitin is also present in several diatom species in the form of extracellular fibrils. Tesson et al. (2008) reported NMR spectroscopic studies, which hint at the presence of chitin apart from the other, aforementioned biomolecules in extracted, cleaned diatom cell walls. It is, however, unknown whether or not chitin may be involved in cell wall biogenesis as a possible template and become a part of the integer cell wall. According to the latest view, several special biomolecules are involved in diatom silicification and become at least partly embedded into their siliceous cell walls. Three classes of such biomolecules are identified: (i) the silaffins, post-translationally highly modified peptides/proteins with zwitterionic character (Kröger et al. 1999, 2002; Sumper et al. 2007); (ii) long-chain polyamines, positively charged in solution (Brunner and Lutz 2007; Gröger et al. 2008; Kröger et al. 2000; Sumper et al. 2005; Sumper and Lehmann 2006); and (iii) the highly acidic silacidins, negatively charged in solution (Wenzl et al. 2008). The silaffins were found to be capable of self-assembling into supramolecular aggregates due to their zwitterionic character. The same is true for the long-chain polyamines (Brunner et al. 2004; Sumper et al. 2003), provided a properly chosen counterion such as
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Fig. 4.5 SEM images (a, b) of chitinous matrix isolated from the cell walls of diatom Thalassiosira pseudonana (images courtesy: Patrick Richthammer)
orthophosphate, pyrophosphate, or silacidin is present (Sumper and Brunner 2008). In our present study (Brunner et al. 2009) we showed for the first time that the cell walls of the diatom species Thalassiosira pseudonana contain a network-like chitin-based scaffold which exactly resembles the size and shape of the biosilica (Fig. 4.5). These scaffolds consist of interconnected fibers of about 25 nm average diameter containing other yet unknown biomolecules apart from chitin. It is tempting to speculate that the chitin-based networks provide the scaffold structure for silica deposition, while other biomolecules—maybe silaffins—actively deposit silica on these superstructures. It is well known that diatom frustules are mechanically resilient, sophisticated structures made of a tough glass-like composite. Consequently, to break the frustules, predators have to generate large forces and invest large amounts of energy. They need mandibles which are hard, and tough enough to resist high stress and wear, for feeding on biomineralized objects such as not only diatoms but also other biomineralized protists (Hamm 2005). Indeed, many lower crustaceans—copepods—feeding on diatoms possess, in analogy to the enamelcoated teeth of mammals, amazingly complex, silica-laced structures in their mandibles (Beklemishev 1954; Miller et al. 1980). These opaline teeth of calanoid copepods develop early in the premolt phase of the molt cycle and in the apolysis space beneath the old tooth row on the chitinous mandibular gnathobase (Miller et al. 1990). Opal is laid down at the outer periphery of the mold and thickens toward the attachment of the mold with newly formed chitin at its base. Unfortunately, the interrelationship between opaline silica and fibrous chitinous matrix in the mandibular blade of copepods has not yet been fully investigated. We hypothesize that copepods probably developed a specific metabolism to utilize both silica of diatomaceous origin and chitin as the building blocks of their own anatomical structures, teeth and mandibles. Recently, we isolated and identified chitin from skeletal remains of some marine glass sponges for the first time. The presence of chitin within the framework skeleton of Farrea occa (Ehrlich et al. 2007a) and Euplectella aspergillum (Ehrlich and
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Worch 2007a) as well as separate spicules Rossella fibulata (Ehrlich et al. 2008b) was revealed by gentle NaOH-based desilicification technique (Ehrlich et al. 2006a). The structure of the chitin extracted from these sponges turned out to be similar to α-chitin from other invertebrates as shown by Raman spectroscopy and electron diffraction techniques. The finding of silica–chitin natural composites as the component of the glass sponge spicules and their skeletons is in good agreement with results of in vitro experiments on silicification of a β-chitin-containing cuttlebone-derived organic matrix reported by Ogasawara et al. (2000). These authors suggest that silicate ions and silica oligomers preferentially interact with glucopyranose rings exposed at the β-chitin surface, presumably by polar and H-bonding interactions. We believe that chitin is acting as an organic template for silica mineralization in the investigated sponge species. Chitin could also play a crucial role in biosilicification in fungi. Accumulation and deposition of silica by microcolonial fungi associated with desert varnish was reported (Kolb et al. 2004). Chitin molecules are probably part of a very old organic template system involved in biosilicification and established a long time before the origin of the first metazoan (e.g., glass sponges).
4.2.2 Collagen Yamada et al. (1980) defined the collagen family of proteins by their distinctive and common compositional and structural properties, including (i) the presence of glycine residues at every third amino acid; (ii) an abundance of prolines and lysines, many of which are hydroxylated; (iii) the characteristic configuration of the molecule, composed of three chains which interact with each other to form a triplehelical structure; and (iv) the presence of many inter- and intramolecular cross-links which result in higher order structural organization. Tropocollagen is the basic structural unit of collagen and consists of three intertwined peptide chains of approximately 1000 amino acids each that form strong fibrils by aggregation of staggered arrays of the tropocollagen molecules. Each triple helix is 300 nm long—intriguingly the same length as reported for the structural unit of chitin (see above). The tight wrapping of the triple-helical chains provides the tensile strength of the collagen that is greater than that of steel wire of an equal cross section. Thus, collagen provides strength and confers form while allowing flexibility and movement. It is the major structural component of tissues, responding to tension in soft tissues and providing a platform for mineralization in the hard tissues that are subjected to compression (Cowin 2000). So far, 26 genetically distinct collagen types have been described. Based on their structure and supramolecular organization, they can be grouped into fibril-forming collagens, fibril-associated collagens, network-forming collagens, anchoring fibrils, transmembrane collagens, basement membrane collagens, and others with unique functions (Gelse et al. 2003). There are three major fibrillar collagens in jawed vertebrates (Kawasaki and Weiss 2006): collagen type I serves as the scaffold for dentin, bone, and teleost enameloid; type-II collagen is the major protein found in
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mineralized cartilage. In contrast to these mineralization-related collagens, typeIII collagen is found in most soft tissues. Collagen-based extracellular matrices must have evolved over many million years prior to the onset of biomineralization (Simmer 2007). The origin of a collagen-related structural motif (CSM) containing glycine−X−Y repeats found in numerous proteins in prokaryotic and eukaryotic organisms is still unknown. The bacterial proteins containing a CSM that could be functionally useful were either surface structures or spore components, whereas viral proteins are generally considered as structural components of the viral particle (Bamford and Bamford 1990). The limited occurrence of CSMs in eubacteria and lower eukaryotes, and the absence of CMSs in archaebacteria, suggests that DNA-encoding CMSs have been transferred horizontally, possibly from multicellular organisms to bacteria. However, the presence of CMSs in prokaryotes underlines the importance of collagen as a common structural motif in nature (Ehrlich and Worch 2007b), although at present there is no information available on the involvement of bacterial CSMs in biomineralization phenomena. Studies on collagen with respect to its role in biomineralization in vivo as well as in vitro are ongoing. Collagen is present in hard tissues and is a useful biomaterial. The biocompatibility and the safety due to its biological characteristics, such as biodegradability and weak antigenicity, make collagen the primary resource in biomedical applications. It is used for tissue engineering and particularly research on bone substitutes, and as a matrix system for evaluation of tissue calcification (Lee et al. 2001). The properties of collagen in biomineralization phenomena are determined not only by its structure but also by different modifications and interactions with non-collagenous proteins, proteoglycans, glycosaminoglycans, and possibly mono- or oligomeric sugars (Ehrlich et al. 2005). Therefore the detailed mechanism of collagen mineralization has been a very much discussed and researched topic over the years. However, despite the many concerted efforts, a satisfactory understanding of the many and various aspects that must be involved is still far from known. The major mineralized tissues in vertebrates are cartilage, teeth, and bone. Mineralization of cartilage occurs as an essential component in the growth plates of developing skull and long bones, in the antlers of deer, and at tendon and ligament insertion into bone (Boskey 2002). The teeth are comprised of three mineralized tissues: dentine, enamel, and cementum. Enamel is thought to have evolved from enameloid, a highly calcified tissue covering the dentine of fish. Enameloid is predominantly of mesenchymal origin and its matrix is composed mainly of collagen fibers (Line and Novaes 2005). From evolutionary point of view and in phylogenetic terms, bone is first manifest as an acellular, matrix-rich mineralized tissue known as “aspidin” in pteraspidomorphs (Donoghue et al. 2006). The aspidin although acellular is dominated by a rich organic matrix presumably consisting of collagen fibers (Donoghue and Sansom 2002). Most bones are based on collagenous matrix and Ge and George (2004) suggested that the matrix in both bone and dentine structures forms a template for biomineralization by the mineral apatite. There is growing evidence that genes encoding extracellular matrix proteins involved in the biomineralization of bone, dentine, and enamel diverged from a common ancestor gene;
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however the genes encoding collagen-based extracellular matrixes are more ancient (Simmer 2007). Collagen not only is relatively rigid when mineralized but also possesses no domains for acidic proteins which are the main players in the invertebrate calcification phenomena. However, recently, Kawasaki and Weiss (2006) proposed that initial vertebrate tissue mineralization utilized collagen as scaffold and different acidic and calcium-binding proteins were critical as mediators of mineral crystallization. These authors assumed that the genes responsible for these additional proteins evolved after the divergence of the fibrillar collagens. Is it possible that collagen itself can provide preferential sites for nucleation and control the orientation of the resulting mineral crystals? I will explore the possibilities. A. Boskey (2002) defined collagen as “template for mineral deposition.” In Weiner and Wagner (1998) the collagen was described in similar manner: “The collagen constitutes the main component of a three-dimensional matrix into which, and in some cases onto which, the mineral forms.” Collagen fibers pack into bundles with a periodicity that leaves small gaps known as “hole zones” 40 nm in length and 5 nm in width, each of which might provide a suitable molecular-scale locus for nucleation and mineralization. The crystallites align themselves with the {001} or c-axis of the HAP lying along the length of the fiber and the {110} crystallographic axis at right angles implying molecular-scale control by the collagen fibrils over both the orientation and the location of the crystal nuclei (De Yoreo and Vekilov 2003). Other researchers, following the templating properties of collagen, such as George (Ge and George 2004) state: During bone and dentin mineralization, the crystal nucleation and growth processes are considered to be matrix regulated. Osteoblasts and odontoblasts (dentinal cells) synthesize a polymeric collagenous matrix, which forms a template for apatite initiation and elongation. Coordinated and controlled reaction between type I collagen and bone/dentin-specific noncollagenous proteins are necessary for well defined biogenic crystal formation. However, the process by which collagen surfaces become mineralized is not understood.
In summary it appears that collagen defines the space in which the crystallites start and may constrain the space that defines the crystal size and morphology. Olszta et al. (2007) proposed that because the mineral phase is shaped by the collagen prior to crystallization and because of the fact that HAP commonly crystallizes as platelets of preferred orientation, the collagen acts as a highly organized “container” to constrain the growth of HAP along its fast growing [0 0 1] or caxis. This leads to crystals that are extremely small (only a few unit cells thick), which would not normally be thermodynamically stable if it were not for being embedded within the organic matrix. Additional HAP crystallites are found on the exterior of fibrils, probably also assisted by the collagen. This leads to intrafibrillar and interfibrillar nanocrystals. There are several publications which confirm mineral crystal growth directly on collagen fibrils without the presence of some intermediate molecules (e.g., acidic proteins). For example, Cui et al. (2007) suggested that the nucleation sites of HAP crystals on collagen fibers could be through the binding of calcium ions on the negatively charged carboxylate groups of collagen and be one of the key factors for the first-step nucleation of HAP crystals (Kikuchi et al. 2001).
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Girija et al. (2003) suggested that formation of the collagen–apatite composite by biomimetic methods without any previous treatment of the collagen fibrils could be achieved. In his article based on results of proteomic analysis, Zhou (2007) reported that one of the three strands of the collagen molecule known as α2 (I) was for the first time shown to directly interact with calcium phosphate mineral. Increased research in calcification has led to using biomimetic synthesis: the generation of collagen-based or collagen-like materials fabricated according to biological principles and processes of self-assembly and self-organization. Additionally, the materials chemistry aspects of calcification have suggested model systems for biomimetic engineering. For example, my group proposed the carboxymethylation of biopolymers, including collagens, employing glucuronic acid as one of the key reactants in mineralization processes (Ehrlich et al. 2009b). Our results with glucuronic acid could contribute to the interaction between glycosaminoglycans and proteoglycans, with collagen fibrils and fibers in vivo forming carboxymethyl lysine (CML)–collagen which could then be a template for calcification (Ehrlich et al. 2009b, c, d). This is a step in the design of new strategies for the in vitro development of biomaterials. According to the data summarized in Table 4.2, collagen not only seems to be the template for HAP formation and deposition but also shows templating properties with respect to calcite formation, for example, within skeletons of sponges (Calcarea: Porifera) and octocorals. We propose collagen as the “universal” template from our experiments which unambiguously showed that collagen is the main template for silicification within spicules of glass sponges (Hexactinellida: Porifera) (see Table 4.2). Collagen differs from chitin with its acidic polysaccharides or acidic proteins, which were unable to be successfully silicified. Also, collagen differs from the cationic polypeptides like silaffins (well described as templates for silicification in diatoms) (Kröger et al. 1999, 2002), which in turn could not be calcified. Similar results were obtained in in vitro experiments with gelatin (denaturated collagen) and polysaccharide alginate. Alginate, a hydroxyl-bearing polysaccharide, does not significantly activate silica formation in contrast to calcium phosphate phases, whereas gelatins strongly interact with both silicates and different calcium phases (Gautier et al. 2008). As collagen serves as a template for calcium phosphate that contains some CO3 2− ; it is possible that the evolution of silica and bone skeletons shares a common origin (Ehrlich and Worch 2007b). Based on information reported to date, the effect of silicon on the formation of bone and connective tissues can be summarized as follows (Ehrlich and Worch 2007b): – Si may be associated with Ca in the early stage of bone calcification, because as mineralization progresses, the Si and Ca contents rise congruently in osteoid tissue. In the advanced stages of mineralization, Si concentrations fall markedly, whereas Ca concentrations approach proportions found in bone apatite (Carlisle 1974). – Si could be involved in the formation of cross-links between collagen and proteoglycans, and may stabilize bone matrix molecules by preventing their degradation (Schwartz 1973).
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Table 4.2 Collagen as universal scaffold and template for biomineralization Calcification
Silicification
In vivo Example
References
Example
Dahllite and HAP in the bone, teeth, tendon, cartilage
Weiner and Wagner (1998); Olszta et al. (2007)
Glass sponges (Hexactinellida: Porifera)
Boskey (2002) Zhou (2007); Jiang et al. (2008)
(a) Euplectella sp. (b) Hyalonema sieboldii (c) Monorhaphis chuni
Calcite in: (a) Sponges’ skeletons (Calcarea) (b) Corals (c) Gorgonian octocorals
In vitro HAP
Calcite
Vaterite
References
Travis et al. (1967) Ehrlich et al. (2006a); Ehrlich and Worch (2007b) Ehrlich et al. (2008c)
Ledger (1974)
Marks et al. (1949) Goldberg (1974, 1976); Kingsley et al. (1990); Lewis et al. (1992) Girija et al. (2003); Cui Silica–collagen et al. (2007) composites
Coradin et al. (2002); Eglin et al. (2005); Ehrlich and Worch (2007b); Heinemann et al. (2007a, b); Ehrlich et al. (2008d)
Nayar and Sinha (2003; Jiao et al. (2006) Falini et al. (1998)
– Deprivation of Si in rats results in altered bone mineral composition and decreased activity of bone enzymes (Seaborn and Nielsen 1994) and i-deprivation reduced the collagen content in skull and long bone (Carlisle 1980). – Orthosilicic acid stimulates collagen I synthesis by human osteoblast-like cells in vitro and enhances osteoblastic differentiation (Reffitt et al. 2003). These effects of Si did not markedly affect bone calcium content and bone strength, although Si did enhance bone elasticity. This indicates that Si does not have a major effect on bone crystal formation but rather has an effect on bone growth processes prior to the start of mineralization. Thus, we speculate that there is a possible role for silica as silica–collagen composite in the earliest stages of bone formation.
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We suggest that the higher structural organization of multicellular organisms (in contrast to prokaryotes), including the presence of fibrillar collagen, may have stimulated the formation and organization of silica–collagen-based skeletons. However, natural silica–collagen composites are no longer the desired end stage in metazoans, since silica-gel matrices have been superseded by calcium phosphate- and calcium carbonate-mineralized collagen matrices during the course of animal evolution. On the other hand, the twisted plywood collagen fibril orientation which was recently observed within silica layers in basal spicules of glass sponges (Ehrlich and Worch 2007b; Ehrlich et al. 2008c) has been investigated extensively in lamellar bone (Wagermaier et al. 2006) and seems to be an example of a conserved feature. From this point of view, silica–collagen composites may be a preliminary phase in bone development which has been conserved from the time when silica–collagen composites were necessary for the construction of the first metazoan skeletons. That is, they may be synthesized and play a crucial role only during an early phase of the embryonic development of vertebrates. This would be in accordance with Haeckel’s Biogenetic Law (Richardson and Keuck 2002), which states that the rapid and brief ontogeny is a precursor still found in the slow and long phylogeny of species. In other words, the production of ancestral patterns of development in descendant ontogenies is a key aspect of biological systems, including skeletogenesis. The nano-fibrous architecture may serve as superior scaffolding versus solidwalled architecture for promoting biomineralization (Kyung et al. 2007). Both mineralized chitin and collagen offer the nano-fibrous framework for mechanical support and imparted to the skeletons in invertebrates are reservoirs for ions and small molecules; and for strain energy storage. Chitin- and collagen-based biocomposites are highly organized from the molecular and the nano- through micro- to the macroscales, a hierarchical relationship with intricate intimate architectures that serve many different functional needs for both soft and hard tissues. Self-assembling properties of both biopolymers and their templating activity with respect to silicification are in accordance with those reported by Pouget et al. (2007). The feedback actions between the template growth and the inorganic deposition are kinetically important and are probably driven non-enzymatically by mutual electrostatic neutralization. We believe that this concept could become accepted as a fundamental mechanism for growth processes in chitin- and collagen-based biological systems with respect to biomineralization phenomena. The evolution of biomineralizing systems continues to be the subject of extensive investigations (Knoll 2003; Matsushiro and Miyashita 2004). One way to inquire what generalizations can be made about strategies in biomineralization is to ask what has been conserved (Wilt 2005). Have molecules like chitin and collagen been conserved? My personal opinion based on a thorough literature analysis is: yes they are. They answer the questions of useful forms from the evolutionary point of view, and they are both common today with their very similar structural organization and chemical nature. For example, the fossil records of the chitin- and collagen-based glass sponges date back to the late Neoproterozoic, and no major modifications of the body plan have occurred since then (Dohrmann et al. 2008). Structure–function relationships in the skeletal formations of these sponges could
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be explained as follows. Silica protects chitin and collagen distributed within skeletons against exochitinases and proteases secreted by bacteria and fungi occurring in marine environments. Chitin and collagen act as a biological resin; they absorb shocks preventing cracks and failure of the glassy construct (Murray and Neville 1998). Both biopolymers answer the purpose of natural design principles (Ingber 2000) selected over the long time by evolution. Structural efficiency is maximized and evolution accelerated through the use of hierarchical networks, which provide small-scale discrete structures, but at every size and scale, complex properties and functions emerge from the behavior of the ensemble. Any of the individual parts are much less important than how they are joined and positioned three dimensionally. Most of the chemical and enzymatic functions carried out by living systems proceed on insoluble scaffolds employing catalysis with other solid phases. Both chitin and collagen are highly ordered insoluble macromolecules. Their composition and structure increase the efficiency of chemical reactions, forming stable functional networks that allow metabolic systems to self-assemble and associate with others to create hierarchical structures with enhanced functionality. Furthermore, chitin and collagen biomineralization answers the needs of the scientific communities by investigating artificial design principles, called “biomimetic design principles,” prominently described in numerous in vitro experiments presented in this chapter. Dilute suspensions of chitin and collagen are isotropic, i.e., the macromolecules can take on any orientation in the fluid phase. Beyond a critical concentration, an ordered nematic phase appears with a higher volume fraction (Belamie et al. 2006). Subsequent oriented precipitation with HAP and other calcium phases in collagenous and chitinous structures where carboxyl or free amino and guanidine groups on the biomolecules had been blocked (Trautz and Bachra 1963) become available for mineral association and accumulation. A reconstruction of the evolution of biocalcification as well as of biosilicification with respect to chitin and collagen may provide the strongest evidence of ancient, ancestral programs (Livingston et al. 2006) of biomineralization started in marine environment based on these common and alternative macromolecular templates.
4.3 Conclusion Biomineralization is a complex process involving crystal growth, dissolution, reprecipitation, and ageing of different salts encountered in living organisms and in close affinity with the organic matrix. Demineralization is the process of removing the inorganic part, or the biominerals, that takes place in nature via either physiological or pathological pathways in organisms. In vitro demineralization processes, used to obtain mechanistic information, consist in the isolation of the mineral phase of the composite biological materials from the organic matrix. Bioerosion, a more general term for the process of deterioration of the composite biomaterials, represents chemical deterioration of the organic and mineral phase followed by biological attack of the composite by microorganisms and enzymes. Bioerosional organisms are represented by endolithic
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cyanobacteria, fungi, algae, plants, sponges, phoronids and polychaetes, molluscs, fish, and echinoids. Overall, biomineralization, demineralization, and remineralization are rapidly growing and challenging aspects of various scientific disciplines such as astrobiology, paleoclimatology, geomedicine, archaeology, geobiology, dentistry, histology, biotechnology, materials science, and others to mention just a few.
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Wan ACA, Khor E, Hastings GW (1998) Preparation of a chitin–apatite composite by in situ precipitation onto porous chitin scaffolds. J Biomed Mater Res 41:541–548 Wang B, Liu P, Jiang W et al (2008) Yeast cells with an artificial mineral shell: protection and modification of living cells by biomimetic mineralization. Angew Chem Int Ed 47(19):3560– 3564 Watabe N, Bernhardt AM, Kingsley RJ et al (1986) Recalcification of decalcified spicule matrices of the gorgonian Leptogorgia virgulata (Cnidaria: Anthozoa). Trans Am Microsc Soc 105: 311–318 Weaver JC, Morse DE (2003) Molecular biology of demosponge axial filaments and their role in biosilicification. Microsc Res Tech 62:356 Weiner S (1984) Organization of organic matrix components in mineralized tissues. Am Zool 24:945–951 Weiner S, Wagner HD (1998) The material bone: structure mechanical function relations. Ann Rev Mater Sci 28:271–298 Weiss IM, Schönitzer V (2006) The distribution of chitin in larval shells of the bivalve mollusk Mytilus galloprovincialis. J Struct Biol 153:264–277 Wenzl S, Hett R, Richthammer P et al (2008) Silacidins: highly acidic phosphopeptides from diatom shells assist in silica precipitation in vitro. Angew Chem (Int edn in English) 47(9):1729–1732 Wessels JGH, Mol PC, Sietsma JH et al (1990) Wall structure, wall growth and fungal cell morphogenesis. In: Kuhn PJ, Trinci APJ, Jung MJ et al (eds) Biochemistry of cell walls and membranes in fungi. Springer, Berlin, Heidelberg, New York, Tokyo Willenz P (1983) Aspects cinetiques quantitatifs et ultrastructuraux de l’endocytose, la digestion, et l’exocytose chez les sponges. Thesis, Université Libre de Bruxelles, Bruxelles Williams A, Lüter C, Cusack M (2001) The nature of siliceous mosaics forming of the first shell of the brachiopod Discinisca. J Struct Biol 134:25–34 Williams JA, Margolis SV (1974) Sipunculid burrows in coral reef: evidence for chemical and mechanical excavation. Pacif Sci 28:357–359 Willmer P (1990) Invertebrate relationships. Cambridge University Press, Cambridge Wilt FH (2005) Developmental biology meets materials science: morphogenesis of biomineralized structures. Dev Biol 280:15–25 Winterberg A (1898) Zur Theorie der Saurevergiftung. Z Physiol Chem XXV:202–235 Wisshak M, Gettidis M, Freiwald A et al (2005) Bioerosion along a bathymetric gradient in a cold-temperate setting (Koster fjord, SW Sweden): an experimental study. Facies 51:93–117 Wisshak M, Rüggeberg A (2006) Colonisation and bioerosion of experimental substrates by benthic foraminiferans from euphotic to aphtotic depths (Kosterfjord. SW Sweden). Facies 52:1–17 Yamada Y, Avedimento VE, Mudryj M et al (1980) The collagen gene: evidence for its evolutionary assembly by amplification of a DNA segment containing an exon of 54 bp. Cell 22:887–892 Zhou H-Y (2007) Proteomic analysis of hydroxyapatite interaction proteins in bone skeletal biology and medicine, part a: aspects of bone morphogenesis and remodeling. Annu NY Acad Sci 1116:323–326 Ziegler A, Weihrauch D, Hagedorn M et al (2004) Expression and polarity reversal of V-type H+ ATPase during the mineralization-demineralization cycle in Porcellio scaber sternal epithelial cells. J Exp Biol 207:1749–1756 Zundelevich A, Lazar B, Ilan M (2007) Chemical versus mechanical bioerosion of coral reefs by boring sponges?lessons from Pione cf. vastifica. J Exp Biol 210:91–96
Chapter 5
Multiphase Biomineralization
Abstract Recently, a unique biocomposite of amorphous silica, crystalline aragonite, and chitin from the Verongida, a group of marine sponges that were thought to lack a mineral skeleton, has been described for the first time. The presence of both carbonate and silica in these demosponges, templated by the evolutionarily ancient chitin, is similar to biomineralization processes founded in other groups. Examples of multiphase biomineralization occurring in marine invertebrates like sponges, molluscs, brachiopods, and crustaceans are described and discussed. Biomineralization is a rewarding area of biological research, yielding both evolutionary insights and inspiration for biomimetic research. In particular, biocomposite materials are valuable sources of novel structures with unusual physical properties and are very informative for the mechanisms of biomineralization. Recently, we described a unique biocomposite of amorphous silica, crystalline aragonite, and chitin from the Verongida, a group of marine sponges that was thought to lack a mineral skeleton (Ehrlich et al. 2010). The presence of both carbonate and silica in these demosponges, templated by the evolutionarily ancient chitin, is similar to biomineralization processes described in other groups. We therefore proposed that chitin and collagen templates may form a universal basis as scaffolds for much of metazoan biomineralization. Here, I give a brief description of the some examples of multiphase biomineralization occurring in marine invertebrates like sponges, molluscs, brachiopods, and crustaceans.
5.1 Silica–Aragonite–Chitin Biocomposites in Demosponges (Demospongiae: Porifera) According to morphological and biochemical analyses, the Verongida forms a coherent order of keratosan demosponges (Erwin and Thacker 2007). Verongid genera are mainly distinguished by the structure and composition of their fibers (Bergquist and Cook 2002) and the group is distributed worldwide. They are traditionally thought to lack a mineral skeleton, possessing instead a collagenous H. Ehrlich, Biological Materials of Marine Origin, Biologically-Inspired Systems 1, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9130-7_5,
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mesohyl supported by spongin fibers that exhibit a granulated “pith” interior and a laminated “bark” exterior (Bergquist and Cook 2002). Recent re-examination showed that chitin, rather than spongin, forms the main organic component of verongid skeletal fibers and that aragonite is also present (Ehrlich et al. 2007b). Because sponges are often regarded as the most ancient metazoans (630−542 My) (Brasier et al. 1997), the finding of chitin within their skeleton is of major significance. It is well established that chitin can function either directly or indirectly as a template for nucleation in mineral phases in other invertebrates (Falini et al. 2002, 2003). In this case, it was suggested that chitin serves as a template for calcium carbonate deposition in sponges (Ehrlich et al. 2007b). The skeleton of Verongula gigantea (Fig. 5.1) is a three-dimensional construction with anastomosing fibers (Fig. 5.2a, b) producing polygonal reticulation. The chemistry of verongid fibers can be studied by selective dissolution in alkaline or acidic solutions. Alkalis can dissolve the siliceous material, leaving the carbonaceous intact, while acidic solutions can dissolve the carbonate to leave the
Fig. 5.1 V. gigantea sponge possesses three-dimensional chitin-based skeleton (image courtesy Fernando Moraes and Guilherme Muricy)
Fig. 5.2 Light microscopy (a) and SEM (b) images of the V. gigantea skeleton fragments (image courtesy Heike Meissner and Gert Richter)
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siliceous phase. In order to isolate the mineral-free organic matrix we used alkali treatment at 37◦ C as described previously (Ehrlich et al. 2007b), yielding translucent tubular structures after 5 days. The results of chitinase digestion testing and additional physico-chemical analyses performed using FTIR, Raman, and XRD were similar to those reported earlier for α-chitin of poriferan origin (Ehrlich et al. 2007b). Weighing of dried natural fibers, and the same fibers after alkali-mediated demineralization, showed that chitin averaged 60% of the dry weight of the native sponge skeleton. No measurable traces of Ca or Si were measured in alkali-treated chitinous fibers after their dialysis. However, calcium carbonate was identified in lyophilized alkali extracts, as in previous results (Ehrlich et al. 2007b). The treatment of native sponge fibers (Fig. 5.3a) with 3 M HCl solutions resulted in dissolution of the acid-soluble mineral component. This yielded a perforated fiber surface (Fig. 5.3b, c). These 40–60 μm thick structures are visible in a light microscope (Fig. 5.3d) or scanning electron microscopy (SEM) (Fig. 5.3e). Results of the EDX analysis show with strong evidence the siliceous nature of these layers (Fig. 5.3f). Silicon concentrations were determined, after alkali dissolution of isolated layers (Fig. 5.3d, e) by the silicomolybdate method (Ehrlich et al. 2010), to be about 100–150 μg of Si per milligram of dried skeletal fiber. Because autofluorescence is a typical for pure silica, but well known for chitin and naturally occurring silica–chitin composites (Ehrlich et al. 2007a; Ehrlich and Worch 2007a), we used fluorescent microscopy and Calcofluor White staining for preliminary identification of chitin within these acid-resistant structures. The characteristic blue fluorescence of both unstained and stained samples indicated the presence of chitin (Ehrlich et al. 2010). We therefore suggested that the nanofibrillar network in this silica-based matrix (Fig. 5.4a) could be of chitinous nature. Samples of the matrix shown in Fig. 5.4 were subsequently submitted to HR-TEM to examine the nature of this material and locate additional occurrences of chitin. HR-TEM studies of the silicified matrix residues obtained after acid-mediated demineralization of V. gigantea fibers revealed the presence of nanocrystallites with a diameter of 2 nm (Fig. 5.4b). These structures were nearly identical in appearance and size to the chitin crystallites observed when examining an α-chitin standard. They were also extremely similar to previously reported TEM observations of chitinous skeletal structures in insects, crustaceans, and arachnid species (Giraud-Guille 1998; Goodrich and Winter 2007; Neville et al. 1976). The chemical nature of the mineral phases within sponge fibers was then investigated by means of photoemission spectroscopy (PES) and X-ray absorption spectroscopy (XAS), FTIR, and Raman spectroscopy (Ehrlich et al. 2010). The origin of the microscale apertures within the acid-treated matrix visible in SEM images (Fig. 5.5a) could be explained by the presence of aggregated calcium carbonate particles of similar diameter to others observed in extracts obtained from fibers after agate mortar disruption and alkali treatment (Fig. 5.5b–d). Closer examination using STEM and electron diffraction measurements (Ehrlich et al. 2010) indicates that the textured core of these aggregates consists of tightly packed chitin (Fig. 5.6) templating aragonite (Fig. 5.6, arrows). Consideration of the polarity of the −N−C(O)− bond in chitin molecule, in which the negative charge is shifted
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Fig. 5.3 The treatment of native V. gigantea fibers (a) with HCl solutions resulted in dissolution of the acid-soluble mineral component. This yielded a perforated fiber surface (b, c). These structures are visible in a light microscope (d) as well as in SEM (e). Siliceous nature of these layers was confirmed using EDX analysis (f)
toward the oxygen atom, suggests that the formation of calcium carbonate may be initiated through the interaction of Ca2+ ions with the oxygen of the C=O (Manoli et al. 1997). Our proposed model for the micro- and nanostructure of the Verongida sponge skeletal fiber is that of a silica–aragonite–chitin-based biocomposite (Fig. 5.7). In cross section, fibers show an outer, cuticle-like layer up to 10 μm thick (Fig. 5.7a) overlying several intermediate layers. These layers are comprised of a strict biocomposite whose building blocks are nano- (Fig. 5.7b) and micron-size crystals of aragonite (Fig. 5.7b), incorporated within a silica matrix (Fig. 5.7c, d), which is itself
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Fig. 5.4 Nanofibrillar structures are well visible in siliceous acid-resistant layers isolated from V. gigantea fibers using TEM (a) and HR-TEM (b)
Fig. 5.5 The microscale apertures within the acid-treated matrix visible in SEM images (a) could be explained by the presence of aggregated calcium carbonate particles of similar diameter to others observed in extracts obtained from fibers after alkali treatment (b–d)
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Fig. 5.6 STEM image of the nanofibrills of chitin, which templated aragonite nanocrystals (arrows)
bound to chitin nanofibers that act as a template for silicification. It is theoretically possible that inorganic Si binds to sponge chitin after the macromolecular structure has been formed. It has been suggested (Ogasawara et al. 2000) that silicate ions and silica oligomers preferentially interact with glycopyranose rings exposed at the αchitin surface, presumably through polar and H-bonding interactions. Alternatively, and more plausibly from stereochemical considerations (Schwarz 1973), mono-or disaccharide Si derivatives may have been incorporated during polysaccharide chain synthesis. The formation mechanisms of this unique biocomposite are unknown, although certain possibilities can be considered. It is difficult to differentiate between organically mediated incorporation and inorganic precipitation; hence, a combination of different mechanisms in the same species cannot be ruled out. The observed threedimensional morphology of this complex biocomposite can be understood as the result of the crystallization of aragonite crystals in the presence of polymeric silica, a well-known trap for organic compounds (Garcia-Ruiz et al. 2002). Sponges are an ideal subject for biomineralization studies due to their complex range of mineralogy, their phylogenetic position at the base of metazoans (Bavestrello et al. 2003), and their ability to build hierarchically structured skeletons (Aizenberg et al. 2005; Fratzl 2007). Although in the phylum Porifera the skeleton may be composed of a large variety of minerals, calcium carbonate and siliceous structures very rarely co-occur in the same sponge. The only previously known examples are (i) Coralline, or calcified sponges, which possess a solid calcareous skeleton and siliceous spicules; (ii) Hemimycale columella (Demospongiae, Poecilosclerida), in which calcium carbonate spherules occur alongside siliceous spicules (Vacelet et al. 1987); (iii) Cinachyrella alloclada (Demospongiae, Spirophorida), in which the siliceous spicules are complemented by calcareous granules (Rützler and Smith 1992). None of these constitute biocomposites.
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Fig. 5.7 Proposed model of micro- and nanostructural organization of the skeletal fiber of verongid sponges with respect to its multiphase biocomposite nature. (a) The cuticular layer (left part of the SEM image) covers the surface of the mineralized outermost layers of fiber (right part). (b) Nanocrystals of aragonite in a cross section of the mineralized layer, imaged by TEM. (c) SEM images of the three-dimensional structure of the silica–chitin-based matrix, visible after dissolution of micro- and nano-scale crystals of aragonite, leaving perforations (d). The base substance of the mineralized concentric layers distributed directly under the cuticle consists of a three-dimensional matrix of silicified chitin fibrils with regularly distributed aragonite crystals within this siliceous construction. (e) SEM images of the unconsolidated pith core region showing nanofibrillar organization of nonmineralized chitinous matrix within the skeletal fiber and on the inner surface (f) of its axial channel
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The early fossil record is more complicated, with a bilayered structure suggestive of a silica–carbonate biocomposite present in the heteractinid sponge Eiffelia globosa (Botting and Butterfield 2005), which also shows morphological characters of both Calcarea and Hexactinellida. It has been argued that the different secretion mechanisms of spicules of these two phases in modern sponges preclude homologous biomineralization between the sponge classes (Sethmann and Wörheide 2008). However, the recognition of a chitin template for the verongid biocomposite described here shows that the process is potentially flexible. We regard this homology of biomineralization as reflecting a continuous lineage of the same templating pathway inducing or controlling mineral crystallization. The common ancestors of the sponge classes could potentially secrete both carbonate and silica as a result of the shared collagen (spongin)/chitin templates. Different lineages may then have utilized these basic processes in different ways through different cytological secretion arrangements, given the appearance of independent biomineralization, but sharing a fundamental origin. In particular, the contrast between intracellular and extracellular spicule secretion appears now to be less significant. Recent discoveries of collagen (Ehrlich et al. 2006; Ehrlich and Worch 2007b) and evolutionary more ancient chitin (Ehrlich and Worch 2007a; Ehrlich et al. 2007a, 2008) as organic templates involved in sponge biosilicification allow us to hypothesize about their possible role as universal templates for biomineralization in nature. For example, collagen also serves as a template for calcium phosphate and carbonate deposition in bone, suggesting that the evolution of silica and bone skeletons shares elements of a common origin (Ehrlich and Worch 2007b), although the structures themselves need not be homologous. It has been suggested that structured polysaccharide moieties of glycoproteins (Albeck et al. 1996) and chitin itself (Weiss and Schönitzer 2006) are important in controlling aspects of mineral crystal growth in vivo. Thus, we showed for the first time that marine sponges produce a unique biocomposite (Fig. 5.8) wherein chitin templates the formation of amorphous silica
Fig. 5.8 Proposed model of the silica-chitin-aragonite composite (image courtesy Denis Kurek)
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with embedded aragonite crystallites (Ehrlich et al. 2010). This discovery represents a completely novel material. The results indicate that the biochemical processes leading to opal and carbonate mineralization are both deeply embedded in sponge biology and flexible on a cellular scale.
5.2 Radula as Example of Multiphase Biomineralization The radula, one of the characteristic features of molluscan classes (excluding bivalves), is a feeding organ usually consisting of a membranous ribbon to which are attached a series of bilaterally symmetrical-transverse rows of teeth, which are morphologically distinctive for any given species (Rinkevich 1993). Each individual denticle along a radula is recurved and has a multicuspid or narrow-bladed distal tip. The radula lies in the floor of the buccal cavity over a tongue-like projection, the odontophore, and is continuously secreted within a ventral special organ called the radular gland, which opens into the buccal cavity. Three distinct regions in the gland’s epithelium secrete the teeth and the radular membrane, a tough leathery substance on which teeth bases are embedded. When first secreted, the radula consists of chitin and proteins rich in tyrosine which become cross-linked, possibly by a quinine-tanning process (Runham 1961). Subsequently, toward its anterior end, the radula’s teeth cusps and bases become impregnated with inorganic salts (Fe, Si, P, Ca) which precipitate inside the framework of the organic matrix and make them exceptionally hard (Kim et al. 1989; Lowenstam 1962; Mann et al. 1986; Runham 1960, 1961; Runham and Thornton 1967; Runham et al. 1969; Towe and Lowenstam 1967). In Patella vulgata large amounts of iron (1.4−3% dry wt. Fe2 O3 ) and silica (8−7% dry wt. SiO2 ) are found (Jones et al. 1935). It was also reported (Rinkevich 1986) that germanium—a competitive inhibitor of silicon transport— was employed for elucidating pathways of radula silicification in owl limpet Lottia gigantea (Gastropoda: Acmaeidae). The reported results obtained with this mollusc indicate a complex pattern of iron biomineralization and the likely diverse nature of tooth biomineralization by various additional minerals, which hints at an overall heterogeneous system involved in the hardening of the limpet radulae. Calcium, for example, clearly begins to be incorporated at the very early stages of teeth formation (even within the first five rows, mainly within the tooth bases and radular membrane). Iron in mollusc cells is stored in siderosomes consisting of aggregates of ferritin or hemosiderin-like proteins that have a mineralized core with composition and structure similar to ferrihydrite (Fe5 O7 (OH)·4H2 O) and of variable crystallinity (St Pierre et al. 1989). The siderosomes observed in the cells surrounding limpet teeth had mineral cores of about 8 nm in diameter (Sone et al. 2007), similar to what has been observed in ferritin extracted from the hemolymph of a chiton species (St Pierre et al. 1989). Iron first begins to appear within the tooth cusps at the 15th row and significantly accumulates inside the following three rows within the internal domain of the scythe-blade-like structure of the cusp. Iron is incorporated into the outer domain
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of the tooth cusp only from the 25th to the 27th rows onward. Mineralization in this region of the radula is such a fast process that, by ~about row 30, the upper part of the tooth is heavily mineralized by iron. Major silica mineralization, as in other limpet radulae (Runham et al. 1969), occurs at the later stages of tooth maturation (Rinkevich 1986). From the “soft milieu” perspective of biomineralization, the cusp’s organic matrix, which is clearly demarcated from the superior epithelium cells, has a characteristic fibrous framework, divided into two main domains. The iron crystal formation probably coincides with this regularly spaced organic matrix (Mann et al. 1986). In L. gigantea radula, iron is distinguished in the superior epithelium cells and is superimposed on the bowed cusps a few rows before it infiltrates into the tooth cusps. The diversity of amorphous and crystalline mineral phases within radula of different mollusc species is relatively broad. Thus, Jones et al. (1935) summarized as follows: the radular teeth of the Patellidae consist largely of silica and iron, the teeth of the Chitonidae contain large amounts of iron and little or no silica, and the teeth of Dentalium contain small amounts of silica but no iron. Additionally, there are species with radula teeth where neither iron nor silica have been found. The role of silica in two-stage-based biomineralization of the radula was recently discussed by Hua and Li (2007) in their studies on limpet Notoacmea schrenckii (Gastropoda: Acmaeidae). Since silica in the base does not form an ordered crystalline structure but is composed of a less-ordered structure of “scrolls” (Mann et al. 1986), it is possible that the base may serve as either a temporary silica reservoir during the tooth growing process or as a gateway through which silica can be transported to the cusp. Silica particles penetrated with organic filaments observed in the cusp of Notoacmea schrenckii imply the possibility that the organic matrix located in a high silica content environment either may serve as a nucleus or may possess the catalytic ability to induce silica deposition. The intimate association of nanometersized silica particles with the organic matrix revealed in this study suggests that the organic matrix might not only serve as spatial constraints, but may play more active roles in catalyzing the aggregation and deposition of minerals. The decrease in the silica concentration in radulae during the transition from stage I to stage II suggests that the amorphous opal deposits along the organic filaments form the particles observed in at this point of stage II. Silica within these particles may be re-deposited in the cusp to fill in the spaces between goethite crystals and further strengthen the tooth structure. Thanks largely to the work of Spek (1921), Sollas (1907), Jones, and others (1935), and later of Rudall (1955), there is little doubt about the real nature of organic matrix within radula. This matrix can include chitin, proteins, tyrosine, tryptophan, or even mucopolysaccharides (Gabe and Prenant 1951). However, it is the chitin fibers of the organic matrix that are believed to control the orientations of goethite crystals in the limpet tooth, according to Mann et al. (1986). When this work was critically reviewed by Vincent (1980), the radula when first secreted was found to consist of chitin and protein, possibly in the form of a glycoprotein. The acetamide and hydroxyl groups of the chitin are at least in part available for reaction, while the protein gives weak reactions for amino groups and
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tyrosine and very strong reactions for tryptophan. Subsequently, the radula becomes altered chemically in various ways. The end result of these changes is a radula consisting of exceptionally hard radular teeth mounted on hard but brittle bases and these are embedded in a tough, leathery, radular membrane. It can therefore be concluded that the hardness of the radular tooth is not due to the inclusion of minerals, but to the matrix. The anterior part of the tooth may well have a sandpaper-like abrading surface caused by the softer matrix wearing away and leaving the hard silica particles exposed (Vincent 1980). It was suggested that by controlling nucleation and/or growth of inorganic crystals, the organic matrix can determine the size, shape, orientation, and polymorph of biominerals, resulting in composite structures that often have remarkable properties (Addadi and Weiner 1992). However, the precise mechanisms of this control remain relatively poorly understood, in large part due to the difficulty of characterizing the different stages of what is a dynamic process, especially in the case of radula teeth (Sone et al. 2007). There are currently many open questions regarding the nanodistribution of biomineralized compounds in the radular teeth of different molluscs (Brooker and Macey 2001; Farina et al. 1994) including multi-front mineralization (Brooker et al. 2003) as well as characterization and detailed structural organization of the organic matrix (Evans et al. 1990). In the invertebrates, quinone tanning is a common method of hardening (Brown 1950; Brunet and Kent 1955; Vincent and Hillerton 1979). It is therefore of great interest to elucidate the role of quinone tanning, especially in case of chitin, as a possible scaffold or template for heterogenous biomineralization (Rinkevich 1993) with respect to amorphous phases of silica and iron as well as to crystalline phase of iron (like goethite).
5.3 Silica–Chitin–Apatite Biocomposites of Brachiopoda Brachiopods are a phylum of shell-forming sessile marine invertebrates which have existed since the early Cambrian (Williams 1968; Williams et al. 2000). These animals have shown a broad diversity of mineral phases including silica, calcium carbonates, calcium phosphates, and fluorapatite carbonates, as well as different biomaterial strategies for their shell organization (Cusack et al. 2008; Lüter 2004; Williams 2003; Williams and Cusack 1999; Williams et al. 1992, 1994, 1997, 1998a, b, 2001). Within Brachiopoda, Discinidae is the only family where siliceous tablets (Fig. 5.9) are observed, with evidence from the recent discinid species Discinisca lamellosa from Chile (Holmer 1989), the deep-sea inhabiting Pelagodiscus atlanticus (Balinski and Holmer 1999; Williams et al. 1998a, b) and Discinisca tenuis from Namibia (Lüter 2001). The larvae of living discinids are evidently encased in a shell composed of discrete tablets of silica in protein, no more than 100 nm thick and cemented together into valvular mosaics by the outermost films of apatite and glucosaminoglycans of the primary layers. Growth of such larval valves ceases with the secretion of a periostracum (chitin–proteinaceous coat), which serves as a substrate
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Fig. 5.9 SEM image of the siliceous tablets on the surface of D. tenuis (image courtesy Maggie Cusack and Peter Chung)
for postlarval apatitic successions. The association of silica and apatite in discinid larval shells is noteworthy because the silanol (SiOH) groups of the silica gel have a catalytic effect on the precipitation of apatite. The siliceous tablets may likewise induce secretion of the apatitic shell of discinid larvae before the formation of the periostracum on which the mature apatitic shell is seeded (Lüter 2004). A possible explanation for the paillette cover of the shells of pelagic brachiopod juveniles (resembling thousands of small reflecting disks, like sequins) is that the tablets and tablet-like structures may have served as a protection against solar radiation when the ozone layer was more rarefied than today. While swimming in near-surface water, the juvenile brachiopods would have been exposed to UV radiation (Lüter 2004). Siliceous tablets and tablet-like structures, therefore, may have served as protective reflectors. This hypothesis should be tested in future research, especially because similar siliceous structures are unknown from comparable pelagic juveniles of the recent linguloid brachiopods Lingula and Glottidia, which are closely related to Discinisca and Pelagodiscus. The fossil record of Linguloidea also dates back to the Lower Cambrian (Williams 1968). Thus, similar developmental stages of species of both superfamilies Linguloidea and Discinoidea may have inhabited the surface waters of Palaeozoic oceans and hence were equally exposed to UV radiation. I want to note that brachiopods also possess another kind of multiphase biomineralization where β-chitin and both amorphous and crystalline calcium carbonate and calcium phosphate mineral phases are present (Goetz et al. 2009; Griesshaber et al. 2005, 2007; Merkel et al. 2007; Schmahl et al. 2004a, b, 2008). The micromechanical properties and ultrastructure of the shells of the modern brachiopod species Lingula anatina, Discinisca laevis, and Discradisca stella were recently investigated in a report by Schmahl et al. (2008). The shells are composed of two distinct layers, an outer primary layer and an inner secondary layer. Except for the primary layer in L. anatina, which is composed entirely of organic matter, all other shell layers are laminated organic/inorganic composites. The organic matter
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Copepoda Teeth as a Multiphase Biocomposite
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is built of chitin fibers, which provide the matrix for the incorporation of calcium phosphate. Amorphous calcium phosphate in the outer, primary layer and crystalline apatite is deposited into the inner, secondary layer of the shell. There is great variation in hardness values between shell layers and between the investigated brachiopod species. The microhardness of the investigated shells is significantly lower than that of inorganic hydroxyapatite. This is caused by the predominantly organic material component which, in these shells, is developed either as purely organic layers or as an organic fibrous matrix reinforced by crystallites. Reported results show that this particular fiber composite material is very efficient for the protection and support of the soft animal tissue. It lowers the probability of crack formation and effectively impedes crack propagation perpendicular to the shell by crack-deviation mechanisms. The high degree of mechanical stability and toughness is achieved by two design features. First, there is the fiber composite material. This material overcomes some detrimental properties, and enhances some advantageous properties, of the single constituents, combining the softness and flexibility of chitin and the hardness and brittleness of apatite. Second, there is a hierarchical structuring from the nanometer to a micrometer level. At least seven levels of hierarchy within the shells of investigated brachiopods were identified (Griesshaber et al. 2007). Thus, the uniqueness of multiphase-based biocomposites in brachiopods, regardless whether it is chitin, proteins, calcite-, silica- or phosphate-dominated, is due to its hybrid composite nature and its complex hierarchical organization, where every structural level contributes to the function of the resulting material design (Currey 2005; Pérez-Huerta et al. 2007; Schmahl et al. 2008).
5.4 Copepoda Teeth as a Multiphase Biocomposite Possession of glass teeth for eating food in glass cases is surely one of the lyrical symmetries of nature (Sullivan et al. 1975).
Copepods are crustaceans. They are found almost everywhere where water is available and they constitute the biggest source of protein in the oceans. Calanoid copepods are typical particle feeders. Their mouth appendages produce a water current that flows from anterior to posterior. As soon as food receptors detect the approach of a suitable diatom cell, the maxillae are opened. Water within the cell is sucked into the chamber between the maxillae. When the chamber is closed, the water is pressed out again, and the diatom cell is trapped between the bristles of the maxillae. In this chamber particles of 5 μm diameter can be retained. The very hard diatomous shells (Hamm et al. 2003) are, however, broken up using unique mandibular blades which possess teeth. Giesbrecht (1892) discussed the specific distinctness of the mandibular blade of copepods for a wide spectrum of species. The individual teeth have been shown by Beklemishev (1954, 1959) to be mineral elements set in sockets in the surface of the exoskeleton. He demonstrated that the
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teeth are not dissolved by concentrated sulfuric acid, which dissolves the chitinous mandible blade. Beklemishev also showed that the teeth are not birefringent in polarized light. The only reasonable substance which would give these results is opaline silica. Beklemishev’s discovery of siliceous tooth crowns in calanoid copepods was confirmed for most of the species examined. Long, sharp projections on the crowns of herbivorous species, and the deep grooves into which they fit on the teeth of the opposite mandible, suggest a cracking rather than a grinding function for these teeth (Miller et al. 1990). Today, it is well established that Calanoid copepods bear cuspate, opal teeth (Fig. 5.10) on the chewing surfaces of the mandibular gnathobases or jaws. Sullivan et al. (1975) suggested that the hardness of these teeth evolved in parallel with the opal-covered food particles such as diatoms. They described the external appearance of the teeth in several species. Miller et al. (1980) showed that Acartia tonsa obtains silica for teeth directly from the water; a dietary source is not required. Siliceous teeth formed by A. tonsa, when placed in a media of low silicic acid concentration, are weak and crumbly.
Fig. 5.10 Light microscopy imagers clearly show the tooth-like structures that line the edge of the mandibular blade of Neocalanus cristatus (samples courtesy Natalya Dolganova and Marina Yurieva). The same mandible may bear teeth that are structurally complex modifications of the chitinous surface, and others that are relatively simple projections of chitin. Some of the teeth are crowns set in sockets in the chitinous surface (image courtesy Sebastian Hunoldt)
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It was reported (Miller et al. 1990) that silica deposition begins at the distal periphery of the mold and proceeds inward toward the base and center, until the entire mold is filled with amorphous opal that obscures the fibrous inclusions. In a mature tooth, the opal is tightly attached to the underlying chitin. Miller et al. (1990) showed, using X-ray fluorescence analysis, the following mineral content of the Calanus pacificus copepod teeth: silicon, 91.09%; copper, 4.18%; zinc, 4.83%. Their finding of small but significant amounts of copper and zinc in tooth material suggests that these minerals may play a role as cofactors in deposition. Zinc, in particular, forms the highly insoluble orthosilicate mineral willemite (solubility coefficient, [Zn]2 [H4 SiO4 ]/[H+ ]4 = 10–13; free energy of formation = −364 kcal/mol; Hem 1972) and the implied strong affinity for silicic acid may be part of the deposition mechanism. Willemite, per se, is not an important component because there is neither enough zinc nor any sign of crystalline structure. Grime et al. (1985) suggested that copper-containing oxidases may be involved in polymerization of aromatic amino acids into polyaromatic structures in the formation of siliceous deposits in the radular teeth of limpets. The role of oxidases in sclerotization of molluscan radular tissues prior to silicification has been reviewed by Kerth (1983). This, in conjunction with the statement of Mann et al. (1986) that silicic acid binds strongly to catechol-type molecules (usually derivatives of tyrosine), may explain the observation of copper in copepod silica. In copepods, the opal structure of the tooth and its attachment to the exoskeleton are formed by the coordinated, sequential activity of a number of functionally differentiated epithelial cells and a remote gland attached by a duct. The sequence of events in vertebrate tooth formation is comparably complex, but even in this case, no remote glandular source of material for mineralization is involved. Occurrence of such a complex process in copepods alone among the Crustacea speaks to the length of their separate evolution (Miller et al. 1990). It seems that except for copepods, other crustaceans could also possess multiphase-based skeletal structures. For example, it was reported (Becker et al. 2005) that the cuticles of Porcellio scaber and Armadillidium vulgare consist of magnesium calcite, amorphous calcium carbonate, and amorphous calcium phosphate.
5.5 Conclusion We hypothesized previously (Ehrlich et al. 2007a, b) that chitin molecules are perhaps part of a very old organic template system involved in a biosilicification phenomenon, which was established a long time before the origin of sponges, and which has the potential to evolve biosilicification iteratively. Together with other known biomineralization processes, the nature of the biocomposites discussed here also raises the possibility that chitin may be an almost universal template in biomineralization. I anticipate that this provocative suggestion will stimulate a great deal of future research.
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The discovery of nanostructured silica–chitin–aragonite biocomposites as structural scaffolds of Verongida sponge skeletons, as well as examples of similar complex biocomposites in molluscs, brachiopods, and crustaceans, suggests that multiphase mineralization is a common phenomenon in nature and opens many interesting questions relating to biomineralization in general. From this point of view, the question of the origin and biological reason for the coexistence of amorphous and crystalline mineral phases (Aizenberg et al. 2002, 2003) within the same skeletal structure which, moreover, contains a common organic template, seems to currently be a subject of much interest. Most significantly, the phenomenon of multiphase biomineralization offers the possibility of a unifying mechanism for biomineralization among Metazoa and related groups. The underlying mechanisms of biomineralization may be both unexpectedly complex and surprisingly consistent across phyla.
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Part III
Biomineralized Structures and Biocomposites
Chapter 6
Hierarchical Biological Materials
One of the main driving forces in studying biological materials from the viewpoint of Materials Science is to use the discovered natural structures and processes as inspiration for developing new materials. Peter Fratzl
Abstract Today bioinspiration is the first step on the long and hard way to the development of man-made materials and constructs. Marine invertebrates are the inexhaustible source for such inspiration. Cellular materials are multiphase composite material systems that consist of a solid matrix and a fluid phase, the fluid usually being a gas. Morphologically, these cellular solids are classified into 2D solids, such as honeycomb structures containing hexagonal cells, and 3D foams, such as sponges. Different kinds of rigid and flexible cellular materials in such diverse marine systems as radiolarians, diatoms, molluscs, corals, and sponges have been founded. Structurally, their high strength and stiffness, combined with low weight, make cellular materials attractive subjects for mimicking in artificial materials. Hierarchical cellular designs are complex microstructures achieved by placing material where most needed, i.e., in areas of high stress. Siliceous honeycomb cellular structures in diatoms are discussed. Nature develops biological objects by means of growth or biologically controlled self-assembly, adapting to the environmental conditions and using the most commonly found materials. Biological materials are developed by using recipes contained in the genetic code. As a result, biological materials and tissues are created by hierarchical structuring at all levels. In order to adapt form and structure to the desired function, there must be the capability of adaptation to changing conditions and self-healing (Fratzl and Weinkamer 2007; Nosonovsky and Bhushan 2008). The genetic algorithm interacts with the environmental conditions, which provides flexibility. For example, a tree branch can grow in two different ways; in the direction of the wind and opposite to it. The only way to provide this adaptive self-assembly is a hierarchical self-organization of the material. Hierarchical structuring allows adaptation and optimization of the material at each level.
H. Ehrlich, Biological Materials of Marine Origin, Biologically-Inspired Systems 1, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9130-7_6,
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It is apparent that nature uses hierarchical structures, consisting of nanostructures in many cases, to achieve the required performance (Nosonovsky and Bhushan 2008). Understanding the role of hierarchical structure and development of low cost and flexible fabrication techniques would facilitate commercial applications (Christian 2009). Biologically inspired design, adaptation, or derivation from nature is referred to as “biomimetics.” It means mimicking biology or nature. Biomimetics is derived from the Greek word “biomimesis.” The word was coined by polymath Otto Schmitt in 1957, who, in his doctoral research, developed a physical device that mimicked the electrical action of a nerve. Other words used include bionics (coined in 1960 by Jack Steele of Wright-Patterson Air Force Base in Dayton, OH), biomimicry, and biognosis (see for review, Bhushan 2009). The field of biomimetics is highly interdisciplinary. It involves the understanding of biological functions, structures, and principles of various objects found in nature by biologists, physicists, chemists, and material scientists; and the design and fabrication of various materials and devices of commercial interest by engineers, material scientists, chemists, and others. However, I agree with opinion made by George Mayer in his excellent review published in Science (Mayer 2005): “Generally, what has been copied from nature for building synthetic structural composites has been the architectural configurations and the material characteristics rather than the specific natural materials that were originally found. This approach has limitations. A complicating and difficult issue has been the enormous potential problem of copying architectural features that are found in nature, at the micro- and nanoscales, into real, macroscale structural materials at reasonable cost.” The evolution of biological structures can be a complicated combination of historical accidents, developmental constraints, and environmental pressures, and thus may not necessarily represent the simplest or optimal approach to performing a particular function. Nevertheless, the diversity of structures observed may lead to new ideas or clues (Poladian et al. 2009). Thus, today bioinspiration is the first step on the long and hard way to the development of man-made materials and constructs. I believe that marine invertebrates are the inexhaustible source for such inspiration.
6.1 Cellular Structures Increased understanding of natural or biological materials shows that nature has developed ways of achieving high structural efficiency from a rather limited set of non-sophisticated constituents. Among nature’s most common efficient structures are hierarchical cellular sandwich structures. These consist of a complex arrangement of cells of different sizes arranged across the section such that the cells are themselves made from cells (i.e., hierarchical arrangement). This arrangement leads to dense regions, or skins, integrally connected to regions with lower density, or core (Burgueño et al. 2005). Not only are the structures, morphologies, and the marked anisotropies of the cellular materials different from those of conventional
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composites but also are their deformation and failure mechanisms quite unusual. These features additionally include pore size, distribution, surface properties, and interconnectivity. Some features of the structures and morphologies of natural cellular materials have to do with supply channels for ion transfer, nutrition, and growth. This is true as well for their mechanical properties, which often determine the survival of the organism, especially in specific marine environments. Therefore it is not surprising to find different kinds of rigid and flexible cellular materials in such diverse marine systems as radiolarians, diatoms, molluscs, corals, and sponges. Structurally, their high strength and stiffness, combined with low weight, make cellular materials attractive subjects for mimicking in artificial materials (Mayer and Sarikaya 2002). Hierarchical cellular designs are complex microstructures achieved by placing material where most needed, i.e., in areas of high stress. The process of arranging the microstructure (cellular material) can lead to a hierarchy that maximizes the efficiency of the resulting material or load-bearing component (Burgueño et al. 2005). Cellular materials are multiphase composite material systems that consist of a solid matrix and a fluid phase, the fluid usually being a gas. Morphologically, these cellular solids are classified into 2D solids, such as honeycomb structures containing hexagonal cells, and 3D foams, such as sponges (Huang et al. 2002). Honeycomb structures are widely used in structural applications owing to their specific strength. In particular, honeycomb cell structures are very prevalent. The continuing desire for stronger, lighter weight, structural materials for use in aerospace and aircraft applications has made these industries the traditional leaders in the development of honeycomb structures for technological use. However, improved manufacturing processes have made these unique composite materials more affordable and viable for other industries.
6.2 Honeycomb Structures: From Nano- to Macroscale The diversity of honeycomb structures in nature is amazing. The development of light microscopy allowed observation on the microscale and the making of numerous drawings with excellent precision. For example, radiolarians have been so represented in the fundamental work by Haeckel in the nineteenth century (Fig. 6.1). Sir D’Arcy Thompson (1942) made a thorough analysis of the biological structures on these images using mathematical knowledge from 1917 which was correspondingly based on basic works from Plateau, Aristoteles, Euler, and Kepler. Cellular materials including honeycombs have been extensively utilized in a variety of engineering applications of light-weight construction, thermal insulation, and energy absorption during the last hundred years. In contrast to majority of bulk structural materials, which fail mainly under tensile loading, the most dangerous loading for the cellular materials is compression. Therefore in many design applications, they are used under tensile loading. If the parent material is brittle (e.g., ceramics), the dominant failure mode in this condition is brittle fracture caused by a propagating crack. This phenomenon was extensively studied by many authors
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Fig. 6.1 Diversity of radiolarian microarchitecture: (a) Reticulum plasmatique. After Carnoy. (b) A nassellarian skeleton, Callimitra agnesae (0.15 mm diameter). (c) Aulonia hexagona. (d, e). Schematic view and skeleton of Prismatium tripodium, respectively. (f) Skeletons of various radiolarians, after Haeckel (adapted from Thompson 1942)
who addressed the typical problem of evaluating the fracture toughness of cellular material in terms of the rupture stress σ fs of the bulk material. Most of these studies were based on the assumption that cellular material can be adequately modeled by a periodic lattice composed of rigidly connected Euler beams (Lipperman et al. 2007). But, Euler’s mathematics has been used for hundreds of years for analysis of naturally occurring cellular materials (D’Arcy Thompson 1942). This is not surprising, because rules in geometry are similar for both nature- and man-made structures independent of their size and dimension (Ashby et al. 1995) (Fig. 6.2). What is surprising is that nature produces such complex architectural constructs without tools and computers used by teams of engineers and managers. However, progress in engineering science, while in part derived from the pure scientific interest on unique naturally occurring formations, was largely determined by the necessity to use honeycomb structural material for broad field of practical application, and this interest constituted the driving source in analytical studies on these constructs. Thus, it is becoming even more important to develop methods for evaluating the elastic
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Honeycomb Structures: From Nano- to Macroscale
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Fig. 6.2 The stress–strain curve of the solid cell wall material in the honeycombs
properties of various honeycomb materials (Fortes and Ashby 1999; Masters and Evans 1996; Warren and Kraynik 1987). Many studies have been performed on this topic in the last few decades (see for review Chen and Ozaki 2009), including a systematic survey of the various analytical methods by Gibson and Ashby (1988). Gibson and Ashby (1988) published analytical formulations for the in-plane and out-of-plane stiffnesses, as well as the upper and lower limits of the transverse shear moduli, for a regular hexagonal honeycomb. The mechanical properties of cellular materials can be analyzed from a cell-edge bending model, which was also proposed by Gibson and Ashby (1997). The properties are found to be dependent on their relative density, cell geometry, and the material properties of the solid from which they are made. In their modeling, some important assumptions are made: the shape and the size of cells are regular and repeated, the cross section of cell edges is uniform, and the profile of cell edges is a straight two-dimensional hexagonal honeycomb (Swellam et al. 1997). Gibson and Ashby (1988) presented a solution for the single hexagonal cell, which they considered as the repeated unit cell (see Figs. 6.3 and 6.4). Invoking the equilibrium conditions, load deformation relations, and the definition of the stiffness for the loadings in the X- and Y-directions, Gibson and Ashby (1988) obtained the following: t 3 Ey Ex = = 2.3 Es Es l t 3 Ex cos (θ ) = h Es l + sin (θ ) sin2 (θ ) l h t 3 l + sin (θ ) Ey = Es l cos3 (θ )
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cos2 (θ ) h + sin (θ ) sin (θ ) l h + sin (θ ) sin (θ ) l μyx = cos2 (θ ) h + sin (θ ) t 3 Gxy l = 2 Es l h 2h 1+ cos3 (θ ) l l
μxy =
Fig. 6.3 Geometry of honeycomb
Fig. 6.4 Finite element model
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Honeycomb Structures: From Nano- to Macroscale
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where t is the strut depth, l is the length of the inclined strut, h is the height of vertical strut, q is the strut inclination with the horizontal, Es is the Young’s modulus for the strut material, Ex is the Young’s modulus for the foam material in the X-direction, Ey is the Young’s modulus for the foam material in the Y-direction, Gxy is the shear modulus for the foam material in the X–Y plane, μxy is the Poisson’s ratio for loading in the X-direction, and μyx is the Poisson’s ratio for loading in the Y-direction. It should be noted that Gibson and Ashby’s model is limited to regular and irregular but geometrically similar hexagonal cells and for the regular hexagonal cells (l = h) (Swellam et al. 1997). Two-dimensional honeycombs with regular hexagonal cells exhibit a Poisson’s ratio of +1 in the honeycomb plane; the out-of-plane properties differ due to anisotropy. The cell walls have 120◦ angles between walls and all walls must be of equal thickness and composition. As noted by Gibson and Ashby (1997), the mechanism which dominates the linear elastic deformation of honeycombs is that of bending of the cell walls. The approach to determining the mechanical behavior for the overall honeycomb structure begins with the identification of the unit cell structure (the smallest repeatable and oriented structural unit). Most silica-based biological materials (e.g., skeletal formations of radiolarians, diatoms, and sponges) can be related to the brittle honeycombs. The fracture toughness of brittle hexagonal honeycombs has been modeled by relating the crack tip elastic fields of an equivalent continuum to the stress state within the lattice (Quintana Alonso and Fleck 2009). It was assumed that the macroscopic fracture toughness is set by local tensile failure when the maximum stress in any strut of the lattice attains the fracture strength Gf of the cell wall material. It is shown that the fracture toughness of the hexagonal honeycomb scales linearly with Gf , quadratically with relative density, and with the square root of cell size (as demanded by dimensional analysis) (Quintana Alonso and Fleck 2009). Of course, different models for evaluating material properties of honeycomb cellular materials are of huge significance, but those of marine origin are made of biocomposites. Similar to real cellular materials, some microstructural imperfections (e.g., non-periodic microstructure, non-uniform cell-edge cross section, and curved cell edges), which, however, pre-exist due to the manufacturing process, are present in biological materials too. Hence, it can be expected that the mechanical properties of cellular materials are affected by the pre-existing microstructural, as well as nanostructural, imperfections (Yang et al. 2008). In the case of cellular materials of marine invertebrate origin, similar imperfections can be determined due to mutagenesis, chemical pollution, as well as climate changes. Unfortunately, there is a lack of information regarding the features of the regulation for remodeling or remineralization processes used by organisms in the cases when honeycomb cellular structure is damaged. Because of the very detailed analysis of honeycomb cellular structures of radiolarians made by D’Arcy Thompson (1942), I have decided to write a brief analytical essay on these structures observed by different teams in diatoms, one of the most investigated objects in biomimetically oriented nanobionics and nanobiotechnology (Gordon 2009).
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6.3 Siliceous Honeycomb Cellular Structures in Diatoms One of the primary ways in which nature achieves high structural performance with silica-based materials is through hybrid material combinations assembled in optimized hierarchical strategies on the nano- and microscale, and such is the case of diatoms. For example, Triceratium favum (Fig. 6.5) possesses unique hierarchical designs typical of cellular materials. The design of cellular materials represents complex microstructures achieved by placing material where most needed, i.e., in areas of high stress. The process of arranging the microstructure (cellular material) can lead to a hierarchy that maximizes the efficiency of the resulting material or the load-bearing component (Burgueño et al. 2005). Honeycomb structures are widely used in structural applications owing to their specific strength. Silica-based honeycomb cell structures are also very prevalent both in nature (radiolarian, diatoms, and glass sponges) (D’Arcy Thompson 1961) and in man-made materials. For example, ceramic honeycombs are used in catalytic converters and diesel particulate filters for automobiles, in filters for continuous casting plants, in plates for gas burners, and in medical prosthetic implants. Glass honeycombs have been used as lightweight supports for space mirrors as, for example, in the Hubble telescope. In most of these applications, the ceramic lattices are loaded in a sandwich panel configuration with stiff and strong face sheets (Quintana Alonso and Fleck 2009). We suggest that structural feature in diatoms like Triceratium, which was established and optimized during the long process of evolution, possesses several advantageous material properties, such as selection and sieving of nanoparticles as well as photonic and photosynthesis.
Fig. 6.5 Triangular nanoorganized honeycomb cellular structures in the shape of T. favum (image courtesy: Christina Brodie)
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It is well known that in integrating sandwich structures into a structural unity, honeycomb materials assure maximum elastic-strength characteristics while having minimum weight (Gibson et al. 1982; Gibson and Ashby 1988). First of all, the honeycomb construction allows for a more economic use of silica, especially where low levels of dissolved silica are present. It has also been demonstrated that the silica content of diatom cells differs significantly between marine and freshwater species and when the salinity content of water changes (Vrieling et al. 2007). Thus the characteristic honeycomb structures of diatom Triceratium punctigera were much more pronounced at lower salinity than at higher salinity. In both T. punctigera and Triceratium weissflogii, the silica appeared to be denser at the lower salinity, and smaller discernable silica particles were present at the surface of the silica, while for T. weissflogii the surface roughness clearly increased at lower salinity (Vrieling et al. 2007). It is known from classical silica chemistry (Iler 1979) that smaller silica particles aggregate more tightly at a lower salinity, while voids between the coalesced particles disappear. Silicon is also relatively dense, so the structure promotes lightness. Seawater is undersaturated in silica, yet diatoms prevent the dissolution of their silica with an organic layer in the outer frustule. From this point of view, the relationship between siliceous honeycomb structure and organic layer which covers this construct is of fundamental importance. We believe that structural integrity, especially of the silica-based honeycomb construct, determines the survival of diatom under natural conditions. Characteristic perforations in the frustule endow the diatom with considerable compressive strength, which explains the frustules’ ability to survive undamaged under different mechanical challenges, such as abrasive particles and solid sediments or changes in osmolarity, which could lead to swelling or bursting of unprotected cells (Losic et al. 2009). Hamm et al. (2003) studied the potential of the frustules to function as an armor conveying protection against predators by measuring the force necessary to break a single cell. An inverse relationship between frustule size and mechanical strength was observed. Mechanical stresses ranging from 150 to 680 N mm−1 were tolerated depending on the region of the frustule. The variation in mechanical strength is explained by the frustule architecture, particularly by the presence of ribs and pores that disperse mechanical stresses (Hamm et al. 2003). In diatoms, in which honeycomb structural motif is present, the apertures in the frustule collectively take on the role of a sieve, a two-way filtration mechanism across which water and nutrient molecules permeate the cell. The holes can range from several micrometers down to 100 nm in diameter. Structural features of the honeycomb-determined apertures appear to give diatoms microfluidic sorting capabilities for particles at surfaces beyond other capabilities of organisms living on the microscale. The minimum size of the molecules and particles influenced, and the tens of nanometers over which their paths are altered, takes the diatom surface well into the realm of nanofluidics (Losic et al. 2009). For diatoms, sorting combines three processes: control, separation, and sensing. Unlike other organisms with flexible exteriors or extensions that may perform these functions at hundreds of micrometers, the rigid silica exterior of diatoms appears to force the performance
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of these functions to rely on sub-micrometer mechanisms. For control, the exterior diatom microtopography influences particle behavior a few micrometers above a diatom surface, localizing colloidal particles and macromolecules over hexagonally arrayed ridges (Losic et al. 2009). The advantages of honeycomb cellular microstructures in man-made materials with respect to photonics are well known. For example, Saitoa and Kosuge (2007) reported that the quantum efficiency of the honeycomb-textured siliceous solar cells is larger than that of untextured solar cells in the measured wavelength range. They considered the micro-roughness of the etched surfaces as a cause of the wavelength dependence of the absorbed light. Only the light rays with short wavelength, which are hardly diffracted, can enter the fine and deep depressions. If the light is trapped in the deep depressions, the light will be reflected many times. Diatoms are recently described as living, self-reproducing photonic crystals because of the periodic pattern of their cell walls (Fuhrmann et al. 2004). The principal difference between terrestrial biophotonic structures (e.g., chitinous wings of butterfly) and those observed in diatoms is that diatoms are the organisms living in water. Due to this feature, the photonic activity of diatoms must be specialized with respect to stimulating photosynthesis under the limiting conditions. When the background is water, we cannot expect efficient Bragg scatterings because of the small contrast of refractive indices of silica (n =1.46) and water (n =1.33) (Yamanaka et al. 2008). However, such structures seem to have a physiological function in relation to photosynthesis because they evolved the structure for surviving through the course of evolution. According to Yamanaka and co-workers, the diatoms use both blue and red wavelength regions for photosynthesis. An excess supply of blue light, however, gives rise to active oxygen, which is harmful to organisms. Thus, one of the roles of the nanostructures inside the frustules may be the reduction of such light. Furthermore, the blue light may be scattered in the frustules to uniformly irradiate chlorophylls inside diatoms for the purpose of an efficient enhancement of photosynthesis. For example, when the intensity of incident blue light is strong, the harmful excess supply of the blue light is absorbed at the inner nanostructure, and the intensity of the blue light reaching the chlorophylls inside the diatoms is weakened. On the other hand, when the intensity of incident blue light is weak, the chlorophylls inside the diatoms approach the inner nanostructure to enhance an interaction of light for photosynthesis. Thus, the theoretical analyses of optical properties supported the experimental results and suggested a strong interaction between blue light and inner silica materials. It was speculated that the interaction served for partial reduction of the excess blue light irradiation and for enhancement of photosynthesis of the diatom (Yamanaka et al. 2008). Therefore, I assert here that diatomaceous honeycomb cellular structures are one of the definitively successful architectural and engineering decisions occurring in nature. Features in photosynthesis-based metabolism, ecology, and evolution have led to survival and determined the optimization of their structure−functional relationships.
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6.4 Conclusion Biomimetics deals with the application of nature-made “design solutions” to the realm of engineering. In this context, mimicking biological materials with finetuned mechanical properties has been on the agenda of engineering research and development for many years. The premise of biomimetics is that it is possible to reduce diversity and complexity of biological materials to a number of “universal” functioning principles. This requires foremost a deep understanding of the hierarchical structure of biological materials. It now appears that multiscale mechanics may hold the key to such an understanding of “building plans” inherent to entire classes of material.
References Ashby MF, Gibson LJ, Wegst U et al (1995) The mechanical-properties of natural materials. 1. Material property charts. Proc R Soc Lond Ser A Math Phys Sci 450:123–140 Bhushan B (2009) Biomimetics. Philos Trans R Soc A 367:1443–1444 Burgueño R, Quagliata MJ, Mohanty AK et al (2005) Hierarchical cellular designs for load-bearing biocomposite beams and plates. Mater Sci Eng A 390(1–2):178–187 Chen DH, Ozaki S (2009) Analysis of in-plane elastic modulus for a hexagonal honeycomb core: effect of core height and proposed analytical method. Compos Struct 88:17–25 Christian S (2009) Biocomposites for the construction industry. Ph.D. Dissertation, Stanford University, Stanford, CA, USA D’Arcy Thompson W (1942) On growth and form. Cambridge University Press, Cambridge D’Arcy Thompson W (1961) In: Bonner JT (ed) On growth and form, Abridged edn. Camridge University Press, Cambridge Fortes MA, Ashby MF (1999) The effect of non-uniformity on the in-plane modulus of honeycombs. Acta Mater 47:3469–3473 Fratzl P, Weinkamer R (2007) Nature’s hierarchical materials. Prog Mater Sci 52:1263–1334 Fuhrmann T, Landwehr S, El Rharbi-Kucki M et al (2004) Diatoms as living photonic crystals. Appl Phys B 78:257–263 Gibson LJ, Ashby MF (1988) Cellular solids: structure and properties, 1st ed. Pergamon, Oxford Gibson LJ, Ashby MF (1997) Cellular solids: structure and properties, 2nd ed. Cambridge University Press, Cambridge Gibson LJ, Ashby MF, Schajer GS et al (1982) The mechanics of two dimensional cellular solids. Proc R Soc Lond A382:25–42 Gordon R, Losic D, Tiffany MA et al (2009) The glass menagerie: diatoms for novel applications in nanotechnology. Trends Biotechnol 27(2):116–127 Hamm CE, Merkel R, Springer O et al (2003) Architecture and material properties of diatom shells provide effective mechanical protection. Nature 421:841–843 Huang FY, Yan BW, Yang DU (2002) The effects of material constants on the micropolar elastic honeycomb structure with negative Poisson’s ratio using the finite element method. Eng Comput 19:742–763 Iler R (1979) The chemistry of silica. Wiley, New York Lipperman F, Ryvkin M, Fuchs MB (2007) Fracture toughness of two-dimensional cellular material with periodic microstructure. Int J Fract 146:279–290 Losic D, Mitchell JG, Voelcker NH (2009) Diatomaceous lessons in nanotechnology and advanced materials. Adv Mater 21:2947–2958 Masters IG, Evans KE (1996) Models for the elastic deformation of honeycombs. Compos Struct 35:403–422
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Mayer G (2005) Rigid biological systems as models for synthetic composites. Science 310: 1144–1147 Mayer G, Sarikaya M (2002) Rigid biological composite materials: structural examples for biomimetic design. Exp Mech 42:395–403 Nosonovsky M, Bhushan B (2008) Multiscale dissipative mechanisms and hierarchical surfaces: friction, superhydrophobicity, and biomimetics. Springer-Verlag, Berlin, Heidelberg Poladian L, Wickham S, Lee K et al (2009) From photonic crystals and its suppression in butterfly scales. J R Soc Interf 6:S233–S242 Quintana Alonso I, Fleck (2009) The damage tolerance of a sandwich panel containing a cracked honeycomb core. Appl Mech 76:1-061003-8 Saitoa Y, Kosuge T (2007) Honeycomb-textured structures on crystalline silicon surfaces for solar cells by spontaneous dry etching with chlorine trifluoride gas. Solar Energy Mater Solar Cells 91:1800–1804 Swellam M, Yi S, Achmad F et al (1997) Mechanical properties of cellular materials. I. Linear analysis of hexagonal honeycombs. J Appl Polym Sci 63:383–393 Vrieling EG, Sun Q, Tian M et al (2007) Salinity-dependent diatom biosilicification implies an important role of external ionic strength. Proc Natl Acad Sci USA 104:10441–10446 Warren WE, Kraynik AM (1987) The linear elastic response of two-dimensional spatially periodic cellular materials. Mech Mater 6:27–37 Yamanaka S, Yano R, Usami H et al (2008) Optical properties of diatom silica frustule with special reference to blue light. J Appl Phys 103:074701 Yang MY, Huang JS, Hu JW (2008) Elastic buckling of hexagonal honeycombs with dual imperfections. Compos Struct 82:326–335
Chapter 7
Paleodictyon Honeycomb Structure
Abstract The trace fossil Paleodictyon, first described in 1850, consists of a network of hexagonal meshes. According to paleontologists, Paleodictyon belongs to the group of trace fossils called graphoglyptids, which are highly organized trace fossils normally found as casts on the lower surface of distal turbidites. Individual mesh elements of Paleodictyon vary in linear dimensions from millimeters to centimeters, whereas entire mesh patterns can cover areas up to a square meter. The edges or threads that make up the mesh are usually cylindrical or ellipsoid in cross section. Until the real creature is caught, scientists will continue the vigorous debate that began in nineteenth century. The main question is whether the hexagonal patterns are burrows or body parts, vacant residences or animal remains. It is hypothesized that Paleodictyon could be of the glass sponge origin. Our images are strong evidence that the walls of the honeycomb structural unit in these sponges are made of silica-based tubular formations because of the presence of the axial channels. It is believed that formation of such complex cellular structures in hexactinellids must be determined genetically and that these features were conserved during about last 700 Myr. While there exists a broad diversity of honeycomb cellular structures observed and described for unicellular marine organisms on the nano- and microlevels, there are some examples that can be related to the macro-objects. First, I mean the Paleodictyon-related “creatures,” and second, some representatives of the glass sponges (Hexactinellida). In contrast to the currently well-known glass sponges, Paleodictyons are very mysterious. So far it has not been possible to capture one of these deep-sea creatures alive. Its only visible feature consists of tiny holes arranged in six-sided pattern. Until the real creature is caught, scientists will continue the vigorous debate that began in 1850. The main question is whether the hexagonal patterns are burrows or body parts and vacant residences or animal remains. The trace fossil Paleodictyon, first described by Menghini (1850), consists of a network of hexagonal meshes. According to paleontologists, Paleodictyon belongs to the group of trace fossils called graphoglyptids (Fuchs 1895), which are highly organized trace fossils normally found as casts on the lower surface of distal turbidites (Fig. 7.1a). Paleodictyon is usually preserved in turbidite and pelagic rock
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Fig. 7.1 (a) Paleodictyon (Glenodictyon) hexagonum (adapted from Mark 1873). (b) Paleodictyon observed in the deep-sea in recent time (image from the IMAX film “Volcanoes of the Deep Sea,” courtesy Rutgers University and The Stephen Law Company)
layers in convex hyporelief (Garlick and Miller 1993). The known stratigraphic range is from the Early Cambrian through the Miocene and forms attributed to Paleodictyon have been observed in the modern deep Atlantic (Ekdale 1980; Rona et al. 2003; Rona 2004). The morphology of Paleodictyon and its ichnospecies was described in detail by Ksiazkiewicz (1970) and Seilacher (1977). The more familiar ichnospecies are remarkably regular hexagonal grids, such as the appropriately named P. regulare. The ichnogenus also includes less regular forms, such as P. petalodium, characterized by a looping, petal-like meshwork of curved lines connected at seemingly random points. Both irregular and regular nets are known throughout the stratigraphic range of the ichnotaxon (Uchman 2003). During the Palaeozoic, ichnospecies of Paleodictyon characterized by large nets and narrow edges dominated, while in the Mesozoic small- to medium-sized nets with wide edges were more prevalent (Uchman 2003). Individual mesh elements (Fig. 7.1) vary in linear dimensions from millimeters to centimeters, whereas entire mesh patterns can cover areas up to a square meter. The edges or threads that make up the mesh are usually cylindrical or ellipsoid in cross section (Ksiazkiewicz 1970). Seilacher (1977) proposed several possible functions for such a complex structure: as a trap for suspended food, a mechanism for bacterial farming, or a foraging path. Alternatively, Swinbanks (1982) suggested that it may be associated with xenophyophoran protists. Plotnick (2003) modeled the form as resulting from the iterative modular growth of an unknown organism. Recently, Honeycutt and Plotnick (2005) carried out thorough mathematical analysis of Paleodictyon. Graph theory and analysis of the geometry of the regular ichnospecies suggest that if the elements of Paleodictyon are interpreted as microtunnels, then they are of extraordinary length relative to the size of any likely solitary tracemaker. In addition, because each vertex of the mesh is of degree three, any
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possible path through mesh requires revisiting in order to travel through the entire network; this makes the minimum path length even longer. These results suggest that it is unlikely that Paleodictyon is the result of subsurface burrowing. It seems, however, that Paleodictyon is even today surviving. Thus, a field of small (diameter 2.4–7.5 cm) strikingly symmetric hexagonal patterns was imaged in 1976 by a camera towed over a sediment-covered area of the lower east wall of the Mid-Atlantic Ridge (water depth 3,415–3,585 m) in the course of an investigation of the axial valley for hydrothermal activity (Rona and Merrill 1978). The pattern imaged comprises three equidistant sets of rows of black dots in the sediment, inferred to be holes at that time, that intersect at angles of 120◦ . Each set of rows parallels two sides of the bounding equilateral triangle to produce an overall hexagonal outline encompassing a network of hexagonal cells. Thousands of the patterns, typically several per square meter, were imaged in an area 3 km along the ridge axis by 2 km across the axis (NW–SE) at water depths of 3,200–3,600 m. Although the pattern defied clear classification either as a known benthic organism or its product, the symmetry and sizes of the pattern were considered to be closest to a compressed hexactinellid sponge adapted to an unconsolidated sediment substrate (Rona and Merrill 1978). Subsequently, the pattern has been identified as the inferred surface expression of the fossil Paleodictyon nodosum found in flysch sediments of Eocene age near Vienna, Austria, and other areas. Body fossil interpretations of the form, in addition to that of a hexactinellid sponge, are the test of a large foraminiferan, either an astrorhiziid, oraxenophyophore, or part of the protistan super group Rhizaria as recently reviewed in Rona et al. (2009). The characteristics that support interpretation of P. nodosum as a hexactinellid sponge of agglutinated sediment (Reiswig and Mackie 1983) are (1) shield shape (2.4–7.5 cm diameter) projecting above the sediment surface (0.5 cm); (2) an outer wall penetrated by a hexagonal array of narrow tubes (1 mm diameter; pores); connection of the tubes through short (1–2 mm long) vertical shafts to a compressed body cavity consisting of a continuous horizontal network of hexagonal tubes; (3) passive ventilation of the body cavity sieve inflow at margins enhanced by marginal ridges; body wall outflow at center. Fortunately, we carried out some investigations of structural features of glass sponge species called Aphrocallistes beatrix in May 2009. After reading Peter Rona’s paper (Rona et al. 2009), it occurred to me to inform him that honeycombstructured hexactinellids really exist. So, I sent him images of A. beatrix obtained by our lab using stereo and scanning electron microscopy (Fig. 7.2). Our images are strong evidence that the walls of the honeycomb structural unit in this sponges are made of silica-based tubular formations because of the presence of the axial channels. I believe that formation of such complex cellular structures in hexactinellids must be determined genetically and that these features were conserved during about last 700 Myr.
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Fig. 7.2 Honeycomb architecture of the glass sponge A. beatrix is well visible using stereo (a, b) as well as scanning electron microscopy (c–f). Axial channel (g) and axial filaments (h) are also present
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7.1 Conclusion Therefore, we can hypothesize that similar genomes may be present in Paleodictyon species as well. The numerous exemplars of fossilized Paleodictyons and related formations, and the existence of recent glass sponges with similar characteristic honeycomb cellular skeletal motif, all allows us to suggest that the rise and development of these structural forms confer their advantages during evolution of biogenic cellular structures.
References Ekdale AA (1980) Graphoglyptid burrows in modern deep-sea sediment. Science 207:304–306 Fuchs T (1895) Studien über Fucoiden und Hieroglyphen. Denkschriften der Kaiserlichen Akademie der Wissenschaften, Wien, Mathematisch-Naturwissenschaftliche Klasse 62: 369–448 Garlick GD, Miller W (1993) Simulations of burrowing strategies and construction of Paleodictyon. J Geol Educ 41:159–163 Honeycutt CE, Plotnick RE (2005) Mathematical analysis of Paleodictyon: a graph theory approach. Lethaia 38:345–350 Ksiazkiewicz M (1970) Observations on the ichnofauna of the Polish Carpathians. In: Crimes TP, Harper JC (eds) Trace fossils, geological journal, special issue 3, Liverpool (Seal House) Mark W (1873) Neue Beiträge zur Kenntnis der fossilen Fische und anderer Thierreste der jüngsten Kreide Westfalen. Paleontographica 22:55–74 Menghini GG (1850) In: Savi P, Menghini GG (eds) Osservazioni stratigrafische e paleontologische concernati la geologia della Toscana e dei paesi limitrofi. In: Murchinson RI (ed) Memoria sulla struttura geologica delle Alpi degli Apennini e dei Carpazi. Stemparia granucale, Firenze, pp 246–528 Plotnick R (2003) Ecological and L-system based simulations of trace fossils. Palaeogeograph Palaeoclimatol Palaeoecol 192:45–58 Reiswig HM, Mackie GO (1983) Studies on hexactinellid sponges III. The taxonomic status of Hexactinellida within the Porifera. Phil Trans R Soc Lond B 301:419–428 Rona PA (2004) Secret survivor. Natural History 113:50–55 Rona PA, Merrill GF (1978) A benthic invertebrate from the mid-Atlantic ridge. Bull Mar Sci 28:371–375 Rona PA, Seilacher A, Luginsland H et al (2003) Paleodictyon, a living fossil on the deep sea floor. Eos Transactions AGU, Fall Meeting Supplement 84, Abstract OS32A-0241 Rona PA, Seilacher A, Vargas C et al (2009) Paleodictyon nodosum: a living fossil on the deepseafloor. Deep-Sea Res II 56:1700–1712 Seilacher A (1977) Pattern analysis of Paleodictyon and related trace fossils. In: Crimes TP, Harper JC (eds) Trace fossils 2. Geological Journal, Special Issue 9, Liverpool (Seal House), pp 289–334. Swinbanks DD (1982) Paleodictyon; the traces of infaunal xenophyophores? Science 218:47–49 Uchman A (2003) Trends in diversity, frequency and complexity of graphoglyptid trace fossils: evolutionary and palaeoenvironmental aspects. Palaeogeograph Palaeoclimatol Palaeoecol 192:123–142
Chapter 8
Peculiarities of the Structural Organization of the Glass Sponges’ (Hexactinellida) Skeletons
Abstract Hexactinellida (Porifera) are marine sponges defined by their production of siliceous spicules of hexactinic, triaxonic (cubic) symmetry, or shapes clearly derived from such forms by either reduction of primary rays or addition of terminal branches to the ends of primary rays. They lack calcareous minerals and sclerified organic spongin as skeletal components. Skeletal formations of hexactinellids display an astounding amount of diversity, complexity, and sizes. In contrast to cellular forms made by silica presented in radiolarians and diatoms, glass sponges are examples of more complex structures which correspondingly represent the macrolevel of structural organization for these biological materials. Interestingly, similar to diatoms, two principal forms of the cellular materials—the honeycomb and tetrahedron—are found in these metazoans. The peculiarities of hierarchical structural organization in the spicular stalk and body spicules of the Caulophacus sp. glass sponge are described. Eiffel’s design in skeletal frameworks of glass sponge is discussed too. Mechanism of silicification in sponges with respect to the formation of skeletal frameworks, as well as spicules, is of principal interest for both materials science and bioinspired materials chemistry because of the possible future applications for the corresponding processes using biomimetic in vitro synthesis. Corresponding hypotheses are proposed and discussed. Sponges (Porifera) are the most simple and ancient multi-cellular animals on earth and live attached to the seabed or another substratum. Sponges diverged from other animals earlier in evolutionary history compared to any other known animal group, extant or extinct, with the first sponge-related record in earth history found in 1.8 billion-year-old sediments (Nichols and Wörheide 2005). The huge diversity with respect to their natural habitat is probably the reason for the estimated number of approximately 15,000 different sponge species (Hooper et al. 2002). To support life, sponges pump huge amounts of seawater (170–72,000 × their own body volume per day) through their bodies (Simpson 1984), filtering it to capture food particles, such as bacteria, microalgae, other unicellular organisms, and dead organic particles. The whole sponge body is designed for efficient filtration of the surrounding seawater, which is essential because of the low nutrient availability at the seafloor.
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The phylum Porifera (sponges) is divided into three classes. Hexactinellida and Demospongiae are comprised of a siliceous skeleton, and the Calcarea have a calcareous skeletal network (Bergquist 1978). Significant separation of the Calcarea from the Hexactinellida/Demospongiae (Silicispongea) clade is also implied by fundamental differences in morphology and development; unlike Silicispongea, calcareans lack morphologically distinct microscleres, and their calcitic spicules are secreted intercellularly within an organic sheath (vs. formation of silica spicules onto an intracellularly secreted axial organic filament in Silicispongea) (Botting and Butterfield 2005). Sponges bear only a few different cell types of which the sclerocytes produce the spicules, i.e., siliceous structures, which are often needleshaped. These spicules often form a distinct skeleton, but occasionally they are loosely distributed throughout the sponge body without identifiable order, or are not present. Size, type, shape, combination of spicules, and their skeletal arrangements are the fundamentals of current sponge systematics (Boury-Esnault and Rützler 1997; Erpenbeck et al. 2006). Among the siliceous sponges, most important are the monophyletic class Hexactinellida and the polyphyletic “Lithistida,” which includes taxa belonging to different groups within the class Demospongiae. Both classes are characterized by rigid skeletons of fused spicules (Fig. 8.1). Among the class Hexactinellida, the order Hexactinosida (subclass Hexasterophora) (Schulze 1886), known since the Late Devonian (Rigby et al.
Fig. 8.1 SEM images: diverse structural networks can be observed in glass sponges like F. occa (a, d) as well as in lithistid sponges like Neopelta sp. (b, c)
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1981, 2001), developed a rigid and, to a certain extent, firm skeleton that consists of fused hexactins. This fused, three-dimensional, generally right-angled skeletal network stays intact even after the death of the sponge.
8.1 Glass Sponges (Hexactinellida) Hexactinellida Schmidt (Porifera) are marine sponges defined by their production of siliceous spicules of hexactinic, triaxonic (cubic) symmetry, or shapes clearly derived from such forms by either reduction of primary rays or addition of terminal branches to the ends of primary rays. They lack calcareous minerals and sclerified organic spongin as skeletal components. Siliceous spicules may be entirely loose or partially fused to form a rigid basal and choanosomal framework. Their living tissues are mainly syncytial, with distinctive porous plugs joining differentiated regions of the syncytium to each other or to discrete cellular components. Flagellated collar units are anucleate. Hexactinellids are viviparous and, from detailed study of a single species, produce distinctive trichimella larvae. Two subclasses are recognized by different microsclere forms—amphidiscs and hexasters. Hexactinellids include about 600 described species, 7% of all Porifera, distributed in 5 orders, about 17 families, and about 118 genera (Lévi et al. 1989; Leys 2003; Mehl 1992; Okada 1928; Reiswig 1971, 2002a, b; Schulze 1886, 1904; Sollas 1888). The spicules of most hexactinellids are larger (see Figs. 8.2 and 8.3) and more luxuriously architectured than those in demosponges (Tabachnick 2002). It was strongly suggested (Maldonado et al. 2005) that the role of sponges in the benthopelagic coupling of the Si cycle is significant. For example, Antarctic giant hexactinellids, such as Rossella nuda and Scolymastra joubini, which may be up to 2 m in length, 1.4 m in diameter, and up to 600 kg wet weight, contain up to 50 kg biogenic silica each (Maldonado et al. 2005). They are extremely slow growing, seemingly reproduce only over long time intervals (Dayton et al. 1974), and have an incredible lifespan, probably more than 1500 years (Dayton 1979; Gatti 2002). While their living tissues represent only a modest biomass, the siliceous spicules of hexactinellids become an important ecological factor. After the death of the sponges, the megascleres do not dissolve but accumulate at the bottom and over large areas to form spicule mats commonly about 50 cm thick but occasionally exceeding 2 m (Dayton et al. 1974; Koltun 1968). The mats structure the fauna living in and on them. Sponges seem to be important players in numerous geobiological processes (Reitner 2004). For example, siliceous sponge reefs had a wide distribution in prehistoric times and once constructed the largest reefs known on earth. During the Upper Jurassic there existed a deepwater reef belt on the northern Thetis shelf that was 7000 km long (Krautter et al. 2001). In present times, hexactinellid sponges of the order Hexactinosida have constructed reefs at several localities off the coast of British Columbia, Canada. The reefs occur on relict glaciated seafloor areas with a low sedimentation rate and a high dissolved silica concentration, such as in the Queen Charlotte Basin and in the Georgia Basin (Conway et al. 2004, 2005). These
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Fig. 8.2 Huge spicular formations of Hyalonema (Corynonema) populiferum Schultze 1899 (samples courtesy: Dan Kamikawa)
reefs represent stable communities that have been growing for up to 9000 years. The main frame builders include the hexactinosidan species Aphrocallistes vastus, Farrea occa, and Heterochone calyx in the Queen Charlotte Basin, and A. vastus and H. calyx in the Georgia Basin (Conway et al. 2004). The framework of these reefs is constructed through several processes of frame building, and the reef matrix is derived from the trapping of suspended sediments (Krautter et al. 2001). Similar frame-building processes are thought to have contributed to the formation of the ancient reefs (Krautter et al. 2006). Upwelling and downwelling oceanographic processes in a biologically productive coastal sea have contributed to the development of large reef complexes. The reefs form as bioherms (mounds) and biostromes (beds or sheets) that may rise up to 21 m above the seafloor and they cover around 1000 km2 on the continental shelf. Individual reef complexes may cover areas of more than 300 km2 and occur in 90–240 m depth range (Conway
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Fig. 8.3 (a) Monorhaphis chuni possess the longest siliceous spicule (image courtesy: Werner E.G. Müller). (b, c) Delicate work is needed to collect individual Monorhaphis spicules from the sea bottom (images courtesy: Konstantin Tabachnick)
et al. 2001, Lehnert et al. 2005). Trapping fine-grained siliciclastic sediments in suspension in near-bottom currents is an important part of the mound-forming process, as it prevents the reef framework from collapsing under its own increasing weight.
8.2 Demosponges (Demospongiae) In demosponges (Demospongiae Sollas 1885) we have much the largest group, with 95% of all extant sponges. They are also the most diverse group. Some are freshwater, but predominantly they are marine species living from the intertidal zones to the deepest seas, with around 15 orders, 88 families, and about 500 valid genera (Hooper and Van Soest 2002). The skeleton is composed of monaxonic or tetraxonic siliceous spicules (never triaxonic) bound together with collagen-like protein spongin in discrete fibers or loosely aggregated, and ubiquitous collagenous filaments forming the ground substance of the intercellular matrix. Because the morphology of the spicules differs species specifically, they are used as a major taxonomic character. However, very little is known about the molecular mechanism(s) that determine the position and arrangement of the spicules within the demosponge skeleton (Uriz et al. 2003; Uriz 2006).
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8.3 Lithistid Sponges Like hexactinellid sponges, lithistid sponges also contain siliceous skeletons comprising spicules. These spicules (desmas) interlock, rendering the skeleton rigid and often stony (Kelly 2000, 2003; Pisera 2003). At present, little is known about the deposition of silica within this taxonomic order. Lithistid sponges are commonly found on tropical and temperate seamounts and continental margins down to depths of about 1000 m (Kelly 2000). Several southwest Pacific species form large cups, bowls, and plates that extend linearly from the sponge margin. Close inspection of the lamellae shows irregular or concentric ridges, indicating that silica deposition is not continuous but rather variable in nature. Ellwood et al. (2007) presented detailed records of trace metals and carbon isotopes to understand siliceous spicule formation in the deep-sea lithistid sponge Corallistes undulatus (Demospongiae: Corallistidae) (Lévi and Lévi 1983). X-ray analysis of two longitudinal sections removed from the lamellae of the cup-shaped sponge revealed 144 and 137 light- and dark-density band pairs, respectively, within the siliceous skeleton. Although there was some variability in the 32 Si data, the overall age established using these data indicated that the sponge was between 135 and 160 years old.
8.4 Cellular Structures in Glass Sponges Skeletal formations of hexactinellids display an astounding amount of diversity, complexity, and sizes. For example, the glass sponge Aspidoscopulia sp. (Fig. 8.4) possesses a quadrangular-meshed siliceous skeleton which can reach up to a height of 2 m and a width of about 75 cm. With regard to Aspidoscopulia, which we recently started to investigate in our laboratory, I can confirm that “structure in Nature is a strategy for design” (Pearce 1978). However, before “stealing Nature’s best ideas” (Oliver et al. 1995) for hierarchical biocomposite-based materials, I propose to analyze in more detail the state-of-the-art research on this topic. Thus, in contrast to cellular forms made by silica presented in radiolarians and diatoms, glass sponges are examples of more complex structures which correspondingly represent the macrolevel of structural organization for these biological materials. Interestingly, the similar to diatoms, two principal forms of the cellular materials—the honeycomb and tetrahedron—are found in these metazoans. Throughout biological morphology, the most common arrangement of cellular monodispersions (Ozin 1997) seems to be the hexagonal pattern. When equal tensions exist in all contact faces, the cellular array will equilibrate to one where all shared faces meet at angles of 120◦ and all vertices are joined equally at angles of 109◦ 28 . In Thompson’s description of the origin of hexagonal structures of diatoms and radiolaria, he proposed a layer of roughly equal-sized cells (i.e., a protoplasmic froth) fashioned through their mutual tensions into a regular meshwork of hexagons (Thompson 1917). This serves as a mold for the deposition of silica into the intercellular spaces.
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Fig. 8.4 Giant glass sponge Aspidoscopulia sp. (a) (image courtesy: Konstantin Tabachnick) possess complex hierarchical skeleton architecture, which is well visible using stereo (b), light (c), as well as fluorescence (d) microscopy
The first geometrical description of structure in nature appears in Plato’s Timaeus, which provides an inventory of four of the regular solids and their association with the four elements: fire with the tetrahedron, earth with the cube, air with the octahedron, and water with the icosahedron. Plato considered the dodecahedron to be the shape that encompasses the entire universe. These five regular polyhedra became known as the Platonic solids (see for review, Ozin 1997). This geometrical theme is embodied in Sir D’Arcy Thompson’s classic 1917 text On Growth and Form. He proposed a physicogeometrical hypothesis of causation to explain the theory of formation of many beautiful protozoan agglutinated forms. In this text, he invoked the basic laws of close packing and surface energy minimization of cellular assemblies, subjected to intra- and intermolecular force fields, to explain the occurrence of a variety of single-cell microskeletons. The physico-mathematical laws and theorems that govern the arrangements and surface tension forces of grouping of cells appeared to somehow control the adsorption–deposition of inorganics into complex patterns and led to the large number of microskeletal forms in biological systems. D’Arcy Thompson noted how protoplasmic organisms exploited
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surface energy minimization to localize and arrest solid particles as an agglutinated shell (Thompson 1917, 1992). As reviewed by Ozin (1997), he considered the sequestration, confinement, and ordering of inorganic particles to a position of equilibrium within a protoplasmic surface boundary layer to be a general phenomenon of considerable biological significance. In the particular case of the multitude of diatom and radiolarian microskeletons, Thompson visualized a protoplasmic froth of contiguous alveoli or vacuoles as the polygonal organic meshwork that directed the silicification process. As far back as 1887, Kelvin published an essay On the Division of Space with Minimum Partitional Area, in which he described a 14-sided figure that he called a tetrakaidecahedron. A similar figure had been known to the crystallographers even before Kelvin’s publication. Kelvin apparently arrived at his tetrakaidecahedron form by studying the cubic skeleton frame of Plateau (1873). Kelvin’s suggestion lay fallow for 36 years and resurfaced in 1923 when Lewis showed that cells of elder pith tend to be 14 sided and at times show an alternation of hexagonal and square faces, suggestive of Kelvin’s figure (Kelvin 1894). Lewis (1925, 1928) has since extended his observations and gave data showing the primarily tetrakaidecahedral form of such diverse tissues as the stellate cells of Juncus (Fig. 8.5), cells of human adipose and oral epithelial tissues, and cork cells, while Hein (1930a, b) came to similar conclusions when studying sclerotial tissue of the fungi. From a mathematical standpoint the orthic tetrakaidecahedron has been considered in a publication by Matzke (1927). Thus, the cells in the pith of Juncus are stellate, and the tissue has the appearance in section of a network of six-rayed stars (Fig. 8.5), linked together by the tips of the
Fig. 8.5 Stellate cells in the pith of Juncus (adapted from Thompson 1942)
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rays and separated by symmetrical, air-filled intercellular spaces, which give snowlike whiteness to the pith. In thick sections, the solid 12-rayed “star dodecahedra” may be seen with wonderful clarity under the binocular microscope. According to Thompson (1917), they are not difficult to understand. Imagine, as before, a system of equal spheres in close contact, each one touching its 12 neighbors, six of them in the equatorial. What actually occurs in the rush is tantamount to this, but not absolutely identical. It is not pith cells which tend to shrink within a boundary of constant size, but rather the boundary wall which continues to expand after the pith cells which it encloses have ceased to grow or to multiply. The points of attachment on the surface of each little pith cell are drawn asunder, but the content of the cell does not correspondingly increase and the remaining portions of the surface shrink inward. This shrinking gradually constitutes the complicated figure which Kepler called a star dodecahedron, which is still a symmetrical figure and is still a surface of minimum area under the new and altered conditions (Matzke 1935). Despite the fact that the stellate cell construct described above is related to the plant world, I inserted the image because of the structural similarity with skeletal meshworks. We observe such structures in some representatives of glass sponges, especially those related to the Farreidae family, for example, the Sarostegia (Fig. 8.6). Farreidae Gray (Hexactinellida: Hexactinosida) contains a total of 21 species in five genera. Individuals of the family have been recovered from depths of 82 to over 5200 m (Reiswig 2002b). Body form within the family is variable and ranges from a typical thin-walled tubular branching and anastomosing stock to cup, funnel, flat blade or solid branching forms. The primary dictyonal skeleton is never channelized, but accreted secondary layers may contain shallow, extradictyonal epirhyses and/or aporhyses. Primary framework is fundamentally one to three layers of fused, quadrangular-meshed dictyonalia (Fig. 8.7), with all nodes being true central (with axial cross), while secondary layers have dictyonalia attached in indefinite orientation, resulting in false (non-central bearing) nodes and triangular meshes. Recently, we investigated F. occa as representatives of the Farreidae family (Ehrlich et al. 2007). This sponge is a typical deep-sea dweller with a depth range from 80 to more than 5000 m and has a global distribution, with a maximum population in the northern hemisphere (Krautter et al. 2006). This organism is able to form tremendously large deepwater reefs larger than 30 m in diameter, such as those on the continental shelf of British Columbia (Canada). Its body shape is a dichotomously branching and anastomosing system of subcircular to ellipsoidal and terminal open tubes. The tubes increase in diameter distally, not reaching more than 2.5 cm at the osculum. A single tube is up to 10–15 cm long (Fig. 8.8). Farrea occa is very thin walled, not thicker than 2 mm, consisting maximally of two layers of fused hexactin spicules. This results in a regular rectangular-meshed, and very brittle, skeleton with dictyonal strands oriented longitudinally. Farrea occa is attached, as all hexactinosidan sponges are, to a solid substrate by a basal plate enveloping or encrusting predominately rocks or other hard substrates (Krautter et al. 2006).
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Fig. 8.6 (a) Glass sponge Sarostegia oculata (arrows) is probably a unique reef-building hexactinellid, which possesses mechanically very rigid skeletons (image courtesy: Konstantin Tabachnick). (b) Hierarchically structured three-dimensional skeleton of this sponge (c, d) is well visible even using light microscopy
Fig. 8.7 From simple to complex: (a) principle structural unit observed in radiolarians (adapted from Thompson 1942); (b) thickened farreid dictyonal frame with two regular, quadrangular, primary layers (1◦ ) and irregular additional secondary layers (2◦ ) on the dermal side is typical for glass sponge skeletons of Farreidae family (adapted from Reiswig 2002)
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Fig. 8.8 Hierarchical structural skeleton of the reef-building Farrea sp. glass sponge (a). Different kinds of geometrical proportions can be observed using SEM (b–d)
We carried out a detailed study of the structural and physico-chemical properties of skeletal fragments of F. occa with respect to identification of the nature and origin of the organic matrix. Our results show unambiguously that α-chitin is an essential component of the skeletal structures of this hexactinellid. We showed that skeletal glassy fibers have a layered design with specific compositional variations in the chitin–silica composite. According to the fluorescence microscopy images, chitin is mostly located in the places where spicular structures join each other (Fig. 8.9). Chitin was also found by us within skeletal structures of such glass sponges as Euplectella aspergillum (Ehrlich and Worch 2007b) as well as in Antarctic sponge Rossella fibulata (Ehrlich et al. 2008a). Does chitin play a key role in the formation of such geometrically complex hierarchical structures in glass sponges, as reported above? It is a challenging and complex question. However, the good news is that now, for the first time, we have two players: crystalline aminopolysaccharide chitin and amorphous silica. Chitin is reported to be a scaffolding material in hierarchically organized skeletal structures in the diatom Thalassiosira pseudonana (Brunner et al. 2009) as well as in molluscs, crustaceans, and insects. The next step is to obtain experimental results for understanding how such silica-containing complex
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Fig. 8.9 Chitin microparticles within silicate-based composites are well visible using fluorescence microscopy both prior to (a) and after staining with calcofluor white reagent (b). We used this simple method for localization of chitinous residues on the surface of F. occa skeletal fragments (c, d, stained). After 6 months of alkali treatment, residual chitin is still well visible (e and f, both stained). Chitin prevents silica against dissolution in alkali (images courtesy: Thomas Hanke)
structures arise. It is well known that chitin may self-assemble on different levels of its structural organization. Therefore, I suggest that this phenomenon determines the initial formation of the meshwork skeleton of glass sponges. It was suggested that silicate ions and silica oligomers preferentially interact with glucopyranose rings exposed at the chitin surface, presumably by polar and H-bonding interactions
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(Ehrlich et al. 2008a; Ogasawara et al. 2000). Moreover, silicon was found associated with glycosaminoglycans as an ether- or ester-like silicate with C–O–Si or C–O–Si–O–Si–O–C bonds, in amounts of one Si atom per 130–280 repeating units of the organic (Schwartz 1973). I also want to note here that skeletogenesis of Farreidae sponges takes place at temperatures above 0◦ C and lower. From this point of view, the possible role of enzymatically determined (silicatein-based) silicification of chitinous scaffolds seems to be unrealistic. Because chitin could play a crucial role also in biosilicification in fungi (Kolb et al. 2004), it was hypothesized (Ehrlich et al. 2007) that chitin molecules are probably part of a very old organic template system involved in a biosilicification phenomenon, which was established a long time before the origin of first metazoan (e.g., glass sponges).
8.5 Eiffel’s Design in Skeletal Frameworks of Glass Sponges Disagreement is necessary in science. It’s good because it forces you to find new arguments and more arguments. Adolf Seilacher
The idea of macroscopic hierarchical frameworks can be traced back at least to Eiffel’s design for his tower (Fig. 8.10). The Eiffel tower is third order and has a relative density p/po (density p as mass per unit volume of the structure divided by density po of material of which it is made) that is 1.2 × 10−3 times that of iron, which is weaker than structural steel (Lakes 1993). The rationale for the use of small girders in such a large structure was attributed to the ease of construction. Thus, this renowned construct proposed by Alexandre-Gustave Eiffel possess 2,500,000 rivets and 18,038 metallic pieces. To my best knowledge, Eiffel was not inspired by any kind of glass sponge skeleton; however, at first glance, constructs which are visually similar to Eiffel tower really exist in Hexactinellida. Not only there exists very impressive images of such structures, for example, in fundamental work by Isao Iijima in 1901 (Iijima 1901), but also there is a very detailed description of the diversity of their spicular building blocks in different species of Euplectella glass sponge (Fig. 8.11). Today, the “Eiffel towerlike” sponge Euplectella aurantiacum has become very popular and is an object of fascination as well as bioinspiration for the materials science community by virtue of a publication by Joanna Aizenberg and co-workers in Science (Aizenberg et al. 2005). One year later, James Weaver and co-authors reported in more detail about the hierarchical assembly of the siliceous skeletal lattice of this hexactinellid (Weaver et al. 2007). However, I could not find in either paper any reference on Iijima’s paper. It is a pity, because Iijima described and discussed spicule-based frameworks of Euplectella species in a very similar manner to that in the modern works listed above, however without using such terms as “hierarchical,” “nano- and microscale.”
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Fig. 8.10 The Eiffel tower—an example of man-made hierarchical construct (photos courtesy: Vasily V. Bazhenov)
I could see little reason to study Euplectella sponges in more detail after the thorough job done by Iijima, Aizenberg, Weaver, and coworkers. Therefore, we focused our attention on the poorly investigated mushroom-like deep-sea glass sponge Caulophacus sp. (Fig. 8.12). These sponges are attached to hard substrates by a long, rigid stalk. Their atrial cavity is everted, being represented by the upper
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Fig. 8.11 Eiffel tower motive in drawings of Euplectella glass sponge made by Iijima in 1901
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Fig. 8.12 Mushroom-like deep-sea glass sponge Caulophacus sp. (image courtesy: NOAA)
surface of the cap (Fig. 8.13). The tubular stalk contains mostly megascleres, long diactine spicules that are connected to their neighbors by numerous articulations (Fig. 8.13b, d) or by bridges (Fig. 8.13f), as a result of secondary silica deposition. The stalk is fixed to the substratum by a basydictional plate which contains mostly hexactins fused to each other at points of mutual contacts. The cap mostly contains loose spicules, megascleres, and microscleres. The subgenus is cosmopolitan, being distributed in all oceans and in some seas with “normal” salinity at a depth of 130–6770 m. It currently contains about 15 species. The reason to investigate especially this sponge was the following. Most people think of the deep sea as a quiet place where currents are sluggish, which explains why the ocean basins are muddy. However, at intermediate depths (1000–3000 m) in the ocean, the water moves at a steady pace, speeding up and slowing down as it encounters various obstacles, or moves through undersea mountain passes. Since sponges are sessile organisms that must filter great volumes of seawater to collect food nanoparticles and resolved organic matter, they therefore rely on water currents, where the potential to be directly and severely impacted by adverse environmental conditions is rather high. Photographs of deep-sea sponges, exposed to the natural deep-sea environment, demonstrate remarkable flexibility of stalks in glass sponges (Heezen et al. 1966). Taking into account that the sponge architecture is made of a brittle material, one may ask the question about the structural background responsible for such a flexibility. The goal of the study was to investigate the peculiarities of structural organization in the spicular stalk and body spicules of the
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Fig. 8.13 There are several similarities between structural motives in Eiffel tower (a, c, e) and within skeletal framework of Caulophacus sp. stalk (b, d, f) observed using light microscopy
Caulophacus sp., because our specimen was collected by Konstantin R. Tabachnick from the depth of about 4200 m. One additional reason to investigate this glass sponge was determined by the doubts I had after reading the explanation for the integrity and consolidation of the skeletal lattice within such complex glassy constructs, as described for Euplectella sponges in the recent papers (Aizenberg et al. 2005; Weaver et al. 2007). According to Weaver et al. (2007), spicules in E. aspergillum are embedded in silica matrix that serves as a cement to consolidate and strengthen the entire skeletal system. The smaller spicules play a critical role in filling the gaps between the larger spicules, prior to cementation of the skeletal lattice via deposition of
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multiple layers of silica/organic composite. The use of multiple small spicules cemented as reinforcing filler was also observed at the junctions of the underlying cruciform spicules. These authors believe that the consolidating silica cement precisely follows the contours of the underlying spicules, apparently enhancing the strength of this fiber-reinforced composite, whose structure is similar to armored concrete. However, at this point I have a disagreement with these researches because of the following questions. Is it really possible that these siliceous constructs will be unbreakable under submarine currents if the spicules are only agglutinated or cemented with each other? And what happens in the case of biocorrosion of those cements and glues? Why did Eiffel propose rivets and not some kind of cement in his tower? Surprisingly, we obtained answers to these questions by studying hierarchically structured stalks of Caulophacus sp. (Fig. 8.14). Individual spicules were isolated from the Caulophacus sp. stalk using pincers. We observed club-like constructions (Fig. 8.14b, arrow) at the ends of separated spicules using scanning electron microscopy (SEM). To obtain more detailed information about the localization of these specific structures within the spicular articulations, we employed gentle desilicification using 2.5 M NaOH solution at 37◦ C as described by us previously (Ehrlich et al. 2007). After 14 days of immersion, the club-like constructions could be observed as being located within articulations using SEM (Fig. 8.14c, d). Detailed analysis of the SEM images revealed that the spiny surface on these spicules could be coupled with each other, preventing slippage against the corresponding silica-based articulation (Fig. 8.15). This structure is entirely unique, having evolved as a natural engineering solution to structural integrity problems in the construction of the hierarchical skeletons of glass sponges. Here, I represent comparative figures to show two different approaches for connecting tubular materials in the case of man-made (Fig. 8.16a, b) and Nature-made materials (Fig. 8.16c). Furthermore, during alkali-based desilicification experiments we observed that the spines of the club-like structures were highly resistant to alkali treatment, in contrast to the surface silica layers which are deeply corroded under these conditions (Fig. 8.14d–f). In order to proceed with more detailed studies, we mechanically disrupted several spicules showing the club-like constructions, as shown in Fig. 8.17. SEM images of these broken elements show that the spines possess central structures, covered with a layer of silica (Fig. 8.17a, b). Elemental mapping (data not shown here) and EDX analysis (Fig. 8.17c) unambiguously show that calcium and not silicon is the main component of this central structure. Atomic absorption spectroscopy also confirmed the presence of calcium in this spicular material, at an average concentration of 37.8 ± 9.0 ng/mg, corresponding to 3.78 ± 0.90 mass percent of calcium in the whole spicule. Additional results obtained using photoemission spectroscopy (PES) and X-ray absorption spectroscopy (XAS) performed on untreated spicules of Caulophacus sp. showed the presence of calcium carbonate within club-like spicules. Transmission electron microscopy (TEM) and electron diffraction analysis (EDA) of the untreated club-like structures (Fig. 8.17d) further confirmed these results. Thus, electron
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Fig. 8.14 SEM images of the spicular network observed in the stalks of Caulophacus sp. (a). Individual club-like spicules (b, arrow) were isolated using pincers. After 14 days of alkali treatment, the club-like constructions could be observed as being located within articulations (c, d). Interestingly, only siliceous layer that covers the club-like spicule is corroded (e, f). The spines were still resistant to alkali
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Fig. 8.15 Different kinds of articulations between spicules of Caulophacus sp. (a, c, e) were observed using light microscopy. We developed corresponding computer representations (b, d, f) including the presence of club-like formations (images courtesy: Denis Kurek)
diffraction patterns for the spines correspond to the (0001) zone axis of calcite with the following d spacings: 100 (1010) 4.32 Å; 010 (0110) 4.32 Å, and 210 (2110) 2.49 Å. Also, obtained FTIR spectra as well as Raman spectra unambiguously confirm the calcite nature of the crystals located within club-like spicules of Caulophacus. The challenge now is to understand the mechanisms by which this unique biocomposite is initially formed and how this process can occur at high pressure (note, this sponge was collected from the 4200 m depth) and at low temperature (between 2 and 4◦ C). In most cases, calcification in sponges is confined to an extracellular space
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Fig. 8.16 Two different approaches against the slippage: (a, b) man-made, (c) made by Caulophacus sp. glass sponge (images courtesy: Paul Simon)
delineated by tightly adhering sclerocyte cells (Uriz 2006). The sclerocytes secrete a macromolecular matrix to the extracellular space, which comprises a genetically programmed, three-dimensional framework that guides spicule formation. It is difficult to differentiate between organically mediated and inorganic precipitation, and a combination of different mechanisms in the same species cannot be ruled out. A decisive factor for the control of nucleation of calcium carbonate is the development
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Fig. 8.17 SEM images of the broken spine of Caulophacus sp. show that they possess central seed-like structures, covered with a layer of silica (a, b). EDX analysis (c) and electron diffraction (d) unambiguously show that crystalline calcium and not silicon is the main component of this central structure. Calcite crystals can be visible as attached microparticles on the surface of axial filaments after demineralization in alkali (e, f)
of local supersaturation with respect to calcite, or to any other calcium carbonate polymorph. This in turn depends on the levels of calcium and carbonate ion activities. The latter depend strongly on local pH, as the dissolution of CO2 is favored at higher pH values. Seawater may become supersaturated with respect to calcium carbonate, especially in spatially confined structures. The inner space of the spicule of Caulophacus sp. represents a microchannel of approximately 1 μm in diameter
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Fig. 8.18 Computer-made representation of the club-like spicule of Caulophacus sp. (a, b) allows us to represent also the cross-sectional view (c, d) of this formation. Calcite crystals are connected with both axial filament and axial channel of the spicule (images courtesy: Denis Kurek)
flooded with ionic solution, while one extremity of the spicule is sealed by silica (Fig. 8.18). Under these conditions, contact with silicic surfaces is expected to favor heterogeneous nucleation of calcite, as in this case lower supersaturation is needed in comparison with the value required for homogeneous nucleation. More specifically, for example, calculations concerning the saturation with respect to calcite at 2000 m, temperature 5◦ C, and pH in the range of 7.62–8.22 yielded saturation ratio values between 49 and 184 (Levendekkers 1975). The observed morphology of this silica–calcite biocomposite can be understood as the result of the crystallization of calcite crystals on the surface of organic filament in the presence of polymeric silica (Fig. 8.17e, f). It has also been shown in vitro that complex crystalline architecture can be induced through the miniaturization of the growth subunits by covering the surface of carbonate crystals with silicate anions and by the self-organized assembly of the miniaturized subunits (Kotachi et al. 2003). The silica–calcite-based club-like structure made by Caulophacus sp. is the first natural example of this principle and confirms that biomineralization is a process in which organisms produce mineral solutions for their own functional requirements (De Yoreo and Vekilov 2003), starting at nano- and microlevels of structural hierarchy. Thus, we have found that the stalk and body skeleton of the deep-sea glass sponge Caulophacus sp. is based on a silica–calcite biocomposite. Siliceous spicules are
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Fig. 8.19 Intriguingly, also hierarchically structured skeleton of Euplectella sp. glass sponge (b) possesses club-like spicules (a, c–g), which definitively are responsible for skeletal integrity of this unique naturally occurring construct
modified by the addition of conical calcite seeds, which then form the basis for further silica secretion to form a spinose region. Spinose regions on adjacent spicules are then joined by siliceous cross-links, leading to unusually strong cross spicule linkages. The calcite component represents the first record of biominerals other than silica in hexactinellid sponges. I take here the liberty to remark about the functional similarity between calcitic crystals within the club-like spicules of glass sponge and rivets which were used to make Eiffel tower stable. And what is the situation with Euplectella hierarchical “glass tower?” We carried out experiments with this sponge as well, due to the significant scientific interest. In this case, we also found the club-like spicules, which are definitively responsible for making this skeletal construct stable (Fig. 8.19e, f). Therefore, we hypothesized that the use of club-like structures to maintain the integrity of the filigrane hierarchically structured skeletons may be widely distributed within different species of Hexactinellida. Note that Caulophacus and Euplectella are representatives of two different families!
8.6 Spiculogenesis Skeletal frameworks, desmas and especially spicules, are the main forms of the silica deposition in sponges. The history of the discoveries related to sponge spicules is reviewed in great detail by Vosmaer and Wijsman (1905). As reviewed by these authors, after Schweigger in 1819 demonstrated that in some cases the spicules of
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sponges do not consist of calcium carbonate, Grant in 1826 found them to contain silica, and Bowerbank in 1841 showed that, in addition to the silica, some organic matter is present. Sollas in 1885 likewise found that the sponge silica resembles opal. He stated that in general the refractive index of sponge silica is more similar to opal or colloidal silica, and not to quartz. This kind of opal was named spicopal by Vosmayer and Wijsman; however, the organic matter of the spicule was called spiculine by F.E. Schulze. As for the structure of the spicules, in 1835 Gray found that Hyalonema glass sponges consist of layers which become conspicuous by heating. These layers concentrically surround a “central canal,” which is filled out, as Kölliker showed in 1864, by an organic mass, the axial rod. Claus in 1868 found that the silica which directly surrounds this central rod is homogenous. He called this homogenous cylinder the axial cylinder. The sponge spicule thus consists of a central organic axis, surrounded by concentric layers of opal, the outermost of which is invested in a spicule sheath of organic matter, or rather of organic matter in intimate association with silica (Vosmaer and Wijsman 1905) (see Fig. 8.20). Using the freshwater sponge Ephydatia fluviatilis, Weissenfels and Landschoff (1977) and Weissenfels (1989) demonstrated that the formation of spicules starts in sclerocytes within a specific vesicle. After the production of an axial organic filament, silicon is deposited around it, and the whole process of forming a spicule (190 μm in length and 6–8 μm in diameter) is completed after 40 h at 21◦ C. Siliceous sponge spicules have traditionally been divided into two categories termed, according to their size, megascleres and microscleres (e.g., Lévi 1973). They are highly diverse in sponges and the selection pressures responsible are difficult to envision. There are over 12 basic types of megascleres and 25 types of microscleres reported in Demospongiae and 20 basic types of megascleres and 24 types of microscleres in Hexactinellida, an additional long list of variations of the basic types (Bavestrello et al. 1993; Boury-Esnault and Rützler 1997; De Vos et al. 1991; Garrone et al. 1981; Lévi 1973, 1993; Simpson 1984, 1990; Tabachnick and Reiswig 2002). Desmas differ from typical demosponge spicules; first, they are joined by articulation; second, they always display irregularity and often complex morphology and sculpture. The most basic difference, perhaps, is in the fact that the crepis or axial filament is usually very short (or even invisible) and extends only a short distance from the spicule center. Growth ceases very rapidly, and thus an important part of a desma, including secondary branches as well as elements of sculptures, does not depend on the axial filament (Lévi 1991; Sollas 1888). Recently, together with Andrzej Pisera we started thorough investigations on isolation and identification of organic matrix from a 37-Myr-old fossil lithistid sponge. Because of the intensive autofluorescence, an organic matrix of unknown nature is highly visible within desmas of this sponge (Fig. 8.21) using fluorescence microscopy. Experiments are now in progress in our lab. In contrast to the high diversity of spicules, there are relatively few basic types of skeletal frameworks in demosponges. Six elemental types of skeletons with intermediate forms can be differentiated: hymedesmoid, plumose, axial, radiate, and reticulate (Boury-Esnault and Rützler 1997). Without doubt, all of these structures are also of interest for materials science.
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Fig. 8.20 Computer-made representation of the multi-layered structure of the glass sponge spicule (a) is based on the observation which has been carried out using SEM (b–e). No organic material is, however, visible
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Fig. 8.21 Light microscopy image of the fossilized Neopelta sp. (a) represents typical structures of lithistid desmas. However, fluorescence microscopy image (b) of the same structure shows the presence of organic material with characteristic autofluorescence (arrows) (images courtesy: Andrzej Pisera)
Thus, the main elements of the skeletons of the sponges are termed spicules, which serve as major systematic characters for a given sponge species (Schulze and v Lendenfeld 1889). Since the comprehensive studies of Bütschli (1901) and Minchin (1909), a descriptive view on the synthesis of the siliceous spicules has been well established. However, because of the wide diversity in size and shape of siliceous spicules in different poriferan classes and species, it seems it is too early to make conclusion about the existence of some common mechanisms of spicule formation. Numerous points in this story have been unclear until recently. Below, I discuss some opinions existing in modern sponge science. According to Werner E.G. Müller, a renowned follower of the silicatein-based theory for biosilicification in all sponges (see below), in Demospongiae, initiation of spicule formation starts intracellularly within sclerocytes. There, an axial filament is assembled in organelles around which the first siliceous deposits are layered (Müller et al. 2006), thus forming the nucleus and initial growth form of a spicule. The first siliceous layers surrounding the axial canal measure 1–2 μm in diameter (Uriz et al. 2000). When reaching lengths of about 5–10 mm, spicules are extruded from the cells into the mesohyl, where their final sizes and shapes are completed. Thickening of spicules proceeds by the apposition of concentric silica layers (Müller et al. 2006; Uriz et al. 2000). It has been proposed that silicon is actively taken up by demosponges through a membrane-bound transporter (Schröder et al. 2004). Intracellularly, polycondensation of silicic acid is mediated enzymatically through the enzyme silicatein (Cha et al. 1999; Shimizu et al. 1998; Weaver and Morse 2003). Thus, according to Müller, the process of spicule formation can be divided into an initial intracellular step and a subsequent extracellular shaping phase: 1. Intracellular phase (initial growth): It has been demonstrated that silicic acid is actively taken up by cells (sclerocytes) via the Na+ /HCO3 − [Si(OH)4 ] cotransporter (Schröder et al. 2004). In parallel, mature silicatein is synthesized/
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processed and subsequently deposited together with silicic acid in special organelles of the sclerocytes, the silicasomes. Within silicasomes, axial filaments are formed around the silica, which is subsequently deposited enzymatically. After the formation of a first layer (or a few layers), juvenile spicules are released into the extracellular space, where they grow in length and diameter by appositional layering of silica lamellae (Müller et al. 2005). There, spicules obtain their final shape, e.g., Suberites domuncula tylostyles are characterized by a terminal globular swelling. 2. Extracellular phase (consecutive appositional growth): Silicatein is present in an enzymatically active form in the extracellular space (Müller et al. 2005). There, silicatein molecules are organized into larger entities, as demonstrated by immunogold electron microscopic analysis. These molecules are arranged along filamentous strings, which are organized concentrically to the spicule surface (Schröder et al. 2006) and consist of the protein galectin, which oligomerizes in the presence of Ca2+ . Within this organic cylinder that enfolds the growing spicule, the siliceous mantel grows stepwise by appositional layering of lamellae. Not only centrifugal growth (“thickening”) but also axial growth (“elongation”) of spicules is driven by extraspicular silicatein. In the extracellular space, both axial and radial growth of the spicules are driven by silicatein that surrounds the surface of the already existing silica lamellae. In hexactinellids, appositionally layered silica lamellae can reach 1000 in number (Wang et al. 2009). However, in demosponges, the individual lamellae fuse and form a “solid” siliceous shell, which surrounds the axial filament. 3. Extracellular phase (final morphogenesis): So far, the processes described above do not explain the species-specific shaping of spicules. This final step of spiculogenesis (i.e., morphogenesis) still remains mysterious. However, since spicules of both demosponges (e.g., S. domuncula) (Eckert et al. 2006) and hexactinellids (e.g., Monorhaphis chuni) (Müller et al. 2008) are surrounded or even embedded into a fibrous network of collagen and other proteins, it is safe to assume that these molecules (released by specialized sponge cells) provide a scaffold within which the galectin-containing strings are organized. Data suggest that the galectin-containing strings are organized by collagen fibers to net-like structures (Schröder et al. 2006). Those fibers that are released by the specialized cells, the collencytes, provide the organized platform for the morphogenesis of the spicules. The longitudinal growth of the spicules can be explained by the assumption that at the tips of the spicules, the galectin–silicatein complexes are incorporated into deposited biosilica under formation and elongation of the axial canal. Here, I must note, however, that the universal role of silicateins in spiculogenesis as well as in silicification seems to be grossly exaggerated. Recently, silicatein genes were found within spicule-free demosponges also (Kozhemyako et al. 2009). In contrast to reports by Müller’s group, we as well as the group of Prof. Gert Wörheidecan did not find silicateins in glass sponge Hyalonema sieboldii using similar techniques. Interestingly, the intra- versus extracellular secretion of spicules in demosponges has been extensively discussed over the last century and is still a matter of
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speculation (Simpson 1984). Microscleres could be secreted both intra- and extracellularly (Simpson 1968) and the most recent studies seem to support the intracellular secretion of megascleres (Garrone 1969; Garrone et al. 1981; Simpson and Vaccaro 1974). However, our current knowledge of spiculogenesis of siliceous sponges is mostly based upon the secretion of microscleres and small megascleres in a few species. It seems physically impossible for a single sponge cell, 20 μm in diameter, to include a large megasclere such as those of Astrophorid, Spirophorid, Hadromerid, and some Axinellid sponges (Uriz et al. 2000). At least in these extreme cases, the involvement of several sclerocytes in secreting a single spicule seems to be necessary and, thus, extracellular secretion cannot be ruled out. For example, it was shown that megasclere secretion takes place in sponge Crambe crambe extracellularly (Uriz et al. 2000). Once the axial filament is extruded to the mesohyl, silicification is accomplished in an extracellular space delimited by the sclerocyte pseudopodia. The silicalemma appears to be nothing more than the plasmalemma, as suggested by Simpson (1984). According to the results of the microanalyses, Si appears to be concentrated in the cytoplasm of the sclerocyte close to the growing spicule. It may be transferred from the sclerocyte to the perispicular space through the cell plasmalemma (Uriz et al. 2000). By investigating the homosclerophorid Corticium candelabrum, traditionally included in the class Demospongiae, Maldonado and Riesgo (2007) showed that two abundant cell types of the epithelia (pinacocytes), in addition to sclerocytes, contain spicules intracellularly. The small size of these intracellular spicules, together with the ultrastructure of their silica layers, indicates that their silicification is unfinished and supports the idea that they are produced “in situ” by the epithelial cells, rather than being incorporated from the intercellular mesohyl. The origin of small spicules that also occur (though rarely) within the nucleus of sclerocytes and the cytoplasm of choanocytes is more uncertain. The location, as well as the structure, of spicules is unconventional in this sponge. Cross-sectioned spicules show a subcircular axial filament externally enveloped by a silica layer, followed by two concentric extraaxial organic layers, each being in turn surrounded by a silica ring. Maldonado and Riesgo (2007) interpret this structural pattern as the result of a distinctive three-step process, consisting of an initial (axial) silicification wave around the axial filament and two subsequent (extra-axial) silicification waves. These findings indicate that the cellular mechanisms of spicule production vary across sponges and reveal the need for a careful re-examination of the hitherto monophyletic state attributed to biosilicification within the phylum Porifera.
8.7 The Role of the Organic Matrix in Biosilica Formation by Sponges Mechanism of silicification in sponges with respect to formation of skeletal frameworks, as well as spicules, is of principal interest for both materials science and bioinspired materials chemistry because of the possible future applications for the corresponding processes using biomimetic in vitro synthesis.
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Silica deposition is a fundamental process in sponges. I hypothesize that either there is one common mechanism which is based on enzymatic activity for silica formation and deposition of silica nanoparticles into surrounding organic matrices or that there are several mechanisms and several templates depending on specific environmental conditions. I am very skeptical of the proposed universal templating activity of silicateins because (i) of the differences in pH and salinity (ionic strength) between freshwater and marine water in different environments; (ii) they must be active also at very low temperatures (between –1.5 and 2◦ C); (iii) they must possess scaffolding properties of structural biopolymers like chitin or collagen to build the simultaneously rigid, twisted, and plywood-like, mechanically stable structures to be responsible for deposition of hundred grams of silica within, for example, up to 3-m-long but up to 3-cm-thick Monorhaphis spicule, or 600-kg-heavy and about 2-m-long Scolymastra skeleton. The difference in molecular weights between silicateins (about 30 kDa) and collagens (about 300 kDa) is amazing. Imagine the astonishment of the engineer who has been assigned the task of building a 5-m-high reinforced concrete-based pillar of diameter 0.5 m, who is told to choose between 3-cm-long nails or 3-m-long reinforcing steel rods which must be used as additives to concrete. Do you have some doubts about his decision? Therefore, from materials point of view, I cannot believe that giant silica-based constructs, for example, like 2-m-tall glass sponge Aspidoscopulia (Fig. 8.4) or 3-m-long Monorhaphis, have been biologically synthesized using only low molecular weight silicateins. The more realistic scenario seems to be that of the silicatein playing a role in sponges similar to that of acidic proteins in calcification or silaffins in silicification in diatoms, where chitinous networks are believed to be the main scaffolding material (Brunner et al. 2009). We also hypothesized early (Ehrlich and Worch 2007a) that silicateins are proteins responsible for the reconstruction of collagen, which forms templates necessary for the subsequent silicification. Despite my opponents’ desire to ignore the reality of the differing opinions in modern science regarding silicification phenomenon in sponges, I want to discuss here two different mechanisms of silicification in sponges which have been proposed during the last decade: enzymatic (silicatein-based) and non-enzymatic, or self-assembling (chitin- and collagen-based).
8.7.1 Silicatein-Based Silicification The group of Daniel E. Morse (Cha et al. 1999; Shimizu et al. 1998) first discovered that organic filaments in demosponge Tethya aurantium are composed of a cathepsin L-related enzyme, termed silicatein. They cloned two of the proposed three isoforms of silicateins, the α- and β-forms, from this demosponge (Cha et al. 1999). In subsequent years, these molecules were cloned from other sponges as well, for example, S. domuncula and Lubomirskia baicalensis (as reviewed in Müller 2003; Müller et al. 2007). Cathepsin L is an endopeptidase which cleaves peptide bonds with hydrophobic amino acid residues in P2 and P3 positions, and occurs in lysosomes as well as extracellularly as secreted enzymes. The silicateins are distinguished from
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the cathepsins by the replacement of the first amino acid residue in the catalytic triad, cysteine by serine (Shimizu et al. 1998). It has been proposed that serine increases the nucleophilicity during the nucleophilic attack at the silicon atom. The polymerization-promoting activity of the silicateins has been shown to be catalytic and not stoichiometric. It is possible that the controlled, punctuated secretion of low concentrations of monomeric silicateins, pulses in the transitory flux of silica– precursor molecules, oscillations in the pH, ionic strength or other conditions of the condensation environment, or a combination of these factors may be responsible for the annular patterning and continued deposition of silica in spicule biosynthesis in vivo once the axial filament has become completely covered during the early stages of silica deposition around this proteinaceous core. Alternatively, the continued growth of silica may be a result of dangling bonds at the growth surface of the newly deposited silica (Saito et al. 1995) There are two possible mechanisms for enzyme catalysis (Fairhead et al. 2008): (1) stabilize at the active site one molecule of deprotonated silicic acid (the nucleophile) which will then react with another molecule of silicic acid or (ii) stabilize a protonated silicic acid (the electrophile) which will then react with another molecule of silicic acid. In the proposed mechanism (Fairhead et al. 2008), the roles of C25S location and flanking mutations are simply to create a sufficiently sized pocket that will allow recognition of the tetrahedral Si(OH)4 molecule in such a way that the H163 can deprotonate it. The deprotonated Si(OH)4 protein complex can be thought of as a template for a condensation reaction. In this proposal there is no need to involve a high-energy covalent intermediate. The presence of a specific Si(OH)4 transporter in sponge suggests that the true substrate in vivo is indeed silicic acid, not high-energy silicon alkoxides. This being so, the simple acid–base activation mechanism authors propose seems a good model for the biological process. According to Müller and co-workers, formation of spicules is a biologically controlled, extracellular process (Müller et al. 2007; Schröder et al. 2006) (see above).
8.7.2 Chitin- and Collagen-Based Silicification Aminopolysaccharide chitin has a nanofibrous structure and the chain of pyranose ring arranges almost parallel to the (100) plane and extends along the fiber axis (Iijima and Moriwaki 1990). The chitin molecule has C=O, O–H, and N–H groups and oxygen atoms, which have affinity for the calcium, phosphate, carbonate, and hydroxyl ions of the corresponding calcium phases. However, the same functional groups possess an affinity for silicate ions. Because there is a possibility that such an oriented organic matrix may act as a template, or an ordered structural framework, the existence of naturally occurring silica–chitin composites was hypothesized (Ehrlich et al. 2007). Some of them were even discovered in sponges as well as diatoms (Brunner et al. 2009). The role of chitin in biosilicification is still not completely understood. Although chitin is one of the most important biopolymers in nature, knowledge of its
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interaction with silicon in vivo was largely absent. Only recently did we isolate and identify chitin from skeletal formations of marine glass sponges for the first time. The presence of chitin within the framework skeleton of F. occa (Ehrlich et al. 2007) and E. aspergillum (Ehrlich and Worch 2007b), as well as separate spicules of R. fibulata (Ehrlich et al. 2008b), could also be revealed by the gentle NaOH-based desilicification technique established by Ehrlich and co-workers (Ehrlich et al. 2006; Ehrlich and Worch 2007b). The structure of the chitin extracted from these sponges turned out to be similar to α-chitin from invertebrates. In 2006, we reported for the first time the example of a H. sieboldii glass sponge, whose spicules are a biocomposite containing a silicificated collagen matrix, and the high collagen content is the origin of the unique mechanical flexibility of the spicules (Ehrlich et al. 2006; Ehrlich and Worch 2007b). Recently (Ehrlich et al. 2010), we reported results of a thorough study on the nature of this collagen, which has been carried out by more than 20 scientists from different international scientific groups. Now we use a diverse suite of modern techniques to identify a hydroxylated fibrillar collagen that contains both a common and a [Gly-3Hyp-4Hyp] motif. The collagen motif determined in Hyalonema is consistent with the model of Schumacher et al. (2006) which describes 3(S)-hydroxyproline residues in the Xaa position of the collagen triple helix. This structure offers a plausible molecular model for the interaction between polysilicic acid and Gly-3Hyp-4Hyp polypeptides of isolated glass sponge collagen (Fig. 8.22). It is established that the interaction
Fig. 8.22 Model of interaction between polysilicic acid and Gly-3Hyp-4Hyp polypeptide (a) and Gly-Pro-4Hyp polypeptide (b) located in collagen triple helix. In the selected amino residues, carbon atoms are colored green, oxygen red, nitrogen blue, and silica white. Mechanisms for these interactions are schematically represented in (c) and (d) (image courtesy: Denis Kurek)
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occurring between orthosilicic acid and hydroxyl groups is likely to be a hydrogen bond (Tilburey et al. 2007). Our model shows the possibility of stable complex formation on the basis of hydrogen bonding between hydroxyl groups of polysilicic acid and surface-exposed hydroxyls of 3Hyp and 4Hyp. Our model also proposes a functional role for 3Hyp in sponge collagen silicification. Collagen will present a layer of hydroxyl groups, which can undergo condensation reactions with silicic acid molecules with a consequent loss of water. As a result, the initial layer of condensed silicic acid will be held fixed to the collagenous template, in a geometric arrangement that will favor further polymerization of silicic acid similar to the model proposed by Hecky et al. (1973). Hydroxylated collagen appears to form the basis for the extraordinary mechanical and optical properties of hexactinellid spicules (Kulchin et al. 2008). The self-assembly properties of collagen and its templating activity with respect to silicification are consistent with recent ideas on the development of hierarchical silica-based architectures (Pouget et al. 2007). Macroscopic bundles of silica nanostructures result from the kinetic cross-coupling of two molecular processes: a dynamic supramolecular self-assembly and a stabilizing silica mineralization. The feedback interactions between template growth and inorganic deposition are driven non-enzymatically with hydrogen bonding. We speculate that the hydroxylated glass sponge collagen may change the nature of silica in aqueous solution by converting the distribution of oligomers to a more uniform and useful set of nanoparticle precursors for assembly into the growing solid. Increased atmospheric oxygen levels became a key factor during the Proterozoic in the synthesis of collagen through its involvement in the post-translational hydroxylation of proline and lysine residues (Exposito et al. 2002), and it is tempting to speculate that the occurrence of silica- and hydroxylated collagen-based composites in skeletal structures of the first metazoan might be a co-evolutionary event. The procedure of alkali slow etching opens up the possibility to observe the forms of collagen fibrils located within silica layers of sponge spicules and their distribution. For example, the results obtained by SEM observations of the desilicified spicular layers of M. chuni provide strong evidence that collagen fibrils’ orientation within spicules possesses twisted plywood architecture (Ehrlich et al. 2008a). Typical fibrillar formations were observed within the tubular silica structures of M. chuni as well as H. sieboldii in all layers, starting from the inner axial channel containing axial filament (Fig. 8.23) up to the outermost surface layer of the spicules as shown in Fig. 8.23c. The fibrils in each cylinder form individual concentric 2D networks with the curvature of the corresponding silicate layers. These layers are about 1 μm in thickness and are connected to each other by protein fibers (Fig. 8.23b, e), which possess a characteristic nanofibrillar organization. Partially desilicificated nanofibrillar organic matrix observed on the surface of silica-based inner layers (Fig. 8.23d) of the demineralized spicule provides strong evidence that silica nanoparticles with a diameter of about 35 nm are localized on the surface of corresponding nanofibrils (Ehrlich et al. 2008a). This kind of silica nanodistribution is very similar to the silica distribution on the surface of collagen fibrils in the form of nanopearl necklace, observed first in the glass sponge H. sieboldii (Ehrlich
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Fig. 8.23 Computer-made representation of the multi-layered structure of the glass sponge spicule after partial demineralization using gentle alkali treatment (a) is based on the observation which has been carried out using SEM (b–e). Organic material is well visible in contrast to intact spicule represented in Fig. 8.20
et al. 2006; Ehrlich and Worch 2007a). We suggest that the nanomorphology of silica on proteinaceous structures described above could be determined as an example of biodirected epitaxial nanodistribution of the amorphous silica phase on oriented collagenous fibrillar templates (Ehrlich et al. 2008a). From this point of view, basal
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spicules of Monorhaphis- and Hyalonema-related sponges could be also defined as natural plywood-like silica ceramics, similar in organization to the crossed-lamellar layers of seashells. We also suggest that the matrix of the both kinds of anchoring spicules is silicificated fibrillar collagen, rather than collagen-containing silica, which is the reason for their remarkable mechanical flexibility. In 2005 I started my examination of numerous sponges’ spicular formations using SEM together with Carsten Eckert from Museum of Natural History in Berlin. We succeeded in finding unique cells while investigating the spicules of S. domuncula (Fig. 8.24). One year later Carsten and co-authors published some of these results (Eckert et al. 2006), but I did not agree with their interpretation of the
Fig. 8.24 Monitoring spicule growth in sponges using SEM. First, the unique, collagen-producing cells are seen to line up along the surface of the spicule (a), which forms a characteristic nanofibrillar structure (b). The line of cells can move from left to right along the spicule (c and d), depositing a rough, collagenous nanofibrillar layer in their wake (e and f)
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obtained results. Now, a few years later, I suggest that phenomenon observed by us at that time is of tremendous significance for both biologists and materials scientists. Initially these cells line up on the surface of spicules, as can be well seen on the SEM image (Fig. 8.24a). The characteristic structural feature of the cells is that they produce collagenous nanofibrils (Fig. 8.24b). Interestingly, the cells once aligned move from left to right (Fig. 8.24c, d) leaving behind a collagenous nanofibrillar layer. Thus, the initially smooth surface of the spicule became rough due to the presence of the new collagenous nanolayer (Fig. 8.24e, f). The diameter of these unique cells is about 1 μm. However, the axial channel of the S. domuncula spicule possesses the same diameter (Fig. 8.25). Moreover, the diameter of the nanofibrils of the axial filament is identical with that of the collagenous nanofibrils produced by the cells. Based on these observations, we propose the following hypothetical mechanism of spicule formation in the case of S. domuncula (Fig. 8.26). The collagen-producing cells find a space where they can line up and start to form the “embryonal” axial filament. Initially it can exist in the form of bundle of collagenous fibrils, with strong axial orientation between two cells (Fig. 8.26I). Each of the cells carries out the fission and the embryonal axial filament can grow divergently in this way. Around this time, the silicification must start and the first silica-based layer must be established around the “embryonal” axial filament (Fig. 8.26II). The cells, which on this step are located within the initial siliceous microtube, use the possibility to fission and, correspondingly, move out from the tube and land on the siliceous surface (Fig. 8.26 II, III). After that, a new generation of the collagen-producing cells arise. They line up on the surface of the prototype spicule (Fig. 8.26IV) similarly to as it has been shown in Fig. 8.24a. After that the cycle repeats again and again. Because of this phenomena, layer-by-layer structures, which are characteristic of siliceous sponge spicules, can be and has been observed using SEM (Uriz et al. 2003; Uriz 2006).
Fig. 8.25 The diameter of the nanofibrils of the axial filament of S. domuncula is identical to that of the collagenous nanofibrils produced by the cells (see Fig. 8.24) (image courtesy: Carsten Eckert)
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Fig. 8.26 A hypothetical mechanism for spicule formation in S. domuncula. A bundle of collagenproducing cells forms into an “embryonal” axial filament, growing by fission of the cells along a linear axis (I). Next, a silica layer is deposited around the cellular filament (II). The cells fission again and emerge from the new silica tube onto its surface (III), where the axial growth is then continued (IV) and repeated so that layers are formed (V) (image courtesy: Vasily V. Bazhenov)
According to our suggestion, the axial filament is nothing but collagen nanofibrils which were made by the initial collagen-producing cells. But it is well known that axial filaments of sponges usually appear as very dense filaments, sometimes triangular or quadrangular in cross section. It is, however, not surprising for us, because axial filaments are located within axial channels, which in turn may be used for ion transport. The life span of silica-forming sponges is reported to be between 100 and 15,000 years, therefore it is no wonder that during this time, axial threads could be mineralized, including the formation of the crystalline phases. Thus, not the organic, but the inorganic components of the axial filaments determine the formation of the triangular or quadrangular structures, which seems so unusual for biologists. However, within freshly formed spicule (Fig. 8.25), only well-defined nanofibrils of collagen are easily visible. Glass sponges are largely restricted to deep, cold-water (between 0 and 4◦ C) habitats (Dohrmann et al. 2008; Janussen et al. 2004; Tabachnick 1991). Therefore from the ecological point of view, collagen (as well as chitin-based)-based silicification which occurs in spicules and other skeletal formations of these sponges is an example of unique cold-water biomineralization. As I noted above, there is little work done in the literature on silicatein activities at this temperature level. Skeletal biomineralization requires energy and so imposes a metabolic cost on skeleton-forming organisms (Knoll 2003). I suggest that phosphate moieties and ATPase-based mechanisms are likely to be in some way involved in biosilicification at temperatures near zero. Probably, cells similar to those described above (Fig. 8.24) perform this job. Moreover, it has been established in the literature for
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a long time that sponges use only silica which is dissolved in water. But all glass sponges are sessile organisms with anchoring spicules or some kind of holdfasts. It is also known that spicules are tubular capillaries, which could be useful for the transport of different ions, including dissolved silica, within axial channels. But perhaps not only the silica dissolved in water is used. By this, I mean the silica from the sandy bottom or from a silicate-based mineral platform which is used by sponge larvae for attachment and initial growth. Unfortunately, the larvae in Hexactinellida were collected and investigated only in small Oopsacas sponge. Therefore, there is complete lack of information on possible bioleaching (demineralization) activity of glass sponges and their larvae as possible alternative sources of silicon.
8.8 Conclusion Biomimetics deals with the application of nature-made “design solutions” to the realm of engineering. In this context, mimicking biological materials with fine-tuned mechanical properties has been on the agenda of engineering research and development for many years. The premise of biomimetics is that it is possible to reduce the diversity and complexity of biological materials to a number of “universal” functioning principles. This requires foremost a deep understanding of the hierarchical structure of biological materials. It now appears that multi-scale mechanics may hold the key to such an understanding of “building plans” inherent to entire classes of material.
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Chapter 9
Phenomenon of Interspace Mineralization in the Bilayered Organic Matrix of Deep-Sea Bamboo Coral (Anthozoa: Gorgonacea: Isididae)
Abstract Isidids are octocorals in the order Gorgonacea, commonly known as bamboo corals. They are the deepest occurring habitat-forming deep-sea coral family, with a maximum depth of over 3,800 m. In contrast to Scleractinia corals, where the biomineralization phenomena have previously been well described, the peculiarities of mineralization for Isididae corals have remained unknown until now. Phenomenon of interspace mineralization in the bilayered organic matrix of deep-sea bamboo coral is described and analyzed. Mineralization in corals is generally considered as an organic matrix-mediated process in which the animal constructs an organic framework that induces nucleation and crystal growth, as well as subsequent crystallographic orientation and skeletal microarchitecture (Goldberg 2001). In contrast to Scleractinia corals, where the biomineralization phenomena have previously been well described (Cuif and Sorauf 2001; Dauphin and Cuif 1997), the peculiarities of mineralization for Isididae corals have remained unknown until now. Isidids are octocorals in the order Gorgonacea, commonly known as bamboo corals. They are the deepest occurring habitat-forming deep-sea coral family, with a maximum depth of over 3,800 m. More than 50% of Northeast Pacific occurrences of these corals are at a depth greater than 1,000 m (Etnoyer and Morgan 2003). F. Bayer and S. Cairns of the Smithsonian Institute in Washington, DC estimate 38 genera and 138 recent species within the family Isididae worldwide (Bayer 1990). Some taxa are unbranched, while others are copiously branched. Branching in some genera can occur at the nodes and in other genera at the internodes (Fig. 9.1a). The skeleton is covered with the colonial living tissue, the coenenchyme. The coenenchyme and polyps consist of epidermal layers enclosing mesoglea and calcareous sclerites. Sanchez (2002) characterized Isidids as typical representatives of suborder Calcaxonia (branching tree-like networks, calcareous joints, continuous multilayered axis (Fig. 9.1c, d)), but the nature of the proteinaceous axis and its homology to the gorgonin were unclear to him. Recently, (Ehrlich et al. 2006) we carried out studies to obtain some understanding of the nanotopography of the coral axial internode, of gorgonin node surfaces and intersurfaces, as well as the nature of nanoscale mineralized morphologies and observed textures. The role
H. Ehrlich, Biological Materials of Marine Origin, Biologically-Inspired Systems 1, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9130-7_9,
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Fig. 9.1 Bamboo corals of Isididae family possess finger-like architecture (a). Continuous multilayered axis is well visible on the cut surface of the skeletal nodes (c, d). Organic matrix can be easily isolated using osteosoft treatment (b, e, f, g). It possesses nanofibrillar, but not collagenous, organization (h). Gorgonin layers are usually visible in fluorescence (i) as well as light (j) microscopy
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of gorgonin-associated mineralization and the biomimetic potential of these bamboo corals for the future development of new biologically inspired materials for biomedical applications was also investigated. The decalcification procedure by Osteosoft treatment was used to gain understanding of the nature and nanostructure of the coral axial organic matrix (Fig. 9.1b). On the seventh day of decalcification, we observed only the presence of a transparent gelatinous pellicle after complete dissolution of the calcite-based axis internode (Fig. 9.1e, f, g). The identification of the organic matrix by means of SEM, TEM (Fig. 9.1 h), and AFM, respectively, shows fibrillar protein behavior. The results of the amino acid analysis of this organic matrix show glutamine and proline (28.9 and 24.0%, respectively) as the dominant components among other amino acids detected (Ehrlich et al. 2006). The very low content of glycine (2.5%) rules out the possibility that the fibrillar matrix had a collagenous nature. This is in contrast to the finding of collagen in the insoluble organic matrix of red coral Corallium rubrum reported previously (Allemand et al. 1994). Therefore, we arrived at the conclusion that the organic matrix of the Isididae axial internode is an example of an acidic fibrillar protein. These results are in accordance with the well-known postulate of Lowenstam and Weiner that acidic macromolecules fulfil important functions in biomineralization (Lowenstam and Weiner 1989). Remarkably, no disruption effects were obtained after an analogous decalcification procedure with a distal gorgonincontaining node, even after 3 months with OsteosoftTM incubation. However, alkali treatment resulted in disruption and partial dissolution of the same sample after 72 h. Light microscopy images of the transverse view of the distal dark brown colored Isidid node (Fig. 9.1i, j) show the presence of annual alternating rings of horny (gorgonin) and mineral nature. Elemental analysis (EDX/ESEM) shows the presence of C—57.73, N—20.91, O—10.94, P—1.05, S—1.96, Cl—1.65, Br—2.54, Zn—0.08, Na—1.86, Mg—0.87, Ca—0.21, Fe—0.20 (At/%). Until now, gorgonin (also named tanned-collagen, bromine-, iodine- or mucopolysaccharide-containing protein, or chitin–protein (Ehrlich et al. 2003)) had no clear chemical definition. Valenciennes first pointed out in 1855 that the internal skeleton of certain Mediterranean gorgonia was composed of a protein similar to horn and he called this gorgonin (Block and Bolling 1939). The unusually large amounts of tyrosine (13%), cystine (9%), and phenylalanine (6.5%) were demonstrated in gorgonin obtained by Block and Bolling in 1939 from Gorgonia flabellum. The organic matrix covering of the middle channel of the gorgonin distal node observed by means of SEM (Fig. 9.2a, b) appears brownish in light microscopy, which differs drastically from the transparent organic matrix of the axial channel. Differences between the two structures arise from a nanostructural point of view (Figs. 9.3a, b and 9.4a, b) as well. Mineralized fibrous structures can be observed on both interfaces of bilayered (layer thickness about 150 nm) epithelium from the middle channel of the gorgonin-containing node. The ca. 70 nm large spherical calcium carbonate formations are tightly distributed on microfibrils, which are placed or formed on the gorgonin intersurface (Fig. 9.5a, b). No mineral phase formations were observed on the surface of the gorgonin layers, which were free of
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Fig. 9.2 SEM image: transverse view of the distal gorgonin-containing node
Fig. 9.3 SEM images of the bilayered gorgonin-containing epithelium covering the middle channel of distal node
Fig. 9.4 SEM images of the bilayered gorgonin-containing epithelium covering the middle channel of distal node
microfibrils. Thus, gorgonin layers themselves could be templates for the formation of protein microfibrils, on which the biomineralization process could correspondingly start. SEM images obtained were similar to those submitted by Bayer and Macintyre (2001), where carbonate-containing hydroxyapatite was deposited in the form of “submicron spheres” in the chambered axial core in some gorgonians. We
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Fig. 9.5 (a) SEM Image of gorgonin-containing epithelium; (b) Gorgonin-containing epithelium as template for microfibrils on which mineralization takes place
surmised that the phenomenon of interspace biomineralization visualized here on the nanolevel may be common for octocorals. The identification of the pigment responsible for the dark brown color of gorgonin-containing internodes was carried out using UV/Vis- and FTIR spectroscopy. UV/Vis spectra of the saturated yellow-colored viscous supernatant (Fig. 9.6, right below) obtained after centrifugation (15,000g, 30 min) of the alkali-treated gorgonin-containing node (data not shown) were identical to
Fig. 9.6 Selective chemical treatment of Isididae sp. coral fragment lead to obtaining of fibrillar organic matrix (right above) or gorgonin-containing extracts (right below)
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spectral data reported by Koob and Cox (1990) for quinone-containing compounds in the dark pigment of skate egg capsules. FTIR results (Ehrlich et al. 2006) correlate exactly with the IR spectra obtained by Suci and Geesy (2001) for the well-known quinone-tanned Mefp-1 foot protein isolated from the sea mussel Mytilus edulis. A possible mechanism of gorgonin tanning may be as follows: the quinones react with terminal amino groups and sulfhydryl groups to produce a cross-linked stable protein. The brown color of quinone-tanned Isidid coral nodes is due in part to the polymerization of excess quinones to give melanin-like biopolymers. A schematic view (Fig. 9.7) of possible cross-linking in gorgonin by means of polyphenolic compounds illuminates the unavailability of these non-reactive oxygen residues for any interaction with gorgonin’s Ca ions. Comparative analysis of a broad spectrum of references related to other quino-proteins of marine origin such as byssus (Coyne and Waite 2000; Zhao and Wait 2005) or antipathin (Goldberg et al. 1994) confirms our assumption that most polyphenol-containing proteins are hydrophobic and
Fig. 9.7 Schematic representation of the gorgonin-based cross-links in Isididae corals (image courtesy Denis Kurek)
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basic and cannot be mineralized under conditions in nature. Therefore, gorgonincontaining nodes of Isididae corals are responsible for flexibility, not for hardness, in contrast to calcite-containing axial internodes.
9.1 Conclusion Thus, the results of the present study provide, for the first time, evidence that quinones are an important compound in the gorgonin-containing nodes investigated in samples from the Gorgonacean family Isididae. A comprehensive understanding of gorgonin-containing nodes with respect to chemical composition, structure, and mineralization behavior may prove to be a novel model for biomimetic synthesis of composites with specific bioelastomeric properties for biomedical applications. Polyphenol-containing biopolymers may be useful for the development of blood vessel implants and new biomaterials resistant to calcification.
References Allemand D, Cuif JP, Watabe N et al (1994) The organic matrix of skeletal structures of the Mediterranean Red Coral Corallium rubrum. Bulletin de l’Institute oceanographique, Monaco 14(1):129–139 Bayer FM (1990) A new Isidid octocoral (Anthozoa: Gorgonacea) from New Caledonia, with descriptions of other new species from elsewhere in the Pacific Ocean. Proc Biol Soc Washington 103(1):205–228 Bayer FM, Macintyre IG (2001) The mineral component of the axis and holdfast of some gorgonacean octocorals (Coelenterata: Anthozoa), with special reference to the family Gorgoniidae. Proc Biol Soc Washington 114(1):309–345 Block RJ, Bolling D (1939) The amino acid composition of keratins. The composition of gorgonin, sponging, turtle scutes, and other keratins. J Biol Chem 127:685–693 Coyne KJ, Waite JH (2000) In search of molecular dovetails in mussel byssus: from the threads to the stem. J Exp Biol 203:1424–1431 Cuif JP, Sorauf JE (2001) Biomineralization and diagenesis in the Scleractinia: part I, biomineralization. Bull Tohoku Univ Museum 1:144–151 Dauphin Y, Cuif JP (1997) Isoelectric properties of the soluble matrices in relation to the chemical composition of some Scleractinian skeletons. Electrophoresis 18:1180–1183 Ehrlich H, Etnoyer P, Litvinov S et al (2006) Biomaterial structure in deep-sea bamboo coral (Anthozoa: Gorgonaceae: Isididae): perspectives for the development of bone implants and templates for tissue engineering. Ma-wiss u Werkstofftech 37(6):552–557 Ehrlich H, Etnoyer P, Meissner H et al (2003) Nanoimage and biomimetic potential of some Isodidae corals. Erlanger Geol Abh 4:34 Etnoyer P, Morgan L (2003) Occurrences of habitat forming deep-sea corals in the Northeast Pacific Ocean. NOAA’s office of protected resources, Silver Spring, MD Goldberg WM (2001) Acid polysaccharides in the skeletal matrix and calicoblastic epithelium of the stony coral Mycetopyllia reesi. Tissue Cell 33(4):376–387 Goldberg WM, Hopkins TL, Holl SM et al (1994) Chemical composition of the sclerotized black coral skeleton (Coelenterata: Antipatharia): a comparison of two species. Comp Biochem Physiol 107B:633–643 Koob TJ, Cox DL (1990) Introduction and oxidation of catehols during the formation of the skate (Raja erinacea) egg capsule. J Mar Biol Ass UK 70:395–411
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Lowenstam HA, Weiner S (1989) On biomineralization. Oxford University, New York Sanchez JA (2002) Dynamics and evolution of branching colonial form in marine modular organisms. Ph.D. Thesis, University of New York at Buffalo, USA, New York Suci PA, Geesy GG (2001) Use of attenuated total internal reflection Fourier transform infrared spectroscopy to investigate interactions between Mytilus edulis foot proteins at a surface. Langmuir 17:2538–2540 Zhao H, Wait JH (2005) Coating proteins: structure and cross-linking in fp-1 from the green shell mussel Perna canaliculus. Biochemistry 44(48):15915–15923
Chapter 10
Bamboo Corals as Living Bone Implants
Abstract The structure of the commonly used coral, Porites, is similar to that of cancellous bone and its initial mechanical properties resemble those of bone. The exoskeleton of these high content calcium carbonate scaffolds has since been shown to be biocompatible, osteoconductive, and biodegradable at variable rates depending on the exoskeleton porosity, the implantation site, and the species. Also octocorals of Isididae family possess high biomimetic potential with respect to biomedicine. It is proposed that biotechnological processes for the aquacultural cultivation of Isididae corals as “living bone implants” should be developed in the near future. Natural coral (calcium carbonate) as well as bioactive glass, calcium sulfate, and calcium phosphates of biologic (derived from bovine bone, coral, and marine algae) or synthetic origin have recently been associated with bioactive bioceramics as an alternative to autografts and allografts (Hertz and Bruce 2007; Le Geros et al. 2008). These bioceramics are available as granules or blocks (dense or porous), specially designed shapes (wedges, cylinders) or cements and as coatings on orthopedic or dental implants. Researchers first started evaluating corals as potential bone graft substitutes in the early 1970s in animals and in 1979 in humans (see for review Demers et al. 2002). The structure of the commonly used coral, Porites, is similar to that of cancellous bone and its initial mechanical properties resemble those of bone. The exoskeleton of these high content calcium carbonate scaffolds has since been shown to be biocompatible, osteoconductive, and biodegradable at variable rates depending on the exoskeleton porosity, the implantation site, and the species. Although not osteoinductive or osteogenic, coral grafts act as an adequate carrier for growth factors and allow cell attachment, growth, spreading, and differentiation. When applied appropriately and when selected to match the resorption rate with the bone formation rate of the implantation site, natural coral exoskeletons have been found to be impressive bone graft substitutes. Coralline hydroxyapatite (CHA) is manufactured from marine corals by the hydrothermal conversion of the calcium carbonate skeleton of coral to hydroxyapatite, a calcium phosphate (see for review Damien and Revell 2004). The important steps in the preparation of sea coral for bone grafting and coralline hydroxyapatite
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Fig. 10.1 General schemes for the development of sea coral for bone grafting (adapted from Damien and Revell 2004)
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for bone replacement in clinical situations are given in Fig. 10.1. While many studies have demonstrated promising biocompatible properties and osteogenic results, as a bone graft substitute and bone void filler, the use of CHA may be limited owing to its inherent mechanical weakness and reduced biodegradation. The benefits of CHA as a bone graft material are predominantly its safety, biocompatibility, and osteoconductivity so that it can be used as a substitution biomaterial for bone in many clinical situations. CHA can also be used as an efficient carrier system for the local delivery of growth factors to enhance osteointegration and implant fixation into peri-implant osseous tissue. (Coughlin et al. 2006). Coralline hydroxyapatite was used also for development of the so-called bio-eye (Jordan et al. 1998). When implanted into the eye socket, porous hydroxyapatite is not treated as a foreign substance by the body, in contrast to most other implants used in the past, but becomes ingrown with blood vessels and tissue (Dutton 1991; Jordan et al. 2004). As a result of this integration with the orbital tissues, the implant resists migration and rejection. In the classical work by Guillemine et al. (1987) coral fragments of different sizes were implanted in the dogs’ bones. For example, in 15 dogs a cortical defect (length 8 mm, width 5 mm; see Fig. 10.2) was created in both ulnae (30 defects) and grafted with Porites coral fragments (open porosity). To investigate the possibility of using the calcite-containing internodes of Isididae octocorals, we implanted a corresponding fragment into the tibia of an experimental animal (Ehrlich et al. 2006). Under conditions of intravenous anesthesia, the oval aperture within the dog’s tibia was made using a milling cutter on low circulations. The sample was implanted into the hole and was quite visible after 24 h in the X-ray image, as its optical density is higher than that of the bone cortical layer. After 10 months the region where the coral sample was implanted was no longer visible, possibly owing to the complete resorption and biotransformation of the implant into the bone tissue. It has been suggested that the main factor in the coral resorption process is carbonic anhydrase, an enzyme abundant in osteoclasts (Ehrlich et al. 2009; Guillemine et al. 1987). The enzyme lowers the pH at the osteoclast–implant interface, dissolving the calcium carbonate matrix. Resorption of other natural coral fragments investigated was most active in the bone–matrix contact areas and proceeds centripetally.
Fig. 10.2 Schematic drawing of a cortical defect (length 188 mm, width 5 mm) grafted with a coral fragment and stabilized with a bone plate (adapted from Guillemine et al. 1987)
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Fig. 10.3 One fragment of the jointed, branched section of bamboo coral Isidella tentaculus (a) has been implanted with the aim to repair the mandible defect of a patient (b). X-ray images (c, d, e) show more detailed view
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Because of the impressive results obtained using dogs, the Russian team from Samara State Technical University and Kalinin-Hospital, (Samara, Russia) on February 22, 2006, successfully implanted a centimeter large bamboo coral fragment into the dental plate of the 17-year-old male patient as represented in Fig. 10.3. No infection or rejection of the coral implant was observed in this case. The resorption of the calcite-containing fragment of the coral implant was faster compared to that of bioceramics (personal communication from Russian colleagues).
10.1 Conclusion On the basis of the high biomimetic potential for the results reported above, we propose that biotechnological processes for the aquacultural cultivation of Isididae corals as “living bone implants” should be developed in the near future. Recently published data related to Isididae growth under natural conditions suggest that the growth rate of these corals was estimated to be between 19 and 120 cm/year (Ehrlich et al. 2006). The establishment of biotechnological approaches for the cultivation of bamboo corals will open a new and unique path for the development of natural bone implants.
References Coughlin MJ, Grimes JS, Kennedy MP (2006) Coralline hydroxyapatite bone graft substitute in hindfoot surgery. Foot Ankle Int 27:19–22 Damien E, Revell PA (2004) Coralline hydroxyapatite bone graft substitute: a review of experimental studies and biomedical applications. J Appl Biomater Biomech 2:65–73 Demers C, Hamdy R, Corsi K et al (2002) Natural coral exoskeleton as a bone graft substitute: a review. Biomed Mater Eng 12:15–35 Dutton J (1991) Coralline hydroxyapatite as an ocular implant. Ophthalmology 98:370–377 Ehrlich H, Etnoyer P, Litvinov S et al (2006) Biomaterial structure in deep-sea bamboo coral (Anthozoa: Gorgonaceae: Isididae): perspectives for the development of bone implants and templates for tissue engineering. Ma-wiss u Werkstofftech 37(6):552–557 Ehrlich H, Koutsoukos P, Demadis K et al (2009) Principles of demineralization: modern strategies for the isolation of organic frameworks. Part II. Decalcification. Micron 40:169–193 Guillemine G, Patat JM, Fournie J et al (1987) The use of coral as a bone graft substitute. J Biomed Mater Res 21:557 Hertz A, Bruce IJ (2007) Inorganic materials for bone repair or replacement applications. Nanomedicine 2:899–918 Jordan DR, Gilberg S, Bawazeer A (2004) Coralline hydroxyapatite orbital implant (bio-eye): experience with 158 patients. Ophthal Plast Reconstr Surg 20(1):69–74 Jordan DR, Gilberg S, Mawn L et al (1998) The synthetic hydroxyapatite implant: a report on 65 patients. Ophthal Plast Reconstr Surg 14:250–255 Le Geros RZ, Daculsi G, LeGeros JP (2008) Bioactive bioceramics. In: Pietrzak WS (ed) Orthopedic biology and medicine. Musculoskeletal tissue regeneration. Humana, Totowa, NJ
Chapter 11
Sand Dollar Spines
Abstract In contrast to other invertebrates, hard tissue in echinoderms generally contains interconnecting cavities and many open spaces. The skeletal plates and the spines of echinoids have a low to high magnesium calcite content depending on genetic and geographical control and each behaves as a single crystal. In contrast to all other echinoids, the test of the sand dollar has lost the globular shape and acquired a disk-like contour. Thus, the most distinctive morphological feature of the sand dollars is very flattened shape adapted to maintaining a stable position against wave action and the presence of enormous numbers of tiny spines. The spines of sand dollar Scaphechinus mirabilis possess a high biomimetic potential and give motivation for development of porous, hierarchically structured bioinspired materials. The diverse arrays of unique porous skeletons that occur in nature, especially in the case of marine invertebrates like echinoderms, possess intricately elaborate morphologies and structures suited to bionic, biomimetic, and materials science. The Echinoidea is a class of the phylum Echinodermata in the branch of the animal kingdom Deuterostomia, which also includes the crown groups Hemichordata and Chordata (Valentine 2004). Common names for members of the class include sea urchins, sand dollars, sea biscuits, and heart urchins. In contrast to other invertebrates, hard tissue in echinoderms generally contains interconnecting cavities and many open spaces (Su et al. 2000). The skeletal plates and the spines of echinoids have a low to high magnesium calcite content (depending on genetic and geographical control) and each behaves as a single crystal when examined by diffraction or polarized light (Politi et al. 2004; Su et al. 2000). The microstructure, however, is quite remarkable and complex, exhibiting a unique fenestrated structure of interconnected trabeculae and pores that are approximately 15 μm in diameter. The porous network and inorganic fractions of the shell occupy approximately equal volumes and display non-crystallographic curved surfaces. Indeed, the pore diameters and pore size distributions are highly controlled and some species of urchin are characterized by highly uniform distributions of pores in very regular structures.
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There is much interest in the preparation of materials with well-defined pore sizes. The use of the skeletal plates of sea urchins to template porous structures with pore sizes of the order of 15 μm was recently reported (Meldrum and Seshadri 2000; Seshadri and Meldrum 2000). The example material chosen in these studies was gold, but the method is quite general. A continuous coating of gold is deposited over the entire surface area of the skeletal plates. Subsequent dissolution of the plate leaves the gold coating in the form of the unique original structure. Thus, rather than attempting to replicate the structure of the echinoid plates by growing crystals de novo, the templating methodology profits from these existing structures (Seshadri and Meldrum 2000). Biocompatibility is the next advantage of the skeletal formations of echinoderms. Fontaine and Hall (1981) described a method for preparing cell-free, sterile echinoderm skeletal plates (ossicles) which were used as porous substrates for cell cultures. Ossicles of the starfish Pisaster ochraceus were evaluated as substrates for the culture of three mammalian cell lines. Each line grew vigorously on ossicles and fibroblasts quickly infiltrated their porous microstructure. More importantly, the preliminary evidence for biocompatibility presented suggests that native echinoderm skeleton has potential use as a biomaterial and because of its microstructure and relative solubility deserves evaluation as a kind of biodegradable ceramic. Spines of the echinoids Heterocentrotus trigonarius and Heterocentrotus mammillatus were converted by the hydrothermal reaction at 180◦ C to bioresorbable Mgsubstituted tricalcium phosphate (β-TCMP) (Vecchio et al. 2007). The converted β-TCMP still maintains the three-dimensional interconnected porous structures of the original spine. In vivo studies using a rat model demonstrated new bone growth up to and around the β-TCMP implants 6 weeks after implantation in rat femoral defects. New bone was found to migrate through the spine structural pores, starting at the outside of the implant and continuing through the pores on the edge of the implants. These results indicate the good bioactivity and osteoconductivity of the porous β-TCMP implants (Vecchio et al. 2007). Sea urchins in particular were intensively investigated due to peculiarities of their biomineralization-related processes (Blake et al. 1984; Kobayashi and Taki 1969; Mooi and David 1997; Smith 1990) like the transformation of amorphous phase into crystalline calcite on early step of morphogenesis of different skeletal formations (Aizenberg et al. 1997; Ameye et al. 2001; Killian and Wilt 2008; MacKenzie et al. 2004; Mann et al. 2008; Politi et al. 2008), the role of organic matrix macromolecules (Weiner 1985), incorporation of magnesium into skeletal calcites (Magdans and Gies 2004; Weber 1969), polycrystalline aggregate of echinoderm calcite (Towe 1967), its crystal orientation (Nissen 1969; Su et al. 2000) and its fracture mechanics (O’Neill 1981), calcite resorption (Märkel and Röser 1983) and regeneration of spines (Dubois and Ameye 2001). The skeletal architecture of echinoids is well described (Smith 1980a, b; Telford 1985), as well as the organization of skeletal tissues in the spines (Pilkington 1969). Mineral-based structures of adult sea urchins include the test, tooth, and spines. The spines of sea urchins consist of magnesian calcite with a typical content of
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Mg varying from 3 to 15 mol%. In addition, a much smaller amount of organic macromolecules are occluded in the structure (about 0.1 wt%) (Weiner et al. 2000). The calcite of the spine has a sponge-like morphology called stereom with wedges running radially about the long axis of the spine (Magdans and Gies 2004). According to Berman et al. (1990) and Aizenberg et al. (1997), a sea urchin spine consists of coherent blocks with dimensions from 100 to 200 nm with an angular spread of about 130 mdeg. Thus, in contrast to most other biogenic calcites, sea urchin spines are thus monocrystalline, but with a much higher mosaicity than inorganic calcite. Sea urchin spines come in a wide range of sizes, shapes, and colors (Burkhardt et al. 1983). Many sea urchins possess fewer spines or are barely covered by the spines. Contrary, sand dollar (animal related to sea urchins) species have a very dense and regular little hair-like structures that traditionally are also termed spines. The outer design of the spines for both echinoids looks similar. Scanning electron microscopic investigations of mineral skeleton of sand dollar spines (Mooi 1986) reveal a similar structure to the calcite skeleton of the large spines of the regular sea urchin Paracentrotus lividus (Aizenberg et al. 1997). However, the proteineous external epithelium enclosed biomineral framework of some sand dollar spines has sacs on its tip (Mooi 1986). In contrast to sea urchin spines, knowledge about the structure and chemistry of sand dollar spines is a very scant (Mooi 1986). Moreover, previous studies on sand dollars were mostly related to their biology (Hyman 1958), to their life in the subsurface of the sandy bottom (Telford et al. 1985, 1987), as well as to the role of spines in feeding behavior (Bell and Frey 1969; Chia 1969; Ghiold 1979; Goodbody 1960; Lane and Lawrence 1982; Mooi and Telford 1982; O’Neill 1978; Parker and Van Alsteyn 1932). These animals dwell in shallow water in a horizontal position. They are usually completely covered by a thin layer of sand, although sometimes they have been found to borrow up to 10 cm beneath the ocean bottom. The sand dollar Scaphechinus mirabilis Agassiz, 1863 (Agassiz 1872) possesses an abundant population in west coast water of Japan Sea (D’yakonov 1949). It is also widely distributed in the northwest of the Pacific Ocean, from southern Japan to the Aleutian Islands. These animals live in fine sandy ocean bottoms from intertidal to sublittoral zone and burrow shallowly on the surface of the sediments (Shigei 1986; Takeda 2008). S. mirabilis has considerable adaptive capabilities and is able to survive for a long time under extreme environmental changes with respect to salinity (Kashenko 2008). This sand dollar species has a diverticulum (Chen and Chen 1994), which consists of a series of tubes and pouches in the peripheral part of the test. When distended with sand grains, the diverticulum can form a weight belt (Mooi and Chen 1996). Interestingly, young sand dollars possess the ability to accumulate in their intestine diverticules significant amounts of sand to increase the weight of precisely the peripheral part of their body to give them stability and allow them to press down to the substrate in surf environment (Chia 1973). In contrast to all other echinoids, the test of the sand dollar has lost the globular shape and acquired a disk-like contour (Seilacher 1979). Thus, the most distinctive morphological feature of the sand dollars is a very flattened shape adapted to
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Fig. 11.1 Sand dollar S. mirabilis (a) possesses numerous spines (b) with different morphological features (c)
maintaining a stable position against wave action (Fig. 11.1a) and the presence of enormous numbers of tiny spines (Fig. 11.1b). It was reported that the aboral spine of sand dollars mechanically blocked sediment particles from falling between the spines, maintaining a required flow of oxygenated water inside their burrow in sediments (Mooi 1986). Observations of the underside of sand dollars, even when buried, show that the surface is perfectly free of fine particulate material: there is none adhering to spines (Telford et al. 1985). These spines are enriched by cells containing the pigment echinochrom A (Fig. 11.1a) (6-ethyl-2,3,5,7,8-pentahydroxy-1,4-naphthoquinone) and the related polyhydroxy-1,4-naphthoquinones (PHNQs) (Kominami et al. 2001; Nishibori 1957). The function of these pigments in adult animals may be related to the bactericidal activity in the coelomic fluid which is spines and blood cells (Lebedev et al. 2003). The sand dollar S. mirabilis is the main source of echinochrome, the pharmocore of cardiological and ophthalmological drugs (Anufriev et al. 1998). The drug histochrome was developed based on the echinochrome structure and has unique therapeutic properties (Fedoreyev et al. 2000). Moreover, this species produced several unique substances, e.g., echinamine A (3-amino-7-ethyl-2,5,6,8tetrahydroxy-1,4-naphthoquinone), the first marine aminated hydroxynaphthazarin (Mischenko et al. 2005), as well as pigments like spinazarin and ethylspinazarin (Yakubovskaya et al. 2007). Research into the efficient extraction of the pigments of S. mirabilis for the preparation of these drugs (Elyakov et al. 2001a, b; Mischenko et al. 2005; Pokhilo et al. 2006; Stonik 2005; Yakubovskaya et al. 2007) is still
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in progress. Considering that sand dollar quinones generally occur in pigment granules associated with Ca2+ , Mg2+ , and proteins, EtOH containing 10% H2 SO4 has been used for extraction. After evaporation of EtOH, the residue is partitioned between H2 O and CHCl3 . Pigments are extracted from the CHCl3 phase with aqueous Na2 CO3 and after acidification and extraction with CHCl3 , an organic layer containing quinines is obtained (Pokhilo et al. 2006). Up until today, however, only pigment-containing extracts have been used in pharmacological applications. The residual skeletons as well as spines left over after the extraction of biologically active pigments from the bodies of S. mirabilis have unfortunately, until now, simply been discarded. Increased understanding of biomineralization has initiated developments in biomimetic synthesis with the generation of synthetic biomimetic materials fabricated according to biological principles and processes of self-assembly and self-organization (Green et al. 2002). From this point of view, the spines of S. mirabilis (Fig. 11.2) possess a high biomimetic potential and give motivation for development of porous, hierarchically structured bioinspired materials. As is true for most irregular echinoids, sand dollar spines have lost their protective function and instead have become effective structures for burrowing and feeding. In sand dollars, these functions are related to sand of a given grain size, so that a certain optimum spine size should ideally be retained throughout the ontogeny. In general terms, the spines have become “sand grain related” structures (Seilacher 1979). All sand dollars are adapted not only to collect particulate food from the substrate
Fig. 11.2 SEM images of S. mirabilis spines (a). Both the tip (b) and the tubercle (c) show unique architecture
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material, but also to maintain their position on or in the bed of mobile sediment in moving water. Their spines have, over thousands of generations, evolved these unique formations with highly specialized function–structural relations. There are several types of spines of sand dollars described in the literature, e.g., club-shaped, aboral military, lunule margin, fringe, locomotory, geniculate, pressure drainage channel, cilium-oral, anal (Telford et al. 1985), ambulatory, frill, and shoe (Seilacher 1979) spines. Some of them play an important role in feeding, others in locomotion. Therefore, an incredibly broad range exists for the selection of corresponding microarchitectural spine-based models suitable for inspiration of material engineers and bionicists (Wilt 2005). Spines have been the subject of numerous morphological and ecological studies (as reviewed in David and Neraudeau 1989). In comparison, the structure of associated tubercles (Fig. 11.2c) has rarely been studied. Intact tubercles are composed of different stereom fabrics, corresponding to their successive concentric zones (Smith 1980b). Such a microstructural organization reflects the functional uses of the different parts of the tubercle: support of the spine, muscle, or catch apparatus attachment (Smith 1980a). In sand dollars, the spine is attached to the tubercle by two types of tissue: smooth muscle and collagenous connective tissue (Mooi 1986). Fibers from the catch apparatus penetrate deeply into the stereom of the spine and tubercle (Motokawa 1984; Smith et al. 1981). Another part of the catch apparatus passes from the central axis of the spine base through a perforation in the mamelon of the tubercle. Tubercles of regular echinoids are usually radially symmetrical and can be broadly separated into fixed-pivot and sliding-pivot systems. In irregular echinoids, spines are usually modified for a particular function and tubercle morphology is correspondingly varied. Spine and tubercle differentiation becomes quite pronounced in certain sand dollars. It is reported (Smith 1980a) that aboral tubercle density is correlated with sediment grain size. Microstructural observations of echinoids tubercles have been provided by Smith (1980a).
11.1 Conclusion The skeletal structures of Echinodermata have the intriguing biomimetic potential to serve as templates for bioinspired materials chemistry, architecture, and materials science (Fig. 11.3). Up to now, sea urchin skeletons have been the most intensively investigated, due to peculiarities of their biomineralization-related processes and the material properties of their calcified teeth, tests, and spines. In contrast to sea urchins, the underlying microstructure and chemistry of the highly perforated spines of sand dollars, especially S. mirabilis (Agassiz 1863), have not been subject to much previous investigation. This sand dollar is widely distributed in the northwest of the Pacific Ocean from southern Japan to the Aleutian Islands and is known as the main source of naphthoquinone-based substances, which have recently drawn medical attention for their use as cardiological and ophthalmological drugs. Unfortunately, after extraction of the naphthoquinones, the residual skeletons and spines of the sand dollars were usually discarded.
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Fig. 11.3 SEM imagery: nanostructural organization of tubercle part of the S. mirabilis spine (a, b) differs from that in the spinal ribs (c, d)
From an engineering and materials science point of view, the calcium carbonatebased skeletal formations of sand dollars have the potential to inspire a next generation of composites with remarkable microporosity, strength, and toughness. Undoubtedly, novel, environmentally friendly technologies that do not waste the remarkable skeleton will be discovered in the near future. This will clear the way for the use of sand dollars species as sources of products, which could find practical applications in pharmacology, medicine, and technologies. Investigations into marine ranching systems that eliminate the necessity of extraction from the natural environment are a top priority, as well as developing sand dollar embryonic cell cultures.
References Agassiz A (1872) Revision of the Echini. University Press, Cambridge Aizenberg J, Hanson J, Koetzle TF et al (1997) Control of macromolecule distribution within synthetic and biogenic single calcite crystals. J Am Chem Soc 119:881–886 Ameye L, De Becker G, Killian C et al (2001) Proteins and saccharides of the sea urchin organic matrix of mineralization: characterization and localization in the spine skeleton. J Struct Biol 134:56–66 Anufriev VP, Novikov VL, Maximov OB et al (1998) Synthesis of some hydroxynaphthazarins and their cardioprotective effects under ischemia-reperfusion in vivo. Med Chem Lett 8:587–592
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Bell BM, Frey RW (1969) Observations on ecology and the feeding and burrowing mechanisms of Mellita quinquiesperforaza (Leske). J Paleontol 43:533–560 Berman A, Addadi L, Kvic A et al (1990) Intercalation of sea urchin proteins in calcite: study of a crystalline composite material. Science 250:664–667 Blake DF, Peacor DR, Allard LF (1984) Ultrastructural and microanalytical results from echinoderm calcite: implications for biomineralization and diagenesis. Micron Microscopica Acta 15:85–90 Burkhardt A, Hansmann W, Märkel K et al (1983) Mechanical design in spines of diadematoid echinoids (Echinodermata, Echinoidea). Zoomorphology 102:189–203 Chen C-P, Chen B-Y (1994) Diverticulum sand in a miniature sand dollar Sinaechinocyamus mai (Echinodermata: echinodea). Mar Biol 119:605–609 Chia F-S (1969) Some observations on the locomotion and feeding of the sand dollar, Dendraster excentricus (Eschscholtz). J Exp Mar Biol Ecol 3:162–170 Chia F-S (1973) Sand dollard: a weight belt for the juvenile. Science 181:73–74 D’yakonov AM (1949) Taxonomy of Echinodermata in Far East seas. Vladivostok, Izv TINRO 30:1–132 David B, Neraudeau D (1989) Tubercle loss in Spatagoids (Echinodermata, Echinoides): original skeletal structures and underlying processes. Zoomorphology 109:39–53 Dubois P, Ameye L (2001) Regeneration of spines and pedicellariae in echinoderms: a review. Microsc Res Tech 55:427–437 Elyakov GB, Maximov OB, Mischenko NP et al (2001a) Histochrom and its therapeutic use in ophthalmology disease. Patent 6,384,084 Elyakov GB, Maximov OB, Mischenko NP et al (2001b) Histochrom and its therapeutic use in myocardial infarction and ischemic heart desease. US Patent 6,410,601 Fedoreyev SA, Mischenko NP, Koltsova EA (2000) A new drug, histochrome, from the sea urchin. Abstr. 5th Int Marine Biotech Conf, Townsville, Australia, 29 Sept–4 Oct 2000 Fontaine AR, Hall BD (1981) Biocompatibility of echinoderm skeleton with mammalian cells in vitro: Preliminary evidence. J Biomed Mater Res 15:61–71 Ghiold J (1979) Spine morphology and its significance in feeding and burrowing in the sand dollar, Mellita quinquiesperforata (Echinodermata: Echinoidea). Bull Mar Sci 29:481–490 Goodbody I (1960) The feeding mechanism in the sand dollar, Mellita sexiesperforata (Leske). Biol Bull 119:80–86 Green D, Walsh D, Mann S et al (2002) The potential of biomimesis in bone tissue engineering: lessons from the design and synthesis of invertebrate skeletons. Bone 30:810–815 Hyman LH (1958) Notes on the biology of the five-lunuled sand dollar. Biol Bull 114:54–56 Kashenko SD (2008) Responses of the sand dollar Scaphechinus mirabilis to extreme environmental changes. Russian J Mar Biol 34(3):166–169 Killian CE, Wilt FH (2008) Molecular aspects of biomineralization of the echinoderm endoskeleton. Chem Rev 108(11):4463–4474 Kobayashi S, Taki J (1969) Calcification in sea urchins. Calcified Tissue Res 4:210–223 Kominami T, Takata H, Takaichi M (2001) Behavior of pigment cells in gastrula-stage embryos of Hemicentrotus pulcherrimus and Scaphechinus mirabilis. Dev Growth Differ 43:699–707 Lane JM, Lawrence JM (1982) Food, feeding and absorption efficiencies of the sand dollar, Mellita quinquiesperforata (Leske). Estuarine Coastal Shelf Sci 14:421–431 Lebedev AV, Ivanova MV, Ruuge EK (2003) How do calcium ions induce free radical oxidation of hydroxy-1,4-naphthoquinone? Ca2p stabilizes the naphthosemiquinone anion-radical of echinochrome A. Archiv Biochem Biophys 413:191–198 MacKenzie CR, Wilbanks SM, McGrath KM (2004) Superimposed effect of kinetics and echinoderm glycoproteins on hierarchical growth of calcium carbonate. J Mat Chem 14:1238–1244 Magdans U, Gies H (2004) Single crystal structure analysis of sea urchin spine calcites: systematic investigations of the Ca/Mg distribution as a function of habitat of the sea urchin and the sample location in the spine. Eur J Mineral 16:261–268 Mann K, Poustka AJ, Mann M (2008) The sea urchin (Strongylocentrotus purpuratus) test and spine proteomes. Proteome Sci 6:22
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Chapter 12
Molluscs Spicules
Abstract Spicule as a morphological form of biomineral-containing structures is widely distributed in marine invertebrates. Moreover, this structure often possesses a nanoscale organic matrix which serves as a template for formation of calcium carbonates or silica phases. There are more than a dozen open questions with regards to the chemical composition and structural features of the spicular formations, both calcareous and siliceous, in molluscs. The nanoarchitecture of the organic templates within diverse spicules, as well as their function, is also not clear. Because of the large diversity of spicules, different mechanisms of their formation may exist. Unique spicular formations in different species of Nudibranchia, Aplacophora, Polyplacophora, and Onchidella molluscs are described and discussed. Many of the marine invertebrates possess calcium carbonate spicules. Kingsley (1984) published a review of the formation of these structures reported by other authors in the Porifera (Jones 1970, 1978; Ledger and Jones 1977; Woodland 1905), Coelenterata, Platyhelminthes, Mollusca, Echinodermata, and Ascidiacea (Giard 1872; Kniprath and Lafargue 1980; Lambert 1979; Loewig and Kölliker 1846; Woodland 1907). He writes as follows: “Mature spicules appear to be extracellular structures. Sponge spicules initiate intercellularly then become extracellular. Alcyonarian, turbellarian, echinoid, and ascidian spicule deposition begins intracellularly and then becomes extracellular. The continuation of growth in the extracellular environment has not been documented except for the echinoids. Placophoran spicules initiate and remain as extracellular structures. Early spicule growth seems to occur from or within a single cell. However, cell aggregation and/or neighboring cells appear to be important to the process of spicule formation. The spicule forming cells, in general, are found in a collagenous medium which may be associated with spicule growth.” Especially, he paid attention in his work to the organic matrix from the spicules of the gorgonian Leptogorgia virgulata, which is a glycoprotein. Autoradiography reveals that this matrix is apparently synthesized in the rough endoplasmic reticulum and Golgi complexes and then transported to the spicule forming vacuole via Golgi vesicles (Kingsley 1984). Molluscs are classified into seven different taxonomic classes, six of which are shell bearing: the Bivalvia, Gastropoda (except order Nudibranchia), Cephalopoda,
H. Ehrlich, Biological Materials of Marine Origin, Biologically-Inspired Systems 1, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9130-7_12,
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Scaphophoda, Monoplacophora, and Polyplacophora (Hyman 1967; Treves et al. 2003). The Aplacophora do not have a shell, but may have spicules embedded in their mantle tissue. All the shells are composite materials formed of mineral deposited inside an organic matrix framework. Because of the very broad and recent literature on shells as well as on nacre and mother pearl’s biomineralization and structural biology (e.g., reviews by Evans 2008; Gilbert et al. 2008; Marin et al. 2007; Paula and Silveira 2009), I want to focus the attention of the readers in this book to just the spicular formations within molluscs. Spicules of sponges are also discussed above in corresponding chapter. In most of marine invertebrates taxa, spicule mineralization is usually mediated by a single cell type (scleroblast) and involves filamentous collagen-like proteins, and mature spicules form extracellular structures that may undergo further growth and remodeling (Mount et al. 2004). The involvement of cells in shell formation may have evolutionary significance for the Mollusca. Scheltema’s phylogeny of extant Mollusca proposes two separate evolutionary lineages: Aculifera (spicule formers) and Conchifera (shell formers) (Scheltema and Schander 2000). Although any cellular processes of mineralization in the two groups may be strictly homologous, it is possible that they have a common origin. Of the two, spicules seem to be the more ancient form of mineral structures in molluscs. For example, calcium carbonate-based spicules were described in primitive molluscs like Solenogasters (Aplacophora) (see below in this chapter) which are placed at the base of phylogenetic tree (Kingsley and Marks 2008). Although these minute molluscs lack a continuous shell, they do have a discontinuous array of calcareous spicules embedded in, and extending beyond, their cuticle. Spicule as a morphological form of biomineral-containing structures is widely distributed in marine invertebrates. Moreover, this structure often possesses a nanoscale organic matrix which serves as a template for formation of calcium carbonates or silica phases. Therefore, the features listed above determine the high interest of spicular formations occurring in nature within the biomaterials science community.
12.1 Spicules of Nudibranchia The Nudibranchia (more than 3,000 species worldwide) stands at the pinnacle of the assemblage of “sea slugs” (Fig. 12.1) which are known variously as the Opisthobranchia, Euthyneura, or Heterobranchia (Alder and Hancock 1845–1855; Behrens 1991, 2004; Garstang 1890; Gosliner 1994; Todd 1981). According to Wägele and Willan (2000), nudibranchs are diagnosed by loss of the shell (and operculum) and simultaneous expansion of the notum over the dorsal body surface during metamorphosis, presence of papillae on the notum, detorsion so extensive as to result in virtually complete (external) bilateral symmetry, head distinct from foot, paired gustatory oral tentacled veil, paired dorsal chemosensory tentacles (rhinophores), multiple bilaterally symmetrical gills, hermaphroditic reproductive system with simultaneous maturation of gametes, obligate cross-fertilization
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Fig. 12.1 Nudibranch species (a, b) on the sea bottom (images courtesy A.V. Ratnikov)
involving copulation, semelparity, and 13 haploid chromosomes. The soft body is protected by elaborate defenses—morphological (spicules and kleptoplasty of cnidarian nematocysts), chemical (direct utilization of toxins from prey and in situ synthesis of toxins as repugnatory fluids), and behavioral (autotomy, crypsis). The group is exclusively free living and marine (except for the freshwater Anylodoh baicalensis), occurring in all habitats. Their diets consist of many major animal groups (Porifera, Cnidaria, Bryozoa, Crustacea, Mollusca, Ascidiacea), with individual species often displaying great specificity. Nudibranchs range in size from Amiwa cinnabareu, with an adult length of 4 mm, to Hexabrunchus sanguineus, with an adult length of 600 mm, though most species are under 30 mm (Wägele and Willan 2000). One of the first detailed descriptions of the spicular formation in nudibranchs was made by Woodland in 1907. He investigated Proneomenia aglaopheniee and made important remarks about localization and formation of the calcareous spicules as well. Woodland’s best approximation of the spherical concretion stage of the spicule is that represented in Fig. 12.2. The granule becomes a rod, and the rod assumes
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Fig. 12.2 Semi-diagrammatic drawing of Hiehypodermis and cuticle of P. aglaopheniee, illustrating the “carrying up” of the spiculiferous cells and spicules by the hypodermal papillae. The figure (× 400 diam.) is composed of drawings of the actual objects, brought together into one field. In (a) the spicule is in its normal position; in (b) the scleroblast is being detached from the hypodermis; in (c) the scleroblast has lost its spicule, this lying free as in K; in (d) a scleroblast with a well-grown spicule has been caught up in the young papillary elevation; in (e) a fairly young spicule has been carried up some distance by a papilla; in (f) the spicule has similarly been brought up, but is older; (g and h) represent young papillae; (j) a full grown papilla, with its pigmented swollen extremity lying just below. (h) is the outer limit of the cuticle. It must be understood that normally the papillae and spicules are quite distinct and are not in any way associated. (i) represents debris of the exterior of the thick cuticle; (m) is the hypodermis (adapted from Woodland 1907)
the form of the adult monaxon, growing over its entire surface by the deposition of calcareous matter derived from the scleroblast cytoplasm which entirely surrounds the spicule. According to Woodland (1907), the median portion of the adult spicule is formed first (evident when this is thickened), and the tapering extremities growing out from this. The scleroblast, i.e., single nucleus, never divides, so that the spicules are purely unicellular products. In most cases the body of the scleroblast (the small mass of protoplasm immediately surrounding the nucleus) is constantly situated midway in the length of the spicule, i.e., in the vicinity of where the spicules will be the thickest. Prenant (1924) and Schmidt (1924) investigated physico-chemical properties of Doris verucosa calcareous spicules using different techniques including demineralization, heating, and X-ray analysis. It was established that these formations are of amorphous calcium carbonate as well as of calcite, but never of aragonite (Schmidt 1944). Shell-forming organic matrix isolated from spicules of D. verucosa was surprisingly resistant to alkali treatment. This procedure, however, led to formation of micropores on the surface of the calcareous spicules. The optical properties of the spicules were also investigated and described for the first time by Schmidt (Schmidt 1924, 1944). More information about nudibranchs spicules has been reported during the 1960s and –1970s. Among opisthobranchs, and particularly doridacean nudibranchs, calcium carbonate skin spicules of an inter- or intracellular origin (Franc 1968; Rieger and Sterrer 1975b) are found to be rather common in the mantle, foot, gills,
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and rhinophores (Cattaneo-Vietti 1991; Cattaneo-Vietti et al. 1993; Kress 1981). Despite the fact that the presence of the spicules is well known, there is a surprising paucity of information on their formation and structure (Kingsley 1984). They appear early in the notum: in a 1 mm long Cadlina laevis, they were visible 2–3 days after hatching (Thompson 1967). Generally these spicules are fusiform, tri- or quadri-radiate, or spheroidal. A spiculose notum generally shows blunt tubercles of various sizes supported by densely packed spicules covered by the epithelium: in the Mediterranean Discodoris atromaculata they look like Native American tepees, while others lie in the intertubercle spaces (Cattaneo-Vietti et al. 1993). These carbonate spicules have different functions, determining the firmness, structure, and architecture of the body, and are generally considered as defensive (Ros 1976; Thompson 1960a), probably diminishing the energetic value of the prey (Todd 1981). In several species, the spicules protrude from the epidermis, forming a complex structure (caryophyllidia) to defend a specialized area, capping a sub-epithelial ganglion (Foale and Willan 1987). Little information is available on the type of mineralization of these kinds of spicules. Very high concentrations of calcium and magnesium were found in Archidoris pseudoargus (=britannica) and Jorunna tomentosa (as reviewed by Cattaneo-Vietti et al. 1995). Odum (1951) recorded “amorphous calcium carbonate” in the spicules of Archidoris, but actually they are considered to be composed of “amorphous” monohydrocalcite and “amorphous” fluorite (Lowenstam and Weiner 1989). Spicule features of several nudibranch species were thoroughly investigated by Cattaneo-Vietti et al. (1995). All the species examined showed fusiform crystalline calcium carbonate spicules in the mantle. In D. atromaculata, Discodoris fragilis, C. laevis, and A. pseudoargus, they were also found in the foot. In Anisodoris tessellata, spicules were found only in the foot. In all the species, the notal and pedal spicules are quite similar in shape; being slender, slightly curved, and sometimes spiny (Fig. 12.3). Generally they are very brittle, being hollow in the center (Fig. 12.3a), but others show quite complex concentric layers inside (Fig. 12.3b). These authors reported that the fusiform spicules are mainly composed of calcite (CaCO3 ) and brucite (Mg(OH)2 ), with a small percentage of fluorite (CaF2 ). The smaller spherules are almost pure calcite. Furthermore, the mineral composition of fusiform spicules of species collected at different latitudes showed inter- and intraindividual differences, but the Ca/Mg ratio does not seem to vary according to the environmental temperature. In fact, this ratio reflects the volume percent of calcite and brucite in a crystalline aggregate, and consequently, temperature has a poor influence on spicule composition. On the other hand, differences between foot and mantle, not only within the same species but also in the same specimen, suggest that the animal itself can influence the ratio of calcite and brucite nucleation. In this case, the different pH in mantle and foot tissue could induce a different uptake and use of calcium and magnesium. However, the presence of pure calcite spherules suggests that they could be related to variations of the ion concentration in the skin caused by pH variations
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Fig. 12.3 (a) D. atromaculata, empty fusiform spicule; (b) Austrodoris kerguelenensis, internal structure of a fusiform spicule, with concentric layers and radial pillars (c) A. tessellata, internal structure of a fusiform spicule, with concentric layers (adapted from Catteneo-Vietti et al. 2005)
due to the dermal gland activity or, more simply, could be considered as a calcium reservoir and source for the production of the slender spicules (Cattaneo-Vietti et al. 1995). More recently, recent reports about the presence of spicule networks in cryptobranch dorid nudibranchs have shown that they play two possible roles: defense against predators and structural support (Penney 2006). In one dorid, Cadlina luteomarginata, whole-mount and thin-section staining revealed an intricate network of spicule tracts and connective tissue ramifying throughout the body, with muscle fibers associated with this spicule/connective tissue matrix and inserting into it. Spicules were present in high concentrations in all areas of the body, but highest in exterior mantle tissue. Relative investment increased isometrically with body size for most body regions, in contrast to the positively allometric investment seen in prosobranch shells. Bioassays with artificial food
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indicated that spicules alone did not deter generalist crabs and anemones, and only weakly increased the deterrence of secondary chemicals to anemones. Thus, while nudibranch spicules may serve as a defense against other predators, their primary role may be in body support. Of course, characterizing these networks is crucial for understanding their functional roles and may also prove useful as a phylogenetic character (Wägele and Willan 2000). Many species of dorid nudibranchs bear, on the dorsal surface of their mantle, minute papillae or tubercles that are supported internally by calcareous spicules. In some dorids the papillae are simple, while in others they are highly organized and bear apical sensory organs (Foale and Willan 1987). Caryophyllidia belong to the latter category and appear to represent the most advanced form of spiculose mantle differentiation within the suborder Anthobranchia of the order Nudibranchia. In dorid nudibranchs Rostanga arbutus and Jorunna sp. caryophyllidia occur in large numbers (several thousand) uniformly distributed over the entire upper surface. Caryophyllidia are minute (40–50 μm diameter) and erect tubercles supported internally by 4–7 vertical, calcareous spicules which emerge in a crown surrounding an apical knob (Fig. 12.4). These structures display a high level of spicular organization and incorporate a complex muscle system at their base. The apical knob is formed from a specialized epidermis capping a sub-epithelial ganglion. The highly organized structure of the caryophyllidium indicates its potential importance as a new character in dorid taxonomy and phylogeny (Foale and Willan 1987). Each caryophyllidium appears to be formed by the stretching of epidermis over a framework of spicules. The interior of the caryophyllidium is therefore separated
Fig. 12.4 SEM of spicular structure of R. arbutus. (a) Epidermal cap from broken tip of emergent spicule. (b) Diagrammatic longitudinal section of a caryophyllidium showing spicule organization with respect to the epidermis (muscles not represented) (adapted from Foale and Willan 1987)
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Fig. 12.5 SEM images of nudibranchs spicular formations. (a) Epidermal cap from broken tip of emergent R. arbutus spicule. (b, c) Crystalline forms of these spicules (adapted from Foale and Willan 1987)
from the epidermis by the epidermal basement membrane and is continuous with the underlying connective tissue of the mantle (Fig. 12.5). The supporting spicules apparently never penetrate this basement membrane. Even after they emerge at the apex, all spicules remain ensheathed by a thin layer of epidermis. Each spicule is contained within a cell. This cell’s outer membrane is usually separated from the spicule by a layer of highly vacuolated cytoplasm. During analysis of literature related to spicules in molluscs, one unusual phenomenon arrested my attention, especially because of its nonsense from the first point of view. I mean the acid secretion by molluscs which at the same time possess calcareous spicules (Fig. 12.5). Analysis of the literature pertaining to spicules reveals an unusual phenomenon, which at first seems nonsensical: molluscs which possess calcareous spicules can also secrete acid (Fig. 12.6a, b). Defensive acid secretion is known to occur in a variety of opisthobranch and prosobranch molluscs. It was first described by Garstang (1890) from the opisthobranch Pleurobranchus membranaceus. Thompson and Slinn (1959) found that this fluid is of pH 1 and appears to be secreted by cells of the mantle as well as by a specific acid gland. A fluid of pH 1 or 2 is also secreted from the skin of the numerous molluscs species, from the polyclad turbellarians Cycloporus papillosus (Lang) and Stylostomum ellipse (Dalyell) (Thompson 1965), and from the ophiuroid Ophiocomina nigra Abildgaard (Fontaine 1964). In O. nigra, the acid is a highly sulfated mucopolysaccharide which can be stained readily with suitable histological and histochemical reagents. In the molluscs and polyclads studied by Thompson, the glands apparently responsible for secretion of acid appear clear in histological sections. There is strong evidence in the case of Pleurobranchus, Lamellaria, and Trivia that the acid contains free sulfuric acid (Thompson and Slinn 1959; Thompson 1961). The acid may play a part in burrowing, as in the date mussel Lithophaga, and in certain gastropods, sponges, and polychaetes in feeding or in defense (Thompson
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Fig. 12.6 Epithelial acid glands of gastropod molluscs. (a) Araldite section, viewed by electron microscopy, through the epidermis of Pleurobranchus. (b) Surface view of the living acid epidermis of Pleurobranchus, showing, through the transparent epidermal cells of the deeper lying calcareous spicules (adapted from Thompson 1983)
1959, 1960a, b, c, 1961). In this last category are examples from subgroups within both the opisthobranch and the prosobranch gastropods so diverse that it seems the phenomenon of acid secretion must have arisen independently many times. In the cases of Discodoris pusae and Onchidoris fusca, indicator paper was also applied to the dorsal surface of the animal under water. The result was a neutral pH, but as soon as the animal was prodded, the paper indicated pH 2 (Edmonds 1968). Thus these dorids can secrete a fluid of pH 2 or 1, or perhaps even less, when the animal is molested, suggesting that acid secretion is a defensive response (Thompson 1983). In spite of the neighboring localization of the calcareous spicules and acid sacs (Fig. 12.6a, b) within epidermal layers of dorid nudibranchs, they do not come into contact when internal cellular structures are intact. Probably, spicules are covered with ultrathin organic layer as previously reported by Schmidt (1944). From the biological point of view, however, molluscs which can protect themselves using both sharp spicules and the acidic “chemical weapon” should have advantages for survival in marine environments, even while being easy available for predators because of their limited mobility.
12.2 Spicules in Aplacophora Aplacophora is vermiform, spicule-covered molluscs that are most numerous and have the greatest diversity of species at depths greater than 200 m in the sea. Numerous works on their morphology, biology, and ecology has been published during the last 130 years (Baba 1938, 1940, 1951, 1999; Hansen 1889; Heath
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1911, 1918; Hubrecht 1881; Koren and Danielssen 1879; Kowalewsky and Mahion 1887; Nierstrasz 1902; Thiele 1897, 1913; Salvini-Plawen 1968, 1972, 1978; Wiken 1892). Their internal anatomy seems to be primitive for the Mollusca, and the vermiform shape derived, a combination thought to be the result of progenesis (Scheltema 1993, 1996). Aplacophora comprise two taxa, the Neomeniomorpha (Solenogastres) and the Chaetodermomorpha (Caudofoveata) (Scheltema et al. 1994). Most neomenioids creep by means of a narrow foot on mud bottoms or on hydroids and octocorals upon which they feed. They are also hermaphrodites. Chaetoderms are burrowers feeding upon foraminifera or organic detritus; they are dioecious. Recent accounts of aplacophoran anatomy are given in Salvini-Plawen (1985) and Scheltema et al. (1994). Aplacophoran spicules are aragonite, with the long axis of the crystals aligned with the long axis of the spicules. Development of the spicules in aplacophoran molluscs has been observed and described also for both larval and postlarval forms. Okusu (2002) reported the results of investigations on embryogenesis and spicule formation in Epimenia babai (Neomeniomorpha). It was observed that a ciliated foot, a pedal pit, and aragonitic spicules in this species develop from the definitive ectoderm of the embryo. A spicule begins as a solid tip, continues to be an openended hollow spicule, and finally becomes a closed-ended hollow spicule. Different forms of spicules are well visible on the surface of E. babai larvae using SEM (Fig. 12.7). More complex structural morphology is evident in some aplacophoran postlarvae. Scheltema and Ivanov (2002) described a tiny neomenioid postlarva collected from the water column 3–6 m above the east Pacific seamount Fieberling Guyot which has six iterated, transverse groups of spicules and seven regions devoid of spicules between the transverse groups and the anterior and posterior most spicules (Fig. 12.8). Three pairs of ventral, longitudinal zones with columns of single spicules, each pair with its own distinctive spicule morphology, lack transverse iteration. The seven regions bare of spicules are compared to shell fields in developing polyplacophorans, and spicule arrangement is compared to sclerite arrangement on the Cambrian fossils Wiwaxia corrugata and Hulkieria evungelista and to the spines and shell plates of the Silurian Acaenoplax hayae. Some remarkable similarities in sclerite and spicule arrangements between the two postlarvae and Paleozoic fossils open possibilities for developing new characters useful in phylogenetic analysis (Scheltema and Ivanov 2002). The authors hypothesized that the morphological and structural features observed by them are similar to those described by Kowalevsky (1883) on embryos of polyplacophorans. Thus, the aplacophoran postlarvae seems to be an evolutionary stage preceding the development of shell plates by the coalescence of spicules in Polyplacophora (Salvini-Plawen 1972, 1985). Unfortunately, there is no data about any kind of organic templates in these postlarval spicules and about their ultrastructural features. Adult forms of Aplacophora are diverse in structural features of the aragonitic spicular formations. For example, the epidermal spicules can be (Scheltema and Schander 2000):
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Fig. 12.7 (a) SEM images: postmetamorphic juvenile of E. babai possess larval spicules (Sp). Three types of larval spicules right after metamorphosis. (b) Dorsal view. Two types of spicules are equally abundant over the entire body of the juvenile: hollow upright spicules (Sp1) lie against the body, while blade-like solid spicules (Sp2) project outward from the body. (c) The third type of larval spicule (Sp3) is broad and solid, forming a single row on each side of the pedal groove (adapted from Okusu 2002)
(1) skeletal (5 tangential)—the spicules lie within the cuticle, at right angles to each other, in one or more layers, spiraling from ventroanterior to dorsoposterior and from dorsoanterior to ventroposterior; (2) upright (5 radial)—arranged in a single layer, more or less erect, usually with the distal ends extending beyond the cuticle (Fig. 12.19); and (3) adpressed, with a single layer of overlapping spicules lying flat against the body wall cuticle (e.g., Tegulaherpia; see Scheltema 1999a). Species may exhibit one arrangement, a combination of (1) and (2) or a combination of (2) and (3) (e.g., Acanthomenia; see Scheltema 1999b). Spicules may
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Fig. 12.8 Postlarva (a) of a neomenioid aplacophoran from Fieberling Guyot showing transverse groups of spicules; anterior to the left. Light areas are cuticle devoid of spicules. The arrow indicates a barbed spicule. (b, c) Drawings of the same postlarva (adapted from Scheltema and Ivanov 2002)
be rounded and hollow with a wall surrounding a single space, or solid and flat or rounded, and encompass a variety of shapes and sizes; several types may occur within a single species. The spicules beside the pedal groove are somewhat arcuate, or at least convexly curved on one side, and are arranged in a single longitudinal row on each side of the pedal groove; they often bear a “handle,” or root. Additionally, the aragonitic spicules of the Aplacophora are ornamented to varying degrees. In the family Chaetodermatidae they generally lie tangential to the body wall and are overlapped, with the distal, pointed ends directed posteriorly, or, in particular regions of some species, they are bent at their proximal ends and held erect, perpendicular to the contracted body wall (Scheltema 1976). Ornamentation is on the outer side; if the inner side is grooved, the base of the spicule appears indented (Fig. 12.9). The spicules very often have a median keel. They differ morphologically along the body from anterior to posterior, and any part of the body may bear more than one type of spicule. Within a species the predominance of type can change with age. Solenogaster molluscs (called Crystallophricon) which were thoroughly described by Schwabl (1961a, b) possess different kinds of spicules within one species (Fig. 12.10). Recently, in collaboration with Dmitry Ivanov we started investigations on other Solenogaster species and related molluscs (Fig. 12.11). Most neomenioids have a few to numerous specialized spicules at the entrance to the mantle cavity; these are presumably used in copulation. One or more copulatory
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Fig. 12.9 Diversity of the spicules isolated from Chaetoderma abidjanense n. sp (adapted from Scheltema 1976) Fig. 12.10 Crystallopbrisson recisum (a) The entire animal is shown in side view, showing the body regions. a, b, c, d, from which are taken the spines represented in (b). Spines from body regions, showing characteristic ones from regions a, b, c, and d (adapted from Schwabl 1961)
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Fig. 12.11 Spicule-forming molluscs (images courtesy Dmitry Ivanov)
spicules are found in many species of neomenioids. They are largely or entirely calcitic (Fig. 12.12) and are secreted in deep, usually paired pockets of the mantle cavity; they are thus of epidermal origin. The exact function of copulatory spicules has never been determined. They are deciduous or become resorbed in some species, and some may be one-third the total length of an individual. Recently, Kingsley and Marks (2008) reported the unusual hydrothermal Solenogaster Helicoradomenia acredema collected by Dr. Cindy Lee Van Dover (The College of William and Mary) from hydrothermal vents of the northern East Pacific Rise. Scanning electron microscopy of its spicules, which cover the entire body surface, reveal a shaggy appearance at low magnification. At higher magnifications, it is apparent that there are multiple geometries of spicules, and many of them have hollow distal tips. Further examination by both SEM and transmission electron microscopy (TEM) reveals both epibiotic and endocuticular bacteria along the body. Of the identifiable prokaryotes, the majority were found to be
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Fig. 12.12 Schematic organization of the posterior body of Dorymenia troncosoi (As—abdominal spicule; Cs—copulatory spicule; Cu—cuticle; Dso—dorsoterminal sense organ; Go—gonad; Ht—hearth; Mg—midgut; Pc—pallial cavity; Pd—periocardioduct; Pr—pericardium; Re— rectum; Sc—suprarectal commissure; Sd—spawning duct; Sr—seminal receptacle; Vs—seminal vesicle
α-Proteobacteria or γ-Proteobacteria, and are believed to be in symbiotic association with Helicoradomenia. The bacteria were closely associated with the spicules both on, and within, the hollow portions of the spicules. The authors also observed bacteria emerging from the tips of spicules. TEM reveals a mantle epidermis consisting of a cuticle embedded with spines. When decalcified, an organic matrix is found within the spines. Early spine formation begins within a crystallization chamber in the mantle epithelium. The crystallization chamber appears to be formed by the deep invagination of the microvillus epithelium. Nucleation and growth are thus extracellular. Neighboring cells adjacent to the epidermal cells containing the developing spines are involved in spine growth. During this growth, the spines emerge from the mantle. Mature spines break through the epithelium into the external environment (Kingsley and Marks 2008).
12.3 Spicules in Polyplacophora (Chitons) The chitons (Polyplacophora) have a shell composed of eight aragonitic plates, hence the name Polyplacophora (bearer of many plates) (Fig. 12.13). The shell covers only part of the body, while the remaining part of the body, the girdle, may be protected by spicules, scales, or may not be mineralized (Pilsbry 1892–1894; Thiele 1909a, b; Van Belle 1999). The Polyplacophora often possess spicules which are either embedded in the cuticula or protrude from it (Rieger and Sterrer 1975b). The site of formation of these spicules is vague. Rieger and Sterrer (1975b) report that each spicule is a secretion of a single epidermal cell. This statement, as well as
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Fig. 12.13 Chiton under sea bottom (image courtesy A.V. Ratnikov). Note the dorsal plates, forming the shell, and the girdle, decorated by microspicules, surrounding them
examination of their micrograph, suggests an extracellular origin. However, they state that a spicule starts intracellularly but eventually will protrude from the cell. In either case, however, the spicules ultimately become extracellular. These structures are suspected to be aragonite (Rieger and Sterrer 1975b). Spicules are also found in the Acochlidiacea. In Hedylopsis, spicules are embedded in the connective tissue layer underlying the epidermis (Morse 1976). There have been no direct observations of spicule formation. Rieger and Sterrer (1975a) did observe, however, that each spicule is completely enclosed by one cell which they believe is also responsible for its formation. The crystal type of these spicules has not been determined. Development of the spicular formations during metamorphosis in Mopalia muscosa was described in detail by Leise (1984) as follows. A 5-day-old trochophore is barrel shaped and about 350 μm long. Its epidermis is a simple columnar epithelium, without the papillae of the adult epidermis. No spicules are present at this stage. All of the epidermal cells, including the ciliated ones, have an apical fringe of uniform microvilli as a brush border. The epidermal cells retain this brush border until metamorphosis. A cuticle as is seen in the juvenile is not present. Large goblet cells containing mucus are scattered throughout the epidermis. Some of the cells around the edge of the mantle field of 6-day-old larvae bear calcareous primary spicules, a characteristic that distinguishes the presumptive girdle from the anlagen of the shell glands. These primary spicules are intracellular, about 6 × 1 μm, conical, and supranuclear. A spiniferous cell produces only one spicule. Five to six rows of spiniferous cells ring the entire mantle field. The spicules remain intracellular until the larva settles, during day 9 or 10. The spicules increase in size until they are about 9.0 μm long and 3.0 μm wide just before settlement. Although the cells of the girdle are still columnar at 6 days, they become shorter (20 μm) until papillae differentiate. After 9 or 10 days, the free-swimming trochophore is ready to settle and metamorphose. At this stage the larva is about 400 μm long and dorsoventrally flattened.
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Within 24 h after settlement, the larva shortens to about 310 μm and transforms into a juvenile. The animal loses the prototroch and apical tuft, secretes a cuticle, the shell plates and hairs, and extrudes the primary spicules. Shell is deposited below the cuticle in the central part of the mantle field and peripherally the tips of the girdle spicules protrude into it. The cuticular surface of young juveniles is scalloped. Some crenulations correspond to spicule tips or hairs. At first, only the tips of the spicules lie within the cuticle, but by 2 days after settlement the spicules are completely extruded and enveloped by the cuticle. The cuticle is thick dorsally and thin ventrally. Primary spicules have thin cups and do not possess the dense chitinous cup, shaft, or central pigmented granules of adult spicules (Leise and Cloney 1982). Lia Addadi and co-workers (Treves et al. 2003) investigated aragonite formation in adult chitons girdle. Four different genera of chitons were examined and compared: Acanthopleura uaillantii, Acanthopleura spinigera, Nuttalina fluxa, and Ischnochitonina sp. The girdles of the first three are decorated by spicules with a typical sturdy elongated shape. A. uaillantii has bands of light and dark spicules, which are different in color, and these were examined separately. The only mineral component in the spicules and plates is aragonite, based on X-ray diffraction and IR spectroscopy. Individual spicules or scales behave under polarized light and X-ray diffraction as polycrystalline materials. Indeed, broken sections of the spicules observed in the scanning electron microscope have a morphology characteristic of polycrystalline aragonite. The single crystallites appear as discrete, elongated prisms, a few micrometers in length and are of sub-micrometer thickness. The individual crystallites are tightly packed in bundles. Within a bundle, the elongated prisms have their long axes (the c-axis of aragonite) aligned, thus forming a compact material. Interestingly, the insoluble matrices of the spicules appear to be composed, in all samples, of heavily glycosylated proteins (Treves et al. 2003). These form a meshwork of fibers that are morphologically very different one from the other in the samples studied (Fig. 12.14). Specifically, A. uaillantii white spicules and Ischnochitonina sp. scale matrices appear in dried samples as lamellae and do not have a preferred spatial orientation. In contrast, the fibers comprising the matrix of A. spinigera spicules (Fig. 12.14a) and A. uaillantii black spicules (Fig. 12.14b) form a meshwork with a clearly defined preferred orientation. To obtain a better diffraction pattern from the organic material, spicules were partially dissolved, and, indeed, these diffraction patterns show much clearer components from the organic matrix fibers. The matrix reflections include arcs and dots. The arcs are parallel to the (002) reflection of the aragonite crystals, while the dots are well aligned with the (111) and the (021) reflections of aragonite. Thus, the authors reported a well-defined spatial relationship between matrix and mineral. Surprisingly, these reflections do not correspond to chitin or to any identifiable fiber structure; however, the organic matrix and mineral are clearly related (Treves et al. 2003). The authors also note that the amount of organic matrix, 2 ± 3% by weight of dry mineral, is higher than in other mollusc mineralized tissues, which is normally ca. 1% .The amounts of protein in the spicules and scales, on the other hand, are
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Fig. 12.14 SEM images of demineralized girdle hard parts of chitons. (a) A. spinigera spicule matrix. (b) A.ualantii black spicule nanofibrillar matrix (adapted from Treves et al. 2003)
remarkably low, between 0.07 and 0.23%, indicating that most of the matrix consists of polysaccharides. The organic material also forms a dense, massive meshwork of fibers or lamellae, very different from the framework commonly observed in mollusc shells. All the above evidence indicates that the mineralized parts of the chiton girdle have mechanisms of formation different from those of the other mineralized parts of the same organisms, and probably of other molluscs as well. It is, thus, of interest to try to better understand the modes of control involved in girdle spicule and scale formation.
12.4 Onchidella Spicules Onchidium is a small, naked, pulmonate mollusk which is remarkable because of several conspicuous peculiarities. The habitat of Onchidium is littoral-marine and the animal divides its existence between life in the water and in the air. Leslie B. Arey
During my work on this book, Konstantin R. Tabachnick focused my attention on the brief remark which he found in a Russian Encyclopedia about siliceous spicules in molluscs, which were reported for the first time by French researcher Labbé in 1933. Surprisingly, I cannot find any other references about this in the existing literature, except those from Alphonse Labbé (Labbé 1933a, b, 1934a, b, c, 1935). Recently, reading the review by Benoit Dayrat (2009) on the current knowledge of the systematic of these molluscs, I understood that these animals possess more than “several conspicuous peculiarities.” Unusual molluscs such as these are related to the taxon Onchidiidae (Gastropoda: Pulmonata). Although the first finding of Onchidium was reported by Buchannan in 1800, Onchidiidae, according to Dayrat (2009), is a poorly known taxon in many regards. Its systematics, which is in a state of confusion, needs to be revised: the status of available species names needs to be addressed so that species diversity can be estimated; the supra-specific relationships need to be reconstructed and the status of current genera needs to be tested using modern phylogenetic methods. The first known species of onchidiacean, Onchidium typhae, was described in 1800 by
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Buchanan. In 1804, Cuvier added a second species and placed the group within the pulmonates. This was challenged by Blainville in 1818, who maintained that they were opisthobranchs. From that time, almost every paper which mentioned the group altered their taxonomic position and they were reassigned between these two subclasses at least once every 10 years. Our anatomical knowledge of onchidiids is also poor. Thus, I found first anatomical data in the works by von Plate dated 1893 and 1894 (Plate 1893, 1894), by von Wissel (1898), and by Stantschinsky (1908). Some unusual anatomical findings were reported. These include the mantle of some species bearing eyes with retinulae of the inverted type—a condition unique among gastropods (Hirasaka 1912; Semper 1877; Stantschinsky 1908). The presence of lungs, except as analogues adapted from a portion of the kidney, was long denied, but this error has since been corrected. Cuvier (1805) was the first to describe lung cavities in Onchidium, but this observation later gave way to other interpretations. Milne-Edwards (1857) considered the pouch hitherto described as lung to be kidney, like that of other gastropods. This view was revived by von Ihering (1877), who interpreted the so-called lung of Onchidium as being comparable to the broadened cloacal portion of the kidney of some other marine forms. This he thought had undergone a partial functional change from a primarily secreting organ to a respiratory sac. The conclusions of von Ihering were enthusiastically supported by JjoyeuxLaffuie (1882). On the other hand, Semper (1876) argued for the existence of two separate though juxtaposed organs. This opinion was shared by Bergh (1895), and especially by von Wissel (1898). As the results of the investigations of these latter workers, the Onchidium family became removed from the nudibranchs and now rests securely once more among the pulmonates. However, respiratory behavior of onchidiids is more specific than those of terrestrial pulmonates. For example, Onchidium floridanum respires cutaneously, and chiefly through its numerous though short mantle papillae, both when submerged and when quiescent in air (Arey and Crozier 1921). Under both of these environmental conditions the pulmonary aperture remains closed and the lungs are inoperative. When given a choice, most animals select the air for long periods of quiescence. When an animal is actively crawling out of water, the posterior portion of the mantle is raised from the substrate to expose the opened pulmonary orifice and facilitate pulmonary respiration, the existence of which is demonstrable. The lungs could be of considerable service during such periods of activity, and it may be that they actually dominate the terrestrial respiration of active animals. Such a conclusion, however, does not rest on any sound evidential basis and the lungs may serve merely as accessory respiratory organs when the animals are out of water and active. If the lungs be subordinate in the terrestrial respiration of active Onchidia, in addition to their disuse during aquatic life and inactive air existence, then the lungs of this pulmonate (which has secondarily acquired marine habits) have indeed sunk to a low-functional state (Arey and Crozier 1921): Thus, most of the literature on onchidiids was published before the 1940s. Few studies have been published since then (e.g., Awati and Karandikar 1948; Britton 1984; Marcus 1978, 1979; Tillier 1983; Weiss and Wägele 1998). Most species were
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succinctly described and distinguished based on few characters, such as the appearance of the dorsal notum, and, occasionally, the radular formula and the anatomy of the male genital organs (penis and accessory gland). The internal anatomy of the vast majority of the species is still unknown. Scanning electron microscopy (SEM) pictures of the radula, one of the most standard features in gastropod systematics, have been published for only two species: Onchidella celtica and O. indolens (Weiss and Wägele 1998). No SEM pictures have been published for features that are critical at the species level, such as the penis and the spine of the accessory penial gland. Onchidiidae, one of the nine major extant taxa of Pulmonata (Mollusca, Gastropoda), has been understudied since the last onchidiid experts were active more than 70 years ago (e.g., Hoffmann 1928a, b, 1929; Labbé 1934a, 1934b, 1934c, 1935; Plate 1893). In fact, there is no living expert able to reliably identify species of onchidiids, and museum lots are often labeled as “Onchidiidae” (Dayrat 2009). A few large monographs focused on parts of European collections: London, Frankfurt, and Berlin (Plate 1893); Copenhagen and Stockholm (Hoffmann 1928a, b); Paris (Labbé 1934a). Onchidiids are true slugs: they lack an internal shell. Most species are marine and live in the upper intertidal zone, either in rocky, sandy, or muddy habitats, including mangroves. However, two species live in brackish habitats and tolerate fresh water: O. typhae (Buchannan 1800) and Labella ajuthiae (Labbé 1935). Also, three terrestrial species have been described from high-elevation rainforests: Semperella montana (Plate 1893), from Sibugan Island, Philippines; Platevindex ponsonbyi (Collinge 1901), from Borneo; and Platevindex apoikistes (Tillier 1983), from Mindoro, Philippines. The highest elevation record of terrestrial onchidiids found in the literature is the original description of P. ponsonbyi (850–1060 m), although the author has undertaken the study of onchidiids collected up to 1850 m from Mindoro and Panay Islands (Phillippines). Most species tend to be seasonal with a population peak in the summer; when present, onchidiids can be very abundant (Dayrat 2009). Onchidiids have a worldwide distribution, with the exception of the Arctic and Antarctic (Hoffmann 1928a, b, 1929).
12.4.1 Onchidella celtica: Silica-Containing Slug or Mystery? O. celtica is commonly known as the celtic sea slug (Fig. 12.15). It was first identified and described (as Oncidium celticum) in 1817 by Cuvier, from the Brittany coast. It seems reasonable to suppose (Peter Barfield, communication) that Cuvier gave it the species name “celticum” because in Latin this adjective describes an inhabitant of Western Europe, particularly Middle Gaul, better known today as France. However, “Celt,” may mean “a warrior,” and with its armored appearance O. celtica might be mistaken for something of a warrior, though this little celt keeps a low profile and lives on a diet of small and microscopic algae. Celtic sea slugs live gregariously in the shelter of rock crevices protected from both strong wave action and sunlight. These animals live during high tide in “nests,” cavities in the rock
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Fig. 12.15 Celtic sea slugs (a, b, c, d) in their natural environment (images courtesy Peter Barfield)
containing a number of individuals, from which the molluscs emerge at low tides to feed upon exposed shore surfaces (Russell 1925; Tween 1987). The individuals emanating from any one nest return simultaneously to that nest before the tide rises again (Arey and Crozier 1918). The numbers vary from 2–3 to up to 60 individuals. They will emerge from their nests with an empty stomach when uncovered by a retreating tide and forage for diatoms and small algae. Often they can be found rasping these algae off mussel beds or encrustations of barnacles. While covered by the tide, an Onchidella will digest the contents of its full stomach; however, it lacks cellulase and so algal filaments remain largely unaffected. Diatoms, on the other hand, form a major part of its diet and most are digested. A lot of sand and detritus is also engulfed, along with bits of sponges and foraminifera. The sand helps to grind the food and as such is a physical aid to digestion. Here I want to note that initially we
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were very optimistic by carrying out HNO3 -based chemical treatment of the whole exemplars of O. celtica obtained from collection of Klaus Groh in our laboratory because of the findings of numerous spicule-like formations and siliceous microparticles. However, later we were disappointed with these results, because in the case of O. celtica, such parts as stomach, derma, genital apparatus must be separated from each other prior to chemical treatment. Thus, in our case spicular formations were originally located in the stomach. Indeed, it was very intriguing for us to know what kind of siliceous materials has been found previously by Labbé in O. celtica. A detailed description of how he isolated and identified siliceous material from molluscs is presented in one of his largest works published in 1934 (Labbé 1934c). He wrote: “The silica is here mostly in the form of spicules: subspherical, elongated, cylindrical, even or dented sticks with an average length of 40–60 μm for a width of 8–12 μm. Some are smaller and don’t exceed 10–12 μm, but they are less common. Each spicule is wrapped by a thin conjunctive capsule; packed parallel to each other, these spicules form a kind of a palisade tissue with two or more layers” (Fig. 12.16).
Fig. 12.16 Spicules of Oncidium leopoldi. (a) Section of integuments of O. leopoldi showing the arrangement of the spicules. (b) Spicules observed in O. maculate. (c) Silica gland. (d) Various forms of spicules observed in O. leopoldi (adapted from Labbe 1934)
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Fig. 12.17 External extra-epidermal spicules of Oncidium peronii surrounded by pigmentcontaining cells (adapted from Labbe 1934)
I find more spectacular the silica identification methods described by Labbé as follows: “With the help of Prof. Lassausse I made sure that these spicules were resistant and not deformable by concentrated nitric acid and strong acids, and the “Villiers”-reaction that we applied to them proved to be positive. It is therefore, indisputably, silica.” According to Labbé, siliceous structures, including very large ones (Fig. 12.17) which he observed in O. peronii, are produced by specific big “silicigene” glands: “I’ve found rare big spicules that seem to be derived from these glands and are probably composed of silica (type II). Since then I’ve found these in all the ‘Onciadés’ studied.” He reported that these glands contain very small granules (max. 1 μm). And again: “A treatment with pure nitric acid doesn’t alter the shape or the size of these granules, even after several days of treatment: I consider them as silica pellets.” Labbé suggested that the intracellular formation of the siliceous spicules of the “Onciadés” is approximately the same as the formation of the calcareous spicules of the “Chitonidés” or of the “Néomeniens.” He wrote: “But I’ve found in Onciidium peronii in addition to the big spicules described above, some rare and more complex spicular formations: these are big elongated spicules that lie in a epithelial crypt of the teguments and are therefore external; their formation is alike that of the horny or calcareous spicules of the ‘Chitons’, those have been shown many times, bur their nature, calcareous or horny hasn’t been specified. So, like the spicules of
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the ‘Chitons’, those of O. peronii are secreted by the cuticle part of the skin; the secretion is therefore external. But I can only deduce that these spicules are made of silica. Moreover, despite the fact that they were outside the skin, these were surrounded by chromatophores that had probably crossed the skin. I only report these curious formations that are presently difficult for me to explain, without comparing them with their ‘chitons’ analogue. But can we credit the skin cells with a ‘siligène’ function?” The next finding made by Labbé is more spectacular. All known species of Onchidiidae are hermaphrodite, with widely separated male and female genital pores. The male pore (or, in exceptional cases pores) opens near the right cephalic tentacle, while the female pore is at the rear end of the body, slightly or markedly to the right of the midline. In some species the penis gland is absent and the penis bears spines in distinct proximal and distal groups, sometimes almost merging on the outer surface of the curve, while the inner edge of the curve is always devoid of spines (Britton 1984). In the old but very detailed anatomical work by Ludwig von Plate (1893), I found several illustrations of penial glands as well as of penial structures of different onchidiids species. This author describes hooks as well as mineral structures within and on the surface of molluscs “chondroid” (cartilage-like) penises as dark colored and highly visible under light microscope (Fig. 12.18). Plate has no doubts that these minerals are of “urine calcareous” origin. But, in 1934, Labbé definitively showed doubts in regard to the same formations. Thus, he wrote: It is even more striking that in a certain number of “Onciadés” the penis is equipped with “cartilaginous” (Semper) or “chondroid” (Plate) parts. And yet I have been able to verify that these are silica plates that come from flattened cellular elements having the function of siliceous glands and that are filled with siliceous spicules.
Fig. 12.18 Mineral structures (arrows) within and on the surface of molluscs “chondroid” (cartilage-like) penises as dark colored and highly visible under light microscope has been described by Plate in 1893 (adapted from Plate 1893)
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Labbé not only criticizes other authors but also gives his arguments as follows: “The penis, more or less big, can be equipped, and that’s quite common for gastropods, by various frames, in particular hooks, directed from bottom to top, in the flue of the canal, and in the posterior part by elements called ‘chondroids’ (Plate) that were discovered by Semper and likened by him to cartilaginous cells, this word being of course inappropriate. As we’ve seen, it is noticeable that this so-called chondroid tissue is in reality made of siliceous plates. I’ve left chondroid tissue of Oncidium staelenii several weeks in nitric acid without it to be modified. The ‘Villiers’-reaction is positive. These are no regular cells, like Semper and the former authors said, but polymorphous cells, having a core and filled with spicules, or rather small siliceous plates of various size and form. I consider them as flattened cells transforming into sheathing siliceous plates; each cell, with or without a central core, produce silica, grow and fills itself with siliceous spicules, flattened or not, with various sizes. It is noticeable that siliceous spicules exist in species, like O. leopoldi, that don’t have chondroid elements.” (Fig. 12.19). In spite of the unique spinose morphology of the penial structures in onchidiids (Fig. 12.20) the real inorganic nature of hook-like formations is still unknown.
Fig. 12.19 Genital system of Oncidium straelenii (P—penis; gp—penial gland), C—siliceous microplates, which cover the penis (adapted from Labbe 1934)
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Fig. 12.20 The penis of onchidiids bears spines in distinct proximal and distal groups, sometimes almost merging on the outer surface of the curve, while the inner edge of the curve is always devoid of spines (a) (images adapted from Britton 1984); (b and c) (images adapted from Plate 1893)
It seems that Labbé found siliceous structures everywhere in Onchidiidae, including dorsal eyes which were described in detail first by Hirasaka (1912). There are no references to the works of Labbé in recent papers related to photoreceptors of onchidiids published by Gotow and Nishi (2002, 2007, 2008), probably because it is unrealistic to suggest the presence of silica-based eyes from the modern point of view. However, Labbé does this and reported as follows: Indeed I believe that considering their resistance to the strong acids the lens cells of the dorsal eyes are also made of silica glands. Semper had already insisted on the similar nature of the cells in the lens and the skin glands, to which he gave a role in the development of the eye. Although it is the opinion of Hirasaka, I don’t think that these are optoblasts that transformed in lens cells; these cells have a great similarity with the siliceous skin glands that are probably also ectodermic. It would anyway be noteworthy if the crystalline lens was formed by a substance close to glass. What seems certain is that there’s a connection between the siliceous spicules and the dorsal eyes: in O. leopoldi that has a dorsal shell of spicules and eyes, there are no spicules in the small areas where the eyes are.
Labbé reported also about quantities of silica founded in O. celtica. He calculated that a 1 cm long O. celtica weighting 0.37 g contains 0.044 g of silica, that is, one-tenth of the weight of the animal (Labbé 1934c). Of course, results and suggestions from Labbé listed above provoke some doubts today. However, I personally find some of his ideas as those which alternatively can stimulate our research today. For example, those formulated by Labbé as follows:
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On the other hand it is possible that the “Oncidies” get their silica from the aluminium silicates of the clay. A ratio must exist between silicic acid and carbonic acid but we don’t know what their equilibriums with silicates are. The respiratory CO2 may play a part here. As for the silica it lies probably in the form of opals in the spicules; like in sponges it could be combined with proteins or in a colloidal form. The spicules of the “Oncidies” are not formed like those of sponges, in concentric layers; they seem at first formed by small clustered granules, and later they show a compact and homogenous structure at least outwardly. These are matters that we will try to solve later.
However, it seems that there was nobody who continued to do this job “later,” because of the absence of corresponding references in the modern literature. Therefore, we made the decision to carry out a thorough re-examination of the very intriguing data reported previously by Alphonse Labbé. Corresponding experiments are in progress today in our laboratory.
12.5 Conclusion There are more than a dozen open questions with regard to the chemical composition and structural features of the spicular formations, both calcareous and siliceous, in molluscs. The nanoarchitecture of the organic templates within diverse spicules, as well as their function, is also not clear. Because of the large diversity of spicules, different mechanisms of their formation may exist. For example, a new mechanism of demosponge spicule formation was recognized during taxonomic studies of bioeroding sponges (Porifera: Demospongiae: Clionidae) by Schönberg (2001). To date different spicule types have been explained by matching structures to their organic matrix and the axial thread. Bulbous structures, however, do not have an organic counterpart. For example, immature spicules of Cliona tinctoria and Pione caesia have irregular, rough heads. Higher magnification during scanning electron microscopy shows that silica granules are deposited regionally to form bulbs. Later silica secretion smoothens the bulb surface. Silica deposition in the form of granules, rather than in layers, appears to be a structuring tool in tylostyles. Similar suggestion, however, was reported by Labbé (1934c) for siliceous spicules in molluscs. Does this represent a very ancient mechanism of biosilicification? Or, there are two different ways in spicule formation? I trust that future work will reveal the answers on these and other interesting questions listed in this chapter.
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Part IV
Non-mineralized Structures
Chapter 13
Spongin
Sponges appeared to me only as skeletons Peyssonel 1757
Abstract The siliceous skeleton that characterizes most sponges in the class Demospongiae is replaced by a skeleton of spongin fibers in a group of sponges that comprises the order Verongida, Dictyoceratida, and Dendroceratida. To provide skeletal support to the bulk of sponge cell tissue, spongin fibers may be either anastomosed to form a network of organized as sets of diversily dendritic, unconnected structures. The history of spongin’s discovery as well as its chemical definition is very similar to those of gorgonin and antipathin described in this book. Therefore, it is not surprising to know that spongin was defined previously as silk, pseudokeratin, eukeratin, gelatinous matter, horny protein, iodospongin, sclerotized collagen, etc. Biological functions, diversity, and material properties of spongin are described and discussed. Studies on chemistry, biochemistry, and the material properties of spongin-based skeletal formations in sponges are in trend now; in particular due to the poorly understood basis of sponge diseases and perspectives of direct applications of sponge skeletons in tissue engineering. Bath sponges have been used for more than 3,000 years (de Laubenfels and Storr 1958; Schulze 1879) for bathing, painting, cleaning, medical uses, padding for battle armor, and as a vessel for drinking water (Cresswell 1922). This broad variety of applications is determined by structural, chemical, and material features of the fibrous skeletons of these animals. In the family Spongidae, to which the commercial sponges belong, the fine structure of the skeleton is difficult to observe because of the very close packing of collagen microfibrils and it has not been elucidated whether these microfibrils have a special arrangement or whether they constitute an irregular network. Skeletal growth and rhythm are unknown. The siliceous skeleton that characterizes most sponges in the class Demospongiae is replaced by a skeleton of spongin fibers in a group of sponges that comprises the order Verongida, Dictyoceratida, and Dendroceratida. To provide skeletal support to the bulk of sponge cell tissue, spongin fibers may be either anastomosed to form a network of organized as sets of diversily dendritic, unconnected
H. Ehrlich, Biological Materials of Marine Origin, Biologically-Inspired Systems 1, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9130-7_13,
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structures. Dendritic skeletons occur in some Verongida and some Dendroceratida, whereas reticulate skeletons occur in all three orders (Bergquist 1980; Maldonado 2009). Recently, we discovered chitin within skeletal fibers of Verongida sponges (Brunner et al. 2010; Ehrlich et al. 2007). This finding suggested the presence of both spongin and chitin as scaffolding materials responsible for the material properties of Verongida sponges. The history of spongin’s discovery and its chemical definition is very similar to those of gorgonin and antipathin described in this book. Therefore, it is not surprising to know that spongin was defined previously as silk, pseudokeratin, eukeratin, gelatinous matter, horny protein, iodospongin, sclerotized collagen, etc.
13.1 Spongin as a Halogenated Scleroprotein Geoffroy was the first who reported about the similarity between silk and bath sponge skeletal fibers (Geoffroy 1707). This suggestion was based on chemical experiments which he carried out. However, during following years, the interest on sponge chemistry was not significant. More attention has been paid to pharmacological applications of sponges. The seminal discovery provided the explanation for the therapeutic value of marine sponge, Spongia usta, the “Coventry Remedy,” which was used by the ancient Chinese. The sponge was shown to contain large quantities of iodine by Andrew Fyfe, a professor of chemistry in Aberdeen in December 1819 (Fyfe 1819). In 1841 bath sponges were described as “keratose sponges,” in which the essential base of the skeleton consists of keratose fibrous matter (Bowerbank 1841). At that time, Croockewit suggested, as before, that horny fibers of sponges are chemically similar to silk. It would appear that sponge is closely analogous to, if not identical with, the fibroin and sericin of silk, differing from it only by containing iodine, sulfur, and phosphorus. According to Croockewit the chemical formula of horny matter must be as follows: 20(C39 H62 N12 O17 ) + J2 S3 P10 (Croockewit 1843). Schlossberger (1859), however, showed that the sponge fiber is distinct in its very slight solubility in ammonical solution of copper hydroxide. Additionally, it yields leucine and glycocoll when treated with diluted sulfuric acid, while under similar treatment sericin yields tyrosine and serine. Similar results to Schlossenberger were also obtained by Städeler (1859) who introduced the name spongin for this matter. The first histological studies on sponges were carried out by von Kölliker (1864). He also investigated structural features of spongin fibers. Hundeshagen (1895) discussed iodine-containing sponges and “jodospongin.” Harnack (1898) examined ordinary bath sponges which contained 1.1–1.2% of iodine. He demonstrated that in these, too, the halogen must exist in combination with organic components. In attempting to isolate this organic complex he learned that superheated steam destroys the organic portion completely, liberating iodine.
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He succeeded in obtaining a more concentrated product, albuminoid in character, which contained over 8.5% iodine and 9.4% nitrogen, and named it “Jodspongin.” Interestingly enough Minchin in 1900 claimed the existence of two different types of spongin, nearly identical in composition, one being produced intracellularly and secreted and the other that formed the fibers of the internal skeleton. In 1926, Clancey prepared a critical analysis summarizing the results to date related to spongin identification. It was the belief of this author that the constitution of spongin varies with the source of its origin and that the substances which form the main framework of the Euceratosa and which surround the spicules in the Pseudoceratosa are not identical, as appears to have been assumed by many physiologists (Clancey 1926). Many workers had published results for the constitution of spongin, usually obtained from either Hippospongia equina, the common bath sponge, or Euspongia officinalis, the “Turkey cup sponge.” These results showed remarkable differences due to defective methods of analysis and working with commercial sponges that have been variously macerated and bleached (Abderhalden and Strauss 1906; Strauss 1904). The cause of the differences in the results of previous workers appears to be the presence of the iodine complex. Wheeler and Mendel (1909) isolated this complex from a barium hydroxide hydrolysis and showed that it was identical with the iodogorgonic acid first obtained from Gorgonia cavolinii by Drechsel (1896), the structure of which was shown to be that of 3,5-diiodotyrosine. They also showed that it does not give all the reactions usually associated with tyrosine; for example, it gives the xanthoproteic reaction owing to the presence of the benzene ring, but the iodine in the ring apparently prevents it from reacting with Millon’s reagent. In order to obtain complete hydrolysis and comparative values for the amino acids, the following method was adopted. A weighed portion of the spongin, about 3 g, was introduced into boiling 20% hydrochloric acid and boiled under a reflux for about 30 h until no further increase of amino acids in the solution could be observed. Spongin after acid hydrolysis yields, besides the other amino acids, iodogorgonic acid. The iodogorgonic acid yielded on hydrolysis is converted into tyrosine and silver iodide by the treatment with acid silver nitrate. The yield of iodine is equivalent to 2–1% tyrosine, while 2–8% was found. This yield is equivalent to 4–7% iodogorgonic acid in the protein (Clancey 1926). Clancey (1926) has also reported the absence of hydroxyproline in the skeleton of Hippospongia equina which had been purified by water, acid, and alkali washings. He found 14% glycine, 5.7% proline, a trace of cystine, 2.8% tyrosine, 11% tryptophan, or histidine and a remarkably high amount, 18.4%, of glutamic acid. Thus, until the publication on two different spongins by Gross et al. in 1956, spongin was established, for the most part, as a halogenated scleroprotein (Ackermann and Burchard 1941; Ackermann and Müller 1941; Low 1951; Roche 1952).
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13.2 Spongin as a Collagenous Protein Most authors agree that the cells which form spongin, the spongioblasts, are derived from the epithelium. Minchin (1900) claims that the cuticular spongin fibrils are formed intracellularly, and the skeletal spongin fibers are secreted to form the large fiber extracellularly. Two morphologically distinct forms of spongin fibers, designated spongin “A” and spongin “B,” were demonstrated by Gross et al. (1956) to be members of the collagen class. This finding was supported structurally by X-ray diffraction and electron microscopy, and chemically by their hydroxyproline amid glycine content as well as by the general amino acid pattern. Ratios of glycine to hydroxyproline were 1.6 and 1.8 for spongin “A” and “B,” respectively. Spongin “A” is a long unbranched fibril of uniform width on the order of 20 nm revealing an axial period of 625 Å by small-angle X-ray diffraction and of about 650 Å by electron microscopy. Spongin “B” is a large branched fiber, 10–50 μm in width, composed primarily of bundles of thin unbranched filaments less than 10 nm wide. “B” fiber fragments occasionally showed an axial period in the collagen range, although X-ray preparations did not yield a low-angle pattern. The content of hexosamine, hexose, pentose, and uronic acid was determined in both fiber types and in the amorphous matrix. Glucosamine, galactosamine, glucose, galactose, mannose, fucose, arabinose, and uronic acid were identified chromatographically in both spongin “A” and in the amorphous substance. A very small amount of amino sugar plus glucose and galactose were identified in spongin “B.” Both spongins differed from mammalian collagen in that they were not dissolved at all by pepsin or collagenase (Clostridium hystolyticum) nor were they dissolved to any appreciable extent in dilute acid or alkali solutions (Gross et al. 1956) Recent views of spongins have been well documented by Exposito et al. in 2002, as follows. Thus, in view of the absence of basement membranes in almost all of the sponges and the similarities between spongin, nematode cuticular, and basement membrane type IV collagens, it is possible that the spongin family reflects two lines of evolution. One line might have been exocollagens (such as spongins) attaching sponges to their substrata (such as worm cuticles, mussel byssus threads, and the egg capsule of Selacians). The second might have been internalization of such collagens, leading to the differentiation of basement membrane collagens. Finally, vertebrate FACIT and FACIT-related collagens appear to be evolutionarily related to nematode cuticular collagens. All consist of several short collagenous domains, with similar C-terminal noncollagenous (NC1) domains as well as conserved cysteine residues at the COL1–NC1 junctions. In sponges, collagens serve several functions (Exposito and Garrone 1990; Exposito et al. 2002; Simpson 1984). Fine fibrils made of fibrillar collagen are involved in construction of the mesohyl, while spongins help comprise the skeleton and permit the adhesion of the sponge to its substratum. The collagen fibrils not only provide the structure of the ECM, but also mediate cell–matrix interactions via membrane receptors, a situation observed in vertebrates. During the process of
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evolution, the increase in diversity of fibrillar collagen chains, their different forms of maturation, and interactions with other ECM components allowed the formation of fibrils with distinct diameters and functions (Garrone and Pottu 1973). Hence, vertebrate fibrils are involved in body protection in the dermis, in providing protection against mechanical strength in tendons, and in assuring the physical properties of cornea. Another interesting feature is the presence of collagen fibrils in the vertebrate skeleton, which is rich in calcium phosphate and nonfibrillar collagens (IX and X). In sponges, spongins are also incorporated into the skeleton with or without an inorganic compound made of calcium carbonate. Hence, the collagen-based skeleton has not been successful during evolution, except in vertebrates (Garrone 1999). Spongins also act as exocollagens which attach the sponge to its substratum, a function which seems necessary since the pinacoderm cells are loosely arranged. During evolution, the exocollagens had a protective role, such as in the cuticle of nematodes and annelids. The protective effect of collagen can be related to its relative resistance to a broad range of proteases. Moreover, although collagen molecules are trimeric, they are involved, either alone or with other collagenous and noncollagenous components, in the formation of polymers. This arrangement not only increases their resistance to proteases, but also promotes the organization of the ECM. Spongins are evolutionarily related to basement membrane collagens and type IV collagens have been described in sponges. Type IV collagens, which form the primary scaffold of basement membranes, are involved in the attachment of epithelial and endothelial layers (Exposito et al. 2002). I take the liberty here to present a classification of spongins proposed by R. Garrone in his fundamental work published in 1978, in the form of a scheme (Fig. 13.1). First, there is spongin of the spiculated fibers, which is always associated with the endogenous inorganic skeleton of the sponge. For example, in the Reniera group, spongin is present only at the nodes of a delicate network of spicules or forms wide fibers which only include a very thin mineral element in the core. This kind of spongin is resistant to diverse bacterial collagenases, pepsin, and mild acid or alkaline
Fig. 13.1 Schematic view: diversity of spongins
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hydrolysis. Only a solution of cuprammonium hydroxide appears to be able to attack spongin at room temperature. Second, the spongin fibers which constitute the skeleton of the horny sponges: the abundance and compactness of the spongin and the almost complete disappearance of its own inclusions, which are replaced to foreign particles, testify to the originality of the spongin in this group. The basal spongin. Because most sponges are sessile animals, the importance of the basal spongin is evident. In sponges with no organized internal skeleton, the animal is attached to the substratum by a more or less continuous layer of external spongin secreted by the basopinacocytes. In sponges with an organized skeleton, formed either of speculated fibers or spongin fibers, the basal spongin is continuous with the internal spongin. In Chondrosia reniformis, a species lacking spicules and internal spongin, the basal spongin fixes the animal so strongly to its substratum that the sponge cannot be removed entirely; this is the case for most sponges. In erect sponges, the basal spongin is discontinuous and forms the starting points of the internal organized skeleton. The spiculoids are organic elements whose shape, often regular, is strikingly similar to that of inorganic spicules. They are encountered in certain Dendroceratida belonging essentially to the genera Darwinella and Igernella where they are either free or partly joined to the fibers of the skeleton. Spiculoids are extremely flexible and elastic (Dandy, 1916). They are compressible and can be easily torn apart. Dandy in 1926 described spiculoids from Darwinella species as follows: “The spicules consist of three or four slender, tapering rays diverging from a common centre, and they are quite detached from the ordinary spongin skeleton, which coexists with them. No axial thread or canal has been observed, though I have examined them carefully, by different staining methods, from this point of view. Nevertheless it seems almost certain that the spongin must be deposited around some axis, and the fact that this has not yet been shown to exist may perhaps be explained by its close agreement in chemical and physical properties with the enveloping spongin. The spongin appears to be secreted by a, surrounding sheath of spongoblasts as in the case of the ordinary fibres of the horny skeleton. There is no evidence at all of origin within, or envelopment by a single ‘mothercell’. ” The skeleton of Igernella species mainly consists of abundant horny spiculoids with a fiber network reduced to the sponge periphery. Fibers are laminated, pithed, and totally devoid of foreign material. No differentiation into primary or secondary fibers can be established. Their thickness varies in the different specimens, ranging from 7 to 67 μm in diameter. The spiculoids are diactines, triactines, or/and tetractines with rays of about 400–1,000 μm in length (Maldonado and Uriz 1996). Finally, there is the shell of the gemmules which is in fact more a protective envelope than a true internal skeleton. Gemmules are asexual reproductive bodies which are formed within the tissues of most freshwater and some marine sponges. They are spherular, from a few tenths of a millimeter to more than 1 mm in diameter, composed of a dense mass of identical cells and surrounded by an organic coat, the shell, which is sometimes fortified with spicules. The shell of gemmules is composed of chitin and a collagenous protein which may be referred to as spongin.
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13.3 Function of Spongins in Natural Environments In addition to spicules, most demosponges are supported by an internal matrix of spongin-based fibers (Bergquist 1978). In some sponges, foreign spicules or sand grains may be embedded in the spongin fibers (Bergquist 1978; Wainwright et al. 1982). The rigidity and toughness of sponge tissue are dependent not only on the density and thickness of spongin fibers (Storr 1964) but also on spicule morphology and orientation (Koehl 1982; Wainwright et al. 1982). Spongin can constitute a significant portion of total colony biomass; for example, Reiswig (1973) reported that spongin accounted for –30% of the total dry mass of Mycale sp. In addition to its structural role, spongin is also believed to be difficult to digest (Chanas and Pawlik 1996). It stands to reason, therefore, that the combination of poorly digestible spongin and indigestible silica may result in tissue of sufficiently low nutritional quality that predators are deterred from eating sponges (Chanas and Pawlik 1995). The biomechanical basis of morphological acclimation to wave force by sponges has been reported to be largely determined by spongin as well (Bell et al. 2002; Maldonado and Young 1998; Palumbi 1986). Colonies of Halichondria panicea were found to be stronger and stiffer in high-wave force habitats than in low-wave force habitats. These biomechanical changes are due to increased spicule number and size in sponges from areas of high-wave action. The spicule changes follow the predictions of theories developed for particulate composite materials (e.g., those comprised of a flexible matrix with ridge imbedded stiffeners), suggesting that the habitat-dependent changes observed in H. panicea are engineering solutions to environmental stresses. An additional constraint imposed upon the basic Porifera body plan is the necessity of pumping water through the skeleton for feeding and respiration. In H. panicea, piping elements decrease in diameter in high-wave force environments. This increases the resistance of oscular systems to water flow, thereby increasing the costs of water pumping. Environments with extremely high-wave force are not inhabited by H. panicea, possibly because the high cost of pumping water through a skeleton dense enough to persist would limit or preclude growth. This limitation, however, is peculiar to the engineering trade-offs required by the body plan and feeding mode of H. panacea (Palumbi 1986). I reported above that spongins were characterized by numerous researches as especially resistant to different enzymes including proteases, collagenases, amylases, lysozymes (Junqua et al. 1974; Junqua 1978). However, in natural environments, the biomechanical properties of spongin-based sponge skeletons dramatically change when a corresponding pathogen possesses the ability to develop a special mechanism for spongin digestion. For example, Webster et al. (2002) reported the first isolation and characterization of a pathogen causing disease in a marine sponge. The bacterial pathogen digests the spongin fibers in the skeleton of a sponge on the Great Barrier Reef, Rhopaloeides odorabile. The symptoms include initial fouling of the surface of the sponge with epiphytic algae and the sponge skeleton being soft and fragile once infected. They state that the symptoms were similar to that in commercial sponges in the Mediterranean (where sponges had fragile and brittle skeletons that crumbled under water), with the bacteria tunneling inside the
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spongin fibers. The similarity of symptoms and the microscopic evidence between the Australian sponge and the Mediterranean sponges indicate that bacterial sponge disease may be a global phenomenon. The similarity to the weakening and disintegration of the skeleton of Xestospongia muta (which may in the last stages be held together by a large quantity of spicules), the softening of diseased Verongula gigantea and the algal epiphytes on Geodia sp. at an initial stage, suggests that the cause may be bacteria burrowing in skeletal fibers.
13.4 Mechanical Properties of Spongin-Based Skeletons Due to the diverse morphological variation that occurs in sponges between and within species, protocols for quantitative quality testing are required to select sponges and optimize conditions for the aquaculture of high-quality bath sponges (Cresswell 1922). Recently, Louden et al. (2007) developed a protocol to assess the quality of sponges using mechanical engineering techniques. It quantified the physical properties of sponges (density, fiber width, fiber length, absorbency, and water retention efficiency) and their mechanical properties (firmness, compression modulus, compressive strength, tensile strength, elastic limit, elastic strain, modulus of elasticity, and modulus of resilience). To demonstrate these methods and provide a relative comparison, these qualities were measured for Rhopaloeides odorabile and Coscinoderma sp. and three commercial species, Hippospongia lachne, Spongia 1, and Spongia 2. There were significant differences between species for all quality parameters, creating a unique profile for each species. R. odorabile was the firmest (37.8 ± 4.3 kPa), strongest (157.4 ± 17.3 kPa), and most rigid (838.7 ± 53.5 kPa) species tested, while Coscinoderma sp. was one of the softest sponges (7.3 ± 1.1 kPa) and had the highest elastic energy (30.5 ± 3.5 kJ/m3 ) and water retention efficiency (40.1 ± 1.4%) of all the species. Of the commercial species, H. lachne was the softest (3.2 ± 0.3 kPa), weakest (36.3 ± 3.1 kPa), and most absorbent sponge (31.0 ± 1.1), while Spongia 1 and Spongia 2 had intermediate quality characteristics for all measured parameters. These tests enable scientifically rigorous comparisons of quality between and within species regardless of origin or post-harvest treatment. Comparisons between species may be used to select species for aquaculture and as a marketing tool to promote aquaculture products for specific applications. Within-species testing will allow quantification of differences in quality caused by genetic or environmental factors (Louden et al. 2007).
13.5 Spongin as a Three-Dimensional Scaffold for Tissue Engineering Natural skeletons are highly optimized structures that support and organize functional tissues. They provide important design information for the fabrication of synthetic tissue engineering scaffolds and may prove efficacious in tissue
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engineering strategies (Green et al. 2002). The provision of affordable, readily available scaffolds for specifiable nonimmunogenic transplantable tissues and high surface area cell carriers for liver, kidney, bladder, esophagus, muscle, bone, and connective tissue remains a major clinical demand (Langer and Vacanti 1993). The current strategy in scaffold design and synthesis of biodegradable synthetic sponge-like constructs and foams for tissue engineering is to produce uniform threedimensional interconnected macroporosity, the aim being to create specifiable pore dimensions and interconnections during scaffold synthesis. Generating sufficient structural support for tissues at the implantation site is a major issue and tissue engineering and biomimetic approaches may offer alternative solutions (reviewed in Green et al. 2002). The skeletons of spongin-based sponges appear to possess a number of unique and suitable properties, including (i) the ability to hydrate to a high degree, which is favorable to cell adhesion, (ii) the possession of open interconnected channels created by the fiber network, and (iii) the tremendous diversity of skeletal architecture and fiber constructs in this phylum (Green 2008). The practical value of the rigid sponge skeletons is given by their large internal surface area which is estimated to be 25–34 m2 for a skeleton sample of 3–4 g dry weight. This enables considerable liquid absorption taking place via capillary attraction (Garrone 1978). Most successes with respect to tissue engineering have been achieved using collagenous marine sponges as templates for musculoskeletal tissue (principally bone tissue) formation and support in vitro and in vivo (Green 2008). Collagenous marine sponges Spongia (class Demospongiae: order Dictyoceratida: family Spongiidae) are bonded fibrous networks with large surface areas and interconnected voids (Green et al. 2003). A complex mixture of collagens makes them highly compatible with co-cultured human cells where strong associations form that promote attachment, proliferation and provide cues that maintain cell phenotype and lead to lineage differentiation. Structurally marine sponges possess an advantage over existing synthetic fibrous tissue engineering constructs such as poly-glycolic acid (PGA) fibrous fleece used in cartilage repair (Freed et al. 1993), with a more coherent organization and direct bonding between fibers. Research using collagenous marine sponges for tissue engineering of bone demonstrated that human cells preferentially adhere to sponge fibers and align along the c-axis. The skeletal network of marine sponges provides structural environments conducive for proliferation, as demonstrated by bridging of the interfiber spaces, and differentiation into pre-mineralized human osteoid tissue. Equivalent cell responses also occur using human liver, kidney, cartilage, fat, mesenchymal stem cells, and fetal cell cocultures (Green et al. 2003). There are two explanations for the success of marine sponges as templates for tissue regeneration, those being the porous and fibrous structure, and because of the presence of collagen (spongin) as the main structural biopolymer which is well known to be biocompatible (Green 2008).
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13.6 Conclusion Within the class Demospongiae, which contains about 95% of extant sponges, there are 14 taxonomic orders. Three of them—Verongida, Dictyoceratida, and Dendroceratida—are characterized by skeletons lacking silica spicules and consisting of networks of spongin fibers. Spongin is a protein resulting from a supercompaction of collagen fibrils and collagen filaments. To date the mechanism through which chitin becomes associated with the collagen is not well understood for the spongin fibers of verongid sponges. Moreover, diversity of spongin forms observed in different sponge species makes it difficult to prepare a classification system based on chemical composition. The relationship between spongin and iodine, as well as bromine, remains unclear. Definitively, studies on chemistry, biochemistry, and the material properties of spongin-based skeletal formations in sponges, are trendy now; in particular due to the poorly understood basis of sponge diseases and perspectives of direct applications of sponge skeletons in tissue engineering.
References Abderhalden E, Strauss E (1906) Die Spaltprodukte der Spongine mit Säuren. Ztschr f physiol Chem 48:49–53 Ackermann D, Burchard C (1941) Zur Kenntnis der Spongine. Hoppe-Seylers Ztschr f physiol Chem 271:153–159 Ackermann D, Müller (1941) Über das Vorkommen von Dibromtyrosin neben Dijodtyrosin im Spongin. Hoppe-Seylers Ztschr f physiol Chem 269:146–157 Bell J, Barnes D, Turner J (2002) The importance of micro and macro variation in the adaptation of a sublittoral demosponge to current extremes. Mar Biol 140:75–81 Bergquist PR (1978) Sponges. University of California, Berkeley Bergquist PR (1980) A revision of the supraspecific classification of the orders Dictyoceratida, Dendroceratida and Verongida (class Demospongiae). NZ J Zoo1 7:443–503 Bowerbank JS (1841) Observations on a Keratose Sponge from Australia. Ann Mag VII:129 Brunner E, Richthammer P, Ehrlich H et al (2010) Chitin-based organic networks – an integral part of cell wall biosilica from the diatom Thalassiosira pseudonana. Angew Chem Int Ed 48:9724–9727 Chanas B, Pawlik JR (1995) Defenses of Caribbean sponges against predatory reef fish. II. Spicules, tissue toughness, and nutritional quality. Mar Ecol Prog Ser 127:195-2t 1 Chanas B, Pawlik JR (1996) Does the skeleton of a sponge provide a defense against predatory reef fish? Oecologia 107:225–231 Clancey VHJ (1926) CL. The constitution of sponges. 1. The common bath Sponge, Hippospongia equine. Biochem J 20:1186–1189 Cresswell E (1922) Sponges: their nature, history, modes of fishing, varieties, cultivation, etc. Sir Isaac Pitman & Sons, London Croockewit JH (1843) Zamenstelling van Spus. In Schenik. Orderzock Labor Utrecht II, 1 Dandy A (1916) On the occurrence of gelatinous spicules, and their mode of origin, in a new genus of siliceous sponges. Proc Roy Soc, Ser B lxxxix:315–322 Dandy A (1926) On the origin, grow thans arrangement of sponge spicules: a study in symbiosys. Q J Micr Sci 70:2–72 Drechsel E (1896) Contribution to the chemistry of a sea animal. Z Biol 33:85–107 Ehrlich H, Krautter M, Hanke T et al (2007) First evidence of chitin in skeletons of marine sponges. Part II. Glass Sponges (Hexactinellida: Porifera). J Exp Zool (Mol. Dev. Evol.) 308b:473–483
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Exposito J-Y, Cluzel C, Garrone R et al (2002) Evolution of collagens. Anat Records 268:302–316 Exposito J-Y, Garrone R (1990) Characterization of a fibrillar collagen gene in sponges reveals the early evolutionary appearance of two collagen gene families. Proc Natl Acad Sci USA 87:6669–6673 Freed LE, Marquis JC, Nohria A et al (1993) Neocartilage formation in vitro and in vivo using cell cultured on synthetic biodegradable polymers. J Biomed Mater Res 27:11 Fyfe A (1819) An account of some experiments, made with the view of ascertaining the different substances from which iodine can be procured. Edinb Phil J 1:245–258 Garrone R (1978) Phylogenesis of connective tissue. In: Robert L (ed) Morphological aspects and biosynthesis of sponge intercellular matrix. S. Karger, Basel Garrone R (1999) Evolution of metazoan collagens. Prog Mol Subcell Biol 21:119–139 Garrone R, Pottu J (1973) Collagen biosynthesis in sponges: elaboration of spongin by spongocytes. J Submicrosc Cytol 5:199–218 Geoffroy CJ (1707) Analyse chimique de l’eponge de la moyenne espece. Mem., p. 507 Green D (2008) Tissue bionics: examples in biomimetic tissue engineering. Biomed Mater 3:034010 (11 pp) Green D, Howard D, Yang X et al (2003) Natural marine sponge fibre skeleton: a biomimetic scaffold for human osteoprogenitor cell attachment, growth and differentiation. Tissue Eng 9:1159–1166 Green D, Walsh D, Mann S et al (2002) The potential of biomimesis in bone tissue engineering: lessons from the design and synthesis of invertebrate skeletons. Bone 30:810 Gross J, Sokal Z, Rougvie M (1956) Structural and chemical Studies on the connective tissue of marine sponges. J Histochem Cytochem 4:227–246 Harnack E (1898) Ueber das Jodospongin, die jodhaltige, eiweissartige Substanz aus dem Badeschwamm. Zeitschrift für Physiologische Chemie 24:412–424 Hundeshagen F (1895) Über jodhaltige Spongien und Jodospongin. Z Anorg Chemie 16:473–476 Junqua S (1978) Les constituants moleculaires de la matrice intercellulaire des spongiaires. These, Universite de Lille I-XXIX Junqua S, Robert L, Garrone R et al (1974) Biochemical and morphological studies on collagens of horny sponges. Irciniafilaments compared to spongins. Connect Tissue Res 2:193–203 Koehl MAR (1982) Mechanical design of spicule-reinforced connective tissue: stiffness. J Exp Biol 98:239–267 Langer R, Vacanti JP (1993) Tissue engineering. Science 260:920 Laubenfels de M, Storr J (1958) The taxonomy of American commercial sponges. Bull Mar Sci Gulf Caribb 8:99–117 Louden D, Inderbitzin S, Peng Z, de Nys R (2007) Development of a new protocol for testing bath sponge quality. Aquaculture 271:275–285 Low EM (1951) Halogenated amino acids of the bath sponge. J Mar Res 10:239–245 Maldonado M (2009) Embryonic development of verongid demosponges supports the independent acquisition of sponging skeletons as an alternative to the siliceous skeleton of sponges. Biol J Linnean Soc 97:427–447 Maldonado M, Uriz J-M (1996) The genus Igernella (Demospongiae: Dendroceratida) with description of a new species from the central Atlantic. Bull Inst Roy Sci Nat Belg 66:153–163 Maldonado M, Young C (1998) Limits on the bathymetric distribution of keratose sponges: a field test in deep water. Mar Ecol, Prog Ser 174:123–139 Minchin FA (1900) Sponges. In: Lankester FR, Black A, Black C (eds) A treatise on zoology. Brill, London Palumbi S (1986) How body plans limit acclimation: responses of a demosponge to wave force. Ecology 67:208–214 Peyssonel JA (1757) New observations upon the worms that form sponges. In: Phil Trans 50: 590–594 Reiswig HM (1973) Population dynamics of three Jamaican Demospongiae. Bull Mar Sci 23: 191–226
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Roche J (1952) Biochimie comparée des scléroprotéines iodées des anthozoaires et des spongiaires. Experientia 8:45–54 Schlossberger JE (1859) Über die Unterscheidung des Fibroins von der Substanz des Badeschwammes. In: Amtlich Bericht 34. Versamml Deutsch Naturf, p 164 Schulze FE (1879) Untersuchungen über den Bau und die Entwicklung der Spongien. Siebente Mitteilung. Die Familie der Spongida. Z Wiss Zool 32:593–660 Simpson TL (1984) The cell biology of sponges. Springer, Berlin Städeler G (1859) Untersuchungen über das Fibroin, Spongin und Chitin, nebst Bemerkungen über den tierischen Schleim. Ann Chem Pharm 111:12–28 Storr JF (1964) Ecology of the Gulf of Mexico commercial sponges and its relation to the fishery (Special scientific report—fisheries 466). U.S. Fish and Wildlife Service, Washington Strauss E (1904) Studien uber die Albuminoide mit besonderw Berucksichtigung des Spongins und der Keratine, Heidelberg, 1904; quoted from Maly’s Jahresbericht jiir Tierchemie, xxxiv, 34 von Kölliker A (1864) Icones histologicae. 1. Abth Protozoen, Leipzig Wainwright SA, Biggs WD, Currey JD, Gosline JM (1982) Mechanical design in organisms. University Press, Princeton Webster NS, Negri AP, Webb RI et al (2002) A spongin-boring a-proteobacterium is the etiological agent of disease in the Great Barrier Reef sponge Rhopaloeides odorabile. Mar Ecol Progr Ser 232:305–309 Wheeler HL, Mendel LB (1909) The iodine complex in sponges (3,5-diiodotyrosins). J Biol Chem 7:1–9
Chapter 14
Gorgonin
Abstract Gorgonians inhabit the world’s oceans from the equator to the poles, from the shallows to great depths. Unlike the scleractinia, gorgonian coral skeletons are formed of the proteinaceous material known as gorgonin. While calcite provides stability, gorgonin delivers flexibility, enabling gorgonian corals to survive in highenergy, current-affected environments. Gorgonin was described during its history by the different workers as eukeratin, pseudokeratin, jodotyrosine-based protein, sclerotin, tanned collagen, etc. Unfortunately, there is currently no progress in the serious analytical investigations with respect to chemical definition of gorgonin. In recently published papers gorgonin is called as polyphenol-containing fibrillar protein or fibrillar scleroprotein. Materials properties of gorgonin as well as its role in paleoceanographic dynamics are discussed. Gorgonian and antipatharian coral skeletons are among the most resilient and chemically resistant proteinaceous materials known (Goldberg 1976). Gorgonians inhabit the world’s oceans from the equator to the poles, from the shallows to great depths. Unlike the scleractinia, gorgonian coral skeletons are formed of the proteinaceous material known as gorgonin. Within the gorgonin mineralized components, usually calcite, are embedded in a locular form (Lewis et al. 1992). Therefore, gorgonian skeletons are examples of unique biocomposite-based biological materials, whose properties determine the development and survival of the organism under specific marine environmental conditions. While calcite provides stability, gorgonin delivers flexibility, enabling gorgonian corals to survive in high-energy, current-affected environments. To capture enough food in a given time, these filter-feeding animals require a large volume of water to pass through the colony. Therefore, gorgonians commonly occur in benthic habitats that are subject to persistent currents. The function of the gorgonian skeleton is essentially to provide efficient filtering of food particles and to withstand strong currents. The pattern and velocity of currents are important factors controlling the abundance and shape of gorgonians (Genin et al. 1986; Grigg 1972; Wainwright and Dillon 1969; Weinbauer and Velimirov 1995). Interpretation of the growth form and orientation of gorgonian corals may give valuable information about local near-bottom current
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patterns and help us understand their relation to the hydrodynamic conditions in cases where the near-bottom currents are known. (Mortensen and Buhl-Mortensen 2005). After the skeleton has reached a critical size, the coral colony accretes calcitic increments around the entire skeleton. These accretions form carbonate crusts, enveloping the nodes in order to increase the stability of the axis (Grasshoff and Zibrowius 1983). This hyper calcification may be an adaption to increasing hydrodynamic energy during late stage coral growth. Both the internodes and the nodes appear in lateral increments that are concentrically arranged around a hollow organomineralic central axis. Thus, mineral phases of gorgonian corals are represented by calcium carbonates. However, what is gorgonin? Unfortunately, the answer to this question from the chemical point of view is currently unknown, because gorgonin is not a homogenous substance itself. The history of gorgonin discovery and studies on its chemical identification, which were carried out over the last 150 years, are the best confirmation for this.
14.1 Introduction into the History and Chemistry of Gorgonin Descriptions of gorgonian corals have been available since the thirteenth century. Thus, Gorgonia viminalis was described for the first time in 1766 by Pallas (Pallas 1766). However, investigations on gorgonin as a substance started later. Balard (1825) reported the presence of organically complexed iodine in gorgonians, which he called iodogorgic acid. Duchassaing and Michelotti (1864) described gorgonians collected near Antilles. Valenciennes (1855) introduced the term “gorgonin” in his monograph on gorgonians. In 1887, von Koch detailed descriptions of gorgonians from the gulf of Naples. In 1896 Drechsel isolated an iodinated amino acid from Gorgonia cavolinii and named it “Jodgorgonsäure.” The preceding observations support the assumption of Drechsel that the gorgonians have a specific iodine metabolism which is essential to the building up of the framework of the axial skeleton and also give further justification for the belief already stated by Mendel (1900) that for many organisms iodine may be an essential element as chlorine is for others. These classic investigations awakened a new interest in the study of the organic iodine compounds in organisms. Henze (1907) made an examination of the substance described by Drechse1 and claimed that it was not iodoaminobutyric acid as had been assumed by its discoverer. Mendel (1900) and Cook (1904) studied the chemical composition of some gorgonian corals and described the presence of iodine. However, the identification of the so-called iodogorgoic acid was completed for the first time by Wheeler and Jamieson (1905), who established the fact that it is identical with the 3,5-diiodotyrosine that had been synthetically prepared by them. Mörner (1907, 1908) as well as Oswald (1908) investigated the chemistry of the organic matrices of numerous anthozoans, especially with respect to the identification of iodine-based substances. At around the same time, studies on axial skeleton formation in gorgonians were also carried out (Neumann 1911). Kükenthal
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described the zoology and systematic of “Gorgonaria” correspondingly in 1915, 1919, and 1924. In 1928 Sugimoto, a chemist re-investigated several species of gorgonian corals. He found that iodine was a constituent of the horny axial skeletons, but not of the adherent concretions. The content of this element in most of the investigated corals approximates 1.0–1.5% (Sugimoto 1928). The definition of gorgonin as a substance changed in 1931 when Block and Vickery (1931) analyzed undigested Gorgonia flabellum and Plexaurella dichotoma and found that G. flabellum yielded histidine, lysine, and arginine in the molecular ratios of 1:6:12 while P. dichotoma contained these amino acids in the molecular ratios of 1:8:12. In view of the general similarity of these ratios to that assigned to the eukeratins (1:4:12), it was tentatively assumed at that time that gorgonin may be a eukeratin. Analyses of somewhat more highly purified gorgonin preparations with an improved method for detecting the basic amino acids yielded comparatively more histidine and lysine, but less arginine. The new results indicated that histidine, lysine, and arginine are in the molecular ratios of approximately 1:4:6 and not 1:4:12, as was earlier thought. It was recognized in 1931 that the amounts of lysine obtained from gorgonin were somewhat high and the quantities of arginine somewhat low for an eukeratin but the histidine to arginine ratio of 1:12 was considered as sufficient evidence for calling this protein an eukeratin. Since that time, other keratins have been analyzed and were characterized by a rather constant lysine to arginine ratio of approximately 4:6. These have been called pseudokeratins (Block and Boling 1939). However, 10 years later, Marks et al. (1949) suggested that gorgonin is a fibrillar collagen-type protein because of its X-ray diffraction properties. But this collagenlike protein was digested neither by gastric nor by pancreatic juice, probably because of a very specific cross-linkage determined by iodine. Thus, Roche and his collaborators (Roche and Eysseric-Lafon 1951; Roche 1952; Roche et al. 1959, 1963) found monoiodotyrosine, diiodotyrosine, and traces of thyroxine in the axial skeletons of several gorgonians (see also Table 14.1) The amount of iodine present varied with species and depended on the tyrosine content of the axial skeleton (Roche and Eysseric-Lafon 1951). Roche (1952) also reported that the iodine content was less in the older parts of the skeleton. Possibly, the iodinated tyrosine residues crosslink with other iodinated tyrosine or phenolic molecules, causing a loss of iodine. This would account for the decrease in iodine in the older, slower growing parts of the colony which remain in contact with the axial epithelium for a longer time and have more time to become tanned (i.e., cross-linked). Material in faster growing younger parts soon becomes separated from the axial epithelium by new depositions; thus, additional tanning is prevented. Roche et al. (1959) further tried to show by radiochromatography that the iodination of the tyrosine residues occurred in the axial epithelium of these corals before the amino acids were assembled into proteins. According to Roche et al. (1963) the iodination in gorgonians takes place in the living tissue, which can concentrate iodide to a high degree. Iodine occurs in many invertebrate scleroproteins: annelid cuticles, hydroid perisarcs, sponge proteins, mollusc periostracum, and arthropod cuticles (Gorbman et al. 1954), but little is known about its function. One wonders why these particular
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Table 14.1 Possible modifications of tyrosine in antipathins, gorgonins, and spongins Name
Chemical structure
Proteins
I
Monoiodotyrosine
Antipathins Gorgonins Spongins
OH HO
CH2
CH
C O
NH2
Diiodotyrosine
Antipathins Gorgonins Spongins
I OH HO
CH2
CH
C O
NH2 I I
Thyroxine
Gorgonins
I OH
HO
O
CH2
CH NH2
I
O
I
Br
Monobromotyrosine
C
Gorgonins OH
HO
CH2
CH
C O
NH2
Dibromotyrosine
Gorgonins Spongins
Br OH HO
CH2
CH
C O
NH2 Br
proteins are heavily iodinated, while those present in the rest of the animal remain relatively free of iodine. Since all of the above iodinated invertebrate scleroproteins are tough structural materials, it can be speculated that the iodine may be directly involved in promoting inter-molecular bonds between the protein chains (i.e., during the tanning or hardening process). This tanning process is fairly well understood from some of the arthropod groups, where polyphenols and quinones are the crosslinking agents (Brunet 1967), but little is known about other invertebrate groups with scleroproteins.
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It is assumed that the phenol oxidase present in the insect and crustacean cuticle oxidizes diphenols to quinones, which then react with amino groups in the proteins, thereby cross-linking them (Welinder 1972). The explanation for the presence of the halogenated tyrosines could therefore be that an intermediate formed in these oxidation processes is capable of oxidizing chloride to chlorine and bromide to bromine, thereby giving rise to the formation of the halogenated tyrosines. A similar suggestion has been made for the iodination of iodo-containing scleroproteins. The formation of mono- and diiodotyrosines would be due to the oxidizing effect of quinones, which function in the “quinone tanning process.” Iodide would thereby be oxidized to iodine, which then reacts with tyrosine. An understanding of the evolution of the tanning process from the sponges through the corals and crustaceans might elucidate the evolution of structural support systems in the marine invertebrates. In spite of Leversee’s (1969) work in which he reported that the major component of the axial skeleton is the gorgonin, which is composed mainly of collagen fibers in a proteinaceous matrix, the chemical composition of the gorgonian axial skeleton remained poorly understood by the scientific community. Szmant (1970) reported that the skeletal material of Muricea californica (Gorgonacea) is inert and persists long after the death of the animal. The axial skeleton also contains, at approximately 1% by weight, the saccharides glucose and galactose (Szmant 1970) and varying amounts of non-spicular inorganic material (Bayer 1956). Limited attempts to disperse the skeletal material into solution with agents such as 8 M urea, 0.1 M mercaptoethanol, 2 M hydroxylamine, and anhydrous formic acid resulted in failure. The banded fibers in M. californica indicate the probable presence of a collagenlike protein in the axial skeleton, but they in no way exclude the presence of other types of structural and non-structural proteins. Because of these considerations Szmant-Froelich (1974) proposed for the first time that the term gorgonin not be used as before to identify a particular protein structure in gorgonians, but rather to identify the mixture of chemical species comprising the axial skeleton of the gorgonians. Since the publications of Goldberg (1974, 1976, 1978), gorgonin became more established as a collagenous matrix and as a sclerotized collagen. The three-dimensional fibrous texture of gorgonin shows a preferred axial orientation (Goldberg 1973). Tidball JG (1982) described an ultrastructural and cytochemical analysis of the cellular basis for tyrosine-derived collagen cross-links in gorgonin of Leptogorgia virgulata. Unfortunately, there is currently no progress in the serious analytical investigations with respect to chemical definition of gorgonin. In recently published papers gorgonin is called as polyphenol-containing fibrillar protein (Ehrlich et al. 2006), or fibrillar scleroprotein (Noé and Dullo 2006; Noé et al. 2008). Thus, “Gorgonin” was described during his history by the different workers listed above as an eukeratin, pseudokeratin, jodotyrosine-based protein, sclerotin, tanned collagen, etc. However, the following properties can be defined as common for this unique biological material of gorgonian origin:
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• • • • • •
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it possess iodine; it possess basic amino acids; it is hydrophobe; it is fibrillar; it is rigid and resistant to variety of enzymes and chemicals; it has never been reported to promote calcification.
In spite of the lack of knowledge on chemistry of gorgonin, its role in mechanical properties of gorgonian axial skeletons is well investigated.
14.2 Mechanical Properties of Gorgonin-Based Skeletons According to Szmant-Froelich (1974), the axial skeleton of gorgonian Muricea can be described as a tubular structure with two parts: an outer solid cortex, and an inner hollow core which is divided into chambers by convex cross-walls. The axial epithelium is presumed to synthesize the axial skeleton. The outer cortex is made up of many thin laminae of dark colored gorgonin. Each lamina extends only partially around the circumference of the axial skeleton; it overlaps and is overlapped by adjacent laminae. The density of these laminae varies: they are more numerous in the lighter colored layers of the axial skeleton, but are less numerous and more closely packed in the darker layers. These light and dark layers alternate, forming bands which represent annual growth rings (Grigg 1970) analogous to those found in tree trunks. These layers also confirmed the biocomposite nature of the axial skeleton of gorgonians, which were particularly visible using a stereomicroscope. Based on the similarity of the molecular structure of gorgonin and collagen, the following biomineralization model was recently proposed for the isidid (bamboo corals) gorgonian endoskeleton (Noé et al. 2006): gorgonin produced by cells of the endodermal epithelium acts as an insoluble structural framework in the mineralization process, while the viscous slimes surrounding the skeleton represent the soluble polypeptide β-sheet. If this assumption proves to be correct, gorgonin does not only construct the nodes, but occurs throughout the calcitic internodes in form of organic seams surrounding the fiber crystals, which represent the remains of the insoluble matrices incorporated into the growing skeleton. Here, I want to note that gorgonin plays a scaffolding role, and not a templating role with respect to biomineralization. Unfortunately, little is known about the calcium carbonate secreting skeletogenic cells of gorgonian axes, or about their ability to control crystal deposition. Kingsley and Watabe’s (1984) work on L. virgulata (a related gorgonian), showed that calcium ions are transported from the external environment to the axis. Calcium ion transport out of the axis through the axial epithelium was mediated by Ca-ATPase (Kingsley and Watabe 1984). In 1987, Kingsley and Watabe isolated carbonic anhydrase activity adjacent to calcifying structures in the axis. Thus, a mechanism for fluid supersaturation exists in gorgonians. The mineral crystals probably grow on a framework of extracellular proteins and polysaccharides.
14.2
Mechanical Properties of Gorgonin-Based Skeletons
263
In Goldberg’s (1976) analysis of the chemistry of gorgonian axial skeletons, the presence of large amounts of glycosaminoglycans (GAGS) was observed in a heavily mineralized gorgonian. The GAGS may coat the individual collagen (gorgonin) fibers and thus initiate nucleation of the fine granular crystals. There is diversity with respect to the mineralization step in different species of octocorals. Thus, different regions of their skeleton may be composed of “massive” (100%) calcite, 100% gorgonin, or a combination of both (Sherwood and Risk 2007). For example, the Bamboo Corals (family Isididae) deposit gorgonin nodes, like the joints of a finger, between internodes of massive calcite. Red tree corals (family Primnoidae) deposit a two-part calcite-gorgonin “horny axis” toward the inner part of the axial skeleton and a massive calcite cortex later on. Therefore, skeletons of different gorgonian species drastically differ from each other in their mechanical and material properties. Flexibility can apparently be controlled or modulated by sclerotization of the collagen within the axial skeleton. A widely used method of stiffening axes in gorgonians is extracellular deposition of carbonates within the collagen interstitial spaces as reported by Jeyasuria and Lewis (1987). The axial skeleton in gorgonians is the main support structure for the colony. While the rind may play a role, the mechanical properties of the axis might be expected to reflect, at least, the major parameter of water movement associated with various species’ ecological niches. The ultimate tensile strength (around 270 MN/m2 ) of the gorgonian axes tested by Goldberg et al. (1984) is more than twice that of vertebrate tendon (around 100 MN/m2 ) encountered by Elliot (1965). Explanations offered for this are extensive sclerotization noted by Tidball (1981, 1982), Szmant-Froelich (1974), and Goldberg (1978) and the mineralization noted by Bayer (1955, 1961) among others and obvious to anyone who has examined a few different species. The results reported by Jeyasuria and Lewis (1987), demonstrate a surprisingly wide range of mechanical properties for gorgonian axial skeletons. Significant interspecific differences are evident from the figures for Young’s modulus, as well as torsion modulus tests that determine resistance to tensile and shear strain, respectively. The Young’s modulus range lies between 2 × 108 and 9 × 109 N/m2 , is above that for tendon, and suggests that axial skeleton collagen is a stiffer form of collagen (Jeyasuria and Lewis 1987). This difference is of functional significance since axial skeletons of octocorals, unlike tendons and ligaments of vertebrates, are the primary support structures that maintain both the elevation and the separation of the branches of the colony. Even with the support provided by water, a material with the stiffness of most ordinary collagens would be too flexible to hold the branches of the colony off an abrasive substratum and to maintain proper separation of the branches. The range of the torsion modulus is between 6.7× 107 and 8.55 × 108 N/m2 (Jeyasuria and Lewis 1987). The resistance to shear varied quite drastically but did not completely correlate interspecifically with Young’s modulus. In other words, a high Young’s modulus for a species did not necessarily mean a high torsion
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modulus for the same species. This may be related to the arrangement of polyps on the branches and the feeding technique employed by individual species. The authors assumed that the calcareous content is directly proportional to the tensile stiffness of the axial skeleton along its axis. The torsion modulus of the axial skeleton is also related to calcareous content. This, however, is not a simple direct relationship, but a more complex polynomial function. It should be borne in mind that the axis of the gorgonian colony is not the only component of the colony that determines the stiffness and torsion resistance of the colony. Some of the rinds of the colonies contain dense aggregations of interlocking spicules that can produce quite stiff materials of considerable thickness, whereas other rinds are extremely thin and are composed of spicules that appear capable of rolling over one another (Thibaudeau 1983). These latter types of rinds would contribute little to the mechanical characteristics of the axis. These relationships are not intuitively obvious and were thus somewhat surprising to the authors. However, once the relationship of greater stiffness with decreasingly violent or rapid change in water movement becomes apparent, it is seen to be eminently sensible. Stiffness and therefore brittleness generated by high carbonate can be used to maintain a slender organism in an optimally favorable position for biological activity for the maximum length of time in deeper water where water movement is usually not violent, rapid, and unpredictable and where stresses are not overly high. Flexibility is one of the only attributes a slender organism can utilize for accommodation to the high-stress, low-turbulence environment produced by the wave surge characteristics of moderate (10–30 m) depths. Before forces become high enough to cause damage, parts bend and relieve pressures. The majority of the shallow-water gorgonians examined in the study by Jeyasuria and Lewis (1987) fit this category. Moderate stiffness and flexibility are combined for accommodation to the high-stress, high-turbulence environment characteristics of many shallow water (1–10 m) areas. Some stiffness is necessary to prevent the floppiness that comes with great flexibility, which would result in destruction of the organism by abrasion against the substratum and probably also to induce less dangerous laminar flow from turbulent water flow. Flexibility is necessary for force reduction by bending before the very high stresses generated by wave action. Though these explanations are obvious oversimplifications, it appears that the broad ecological niche for which an individual gorgonian species is adapted may be determined by the mechanical properties of its axial skeleton. Finally, Jeyasuria and Lewis (1987) in their amazing study determined the Young’s modulus of the axial skeletons of 13 species of holaxonian octocorals, representing 12 genera, and found that they range from 0.2 to 90 Gdynes/cm2 . Axial stiffness also correlated well with zone-related water movement. Relative quantities of calcareous material in the axial skeletons were strongly correlated with Young’s modulus, suggesting an important role for calcareous material in modulating the mechanical properties of the axial skeleton. Modulation of axial stiffness through calcification would be an effective mechanism for dealing with the different hydrodynamic forces encountered at various depths.
14.3
Gorgonin-Based Skeletons and Paleoceanographic Dynamics
265
14.3 Gorgonin-Based Skeletons and Paleoceanographic Dynamics Much of what we know about the history of Earth’s climate system is derived from the chemical composition of biogenic skeletons preserved in the geologic record (Bond et al. 2005). The isotopic and elemental composition of scleractinian corals, including massive reef corals and deep-sea species, have proven to be useful tools for paleoceanographic reconstruction, providing information about the surface and deep oceans on timescales of decades to millennia (e.g., Dunbar and Cole 1999). The banded skeletons of gorgonian corals are also potentially valuable archives of past climate conditions. Furthermore, the mixture of organic and inorganic materials within the gorgonian skeleton provides a unique opportunity to develop novel tracers of ocean circulation. Primnoid gorgonians as well as bamboo corals were recently investigated as useful model organisms in studies on paleoceanographic dynamics, growth rates, and age estimation (Andrews et al. 2002; Noé et al. 2007, 2008; Sherwood et al. 2005a, 2005b; Thresher et al. 2004). It was established that high temporal resolution, geochemical-based climate reconstructions of both surface and intermediate/deepwater processes may be made from the gorgonin-based skeletons of deep-sea octocorals (Heikoop et al. 2002; Sherwood et al. 2005a, 2005b; Thresher et al. 2004). For example, reported growth rates of bamboo coral skeletons may fluctuate by about two orders of magnitude—from 0.05–0.1 mm/year (Roark et al. 2005) to 1.9–4.4 mm/year (Andrews et al. 2005). These data prove that growth rates may vary considerably between individual skeletons, possibly controlled by changing oceanographic conditions at the coral habitat. Growth rings in octocorals may be observed in either the massive calcite or horny regions of the skeleton. In the massive calcite, the rings probably relate to the repetition of crystal growth and nucleation events. In the horny regions, the rings are produced by alternations in the ratio of gorgonin:calcite (Marschal et al. 2004; Risk et al. 2002; Sherwood 2002), perhaps compounded by variations in the extent of protein tanning (Szmant-Froelich 1974). Using bomb-14 C (Sherwood et al. 2005c; Sherwood 2006) and 210 Pb-dating (Andrews et al. 2002) annual ring periodicity in the red tree coral Primnoa resedaeformis has been proven. Finer scale growth rings, possibly lunar in origin, have also been described in P. resedaeformis (Risk et al. 2002; Sherwood 2002) making this species perhaps the highest resolution deep-water archive in the world. Other studies report more diffuse banding patterns, with ambiguous periodicities (Andrews et al. 2005; Druffel et al. 1990; Roark et al. 2005). As with scleractinians, it is highly likely that the appearance and timing of rings depend on local environmental factors, such as the downward flux of organic matter from the spring plankton bloom. Life spans of octocorals often exceed several hundreds of years (Andrews et al. 2002; Druffel et al. 1990; Risk et al. 2002; Roark et al. 2005; Thresher et al. 2004), the oldest reported life span being a 700 year old specimen of P. resedaeformis (Sherwood et al. 2006). The paper by Sherwood et al. (2006) explores aspartic acid racemization dating of the gorgonin fraction in modern and
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fossil specimens of primnoid gorgonians collected from the NW Atlantic Ocean. Interestingly, gorgonin amino acid compositions were identical in the fossil and modern specimens, indicating resistance to organic diagenesis. Similar to bone collagen, the fibrillar protein of gorgonin may impose conformational constraints on the racemization of Asp at low temperatures. The rate of racemization of aspartic acid (D/L-Asp) was similar to previously published results from an 1800 year old anemone (Drüffel et al. 1995). Compared with deep-sea scleractinian corals, obtaining centuries-long geochemical records from octocorals, is simplified by the ability to sample across the growth rings of the axial skeletal. Bamboo coral colonies have a distinctive appearance. The skeleton consists of an articulated axis of alternating calcitic internodes and proteinaceous nodes, surrounded in life by non-retractile polyps with rod-shaped sclerites and large needles projecting prominently from the septa at the base of the tentacles. Species of Keratoisidinae are well adapted to bathyal and abyssal depths; they are known in all seas but the Arctic Ocean (Bayer 1956; Kükenthal 1924). They can be abundant on abrupt topographies like seamounts and canyons, where large, suspension feeding colonies provide an important habitat to associated species of fish, invertebrates, and microbial fauna. The subfamily Keratoisidinae consists of four genera (Acanella, Isidella, Lepidisis, and Keratoisis) and two genera (Tenuisis and Australisis) that may belong elsewhere in Isididae (Bayer 1990). Isidid skeleton provides a highresolution archive of paleoceanographic dynamics in deeper water masses (Noé and Dullo 2006). Concentric incremental accretion around the central axis in the early growth stages changed into a unilaterally asymmetric growth during late-stage evolution, probably triggered by the establishment of a stable system of unidirectional currents and nutrient flux. The nodes of isidids are also useful for dendrochronology. Stable isotope signatures of Pb and U/Th from growth rings in the basal nodes were used to estimate that the ages of colonies of Isidella tentaculum (Etnoyer 2008) are between 75 and 100 year (Andrews et al. 2005; Roark et al. 2005). These rings indicate lunar growth cycles but also record oceanic events, such as the testing of nuclear bombs (Roark et al. 2005). Skeletal axes of the Keratoisidinae are durable and distinctive, one of few octocorals with a fossil history (Di Geronimo et al. 2005).
14.4 Conclusion Gorgonian octocorals possess a branched endoskeleton, which is precipitated by the surrounding coenenchyme of the octocoral colony. The skeletons are characterized by compositional heterogeneity: a vertical alternation of bright calcified internodes and dark organic nodes composed of the gorgonin were described for these marine invertebrates. Gorgonin, a halogenated cross-linked proteinaceous fibrous biopolymer, was proved to form a major part of the insoluble matrix of the gorgonians. It provides flexibility, enabling these corals to survive in high-energy, current-affected environments. The biomimetic potential of gorgonin-based natural materials is
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high, especially because most deep-sea gorgonians are distributed within the coldwater coral ecosystems, which suggests that gorgonin made biological materials are developed under temperatures between 0 and 4◦ C.
References Andrews AH, Cailliet GM, Kerr LA et al (2005) Investigations of age and growth for three deepsea corals from the Davidson Seamount off central California. In: Freiwald A, Roberts JM (eds) Cold-water corals and ecosystems. Springer, Heidelberg Andrews AH, Cordes EE, Mahoney MM et al (2002) Age, growth and radiometric age validation of a deep-sea, habitat-forming gorgonian (Primnoa resedaeformis) from the Gulf of Alaska. Hydrobiologia 471:101–110 Balard M (1825) Note pour servir a l’histoire naturelle de l’iode. Annls Chim Phys, Ser 2 28: 178–181 Bayer FM (1955) Contributions to the nomenclature, systematics, and morphology of the octocorallia. Proc U S Natl Mus 105:207–220 Bayer FM (1956) Octocorallia. In: Moore RC (ed) Treatise on invertebrate paleontology, Part P. University of Kansas, Kansas, Lawrence Bayer FM (1961) The shallow water Octocorallia of the West Indian Region. A manual for marine biologists. Martinus Nijhoff, The Hague Bayer FM (1990) A new isidid octocoral (Anthozoa: Gorgonacea) from New Caledonia, with descriptions of other new species from elsewhere in the Pacific Ocean. Proc Biol Soc Washington 103:205–228 Block RJ, Boiling D (1939) The amino acid composition of keratins. The composition of gorgonin, spongin, turtle scutes and other keratins. J Biol Chem 127:685–693 Block RJ, Vickery HB (1931) The basic amino acids of proteins. A chemical relationship between various keratins. J Biol Chem 93:113–118 Bond ZA, Cohen AL, Smith SR et al (2005) Growth and composition of high-Mg calcite in the skeleton of a Bermudian gorgonian (Plexaurella dichotoma): potential for paleothermometry. Geochem Geophy Geosy 6. doi:10.1029/2005GC000911 Brunet PCJ (1967) Sclerotins. Endeavor 26:68–74 Cook FC (1904) The chemical composition of some gorgonian corals. Am J Physiol 12:95–98 Di Geronimo I, Messina C, Rosso A et al (2005) Enhanced biodiversity in the deep: early Pleistocene coral communities from southern Italy. In: Freiwald A, Roberts JM (eds) Coldwater corals and ecosystems. Springer, Berlin, Heidelberg Dreschel HFE (1896) Beitrgge zur Chemie einiger Seetiere. II. Uber das Achsenskelett der Gorgonia cavolini. Z Biol 33:85–107 Drüffel ERM, Griffin S, Witter A et al (1995) Gerardia: Bristlecone pine of the deep-sea? Geochimica et Cosmochimica Acta 59:5031–5036 Druffel ERM, King LL, Belastock RA et al (1990) Growth rate of a deep-sea coral using 210Pb and other isotopes. Geochimica et Cosmochimica Acta 54:1493–1500 Duchassaing P, Michelotti J (1864) Supplément au mémoire sur les coralliaires des Antilles. Extrait des mémoires de l’Académie des Sciences de Turin 2(23):1–112 Dunbar RB, Cole JE (1999) Annual records of tropical systems (ARTS), PAGES workshop report, Series 99-1, pp 71, Switzerland, Bern Ehrlich H, Etnoyer P, Litvinov SD et al (2006) Biomaterial structure in deep-sea bamboo coral (Anthozoa: Gorgonacea: Isididae): perspective for the development of bone implants and templates for tissue engineering. Materialwissenschaft und Werkstofftechnik 37(6):552–557 Elliot DZ (1965) Structure and function of mammalian tendon. Biol Rev 40:392–421 Etnoyer P (2008) A new species of Isidella bamboo coral (Octocorallia: Alcyonacea: Isididae) from northeast Pacific seamounts. Proc Biol Soc Washington 121(4):541–553
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Chapter 15
Antipathin
Abstract Antipatharians have a rigid, erect chitin skeleton that forms a branched, tree-like colony, or a long unbranched whip-like coil. The structure of the black coral skeleton is that of a laminated composite; its specific chemical composition may vary greatly from one species to another. Other components that are also present in the skeleton are lipids, carbohydrates, phenols, and sterols. Iodine and bromine appear to be the dominant single elements. The main structural components of the antipatharian skeletal formations are represented by a halogenated scleroprotein, called antipathin, and chitin. Antipathin seems to be responsible for specific material properties of black coral skeletons. The skeletons of antipatharians are more elastic and less rigid than some other biomaterials selected and used in the nature as structural components, including wood, bone, mollusc shell, and the insect cuticle. At the same time, the density of antipathin is higher than wood, lower than shell or bone, but nearly the same as insect cuticle.
15.1 Brief Introduction into Black Corals Black corals (Antipatharia) are colonial cnidarians found throughout the world’s oceans. The order Antipatharia (Milne-Edwards and Haime (1857) (Cnidaria, Anthozoa) currently possesses about 230 valid species (Brugler and France 2007; Schultze 1896; Totton 1923) belonging to 39 genera and 6 families, even if there is some debate on the precise number of valid genera (Opresko 1974). Systematic studies concerning black corals date from the 1700s and several taxonomic monographs were published at the end of the following century and early 1900s. These include those manuscripts written by Brook (1889), Schultze (1896), and van Pesch (1914), many of which were associated with long-range oceanographic expeditions (Cooper 1909; Delage and Hérouard 1901; Echeverria 2002; Grigg and Opresko 1977; Kinoshita 1910; Loiola 2007; Pasternak 1977; Warner 1981). All species demonstrate slow growth, compensated for by a long life and low natural adult mortality. They reach reproductive maturity after about 10 years, which is much later than for other similar organisms. The output of larvae (called planula) is small. The recovery occurs at a slow rate. Particular fragments (polyps) reach H. Ehrlich, Biological Materials of Marine Origin, Biologically-Inspired Systems 1, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9130-7_15,
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dimensions ranging from 0.5 to 5 mm; however, the majority are sized between 1 and 2 mm (Nowak et al. 2009). Recent radiocarbon results show the age of deepwater black coral Leiopathes sp. to be on the order of thousands of years. The longest-lived Leiopathes sp. specimens was 4265 years (Roark et al. 2009). Antipatharians have a rigid, erect chitin skeleton that forms a branched, tree-like colony or a long unbranched whip-like coil (wire coral) (Fig. 15.1). The structure of the black coral skeleton is that of a laminated composite, constituted primarily of chitin fibrils and non-fibrillar protein; however, its specific chemical composition may vary greatly from one species to another. Other components that are also present in the skeleton are lipids, carbohydrates, phenols, and sterols. Iodine and bromine appear to be the dominant single elements (Juárez de la Rosa et al. 2007). The skeleton is arranged in the shape of a clear ring structure, which can be used to estimate the age of corals. Growth rings in the skeletons of antipatharian corals have indeed been correlated with age (Goldberg 1978). This suggests that, as in the case of some red corals, additional information potentially stored in the rings (e.g., on the chronology, the water temperature, salinity) can be extracted and deciphered.
Fig. 15.1 Antipatharians, or black corals, have a rigid, gorgonin-, and chitin-based skeleton (a, b) that forms a branched, tree-like colony or a long meters long unbranched whip-like coil (c, d, e) (images courtesy Marzia Bo, Giorgio Bavestrello, and Mark Spencer)
15.2
Chemistry of Black Corals
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Black corals are well known as articles of commerce in the jewelry and curio trade dating from at least the of the ancient Greeks (Hickson 1924). The skeleton can be polished to an onyx-like luster, but because it is organic, it can also be bent and molded when heated. Consequently, this material is highly prized in the jewelry trade and collective pressure has resulted in the listing of black corals in the Convention for the International Trade in Endangered Species (CITES) (Wood and Wells 1988).
15.2 Chemistry of Black Corals The main structural components of the antipatharian skeletal formations are represented by a halogenated scleroprotein, called antipathin and chitin. For example, NMR analysis estimates the organic content of the load-bearing skeletal base of the New Zealand black coral Antipathes fiordensis as 70% protein, 10% chitin, 15% diphenol, and 5% lipid by weight (Holl et al. 1992). The younger pinnules or tips of A. fiordensis are less than 3% diphenol by weight. The only diphenols extracted from coral skeleton by hydrochloric acid were 3-(3,4-,dihydroxyphenyl)-IX-alanine (DOPA) and 3,4-dihydroxybenzaldehyde (DOBAL). More DOPA is found in the base than in the tips of A. fiordensis and it appears to be a peptidyl component of coral skeletal protein. The oxidation of DOPA and DOBAL to quinones may provide mechanical stabilization of the coral skeleton by the cross-linking of structural proteins, either to other proteins or to chitin (Holl et al. 1992; Kim et al. 1992). The term “antipathin” was used to distinguish these proteins from the “gorgonins.” This term was coined for this distinctive material (Roche and EyssericLafon 1951) prior to the discovery of the chitin component. The high histidine levels shown in the antipathins confirm the results of Roche and Eysseric-Lafon (1951) who originally characterized them as having the highest histidine content of any known protein. It is interesting to note that a significant quantity of iodohistidine was detected in the antipatharian skeleton (Goldberg 1976). Antipathin, like gorgonin, has all the attributes of a sclerotized protein (Brown 1950): it is unaffected by proteolytic agents, deep brown to black in color, high in tyrosine, and soluble only in chlorine bleach. Antipathin is not solubilized by autoclaving; however, the amino acid composition of this material does not suggest collagen (Goldberg 1976). The effect of the large amount of histidine is not known, but the presence of iodinated histidine could be an indication that histidine crosslinks are being formed in addition to links with tyrosine. This could explain the extremely stable nature of antipathin. It will not stain with typical connective tissue stains such as van Gieson’s, Mallory’s, or Gomori’s. The only histochemically demonstrable polysaccharide appears to be a sialic acid, the significance of which is not known. Thus, the composition, structure, and reactivity of this material suggest that it contains neither collagen nor keratin (Goldberg 1976). To my best knowledge, there is no information in the available literature about the role of antipathin in any kind of biomineralization.
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The relative abundance of iodine in a number of antipatharian species has been traced to tyrosyl and histidyl residues (Goldberg 1976; Roche et al. 1963). The considerably higher concentration of iodine in Antipathia salix suggests the possibility of increased cross-linking in this species by an iodine-mediated mechanism. Considering the level of bromide in seawater (about 0.8 mM) and in the antipatharian skeletons, bromotyrosine (and perhaps bromohistidine) may also be present as in gorgonian coral and sponge skeletons (Roche et al. 1963), but these compounds have yet to be discovered in black corals. All the species of black corals have considerable chitin content, ranging from 6 to 18% of the total mass of the skeleton (Nowak et al. 2009). In the most complete experimental study, published by Goldberg (1976), it was estimated that the chitin represents 14.5% of the skeletal mass in A. salix. However, there are big differences in the content of both antipathin and chitin reported for different black coral species. For example, the chitin content of A. salix measured by NMR was about twice that of A. fiordensis (Holl et al. 1992). Using glucosamine levels to estimate chitin, Kim et al. (1992) found that A. salix tips contained ca. 29% more chitin than A. fiordensis. Recently, Nowak et al. (2009) reported that black corals collected from three different regions contain morphologically distinctive multilayer hybrid structures. The matrix is composed of α-chitin, which constitutes approximately 15% of its weight, supplemented by a set of proteins and carbohydrates. The orientations of the chitin crystallites were different in different zones; in the interior, the chitin fibrils were 4 μm wide and oriented along the long axes of the cells. Although tyrosine is not prominent in the skeletal hydrolysates of the antipathins studied previously (Goldberg 1976), a relatively much larger amount of this amino acid remains closely associated with the skeletal chitin, suggesting a role of covalent bonding between chitin and protein. Additional tyrosine may also be halogenated or involved in stable cross-links. Morphological observations showed that the axial structure of black corals consisted of a core encased in a number of structural cells bonded with intermediate gluing layers (Nowak et al. 2009). Previously, these layers were described as “layers of organic cement” (Holl et al. 1992). The periodic clusters of opaque cement lines between skeletal layers are suggested to be responsible for the growth-ring pattern (Goldberg 1991). The periodicity of the black coral structure, and variations in its chemical composition, may be attributable to environmental fluctuations and hence may serve as a record of local conditions (Goldberg 1978; Holl et al. 1992). The elemental analysis of gluing cementous layer showed high I, K, Zn, and Ca concentrations. These elements are probably in contact with the carboxyamino acids and lipids which also concentrate in the gluing zones, according to independent studies. The material of gluing zone seems to be the most interesting among all the constituents of the black corals—it involves highly concentrated iodine inside the organic zone (Nowak et al. 2009). An interesting metabolite was also isolated from black coral Leiopathes sp. Guerriero et al. (1988) reported on the identification of leiopathic acid (hydroxydocosapentaenoic acid). However, the possible role of this fatty acid with respect to structural feature of antipatharians is still unknown.
15.3
Material Properties of Antipathin-Based Skeletons
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15.3 Material Properties of Antipathin-Based Skeletons From the morphological and mechanical point of view, black coral skeletons are laminated composites, composed primarily of chitin fibrils and antipathin. However, in antipatharian skeleton, the layers are not simple laminated structures. Because the spines are cemented and inserted layer upon layer (Goldberg and Taylor 1989a, b), the helically wound skeleton is fixed at multiple points. Kim et al. (1992) hypothesized that the spines increase the surface area for cementing one skeletal layer to the next. Moreover, they may play an important role as continuous rivets, preventing delamination from shear forces produced by skeletal bending and torsion. If this suggestion is correct, the presence of spines should reduce or eliminate the requirement of small fibril biases between helically wound layers. The skeletons of antipatharians are more elastic and less rigid than some other biomaterials selected and used in the nature as structural components, including wood, bone, mollusc shell, and the insect cuticle. At the same time, the density of antipathin is higher than wood, lower than shell or bone, but nearly the same as insect cuticle (Wainwright et al. 1976). The ratio of Young’s modulus to density (E/p) is the specific modulus and serves as a means of assessing the stiffness per unit weight of materials. High values of specific stiffness are often considered superior to low values because they enable construction of stiffer and lighter structures. However, for antipatharians, greater flexibility per unit of density should be more important than stiffness (Goldberg 1976; Kim et al. 1992). Thus antipatharians have a lower specific modulus compared to insect cuticle values given in Wainwright et al. (1976), achieved by having a lower modulus with about the same density (E/p = 5.1–7.9 for two insects and 1.0–2.3 for A. fiordensis and A. salix, respectively). There is a growing body of evidence showing a relationship between skeletal mechanics and ecological function (Kim et al. 1992). In organisms with flexible skeletons, orientation to flow can maximize efficiency of suspension feeding and minimize drag forces (reviewed by Wainwright et al. 1976). In certain gorgonian corals, adaptation to flow may be recorded in the skeleton as a change in preferred orientation of fan-like species (Grigg 1972; Wainwright and Dillon 1969). In branched gorgonians the skeleton can be reinforced perpendicular to the direction of flow, by deposition of carbonate (Wainwright and Koehl 1976; Wainwright et al. 1976). Preferred orientation occurs in the Antipatharia (Warner 1981), and there is a degree of it exhibited in A. fiordensis. Colonies near the mouths of the fiords are subjected to more consistent current fields, resulting in more fan-shaped colonies. Otherwise, this species tends to branch in multiple planes (Grange 1988). In addition, the skeleton often has an elliptical cross section, especially in the thicker branches, with the compressed sides facing the predominant current flow. In A. salix, there is no obvious structural asymmetry and the colonies tend to be branched in many planes. Antipatharians generally require low rheological environments. Unlike other cnidarians, the polyps have no structural protection from the abrasive forces associated with strong current. The muscular systems of the polyps and tentacles are so poorly developed that a modest contraction is their only apparent
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defense against such forces (Goldberg and Taylor 1989a, b). Transplant experiments into relatively shallow water further suggest that abrasion is a major source of mortality (Grigg 1965). Thus the substantial structural and mechanical properties of the black coral skeleton seem to be overdesigned for the deeper and hydrodynamically more docile zones in which antipatharians are generally found. While the fit between ecological function and skeletal design is unclear, the distinction between the two species studied has shown that A. salix is darker, harder, denser, more hydrophobic, and stiffer than A. fiordensis (Holl et al. 1992; Kim et al. 1992). These material differences appear to reflect the more considerable commercial value of A. salix in the jewelry trade. High stiffness is often a bonus, since it enables the construction of strong and light skeletons. The hardness of the skeleton (A. fiordensis and A. salix) on the Mohs scale is 3. It is sufficiently hard to scratch calcite (Nowak et al. 2009). It is little strange that organisms living in conditions that provide them with only a small chance to obtain a lot of energy have decided to form such a refined and energy-consuming skeleton constructed of chitin instead of the simple, aragonitebased and nearly self-sedimented inorganic skeleton. The probable explanation is that the organic skeleton, due to its elasticity, obeys the natural wave movements of the water streams. This makes hunting strategies easier. In addition, after the initial expending of material and energy on construction of the skeleton, the organism saves the energy later on, with a large total surplus over the scale of a lifetime (~40 years for colonies reaching a maximum of 1.8 m in height) (Nowak et al. 2009).
15.4 Conclusion The protein component of the black coral skeleton is complex and incompletely characterized; because it is amorphous, its architecture cannot be visualized by microscopy and compared with that of chitin. Because of its compositional dominance and by analogy with insect cuticle, many of the physical properties of black coral skeletons are likely to be related to the protein components, including the extent to which they are cross-linked to one another and to chitin (Hillerton 1984; Hopkins and Kramer 1992). However, cross-linking between halogenated tyrosyl residues in proteins may also play a prominent role in stabilizing black coral skeleton, particularly among species such as A. salix that have a low diphenol content. Physical and mechanical properties of black coral skeletons are likely to reflect the nature, relative abundance, and architecture of their structural polymers, as well as the type and number of intra- and intermolecular bonds that stabilize them. Understanding the high degree of diversity with respect to the chemical and structural properties in antipatharians is an intriguing question. For example, why is the skeleton of the Caribbean black coral, A. salix, is stiffer, darker, harder, denser, and more hydrophobic than that of the New Zealand species (Kim et al. 1992)? The management and conservation of deep-sea coral communities including black corals is challenged by their commercial harvest for the jewelry trade and
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damage caused by deep-water fishing practices. I absolutely agree with Brendan Roark, who recently noted (Roark et al. 2009) that “in light of the unusual longevity of black corals, a better understanding of deep-sea coral ecology and their interrelationships with associated benthic communities is needed to inform coherent international conservation strategies for these important deep-sea habitat-forming species.” There are no doubts that investigations on the unique chemical as well as the biomaterial features of these thousands of years old antipathin-based structures must continue in the future.
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Hopkins TL, Kramer KJ (1992) Insect cuticle sclerotization. Annu Rev Entomol 37:273–302 Juárez de la Rosa BA, Ardisson P-L, Azamar-Barrios JA et al (2007) Optical, thermal, and structural characterization of the sclerotized skeleton of two antipatharian coral species. Mater Sci Eng C 27:880–885 Kim K, Goldberg WM, Taylor GT (1992) Architectural and mechanical properties of the black coralskeleton (Coelenterata: Antipatharia): a comparison of two species. Biol Bull Mar Biol Lab, Woods Hole 182:195–209 Kinoshita K (1910) On a new antipatharian Hexapathes heterosticha, n.g. et n.sp. Annotationes Zoologicae Japonensis 7(4):231–234 Loiola LL (2007) Black corals (Cnidaria: Antipatharia) from Brazil: an overview. In: George RY, Cairns SD (eds) Conservation and adaptive management of seamount and deep-sea coral ecosystems. Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami Milne-Edwards H, Haime J (1857) Histoire Naturelle des Coralliairesou Polypes Proprement Dits, Paris Nowak D, Florek M, Nowak J et al (2009) Morphology and the chemical make-up of the inorganic components of black corals. Mater Sci Eng C 29:1029–1038 Opresko DM (1974) A study of classification of the Antipatharia (Coelenterata: Anthozoa) with description of eleven species. Ph.D. Dissertation, University of Miami, Coral Gables, FL Pasternak FA (1977) Antipatharia. Galathea Rep 14:157–164 Roark EB, Guilderson TP, Dunbara RB et al (2009) Extreme longevity in proteinaceous deep-sea corals. PNAS 106:5204–5208 Roche J and Eysseric-Lafon M (1951) Biochemie comparte des scltroprotines iodes des anthozoaires (Gorgonaires, Antipathaires, Gtrardiidts) et sptcificit des gorgonines. Bull Stk Chim Biol 33:1437–1447 Roche J, Fontaine M, Leloup L (1963) Halides. In: Florkin M, Mason HS (eds) Comparative biochemistry, vol VI. Academic, New York Schultze LS (1896) Beitrag zur Systematik der Antipatharien. Abhand Senckenb naturf Gesellsch 23:1–40 Totton AK (1923) Coelenterata. Part III. Antipatharia (and their cirripede commensals). British Antarctic (“Terra Nova”) Expedition, 1910. Nat History Rep, Zool 5:97–120 van Pesch AJ (1914) The antipatharia of the siboga expedition. Siboga Exped Monogr 17:1–258 Warner GF (1981) Species descriptions and ecological observations of black corals (Antipathatia) from Trinidad. Bull Mar Sci 31:147–163 Wainwright SA, Biggs WD, Currey JD, Gosline JM (1976) Mechanical desing in organisms. Edward Arnold, London Wainwright SA, Dillon JR (1969) On the orientation of sea fans (genus Gorgonia). Biol Bull 136:130–139 Wainwright SA, Koehl MAR (1976) The nature of flow and the reaction of benthic Cnidaria to it. In: Mackie GO (ed) Coelenterate ecology and behavior. Plenum, New York Wood EM, Wells SM (1988) The marine curio trade: conservation issues. A report for the Marine Conservation Society, Herefordshire, England, pp 1–120
Chapter 16
Rubber-Like Bioelastomers of Marine Origin
Abstract The hinge ligament of bivalve molluscs is a proteinaceous structure secreted by the mantle isthmus in the region where the two shells articulate. The hinge ligament consists of two parts. The outer part holds the two valves closely together along the hinge line. It is flexible and acts as a hinge. The inner part is a block of elastic material which acts as a compression spring, making the shell gape when the adductor muscle relaxes. The chemistry and structural features of molluscs hinge ligaments as examples of marine bioelastomers are described and discussed. Elastic proteins also perform unusual and unique functions, for example, abductin ligaments in clams (bivalve molluscs) open the shell when the muscles relax, generating a primitive swimming action; while byssus threads bind mussels to rocks and possess elastic and rigid domains to resist wave action. The recent rapid explosion of our understanding of protein elasticity has been based on our ability to determine the molecular sequences and three-dimensional structures of the proteins and to relate them to the mechanical properties of macromolecular arrays and single molecules, the latter using new techniques, such as laser tweezers and atomic force microscopy. These techniques have led to rapid advances, but have inevitably also raised many more intriguing questions. Several bioelastomers are described and discussed in the book edited by Peter R. Shewry, Arthur S. Tatham, and Allen J. Bailey and entitled Elastomeric Proteins: Structures, Biomechanical Properties, and Biological Roles (Cambridge University Press, 2001). Therefore, following chapters contain only bioelastomers of marine invertebrate origin-like hinge ligaments of molluscs, the unusual biopolymer isolated from whelk egg capsules as well as crustaceans resilin.
16.1 Hinge Ligament Bivalved shells, especially those of molluscan origin, act as a system of choice for biomineralization, bionic as well as biomimetic research (David 1998). Compared to other composite materials, shell presents superior mechanical properties (stiffness, fracture toughness, tensile strength) due to a complex architecture and the H. Ehrlich, Biological Materials of Marine Origin, Biologically-Inspired Systems 1, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9130-7_16,
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involvement of biological macromolecules like chitin, silk, and acidic proteins. At present, many reports are available about the intricate organization of the mineral phases, the crystallographic orientation within individual layers; as well as the influence of the biological phase on crystalline species and on the properties of biological components (see for review Marin et al. 2008; de Paula and Silveira 2009). However, there are two additional topics related to bivalved shells of molluscs—the hinge ligament and abductin, both of interest for materials science. The bivalved shell is the simplest possible lever skeleton. It consists of two valves, rigid enough to provide skeletal support and protection, which are free to rotate about a fixed hinge axis. Except in Calceola and almost all the rostroconchs, the valves are closed by one or two adductor muscles. The opening mechanisms are much more diverse. In conchostracans and living phyllocarids, the shell as a whole is flexible and there is no discrete hinge or ligament structure. Most of these animals are small (the carapace of Nebaliopsis, one of the largest, reaches a length of 2 cm) and the shells are opened by their own elasticity (Thomas 1988). In contrast, most pelecypods and the bivalved gastropods have rigid valves that are united and opened by specialized elastic ligaments, often with the aid of the foot. Ostracode shells are similarly designed, but elasticity of the ligament does not appear to have been demonstrated directly. Bivalve armor is common in attached nuts and seed-pods, on land. However, the bivalved shell of an animal cannot be simultaneously closed and clamped to a surface, in contrast with the shell of a snail. These are general limitations of the bivalved shell and limit its suitability for adaptation to life on the land (Thomas 1988). The morphology, function, and materials properties of hinge ligament in different mollusc species are described in detail in papers by Trueman (1949, 1950a, b, 1951, 1953, 1969), Owen et al. (1953), and Alexander (1966, 1968, 2002). The hinge ligament of bivalve molluscs is a proteinaceous structure secreted by the mantle isthmus in the region where the two shells articulate (Fig. 16.1). Briefly, the hinge ligament consists of two parts (Trueman 1953). The outer part holds the two valves closely together along the hinge line. It is flexible and acts as a hinge. The inner part is a block of elastic material which acts as a compression spring, making the shell gape when the adductor muscle relaxes. Dall (1889) proposed the name “resilum” for the inner ligament in an attempt to convey its compressional function. It is calcified at its lateral ends where it is attached to the valves, but its main part is an uncalcified protein. It breaks with a conchoidal fracture and shows no birefringence in sections (of Pecten, Pectinidae (Lamellibranchiata)) hand-cut in three planes mutually at right angles. It is therefore amorphous, at least when unstrained. The excised ligament bounces like rubber when dropped on a hard surface. The function of hinge ligament is also significant for swimming molluscs. For example, Scallops (Pecten, etc.) are exceptional among bivalve molluscs in being able to swim. They do this by rapidly and repeatedly opening and closing their shells, ejecting jets of water on either side of the hinge. The shell is closed by means of a muscle, but there is no muscle to open it; instead, there is an elastic hinge
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Chemistry of the Hinge Ligament
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Fig. 16.1 SEM images (a, b, c, d) of the hinge ligament (white arrows) of Solen sp. collected in Tunisia (image courtesy Lothar Brehmer)
ligament just inside the hinge. This is a block of the rubber-like protein abductin. Kelly and Rice (1967) proposed the name “abductin” for the protein of the internal ligament of Aequipecten irradians and Placopecten magellanicus. It is compressed when the shell closes and recoils elastically to open it. The resonant frequency of the animal, due to the interaction of the compliance of the hinge ligament with the masses of the valves of the shell and the added masses of water that move with them, matches the frequency of the swimming movements (Alexander 2002). Bivalve molluscs have adductor muscles to close their shells, but no muscles to open them. Instead, the shell springs open by elastic recoil of the hinge ligament. In other bivalves, which do not swim the hinge ligament functions simply as a return spring that enables the animal to open the closed shell. The hinge ligament of scallops has low hysteresis, suiting its function in swimming, but the hinge ligaments of other bivalves show much more marked hysteresis (Trueman 1953, 1969).
16.2 Chemistry of the Hinge Ligament Data on chemistry of hinge ligaments in molluscs are very diverse and sometimes curious.
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Thus, Trueman reported birefringence in portions of the ligaments of Tellina tenuis (Trueman 1949) and Mytilus edulis (Trueman 1950b) and speculated that the birefringence of the Tellina ligament was due to some substance in the ligament of lipoid nature. Generally, the ligament, like the shell, is composed of some combination of protein and calcium carbonate, although the latter phase may be absent in the ligament of some species, e.g., Aequipecten. The protein phase of portions of Anodonta ligament has been characterized as a quinone-tanned protein (Trueman 1950a). Unusually high glycine content has been reported in the ligaments of A. irradians, P. magellanicus, and Mytilus californianus (Hare 1963; Kelly and Rice 1967). Kahler et al. (1976 a, b) investigated the composition of the inner hinge ligament of the marine bivalve mollusc, Spisula solidissima. These authors reported that calcium carbonate is embedded in a hydrated protein matrix. Amino acid analysis of this matrix indicates that more than 62% of the residues are glycine. The possibility exists that hinge ligament proteins are related to shell-soluble proteins. It was reported (Kahler et al. 1976a) that the amino acid composition of the crystal sheaths from the Mercenaria ligament is more closely related to that of the Mercenaria shell soluble protein, than it is to the bulk ligament protein in which they are located. The shell protein contains 29.7% aspartic acid and 15.6% glycine, compared to 20.0% aspartic and 16% glycine in the ligament crystal sheaths and 11% aspartic and 29% glycine in the bulk ligament protein. Such similarities indicate that the proteins from both shell and ligament crystal sheaths are involved in similar phases of mineralization which may proceed by the same mechanism and that the bulk protein elastomer which comprises the ligament is uninvolved in the mineralization process. Unlike Mercenaria shell protein, however, the ligament crystal sheaths contain only 17% of the acid residues in the amide form, which compares well with 16% amide residues in the ligament protein (Kahler et al. 1976a). The Mercenaria shell protein, on the other hand, is fully amidated (Crenshaw 1972). The ligament crystal sheaths contain a net of 20.2% acidic residues after subtract of the amide residues, which leaves a nearly twofold excess over the basic residues. In the Spisula crystal sheaths, 50% of the acid residues are amides. After subtracting the amides this leaves the Spisula sheaths with only 5.5% acidic residues, a rather modest amount and comparable to the total basic residues of 6.2%. The carbohydrate component of the sheaths is small, less than 1% for Spisula sheaths and about 5% for Mercenaria sheaths. Crenshaw (1972) found 20.5% carbohydrate in the soluble Mercenaria shell protein and Tompa et al. (1977) found about 26% carbohydrate in the insoluble matrix of the giant land snail (Strophocheilus oblongus) egg. The Mercenaria ligament sheath carbohydrate has a weight ratio of hexosamine/uronic acid/ester sulfate/neutral carbohydrate of 1:1:4.8:4. The ratio in the land snail egg is similar, 1:1:6.5:4.5. However, in the Mercenaria soluble shell matrix the hexosamine level is much higher, accounting for 50% by weight of the carbohydrate component.
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Chemistry of the Hinge Ligament
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Many authors suggested the presence of different kinds of cross-links within hinge ligaments of molluscs. Thus, Andersen (1967) suggested that hinge ligaments contain phenolic compounds which in several respects resemble dityrosine. He isolated 3,3 -methylene-bistyrosine from of M. edulis by decalcification of the ligaments in 1 M acetic acid, followed by hydrolysis in 6 M HCl. In his Nature publication, Anderson assumed that the function of dityrosine-based compounds is to link the protein chains in the ligament together in a three-dimensional network. However, Kikuchi and co-workers (1982, 1987, 1988) re-examined results reported by Anderson and showed the isolated compound to be an artifact. These authors investigated the cross-linking components of the hinge ligament of a surf clam species, Sakhalin surf clam (Pseudocardiurn suchalinensis), in family Mactridae. It was shown that the resilium of this species consists of about 35% protein and 65% aragonite crystals by the dry weight. The resilium protein is composed of about 50 mol glycine and 20 mol methionine per 100 mol total amino acids. Almost all of the methionine is present in an oxidized form, methionine-S-oxide. This characteristic resilium protein is common among the species in family Mactridae. A preliminary examination by hydrazinolysis showed that the protein had glycine at its carboxyl terminal and that it contained about 1800 amino acid residues. The results of this work showed that the resilium protein contained isodesmosine and desmosine as cross-linking components. 3,3 -Methylenebistyrosine was also isolated from HC1 hydrolysates of the protein. It was shown, however, that the tyrosine derivative could be formed as an artifact from tyrosine and formaldehyde during the hydrolysis process (Kikuchi et al. 1987, 1988). Molluscan hinge ligament is strongly resistant to chemical and enzymatic treatment. For example, in contrast to collagen, Spisula inner ligament protein is not affected by 0.5 N acid or alkali at room temperature and undergoes no visible change when heated to 100◦ C in water for 5–6 h (Kahler et al. 1976a, b). However, some species of marine bacteria developed mechanisms to digest hinge ligaments. Of the two structural elements composing the oyster hinge ligament, the resilium is responsible for opening the valves and is also the most common site of erosive lesions. Thus, several strains of cytophaga-like gliding bacteria (CLB) were isolated as numerically dominant or codominant components of bacterial populations associated with proteinaceous hinge ligaments of cultured juvenile Pacific oysters, Crassostrea gigas (Dungan 1987). These bacteria were morphologically similar to long, flexible bacilli occurring within degenerative lesions in oyster hinge ligaments. Among bacteria isolated from hinge ligaments, only CLB strains were capable of sustained growth with hinge ligament matrix as the sole source of organic carbon and nitrogen. In vitro incubation of cuboidal portions of ligament resilium with ligament CLB resulted in bacterial proliferation on the surfaces and penetration deep into ligament matrices. Bacterial proliferation was accompanied by loss of resilium structural and mechanical integrity, including complete liquefaction at incubation temperatures between 10 and 20◦ C (Dungan and Elston 1988; Dungan et al. 1989). Interestingly, electron microscopy of eroding ligament surfaces from areas deep within degenerative lesions revealed the presence of a dense and morphologically
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homogeneous population of CLB bacteria oriented at right angles to intact resilium and surrounded by remnants of degraded resilium (Dungan 1987).
16.3 Structural Features of Hinge Ligaments Pioneering studies by Trueman (1949, 1950a, b, 1951, 1964) represent the first morphological and structural examination of bivalve ligaments. A lamellar continuity appears to exist between the shell and adjacent ligament (Owen et al. 1953). The lamellae of the ligament were considered to be a local modification of the corresponding shell lamellae (Trueman 1964). Galtsoff (1964) published electron micrographs of Crassostrea virginica inner ligament and reported that images from sections tangent to the growth lamellae revealed 500 Å holes in a hexagonal array. Another image from sections, apparently at right angles to these holes, revealed fibrils varying in diameter from 370 to 500 Å. The organic phase of ligament is most often associated with an inorganic phase, CaCO3 . Stenzel (1962) identified aragonite as the crystalline phase in the ligament of C. virginica as did Hare (1963) in M. californianus. Bevelander and Nakahara (1969), in a paper concerning the synthesis of the ligament matrix, published electron micrographs indicating the presence of aragonite crystals in the ligament of M. edulis. Calcite has never been found in the ligament, even in species with a shell composed totally of calcite (Kahler et al. 1976b). Electron microscopic, X-ray diffraction, and polarized light data reported by Kahler et al. (1976b) and Marsh et al. (1976) show the calcium carbonate phase to be single crystals of aragonite, about 1000 Å in diameter, and several thousand angstroms in length. The crystals are oriented with the crystallographic c-axis normal to the visible growth lamellae. The crystallographic a- and b-axes are in random orientation (Fig. 16.2). A detailed model for the ligament of Pinctada maxima is given recently by Zhang (2007), who analyzed the ultrastructure and light reflectivity of dry and wet samples. The author arrived at a model of lamellate structure. The lamellate is about 35 μm thick with aragonite fibers (ca. 78 nm) regularly embedded every 127 nm in the protein matrix, in such a way to behave as a photonic crystal. Being about 6.5 cm long, 2 cm wide, and 0.2 cm thick, the natural dry ligament of P. maxima is coated by a horny layer and appears black. However, when polished and wetted by water, it becomes brilliantly blue, which will return to black spectra of ligament. For wet ligament, the dominant peak is centered at blue wavelengths (453 nm), which gradually shifts to ultraviolet (374 nm) with prolonging drying time. Concordantly, the color changes from blue to black. Note that the above process is reversible. In addition, another secondary peak at about 495 nm is weak and contributes little to ligament coloration. Based on the above results, the author predicted that the blue color in wet ligament arises from structures. The dry ligament is made of about 70 wt% of aragonites fibers and remaining proteins. As is evident in Fig. 16.3 (a, b) the ligament is made of lamellae about 35 μm in thickness. In single lamella, the fibers are highly aligned, but slightly
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Fig. 16.2 Diagrammatic representation of aragonite crystal orientation on the growth lamellae in the inner hinge ligament of Spisula solidissima, (a) Transverse and (c) sagittal fascism. Electron micrographs of transverse sections through the inner ligament of S. solidissima (b) show parallel aligned single aragonite crystals measuring about 1,000 Å in diameter and many thousands of angstroms in length. The protein matrix appears to be amorphous. Electron micrographs of ligaments sectioned at right angles to the long axis of the crystals show typical hexagonal cross sections of single aragonite crystals (d). The population of these crystals in the ligament is consistent with the weight percentage of CaCO3 in the ligament (adapted from Kahler et al. 1976)
misaligned across different lamellae. In addition, the fibers generally lie in observation planes, consistent with the fact that fibrous composite-like ligament is easily fractured along the long axes of fibers. Therefore the colors and reflective spectra described by Zhang (2007) apply when incident light is normal to the long axes of fibers (or observation planes) after being re-dried. Observations of rigidification properties of hinge ligaments were also reported previously. Hydrated inner hinge ligament powder from S. solidissima achieved constant weight when dried in vacuo over CaC12 for 24 h at 20◦ C with a weight loss was 11.9+0.44% (Kahler et al. 1976a). Drying at 98◦ C at atmospheric pressure resulted in a further loss of 2.6±0.1% during the first 24 h with no additional weight loss after 72 h. Whole ligament becomes quite hard and brittle upon drying at room temperature. Thus, the characteristic resilience of the hinge protein is restored upon
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Fig. 16.3 (a) FE-SEM image of cross section of ligament fibers; (b) schematic diagram showing the two-dimensional lattice model for theoretical simulation. Z indicates the propagation direction of light. Double arrowhead indicates axes of fibers (adapted from Zhang 2007)
rehydration. The ligament of S. solidissima shrinks markedly and loses its resilience when exposed to glycerol for several days (Kahler et al. 1976b). Materials properties of molluscan hinge ligament with respect to thermodynamics of elasticity and resilience in comparison with other bioelastomers (elastin, resilin) are discussed in detail in an excellent work by McNeil Alexander cited below.
16.4 Conclusion Interest in elastomeric biocomposites like hinge ligaments of molluscs is currently high for several reasons—first, because of their biological and medical significance, particularly in human diseases. Second, the unusual properties of these marine biological materials provide opportunities to develop novel artificial materials. Third, the development of modern structural and bioanalytical methods makes it possible to study structures and biomechanical properties of these structured bioelastomers at the single molecule level. To effectively understand the mechanical properties of the bivalve hinge ligament, there is a definite need for more detailed information on the composition of the organic matrix, as well as the composition and structure of the inorganic crystalline phase in that matrix.
References Alexander RMcN (1966) Rubber-like properties of the inner hinge-ligament of pectinidae. J Exp Biol 44:119–130 Alexander RMcN (1968) Animal mechanics. University of Washington, Seattle, Washington, pp 346
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Alexander RMcN (1966) Rubber-like properties of the inner hinge-ligament of pectinidae. J Exp Biol 44:119–130 Alexander RMcN (2002) Functions of elastomeric proteins in animals. In: Shewry PR, Tatham AS, Bailey AJ (eds) Elastomeric proteins: structures, biomechanical properties, and biological roles. University Press, Cambridge Andersen SO (1967) Isolation of a new type of cross link from the hinge ligament protein of molluscs. Nature 216:1029–1030 Bevelander G, Nakahara H (1969) An electron microscope study of the formation of the ligament of Mytilus eduliis and Pinctada radiata. Calcified Tissue Res 4:101–112 Crenshaw MA (1972) The soluble matrix from Mercenaria mercenaria shell. Biomineralization 6:6–11 Dall WH (1889) On the hinge of the pelecypods and its development, with an attempt toward a better subdivision of the group. Am J Sci 138:445–462 David L (1998) Mollusc shell structures: novel design strategies for synthetic materials. Curr Opin Solid State Mater Sci 3:232–236 de Paula SM, Silveira M (2009) Studies on molluscan shells: contributions from microscopic and analytical methods. Micron 40:669–690 Dungan CF (1987) Pathological and microbiological study of bacterial erosion of the hinge ligament in cultured juvenile Pacific oysters, Crassostrea gigas. Master’s thesis. University of Washington, Seattle Dungan CF, Elston RA (1988) Histopathological and ultrastructural characteristics of bacterial destruction of hinge ligaments in cultured juvenile Pacific oysters, Crassotrea gigas. Aquaculture 72:1–14 Dungan CF, Elston RA, Schiewe M (1989) Evidence for colonization and destruction of hinge ligaments in cultured juvenile pacific oysters (Crassostrea gigas) by cytophaga-like bacteria. Appl Environ Microbiol 55:1128–1135 Galtsoff PS (1964) The American oyster Crassostrea virginica (Gmelin). US Fish Wildlife Serv Fish Bull 64:1–480 Hare PE (1963) Amino acids in the proteins from aragonite and calcite in the shells of Mytilus californianus. Science 139:216–217 Kahler GA, Fisher FM, Sass RL (1976a) The chemical composition and mechanical properties of the hinge ligament in bivalve molluscs. Biol Bull 151:161–181 Kahler GA, Sass RL, Fisher FM Jr (1976b) The fine structure and crystallography of the hinge ligament of Spisula solidissima (Mollusca: Bivalvia: Mactridae). J Comp Phys 109:209–220 Kelly RE, Rice RV (1967) Abductin: a rubber-like protein from the internal triangular hinge ligament of pecten. Science 155(3759):208–210 Kikuchi Y, Higashi K, Tamiya N (1988) Diastereomers of methionine S-oxide in the hingeligament proteins of molluscan Bivalve Species. Bull Chem Soc Jpn 61:2083–2087 Kikuchi Y, Tamiya N, Nozawa T et al (1982) Non-destructive detection of methionine sulfoxide in the resilium of a surf clam by solid-state 13C-NMR spectroscopy. Eur J Biochem 125: 575–577 Kikuchi Y, Tsuchikura O, Hirama M et al (1987) Desmosine and isodesmosine as cross-links in the hinge-ligament protein of bivalves 3,3 -methylene-bistyrosine as an artefact. Eur J Biochem 164:397–402 Marsh M, Hopkins G, Fisher F et al (1976) Structure of the molluscan bivalve hinge ligament, a unique calcified elastic tissue. J Ultrastruct Res 54:445–450 Marin F, Luquet G, Marie B et al (2008) Molluscan shell proteins: primary structure, origin and evolution. Curr Top Dev Biol 80:209–276 Owen G, Trueman ER, Yonge CM (1953) The ligament in the lamellibranchia. Nature (London) 171:73–75 Stenzel HB (1962) Aragonite in the resilum of oysters. Science 136:1121–1122 Thomas RDK (1988) Evolutionary convergence of bivalved shells: a comparative analysis of constructional constraints on their morphology. Am Zool 28:267–276
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Tompa AS, Wilbur KM, Waite JH (1977) Structural proteins in the calcified egg shell of the giant land snail Strophocheilus oblongus (Becquaert). Comp Biochem Physiol B Biochem Mol Biol 56:279–283 Trueman ER (1949) The ligament of Tellina tenuis. Proc Zool Soc London 119:717–742 Trueman ER (1950a) Observations on the ligament of Mytilus edulis. Q J Micro Sci 91:225 Trueman ER (1950b) Quinone-tanning in the mollusca. Nature (London) 165:297–398 Trueman ER (1951) The structure, development, and operation of the hinge ligament of Ostrea edulis. Q J Micr Sci 92:129–140 Trueman ER (1953) Observations on certain mechanical properties of the ligament of Pecten. J Exp Biol 30:453–467 Trueman ER (1964) Adaptive morphology in paleoecological interpretation. In: Embrie J, Newell N (eds) Approaches to paleoecology. Wiley, New York Trueman ER (1969) Ligament. In: Moore RC (ed) Treatise on invertebrate paleontology, part N, vol 1. Geological Society of America, Boulder, and University of Kansas, Lawrence Zhang G-S (2007) Photonic crystal type structure in bivalve ligament of Pinctada maxima. Chin Sci Bull 52(8):1136–1138
Chapter 17
Capsular Bioelastomers of Whelks
The eggs of the whelks are laid enclosed in protective capsules which, after the hatching of the eggs, are frequently found cast up by the sea along the shoreline. These capsules have a horny sclerotized appearance and are semitransparent and brownish in colour. S. Hunt, Nature, 1966.
Abstract Many marine caenogastropod snails (i.e., whelks) deposit their embryos in egg capsules constructed from a highly resilient, multilaminate biological material. Capsular protein polymer exhibits long-range elasticity with an interesting recoverable yield evidenced by an order of magnitude decrease in elastic modulus (apparent failure) that begins at 3–5% strain. This material differs significantly from other common structural proteins such as collagen and elastin in mechanical response to strain. It is suggested that the answers to numerous questions in regard to the development of hierarchical structures, as well as features of the chemical composition and material properties of egg capsules, can be found in the near future if materials scientists reach an understanding of the biological function of these capsules. Many marine invertebrates whose eggs undergo partial or complete development in the benthos deposit them in enclosing structures, which range from multilaminated capsules to fragile gelatinous masses and belts (Fretter and Graham 1994). The encapsulation allows benthic development of the embryos and is particularly common in polychaetes and gastropod molluscs (Pechenick 1979). As reviewed by Ojeda and Chaparro (2004), capsules protect embryos against desiccation and predation, osmotic stress, microorganisms, and UV irradiation and can be very large in size. For example, Busycon eggs are released in meter-long helical strands known as mermaid necklaces that link up to 160 capsules (Fig. 17.1). These are buffeted about for months by breakers at velocities of up to 10 m/s along the seashores of the east coast of North America (Miserez et al. 2009). The egg capsules are acellular and their morphology and structure are related to their functional roles. Knowledge of the composition of the capsule walls is important in
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Fig. 17.1 Whelks produce unique egg capsules (a, c). Some of these capsules possess interesting mechanical properties (b) (image courtesy Ali Miserez)
interpreting the origin, function, and evolutionary relationships among the different structural patterns observed, as well as for bimimetics and materials science. As assumed in the literature (Gathercole 1969; Rapoport 2003), egg capsules are typically formed in the following fashion. The embryos are encapsulated in a soft pliable sack (precapsule) of proteinaceous biomaterial by the nidamental (capsule) gland mesodermal secretory cells before leaving the genital tract. Encapsulated embryos are then passed to a pedal pore along a temporary groove in the anterior portion of the foot. In the pedal pore, the precapsule is thought to undergo enzymatically catalyzed post-translational modifications as well as physical shaping processes that create the final morphology and chemical properties of the hard rubbery capsule that is observed in situ. The fully formed capsules are then mounted individually to the substratum or joined in a growing strand to other capsules that are then attached as a unit to the substratum. Early descriptive work on these egg capsules has demonstrated a high degree of species-specific morphological variation and this has been attributed to morphological variation in the pedal gland itself. That is, differently shaped pedal glands ultimately produce unique egg capsule morphologies, while gradations in snail size within a species produce egg capsules that are scaled versions of similar morphology (Rapoport and Shadwick 2007). The egg capsule is thus the product of an elaborate exocrine secretory activity. However, it was suggested (Tamarin and Carriker, 1967) that in contrast to the usual amorphic type of secretion, the capsule wall represents a special kind of exocrine secretory product which forms a stable organized structure with a species-specific architectural design. For example, in case of the snail UrosaIpinx cinerea, the
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vase-shaped egg capsule consists of four laminae, three of which are anisotropic but with different patterns in the transverse as compared to the longitudinal plane. The third lamina is completely isotropic. Ultrastructurally, the birefringent matrix reveals a precise periodicity of alternating light (290 Å) and dark (240 Å) zones, each subdivided into five subzone striae (Fig. 17.2a). The third lamina and the basal core consists of dispersed filaments (50 Å wide), which in some places aggregate into characteristic periodic fibers (Tamarin and Carriker 1967). In studies by Goldsmith et al. (1978) the visually apparent major component (striated filaments) was given the names “pre-capsulin” and “capsulin” based on observed solubility differences between the respective pretranslationally modified material and the final sclerotized end product. It is highly probable that the cross-linking mode and density may determine the mechanical properties of the egg capsule biomaterial (Rapoport 2003). Many marine caenogastropod snails (i.e., whelks) deposit their embryos in egg capsules constructed from a highly resilient, multilaminate biological material; however, the architecture of these constructs differ from those of U. cinerea. Embryos can develop within the whelks capsules for extended periods of time (on the order of months), thus requiring the egg capsule biomaterial to persist in the hostile marine
Fig. 17.2 Structural features of the whelks ribbon-like fibers. (a) Electron micrograph of stripped Urosalpinx cinerea ribbons negatively stained with uranyl acetate, × 158,000. (Adapted from Flower et al. 1969). (b) SEM image of the similar fibers observed in the egg capsule from B. canaliculatum (image courtesy Ali Miserez)
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environment without loss of integrity. In his Nature publication, Hunt investigated the egg capsules of the whelk Buccinum undatum which consist of a tough fibrous protein, which is extremely resistant to chemical (0.5 M NaCl, 4 N NaOH, 45% phenol at 90◦ C, 4% trichloroacetic acid at 60◦ C) and enzymes (pepsin, trypsin, chymotrypsin, bacterial proteinases) attack (Hunt 1966). This protein has been shown to have a clearly defined alpha configuration (Flower et al. 1969; Rudall 1968) and, in certain of its properties, resembles the connective tissue proteins of both vertebrates and other invertebrates (Price and Hunt 1973). Chemically, studies have shown the egg capsules to be comprised of predominantly protein with minor amounts of carbohydrate and lipid (e.g., Hunt 1966). Protein hydrolysis of egg capsules has shown high concentrations (>100 per 1000 residues) of the following amino acids: aspartic acid, glutamic acid, leucine, and lysine (see for review Rapoport 2003). Like elastin and some other structural proteins, the capsule protein from the egg cases of B. undatum is both yellow in appearance and exhibits a striking blue-white fluorescence in UV light, both in the native state and when solubilized. The possibilities of the role of the fluorophore–chromophore, which contains aldehydic functional groups, in the cross-linking of the capsule protein were discussed by Price and Hunt (1974). There are no reports on the presence of di- or trityrosines in the egg capsules of the whelks. Recently, Shadwick and co-workers (Rapport and Shadwick 2000, 2001, 2002, 2007) characterized basic biochemical and mechanical properties of egg capsules from several species of whelk snails, including the channeled whelk snail, Busycon canaliculatum, native to the East Coast, and the Kellet’s whelk snail, Kelletia kelletii, native to the West Coast of the USA. Tests focused on quantifying the material’s response to being stretched. Of particular interest was measuring the amount of energy dissipated as the material returned to its original shape. Other tests involved the repeated stretching and relaxing of the material. Shadwick and Rapoport, found that capsule material was virtually identical in all seven species of snails examined, meaning the material has been highly conserved during evolution and increases snail survivorship. According to Rapoport and Shadwick (2002, 2007) capsular protein polymer exhibits long-range elasticity with an interesting recoverable yield evidenced by an order of magnitude decrease in elastic modulus (apparent failure) that begins at 3–5% strain. This material differs significantly from other common structural proteins such as collagen and elastin in mechanical response to strain. Qualitative similarities in stress/strain behavior to keratin, another common structural protein, are more than coincidental when composition and detailed mechanical quantification are considered. This suggests the possibility of alpha-helical structure and matrix organization that might be similar in these two proteins. Indeed, the egg capsule protein may be closely related to vertebrate keratins such as intermediate filaments. Scanning electron microscopy (Rapoport and Shadwick 2007) revealed that the capsules of B. canaliculatum and K. kelletii possess fibrous hierarchical arrangements at all stages during the development of mechanical integrity. This suggests
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Fig. 17.3 Model of WECB mechanics through maturation. Blue arrows denote sequential movement from generalized structure to modified structure as described (a) generalized structure; (b) pre-ventral pedal gland—organ located in the foot where a final stabilization process renders WECB insoluble; (c) during ventral pedal gland, and (d) post-ventral pedal gland. For detailed description see text (adapted from Rapoport and Shadwick 2007)
that an as yet uncharacterized sclerotization mechanism that occurs in the ventral pedal gland is the primary action that binds these fibrous components together. Decomposing the mechanical behavior of whelk egg capsule biopolymer (WECB) through various physical and chemical treatments led these authors to develop a model (Fig. 17.3 for the structure and mechanical properties of this material that supports its designation as a keratin analogue. Below, I present a brief description of this model. (a) In general WECB consists of sheets (foils) of 1·μm diameter macrofibrils consisting of hierarchical assemblages of intermediate filament (IF)-type structures arranged in parallel (cylindrical structures). There is presumably a matrix associated with the IFs, but it is not well characterized at this time. Successive layers are arranged in varying orientations throughout the thickness of the material and are presumably responsible for bulk orthotropic mechanical behavior in the plane formed by foils. As the inset illustrates, each macrofibril comprises the staggered head to tail arrangement of coiled-coil molecules at its smallest level of organization (Flower et al. 1969; Gathercole 1969). Alignments of coiledcoils favored by charge-based self-assembly are responsible for repeat striation patterns. For simplicity, IF structures, the next hierarchical level of organization, are not shown in the model. (b) Tensile loading results in shearing and sliding of successive layers, leading to material failure. Immediately following formation in the nidamental gland
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(NG), WECB is white in color and still soluble, indicating lack of sclerotization. Bulk mechanical measurements at this stage are hampered by a lack of cohesiveness in the material, but noncovalent interactions are probably responsible for maintaining its structure and rudimentary cohesiveness. Authors have conjectured that the application of tensile force allows the foils to slide apart fairly easily. Since the individual foils appear to be discontinuous and interdigitated with other layers (foils appeared to be laid down in a fashion analogous to disordered strokes of a broad painting brush; thus, the foils do not necessarily span the entire length of the egg capsule), the loose material associations resulting from self-assembly are not sufficient to hold the material together and are difficult to measure at the bulk material scale. (c) Shearing of successive sheets with restoring force provided by sporadic linkages, primarily among macrofibrils (idealized cross-links, illustrated as black lines in inset). During treatment in the ventral pedal gland (VPG), cross-links begin to stabilize WECB by linking successive foils as the muscular action of the VPG brings them closer together. These cross-links are hierarchically ubiquitous, stabilizing IFs, macrofibrils and matrix. At early stages of cross-linking, the density of the cross-linking is minor (see inset as well) and the material behaves much like a pliant rubber with a restoring force provided by bulk deformations of matrix. Mechanics are dominated by features on the bulk material scale (i.e., changes in foil position and perhaps some warping of foils). (d) Tightly cross-linked material transfers stresses down to level of coiled-coils. Following processing in the VPG, the cross-link density is now sufficient to transfer mechanical stress down to the smallest hierarchical level of the material, thus adding a level of complexity to the witnessed mechanical response. Here, we see the development of the Hookean region that results from strain directed to a network of stiff coils. Unraveling of coiled-coils into random coiled configurations (see inset) begins at the transition between the Hookean and yield regions. In the yield region, extensive unraveling of coiled-coils occurs. Under physiological conditions this is not a stable conformation, so recovery of the initial state occurs when the strain is removed. The restoring force is provided by a combination of matrix contributions and entropic mechanisms, but not necessarily due to the IFs themselves (Kreplak et al. 2005). Recently, interesting results were reported from the laboratory of Herbert Waite (Miserez et al. 2009). The egg capsule wall of the channeled whelk B. canaliculatum was investigated as an effective shock absorber with high reversible extensibility and a stiffness that changes significantly during extension. The authors showed that poststretch recovery in egg capsules is not driven by entropic forces as it is in rubber. Indeed, at fixed strain, force decreases linearly with increasing temperature, whereas in rubber elasticity the force increases. Instead, capsule wall recovery is associated with the internal energy arising from the facile and reversible structural alpha-helix beta-sheet transition of egg capsule proteins during extension. This behavior is extraordinary in the magnitude of energy dissipated and speed of recovery and is
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reminiscent of strain-induced crystallization in some polymeric fibers and superelastic deformations associated with diffusionless phase transitions in shape-memory alloys. I suggest that the answers to numerous questions in regard to the development of hierarchical structures, as well as features of the material properties of egg capsules, can be found in the near future if materials scientists reach an understanding of the biological function of these capsules. Two phenomena may be in my opinion of particular significance in this case. The first one is determined by the diffusion of gases within egg capsules, especially with regards to oxygen. Thus, designed to protect embryonic stages from predation, egg capsules also can provide severe physiological challenges to their inhabitants, as the egg wall represents a barrier to diffusion of gases (Gutowska and Melzner 2009). Previous work has demonstrated that oxygen diffusion coefficients of marine animal egg capsules are typically 10–20% that of pure water (e.g., Brante 2006). In molluscs, oxygen consumption rates rise dramatically during development (e.g., Brante 2006; Cronin and Seymour 2000). Thus, in order to enable rising oxygen fluxes by means of diffusion, many molluscan eggs swell during development, leading to enhanced surface areas, reduced egg wall thicknesses (e.g., Cronin and Seymour 2000), and consequently increased oxygen conductance. In addition, embryos inhabiting fluid filled capsules often produce convective currents that prevent the formation of pO2 gradients within the egg fluid (Cronin and Seymour 2000). Finally, in species without active brooding behavior, egg masses or capsules may be deposited in sites where water exchange and oxygen concentration is high (Gutowska and Melzner 2009). The second phenomenon is determined by the hatching mechanism and osmosis. From biological point of view, the capsule must be constructed so that it is possible for the encapsulated embryos not only to survive and grow, but ultimately to escape. An understanding of the fulfillment of these different requirements can be furthered by examining the physicochemical properties and composition not only of capsules but also of their contents, including gas exchange during development. It was proposed that, for example, the hatching process in Ocenebra erinacea is initiated by embryonic secretion of proteolytic substances into the gel matrix (Hawkins and Hutchinson 1988). Osmotic concentration is a colligative property; therefore the fragmentation of matrix proteins increases the number of particles in the gel matrix, increasing the matrix osmotic concentration. Furthermore, the close correspondence between sections of wall from unhatched capsules exposed to proteolytic enzymes and hatched capsules indicates that proteolysis extends into the wall structure, thereby increasing its permeability and facilitating the osmotically induced uptake of water from the medium. This proteolysis would also be presumed to weaken the “cementum” described by Hancock (1956) that binds the mucus plug to the rest of the capsule. The rise in internal pressure caused by the influx of water will tend to force out the loosened plug and so liberate the embryos. Where the rise in internal pressure proves to be insufficient to force out the plug, the hatching mechanism will fail as no further increase in the number of particles can be produced to offset the dilution caused by the influx of water. The capsule
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weight will therefore return to its prehatching value as an osmotic equilibrium is reestablished. This is the same situation that is seen when developing capsules adjust to a new osmotic environment. To summarize, the reported results (Hawkins and Hutchinson 1988) support the view that the hatching mechanism of O. erinacea is a combination of chemical dissolution of susceptible elements in the capsule, leading to an osmotically induced uptake of water that finally disrupts the continuity of the structure by forcing out the capsule plug. It seems that isolation followed by identification of the molluscan capsular proteolytic enzymes could help to obtain information about chemistry and cross-linking of the egg capsule biopolymers as well as on the rate of hydroxylation of corresponding amino acids involved in capsule formation.
17.1 Conclusion Whereas α–β structural transitions have previously been described in α-keratin fibers, molluscs egg capsules (e.g., Busycon species) show that it can be exploited to create bioelastomers with higher extensibility and much less time-dependent recovery than previously recognized. This paradigm could prove useful in designing new bioencapsulants for delicate tissue implants (Miserez et al. 2009). Engineering versatile encapsulants for pharmaceuticals and the transplantation of cells and tissues is a very active area of medical research. Surprisingly, engineers are largely unaware of how exquisitely well-tuned naturally occurring encapsulation strategies are. Definitively, complete characterization and recombinant expression of Busycon egg capsule proteins should ultimately allow the engineering of capsule-like materials that combine high modulus, reversible extensibility (>100%), and impactabsorbing properties for the insulation of damage-prone tissues.
References Brante A (2006) An alternative mechanism to reduce intracapsular hypoxia in ovicapsules of Fusitriton oregonensis (Gastropoda). Mar Biol (Berl) 149:269–274 Cronin ER, Seymour RS (2000) Respiration of the eggs of the giant cuttlefish Sepia apama. Mar Biol (Berl) 136:863–870 Flower NE, Geddes AJ, Rudall KM (1969) Ultrastructure of the fibrous protein from the egg capsules of the whelk Buccinum undatum. J Ultrastruct Res 26(3–4):262–273 Fretter V, Graham A (1994) British prosobranch molluscs. Their functional anatomy and ecology. Ray Society, London Gathercole L (1969) Studies on the protein of the egg capsule of whelks. Ph.D. thesis, University of Leeds Goldsmith LA, Hanigan HM, Thorpe JM et al (1978) Nidamental gland precursor of the egg capsule protein of the gastropod mollusc Busycon carica. Comp Biochem Physiol 59B:133–138 Gutowska MA, Melzner F (2009) Abiotic conditions in cephalopod (Sepia officinalis) eggs: embryonic development at low pH and high pCO2 . Mar Biol 156:515–519 Hancock DA (1956) The structure of the capsule and the hatching process in Urosulpinx cinerea (Say). Proc Zool Soc London 127:565–571
References
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Hawkins LE, Hutchinson S (1988) Egg capsule structure and hatching mechanism of Ocenebra erinacea (L.) (Prosobranchia: Muricidae). J Exp Mar Biol Ecol 119:269–283 Hunt S (1966) Carbohydrate and amino-acid composition of the egg capsule of the whelk Buccinum undatum L. Nature 210:436–437 Kreplak L, Bar H, Leterrier JF et al (2005). Exploring the mechanical behavior of single intermediate filaments. J Mol Biol 354:569–577 Miserez A, Wasko SS, Carpenter CF et al (2009) Non-entropic and reversible long-range deformation of an encapsulating bioelastomer. Nat Mater 8:910–916 Ojeda JA, Chaparro OR (2004) Morphological, gravimetric, and biochemical changes in Crepidula fecunda (Gastropoda: Calyptraeidae) egg capsule walls during embryonic development. Mar Biol 144:263–269 Pechenick JA (1979) Role of encapsulation in invertebrate life histories. Am Nat 114:859–870 Price NR, Hunt S (1973) Studies of the cross linking regions of whelk egg capsule proteins. Biochem Soc Trans 1:158–159 Price NR, Hunt S (1974) Fluorescent chromophore components from the egg capsules of the gastropod mollusc Buccinum undatum (L.), and their relation to fluorescent compounds in other structural proteins. Comp Biochem Physiol 47B:601–616 Rapoport HS (2003) Biomechanics, biochemistry, and molecular biology of a molluscan scleroprotein elastomer: whelk egg capsules. Ph.D. thesis, University of California, San Diego, USA Rapoport HS, Shadwick RE (2000) Investigations into the selfhealing behavior of whelk egg capsule biomaterial, genus Busycon. Comp Biochem Physiol 126B(Suppl 1):S81 Rapoport HS, Shadwick RE (2001) A keratin-like gastropod biomaterial used to clarify the mechanical models of keratin. Am Zool 41:1563 Rapoport HS, Shadwick RE (2002) Mechanical characterization of an unusual elastic biomaterial from the egg capsules of marine snails (Busycon spp.). Biomacromolecules 3:42–50 Rapoport HS, Shadwick RE (2007) Reversibly labile, sclerotization-induced elastic properties in a keratin analog from marine snails: Whelk egg capsule biopolymer (WECB). J Exp Biol 210: 12–26 Rudall KM (1968) Intracellular fibrous proteins and the keratins. In: Florkin M, Stotz EH (eds) Comprehensive biochemistry, vol 26B. Elsevier, New York Tamarin A, Carriker M (1967) The egg capsule of the Muricid gastropod Urosalpinx cinerea: an integrated study of the wall by ordinary light, polarized light, and electron microscopy. J Ultrastruct Res 21:26–40
Chapter 18
Byssus: From Inspiration to Development of Novel Biomaterials
Abstract Attachment of marine molluscs is mediated by a fibrous shock-absorbing structure known as the byssus. The byssus is an extra-corporeal bundle (thread) of tiny tendons attached distally to a foreign surface and proximally by insertion of the root into the byssal retractor muscles. It is deposited outside the boundaries of living tissue and contains no cells for maintenance or repair. The byssus is, nowadays, regarded as an inspirational material for the development of advanced biomimetic fabrics, as well as for adhesives that cure effectively underwater, but their exploitation began as far back as ancient cultures where byssal threads were woven into fine clothing. Chemistry of byssus and related proteins as well as their materials properties are described and discussed here. Molluscs developed during their evolution two different attachment strategies— cementation and adhesion using byssal threads. The last one is characteristic for marine mussels, which are superbly successful inhabitants of wind- and wave-swept rocky shores. This success is due in a large part to their strong and opportunistic attachment to hard surfaces. Attachment is mediated by a fibrous shock-absorbing structure known as the byssus. The byssus is an extra-corporeal bundle (thread) of tiny tendons attached distally to a foreign surface and proximally by insertion of the root into the byssal retractor muscles (Fig. 18.1). It is deposited outside the boundaries of living tissue and contains no cells for maintenance or repair. The function of the byssus lies in providing mussels with secure attachment to rocks and pilings. It mediates contact between very soft living tissue and a very stiff inert material such as rock or ship hull. Every individual mussel byssus thread represents a unit of attachment, with the distal end bonded to rock and proximal end inserted into living mussel tissue. Each thread measures between 2 and 6 cm in length and 50 μm in diameter, dependent on species (Fig. 18.2). The investigation of byssus as a marine biomaterial is an interdisciplinary endeavor encompassing the fields of biochemistry, polymer chemistry, materials science, biomimetics, and biomedicine. Here, the main scientific directions regarding the properties of the byssus, which have been developed since the early 1950s of twentieth century and continues in current research, are as follows:
H. Ehrlich, Biological Materials of Marine Origin, Biologically-Inspired Systems 1, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9130-7_18,
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Fig. 18.1 Mollusc attached to the surface of dead wood using byssus threads
Fig. 18.2 Naturally occurring byssal threads from marine abyssal molluscs (samples courtesy Anatoly Drosdov)
• • • • • •
material performance of mussel byssus; biochemistry and molecular biology of byssus precursor proteins; cross-linking reactions and adhesion mechanisms of mussel adhesive protein; surface chemical properties of mussel adhesives; antifouling strategies based on the surface chemical properties of the substrata; tissue adhesives for modern surgery and dentistry.
It’s not surprising that the literature sources, including patents, related to byssus are amazing. Therefore, I recommend readers some excellent review articles and books published by J. H. Waite (1985, 1995, 2008), J. H. Waite et al. (2002, 2004), S. Haemers (2003) and Phillip B. Messersmith (see references to this chapter) for obtaining more detailed information regarding to the state of the art.
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Byssus—An Ancient Marine Biological Material
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18.1 Byssus—An Ancient Marine Biological Material The byssus is, nowadays, regarded as an inspirational material for the development of advanced biomimetic fabrics, as well as for adhesives that cure effectively underwater, but their exploitation began as far back as ancient cultures where byssal threads were woven into fine clothing. Mussel attachment was the subject of one of the earliest recorded observations of bioadhesion. Aristotle (transl. 1910) noted that the holdfast in the fan mussel (Pinna) consisted of a robust bundle of fibers with sticky tips. The term byssus (Greek “bysso” for flax linen) was accidentally coined by him for the holdfast (van der Feen 1949) and has since gained universal acceptance. However, this term was known prior to Aristoteles. I give below a very short overview on history of byssus. More detailed information can be found in numerous papers by F. Maeder (1999, 2002) and F. Maeder and M. Halbeisen (2001, 2002) listed in the references. The byssus is called “bus” in the aramaic, from which the greek name “bussos” originated. Like literature or a work of art, a myth is subject to interpretation, whose meaning is malleable through time as context changes, cultures evolve and writers become cleverer: “byssus” translated by the latin word “sericum” (silk), means the fine sea silk. A cloth of exceedingly fine texture made of byssal fibers obtained from the Pinna nobilis or pen shell (Fig. 18.3) was also known to the Egyptians as sea silk. Only royalty were allowed to wear the cloth made from byssus. In a religious Egyptian text there is a passage that describes a deity who appears in the likeness of a priest dressed in byssus “a gauzelike cloth of a golden hue, which is silky, like the fine threads of many molluscs.” Herodotus, the Greek historian, who personally visited Egypt and the pyramids in 500 BC speaks of a tunic found in a sarcophagus at Thebes and seen by him, tells us that it is “made of a loose fabric of exceedingly fine thread, as thin as that used in the manufacture of lace. It is finer than a hair, twisted and made of two strands, implying either an unheard of skill in hand-spinning, or else machinery of great perfection.” The Egyptologist Sir John Gardner Wilkinson
Fig. 18.3 Mollusc P. nobilis (a) a well-known producer of sea silk (byssus) (b)
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found byssus in the tombs of the pharaohs and counted the threads in order to ascertain the fineness of the weave. He found along a 25.4 mm length, 152 threads in the warp and 71 threads in the woof. The finest cotton produced today with the best technical methods contains in comparison only about 88 threads. It’s thought that the “golden fleece,” sought by the legendary Greek hero Jason, was woven from the pen shell’s threads. In Greek mythology’s most famous legend
Fig. 18.4 Byssus manufacture. (a, b, c) after the harvest the raw byssus fibers were washed several times, dried, and combed; (d) byssus filaments—finer than a hair—are twisted together to form a single thread; (e, f, g) a spindle—a tapered stick of wood was the earliest spinning tool. In order to handle the threads it is first necessary to spin the yarn to insert sufficient twist to bind the fibers together; (h) the typical gold-brown shining color, which made “sea silk” so famous, receives it by inserting it into lemon juice; (i) raw byssus threads and byssus filaments; (j) sea byssus spindles; (k) detail of byssus tie (adapted from www.designboom.com)
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Why Molluscs Produce Different Kinds of Byssus
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of gallantry and heroism Jason sets sail in the argo in search of the “golden fleece” to avenge his father’s death and to claim his legitimate birthright to the throne as king. The fine, diaphanous fabrics were commonly used in making the apparel of the queen and the princesses and the wives and daughters of rich men and high officials. Also, as mentioned by Homer, hair-nets were frequently made of the goden elean byssus. Females with this kind of head-dress frequently occur in paintings found at Pompeii. 200 A.D. Tertullian has written about the legendary byssus in a short humorous speech on why he has stopped wearing the roman “toga” and started instead to wear the “pallium.” Nor was it enough to plant and sow your tunic, unless it had likewise fallen to your lot to fish for raiment. For the sea withal yields fleeces, inasmuch as the more brilliant shells of a mossy wooliness furnish a hairy stuff.
Until the Middle Ages these fibers were used to weave a strong but supple fabric called “cloth of gold.” Some places in Italy (Golfodi Taranto/Calabria, Sardinia, Sicily), France (Corsica), Greece, Turkey (Smyrna/Today Izmir), manufactured byssus textiles as stockings and gloves. Byssus is a very light and transparent material, this cloth was so fine that a pair of gloves made from it could be folded and packed inside a walnut shell. The eighteenth century saw a revival of byssus manufacture in the south of France and in south Italy. In 1870 the French novelist Jules Verne published his novel “20,000 leagues under the sea,” he writes “. . .I felt so great a heat that I was obliged to take off my coat of byssus!” Note that a pen shell P. nobilis produces ca. 1–2 g of raw byssus threads 1,000 mussels were needed for 200–300 g of fine byssus silk. The art of making cloth of gold has been lost to time and the pen shell is now much less common. There are still a few examples (ca. 30) of the cloth in European museums. As far as we know there remain today only a few women in Sardinia who spin and weave these fine linens. Their extremely ancient tradition dates back to the era of the Phoenicians. Because of the very simple (and unique today) technology of the spinning of the byssus threads, I take a liberty to represent here several images which, to my opinion, will astonish our materials research community (Fig. 18.4).
18.2 Why Molluscs Produce Different Kinds of Byssus Mussels (Mytilus edulis and other Mytilidae) are known as the main source of byssus, are also the most common marine bivalve molluscs, of importance to commercial fisheries and the anti-biofouling sector. The diversity of molluscs species that produce byssus as well as the diversity of byssus-based biopolymers in nature are of great scientific importance because of ontogenesis, evolutionary science, ecology, and biomechanics. Recently, Pearce and LaBarbera (2009a) critically analyzed these topics from a biomechanical point of view as follows.
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Although the byssus first evolved to aid in postlarval dispersal and settlement, a recent catalogue of tropical marine bivalves revealed that about a quarter of the genera surveyed are byssally attached as adults. With more biomechanical data on the threads of both epifaunal and semi-infaunal bivalves from a variety of pteriomorph orders, one would be able to sort out whether life habits are correlated with the mechanical properties of byssal threads. Members of the pteriomorphian superfamily Pinnoidea live with their tapered anterior end buried to varying degrees in sediment. The fan shell Pinna may be buried up to one-third of its length and Atrina is even more completely buried. This is an especially interesting question, as both endobyssate (infaunal or semiinfaunal with byssal attachment) and epibyssate (epifaunal with byssal attachment) groups declined during the Paleozoic and Mesozoic, perhaps due to increased predation pressure. The surviving byssate groups live in a variety of environments and their survival probably involved adjustments of their thread mechanics. There are certainly interesting chemical differences between the threads of different bivalve groups, which may translate into differences in mechanical properties. For example, mytilid threads are collagenous, whereas the threads of pinnids, anomiids, and dreissenids are not (Jackson et al. 1953; Ohkawa et al. 2004; Pujol 1967; Pujol et al. 1970). Pearce and LaBarbera (2009b) measured the mechanical properties of the byssal threads of two species outside the Mytilidae, the pen shell Atrina rigida Lightfoot and the flame “scallop” Ctenoides mitis Lamarck. The mechanical properties of their byssal threads were significantly different from those of mytilids. For instance, the byssal threads of both species were significantly weaker than mytilid threads. A. rigida threads were less extensible than mytilid threads, while C. mitis threads exhibited the highest extensibility ever recorded for the distal region of byssal threads. However, there were also interesting similarities in material properties across taxonomic groups. For instance, the threads of A. rigida and Modiolus modiolus Linnaeus both exhibited a prominent double-yield behavior, high stiffness combined with low extensibility, and similar correlations between stiffness and other thread properties. These similarities suggest that the thread properties of some semi-infaunal species may have evolved convergently. Henrik Birkedal and co-workers has recently reported a non-typical kind of byssus that is mineralized, in contrast to non-mineralized byssus known from representatives of Mytilidae. In contrast to the mussels, the bivalves Anomiidae have but a single byssus thread, which intriguingly is mineralized (with the exception of Enigmonia) (Eltzholtz and Birkedal 2009). The jingle shell, Anomia sp., is the only member of the Anomiidae whose byssus has been studied in any detail. It is highly mineralized ( > 90%) by calcium carbonate, and both of the CaCO3 polymorphs aragonite and calcite are present, although their distribution still has not been investigated. The animal attaches to small stones or shells from other molluscs and lies on the side so that the right shell is turned toward the substrate, while the left shell is presented to the environment (Eltzholtz et al. 2009). The diversity in forms of byssus threads, their nature as well in their quantities, seems to be determined due to specific environmental conditions. As previously
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Chemistry of Byssus and Related Proteins
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observed among mytilids (Meadows and Shand 1989), semi-infaunal species seem to produce a very large number of thin threads, whereas epifaunal species produce a smaller number of thicker threads. One reason for this difference might be that having a larger number of thin threads is more effective in anchoring semi-infaunal animals within a particulate substrate, as the threads can create an extensive network of individual attachments to small particles. The M. modiolus examined in a previous study tended to leave the glass plates to which we tried to confine them and bury themselves in the gravel, from which they were difficult to extricate without digging (Pearce and LaBarbera 2009b). For Mytilus species, on the other hand, which attach to rocks and other hard substrates, a smaller number of thick threads may provide a more reliable tether against wave action or predation (Bell and Gosline 1996, 1997; Carrington 2002; Carrington and Gosline 2004). The stalk-like byssus of some arcoids and pterioids may be an extreme form of this tendency to consolidate material into a smaller number of thicker threads (Oliver and Holmes 2006; Tëmkin 2006). C. mitis appears to represent a somewhat different case, as it only uses byssal threads for temporary attachment and not for predator resistance; thus a set of weak but stretchy threads of intermediate thickness allows it to hang inside crevices, ready to drop the threads and swim away on disturbance. It was established (Selin and Vekhova 2004) that the process of repeated attachment to a substrate in bivalve molluscs Crenomytilus grayanus (Gray’s mussel) and M. modiolus (the northern horse mussel) involved several successive stages, which in vitro required about 1 month at a water temperature of 19◦ C. Comparison with Gray’s mussel revealed that the northern horse mussel had a higher rate of byssal thread production and a greater thread number by the end of the complete formation of the byssus complex. The observed differences are explained by the adaptation of molluscs to habitation in different biotopes. Further research on specific patterns, along with biochemical analysis of both non-mineralized and mineralized byssal threads which exhibit unusual properties, promises to contribute to both evolutionary biology and ecology, as well as materials engineering.
18.3 Chemistry of Byssus and Related Proteins The first published results related to the chemistry of byssus were made, probably, by Italian Professor Lavini in 1835 (Lavini 1835). He found J, Br, Na, Mg as well as residual Si and Fe after alkali-based hydrolysis of byssus (“gnaccara” ital.) obtained from P. nobilis. Initially, byssus has been classified to something between chitin and proteins (Carriere 1879; Müller 1837; Tallberg 1877). Later, Aderhalden (1903) reported about byssus as a silk-like protein. Brown (1952) started with more detailed investigations on structural proteins of Mytilidae. Today, it is known that byssal threads are composed primarily of six proteins (termed mefp (M. edulis foot protein) 1–5) and byssal collagen (PreCol D). They are divided broadly into three areas (Harrington and Waite 2007; Waite et al. 1998, 2005):
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(1) the root that is embedded in the base of the muscular foot; (2) the threads (proximal and distal regions) that are produced along a ventral groove that runs the length of the foot; and (3) the attachment disk, or plaque, which mediates adhesion to (4) the substratum. Thus, a thin protective protein coating, dubbed mefp-1 (M. edulis foot protein-1), protects the threads from abrasion by sand as well as degradation by bacteria and other microorganisms. The presence of mefp-1 to mefp-5 is also located at the end of each thread, in an adhesive plaque, allowing plaque to anchor to wet solid surfaces (Suci and Geesey 2001). Based on X-ray diffraction, it is estimated that the distal portion of the byssal thread consists of at least approximately 50% collagen triple helices and approximately 50% unidentified ß-pleated sheets that could be a separate phase of the byssal collagen (Rudall 1955). Qin and Waite (1995) partially sequenced this material (named (pre)Col-D) and isolated a homotrimer with α-chain mass approximately 60 kDa, although this monomer is now thought to have a mass in the range of 95–97 kDa. PreCol is a block copolymer that contains a central collagen domain, which occupies roughly half of the preCol. Its other domains are the N- and Cterminal His-rich domains (HIS), the flanking domains, and an acidic motif. The collagen domains are highly homologous in the three known variants, preCol-P, -D, and -NG in which the postscripts P, D, and NG denote proximal distal and non-graded, respectively (Coyne 1997). Mefp proteins possess following characteristics as recently reviewed by Deshmukh (2005). M. edulis adhesive protein-1 (also known as “polyphenolic protein” and “mussel adhesive protein”) has a molecular weight of 115 kDa based on mass spectroscopy and of 130 kDa on the basis of gel chromatography. Mefp-1 protein is mainly built from two building blocks 71 deca-peptides containing the residues Ala1 -Lys2 -Pro3 Ser4 -Tyr5 -Pro6 -Pro7 -Thr8 -Tyr9 -Lys10 and 12 hexa-peptides containing Ala1 -Lys2 Pro3 -Thr4 -Tyr5 -Lys6 (Waite et al. 1985). The deca-pepetide part contains three post-translation modifications. First being at the sixth residue in which a Pro converts to dihydroxyproline, second at the seventh position where a Pro gets modified into a hydroxyproline (Taylor et al. 1994). The third modification, which is more important as we will see in the following sections, leading to a conversion of Tyr to 3,4-dihydroxyphenylalanine (DOPA) (Waite and Tanzer 1981). Tyr at the fifth position in the hexa-peptide also gets modified into DOPA. These hydroxylated amino acids constitute a strikingly high percentage of mefp-1 protein. Like the deca and hexa peptide, the N-terminus non-repetitive domain is hydrophobic and rich in Lys residues. Between different populations of mussels, the composition of the decapeptide itself remains roughly the same. The presence and location of the Tyr and/or DOPA and Lys in this sequence are completely invariant. Although hydroxyproline is erroneously assumed to be unique to collagenous proteins, in the polyphenolic protein most or all of the hydroxyproline remains associated with the Gly-deficient collagenase-resistant fragment (Waite 1983).
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Fig. 18.5 In the presence of ferric iron, mefp-1 (a) forms an extremely complicated macromolecular coordination of ferric ion (b)
Mefp-1 is applied to the byssus as a lacquer-like coating; the addition of ferric iron may render it an “ironclad” finish and contribute to the notorious intractability of this material (Taylor et al. 1996) (Fig. 18.5a, b). In the presence of ferric iron, mefp-1 forms an extremely complicated macromolecular coordination of ferric ion. Notwithstanding the current study that mefp-1 behaves very similarly to low molecular weight catecholates like the siderophores, the protein can solubilize ferric iron even when present in polynuclear hydrolytic species inaccessible to simple complex agents.
18.3.1 M. edulis Adhesive Protein-2 (Mefp-2) Mefp-2 is a second major DOPA protein of the blue mussel and it appears to be a structural component exclusively of the plaque, contributing up to 25% of plaque protein (Rzepecki et al. 1992). It is a Cys rich, tandemly repetitive 45 kDa protein. It is a multi-domain protein, with short, acidic, DOPA containing N-and C-terminal regions and a large central domain constrained by quasi-periodic internal disulfide bridges to compact conformation resist proteolytic degradation. The peptide motifs of mefp-2 are quite unlike those of any other known structure. However, in tandemly repetitive proteins, the correct order of short peptide motifs over an entire protein cannot be deduced by standard peptide mapping techniques. The composition of mefp-2, incorporating Cys and DOPA, implies some role involving the stabilization of the plaque matrix by covalent disulfide and quinine-derived cross-links. Mefp-2 can form oligomeric aggregates that might be stabilized by rearrangement of disulfide bonds to form inter-molecular cross-links. If mefp-2 indeed constitutes 25% of plaque protein, then it would incorporate 90% of the plaque Cys residues and thus would be virtually its own sole potential disulfide cross-link partner. The DOPA residues might then serve to cross-link the resulting disulfide-linked mefp-2 homopolymer to other protein components of the plaque. Consideration of the amino acids composition of mefp-1 and 2 and of the terminal adhesive plaques of byssal threads suggests that mefp-2 makes up about 25% of plaque protein, whereas mefp-1 content is about 5%. Mefp-2 has minimal sequence
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homology with known structural proteins and may have a properly folded structure in the plaque matrix. M. edulis adhesive protein-3 (mefp-3) is a small (6 kDa), non-repetitive protein. Mefp-3 resembles to other byssal precursor proteins in basicity and DOPA content. Specific variants of the mefp-3 family may be preferentially deposited onto glass, stainless steel, and polyethylene. Mass spectrometry with time-of-flight suggests that mefp-3 is the only detectable protein family near the plaque–substrate interface. It is also Arg rich and many of these residues are modified to 4-hydroxy-L-arginine (Papov et al. 1995). Like DOPA, Arg and presumably its hydroxylated derivative is also an asset for the molecular interactions indispensable for adhesion (Vreeland et al. 1998). In contrast to other mussel adhesive proteins such as mefp-1 and -2, which have large number of highly conserved, tandemly repeated peptide motifs, the function of mefp-3 in byssal adhesion is unknown (Vreeland et al. 1998)
18.3.2 M. edulis Adhesive Protein-4 (Mefp-4) The mefp-4 protein belongs to this family of proteins and has a mass of 70–80 kDa. All have a common N terminus and contain elevated levels of G, R, and H. It has been assumed that mefp-4 is located in the bulk adhesive of the plaque. Occurrence of the DOPA in this protein is 5 mol% (Vreeland et al. 1998). M. edulis adhesive protein-5 (mefp-5) is an adhesive protein derived from the foot of the common mussel, M. edulis, and is deposited into the byssal attachment pads (Waite and Qin 2001). Purification and primary structure of mefp-5 were determined by peptide mapping and cDNA sequencing. The protein is 74 residues long and has a mass of about 9500 Da. Mefp-5 composition shows a strong bias for nonaromatic amino acids, Lys and Gly, represent 65 mol% of the composition. More than one-third of all the residues in the protein are post-translationally modified by hydroxylation or phosphorylation. The conversion of Tyr to DOPA and serine to o-phosphoserine accounts for the hydroxylation and phosphorylation (Waite and Qin 2001). At over one in every four amino acid, mefp-5 contains the highest level of DOPA discovered thus far. Seven or eight Ser residues in mefp-5 are phosphorylated. The adhesive proteins mefp-1 to -5 are difficult to produce biochemically and it is very difficult to extract them from the biological source (i.e., marine mussels) because of poor yields. This situation makes synthetic approaches (Taylor and Weir 2000) unique and demanding for accessing more quantities of these proteins. As it has been shown, certain residues like DOPA play a very important role in the adhesive properties of MAPs thus making it a plausible constituent of the synthetically prepared adhesive polymer. PreCol-D, also described as the sixth mussel adhesive protein by Waite (2002), has 175 Gly-XY repeats in its collagen domain and six polyalanine clusters in the flanking silk-like domains (Qin et al. 1997). Besides protein, significant amounts of fatty acids (8% w/w) have been detected in byssal threads of M. edulis (Cook 1970) and Mytilus galloprovincialis
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(Holten-Andersen and Waite 2008). Other known constituents are metal ions such as Fe, Al, Si, and Ca. The metal composition of byssus appears to be highly variable and often reflects the chemistry of the water column and/or sediment. The functional significance of iron binding is still in question, since many byssal threads of mussels collected from the field are devoid of iron, but seems mechanically robust in tension. Similar to marine scleroproteins like gorgonin, antipathin, or spongin, byssus is known as a chemically and enzymatically very resistant biopolymer. However, some microorganisms can destroy and digest also this polyphenol-based biomaterial. Studies have investigated (Kohlmeyer 1972) the proteolytic activity of some marine fungi against keratin-based materials. Some ascomycetes were found to be localized deep within byssus threads of M. galloprovincialis by Vitellaro-Zuecarello (1973). As for the relationship between the byssal matrix and the fungus, it was assumed that the former provides a preferential substratum for fungal development, the hyphae living as saprophytes in the byssus, and feeding on some of its constituents. The histochemical reaction for fungi revealed the presence of fungal hyphae in the intra-organismal part of the byssus apparatus in about 75% of the mussels examined (Franchini et al. 2005). To date, the fungal pathogen has not been identified. Also, some marine bacteria are able to destroy byssus. Thus, novel marine mussel-thread-degrading bacterium Pseudoalteromonas peptidolytica sp. nov isolated from the Sea of Japan has been reported (Venkateswaran and Dohmoto 2000). This microorganism secretes specific proteases that degrade the protein compound of the M. edulis foot.
18.4 Biomechanics and Materials Properties of Byssus Organisms build a variety of load-bearing materials with unique mechanical properties chiefly by adjusting two parameters, composition and architecture. In principle, composition and architecture in tissues can be adjusted either abruptly or gradually, but it is becoming increasingly evident that nature prefers gradients. Manufactured materials with graded mechanical properties show superior resistance to contact deformation and damage (Sun and Waite 2005). The function of the mussel byssus thread is to hold the animal firmly to the substratum against the activity of waves and predators. It is important, therefore, not only that it is strong, but also that it can absorb the energy imparted by breaking waves and strong surges. Dynamic as well as static considerations are therefore important. According to Smeathers and Vincent (1979), each byssus thread is capable of withstanding a tensile force of about 0.25 N, nearly double this if dry, so a mussel secured by many threads will have a strong attachment. The effective anchorage strength of a mussel with 50 threads (a conservative estimate), assuming a radial arrangement of threads, a contact angle of 20◦ with the substratum and an ultimate strain of 0.4 is 9–10 N normal to the substratum and about 4 N parallel to the substratum. Calculations on drag forces for a shell with a length of 60 mm indicate that
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to produce a drag of 4 N water flow over the shell must exceed 5.7 m/s. Velocities of this magnitude could occur in winter storms, but normal currents are less than 3–4 m/s above the mussel beds and flow directly over the shells will be considerably less due to the velocity gradient. Damaged byssus material can be replaced readily by secretion of new threads at the base of the stem. However, byssus is able to recover from the stress softening effects of previous strains if it is allowed to relax over a period of time of several hours as would occur in intertidal zones. Thus, byssus can remain an effective energy absorber over several exposures to stressing (Smeathers and Vincent 1979). The composition–structure–function relationship was excellently described for the byssus (Sun 2002). Briefly, the byssal thread of a mussel can be accepted as an extraorganismic connective tissue that exhibits a striking end-to-end gradient in mechanical properties and thus provides a unique opportunity for studying how gradients are made. Thus, mefp-1 is a conspicuous component of the protective outer cuticle of byssal threads given its high 3,4 dihydroxyphenylalanine (DOPA) content at 10–15 mol%. Amino acid analysis of mefp-1 extracted from successive foot sections of M. galloprovincialis reveals a post-translationally mediated gradient with highest DOPA levels present in mefp-1 from the accessory gland near the tip of the foot, which decrease gradually toward the base. The DOPA content of successive segments of byssal threads decreases from the distal to the proximal end and thus reflects the trend of mefp-1 in the foot (Sun and Waite 2005). Inductively coupled plasma analysis indicates that certain metal ions, including iron, follow the trend in DOPA along the thread. Energy-dispersive X-ray spectrometry showed that iron, when present, was concentrated in the cuticle of the threads but sparse in the core. The axial iron gradient appears most closely correlated with the DOPA gradient. The direct incubation of mussels and byssal threads in Fe3+ supplemented seawater showed that byssal threads are unable to sequester iron from the seawater. Instead, particulate/soluble iron is actively taken up by mussels during filter feeding and incorporated into byssal threads during their secretion. These results suggest that mussels may exploit the interplay between DOPA and metals to tailor the different parts of threads for specific mechanical properties (Harrington and Waite 2007). There are also reports in the literature of mussel tenacity, of byssal tensile strength, and of adhesive tensile strength. Previously, workers have been restricted to simple descriptions of byssal mechanical behavior, being linear at low strain followed by a yield, post-yield plateau, and stiffness before breaking (Vaccaro and Waite 2001). Using Mytilus californianus, Mytilus trossulus, and M. galloprovincialis, Bell and Gosline (1996) showed that the tough distal region of a byssal thread is of high strength and somewhat “over designed.” This excessive strength, it was proposed, may be a by-product of its yielding character. This conclusion was drawn since threads invariably give way at the weaker proximal endor in the plaque before the distal portion approaches its ultimate strain. On average, the recorded yield force ranged from 52 to 81% of the force required for structural failure and this, theoretically, outperforms an equivalent byssus of steel threads. Byssi achieve this by exploiting more than twice as much of their potential maximum strength as
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elasticity, as compared to steel. The resulting compliance of byssi allows transfer of load between threads; something the stiffer steel threads could not achieve. The yield of the distal byssus is manifested in a sharp loss of stiffness at approximately 35 MN/m2 , where 0.5 cm of extension can be achieved with essentially no increase in force. Price (1981) showed an ultimate stress of 83 G (15 MN/m2 ) for M. edulis distal byssus and Smeathers and Vincent (1979) an ultimate strain of 0.66–0.01 before breaking. Waite et al. (1998) provided further data for the ultimate tensile stress and strain of the distal portion of the byssus, suggesting a behavior that is consistent with a multi-component material, in which one component deforms semipermanently but later recovers when the load is transferred to another, more elastic, component (although no direct evidence for this was presented). That is for this material, “yield,” contrary to the accepted definition of the term is not necessarily permanent. This phenomenon could be the result of a reversible cross-linking mechanism involving metal complexion binding or covalent bonding. Peptidyl-dopa provides excellent metal-binding sites and the histidine-rich sequences in the terminal regions of preCols are strongly reminiscent of other metal-binding peptides, as described above. Gosline et al. (2002) provide a comprehensive study on this topic, clarifying several important points. In a reappraisal of previous data, they drew the conclusion that more work is clearly needed before a useful understanding of these structures can be claimed. Further, they make the point that no studies have yet investigated the changes in yield properties that occur when the byssi dry out; an obvious requirement for an intertidal organism (all previous studies have used hydrated byssi). This, it is proposed, may cause them to “achieve greater strength with little compromise in extensibility.” Indeed, Qin and Waite (1995) suggested that the mussel is in a paradoxical position of needing to produce a thread that is both strong under tension and able to effectively absorb shock. Vaccaro and Waite (2001) described specific yield points on their stress/strain curves as “elusive,” implying that identification of any points of yield except the final, catastrophic one was difficult, a restriction probably imposed by the apparatus used (an MTS Bionix tensile tester). Aldred et al. (2007), however, used in their study dynamic mechanical analysis (DMA), a more sensitive technique, the resolution of which allows a somewhat more detailed analysis of the mechanical behavior of byssi. DMA involves tensile analysis of materials over a temperature range and, when used in combination with its tensile mode, allows conclusions to be drawn regarding the strength, modulus, glass transition point, and polymerization/cross-linking of materials. Fresh byssi, and byssi aged 2 weeks prior to testing, were used to further study the effects of age on the mechanical properties of this material. It was found that while older threads demonstrated increased stiffness, age did not necessarily affect their ultimate tensile strength. Dehydration had a more pronounced effect on thread stiffness and also increased the ultimate strength of the material. In their dry state, byssal threads displayed multiple yield points under tension and these, it is suggested, could equate to different phases within the bulk of the material. Dynamic analysis revealed glass transition (Tg ) and ecologically relevant operational temperatures for byssi, where their modulus (E0 ) remained constant. These
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discoveries are related to the ecological function of byssal threads and to the emerging field of biomimetics (Aldred et al. 2007). Recently, interesting results on mechanical properties of byssal cuticles has been reported by Holten-Andersen and Waite (2008). The outer surface of byssal threads is coated by a thin (5 μm) but distinctive cuticle in Mytilus (Vitellaro-Zuecarello 1981)—the distinctive feature being the presence of densely packed mottled granules in M. edulis and M. galloprovincialis byssal cuticles. Viewed by scanning electron microscopy (SEM), the cuticle on M. galloprovincialis mussel threads resembles sandpaper. In thin sections for transmission electron microscopy (TEM), the mottled granules, which are embedded in a continuous homogeneous matrix, reveal a distinct biphasic structure, a feature that is greatly enhanced when viewed by atomic force microscopy (AFM). The granules are about 0.8 μm in diameter and constitute about 50% of the cuticle volume. The phase-separated morphology within each granule has a domain size of 20–40 nm. The cell and molecular processes mediating formation of this unusual microstructure remain unknown. The hardness (H) and stiffness (Ei ) of the hydrated cuticle coating M. galloprovincialis threads have been measured by nanoindentation (Holten-Andersen et al. 2005, 2007). These parameters are of particular interest, because H3/2 /E is empirically proportional to wear resistance in ceramics. Consistent with its function to protect the collagens in the core, cuticle H and Ei are comparable with those of engineering epoxies. The H and Ei of the cuticle are 4–5 times higher than those of the core collagens. Despite its high stiffness, the cuticle remains extensible, with a tensile failure strain as high as 70% (Holten-Andersen et al. 2007). Large-scale catastrophic failures are prevented from propagating through the cuticle at strains less than 70% by controlled micro-tearing localized to the interface between the granules and the matrix and within the matrix itself. The granular composite coating thereby “absorbs” strain-induced damage by redistributing it to a large volume, thereby enabling coat strains up to 70% before rupture (Holten-Andersen et al. 2007). Micro-tears are presumed to be reversible, since threads can be repeatedly extended with no apparent structural changes. The significance of the micro-granular structure in strain tolerance is corroborated by the results of two comparative investigations. The cuticle on P. canaliculus threads is homogenous instead of granular; despite having H and Ei similar to those of the M. galloprovincialis cuticle, it shattered at strains of 30% or lower (Holten-Andersen et al. 2007). Moreover, the cuticle of M. californianus, with granule diameters at only 200 nm (25% of those in M. galloprovincialis), exhibits catastrophic cracking only at strains greater than 120%.
18.5 Biocomposite-Based Byssus Holdfast biochemistry as an area of study is still early in its evolution (Sagert et al. 2006). Byssal threads are example of “permanent holdfasts” in that they cannot be undone by the organisms even though the holdfasts may break, be abandoned, or deteriorate in time. A holdfast for tenacious attachment to the hard substratum
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is a prerequisite for exposed organisms and the energy allocation for making and maintaining a holdfast can be considerable. In the blue mussel and related species the byssal system consists of a series of byssus threads that are organic in nature. In contrast hereto, the jingle shell, Anomia sp., has a single large mineralized byssus that extends through the bottom shell. Recently, Eltzholtz et al. (2009) investigated the architecture and composition of the Anomia simplex byssus using scanning electron microscopy and energy dispersive X-ray spectroscopy. They showed that this byssus is organized into a hierarchical assembly of crystals and organic matrix. There is a distinct magnesium distribution reported that is likely to reflect a combination of polymorph and chemical composition control. Sulfur is found to be distributed in distinct zones and sulfur-containing organic matrix provides interconnections between soft tissue and the mineralized byssus. Powder X-ray diffraction shows that calcite and aragonite are present in roughly equal ratios: 55.5(5) wt% aragonite by Rietveld refinement. The mechanical properties of the Anomia byssus were probed by nanoindentation. It was found that the mineralized part of the byssus is very stiff with a reduced modulus of about 67 GPa and a hardness of ∼3.7 GPa (Eltzholtz and Birkedal 2009). The complex microstructure revealed in this study brings to light an advanced hierarchical architecture. The presence of holes surrounded by a layered organic matrix in the bottom porous part, which is extended outside the animal, is suggestive of a fracture stopping mechanism. This is further supported by the observation of the lamellar structure of the organic lining surrounding the pore’s cavities. The lamellar nature of the top half of the byssus clearly provides a possibility for interconnection with the soft tissue through interweaving. The hard, stiff nature of the highly mineralized byssus makes one ponder how the animal copes with the build up of stress at the soft tissue byssus interface where a presumably very stiff, highly mineralized structure meets soft muscular tissue. Whether this holds will be studied by investigations of the mechanical properties of the byssus. Preliminary nanoindentation measurements have revealed that the average indentation hardness and modulus are respectively 20% higher (hardness) and lower (modulus) than those of single crystal calcite (Eltzholtz and Birkedal 2009).
18.6 Conclusion Wave and wind-swept rocky shores are turbulent and punishing marine habitats. Despite this, many organisms prefer to live there, often at extremely high densities like mussels. Nutrient mixing, waste product removal, and dissolved gases are important habitat benefits, but the risks include high drag, abrasion, desiccation, and anoxia. Molluscs developed corresponding effective strategies to survive under these conditions, partially because of their elastic holdfasts based on byssal threads as well as on mineral-containing byssus. Findings of very specific mef-proteins as well as DOPA related compounds in byssus determined a broad variety of inspiration for both chemical synthesis and biomimetics. I much prefer the terminology proposed by Herbert Waite in this special case, who spoke of “biomimetic attempts.”
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Thus, availability of the complete primary sequence of several plaque-derived proteins has inspired attempts to produce complete or partial biomimetic analogues. All efforts to date pertain to mefp-1 and its tandemly repeated decapeptide. The field abounds with reports of the synthesis of “approximate” DOPA- and hydroxyprolinecontaining decapeptides (see Olivieri et al. 1989; Swerdloff et al. 1989); however, Yamamoto (1987) significantly advanced the field by using a fragment condensation strategy to make a polymer with ten decapeptide repeats (10 mer). A more fastidious synthesis of the decapeptide replete with DOPA and hydroxyproline and dihydroxyproline has been reported (Taylor and Weir 2000). The availability of mefp-1 and synthetic mefp-1-inspired decapeptides has spurred numerous initiatives in surface science as well. There is general agreement (Holten-Andersen and Waite 2008) that: (1) mefp-1 adsorbs rapidly and strongly to a variety of surfaces; (2) the strength of adsorption is closely linked to the presence of DOPA; (3) the oxidation of DOPA before adsorption leads to weaker adsorption to mineral surfaces, but to stronger interactions with other organic macromolecules; and (4) the oxidation of DOPA after adsorption provides an effective platform for adlayer formation. Commercial suppliers of various grades of mefp-1 described their product (e.g., CellTakTM , Becton-Dickinson, Bedford, MA, USA) as an adhesive protein, because it improved attachment of cells and tissues in culture. While mussel byssus possess except adhesive properties, many potentially useful features including, abrasionresistant coatings, self-healing polymers, giant self-assembling mesogens, and pHtriggered cross-linking (Hassenkam et al. 2004; Harrington and Waite 2007; HoltenAndersen et al. 2007).
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Pearce T, LaBarbera M (2009b) A comparative study of the mechanical properties of Mytilid byssal threads. J Exp Biol 212:1442–1448 Price HA (1981) Byssus thread strength in the mussel, Mytilus edulis. J Zool London 194:245–255 Pujol JP (1967) Le complexe byssogène des mollusques bivalves: histochimie comparée des sécrétions chez Mytilus edulis L. et Pinna nobilis L. Bull Soc Linn Normandie 10:308–332 Pujol JP, Rolland M, Lasry S et al (1970) Comparative study of amino acid composition of byssus in some common bivalve molluscs. Comp Biochem Physiol 34:193–201 Qin XX, Coyne KJ, Waite JH (1997) Tough tendons – mussel byssus has collagen with silk-like domains. J Biol Chem 272:32623–32627 Qin XX, Waite JH (1995) Exotic collagen gradients in the byssus of the mussel Mytilus-edulis. J Exp Biol 198:633–644 Rudall KM (1955) The distribution of collagen and chitin. Symp Soc Exp Biol 9:49–72 Rzepecki LM, Hansen KM, Waite JH (1992) Bioadhesives: DOPA and phenolic proteins as composite materials. In: Richardson PD, Steiner M (eds) Principles of cell adhesion. CRC, Boca Raton, FL Sagert J, Sun C, Waite JH (2006) Chemical subtleties of mussel and polychaete holdfasts. In: Smith AM, Callow JA (eds) Biological adhesives. Springer, Berlin Selin NI, Vekhova EE (2004) Dynamics of byssal thread production in Crenomytilus grayanus and Modiolus modiolus (Bivalvia) upon reattachment to substrate. Rus J Mar Biol 30:418–420 Smeathers JE,Vincent JFV (1979) Mechanical properties of mussel byssus threads. J Molluscan Stud 49:219–230 Suci PA, Geesey GG (2001) Comparison of adsorption behavior of two Mytilus edulis foot proteins on three surfaces. Coll Surf 22:159–168 Sun CJ (2002) Matrix protein PTMP1 and its possible role in the biomechanics of mussel byssal thread. Ph.D. Dissertation, University of California, Santa Barbara, CA Sun CJ, Waite JH (2005) Mapping chemical gradients within and along a fibrous structural tissue: mussel byssal threads. J Biol Chem 280:39332–39336 Swerdloff MD, Anderson SB, Sedgwick RD et al (1989) Solid-phase synthesis of bioadhesive analogue peptides with trifluoromethanesulfonic acid cleavage from PAM. Int J Peptide Protein Res 33:318–327 Tallberg T (1877) Über die Byssus des Mytilus edulis. Nova acta Regiae Societatis Scientarum Upsaliensis 18:1–9 Taylor CM, Weir CA (2000) Synthesis of the repeating decapeptide unit of Mefp1 in orthogonally protected form. J Org Chem 65:1414–1421 Taylor SW, Chase DB, Emptage MH et al (1996) Ferric ion complexes of a DOPA-containing adhesive protein from Mytilus edulis. Inorg Chem 35:7572–7577 Taylor SW, Waite JH, Ross MM et al (1994) trans-2,3-cis-3,4-Dihydroxyproline in the tandemly repeated consensus decapeptides of an adhesive protein from Mytilus edulis. J Am Chem Soc 116:10803–10804 Tëmkin I (2006) Morphological perspective on the classification and evolution of recent Pterioidea (Mollusca: Bivalvia). Zool J Linn Soc 148:253–312 Vaccaro E, Waite JH (2001) Yield and post-yield behaviour of mussel byssal thread: a self-healing biomolecular material. Biomacromolecules 2:906–911 Van der Feen PJ (1949) Byssus. Basteria 13:66–71 Venkateswaran K, Dohmoto N (2000) Pseudoalteromonas peptidolytica sp. nov., a novel marine mussel-thread-degrading bacterium isolated from the sea of Japan. Int J Syst Evol Microbiol 50:565–574 Vitellaro-Zuecarello L (1973) Uhrastructure of the Byssal Apparatus of Mytilus galloprovincialis. I. Associated Fungal Hyphae. Mar Biol 22:225–230 Vitellaro-Zuecarello L (1981) Ultrastructural and cytochemical study on the enzyme gland of the foot of a mollusc. Tissue Cell 13:701–713 Vreeland V, Waite JH, Epstein L (1998) Polyphenols and oxidases in substratum adhesion by marine algae and mussels. J Phycol 34:1–8
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Chapter 19
Abductin
Another protein rubber is abductin found in the shell-opening ligaments of bivalve mollusks. One or two adductor muscles hold the two half shells or valves of a bivalve closed (the edible part of a scallop is one of these muscles). Closing compresses the ligament, so its elastic resiliency can reopen the shell if the muscles relax. Interestingly, scallops, which swim by repeatedly clapping their valves together, recover a greater fraction of the work done on their abductin than do clams and other more sedentary forms. Steven Vogel, 2003
Abstract Abductin is a unique protein as it is the only elastomer identified in nature that possesses compressible elasticity. Amino acid analyses of abductin derived from the swimming scallop, Placopecten magellanicus, revealed the presence of three prominent amino acids: glycine, methionine, and phenylalanine. In addition, the primary sequence of Argopecten abductin shows the presence of a repeating pentapeptide sequence, FGGMG, throughout the molecule. The main sequence feature of abductin is the presence of many repeating sequences, all of them containing glycyl residues, in a similar way to elastin. One obvious application for abductin research is the production of abductin-like biomaterials to be used, for example, as vascular prostheses. The abductin polypeptides and their derivatives, including recombinant forms, also can be used in the manufacture of a broad range of biomaterials ranging from light-weight durable fabric for clothing to matrices useful for human tissue. Abductin is a natural elastomer that serves as the primary building block for the abductor ligament in bivalves. The hydrated abductor ligament is triangular in shape with dark brown tint (Thornhill 1971) and rubber-like consistency (Kelly and Rice 1967). It is located in the hinge region of the bivalve and functions similarly to a coil spring to open the shell upon relaxation of the adductor muscle that keeps the shell tightly closed. In addition, the ligament plays a role in the swimming of scallops. The mechanics and energetics of scallop adductor muscle during swimming has been thoroughly investigated (Denny and Miller 2006; Marsh et al. 1992; Marsh and
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Olson 1994; Morton 1980; Rall 1981; Vogel 1997). The abductor ligament allows the shell to repeatedly open after relaxation of the adductor muscle at a frequency of 4 Hz, causing expulsion of water dorsally. This enables the scallop to swim several meters at a time to escape slow-moving predators such as starfish. By rapidly clapping their valves together, these clams expel jets of water from the dorsal edge of the shell. The resulting thrust propels the animals ventrally (Cheng and DeMont 1996; Gould 1971), allowing them to escape from both predators and environmental stress and potentially allowing them to migrate (Morton 1980). As with all jet-propelled animals, swimming in scallops and file shells depends on the ability to rapidly expel fluid (thereby producing thrust) and then to re-inflate. The more frequently the animal can perform this cycle, the more thrust is produced in a given time, and the more power is available to propel the body. The consequences of increased power of thrust are potentially valuable: the larger the power, the larger the mass that can be lifted against gravity, and the faster the animal moves. Thus, the thrust power available to a jet-propelled bivalve increases if the mass or the damping coefficient is decreased or the stiffness of the springs is increased. The adductor muscle of a scallop (the muscle responsible for clapping the valves together) forms an unusually large proportion of the overall mass of the animal (typically ~25%) and has striated fibers that contract rapidly relative to the muscles found in other bivalves (Marsh et al. 1992; Marsh and Olson 1994; Rall 1981). The shell mass in swimming bivalves is reduced relative to their sedentary cousins. This adaptation both increases the power of thrust and simultaneously reduces the need for thrust by reducing the weight that must be lifted against gravity (Gould 1971). Lastly, the resilium is formed from a stiff elastic abductin that causes the shell to open rapidly after it has clapped shut (Cheng and DeMont 1996; Gould 1971) and the mechanical resilience of abductin (its ability to store the potential energy of deformation with little loss to viscous processes) reduces the damping of the system. Although these adaptations allow scallops to swim, these bivalves are nonetheless on the verge of failure. If scallops’ shells were slightly bigger, if their muscles were capable of producing slightly less power, or if their abductin were less resilient, these animals might never get off the seafloor. Abductin is a unique protein as it is the only elastomer identified in nature that possesses compressible elasticity. The conceptual amino acid sequence, derived from Argopecten irradians, has been published (Cao et al. 1997). Amino acid analyses of abductin derived from the swimming scallop, Placopecten magellanicus, revealed the presence of three prominent amino acids: glycine, methionine, and phenylalanine (Cao et al. 1997). In addition, the primary sequence of Argopecten abductin shows the presence of a repeating pentapeptide sequence, FGGMG, throughout the molecule. Tetrapeptides such as MGGG and GGMG and tripeptides such as FGG are also repeated at a lower frequency, while the decapeptide FGGMGGGNAG and the nonapeptide GGFGGMGGG seem to be the major repeating sequences. In general terms, the main sequence feature of abductin is the presence of many
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repeating sequences, all of them containing glycyl residues, in a similar way to elastin (Bochiccio et al. 2005). A United States Patent entitled, “Molluscan ligament polypeptides and genes encoding them,” describes a mollusc protein based on the repeat sequences in abductin which can be used as a novel biomaterial. The gene encoding abductin is not related to the resilin gene (<30% identity) and the formation of beta-turns is not predicted. The repeat sequence identified for abductin is GGFGGMGGGX, which does not contain tyrosine and therefore cannot cross-link through the formation of dityrosine links, as resilin does (see Chapter 20). Here is one additional example for “extreme biomimetics”—a novel scientific direction I will discuss in the separate chapter of this book. The ability of scallops to swim is put to a stringent test by the Antarctic scallop, Adamussium colbecki. In this animal’s frigid habitat (–1.8◦ C), water is approximately 43% more viscous than at 10◦ C, where most temperate scallops live. Increased viscosity amplifies the power required both to form a propulsive jet and to push the animal through the water. At the same time, low temperature has the potential to decrease the power output of the adductor muscle. Furthermore, the resilience of rubbery materials (such as abductin) typically decreases at low temperatures. For example, the decrease in resilience of a rubber O-ring contributed to the loss of the space shuttle Challenger when the craft was launched at temperatures near freezing (Denny and Miller 2006). A decrease in the resilience of its abductin pad would increase the damping coefficient of the Antarctic scallop’s spring-mass system, potentially reducing the power available for thrust. Despite these potential problems, A. colbecki is capable of swimming. However, it is evident that the Antarctic scallop is on the edge: A. colbecki swims at speeds of only 25–50% of those found in temperate scallops (Ansell et al. 1998), barely above the minimal speed required to stay aloft (Cheng and DeMont 1996). Regardless of its evolutionary history, the increased resilience of A. colbecki abductin is intriguing (Denny and Miller 2006). A rubber that retains its resilience at low temperature would have great practical value in human technology and it would therefore be useful to determine the molecular basis for the increased resilience in A. colbecki abductin. However, the amino acid composition of A. colbecki abductin is very similar to that of temperate scallops, indicating that compositional adjustments to cold temperatures are subtle. For example, it is possible that the ratio of methionine to methionine sulfoxide is higher in A. colbecki than in temperate scallops: methionine is more hydrophobic than methionine sulfoxide, and the weakening of any hydrophobic interactions at low temperature would help maintain the mobility of protein chains. However, the analysis performed by Denny and Miller (2006) does not distinguish between methionine and methionine sulfoxide: both are converted to methionine sulfone before hydrolysis. One other protein rubber, elastin, is known to maintain high resilience at low temperature (Gosline and French 1979). As temperature decreases, hydrophobic bonds within the elastin rubber network weaken and the rubber swells drastically, thereby reducing viscous interactions among chains. However, if this mechanism is
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present in A. colbecki abductin, its action must be relatively subdued: unlike elastin, A. colbecki abductin volume decreases at low temperatures.
19.1 Conclusion Due to the low complexity associated with their sequences, uncovering the evolutionary and functional relationships in highly repetitive proteins like abductin represents a significant challenge. One obvious application for abductin research is the production of abductin-like biomaterials to be used, for example, as vascular prostheses. The abductin polypeptides and their derivatives, including recombinant forms, can also be used in the manufacture of a broad range of biomaterials ranging from light-weight durable fabric for clothing to matrices useful for human tissue and organ prostheses (Shewry et al. 2003). There are no doubts that, due to the high resilience to cold water by the A. colbecki abductin, this material may be of interest to materials engineers.
References Ansell AD, Cattaneo-Vietti R, Chiantore M (1998) Swimming in the Antarctic scallop Adamussium colbecki: analysis of in situ video recordings. Antarct Sci 10:369–375 Bochiccio B, Jimenez-Oronoz F, Pepe A et al (2005) Synthesis of and structural studies on repeating sequences of abductin. Macromol Biosci 5:502–511 Cao Q, Wang Y, Bayley H (1997) Sequence of abductin, the molluscan “rubber” protein. Curr Biol 7:R677–R678 Cheng J-Y, DeMont ME (1996) Jet-propelled swimming in scallops: swimming mechanics and ontogenic scaling. Can J Zool 74:1734–1748 Denny M, Miller L (2006) Jet propulsion in the cold: mechanics of swimming in the Antarctic scallop Adamussium colbecki. J Exp Biol 209:4503–4514 Gosline JM, French CJ (1979) Dynamic properties of elastin. Biopolymers 18:2091–2103 Gould SJ (1971) Muscular mechanics and the ontogeny of swimming scallops. Paleontology 14:61–94 Kelly RE, Rice RV (1967) Abductin: a rubber-like protein from the internal triangular hinge ligament of pecten. Science 155:208–210 Marsh RL, Olson JM (1994) Power output of scallop adductor muscle during contractions replicating the in vivo mechanical cycle. J Exp Biol 193:136–156 Marsh RL, Olson JM, Quzik SK (1992) Mechanical performance of scallop adductor muscle during swimming. Nature 357:411–413 Morton B (1980) Swimming in Amusium pleuronectes (Bivalvia: Pectinidae). J Zool Lond 190:375–404 Rall JA (1981) Mechanics and energetics of contraction in striated muscle of the sea scallop. Placopecten magellanicus. J Physiol Lond 321:287–295 Shewry PR, Tatham AS, Bailey A (2003) Elastomeric proteins. Structures, biomechanical properties, and biological roles. University Press, Cambridge Thornhill (1971) Abductin, locus and spectral characteristics of a brown fluorescent chromophore. Biochemistry 10:2644–2649 United States Patent “Molluscan ligament polypeptides and genes encoding them” No.6.127.166 Vogel S (1997) Squirt smugly, scallop. Nature 385:21–22 Vogel S (2003) Comparative biomechanics: life s physical world. University Press, Princeton
Chapter 20
Resilin
Abstract Resilin-like proteins contain distinct repetitive domains consisting of tyrosine residues and, in nature, cross-linking occurs between tyrosine residues, generating di- and trityrosine. In spite of that most research on resilin is dedicated to terrestrial arthropods, investigations on its features as well as role in marine crustaceans are in progress. The joint in investigated crustaceans is controlled by a single abductor muscle operating against a spring in which the elastic properties of resilin play a key role. The outstanding mechanical properties of high resilience and high fatigue lifetime make resilin or resilin-like materials attractive for a number of biomedical applications. Therefore, obtaining recombinant resilin-like proteins is of importance today. Resilin, an almost perfect elastic, is found in many places in insects and in some crustaceans where energy must be stored or where rapid and full recoil is needed (Burrows et al. 2008). Recently, Bennet-Clark (2007) discussed this unique protein from historical point of view. Weis-Fogh had originally found that resilin shows perfect elasticity: even when strained to over twice its original length for 2 weeks, a dragon fly’s resilin tendon snaps back perfectly when the stress is relieved (hence the name he gave it) and that it showed neither tearing norm fatigue when stressed within its natural limits (Weis-Fogh 1960). He pointed out that resilin was an ideal material for making elastic joints, such as hinges, that were subjected to repeated cyclical stress. In later studies, with Sven Olav Andersen and others, came confirmation of the rubber-like nature of this protein and identification that the cross-links were the fluorescent amino acids, dityrosine and trityrosine (Andersen 1964). Dityrosine fluoresces in UV light, being maximally excited with light at 315·nm and radiating maximally at 430·nm (Andersen and Weis-Fogh 1964; Elvin et al. 2005): this provides a useful way of identifying resilin non-invasively (see Fig. 20.1). Chris Elvin and his colleagues (Elvin et al. 2005) have successfully inserted the gene for pro-resilin into Escherichia coli, obtaining the gene product and then crosslinking this product and casting it into quite large structures with remarkably high resilience: in other words, they have been able to produce resilin in potentially useful quantities and with the potential to form it into structures. Elvin suggests that
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Fig. 20.1 Identification of the resilin localization in crayfish using UV (a, b) (images courtesy Malcolm Burrows)
applications could range from spinal disk implants and heart and blood valve substitutes to high-efficiency industrial rubbers, microactuators, and nanosprings. There are serious practical problems to overcome, however, the most serious of which appear to be the ease with which resilin can be de-natured by proteases, the effects of pH and hydration on its mechanical properties, and, in the context of a prosthesis, that it could create an immune response (Bennet-Clark 2007). In spite of that most research on resilin is dedicated to terrestrial arthropods (Bennet-Clark and Lucey 1967; Haas et al. 2000), investigations on its features as well as role in marine crustaceans are in progress. For example, interesting results were recently reported by Malcolm Burrows and his team from the University of Cambridge, UK (Burrows et al. 2008). In crustaceans, the joint between the ischus and merus of a walking leg has only a flexor muscle and is said to operate against a pad-like ligament containing resilin, which brings about extension. Resilin has two key signatures that enable it to be recognized. First, it fluoresces in the blue when illuminated by a narrow band of near ultraviolet (UV) light and, second, the fluorescence is reversibly dependent on pH. This principle is analyzed and illustrated by striking photographs and high-speed video footage, published in the open access journal BMC Biology, of the movements of the mouthparts of crabs and crayfish where resilin is very visible because of the fluorescence (Burrows 2009). The limbs studied, called maxillipeds, move rhythmically to deflect the exhalent water currents emerging from the gills. According to Burrows, the water currents created by these movements have two important roles. First, as an active sensor; water is drawn from a wide area in front of the animal over the sensory neurons around its mouth. The flow created in this way mixes odor molecules in the water and thus enables better odor acquisition and sampling. It may also allow an assessment of the amount of particulate matter in the exhalent current that might indicate when the gills need cleaning. Second, as an active communication and signaling mechanism; the currents created distribute odor molecules in the urine released into them and act as a signal to other animals, particularly of their own species. The results obtained by Burrows can be summarized as follows: the joint in investigated crustaceans is
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therefore controlled by a single abductor muscle operating against a spring in which the elastic properties of resilin play a key role. The outstanding mechanical properties of high resilience and high fatigue lifetime make resilin or resilin-like materials attractive for a number of biomedical applications. Therefore, obtaining recombinant resilin-like proteins is of importance today. Resilin-like proteins contain distinct repetitive domains consisting of tyrosine residues and, in nature, cross-linking occurs between tyrosine residues, generating di- and trityrosine. Using a recursive stepwise approach, Elvin and co-workers (Lyons et al. 2009) recently generated oligomeric expression constructs encoding periodic polypeptides based upon consensus repeat motifs of either the Drosophila melanogaster resilin gene or a putative mosquito resilin gene. These 16-repeat polypeptides were observed to exhibit heat stability and hydrophobic properties similar to that of recombinant Rec1-resilin. Two novel recombinant proteins, An16 and Dros16, have recently been generated. These recombinant proteins contain, respectively, 16 copies of an 11 amino acid repetitive domain (AQTPSSQYGAP), observed in a resilin-like gene from Anopheles gambiae, and 16 copies of a 15 amino acid repetitive domain (GGRPSDSYGAPGGGN) observed in the first exon of the D. melanogaster CG15920 gene. The authors compared the structural characteristics of the proteins and material properties of the resulting biopolymers relative to Rec1-resilin, a previously characterized resilin-like protein encoded by the first exon of the D. melanogaster CG15920 gene. While the repetitive domains of natural resilins display significant variation in terms of both amino acid sequence and length, reported synthetic polypeptides have been designed as perfect repeats. Using techniques including circular dichroism, atomic force microscopy, and tensile testing, it was demonstrated that both An16 and Dros16 have similar material properties to those previously observed in insect and recombinant resilins. Modulus, elasticity, resilience, and dityrosine content in the cross-linked biomaterials were assessed. Despite the reduced complexity of the An16 and Dros16 proteins compared to natural resilins, the authors have been able to produce elastic and resilient biomaterials with similar properties to resilin.
20.1 Conclusion It is not surprising that numerous processes related to the development of resilinlike biopolymers as well as “hybrid resilin” polymers are the base of corresponding inventions and patents (Bollschweiger et al. 2008). A hybrid resilin may be comprised of a pro-resilin fragment capable of forming a plurality of β-turns and able to cross-link through dityrosine formation and a second polymeric molecule, preferably selected from the group consisting of mussel byssus protein, spider silk protein, collagen, elastin, glutenin and fibronectin, or fragments thereof. These “hybrid resilin” polymers will display new properties including resilience with high tensile strength, adhesion properties, and cell interaction and adhesion. It seems that resilin
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as a novel biomimetic nanospring will possess a future, especially in protein-based elastomer for engineering applications.
References Andersen SO (1964) The cross links in resilin identified as dityrosine and trityrosine. Biochim Biophys Acta 93:213–215 Andersen SO, Weis-Fogh T (1964) Resilin. A rubber-like protein in arthropod cuticle. Adv Insect Physiol 2:1–65 Bennet-Clark HC (2007) The first description of resilin. J Exp Biol 210:3879–3881 Bennet-Clark HC, Lucey ECA (1967) The jump of the flea: a study of the energetics and a model of the mechanism. J Exp Biol 47:59–76 Bollschweiger C, Liebmann B, Fehr M et al (2008) Patent WO/2008/055931 Use of natural, recombinant and synthetic resilins in cosmetics Burrows M (2009) A single muscle moves a crustacean limb joint rhythmically by acting against a spring containing resilin. BMC Biology 7:27 Burrows M, Shaw SR, Sutton GP (2008) Resilin and cuticle form a composite structure for energy storage in jumping by froghopper insects. BMC Biol 6:41 Elvin CM, Carr AG, Huson MG et al (2005) Synthesis and properties of crosslinked recombinant pro-resilin. Nature 437:999–1002 Haas F, Gorb S, Wootton RJ (2000) Elastic joints in dermapteran hind wings: materials and wing folding. Arthropod Struct Develop 29:137–146 Lyons RE, Nairn KM, Huson MG et al (2009) Comparisons of recombinant resilin-like proteins: repetitive domains are sufficient to confer resilin-like properties. Biomacromolecules 10(11):3009–3014 Weis-Fogh T (1960) A rubber-like protein in insect cuticle. J Exp Biol 37:889–907
Chapter 21
Adhesion Systems in Echinodermata
Abstract Adhesives of aquatic organisms have to fulfill several functions, including prevention of random aggregation in the secretory glands and during transport, priming underwater surfaces, dispersion of adhesive proteins and adsorption to various materials, self-organization and shielding from aqueous erosion, and microbial degradation. Permanent, transitory, temporary, and instantaneous adhesion systems are known in marine environments. All these systems rely on different types of adhesion and therefore differ in the way they operate, in their structure, and in the composition of their adhesive. Different bioadhesive systems of echinoderm origin are discussed here. Their applications cover two broad fields of applied research: design of water-resistant adhesives and development of new antifouling strategies. Rocky intertidal shores occur throughout the world and are home to a profusion of sessile plants and animals. Organisms in the intertidal region experience significant drag and lift forces because water velocities are often in excess of 10 ms−1 ; thus, strong and environmentally durable adhesion is a prerequisite for survival. One factor that makes the adhesive strategies of most of these organisms especially interesting is that they adhere underwater and attach to virtually any available hard surface (Waite 1987). Adhesives of aquatic organisms have to fulfill several functions, including prevention of random aggregation in the secretory glands and during transport, priming underwater surfaces, dispersion of adhesive proteins and adsorption to various materials, self-organization and shielding from aqueous erosion, and microbial degradation (Kamino 2003). The density of seawater denies gravity the power to hold organisms to the bottom; thus, if they want to withstand the hydrodynamic forces, marine organisms must have adhesive mechanisms. Biological mechanisms of attachment, the comparative morphology and bioengineering of organs for linkage, suction, and adhesion are therefore of significant interest (Nachtigall 1974). The following adhesion systems displayed by marine organisms are established in the literature (see for review Flammang 2006).
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(a) Permanent adhesion. This involves the secretion of a cement and is characteristic of sessile organisms that remain in the same place throughout their adult life (e.g., the attachment of barnacles on rocks). (b) Transitory adhesion. This allows simultaneous adhesion and locomotion: the animals attach using a viscous film they lay down between their body and the substratum and creep on this film which they leave behind as they move (e.g., the ventral secretions of turbellaria plathyhelminthes). (c) Temporary adhesion. This allows organisms to attach firmly but momentarily to a substratum (e.g., the adhesion of echinoderm podia). (d) Instantaneous adhesion. This comprises invertebrate adhesive systems that do not fit into the three types of adhesion described above. The boundary between transitory and temporary adhesion is not always clear, however. Indeed, gastropod molluscs may use either transitory adhesion (in conjunction with suction) when they are moving or temporary adhesion when stationary for a long period of time, the latter giving by far the greatest adhesive strength to the animal (see, e.g., Smith et al. 1999). Echinoderms are also quite exceptional in the sense that most species belonging to this group use adhesive secretions extensively. Moreover, according to the species or to the developmental stage considered, different adhesive systems may be employed. These include (Flammang 2006) (1) tube feet or podia, organs involved in attachment to the substratum, locomotion, food capture, or burrowing; (2) larval adhesive organs allowing attachment of larvae during settlement and metamorphosis; and (3) Cuvierian tubules, sticky defense organs occurring in some holothuroidea species. All these systems rely on different types of adhesion and therefore differ in the way they operate, in their structure, and in the composition of their adhesive. Traditionally, most studies have focused on the characterization of permanent adhesives characteristic of sessile organisms staying in the same place throughout their adult life such as mussels, tube-dwelling polychaetes, and barnacles. Comparatively, nonpermanent adhesives that allow the organisms both to attach and to move have received much less attention. They are typically more hydrated and consist of a mixture of proteins and polysaccharides. Unfortunately, there is still a considerable gap in the knowledge on the nanostructural organization, as well as the biochemistry, of permanent adhesives. Here I analyze several examples of echinoderm adhesives.
21.1 Sea Urchins Sea urchins attach by employing a multitude of independent adhesive organs, the adoral tube feet, which are extremely well designed for temporary adhesion. They
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possess an enlarged and flattened apical disk that produces an adhesive secretion to fasten the sea urchin to the substratum, as well as a deadhesive secretion to allow voluntary detachment. In addition, this disk is connected to an extensible stem that bears the tensions placed on the animal by hydrodynamic forces (Flammang and Jangoux 1993; Flammang 1996). The adhesive secretion is delivered through the disk cuticle onto the tube foot distal surface, where they form a thin film that binds the disk to the substratum (Flammang et al. 2005). Deadhesive secretions are released within the cuticle where they are believed to remove its outermost layer, the so-called fuzzy coat, due to their enzymatic activity. Thus, after detachment, most of the adhesive material remain strongly attached to the substratum as a footprint (Flammang 1996; Flammang and Jangoux 1993; Flammang et al. 1998). The properties of sea urchin temporary adhesive are remarkable for several reasons. Tube feet (1) have a high adhesive strength (force per unit area) ranging from 0.09 to 0.54 MPa (Santos and Flammang 2006, 2008; Santos et al. 2005), in the range of values measured in other marine invertebrates (0.1–0.5 and 0.5–1 MPa for nonpermanent and permanent adhesives, respectively; Smith 2006) and matching the technological requirements for underwater synthetic adhesives (0.2–0.7 MPa; Waite 2002); (2) attach efficiently to substrata with various chemistries and roughness (Santos and Flammang 2006; Santos et al. 2005); and (3) have highly specialized epidermal adhesive areas made up of different secretory cells that separately release the adhesive and deadhesive secretions, thus enabling repeated attachment–detachment cycles (Santos and Flammang 2006). The only available information on the nature and composition of the sea urchin adhesive comes from histochemical studies showing that echinoid footprints (circular prints of secreted adhesive that remain on the substratum after detachment) stain for acid mucopolysaccharides, but not for proteins (Flammang and Jangoux 1993). Aside from inorganic residues apart (45.5%), the footprints are composed of proteins (6.4%), neutral carbohydrates (1.2%), and lipids (2.5%). At present, data on the biochemical composition of echinoderm adhesive footprints are only available for one sea star species, Asterias rubens (Flammang et al. 1998). In this species, footprints also contain a significant amount of inorganic residues (40%), similar values of neutral sugars (3%) and lipids (5.6%) but a much higher amount of proteins (20.6%). Other carbohydrates such as amino sugars (1.5%) and uronic acids (3.5%) were also found in A. rubens footprints. The absence of DOPA in the footprint material indicates that contrary to the cements of mussels and polychaetes, in sea urchin, DOPA cross-links do not seem to contribute for the adhesive cohesiveness and insolubility (Santos et al. 2009).
21.2 Sea Cucumbers Several species of sea cucumbers, all belonging to a single family, possess a peculiar and specialized defense system, the Cuvierian tubules (Flammang et al. 2002). The system is mobilized when the animal is mechanically stimulated, resulting in the discharge of a few white filaments, the tubules (Fig. 21.1). In seawater, the expelled
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Fig. 21.1 Cuvierian tubules in Bohadschia argus sea cucumber (image courtesy Patrick Flammang)
tubules lengthen considerably and become sticky upon contact with any object. The adhesiveness of their outer epithelium combined with the tensile strength of their collagenous core makes Cuvierian tubules very efficient at entangling and immobilizing most potential predators. Cuvierian tubule adhesion is a typical example of instantaneous adhesion, adhesion being achieved in a matter of seconds (less than 10s) (Flammang 2006). Flammang and co-workers designed a method to measure the adhesion of holothuroidea Cuvierian tubules (Flammang et al. 2002). Tubule adhesive strength was measured in seven species of sea cucumbers belonging to the genera Bohadschia, Holothuria, and Pearsonothuria. The tenacities (force per unit area) varied from 30 to 135 kPa, falling within the range reported for marine organisms using nonpermanent adhesion. Two species, Holothuria forskali and H. leucospilota, were selected as model species to study the influence of various factors on Cuvierian tubule adhesive strength. Tubule tenacity varied with substratum, temperature, and salinity of the seawater, and time following expulsion. These differences give insight into the molecular mechanisms underlying Cuvierian tubule adhesion. Tenacity differences between substrata of varying surface free energy indicate the importance of polar interactions in adhesion. Variation due to temperature and time after expulsion suggests that an increase of tubule rigidity, presumably under enzymatic control, takes place after tubule elongation and reinforces adhesion by minimizing peeling effects. In H. forskali, tubule print material—i.e., the secreted adhesive left on the substratum after mechanical detachment of the tubule—is composed of 60% protein and 40% neutral carbohydrate (De Moor et al. 2003). The proteinic nature of the adhesive material is confirmed by the observation that proteolytic enzymes reduce the adhesive strength of Cuvierian tubules in H. forskali (Zahn et al. 1973). The amino acid compositions of the protein fraction in H. forskali, H. leucospilota,
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Bohadschia subrubra, and Pearsonothuria graeffei indicate that their adhesives are closely related. All are rich in small side-chain amino acids, especially glycine, and in charged and polar amino acids. Only a small fraction of the secreted Cuvierian tubule adhesive (tubule prints) can be extracted using denaturing buffers containing both chaotropic and reducing agents. This soluble fraction contains about ten different proteins with molecular masses ranging from 10 to 220 kDa, but with closely related amino acid compositions, resembling that of the whole adhesive (De Moor et al. 2003).
21.3 Sea Stars In sea stars (Fig. 21.2), on the other hand, adhesion is temporary. If the substratum does not allow the formation of a firm holdfast (like Teflon), the sea star presumably detaches voluntarily at an early stage, leaving only a low amount of adhesive material on the substratum (Hennebert et al. 2008). Previous investigations have clearly demonstrated that the adhesive material in sea stars is released by the two types of adhesive secretory cells occurring in the disk epidermis of the tube feet (Flammang et al. 1994, 1998). However, how these secretions form the complex micro-structure of the footprint is not known. The morphology of type 1 adhesive cells with an empty process varies from that of cells with an apical process packed with intact granules. In all cases, however, these cells are conspicuous and easily distinguishable on the sections. On the other hand, type 2 adhesive cells are always much less conspicuous than in unattached tube feet, their apical processes being always smaller and containing fewer granules. This suggests that type 2 cells would be the first ones to release their contents and
Fig. 21.2 In sea stars adhesion is temporary (image courtesy Zoran Kljajic)
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are therefore responsible for the formation of the homogeneous film covering the substratum. Indeed, the material released by these cells has the same appearance as the one constituting this film. As discussed previously, this priming film could displace water and bind strongly to the surface. Meanwhile, type 1 cells start to release their contents, a heterogeneous electron-dense material. At the beginning of this stage, the two constituents, secreted by their respective cells, alternate in the adhesive secretion (first pattern). The material from type 1 adhesive cells, which undoubtedly derives directly from the rods constituting the granules, seems to expand gradually either inside the cells or after its release. Indeed, in some cases, it is secreted as spheroidal structures, i.e., clusters of granules released together, visible in both SEM and TEM. In some areas on the disk surface of the detached tube feet, the secreted material expands and fuses with the one released from other cells, initiating the formation of a meshwork. Type 1 cells would be therefore the origin of the meshwork pattern, and the arrangement of their secretory pores on the disk surface could act as a template for formation of this pattern. The third constituent of the adhesive layer, the loose electron-lucent material, is always observed in-between the different materials secreted by the two types of adhesive cells. This material could originate directly from the adhesive cells, in which a similar material is observed inside and around modified type 1 granules; or, alternatively, it could correspond to the outermost layer of the cuticle covering the disk epidermis, the so-called fuzzy coat. It is that gelatinous fuzzy coat, presumably made up of proteoglycans, that could collapse when footprints are dried. According to the model proposed by Flammang et al. (1998), this layer is indeed detached from the disk surface and incorporated into the footprints through the release of the deadhesive secretion. The fine structure of the adhesive material deposited on various substrata by the sea star A. rubens was observed using LM, SEM, TEM, and AFM (Hennebert et al. 2008). Whatever the method used, it always appears as a sponge-like meshwork deposited on a thin homogeneous film. The thickness of the adhesive layer varies between different areas in a same footprint, giving different aspects to the adhesive material. In thin areas, meshes ranging between 1 and 5 μm in diameter are clearly distinguishable; whereas in thick areas, these meshes are obscured because of the accumulation of material. It was demonstrated that the structure of the adhesive is altered neither by release of the deadhesive secretion which occurs at tube foot detachment (Flammang et al. 1998, 2005) nor by separation instabilities such as fingering instabilities or cavitation. The appearance of the adhesive material does not differ according to whether the footprints are fixed or not, and whether they are observed partially hydrated or dry. The thickness of the footprint is influenced by the substratum on which it is deposited. Indeed, although footprints left on glass, mica, and Teflon have the same shape and diameter, the quantity of material deposited by the tube feet varies according to the substratum tested. Tube feet produce more adhesive material on glass and mica (two high-energy surfaces) than on Teflon (a low-energy surface). At the nanometer scale, both the homogeneous film and the material forming the
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meshwork are composed of a succession of globular nanostructures. The diameter of these structures is almost the same for dry footprints observed either with SEM or AFM (50–100 and 50–90 nm, respectively) (Hennebert et al. 2008). On the other hand, AFM investigations on partially hydrated footprints reveal a globule diameter ranging between 80 and 200 nm. This slight difference in the size of the globular structures between dry and hydrated footprints suggests once again that the adhesive material may shrink upon drying.
21.4 Conclusion In addition to fundamental interests in echinoderm bioadhesives, a substantial impetus behind understanding these adhesives are the potential technological applications that can be derived from their knowledge. These applications cover two broad fields of applied research: design of water-resistant adhesives and development of new antifouling strategies. In this context, echinoderm adhesives could offer novel features or performance characteristics for biotechnological applications. For example, the rapidly attaching adhesive of Cuvierian tubules, the releasable adhesive of tube feet, or the powerful adhesive of asteroid larvae could each be useful to address particular bioadhesion problems (Flammang 2006; Flammang et al. 2005).
References De Moor S, Waite JH, Jangoux M et al (2003) Characterization of the adhesive from the Cuvierian tubules of the sea cucumber Holothuria forskali (Echinodermata, Holothuroidea). Mar Biotechnol 5:37–44 Flammang P (1996) Adhesion in echinoderms. In: Jangoux M, Lawrence JM (eds) Echinoderm studies, vol 5. Balkema, Rotterdam, p. 1–60 Flammang P (2006) Adhesive secretions in echinoderms: an overview. In: Smith AM, Callow JA (eds) Biological adhesives. Springer, Berlin, Heidelberg Flammang P, Demeulenaere S, Jangoux M (1994) The role of podial secretions in adhesion in two species of sea stars (Echinodermata). Biol Bull 187:35–47 Flammang P, Jangoux M (1993) Functional morphology of coronal and peristomeal podia in Sphaerechinus granularis (Echinodermata Echinoida). Zoomorphology 113:47–60 Flammang P, Michel A, Van Cauwenberge A et al (1998) A study of the temporary adhesion of the podia in the sea star Asterias rubens (Echinodermata, Asteroidea) through their footprints. J Exp Biol 201:2383–2395 Flammang P, Ribesse J, Jangoux M (2002) Biomechanics of adhesion in sea cucumber Cuvierian Tubules (Echinodermata, Holothuroidea). Interg Comp Biol 42:1107–1115 Flammang P, Santos R, Haesaerts D (2005) Echinoderm adhesive secretions: from experimental characterization to biotechnological applications. In: Matranga V (ed) Marine molecularbiotechnology: echinodermata. Springer, Berlin, Heidelberg, New York Hennebert E, Viville P, Lazzaroni R et al (2008) Micro- and nanostructure of the adhesive material secreted by the tube feet of the sea star Asterias rubens. J Struct Biol 164:108–118 Kamino K (2003) Barnacle underwater adhesive – biochemistry of self-organized multi-functional complex. Abstract, Workshop on New Perspectives in Marine Biofouling and Biofouling Control, Fiskebäckskil, Sweden. http://marinpaint.org.gu.se,pdf:10
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Nachtigall V (1974) Biological mechanisms of attachment. The comparative morphology and bioengineering of organs for linkage, suction, and adhesion. Springer, Berlin Santos R, da Costa G, Franco C et al (2009) First insights into the biochemistry of tube foot adhesive from the sea urchin Paracentrotus lividus (Echinoidea, Echinodermata). Mar Biotechnol. doi:10.1007/s10126-009-9182-5 Santos R, Flammang P (2006) Morphology and tenacity of the tube foot disc of three common European sea urchin species: a comparative study. Biofouling 22:187–200 Santos R, Flammang P (2008) Estimation of the attachment strength of the shingle sea urchin, Colobocentrotus atratus, and comparison with three sympatric echinoids. Mar Biol 154:37–49 Santos R, Gorb S, Jamar V et al (2005) Adhesion of echinoderm tube feet to rough surfaces. J Exp Biol 208:2555–2567 Smith AM (2006) The biochemistry and mechanics of gastropod adhesive gels. In: Smith AM, Callow JA (eds) Biological adhesives. Springer, Berlin Smith AM, Quick TJ, St Peter RL (1999) Differences in the composition of adhesive and nonadhesive mucus from the limpet Lottia limatula. Biol Bull 196:34–44 Waite JH (1987) Nature’s underwater adhesive specialist. Int J Adhes Adhes 7:9–14 Waite JH (2002) Adhesion à la moule. Integr Comp Biol 42:1172–1180 Zahn RK, Müller WEG, Michaelis M (1973) Sticking mechanisms in adhesive organs from a Holothuria. Res Mol Biol 2:47–88
Chapter 22
Adhesive Gels from Marine Gastropods (Mollusca)
Abstract The structure and properties of gastropod gels are strikingly different from common commercial glues. Commercial glues are generally solids; they may be applied in liquid form and then solidify or they may be deformable, tacky solids. In either case, their final form consists entirely of polymers or cross-linked materials. In contrast, adhesive gels typically consist of dilute polymer networks that contain more than 95% water. These gels are highly deformable. The mechanism of detachment of marine gastropods may or may not involve biochemical changes. In limpets, the glue forms a thin layer between the foot and substratum, so one possibility is that they secrete a layer of non-adhesive mucus over the top of the glue. This mucus could include molecules that compete for binding sites and block them or it could include an enzyme that breaks bonds in the glue. Alternatively, the animal may break the bonds mechanically, by generating sufficient shear. Among the more extreme examples of biological glues with high water contents are the adhesive gels produced by gastropod molluscs. Representatives of the class Gastropoda—snails, slugs, whelks, and limpets—are animals with a long, flat foot; a distinct head with eyes and tentacles; and a dorsal visceral mass usually housed in a spiral shell. Gastropods comprise the largest and most successful class of molluscs. Ecologically, they are the most versatile molluscs with freshwater, marine, and terrestrial species. Some gastropods are carnivores (feed on animal tissues), some are herbivores (feed on plant material), and still others are parasites. Their gels often consist of 97% water, yet they can produce attachment forces strong enough that animals such as limpets can be remarkably difficult to detach by hand. Adhesive forces per unit area for limpets are often in the range of a few hundred kilopascals or more (Smith 2002, 2006). They use gels to create tenacities (attachment force per unit area) ranging from 100 to 500 kPa (Smith 1992; Smith and Morin 2002). This approaches the adhesive strength of solid cements of mussels and barnacles, which is typically 500–1000 kPa (Waite 1983; Yule and Walker 1987). The structure and properties of gastropod gels are strikingly different from common commercial glues. Commercial glues are generally solids; they may be
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applied in liquid form and then solidify or they may be deformable, tacky solids (Wake 1982). In either case, their final form consists entirely of polymers or crosslinked materials. In contrast, adhesive gels typically consist of dilute polymer networks that contain more than 95% water. These gels are highly deformable. One would not expect such a dilute hydrogel to be suited for adhesion. In fact, dilute polymer gels are often excellent lubricants (Smith 2002). Because they are gels, these glues have a variety of interesting and useful properties. Foremost among these are their great flexibility and their ability to bond to wet, untreated surfaces, a feature which is important to survive under specific conditions in marine environment.
22.1 The Role of Mucus in Gastropod Gels It is now becoming clear that there are many ways to construct gels and these differences give rise to substantial functional differences (Smith 2002). The normal mucus covering their outer surface of the gastropod (e.g., limpets) is not inherently sticky. This type of mucus appears to be used during the suction adhesion of limpets. If suction is eliminated through a leak or because the animal is not forcibly contracting the musculature that creates suction, the adhesive strength in shear is low to non-existent (Smith 1991, 2002). Thus, the mucus that the animals normally crawl on provides virtually no adhesive strength on its own. The mucus that is used in adhesion, though, is different. When limpets glue down in an aquarium, they are easily distinguished from limpets that are not glued down (Smith 1992). In addition to having a high shear tenacity, detachment of limpets that are glued down occurs abruptly and usually leaves a thin film of gel stuck firmly to the glass. One can remove this gel with a razor blade to get an elastic mass that is unlike the loose slime that many snails produce across their general body surface. Thus, it is likely that there are substantial structural differences between these gels. The attachment behavior of limpets as model organisms has been studied for the greatest length of time. Limpets are commonly found on wave-swept rocky shores, where they may be subjected to water velocities in excess of 20 m/s (Denny 2000). These extreme flows can impose large forces (lift and drag), challenging the animal’s ability to adhere to the substratum (Fig. 22.1). Limpets in the genus Lottia use their adhesive gel to glue down when they are exposed and inactive during low tide (Smith 1992). The adhesive strength protects them from dislodgement by predators such as shorebirds. When the tide returns, the limpets typically become active and at this point rely on suction for adhesion (Smith 1992). The adhesive might also be used instead of suction to attach when wave surge is particularly strong. It is likely that other limpets also alternate between attachment mechanisms, though the cues may be different. The marsh periwinkle, Littoraria irrorata, can also produce adhesive and nonadhesive gels. These snails forage along mud flats, but when the tide returns they climb marsh grass stems and glue the lip of their shell down. In this way, they avoid
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Fig. 22.1 Limpets are well-known specialists on adhesion to surfaces
aquatic predators such as crabs and fish (Warren 1985). When the tide recedes, they break their adhesion and return to the mud flats. The shear tenacity created by their adhesive gel can exceed 100 kPa. This is an order of magnitude greater than the tenacity these snails create using suction and any viscous contributions from the mucus they crawl upon (Smith and Morin 2002). As with limpets, the adhesive gel is surprisingly elastic and significantly firmer than the mucus the animal crawls upon. One difference from limpets is that the glue forms a thin strip along the edge of the shell, while limpets secrete the glue under the sole of the foot. This means that periwinkle glue, unlike limpet glue, is exposed to the elements. Thus, it may dry into a solid sheet in warm, dry weather. In some species of periwinkle, such as L. aspera, the glue will always dry (Denny 1984), while in others it typically stays gelled. The peak force required to detach marsh periwinkles using the gelled glue, though, is not significantly different from that of the dried film. If anything, the flexibility of the gel may provide better adhesive performance by absorbing energy during detachment rather than failing as a brittle solid. While L. irrorata has been studied in-depth, many other periwinkles also use glues to attach to rocks or vegetation, often switching between active and inactive states. The mechanism of detachment of marine gastropods may or may not involve biochemical changes. In limpets, the glue forms a thin layer between the foot and substratum, so one possibility is that they secrete a layer of non-adhesive mucus over the top of the glue. This mucus could include molecules that compete for binding sites and block them or it could include an enzyme that breaks bonds in the glue. Alternatively, the animal may break the bonds mechanically, by generating sufficient shear. It is worth noting that there is a 68 kDa protein that is unique to the nonadhesive mucus of the limpet L. limatula (Smith et al. 1999). This may play a role in detachment, though it is also found in the pedal mucus used during locomotion, not solely during detachment.
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22.2 Chemistry of Gastropod Gels According to Smith and co-workers (Smith and Cawlow 2006; Smith et al. 1999, 2009), the glue proteins of different gastropod species may be structurally similar. They typically have acidic isoelectric points and a large proportion of charged and polar amino acids. For the limpet L. limatula, the proteins have isoelectric points that are typically between 4.7 and 5.3, and 65% of the amino acids would be polar or charged at neutral pH (Smith et al. 1999). For marsh periwinkles the two glue proteins have an isoelectric point of 4.75 and contain 49 and 52% charged or polar amino acids (Smith and Morin 2002). For land snails (Helix aspersa) and slugs (Arion subfuscus), the isoelectric points fall in the same range. The glue proteins differ in size, which is likely to be functionally significant. The glue proteins range in mass from 14 kDa for the primary slug glue protein to 118 kDa for the primary limpet glue protein (Pawlicki et al. 2004; Smith et al. 1999). While most of the proteins in limpet glue share similar amino acid compositions, Smith et al. (1999) found other differences between them. One of the two most common proteins in limpet glue, the 140-kDa protein, is glycosylated, unlike the other proteins in the glue. It also has substantially more proline than the other proteins. One relatively less common protein, at 53 kDa, has a basic isoelectric point (8.6), while the pIs of the other proteins typically fall between 4.7 and 5.3. These differences may relate to their function (Smith et al. 2009).
22.3 Possible Mechanism of Cross-Linking The mechanism of cross-linking for gastropod adhesive gels is still unknown and it would have to be a mechanism that worked effectively in the presence of water. Pawlicki et al. (2004) found that the glue proteins stiffen gels made of negatively charged polymers. Based on this and the fact that the glue proteins carried a relatively high percentage of charged amino acids, the authors initially suggested that electrostatic interactions may be involved in cross-linking. These interactions are relatively weak underwater, however, due to the high dielectric constant of the medium. Werneke et al. (2007) showed using atomic absorption spectrometry that glue from the slug A. subfuscus contains substantial quantities of zinc (46±7·and 189±80·ppm in two different sets of experiments), as well as iron, copper, and manganese (2–7·ppm). Iron-specific staining demonstrated that iron is bound specifically to the 15·kDa glue protein. Several approaches were used to show that these metals have important functional effects. Adding iron or copper to dissolved glue causes the proteins to precipitate rapidly, although zinc has no effect. Removing iron and related transition metals with a chelator during secretion of the glue causes a sixfold increase in the solubility of the glue. Once the glue has set, however, removing these metals has no effect. Finally, the gel-stiffening activity of the glue proteins was measured in the presence and absence of the chelator. The chelator eliminated the
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gel-stiffening effect of the proteins, suggesting that transition metals were necessary for the proteins to act on the gel. Thus, the glue contains transition metals and these metals play an essential role in glue function. Recently, Smith et al. (2009) suggested that unlike the situation in many wellknown gels, electrostatic and hydrophobic interactions do not appear to play a significant role in cross-linking slug glue. High salt or non-ionic detergent concentrations that normally break electrostatic or hydrophobic interactions, respectively, had no detectable effect on the solubility of proteins in the glue of A. subfuscus, their ability to stiffen gels, or the tendency of specific proteins to aggregate. This was true even at treatment concentrations significantly above what has been reported to break interactions in other systems. The results also identify a 40 kDa protein as a potentially key component of the cross-linking mechanism. This protein is linked into complexes of more than a megadalton. This suggests that it either links together, or binds to, giant polymers such as polysaccharides or proteoglycans. Such giant polymers are common components of biological gels due to the structural framework they provide by tangling (Smith 2002). Any protein that bound tightly to them could potentially cross-link them into a more rigid, tougher network. The strength of the interactions is surprising, as the 40 kDa protein isolated from A. subfuscus remained linked in large complexes even in the presence of strongly dissociating conditions. Even 8 M urea with non-ionic detergent was insufficient to disrupt these aggregations; only denaturing the proteins in SDS sufficed (Smith et al. 2009). Metal removal from the mature glue does not, however, affect the ability of the 40 kDa protein to cross-link into large complexes. Instead, these complexes are stable unless metals are chelated at the time of secretion, before the glue sets. In this case the aggregations do not consistently form. Thus, metals may directly link some polymers together and may also contribute to the formation of other cross-links. As there are several different metals in the glue, it seems likely that different metals may have different roles. It is worth noting that transition metal-specific chelation with deferoxamine affected gel setting, but not the mature glue (Werneke et al. 2007). Meanwhile, the more general divalent ion chelator EDTA did affect the solubility of the mature glue. In conclusion, it was suggested (Smith et al. 2009) that the adhesive gels of the slug A. subfuscus depend on robust cross-links. The extent to which these interactions are insensitive to disruptive treatments is surprising, especially compared to other gels (Smith et al. 1999; Smith 2006). It appears that metals are primarily responsible for the strong cross-links in these glues and this may involve more than one cross-linking mechanism.
22.4 Conclusion Many molluscs use similar gels to glue themselves to the substratum when they are inactive. Limpets use such gels to glue themselves to rocks and violent wave surge. Similarly, a thin line of gel along the lip of a marsh snail’s shell can firmly attach it to the top of a wet marsh grass stem, despite the windswept movements of the grass.
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Adhesive Gels from Marine Gastropods (Mollusca)
The research on gastropod adhesive gels has clear practical implications. The adhesive gels produced by molluscs form strong attachments in wet, irregular environments, using a minimum of organic material. Adhesives used by marine animals are especially likely to have unusual and useful characteristics because of the demands of adhesion underwater (Waite 2002). Furthermore, there is substantial interest in the development of gels with unusual properties, particularly for applications such as drug delivery, biomedical adhesives for surgery and dentistry, as well as in food science. Despite this biomimetic potential, there has been little research on the gels produced by these marine invertebrates.
References Denny MW (1984) Mechanical properties of pedal mucus and their consequences for gastropod structure and performance. Am Zool 24:23–36 Denny MW (2000) Limits to optimization: fluid dynamics, adhesive strength and the evolution of shape in limpet shells. J Exp Biol 203:2603–2622 Pawlicki JM, Pease LB, Pierce CM et al (2004) The effect of molluscan glue proteins on gel mechanics. J Exp Biol 207:1127–1135 Smith AM (1991) The role of suction in the adhesion of limpets. J Exp Biol 161:151–169 Smith AM (1992) Alternation between attachment mechanisms by limpets in the field. J Exp Mar Biol Ecol 160:205–220 Smith AM (2002) The structure and function of adhesive gels from invertebrates. Integr Comp Biol 42:1164–1171 Smith AM (2006) The biochemistry and mechanics of gastropod adhesive gels. In: Smith AM, Callow JA (eds) Biological adhesives. Springer, Berlin Smith AM, Callow JA (2006) Biological adhesives. Springer, Berlin Smith AM, Morin MC (2002) Biochemical differences between trail mucus and adhesive mucus from marsh periwinkles. Biol Bull 203:338–346 Smith AM, Quick TJ, St Peter RL (1999) Differences in the composition of adhesive and nonadhesive mucus from the limpet Lottia limatula. Biol Bull 196:34–44 Smith AM, Robinson TM, Salt MD et al (2009) Robust cross-links in molluscan adhesive gels: testing for contributions from hydrophobic and electrostatic interactions. Comp Biochem Physiol, Part B 152:110–117 Waite JH (1983) Adhesion in bysally attached bivalves. Biol Rev Camb Philos Soc 58:209–231 Waite JH (2002) Adhesion à la moule. Integr Comp Biol 42:1172–1180 Wake WC (1982) Adhesion and the formulation of adhesives. Applied Science, London Warren JH (1985) Climbing as an avoidance behaviour in the salt marsh periwinkle, Littorina irrorata (Say). J Exp Mar Biol Ecol 89:11–28 Werneke SW, Swann C, Farquharson LA et al (2007) The role of metals in molluscan adhesive gels. J Exp Biol 210:2137–2145 Yule AB, Walker G (1987) Adhesion in barnacles. In: Southward AJ (ed) Crustacean issues: barnacle biology, vol 5. Balkema, Rotterdam
Chapter 23
Barnacle Cements
Abstract Barnacles are small, shrimp-like crustaceans that live in volcano-shaped shells, which are composed of calcareous plates. Their body is positioned upsidedown so that feathery filter feeding appendages protrude from the top of the shell. Free-living barnacles attach to rocks, shells, corals, or other objects, while commensal species attach on whales, turtles, fish, and other animals. Barnacles have elicited considerable scientific attention because of the strength and durability of their adhesive and because of practical concerns initially related to marine fouling. Therefore, the first motivation for understanding the biology of barnacle cement secretion in detail has the goal of finding new techniques to control their settling on man-made marine surfaces. The second reason is related to biomimetics and materials science. Undoubtedly, such a natural adhesive as that used in barnacle cements can be utilized for potential medical and engineering applications. Due to its effectiveness in a saline, wet environments, these natural glues may be promising biodegradable adhesives for various medical procedures including surgery. Numerous marine invertebrates, including barnacles, use various unique strategies to “glue” themselves to hard surfaces found in seawaters using different adhesives. These adhesives are able to displace water, spread and form adhesive bonds with the substrate, as well as coagulate/cross-link, which impart stability to the adhesive (Waite 1987). Underwater adhesives of marine organisms are currently investigated as example of the vital link between biological science and materials science (Kamino 2008). Also, the development of novel antifouling technologies is currently quite trendy. The adhesive properties of barnacle cement are currently one of the most studied.
23.1 Barnacles—Crustaceans That Mimic Molluscs In general, barnacles (Cirripedia: Maxillopoda: Crustacea: Arthropoda) are small, shrimp-like crustaceans that live in volcano-shaped shells, which are composed of calcareous plates (Fig. 23.1). Because of their unusual body plan, barnacles were
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Fig. 23.1 Adult form of barnacle (about 3 cm in diameter) (image courtesy Gary H. Dickinson)
thought to be molluscs until 1830 (Ruppert and Barnes 1994). Their free-living larval stage is what defined them as crustaceans. There are more than 1280 species currently (Pitombo 2004). Their body is positioned upside-down so that feathery filter feeding appendages protrude from the top of the shell. The two main groups of barnacles are the stalked and stalkless barnacles. Stalked barnacles (up to 75 cm in length), also called gooseneck or goose barnacles, have a muscular, flexible stalk (peduncle) that attaches to the substrate at one end. The other end bears the major part of the body, the capitulum. The peduncle contains the remnants of the larval stage, the antennae, and the cement glands. Stalkless barnacles (up to 23 cm high and 8 cm in diameter), also called acorn barnacles, are permanently attached to a substrate. A vertical wall of plates completely envelopes the animal, and within this wall is a second layer of protection, an operculum, that also covers the animal. The plates composing the wall are made of either living tissue, interlocking teeth, or may actually be fused to some extent. Barnacles extend and retract their feathery appendages to filter feed. Small food particles are trapped by the fine bristles of the appendages. Free-living barnacles attach to rocks, shells, corals, or other objects, while commensal species attach on whales, turtles, fish, and other animals (Fig. 23.2). Because of their behavior, it is not surprising that barnacles also encrust man-made materials and constructs like ship bottoms, buoys, and pier pilings, and therefore can create many problems. For instance, the speed of a badly encrusted ship may be reduced as much as 30%, increasing fuel consumption. Also, many species of barnacles have become “invasive.” It is believed that larvae settle on ships at sea or are contained in ballast water,
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“First-Kiss” Adhesion Behavior in Barnacles
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Fig. 23.2 Photographs of the barnacle base (images courtesy Gary H. Dickinson)
allowing them to be transported all over the world. The life span of these animals is between 1 and 10 years (Ruppert and Barnes 1994).
23.2 “First-Kiss” Adhesion Behavior in Barnacles Most sessile marine invertebrates have a planktonic larvae stage. The transition from a planktonic to a benthic existence is a critical event in their survival. Settlementcompetent larvae start their searching behaviors for suitable substrata to attach, which is then followed by metamorphosis to juveniles. Larval settlement is influenced by many environmental factors in fields, among which chemical cues are thought to be most important (Morse 1990; Pawlik 1992). The life cycle of Balanus improvisus has seven planktonic larval stages before it metamorphoses into a sessile organism (Ödling et al. 2006). Between the sixth and seventh transitions, the larva transforms from a nauplii larva into a cyprid larva. Barnacle larvae swim around freely in the water column, but in order to complete the transition to adult life, the cyprid form must attach to a hard substrate (Walker et al. 1987). During a series of these processes, cyprids do not feed but use as an energy source, cyprid major protein (CMP), which appears mainly in the cyprid stage (Satuito et al. 1996). A wide range of environmental and synthetic factors have been reported to affect larval attachment (settlement) and metamorphosis: surface color (Yule and Walker 1984), water movement (Rittschof et al. 1984), conspecific adult-derived proteins, soluble pheromones (Dreanno et al. 2006), synthetic peptide analogs of barnacle attachment pheromones, footprints by cyprid antennular secretion, and bacterial films (as reviewed by Yamamoto et al. 1999). In addition to these exogenous factors, endogenous factors including those involved in signal transduction systems also have been reported to influence this
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process, although little is known about the systems involved. The results obtained by Yamamoto et al. (1996, 1999) in experiments with Balanus amphitrite suggested that the promotion effect on larval searching behavior was derived from a combination of the activities of serotonin and dopamine. Thus, cyprids explore a surface by “walking” using a pair of attachment organs, or antennules (Walker and Yule 1984; Yule and Walker 1985), which secrete an adhesive from unicellular glands (Okano et al. 1996). Briefly, this event can be described as follows. The adhesive-secreting cells are located within a pair of cement glands, which are connected by cement ducts that widen into muscular cement sacs, the presumed temporary storage location during cement secretion (Walker 1971). Cement ducts connect the sacs to an antenna, which is composed of four segments. The cement duct extends into the third segment, the adhesive disks. The adhesive is secreted through the disks, and the cyprid larva is able to attach itself to the surface and begin metamorphosis (Harrison and Sandeman 1999). Recently, Ödling et al. (2006) investigated cement secretion by cyprid larvae of B. improvisus and the morphology of their cement glands using modern techniques. The authors defined the process of cement secretion in vivo as the exocytotic and discovered that cement granules undergo a dramatic swelling during secretion. Such swelling might be due to an increased osmotic activity of granule contents, following a process of hydration. It was hypothesized that this hydration is essential for exocytotic secretion and concluded that cement protein exocytosis is a more complex process than previously thought and is similar to exocytotic secretion in vertebrate systems, such as histamine secretion from mast cells and exocrine secretion in the salivary gland and the pancreas (Ödling et al. 2006). In the exploratory phase, the cyprids have to be capable of detaching, leaving behind blobs of temporary adhesive “footprints.” The temporary adhesive does not disperse in water; it is resistant to biodegradation and also operates as a signaling molecule to induce the settlement of additional cyprids. Cyprids showed a clear pheromone-induced settlement response in the presence of polymerizing cement, with the proportion of settled cyprids being dramatically higher in cement treatments than in controls. Evidence that settlement was in response to a waterborne peptide pheromone rather than a substrate-bound cue was provided by the position of cyprid settlement (cyprids did not settle directly on cement droplets, which may contain surface-bound settlement cues) and through settlement assays conducted on cement separated by molecular mass (Dickinson 2008). This suggests that the process of cement polymerization, which is necessary for barnacle growth, results in the release of peptide pheromones that induce cyprid settlement and attract predators. Hence, barnacle growth and the associated release of cement may play an important role in structuring intertidal communities (Dickinson et al. 2009). After selection of an appropriate site on which to settle, the cyprid stands on its head and releases proteinaceous cement onto the paired antennules. Initially fluid, this permanent cyprid cement flows around and embeds the attachment organs, curing within 1–3 h to form a discrete matrix. The firmly attached organism subsequently metamorphoses into the calcified adult barnacle (Callow and Callow 2002).
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23.3 Barnacle Cements The inherent insolubility of the barnacle cement is the main limiting factor in biochemical investigations related to its nature. Polymerized barnacle cement has not been rendered fully soluble under any conditions (Kamino 2006). As recently reviewed by Dickinson et al. (2009), barnacle cement is composed of approximately 90% protein (Kamino et al. 2000; Naldrett 1993; Walker 1972) with the remainder as carbohydrate (1%), lipid (1%), and inorganic ash (4%; 30% of the inorganic ash is calcium) (Walker 1972). Barnacle cement is an aggregate of at least 10 major proteins, a portion of which have been isolated and sequenced (for reviews, see Kamino 2006, 2008). Although progress has been made toward understanding the chemical properties of cement proteins, the biochemical mechanisms of cement polymerization remain largely unknown. Recently, Dan Rittschof and co-workers (Barlow et al. 2009; Dickinson et al. 2009) proposed interesting mechanisms for barnacle cement polymerization that may be a theme for many other marine animal glues. The barnacle Amphibalanus amphitrite (previously Balanus amphitrite) (Pitombo 2004) was used in these studies. The scientists tested the broad hypothesis that barnacle cement polymerization is biochemically similar to blood clotting. Based on experimental evidence, they suggested that barnacle cement polymerization involves proteolytic activation of enzymes and structural precursors, transglutaminase cross-linking, and assembly of fibrous proteins. Peptides and/or epitopes homologous to vertebrate trypsin and transglutaminase were identified in barnacle cement with tandem mass spectrometry and/or western blotting. The peptides generated during proteolytic activation functioned as signal molecules, linking a molecular level event (protein aggregation) to a behavioral response (barnacle larval settlement). The application of evolutionary concepts and a multidisciplinary approach has helped to elucidate barnacle cement polymerization phenomenon: the presence of biochemically similar proteins in the two systems suggests that these processes may be derived from common ancestral elements (Dickinson et al. 2009). Thus, barnacle growth appears to be a specialized form of wound healing. The presence of hemocytes and proteins previously identified in hemolymph (Dreanno et al. 2006) in barnacle cement leads researchers to believe that barnacle hemolymph functions as a cement (Dickinson 2008). In the proposed wound healing model for barnacle cement polymerization, cement is released during growth and repair. The cement contains structural precursors, inactive trypsin, and inactive transglutaminase (contained within hemocytes). Trypsin-like serine proteases activate structural and proteolytic precursors within the cement. Activation of cement structural proteins maximizes bonding interactions, facilitating their assembly and rearrangement with the surface. Covalent cross-linking, brought about by hemocyte-released transglutaminase, reinforces the cement (Fig. 23.3). Reorganization and covalent cross-linking of activated structural proteins result in an insoluble mesh of interwoven fibrous proteins (Fig. 23.4).
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Fig. 23.3 ε-(γ-Glutamyl)lysine cross-link, which results from transglutaminase activity; 5% of Lys residues in barnacle cement were shown to be bound in ε-(γ-glutamyl)lysine cross-links (adapted from Dickinson et al. 2009)
Fig. 23.4 AFM imaging revealed a fibrous ultrastructure of the unpolymerized cement of Amphibalanus amphitrite (adapted from Dickinson et al. 2009)
23.4 Conclusion Biofouling, the undesirable accumulation of microorganisms, algae, and animals on submerged artificial surfaces, causes tremendous technical and economic problems to maritime operation worldwide (Townsin 2003). Governments and industries spend more than US $5.7 billion annually to prevent and control marine biofouling (Sur 2008). It is a worldwide problem in marine systems, costing the US Navy alone an estimated $1 billion per annum (Callow and Callow 2002). Barnacles have elicited considerable scientific attention because of the strength and durability of their adhesive and because of practical concerns initially related to marine fouling. Therefore, the first motivation for understanding the biology of barnacle cement secretion in detail has the goal of finding new techniques to control their settling on man-made marine surfaces. Unfortunately, current methods of control mainly use biocide-doped paints on surfaces, and such biocides leach in significant quantities to cause serious toxicity to the marine environment. The second reason is related to biomimetics and materials science. Undoubtedly, such a natural adhesive as that used in barnacle cements can be utilized for potential medical and engineering applications. Due to its effectiveness in a
References
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saline, wet environment, these natural glues may be promising biodegradable adhesives for various medical procedures including surgery. Thus, a new challenge is to discover uses for the barnacle-produced adhesives in potential medical applications.
References Barlow DE, Dickinson GH, Orihuela B et al (2009) In situ ATR-FTIR characterization of primary cement interfaces of the barnacle Balanus amphitrite. Biofouling 25:359–366 Callow ME, Callow JA (2002) Marine biofouling: a sticky problem. Biologist 49(1):1–5 Dickinson GH (2008) Barnacle cement: a polymerization model based on evolutionary concepts. Ph.D. thesis, Duke University, USA Dickinson GH, Vega IE, Wahl KJ et al (2009) Barnacle cement: a polymerization model based on evolutionary concepts. J Exp Biol 212:3499–3510 Dreanno C, Kirby RR, Clare AS (2006) Locating the barnacle settlement pheromone: spatial and ontogenetic expression of the settlement-inducing protein complex of Balanus amphitrite. Proc Biol Sci 273:2721–2728 Harrison PJH, Sandeman DC (1999) Morphology of the nervous system of the barnacle cypris larva (Balanus amphitrite Darwin) revealed by light and electron microscopy. Biol Bull 197:144–158 Kamino K (2006) Barnacle underwater attachment. In: Smith AM, Callow JA (eds) Biological adhesives. Springer, Berlin Kamino K (2008) Underwater adhesive of marine organisms as the vital link between biological science and material science. Mar Biotechnol 10:111–121 Kamino K, Inoue K, Maruyama T et al (2000) Barnacle cement proteins: importance of disulfide bonds in their insolubility. J Biol Chem 275:27360–27365 Morse DE (1990) Recent progress in larval settlement and metamorphosis: closing the gaps between molecular biology and ecology. Bull Mar Sci 46:465–483 Naldrett MJ (1993) The importance of sulfur cross-links and hydrophobic interactions in the polymerization of barnacle cement. J Mar Biol Assoc UK 73:689–702 Ödling K, Albertsson C, Russell JT et al (2006) An in vivo study of exocytosis of cement proteins from barnacle Balanus improvisus (D.) cyprid larva. J Exp Biol 209:956–964 Okano K, Shimizu K, Satuito CG et al (1996) Visualization of cement gland of the barnacle Megabalanus rosa. J Exp Biol 199:2131–2137 Pawlik JR (1992) Induction of marine invertebrate larvae settlement: evidence for chemical cues. In: Paul VJ (ed) Ecological roles of marine natural products. Cornell University, New York, NY Pitombo FB (2004) Phylogenetic analysis of the Balanidae (Cirripedia, Balanomorpha). Zool Scr 33:261–276 Rittschof D, Branscomb ES, Costlow JD (1984) Settlement and behavior in relation to flow and surface on larval barnacles, Balanus amphitrite Darwin. J Exp Mar Biol Ecol 82:131–146 Ruppert EE, Barnes RD (1994) Invertebrate zoology. Harcourt Brace College, San Diego Satuito CG, Shimizu K, Natoyama K et al (1996) Aged-related settlement success by cyprids of the barnacle Balanus amphitrite Darwin, with special reference to consumption of cyprid storage protein. Mar Biol 127:125–130 Sur UK (2008) Nature’s strongest glue: a potential alternative to commercial super glue. Curr Sci 94:1563–1564 Townsin RL (2003) The ship hull fouling penalty. Biofouling 19:9–15 Waite JH (1987) Natures underwater adhesive specialist. Int J Adhes Adhes 7:9–14 Walker G (1971) A study of the cement apparatus of the cypris larva of the barnacle Balanus balanoides. Mar Biol 9:205–212 Walker G (1972) The biochemical composition of the cement of the two barnacle species, Balanus hameri and Balanus crenatus. J Mar Biol Assoc UK 52:429–435 Walker G, Yule AB (1984) Temporary adhesion of the barnacle cyprid: the existence of an antennular adhesive secretion. J Mar Biol Assoc UK 64:679–686
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Walker G, Yule AB, Nott JA (1987) Structure and function in balanomorph larva. In: Southward AJ (ed) Barnacle biology. AA Balkema, Rotterdam Yamamoto H, Shimizu K, Tachibana A et al (1999) Roles of dopamine and serotonin in larval attachment of the barnacle, Balanus amphitrite. J Exp Zool 284:746–758 Yamamoto H, Tachibana A, Kawaii S et al (1996) Serotonin involvement in larval settlement of the barnacle, Balanus amphitrite. J Exp Zool 275:339–345 Yule AB, Walker G (1984) The temporary adhesion of the barnacle cyprids: effects of some differing surface characteristics. J Mar Biol Assoc UK 64:429–439 Yule AB, Walker G (1985) Settlement of Balanus balanoides: the effect of cyprid antennular secretion. J Mar Biol Assoc UK 65:707–712
Part V
Suction-based Adhesion in Marine Invertebrates
Sucker attachment systems are available commercially and have found broad application in a variety of industries. These technologies suffer from two limitations (Bandyopadhyay et al. 2008). First, their adhesive force depends on the availability of a vacuum generator to produce and maintain useful attachment. This means that they are noisy and not very portable, both of which can be undesirable in some applications. Second, the surface is a cup of fixed shape. This means that attachment quality for a variety of surfaces requires changing to a cup that matches the surface geometry. The marine invertebrates’ sucker systems prove by their existence that a single solution is possible to both these problems. The contact elements use a mushroom-shaped geometry and are completely independent, staying “passively” adhered without any external support such as muscular expenditure. Along with the high pull-off forces that are required to rupture the contact, this makes the mushroom-shaped geometry optimal for a long attachment required for life in wave-swept seashores or time-consuming pairing process. This type of contact element geometry can also be used in synthetic patterned dry adhesives. Recent achievements in adhesive technologies showed that microstructures consisting of mushroom-shaped contact elements produce much more effective attachment than earlier adhesive micropatterns based on flat punch geometry (Gorb and Varenberg 2007). Animals use mechanisms such as suction adhesion, capillary adhesion, and mechanical devices (such as minute toothed plates) to attach to a surface or another animal. Here, I want to discuss some examples of suction adhesion described in the literature. Most species have only one type of so-called sucking tentacles, which usually bear a knob-like broadening at the distal end of the tentacle’s shaft.
References Bandyopadhyay PR, Hrubes JD, Leinhos HA (2008) Biorobotic adhesion in water using suction cups. Bioinsp Biomim 3:016003, pp 11 Gorb SN, Varenberg M (2007) Mushroom-shaped geometry of contact elements in biological adhesive systems. J Adhesion Sci Technol 21:1175–1183
Chapter 24
Suctorian Protozoa
Abstract Marine animals use mechanisms such as suction adhesion, capillary adhesion, and mechanical devices (such as minute toothed plates) to attach to a surface or another animal. Here, some examples of suction adhesion in suctorian protists are described and discussed. No other protozoan derives its nourishment in a manner comparable to the “sucking through a straw” mechanism characteristic of Suctoria. Their consumption of live ciliates, which are their sole food source, is additional evidence of the specialized mechanisms involved. The occurrence of adhesive tentacles in different species of marine invertebrates, from microscopic protists to giant cephalopod molluscs, is well known. They are diverse in their morphology, structure, and functions (Williams 1991). However, one feature, adhesion due to suction mechanisms, is common on nano-, micro-, as well as macrolevels of the structural organization of both unicellular and metazoan invertebrates. In the interests of brevity, I will present only a few examples of the suction phenomenon. These tentacles possess amazing biomimetic potential for nanobionics, as well as for other disciplines, of modern materials science.
24.1 Suctorian Ciliates Some of these creatures are really tiny vampires. They are suctorians, sessile protists, Phylum Ciliophora, Class Kinetofragminophora; some of them live in a rigid house called a lorica, and the main species affix to objects like algae by a non-contractile stalk. These protozoa do not have a cytostome (mouth) to eat their prey but do possess two bundles of straight tentacles – each one terminated by a rounded structure like a disk and probably adhesive. Jean-Marie Cavanihak
Suctorians are a small specialized subclass of the protozoan class Ciliatea whose members were long considered entirely separate from the “true” ciliates. The name of the group stems from suction, a method of feeding through extremely fine, stiff, and long tubes called tentacles (Fig. 24.1). The sole order of this subclass is Suctorida. No other protozoan derives its nourishment in a manner comparable H. Ehrlich, Biological Materials of Marine Origin, Biologically-Inspired Systems 1, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9130-7_24,
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Fig. 24.1 Light microscopy images of different suctorian protists: (a, b) Ephelota sp., (c, d) Acineta sp. (images courtesy Jean-Marie Cavanihac)
to the “sucking through a straw” mechanism characteristic of Suctoria. Their consumption of live ciliates, which are their sole food source, is additional evidence of the specialized mechanisms involved. These protist forms show a number of highly specialized features. Adults are generally found attached to the surface of marine or freshwater invertebrates, or occasionally to inanimate substrates, by means of a non-contractile stalk. Most conspicuous are their tentacles, often numerous, which serve as mouths. These multiple organelles of ingestion fasten to the pellicle of prey organisms, generally passing ciliates. By forces not entirely understood, the tentacles are used to suck out the prey’s protoplasm to provide sustenance for the suctorian. Nearly all species are stalked, and the sedentary, mature forms are devoid of any external ciliature. Young larval forms are produced by both endogenous and exogenous budding. These forms bear locomotor cilia and serve, as in the case of species of the Peritrichia, for dissemination. Investigations on adhesive tentacles of suctorians have a long history. The actual mechanism of food intake using adhesion, widely considered to be a suctorian
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process, has puzzled protozoologists for more than a century (Bütschli 1887; Eismond 1890; Hartog 1901; Hertwig 1876) and is still in progress today. The tentacles of a suctorian are described as consisting of two concentric tubes (Collin 1912), the walls of which unite at the distal end. The wall of the outer tube appears to originate in and to be continuous with some component of the body surface of the suctorian (Noble 1932). The inner tube projects inward from the tentacle for a short distance into the cytoplasm and ends abruptly. During feeding the contents of the prey pass through the inner tube. The elongated shape could not be maintained without some rigidity, and the disintegration of suctorian tentacles under the influence of high hydrostatic pressure (Kitching and Pease 1939) suggests the collapse of a gel structure. According to Kormos (1938), the tentacles emerge through holes in the cuticle of the suctorian. The tentacles terminate, at their distal ends, either in a knob or in an open funnel. Capture of prey is attributed by some authors to the development of a sudden suction. However, according to Kormos (1938) tentacles with open funnels are incapable of capturing live prey; he attributes capture to a stickiness of the knob. There is evidence to suggest that the body of the prey may be subject to some digestive influence while the suctorian is feeding upon it. Iziumov (1947) colored the ciliate Glaucoma with congo red and then offered it to the suctorian Tokophrya infusionum. A change in color, indicating increased acidity, was observed in the prey in the region where the tentacles of the suctorian were attached. He also observed the dissolution of grains of paramylum in a captured Chilomonas. The mechanism of the suction of Suctoria has provoked much speculation. Early work is summarized in Collin’s fine monograph (Collin 1912), which contains full references. Collin rejected the idea that material flows along the tentacles of the suctorian because of the hydrostatic pressure within the body of the prey, but this explanation has more recently been reaffirmed by Pestel (1931). Collin (1912) considered that the pressure required would be too great and believed that the liquid contents of the prey were driven along by peristalsis of the inner tube of the tentacle. Other authors refer to suction but do not explain it. Kahl (1931) rejected suction on the grounds that it would involve an expansion of the surface, which he believed to be impossible. Kitching (1954) gave a very detailed description of the mechanism of feeding in the suctorian Podophrya, as follows. The tentacles of the Podophrya normally remained motionless and extended. When a suitable ciliate collided with the Podophrya, the tentacles which the ciliate happened to touch at the tip adhered to it. Adhesion of tentacles only occurred with certain ciliates—for instance, Paramecium caudatum, P. aurelia, and Colpidium sp., but not Spirostomum, Euplotes, or Vorticella. Within a few minutes, the cilia of the prey in the region where the Podophrya was attached stopped beating. The stoppage spreads from that point outward over the surface of the prey. In some cases the surface of the Podophrya became wrinkled. The prey was broken down locally in the region of attachment, with material from the prey flowing up the tentacles into the Podophrya. As the Podophrya got bigger, with any wrinkles that may have formed smoothed out in the process.
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Wrinkling of the Podophrya is shown by observations and experiments to be due to an expansion or growth of the body surface. This expansion serves to reduce the hydrostatic pressure within the Podophrya, so that feeding may proceed. It is suggested by the author that expansion of the body surface, coupled with a supposed resistance to inward collapse, might provide suction for feeding. Wrinkling of the surface is ascribed to a local collapse, which might occur when uptake of food material from the prey fails to keep pace with the expansion. During food intake the tentacle broadens and in some species is also retracted. The predator ingests a tremendous amount of food, and some species are able to increase their volume up to 10-fold compared with the size of the starved animal (Bardele 1972). Note that these dramatic events, from human point of view, take place at the microlevel. Small suctorians feed on prey that measure 40–60 μm, such as Qvcilidium; large suctorians feed on prey that measure more than 200 μm, such as Spirostomum (Verni and Gualtieri 1997). For example, typical rectilinear knobbed tentacles of Solenopkya nzicraster are 1–1.5 μm in diameter and vary in length from one to five times the diameter of the lorica of the suctorian (average 44.1 μm). Tentacles are deployed both above and lateral to the attached organism, so that their ends may cover a hemispherical area as great as 0.392 mm2 (Hull 1961). If an acceptable food ciliate contacts any of these tentacles, there is immediate adhesion. Thus even the length of the tentacles represents a striking adaptation for foraging by these sessile organisms. Most species have only one type of so-called sucking tentacles, which usually bear a knob-like broadening at the distal end of the tentacle’s shaft. Close light microscopical observation shows that the shaft of the tentacle consists of two tubes, one lying in the other. The wall of the outer tube is continuous with the animal’s pellicle; the inner tube ends deep within the body of the predator. If a suitable food organism touches the knob of the tentacle, it is held fast and has little chance to escape. Usually the attachment of the food organism to the knob of the tentacle leads to the paralysis of the prey. Immediately after the attachment to the prey, a firm connection is established by the anchoring of the tentacle’s knob within the prey (Bardele 1972). At the same time, a stream of small granules up along the periphery of the tentacle is observed, and shortly thereafter the content of the prey begins to flow through the inner tube to the body of the predator. The food capture strategy of suctorians is a parallel process since either several small prey can be captured simultaneously or a single large prey can be captured by several tentacles (Verni and Gualtieri 1997). The ultrastructure of adhesive tentacles of suctorians is well known today because of the excellent works made by Rudzinska (1965, 1967, 1970) as well as Bardele (Bardele 1968, 1970, 1971, 1972) and Bardele and Grell (1967). Furthermore, several works have been related to investigation of the role of actin within tentacles. For example, Hackney et al. (1982) described the action in Discophrya collini as follows. This suctorian protozoan possesses contractile tentacles which are covered by a thick fibrous cortex and which contain a central canal composed of two concentric rings of microtubules. The canal extends from the base of the apical knob down into the cell body, where it is surrounded by a fibrous
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collar of 2.5–6.0 nm filaments. Heavy meromyosin treatment of glycerinated tentacles indicates that the components of the fibrous collar and cortex may be actin like, providing the motive force for tentacle contraction. The tentacles contract in response to a range of stimuli including UV irradiation, mechanical or electrical stimulation, low temperature, high pressure, and ether. There is considerable evidence that cations can also affect the mechanism of suctorian tentacle contraction (Hackney et al. 1982).
Fig. 24.2 Suctorian protozoa. (a) SEM image of a lorica with tentacles in Flectacineta isopodensis sp.n. (b) Scheme of F. isopodensis sp.n. (cv—contractile vacuole, lf—lateral furrow, ma—macronucleus, mi—micronucleus, s—stalk, t—tentacles) (adapted from Fernandez-Leborans et al. 2002). (c) Reconstruction of the microtube array in Acineta tuberosa. The two states show represent the coming back and forth of the microtubule ribbons (number reduced), which is suggested to play the most important part in the ingestion mechanism. The membrane of the knob and the pellicle of the shaft were omitted for the sake of clarity and the white material in the knob was used for support only (adapted from Bardele 1972). (d) Schematic view of the suction mechanism of A. tuberosa according to Bardele and Grell (1967)
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Numerous suctorian species have been described living as epibionts on marine crustaceans (Fernandez-Leborans and Tato-Porto 2000). New marine suctorian species have also been discovered during the last decade. As an example, I will describe Flectacineta isopodensis sp.n. because of specific localization, as well as the microstructure of their adhesive, about 1 μm large, tentacles. FernandezLeborans et al. (2002) described this novel species as follows. These suctorians were small in size (10.3–17.2 μm in length; 8.6–10.8 μm in width), oval- or bell shaped (Fig. 24.2). The body was surrounded completely by a lorica, which showed the rim curved outward. The tentacles were sticking out through the opening of the lorica. Laterally, the lorica presented a prolongation as a furrow, which terminated in an acute angle. This furrow was 3.4–4.3 μm long, with a maximum width of 2–2.2 μm. There were —five to nine capitate tentacles in one apical group. The distal end of the tentacles had a diameter of 0.9–1.1 μm. The macronucleus was located centrally in the body and was 3–4.5 in length and 2.5–3.8 μm in width. Near the macronucleus, laterally on the top, was a micronucleus (0.6–0.9 μm diameter). The contractile vacuole, rounded, about 1.1–1.7 μm diameter, was located in the middle of the body, behind the macronucleus. The stalk was 8.9–25.2 μm in length and its outer surface was covered by thin transverse striations. Inside the stalk, in several specimens, there was a central channel (Fig. 24.2).
24.2 Conclusion I suggest that the next step in better understanding of the unique adhesion mechanisms of the suctorian microscaled tentacles must be on the nanolevel, and more sophisticated structural and bioanalytical methods must be used for these aims.
References Bardele CF (1968) Acineta tuberosa. I. Der Feinbau des adulten Suktors. Arch Protistenk 110: 403–421 Bardele CF (1970) Budding and metamorphosis in Acineta tuberosa. An electron microscopic study on morphogenesis in Suctoria. J Protozool 17:51–70 Bardele CF (1971) Microtubule model systems: cytoplasmic transport in the suctorian tentacle and the centrohelidian axopod. 29th Ann Proc Electron Microscopy Soc Amer. Boston, MA, p. 334–335 Bardele CF (1972) A microtubule model for ingestion and transport in the suctorian tentacle. Z Zelllorsch 126:116–134 Bardele CF, Grell KG (1967) Elektronenmikroskopische Beobachtungen zur Nahrungsaufnahme bei dem Suktor Acineta tuberosa Ehrenberg. Z Zellforsch 80:108–123 Bütschli O (1887–1889) Protozoa. Bronn’s “Klassen und Ordnungen des Thierreichs”. Bd. I. 3 Collin B (1912) Etude monographique sur les Acine’tiens. II. Morphologie, Physiologie, Syste’matique. Arch Zool Exp Gin 51:1–457 Eismond J (1890) Zur Frage fiber den Saugmechanismus bei Suctorien. Zool Anz 13: 721–723
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Fernandez-Leborans G, Hanamura Y, Nagasaki K (2002) A new suctorian, Flectacineta isopodensis (Protozoa: Ciliophora) epibiont on marine isopods from Hokkaido (Northern Japan). Acta Protozool 41:79–84 Fernandez-Leborans G, Tato-Porto ML (2000) A review of the species of protozoan epibionts on crustaceans. I. Suctorian ciliates. Crustaceana 73:1205–1237 Hackney C, Al-Khazzar AR, Butler RD (1982) Tentacle contraction and ultrastructure in Discophrya collini: the response to cations. Protoplasma 112:92–100 Hartog MM (1901) Notes on suctoria. Arch Protistenk 1:372–374 Hertwig R (1876) Über Podophrya gemmipara nebst Bemerkungen zum Bau und zur systematischen Stellung der Acineat. Morph Jb 1:20–82 Hull RW (1961) Studies on suctorian protozoa: the mechanism of prey adherence. J Protozool 8:343–350 Iziumov GJ (1947) The digestive processes in Tokophrya infusionum. Zool J Moscow 36:263–268 (in Russian) Kahl A (1931) Über die verwandtschaftlichen Beziehungen der Suctorien zu den prostomen Infusorien. Arch Protittenk 73:423–481 Kitching JA (1954) On suction in suctoria. Proc Syrup Colston Res Soc 7:197 Kitching JA, Pease DC (1939) The liquefaction of the tentacles of suctorian protozoa at high hydrostatic pressures. J Cell Comp Physiol 14:1–3 Kormos J (1938) A szlvokasok (Suctoria) szivdcsdveinek szerkezete is mukSdese. Allatt Koslem 35:130–153 Noble AE (1932) On Tokophrya lemnarum Stein (Suctoria) with an account of its budding and conjugation. Univ Calif Publ Zool 37:477–520 Pestel B (1931) Beiträge zur Morphologie und Biologie des Dendrocometet paradoxus Stein. Arch Protistenk 75:403–471 Rudzinska MA (1965) The fine structure and function of the tentacle in Tokophrya infusionum. J Cell Biol 25:459–477 Rudzinska MA (1967) Ultrastructures involved in the feeding mechanism of suctoria. Trans NY Acad Sci 29:512–525 Rudzinska MA (1970) The mechanism of food intake in Tokophrya infusionum and ultrastructural changes in food vacuoles during digestion. J Protozool 17:626–641 Verni F, Gualtieri P (1997) Feeding behaviour in ciliated protists. Micron 28:487–504 Williams RB (1991) Acrorhagi, catch tentacles and sweeper tentacles: a synopsis of ‘aggression’ of actiniarian and scleractinian Cnidaria. Hydrobiologia 216/217:539–545
Chapter 25
Trichodina Sucker Disk
Abstract Trichodinids are characterized by a sucker-like disk that enables them to adhere to host surfaces. The disk is reinforced by a band of radially arranged ribs or striae which are attached to a narrow pellicular band about the periphery. The skeletal or denticulate ring is 44–62 μm in outside diameter, 29–46 μm in ring diameter, and lies immediately posterior to the striae of the disk. The ring is composed of a series of articulated elements or denticles ranging in number from 26 to 36 with a mode at 29. Each denticle consists of a conical centrum, a proximal spine or ray, and a distal hook or blade. Marine trichodinids are generally found on the gills of marine fish species. Trichodinids are a diverse group of ecto- and endoparasitic forms, consisting mostly of fish, amphibians, molluscs, and copepods in marine, freshwater, and terrestrial environments (Basson and Van As 1991; Green and Shiel 2000; Fantham 1930; Lom 1958, 1962, 1970; Lom and Laird 1969; Nilsen 1995). Trichodinids are characterized by a sucker-like disk that enables them to adhere to host surfaces (Van As and Basson 1990). According to Lom, marine trichodinids are generally found on the gills of marine fish species (Grupcheva et al. 1989; Lom 1962). The general morphology of trichodinids has been reviewed in the excellent and comparative studies of Fauré-Frémiet (1949), Uzman and Stickney (1954), Lom (1973), and Hausmann and Hausmann (1981a, b).These authors have emphasized and demonstrated the taxonomic importance of the skeletal ring in the problem of specific determination. The sucker disk is supported by an intracellular skeletal ring of interlinking denticles, the number, shape, and dimensions of which are used in trichodinid taxonomy (e.g., Lom 1958). Ultrastructure of adhesive disk is described in detail for several species of trichodinids (e.g. Gaze and Wootten 1999; Kruger et al. 1993, 1995). Here, I present a brief description of this unique structure made by Uzmann and Stickney (1954) for the peritrich Trichodina myicola. This organism is an epizoic commensal which attaches strongly to epithelial surfaces in the oral region of its host marine bivalve Mya arenaria. The greatest concentrations of these peritrichs are found on the outer faces of the palps where numbers may run as high as 100 or more to each of the four palps. Occasional specimens are found on the wall of the visceral body and on the internal face of the pallial muscle. Frequency of infection
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with T. myicola in samples of Mya from endemic areas ranges from 0 to 62%, with the highest percentages and degrees of infection occurring in late spring. The adhesive or basal disk of T. myicola is concave, circular in outline, and varies from 42 to 79 μm in diameter. The disk is reinforced by a band of radially arranged ribs or striae which are attached to a narrow pellicular band about the periphery (Fig. 25.1). The free ends
Fig. 25.1 Trichodina myicola, ventral aspect (a). Semi-diagrammatic reconstruction of entire organism from living and stained material, silver preparations, and sections. Cutaway shows detail of basal disk and associated ciliature in section. The ciliary girdle is depicted with every third membranelle attached; the membranelles are discontinued at the right of center in order to indicate more clear the relative disposition of the three ciliary systems. (b) T. myicola, posterior aspect. Semi-diagrammatic representation of basal disk showing details of denticulate ring, striated band, and border membrane. Ad, adoral cilia; BM, border membrane; CG, ciliary girdle; CV, contractile vacuole; Cy, cytopharings; D, denticle; DR, denticulate ring; FV, food vacuole; IC, inner cilia; Ma, macronucleus; Mi, micronucleus; MC, marginal cilia; My, myoneme; SR, striated band (adapted from Uzman and Stickney 1954)
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are finely tapered and terminate half-way into the center of the disk. A system of radiating myonemes is present in conjunction with the striae; these myonemes extend from the mid-point of the anterior side of the striae to the bases of the membranelles of the ciliary girdle (Fig. 25.1). There are five to nine striae to each element of the skeletal ring, the number varying directly with the size of the disk. The skeletal or denticulate ring is 44–62 μm in outside diameter, 29–46 μm in ring diameter, and lies immediately posterior to the striae of the disk. The ring is composed of a series of articulated elements or denticles ranging in number from 26 to 36 with a mode at 29 (Fig. 25.2). Each denticle consists of a conical centrum,
Fig. 25.2 Trichodina domerguei. (a) Adoral view of denticle ring; (b, c) aboral views of denticle ring; (d) Diagrammatic representation of denticle form apparent in silver stained and SEM preparations. Solid outline represents actual form observed in SEM preparations; solid internal lines define extent of thickened part of denticle. Broken line indicates approximate silver-stained appearance. Hatched area represents the delicate anterior part of blade prone to mechanical damage and imperfect silver impregnation. Left: Trichodina intermedia, right: Paratrichodina incissa (adapted from Gaze and Wootten 1999)
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a proximal spine or ray, and a distal hook or blade. The blades are convex and very thin on one edge and slightly concave on the other; the concave edge is relatively thicker and is further strengthened by a posterior incurved flange. Gaze and Wootten (1999) also described these structures in T. intermedia. The denticle blades were with a curved anterior margin, semi-lunar in shape. The anterior portion of the blade is extremely thin. The posterior part of the blade is considerably thicker, and the anterior margin integral with the central part of the denticle sometimes gives the appearance of a small anterior projection. The size and shape of the central conical part of trichodinid denticles, in addition to the blade, ray, and supporting apophyses, appear to be highly variable between species in which isolated skeletal structures have been observed (Gaze and Wootten 1999; Kruger et al. 1993, 1995; Nilsen 1995). This gives additional species-specific characteristics which can be used in species discrimination. Unfortunately, to my best knowledge there is a dearth of information regarding the chemistry, origin, and material properties of these very complex structures.
25.1 Conclusion Therefore no protype currently exists for the development of human made apparatus with both adhesive and drilling functions. I have no doubts that unique constructs based on these adhesive disks in combination with the denticulate ring, which has been observed in trichodinids, will be a source of inspiration and high biomimetic potential for bionicists, engineers, and materials scientists.
References Basson L, Van As JG (1991) Trichodinids (Ciliophora: Peritrichia) from a calanoid copepod and catfish from South Africa with notes on host specificity. Syst Parasitol 18:147–158 Fantham HB (1930) Some parasitic protozoa found in South Africa. XIII. South African J Sci 27:376–390 Fauré-Frémiet E (1949) Deux espèces commensales des Conochilus. Hydrobiologia 1:126–132 Gaze WH, Wootten R (1999) An SEM study of adhesive disc skeletal structures isolated from trichodinids (Ciliophora: Peritrichida) of the genera Trichodina Ehrenberg, 1838 and Paratrichodina Lom, 1963. Syst Parasitol 43:167–174 Green JD, Shiel RJ (2000) Mobiline peritrich riders on Australian calanoid copepods. Hydrobiologia 437:203–212 Grupcheva G, Lom J, Dykova I. (1989) Trichodinids (Ciliata: Urceolariidae) from gills of some marine fishes with the description of Trichodina zaikai sp.n. Folia Parasitol 36: 193–207 Hausmann K, Hausmann E (1981a) Structural studies on Trichodina pediculus (Ciliophora, Peritricha). I. The locomotor fringe and the oral apparatus. J Ultrastruct Res 74:131–143 Hausmann K, Hausmann E (1981b) Structural studies on Trichodina pediculus (Ciliophora, Peritricha). II. The adhesive disc. J Ultrastruct Res 74:144–155 Kruger J, Basson L, Van As JG (1993) On the ultrastructure of the adhesive disc of Trichodina xenopodus Fantham, 1924 and T. heterodentata Duncan, 1977 (Ciliophora: Peritrichida). Acta Protozoologica 32:245–253
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Kruger J, Van As JG, Basson L (1995) Observations on the adhesive disc of Trichodina xenopodus Fantham, 1924 and T. heterodentata Duncan, 1977 (Ciliophora: Peritrichida) during binary fission. Acta Protozoologica 34:203–209 Lom J (1958) A contribution to the systematics and morphology of endoparasitic trichodinids from amphibians, with a proposal of uniform specific characteristics. J Protozool 5:251–263 Lom J (1962) Trichodinid ciliates from fishes of the Rumanian Black sea coast. Parasitol 52:49–61 Lom J (1970) Trichodinid ciliates (Peritrichida: Urceolariidae) from some marine fishes. Folia Parasitologica 17:113–125 Lom J (1973) The adhesive disc of Trichodinella epizootica – ultrastructure and injury to the host tissue. Folia Parasitologica 20:193–202 Lom J, Laird M (1969) Parasitic protozoa from marine and euryhaline fish of Newfoundland and New Brunswick. Can J Zool 47:1367–1380 Nilsen F (1995) Description of Trichodina hippoglossi n. sp. from farmed Atlantic halibut larvae Hippoglossus hippoglossus. Dis Aquat Org 21:209–214 Uzmann JR, Stickney AP (1954) Trichodina myicola n. sp., a peritrichous ciliate from the marine bivalve Mya arenaria L. J Protozool 1:149–155 Van As JG, Basson L (1990) An articulated internal skeleton resembling a spinal column in a ciliated protozoan. Naturwissenschaften 77:229–231
Chapter 26
Giardia Suction
Abstract Giardia intestinalis is a microbial eukaryotic parasite and is prevalent in marine ecosystems. A wide range of marine hosts capable of harboring zoonotic forms of this parasite exist. The outstanding anatomical feature of Giardia is its large sucking disk that covers almost the entire ventral surface of the organism. The animal uses the ventral disk to attach itself to the intestine wall of its host. The ventral disk, a structure unique to Giardia, is a 9 μm diameter concave spiral of cross-linked microtubules and associated proteins that run across the anterior underside of the cell. This disk is rigid and wedges between microvilli of the host’s intestinal columnar cells during attachment. Giardia intestinalis is a microbial eukaryotic parasite that causes diarrheal disease in humans and other vertebrates worldwide (Fayer et al. 2004). Giardia are shaped like flattened tear drops, 5–7 μm wide, 10- to 12 μm long, and about 1–2 μm thick (Owen 1980) (Fig. 26.1). In general contour, Giardia resemble king (horseshoe) crabs with a hydrodynamically efficient form for forward motion through the liquid small intestinal contents. The dorsal surface is rounded and smooth or pebbled like a peach stone, depending on hydration and subsurface vesicular activity. The ventral surface is concave with a prominent anterior adhesive disk. Giardia lamblia is the most common human intestinal parasite worldwide and Giardia sp. cysts are common in agricultural animals such as cattle, horses, sheep, and pigs (Adam 1991; Wolfe 1992). There are recent reports of G. intestinalis in shellfish, seals, sea lions, and whales (Deng et al. 2000; Gaydos et al. 2008; Measures and Olson 1999; Olson et al. 1997) suggesting that marine animals are also potential reservoirs of human disease. However, the prevalence, genetic diversity, and effect of G. intestinalis in marine environments and the role that marine animals play in transmission of this parasite to humans are relatively unexplored. Using a multi-locus sequencing approach, Lasek-Nesselquist et al. (2008) identified human-infecting G. intestinalis haplotypes of both assemblages A and B in the fecal material of dolphins, porpoises, seals, herring gulls Larus argentatus, common eiders Somateria mollissima, and a thresher shark Alopias vulpinus. These results indicate that G. intestinalis is prevalent in marine ecosystems and a wide range of marine hosts capable of harboring zoonotic forms of this parasite exist. The
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Fig. 26.1 SEM imagery: (a) G. lamblia trophozoites attached to the surface of polysterene chip. (b) Individual G. lamblia trophozoite adhering to surface of pillar arrays observed at higher magnification
presence of G. intestinalis in marine ecosystems raises concerns about how this disease might be transmitted among different host species. The outstanding anatomical feature of Giardia is its large sucking disk that covers almost the entire ventral surface of the organism. The animal uses the ventral disk to attach itself to the intestine wall of its host (Elmendorf et al. 2003). The ventral disk, a structure unique to Giardia, is a 9 μm diameter concave spiral of crosslinked microtubules and associated proteins that run across the anterior underside of the cell. This disk is rigid and wedges between microvilli of the host’s intestinal columnar cells during attachment (Owen 1980). This attachment can cause lesions in the intestine mucosa. The basic structure shared by all Giardia strains appears to be remarkably efficient for survival and proliferation in the intestinal niche under near anaerobic conditions. There have been extensive experimental and electron microscope studies on attachment in G. muris, a species almost morphologically identical to G. lamblia (Friend 1966; Holberton 1973). In the first of these, Friend (1966) introduced an interesting puzzle. He noticed that the cytoskeleton formed such a rigid structure that it was not possible to deform the sucking disk to obtain a vacuum. He suggested that possibly the disk was not responsible for adhesion. Holberton (1973) corroborated Friend s findings on the rigid structure of the cytoskeleton. Additionally, using light microscopy, he observed living trophozoites attached to the microscope slide. He noticed that they became detached when the ventral flagella ceased oscillating. Holberton proposed a mechanism for attachment that depended on the ventral flagella for suction in the ventral disk (Holberton 1974).
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Fig. 26.2 (a) Schematic of Giardia. Displayed are the ventral disk, VD, marginal grooves, MG, portal, P, ventrocaudal groove, VG, and the two flagella, F, located in the ventrocaudal groove. The dorsal flagella are not displayed. The fluid flow is indicated by the arrows (adapted from Jones et al. 1983) (b) SEM image of Giardia sp.
Jones et al. (1983) proposed a schematic of Giardia (Fig. 26.2a). Running laterally along the ventral disk are two tubes (marginal grooves). These tubes come together at the caudal side of the disk and form a single tube (ventrocaudal groove) that emerges caudally. In the region where the marginal grooves meet the ventrocauadal groove, there is an opening (portal) from the grooves into the ventral disk. Emerging from a position caudal to this opening and lying within the ventrocaudal groove are two flagella that seem to beat coherently. In the Holberton scheme (Holberton 1974), the beating flagella draw fluid through the marginal grooves and out through the ventrocaudal groove. The flow induces a pressure drop along the marginal grooves, which lowers the pressure in the ventral disk. Attachment is due to the pressure differential between the interior of the disk and the exterior of the organism. Additionally, when the organism is moving away from the wall, there is another mechanism that tends to keep the organism attached. For physical dimensions on the order of a few micrometers, fluids, such as water, are very viscous. This viscosity produces a force on the organism that limits the speed of detachment. If this speed is very small, then the organism can remain in the vicinity of the wall for long periods of time. Jones et al. (1983) calculated the force of adhesion in Giardia using a low Reynolds number hydrodynamic model. The adhesive force on Giardia was a few tenths of a microdyne. These authors compared the adhesive force with typical forces that would tend to detach the organism. They found that the detachment forces are not strong enough to overcome the force of adhesion. More recent mechanisms of attachment to hard surfaces have been reviewed by Hansen et al. (2006) and can be grouped into two categories: (i) binding and (ii) suction mechanisms.
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Binding models require close contact between a portion of the cell (such as the ventral disk or the cell periphery) and the substrate in order to generate attachment force via molecular-level interactions. Possible binding-based mechanisms include non-specific interactions (electrostatic and van der Waals), hydrophobic interactions, or ligand-mediated specific binding. In contrast, suction-based mechanisms are those requiring a pressure differential under the ventral disk for generation of attachment force. Erlandsen et al. (2003) proposed that the ventral adhesive disk has contractile proteins arranged around its circumference, and it has been suggested that contraction of this area might function like a purse-string suture. The resemblance of the ventral adhesive disk to a suction cup has led to the hypothesis that suction or a negative pressure produced under the disk by a grasping action might produce the adhesive force regulating attachment. Hansen et al. (2006) developed a centrifugation technique to measure the force of attachment of Giardia cell populations on plain glass and chemically modified substrates. The use of this simplified system allows separation of physical attachment from molecular interactions with host cells, enabling greater control of substrate surface properties, ease of modeling, and comparison to other eukaryotic cell-glass adhesion results. Additionally, centrifugation offers the advantage of being able to apply uniform normal forces to large populations of cells. The authors have presented detachment force measurements based on centrifugation indicating that Giardia can resist detachment forces of 1.5 nN on plain glass. Their results challenge the idea that specific or non-specific binding is essential for generating attachment force and are consistent with suction-based models, in which the cell would generate attachment force by lowering the pressure underneath its ventral disk.
26.1 Conclusion A suction-based attachment mechanism may explain how Giardia cells are able to attach to a wide variety of substrates in vitro and infect a diverse population of vertebrate hosts, including in some cases transmission between different host species. Additionally, use of suction by a single-celled organism would represent a unique mechanism of cellular attachment to surfaces.
References Adam RD (1991) The biology of Giardia spp. Microbiol Rev 55:706–732 Deng M, Peterson RP, Cliver DO (2000) First findings of Cryptosporidium and Giardia in California Sea Lions (Zalophus californianus). J Parasitol 86:490–494 Elmendorf H, Dawson SC, McCaffery JM (2003) The cytoskeleton of Giardia lamblia. Int J Parasitol 33:3–28 Erlandsen SL, Russo AP, Turner JN (2003) New adhesion mechanism in Giardia: role of the ventrolateral flange in the attachment of trophozoites. Microsc Microanal 9(Suppl 2):230–231 Fayer R, Dubey JP, Lindsay DS (2004) Zoonotic protozoa: from land to sea. Trends Parasitol 20:531–536
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Friend DS (1966) The fine structure of Giardia muris. J Cell Biol 29:317–332 Gaydos JK, Miller WA, Johnson C et al (2008) Novel and canine genotypes of Giardia duodenalis in harbor seals (Phoca vitulina richardsi). J Parasitol 94:1264–1268 Hansen WR, Tulyathan O, Dawson SC et al (2006) Giardia lamblia attachment force is insensitive to surface treatments. Eukaryotic Cell 5:781–783 Holberton DV (1973) Fine structure of the ventral disk apparatus and the mechanism of attachment in the flagellate Giardia muris. J Cell Sci 13:11–14 Holberton DV (1974) Attachment of Giardia – a hydrodynamic model based on flagellar activity. J Exp Biol 60:207–221 Jones RD, Lemanski CL, Jones TJ (1983) Theory of attachment in Giardia. Biophys J 44:185–190 Lasek-Nesselquist E, Bogomolni AL, Gast RJ et al (2008) Molecular characterization of Giardia intestinalis haplotypes in marine animals: variation and zoonotic potential. Dis Aquat Organ 81(1):39–51 Measures LN, Olson M (1999) Giardiasis in pinnipeds from Eastern Canada. J Wildlife Dis 35:779–782 Olson ME, Roach PD, Stabler M et al (1997) Giardiasis in ringed seals from the Western Arctic. J Wildlife Dis 33:646–648 Owen RL (1980) The ultrastructural basis of Giardia function. Trans Roy Soc Trop Med Hyg 74:429–433 Wolfe MS (1992) Giardiasis. Clin Microbiol Rev 5(1):93–100
Chapter 27
Suction in Molluscs
Abstract Molluscs, like limpets or cephalopods, possess markedly different mechanisms of suction as well as sucker types. Some of them are described here. Most cephalopods can be categorized either as decapods (cuttlefish and squid, orders Sepioidea and Teuthoidea) or octopods (order Octopoda). Decapods have stalked suckers (or sometimes just hooks), while octopods have unstalked, sessile suckers. Stalked suckers consist of a rigid cylinder, a muscular piston that fits into this cylinder, and a thin, tough stalk that connects the piston to the arm or tentacle club. Proteinaceous sucker rings of Dosidicus gigas exhibit a unique set of characteristics not reported previously for any other biological structural material. They consist of a nanoscale network of parallel tubular elements that are presumably almost entirely stabilized by hydrogen bonding and hydrophobic interactions. It is remarkable that the sucker rings do not contain chitin, which is common in molluscs, and is an integral part of the pen and beak of squids. Sucker systems of cephalopods determine future progress in development of biorobotics.
27.1 Limpets Limpets use suction when they are under water; when they are high and dry, they employ sticky mucus (Cook et al. 1969). Hydrodynamics, shell shape, adhesion behavior, and survivorship in limpets have been investigated in detail (Denny 1988, 1989, 2000; Denny and Blanchette 2000; Smith 1991b, 1992). Smith (1991b) performed an experiment that positively identified suction as an adhesion mechanism in limpets. In the process of this experiment, he developed an apparatus that was able to measure the pressure under the foot of the limpet in comparison with a vertically applied force. Smith (1991b) showed that the force with which limpets adhere to the substratum is often greater than the force applied to the shell during detachment. The findings of Smith (1991b) and the results reported by Ellem et al. (2002) using Cellana tramoserica support the idea that limpets cannot be approximated by the simple mechanical analogue of a suction cap. A suction cap is a passive device in which the pressure drop beneath the cap is a direct result of the detachment force H. Ehrlich, Biological Materials of Marine Origin, Biologically-Inspired Systems 1, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9130-7_27,
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Fig. 27.1 A force diagram describing the forces at work during wave action. The model assumes that the limpet clamps its shell against the substratum in order to generate friction to resist horizontal shear (adapted from Ellem et al. 2002)
placed on the cap. The force of adherence in this instance is therefore equal to the applied force. The individuals of C. tramoserica observed in experiment used the muscular tissue of the foot actively to develop pedal adhesion in excess of the force applied to them (Ellem et al. 2002). This process of active adhesion is a conceptual departure from previous adhesion models that treat limpets as mechanical devices and, as such, exclude consideration of muscular activity and associated metabolic costs (Denny 2000). Smith (1992) found that limpets use suction while foraging at high tide. This is a problem for limpets because, although suction provides good resistance to hydrodynamic lift, it provides poor resistance to shear forces. Elem et al. (2002) proposed that limpets clamp their shell against the substratum to generate a frictional force that resists horizontal shear. Shell clamping against the substratum uses the vertical adherence strength of the suction mechanism to create a frictional force that resists the shear force of hydrodynamic drag. Figure 27.1 outlines the forces exerted on the limpet while exposed to fluid flow. The results obtained by Ellem et al. (2002) show that C. tramoserica clamps its shell in a closely regulated manner consistent with an active role in the limpet adhesion mechanism. Limpets clamped sharply for several seconds in response to single disturbances such as tapping the shell. In response to more continuous disturbance simulating a concerted predator attack, limpets clamped tightly for several minutes. In response to lifting forces applied to the shell, limpets clamped at a set proportion of the lifting force, even if the lift force was a highly dynamic wave profile.
27.2 Cephalopods Molluscs, like cephalopods, possess two markedly different sucker types (Smith 1996). Most coleoid cephalopods can be categorized either as decapods (cuttlefish and squid, orders Sepioidea and Teuthoidea) or octopods (order Octopoda).
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Cephalopods
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Decapods have stalked suckers (or sometimes just hooks), while octopods have unstalked, sessile suckers (Nixon and Dilly 1977). Stalked suckers consist of a rigid cylinder, a muscular piston that fits into this cylinder, and a thin, tough stalk that connects the piston to the arm or tentacle club. Pulling on the stalk pulls the piston back against the resistance of the enclosed water. This reduces the pressure of the water. The harder the stalk is pulled, the greater the pressure differential, and the harder the sucker holds on (Naef 1921–1923). This can continue until the stalk tears. Sessile suckers, in contrast, are flexible, muscular cups. The musculature of the wall of the cup generates an expansive force that decreases the pressure of the enclosed water (Kier and Smith 1990). They are connected to the arm by a wide, fleshy base. The muscles attaching the cup to the arm insert near the rim rather than the base of the cup. Therefore, pulling the sucker away from the arm will not augment the pressure differential (Kier and Smith 1990). Only the musculature of the cup contributes to the pressure differential. These differences in sucker anatomy suggest that stalked suckers may be capable of producing larger pressure differentials than sessile suckers. The octopus is a famous subject for inspiration, probably because the strongest suckers belong to the fast-swimming, open-water species in the decapod suborder Oegopsida (Smith 1996). Their arms house 200–300 independently controlled suckers that can alternately afford an octopus fine manipulation of small objects and produce highadhesion forces on virtually any non-porous surface (Hanlon and Messenger 1996). Octopuses use their suckers to grasp, rotate, and reposition soft objects (e.g., octopus eggs) without damaging them and to provide strong, reversible adhesion forces to anchor the octopus to hard substrates (e.g., rock) during wave surge. The structure and adhesive mechanism of octopus suckers has been widely investigated (Kier and Smith 1990, 2002a, b; Parker 2005). Many of the previous results motivated further investigations with respect to development of different biorobotic adhesion devices (Bandyopadhyay et al. 2008; Sumbre et al. 2001, 2006). The biological “design” of the sucker system in cephalopods is understood to be divided anatomically into three functional groups: the infundibulum that produces a surface seal that conforms to arbitrary surface geometry; the acetabulum that generates negative pressures for adhesion; and the extrinsic muscles that allow adhered surfaces to be rotated relative to the arm (Grasso and Setlur 2007). The effector underlying these abilities is the muscular hydrostat. Guided by sensory input, the thousands of muscle fibers within the muscular hydrostats of the sucker act in coordination to provide stiffness or force when and where needed. Kier and Smith’s (2002a, b) scanning electron micrography of suckers of Octopus bimaculoides shows that the circular contact lip has a periodic small-scale structure. The contact lip has concentric layers that are composed of radial grooves (5 per mm), ridges, and a rim of loose material. Such design should allow the lip to expand and contract azimuthally without losing any contact, thereby maintaining a leak-proof seal when subjected to unsteady release forces. Octopus suckers are unique manipulation devices and may be the most structurally complex organ to which cephalopod muscular hydrostats have been applied. Octopus suckers are radially symmetric structures suspended from the oral surface
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of the octopus arm that incompletely enclose a volume of ambient fluid; seawater for an octopus in the ocean, or air for an out-of-water octopus. It is the ambient fluid medium that the sucker acts upon to reversibly attach an object to the octopus arm or the octopus arm to a surface. The mechanisms by which they facilitate grasping in octopus have been inferred from anatomy (Kier and Smith 2002a, b) and their force generation capabilities have been studied in the laboratory (Smith 1996). Octopus suckers are reported to produce substantially lower negative pressures on wettable surfaces in marine water than the well-known 0 MPa limit in pure water (Smith 1991a, 1996). This finding has led to a focus on suckers’ ability to produce vacuum and wettability as the important mechanisms that allow octopi to achieve their attachment prowess. Crum (1979) has reported that decreasing surface tension decreases the cavitation threshold of a liquid. In the laboratory, cephalopod suckers produce pressure differentials of 100–200 kPa at sea level. As depth increases, greater pressure differential needs to be created for cavitation onset and this is why greater pressure differentials have been reported below sea level (100–1000 m), such as 250–300 kPa with suckers that are >7.5 mm2 and up to 800 kPa with suckers of size <7.5 mm2 (Smith 1996). It is not clear how scientists can build and hold such suction prowess with low penalty and the mechanism is under investigation. Recently, investigations on the unusual sucker system of Humboldt squid (Dosidicus gigas) have been carried out in the groups of David Kisailus and Henrik Birkedal. The species used in their study is a large, aggressive, and predatory species commonly encountered throughout the Eastern Pacific. They can reach lengths of nearly 2 m and a mass up to 50 kg (Miserez et al. 2009). In this species, each of the eight arms and two tentacles is lined with suckers that contain an interior rigid ring equipped with formidable triangular teeth (Fig. 27.2). The presence of the sucker rings increases the functionality of the suckers, most likely by increasing the shear forces (created by struggling prey) that are required to break the seal created by the infundibulum of the sucker. As the circular muscles of each sucker contract, the
Fig. 27.2 SEM image of the proteinaceous sucker rings of Loligo sp. (adapted from Miserez et al. 2009)
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Cephalopods
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teeth are bent inward and subsequently penetrate the skin or scales of strong fast prey, such as fishes. The sucker-ring teeth are very sharp and even moderately sized D. gigas (1 m total length) can easily lacerate a human arm. Each sucker ring consists of a basal ring and a series of dentitions (Fig. 27.2). Scanning electron microscopy (SEM) images of fracture surfaces reveal that they are composed of a series of parallel tubular elements (Fig. 27.3). The channels initially have an average orientation parallel to the dentitions and then spread out through the basal ring. Closer examination of the channel organization reveals that the pore fraction across a given tooth is not uniform and that there is a gradual decrease in pore fraction from the tooth core to the periphery. The parallel channel-like ultrastructural organization of the sucker rings has a direct effect on their mechanical properties, which were investigated via nanoindentation (Miserez et al. 2009). Peak modulus and hardness values near the tooth periphery are approximately 7–7.5 and 0.7GPa, respectively, while in the tooth core they reach minimum values of 4.5–5 and 0.4 GPa, respectively, that is, an Ep /Ee ratio of 0.65. The orientation of the channels that run parallel to the long axis of the teeth probably increase their bending stiffness, a feature that is most important where they are likely to be subjected to large bending or shear forces as the suckers themselves are soft actuators that flex in many directions. An additional advantage of the porous architecture is that it reduces the probability of catastrophic structural failure by introducing a potential crack-arresting mechanism at the boundaries between the two constituent materials (in this case, protein and seawater). Complete amino acid compositional analysis of the sucker rings reveal a high Gly (37 mol%), Tyr (14 mol%), and His (13 mol%) content. This distinctive amino acid composition is a notable feature, given the recurring presence of Gly- and Hisrich proteins in load-bearing and impact-resistant tissues (Broomell et al. 2007). In such structures, the imidazole side chain has been shown to be particularly versatile in its ability to couple with various chemical entities. This can be in the form of
Fig. 27.3 Ultrastructural features of Dosidicus gigas sucker rings. The SEM image reveal the highly parallel nature of the fused tubular elements within a tooth tip (image courtesy James Weaver)
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coordination bonds with transition metals (as in the jaws of Nereis, a predatory marine worm) (Birkedal et al. 2006; Broomell et al. 2006; Lichtenegger et al. 2003) or as covalent cross-links with peptidyl dihydroxy phenylalanine (DOPA) in squid beaks (Miserez et al. 2008). Although it is tempting to attribute the mechanical robustness of the sucker rings to the high His content, none of the aforementioned components were detected; metal ions, mineral, DOPA, and melanin are all absent in the sucker rings. Significantly, the rigidity of the sucker rings decreases with increasing water temperature, becoming noticeably pliable at temperatures as low as 10◦ C over ambient, suggesting that structural stabilization is predominantly through hydrogen bonding (Miserez et al. 2009). This is further supported by the observations that the sucker rings completely dissolve in concentrated formic acid, a known disrupter of hydrogen-bonding interactions, and that the heat-induced pliability is a reversible phenomenon. The authors suggested that His residues take part in forming a stable hydrogen-bonded network. The large quantities of Tyr could be related to sclerotization, which would also help explain the brown coloration in the sucker rings. It is likely that Tyr and possibly also His are partially chlorinated, as has been observed in Nereis worm jaws (Birkedal et al. 2006). Another remarkable feature of the amino acid composition of these squid rings is the large abundance of hydrophobic amino acids other than Tyr (25.5% total for Leu, Ala, Val, Phe, Ile, Met). Hence, hydrophobic interactions can be expected to play an additional stabilizing role for the sucker rings.
27.3 Conclusion As revealed from the studies by David Kisailus and Henrik Birkedal, the chlorinated, wholly proteinaceous sucker rings of D. gigas exhibit a unique set of characteristics not reported previously for any other biological structural material. They consist of a nanoscale network of parallel tubular elements that are presumably almost entirely stabilized by hydrogen bonding and hydrophobic interactions. It is remarkable that the sucker rings do not contain chitin, which is common in molluscs and is an integral part of the pen and beak of squids. The mechanical properties can be understood from the porous nature of the ring material, suggesting that microstructural characteristics rather than biochemical or elemental gradients are primarily responsible for the localized differences in mechanical response. Sucker systems of cephalopods determine future progress in development of biorobotics. Recently, Grasso and Setlur (2007) developed a dynamic simulator (ABSAMS) that models the general functioning of muscular hydrostat systems built from assemblies of biologically constrained muscular hydrostat models. They reported on simulation studies of octopus-inspired and artificial suckers implemented in this system. These simulations reproduce aspects of octopus sucker performance and squid tentacle extension. Simulations run with these models using parameters from man-made actuators and materials can serve as tools for designing soft robotic implementations of man-made artificial suckers and soft manipulators.
References
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References Bandyopadhyay PR, Hrubes JD, Leinhos HA (2008) Biorobotic adhesion in water using suction cups. Bioinsp Biomim 3:016003, pp 11 Birkedal H, Khan RK, Slack N et al (2006) Halogenated veneers: protein cross-linking and halogenation in the jaws of Nereis, a marine polychaete worm. ChemBioChem 7:1392–1399 Broomell C, Mattoni MA, Zok FW et al (2006) Critical role of zinc in hardening of Nereis jaws. J Exp Biol 209:3219–3225 Broomell CC, Khan RK, Moses DN et al (2007) Mineral minimization in nature’s alternative teeth. J R Soc Interface 4:19–31 Cook A, Bamford O, Freeman J et al (1969) A study of the homing habit of the limpet. Anim Behav 17:330–339 Crum LA (1979) Tensile strength of water. Nature 278:148–149 Denny M (1988) Biology and the mechanics of the wave-swept environment. University Press, Princeton, NJ Denny M (1989) A limpet shell shape that reduces drag: laboratory demonstration of a hydrodynamic mechanism and an exploration of its effectiveness in nature. Can J Zool 67:2098–2106 Denny MW (2000) Limits to optimization: fluid dynamics, adhesive strength and the evolution of shape in limpet shells. J Exp Biol 203:2603–2622 Denny MW, Blanchette CA (2000) Hydrodynamics, shell shape, behavior and survivorship in the owl limpet Lottia gigantea. J Exp Biol 203:2623–2639 Ellem GK, Furst JE, Zimmerman KD (2002) Shell clamping behaviour in the limpet Cellana tramoserica. The J Exp Biol 205:539–547 Grasso FW, Setlur P (2007) Inspiration, simulation and design for smart robot manipulators from the sucker actuation mechanism of cephalopods Bioinsp. Biomim 2:S170–S181 Hanlon RT, Messenger JB (1996) Cephalopod behaviour. University Press, Cambridge Kier WM, Smith AM (1990) The morphology and mechanics of octopus suckers. Biol Bull Mar Biol Lab, Woods Hole 178:126–136 Kier WM, Smith AM (2002a) Symposium on the biomechanics of adhesion. Society for Integrative and Comparative Biology, Anaheim, CA Kier WM, Smith AM (2002b) The structure and adhesive mechanism of octopus suckers. Integr Comp Biol 42:1146–1153 Lichtenegger HC, Schöberl T, Ruokolainen JT et al (2003) Zinc and mechanical prowess in the jaws of Nereis, a marine worm. Proc Natl Acad Sci USA 100:9144–9149 Miserez A, Schneberk T, Sun C et al (2008) The transition from stiff to compliant materials in squid beaks. Science 319:1816–1819 Miserez A, Weaver JC, Pedersen PB et al (2009) Microstructural and biochemical characterization of the nanoporous sucker rings from Dosidicus gigas. Adv Mater 21:401–406 Naef A (1921–1923) Cephalopoda. In Fauna and Flora of the Bay of Naples, no 35, pp 1–917, Jerusalem: Israel Program for Scientific Translation Nixon M, Dilly PN (1977) Sucker surfaces and prey capture. Symp Zool Soc, London 38:447–511 Parker GH (2005) The power of adhesion in the suckers of Octopus bimaculatus Verrill. J Exp Zool 33:390–394 Smith AM (1991a) Negative pressure generated by octopus suckers: a study of the tensile strength of water in nature. J Exp Biol 157:257–271 Smith AM (1991b) The role of suction in the adhesion of limpets. J Exp Biol 161:151–169 Smith AM (1992) Alternation between attachment mechanisms by limpets in the field. J Exp Mar Biol Ecol 160:205–220 Smith AM (1996) Cephalopod sucker design and the physical limits to negative pressure. J Exp Biol 199:949–958 Sumbre G, Fiorito G, Flash T et al (2006) Octopuses use a human-like strategy to control precise point-to-point arm movements. Curr Biol 16:767–772 Sumbre G, Gutfreund Y, Fiorito G et al (2001) Control of octopus arm extension by a peripheral motor program Science 293:1845–1848
Chapter 28
Halogenated Biocomposites
Abstract Metal–halogen biological materials appear to be part of a distinctly different system that is widely found among small organisms. In contrast to biomineral-based natural materials, very little is known about the form and function of these biological materials or their role in the behavior, ecology, and evolution of marine invertebrates. It is suggested that interaction between halogens and proteins as well as chitin is one of the key reactions which have determined development of naturally occurring halogen-based biological materials in marine organisms. Polychaetes jaws as well as crustaceans alternative cuticles are described and discussed here as examples of halogenated biocomposites. The proportion of mineral and organic phases in biological formations is an evolutionarily adjustable continuum. Nature undoubtedly possesses examples for every step of this continuum, between all-mineral and all-organic. Recently, Broomell et al. (2007) described the biochemical, structural, and mechanical characteristics of three impact structures: the jaws from two marine polychaetes (Glycera dibranchiata and Nereis species), and the beak of the jumbo squid (Dosidicus gigas). Of these, only the Glycera jaws contain a small amount of mineral. The Nereis jaws contain Zn ions but no mineral, and the Dosidicus beak has no detectable inorganic content (Broomell et al. 2007). Highly mineralized (greater than 50%) impact structures, particularly teeth, have been extensively studied in structural, chemical, mechanical, and clinical detail. However, much less is known about the more diverse non- or slightly mineralized structures. Indeed, given the widely held view that mechanical robustness is imparted by the presence of hard minerals, non-mineralized structures have typically been dismissed—at least as bio-inspired design paradigms for hard, abrasion-resistant materials. In nature, tiny amounts of inorganic impurities, such as metals, are incorporated in the protein structures of some biomaterials and lead to unusual mechanical properties of those materials (Lee et al. 2009). The jaws, tarsal claws, stings, and other “tools” of a large fraction of arthropods, some worms, and members of other phyla contain extraordinary amounts of heavy metals (e.g., zinc, manganese, copper) and halogens (bromine, chlorine). Furthermore, the concentration of the metal or
H. Ehrlich, Biological Materials of Marine Origin, Biologically-Inspired Systems 1, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9130-7_28,
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halogen is usually lower than that of calcium in calcified cuticle (Schofield et al. 2009). Although the measured concentrations reach 25% of dry mass, the tissue is not filled with a biomineral, like calcified tissues are. Instead, metal–halogen biological materials appear to be part of a distinctly different system that is widely found among small organisms (Schofield 2005). In contrast to biomineral-based natural materials, very little is known about the form and function of these biological materials or their role in the behavior, ecology, and evolution of invertebrates. When metals met halogens and when metal–halogene complexes and biocomposites arose remains unclear. However, hypothesises about the role of halogens in ancient unicellular organisms exist in the literature. Cyanobacteria, are the most primitive of the oxygenic photosynthetic organisms and are the ancestors of multicellular eukaryotic algae that contain the highest amount of iodine. More than 3 billion years ago, Cyanobacteria were the first living cells to produce oxygen, which was toxic at that time, in the terrestrial atmosphere (Venturi et al. 2000). So, algal cells required a protective antioxidant action in which iodides might have had this specific role. In fact, iodides are present and readily available in seawater, where algal phytoplankton acts as a biological accumulator of iodides. As of approximately 700 million years ago, thyroxine (T4 ) has been present in fibrous exoskeletal scleroproteins of the lowest marine invertebrates like Porifera and Anthozoa, without showing any hormonal action. However, thyroxine-induced metamorphosis in Aurelia jellyfish. This organism has gastrodermal and epidermal gland cells, but no thyroid gland (Spangenberg 1967). During strobilation jellyfish polyps (strobilae) become segmented and metamorphose to give rise to several young medusae (ephyrae) in sequential order. Spangenberg (1967) discovered that the metamorphic process can be initiated by iodide and thyroxine, especially when the organisms were preconditioned by low temperatures. When some primitive marine vertebrates started to emerge from the iodine-rich sea and transferred to iodine-deficient freshwater, and finally land, their diet became iodine deficient and also harbored vegetable iodine-competitors such as nitrates, nitrites, thiocyanates, and some glycosides (Venturi et al. 2000). In water, the iodine concentration decreases step by step, from seawater (about 50–60 μg/L) to estuary and source of rivers (less than 0.26 μg/L). Hence, these animals needed an efficient thyroid gland as reservoir of iodine. Therefore, it was suggested (Venturi et al. 2000) that during progressive slow adaptation to terrestrial life, the first chordates started to use the primitive, but not antagonized, T4 in order to transport antioxidant iodide into the cells. So, the remaining triiodothyronine, the real active hormone that stimulates cellular oxidations, became active in the metamorphosis and thermogenesis for a better adaptation of the organisms to terrestrial environment (freshwater, atmosphere, gravity, temperature, and diet). Gorbman and co-workers (1954) investigated utilization of radioiodine by several marine invertebrates like worms (annelida) and molluscs. They found that most of the invertebrates studied took up iodide in the hard, horny, or fibrous protein structures such as the skeleton of sponges and corals; the exoskeleton of arthropods; the shell and periostracum of molluscs; and the teeth, setae, and cuticle of annelids.
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I suggest that interaction between halogens and proteins as well as chitin is one of the key reactions which have determined development of naturally occurring halogen-based biological materials in marine invertebrates. Why were halogen bonds successful in biological macromolecules during evolution? Auffinger et al. (2004) proposed a very plausible explanation, which I will summarize here. As an example, they analyzed the situation with thyroid hormones. These represent a class of naturally iodinated molecules for which halogen bonds appear to play a role in their recognition, as evident by the short I–O contacts between tetraiodothyroxine and its transport protein transthyretin. These short contacts, originally called charge transfer bonds, were attributed to the transfer of negative charge from an oxygen, nitrogen, or sulfur (a Lewis base) to a polarizable halogen (a Lewis acid). They are now referred to as halogen bonds (Fig. 28.1) by analogy to classical hydrogen bonds with which they share numerous properties and are currently being exploited to control the crystallization of organic compounds in the design of new materials as well as in supramolecular chemistry. Extensive surveys of structures in the Cambridge Structural Database coupled with ab initio calculations have characterized the geometry of halogen bonds in small molecules and show that the interaction is primarily electrostatic, with contributions from polarization, dispersion, and charge transfer. The stabilizing potential of halogen bonds is estimated to range from about half to slightly greater than that of an average hydrogen. The present survey of protein and nucleic acid structures reveals similar halogen bonds as potentially stabilizing inter- and intramolecular interactions that can affect ligand binding and molecular folding. A halogen bond in biomolecules can be defined as a short C–X···O–Y interaction (C–X is a carbon-bonded chlorine, bromine, or iodine, and O–Y is a carbonyl, hydroxyl, charged carboxylate or phosphate group), where the X···O distance is less than or equal to the sums of the respective van der Waals radii (3.27 Å for Cl···O, 3.37 Å for Br···O, and 3.50 Å for I···O) and can conform to the geometry seen in small molecules, with the C–X···O angle ca.165◦ (consistent with a strong directional polarization of the halogen) and the X···O–Y angle ca.120◦ . Alternative geometries can be imposed by the more
Fig. 28.1 Schematic of short halogen (X) interactions to various oxygen-containing functional groups (where O–Y can be a carbonyl, hydroxyl, or carboxylate when Y is a carbon; a phosphate when Y is a phosphorus; or a sulfate when Y is a sulfur). The geometry of the interaction is defined by the normalized RX·O distance [RX·O = dX·O /RvdW(X·O) ], the 1 angle of the oxygen relative to the C–X bond, and the 2 angle of the halogen relative to the O–Y bond (adapted from Auffinger et al. 2004)
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complex environment found in biomolecules, depending on which of the two types of donor systems are involved in the interaction (Auffinger et al. 2004): (i) the lone pair electrons of oxygen (and, to a lesser extent, nitrogen and sulfur) atoms or (ii) the delocalized π-electrons of peptide bonds or carboxylate or amide groups. Thus, the specific geometry and diversity of the interacting partners of halogen bonds explain the diversity of halogen-based biocomposites in nature, as well as offering new and versatile tools for the design of ligands as drugs and materials in nanotechnology. Hard, but non-mineralized, marine biological materials are peculiarly rich in halogens, which are associated with a variety of post-translationally modified amino acids, many of which are multiply halogenated by chlorine, bromine, and/or iodine (Birkedal et al. 2006). Several of these modified amino acids, namely dibromohistidine, bromoiodohistidine, chloroiodotyrosine, bromoiodotyrosine, chlorodityrosine, chlorotrityrosine, chlorobromotrityrosine, and bromoiodotrityrosine, have been recently reported in numerous publications by Henrik Birkedal and Herbert Waite (see references to this chapter). Researches from their groups also made a valuable contribution to the topics related with metal–halogen-based biocomposites (Broomell et al. 2006, 2007; Khan et al. 2006; Lichtenegger et al. 2002, 2003). Several milestone papers were published also by Robert Schofield and coworkers (Schofield 1990, 2001, 2005; Schofield et al. 2002, 2003) especially with respect to “heavy-element biomaterials” (an intriguing term proposed by him!) in marine crustaceans. I decided to discuss here only two main examples of these particular biological materials, which have been found in polychaetes and crustaceans.
28.1 Polychaetes Jaws Polychaetes (Annelida) are among the most abundant metazoans in modern marine environments. They display a wide variety of modes of life, from vagrant benthos through sessile, pelagic, and parasitic forms, and are among the most species-rich groups in nearly all marine environments, from bathyal and abyssal through sublittoral environments, including harbors and coral reefs (Eriksson and Elfman 2000). Many representatives of the orders Phyllodocida and Eunicida are equipped with powerful jaws, which are used for feeding and digging. In contrast to the soft parts of the worm body, the jaws generally have very good fossilization potential. Their fossil record dates back to the early Cambrian (ca. 540 Ma), possibly even further. Jawed polychaete worms appeared in the early Ordovician (ca. 480 Ma) and preserved jaws (scolecodonts) are abundant in many Paleozoic rocks (Bergman 1979). Here, I want to note that these exceptionally well-preserved fossilized jaws additionally confirms certain unique material properties of these structures with respect to thermal metamorphism, as well as the corresponding high pressure that occurs during diagenetic processes.
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Polychaete jaws are normally 0.1±1.5 mm, brownish or reddish to black in color, hollow, and vary in thickness, with a denticulated dorsal side (Elfman et al. 1999). In some cases, jaws function as a part of the venomous apparatus. Thus, the venomous apparatus of the polychaetous annelid, Glycera convoluta Keferstein, is situated at the mouth of the everted proboscis. It is composed of four very hard jaws (Michel et al. 1973), with each of them having running through its length the excreter canal of the associated venomous gland. With this apparatus the annelid can bite small interstitial crustaceans, which are then paralyzed by the venom prior to ingestion. However, the North American Pacific bloodworm, G. dibranchiata Ehlers, whose length reaches up to 37 cm, can inflict on a human a bite as painful as the sting of a bee (Klawe and Dickie 1957). It is obvious that both recent polychaetes jaws and scolecodonts, apart from their suggested protein composition, also contain various inorganic components (Elfman et al. 1999). The chemistry of the recent specimens differs from that of the fossil specimens (Fig. 28.2). Scolecodonts contain silicon, sulfur, chlorine, potassium, calcium, iron, copper, nickel, titanium, chromium, and sometimes phosphorus, manganese, bromine, and iodine. The elemental distribution in the jaws is generally homogeneous. Some elements (e.g., zinc and iron) tend to be patchily distributed and concentrated in certain regions (Eriksson and Elfman 2000).
Fig. 28.2 The elemental distribution in the jaws of the recent (a) and fossilized (b) polychaete jaws (adapted from Eriksson and Elfman 2000)
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The jaws of Nereis virens are complex with respect to architecture and composition (Broomell et al. 2007). Structurally, they approximate fiber-reinforced composites with bundles of fibers arranged parallel to the contour of the jaw’s long axis. SAXS analysis indicates that the fibers are themselves arrays of tightly packed fibrils, each with a diameter of roughly 50–100 nm (Lichtenegger et al. 2003). The jaw is encased within an amorphous coating (3–10 mm thick) of unknown composition. Nereis jaws contain protein, metal ions, and the halogens Br, Cl, and I; each differentially distributed throughout the structure (Birkedal et al. 2006; Khan et al. 2006; Lichtenegger et al. 2003). The distributions of Br, I, and Cl were explored near the jaw tip using SIMS from the surface to a depth of about 10 mm and found to be deficient in the first 1–2 mm. Protein is the most abundant constituent, comprising between 70 and 90% of the total mass. It was reported that histidine concentration increases toward the tip and toothed-edge of the jaw (Birkedal et al. 2006). The opposite trend is observed for alanine; levels increase toward the jaw base. Concentrations of other predominant amino acids (tyrosine and aspartate) do not vary within the jaw. These results suggest that the jaw is made up of at least two, but probably more, proteins with distinct compositions and distribution profiles. Extractable protein accounts for only 0.1% of the total jaw mass. The major protein extractable from the jaw has a mass of about 35 kDa and a composition in which both glycine and histidine approach 30 mol% (Broomell et al. 2007). This is likely to predominate at the tip. Recent evidence suggests considerable post-translational modification of nereid jaw proteins. Halogenation of both tyrosine and histidine is prevalent, but DOPA and aryl coupling products, e.g., di- and trityrosines, are also detectable (Birkedal et al. 2006). The biochemical reactions responsible for these modifications in the jaws have not yet been determined. Both halogenation and cross-linking of tyrosine residues are often mediated by peroxidase activity, which has been histochemically detected in situ (Broomell et al. 2007). It is possible that either modification is a by-product of the other (i.e., intentional cross-linking results in inadvertent halogenation or vice versa). However, the distinct distribution of each halogen suggests that jaw protein modification involves multiple specific processes. Comparisons of the insects (termite) data with that for Nereis, recently carried out by Cribb et al. (2008), indicate that a change in the Cl/Zn ratio does not disrupt the correlation of both elements with hardness. Presumably, the molecular incorporation is similar with increasing concentration, as the ratio is constant over a variety of concentrations: the Zn is not simply loading into the system at higher concentrations without concomitant Cl. Therefore, both halogen and metal appear integral to the biochemistry and mechanical effects (Cribb et al. 2008). Since early studies on the composition of Nereis jaws were conducted on specimens from polluted estuaries in the UK, it was initially proposed that the jaws might serve as a metalsink, sequestering toxic levels of Zn absorbed from the sediment away from the living tissue (Bryan and Gibbs 1979). Further observations demonstrated that Zn levels in jaws were high, regardless of environmental context, leading to the hypothesis that metals might contribute to their mechanical properties.
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Several lines of evidence support this hypothesis. Zn levels increase (approaching 10%) toward the tip and toothed-edges of the jaw—both regions that need to be hard in vivo. Khan et al. (2006) found that in the jaws of Nereis, while Br and I were present with single chemical environments, Cl was present in two modes, Cl–Zn and Cl–C, with the latter comprising 40% of the Cl content. This points to at least one halogen possibly forming an important structural component in the absence of a metal. Both hardness and modulus increase with Zn content in jaws (Lichtenegger et al. 2003). Most importantly, removal of Zn by chelation with EDTA causes a nearly 80% reduction in both H (hardness) and E (Young modulus) (Broomell et al. 2006). Both properties are almost completely restored following reintroduction of Zn into the jaw matrix. Taken together, these data support the view that Zn is an essential ingredient for endowing the Nereis jaw with its mechanical robustness. Thus, it was assumed (Broomell et al. 2007) that in Nereis, a major matrix protein with histidine levels at 30 mol% appears to be a polydentate ligand for Zn2+ . Both hardness and stiffness are greatly reduced by the removal of Zn2+ and restored by re-exposure to Zn2+ . Zn2+ is proposed as a reversible cross-linker, in contrast to the dityrosines also present in the structure. In contrast to Nereis jaws, Glycera jaws are a composite of protein, melanin, mineral and metal ions. These jaws consist of melanin, protein, atacamite mineral [Cu2 Cl(OH)3 ], and Cu ions (Gibbs and Bryan 1979; Lichtenegger et al. 2002, 2005; Moses et al. 2006). Melanin comprises about 40% of the jaws by dry weight. Moreover, it exists as a contiguous phase throughout the jaw and is arranged in sheets approximately 200 nm thick, oriented perpendicular to the long axis of the jaw. Protein comprises 40–45% of the jaws by dry weight (Broomell et al. 2007). Although the protein is distributed throughout the jaws, the His content is elevated near the jaw tips, where it approaches 25 mol%. Atacamite mineral accounts for less than 10% of the jaws by dry weight. The mineral is arranged as fine fibers, located near the outer surfaces of the jaw tip, and oriented parallel to the outer surface (Lichtenegger et al. 2002). Distinct from atacamite, Cu ions also appear to be localized to the near-surface layers in the jaw tips (Lichtenegger et al. 2005). Nanoindentation tests indicate that the highest values of H (0.8–0.9 GPa) and E (9–10 GPa) are obtained in regions with high Cu and Cl content (Broomell et al. 2007). The implication is that atacamite fibers and Cu ions near the jaw tip play a significant structural role (Moses et al. 2006). Data on materials properties of jaws from both Nereis and Glycera species were compatible with those known for mineral-based biomaterials. Lichtenegger et al. (2002) reported that the abrasion resistance of Glycera jaws was tantamount to that of tooth enamel, yet with only a tenth as much mineral. This was followed by another report that hardness and stiffness in the completely non-mineralized jaw of Nereis approach those of tooth dentin (Lichtenegger et al. 2003). Both examples discussed above confirm the research hypothesis that factors other than mineral content can endow biological materials with stiffness, hardness, and abrasion resistance (Broomell et al. 2007).
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In spite of numerous open questions in regard to the phenomena discussed here, some key conclusions and implications about the role of transition metals in sclerotization of biological tissues of marine invertebrates have been proposed as follows (Broomell et al. 2008): (i) The metal is involved in the formation of strong Zn(His)3 Cl cross-linking complexes between the constituent proteins, both displacing water molecules that would otherwise bind to polar or charged residues and inhibiting the matrix swelling necessary for water imbibition. Furthermore, the reversible nature of water plasticization of the Zn-free jaw tip (through treatment with ZnCl2 solutions) demonstrates that the chemical affinity of Zn for the protein matrix is significantly stronger than that of water. (ii) The hardening of a specific proteinaceous material is not restricted to the metals that are preferentially incorporated in the native environment. In the nereid jaws, comparable elevations in hardness and modulus are obtained with the addition of Cu or Zn. The slightly lower efficacy of Cu is consistent with its absence in the native jaws and suggests a stronger affinity of Zn for the protein matrix. (iii) Additionally, Mn possesses potential as a hardening agent in biological tissue. Even low concentrations (<2 wt.%) are sufficient to double both stiffness and hardness of the hydrated nereid jaw tip. Indeed, the low Mn levels in combination with the property elevations indicate that, in some sense, Mn has greater efficacy than Zn in hardening. (iv) The mechanism of metal hardening must involve factors other than dehydration alone. Even with the addition of 12 wt.% Zn into the jaw base (50% greater than that occurring naturally at the jaw tip), no significant changes in properties are obtained in the hydrated state. At this concentration, Zn would be expected to displace a significant amount of water and bind with matrix proteins. The lack of hardening suggests that the proteins at the jaw base may not support metal-based sclerotization. There is no a priori basis for hardening of biological systems by metal additions alone and this underscores the sensitivity of mechanical properties to composition and organization of the constituent proteins.
28.2 Crustaceans Alternative Cuticles The grapsid, or grasping crab, Metopograpsus frontalis uses the tips of the chelipeds to scrape and tug algae from intertidal substrate surfaces, which it then eats (Shaw and Tibbetts 2004). These chelipeds and legs are tipped with a translucent light brown colored material that is readily distinguished from the rest of the carapace. These characters are potentially indicative of a lack of typical mineralization in “alternative cuticles” (Cribb et al. 2009). Previously, Schofield et al. (2003) indicate that as calcium levels drop, the predominant inorganic element in the tips of tarsal claws in crabs becomes chlorine (Cl). The non-calcified regions of M. frontalis are less hard and less stiff than the carapace, but are equivalent to values found for insect
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Crustaceans Alternative Cuticles
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cuticle lacking metals. Cheliped tips showed a complex morphological layering that differed from leg tips. The exoskeleton of M. frontalis has provided a useful model to examine the correlation of halogens with physical properties of hardness and elastic modulus within a chitin-based matrix in the absence of metals. Neither Cl nor Br concentration was found to correlate with H and/or E in this tissue type. These data suggest that halogens, and the forms in which they occur in the absence of metals, do not correlate specifically with harder or stiffer cuticle. The question remains as to what advantage this non-calcified material may confer. Instead of differences in H and E, the role of non-calcified tissues may be functionally related to friction or wear resistance. Khan et al. (2006) found that halogens are scarce at the tip of the jaws of Nereis, where wear resistance properties are most needed, while present in high concentrations at the base. From these data they argued that halogens are not contributors to properties of wear and hardness. However, the unpublished, and perhaps more directly relevant, work of Schofield and Nesson (as reported in Schofield 2005) stated that cheliped tips of Pachygrapsus crassipes are more resistant to “chipping” than the surrounding calcified tissue, when exposed to bead blasting, suggesting some improvements in wear. Recently, Schofield et al. (2009) showed that Br-rich tips of calcified crab (P. crassipes) claws are less hard but more fracture resistant. They measured a broad array of mechanical properties of a heavy-element biomaterial (abrasion resistance, coefficient of kinetic friction, energy of fracture, hardness, modulus of elasticity, and dynamic mechanical properties) for the first time and they carried out a direct comparison with a mineralized tissue. The results suggested that the greatest advantage of bromine-rich cuticle over calcified cuticle is resistance to fracture (the energy of fracture is about an order of magnitude greater than for calcified cuticle). The spoon-like tips gain increased fracture resistance from the orientation of the constituent laminae, and from the viscoelasticity of the materials. Schofield et al. (2009) suggest that fracture resistance is of greater importance in smaller organisms and they speculate that one function of heavy elements in mechanical biomaterials is to reduce molecular resonant frequencies and thereby increase absorption of energy from impact. While some authors have suggested that halogenated phenyl rings are an incidental by-product of sclerotization in seawater, Pryor (1962) suggested a functional role for halogenated tyrosine derivatives: that the electrophilic halogens would generate a partial positive charge on the phenolic hydroxyl groups, which might then participate in cross-linking proteins. The suggestion that halogenation is a non-functional by-product of sclerotization in seawater is contradicted by the presence of bromine in tarsal claws of land-based crustaceans, such as isopods (Schofield 2001). A functional role for bromine is further supported by the observation that the concentration of bromine is often higher in the contact regions of cuticular “tools” and a comparison of the mechanical properties of bromine-rich and unenriched arthropod cuticle also supports a functional role (Schofield et al. 2009). The main advantage of brominated cuticle in crustaceans over unenriched cuticle is that it has a higher modulus of elasticity and hardness (though not as high as
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for calcified cuticle). Reported data (Schofield et al. 2009) suggest no advantage in using brominated cuticle in regions susceptible to abrasion, that an exoskeleton of brominated rather than calcified cuticle could resist a factor of 1.4 greater force before rupture during attack, that calcified cuticle would be best for regions designed to apply large pressures, but that were not sensitive to fracture, and that the brominated cuticle would be optimal for sharp edges and tips that must keep their shape under pressure and not fracture. A comparison with other natural and man-made materials indicated that the brominated cuticle was harder and stiffer than the hardest tested plastic, acrylic (PMMA). Unenriched mandible cuticle was about the same hardness as acrylic. Brominated cuticle was comparable to acrylic in low frequency dynamic mechanical properties. Salmon tooth was harder, but less fracture resistant than brominated material (Schofield et al. 2009). According to Robert Schofield, bromine and other heavy elements employed in “heavy-element biomaterials” may improve absorption of energy from impacts by virtue of their high mass density, lowering the resonant frequencies of lowfrequency molecular motions to give more overlap with the range of vibration frequencies produced by impacts.
28.3 Conclusion Halogens do play important roles in natural systems. In addition, ca. 3500 halogencontaining metabolites, including the important antibiotics chloramphenicol, 7chlorotetracyclin, and vancomycin are currently known (Auffinger et al. 2004). Moreover, direct halogenation of proteins and nucleic acids can result from oxidative halogenation by a number of peroxidases. Existence of halogenated biocomposites in structural formations of numerous invertebrates species, which possess materials properties similar to mineralized structures, suggest that key alternatives have been established during evolution with respect to survival of the functional loading of the hard tissues. Metal accumulation is referred to as biomineralization when it involves deposition of mineral phases; however, a number of these metals do not show mineral formation. In the cases discussed above, we are confronted with very complex and evolutionary ancient phenomenon where “metal–protein–halogen”, “metal– protein,” and “protein–halogen” systems exist either separately or within the same organism. It seems that only very sophisticated approaches based on step-by-step selective and gentle dissolution technique will help us to resolve this puzzle.
References Auffinger P, Hays FA, Westhof E et al (2004) Halogen bonds in biological molecules. Proc Natl Acad Sci USA 101(48):16789–16794 Bergman C (1979) Polychaete jaws. In: Jaanusson V, Laufeld S, Skoglund R (eds) Lower Wenlock faunal and floral dynamics – Vattenfallet section, vol C762. Gotland Sver Geol Unders
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Birkedal H, Khan RK, Slack N et al (2006) Halogenated veneers: protein cross-linking and halogenation in the jaws of Nereis, a marine polychaete worm. Chembiochem 7:1392–1399 Broomell CC, Mattoni MA, Zok FW et al (2006) Critical role of zinc in hardening of Nereis jaws. J Exp Biol 209:3219–3225 Broomell CC, Khan RK, Moses DN et al (2007) Mineral minimization in nature’s alternative teeth. J R Soc Interface 4(12):19–31 Broomell CC, Zok FW, Waite JH (2008) Role of transition metals in sclerotization of biological tissue. Acta Biomaterialia 4:2045–2051 Bryan GW, Gibbs PE (1979) Zinc – a major inorganic component of nereid polychate jaws. J Mar Biol Assoc UK 59:969–973 Cribb BW, Rathmell A, Charters R et al (2009) Structure, composition and properties of naturally occurring non-calcified crustacean cuticle. Arthropod Struct Develop 38:173–178 Cribb BW, Stewart A, Huang H et al (2008) Insect mandibles – comparative mechanical properties and links with metal incorporation. Naturwissenschaften 95:17–23 Elfman M, Eriksson M, Kristiansson P et al (1999) Application of mikroPIXE and STIM in analanalyses of fossil and recent polychaete jaws (scolecodonts). Nucl Instrum Methods Phys Res, B Beam Inter Mater Atoms 158:287–291 Eriksson M, Elfman M (2000) Enrichment of metals in the jaws of fossil and extant polychaetes— distribution and function. Lethaia 33:75–81 Gibbs PE, Bryan GW (1979) Copper—the major metal component of glycerid polychaete jaws. J Mar Biol Ass UK 60:205–214 Gorbman AM, Clements M, O’Brien R (1954) Utilization of radioiodine by invertebrates with special study of several Annelida and Mollusca. J Exp Zool 127:75–92 Khan RK, Stoimenov PK, Mates TE et al (2006) Exploring gradients of halogens and zinc in the surface and subsurface of Nereis jaws. Langmuir 22:8465–8470 Klawe WL, Dickie LH (1957) Biology of the bloodworm, Glycera dibranchiata Ehlers and its relation to the bloodworm fishery of the maritime provinces. Bull Fish Res Bd Can 115:1–136 Lee S-M, Pippel E, Gösele U et al (2009) Greatly increased toughness of infiltrated spider silk. Science 324(5926):488–492 Lichtenegger HC, Birkedal H, Casa DM et al (2005) Distribution and role of trace transition metals in Glycera worm jaws studied with synchrotron microbeam techniques. Chem Mater 17: 2927–2931 Lichtenegger HC, Schoberl T, Bartl MH et al (2002) High abrasion resistance with sparse mineralization: copper biomineral in worm jaws. Science 298:389–392 Lichtenegger HC, Schoberl T, Ruokolainen JT et al (2003) Zinc and mechanical prowess in jaws of Nereis, a marine worm. Proc Natl Acad Sci USA 100:9144–9149 Michel C, Fonze-Vignaux M-T, Voss-Foucart M-F (1973) Donnes nouvelles sur la morphologie, l’histochimie et la composition chimique des machoires de Glycera convoluta Keferstein Ann61ide Polychete. Bull Biol Fr Belg 107:301–321 Moses DN, Mattoni MA, Slack NL et al (2006) Role of melanin in mechanical properties of Glycera jaws. Acta Biomaterialia 2:521–530 Pryor MGM (1962) Sclerotization. In: Florkin M, Mason HS (eds) Constituents of Life—Part B (IV), Comparative Biochemistry. Academic, New York Schofield RMS (1990) X-ray microanalytic concentration measurements in unsectioned specimens: a technique and its application to zinc, manganese, and iron enriched mechanical structures of organisms from three phyla. Ph.D. Dissertation, University of Oregon Schofield RMS (2001) Metals in cuticular structures. In: Brownell P, Polis G (eds) Scorpion biology and research. University Press, UK, Oxford Schofield RMS (2005) Metal–halogen biomaterials. American Entomologist 51:45–47 Schofield RMS, Nesson MH, Richardson KA (2002) Tooth hardness increases with zinc-content in mandibles of young adult leaf-cutter ants. Naturwissenschaften 89:579–583 Schofield RMS, Nesson MH, Richardson KA et al (2003) Zinc is incorporated into cuticular “tools” after ecdysis: the time course of the zinc distribution in “tools” and whole bodies of an ant and a scorpion. J Insect Physiol 49:31–44
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Schofield RMS, Niedbala JC, Nesson MH et al (2009) Br-rich tips of calcified crab claws are less hard but more fracture resistant: a comparison of biomineralized and heavy-element biomaterials. J Struct Biol 166(2009):272–287 Shaw M, Tibbetts IR (2004) Grazing by Metopograpsus frontalis (Decapoda: Grapsidae) on intertidal rock walls of Moreton Bay. Proc Roy Soc Queensland 111:95–101 Spangenberg DB (1967) Iodine induction of methamorphosis in Aurelia. J Exp Zool 165:441–450 Venturi S, Donati FM, Venturi A et al (2000) Letter to editor. Environmental iodine deficiency: a challenge to the evolution of terrestrial life? Thyroid 10:727–729
Chapter 29
Chitin–Protein-Based Composites
Abstract There are several marine crustaceans’ species which possess very dense setae, so in some cases researches spoken about a fur. Examples of two poorly investigated representatives of the hydrothermal vent fauna (Kiwa hirsuta and Shinkaia crosnieri) as well as two representatives of mitten crabs (Eriocheir sinensis and E. japonica) are described here. The last two are well investigated from ecological point of view, but there is no information on structural biology of their setae. Furthermore, the possible function of these very specialized setae as some kind of mechanoreceptors or sensilla-like structure has never before been reported. It is suggested about the presence of silk-like fibrillar protein within chitinous setae of these crustaceans. Any living animal receives a constant flow of information from its environment. This information is involved in adjusting the animal’s metabolism, possibly through the endocrine system, and changing its behavior so that the animal can optimally cope with the environment. During the flow of information through the animal, information is also released to the environment and to neighboring animals (Kouyama et al. 1981). To perceive a given type of information adequately, an animal must have a sufficient number of sensors. An understanding of the operation of such sensors is necessary for one to comprehend the mechanics of animal behavior (Weatherbya and Lenz 2000). In 1851 Leydig described the structure of crustacean setae and concluded that they must serve a sensory function because they are innervated, and because crustaceans are closely related to insects, which recognize their food by touching it with their antennae. This inference was neither challenged nor directly proven, though Claus (1891) disagreed strongly with vom Rath (1891, 1892) on the structure of the innervations of the setae. Crustacea and insects are the principal arthropods whose external surface is protected by sclerotized chitinous cuticle. Such animals therefore possess a cuticular structure as a sensory interface with the environment. For instance, the antennular basal segment of the crayfish carries several groups of mechano-sensory hairs. A group of hairs at the dorso-anterior edge of the segment has been identified as the particular input of the semi-giant interneuron C4 in the ventral cord (Kouyama et al. 1981). H. Ehrlich, Biological Materials of Marine Origin, Biologically-Inspired Systems 1, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9130-7_29,
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The specialized cuticular structures which are sensitive to external forces are called cuticular mechanoreceptors, or sensory hairs (Kouyama and Shimozawa 1982). Companiform and lyriform sensilla are derived from this type of cuticular mechanoreceptor; these sensilla detect not only external forces but also mechanical stress within the cuticle wall. The stretch receptor is another type of mechanoreceptor sensitive to the position or movement of the exoskeleton. As it has no specialized structure on the cuticular surface, it is called a subcuticular mechanoreceptor, or chordotonal organ. Some of the subcuticular mechanoreceptors lie beneath a thin cuticle and are sensitive to external vibratory or auditory stimuli. Examples are Johnston’s organ and the tympanal organs of insects. A special mechanosensory transducing element called scolopidium is known only in subcuticular chordotonal organs (e.g., for review, Howse 1968). The scolopidium of the chordotonal organ seems to work as a stretch-sensitive element (Young 1970). There are several crustaceans species which possess very dense setae, so in some cases researches spoken about a fur. I chosen as examples two poorly investigated representatives of the hydrothermal vent fauna (Kiwa hirsuta and Shinkaia crosnieri) as well as two representatives of mitten crabs (Eriocheir sinensis and E. japonica). The last two are well investigated from ecological point of view, but there is no information on structural biology of their setae. Furthermore, the possible function of these very specialized setae as some kind of mechanoreceptors or sensilla-like structure has never before been reported.
29.1 The Highly Flexible Setae of Hairy Lobster K. hirsuta The decapod crustacean fauna associated with the hydrothermal vents is represented by about 70 species (Wolff 2005), including 15 species of Anomura crustaceans belonging to four families. Some of these animals possess well developed setae, which are of interest because of their structural features as well as their nature and origin. Three years ago, while attending the International Sponge Symposium in Brazil, I received and email that contained an image of a very unusual hairy lobster called the “Yeti” crab. Fortunately, I met Professor Jean Vacelet there, who had previously published a book together with Michel Segonzac, one of the discoverer of the “Yeti” crab. Several months later, Michel sent me about 20 setae from the only animal in captivity, which was collected during an expedition in 2005 for structural analysis. Until now, there are only two papers (Goffredi et al. 2008; Macpherson et al. 2005) and one catalogue (Baba et al. 2008) published on this unique crustacean. Thus, during March–April 2005 a research cruise, PAR 5 (Pacific–Antarctic Ridge 2005 http://www.mbari.org/expeditions/eastermicroplate) organized by MBARI (RV Atlantis and DS Alvin, R. Vrijenhoek, Chief scientist), was carried out along four hydrothermal vent areas of the Easter Microplate and Pacific– Antarctic Ridge between 23◦ S and 38◦ S. The 38◦ S site is the southernmost vent
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The Highly Flexible Setae of Hairy Lobster K. hirsuta
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area known on the complex East Pacific Rise/Pacific–Antarctic Ridge and it exhibits the highest known spreading rates, c. 100 mm/year vs. 160 mm/year at 31◦ S, which are expected to affect the turnover rate off local vent communities. The substratum consists of very glassy basalt with no sediment cover, suggesting the lava emissions are very young. On several occasions, scientists diving in the submarine Alvin on board the RV Atlantis observed a number of large (ca. 15 cm length), white, “hairy” crustaceans (Fig. 29.1a), and collected one of them. The specimen belongs to the superfamily Galatheoidea, a clade that includes the families Galatheidae, Chirostylidae, and Porcellanidae (Henderson 1888 and Aeglidae Dana 1852), but does not fit within the morphological or genetic boundaries of any of these known families. The extraordinarily setose nature of the chelipeds and walking legs led to adoption of the common name, “Yeti” crab. The scientific name of this animal was given K. hirsuta (from the Latin, hirsutus, hairy, in reference to the abundance of setae on pereopods) (Macpherson et al. 2005).
Fig. 29.1 Kiwa hirsuta, or “Yeti” lobster, possesses extraordinarily setose chelipeds and walking legs (a). Setae (b) are made of chitin and fibrillar protein. Chitinase treatment of the setae leads to dissolution of the chitinous outermost layer (c) and numerous proteinaceous microfibrils became free and visible using SEM (d) (samples courtesy Michel Segonzac)
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Its chelipeds, walking legs, and the ventral surface of its cephalothorax are covered with dense setae (Fig. 29.1b) that, in turn, are covered with clusters of filamentous bacteria, making the crab appear extraordinarily “hairy.” Electron microscopy revealed dense bacterial clusters attached to the chitinous outer layer of the setae. Molecular phylogenetic analyses revealed the setae-associated bacteria to be dominated by Epsilonproteobacteria (~56% of the recovered ribotypes), Gammaproteobacteria (~25%), and Bacteroidetes (~10%) (Goffredi et al. 2008). Fluorescence in situ microscopy confirmed the attachment of filamentous Epsilonproteobacteria on setae, but no specialized morphological structures appeared to exist for bacterial attachment. Key enzymes involved in the reductive tricarboxylic acid cycle (ATP-dependent citrate lyase) and sulfite oxidation or dissimilatory sulfate reduction (bidirectional APS reductase) were detected. Consequently, the potential for carbon fixation and cycling of reduced and oxidized sulfur appear to exist in the dense microflora that grow on the crab’s setae (Goffredi et al. 2008) The new species occur at densities of one to two individuals per 10 m2 , more or less regularly spaced on the zone of pillow basalt surrounding active hydrothermal vents. Specimens of K. hirsuta n. gen., n. sp. were also observed on extinct chimneys and at the base of black smokers, among vent mussels, where shimmering milky water emanates. Like other vent decapod crustaceans K. hirsuta n. gen., n. sp. is probably omnivorous. The setae themselves are about 10–20 mm in length and made of two distinct biological materials: chitin and protein (Fig. 29.1c). Chitinase treatment led to the enzymatic digestion of the outermost layer (Fig. 29.1d) while alkali treatment, a test for proteinaceous materials, led to the complete dissolution of only the inner layers. Thus, the core of the setae is made of a fibrillar protein of undefined nature while the external material is primarily chitin (Fig. 29.2). Results of analyses using Calcofluor White staining and chitinase treatment, as well as additional Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy (carried out as described previously in Ehrlich et al. 2007), provided further evidence that the outermost layer of the setae is made of α-chitin. Microscopic examination revealed the bacteria attached, in clusters, only to the chitinous outermost layer and primarily at the distal end of the setae. Results of the preliminary experiments using FTIR, Raman, and X-ray diffraction analysis, which have been carrying out in our laboratory, showed that protein of nanofibrillar organization localized within setae of K. hirsuta is very similar to silk. However, it is well known that crustaceans, in contrast to Insecta and Arachnoidea, produce no silk. Recently, Gorb et al. (2006) reported that the zebra tarantula Aphonopelma seemanni secretes silk from its feet, which might improve its ability to climb on vertical surfaces. Therefore, we hypothesize that silk production from the chelipeds of decapod crustaceans, which are evolutionary older than arachnids, is possible. Moreover, we cannot find up to now any evidence on the presence of microtubuli within K. hirsuta setae, which are typical for the well-described mechanoreceptors in insects (Kouyama and Shimozawa 1982). It seems that these setae possess a very specific function, other than sensoric.
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S. crosnieri
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Fig. 29.2 Schematic view of the structural features of K. hirsuta individual setae (a) proposed by Denis Kurek is based on SEM observations, which show nanostructural organization (b, c, d) of the fibrillar core protein
Now, we are starting with genomics and proteomics experiments to confirm our suggestion about the silk nature of the inner part of setae.
29.2 S. crosnieri Baba and Williams (1998) first identified hairy crab S. crosnieri at the Bismarck Archipelago and in the Okinawa Trough in the west Pacific Ocean. The discoverers placed it into Decapoda: Anomura: Galatheidae according to its morphological features. Chan et al. (2000) also mentioned this species as the first known hydrothermal crustacean in Taiwan. The recent S. crosnieri was described as having small spines on the lateral margins of the rostrum (Fig. 29.3a, b); however, Baba and Williams (1998) noted that spines of the carapace could become worn down over time, as did Chan et al. (2000), whose specimens lacked the lateral spines described for the type
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Fig. 29.3 S. crosnieri was described as having small spines (arrows) on the lateral margins of the rostrum (a, b). This species forms dense colonies (c) in deep-sea hydrothermal vent fields
material of S. crosnieri. Gathering near the top of the vents, S. crosnieri probably feed on polychaetes and “culture” filamentous bacteria on their abdominal surface (Ohta and Kim 2001), similar to K. hirsuta. Thus, S. crosnieri forms dense colonies (Fig. 29.3c) in deep-sea hydrothermal vent fields. Recently, Watsuji and Takai (2009) investigated compositional and functional transition in epibiotic symbionts of S. crosnieri collected from the Hatoma Knoll and Iheya North fields in the Okinawa Trough. They had numerous morphologically novel ventral setae covered with filamentous epibiotic microorganisms, like in K. hirsuta. Phylogenetic analyses using 16S rRNA gene cloning and fluorescence in situ hybridization revealed that filamentous microbial communities
29.2
S. crosnieri
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were heterogeneous and consisted of previously uncultivated phylotypes within the Epsilonproteobacteria (the Sulfurovum group) and the Gammaproteobacteria (thioautotrophic and metanotrophic symbiont groups) in both populations from the different fields. Experimental results clearly implies that strictly sulfur-oxidizing chemolithoautotrophic and methanotrophic production of the epibionts supports the trophic basement of S. crosnieri. The populations of S. crosnieri collected from both fields were successfully reared in sulfidic and aerobic environment in an aquarium for more than 5 months. No genetic data have been characterized for this crab species so far. Recently, Yang and Yang (2008) investigated the complete mitochondrial genome sequence of the S. crosnieri. These authors reported that nucleotide composition and bias of S. crosnieri mitogenome are similar with other decapods. Phylogenetic analysis shows a close relationship with another anomuran Pagurus longicarpus, supporting the taxonomic classification by the morphology. No trend of evolutionary acceleration or retardation was observed. An exterior origin may explain these conserved features found in S. crosnieri (Yang and Yang 2008). Interesting results about the evolution of Shinkaia species were recently reported by Schweitzer and Feldmann (2008). A new decapod crustacean species, Shinkaia katapsyxis, from the Eocene Humptulips Formation of western Washington, USA, has been described. The specimens were collected from a hydrocarbon seep deposit that has been well documented and contains a well-described molluscan fauna. The new occurrence extends the geologic range of the genus Shinkaia (Baba and Williams 1998), and subfamily Shinkaiinae (Baba and Williams 1998), into the Eocene from its only other known occurrences in hydrothermal vent environments in the Pacific Ocean. The range extension of an extant decapod genus into the Eocene is not uncommon and adds to the evidence that the Decapoda may be unusually resistant to extinction and are distinctly conservative evolutionarily. The new fossil specimens attributed to Shinkaia are remarkably similar to the current species, especially given the approximately 40 million year age difference between them. The fossils possess all of the diagnostic features of the genus. The extant species was described as being covered with setal hairs, especially on the ventral surface. The ventral surface is not preserved in the fossils and only one incomplete abdomen is present; however, the authors noted the presence of setal pits on many of the carapace surfaces on the fossil specimens. The new fossils lack spines on the rostrum. S. crosnieri narrows considerably posteriorly, much more than the new fossils described herein. In addition, the new fossils do not share the spines on the outer surface of the manus with S. crosnieri. Seep and vent faunas appear to be resistant to extinction. The depth of these communities and their dependence on chemosynthesis rather than photosynthesis makes them a natural contender as refugia from impact events or volcanic eruptions clouding the sky with debris and blocking the sun for long time periods. It was hypothesized that the offshore transitions of taxa into deeper water is determined presumably to escape predation and competition (Schweitzer and Feldmann 2008). Therefore, we cannot exclude the existence of crustaceans communities similar to those of K. hirsute and Shinkaia species in other locations of hydrothermal vents.
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Unfortunately, there is a lack of any kind of information about structure and composition of the Shinkaia crabs setae. Because of the problems with obtaining of setae from these unique crustaceans, we decided to start investigate setae of the Mitten Chinese Crab E. sinensis, which was easy to obtain because of the very large population in Germany. However, it was initially unclear to us how similar the structure of its setae will be to those from K. hirsuta.
29.3 Structural Features of E. sinensis Setae Mitten crabs are limnic and intertidal crabs distributed in Asia and are one of the most important groups of crabs in the world. They have adapted to a wide variety of semi-terrestrial, freshwater, and marine habitats. Brachyuran crabs in the genus Eriocheir are assigned to the Varunidae, which are one of six families in the superfamily Grapsoidea. The mitten crabs of the varunid genus Eriocheir de Haan, 1835, as presently recognized, consist of five taxa, viz., Eriocheir japonica de Haan, 1835, E. sinensis H. Milne Edwards, 1853, Eriocheir hepuensis Dai, 1991, Eriocheir leptognatha Rathbun, 1913, and Eriocheir formosa Chan et al. (1995), as reviewed by Tang et al. (2003). The species E. leptognatha has now been assigned to the genus Neoeriocheir (Sakai 1983), but there is still controversy about this revision and about the taxonomy of mitten crabs in general (Tang et al. 2003). Mitten crabs are mostly restricted to East Asian waters except E. sinensis, which naturally occurs in eastern and northern China, but has been introduced into Europe and North America (Cohen and Carlton 1997; Dittel and Epifanio 2009; Ingle and Andrews 1976). Taxonomic boundaries between genera and species and species and subspecies of the mitten crabs have received some attention from systematists (DeLeersnyder 1967; Hoestlandt 1948). Adults and juveniles (Fig. 29.4) in the genus Eriocheir are characterized by the presence of patches of brown setae on the inner and outer surface of their whitetipped chelae. Males have a denser mat of setae, but there is no gender-based dimorphism in claw size. Juvenile crabs resemble adults, except for the lack of setae on the claws of crabs smaller than 20 mm in carapace width (Veldhuizen 2001). The claws are approximately equal in size. The carapace is slightly wider than long and has four spines on its anterior lateral margins. The maximum carapace width of the adult mitten crab in California is approximately 80 mm, although occasionally larger individuals are found (Siegfried 1999). The crabs’ pigmentation varies from a brownish orange, particularly among juvenile crabs, to a more greenish-brown seen in adult crabs and in newly molted crabs (Zhao 1999). The legs of the mitten crab are generally twice as long as the width of the carapace, and, among older juveniles and adult crabs, the distal segments of the legs exhibit hairs along the lateral margins (Fig. 29.5). After reaching a size exceeding approximately 10 mm in carapace width, the male and female crabs can be differentiated by the shape of the abdomen, which in the female is rounded and occupies most of the area of the thorax; while the male exhibits a narrower abdomen shaped like an inverted funnel.
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Structural Features of E. sinensis Setae
399
Fig. 29.4 Adult and juvenile Chinese mitten crab (E. sinensis)
Fig. 29.5 E. sinensis is characterized by the presence of patches of brown setae (a, b, c). Fluorescence microscopy image (d) of the setae stained with Calcofluor White confirm the chitinous nature of these unique structural formations (image courtesy Thomas Hanke and Sascha Heinemann)
Female mitten crabs carry from 250,000 to 1,000,000 eggs attached to the pleopods (abdominal appendages) on the underside of their abdomen. China mitten crabs reproduce only once; most crabs reproduce in the fall but some hold over until the spring (Hymanson et al. 1999). Post-reproductive mitten crabs return to the coastal banks; the males die after mating and the females die after the eggs hatch (Zhao 1999).
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Mitten crabs begin their lives as marine pelagic zoeae. The crabs undergo five zoeal and one megalopal stages, and, under laboratory conditions, require an average of 15 days to reach the megalopal stage (Kim and Hwang 1995). In the megalopal stage, the crabs begin migrating inland toward fresh water. The megalopal crabs swim quickly, cueing toward a lower salinity to reach brackish water (Zhao 1999). Following the megalopal stage, the crabs settle to the substrate and continue their migration as juveniles. Juvenile mitten crabs undertake enormous journeys during their migration and have been found as far as 1,250 km from the coast in freshwater streams (Dan et al. 1984). Juvenile crabs spend from 1 to 5 years feeding and growing in freshwater streams in China, with the vast majority maturing in 1–2 years, before beginning their migration downstream as adults (Hymanson 1999; Zhao 1999). According to European studies of this species, adult mitten crabs begin to migrate downstream in late fall and winter, with the males descending to brackish waters first and the females following a month after (Anger 1991). The crabs live in burrows in the banks of rivers. They feed on a wide variety of plants, invertebrates, fishes, and also detritus, with snails and clams being the main food. During their life E. sinensis are confronted with a variety of different ambient salinities (Olsowski et al. 1995; Shaw 1961). As juveniles, the animals migrate from the sea upstream to freshwater. As adults, Mitten Chinese Crabs spend the major proportion of their life in fresh water. However, for reproduction the crabs return to the sea. On their way to and from the sea Mitten Chinese Crabs pass brackish waters of different salt contents (Onken 1999). As already observed by Krogh (1938) on whole animals, these crustaceans are able to absorb Na+ and Cl− independent from each other. These animals can also live in the waters with high level of pollution with respect to metals (e.g., cadmium) (Silvestre et al. 2004, 2005a, b). The most works published on E. sinensis are, however, related to ecology, because this species is one of the main players in “ecological roulette” (the term proposed in Science paper by Carlton and Geller in 1993). Mitten crabs were first introduced to Europe almost a century ago, most likely via the release of ballast water (Panning 1939). The first invasion was documented in Germany in 1912 and the species spread rapidly throughout northern Europe (Marquard 1926; Peters and Panning 1933). According to the excellent review by Dittel and Epifanio (2009), by the 1940s the mitten crab had been reported in Denmark, Sweden, Finland, Poland, the Netherlands, Belgium, France, and the UK. Forty years later, the European range of Chinese mitten crabs stretched from the Bay of Biscay to the Baltic Sea. Nepszy and Leach (1973) reported about the first records of the Chinese Mitten Crab from North America. By the 1990s, the invasion had reached Spain and Portugal and Eastern Europe. Crabs were reported from the Volga River delta, the Serbian part of the Danube River, and Lake Ladoga in Russia. Other recent reports include two different areas in western Asia—the River Tazeh Bekandeh in Northern Iran and the Shatt Al-Basrah Canal in Iraq (Dittel and Epifanio 2009). The presence of this alien species is not without significance in the food chain, because E. sinensis is also prey for wading birds, fishes, and seals. In the first
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Structural Features of E. sinensis Setae
401
6 months of 1935, Panning (1938) reported capturing over 12,000 kg (3,444,680 individuals) at a dam on the Weser River in Germany. At this time, the infestation of Mitten Crabs was so intense that the crabs swarmed out of the rivers and were found walking down city streets and into houses! These crabs are also a potential source of food for humans and a recent study revealed that E. sinensis is being consumed more often. The price per kilogram of this crustacean in Asian markets is about US$ 30 (Normant et al. 2002). It seems that in spite of their meat, E. chinensis could be a potential candidate for production of chitin and chitosan from the carapaces. But, we also suggest that setae of these animals will find practical application in materials science because of the following results obtained in our laboratory. We found that the inner structure as well as composition of E. chinensis setae is very similar to those from K. hirsuta reported above. Mitten crab setae possess both biopolymers, chitin and fibrillar protein, with characteristic nanostructural organization (Fig. 29.6). Nanofibrils also became visible after chitinase treatment (Fig. 29.6c, d). The light microscopy images of naturally occurring setae show the presence of unidentified bacteria attached to their surface (Fig. 29.7a). Cleaning of the E. chinensis setae using 35% H2 O2 at room temperature and under intensive
Fig. 29.6 SEM imagery: E. sinensis setae (a, b) possess both chitin and fibrillar protein with characteristic nanostructural organization. Nanofibrils became visible especially after chitinase treatment (c, d). Similar features possess setae from K. hirsuta (see Fig. 29.2)
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Fig. 29.7 Light microscopy observations: (a) bacteria (arrows) colonized the surface of E. sinensis setae like in the case of K. hirsuta (Goffredi et al. 2008). Treatment with 35% hydrogen hydroxide leads to discoloration (b) and to depolymerization (c) of the chitinous parts
lighting resulted in not only the disappearance of the brownish pigmentation but also the dissolution of the setae (Fig. 29.7c). Similar phenomenon was recently observed by us with chitinous fibers isolated from marine Verongida sponges.
29.4 Conclusion Decapod marine crabs possess not only very specific ecological behavior but also some unusual features with respect to their setae. Investigations on these chitin–protein-based formations are very important for ecology, evolutionary, and
References
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Fig. 29.8 Stony particles (arrows) are attached to the setose surface of the spider crab
biomaterials sciences. I am very optimistic about the potential of information gathered on these fibrillar proteins within setae, which we wish to obtain in the near future. If this protein is really a silk, then several postulates in biology of arthropods must be re-examined. The really function of setae of crustacean species described above is unknown. Interestingly some spider crabs (Fig. 29.8) use their setose surface for attachment of microstones. Correspondingly, they seem to be invisible for predators being cover with these stony particles on the sea bottom.
References Anger K (1991) Effects of temperature and salinity on the larval development of the Chinese mitten crab Eriocheir sinensis (Decapoda: Grapsidae). Mar Ecol Progr Ser 72:103–110 Baba K, Macpherson E, Poore GCB et al (2008) of squat lobsters of the world (Crustacea: Decapoda: Anomura—families Chirostylidae, and Kiwaidae) (Zootaxa 1905) Baba K, Williams AB (1998) Galatheoidea (Crustacea, Decapoda, Anomura) from hydrothermal systems in the west Pacific Ocean: Bismarck Archipelago and Okinawa Trough. Zoosystema (2):143–156 Carlton JT, Geller JB (1993) Ecological roulette: the global transport of nonindigenous marine organisms. Science 261:78–82 Chan TY, Hung M, Yu H (1995) Identity of Eriocheir recta (Stimpson, 1858) (Decapoda: Brachyura), with description of a new mitten crab from Taiwan. J Crustacean Biol 15(2): 301–308 Chan TY, Lee DA, Lee CS (2000) The first deep-sea hydrothermal animal reported from Taiwan: Shinkaia crosnieri Baba and Williams, 1998 (Crustacea: Decapoda: Galatheidae). Bull Mar Sci (2):799–804 Claus C (1891) Ueber das Verhalten des nervosen Endapparates an den Sinneshaaren der Crustaceen. Zool Anz 14:363–368 Cohen AN, Carlton JT (1997) Transoceanic transport mechanisms: introduction of the Chinese mitten crab, Eriocheir sinensis, to California. Pacific Sci 51(1):1–11 Dan QK et al (1984) The ecological study on the anadramous crab Eriocheir sinensis going upstream. Tung wu hsueh tsa chih (Chinese J Zool) 6:19–22 DeLeersnyder M (1967) Le Milieu intérieur d’Eriocheir sinensis Milne–Edwards et ses variations. I. Etude dans le milieu naturel. Cahier Biol Mar 8:195–218 Dittel AI, Epifanio CE (2009) Invasion biology of the Chinese mitten crab Eriochier sinensis: a brief review. J Exp Mar Biol Ecol 374:79–92
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Ehrlich H, Krautter M, Hanke T et al (2007) First Evidence of the presence of chitin in skeletons of marine sponges. Part II. Glass sponges (Hexactinellida: Porifera). J Exp Zool (Mol Develop Evol) 308B:473–483 Goffredi SK, Jones WJ, Ehrlich H et al (2008) bacteria associated with the recently Yeti crab, Kiwa hirsuta. Environ Microbiol 10(10):2623–2634 Gorb et al (2006) Silk-like secretion from tarantula feet. Nature 443:407 Hoestlandt H (1948) Recherches sur la biologie de l’Eriocheir sinensis H. Milne Edwards (Crustacé Brachyoure). Annales de l’Institut Océanographique 24:1–36 Howse PE (1968) The fine structure and functional organization of chordotonal organs. Syrup Zool Soc London 23:167–198 Hymanson Z (1999) The Chinese mitten crab in its native range. Report presented before the Chinese mitten crab Project Work Team, California, Stockton Hymanson Z, Wang J, Sasaki T (1999) Lessons from the home of the Chinese mitten crab. IEP Newslett 12(3):25–32 Ingle RW, Andrews MJ (1976) Chinese mitten crab reappears in Britain. Nature 263:638 Kim CH, Hwang SG (1995) The complete larval development of the mitten crab Eriocheir sinensis H. Milne-Edwards, 1854 (Decapoda, Brachyura, Grapsidae) reared in the laboratory and a key to the known zoeae of the Varuninae. Crustaceana 68(7):703–812 Kouyama N, Shimozawa T (1982) structure of a hair mechanoreceptor in the antennule of crayfish (Crustacea) Cell Tissue Res 226:565–578 Kouyama N, Shimozawa T, Hisada M (1981) Transducing element of crustacean mechano-sensory hairs. Experientia 37:379–380 Krogh A (1938) The active absorption of ions in some freshwater animals. Z Vergl Physiol 25: 335–350 Leydig F (1851) Ueber Artemia salina und Branchipus stagnalis. Z Wiss Zool 3:280–307 Macpherson E, Jones W, Segonzac M (2005) A new squat lobster family of Galatheoidea (Crustacea, Decapoda, Anomura) from the hydrothermal vents of the Pacific-Antarctic Ridge. Zoosystema 27:709–723 Marquard O (1926) Die Chinesische Wollhandkrabbe, Eriocheir sinensis MILNE-EDWARDS, ein neuer Bewohner deutscher Flüsse. Fischerei 24:417–433 Nepszy SJ, Leach JH (1973) First records of the Chinese mitten crab, Eriocheir sinensis, (Crustacea: Brachyura) from North America. J Fish Res Bd Canada 30(12):1909–1910 Normant M, Chrobak M, Szaniawska A (2002) Energy value and chemical composition (CHN) of the Chinese mitten crab Eriocheir sinensis (Decapoda: Grapsidae) from the Baltic Sea. Thermochimica Acta 394:233–237 Ohta S, Kim D (2001) Submersible observations of the hydrothermal vent communities on the Iheya Ridge, mid Okinawa trough, Japan. J Oceanogr 57:663–677 Olsowski A, Putzenlechner M, Böttcher K et al (1995) The carbonic anhydrase of the Chinese crab Eriocheir sinensis: effects of adaptation from tap to salt water. Helgol Meeresunters 49: 727–735 Onken H (1999) Active NaCl absorption across split lamellae of posterior gills of Chinese crabs (Eriocheir sinensis) adapted to different salinities. Comp Biochem Physiol Part A 123:377–384 Panning A (1938) The Chinese mitten crab. Annual Report of the Board of Regents of the Smithsonian Institution, Washington, DC Panning A (1939) The Chinese mitten crab, Annual Report Smithsonian Institution, 1938. Washington, DC Peters N, Panning A (1933) Die Chinesische wollhandkrabbe (Eriocheir sinensis H. Milne Edwards) in Deutschland. Zoologischer Anzeiger Supplement 104:1–180 Rath vom O (1891) Zur Kenntnis der Hautsinnesorgane der Crustaceen. Zool Anz 14:195–200, 205–214 Rath vom O (1892) Ueber die von C. Claus beschriebene Nervenendigung in den Sinneshaaren der Crustaceen. Zool Anz 15:96–101 Sakai T (1983) Descriptions of new genera and species of Japanese crabs, together with systematically and biogeographically interesting species, 1. Res Crust 12:3–23
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Part VI
Macromolecular Biopolymers
Chapter 30
Chitin
Abstract The skeleton of some marine sponges (Verongida: Porifera) appears to possess a number of unique and suitable properties, including the chitinous nature and the possession of open interconnected channels created by the fibrous twoand three-dimensional networks. Verongid sponges are biochemically characterized by the production of brominated tyrosine derivatives as well as aplystane sterol products. Unfortunately, the residual skeletons obtained after extraction of bromotyrosines from the verongid sponges were usually discarded. Here, a non-waste technology for the use of Verongida sponges as sources of products, which could find practical applications in pharmacology, medicine, and technologies, is proposed. Modern view on toxicity, immunology, biodegradation, and biocompatibility of chitin from marine sources is also discussed.
30.1 Two- and Three-Dimensional Chitinous Scaffolds of Poriferan Origin Sponges are probably the earliest branching animals (Philippe et al. 2009) and their fossil record dates back to the Precambrian (Love et al. 2009; Reitner and Wörheide 2002). In former studies, chitin was only identified in the inner layer of so-called gemmulae and not in skeletal formations. These microbodies are produced by fresh water sponges prior to their seasonal disappearance under extreme environmental conditions (Simpson 1984). Previously, the presence of chitin in some sponge macerates was ascribed to “contamination” by a variety of microinvertebrates harbored by the sponges (Dauby and Jeuniaux 1986). First observations of chitin-based scaffolds as an integral part of skeletal elements of a number of sponges were recently reported by our group (Ehrlich et al. 2003; 2007a, b; Ehrlich and Worch 2007). A systematic study among the various sponge species is in progress now. Thus, chitin-based scaffolds could be isolated from the sponge skeletons using a stepwise extraction procedure mainly based on the use of NaOH (Ehrlich et al. 2007b). Intriguingly, it is possible to isolate three-dimensional, as well as twodimensional chitinous scaffolds, for example, from Ianthella basta (Figs. 30.1 and 30.2). This procedure results in the removal (hydrolysis) of biomolecules other than H. Ehrlich, Biological Materials of Marine Origin, Biologically-Inspired Systems 1, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9130-7_30,
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Fig. 30.1 Giant two-dimensional I. basta demosponge possesses chitin-based skeleton (image courtesy Mark Spencer)
Fig. 30.2 Photographs of I. basta: (a) dried skeleton; (b) partially demineralized sample after alkali treatment; (c) purified chitinous matrix. (d) SEM image of the sponge chitin (samples courtesy Peter Schupp)
chitin from the skeletal formations whereas the chitin-based scaffolds withstand this treatment (Ehrlich et al. 2007a, b; Brunner et al. 2009). It should be noted that the NaOH concentration of 2.5 M is well below the critical concentration where the base-induced transformation of β-chitin into α-chitin starts to take place (Noishiki et al. 2003). Therefore, the crystal structure of the chitin-based scaffolds is not influenced by this kind of extraction. The effect of the procedure is demonstrated in
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Fig. 30.2 for the two-dimensional chitin-based scaffold of the marine demosponge I. basta. While the untreated sponge skeleton consists of a variety of organic compounds, the characteristic signals due to chitin dominate the NMR spectrum after the three extraction steps (Brunner et al. 2009). After a final H2 O2 purification, clean white scaffolds remain which mainly consist of chitin. The identification and characterization of these scaffolds relies on a variety of bioanalytical techniques such as solid-state 13 C NMR spectroscopy, Raman and IR spectroscopy, chitinase digestion, fluorescence microscopy using Calcofluor White staining as detailed described by Brunner et al. 2009. It is important to note that the species-specific shape and morphology of these chitin-based scaffolds closely resembles the shape and morphology of the sponge species. At the end of the isolation procedure, also fibers of the sponges with threedimensional skeletons (e.g., Verongula or Aplysina species) became hollow, pipelike, translucent structures. The current strategy in scaffold design and synthesis of biodegradable synthetic sponge-like constructs and foams for tissue engineering is to produce uniform threedimensional interconnected macroporosity. The aim of this strategy is to create specific pore dimensions and interconnections during scaffold synthesis. Generating sufficient structural support for tissues at the implantation site is a major issue and tissue-engineering and biomimetic approaches may offer alternative solutions (reviewed in Green et al. 2002). The skeleton of sponges (Porifera) appears to possess a number of unique and suitable properties, including (i) the ability to hydrate to a high degree, which is favorable to cell adhesion, (ii) the possession of open interconnected channels created by the fiber network, and (iii) the tremendous diversity of skeletal architectural elements and fiber constructs in this phylum (Green 2008). Here, I represent a schematic view (Fig. 30.3) of strategies for practical application of three-dimensional chitinous scaffolds which can be isolated from Verongida sponges as described above.
Fig. 30.3 Strategies for practical application of three-dimensional chitinous scaffolds of poriferan origin
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In addition to collagen, chitin has been established as a very attractive biomaterial for practical application in tissue engineering and biomedicine during the last decade (see reviews by Khor 2001, 2002; Khor and Lim 2003). Chitin as a biomaterial can be exploited in two main manners, as bio-stable chitin or as a biodegradable material (Khor 2001). Because of the previous absence of “naturally prefabricated” three-dimensional chitinous scaffolds, corresponding complex and cost-depended technologies had been developed to produce three-dimensional sponge-like chitin scaffolds for applications in tissue engineering. Thus, Abe et al. (2004) produced a bioresorbable β-chitin sponge-like construct and used it as a scaffold for three-dimensional cultures of chondrocytes. β-Chitin was obtained from the pens of Loligo squid. Biochemical analyses along with histochemical and immunohistochemical studies and RT-PCR analyses indicated that the cartilage-like layer in the chondrocyte-βchitin sponge-like composite was similar to hyaline cartilage. Electron microscopy revealed that the cell layer at the surface of the β-chitin sponge-like material was filled with chondrocytes and abundant extracellular matrix. Since this method produced pillar-shaped composites, it is possible to press-fit the material into articular cartilage defects without covering the periosteum or suturing the implant. Only recently Suzuki and colleagues (2008) described the preparation of sponge-like
Fig. 30.4 Light microscopy images of V. gigantea chitinous matrix prior (a) and after mineralization in vitro with respect to obtaining of different calcium phosphate phases (e.g., brushite (b) or hydroxyapatite (c and d))
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Fig. 30.5 SEM images of V. gigantea chitinous matrix show features of its structural organization on micro- (a, b) and nanolevels (c). Nanofibrils of chitin are also well visible after mechanical damage of the sponge skeletal fibers (d)
chitinous material. Pure β-chitin, pure chitosan, 3:1, 1:1, and 1:3 β-chitin:chitosan sponge-like scaffolds were created from mixtures of β-chitin hydrogels and chitosan hydrogels. The hydrogels were frozen and vacuum-dried for 24 h to form sponges of pillar-like shape (5 mm diameter, 10 mm length). The provision of affordable, readily available three-dimensional scaffolds for technical applications remain a major practical demand in other fields in addition to tissue engineering. Chitinous matrices as isolated by us can be proposed, for example, for the development of novel filtering systems as well as biomaterials. Moreover, some material properties of chitin open new perspective with respect to obtaining mineralized (Figs. 30.4, 30.5, 30.6, and 30.7) as well as metalized surfaces with high catalytic activity. This could allow reactions under conditions, which are known to be too extreme for most other biopolymers. Thermal stability of chitin depends on the size and perfection of crystallites as well as on the crystalline form of the chitin. Therefore thermodynamically stable α-chitins (arthropods, sponges) are thermally more stable than β-chitins (diatoms, squids) (Stawski et al. 2008). Thermogravimetric analysis of purified non-mineralized chitin revealed that this aminopolysaccharide is stable up to 210◦ C (Köll et al. 1991) or even up to 360◦ C (Stawski et al. 2008) as shown in in vitro experiments. Based on these properties, novel ways for the development of chitin-based two- and three-dimensional
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Fig. 30.6 SEM images of the sample represented in Fig. 30.6b. Brushite crystals are formed within (a) as well as on the surface (b, c) of chitinous skeletal fibers of V. gigantea. Different growth phases of nano-organized brushite are also observed (d)
constructs with functionalized (e.g., metalized) surfaces could be proposed and carried out in the nearest future, allowing synthesis temperatures up to 300◦ C and pH diapason between 1 and 12 (see also Chapter 35). The biotechnological and biomimetic potential of marine sponges as a goldmine to chemists, pharmacologists, bioengineers and materials scientists seems to be amazing. Over the past 40 years, an ever increasing number of biologically active secondary metabolites have been isolated from marine sponges, among them many from the order Verongida. Verongid sponges are biochemically characterized by the production of brominated tyrosine derivatives as well as aplystane sterol products. Unfortunately, the residual skeletons obtained after extraction of bromotyrosines from the verongid sponges were usually discarded. Here, we propose a non-waste technology for the use of Verongida sponges (Fig. 30.8) as sources of products, which could find practical applications in pharmacology, medicine, and technologies. One of the remarkable advantages of Verongida sponges is their potential to grow in marine ranching systems as well as in fragmental tissue cultures that exclude the necessity of their harvest from the natural environment.
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Fig. 30.7 Mineralization of sponge chitin in vitro. SEM images (a, b) of the hydroxyapatite phase represented in Fig. 30.4c and d
Fig. 30.8 Schematic view of the proposed non-waste technology based on the utilization of Verongida sponges
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30.2 Modern View on Toxicity, Immunology, Biodegradation, and Biocompatibility of Marine Chitin Chitin as a biomaterial can be exploited in two main manners, as bio-stable chitin or as a biodegradable material (Khor 2001). Wound dressing and coatings onto blood-contact tubing are external device situations where chitin is used in a biostable form. Bone substitutes and tissue engineering applications are classified in the implant category. These applications require chitin to participate in a biodegradation role, the purpose being to avoid the necessity for a second operation to retrieve the implant. The wound dressing applications for chitin is designed in most instances to be an external device that does not biodegrade (Khor 2002). While it is claimed that lysozyme abundantly present in wound beds will degrade chitin wound dressings, this may only happen at the interface, leaving the dressing mostly intact. When used as a coating for blood-contact tubing, the substituted chitins are expected to remain on the inner surface of the lumen, provided of course, proper anchoring of the chitin to that surface was achieved.
30.2.1 Toxicity The low toxicities of chitin even in large doses have been noted. The addition of 8.5% chitin to mouse feed does not affect the growth and the health of the animals (Knorr 1984). When 5 mg of chitin were injected intraperitoneally every 2 weeks over a 12-week period, the mice were apparently normal, but histologically, many macrophages with hyperplasia were observed in the mesenterium and foreign body giant cell-type polykaryocytes were observed in the spleen (Tanaka et al. 1997). The polykaryocytes were also observed in the spleen of the mice injected subcutaneously with 5 mg of chitin, but no other changes were observed. However, when 5 mg of chitosan was injected intraperitoneally, the body weights of the mice decreased significantly and inactivity was observed in the fifth week (Tanaka et al. 1997). Recently, Kwak et al. (2005) investigated chitin-based embolic materials in the renal artery of rabbits. The safety of various kinds of chitin as embolic material was demonstrated in this experiment, as none of the rabbits died of causes related to the embolic material. Furthermore, no substantial abnormality was observed at hematologic or blood chemical examination when these various chitin embolic materials were compared with polyvinyl alcohol. A toxicologic examination also supported the compatibility and safety of chitin. Application of chitin in veterinary practice is detailed described by Okamoto et al. (1993).
30.2.2 Immunology Chitin deposition in nature is regulated by biosynthesis and degradation. Chitinases are endo-β-1,4-N-acetylglucosamidases. They have been studied most intensively
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in lower life forms, where they are produced in significant quantities by hosts defending against infections with chitin-containing organisms (Elias et al. 2005). This can be appropriately considered part of the innate immune response against a chitin-containing pathogen. Paradoxically, while chitin and chitin synthase do not exist in mammals, human chitinase family members such as acidic mammalian chitinase (AMCase) have recently been described (Zhu et al. 2004). Mammalian chitinase-like proteins are induced at sites of inflammation (e.g., parasitic infections) and remodeling. This raises the possibility that these molecules play active roles in human antiparasite and anti-infective defense and repair responses. Elias and co-workers (2005) postulate that chitinases can also play a role as sentinels that trigger responses to parasites, infections, and/or antigen challenge, function directly as chemotactic agents or indirectly by (1) inducing other chemokines that attract eosinophils and T cells to sites of parasitic infection; (2) and/or modulate tissue inflammation, immunity, and/or remodeling. It was reported that chitin induces accumulation in tissue of innate immune cells associated with allergy (Reese et al. 2007). Chitinmediated alternative macrophage activation in vivo and the production of leukotriene B4, which was required for optimal immune cell recruitment. It was suggested (Reese et al. 2007) that chitin is a recognition element for tissue infiltration by innate cells implicated in allergic and helminth immunity and this process can be negatively regulated by a vertebrate chitinase. Da Silva et al. (2009) reported that chitin contains size-dependent pathogen-associated molecular patterns that stimulate TLR2, dectin-1, and the mannose receptor; differentially activate NF-kB and spleen tyrosine kinase; and stimulate the production of proand anti-inflammatory cytokines. However, the authors showed that in these experiments, large chitin fragments (α-chitin from Sigma-Aldrich) were inert, while both intermediate-sized chitin (40–70 μm) and small chitin (2–10 μm) stimulated tumor necrosis factor elaboration. In contrast to above-mentioned papers, Ozdemir et al. (2006) published results which showed that treatment with chitin microparticles is protective against lung histopathology in a murine asthma model. The authors used intranasal application of chitin microparticles in new born mice before and after the establishment of a model of allergic asthma. Intranasal application of microgram quantities of this kind of chitin has a beneficial effect in preventing and treating histopathologic changes in the airways of asthmatic mice. Because of the different interpretations regarding to the role of chitin in immune response I took a liberty to ask Prof. Jack Elias (Yale University School of Medicine, USA), a renowned expert on this topic, about his opinion. He answered as follows: I wish I could give you a simple answer. There is no question that chitin has been used in implants and has been reported to be inert in that setting. On the other hand, our studies and the work of others suggest that chitin can drive inflammation. Our best guess is that, once we know enough, we will have defined the criterion that determines these different results. One appears to be the size of the chitin fragment with very large polymers tending to be inert and smaller fragments inducing cytokine production and inflammation. We also have evidence that chitin can act as an adjuvant and an antigen.
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Therefore, the mechanisms that chitin uses to regulate innate and adaptive immunity and the utility of therapies that alter these pathways in the treatment of asthma and other pathogen/parasite and chronic inflammatory diseases is worthy of additional investigations (Da Silva et al. 2009).
30.2.3 Biodegradability The term biodegradable normally refers to a material being susceptible and degraded by enzymes and other bio-based reactions when placed in the biological system. There are several enzyme systems present in humans that can break chitin down. First, macrophages in the body do contain chitinases and lysozyme as part of their phagocytic arsenal (Boot et al. 1995). In addition, the terminal non-reducing N-acetylglucosamine residue of chitin is also exposed to hydrolysis by N-acetyl-βD-glucosaminidase. Escott and Adams (1995) have reported chitinase-type activity in human serum and leukocytes. The chitinolytic activity was demonstrated to be distinct from that of lysozyme. Also, corresponding patent by J. Aerts was published in 1996 and entitled as “A human chitinase, its recombinant production, its use for decomposing chitin, its use in therapy of prophylaxis against infection diseases.” Following studies highlight the ready biodegradability of chitin in vivo using animal model studies. In the report by Saimoto et al. (1997), chitin from squid pen were very sparingly digested by lysozyme, yet were completely adsorbed in 14 days when implanted subcutaneously in dogs. Tomihata and Ikada (1997) investigated the degradation of chitin and chitosan films by lysozyme action and subcutaneous implant in a rat model. The rate of in vivo degradation was high for chitin reducing as the degree of deacetylation increased. Onishi and Machida (1999) reported that 50% deacetylated chitin delivered by intraperitoneal injection into mice was excreted as small molecular weight materials in the urine. These results clearly indicate that chitin is degraded in vivo and does not accumulate in the body.
30.2.4 Biocompatibility Biocompatibility is defined as the ability of a biomaterial to perform with an appropriate host response in a specific application. Biocompatibility is concerned with the interactions that occur between biomaterials and host tissue (Khor 2001). Cell culture methods have been developed to elicit both toxicological aspects as well as cell–biomaterial interactions of biomaterials. Schmidt et al. (1993) investigated the effect of chitin on fibroblast cells proliferation including a macrophage activity assay. Chitin gave a low cell yield relative to control at day 3, decreasing further at day 6 indicating chitin did not support cell proliferation. However, cells exposed to chitin were viable, therefore chitin was non-cytotoxic. Furthermore, chitin was found to have no inhibitory effect on macrophage activity.
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In the use of a feline in vivo model in wound healing, Kojima et al. (1998) investigated the formation of granulation tissue on wounds stimulated by chitin and chitosan-containing implants after a 14-day implant period. Granulation tissue excised from the wound mediated by the chitin-containing implant was thin and displayed minimal foreign body reaction. In comparison, the chitosan exposed wound site was found to produce excessive granulation tissue, extending into the surrounding tissue. The results suggest better stimulation of the wound site by chitin to produce preliminary collagen followed by digestion of this collagen as the wound site reorganized the collagen assembly while chitosan elicited a greater and continuous inflammatory response unfavorable for proper wound healing. Recently, Morganti and Morganti (2008) represented very speculative images (Fig. 30.9) of the practical application of nanofibrillar chitin in wound healing. The authors suggested that chitin nanofibrils exhibit an enormous surface development that allows them to interact with enzymes, platelets, and other cell compounds present in living tissues. Thus, the recovered peculiarity and the ability for faster adequate granulation tissue formation are accompanied by angiogenesis and regular deposition of collagen fibers, with the consequent enhanced and correct repair of dermoepidermal lesions (Fig. 30.9). These nanocrystals therefore have a relevant biologic significance, activating the polymorphonuclear cells and fibroblasts, increasing cytokine production, favoring giant cells migration, and stimulating type IV collagen synthesis. In the case reported above, it seems that chitin induces a type 2 immune response. These responses are known to be associated with tissue fibrosis/healing.
Fig. 30.9 Repairing activity of an innovative medical device based on chitin nanofibrils (adapted from Morganti and Morganti 2008)
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30.2.5 Wound Dressing The wound dressing application is by far the most comprehensively evaluated biomedical application for chitin, touted as one of many natural materials with wound healing augmentation properties. The monomeric unit of chitin, N-acetylglucosamine (NAG), occurs in hyaluronic acid, which is responsible for the formation of fibrous network for protein attachment during wound healing. The positive effect of NAG on wound healing is well known (Yano et al. 1985). Therefore chitin may possess the characteristics of a growth factor and may act as a favorable scaffold for cell attachment and proliferation (Yusof et al. 2001). The origins for chitin being propounded as a candidate for wound healing can be traced back to the breakthrough paper by Prudden et al. (1970). Based on their study of the use of cartilage in accelerating wound healing, they deduced that the active component was NAG. To verify their hunch, chitin obtained from shrimp and fungal sources was applied as topical powders on wounds. Eventually, results confirmed chitin’s accelerating effect in wound healing. The authors proposed that the chitin powders released NAG as a consequence of the breakdown of chitin by the enzyme lysozyme, abundantly present in fresh and healing wounds. It was reported that fungal chitin was resorbed twice as fast as shrimp chitin. In addition, Kishimoto and Wakabayashi (1985) suggested that chitin, when applied to wounds, may attract histiocytes containing abundant lysozyme. At early stage of wounds, the chitin dressing possibly increases fibroblasts which produce the fine type III collagen through histiocytes (Kishimoto and Tamaki 1987). It was reported that chitin also induced type IV collagen and elastic fiber in implanted non-woven fabric used as tendon prosthesis model (Minami et al. 1996). Kojima et al. (2004) investigated effects of chitin and chitosan on collagen synthesis in wound healing. Compared to chitosan, chitin at the higher concentration was found to induce stable collagen synthesis in the early wound healing process. The regular chitin dressing has previously been studied for many donor sites and local burn therapy and the advantages of pain relief and protection of the wound have been acknowledged (Kishimoto and Wakabayashi 1985; Ohshima et al. 1986, 1988, Ohura et al. 1988). Also good results were obtained in the studies that utilize chitin as a non-woven fabric-type dressing (Ohshima et al. 1987). The non-woven dressing was prepared by first making chitin fiber, cutting the fiber to desired length, dispersing the cut fibers in water and binders giving non-woven sheets that were cut to dimensions suitable for a dressing. This chitin dressing was shown to be effective in treating burns and skin ulcers, skin-graft areas, and dressing of donor sites, in some instances accelerating epithelialization and granulation in a sampling of 91 human subjects. In addition, the wounds were kept dry and the dressing adhered to the wound well. It was also possible to apply chitin-based sponge-like material to deep ulcers for longer period. The sponge-sheet reinforced gauze dressing absorbs exudates well; allowing it to permeate to the outside, and its increased thickness resulted in satisfactory hemostasis under pressure on convex surfaces, such as on the thigh or buttock (Ohshima et al. 1991). Dr. Collini described his experience with chitin-based therapy as follows (Collini 1991):
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While at the Mayo Clinic in 1988, I had the opportunity to use the original form of chitin on the skin graft donor sites of 4 patients. My experience was favourable. Each patient had adequate pain relief, the wounds showed suitable drying and complete healing took place in 5–7 days. I found that if topical thrombin was first applied to the donor site, adherence of chitin was almost immediate and no further dressing was necessary. As re-epithelization took place, the chitin painlessly separated from the wound. No side effects were observed and patient satisfaction was dramatic.
Wound healing evaluation of chitin granules referenced to untreated controls has been reported by Okamoto et al. (1995). Macroscopic inspection of the wound site showed complete re-epithelization at 28 days post-procedure in 100% of sites treated with chitin and less that 50% for the controls. The histology assessment based on the presence of inflammatory cells, fibroblasts, and neovasculature detected at the wound site was very low for chitin compared to controls, indicating normal healing occurring. Very recently, Singh et al. (2008) investigated chitin membrane for wound dressing applications. It was shown, that chitin membrane-based dressing provided an effective barrier to microbial penetration and exerted a broad bacteriostatic action against Gram-positive and Gram-negative organisms. γ-irradiation at 25 kGy was found suitable for sterilization of the dressing. No significant change in the thermal behavior because of irradiation at 25 kGy was also observed. In vitro biodegradation of unirradiated and irradiated chitin membranes showed the susceptibility of the chitin dressing to lysozyme. Subcutaneous and scarification test in guinea pigs showed no signs of inflammation. This was further supported by the Finkelstein’s test performed in rabbits (Singh et al. 2008). The chitin membranes were found to have optimal performance characteristics of a wound dressing and showed no toxicity or possible adverse reactions. This modern study shows the chitin dressing as useful adjunct in wound care. For the distribution of chitin-based dressings in Japan following company was responsible: Unitika Ltd., JP Building, 3-4-4, Muromachi, Nihonbashi, Chuo-ku, Tokyo 103, Japan. Also other different chitin-based materials, for example, Beschitin W and F, Chitipack S and F, produced by Eisai, (Japan) are distributed in Japan. The first patents to utilize chitin in wound dressing are published in the 1970s. Here are the main of them Balassa LL, Process for facilitating wound healing with N-acethylated partially depolymerized chitin materials. US Patent 3914413, October 21st, 1975. Balassa LL, Process for promouting wound healing with chitin derivatives, US Patent 3911116, 7th October, 7th, 1975. Balassa LL, Chitin and chitin derivatives for promoting wound healing, US Patent 3903268, 2nd September 2nd, 1975. In a 1990 invention, the dispersion of chitin to form wound dressing was described where the mild shearing of chitin with aqueous hydrogen peroxide or
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bleaches followed by treatment with sodium hydroxide, washed and finally dispersed in water gave a paper like dressing upon drying (Albisetti and Castle 1990). The use of β-chitin derived from squid, laminated to a fish-derived collagen, formed a wound dressing that encouraged cell adherence and proliferation (Takai et al. 1997).
30.2.6 Tissue Engineering Chitin has been applied both neat as well as in combination with calcium compounds in orthopedic applications. Maeda et al. (1984) used chitin in the form of braided filaments, rods, and powders. These substitutes were found to be potentially suitable for sutures and temporary artificial ligaments for the knee joint. Wan et al. (1998) have prepared calcium-containing chitin composites by inducing the precipitation of calcium phosphate from solution onto porous chitin scaffolds. Up to 55% by mass of calcium was deposited onto the chitin scaffold and this approach could be a useful method for the preparation of materials containing chitin and calcium for tissue engineering. One of the strategies in tissue engineering is the use of biodegradable polymers to form a porous matrix or scaffold onto which cells are seeded. In time, the cells proliferate the scaffold to form a tissue system. In the ideal situation, the tissue system after transplantation into the body becomes integrated with the host tissue as the scaffold gradually biodegrades. The preparation of chitin scaffolds for these aims is described by Chow and Khor (2000a, b). Production of chitin scaffolds with controlled pore size and interconnectivity for tissue engineering was described by Weng and Wang (2001). Recently, Freier et al. (2005) reported that chitin supports nerve cell adhesion and neurite outgrowth, making this material a potential candidate for matrices in neural tissue engineering.
30.3 Conclusion Taking above-listed results on poriferan chitin together, our investigations show that the uniquely structured, two- and three-dimensional chitin-based scaffolds found in Verongida skeletons consist of a special type of chitin. Since the processing of chitin is technologically difficult, such chitin-based sponge scaffolds may be of interest for various applications, in particular because it is possible to generate the required amount of material from natural sources, e.g., marine ranching or of sponges. The data compiled above support the claim that chitin of marine origin is biodegradable and biocompatible both in terms of a low-level toxicity to animals and humans and performance in various applications. There are several recent reviews on biomedical applications of chitin which could be recommended. For example, about
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• the biomaterial properties of chitin by Singh and Ray (2000) and by Khor (2001, 2002); • the implantable applications of chitin by Khor and Lim (2003); • a chitin as functional biopolymer by Kurita (2006); • The global requirements for medical applications of chitin by Struszczyk (2006).
References Abe M, Takahashi M, Tokura S et al (2004) Cartilage-scaffold composites produced by bioresorbable β-chitin sponge with cultured rabbit chondrocytes. Tissue Eng 10:585–594 Aerts JMFG (1996) A human chitinase, its recombinant production, its use for decomposing chitin, its use in therapy of prophylaxis against infection diseases, WO/1996/040940 Albisetti CJ, Castle JE (1990) Dispersion of chitin and product therefrom, US Patent 4931551, June 5th Boot RG, Renkema GH, Strijland A et al (1995) Cloning of a cDNA encoding chitotriosidase, a human chitinase produced by macrophages. J Biol Chem 44:26252–26256 Brunner E, Ehrlich H, Schupp P et al (2009) Chitin-based scaffolds are an integral part of the skeleton of the marine demosponge Ianthella basta. J Struct Biol 168:539–547 Chow KS, Khor E (2000a) Novel fabrication of open-pore chitin matrixes. Biomacromolecules 1:61–67 Chow KS, Khor E (2000b) Fabrication of porous chitin matrices. In: Peter MG, Domard A, Muzzarelli RAA (eds) Advances in chitin science, vol 4. Universität Potsdam, Germany, Potsdam Collini FJ (1991) Invited comments. Eur J Plat Surg 14:209–210 Da Silva CA, Chalouni C, Williams A et al (2009) Chitin is a size-dependent regulator of macrophage TNF and IL-10 production. J Immunol 182:3573–3582 Dauby P, Jeuniaux C (1986) Origine exogene de la chitine d’cel’e chez les spongiares. Cah Biol Mar 28:121–129 Ehrlich H, Worch H (2007) Sponges as natural composites: from biomimetic potential to development of new biomaterials. In: Hajdu E (ed) Porifera research: biodiversity, innovation & sustainability. Rio de Janeiro: Museu Nacional Ehrlich H, Krautter M, Hanke T et al (2007a) First evidence of the presence of chitin in skeletons of marine sponges. Part II. Glass sponges (Hexactinellida: Porifera). J Exp Zool (Mol Dev Evol) 308B:473–483 Ehrlich H, Maldonado M, Hanke T et al (2003) Spongins: nanostructural investigations and development of biomimetic material model. VDI Berichte 1803:287–292 Ehrlich H, Maldonado M, Spindler K-D et al (2007b) First evidence of chitin as a component of the skeletal fibers of marine sponges. Part I. Verongidae (Demospongia: Porifera). J Exp Zool (Mol Dev Evol) 308B:347–356 Elias JA, Homer RJ, Hamid Q et al (2005) Chitinases and chitinase-like proteins in Th2 inflammation and asthma. J Allergy Clin Immunol 116:497–500 Escott GM, Adams DJ (1995) Chitinase activity in human serum and leukocytes. Infect Immun 63(12):4770–4773 Freier T, Montenegro R, Koh HS et al (2005) Chitin-based tubes for tissue engineering in the nervous system. Biomaterials 26:4624–4632 Green D (2008) Tissue bionics: examples in biomimetic tissue engineering. Biomed Mater 3:034010 Green D, Walsh D, Mann S et al (2002) The potential of biomimesis in bone tissue engineering: lessons from the design and synthesis of invertebrate skeletons. Bone 30(6):810–815 Khor E (2001) Chitin: fulfilling a biomaterials promise. Elsevier Khor E (2002) Chitin: a biomaterial in waiting. Curr Opin Solid State Mater Sci 6:313–317
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Khor E, Lim LY (2003) Implantable applications of chitin and chitosan. Biomaterials 24: 2339–2349 Kishimoto S, Tamaki K (1987) Immunohistochemical and histochemical observations in the process of burn wound healing in guinea pig skin under chitin membrane dressing. Acta Dermatol (Kyoto) 82:471–479 Kishimoto S, Wakabayashi S (1985) The clinical effect of chitin dressing to donor site and burned ulcer. Kiso to Rhinsyo 19:352–366 Knorr D (1984) Use of chitinous polymers in food. Food Technol 38:85–89 Kojima K, Okamoto Y, Kojima K et al (2004) Effects of chitin and chitosan on collagen synthesis in wound healing. J Vet Med Sci 66:1595–1598 Kojima K, Okamoto Y, Miyatake K et al (1998) Collagen typing of granulation tissue induced by chitin and chitosan. Carbohydrate Polymers 37:109–113 Köll P, Borchers G, Metzger JO (1991) Thermal degradation of chitin and cellulose. J Anal Appl Pyrolysis 19:119–129 Kurita K (2006) Chitin and chitosan: functional biopolymers from marine crustaceans. Mar Biotechnol 8(3):203–226 Kwak BK, Shim HJ, Han SM et al (2005) Chitin-based embolic materials in the renal artery of rabbits: pathologic evaluation of an absorbable particulate agent. Radiology 236:151–158 Love GD, Grosjean E, Stalvies C et al (2009) Fossil steroids record the appearance of demospongiae during the cryogenian period. Nature 457(7230):718–721 Maeda M, Iwase H, Kifune K (1984) Characteristics of chitin for orthopedic use. In: Zikakis JP (ed) Chitin, chitosan and related enzymes. Academic, Orlando, FL Minami S, Okamoto Y, Miyatake K et al (1996) Chitin induces type IV collagen and elastic fiber in implanted non-woven fabric of polyester. Carbohydrate Polymers 29:295–299 Morganti P, Morganti G (2008) Chitin nanofibrils for advanced cosmeceuticals. Clinics Dermatol 26:334–340 Noishiki Y, Takami H, Nishiyama Y et al (2003) Alkali-induced conversion of β-chitin to α-chitin. Biomacromolecules 4:869–899 Ohshima Y, Nishino K, Okuda R et al (1988) Clinical experience of chitin non-woven fabric as wound dressing. Med J Kyoto RCH 9:157–165 Ohshima Y, Nishino K, Okuda R et al (1991) Clinical application of new chitin non-woven fabric and new chitin sponge sheet as wound dressing. Eur J Plat Surg 14:207–211 Ohshima Y, Nishino K, Yonekura Y et al (1986) The clinical application of chitin non-woven fabric in the topical treatment of burns. Jpn J Burn Inj 12:31–36 Ohshima Y, Nishino K, Yonekura Y et al (1987) Clinical applications of chitin non-woven fabric as wound dressing. Eur J Plastic Surg 10:66–69 Ohura T, Urabe H, Ohshima Y (1988) Clinical evaluation of chitin wound dressing for donor sites. Nishi Niti Hifu 50:712–724 Okamoto Y, Minami S, Matsuhashi A et al (1993) Application of polymeric N-acetyl-Dglucosamine (chitin) to veterinary practice. J Vet Med Sci 55:743–743 Okamoto Y, Shibazaki K, Minami S et al (1995) Evaluation of chitin and chitosan on open wound healing in dogs. J Vet Med Sci 57(5):201–205 Onishi H, Machida Y (1999) Biodegradation and distribution of water-soluble chitosan in mice. Biomaterials 20:175–182 Ozdemir C, Yazi D, Aydogan M et al (2006) Treatment with chitin microparticles is protective against lung histopathology in a murine asthma model. Clin Exp Allergy 36:960–968 Philippe H, Derelle R, Lopez P et al (2009) Phylogenomics revives traditional views on deep animal relationships Curr Biol 19(8):706–712 Prudden JF, Migel P, Hanson P et al (1970) The discovery of a potent pure chemical wound-healing accelerator. American J Surg 119:560–564 Reese TA, Liang H-E, Tager AD et al (2007) Chitin induces accumulation in tissue of innate immune cells associated with allergy. Nature 447:92–97
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Reitner J, Wörheide G (2002) Non-lithistid fossil demospongiae? Origins of their palaeobiodiversity and highlights in history of preservation systema porifera. In: Hooper JNA, Rob WM (eds) A guide to the classification of sponges. Van Soest Kluwer Academic/Plenum, New York Saimoto H, Takamori Y, Morimoto M et al (1997) Biodegradation of chitin with enzymes and vital components. Macromol Symp 120:11–18 Schmidt RJ, Chung LY, Andrews AM et al (1993) Biocompatibility of wound management products: a study of the effects of various polysaccharides on Murine L929 fibroblast proliferation and macrophage respiratory burst. J Pharm Pharmacol 45:508–513 Simpson TL (1984) The cell biology of sponges. Springer, New York Singh DK, Ray AR (2000) Biomedical applications of chitin, chitosan, and their derivatives. Rev Macromol Chem Phys C40(1):69–83 Singh R, Chacharkar MP, Mathur AK (2008) Chitin membrane for wound dressing application – preparation, characterisation and toxicological evaluation. Int Wound J 5:665–673 Stawski D, Rabiej S, Herczynska L et al (2008) Thermogravimetric analysis of chitins of different origin. J Therm Anal Calorim 93:489–494 Struszczyk MH (2006) Global requirements for medical applications of chitin and its derivatives. Polish Chitin Society, Monograph XI 95–102 Suzuki D, Takahashi M, Abe M , Sarukawa J, Tamura H, Tokura S, Kurahashi Y, Nagano A (2008) Comparison of various mixtures of beta-chitin and chitosan as a scaffold for three-dimensional culture of rabbit chondrocytes. J Mater Sci Med 19:1307–1315 Takai M, Shimizu Y, Shimizu J et al (1997) Wound healing composition using squid chitin and fish skin collagen, US Patent 5698228, December 16th Tanaka Y, Tanioka S, Tanaka M et al (1997) Effects of chitin and chitosan particles on BALB/c mice by oral and parenteral administration. Biomaterials 18:591–595 Tomihata K, Ikada Y (1997) In vitro and in vivo degradation of films of chitin and its deacetylated derivatives. Biomaterials 18:567–575 Wan ACA, Khor E, Hastings GW (1998) Preparation of a chitin-apatite composite by in situ precipitation onto porous chitin scaffolds. J Biomed Mater Res: Appl Biomater 41:541–548 Weng J, Wang M (2001) Producing chitin scaffolds with controlled pore size and interconnectivity for tissue engineering. J Mat Sci Lett 20:1401–1403 Yano H, Iriyama K, Nishiwaki H et al (1985) Effect of N-acetylglucosamine on wound healing in rats. Mie Med J 35:53–56 Yusof NLBM, Lim LY, Khor E (2001) Preparation and characterization of chitin beads as a wound dressing precursor. J Biomed Mater Res 54:59–68 Zhu Z, Zheng T, Homer RJ et al (2004) Acidic mammalian chitinase in asthmatic TH2 inflammation and IL-13 pathway activation. Science 304:1678–1682
Chapter 31
Marine Collagens
Abstract As a family of proteins with unique structural features, marine invertebrate collagens have been a focus of structure–function correlation studies as well as studies interrelating successive levels of structural organization, from the amino acid sequence to the anatomically defined fibril. Structural and biochemical peculiarities of marine invertebrates collagens isolated from sponges, jellyfishes, molluscs, and echinoderms as well as perspectives of their applications are described and analyzed here. Collagens share a rod-like triple-helical segment as a typical structural element but differ otherwise in their size, the presence of triple helix imperfections and globular domains, self-assembly patterns, and functional roles. These structural proteins of fundamental evolutionary significance are present both in marine invertebrates as well in vertebrate taxa; however, here I will discuss only collagens of the invertebrates origin. As a family of proteins with unique structural features, marine invertebrate collagens have been a focus of structure–function correlation studies as well as studies interrelating successive levels of structural organization, from the amino acid sequence to the anatomically defined fibril. Several review papers (e.g., Adams 1978; Bailey 1968; Engel 1997; Exposito et al. 2002; Garrone 1999; Gross et al. 1956; Tanzer 1978) and books (e.g., Bairati and Garrone 1985; Garrone 1978) are dedicated especially to collagens of marine invertebrate organisms, including sponges, corals, worms, molluscs, echinoderms, and crustaceans. The supporting frameworks provided by collagen assume a variety of forms and, indeed, the protein molecule itself shows remarkable variation in size, shape, and chemical nature, albeit within the bounds of a characteristic wide-angle X-ray diffraction pattern and somewhat restricted amino acid composition (Tanzer 1978). While, in general, similarities between vertebrate and invertebrate collagens appear more impressive than the differences, it should be noted that unique features of collagen structure and synthesis have been described in specific groups of invertebrates (Adams 1978). According to the modern point of view (Exposito et al. 2008) ancestral fibrillar collagen gene arose at the dawn of the Metazoa, before the divergence of sponge and eumetazoan lineages. The duplication events leading to the formation
H. Ehrlich, Biological Materials of Marine Origin, Biologically-Inspired Systems 1, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9130-7_31,
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of the three fibrillar collagen clades (A, B, and C) occurred before the eumetazoan radiation. Interestingly, only the B clade fibrillar collagens preserved their characteristic modular structure from sponge to human. This observation is compatible with the suggested primordial function of type V/XI fibrillar collagens in the initiation of the formation of the collagen fibrils. In the 1970s through 1980s, academics and commercial researchers began to use collagen as a biomaterial in a variety of connective tissue applications because of its excellent biocompatibility; low antigenicity; high biodegradability; and good mechanical, hemostatic, and cell-binding properties (Stenzel et al. 1974). Recently, Uzel and Buechler (2009) make a link between biochemical parameters (amino acid sequence) and the associated biologically relevant functional properties (elasticity, stiffness, energy storage capacity) of collagen. The key insight reported in their work is that the type of amino acid motif that defines the tropocollagen molecule has significant effects on its mechanical properties. Therefore, it can be hypothesized that diversity of collagen polyforms determined the futures of their function, even within the same organism. The relative complexity of the marine invertebrate collagens and the difficulty in their purification and characterization has hindered continued progress in their research. However, there are more than enough examples for inspiring materials scientists, practically in each order of marine invertebrates. Thus, here I will try to represent and discuss the most spectacular events that are useful for biomimetic and materials science, starting with sponges as the lowest marine invertebrates.
31.1 Poriferan Collagens Sponges are usually considered as representing the lowest level of multicellular invertebrates. They contain a quantitatively well-developed connective tissue rich in different forms of collagen. The detailed study of these intercellular macromolecules may lead to a better understanding of the role that such macromolecules play in the spatiotemporal organization of cells and tissues. Sponges also display considerable polymorphism with respect to their collagenous structures. The collagen in sponges is highly insoluble, and therefore it has been impossible to carry out any detailed biochemical analysis up today. According to the morphological studies, the collagenous fibrils dispersed throughout the intracellular matrix form the skeletal material of sponges. Cuticular structures have been found in some sponges, but their molecular composition has not been determined (Garrone 1978). It was recognized very early that collagen fibers can present quite different morphological aspects in sponges (Junqua et al. 1974). Gross and co-workers isolated from Spongia graniniea two distinct forms of collagen, which they called spongin A and spongin B (Gross et al. 1956). The first corresponds to fine intercellular collagen fibrils, visible only by electron microscopy. The second, spongin B, forms macroscopically visible ramified fibers, which are characteristic of horny sponges (Garrone 1978). Above (see Chapter 13) I discussed the state of the art knowledge regarding spongins.
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In particular, the collagen from demosponge Chondrosia reniformis has received the focused attention of researches since 1970, as reviewed by Garrone et al. (1975). C. reniformis collagen possesses some interesting features. Slices incubated with collagenase are not modified; even after 48 h of incubation they do not show any changes in the aspect, consistency, or fine structure of fibrils. After collagenase treatment, the isolated collagen fibrils are not digested either, and they do not show any damaged aspects when observed with the electron microscope. Garrone et al. (1975) described the mechanical properties of this collagen as follows. Chondrosia cortex, though less resistant than calf skin, for example, shows mechanical properties of the same order as those of bovine nasal cartilage (Young modulus 150–250 kg/cm2 and 100–250 kg/cm2 , respectively). The outside cell layer that covers sponges is often loose and in many circumstances the collagen fibrils can be in direct contact with water. Moreover, ionic extracellular concentrations are not very different from those of the external milieu. One could expect that in such an environment, the extracellular molecular assemblies would be strengthened. The insolubility of sponge collagens, as of other collagens that are in contact with seawater such as sea-urchin skeletons, could reflect this reinforcement. This must be even stronger in Chondrosia, where the functions responsible for shape maintenance and tissue compactness are due to a great extent to collagen fibrils. Within a bundle, the individual fibrils appear tightly packed and thus the mechanical strength of the whole tissue is then enhanced. Lastly, what makes this highly resistant framework most original is its great malleability when subjected to progressive tensions. The whole sponge can slowly become flat and slide to avoid a compression or stretch itself to a slender thread under a continuous stress. Such a creeping behavior of a fibrous and living material constitutes a remarkable example for the study of mechanical stresses as morphogenetic factors (Garrone et al. 1975). In contrast to horny sponges (Demospongiae), where the silica spicules are “glued together” by “collagenous cement” made of microfibrils, fibrillar collagen is also present within the spicules of the older glass sponges (Hexactinellidae) and acts as a template for the biosilicification process. The fact that fibrillar collagen is present in glass sponges, which date back to the Cambrian (600 million years ago), highlights collagen’s fundamental role in the evolution of the earliest metazoan and their skeletons. It can be deduced that living organisms at the earliest stages of metazoan evolution were the first to form highly structured silica networks controlled by collagen; the same protein was later involved in bone and tooth formation. Owing to its low immunogenicity, natural collagen today is applied in medicine, surgery, and cosmetics, e.g., as shields, injectable dispersions, sponges, and microparticles. For these purposes, generally an acid-supported isolation of the collagen from calf skin is performed. However, bovine collagen may elicit antigenic responses and varies from batch to batch. Reconsideration of bovine collagen as a main source arose with the risks of BSE (bovine spongiform encephalopathy) and TSE (transmissible spongiform encephalopathy). In 2007 in our paper related to
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Fig. 31.1 AFM images: nanotopography of C. reniformis collagen fibers (a, b, c) (images courtesy Sascha Heinemann)
ultrastructure of C. reniformis collagen, we made the following remark (Heinemann et al. 2007a): “Excepting porcine collagen for example, maybe alternatives can be found in sponge collagens.” However, today because of the Swine Influenza, I have significant doubts that porcine collagen is greatly welcomed by the people. Recent interest arose with the application of Chondrosia collagen (Fig. 31.1) as an organic template for silicification in vitro. (Heinemann et al. 2006, 2007a, b, c, 2008). It has been recognized that the mechanical properties of the biomimetically inspired hybrid xerogels can be improved remarkably with the presence of the collagen. Different approaches of application of sponge collagens in tissue engineering are recently reported by David Green (2008).
31.2 Coelenterates Collagens Representatives of different Coelenterata phyla, including Cnidaria and Ctenophora, possess collagen polymorphism. Cnidaria: invertebrate phylum that contains animals such as anemones and corals, as well as a range of jellyfish, including large scyphozoan jellyfish (up to 2 m in diameter) and smaller hydromedusae (only a few millimeter in diameter). These jellyfish generally have alternating polyp and medusa life stages. Stinging cells or cnidoblasts (nematocysts) concentrated in the tentacles and mouth appendages are used to poison or stun prey. Ctenophora are an invertebrate phylum, sometimes called comb jellies or sea gooseberries, that propel themselves through the sequential beating of their rows of cilia (comb rows). This phyla has colloblasts, cells which discharge a glue to ensnare prey. Ctenophores are holoplanktonic, remaining in the plankton their entire life (Richardson et al. 2009). Coelenterate collagens have been reviewed in detail by Franc (1985). Collagens of sea anemones, however, have been examined several times, both because this group contains a thick extracellular layer, the mesoglea, composed primarily of collagen and water, and because of the presumed primitive stage in animal evolution of the coelenterates (Piez and Gross 1959)
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The anthozoan collagens differ from other invertebrate collagens in the proportions of hydroxylated proline and lysine, the total of basic amino acids, and the lower total of imino acids (Young 1971) These differences may be true of all coelenterate collagens if the studies of Piez and Gross (1959) on the float of the siphonophore Physalia and of Chapman (1973) on the mesoglea of the hydroid Corynactis are representative of other coelenterate groups. The axial skeletal rod of Veretillum cynomorium was the subject of an extensive study by Franc et al. (1974). These authors demonstrated that the organic phase of this calcified structure is at least partly collagenous in structure, as earlier X-ray diffraction studies (Marks et al. 1949) had shown for two other pennatulid species. Ultrastructural investigations of the fibrillar matrix showed that although some banded fibrils were present, the most abundant fibril type only rarely showed a transverse banding and therefore could not be unequivocally identified with the physicochemically identified collagen. However, the collagenous nature of these fibrils has since been confirmed by negative staining, which reveals a distinct banding in isolated fibrils, and by electron diffraction of frozen hydrated fibrils, which gives a diffraction pattern comparable to that of vertebrate collagen. The axial rod of Veretillum is the only authenticated example of a collagenous matrix calcified with calcite, as fibrillar collagen previously thought to be present in sponge and echinoderm skeletons (Travis et al. 1967) was undoubtedly extraskeletal in origin (Ledger and Jones 1977; Wilbur 1976). The axis is thus of immediate interest as a naturally occurring contrast to the collagen–hydroxyapatite association typical of vertebrate mineralized tissues (Ledger and Franc 1978). Octocoral axes are composed of a limited number of structural elements. Principally they contain variable amounts of flexible collagen fibers, embedded in a pliant, proteinaceous matrix, and minerals that exist in a variety of crystal forms and aggregate shapes (Kingsley and Watabe 1984). Usually the major component of the axial skeleton of gorgonians (Coelencerata: Octocorallia) is the gorgonin, composed mainly of collagen fibers in a proteinaceous matrix (Leversee 1969). The protein matrix is largely uncharacterized, but the collagen, though modified, (Goldberg 1974) is characterized as collagen (see also Chapter 14). Unlike vertebrate osseous tissues that consist of hydroxyapatite and collagen, collagen has not been associated with the formation of invertebrate calcium carbonate structures. However, decalcification of the calcareous spicules from the gorgonian Leptogorgia virgulata reveals an organic matrix that may be divided into water insoluble and soluble fractions. As reported by Kingsley et al. (1990), the insoluble fraction displays characteristics typical of collagen, which is an unusual component of an invertebrate calcium carbonate structure. This matrix fraction exhibits a collagenous amino acid profile and behavior upon SDS-PAGE. Furthermore, the reducible cross-link, dihydroxylysinonorleucine (DHLNL), is detected in this fraction. The composition of the matrix varies seasonally; i.e., the collagenous composition is most prevalent in the summer. These results indicate that the insoluble matrix is a dynamic structure.
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Of course, it very hard to believe that there are some correlations between coelenterates collagen and climate changes, but in the case of giant jellyfish species Nemopilema nomurai, it is true (Pauly et al. 2009; Purcell et al. 2007). According to excellent review recently published by Richardson et al. (2009), human-induced stresses of overfishing, eutrophication, climate change, translocation, and habitat modification appear to be promoting jellyfish (pelagic cnidarian and ctenophore) blooms to the detriment of other marine organisms. Mounting evidence suggests that the structure of pelagic ecosystems can change rapidly from one that is dominated by fish (that keep jellyfish in check through competition or predation) to a less desirable gelatinous state, with lasting ecological, economic, and social consequences. Jellyfish is a well-known source of collagen and especially of edible collagen. Regardless of their size or shape, most jellyfish are very fragile and contain more than 95% of water, with more than 40% of the dry weight of the edible jellyfish being collagen (Miura and Kimura 1985; Nagai et al. 1999). Edible jellyfish comprise five to seven species, including Lobonema smithi, Lobonemoides gracilis, Rhopilema esculentum, Stomolophus meleagris, and N. nomurai (Nishimoto et al. 2008). A large portion of the body, called the umbrella, is composed of both mesogloea and outer skin and the latter is separated into outer and inner membranes, known as exumbrella and subumbrella, respectively (Nagai et al. 1999). Different polymorphs of collagen can be isolated from exumbrella and subumbrella. Jellyfishes have been eaten by humans since 300 AD in China, and about 425,000 tonnes/year are harvested globally (1996–2005) for human consumption in Southeast Asia. Jellyfish fisheries exist in 15 countries, including China, India, Indonesia, Japan, Malaysia, and the Philippines, with export industries in Australia and the USA (Richardson et al. 2009). The giant jellyfish N. nomurai is a large Scyphozoan that is capable of attaining a bell diameter of up to 1.5 m (Fig. 31.2). It mainly inhabits the Bohai Sea, Yellow Sea, and northern East China Sea and was only rarely reported in the Japanese sea area during the twentieth century. However, each year between 2002 and 2007, dense aggregations of this jellyfish have been reported in the seas surrounding Japan (Honda et al. 2005, 2009; Honda and Watanabe 2007; Iizumi 2004). If considerable amounts of collagen were obtainable from such jellyfish, which can weigh up to 200 kg, they would have potential as an important source for collagen and development of this unutilized resource would follow. Jellyfish processing is carried out as follows (Hsieh et al. 2001). Fresh jellyfish readily spoil at ambient temperature. Therefore, processing of jellyfish is carried out preferably within a few hours of capture, while the animals are still alive. The body of jellyfish consists of a hemispherical transparent umbrella. The mouth is on the undersurface of the umbrella and is protected by fused oral arms, commonly known as “legs.” The umbrella and oral arms of jellyfish are separated immediately after catching. Jellyfish are cleaned with seawater, scraped to remove mucus membranes and gonadal material. Both umbrella and oral arms are used in processing. Traditional methods of processing involve a stepwise reduction of the water content using salt (NaCl) and alum (AlK[SO4 ]2 ·12H2 O). A salt mix
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Fig. 31.2 The “living collagen bomb” Nemopilema nomurai (a, b) is a large jellyfish that is capable of attaining a bell diameter of up to 1.5 m (c) (images courtesy Naoto Honda)
containing about 10% alum is used for initial salting of jellyfish, using about 1 kg salt–alum mix for 8–10 kg of jellyfish. Salted jellyfish are then left in the brine for 3–4 days, followed by several transfers to another container salted with a fresh mix containing a smaller amount of alum. The salted jellyfish can then be heaped and left to dry on a draining rack at room temperature for 2 days and the heap is turned upside down several times during that period to allow excess water to drain out through compression from its own weight. The entire process requires 20–40 days to produce a salted final product with 60–70% moisture and 16–25% salt. The processed jellyfish has a yield of about 7–10% of the raw weight depending on the species and processing formula. Preservation of jellyfish in a mixture of salt and alum is necessary to obtain products of desirable structure and texture. Alum reduces pH, acts as a disinfectant and a hardening agent, giving and maintaining a firm texture by precipitating protein. Salt aids in reducing the water content and in keeping the product microbially stable. Cured jellyfish has a special crunchy and crispy texture that makes it unique. The salted jellyfish has a stable shelf life of up to 1 year at room temperature. The shelf life can be increased to more than 2 years if the product is kept cool; however, freezing spoils the product, which dries out completely and becomes covered with wrinkles (Hsieh et al. 2001). Numerous modern separation and purification technologies for jellyfish collagen have also been recently reported (Jin 2008; Shen et al. 2009). Except use as a food, jellyfish collagen has been investigated for application in tissue engineering. Thus, Song et al. (2006) reported on porous scaffolds composed
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of the jellyfish collagen from an edible jellyfish, Stomolophus nomurai meleagris. Scaffolds were prepared by freeze-drying and cross-linking with 1-ethyl(3-3 dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide. Also the hybrid scaffolds with an average pore size of 150–750 μm, composed of a porous collagen matrix and a fibrous polylactide-co-glycolide (PLGA) layer, has been developed (Jeong et al. 2007). The results indicated that jellyfish collagen exhibited higher cell viability than other naturally derived biomaterials, including bovine collagen, gelatin, hyaluronic acid, and glucan. Jellyfish collagen scaffolds also had a highly porous and interconnected pore structure, which is useful for a high-density cell seeding, serving as an efficient nutrient and oxygen supply to the cells cultured in the three-dimensional matrices. This collagen was found to induce an immune response at least comparable to those caused by bovine collagen and gelatin. Recently, Nishimoto et al. (2008) have demonstrated that collagen from giant jellyfish N. nomurai simulated immunoglobulin and cytokine production by human– human hybridoma line HB4C5 cells and by human peripheral blood lymphocytes (hPBL). These results also suggest that collagen from jellyfish stimulated not only the transcription activity but also the translation activity for enhanced immunoglobulin and cytokine production. Hsieh et al. (2001) reported experimental results which demonstrated that laboratory rats fed with low doses of jellyfish collagen had significantly (p < 0.05) reduced incidence, onset, and severity of antigen-induced arthritis, in a model that shares clinical, histological, immunological, and genetic features with human rheumatoid arthritis. Unfortunately, attention to jellyfish collagen and its application in recent time are determined mostly by the dramatic changes in marine environments. Jellyfish have a suite of successful attributes that enable them to survive in disturbed marine ecosystems and to rebound rapidly as conditions improve. These attributes include a broad diet, fast growth rates, the ability to shrink when starved, the capacity to fragment and regenerate, and the ability to tolerate hypoxia. These are characteristic of opportunistic “weed species” and would appear to give jellyfish an edge over fish in environments stressed by climate change, eutrophication, and overfishing (Richardson et al. 2009). However, these animals have no risk of infection with BSE (bovine spongiform encephalopathy) or FMD (foot and mouth disease) and therefore jellyfish collagens definitively have advantages in biological safety, as well as the relatively simple procedure for their isolation.
31.3 Molluscs Collagens Among the invertebrates, the molluscs exhibit a wide evolutionary history and possess connective tissues typical of vertebrates, which define the shape of the body (Bairati 1985). Different types of collagens were isolated and described from bivalve molluscs (Mizuta et al. 2004, 2005) as well as from cephalopods (Bairati et al. 1989,
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1999; Mizuta et al. 1994a, 1994b, 2009; Nagai et al. 2001; Rigo and Bairati 1998, 2002; Shadwick 1985; Sivakumar and Chandrakasan 1998; Sivakumar et al. 2003). The cephalopods have evolved along the same lines as that of the fast moving predatory aquatic vertebrates and share substantial structural similarities with vertebrates, which include the development of a complex nervous system, a brain protected by a cartilaginous case, and an image-perceiving eye. For these reasons, the cephalopod connective tissue is ideal to understand collagen in relation to structure–function and evolution (Sivakumar et al. 2003). Different polymorphic collagen chains have been described for various representatives of Cephalopoda. Thus, it was reported the presence of a heterotrimeric collagen in cranial cartilage of the cuttlefish, Sepia officinalis, and its possible similarity to vertebrate minor collagens (Sivakumar and Chandrakasan 1998). Very unusual collagen was also isolated by Rigo and Bairati (2002) from the extracellular matrix of S. officinalis cartilage. Recently, two genetically distinct types of collagen have been identified in the mantle muscle and skin of the common squid Todarodes pacificus by Mizuta and coworkers (Mizuta et al. 2009). The major collagen type, based on quantity, has been denoted Type SQ-I and accounts for about 90% of the total collagen in the common squid. This collagen type shows properties similar to those of vertebrate Type I collagen, while the relatively minor collagen type, denoted Type SQ-II, resembles vertebrate Type V collagen in terms of solubility and chemical composition. Both SQ-I and SQ-II were isolated from pepsin-solubilized collagen preparations and found to possess heterotrimeric chain compositions, i.e., [α1(SQI)]2α2(SQ-I) and [α1(SQ-II)]2α2(SQ-II), respectively. In contrast to acid-based isolation of collagens from jellyfish (Nishimoto et al. 2008), the same procedure using cephalopods has been carried out with alkali extraction. For example, the mantle muscles of five cephalopod species, T. pacificus, Photololigo edulis, Sepioteuthis lessoniana, Sepia esculenta, and Sepia longipes, were extracted with 0.1 M NaOH to prepare crude collagen fiber, called the “residue after alkali extraction” (RS-AL) (Mizuta et al. 2009). The collagens showed a similar tendency in solubility, which gradually increased depending on the treating temperature, and the values at 40–90◦ C were constantly less than 47% for all the species examined. In addition, the collagens were estimated to denature in the approximate temperature range of 37.5–42.5◦ C. These results suggest that the collagens in RS-AL from these species may have relatively high resistance to hot water extraction even after their denaturation.
31.4 Echinoderm Collagens During analysis of the literature related to the collagens of Echinodermata origin, I leapt to the conclusion that two scientific directions dominate in this topic: food chemistry and biomechanics. The first one is traditionally in the focus of our colleagues from China and Japan. There are more than 100 species of sea cucumbers belonging to the phylum Echinodermata, growing along Japanese and Chinese
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coasts. Among them, over 20 species are edible (Saito et al. 2002). In general, the body wall of uncooked sea cucumber that is utilized as a vinegared dish is very hard to masticate. After cooking, however, it becomes as tender as jelly. The body wall of Stichopus japonicus is enjoyed by people in Japan and China as a hors d’oeuvre or a main dish, owing to its unique elasticity and palatability. S. japonicus has a strong potential for commercial use, because it is easily obtainable, inexpensive, and easy to cook. The intestine of S. japonicus is also consumed as one of the fermented seafoods, called konowata in Japanese. Moreover, the boiled-dried sea cucumber, called iriko in Japanese, is produced as a preserved food. The major component of these edible portions is a collagenous fiber (Bailey et al. 1982; Cui et al. 2007; Matsumura 1974), but little is known about the chemical properties and subunit structure of the collagen molecule because of its extreme insolubility. Features of the collagens isolation and solubilization from such representatives of echinoderms as starfish Asterias amurensis (Kimura et al. 1993) or purple sea urchin (Anthocidaris crassispina) test (Nagai and Suzuki 2000) have also been reported. The second direction that is related to materials properties of echinoderms collagens is dominated by scientists from the USA and Europe. Echinoderms have the ability to change the mechanical properties of their collagen fibrillar networks in a way that is interfaced with their neural systems over physiologically relevant timescales. As recently reviewed by Eppell et al. (2006), the major difference between echinoderm collagen fibrils and other animals is therefore at the systemic level (Thurmond and Trotter 1996; Trotter et al. 1994). Sea cucumber fibrils are similar to those found in vertebrates having the same length, assembling with the same repeat period, possessing the same gap/overlap ratio (Trotter et al. 1994) and possessing the same cross-linking chemistry (Butler et al. 1987). The habit of echinoderm fibrils is spindle shaped, rather than cylindrical as found in mammals. In addition, the collagen amino and carboxy termini are arranged in a bipolar manner with the center of symmetry existing at the middle of the long axis of the spindle, rather than monopolar as found in mammals. This spindle structure can also be obtained using mammalian collagen monomers to reconstitute synthetic fibrils (Rainey et al. 2002). Clear evidence of proteoglycans utilized to aggregate echinoderm fibrils has been found, but these molecules are all removed in the purification procedure used to obtain the fibrils we measured (Graham et al. 2004). Thus, sea cucumber fibrils possess the characteristics typical of vertebrate collagen fibrils; therefore cucumber fibrils offer a suitable model for the analysis of fibril properties which can be applied to higher ordered structures based on fibril subunits. Because of the specific structural features of a single type I collagen fibril isolated from the sea cucumber, Cucumaria frondosa, it was possible to carry out nanomeasurements with micro-devices as reported by Eppell et al. (2006). They used the device to obtain the first stress–strain curve of an isolated collagen fibril producing the modulus and some fatigue properties of this soft nanofibril.
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Remarkably, these preliminary true stress–Eulerian strain curves suggested a tensile strength of the fibrils that may be greater than 1.0 GPa (corresponding to a nominal stress of 0.7 GPa). The elastic modulus of the native fibrils was measured also by radial nanoindentation and found to be 1–2 GPa (Heim et al. 2006). These data are higher than previously reported values, most likely resulting from the partially dehydrated conditions under which the measurements were performed. The mutable collagenous tissue (MCT) is an another intriguing topic that is related to echinoderms collagens. In 1884, C. F. Jickeli described the curious, mutable properties of echinoderm connective tissues (Jickeli 1884). A slow-moving debate ensued over the next four decades as to whether these tissues were primarily extracellular connective tissues, which is how they appear under the microscope, or some new type of muscle. The reason for the confusion was that echinoderms can rapidly and reversibly alter the stiffness of their collagenous connective tissues, prompting later researchers to dub these tissues “mutable collagenous tissues” or MCTs (see for review Szulgit 2007). The mutable collagenous tissue (MCT) of echinoderms has the capacity to change its mechanical properties in a timescale ranging from less than 1 s to a few minutes under the influence of the nervous system (Wilkie 1984). Although accumulating evidence indicates that the mechanical adaptability of MCT is due primarily to the modulation of interactions between components of the extracellular matrix, the presence of muscle in a few mutable collagenous structures has led some workers to suggest that contractile cells may play an important role in the phenomenon of variable tensility and to call for a re-evaluation of the whole MCT concept (Wilkie 2002; Wilkie et al. 2004). The most spectacular manifestation of MCT variable tensility—the irreversible destabilization that occurs during autotomy and holothurian dermis “melting”— cannot possibly be attributed to the activities of muscle cells. Muscle cells have not been detected in most confirmed mutable collagenous structures that show reversible stiffening and de-stiffening. Investigations of the few mutable collagenous structures in which muscle has been detected have demonstrated that the latter cannot account for the variability of their passive mechanical properties. For example, to be responsible for the maximally stiffened state of these structures, intraligamental muscle fibers would have to develop a tensile strength many times greater than that of the strongest muscle known heretofore. It is possible, however, that the muscle fibers affect the passive mechanical properties of these structures when the extracellular matrix is in its low-stiffness state. Pharmacological data provide no evidence for the involvement of muscle in the variable tensility of MCT, although they reveal features common to the control pathways regulating contractile and collagenous components. It was suggested (Wilkie 2005) that there are no grounds for reformulating the current concept of mutable collagenous tissue to include a role for intraligamental muscle.
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31.5 Conclusion To describe diversity as well as structural and biochemical peculiarities of marine invertebrates collagens, I would need space for a separate book. Therefore, I perform only brief analysis of selected examples listed above. Collagens of annelids origin or those discovered in glass sponges are described in corresponding chapters as well. Collagens are of interest for materials science, especially from biomechanics point of view. The mechanical response of a biological material to applied forces reflects deformation mechanisms occurring within a hierarchical architecture extending over several distinct length scales. Characterizing and in turn predicting the behavior of such a material requires an understanding of the mechanical properties of the substructures within the hierarchy, the interaction between the substructures, and the relative influence of each substructure on the overall behavior (Eppell et al. 2006). Progress in development of highly sensitive methods like different nanoindentation techniques allow us today to use even individual collagen fibrils isolated from marine invertebrates as models for the better understanding of unique mechanisms of biomechanics. Due to the ease at which disease spreads in the higher order animals, the biotechnological and biomimetical potential for marine invertebrates collagens is beyond doubt.
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Kimura S, Omura Y, Ishida M et al (1993) Molecular characterization of fibrillar collagen from the body wall of starfish Asterias amurensis. Comp Biochem Physiol 104B(4):663–668 Kingsley R, Tsuzaki M, Watabe N et al (1990) Collagen in the spicule organic matrix of the gorgonian Leptogorgia virgulata. Biol Bull 179:207–213 Kingsley RJ, Watabe N (1984) Calcium uptake in the gorgonian Leptogorgia virgulata. The effects of ATPasc inhibitors. Comp Biochem Physiol 79A:487–491 Ledger PW, Jones WC (1977) Spicule formation in the calcareous sponge Sycon ciliatum. Cell Tiss Res 181:553–567 Ledger PW, Franc S (1978) Calcification of the collagenous axial skeleton of Veretillum cynomorium Pall. (Cnidaria: Pennatulacea). Cell Tissue Res 192:249–266 Leversee GJ (1969) Composition and function of the axial skeleton in the gorgonian coral Leptogorgia virgulata. Am Zool 9:11–15 Marks MH, Bear RS, Blake CH (1949) X-ray diffraction evidence of collagen-type protein fibres in the Echinodermata, Coelenterata and Porifera. J Exp Zool 111:55–78 Matsumura T (1974) Collagen fibrils of the sea cucumber, Stichopus japonicus: purification and morphological study. Conn Tiss Res 2(2):117–125 Miura S, Kimura S (1985) Jellyfish mesogloea collagen. Characterization of molecules as a1a2a3 heterotetramers. J Biol Chem 260:15352–15356 Mizuta S, Miyagi T, Yoshinaka R (2005) Characterization of the quantitatively major collagen in the mantle of oyster Crassostrea gigas. Fisheries Sci 71:229–235 Mizuta S, Miyagi T, Nishimiya T et al (2004) Partial characterization of collagen in several bivalve mollusks. Food Chem 87:83–88 Mizuta S, Tanaka T, Yokoyama Y et al (2009) Hot-water solubility of mantle collagens in several cephalopod molluscs. Fish Sci 75:1337–1344 Mizuta S, Yoshinaka R, Sato M et al (1994a) Isolation and partial characterization of two distinct types of collagen in the muscle and skin of the squid Todarodes pacificus. Fish Sci 60(4): 467–471 Mizuta S, Yoshinaka R, Sato M et al (1994b) Subunit composition of two distinct types of collagen in the muscle of the squid Todarodes pacificus. Fish Sci 60(5):597–602 Nagai T, Suzuki N (2000) Partial characterization of collagen from purple sea urchin (Anthocidaris crassispina) test. Int J Food Sci Technol 35(5):487–501 Nagai T, Ogawa T, Nakamura T et al (1999) Collagen of edible jellyfish exumbrella. J Sci Food Agric 79:855–858 Nagai T, Yamashita E, Taniguchi K et al (2001) Isolation and characterisation of collagen from the outer skin waste material of cuttlefish (Sepia lycidas). Food Chem 72:425–429 Nishimoto S, Goto Y, Morishige H et al (2008) Mode of action of the immunostimulatory effect of collagen from jellyfish. Biosci Biotechnol Biochem 72 (11):2806–2814 Pauly D et al (2009) Jellyfish in ecosystems, online databases and ecosystem models. Hydrobiologia 616:67–85 Piez KA, Gross J (1959) The amino acid composition and morphology of some invertebrate and vertebrate collagens. Acta 34:24–39 Purcell JE et al (2007) Anthropogenic causes of jellyfish blooms and their direct consequences for humans: a review. Mar Ecol Prog Ser 350:153–174 Rainey JK, Wen CK, Goh MC (2002) Hierarchical assembly and the onset of banding in fibrous long spacing collagen revealed by atomic force microscopy. Matrix Biol 21:647–660 Richardson AJ, Bakun A, Hays GC et al (2009) The jellyfish joyride: causes, consequences and management responses to a more gelatinous future. Trends Ecol Evol 24:312–322 Rigo C, Bairati A (1998) Use of rotary shadowing electron microscopy to investigate the collagen fibrils in the extracellular matrix of cuttle fish (Sepia officinalis) and chicken cartilage. Tissue Cell 30:112–117 Rigo C, Bairati A (2002) A new collagen from the extracellular matrix of Sepia officinalis cartilage. Cell Tissue Res 310:253–256 Saito M, Kunisaki N, Urano N et al (2002) Collagen as the major edible component of sea cucumber (Stichopus japonicus). J Food Sci 67:1319–1322
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Shadwick RE (1985) Crosslinking and chemical characterization of cephalopod collagens. In: Bairati A, Garrone R (eds) Biology of invertebrate and lower vertebrate collagens. Plenum, New York Shen J, Li D, Jiang F et al (2009) Purification and concentration of collagen by charged ultrafiltration membrane of hydrophilic polyacrylonitrile blend. Sep Purif Technol 66:257–262 Sivakumar P, Chandrakasan G (1998) Occurrence of a novel collagen with three distinct chains in the cranial cartilage of the squid, Sepia officinalis: comparison with shark cartilage collagen. Biochim Biophys Acta 1381:161–169 Sivakumar P, Suguna L, Chandrakasan G (2003) Similarity between the major collagens of cuttlefish cranial cartilage and cornea. Compar Biochem Physiol Part B 134:171–180 Song E, Kim SY, Chun T et al (2006) Collagen scaffolds derived from a marine source and their biocompatibility. Biomaterials 27:2951–2961 Stenzel KH, Miyata T, Rubin AL (1974) Collagen as a biomaterial. Ann Rev Biophys Bioeng 3:231–253 Szulgit G (2007) The echinoderm collagen fibril: a hero in the connective tissue research of the 1990s. BioEssays 29:645–653 Tanzer ML (1978) The biological diversity of collagenous proteins. TIBS 3:15–17 Thurmond F, Trotter J (1996) Morphology and biomechanics of the microfibrillar network of sea cucumber dermis. J Exp Biol 199:1817–1828 Travis DF, Francois CJ, Bonar LC et al (1967) Comparative studies of the organic matrices of invertebrate mineralized tissues. J Ultrastruct Res 18:519–550 Trotter JA, Thurmond FA, Koob TJ (1994) Molecular-structure and functional-morphology of echinoderm collagen fibrils. Cell Tissue Res 275:451–458 Uzel SGM, Buechler MJ (2009) Nanomechanical sequencing of collagen: tropocollagen features heterogeneous elastic properties at the nanoscale. Integr Biol 1:452–459 Wilbur KM (1976) Recent studies of invertebrate mineralization. In: Watabe N, Wilbur KM (eds) The mechanisms of mineralization in the invertebrates and plants. University of South Carolina, South Carolina Wilkie IC (1984) Variable tensility in echinoderm collagenous tissues: a review. Mar Freshwater Behav Physiol 11:1–34 Wilkie IC (2002) Is muscle involved in the mechanical adaptability of echinoderm mutable collagenous tissues? J Exp Biol 205:159–165 Wilkie IC (2005) Mutable collagenous tissue: overview and biotechnological perspective. In: Matranga V (ed) Marine molecular biotechnology. Springer, Berlin, Heidelberg Wilkie IC, Candia Carnevali M, Trotter J (2004) Mutable collagenous tissue: recent progress and an evolutionary perspective. In: Heinzeller T, Nebelsick J (eds) Echinoderms. Taylor & Francis, London Young SD (1971) Organic material from scleractinian coral skeletons. I. Variation in composition between several species. Camp Physiol 40:113–120
Part VII
Self-Made Biological Materials
Organisms living in the marine environment contain a number of primary and secondary metabolites which are involved in bioadhesive processes as well as in construction of shells and tube-like shelters. Recently, much progress has been made regarding the characterization of underwater adhesive structures utilized by marine invertebrates such as siphonous green algae, foraminifera, barnacles, mussels, tube worms, echinoderms, and crustaceans. The structural components and biochemical mechanisms involved in naturally occurring processes, which lead to development of self-made biological materials, are current topics in modern biomimetics, bionics as well as materials science.
Chapter 32
Self-Made Biological Materials of Protozoans
Abstract The building of shells, tests, and loricae from bits of sand and other kind of mineral particles picked up from the environment is a widely distributed survival strategy in marine environments. As examples of these self-made biological materials of protozoan origin, numerous constructs from testate amoeba, gromiids, tintinnids, and xenophyophores are described and discussed here. Unicellular marine protists have developed during their evolution different survival strategies for adaptation to changes in the environment, particularly by providing protection against mechanical and chemical disturbances, as well as by disguising the organism from predators. The building of shells, tests, and loricae from bits of sand and other kind of mineral particles picked up from the environment is a widely distributed survival strategy. As examples of these self-made biological materials of protozoan origin, I will analyze in this chapter the corresponding constructs from testate amoeba, gromiids, tintinnids, and xenophyophores.
32.1 Testate Amoeba Testate amoebae (also referred to as rhizopods and thecamoebians andarcellaceans) are single-celled organisms in which the cytoplasm is enclosed within an external shell (the test). They live in a wide range of terrestrial and aquatic habitats, including wet soils, lakes, saltmarshes, and in marine environments (Charman 2001; Dujardin 1835). The test is the outer shell that encloses the living cell. There are several types of test that can be referred to as xenosomic or idiosomic tests, depending on the source of the materials used in their construction. Xenosomic tests are composed of particles derived from the environment, such as small grains of silica, and sometimes including other detritus such as pollen and diatom frustules. These are fixed together with secreted cement of variable composition. Idiosomic tests are composed of materials produced by the parent amoebae at reproduction, such as pre-formed, regularly shaped overlapping plates that are usually round or oval. Spines may also be H. Ehrlich, Biological Materials of Marine Origin, Biologically-Inspired Systems 1, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9130-7_32,
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formed by this process, although some are also continuous with test walls and are not separate idiosomic structures. Some tests do not have plates but are formed entirely of a smooth proteinaceous secretion. Other tests are intermediate between idiosomic and xenosomic. For example, taxa in the genus Nebela often form tests that include plates of other smaller taxa, which they have consumed, while also employing secretion and perhaps preformed plates. While some taxa appear to be entirely idiosomic, many xenosomic taxa may be capable of producing tests entirely of secretion when deprived of suitable materials (Medioli et al. 1987).
Fig. 32.1 SEM images of different testate amoeba: (a) Centropyxiella arenaria; (b) Psammonobiotus linearis (dorsal view); (c) O. elegans; (d) Alepiella tricornuta (dorsal view); (e) Psammonobiotus linearis (images courtesy Vassil Golemansky)
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Testate amoebae have been used as a new method of reconstructing paleoclimates from ombrotrophic peats; they may also be useful as indicators of pollution and temperature. Additionally, they can now be added to diatoms and foraminifera as potential indicators of sea-level change (Charman 2001). The marine interstitial testate amoebae form a specific taxocenose in the marine sand supralittoral. They were found and described during the last three decades. So far, 129 species of testate amoebae from the orders Arcellinida and Gromida have been established in the studied seas and oceans (Golemansky and Todorov 2004). Eighty psammobiotic interstitial testate amoebae from the marine sand supralittoral have been established so far, but only 26 have been extensively studied to provide more detailed information about their structure and ultra-morphology (Golemansky 1980; Golemansky and Coûteaux 1982; Golemansky and Ogden 1980; Golemansky and Todorov 1996, 2004, 2006; Ogden and Coûteaux 1986, 1989). A morphological feature shared by many species is a thin organic test that is covered either in endogenously produced, small, siliceous scales (Cyphoderiidae) or in thin, irregularly shaped quartz particles assembled by the amoeboid organism (Psammonobiotidae). For example, examination of the shell structure of marine species Ogdenia elegans by scanning electron microscopy shows that the whole shell surface is covered with a mixture of small to medium flattish pieces of quartz, so arranged to overlap each other, and to give a relatively smooth and regular outline. The shell has a thin wall and is extremely fragile. It usually collapses when is air-dried in preparation for scanning electron microscopy (Golemansky and Todorov 2006) (Fig. 32.1).
32.2 Gromiids Gromiids are large benthic amoeboid protists, with a monothalamous (singlechambered) proteinaceous test and filose pseudopodia (Jepps 1926). Proteinaceous tests (Hedley 1960) includes an inner layer of “honeycomb membranes,” a feature unique to this genus (Hedley and Bertaud 1962). One species, Gromia oviformis, is the best-known representative of this group. It is a cosmopolitan species that inhabits coastal intertidal and sublittoral waters and is found on the weed of coralline pools, on Cladophora, on the walls of rock crevices, undersurfaces of stones, holdfasts of kelp and the surface layer of sandy and muddy sediments (Bowser et al. 1996; Hedley and Bertaud 1962; Jepps 1926). Although this group has well-known habitats in shallow water, it was unknown in the deep-sea until the first species was discovered at bathyal depths (between 1,200 and 1,700 m water depth) on the Oman margin of the Arabian Sea (Gooday et al. 2000). This deep-sea species, characterized by a spherical test and multiple apertures, was identified as a gromiid on the basis of its wall structure and described as Gromia sphaerica (Gooday et al. 2000). Today, an increasing number of deep-water species have been identified and described, based on both morphological and molecular characteristics (Gooday and Bowser 2005). Recently, new species were observed in the abyssal zone of the Weddell Sea in Antarctica (Rothe et al. 2009). Authors reported that the coarsely agglutinated case enclosing the test in Gromia winnetoui sp. nov. represents the first
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report of a secondary agglutinated structure in Gromia. It is composed of loosely cemented quartz grains, which press closely against the test and leave a clear imprint on the flexible underlying test surface. The function of the agglutinated case in G. winnetoui sp. nov. is not clear.
32.3 Tintinnids The classification of the about 1200 tintinnid choreotrichid species is merely based on lorica morphology, as cytological features, including the ciliary pattern, are only known in 17 species (Agatha and Riedel-Lorjé 2006; Kofoid and Campbell 1929). Tintinnids are ubiquitious in marine systems and a very specious group (Wasik 1998) (Fig. 32.2). Ciliates of the suborder Tintinnina produce loricae which are either hyaline or agglutinated by biogenic (coccoliths, diatom frustules) (Fig. 32.3) or non-biogenic (sand grains, mineral flakes) particles (Wasik et al. 1996). They may cover the entire outer surface of the lorica (Tintinnopsis, Leprotintinnus) or only its bowl, thus leaving the collar hyaline (Codonellopsis, Stenosemella, Laackmanniella). The lorica, formed of one or more layers, is composed primarily of proteinaceous material. Although the general shape of a lorica is a tube or vase tintinnids can show very distinct lorica morphologies (Davis 1981; Dolan et al. 2006). Gold and Morales (1976) proposed following terminology to describe particle accumulation in the lorica development process: agglutination refers to the process of accumulation of particles regardless of the source and types of materials utilized. Arenaceous refers to the appearance of a lorica that was produced predominantly of mineral grains. The grains appear to be cemented together by what may be an active process; such forms could have an underlying organic matrix present. A lorica is considered to be agglomerated if it has both mineral grains and a large proportion of fragments of biological origin adhering to it. Agglomerated loricae differ from the arenaceous types in the amount of biogenic material present and in their appearance.
Fig. 32.2 Tintinnids produce loricae which are either hyaline, or agglutinated by biogenic or non-biogenic particles (light microscopy image courtesy Joachim Henjes and Philipp Assmy)
32.3
Tintinnids
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Fig. 32.3 SEM image shows Codonelopsis pusilla that agglutinated biogenic particles like debris of diatoms frustules (image courtesy Joachim Henjes and Philipp Assmy
Additionally, the particles on the loricae of agglomerated species seem to have been taken up randomly. According to Rassoulzadegan (1980), the origin and size of adhered particles reveal the nature of the ecosystem in which the lorica was built. Mechanisms of agglutination and particle selection are still unclear, but Gold and Morales (1976, 1977) suggest that arenaceous loricae (e.g., Stenosemella) can be built in sediments, while agglomerated forms might be produced in the water column. Gowing and Garrison (1992), however, consider lorica building in sediments to be unlikely; cells of species living in deep waters, for example, would have to descend –2,000 m, adhere particles from the sediment, and then move to the surface (Fig. 32.4). This is supported by Wasik et al. (1996) who found loricae of Tintinnopsis lobiancoi Daday 1887, that were classified as arenaceous, agglutinated with diatoms from one particular genus. These mostly surface dwelling species would have to descend between 2,000 and 4,000 m to attach particles from the sediment and then ascend to the surface water again. The mechanisms of agglutination and selectivity for certain particles (e.g., monospecific coccoliths or diatoms) within the agglomerated tintinnids are still poorly known. According to observations of some pelagic tintinnid taxa from low latitudes, particles are taken up randomly (Gold and Morales 1976). A study of Winter et al. (1986) suggests that tintinnids are not strongly selective, but restricted by particle size when agglutinating coccoliths from their environment. Conversely, studies by Gold and Morales (1977), Takahashi and Ling (1984), Gowing and Garrison (1992) and Wasik et al. (1996) imply that tintinnids have the capability to select biogenic particles specifically for the agglutination of their lorica since they do not single out only those that are dominant in the water column. Furthermore, these authors hypothesised that tintinnids might ingest their prey organisms before utilising them in lorica construction. In his early experiments with Tintinnopsis pawa, Gold (1979) showed that this species, which agglutinates siliceous matter in situ, also produced well-developed loricae with only CaCO3 particles present in the culture medium. Moreover, there
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Fig. 32.4 SEM images of Stenosemella pacifica (images courtesy Sabine Agatha)
was no clear preference for one type of material over the other when a mixture of the two experimental particles was offered. It appears, therefore, that the abundance of one mineral type rather than an inherent chemical preference is the principal factor influencing particle agglutination. Recently, Henjes and Assmy (2008) reported experimental results in the Southern Ocean obtained using Stenosemella spp. that clearly indicated particle availability seems to be the driving mechanism in the agglutination tintinnids.
32.4 Xenophyophores Xenophyophores are giant, multinucleate, agglutinated rhizopods which are found exclusively in the deep-sea (Gooday and Tendal 2000; Tendal 1972, 1996). A characteristic feature of xenophyophores is that they construct a sort
32.4
Xenophyophores
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of artificial exoskeleton by gluing together particles of sediment and the microscopic shells of foraminifera. (The name “xenophyophore” is Greek for “foreign body bearer.”). The Xenophyophorea, like many Eukarya, have gone by a variety of names: Arxenophyria, Domatocoela, Psamminidea, Psammininae, Xenophiophorae, Xenophyophora, Xenophyophoria, Xenophyophorida, and Xenophyophoridae. They are rather unevenly divided between two easily distinguishable groups (Tendal 1972). Although some information on their role in deep-sea ecology has recently become available, many aspects of their biology are still poorly understood (Tendal 1996). In recent years, photographic surveys have shown xenophyophores to be a dominant and conspicuous component of the megabenthos in certain deep-sea regions (Hughes and Gooday 2004). They are often found in areas with enhanced organic carbon fluxes, such as beneath highly productive surface waters, on sloped topography, or near certain topographic features such as cauldera walls, basalt outcrops, or on the sides of sediment mounds (Tendal 1972) (Fig. 32.5). Syringammina fragilissima Brady was the first xenophyophore to be described, albeit as a large agglutinated foraminiferan (Brady 1883). The description was based on specimens obtained during the 1882 Triton expedition from a station at 1000 m water depth, slightly to the southeast of the present study area. The species has a hemispherical test which consists of a complex system of anastomosing branches composed of agglutinated foreign particles (xenophyae), mainly mineral grains and dead foraminiferal tests (Tendal 1972). Like other xenophyophores, the test contains strands of protoplasm encased within a branched organic tube system and waste pellets (stercomata) which accumulate as strings and masses (stercomare) enclosed within an organic membrane. The cell is multinucleate, with nuclei evenly distributed throughout the cytoplasm. The other obvious feature of the cell is the presence of numerous crystals (called granellae) of barite (BaSO4 ) probably secreted by the xenophyophore itself. The point of all
Fig. 32.5 Representatives of Xenophyophores on the sea bottom (image from NOAA Ocean Explorer)
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this is unknown, though it may be to remove toxic barium solutions ingested while feeding (Tendal 1972). Until 2004 only 14 S. fragilissima individuals had been recovered (Hughes and Gooday 2004). In 2006 in an area called the Darwin Mounds, northwest of Scotland, researchers discovered specimens of S. fragilissima with tests as large as 20 cm in diameter. How can they grow so big? Usually, the size of protists is limited by the surface–volume relationship. Multicellular creatures have specialized structures for transporting nutrients and wastes. Diffusion is the main mode of transport in one-celled organisms and at large sizes diffusion is not enough to sustain the cell s interior. Some xenophyophores minimize the problem by having a coin-like shape; Stannophyllum venosum is 25 mm across, but only 1 mm thick. S. fragilissima is more of a blob. However, its surface area is increased by having a structure of branching tubes, supported by the test. Another factor that helps to account for its gigantic size is that it lives in water. It is thought that xenophyophores are filter feeders. The large specimens are found on sea bottoms where there is a current and lie beneath surface waters rich in food. In some areas of the deep, they are the dominant species with over 2000 individuals per 100 m2 .
32.5 Conclusion Numerous taxa of marine protists have developed during their evolution intriguing survival strategies for adaptation to changes in the environment, particularly by providing protection against disturbances, as well as by disguising the organism from predators. These species use both biogenic and diagenic (or artificial) materials for the aims listed above. The advantages of their engineering strategies in contrast to biomineralization are, however, still unknown.
References Agatha S, Riedel-Lorjé JC (2006) Redescription of Tintinnopsis cylindrical Daday, 1887 (Ciliophora: Spirotricha) and unification of tintinnid terminology. Acta Protozool 45:137–151 Bowser SS, Marko M, Bernhard JM (1996) Occurrence of Gromia oviformis in McMurdo Sound. Antarct J US 31:122–124 Brady HB (1883) Note on Syringammina, a new type of arenaceous rhizopoda. Proc Roy Soc London 35:155–161 Charman DJ (2001) Biostratigraphic and palaeoenvironmental applications of testate amoebae. Quaternary Sci Rev 20:1753–1764 Davis CC (1981) Variations of lorica shape in the genus Ptychocylis (Protozoa: Tintinnia) in relation to species identification. J Plank Res 3:433–443 Dolan JR, Jaquet S, Torreton J-P (2006) Comparing taxonomic and morphological biodiversity of tintinnids (planktonic ciliates) of New Caledonia. Limnol Oceanogr 51(2):950–958 Dujardin F (1835) Recherches sur les organisms inférieurs. Ann des Sci Nat 2:345–351 Gold K (1979) Scanning electron microscopy of Tintinnopsis pawa: studies on particle accumulation and the striae. J Prolozool 26(3):415–419
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Gold K, Morales EA (1976) Studies on the sizes, shapes, and the development of the lorica of agglutinated tintinnina. Biol Bull 150:377–392 Gold K, Morales EA (1977) Studies on the tintinnida of Enewetak Atoll. J Protozool 24:580–587 Golemansky V (1980) La faune técamoebienne interstitielle du psammal supralittoral des mers. DSc Thesis, Sofia Golemansky V, Coûteaux M-M (1982) Etude en microscopie éléctronique a balayage de huit espèces de thécamoebiens interstitiels du supralittoral marin. Protistologica 18:473–480 Golemansky V, Ogden C (1980) Shell structure of three littoral species of testate amoebae from the black sea (Rhizopodea: Protozoa). Bull Br Mus Nat Hist (Zool) 38:1–6 Golemansky V, Todorov M (1996) Interstitial Rhizopods (Rhizopoda: Testacea & Foraminiferida) from the Antarctic Region of Chile and Valparaiso in the Pacific. In: Golemansky V, Chipev N (eds) Bulgarian Antarctic research. Life Sciences. Pensoft, Sofia Golemansky V, Todorov M (2004) Shell morphology, biometry and distribution of some marine interstitial Testate Amoebae (Sarcodina: Rhizopoda). Acta Protozool 43:147–162 Golemansky V, Todorov M (2006) New data to the shell ultrastructure and the biometry of the marine interstitial Testate Amoebae (Rhizopoda: Testaceafilosia). Acta Protozool 45:301–312 Gooday AJ, Bowser SS (2005) The second Gromia species (testate amoeba) from the deep sea: its natural history and association with the Pakistan margin oxygen minimum zone. Protist 156:113–126 Gooday AJ, Bowser SS, Bett BJ et al (2000) A large testate protist, Gromia sphaerica sp nov (Order Filosea), from the bathyal Arabian Sea. Deep-Sea Res II 47:55–73 Gooday AJ, Tendal OS (2000) Class xenophyophorea. In: Lee JJ, Leedale GF, Bradbury P (eds) The illustrated guide to the protozoa, 2d ed. Allen, Lawrence, KS Gowing MM, Garrison JJL (1992) Abundance and feeding ecology of larger protozooplankton in the ice zone of the Weddell and Scotia Seas during the austral winter. Deep-Sea Res 39:893–919 Hedley RH (1960) The iron-containing shell of Gromia oviformis (Rhizopoda). Q J Microsc Sci 101:279–293 Hedley RH, Bertaud WS (1962) Electron-microscopic observations of Gromia oviformis (Sarcodina). J Protozool 91:79–87 Henjes J, Assmy P (2008) Particle availability controls agglutination in pelagic tintinnids in the Southern Ocean. Protist 159(2):239–250 Hughes JA, Gooday AJ (2004) Associations between living benthic foraminifera and dead tests of Syringammina fragilissima (Xenophyophorea) in the Darwin Mounds region (NE Atlantic) Deep Sea Research Part I. Oceanogr Res Papers 51:1741–1758 Jepps MW (1926) Contribution to the study of Gromia oviformis Dujardin. Q J Microsc Sci 70:701–719 Kofoid CA, Campbell AS (1929) A conspectus of the marine and fresh-water ciliata belonging to the suborder tintinnoinea, with descriptions of new species principally from the Agassiz expedition to the eastern tropical Pacific 1904–1905. Univ Calif Publ Zool 34:1–403 Medioli FS, Scott DB, Abbott BH (1987) A case study of protozoan intraclonal variability: taxonomic implications. J Foraminiferal Res 17:28–47 Ogden CG, Coûteaux M-M (1986) The nature of the shell of Alepiella tricornuta, a marine testate amoeba (Sarcodina: Rhizopoda). Protistologica 22:213–220 Ogden CG, Coûteaux M-M (1989) Interstitial marine rhizopods (Protozoa) from littoral sands of the east coast of England. Eur J Protistol 24:281–290 Rassoulzadegan F (1980) Granulometric analysis of the particles used by a tintinnid Stenosemella ventricosa (Clap. & Lachm.) Jorg., during lorica building. Protistologica 16:507–510 Rothe N, Gooday AJ, Cedhagen T et al (2009) Three new species of deep-sea Gromia (Protista, Rhizaria) from the bathyal and abyssal Weddell Sea, Antarctica. Zool J Linnean Soc 157: 451–469 Takahashi K, Ling HY (1984) Particle selectivity of pelagic tintinnid agglutination. Mar Micropaleontol 9:87–92 Tendal OS (1972) A monograph of the Xenophyophoria (Rhizopodea, Protozoa). Galathea Rep 12:7–103
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Tendal OS (1996) Synoptic checklist and bibliography of the Xenophyophorea (Protista), with a zoogeopgraphical survey of the group. Galathea Rep 17:79–101 Wasik A (1998) Antarctic tintinnids: their ecology, morphology, ultrastructure and polymorphism. Acta Protozool 37:5–15 Wasik A, Mikolajczyk E, Ligowski R (1996) Agglutinated loricae of some Baltic and Antarctic Tintinnina species (Ciliophora). J Plankton Res 18:1931–1940 Winter A, Stockwell D, Hargraves PE (1986) Tintinnid agglutination of coccoliths: a selective or random process? Mar Micropaleontol 10:375–379
Chapter 33
Foraminifera
Abstract There are three basic types of foraminiferan test. First, the purely organic type. Second, the arenaceous or agglutinate type, composed of extraneous material is held together by a secreted cement containing both organic and inorganic components. These types can be referred to as ferruginous, calcareous, or siliceous. Third, the inorganic and usually calcified type composed of secreted calcareous material together with an organic basis. Therefore, structural formations of foraminiferans are of great interest for materials scientists because of an example of self-made constructs achieved on the micro-bioengineering level. Moreover, these primitive unicellular organisms are superb builders of their houses, possessing the know-how to build using naturally occurring “bricks” and “cements” with enviable skill.
33.1 Foraminifera: Agglutination Versus Biomineralization Foraminifera (Rhizopoda: Granuloretriculosea) are known as an exclusively marine group of testate sarcodic protozoans, most of them intolerant to variations in salinity. Since the first publications, e.g., those by Fichtel and Moll (1798) and Dujardin (1835), as many as 30,000 modern and fossil species have been discovered to date (Cushman 1948; Galloway 1933; Hemleben et al. 1989; Loeblich and Tappan 1987; Sen Gupta 1999). Cushman (1948) in his fundamental work on foraminifera gives them following common description: They are single celled animals. Except for a few of the simplest types, there is developed a test, either of agglutinated foreign material, or chitin, or of calcareous material secreted by the animal itself. These tests are preserved as fossils in many of the geologic formations since Cambrian time. In the existing oceans, foraminifera occur in enormous numbers, and in water from the continental shelf out to about 2000 fathoms or more their tests form the thick Globigerina ooze of the ocean floor. In general, so far as known, the usual food of the foraminifera consists of vegetable material, the diatoms and various other algae furnishing the greater part. In some of the pelagic forms it has been observed that copepods are captured and eaten as well as other protozoa. As fossils they are often very abundant, and in the Palaeozic as well as in the younger formations they have formed thick limestones. The great pyramids of Egypt are constructed from limestones made largely of fossil foraminifera. In
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the tropics, the sands of the beaches are often largely composed of the tests of foraminifera, and in shallow water their great numbers actually form obstructing shoals.
Test characteristics have been used for approximately 150 years as the main criterion for classification of both fossil and modern representatives of the group (Lipps 1973; Sen Gupta 1999). The consensus view has been that the organic-walled and atestate forms are primitive, with agglutination, polythalamy (i.e., the ability to make multiple chambers), and calcification representing successive innovations within the lineage (Tappan and Loeblich 1988). Structural formations of foraminiferans are of great interest for materials scientists because of an example of self-made constructs achieved on the microbioengineering level. Moreover, these primitive unicellular organisms are superb builders of their houses, possessing the know-how to build using naturally occurring “bricks” and “cements” with enviable skill. The covering of the animal in the foraminifera is usually referred to as a test, rather than a shell such as those secreted by special organs in the Mollusca, etc. In most of the earlier and more primitive groups of the foraminifera there is an arenaceous or agglutinated. Test composed of foreign material, sand grains, sponge spicules, mica flakes, etc., loosely or firmly cemented together over a thin chitinous inner layer which is related to the primitive chitinous test of the still simpler groups. The only purpose of the test seems to be to form a somewhat rigid protection about the softer protoplasmic body (Cushman 1948). Test construction in agglutinated foraminifera requires two main components: cement and grains. There are three basic types of foraminiferal test (Hedley 1963). First, the purely organic type, currently considered to be composed of tecnin. The term tecnin is now used in preference to pseudochitin, which has become objectionable because of the implied affinity to chitin, e.g., the tests of Gromia, Allogromia, and Myxotheca. Second, the arenaceous or agglutinate type, composed of extraneous material is held together by a secreted cement containing both organic and inorganic components. These types can be referred to as ferruginous, calcareous, or siliceous, depending on the color or reaction with dilute mineral acids, e.g., the tests of Astrorhiza, Pilulina, Rhabdammina, and Cyclammina. Third, the inorganic and usually calcified type composed of secreted calcareous material together with an organic basis, e.g., the tests of Elphidium, Globigerina, and Homotrema (Hedley 1963). According to Bender (1989), the organic cement can be organized into four species-specific morphotypes: (1) either as single strands, which may gradually become (2) a fibrous meshwork of strands, in (3) a foam-like mass, or (4) an undifferentiated substance at the inter-particle contacts. The cement contains an accessory, the ferruginous-rich mineral component, which is not biomineralized by the foraminifer itself but is passively incorporated. The mineralogical component stabilizes the organic substance and after its post-mortem bacterial decomposition retains its globular condition (50 nm) within the intergranular space. It is likely that the amount of the mineralogical component is controlled by the local sedimentary
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composition and its affinity in adsorbing anorganic compounds. Organic phases of foraminiferan cements can contain not only the pseudochitin-like material tectin, glycoproteins, or mucopolysaccharides, but also collagen (Hedley and Wakefield 1967). Calcitic cement exhibits a characteristic microstructure which differs from all previously described types of foraminiferal biomineralizate (Bender and Hemleben 1988). Individual, organically enveloped crystals (0.1–0.3 μm) are aligned into rods, which are oriented in parallel to form bundles (1–2 μm). In the intergranular space, the bundles are arranged randomly. An intracellular biomineralization process and a transporting system within vesicles, which also exist in miliolid foraminifers, are inferred. The presence of an organically lined pore system is characteristic for all species with calcitic cement. It has a species-specific structure, pore diameter, and spatial distribution within the chamber wall (Bender 1989). Calcitic as well as organic cemented foraminifers shows agglutinated particles enveloped with an organic layer, suggesting a former incorporation into the cytoplasmic system. The particles are incorporated into the test wall and oriented in a selective manner that accounts for both particle size and form. The “inner organic lining” is also fundamental to the test structure in all textulariid foraminifers. It either may be one-layered throughout the entire test or it may become increasingly thicker in older chambers due to a multilayered structure (Bender 1989). The diversity of wall cements in foraminifera is well described (Loeblich and Tappan 1987). However, there are still numerous open questions related to both organic and inorganic phases of these biologically produced (secreted) materials as well as the influence of environmental conditions. For example, it was reported (Roberts and Murray 1995) that the mineral phase in Spirorutilis wrightii cement is calcite; however, the agglutinated foraminiferan Texrularia crenara use aragonite within biomineralized cement. It is not clear where are the advantages of biomineralization in comparison with a “self-made-house” strategy using agglutination of mineral-based microparticles, because the goal—to protect “naked body” of the animal and to survive under environmental conditions—will be achieved in both cases. Taxonomically, the presence of an agglutinated, as opposed to a mineralized, test wall in foraminifera appears to be less phylogenetically significant than does the overall arrangement of test chambers (Pawlowski et al. 2003). This suggests that chamber arrangement is a fundamental developmental characteristic in at least some polythalamous clades and that the composition of the test can be altered over time, possibly reflecting adaptation to changing environmental conditions (Habura et al. 2006). The evolutionary advantage of constructing an agglutinated test rather than a mineralized one is not immediately apparent. Based on physical manipulation, for example, Miliammina fusca’s agglutinated test appears to be structurally weaker, for its mass, than are those of its calcareous relatives. It is likely that the relatively low pH and oxygen levels and reduced salinity of corresponding marine environments (e.g., Sen Gupta 1999) disfavor calcification in foraminiferans. Speed of calcification in these organisms is dependent on the local carbonate concentration, which decreases in environments that are acidic, dysoxic, low salinity, or unusually cold. Most calcareous foraminiferans also experience
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substantial decalcification and test deformation in conditions more acid (pH < 7.8) than is typical for seawater (LeCadre et al. 2003). Therefore, modern calcareous foraminifera occurring high in estuaries or in other low-carbonate environments are often found in a so-called chitinous form, in which only the organic lining of the test is present (Habura et al. 2006). The next intriguing phenomenon is related to the feature of some foraminiferans to select corresponding building blocks. Cases for random grain selection according to grain size, shape, and mineralogy during test construction have been suggested previously (Allen et al. 1998). Certain species have tests consisting of clasts (fragments) embedded in a matrix of finer clasts, which are in turn set in an even smaller assembly of clasts and so on, down to extremely small grain sizes. The dimensions acquired from the foraminiferal studies (Bender 1989) correspond well with those previously obtained from natural fractal geological structures and ideal fractures. The self-similar grain arrangement within walls of the foraminifera exists over three orders of magnitude, after which alternative methods of test wall construction are evident. This suggests that a limit exists where grain selection terminates. A self-similar grain distribution limits the amount of biologically produced adhesive material required by the foraminifera for constructing their tests (Allen et al. 1998). A more complex situation with respect to selectivity of mineral particles occurs in very specific marine environments, for example, as in the case of some regions of Pacific basins with high concentrations of so-called manganese nodules (Graham and Cooper 1959). Manganese nodules are heterogenous mixtures of very fine-grained iron and manganese oxides, detrital mineral grains, and biogenic components deposited in semiconcentric layers around a central nucleus of variable composition. These variations are believed to reflect changes in the environment of formation (Margolis and Burns 1976). Biogenic sediment grains often are incorporated in the nodule matrix during periods of low metal oxide accretion or of higher pelagic sedimentation. Variable amounts of biogenic silica, calcium carbonate, and phosphate may be found in manganese nodules, diluting the metal oxide phases. Works by Greenslate (1974) and Wendt (1974) have suggested that benthic agglutinated foraminifera may play an active role in the actual construction of the nodules on which they live. These animals attach themselves to the surface of manganese nodules, which frequently present the only hard, stable substrate found on the deep-ocean floor. They construct their tubular tests out of biogenic and mineral debris found on and adjacent to the nodules; these particles are then held together by a cementing agent secreted by the foraminiferan (Riemann 1983). The shape and composition of these tests, which eventually get incorporated into the nodule matrix, are a function of individual species. However, until recently only a few genera (e.g., Tolypammina sp.) had been identified on manganese nodules and no chemical work had been done to test compositions (Margolis and Burns 1976). The greatest number and highest diversity of foraminiferans were found in the equatorial northwestern Pacific, where nodules are enriched in copper and nickel. The majority of specimens were attached to the equatorial rim of the nodule, which is actually at the sediment–water interface. The foraminiferal tubes all showed higher Fe, Co, and Ti and lower Mn, Cu, and Ni than the nodule surface on which they were
33.2
Silk-Based Shell of Stannophyllum zonarium
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growing. In addition, the interior linings of the tubes show the highest concentrations of iron and micronodules are occasionally found incorporated in the foraminiferal tube (Dudley 1976, 1979). How do prompt foraminifera build their constructs? Observations were reported (Bender and Hemleben 1988) that indicate chamber formation is completed in 24 h in laboratory cultures. Prior to the chamber formation the foraminifer forms a protective cyst of detrital particles in which the chamber formation takes place. The constituents for the new chamber are collected from the cyst material by pseudopods, then organically enveloped and finally cemented together with a calcareous or an organic cement. It was not easy for me to decide what foraminiferan species must be definitively discussed in this book as examples of unique structural formations due to the very broad diversity of species and the unique architecture of their test forms. But two of them, it seems to me, are very interesting from both the biochemical and the materials science point of view. The first one is regarding to the discovery of silklike protein within the agglutinated shell of one giant foraminiferan species, and the second, representing the sponge-imitating species, is also a giant foraminifer.
33.2 Silk-Based Shell of Stannophyllum zonarium S. zonarium, Haeckel 1889, is a gigantic marine rhizopod—3–19 cm long and 1–2 mm thick—reported from various localities in the Pacific Ocean at depths between 981 and 5307 m (Fig. 33.1). Along with other members of the relatively little known sub-class Xenophyophoria, S. zonarium has been the subject of a detailed taxonomic revision by Tendal (1972). The present account is concerned with the numerous extracellular fibers called lineallae which ramify and form a significant part of the agglutinated shell. Basically S. zonarium is a multinucleate plasmodium surrounded by extraneous and agglutinated material, consisting of radiolarian shells, sponge spicules, and mineral particles, through which the linellae interweave. The linellae have a supporting and strengthening role in the agglutinated shell and impart to it a characteristic flexibility. These structures are not known in protozoans (other than certain xenophyophores). The composition of the linellae was referred to by Schulze (1907) who detected nitrogen, sulfur, and iodine and noted a positive reaction to protein tests. Hedley and Rudall (1974) investigated the shell of this animal in more detail. The whole agglutinated shell is shown in Fig. 33.1a. When the extraneous materials have been removed, the matrix of linellae can be clearly seen (Fig. 33.1b). The linellae are 2–3 μm in diameter and several millimeter in length, possess weakly positively birefringent, and can be seen in cross section and longitudinal section to be constructed of approximately 20–30 concentrically arranged lamellae. Each lamella is about 55 nm thick and has a strongly electron dense band 10 nm thick at its outer surface. Examination by X-ray diffraction gives an amorphous type of diagram with a single diffraction ring in the region with a spacing of 4.5 Å. The
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Fig. 33.1 Silk-based shell of S. zonarium foraminiferan. (a) Part of the 2 mm thick specimen of S. zonarium. (b) Cross section of a linella showing concentrically arranged lamellae, (×30,000). (c) Part of S. zonarium after ultrasonic vibration to reveal mass of linellae fibers (×140) (images adapted from Hedley and Rudall 1974)
linellae are resistant to digestion in pronase but rapidly disperse in sodium hypochlorite solution. Its general resistance to reagents suggests it is cross-linked or tanned. Attempts to induce some intramolecular change by heating or treating in acid and alkali have produced no definable structure. The amino acid composition of the linellae in Stannophyllum presents a picture which is very like the general situation
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Sponge-Imitating Giant Foraminifer
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found in silk proteins of arthropods, revealing a high content of glycine, alanine, and serine (Hedley and Rudall 1974). Without doubts, modern genomics and proteomics techniques could be useful to re-examine these previously mentioned results today, because several principle questions relating to the origin of the silk genome, as well as the very unusual biomaterial discovered in this foraminiferan.
33.3 Sponge-Imitating Giant Foraminifer Although presence of siliceous spicules generally points to sponges, many invertebrates (including sponges) incorporate foreign mineral particles such as siliceous sponge spicules to serve as skeletal support. On the June 11, 1966, Klaus Rützler (now in the Smithsonian Institute, Washington, DC) discovered at the roof of a reefcave one organism that superficially resembles a gorgonian (Trichogorgia viola); but is entirely composed of siliceous sponge spicules cemented together like the skeleton of a raspailiid sponge (Rützler and Richardson 1996). Later, more careful examination of several samples from different locations and subsequent detailed light and electron microscope study clearly showed that the spicules were taken up from the sediment and cemented together to support a tiny mass of protoplasm, an agglutinated, arborescent member of Foraminifera. Thus, a new genus and species, Spiculidendron corallicolum, was established in the Textulariina family Astrorhizidae (Rützler and Richardson 1996). The new taxon is characterized by a complexly branching tubular test that is attached to hard substrate and has a simple wall lacking septae and apertures. S. corallicolum may well be one of the largest species of Foraminifera—indeed of Protozoa—ever recorded. The tallest test among the type specimens measures 61 mm (Fig. 33.2), not counting the portion of the organism that resides in cavities of the substrate. The ultrastructure of the bioadhesive (Bowser and Bernhard 1993), or organic cement, appears in electron micrographs as a dense fibrillar substance similar to spongin, the collagen-like protein that forms intercellular matrix and fibers in Porifera. Indeed, in a study of a comparable species in the foraminiferan genus Halyphysema (Hedley and Wakefield 1967) the structural similarity of the cement with collagen has been confirmed. It was assumed therefore that protozoan cement was deposited secondarily inside the small spaces of the central canals of broken and eroded spicules. S. corallicolum is clearly chemoselective for silica and incorporates a considerable size range of sponge spicules. In the habitats studied by Rützler and Richardson, calcareous sediment is plentiful in a great size range (from sponge Cliona-excavated chips of a few micrometer to Halimeda-derived plates of several millimeter) but never accepted for test building, although small carbonate particles initially attach to pseudopodia in the distal branch region of the test. Furthermore, S. corallicolum is somewhat unusual because it does not have distinctive apertures where pseudopodia extrude to capture food. It was presumed that minute pores through the spicule cement in distal areas of branches serve as an exit for cytoplasm, for obtaining food, and for trapping spicules to expand the test.
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Fig. 33.2 Giant foraminifer S. corallicolum is characterized by a complexly branching tubular test that is attached to hard substrate and has a simple wall lacking septae and apertures (adapted from Rützler and Richardson 1996)
S. corallicolum occurs singly or in clusters in semishaded clear-water reef locations, 20–30 m and below. The species is common just inside the entrance of crevices and caves, under ledge overhangs, and along vertical walls where sediment exposure is low. Common associates are crustose red algae, sponges and foraminifers, reteporid bryozoans, algal turfs, and hydroids. Smaller specimens are
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difficult to detect in the field because they blend well with similarly structured algae and invertebrates. Distribution of the species is presumed to be Caribbean-wide; so far it has been found in the Bahamas (San Salvador), Turks and Caicos Islands, Leeward Islands (Dominica), Colombia, Honduras (Roatan), Belize (Carrie Bow Cay, Lighthouse Reef), and Cayman Islands (Rützler and Richardson 1996).
33.4 Conclusion Here I want to note that in spite of the difficulties of obtaining abyssal foraminiferans from such very specific environments, studies using modern analytical and structural methods must be carried out more intensively because of the possibility for the isolation of novel biological materials like iron–chitin or iron–collagen biocomposites of foraminiferan origin with very specific physico-chemical and material properties.
References Allen K, Roberts S, Murray JW (1998) Fractal grain distribution in agglutinated foraminifera. Paleobiology 24(3):349–358 Bender H (1989) Gehäseaufbau, Gehäusegenese und Biologie agglutinierter Foraminiferen (Sarcodina: Textulariina). Jahrb GeBundesanst 132:259–347 Bender H, Hemleben C (1988) Calcitic cement secreted by agglutinated foraminifers grown in laboratory culture. J Foram Res 18:42–45 Bowser SS, Bernhard JM (1993) Structure, bioadhesive distribution and elastic properties of the agglutinated test of Astrammina rara (Protozoa: Foraminiferida). J Eukariotic Microbiol 40:121–131 Cushman JA (1948) Foraminifera, their classification and economic use, 4th ed. Harvard University, Cambridge, MA Dudley WC (1976) Cementation and iron concentration in foraminifera on manganese nodules. Foram Res 6:202–207 Dudley WC (1979) Biogenic influence on the composition and structure of marine manganese nodules. In: La genese des nodules de manganese. Colloq Int CNRS, Paris 289:227–232 Dujardin F (1835) Observations nouvelles sur les prétendu Céphalopodes microscopiques. Ann des Sci Nat, series 2 3:312–314 Fichtel L, Moll JPC (1798) Testacea microscopica aliaque minuta ex generibus. Argonauta et Nautilus ad naturam delineata et descripta I–XII:123 Galloway JJ (1933) A manual of foraminifera. Principia, Bloomington, IN Graham LW, Cooper SC (1959) Biological origin of manganese-rich deposits of the seafloor. Nature 183:1050–1051 Greenslate JL (1974) Manganese and biotic debris associations in some deep-sea sediments. Science 186:529–531 Habura A, Goldstein ST, Parfrey LW et al (2006) Phylogeny and ultrastructure of Miliammina fusca: evidence for secondary loss of calcification in a Miliolid Foraminifer. J Eukaryot Microbiol 53(3):204–210 Hedley RH (1963) Cement and iron in the arenaceous foraminifera. Micropaleontology 9:433–441 Hedley RH, Wakefield JSTJ (1967) A collagen-like sheath in the arenaceous foraminifer Haliphysema (Protozoa). J Roy Microsc Soc 87:474–481 Hedley RM, Rudall KM (1974) Extracellular silk fibres in Stannophyllum (Rhizopodea: Protozoa). Cell Tissue Res 150:107–111
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Hemleben C, Spindler M, Anderson OR (1989) Modern planktonic foraminifera. Springer, New York LeCadre V, Debenay J-P, Lesourd M (2003) Low pH effects on Ammonia beccarii test deformation: implications for using test deformation as a pollution indicator. J Foraminiferal Res 33:1–9 Lipps JH (1973) Test structure in foraminifera. Ann Rev Microbiol 27:471–488 Loeblich AR, Tappan H (1987) Foraminiferal genera and their classification. Van Nostrand Reinhold Margolis SV, Burns RG (1976) Pacific deep-sea manganese nodules: their distribution, composition, and origin. Annu Rev Earth Planet Sci 4:229–263 Pawlowski J, Holzmann M, Berney C et al (2003) The evolution of early foraminifera. Proc Natl Acad Sci USA 100:11494–11498 Riemann F (1983) Biological aspects of deep-sea manganese nodule formation. Oceanol Acta 6:303–311 Roberts S, Murray JW (1995) Characterization of cement mineralogy in agglutinated foraminifera (Protista) by Raman spectroscopy. J Geol Soc 152:7–9 Rützler K, Richardson S (1996) The Caribbean spicule tree: a sponge-imitating foraminifer (Astrorhizidae). Bull de l’Institute Roy des Sci de Belgique, 66(Suppl):143–151 Schulze FE (1907) Die Xenophyophoren, eine besondere Gruppe der Rhizopoden. Wiss Ergebn d Tiefsee-Exped “Valdivia” 11:1–55 Sen Gupta BK (1999) Modern Foraminifera. Kluwer Academic, Dordrecht, The Netherlands Tappan H, Loeblich AR (1988). Foraminiferal evolution, diversification, and extinction. J Paleontol 62:695–714 Tendal OS (1972) A monograph of the Xenophyophoria (Rhizopodea, Protozoa). Galathea Rep 12:1–103 Wendt L (1974) Encrusting organisms in deep-sea manganese nodules. Spec Publ Int Assoc Sedimentol 1:437–447
Chapter 34
Polychaete Worms: From Tube Builders to Glueomics
Abstract Among polychaetes, calcareous tubes occur in serpulids, sabellids, and cirratulids. These worms are of interest because they form two different types of dwelling tubes. The members of the family Serpulidae precipitate cylindrical tubes of calcium carbonate on an organic matrix. Members of the family Sabellariidae construct similarly shaped, non-calcified tubes that consist of mineral grains cemented with a mucous secretion. Like Sabellariidae, the Pectinariidae cement adventiously gathered particles into a solitary tube, but do not form colonies. The chemistry and possible mechanisms of the polychaetes self-made biological materials are discussed here. Marine polychaete worms are common members of hard substratum communities in all of the world’s oceans. They construct different kinds of external tube-like structures during their ontogenesis using both biomineralization and self-made adhesive-based mechanisms. I will now briefly present an overview recently made by Estonian researcher Olev Vinn (Vinn et al. 2009). Among polychaetes, calcareous tubes occur in serpulids, sabellids, and cirratulids (Fischer et al. 2000; Vinn et al. 2008a). These worms are of interest because they form two different types of dwelling tubes. The members of the family Serpulidae precipitate cylindrical tubes of calcium carbonate on an organic matrix. Members of the family Sabellariidae construct similarly shaped, non-calcified tubes that consist of mineral grains cemented with a mucous secretion (Vovelle 1965). Sabellariids (family Sabellaridae: suborder Sabellida) have an unusual strategy for constructing their mineralized tubes (Stewart et al. 2004). Rather than synthesizing a complete mineralized structure by the controlled precipitation of concentrated ions with matrix proteins, they use a strategy shared by many other tube or shelldwelling marine invertebrates. Sabellariids gather the mineral phase adventitiously as preformed particulates from the water column, usually sand and bits of calcareous shell of the right size, and secrete only a proteinaceous cement for joining the particles. Captured particles are conveyed along the tentacles of the crowns to the building organ, a U-shaped invagination near the mouth, where they are held, turned, and evaluated for size, shape, and composition.
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Sabellariids are commonly called sandcastle worms because individual tubes with their resident worm are honeycombed together into large reef-like mounds, although the mounds more closely resemble proletarian apartment buildings than castles (Stewart et al. 2004). Their gregariousness is due to a component of the tube cement that induces larvae to settle, metamorphose, and build a new tube on existing conspecific tubes (Jensen 1992). The three-dimensional colonies occur in the intertidal zone where there is sufficient wave action to suspend food and appropriate particles for tube building and repair. The sandcastle construction, and in particular the cement bonds, must therefore be robust enough to withstand the siege of a turbulent, high-energy environment. Munro Fox published in his Nature paper (Munro Fox 1938) very interesting observations on two sabellariid species. Spirographis spallanzanii Viviani and Sabella pavonina Savigny live in marine environment in tubes made of a mucoid secretion mixed with mud. The tubes, after the animals have been removed from them, cannot be stretched, but they can easily be bent. The tubes, however, are completely elastic in that they return at once to their initial shape after bending. In spite of this, the animals can bend their tubes permanently into new curves in course of their various tropisms. This author suggested that to bend such a tube permanently it would be necessary to soften it; this is probably the function of the external digestion of the tube. The innermost living of the tube is more elastic than the rest, and the new curvatures would be produced by softening the deposition of new elastic tube substance within the concave side of the bend. Removed from their tubes, Spirographis and Sabella are incapable of making new ones. Tubeless worms can be kept alive indefinitely in aerated seawater, but in water deficient in oxygen they die. Interestingly, worms with their tubes live in the non-aerated water. We thus have the apparent paradox that a worm exposed on all sides to the water dies of asphyxia, while a worm whose body is enclosed in a rube can breathe and live. Thus, the following experiment was carried out (Munro Fox 1938). Sabella and Spirographis were removed from their own tubes and put into glass tubes. They lined the glass tube with a secretion and lengthened it with normal tube material. Worms in these glass tubes will live in low oxygen conditions which kill naked worms. Observation of Spirographis in glass tubes has given a probable reason why naked worms are less viable than those in tubes. Inside its tube the worm executes rhythmic movements, which must continually renew the water in contact with the body. At regular intervals a swelling of the body wall, completely filling the tube, forms at the hind end of the worm; this swelling moves forward. In the case of tubeless worms there are scarcely any such movements, and the animals are probably in immediate contact with the body. Tubeless worms can live in aerated water, presumably they are able to do so in spite of the absence of vigorous body movements because there is more than enough oxygen for them in the water. Worms in their tubes execute the rhythmic movements even in fully aerated water; the result of this must be that the small amount of water in the tube is continually renewed, and presumably it is necessary for the worm to renew this water even when the surrounding seawater is fully aerated. These observations show that the tube has a second function besides that of protection:
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it provides the mechanical stimulus for the reflex body movements necessary for respiration (Munro Fox 1938). I suggest that, in the future, corresponding biotechnological approaches will be developed where these worms will produce tube-like biomaterials under laboratory conditions from mineral sources given from human. Serpulids construct their tubes from a mixture of calcium carbonate and an organic matrix (Hedley 1956a; Neff 1971a, b). Swan (1950) was the first to identify the tissues responsible for the secretion of calcium carbonate in serpulids. He described a pair of exocrine glands embedded in the subepithelial connective tissue of the ventro-lateral peristomium under the fold of the collar in Ficopomatus enigmaticus (Fauvel 1923) (as Mercierella). Later, detailed histological and histochemical studies of these glands in Pomatoceros triqueter Linnaeus 1758 were made by Hedley (1956a) and Vovelle (1956), who named them calcium-secreting glands (respectively “glandes a calcaire”). The other glandular area is the epithelium of the ventral shield surrounding the opening of the calcium-secreting glands (Hedley 1956b; Neff 1971a, b; Simkiss and Wilbur 1989). The calcium-secreting glands and their functioning have been described in detail for various serpulid species (Hedley 1956b; Neff 1971a; Nott and Parkes 1975; Vovelle et al. 1991). The study by Neff (1971a, b) has been used as a “standard” model of serpulid tube formation; he described the secretion of calcium carbonate in Pomatoceros americanus Day (1973) (as P. caeruleus (Schmarda 1861)) using a transmission electron microscope (TEM). According to Neff (1971a), the secretory products of the calcium-secreting glands in P. americanus have the form of cubic or rhombohedral granules with average dimensions of 0.15–0.2 μm on a side. The granules are composed of a fibrous organic matrix in which needle-like calcite crystals with low magnesium content are deposited (Neff 1971a). According to this model, the calcareous granules are an important contributor to the formation of the tube in which the animal lives. The granules reach the exterior of the animal as a slurry that solidifies sufficiently slowly to allow the undersurface of the collar, which is folded back over the aperture of the tube, to mould the calcite-saturated mucus, shaping the end of the tube. This appears to invoke two new phenomena that are more generally associated with the building industry, namely, the solidification of previously prepared granules and the controlled setting of this material. The resulting mineral tube is largely lacking orientation in its fine structure (Simkiss and Wilbur 1989). Tube formation in sabellids and cirratulids takes place by a mineralization system, in which an organic matrix and calcium ions are secreted by an epithelium. The serpulid opercular plate is also secreted by an organic matrix-mediated system (Bubel 1983; Vinn et al. 2008b). This plate consists of an outer cuticle and two calcified layers, all formed by a single layer of epithelial cells; the organic components of the opercular plate play a major role in the organization of inorganic components. Oriented structures of the opercular plate can be explained by the control of an organic matrix (Bubel 1983). Indeed, the majority (54%) of the 44 serpulid species studied (out of a total number of about 350 species) have an unoriented tube ultrastructure (Vinn et al. 2008c), which could be considered as in concordance with the standard granular secretion model. However, serpulids possess not exclusively unoriented, but very diverse oriented tube ultrastructures as well (Vinn et al. 2008c).
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These oriented tube structures are present in many other serpulid species and cannot be explained by the standard carbonate slurry model (e.g., Weedon 1994). Vinn et al. (2008a) have hypothesized that oriented structures in serpulid tubes have been secreted in the same way as in mollusc shells, based on their ultrastructural similarity. In simple oriented prismatic structures the crystallization axis has a uniform orientation and is continuous through successive growth increments (Vinn et al. 2008c). In complex oriented structures the crystallization axis of crystals has a uniform orientation, which is not continuous through successive growth increments (Vinn et al. 2008c). Trends in the evolution of tube ultrastructure in serpulids implicate that complex-oriented structures have evolved from unoriented structures (Vinn et al. 2008c). Vinn et al. (2009) proposed alternative ways to explain the calcified secretory granules described by Neff (1971a) in the lumen of the calcium-secreting glands in P. americanus: (1) The worm actually produces calcium-saturated mucus in the glands. The mucus is then deposited on the tube aperture, where crystallization of the structure is controlled by an organic matrix, as in molluscs. The calcified granules may only be an artifact of fixation and formed after the death of the worm. (2) If calcified secretory granules are not an artifact of fixation, then they must be dissolved and recrystallized before deposition of the material on the tube aperture. Consecutively, an oriented structure is formed from the mucus and regulated by the organic matrix.
34.1 Larvae Metamorphosis and the Initial Phases of Tube Formation Unlike insects, which begin metamorphosis to the adult form on a timetable determined for the most part by changes in endogenous hormonal levels, the planktonic larvae of many species of benthic marine invertebrates often delay metamorphosis to the adult form until triggered by external environmental cues (Morse 1984). For insects, the dispersing adults generally possess the external receptors necessary for recognition of the appropriate habitat for the more sedentary larval stages. The reverse is true for many species of marine invertebrates, where the dispersing larvae are responsible for recognizing the habitat that is optimal for survival and reproduction of the more sedentary adult forms (Butman et al. 1988; Chia and Rice 1978). Observations on larvae in natural marine environments are difficult. However, some interesting information was obtained in experiments using laboratory marine aquaria. Probably the first observations which were carried out on polychaetes larvae were made by Giard (1876). Charles Zeleny in 1905 described the rearing of serpulid larvae (Hydroides uncinata and H. pectinata) with respect to the formation of their tubes (Zeleny 1906) as follows:
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Larvae Metamorphosis and the Initial Phases of Tube Formation
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As soon as the pre-oral band of cilia is well developed the young larvae swim toward the surface of the water and collect there in great numbers, especially at the edges near the glass sides of the jar. The young serpulids were found to be fairly uniformly distributed, though there were groupings at several different places. One of these consisted of a large number collected on the glass just below the surface film of the water. These formed a band surrounding the jar, but it is interesting to note that they did not grow as rapidly as—those lower down, although fresh sea water was added in each case to raise the level slightly and make up for evaporation. Going down from this band-like zone of greatest frequency the number of individuals decreased until the bottom of the vessel was reached, where again there was a considerable number, especially in the corner between the bottom and the sides of the jar. The tube when first formed is a very narrow, almost transparent ring of calcareous matter, the body of the short larva extending out of it at both ends. This ring is secreted by the region just back of the free anterior end of the thoracic membrane and as its formation goes on the animal can be seen to extend its thoracic membrane over the anterior edge of the tube in order apparently to smooth the edges and get the material in shape to fit the body. At such a time the body may project a considerable distance from the anterior end of the tube. The tube is deposited quite rapidly. In the case shown in Fig. 34.1. the amount of growth in the course of twenty hours is given. This is equal to 0.29 mm. or 0.35 mm. per day.
Detection of environmental conditions by many invertebrate larvae has been shown to involve the recognition of very specific chemical cues associated with the surfaces on which the organisms settle or attach (Jensen 1987). For a variety of organisms, extracts prepared from substrata that induce metamorphosis of larvae upon contact have been shown to act as morphogens in solution. Evidence suggests that chemically induced metamorphosis of many species of marine invertebrate larvae is under the control of the larval nervous system with
Fig. 34.1 Drawings made by Zeleny in 1905: (a) serpulid larvae (Hydroides uncinata) attached to the glass surface in aquarium. (b and c) formation of the tubes (see the text) (adapted from Zeleny 1906)
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specific sensory receptors acting, at least in part, to interface the nervous system with the environment (Morse et al. 1979; Morse and Morse 1984; Morse 1984, 1985). As reviewed by Morse (1985), several cases of chemically induced settlement and metamorphosis of marine invertebrate larvae involve neurotransmitter-like inducers. Larvae of the sandcastle worm Phragmatopoma californica metamorphose into the adult form upon contact with tube material cemented together by conspecifics (Jensen and Morse 1984). The adhesive material is comprised largely of a DOPA cross-linked protein that otherwise resembles silk in amino acid composition (Jensen and Morse 1988; Waite 1987). Of the component amino acids found in the adhesive, only the aromatic amino acids, especially DOPA, were observed to have metamorphosis-inducing activity when exposed to larvae in the laboratory, albeit at a low level (10–15% at 10–5 M), higher concentrations often resulting in incomplete metamorphosis, with evidence of toxicity (Jensen 1987). It has been shown that an increase in the concentration of K+ in defined seawater medium induces settlement and metamorphosis of P. californica larvae, presumably by causing the depolarization of externally accessible, excitable cells (Yool et al. 1986). These results implicate ionic depolarization and possibly cAMP in the metamorphic process, suggesting involvement of the larval neural system in the induction of metamorphosis by the native tube cement. Possible roles of these compounds and the dependence upon receptor activation await resolution by further neurophysiological and in vitro analyses. It appears likely that the inducing chemical component(s) of native tube cement interact with receptors at the cell surface, which in turn activate adenyl cyclase to convert ATP to cAMP, a second messenger. This could act indirectly to open ion channels, causing excitatory depolarization of the membrane, initiating the cascade of behavioral and morphogenic events occurring during metamorphosis. Tufts of sensory cilia on tentacles of larvae of Phragmatopoma lapidosa have been implicated in substratum selection (Chia and Rice 1978). Similar ciliary tufts, also presumably sensory in function, have been described for Sabellaria cementarium and P. californica (Amieva et al. 1987). Upon encountering native tube-cement P. californica larvae increase contact with substrata by rubbing their tentacles, mouth, and head region against them. It is likely that chemosensory cells are located primarily in these regions. It can be assumed here that the larvae have a sequence of movements during exploration of the substratum, with each activity initiated by a hierarchy of stimuli. Given a positive set of responses to a suitable habitat, the distances moved on the substratum are limited progressively so that finally the larva investigates a very restricted area. When the exact site for settlement is determined the larva ceases to move, attaches permanently, and immediately commences metamorphosis into the adult form. However, if certain stimuli are lacking or obnoxious to the searching larva, it recommences the initial free-swimming activity, in order to reach alternative substrata.
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The Chemistry of Tube Construction
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34.2 The Chemistry of Tube Construction The construction process of the tubes is meticulous. The worm’s tentacles select an individual sand grain and move it on to a remarkable appendage which puts two small dabs of glue on the grain and places it where the next piece of construction is needed. It holds it there for around 25 s, moving it a bit to ensure that it is snuggly in place, just as we do when gluing two things together. Stewart et al. (2004) proposed a model for the structure and bonding mechanism of the cement isolated from P. californica that has the following major features: (1) within the secretory pathway of cement gland cells, the electrostatic association of the oppositely charged proteins and divalent cations (Ca2+ and Mg2+ ) condense the cement proteins into dehydrated secretory granules; (2) the condensation of the cement leads to the separation of the solution into two aqueous phases (complex coacervation) that creates the closed cell foam structure of the cement; (3) rehydration of the condensed cement granules after deposition onto tube particles contributes to the displacement of water from the mineral substrate to facilitate underwater adhesion; and (4) after secretion, covalent cross-linking through oxidative coupling of DOPA gradually solidifies the continuous phase of the cement to set the porous structure. According to the results recently obtained in the laboratory of Herbert Waite (Sun et al. 2009), the glue is a mixture of post-translationally modified proteins, notably the cement precursor proteins Pc-1 and Pc-2 with the amino acid, 3,4-dihydroxyphenyl-L-alanine (DOPA). Interestingly, both of these precursor proteins are basic (predicted pI values of 9.7 and 9.95), in contrast to numerous acidic proteins responsible for calcification phenomenon in a broad variety of marine invertebrates. The sequences of the P. californica glue proteins, referred to as Pc1-3, are highly repetitive and relatively simple (Zhao et al. 2005; Shao et al. 2009). Pc1 is comprised mostly of just three residues, glycine (45 mol%), lysine (14 mol%), and tyrosine (19 mol%), which occur as 15 repeats of the decapeptide VGGYGYGGKK. The Pc2 sequence is comprised of several degenerate copies of the dodecapeptide HPAVHKALGGYG. The sequence of Pc3, which exists as two major variants (A and B) and several minor variants, is distinguished by runs of 4–13 serine residues punctuated with single tyrosine residues. Significant amounts of a halogenated derivative of DOPA were isolated from the worm cement following partial acid hydrolysis and capture of catecholic amino acids by phenylboronate affinity chromatography. Analysis by tandem mass spectrometry and 1 H NMR indicates that the DOPA derivative is 2-chloro-4, 5-dihydroxyphenyl-L-alanine (Sun et al. 2009).
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When individual P. californica in a short segment of their tube were laid on a bed of glass beads (0.5·mm diameter), they worked compulsively to rebuild their tube by cementing the available glass beads to the tube’s anterior end (Jensen and Morse 1988). In a 24-h period, a typical adult animal extended its tube by up to 1·cm with glass beads. The cement of freshly deposited glass beads was creamy white. Over a period of about 6·h the cement spots became distinctly reddish brown in color. Old cement spots ( > 48·h) were dark reddish brown. SEM examination of cemented glass beads less than 24·h old revealed cement disks with uniform diameters (about 200·μm) that were positioned at uniform distances from one another on the beads (Fig. 34.2). P. californica tube cement has an internal structure reminiscent of a solid foam with closed cells that is covered by a less porous skin (Fig. 34.3). In this respect, it closely resembles the foam-like structure of the byssal adhesive plaques of mussels (Benedict and Waite 1986; Tamarin et al. 1976). Based on sound engineering principles, foamed adhesives offer several benefits for tubeworms (Stewart
Fig. 34.2 When individual P. californica in a short segment of their tube were laid on a bed of glass beads (500 μm diameter), they worked compulsively to rebuild their tube by cementing the available glass beads to the tube’s anterior end. SEM examination of cemented glass beads less than 24-h old revealed cement disks with uniform diameters that were positioned at uniform distances from one another on the beads (image courtesy Dan Morse)
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The Chemistry of Tube Construction
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Fig. 34.3 Sandcastle worm Phragmatopoma californica (a). The worm’s tentacles select an individual glass particles (b) or sand grain (c) and move it on to a remarkable appendage which puts two small dabs of glue on the grain and places it where the next piece of construction is needed
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et al. 2004). First, the foam-like structure would increase the cement’s elasticity and toughness—the amount of energy a material can absorb before failing (Gibson and Ashby 1997). The more flexible cement junctions would absorb and dissipate the energy of the impinging surf in the inter-tidal environment to minimize damage to the tube, like foam packing material. A related benefit is the demonstrated crackstopping behavior of foams. Second, the cellular structure of the cement would save material and metabolic energy. If the cohesive strength of the solid cement were much greater than the adhesive strength of the cement to the substrate, then the extra material in a solid cement would be wasted. Matching the adhesive and cohesive strengths of the cement is more economical. Third, a foam structure could minimize the abruptness of the elastic modulus mismatch between the rigid particles and the flexible cement. A gradient in the cell size from the interface with the particle to the center of the cement, with the smallest cells being close to the interface, would create a gradient in the elastic modulus with the highest cement modulus being at the interface (Stewart et al. 2004). The serpulid Mercierella enigmatica secretes a hard, calcareous tube shortly after it settles from the plankton. Initially, tube growth is relatively fast, but as the tube increases in length the rate of growth declines. However, the tube of the adult worm can be as much as 80 mm in length or more than four times the length of its body. Large aggregates of calcareous serpulid worm tubes have been recorded at paleoclimate conditions. Observation of modern serpulid reefs in Baffin Bay, Texas, suggests serpulids are a euryhaline animal, able to withstand variable environmental conditions (Glumac et al 2004). Tubes can grow to ∼2 cm in length and form bell-shaped accretionary ridges. In Baffin Bay, these filter-feeding worms colonize waters at depths ranging from 0.5 to 2.5 m and are generally not exposed at high tide (Andrews 1964). Geologically, sabellariids are of interest because they select only certain mineral grain sizes and thus effect a natural sorting of the sediment (Multer and Milliman 1967). Heavy minerals are concentrated in the tubes relative to their abundance in the adjacent sediment (Gram 1968). After the worm’s death the tubes are readily disaggregated; hence, sabellariid reefs are only rarely known to be preserved as fossils. Calcareous serpulid tubes, however, do contribute considerably to the fossil record.
34.3 Features of the Pectinariid Tubes The foam-like cement of a tubicolous polychaete was noted as early as 1903 (Fauvel 1903), who described the “ciment bulbous,” or “bubbly cement” of a pectinariid polychaete. Like Sabellariidae, the Pectinariidae cement adventiously gathered particles into a solitary tube (Fig. 34.4), but do not form colonies. Recently, Dean et al. (2009) reported results of comparative studies which have been carried out using two different species of polychaetes. Pectinaria gouldii
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Features of the Pectinariid Tubes
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Fig. 34.4 Overview of the solitary tube of Pectinaria sp. (a) collected in the Sea of Japan (samples courtesy Marina Yurieva). Light (b) and fluorescence microscopy (c) images show how siliceous microparticles are cemented (arrows) with each other. Treatment using 35% H2 O2 leads to dissolution of organic-based cements (d). The siliceous grains that comprise the main building blocks of the tube were dissolved, however, using 48% HF during 24 h at room temperature (e)
(Verrill) and Phragmatopoma lapidosa (Kinberg) are small polychaetous annelids found in intertidal and subtidal regions along the Atlantic coastline. The benthic, solitary living P. gouldii is also known as a trumpet worm or ice cream cone worm and can grow up to 5 cm in length (Remsen 2007). It uses sand grains to construct its tube which has an ice cream cone-like structure that is open at both ends. Sand grains are selected by size, then fitted and cemented together in a single layer. The
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smaller, gregarious P. lapidosa form reef-like mounds that can vary in size from a softball to large boulder-like formations that can be a part of a network that stretches for miles (Kirtley and Tanner 1968). These large structures form reefs that help shelter beaches by absorbing the force of the waves. Both polychaetes utilize a type of cement that is secreted from specialized glands to attach sand particles together in order to build their dwellings. The properties of cellular solids (Gibson and Ashby 1997) are advantageous to the dwellings of P. gouldii and P. lapidosa. One of these properties is the ability to absorb the energy of impacts, which allows P. lapidosa to handle wave action along the Atlantic coastline and allows P. gouldii to maintain structural integrity under benthic conditions. A second property may be reliable thermal insulation, which helps them to tolerate temperature fluctuations of the ocean environment. Like most natural foams, both biocements are anisotropic, which occurs as these compartments elongate in a given direction. A variation in foam bubble compartment size for P. lapidosa (0.1–5 μm) was seen consistently in SEM scans. P. gouldii had foam bubble compartment sizes in the range of 5–7 μm (Dean et al. 2009). Both biocements present high levels of magnesium and phosphorus, indicating similarities in the composition between the two biocements. However, differences in calcium levels suggest that the biocements accomplish their function by slightly different mechanisms (Dean et al. 2009). Metal ions are commonly used to help cross-link proteins noncovalently. The high levels of phosphorus in both biocements correlate well with published data on the sequence of a suspected P. gouldii cement protein, which shows high levels of serine (Briggs et al. 2004). Serine is a commonly phosphorylated amino acid, therefore when high levels of serine are seen, high levels of phosphorus can be expected. Additionally, P. californica has already been reported to also have high levels of serine associated with cement proteins (Zhao et al. 2005). Recently, we also carried out some experiments with solitary tubes obtained from one unidentified Pectinaria species to investigate the chemical resistance of cementous material against hydrofluoric acid and hydrogen peroxide. We monitored these experiments using light and fluorescence microscopy. Intriguingly, biocement was absolutely resistant to treatment using 48% HF during 24 h at room temperature. However, the siliceous grains that comprise the main building blocks of the tube were dissolved under these conditions (Fig. 34.4e). Treatment using 35% H2 O2 , under the same conditions, leads to dissolution of organic-based cements (Fig. 34.4d). Further investigations to better understand the mechanisms of phenomena listed above are currently underway in our laboratory.
34.4 Biomimetic Potential of Polychaetes Bioadhesives The polychaetes cement is an important model for biomimetic adhesives because of its apparent toughness, because it adheres strongly to a variety of materials and because it bonds rapidly to these materials in seawater.
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Biomimetic Potential of Polychaetes Bioadhesives
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Orthopedic medicine, in particular, has a striking reliance on metal hardware and power tools for repairing damaged bones. Bonding living tissues is challenging because of the wet environment, aggressive immune surveillance for foreign materials, restricted temperature range. An additional factor includes the requirement for non-poisonous reactants, reaction by-products, and degradation products. There are no suitable adhesives that are currently in widespread use in orthopedic surgery. Modern high-performance adhesives including epoxies, polyurethanes, cyanoacrylates, and polyacrylics have been tested and in some cases are used to repair superficial wounds, but have not met the demands of deep tissue bonding or bone fixation. Guided by a natural adhesive produced by a shell-dwelling marine worm, Shao et al. (2009) have taken an alternative approach to developing adhesives for orthopedic surgery and other medical applications. Polyacrylate glue protein analogs of the glue secreted by P. californica were synthesized with phosphate, primary amine, and catechol side chains with molar ratios similar to the natural glue proteins. Aqueous mixtures of the mimetic polyelectrolytes condensed into liquid complex coacervates at roughly neutral pH. Wet cortical bone specimens bonded with the coacervates, oxidatively crosslinked through catechol side chains, had bond strengths nearly 40% of the strength of a commercial cyanoacrylate. The unique material properties of complex coacervates, proposed by the authors, may be ideal for development of clinically useful adhesives and other biomaterials. A conceptual model (Shao et al. 2009) of the phase behavior and bonding mechanism of the mimetic coacervated adhesive is presented in Fig. 34.5. At low pH the oppositely charged polyelectrolytes associate electrostatically into nanocomplexes with a net positive surface charge that stabilizes the suspension (Fig. 34.5a). With increasing pH, the net charge of the complexes changes from positive to negative but remains near net neutrality. The complexes form loose precipitates (Fig. 34.5a) over the pH range where the phosphate side chains have a single negative charge. As the
Fig. 34.5 Model of pH-dependent coacervate structure and adhesive mechanisms (adapted from Shao et al. 2009). (a) The polyphosphate with low charge density paired with the polyamine form nanometer scale complexes. The complexes have a net positive charge. (b) Extended high charge density polyphosphates form a network connected by more compact lower charge density polyamines and, when present, divalent cations. The net charge on the copolymers is negative. (c) Oxidation of 3,4-dihydroxyphenol (D) by O2 or an added oxidant initiates cross-linking between the quinone (Q) and primary amine side chains. The coacervate can adhere to the hydroxyapatite surface through electrostatic interactions, 3,4-dihydroxyphenol side chains, and quinone-mediated covalent coupling to matrix proteins
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phosphate side chains undergo a second ionization with rising pH, the polyphosphates become more extended and rigid due to the increasing double-negative charge density. The excess negative charges (Fig. 34.5b) are shielded only by monovalent ions in the low ionic strength solution. Eventually the densely charged polyphosphates extend to form a cohesive liquid network with dynamic ionic junctions formed by the more compact polyamines and divalent cations (Fig. 34.5b). The liquid coacervate has low initial viscosity, a specific gravity greater than 1, and being mostly water by weight, low interfacial tension in an aqueous environment. All of these factors contribute to its ability to wet the bone surface, a prerequisite for effective underwater adhesion. The coacervate adheres to bone (and other minerals) through several mechanisms (Fig. 34.5c). The surface of the bone’s hydroxyapatite mineral phase [Ca5 (PO4 )3 (OH)] is an array of both positive and negative charges. The negative polyphosphate can interact directly with the positive surface charges or it can be bridged to the negative surface charges through the positive polyamine and/or divalent cations. Likewise, direct interaction of the polyamine with the negative surface charges would contribute to adhesion. Molecules containing catechol moieties have been shown to have strong absorptive properties and to readily wet hydroxyapatite. Further contributions to strong interfacial adhesion may therefore come from direct bonding of unoxidized dopamide side chains to hydroxyapatite. Side chains oxidized to dopaquinone could covalently couple to nucleophilic side chains of bone matrix proteins as another factor in strong adhesion. Cohesive strength develops through intermolecular covalent coupling between the dopaquinone side chains on the polyphosphate and the primary amine side chains of the polyamine (Fig. 34.5c). The increased density of the cation containing coacervate phases may be due to more compact junctions between polyphosphates and divalent cations compared to polyamine junctions. Divalent cation bridging between the polyphosphate and hydroxyapatite surface may be more effective than polyamine bridging or direct polyphosphate binding contributing to stronger adhesion. The divalent cations may also affect polyphosphate conformation, the number of effective intermolecular cross-links, as well as polyphosphate hydration and solubility. The first-generation mimetic adhesive described by Stewart and co-workers (Shao et al. 2009) qualitatively reproduced several observed or predicted features of the natural glue: the nanoparticulate structure, pH-dependent phase behavior, ability to bond wet mineral substrates, and oxidative DOPA-mediated curing. As the composition, application methods, curing trigger, and kinetics are optimized it seems reasonable to expect significant improvements in the adhesive bond strength to hydroxyapatite. In an iterative cycle of discovery and implementation, additional features of the P. californica adhesive, both already known and yet to be appreciated, will be built into the mimetic adhesive system for the dual purposes of testing hypotheses about natural glue structure and bonding mechanisms and to further improve the synthetic adhesive.
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Conclusion
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Hydroxyapatite, or something like it, therefore sounds ideal for repairing bones. The resulting glue not only sticks bits of bone together in watery environments but also does so with twice the strength of the glue used by the worm. And, although it is still early days, preliminary tests suggest it is both non-toxic and biodegradable. If further testing confirms this, it means that, as the broken bone heals, the glue will disappear naturally. Complex fractures will thus heal more easily. Industrial demand in novel biomimetic adhesives seems to be a driving force for the establishment of a new scientific direction called glueomics (Endrizzi and Stewart 2009). Thus, random clones were sequenced from a cDNA library constructed from the adhesive gland of P. californica. As many as 14 new proteins and 2 phenoloxidase enzymes were found that may be structural components of the bioadhesive or involved in its processing. Glue protein classification was based on the following criteria: (i) the presence of predicted secretion signal peptides; (ii) low complexity sequences; (iii) strongly skewed amino acid compositions enriched with G, Y, K, H, A, or S; (iv) repeating peptide motifs; and (v) homology to known glue proteins, other structural proteins, or enzymes. The new genes provide probes for further characterization of the adhesive gland as well as potential biotechnological resources and insight (Endrizzi and Stewart 2009).
34.5 Conclusion We are not the only species that builds homes and condominium complexes from sand. The sandcastle worm lives in a mineral shell. It does not, however, secrete this shell directly in the way that, for example, a mollusc would. Instead, it secretes a glue or biocement and uses this to stick bits of sand together to form its casing, in the way that, for example, a freshwater caddis fly larva does. The glue does not dissolve in water. Indeed, it is able to displace water and thus adhere to surfaces in aqueous solutions, and it solidifies soon after being secreted. Some fascinating properties of these biocements are their ability to harden in seawater and withstand ocean waves, tide cycles, and other forces prevalent along the coast and in extreme marine environments. Moreover, some of these biocements-based constructs are reefs and can give us information as a paleoarchives. Above, we discussed several examples where biology employs different biopolymers to perform mechanical functions using very specific covalent-based cross-links to confer elasticity and strength. The chemistry of annelids bioadhesion is well described in the numerous pioneering and fundamental works by Dan Morse cited here, as well as those by Herbert Waite, which I definitively recommend to the reader as follows (Waite 1991, 1995, 1999, 2002; Waite et al. 1992; Waite and Qin 2001). Finally, the first attempts to mimic these evolutionary ancient and biochemically complex processes for applications in biomedicine has optimistic preliminary results.
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References Amieva MR, Reed CG, Pawlik JR (1987) Ultrastructure and behavior of the larva of Phragmatopoma californica (Polychaeta: Sabellariidae): identification of sensory organs potentially involved in substrate selection. Mar Biol 95:259–266 Andrews PB (1964) Serpulid reefs, Baffin Bay, Southeast Texas. Depositional Environments, South-Central Texas Coast. Field Trip Guidebook, October 30–31, Gulf Coast Association of Geological Societies, Annual Meeting in Corpus Christi Benedict CV, Waite JH (1986) Composition and ultrastructure of the byssus of Mytilus edulis. J Morphol 189:261–270 Briggs DT, Edwards HD, Watson AM et al (2004) Pectinaria gouldii clone A10-802 hypothetical protein mRNA, 3’ partial cds. Accepted Jan 2004. GenBank Accession Number: AY534619 Bubel A (1983) A fine structural study of the calcareous opercular plate and associated cells of a polychaete annelid. Tissue Cell 15:457–476 Butman CA, Grassle JP, Webb CM (1988) Substrate choices made by marine larvae settling in still water and in a flume flow. Nature 333:771–773 Chia FS, Rice ME (1978) Settlement and metamorphosis of marine invertebrate larvae. Elsevier, New York Day JH (1973) New Polychaeta from Beaufort, with a key to all species recorded from North Carolina. National Oceanic and Atmospheric Administration Technical Report. National Marine Fisheries Service. Circular 375:1–140 Dean M, Welch J, Brandt C et al (2009) Surface analyses of biocements from Pectinaria gouldii (Polychaeta: Pectinariidae) and Phragmatopoma lapidosa (Polychaeta: Sabellariidae). Zoosymposia 2:329–337 Endrizzi BJ, Stewart RJ (2009) Glueomics: an expression survey of the adhesive gland of the Sandcastle Worm. J Adhesion 85:546–559 Fauvel P (1903) Le tube des Pectinaires. Mem Pontif Ac nuov Lin 21:28–57 Fauvel P (1923) Un nouveau serpulien d’eau saumatre Mercierella n. g. enigmatica n. sp. Bull Soc Zool France 47(1922):424–430 Fischer R, Pernet B, Reitner J (2000) Organomineralization of cirratulid annelid tubes – fossil and recent examples. Facies 42:35–50 Giard A (1876) Note sur l’embryoginie de la Salmacina Dysteri HuxI. Compt Rend 82:233–235, 82:285–288 Gibson LJ, Ashby MF (1997) Cellular solids: structure and properties. University Press, Cambridge, UK Glumac B, Berrios L, Greer L et al (2004) Holocene tufa-coated serpulid mounds from the Dominican Republic: depositional and diagenetic history, with comparison to modern serpulid aggregates from Baffin Bay, Texas. Proceedings of the 11th Symposium on the Geology of the Bahamas and Other Carbonate Regions: San Salvador, Gerace Research Center, pp 49–65 Gram R (1968) A Florida Sabellariidae reef and its effect on sediment distribution. J Sediment Petrol 38:863–868 Hedley RH (1956a) Studies on serpulid tube formation. I. The secretion of the calcareous and organic components of the tube by Pomatoceros triqueter. Q J Microsc Sci 97:411–427 Hedley RH (1956b) Studies on serpulid tube formation. II. The calcium-secreting glands in the peristomium of Spirorbis, Hydroides and Serpula. Q J Microsc Sci 97:421–427 Jensen RA (1987) Factors affecting the settlement, metamorphosis and distribution of larvae of the marine polychaete Phragmatopoma californica (Fewkes). Ph.D. Dissertation. University of California, Santa Barbara, CA Jensen RA (1992) Marine bioadhesive: role for chemosensory recognition in a marine invertebrate. Biofouling 5:177–193 Jensen RA, Morse DE (1984) Intraspecific facilitation of larval recruitment: gregarious settlement of the polychaete Phragmatopoma californica (Fewkes). J Exp Mar Biol Ecol 83: 107–126
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Jensen RA, Morse DE (1988) The bioadhesive of Phragmatopoma californica tubes: a silk-like cement containing L-DOPA. J Comp Physiol B 158:317–324 Kirtley DW, Tanner WF (1968) Sabellariid worms; builders of a major reef type. J Sediment Res 38:73–78 Linnaeus C (1758) Systema Naturae, 10th edn, vol 1. L. Salvius, Holmiae Morse DE (1984) Biochemical control of larval recruitment and marine fouling. In: Costlow JD, Tipper RC (eds), Marine biodeterioration. Naval Institute, Annapolis, MD Morse DE (1985) Neurotransmitter-mimetic inducers of larval settlement and metamorphosis. Bull Mar Sci 37:697–706 Morse DE, Hooker N, Duncan H, Jensen L (1979) T-Aminobutyric acid, a neurotransmitter, induces planktonic abalone larvae to settle and begin metamorphosis. Science 204:407–410 Multer HG, sMilliman JD (1967) Geologic aspects of sabellarian reefs Southeastern Florida. Bull Mar Sci 17:257–267 Munro Fox H (1938) Function of the tube in sabellid worms. Nature 141:163 Neff JM (1971a) Ultrastructural studies of the secretion of calcium carbonate by the serpulid polychaete worm, Pomatoceros caeruleus. Zeitschrift fur Zellforschung 120:160–186 Neff JM (1971b) Ultrastructure of calcium phosphate-containing cells in the serpulid Pomatoceros caeruleus. Calcified Tissue Res 7:191–200 Nott JA, Parkes KR (1975) Calcium accumulation and secretion in the serpulid polychaete Spirorbis spirorbis L. at settlement. J Mar Biol Assoc UK 55:911–923 Remsen D (2007) Marine organisms database, Pectinaria. MBL Woods Hole, Massachusetts. http://www.mbl.edu/marine_org/marine_org.php?func=detail&myID=ITA-67706 Accessed 31 August 2008 Schmarda LK (1861) Neue wirbellose Tiere beobachtet und gesammelt auf einer Reise um die Erde 1853 bis 1857, I: Turbellarien, Rotatorien und Anneliden (2). Verlag von Wilhelm Engelmann, Leipzig Shao H, Bachus KN, Stewart RJ (2009) A water-borne adhesive modeled after the sandcastle glue of P. californica. Macromol Biosci 9:464–471 Simkiss K, Wilbur KM (1989) Biomineralization: cell biology and mineral deposition. Academic, New York Stewart RJ, Weaver JC, Morse DE et al (2004) The tube cement of Phragmatopoma californica: a solid foam. J Exp Biol 207:4727–4734 Sun CJ, Srivastava A, Reifert JR et al (2009) Halogenated DOPA in a marine adhesive protein. J Adhesion 85:126–138 Swan EF (1950) The calcareous tube secreting glands of the serpulid polychaetes. J Morphol 86:285–314 Tamarin A, Lewis P, Askey J (1976) The structure and formation of the byssal attachment-forming region in Mytilus californianus. J Morph 149:199–221 Vinn O, Kirsimäe K, ten Hove HA (2009) Tube ultrastructure of Pomatoceros americanus (Polychaeta, Serpulidae): implications for the tube formation of serpulids Estonian. J Earth Sci 58:148–152 Vinn O, Mutvei H, ten Hove HA et al (2008a) Unique Mg-calcite skeletal ultrastructure in the tube of the serpulid polychaete Ditrupa. Neues Jahrbuch fur Geologie und Palaontologie, Abhandlungen 248:79–89 Vinn O, ten Hove HA, Mutvei H (2008b) On the tube ultrastructure and origin of calcification in sabellids (Annelida, Polychaeta). Palaeontology 51:295–301 Vinn O, ten Hove HA, Mutvei H et al (2008c) Ultrastructure and mineral composition of serpulid tubes (Polychaeta, Annelida). Zool J Linnean Soc 154:633–650 Vovelle J (1956) Processus glandulaires impliques dans la reconstitution du tube chez Pomatoceros triqueter (L.) Annelida Polychete (Serpulidae). Bull Lab Maritime Dinard 42:10–32 Vovelle J (1965) Le tube de Sabellaria alveolata (L) – Annelide Polychète Hermellidae – et son ciment. Thèse, Université de Paris; Centre National de la Recherche Scientifique
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Vovelle J, Grasset M, Truchet M (1991) Sites of biomineralization in the Polychaete Pomatoceros triqueter (Serpulidae) with comments on some other species. Ophelia, Supplement 5:661–667 Waite JH (1987) Nature’s underwater adhesive specialist. Int J Adhes Adhes 7:9–14 Waite JH (1991) Detection of peptidyl-3,4 dihydroxyphenylalanine by amino acid analysis and microsequencing techniques. Anal Biochem 192:429–433 Waite JH (1995) Precursors of quinone tanning: dopa-containing proteins. Meth Enzymol 258: 1–20 Waite JH (1999) Reverse engineering of bioadhesion in marine mussels. Ann NY Acad Sci 875:301–309 Waite JH (2002) Adhesion a la moule. Integrat Comp Biol 42:1172–1180 Waite JH, Qin X (2001) Polyphosphoprotein from the adhesive pads of Mytilus edulis. Biochemistry 40:2887–9283 Waite JH, Jensen RA, Morse DE (1992) Cement precursor proteins of the reef-building polychaete Phragmatopoma californica (Fewkes). Biochemistry 31:5733–5738 Weedon MJ (1994) Tube microstructure of recent and Jurassic serpulid polychaetes and the question of the Palaeozoic ‘spirorbids’. Acta Palaeontol Pol 39:1–15 Yool AJ, Grau SM, Hadfield MG et al (1986) Excess potassium induces larval metamorphosis in four marine invertebrate species. Biol Bull 170:255–266 Zeleny C (1906) The rearing of serpulid larvae with notes on the behaviour of the young animals. Contributions from the Zoological Institute of Indiana University 65:308–312 Zhao H, Sun C, Stewart RJ et al (2005) Cement proteins of the tube-building polychaete Phragmatopoma californica. J Biol Chem 280:42938–42944
Part VIII
Extreme Biomimetics
Chapter 35
Life in Extreme Environments: From Bacteria to Diatoms
Abstract Temperature variation is an intrinsic property of almost all ecosystems and many environments feature large temporal and/or spatial temperature gradients. Several species of eurythermal marine invertebrates dwell in environments with extreme physico–chemical conditions, e.g., temperatures exceeding 100◦ C, concentrations of hundreds of micromolar in sulfide, oxygen depletion, acidic pH, CO2 -rich conditions, and the enrichment of ocean waters by several order of magnitude in various metals. Another example of extreme environments is related to hydrothermal systems, such as terrestrial hot springs and hydrothermal vents on the seafloor, where biomineralization and especially silica precipitation occurs. Thus, Nature opens for us new, still poorly investigated exotic niches with extreme physicochemical conditions. Also in these niches numerous taxa of marine invertebrates dwell in, as well as produce, biological materials with unique properties. Extreme Biomimetics is proposed here as a new direction in materials science with respect to develop a new generation of biomaterials. I already reported in this work on marine organisms which dwell in the extreme cold environments. Unfortunately, the mechanisms and principles of biological materials formation in these animals, including cuticles, skeletons, and skeletal formations like spicules, at 0◦ C and lower are still unknown. Temperature variation is an intrinsic property of almost all ecosystems and many environments feature large temporal and/or spatial temperature gradients. Organisms adapted to such wide ranges of temperatures are termed eurythermal (Lee et al. 2008). Several species of such eurythermal marine invertebrates dwell in environments with extreme physico–chemical conditions, e.g., temperatures exceeding 100◦ C, concentrations of hundreds of micromolar in sulfide, oxygen depletion, acidic pH, CO2 -rich conditions, and the enrichment of ocean waters by several order of magnitude in various metals (Le Bris and Gaill 2007). The best known examples of animals from such extreme environments are vestimentiferan worms from hydrothermal vents (Blake 1985) (Fig. 35.1). Thus, the high-temperature diffuse flow surrounding the worms’ tubes is acidic (pH 4.2–6.1) carrying high levels of total hydrogen sulfide (>1 mM), ammonia (3.8–10 μM), and reactive heavy metals (0.3–200 μM) including ferrous iron (290–840 μM) (Grzymski et al. 2008). H. Ehrlich, Biological Materials of Marine Origin, Biologically-Inspired Systems 1, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9130-7_35,
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Fig. 35.1 Deep-sea images of hydrothermal vent (a) as well as of vestimentiferan fauna (b, c, d), which is well adapted to these extreme environment (images from the IMAX film “Volcanoes of the Deep Sea,” courtesy Rutgers University and The Stephen Law Company)
Another example of extreme environments is related to hydrothermal systems, such as terrestrial hot springs and hydrothermal vents on the seafloor, where biomineralization and especially silica precipitation occurs (Tazaki et al. 1996, 1999; Tazaki 1999). The diversity of species in these extreme ecosystems is restricted. Where the pH of the system is low, the diversity is even more restricted. Silicified microbial mats (biomats), from modern to Precambrian aqueous settings, have been investigated in the past (Akai et al. 1995; Asada and Tazaki 1999; Tazaki et al. 1998; Walter et al. 1972). However, the mechanism of silicification of the surface of corresponding microorganisms is poorly understood. I believe, that from biomaterials science point of view, investigations of biocomposites, which are formed under extreme physicochemical conditions listed above, are absolutely necessary. Moreover, I propose the introduction of the new term, “Extreme Biomimetics,” to describe this novel direction in materials science. Below, I will make an attempt to explain why an establishing of Extreme Biomimetics is necessary today.
35.1 Eurythermal Marine Biota as Source for Development of Novel Biomaterials Discoveries of the unique hydrothermal vent fauna are indebted to the progress made by underwater expeditions using deep-sea vessels. The history of these discoveries is well described by Francheteau and Laubier (1982); Rona et al. (1983);
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Laubier and Desbruykes (1985). Briefly, in the early 1950s the Danish research vessel Galathea had established that life was present at the greatest depths known, more than 10,000 m below the surface. Since then, it had been established that, though life might have no lower limit in the oceans, the primary characteristic of the fauna of the great depths was its sparse distribution. However, despite popular belief, the almost deserted appearance of the great depths is the result neither of the water temperature, barely above 0◦ C, nor the total absence of light, nor yet the pressure of around 1,000 bars—life is scarce for the simple reason that food is likewise scarce. During the spring of 1977 the US Navy submersible Alvin, diving over the Galapagos Ridge on the Equator at about 86◦ W where, 1 year previously, the American Pleiad Expedition organized by the Scripps Institution of Oceanography had brought back remarkable photographs of the ocean floor showing life flourishing around hot water springs, confirmed the importance of this discovery. Entire animal communities were seen growing around the outlets of hot springs in exuberant colonies of creatures with stunning morphology and size. These were nothing less than oases, in stark contrast to the usual desert of the ocean depths. In 1978 the French submersible Cyana began exploring a section of the Eastern Pacific Ridge around 21◦ N. There, some distance from massive polymetallic sulfur structures like anthills, observers discovered a number of zones covered with the abandoned shells of one of the two large bivalve molluscs found at the Galapagos. One year later, the same teams on board the Alvin discovered, only a few kilometres away, the famous black smokers: jets of superheated black water ejected from chimneys formed from deposits of polymetallic sulfides. Around the smokers, the animal colonies were reminiscent of those of the Galapagos, but extended over even wider areas. American biologists and French researchers made a series of dives in the Alvin over this site during the spring of 1982. Two years previously, research by Jean Charcot had established that hydrothermal phenomena were widespread along the axis of the Eastern Pacific Ridge, from 21◦ N over a distance of 2,400 nautical miles as far as Easter Island at almost 20%. One of the sites thus identified at 13◦ N was the scene of the first French research programme organized by biologists (Biocyatherm), in March 1982, where animal populations associated with hydrothermal phenomena proved to be particularly abundant. In another geological environment American biologists were carrying out observations in the Guaymas Basin in the Gulf of California, where basalt bedrock is covered by almost 400 m of sediment. There, the ocean floor is covered with a thick layer of filament bacteria of the genus Beggiatoa, with the main species of hydrothermal invertebrates likewise being present. During the summer of 1983, a Canadian team using the Canadian vessel Pisces IV studied a new type of hydrothermal population associated with emissions centered on a volcano on the Juan de Fuca Ridge of British Colombia at 46◦ N and at a depth of 1,570 m. Finally, in March 1984, a French team made a second visit, 2 years after its discovery, to the Pacific hydrothermal site situated at 13◦ N. Comparison with the detailed observations carried out in March 1982 showed the scale of fluctuations in the flow of hydrothermal fluids around a single spring, or even a group of springs, and provided original data on the various species’ tolerance of variations in the physico-chemical environment.
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In March 1984, at a depth of 3,270 m in the Gulf of Mexico off Florida, Alvin discovered a population of the hydrothermal type, including large pogonophorous worms and two large bivalves. The hydrothermal fluid had little apparent similarity with the more or less dilute fluids emitted by the black smokers of the Eastern Pacific; this was a supersaline ammonia-rich fluid which resulted in the deposition of sediments rich in iron sulfide. Since the Galapagos Rift discovery, numerous hydrothermal vent sites have been found throughout the world’s oceans and over 500 new species have been described from these regions. Also fluids with temperatures as high as 403◦ C exit from polymetallic sulfide chimneys have been reported in many of these regions. Since their discovery in the 1970s, manned-submersibles like the actual Nautile (Ifremer, France), Alvin (WHOI, USA), Shinkai (Jamstec, Japan), and “Mir-2” (Russia) have allowed to access these unique habitats at depths ranging from 1,500 to 4,000 m. In the last decade, the capacity to characterize these habitats has been further expanded not only by ROVs (Remote Operated Vehicles), that substantially enlarged dive time, but also by the development of a new set of dedicated instruments. Thus, the hydrothermal environment is harsh, considering the pressure (260 atm), temperature (350◦ C), and toxicity of the hydrothermal fluid (acid, anoxic, and rich in metallic sulfides) (Gaill 1993). The most attention of researches was focused on two representatives of the hydrothermal vent fauna, the Alvinella and Riftia species. Pompeii worms (Alvinella pompejana) (christened Pompeii worm by the geologists because it can tolerate a permanent shower of metallic particles) inhabit the hottest part of the hydrothermal ecosystem on the walls of chimney-like structures (Desbruyères et al. 1998). Capable of withstanding temperatures up to 105◦ C (Chevaldonné et al. 1992), these animals are considered as the most eurythermal metazoans known (Cary et al. 1998). At their surface, where the plumes of the worm’s four pairs of branchiae develop, the temperature is between 20 and 30◦ C; it rises to over 100◦ C when the probe is inserted to a depth of some 12 cm in the mass of tubes and 250◦ C when inserted to its maximum length, 20 cm! The appearance of hydrothermal fluid emissions varies according to its temperature. Apart from being possibly the most eurythermal eukaryote discovered to date, A. pompejana is also likely to be highly thermotolerant, since frequent temperature spikes of 80◦ C or more have been observed inside tubes in which it resides (Cary et al. 1998). Two biological materials of A. pompejana that arise under these extreme physicochemical conditions must attract attention of materials scientists: the exoskeleton of the worm tube and the collagen (Le Bris and Gaill 2007). While the other species are embedded in the mucus layer they secrete, Alvinella spp. are the only species that forms such an exoskeleton. This concentrically multilayered structure secreted by the animal has a considerable thermal and chemical stability, as compared to other annelid tubes (Gaill and Hunt 1986). This assemblage of biopolymers is composed of about 50% of proteins, forming a liquid crystallinelike organization (Gaill and Bouligand 1987). This exopolymer cannot be destroyed within the 0–100◦ C range and neither strongly acidic nor alkaline solution causes major degradation (Gaill and Hunt 1986). Such a hydrophobic extracellular
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structure was hypothesized to protect the worm from the fluids migrating around their outer environment (Gaill and Hunt 1991). The micro-analysis of mineral precipitates formed at the outer and inner faces of the tube have emphasized a strong mineralogical gradient and were the first confirmation that the tube acts as a very robust barrier against the vent fluids (Zbinden et al. 2003). The collagen of these worms is also very unusual. The triple helices of cuticular collagen belong to the longest collagen molecules known thus far. They reach between 2,400 and 2,600 nm in A. pompejana, Alvinella caudata, Paralvinella grasslei (Gaill et al. 1991, 1995). Whereas the interstitial collagen of coastal polychaete worms (Arenicola marina) is denatured at 28◦ C, the collagen of A. pompejana remains stable at 45◦ C and is thus the most thermostable fibrillar collagen currently known (Gaill et al. 1991, 1995). Conversely, its northern Pacific relative, Paralvinella sulfincola, was very recently confirmed to be tolerant to temperature of 50–55◦ C (Girguis and Lee 2006), the highest ever found for a marine metazoan. Interestingly, Lepescheux (1988) reported in detail about features of spatial organization of collagen fibrils in P. grasslei cuticle (skin). Collagen fibrils formed trigonal as well as pentagonal lattices (Fig. 35.2). Moreover, it has been reported about the presence of two superimposed layers of collagen fibrils in the skin of P. grasslei. (Fig. 35.3). Probably, because of these superstructures cuticular collagens of Paralvinella species are especially resistant to high temperatures. Several features of the Alvinella collagen suggest an adaptation to the hydrothermal vent environment: thermostability (Gaill et al. 1995), but also barostability
Fig. 35.2 Schematic representation in horizontal projection of the perfect lattice of collagen fibrils (a) and the two main irregularities pentagonal (b) and trigonal (c) (images courtesy Denis Kurek, adapted from Lepescheux 1988)
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Fig. 35.3 Schematic drawing of two superimposed layers of collagen fibrils in the skin of P. grasslei. The coiled bundles are parallel to the skin and left-handed, as is the twist within bundles, whereas collagen molecules follow right-handed helices. Microvilli are shown penetrating between fibrils (blue colour) (image courtesy Denis Kurek, adapted from Lepescheux 1988)
(Auerbach et al. 1995), and the associated enzymatic processes which appear to be optimized under anoxic conditions (Kaule et al. 1998). For example, the A. pompejana prolylhydroxylase are only active in hypoxic media and, above 10% of atmospheric saturation, oxygen appears as a poison for the metabolic machinery of collagen synthesis. This indicates that the worm is not only facing the highest temperature ever known for marine invertebrates, but has a metabolic machinery adapted for working in low-oxygen environments. Riftia pachyptila also possess unusual cuticular collagen. In this organism the glycosylated threonine is located at the Y-position of the Gly-X-Y triplets of the amino acid sequence and presumably enhances the thermal stability of the triple helices of collagen as an adaption to hydrothermal environment of the species (Bann and Bächinger 2000; Bann et al. 2000; Mann et al. 1996; Sicot et al. 2000). The role of chitin in survival of Riftia species under extreme environmental conditions is also very significant. Pogonophora are the only polychaete group where chitin has been detected within the tubes (Blackwell et al. 1965; Gaill and Hunt 1986). In R. pachyptila many microfibrils or crystallites of β-chitin are embedded in parallel within a protein matrix and together form flat ribbon like structures (Gaill et al. 1992a, b). Several criss-crossing layers of these ribbons build up the tube wall. The huge crystallites are composed of up to 6000 β-chitin chains and are secreted by specialized multicellular so-called pyriform glands. In R. pachyptila the secreting cells of these glands bear many cup-shaped microvilli-like structures, which presumably are the sites of a highly regulated microfibril formation. Two proteins from the tube of R. pachyptila, which are thought to tighten the different parts of the tube by protein–protein and specific β-chitin–protein interactions, were sequenced and characterized (Chamoy et al. 2000, 2001). Its mRNA is detectable in special epidermal cells, but never within the chitin-secreting pyriform glands. A stabilization process by disulfide bonds of the protein–chitin link, rather than the crystalline form of the chitin, accounts for the resistance of Riftia tubes to
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Biosilicification in Geothermal and Hydrothermal Environments
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enzymatic attacks. Riftia tube was previously shown to be very resistant to aggressive in vitro chemical and physical treatments (Gaill and Hunt 1986). Moreover, empty tubes were often observed in situ after an animal’s death (Fustec et al. 1987; Roux et al. 1989). It was reported (Le Bris and Gaill 2007) that after about 6 months of exposure the tube samples appeared slightly altered, in contrast to exoskeleton fragments of the vent crab Bythograea thermydron. The estimated degradation rate of Riftia tube organic material is less than 4% month (40% year), when extrapolated from the results obtained after 180 days. These data suggest that Riftia tubes would be degraded within 2.5 years, while crustaceans chitinous carapaces would be degraded in about 1 month (36 days). Histochemical studies on Riftia tubes demonstrate the presence of disulfide bonds in vestimentiferan tube proteins. Gaill and Hunt (1986) reported a high cystein content (10% of the amino acids) in Riftia tubes. The breakage of disulfide bonds seems to increase the accessibility of the chitin polymers to enzymatic hydrolysis. This leads to the conclusion that the stability of the vestimentiferan tubes is not due to the crystallographic form of the chitin, but results rather from some properties of its associated proteic fraction (Le Bris and Gaill 2007). Thus, both chitin and collagen of vestimentiferan origin as well as their exoskeletal structures possess intriguing biomimetic potential, which can be very useful for developing new technological processes and correspondingly new biomaterials.
35.2 Biosilicification in Geothermal and Hydrothermal Environments It has been well recognized that thermophilic and hyperthermophilic microorganisms are widely distributed around geothermal and hydrothermal environments. For example, Pyrolobus fumari is the most hyperthermophilic archaeon and can grow at up to 113◦ C (Blöchl et al. 1997). Inagaki et al. (2003) used molecular phylogenetic analyses and obtained results which suggested that extreme thermophilic bacteria within the genera Thermus and Hydrogenobacter are predominant components among the indigenous microbial community in siliceous deposits formed within the pipes and equipment of Japanese geothermal power plants. These bacteria seem to actively contribute to the rapid formation of huge siliceous deposits (Fig. 35.4). Additionally, in vitro examination suggested that Thermus cells induced the precipitation of supersaturated amorphous silica during the exponential growth phase, concomitant with the production of a specific cell envelope protein. Dissolved silica in geothermal hot water may be a significant component in the maintenance of position and survival of microorganisms in limited niches (Inagaki et al. 1997, 1998, 2001, 2003). The cell walls of microbes generally include, more or less, polysaccharides and proteins, and have an affinity for silica (Inagaki et al. 2003). Mizutani et al. (1998) also reported that amines, notably polyamine, catalyze the polycondensation of silicic acid in water.
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Fig. 35.4 Examples of inspiration for “extreme biomimetics”: (a) silica microparticles of geyserites from Kamchatka surrounding by organic matter; (c and d) different kind of silicified microorganisms observed using SEM within these formations (images courtesy Gennady Karpov); (b) Thermus and Hydrogenobacter are predominant components among the indigenous microbial community in huge siliceous deposits formed within the pipes and equipment of Japanese geothermal power plants
Asada and Tazaki (2000) reported the formation of silica crusts by thermophilic unicellular red algae Cyanidium caldarium under extreme hydrothermal environment in hot springs (pH < 2, sulfuric acid, temperature more than 60◦ C). They observed formation of a double-layer silica crusts, which have a double-layer cell wall, using SEM and TEM. One must note that C. caldarium has a specific type of cell wall and a great ability to regulate pH to permit its tolerance to acid. Examples of specificity of the cell walls are that the protein is rich in the amino acids serine and threonine and that the polysaccharide is rich in hemicelluloses. This great ability to regulate pH also induces a strong gradient in pH across the external walls of the cell with fluctuations in pH. It is necessary for C. caldarium to show tolerance not only to acid, but also to silica in sites near the spouts of hot springs. The nature of proteins and polysaccharides in cell walls of microbes can determine the affinity for silica in various environments. Therefore, the cell walls of C. caldarium may also have the proper structure for the grade of silicification or the potential for change. The double-layer silica crust seems to offer protection against silicification of the cytoplasm. A schematic model for the formation of silica crust under strongly acidic conditions has been proposed (Asada and Tazaki 2000) (Fig. 35.5).
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Fig. 35.5 A schematic model to explain the formation of a silica crust by C. caldarium under strongly acidic conditions (adapted from Asada and Tazaki 2000)
Diatoms occupy the most varied of water biotopes, including warm and hot waters. For convenience it is possible to divide such diatoms into two groups, the thermophilic and the thermotolerant. The first prefer to live in warm water. Optimum temperatures for them are 20–30◦ S. Such species occupy tropic and subtropic reservoirs and also the heated summer reservoirs of temperate and boreal latitudes. The second group includes microalgae, which are capable of existing at temperatures above 30◦ S. Specific thermophiles, preferring hot water for their entire lives, are not found among diatoms (Round 1965). There are not too many thermotolerant diatoms. To date, only a few dozen of these species have been found in the various geothermal springs of the planet. Limiting factors for the number of species are temperature and the rN of water. The higher the values of these factors, the lower the concentration of diatoms. Peterson (1946) observed chloroplasts in diatoms Pinnularia lagerstedtii var. minuta from preserved material taken from waters at temperatures as high as 70◦ C in collections taken by Eric Hulten from Hot Springs on the Kamchatka Peninsula, Siberia (Stockner 1967). When processing samples of the Institute of Microbiology Russian Academy of Sciences, taken in one of the hot springs of Kamchatka in 1994, the mass development of diatoms Amphora veneta (Fig. 35.6) and four species of cyanobacteria was found (personal communication by Philipp Sapozhnikov). The temperature of this spring reached 80◦ C. Jana et al. (1982) note the appearance of diatoms in thermal communities when water temperature decreases below 60◦ C. In seminatural thermal springs in Karlovy Vary, sustainable development of diatoms Amphora coffeaeformis and Pinnularia microstauron at 55◦ S (Kaštovský and Komárek 2001) has been described.
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35 Life in Extreme Environments: From Bacteria to Diatoms
Fig. 35.6 Light microscopy images (a, b) of A. veneta isolated from hot spring (80◦ C) in Kamchatka region (images courtesy Philipp Sapozhnikov)
An environment with high alkalinity lends itself more readily to variety in bluegreen algae. Diatom flora is not rich in such conditions. Thomas and Gonzalves (1965) indicate 7 species of diatoms and 5 species of cyanobacteria for a hot spring Tooral in Western India (60–61◦ S). In the cooler, but more alkaline spring in Aravali, West India, of the 31 species of algae, diatoms comprised only 2 (other algae were blue-green). In contrast, in a nearby spring Rajewadi with acidified water at a temperature of 48.5–49◦ C, 41 species of diatoms were found and only 11 of cyanobacteria. Diatoms prefer a more acidic environment (Jana et al. 1982). In turn, species composition and structure of diatom communities in hot springs are determined by the ratio of mineral salts (Mpawenayo et al. 2005). The discovery of chitinous networks within the cell walls of diatom Thalassiosira pseudonana recently made in our laboratory (Brunner et al. 2009) suggests existence of silica–chitin composites which could be also arising under extreme physicochemical conditions similar to those of hot springs. The most common in the hot springs are the following species: P. microstauron, Rhopalodia gibberula,
35.2
Biosilicification in Geothermal and Hydrothermal Environments
495
Navicula cincta, Achnanthidium minutissimum, Gomphonema parvulum, A. coffeaeformis, Nitzschia amphibian, and Achnanthidium exiguum. Our future task is to monitor the presence of chitin within cell walls of these species. Thus, polysaccharides as well as specific proteins seem to be involved in the phenomenon described above of silicification at very low pH and temperatures near the boiling point. They open the way for attempts to develop novel silica-based composites under similar conditions in vitro. In preliminary experiments we decided to use chitin as appropriate thermostable biological material which can be effectively silicified as well as used for calcification. Preliminary thermogravimetric experiments, which has been carried out by Dawid Stawski showed the following results (Fig. 35.7). ◦ The main area of chitin degradation is between 250 and 390 C. As it is seen in Fig. 35.7, the marine sponge chitin sample is less thermally stable than mineralized chitin-based sponge skeleton. α-Chitin of crustacean origin is more thermostable that that from sponges, probably because of the difference in the structural organization. The first experiment on silicification of chitinous scaffolds that we isolated from demosponge Aplysina cauliformis was recently carried out using tetramethoxysilane as silica precursor at pH 1.5 and 85◦ C. We obtained three-dimensional silicified chitinous scaffolds. SEM observations definitively showed that monolithic silica was formed within fibers (of about 120 μm in diameter) of the spongeformlike scaffold. Thus, microtubular structured chitinous scaffolds were silicified from within. Investigations regarding the application of these scaffolds, obtained using
Fig. 35.7 The representative thermogravimetric curve of the crab chitin, Verongida sponge chitin and Verongida chitinous sponge skeleton (courtesy Dawid Stawski)
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35 Life in Extreme Environments: From Bacteria to Diatoms
this “extreme biomimetic” procedure, in tissue engineering are in progress now. We also started with studies on metallization of 3D chitinous matrixes using Mo-, W- as well as Zr-containing compounds at temperature diapason between 180 and 250◦ C.
35.3 Conclusion There are currently numerous papers that have been published where different kinds of biomaterials and composites were developed under so-called biomimetic conditions or biomimetically. However, all of these experiments have been carried out at temperatures between 20 and 30◦ C, because traditionally researches thought that these temperatures are only ones appropriate from a biological point of view. However, Nature opens for us new, still poorly investigated exotic niches with extreme physico-chemical conditions. Also in these niches numerous taxa of marine invertebrates dwell in, as well as produce, biological materials with unique properties. I very much hope that Extreme Biomimetics will help us to develop a new generation of biomaterials.
References Akai K, Kurokawa K, Akai J (1995) Growing stromatolites at the Onikobe and Akakura hotsprings, Japan. Earth Science (Chikyu Kagaku) 49:292–297 (in Japanese) Asada R, Tazaki K (1999) Biomineralization of silica under strong acidic condition. Proc Int Symp Kanazawa, –Earth–Water–Humans, Kanazawa University, Kanazawa Asada R, Tazaki K (2000) Biomineralization of silica associated with colonization of an unicellular alga, Cyanidium caldarium, in an acidic hot spring. J Geol Soc Japan 106:597–608 Auerbach G, Gaill F, Jaenicke R et al (1995) Pressure dependence of collagen melting. Matrix Biol 14:589–592 Bann JG, Bächinger HP (2000) Glycosylation/Hydroxylation-induced Stabilization of the Collagen Triple Helix. J Biol Chem 275:24466–24469 Bann JG, Peyton DH, Bächinger HP (2000) Sweet is stable: glycolysation stabilizes collagen. FEBS Lett 473:237–240 Blackwell J, Parker KD, Rudall KM (1965) Chitin in pogonophore tubes. J Mar Biol Assoc UK 45:659–661 Blake JA (1985) Polychaeta from the vicinity of deep-sea geothermal vents in the Eastern Pacific. I: Euphrosinidae, Phyllodocidae, Hesionidae, Nereididae, Glyceridae, Dorvilleidae, Orbiniidae, and Maldanidae. Bull biol Soc Washington 6:67–101 Blöchl E, Rachel R, Burggaraf S et al (1997) Pyrolobus fumarii, gen. and sp. nov. represents a novel group of archaea, extending the upper temperature limit for life to 113 degrees C. Extremophiles 1:14–21 Brunner E, Richthammer P, Ehrlich H et al (2009) Chitin-based organic networks—an integral part of cell wall biosilica in the diatom Thalassiosira pseudonana. Angevante Chemie. doi:10.1002/anie.200905028 Cary SC, Shank T, Stein J (1998) Worms bask in extreme temperatures. Nature 391:545–546 Chamoy L, Nicolai M, Quennedy B et al (2000) Characterization of a cDNA encoding RP43, a CUB-domain-containing protein from the tube of Riftia pachyptila (Vestimentifera) and distribution of its transcripts. Biochem J 350:421–427
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Chamoy L, Nicolai M, Ravaux J et al (2001) A novel chitin-binding protein from the vestimentiferan Riftia pachyptila interacts specifically with β-chitin. J Biol Chem 276(11):8051–8058 Chevaldonné P, Desbruyéres D, Childress JJ (1992) . . . and some even hotter. Nature 359:593–594 Desbruyères D, Chevaldonne P, Alayse-Danet A-M et al (1998) Biology and ecology of the “Pompeii worm” (Alvinella pompejana Desbruyères et Laubier), a normal dweller of an extreme deep-sea environment: a synthesis of current knowledge and recent developments. Deep Sea Res 45:383–422 Francheteau J, Laubier L (1982) Naissance des oceans, l’hydrothermalisme profond et la faune associte. Rev Palais Decouverte 10 Fustec A, Desbruye`res D, Juniper K (1987) Deep-sea hydrothermal vent communities at 13_N on the east pacific rise: microdistribution and temporal variations. Biol Oceanogr 4:121–163 Gaill F (1993) Aspects of life development at deep sea hydrothermal vents. FASEB J 7:558–565 Gaill F, Bouligand Y (1987) Supercoil of collagen fibrils in the integument of Alvinella, an abyssal annelid. Tissue Cell 19(5):625–642 Gaill F, Hunt S (1986) Tubes of deep sea hydrothermal vent worms Riftia pachyptila (Vestimentifera) and Alvinella pompejana (Annelida). Mar Ecol Progr Ser 34:267–274 Gaill F, Hunt S (1991) The biology of annelid worms from high temperature hydrothermal vent regions. Rev Aquat Sci 4:107–137 Gaill F, Mann K, Wiedemann H et al (1995) Structural comparison of cuticle and interstitial collagens from annelids living in shallow sea-water and at deep-sea hydrothermal vents. J Mol Biol 246:284–294 Gaill F, Persson J, Sugiyama J et al (1992a) The chitin system in the tubes of deep sea hydrothermal vent worms. J Struct Biol 109:116–128 Gaill F, Shillito B, Lechaire JP et al (1992b) The chitin secreting system from deep sea hydrothermal vent worms. Biologie Cellulaire 76:201–204 Gaill F, Wiedemann H, Mann K et al (1991) Molecular characterization of cuticle and interstitial collagens from worms collected at deep sea hydrothermal vents. J Mol Biol 221:209–223 Girguis PR, Lee RW (2006) Thermal preference and tolerance of alvinellids. Science 312:231 Grzymski JJ, Murray AE, Campbell BJ et al (2008) Metagenome analysis of an extreme microbial symbiosis reveals eurythermal adaptation and metabolic flexibility. Proc Natl Acad Sci USA 105:17516–17521 Inagaki F, Hayashi S, Doi K et al (1997) Microbial participation in the formation of siliceous deposits from geothermal water and analysis of the extremely thermophilic bacterial community. FEMS Microbiol Ecol 24:41–48 Inagaki F, Motomura Y, Doi K et al (2001) Silicified microbial community at Steep Cone hot spring, Yellowstone National Park. Microb Environ 16:125–130 Inagaki F, Motomura Y, Ogata S (2003) Microbial silica deposition in geothermal hot waters Appl Microbiol Biotechnol 60:605–611 Inagaki F, Yokoyama T, Doi K et al (1998) Biodeposition of amorphous silica by an extremely thermophilic bacterium Thermus spp. Biosci Biotechnol Biochem 62:1271–1272 Jana BB, Pal DN, Sarkar HL (1982) Spatial distribution of the biotic community in the thermal gradients of the two hot springs. Acta hydrochimica et hydrobiologica 10(1):101–108 Kaštovský J, Komárek J (2001) Phototrophic microvegetation of thermal springs in Karlovy Vary, Czech Republic. In: Elster J, Seckbach J, Vincent W, Lhotský O (eds) Algae and extreme environments – ecology and physiology. Nova Hedvigia, Beiheft 123:107–119 Kaule G, Timpl R, Gaill F et al (1998) Prolyl activity in tissue homogenates of annelids from deep sea hydrothermal vents. Matrix Biol 17:205–212 Laubier L, Desbruykes D (1985) Oases at the bottom of the ocean. Endeavour 9:67–76 Le Bris N, Gaill F (2007) How does the annelid Alvinella pompejana deal with an extreme hydrothermal environment? Rev Environ Sci Biotechnol 6:197–221 Lee CK, Cary SG, Murray AE et al (2008) Enzymic approach to eurythermalism of Alvinella pompejana and its episymbionts. Appl Environ Microbuiol 74:774–782 Lepescheux L (1988) Spatial organization of collagen in annelid cuticle: order and defects. Biol Cell 62:17–31
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Mann K, Mechling E, Bächinger HP et al (1996) Glycosylated threonine but not 4-hydroxyproline dominates the triple helix stabilizing positions in the sequence of a hydrothermal vent worm cuticle collagen. J Mol Biol 261:255–266 Mizutani T, Nagase H, Ogoshi H (1998) Silicic acid polymerization catalized by amines and polyamines. Chem Lett 2:133–134 Mpawenayo B, Cocquyt C, Nindorera A (2005) Diatoms (Bacillariophyta) and other algae from the hot springs of Burundi (Central Africa) in relation with the physical and chemical characteristics of the water. Belgian J Bot 138(2):152–164 Peterson JB (1946) Algae collected by Eric Hulten on the Swedish Kamchatka Expedition, 1920–22, especially from hot springs. Kgl. Danske Videnskab. Selskab. Biol Medd 20:3–122 Rona PA, Bostrom K, Laubier L et al (1983) Hydrothermal processes at seafloor spreading centres. NATO Conference Series IV, Marine Sciences, Plenum Round FE (1965) The biology of the algae. St Martin’s, New York Roux M, Rio M, Schein E et al (1989) Mesures in situ de la croissance des bivalves et des vestimentiferes et de la corrosion des coquilles au site hydrotermal de 13◦ N (dorsale du Pacifique oriental). C R Acad Sci Paris 308:121–127 Sicot FX, Mesnage M, Masselot M et al (2000) Molecular adaption to an extreme environment: origin of the thermal stability of the pompeij worm collagen. J Mol Biol 302:811–820 Stockner JG (1967) Observations of thermophilic algal communities in mount rainier and yellowstone national parks. Limnol Oceanogr 12(1):13–17 Tazaki K (1999) Microorganisms design the Earth history. Proc Int Symp, –Earth–Water–Humans, Kanazawa University, Kanazawa Tazaki K, Aoki A, Asada R et al (1998) A new world in the science of biomineralization. In: Tazaki K (ed) Environmental biomineralization in microbial mats in Japan. Sci Rep Kanazawa University 42:65 Tazaki K, Sato T, Tawara K et al (1999) Biomineralization in hydrothermal systems. Gekkan Kaiyo 19:211–216 (translation from Japanese) Tazaki K, Yamamura T, Nagai H et al (1996) Banded architecture of bacterial control. Gekkan Chikyu 18:9–16 (translation from Japanese) Thomas J, Gonzalves EA (1965) Thermal algae of Western India. IV. Algae of the hot springs at Aravali, Tooral, and Rajewadi. Hydrobiologia 26(1–2):29–40 Walter MR, Bauld J, Brock TD (1972) Siliceous algal and bacterial stromatolites in hot spring and geyser effluents of Yellowstone National Park. Science 78:402–405 Zbinden M, Le Bris N, Compère P et al (2003) Mineralogical gradients associated with Alvinellids at deep-sea hydrothermal vents. Deep-Sea Res I 50:269–280
Epilogue
My efforts in this work were to propose the very first classification of biological materials of marine origin and to show their structural and chemical diversity. The scientific history of the discovery of these materials spans the last 150 years and our own results may stimulate other researchers to do better. Because of the space limitations, my book is dedicated to biological materials isolated, observed, or described only in marine invertebrate organisms. The second work, which covers the biological materials of marine chordate animals including hagfishes, lampreys, fishes (e.g., sharks, scats, tarpons) as well as reptilian (turtles) and mammals (dolphins, whales), is in progress now. In these conclusion remarks, I want to focus the attention of readers on the three principal aspects, as follows: (i) the evolution of biological materials and Earth history, (ii) the state of the art of marine biological materials in science today, and (iii) the clash between marine biological and man-made materials in the open sea. The term biological material is very common, because it includes composites of both organic (e.g., protein–protein, protein–polysaccharide) and inorganic (e.g., mineral–protein, mineral–polysaccharide) phases and in this way covers pure organic-, inorganic-, or biocomposite-based exo- and endoskeletons with amazing diversity of forms, shapes, and dimensions. Moreover, all of them possess materials properties. The arising of biological materials is doubtless related to the origin of life on Earth and the first prokaryotic organisms. Even nano-organized bacterial S-layers are investigated today as biological materials (Göbel et al. 2010). From a scientific point of view, the origin of life is seen as a chemical process that is simultaneously biotic and non-biotic and that generates the first organized, evolving entity, which constitutes the starting point of evolution for our biosphere (Wähtershäuser 2007). It is very likely that some inorganic minerals served as templates and matrices for the formation of the first self-replicating organic compounds, predecessors of the cellular organization of life. This may be an explanation of their chiral selectivity, which would be unlikely in the case of the non-matrix-mediated abiogenic synthesis (Barskov 1982). Thus the interaction between the mineral and biological worlds can be traced throughout the entire period of their existence that is available for study. For the world of minerals, this co-evolution enabled the existence of some minerals in
H. Ehrlich, Biological Materials of Marine Origin, Biologically-Inspired Systems 1, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9130-7,
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the thermodynamically unbalanced conditions of the biosphere and allowed minerals to acquire functions other than as the substrate for life. For the biological world, biomineralization was a powerful means for conditioning its environment, facilitated the acceleration of the biological evolution and growth of diversity (Lowenstam 1981, 1984; Lowenstam and Weiner 1989; Mann 2001) and allowed the acquisition of new organs responsible for various biological functions. Biological materials of marine invertebrates, which I have analyzed here, are the results of natural selection. According to de Duve (2007), selection was first added to chemistry in the development of life by molecular replication. This process automatically entails two concomitants of paramount importance that have dominated the history of life ever since. As long as replication is perfect, the information is perpetuated unchanged, generation after generation, providing genetic continuity. When, as must inevitably occur, imperfect copies are made, the resulting variants compete with one another for available resources. The obligatory consequence of this competition, as first divined by Darwin, is the natural selection of those variants most able to survive and reproduce under prevailing conditions (de Duve 2007; Knoll 2004). The chemical and structural features of some of these naturally selected biological materials are unique and specific. The morphological and chemical uniformity of the corresponding group of organisms is so marked that they should be ranked only as phylum (Cavalier-Smith 1998). Furthermore, each skeletal structure, as well as the skeleton, is suggested to be an embryological and phylogenetic composite (Donoghue and Sansom 2002). Since modern science can only hypothesize the existence of a single ancestral form of life, the so-called Last Universal Common Ancestor (de Duve 2007), we attempt to understand the principles of organization and function of the more recently evolved organisms, some of which possess conserved unique features since Cambrian explosion (see for review Cavalier-Smith 2006; Conway-Morris 2003). The marine biological materials of the deep-sea invertebrates are replete with examples of these features. The deep-sea is colder, darker, and less nutrient rich than is the ocean surface, and as a result, deep-sea life tends to be exceedingly slow growing and late to mature. Deep-sea fish such as orange roughy (Hoplostethus atlanticus) live for more than a century, and deep-sea corals much longer—5,000 years or more, while glass sponges can live more than 15,000 years. Regarding the second aspect I mentioned above, I hope that this monograph gives enough information to the readers about the state of the art in biological materials of marine origin. Unfortunately, there are some absolutely negative events which are also related to the state of the art. The incredible potential of undersea “biological materials sources” is threatened by an extremely destructive fishing practice known as bottom trawling. This fishing method drags large weighted nets across the ocean floor to catch fish that dwell on or near the bottom. Weighted nets act like bulldozers, ripping up sediments, upending boulders, pulverizing fragile corals and sponge fields, and crushing, burying, or exposing to predators organisms that cannot move out of the path of the net. In the Tasman Sea, for example, bottom trawlers fishing for orange roughly in 1997 pulled up approximately of 10 tons of coral per tow. In that year, an estimated 10,000 tons of coral were destroyed in the capture of 4,000
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tons of fish. By some estimates, nearly 15,800 miles2 (40,500 km2 ) of ocean floor are trawled per day around the globe (Maxwell et al. 2005). The unique characteristics of deep-sea organisms make them exceptionally vulnerable to this type of disturbance. In 2004, I as a member of a group of 1,136 marine scientists from all over the world who signed a statement urging the United Nations to adopt a moratorium on high-seas bottom trawling. Deep-sea biodiversity in international waters represents an extraordinary resource that all nations have an interest in protecting and managing sustainably. Marine Conservation Biology Institute (MCBI) and Natural Resources Defense Council (NRDC), together with dozens of governments and conservation organizations from around the world, are calling for such a moratorium until the biodiversity of the deep-sea is assessed and a regimen to manage and protect it is developed. It was a honor to me to be invited as co-author in corresponding paper initiated by Sarah Maxwell from MCBI (Maxwell et al. 2005). The third aspect listed above is also related to ecological problems. We are far away from any understanding of the complex phenomena related to biological materials of marine origin, yet even now new, disturbing phenomena are developing. What happened when man-made materials meet biological materials? From scientific point of view, we can congratulate ourselves with a new task, that of research into so-called marine debris (Fig. 1) (Moore et al. 2001, 2002).
Fig. 1 Man-made materials meet biological materials of marine origin. Plastic trash in the ocean (a) is a source of anxiety for Captain Charles Moore from Algalita Marine Research Foundation (c). Nature struggles to adapt and survive to the changes forced on it by these man-made materials (b), but is this truly an image for inspiration?
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Marine debris is any man-made, solid material that enters waterways directly through littering or indirectly via rivers, streams, and storm drains. Marine debris can be simple items such as a discarded soda can, cigarette butts, plastic bags, or a lost fishing net that ends up in the ocean, potentially harming marine life. Nearly 80% of marine debris results from land-based sources. In particular, the most dangerous are “microplastics,” because they can attract high levels of toxic molecules such as polychlorinated biphenyls, phenanthrene, polystyrene-based (PS) oligomers, styrene monomers, di- and trimers. I recommend the readers to read such documents as “Plastic Debris in the World’s Oceans” published by Greenpeace on the November 2, 2006, and visit the web site of the Algalita Marine Research Foundation (USA). Thus, I am very afraid that in the future somebody and somewhere will submit a paper about finding of the “unique and unusual” marine invertebrate biocomposite made from some kind of toxic polymer and a biopolymer with a very ancient origin. Hopefully, with global cooperation on conservation efforts, such a tragic scenario will never occur.
References Barskov IS (1982) Biomineralization and evolution. Paleontol Zh (4):5–13 Cavalier-Smith T (1998) A revised six–kingdom system of life. Biol Rev Cambridge Philos Soc 73:203–266 Cavalier-Smith T (2006) Cell evolution and Earth history: stasis and revolution. Phil Trans R Soc B 361:969–1006 Conway-Morris S (2003) The Cambrian “explosion” of metazoans and molecular biology: would Darwin be satisfied? Int J Dev Biol 47:505–515 Donoghue PCJ, Sansom IJ. 2002. Origin and early evolution of vertebrate skeletonization. Microsc Res Tech 59:352–372 Duve de C (2007) Chemistry and Selection. Chem Biodiversity 4:574–583 Göbel C, Schuster B, Baurecht D et al (2010) S-layer templated bioinspired synthesis of silica. Colloids Surfaces B: Biointerfaces 75:565–572 Knoll AH (2004) Biomineralization and evolutionary history. In: Dove PM, DeYoreo JJ, Weiner S (eds) Reviews in mineralogy and geochemistry, 54(1), pp 329–356 Lowenstam HA (1981) Minerals formed by organisms. Science 221:1126–1131 Lowenstam HA (1984) Processes and products of biomineralization. Evolution of biomineralization. In: Reports 27 international geological congress USSR Moscow paleontology Sect. C.02, 2, pp 51–56 (in Russian) Lowenstam HA, Weiner S (1989) On biomineralization. University Press, Ox–ford, New York Mann S (2001) Biomineralization. University Press, Oxford, New York Maxwell S, Ehrlich H, Speer L (2005) Medicines from the deep: the importance of protecting the high seas from bottom trawling. Natural Resources Defense Council Issue Paper Moore CJ, Moore SL, Leecaster MK et al (2001) A comparison of plastic and plankton in the North Pacific Central Gyre. Mar Pollut Bull 42:1297–1300 Moore CJ, Moore SL, Weisberg SB et al (2002) A comparison of neustonic plastic and zooplankton abundance in Southern California’s Coastal Waters. Mar Poll Bull 44:1035–1038 Wähtershäuser G (2007) On the chemistry and evolution of the pioneer organism. Chem Biodiversity 4:584–602
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Additional Sources Amos AF (1993) Solid waste pollution on texas beaches: a post-MARPOL annex V study: OCS study MMS 93-0013. U.S. Department of the Interior, Minerals Management Service, Gulf of Mexico OCS Region, vol 1. New Orleans, LA Center for Marine Conservation (1994) A citizen’s guide to plastics in the ocean: more than a litter problem. Center for Marine Conservation, Washington, DC Coe JM, Rogers DB (1996) Marine debris: sources, impacts, and solutions. Springer, New York Committee on Shipborne Wastes, Marine Board Commission on Engineering and Technical Systems, National Research Council. Clean Ships, Clean Ports, Clean Oceans: Controlling Garbage and Plastic Wastes at Sea. National Academy, Washington, DC, 1995
Internet Resources Assessing and Monitoring Floatable Debris. U.S. Environmental Protection Agency, Oceans and Coastal Protection Division. http://www.epa.gov/owow/oceans/debris/floatingdebris/toc.html Marine Debris Abatement: Trash in Our Oceans—You Can Be Part of the Solution. U.S. Environmental Protection Agency, Ocean and Coastal Protection Division. http://www. epa.gov/owow/oceans/debris/index.html Marine Debris. The Ocean Conservancy. http://www.oceanconservancy.org/dynamic/issues/threats/ debris/ebris.htm Pollution of the Ocean by Plastic and Trash – sea, oceans, effects, types, source, effect, marine, human http://www.waterencyclopedia.com/Po-Re/Pollution-of-the-Ocean-by-Plasticand-Trash.html-ixzz0a2lzUwHe
TEM images of Phormidium from mat on surface of stromatolite. Longitudinal section through filament showing external epicellular silicification and intact cytoplasm (scale bar: 2 μm) (Adapted from Jones et al. (2005))
Bacteria
Image Gallery
Addendum
Biofilm community
Nostoc-type cyanobacteria
Cyanobacteria Filamentous microorganisms Hyperthermophilic microorganisms
Organism
Fujino et al. (2008) Inagaki et al. (1998, 2003)
Handley et al. (2008)
McKenzie et al. (2001) Pancost et al. (2005), Jones et al. (1999) Aubrecht et al. (2008)
Siliceous sinters Siliceous oncoids “Biospeleotherms” and shrubs Siliceous microstromatolite laminae Silica scale from geothermal power plant
Jones et al. (2001) Chafetz and Guidry (1999)
References
Siliceous “geyser eggs” Siliceous shrubs
Structure/form of biosilica
Table 1 Structural diversity of biosilica
Large microbial speleothem forms called “champignons” are frequently more than 30 cm in size (Cueva Charles Brewer, Chimantá). Photo: B. Šmída (scale bar: 30 cm) (Adapted from Aubrecht et al. 2008)
Image Gallery
Jones and Renaut (2006)
Siliceous spicules (up to 3 cm high and up to 5 mm in diameter) Granular silica spherules
Asada and Tazaki (2001)
Jones et al. (2005)
Lilypad stromatolites (up to 3 m long and 1.5 m wide)
Thermus thermophilus TMY Thermus spp., Hydrogenobacter spp. Phormidium, Fischerella
Cyanidium, Alicyclobacillus Cyanidium caldarium
References
Structure/form of biosilica
Organism
Table 1 (continued)
506 Addendum
Silica-encrusted shrubs forming an initial mat of Nostoc-type microbes on the quartzite substrate (Cueva Cañon Verde, Chimantá). Photo: B. Šmída (scale bar: 1 cm) (Adapted from Aubrecht et al. (2008))
Image Gallery
Organism
Table 1 (continued) Structure/form of biosilica
References
Addendum 507
Fungi
Yeast
Image Gallery
Organism
Table 1 (continued)
Siliceous coated grains Desert varnish
Silicified cell walls
Structure/form of biosilica
Jones et al. (1999) Kolb et al. (2004)
Brasser et al. (2006, 2008)
References
508 Addendum
Netzelia tuberculata. Scanning electron micrograph of shells showing the lobed apertures and arrangement of the siliceous particles within the test (scale bar: 25 μm) (Adapted from Anderson (1992))
Whole mounts of teratological scales, showing some of the abnormalities that may be encountered. See the text for details. × 10,000 (scale bar: 2.5 μm) (Adapted from Patterson (1988))
Protozoa
Image Gallery
Sarcodines Gymnamoebae Testate amoebae Heliozoa Radiolaria
Organism
Table 1 (continued)
Siliceous boat-shaped scales Siliceous curved rods Siliceous body plates Siliceous particles Siliceous needles, spines Siliceous spicules Siliceous porous shells Siliceous spongiose shells
Structure/form of biosilica
Anderson (1992) Patterson (1988) Ogden (1991)
References
Addendum 509
Image Gallery
Silicoflagellates Thaumatomastigids Chlorophytes Xanthophytes
Anderson (1994)
Preisig-Mueller et al. (1995) Ludwig et al. (1996) Smol (2008)
Stomatocysts, scales, spines, bristles Siliceous loricae Siliceous skeletons, cysts, granules, external skeleton, scales Statosphores (siliceous resting cysts) Silicified cell walls (quartz-cellulose-calcite composite) Silicified cell walls
Flagellates chrysophytes, synurophytes Choanoflagellates Dinoflagellates Ebridians
References
Structure/form of biosilica
Organism
Table 1 (continued)
510 Addendum
Diatoms
Foraminifera
Image Gallery Silicosigmoilina futabaensis
Organism
Table 1 (continued)
Frustule Spines Heavily silicified resting spores Nanogranules in mitochondria
Siliceous cement
Structure/form of biosilica
Mehard et al. (1974)
Kröger (1999) Round et al. (1990) Hasle and Sims (1985)
Asano (1950)
References
Addendum 511
Sponges
Image Gallery
Demospongiae
Hexactinellida
Organism
Table 1 (continued)
Skeletal frameworks and spicule (macrosclerae) Microsclerae
Structure/form of biosilica
Uriz et al. (1997) and Uriz (2006)
Boury-Esnault and Rützler (1997)
References
512 Addendum
Mollusca
Image Gallery
Patellacea spp.(Gastropoda) Onchidella celtica
Organism
Table 1 (continued)
Lowenstam (1971) Labbé (1933a, b, 1934a, b)
Siliceous spicules and penial spines
References
Silica in radula teeth
Structure/form of biosilica
Addendum 513
Styela clava. Silica granules (SEM) in ovary. Details of granules in 7 μm section shown in (A) (scale bar: 1 μm). (A) Interstitial ovarian tissue in 7 μm section; (Adapted from Monniot et al. (1992))
Ascidians
Image Gallery
Styela clava
Organism
Table 1 (continued)
Intracellular granules
Structure/form of biosilica
Monniot et al. (1992)
References
514 Addendum
Calanus pacificus. Section perpendicular to tooth row, showing a tooth mold early in opal deposition stage; opal is laid down sequentially from distal surface to tooth base; section also shows passage of a salivary duct adjacent to remains of central cell (scale bar: 6.0 μm) (Adapted from Miller et al. (1990))
Crustaceans
Image Gallery
(Copepoda) Acartia tonsa Neocalanus spp. Calanus pacificus
Organism
Table 1 (continued)
Opal and willemite-based teeth
Structure/form of biosilica
Beklemishev (1954, 1959) Miller et al. (1980, 1990)
References
Addendum 515
Siliceous tablets on the larval surface of Discinisca tenuis. Secondary electron image of siliceous tablets on untreated surface of phosphatic-shelled brachiopod, Discinisca tenuis. The siliceous tablets form a regular mosaic (scale bar: 1 μm) (Adapted from Cusack and Freer (2008))
Brachiopods
Image Gallery
Discinisca tenuis
Organism
Table 1 (continued)
Siliceous tablets
Structure/form of biosilica
Cusack and Freer (2008)
References
516 Addendum
Photomicrographs of a cytoplasmic extract in crossed-polarizers, the presence of silica following the previous morphology. Photographs are with crossed-polarizers showing SiO2 replacements in grey and white arrangements (chalcedony) (scale bar: 50 μm) (Adapted from Prado Figueroa et al. (2008))
Fishes
Image Gallery
Psammobatis extenta (Rajidae)
Organism
Table 1 (continued)
Chalcedony in electrocytes and cholinergic nerves
Structure/form of biosilica
Prado Figueroa et al. (2005, 2006, 2008)
References
Addendum 517
This calculus was broken open to show that the interior consisted of a large number of small, well-formed calculi in a fine-grained white matrix enclosed in a thin white envelope (scale bar: 1 mm) (Adapted from Forman et al. (1959))
Mammals
Image Gallery
Dog Cattle Monkey (Macaca fuscata) Rat liver mitochondria
Organism
Table 1 (continued)
Silica calculi and stones Silica calculi and stones Dental calculi Silica nanogranules
Structure/form of biosilica
Legendre (1976) Baily (1972) Hidaka et al. (1994) Mehard and Volcani (1976) Policard et al. (1961)
References
518 Addendum
Peculiar leaf shaped deposits on the surface of a brushite stone (not shown). X-ray analysis of the leaves yielded only silicon (scale bar: 10 μm) (Adapted from Kim et al. (1983))
Human
Image Gallery
Human glial malignant tumors
Cerebral cortex Urinary bladder and urethra Kidney
Organism
Table 1 (continued)
Prado Figueroa et al. (2007)
Jokes et al. (1973)
Renal silica calculi
Chalcedony
Prado Figueroa et al. (2006, 2008)Levison et al. (1982)
References
Chalcedony Silica calculi and stones (urolithiasis)
Structure/form of biosilica
Addendum 519
STEM imaging of a silica body. Bright field image (scale bar: 0.5 μm) (Adapted from Laue et al. (2007))
Plants
Image Gallery
Chaetoceros gracilis (Bacillariophyceae) Canary grass (Phalaris canariensis) Foxtail millet (Setaria italica) Pleioblastus chino (Poaceae, Bambusoidea) Heath grass (Sieglingia decumbens)
Organism
Table 1 (continued)
Rogerson et al. (2008) Bhatt et al. (1984) Hodson et al. (1982) Motomura et al. (2006)
Sangster (1970)
Silica fiber Inflorescence bristles Silica cells
Silica bodies (phytoliths)
References
Siliceous setae
Structure/form of biosilica
520 Addendum
Image Gallery
Structure/form of biosilica Tabasheer Siliceous nodular deposits
Organism Bambusoideae Higher plants
Table 1 (continued)
Sangster (1978)
Judd (1887)
References
Addendum 521
522
Addendum
References Anderson OR (1994) Protoplasma 181:61 Anderson OR (1992) The effects of silicate depletion and subsequent replenishment on the cytoplasmic fine structure of the silica-secreting testate amoeba Netzelia tuberculata in laboratory culture. J Morphol 211:285–293 Asada R, Tazaki K (2001) Can Miner 39:1 Asano K (1950) Pacif Sci 4:158 Aubrecht R, Brewer-Carías Ch, Šmída B, Audy M, Kováˇcik’ (2008) Sed Geol 203:181 Baily CB (1972) Invest Urol 10:178 Beklemishev KV (1954) Dokl Akad Nauk SSR 97:543 Beklemishev KV (1959) Trudy Inst Okeanol 30:148 Bhatt TM, Coombs M, O’Neil C (1984) Int J Cancer 34:519 Boury-Esnault N, Rützler K (1997) Thesaurus of sponge morphology. Smithsonian contributions to zoology, no 596, Smithsonian Institution, Washington DC, p 55 Brasser HJ, Krijger GC, Van Meerten TG, Wolterbeek HT (2006) Biol Trace Elem Res 112:175 Brasser HJ, Krijger GC, Wolterbeek HT (2008) Biol Trace Elem Res 125:81 Chafetz HS, Guidry SA (1999) Sed Geol 126:57 Cusack M, Freer A (2008) Biomineralization: elemental and organic influence in carbonate systems. Chem Rev 108:4433–4454 Forman SA, Whiting F, Connell R (1959) Silica urolithiasis in beef cattle 3. Chemical and physical composition of the uroliths. Can J Comp Med XXIII(4):157–162 Fujino Y, Kawatsu R, Inagaki A, Umeda YT, Okaue Y, Iwai S, Ogata S, Oshima T, Doi KJ (2008) Appl Microb 104:70 Handley KM, Turner SJ, Campbell KA, Mountain BW (2008) Astrobiology 8:747 Hasle GR, Sims PA (1985) Eur J Phycol 20:219 Hidaka S, Okamoto Y, Oga Y, Hirose T, Abe K (1994) Arch Oral Biol 39:595 Hodson MJ, Sangster AG, Parry DW (1982) Ann Bot 50:843 Inagaki F, Motomura Y, Ogata S (2003) Appl Microbiol Biotechnol 60:605 Inagaki F, Yokoyama T, Doi K, Izawa E, Ogata S (1998) Biosci Biotech Biochem 62:1271 Jokes AM, Rose AG, Sutor J (1973) Br Med J 1:146 Jones MS, Wakefield RD, Forsyth G (1999) Materiales de Construccion (Madrid) 49:3 Jones B, Renaut RW (2006) Palaios 21:406 Jones B, Renaut RW, Konhauser KO (2005) Sedimentology 52:1229 Jones B, Renaut RW, Rosen MR (1999) Palaios 14:475 Jones B, Rosen MR, Renault RW (2001) J Sed Res 71:190 Judd JW (1887) Nature 35:396 Kim KM, David R, Johnson FB (1983) Siliceous deposits in human urinary calculi – an E. M. Study. Urol Res 11:155–158 Kolb VM, Philip AI, Perry RS (2004) Testing the role of silicic acid and biochemical materials in the formation of rock coatings. In: Hoover RB, Levin GV, Rozanov AY (eds) Instruments, methods, and missions for astrobiology VIII. Bellingham, Washington, SPIE, p 312 Kröger N, Deutzmann R, Sumper M (1999) Science 286:1129 Labbé A (1933a) Notes des Comptes-Rendus de l’Académie des Sci 197:697 Labbé A (1933b) Comptes-Rendus de l’Académie des Sci 114:1002 Labbé A (1934a) Annales de l’InstitutOceanographique de Monaco, 14:173 Labbé A (1934b) Opisthobranches et Silicodermés (Oncidiadés) In Memoires du Musée royal dhistoire naturelle de Belgique. Hors série. Résultats scientifiques du voyage aux Indes Orientales Néerlandaises de LL. AA. RR. le Prince et la Princesse Léopold de Belgique, vol 2. Musée Royal dHistoire Naturelle de Belgique,Brussels
Addendum
523
Laue M, Hause G, Dietrich D, Wielage B (2007) Ultrastructure and microanalysis of silica bodies in Dactylis Glomerata L. Microchim Acta 156:103–107 Legendre AM (1976) J Am Vet Med Assoc 168:418 Levison DA, Crocker PR, Banim S, Wallace DMA (1982) Lancet 1(8274):704 Lowenstam HA (1971) Science 171:487 Ludwig M, Lind JL, Miller EA, Wetherbee R (1996) Planta 199:219 McKenzie EJ, Brown KL, Cady SL, Campbell KA (2001) Geothermics 30:483 Mehard CW, Sullivan CW, Azam F, Volcani BE (1974) Physiol Plant 30:265 Mehard CW, Volcani BE (1976) Cell Tissue Res 174:315 Miller CB, Nelson DM, Weiss C, Soeldner AH (1990) Morphogenesis of opal teeth in calanoid copepods. Mar Biol 106:91 Miller CB, Nelson DM, Guillard RL, Woodward BL (1980) Biol Bull Mar Biol Lab 159:349 Monniot E, Martoja R, Truchet M, Fröhlich E (1992) Opal in ascidians: a curious bioaccuinulation in the ovary. Mar Biol 112:283–292 Motomura H, Fujii T, Suzuki M (2006) Ann Bot 97:513 Ogden CG (1991) Protoplasma 163:136 Pancost RD, Pressley S, Coleman JM, Benning LG, Mountain BW (2005) Environ Microbiol 7:66 Patterson DJ, Dürrschmidt M (1988) J Cell Sci 91:33 Policard A, Collet A, Daniel-Moussard H, Pregermain S (1961) J Biophys Biochem Cytol 9:236 Prado Figueroa M, Barrera F, Cesaretti NN (2008) Chalcedony (a crystalline variety of silica): biogenic origin in electric organs from living Psammobatis extenta (family Rajidae). Micron 39:1027–1035 Prado Figueroa M, Barrera F, Cesaretti NN (2005) Si4+ and chalcedony precipitation during oxidative stress in rajidae electrocyte: a mineralogical study. In: 41th annual meeting. Argentine Society for Biochemistry and Molecular Biology Research, Pinamar, Argentina Prado Figueroa M, Casavilca S (2007) Tumores Malignos de la Glıa: Formacion de Calcedonia (sılice cristalina) y Posible Rol de la Anhidrasa Carbonica. VI Encuentro Cientıfico Internacional de Invierno. ECI 2007i. http://www.cienciaperu.org/eci2007i/ Libroderesumenes. July 2007, Lima, Peru Prado Figueroa M, Flores L, Sanchez J, Cesaretti NN (2008) Micron 39:859 Prado Figueroa M, Sanchez J, Cesaretti NN (2006) Chalcedony (incipient fossilization process) in human brain cortex and cerebellum from aged patients. In: 42th annual meeting, Argentine Society for Biochemistry and Molecular Biology Research, Rosario, Santa Fe, Argentina Preisig-Mueller R, Gühnemann-Schäfer K, Kindl H (1994) J Biol Chem 269:20475 Rogerson A, DeFraitas ASW, McInnes AG (2008) J Phycol 22:56 Round FE, Crawford RM, Mann DG (1990) The diatoms: biology and morphology of the genera. University Press, Cambridge, p 757 Sangster AG (1970) Ann Bot 34:557 Sangster AG (1978) Am J Bot 65:929 Smol JP (2008) Pollution of lakes and rivers: a paleoenvironmental perspective, 2nd edn. Blackwell, Oxford, p 383 Uriz M-J (2006) Can J Zool 84:322 Uriz M-J, Turon X, Becerro MA, Agel G (2003) Microsc Res Tech 62:279
“Everything that a scientist does is a function of what others have done before him; the past is embodied in every conception and even in the possibility of its being conceived at all” (Medawar, 1979)
524
Addendum
Table 2 History of biomineralization, demineralization, and remineralization research Year
Events and discoveries
Sixteenth century 1552 “Tooth-worm” de-worming technique is first described Seventeenth century 1670 The Vain Speculation Undeceived by Senses. Response Letter about the Petrified Marine Objects that are Found in Different Inland Locations is published Eighteenth century 1728 Pierre Fauchard writes the Le Chirurgien Dentiste ; he rejects the tooth-worm theory of dental caries and describes enamel hypoplasia as “an erosion of the enamel” 1754 Tooth-worm in Onomatologia Medica 1757 Images of tooth-worms are published 1766 Alexander Blackrie develops Blackrie’s Lixivium for dissolution of kidney stones 1771 The Natural History of Human Teeth is published 1777 First observations of stone deterioration through biological processes 1784 First description of morphology of the fish (Anarchichas lupus) teeth; author considers the dentine as a variety of bone Nineteenth century 1800 The history of teeth is published 1823 1826
1829
1829
Discovery of chitin in cuticles First description of the sponge-like boring organism within the valves of oysters Anatomy, Physiology and Disorders of the Teeth is published Amphibian dermal scales and osteoderms, description, demineralization, and fine structure studies start
References Boorde (1552)
Scilla (1670)
Cited by W. Hoffman-Axthelm in 1981
Von Haller (1754, 1756) Schäffer (1757) Blackrie (1766)
Hunter (1771) Hunter (1778) Knight (1777) Liebeg (1853) Andre (1784)
Schreger (1800) Lehner, Plenk (1936) Odier (1823) Osler (1826)
Bell (1829)
Mayer (1829a, b) Cockerell (1912) Zylberberg, Castanet, de Ricqles (1980) Zylberberg and Wake (1990)
Addendum
525 Table 2 (continued)
Year
Events and discoveries
References
1840
Odontography including classification of the different types of dentine is published First description of endolithic boring foraminifera
Baillere (1840)
1845
1845
1849
1849
1850 1852
1852 1854 1856
1857
1859
1859 1859 1859
1863
Review on demineralization of invertebrates skeletons using acids and alkali Studies on microscopic structure of the scales and dermal teeth of some fishes Studies on the “excavating powers of certain sponges” Microscopic anatomy of human teeth Decalcification of mollusc shells using hydrochloric, acetic, and formic acids Handbook of human tissue study is published First description of the accessory boring organ of molluscs Discovery of Sharpey’s fibers—as fibers which had perforated into lamellar bone from the surrounding periosteum Discovery of “halisteresis” as the possibility of calcium loss by living bone without its obligatory and simultaneous resorption First evidence of the presence of boring “unicellular fungi” (algae) in hard tissues of molluscs, balanids, corals, and other animal groups Demineralization of spongin and chitin The physiological anatomy and physiology of man is published Microscopic structure of the skeleton of osseous fishes is described First report on demineralization of diatoms
Quenstedt (1845–1849) Venec-Peyre (1996) Wisshak and Rüggeberg (2006) Bromley et al. (2007) Schmidt (1845)
Williamson (1849)
Hancock (1849) Nassonov (1883) Leidy (1889) Czermak (1850) Leydolt (1852, 1856) Schmidt (1924, 1928) Von Kölliker (1852) Troschel (1854) Sharpey (1856)
Kilian (1857)
Von Kölliker (1859a) Von Kölliker (1860a, b)
Heintz (1859) Todd and Bowmann (1859) Von Kölliker (1859)b
Schultze (1863)
526
Addendum Table 2 (continued)
Year
Events and discoveries
References
1864
Desilicification of hyalonema glass sponge Discovery of canaliculi boring by fungus in both recently deposited and fossil bones Crystal formations in plant cells are described Overview of Crystalline Minerals in Table Form is published Fungoid sporangia with filamentous processes are found in shells of molluscs Discovery of “spiculin,” collagen and other organic components after demineralization of calcareous sponge spicules
Von Kölliker (1864)
1864
1865 1866 1872
1872
1873
1875 1877
1877
1878 1878 1880 1881
1881 1882
Discovery of osteodentine in the teeth of some of the lower vertebrates Treatise on Dental Caries is published Dissertation on studies of the origin of calcareous minerals in plants is published Discovery of plant cystoliths
Thermochemical studies on water-containing salts On the Caries of the Teeth is published On the Action of a Lichen on a Limestone is published Willoughby D. Miller finds that acid produced by microorganisms causes caries of the enamel Studies on the skeleton of radiolaria Discovery of vasodentine in the teeth of pike (Esox lucius)
Wedl (1864)
Rosanoff (1865, 1867) Bütschli (1866) Stirrup (1872)
Haeckel (1872) Von Ebner (1887) Sollas (1885) Weinschenk (1905) Travis et al. (1967) Ledger (1974) Aizenberg et al. (1995, 1996) Sethmann and Wörheide (2007) Heincke (1873)
Magitot (1875) Magitot (1867) Melnikoff (1877)
De Bary (1877) Molisch (1882) Chareyre (1883) Thomsen (1878) Leber and Rottenstein (1878) Sollas (1880) Miller (1883)
Bütschli (1881) Sternfeld (1882)
Addendum
527 Table 2 (continued)
Year
Events and discoveries
References
1883
Chemical theory of hard substrates’ dissolution by boring sponges first established
1885
First observation of “an inner gelatinous uncalcified nucleus” after the decalcification of ascidian spicules Dental caries is recognized as a process that may show “decalcification” Histology of the teeth Mycelytes ossifragus—fungus producing bored channels in bone Desilicification of glass sponge skeletons using HF and KOH Evidence of the presence of fungi-mediated bored channels in bone and teeth Discovery of “hyalodentine—an osseous layer on elasmoid fish scales Chemo-parasitic theory of the etiology of dental caries First evidence of the presence of carbonate-boring lichens First suggestion about the role of chemolithotrofic microorganisms in stone deterioration Discovery of cellulose, pectin, and “callose” in plant cystoliths Morphology of the fish scales including history of hardtissue is described First suggestion that accessory boring organ secretes an acid, chemical theory of the boring mechanism by molluscs Endolithic fungi in shells are recognized and described Histological studies on coelencerates Botanische Mikrotechnik (botanical microtechnique) is published
Nassonow (1883) Cotte (1902) Warburton (1958) Cobb (1969) Sluiter (1885)
1886
1887 1887
1888 1889
1889
1890 1890 1890
1890 1890
1891
1891 1892 1892
Magitot (1886)
Weil (1887) Roux (1887)
Sollas (1888) Schaffer (1889, 1890, 1894)
Hofer (1889) Meunier (1984) Miller (1890) Zahlbruckner (1890) Müntz (1890)
Mangin (1890a, b) Klaatsch (1890a, b) Nickerson (1893) Schiemenz (1891) Turner (1953)
Bornet (1891) Bornet and Flahaut (1889) Schneider (1892a, b) Zimmermann (1892)
528
Addendum Table 2 (continued)
Year
Events and discoveries
References
1897
Pathology of enamel
1898
Winterberg shows that rabbits fed on oats can protect themselves against ingested mineral acids by coupling these with ammonia Introduction of decalcified bone as a bone grafting material Susceptibility and immunity in dental caries
Williams,1897 Black (1897) Winterberg (1898)
1899 1899
Twentieth century 1900 Studies on microstructure of artificial and natural silica (tabaschir, hydrophane, opal) 1901 Dissociation of calcium citrate 1901 Isolation of osseomucoid from ox bone 1902 Boring algae and disintegration of corals 1903 Review on demineralization of skeletons of lower invertebrates 1906 Analysis of dentin and enamel of human teeth 1906 Demineralization of acantharia skeletons 1906 Studies on influence of KOH on spicules of calcareous sponges 1907 Studies on nature of the crystals isolated from crustaceans test and blood 1908 The solvent action of soil bacteria upon the insoluble phosphates of raw bone meal and natural rock phosphates 1908 Reptilian osteoderms, description, demineralization, and fine structure 1908 Demineralization of the fish otoliths and isolation of gelatinous organic matrix 1909 A History of Dentistry is published 1911 Bacterial–chemical study of dental caries 1911 Fish scales, demineralization, and fine structure, fish scale collagen
Senn (1899) Black (1899)
Bütschli (1900)
Sabbatani (1901) Hawk & Gies (1901) Duerden (1902) Von Fuerth (1903) Hinkins (1901) Bütschli (1906a) Bütschli (1906b) Bütschli (1907)
Sackett et al. (1908)
Otto (1908) Schmidt (1912) Zylberberg and Castanet (1985) Immermann (1908) Maier (1908) Lissner (1925) Prinz (1909) Lothrop (1911) Cockerell (1911) Waterman (1970) Onozato and Watabe (1979) Schonborner, Meunier, Castanet (1981) Zylberberg and Meunier (1981) Zylberberg, Bereiter-Hahn, Sire (1988)
Addendum
529 Table 2 (continued)
Year
Events and discoveries
References
1914
High ingestion of acid-forming foods appeared to cause decalcification The role of phosphoric esterase in decalcification Kalklösende Algae The origin, growth, and fate of osteoclasts and their relation to bone resorption Diaphanol (ClO2 in acetic acid) as demineralizing agent for animal hard tissues Development of decalcification solutions containing organic solvents Use of hematoporphyrin for identification of decalcification in bone Demineralization of marine invertebrates Coral sclerites as biocrystals Study on endolithic limestone lichens Review on microchemistry of animal skeleton substances is published Demineralization of plant encrustations Enamel and parasitic processes
Steenbock et al. (1914)
1914 1915 1920
1921
1921
1922
1922 1922 1922 1923
1923 1924 1924 1925
1925 1926
1926 1927 1928
Lichenes mediate biodeterioration of historical glass Review on demineralization properties of cellulose, chitin, conchin, spongin, and cornein Classis “osteoclasis” hypothesis is proposed First studies on morphology of scleral ossicles (bony plates within vertebrates’ eyes)
Study on pathological chemistry of the teeth Bacteria as agents of chemical denudation The Normal and Pathological Physiology of Bone is published
Bergeim (1914) Bachmann (1915) Arey (1920)
Schulze (1921)
Jenkins (1921) Scott and Kyffin (1978) McCollum et al. (1922)
Clarke and Wheeler (1922) Schmidt (1922a, b) Fry (1922) Schulze and Kunike (1923)
Schmidt et al. (1923) Faber (1924) Faber (1928) Mellor (1924) Kunike (1925)
Pommer (1925) Yano (1926) Edinger (1929) Lemmrich (1931) Franz-Odendaal and Hall (2006) Franz-Odendaal and Vickaryous (2006) Toverud (1926) Thiel (1927) Leriche and Policard (1928)
530
Addendum Table 2 (continued)
Year
Events and discoveries
References
1929
Use of magnesium citrate for decalcification of bone Use of X-rays for determining when the decalcification is complete Microscopical observation of disorganized bone fibrils after decalcification The Resorption of Bone is published X-ray and histological evidence of decalcification of bones Decalcifying action of ammonium chloride could be reduced by administration of calcium salts Studies on the cause and nature of dental caries Comparative study of histological preparations of bone with different decalcifying fluids The participation of the carbonates of bone in the neutralization of ingested acid: bone demineralization occurs in response to chronic acidosis Decalcification of rats’ teeth using 3% HNO3 in 80% alcohol The relationship of microorganisms to decay of stone The role of the parathyroid glands in deseasis associated with demineralization of the human skeleton is discussed Dissolution of silica-containing plant cystoliths is described Demineralization of bone using 3% KOH in glycerol Inorganic calcium and phosphate of blood appear to be in equilibrium with the bone salts Ancient biosignatures First review on boring (endolithic) algae is published Studies on bone tumors and osteolytic sarcomas started
Kramer and Shipley (1929)
1930
1930
1930 1931 1932
1932 1932
1932
1933 1933
1933
1933 1934 1935
1935 1936 1936
Hagens (1930)
Bodansky et al. (1930)
Jaffe (1930) Shelling (1931) Jaffe et al. (1932)
Enright, Friesell, Trescher (1932) Gooding and Stewart (1932)
Irving & Chute (1932) Bettice (1984)
Templin and Steenbock (1933) Paine et al. (1933)
Compere (1933)
Freiserleben (1933) Crowell et al. (1934) Schmidt and Greenberg (1935)
Abel (1935) Fremy (1936) Geschickter and Copeland (1936) McInnes and McCullough (1953) Lesure (1958) Guise (2000) Goltzman (2001)
Addendum
531 Table 2 (continued)
Year
Events and discoveries
References
1937
First postulation of the presence of “calcase—enzyme secreted by accessory boring organ and responsible for demineralization of mollusc shells Osteoporotic rat bone is produced by a diet containing calcium carbonate Calcium carbonate-dissolving algae Chemical constitution of enamel and dentine The Dissociation of Some Calcium Salts is published Decalcification of crustaceans’ cuticles using 30% aqueous solution of sodium hexamethaphosphate Lactic acid associated with the caries process Preparation of the enamel organic matrix Plant cystolith skeletons are described and reviewed Histology and regeneration of the fish scale are described Discovery of accessory boring organ by Muricidae and suggestion of chemo-mechanical theory of penetration Chemolysis of renal calculi by direct irrigation
Ankel (1937) Ankel (1938)
1937
1937 1937 1938 1938
1939 1940 1940 1940 1941
1943
1944 1945
1945 1945
1948
The Chemistry of Bone Formation is published Formic acid–sodium citrate decalcification of teeth and bones The pH of the carious lesion X-ray study on mineral formations of plant, animal, and human origin An Improved Method of Decalcification using formic acid is published
Harrison (1937)
Von Pia (1937) Armstrong and Brekhus (1937) Greenwald (1938) Wilks (1938)
Miller and Muntz (1939) Dimond and Weinmann (1940) Wieler (1940) Neave (1940) Fretter (1941) Carriker (1943)
Suby and Albright (1943) Keyser, Scherer, Claffey (1947) Dretler and Pfister (1983) Kuyper 1944 Morse (1945)
Stephan (1945) Branderberger (1945)
Kristensen (1948)
532
Addendum Table 2 (continued)
Year
Events and discoveries
References
1948
First evidence that microorganisms in rhizosphere can dissolve sparingly soluble inorganic phosphate Decalcification of the mother-of-pearl (nacre), isolation of organic components, and discovery of stratified membranes of conchiolin Demineralization of enamel and isolation of eukeratin Demineralization and classification of diseases in bones Bacterial chemistry of dental plaques EDTA (Versene) as organic chelating agent for demineralization of hard tissues Collagen fibers of bony tissue in the electron microscope Acid-mediated demineralization of dental tissues for electron microscopy EDTA-mediated demineralization of bone for electron microscopy Isolation of collagen from mammalian bone using dilute HCl First report about the presence of amino acids in fossil bones and shells up to approximately 350 Myr old Control of endpoint of decalcification by fluoroscopy The organic content of chalky enamel is described Preparation of the inorganic matrix of bone is described Demineralization against atherosclerosis; chelation therapy Electron microscopy studies on normal and caries teeth Bone is published Discovery and study on organic matrix of urinary concretions
Gerretsen (1948)
1949
1949 1950 1950 1951
1951 1952
1952 1953
1954
1954 1954 1954 1955
1955 1955 1956
Gregoire, Duchateau, Florkin (1949, 1950, 1954, 1955) Gregoire (1957) Gregoire (1959) Block, Horwitt, Bolling (1949) Haldeman (1950) Stralfors (1950) Nikiforuk (1951) Sreebny and Nikiforuk (1951) Nikiforuk and Sreebny (1953) Huber and Rouiller (1951) Albright et al. (1952) Scott (1952) Robinson and Watson (1952) Eastoe and Eastoe (1953)
Abelson (1954)
Waerhaug (1954) Stack (1954) Williams and Irvine (1954) Clarke et al. (1955) Ernst (2000) Helmcke (1952) McLean and Urist (1955) Boyce and Sulkin (1956) Boyce and Garvey (1956) King and Boyce (1957) Boyce et al. (1958) Boyce (1968)
Addendum
533 Table 2 (continued)
Year
Events and discoveries
References
1956
The basic factors of bone demineralization are published Decalcification of serpulid worms’ tubes A comparative histological study of fossil and recent bone tissue is published General Anatomy and Histology of Bone is published A Histochemical Study of the Organic Matrix of Hen Egg-Shells is published Fluoridization of calcium carbonate microfossils A quantitative study of decalcification methods The Chemical Dynamics of Bone Mineral is published Study on nature and chemical analysis of ossicles—holothurian calcium carbonate-containing sclerites First evidence of the presense of collagen in human cementum as shown by electron microscopy Osteolytic bone is dissolved by aminopeptidase secreted by osteocytes Specificity of the Molecular Structure of Organic Matrices in Mineralization is published Histopatological Technic and Practical Histochemistry is published Rapid complexometric method for the estimation of calcium in bone, dentine, and enamel Method for studying the breakdown of synthetic and natural silicates by soil bacteria is developed The mechanism of silica dissolution from diatom walls is described An osteolytic mucor mycosis in a penguin is described
Morris and Benton (1956) Benton and Morris (1956) Hedley (1956) Bernhardt, Manyak, Wilbur (1985) Enlow and Brown (1956)
1956 1956
1956 1957
1957 1958 1958 1958
1958
1959
1960
1960
1960
1960
1961
1961
Bourne (1956) Simkiss and Tyler (1957)
Upshaw, Todd, Allen (1957) Vardenius and Alma (1958) Neuman and Neuman (1958) Hampton (1958)
Tonge and Boult (1958)
Lipp (1959)
Glimcher (1960)
Lillie and Fuller (1960)
Weatherell (1960)
Webley, Duff, Mitchell (1960)
Lewin (1961)
Bigland et al. (1961)
534
Addendum Table 2 (continued)
Year
Events and discoveries
References
1961
First report on amino acid composition of the organic matrix of decalcified fetal bovine dental enamel Report on the regular occurrence of demineralized collagen fibers at the resorbing bone surface Decalcification of the sections of calcified tissue on the grids with potassium permanganate, uranyl acetate, or phosphotungstic acid for electron microscopy Comparative studies of bone matrix in normal and osteoporotic bone Kinetics of acid demineralization are described Decalcification of chicken egg shell and isolation of glycosaminoglycans
Glimcher et al. (1961)
1961
1961
1962
1962 1962
1963
Collagen and a cellulose-like substance in fossil dentine and bone
1963
Principles of Bone Remodelling is published Mechanism of Hard Tissue Destruction is published Comparative Biology of Calcified Tissue is published Macromolecular organization of dentine matrix collagen Lipids in demineralized dentine, proteolipids, phospholipids, and lipids in demineralized bone and kidney stone matrices
1963 1963 1964 1964
1965
1965 1966
Intramuscular implantation of demineralized bone matrix elicits new bone formation, discovery of Bone Morphogenetic Protein Phenomenon of focal calciolysis in exhumed bones is described Preparation of Decalcified Sections is published
Hancox and Boothroyd (1961)
Dudley and Spiro (1961)
Little et al. (1962)
Gray (1962) Birkedal-Hansen (1974) Baker and Balch (1962) Bronch and Diamantstein (1965) Heaney and Robinson (1976) Nakano, Ikawa, Ozimek (2001) Isaacs (1963) Shackleford and Wyckoff (1964) Wyckoff et al. (1964) Ho (1966) Pawlicki et al. (1966) Enlow (1963) Sognnaes (1963) Moss (1963) Veis and Schlueter (1964) Dirksen and Ikels (1964) Ennever et al. (1977) Nefussi et al. (1992) Khan et al. (1996) Goldberg and Septier (2002) Urist (1965) Urist and Nogami (1970) Urist, Mikulski, Lietze (1979)
Thurner et al. (1965) Brain (1966)
Addendum
535 Table 2 (continued)
Year
Events and discoveries
References
1966
Interactions in Electrolyte Solutions is published Kinetics of enamel dissolution Bacteria can penetrate rock Historadiographic studies on calciolysis as the initial stage of bone resorption Structural and Chemical Organization of Teeth is published Discovery of the first acidic protein in vertebrate dentin Structural and Chemical Organization of Teeth is published Scanning electron microscopy studies of resorbing surfaces of dental hard tissues Isolation of proteins from modern and fossil molluscan shells Isolation of lipids and phospholipids from mineralized tissues of fish and other animals Dentine and Pulp: Their Structure and Reaction is published Phosphoprotein phosphatase catalyzes the rapid demineralization of tooth enamel Fungi are considered to be agents of carbonate deterioration for the first time Calcibiocavitology—the science dealing with the hollowing out of spaces in hard calcareous substrata by organisms Carbonic anhydrase is responsible for in vivo demineralization of the valves of lamellibranches by molluscs Evidence of the chemical nature of the boring mechanism by Polydora “mud worm” in calcareous substrates Biological Calcification: Cellular and Molecular Aspects is published Fungal attack on rock: solubilization mechanisms
Nancollas (1966)
1966 1966 1966
1967
1967 1967
1967
1968 1968
1968 1969
1969
1969
1969
1969
1970
1970
Gray (1966) Myers and McCready (1966) Bohartirchuk (1966)
Miles (1967)
Veis and Perry (1967) Miles (1967)
Boyde and Lester (1967)
Bricteux-Gregoire et al. (1968) Shapiro (1968) Wuthier (1968) Symons (1968) Kreitzman et al. (1969) (1970)
Krumbein (1969)
Carriker and Smith (1969)
Chetail and Fournie (1969)
Haigler (1969) Blake and Evans (1973) Zottoli and Carriker (1974) Schraer (1970)
Silverman and Munoz (1970)
536
Addendum Table 2 (continued)
Year
Events and discoveries
References
1970
The demineralization in the bone of the teleost fish can be produced in three different ways: osteoclastic, osteolytic, and halastatic The Metals of Life. The Solution Chemistry of Metal Ions in Biological Systems is published First ultrastructural study on osteodentin in the pike (Esox lucius) Organic acids and chemical weathering Chemical Zoology is published Uronic acid containing soluble intracrystalline polysaccharides isolated from algal coccoliths for the first time Studies on morphology and ultrastructure of shark enamel Handbook of histopatology and histochemical techniques is published 370 MYO devonian boring algae were described Fungal osteoclasia: a model of bone resorption Biodegradation and utilization of silica in nature Demineralization of bone matrix: observations using the electron microscope The Study of Trace Fossils is published Mineral–tetracycline reactions and tetracyclines as demineralization agents in bone, teeth, and hard tissues Decalcification techniques in electron microscopy Isolation of 80-million-year-old mollusc shell proteins Demineralization in forensic science Oldest (Upper Silurian) organic remains of boring algae are found
Lopez (1970)
1971
1971
1972 1972 1973
1973 1974
1974 1974 1974 1975
1975 1975
1975 1976 1976 1976
Williams (1971)
Herold (1971)
Huang and Keller (1972) Florkin (1972) Westbroek (1973)
Reif (1973) Culling (1974)
Kobluk and Risk (1974) Marchiafava, Bonucci, Ascenzi (1973) Lauwers and Heinen (1974) Thorogood and Gray (1975)
Frey (1975) Skinner and Nalbandian (1975) Wikesjö et al. (1986) Sterrett et al. (1997) Dietrich and Fontaine (1975) Weiner, Lowenstam, Hood (1976) Helfman and Bada (1976) Waite et al. (1999) Kazmierczak and Golubic (1976)
Addendum
537 Table 2 (continued)
Year
Events and discoveries
References
1976
SEM study on dentin: demineralization results in shrinkage of the dentin structure Forensic Dentistry is published Decalcified bone as a substrate for osteogenesis Caries and the remineralization phenomena Phosphatic shell formation in brachiopod molluscs and isolation of their shell proteins EDTA demineralization of calcium oxalate stones and discovery of a soluble gamma-carboxyglutamic acid-containing protein in renal calculi Dissolution of biominerals: a constant composition method Anatolepis—the earliest (520 MYA) presumed vertebrate known to possess a mineralized skeleton is found Osteoclast-mediated demineralization and molecular mechanisms of bone resorption
Garberoglio and Brännström (1976)
1976 1977 1977 1977
1977
1978 1978
1978
1978
1978 1978 1979
1979
1979
Discovery of calcareous deposits in the renal sac of ascidians and isolation of organic matrix from uric-acid-based spherulites Direct resorption of bone by cancer cells in vitro Electron microscopy studies on demineralized osteodentine Creation of Mutvei’s solution as an ideal agent for the dissolution of biogenic carbonates Discovery of aspartic acid-rich proteins in the soluble organic matrix of mollusc shell The Chemistry of Silica—Solubility, Polymerization, Colloid and Surface Properties, and Biochemistry is published
Sopher (1976) Nade and Burwell (1977) Silverstone (1977) Jope (1977) Watabe and Pan (1984) Lian et al. (1977) Warpehoski et al. (1981)
Tomson and Nancollas (1978) Repetski (1978) Smith, Sansom, Repetski (1996)
Heersche (1978) Baron (1989) Titelbaum (2000) Väänänen et al. (2000) Titelbaum (2007) Saffo and Lowenstam (1978) Lambert et al. (1998)
Eilon and Mundy (1978) Kerebel et al. (1978) Mutvei (1979) Schöne et al. (2005) Weiner (1979)
Iler (1979)
538
Addendum Table 2 (continued)
Year
Events and discoveries
References
1979
Demineralization of pancreatic stone and isolation of an acidic-rich phosphoglycoprotein Etching cells of boring sponges can effect chemical dissolution of calcium carbonate substrates by enzymic digestion via the lysosomal system and membranes of etching cell processes Skeletal Growth of Aquatic Organisms is published Biogeochemistry of Amino acids is published Demineralization of ganoid fish scales and isolation of ganoine—a superficial hypermineralized layer that lacks collagenous fibers and is true enamel whose organic matrix contains amelogenin Theory and Practice of Histotechnology is published The implication of carbonic anhydrase in the physiological mechanism of penetration of carbonate substrata by the marine burrowing sponge Cytological mechanisms of calcium carbonate excavation by boring sponges are described Desilicification techniques are discussed in Silicon and Siliceous Structures in Biological Systems Biological Mineralization is published Symbiotic zooxanthellae enhances boring activity of host sponges Biological Mineralization and Demineralization is published Antharctic cryptoendolithic microorganisms are described Precambrian endoliths discovered
De Caro, Lohse, Sarles (1979) Lohse, Vernie, Sarles (1981) Multinger et al. (1983) Pomponi (1979)
1979
1980 1980 1980
1980 1980
1980
1981
1981 1981 1982 1982 1982
Rhoads and Lutz (1980) Hare, Hoering, King (1980) Meunier (1980) Sire et al. (1987) Daget et al. (2001)
Shenaan and Hrapchak (1980) Hatch (1980)
Pomponi (1980)
Simpson and Volcani (1981)
Nancollas (1981) Vacelet (1981) Hill (1996) Nancollas (1982) Friedmann (1982) Campbell (1982)
Addendum
539 Table 2 (continued)
Year
Events and discoveries
References
1982
Demineralization of fish otoliths: chemistry, composition, microstructure, organic matrix proteins (OMP-1, Oltolin, zOtolin, otopetrin)
1982
Demineralization of fish scales and isolation of isopedine—a tissue consisting of collagen fibrils organized into an orthogonal plywood-like structure Biomineralization and Biological Metal Accumulation is published Demineralization of biomaterials: biodegradation that takes place by solution-driven and cell-mediated processes
Watabe et al. (1982) Campana and Neilson (1985) Campana (1999) Murayama (2002) Dauphin and Dufour (2003) Hugles et al. (2004) Murayama et al. (2005) Meunier and Castanet (1982) Meunier (1987)
1983
1983
1983
1984 1984
1984 1984 1985 1985
1986
1986
Electron microscopy studies on fossil proteins in vertebrate calcified tisssues Calcium and its Role in Biology is published Development of demineralization tests and methods for determining the cariogenic potential of foods Rapid nitric acid decalcification method Methods of Calcified Tissue Preparation is published Chemical activity of lichens on mineral surfaces Rate of dissolution of carbonate sediments by microboring organisms is calculated Factors Relating to Demineralization and Remineralization of the Teeth is published Demineralization–remineralization phenomena and human dental decay
Westbroek and Jong (1983)
Klein and de Groot (1983) Nagai and Takeshita (1984) Frayssinet et al. (1993) Koerten and van der Meulen (1999) Lu et al. (2002) Xia and Triffitt (2006) Armstrong et al. (1983)
Sigel (1984) Brudevold et al. (1984) Imfeld (1994)
Mawhinney et al. (1984) Dickson (1984) Jones and Wilson (1985) Tudhope and Risk (1985)
Leach (1986)
Loesche (1986)
540
Addendum Table 2 (continued)
Year
Events and discoveries
References
1986
Studies on organic matrix of the skeletal spicules of sea urchins and other echinodermates
1986
Demineralization of coccoliths and isolation of polysaccharides The microstructure of dentine in taxonomic and phylogenetic studies
Benson et al. (1983) Benson, Benson, Wilt (1989) Berman et al. (1990) Kilian and Wilt (1996) Ameye et al. (1998) Wilt (1999, 2002) Seto et al. (2004) Bottjer et al. (2006) Kok et al. (1986)
1986
1987
1987 1987 1987
1987
1987
1988 1988
1989
The oldest microboring cyanobacteria are found in 1500 MYO rocks Biogenic etching in amorphous and crystalline silicates Biodeterioration of Constructional Materials is published Isolation of intricately patterned organic matrix from ascidian spicules and investigation of factors involved in the formation of amorphous calcium carbonate Coupled diffusion as a basis for subsurface demineralization in dental caries Demineralization of human calcium oxalate renal stones and isolation of nephrocalcin glycoprotein Review on Dental Anthropology is published The Testimony of Teeth: Forensic Aspects of Human Dentition is published Origin, Evolution and Modern Aspects of Biomineralization in Plants and Animals, On Biomineralization, Biomineralization: Cell Biology and Mineral Deposition, Biomineralization: Chemical and Biochemical Perspectives, and Skeletal Biomineralization: Patterns, Processes and Evolutionary Trends are published
Hildebolt et al. (1986)
Zhang and Golubic (1987) Golubic et al. (2005) Callot et al. (1987) Morton (1987) Lambert and Lambert (1987) Lambert (1992) Aizenberg et al. (2002)
Anderson and Elliott (1987, 1992, 2003) Anderson et al. (2004) Nakagawa et al. (1987)
Scott and Turner (1988) Rogers (1988)
Crick (1989) Lowenstam and Weiner (1989) Simkiss and Wilbur (1989) Mann, Webb, Williams (1989) Carter (1990)
Addendum
541 Table 2 (continued)
Year
Events and discoveries
References
1989
Demineralization of cholesterol gallstones and isolation of 30 kD acidic protein which regulates the precipitation and accretion of calcium salts Osteoclastic bone resorption by a polarized vacuolar proton pump Ultrastructure of Skeletal Tissues is published Calcium Phosphates in Oral Biology and Medicine is published Bioerosion of coral reef—chemical approach Biologically mediated corrosion of synthetic glass Mechanisms and Phylogeny of Mineralization in biological systems is published Perfusion of demineralization agents by the blood vessels could help to shorten the decalcification process Studies on calcium oxalate monohydrate renal uroliths and kinetics of their dissolution Isolation of cement precursor and cement proteins from the concrete tubes of sandcastle worms Acidolysis, complexolysis, redoxolysis, and mycelial metal accumulation as main mechanisms of fungal-mediated mineral dissolution Black fungal colonies induce decay phenomena of antique marbles Discovery of DMP1—novel dentin matrix acidic phosphoprotein Decalcification of otoconia and isolation of organic matrix proteins (otoconin-22, 90, calbindin D28K)
Shimizu et al. (1989)
1989 1990 1991
1991 1991 1991
1991
1991
1992
1993
1993
1993 1993
Blair et al. (1989) Bonucci and Motto (1990) LeGeros (1991)
Lazar and Loya (1991) Krumbein and Urzi (1991) Suga and Nakahara (1991)
Nilsson, Hellstrom, Albiin (1991)
March, Costa-Bauza, Grases (1991) Grases et al. (1995) Waite, Jense, Morse (1992) Zhao et al. (2005) Sun et al. (2007) Burgstaller and Schinner (1993) Burford, Fomina, Gadd (2003) Gadd (2007)
Krumbein and Urzi (1993)
George et al. (1993) He at al. (2003) Pote et al. (1993) Davis et al. (1995) Wang et al. (1998) Verpy, Leibovici, Petit (1999) Merchan-Perez et al. (1999) Thalmann et al. (2001) Piscopo et al. (2003) Huss and Dickmann (2003)
542
Addendum Table 2 (continued)
Year
Events and discoveries
References
1993
Discovery of hyaloine—highly mineralized tissue of the fish bony scutes composed of thin vertical fibrils AFM appeared to offer a powerful new tool to directly evaluate demineralization threatments for dentin The Review on Dentinogenesis is published Demineralization of diatoms’ cell walls and isolation of frustulins (glycoproteins) EDTA- and EGTA-based method for gentle decalcification of the algal cell walls Selective extractability of noncollagenous proteins from chicken bone is described Handbook of Metal-Ligand Interactions in Biological Fluids: Bioorganic Chemistry is published Discovery of quartz dissolution by sponges Demineralization of calcium oxalate crystals in plants and isolation of organic matrix
Sire (1993) Sire and Akimenko (2004)
1993
1993 1994
1994
1994
1995
1995 1995
1996 1996
1996
1996
1996
Mechanisms of microbially mediated mineral dissolution Theory and Practice of Histological Techniques is published Bacterial osteolytic factors and mechanism of bacterially induced bone destruction Biotechnological approach for chitin demineralization from shellfish waste by lactic acid fermentation First evidence that protein-containing material is trapped within biologically precipitated silica in plants
Marshall et al. (1993)
Linde and Goldberg (1993) Kröger, Bergsdorf, Sumper (1994) Kröger, Bergsdorf, Sumper (1996) Kröger et al. (1997) Morse et al. (1994)
Gerstenfeld et al. (1994)
Berthon (1995)
Bavestrello et al. (1995) Webb et al. (1995) Bouropoulos, Weiner, Addadi (2001) Li et al. (2003) Nakata (2003) Ehrlich (1996) Bancroft and Stevens. (1996)
Nair et al. (1996)
Zakaria, Hall, Shama (1996, 1998, 2005) Jung et al. (2005) Harrison (formely Perry) (1996) Perry and Keeling-Tucker (2003)
Addendum
543 Table 2 (continued)
Year
Events and discoveries
References
1996
Mineralized tissue is shown to be important in buffering lactic acid during anoxic submergence in reptiles and amphibians
1996
Demineralization of enamel: gastric juice as erosive agent Review on Scanning Electron Microscopy of Natural and Demineralised Bone is published An ion exchange method using Dowex ion exchange resin is developed and applied for demineralization of biogenic minerals Demineralization of molluscs’ shells and nacres and isolation of proteins (nacrein, lustrin, perlustrin, pearlin, perlucin, perlwapin, aspein, perlinhibin, perlbikunin, mucoperlin, prismalin, caspartin, calprismin), glycoproteins, and acidic polysaccharides
Jackson et al. (1996) Jackson, Crocker, Ultsch (2000) Jackson, Andrade, Abe (2003) Warren and Jackson (2005) Davis and Jackson (2007) Jackson et al. (2007) Bartlett, Evans, Smith (1996) Bartlett and Coward (2001) Boyde and Jones (1996)
1996
1996
1997
1997
1998 1998
1998
Geomicrobiology: Interaction Between Microbes and Minerals and Biological Impact on Mineral Dissolution are published Silicates: principles of dissolution Desilicification of demosponge spicules and isolation of silicatein filaments Decalcification of Bone: Literature Review and Practical Study of Various Decalcifying Agents, Methods, and Their Effects on Bone Histology is published
Albeck, Weiner, Addadi (1996) Gotliv, Addadi, Weiner (2003)
Matsushiro et al. (1997) Shen et al. (1997) Sudo et al. (1997) Mutsushiro (1999) Mann et al. (2000) Weiss et al. (2000) Miyashita et al. (2000) Marin et al. (2000) Gotliv, Addadi, Weiner (2003) Marxen et al. (2003) Suzuki et al. (2004) Tsukamoto et al. (2004) Marin et al. (2005) Marin and Luquet (2005) Dauphin (2006) Marie et al. (2007) Banfield and Nealson (1997) Banfield et al. (1999)
Dietzel (1998) (2000) Shimizu et al. (1998) Cha et al. (1999) Callis and Sterchi (1998)
544
Addendum Table 2 (continued)
Year
Events and discoveries
References
1998
Organic matrix-mediated remineralization process based on interaction between self-assembled mussel adhesive protein vesicles and apatite Qualitative and quantitative measurement of enamel demineralization using AFM for the first time Digestive degradation of a king-sized theropod coprolite is described Dental Anthropology: Fundamentals, Limits, and Prospects is published Desilicification of diatoms and isolation of unusual phosphoproteins termed silaffins and long chain polyamines
Shirkhanzadeh (1998)
1998
1998
1998
1999
1999
Decalcification of bony samples by EDTA is highly recommended for application in DNA in situ hybridization and comparative genomic hybridization techniques 1999 Kinetics of enamel demineralization in vitro are described Twenty first century 2000 Phenomena of “dark decalcification” in coralline algae and soft corals 2000 The Biomineralization of Nanoand Micro-structures and Biomineralization: Principles and Concepts in Bioorganic Material Chemistry are published 2000 Assessment of decalcifying protocols for detection of specific RNA 2000 Review on Phosphate-Solubilizing Fungi is published 2000 Demineralization of bone and calcium regulation during space flight
Parker et al. (1998) Finke, Jandt, Parker (2000)
Chin et al. (1998)
Alt et al. (1998)
Kröger, Deutzmann, Sumper (1999) Kröger et al. (2002) Poulsen, Sumper, Kröger (2003) Poulsen and Kröger (2004) Sumper and Brunner (2006) Poulsen et al. (2007) Alers et al. (1999) Yamamoto-Fukud et al. (2000) Sarsfield et al. (2000) Brown et al. (2002) Gilbert et al. (2005) Margolis et al. (1999)
Chisholm (2000) Tentori and Allemand (2006) Bäuerlein (2000) Mann (2001)
Shibata et al. (2000)
Whitelaw (2000) Doty and Seargrave (2000)
Addendum
545 Table 2 (continued)
Year
Events and discoveries
References
2000
Similarities between the accessory boring organ, osteoclasts, and the mantle of freshwater bivalves suggest that the mechanism for decalcification of calcareous substrates is conserved Review: The Chemistry of Enamel Caries is published Crystal dissolution stepwave model Method for estimation of the extent of endolithic tissue of the bioeroding sponges Biotechnology on the rocks: chrysotile asbestos is converted into amorphous material by chelating action of fungi and lichen metabolites Nanoindentation of dental enamel demineralization and demineralization/remineralization cycles on human tooth enamel surfaces “Adhesion-Decalcification Concept” relating to adhesion to and decalcification of hydroxyapatite by carboxylic acids is published Geomicrobiology is published Nanosized particles: new understanding of demineralization, surface energetic control in dissolution of crystallites and a new model for nanoscale enamel dissolution are described Mineralization–demineralization cycle in terrestrial isopods and architecture of organic matrix in sternal CaCO3 deposits The demineralization process inactivates infectious retrovirus in infected bone Silicase, an enzyme which degrades biogenous amorphous silica The Experimental Determination of Solubilities is published
Clelland and Saleuddin (2000)
2000 2001 2001
2001
2001
2001
2002 2003
2003
2003
2003
2003
Robinson et al. (2000) Lasaga and Lüttge (2001) Schönberg (2001)
Fenoglio, Tomatis, Fubini (2001) Martino et al. (2003) Favero-Longo et al. (2005)
Finke et al. (2001) Barbour, Parker, Jandt (2003) Lippert et al. (2004a, b) Barbour and Shellis (2007) Yoshida et al. (2001) Yoshioka et al. (2002)
Ehrlich (2002) Tang et al. (2003, 2004) Wang et al. (2005) Wang, Nancollas, Henneman. (2006)
Fabritius and Ziegler (2003) Ziegler et al. (2004) Fabritius, Walther, Ziegler (2005) Ziegler et al. (2006) Swenson and Arnoczky (2003)
Schroeder et al. (2003)
Tomkins and Hefter (2003)
546
Addendum Table 2 (continued)
Year
Events and discoveries
References
2003
Discovery of AP7 and AP24—two aragonitic proteins isolated from nacre of the red abalone The use of bacterial oxalate-degrading enzymes to coat urinary biomaterials represents a novel paradigm to reduce biomaterial-related encrustation Discovery of bacteriomorphic nature of mineral formation in cardiolytes (human heart valves) Silicon Biomineralization is published Review on Palaeoecology and Evolution of Marine Hard Substrate Communities Including Bioerosion is published HF/HCl demineralization of a 3.5 billion year old Archean chert and isolation of the organic matter Biologically produced alginic acid affects calcite dissolution and determines microbial deterioration of historic stone Antarctic cryptoendolitic microorganisms could be suitable models for investigations on extinct or extant life on Mars 3.5 billion year old biosignatures discovered in Archean pillow lavas Enamel dissolution and self-preservation of biominerals The mineralization index as a new approach to the histomorphometric appraisal of osteomalacia Demineralization of fossil hard tissues reveals the preservation of original tissues, as well as apparent cells and blood vessels
Michenfelder et al. (2003)
2003
2003
2003 2003
2004
2004
2004
2004
2004 2004
2005
Watterson et al. (2003)
Gilinskaya et al. (2003)
Müller (2003) Taylor and Wilson (2003)
Derenne (2004) Skrzypczak et al. (2004, 2005)
Perry et al. (2004, 2005) Mc Namara and Mitchell (2005)
Onofri et al. (2004) Onofri, Zucconi, Tosi (2007)
Furnes et al. (2004)
Tang et al. (2004) Parfitt et al. (2004)
Schweitzer et al. (2005) Schweitzer, Wittmeyer, Horner (2005) Asara et al. (2007) Schweitzer et al. (2007)
Addendum
547 Table 2 (continued)
Year
Events and discoveries
References
2005
Desilicification of glass sponge spicules and the first evidence of the presence of collagen and chitin in their skeletal formations Microbial interaction with silica and mineralogical footprints of microbial life Discovery of asprich—a novel aspartic acid-rich protein family from mollusc shell and acidic 8-kDa protein from aragonitic abalone shell nacre Coralline alga: cell wall decalcification as part of epithelial cell replacement Biominerals is published EDTA-mediated calcite dissolution demonstrates that, after penetration through a critical pit depth barrier, step velocity increases linearly with the pit depth Mechanism of classical crystal growth theory explains quartz and silicate dissolution behavior Biosilicified structure–function relationship is described Plausible mechanism for the bioboring on carbonates proposed Boring sponges: establishment of method for measurement of the rate of chemical bioerosion Comparison of six different methods for extracting amino acids and proteins from marine sediments Modern review of methodologies for extracting plant-available and amorphous silica from soils and aquatic sediments Acid-induced demineralization in vitro and dissolution kinetics of primary and permanent tooth enamel Biomineralization-Medical Aspects of Solubility is published
Ehrlich et al. (2005) Ehrlich et al. (2006), Ehrlich and Worch (2007) Ehrlich et al. (2007)
2005
2005
2005
2005 2005
2005
2005 2006
2006
2006
2006
2006
2007
Douglas (2005) Perry (2003) Gotliv et al. (2005) Fu et al. (2005)
Pueschel, Judson, Wegeberg (2005) Skinner (2005) Perry et al. (2005b)
Dove, et al. (2005)
Wang et al. (2005) Garcia-Pichel (2006)
Zundelevich, Lazar, Ilan (2006)
Nunn and Keil (2006)
Sauer et al. (2006)
Wang et al. (2006)
Königsberger and Königsberger (2007)
548
Addendum Table 2 (continued)
Year
Events and discoveries
References
2007
Function of Eggshell Matrix Proteins, Biological Calcification: Normal and Pathological Processes in the Early Stages and Handbook of Biomineralization are published Endolithic microborings on early Earth and applications to astrobiology Osteoclasts have the ability to demineralize calcified elastin Differentiating Human Bone from Animal Bone: A Review of Histological Methods is published HCl-mediated demineralization and studies on homology and phylogeny of chondrichthyan tooth enameloid Biomineralization: From Nature to Application will be published The paper “Kinetics of amorphous silica dissolution and the paradox of the silica polymorphs” published in PNAS Numerous review papers on biomineralization are published in Chemical Reviews Peptides Enhance Magnesium Signature in Calcite 10th International Symposium on Biomineralization held in Lianyungang, China (September 2009) Overview of the Amorphous Precursors Phase Strategy in Biomineralization is published First evidence of the chitin-based organic networks within cell walls of diatoms “The initial stages of template-controlled CaCO3 formation revealed by Cryo-TEM” is published in Science
Huopalahti et al. (2007) Bonucci (2007) Bäuerlein (2007)
2007
2007 2007
2007
2008 2008
2008
2008 2009
2009
2009
2009
Mc Loughlin et al. (2007)
Simpson et al. (2007) Hiller and Bell (2007)
Gillis and Donoghue (2007)
Sigel and Sigel (2008) Dove et al. (2008)
Chem Rev (2008), 108(11)
Stephenson et al. (2008) Front Mater Sci China (2009), 3(2)
Weiner et al. (2009)
Brunner et al. (2009)
Pouget et al. (2009)
Addendum
549 Table 2 (continued)
Year
Events and discoveries
References
2009
Mechanisms of gold biomineralization in the bacterium Cupriavidus metallidurans Biomineralization by Photosynthetic Organisms is published Biomineralization under microgravity conditions The term “Phenomenon of multiphase biomineralization” is proposed and discussed
Reith et al. www.pnas.org_cgi_doi_10.1073 _pnas.0904583106
2009
2009 2010
Raven and Giordano (2009)
Sinha et al. (2009) Ehrlich et al. (2010)
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Addendum
Index
A Abductin, 279–281, 319–322 Accessory boring organ, 66 Acineta, 352, 355–356 Adhesion, 5, 248, 253, 299–301, 306, 308, 327–333, 336–337, 343–344, 349, 351–356, 359–362, 365–368, 371–376, 379–388, 391–403, 411, 422, 471, 478 systems, 327–333 Adhesive gels, 335–340 Agglutination, 448–450, 455–459 Alternative cuticles, 379, 386–388 Amphora veneta, 493 Annelids collagen and chitin, 547 Antipatharia, 271–272, 274–276 Antipathin, 192, 245–246, 260, 271–277, 309 Aphrocallistes beatrix, 139 Aplacophora, 211–212, 219–225 Aragonite, 26, 29, 31, 52, 68, 79–80, 82, 103–111, 118, 214, 220, 226–227, 276, 283–285, 304, 313, 457 Argopecten, 319–320 Aspidoscopulia, 148–149, 172 Attachment, 19, 53, 60, 79, 83, 117, 151, 180, 195, 206, 249, 255, 299, 301, 304–306, 308–309, 312, 314, 327–329, 335–336, 340, 343–344, 349, 353–354, 365–368, 374, 394, 403, 420 B Balanus, 343–346 Bamboo corals, 187–193, 195–199, 262–263, 265–266 Barite-based biominerals, 32–33 Barnacle cement, 341–347 Barnacles, 231, 328, 335, 341–347, 443 Bioadhesion, 301, 479
Biocement, 476, 479 Biocompatibility, 7, 9, 11, 18, 85, 197, 202, 409, 416–422, 428 Biocomposite, 35, 40, 42, 60, 82, 89, 103–111, 113–115, 125–135, 137–141, 143–180, 187–193, 195–199, 201–207, 211–237 Biodegradation, 197, 344, 409, 416–422 Bioelastomer, 279–286, 289–296 Bioerosion, 59–60, 65, 90 Biofouling, 303 Biogenic cellular structures, 141 Bioglue, 335 Bioinspiration, 125–126, 155 Biological materials, 1, 3–21, 125–135, 148, 257, 261, 291, 301–303, 379, 381–382, 385, 394, 445–452, 455–463, 465–479, 485, 488, 495, 499–501 Biomaterials market, 20–21 properties, 423 Biomechanics, 10–13, 303, 309–312, 435, 438 Biomedical adhesives, 340 Biomedicine, 5, 8, 43, 195, 299, 412, 479 Biomimetics, 43, 52, 63, 70, 73, 87, 90, 103, 126, 131, 143, 171, 189, 195, 201, 205, 253, 279, 299, 301, 312–314, 321, 341, 411, 414, 428, 430, 443, 476–479, 485–496 Biomineralization, 25–43, 51–56, 59–91, 103–118, 165, 179, 189–191, 202, 205, 212, 262, 273, 279, 455–459, 465, 486, 500 Biominerals, 25–43, 51–56, 59–91, 103–118, 166, 203, 212, 380 Bionics, 126, 201, 279, 443 Biorobotics, 371, 373, 376 Biosilica, 35, 83, 170–180
565
566 Biosilicification, 54, 82, 84, 90, 110, 155, 169, 171, 173, 179, 429, 491–496 Bivalvia, 211 Black corals, 53, 271–276 Bone, 3–8, 11, 13–14, 16–17, 20, 26–27, 52, 60, 63, 84–89, 110, 195–199, 202, 253, 266, 275, 416, 429, 432, 477–479 Boron, 35, 38 Brachiopoda, 79–80, 113–115 Bromine, 189, 261, 271–272, 379, 381–383, 387–388 Bromotyrosine, 260, 274, 414 Busycon, 289, 292 Byssus, 192, 248, 299–314 C Caenogastropod, 291 Calcification, 28, 31, 43, 61–63, 67–68, 73–74, 76, 78, 80–82, 85–88, 90, 162, 172, 189, 258, 262, 264, 283, 431, 456–458, 471, 495 Calcite, 26, 29–33, 52–53, 65–66, 71, 77, 79–82, 87–88, 115, 117, 162, 164–166, 189, 197, 199, 201–203, 214–215, 257, 263, 265, 276, 284, 287, 304, 313, 431, 457, 467 Calcium-based biominerals, 26, 28–30 Capsular protein, 289, 292 Caulophacus, 143, 156, 158–166 Cellular structures, 74, 125–127, 131–135, 137, 139, 148–155, 211–212, 219, 474 Cephalopods, 211, 372–376, 434–435 Chinese mitten crab, 399–400 Chitin, 74–84, 87, 103–111, 113–115, 154, 173–180, 391–403, 409–423 Chitin-protein-based composites, 391–403 Ciliary girdle, 360–361 Ciliatea, 351 Ciliates, 54, 220, 226, 351–356, 448 Cirratulids, 465, 467 Classical nucleation theory, 52 Classification, 3–21, 139, 249, 397, 448, 456, 479, 499 Coelenterates collagen, 430–434 Collagen, 84–90, 173–180, 248–250, 427–438, 489–490 Composite materials, 4, 11–12, 16, 25, 42, 103, 115, 125, 127, 212, 251, 279, 382 Copepoda, 115–117 Copper-based biominerals, 36 Copulatory spicules, 224–225 Coralline hydroxyapatite, 195, 197
Index Corals bamboo, 187–189, 195–199, 262–263, 265 black, 53, 271–276 gorgonian, 257–259, 265, 275 sea, 500 water, 267 Corynonema, 146 Cross-linked microtubules, 365 Cross-linking, 35, 76, 192, 261, 273–274, 283, 291–292, 294, 296, 300, 311, 314, 325, 338–339, 345, 384, 386–387, 434, 436, 471, 477 Crustaceans, 30, 76, 79, 81, 83, 103, 105, 115, 117–118, 153, 261, 279, 323–324, 341–343, 356, 382–383, 386–388, 391–394, 397–398, 400, 427, 443, 491 D Decalcification, 61–63, 68, 73, 189, 283, 431, 458 Decapod marine crabs, 402 Decapods, 372–373, 397 Deep-sea ecology, 451 Deep-sea expedition, 451, 486 Definitions, 3–21 Demineralization, 59–91, 164, 176 Demosponges, 33–41, 103–111, 145, 147, 167, 169, 170, 251 Dendroceratida, 245–246, 250 Denticle blades, 362 Denticulate ring, 359–362 Desilicification, 69, 71–73, 84, 160, 174 Diatoms, 17, 29, 31–33, 35, 37, 39, 72, 76, 83, 87, 116, 125–127, 131–134, 143, 148, 172–173, 231, 413, 447, 449, 455, 485–496 Dictyoceratida, 245, 253 Dityrosine, 283, 321, 323, 325, 382, 385 DOPA, 273, 306–308, 310–311, 313–314, 329, 374, 376, 384, 470–471, 478 Dosidicus gigas, 371, 374–375 E Echinochrome, 204 Echinodermata, 26, 33, 201, 327–333, 435 Echinoderms collagens, 436–437 Egg capsules, 192–193, 248, 279, 289–296 Eriocheir sinensis, 391–392 Extreme biomimetics, 485–496 F Farrea, 80, 83, 146, 151, 153 Fibres, 250 Food collagens, 87
Index Foraminifera, 26, 34–35, 62, 64, 139, 220, 231, 443, 447, 451, 455–463 Fossils, 53, 76, 137, 220, 397, 455, 474 Frustules, 32–33, 38, 83, 133–134, 445, 448–449 G Geothermal and hydrothermal environments, 491–496 Germanium-based biominerals, 38–39 Giardia, 365–368 Giardia lamblia, 365 Glass sponges, 26, 41, 53, 71, 83–84, 87–89, 132, 137, 143–180, 429, 500 Glueomics, 465–479 Glycera, 36, 379, 383, 385 Gorgonaceae, 193 Gorgonin, 67, 187–193, 245, 257–267, 273, 309, 431 Graft material, 197 Gromiids, 445, 447–448 H Hairy lobster, 392–395 Hexactinellida, 71, 87–88, 110, 137, 143–180, 429 Hexactinellids, 137, 139, 143, 145, 148, 170 Hierarchical biomaterials, 125–135, 148 Hinge ligament, 279–286 History, 3–21, 60, 64, 143, 166, 177, 246, 258–262, 265–266, 301, 321, 352, 434, 486 History of biomaterials, 3, 5–9 Holdfast structures, 53, 180, 301, 312–313, 331, 447 Honeycomb architecture, 140 Honeycomb structural motif, 133 Honeycomb structure, 127–133, 137–141 Hot-springs microfauna, 33, 485–487, 492–494 Hydrothermal vent fauna, 391–392, 486, 488 I Ianthella basta, 409 Immunology, 416–422 Implants, 4, 6–9, 13, 15–16, 19, 132, 195–199 Instantaneous adhesion, 328, 330 Interface, 4, 8, 12, 14, 16, 52, 65, 74, 189, 197, 308, 312–313, 391, 416, 436, 458, 470, 474 Interspace mineralization, 187–193 Iodine, 189, 246–247, 258–262 Iron-based biominerals, 42 Isidella tentaculum, 266 Isididae, 187–193, 197, 263, 266
567 J Jaws, 36–37, 116, 376, 379, 382–387 Jellyfish collagen, 433–434 K Keratose sponges, 33, 246 Kiwa hirsuta, 391–393 L Laminated biocomposite, 114, 272, 275 Larvae, 41, 52, 66, 82, 113–114, 145, 180 Larvae metamorphosis, 468–470 Limpets, 33, 117, 335–337, 371–372 Lithistida, 144 Loricae, 448–449 M Magnesium-based biominerals, 30–32 Manganese oxides, 38, 458 Marine gastropods, 335–340 Marine invertebrates, 5, 25, 28–43, 51, 53, 60, 103, 113, 125–126, 131, 211–212, 261, 279, 289, 329, 341, 343, 349, 351–356, 359–362, 365–368, 371–376, 379–388, 427–428, 443, 445–452, 455–463, 465–479, 485, 490 Marine parasites, 335, 359, 365, 382, 417–418 Marine protists, 445, 452 Material properties, 129, 131–132, 246, 263, 275–276, 295, 304, 325, 362, 382, 413, 477 Mechanical properties, 7, 9, 11–12, 16, 19, 37, 52, 60, 114, 127, 129, 131, 251, 262–264, 276, 279, 290–292, 304, 309–313, 323–325, 375, 379, 384, 386–388, 428–430, 436–437 Mechanisms biological, 8, 327 bonding, 471, 477–478 chemical, 65, 345, 443 mechanical, 65 molecular, 147, 330 Mefp-proteins, 292, 305–308, 310, 314 Melanin, 36, 192, 385 Metal-halogen-based biocomposites, 382 Microtubules, 354, 365–366 Military biomaterials, 20 Mollusca, 26, 29, 35, 41, 68, 78, 111, 117, 211–213, 220, 230, 279, 283, 286, 295–296, 321, 335–340, 397, 456, 513 Molluscs collagens, 427–428, 431, 434–435 spicules, 211–237
568 Mucus, 226, 295, 336–337, 371, 432, 467–468, 488 Multiphase biomineralization, 103–118 N Naphthoquinone, 204, 206 Nemopilema nomurai, 432–433 Nereis, 36–37, 376, 379, 384–385, 387 Nudibranchia, 211–219 O Octocorals, 53, 62, 87–88, 187, 191, 195, 197, 220, 263–266, 431 Octopods, 372–373 Onchidella, 31, 41, 228–237 Organic cement, 274, 456–457, 459, 461 Organic matrix, 25, 30, 52–53, 59–61, 63, 67–69, 72–73, 78–79, 81, 84–86, 105, 111–113, 153, 167, 171–180, 187–193, 202, 211–212, 214, 225, 227, 313, 431, 448, 465, 467–468 Osteoblasts, 16, 86, 88 Osteoclasts, 197 P Paleoceanography, 64, 257, 265–266 Paleodictyon, 137–141 Parasites, 335, 417–418 Pathological biomineralization, 51 Pectinariid tubes, 474–476 Permanent adhesion, 328, 330 Pinna, 301, 304 Placopecten, 281, 320 Polychaeta, 66 Polyphenol, 53, 192, 257, 260–261, 306, 309 Porifera, 33, 64, 71, 87–88, 103–111, 143–145, 169, 171, 211, 213, 251, 380, 409–415, 428–430, 461 Poriferan collagens, 428–429 Precursor phase, 52 Principles of demineralization, 63–73 Protein acidic, 54, 86–87, 172, 280, 471 capsule, 292, 294 collagenous, 74–75, 85–86, 248–250, 306 elastomeric, 279 fibrillar, 189, 257, 261, 266, 272, 391, 393–394, 401, 403 fibrous, 74, 79, 292, 345, 380 glue, 338, 471, 477, 479 ligament, 282–283 matrix, 68, 85, 295, 385–386, 465, 477–478 resilium, 283 shell, 282
Index sponge, 69, 259 tissue, 292 viral, 85 Protozoa, 34, 41, 54–55, 77, 82, 149, 351–356, 445–452, 455, 459, 461, 509 Pseudochitin, 456–457 Pseudokeratin, 245–246, 259, 261 R Radula, 25–26, 33, 41, 66–67, 78, 111–113, 117, 230 Recombinant proteins, 325 Remineralization, 59–91, 131 Requirements of biomaterials, 17–19 Resilin, 279, 286, 321, 323–326 Resilium, 283–284, 320 Ribbon-like fibers, 291 S Sabellids, 465, 467 Sand dollar, 201–207 Sarostegia, 151–152 Scaffolds, 17, 21, 29, 54, 74, 76, 80, 82–84, 86, 88–90, 103, 113, 118, 153, 155, 170, 172, 195, 246, 249, 252–253, 262, 409–412, 420, 422, 433–434, 495 Scaphechinus mirabilis, 201, 203 Scleroprotein, 246–247, 257, 259–261, 273, 309, 380 Sclerotization, 117, 263, 293–294, 376, 386–387 Sea cucumbers, 329–331, 435 star, 329, 331–333 urchin, 52, 201–203, 328–329, 429, 436 Self-made biomaterials, 445–452, 455–463, 465–479 Serpulids, 465, 467–469, 474 Setae, 65, 380, 391–396, 398–399, 401–403 Shinkaia crosnieri, 391–392 Silica -based biominerals, 25, 28, 40–42 -based tubular formations, 137, 139 Silicase, 69–70 Silicateins, 69, 72, 155, 169–170, 172–173, 179 Silk-based shell, 459–461 Skeletal frameworks, 143, 155–167, 171 Skeleton, 27, 29, 31, 33, 53, 61, 66–67, 71, 74, 79–80, 83, 103–105, 108, 128, 144–145, 147–155, 165–166, 172, 174, 179, 187, 189, 195, 202–203, 207, 245–253, 257–259, 261–265, 272–276, 280, 380, 410–411, 431, 461, 495
Index Solenogaster, 212, 222, 224 Spicule anchoring, 42, 177, 180 ascidian, 30, 211 basal, 89 carbonate, 31, 211, 215 cruciform, 160 fusiform, 31, 215–216 hexactin, 151 larval, 221 molluscs, 211–237 monorhaphis, 172 nudibranch, 31, 217 onchidella, 228–237 siliceous, 31, 39, 71–72, 108, 143, 145, 147–148, 165, 169, 228, 233–236, 461 sponge, 41, 71, 80, 84, 166–168, 175–176, 178, 211, 456, 459, 461 Spiculidendron corallicolum, 461 Spiculogenesis, 166–171 Spines, 40, 77, 160–162, 201–207, 220, 223, 225, 234, 236, 275, 395–398, 445 Sponge-imitating giat foraminifer, 461–463 Sponges calcified, 108 Euplectella, 156, 159 freshwater, 39, 167 glass, 26, 41–42, 53, 63, 71, 80, 83–84, 87–89, 132, 137, 139–140, 143–180, 429, 500 hexactinellid, 139, 145, 148, 166 keratose, 33, 246 lithistid, 144, 148, 167 marine, 33, 42, 69, 103, 110, 145, 246, 250–251, 253, 414, 495 siliceous, 144–145, 167, 171, 178, 461 verongida, 106, 118, 246, 402, 411, 414–415, 495 Spongin, 33, 104, 110, 145, 147, 245–254, 260, 309, 428, 461 Stannophyllum zonarium, 459–461 Strontium-based biominerals, 34 Structure-functional relationships, 134 Sucker disk, 359–362 rings, 374–376 systems, 349, 371, 373–374, 376
569 Suction, 327–328, 336–337, 349, 351–356, 359–362, 365–368, 371–376, 379–388, 391–403 Suctorian protists, 351–352 Swimming scallop, 319–320 T Tanning, 111, 113, 192, 259–261, 265 Tecnin, 456 Temporary adhesion, 328–329 Tentacles, 78, 212, 266, 275, 335, 351–356, 374, 430, 465, 470–471, 473 Testate amoeba, 445–447 Thermophyle microorganisms, 493 Thermotolerant diatoms, 493 Threads, 137–138, 179, 248, 279, 299–313 Tintinnids, 445, 448–450 Tissue engineering, 5, 8, 14, 17, 19, 85, 252–253, 411–413, 416, 422, 430, 433, 496 Titanium-based biominerals, 35–36 Toxicity, 42, 346, 409, 416–422, 470, 488 Transitory adhesion, 328 Trichodina, 359–362 Trichodinids, 359–362 Trityrosine, 292, 323, 325, 382, 384 Tube builders, 465–479 formation mechanisms, 467–470 Tubercles, 206, 215, 217 V Vanadium, 6, 25, 34 Ventral disk, 365–368 Verongida, 103, 106, 118, 245–246, 402, 409, 411, 414–415, 495 Verongula gigantea, 104, 252 W Whelks, 289–296, 335 X Xenophyophores, 450–452, 459 Z Zinc-based biominerals, 36–38
