Advances in Insect Physiology
Volume 37
Advances in Insect Physiology edited by Stephen J. Simpson School of Biological Sciences, The University of Sydney, Sydney, Australia
Je´roˆme Casas Universite´ de Tours, Institut de Recherche en Biologie de l’Insecte UMR, CNRS, Tours, France
Volume 37
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ISBN: 978-0-12-374829-4 ISSN: 0065-2806 For information on all Academic Press publications visit our website at elsevierdirect.com
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Contents Contributors
vii
Preface
ix
Orientation Towards Hosts in Haematophagous Insects: An Integrative Perspective CLAUDIO R. LAZZARI
1
From Sialomes to the Sialoverse: An Insight into Salivary Potion of Blood-Feeding Insects ` JOSE´ M. C. RIBEIRO, BRUNO ARCA
59
The Enemy Within: Interactions Between Tsetse, Trypanosomes and Symbionts DEIRDRE P. WALSHE, CHER PHENG OOI, MICHAEL J. LEHANE, LEE R. HAINES
119
Interactions of Trypanosomatids and Triatomines ¨ NTER A. SCHAUB GU
177
Lyme Disease Spirochete–Tick–Host Interactions KATHARINE R. TYSON, JOSEPH PIESMAN
243
Epidemiological Consequences of the Ecological Physiology of Ticks SARAH E. RANDOLPH
297
Index
341
Preface How do insect vectors of disease find their animal hosts? Once a host is located, how do insects deploy their intricate mouthparts and the extraordinary complexities of salivary chemistry to secure a blood meal, and in so doing cause transmission of disease organisms? What are the critical molecular components that mediate the interactions between insect vectors and disease organisms? How do insect physiology and life history interact with environmental conditions to shape patterns of disease incidence? Given the enormous health and socioeconomic impacts of insect-borne diseases, the study of insect physiology acquires special significance when applied to questions such as these. In addition to reviewing the progress made towards answering these questions, the papers in this special issue of Advances in Insect Physiology give rise to key themes for the future. Hence, the field has benefited enormously from recent advances in molecular biology and protein biochemistry, yet in order to be fully effective, these tools need to be used within the context of a deep understanding of vector physiology, behaviour and ecology. In turn, an understanding of ecophysiology, behaviour and life-history is necessary for explaining and predicting the biogeography and epidemiology of insect vector-borne diseases - especially in a world experiencing global warming, population growth and changing patterns of land use. STEPHEN J. SIMPSON JE´ROˆME CASAS
Contributors Bruno Arca` Department of Structural and Functional Biology, University ‘‘Federico II’’, Naples, Italy; and Parasitology Section, Department of Public Health, University ‘‘La Sapienza’’, Rome, Italy
Lee R. Haines Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom
Claudio R. Lazzari Institut de Recherche sur la Biologie de l’Insecte, UMR 6035 CNRS – Universite´ Franc¸ois Rabelais, Faculte´ des Sciences et Techniques, Av. Monge, Parc Grandmont, 37200 Tours, France
Michael J. Lehane Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom
Cher Pheng Ooi Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom
Joseph Piesman Centers for Disease Control and Prevention, Coordinating Center for Infectious Diseases, National Center for Zoonotic, Vector-Borne and Enteric Diseases, Division of Vector-Borne Infectious Diseases, Fort Collins, Colorado
Sarah E. Randolph Department of Zoology, University of Oxford, Oxford OX1 3PS, United Kingdom
Jose´ M. C. Ribeiro Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, 12735 Twinbrook Parkway room 2E-32D, Rockville, Maryland 20852, USA
Gu¨nter A. Schaub Zoology/Parasitology Group, Ruhr-Universita¨t Bochum, 44780 Bochum, Germany
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Contributors
Katharine R. Tyson Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA
Deirdre P. Walshe Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom
Orientation Towards Hosts in Haematophagous Insects: An Integrative Perspective Claudio R. Lazzari Institut de Recherche sur la Biologie de l’Insecte, UMR 6035 CNRS – Universite´ Franc¸ois Rabelais, Faculte´ des Sciences et Techniques, Av. Monge, Parc Grandmont, 37200 Tours, France
1 Introduction 2 2 Functional neuroanatomy 3 3 A brief history of haematophagy 5 3.1 The relationship between insects and vertebrate hosts 6 3.2 Feeding on blood 6 4 The host signals 7 4.1 Odours 7 4.2 Heat 8 4.3 Water vapour 10 4.4 Visual cues 12 5 Looking for food 14 5.1 Activation 15 5.2 Appetitive search 15 5.3 Host detection 16 5.4 Host finding 16 5.5 Host contact 17 5.6 Host biting 17 5.7 Food recognition and feeding 18 5.8 Leaving the host 20 6 Stimulus propagation and sensory reception 20 7 Orientation mechanisms 23 8 Thermal sensing in kissing bugs 26 9 Sensory parsimony 34 9.1 Parsimonious use of information in blood-sucking insects 34 9.2 Practical consequences 35 10 State-dependency of host-seeking behaviour 36 10.1 The temporal modulation of the response to odours 37 10.2 Maturation and responsiveness 38 10.3 The modulation of host-seeking activity by reproduction 39 10.4 Feeding conditions and host searching 40 11 Why some people are bitten more than others? 41 12 Learning and memory 42 ADVANCES IN INSECT PHYSIOLOGY VOL. 37 ISBN 978-0-12-374829-4 DOI: 10.1016/S0065-2806(09)37001-0
Copyright # 2009 by Elsevier Ltd All rights of reproduction in any form reserved
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CLAUDIO R. LAZZARI
13 Repellents, how they work 42 14 Conclusions and perspectives 43 Acknowledgements 48 References 48
1
Introduction
Neuroethology (‘neuro’ Greek; related to nerve cells, ‘ethos’ Greek; habit or custom) addresses the neural basis of animal behaviour, through an evolutionary and comparative approach. Its main focus is understanding how the central nervous system translates biologically relevant stimuli into behavioural activity (Ewert, 1980). Various notions about the origins and goals of neuroethology exist (Ewert, 1980; Hoyle, 1984; Bullock, 1990; Pfluger and Menzel, 1999). However, the main questions addressed in this area of study, by means of experimental exploration, are as follows (Ewert, 1980): (1) Which sensory processes are responsible for distinguishing between behaviourally relevant and irrelevant stimuli? (2) How are signals localized in space and time? (3) How is information acquired, stored and recalled? (4) What is the neurophysiological basis for the motivation of a behavioural pattern? (5) How is behaviour coordinated and controlled by the central nervous system? (6) How is behaviour ontogeny related to neuronal mechanisms? Insect neuroethology is a well-developed field, thanks to advances in the development of several model systems. Studies on some of the major topics, such as wind-triggered escape, the recognition of acoustic signals, learning and memory and others, have provided considerable insight into how the nervous system controls adaptive behavioural responses in insects. The particular species chosen for detailed analysis in such studies include honeybees, cockroaches, crickets, flies and a few others. However, no blood-sucking insects are included in this ‘‘select’’ group, even though some of them are classical models in insect physiology (e.g. Rhodnius prolixus) or the subject of intense study due to their impact on human health (mosquitoes). This does not mean that we lack information on their behaviour and neurobiology. On the contrary, important aspects of their behaviour, sensory physiology and functional neuroanatomy have been extensively studied. Nevertheless, very few studies have analysed this aspects with an integrative view in haematophagous insects.
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In this chapter, I summarize the current understanding of the physiological mechanisms that underlie host-seeking in blood-sucking behaviour, with an integrative view, to elucidate some of the issues described above.
2
Functional neuroanatomy
To understand the neurobiological basis of behaviour, we firstly need to understand the organization of the nervous system. Functional neuroanatomy provides the basis for focusing physiological studies on the neural elements associated to a particular behaviour. A series of detailed studies have been published over the past few years on the functional neuroanatomy of mosquitoes, particularly Aedes aegypti and Anopheles gambiae. These studies have revealed the neural architecture of the olfactory brain and the organization of sensory pathways from the antennae, maxillary palps and labium (Anton, 1996; Anton et al., 2003; Anton and Rospars, 2004; Ignell and Hansson, 2005; Ignell et al., 2005; Ghaninia et al., 2007a,b; Siju et al., 2008). Some of the most significant findings of these studies concerning these malaria and yellow fever mosquitoes (A. gambiae and A. aegypti, respectively) are described below: In A. gambiae, antibody labelling and subsequent three-dimensional reconstructions of the antennal lobes showed that males have 61 glomerular neuropils and females have 60. The size of the antennal lobe and of individual glomeruli was also tested for sexual dimorphism (Ghaninia et al., 2007b). In A. aegypti, sexual dimorphism has been demonstrated both for the number of total glomeruli (49 in males and 50 in females) and size of certain glomeruli (Ignell et al., 2005). Maxillary palp projections in A. aegypti are restricted to two posteromedial glomeruli, which do not receive antennal afferents. These include nerve projections from carbon dioxide receptors, which project to a single glomerulus (Anton, 1996; Anton et al., 2003). Five non-overlapping projection zones were identified within the antennal lobe of A. gambiae, with one zone receiving input exclusively from maxillary palp sensilla and two zones each receiving input exclusively from trichoid or grooved-peg antennal sensilla (Anton and Rospars, 2004). Extensive serotonergic neurohemal plexi have been observed in the peripheral chemosensory organs of A. aegypti and A. gambiae, that is in the antenna, the maxillary palp and the labium, suggesting a potential role of serotonin as a neuromodulator in the chemosensory system (Siju et al., 2008).
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CLAUDIO R. LAZZARI
The central projections of the contact chemoreceptors in the labium and cibarium of A. gambiae and A. aegypti have been described in detail (Ignell and Hansson, 2005). Notably, despite the differences in feeding habits between male and female mosquitoes, sexual dimorphism in the olfactory brain is not very marked, at least in terms of anatomical differences. Given the specificity and sensitivity of sensory organs dedicated to perceiving specific host-associated cues in females but not in males, one would expect a greater divergence between the sexes, but this does not seem to be the case. This could be related to the fact that both sexes feed on nectar, with females additionally sucking on blood. Even assuming that both males and females detect the same plant-associated volatiles and that certain chemical cues are common to both plants and animals (Syed and Leal, 2007), one would still expect a greater difference between the olfactory brains of males and females. It is possible that the glomeruli responsible for processing host-specific signals in females are, in males, involved in processing signals specific to this sex. This would thus allow for physiological dimorphism that it is not anatomically visible. Neuroanatomical studies of Hemiptera have focused on bedbugs and kissing bugs. Whereas studies on bedbugs have been mostly limited to the general anatomy of the central nervous system (Singh et al., 1996), more complete sets of data are available on the neuroanatomy of kissing bugs. Indeed, following on from the classical studies of V.B. Wigglesworth on the histology of the peripheral and central nervous systems in R. prolixus (Wigglesworth, 1953, 1959a,b), further studies described the general anatomy of their central nervous system, the organization of antennal lobes and the ocellar and mechanosensory pathways (Barth, 1952, 1976; Insausti, 1994; Insausti and Lazzari, 1996, 2000a; Barrozo et al., 2009). Recent studies on triatomines suggest that there is no sexual dimorphism in the organization of the antennal lobes (Barrozo et al., 2009). This is not surprising, given that, in contrast to mosquitoes, both sexes feed on vertebrate blood, and no marked difference has been found in the number and type of sensory organs between males and females. Another interesting finding, meriting further analysis, is the fact that antennal inputs do not only project into the deutocerebrum, but descend further along the ventral nervous chain, synapsing nervous elements in the suboesophageal, porthoracic and posterior (mesoþmethathoracicþabdominals) ganglia (Barrozo et al., 2009). These kind of direct connections had been previously described for ocellar interneurons and cephalic mechanoreceptors in kissing bugs (Insausti and Lazzari, 1996, 2000a), thought to be associated with the direct control of motor patterns. These neuroanatomical findings provide a good basis for further analyses of the central processing of sensory signals. Such analyses should address haematophagy-related traits, such as the representation of host odours in the olfactory
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5
brain of blood-sucking insects or the potential involvement of specific glumeruli in host recognition. Haematophagous insects may be good models for the investigation of more general aspects, such as the mechanisms allowing multimodality to be represented in the insect brain. The central processing of odours is a major aspect of insect neurobiology and is studied intensively in non-haematophagous experimental models (e.g. honeybees, moths, locusts and Drosophila). Such studies require the use of electrophysiological and optophysiological (most frequently calcium imaging) methods that need to be supported by accurate neuroanatomical data (Galizia et al., 1997, 1999a,b; Galizia and Menzel, 2000; Zube et al., 2008). The brain neuroanatomy of mosquitoes is the best known of the bloodsucking insects, making them the most suitable model for studying the central processing of sensory information. However, in vivo optical recordings could be technically difficult due to their small size. Kissing bugs may thus be a good alternative model because of their larger size, robustness and easy access to their brain and rest of the nervous system. Even though mosquitoes and bugs differ considerably in terms of phylogeny, they exploit the same source of food (vertebrates) and respond to similar spectra of signals.
3
A brief history of haematophagy
Haematophagy is believed to have evolved independently many times in insects (Lehane, 2005). Blood provides a rich and usually sterile source of many nutrients needed for survival and reproduction. The circulation of blood within vessels hidden under the skin of moving animals, however, makes it difficult to obtain. Additionally, hosts exhibit defensive behaviours to avoid being bitten or may even play the dual role of prey and predator at the same time. The ability to feed on blood has thus evolved under the control of selective pressures, with blood-sucking insects able to detect and locate a potential host, locate in space, identify the best site to land on, locate the blood vessels, pierce the skin and find the blood. These processes must all be accomplished minimizing the risk of being detected and killed by the antiparasitic behaviour of the host or of being predated on by the host itself. With more than 14,000 species and 400 genera of arthropods feeding on blood, haematophagy would appear to confer certain advantages (Ribeiro, 1995; Lehane, 2005). The ability to feed on the blood of vertebrate hosts may have developed through one of two possible routes. The first possibility involves a gradually closer association with vertebrates, first to their nest, and later to their body, leading to morphological changes to allow the insects to feed on their hosts’ blood. The second possibility is based on pre-adaptation, whereby insects were able to pierce animal or vegetal tissues and feed on fluids before developing the ability to suck blood (Lehane, 2005).
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3.1
CLAUDIO R. LAZZARI THE RELATIONSHIP BETWEEN INSECTS AND VERTEBRATE HOSTS
Blood-sucking arthropods and their vertebrate hosts exhibit various degrees of association. In some cases, the association between the host and haematophagous insect is limited to the nutritional aspect, with the arthropod contacting the vertebrate just briefly, for the time necessary to feed on its blood. Each partner thus lives independently, even exploiting different habitats. This is seen, for instance, in the relationship between haematophagous flies and vertebrate hosts, and for many mosquito species. Some insect–host pairs share the same habitat, usually the burrow or nest of the vertebrate, or the house of human hosts. Blood-sucking Hemiptera (kissing bugs, bed bugs) are typical examples of insects that occupy the habitat of the host, exploiting the habitat’s protective aspect, its microclimate and the ready access to food sources. The closest relationships between insects and their hosts are found when the insect becomes a true ectoparasite, living on the host’s body. This is observed for fleas, lice and ticks, all three displaying different extents of association with their host. Fleas leave their host to reproduce and their larvae obtain blood from vertebrate only indirectly, by eating the excrement of adults; lice reproduce on their hosts, but may move from one host to another; and ticks only leave their hosts for moulting, remaining fixed to the host skin by their mouthpieces the rest of the time. The sensory machinery used to gather information required to identify a host, and to locate it in space and time, is determined by the nature of the insect–host relationship. The sensory organs of blood-sucking insects involved in receiving signal from their hosts exhibit patterns of organization typically seen in most insects. The structure of haematophagous sensilla does not seem to particularly differ between the species, but the number present does appear to be related to the nature of the relationship of each species with their host: the closer the relationship with their host, the fewer sensory organs are present (Chapman, 1982; Lehane, 2005). Thus, insects closely associated to the host have a smaller number of sensory organs than those that search for a potential host across a large area: lice have 10 sensory organs; sensilla, 20; fleas, about 50; bedbugs, 56; kissing bugs, 2900; and stable flies, nearly 5000 (Lehane, 2005). 3.2
FEEDING ON BLOOD
A blood-based regime offers a number of advantages. Blood is rich in nutrients, sources of blood can be found almost everywhere and, except for the incidental presence of parasites, it is otherwise sterile. The exploitation of vertebrate blood as food has led to many specific morphological, physiological and behavioural adaptations. To feed on blood, a haematophagous insect needs to be able to identify a vertebrate source, approach it at the right moment and locate a site on
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the host skin where the blood supply is assured; it then needs to cut or pierce the skin, locate a vessel hidden under the skin, identify the fluid, suck up a sufficient amount of blood as quickly as possible, avoiding the coagulation of blood within the vessel or the mouthparts, and then escape, carrying the extra weight. These actions all require the input of multimodal signals from the host, environment and within the arthropod itself.
4
The host signals
Vertebrates, particularly endothermic vertebrates, emit a large number of informational cues of different types. Blood-sucking insects can then exploit these signals, using their various sensory systems. The human skin emits around 350 volatile compounds potentially exploitable by haematophagous insects (Bernier et al., 2000); many other compounds are released with breathing. Vertebrates able to physiologically or behaviourally regulate the temperature of their bodies (e.g. by basking) also act as sources of heat. When active, their movements and contrast against the background may be detected by insects’ visual systems, and the vibrations produced may be detected by the mechanoreceptive organs of insects. Given that many of these signals (e.g. heat and CO2) are emitted by most, if not all, warm-blooded vertebrates, we would expect them to be general cues that are perceived by most haematophagous insects. The way they use this information, however, may differ from one species to another. As observed for other insects, the different types of chemo-, mechano-, thermoand hygrosensitive sensilla are mainly concentrated on the antennae. Antennae are not the only bearers of sensilla (sensilla are present in other regions of the body as well), nor is their function limited to providing support. The antennae have an important role in the active gathering of sensory information. The visual organs are also involved in the detection of moving hosts by some insects. Compound eyes are the main organs dedicated to detailed analysis of the contrast, form and colour of objects in the visual field. Simple eyes or ocelli are probably not involved at all, but cannot be excluded as potential inputs of host-related information given the wide variety of functions that these organs are involved in, in other species. 4.1
ODOURS
Virtually every haematophagous insect modifies its behaviour in the presence of carbon dioxide, either increasing their activity or responsiveness to other stimuli or orienting itself towards the source (Guerenstein and Hildebrand, 2008). Carbon dioxide is produced intermittently (animal breath) and, as with other odours, travels through the air in discrete packets interspersed with clean air,
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being detected as intermittent stimuli. Thus, mosquitoes only orientate towards a source of CO2 that emits an intermittent (pulsed) signal (Geier et al., 1999a). In contrast, Triatoma infestans is attracted by a CO2 source only under continuous or low-rate pulsed stimulation, and is repelled by CO2 signals delivered at high frequency (Barrozo and Lazzari, 2006). This difference between two haematophagous species exploiting the same cue and the same CO2 sources may be explained by differently adapted strategies to approach their hosts. For a flying insect, such as the mosquito, the frequency of encountering CO2 packets can be relatively high, with an increased rate with proximity to the emitting source. Triatomines, in contrast, search for food by walking in a relatively closed habitat, in which air currents have a smaller effect on the propagation of CO2 than in open spaces. Additionally, they feed mostly during the night on sleeping hosts, which do not generate air turbulence by movement. Thus, odours may disperse more homogeneously and the rate of release is more stable in such environments. Despite being sufficient to attract most blood-sucking insects, CO2 is not an indispensable stimulus for triatomines (Nu´n˜ez, 1982; Taneja and Guerin, 1995; Barrozo and Lazzari, 2004a), or for some mosquito species (Gibson and Torr, 1999; Bosch et al., 2000; Bernier et al., 2003; Smallegange et al., 2005). Previous studies on tsetse flies have demonstrated that CO2, due to its high diffusion rate, is only useful for insects when they are relatively close to the host (Torr, 1990; Leak, 1999). As well as responding to CO2, different haematophagous insects appear to respond to similar or the same host odorants. Such odorants include nonanal (triatomines: Guerenstein and Guerin, 2001; mosquitoes: Du and Millar, 1999), lactic acid (triatomines: Barrozo and Lazzari, 2004b; mosquitoes: e.g. Geier et al., 1999b), ammonia (triatomines: Taneja and Guerin, 1997; mosquitoes: e.g. Meijerink et al., 2001), 1-octen-3-ol (triatomines: Barrozo and Lazzari, 2004b; mosquitoes: Takken and Knols, 1999), short-chain carboxylic acids such as butyric acid (triatomines: Guerenstein and Guerin, 2001; Barrozo and Lazzari, 2004a; mosquitoes: e.g. Pappenberger et al., 1996), C4–5 aliphatic amines such as isopentylamine (triatomines: Diehl et al., 2003; mosquitoes: Pappenberger et al., 1996) and terpenes including a(þ) pinene (triatomines: Guerenstein, 1999; mosquitoes: Bowen, 1992). 4.2
HEAT
It is well established that blood-sucking insects respond to heat emanated from the body of warm-blooded vertebrates. The mechanisms underlying their use of thermal information to find food have received much less attention than the response to odours. In-depth studies have been carried out investigating the morphology and physiology of thermosensilla in insects (Altner et al., 1981; McIver and Siemicki, 1985; Altner and Loftus, 1985; Gingl and Tichy, 2001; Tichy et al., 2008), including those of haematophagous insects (McIver and
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Siemicki, 1985; Gingl et al., 2005). However, the use of thermal information by blood-sucking insects has been analysed only partially or not at all in most species. The transfer of heat between two bodies at different temperatures may occur through three different physical processes, conduction, convection and radiation (Fig. 1). When heat is conducted, atoms are energized, increasing their vibration rate. This causes neighbouring atoms to vibrate more vigorously, and the signal thus spreads through the material. In our case, the material is the air, and the result is the formation of a temperature gradient around the body of a host. This gradient may then be used as an orientation cue by the insects. Convection concerns heat exchange with a moving fluid. When the fluid (air) is heated by conduction from the body, its temperature increases and it becomes less dense. It then starts to ascend away from the heat source. When its temperature drops back, its density increases and it descends. For some haematophagous insects, convection is not only a matter of heat transfer, but also of the production of ascending airstreams that transport host odours. Mosquitoes, for example, seem to make use of convection currents to approach a host (Lehane, 2005). Convection currents are only useful as a cue when approaching from just above a host.
Conduction Heat source
Convection
Heat source
Heat source Radiation
FIG. 1 Diagrams representing the three mechanisms for heat exchange. Conduction: atoms are energized and vibration increases; vibration spreads through the material. Convection: air is heated and rises, and descends when cool. The air movement from a host transports odorants that can be detected by haematophagous insects. Radiation: any object at a temperature higher than 0 K emits infrared radiation of a wavelength dependent on its temperature. Radiation spreads in all directions and is not affected by air movements.
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The third mechanism of heat transfer, radiation, involves the emission and absorption of radiant heat at wavelengths corresponding to the infrared region of the electromagnetic spectrum. This exchange does not require a conducting material or the movement of fluid. Any object at a temperature above absolute zero (0 K or 273 C) emits infrared radiation (IR) of a wavelength corresponding to its temperature. The perception of radiant heat has only been observed for a few groups of animals and in insects belonging to the orders Coleoptera and Hemiptera. For a blood-sucking insect to detect radiation, it needs to be able to assess, from any relative position, the heat emitted by a potential host, without the effects of wind, which can disrupt conduction gradients and convective currents. Experimental evidence has been obtained over many years for the role of heat in the orientation of blood-sucking insects, for example in mosquitoes (Peterson and Brown, 1951; Khan et al., 1966), bed bugs (Usinger, 1966; Reinhardt and Siva-Jothy, 2007), kissing bugs (Wigglesworth and Gillet, 1934a,b; Nicolle and Mathis, 1941) and lice (Wigglesworth, 1941). However, since the initial discovery of a sensory basis for host orientation, relatively few studies have been carried out on this topic. The responses to radiant heat and convection currents were first studied more than 50 years ago in mosquitoes (Peterson and Brown, 1951). When an infrared transparent filter was placed between the heat source and the insects, the response of the insects was weaker than in the absence of the filter. It was thus concluded that convection currents, but not radiant heat, constitute the main thermal cue for mosquitoes. Further work reinforced the notion of ascending convection currents acting as carriers of water vapour and odours (Eiras and Jepson, 1991, 1994). Thus, mosquitoes flying over a host perceive ascending air currents that are warm, humid and charged with host odours. Heat plays a major role in the orientation of kissing bugs (Heteroptera: Reduviidae). The first experiments on the responses of these bugs were conducted on R. prolixus by Wigglesworth and Gillet (1934a,b). They showed that bugs orientate themselves towards a heat source by a mechanism of telotaxis. When the bugs reach a distance of 1–2 cm from the source, they extend their proboscis and switch to tropotaxis (Fig. 2). 4.3
WATER VAPOUR
Water vapour has been suggested to play a role in the location of vertebrate hosts by blood-sucking insects (Bernard, 1974; Altner and Loftus, 1985). However, most of the studies were restricted to examining the electrophysiological or morphological characteristics of sensory organs. Given the small number of behavioural studies on this topic, the role of water vapour in shortand long-range orientation remains unclear in most cases. The mosquito A. aegypti moves upwind towards humid and warm airstreams in conditions
ORIENTATION TOWARDS HOSTS IN HAEMATOPHAGOUS INSECTS A
5 cm C
5 cm
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B
5 cm D
5 cm
FIG. 2 Trajectories of unilaterally antennectomized and control insects towards a thermal source in the absence of sensory cues other than heat. (A) An intact insect in a big size arena and (B) in a small one. (C) Right antennectomized insect in a big size arena and (D) left antennectomized bug in a small one. Note that the insects approached the source using a direct path, but when close to the source, antennectomized bugs deviated in the direction of the intact antennae and missed the goal. Two orientation mechanisms seem to be involved: telotaxis up to 1.5–2 cm from the source and tropotaxis when in close proximity, at the same time as the proboscis is extended (not shown). The switch from telo- to tropotaxis occurred before any physical contact with the heat source. This and the fact that insects were not exposed to either visual or chemical information suggest that thermal cues provided enough information for both the intact and unilaterally antennectomized bugs to estimate their position relative to the source (modified from Flores and Lazzari, 1996).
of water deprivation; Culex quinquefasciatus also seems to use water vapour as a cue to orientate itself towards human hosts (Mboera et al., 1998). In kissing bugs, the presence of a humid source alone is sufficient to elicit short-range, but not long-range, orientation in T. infestans. The humidity of an airstream has no visible effect on the orientation behaviour of this species, regardless of the physiological state of the bugs (Barrozo et al., 2003). Interestingly, the response to thermal sources seems to be enhanced by moisture, leading to stronger responses at greater distances (Barrozo et al., 2003). This enhancement is probably due to a combination of factors: (1) an increased amount of heat reaching the insect thermoreceptors, the enthalpy of moist air being greater than that of dry air; (2) bimodal convergence of peripheral inputs
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onto thermosensitive cells, the activity of which varies not only as a function of air temperature, but also relative humidity (Altner and Loftus, 1985); (3) the potential bimodal convergence of thermosensitive and hygrosensitive inputs in the central nervous system (Horn, 1985). 4.4
VISUAL CUES
Insects have a highly developed visual system, composed of simple and compound eyes (Warrant and Nilsson, 2006). Two types of simple eye, stemmata and ocelli, are found in larvae of endopterygota and most adult insects, respectively. Compound eyes are present in adults of all groups and in the immature instars of exopterygota. Each type of eye fulfils different functions at different phases of the insect’s life. Stemmata lie laterally in the head of larvae. The number of stemmata varies between species. The capacity of these structures to gather visual information – determining changes in light intensity, shape detection and spectral discrimination – is also variable (Gilbert, 1994). Ocelli are not able to form images; indeed the light entering their optical apparatus is focused beyond the retina, eliminating any structural detail of the visual panorama. Each ocellar interneuron gathers information from a large number of photoreceptors, that is from large areas of the visual field. In some insects, ocellar photoreceptors are sensitive to either green or UV light. Ocellar inputs are transmitted through giant descending neurons, which form synapses at different nervous centres in the brain and in the ventral nervous chain. In most insects, they function as horizon detectors. The characteristics of this system are well suited to its role in horizon detection (the green earth/UV sky boundary not interrupted by structural details), with the rapid conduction of nervous signals directly to motor centres allowing rapid flight stabilization of the insect (Mizunami, 1995). Compound eyes constitute the most developed and sophisticated sensory system in insects. They are composed of hundreds or thousands of visual units (ommatidia) and are capable of transmitting detailed spatial information, object shapes and, in many cases, colour and polarization vision (Stavenga and Hardie, 1989). Whereas other sensory systems exhibit particular characteristics adapted to haematophagy – for example numbers of thermoreceptors or the specificity of chemoreceptors – the visual system of blood-sucking insects does not seem to be related to their particular lifestyle. Tsetse flies, for instance, detect and follow potential hosts visually (Brady, 1972a; Gibson, 1992; Leak, 1999; Lehane, 2005), but their compound eyes do not display any particular specialized features. The ocelli and compound eyes of kissing bugs are both involved in negative phototaxis (Lazzari et al., 1998), but this seems to be related more closely to the central processing of information than to structural or physiological adaptations in the periphery. Vision is directly involved in host location in many diurnal haematophagous insects and plays an indirect role in nocturnal insects. Compound eyes are characterized by their high sensitivity to the movement of objects (Stavenga
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and Hardie, 1989). Tsetse flies exploit this capacity to detect and react to the moving elements in their visual field, exhibiting a stronger response to movement with increasing starvation time (Brady, 1972b; Torr, 1988). The detection of movement also plays an important role in the orientation of haematophagous insects when they follow an odour plume, notably during flight. As with other insects in flight, their small mass allows them to be easily displaced from their intended path by the wind (Fig. 3). Additionally, being submerged in a moving medium and without any contact with the substrate, flying insects do not extract reliable information about their displacement from mechanoreceptors, but from visual inputs. Retinal image motion is elicited when a moving object crosses the visual field (object motion); even if the outside world is stationary, there is a continuous image flow on the retina when the insect moves. The optic flow is a key source of information about the three-dimensional layout of the environment as well as the path and speed of displacement (Egelhaaf, 2006). It is thus the optic flow of stationary objects
Active flight direction Actual flight path
Translational flow field
Wind Rotational flow field
FIG. 3 Flight direction of insects in the presence of wind blowing from the side. The actual path depends on both the forward impulse of the flight motor system and the wind force. Insects make use of the movement of objects over the retina to extract information about the real displacement. During linear translation, objects move differently depending on their position on the retina, relative to the direction of displacement. Objects in the regions of the visual field perpendicular to the translation direction move faster than those in the direction of flight. If the insect turns, objects move uniformly over the whole retina (i.e. at the same speed). By distinguishing translational from rotational components in the flow field, they may compensate for rotational movements (i.e. optomotor reaction) to correct for deviations in their flight path. In the example (right bottom), the animal turned clockwise and the retinal image moved in the opposite direction. Thick arrows over the insect heads indicate the direction of the movement, and thin arrows indicate the apparent movement of the objects in the visual field.
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through their visual field which allows the insect to determine flight direction and speed, and to avoid collision. When insects fly towards a source of odour, the motor responses to visual and olfactory stimuli are superimposed. Olfactorymediated changes in muscle mechanics, wing kinematics and aerodynamics are somehow gated by visual feedback in a context-dependent manner to maintain a constant heading (Frye, 2007). The overall concept of such a system may also explain, for instance, how mechanosensory, chemosensory and visual information combine to allow tsetse flies and other haematophagous insects to track an odour source (Colvin et al., 1989; Hardie et al., 2001; see also Lehane, 2005). Compound eyes undergo dynamic changes in the distribution of pigment and in the position of photosensitive regions of the visual unit, the rhabdom. These changes allow the photon flux reaching the rhabdom to be controlled, enabling the eye to operate across a wide range of light intensities, particularly in crepuscular and nocturnal insects. Nocturnal mosquitoes and kissing bugs possess compound eyes capable of adjusting both their absolute sensitivity and spatial resolution (Land et al., 1999; Reisenman et al., 2002). During the day, their sensitivity to light is lower, but spatial resolution higher, than during the night. During the dark period, the eye becomes more sensitive, but sacrifices spatial resolution. These changes are linked to daily variation in the behavioural sensitivity to light, and, in kissing bugs at least, are under the dual control of circadian clocks and environmental light intensity (Reisenman et al., 1998, 2002). Several species of blood-sucking insects respond to the colour of objects by approaching or landing on them. This behaviour has been interpreted as evidence of colour vision and is exploited to attract them to targets impregnated with insecticides (Lehane, 2005). The range of wavelengths to which insects are sensitive varies with species. A common belief, originating from early classical studies of honeybees, is that insects are blind to red, or more precisely, that they are not sensitive to wavelengths of around 700 nm. This is true for honeybees and certain other insects, but not for all insects (Briscoe and Chittka, 2001). Indeed, spectral sensitivity, reminiscent of the capacity to distinguish between colours, has been modelled by the selective pressures acting on each species. The spectral sensitivity of the haematophagous insect Aedes aegypty ranges from 327 (UV) to 621 nm (orange) (Muir et al., 1992), whereas that of the kissing bug T. infestans ranges from 357 (UV) to 695 nm (red) (Reisenman et al., 2000; Reisenman and Lazzari, 2006).
5
Looking for food
As described above, various types of information are used by haematophagous insects to detect, localize and recognize a potential host. Each step of the feeding behaviour of blood-sucking insects may be associated with a different strategy used to acquire the relevant information and with specific mechanisms
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allowing the insect to reach its food source. The strategies used to acquire information consist of particular behavioural patterns guiding the activity and displacements of the insect and are under the temporal control of endogenous clocks. The mechanisms involved determine which parameter of a given stimulus the insect responds to and underlie the way insect behaviour is then modified. Different phases involved in location of a host by haematophagous insects have been described in several studies. Most of the phases identified were defined according to the particular behavioural characteristics of the species studied. However, the same or similar general components can be recognized across different groups of blood-sucking insects, particularly in Diptera. I will describe these phases below, based on a modified version of the model proposed by Lehane (2005), taking into account recent findings from different groups of haematophagous insects. These different phases are thus referred to as: activation, appetitive search, host detection, host finding and host contact. 5.1
ACTIVATION
As in other animals, the daily life of blood-sucking insects is organized as a function of time and is controlled by circadian clocks (Barrozo et al., 2004b). When hosts are less active or the environmental conditions become adequate for food search, the insects start moving spontaneously. This activity may be restricted to just one part of the day or split into two periods (bimodal daily activity) (Brady, 1972c; Lazzari, 1992; Barrozo et al., 2004b). 5.2
APPETITIVE SEARCH
As the spontaneous activity increases and the insect starts walking or flying spontaneously, it leaves its initial refuge and moves over a progressively larger area. The non-oriented movements subside, to give way to movements that enhance the probability of the insect encountering signs that indicate the proximity of a host. This active behaviour may last for the whole active period (or periods, if bimodal activity), as observed for tsetse flies (Barrozo et al., 2004b), or during only a part of the daily activity period. Kissing bugs, for example, only devote their first period of bimodal activity to food search, whereas the second activity period is concerned with the return to their refuge (Lazzari, 1992; Lorenzo and Lazzari, 1998; Barrozo et al., 2004b). Bugs experience their maximal motivation to feed and exhibit their strongest responses to host-associated stimuli at the start of the night (Barrozo et al., 2004a; Bodin et al., 2008). In the presence of air currents, insects orientate their movements according the direction of the wind to optimize their search efforts (Fig. 4). If the wind direction is fairly constant (not differing by more than 30 in either direction),
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CLAUDIO R. LAZZARI <30°
>30°
Upwind
Crosswind
Downwind
FIG. 4 During the exploratory phase of appetitive behaviour, insects need to adjust the direction of their displacement relative to the wind to increase their chance of encountering host-derived cues transported by air currents. For wind coming always from the same direction or shifting within a range of less than 30 , the most efficient way to increase the probability of encountering a stimulus is moving crosswind. When fluctuations in wind direction exceed 30 on both sides, the optimal strategy involves moving either upwind or downwind (Sabelis and Schippers, 1984; Dusenbery, 1990).
the most effective strategy is to move crosswind; however, for conditions in which changes in wind direction are greater than 30 , insects are more likely to encounter a chemical cue transported by the wind by moving either upwind or downwind (Sabelis and Schippers, 1984; Dusenbery, 1990). 5.3
HOST DETECTION
The detection of signals revealing the presence of a host may not be informative enough to elucidate the direction of the source. In such cases, the signal may exert an effect on the insect’s movements, enhancing the insect’s general locomotion activity or making the trajectory more or less direct. By moving faster and scanning a wider area, the insect eventually encounters a stimulus or combination of stimuli able to direct it towards the source and to determine whether the sensory signals detected belong to an adequate host (host selection or recognition). 5.4
HOST FINDING
As mentioned above, orientation towards a host is guided by direct and indirect cues (i.e. not associated to the host) and is mediated by a combination of kinetic and directional mechanisms. Orientation towards a distant host mostly relies on the combination of multimodal information. When host-associated odours are dispersed by the wind, they may reach blood-sucking insects looking for food and, if intense enough, may stimulate specific olfactory receptors. The chemical gradient established between the host and the insect is usually neither strong nor
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stable enough to guide the insect as far as the odour source itself. Nevertheless, air currents loaded with host odours may provide insects with the information they need to locate a potential food source. Indeed, haematophagous insects, as observed for other insects, follow odour-laden airstreams moving upwind (odour-triggered anemotaxis). The orientation of insects following odour plumes has been analysed in depth, for moths tracking pheromones (e.g. Carde´, 1996). Available data on blood-sucking insects mostly concern tsetse flies, which perform odour-triggered anemotaxis (Warnes, 1990; Lehane, 2005). This orienting mechanism depends on wind direction and insect behaviour is subject to modulation by chemical stimulation. Even though airstreams provide directional information, neither wind nor odours provide signals precise enough for the insect to maintain a straight trajectory to the odour source. Integration of a third system is thus required, that is vision. The visual flow field helps by compensating for any rotational movement during displacements of the insect, allowing only translational components (Fig. 3). This is particularly important for flying insects, which lack any contact with a solid substrate and need to keep their path suspended in the air, at the same time being subjected to turbulence that causes them to deviate from their intended trajectories. The interplay of all these components, together with modulation of the tendency to turn (klinotaxis) with the odour concentration within an odour plume and upon intermittent contact with pulsed odours in turbulent air, gives rise to characteristic zigzagging trajectories during displacement towards the origin of the odour (Mafra-Neto and Carde´, 1994; Carde´, 1996). 5.5
HOST CONTACT
During the final approach to the host, odours gradually become more concentrated and chemical gradients are more easily perceived. These cues thus become more useful for directional orientation. At the same time, air currents and anemotaxis becomes less important and other signals, such as visual contrast, colour or movement, and heat and water vapour, can be perceived, bringing the insect in to land or into contact with the host body. Some haematophagous species exhibit characteristic preferences for biting at particular sites on the host body (Lehane, 2005), and orientate themselves using specific kairomones produced locally by the animal itself or by micro-organisms living in the preferred part of the body (Knols et al., 1994; De Jong and Knols, 1995). Arrival at these sites is the final step in the approach to the host, usually taking place before any physical contact is established. 5.6
HOST BITING
Most studies on food searching in haematophagous insects have focused on how the insects detect and follow host-emitted signals to find their food source. However, this is only part of the challenge faced by insects feeding on blood.
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These insects confront another, largely neglected, problem: the localization of blood vessels. Once in contact with a host, the insect must find its food circulating in vessels below the skin surface. Regardless of the feeding strategy used (solenophagy or telmophagy), insects need to obtain blood as quickly as possible to avoid detection by the host. Two possible approaches can be envisaged: biting repeatedly at random until a vessel is located by chance or confining bites to the most irrigated areas. Biting at random sites does not require any particular ability to detect vessels. However, the insect runs a higher risk of being detected by the host if the host is repeatedly bitten. This is particularly relevant for telmophagous insects, which cause skin lacerations and pain, but also applies in the case of solenophagous insects, which cannulate blood vessels with their piercing mouthparts. A study by Ferreira et al. (2007) suggested that changes in temperature over the host skin, due to variation in irrigation, may help blood-sucking insects to locate blood vessels. The authors analysed the distribution of bites over the inner face of a rabbit ear. Most bites occurred over or close to blood vessels. The authors then tested the biting activity of bugs over a surface maintained at a given temperature, crossed by a linear heat source, which was slightly warmer than the background temperature. In this experiment, most bites were directed to the linear heat source (Ferreira et al., 2007). In both the living host and artificial model, the insects extended their proboscis or rostrum directly to the warmest place. This suggests that the signal used by the insects for directing bites is perceived and evaluated before any physical contact between the rostrum and the host skin. The crucial role of the antennae in the detection of heat emanated by the host suggests that antennal thermoreceptors, rather than rostral thermoreceptors, are involved in this process. Bugs with one antenna removed systematically miss the warmest area, directing their bites to the side of the remaining antenna. This is a typical behaviour, indicative of the tropotactic use of sensory information. When both antennae were removed, no response could be evoked, suggesting that only antennal thermoreceptors are involved in guiding biting (Ferreira et al., 2007). 5.7
FOOD RECOGNITION AND FEEDING
Once the skin is pierced, haematophagous insects start a probing phase, in which stylets are moved inside the skin to pierce a vessel, usually a venule. Having located the circulating fluid, the insect starts sucking it through the alimentary channel. Sensory receptors located near the pharynx recognize the blood through the detection of its key properties (Friend and Smith, 1977). Some haematophagous insects are sensitive to ATP and other adenine nucleotides and related compounds (e.g. GTP and GDP). These factors differ in their phagostimulant power (Galun and Margalit, 1970; Friend and Smith, 1975; Friend and Smith, 1977; Galun et al., 1988). For the sandfly Lutzomyia longipalpis and certain anopheline mosquitoes, the most important feature seems to be the tonicity of the solution, with no effect of ATP on feeding in these insects
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(Lehane, 2005). This has also been observed for T. infestans, which is particularly sensitive to food osmolarity (Guerenstein and Nu´n˜ez, 1994). The use of phagostimulant compounds or osmolarity to trigger gorging seems to be dependent on thresholds in some species. For example, R. prolixus shows an all-ornothing gorging response to phagostimulants, which switches after a long starvation period to a graduated response to food osmolarity, similar to that seen in T. infestans (Guerenstein and Nu´n˜ez, 1994). The comparison of the strategies adopted by these two closely related triatomines is particularly interesting because their differences in feeding behaviour may be related to the type of habitat and host exploited by each species. Wild R. prolixus associates with sylvatic hosts, whereas T. infestans almost exclusively targets human dwellings. Access to a potential host is much less predictable in wild habitats than in anthropic habitats. Thus, less selective strategies and partial meals may be advantageous for survival in areas of permanent proximity to potential food sources (e.g. T. infestans) or during prolonged periods of food deprivation (e.g. the long starvation periods of R. prolixus); however, when food sources are less predictable, it may be more advantageous to be selective and to have large meals (e.g. the all-or-nothing R. prolixus response). The sensory basis for the perception of phagostimulants and osmorality seems to differ between different blood-sucking species. In mosquitoes, phagostimulants activate receptors located in the mouthparts (Werner-Reiss et al., 1999a,b). In triatomines, although both the maxillae and mandibles are innervated, their sensory nerve endings appear to be only mechanoreceptive, with their response to phagostimulants mediated by sensilla within the food canal, probably by the eight peglike sensilla located in the epipharynx (Bernard, 1974; Friend and Smith, 1977). For some haematophagous insects the contact with host skin triggers another process associated with blood ingestion, the change in the mechanical properties of the abdominal cuticle. This ‘‘plasticization’’ process is similar to the process allowing extension of the new cuticle in the period between ecdysis and the sclerotization of the exoskeleton. In triatomine bugs, at least, plasticization occurs each time a larva has a blood meal. Plasticization is triggered by the simple contact of the proboscis with a warm object, feeding not being necessary (Ianowski et al., 1998). During feeding, serotonin is released from neurohaemal organs located at the abdominal wall. The released serotonin acts on many targets throughout the body to coordinate the physiological changes associated with feeding, probably including plasticization (Orchard et al., 1988; Lange et al., 1989; Barrett and Orchard, 1990; Orchard, 2006). Changes in the molecular structure of the endocuticle (disappearance of low-energy bonds between protein chains) allow the endocuticle to extend during the feeding period. These changes are then reversed, with the original stiffness recovered within about 1 h (Melcon et al., 2005). If a bug cannot complete a meal, plasticization may occur repeatedly during the same larval instar once the cuticle has recovered its normal stiffness (Melcon et al., 2005). The sensory mechanism responsible for triggering this process is only partially understood.
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As mentioned above, mechanical contact with a warm object is sufficient for evoking a response. However, given that heat is necessary to evoke contact in the first place, it is difficult to separately examine the effects of the individual stimuli. It is possible that mechanoreception alone is linked to plasticization, with heat evoking proboscis extension. Indeed, thermoreceptors have not been conclusively shown to exist in the proboscis, but mechanosensory sensilla are abundant (Bernard, 1974; Catala, 1996). Despite the key role of heat in the various processes of host localization, biting and plasticization, blood temperature is not involved in food recognition by triatomines. Indeed, blood as cold as 3 C can be ingested by haematophagous bugs if their antennae are sufficiently stimulated by heat for the bugs to extend their proboscis and to bite (Lazzari and Nu´n˜ez, 1989b). 5.8
LEAVING THE HOST
The end of the gorging process is marked by irregular activity of the ingestion pump, which decreases rapidly to eventually stop once the abdomen is full of blood. Two different mechanisms may cause feeding to stop, one based on the increase of mechanical resistance to blood pumped into the intestine (BennetClark, 1963) and another based on a neural pathway involving stretch receptors in the abdominal wall (Anwyl, 1972; Nijhout and Sheffield, 1979; Belzer, 1979; Chiang and Davey, 1988). These mechanisms are not necessarily exclusive and the process may involve a combination of factors. Once gorged, blood-sucking insects leave their host and look for a protected place. Many haematophagous insects ingest large meals, meaning that they then have to displace an extra weight that may be many times their own body weight. Triatomines are particularly vulnerable to aggressive hosts, due to the plasticization of their abdominal cuticle. The most effective behavioural response is to move away from the host as quickly as possible to escape from any dangerous antiparasitic reaction or from predation by their food source. Theoretically, the same cues serving to approach a potential host can be used to move away from it. Host-associated volatile compounds and heat can guide the insects just by inverting the direction of the orientation setting mechanism (Jander, 1963). As I will described below, a switch in the response to carbon dioxide from attraction to repulsion occurs in triatomine bugs some time after feeding (Bodin et al., 2009b); it remains unclear whether this is the case just after gorging.
6
Stimulus propagation and sensory reception
The location in space of the source of a stimulus depends on several factors, including: (1) the specific nature of the signal and the way it is propagated, (2) the ability of sensory organs to gather directional information about the
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source and (3) the way in which the animal uses the sensory information or the orientation mechanism (Fig. 5). Insects, as observed in other animals, are also able to use indirect cues to orientate themselves to a particular site. However, haematophagous insects do not seem to use indirect cues to locate a host. Eyes are able to provide precise spatial information, thanks to both the discrete nature of light and the arrangement of photoreceptors in the retina. Other sensory systems, such as the wind-sensitive cercal system of Orthoptera and cockroaches, which uses directional information from the deflection of hairs, are able to determine the direction that the stimulus came from, but not the exact location of its source in space. In other sensory systems, the sensory information must be actively gathered. In some cases, the location of the source of a stimulus in space requires successive comparisons of sensory information. This is usually accompanied by active movement, either of the whole individual, or of the organs bearing sensory receptors, that is the antennae, to detect the stimulus intensity gradient. Movements concerning the whole individual are characterized by turning motion, associated with klinotaxic orientation.
A
B
C
D
FIG. 5 Examples of factors determining the acquisition of sensory information. (A) An animal with an unpaired sensory organ can locate the source of a stimulus diffusing in the form of a gradient (e.g. chemical volatiles, water vapour) by comparing the intensity of the stimulation at different moments and spatial positions. (B) When sensory organs are distributed bilaterally, as in insect antennae, directional information about the same type of source can be obtained by comparing stimulation both simultaneously (between bilateral inputs) and successively, as a function of displacement. (C) Stimuli propagating radially (e.g. light) provide more precise directional information, which can be identified by animals by successive comparison of stimulation of unpaired sense organs or by comparing stimulation of bilateral organs; in the case of spatial vision, the pattern of stimulation of different regions of the retinal lattice of one eye is sufficient. (D) Some radially propagating stimuli, such as radiant heat, could be perceived using the stimulation of specific sensory organs distributed over the antennae (e.g. kissing bugs) or other regions of the body (e.g. pyrophilic beetles).
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The antennae can also be moved actively to collect information. Antennal movements occur when insects are confronted with a source of stimuli. For example, kissing bugs, T. infestans and R. prolixus display typical antennal movements when exposed to a heat source (Wigglesworth and Gillet, 1934a; Flores and Lazzari, 1996). These movements differ depending on whether the insect is walking or standing during its approach to the source. During the standing phase, antennae move synchronously and in a saccadic fashion, across wide angles; when walking, both antennae are kept at a constant angle in the horizontal plane, moving smoothly up and down in the vertical plane (Flores and Lazzari, 1996). Saccadic and smooth antennal movements can be interpreted as the ‘‘scanning’’ (of the environment) and ‘‘fixation’’ (of the source) phases. Thus, insects stop walking to scan wide regions of the environment with antennal saccades and, when the direction to the thermal source is established, the insect starts walking, fixing the source with the antennae (Flores and Lazzari, 1996; Flores, 2001). We currently have a relatively good understanding of the physiology, transduction mechanisms and molecules involved in reception of the signal, the first level of sensory information processing. This concerns all the sensory systems, vision (e.g. Warrant and Nilsson, 2006), olfaction (e.g. Qiu et al., 2006; Xia et al., 2008) and thermoreception (e.g. Gingl et al., 2005). Considerable efforts have been made to try to understand how haematophagous insects respond to sensory cues (e.g. Takken and Knols, 1999; Zwiebel and Takken, 2004; Barrozo and Lazzari, 2004a). Relatively little is known, however, about how sensory information is acquired and how different systems are integrated to locate a host. Even though it is widely accepted that a given behavioural input results from the interplay of many external and internal factors, the roles of multimodality, state-dependency and active gathering of information remain unclear in most cases. In this section, we will discuss some of the processes that occur in an intermediate stage, between sensory reception and behaviour. We will firstly define the three levels of information processing, determining how animals acquire and make use of information to locate resources. The first level of processing is ‘‘reception,’’ which refers to peripheral events in the sensory organs, occurring as a consequence of a change in the physical or chemical properties of the environment. In physiological terms, this phase determines the type of variables the sensory system of a given animal is potentially able to detect. Reception is usually studied using molecular tools and electrophysiological methods to analyse the transduction of external signals as nervous activity. The main output from such studies is obtained in the form of spectra showing the type and intensity of stimuli received and transduced into nervous signals by a given sensory organ. The second level is ‘‘perception,’’ which we use here to refer to the central processing of sensory information, that is filtering and multimodal integration of sensory inputs and endogenous signals. The analysis of these processes requires several approaches, including electrophysiological
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and optophysiological techniques to study central nervous activity and behavioural methods (black-box analysis of inputs and outputs). The final level involves behavioural output, revealing how the animal makes use of the information gathered from the environment. The distinction between these three levels helps us to determine the type of information we expect to obtain from the different types of experiments and to interpret the results obtained. As an example, the fact that a given chemical compound stimulates a particular olfactory receptor neuron does not necessarily mean that such a compound will modify the behaviour of the insect. This input may require integration with other inputs from the same or different systems and with endogenous signals defining a particular physiological state (determined by circadian clocks, nutritional and reproductive status, and other physiological processes), to cause a behavioural effect. Similarly, the fact that an insect possesses two or more different types of photoreceptor sensitive to different wavelengths does not mean that it is able to use colour vision to solve different visual tasks (object recognition, movement detection etc.). Conversely, the preference for landing on particular colours of targets cannot be considered as evidence of colour vision either; indeed, other possibilities such as intensity-guided choice cannot be excluded (Kelber et al., 2003; Kelber, 2006). In summary, the behavioural output shows us how the information is actually used by the insect. However, behavioural tests can be time and effort consuming, and a large number of individuals usually need to be tested to compensate for individual variability and obtain sufficient statistical power. Studies of reception and perception processes usually require expensive equipment. However, the independence of data and rigorous statistical analysis are not always required for publication, because of the limited number of experimental units that can be obtained in some cases (e.g. the same neuron in different individuals). The combination of different approaches may be the most effective strategy. For example, in studies aimed at identifying biologically relevant compounds in a complex mixture, initial screening using an electroantennogram (EAG) may exclude compounds that do not evoke sensory activity.
7
Orientation mechanisms
The way in which sensory information is made available and becomes exploitable for an animal depends on the nature of three main factors: signal propagation, the morphology and function of sensory tools and processing of the information by the central nervous system. Figure 5 illustrates the effects of signal propagation and sensory tool morphology and function on the orientation of animals. Two types of stimulus are represented. The first type includes stimuli that disperse through the medium, giving rise to intensity gradients emanating from the source. Dispersion of these signals is dependent on turbulence and flow and the stability of the signal is variable, depending on
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movements of the air. Examples include the dispersion of chemicals or water vapour and warm air gradients generated through conduction. The second type includes discrete stimuli that disperse radially. They are not affected by movements of the medium and thus provide accurate directional information. This group includes light and infrared radiation. The sensory organs detecting these stimuli may or may not able to discriminate the potential spatial information provided. Eyes, for instance, generate an image on each retina allowing the location of objects to be determined, even using just one organ. The lattice of receptive units on each eye provides sufficient directional information to locate a source. Other sensory organs are organized differently, requiring comparisons of stimulation intensity between bilateral inputs to elucidate the direction the stimuli are coming from. Insect ears are classical examples, in which the comparison of bilateral inputs allows insects to locate the source of a sound. The animal turns around and, when the bilateral inputs equalize, the insect heads towards the source. To reach a source, information from the environment can be used in different ways, requiring different orientation mechanisms. The nature of the stimuli and the properties of the sensory organs are two major factors determining the orientation mechanism used. A third factor is how the sensory inputs are integrated in the central nervous system. Indeed, the orientation in space of an animal using the same information can be controlled by different mechanisms (Fraenkel and Gunn, 1961; Jander, 1963; Dusenbery, 1992). Some mechanisms modulate activity as a function of the intensity of a given stimulus, independent of the direction the stimulus is coming from kinesis. In other words, the individual can reach the source just by modulating the velocity of its displacement (orthokinesis) or by modulating its tendency to turn (kinokinesis), without using directional information at all. In other animals, directional information is taken into account and the animal moves as a function of the spatial position of the stimulus source (taxis). These mechanisms are not mutually exclusive. Haematophagous insects use these mechanisms in combination when approaching a host. The traditional classification of orientation mechanisms (Fraenkel and Gunn, 1961; Jander, 1963; Dusenbery, 1992) may be considered out of date or too restrictive. However, it is not only very useful, but essential, for designing experiments, interpreting results and understanding how information is acquired and used to find resources. According to the classical definition (Fraenkel and Gunn, 1961; Dusenbery, 1992), stimuli may affect behaviour in two ways, affecting either kinetics or direction. In the first case, called ‘‘kinesis,’’ the intensity of the stimulus (and not the direction that it comes from) modulates either locomotion speed (orthokinesis) or the turning tendency of the animal (klinokinesis). In the second case, the animal extracts directional information from the signal (taxis). The direction of the source may be evaluated by successive comparisons of stimulation received during a programmed pattern of alternate left and right movements (klinotaxis) or by the simultaneous comparison of bilateral inputs (tropotaxis) or
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of receptive units of a single organ (telotaxis); in cases involving receptive units of a single organ, each component of a bilateral pair of sensory organs (eyes, antennae) provides enough information to determine the direction of the source. In most cases, both elements, stimulus structure and spatial resolution of sensory organs, are linked by the fact that sensory organs devoted to detecting stimuli providing poor directional information, such as chemicals, are not able to extract directional information unilaterally, so that telotaxis is not possible. It should be noted, however, that the relationship between the signal, sensory organs and orientation mechanism varies, even for the same individual. Kissing bugs approaching a source of heat orientate by telotaxis until they are about 2 cm from the source, switching to tropotaxis to bite the host at the site of a blood vessel, for instance (Wigglesworth and Gillet, 1934a; Flores and Lazzari, 1996; Ferreira et al., 2007). Thus, insects with just one antenna (i.e. unilaterally antennectomized) are able to approach a heat source, but miss the goal when they try to extend their proboscis towards it. The orientation mechanism associated with the response of a particular insect to a particular cue may provide important information about how sensory information is acquired and employed. Taking the example of kissing bugs, they approach heat sources by telotaxis from relatively long distances (Flores and Lazzari, 1996), walking in the same horizontal plane as the source or below it. Ascending convection currents are therefore rarely involved, being effective only when the insects approach from above, as shown in mosquitoes by Eiras and Jepson (1994). Given that animals with just one antennae approach the source by walking in a straight path (Wigglesworth and Gillet, 1934a; Flores and Lazzari, 1996), they are using neither bilateral simultaneous comparison (if this were the case, the path would deviate towards the intact antennae, Jander, 1963), nor successive comparisons (otherwise, the path would meander, Dusenbery, 1992). The ability to approach a source using directional information from just one sensory organ depends on two criteria being met: first, that the stimulus provides reliable directional information over the required distance, and second, that the sensory organs provide spatial information unilaterally. As described above, both of these criteria are met in the normal functioning of the eyes, the classical example of telotaxis, by forming images on the retina; but this is not the case for insect antennae. We can now evaluate the effects of each of the three mechanisms of heat exchange – convection, conduction and radiation – on the potential use of thermal telotaxis by an insect walking towards a source of heat. Convection currents propagate upwards and cannot be perceived by an insect located tens of centimetres away from, and at the same level or below, the source, and thus are not likely mediators of thermal telotaxis. Warm air gradients extend in all directions and could reach the insect. However, not only are gradients of air temperature disrupted by externally induced turbulence, but they themselves create turbulence in the form of ascending convention currents. Thus, the resulting signal may not be sufficiently stable to provide precise directional
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CLAUDIO R. LAZZARI
information. Convection currents and warm air gradients may still provide some directional information; however, the signal is in a non-discrete form and does not provide a sufficient level of directional information to promote thermal telotaxis. The third mechanism, heat radiation, being independent of air turbulence and propagated in the form of waves in all directions, seems to be the only signal that provides information in an appropriate form and at a sufficient level for thermal telotaxis to take place. Kissing bugs are the only group of haematophagous insects for which IR perception has been demonstrated (Lazzari and Nu´n˜ez, 1989a; Schmitz et al., 2000), although IR perception has been observed for other heteropterans displaying phytophagous and pyrophilous activity, as well as in some Coleoptera (Schmitz and Bleckmann, 1997; Schmitz et al., 2008; Taka´cs et al., 2009). It remains less clear how insects with just one antenna are able to determine the direction of a heat source from a distance, with sufficient precision to maintain a straight path (i.e. without klinotaxic turns). One possibility is that the insect recognizes the spatial pattern of thermoreceptor stimulation along the length of the antenna or even the body. The insect does not need to create a thermal ‘‘image’’; rather, it must simply recognize which side of the antenna (or body) is receiving more stimulation. This example thus demonstrates the importance of taking into account the orientation mechanism in determining the nature of the biological signal detected and how the sensory information is used.
8
Thermal sensing in kissing bugs
As mentioned above, triatomine bugs rely on various sensory cues to search for a host. These cues are mainly olfactory and thermal. The thermal cue is the only one capable to induce biting by the insect (Wigglesworth and Gillet, 1934a; Flores and Lazzari, 1996). Additionally, triatomine bugs are the only haematophagous insects that have been demonstrated to perceive radiant heat (Lazzari and Nu´n˜ez, 1989a; Schmitz et al., 2000). When confronted with heat sources of different sizes and located at various distances from the source, triatomine bugs respond only to objects with a temperature corresponding approximately to that of a warm-blooded vertebrate host (Nicolle and Mathis, 1941; Fujita and Kloetzel, 1976; Lazzari and Nu´n˜ez, 1989a). Generally, a distant (or small) burning source and a close (or big) tepid source are easily identified by these bugs. The amount of heat reaching the thermoreceptors depends not only on the temperature of a heat source, but also on the distance between the insect and the object, the surface area of the emitting object and the environmental temperature. Thermal sensory organs alone therefore do not provide enough information to determine the temperature of the source. To achieve this, the insects need to be able to determine the temperature and distance from a heat source, independently of the surface area of the source, using only thermal information (Flores and Lazzari, 1996).
ORIENTATION TOWARDS HOSTS IN HAEMATOPHAGOUS INSECTS
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This capacity may be related to their ability to perceive radiant heat (Lazzari and Nu´n˜ez, 1989a). Radiation is a more precise cue than heat conduction or convection; consequently, the distance to a thermal source may be estimated using information derived from the different levels of stimulation at different sites on the antennae. In particular, the angle of incidence, surface area and temperature, would provide useful information. The importance of heat in triatomines is highlighted not only by the high level of sensitivity of their thermal sensing and by their ability to evaluate the thermal properties of a distant source, but also by its various the biological effects. Indeed, heat is the only host-associated signal, both necessary and sufficient to induce the proboscis extension response (PER) in these bugs and to induce them to bite an object. As mentioned, odours are important cues in the orientation of the bugs. However, an object emitting odours at room temperature is never bitten. Only objects at temperatures between a few degrees above ambient levels and about 47 C are considered to be potential hosts (Lazzari and Nu´n˜ez, 1989a). The overall behaviour of these insects changes dramatically in the presence of a thermal source. Insects in a state of akinesis (immobility) ‘‘wake up’’ (Wigglesworth and Gillet, 1934a) and display characteristic movements of their antennae, sweeping the air within the horizontal and vertical planes at particular angles (Wigglesworth and Gillet, 1934a; Flores and Lazzari, 1996; Flores, 2001). When active, insects typically alternate between standing and bouts of walking, displaying two different patterns of antennal movements (Figs. 6 and 7). While standing, the movements are jerky and the antennae sweep at wide angles, but while walking, the movements become smooth, and the angles between the antennae remain largely constant, as described earlier. The insect’s standing phases can be interpreted as phases during which the insect scans for odours and heat, whereas during phases of walking, the antennal positions may function as a possible means of detecting the edges of a warm object (Flores and Lazzari, 1996). These different antennal movements are observed in particular when the insect is approaching a thermal source. These observations suggest that the antennae, which bear thermoreceptors (Bernard, 1974), play a central role in the insect’s search for blood. However, how the information conveyed is then used by the insect remains unclear. The basic laws of thermodynamics predict that the information received by the insect upon delivery of an amount of heat is ambiguous. A large, hot, distant object could produce the same local warming effect on the insect’s antennae as a small tepid object placed at a shorter distance. Thus, other characteristics of a thermal source may provide additional and useful information about the source. To recognize a host using temperature (between 32 and 42 C at the body surface of most warm-blooded vertebrates), the insect must integrate information based on the amount of heat reaching the antennae with information concerning the distance of the source and its size. Together with temperature, the source and size of the source directly affect the amount of heat reaching the
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Angle (degrees)
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FIG. 6 Sample records of antennal movements performed by blood-sucking bugs during their approach to a heat source. The insects alternated between walking and standing, performing characteristic antennal movements in both horizontal and vertical planes (A) horizontal and (B) vertical antennal movements during walking; (C) horizontal and (D) vertical sample records of saccadic antennal movements in a standing insect. Positive values in (A) and (C) correspond to the left antenna and negative values correspond to the right antenna. In (B) and (D), positive values correspond to angles above the horizontal plane and negative values correspond to angles below the horizontal plane. During both phases, antennae are moved in coordination (modified from Flores and Lazzari, 1996). −20
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FIG. 7 Coordination of the movement of both antennae during stopping and walking periods of a haematophagous bug approaching a thermal source. Movements are coordinated and the positions of the right and left antennae are significantly correlated in both cases (Flores and Lazzari, unpublished).
ORIENTATION TOWARDS HOSTS IN HAEMATOPHAGOUS INSECTS
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insect’s thermoreceptors. Thus, several possible combinations of these three variables may give rise to the same amount of heat. T. infestans is able to estimate the distance to a thermal source in the absence of visual information or physical contact with the object, relying only on thermal information (Lazzari and Nu´n˜ez, 1989a; Flores and Lazzari, 1996). The lack of one antenna causes errors in targeting the goal, but does not affect the distance at which the insects try to bite (Flores and Lazzari, 1996). So far, there are no data available showing the ability of any insect to estimate the size of an object using only thermal information. This possibility was investigated in kissing bugs (Flores and Lazzari, 1996; Lazzari and Flores, unpublished). Insects were placed under open-loop conditions (i.e. at a fixed position). Heated aluminium square plates (34 C) of three different sizes were placed at a fixed distance from the insect, or plates of a given size were placed at three different distances (Fig. 8). Each assay was carried out using a single plate placed at a particular distance. Plate distances and sizes were chosen to give three different subtended angles (i.e. 14 , 21 and 28 ) at the insect position. The angle formed by the antennae during locomotion (i.e. tendency to approach the source) was recorded during each experiment. In both experimental series, the mean angle formed by the antennae significantly increased with the size of the source (ANOVA, p < 0.0001 in both cases). It was also observed a direct relationship between the antennal angles and the angles subtended by the source at the position of the insect, regardless of the size or distance of the source used in each experimental series (Fig. 9) (Flores, 1996; Lazzari and Flores, unpublished). We compared data sets from the two series using a paired t-test. We did not observe any difference in the mean antennal angle between groups of insects exposed to objects displaying the same subtended angle, regardless of the actual distance or size of the source.
14⬚
40 cm
21⬚
27 cm
28⬚
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FIG. 8 Experimental conditions for testing the relationships between the position of the antennae (antennal angle) and the size and distance of the thermal source. In a first series of experiments, a square (10 10 cm) plate heated to 34 C was placed at 20, 27 or 40 cm, at solid angles of 14 , 21 and 28 subtended by the insect position (open-loop conditions). In a second series, the same subtended angles were obtained by placing plates of different sizes (5 5, 7.5 7.5 and 10 10 cm) at 20 cm of the insect. The position of the antennae during the walking phases of independent groups of insects was recorded for a period of 2 min and the mean antennal angle was determined for each individual.
30
CLAUDIO R. LAZZARI 80
Antennal angle (degrees)
n.s.
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27 cm 20 cm n.s.
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n.s. Constant distance, variable size Constant size, variable distance
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FIG. 9 Antennal angles in insects exposed to sources subtending various solid angles. Significant differences in the position of the antennal angle as a function of the apparent size of the source were found for both experimental series (i.e. varying distance or actual size, ANOVA p < 0.0001) However, no differences were observed between assays for plates differing in size or distance to the insect, but subtending the same solid angle. Black, grey and white rectangles represent the differently sized plates that were placed at different distances from the insect to obtain similar subtended angles.
These findings reveal several important features of thermal sensing in haematophagous bugs. First, the insects can actively control the position of their antennae according to the apparent size of a thermal source. Second, the insects can integrate thermal information from both antennae in such a way that enables them to maintain a constant angle. This finding confirms previous findings from orientation experiments (Wigglesworth and Gillet, 1934a; Flores and Lazzari, 1996). Third, these insects integrate proprioceptive inputs (probably from mechanoreceptors located at the joints of the antennae) with thermal information to produce the appropriate motor output to control antennal movements. According to the laws of thermodynamics, the amount of heat reaching a thermoreceptor depends on three variables: the distance to the thermal source, the surface area of the source and its temperature (more precisely, the difference between the temperature of the source and that of the environment). The processes allowing triatomine insects to estimate distance remain unclear, but may involve simple mechanisms. Previous experiments on the orientation of bugs towards thermal sources suggest that the insects integrate bilateral inputs
ORIENTATION TOWARDS HOSTS IN HAEMATOPHAGOUS INSECTS
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from the antennae through triangulation, i.e. by comparing the amount of heat received simultaneously by two thermoreceptors located at different sites on the body (e.g. on each antenna) (Flores and Lazzari, 1996). The insects may also be comparing the increases in heat energy as they approach the thermal source using internal representations of these increases. Studies on the response to heat in unilaterally antennectomized insects (Wigglesworth and Gillet, 1934a; Flores and Lazzari, 1996), together with the fact that heat increases at a constant rate with decreasing distance for objects of different temperatures and sizes (Fig. 10), are consistent with a second possibility. In this case, the insects compare the amount of heat received by the same thermoreceptor at successive time points during their approach to the thermal source or use the temperature gradient along the length of the antennae and the rest of the body (Fig. 11).
0.06 30 °C 35°C 40 °C 45 °C 50 °C
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FIG. 10 A possible mechanism used to estimate the distance to a heat source, based on the gradient of heat energy decrement around a heat source. The figure depicts the gradient of thermal energy formed around a heat source of 1 cm2 area in an environment at 25 C. Each curve represents a different source temperature. The relative rate of decrement is independent of the temperature of the source. Similar curves can be obtained for sources differing in size, but sharing the same temperature. During the approach, the insect experiences heat energy increasing at different rates, as a function of the insect’s relative position. Successive comparisons of thermal stimulation during the approach or simultaneous comparisons between two regions of the body could allow bugs to estimate the distance to the source. These mechanisms could underlie thermotelotaxis in triatomines, a process that requires evaluation of the direction of a source even if bilateral integration is abolished by atennectomy. They may also determine how triatomines estimate their position relative to a heat source when only thermal information is available (Wigglesworth and Gillett, 1934a; Flores and Lazzari, 1996).
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CLAUDIO R. LAZZARI 32.2 ⬚C
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FIG. 11 Thermographic images of a haematophagous bug exposed to a heat source (35 C; diameter 2.5 cm). A temperature gradient is established along the antennae. The form and intensity of the gradient over each antenna varies as a function of the exact position of the organs during active movement.
Preliminary experiments suggest that the way in which distance is estimated differs depending on whether the insect itself approaches a heat source or whether the source appears suddenly at a fixed position. In the second case, the insect reacts extending its proboscis when the source is at a much shorter distance. These results seem to suggest that these insects use successive rather than simultaneous comparisons. The exact mechanism, however, remains to be confirmed, since the thermal information received in both cases theoretically provides sufficient information to determine the distance to the source. Based on our findings, previous experimental evidence and basic thermodynamics, a simple model of the multimodal integration of thermal and proprioceptive information can be proposed as a hypothesis to explain how triatomines recognize a host using thermal cues. The insects may elucidate the actual size of a thermal object by integrating information on distance to the thermal source and the subtended angle (the apparent size of the source). The temperature of the object could then be estimated using information on the amount of heat energy reaching the thermoreceptors and the distance to and size of the source. This would allow a host to be recognized by its temperature alone, allowing the insect to react accordingly. This model (Fig. 12) is based on the multimodal integration of proprioceptive and thermoreceptive inputs, the comparison of thermal stimuli and basic thermodynamics. The heat emanating from the source stimulates thermoreceptors. When the insect moves its antennae under proprioceptive control, the position of the thermoreceptors relative to the source changes, causing a change in the intensity of thermal stimulation. By integrating these changes with proprioceptive inputs to determine the position of the antennae, the insect could detect the edges of the source and thus estimate its apparent size. The insects may also determine the distance to the source based on the inverse square relationship between heat energy and distance. This can be achieved either by simply
ORIENTATION TOWARDS HOSTS IN HAEMATOPHAGOUS INSECTS Heat
Antennal mov.
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Energy amount
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E1 ≈ (Tsource4-Tenv4) A d2
central integration
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Approach
FIG. 12 Model of integration of thermal and proprioceptive information. Heat emanating from the source stimulates thermoreceptors. The intensity of the thermal stimuli and the position of the thermoreceptors change when the insect moves its antennae under proprioceptive control. The insect may estimate the apparent size of the source through integration of information on both the intensity of the stimuli and the position of the antennae. Alternatively, the insect may use the inverse square relationship between energy and distance to determine the distance to the source, either through simultaneous comparisons of the heat energy reaching different parts of its body or through successive comparisons of the heat energy reaching a certain part of its body during its approach to the thermal source. By integrating the distance with the apparent size of the source, the insect could easily determine the actual size. Thus, the insect would possess all the required variables, as described in the Stephan–Bolzmann law (inset), to estimate the temperature of a distant source, that is the heat energy reaching thermoreceptors, the surface area and the distance of the source, and the environmental temperature directly measured by the thermoreceptors. The insect could then use this information to ‘‘decide’’ whether a warmth object qualifies as a host, allowing it to react accordingly (Lazzari and Flores, unpublished).
comparing the heat energy reaching different parts of its body simultaneously or through successive comparisons of the heat energy reaching a certain part of its body as it approaches the thermal source. The insect could then easily compute the actual size of the source by integrating information on the distance with the information on the apparent size of the source. Thus, according to the Stefan– Bolzmann law, the insect would have all the information needed to estimate the
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temperature of a distant source, that is the heat energy reaching thermoreceptors, the surface area of the emitting source, the distance to the source and the environmental temperature measured directly by the thermoreceptors. The insect could then use this information to determine whether a warm object qualifies as a potential host and whether or not to approach it. Triatomines display a very high level of behavioural sensitivity to heat, reacting to very low amounts of heat energy that reach them. In the experiments by Lazzari and Nu´n˜ez (1989a), strong responses were obtained to a blackpainted circular object, with a 2 mm diameter and a temperature of 30 C (room temperature 20 C). These responses remained statistically significant when three sheets (250 mm each) were placed between the source and the insects, preventing detection of any warm air gradients or convection current and reducing the amount of IR radiation reaching the insects by 64%. Under these conditions, given that the amount of thermal energy decays with the square of the distance, the Stefan–Bolzmann law gives a value of about 3.3 mWatt/cm2 for the amount of heat energy that should have reached the insect in these experiments (Lazzari and Nu´n˜ez, 1989a; Lorenzo et al., 1999). This would imply that the insects can perceive the heat emitted from a host located several meters away. This value is lower than the thresholds computed for other animals that respond to radiant heat, such as snakes (10 mWatt/cm2), vampire bats (50 mWatt/cm2) and Melanophila beetles (60 mWatt/cm2) (Campbell et al., 2002). If these results are confirmed, triatomines would possess one of the most sensitive thermal sensing systems in animals.
9
Sensory parsimony
Despite the fact that host-related information can be relatively specific and that insects undergo a process of selection of both their hosts and the sites on the vertebrate body at which they prefer to bite, specific sensory cues are not necessarily involved. Many haematophagous insects seem to make use of the same cues to locate different resources, through parsimonious use of sensory information. In such cases, interpreting a particular type of such information does not depend on specific cues, but is dependent on the biological context. 9.1
PARSIMONIOUS USE OF INFORMATION IN BLOOD-SUCKING INSECTS
Tsetse flies spent much of their time resting on plants, which provide refuge and protection from excessive heat (Leak, 1999). Some of these plants release chemicals that attract the insects. The combined study of the volatile substances released by the invasive plant species in Africa, Lantana camara, and of the sensory and behavioural responses of tsetse flies, led to the identification of the compounds involved in this biological process (Syed and Guerin, 2004). The study showed that, in addition to plant-specific compounds, Lantana
ORIENTATION TOWARDS HOSTS IN HAEMATOPHAGOUS INSECTS
35
releases 1-octen-3-ol (octenol). Octenol is part of the mixture of volatile compounds released in the breath of vertebrate hosts and is a powerful attractant for tsetse flies and other haematophagous insects (Hall et al., 1984; Vale and Hall, 1985; Takken and Kline, 1989; Barrozo and Lazzari, 2004b). Octenol can thus be used as a chemical lure for trapping tsetse flies in the field (Leak, 1999). Mosquitoes, as observed for many other haematophagous insect, use carbon dioxide to locate potential hosts (Gillies and Wilkes, 1969; Gillies, 1980; Takken and Kline, 1989). The sensitivity of several species to CO2 and the synergic interaction between CO2 and other volatile compounds have been characterized (Acree et al., 1968; Geier et al., 1999b). Carbon dioxide is thus widely used as an attractant in different kinds of devices to capture mosquitoes (Burkett et al., 2001). Carbon dioxide is also released by opening flowers, where it is possibly used by insects sensitive to CO2 to find nectar (Guerenstein and Hildebrand, 2008). In mosquito maxillary palps the same sensilla house both olfactory receptor neurons (ORN) sensitive to CO2 and ORNs sensitive to 1-octen-3-ol and to plant odours (Syed and Leal, 2007). With a dual diet of blood and nectar, female mosquitoes may use the same sensory receptors to locate both food sources. Some haematophagous arthropods seem to use volatile compounds that are also released by their hosts for intraspecific communication. Thus, the same compound acts as a host-associated kairomone, possibly as part of a pheromonal blend. Several volatile compounds released by the skin act in this way and have been identified in alarm and sexual blends of kissing bugs (Bernier et al., 2000; Manrique et al., 2006; Pontes et al., 2008). These findings demonstrate that at least some haematophagous insects seem to make parsimonious use of their sensory system and the information provided. This has several implications. The first and most obvious is the efficient use of sensory organs, for which sensitivity to the same cues may help to identify and locate different resources. The second is that a given cue (e.g. a particular volatile compound) does not have an unequivocal biological meaning for an individual; rather, the information it provides is ambiguous until it is put into context via integration with cues targeting the same or different sensory systems. The third major implication is that a given cue involved in different behaviours may be associated with quite different responses; for instance, signals that incite attraction when the source is identified as a potential host may cause a repellent response if integrated as part of an alarm signal. So, the function of at least some cues is determined by the context in which they are detected, rather than being associated with one particular biological response. 9.2
PRACTICAL CONSEQUENCES
Sensory parsimony has important practical consequences for attracting and capturing haematophagous insects, given that attractants do not act in an additive fashion. Thus, combining different attractive compounds does not
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CLAUDIO R. LAZZARI
necessarily increase the chances of finding a better lure. Indeed, different elements need to be combined in specific ways to make a bait biologically effective, in terms of not only the components used but also their specific proportions in the mixture (e.g. Barrozo and Lazzari, 2004a). Mixing hostassociated cues with assembling or sexual pheromones may therefore not have the expected effect, simply because the combination does not resemble a particular source of interest for the insect, despite individual components being potential attractants. Similarly, capture devices may prove ineffective if based on exploiting a behavioural characteristic that is linked to a specific context. This point was well illustrated by a trap designed to exploit the tendency of kissing bugs to fall down onto their hosts from walls or ceilings (Guerenstein et al., 1995). The trap was associated to a culture of baker yeast as lure. The bugs only jumped inside the trap (they could not climb away) when odours came from below. If the odours just diffused freely (i.e. not reaching the insect from below), the insects were attracted, but the jumping down behaviour was not evoked. This same effect – insects being attracted but not displaying jumping behaviour – was observed when assembly pheromones from faeces were used as lure. This variable jumping down behaviour was interpreted as being specifically associated to host-seeking when the insect walks on the roof and perceives events below it, but not when walking on the ground or when the attractant is perceived in a different context. Aggregation pheromones also include some volatile components emitted by vertebrate hosts, but are mostly associated with insect refuges, and jumping down into places of refuge is not a usual behaviour (Guerenstein et al., 1995; Lorenzo and Lazzari, 1996)
10
State-dependency of host-seeking behaviour
The response of blood-sucking insects does not only depend on external signals and their interactions outside (i.e. physical interaction) and inside the insect (multimodal convergence). Various signals originating from within the insect also play major roles in determining if, how and when a given behaviour will be evoked in the presence of environmental stimuli. These internal signals may exist in different forms (i.e. nervous or endocrine signals) and involve different physiological processes, which modulate the behaviour of the insect. The most widely recognized physiological determinants affecting the response to host signals include nutritional state (or feeding condition), moulting, reproductive state (mating and oviposition) and the circadian cycle. These factors not only determine whether or not insects respond to a given signal, but also how they respond. As we will discuss below, the identical stimulus may be attractive our repellent depending on the physiological state of the insect. Before analysing in detail how endogenous factors modulate the response to the presence of a host, we will briefly discuss the biological function or adaptive
ORIENTATION TOWARDS HOSTS IN HAEMATOPHAGOUS INSECTS
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value of such modulation. These factors minimize the costs and risks associated with feeding on blood, by making the insect feed only when necessary. Ideally, the insect should feed quickly, on an inactive host and only when the energetic expenditure for contacting the host is at a minimum. The insect therefore needs to be mature enough (or their mouthparts sufficiently developed) to pierce the host skin and feed during the host’s resting period at a time when all mature eggs have been laid and no extra weight due to stored food is being carried. The insect thereby minimizes the number of contacts with the host and the associated risks. 10.1
THE TEMPORAL MODULATION OF THE RESPONSE TO ODOURS
Biological rhythms are expressed ubiquitously in almost every organism as temporal oscillations in the course of biochemical, physiological and behavioural processes, from the cellular to the population level and even in multitrophic interactions. The host–vector–parasite interaction offers an excellent example of the adaptive value of biological rhythms (Pittendrigh, 1974; Aschoff, 1989; Barrozo et al., 2004b). In particular, most haematophagous insects synchronize their daily activity to seek food during host resting period, thus during a period when the host is less able to defend itself (Barrozo et al., 2004b). Haematophagous insects do not necessarily search for food for the duration of their activity period every day. Actively searching for something does of course require some activity, but the inverse is not true. Even in insects in which their physiological state promotes feeding, activity is devoted to a variety of behaviours, not only to obtaining food. For example, in insects with extended or bimodal activity periods every day, food-searching activity may be limited to just a narrow window of time. Many examples demonstrate the temporal control of insect responsiveness to external stimuli. Examples include the daily modulation of olfactory responses (Saunders et al., 2002) and the modulation of chemoreceptor sensitivity by the circadian system (van der Goes van Naters et al., 1998; Krishnan et al., 1999; Page and Koelling, 2003; Zhou et al., 2004; Merlin et al., 2007; Saifullah and Page, 2009). Paradoxically, in some cases, olfactory sensitivity seems to be maximal during the insect resting period. Various explanations have been proposed, but further studies are needed to fully understand the biological relevance of this phenomenon. Blood-sucking insects are no exception, both their sensory and behavioural responsiveness to odours being under the control of the circadian system (Van der Goes van Naters et al., 1998; Barrozo et al., 2004a). It remains unclear how the responsiveness to specific odours in particular behavioural contexts is controlled within certain temporal windows. The general hypothesis, based on studies carried out in Drosophila and cockroaches, suggests that the overall sensitivity of the antennae is modulated for all chemical stimuli at the same time
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(Krishnan et al., 1999; Page and Koelling, 2003; Zhou et al., 2005; Merlin et al., 2007). However, this method of modulation may not be the most beneficial for insects that exhibit bimodal activity and perform different tasks during different temporal windows, requiring different sensitivities for the different activity periods of the day. This has been addressed recently in triatomine bugs, which display bimodal daily activity, looking for a host at dusk and for a refuge at dawn, both activities guided by different chemical cues (Lazzari, 1992; Lorenzo and Lazzari, 1998). Experimental analysis of the temporal modulation of the bugs’ responsiveness to carbon dioxide, a host-associated cue, and to refugeassociated chemical cues in aggregation pheromones showed that these insects respond to carbon dioxide only during the first hours of the night and are attracted by the pheromones only at sunrise (Barrozo et al., 2004a; Bodin et al., 2008). Further analysis of the chronobiological basis of this differential modulation showed that an endogenous circadian system controls responsiveness to carbon dioxide, whereas the responsiveness to aggregation pheromones depends on environmental signals (Bodin et al., 2008). This shows that endogenous clocks and exogenous cycles may act together to adaptively modulate sensory responses. 10.2
MATURATION AND RESPONSIVENESS
The moulting process involves profound changes in the insect’s body, which persist beyond ecdysis and the appearance of a new instar. Indeed, completion of the changes in the insect morphology, physiology and behaviour of the insect takes a certain amount of time. In many teneral insects, the sclerotization of their exoskeleton may remain incomplete for a time following ecdysis. In other insects, the development of certain sensory organs continues for a few days after the ecdysis (Insausti and Lazzari, 2000b). This post-ecdysis delay in completing the development of certain organs is usually associated with a period of maturation of behaviours linked to the use of these structures. Haematophagous insects (both holometabolous and hemimetabolous groups) show a delay before they start responding to host-associated signals. In holometabolous blood-sucking insects, moulting and other activities occurring after the emergence of the adult, such as reproduction, affect feeding behaviour. Blood meals taken before a certain degree of ovarian maturation do not increase the reproductive success of these insects, but visiting the host increases the insect’s chances of being damaged or killed. In mosquitoes, there is period after adult emergence during which insects do not seek for a host. Blood-feeding behaviour begins between 24 and 96 h after a female mosquito emerges (Seaton and Lumsden, 1941; Bishop and Gilchrist, 1946; Laarman, 1955; Bowen and Davis, 1989). A similar period of maturation appears to be required before the peripheral sensory organs are fully responsive (Davis, 1984a). The activation of lactic acid-sensitive neurons in newly emerged virgin female A. aegypti is dependent on age and correlated to the
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development of host-seeking behaviour (Davis, 1984a). The receptors for lactic acid develop faster (12 h) in Aedes atropalpus. The absence of attractive behavioural response to host components between the time of emergence and the end of the first gonotrophic cycle in this species cannot be attributed to the delay in the development of the peripheral sensory system only (Bowen et al., 1994a,b). The hemimetabolous insect R. prolixus is an obligate blood feeder throughout its lifetime. This insect has thus been used to follow post-ecdysis development of the response to host-associated cues in larvae, that is without the effects of physiological processes associated with reproduction (Bodin et al., 2009a). The development of the insect responses to carbon dioxide and heat, and their motivation to feed, is age-dependent; all three types of response reach a plateau more or less at the same time, but show different development patterns and rates. The response to CO2 does not increase gradually with age, but is an all-ornothing response. In other words, insects remain indifferent to this cue during the first week post-ecdysis and are highly attracted afterwards. In contrast, the response to heat progresses gradually, with the responsiveness to heat increasing steadily for about 10 days before reaching a maximum level. These observations suggest that each of the three responses – attraction to CO2 or heat, and feeding – follows its own pattern of maturation. This is probably related to the roles of different physiological processes, such as the maturation of sensory organs, sclerotization of the mouthparts, modulation of the biochemical machinery associated with food intake, handling and digestion, and others that still need to be elucidated. Before the behavioural response to CO2 in R. prolixus becomes properly established, bugs display a bimodal response, either approaching the odour source or walking away from it. This phenomenon has also been observed under similar stimulation conditions, when insects were exposed to CO2 at a time of the day when they normally search for a refuge, rather than for food (Bodin et al., 2008). So, three different and successive phases have been observed for the post-ecdysis development of the response to CO2 in R. prolixus: indifference, bimodal response and attraction. This particular pattern of development suggests that maturation processes also occur at the central level. During the second phase responses, the insects seem to perceive the signal, but their behaviour is ambiguous. In fed insects, a new period of repulsion to CO2 appears 2 days before completing ecdysis (Bodin et al., 2009b). 10.3
THE MODULATION OF HOST-SEEKING ACTIVITY BY REPRODUCTION
The relationship between reproduction and feeding in mosquitoes, underlying the generation of gonotrophic cycles, is a classical topic in the study of insect physiology. It has been mostly analysed in female mosquitoes, in which oogenesis depends on a blood meal, the relationship between blood feeding and reproduction, and endocrine factors (Klowden, 1997). Once the gonadotrophic
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cycle has started, host-seeking activity is inhibited until the oocytes have matured or oviposition has occurred, depending on the species (Klowden and Briegel, 1994). Two processes mediate this inhibition. The first depends on the stimulation of abdominal mechanoreceptors due to abdominal distension during feeding (Klowden and Lea, 1978, 1979a; Klowden, 1990). The second process occurs after the first one and is induced by a humoral response through a complex mechanism involving signals from the ovaries, fat body and neurosecretory cells of the insect (Klowden and Lea, 1979b; Klowden, 1981; Klowden et al., 1987; Brown et al., 1994; Takken et al., 2001). The absence of host-seeking behaviour in fed mosquitoes may be due to the inhibition of the response of ORN sensitive to lactic acid by humoral factors. This inhibitory effect would thus act on peripheral sensory neurons, rather acting centrally (Klowden and Lea, 1979a,b; Davis, 1984b; Davis et al., 1987), resulting in a 10-fold reduction in the sensitivity of olfactory neurons to lactic acid (Davis, 1984b). Host-seeking behaviour remains inhibited in A. aegypti until oviposition (Klowden, 1981, 1990) and, in A. gambiae, until oocyte maturation (Takken et al., 2001). Gonotrophic cycles have not been defined for triatomine bugs. However, after feeding, modulation of the responsiveness to host cues differs between males and females. Whereas males remain indifferent to CO2 for at least 20 days after feeding, females experience a phase of repulsion to the same stimulus (Bodin et al., 2009a). Thus, even though neither sex shows an attraction response, females do not remain indifferent to the stimulus, suggesting that the mechanism does not operate at the periphery, but acts centrally (Bodin et al., 2009b). It also suggests that egg production in females is probably involved in modulating the response to host-associated cues in these insects. 10.4
FEEDING CONDITIONS AND HOST SEARCHING
As discussed in the previous section, ovarian activity is involved in the modulation of haematophagous insect responses to host-associated cues. However, the link between feeding and reproduction prevents independent analyses of these two types of process. In a recent study on R. prolixus (Bodin et al., 2009b) the responses to CO2 and to heat and the motivation to feed were analysed in male and female larvae, the use of larvae allowing separation of these responses from the effects of reproductive processes. R. prolixus larvae do not respond to carbon dioxide for the first 2 days that follow feeding. On the third day, they start responding to the direction of the CO2 laden air-current, but by orientating themselves downwind. This tendency to be repelled by CO2 is gradually reduced, and insects become indifferent again for several days, with another period of repulsion appearing before ecdysis (Bodin et al., 2009b). Host-associate odours exert allomonal effects on mosquitoes (Mukabana et al., 2004; Logan et al., 2008). Repulsion to an otherwise attractive odour only seems to have been observed for kissing bugs.
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Additionally, whereas the allomonal effects observed in mosquitoes are associated with specific volatile compounds, it is the physiological state of the insect that seems to determine the type of response observed in bugs. Carbon dioxide exerts only kairomonal effects, such as repulsion to CO2 when the animal is full of undigested blood, constituting an adaptive response that benefits the insect and not its host. The neural mechanism underlying this modification of the response to host stimuli takes place centrally in R. prolixus, but involves the modification of peripheral sensitivity in mosquitoes (Davis, 1984b). The fact that the insects do not stop responding, but rather modify their response (Bodin et al., 2009b), suggests that they perceive the stimulus normally, with the central integration of exogenous and endogenous information determining the direction of oriented behaviour in a state-dependent way. It should be noted that the available data on both mosquitoes and bloodsucking bugs only concern responses to single chemical cues. We thus cannot assume these observations to be true for the general modulation of any hostassociated cue or conclude that modulation takes place at the periphery for one group of insects and in the central nervous system for another group of insects. Both mechanisms probably occur together in both groups of insects for a better control of host-seeking behaviour. In both mosquitoes and bugs, humoral factors, rather than nervous signals, seem to be involved (Klowden, 1995; Bodin et al., 2009b). Indeed, the transfer of haemolymph from recently fed individuals to starved individuals inhibits the response to host-associated signals in the receivers.
11
Why some people are bitten more than others?
This is a central question in the epidemiology of diseases transmitted by arthropods. It has been addressed using various approaches, including evolutionary and ecological methods and physiological studies of insect sensory systems (Kelly and Thompson, 2000; Kelly, 2001; Mukabana et al., 2002, 2004). Many inductive studies, based on statistical surveys, have also been carried out. The differences in host attractiveness have been related to age, sex, pregnancy, hygiene, blood group, physiological state and others. However, the major factors and mechanisms involved remain unclear: predictions have not been tested and experimental studies have mostly been based on small numbers of human subjects. Indeed, the insects are generally considered as the experimental unit, despite the fact that the question concerns human subjects. So, independency is kept for the insects, but not for the source the odours, introducing pseudoreplicative bias (Hurlbert, 1984; Ramirez et al., 2000). However, despite these limitations, these studies have introduced the idea that differential repulsion rather than attractiveness may underlie the differences observed. Recently, Logan et al. (2008)
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identified 33 physiological active people from a group of volunteers exhibiting variable degrees of attractiveness to A. aegypti. Chemical analyses, combined with electrophysiological and behavioural tests, revealed allomonal effects of some volatile compounds. This is consistent with the notion that unattractiveness of individuals may result from a repellent, or attractant ‘‘masking’’ mechanism.
12
Learning and memory
Some insect species exhibit highly complex cognitive abilities. Honey bees, for instance, demonstrate complex non-elementary forms of learning, being able to learn concepts such as ‘‘symmetry,’’ ‘‘asymmetry,’’ ‘‘sameness’’ or ‘‘difference’’ (Giurfa et al., 1996, 2001; Giurfa, 2003). Their evolutionary history and their exploitation of the environment have led them to acquire capacities that were until only recently believed to be exclusive to superior vertebrates. Bloodsucking insects exhibit various behaviours that have also been subject to particular selective pressures, notably associated with obtaining blood from vertebrate hosts, a food source much more reactive and unpredictable than flowers. Given that many insects modify their behaviour adaptively through experience, and given the potential impact that vector learning and memory could have on the transmission of parasites, the cognitive abilities of blood-sucking insects are of particular interest (McCall et al., 2001; McCall and Eaton, 2001; McCall and Kelly, 2002; Bouyer et al., 2007). Currently available data on the learning capacity of these insects, however, are inconclusive for most haematophagous insects. The title of a relatively recent article by Alonso et al. (2003) seems illustrates the lack of clarity on this topic: ‘‘Are vectors able to learn about their hosts?’’ The chapter describes a series of unsuccessful attempts at inducing olfactory conditioning in mosquitoes. More recently, a critical analysis of the available evidence on learning and memory in mosquitoes demonstrated the lack of conclusive findings in most of the published work analysed, with a few exceptions (Alonso and Schuck-Paim, 2006, including Mwandawiro et al., 2000; McCall and Eaton, 2001). In most of these studies, the experiments were not designed to test learning and thus lacked adequate controls; in other studies, alternative mechanisms appear to be more likely explanations than learning.
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Repellents, how they work
A repellent has been defined as a chemical that, in the vapour phase, prevents an insect from reaching a target to which it would otherwise be attracted (Browne, 1977). Among them, DEET ([N-N]-diethyl-mtoluamide) is widely used around the world as a repellent for mosquitoes and other biting insects. A number of other compounds with a similar activity have been identified, but DEET
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remains the gold standard. However, and despite a large number of studies (Debboun et al., 2007), a plausible and evidence-based mechanism for DEET’s action remains to be elucidated (Pickett et al., 2008). Two recent studies have shed some light on how DEET works (Syed and Leal, 2008; Ditzen et al., 2008), demonstrating two major effects of DEET. Firstly, DEET seems to act as a ‘‘fixative’’ of molecules in the main mosquito attractant, 1-octen-3-ol, reducing the stimulation of ORN and giving the impression of repellency. Secondly, it stimulates olfactory neurons that normally detect linalool and other plant odours, inducing true behavioural repellency to chemical and physical hostassociated cues (Syed and Leal, 2008; Pickett et al., 2008) (Fig. 13). These findings give rise to additional questions, such as how DEET repels insects in the absence of chemical attractants, for example kissing bugs, which have no biological relationship with plants (Sfara et al., 2006, 2008).
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Conclusions and perspectives
The study of how blood-sucking insects locate their host is largely biased towards chemoreception-related aspects and the identification of biologically significant compounds. Other aspects, such as the neurobiological and molecular bases of olfaction, are also being intensively studied. This bias is justified for a number of reasons: (1) odours are able to attract insects from relatively long distances; (2) chemoreception is a well studied subject in insect sensory physiology, with data from Drosophila, moths, honeybees and other classical models being easily transferred to understand odour reception in haematophagous;
1-octen-3ol ONR
DEET/terpenoids ONR
1-octen-3-ol
Activation
No-activation
Attraction
1-octen-3-ol + DEET
No-activation
Activation
Repellency
DEET + 1-octen-3-ol
Activation
Activation
Repellency
Behaviour
FIG. 13 Sensory and behavioural effects of DEET on mosquitoes. DEET interacts with some attractants, reducing or abolishing their effect on specific olfactory neurons (ORNs). The mechanism underlying such interactions remains unknown. Furthermore, DEET specifically stimulates ORNs sensitive to plant-derived terpenoids. The combination of these two effects abolishes attraction and causes repellency (Syed and Leal, 2008; Pickett et al., 2008; Ditzen et al., 2008).
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(3) mosquitoes, the most important group of disease vector insects, seem to rely mainly on host-associated odours to find food; (4) the potential manipulation of the behaviour of disease vectors using chemical baits or by blocking odour perception provides a clear objective, despite the difficulties in applying this approach at levels beyond the individual. How sensory systems other than olfaction are used to locate a food source is poorly understood. As described above, olfaction plays a key role in the feeding behaviour of mosquitoes, whereas visual and thermal cues are relatively less important in host-seeking. For many haematophagous insects, however, other sensory signals play a major role, for example vision in tsetse flies or heat for kissing bugs, bed bugs and lice (Wigglesworth, 1941; Brady, 1972b; Flores and Lazzari, 1996; Anderson et al., 2009). A large amount of data has been, and continues to be, collected on the individual roles of attractive stimuli. Hosts provide an abundant source of a complex mixture of stimuli. The analysis of the individual components of these mixtures seems indispensable in understanding how host-insect interactions work. However, in natural conditions, stimuli are presented to their receivers as part of a multimodal complex and are subjected to various interactions. Indeed, the integration of these signals via unimodal, multimodal and exogenous/endogenous mechanisms is involved in modifying not only response thresholds, but also the way an insect reacts at a given moment. Kissing bugs demonstrate the use of all these different forms of integration. Compounds such as lactic acid and short-chain fatty acids have no effect on the behaviour of these bugs when tested alone, but their combination in the presence of sub-threshold amounts of carbon dioxide show the same level of attractiveness as a live host (Barrozo and Lazzari, 2004a). Multimodal mechanisms are used to integrate information from heat and water vapour (Barrozo et al., 2003), from mechanical and chemical cues (Barrozo and Lazzari, 2006) and from thermal and proprioceptive inputs (Flores, 2001; this chapter). Endogenous circadian clocks and exogenous temporal cues modulate responsiveness to odours associated with specific biological contexts (Barrozo et al., 2004a; Bodin et al., 2008). Finally, physiological state-dependency (i.e. moulting, reproduction and nutritional state) modulates their response to chemical and thermal stimuli, as well as their motivation to have a blood meal (Bodin et al., 2009a,b). Given such modifications of the bugs’ responses, the separate analysis of individual components, such as particular volatile compounds or signals, is only the first step in understanding their biological role. It should be noted that these multilevel interactions (i.e. unimodal, multimodal and endogenous modulation) need to be considered not only to understand how blends of volatile compounds or multimodal cues work together or how their biological effectiveness is modulated. Their consideration is also crucial in the identification of individual components in the laboratory, particularly in behavioural bio-essays. The classical example is the bimodal interaction of mechanical and chemical cues in odour-triggered anemotaxis, with neither
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the wind nor the odour evoking a response when presented separately. In other cases, the inverse can be true, for example when the presence of wind disrupts the chemical gradient created by an odour, preventing chemotaxis. In both cases, the same modalities are involved but opposite outcomes can be achieved depending on the experimental design used in the bio-essay. The choice of experimental design may be associated with a specific objective, for example testing whether a given volatile compound attracts insects located some distance away. In this case, if the candidate compound is found to be ineffective, the result will be negative in relative terms, concerning the original hypothesis tested. However, to exclude any behavioural response to a given cue, it needs to be tested under different contexts. If such experiments are not carried out, or the biological role of a particular signal is not the main focus of the study, caution should be taken in the interpretation of negative results. False-positive results mainly arise due to experimental problems and can be controlled with adequate control experiments and by avoiding pseudoreplication (Hurlbert, 1984; Ramirez et al., 2000). Similar considerations need to be taken into account when interpreting statedependency or endogenous effects. A recent analysis of a representative number of papers on the chemical ecology of blood-sucking insects showed that temporal effects (i.e. the time of the day when the experiments were conducted) were rarely taken into account (Lazzari et al., 2004). The reproducibility of such experiments is thus compromised, as are the validity of negative results or the reliability of response thresholds measured. The role of circadian clocks, exogenous temporal signals and the state-dependency of behavioural responses seem to be receiving increasing interest in studies. The same rigour applied to the statistical treatment of data should be applied to the standardization of the conditions of the subjects tested (Takken, 2005). This does not only require the synchronization of individuals and their physiological state: the relevant temporal and physiological context for the response studied, or signal tested must also be taken into account. Indeed, synchronization of the insects studied is not helpful if the experiments are carried out during a daytime period that does not correspond to the temporal window normally associated with the biological signal. This is also true for the physiological state of the insects. Thus, rather than homogeneity, it is the general context of the experiment that determines whether meaningful results may be obtained. The study of the cognitive abilities of haematophagous insects has important fundamental and epidemiological implications. It is difficult to conduct experiments on learning in a natural context due to limitations in the adequate control of many of the variables. Experimental conditions can be better controlled in the laboratory, but some common protocols, such as those used to establish associations between stimuli and positive reinforcements are not as easily tested in haematophagous insects as they are in other insects. In particular, in contrast to insects that can be rewarded with a drop of sugar solution, haematophagous insects need to bite to obtain blood. Artificial feeders may be used in cases
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where the insects pierce a membrane to obtain blood (e.g. Nu´n˜ez and Lazzari, 1990). These devices can be relatively simple, but the reward offered needs to be carefully controlled, to ensure that a known amount of reward is given and, more importantly, to control the motivational state of insects subjected to several consecutive trial sessions. Insects should thus be allowed to take only small volumes of blood, without abruptly interrupting feeding, which could otherwise cause an aversive effect. The study of non-associative forms of learning, such as habituation, does not require rewards. The insect is repeatedly exposed to a stimulus (unconditional stimulus, UCS) able to evoke an automatic response (unconditional response, UCR). The repeated stimulation causes the response to decrease gradually to no response. Such experiments require an in-depth knowledge of the behaviour of the species being studied and an appropriate set of UCS–UCRs. For instance, kissing bugs extend their proboscis when exposed to an object with a temperature close to that of a potential host. Thus, the PER is the UCR, which is associated to thermal stimulation, the UCS (Fig. 14). The PER can be habituated or rapidly extinguished by associating with negative reinforcement (Lazzari et al., unpublished). Thus, most efforts are devoted to the study of learning and memory in mosquitoes, which is perfectly justified by their paramount importance in health. However, the study of other haematophagous insects allows the use of training protocols that have been mostly validated in classical models, such as Drosophila or honeybees. More attention should be paid to similarities between most haematophagous insects and the most suitable model species, in terms of factors such as size, particular behaviour or type of locomotion, should be used for each analysis. As summarized in Fig. 15, host-seeking by blood-sucking insects is a complex behaviour, based on the exploitation of multimodal sensory cues emitted
25 °C
35 °C
FIG. 14 Proboscis extension response (PER) in blood-sucking bugs. Triatomine bugs perform PER when exposed to an object with a temperature similar to that of a warmblooded vertebrate host. PER is an unconditional response evoked by heat, an unconditional stimulus. The response habituates and can be inhibited by aversive conditioning (Lazzari et al., 2009).
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Mechanical contact aggregation signals Resting Visual cues Odours CO2 Heat Water vapour
Host leaving
Activation Stretch receptors
Circadian clocks Appetitive search
Feeding Feeding status reproduction moult
Phagostimulants Biting
Wind Host detection
Experience Heat Host finding
Visual cues Odours CO2 Heat Water vapour
FIG. 15 Factors affecting the haematophagous act in a generalized blood-sucking insect. Outside the circle of events, factors exogenous to the insect are represented, and inside it, the influences of signals originating inside the insect’s body. The insect activates at a particular moment under the influence of internal clocks. If its physiological state is the appropriate (starved, no reproductive activity) it starts searching for appetitive signals using the wind direction as reference. The presence of host-associated directional cues allows the insect detecting a host and to locate it. The sensitivity and responsiveness to this signals is modulated by endogenous factors (state-dependency). The individual experience may affect the final approach mediating host selection if more than one is present. Once on the host, the insect should chose the most appropriate place to bite, guided probably by thermal gradients over the skin indicating the degree of irrigation and proximity of blood vessels. Once a potential food is contacted, chemoreceptors located in the alimentary channel sense to presence of phagostimulants. The insect feeds until mechanical proprioreceptors indicate that the digestive tube is full of blood. Mouthparts detach then from the host skin and the gorged insect goes moves away from its host guided probably by the same cues involved in the approach. Finally, it returns to a refuge or a protected place for resting thanks to specific cues, assembling pheromones and physical contact with the substrate (thigmotaxis).
by vertebrate hosts. Additionally, the behavioural response of haematophagous insects to host signals is dependent on the motivational state of the insects, which results from the interplay of diverse physiological signals. Specific links between particular external cues and the physiological state of the insects may depend on individual experience (e.g. Pompilio et al., 2006), adding a subjective element. Further studies should now address how these elements interact, to gain a better understanding of the real abilities of haematophagous insects and to identify novel targets for controlling them.
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Acknowledgements The author wish to thank Stephen Simpson and Je´roˆme Casas for their kind invitation to contribute this chapter and for their valuable comments on the manuscript, and the University of Tours, the CNRS and the ANR (France) for their support. References Acree, F. Jr, Turner, R. B., Gouck, H. K., Beroza, M. and Smith, N. (1968). L-Lactic acid: a mosquito attractant isolated from humans. Science 161, 1346–1347. Alonso, W. J. and Schuck-Paim, C. (2006). The ‘ghosts’ that pester studies on learning in mosquitoes: guidelines to chase them off. Med. Vet. Entomol. 20, 157–165. Alonso, W. J., Wyatt, T. D. and Kelly, D. W. (2003). Are vectors able to learn about their hosts? A case study with Aedes aegypti mosquitoes. Mem. Inst. Oswaldo Cruz 98, 665–672. Altner, H. and Loftus, R. (1985). Ultrastructure and function of insect thermoreceptors and hygroreceptors. Annu. Rev. Entomol. 30, 273–295. Altner, H., Routil, C. and Loftus, R. (1981). The structure of bimodal chemo-, thermo-, and hygroreceptive sensilla on the antenna of Locusta migratoria. Cell Tissue Res. 215, 289–308. Anderson, J. F., Ferrandino, F. J., McKnight, S., Nolen, J. and Miller, J. (2009). A carbon dioxide, heat and chemical lure trap for the bedbug, Cimex lectularius. Med. Vet. Entomol. 23, 99–105. Anton, S. (1996). Central olfactory pathways in mosquitoes and other insects. Ciba Found. Symp. 200, 184–192. Anton, S. and Rospars, J. P. (2004). Quantitative analysis of olfactory receptor neuron projections in the antennal lobe of the malaria mosquito, Anopheles gambiae. J. Comp. Neurol. 475, 315–326. Anton, S., van Loon, J. J. A., Meijerink, J., Smid, H. M., Takken, W. and Rospars, J. P. (2003). Central projections of olfactory receptor neurons from single antennal and palpal sensilla in mosquitoes. Arthropod Struct. Dev. 32, 319–327. Anwyl, R. (1972). The structure and properties of an abdominal stretch receptor in Rhodnius prolixus. J. Insect Physiol. 18, 2143–2145. Aschoff, J. (1989). Temporal orientation: circadian clocks in animals and humans. Anim. Behav. 37, 881–896. Barrett, M. and Orchard, I. (1990). Serotonin-induced elevation of cAMP levels in the epidermis of the bloodsucking bug, Rhodnius prolixus. J. Insect. Physiol. 36(625– 627), 629–633. Barrozo, R. B. and Lazzari, C. R. (2004a). Orientation behaviour of the blood-sucking bug Triatoma infestans to short-chain fatty acids: synergistic effect of L-lactic acid and carbon dioxide. Chem. Senses 29, 833–841. Barrozo, R. B. and Lazzari, C. R. (2004b). The response of the blood-sucking bug Triatoma infestans to carbon dioxide and other host odours. Chem. Senses 29, 319–329. Barrozo, R. B. and Lazzari, C. R. (2006). Orientation response of haematophagous bugs to CO2: the effect of the temporal structure of the stimulus. J. Comp. Physiol. A 192, 827–831. Barrozo, R. B., Manrique, G. and Lazzari, C. R. (2003). The role of water vapour in the orientation behaviour of the blood-sucking bug Triatoma infestans (Hemiptera, Reduviidae). J. Insect Physiol. 49, 315–321.
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From Sialomes to the Sialoverse: An Insight into Salivary Potion of Blood-Feeding Insects Jose´ M. C. Ribeiro* and Bruno Arca`†,‡ *Laboratory
of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, 12735 Twinbrook Parkway room 2E-32D, Rockville, Maryland 20852, USA † Department of Structural and Functional Biology, University ‘‘Federico II’’, Naples, Italy ‡ Parasitology Section, Department of Public Health, University ‘‘La Sapienza’’, Rome, Italy
1 Insects discover blood as food 60 2 Blood feeders like fast food: A historical perspective 63 3 Problems faced by arthropods when taking blood 64 3.1 Haemostasis 64 3.2 Inflammation 66 3.3 Annoying itching 67 3.4 . . . and pain . . . 70 3.5 The attacked endothelium fights back 70 3.6 Microbiological concerns 70 4 Toward a longitudinal definition of the salivary components of blood-feeding insects 70 4.1 Enzymes 71 4.2 Receptor antagonism and platelet aggregation inhibitors 75 4.3 Physiological antagonists, primarily vasodilators 76 4.4 Kratagonists 77 4.5 Protease inhibitors 80 4.6 Anaesthetics 82 4.7 Antigen (Ag5) family members 87 4.8 Immunity-related products 87 4.9 The unexpected 88 5 Salivary diversity 88 6 The evolutionary scramble 90 7 On the odd, the paradoxical, the bizarre and the bias 94 8 Measuring the size of our ignorance 95 8.1 A forecast of the costs and time required for acquiring sialome wisdom 99 9 Salivary antigens: Epidemiological tools? 99 Acknowledgements 100 References 100
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1
JOSE´ M. C. RIBEIRO AND BRUNO ARCA`
Insects discover blood as food
The habit of blood feeding evolved several times within the Insecta, including several instances within the Hemiptera and Diptera, as well as in the Anoplura, Siphonaptera and even in the Lepidoptera (Table 1) (Ribeiro, 1995). Several scenarios can be envisioned to understand the evolution of such diet. Insects may have been nest parasites and initially predators on other insects, as proposed for triatomine bugs (Cobben, 1979; Schofield, 1979; Sweet, 1979), and still extant on the Blephariceridae, Nematocera midges the adults of which can feed on insect haemolymph (Grimaldi and Engel, 2005), or they may have approached vertebrate eyes or other mucosal membranes to feed on their secretions, as eye gnats and some moth species do. This habit may well have been the stepping stone for individuals to obtain further nourishment by piercing the skin and acquiring blood. Indeed this may have happened with all or most blood-feeding Diptera, as well as with the few blood-feeding moth species, which include close relatives that do not blood feed but lick the eyes of vertebrates (Ba¨nziger, 1970, 1975, 1979; Hilgartner et al., 2007; Zaspel et al., 2007). Many lice species, even today, do not blood feed, but live on their host’ skin feeding on dander. Obviously, insects that were already equipped with either piercing or cutting mouthparts were provided with an important preadaptation to remove blood from vertebrates and were favoured by natural selection. The insect orders feeding on blood include both holometabolous (those having full metamorphosis, with immature instars that are very different and occupy a different niche as compared to adults) and hemimetabolous organisms (where immature forms and adults inhabit the same niche and are not very different from each other). Accordingly, all haematophagous hemimetabolous orders feed exclusively on blood and nothing else. In the case of holometabolous orders (all flies and fleas), only the adults take blood meals, except for the Congo floor maggot. Within the Nematocera, it is also common that only the adult female will feed on blood whereas both adult sexes will feed also on sugar solutions. The adults of haematophagous Brachycera flies feed exclusively on blood. Blood is not an ideal meal because it is heavily unbalanced towards proteins, produce dangerous amounts of pro-oxidating haem (Dansa-Petreski et al., 1995), and lacks or has little amount of some vitamins. However, it can provide, in the form of proteins, the large amounts of amino acids needed for egg development. In mosquitoes and sand flies, only adult females are haematophagous and blood feeding is promptly followed by egg development. Those insects that feed solely on blood, such as the bugs and tsetse, require the presence of bacterial endosymbionts to survive. Killing the symbionts with antibiotics prevents bugs from developing to adults and suppresses tsetse reproduction and immunity, a deleterious effect that can be reversed with B vitamin supplements (Wigglesworth, 1936; Baines, 1956; Hill et al., 1973, 1976; Pais et al., 2008).
TABLE 1 List of BFI Families Order Diptera
Sub-order
Common name
Genera
Species
References
Nematocera
Sand flies Mosquitoes Black flies Biting midges Frog-biting midges Tsetse Horse flies Stable flies Keds Bat flies Blowfly Bed bugs Bat bugs Kissing bugs Lice Fleas Moth 17
6 36 24 4 1 1 5 3 19 31 1 23 5 14 42 239 1 455
70 3450 1571 1000 97 23 4400 50 130 520 5 91 32 118 490 2500 8 14,555
Lane and Crosskey (1993) Lane and Crosskey (1993) Lane and Crosskey (1993) Lane and Crosskey (1993) Borkent (2008) Lane and Crosskey (1993) Lane and Crosskey (1993) Lane and Crosskey (1993) Maa (1964) Maa (1965) Lane and Crosskey (1993) Lane and Crosskey (1993) Lane and Crosskey (1993) Lane and Crosskey (1993) Lane and Crosskey (1993) Lane and Crosskey (1993) Zaspel et al. (2007)
Brachycera
Hemiptera
Anoplura Siphonaptera Lepidoptera Total
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The surface availability of blood varies considerably among vertebrates. There, blood not only serves a nourishing tissue function but also contributes to the organism’s thermoregulation. Fresh water turtles, for example, have a venous plexus on the basis of their carapace that serves a heat exchange function with the environment. On the other extreme, humans can use their whole body skin as a thermoregulatory organ. Surface capillary loops are attended by a plexus of arterioles and venules and blood flow to these capillaries is controlled by the arteriolar precapillary sphincter. There are 20 times more vessels in the skin than needed for nourishment of its cells, so only 5% of these capillaries need to be with active blood flow, but they can all be turned on when the organism needs to lose heat. The normally closed state of the precapillary sphincter is affected by a sympathetic tonus, through the neural release of the smooth muscle contracting agent norepinephrine. Arteriolar-to-venous anastomosis serves as bypass to conduct blood directly from arterioles to venules in the case there is no flow to the capillaries (Fig. 1) (Abramson, 1989; Braverman, 1997). Other mammalians use specialized skin regions for thermoregulation: this is the case for rodents, whose tail and ears can be more vascularized as compared to other skin regions, as those in their furry backs or abdomens. Accordingly, the blood sources that can be tapped by haematophagous insects can vary from surface capillaries to deeper venules and arterioles. The accessibility to the skin surface and their vessels can also be turned more difficult to blood suckers by the presence of scales, feathers and fur, which can completely prevent access to some blood-feeding insects (BFI). The mechanics of blood feeding by insects have been described in classical studies using histology of fixed insects while feeding on the skin of their hosts (Short and Swaminath, 1928), or directly by live observations using frog feet or the transilluminated ear of the mouse (Gordon and Lumsden, 1939; Gordon and Crewe, 1948; O’Rourke, 1956; Dickerson and Lavoipierre, 1959; Lavoipierre et al., 1959). From these studies two modes of feeding became apparent. Feeding can either occur from haemorrhagic pools or haematomas that accumulate in the tissues following skin lacerations (pool feeders or telmophagous insects), or directly from a cannulated venule or arteriole (vessel feeders or solenophagous insects) (Lavoipierre, 1964). In some cases, both modes of feeding can be observed in the same species (O’Rourke, 1956). Some insects, such as the sand flies, have very short mouthparts that penetrate no more than 0.5 mm into their host skin (Adler and Theodor, 1926; Lewis, 1975). Accordingly, they can only feed from superficial pools of blood (haematomas) formed from haemorrhages following the laceration of capillaries. Other insects like the bugs and mosquitoes can penetrate their host skin for several millimetres and cannulate the arterioles and venules deeper in the skin circulatory plexus. The tip of the mouthparts of triatomine bugs have only 10 mm in diameter (Lavoipierre et al., 1959); accordingly, only one erythrocyte can pass at a time, showing the limits achieved by evolution in developing a fine blood drawing needle. On the other hand, Tabanids have veritable cutting
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scissors that can lacerate several vessels simultaneously, potentially forming a large haematoma, as in cattle feeding horse flies, or tabanid species that specialize in feeding on the venous plexus of turtles (DeGiusti et al., 1973). It is important to notice that blood feeding does not initiate immediately after the insect pierces their host skin. Sometimes blood ingestion may start in a few seconds, but most commonly it may take 1 min or more for the insect to find blood, and then several more minutes to feed to repletion. The period of time from the initial penetration of the skin to the initial ingestion of blood is known as the exploratory phase or probing time (Gillett, 1967; Ribeiro et al., 1985a). Saliva is continuously ejected during this exploratory phase of probing, while the insect mouthparts move actively inside their host skin (Friend and Smith, 1971; Soares et al., 2006). It is during this phase that saliva interacts with host tissues. Saliva is also continuously discharged during the blood ingestion phase, which ends up mostly in the insect gut, reingested with the food (Ribeiro and Garcia, 1980).
2
Blood feeders like fast food: A historical perspective
Although saliva of blood-sucking arthropods (BFA) was known to contain anti-clotting activities near a century ago (Cornwall and Patton, 1914), its role was postulated to have a post-meal effect by preventing blood clotting in the insect mouthparts and gut (Lester and Lloyd, 1928), or to have no positive function at all, since allergic reactions to insect bite were obviously nonadaptive (De Meillon, 1949). The classical method to study the role of any gland in animal physiology was to remove it, or prevent its juices from reaching their target, and observe its effect on the impaired animal (Pavlov, 1902). Such protocols applied to insects led to the conclusion that saliva played no important role in blood feeding because insects (tsetse and mosquitoes) could still feed after such treatment (Lester and Lloyd, 1928; Hudson et al., 1960; Hudson, 1964; Rossignol and Spielman, 1982). Indeed, the last sentence of a 1982 paper studying salivation in a mosquito was ‘‘We conclude that saliva is not prerequisite to blood feeding’’ (Rossignol and Spielman, 1982). These studies, however, allowed a long contact time between the surgically treated insects and their hosts, and did not measure the feeding kinetics. When similar experiments were repeated with mosquitoes and triatomine bugs, but at this time measuring the effect of salivation on the speed of blood meal acquisition, a significant role of saliva in feeding was discovered (Mellink and Van Den Boven Kamp, 1981; Ribeiro and Garcia, 1981; Ribeiro et al., 1984a). Notably, salivation significantly reduced the time from the initial mouthpart penetration of the skin to the first observed ingestion of blood, the so-called probing time (Gillett, 1967; Mellink and Van Den Boven Kamp, 1981; Ribeiro et al., 1985a; Ribeiro, 1988). Speed of blood meal acquisition is important to insects because they are at a great risk of predation during their stealing act. The role of saliva was
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then postulated to function by antagonizing host haemostasis, allowing for haematomas to form at the feeding site, which could themselves be the source of the blood meal in pool-feeding insects, or, when the haematomas were rapidly sucked, their walls collapsed and drove the mouthparts towards the ruptured vessel, in the case of vessel-feeding insects (Ribeiro, 1987, 1988, 1995). In the past 25 years anti-platelet and vasodilatory activities, in addition to anti-coagulants, were described in several blood-sucking insects (Champagne, 2004; Valenzuela, 2004); vector saliva was also discovered to enhance pathogen transmission and thus became an attractive target for developing vaccines against vector borne diseases (Mejia et al., 2006; Oliveira et al., 2006; Gomes et al., 2008). At less than a decade ago, the omics revolution kicked in, in the form of whole genomes, salivary gland transcriptomes and tissue proteomes, generating the sialomes (from the Greek, sialo ¼ saliva) which changed the direction we now do science: pre-revolutionary times started with a bioactivity identified in an organ homogenate and ended with the characterization of the molecule responsible for the biological effect, but now starts with a recombinant molecule identified in a transcriptome, often without any clue regarding its function, and ends with the determination of the molecule’s function and verification of its presence in saliva (Ribeiro and Francischetti, 2003). This chapter will concentrate on the past 5 years advances on the role of haematophagous insect saliva in antagonizing their hosts’ haemostasis and inflammation, specifically the processes that occur within 15 min of skin injury. An attempt will also be made to classify the modes of action of the various antagonists so far characterized, and of the genomic and evolutionary mechanism that allowed the concoction of the haematophagous ‘‘magic potion’’. Immunity-related products and the potential to use salivary proteins as markers of exposure to bites of insect disease vectors will be mentioned only briefly. The reader is directed to other reviews related to the physiological role of saliva in ticks (Bowman and Sauer, 2004; Steen et al., 2006; Hovius et al., 2008; Francischetti et al., 2009), specifically those related to host immunity (Brossard and Wikel, 2004), and to the investigations related to vaccine development using salivary proteins as antigens to disrupt tick feeding or modify transmission of vector borne diseases, such as leishmaniasis (Valenzuela, 2004; Mejia et al., 2006; Nuttall et al., 2006; Titus et al., 2006).
3 3.1
Problems faced by arthropods when taking blood HAEMOSTASIS
The idea that saliva of BFA counteracted their hosts’ blood clotting, platelet aggregation and vasoconstriction drove much of the early research on this area. Earlier reviews focused on the components of this haemostasis tripod (Ribeiro, 1987; Law et al., 1992) to develop experiments leading to the discovery first of
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the biological activities of saliva or salivary gland homogenates, followed then by the isolation of the responsible molecules. The recognition of the physiological redundancy of haemostasis led to the consequent idea that a ‘‘magic bullet’’ is virtually impossible to function against such a system, and that BFA opted instead for a ‘‘magic potion’’ to accomplish such a pharmacological task (Ribeiro, 1995). This salivary anti-haemostasis paradigm was enlarged to include the contributions of inflammation, tissue repair or angiogenesis and immunity (Ribeiro and Francischetti, 2003; Francischetti et al., 2009), mostly to account for the effects of tick saliva. Results from the last 7 years, mostly driven by unexpected findings deriving from sialome discovery projects, are pointing out that fast BFI have a plethora of antagonists targeting neutrophils or their products, as well as directed against other arms of inflammation that have been previously neglected. Some of them also have enzymes that attack the extracellular matrix (ECM), which would be natural for ticks, but unexpected in fast-feeding insects. Within this framework, we will attempt in this section to provide an expanded (but simplified) description of the haemostasis process to include the role of inflammation, including the participation of neutrophils, mast cells and complement, as they may affect those critical 15 min taken by the majority of haematophagous insects to blood feed. It is not our goal in this chapter to produce a detailed account of these processes, which are the subject of textbooks, but rather to describe their general outlines in the context of the vertebrate barriers to blood feeders, and to direct the reader to more detailed literature. Haemostasis is the natural response to vascular injury that serves the purpose of arresting bleeding and it is based on the triad of platelet aggregation, blood clotting and vasoconstriction (Colman et al., 2005). Inflammation comprises the larger spectrum of physiological responses following tissue (not only vascular) injury, and it was classically described by Cornelius Celsus in the first century (Collier, 2008) as rubor (redness) and tumour (swelling) with calor (heat) and dolour (pain). Notice that redness and heat both result from vasodilation: actually, while at the site of vascular injury there is vasoconstriction due to haemostasis, injured tissue in the vicinity of non-injured vascular tissue brings about vascular changes that are of a vasodilatory nature. Accordingly, a puncture wound shows vasoconstriction at the site of the injury due to haemostasis reactions, and vasodilatation in the nearby areas due to inflammation. We will proceed to describe the events occurring at the site of tissue injury, such as one being created by a hungry kissing bug (Fig. 2). To our purposes, the immediate relevant events following vascular and tissue injury are the mixing of blood components with extravascular tissues, and the release of ATP and ADP by broken cells to the extracellular milieu. These nucleotides have cytoplasmic concentrations at the millimolar level and only sub-micromolar concentrations extracellularly. Within seconds of exposure to micromolar concentrations of ADP, platelets expose surface receptors to fibrinogen, a multi-domain protein that clamps platelets together. The mixing of extra
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66 EP D
PCS A V
FIG. 1 Diagram of human skin showing the epidermis (EP), dermis (D), arterioles (A), venules (V) and precapillary sphincter (PCS). Based on Abramson (1989).
and intravascular compartment components allows platelets to interact with collagen, which by a specific signalling pathway also induce fibrinogen receptors to be expressed in the platelet surface. Exposure of plasma components to membrane bound tissue factor, present in most extravascular cells, promotes activation of the extrinsic pathway of blood clotting, with activation of prothrombin to thrombin, which cleaves fibrinogen into fibrin, leading to blood clotting. Thrombin activates the platelet protease-activated receptor (PAR) which independently of the ADP or collagen receptors, also activate platelets to expose their fibrinogen receptors. The ATP released by broken cells also activates neutrophils to aggregate and to adhere to the endothelium (Colman et al., 2005). Accordingly, within the first minute of vascular injury, platelets had acquired the signalling of three different receptors that induce platelet aggregation, and neutrophils are also being warned. 3.2
INFLAMMATION
But not only platelet aggregation occurs during this first minute. Platelets under those three strong stimuli (ADP, collagen and thrombin) release their granule contents, which include 5-hydroxytriptamine (5-HT, serotonin), epinephrine and norepinephrine (NE), ADP, inorganic polyphosphate and the chemokine precursors platelet factor 4 (PF-4) and b-thromboglobulin (von Hundelshausen et al., 2007). ADP, collagen and thrombin, and also less effectively 5-HT and NE, activate platelet membrane phospholipases that release arachidonic acid (AA) from membrane phospholipids. AA is converted by platelet cyclooxygenase to prostaglandin H2 (PGH2) which is then transformed by platelets into thromboxane A2 (TXA2) and by endothelial and smooth muscle cells to prostacyclin (PGI2). During the granule exocytosis process, the platelet membrane inverts its membrane polarity, exposing negatively charged phospholipids in its outer surface. The polyphosphates and the negatively charged membrane phospholipid surface serve as scaffolds for the assembly and activation of the Xase and prothrombinase complexes of the blood coagulation cascade, thus connecting platelets to blood clotting. TXA2, NE and 5-HT are potent vasoconstrictors, thus decreasing the
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blood flow to the site of injury. Aggregated platelets also expose the P-selectin protein on their outer surface. P-selectin promotes neutrophil binding to platelets and neutrophil activation. The platelet-derived chemokine precursors are processed within minutes by neutrophil proteases, cathepsin G (CG) in particular, which further activate neutrophils. Neutrophil CG can also activate platelets in a similar way as thrombin does, by activation of PAR. Activated neutrophils expose additional adhesive surface molecules, release their granules, which include ATP and proteases, and produce the lipidic mediators PAF (platelet-activating factor), and leukotriene B4 (LTB4). PAF is a potent activator of platelet aggregation and neutrophil activation. LTB4 is a potent neutrophil activator. It is to be noted also that activated neutrophils release antimicrobial peptides (AMP) and induce a respiratory burst producing superoxide radicals, toxic to microbes. Taking it all together, platelets and neutrophils are very actively at work within the first minute of vascular injury. Within 1 min, a platelet plug containing adhered neutrophils is formed in the vicinity of the injury, this platelet plug is consolidated by a fibrin network that gives more rigidity to the plug; 5-HT, NE and TXA2 contracts the arterioles’ and venules’ smooth muscle (capillaries have no smooth muscle). Within 1 min, the full haemostasis triad is in place. (For a review on inflammation, see Chapter 2 of Kumar et al., 2004.) It is interesting to point out that the most immediate triggers of haemostasis, ADP and ATP, are themselves products of degranulating platelets and neutrophils, representing a positive feed back system (shown by the double arrows in Fig. 2). Notice also that the agonists TXA2, NE, 5-HT, PAF and LTB4, produced by platelets and neutrophils, are also self-activating agonists, representing another group of molecules in the positive feed back loop of platelet and neutrophil activation. Neutrophil proteases also represent a positive feed back loop because they activate platelet-derived chemokine precursors. With such potent feed back loops, how come a needle puncture does not lead to clotting and platelet aggregation of the whole vascular system? First, the ADP and ATP concentrations produced by injured cells decrease as one moves away from the injury site. Secondly, endothelial cells and smooth muscle cells convert platelet produced PGH2 to PGI2 which is a very potent platelet aggregation inhibitor and vasodilator. Third, the activated clotting enzymes all have a relatively brief life, as there are many endogenous inhibitors (such as anti-thrombin III, a2-macroglobulin, thrombomodulin, etc.) that regulate their activity, so clotting will stay active only in the vicinity of a strong promoter. This demonstrates the criticality of the initial mix of agonists and haemostasis mediators in the propagation of the haemostatic reaction, these critical agonists being targets of salivary components of haematophagous insects. 3.3
ANNOYING ITCHING
A vast literature exists on the annoying allergic effects of insect saliva (Peng and Simons, 2007; Hoffman, 2008). The salivary potion injected by BFA into the vertebrate host skin can induce cutaneous reactions that are immunological in
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ATP
TF Collagen ADP
Neutrophil activation
CG Platelet aggregation Thrombin PPi Blood clotting
Salivary antigens Tryptase
PF-4 β-TG
PAF
NE TXA2 5HT ULvWF Vasoconstriction
LTB4 PSelec
Complement Mast cell activation degranulation C5a
CysLT H
Endothelium activation
Bk Edema B
H + ATP + 5-HT + BK + LTB4 =
Itch, pain
FIG. 2 (A) Simplified diagram of vertebrate haemostasis and inflammation and (B) agonists of pain. Abbreviations: 5-HT, serotonin; ADP, adenosine diphosphate; ATP, adenosine triphosphate; BK, bradykinin; b-TG, beta-thromboglobulin; C5a, anaphylatoxin; CG, cathepsin G; CysLT, cysteinyl leukotrienes; H, histamine; LTB4, leukotriene B4; NE, norepinephrine; PAF, platelet-activating factor; PF-4, platelet factor 4; PPi, inorganic pyrophosphate; PSelec, P-selectin; TF, tissue factor; TXA2, thromboxane A2; ULvWF, ultra-large von Willebrand factor. (Photo of Rhodnius prolixus feeding on the arm of Andrew Spielman taken by Philippe Rossignol).
nature and may vary, in different individuals, from no reaction to severe hypersensitivity allergic responses. Several factors, including host genetic background, immunological profile, age and history of exposure can contribute to type and intensity of the response. Most typically, the reaction to insect bites of humans and experimental animals evolves with repeated exposure and can be described as a five stage sequence of skin reactivity (Mellanby, 1946; Feingold, 1968; Lehane, 2005). Initially, after exposure to bites of naı¨ve hosts, no reaction is observed (stage I). This is followed by a delayed-type reaction that appears 12–24 h after the bite and consists of erythema, often associated with papules and pruritus (stage II). As exposure continues an immediate response causing erythema and itching appears within 15–20 min, and it is followed by the delayed response (stage III). This delayed reaction tends then to disappear leaving only an immediate response lasting for a couple of hours (stage IV). Finally, after repeated long-term exposure, the host may develop desensitization and no skin reaction is observed anymore (stage V). Desensitization to mosquito bites has been observed to occur in experimental conditions in rabbits and
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humans (Peng and Simons, 1998). In natural conditions desensitization is also acquired, but it may take long time or never occur, and the underlying immune mechanisms are still not completely understood. These cutaneous reactions are associated, in humans and other vertebrates, to both humoral and cellular responses that result in circulating anti-saliva IgG and IgE antibodies. Understanding the mechanisms involved in these immune responses may be relevant both for the development of vaccines based on salivary antigens (Titus et al., 2006) and for the diagnosis and immunotherapy of severe allergic reactions (Peng and Simons, 2007). Within the limits of our chapter, aimed at the problems insects face while feeding, immediate-type hypersensitivities are of importance because they can add more mediators to the haemostasis reaction, thus drying up the source of the meal, and perhaps more importantly, by promoting host behavioural responses that can minimally disrupt feeding and maximally leading to the insect’s death. It is indeed quite amazing that some plump triatomine bug species inhabit the nests of insectivorous rodents, or bats, or lizards, from which they obtain their sole source of nourishment. Figure 2, above, lists two host immune-mediated mechanisms producing effects within minutes of antigen contact; these are of humoral (complement) and cellular (mast cell) natures. The complement system comprises a complex cascade of proteases that may be activated by four different pathways, the so-called classic (antibody dependent), alternative (involving C3 recognition of carbohydrate surfaces), the colectin pathway (also involving carbohydrates, such as mannan), and the recently discovered activation of C3 by thrombin, linking the clotting and the complement pathways (Paul, 2008). The final result of complement activation is the formation of a lytic complex on the surface of invading organisms, but it can also occur in pure soluble form, without such substrates, as for example with antigen–antibody complexes (dependent on the IgG subclass) or soluble polysaccharides and plasma. It is of our concern that during the proteolytic activation of complement the C3 and C5 components are hydrolysed producing the relatively small peptidic fragments C3a and C5a, which are potent activators of endothelial cells as well as mast-cell degranulators. Skin mast cells appear to have evolved with the sole purpose to make the life of a blood sucker miserable and dangerous, and perhaps to provide a vain attempt to protect their owners from infection of the various lethal or disabling vector borne pathogens. When specific IgE or other subclasses of IgG on the surface of mast cells meet a divalent antigen that can crosslink two Ig receptors, a signalling cascade starts that can degranulate the whole contents of the cell (Paul, 2008). This is quite an amazing amplification, as a single molecule can trigger an event that can cause a macroscopic effect. A single degranulating mast cell can be felt by itching and its resulting small urticaria is quite visible to the naked eye. In addition to histamine (H) mast cells also release proteases, including tryptase that can activate PAR on platelets and endothelium, and they also synthesize the arachidonate-derived cysteinyl leukotriene LTC4, which is metabolized to the
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still active LTD4 and LTE4 leukotrienes. These leukotrienes are potent vasodilators, and additionally activate the endothelium, which is also redundantly activated by various other agonists described above, such as histamine, serotonin, bradykinin (BK), thrombin, cathepsin G, tryptase and anaphylatoxins. 3.4
. . . AND PAIN . . .
Pain and itching can be produced by direct stimulation of certain nerves, as well as by the action of several inflammation agonists which initiate a nerve impulse interpreted as pain or itch by the central nervous system. Among these agonists are BK, ATP, 5-HT, H and LTB4 all of which described above as mediators of haemostasis and inflammation (Fig. 2B) (Millan, 1999). 3.5
THE ATTACKED ENDOTHELIUM FIGHTS BACK
Activated endothelial cells expose on their surfaces P-selectin, and E-selectin, that increase the adhesion of neutrophils (Kumar et al., 2004), and also secrete ultra-high molecular weight von Willebrand factor (vWF), that can form heavy complexes functioning as platelet activators. Importantly, in the activated endothelium the connections between neighbouring endothelial cells are modified, allowing larger spaces between them. Fluid then flows from intra- to extravascular space creating oedema. Neutrophils go with the flow, following the chemical gradient created by the anaphylatoxins, LTC4 and LTB4 which are potent chemotactic substances. Neutrophil and platelet products can also activate resident macrophages to produce PGE2, a potent skin vasodilator. This process of endothelium activation and oedema formation takes a few minutes to occur, but is within the time frame of most insect blood feeders. 3.6
MICROBIOLOGICAL CONCERNS
To obtain blood, insects necessarily have to break their host’s skin, which is a barrier to pathogen infection. The opportunity arises for skin surface microbes to contaminate their host circulation, and the ingested blood meal. The insect digestive system may also be previously contaminated by microbes that might develop with the blood meal. Perhaps for this reason, a common finding in sialotranscriptomes of BFA is a plethora of proteins known to have antimicrobial activity.
4
Toward a longitudinal definition of the salivary components of blood-feeding insects
From the analysis of the several spitomes done so far (Table 3), some generalizations can be made regarding the composition of the salivary potion of BFI. These can be summarized in the following arbitrary categories: (a) enzymes;
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(b) receptor antagonists; (c) physiological antagonists, with an important subcategory of vasodilators; (d) kratagonists; (e) protease inhibitors; (f ) antigen 5 proteins; (g) immunity-related products, including antimicrobial peptides; (h) anaesthetics and (i) the unexpected. 4.1
ENZYMES
Salivary enzymes can help haematophagy by destroying agonists of haemostasis and inflammation, by destroying the final products of clotting, such as fibrin, by activating their host plasminogen, by enlarging the feeding cavity in telmophagous insects and helping the spread of salivary components into the skin matrix, and by potentially interfering the signalling pathway of their hosts. 4.1.1
Apyrase
All mammalian BFI studied so far have large amounts of salivary apyrase (ATP diphosphohydrolase) activity. The apyrase reaction degrades both ATP and ADP to AMP and orthophosphate, thus inhibiting platelet aggregation. This activity was associated with the first papers describing platelet aggregation inhibitors in the saliva of Rhodnius and tsetse (Ribeiro and Garcia, 1980; Smith et al., 1980; Mant and Parker, 1981) and later was described in several other BFA (Ribeiro et al., 1984b, 1985b, 1986, 1989, 1990, 1991; Kerlin and Hughes, 1992; Cupp et al., 1993, 1995; Marinotti et al., 1996; Valenzuela et al., 1996; Cheeseman, 1998). Interestingly, lizard (Ribeiro et al., 1989) or birdfeeding (Ribeiro, 2000) species have very small amounts of the enzyme activity, which is in accordance with platelets being a mammalian invention that uses ADP as a main agonist; accordingly those insects selecting mammals on their menu were better off if they found ways of destroying it. Male mosquitoes, which do not blood feed, and mosquito species that lost their blood-feeding ability also have very little amount of salivary apyrase activity (Ribeiro et al., 1985b; Calvo et al., 2008). Mosquitoes and triatomine bugs of the Triatoma genus (but probably not Rhodnius genus) elected the 50 -nucleotidase family to take care of host ADP and ATP (Champagne et al., 1995b; Faudry et al., 2004; Sun et al., 2006). This appears also to be the case with tabanids, where a collagen-induced platelet aggregation inhibitor named chrysoptin is a member of the 50 -nucleotidase family, although the authors reported this protein, possibly erroneously, as a specific collagen receptor inhibitor (Reddy et al., 2000). Bed bugs and sand flies (Valenzuela et al., 1998, 2001b) selected the Cimex type of apyrase, then a novel protein family that was found later to exist ubiquitously in eukaryotes (Failer et al., 2002). Fleas appear to have elected the CD-39 family of nucleotidases (Andersen et al., 2007), although the evidence is so far circumstantial. Salivary apyrase are so abundant on these organisms that, unusually for enzymes, they are displayed as a strong protein band in SDS–PAGE experiments (Francischetti et al., 2002c;
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Valenzuela et al., 2002b, 2004). The presence of different protein families to serve the apyrase function in BFA is an example of convergent evolution that occurred when unrelated BFI had to adapt to the menu change that occurred with the extinction of dinosaurs and irradiation of mammals, at 60 millions years ago (MYA). 4.1.2
Additional nucleotidases
In addition to apyrase, Lutzomyia longipalpis sand flies have been found to contain secreted 50 -nucleotidases (Charlab et al., 1999; Ribeiro et al., 2000b), thus further degrading AMP, the final product of the apyrase reaction, into adenosine. Also mosquito saliva carries the product of a second 50 -nucleotidase family member (Arca` et al., 1999; Lombardo et al., 2000; Ribeiro et al., 2004b, 2007) that may function either as an alternative apyrase or like in L. longipalpis, as secreted salivary 50 -nucleotidase. Adenosine is a potent vasodilator (Collis, 1989) and platelet aggregation inhibitor (Dionisotti et al., 1992). However, adenosine is also a powerful degranulator of mast cells and basophils (Lohse et al., 1987; Tilley et al., 2000), and perhaps for this reason, salivary adenosine deaminase has also been found in sialotranscriptomes of sand flies (Charlab et al., 1999; Valenzuela et al., 2004; Anderson et al., 2006; Kato et al., 2006), mosquitoes (Ribeiro et al., 2001; Valenzuela et al., 2002b) and tsetse (Li and Aksoy, 2000; Li et al., 2001). In one case, the recombinant enzyme from Phlebotomus duboscqi was produced and its kinetics characterized (Kato et al., 2007). The function of this enzyme may be related to conversion of adenosine to inosine, which is a weaker agonist of the adenosine receptor (Tilley et al., 2000). Perhaps because inosine is still a mast-cell agonist, Aedes aegypti contains large amounts of a salivary purine hydrolase, which hydrolyses inosine to hypoxanthine and ribose (Ribeiro and Valenzuela, 2003), thus completely destroying any remaining purinergic activity. No such activity was found in Culex quinquefasciatus, Anopheles gambiae or Lutzomyia longipalpis sand flies. 4.1.3
Peroxidase
So far a true vasodilator agonist was not yet found in anopheline mosquitoes. However, their saliva inhibits norepinephrine-induced aortic ring contractions with a peroxidase enzyme that destroys catecholamines (Ribeiro and Nussenzveig, 1993; Ribeiro et al., 1994; Ribeiro and Valenzuela, 1999). Norepinephrine is responsible to keep more than 95% of human skin capillaries normally closed, thus destruction of norepinephrine may bring about vasodilatation. In the presence of hydrogen peroxide, this peroxidase can also destroy 5-HT but it is not clear whether this is physiologically relevant, since the concentration of H2O2 is low in tissues, but could be high near activated neutrophils. This enzyme is not found in Aedes or in Culex sialomes.
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73
PAF hydrolases and phospholipases
Two insects so far have salivary enzymes that hydrolyse PAF. A phospholipase C activity-inhibiting PAF-induced platelet aggregation was found in saliva of C. quinquefasciatus (but absent of Ae. aegypti and An. gambiae) (Ribeiro and Francischetti, 2001), and a PAF acetylhydrolase was found in cat flea salivary gland homogenates (Cheeseman et al., 2001). This flea enzyme also displays an esterase activity. None of the enzyme activities have been molecularly characterized. In Ae. aegypti, an esterase was demonstrated to be secreted in saliva from both males and female mosquitoes (Argentine and James, 1995), suggesting it is not related to obtaining a blood meal. Its biological substrate remains unknown. cDNAs coding for diverse phospholipases with signal peptide indicative of secretion have been found in insect sialotranscriptomes, but none have been functionally characterized so far. Among these phospholipases are included phospholipase A2, which is known to have pro-inflammatory effects in the skin, with the production of free arachidonic acid (AA) and lysophosphatidylcholine (LPC). Most tissues have cyclooxygenases that can convert AA to PGH2, but the final product of the unstable PGH2 depends on the tissue, as indicated above. Mammalian skin has a PGD2 synthase that converts PGH2 to PGD2, a potent vasodilatory and platelet aggregation inhibitor (Ujihara et al., 1988; Braun and Schror, 1992; Morrow et al., 1992; Warren et al., 1994). Phospholipases can also have haemolytic activity. In ticks, a salivary phospholipase A2 activity has been identified and proposed to be important in the lysis of erythrocytes (Bowman et al., 1997; Zhu et al., 1997, 1998). Interestingly, saliva of Rhodnius prolixus contains LPC which is proposed to have an antihaemostatic function (Golodne et al., 2003), but LPC is also a haemolysin, as its name testifies. 4.1.5
Inositol phosphatase
The sialotranscriptomes of the kissing bugs R. prolixus, T. infestans and T. brasiliensis revealed transcripts coding for a protein with high similarity to inositol phosphatases. This finding is so far unique among kissing bugs. We initially thought that the enzyme could represent the salivary apyrase of Rhodnius but ADPase activity of the recombinant protein was not found, but rather the predicted inositol phosphatase activity (Andersen and Ribeiro, 2006). Vertebrate homologues of this enzyme are important regulators of the inositol triphosphate signalling cascade, because the enzyme activity hydrolyses phosphoinositolphosphate(3,4,5)P3 (PIP3). Indeed the enzyme is an important regulator of the activity of cytokines and immune cells (Kalesnikoff et al., 2003; Kashiwada et al., 2007; Sly et al., 2007; Harris et al., 2008; Leung et al., 2008). There is one problem only, the salivary enzyme is on the wrong side, PIP3 is an intracellular mediator. We here raise the speculation that saliva of kissing bugs,
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by its content of LPC may actually transfect cells with some of their salivary components, for example Rhodnius LPC may induce the salivary inositol phosphatase to become intracellular and inactivate platelets, mast cells and other immune cells. Indeed Triatoma infestans saliva is very soapy in nature, foaming prolifically if air is flowed in a solution containing it (unpublished observations). 4.1.6
Other hydrolases
Transcripts coding for hydrolases, in particular serine proteases, have been regularly found in sialotranscriptomes of BFA. These enzymes could have a role in degrading fibrin, or in activating plasminogen, a vertebrate protein that is proteolytically activated to an enzyme that has high specificity for fibrin, and is part of the self-regulation of the clotting process. Tabfiglysin from Tabanus yao is one of such BFI salivary enzymes shown to degrade fibrinogen, and possibly fibrin (Xu et al., 2008), an activity similar to that of tick metalloproteases (Francischetti et al., 2003). Bioinformatic analysis of many mosquito and Triatoma salivary serine proteases revealed enzymes similar to chymotrypsin, suggesting they could have a role as an elastase, or in degrading other components of the ECM. Support for a salivary function associated with ECM degradation is also found in the form of hyaluronidases that have been described in C. quinquefasciatus mosquitoes (but absent in Aedes or Anopheles), in sand flies, black flies, Culicoides and tabanids (Charlab et al., 1999; Ribeiro et al., 2000a; Cerna et al., 2002; Campbell et al., 2005; Volfova et al., 2008; Wilson et al., 2008; Xu et al., 2008). Except for Culex, which may be a vessel feeder, all other insects are strict pool feeders, indicating that telmophagy may promote evolution of enzymes that digest ECM constituents, with the benefit of creating an enlarged feeding cavity. Hyaluronidase decreases the viscosity of the ECM, thus favouring the spread or diffusion of salivary agonists in the host skin and, for example, favouring diffusion of vasodilators from the surface of the skin to the deeper precapillary sphincter in the arteriolar plexus. C. quinquefasciatus also possess a salivary endonuclease that was functionally characterized (Calvo and Ribeiro, 2006), and found to be abundantly secreted in saliva. Endonucleases may help reduce skin viscosity caused by DNA released from broken cells, and may additionally produce pharmacologically active DNA products. Transcripts coding for endonucleases have also been found in sand fly and tsetse sialotranscriptomes, but their recombinant forms have not yet been characterized. Ticks have a salivary dipeptidyl peptidase that is quite active on hydrolysing bradykinin (Ribeiro and Mather, 1998). The same enzyme or a different one is also capable of completely inactivating the anaphylatoxins (Ribeiro and Spielman, 1986). It is possible that similar enzymes may be found in insect saliva in the future. Sand fly sialotranscriptomes have revealed the presence of transcripts coding for pyrophosphatases with a signal secretion, but the enzymatic activity was
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never reported. This enzyme could have a possible role in degrading the polyphosphates secreted by platelets, which activate blood clotting (Ruiz et al., 2004). Since this is quite a novel discovery in platelet physiology, the discovery that a sand fly cares about it would be a confirmation of an ancient role of platelet polyphosphates in mammalian haemostasis. The reader should be cautioned, though, that not all serine proteases found in sialotranscriptomes should have a blood-feeding role. They could represent proximal serine proteases associated with innate immunity, either the prophenoloxidase or the TEP cascades. This is reinforced in mosquitoes and other nematocera by the finding of other proximal members of this cascade, such as pattern recognition molecules of the ficolin and C-lectin families, for example (although some lectins of both types were found to be selectively expressed in adult female salivary glands suggesting a role in blood feeding) (Arca` et al., 2005, 2007; Ribeiro et al., 2007). Non-blood-feeding male mosquitoes and mosquitoes of the genus Toxorhynchites also have many of these salivary enzymes, but lack those associated with blood feeding such as the D7 proteins (Calvo et al., 2006b, 2008). 4.2
RECEPTOR ANTAGONISM AND PLATELET AGGREGATION INHIBITORS
Although receptor antagonists constitute an important chapter of pharmacological antagonists designed by humans, only one example was found so far in blood-sucking insects. The tripeptide Arg-Gly-Asp (RGD), flanked by cysteines, binds to integrin receptors on platelets that would otherwise bind fibrinogen and produce platelet aggregation. In the presence of RGD peptides, platelets cannot aggregate by any agonist, because fibrinogen cannot crosslink them. Although this type of domain is very prevalent in tick salivary proteins (Mans et al., 2008; Francischetti et al., 2009) and snake venom (Lu et al., 1993; Suehiro et al., 1996), in insects it has been recognized solely in tabanids, where a member of the CAP/antigen 5 protein family (tabinhibitin) acquired such a domain, possibly by exon shuffling. This RGD-containing antigen 5 protein is a potent platelet inhibitor, as expected (Xu et al., 2008). No such domain has been found so far in fleas, sand flies or bugs, although recently it has been found by transcriptome analysis of a New World anopheline mosquito (Anopheles darlingi) (Calvo et al., 2009b), within an ubiquitous mosquito protein family (30-kDa antigen/Aegyptin) that has not such domain in at least six other species studied in three genera. However, confirmation of An. darlingi anti-platelet activity of this protein is lacking. It is interesting to note that while plants produce a multitude of receptor antagonists in the form of alkaloids (small, basic, nitrogen-containing organic compounds), this is quite rare in insects. We can here speculate that plants are primarily ‘‘organic chemists’’, while insects, or animals, are ‘‘biochemists’’ in their pharmacological ‘‘schools’’. Indeed, plant genomes reveal a greater number of enzymes than found in animals, largely those associated with intermediary metabolism leading to a
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‘‘combinatorial organic synthesis’’ function, of adaptive value in their biological warfare with ruminants, for instance, which would digest any potent peptidic protein produced by plants, a problem not relevant for BFI that inject proteins directly into the skin. 4.3
PHYSIOLOGICAL ANTAGONISTS, PRIMARILY VASODILATORS
Physiological antagonism is the process where one agonist is antagonized by another agonist of a different receptor that triggers a contrary or neutralizing signal transduction cascade. For example, serotonin and TXA2 contract vascular smooth muscle while PGI2 is a potent vasodilator. Accordingly, PGI2 is a physiological antagonist of serotonin (and vice versa). Of course all these physiological antagonists are themselves agonists of different receptors. Vasodilatory agonists are commonly found in the saliva of BFI. The variety of these compounds is enormous. From the small to the big, it starts with nitric oxide (NO) produced by bed bugs and Rhodnius (but not Triatoma), carried by different haem proteins that transport and stabilize the NO, named nitrophorins (NP). In Rhodnius, the NP is a member of the lipocalin family and in Cimex it is a truncated member of the inositol phosphatase family of enzymes. Continuing with small molecules, adenosine was identified as the main vasodilator in the Old World sand fly Phlebotomus papatasi and Phlebotomus perniciosus (Ribeiro et al., 1999; Katz et al., 2000; Ribeiro and Modi, 2001), but not in P. duboscqi (a West African species) or in the New World sand flies. Members of the New World sand fly genus Lutzomyia possess a unique protein, maxadilan, the most potent vasodilator known and an agonist of the PACAP receptor (Lerner et al., 1991; Lerner and Shoemaker, 1992; Moro and Lerner, 1997). Mosquitoes of the genus Aedes have a salivary vasodilator member of the tachykinin family, named sialokinin (Champagne and Ribeiro, 1994; Beerntsen et al., 1999), which act by stimulating the endothelial cells to produce NO. These peptides are not found in mosquitoes of the genus Culex or Anopheles. Tabanids (deer flies and horse flies) have a protein member of the Kazal family acting as a vasodilator (Taka´cˇ et al., 2006; Xu et al., 2008), but its receptor is still undefined. Normally Kazal domain proteins are protease inhibitors, but the domain was hijacked in tabanids to serve another function. The black fly Simulium vittatum has a peptidic vasodilator named SVEP, which is of unique sequence. It activates ADP-dependent Kþ channels, possibly using the PACAP receptor as well (Cupp et al., 1998). Ticks produce abundant amounts of prostaglandins, specifically PGE2 and PGF2a in their saliva, potent skin vasodilating substances (Dickinson et al., 1976; Higgs et al., 1976; Ribeiro et al., 1992; Inokuma et al., 1994; Bowman et al., 1996). It is quite possible that insects also produce salivary PGs, but perhaps because collection of insect saliva is not always as easily done as with ticks, it remains undiscovered. Scrambling convergent evolution appears here to counteract the vasoconstrictor component of haemostasis using diverse vasodilatory compounds, acting on a variety of receptors.
FROM SIALOMES TO THE SIALOVERSE
77
Although we mentioned above that there can be no magic bullet to prevent haemostasis and inflammation, many vasodilators described in this section are closer to this ideal design, because many of them can also prevent platelet aggregation, when platelets have the same type, or similar receptors as the smooth muscle cell. This is the case with NO, for example, that not only is a powerful vasodilator but is also a platelet aggregation inhibitor, in both cases by activation of cytosolic guanylate cyclase, existing in both type of cells. Similarly, adenosine leads platelets to increase cytosolic cyclic AMP that prevents also platelets from aggregating. The same is true for PACAP agonists. These molecules that are simultaneously vasodilators and platelet aggregation inhibitors must be of a high benefit/cost in terms of adaptive value to their bearers. It will not be surprising if agonists targeting the receptors of the atrial natriuretic factor (ANF), vasoactive intestinal peptide (VIP), calcitonin generelated peptide (CGRP) and adrenomedullin (ADM) are discovered in the future, as these are potent vasodilators and platelet inhibitors. The pleiotropic effects of these vasodilators are not limited to smooth muscle cells and platelets. Immune cells are also susceptible to many of these agonists. The vasodilator maxadilan is known to be a potent suppressor of macrophage function (Soares et al., 1998a), as is PGE2 for dendritic cells (Sa´-Nunes et al., 2007), for example. 4.4
KRATAGONISTS
No textbook of pharmacology could forecast the unique type of agonist inhibition that we are finding over and over again in insects (and ticks). We are discovering salivary proteins that have very high affinity (usually on the low nM range) for 5-HT, H, NE, ADP, TXA2, LTB4 and cysteinyl leukotrienes. Notice that these agonists are all represented by double arrows in Fig. 2, providing an important role in the propagation of the haemostasis response. The first kratagonist was discovered accidentally. It is known for over 25 years that saliva of R. prolixus has anti-histaminic activity (Ribeiro, 1982). Twelve years later, having discovered the salivary nitrophorins of the same insect (in the plural because at the time we have purified four similar proteins from the saliva, having similar NO-carrying properties) (Champagne et al., 1995a), we became interested to verify their interactions with histamine, an imidazolic compound known to interact with haem proteins. To our surprise, not only histamine binding to the haem moiety dislodged the NO from the nitrophorin, but this binding was of very high affinity, larger than any imidazolic compound so far investigated with haem proteins (Ribeiro and Walker, 1994). Crystallization of NP-1 with histamine revealed very specific interactions of histamine not only with the haem iron, but with specific amino acids strategically located to clamp H in place (Andersen et al., 1998). Moreover, this histamine mopping effect accounted for all the anti-histaminic activity of Rhodnius saliva. At first sight, this type of inhibition appears to be of very ‘‘stupid design’’, because a
78
JOSE´ M. C. RIBEIRO AND BRUNO ARCA`
19,000-Da molecule is being synthesized to scavenge a 100-Da agonist. It does not make reasonable sense. However, the math works. Histamine can accumulate to 1 mM amounts in inflamed tissues, a concentration that saturates the H1 receptor. If we take in consideration that a fifth instar nymph of Rhodnius has 50 mg of salivary nitrophorins, and can inject 90% of it in a blood meal that amounts to 300 ml, the final concentration of NPs in the blood meal are 8 mM, more than sufficient to chelate inflammatory concentrations of histamine. The stupidity of the design is thus compensated by brute force, or a large mass. In the absence of a name for this kind of inhibition, we propose the name kratagonist to characterize it, from the Greek word representing to arrest or to seize. In addition to the nitrophorins exquisite histamine kratagonist function, other abundant lipocalins in Rhodnius bind 5-HT and catecholamines (Andersen et al., 2003), explaining also earlier work demonstrating anti-5-HT activity in Rhodnius saliva (Ribeiro, 1982). Yet another Rhodnius family of lipocalins, named RPAI, initially described as inhibitors of collagen-induced platelet aggregation was shown to be ADP kratagonists (Francischetti et al., 2000, 2002a). Their inhibition of collagen-induced aggregation happens because after exposure to collagen, platelets secrete ADP, and the combination of the collagen signal plus the ADP signal triggers aggregation. Removal of ADP aborts platelet aggregation by collagen. It appears that Rhodnius co-opted the lipocalin family of proteins (Andersen et al., 2005), which are known carriers of small compounds in metazoa (Flower et al., 2000; Schlehuber and Skerra, 2005), to exercise various kratagonist functions. Salivary lipocalins were also discovered and used by ticks as kratagonists of histamine, serotonin, NE, TXA2 and leukotrienes (Paesen et al., 1999, 2000; Sangamnatdej et al., 2002; Mans and Ribeiro, 2008a,b), so this represents another example of convergent evolution, as ticks and insects are over 400 MYA from a common ancestor, and do not share a common blood-feeding ancestor. In mosquitoes, D7 proteins are among the most abundant salivary components and the first member of the family was identified in the culicine mosquito Ae. aegypti (James et al., 1991). It was later shown that the D7 is a multi-gene family in An. gambiae (Arca` et al., 1999, 2002), and that it is widely spread in blood-feeding Nematocera (Valenzuela et al., 2002a). The D7 are distantly related to the insect odorant-binding protein (OBP) family that, similarly to lipocalins, functions in carrying small organic, usually hydrophobic substances (Hekmat-Scafe et al., 2000). However, the function of the D7 family was elusive, until their role as kratagonists was identified. The D7 family comes in two sizes, long and short, the long having two similar domains, possibly representing an exon duplication event. An. gambiae has five genes coding for short and three genes coding for long D7 proteins, the most abundantly expressed being members 1–4 of the short family, member 5 perhaps being a pseudogene (Arca` et al., 2005). The first two proteins of the long family are expressed at relatively low levels, the third hardly so. Recombinant forms of the abundant short D7 proteins of An. gambiae were shown to bind the biogenic
FROM SIALOMES TO THE SIALOVERSE
79
amines H, 5-HT and NE (Calvo et al., 2006a). Although most can bind all amines with high affinity, each has its preference for a particular agonist, a typical process of divergence of function following gene duplication. Interestingly, in Aedes the expression pattern of the family is opposite to that seen in Anopheles: the large D7 proteins are highly expressed, while the short ones are poorly expressed (Ribeiro et al., 2007). The carboxy domain of the large D7 protein of Aedes was shown to bind biogenic amines similarly to the short D7 proteins of Anopheles (Calvo et al., 2006a). The crystal structures of one long D7 protein of Aedes (Calvo et al., 2009a) and one short protein of Anopheles (Mans et al., 2007) have been obtained, showing that the D7 domain has two additional helices as compared to the OBP family. Solution of the crystal structure and their binding pockets revealed that the amino-terminal domain of the long D7 protein of Aedes could bind a hydrophobic compound. After testing several potential agonists using isothermal calorimetry, it was discovered that the N-terminal domain of the long Aedes D7 binds cysteinyl leukotrienes, making this protein actually to look intelligent in targeting mastcell agonists (Calvo et al., 2009a). In Anopheles, both domains of the long D7 proteins appeared also to be suitable to high affinity interaction with hydrophobic ligands. These discoveries also can explain the relative abundance of the proteins in the salivary glands: H and 5-HT accumulate and saturate their receptors at one order of magnitude above the levels required for TXA2 and LTs. According to the brute force needed with the kratagonist design, one needs 10 times less force, or protein mass, to be effective with TXA2 or LTs as compared to biogenic amines. Possibly for this reason, the long D7 of Aedes are in high concentrations, and the small D7 of Anopheles are in higher concentrations, these being the D7 that bind 5-HT and H. We still do not know the ligands of the short D7 of Aedes, but it will be probably lipidic ligands, possibly TXA2. Another ubiquitous protein family found in mosquitoes, as well as in other blood-sucking Nematocera, comprises the 30-kDa antigen/Aegyptin family. It is as abundant as D7 protein members, and in some mosquito species it is the most abundant salivary protein. It was first recognized as an important salivary antigen in Aedes (Simons and Peng, 2001), but its function was elusive. More recently, members of this family have been shown to be potent inhibitors of platelet aggregation by collagen (Calvo et al., 2007b; Yoshida et al., 2008). This protein interacts with collagen at high affinity, masking its sites that are recognized by platelet receptors, and also by vWF. Aegyptin family members thus illustrate that kratagonists are not limited to binding small agonists, but proteins far larger than the kratagonist itself. Following the discovery that kratagonists for biogenic amines may be widespread in BFA, we can hypothesize that a kratagonist function for 5-HT and H, or perhaps collagen should be found among the most abundant salivary proteins of arthropods, because of the ‘‘brute force’’ needed against these agonists. Unpublished results from Dr. Valenzuela’s laboratory appear to support the validity of this law: indeed, a sand fly salivary member of the Yellow family of
JOSE´ M. C. RIBEIRO AND BRUNO ARCA`
80
proteins, which constitutes the most intense protein band in SDS–PAGE analysis of salivary extracts, is a 5-HT kratagonist. As mentioned above salivary kratagonists against LT and TXA2 should be present at 1/10 or less of the concentration of kratagonists for H or 5-HT, because these agonists saturate receptors at 1/10 of the concentrations needed for 5-HT or H. A corollary of the idea of kratagonists is that this mechanism should be found abundantly within animals, perhaps related to immunity or neurobiology, from where the prototype genes must have been recruited to serve a salivary function. An approximation of the mechanism in immunity is the idea of ‘‘soluble receptors’’ for cytokines, which function as scavengers of cytokines. Soluble receptors appear following the proteolytically cleavage of cytokine receptors on the surface of cells that, when released to the environment, chelates and neutralizes cytokines that could otherwise interact with intact receptors and thus trigger the signalling cascade (Horuk et al., 1993; Tsimanis et al., 2005). Soluble receptors, accordingly, are fragmented receptors and not products of a different gene that evolved with the function of modulating the effect of a particular agonist. We propose that many regulators of cytokines and neuromediators may be promoted by specialized proteins adapted to be their endogenous kratagonists, and these may have not been discovered yet due to the ‘‘stupidity’’ of its mechanism, and scientist bias. In entomology, for instance, many of the juvenile hormone (JH)-binding proteins that were discovered, falsely, as the elusive JH receptor may actually be JH kratagonists that modulate JH availability in the vicinity of particular cells. 4.5
PROTEASE INHIBITORS
The clotting and complement cascades of vertebrates, as the name implies, represent a system of proteases with, in one end, a proximal protease acting on intermediate inactive protease precursors and, at the other end, a terminal protease that finally produces the effector molecule(s), thrombin in the case of clotting or the lytic complex in the case of complement. Because one enzyme produces another enzyme that produces another enzyme, with a single activated protein in the beginning of the process, millions can be produced at the end, thus the apt name of an amplification cascade. In these amplification steps there are many non-enzymatic proteins that serve as modulators of the system, either by activating it or by tuning it down. These multiple steps of control allow the system to go forward in certain states, or to be deactivated, preventing their unregulated and noxious spread throughout the body. Examples of nonenzymatic activators are factor VIII of the clotting cascade and factor B of the complement pathway, while endogenous inhibitors are, for example, antithrombin III of the clotting cascade and factor H of the complement system. Proteins of the serpin and Kunitz family serve as endogenous inhibitors of activation of these proteolytic cascades. Insects have their proteolytic cascade equivalent in the prophenoloxidase activation cascade, in their own clotting
FROM SIALOMES TO THE SIALOVERSE
81
cascade, and in the activation of thioester proteins (TEP) on pathogen surfaces, which is analogous to the colectin pathway of complement activation in vertebrates (Thiel, 2007). Insects also use regulators of these proteases, the serpin and Kunitz families of proteins being abundant in insect genomes, and known to play a role in regulation of insect proteolytic cascades (Kanost et al., 2004). For a review on blood clotting and complement cascades, the reader is directed to recent reviews and books (Colman et al., 2005; Paul, 2008). We add also to this section the proteases from neutrophils and mast cells as possible targets of disruption for inhibitors to be found in the saliva of BFI. The various proteases thus involved in clotting, complement activation and inflammation provide a variety of targets for BFI. Toward this goal, insects recruited traditional genes as well-adapted novel protease inhibitor families to be expressed in their saliva. While clotting inhibitors are ubiquitously described in the salivary gland homogenates of BFI for almost one century (Cornwall and Patton, 1914; Yorkee and Macfie, 1924; Metcalf, 1945), only a few have been so far characterized molecularly. Belonging to traditional families are the anti-factor Xa serpin of the mosquito Ae. aegypti (Stark and James, 1995, 1998), and tabkunin, the Kunitz anti-thrombin of T. yao (Xu et al., 2008). Transcripts coding for members of the Kunitz family of peptides have also been described in sialotranscriptomes of Culicoides sonorensis and the black fly Simulium vittatum (Table 3), but no recombinant protein has been characterized so far. Ticks have also co-opted the Kunitz family to serve as anti-clotting agents (Francischetti et al., 2002b, 2004; Lai et al., 2004). The remaining molecularly characterized salivary clotting inhibitors from insects are of non-traditional protease inhibitor families, or belong to completely novel families. Triabin and prolixin-S are anti-thrombin and anti-factor VIII/IXa lipocalins from Triatoma pallidipennis (Noeske-Jungblut et al., 1995; Fuentes-Prior et al., 1997) and Rhodnius prolixus (Hellmann and Hawkins, 1965; Ribeiro et al., 1995; Sun et al., 1996; Zhang et al., 1998; Isawa et al., 2000; Gudderra et al., 2005), respectively. These anti-clotting roles of lipocalins are unique, and may have evolved by accretion of domains (exon shuffling) or acquired by classical evolution of a particular existing protein domain. For example, in the case of R. prolixus the lipocalin NP-2 has a unique anti-clotting activity, but it is also a NO-carrying protein, and an H kratagonist. It is reasonable to assume that the ancient function of NP-2, similarly to the other nitrophorin paralogues, was to carry NO and bind histamine. These functions require a specific conformation inside the lipocalin barrel where the haem, NO and histamine are bound. Amino acid side chains outside the loops are free to evolve neutrally or perhaps fast to avoid host immunity, and eventually may acquire a new function. While serpins serve as an anti-clotting in Aedes mosquitoes (transcripts for serpins also being found in sialotranscriptomes of Aedes albopictus, the close related Ochlerotatus triseriatus (submitted) and in Culex quinquefasciatus), the salivary anti-clotting of Anopheles albimanus is a very negatively charged peptide uniquely found in anophelines, named anophelin. It functions by tightly binding to
JOSE´ M. C. RIBEIRO AND BRUNO ARCA`
82
thrombin (Francischetti et al., 1999; Valenzuela et al., 1999). Sialotranscriptomes of An. darlingi, An. gambiae, An. funestus and An. stephensi reveal abundant transcription of homologues (Arca` et al., 1999, 2005; Valenzuela et al., 2003; Calvo et al., 2004, 2007a). A novel and relatively short peptide ( 3 kDa) also serves as an anti-thrombin in tsetse (Cappello et al., 1998), and yet another, named simulidin in black flies (Abebe et al., 1995; Cupp and Cupp, 2000), although this may be a distant and truncated member of the D7 protein family. The muscid Haematobia irritans (the face fly) has a unique peptidic anti-thrombin, named thrombostasin (Zhang et al., 2002). The related stable fly Stomoxys calcitrans has a homologue, albeit its primary structure is only 41% identical. Activated factor XII of clotting activates pre-kallikrein to kallikrein, and this in turn acts on blood kininogens to produce BK, an inflammatory and painproducing peptide. Because the most important physiological activator of FXII is thrombin, inhibition of clotting should also decrease BK production. Nonetheless, saliva of anopheline mosquitoes possess two inhibitors of the FXII–kallikrein axis, one promoted by one member the short D7 protein from An. stephensi, which was named hamadarin (Isawa et al., 2002), and the other by a protein found specifically in anopheline mosquitoes, named anophensin (Isawa et al., 2007). Molecularly characterized salivary anti-complement activities have been described in soft and hard ticks (Valenzuela et al., 2000; Nunn et al., 2005); moreover, anticomplement activity has been reported in salivary homogenates of sand flies and triatomine bugs (Cavalcante et al., 2003) although their molecular nature has been so far elusive. Soon to be published results from Dr. Valenzuela’s lab will present some novel protein families with such a function on sand flies (J. G. Valenzuela, personal communication). Similarly, although ticks have Kunitz proteins inhibitory of tryptase (Paesen et al., 2007), no such activity has been so far discovered in insect saliva, although transcriptome analysis has revealed such protein families in biting midges and sand flies. No inhibitors of cathepsin G have been so far described in either insect of tick saliva (Table 2). From the above account, it can be observed that the nature of insect salivary anti-clotting is very diverse, even within insects sharing a common haematophagous ancestor, as is the case with Triatoma and Rhodnius or culicines and anopheline mosquitoes. We can also observe that we lack the knowledge of the anti-clotting nature for whole orders or families of BFA, such as the fleas, bed bugs, biting midges, lice, etc. Actually, some of these orders/families do not have a single functionally characterized protein. When considering that considerable diversity is found at the genus level, with 455 genera accounted for in Table 1, the extent of our ignorance on this subject becomes quite apparent. 4.6
ANAESTHETICS
Following nerve activation by pain agonists (ATP, H, 5-HT, NE and BK), the nervous signal is conduced from the periphery to the spinal chord by special neurons using a specific Naþ channel. Traditional anaesthetics block nerve
TABLE 2 Molecularly characterized salivary proteins of blood-feeding insects (excluding immunity related) Name Apyrase Cimex nitrophorin
Sialokinin
Apyrase
Biological action
Activity
Molecular family
Type
Naturea
Characterizationb
Organism
Family or order
References
Degrades ADP and ATP Carriers of NO
Anti-platelet
Cimex type
Enzyme
1
1, 2, 3, 4, 5
Cimex lectularius
Cimicidae
Vasodilator, anti-platelet
Kratagonist
5
1, 2, 3, 4, 5
Cimex lectularius
Cimicidae
Endotheliumdependent vasodilator Degrades ADP and ATP
Vasodilator
Inositolphosphate phosphatase, truncated Tachykinins
Agonist
4
1, 2, 3, 4, 5
Aedes aegypti
Culicidae
Anti-platelet
50 -nucleotidase
Enzyme
1
1, 2, 3, 4, 5
Aedes aegypti
Culicidae
Purine nucleosidase
Enzyme
1
1, 2, 3
Aedes aegypti
Culicidae
Ribeiro and Valenzuela (2003)
Serpin
Protease inhibitor
2
1, 2, 3, 4, 5
Aedes aegypti
Culicidae
Stark and James (1998) Calvo et al. (2007b) and Yoshida et al. (2008) Ribeiro and Valenzuela (1999)
Anti-Xa serpin
Factor Xa inhibitor
Destroys mastcell degranulating adenine and inosine Anti-clotting
Aegyptin
Collagen inhibitor
Anti-platelet
Unique
Kratagonist
2
1, 2, 3, 5
Aedes aegypti, Anopheles stephensi
Culicidae
Peroxidase
Degrades catecholamines
Vasodilatory
Peroxidase
Enzyme
1
1, 2, 3, 4, 5
Anopheles albimanus
Culicidae
Purine Degrades adenine nucleosidase to hypoxanthine
Notes
Valenzuela et al. (1998) Valenzuela and Ribeiro (1998) Champagne and Ribeiro (1994) Champagne et al. (1995b)
So far found only in Aedes aegypti Several other mosquito species have been enzymatically characterized to have salivary apyrase activity
Found in other culicines, not anophelines Family pervasive in blood-sucking Nematocera Found only in anophelines, not culicines
(continues)
TABLE 2 (Continued) Name
Biological action
Activity
Molecular family
Type
Nature
a
Characterization
b
Organism
Family or order
References
Anophelin
Thrombin inhibitor
Anti-clotting
Unique
Protease inhibitor
2
1, 2, 3, 4, 5
Anopheles albimanus
Culicidae
D7 large
Sequesters leukotrienes
Antiinflammatory
D7–OBP superfamily
Kratagonist
5
1, 2, 3, 5
Anopheles gambiae
Culicidae
D7 small
Sequesters biogenic amines
Vasodilatory, antiinflammatory
D7–OBP superfamily
Kratagonist
5
1, 2, 3, 5
Anopheles gambiae
Culicidae
Hamadarin
Factor XII inhibitor Factor XII inhibitor Degrades doublestranded DNA
Anti-bradykinin
Kratagonist
2
1, 2, 3, 5
Anopheles stephensi
Culicidae
Anti-bradykinin
D7–OBP superfamily gSG7
2
1, 2, 3, 5
Anopheles stephensi
Culicidae
Spreading factor
Endonuclease
Protease inhibitor Enzyme
1
1, 2, 3, 5
Culex quinquefasciatus
Culicidae
Hyaluronidase
Degrades skin matrix components
Spreading factor
Hyaluronidase
Enzyme
1
1, 2, 3, 4, 5, 6
Tabanus yao
Diptera
Xu et al. (2008)
Adenosine deaminase
Converts adenosine to inosine
Destroys mastcell degranulating adenosine Anti-clotting
Adenosine deaminase
Enzyme
1
1, 2, 3
Glossina morsitans
Glossinidae
Li and Aksoy (2000)
Unique
2
1, 2, 3, 4, 5
Glossina morsitans
Glossinidae
Anti-clotting
Thrombostasin
3
1, 3, 5
Haematobia irritans
Muscidae
Binds Ig
Antigen 5
Protease inhibitor Protease inhibitor Kratagonist?
1, 2, 3, 4
Stomoxys calcitrans
Muscidae
Cappello et al. (1998) Zhang et al. (2002) Ameri et al. (2008)
Anophensin Endonuclease
Tsetse thrombin Thrombin inhibitor inhibitor Thrombostasin Anti-thrombin Stomoxys Ag5
Unknown target (complement activation?)
Francischetti et al. (1999) and Valenzuela et al. (1999) Calvo et al. (2009a) Calvo et al. (2006a) and Mans et al. (2007) Isawa et al. (2002) Isawa et al. (2007) Calvo and Ribeiro (2006)
Notes Found only in anophelines, not culicines
Family pervasive in blood-sucking Nematocera Family pervasive in blood-sucking Nematocera
Not found in culicines Not found in Aedes or Anopheles genera Activity also found in black flies, sand flies, tabanids, Culex quinquefasciatus
Charlab et al. (2000), Ribeiro et al. (2001) and Kato et al. (2007) Lerner et al. (1991)
Adenosine deaminase
Converts adenosine to inosine
Destroys mast-cell degranulating adenosine
Adenosine deaminase
Enzyme
1
1, 2, 3
Aedes aegypti, Phlebotomus duboscqi
Nematocera
Maxadilan
PACAP receptor agonist
Vasodilator
Unique
Agonist
4
1, 2, 3, 4, 5
Lutzomyia longipalpis
Psychodidae
50 -nucleotidase
Degrades AMP to adenosine
Produces vasodilatory and antiplatelet adenosine Anti-platelet
50 -nucleotidase
Enzyme
1
1, 2
Lutzomyia longipalpis
Psychodidae
Ribeiro et al. (2000b)
Cimex type
Enzyme
1
1, 2, 3, 5
Psychodidae
Vasodilator, anti-platelet
Nucleotides
Agonist
4
1, 3, 4, 5
Phlebotomus papatasi Phlebotomus papatasi, P. argentipes
Valenzuela et al. (2001b) Ribeiro et al. (1999)
LysophospInhibits platelet hatidylcholine aggregation Rhodnius Carriers of NO Nitrophorins
Anti-platelet
Lipid
Agonist
4
1, 3, 4, 5
Rhodnius prolixus
Reduviidae
Vasodilator, anti-platelet
Kratagonist
5
1, 2, 3, 4, 5
Rhodnius prolixus
Reduviidae
Inositol Degrades inositol phosphatase phosphates
Unknown
Lipocalin of the nitrophorin family SHIP
Enzyme
1
1, 2, 3, 5
Rhodnius prolixus
Reduviidae
RPAI
Sequesters ADP
Anti-platelet
Kratagonist
3
1, 2, 3, 5
Rhodnius prolixus
Reduviidae
BABP
Anti-serotonin, anti-adrenergic Inhibits factor VIII
Vasodilatory
Lipocalin of the triabin superfamily Lipocalin
Kratagonist
5
1, 2, 3, 4, 5
Rhodnius prolixus
Reduviidae
Protease inhibitor
2
1, 2, 3, 4, 5
Rhodnius prolixus
Reduviidae
Reduces nerve sodium current Degrades sialic acid
Anesthetic
Lipocalin of the nitrophorin family Unknown
?
1
Triatoma infestans
Reduviidae
Dan et al. (1999)
Anti-neutrophil?
Unknown
Channel blocker Enzyme
1
1
Triatoma infestans
Reduviidae
Amino et al. (1998)
Apyrase
Degrades ADP and ATP Adenosine, AMP Agonist of purinergic receptors
Prolixin-S
Triatoma anesthetic Sialidase
Anti-clotting
Psychodidae
Golodne et al. (2003) Andersen et al. (2005) Andersen and Ribeiro (2006) Francischetti et al. (2000, 2002a) Andersen et al. (2003) Ribeiro et al. (1995)
Protein not found in Old World sand fly genera
Not found in Lutzomyia, not found in P. duboscqi
Multi-gene family
At least two members in Rhodnius
Also carries NO and binds histamine
(continues)
TABLE 2 (Continued) Name Apyrase Triapsin
Triplatin
Pallidipin
Biological action Degrades ADP and ATP Serine protease
Possible GPVI antagonist or ADP binder Possible ADP binding
Activity
Molecular family
Type
Naturea
Characterizationb
Organism
Family or order
References
Anti-platelet
50 -nucleotidase
Enzyme
1
1, 2, 3, 4, 5
Triatoma infestans
Reduviidae
Unknown – matrix degradation? Anti-platelet
Trypsin
Enzyme
1
1, 2, 3, 4, 5
Triatoma infestans
Reduviidae
Lipocalin of the triabin superfamily Lipocalin of the triabin superfamily Lipocalin of the triabin superfamily Ricin lectin family
Kratagonist
3
1, 2, 3, 5
Triatoma infestans
Reduviidae
Morita et al. (2006)
Kratagonist
3
1, 2, 3, 5
Triatoma pallidipennis
Reduviidae
Noeske-Jungblut et al. (1994)
Protease inhibitor
3
1, 2, 3, 5
Triatoma pallidipennis
Reduviidae
Noeske-Jungblut et al. (1995)
Agonist
4
1, 2, 3, 4, 5
Simulium vittatum
Simuliidae
Cupp et al. (1998)
Protease inhibitor
2
1, 2, 3, 4, 5
Simulium vittatum
Simuliidae
Abebe et al. (1995)
Enzyme
1
1, 2, 3
Xenopsylla cheopis
Siphonaptera
Anti-platelet
Faudry et al. (2004) Amino et al. (2001)
Triabin
Thrombin inhibitor
Anti-clotting
SVEP
Possible PACAP receptor agonist Anti-thrombin
Vasodilator
Degrades ADP and ATP Degrades ADP and ATP Possible ion channel effect
Anti-platelet
Possible D7 distant member CD-39
Anti-platelet
50 -nucleotidase
Enzyme
1
1, 3, 4
Chrysops spp.
Tabanidae
Vasodilator
Kazal
4?
1, 2, 3, 4, 5
Tabanidae
2
1, 2, 3, 4, 5, 6
Hybomitra bimaculata, Tabanus yao Tabanus yao
Tabanidae
Andersen et al. (2007) Reddy et al. (2000) Taka´cˇ et al. (2006) and Xu et al. (2008) Xu et al. (2008)
3
1, 2, 3, 4, 5, 6
Tabanus yao
Tabanidae
Xu et al. (2008)
1
1, 2, 3, 4, 5, 6
Tabanus yao
Tabanidae
Xu et al. (2008)
1
1, 2, 3, 4, 5, 6
Tabanus yao
Tabanidae
Xu et al. (2008)
Simullidin
Apyrase Chrysoptin
Anti-clotting
Vasotab, Vasotab TY Tabkunin
Anti-thrombin
Anti-clotting
Kunitz
Tabinhibitin
RGD disintegrin
Anti-platelet
Antigen 5
Tabserin
Serine protease
Trypsin
Tabfiglysin
Serine protease
Anti-clotting, target unknown Fibrinogenolytic
Agonist, receptor unknown Protease inhibitor Receptor antagonist Enzyme
Trypsin
Enzyme
a b
Notes
Not found in Rhodnius
1, enzymatic; 2, enzyme or target inhibitor; 3, receptor antagonist; 4, receptor agonist; 5, kratagonist. 1, enzymatic activity or bioassay; 2, candidate found in sialotranscriptome; 3, proteomic or mass confirmation; 4, purified compound confirms activity; 5, recombinant/synthetic compound confirms activity.
FROM SIALOMES TO THE SIALOVERSE
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conduction by preventing nerve impulse conduction, and thus the pain signal from reaching the brain. Saliva of T. infestans blocks nerve conduction in the sciatic nerve and the activity of Naþ channels in cell cultures (Dan et al., 1999), but it is still molecularly uncharacterized. No other aesthetic of BFA has been reported so far. Notice that the efficient removal of pain agonists with enzymes, protease inhibitors and kratagonists may prevent pain from being elicited, making an aesthetic not necessary. 4.7
ANTIGEN (AG5) FAMILY MEMBERS
All BFA sialotranscriptomes analysed so far, including those from ticks, indicate the expression of one or more members of the antigen 5 protein family, which was originally described in the venom of ants and wasps (King and Spangfort, 2000). Insect genomes encode several divergent members of this widely spread protein family that is part of the larger superfamily of cysteine-rich extracellular proteins ubiquitously found in animals and plants (Schreiber et al., 1997; Megraw et al., 1998). The Drosophila melanogaster genome encodes 25 different Ag5 genes (Kovalick and Griffin, 2005) and 19 family members sharing 32–51% amino acid identity can be identified in the An. gambiae genome. At least one member of the Ag5 family was recruited by haematophagous insects to play some essential blood-feeding role as suggested by the abundant and tissue-enriched expression in salivary glands (Arca` et al., 2005, 2007; Ribeiro et al., 2007). Members of this family in snake venoms are associated with ion channel inhibitors and toxins (Yamazaki et al., 2003; Yamazaki and Morita, 2004). In Conus snails, an Ag5 protein was found to have a specific protease activity (Milne et al., 2003), and as seen above, a protein of this family acquired a RGD domain and acts as a salivary inhibitor of platelet aggregation (Xu et al., 2008). Recently, a recombinant Ag5 protein expressed in the salivary glands of the stable fly S. calcitrans was found to bind immunoglobulins strongly, particularly their Fc fragment (Ameri et al., 2008). The action of this protein on the activation of the classical pathway of complement was not described. Besides the Tabanus and Stomoxys proteins, no other salivary Ag5 member from BFA has been so far functionally characterized. It is difficult to predict the function of these proteins in the saliva of BFA because of the very diverse functions found for members of this protein family. 4.8
IMMUNITY-RELATED PRODUCTS
A common feature of insect sialotranscriptomes is the presence of AMP such as defensins, cecropins, gambicin and lysozyme, as well as pattern recognition molecules (Gram-negative binding proteins, C-type lectins and ficolins) and serine proteases that may act as proximal activators of the prophenoloxidase or TEP cascades. Many AMP are lytic to microbes, and some can also act as eukaryotic cytolysins. The salivary trialysin of T. infestans is of a unique family that can
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lyse bacteria and protozoa (Amino et al., 2002; Martins et al., 2006). Recently, novel AMP were isolated from the saliva of T. yao (Xu et al., 2008). Saliva of BFA must have a large number of still unknown antimicrobial families, an area virtually untapped by the scientific community. 4.9
THE UNEXPECTED
As will be discussed in more detail below, the sialome studies from different genera reveals many proteins that are unique to the genus, and this includes whole families of related proteins that are exclusively found expressed in the salivary glands of BFA. Most of these protein families have no known function, but may function as members of several generic categories above, such as physiological antagonists (agonists), kratagonists, enzymes, antimicrobials or protease inhibitors.
5
Salivary diversity
It has become apparent within the last decade that molecular evolution of genes coding for salivary proteins in BFA is at a very fast pace as compared to housekeeping genes. For example, when comparing several anopheline mosquitoes of the subgenus Cellia with the African mosquito An. gambiae, for which there is available a genome draft that allows reasonable identification of the orthologs, it is verified that orthologous salivary proteins have much less identity than housekeeping ones. For the Asiatic An. stephensi the % identity ( SE) is 62.4 15 and 93.1 6 (Valenzuela et al., 2003) and for the African An. funestus the values are 66.7 1.9 and 96 0.84 for salivary gland and housekeeping proteins, respectively (Calvo et al., 2007a). These differences are highly significant in both cases. In another example, species within the Rhodnius genus are very similar and differentiation is morphologically feasible only by looking at the male genitalia characters (Lent and Wygodzinsky, 1979), but the electrophoresis pattern of the salivary gland proteins are very distinct among these morphologically very similar species (Soares et al., 1998b, 2000). Few studies have tackled the issue of polymorphism of the salivary gland genes from wild populations, but in one such instance the vasodilatory maxadilan from L. longipalpis was found to be polymorphic (Lanzaro et al., 1999). Interestingly, all forms studied were equally powerful vasodilators but had different antigenic properties. Antibodies against a particular form neutralized the vasodilatory effect of that form, but not of all forms (Milleron et al., 2004), suggesting that the gene variation may represent a form of antigenic variation, and that variation could be maintained in the fly population by balanced polymorphism through frequency-dependent selection. However, it should be pointed out that high degree of intra-specific conservancy of salivary proteins was found when Phlebotomine sand flies from two different geographic locations were
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compared (Kato et al., 2006), suggesting that intra-specific variation (both within and between populations) deserves a deeper evaluation with extension of the analysis to other insect species. It should be noted that the invariance in the salivary proteins of P. papatasi may be related to the exceptionally strong DTH response following this fly’s bite, a response that facilitates feeding, thus making advantageous to the fly to conserve its antigenicity (Belkaid et al., 2000). When more distantly related species are compared, such as mosquitoes belonging to different subfamilies, qualitative, not quantitative genetic differences are manifested. As indicated above, the anti-clotting function in Ae. aegypti saliva is provided by a member of the serpin family, and serpins have also been found in sialotranscriptomes of Ae. albopictus and C. quinquefasciatus, both mosquitoes belonging to the Culicine subfamily. However, in the anopheline subfamily a completely novel polypeptide, named anophelin, serves this function. Why is this the case? While anophelines and culicines share many unique protein families, such as the 30-kDa antigen/Aegyptin and D7 proteins, suggesting the ancestral mosquito ( 150 MYA) already possessed these salivary proteins, could it be that this ancestor did not have an anti-clotting and its acquisition occurred only after the split of the lineages? While this could be the case, another possibility is that in the early anophelines the anti-clotting may have become ineffective, creating the conditions for the evolution of an alternative gene to serve the lost function. In an analogous manner to the anti-maxadilan antibodies, host immunity may have neutralized the ancestral anopheline anti-clotting peptide. This is supported by the author’s unpublished observation that salivary gland homogenates of Ae. aegypti show no anti-clotting activity if the assay is done with the plasma of previously exposed Guinea pigs or humans, but not when plasma of naive animals are used. The vertebrate host immune pressure may thus be a powerful driving force for the fast evolution of the salivary potion, not only by conventional, point mutational modes (as seems to be occurring with maxadilan), but also by promoting complete gene loss of function, the opportunity arising for a completely new gene recruitment to occupy the vacuum (as may have been the case with anopheline’s anti-clotting proteins). It is apparent that the inflammatory, haemostatic and immune pressure from hosts are driving the evolution of the salivary magic potion of BFA. Adaptive host immunity may prevent reaching a ‘‘perfect’’, immutable potion, creating the engine for the fast, perpetual evolution of these molecules, or at best reaching a balanced polymorphism state where genes are under frequency-dependent selection (the gene with the lower frequency has the best fitness, because it codes for a rare epitope). Short lived hosts, may well favour a balanced polymorphism, while long lived hosts, such as humans, may well lead to BFA gene function loss. For example, mice and other small rodents live on average less than 1 year in the wild, equivalent to less than 10 generations of sand flies. Because newborn mice do not inherit the memory of sand fly antigens circulating in their parent’s generation, a gene driven to low frequency in the end of the previous year may become a better gene when a new cohort of mice arises.
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Conversely, humans and other mammals and birds can accumulate their immunological memory over decades, representing hundreds of sand fly or mosquito generations, which could lead to local extinction of alleles in a finite population. In this case a gene could lose its function, and the genome will have to scramble for a new solution. The still unanswered question is to what extent the BFA salivary proteins are driving the evolution of vertebrate inflammatory, haemostatic and immune systems? If this is the case, we have a true scenario of coevolution of vertebrates and BFA. Coevolution is not the mere fact of evolving together, but rather that characters of one organism influence another organism to change, and this in turn changes the characters of the first organism, as is the coevolution of pollinators and flowers. The case of the evolution of mammalian skin mast cells appears to be one such case in point. In the absence of BFA and the burden of the diseases that they transmit, mast cells appear to be of negative fitness value, as they are effectors of many disabling dysfunctions. Extrapolating from our scope on insects, the evolution of basophils, which are very similar to mast cells, appear to have been the response of some mammals to prevent ticks from feeding (Allen, 1973), by their ability to congregate in much larger numbers than mast cells and thus release large amounts of their mediators. One of the characteristics of coevolution and interspecies arms race is phenotypic exaggeration, such as the elaborate flowers and bird beak adaptation, for example (Thompson, 2005; Hanifin et al., 2008). The load of histamine/ serotonin granules in a mast cell and the large amounts of H/5-HT kratagonists in insects are indicative of these coevolutionary exaggerations. Under this light, the evolution of the BFA salivary magic potion and vertebrate skin is represented by an arms race scenario resulting from coevolving organisms.
6
The evolutionary scramble
Gene duplication is responsible for more than 50% of the genes in eukaryote genomes (Holland, 1999; Ohno, 1999; Sankoff, 2001; Mazet and Shimeld, 2002; Nei and Rooney, 2005), and it has been proposed as an important mechanism for the evolution of salivary genes in ticks (Mans et al., 2002). Sialotranscriptome analysis of >20 species (Table 3) so far studied indicates that this mechanism is surely occurring in BFI, not only with genes belonging to ubiquitous families, but also within genes uniquely found associated with particular BFI families or subfamilies, such examples being found in all papers associated with Table 3. As mentioned above, the D7 protein family has eight genes in Anopheles, and is polygenic in all Nematocera so far studied. There are two salivary serpins in culicines, one of which is a factor Xa inhibitor, as indicated above, the other being now identified as a tryptase inhibitor (M. Kotsyfakis, personal communication). Perusal of the papers cited in Table 3 will demonstrate many other examples, including several families so
TABLE 3 Sialotranscriptomes of blood-feeding insects Order
Sub-order
Family
Tribe
Subgenus
Species
References Francischetti et al. (2002c), Arca` et al. (2005) and Calvo et al. (2006b) Valenzuela et al. (2003) Calvo et al. (2007a) Calvo et al. (2004, 2009b) Valenzuela et al. (2002b) and Ribeiro et al. (2007) Arca` et al. (2007) In process Ribeiro et al. (2004b)
Diptera
Nematocera
Culicidae
Anophelini
Cellia
Anopheles gambiae
Diptera
Nematocera
Culicidae
Anophelini
Cellia
Diptera Diptera
Nematocera Nematocera
Culicidae Culicidae
Anophelini Anophelini
Cellia Nyssorhynchus
Anopheles stephensi Anopheles funestus Anopheles darlingi
Diptera
Nematocera
Culicidae
Culicini
Stegomyia
Aedes aegypti
Diptera Diptera Diptera
Nematocera Nematocera Nematocera
Culicidae Culicidae Culicidae
Culicini Culicini Culicini
Stegomyia Culex Culex
Diptera
Nematocera
Culicidae
Toxorhynchitini
Diptera
Nematocera
Psychodidae
Diptera
Nematocera
Psychodidae
Aedes albopictus Culex tarsalis Culex quinquefasciatus Toxorhynchites amboinensis Lutzomyia longipalpis Phlebotomus ariasi
Calvo et al. (2008) Charlab et al. (1999) Oliveira et al. (2006) (continues)
TABLE 3 (Continued) Order
Sub-order
Family
Tribe
Diptera
Nematocera
Psychodidae
Diptera
Nematocera
Psychodidae
Diptera
Nematocera
Psychodidae
Diptera
Nematocera
Psychodidae
Diptera
Nematocera
Ceratopogonidae
Culicoidini
Diptera Hemiptera Hemiptera
Nematocera
Simuliidae Reduviidae Reduviidae
Rhodnini Triatomini
Reduviidae
Triatomini
Hemiptera Siphonaptera
Subgenus
Culicoides
Species Phlebotomus papatasi Phlebotomus duboscqi Phlebotomus argentipes Phlebotomus perniciosus Culicoides sonorensis Simulium vittatum Rhodnius prolixus Triatoma infestans Triatoma brasiliensis Xenopsylla cheopis
References Valenzuela et al. (2001a) Kato et al. (2006) Anderson et al. (2006) Anderson et al. (2006) Campbell et al. (2005) Andersen et al. (2009) Ribeiro et al. (2004a) Assumpc¸a˜o et al. (2008) Santos et al. (2007) Andersen et al. (2007)
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far not mentioned because their function is still unknown. One of the immediate outcomes of gene duplication is an increased dosage of the gene product, because there are more templates available for translation. Following this ‘‘saltatory’’ evolutionary event, traditional mutations can both change the promoter region of the gene, changing tissue expression levels of the message, as well as allowing functional divergence, as exemplified above by the specialization of the anopheline short D7 proteins regarding their different affinities to biogenic amines (Calvo et al., 2006a). While the normal process of gene duplication is through segmental chromosome duplication creating tandem duplications (Eichler and Sankoff, 2003), a more unusual process is by genomic insertion of reverse transcribed mRNA by the machinery of transposable elements. While the vast majority of eukaryotic genes contain two or more exons separated by introns, these novel retrotransposed genes are uniexonic. Although this mechanism of gene duplication is rare, analysis of the sialogenomes (the organization of the salivary genes in the organism’s genome) of the mosquitoes An. gambiae (Arca` et al., 2005), Ae. aegypti (Ribeiro et al., 2007) and C. quinquefasciatus (unpublished observations) indicates the presence of many uniexonic genes coding for salivary proteins, including novel protein families larger than 60 kDa, a rare occurrence of single exon genes in metazoa. This is the case with the following families: SGS/Wolbachia-like genes and 56-kDa family found in the three mosquitoes, and the 62- and 34-kDa families of Ae. aegypti (not found in Anopheles). In Culex, the 16.8-kDa family (Ribeiro et al., 2004b) has more than 30 genes, most of which are single exonic (unpublished observations), suggesting its genomic expansion by retrotransposition, and perhaps later gene duplication of uniexonic copies. Horizontal gene transfer (HGT), or lateral gene transfer, is an unusual mechanism of novel gene acquisition in eukaryotes (Andersson, 2005; Keeling and Palmer, 2008), although relatively common in prokaryotes (Jain et al., 2002; Gogarten and Townsend, 2005). Interestingly, horizontal transfer in eukaryotes is more common in organisms that have intracellular endosymbionts, or organisms associated with particular ecological adaptations (Keeling and Palmer, 2008), the two conditions being true for most blood-sucking insects and ticks (Wigglesworth, 1936; Hill et al., 1973; Nogge, 1978; Shaw and Moloo, 1991; Hypsa, 1993; Ricci et al., 2002; Sinkins, 2004; Aksoy and Rio, 2005; Mattila et al., 2007; Pais et al., 2008). Remarkably, the mosquito salivary families SGS/ Wolbachia and the 56-kDa family (found in Aedes, Culex and Anopheles), as well as the 62- and 34-kDa families (found only in culicines) and the exclusive 16.8kDa family of Culex represent uniexonic protein families matching bacterial proteins when compared to the GenBank non-redundant (NR) protein database using the tool Psiblast (Altschul et al., 1997), suggesting their acquisition by HGT into the mosquito genomes. We have mentioned above several instances of salivary genes coding for novel proteins. The label ‘‘novel’’ is imposed because comparison of the deduced primary sequence of these proteins to available databases, such as the
94
JOSE´ M. C. RIBEIRO AND BRUNO ARCA`
NR database of GenBank (now amounting to over 6 million proteins), fails to retrieve matches with convincing statistical values of significance. It is possible that these ‘‘novel’’ genes originated from an ancestor of a recognizable family, but that their fast evolution led to obscuring any residual primary sequence similarity. This is the case, for instance with the salivary nitrophorins from Rhodnius, which give only limited sequence similarity to other salivary triatomine proteins when their primary structure is used to find similar proteins. However, their crystal structure revealed unequivocally that nitrophorins belonged to the lipocalin superfamily (Andersen et al., 1997, 1998; Weichsel et al., 1998), which is a well known and very diverse protein family (Flower et al., 2000). Similarly, the D7 proteins produce strong primary similarities only to other BFI proteins, but have motifs identifiable by the tool Rpsblast (Marchler-Bauer et al., 2002) as belonging to the OBP family. Crystal structure of a D7 protein allowed identification of two extra helices in D7 proteins, in addition to the normal six helices of canonical OBPs (Mans et al., 2007), confirming its origin as an OBP, possibly evolving by exon duplication. More recently, the crystal structure of SVEP, the salivary vasodilator of S. vittatum, revealed that this protein belongs to a classical trefoil structure found in lectins, such as ricin and many other bacterial proteins (J.F. Andersen, personal communication). The primary structure of SVEP does not reveal any relatives when compared to the NR database. The solution of the crystal structure of these novel proteins will certainly help to classify many of these ‘‘novel’’ proteins into already known families, or help to confirm then as real novel structures.
7
On the odd, the paradoxical, the bizarre and the bias
Although many BFA studied to date have salivary anti-histaminic activity, there are reports of the existence of histamine in the salivary glands and saliva of black flies (Wirtz, 1988, 1990). Although confirmation by mass spectrometry would be desirable, it is possible that some BFI actually use this inflammatory amine to assist haematophagy. Histamine is known to have both vasoconstrictory and vasodilatory properties, depending on the nature of the vascular bed (Carroll and Neering, 1976; Kelm et al., 1993). In conjunction with other salivary components, histamine may constitute an odd ingredient for these flies magic potion. Histamine may also be recruited to the feeding site by active mast-cell degranulation, as shown to occur with saliva of the mosquito An. stephensi in non-immune mice (Demeure et al., 2005). Another group of flies that might exploit host inflammatory and immune reactions are sand flies of the Phlebotomus genus. Their saliva can rapidly induce strong delayed-type hypersensitivity in mammals. Flies feeding in these DTH skin sites feed much faster, in accordance with their higher blood flow, as measured by a laser Doppler apparatus (Belkaid et al., 2000). As mentioned above, P. papatasi is also odd in its recruitment of AMP and adenosine to their salivary glands, in
FROM SIALOMES TO THE SIALOVERSE
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opposition to the reverse strategy of many other BFI that specifically destroy adenosine. Because the mediators of haemostasis, inflammation and immunity are many, and the possible manner to antagonize them is still higher (there are many ways of antagonizing each agonist), the product resulting from many independent and bifurcating evolutionary paths for the creation of the salivary potion may attain odd and apparently paradoxical compositions. While transcriptome studies point to putative secreted proteins largely by comparison of their primary sequence with known databases, and by the existence of a leader sequence indicative of secretion (Nielsen et al., 1997), proteomics studies done with saliva (not salivary gland homogenates) of Ae. aegypti and An. gambiae (Orlandi-Pradines et al., 2007) indicated the presence of salivary peptides deriving from the cleavage of membrane proteins, specifically fragments from a mosquito-exclusive protein family postulated to be a salivary gland receptor for Plasmodium parasites (Korochkina et al., 2006). It is possible that these proteins are expressed in the membrane of secretory vacuoles from where they are processed and make their way to the secreted saliva. Perhaps due to the power of the ‘‘omics’’ revolution, this chapter is biased to the description and findings of bioactive polypeptides as components of the salivary potion of BFI. We have mentioned, however, several examples of nonpeptidic components such as NO, adenosine, AMP and LPC that are pharmacologically active compounds found in the saliva of bugs and flies. Advances in mass spectrometry, reaching fentomol levels of detection, make it ripe right now to investigate the presence of small agonists in the saliva of BFA. Because many of these compounds, such as prostaglandins or other eicosanoids, are produced during salivation and thus not previously stored, experimental collection of insect saliva should be used instead of salivary gland homogenates.
8
Measuring the size of our ignorance
Biology has reached an interesting point in its history that makes it quite different from other natural sciences. Genomics and transcriptomic analysis can give us a good measure of what is there to learn. Imagine if in physics there was an experimental way to measure the number of atomic sub-particles that remain undiscovered! Physicists are thus unaware of their ignorance’s extent but biologists can now have a measure of it, or at least part of it, with some precision. For example, the genome of An. gambiae has approximately 16,000 genes coding for proteins, and humans have less than 10,000 more. Within these 15,000–30,000 genes found in eukaryotes, there is a substantial proportion coding for proteins that fit in the class of conserved hypothetical proteins (Galperin and Koonin, 2004), representing a measure of our ignorance. Even when we have an idea of the biochemical function for a protein, indicated by its sequence signatures, such as those for serine protease, or a G-coupled receptor, we may still not know when and where the protein is expressed and its role in
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the organism’s physiology, thus our physiological ignorance can be considered greater than that of the biochemist. Sialotranscriptomic analyses done so far are giving us a measure of our ignorance, or how much more we need to learn (Tables 3 and 4). Of the 21 sialotranscriptomes listed in Table 3, nine derive from mosquitoes, including two subfamilies, four genera and five subgenera. Additionally, there are six transcriptomes from sand flies (two genera) and one transcriptome each from a biting midge and a black fly. Mosquitoes, biting midges, black flies and sand flies belong to the Diptera sub-order Nematocera, which, perhaps except for sand flies, may have had a common blood-feeding ancestor (Grimaldi and Engel, 2005). Three transcriptomes are from triatomine bugs, including two different tribes (Rhodnini and Triatomini). A single flea transcriptome has been done and one more is on the pipeline, from two different genera. With this limited data set, we have found (references on Table 3) that insects have from 70 to 150 putative salivary secreted proteins, as indicated by polypeptides containing a signal secretory leader sequence (Nielsen et al., 1997), and excluding those of obvious endoplasmic reticulum function. Of these nearly 100 secretory products, we can assign the function of less than 20 proteins per transcriptome. Most transcriptomes listed in Table 3 have been done by randomly sequencing 1000–2000 clones from salivary gland cDNA libraries. It has been our experience that, perhaps due to the relatively low complexity of the salivary gland proteome (when compared to organs like the mammalian liver or brain, for example), 1000–2000 sequenced clones are enough to display the majority of the sialome. Indeed, 2000 clones sequenced from an Ae. aegypti salivary gland cDNA library (Ribeiro et al., 2007) discovered virtually all those found in another effort that obtained 20,000 sequences (Nene et al., 2007). A similar situation was encountered with the An. gambiae sialome, where 4000 sequences identified basically all those in a large sequencing effort, also 20,000 sequences done by the Pasteur Institute (Arca` et al., 2005). Sequencing of normalized libraries, or more intensive sequencing of existing libraries, perhaps with newer generation of pyrosequencing methodology, may increase the number of salivary secreted peptides, but possibly to no more than 10% of TABLE 4 Relationship between number of known blood-feeding insect species and sialotranscriptome knowledge
Orders Family Genera Species
From Table 1
From Table 3
Percentage analysed
5 17 455 14,555
3 6 11 21
60.00 35.29 2.42 0.14
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the number found with random sequencing of 2000 clones. It should be indicated, however, that these additional low abundance transcripts may account for pharmacologically potent peptides. If we use the rough estimate of 100 polypeptides as the ballpark number for the constitution of the BFI’ salivary potion, how much of these vary from one organism to another, and what is the extent of the universe of the BFI’ salivary proteins, or using the word Ben Mans coined, the sialoverse? First of all, there is virtually no molecular overlap between the magic potions from one order of insects when compared to another order. The sialome of Rhodnius is completely different from that of mosquitoes (as indicated above, even their apyrase activities derive from different enzyme families), or any other non-triatomine listed in Table 1, so we can assign 200 for the number of unique proteins in the sialoverse of triatomine bugs and mosquitoes. Table 1 lists 17 BFI families, five of which are from Nematocera that may have evolved to haematophagy from a common ancestor, sand flies excepted (Grimaldi and Engel, 2005). From these considerations we can number 14 families that evolved independently to haematophagy, and thus a rough maximum number of 1400 unique polypeptides. This number may be somewhat reduced, but not to less than one-half. For example, if we compare sand flies with mosquitoes and black flies, the majority of the sand fly protein families are still unique, or if related, they are very divergent. As mentioned above, sand flies have members of the D7 protein family, which are quite distinct from those of mosquitoes (Valenzuela et al., 2002a) and black flies (Andersen et al., 2009). Sand flies also recruited proteins from the Yellow family to their sialome, not found in any other blood-feeding Nematocera studied so far. But they recruited the Cimex type of apyrase, in complete distinction of their Nematoceran relatives. For the possible overlaps, we can lower our estimate for the family level sialoverse to 1400/2 ¼ 700 unique polypeptides. What is the sialome overlap between same family organisms belonging to different genera, or even at subgenus level? Mosquito sialotranscriptomes of three different genera (excluding the non-blood-feeding Toxorhynchites amboinensis) (Table 3) indicate the presence of several unique protein families belonging to each genus. For example, in Anopheles we mentioned earlier the unique anopheline proteins anophelin, anophensin and the salivary peroxidase, but we here may add the unique families SG1 (a protein family with four members) and SG6, all with unknown function (Arca` et al., 2005). Aedes and Culex share several protein families unique to their culicine subfamily level, namely the 62, 41.9, 34, 27, 23.4, 8.9, basic 3.8 and basic 7.6-kDa families (Ribeiro et al., 2004b, 2007; Arca` et al., 2007), thus eight families of unknown function not found in anophelines, or any other organism. C. quinquefasciatus sialome, in its turn, has one large unique tryptophan-rich protein family, with over 30 genes, and 14 additional putative secreted polypeptides with no known function (Ribeiro et al., 2004b). Similarly, comparison between Ae. albopictus and Ae. aegypti both from the same subgenus Stegomyia reveals eight novel
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peptides in Ae. albopictus, including four that are of a novel multi-gene family (Arca` et al., 2007). Comparison of Rhodnius and Triatoma sialomes, insects from different tribes, reveals various non-overlapping protein families, including the unique nitrophorin family of haem-binding lipocalins carriers of NO, found only in Rhodnius, a multi-gene family coding for at least 20 different proteins (Ribeiro et al., 1998, 2004a). Although members of the triabin lipocalin superfamily are expressed in both Rhodnius and Triatoma, they are very divergent, and many are functionally distinct, as exemplified above with the T. pallidipennis salivary anti-thrombin named triabin that gave its name for this lipocalin family (Noeske-Jungblut et al., 1995; Fuentes-Prior et al., 1997; Glusa et al., 1997). Rhodnius does not have a salivary anti-thrombin, and instead has a lipocalin of the nitrophorin family that blocks the Xase complex of clotting (Gudderra et al., 2005). These considerations indicate that sialome novelty can be reasonably expected between two different genera of the same blood-feeding insect family, or even within different species of the same genus. It is not unreasonable at this point to extrapolate that we should find at least two novel proteins per genus of BFI, or, according to Tables 1 and 4, another 890 novel proteins from 445 genera, reaching a limit of 890 þ 650(700) ¼ 1500 proteins within the BFI sialoverse. On the other hand, if we presume there is a single novel salivary protein in every species of BFI, we should expect the sialoverse to contain near 15,000 proteins. We have to be careful, though, with the extrapolation above regarding the number of different genera and species. First, we have many knowledge gaps at high levels, including nearly one-half of the orders and families still unexplored (Table 4). Only 2.4% of the genera listed in Table 1 have been studied (Table 4). It is clear that two mosquito genera that have a common ancestor at >150 MYA, as are Aedes and Anopheles (Krzywinski et al., 2006) should be quite divergent, as their sialomes bifurcated at the time dinosaurs were still irradiating, and nearly 90 million years before mammal irradiation. Two more closely related genera, such as Ochlerotatus and Aedes (Shepard et al., 2006) should provide more overlapping sialomes, as they probably share a common ancestor within the time frame of mammalian irradiation. Taxonomists are classified into lumpers and splitters, lumpers being those that aggregate many species into a single genus. Observation of Table 1 may lead to the conclusion that flea taxonomists, Miriam Rothschild in particular, may be accused of being a splitter (Rothschild, 1975). One-half of all genera listed in Table 1 indeed derive from the Siphonaptera. However, the Siphonaptera is the youngest of the haematophagous orders, and are thought to have irradiated with the mammals and birds to which they are closely associated. In other words, if a rat flea should be in the same genus as a rabbit flea, rats and rabbits should be, nonsensically, promoted to the same genus. What may be more indicative of the true level of salivary diversity, but the data are not there yet, is to determine how many genera are at least 50 million years apart, a time estimate that would lead as much time to evolve the sialome as it did to evolve mammals.
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A FORECAST OF THE COSTS AND TIME REQUIRED FOR ACQUIRING SIALOME WISDOM
If we consider a low estimate number for the sialoverse as consisting of 1000 proteins, and that for each protein it takes the effort of one scientist for the period of 2 years, including its recombinant expression and functional evaluation, an estimate of 2000 scientist years are to be expected to do this job. A group of 10 scientists accordingly would take 200 years and 100 scientists would take 20 years to this effort. These numbers are clearly an underestimate of the work ahead. Our experience in these novel proteins indicate that they end up generating many more publications than the single one used in the calculation. For example, papers with maxadilan in their title now number 34, nitrophorin number 35, five have D7 in their title and relate to mosquitoes, ixolaris appears in four titles and triabin in three. It is thus possible that 1 in 10 salivary proteins become the target of further pharmacological/vaccine/epidemiological investigations, leading each selected case to the additional production of dozens of additional papers, thus at least doubling the amount of research time needed, to over 40 years for a group of 100 scientists. Prioritization of the type of research to be pursued will probably follow the haphazard motions of scientist initiated research, unless organized departments or institutes decide to attack the subject in a coordinated way, as has been done, for example, with the study of snake venoms. Many generations of scientists will be necessary to decipher the magic potion of BFA, and to collect its fruits.
9
Salivary antigens: Epidemiological tools?
While we may speculate on the size of the task ahead to decipher the magic potion of BFI, a more immediate use of this knowledge is becoming available to help evaluate parameters associated with the transmission of vector borne diseases. Indeed the host immune response to arthropod saliva may also be used as an epidemiological marker of exposure to vector bites. This may represent a very useful serological indicator of risk of disease transmission by a given vector and a convenient tool for surveillance and for evaluation of vector control interventions. After the first evidence that anti-tick saliva antibodies could be a useful epidemiological marker of tick exposure and Lyme disease risk (Schwartz et al., 1990, 1991), several additional reports described the possible use of saliva or salivary extracts as potential markers of exposure. This is the case for sand fly vectors of Leishmania (Barral et al., 2000), triatomine vectors of Chagas disease (Nascimento et al., 2001), tsetse vectors of African trypanosomiasis (Van Den Abbeele et al., 2007) and for mosquito vectors (Trevejo and Reeves, 2005; Trevejo et al., 2005; Remoue et al., 2006; Waitayakul et al., 2006; Orlandi-Pradines et al., 2007). These studies established the interesting principle that the host immune response to BFA saliva
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may represent a reliable indicator of vector density and, therefore, an indirect measure of disease risk, not only at population but also at individual level. However, as we just learned, the salivary potion of BFI is a complex cocktail consisting both of species-shared and of species-specific components and therefore cross-reactivity may significantly contribute to the observed response. This potential cross-reactivity of salivary antigens from different BFI may represent an advantage, for example for vaccine development (Mejia et al., 2006) or for potential use in immunotherapy of hypersensitivity reactions (Peng and Simons, 1997; Peng et al., 1998); however, on the contrary, it may be misleading if the response to salivary antigens is to be used for epidemiological studies. Proof of principle that individual recombinant salivary antigens may be efficient substitutes for saliva or salivary extracts has been obtained for different disease vectors such as ticks (Sanders et al., 1998, 1999), sand flies (Barral et al., 2000), tsetse (Caljon et al., 2006) and mosquitoes (Poinsignon et al., 2008; Lombardo et al., 2009). In this respect, the BFI sialotranscriptomes available so far (Table 3) started revealing the complexity of the sialoverse, providing detailed information on the salivary repertoires of important disease vectors, as mosquitoes and sand flies, and offering the opportunity to carefully select species- or genus-specific antigens for recombinant expression and for the development of novel epidemiological tools for improved vector and disease control (Billingsley et al., 2006). Acknowledgements This work was supported by the Intramural Research Program of the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. We are grateful to Drs. Michalis Kotsyfakis and Babis Savakis for independently suggesting the name kratagonist. Because JMCR is a government employee and this is a government work, the work is in the public domain in the United States. Notwithstanding any other agreements, the NIH reserves the right to provide the work to PubMedCentral for display and use by the public, and PubMedCentral may tag or modify the work consistent with its customary practices. You can establish rights outside of the US subject to a government use license. References Abebe, M., Cupp, M. S., Champagne, D. and Cupp, E. W. (1995). Simulidin: a black fly (Simulium vittatum) salivary gland protein with anti-thrombin activity. J. Insect Physiol. 41, 1001–1006. Abramson, D. I. (1989). Dermal blood vessels and Lymphatics. In: Handbook of Experimental Pharmacology: The Pharmacology of the Skin, Vol. 1 (eds Greaves, M. W. and Shuster, S.), pp. 89–116. Springer-Verlag, Berlin.
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Braverman, I. M. (1997). The cutaneous microcirculation: ultrastructure and microanatomical organization. Microcirculation 4, 329–340. Brossard, M. and Wikel, S. K. (2004). Tick immunobiology. Parasitology 129, S161–S176. Caljon, G., Van Den Abbeele, J., Stijlemans, B., Coosemans, M., De Baetselier, P. and Magez, S. (2006). Tsetse fly saliva accelerates the onset of Trypanosoma brucei infection in a mouse model associated with a reduced host inflammatory response. Infect. Immun. 74, 6324–6330. Calvo, E. and Ribeiro, J. M. C. (2006). A novel secreted endonuclease from Culex quinquefasciatus salivary glands. J. Exp. Biol. 209, 2651–2659. Calvo, E., Andersen, J., Francischetti, I. M. B., de Lara Capurro, M., de Bianchi, A. G., James, A. A., Ribeiro, J. M. C. and Marinotti, O. (2004). The transcriptome of adult female Anopheles darlingi salivary glands. Insect Mol. Biol. 13, 73–88. Calvo, E., Mans, B. J., Andersen, J. F. and Ribeiro, J. M. C. (2006a). Function and evolution of a mosquito salivary protein family. J. Biol. Chem. 281, 1935–1942. Calvo, E., Pham, V. M., Lombardo, F., Arca`, B. and Ribeiro, J. M. C. (2006b). The sialotranscriptome of adult male Anopheles gambiae mosquitoes. Insect Biochem. Mol. Biol. 36, 570–575. Calvo, E., Dao, A., Pham, V. M. and Ribeiro, J. M. C. (2007a). An insight into the sialome of Anopheles funestus reveals an emerging pattern in anopheline salivary protein families. Insect Biochem. Mol. Biol. 37, 164–175. Calvo, E., Tokumasu, F., Marinotti, O., Villeval, J. L., Ribeiro, J. M. C. and Francischetti, I. M. B. (2007b). Aegyptin, a novel mosquito salivary gland protein, specifically binds to collagen and prevents its interaction with platelet glycoprotein VI, integrin a2b1, and von Willebrand factor. J. Biol. Chem. 282, 26928–26938. Calvo, E., Pham, V. M. and Ribeiro, J. M. C. (2008). An insight into the sialotranscriptome of the non-blood feeding Toxorhynchites amboinensis mosquito. Insect Biochem. Mol. Biol. 38, 499–507. Calvo, E., Mans, B. J., Ribeiro, J. M. C. and Andersen, J. F. (2009a). Multifunctionality and mechanism of ligand binding in a mosquito antiinflammatory protein. Proc. Natl. Acad. Sci. USA 106, 3728–3733. Calvo, E., Pham, V. M., Marinotti, O., Andersen, J. F. and Ribeiro, J. M. C. (2009b). The salivary gland transcriptome of the neotropical malaria vector Anopheles darlingi reveals accelerated evolution of genes relevant to hematophagy. BMC Genomics 10, 57. Campbell, C. L., Vandyke, K. A., Letchworth, G. J., Drolet, B. S., Hanekamp, T. and Wilson, W. C. (2005). Midgut and salivary gland transcriptomes of the arbovirus vector Culicoides sonorensis (Diptera: Ceratopogonidae). Insect Mol. Biol. 14, 121–136. Cappello, M., Li, S., Chen, X., Li, C. B., Harrison, L., Narashimhan, S., Beard, C. B. and Aksoy, S. (1998). Tsetse thrombin inhibitor: bloodmeal-induced expression of an anticoagulant in salivary glands and gut tissue of Glossina morsitans morsitans. Proc. Natl. Acad. Sci. USA 95, 14290–14295. Carroll, P. R. and Neering, I. R. (1976). Relationship between vasoconstriction and histamine induced vasodilation. Aust. J. Exp. Biol. Med. Sci. 54, 197–202. Cavalcante, R. R., Pereira, M. H. and Gontijo, N. F. (2003). Anti-complement activity in the saliva of phlebotomine sand flies and other haematophagous insects. Parasitology 127, 87–93. Cerna, P., Mikes, L. and Volf, P. (2002). Salivary gland hyaluronidase in various species of phlebotomine sand flies (Diptera: Psychodidae). Insect Biochem. Mol. Biol. 32, 1691–1697.
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Valenzuela, J. G., Pham, V. M., Garfield, M. K., Francischetti, I. M. B. and Ribeiro, J. M. C. (2002b). Toward a description of the sialome of the adult female mosquito Aedes aegypti. Insect Biochem. Mol. Biol. 32, 1101–1122. Valenzuela, J. G., Francischetti, I. M. B., Pham, V. M., Garfield, M. K. and Ribeiro, J. M. C. (2003). Exploring the salivary gland transcriptome and proteome of the Anopheles stephensi mosquito. Insect Biochem. Mol. Biol. 33, 717–732. Valenzuela, J. G., Garfield, M., Rowton, E. D. and Pham, V. M. (2004). Identification of the most abundant secreted proteins from the salivary glands of the sand fly Lutzomyia longipalpis, vector of Leishmania chagasi. J. Exp. Biol. 207, 3717–3729. Van Den Abbeele, J., Caljon, G., Dierick, J. F., Moens, L., De Ridder, K. and Coosemans, M. (2007). The Glossina morsitans tsetse fly saliva: general characteristics and identification of novel salivary proteins. Insect Biochem. Mol. Biol. 37, 1075–1085. Volfova, V., Hostomska, J., Cerny, M., Votypka, J. and Volf, P. (2008). Hyaluronidase of bloodsucking insects and its enhancing effect on leishmania infection in mice. PLoS Negl. Trop. Dis. 2, 1–8. von Hundelshausen, P., Petersen, F. and Brandt, E. (2007). Platelet-derived chemokines in vascular biology. Thromb. Haemost. 97, 704–713. Waitayakul, A., Somsri, S., Sattabongkot, J., Looareesuwan, S., Cui, L. and Udomsangpetch, R. (2006). Natural human humoral response to salivary gland proteins of Anopheles mosquitoes in Thailand. Acta Trop. 98, 66–73. Warren, J. B., Loi, R. K. and Wilson, A. J. (1994). PGD2 is an intermediate in agoniststimulated nitric oxide release in rabbit skin microcirculation. Am. J. Physiol. 266, H1846–H1853. Weichsel, A., Andersen, J. F., Champagne, D. E., Walker, F. A. and Montfort, W. R. (1998). Crystal structures of a nitric oxide transport protein from a blood-sucking insect. Nat. Struct. Biol. 5, 304–309. Wigglesworth, V. B. (1936). Symbiotic bacteria in a blood sucking insect, Rhodnius prolixus Stal. Parasitology 28, 284–289. Wilson, A. D., Heesom, K. J., Mawby, W. J., Mellor, P. S. and Russell, C. L. (2008). Identification of abundant proteins and potential allergens in Culicoides nubeculosus salivary glands. Vet. Immunol. Immunopathol. 122, 94–103. Wirtz, H. P. (1988). Quantitating histamine in the saliva and salivary glands of two Palaearctic blackfly species (Diptera: Simuliidae). Trop. Med. Parasitol. 39, 309–312. Wirtz, H. P. (1990). Bioamines and proteins in the saliva and salivary glands of Palaearctic blackflies (Diptera: Simuliidae). Trop. Med. Parasitol. 41, 59–64. Xu, X., Yang, H., Ma, D., Wu, J., Wang, Y., Song, Y., Wang, X., Lu, Y., Yang, J. and Lai, R. (2008). Toward an understanding of the molecular mechanism for successful blood feeding by coupling proteomics analysis with pharmacological testing of horsefly salivary glands. Mol. Cell. Proteomics 7, 582–590. Yamazaki, Y. and Morita, T. (2004). Structure and function of snake venom cysteine-rich secretory proteins. Toxicon 44, 227–231. Yamazaki, Y., Hyodo, F. and Morita, T. (2003). Wide distribution of cysteine-rich secretory proteins in snake venoms: isolation and cloning of novel snake venom cysteine-rich secretory proteins. Arch. Biochem. Biophys. 412, 133–141. Yorkee, W. and Macfie, J. W. S. (1924). The action of the salivary secretion of mosquitoes and Glossina tachinoides on human blood. Ann. Trop. Med. Parasitol. 18, 103–108. Yoshida, S., Sudo, T., Niimi, M., Tao, L., Sun, B., Kambayashi, J., Watanabe, H., Luo, E. and Matsuoka, H. (2008). Inhibition of collagen-induced platelet aggregation by anopheline antiplatelet protein, a saliva protein from a malaria vector mosquito. Blood 111, 2007–2014.
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Zaspel, J. M., Kononenko, V. S. and Goldstein, P. Z. (2007). Another blood feeder? Experimental feeding of a fruit-piercing moth species on human blood in the Primorye Territory of Far Eastern Russia (Lepidoptera: Noctuidae: Calpinae). J. Insect Behav. 20, 437–451. Zhang, Y., Ribeiro, J. M. C., Guimara˜es, J. A. and Walsh, P. N. (1998). Nitrophorin-2: a novel mixed-type reversible specific inhibitor of the intrinsic factor-X activating complex. Biochemistry 37, 10681–10690. Zhang, D., Cupp, M. S. and Cupp, E. W. (2002). Thrombostasin: purification, molecular cloning and expression of a novel anti-thrombin protein from horn fly saliva. Insect Biochem. Mol. Biol. 32, 321–330. Zhu, K., Dillwith, J. W., Bowman, A. S. and Sauer, J. R. (1997). Identification of hemolytic activity in saliva of the lone star tick (Acari: Ixodidae). J. Med. Entomol. 34, 160–166. Zhu, K., Bowman, A. S., Dillwith, J. W. and Sauer, J. R. (1998). Phospholipase A2 activity in salivary glands and saliva of the lone star tick (Acari: Ixodidae) during tick feeding. J. Med. Entomol. 35, 500–504.
The Enemy Within: Interactions Between Tsetse, Trypanosomes and Symbionts Deirdre P. Walshe, Cher Pheng Ooi, Michael J. Lehane and Lee R. Haines Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom
1 Background 120 1.1 Human and animal trypanosomiases 120 1.2 Trypanosome species 121 1.3 Tsetse identification and distribution 122 1.4 Tsetse life cycle and physiology 123 1.5 Trypanosome (T. brucei sspp.) life cycle: Development and differentiation 126 2 Tsetse–trypanosome interactions 129 2.1 Parasite surface coat 130 2.2 Host blood factors 131 2.3 Tsetse midgut environment and signals for differentiation 133 2.4 Trypanosomes and tsetse digestive enzymes 134 2.5 Tsetse immune system 135 2.6 Effects of trypanosome infection on tsetse physiology 148 2.7 Fly sex, age and starvation and trypanosome transmission 149 2.8 Environmental temperature and trypanosome transmission 151 3 Symbiont–tsetse–trypanosome interactions 152 3.1 Wigglesworthia glossinidius 152 3.2 Wolbachia pipientis 153 3.3 Sodalis glossinidius 153 4 Towards new methods of disease control 156 4.1 Gene knockdown in Glossina 156 4.2 Paratransgenesis 158 5 Conclusion 159 Acknowledgements 160 References 160
ADVANCES IN INSECT PHYSIOLOGY VOL. 37 ISBN 978-0-12-374829-4 DOI: 10.1016/S0065-2806(09)37003-4
Copyright # 2009 by Elsevier Ltd All rights of reproduction in any form reserved
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Background HUMAN AND ANIMAL TRYPANOSOMIASES
African trypanosomiasis refers to a set of diseases of humans and their domesticated animals, which have devastating consequences for Africa. Tsetse flies (Diptera: Glossinidae) are the sole insect vectors responsible for cyclical transmission of African trypanosomes, the protozoan parasites responsible for Human African Trypanosomiasis (HAT ¼ sleeping sickness) and African Animal Trypanosomiasis (AAT ¼ nagana). In the early part of the last century several HAT epidemics occurred on the African continent, but by the early 1960s the disease was controlled and had almost disappeared (Steverding, 2008). However, relaxation of surveillance and control measures led to resurgence in HAT, peaking in 1997 at an estimated 450,000 cases (Barrett, 2006) with an estimated 60 million people at risk in 37 countries of sub-Saharan Africa (corresponding to one third of Africa’s total land area) (WHO, 2000). Since that date, case detection and treatment have been increased, and by 2007, the reported number of new HAT cases had dropped to 10,769 (WHO, 2007), which probably equates to 50–70,000 total human cases. HAT can take two forms depending on the parasite involved. Trypanosoma brucei rhodesiense and T. b. gambiense are the causative agents of HAT in East/Southern Africa and Central/West Africa, respectively. Typically, the T. b. rhodesiense transmission cycle involves wild and domestic animals, but intensified human to human transmission may occur during epidemics. The T. b. gambiense transmission cycle is mostly from human to human, involving animals to a much lesser extent. In humans, T. b. rhodesiense infections are acute, lasting from a few weeks to several months, while T. b. gambiense infections are chronic, generally lasting for several years, often without any major signs or symptoms. There are no prophylactic drugs or vaccines available to prevent HAT. In both cases, without proper diagnosis and treatment, the outcome is fatal. Therefore, the earlier the disease is detected the better the chance of survival. However, all four drugs currently used to treat HAT exhibit toxicity and, in many countries, drug resistance is beginning to emerge (Legros et al., 2002; Delespaux and de Koning, 2007; Balasegaram et al., 2009). Other parasite species and sub-species of the Trypanosoma genus are pathogenic to many wild and domestic animal species. In particular, T. b. brucei, T. congolense and T. vivax are major causes of the animal form of trypanosomiasis. Disease severity is dependent on both the pathogenicity of the parasite strain and the genetics of the mammalian host (Courtin et al., 2008). While most African wildlife is tolerant of the parasites, domesticated livestock are highly susceptible to disease, particularly if they originate from European stock. Despite implementation of many tsetse control strategies,
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nagana still has a large impact on agricultural and livestock production systems and land use. The disease has an estimated annual economic cost of approximately US $4.5 billion to the African economy due to losses in milk, meat and wool yields through adult mortality, calf mortality and subsequent depressed herd growth (Kristjanson et al., 1999). Furthermore, nagana is a major restriction to the development of arable agricultural in sub-Saharan Africa, limiting the use of draught and pack animals and preventing the development of mixed agricultural practices (Jordan, 1986). Currently, nagana is managed predominantly by use of trypanotolerant breeds of cattle and use of chemoprophylactic and trypanocidal drugs (Miruk et al., 2008).
1.2
TRYPANOSOME SPECIES
Two distinct groups of insect-transmitted trypanosomes are generally recognized. The Stercocaria (subgenus Megatrypanum, Schizotrypanum and Herpetosoma) and the Salivaria (subgenus Nannomonas, Duttonella and Trypanozoon). Stercorarian trypanosomes develop in the hindgut of the insect and are transmitted in the faeces. The predominant vectors of stercorarian trypanosomes are tabanids, triatomines, leeches and ticks. It is the salivarian trypanosomes which cause nagana and African sleeping sickness. The salivarian trypanosomes develop in the anterior part of the tsetse fly alimentary canal and are transmitted via the mouthparts. Tsetse flies are the major vectors of T. brucei trypanosomes but mechanical transmission of several salivarian trypanosome species, by tabanids and Stomoxys vectors, also occurs. Only the salivarian trypanosomes exhibit antigenic variation, a unique form of parasite immune evasion within the mammalian host (Cross, 1996) and it is several of these species which are responsible for the complex of diseases known as African trypanosomiasis. T. vivax (subgenus Duttonella), believed to be the most ancient of the salivarian trypanosomes, produce a high incidence of infection in the tsetse proboscis (Haag et al., 1998) and are serious pathogens of cattle. The Nannomonas subgenus is comprised of three species: T. congolense (Broden, 1904), T. simiae (Bruce et al., 1912) and T. godfreyi (McNamara et al., 1994). Of these, T. congolense is the most economically important, with a broad host range and wide geographical distribution. T. simiae and T. godfreyi are primarily associated with suid infections; the former being extremely pathogenic (acute) and the latter producing a chronic, sometimes fatal, infection in pigs. The Trypanozoon subgenus contains the trypanosomes causing HAT (T. b. gambiense and T. b. rhodesiense) and T. b. brucei which is one of the parasites causing nagana. T. b. brucei is unable to infect humans as it is sensitive to a trypanolytic factor in human serum (Oli et al., 2006) and is thus restricted to development in domestic animals and many species of wildlife.
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Of the African trypanosomes, T. congolense, T. b. brucei, T. b. gambiense and T. vivax have been selected for partial or complete genome sequencing by a consortium of institutes: The Institute for Genome Research (TIGR), now the J. Craig Venter Institute (Rockville, MD), and the Wellcome Trust Sanger Institute (Hinxton, UK). All genomes are in various states of completion. At the time of writing, the nuclear genome of T. brucei (TREU927 GUTat 10.1), the model species most often used for studying trypanosome biology, had been completed and the T. b. gambiense (MHOM/CI/86/DAL972) genome is in the last stages of finishing, with 8 coverage. Also, the partial nuclear genome sequence (5 coverage) of T. congolense (IL3000) and T. vivax are being assembled (http://www.sanger.ac.uk/Projects/Protozoa/).
1.3
TSETSE IDENTIFICATION AND DISTRIBUTION
The name tsetse (pronounced tsee–tsee) is derived from the noise the fly creates when it raises its body temperature by contraction of flight muscles decoupled from the wings, prior to energetically demanding events (e.g. birth of the larva, flight, rapid dehydration of the bloodmeal). Interestingly, tsetse means ‘‘fly’’ in the Tswana language and in Sechuana it is interpreted as ‘‘fly destructive to cattle’’. Tsetse flies are easily distinguishable from other insects (Fig. 1). They are light brown to black in colour and, dependant on the species, are roughly twice the size of a housefly. Characteristic aristae are present on the third antennal segment (Fig. 1A). In addition, the unique ‘‘hatchet’’ wing cell is found in the centre of each wing between the fourth and fifth veins (Fig. 1B). Also, tsetse flies adopt a characteristic resting attitude with their single pair of wings folded scissor-like over the dorsal surface of the abdomen. A
B
FIG. 1 Characteristic anatomical features of Glossina sspp. (A) Side view of an engorged female Glossina morsitans morsitans resting during diuresis. An anal droplet has started to form within minutes of feeding. The characteristic arista (arrow), a thin structure bearing a single unidirectional row of branched setae, is used for species identification. Photo: R. Wilson http://www.raywilsonbirdphotography.co.uk. (B) The middle of the wing contains a unique venation pattern resembling a hatchet, which is also characteristic of tsetse flies. Photo: L. Rafuse Haines.
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Tsetse flies fall into a single genus, Glossina, and are restricted to subSaharan Africa except for two localities in the Arabian peninsula. Twentythree species and eight sub-species of tsetse fly are currently recognized (Leak, 1999; Krafsur, 2009). These are divided into three distinct clades: Morsitans, Palpalis and Fusca, which are named after the best known species in each subgenus. Commonly these groups are described by the ecological niches they occupy; savanna (Morsitans), riverine (Palpalis) or forest (Fusca) groups. In Central and West Africa, the riverine species (Palpalis group) tend to feed predominantly on reptiles and ungulates. Humans regularly encounter these flies, particularly when visiting water sources, and these species are important vectors of human sleeping sickness. The savannah-woodlands species (Morsitans group) are the most economically important, as they preferentially feed on livestock and wildlife and are the major vectors of nagana. Both the Palpalis and Morsitans groups are vectors of T. brucei sspp. Most tsetse from the third clade (Fusca group) inhabit the damp, evergreen forests. The exception is G. brevipalpis, which is more regularly found in association with livestock. With the exception of G. brevipalpis, flies in the Fusca group are not considered to be medically or agriculturally important. Whether this will change when further pressure on land use drives them into more regular encounters with humans and their domesticated animals remains to be seen.
1.4
TSETSE LIFE CYCLE AND PHYSIOLOGY
Tsetse are unique among insect disease vectors in that they possess a viviparous lifestyle. Consequently, flies have a very low rate of reproduction, typically producing 8–10 offspring in their lifetime in optimal laboratory conditions (Leak, 1999; Attardo et al., 2006). The tsetse reproductive tract possesses extensive modifications to permit intrauterine larval development, which include a reduced number of ovarioles per ovary (two), a highly tracheated and muscular uterus and a modified uterine accessory gland (milk gland) to supply nutrients to the developing larvae (Leak, 1999; Attardo et al., 2006). Oogenesis begins before tsetse eclosion. A single oocyte develops at a time, starting with one of the two ovarioles in the right ovary, and takes 6–7 days to complete. Oogenesis appears to be regulated by the presence of a developing embryo or larvae in the uterus. In most Glossina species, the female is sexually mature 48–72 h posteclosion (i.e. emergence of the adult insect from the puparium), while males become fertile several days after eclosion. Female flies generally mate only once and can store sperm for the duration of their life. Upon completion of oogenesis, an oocyte is synchronously ovulated and fertilized in the uterus where it undergoes embryonic and larval development. The larva is solely nourished by a milk-like secretion rich in proteins and lipids produced by a pair of milk glands. A fully developed third instar larva is deposited by the
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female approximately 16 days after fertilization in the case of the first offspring, and every 9 days thereafter. The larva burrows into the ground where rapid pupariation takes place, and the adult fly emerges from the puparium approximately 4 weeks later. In the tsetse field, the term teneral is used to describe the period of time following emergence of the adult fly from the puparium until it takes its first bloodmeal. Both male and female adult tsetse flies are obligate haematophages capable of transmitting trypanosomes. Tsetse flies are pool feeders and the repeated penetration of mammalian host tissue by the tsetse proboscis results in the formation of a sub-surface blood pool. Saliva is expressed into the wound and trypanosomes are transmitted to the mammalian host at this stage. The bloodmeal is sucked up through the proboscis and oesophagus and propelled into the rest of the alimentary canal by the rhythmic pumping of the cibarial pump aided by the contraction of circular muscles that encompass the oesophagus. The proventriculus (¼cardia) (Fig. 2C) lies at the junction of the oesophagus, midgut and crop duct. Blood may pass directly into the midgut or into the extension of the foregut known as the crop (Fig. 2A), before regurgitation into the midgut (Moloo and Kutuza, 1970). The proventriculus acts as a valve regulating the directional flow of blood and is also the organ responsible for producing the peritrophic matrix (PM) (see Section 2.5.1). The tsetse midgut is a simple tube, lacking diverticula, running from the proventriculus to the junction with the hindgut, which is marked by the entrance of the Malpighian tubules into the alimentary canal (Fig. 2, top panel and Fig. 2J). Although more complex divisions exist (Bo¨hringer-Schweizer, 1977), the midgut can be crudely separated into three functional regions: the anterior midgut, bacteriome (¼mycetome) and posterior midgut. The first part of the anterior midgut is a linear tube running though the thorax. Once it enters the abdomen, the anterior midgut becomes distended and here the blood is stored and dehydrated prior to digestion. Epithelial cells of the anterior midgut possess extensive infoldings of the basal plasma membrane with associated mitochondria, enabling the fly to achieve this rapid dehydration (Bo¨hringerSchweizer, 1977). The anterior midgut is interrupted approximately in its middle section by a region of cells called the bacteriome (Fig. 2E). The bacteriome houses the intracellular symbiotic bacteria Wigglesworthia glossinidius. Haemolysis and bloodmeal digestion commence at the very beginning of the posterior midgut, where haemolytic agents and digestive enzymes are produced. Virtually all proteolytic enzymes are restricted to the posterior midgut (Gooding, 1974a). The junction of the anterior and posterior midguts (Fig. 2F) is obvious and tightly delineated in fed flies, as the bloodmeal changes in colour from red to brown/black as haemolysis and digestion progesses. Cells of the proximal part of the posterior midgut possess extensive rough endoplasmic reticulum, large numbers of Golgi bodies and secretory vesicles allowing efficient production, storage and secretion of proteins (Bo¨hringer-Schweizer, 1977).
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A Posterior Midgut
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FIG. 2 Anatomy of the alimentary canal of Glossina morsitans morsitans. Top panel represents a dissected alimentary canal aligned anterior (left) to posterior (right). The letters (A–L) correspond to the lower panels, which are magnified regions of the digestive system visualized using light microscopy; (A) the crop, (B) salivary glands, (C) the proventriculus capped by a modified fat body crown (oenocytes), (C–D) the most proximal part of the anterior midgut that lies in the thorax, (D) the point where the anterior midgut enters the abdomen, (E) the bacteriome that contains the obligate symbiont Wigglesworthia glossinidia, (F–G) the most distal part of the anterior midgut and the junction where the anterior and posterior midgut meet, demarked by a bloodmeal colour change (from red to black), (H) fat body attached to the wall of the posterior midgut, (I) the distinctive posterior midgut epithelial cells (luminal surface), (J) the bases of the Malpighian tubules leave the alimentary canal at the junction of the midgut and hindgut. The two Malpighian tubules each branch near their base to form a total of four tubules, (K) ileum (severed from the MT and midgut) and the junction with the colon, (L) colon ending in the bulbous rectum; note peritrophic matrix (*) exuding from a breach in the hindgut wall. Photo: L. Rafuse Haines.
Cells of the distal part of the posterior midgut are involved in absorption of digested products. They possess extensive smooth endoplasmic reticulum and also lipid droplets and glycogen at various times of the digestive cycle (Bo¨hringer-Schweizer, 1977).
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DEIRDRE P. WALSHE ET AL. TRYPANOSOME (T. BRUCEI SSPP.) LIFE CYCLE: DEVELOPMENT AND DIFFERENTIATION
T. brucei has the most complex, but perhaps the best characterized, life cycle of all African trypanosome species. The trypanosome life cycle was first described in detail by Muriel Robertson who described the successive stages of parasite establishment and maturation within the insect and mammalian hosts, demonstrated the migration of parasites through the fly midgut and proved that only salivary gland forms were capable of producing a mammalian infection (Robertson, 1913). Since then, a more complete understanding of trypanosome development has been achieved, with an agreed parasite nomenclature adopted (Roditi and Clayton, 1999) and a consensus achieved on many of the barriers present in the fly that the trypanosome must overcome to survive and develop in order to complete cyclical transmission. Within the vertebrate bloodstream at least two different major forms of trypanosomes are found; a long slender form, which replicates by asexual division, and a short stumpy, non-replicating form (Fig. 3(1)). These extracellular parasites are covered with an immunogenic surface coat composed of approximately 107 identical variant surface glycoprotein (VSG) molecules (Vickerman, 1969; Barry and McCulloch, 2001; Barry et al., 2005). The VSG coat physically shields underlying membrane proteins from host immune responses and is central to antigenic variation and survival in the mammalian host. The consecutive, but mainly unpredictable, expression of a large repertoire of VSG genes permits expansion of antigenically distinct trypanosome populations within the host. After activation of host immune responses (in reaction to high parasitaemia), the majority of the parasite population is destroyed. A small number of trypanosomes survive because they express an antigenically distinct VSG coat, and proceed to expand in numbers. The continuous cycles of trypanosome replication and destruction result in waves of fluctuating parasitaemia. The differentiation of the long slender bloodstream form (BSF) into the non-dividing stumpy BSF occurs in high density populations of long slender BSFs (Vassella et al., 1997; Seed and Wenck, 2003). The switch to stumpy BSF involves changes in metabolism within the trypanosome, but the molecular signals involved are not yet known. Short stumpy BSFs are believed to be pre-adapted for survival within the insect midgut due to the presence of a functional mitochondrion. In the vertebrate bloodstream, trypanosomes utilize glucose as an energy source. However, in the fly midgut, glucose is limiting and a more efficient utilization of glucose and amino acids occurs via the Krebs cycle and oxidative phosphorylation in the mitochondrion. To study the early events of trypanosome establishment in the tsetse midgut, T. b. brucei trypanosomes expressing green fluorescent protein (GFP) under the control of a procyclin promoter have been created (Gibson and Bailey, 2003). It is evident that differentiation of the BSF to the procyclic form (i.e. the insect
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FIG. 3 Diagram of the life cycle stages of T. b. brucei within the tsetse fly. Trypanosome morphology was based on observations by Van den Abbeele et al. (1999) and Sharma et al. (2008). Stages of the trypanosome life cycle within the tsetse are indicated within circles joined by the red arrow; the grey arrow represents transit between insect and mammalian host. 1. Short stumpy (SS) and long slender (LS) bloodstream forms (BSFs) ingested with the infective bloodmeal; 2. Transformation of short stumpy (SS) BSF into procyclics (P) within the midgut endoperitrophic space; 3. Procyclics differentiate into mesocyclics (Ms) within the ectoperitrophic space; 4. Transformation of mesocyclics (Ms) into long trypomastigotes (LT), which give rise to asymmetrically dividing trypomastigotes (ADT); 5. Trypomastigotes (ADT) divide into long epimastigotes (LE) and short epimastigotes (SE). These three stages are present in both the proventriculus and salivary glands; 6. Short epimastigotes (SE) interdigitate within the salivary gland epithelium at their anterior ends, and subsequently mature into the nondividing, mammalian infective metacyclics (M). Note the gradual migration of the kinetoplast in the fly stages of the parasite, culminating in a position anterior to the nucleus in the trypomastigote and epimastigote forms. The reversion of this position to the posterior end of the parasite is a signature of mature metacyclics.
midgut-adapted form), which involves replacement of surface VSGs by procyclins, occurs rapidly after bloodmeal ingestion by the fly (Fig. 3(2)) (Vassella et al., 2001; Acosta-Serrano et al., 2001; Gibson and Bailey, 2003). Most flies successfully kill all invading trypanosomes in a process termed self-cure.
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For the first 3 days, trypanosomes are mostly contained within the bloodmeal as it is being digested. The critical events in parasite establishment appear to occur approximately 3 days after infection, when the relatively small proportion of surviving trypanosomes ( 10%) either die or rapidly multiply in number (Gibson and Bailey, 2003). Typically, from 8 days after the infected bloodmeal, dissected flies can be confidently divided into two groups; the first in which most flies will have self-cured (having completed the clearing of ingested trypanosomes from their midguts) and the second which have established midgut infections. Trypanosomes in an established infection migrate to the ectoperitrophic space 3–5 days post-infection (Gibson and Bailey, 2003) (Fig. 3(3)). It is believed that this occurs by direct penetration through the PM (Ellis and Evans, 1977; Gibson and Bailey, 2003) although an alternative but less likely, suggestion is that it occurs by circumnavigation around the open, posterior end of the PM in the hindgut (see Section 2.5.1). Typically the midgut population in an established infection reaches approximately 5 105 trypanosomes (Van den Abbeele et al., 1999; Gibson and Bailey, 2003). From 6 to 8 days post-infection, large numbers of trypanosomes congregate within the proventriculus (Van den Abbeele et al., 1999; Gibson and Bailey, 2003; Sharma et al., 2008) (Fig. 3(4)). Here they appear to cease division, elongate to mesocyclic forms and later differentiate into long trypomastigotes (Fig. 3(4)) (Van den Abbeele et al., 1999). Trypanosomes then migrate back into the endoperitrophic space by actively penetrating the PM and move anteriorly in the lumen of the foregut to the opening of the hypopharynx at the tip of the proboscis. An alternative theory of migration involves the direct penetration of the tsetse salivary glands after trypanosomes have traversed the fly haemolymph (Mshelbwala, 1972). It is generally accepted that this is unlikely, as trypanocidal factors known to be present in the haemolymph (Croft et al., 1982) would act as a major barrier for trypanosomes attempting to traverse it. Early positioning of trypanosomes in the anterior midgut and proventriculus should also favour passage along the foregut to the salivary glands (Peacock et al., 2007). Asymmetric division of the proventricular epimastigote form generates both long and short parasites (Fig. 3(5)) and it is either the asymmetrically dividing trypanosome or the short epimastigote that arrives at the salivary gland (Sharma et al., 2008). Each tsetse fly has two salivary glands. Evidence suggests that each gland is invaded and colonized separately, with few epimastigotes constituting the founder populations (Peacock et al., 2007). The short epimastigote forms are believed to attach to the salivary gland epithelium by interdigitation of their membranes (Fig. 3(6)). Upon binding, the non-infective epimastigotes complete several rounds of replication and differentiate into the metacyclic form. Differentiation (metacyclogenesis) includes the appearance of a VSG surface coat. Metacyclic VSGs display a specific VSG repertoire subset and their expression is regulated differently to bloodstream VSGs (Barry et al., 1998; Graham et al., 1999). Mitochondrial changes also occur, including loss of mitochondrial cristae and Krebs cycle enzymes. The biochemical changes
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accompany the posterior migration of the kinetoplast before the parasite detaches into the lumen as a mature, free-form, infective metacyclic trypomastigote. At this point, each mature metacyclic parasite has undergone the transformations necessary for survival in a mammalian host. The above is an account of the most complex trypanosome life cycle completed in the tsetse fly and it should be noted that there are distinct differences between the life cycle of T. brucei sp. trypanosomes and other African trypanosome species. T. vivax completes its entire life cycle in the proboscis of the fly while T. congolense infections develop in the fly midgut and mature in the hypopharynx. The number of flies that develop a mature infection and the length of time required for an infection to establish and mature into a transmissible form can vary depending on several factors, including fly species (Welburn et al., 1989, 1994), fly sex (Distelmans et al., 1982; Dale et al., 1995) (see Section 2.7) and parasite strain (Dale et al., 1995). Recent evidence suggests migration of trypanosomes from the proventriculus to the salivary glands may occur continuously (Peacock et al., 2007; Sharma et al., 2008), albeit at low numbers, as opposed to being restricted to a limited time ‘‘window’’ (Van den Abbeele et al., 1999). Once a fly is infected it will produce infective metacyclics for the duration of its life, which can be 150 or more days for females and about half that for males (Lehane and Mail, 1985; Msangi et al., 1998). Thus, there is potential for infective parasites to be transmitted every time a fly feeds on a new host. In addition, flies are capable of harbouring mixed infections of two or more species of trypanosome (Lehane et al., 2000; Van den Bossche et al., 2004; Kubi et al., 2005; Peacock et al., 2007). Sexual reproduction has been reported in T. b. brucei in the salivary glands and may exist in other trypanosome species (Jenni et al., 1986; Peacock et al., 2007; Gibson et al., 2008). However, mating is not an obligatory part of the trypanosome life cycle and occurs in only a proportion of flies co-infected with two different strains of T. b. brucei (Jenni et al., 1986; Schweizer et al., 1988). It is suggested that the unattached epimastigote is the mating stage (Gibson et al., 2008) (Fig. 3) but the mechanism of genetic exchange is not yet known. Under field conditions mating may be a rare event (Koffi et al., 2009).
2
Tsetse–trypanosome interactions
It appears from the established literature that all tsetse species are susceptible, to some degree at least, to trypanosome infections. In general, tsetse in the Palpalis group species tend to be poor vectors of congolense-type trypanosomes compared to the Morsitans group flies (Harley and Wilson, 1968; Moloo and Kutuza, 1988a; Ndegwa et al., 1992). Conversely, tsetse of the Morsitans group are poorer vectors of T. b. gambiense than the Palpalis group (Richner
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et al., 1988). Care needs to be taken with much of the data on susceptibility, as often fly and trypanosome strains used in experiments are from widely divergent geographical origins. Many factors influence fly susceptibility to trypanosome infection. Our understanding of these factors and their underlying mechanisms is still rudimentary. In this review, we concentrate on the physiological factors influencing tsetse fly susceptibility to trypanosomes. 2.1
PARASITE SURFACE COAT
Throughout their life cycle in the tsetse fly, trypanosomes are covered by different surface molecules. Successful differentiation of BSF to procyclic forms involves the shedding of surface VSG and replacement with a set of new surface molecules, the best known being the procyclins (Roditi and Pearson, 1990; Beecroft et al., 1993). The procyclins are major GPI-anchored proteins possessing extensive C-terminal glu-pro (EP) or gly-pro-glu-glu-thr (GPEET) repeats, and comprise a surface coat of approximately three million procyclin molecules per cell (Roditi et al., 1998; Pays and Nolan, 1998; Roditi and Lehane, 2008). Three isoforms of EP procyclin, EP1, EP2 and EP3, are known, which differ in the length of their repeats, the sequence of their N-terminal domains and the presence or absence of N-glycosylation sites. Analysis of procyclin expression by mass spectrometry indicated that trypanosomes exhibit distinct procyclin expression profiles in vivo. All four procyclins are present at similar levels within a few hours of differentiation to procyclics (Vassella et al., 2001). However, 3 days post-infection, the procyclic coat consisted primarily of GPEET with lower amounts of EP forms (AcostaSerrano et al., 2001). This GPEET was 11 residues shorter than that of in vitro cultured procyclic culture forms (PCFs). From 7 days post-infection, GPEET procyclin disappeared and expression switched to the glycosylated isoforms EP1 and EP3. EP1 and EP3 were also truncated in comparison to these isoforms expressed by in vitro cultured PCF. Transcripts of EP2 are quite abundant in fly-derived procyclic forms but EP2 proteins have not yet been confirmed in vivo (Urwyler et al., 2005). While tsetse procyclins exhibit complex expression profiles (suggesting a selective advantage in their production) their function remain unknown. Proteolysis in the fly removes the N-terminal domains of all procyclins, but the acidic amino acid repeat sequences are largely resistant to proteolysis (Acosta-Serrano et al., 2001). Thus procyclins may serve a protective role, aid parasite development and/or influence ligand-associated parasite–vector signalling (Roditi and Pearson, 1990; Ruepp et al., 1997). Interestingly, parasites expressing procyclins with truncated N termini can still establish midgut infections to similar levels as wild-type parasites under laboratory conditions and form mature metacyclics within the salivary glands (Liniger et al., 2004). Thus, procyclins are not crucial for migration of procyclic forms from the tsetse midgut to the salivary glands (Vassella et al., 2009). However, in co-infection experiments,
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procyclin null mutants were rapidly outgrown in the midgut by wild-type parasites. This suggests that under field conditions, where mixed infection in flies is common (Lehane et al., 2000), surface procyclins probably play a significant role in trypanosome fitness (Vassella et al., 2009). As trypanosome epimastigotes within the tsetse salivary glands typically lack a procyclin coat (Urwyler et al., 2005), the function of the procyclin coat molecules probably occurs earlier in trypanosome development in the fly. T. congolense PCFs express several major stage-specific surface molecules different to those of T. brucei. In the early stage of T. congolense midgut infection, a protease-resistant surface molecule (PRS) is expressed, while later, a heptapeptide-repeat containing molecule (Butikofer et al., 2002) and a glutamic acid–alanine-rich protein (GARP) are expressed (Beecroft et al., 1993; Bayne et al., 1993; Utz et al., 2006). Interestingly, T. congolense procyclins bear similarity to T. brucei procyclins in being acidic and having repeat sequences rich in glutamic acid, glycine, threonine and proline (Utz et al., 2006). Also, genes related to GARP have been identified in both T. simiae and T. godfreyi. Epimastigote forms of T. congolense and T. brucei, found in the proboscis and salivary glands respectively, express related surface glycoproteins named congolense epimastigote specific protein (CESP) (Inoue et al., 2000) and brucei alanine-rich proteins (BARP) (Urwyler et al., 2005). The function of these different parasite surface molecules is still unknown. In the salivary glands nascent and mature metacyclics (re)acquire a VSG coat in preparation for transfer to the next mammalian host (Tetley and Vickerman, 1985). 2.2
HOST BLOOD FACTORS
Different trypanosome species are capable of infecting different mammalian hosts, which implies that host blood factors can affect trypanosome survival (Vickerman, 1985; Masaninga and Mihok, 1999). An intriguing question is whether or not the influence of these host blood factors extends to trypanosome establishment in the tsetse midgut. T. brucei developmental forms are present in the midgut for at least 8 days, before commencing migration and maturation in the foregut and salivary glands. As tsetse flies ingest a new bloodmeal every 24–48 h, there is ample opportunity for an ingested bloodmeal to have an impact on trypanosome survival. The mammalian host blood in which the trypanosome infection is acquired by tsetse affects both differentiation (Nguu et al., 1996) and multiplication of parasites in the fly midgut (Olubayo et al., 1994; Mihok et al., 1995). Buffalo and eland blood support establishment of T. congolense infections in G. m. morsitans less well than goat blood (Olubayo et al., 1994). This was apparent from 3 days after the infective bloodmeal and occurred regardless of whether flies were maintained on rabbit blood or the blood of the respective mammalian hosts in which the infective meal was given (Olubayo et al., 1994).
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This suggests that the trypanocidal event in the fly midgut occurs early in the infection process. Serum components of the blood may be either conducive or detrimental to survival of trypanosomes in the fly (Reduth et al., 1994; Mihok et al., 1995; Muranjan et al., 1997). The serum of the Cape buffalo, for example, is trypanocidal to most species of trypanosomes (Reduth et al., 1994). Using T. b. brucei BSF, the primary cause of parasite death was inhibition of glycolysis by host hydrogen peroxide (H2O2) generated by xanthine oxidase (Muranjan et al., 1997). Whether or not this effect holds true for procyclic forms if a recently infected fly subsequently feeds on Cape buffalo merits a more detailed study. However, it is unlikely the same trypanocidal mechanism would occur in vivo since procyclic trypanosomes metabolize proline, rather than glucose, as a major energy source (Muranjan et al., 1997). While xanthine oxidase is present in the serum of other mammals, it does not cause death of different trypanosome species in these hosts (Cruz et al., 1983; Reduth et al., 1994). Therefore, it is possible that generation of H2O2 to trypanocidal levels by xanthine oxidase in Cape buffalo may be part of a wider metabolic cascade, including purine catabolizing enzymes (Muranjan et al., 1997). Thus, experiments using the serum of different mammalian species, which show seemingly little correlation between serum concentration and BSF trypanosome survival, may simply differ in the molecular constituents of different mammalian host serums (Black et al., 1999). Host serum complement (an immunological cascade present in mammalian serum, which, upon activation, induces lysis of target microbes) may also be involved in causing trypanosome mortality (Ferrante and Allison, 1983; Black et al., 1999). A BSF trypanocidal factor, which is heat labile (inactivated following 30 min at 56 C), has been documented in the serum of mammalian species lacking any xanthine oxidase activity (Black et al., 1999). Also, T. congolense and T. b. brucei PCFs are killed by another heat sensitive factor in human serum (Ferrante and Allison, 1983). Similar PCF lytic activity was observed in human serum deficient in complement C2 (part of the classical complement pathway), as well as in ethylene glycol tetra-acetic acid (a preferential chelator of calcium ions, a crucial substrate for initiation of the classical pathway). Consequently, the trypanocidal factor may rely on the alternative complement pathway for activation (Ferrante and Allison, 1983). The infective bloodmeal origin also appears to play a key role in determining the virulence and infectivity of the resulting salivary gland metacyclics (Masaninga and Mihok, 1999). In vitro grown T. congolense BSFs were mixed with either goat, eland or zebra blood and fed to G. m. centralis flies. The resulting metacyclics used to infect BALB/c mice varied in their prepatent periods and resulted in differences in mouse mortality rates (Masaninga and Mihok, 1999). A strong selection pressure appears to be exerted by host blood, most likely on the procyclic parasite stage exposed to incoming bloodmeals, which may lead to differential killing of parasites.
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TSETSE MIDGUT ENVIRONMENT AND SIGNALS FOR DIFFERENTIATION
Efforts to determine the triggers responsible for trypanosome switching from bloodstream to procyclic forms have primarily been conducted in vitro and have occasionally ignored the realities of tsetse fly biology. A variety of methods have been used to define differentiation including morphological markers (Hunt et al., 1994), the release of VSG proteins and concomitant detection of procyclins, or the detection of DNA synthesis (Matthews and Gull, 1997). Other novel approaches include the use of transgenic trypanosomes carrying the E. coli glucuronidase (GUS) gene under the control of procyclin expression elements (Sbicego et al., 1999) and the use of GFP-expressing trypanosomes under the regulation of a procyclin-linked promoter (Sheader et al., 2004). Reduction of temperature alone from 37 to 27 C can initiate the differentiation of BSFs to PCFs (Brown et al., 1973; Bienen et al., 1980; Overath et al., 1986) and is conducive to growth and proliferation of a transformed trypanosome population. Using the GUS system, Sbicego et al. (1999) found that both citrate and cis-aconitate (Krebs cycle intermediates) could induce trypanosome differentiation to PCF in vitro, in a manner independent of the commonly used temperature shift (from 37 to 27 C). This phenomenon appears to be highly specific as other Krebs cycle intermediates, even when present at relatively high concentrations in vitro (10 mM), did not trigger parasite differentiation (Sbicego et al., 1999). Also, compounds closely resembling citrate and cis-aconitate, for example trans-aconitate and 5-fluoro-citrate, were trypanocidal (Hunt et al., 1994). To date, the citrate concentration of the fly midgut lumen has not been reported. However, if similar to the low citrate concentration estimated within the fly haemolymph (15.9 mM), this may be insufficient to trigger BSF differentiation (Hunt et al., 1994). However, BSFs exhibit hypersensitivity towards cis-aconitate when they are exposed to a cold shock (Engstler and Boshart, 2004). Thus lower concentrations of citrate and cis-aconitate may be sufficient to trigger differentiation of BSFs in vivo when used in combination with a temperature drop (Hunt et al., 1994; Rolin et al., 1998; Fenn and Matthews, 2007). Trypanosome differentiation may also be initiated in vitro by growth of BSFs in low glucose medium (Milne et al., 1998). This may reflect what occurs in vivo, as changes in glucose concentration occur as the trypanosomes transfer from a high glucose environment (mammalian bloodstream) to a lower glucose environment (fly midgut). Furthermore, glucose concentration may be a factor involved in controlling switches in procyclin expression at later stages of trypanosome development (Morris et al., 2002). Incubation of BSFs under mildly acidic conditions (pH 5.5) was shown to cause differentiation to PCFs in vitro (with corresponding shedding of the VSG coat and expression of procyclins) at 27 C, even in the absence of cis-aconitate (Rolin et al., 1998). However, this in vitro work seems to have ignored conditions in the tsetse fly, where the best available data suggest that the pH of the midgut is highly alkaline. The use of microelectrodes to measure the midgut pH
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in unfed flies and fed flies 48 and 72 h post-feed has shown that the tsetse digestive tract average pH lies between 9 and 9.5, with the lowest pH value (pH 7.9) occurring in the posterior midgut 72 h post-feeding (Liniger et al., 2003). The enzymes trypsin (see Section 2.4) and thermolysin stimulate trypanosome differentiation in vitro. In the case of trypsin, at least, this occurs independently of any slender BSF attrition effects (Sbicego et al., 1999). Pronase was also shown to be an efficient trypanosome differentiation trigger, with up to 95% differentiation to PCFs achieved (Hunt et al., 1994). However, as far as we are aware, of the proteases above only trypsin is found in the tsetse midgut (Gooding and Rolseth, 1976). Another molecular trigger native to the tsetse midgut, the Glossina proteolytic lectin serine protease (Gpl) isolated from Glossina fuscipes fuscipes, has been identified as having a role in trypanosome differentiation in vitro (Abubakar et al., 2006). To date, while several triggers of differentiation from BSF to PCFs have been identified, the signals that cause differentiation to epimastigote and metacyclic forms in vivo are unknown. 2.4
TRYPANOSOMES AND TSETSE DIGESTIVE ENZYMES
Tsetse flies are obligate haematophages and thus are completely specialized for digesting blood. The majority of their nutritional resources are obtained from the protein content of the bloodmeal (Moloo, 1976; Kabayo and Langley, 1985). Consequently, the fly has a range of proteolytic digestive enzymes in the posterior, digestive portion of the midgut (Gooding and Rolseth, 1976; Cheeseman and Gooding, 1985). The anterior portion is virtually free of proteolytic activity and indeed the epithelium of the anterior midgut actively secretes proteinase inhibitors into the gut lumen (Gooding, 1974b; Houseman, 1980; Stiles et al., 1991). It has previously been suggested that midgut proteases are a major barrier to trypanosome establishment within the tsetse midgut (Imbuga et al., 1992). Given the above, while this may be the case, it probably only refers to trypanosomes entering the posterior midgut. In addition, fly midgut trypsin levels do not differ in infected and refractory flies, suggesting that digestive enzymes are not part of a protective response to trypanosome infection. Neither feeding excess trypsin nor inhibition of trypsin results in any difference in infection phenotype within the fly (Welburn and Maudlin, 1999). Studies using tsetse midgut extracts have found no correlation between tsetse gut protease activity and trypanosome infection rates (Mihok et al., 1994). Collectively these data indicate that it is questionable whether tsetse proteases have any major bearing on trypanosome infections within the fly gut. Curiously, an increase in mRNA transcript levels of certain digestion related enzymes, namely the tsetse cathepsin B, zinc carboxypeptidase and zinc metallo-protease, have been reported when a trypanosome infection is present (Yan et al., 2002). It should be noted however that protein levels, particularly in the intestine of insects taking occasional meals, may not be proportional to transcript levels.
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135
TSETSE IMMUNE SYSTEM
Despite their obvious efficiency in maintaining large burdens of trypanosomebased disease in Africa, tsetse flies exhibit a considerable level of refractoriness to trypanosome infection. Even under optimal laboratory conditions, where flies are fed at regular intervals, only a proportion of flies will establish midgut infections and the number decreases dramatically after the adult fly has taken three to four bloodmeals (Fig. 4) (Distelmans et al., 1982; Welburn and Maudlin, 1992; Kubi et al., 2006). Furthermore, less than half of the infections that become established in the midgut will mature (Van den Abbeele et al., 1999; Gibson and Bailey, 2003; Peacock et al., 2006). A key factor in this refractoriness is the fly immune system (Hao et al., 2001). Immune stimulation, by injection of live E. coli or lipopolysaccharide (LPS) into the haemocoel of the fly prior to feeding an infective bloodmeal, leads to a statistically significant decrease in trypanosome midgut infection rates (Hao et al., 2001). Identification of the tsetse immune molecule(s) responsible for conferring resistance to trypanosome infection has been hampered by the lack of an annotated Glossina genome (scheduled for completion in 2011). However, the sequencing and annotation of EST libraries from several tissue sources,
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Trypanosome prevalence (%)
90 80 70 60 50 40 30 20 10 0
1st BM n = 307 r = 11
2nd BM n = 165 r=5
3rd BM n = 333 r = 12
4th BM n = 117 r=4
5th BM n = 100 r=2
8th BM n = 52 r=2
10th BM n = 35 r=1
FIG. 4 G. m. morsitans teneral phenomenon. The timing of the infective bloodmeal (BM) affects the prevalence of establishment of trypanosomes in the tsetse midgut. The infective BM containing T. b. brucei (TSW196) BSF given at one of the stated bloodmeals (x-axis) results in the indicated prevalence of infection (mean SE). N ¼ cumulative sample size number (male), R ¼ number of replicates (Lehane laboratory, unpublished results).
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including the major immunoresponsive tissues of midgut (Lehane et al., 2003) and fat body (Attardo et al., 2006), has provided the foundation for more extensive studies of the Glossina innate immune system at a molecular level (http://www.genedb.org/genedb/glossina/index.jsp). Based largely on work on Drosophila melanogaster, it is known that insects possess a complex, interacting, innate immune system. This system is comprised of physical barriers (such as the cuticle and the PM), cellular responses (such as encapsulation and phagocytosis), and humoral responses, such as the generation of host defence peptides (HDP, previously called antimicrobial peptides), reactive oxygen species (ROS) and melanization by the phenoloxidase pathway (Lemaitre and Hoffmann, 2007). The immune response depends not only on the nature of the immune stimulus, but also its route of delivery, with quite distinct epithelial and systemic immune response profiles generated to the same pathogen (Hao et al., 2001). Clearly, in tsetse–trypanosome interactions, it is the epithelial immune responses of the alimentary canal and salivary gland tissues that are likely to be of major importance, as trypanosomes involved in the natural life cycle are exposed only to epithelial surfaces throughout the parasite life cycle. Trypanosomes have been reported in the tsetse haemocoel (Mshelbwala, 1972; Otieno et al., 1976), but these are almost certainly not important to the completion of the normal life cycle. Those trypanosomes that do traverse the midgut epithelium are rapidly killed by an unidentified systemic immune response (Croft et al., 1982), which effectively confines the trypanosomes to the lumen of the alimentary canal.
2.5.1
Peritrophic matrix
The insect midgut epithelium is physically protected from abrasion by food (and the pathogens it may contain) by the peritrophic matrix (PM, previously known as the peritrophic membrane) (Lehane, 1997). The PM is composed of a highly organized, glycosaminoglycan-rich layer reinforced with chitin (Tellam et al., 1999). In tsetse, the PM is constitutively expressed, forming a protective sleeve along the entire length of the midgut. Type II PM, such as that occurring in tsetse flies, physically separates the midgut lumen into two compartments. The endoperitrophic space is where the food lies and the ectoperitrophic space is the region between the PM and the midgut epithelium (see inset, Fig. 3). Clearly, by separating the food from the midgut epithelium, the PM must act as a molecular sieve through which digestive enzymes and nutrients destined to be absorbed must pass. The molecular sieving properties of the PM vary with changes in the surrounding ionic environment (Miller and Lehane, 1993). Typically, the tsetse PM has a pore size of 9 nm, making the PM permeable only to globular molecules less than 150 kDa (Miller and Lehane, 1990). Consequently, the tsetse PM presents a barrier to procyclic trypanosomes that need to get into the ectoperitrophic space to continue their development.
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Whether trypanosomes penetrate the PM, or circumnavigate around its broken ends in the hindgut to get to the ectoperitrophic space, is still the subject of speculation. There is little dispute that trypanosomes returning to the endoperitrophic space, from 6 to 8 days post-infection, actively penetrate the PM. Given the data obtained mainly by transmission electron microscopy (Ellis and Evans, 1977; Gibson and Bailey, 2003), it seems probable that trypanosomes also actively penetrate the PM when entering the ectoperitrophic space 3–5 days post-infection. Penetration is likely to be preceded by a specific attachment event, so it is interesting to note that GFP-expressing T. b. brucei PCFs tend to line up parallel with the PM and become associated with tsetse gut fragments upon dissection (Gibson and Bailey, 2003). How trypanosomes achieve PM penetration is unknown, and no chitinase gene has been identified from the trypanosome genome. However, chitin can be a relatively minor component of dipteran PM (Tellam et al., 1999) and other enzymes capable of dealing with glycosaminoglycans may be more important in the penetration event. In support of penetration rather than circumnavigation via the hindgut, only small numbers of procyclics were observed in the posterior region of the midgut when GFP-expressing parasites were used. These parasites were considered to be an indication of a failed infection cycle in its terminal stages (Gibson and Bailey, 2003).
2.5.2
Immune signalling
Genetic studies have demonstrated that Drosophila uses two main immune signalling pathways to control immune responses to pathogen challenge. The Toll and Imd (immunodeficiency) pathways respond to different classes of microbes and are used differently in various fly tissues (De Gregorio et al., 2002). The Imd pathway is predominantly involved in regulating epithelial immune responses and is most strongly utilized following Gram-negative bacterial challenge. The Toll pathway is most heavily involved in systemic immune responses and is activated most by fungal and Gram-positive bacterial infections (Fig. 5). These pathways are highly conserved among insect species and bear similarity to the vertebrate Toll-like receptor (TLR) and tumour necrosis factor (TNF) pathways. The Drosophila immune system is also highly regulated as over- or underexpression of immune responses is detrimental to host health (Lemaitre and Hoffmann, 2007; Ryu et al., 2008; Aggarwal and Silverman, 2008). The immune response may be divided into three key stages: recognition of pathogens, activation of the signal transduction pathway(s) and production of immune effector and regulator molecules. Orthologous intracellular and extracellular members of both pathways have been identified from Glossina EST databases (Table 1), although very little experimentation to gather functional evidence for their role in tsetse flies has been undertaken.
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Toll
Imd
Yeast
Fungi
Gram-positive bacteria
Gram-negative bacteria
GNBP3 Lysine-type peptidoglycan
DAP-type peptidoglycan PGRP-LE
Haemolymph
Necrotic GNBP1
PGRP-SD
Monomeric peptidoglycan
SPE
Persephone
Polymeric peptidoglycan
PGRP-SA Pro-Spätzle PGRP-LCx PGRP-LCa PGRP-LCx PGRP-LCx
Spätzle
PGRP-LF Imd
Toll
MYD88
dIAP2 Tube
Pelle
Caspar
dFADD
Cytoplasm
dTAB2 dTAK1
Dredd Dnr1
Cactus WntD
Dorsal
DIF
JNK pathway
Nucleus
DaPKC Drosomycin + other HDPs
Relish Kenny Ird5 (IKKγ) (IKKβ) Caudal Diptericin + other HDPs
FIG. 5 Schematic representation of the Toll and Imd signalling pathways. The function of the Toll and Imd signalling pathways has been intensively investigated in Drosophila melanogaster and has been reviewed in detail by Lemaitre and Hoffmann (2007) and Aggarwal and Silverman (2008). This figure is modified from these references. The omniBLAST search tool was used to identify putative G. m. morsitans orthologs (at 1e 20 or less) of all identified Drosophila Toll and Imd pathway genes in all library clustered EST translated sequences of G. m. morsitans (http://www.genedb.org). Orthologs of all Toll and Imd pathway members depicted were identified (except WntD) and are summarized in Table 1.
2.5.3
Recognition and activation
Differential regulation of immune effector HDP indicates that the tsetse immune system can distinguish between virulent and avirulent strains of bacteria (Weiss et al., 2008), between trypanosome and bacterial infections and also between different trypanosome life stages, that is procyclic and bloodstream forms (Hao et al., 2001). In Drosophila, pathogen recognition is achieved by pattern recognition receptors (PRRs), which each recognize a certain class of pathogen-associated molecular pattern (PAMP). Two families of PRRs, the peptidoglycan recognition proteins (PGRPs) and Gram-negative binding proteins (GNBPs), are known. Interestingly, PRRs are proteins originally derived from enzymes known to degrade microbial cell wall components, thus evolving
TABLE 1 Identification of Toll and Imd immune pathway orthologs in G. m. morsitans Toll Drosophila Molecule
Imd a
b
Ortholog
Drosophila Molecule
GNBP1 (Gram-negative FBgn0040323 binding protein 1) GNBP3 (Gram-negative FBgn0040321 binding protein 3) Persephone FBgn0030926
cn16500
Necrotic
FBgn0002930
cn8210
SPE (Spa¨tzle processing FBgn0039102 enzyme) Spa¨tzle FBgn0003495 Toll FBgn0003717
cn8446
Pelle
FBgn0010441
cn1855
Tube
FBgn0003882
Gmsg-10360
MyD88
FBgn0033402
cn12871
PGRP-LC (peptidoglycan recognition protein LC) PGRP-LE (peptidoglycan recognition protein LE) PGRP-LF (peptidoglycan recognition protein LF) PGRP-SA (peptidoglycan recognition protein SA) PGRP-SD (peptidoglycan recognition protein SD) Imd (immune deficiency) dTAK1 (TGF-beta activated kinase 1) Kenny (IKKg) (IkappaB kinase) Dredd (Death released ced-3/NeddZ-like protein) Dnr1 (defence repressor 1)
FlyBase ID
cn16500 cn353
Gmsg-10321 cn14933
FlyBase IDa
Orthologb
FBgn0035976
cn1360
FBgn0030695
Gmm-3156
FBgn0035977
cn1360
FBgn0030310
Tse122g03
FBgn0035806
cn1360
FBgn0013983 FBgn0026323
cn3932 Gmsg-7119
FBgn0041205
Gmsg-7350
FBgn0020381
cn13356
FBgn0260866
Tse104g08.qlc (continues)
TABLE 1
(Continued)
Toll Drosophila Molecule
FlyBase IDa
Imd Orthologb
Cactus
FBgn0000250
Gmsg-2191
Dorsal
FBgn0260632
cn12921
Dif (dorsal-related immunity factor) DaPKC (Drosophila atypical protein kinase C) WntD (Wnt inhibitor of Dorsal)
FBgn0011274
Gmsg-2191
FBgn0022131
cn10314
FBgn0038134
Not identified
a b
Drosophila Molecule DIAP2 (inhibitor of apoptosis-2) Ird5 (IKKb) (Immune response deficient 5) dFADD
FlyBase IDa
Orthologb
FBgn0015247
cn3040
FBgn0024222
cn5893
FBgn0038928
cn6552
dTAB2 (TAK1-associated binding protein 2)
FBgn0086358
Gmsg-6058
Caspar
FBgn0034068
GLAEJ30TV
Relish Caudal
FBgn0014018 FBgn0000251
cn7095 cn9066
Drosophila melanogaster accession numbers from genome data assembled by Flybase http://flybase.org. Glossina orthologs obtained from G. m. morsitans EST libraries clustered by GeneDB http://www.genedb.org/gendb/glossina.
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from a microbicidal to a recognition role (Lemaitre and Hoffmann, 2007). A role for PGRP-LB, a known negative regulator of the Imd pathway in Drosophila, has been noted in downregulating tsetse immune responses to virulent but not avirulent bacteria (Weiss et al., 2008). Notably, no PRR has yet been identified for African trypanosomes, although it is clear that there is a tsetse immune response to their presence (Hao et al., 2001). Binding of PRRs to a pathogen leads to activation of Toll/Imd downstream signalling cascades. Signalling culminates in the activation and nuclear translocation of NFkB transcription factors Dorsal/Dif (Toll pathway) or Relish (Imd pathway). This in turn causes transcription of immune effector genes such as HDPs. Importantly, molecular crosstalk may occur between the Toll and Imd pathways, thus giving rise to a wide spectrum of possible immune responses to pathogen invasion (Tanji and Ip, 2005; Tanji et al., 2007). In Drosophila, control of induced epithelial immune responses is associated with the Imd signalling pathway (Tzou et al., 2000). Gene knockdown of the Imd pathway transcription factor Relish in tsetse flies has been demonstrated to cause an increase in midgut and salivary gland infection rates with trypanosomes (Hu and Aksoy, 2006). This evidence implicates the Imd pathway in regulating fly susceptibility to trypanosome infection. Furthermore, knockdown of Relish leads to downregulation of attacin, a HDP associated with tsetse– trypanosome interactions (Hao et al., 2001; Hu and Aksoy, 2005, 2006; Nayduch and Aksoy, 2007).
2.5.4
Effector molecules
HDPs are evolutionarily conserved effector molecules of the humoral defence system and are found among all classes of life. They exhibit a broad spectrum of activity against bacteria, fungi, viruses and transformed cells (Lemaitre and Hoffmann, 2007). In addition, the anti-parasite activity of HDPs has been illustrated in several vector–parasite systems (Durvasula et al., 1997; Shahabuddin et al., 1998; Boulanger et al., 2002b) including a tsetse– trypanosome system (Hu and Aksoy, 2005; Hu and Aksoy, 2006). Many HDPs target pathogens by disturbing the pathogen membrane potential or by disrupting internal cell functioning leading to cell death by apoptosis or necrosis. Early research into these immune mediators in G. m. morsitans identified four HDPs: an attacin (AttA1), a cecropin, a defensin and a diptericin (Hao et al., 2001; Boulanger et al., 2002a). More recently, characterization of the G. m. morsitans attacin loci has recognized that attacin genes are organized in three clusters encoding three different attacins: attA, attB and attD. The amino acid sequences of AttA and AttB are almost identical while AttD is only 69% identical to the AttA/B form. These genes are differentially regulated (Wang et al., 2008). Interestingly, two additional HDPs, one with anti-Gram-negative activity and the other with anti-Gram-positive activity, have also identified
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following trypanosome challenge (Boulanger et al., 2002a). These HDPs remain uncharacterized. In vivo analysis of HDP transcript expression during trypanosome infection has indicated that these peptides are differentially regulated in the haemolymph and in the major immunoresponsive tissues of midgut, fat body and proventiculus (Hao et al., 2001, 2003; Hu and Aksoy, 2006). Early in the infection process, the presence of trypanosomes in the midgut or haemolymph does not lead to activation and increased transcription of midgut or fat body HDP genes (Hao et al., 2001). However, by day 6, as parasite numbers increase, attacin (AttA/B and AttD) and defensin transcript expression is high in the fat body (Hao et al., 2001, 2003; Wang et al., 2008). In selfcured flies HDP transcript expression levels fall, but in flies with established midgut infections expression levels remain high in the fat body and proventriculus (Hao et al., 2001, 2003). This does not appear to affect the viability of the parasite population within the midgut, although it remains to be seen if individual parasites are affected, thus resulting in a change in the nature of the parasite population. It is possible that trypanosomes exhibit a stage-specific sensitivity to particular immune molecules, with procyclics exhibiting higher resistance to the trypanocidal activity of HDPs than BSF trypanosomes as was observed in vitro by Haines et al. (2003). Alternatively fat body synthesized peptides circulating in the fly haemolymph may fail to reach parasites located in the midgut environment. The trypanocidal activity of HDPs has been recognized. Stomoxyn, isolated from the facultative hematophagous fly Stomoxys calcitrans, exhibits trypanocidal activity (Boulanger et al., 2002b). In addition, there is direct evidence of trypanosome killing by Glossina HDPs themselves (Hu and Aksoy, 2005; Hu and Aksoy, 2006; Nayduch and Aksoy, 2007). Recombinant attacin (AttA1) inhibited both BSF and PCF growth in vitro (Hu and Aksoy, 2005). In vivo, gene knockdown of the AttA1 peptide, or its transcriptional regulator Relish, led to a statistically significant increase in midgut and mature salivary gland trypanosome infection rates (Hu and Aksoy, 2006). Relish also regulates cecropin expression in Glossina, but whether cecropin possesses trypanocidal properties is yet to be directly investigated. More recently, differential expression of AttA1 transcripts in a trypanosome susceptible species (G. m. morsitans) and two comparatively trypanosome-refractory species (G. pallidipes and G. p. palpalis) has been reported (Nayduch and Aksoy, 2007). Refractory species showed higher attacin transcript expression in fat body (systemic) and proventiculus/ midgut (local) tissues in comparison to susceptible flies in both teneral and blood-fed states. Knockdown of attacin expression in G. pallidipes led to increased trypanosome susceptibility, although the possible confounding effects of high mortality rate, starvation, wounding and low sample number in this study should be noted (Nayduch and Aksoy, 2007). Nevertheless, the evidence suggests that attacin may be an important regulator of tsetse–trypanosome interactions.
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Notably, several investigations of HDP function in tsetse–trypanosome interactions bypass a natural trypanosome developmental stage by using PCF rather than BSF trypanosomes to infect the flies (Hao et al., 2001, 2003). It is too early to say how this experimental approach to infection affects the tsetse– trypanosome interaction but it clearly runs the risk of bypassing part of the natural fly immune response. In some cases, the authors do not state the trypanosome life cycle stage used (Nayduch and Aksoy, 2007). Furthermore, supplementation of bloodmeals with glucosamine (Hao et al., 2001, 2003) may disrupt the natural infection process and confound the interpretation of results (Peacock et al., 2006). The HDP diptericin is constitutively expressed in the proventriculus and fat body of tsetse (Hao et al., 2001). This HDP expression profile has been attributed to the presence of symbionts, in particular to the presence of Sodalis in the gut and haemolymph. Interestingly, Sodalis is resistant to the Gramnegative bactericidal activity of mature synthetic diptericin in vitro (Hao et al., 2001), whereas the non-native bacterium E. coli is not. Sodalis is also more resistant to recombinant attacin (recGmAttA1) than E. coli (Hu and Aksoy, 2005) and to a battery of other Gram-negative and Gram-positive bacteriolytic HDPs (Haines et al., 2003). Thus, it is possible Sodalis may have evolved HDP resistance traits permitting survival in the hostile tsetse midgut environment while invading pathogens are eliminated. 2.5.5
Antioxidants
In addition to the NFkB pathway-mediated defense systems, the NFkB-independent production of microbicidal ROS is a key component of insect epithelial immune responses (Ha et al., 2005a; Lemaitre and Hoffmann, 2007). In Drosophila, natural gut infections with bacteria are associated with rapid synthesis of ROS. Studies have demonstrated that it is Drosophila dual oxidase (dDuox), and not NADPH oxidase (dNox), that provides the main source of ROS that limits bacterial proliferation in the midgut (Ha et al., 2005a,b). The infection-inducible nature of intestinal dDuox suggests the transcriptional role of dDuox may play a pivotal role in fly midgut protection against invading pathogens. dDuox is capable of generating H2O2, and the highly microbicidal HOCl is derived from H2O2 by neutrophil-derived myeloperoxidase (MPO) type activity. Physiological regulation of ROS levels in the midgut is achieved via dDuox-dependant ROS generation and immune-regulated catalase (IRC)dependant ROS removal (Ha et al., 2005a,b). A fine redox balance is critical, as knockdown of either dDuox or IRC leads to higher mortality rates due to either insufficient or prolonged oxidative stress responses respectively. It is known that ROS activates a cell death pathway in procyclic T. b. brucei trypanosomes (Ridgley et al., 1999). In tsetse, increased ROS and nitric oxide (NO) transcript expression have been reported in the proventriculus in response to trypanosome challenge (Hao et al., 2003). Additionally, upregulation of
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oxidative stress genes has been demonstrated in the midgut transcriptome of infected flies (Lehane et al., 2003; Munks et al., 2005) and self-cured flies (Lehane et al., 2008). Hao et al. have hypothesized that reactive intermediates such as NO or H2O2 may function as chemical signals to mediate communication between different immunoresponsive tissues (Hao et al., 2001, 2003). Indirect evidence suggests a role for antioxidants in signalling during parasite development in the fly and protecting tsetse flies against trypanosomes (Hao et al., 2001, 2003; Macleod et al., 2007a,b). Recently, Macleod et al. (2007b) demonstrated the feeding of different antioxidants (glutathione, cysteine, N-acetyl-cysteine, ascorbic acid or uric acid) in the infective bloodmeal led to significant increases in midgut infection rates. Also, glucosamine, which has routinely been used to boost trypanosome midgut infection rates, has been shown to be an antioxidant molecule (Xin et al., 2006). The original interpretation of increased infection rates in flies fed glucosamine was that this sugar neutralized specific trypanocidal lectins in the midgut, permitting higher infection rates to occur (Maudlin and Welburn, 1987; Welburn et al., 1989; Murphy and Welburn, 1997) (see Section 2.5.6). However, with this more recent demonstration that N-acetyl glucosamine can scavenge ROS, superoxide and hydroxyl ions (Xin et al., 2006), an effect on trypanosome infection rates through reduction of ROS challenge is a strong alternative hypothesis. Oxidative stress is believed to be involved in control of parasites in other vector–parasite systems, including the Plasmodium–mosquito and Trypanosoma–Rhodnius systems. Augmented production of NO has been noted in Anopheles stephensi, An. gambiae and An. pseudopunctinpennis following P. berghei infection (Luckhart et al., 1998; Herrera-Ortiz et al., 2004). The R. prolixus NO system responds to T. rangeli and T. cruzi, implicating involvement in trypanosome infection regulation (Whitten et al., 2001, 2007). Furthermore, the stable suppression of trypanosome parasitaemia in Cape buffalo is associated with increased levels of serum ROS (Wang et al., 2002). A role for transferrin in immune signalling and the upregulation of NO in vertebrates has been suggested (Stafford and Belosevic, 2003). Recently, knockdown of transferrin in G. m. morsitans was demonstrated to have a statistically significant impact on trypanosome prevalence, resulting in almost a doubling of trypanosome midgut infection rate (Lehane et al., 2008). The mechanism of involvement of tsetse transferrin in mediating tsetse– trypanosome interactions is still unknown, but there is the intriguing possibility that it may be analogous to the function in vertebrates. 2.5.6
Lectins and programmed cell death
Susceptibility, defined as midgut establishment of T. congolense or T. b. brucei in G. m. morsitans infected at the first bloodmeal, can be selected for with results apparent in the F1 generation (Maudlin, 1982). This susceptibility is an extra-chromosomal trait inherited through the female line (Maudlin and
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Dukes, 1985). G. m. centralis infected with T. congolense show similar susceptibility phenotypes. It appears the effect operates in both the midgut and foregut, because selected lines showed similar phenotypes to T. vivax infection (Moloo and Kutuza, 1988a). The lectin hypothesis was proposed as the mechanism underpinning this phenotype by Maudlin and Welburn (1987). They argued that midgut-expressed lectins were the predominant factor killing trypanosomes in vivo in the tsetse fly, with lectin levels modulated by the changing number of the symbiont Sodalis glossinidius in the fly midgut (Welburn and Maudlin, 1999). This argument was based primarily on evidence that feeding of sugars capable of inhibiting lectins increased trypanosome midgut infection rates (Maudlin and Welburn, 1987; Ingram and Molyneux, 1988; Welburn et al., 1994). Additionally, based on the effects of the plant lectin concanavalin A (Con A) on trypanosomes in vitro, lectin-mediated killing of trypanosomes was believed to occur by a process termed proto-apoptosis (Welburn and Maudlin, 1999; Pearson et al., 2000) (see below). A lectin specific for D-glucosamine and with lesser affinity for N-acetyl-D-glucosamine has been tentatively identified in tsetse midgut (Ibrahim et al., 1984; Ingram and Molyneux, 1988). Most of this lectin is found attached to the PM (Lehane and Msangi, 1991). Inhibition of midgut lectins was hypothesized to occur naturally to the greatest extent in newly emerged flies, where lectins were neutralized by the build-up of inhibitory sugars released from chitin by the endochitinase of the symbiont Sodalis during the pupal period (Welburn and Maudlin, 1999). The increased potential of older starved flies to be infected when compared with age-matched non-starved flies (Kubi et al., 2006) is in disagreement with the lectin hypothesis, as both are reported to have similar midgut lectin levels (Lehane and Msangi, 1991). More recently, the multiple effects of N-acetyl-Dglucosamine or D-glucosamine inclusion in the bloodmeal upon trypanosome growth and tsetse physiology have been reported (Peacock et al., 2006; Ebikeme et al., 2008). Both sugars slowed bloodmeal movement along the midgut. Glucosamine significantly increased the size of bloodmeal taken, as well as increasing the fly mortality rate. Interestingly, N-acetyl-D-glucosamine stimulated trypanosome growth in the midgut and in vitro in the absence of any fly-derived factors. The ability of N-acetyl-D-glucosamine to enhance trypanosome PCF growth in vitro does not seem to involve any direct effect of N-acetyl-D-glucosamine upon metabolic processes within the trypanosome (Ebikeme et al., 2008). In vitro experiments have instead shown that N-acetylD-glucosamine indirectly enhances the rate of L-proline metabolism by inhibiting the trypanosome hexose transporter, thus depriving the parasite of D-glucose (Ebikeme et al., 2008). However, PCFs cultured in D-glucose free medium, and thus possessing a metabolism primed for L-proline use, do not appear to have a higher infection rate when used to infect tsetse flies. This suggests that the metabolic impact of N-acetyl-D-glucosamine on trypanosomes has no bearing on the success of trypanosome establishment within the tsetse midgut (Ebikeme et al., 2008). These off-target effects of feeding glucosamine complicate the
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interpretation of experiments on the trypanosome prevalence phenotype observed (Peacock et al., 2006). Above all, the demonstration that glucosamine (which has routinely been included in infected bloodmeals to artificially boost trypanosome midgut infection rates) is an antioxidant molecule (Xin et al., 2006) suggests a simpler explanation (see Section 2.5.5) for the effect of this sugar on tsetse–trypanosome interactions. In addition, the lectin hypothesis contends that higher symbiont densities lead to increased susceptibility. However, a correlation between symbiont density and susceptibility does not occur in all instances (Shaw and Moloo, 1991; Weiss et al., 2006). Successfully established midgut infections show an average parasite density of approximately 5 105 cells per midgut (Van den Abbeele et al., 1999; Gibson and Bailey, 2003), but this number can vary with clone and species of trypanosome. Procyclic trypanosomes share a common energy source (proline) with their tsetse hosts (Welburn and Maudlin, 1999), which may become a limiting factor if trypanosome numbers expand too far. It is possible that trypanosomes self-regulate their numbers and that quorum sensing may be involved in the process (Acosta-Serrano et al., 2001; Roditi and Lehane, 2008). More specifically, it has been suggested that the cleaved N-terminal fragments of trypanosome procyclins might be involved in a density-sensing mechanism, which the trypanosomes use to control their population densities (Acosta-Serrano et al., 2001). However, this mechanism may not be essential as procyclin knockout mutants are still fly-transmissible (Vassella et al., 2009). A novel form of cell death is believed to occur in trypanosomes, known as proto-apoptosis, and it is suggested that this is part of the mechanism by which trypanosomes regulate numbers within the midgut (Welburn et al., 1996; Murphy and Welburn, 1997; Welburn and Maudlin, 1997). In support of this idea, incubation of PCFs of T. b. brucei, T. b. rhodesiense or T. congolense trypanosomes (but not their corresponding BSFs) with the plant lectin Con A in vitro induces growth arrest and death, with many of the characteristics of programmed cell death (PCD) (Welburn and Maudlin, 1997; Murphy and Welburn, 1997). In tsetse, with some trypanosome infections, cyst-like forms have been observed embedded in the PM (Robertson, 1913; Evans and Ellis, 1983; Gibson and Bailey, 2003). Some cysts contain highly motile forms while others appear to consist of aggregates of rounded up trypanosomes. Whether these cysts represent an unknown life cycle stage or degenerating trypanosome forms is unknown. Gibson and Bailey (2003) noted that these forms resembled the morphologically altered PCF trypanosomes treated with Con A in vitro to induce cell death. Proteins upregulated during the process of PCD include the trypanosome protein kinase C receptor, which is similarly upregulated in the short stumpy BSF. This suggests that stumpy form BSFs are removed from the trypanosome population within the mammalian hosts (should ingestion by a tsetse host not occur). This would avoid triggering a wider immune response within the
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mammalian host, which could jeopardize the survival of the long slender BSF population (Murphy and Welburn, 1997). 2.5.7
Other potential effector molecules
Melanization plays an important role in insect defence reactions such as wound healing, sequestration of microorganisms, encapsulation and the production of intermediates toxic to invading pathogens (Lemaitre and Hoffmann, 2007). Prophenoloxidases (ProPOs) are oxidoreductases related to hemocyanins that mediate melanization. These molecules have been implicated in tsetse– trypanosome interactions as inhibition of phenoloxidase (PO) activity with phenylthiourea (PTU) increases midgut trypanosome infection rates (Nigam et al., 1997). Furthermore, T. b. rhodesiense has been shown to significantly activate haemolymph proPO of female G. m. morsitans (Nigam et al., 1997). Such a complex biochemical cascade as occurs in the ProPO system is almost certainly highly reliant on the controlled environment of the haemolymph for its successful operation, and it is hard to see how it could operate effectively in the midgut lumen, with the possible exception of the immediate surface of the epithelium. However, it is possible that proPO may be a means of killing those parasites which do enter the haemocoel of the fly. TsetseEP protein may also play a role in tsetse–trypanosome interactions. This immunoresponsive protein is expressed strongly in the midgut of Glossina (Chandra et al., 2004) and is upregulated in response to immune stimulation with Gram-negative bacteria (Haines et al., 2005). This protein exhibits a high sequence identity to the carboxy terminal region of the EP form of procyclins, which is found on the surface of procyclic T. b. brucei. The glutamic acid– proline (EP) repeats found on both molecules are extremely unusual and it seems unlikely that they coincide in the tsetse midgut solely by chance. The potential role of tsetseEP in mediating fly responses to trypanosome infection merits further investigation. 2.5.8
Adaptive immunity
The existence of immunological memory has long been known in vertebrates, with humans utilizing vaccination before key mechanisms of adaptive immunity were known or understood. Vertebrate adaptive immunity is characterized by long-term protection against specific antigens achieved in part by antibodies that consist of a large diversity of somatically rearranged immunoglobulin receptors. The possibility that invertebrates are capable of adaptive immunity to pathogen challenge has recently re-emerged as a possibility (Watson et al., 2005; Dong et al., 2006; Dong and Dimopoulos, 2009). For example, Dong et al. (2006) identified Down syndrome cell adhesion molecule (Dscam) as an alternatively spliced, hypervariable immunoglobulin domain containing gene responsible for generating a broad range of pathogen recognition receptors
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(PRRs) in Anopheles gambiae mosquitoes. The gene contains 101 exons including three variable Ig exon cassettes. Alternative splicing of this gene produces a highly diverse set of over 31,000 potential splice forms. Challenge with different pathogens leads to production of specific AgDscam splice-form repertoires. Interestingly, an ortholog of Dscam has been identified from a Glossina EST library (cn9121, e-value 6.0 10 21). Whether Glossina uses this gene in immune responses to trypanosomes or bacteria remains to be elucidated. 2.5.9
Fly immune system and microbial balance
Glossina carries a range of microorganisms (see Section 3) and hence, must manage to coexist with these symbionts while still protecting themselves from the onslaught of pathogens. The host factors that maintain the homeostatic relationship between the tsetse host and its symbionts are largely unknown, and our current insight is based primarily on studies in Drosophila. Recently, the intestinal homeobox gene Caudal (Cad) has been identified as a key factor in repressing NFkB-dependant HDP genes in Drosophila (Ryu et al., 2008). Thus, although Drosophila commensal organisms can induce a high level of local Imd-regulated NFkB activation, only a subset of target genes is activated. Regulation of the immune system is tightly controlled, as overexpression of HDP genes in Caudal knockdown flies caused a shift in the commensal population in the midgut. In particular, the dominance of Gluconobacter sp. strain EW707 eventually led to gut apoptosis and host mortality in Drosophila. The model proposed by Ryu et al. (2008) involves interplay between Caudal and the NFkB transcription factor Relish to regulate the expression of HDPs, which in turn defines the microbial community and insect health. Whether this is also the case in Glossina has not yet been investigated, although an ortholog of Caudal has been identified in the EST libraries (cn9066, e-value 1.2 10 47) making this a possibility. 2.6
EFFECTS OF TRYPANOSOME INFECTION ON TSETSE PHYSIOLOGY
Trypanosome infection may have multiple effects on the tsetse host. A suppression subtractive hybridization study (Lehane et al., 2008) identified molecules differentially expressed in established versus self-cured T. b. brucei infections in G. m. morsitans. Analysis of the gene fragments generated suggested that trypanosome infection has a marked effect upon the metabolism of the fly host. Flies self-cured of trypanosome infection displaying the signals of increased energy usage and an oxidative stress response compared to infected flies. Within the tsetse fly, procyclic trypanosomes employ the amino acid proline as a source of energy, while the fly typically uses proline reserves for flight. In infected male flies, trypanosomes infection can reduce the flight potential by 15% (Bursell, 1981). Trypanosome infection in female flies would be far more
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costly in terms of flight potential as females have smaller proline reserves due to the energy required for larval development. Recently, Hu et al. (2008) investigated the cost of trypanosome infection on fly reproductive fitness. Two trypanosome strains were selected that differentially activated the host immune system: a T. b. rhodesiense wild-type strain that induced expression of attacin and defensin in trypanosome infected flies and a mutant strain that did not. Only infection with the wild-type strain led to a significant increase in larval deposition periods and a decrease in milk gland protein expression. This suggests that activation of tsetse immune responses by infection with immunogenic trypanosomes delays larvigenesis via decreased expression of the milk gland protein vital for larval growth. Trypanosome infection may also impact the feeding behaviour of infected tsetse hosts. Infection of G. m. morsitans and G. austeni with T. b. brucei reportedly results in increased probing behaviour and more voracious feeding (Jenni et al., 1980). Accumulation of large numbers of trypanosomes in the salivary glands or proboscis has been observed, and may impede function of labral mechanoreceptors as detectors of blood flow rate in the gut (Molyneux and Jenni, 1981). Thus an infected fly may take several smaller meals or feed for longer, either scenario potentially leading to increased trypanosome transmission to the vertebrate host.
2.7
FLY SEX, AGE AND STARVATION AND TRYPANOSOME TRANSMISSION
Susceptibility to parasite infection is also influenced by the sex of the fly. This is evident in midgut infections with T. congolense and T. brucei, but not in infections with T. vivax. Most laboratory studies (Burtt, 1946b; Harley, 1971; Distelmans et al., 1982; Mwangelwa et al., 1987; Hide et al., 1991; Moloo, 1993; Welburn et al., 1995; Dale et al., 1995), but not all (Welburn and Maudlin, 1992; Moloo et al., 1992; Moloo, 1993), suggest that male flies are more susceptible. Female G. p. palpalis are more resistant to developing mature T. congolense infections (Distelmans et al., 1982) than males. Unexpectedly, this sex-dependent phenomenon was not observed with T. congolense infections in G. m. morsitans (Dale et al., 1995). Of interest, compared to a virgin G. m. morsitans, a mated female is twice as refractory to T. b. brucei infection (Macleod et al., 2007a). Although both sexes are capable of transmitting parasites, both male and female flies are innately resistant to parasite infection and this resistance increases rapidly with age (Fig. 4). This natural refractoriness to trypanosome maturation and/or establishment is seen in both laboratory and field populations (Distelmans et al., 1982; Leak 1999) and is termed the teneral phenomenon (Welburn and Maudlin, 1992). In the tsetse community the term teneral is used to describe a newly emerged fly that has not yet had its first bloodmeal. The fly during this time is marked by
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a soft, often described as soapy-feeling, exoskeleton and lighter body coloration. While tsetse researchers universally use the word ‘‘teneral’’, they seem to have forgotten that the term represents a highly variable physiological state, particularly in terms of fly susceptibility. Depending on tsetse species and the nutritional status of the newly eclosed adult fly, teneral flies can survive for up to 1 week before dying of starvation (Kubi et al., 2006; Lehane lab, unpublished observations). Van Hoof et al. (1937) originally demonstrated the ‘‘teneral phenomenon’’, that is the higher susceptibility of a newly emerged fly to trypanosome infection compared to an older fed fly, using G. f. fuscipes challenged with T. b. gambiense. The highest midgut establishment rates were obtained in flies fed the infective meal 0–1 days post-emergence (p.e.). This work was later confirmed by Wijers (1958), who observed that G. p. palpalis emergents less than 30 h p.e. were more susceptible to a first infective bloodmeal infection than flies 30–78 h p.e. In recent years, the teneral definition has shifted to a nutritional focus and teneral has become synonymous with ‘‘unfed’’. Many published papers ignore the fact that parasite susceptibility during this teneral period can vary widely, and consequently flaws in experimental design may confound data interpretation. Speculation on why newly emerged flies are so susceptible to trypanosomes is profuse. Incomplete formation of the PM, variable antioxidant or lectin concentrations, molecular maturity of the midgut (i.e. proteases), immaturity of the immune response, larval meal turnover (preand post-meconium expulsion), pH gradient differences and variation in symbiont loads may, either singly or as a combination, play a role in creating the teneral phenomenon. Although further research is required to confirm which of these factors (if any) contribute to this age-related susceptibility to infection, it is crucial to standardize trypanosome infection experiments by redefining the definition for teneral as ‘‘hours post-eclosion’’ instead of simply ‘‘unfed’’. Starvation is a constant risk the tsetse fly faces throughout its lifetime. Flies deprived of a bloodmeal utilize energy reserves stored in the fat body and risk death when fat reserves drop to approximately 6% of the total dry body-mass (Rogers et al., 1994). Male flies, which use most of each bloodmeal for lipid production, are more resistant to starvation than females, which use the bloodmeal for both lipid production and growth of the larva (Randolph and Rogers, 1981). Starvation may influence feeding behaviour with the range of host choice expanding as the fly’s hunger intensifies (Bouyer et al., 2007) with obvious consequences for the epidemiology of disease. Environmental conditions, such as temperature and relative humidity, also influence fly starvation with many Glossina species becoming hungrier during the dry season (Jackson, 1933). Starvation is a key factor in tsetse–trypanosome interactions because the nutritional status of the fly at the time of the infective bloodmeal is a key determinant of fly susceptibility to trypanosome infection (Gingrich et al., 1982; Mwangelwa et al., 1987; Gooding, 1988; Kubi et al., 2006).
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Under laboratory conditions with bloodmeals given every 4 days, newly emerged male tsetse flies are highly susceptible to trypanosome infection at the first bloodmeal while older flies exhibit a significantly lower susceptibility to trypanosome infection (Fig. 4) (Distelmans et al., 1982; Welburn and Maudlin, 1992; Kubi et al., 2006). However, field studies of trypanosome prevalence with fly age suggest a significantly higher proportion of the older adult tsetse population than predicted develop a mature trypanosome infection (Woolhouse et al., 1993; Msangi et al., 1998; Lehane et al., 2000). Recently, laboratory-based studies by Kubi et al. (2006) demonstrated a period of starvation (3–4 days for teneral or 7 days for adult flies) increased fly susceptibility to T. b. brucei or T. congolense infection, resulting in a significantly higher number of T. congolense or T. b. brucei mature salivary gland infections. In the case of T. congolense, a significantly higher number of midgut infections were also observed. It is likely that the increased infection rates noted in older field flies compared to laboratory flies is explained by starvation events in the field. An explanation for this observation may be that under high nutritional stress the physiological barriers to trypanosome infection are suppressed because the energy cost of an immune response is high (Schmid-Hempel, 2005; Ye et al., 2009). Trypanosome infection may have a negative impact on fly longevity (Bursell, 1981). More immunogenic lines of trypanosomes adversely affect tsetse reproductive fitness (Geiger et al., 2008), which suggests that trypanosome infection places a selection pressure on tsetse flies.
2.8
ENVIRONMENTAL TEMPERATURE AND TRYPANOSOME TRANSMISSION
As puparial incubation temperature rises (within biological limits), so does the ability of the corresponding adult fly to develop a mature infection (Burtt, 1946a; Ndegwa et al., 1992). This is not attributed to an increase in the number of flies that establish a midgut infection, but rather to an increase in the proportion of these flies that go on to develop mature infections (Dipeolu and Adam, 1974). The mechanism responsible is unknown. However, this observed trend may have consequences in the field for the distribution of trypanosomiasis. There is a positive correlation between latitude relative to 7 S (the median of tsetse distribution) and infection rate (Ford and Leggate, 1961). Although the factors involved in the field are undoubtedly complex (Jordan, 1965), ambient temperature also increases towards the 7 S median, implying that it may be puparial incubation temperature that influences this trend. If that is the case, then temperature plays a central role in determining the geographical distribution and epidemiology of trypanosomiasis.
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Symbiont–tsetse–trypanosome interactions WIGGLESWORTHIA GLOSSINIDIUS
All Glossina sp. harbour W. glossinidius, a primary obligate (beneficial) Gramnegative endosymbiont. Based on concordant evolutionary analyses, it is estimated the Wigglesworthia-Glossina symbiosis evolved in ancestral forms between 50 and 80 million years ago (Chen et al., 1999). This bacterium resides within the cytoplasm of specialized epithelial midgut cells, bacteriocytes, which form a horseshoe shaped organ, the bacteriome (Fig. 2E), located in the anterior midgut (Aksoy et al., 1995; Aksoy, 1995). The Wigglesworthia genome is grossly reduced in size to less than 700 kb and is predicted to encode for 617 proteins (Akman et al., 2002). This streamlined genome has undergone massive gene erosion as genes required for defense mechanisms, metabolic pathways, DNA repair mechanisms and so on, are no longer essential as this endosymbiont exists in a stable host environment. In fact, even a gene involved in chromosome replication, dnaA, which is considered essential for bacterial survival, has been lost in Wigglesworthia (Akman et al., 2002). This member of the Enterobacteriacae family, first observed over a century ago (Stuhlmann, 1907), was thought to produce metabolites to compensate for nutritional deficits in the host’s haematophagous diet and appears to be partially associated with the metabolism of B-complex vitamins essential for tsetse survival (Wigglesworth, 1929; Nogge, 1981; Akman et al., 2002). The elimination of Wigglesworthia reduces fly longevity, rate of bloodmeal digestion and fecundity by making females sterile (Nogge, 1976; Dale and Welburn, 2001; Pais et al., 2008), although supplementation of the host bloodmeal with vitamins partially reverses this infertility phenotype (Nogge, 1981). It has been suggested that the bacterium may also influence host digestive processes through either synthesis of protoheme or glycerophospholipids, fatty acids and steroids (Pais et al., 2008). Wigglesworthia is vertically transmitted from the female fly to her progeny via milk gland secretions (Denlinger and Ma, 1975). More specifically, in situ hybridization has identified Wigglesworthia exclusively associated with the milk gland lumen and the canals leading to the secretory reservoirs (Attardo et al., 2008). Recently, studies by Pais et al. (2008) have demonstrated that selective elimination of Wigglesworthia symbionts by ampicillin antibiotic treatment leads to increased susceptibility to trypanosome midgut infection in non-teneral flies. This implies that symbiont clearance may lead to a decrease in fly basal immunity, thus affecting host immune responses to trypanosome infection. However, these Wiggleworthia-cleared flies also had a compromised ability to digest their bloodmeals and increased trypanosome susceptibility was only observed in older flies. Thus, neither the effect of ‘‘starvation’’, known to cause increased parasite susceptibility (Kubi et al., 2006), nor the effects of antibiotic treatment on the fly and its other symbionts can be discounted.
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WOLBACHIA PIPIENTIS
A secondary tsetse symbiont, Wolbachia pipientis, is also maternally inherited. Unlike Wigglesworthia, it colonizes the female reproductive organs (not the milk gland) and is transovarially transmitted to progeny. Wolbachia are the most frequently described symbiotic association in arthropods; surveys have indicated that more than 70% of all insect species are infected with Wolbachia (Werren and Windsor, 2000). Wolbachia belong to the subdivision of Gramnegative intracellular alpha-proteobacteria and are most famous for their reproductive manipulations of the host including parthenogenesis, feminization, male-killing and cytoplasmic incompatibility (for a review of Wolbachia insect symbioses see Siozios et al., 2008). It has been reported that 100% of laboratory-reared tsetse colonies are infected with Wolbachia, while infections in wild populations vary significantly (Cheng et al., 2000). Within infected tsetse, Wolbachia colonizes several tissues, depending on the species of Glossina. Using a Wolbachia-specific PCR-based assay, Wolbachia was found in the reproductive tissues of all infected tsetse species examined. However, a wider tissue tropism was observed in the somatic tissues (gut, head, muscle, fat body, milk gland and salivary gland) in certain populations of G. austeni (Cheng et al., 2000). The role Wolbachia plays in the tsetse is unknown and whether its presence influences trypanosome infection rates by stimulating the fly immune system remains unknown. There are strong incentives to investigate whether or not Wolbachia induce cytoplasmic incompatibility in tsetse because it is one means by which selective traits can be driven through insect populations. As such, it is a highly desirable tool for promoting the success of paratransgenesis, a potential disease control strategy involving engineered symbionts and alteration of fly susceptibility to parasite infection – see Section 4.2 (Aksoy et al., 2003; Aksoy and Rio, 2005). 3.3
SODALIS GLOSSINIDIUS
The third tsetse symbiont, S. glossinidius, is a Gram-negative, microaerophilic, non-motile bacterium that forms a new bacterial taxon and species in the family Enterobacteriaceae (Dale and Maudlin 1999). Reinhardt et al. (1972) first described Sodalis as small rickettsia-like organisms (RLO) that were separated from the cytoplasm of midgut cells by a clear lytic zone. Pinnock and Hess (1974) confirmed these observations and reported the presence of this pleomorphic microbe in other tissues such as the fat body and ovaries. The development of a Sodalis-specific PCR-assay further established that this symbiont resided in the midgut, haemolymph and milk gland of teneral flies (Cheng and Aksoy, 1999; Attardo et al., 2008). Sodalis can live both intracellularly and as free-living forms in the gut lumen. This bacterium is one of the only insect symbionts that has been adapted to
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in vitro culture (Dale and Maudlin, 1999; Matthew et al., 2005), making it a model organism particularly for research on host cell invasion, evolutionary biology (the transition from free-living to obligate endosymbiosis) and phylogenetic associations. In recent years, two new symbionts belonging to the Sodalis lineage have been identified, a bacterial isolate from the bloodsucking fly, Craterina melbae (Novakova and Hypsa, 2007) and a bacteriocyteassociated symbiont from the chewing louse, Columbicola columbae (Fukatsu et al., 2007). Unlike other bacterial symbionts, Sodalis does not produce catalase (characteristic of several microaerophiles) which, when combined with the results of additional phenotypic tests, indicates that Sodalis has a reduced biochemical profile when compared to other members of the family Enterobacteriaceae (Dale and Maudlin, 1999). Initially, 85% of the Sodalis genome was characterized by hybridization to E. coli gene macroarrays (Akman and Aksoy, 2001) and many potential gene products were identified. The use of E. coli arrays of course did not allow the identification of molecules unique to Sodalis, as exemplified by the failure to detect genes of the type III secretion system (Dale and Welburn, 2001) and genes encoding two chitinases. In 2006, the genome of Sodalis was published (Toh et al., 2006). Unlike Wigglesworthia, Sodalis has a much larger genome (4.1 Mb), but surprisingly it is predicted to encode only 2432 proteins (which is approximately 50% of what would be expected of a free-living organism). The presence of 972 pseudogenes indicates this genome is gradually eroding, although a previously designated pseudogene (carA) has now been shown to be functional and transcriptionally responsive despite its truncation (Pontes et al., 2008). Extrachromosomal DNA revealed another 160 potential ORFs located on four circular elements (Darby et al., 2005). While this would indicate a general transition to an obligate mutualistic association with the tsetse, several virulence related-factors such as haemolysin, phospholipases and invasion proteins have maintained functionality, implying that pathogenic genes are being retained (Dale and Welburn, 2001; Toh et al., 2006; Feldhaar and Gross 2009). Observations by Pinnock and Hess (1974) support this virulence concept as originally Sodalis was described as a pathogen based on electron microscopy of infected tsetse tissues; the host lytic zones surrounding Sodalis might be indicative of an adverse reaction between the tsetse host and the bacteria. In addition, the highly infected fat body in Glossina pallipides showed signs of cellular disruption and degeneration that could not be explained as procedural artefacts. This cellular disruption was also observed when Sodalis, isolated from the haemolymph of tsetse, were cultured on a layer of Aedes albopictus feeder cells (Welburn et al., 1987). Since Sodalis exists both extracellularly in the haemolymph and intracellularly within tsetse midgut epithelial cells and other tissues, the invasive form may represent a pathogenic remnant. Analysis of the Sodalis transcriptome indicates that quorum sensing may be used to alter gene expression in response to intracellular symbiont density (Pontes et al., 2008). It is further suggested that this may be a key adaptation
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strategy, with Sodalis and host tissues both modulating gene expression profiles and metabolic activities so that immune responses (in particular oxidative stress responses) are minimized. When the impact of different stresses on all three symbiont communities in the tsetse was assessed, the authors concluded that there was a distinct flexibility in symbiont density (with the exception of the teneral stage of the insect), which may help reduce friction between symbionts and host tissues (Rio et al., 2006). The biological contribution, if any, of Sodalis to the tsetse is unknown. A mutualistic relationship has been postulated (and generally accepted) as functional genes encoding enzymes required for vitamin synthesis (Akman and Aksoy, 2001; Akman et al., 2002) (as observed in Wigglesworthia) are present. Such a facultative association is supported by research showing that tsetse longevity is reduced in flies lacking Sodalis (Dale and Welburn, 2001). In addition, puparia that are heavily infected with Sodalis display increased survival under adverse conditions (Baker et al., 1990). It has been suggested that Sodalis may influence tsetse vector competence. Increased trypanosome midgut infections were reported in both lab-reared and wild tsetse that hosted high densities of rickettsia-like organisms (RLOs) (Maudlin and Ellis, 1985; Maudlin et al., 1990). By comparing genetically susceptible and refractory lines of G. m. morsitans, it was observed that the midguts of the susceptible line were prone to the heaviest symbiont infections. To further investigate the role of symbionts (the involvement of Sodalis is inferred) in vector competency, comparisons between refractory lines of G. m. morsitans (Maudlin, 1982) and G. m. centralis (Moloo and Kutuza, 1988a) showed that parasite susceptibility is a maternally inherited factor. In addition, puparial development at lower temperatures revealed a drastic reduction of RLO numbers in teneral flies. This symbiont-depressed tsetse population subsequently produced flies with a greater resistance to trypanosome infection (Welburn and Maudlin, 1991). Examination of the midguts of wild caught tsetse also revealed a direct correlation between trypanosome infections and the density of RLOs. Indeed, it was stated that a wild fly carrying RLOs was six times more likely to be infected with trypanosomes than a RLO-free fly (Maudlin et al., 1990). However, other populations of wild fly do not reflect such a direct relationship between bacterial densities and tsetse refractoriness or susceptibility (Moloo and Shaw, 1989; Geiger et al., 2005b). The vectorial competence between different species of Glossina is highly variable (Harley and Wilson, 1968; Moloo and Kutuza, 1988a,b; Reifenberg et al., 1997; Kazadi et al., 2000). Using this fact as a basis, Geiger et al. (2005b) demonstrated no correlation between Sodalis prevalence and maturation of a Trypanosoma congolense infection in two distinct species of tsetse. However, they could not rule out a potential role for Sodalis in the initial parasite establishment phase in the fly midgut. Further work from the same group investigated the genetic diversity between Sodalis isolated from the same two species of flies using amplified fragment length polymorphism markers (AFLP) (Geiger et al., 2005a). The data
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(albeit weak) implied that vector competence was related to the genetic diversity of Sodalis (not merely the absence/presence of the symbiont) as the symbiont populations from each fly were distinct. The genetic diversity of Sodalis was further interrogated by screening symbionts isolated from trypanosome infected and uninfected flies (Geiger et al., 2007). The ability of a specific parasite species to establish in the insect midgut is statistically linked to the Sodalis genotype present. To address host-specificity among Sodalis, Weiss et al. (2006) transinfected two species of tsetse (previously cleared of native Sodalis with ampicillin) with reciprocal Sodalis strains. Equal symbiont densities were obtained in surrogate hosts without seriously deleterious effects on the fly. Regrettably, determining the effect of Sodalis transfection on the flies’ ability to vector trypanosomes was not reported. Although the above research does not unequivocally link the tsetse symbionts to vector competence it does suggest that symbionts are involved and forms a strong foundation for future investigations.
4
Towards new methods of disease control
Current interventions for management and control of trypanosomiasis are focused predominantly on drug chemotherapy and tsetse fly control especially for the control of nagana (Torr et al., 2005). While both these existing methods are successful, their implementation over extended time periods has not always been sustainable (Vale 1982; Aksoy et al., 2003). Additionally, concern over the development of resistance to available drugs and their toxicity is mounting, and the prospects for new drugs to treat HAT and nagana are bleak (Legros et al., 2002; Fevre et al., 2006). New means of controlling HAT are urgently required. We may find new ways of controlling trypanosomiasis by exploiting the new information becoming available from the genome projects on both tsetse and trypanosomes. To exploit these resources to their fullest we need a means of studying gene function in tsetse and means of expressing transgenic constructs in the fly. 4.1
GENE KNOCKDOWN IN GLOSSINA
The very slow reproductive rate in tsetse (one offspring every 9 days; 40 days adult to adult) and the high costs of maintaining colonies have prevented maintenance of multiple mutant fly lines. In addition, the unusual, viviparous reproductive system of tsetse flies prevents the development of germline transgenesis. Consequently, gene knockdown through RNA interference (RNAi) (Fire et al., 1998) has been of particular value in the study of tsetse biology and gene function. RNAi operates as a means of gene-specific post-transcriptional knockdown and is triggered by double-stranded RNA (dsRNA) mediated degradation of homologous mRNA sequences in the target organism. In the
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current model of RNAi two major steps are involved. The initiator phase involves recognition of dsRNA (that is expressed in or introduced into the cell) and cleavage into short interfering RNA (siRNA) fragments 21–23 bp in length by the enzyme Dicer (Zamore, 2000; Hammond, 2005). The subsequent effector phase involves the incorporation of cleaved dsRNA into a multi-protein complex, known as the RNA-induced silencing complex (RISC), capable of silencing homologous mRNA transcripts (Siomi and Siomi, 2009). Although several methods exist to administer dsRNA to the organism of interest, these are often very species specific. Stable transgenic expression of RNA hairpin constructs (Tavernarakis et al., 2000; Kennerdell and Carthew, 2000), and the use of recombinant viruses to deliver the dsRNA (Travanty et al., 2004) have been used in some Diptera. However, the most commonly used method for dsRNA delivery to insects is the direct injection of dsRNA into the haemocoel. This was first demonstrated in A. gambiae by Blandin et al. (2002), who showed successful gene knockdown of the HDP defensin. This technique has been successfully extended to the analysis of Glossina genes (Hu and Aksoy, 2006; Lehane et al., 2008; Attardo et al., 2008). To date three G. m. morsitans genes, a transferrin, an attacin and tsetseEP protein, have been implicated in tsetse–trypanosome interactions through use of this technique. Knockdown of these genes by dsRNA injection resulted in a statistically significant increase in trypanosome midgut prevalence (Hu and Aksoy, 2006; Lehane et al., 2008; Haines et al., 2009, submission pending). While it is universally practiced, gene knockdown by direct injection of dsRNA into the tsetse haemocoel (and the physical damage that it causes) is clearly not ideal when studying fly immunity. For example, control injections with PBS result in sustained upregulation of attacin and defensin transcripts in tsetse fat body until 18–30 h post-injection (Hao et al., 2001). Cuticular damage is known to stimulate immune responses in other insects including Bombyx mori and A. gambiae (Brey et al., 1993; Han et al., 1999). Thus any injection based RNAi studies on immunity, particularly those of tsetse–trypanosome interactions, should be interpreted with caution as it is known that the establishment of trypanosomes in the tsetse midgut is influenced by, among other factors, the fly immune system (Hao et al., 2001; Hu and Aksoy, 2006; Lehane et al., 2008). In addition, high mortality rates can occur in flies following injection (Walshe et al., 2009) and this too can complicate the interpretation of experimental results because gene knockdown may have occurred to differing extents in the surviving and killed flies. More recently, successful local gene knockdown in Glossina has been demonstrated by ingestion of dsRNA in a bloodmeal (Walshe et al., 2009). Feeding dsRNA may serve as an alternative and preferable means of delivering dsRNA as oral administration is a more natural route of delivery and less invasive than dsRNA injection. In this study, a comparative analysis of knockdown of the immunoresponsive protein tsetseEP (Haines et al., 2005), at both the transcript and protein level in the midgut, was followed after either feeding or injecting dsRNA. High knockdown efficiency was observed using either method of dsRNA delivery, but a
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significantly lower mortality rate was observed following feeding dsRNA compared to injection. Although the midgut specific tsetseEP protein was successfully knocked down by feeding dsRNA, knockdown of the fat bodyexpressed transferrin gene failed (Walshe et al., 2009). However, this gene can be knocked down by dsRNA injection (Lehane et al., 2008). Failure of the RNAi signal to spread beyond the midgut epithelium may be due to the apparent absence of an ortholog to SID-1, the gene responsible for import of circulating RNAi silencing signals in C. elegans, from available Glossina EST databases (and from the Drosophila genome). So, it is possible that the RNAi signal is not distributed systemically in Diptera (Van Roessel et al., 2002; Winston et al., 2002; Roignant et al., 2003; Dietzl et al., 2007; Jose et al., 2009). Thus, while fed dsRNA can clearly enter midgut cells, it may be unable to cross the midgut epithelial barrier in order to cause gene knockdown in tissues beyond the midgut. If this proves to be the case, it would be a valuable tool in itself because the use of two different methods of dsRNA delivery may permit dissection of tissue-specific gene function by permitting tissue specific gene knockdown. This study was the first demonstration of successful gene knockdown in a dipteran by feeding dsRNA. As gene knockdown by feeding dsRNA has been achieved in two other blood feeding arthropods, Ixodes scapularis and Rhodnius prolixus (Soares et al., 2005; Araujo et al., 2006), it is possible this useful molecular tool may be more widely available in blood feeding insects. 4.2
PARATRANSGENESIS
Transgenesis (i.e. the introduction of foreign genes into a living organism) is currently impossible in tsetse flies due to the viviparous lifecycle. So, there is no prospect of disease control through direct transgenic means. However, one proposed approach to trypanosomiasis control involves the modulation of vector competence through paratransgenesis (Aksoy et al., 2003; Weiss et al., 2008). This involves the genetic transformation of symbiotic bacteria residing in the insect to produce anti-parasitic molecules into the midgut. This approach has proven successful in the laboratory, reducing T. cruzi transmission by R. prolixus via expression of a HDP, cecropin A, by its endosymbiont Rhodococcus rhodnii (Durvasula et al., 1997). The foreign genes expressed by the symbiotic bacterium would have trypanocidal properties that would in principal reduce or eliminate trypanosome midgut establishment, thus breaking the parasite transmission cycle (Durvasula et al., 1997; Aksoy et al., 2003). The endosymbiont Sodalis is the target expression vector in the tsetse fly. This bacterium resides in the tsetse midgut as well as other body sites. Consequently, invading brucei and congolense group trypanosomes would encounter trypanocidal secretion products before the trypanosomes could establish in the midgut. Already, recombinant Sodalis produced in vitro may be reintroduced by microinjection into the mother’s haemolymph and subsequently passed to intrauterine progeny
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in utero (Cheng and Aksoy, 1999). As Sodalis can be cultured in vitro (Matthew et al., 2005) and a genetic transformation system has already been developed (Beard et al., 1993), the potential for successful paratransgenesis is high. To date, several candidate anti-trypanosomal molecules have been identified which could be introduced into Sodalis to control trypanosome midgut establishment. One candidate is tsetse attacin (Hu and Aksoy, 2006). Less obvious, but potentially more powerful candidate molecules are also under investigation. For example, BMAP-18 is a truncated form of the bovine myeloid antimicrobial peptide-27 (BMAP-27). BMAP-27 (Skerlavaj et al., 1996) is expressed by bovine neutrophils and exhibits low toxicity to mammalian cells, insect cells and Sodalis, yet causes rapid death to both BSF and PCF trypanosomes (Haines et al., 2003). The truncated BMAP-18 peptide can kill a variety of kinetoplastid parasites, including trypanosomes and Leishmania, yet exhibits reduced cytotoxicity to Sodalis, mammalian and insect cell lines (Haines et al., 2009). Thus BMAP-18 is also a strong candidate for use in a paratrangenesis approach. Several technical problems will need to be overcome before this technology can be applied to disease control. A suitable biological drive system, such as use of Wolbachia (Aksoy et al., 2003), would be required to ensure replacement of the susceptible wild tsetse population with the refractory population. Additionally, this approach would only be suitable for trypanosome species (brucei and congolense groups) that establish in the fly midgut. Therefore, another tactic would be required to control trypanosome species such as T. vivax, which establish in the mouthparts and would not be affected. How rapidly trypanosome resistance would appear with such a system in place is an unknown and a concern. Therefore, it would probably be advantageous to have a selection of effector molecules available for use in a managed control strategy.
5
Conclusion
This review has come at an important time. The genome resources now available for tsetse flies, their symbionts and the trypanosomes they transmit have dramatically increased possible lines of scientific enquiry. Currently, the GeneDB and Vectorbase data repositories contain comprehensive, annotated Glossina EST libraries and completed annotation of the full tsetse genome is projected for 2011. That will mean the complete repertoire of fly, symbiont and trypanosome genomes will soon be available. There are also a range of molecular tools, including RNAi, proteomics, mutant trypanosome lines and an in vitro culture system for Sodalis, that will further our research endeavours on the enemy within. We believe that the technical resources now available mean that it is time for a more integrated approach in research on tsetse–trypanosome interactions rather than the traditional, more fragmentary, approach based on entomology, bacteriology or trypanosome biology. To this end we have used this review to try to make the pertinent entomological information more available to a wide audience across
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the subject. We have taken an entomological viewpoint to examine the interacting biology of the three major components of the tsetse–trypanosome interaction, the fly, its symbiotic bacteria and the invading trypanosome. We have also tried to highlight the problems we believe are associated with some experimental approaches currently adopted in the entomological field. For example, routinely feeding trypanosomes in a blood meal containing glucosamine; failure to precisely define the starvation period prior to the infective bloodmeal; infecting flies with procyclic rather than blood stream form trypanosomes; the inappropriateness in some studies, particularly susceptibility studies, of using long established fly and trypanosome laboratory cultures with no view to the geographical origins of either. Continued use of these experimental approaches may well create increasing problems in interpretation of data. We believe that more field-based studies, despite their difficulties, would be a valuable addition to the understanding of the tsetse–trypanosome interaction. Also, the majority of studies on this vector–parasite system have centered on T. brucei, with T. congolense and T. vivax interactions comparatively lightly investigated. Given the economic importance of both T. vivax and T. congolense and the available genomic resources more emphasis on relations of these trypanosomes with Glossina is also called for. Finally, we are of the opinion that with major improvements in the technical resources available for experimentation of tsetse–trypanosome interactions the remaining major brake slowing progress in the tsetse–trypanosome research field continues to be the high cost and difficulty of tsetse fly colony maintenance. This difficulty has resulted in only a small number of laboratories studying this phenomenon. It is important that funding bodies are fully aware of this fundamental problem if this field of research is to expand. Acknowledgements The authors thank Alvaro Acosta-Serrano, Leyla Akman, Geoffrey Attardo, Lori Peacock and Terry Pearson for their critical reading of the manuscript and helpful comments. Any remaining errors in the manuscript are the responsibility of the authors. We are also grateful to Ray Wilson for kindly granting permission to use his image of the tsetse fly. References Abubakar, L. U., Bulimo, W. D., Mulaa, F. J. and Osir, E. O. (2006). Molecular characterization of a tsetse fly midgut proteolytic lectin that mediates differentiation of African trypanosomes. Insect Biochem. Mol. Biol. 36, 344–352. Acosta-Serrano, A., Vassella, E., Liniger, M., Renggli, C. K., Brun, R., Roditi, I. and Englund, P. T. (2001). The surface coat of procyclic Trypanosoma brucei: programmed expression and proteolytic cleavage of procyclin in the tsetse fly. Proc. Natl. Acad. Sci. USA 98, 1513–1518.
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Aggarwal, K. and Silverman, N. (2008). Positive and negative regulation of the Drosophila immune response. BMB Rep. 41, 267–277. Akman, L. and Aksoy, S. (2001). A novel application of gene arrays: Escherichia coli array provides insight into the biology of the obligate endosymbiont of tsetse flies. Proc. Natl. Acad. Sci. USA 98, 7546–7551. Akman, L., Yamashita, A., Watanabe, H., Oshima, K., Shiba, T., Hattori, M. and Aksoy, S. (2002). Genome sequence of the endocellular obligate symbiont of tsetse flies, Wigglesworthia glossinidia. Nat. Genet. 32, 402–407. Aksoy, S. (1995). Wigglesworthia gen. nov. and Wigglesworthia glossinidia sp. nov., taxa consisting of the mycetocyte-associated, primary endosymbionts of tsetse flies. Int. J. Syst. Bacteriol. 45, 848–851. Aksoy, S. and Rio, R. V. M. (2005). Interactions among multiple genomes: tsetse, its symbionts and trypanosomes. Insect Biochem. Mol. Biol. 35, 691–698. Aksoy, S., Pourhosseini, A. A. and Chow, A. (1995). Mycetome endosymbionts of tsetse flies constitute a distinct lineage related to Enterobacteriaceae. Insect Mol. Biol. 4, 15–22. Aksoy, S., Gibson, W. C. and Lehane, M. J. (2003). Interactions between tsetse and trypanosomes with implications for the control of trypanosomiasis. Adv. Parasitol. 53, 1–83. Araujo, R. N., Santos, A., Pinto, F. S., Gontijo, N. F., Lehane, M. J. and Pereira, M. H. (2006). RNA interference of the salivary gland nitrophorin 2 in the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae) by dsRNA ingestion or injection. Insect Biochem. Mol. Biol. 36, 683–693. Attardo, G. M., Guz, N., Strickler-Dinglasan, P. and Aksoy, S. (2006). Molecular aspects of viviparous reproductive biology of the tsetse fly (Glossina morsitans morsitans): regulation of yolk and milk gland protein synthesis. J. Insect Physiol. 52, 1128–1136. Attardo, G. M., Lohs, C., Heddi, A., Alam, U. H., Yildirim, S. and Aksoy, S. (2008). Analysis of milk gland structure and function in Glossina morsitans: milk protein production, symbiont populations and fecundity. J. Insect Physiol. 54, 1236–1242. Baker, R. D., Maudlin, I., Milligan, P. J. M., Molyneux, D. H. and Welburn, S. C. (1990). The possible role of rickettsia-like organisms in trypanosomiasis epidemiology. Parasitology 100, 209–217. Balasegaram, M., Young, H., Chappluis, F., Priotto, G., Raguenaud, M. E. and Checchi, F. (2009). Effectiveness of melarsoprol and eflornithine as first-line regimens for gambiense sleeping sickness in nine Me´decins Sans Frontie`res programmes. Trans. R. Soc. Trop. Med. Hyg. 103, 280–290. Barrett, M. P. (2006). The rise and fall of sleeping sickness. Lancet 367, 1377–1378. Barry, J. D. and McCulloch, R. (2001). Antigenic variation in trypanosomes: enhanced phenotypic variation in a eukaryotic parasite. Adv. Parasitol. 49, 1–70. Barry, J. D., Graham, S. V., Fotheringham, M., Graham, V. S., Kobryn, K. and Wymer, B. (1998). VSG gene control and infectivity strategy of metacyclic stage Trypanosoma brucei. Mol. Biochem. Parasitol. 91, 93–105. Barry, J. D., Marcello, L., Morrison, L. J., Read, A. F., Lythgoe, K., Jones, N., Carrington, M., Blandin, G., Bo¨hme, U., Caler, E., Hertz-Fowler, C. Renauld, H., et al. (2005). What the genome sequence is revealing about trypanosome antigenic variation. Biochem. Soc. Trans. 33, 986–989. Bayne, R. A. L., Kilbride, E. A., Lainson, F. A., Tetley, L. and Barry, J. D. (1993). A major surface antigen of procyclic stage Trypanosoma congolense. Mol. Biochem. Parasitol. 61, 295–310. Beard, C. B., Oneill, S. L., Tesh, R. B., Richards, F. F. and Aksoy, S. (1993). Modification of arthropod vector competence via symbiotic bacteria. Parasitol. Today 9, 179–183.
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Walshe, D. P., Lehane, S. M., Lehane, M. J. and Haines, L. R. (2009). Prolonged gene knockdown in the tsetse fly Glossina by feeding double stranded RNA. Insect Mol. Biol. 18, 11–19. Wang, J., Van Praagh, A., Hamilton, E., Wang, Q., Zou, B. X., Muranjan, M., Murphy, N. B. and Black, S. J. (2002). Serum xanthine oxidase: origin, regulation, and contribution to control of trypanosome parasitemia. Antioxid. Redox Sig. 4, 161–178. Wang, J., Hu, C., Wu, Y., Stuart, A., Amemiya, C., Berriman, M., Toyoda, A., Hattori, M. and Aksoy, S. (2008). Characterization of the antimicrobial peptide attacin loci from Glossina morsitans. Insect Mol. Biol. 17, 293–302. Watson, F. L., Puttmann-Holgado, R., Thomas, F., Lamar, D. L., Hughes, M., Kondo, M., Rebel, V. I. and Schmucker, D. (2005). Extensive diversity of Ig-superfamily proteins in the immune system of insects. Science 309, 1874–1878. Weiss, B. L., Mouchotte, R., Rio, R. V. M., Wu, Y. N., Wu, Z. Y., Heddi, A. and Aksoy, S. (2006). Interspecific transfer of bacterial endosymbionts between tsetse fly species: infection establishment and effect on host fitness. Appl. Environ. Microbiol. 72, 7013–7021. Weiss, B. L., Wu, Y. N., Schwank, J. J., Tolwinski, N. S. and Aksoy, S. (2008). An insect symbiosis is influenced by bacterium-specific polymorphisms in outer-membrane protein A. Proc. Natl. Acad. Sci. USA 105, 15088–15093. Welburn, S. C. and Maudlin, I. (1991). Rickettsia-like organisms, puparial temperature and susceptibility to trypanosome infection in Glossina morsitans. Parasitology 102, 201–206. Welburn, S. C. and Maudlin, I. (1992). The nature of the teneral state in Glossina and its role in the acquisition of trypanosome infection in tsetse. Ann. Trop. Med. Parasitol. 86, 529–536. Welburn, S. C. and Maudlin, I. (1997). Control of Trypanosoma brucei brucei infections in tsetse, Glossina morsitans. Med. Vet. Entomol. 11, 286–289. Welburn, S. C. and Maudlin, I. (1999). Tsetse-typanosome interactions: rites of passage. Parasitol. Today 15, 399–403. Welburn, S. C., Maudlin, I. and Ellis, D. S. (1987). In vitro cultivation of Rickettsia-like organisms from Glossina spp. Ann. Trop. Med. Parasitol. 81, 331–335. Welburn, S. C., Maudlin, I. and Ellis, D. S. (1989). Rate of trypanosome killing by lectins in midguts of different species and strains of Glossina. Med. Vet. Entomol. 3, 77–82. Welburn, S. C., Maudlin, I. and Molyneux, D. H. (1994). Midgut lectin activity and sugar specificity in teneral and fed tsetse. Med. Vet. Entomol. 8, 81–87. Welburn, S. C., Maudlin, I. and Milligan, P. J. M. (1995). Trypanozoon: infectivity to humans is linked to reduced transmissibility in tsetse. 1. Comparison of human serum-resistant and human serum-sensitive field isolates. Exp. Parasitol. 81, 404–408. Welburn, S. C., Dale, C., Ellis, D., Beecroft, R. and Pearson, T. W. (1996). Apoptosis in procyclic Trypanosoma brucei rhodesiense in vitro. Cell Death Diff. 3, 229–236. Werren, J. H. and Windsor, D. M. (2000). Wolbachia infection frequencies in insects: evidence of a global equilibrium? Proc. Soc. Biol. Sci. 267, 1277–1285. Whitten, M. M. A., Mello, C. B., Gomes, S. A. O., Nigam, Y., Azambuja, P., Garcia, E. S. and Ratcliffe, N. A. (2001). Role of superoxide and reactive nitrogen intermediates in Rhodnius prolixus (Reduviidae)/Trypanosoma rangeli interactions. Exp. Parasitol. 98, 44–57. Whitten, M. M. A., Sun, F., Tew, I. F., Schaub, G., Soukou, C., Nappi, A. and Ratcliffe, N. A. (2007). Differential modulation of Rhodnius prolixus nitric oxide activities following challenge with Trypanosoma rangeli, T. cruzi and bacterial cell wall components. Insect Biochem. Mol. Biol. 37, 440–452.
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WHO (2000). WHO Report on Global Surveillance of Epidemic-prone Infectious Diseases. WHO, Geneva. WHO (2007). Report of a WHO Informal Consultation on Sustainable Control of Human African Trypanosomiasis. WHO, Geneva. Wigglesworth, V. B. (1929). Digestion in the tsetse fly: a study of structure and function. Parasitology 21, 288–321. Wijers, D. J. (1958). Factors that may influence the infection rate of Glossina palpalis with Trypanosoma gambiense. 1. The age of the fly at the time of the infective feed. Ann. Trop. Med. Parasitol. 52, 385–390. Winston, W. M., Molodowitch, C. and Hunter, C. P. (2002). Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1. Science 295, 2456–2459. Woolhouse, M. E. J., Hargrove, J. W. and McNamara, J. J. (1993). Epidemiology of trypanosome infections of the tsetse fly Glossina pallidipes in the Zambezi valley. Parasitology 106, 479–485. Xin, R. G., Liu, S., Guo, Z. Y., Yu, H. H., Li, C. P., Ji, X., Feng, J. H. and Li, P. C. (2006). The antioxidant activity of glucosamine hydrochloride in vitro. Biol. Med. Chem. 14, 1706–1709. Yan, J., Cheng, Q., Li, C. B. and Aksoy, S. (2002). Molecular characterization of three gut genes from Glossina morsitans morsitans: cathepsin B, zinc-metalloprotease and zinc-carboxypeptidase. Insect Mol. Biol. 11, 57–65. Ye, Y. H., Chenoweth, S. F. and McGraw, E. A. (2009). Effective but costly, evolved mechanisms for defense against a virulent opportunistic pathogen in Drosophila melanogaster. PLoS Pathog. 5, e1000385. Zamore, P. D. (2000). RNAi: an in vitro approach to understanding mechanism. In ‘‘Regulatory Mechanisms Involving RNA Session. Biological Regulatory Mechanisms Conference’’ Gordon Research Conferences, Holderness’’, NH, July 30–August 4.
Interactions of Trypanosomatids and Triatomines Gu¨nter A. Schaub Zoology/Parasitology Group, Ruhr-Universita¨t Bochum, 44780 Bochum, Germany
1 Introduction 177 2 Triatomines 178 2.1 Distribution 178 2.2 Development 179 2.3 Intestinal tract, digestion and excretion 182 2.4 The intestinal microenvironment 183 3 The trypanosomatids 190 3.1 Distribution of species and strains 190 3.2 Developmental cycle in the triatomines 195 4 Effects of the host on trypanosomatids 198 4.1 Susceptibility and refractoriness 198 4.2 Effects of pH, osmolality and ionic composition 200 4.3 Effects of the border face 200 4.4 Effects of microorganisms and antimicrobial compounds 203 4.5 Effects of digestion, digestion products and excretion 204 4.6 Effects of other soluble factors 209 5 Effects of trypanosomatids on triatomines 210 5.1 Classification of pathogenicity and action of secondary stressors 210 5.2 Pathogenicity of Blastocrithidia triatomae and Trypanosoma rangeli 211 5.3 Subpathogenicity of trypanosomatids in triatomines 217 6 Interactions in double infections 219 7 Conclusions 220 Acknowledgements 220 References 220
1
Introduction
Investigations on the interactions of trypanosomatids and triatomines mainly consider Trypanosoma cruzi because this protozoon is the etiologic agent of Chagas disease, one of the ‘‘big six’’ which were selected in 1975/1976 by the World Health Organization for the ‘‘Special Programme for Research and Training in Tropical Diseases’’ (Schaub and Wu¨lker, 1984). Chagas disease is the only ADVANCES IN INSECT PHYSIOLOGY VOL. 37 ISBN 978-0-12-374829-4 DOI: 10.1016/S0065-2806(09)37004-6
Copyright # 2009 by Elsevier Ltd All rights of reproduction in any form reserved
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important tropical disease in which the parasite was first detected in the vector, namely triatomines (Chagas, 1909, 1922). The protozoon is mainly transmitted by these haematophagous bugs, but also through blood transfusions, organ implantations, parasite-contaminated food, etc. (Schmunis, 2004; Coura, 2007). Initially, Try. cruzi circulated exclusively between wild mammals and was transmitted by sylvatic triatomines (WHO, 2002). When man entered the natural foci and altered the ecosystem equilibrium, the domestic triatomines cycle became established (da Silva, 1986). The pathology of the disease is very variable, ranging from a sudden death within several weeks to nearly no symptoms for more than 70 years (Lana et al., 1996; Coura, 2007). Since only two compounds are available for therapy, which often results in severe side effects, the main target for control is the vector. Although house improvements can limit the colonization of the houses by triatomines (Schofield, 1994), mainly intense insecticide campaigns against the domestic populations of the most important vector, Triatoma infestans, has reduced the prevalence, according to estimates, from about 20 million chronically infected people in 1982 to about 12 million in 2007 (WHO, 1982; Dias, 2007). However, since Chagas disease is an anthropozoonosis circulating also in many wild mammals as reservoir hosts, an eradication is impossible. In addition to Try. cruzi, three other species of trypanosomatids develop in the triatomines. Besides the insect flagellate Blastocrithidia triatomae, these are species of the genus Trypanosoma: the rat trypanosomatid Try. conorhini and Try. rangeli. The latter is also regularly found in humans, but often in double infections with Try. cruzi. In single infections it is non-pathogenic, developing only a low parasitemia and persisting at detectable levels in the blood only for about 1–3 weeks (D’Alessandro, 1976; Guhl et al., 2002). The chapter does not cover bat trypanosomes which also develop in triatomines, because for these infections mainly epidemiological and systematic aspects are considered. Previous reviews focused only on interactions of Try. cruzi or Try. rangeli on triatomines (Zeledo´n et al., 1977; Garcia and Azambuja, 1991; Gonzalez et al., 1998; Azambuja et al., 2005a,b; Garcia et al., 2007). Above and beyond that, a comparison of the different trypanosomatids provides an understanding of the individual adaptations and general aspects and either of factors acting on the flagellate inside the vector or of protozoon-derived factors acting on the triatomine.
2 2.1
Triatomines DISTRIBUTION
According to recent monographs, the current taxonomy of Triatominae recognizes 140 species (Galva˜o et al., 2003; Schofield and Galva˜o, 2009). However, this number will change in the near future, because molecular biological methods will identify new species and subspecies (Mas-Coma and Bargues, 2009).
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Of the six tribes and 18 genera only species of the genus Linshcosteus are confined to the Indian subcontinent. Eight species forming the rubrofasciata group are associated with rats. Originating from the New World, Triatoma rubrofasciata has been distributed by sailing ships to many ports in tropical and subtropical regions especially in Southeast Asia (Haridass and Ananthakrishnan, 1980; Gorla et al., 1997). The other triatomines occur only on the American continent from latitude 42 N to 46 S, that is, between the Great Lakes of North America and Argentina (Lent and Wygodzinsky, 1979; Schofield, 1994; Gorla et al., 1997). Triatomines colonize all terrestrial habitats, but prefer to stay near the host. This is easy in caves of bats, burrows of rodents or preferred resting places of other animals, but more complicated in nests of birds which only breed once a year. Nests are often colonized by species of the tribe Rhodniini which prefer palm trees, whereas species of the genus Triatoma often prefer rocky habitats and rodent burrows and species of the genus Panstrongylus tree cavities and burrows (Gaunt and Miles, 2000; Schofield and Galva˜o, 2009). Since the dwellings of the indigenous humans of Latin America were caves or were made of material from the forest, the transition from animals to humans was not a big step. The construction of houses as a wooden frame covered with mud or adobe still offered a good habitat for triatomines since the cracks in the mud or adobe are optimal hiding places during the day for the night active bugs (Figs. 1A, B and 2A, B). In addition, the use of palm leaves for the roof provides a direct access to the house if eggs have been glued to the leaves by Rhodnius sp. According to their proclivity to approach the houses, the species of triatomines can be classified into sylvatic species, those of the peridomestic regions, mainly using farm animals near the houses as hosts, and domestic species, sucking blood of humans and animals inside the house. The most important domestic species is Tri. infestans, presumably originating from sylvatic populations in the Andean valleys of Bolivia (Noireau et al., 2005). This species has successfully displaced many other species of Triatominae (Pereira et al., 2006) and is nowadays threatened back in its distribution (see Section 1). Also, the domestic species Rhodnius prolixus is a very important vector. This species was also found on palm trees, an argument against the suggestion that it is restricted to the house and evolved from the almost morphologically indistinguishable but strictly sylvatic R. robustus (Feliciangeli et al., 2007). 2.2
DEVELOPMENT
All postembryonic stages of these hemimetabolous insects are obligate blood suckers, developing from eggs through five instars to the adults (Schaub, 2008). In each of the instars, the bugs require one full feeding engorgement – of about 6–12 times their own body weight – or several smaller ones. (Most scientists in Central Europe call the first four instars of Hemiptera ‘‘larvae’’ and the fifth a
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FIG. 1 cover.
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(A) Rural wooden framed mud covered house in Brazil. (B) Cracks in the mud
nymph while others use the term ‘‘nymph’’ for all five pre-adult instars.) The full engorgement activates via distension receptors the secretion of hormones – finally of ecdysone – and thereby the development of the new cuticle and the moult to the next instar or to the adult (Wigglesworth, 1940; Anwyl, 1972;
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FIG. 2 (A) Rural adobe brick house in Bolivia. (B) Gaps between the adobe bricks.
Chiang and Davey, 1988). Without a full engorgement, more blood meals are necessary to obtain a specific level of reserve compounds for the new cuticle, and even with a full engorgement pathogenic flagellates may reduce the reserve level, resulting in delays of moulting (Schaub, 1988a). Therefore, the duration of the different stages is determined by the availability of blood, parasites and
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characteristics of the respective species, but also by temperature and relative humidity (rH). The source of blood also affects the development and/or egg production, blood of mice being superior to blood of pigeons and chickens (summarized by Emmanuelle-Machado et al., 2002). In addition, development depends on symbiotic bacteria which colonize the anterior regions of the midgut and deliver essential compounds (Eichler, 1998; Eichler and Schaub, 2002) (see Section 2.4.3). The availability of hosts is a limiting factor for populations of triatomines (Ceballos et al., 2005). In wild populations many insects are starved, and only about 14% of the offspring are estimated to reach the adult stage (Schofield, 1980a,b). If no host is available, bugs can starve for long periods of time, increasingly from instar to instar up to the fourth. In Tri. infestans, at 90% rH this instar can survive 250 70 days after last feeding in the third instar, up to 14 months (Schaub and Lo¨sch, 1989). Fifth instars possess a lower, in other species a stronger starvation resistance than fourth instars, adults always a lower one (e.g. Zeledo´n et al., 1970; Feliciangeli et al., 1980; Schaub and Lo¨sch, 1989; Corte´z and Gonc¸alves, 1998; Cabello, 2001; Almeida et al., 2003). Some days after the moult, copulation occurs, males depositing the spermatophore with the sperms in the vagina (Wigglesworth, 1972). Females lay 100–600 eggs, for most species loosely on the ground, but species inhabiting trees glue them to the leaves or feathers of birds (Schaub, 2008). Adults live 3–12 months, depending on the species, abiotic factors and availability of blood. 2.3
INTESTINAL TRACT, DIGESTION AND EXCRETION
The intestinal tract of triatomines is a relatively simple tube without diverticula (Kollien and Schaub, 2000). Of the foregut, the cibarium and pharynx are located in the head and the oesophagus with the transverse rings of the wall in the thorax (Wenk and Renz, 2003). The ducts of the salivary glands – two pairs in species of the genus Rhodnius and three pairs in those of the genera Triatoma, Dipetalogaster and Panstrongylus – end in the foregut (Barth, 1954; Lacombe, 1999). The midgut begins with the cardia, followed by the strongly distensible stomach and then the small intestine. (Other groups use the terms ‘‘crop’’ or ‘‘anterior midgut’’ for the stomach and ‘‘posterior midgut’’ for the small intestine.) The latter is subdivided by a central narrow region into the anterior, middle and posterior small intestine. The beginning of the hindgut is a very short pylorus/ileum region in which the Malpighian tubules end, followed by the big rectal sac. After feeding, several layers of membranes are shed from the microvilli, building the perimicrovillar membranes (also named extracellular membrane layers) (Billingsley, 1990; Terra, 1990). The functions of the different gut regions differ: In the foregut are located the pumps giving the saliva into the wound and blood vessels and pumping the blood into the oesophagus (Wenk et al., 2009). The short cardia seems to act as a sphincter and possesses deep enfoldings, separating the symbionts from the
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blood flow (Kollien and Schaub, 2000). In the stomach, the blood is stored essentially undigested and concentrated, followed by a lysis of erythrocytes (Azambuja et al., 1983). Then the haemoglobin of some hosts, for example, guinea pigs, crystallizes in the stomach (Bauer, 1981). The stomach wall is not only a simple transport epithelium, pumping ions into the haemolymph and thereby passing the water to it and concentrating the blood, but also serves for the intracellular storage of lipids (Kollien and Schaub, 2000). In addition, after blood ingestion the number of symbionts increases there enormously (see Section 2.4.3). Small portions of the blood meal are passed into the small intestine. There the blood is digested, the symbionts are killed, and the nutrients are absorbed (Bauer, 1981). The remains of the blood meal are stored in the rectum for further absorption processes before being defecated. The ingestion of blood initiates a rapid release of diuretic hormones from the storage in the prothoracic and mesothoracic ganglionic masses (Maddrell, 1966; Maddrell et al., 1991). These hormones regulate the transport of fluid across the gut wall and induce a 1000-fold increase of the diuresis rate by the four Malpighian tubules, in R. prolixus and Tri. infestans to a rate as high as 3.3 ml/min/cm2 tubule (2.9 nl/min/mm) (Maddrell, 1969; Maddrell and Gardiner, 1980; Schnitker et al., 1988). Thereby, the conditions in the rectum change rapidly. The remnants of digestion are often eliminated at the end of blood ingestion, followed by clear colourless urine. Within 24 h after blood ingestion, the bugs excrete about 50–60% of the weight of the total ingested blood (76% of the imbibed fluid) (Wigglesworth, 1931). Then the yellow uric acid granules are eliminated for about 10 days, followed by a mixture with the dark remains of digestion (Wigglesworth, 1931; Hase, 1932). After the moult, increasingly less remains are defecated. 2.4 2.4.1
THE INTESTINAL MICROENVIRONMENT
The intestinal border face
According to the ectodermal origin, foregut and hindgut are lined by a cuticle, and the cells of the midgut are bordered by the microvilli. The microvilli of the epithelium of starved insects are covered by a single perimicrovillar membrane which develops to thick staples after feeding (Billingsley and Downe, 1983, 1986). This is very intensive after feeding of starved adults of R. prolixus, whereas in larvae of Tri. infestans starvation does not induce such a strong breakdown of the membranes and not such a synchronous development of the membranes after feeding (Jensen et al., 1990). The full development of the perimicrovillar membranes depends on the distension of the abdomen (see Section 2.2), blood components (especially haemoglobins) and the release of ecdysone (Albuquerque-Cunha et al., 2004). In detailed investigations especially the group of P. Azambuja and E.S. Garcia elucidated these mechanisms. The development of the membranes can be inhibited or reduced by decapitation,
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gamma irradiation, cutting of the nerve cord transmitting the signals of the distension receptors, anti-ecdysone compounds (e.g. azadirachtin) or a saline diet without proteins (e.g. Cortez et al., 2002; Gomes et al., 2002; AlbuquerqueCunha et al., 2004). The membranes show a strong reaction of non-specific carbohydrate staining (Bauer, 1981). The hindgut is bordered by an extracellular cuticle. This cuticle has the same basic architecture as the external integument of insects (Schmidt et al., 1998). The cell membrane is covered by the procuticle. Only this region reacts with wheat-germ lectin, indicating the presence of N-acetyl-D-glucosamine of the chitin. The next layer is the epicuticle, in electron microscopy subdivided into the electron-dense inner epicuticle and the outer epicuticle built by a cuticulin layer. The latter is covered by the superficial layer, which reacts with the lipidspecific Nile red stain (Schmidt et al., 1998). At the entrance into the rectal sac, the four rectal pads – also called rectal glands – possess a much thinner cuticle than the other regions of the rectum (Wigglesworth, 1972). In other insects, the rectal glands absorb water, ions and amino acids (Wigglesworth, 1984). 2.4.2
pH, osmolality and ions
Directly after blood ingestion, the blood determines the conditions in the stomach, that is, pH 7.4 (Billker et al., 2000). According to pH determinations via pH indicators, the pH in the stomach of larvae of R. prolixus increases after feeding to pH 7.4 and within 1 week decreases slowly to pH 6.5, in the small intestine from pH 7.2 to 5.5 (J. M. C. Ribeiro and E. S. Garcia, personal communication). According to a more detailed investigation using microelectrodes, pH 6.2 in the stomach of starved fifth instars of Tri. infestans changes to pH 7.3 after blood ingestion, decreases slowly within the following 10 days to pH 5.2, and then increases to pH 5.6 at 20 days after feeding (M. Oldenburg and G. A. Schaub, unpublished data). In the small intestine, pH 6.8 in starved insects directly decreases and increases then to similar levels as in the stomach, reaching pH 6.1 at 20 days after feeding. The conditions in the rectum show a much stronger variation after feeding. Whereas the first deposited drop, mainly remains of digested blood, is acidic, pH 5.9, the pH increases to over pH 8.0 in the following four drops. For 24 h the pH of the urine is alkaline, reaching pH 8.9. Then the pH drops to pH 6.5, remaining at this level at least up to 96 h after feeding (Kollien et al., 2001). The osmolality changes from 320 mosmol/kg H2O in the first drop to 370 in the second and 410 mosmol/kg H2O in the fourth. At 1 and 3 days after feeding, the rectal contents possess an osmolality of 370 and 760 mosmol/kg H2O, respectively (Kollien et al., 2001). In determinations of the concentrations of anions and cations, sulphate and phosphate dominate in the rectal content of unfed bugs, and sodium and chloride in the clear urine; strong individual variations of the individual ions begin at 1 day after feeding (Kollien et al., 2001).
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The microorganisms
Triatomines possess symbionts, mainly actinomycetes, which colonize the anterior regions of the midgut and deliver essential compounds (Eichler, 1998; Eichler and Schaub, 2002; Durvasula et al., 2008). Symbionts are transmitted within a population of bugs via coprophagy (Schaub, 1988c; Schaub et al., 1989a). Therefore, ammonia and pheromones deposited in the faeces and urine after blood ingestion and attracting other hungry bugs (Taneja and Guerin, 1997; Guerenstein and Lazzari, 2009) indicate not only the presence of a host but also of symbionts. Since dry faeces needs to be redissolved by fresh faeces (Schaub et al., 1989a), pheromones in dry faeces only mark the location of refuges (Lorenzo and Lazzari, 1996; Vitta et al., 2007), but should not attract for the transmission of symbionts. During coprophagy, also air-borne microorganisms get access to the intestinal tract. A minor risk is the contact of the mouthparts with the skin before penetration, but like all terrestrial insects, triatomines swallow air before moulting (Kollien et al., 2003). In addition, an uptake of tap water (Wiesinger, 1956) can be a source of bacteria. After the first description of a Gram-positive bacillus in the intestine of R. prolixus (Duncan, 1926), many different microorganisms were isolated from this and other species of triatomines, not only bacteria but also fungi (Figueiredo et al., 1990; Eichler et al., 1996; Vallejo et al., 2009). [The intestinal bacteria are classified as symbionts (i.e. they support the growth of the host), commensals (i.e. they develop in the intestine without positive or negative effects on the host) or as parasites if they affect the insect.] Upon incubating the intestinal contents from Tri. infestans from the field on agar plates, 16 morphologically different bacteria were isolated. These were mainly bacteria colonizing the soil or the skin (N. Reintjes and G. A. Schaub, unpublished data). In Tri. sordida from the field, a Dictyostelium-like slime mould occurred, and this bug contained no other bacteria (Eichler, 1998). Focusing on actinomycetes, seven morphologically different isolates were separated from Tri. sordida (Eichler et al., 1996). The stomach of R. prolixus contained a bacterium so far unknown from triatomines, Serratia marcescens (Azambuja et al., 2004). This Gram-negative bacterium possesses haemolytic and cytotoxic factors – at least one of them being a metalloprotease (Hertle et al., 1999; Marty et al., 2002) – and is pathogenic for immune-suppressed humans and some insects (summarized by Flyg and Xanthopoulos, 1983; Hejazi and Falkiner, 1997), but has no obvious effects on the triatomine. Also the majority of the other intestinal bacteria of triatomines can be classified as commensals of triatomines. The identification of the symbionts is mainly based on simple microbiological criteria, Gram-staining, development and morphology, with the identification of Rhodococcus rhodnii as a symbiont of many species of triatomines (summarized by Vallejo et al., 2009). However, the slow growth and the colour change of old cultures are not sufficient to differentially distinguish species of
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actinomycetes. After sequencing the 16S rDNA and determining the symbiotic function by infections of aposymbiotic bugs, in Tri. infestans from Bolivia only a Nocardia sp. and in Panstrongylus megistus a Rhodococcus equi-like species fulfilled the criteria of a symbiont, that is, they establish and multiply in the gut, some survive the passage through the small intestine, the larvae develop without retardations or increased mortality rates, and the reproductive rates of the adults and the emergence rate of the offspring are in the normal range (Eichler et al., 1996; Vallejo et al., 2009; G. A. Schaub, unpublished data). In Tri. infestans from Argentina, a corynebacterial species fulfilled these criteria (Durvasula et al., 2008). After infection of first instars by feeding a mixture of blood and symbionts – R. rhodnii for R. prolixus and Nocardia sp. for Tri. infestans – followed by an axenic maintenance including in vitro feeding with defibrinated sheep blood, the number of colony-forming units of symbionts differs in the different regions of the intestinal tract of fifth instars and is affected by blood ingestion and digestion (Eichler and Schaub, 2002). After blood ingestion, the concentration of the respective symbiotic bacteria decreases due to the dilution of the populations by the blood. Within 10 days, the total population/bug increases 15- or 18-fold to about 0.8 109 colony-forming units in R. prolixus and 1.8 109 colony-forming units in Tri. infestans. About 95–99% of the total population of both symbionts develop in cardia and stomach. Directly after the passage from the blood-storing stomach into the digesting small intestine about 99% of the symbiont populations are killed, and only about 0.01% of the total population is present in the rectum. A lysis of bacteria in the small intestine is also visible using electron microscopy (Bauer, 1981). After blood ingestion the urine washes out the rectal population, which is thereby mainly present in the remains of blood digestion, that is, in the first drops of defecation, not in the urine (Eichler and Schaub, 2002). A first suggestion of a symbiotic function was published by Dias (1934, 1937). Experiments of Wigglesworth (1936) indicated that the symbionts support the triatomines with vitamin B. In larvae, aposymbiosis results in a retarded development and increased mortality rate and disturbances of cuticle melanization, digestion and excretion; if adults develop, their fecundity is strongly reduced (summarized by Vallejo et al., 2009). Supplying aposymbiotic larvae which show the aposymbiosis syndrome with a mixture of B vitamins or symbionts reduces the effects in the following instar. The hypothesis that the symbionts supply compounds of the vitamin B complex requires new investigations, since variable results were obtained using auxotrophic bacteria in determinations of the vitamin production by cultures of the symbionts (Geigy et al., 1953; Harington, 1960). In addition, after supplying aposymbiotic bugs with auxotrophic symbiont mutants no aposymbiosis effects occurred (Hill et al., 1976). Therefore, not supplying with B vitamins, but with sugar components might be the function of symbionts (Ribeiro and Pereira, 1984).
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Antimicrobial factors
Like vertebrates, insects possess an innate immunity of cellular and humoral responses (Dillon and Dillon, 2004; Ratcliffe and Whitten, 2004; Mu¨ller et al., 2008). In triatomines the investigations focused very early on responses in the haemocoel after injection of bacteria (Azambuja and Garcia, 1987; Azambuja et al., 1989b) and rarely on the intestinal homeostasis (Ribeiro and Pereira, 1984; Kollien et al., 2004). Since triatomines require symbionts for their development, but are killed after ingestion of blood contaminated with airborne bacteria (G. A. Schaub, unpublished data), their intestinal immune system is even more important than that of the haemolymph and must distinguish between the symbionts and the pathogenic bacteria. In addition, the symbionts must resist the antibacterial activities. Antibacterial activities in the gut of triatomines were recognized very early by Duncan (1926). Later this activity was attributed for other insects to lysozyme, a widespread antibacterial compound (Mohrig and Messner, 1968). In R. prolixus, the antibacterial activity varies according to the region of the gut and the period of time after feeding. In the stomach, the activity increases after feeding and is much higher than in the small intestine (Ribeiro and Pereira, 1984). Although lysozyme has not been isolated and identified from the intestinal contents, the production in the intestinal wall is proved by the characterization of the cDNA and the determination of the expression levels in the different regions of the gut (Kollien et al., 2003; Arau´jo et al., 2006; Balczun et al., 2008; Ursic-Bedoya et al., 2008). In the cardia and stomach, the concentration of mRNA-encoding lysozymes increases after feeding, reaching a maximum before moulting, and is much higher than in the small intestine (Kollien et al., 2003). The difference is also visible in whole mount in situ hybridizations using Tri. brasiliensis and sense and antisense digoxigenin-labelled RNA (Arau´jo et al., 2006). Whereas all these lysozymes belong to the chicken-type lysozymes (c-type), invertebrate-type lysozymes (i-type) – originally found in a starfish – also occur in insects (Paskewitz et al., 2008) and have to be considered for triatomines. In addition to lysozyme, defensin appears to be present in the intestine. After injection of bacteria into the haemocoel, a rapid induction of transcription was found in the haemocoel and a delayed induction in the gut (Lopez et al., 2003). Without such an immunization, the mRNA encoding defensins is distributed uniformly throughout the cardia and stomach and to a much lower extent in the small intestine of Tri. brasiliensis (Arau´jo et al., 2006). Using quantitative realtime PCR, the expression level of the defensin gene in the stomach is found to be about 10-fold higher than that of the lysozyme gene. Comparing the different regions of the intestine, it is 500–2500 times higher in the stomach than in the cardia and very low in the small intestine. The time course of the expression is similar to that of the lysozyme gene, that is, a strong increase after feeding and a decrease between 5 and 10 days after feeding (Arau´jo et al., 2006).
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Most recently, two additional defensin-encoding genes of Tri. brasiliensis were characterized (Waniek et al., 2009). The mRNA of defensin3 is present in the small intestine, but not in the stomach, that of defensin4 shows higher concentrations in the stomach than in the small intestine. Other antimicrobial factors of 7 and 25 kDa occur as humoral response after injection of bacteria into the haemocoel of R. prolixus (Azambuja et al., 1986, 1989b), but have not been identified nor found in the gut. According to a suppressive subtractive hybridization approach for the identification of the upregulation of immune-related genes after intracoelomic inoculation of Try. cruzi, also genes for transferrins are upregulated, which seem to be components of the humoral immune system and sequester iron away from the bacteria (Ursic-Bedoya and Lowenberger, 2007). In addition to these proteins/peptides, a low molecular weight compound, nitric oxide, is produced in the intestinal wall and may regulate the populations of microorganisms in the intestinal tract of triatomines (Whitten et al., 2001, 2007). The concentration of mRNA encoding for the nitric oxide synthase increases in the stomach within the first 24 h after feeding and then decreases. This is also evident for the small intestine in which the expression level is higher than in the stomach up to 12 h after feeding (Whitten et al., 2007). Also host-derived factors, the complement system and antibodies against components of the saliva and the Trypanosoma species have to be considered since these are ingested together with the blood of the host. However, salivary compounds of triatomines inhibit the classical pathway of complement activation (Cavalcante et al., 2003). Antibodies against the components of the saliva are produced rapidly by birds and mammals (summarized by Schwarz et al., 2009a,b). A component of the immune system of insects which is not present in the lumen of the intestine but in the cells of the intestinal wall and the haemocytes is the phenoloxidase (summarized by Ratcliffe and Whitten, 2004; Mu¨ller et al., 2008). This enzyme catalyses the oxidation of phenolic amino acids, resulting in melanin, which is used to encapsulate big parasites in the haemolymph or nodules of haemocytes that have ingested high numbers of microorganisms. Immune responses using this enzyme are regulated by juvenile hormones (Nakamura et al., 2007).
2.4.5
The enzymes and digestion products
Different enzymes originate from the intestinal tract. The saliva contains many different compounds, also serine proteases for the activation of some of these compounds (Assumpc¸a˜o et al., 2008). Although the major digestion occurs in the small intestine, during storage in the stomach some enzymes act on the blood. A sialidase, and alkaline and acid phosphatases are found in the stomach contents, and b-acetylglucosaminidase, a-galactosidase, a-glucosidase, a-mannosidase and
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b-mannosidase are involved in the metabolism of carbohydrates (Terra et al., 1988; Amino et al., 1995). In the small intestine, alkaline and acid phosphatases and a-glucosidase and a-mannosidase are also active, but with a higher activity than in the stomach (Terra et al., 1988). Digestion of haemoglobin starts immediately, as indicated by the colour change from red to brown. Whereas most insect groups use serine proteases – trypsins and chymotrypsins – as their principal proteases, Hemiptera depend on cysteine and/or aspartic proteases. This correlates to the pH of the insect midgut lumen, which is neutral or alkaline in the majority of insects, but acidic in Hemiptera (Lehane, 1994). The activity of cysteine proteases was attributed to cathepsin B (Terra et al., 1988; Terra, 1990), until the characterization of a cathepsin L cDNA from the intestine of R. prolixus and then from Tri. infestans (Lopez-Ordon˜ez et al., 2001; Kollien et al., 2004). Carboxypeptidases and aminopeptidases continue the digestion of the blood (Garcia and Guimara˜es, 1979; Garcia, 1987; Terra et al., 1988). Whereas cathepsin B is active in the lumen of the gut, aminopeptidases and acid phosphatases are localized on the perimicrovillar membranes (Bauer, 1981; Billingsley and Downe, 1985, 1988). Since one of the components of haemoglobin, free haem, can cause oxidative damage to the tissues, triatomines sequester it into haemozoin (Oliveira et al., 2000). Not only proteolysis, but also the digestion of lipids is restricted to the small intestine (Rimoldi et al., 1985; Canavoso et al., 2004; Grillo et al., 2007). In the lumen, the triacylglycerols are hydrolysed to free fatty acids, which are absorbed by the epithelium, used for the synthesis of lipids and stored in the fat body and the wall of the gut. Detailed investigations of the digestion products have only been performed for the rectum and the drops of faeces and urine, the latter reflecting the situation in the rectum immediately before (A. H. Kollien et al., unpublished data). Due to dilution by the urine, in the brown rectal contents of Tri. infestans, the total concentrations of free and protein-bound amino acids are higher than in the first drop of faeces, but the percentages of amino acids are similar. The first dark drop of faeces contains 4 mM free and 18 mM bound amino acids. These concentrations are reduced to 0.15 mM total free and bound amino acids in the subsequent drops of urine. The reduction in free amino acids is significantly different for nearly all amino acids. Thus, whereas in the faeces taurine predominates, followed by proline, histidine, tyrosine, alanine and valine, in the urine proline predominates, followed by alanine, glycine and valine. 2.4.6
Other soluble factors
In the stomach of the triatomines, different factors must be considered, first of all compounds from the saliva which are continuously injected into the capillaries during blood sucking and ingested together with the blood (Ribeiro and Garcia, 1980). The saliva contains compounds that, for example, counteract host pain, dilate the capillaries and inhibit clotting of platelets (Stark and James, 1996;
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Ribeiro and Francischetti, 2003; Champagne, 2005). According to the detailed investigations of the sialome of the blood-sucking triatomines R. prolixus, Tri. infestans and Tri. brasiliensis by Ribeiro et al. at NIH (Ribeiro et al., 2004; Santos et al., 2007; Assumpc¸a˜o et al., 2008), the most abundant secreted proteins are lipocalins, which are not only transport proteins, but act as inhibitors of collageninduced platelet aggregation and thrombin (Assumpc¸a˜o et al., 2008). Also in the lipocalin family are the cherry red nitrophorins, which transport the vasodilatory nitric oxide (Ribeiro, 1996). This is evident for sucking blood from capillaries since a knockdown of nitrophorins by RNAi affects the feeding on the skin, but not on the relatively large tail vein (Araujo et al., 2009). Only triatomines of the tribe Rhodniini possess nitrophorins (Pereira et al., 1998; Soares et al., 1998, 2000; Ribeiro et al., 2004). Beside apyrases, which degrade ADP and thereby inhibit platelet aggregation and inflammation, saliva contains serine protease inhibitors (thrombin inhibitors), serine proteases (presumably for the activation of enzymes), defensins as antimicrobial peptides, and in Tri. infestans the pore-forming molecule trialysin (Ribeiro and Francischetti, 2003; Assumpc¸a˜o et al., 2008). In the stomach Kazal-type inhibitors prevent the clotting of the ingested blood, for example, by reaction with thrombin (e.g. Hellmann and Hawkins, 1965; Friedrich et al., 1993; Mende et al., 1999; Campos et al., 2002; Araujo et al., 2007). The siRNA knockdown of an intestinal thrombin inhibitor or the administration of thrombin reduces the amount of blood ingested by Tri. brasiliensis (Araujo et al., 2007). In the initial phase after blood ingestion, clotting is inhibited; in a subsequent phase, erythrocytes are lysed by a haemolytic factor that has been semi-purified from R. prolixus (Azambuja et al., 1983, 1989a) (see Section 4.6). In the interaction of blood cells and the intestinal tract also agglutinins and lectins have to be considered. Different lectins seem to be present in the stomach and the small intestine of R. prolixus (Pereira et al., 1981). In a follow-up investigation, especially the lectins in the small intestine were not verified (Grego´rio and Ratcliffe, 1991a; Ratcliffe et al., 1996). However, the levels of agglutinins seem to be affected by the blood source, and the agglutinins in the small intestine might be enzymes with lectin-like properties and not a normal type of lectin (Ratcliffe et al., 1996).
3
The trypanosomatids
3.1 3.1.1
DISTRIBUTION OF SPECIES AND STRAINS
Blastocrithidia triatomae
The homoxenous flagellate B. triatomae was first described from Argentine laboratory colonies of Tri. infestans (Cerisola et al., 1971). In such colonies high infection rates can occur (Schaub, 1988d). Despite the high numbers of wild
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triatomines dissected for determinations of the epidemiology of Chagas disease, only very low numbers of bugs were found infected in natural populations, in Brazil 0.03% and 0.08% of Panstrongylus megistus, 0.36% of Triatoma sordida and 0.02% of different triatomines (mainly Tri. sordida), in Argentina 2% of Triatoma guasayana and 1.5% of Triatoma garciabesi and in Venezuela 5–10% of Triatoma maculata (Correˆa et al., 1977; da Rocha e Silva et al., 1977; de Hubsch et al., 1977; Luz and Silveira, 1984; Schijman et al., 2006). These low rates indicate that triatomines are not the natural host of B. triatomae. Since all postembryonic instars in all populations of the insectivorous reduviid bug Zelus leucogrammus were infected with a very similar B. triatomae and since flagellates from the insectivorous bug were infective for Tri. infestans (Carvalho, 1973; Carvalho and Deane, 1974), this can be the origin of the infections of triatomines. Z. leucogrammus colonizes citrus plantations all over Brazil (Carvalho, 1973). The variability of B. triatomae has not been investigated, but subspecies have been denominated (Carvalho, 1973; da Rocha e Silva et al., 1977; de Hubsch et al., 1977). However, the original description was based on morphometrics of 10 specimens (20 specimens in length measurements). Using the original strain and measuring about 45 epimastigotes, the total length was much smaller, due to the length of the free flagellum (Reduth, 1986). After the classification of hundreds of flagellates into the different stages, I emphasize that the occurrence and arrangement of vacuoles and the length of the free flagellum are very variable. Especially in the stomach, epimastigotes with a short free flagellum develop. Comparing all morphometric data (see Table 1), the separation of subspecies according to morphological criteria is doubtful and molecular biological tools should be used. 3.1.2
Trypanosoma conorhini
Try. conorhini has only been found naturally in the triatomine Triatoma rubrofasciata (Hoare, 1972). This triatomine is very closely associated with rats (Rattus rattus), and thereby is present in tropical and subtropical ports of Africa, Asia and South America (see Section 2.1). In Southeast Asia similar parasites are present in monkeys (Hoare, 1972). The flagellate has from time to time been considered in different aspects, for example, in investigations of the cultivation and ultrastructure (cf. Deane and Deane, 1961; Milder and Deane, 1967; Deane and Milder, 1972), but no recent investigations considered a variation of strains. 3.1.3
Trypanosoma cruzi
Try. cruzi has been found in many triatomines. In the United States, it mainly infects dogs, in Latin America from Mexico to Argentina it infects all mammals including bats, correlated to the distribution of triatomines. Initially two groups were separated according to isoenzyme patterns, named zymodeme
TABLE 1 Morphometric data (mm) of Blastocrithidia triatomae from different insects or in vitro cultures Tri. infestans
Length Total Body Free flagellum Width Width at nucleus Width at vacuole Post.– Kinetopl.g Kinetopl. –Nu.h Post.–Nu. (P–N)i
Tri. infestans
In vitro
T. inf./P. meg.a
P. meg.b
Tri. maculatab
Z. leucogrammus
Mean Min
Max
Mean Min
Max
Mean Min
Max
Mean
Mean
Mean Min
53.5 25.0 22.5
48.5 14.8 19.5
62.5 32.0 28.5
35.7c 26.2 9.5
– 15.0 3.3
– 39.3 19.4
32.2c 22.0 10.2
– 33.5 22.7
51.8 23.9 23.3
54.6 32.5 18.6
26.9 24.0 34.5 28.9 12.1 8.0 15.0 15.6 14.8d 5.0d 19.5d 3.3
10.5 9.1 1.4
45.6 22.8 22.8
– 2.6
– 2.2
– 2.9
2.1e –
1.6e –
3.2e –
2.1e –
–
–
– 2.8
– 3.2
1.6f –
1.0f –
2.0f –
2.1e –
1.1e –
3.7e –
3.4
2.5
3.8
–
–
–
–
–
–
–
–
–
–
–
–
–
–
14.5
10.2
18.5
28.6
13.8
22.1
–
–
–
–
–
–
–
–
5.7
4.3
7.9
3.9c
–
–
–
0.8
0.1
3
–
–
–
10.7j
10.1j
11.8j
12.9k
16.9k
11.7j
13.1j
6.4k
4k
9k
–
–
–
16.8
8.4 – 5.6k
– 21.8k
3.2c 10.5k
– 13.0 3.4 1.6e
8.4 – 6k
3.6e
Max
Mean Min
Max
Nu.–Ant. 13.6j 12.8j 14.2j 13.2k 7.4k 23.7k (N–A)l 0.60 1.50 Nuclear index 0.79 0.79 0.83 1.00 (P–N)/ (N–A) Authorsm Cerisola et al. (1971) Reduth (1986) and Del Prado (1972)n
11.7k 0.91
7.6k
18.9k
0.60 1.50
12.8j
18.4j
0.91
0.71
5.7k
3.5k
9k
1.1
0.7
1.8
da Rocha e Silva et al. de Hubsch et al. (1977) (1977)
0.9
0.5
1.8
Carvalho (1973)
T. inf. and P. meg. ¼ Triatoma infestans and Panstrongylus megistus. Triatomines from the field, otherwise laboratory colonies or cultures. Calculated from the means. d No clear statement, whether measurement of length of free or total flagellum. e Measurement at broadest point. f Position of measurement not specified. g Post.–Kinetopl. ¼ posterior end to kinetoplast. h Kinetopl.–Nu. ¼ kinetoplast to nucleus. i Post.–Nu. ¼ posterior end to nucleus. j No clear statement of the position of measurement. k Up to centre of the nucleus. l Nu.–Ant. ¼ nucleus to anterior end. m Numbers of measured flagellates: Cerisola et al. (1971): 10 stained specimens and 10 snapshots (of the latter presumably only length measurement); Carvalho (1973): no data; de Hubsch et al. (1977): no data; da Rocha e Silva et al. (1977): no data; Reduth (1986): 45/46 flagellates from Tri. infestans (width only 21) and 57 flagellates from cultures (width only 25). n Gives only data for total length and length of the free flagellum. a b c
194
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1 and 2 (Z1 and Z2) (Miles et al., 1978; Ebert and Schaub, 1983). The comparison of many strains indicates a predominantly clonal genetic structure of Try. cruzi and only restricted recombinations (Tibayrenc et al., 1986; Tibayrenc and Ayala, 1988). Strains showing a pattern differing from both group criteria are classified into zymodeme 3. Similar, but not identical groupings are obtained using other methods, for example, restriction enzymes for kinetoplast DNA and molecular biology tools, and the two groups are denominated Try. cruzi I and Try. cruzi II (Anonymous, 1999). Genetic recombinations may be the cause why some strains have to be classified into a third lineage. Discrepancies even when considering the same strains may be due to the fact that under natural conditions, double infections with parasites belonging to both groups are found and after prolonged in vitro cultivation under different conditions different populations dominate (summarized by Carneiro et al., 1990; Solari et al., 1998). Changes from one zymodeme and schizodeme to another seem to occur (Carneiro et al., 1990) also if using cloned parasites (Alves et al., 1993). Recently, subpopulations of Try. cruzi I and Try. cruzi II have been classified (Brisse et al., 2000; O’Connor et al., 2007). In some regions, these haplotypes and/or lineages of Try. cruzi are associated with infections in humans and domestic vectors, humans and sylvatic vectors, and wild mammals and sylvatic vectors (Fernandes et al., 1998, 1999; Herrera et al., 2007; summarized by Vallejo et al., 2009). In Brazil, the generalizing correlation of Try. cruzi I and Try. cruzi II to the sylvatic and domestic transmission cycles, respectively, might be changed by the increasing contact of humans to sylvatic cycles, especially in the Amazon, through which originally sylvatic Try. cruzi strains are introduced into the domestic cycle and originally domestic Try. cruzi into the sylvatic cycles. However, it is possible that the introduced strains do not establish on a long-term scale since they are not only selected by the mammalian host but also by the triatomine (Arau´jo et al., 2009). Evolutionarily, Try. cruzi I seems to originate from an association with opossums and predominates north of Amazonia while Try. cruzi II arose from an association with armadillos and predominates in Southern Cone countries of South America (Yeo et al., 2005). The dominance of Try. cruzi I in human infections north of the Amazon basin is supported by several investigations (e.g. del Carmen Sa´nchez-Guille´n et al., 2006; Falla et al., 2009). This might be an over-simplification, as, for example, in Chile both groups of Try. cruzi occur in sylvatic areas but circulate in different species of rodents (Galuppo et al., 2009). After infection with mixed populations of strains belonging to Try. cruzi I and Try. cruzi II, also in humans and reservoirs a host-dependent selection towards one of the groups seems to occur (Steindel et al., 2008). Clone or strain specificities are not only indicated by different numbers of parasites in the gut, but also by different percentages of metacyclic trypomastigotes (e.g. Schaub, 1989a; Perlowagora-Szumlewicz et al., 1990; Perlowagora-Szumlewicz and Moreira, 1994; Alvarenga and Bronfen, 1997; de Silveira Pinto et al., 1998; Lana et al., 1998; Lima et al., 1999).
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Using two strains from the same locality and Tri. infestans also originating from there, the number of metacyclics differed strongly (Schaub, 1989a). 3.1.4
Trypanosoma rangeli
Try. rangeli is usually restricted to species of the genus Rhodnius (Guhl and Vallejo, 2003), although it has been found in other triatomines (Cuba, 1998). Thereby, its distribution is limited from Mexico in the north to southern Brazil. Similarly to Try. cruzi, strains of Try. rangeli are classified into two groups, KP1þ and KP1, but according to the presence and absence of a specific type of kinetoplast minicircles (Vallejo et al., 2002, 2003; Marquez et al., 2007; Cabrine-Santos et al., 2009). Again similarly, there seem to be specific associations of subpopulations with specific vectors (see Section 4.1). 3.2
DEVELOPMENTAL CYCLE IN THE TRIATOMINES
The cycle of the four species of trypanosomatids in the vector is for the most part very similar, but contains species-specific peculiarities. 3.2.1
Blastocrithidia triatomae
In the homoxenous flagellate B. triatomae, which only colonizes the intestinal tract and the Malpighian tubules, the cycle starts with the ingestion of the droughtresistant cyst. Such a stage is not present in the developmental cycle of the other three trypanosomatids – although it was reported for Try. cruzi before the description of B. triatomae (Silva, 1958) – and is correlated to ultrastructural peculiarities (Schaub and Pretsch, 1981; Schaub et al., 1990a). The encystation, indicated by the outgrowth of the flagellum, does not occur in the stomach, but requires the conditions in the small intestine (Schaub and Pretsch, 1981; Reduth, 1986). Usually more flagellates colonize this region than the rectum (Kollien and Schaub, 1999, 2002, 2003). In established infections, the small intestine harbours about 7 million B. triatomae, the rectum about 3.5 million parasites, of which at most 130,000 are attached at the rectal wall (Kollien and Schaub, 1999). During the growth of the cell to the epimastigote stage, a first encystation can occur. Usually this is a development of full grown epimastigotes and is an unequal division. The resulting daughter cell remains attached to the flagellum and undergoes further divisions. The final encystation stages detach and condense to the cyst. The enormous multiplication of the population in the small intestine and rectum is accomplished by equal divisions of the epimastigotes. Spheromastigotes, their intermediates to and from epimastigotes, and ring-like forms develop in low percentages. The ring form sometimes resembles a trypomastigote with connected ends and can only be identified without doubt in living specimens (G. A. Schaub, unpublished data).
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B. triatomae is transmitted within a population of triatomines via coprophagy, a behaviour necessary to obtain the symbionts. Usually, fresh faeces is ingested, but the drought-resistant cysts in dry faeces can also be the source, if this faeces is liquefied by fresh faeces. In addition, during cannibalism the epimastigote population from the stomach can also be the origin of a new infection (Schaub et al., 1989a). 3.2.2
Trypanosoma conorhini
The development of Try. conorhini, a parasite of rats, is mainly known through the monograph of Morishita (1938). In the vector it starts with the ingestion of blood containing blood trypomastigotes. Generally within 24 h, they transform in the stomach into long epimastigotes and multiply by equal and unequal divisions. After 5 days they colonize the small intestine (following the textbooks of his time, Morishita denominated the small intestine ‘‘hindgut’’), and after 10 days the rectum. During the early stages of infection, the prevailing forms are the epimastigotes, then a-, sphero-, trypomastigotes and all the intermediate forms appear. Metacyclic trypomastigotes originating from epimastigotes and – according to the drawings – presumably also from spheromastigotes develop in the small intestine and rectum 5 and 14 days after infection, respectively, and less frequently in the stomach. They are transmitted to the rat via the infectious faeces or if a bug is the prey of the rat. The transmission to triatomines should apparently follow the same routes as in Try. cruzi (see Section 3.2.3). The importance of cyst-like bodies in the faeces is emphasized (Hoare, 1972), but the drought resistance has not been proved. 3.2.3
Trypanosoma cruzi
Like B. triatomae, Try. cruzi can be transmitted in a population of bugs via coprophagy – but only via fresh faeces – and by cannibalism (Schaub, 1988c). Usually, the development of Try. cruzi in the vector starts with the ingestion of blood trypomastigotes. In the stomach, they immediately are aggregated (Brener, 1972, 1973; Alvarenga, 1974), shorten and develop to spheromastigotes (Brack, 1968). After aggregation, some parasites seem to fuse, enabling a genetic exchange (Brener, 1972). The flagellates are given together with the blood into the small intestine where epimastigotes develop from blood trypomastigotes and spheromastigotes. Small and long epimastigotes multiply by unequal and equal divisions. According to the flow of the intestinal contents, epi- and spheromastigotes are transported within 2–3 days to the rectum, where they also multiply intensively. Epimastigotes take up nutrients in the flagellar pocket. Beside proteases for the intracellular digestion they possess membranebound cysteine proteases and the respective inhibitors (Souto-Padro´n et al., 1990; Santos et al., 2005; Sant’Anna et al., 2008).
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Although only remains of blood are present in the rectum, in regularly fed fifth instars of Tri. infestans 1.5 million flagellates/bug develop there, about three times more parasites than in the small intestine, and about two-thirds are attached to the rectal wall (Schaub and Lo¨sch, 1988; Schaub, 1989a; Kollien and Schaub, 1998a,b). After infecting fourth instars of Tri. brasiliensis and feeding the fifth instar, the rectum even contained up to 10 times more parasites than the small intestine (Arau´jo et al., 2008). Using another parasite/vector system, the isolate mainly colonizes the small intestine (Arau´jo et al., 2007). The infection extends to the Malpighian tubules and the final enlargements, the ampullae (Schaub and Lo¨sch, 1988; Schaub et al., 1989b). In established infections and after ingestion of uninfected blood, the stomach is rarely colonized. However, if nearly all blood has passed to the small intestine, the red colour of the remaining blood changes to brown, indicating a re-flux of digestive enzymes from the small intestine into the stomach. Only then, a population of Try. cruzi re-establishes in the stomach. Although intermediate stages may develop in the small intestine, metacyclogenesis seems to be restricted to the rectum, in established infections yielding up to 50% of the rectal population (e.g. Dias, 1934; Schaub and Lo¨sch, 1988; Schaub, 1989a; Kollien and Schaub, 1997, 1998a,b; Cabral et al., 2001). Metacyclic trypomastigotes develop from spheromastigotes via drop-like forms, from epimastigotes via a translocation of the kinetoplast to the posterior end and – similarly to the encystment of B. triatomae (see Section 3.2.1) – after unequal divisions resulting in an epi- and a trypomastigote daughter cell (Brener and Alvarenga, 1976; Schaub, 1989a). They also seem to develop from spheromastigotes via a ring form that usually is an intermediate form between round forms and epimastigotes (Schaub, 1989a) and might have been classified as vacuolized spheromastigotes in stained smears (Alvarenga, 1974). During metacyclogenesis, the surface coat changes. Whereas the events of the cell cycle of epimastigotes are precisely coordinated (Elias et al., 2007), the change of the surface coat and the internal processes – the transition of the kinetoplast – is not a strictly regulated step-by-step process since the surface labelling with wheat germ agglutinin–bovine serum albumin–gold conjugates varies strongly in the early transition stages (Schaub et al., 1989b). The new surface coat contains different glycoproteins, for example, those which protect the parasite against the complement system in the blood or are involved in the penetration of the mammalian cell (summarized by Nogueira et al., 1975; Gentil et al., 2009). The level of the expression of the respective genes by Try. cruzi can also be quantified in samples from the vector (Cordero et al., 2008). 3.2.4
Trypanosoma rangeli
Usually the infection with Try. rangeli starts via the ingestion of blood trypomastigotes. In the stomach, they transform to epimastigotes and are transported with the blood to the small intestine, where they multiply. Later in the infection,
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they cross the intestinal wall, presumably in the anterior region of the small intestine (de Oliveira and de Souza, 2001). In the haemocoel, they multiply initially as short and then as long epimastigotes, also developing amastigotes within the haemocytes and metacyclic trypomastigotes. They only take up lipophorins and no other proteins (Folly et al., 2003). Only trypanosomes in the haemolymph – not those in the midgut and salivary glands – react with the lectin of Canavalia ensiformis (Con A) (Rudin et al., 1989). Short and long epimastigotes possess different cell surface polypeptides and ecto-phosphatase activities (Gomes et al., 2006). Finally, they penetrate the epithelium of the salivary glands – presumably using a pore-forming haemolytic compound – and are transmitted via the saliva to the new mammalian host (Tobie, 1970; Cuba, 1975; D’Alessandro, 1976; Ellis et al., 1980; Mello et al., 1995; Meirelles et al., 2005). Since Try. rangeli rarely colonizes the rectum, transmission via coprophagy must be less important. A transmission between triatomines occurs via cannibalism to an uninfected bug after ingestion of parasites present in the haemolymph and/or stomach of an infected bug (An˜ez, 1982). Although not demonstrated, Try. rangeli should also be transmitted during cannibalism by an infected bug if parasites are injected with the saliva into the attacked bug.
4 4.1
Effects of the host on trypanosomatids SUSCEPTIBILITY AND REFRACTORINESS
Investigations of the interactions of trypanosomatids and triatomines have to consider the susceptibility and refractoriness of triatomines, that is, whether or not a trypanosomatid establishes in a host and develops metacyclic forms. In all triatomines given a mixture of blood and cysts of B. triatomae or the possibility to acquire the infection via coprophagy, the flagellate establishes and encysts. According to field investigations, these include Tri. infestans, Tri. maculata, Tri. guasayana, Tri. garciabesi and P. megistus (see Section 3.2.1) and in the laboratory Tri. pallidipennis, Tri. sordida, P. megistus, P. lignarius, Dipetalogaster maxima, R. prolixus, R. robustus, R. neglectus and Mepraia spinolai (Correˆa et al., 1977; da Rocha e Silva et al., 1977; de Hubsch et al., 1977; Luz and Silveira, 1984; Schaub, 1988d; Schaub and Breger, 1988, Schijman et al., 2006; G. A. Schaub, unpublished data). In all triatomines, cysts develop. Try. conorhini has been found naturally only in species of the Tri. rubrofasciata complex. However, upon feeding R. prolixus, Tri. infestans, Tri. vitticeps and P. megistus on Try. conorhini-infected rats, the flagellate also develops in these triatomines (Morishita, 1938; Dias and Seabra, 1943). Over 60 species of triatomines have been found naturally infected or have been experimentally infected with Try. cruzi, indicating that probably all species are potential vectors (Schofield, 1994). However, susceptibility varies and
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depends on various factors (Garcia and Azambuja, 1991). In selections for susceptibility and refractoriness of R. prolixus, the third generation shows differences in the intensity of infection, but the percentage of uninfected bugs remains similar (Maudlin, 1976). Using Try. cruzi and Tri. infestans from the same village, even after eight generations of selections Try. cruzi developed in all larvae of the refractory strain, but with fewer parasites than in the susceptible strain (G. A. Schaub, unpublished data). The susceptibility is very important in xenodiagnosis, in which laboratorybred triatomines ingest blood of patients suspected to be infected but with such a low parasitemia that the parasite cannot be found in microscopical blood examinations (Dias, 1940). If the blood contains Try. cruzi, the parasites multiply and can more easily be found after about 3 weeks. However, sometimes parasites do not establish and multiply. Therefore, improvements of the methodology are tested. Using different combinations of triatomine species and/or parasite strains, the prevalences vary (e.g. PerlowagoraSzumlewicz and Mu¨ller, 1982; Perlowagora-Szumlewicz et al., 1988, 1990; Sousa, 1988; de Silveira Pinto et al., 1998; Kollien et al., 1998b; Lana et al., 1998). Feeding epimastigotes of a Try. cruzi strain isolated from Panstrongylus geniculatus to three species of Rhodnius resulted in no infections (Mejı´a-Jaramillo et al., 2009). Also comparisons of xenodiagnoses indicate the superiority of the local vector (Dias, 1940), especially if infection rates of naturally infected rodent hosts are determined (Campos et al., 2007). So far, the only exception is Tri. dimidiata from Costa Rica which sometimes loses the naturally acquired infection (Vargas and Zeledo´n, 1985). A loss of infection also occurs after experimental infections of triatomines (e.g. Brener, 1971; Alvarenga and Bronfen, 1997), but might be caused not only by the insusceptibility of the triatomine, but also by the use of attenuated old laboratory strains of Try. cruzi. In human chronic infections, the locality of the infection is sometimes unknown. Therefore, for xenodiagnosis the largest and most aggressive triatomine, D. maxima, has been suggested (Marsden et al., 1979). However, at least a parallel use of the local vector should be recommended. The interaction of Try. cruzi and the triatomine increases the virulence, not only the passage in the vector from the same locality as the parasite, but also in triatomines foreign to the area of the respective Try. cruzi strain (Lammel et al., 1985; Magalha˜es et al., 1996). However, in such investigations the importance of Try. cruzi strain peculiarities can hardly be separated from effects of the vector (see Section 3.1.3). Try. rangeli develops nearly exclusively in species of the genus Rhodnius (see Section 3.1.4). An invasion of the haemocoel of several species of the genus Triatoma (de Stefani Marquez et al., 2006) indicates a broader range of susceptible species. However, a suboptimal supply with symbionts might have weakened the gut, enabling the invasion. Such an effect also has to be considered in invasions of the haemocoel by Try. cruzi (Lacombe and dos Santos, 1984).
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The refractoriness of Tri. infestans for infections by Try. rangeli is based on several mechanisms, for example, the salivary glands possess compounds which lyse Try. rangeli and are absent in the salivary glands of R. prolixus (Grego´rio and Ratcliffe, 1991a). Investigations of susceptibility are more complicated with Try. rangeli, because in contrast to Try. cruzi, long in vitro cultivation often induces an attenuation. Thereby, the parasites do not penetrate the intestinal wall or do not invade the salivary glands and have to be inoculated intracoelomically. Similarly to Try. cruzi, a high adaptation between the flagellate and the local vector seems to exist, resulting in higher rates of infection and invasion of the salivary glands (Machado et al., 2001). In addition, the Try. rangeli KP1 subpopulations seem to be specifically adapted to Rhodnius pallescens, R. colombiensis and R. ecuadoriensis, those of the KP1þ subpopulations to R. prolixus and R. robustus (summarized by Vallejo et al., 2009). 4.2
EFFECTS OF PH, OSMOLALITY AND IONIC COMPOSITION
Although strong changes of these parameters occur in stomach and rectum (see Section 2.4.2), changes in the developmental stage of trypanosomatids could not be correlated to a single factor (see Section 4.5.4). 4.3 4.3.1
EFFECTS OF THE BORDER FACE
Effects of the border face of the intestine
The trypanosomatids of triatomines do not colonize the foregut, only the midgut and hindgut. In ultrastructural investigations of infected small intestines, all trypanosomatids are in intimate contact with the perimicrovillar membranes (Jensen et al., 1990; Kollien et al., 1998a; Gonzalez et al., 1999; de Oliveira and de Souza, 2001). In these interactions, no ultrastructural modifications of the flagellum of trypanosomatids are evident. A direct attachment to the cells of the midgut wall is only achieved by B. triatomae. Then the flagellum is enlarged, sometimes resembling a flagellapodium, enabling an interdigitation with the microvilli (Jensen et al., 1990). The importance of the perimicrovillar membranes for the development of Try. cruzi and Try. rangeli is shown in the detailed investigations of the group of P. Azambuja and E.S. Garcia (summarized by Garcia et al., 2007). Epimastigotes of Try. cruzi attach via glycoinositolphospholipids at their surface to the luminal surface of the small intestine of R. prolixus (Alves et al., 2007; Nogueira et al., 2007). Decapitation and giving antiserum against the membranes and midgut tissue strongly affects Try. cruzi, and after 10 days parasites are present only occasionally (Gonzalez et al., 2006; Alves et al., 2007). The flagellate develops only up to 15 days in the intestine of decapitated insects, and no metacyclics are present in the rectum. The effect on the population is partly
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rescued by a subsequent feeding of ecdysone, but again, no metacyclogenesis occurs (Cortez et al., 2002). The effects of hormonal disturbances are also emphasized using phytochemicals, for example, lignoids and azadirachtin (Rembold and Garcia, 1989; Azambuja and Garcia, 1992; Gonzalez and Garcia, 1992; Cabral et al., 1999; Gonzalez et al., 1999; Garcia and Azambuja, 2004). In the rectum, peculiarities of colonization are evident. Try. cruzi prefers the cuticle and only about 30% of the rectal population are free in the lumen (Schaub and Lo¨sch, 1988). In infections with B. triatomae and Try. cruzi, both parasites initially colonize the four rectal pads (Zeledo´n et al., 1977, 1984, 1988; Bauer, 1984; Bo¨ker and Schaub, 1984; Schaub and Bo¨ker, 1986a). In established infections, about 30–60% of the attached population of B. triatomae and about 50% of Try. cruzi are localized on the rectal pads (Schaub and Lo¨sch, 1988; Kollien and Schaub, 1997, 1998b, 1999), which cover – roughly estimated – only about 20% of the rectal surface. This preference could be due to the location directly at the entrance of the pylorus/ileum into the rectal sac, thereby offering direct access to new intestinal contents after the passage into the rectum. However, this preference is also evident in longterm starved bugs (see Section 4.5.2), in which content is rarely forwarded. Therefore, other factors seem to be responsible for this colonization. One factor might be also of nutritional nature. Although the absorption processes in the rectum of triatomines have not been investigated, the very thin epicuticle of that region and absorption processes at similar cells of other insects suggest this (see Section 2.4.1). Thereby, the flagellates would colonize a region with a very intense flow of amino acids. Another factor might be the relative stability. Whereas the cuticle of the main rectal sac is folded and unfolded due to the filling state of the rectum and defecation, the region of the four rectal pads remains relatively unchanged, since the four flat ampullae at the end of the Malpighian tubules are of similar size as the pads and are located at the haemocoelic side of the rectal wall (Lacombe, 1957). This should stabilize the rectal pads. Several mechanisms have been proposed to explain attachment of flagellates to the cuticle, for example, via lectins to chitin residues (summarized by Schmidt et al., 1998). Using wheat-germ lectin for the detection of chitin, asialofetuin for galactose-binding lectins, and heparin for heparin-binding receptors, chitin is proved only in the procuticle, which is very thin at the rectal pads and covered by the epicuticle and a superficial layer. Therefore, chitin is not accessible for Try. cruzi. In addition, no carbohydrate–lectin interaction between the rectal wall and the flagellates could be detected. Using the fluorochrome Nile red for staining of lipids and hexane for the extraction of lipids, the superficial layer at the luminal surface of the rectum was shown to be covered by a wax layer (Schmidt et al., 1998). The use of hexadecane droplets identifies a so far unrecognized small region near the tip of the flagellum of epimastigotes (Kleffmann et al., 1998). Thus, the initial
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attachment is based on the hydrophobicity of the wax layer and is restricted to the small region of the flagellum. This was verified using different hydrophobic and hydrophilic substrates. The mechanism of attachment allows a gentle separation of trypomastigotes from epimastigotes (Kleffmann et al., 1998). Furthermore, the attachment enhances the transformation of epimastigotes to trypomastigotes in hydrophobic, wax-coated culture vessels. The importance of attachment in this crucial step of the development of Try. cruzi is also emphasized by Bonaldo et al. (1988). Strain-dependant differences in the attachment of Try. cruzi to the rectum of different triatomines seem to be caused by differences in the composition of the rectal cuticle and result in different metacyclogenesis rates. Upon incubating recta of two species of triatomines, R. neglectus and Tri. pseudomaculata, with epimastigotes of Try. cruzi strain Y and strain Berenice, epimastigotes of the latter strain adhere better to the recta from R. neglectus than to recta from Tri. pseudomaculata, corresponding to a higher metacyclogenesis rate in vivo (Carvalho-Moreira et al., 2003). The Try. cruzi strain Y also develops more metacyclic trypomastigotes in R. neglectus but shows no differences in the attachment rate to the rectum of the two triatomines in vitro. After the initial attachment to the rectal wall, the flagellum of epimastigotes of B. triatomae and Try. cruzi is modified. Located at the top of a carpet of flagellates, the flagellum of epimastigotes of B. triatomae is enormously elongated (Schaub and Bo¨ker, 1986a). At the attachment site to the rectal cuticle, epimastigotes of both species develop enlargements of the flagellum (Schaub and Bo¨ker, 1986a,b; Zimmermann et al., 1987; Kollien et al., 1998a).
4.3.2
Effects of the border face of the salivary glands
Only Try. rangeli is transmitted via the saliva and has to reach the lumen of the salivary glands. Therefore, the first border face is the basal lamina. Investigating the distribution of carbohydrate moieties on the tissues of R. prolixus and Try. rangeli and the interactions of both via incubations in different sugars, N-acetylD-glucosamine, N-acetyl-D-galactosamine and galactose show the highest inhibitory effect (Basseri et al., 2002). Thus, lectins or these sugars on the surface of the long epimastigotes of Try. rangeli might interact with the corresponding sugars or lectins of the basal lamina of the salivary glands in the adhesion before invasion. Then the epimastigotes penetrate the host cell with the flagellum (Meirelles et al., 2005), similarly to the interaction of B. triatomae with host cells (Reduth et al., 1989; Schaub et al., 1990a). The molecular interactions with the inner surface of the salivary glands have not been described in detail. After penetration, the long epimastigotes attach to the microvilli developing no flagellar adaptations. The short metacyclic trypomastigotes swim free in the saliva (Hecker et al., 1990; Meirelles et al., 2005).
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203
EFFECTS OF MICROORGANISMS AND ANTIMICROBIAL COMPOUNDS
Although the intestinal tract is colonized by many different bacteria and fungi, an effect of non-symbiotic bacteria on the flagellates has only been considered for S. marcescens. Using a colony of R. prolixus which is infected with the bacterium and a subsequent infection with epimastigotes of Try. cruzi strain Y and strain Dm28c, the population density of strain Dm28c in the stomach remains unchanged, whereas the density of strain Y decreases (Azambuja et al., 2004, 2005a). In incubations of the bacterium with these Try. cruzi strains in vitro, the same differences arise. Using different strains of S. marcescens, only the strain which produces the red pigment kills Try. cruzi strain Y. Also Try. rangeli is lysed after the development of long filamentous structures that connect the bacteria with the parasites (Castro et al., 2007a,b). The effects of symbionts on the trypanosomatids are totally under-investigated. According to the only investigation of the development of Try. cruzi in aposymbiotic and symbiotic R. prolixus, the initial development of the flagellates is stronger in bugs containing symbionts, but after a longer period stronger in aposymbiotic bugs (Mu¨hlpfordt, 1959). However, the percentages of the broad and slender epimastigotes, amastigotes and metacyclic trypomastigotes remain unaffected. The importance of symbionts is also indicated using B. triatomae. In Tri. infestans, a supplementation of blood with B-group vitamins (folic acid, nicotinic acid, pantothenic acid, pyridoxine, riboflavin and thiamine) supports the initial development of B. triatomae in the small intestine of young instars, but not in the rectum (Jensen and Schaub, 1991). Possible effects of antibacterial compounds of triatomines on their respective trypanosomatid have not been investigated (Azambuja et al., 1998). However, antibacterial compounds not belonging to the repertoire of triatomines affect the development of Try. cruzi in vitro, for example, mellitin (Azambuja et al., 1989a). In addition, the production of the lepidopteran cecropin by transformed symbionts kills all Try. cruzi in the gut (Durvasula et al., 1997; Beard et al., 2002). The development of flagellates in bugs with a knockdown or overexpression of antimicrobial compounds like lysozymes or defensins or the 7- and 25-kDa compounds of the haemolymph or enzymes of the immune system like the prophenoloxidase or nitric oxide synthase remains to be considered. It is unknown whether or not compounds of the mammalian host ingested in the blood, for example, complement and antibodies, act on the flagellates during the initial transformation in the stomach after the ingestion of infectious blood. However, in old established infections, in which the stomach has been re-colonized from the small intestine (see Section 3.2.3), the epimastigotes there are killed by the complement system in the blood of chicken, but not by the weak complement system in the blood of mice (Schaub, 1988c). After such a feeding on mice, epimastigotes of the stomach population are able to bind plasminogen from the blood meal (Rojas et al., 2008). Whether or not this acquired proteolytic activity is advantageous for the parasite remains to be
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investigated. In vitro, decomplemented sera of mice or rabbits previously immunized with homogenates of epimastigotes of Try. cruzi, agglutinate epimastigotes and induce ultrastructural damages (Ferna´ndez-Presas et al., 2001). 4.5 4.5.1
EFFECTS OF DIGESTION, DIGESTION PRODUCTS AND EXCRETION
Effects of digestion
In the initial development, all four trypanosomatids strongly colonize the small intestine, and the excystation of B. triatomae is restricted to this part of the intestine (see Section 3.2.1), the main region of digestion of triatomines. Thus, the trypanosomes must possess a refractory surface, a rapid shedding of attached proteases or inhibitors of digestive enzymes. An indication for the latter is the identification of chagasin, a cysteine protease inhibitor, at the surface of Try. cruzi (Monteiro et al., 2001; Ljunggren et al., 2007). A recombinant form inhibits cysteine proteinases of an insect pest of beans (Monteiro et al., 2008). Chagasin might act against cathepsins in the direct neighbourhood of the parasite or in the lumen of the gut (see Section 5.3.4). However, the developmentally regulated expression of chagasin is inversely correlated with that of papain-like cysteine proteases, cruzipains, and the inhibitor is present at lower levels in the epimastigotes, the major form in the gut, than in tissue culture trypomastigotes (Monteiro et al., 2001). In epimastigotes, it might mainly regulate the endogenous cruzipain. Inhibition of the activity of cathepsin B of R. prolixus by a supplementation of blood with pepstatin does not affect the population density or metacyclogenesis of Try. cruzi (Garcia and Gilliam, 1980), and it remains to be investigated whether or not proteases of Try. cruzi are upregulated in these triatomines. Whereas triatomines and Try. cruzi seem not to compete for major nutrients of the blood, there is a strong competition for sialic acid (summarized by Amino et al., 1995). The acquisition of sialic acid appears to be important for the survival in the mammalian host, and Try. cruzi possesses a trans-sialidase to transfer sialic acid to its surface. However, in Tri. infestans, a strong sialidase is mainly active in the stomach, rapidly desialylating the blood cells. Thereby, the epimastigotes in the midgut are poorly sialylated, an indication that sialic acid is not required for the development in the intestine (Amino et al., 1995). The reason for the high levels of the trans-sialidase in stationary phase epimastigotes in the rectum remains to be clarified. 4.5.2
Effects of starvation
Strong effects on the flagellates are induced by the concentration of digestive products due to starvation or blood ingestion. Starvation not only affects the number of parasites, but also the development of stages. In established B. triatomae infections of fifth instars of Tri. infestans, which have been totally
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engorged in the fourth instar, a short-term starvation of 1 month induces no strong effects (Kollien and Schaub, 2002). However, after an additional 3 months, the population in the small intestine is reduced by about 45%, and 30% of the mastigote stages in the small intestine and 99% in the rectum are dead. Whereas the composition of the population in the small intestine – 90% epimastigotes, 7% cysts and 3% spheromastigotes – does not change, in the rectum starvation induces a strong encystation and a change from about 30% to nearly 90% cysts, 10% epimastigotes and some spheromastigotes (Kollien and Schaub, 2002). For species of the genus Trypanosoma, a general observation for Try. conorhini is mentioned that the number of flagellates decreases without a second feeding (Morishita, 1938). More details are only available for Try. cruzi. In the small intestine of Tri. infestans, starvation periods of 3 or 4 weeks reduce the number of Try. cruzi, and dead flagellates are present (Schaub, 1989a; Schaub and Lo¨sch, 1989). However, even in bugs which have died of starvation, some Try. cruzi are still alive (Schaub and Lo¨sch, 1989). Upon a later investigation, 2 months after the last feeding, the population in the small intestine was eliminated (Kollien and Schaub, 1998a). At that time, the rectum still contained 130,000 parasites, but 12% were dead. The population there decreased, 4 months after the last feeding to only 1% of the initial population. However, a total elimination never occurred. In Tri. dimidiata from the field, starvation induced an elimination of the infection in more insects than in the group fed regularly (Vargas and Zeledo´n, 1985). A decrease of the population density of Try. cruzi is also evident in scanning electron microscopy (Schaub and Bo¨ker, 1986b): Whereas the colonization pattern throughout the first 16 weeks after feeding is similar (minimal around the entrance into the rectum, highest on the rectal pads and similar in the other regions of the rectal sac), 4 weeks later, many regions are free of flagellates. However, a residual population always remains attached to the rectal pads. In the addition, the composition of the population changes (Kollien and Schaub, 1998a). Whereas in well fed bugs only up to 2% of the population are spheromastigotes, the percentage of this stage and its intermediate forms increases to about 20% at 2 and 3 months after the last feeding (Kollien and Schaub, 1998a). An effect of starvation on the metacyclogenesis rate occurs if the bugs are fed once on an infected mammal and subsequently starved. Then, metacyclics are scarce (Piesman and Sherlock, 1985). 4.5.3
Effects of blood ingestion and excretion
Not only a reduction of the available nutrients affects the flagellates in the intestine, but also blood ingestion and the resulting excretion. In B. triatomaeinfected fifth instars of Tri. infestans, the number of parasites in the small intestine increases 16-fold within 15 days after feeding, but only 1.5-fold in the rectum (Kollien and Schaub, 2003). However, in the rectum feeding initially
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induces a washing out of 93% of the population present there before feeding. This reflects the high population density of this flagellate in the lumen – 2 million flagellates – and the fact that only 4% of the population are attached to the rectal wall. Since the ‘‘carpet’’ of flagellates consists of about five layers (Schaub and Bo¨ker, 1986a), there seems to be no free attachment area. In Try. cruzi-infected Tri. infestans, feeding might affect the population in the stomach (see Section 4.4); in the small intestine, the population density increases (Schaub, 1989a). In bugs ingesting different amounts of blood, more epimastigotes develop in those having ingested more blood (Asin and Catala´, 1995). Direct effects of blood ingestion were only investigated for the rectal population. Similarly to B. triatomae, the majority of the population in the lumen is washed out by the urine but only a lower percentage of the attached population (Schaub and Bo¨ker, 1987; Schaub and Lo¨sch, 1988; Kollien and Schaub, 1997). However, Try. cruzi never develops such high densities as the homoxenous flagellate and 50–70% of the population are attached to the rectal wall. The phenomenon that the percentage of metacyclic trypomastigotes is low in the first drop of faeces (which however in total contains 1000–25,000 metacyclics) and that the urine often contains pure populations of metacyclics presumably is based on the inability of trypomastigotes to attach (Zeledo´n et al., 1977, 1984, 1988; Bo¨ker and Schaub, 1984; Schaub and Bo¨ker, 1987; Schaub and Lo¨sch, 1988; Zeledo´n, 1997). After the development of a thick surface coat also Try. brucei does not attach to the microvilli of the salivary gland of tsetse (Tetley and Vickerman, 1985). Metacyclics of Try. cruzi lying on the carpet or in the upper layers of the carpet are washed out by the urine. These metacyclics invade the skin lesions or mucous membranes of the mammalian host and initiate the infection (Schuster and Schaub, 2000). In addition to the changes of the population density, blood ingestion by the bug also strongly affects the composition of the population. The percentages of spheromastigotes and their intermediate forms is reduced from about 20% – the starvation effect – to the normal level of about 2–3% (Schaub and Lo¨sch, 1988; Kollien and Schaub, 1997). In addition, metacyclogenesis is induced (see Section 4.5.4). The reduction of the population density by the washing out of the population in the lumen also occurs after feeding of long-term starved bugs, that is, in fifth instars which have starved for 60 days (Kollien and Schaub, 1998b). More evident are the changes of the composition of the population. In the starved bugs about 30% are spheromastigotes and the respective intermediate stages, 20% epimastigotes and 30% trypomastigotes. One day after feeding, these forms represent 2%, 70% and 10%, respectively, and a new stage, giant cells, has appeared, so far rarely seen. In the following 2 days, the percentage of this form increases to 30–50% of the total population, and then it disappears nearly completely. Giant cells contain many nuclei, kinetoplasts and flagella and have been found in the initial development after the infection (Brack, 1968; Alvarenga, 1974). The strong improvement in the nutritional conditions seems to induce the generation of cytoplasm and cell organelles without cell division
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processes. In vitro, such an uncoupling was induced by vinca alkaloids (Grellier et al., 1999). The strong decrease of the percentages of spheromastigotes in the first day after feeding without an adequate increase of the percentages of giant cells but with an increase in those of epimastigotes and the following decrease of the percentages of epimastigotes and an increase of those of giant cells indicate that the majority of giant cells or all originate from epimastigotes (Kollien and Schaub, 1998b). 4.5.4
Induction of metacyclogenesis
In the triatomines, metacyclogenesis already starts a short period of time after colonization of the rectum, about 1–2 weeks after infection, and the percentages increase with prolonged periods of infection (e.g. Schaub, 1989a). An interesting phenomenon is the induction of metacyclogenesis after blood ingestion (Schaub and Lo¨sch, 1988; Kollien and Schaub, 1997). This is important for the population of Try. cruzi. Since the urine washes out the population in the lumen, only those parasites can continue the development in the mammalian host which possess the surface of the metacyclic trypomastigote. A rapid induction of metacyclogenesis increases the chance for the continuation of development. Whereas unequal divisions and ring forms are rarely found in stained smears of the rectal content before blood ingestion, ‘‘drop-like’’ and slender forms each make up about 50% of the intermediate forms. These percentages change rapidly within the first four drops of faeces, and then only slender intermediate forms originating from epimastigotes are present (Schaub and Lo¨sch, 1988). This also occurs in vitro in incubations of the isolated complex of the rectum and the four Malpighian tubules in physiological saline and the induction of diuresis by the artificial diuretic hormone 5-hydroxytryptamine (Kollien and Schaub, 1997). Hence, the inducing factors must originate from the urine rather than from the haemolymph or small intestine. This interpretation is supported by incubations of pieces of recta with attached Try. cruzi either in saline, or with faeces or urine of triatomines. Within 4 h, metacyclogenesis is increased only in incubations with urine, not with a mixture of remains of digestion and urine deposited in the first drop after feeding (Kleffmann, 1999). The elucidation of the mechanisms in the crucial step in the development of Try. cruzi, metacyclogenesis, has been the topic of many in vitro investigations. Upon supplementing Grace medium with extracts of the small intestine or stomach of adult Tri. infestans, dissected 24–48 h after feeding, metacyclogenesis is induced between the fourth and sixth day of incubation (Isola et al., 1981). Extracts of adults fed 3 weeks before the dissections are inactive. Metacyclogenesis is also induced within 15 min after incubation in Grace medium supplemented with extracts of the rectum, but inhibited in the presence of ADP-ribosyltransferase inhibitors (Isola et al., 1986, 1987). Fraidenraich et al. (1993) identified a 10-kDa peptide, which is present in the rectum of
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fifth instars and adults 2 days after feeding on chicken and increases the activity of the adenylate cyclase of Try. cruzi and thereby induces metacyclogenesis, being identified as a fragment from the amino terminus of chicken aD-globin. The concentration of this peptide in the rectum decreases during the subsequent days after feeding. In addition, after feeding on mice, an active compound of similar capacity is present in the rectum. The origin of the aD-globin requires further investigations since 24–48 h after feeding urine or uric acid granules are present in the rectum, but no dark remains of digestion of haemoglobin (see Section 2.3). Using synthetic peptides corresponding to different parts of the 90 amino acid residues of the aD-globin fragment, the peptide corresponding to residues 1–40 at the amino terminus possesses the highest activity at concentrations higher than 10 10 M. Pure chicken haemoglobin is inactive. A peptide with residues 41–73 is also inactive, but enhances the effect of the other peptide (Fraidenraich et al., 1993). Upon feeding blood or plasma with different concentrations of haemoglobin and the synthetic peptides to infected R. prolixus, the percentage of metacyclics increases at higher concentrations of haemoglobin (Garcia et al., 1995). However, pure blood is more efficient. The peptide with residues 41–73 which is inactive in metacyclogenesis in vitro inhibits the rate of metacyclogenesis in the bug. Peptides with the residues 30–49 and 35–73 induce a lower rate of metacyclogenesis than blood, but to the same extent if fed together. Since the aD-globin should also be present in the small intestine in which haemoglobin is digested, but where metacyclic trypomastigotes rarely develop (Schaub, 1989a), other factors must also be necessary in the rectum. One possibility is the attachment to the cuticle, since attachment is necessary for supporting metacyclogenesis in vitro (see Section 4.3). Explaining these differences in the experiments using urine of triatomines and rectal content containing aD-globin will require further investigations. Presumably urine enhances rapid metacyclogenesis in those epimastigotes which have started it. The aD-globin might be responsible for metacyclogenesis of epimastigotes after recovery of the population from the loss of a major part due to defecation. The effects of urine were mimicked in detailed investigations of the group of S. Goldenberg. Harvesting epimastigotes in the late exponential growth phase, short before an increase in the number of metacyclics, and incubating them for 2 h in ‘‘artificial urine’’ and then in this medium supplemented with glutamate, aspartate, proline and glucose, strongly increases metacyclogenesis rates (Contreras et al., 1985a,b), most intensively in a specific strain of Try. cruzi (Dm28c) (Contreras et al., 1988) and less strongly in other strains (G. A. Schaub, unpublished data). The ‘‘artificial urine’’ mimicks the nutritional stress induced in the triatomine by the urine, but has another pH, other ionic strengths and contains neither amino acids nor peptides (Kollien et al., 2001). In addition, in the in vitro assays glucose is necessary (Tyler and Engman, 2001). The presence of this carbohydrate in the rectum of a triatomine is doubtful.
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An important difference is the period of time: urine induces metacyclogenesis within 15 min (Schaub and Lo¨sch, 1988), but the in vitro system requires an incubation for 2 h in the ‘‘artificial urine’’ and a subsequent incubation in the enriched medium. However, the reproducibility and good timing of events enables the identification of steps in this cAMP-mediated process and of genes specifically expressed during metacyclogenesis (e.g. Gonzales-Perdomo ´ vila et al., 2001). et al., 1988; Krieger and Goldenberg, 1998; A In addition to these peptides and chemically defined media, also free fatty acids induce metacyclogenesis, especially oleic acid (Wainszelbaum et al., 2003). Using concentrations similar to those found in the intestinal tract, not the usual adenylate cyclase pathway (Parsons and Ruben, 2000), but protein kinase C isoenzymes are translocated to the membrane of culture-derived epimastigotes (Belaunzara´n et al., 2009). Also in these investigations, metacyclogenesis is induced in epimastigotes. Whereas several factors have been found to induce metacyclogenesis in epimastigotes, so far there is no indication which factors might induce metacyclogenesis via the other routes from spheromastigotes, ring-like forms and the unequal divisions. 4.6
EFFECTS OF OTHER SOLUBLE FACTORS
Detecting a possible effect of factors of components from the saliva or the Kazal-type inhibitors from the stomach on Try. cruzi will require investigations using knockdown or overexpressing triatomines. So far experiments using the RNAi technique have not considered infected bugs. However, some effects in the stomach are striking and perhaps are responsible for the susceptibility or refractoriness of the respective species or population of triatomines for a specific strain of Try. cruzi (see Section 4.1). Comparing the agglutination and lysis of three strains of Try. cruzi by extracts of the stomach of R. prolixus, two strains, Dm28c and Cl, are agglutinated but not lysed and establish in the bugs. Strain Y is not agglutinated but is lysed and is unable to establish (Mello et al., 1996). Since epimastigotes of the strains Cl and Y do not react with peanut agglutinin – a criterion for the classification into Try. cruzi Z1 (Schottelius, 1982) – the agglutination is not correlated to strains of one of the two major groups of Try. cruzi. A lectin of the intestinal tract is suggested to interact with a major surface glycoprotein, GP72, since monoclonal antibodies against GP72 strongly inhibit metacyclogenesis of epimastigotes (Snary, 1985). After deletion of the gp72 gene, the faeces of Tri. infestans contains less than 1% of parasites present in the faeces of bugs infected with a wild-type strain (Basombrı´o et al., 2002). In infections of Rhodnius with Try. rangeli, trypanolytic factors in the haemolymph interact with the parasite (Grego´rio and Ratcliffe, 1991a,b; Mello et al., 1995). In addition, the differences of the susceptibility of different species of Rhodnius for Try. rangeli KP1þ and KP1 isolates depend on these factors, for example, haemolymph of R. prolixus lyses Try. rangeli
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KP1 isolates but not Try. rangeli KP1þ isolates (Pulido et al., 2008). A haemolymph galactoside-binding lectin from R. prolixus affects the survival of short, but not long epimastigotes (Mello et al., 1999).
5 5.1
Effects of trypanosomatids on triatomines CLASSIFICATION OF PATHOGENICITY AND ACTION OF SECONDARY STRESSORS
The classification that ‘‘the trypanosomatids are probably all parasitic’’ (Vickerman, 1976) and the definition of parasites as organisms that affect the host which belongs to another species (Wu¨lker and Schaub, 2002) implies pathological effects of parasites on the host. However, pathological effects are evident in less than 30 trypanosomatid-insect systems (summarized by Schaub, 1992) and not even all Trypanosoma sp. are pathogenic for the respective vertebrate host, for example, Try. rangeli is classified to be a ‘‘harmless parasite of man and a variety of wild and domestic animals’’ (D’Alessandro, 1976). This discrepancy can be solved by using the term ‘‘subpathogenic’’ instead of ‘‘apathogenic’’ or ‘‘non-pathogenic’’ for those species of trypanosomatids for which no pathological effects are known. According to the definition of this term, no effects are obvious under optimal conditions and infections induce only adverse effects if a second synergistic stressor is present (Schaub, 1989b, 1992). Under optimal feeding conditions, an increase in the number of blood feeds and/ or the volume of ingested blood compensates the metabolite losses of triatomines to the parasite. Secondary stressors regularly occur in natural populations. Such populations are often subjected to adverse biotic and abiotic stress factors, for example, predators, competitors, availability of food, temperature and humidity. Effects of pathogenic trypanosomatids can be enhanced by such stressors, whereas effects of subpathogenic trypanosomatids can only be recognized under stress conditions. Synergistic actions have to be considered, if the mortality rates in groups of larvae of triatomines are >20%. Handling stress, a factor to which bugs react very sensitively, is especially important (summarized by Schaub, 1988d; Schaub and Breger, 1988). Effects of the pathogenic B. triatomae on Tri. infestans are increased by a maintenance in critical group sizes. Since triatomines often stay close together like many non-predatory Hemiptera, a maintenance isolated singly should affect them. Whereas the development of only a minor proportion of uninfected singly isolated bugs is delayed in comparison to larvae maintained in groups of 20, 30 or 40 bugs, more infected isolated bugs show a delayed development than infected bugs maintained in groups (Schaub, 1990b). Beside this isolation effect, a much stronger crowding effect on development is evident by a maintenance of groups of 50 larvae in 1-l beakers. Also the mortality rates increase by about 20–75%.
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An enhancement of the pathogenicity also reduces the starvation capacity. After an infection in the first instar and a last feeding in the second, third or fourth instar, the mean starvation resistance period is reduced, respectively, by 51%, 55% and 32% relative to uninfected bugs (Schaub and Lo¨sch, 1989). In the most resistant stage, the fourth instar, uninfected larvae survived up to 432 days after feeding in the third instar, whereas the last bug in the B. triatomae-infected group survived 140 days. The effect on starvation resistance is the only unequivocal case of a synergistic effect of Try. cruzi and a second stressor. In the same experimental design as in the study with B. triatomae, after the last feed in the second, third and fourth instar, which allows the development to the next instar, the mean starvation resistance of third, fourth and fifth instar larvae is reduced, respectively, by 3%, 14% and 32% relative to uninfected bugs. Again more food remnants are present in the intestine of infected than in uninfected bugs. This indicates that not the availability of proteins like haemoglobin determines the starvation capacity, but the concentration of essential metabolites for which trypanosomatid and vector compete (see Section 4.4). 5.2
PATHOGENICITY OF BLASTOCRITHIDIA TRIATOMAE AND TRYPANOSOMA RANGELI
Both parasites induce a complex sickness syndrome in triatomines, but are pathogenic or apathogenic to just the opposite species of triatomines. Whereas B. triatomae strongly affects species of the genus Triatoma, Tri. rangeli is only pathogenic for species of the genus Rhodnius (Schaub, 1988d, 1992; Schaub and Breger, 1988). In the latter system, this can be explained by the limited development of Try. rangeli in species outside of the genus Rhodnius, but B. triatomae also develops well in R. prolixus without affecting it (Schaub, 1988d). (Since the effects of B. triatomae are obvious and no effect is mentioned by Carvalho, 1973, the insectivorous Hemiptera Z. leucogrammus is presumably not affected by this flagellate.) It should be emphasized that apart from the effects on feeding behaviour, the other effects of Try. rangeli have been recognized not in animals from the field, but in experimental infections. An artificial combination of strains of parasite and vector may induce effects which do not occur in field infections (summarized by Schaub, 1992). In addition, in laboratory infections handling stress may increase the pathological effects on triatomines (summarized by Schaub and Breger, 1988). 5.2.1
Pathology: Effects on fitness, development and mortality rates
For B. triatomae and Try. rangeli, both trypanosomatids affect the general fitness, resulting in sluggish movements (Grewal, 1957, 1969; Schaub and Schnitker, 1988). Both also strongly increase the period of time required until moulting especially in the older larval stages. Under the respective feeding
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schedule and at 27 C, 50% of B. triatomae-infected first instar larvae of Tri. infestans need 150 days to moult to the adult stage, whereas uninfected same stage larvae need 130 days (Schaub, 1990a). Try. rangeli infections prolong the period of time until this final larval moult of R. prolixus by 10–40% (An˜ez et al., 1987). Also the mortality rate is strongly increased. In groups of Tri. infestans with differential exposure to coprophagic infections with B. triatomae, the larval mortality rates are correlated to the infection rates and range from 20% to 50% in groups with 10% to 50% infected bugs, respectively (Schaub and Jensen, 1990). Since mortality rates in control groups sometimes exceed 20% (summarized by Schaub, 1992) it is not surprising that no pathological effects were recognized in groups with an infection rate of 10% (da Rocha e Silva et al., 1977). The correlation of larval mortality and infection rates is also evident after experimental infections with different concentrations of cysts in the blood (Schaub et al., 1992a). After experimental infections of first instars, only 5– 39% (mean 16%) reach the adult stage (Schaub, 1990a). Especially the older instars are affected. In Try. rangeli infections of R. prolixus, the mortality rates are 18–56% higher than in uninfected controls, and not only the older instars are killed by the infections, but also the first instar (Tobie, 1965; Go´mez, 1967; An˜ez, 1984; An˜ez et al., 1987). A fivefold higher infection dose doubles the mortality rates (Grewal, 1957). B. triatomae also affects the life span of adult Tri. infestans as well as the reproduction rate. Whereas uninfected males and females have mean life spans of 35 and 30 weeks, respectively, the data for infected adults are 12 and 9 weeks, respectively (G.A. Schaub, unpublished data). The number of eggs laid per day, egg weight, hatching rate and weight of the first instars are also reduced by the infection. Together with the reduced life span, the reproduction rate is 95% lower than in uninfected Tri. infestans (G. A. Schaub, unpublished data). After infection of adult R. prolixus with Try. rangeli, the mortality rate in the following 3 months is similar to that of uninfected bugs (Tobie, 1965). In the same observation period, none of 10 uninfected, but four of 30 infected adults died (An˜ez et al., 1987). An intracoelomic inoculation of Try. rangeli, but not of Try. cruzi, reduces the egg production by 60% and the hatching rate by 27% (Watkins, 1969). 5.2.2
Pathology: Effects on behaviour
Such effects have not been considered in investigations of B. triatomae. In Try. rangeli infections of R. prolixus and R. robustus, the bugs probe more often before blood ingestion, feed less frequently, and ingest less blood and at a slower rate than uninfected bugs (D’Alessandro and Mandel, 1969; An˜ez and East, 1984; Garcia et al., 1994). This resembles effects of other parasite-infected vectors, for example, infections of fleas with Yersinia pestis, of Anopheles with
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Plasmodium, of tsetse with salivarian African trypanosomes and of phlebotomines with Leishmania (reviewed in Schaub, 1992, 1994, 1996, 2006). The mechanisms for this parasite-advantageous change of behaviour differ, for example, a blockage or reduction of the diameter of the foregut or an attachment to mechanoreceptive sensilla in the labrum which measure the velocity of the blood flow. In Try. rangeli-infected R. prolixus, the invasion of the salivary glands by the parasites destroys salivary gland tissue – indicated by a less intensive cherry red colour – and reduces the concentration of salivary antihaemostatic components, apyrases and nitrophorins (Garcia et al., 1994). Whereas the apyrases hydrolyse ADP, released from destroyed host cells, and thereby inhibit platelet aggregation, the cherry red nitrophorins act as vasodilators and also inhibit platelet aggregation (see Section 2.4.6). Therefore, the parasitogenic changes of the behaviour can be caused by difficulties in the location of the blood vessel and/or the difficulties during blood ingestion (Garcia et al., 1994). 5.2.3
Pathology: Effects on the composition of haemolymph and intestinal contents
B. triatomae strongly affects the free amino acids in the haemolymph (Schaub et al., 1990b). The concentrations of the majority of amino acids are lower in infected bugs. Most remarkable is the decrease for phenylalanine and tyrosine, the occurrence of detectable concentrations of b-alanine and the increase in the concentration of its possible precursor aspartate. These amino acids are necessary for the sclerotization and melanization of the cuticle (see Section 5.2.4). The changes in the concentrations of the amino acids in the gut do not offer such a direct connection to a developmental process (summarized by Schaub, 1992). In strong infections of R. prolixus with Try. rangeli, the haemolymph is whitish and more copious (Grewal, 1969). After inoculation of Try. rangeli into the haemocoel of R. prolixus, concentrations of total proteins, carbohydrates and total free amino acids decreases, the latter by 27%, whereas it increases in uninfected bugs (Zeledo´n and de Monge, 1966). Similarly to B. triatomae, the changes in the concentrations of individual amino acids varied, some of them increasing strongly, others being reduced (summarized by Schaub, 1992). Noteworthy is the decrease in the concentration of tyrosine (Ormerod, 1967; Watkins, 1969) since also Try. rangeli affects tanning processes (see Section 5.2.4). Interestingly, there are different effects according to the developmental stage of the flagellate. Whereas the short epimastigotes in the initial colonization of the haemolymph induce the activity of metalloproteases, such an activity is suppressed by long epimastigotes (Feder et al., 1999). At the interface with the salivary glands, short epimastigotes inhibit ecto-phosphatase activities more strongly than long epimastigotes (Gomes et al., 2008).
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5.2.4
Pathology: Effects on cuticle and tracheal system
Both flagellates reduce the tanning and sclerotization of the cuticle and in Try. rangeli infections also the pigmentation of the eyes (reviewed by Schaub, 1992). Directly after the moult, the cuticle is soft and pink coloured. Whereas the cuticle of uninfected bugs becomes stiff and dark within 15 min, in some B. triatomae-infected bugs it changes only slightly within 1 day, and all intermediate stages to a normal tanning are present (Schaub et al., 1990b). The retarded or missing sclerotization and tanning presumably result from the competition of flagellate and insect host for the phenylic amino acids (see Section 5.2.3). In Try. rangeli infections, the pale and translucent cuticle can also be caused by the parasite’s development in the epidermal cells (Watkins, 1971a). In B. triatomae infections, effects on the tracheal system of Tri. infestans are indicated by the density of stellate cells located at the end of the tracheal system directly before the tracheoles. In bugs supplemented with the symbiont Nocardia sp. this density is reduced by 5–60%, most strongly at the rectum and least at the small intestine (Eichler and Schaub, 1998). Similar effects are evident in bugs having been infected with the symbiont of R. prolixus, R. rhodnii, and in aposymbiotic bugs. In the latter a supplementation of the blood with vitamin B reverses the effect. However, under resting conditions the oxygen consumption of infected and uninfected bugs is similar (Eichler, 1998). Therefore, an increased ventilation rate compensates the less developed tracheal system. However, this is not efficient enough during energy-dependent processes such as diuresis and moulting. Then, the oxygen consumption of infected bugs is reduced (Eichler, 1998). In Try. rangeli infections, the effect on the tracheal system is attributed to the intracellular development (Watkins, 1971a,b; Schwarzenbach, 1987), but can also be due to the same mechanisms as just mentioned (see Section 5.2.7). 5.2.5
Pathology: Effects on intestine and excretion
Infections with B. triatomae strongly affect the intestine (Jensen et al., 1990; Schaub et al., 1992b). Electron microscopy shows less developed perimicrovillar membranes and a penetration of the unprotected cells of the intestinal wall by the parasite. Finally gut cells are destroyed. Also in cultivations in vitro, which are easily possible in a co-cultivation with a triatomine cell line, the host cells are surrounded by a corona of flagellates which penetrate and destroy the cell (Reduth et al., 1989; Schaub et al., 1990a). Infected bugs often possess a dilated small intestine with red content – an indication of a missing digestion – and the red colour of the haemolymph indicates a leakage of undigested haemoglobin into the haemocoel (Schaub and Meiser, 1990). The effects of Try. rangeli can be attributed to the destruction of the cells through the intracellular development. A multiplication in nerves and muscle
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cells of the gut affects peristalsis, and the gut may burst (Watkins, 1971a). Similar effects occur after blocking the abdominal spiracles. According to ultrastructural investigations the cells of the midgut of infected and uninfected bugs are covered by similar dense layers of perimicrovillar membranes (Schwarzenbach, 1987). Sometimes these cells also show an electron-lucent cytoplasm after penetration of the cells of the anterior small intestine by Try. rangeli (Hecker et al., 1990; de Oliveira and de Souza, 2001). Both trypanosomatids affect the excretion, indicated by a swollen abdomen even days after blood ingestion (Watkins, 1971a,b; Schnitker et al., 1988; Eichler and Schaub, 1998). During the first 24 h after feeding, B. triatomaeinfected fifth instars of Tri. infestans excrete approximately a 2.5-fold smaller volume of urine (Schnitker et al., 1988). Ultrastructural alterations are evident, but neither secretion rates of isolated tubules nor the storage and release of diuretic hormones in the prothoracic and metathoracic ganglionic mass are affected (Schaub and Schnitker, 1988; Schnitker et al., 1988). The discrepancy between the in vivo and the in vitro results might be due to the effects on the tracheal system in the bug and the good oxygen supply in the in vitro investigations (see Section 5.2.4). In Try. rangeli-infected R. prolixus, diameters and in vitro excretion rates of the Malpighian tubules are affected (Watkins, 1971a,b). The latter is caused by damage of the tissue, but also by a reduction of the concentration of diuretic hormones in the meso-metathoracic ganglia or the presence of inhibitors. 5.2.6
Pathology: Effects on the immune system
An effect on cellular immunity in the haemocoel is evident in old B. triatomae infections in which cells rarely attach to pieces of nylon thread for an encapsulation, and no melanization is evident (G. A. Schaub, unpublished data). Also the intestinal homeostasis is affected. After an infection of first instar larvae of Tri. infestans with the symbiont and B. triatomae and feeding with a mixture of blood and Candida sp., Dietzia maris, Escherichia coli or Gordonia rubropertinctus to third instar larvae, the non-symbiotic microorganisms are rarely present in the different regions of the gut of B. triatomae-uninfected fifth instars and regularly in B. triatomae-infected larvae (Eichler, 1998). Also, Try. rangeli affects the cellular immune system (Azambuja and Garcia, 2005). After an injection into the haemocoel of R. prolixus, the number of phagocytic cells increases (Zeledo´n and de Monge, 1966; Mello et al., 1995). In old and heavy infections, the intracellular development results in a decrease of the number of haemocytes (Grewal, 1957; Gomes et al., 2002). After oral infection, Try. rangeli seems to inhibit the release of arachidonic acid and reduces the concentration of an insect platelet-activating factor (iPAF). Thereby, pathways of cellular immunity via eicosanoid and iPAF are depressed (Garcia et al., 2004a,b; Machado et al., 2006; Figueiredo et al., 2008). A component at the interface of cellular and humoral immunity, the
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prophenoloxidase system, is not activated by Try. rangeli, presumably due to an immune suppression (Grego´rio and Ratcliffe, 1991a,b). However, this is only evident after an inoculation of long epimastigotes – resembling the late phase of an infection – whereas the inoculation of short epimastigotes activates the prophenoloxidase system (Gomes et al., 1999, 2003). Agglutinating and trypanolytic factors seem to be more widely distributed in the tissues of the refractile Tri. infestans, than in those of R. prolixus (Grego´rio and Ratcliffe, 1991a,b). An indication for another effect on humoral immunity is the increased lysozyme level in the haemolymph. However, the synthesis of antibacterial peptides is not induced (Mello et al., 1995). After haemocoelic inoculation of a non-attenuated strain of Try. rangeli and a strain, that had lost the capacity to multiply in the vector and to invade the salivary glands, the latter stimulates higher levels of prophenoloxidase and superoxide and nitrites than the non-attenuated strain (Whitten et al., 2001). In both strains, the long epimastigotes induce less prophenoloxidase and superoxide than short epimastigotes. According to the effects of inhibitors, NADPH oxidases and nitric oxide synthases seem to be involved in these immune reactions. Similarly to B. triatomae (see above), Try. rangeli disturbs the intestinal homeostasis, and the numbers of usually eliminated bacteria and fungi increases (Eichler, 1998). In addition, the intestinal levels of nitrite and nitrate – metabolites of nitric oxide – and the expression rate of the gene of the respective enzyme, the nitric oxide synthase, are modulated after infections with blood trypomastigotes of Try. rangeli (Whitten et al., 2007). Already in early-stage infections, Try. rangeli represses the expression of the gene encoding the nitric oxide synthase in the stomach. Then and in mid-stage infections, the expression levels in fat body and haemocytes are reduced. After invasion of the haemocoel, the concentrations of nitrite in the small intestine decrease. In late-phase infections, the expression rate is strongly induced in the rectum (Whitten et al., 2007). In the interaction of Try. rangeli and antimicrobial compounds, not only the effects of nitrogen radicals on the flagellate have to be considered, but also an effect of reactive oxygen species like hydrogen peroxide (H2O2) (CosentinoGomes et al., 2009).
5.2.7
Pathology: Effects on symbionts
In fifth instars of Tri. infestans infected with B. triatomae, the number of colony-forming units of the symbiont Nocardia sp. in the cardia, stomach, small intestine and rectum are reduced by about 90%, 15%, 80% and 80%, respectively, in comparison to uninfected larvae and those infected with Try. rangeli (Eichler and Schaub, 2002). Try. rangeli seems to act directly on the symbionts, since the supernatants of culture media of Try. rangeli inhibit the growth of the R. prolixus symbionts on
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agar (Watkins, 1969). However, media from in vitro cultures of other trypanosomatids, for example, Try. cruzi, must be tested to verify that these effects are specific for Try. rangeli. In infected fifth instars of R. prolixus, 6–10 days after feeding the number of colony-forming units of R. rhodnii is reduced by about 45% in the cardia, 30% in the stomach, 20% in the small intestine and 25% in the rectum in comparison to uninfected larvae and larvae infected with B. triatomae (Eichler and Schaub, 2002). 5.2.8
Mechanism of pathology
The mechanisms of pathology of B. triatomae and Try. rangeli seem to be identical. Both affect the respective symbiont, B. triatomae acting on Nocardia sp. in Tri. infestans and Try. rangeli on R. rhodnii in R. prolixus (Eichler and Schaub, 2002). The sickness syndrome of both parasites is very similar to the effects of aposymbiosis (see Section 2.4.3). At least B. triatomae and Tri. infestans compete for vitamin B (see Section 4.4), and a supplementation of the diet with vitamin B reduces the pathogenicity of B. triatomae (Eichler and Schaub, 1998). However, the number of symbionts is also reduced by a blockage of the spiracles (Eichler, 1998), indicating the strong dependence of the development of symbionts on an adequate oxygen supply. Since the number of end cells of the tracheal system is also reduced in the respective triatomine infected with B. triatomae or Try. rangeli (see Section 5.2.4), the reduced oxygen supply in long-term infected larvae could induce a vicious circle in the pathology of both trypanosomatids. 5.3
SUBPATHOGENICITY OF TRYPANOSOMATIDS IN TRIATOMINES
Since the pathological effects of B. triatomae and Try. rangeli are obvious and since no pathological effects of infections with Try. conorhini were evident in infected bugs, I suggest that this species can be classified as subpathogenic like Try. cruzi. The old investigations have been described in detail previously (Schaub, 1989b), so the present chapter only covers those published afterwards or those connected to recent publications. 5.3.1
Effects on fitness, development and mortality rates
Fitness of infected bugs has not been investigated, but according to the subpathology classification, an effect should only occur after exhaustion periods. Whereas effects on the developmental rate were found only once, three investigations observed no retardation of development of Try. cruzi-infected bugs in relation to uninfected triatomines (summarized by Schaub, 1992). Also, the mortality rates are not affected by Try. cruzi if the groups are maintained under optimal conditions as indicated by having <10% mortality in the controls (Schaub, 1988a,b).
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5.3.2
Effects on behaviour
Since the disturbances of probing increase the transmission rate in different parasite–vector system, this aspect has recently been reconsidered for Try. cruzi-infected R. prolixus and M. spinolai (Takano-Lee and Edman, 2002; Botto-Mahan et al., 2006). However, the data presented should not be used to estimate an increased risk of infection. As emphasized previously (Schaub, 1992), such studies have to consider the competition of trypanosomatids and insect host for the ingested food. Therefore, if infected and uninfected bugs are used at the same time after the moult to the respective instar, the infected bugs are in a more progressive state of starvation. Since starved bugs probe more often and long-term starved bugs ingest less or no blood (G. A. Schaub, unpublished data), the effects attributed to Try. cruzi might have been effects of starvation. Under natural conditions, infected larvae should approach a host earlier than uninfected larvae do. If they ingest blood only once per larval instar, the risk of infecting mammals is identical. However, we should also consider that the vector–subpathogenicity classification of Try. cruzi is based on investigations on Tri. infestans and R. prolixus, and a generalization for all triatomines will require much more investigations. 5.3.3
Effects on composition of haemolymph and intestinal contents
The haemolymph of bugs infected with Try. cruzi has a normal appearance. However, not only gut infections with the pathogenic trypanosomatids affect the composition of the haemolymph, but also those with Try. cruzi (summarized by Schaub, 1992). Although concentrations of individual amino acids are changed, those of phenolic amino acids seem to be unaffected, and therefore tanning and sclerotization are not affected in Try. cruzi infections. After infection the composition of the rectal contents is changed. However, Try. cruzi infections reduce the concentrations of all amino acids in the rectal contents (A. H. Kollien, unpublished data). 5.3.4
Effects on digestion, intestine and excretion
In the midgut, no effects are evident, nor is the crystallization of haemoglobin in the stomach affected, nor correlates the protein digestion by cathepsin B in the small intestine with the density of Try. cruzi (Pick, 1952; Garcia and Gilliam, 1980). However, in a young infection with epimastigotes of Try. cruzi the level of cathepsin D activity is increased 1 and 3 days after infection but thereafter remains on the level of the control (Borges et al., 2006). Not only the triatomine possesses serine and aspartate proteases and the respective inhibitors, but also the flagellate synthesizes these enzymes and inhibitors as membrane-bound proteases (see Section 4.5.1), and the complex interaction of both can only be elucidated in detailed investigations. Another effect in the intestinal tract is evident in the rectum. If during the preparation of samples for electron microscopy the waxy layer is not removed
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by incubations in acetone or ethanol, an insertion of the flagellum between the layers of the wax is visible (Schmidt et al., 1998). Investigations of the composition of the waxy layer in the rectum of infected and uninfected bugs are necessary to recognize whether or not the parasite modifies the attachment site. 5.3.5
Effects on the immune system and symbionts
Implantation experiments by others indicate a strongly reduced cellular immune response of Try. cruzi-infected Tri. infestans (Bitkowska et al., 1982). However, such effects are not evident in our Try. cruzi/Tri. infestans system (G. A. Schaub, unpublished data). Strong effects are also evident for the humoral immune system of the gut. After infections with blood trypomastigotes of Try. cruzi, the expression rate of the gene of the nitric oxide synthase is upregulated in the stomach (Whitten et al., 2007). In addition, the levels of nitrite are increased in the midgut. After an experimental infection via a mixture of blood and the different microorganisms, in long-term Try. cruzi-infected Tri. infestans high numbers of bacteria and fungi develop which are unable to multiply in uninfected bugs (Eichler, 1998). Since the numbers of colony-forming units of the symbionts are similar in infected and uninfected Tri. infestans and R. prolixus (Eichler and Schaub, 2002), the immune mechanisms which regulate the development of symbionts and are active against air-borne bacteria seem to be different for both groups of microorganisms; alternatively, the symbionts are insensitive to these compounds. In the interaction with iPAF (see Section 5.2.6), the production of PAF by Tryp. cruzi has to be considered which triggers in vitro growth of epimastigotes, the secretion of phosphatases and the differentiation of epimastigotes into metacyclic trypomastigotes (Rodrigues et al., 1969, 1999; Gomes et al., 2006).
6
Interactions in double infections
Double infections have rarely been considered. Since B. triatomae multiplies faster in the bug, it suppresses the development of Try. cruzi, even if the bugs have been infected with Try. cruzi in the first instar and with B. triatomae in the second. However, even with the converse infection schedule, some metacyclic trypomastigotes develop (G. A. Schaub and H. Mehl, unpublished data). An interaction between different clones of Try. cruzi in the vector is indicated by the fact that after an infection with a mixture of clones there is a much higher or lower number of flagellates than in single infections (de Silveira Pinto et al., 1998). In such interactions, the selection phenomena of the triatomines have to be considered (see Section 3.1.3). Double infections of Try. cruzi and Try. rangeli regularly occur in bugs from the field (D’Alessandro, 1976). Since both species finally colonize different regions in the triatomine – haemocoel versus gut – a competition is unlikely.
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7
Conclusions
During the last decade, investigations of the physiology of triatomines and trypanosomatids have benefited from new molecular biology and protein biochemistry tools and presented interesting results. However, the present chapter clearly points out the missing investigations of the intestinal milieu in which the parasites develop. Only with these can the effects of trypanosomatids on triatomines and the effects of trypanosomatids on the triatomines be elucidated. Acknowledgements I thank many colleagues for long periods of discussion of these topics, especially Dr. Patrı´cia de Azambuja, Dr. Eloi S. Garcia and Dr. Fernando Genta from the Department of Biochemistry and Molecular Biology and Dr. Ana Jansen from the Department of Protozoology, Fiocruz, Rio de Janeiro. I am much indebted to Dr. Randy Cassada for reading and correcting the English version. Finally, I am deeply grateful for the funding of my investigations and travels by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, the Volkswagenstiftung, the German Academic Exchange Service (DAAD), European Community programmes, the Humboldt Foundation and the Deutsche Forschungsgemeinschaft. References Albuquerque-Cunha, J. M., Mello, C. B., Garcia, E. S., Azambuja, P., de Souza, W., Gonzalez, M. S. and Nogueira, N. F. S. (2004). Effect of blood components, abdominal distension, and ecdysone therapy on the ultrastructural organization of posterior midgut epithelial cells and perimicrovillar membranes in Rhodnius prolixus. Mem. Inst. Oswaldo Cruz 99, 815–822. Almeida, C. E., Francischetti, C. N., Pacheco, R. S. and Costa, J. (2003). Triatoma rubrovaria (Blanchard, 1843) (Hemiptera–Reduviidae–Triatominae) III: patterns of feeding, defecation and resistance to starvation. Mem. Inst. Oswaldo Cruz 98, 367–371. Alvarenga, N. J. (1974). Evoluc¸a˜o do Trypanosoma cruzi no trato digestivo de Triatoma infestans. Ph.D. Thesis, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais, Brazil. Alvarenga, N. J. and Bronfen, E. (1997). Metaciclogeˆnese do Trypanosoma cruzi como paraˆmetro de interac¸a˜o do parasita com o triatomı´neo vetor. Rev. Soc. Bras. Med. Trop. 30, 247–250. Alves, A. M. B., Tanuri, A., de Almeida, D. F. and von Kru¨ger, W. M. A. (1993). Reversible changes in the isoenzyme electrophoretic mobility pattern and infectivity in clones of Trypanosoma cruzi. Exp. Parasitol. 77, 246–253. Alves, C. R., Albuquerque-Cunha, J. M., Mello, C. B., Garcia, E. S., Nogueira, N. F., Bourguingnon, S. C., de Souza, W., Azambuja, P. and Gonzalez, M. S. (2007). Trypanosoma cruzi: attachment to perimicrovillar membrane glycoproteins of Rhodnius prolixus. Exp. Parasitol. 116, 44–52.
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Schaub, G. A. and Lo¨sch, P. (1989). Parasite/host-interrelationships of the trypanosomatids Trypanosoma cruzi and Blastocrithidia triatomae and the reduviid bug Triatoma infestans: influence of starvation of the bug. Ann. Trop. Med. Parasitol. 83, 215–223. Schaub, G. A. and Meiser, A. (1990). Presence of undigested haemoglobin in small intestine and haemolymph of Triatoma infestans (Reduviidae) infected with Blastocrithidia triatomae (Trypanosomatidae). Parasitol. Res. 76, 724–725. Schaub, G. A. and Pretsch, M. (1981). Ultrastructural studies on the excystment of Blastocrithidia triatomae (Trypanosomatidae). Trans. R. Soc. Trop. Med. Hyg. 75, 168–171. Schaub, G. A. and Schnitker, A. (1988). Influence of Blastocrithidia triatomae (Trypanosomatidae) on the reduviid bug Triatoma infestans: alterations in the Malpighian tubules. Parasitol. Res. 75, 88–97. Schaub, G. A. and Wu¨lker, W. (1984). Tropische Parasitosen im Programm der Weltgesundheitsorganisation. Universitas 39, 71–80. Schaub, G. A., Bo¨ker, C. A., Jensen, C. and Reduth, D. (1989a). Cannibalism and coprophagy are modes of transmission of Blastocrithidia triatomae (Trypanosomatidae) between triatomines. J. Protozool. 36, 171–175. Schaub, G. A., Gru¨nfelder, C. G., Zimmermann, D. and Peters, W. (1989b). Binding of lectin–gold conjugates by two Trypanosoma cruzi strains in ampullae and rectum of Triatoma infestans. Acta Trop. 46, 291–301. Schaub, G. A., Reduth, D. and Pudney, M. (1990a). The peculiarities of Blastocrithidia triatomae. Parasitol. Today 6, 361–363. Schaub, G. A., Schmidt, A. and Ullrich, J. (1990b). The effect of moulting and of infection with Blastocrithidia triatomae (Trypanosomatidae) on the concentration of free amino acids in the haemolymph of the reduviid bug Triatoma infestans. J. Insect Physiol. 36, 843–853. Schaub, G. A., Neukirchen, K. and Golecki, J. (1992a). Attachment of Blastocrithidia triatomae (Trypanosomatidae) by flagellum and cell body in the midgut of the reduviid bug Triatoma infestans. Eur. J. Protistol. 28, 322–328. Schaub, G. A., Rohr, B. and Wolf, S. (1992b). Pathological effects of Blastocrithidia triatomae (Trypanosomatidae) on populations of the reduviid bug Triatoma infestans with different infection rates (Heteroptera: Reduviidae). Entomol. Gen. 17, 21–27. Schijman, A. G., Lauricella, M. A., Marcet, P. L., Duffy, T., Cardinal, M. V., Bisio, M., Levin, M. J., Kitron, U. and Gu¨rtler, R. E. (2006). Differential detection of Blastocrithidia triatomae and Trypanosoma cruzi by amplification of 24S-ribosomal RNA genes in faeces of sylvatic triatomine species from rural northwestern Argentina. Acta Trop. 99, 50–54. Schmidt, J., Kleffmann, T. and Schaub, G. A. (1998). Hydrophobic attachment of Trypanosoma cruzi to a superficial layer of the rectal cuticle in the bug Triatoma infestans. Parasitol. Res. 84, 527–536. Schmunis, G. A. (2004). Medical significance of American trypanosomiasis. In: The Trypanosomiases (eds Maudlin, I., Holmes, P. H. and Miles, M. A.), pp. 355–368. CABI Publishing, Wallingford. Schnitker, A., Schaub, G. A. and Maddrell, S. H. P. (1988). The influence of Blastocrithidia triatomae (Trypanosomatidae) on the reduviid bug Triatoma infestans: in vivo and in vitro diuresis and production of diuretic hormone. Parasitology 96, 9–17. Schofield, C. J. (1980a). Density regulations of domestic populations of Triatoma infestans in Brazil. Trans. R. Soc. Trop. Med. Hyg. 74, 761–769. Schofield, C. J. (1980b). Nutritional status of domestic populations of Triatoma infestans. Trans. R. Soc. Trop. Med. Hyg. 74, 770–778.
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Lyme Disease Spirochete–Tick–Host Interactions Katharine R. Tyson*,1 and Joseph Piesman† * Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA † Centers for Disease Control and Prevention, Coordinating Center for Infectious Diseases, National Center for Zoonotic, Vector-Borne and Enteric Diseases, Division of Vector-Borne Infectious Diseases, Fort Collins, Colorado
1 Introduction 243 2 Tick biology and physiology 244 2.1 Argasidae 245 2.2 Ixodidae 246 2.3 Feeding characteristics of ixodid ticks 247 2.4 Host responses to tick feeding 248 2.5 Anti-haemostatic tick salivary components 249 2.6 Anti-inflammatory tick salivary components 255 2.7 Immunosuppressive tick salivary components 259 2.8 Tick midgut components 267 2.9 Tick haemolymph components 270 3 B. burgdorferi biology and interaction with tick vectors 272 3.1 Genome of B. burgdorferi 272 3.2 B. burgdorferi genes that facilitate tick colonization and persistence 273 3.3 Tick proteins that facilitate Borrelia transmission 277 4 Summary 279 Acknowledgements 279 References 279
1
Introduction
Ticks are obligate parasitic blood-sucking arthropods that feed on a wide variety of vertebrate hosts including mammals, birds, reptiles and some amphibians (Dennis and Piesman, 2005; Sonenshine, 2005). Ticks are distributed on virtually every continent, excluding Antarctica, where they are persistent pests of
1
Current Address: Scynexis, Inc., Research Triangle Park, North Carolina, USA
ADVANCES IN INSECT PHYSIOLOGY VOL. 37 ISBN 978-0-12-374829-4 DOI: 10.1016/S0065-2806(09)37005-8
Copyright # 2009 by Elsevier Ltd All rights of reproduction in any form reserved
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livestock and wildlife (Sonenshine, 1991; Jongejan and Uilenberg, 2004). Tick infestations of livestock can cause severe toxic conditions, including tick paralysis, toxicoses, allergic reactions and severe blood loss, which can lead to major economic losses in several countries. In addition to causing direct pathology via the tick bite, ticks are also important vectors of numerous viral, bacterial and protozoan pathogens that cause various diseases in animals and humans. Tick-borne diseases of public health importance include babesiosis, ehrlichiosis, tularemia, Rocky Mountain spotted fever, tick-borne encephalitis, Crimean–Congo haemorrhagic fever and Lyme disease, the most prevalent vector-borne disease in the United States and Europe (Sonenshine, 1991; Dennis and Piesman, 2005; Sonenshine, 2005). Lyme disease is caused by spirochetes of the genus Borrelia (Steere et al., 2004). Three genospecies of Borrelia are primarily responsible for causing Lyme disease: B. burgdorferi sensu stricto in North America and B. afzelii and B. garinii in Europe and Asia. In the United States, Lyme disease occurs in regions along the east and west coast, as well as the upper Midwest, with the majority of cases concentrated in the northeastern regions of the country. The number of reported cases of Lyme disease in the United States has increased steadily over the past 20 years, with a total of 248,074 cases reported from 1992 to 2006 (Bacon et al., 2008). Symptoms of Lyme disease include erythema migrans, the characteristic ‘‘bullseye’’ rash, fatigue, headache, fever, and muscle and joint pain (Steere, 1989). If the infection is left untreated, more severe symptoms, such as Lyme arthritis, can develop in several tissues, including the joints. Neurologic and cardiac sequelae have also been associated with untreated Lyme disease. In this chapter, we describe the molecular biology and physiology of Borrelia and ticks and discuss the crucial interactions that take place between the spirochetes and the vectors that transmit them.
2
Tick biology and physiology
Approximately 900 species of ticks exist worldwide, which are separated into two major families, the Ixodidae (hard ticks) and the Argasidae (soft ticks) (Sonenshine, 1991; Goddard, 1993; Anderson, 2002; Barker and Murrell, 2004; Dennis and Piesman, 2005). A third family exists, the Nuttalliellidae, containing only a single species. The lifecycle of ticks consists of three developmental stages (instars), the larva, nymph and adult (Sonenshine, 2005). Larval ticks have three pairs of legs like insects, while nymphal and adult ticks possess four pairs of legs; this body pattern is basic in arachnids, which are comprised of ticks, mites, spiders and scorpions (Sonenshine, 1991, 2005; Mans and Neitz, 2004). Tick mouthparts consist of three main appendages: the chelicerae, which are toothed organs used for cutting, ripping and tearing skin; the palps, which are sensory organs used during host attachment; and the hypostome, which is
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the barbed mouthpart inserted into the skin during feeding (Sonenshine, 1991, 2005). Ixodidae, or hard ticks, possess a tough, sclerotized plate, the scutum, on their dorsal body surface, which acts as an attachment site for many vital muscle groups (Sonenshine, 1991, 2005; Goddard, 1993). Argasidae, or soft ticks, lack a scutum but possess a tough leathery cuticle that is highly folded, allowing ample growth during feeding. Due to their feeding and moulting characteristics, ixodid and argasid ticks both exhibit long life spans, lasting several years. Even though they share many common features, drastic differences exist between argasid and ixodid ticks. 2.1
ARGASIDAE
Argasid ticks can be subdivided into four main genera, Argas, Carios, Ornithodoros and Otobius, where Argas, Ornithodoros and Otobius ticks are important medical and veterinary pathogen transmission vectors for relapsing fever spirochetes (Jongejan and Uilenberg, 2004; Dennis and Piesman, 2005). As mentioned previously, argasid ticks lack a scutum, containing only a tough, leathery cuticle with many folds, giving rise to the term ‘‘soft ticks’’ (Sonenshine, 1991, 2005; Goddard, 1993). The mouthparts of argasid nymphs and adults are found at the anterior end of the body, but are covered by the body and cannot be seen from the dorsal view. Argasid ticks are typically found in environments very close to their hosts, such as bird nests, bat caves, burrows and dens of various animals, or dilapidated huts and cabins of humans (Mu¨ller-Doblies and Wikel, 2005; Sonenshine, 2005). When ready to feed, argasid ticks attach to hosts by cutting into the skin with their chelicerae and inserting their barbed hypostome (Sonenshine, 1991, 2005; Goddard, 1993; Anderson, 2002; Mans and Neitz, 2004; Mu¨ller-Doblies and Wikel, 2005). Once attached, they begin to secrete saliva into the host and suck blood from the host. Argasids only feed for short periods of time, usually several minutes. As they feed, their highly folded body cuticle stretches to accommodate the incoming host blood meal, which is approximately 5–10 times their body weight. To maximize blood consumption, the tick concentrates the blood as it feeds by secreting a colourless fluid, termed coxal fluid, consisting of water and salts extracted from the blood from pores in the body. Argasid ticks typically have a multi-host lifecycle (Sonenshine, 1991, 2005; Goddard, 1993; Mans and Neitz, 2004; Dennis and Piesman, 2005; Mu¨llerDoblies and Wikel, 2005). Larvae emerge from eggs and attach to a host, feed to repletion, drop off and moult into nymphs. Nymphs then feed on the same or a different host, drop off and moult. Nymphs usually feed several times on multiple hosts and undergo 3–5 moults before becoming adults. Once adults, females may mate before or after feeding, away from a host. After feeding to repletion, adult females drop off the host and lay several hundreds of eggs. Argasid females, like nymphs, are also capable of feeding numerous times and laying multiple batches of eggs several times during their lifetime.
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KATHARINE R. TYSON AND JOSEPH PIESMAN IXODIDAE
Ixodid ticks, which account for approximately 80% of all tick species worldwide, are subdivided into 13 genera, and the larger genera are Amblyomma, Dermacentor, Haemaphysalis, Hyalomma, Ixodes and Rhipicephalus (Barker and Murrell, 2004; Jongejan and Uilenberg, 2004). Ixodid tick species within each of the seven genera are important medical or veterinary pathogen transmission vectors of several viruses, bacteria and protozoan parasites, including B. burgdorferi, the causative agent of Lyme disease (Dennis and Piesman, 2005). Ixodid ticks possess a scutum, or a tough, sclerotized plate, on their dorsal surface giving them the name ‘‘hard ticks’’ (Sonenshine, 1991, 2005; Goddard, 1993; Anderson, 2002) In adult males, the scutum covers the entire dorsal body surface, while it only covers a portion of the dorsal body surface in nymphs and adult females. As ixodid ticks are sensitive to environmental humidity and temperature conditions, most of the body in nymphs and adult females, excluding the scutum, is covered by a waxy, tough, dense cuticle that prevents desiccation (Sonenshine, 2005). The mouthparts of ixodid ticks are very similar to argasid ticks and located at the anterior end of the body. However, unlike argasids, the body does not cover the mouthparts in ixodids and can be readily seen from the dorsal view (Sonenshine, 1991, 2005; Goddard, 1993; Anderson, 2002). Ixodid ticks are usually found in brushy, wooded or weedy areas populated by medium or large sized mammals (Goddard, 1993; Sonenshine, 2005). The lifecycle and feeding characteristics of ixodid ticks are different from argasid ticks. Ixodid ticks display a two- or three-host lifecycle, as opposed to argasid ticks, which display a multi-host lifecycle (Sonenshine, 1991, 2005; Goddard, 1993; Anderson, 2002; Mans and Neitz, 2004). Larvae emerge from eggs and attach to small animals for their first blood meal. After feeding for several days to repletion, larvae drop off the host and moult into nymphs. In some ixodid species, larvae remain on the host after feeding and moult into nymphs. Nymphs then either seek a new host to feed on or feed from the same host if the larvae remained on the host after the initial feeding. After the second feeding, the nymphs drop off the host and moult into either female or male adults. Adult ixodid ticks attach to a new host and begin feeding, and mating typically occurs during feeding. After feeding to repletion, the mated female adult drops off the host, lays thousands of eggs and dies. When larvae emerge from the eggs, the cycle repeats itself. Unlike argasid ticks, ixodid ticks only ingest three blood meals and moult three times in their lifetime. Since ixodid ticks do not undergo multiple nymphal moults like argasid ticks, the ixodid lifecycle is typically shorter, usually 1–2 years depending on the tick species and environmental conditions, which include humidity and temperature, than the argasid tick lifecycle.
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FEEDING CHARACTERISTICS OF IXODID TICKS
Ixodid ticks exhibit unique feeding characteristics, distinguishing them from argasid ticks and other blood-feeding arthropods including flies, fleas, mosquitoes and mites. Unlike most blood-sucking arthropods, ixodid ticks feed for several days on a single host, consuming large quantities of blood. The majority of ixodid tick species find suitable hosts using a strategy termed ‘‘questing’’ (Sonenshine, 1991, 2005; Goddard, 1993; Anderson, 2002; Dennis and Piesman, 2005), which consists of climbing blades of grass, weeds or bushes and waiting for a host to brush against them. While questing, ticks remain hydrated by moving from their perch to the humid environment of the leafy ground cover and secreting a hygroscopic, salty saliva solution onto their hypostomes that adsorbs water from the surrounding atmosphere. Once rehydrated, the ticks climb back up the vegetation and continue waiting for a host. Ixodid and argasid ticks sense approaching hosts by several factors including shadows, vibrations and odours. Ticks are especially attracted to carbon dioxide, which is found in host breath, and ammonia, which is found in host urine. When they finally sense a host, ticks stretch out their forelegs and cling to the hair or clothing of the host. Some species of ixodid ticks, in particular species of Hyalomma, actively hunt their hosts (Dennis and Piesman, 2005; Sonenshine, 2005). These ixodid ticks bury themselves in sand or dirt, preventing desiccation. When they sense a nearby host, they emerge from the ground and run towards the host. Upon finding an appropriate host, ticks use the sensory organs in their palps to locate an attachment site (Sonenshine, 1991, 2005). Once an attachment site is found, the chelicerae of the tick begin slicing into the skin in an outward motion, creating a small hole (Sonenshine, 1991, 2005; Goddard, 1993; Anderson, 2002; Mu¨ller-Doblies and Wikel, 2005). The tick then inserts the barbed hypostome into the hole and secretes a proteinaceous cement compound. The cement covers the hypostome and host skin, acting as an adhesive to anchor the hypostome in place. Since argasid ticks only feed for a few hours rather than several days, they do not secrete any cement upon attachment. In ixodid ticks, the process of attachment may take hours to days, but once firmly attached, the ticks are hard to remove. After the tick is firmly cemented in place, blood feeding begins from a pool created by tissue and blood vessel damage during attachment (Anderson, 2002; Mans and Neitz, 2004; Mu¨ller-Doblies and Wikel, 2005; Sonenshine, 2005). For the first few days, feeding proceeds slowly as new cuticle is synthesized to allow expansion of the ixodid tick with the incoming blood meal. Once the new cuticle has been synthesized, feeding proceeds rapidly and the tick may increase its weight 10 (larvae and nymphs) to 100 (adult mated females) times its prefed weight (Sonenshine, 1991, 2005; Mans and Neitz, 2004; Mu¨ller-Doblies and Wikel, 2005). Since adult ixodid males are covered by the rigid scutum and cannot expand easily, they usually do not ingest large amounts of blood when
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KATHARINE R. TYSON AND JOSEPH PIESMAN
feeding. Ixodid ticks in the genus Ixodes are termed Prostriate ticks, and the male Ixodes can mate without feeding on a host. All other Ixodid ticks are termed Metastriate, and metastriate males must begin the feeding process on a host to develop mature sperm and successfully mate. Feeding is discontinuous, characterized by periods of blood sucking alternating with periods of tick salivation (Mu¨ller-Doblies and Wikel, 2005; Sonenshine, 2005). To maximize blood consumption, portions of ixodid tick salivary glands function as water-secreting compartments that remove excess water and salts to concentrate the blood meal (Sonenshine, 1991, 2005; Mans and Neitz, 2004; Mu¨ller-Doblies and Wikel, 2005). When the tick is replete, it will drop off the host and either moult (larvae or nymphs) or lay eggs (adult mated females). 2.4
HOST RESPONSES TO TICK FEEDING
Ticks acquire a meal by sucking blood from a pool created during attachment, when the mouthparts cut into the skin, lacerating numerous small blood vessels and causing tissue destruction at the feeding site (Mans and Neitz, 2004; Valenzuela, 2004; Mu¨ller-Doblies and Wikel, 2005). The host is normally capable of detecting and repairing wounds through the processes of haemostasis and inflammation. Haemostatic responses prevent blood loss, potentially making the acquisition of a blood meal difficult for the tick (Schoeler and Wikel, 2001; Ribeiro and Francischetti, 2003; Mans and Neitz, 2004; Valenzuela, 2004). Inflammation results in redness, swelling and irritation at the feeding site, allowing the host to sense the presence of the tick, which potentially leads to host grooming and tick removal (Schoeler and Wikel, 2001; Ribeiro and Francischetti, 2003; Mu¨ller-Doblies and Wikel, 2005). To counteract the haemostatic and inflammatory responses of the host, ticks produce multiple anti-haemostatic and anti-inflammatory mediators that are secreted into the host through the saliva during feeding. In addition to haemostatic and inflammatory responses, other host immune responses may also be triggered during tick attachment and feeding. Host immune responses likely mediate recognition of multiple tick antigens, eventually resulting in rejection of the feeding tick (Ribeiro and Francischetti, 2003). Tick rejection is normally characterized by a reduction in fed tick weights, altered feeding times, a reduction in the viability and number of ova produced, impaired moulting and death of the feeding tick (Wikel, 1996; Schoeler and Wikel, 2001; Ribeiro and Francischetti, 2003). In addition to secreting antihaemostatic and anti-inflammatory mediators, ticks also secrete immunosuppressive molecules in their saliva to prevent host immune recognition and rejection (Schoeler and Wikel, 2001; Ribeiro and Francischetti, 2003; Mans and Neitz, 2004; Mu¨ller-Doblies and Wikel, 2005).
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2.5
249
ANTI-HAEMOSTATIC TICK SALIVARY COMPONENTS
Haemostasis, a host response generated during tissue injury, prevents blood loss from damaged tissues through platelet aggregation, vasoconstriction and blood coagulation (Schoeler and Wikel, 2001; Ribeiro and Francischetti, 2003; Champagne, 2004; Mans and Neitz, 2004; Valenzuela, 2004; Andrade et al., 2005). To obtain a complete blood meal successfully by avoiding host haemostatic responses, many blood-sucking arthropods, including mosquitoes, flies, lice, fleas, mites and ticks, secret salivary anti-haemostatic components into the host during feeding (Gillespie et al., 2000; Ribeiro and Francischetti, 2003; Champagne, 2004, 2005; Andrade et al., 2005; Titus et al., 2006). Argasid, and in particular ixodid ticks, which feed on a host for periods of several days, secrete a wide variety of anti-haemostatic molecules including platelet aggregation inhibitors, vasodilators and anticoagulants, which all aid the tick in overcoming host haemostasis. 2.5.1
Platelet aggregation inhibitors
Tissue damage leads to the exposure of various agonists, including adenosine diphosphate (ADP), thromboxane A2 and collagen, which cause platelet aggregation. To prevent platelet aggregation, many blood-feeding arthropods secrete salivary apyrases, which are enzymes that hydrolyze adenosine triphosphate (ATP) and ADP into adenosine monophosphate (AMP) and inorganic phosphate. Eliminating ADP potentially prevents activation and aggregation of platelets (Schoeler and Wikel, 2001; Champagne, 2004; Mans and Neitz, 2004; Mu¨ller-Doblies and Wikel, 2005). Apyrase activity has been identified in the saliva or SGE of the soft ticks Ornithodoros savignyi, Ornithodoros moubata and Argas monolakensis, as well as the hard tick, Ixodes scapularis (Table 1) (Ribeiro et al., 1985, 1991; Mans et al., 1998, 2008a; Ribeiro and Francischetti, 2003; Mans and Neitz, 2004; Mu¨ller-Doblies and Wikel, 2005). In addition to salivary apyrases, O. savignyi and O. moubata secrete salivary lipocalins that binds thromboxane A2, preventing platelet aggregation (Table 1) (Mans and Ribeiro, 2008a). O. moubata also expresses tick adhesion inhibitor (TAI), a salivary gland protein that specifically inhibits platelet adhesion (Table 1) (Keller et al., 1993; Waxman and Connolly, 1993; Karczewski et al., 1995). Once platelets are activated by various agonists, they express glycoprotein IIbIIIa (GPIIbIIIa) on their surfaces. This integrin binds fibrinogen or von Willebrand’s factor, resulting in platelet cross-linking and aggregation (Mans and Neitz, 2004; Valenzuela, 2004; Andrade et al., 2005). In addition to apyrases, ticks have also developed salivary components that block GPIIbIIIa, preventing fibrinogen binding and platelet aggregation. Dermacentor variabilis,
250
TABLE 1 Anti-haemostatic tick salivary proteins Activity Platelet aggregation inhibitors
Hydrolyzes ATP and ADP into AMP
Apyrase
Bind integrin GPIIbIIIa
MG1 MG2 Variabilin Disagregin
Proteolysis of fibrinogen Inhibits platelet adhesion Binds thromboxane A2
Vasodilators
Protein
Relax smooth muscles
Found in Argas monolakensis Ixodes scapularis Ornithodoros moubata Ornithodoros savignyi Argas monolakensis Dermacentor variabilis Ornithodoros moubata
Savignygrin
Ornithodoros savignyi Ixodes scapularis
TAI
Ornithodoros moubata
Moubatin TSGP3
Ornithodoros savignyi
Prostaglandin E2
Amblyomma americanum
Boophilus microplus
References Mans et al. (2008a) Ribeiro et al. (1985) Ribeiro et al. (1991) Mans et al. (1998) Mans et al. (2008a) Mans et al. (2008a) Wang et al. (1996) Karczewski et al. (1994) Mans et al. (2002a,b) Francischetti et al. (2003) Karczewski et al. (1995) Mans and Ribeiro (2008a) Mans and Ribeiro (2008a) Ribeiro et al. (1992), Bowman et al. (1995) and Aljamali et al. (2002) Dickinson et al. (1976) and Inokuma et al. (1997)
Prostaglandin I2 prostaglandin F2
Bind Ca
Anticoagulants
Inhibit FXa activity
Calreticulin
NTI-1 NTI-2
Haemaphysalis longicornis Ixodes holocyclus Ixodes scapularis
Inokuma et al. (1997)
Amblyomma americanum
Ribeiro et al. (1992), Bowman et al. (1995) and Aljamali et al. (2002) Jaworski et al. (1995) and Sanders et al. (1998) Ferreira et al. (2002) Xu et al. (2004) Xu et al. (2004)
Amblyomma americanum
Boophilus microplus Dermacentor variabilis Haemaphysalis longicornis Haemaphysalis qinghaiensis Ixodes scapularis Rhipicephalus sanguineus Hyalomma dromedarii Hyalomma truncatum Ixodes holocyclus
Salp14
Inokuma et al. (1997) Sa´-Nunes et al. (2007) Ribeiro et al. (1988)
Gao et al. (2007) Xu et al. (2004) Xu et al. (2004) Ibrahim et al. (2001b) Ibrahim et al. (2001b) Joubert et al. (1995) Anastopoulos et al. (1991)
Ixodes scapularis
251
(continues)
252
TABLE 1 (Continued) Activity
Inhibit thrombin activity
Protein
Found in
TAP
Ornithodoros moubata
Americanin Variegin Monobin Calcaratin
Ornithodoros savignyi Rhipicephalus appendiculatus Amblyomma americanum Amblyomma variegatum Argas monolakensis Boophilus calcaratus
BmAP Microphilin Madanin-1 Madanin-2 NTI-1 NTI-2
Boophilus microplus Haemaphysalis longicornis Hyalomma dromedarii Ixodes holocyclus
Ixin Ornithodorin
Ixodes ricinus Ornithodoros moubata
Savignin
Ornithodoros savignyi
References Narasimhan et al. (2002) Neeper et al. (1990) and Waxman et al. (1990) Gaspar et al. (1996) Limo et al. (1991) Zhu et al. (1997a,b) Koh et al. (2007) Mans et al. (2008a) Motoyashiki et al. (2003) Horn et al. (2000) Ciprandi et al. (2006) Iwanaga et al. (2003) Iwanaga et al. (2003) Ibrahim et al. (2001a) Ibrahim et al. (2001a) Anastopoulos et al. (1991) Hoffmann et al. (1991) van de Locht et al. (1996)
Dermacentor andersoni
Inhibit FVII, FV Inhibit FVIIa/TF complex
Ixolaris
Ixodes scapularis
Penthalaris Inhibit FXII activity
Haemaphysalin
Inhibit extrinsic pathway
BSAP-1 BSAP-2
Haemaphysalis longicornis Ornithodoros savignyi
Nienaber et al. (1999) and Mans et al. (2002a) Gordon and Allen (1991) Francischetti et al. (2002) Francischetti et al. (2004) Kato et al. (2005) Ehebauer et al. (2002) Ehebauer et al. (2002)
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KATHARINE R. TYSON AND JOSEPH PIESMAN
O. moubata, O. savignyi and A. monolakensis, produce salivary gland proteins that bind GPIIbIIIa, inhibiting fibrinogen binding or displacing bound fibrinogen from the receptor (Table 1) (Karczewski et al., 1994; Wang et al., 1996; Mans et al., 2002b,c, 2008a). These activities either prevent platelet aggregation or cause aggregated platelets to disaggregate. I. scapularis saliva is also capable of causing platelet disaggregation through the proteolysis of fibrinogen (Francischetti et al., 2003). The presence of multiple platelet aggregation inhibitors in a variety of ticks indicates the necessity to inhibit this host response during feeding. 2.5.2
Vasodilators
Many blood-feeding arthropods employ a variety of strategies to promote vasodilation, which counteracts vasoconstriction induced by platelet aggregation and increases the rate of blood flow to the feeding site (Champagne, 2004). To maintain blood flow during their extended feeding periods, ixodid ticks produce and secret numerous salivary prostaglandins. As argasid ticks feed for much shorter periods, prostaglandins have not been detected in their saliva. Ixodid salivary prostaglandins are lipid molecules that promote smooth muscle relaxation, causing vasodilation (Bowman et al., 1996; Andrade et al., 2005). Prostaglandin E2 has been identified in the saliva of many ixodid ticks including Rhipicephalus (Boophilus) microplus, Haemaphysalis longicornis, Amblyomma americanum, Ixodes holocyclus and I. scapularis (Table 1) (Dickinson et al., 1976; Ribeiro et al., 1992; Inokuma et al., 1994; Bowman et al., 1995; Aljamali et al., 2002; Sa´-Nunes et al., 2007). In addition, I. scapularis also produces prostaglandin I2, while A. americanum produces prostaglandin F2 (Ribeiro et al., 1988, 1992; Aljamali et al., 2002). Besides their primary functions in vasodilation, prostaglandins also display immunosuppressive effects and prevent platelet aggregation (Bowman et al., 1996; Sa´-Nunes et al., 2007). Tissue damage and platelet activation and aggregation lead to elevated levels of Ca2þ, which potentially triggers vasoconstriction (Andrade et al., 2005). Several ixodid tick species secrete salivary calreticulins, which bind Ca2þ, potentially preventing vasoconstriction (Table 1) (Jaworski et al., 1995; Sanders et al., 1998; Ferreira et al., 2002; Xu et al., 2004; Gao et al., 2007). Similar to prostaglandins, calreticulins may also display anticoagulant and immunosuppressive functions in addition to vasodilatory activities as Ca2þ is required for a variety of host cell responses (Jaworski et al., 1995; Brossard and Wikel, 2004; Champagne, 2004). 2.5.3
Anticoagulants
Blood coagulation is initiated through two different pathways, either the intrinsic or the extrinsic pathway (Champagne, 2004; Mans and Neitz, 2004; Valenzuela, 2004). Both pathways converge at the step of factor X (FX)
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255
activation into FXa. FXa activates prothrombin into thrombin, which then cleaves fibrinogen into fibrin. Fibrin then polymerizes, ultimately resulting in clot formation, which prevents blood loss from damaged tissues. Numerous blood-feeding arthropods, including argasid and ixodid ticks, secrete anticoagulants into the host during feeding, inhibiting blood coagulation. Interestingly, individual ixodid tick species typically express several anticoagulants, as opposed to argasid ticks, likely because of their prolonged feeding times. Most tick salivary anticoagulants inhibit either FXa or thrombin. Several species of Ixodes, Hyalomma, Rhipicephalus and Ornithodoros ticks secrete various salivary proteins that directly bind FXa, preventing it from activating prothrombin (Table 1) (Neeper et al., 1990; Waxman et al., 1990; Anastopoulos et al., 1991; Limo et al., 1991; Joubert et al., 1995; Gaspar et al., 1996; Ibrahim et al., 2001b; Narasimhan et al., 2002). Additionally, species of Ixodes, Rhipicephalus, Haemaphysalis, Hyalomma, Amblyomma and Ornithodoros also secrete salivary proteins that directly bind thrombin, preventing it from cleaving fibrinogen (Table 1) (Anastopoulos et al., 1991; Hoffmann et al., 1991; van de Locht et al., 1996; Zhu et al., 1997a,b; Nienaber et al., 1999; Horn et al., 2000; Ibrahim et al., 2001a; Mans et al., 2002a,c, 2008a; Iwanaga et al., 2003; Motoyashiki et al., 2003; Ciprandi et al., 2006; Koh et al., 2007). Ticks also produce salivary components that inhibit blood coagulation prior to the activation of FX. I. scapularis, Dermacentor andersoni and O. savignyi secrete salivary proteins that inhibit the extrinsic, or tissue factor pathway, and H. longicornis secretes an intrinsic pathway inhibitor (Gordon and Allen, 1991; Ehebauer et al., 2002; Francischetti et al., 2002, 2004; Kato et al., 2005). Presumably, inhibition of blood coagulation by various secreted salivary components is essential for successful argasid or ixodid tick feeding. 2.6
ANTI-INFLAMMATORY TICK SALIVARY COMPONENTS
Tissue damage, platelet activation and aggregation, and activation of blood coagulation all trigger the induction of inflammation, a host response resulting in pain, itch, redness and irritation at the site of tissue damage (Ribeiro and Francischetti, 2003). Inflammation can lead to host grooming, resulting in removal of a feeding tick (Valenzuela, 2004). To obtain a blood meal without premature host removal during their extended feeding periods, ixodid ticks secrete an array of anti-inflammatory proteins including histamine-binding proteins, kininases and anaphylatoxin inhibitors. 2.6.1
Histamine-binding proteins
Histamine and serotonin are essential mediators of inflammation that cause itching sensations, oedema and erythema by increasing vascular permeability (Schoeler and Wikel, 2001; Valenzuela, 2004; Mu¨ller-Doblies and
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KATHARINE R. TYSON AND JOSEPH PIESMAN
Wikel, 2005). As ixodid ticks feed for several days, they have developed histamine-binding proteins (HBPs) to inhibit the activities of histamine and serotonin and prevent inflammation during feeding. Rhipicephalus sanguineus SGE inhibit the activity of histamine, and Rhipicephalus appendiculatus was found to express three salivary HBPs (Chinery and Ayitey-Smith, 1977; Paesen et al., 1999). Homologues of these proteins have been identified in I. scapularis and A. americanum, and Mans et al. have recently demonstrated that several recombinant salivary proteins from I. scapularis, as well as from the soft ticks O. savignyi and A. monolakensis, bind histamine and/or serotonin (Table 2) (Bior et al., 2002; Valenzuela et al., 2002; Mans et al., 2008b). Additionally, Dermacentor reticulatus secretes a salivary protein that binds both histamine and serotonin (Table 2) (Sangamnatdej et al., 2002). These proteins are likely essential for successful tick feeding as RNAi knockdown of A. americanum HBPs prevented successful tick feeding (Aljamali et al., 2003). 2.6.2
Bradykinin inhibitors
Another important mediator of inflammation is bradykinin, which is generated during activation of the intrinsic coagulation cascade. Bradykinin acts similarly to histamine, promoting pain, itch and oedema by increasing vascular permeability (Schoeler and Wikel, 2001; Valenzuela et al., 2002; Mu¨ller-Doblies and Wikel, 2005). I. scapularis secretes a carboxypeptidase that degrades bradykinin, while R. (Boophilus) microplus secretes a serine protease inhibitor that inhibits kallikrein, preventing the formation of bradykinin (Table 2) (Ribeiro and Mather, 1998; Tanaka et al., 1999). Recently, sialostatin L and sialostatin L2 from I. scapularis saliva were demonstrated to bind and inhibit cathepsin L, a protease implicated in kinin generation, preventing inflammation (Kotsyfakis et al., 2006, 2007). Sialostatin L and sialostatin L2 also display immunosuppressive functions. 2.6.3
Anaphylatoxin inhibitors
Anaphylatoxins are inflammatory mediators released during complement activation that induce vascular permeability, cause histamine release and recruit inflammatory cells to sites of tissue damage (Morgan and Harris, 1999; Walport, 2001a,b; Valenzuela, 2004). Ixodes ricinus and I. scapularis secrete salivary proteins that inhibit the alternative complement pathway, preventing the production of anaphylatoxins (Table 2) (Ribeiro and Spielman, 1986; Ribeiro, 1987; Valenzuela et al., 2000; Daix et al., 2007; Tyson et al., 2007). O. moubata and O. savignyi also produce complement inhibitors that bind C5, preventing its cleavage into the anaphylatoxin C5a (Table 2) (Roversi et al., 2007; Mans and Ribeiro, 2008a). By preventing the generation of anaphylatoxins, ticks inhibit the induction of inflammation allowing them to feed successfully.
TABLE 2 Anti-inflammatory tick salivary proteins Activity Histamine-binding proteins
Protein
Bind histamine Monomine Ra-HBP1 Ra-HBP2 Ra-HBP3
Bind histamine and serotonin
SHBP TSGP1
Bind serotonin
Bradykinin inhibitors
Inhibits kallikrein Degrades bradykinin Inhibit cathepsin L
Monotonin IS-14 IS-15 BmTI-A Carboxypeptidase Sialostatin L Sialostatin L2 IRACI
Found in Amblyomma americanum Argas monolakensis Ixodes scapularis Rhipicephalus appendiculatus Rhipicephalus sanguineus Dermacentor reticulatus Ornithodoros savignyi Argas monolakensis Ixodes scapularis Boophilus microplus Ixodes scapularis Ixodes ricinus
References Bior et al. (2002) Mans et al. (2008b) Valenzuela et al. (2002) Paesen et al. (1999) Paesen et al. (1999) Paesen et al. (1999) Chinery and Ayitey-Smith (1977) Bior et al. (2002) and Sangamnatdej et al. (2002) Mans et al. (2008b) Mans et al. (2008b) Mans et al. (2008b) Mans et al. (2008b) Tanaka et al. (1999) Ribeiro et al. (1988) Kotsyfakis et al. (2006) Kotsyfakis et al. (2007) Daix et al. (2007) (continues)
TABLE 2 (Continued) Activity Anaphylatoxin inhibitors
Inhibit the alternative complement pathway Binds C5
Leukotriene-binding proteins
Binds LTB4
Binds LTC4, LTD4 and LTE4
Protein IRACII Isac Salp20 OmCI Ir-LBP Moubatin TSGP2 TSGP3 AM-33 TSGP4
Found in Ixodes scapularis Ornithodoros moubata Ixodes ricinus Ornithodoros moubata Ornithodoros savignyi Argas monolakensis Ornithodoros savignyi
References Daix et al. (2007) Valenzuela et al. (2000) Tyson et al. (2007) Roversi et al. (2007) Beaufays et al. (2008a,b) Mans and Ribeiro (2008a) Mans and Ribeiro (2008a) Mans and Ribeiro (2008a) Mans and Ribeiro (2008b) Mans and Ribeiro (2008b)
LYME DISEASE SPIROCHETE–TICK–HOST INTERACTIONS
2.6.4
259
Leukotriene-binding proteins
Leukotrienes are important lipid inflammatory mediators involved in the recruitment of leukocytes to sites of tissue injury and inflammation (Abbas et al., 2000). Mans et al. demonstrated that salivary components of the soft ticks O. moubata and A. monolakensis bind leukotrienes C4, D4 and E4 (Table 2) (Mans and Ribeiro, 2008b). Similarly, Beaufays et al. (2008a,b) recently characterized a protein from I. ricinus saliva that specifically binds leukotriene B4, Ir-LBP, preventing neutrophil migration and apoptosis in vitro (Table 2). These leukotriene-binding proteins likely aid the inhibition of inflammation during tick feeding. 2.7
IMMUNOSUPPRESSIVE TICK SALIVARY COMPONENTS
The host innate and adaptive immune responses are likely activated when blood-sucking arthropods attach and begin feeding. These immune responses can lead to the induction of inflammation and the activation and production of various components that result in host immune recognition and rejection. Numerous blood-sucking arthropods have evolved various salivary components to inhibit host innate and adaptive immune responses. As ixodid ticks uniquely feed on hosts for longer periods than most blood-sucking arthropods, they have developed large families of salivary proteins that inhibit multiple host innate and adaptive immune mechanisms. 2.7.1
Inhibitors of endothelial cell adhesion molecule expression
During tissue damage or antigen stimulation, activated macrophages release cytokines that promote endothelial cells to express various surface adhesion molecules. These molecules allow circulating leukocytes to attach to endothelial cells and eventually traverse the blood vessel wall, migrating to sites of tissue damage. SGE from D. andersoni and I. scapularis significantly reduce the expression of several endothelial cell adhesion molecules in vitro and in vivo (Table 3) (Macaluso and Wikel, 2001; Maxwell et al., 2005). In addition, expression of adhesion molecules on leukocytes is also reduced by I. scapularis saliva in vitro (Montgomery et al., 2004). Expression of leukocyte adhesion molecules by endothelial cells and leukocytes is likely downregulated during tick feeding, potentially preventing the initiation of innate immune responses and inflammation. 2.7.2
Inhibitors of natural killer cells, neutrophils and macrophages
Activated natural killer (NK) cells are important lymphocytes in innate immunity as they are cytotoxic cells and release interferon-g (IFN-g), a cytokine that activates macrophages (Abbas et al., 2000; Brossard and Wikel, 2004;
260
TABLE 3 Immunosuppressive tick salivary protein Activity Antagonists of leukocyte adhesion
Natural killer cell, neutrophil and macrophage inhibitors
Protein
Reduce expression of ICAM-1, LFA-1, VLA-4 Reduce expression of P-selectin, VCAM1 Reduce expression of LFA-1 Impair phagocytosis and killing activity
Found in Dermacentor andersoni
Macaluso and Wikel (2001)
Ixodes scapularis
Maxwell et al. (2005)
Montgomery et al. (2004) Amblyomma variegatum Dermacentor andersoni Dermacentor reticulatus Haemaphysalis inermis Ixodes ricinus
Ixodes scapularis
Antioxidants
References
Peroxiredoxin
HIPrx
Glutathione peroxidase
Salp25D
Rhipicephalus sanguineus Haemaphysalis longicornis Ixodes scapularis
Kubes et al. (2002) Ramachandra and Wikel (1995) Kubes et al. (1994) Kubes et al. (2002) Kopecky´ and Kuthejlova´ (1998) and Kuthejlova´ et al. (2001) Ribeiro et al. (1990) and Montgomery et al. (2004) Ferreira and Silva (1998) Tsuji et al. (2001) Das et al. (2001)
Cytokine regulators
Inhibition of IL-2, IFN-g, IL-12, IL-1, TNF-a, IL-6; unaffect or enhance IL-4 and IL-10 expression
Dermacentor andersoni
Iris
Ixodes pacificus Ixodes ricinus
Ixodes scapularis
Rhipicephalus appendiculatus Rhipicephalus sanguineus Chemokine inhibitors
Inhibit IL-8, MCP-1, MIP-1a, RANTES, exotaxin
Amblyomma variegatum
Dermacentor reticulatus Haemaphysalis inermis Ixodes ricinus
Ramachandra and Wikel (1992) Schoeler et al. (2000) Ganapamo et al. (1995), Kopecky´ et al. (1999), Kova´rˇ et al. (2001, 2002), Leboulle et al. (2002) and Pechova´ et al. (2004) Urioste et al. (1994), Schoeler et al. (1999), Schoeler et al. (2000), Gillespie et al. (2001) and Mu¨ller-Doblies et al. (2007) Gwakisa et al. (2001) Ferreira and Silva (1998, 1999) Hajnicka´ et al. (2001, 2005) and Vancˇova´ et al. (2007) Hajnicka´ et al. (2001, 2005) Hajnicka´ et al. (2001) Hajnicka´ et al. (2001, 2005)
261
(continues)
262
TABLE 3 (Continued) Activity
Binds CCL3, CCL4 and CCL18
Protein
Evasin-1
Found in Rhipicephalus appendiculatus Rhipicephalus sanguineus
Evasin-2
Inhibitors of dendritic cell migration
Inhibitors of T-cell proliferation
Binds CXCL8 and CXCL1
Evasin-3
Binds CCL5 and CCL11
Evasin-4
Inhibits production of IL-12 and TNF-a by DCs Suppresses DC stimulated CD4þ Tcell activation Downregulates CCR5 and reduces migration towards MIP-1a Binds T-cell CD4 coreceptor
PGE2
Ixodes scapularis
References Hajnicka´ et al. (2001) Frauenschuh et al. (2007) and Deruaz et al. (2008) Frauenschuh et al. (2007) and Deruaz et al. (2008) Frauenschuh et al. (2007) and Deruaz et al. (2008) Frauenschuh et al. (2007) and Deruaz et al. (2008) Sa´-Nunes et al. (2007)
Sa´-Nunes et al. (2007)
Salp15
Rhipicephalus sanguineus
Cavassani et al. (2005) and Oliveira et al. (2007)
Ixodes scapularis
Anguita et al. (2002), Garg et al. (2006) and Juncadella et al. (2007)
Suppresses T-cell activation and proliferation
Inhibitors of B-cell proliferation Immunoglobulinbinding proteins
Anti-complement proteins
Suppresses B-cell activation and proliferation Bind host IgG
Prevent the formation and cause dissociation of C3bBb
Bind properdin
Bind C5
p36
Dermacentor andersoni
Iris
Ixodes ricinus
BIF BIP
Rhipicephalus sanguineus Hyalomma asiaticum Ixodes ricinus
IGBP
IRAC-I IxAC-B2 IxAC-B3 IxAC-B4 IxAC-B5 Isac IxAC-B1 IRAC-II Salp20 OmCI TSGP2 TSGP3
Amblyomma variegatum Ixodes hexagonus Rhipicephalus appendiculatus Ixodes ricinus
Ixodes scapularis Ixodes ricinus Ixodes scapularis Ornithodoros moubata Ornithodoros savignyi
Bergman et al. (1998, 2000) and AlarconChaidez et al. (2003) Leboulle et al. (2002) and Hovius et al. (2007) Ferreira and Silva (1998) Yu et al. (2006) Hannier et al. (2004) Wang and Nuttall (1995b) Wang and Nuttall (1995b) Wang and Nuttall (1994, 1995a,b) Daix et al. (2007) Couvreur et al. (2008) Couvreur et al. (2008) Couvreur et al. (2008) Couvreur et al. (2008) Valenzuela et al. (2000) Couvreur et al. (2008) Couvreur et al. (2008) Tyson et al. (2008) Nunn et al. (2005) and Roversi et al. (2007) Mans and Ribeiro (2008a) Mans and Ribeiro (2008a)
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Andrade et al., 2005). Neutrophils are polymorphonuclear leukocytes (PMN) that are activated during tissue damage and phagocytose invading organisms or cellular debris, inducing the production of reactive oxygen intermediates (ROIs) and releasing various granular constituents, including lysozyme. Macrophages are phagocytic cells important in innate and adaptive immunity. In innate immunity, macrophages function to phagocytose and kill invading microorganisms and secrete proinflammatory cytokines. When SGE of D. reticulatus, Amblyomma variegatum, Haemaphysalis inermis and I. ricinus were incubated with NK cells, the activity of the cells was substantially decreased (Table 3) (Kubes et al., 1994, 2002; Kopecky´ and Kuthejlova´, 1998). I. scapularis saliva also inhibited the activity of neutrophils, preventing the phagocytosis of B. burgdorferi (Ribeiro et al., 1990; Montgomery et al., 2004). SGE of I. ricinus, R. sanguineus and D. andersoni inhibited the killing of intracellular parasites and B. afzelii, a causative agent of Lyme disease in Europe, by activated macrophages in vitro (Ramachandra and Wikel, 1995; Ferreira and Silva, 1998; Kuthejlova´ et al., 2001). These inhibitory activities possibly prevent the activation of detrimental innate immune responses allowing successful tick feeding and facilitating efficient pathogen transmission. 2.7.3
Antioxidants
ROIs generated by activated neutrophils and macrophages promote inflammation, tissue damage and killing of invading microorganisms (Abbas et al., 2000). Even though ixodid ticks have rigid, sclerotized mouthparts that are likely not sensitive to ROIs, antioxidants, which inhibit the activities of ROIs, have been detected in the SGE of I. scapularis and H. longicornis (Table 3) (Das et al., 2001; Tsuji et al., 2001). These antioxidants potentially aid the tick during extended feeding periods by preventing inflammation and activation of innate immune responses and by possibly protecting tick gut tissue. 2.7.4
Cytokine and chemokine regulators
Cytokines and chemokines are vital for the initiation and development of innate and adaptive immune responses. Cytokines mediate various inflammatory responses and stimulate the activation and proliferation of lymphocytes and effector cells, that is, macrophages, while chemokines are chemoattractants for circulating neutrophils, basophils, lymphocytes and monocytes, recruiting them to sites of tissue damage and inflammation (Brossard and Wikel, 2004; Mu¨llerDoblies and Wikel, 2005). Since they are likely exposed to various immune components during their extended feeding periods, ixodid ticks have developed multiple strategies to inhibit the effects of cytokines and chemokines that include limiting their production and directly binding them to prevent their functions.
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Multiple components in the saliva or SGE of I. ricinus, I. scapularis, Ixodes pacificus, D. andersoni, R. appendiculatus and R. sanguineus reduce the expression and secretion of various proinflammatory cytokines, including IFN-g, tumour necrosis factor-a (TNF-a), interleukin-1 (IL-1), and IL-6, thereby limiting the activation of inflammation and cell-mediated immunity (TH1 immune responses) (Table 3) (Ramachandra and Wikel, 1992; Ganapamo et al., 1995; Ferreira and Silva, 1998, 1999; Kopecky´ et al., 1999; Schoeler et al., 1999, 2000; Gwakisa et al., 2001; Kova´rˇ et al., 2001, 2002; Leboulle et al., 2002; Pechova´ et al., 2004; Mu¨ller-Doblies et al., 2007). Interestingly, the production of immunosuppressive cytokines, such as IL-4 and IL-10, is usually unchanged or enhanced by the saliva or SGE from these same tick species. The production of IL-4 and IL-10 induces a TH2, or antibody-dependent, immune response. In addition to producing factors that inhibit cytokines important for innate immunity, I. scapularis saliva also affects adaptive immunity by preventing secretion of IL-2, a cytokine important for T-cell and B-cell proliferation from activated T cells, and by expressing a salivary protein that directly binds IL-2 (Urioste et al., 1994; Gillespie et al., 2001). Besides limiting the production and action of cytokines, components in saliva and SGE from A. variegatum, D. reticulatus, H. inermis, I. ricinus, R. appendiculatus and R. sanguineus also bind and inhibit the activity of multiple chemokines, preventing the recruitment of immune cells to sites of tissue damage (Table 3) (Hajnicka´ et al., 2001, 2005; Frauenschuh et al., 2007; Vancˇova´ et al., 2007; Deruaz et al., 2008). 2.7.5
Inhibition of antigen-presenting cells
Dendritic cells (DCs) and macrophages are important antigen-presenting cells (APCs) in adaptive immunity that stimulate antigen-specific T cells through antigen presentation. DCs and macrophages normally reside in peripheral tissues in an inactive state. Upon antigen or cytokine stimulation, activated DCs and macrophages engulf antigens and migrate to draining lymph nodes where they present antigens to CD4þ T cells. Saliva of R. sanguineus inhibited the activation and migration of DCs in vitro, while I. scapularis saliva prevented CD4þ antigen T-cell stimulation in vitro (Cavassani et al., 2005; Oliveira et al., 2007; Sa´-Nunes et al., 2007). 2.7.6
Inhibition of T-cell and B-cell proliferation
T-cell and B-cell activation is essential for the generation of adaptive immune responses, specifically cell-mediated immunity and antibody responses. APCs present antigens to CD4þ T cells through MHC class II complexes, causing activation, proliferation and differentiation of the T cells. Upon activation, CD4þ T cells secrete multiple cytokines and stimulate B-cell proliferation and maturation, which results in the generation of antibody-secreting plasma cells (Abbas et al., 2000). I. scapularis saliva contains a protein, Salp15, which
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inhibits the activation of CD4þ T cells by directly binding the CD4 coreceptor and inhibiting signalling pathways, resulting in a reduction of IL-2 production and inhibition of T-cell activation and proliferation (Table 3) (Anguita et al., 2002; Garg et al., 2006; Juncadella et al., 2007). In addition, Salp15 also inhibits cytokine production by DCs (Hovius et al., 2008). Recently, Salp15 homologues have been identified in I. ricinus and I. pacificus and recombinant Iris, an immunosuppressive protein from I. ricinus, was found to inhibit T-cell proliferation in vitro (Leboulle et al., 2002; Hovius et al., 2007). D. andersoni and R. appendiculatus saliva also contains components that inhibit T-cell activation and proliferation in vitro (Table 3) (Bergman et al., 1998, 2000; Ferreira and Silva, 1998; Alarcon-Chaidez et al., 2003). B-cell proliferation, which eventually leads to the generation of antibodysecreting plasma cells, is also inhibited by ixodid tick saliva. Since IL-2 secreted by CD4þ T cells is important for B-cell proliferation, the IL-2 inhibitory activities of I. scapularis saliva likely inhibit B-cell proliferation (Gillespie et al., 2001; Anguita et al., 2002). Proteins isolated from the saliva of I. ricinus and Hyalomma asiaticum inhibited lipopolysaccharide (LPS)-induced proliferation of B cells in vitro (Hannier et al., 2004; Yu et al., 2006). Furthermore, D. andersoni and R. sanguineus ticks reduced antibody responses in tick infested animals, suggesting salivary secretions from these ticks suppress antibody production, possibly through inhibition of B-cell activation and proliferation (Wikel, 1985; Inokuma et al., 1997). Interestingly, R. (Boophilus) microplus saliva altered the isotype of antibodies produced in susceptible hosts (Kashino et al., 2005). Ticks successfully avoid immune recognition by limiting T-cell and B-cell responses. In addition, inhibition of adaptive immune responses also potentially facilitates the transmission of multiple pathogens. 2.7.7
Immunoglobulin-binding proteins
Besides limiting the activation and proliferation of B cells, which ultimately prevents the production of antigen-specific antibodies, ixodid ticks also secrete salivary proteins that directly bind immunoglobulins. Immunoglobulin-binding proteins were detected in the haemolymph and SGE of R. appendiculatus, A. variegatum and Ixodes hexagonus (Table 3) (Wang and Nuttall, 1994, 1995a,b). These proteins are speculated to be an important defence mechanism of the tick, allowing excretion of host antibodies that may be detrimental to tick midgut tissues from the tick during feeding through the tick saliva. 2.7.8
Anti-complement proteins
Activation of the alternative complement pathway is an important host innate immune response that results in the production of anaphylatoxins, C3a and C5a, phagocytosis of opsonized invading organisms and formation of the membrane attack complex (MAC) in the outer surface of invading microbes leading to lysis
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of the organism (Morgan and Harris, 1999; Walport, 2001a,b). The alternative complement pathway is initiated when C3b covalently binds to an activating surface (Pangburn and Muller-Eberhard, 1986; Morgan and Harris, 1999; Walport, 2001a,b). Factor B (fB) then binds C3b, and factor D (fD) cleaves bound fB, generating the alternative pathway C3 convertase (C3bBb). The C3 convertase then cleaves additional molecules of C3 into C3b and the anaphylatoxin C3a, amplifying the complement cascades. C3b covalently binds more surfaces, forming more C3 convertases or causing opsonization. C3b also binds to C3 convertases to form C5 convertases (C3bBbC3b) and initiate the late steps of complement activation, which ultimately result in the formation of the MAC. Ixodid and argasid ticks have evolved various mechanisms to inhibit complement activation during feeding, preventing the recruitment of inflammatory mediators and the induction of an inflammatory response at the feeding site. Two members of a large family of I. scapularis anti-complement proteins, Isac and Salp20, specifically inhibited the alternative complement pathway by preventing the assembly of C3 convertases (C3bBb) on activating surfaces and dissociating preformed C3 convertases (Table 3) (Valenzuela et al., 2000; Soares et al., 2005; Ribeiro et al., 2006; Tyson et al., 2007). Recently, Tyson et al. (2008) demonstrated that Salp20 specifically binds properdin, a positive regulator of the alternative complement pathway, to prevent assembly and cause dissociation of the C3 convertase. Similarly, Couvreur et al. also demonstrated that IxAC-B1 and IRAC-II, members of a large family of I. ricinus anticomplement proteins, inhibited the alternative complement pathway C3 convertases by specifically interacting with properdin (Table 3) (Daix et al., 2007; Couvreur et al., 2008). In addition to the anti-complement activity present in I. scapularis and I. ricinus saliva, Lawrie et al. (1999) demonstrated that other ixodid tick saliva, specifically I. hexagonus and I. uriae saliva, inhibited the alternative complement pathway. The argasid ticks, O. moubata and O. savignyi also express salivary lipocalins that directly bind C5 (Table 3) (Nunn et al., 2005; Roversi et al., 2007; Mans and Ribeiro, 2008a). By binding C5, OmCI from O. moubata inhibited the classical and alternative complement pathways, preventing the generation of C5a in the presence of C5 convertases (Nunn et al., 2005; Roversi et al., 2007). 2.8
TICK MIDGUT COMPONENTS
During tick feeding, the host blood meal within the tick midgut is digested, producing energy and nutrients that are essential for moulting, oogenesis and vitellogenesis (Grandjean, 1983). Digestion in ticks is unique from other haematophagous arthropods because most of the host blood components are endocytosed by midgut epithelial cells and digested intracellularly (Coons et al., 1986; Sonenshine, 1991; Lara et al., 2003, 2005). Since digestion is intracellular, the lumen of the midgut is a relatively neutral environment that is free of various digestive proteases (Sonenshine, 1991). However, as potential viral,
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bacterial and protozoan pathogens are ingested with the incoming host blood meal, several antimicrobial components have been identified in the lumen of the midgut, potentially serving to protect the tick from infection (Fogac¸a et al., 1999; Kopa´cˇek et al., 1999; Nakajima et al., 2002a,b, 2003; Sonenshine et al., 2005; Ceraul et al., 2008). 2.8.1
Midgut anticoagulants
To fully digest the incoming host blood meal, ticks express a variety of enzymes, including protease inhibitors that may prevent host blood clotting (Anderson et al., 2008). In particular, two thrombin inhibitors, BmGTI and haemalin, have been identified and characterized from the midguts of the hard tick species, R. (Boophilus) microplus and H. longicornis, respectively (Ricci et al., 2007; Liao et al., 2009). BmGTI purified directly from R. (Boophilus) microplus gut extracts and recombinant haemalin delayed the clotting time of bovine plasma by inhibiting thrombin-induced fibrinogen clotting and platelet aggregation. Furthermore, silencing of haemalin by RNAi prevented successful tick feeding, indicating the importance of this protein during feeding and blood meal digestion (Liao et al., 2009). Since thrombin is an essential component for clot formation, expression of thrombin inhibitors in the midgut potentially prevents host blood clotting, allowing effective digestion of the blood meal. In addition to BmGTI and haemalin, a five-domain Kunitz-type serine protease inhibitor (KPI) that possesses anticoagulant activity and likely aids in host blood meal digestion has also been identified from D. variabilis (Ceraul et al., 2008). 2.8.2
Digestive proteases
Multiple proteases, including serine proteases, cysteines peptidases, aspartic peptidases, asparaginyl endopeptidases and leucine aminopeptidases, are expressed in the tick midgut to facilitate degradation of host blood proteins in the incoming blood meal once the tick initiates feeding (Vundla et al., 1992; Mendiola et al., 1996; Renard et al., 2000; Miyoshi et al., 2004; Boldbaatar et al., 2006; Hatta et al., 2006; Abdul Alim et al., 2007; Sojka et al., 2007, 2008; Alim et al., 2008, 2009; Anderson et al., 2008). Interestingly, expression of many of these proteases is upregulated during the feeding process, indicating their crucial role in host blood meal digestion (Miyoshi et al., 2004; Boldbaatar et al., 2006; Hatta et al., 2006; Abdul Alim et al., 2007; Alim et al., 2008). Cysteine and aspartic peptidase activity have been detected in the midguts of I. ricinus, R. (Boophilus) microplus, H. longicornis and R. appendiculatus (Vundla et al., 1992; Mendiola et al., 1996; Renard et al., 2000; Boldbaatar et al., 2006; Sojka et al., 2008). When purified or expressed in vitro, several of these cysteine and aspartic peptidases hydrolyzed and degraded a major host blood component, haemoglobin, at low pHs, consistent with intracellular host blood meal digestion (Vundla et al., 1992; Renard et al., 2000; Boldbaatar et al., 2006).
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Legumains, asparaginyl endopeptidases, have been detected in the midguts of H. longicornis and I. ricinus (Abdul Alim et al., 2007; Sojka et al., 2007, 2008; Alim et al., 2008, 2009). When expressed from E. coli or yeast, recombinant legumains degraded host haemoglobin and serum albumin (Abdul Alim et al., 2007; Alim et al., 2008). Similarly, a recombinant serine protease from H. longicornis degraded bovine serum albumin (Miyoshi et al., 2004). RNAi of the legumains in H. longicornis resulted in a reduction in tick feeding times and weights, ultimately interfering with oviposition and causing tick death (Alim et al., 2009). These results indicate the importance of legumains in successful tick feeding and host blood meal digestion. In addition to the multiple digestive proteases mentioned, a tick protective antigen, subolesin, controls multiple cellular pathways and may present a tempting target for anti-tick vaccines directed at various midgut components (Galindo et al., 2009). 2.8.3
Midgut antimicrobials
Antimicrobial activity has been detected in the midguts of several tick species, including O. moubata, D. variabilis and R. (Boophilus) microplus (Fogac¸a et al., 1999; Nakajima et al., 2003; Sonenshine et al., 2005). When directly isolated from midgut extracts, antimicrobial proteins were identified as degradation products of host haemoglobin. Presumably, during digestion, midgut proteases cleave host haemoglobin in the incoming blood meal, generating haemoglobin peptide fragments that may serve as an immune defence mechanism in the midgut during feeding. In addition to host haemoglobin degradation products, defensin, an antimicrobial peptide, has also been identified in the midguts of the argasid tick O. moubata (Nakajima et al., 2002a,b) Defensins are small, cysteine-rich cationic peptides that exhibit antimicrobial activity by interacting with microbial membranes and forming pores, which eventually leads to death of the microorganism (Ganz, 2003). Even though defensin peptides have not been directly detected in the midguts of ixodid ticks, expression of defensin transcripts have been detected in H. longicornis, D. variabilis and I. scapularis midguts (Hynes et al., 2005; Ceraul et al., 2007; Zhou et al., 2007), suggesting the peptides are possibly present in the midgut to aid in immunity against microorganisms in the incoming host blood meal. Besides haemoglobin degradation products and antimicrobial peptides, a serine protease inhibitor in the midgut of D. variabilis, KPI, also displayed bacteriostatic activity against Rickettsia montanensis, a potential bacterial pathogen ingested by ticks during feeding (Ceraul et al., 2008). Even though ticks possess numerous midgut components that potentially destroy incoming microorganisms, some parasites are capable of evading the immune response and establishing an infection, including B. burgdorferi in I. scapularis ticks.
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2.9
KATHARINE R. TYSON AND JOSEPH PIESMAN TICK HAEMOLYMPH COMPONENTS
The tick haemolymph, or the circulating body fluid in the haemocoel, is composed of cell-free plasma and haemocytes (Sonenshine, 1991). Since ticks are incapable of producing haem, an essential constituent of many proteins and cellular processes, major proteins present in the cell-free haemolymph are haem-binding proteins, which likely sequester haem from the host blood meal (Gudderra et al., 2002a; Donohue et al., 2009). Haemocytes in the haemolymph contribute to nutritional functions of the tick and also act as the immune cells, engulfing microorganisms and secreting various antimicrobial components into the haemolymph, including lectins, antimicrobial peptides and lysozyme (Inoue et al., 2001; Grubhoffer et al., 2004; Borovicˇkova´ and Hypsˇa, 2005). These antimicrobial components aid in haemolymph tick immune responses against various pathogenic organisms, preventing infection of the tick. However, in specific tick species, certain microorganisms are able to evade the triggered immune response, which leads to colonization of the tick and potential transmission to a new host. 2.9.1
Haem-binding proteins
Within the haemolymph of ixodid and argasid ticks, two major haem-binding proteins exist, vitellogenin and haem-carrier proteins. During the digestion of host haemoglobin in the midgut epithelial cells, haem, an essential component of some proteins and specific cellular pathways, is produced. As ticks are incapable of synthesizing haem, it is sequestered and concentrated into haemosomes within midgut epithelial cells (Lara et al., 2005; Donohue et al., 2009). Midgut epithelial cells directly in contact with the haemolymph then transfer haem to haem-carrier proteins (Donohue et al., 2009). Expression of haemcarrier proteins (CPs), which bind and sequester haem, has been identified in several tick species including D. variabilis, R. microplus, H. longicornis, R. appendiculatus, A. americanum, A. variegatum and I. scapularis (MayaMonteiro et al., 2000; Gudderra et al., 2001; Nene et al., 2004; Donohue et al., 2008, 2009). A similar carrier protein was also partially characterized from the argasid tick O. parkeri, but the protein does not appear to bind haem (Gudderra et al., 2001). Within D. variabilis adult females, CP expression began shortly after host attachment and continued through the duration of blood feeding (Gudderra et al., 2002b; Donohue et al., 2008), suggesting CPs bind haem in the haemolymph during a blood meal. Similar to CPs, tick vitellogenin (Vg) also binds and sequesters haem (Logullo et al., 2002). During vitellogenesis, haem is transferred to Vg, which is then absorbed into developing oocytes and converted to vitellin (Gudderra et al., 2002a; Thompson et al., 2007; Donohue et al., 2009). Vitellin eventually serves as a nutritional source for developing embryos. Vg expression has been
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detected in D. variabilis, R. microplus, H. longicornis, I. scapularis, O. parkeri and O. moubata (James and Oliver, 1996; Gudderra et al., 2002a; Donohue et al., 2009). Unlike CP, Vg expression initiated in adult D. variabilis females during mating, which correlates with the function of Vg (Thompson et al., 2005, 2007). 2.9.2
Haemolymph antimicrobials
Small, cationic antimicrobial peptides that kill a wide range of parasites have been detected in the haemolymph of many tick species including D. variabilis, I. ricinus, I. scapularis, A. americanum, A. hebraeum, R. microplus and O. moubata (Johns et al., 2001b; Nakajima et al., 2001; Ceraul et al., 2003; Fogac¸a et al., 2004; Lai et al., 2004a; Hynes et al., 2005; Rudenko et al., 2005; Todd et al., 2007). The most characterized antimicrobial peptide in ticks is defensin. Defensin was initially identified in the argasid tick O. moubata and the ixodid tick D. variabilis (varisin) (Johns et al., 2001b; Nakajima et al., 2001). Production and secretion of varisin was triggered upon injection of D. variabilis with B. burgdorferi, resulting in bacterial death (Sonenshine et al., 2002; Ceraul et al., 2003). These results support the finding that D. variabilis is not a competent vector of B. burgdorferi (Johns et al., 2001a). In addition to defensin, several other novel antimicrobial peptides displaying activity against Grampositive and Gram-negative bacteria have emerged from multiple tick species (Fogac¸a et al., 2004, 2006; Lai et al., 2004a,b). Together, these antimicrobial peptides facilitate immune responses to invading organisms in the haemolymph. Besides antimicrobial peptides, several other tick haemolymph molecules have been characterized that also participate in the haemolymph immune response against invading microorganisms. The serine protease inhibitors, tick a2-macroglobulin (TAM) and IrAM, have been identified in O. moubata and I. ricinus, respectively (Kopa´cˇek et al., 2000; Buresova et al., 2009). In I. ricinus haemocytes, IrAM promoted the phagocytosis of Chryseobacterium indologenes, a Gram-negative pathogen of O. moubata (Buresova et al., 2009). As the phagocytosis was dependent on a C. indologenes metalloprotease, TAM and IrAM may serve to protect the tick from proteolytic attack by foreign proteases. Tick lectins, proteins or glycoproteins that bind carbohydrates, also play a role in tick haemolymph immunity by potentially facilitating pathogen recognition. Lectins from I. ricinus and O. moubata recognize a wide range of carbohydrates with a preference for sialic acid (Kova´rˇ et al., 2000; Grubhoffer et al., 2004). The lectins are typically expressed in haemocytes and secreted into the haemolymph where they potentially act as pattern recognition receptors (Rego et al., 2006). Tick lysozyme, which mediates the lysis and killing of Gram-positive and Gram-negative bacteria, has also been identified as an immune component in the haemolymph of D. variabilis (Johns et al., 1998; Simser et al., 2004). Even though ticks express a variety of immune components that limit potential infections, certain pathogens are capable of evading tick-mediated immunity in specific tick species.
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B. burgdorferi biology and interaction with tick vectors
In the eastern United States, I. scapularis ticks are the primary vector for the transmission of B. burgdorferi while the white-footed mouse, Peromyscus leucopus, is the main reservoir of these spirochetes. Acquisition of the spirochetes occurs when larval ticks feed on an infected host (Barbour and Fish, 1993; Kurtenbach et al., 2006). Once larval ticks ingest spirochetes from the host, B. burgdorferi colonize the midgut and persist transstadially during the moulting periods to the nymphal stage. When nymphal ticks begin ingesting a second blood meal, the spirochetes multiply rapidly within the tick gut, traverse the midgut epithelium, enter the haemocoel and migrate to the salivary glands (Ribeiro et al., 1987; Piesman et al., 1990; De Silva and Fikrig, 1995). B. burgdorferi are then transmitted to new host through the saliva of infected ticks. Transmission of the spirochetes to a new host generally occurs after the tick has been feeding for 48 h (Hojgaard et al., 2008) due to complex changes that take place in B. burgdorferi gene expression in order to facilitate dissemination within the tick and transmission. Although both nymphal and adult female I. scapularis are capable of transmitting B. burgdorferi to human and animal hosts, the nymphal stage is the primary vector of Lyme disease spirochetes to humans due to their small size (<2 mm) and their ability to feed for the required 2 days before they are detected and removed (Falco et al., 1996). 3.1
GENOME OF B. BURGDORFERI
B. burgdorferi sensu stricto strain B31C1 contains a segmented genome that is approximately 1.5 Mb and is composed of a 950-kb linear chromosome and 21 extra-chromosomal linear and circular plasmids between 5 and 56 kb in size, which total approximately 620 kb (Fraser et al., 1997; Casjens et al., 2000). The linear chromosome encodes 853 genes, which primarily encode housekeeping proteins involved in DNA replication, transcription, translation, solute transport and energy metabolism (Fraser et al., 1997). The linear and circular plasmids potentially encode 535 primarily unique genes that do not share similarity to genes outside of the Borrelia genus, indicating that these genes are possibly required for the adaptation of the spirochetes to multiple hosts (Casjens et al., 2000). Interestingly, the plasmids also contain approximately 167 non-functional pseudogenes that are the result of DNA rearrangements, suggesting that the plasmids may still be evolving. Several of the plasmids are unstable and easily lost during in vitro propagation of the spirochetes, supporting the idea that they are required for vector/host adaptation and colonization (Rosa et al., 2005). Genetic manipulation of Borrelia has been difficult due to the unstable genome and the presence of a plasmid-encoded restriction modification system that
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degrades foreign DNA. However, several recent experimental approaches have circumvented these limitations and successfully introduced foreign DNA into the spirochetes (Stewart et al., 2004; Stewart and Rosa, 2008). 3.2
B. BURGDORFERI GENES THAT FACILITATE TICK COLONIZATION AND PERSISTENCE
From the instant B. burgdorferi spirochetes are ingested by larval I. scapularis ticks, profound changes in spirochetal gene expression and protein production begin (Schwan and Piesman, 2000). Multiple spirochetal genes are differentially regulated to allow for successful colonization and persistence within the tick gut. Recent advances in the genetic manipulation of B. burgdorferi have allowed site directed mutagenesis experiments to determine the functional significance of differentially regulated genes in the tick vector (Stewart and Rosa, 2008). Moreover, microarray technology has enabled studies on the upregulation and downregulation of specific B. burgdorferi genes within the tick (Revel et al., 2002), and RNAi technology has allowed the specific inhibition of selected tick genes, demonstrating their importance for successful tick feeding and pathogen transmission (Soares et al., 2005). The intricate changes that occur in both the spirochete and the tick during spirochete–tick colonization and transmission may soon be unravelled at the molecular level. In the future, this information may hopefully lead to the development of new transmission blocking tools. 3.2.1
ospAB operon
Two well-characterized genes that are required for successful tick gut colonization are ospA (outer surface protein A) and ospB, which are located together in an operon on linear plasmid 54 (lp54) (Pal and Fikrig, 2003). More is known about the role ospA plays in the transmission of B. burgdorferi than is known about ospB. OspA is generally not expressed by spirochetes while they reside in the vertebrate host. However, as soon as spirochetes are ingested by larval I. scapularis, they begin expressing ospA (Schwan et al., 1995). Expression of the ospA and ospB genes is regulated in vitro by several physiochemical signals, including temperature, pH and cell density (Indest et al., 1997; Carroll et al., 1999; Ojaimi et al., 2002, 2003; Revel et al., 2002). When grown under ‘‘tick’’ like conditions (low temperature, high pH) in vitro, OspA and OspB are predominantly expressed. Furthermore, spirochetes entering the tick gut from an infected vertebrate host during tick feeding express ospA and ospB, also indicating physiochemical signals control the expression of ospA and ospB in the feeding tick (Schwan et al., 1995; Stevenson et al., 1995). OspA and OspB are important for successful tick colonization by B. burgdorferi as ospAB mutants and ospB mutants, which were fully infectious in mice, were incapable of persisting within the tick gut
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when ingested from an infected mouse (Table 4) (Yang et al., 2004; Neelakanta et al., 2007). Additionally, recombinant OspA and OspB specifically bind to tick gut extracts, and the passive transfer of non-Borreliacidal OspA or OspB antibodies to an infected mouse prevented adherence of spirochetes to the tick gut epithelium during tick feeding (de Silva et al., 1997; Pal et al., 2000, 2001; Fikrig et al., 2004; Neelakanta et al., 2007). Interestingly, constitutive expression of ospA prevented mutant spirochetes from infecting immunocompetent mice, indicating that ospA expression is necessary for tick colonization and not for host infection (Strother et al., 2007). 3.2.2
ospC
Besides OspA and OspB, another outer surface membrane protein, OspC, has been well characterized in B. burgdorferi. Expression of ospC is upregulated during transmission of the spirochetes from the nymphal tick to a new host (Schwan et al., 1995; Schwan and Piesman, 2000). OspC is absolutely required for host infection as null mutants fail to infect mice during transmission (Table 4) (Grimm et al., 2004b; Pal et al., 2004b). When tick blood feeding begins, spirochetes in the tick gut downregulate ospA expression while upregulating ospC expression (Schwan et al., 1995; de Silva et al., 1996; Schwan and Piesman, 2000; Schwan, 2003). Studies have indicated that OspC is necessary for migration of the spirochetes to the salivary glands during transmission as B. burgdorferi and B. afzelii ospC mutants fail to invade tick salivary glands (Pal et al., 2004b; Fingerle et al., 2007). However, separate studies using another B. burgdorferi ospC null mutant strain than the previous studies established that OspC is only required for host infection and not migration or
TABLE 4 Proteins that aid in Borrelia tick colonization, persistence and host transmission Borrelia protein
Target binding protein
OspA and OspB
TROSPA
BptA Dps OspC
– – Salp15
Erps and CRASPs
fH and FHL-1
–
Isac and Salp20
Facilitates Tick gut colonization Tick persistence Tick persistence Transmission to host Transmission to host Transmission to host
References Pal et al. (2004a) Revel et al. (2005) Li et al. (2007) Ramamoorthi et al. (2005) Hartmann et al. (2006) Soares et al. (2005) and Tyson et al. (2007)
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dissemination within the tick during feeding (Grimm et al., 2004b; Stewart et al., 2006; Tilly et al., 2006, 2007). Complementation of the B. afzelii ospC mutant with wild-type ospC in trans restored the ability of the mutant to disseminate and invade the tick salivary glands during feeding, suggesting that ospC expression is necessary for salivary gland invasion during transmission (Fingerle et al., 2007). Furthermore, a tick salivary protein was found to bind OspC, promoting transmission of B. burgdorferi to a new host during feeding (Ramamoorthi et al., 2005). Due to the conflicting results of multiple studies, the role of OspC within the spirochetes during tick blood feeding and transmission is currently inconclusive. In an elegant series of experiments, the central regulatory Rrp2–RpoN–RpoS pathway was shown to control the reciprocal expression of ospA and ospC (Boardman et al., 2008; He et al., 2008). Basically, Rrp2, in concert with RpoN, regulates the alternative sigma factor RpoS. When RpoS is abundantly expressed, it upregulates many Borrelia genes, including ospC. The Rrp2– RpoN–RpoS pathway also downregulates ospA. In flat infected nymphs, the Borrelia Rrp2–RpoN–RpoS network is not activated and the spirochetes express ospA and remain bound to the tick midgut. When the infected nymph begins feeding, the Rrp2–RpoN–RpoS pathway becomes activated and the spirochetes downregulate ospA while upregulating ospC. The spirochetes then detach from and leave the tick midgut, and begin their migration to the tick salivary glands where subsequent transmission to the vertebrate host will occur. In this manner, the spirochete is exquisitely adapted to maximizing its potential for transmission and survival in both the tick vector and vertebrate host (Yang et al., 2003). 3.2.3
Genes of lp25
B. burgdorferi contains numerous linear and circular plasmids that are required for host infection and tick colonization. During culture of the spirochetes in vitro, many of these plasmids are lost, which provides information about non-essential plasmids necessary for either host infection or tick colonization. Linear plasmid 25 (lp25) is required by B. burgdorferi for both host infection and tick colonization (Purser et al., 2003; Grimm et al., 2004a; Strother and de Silva, 2005; Strother et al., 2005). When bbe22, a gene on lp25 that encodes nicotinamidase, was expressed in spirochetes lacking lp25, host infectivity and partial tick infectivity were restored (Purser et al., 2003; Strother and de Silva, 2005). Thus, expression of bbe22 is required for host infection, but other genes on lp25 are also required for tick colonization. BptA (bbe16), another gene on lp25 that potentially encodes an outer surface lipoprotein, was found to be required for tick colonization (Table 4) (Revel et al., 2005). Mutant B. burgdorferi strains lacking bptA failed to colonize ticks, but tick infectivity was restored in complemented mutants, indicating the importance of this gene for tick colonization by the spirochetes.
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Chromosomal genes
In addition to plasmid-encoded genes, several chromosomal genes have also been identified that are necessary for tick colonization and host infectivity. B. burgdorferi gene bb0690, which encodes a protein having similarity to DNA-binding proteins that are induced during starvation (Dps proteins), is highly expressed by the spirochetes during inter-moult periods in the tick (Table 4) (Li et al., 2007). Mutant spirochetes successfully infected mice and were transmitted to naı¨ve larval ticks during the feeding period. However, during the inter-moult period between blood feedings, the dps mutant spirochetes failed to survive and were not transmitted from moulted nymphal ticks to a new host. Since Dps proteins serve to protect DNA against damage during starvation and oxidative stress, BB0690 may protect B. burgdorferi DNA from damage in the tick. Another chromosomally encoded gene, bb0365, also appears to be vital for the persistence of B. burgdorferi within ticks (Pal et al., 2008). Bb0365deficient spirochetes were fully infectious to mice and were transmitted to naı¨ve ticks during feeding, but displayed decreased survival rates when compared to wild-type spirochetes in the fed ticks. These studies suggest that expression of bb0365, like bb0690, facilitates persistence of B. burgdorferi in ticks. 3.2.5
CRASPs
A whole series of proteins are expressed by B. burgdorferi sensu stricto that allow it to resist complement when transmitted to vertebrate hosts. These proteins expressed by B. burgdorferi allow the spirochetes to bind host proteins known as factor H or factor H-like protein-1 (FHL-1), which are natural host complement regulators that limit host complement activation and prevent host complement attack. The B. burgdorferi genes that bind these natural host complement regulators consist of three types: Erps (OspE/F-related proteins), BbCRASP-2 (complement regulator-acquiring surface proteins) and BbCRASP-1 (Table 4) (Hartmann et al., 2006). In an intricate series of adaptations to transmission by ticks, spirochetes in the unfed nymphal tick midgut failed to express detectable levels of CRASPs. When the infected ticks began to feed, Erp proteins were initially expressed while BbCRASP-1 and -2 were not expressed. As the nymphal ticks continued to feed, spirochetes were transmitted through the tick salivary glands and deposited into the host skin. At that point the transmitted spirochetes were expressing Erps and BbCRASP-1. After approximately 2 weeks of mammalian infection, the spirochetes began producing BbCRASP-2 (Bykowski et al., 2008). Even though BbCRASP-2 is produced during host infection, a BbCRASP-2-deficient B. burgdorferi strain was recently found to be insensitive to complement-mediated killing in vitro and retained full host infectivity and pathogenesis in vivo, suggesting that this gene alone does not confer protection from host complement attack
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(Coleman et al., 2008). The spectrum of CRASPs expressed by B. burgdorferi sensu stricto allow the spirochete to resist host complement and infect both mammalian and avian hosts, whereas other genospecies are more restricted in their CRASP expression: B. afzelii only infects rodents and most B. garinii strains only infect birds (Kurtenbach et al., 2002). 3.3
TICK PROTEINS THAT FACILITATE BORRELIA TRANSMISSION
The anti-haemostatic, anti-inflammatory and immunosuppressive activities of tick saliva facilitate the tick during feeding, and may also contribute to salivaactivated transmission (SAT) of various pathogens. SAT is defined as the promotion of tick-borne pathogen transmission through the action of tick salivary components on the host (Nuttall and Labuda, 2004). As the tick is salivating during feeding, it is secreting its anti-haemostatic, anti-inflammatory and immunosuppressive components into the host and creating an immunosuppressed environment in which the pathogen enters. Several tick-borne viruses and bacteria, including various species of Borrelia, have been shown to display enhanced infectivity in the presence of tick SGE (Jones et al., 1992a,b,c; Labuda et al., 1993; Pechova´ et al., 2002; Zeidner et al., 2002; Lima et al., 2005; Ky´ckova´ and Kopecky´, 2006; Macha´ckova´ et al., 2006). In fact, I. scapularis expresses a salivary protein that directly binds OspC of B. burgdorferi, enhancing transmission of the pathogen to a murine host (Ramamoorthi et al., 2005). The results of multiple studies have indicated that tick salivary components enhance Borrelia infectivity. 3.3.1
Salp15
Recently, I. scapularis salivary protein 15 (Salp15), was shown to bind B. burgdorferi OspC and facilitate transmission of the spirochetes to a new host (Table 4) (Ramamoorthi et al., 2005). RNAi of salp15 in I. scapularis nymphs resulted in the transmission of fewer spirochetes to naı¨ve mice when compared to mock-treated ticks, further suggesting a role for Salp15 in spirochete transmission. Salp15 homologues were also identified in a related tick species, I. ricinus, which is the vector of European Lyme borreliosis (Hovius et al., 2007). Salp15 from both I. ricinus and I. scapularis protected serum-sensitive strains of B. burgdorferi from complement-mediated killing, indicating that these proteins may bind spirochetes during transmission and protect them from host immune attack (Schuijt et al., 2008). Amazingly, the presence of B. burgdorferi in the tick salivary glands was shown to enhance Salp15 expression (Ramamoorthi et al., 2005). Thus, the Lyme disease spirochete not only ‘‘usurps’’ the tick protein Salp15 for its own survival, but also appears to increase the amount of Salp15 produced in the tick.
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TROSPA
A tick protein that mediates attachment of B. burgdorferi to the tick gut has been identified in I. scapularis ticks. TROSPA, the tick receptor for OspA, specifically binds spirochetes by directly interacting with OspA, which is expressed by the spirochetes when they enter the tick gut in an infected blood meal (Table 4) (Pal et al., 2004a). The function of TROSPA in the tick remains to be elucidated, but expression of TROSPA increased during B. burgdorferi infection and decreased during tick engorgement, corresponding to the periods when spirochetes downregulate ospA expression, upregulate ospC expression, exit the gut of an infected tick and migrate to the salivary glands for transmission to a new host. RNAi of TROSPA reduced the numbers of spirochetes acquired by nymphal ticks during feeding. Similarly, acquisition of B. burgdorferi by nymphal ticks was reduced during tick feeding on infected mice passively transferred with TRSOPA antisera, indicating this tick protein promotes successful spirochete–tick gut colonization. 3.3.3
Salivary anti-complement proteins
As described previously, the anti-complement protein, Isac, was originally identified in I. scapularis (Valenzuela et al., 2000). Several studies have described additional proteins sharing similarity with Isac in I. scapularis and I. ricinus, which together with Isac, comprise a large family of proteins, the Isac-like protein (ILP) family (Das et al., 2001; Soares et al., 2005; Ribeiro et al., 2006; Daix et al., 2007; Couvreur et al., 2008). Multiple ILP family members inhibit the alternative complement pathway by binding properdin, causing dissociation of the C3 convertase (Couvreur et al., 2008; Tyson et al., 2008) and facilitating the tick during tick feeding. Specific species of Borrelia are effectively killed by the alternative complement pathway of certain hosts (van Dam et al., 1997; Kurtenbach et al., 1998). Salp20 protected serum-sensitive Borrelia strains from complement-mediated killing in vitro, indicating that the ILPs may also protect spirochetes from damage by the host immune system at the vector–host interface (Table 4) (Tyson et al., 2007). RNAi of isac prevented successful feeding of nymphal ticks and resulted in lower numbers of Borrelia compared to mock treated ticks when the ticks were fed on infected mice (Table 4) (Soares et al., 2005). These results support a potential role for Isac, and possibly similar proteins in the ILP family, in the successful transmission of Borrelia from a host. Further characterization of tick salivary proteins will undoubtedly identify additional tick proteins required for successful Borrelia tick and host infectivity.
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Summary
The relationship between the Lyme disease spirochete, its tick vectors and vertebrate hosts is an intricate web of delicate interactions. Ticks produce an assortment of pharmacologically active compounds in their saliva that allow the ticks to acquire a host blood meal while minimizing their chance of being detected by the host and removed. These salivary compounds include antihaemostatic components, anti-inflammatory components and immunosuppressive components. The tick midgut and haemolymph also contain anticoagulants, antimicrobials and haem-binding proteins. Not to be outdone, B. burgdorferi regulates its outer surface protein expression to initially remain in the tick midgut during the moulting process from larva to nymph by expressing OspA, which binds to the tick midgut protein TROSPA. Once the nymphal tick starts to feed, the spirochete downregulates OspA, leaves the midgut and travels to the salivary gland where it actually stimulates the production of a protein (Salp15) that helps it infect vertebrate hosts. Hopefully, the knowledge gained regarding these complex spirochete–tick–host relationships may hopefully lead to the development of new transmission blocking tools to prevent Lyme disease. Acknowledgements The authors would like to thank Dr. Aravinda de Silva for his critical reading of this manuscript, as well as serving as a guide and mentor to one of us (K.R.T.) during her Ph.D. studies. Parts of this chapter overlap with material presented as part of the Ph.D. thesis of K.R.T. at the Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC. The authors would also like to thank Dr. Jose´ M. C. Ribeiro for his incredible body of work and inspiration to those studying vector–host–pathogen relationships. References Abbas, A. K., Lichtman, A. H. and Pober, J. S. (2000). Cellular and Molecular Immunology. 4th ed. W.B. Saunders Company, Philadelphia, PA. Abdul Alim, M., Tsuji, N., Miyoshi, T., Khyrul Islam, M., Huang, X., Motobu, M. and Fujisaki, K. (2007). Characterization of asparaginyl endopeptidase, legumain induced by blood feeding in the ixodid tick Haemaphysalis longicornis. Insect Biochem. Mol. Biol. 37, 911–922. Alarcon-Chaidez, F. J., Mu¨ller-Doblies, U. U. and Wikel, S. (2003). Characterization of a recombinant immunomodulatory protein from the salivary glands of Dermacentor andersoni. Parasite Immunol. 25, 69–77. Alim, M. A., Tsuji, N., Miyoshi, T., Islam, M. K., Huang, X., Hatta, T. and Fujisaki, K. (2008). HILgm2, a member of asparaginyl endopeptidases/legumains in the midgut of the ixodid tick Haemaphysalis longicornis, is involved in blood-meal digestion. J. Insect Physiol. 54, 573–585.
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Urioste, S., Hall, L. R., Telford, S. R. III and Titus, R. G. (1994). Saliva of the Lyme disease vector, Ixodes dammini, blocks cell activation by a nonprostaglandin E2-dependent mechanism. J. Exp. Med. 180, 1077–1085. Valenzuela, J. G. (2004). Exploring tick saliva: from biochemistry to ‘sialomes’ and functional genomics. Parasitology 129, S83–S94. Valenzuela, J. G., Charlab, R., Mather, T. N. and Ribeiro, J. M. C. (2000). Purification, cloning, and expression of a novel salivary anticomplement protein from the tick, Ixodes scapularis. J. Biol. Chem. 275, 18717–18723. Valenzuela, J. G., Francischetti, I. M. B., Pham, V. M., Garfield, M. K., Mather, T. N. and Ribeiro, J. M. C. (2002). Exploring the sialome of the tick Ixodes scapularis. J. Exp. Biol. 205, 2843–2864. van Dam, A. P., Oei, A., Jaspars, R., Fijen, C., Wilske, B., Spanjaard, L. and Dankert, J. (1997). Complement-mediated serum sensitivity among spirochetes that cause Lyme disease. Infect. Immun. 65, 1228–1236. van de Locht, A., Stubbs, M. T., Bode, W., Friedrich, T., Bollschweiler, C., Hoffken, W. and Huber, R. (1996). The ornithodorin-thrombin crystal structure, a key to the TAP enigma? EMBO J. 15, 6011–6017. Vancˇova´, I., Slova´k, M., Hajnicka´, V., Labuda, M., Sˇimo, L., Peterkova´, K., Hails, R. S. and Nuttall, P. A. (2007). Differential anti-chemokine activity of Amblyomma variegatum adult ticks during blood-feeding. Parasite Immunol. 29, 169–177. Vundla, W. R., Brossard, M., Pearson, D. J. and Labongo, V. L. (1992). Characterization of aspartic proteinases from the gut of the tick, Rhipicephalus appendiculatus. Insect Biochem. Mol. Biol. 22, 405–410. Walport, M. J. (2001a). Complement. First of two parts. N. Engl. J. Med. 344, 1058–1066. Walport, M. J. (2001b). Complement. Second of two parts. N. Engl. J. Med. 344, 1140–1144. Wang, H. and Nuttall, P. A. (1994). Excretion of host immunoglobulin in tick saliva and detection of IgG-binding proteins in tick haemolymph and salivary glands. Parasitology 109, 525–530. Wang, H. and Nuttall, P. A. (1995a). Immunoglobulin G binding proteins in male Rhipicephalus appendiculatus ticks. Parasite Immunol. 17, 517–524. Wang, H. and Nuttall, P. A. (1995b). Immunoglobulin-G binding proteins in the ixodid ticks, Rhipicephalus appendiculatus, Amblyomma variegatum and Ixodes hexagonus. Parasitology 111, 161–165. Wang, X., Coons, L. B., Taylor, D. B., Stevens, S. E. Jr. and Gartner, T. K. (1996). Variabilin, a novel RGD-containing antagonist of glycoprotein IIb–IIIa and platelet aggregation inhibitor from the hard tick Dermacentor variabilis. J. Biol. Chem. 271, 17785–17790. Waxman, L. and Connolly, T. M. (1993). Isolation of an inhibitor selective for collagenstimulated platelet aggregation from the soft tick Ornithodoros moubata. J. Biol. Chem. 268, 5445–5449. Waxman, L., Smith, D. E., Arcuri, K. E. and Vlasuk, G. P. (1990). Tick anticoagulant peptide (TAP) is a novel inhibitor of blood coagulation factor Xa. Science 248, 593–596. Wikel, S. K. (1985). Effects of tick infestation on the plaque-forming cell response to a thymic dependent antigen. Ann. Trop. Med. Parasitol. 79, 195–198. Wikel, S. K. (1996). Host immunity to ticks. Annu. Rev. Entomol. 41, 1–22. Xu, G., Fang, Q. Q., Keirans, J. E. and Durden, L. A. (2004). Cloning and sequencing of putative calreticulin complementary DNAs from four hard tick species. J. Parasitol. 90, 73–78.
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Yang, X. F., Alani, S. M. and Norgard, M. V. (2003). The response regulator Rrp2 is essential for the expression of major membrane lipoproteins in Borrelia burgdorferi. Proc. Natl. Acad. Sci. USA 100, 11001–11006. Yang, X. F., Pal, U., Alani, S. M., Fikrig, E. and Norgard, M. V. (2004). Essential role for OspA/B in the life cycle of the Lyme disease spirochete. J. Exp. Med. 199, 641–648. Yu, D., Liang, J., Yu, H., Wu, H., Xu, C., Liu, J. and Lai, R. (2006). A tick B-cell inhibitory protein from salivary glands of the hard tick, Hyalomma asiaticum asiaticum. Biochem. Biophys. Res. Commun. 343, 585–590. Zeidner, N. S., Schneider, B. S., Nuncio, M. S., Gern, L. and Piesman, J. (2002). Coinoculation of Borrelia spp. with tick salivary gland lysate enhances spirochete load in mice and is tick species-specific. J. Parasitol. 88, 1276–1278. Zhou, J., Liao, M., Ueda, M., Gong, H., Xuan, X. and Fujisaki, K. (2007). Sequence characterization and expression patterns of two defensin-like antimicrobial peptides from the tick Haemaphysalis longicornis. Peptides 28, 1304–1310. Zhu, K., Bowman, A. S., Brigham, D. L., Essenberg, R. C., Dillwith, J. W. and Sauer, J. R. (1997a). Isolation and characterization of americanin, a specific inhibitor of thrombin, from the salivary glands of the lone star tick Amblyomma americanum (L.). Exp. Parasitol. 87, 30–38. Zhu, K., Sauer, J. R., Bowman, A. S. and Dillwith, J. W. (1997b). Identification and characterization of anticoagulant activities in the saliva of the lone star tick, Amblyomma americanum (L.). J. Parasitol. 83, 38–43.
Epidemiological Consequences of the Ecological Physiology of Ticks Sarah E. Randolph Department of Zoology, University of Oxford, Oxford OX1 3PS, United Kingdom
This paper is dedicated to the memory of Klaus Kurtenbach, 1959–2009, who contributed many novel ideas to the field of tick-borne diseases, particularly Lyme borreliosis. 1 Introduction 297 2 Physiological adaptations for tick feeding habits 299 2.1 Cuticle structure and function 300 2.2 Adaptive patterns of cuticular wax deposition 301 2.3 Respiration and metabolic rates 304 3 Water balance, defence and consequences for pathogen conveyance 305 3.1 Osmoregulation: Spitting into the host 305 3.2 Immunomodulation: Salivary pharmacology 306 3.3 Pathogen traffic 309 4 Seeking a host – Where and when? 310 4.1 Water balance constraints 311 4.2 Sensory systems 314 4.3 Recruitment of unfed ticks to the questing population 316 4.4 Fat reserves determine lifespan 323 5 Epidemiological consequences of tick phenology 323 5.1 Focal distribution of tick-borne encephalitis 324 5.2 Widespread, but partitioned, distribution of B. burgdorferi s. l. 326 5.3 Sensitivity to climate versus impact of climate change 327 6 Conclusions 328 Acknowledgements 329 References 329
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Introduction
Explanations of the epidemiology of tick-borne diseases (TBDs) can be based on the operation of the tick’s spiracles, or its salivary glands, or its sense organs, or any other physiological system that determines the differential ADVANCES IN INSECT PHYSIOLOGY VOL. 37 ISBN 978-0-12-374829-4 DOI: 10.1016/S0065-2806(09)37006-X
Copyright # 2009 by Elsevier Ltd All rights of reproduction in any form reserved
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survival, phenology and reproduction of the vector tick under variable environmental conditions. A tick, like any other organism, clearly depends on every tissue structure and every organ system for its complete bodily functioning, but certain systems have more direct influence than others on the timing, pace and overall potential of the transmission of microbes between ticks via vertebrates. Furthermore, the quantitative impacts of environmental factors been characterized for only some of these systems. These will be the subject of this review. In a previous review (Randolph, 2004), I emphasized the hierarchy of processes and patterns inherent in the epidemiological risk posed by ticks as vectors. At the base is the physiological ecology of ticks, the adaptation of individuals to their natural habitats. As each tick is born, lives and dies, it contributes to the overall rates of the demographic processes that add up to the next level, population ecology. There I dealt with tick– host relationships as properties of populations, but here I shall put more emphasis on ‘‘ecological physiology’’ at the level of individual ticks as ticks per se, with the unintended but critical consequences for their performance as vectors. On the other hand, if pathogens have any impact on the rates of tick survival and/or reproduction (Davey, 1981; Gray, 1982; Randolph, 1991), then adaptations to cope with this vector–pathogen interaction would be selected for. Epidemiologists strive to describe, explain and therefore predict the variable risk of infection in space and time. In the case of zoonotic vector-borne pathogens, whose cycles are maintained by transmission amongst wildlife reservoir hosts, the probability of human infection depends on the abundance of infected vectors and the exposure of humans to those vectors. Even though the latter may be responsible for significant short- and long-term variations in disease incidence (Randolph et al., 2008; Sˇumilo et al., 2008), it is the former with which we are concerned here and which is largely determined by the vectors’ physiological responses to habitat conditions that drive both their population dynamics and the transmission dynamics of the microbes. Vector-borne pathogens differ from most other indirectly transmitted ones in not having any freeliving stage, as they are typically acquired and delivered directly from and to vertebrates by blood-feeding arthropods. Most insect vectors feed repeatedly as adults, albeit periodically as bingers rather than frequently as sippers to avoid the additional mortality associated with stealing blood from defensive hosts. They can therefore acquire pathogens and go on to transmit them repeatedly within their adult lifespan. In contrast, the key characteristic of ticks as haematophages is their extraordinary biology associated with the adaptation of taking single (in the case of hard ticks, the Ixodidae) or very few (in the case of soft ticks, the Argasidae) extremely large blood meals per life stage, as a larva, nymph and adult. Between each meal, ticks leave their hosts to undergo development and moulting to the next stage on the ground (although some species take both larval and nymphal meals from the same hosts before dropping off, while the one-host Boophilus spp. take all three
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meals from the same host). The consequence for the pathogens is that those acquired from an infected vertebrate by ticks feeding as one stage cannot be transmitted to a new susceptible host until the tick feeds again as the next stage, after a long developmental period biologically distinct from, but functionally equivalent to, the extrinsic incubation period of insect-borne microbes. The consequence for the tick is that they are only periodic parasites, spending the vast majority of their time subject to ambient environmental conditions unprotected by the buffering effects of their hosts. This pattern of feeding demands certain unique physiological systems and also imposes certain constraints on the performance of ticks as vectors.
2
Physiological adaptations for tick feeding habits
It is tempting to explain the habit of taking a few very large meals as the ticks’ solution to the problem of contacting hosts while hampered by the absence of wings. This, however, is a double-edged sword, because the long interval between accesses to energy reserves puts an especially high premium on energetic economy and efficiency in seeking hosts. Some species, particularly amongst the Argasid ticks, occupy their hosts’ living quarters, allowing easy access to nutrition with the minimum of locomotion. The majority of species are sit-and-wait ambushers, positioning themselves on the vegetation from where they catch on to a passing host. Others are hunters, sheltering in cracks and under stones and emerging to hunt hosts actively when the opportunity arises. These tend to be larger species, with the concomitant metabolic advantages (West et al., 2000), for example Hyalomma spp; Sonenshine describes how ‘‘camel ticks, Hyalomma dromedarii, emerge from the barren sand around desert caravansaries and run across the hot, exposed ground to attack people and livestock when these hosts were resting near their shelters’’ (Sonenshine, 1991). Any activity requires respiration sooner or later. Although hungry ticks are able to close their spiracles and survive for long periods without air exchange (Arthur, 1960; Hefnawy, 1970), opening the spiracles during locomotion causes water loss exacerbated by exposure to low humidity outside the shelter of their refuges. Water loss remains the Achilles heel of all terrestrial arthropods, including ticks. Specific adaptations to overcome this (see below) build on the generic foundation of the waterproofed cuticle. For ticks, however, the structure and material of the cuticle must be adapted to accommodate a volume change of one (for immature stages) to two (for adults) orders of magnitude during feeding, compared with a mere doubling of volume during insect blood meals. Given the high water content of blood as a food, the acquisition of enough energy poses even greater problems for ticks than for, say, protein-gorging snakes, so that ticks are faced alternately with over-hydration while attached to their hosts and
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dehydration while off their hosts, both of which are dealt with by the salivary glands (see Section 3). 2.1
CUTICLE STRUCTURE AND FUNCTION
Over half a century ago, one of the pioneers of tick biology, A.D. Lees, described the dynamics of cuticle change in relation to the dynamics of tick feeding (Lees, 1952), focusing on Ixodes ricinus, the most widespread, abundant and multi-potent vector throughout Europe and Eurasia west of the Urals. A combination of deposition and distension of different components of the whole cuticle keeps step with the exponential rate of blood uptake. Over the first 6 days the body weight of an adult I. ricinus increases by one order of magnitude, followed by a further increase of the same magnitude by the end of the ca. 10-day feeding period. This pattern is common for many tick species and conforms only loosely to a steady exponential rate of increase, with the rate of blood intake over the first 6 days slower than the overall average rate, and faster thereafter (Fig. 1A), indicating specific physiological adaptations. In unfed ticks, the epicuticular layer of the alloscutal cuticle is deeply folded, so that it can accommodate the full blood meal by flattening the folds but not increasing its area (Lees, 1952; Hackman and Filshie, 1982). This is permitted by bands of resilin, a rubbery flexible protein, lying systematically in the valleys between the folded lamellae (Dillinger and Kesel, 2002). The distension of the alloscutal cuticle could thus be passive, driven by increased internal pressure (Dillinger and Kesel, 2002). Structural analysis has shown that the near-perfect elasticity of resilin is due to the tendency of the peptide chains to occur in a nearly random conformation, which can be easily and reversibly deformed by external forces, while inter-chain cross-links prevent the peptide chains from sliding past each other when the material is stretched (Andersen and Roepstorff, 2005). It is not known whether the alloscutal cuticle behaves more like rubber or plastic (i.e. flowing under tension) during the tick’s feed. At the same time, the underlying non-sclerotized endocuticle first increases in thickness with the deposition of more material with a more open structure than the initial layer. It is then stretched during the last part of feeding (body weight >60 mg) when most blood is taken in very rapidly but cuticular weight shows no further increase (Andersen and Roepstorff, 2005). Resilin is not unique to ticks, but its distribution throughout so much of the body surface, rather than being confined to body parts such as joints that require special deformability, as in many insects (Weis-Fogh, 1965), is a crucial factor in the exceptional feeding biology of ticks that itself underpins the epidemiological patterns of TBDs. The need for new material to be deposited during the feed must be a factor in prolonging the meal, and that itself has significant consequences for the dynamics of pathogen distribution, migration and replication within the vector, resulting in specific patterns of transmission (see Section 3.3).
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2.2
ADAPTIVE PATTERNS OF CUTICULAR WAX DEPOSITION
Although much of the concentration of the blood meal is achieved by secretion of excess fluid by the tick salivary glands back into the host (see Section 3), water loss across the integument may supplement this route, with the advantage that it requires no energy. It depends upon the functional plasticity of tick
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integument (Amosova, 1983) as its role switches from controlling water loss while the tick is off the host to facilitating water loss while the tick is feeding. A pivotal study on ticks revealed that the quantity and quality of cuticular lipid deposition changed during development both within and between life stages, but that other strategies were used, at least by Amblyomma americanum, to respond to variable atmospheric relative humidity (RH) (Yoder et al., 1997). After moulting, nymphs and adults deposited only modest amounts of surface wax during their pre-feed phase, and none while feeding, and accordingly lost large amounts of water while feeding (Fig. 2). As a result, water loss increased in feeding ticks by >50% in nymphs and >80% in females, just as would aid blood meal concentration. This water loss would also be facilitated by the increase in exposed surface area as the alloscutal cuticle unfolds and stretches (see above). Once engorged and detached from the host, however, both stages increased their cuticular wax nearly threefold, with a corresponding >50% drop in water loss. Interestingly, although only a modest amount of additional wax was laid down by the late pharate female before moulting, water loss decreased sharply after moulting (despite the tick losing one of its protective layers, the outer nymphal exuvium), which suggests a change in the quality, rather than quantity, of the lipid (Yoder et al., 1997), as is true of most arthropods (Hadley, 1994). An obvious question from the ecological viewpoint is to ask to what extent these dynamics are affected by differential environmental moisture stresses. Newly emerged female A. americanum held at 33% or 93% RH (both at 22–24 C) showed no difference in the quantity of their surface lipid, but those in the drier atmosphere lost 10% less lipid in the shed exuvia, and a massive 44% less lipid in their faeces (Yoder et al., 1997). Evidently the engorged nymph can detect and react to moisture stress not by committing additional wax irrevocably to the adult cuticle (which might later impede passive water loss during feeding), but by reclaiming more lipid from the exuvium and faeces. Yoder et al. (1997) suggest that this tick species relies more on behaviour than physical properties to conserve water during host location, a more flexible strategy for an organism faced with such short-term fluctuations in ambient and internal moisture conditions. Some ticks species are known to inhabit unusually dry environments, giving rise to the idea that they are especially tolerant of dehydration. In fact, one of these, the so-called kennel tick Rhipicephalus sanguineus, is thought not to differ from other species in its dehydration tolerance, capacity for water vapour absorption or free water drinking (see Section 4.1.1). Instead, it has been able to colonize human habitation, especially where dogs are present, because of its unusually low rate of water loss, at only about 44, 53 and 67% the rate shown (above) for A. americanum as unfed nymphs, adult females and adult males, respectively, under comparable conditions (Yoder et al., 2006). Indeed, R. sanguineus appears to be restricted to dry habitats because it is intolerant of moisture-rich environments due to its xerophilic (water-conserving) adaptations. The mechanisms underlying these adaptations have not been described,
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but the phenomenon is epidemiologically highly significant. This tick has a global distribution, found in all the major bio-geographical regions of the world (Camicas et al., 1998). Its occupation of peridomestic habitats and use of dogs as primary hosts makes it a medically significant vector of many rickettsial pathogens (Parola et al., 2005, 2008), including the highly virulent Rickettsia rickettsii, causative agent of Rocky Mountain spotted fever (Demma et al., 2005) that is currently undergoing its third known recrudescence in the United States, up to a record 1514 cases in 2004 with a 5–10% case fatality rate
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(Dumler and Walker, 2006) (although over-diagnosis by new sub-specific tests may be partly to blame). The transport of pets by their owners is an obvious potential means of global dispersion of this tick, with a higher than average chance of establishment after arrival within the permissive microhabitats associated with dogs. 2.3
RESPIRATION AND METABOLIC RATES
Concomitant with the adaptation of feeding at such long intervals is the ability to survive long periods of starvation. Indeed, off-host ticks can survive starvation and desiccation longer than any other arthropod, up to several years in the field (Jaworski et al., 1984; Needham and Teel, 1991). Limited mobility obviously helps, but ticks are characterized by exceptionally low metabolic rates, 12% less than those of ants, beetles or spiders (Lighton and Fielden, 1995). In non-locomotory unfed, Amblyomma hebreum and Dermacentor variabilis ticks, such low weight-specific metabolic rates and associated rates of carbon dioxide emission are maintained by intermittent opening of the spiracles and discontinuous bursts of CO2 production lasting ca. 3–5 min at ca. 20–70 min intervals (Fielden et al., 1994, 1999). Once the female D. variabilis tick starts to feed, the interval of CO2 bursts decreases to ca. 2 min by the end of the slow feeding phase (day 6), but with shorter duration and thus little change in burst volume, followed by effectively continuous respiration over the 3 days of rapid engorgement. With continuous opening of the spiracles, absolute water loss rates increases ca. 10-fold during this final phase of feeding, consistent with the tick’s state of over-hydration at this parasitic phase of its life. Water loss by combined cutaneous and respiratory avenues, however, still constituted a tiny fraction of total water loss (1% of body mass – Yoder et al., 1997) compared with that excreted back into the host via the salivary glands (ca. 100% of final engorged weight for Boophilus microplus – Tatchell, 1967). Rather, the enhanced respiratory flow rate seems to be essential for excretion of CO2 that increases exponentially in absolute terms during the feeding period, associated with blood meal digestion (Fielden et al., 1999). CO2 excretion, however, increases faster than body mass early in feeding (Fig. 1B). It would be interesting to know what the metabolic costs of egg production are and whether this involves higher respiration and water loss rates, but this would be at a time when the female is expendable, doomed to die very soon after oviposition is complete. This physiological strategy of reduced metabolic activity and associated water loss via the spiracles except during feeding, although not unique, is taken to extremes in ticks, and again has direct consequences for the epidemiology of TBDs. The pace of pathogen transmission may be very slow, retarded by the long inter-feed interval, but the extended survival of ticks in certain habitats ensures an enduring reservoir of infections. This survival period, however, is inevitably shortened by the ticks’ attempts to find a host and progress to the next stage; the choice is between sitting still to conserve energy (taken to extreme by behavioural
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diapause – see Section 4.3.2) and moving to find a host even if that means dying of exhaustion in the attempt (see Section 4.2). It is, of course, the adaptive balance between the rates of the life-threatening and life-enhancing processes of individual ticks that results in the diverse patterns of vector seasonal population dynamics that in turn supports specific pathogen transmission to different degrees.
3
Water balance, defence and consequences for pathogen conveyance
Salivary glands are undoubtedly the workhorses of tick survival. They provide solutions to the alternating problems of over-hydration and dehydration, and also ensure that the tick can complete its long slow blood meal in the face of host defences. During feeding, salivary secretions carry a great deal of water back into the host as the principal means of osmoregulation, a great many bioactive molecules to facilitate feeding and survival during intimate cellular contact with the host, and also, inadvertently, act as a vehicle for pathogen transport. Salivary glands are therefore still the ‘‘focus of virtually all the medical and veterinary problems associated with ticks’’ (Kaufman, 1989). 3.1
OSMOREGULATION: SPITTING INTO THE HOST
Blood is not a good food. It is nutritionally imbalanced (protein and lipid – rich, but carbohydrate – poor, and lacking certain vital nutrients such as B vitamins) so requiring symbionts in the gut, is hard to digest so that much is wastefully passed undigested through the gut and is excessively dilute. About 70% of the imbibed water and ions are actively excreted directly back into the host by the salivary glands (Kaufman et al., 1980). As befits their biological importance, salivary gland morphology, morphogenesis, function and neural control during tick feeding have all been extensively and intensively investigated and reviewed, most visually by Coons and Alberti (1999) and most recently by Anderson and Valenzuela (2008). The glands are structurally complex and transiently huge, increasing 25-fold in mass and protein content to occupy 30–50% of the adult female ixodid tick’s haemocoel while she feeds (Anderson and Valenzuela, 2008). Once feeding is complete, the glands degenerate through a sequential and highly regulated physiological process of programmed cell death orchestrated by ecdysteroid (Harris and Kaufman, 1985; et sequitor). Bowman et al. (2008) describe the current model as follows: a competent ecdysteroid receptor complex in the gland responds to rising ecdysteroid levels on completion of the blood meal that is somehow stimulated by a protein factor from the male gonad (Weiss and Kaufman, 2004). This protein appears to be of the same material as a factor, named voraxin, that is passed from males to females during copulation and stimulates engorgement, which is one of many naturally occurring substances seen to hold promise as an anti-tick vaccine (Weiss and Kaufman, 2004).
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While the fundamental energetics of tick survival and reproduction clearly depend on blood meal concentration and maintenance of ionic balance, the more specific epidemiological interest arises from the way in which pathogens have exploited this aspect of tick physiology to their advantage, as follows. It hinges on the sheer volume and timescale of fluid secretion, and also the specific constituents. 3.2 3.2.1
IMMUNOMODULATION: SALIVARY PHARMACOLOGY
Battling for a meal
Because of their very large, prolonged meals, ixodid ticks face particularly acute problems in dealing with host defences against haematophagy, including haemostasis, inflammation and immune responses. Early experimental evidence that the host may acquire protective immune and allergic responses after a certain degree of exposure to tick antigens (Trager, 1939) led to the idea that acquired resistance was a feature of a tick association with non-natural host species (Ribeiro, 1989). This was explicitly tested and supported by showing that laboratory mice did, but woodmice (Apodemus sylvaticus) did not acquire resistance and reject feeding ticks (Randolph, 1979). Later, however, differences between species of natural rodent hosts were demonstrated for Ixodes trianguliceps (Randolph, 1994) and I. ricinus (Dizij and Kurtenbach, 1995): the vole Clethrionomys (now Myodes) glareolus is better able than Apodemus spp. to protect itself against tick feeding. Many of the salivary proteins in an ever-growing list catalogued by cDNA library screening, PCR subtraction and transcriptome analysis are now recognized as the key to the tick’s ability to complete its blood meal. Putative assigned functions include anticoagulant, platelet aggregation inhibitor, antimicrobial defence, anti-haemostatic, anti-platelet, anti-inflammatory, vasodilatory, tick mouthpart maintenance and unspecified housekeeping, with still a great many of unknown function (Ribeiro et al., 2006; Anderson and Valenzuela, 2008). Functional genomic tools are urgently needed to disentangle the complexity, redundancy and variability of thousands of tick salivary gland protein fragment sequences now deposited in GenBank (Anderson and Valenzuela, 2008). Given the complexity of vertebrate immune responses thrown at ticks (Wikel, 1996), it is little wonder that ticks have responded counter-punch for punch (Table 9.1 in Brossard and Wikel, 2008) as they run the evolutionary arms race in pursuit of both their dinners and their lives (Dawkins and Krebs, 1979). Salivary gland extract (SGE) has now been shown to produce effective counter-measures to 11 classes of host defensive cells or molecules, most of which appear to show a degree of species-specificity, as would be expected from such intense dynamic duelling, but which may complicate the design of new, generic vaccines or therapeutic products.
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The ability of a tick to complete a blood meal on a host is clearly the sine qua non of that host’s complete role in tick-borne pathogen (TBP) transmission. It is possible to dream up scenarios whereby a tick takes a partial meal, sufficient to acquire infection, before being rejected and then going on to feed on another host, but there is no evidence yet that this is anything other than a rare, minority event under natural field conditions. Host-feeding specificity by ticks is therefore a crucial factor in the patterns of pathogen transmission amongst free-living vertebrates. Its variability and causes are hotly debated; extensive survey data indicate opportunistic generalism (Cumming, 1998), while intensive observational studies provide evidence of specificity (Horak et al., 1991). The immunobattle may play some part. Tick defence against the host alternative complement cascade, that is activated very early against feeding ticks (Wikel and Allen, 1977), has been shown to be specific to tick–host relationships. Anti-complement activity in SGE taken from I. ricinus, I. hexagonus and I. uriae, for example, differed between host species and was correlated with the reported host range of each tick species (Lawrie et al., 1999). This suggests that different elements in the alternative complement cascade are targeted by the anti-complement activity of each Ixodes species; saliva from both unfed and feeding I. ricinus, the species with one of the most catholic host ranges known, specifically inhibits C3a generation and factor B cleavage (Lawrie et al., 2005). Is it this early inhibition near to the start of the alternative complement pathway that confers general host permissiveness to I. ricinus, with such significant vector consequences? Other recent studies support the idea that different ticks display different host complement sensitivities because they interfere with different steps in the pathway. For example, anticomplement proteins of both I. ricinus and I. scapularis inhibit the host alternative complement pathway by interacting with properdin, thereby preventing activation of the pathway and cleavage of C3 and fB (Couvreur et al., 2008; Tyson et al., 2008). Furthermore, amongst the soft ticks, Ornithodoros moubata secretes a complement inhibitor that binds C5, preventing its cleavage and the generation of C5a (Nunn et al., 2005). 3.2.2
Orchestrating pathogen transmission
Clearly, the long evolutionary battle between ticks, pathogens and hosts may result in a species-specific balance (Humair et al., 1999). In the example above, while voles are better able than mice to protect themselves immunologically against ticks, they mount a less effective immune response against tick-transmitted Lyme disease spirochaetes Borrelia burgdorferi s. l. (Kurtenbach et al., 1994) and piroplasms Babesia microti (Randolph, 1995). This differential ability of a pathogen to use particular hosts for transmission is also apparently mediated by saliva as pathogens enter the vertebrate host at a site that is highly modified by the presence of a biting tick. The presence of whole SGE or recombinant saliva protein Salp15 (Ramamoorthi et al., 2005) inoculated alongside the pathogens significantly increases the transmission of many
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TBPs, including viruses (Jones et al., 1989; Alekseev et al., 1991; Labuda et al., 1993a) and bacteria (Pechova´ et al., 2002; Zeider et al., 2002; Krocˇova´ et al., 2003; Macha´ckova´ et al., 2006). This so-called ‘‘saliva-activated (now assisted) transmission’’ (SAT) (reviewed in Nuttall and Labuda, 2008), that varies with the competence of the vector species for the pathogen in question and even the particular pathogen, helps to explain the mechanism behind pathogen transmission between co-feeding ticks in the absence of a systemic infection (Jones et al., 1987; Alekseev and Chunikhin, 1990; Labuda et al., 1993a; Gern and Rais, 1996). No longer are systemic infections above a certain threshold level seen as a necessary condition for transmission of certain pathogens, but rather viraemia and even bacteraemia may occur as an inconsistent, species-specific consequence of infection. Instead, the presence of uninfected ticks co-feeding with an infected tick is sufficient for transmission to take place. TBEV and B. burgdorferi s.s. appear to be recruited preferentially to the skin site of tick feeding, rather than reaching co-feeding ticks by generalized diffusion, because skin biopsies taken from hosts where ticks are not feeding remain negative while feeding ticks at other sites become infected (Labuda et al., 1996; Gern and Rais, 1996). When tested empirically on natural tick–hosts, different species varied inversely in the development of TBEV viraemia and their ability to support co-feeding transmission: amongst rodents, despite developing the lowest viraemic titres, Apodemus spp. mice showed the highest transmission probability (Labuda et al., 1993b). The epidemiological significance for tick-borne encephalitis virus (TBEV) lies in the fact that, by avoiding the higher virulence of systemic viraemia, more hosts survive long enough to allow ticks to complete their blood meals. The consequent ca. 50% increase in transmission potential from tick to tick via vertebrates is highly significant in a system whose conventionally perceived biology up to that point appeared too fragile to permit persistence (Randolph et al., 1996). Even this, however, is not sufficient alone (see Section 5.1). As a result of this dependence on the general but specific immunomodulatory property of tick saliva to permit tick feeding on the one hand and pathogen transmission on the other, several different types of hosts are commonly needed to support the persistence of any one pathogen. Natural cycles of B. microti, for example, require both mice and voles to perform complementary roles, one to sustain the tick population and the other to sustain the microbes. This situation is especially well recognized in systems where the adult reproductive tick stage feeds on large hosts, commonly deer or other ungulates, which are typically not competent to transmit the pathogens between ticks; it applies to most I. ricinusborne pathogens such as B. burgdorferi s. l., TBEV and Louping ill virus (Gilbert et al., 2001). The non-competence of deer in pathogen transmission has given rise to the idea that they introduce some degree of zoo-prophylaxis (i.e. reduction in the abundance of infected ticks) by wasting infected tick bites (LoGiudice et al., 2003). This needs very careful quantitative analysis of field data, not just models, to be upheld, because these same hosts also contribute to maintaining and enhancing tick populations. Hitherto, models suggest that
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zoo-prophylaxis only occurs at unrealistically high densities of deer (Rosa et al., 2003). Indeed, contrary to causing zoo-prophylaxis, an increase in roe deer abundance due to changes in land and wildlife management practices appears to be one of the most crucial factors enhancing the circulation of TBEV and, consequently the risk of TBE emergence in humans in northern Italy, and possibly in many parts of Europe (Carpi et al., 2008; Rizzoli et al., 2009). The sudden increase in deer abundance in southern Sweden in the early 1980s as a result of a crash in fox populations due to the southward spread of sarcoptic mange (Lindstro¨m et al., 1994; Kjellander and Nordstro¨m, 2003) may have contributed to the equally sudden increase in TBE incidence in 1984 (Randolph, 2001). 3.3
PATHOGEN TRAFFIC
Given the passage of such large volumes of saliva from tick to host, it is not surprising that salivary glands are the site of development and replication for many TBPs. Nevertheless, TBPs vary in their degree of cell specificity within ticks. While viruses apparently show little specificity, either within the tick body in general or the salivary glands in particular, and may be transmitted very soon after tick attachment, others infect specific cells within the glands. Theileria annulata, for example, a protozoan of considerable veterinary significance, only infects cell type ‘‘e’’ of type III acini (Bowman et al., 2008). The detailed function of each cell type (a–f) within each acinus type (I, II and III) is beyond the scope of this review, but it is relevant that both types II and III increase greatly in size during feeding. Furthermore, Bowman et al. (2008) describe how ‘‘during blood meal concentration, fluid accumulates in an expanding acinar lumen and is expelled by contraction of the adluminal cell winding in web-like fashion around the apical side of cells in acini II and III.’’ It is thus to be expected that saliva, rather than any of the other potential routes of fluid transfer (leakage of fluid from coxal glands, regurgitation, or faecal excretion) should be the vehicle of TBP transmission, but this was not conclusively shown to be the case until 1996 (Kaufman and Nuttall, 1996). Furthermore, in North America, infective B. burgdorferi s.s. spirochaetes do not appear in salivary glands until >48 h after the attachment of infected nymphal I. scapularis, as shown by inoculating susceptible mice with glands that had been dissected from infected nymphs that had fed for increasing periods (Piesman, 1995) (Fig. 3). This was corroborated by results of feeding infected ticks on mice; the probability of transmission increased rapidly from <0.05 to >0.70 during the third day of tick feeding (des Vignes et al., 2001; Hojgaard et al., 2008). There appears to be some species-specific variation in the timing of transmissibility: while I. ricinus did not transmit European strains of B. burgdorferi s.s. by natural tick bite if attached for only 48 h, B. afzelii was transmitted with increasing probability after 24 h of attachment (0.14 at 24 h and 0.5 at 48 h) (Crippa et al., 2002). Nevertheless, inoculation of homogenates of
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FIG. 3 The timing of the dispersal of B. burgdorferi s.s. to the salivary glands of nymphal I. scapularis as revealed by mouse inoculation bioassay. Drawn from data given in Piesman (1995).
whole ticks that had fed for only 24 h was infective (probability 0.6–0.7 for each strain), showing that spirochaetes are infectious in the tick before they reach the salivary glands. As Hojgaard et al. (2008) explain, this delay in infectivity may be at least partially due to the decreased production of outer surface protein A (OspA) by spirochaetes, and increased production of OspC, that both start when ticks begin to feed (Schwan and Piesman, 2000). This in turn allows spirochaetes to be released from the tick midgut protein TROSPA, migrate to the salivary glands, bind to a tick salivary gland protein Salp15, and achieve transfer to the vertebrate host (Pal et al., 2004; Ramamoorthi et al., 2005; Rosa, 2005; Hovius et al., 2007). Whatever the precise mechanism, the epidemiological consequences are clear. Human patients are at lower risk of infection if a tick is removed soon after attachment, and this may limit the need for prophylactic antibiotic treatment (Sood et al., 1997; Nadelman et al., 2001; Wormser et al., 2006).
4
Seeking a host – Where and when?
Even though ticks feed very rarely, their search for food (hosts) occupies a great deal of their time. Constraints on host-questing behaviour, driven principally by moisture and temperature conditions, are a major determinant of the variable threat they pose to humans, livestock and wildlife. The most common (but see
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Section 2) strategy is to sit on vegetation and await passing hosts, but limited tolerance to water stress and the need to maintain their water balance forces them to interrupt their questing activity to a greater or lesser extent according to environmental conditions. In addition to short-term constraints of water balance, there are longer term constraints imposed by temperature-dependent rates of development from the previous stage, that is rates of recruitment, and also by energy reserves. Questing behaviour therefore varies diurnally and seasonally with climate (Lees and Milne, 1951; Belozerov, 1982). 4.1 4.1.1
WATER BALANCE CONSTRAINTS
Water vapour uptake by unfed ticks
The dehydration that ticks suffer while questing for hosts is reversible, but at a cost. First there is the metabolic cost of active water absorption (Fielden and Lighton, 1996a) as ticks deploy several mechanisms for extracting water vapour from unsaturated atmospheres. Central to this process is secretion of hygroscopic material by the salivary glands, specifically the type I acini, that performs two functions. First, in dehydrated unfed ticks, surplus ions and other substances are temporarily removed from the haemolymph by the production of hypertonic saliva and its deposition on the gnathosoma, where it dries and remains as a crystalline substance while RH is low (Rudolph and Knulle, 1974, 1979). The lowest RH at which water vapour uptake is possible, the so-called critical equilibrium humidity (CEH), is typically 85–90% for ticks. Once RH rises and exceeds the CEH, the crystalline substance dissolves and is swallowed (Needham and Teel, 1991). Second, it appears that water vapour absorbed at sub-saturated humidities by the hydrophilic cuticle in the hypostome can be released as salivary secretions that temporarily reduce the water affinity of this cuticle, allowing the condensed water to be sucked into the gut by a powerful sucking action of the pharynx (Gaede and Knu¨lle, 1997). Ticks do not, however, drink from free water (Kahl and Alidousti, 1997), and indeed have been shown to avoid contact with water and even walking on wet surfaces (Kro¨ber and Guerin, 1999; Guerin et al., 2000). 4.1.2
Questing behaviour and consequent tick–host relationships
Another cost associated with water balance in unfed ticks is the need to interrupt questing and return to the moist parts of their habitat, typically at the base of the vegetation where RH commonly exceeds CEH. Under favourable conditions, ticks may remain in questing positions on the vegetation for periods of several days (Lees and Milne, 1951; Loye and Lane, 1988), but they descend much more frequently in response to increased saturation deficit (SD – a measure of the drying power of the atmosphere that depends on both temperature and RH) so that, commonly, fewer ticks quest during the middle of the day (Fig. 4B).
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FIG. 4 Questing activity and attachment to rodents of Ixodes ricinus nymphs and larvae in relation to degrees of moisture stress in dry (left) and wet (right) experimental arenas. (A) maximum saturation deficit; (B) numbers of nymphs and (C) larvae counted per hour at 09:00 and 21:00 h (filled symbols) and 12:00, 15:00 and 18:00 h (open symbols); (d) numbers of nymphs (closed squares) and larvae (open squares) attached per rodent. Fresh ticks were added to the arenas on days 17 and 24 as indicated, and the dry arena was watered on day 24, both of which showed that quiescence rather than death was the cause of low questing activity. With permission from Randolph (2004).
An early observation that I. ricinus shows positive geo-tropism at saturation deficits above 4.4 mmHg (equal to 80% RH at 24 C, or 71% RH at 18 C) (Macleod, 1935) was corroborated by counts of reduced numbers of questing I. ricinus in Swiss woodlands when maximum SD exceeded 4.4 mmHg
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(Perret et al., 2000), with adults less affected than nymphs. Likewise, experiments in quasi-natural arenas (Randolph and Storey, 1999) revealed that under such dry conditions questing activity was diminished more amongst larvae than nymphs, but for both stages it was reversible once moist conditions were restored, that is high SD, even up to 15–20 mmHg, induced quiescence rather than direct mortality (Fig. 4A–C). Nevertheless, high SD may shorten a tick’s lifespan indirectly. In the dry arena, nymphs used up their fat twice as fast as those in the wet arena, presumably largely related to the increased metabolic costs of walking, which increases in dry conditions (see below), and the active water absorption described above, thereby reducing the estimated maximum questing period from 4 to 2 months (Randolph and Steele, 1985; Randolph and Storey, 1999). If ticks run out of fat before finding a host they will die. The reduced longevity recorded under increasing SD conditions for six species of African ticks housed in glass tubes in the laboratory was partly due to direct desiccation, with ticks from largely drier habitats better able to withstand low humidities. At RH above the CEH, however, death was more likely to be due to energy depletion (Fielden and Lighton, 1996b). Accordingly, longevity at 85% RH increased with tick size, presumably reflecting the energy reserve and metabolic advantages of larger organisms as well as the relatively smaller surface area of larger bodies. The absence of spiracles in the smallest stages, larvae, presumably helps in this respect. Furthermore, aggregation amongst Haemaphysalis longicornis larvae, but not nymphs or adults, increases longevity when exposed to low humidity (Tsunoda, 2008). This, of course, arises naturally as larvae hatch from egg batches, without any additional effort required by the ticks. Presumably as a result of these physical and physiological constraints, a general feature of many tick species is vertical separation in questing positions between life stages, with sub-adults sitting nearest to the base of the vegetation, commonly with larvae lower than nymphs, and adults questing very much higher (Gigon, 1985). Inter-stadially, it seems as if ticks ascend as high as possible within the limitations of their size-related tolerance to moisture stress (Rechav, 1979), locomotory powers and costs, and energy reserves, but the benefits of height are not entirely obvious. Making contact with the larger circumference of the hosts’ upper body parts will certainly facilitate attachment there (Andra´s Lakos, personal communication), but large numbers of nymphal and larval I. ricinus, for example, attach and engorge successfully on the lower body and lower legs, respectively, of large ungulate hosts (Gilot et al., 1994; Talleklint and Jaenson, 1994; Ogden et al., 1998). Any increased ability of later life stages to exploit higher, unoccupied feeding niches is off set by the lost opportunity to exploit smaller hosts, particularly rodents that are abundant and ubiquitous. Such apparently inherent stage-specific host relationships are affected by the impact of climate on questing heights. In the same arena experiments referred to above (Randolph and Storey, 1999), dry conditions forced nymphs of I. ricinus
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to quest lower down the vegetation from where they contacted and attached to small rodents in greater numbers than in wet conditions (Fig. 4D). Very few larvae in the same dry conditions fed on the rodents. Once the dry arena was watered, the same host relationships were established as in the wet arena, with the same low nymph:larva ratio on rodents as is typically seen in the wild. These climatic effects will clearly have an impact on the potential for pathogen transmission between nymphs and larvae feeding on rodents, and perhaps also on other trans-stadial routes via other host species, and involving other tick species. The risk to humans of infection with TBPs thus depends on the outcome of a balance between several extrinsic abiotic and biotic factors acting on individual ticks as they quest for hosts. The population of questing ticks will be adversely affected by hot dry conditions, and also desiccating winds, that impose mortality directly and indirectly on unfed ticks. Conversely, however, under favourable warm, moist conditions, prolonged questing will increase the probability of finding a host, thereby exhausting the questing tick population more rapidly. High host availability will have a similar effect (Randolph and Steele, 1985). Standard field sampling data alone can rarely distinguish between tick quiescence/mortality and tick feeding as a cause for the end of the active questing season (Eisen et al., 2002). 4.2
SENSORY SYSTEMS
As implied above (Section 2), haematophagous arthropods live on an energetic knife-edge; they necessarily minimize the high costs of locomotion by having evolved systems enabling them to find hosts quickly and efficiently at optimal intervals. In the case of tsetse, their sophisticated host-detection system, using a combination of olfactory and visual stimuli (Vale, 1974, 1977; Torr, 1989), gives them a 0.8 per day probability of finding a host under natural conditions of dense bush once they start their search (Randolph et al., 1992). At the same time, their strong flight apparatus permits them to redistribute themselves across large areas in response to environmental conditions; on a short timescale (days) they move between different parts of the habitat that are differentially suitable for feeding and breeding (Randolph and Rogers, 1984), and on longer timescales they track seasonal habitat suitability (Davies, 1967). Ticks are well equipped with sensory apparatus, comprising setiform sensilla, nonsetal sensilla and photoreceptors sensitive to CO2, ammonia, hydrogen sulphide, heat, sound, gravity, humidity, pheromones, host odours and light (described in detail in Coons and Alberti, 1999), but do not have the luxury of long-distance travel independent of their hosts. Recent work, however, suggests that even sit-andwait strategists are not as limited in their directed horizontal (as opposed to vertical, see above) locomotion as is commonly imagined. Ticks in water deficit will orientate towards zones of high humidity (Lees, 1948; Hair et al., 1975) and approach water drops (but avoid contact) (Kahl and
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Alidousti, 1997). Triggered by the onset of darkness, ticks appear to make use of moist night-time conditions to walk, thought to be related to selecting suitable questing locations (Perret et al., 2003). Continuous automated observations of I. ricinus walking up and down within vertical channels were interpreted as the equivalent of horizontal displacement. In these experimental conditions, nymphs walked further after periods of quiescence (median 43 cm, max 9.7 m) than after questing (median 17 cm, max 2.9 m), while those denied atmospheric moisture walked between 5 and 31 m until they died (Perret et al., 2003). Nevertheless, walking occupied only 6.6% of the 10 days of continuous observations. In horizontal arenas, more limited displacement was observed, with 27% of nymphs not walking at all within 24 h and 30–50% of those that did walk not moving beyond 14 cm (Crooks and Randolph, 2006). In field experiments, I. scapularis Say nymphs dispersed average distances of 2–3 m, and adults >5 m, over a period of several weeks, apparently by their own locomotion (Carroll et al., 1996). If questing ticks persistently fail to contact hosts, conserving any remaining energy is clearly an essential part of their sit-and-wait strategy, but ticks that do not move may be caught in a downward spiral towards starvation. Host finding would be more efficient if any locomotory activity were directed towards hosts. When adult I. scapularis were released centrally within a 2 m diameter circle of upright wooden skewers, within 2 weeks they accumulated on skewers anointed with substances rubbed from the external glands on the legs of white-tailed deer to a significantly greater extent than on control skewers (Carroll et al., 1996). Observations on I. ricinus in more controlled laboratory conditions suggested that walking is not entirely random followed by responses to stimuli encountered by chance, but rather the onset and direction of walking is stimulated by both intrinsic and extrinsic factors (Crooks and Randolph, 2006). Comparing ticks with different levels of fat content, those with lower energy reserves were indeed less likely to walk, but overall 66% of even the low-fat nymphs (typical of field ticks in June) did move horizontally over short distances. Intrinsic moisture conditions of ticks also exerted an influence. Whereas a mild degree of dehydration did not stimulate greater walking activity, it did increase the probability of ticks moving up a humidity gradient. Biologically, this is as would be expected. Walking was also directed towards secretions from dog skin, but only when humidity was high (Crooks and Randolph, 2006). Why this should be is still a mystery. High atmospheric vapour content may possibly have impeded the dissemination of the odour molecules and so created a stronger gradient, or alternatively aided the detection of the molecules by the tick’s olfactory sensilla within the Haller’s organ (Guerin et al., 2000; Leonovich, 2004). The volatile rumen metabolites to which ticks are attracted (Donze´ et al., 2004) would reach ticks within a naturally moist air stream. Alternatively, I. ricinus might be more responsive to host odour when the moisture stress of walking is less, consistent with Perret et al. (2003) observations of night-time walking.
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Thus it seems that under certain circumstances host odour can act as a kairomone (a host-produced substance that stimulates tick appetence behaviour), but does so by attracting ticks to move towards the source, rather than merely acting as an arrestant on contact as shown for other tick species (Carroll, 2002). This behaviour may allow adult ticks to adopt species-specific preferred questing heights, loosely correlated with the size of their principal host animal (Lees and Milne, 1951; Gigon, 1985; Loye and Lane, 1988). Laboratory experiments with artificial ‘‘vegetation’’, such as glass rods or wooden dowels, suggest an inherent preference (ibid ), which may have been established under natural conditions through a response to scent-markings from glands on various parts of the host’s body. Alternatively, the ‘‘choice’’ of hosts may be determined purely mechanistically by the tick’s questing height. This does not resolve the debate about whether the variable degree of host-specificity observed amongst tick species is adaptive or merely the result of opportunism operating within differential constraints. Either way, adaptations to maximize tick feeding success on the particular hosts encountered may reinforce stageand species-specific host detection and/or location mechanisms. 4.3 4.3.1
RECRUITMENT OF UNFED TICKS TO THE QUESTING POPULATION
Development rates
The backdrop to the variable host-seeking behaviour in relation to environmental conditions is the seasonal timing of questing determined principally by rates of development from one tick stage to the next. This dictates the timing of recruitment of unfed ticks into the questing population. Development rate is more useful than the birth rate as a demographic input parameter for ticks because a female ixodid tick lays a single large egg mass (typically several thousand eggs) over a short time period, but the annual recruitment of unfed larvae, nymphs and adults must be treated separately. In common with most physiological processes in poikilothermic animals, inter-stadial development rates of ticks increase non-linearly with increasing ambient temperature, with quantitative relationships that vary between stages and species (see figure 2 in Randolph 2004). It might be tempting to interpret the rather slow development rates, and consequent long inter-stadial development periods (typically months), as a trade-off against the advantages of low metabolic rate (see Section 2.3), but some tick species show exceptions indicating that slow development is not a universal constraint. In Slovakia, for example, two sympatric species within the same habitat show very different patterns: I. ricinus develops slowly so that each stage appears in successive years and the whole life cycle takes 3 years, whereas Dermacentor reticulatus develops much more rapidly, passing from engorged larva to moulted nymphs within one month during the summer so that each tick stage follows sequentially and the life cycle is completed within a year. This has significant consequences for the ticks’ role as vector of TBEV
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(see Section 5.1). Such inter-specific differences suggest an element of adaptation evolved by each species, which may extend to each geographical race of the same species. It is essential, therefore, to establish the correct rates for each species, preferably under controlled conditions in the laboratory for application to natural temperature conditions in the field, laborious though this is. Then daydegree summation methods may be used to estimate which ticks, and therefore how many, of one stage give rise to which and how many ticks of the next stage, as counted in the field. Not only does this permit further analysis of the seasonal population dynamics of the tick (Randolph, 1997; Randolph et al., 2002), it also allows the temporal course of pathogen transmission to be constructed. The variable prevalence of infection in ticks or vertebrates at certain points in time may then be attributed to factors operating at the correct interval before. The simple fact of geographical variation in absolute ambient temperatures and their seasonal variability results in contrasting patterns of recruitment to questing tick populations, potentially continuous in the tropics and even in the sub-tropical regions of South Africa (Randolph, 1997), but intermittent in temperate regions. Where development rates drop very low and even to zero for large parts of the year (see figure 3 in Randolph, 2004), development periods are telescoped and so emergence of unfed ticks becomes more synchronized. This immediately raises the question of the potential impact of increased temperatures as part of more general climate change on seasonal patterns of tick abundance, which could be non-linear and not necessarily proportional to any simple acceleration of development. Precise seasonal patterns, that are highly variable in space and time, can be critical to pathogen transmission potential and exposure of humans (see Section 5). 4.3.2
Diapause
Few animals show only simple graded responses to environmental conditions, and ticks are no different. Below certain temperature thresholds neither development of engorged stages nor questing activity of unfed stages occurs at all, and in addition there are day-length triggers of diapause that operate while temperatures would otherwise be permissive. Diapause is particularly important in temperate species as it resets the clock each year according to the highly regular and predictable pattern of day-length change. The physiology of light detection thus becomes a central factor, although it is poorly understood for ticks. Paired eyes of variable structure at the lateral margins of the scutum have been described for Amblyomma and Hyalomma species (Coons and Alberti, 1999), while all stages of I. ricinus have no eyes but up to 21 bilaterally arranged photoreceptors immediately below the cuticle along the dorsolateral margins behind the second coxa (Perret et al., 2003). Tick eyes represent ocelli, capable of responding to shadows and to variation in light intensity and they may vaguely discriminate shapes, but cannot discriminate colour (Kopp and Gothe, 1995; Coons and Alberti, 1999). The preference of I. ricinus for walking at night
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(see above) was shown to be a response to darkness irrespective of climatic conditions (Perret et al., 2003), but the exact light sensitivity of the photoreceptor cells is unknown. In the induction of diapause, ticks have been shown to respond to simulated natural changes in day length (Beattie and Randolph, unpublished observations), which are much more gradual (ca. 20–30 min per week) than those used in most experiments investigating diapause (typically an 8-h difference within a few days, either side of feeding). Defining what a tick perceives as day length (the limiting position of the sun relative to the horizon) is a separate unresolved issue. The latitudinal variation in the date of diapause induction in I. ricinus (see below), however, is best correlated with day length defined by when the sun is 9 below the horizon. Two distinct sorts of diapause are recognized for ticks, so-called behavioural diapause that switches off questing activity in unfed stages, and morphogenetic diapause that delays development in fed ticks (Belozerov, 1982). Both are presumably adaptations to ensure that the exposure of vulnerable stages to unfavourable abiotic conditions is minimized so that overall mean daily survival more than compensates for the fitness costs of a prolonged generation time. Behavioural diapause in the African tick Rhipicephalus appendiculatus illustrates nicely the plastic, presumably adaptive nature of diapause. It is confined to unfed adult ticks, which do not always quest and feed as soon as they have hardened after emergence from the engorged nymphal stage. In South Africa, this diapause appears to be induced by short day lengths so that adults that emerge after July each year (winter solstice in June in the southern hemisphere) do not start questing until some time after November of the same year (Rechav, 1981; Short and Norval, 1981; Pegram and Banda, 1990; Randolph, 1997; Madder et al., 1999). As a result, there are periods of near absence of each tick stage sequentially during the year, and the majority of the vulnerable eggto-larval stage occurs during the warm wet season (December–May), which would favour rapid development and good survival. In contrast, in equatorial Africa, where long cold dry seasons do not occur, the same species does not show any diapause (Branagan, 1973). Here, all tick stages feed throughout the year, interrupted only by abrupt declines in abundance of all stages simultaneously during the dry season, allowing continuous overlapping generations each of ca. 8–9 months duration. The propensity to diapause shows a latitudinal gradient (Madder et al., 1999), and the mechanism of diapause termination is thought to change from a long day-length trigger in the south to gradual physiological ageing further north (Berkvens et al., 1995). This variable pattern of diapause is significant not only for the survival of tick populations, but also for the epidemiology of associated infections. Diapause in southern populations of R. appendiculatus may prevent the circulation of the virulent strain of Theileria parva that causes East Coast Fever in cattle in eastern and central Africa (Norval et al., 1991). Only where there is sufficient overlap between the more or less continuous activity periods of the different tick stages can the virulent strain of T. parva be acquired from infected cattle by larvae and
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nymphs and transmitted onwards by nymphs and adults. The less virulent strain of T. parva that causes January or Corridor disease survives for longer periods in carrier cattle, and so can be acquired by nymphs and transmitted by adults where there is less inter-stadial overlap; hence its occurrence in southern Africa. In the more markedly seasonal environments of temperate zones, ticks show both behavioural and morphogenetic diapause. The inter-stadial periods may be prolonged (i.e. adjusted) by a variable delay in the onset of development after engorgement (Campbell, 1948; Kemp, 1968). In Palaearctic (I. ricinus and I. persulcatus) and Nearctic (I. scapularis) members of this widespread species complex, morphogenetic diapause is evidently triggered by the change from long (pre-feed) to short (post-feed) day length (Belozerov, 1998; Belozerov and Naumov, 2001), either as experienced by the questing stages or as the difference between pre- and post-engorgement conditions. Thus I. ricinus, for example, that quest in the field after July enter diapause whether they feed and are held in quasi-natural field conditions (Chmela, 1969; Cerny´ et al., 1974; Daniel et al., 1976, 1977; Gray, 1982) or in constant laboratory conditions (Campbell, 1948; Kemp, 1968). There is a latitudinal gradient, with the date of diapause onset varying from the end of July in Scotland (ca. 56 N) to the end of August in the Czech Republic (ca. 49 N). This appears to be due simply to the latitudinal gradient in sun position relative to the horizon during late summer (see above), because in light conditions the simulated rapid or slow day length decrease equivalent to July–October at 60 and 49 N, respectively, and under 18 C constant temperature conditions, ticks entered diapause as photoperiods reached the same absolute length, ca. 13 h light, irrespective of the rate of decrease (Beattie and Randolph, unpublished observations). Temperature rather than day length appears to be instrumental in breaking morphogenetic diapause in I. ricinus. Even a brief period of exposure to 0 C induces rapid development without delay similar to that seen in spring-fed (i.e. pre-July) ticks (Campbell, 1948). This ensures that all ticks, whenever they feed after July/August, start development once temperatures increase after the winter (see figure 3 in Randolph, 2004). Under a wide range of conditions across the United Kingdom, the combination of diapause and seasonal temperaturedependent development rates acts to synchronize the emergence of virtually all unfed ticks of each stage to within a couple of months every autumn (Randolph et al., 2002) (which is likely to act as a brake on any impact of climate change). This conclusion was validated when emergence dates of unfed ticks predicted from a day-degree summation development model coincided precisely with the first appearance in the field of ticks with high fat content. Fat is a source of energy derived from each blood meal, which can be used as a marker of physiological ageing in the field (Uspensky, 1995; Walker, 2001). Its natural rate of usage varies with seasonal activity and climatic conditions according to the tick’s locomotory and physiological activities (Steele and Randolph, 1985; Randolph and Storey, 1999), allowing a distinction between the calendar age and physiological age of ticks. The frequency distribution of fat
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contents in questing ticks collected regularly throughout several years at three sites in the United Kingdom indicated clearly that under a wide range of climatic conditions, a single cohort of each stage of I. ricinus is recruited each year in the autumn and then survives for 1 year until the ticks have either fed or died of fat exhaustion. Crucially, however, this timing of recruitment does not coincide with the timing of peak numbers of ticks seen questing in the field. Most newly emergent ticks delay their questing activity until the following spring in many parts of Europe (inter alia Lees and Milne, 1951; Daniel et al., 1976; Gray, 1985; Randolph et al., 2002). This may be true ‘‘behavioural diapause’’ (Belozerov, 1982), similar to that seen in adult R. appendiculatus and I. pacificus (Padgett and Lane, 2001), although the triggers will be species-specific. Again, short day length has been implicated (Belozerov, 1982), but in the field in mild climates increasing numbers of ticks may be active from January onwards (when day length is at its minimum, but increasing) (Fig. 5A), suggesting that decreasing day length beyond a certain level, rather than absolute day length, may be the cue for diapause. If it is indeed true diapause, it is nevertheless not absolute, as a small and variable percentage of I. ricinus do become active in the autumn. It is not yet known whether this represents a low probability of all ticks questing at this time, or the unequal division of the tick population into sub-sets with different physiological behaviour according to, perhaps, their developmental history or some genetic characteristics. 4.3.3
Temperature thresholds
Ticks also vary their questing activity in response to their immediate climatic conditions, which should be interpreted as quiescence rather than true diapause. Interacting with the putative inhibitory effects of decreasing day length, and the supposed converse permissive effects of increasing day length, low winter temperatures in temperate regions inhibit tick activity. Yet even where temperatures are permissive throughout the year, fewer ticks quest before the winter solstice than after it (Fig. 5A). It appears that at the end of each year, decreasing day length reduces the probability of questing and low temperatures may inhibit activity altogether (Fig. 5B), while at the start of each year, increasing day length is permissive, but only if temperatures are high enough. Gradual recruitment to the questing population will occur as temperatures reach the threshold level (see below) in more and more parts of the ticks’ over-wintering micro-habitats (Eisen et al., 2002). Consistent with this, altitude influences the timing of the onset, more than the cessation, of tick activity (Jouda et al., 2004). Climate change may therefore affect tick activity in the spring more than in the autumn, because diapause induced by day length will continue unchanged. In the unusually warm conditions over the winter of 2006/2007, following unusually cold conditions the year before (i.e. a case of weather variability rather than climate change), ticks were seen questing within enclosed arenas in a Berlin forest in January and February, which is earlier than usual (Dautel et al., 2008).
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FIG. 5 Seasonal variation in maximum air temperature (Ta) and numbers of questing larval (○) and nymphal (●) Ixodes ricinus at two sites in the United Kingdom, (A) Dorset, a warm site and (B) Wales, a cooler site (note that larval density is presented at 33% of observed level at site A). Dotted and dashed lines indicate the temperature thresholds for the onset of seasonal activity of larvae and nymphs, respectively. With permission from Randolph (2004).
The threshold temperature for activity by questing nymphs and adults of I. ricinus has been estimated approximately as a weekly mean daily maximum temperature of ca. 7 C (Macleod, 1936; Perret et al., 2000; Randolph, 2004) (Fig. 5). This is a useful estimate for analysis, interpretation and modelling, and appears to apply widely across Europe even at coarse spatial and temporal resolutions (Randolph et al., 2008), but is only approximate for a number of reasons. First, temperature is usually recorded at standard meteorological stations some distance from the tick-monitoring site and virtually never
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precisely as the tick experiences it. In any case, temperature varies considerably over tiny distances within natural microhabitats. Secondly, activity threshold temperatures vary inter-specifically, interstadially, and even intra-stadially, which appears to be size-dependent (Clark, 1995). In the United Kingdom, the onset of larval activity coincides with a threshold of 10 C, rather than 7 C, mean maximum temperature (Fig. 5). Furthermore, smaller nymphs and adults start questing later each year (Randolph et al., 2002). Each new cohort of ticks that appeared on the vegetation in the autumn in the United Kingdom showed a normal distribution of size (fat-free weight), virtually symmetrical about a median of 75–85 mg (range 40–120), which then changed seasonally (Fig. 6 for an example from one site over 1 year). A sub-set of larger nymphs (and adults, data not shown) started questing the following February, with only 15–30% <80 mg and almost none <60 mg. As the year progressed, the questing population comprised increasingly smaller individuals, as small ticks appeared from March onwards and the larger ticks (>100 mg) disappeared first. In addition to arriving sooner, the latter may find hosts more rapidly if their relatively higher energy levels (Van Es et al., 1998; Randolph et al., 2002) permit them to quest for longer periods. Smaller ticks (including all larvae) that arrive later may find hosts more slowly if they are forced into temporary inactivity during the summer by the other major constraint on questing probability, moisture stress; only the smallest ones persist
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FIG. 6 Seasonal changes in the size (i.e. fat-free weight) composition of populations of questing nymphal Ixodes ricinus during the life span of a single cohort on Exmoor, United Kingdom 1998–1999. Sample dates and sizes are shown in the key. With permission from Randolph et al. (2002).
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to September. Thus there will be successive waves of recruits to the spring and summer questing tick populations, ordered by size and stage, the pattern of which varies with the seasonal ambient temperature profile. As early recruits disappear and are replaced by later recruits, the season of pathogen transmission and human risk will be prolonged beyond the questing time-span of any individual tick. Esoteric as this level of detail may seem, it has been shown to underpin the observed focal distribution of TBEV in Europe and may also apply to different B. burgdorferi s. l. strains around the world (see Section 5). 4.4
FAT RESERVES DETERMINE LIFESPAN
The fat body in ticks is a diffuse organ, consisting of highly dispersed strands of cells clinging to branches of the tracheal system and also other organs (Sonenshine, 1991). It has a range of specific functions, including the metabolism of hormones and other messenger molecules, the detoxification of waste and, of most interest here, storage of food reserves. The whole-body fat content of ticks, measured by chloroform extraction, decreased very little, by 0.016 mg/day on average, when ticks were held in cool, dark conditions, but at nearly ca. 9 times this rate for ticks questing freely in quasi-natural damp arenas and twice as fast again in dry arenas (Randolph and Storey, 1999). From this rate and the observed range of fat contents in field nymphs, it is possible to estimate a notional maximum period of survival of 130 days in wet conditions and 64 days in dry conditions before the ticks would die of fat exhaustion. Data from the field under natural but unrecorded conditions suggested a very similar maximum lifespan of about 4 months, but only about 2 months for larvae (derived from more indirect evidence) (Randolph and Steele, 1985). Once recruitment into the questing population, staggered according to stage and size, has ceased each year, the season of tick questing is terminated when all ticks have either found a host or run out of energy reserves. In years with warmer springs, ticks emerge earlier in the year, but the available pool of unfed ticks simply declines faster with no greater total annual abundance of ticks caused by this one environmental change (Randolph et al., 2008). Indeed, higher temperatures in spring and summer may shorten the questing tick’s lifespan through a higher rate of fat depletion. These patterns will be most apparent in temperate zones with more or less discrete tick generations compared to the free-running tick populations in the tropics, but the underlying physiological processes and resultant demographic events still operate in the tropics, with consequences for temporal patterns of potential pathogen transmission.
5
Epidemiological consequences of tick phenology
Because ticks take only one blood meal per life stage, transmission of pathogens necessarily involves ticks of at least two stages, first to acquire and then to transmit. Furthermore, given the high inter-stadial mortality suffered naturally
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by ticks (ca. 80–90% at equilibrium if females lay thousands of eggs – Randolph, 1998), pathogens must be passed backwards through the tick life cycle, from infected ticks of one stage via vertebrates to the more abundant ticks of the previous stage, if transmission cycles are to persist. It is clear, therefore, that the relative timing of peak abundance of each tick stage is more or less critical to TBD epidemiology, depending on the duration of infection in the vertebrate hosts that act as a bridge between transmitting and acquiring ticks. This is the supposed cause of the differential transmission of the virulent strain of T. parva that causes East Coast Fever in cattle in eastern and central, but not in southern, Africa (Norval et al., 1991) (see Section 4.3.2). A more detailed example concerns TBEV in Europe, and evidence is emerging that the same phenomenon may apply to B. burgdorferi in North America. 5.1
FOCAL DISTRIBUTION OF TICK-BORNE ENCEPHALITIS
The principal transmission hosts for TBEV are rodents of the genus Apodemus (Labuda et al., 1993b), upon which only larval and nymphal ticks feed. Whether TBEV establishes a systemic viraemia or not (see Section 3.2.2), the duration of infection in rodents is limited to a few days (Chunikhin and Kurenkov, 1979; Kozuch et al., 1981). Infected nymphs must therefore feed alongside large numbers of infectible larvae to ensure transmission at sufficient levels to support persistent cycles. The ecological requirements imposed by this so-called co-feeding transmission is synchrony in the highly seasonal activity of these two tick stages (Randolph et al., 2000), which dictates the focal distribution of TBEV within the much more widespread distribution of the vector ticks. Synchrony is the outcome of the long slow life cycle of I. ricinus, reset every year by winter conditions and diapause as described above, but only in certain climatic conditions. The degree of synchrony with which larvae and nymphs emerge from diapause in the spring depends on whether temperatures rise sufficiently rapidly to cross the threshold for larval activity (ca. 10 C mean daily maximum) soon after the threshold for nymphal activity (ca. 7 C) (Randolph and Sˇumilo, 2007). The variable existence of such specific thermal conditions has been identified as the key determinant of the focal distribution of TBEV across Europe allowing the predicted risk of TBE to be mapped using climatic surrogates sensed from space (Randolph, 2000). Originally, however, it was the rate of cooling in the autumn that was identified as the best correlate of (a) synchrony and (b) TBE presence (Randolph et al., 2000). Once the time course of the processes underlying the observed phenology of I. ricinus was better understood (Randolph et al., 2002) and the analysis was rerun, the rate of spring warming was correctly identified as the most significant factor both statistically and biologically (Randolph and Sˇumilo, 2007) (Fig. 7). This phenological requirement imposes an ecological specificity on which vectors can be used by TBEV (Labuda and Randolph, 1999). Only I. ricinus and I. persulcatus appear to contribute substantially to natural TBEV cycles, despite
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FIG. 7 Satellite-derived temperature conditions across Europe and Baltic States, related to TBE presence and seasonal synchrony between larval and nymphal I. ricinus ticks. (A) Pixels where TBE is present (dark/red dots) or absent (grey dots), and sites where larval and nymphs show synchrony (large filled squares) or non-synchrony (large open circles), show significantly different rates of spring warming. (B) Results of principal components analysis. PCA axis 2 score: TBE-present, mean ¼ 0.861, st. dev ¼ 0.740, n ¼ 418; TBE-absent, mean ¼ 0.228, st. dev ¼ 0.934, n ¼ 1574; t ¼ 25.23, P < 0.001. With permission from Randolph and Sˇumilo (2007).
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the biological competence of many tick species to transmit this virus in the laboratory. The rapid life cycle of D. reticulatus, each stage following sequentially from late spring to autumn, precludes it role as a vector for TBEV in nature, despite its sympatric occurrence with I. ricinus in Slovakia, for example. D. reticulatus also feeds more on voles that are less transmission competent than mice (Labuda et al., 1993b). 5.2
WIDESPREAD, BUT PARTITIONED, DISTRIBUTION OF B. BURGDORFERI S. L.
The very opposite appears at first glance to be true of B. burgdorferi s. l., which utilizes an exceptional diversity of ticks and vertebrates, matching an increasing knowledge of its genetic diversity. Compared with TBEV, systemic infections endure for relatively long periods in many vertebrate species, measured in months rather than days. Not all Borrelia strains, however, even within B. burgdorferi s.s., show the same degree of prolonged infectivity in their natural rodent host Peromyscus leucopus (Derda´kova´ et al., 2008; Hanincova´ et al., 2008). Their fitness, and therefore relative prevalence in tick populations, was predicted to vary with different degrees of predicted seasonal synchrony of I. scapularis life stages (Ogden et al., 2007, 2008). Empirical observations have started to confirm this across the range of endemic foci in the Northeast and upper Midwest of USA: strains of B. burgdorferi s.s. with shorter periods of infectivity in rodents are more prevalent in populations of I. scapularis that show greater degrees of overlap in the seasonal questing activity of nymphs and larvae (Gatewood et al., 2009). This appears to be driven by the earlier onset of larval activity in the summer under certain conditions of seasonal temperature profiles (Gatewood et al., 2009). Models to predict both the present situation and also any future changes under forecast conditions of climate change must take account of any differences in pathogen-induced host mortality and transmission coefficients associated with duration of infectivity (Ogden et al., 2008), a degree of quantitative detail that is only just emerging. The B. burgdorferi s. l. species complex shows the widest geographical range of any tick-borne (indeed vector–borne) pathogen in the northern hemisphere, with a concomitant ecological diversity. First, there were observations of the differential transmission competence of taxonomically distinct vertebrate species (e.g. birds vs. mammals, rodents vs. ungulates) for four genospecies of this bacterial complex (B. garinii, B. valaisiana, B. afzelii and B. burgdorferi s.s.), and for strains within B. garinii (Humair et al., 1995; Humair and Gern, 1998; Kurtenbach et al., 1998a,b; Huegli et al., 2002). Later, increasingly detailed descriptions of the species-specific battles between host immune responses and the bacteria’s counter-defences, particularly those involving the alternative complement system (Kurtenbach et al., 1998c, 2002), offered some convincing explanations for this diversity (Gilmore et al., 2001; Kurtenbach et al., 2002; Stevenson et al., 2002). The local prevalence of each bacterial strain depends on the relative density of each differentially competent vertebrate transmission
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host (Humair and Gern, 1998; Kurtenbach et al., 1998b, 2001), yet broad-scale patterns in the distribution of each genospecies across the Old World (Kurtenbach et al., 2006) are not obviously constrained by the absence of competent hosts. It remains to be explored whether differential duration of infectivity for each genospecies and even species strain determines their global patterns of circulation under different seasonal temperature conditions. 5.3
SENSITIVITY TO CLIMATE VERSUS IMPACT OF CLIMATE CHANGE
The sensitivity of TBD systems to climatic conditions is indisputable, due to the response of so many of the physiological processes of ticks to temperature and moisture conditions, as discussed in this review. It is this that has allowed the risk of TBDs to be mapping in areas beyond empirical observations of presence, predicted by broad-scale climate data or satellite imagery that comprises surrogate climate information at fine spatial resolution across continental areas (Hay et al., 2000). Examples include tropical, sub-tropical and temperate systems (Rogers and Randolph, 1993; Hugh-Jones, 1989; Randolph, 2000; Brownstein et al., 2003; Estrada-Pen˜a et al., 2007). As well as offering blueprints to help define the level of risk and so direct control strategies, risk mapping offers insight into the critical environmental factors that drive the rates of the underlying demographic and transmission processes. For ticks, and therefore TBDs, moisture-related indices have proved to be significant predictors of presence, with wetter conditions virtually always beneficial. This is entirely consistent with the high CEH typical of most tick species (see Section 4.1.1). High maximum (summer) levels of the Normalized Difference Vegetation Index (NDVI), a measure of photosynthetic activity, are the most significant predictor of TBE foci (Randolph, 2000), because I. ricinus thrives best where woodlands provide shelter from desiccating conditions and also habitats for the essential large hosts for adult ticks (deer). Soil moisture is an important predictor of the presence of I. scapularis in the upper Midwest of USA for similar reasons (Guerra et al., 2002). This, however, is quite distinct from assuming that climate change has already expanded the geographical range of ticks, caused increases in tick abundance, and single-handedly driven the emergence of TBDs, or may do so in the future. The specific changing patterns of temperature and rainfall may, by chance, act either synergistically or antagonistically on the reproduction and mortality rates of ticks, and each may result in either an increase or decrease in the balance between these rates. Where temperatures are currently limiting but moisture is not, at higher altitudes for example, ticks may be able to colonize new areas as has been described in the cold Krkonosˇe mountains of northern Czech Republic (Danielova´ et al., 2008), but this specific situation applies to relatively few places globally. In fact, TBE cases have not been detected at increasingly high altitudes over the past 46 years in the West Bohemian region of the Czech Republic (Pazdiora et al., 2008). The impact of multi-factorial
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climate change on TBD systems is even less easy to predict, given their complexity and dependence for persistent cycles on subtle seasonal climatic factors and also on land cover, land use and host populations. In Europe, analyses of meteorological station data suggest that climate change may possibly have played a minor role in the emergence of TBE over the past two decades (Randolph and Sˇumilo, 2007), but changes in other environmental conditions (land cover, land use and host abundance) and in human activities associated with the socio-economic transition following the end of Soviet rule have been far more significant to varying degrees in different parts of the continent (Sˇumilo et al., 2008; Rizzoli et al., 2009).
6
Conclusions
Each of the three essential steps underpinning cycles of vector-borne pathogens (acquisition, development, transmission of pathogens) depends on the ability of a microbe to overcome the many intrinsic biological (molecular, cellular, physiological and physical) barriers during its passage from vector to vector via the host. Understanding of the cellular and sub-cellular interactions between pathogens, hosts and vectors have burgeoned in the past few decades (Randolph, 2009). Even if biologically possible, however, a cycle may not proceed with sufficient force to allow persistent infection. That depends on the quantitative balance of the rates of all the processes involved in each complete transmission cycle. The majority of these rates are affected principally by extrinsic environmental factors, both biotic and abiotic, although host acquired immunity also plays a significant role. Thus, critical to transmission potential is the behaviour and ecology of the vectors, permitted by physiological adaptations evolved to minimize the limiting effect of the environment on the fitness of each species. First there is haematophagy itself. While its evolution must have been a dream comes true for the lucky microbes that were able to harness its potential for their dissemination amongst vertebrate hosts, the precise pattern of haematophagy nevertheless imposes fundamental and specific constraints on the epidemiology of each resulting disease. The ixodid tick’s unusual habit of taking a single blood meal per life stage defines the framework within which the dynamics of TBP transmission operates. Seasonal phenomena over the tick’s long generation time become of even greater importance for TBDs than for most other vector-borne disease systems. One direct consequence is to limit the pace of change of infection prevalence in the vector population, as it is the product of past events over long and variable periods. As a result, sudden epidemic events, either positive or negative, may be due less to changes in maintenance transmission cycles and more to changes in human exposure to those cycles. From a public health perspective, the growing understanding of the molecular and cellular bases for many of the vital physiological processes underpinning
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tick survival and pathogen transmission potential offer promise of novel control strategies in the form of vaccines or genetic manipulations to disrupt the mechanisms. Deploying any such novel interventions effectively will depend on knowing where the need is greatest and where the likelihood of success is greatest, which, almost by definition, do not usually coincide. The former will be in places of highest transmission potential and the latter in places of lowest transmission potential. Predictive mapping of risk is now a well-established discipline (Hay et al., 2006), at its best taking account of both infection incidence rates and global human population distributions (Hay et al., 2009). While this statistical approach is possible without any knowledge of the underlying biology, a deep understanding of how the biology of the vector, and its sensitivity to environmental forces, determines the epidemiology of the disease will certainly strengthen the reliability, interpretation and future development of broad-scale epidemiological analyses and predictions.
Acknowledgements I am most grateful to Stephen Simpson and Je´roˆme Casas for inviting me to write this review. This work is partially supported by the EU grant GOCE-2003010284 EDEN; it is catalogued by the EDEN Steering Committee as EDEN0150 (http://www.eden-fp6project.net/). The contents are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. References Alekseev, A. N. and Chunikhin, S. P. (1990). Exchange of tick-borne encephalitis virus between Ixodidae simultaneously feeding on the animals with sub-threshold levels of viraemia. Med. Parazitol. Parazit. Bolezni 2, 48–50. Alekseev, A. N., Chunikhin, S. P., Rukhkyan, M. Y. and Stefutkina, L. F. (1991). Possible role of Ixodidae salivary gland substrate as an adjuvant enhancing arbovirus transmission. Med. Parazitol. Parazit. Bolezni 1, 28–31. Amosova, L. I. (1983). The integument. In: An Atlas of Ixodid Tick Ultrastructure (eds Raikhel, A. and Hoogstraal, H.), pp. 23–59. Entomological Society of America, Washington, DC. Andersen, A. O. and Roepstorff, P. (2005). The extensible alloscutal cuticle of the tick, Ixodes ricinus. Insect. Biochem. Mol. Biol. 35, 1181–1188. Anderson, J. F. and Valenzuela, J. G. (2008). Tick saliva: from pharmacology and biochemistry to transcriptome analysis and functional genomes. In: Ticks: Biology, Disease and Control (eds Bowman, A. S. and Nuttall, P. A.), pp. 92–107. Cambridge University Press, Cambridge. Arthur, D. R. (1960). Ticks. Part V. The genera Dermacentor, Anoncenter, Cosmiomma, Boophilus, Margorups. Cambridge University Press, Cambridge. Belozerov, V. N. (1982). Diapause and biological rhythms in ticks. In: Physiology of Ticks (eds Obenchain, F. D. and Galun, R.), pp. 469–500. Pergamon Press, Oxford.
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Belozerov, V. N. (1998). Role of two-step photoperiodic reaction in the control of development and diapause in the nymphs of Ixodes persulcatus. Russ. J. Zool. 2, 414–418. Belozerov, V. N. and Naumov, R. L. (2001). Photoperiodic control of nymphal diapause in the North American tick, Ixodes (Ixodes) scapularis Say (Acari: Ixodidae). In: IVth European Workshop of Invertebrate Ecophysiology (ed Kipyatkov V.E.) p. 69. St Petersburg, Russia. Berkvens, D. L., Pegram, R. G. and Brandt, J. R. A. (1995). A study of the diapausing behaviour of Rhipicephalus appendiculatus and R. zambesiensis under quasi-natural conditions in Zambia. Med. Vet. Entomol. 9, 307–315. Bowman, A. S., Ball, A. and Sauer, J. R. (2008). Tick salivary glands: the physiology of tick water balance and their role in pathogen trafficking and transmission. In: Ticks: Biology, Disease and Control (eds Bowman, A. S. and Nuttall, P. A.), pp. 73–91. Cambridge University Press, Cambridge. Branagan, D. (1973). Observations on the development and survival of the Ixodid tick Rhipicephalus appendiculatus Neumann, 1901 under quasi-natural conditions in Kenya. Trop. Anim. Hlth. Prod. 5, 153–165. Brossard, M. and Wikel, S. K. (2008). Tick immunobiology. In: Ticks: Biology, Disease and Control (eds Bowman, A. S. and Nuttall, P. A.), pp. 186–204. Cambridge University Press, Cambridge. Brownstein, J. S., Holford, T. R. and Fish, D. (2003). A climate-based model predicts the spatial distribution of the Lyme disease vector Ixodes scapularis in the United States. Environ. Hlth. Perspect. 111, 1152–1157. Camicas, J.-L., Hervy, J.-P., Adam, F. and Morel, P.-C. (1998). Les Tiques du Monde. Orstom Editions, Paris. Campbell, J. A. (1948). The life history and development of the sheep tick Ixodes ricinus Linaeus in Scotland, under natural and controlled conditions. Ph.D. thesis, Department of Agricultural and Forest Zoology, University of Edinburgh, pp. 131. Carpi, G., Cagnacci, F., Neteler, M. and Rizzoli, A. (2008). Tick infestation on roe deer in relation to geographic and remotely-sensed climatic variables in a tick-borne encephalitis endemic area. Epidem. Infect. 136, 1416–1424. Carroll, J. F. (2002). How specific are host-produced kairomones to host-seeking ixodid ticks? Exp. Appl. Acarol. 28, 155–161. Carroll, J. F., Mills, G. D. and Schmidtmann, E. T. (1996). Field and laboratory responses of adult Ixodes scapularis (Acari: Ixodidae) to kairomones produced by white-tailed deer. J. Med. Entomol. 33, 640–644. Cerny´, M., Daniel, M. and Rosicky´, B. (1974). Some features of the developmental cycle of the tick Ixodes ricinus (L.) (Acarina: Ixodidae). Folio Parasitol. 21, 85–87. Chmela, J. (1969). On the developmental cycle of the common tick (Ixodes ricinus L.) in the north-Moravian natural focus of tick-borne encephalitis. Folio Parasitol. 16, 313–319. Chunikhin, S. P. and Kurenkov, V. B. (1979). Viraemia in Clethrionomys glareolus – a new ecological marker of tick-borne encephalitis virus. Acta Virol. 23, 257–260. Clark, D. D. (1995). Lower temperature limits for activity of several ixodid ticks (Acari: Ixodidae): effects of body size and rate of temperature change. J. Med. Entomol. 32, 449–452. Coons, L. B. and Alberti, G. (1999). The Acari – ticks. In: Microscopic Anatomy of Invertebrates, vol. 8B, Chelicerae Arthropods (eds Harrison, F. W. and Foelix, R.), pp. 267–514. Wiley-Liss, New York. Couvreur, B., Beaufays, J., Charon, C., Lahaye, K., Gensale, F., Denis, V., Charloteaux, B., Decrem, Y., Prevot, P.-P., Brossard, M., Vanhamme, L. and
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Index Aedes aegypti, 3–4 Amblyomma americanum, 302–303 anaphylatoxin inhibitors, 256 animal trypanosomiasis (AAT), 120–121 Anopheles gambiae, 3–4 anophelin, 81, 84, 89 antioxidants, 143–144 Argasidae, 245 Arg-Gly-Asp (RGD) peptide, 75 Arthropods. see blood-sucking arthropods (BFA) BFA. see blood-sucking arthropods (BFA) Blastocrithidia triatomae, triatomines developmental cycle, 195–196 pathogenicity behaviour, 212–213 cuticle and tracheal system, 214 fitness, development and mortality rates, 211–212 haemolymph composition and intestinal contents, 213 immune system, 215–216 intestine and excretion, 214–215 mechanism, 217 symbionts, 216–217 species and strains distribution, 190–193 blood-feeding insects (BFI), 61. see also haematophagous blood-sucking arthropods (BFA) annoying itching and pain, 67–70 endothelial cell activation, 70 haemostasis, 64–66 inflammation, 66–67 microbiological concerns, 70 blood-sucking insects. see haematophagous Borrelia. burgdorferi, biology and vector interaction
distribution, 326–327 genome, 272–273 immune modulation, 307–308 tick colonization and persistence facilitation genes chromosomal genes, 276 complement regulator-acquiring surface proteins (CRASPs), 276–277 lp25 genes, 275 ospAB operon, 273–274 ospC, 274–275 transmission facilitation genes salivary anti-complement proteins, 278 salivary protein 15 (Salp15), 277 TROSPA receptor, 278 transmission, salvary gland, 309–310 bradykinin inhibitors, 256 coevolution, 90 co-feeding transmission, TBEV, 324 critical equilibrium humidity (CEH), 311 cuticle, ticks, 300–301 DEET. see [N-N]-diethyl-mtoluamide (DEET) diapause, ticks behavioural diapause, 318–319 fat, physiological ageing marker, 319–320 morphogenetic diapause, 319 ocelli eyes, 317–318 [N-N]-diethyl-mtoluamide (DEET) repellent, 42–43 sensory and behavioural effects, 43 epimastigote, 128–129
342 gene knockdown method, 156–158 Glossina spp. see tsetse-trypanosome interactions gly-pro-glu-glu-thr (GPEET) repeats, 130 haematophagous arthropods, blood intake problems annoying itching and pain, 67–70 endothelial cell activation, 70 haemostasis, 64–66 inflammation, 66–67 microbiological concerns, 70 blood as food blood-feeding insects (BFI), 61 deleterious effect, 60 feeding mechanics, 62–63 hemimetabolous, 60 holometabolous, 60 surface availability and thermoregulation, 62 food source selection activation, circadian clocks, 15 appetitive search, 15–16 biting activity, 17–18 detection of, 16 host finding and contact, 16–17 leaving the host, 20 recognition and feeding of, 18–20 functional neuroanatomy, mosquito, 3–4 histamine activity, 94–95 history advantages of, 6–7 degrees of association, 6 feeding routes, 5 sensory machinery, 6 host attractiveness, 41–42 host signals heat, 8–10 odours, 7–8 visual cues, 12–14 water vapour, 10–12 learning and memory, 42 orientation mechanisms directional information, 24 kinesis, 24 kissing bugs, 25–26
INDEX stimulus types, 23–24 taxis, 24–25 repellents, 42–43 salivary gland, components and diversity anaesthetics, 82–87 antigen (AG5) family members, 87 antigens as epidemiological marker, 99–100 coevolution, 90 crystal structure analysis, 93–94 enzymes, 71–75 (see also salivary gland, haematophagous) gene duplication, 90–93 horizontal gene transfer (HGT), 93 immunity-related products, 87–88 intra-specific conservancy, 88–89 kratagonists, 77–80 physiological antagonists, primarily vasodilators, 76–77 polymorphism, 88 protease inhibitors, 80–82 qualitative difference, 89 receptor antagonism and platelet aggregation inhibitors, 75–76 sensory parsimony blood-sucking insects, 34–35 practical consequences, 35–36 sialotranscriptome, 96–97 sialoverse and sialome overlap, 97–98 state-dependency, host-seeking behaviour feeding conditions and host searching, 40–41 maturation and responsiveness, 38–39 odours and temporal modulation of, 37–38 reproduction and modulation, 39–40 stimulus propagation and sensory reception antennae, 22 eyes, 21 factors, 21 information processing levels, 22–23 thermal sensing, kissing bugs
INDEX antennal movements, 27, 28 multimodal integration, proprioceptive and thermoreceptive inputs, 32–34 object size estimation, 29–30 proboscis extension response (PER), 27 radiant heat perceivability, 26–27 source distance estimation, 31 thermoreceptor, 30–32 vector salivation significance, 63–64 haemostasis, 65, 68 heat signals, 8–10 hemimetabolous, 60 holometabolous, 60 Horizontal gene transfer (HGT), 93 human African trypanosomiasis (HAT), 120 immunomodulation, ticks acquired resistance, 306 anti-complement activity, 307 orchestrating pathogen transmission saliva-activated (now assisted) transmission (SAT), 307–308 zoo-prophylaxis, 308–309 pathogen traffic, 309–310 species-specificity, 306 Ixodidae, 246 kairomone, 316 kissing bugs antennal movements, 27, 28 multimodal integration, proprioceptive and thermoreceptive inputs, 32–34 object size estimation, 29–30 orientation mechanism, 10–12 proboscis extension response (PER), 27 radiant heat perceivability, 26–27 source distance estimation, 31 telotaxis, 25–26 thermoreceptor, 30–32 kratagonists, 77–80 learning and memory, 42 lyme disease
343 B. burgdorferi, biology and vector interaction genome, 272–273 tick colonization and persistence facilitation genes, 273–277 transmission facilitation genes, 277–278 ticks, biology and physiology anti-haemostatic salivary components, 249–255 anti-inflammatory salivary components, 255–259 Argasidae, 245 feeding characteristics, ixodid ticks, 247–248 haemolymph components, 270–271 host responses, 248 immunosuppressive salivary components, 259–267 Ixodidae, 246 midgut components, 267–269 mouthparts, 244–245 mesocyclics (Ms), tsetse, 128 mosquito D7 proteins, 78–79 functional neuroanatomy, 3–5 host-seeking behaviour feeding conditions and host searching, 40–41 maturation and responsiveness, 38 reproduction, 39–40 host signals food recognition and feeding, 18–19 heat, 8–10 odours, 7–8 visual cues, 14 water vapour, 10–12 hydrolases and phospholipases in, 75 learning and memory, 42 orientation mechanisms, 25 repellent, 42–43 salivary apyrase activity, 71 sensory parsimony, 35 serpin, 81–82 sialokinin, 76 sialotranscriptomes, 97–98
344 nagana. see animal Trypanosomiasis (AAT) odours, host signals, 7–8 orientation mechanisms directional information, 24 kinesis, 24 kissing bugs, 25–26 stimulus types, 23–24 taxis, 24–25 osmoregulation, ticks, 305–306 paratransgenesis, 158–159 proboscis extension response (PER), 27 resilin, 300 RGD peptide. see Arg-Gly-Asp (RGD) peptide Rhipicephalus sanguineus, 302 rickettsia-like organisms (RLOs), 153, 155 saliva-activated transmission (SAT), ticks, 307–308 salivarian trypanosomes, 121 salivary gland, haematophagous anaesthetics, 82–87 antigen (AG5) family members, 87 enzymes apyrase, 71–72 hydrlases, 74–75 inositol phosphatase, 73–74 nucleotidases, 72 peroxidase, 72 platelet-activating factor (PAF) hydrolases and phospholipases, 73 immunity-related products, 87–88 kratagonists, 77–80 physiological antagonists, primarily vasodilators, 76–77 protease inhibitors, 80–82 receptor antagonism and platelet aggregation inhibitors, 75–76 SAT. see saliva-activated transmission (SAT), ticks saturation deficit (SD), 311–313 sensory system, ticks
INDEX horizontal displacement, 314–315 kairomone, 316 olfactory sensilla, 315 sialotranscriptome, BFI, 91–92 sleeping sickness. see human African trypanosomiasis (HAT); trypanosomes Sodalis glossinidius, tsetse symbiont genetic diversity investigation, 155–156 genome characterization, 154 host-specificity, 156 in vitro culture adaption, 153–154 mutualistic relationship, 155 small rickettsia-like organisms (RLO), 153, 155 transcriptome analysis, 154–155 vector competency, 155 spirochete Borrelia. burgdorferi, biology and vector interaction genome, 272–273 tick colonization and persistence facilitation genes, 273–277 transmission facilitation genes, 277–278 lyme disease, 244 Stercorarian trypanosomes, 121 telotaxis, 25–26. see also kissing bugs teneral phenomenon, tsetse, 149–150 thermal sensing. see kissing bugs tick-borne encephalitis virus (TBEV) co-feeding transmission and synchrony, 324 ecological specificity, 324–326 ticks, biology and physiology anti-haemostatic salivary components anticoagulants, 254–255 platelet aggregation inhibitors, 249–254 vasodilators, 254 anti-inflammatory salivary components anaphylatoxin inhibitors, 256 bradykinin inhibitors, 256
INDEX histamine-binding proteins, 255–256 leukotriene-binding proteins, 257–259 Argasidae, 245 epidemiological consequences B. burgdorferi distribution, 326–327 climate sensitivity and climate change impact, 327–328 focal distribution, tick-borne encephalitis virus (TBEV), 324–326 feeding characteristics, ixodid ticks, 247–248 feeding habit adaptations cuticle structure and function, 300–301 respiration and metabolic rates, 304–305 wax deposition, 301–304 haemolymph components antimicrobials, 271 haem-binding proteins, 270–271 host-questing behaviour development rates, 316–317 diapause, 317–320 fat reserves and lifespan determination, 323 sensory systems, 314–316 temperature thresholds, 320–323 water balance constraints, 311–314 host responses, 248 immunosuppressive salivary components anti-complement proteins, 266–267 antigen-presenting cells inhibition, 265 antioxidants, 264 cytokine and chemokine regulators, 264–265 endothelial cell adhesion molecule expression inhibitors, 259 immunoglobulin-binding proteins, 266 natural killer cells, neutrophils and macrophages inhibitors, 259–264
345 T-cell and B-cell proliferation inhibition, 265–266 Ixodidae, 246 midgut components anticoagulants, 268 antimicrobials, 269 digestive proteases, 268–269 mouthparts, 244–245 pathogen conveyance immunomodulation, 306–309 osmoregulation, 305–306 transmissibility, 309–310 triatomines development, 179–182 distribution, 178–179 intestinal microenvironment antimicrobial factors, 187–188 border face, 183–184 enzymes and digestion products, 188–189 microorganisms, 185–186 pH, osmolality and ions, 184 soluble factors, 189–190 intestinal tract, digestion and excretion, 182–183 trypanosomatids effect Blastocrithidia triatomae and Trypanosoma rangeli pathogenicity, 211–217 pathogenicity classification and secondary stressors action, 210–211 subpathogenicity, 217–219 Trypanosoma cruzi, triatomines developmental cycle, 196–197 species and strains distribution, 191–195 subpathogenicity effects behaviour, 218 digestion, intestine and excretion, 218–219 fitness, development and mortality rates, 217 haemolymph composition and intestinal contents, 218 immune system and symbionts, 219
346 Trypanosoma rangeli, triatomines developmental cycle, 197–198 pathogenicity behaviour, 212–213 cuticle and tracheal system, 214 fitness, development and mortality rates, 211–212 haemolymph composition and intestinal contents, 213 immune system, 215–216 intestine and excretion, 214–215 mechanism, 217 symbionts, 216–217 species and strains distribution, 195 trypanosomatids developmental cycle in triatomines Blastocrithidia triatomae, 195–196 Trypanosoma conorhini, 196 Trypanosoma cruzi, 196–197 Trypanosoma rangeli, 197–198 double infection interactions, 219 effects on triatomines Blastocrithidia triatomae and Trypanosoma rangeli pathogenicity, 211–217 pathogenicity classification and secondary stressors action, 210–211 subpathogenicity, 217–219 host effects blood ingestion and excretion effects, 205–207 border face, 200–202 digestion effects, 204 metacyclogenesis induction, 207–209 pH, osmolality and ionic composition, 200 soluble factors effect, 209–210 starvation effects, 204–205 susceptibility and refractoriness, 198–200 species and strains distribution Blastocrithidia triatomae, 190–193 Trypanosoma conorhini, 191 Trypanosoma cruzi, 191–195 Trypanosoma rangeli, 195
INDEX trypanosomes disease control methods gene knockdown method, 156–158 paratransgenesis, 158–159 human and animal trypanosomiases causative agents, 120 disease severity and impact, 120–121 species, 121–122 life cycle, T. brucei bloodstream form (BSF), 126 epimastigote division, 128 long trypomastigotes (LT), 128 mating stage, 129 metacyclogenesis, 128–129 procyclic differentiation, 126–128 stages, diagramatic representation, 127 symbiont–tsetse–trypanosome interactions Sodalis glossinidius, 153–156 wigglesworthia glossinidius, 152 Wolbachia pipientis, 153 tsetse identification and distribution, 122–123 life cycle and physiology, 123–125 trypanosome interactions, 129–151 (see also tsetse-trypanosome interactions) tsetse-trypanosome interactions digestive enzymes, 134 host blood factors trypanocidal acivity, 131–132 virulence and infectivity determination, 132 immune system, tsetse, 135–136 adaptive immunity, 147–148 antioxidants, 143–144 effector molecules, 141–143 fly immune system and microbial balance, 148 infection refractoriness, 135 innate immune system, 136 lectins and programmed cell death, 144–147 peritrophic matrix, 136–137
INDEX prophenoloxidases (ProPOs), 147 recognition and activation, 138–141 signalling pathway, 137–138 Toll and Imd signalling pathway, 138–140 tsetse glutamic acid–proline (EP) protein, 147 infection and tsetse physiology, 148–149 midgut environment and signals for differentiation, tsetse enzyme activity, 134 glucose concentration, 133 pH conditions, 133–134 temperature reduction, 133 parasite surface coat gly-pro-glu-glu-thr (GPEET) repeats, 130
347 procyclins functions, 130–131 surface molecule functions, 131 trypanosome transmission environmental temperature, 151 sex and age, 149 starvation, 150–151 teneral phenomenon, 149–150 visual cues, host signals, 12–14 water vapour, host signals, 10–12 Wigglesworthia-Glossina symbiosis, 152 Wolbachia pipientis, tsetse symbiont, 153 zoo-prophylaxis, ticks, 308–309
