Oceanography And Marine Biology

OCEANOGRAPHY AND MARINE BIOLOGY

AN ANNUAL REVIEW Volume 27

OCEANOGRAPHY AND MARINE BIOLOGY AN ANNUAL REVIEW Volume 27

HAROLD BARNES, Founder Editor MARGARET BARNES, Editor The Dunstaffnage Marine Research Laboratory Oban, Argyll, Scotland Assistant Editors A.D.Ansell R.N.Gibson T.H.Pearson

ABERDEEN UNIVERSITY PRESS

FIRST PUBLISHED IN 1989 This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.” This book is copyright under the Berne Convention. All rights reserved. Apart from any fair dealing for the purpose of private study, research, criticism or review, as permitted under the Copyright Act, 1956, no part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, electrical, chemical, mechanical, optical, photocopying, recording or otherwise, without the prior permission of the copyright owner. Enquiries should be addressed to the Publishers. © Aberdeen University Press 1989 British Library Cataloguing in Publication Data Oceanography and marine biology: an annual review.—Vol. 27 1. Oceanography—Serials 2. Marine biology—Serials 551.46′005 ISBN 0-203-01629 -7 Master e-book ISBN

ISBN 0-203-19 130-7 (Adobe eReader Format) ISBN 0-08-036397-0 (Print Edition) ISSN 0075-3218

PREFACE

The demand for this series of Annual Reviews continues unabated. Many people are still willing to contribute and we are grateful to them. It is always a pleasure to acknowledge the willingness with which they accede to our editorial requests. The success of the series depends on their co-operation; without it our task would be impossible. We thank them all and also the publishers for maintaining the regular appearance of these Annual Reviews. In the first 27 volumes of the series titles of references have not been included. The reason was the space they would occupy which it was thought could be better spent on text. Present demand, however, indicates that a change is necessary. From Volume 28 onwards it is hoped to include full titles in reference lists.

CONTENTS

page PREFACE

iv

A Comparison of Marine Photosynthesis with Terrestrial Photosynthesis: a Biochemical Perspective GRAHAM J.KELLY

1

The Ecology and Behaviour of Ascidian Larvae IB SVANE AND CRAIG M.YOUNG

28

Egg Production in Cirripedes MARGARET BARNES

62

Biology of Marine Herbivorous Fishes MICHAEL H.HORN

134

The Benguela Ecosystem. Part VI. Seabirds A.BERRUTIN.J.ADAMS AND S.JACKSON

222

Review of Research Relevant to the Conservation of Shallow Tropical Marine Ecosystems B.G.HATCHER, R.E.JOHANNES AND A.I.ROBERTSON

284

AUTHOR INDEX

353

SYSTEMATIC INDEX

386

SUBJECT INDEX

398

Oceanogr. Mar. Biol. Annu. Rev., 1989, 27, Margaret Barnes, Ed. Aberdeen University Press

A COMPARISON OF MARINE PHOTOSYNTHESIS WITH TERRESTRIAL PHOTOSYNTHESIS: A BIOCHEMICAL PERSPECTIVE GRAHAME J.KELLY* CSIRO Marine Laboratories, G. P. O. Box 1538, Hobart, Tasmania 7001, Australia

ABSTRACT The maximum photosynthetic rate of marine microalgae—both cultures and natural populations—is often estimated as being two to four times higher than that of leaves of terrestrial plants, although the average estimate is one to two times. In an attempt to ascertain whether the higher estimates are biochemically feasible, the photosynthetic biochemistry of the two groups of plants is compared. A survey of the literature on the physiology and biochemistry of marine algae shows that: (1) their chlorophyll a content (dry weight basis) is similar to that of leaves of terrestrial plants, (2) the in vitro values obtained for photosynthetic electron transport and ribulosel,5-bisphosphate carboxylase activity have all been low, (3) carboxylation of phosphoenolpyruvate can account for no more than 10% of net carbon fixation, (4) photorespiration, which would have little influence on estimates of primary productivity, is possibly prevented by active uptake of inorganic carbon, (5) photoinhibition is either avoided or protections against it exist, and (6) the extent of dark respiration during photosynthesis is not known. Information in the literature indicates that the actual in situ (light-limited) photosynthesis of marine microalgae is 7% of its potential (at light saturation). Assuming that phytoplankton biomass (chlorophyll) has been correctly estimated, and that the biochemistry of marine algal photosynthesis is similar to that of terrestrial plants, then the current estimate of in situ marine photosynthesis is concluded to be biochemically reasonable.

INTRODUCTION This review is an attempt to put into some order the rambling thoughts of a peas-and-barley biochemist whose head was suddenly immersed in the ocean. Peas-and-barley biochemists are accustomed to expecting maximum rates of photosynthesis (Pmax: photosynthesis at saturating light and carbon dioxide and ambient temperature) of between 200 and 400 µmoles CO2 fixed·mg chlorophyll−1·h−1. These rates are equivalent to

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GRAHAME J.KELLY

assimilation numbers (i.e. photosynthesis expressed as mg carbon fixed.mg chl. a−1·h−1) of between 2.4 and 4.8. I was therefore rather surprised to find that estimates of the Pmax of marine phytoplankton commonly yielded assimilation numbers between 5 and 10 (and sometimes approaching 20), and that investigators of marine photosynthesis often fear that assimilation numbers much below 5 may represent under-estimates of the true values. This difference between the marine and terrestrial sectors is illustrated by the contrast between the following two quotations: “Maximum rates of photosynthesis [by land plant leaves] depend on species and growth conditions; they can surpass 300 µmol CO2 reduced.mg chlorophyll−1·h−1” (i.e. assimilation number of 3.6) (Heber, Neimanis & Dietz, 1988); “Often cited as evidence of low growth rates has been the fact that assimilation ratios reported from open-ocean stations have almost always been <5 g carbon.g chlorophyll a−1·h−1…” (Laws, DiTullio & Redalje, 1987). The core of this review analyses this apparently superior photosynthetic performance of marine phytoplankton over terrestrial plants. Particular attention is given to the known and unknown biochemical aspects of photosynthesis in these two groups of plants, and to the interplay between this biochemistry and the plants’ respective environments. I conclude that, while all but the highest of the claimed rates of Pmax by marine phytoplankton are biochemically feasible, the concern that current measurements of primary productivity in the ocean may be misleadingly low is not necessarily valid, because higher estimates of Pmax would be inconsistent with the known biochemistry of photosynthesis. CURRENT ESTIMATES OF TERRESTRIAL AND MARINE PHOTOSYNTHESIS ACTUAL PHOTOSYNTHESIS Actual (i.e. in situ marine algal photosynthesis is generally agreed to average about 60g carbon fixed·m−2·yr −1 (Ryther, 1969; Whittaker & Likens, 1975; Malone, 1980; Dring, 1982, p. 91; Boynton et al., 1983). Production in the nutrient-poor tropical oceans is usually below this average, while it is above the average in the nutrient-rich cooler waters of the northern and southern oceans, and off the west coasts of Africa and the Americas (Bunt, 1975). Production is relatively high in shallow coastal regions where both macroalgae (seaweeds) and microalgae (phytoplanktonic and benthic) occur, but because these areas are comparatively small, they contribute only 23% of the total marine photosynthesis. The open ocean phytoplankton contributes the remaining 77% (Table I); this review, therefore places most emphasis on marine microalgal photosynthesis. Terrestrial photosynthesis, similarly expressed on an area basis, is about five-fold greater than ocean photosynthesis. Assuming dry matter to be 45% carbon (Walker, 1979), the mean net primary productivity on land is estimated to be 350 g carbon·m−2·yr−1 (Whittaker & Likens, 1975). When photosynthesis is expressed on a chlorophyll basis, a contrasting picture, however, emerges. Photosynthesis expressed in the form of assimilation numbers (i.e., mg carbon fixed·mg chl. a−1·h−1) gives 0.053 for terrestrial and 0.31 for marine photosynthesis. For the open-ocean phytoplankton, the value is 0.43. (These calculations are based on the values in Table I, and assume dry matter to be 45% carbon, and one year to have 4380 hours of daylight). Thus, based upon chlorophyll, marine photosynthesis is six times greater TABLE I

* Present address: Department of Biology, Queensland University of Technology, G.P.O. Box 2434, Brisbane, Queensland, 4001 Australia.

PHOTOSYNTHESIS: A BIOCHEMICAL PERSPECTIVE

3

Global net productivity of terrestrial and marine plants (from Whittaker & Likens, 1975) Production 109 tonnes dry matter·yr−1 (P)

Chlorophyll 107 tonnes (Chl)

Biomass 109 tonnes dry matter

Community Total

%

Total

%

Total

%

Marine Open ocean 42 13 Coastala 118 Terrestrial

24 7 69

1.0 0.8 23

4 3 93

1.0 2.9 1840

0.06 0.16 99.8

a

P·Chl−1 ×103

4.2 1.6 0.51

Continental shelves, estuaries, seaweed beds, reefs.

than terrestrial photosynthesis, although the chlorophyll a contents of both groups of plants, expressed as a percentage of dry weight, are apparently about the same (see p. 17). POTENTIAL PHOTOSYNTHESIS The above actual assimilation numbers are in fact much lower than potential photosynthesis values. Many investigations have been made with phytoplankton, and from a sample of these (Table II), a mean potential assimilation number of 6.4 is obtained. Most values in this Table were measured with high irradiances. The assimilation number of 0.43 for actual open ocean productivity is only 7% of this potential value because (1) the phytoplankton grow at depths where irradiance is not saturating for photosynthesis, (2) irradiance is low early in the morning and late in the afternoon, and (3) respiratory losses occur, probably mainly at night. Unlike land plants, which must cope with periodic water shortage and a less-than-saturating supply of CO2, there seems to be no other major hindrance to phytoplankton photosynthesis (Table III). As emphasised earlier, the measured potential rates of photosynthesis (Pmax) by phytoplankton (Table II) are, however, often well above maximum values measured for terrestrial plants. The mean assimilation number for 49 C3 land plants under saturating irradiance (but in air, and therefore below saturation with respect to CO2) was 1.8 (Björkman, 1981). From a comparable survey (Boardman, 1977) mean Pmax values of 2.0 for sun species, and 0.39 for shade species can be calculated. In a more recent study of 10 C4 plants, the mean Pmax was 2.2 (Usuda, Ku & Edwards, 1984; the terms “C3” and “C4” are explained on p. 28). Bean, pea, spinach, tomato, and wheat leaves, saturated with both light and CO2, gave values between 3.6 and 4.9 (Dietz, Neimanis & Heber, 1984; Dietz, 1986; Stitt, 1986; Chow & Anderson, 1987a; Kobza & Edwards, 1987; Terashima & Evans, 1988). Biochemical experiments on photosynthetic electron transport suggest that an assimilation number in excess of 4.3 (as recorded for sunflower) is unlikely with terrestrial TABLE II A selection of assimilation assimilation numbers in the laboratory and in the field. Some assimilation numbers have been calculated from values of other parameters provided by the authors Alga or locality

Assimilation numbers mg C·mg chl a −1·h−1

Reference

Various oceanic waters Skeletonema sp. Peruvian coast Skeletonema costatum

1–8 4.8 0.4–7.4 5.1

Steemann Nielsen & Hansen, 1961 Cassie, 1963 Strickland et al., 1969 Jørgensen, 1970

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GRAHAME J.KELLY

Alga or locality

Assimilation numbers mg C·mg chl a −1·h−1

Reference

Various oceanic waters

4.9

Nannochloris atomis Glenodinium sp. N. Pacific gyre Three diatoms Two dinoflagellates Gulf of Maine One diatom and one chlorophyte Ten species Ceratium longipes Pyrocystis noctiluca Near Hawaii Narragansett Bay Bedford Basin Near Hawaii

4.5 1.1 8.8 0.2–0.5 3.0 1–18 2.8–5.3 1.6–5.8 2.2–10.4 2.9 5–14.5 1.7–5.8 1.6 11.2–18.9

Tropical Atlantic Near Hawaii Synechococcus spp. Vineyard Sound Thalassiosira fluviatilis Phaeodactylum tricornutum Chlorella sp. Br. Columbian coast South of Tasmania

8.5 10.3 16.0 10–20 16 11.5 7.5 1–19 2.6

Celtic Sea Tropical Pacific Firth of Forth Antarctic N.W.Atlantic Hudson Bay Antarctic Great Barrier Reef Synechococcus N.Pacific gyre Skeletonema costatum

1–6 7.5 3.5 3–8 0.2–7.4 1–9 0.8–4.4 3–14 6–12 9.5 3.3

Gulf of Fos

0.5–14

Eppley, 1972 (average of 17 values, mostly from other investigators) Yentsch, 1974 Burris, 1977 Venrick, Beers & Heinbokel, 1977 Kremer & Berks, 1978 Prézelin & Alberte, 1978 Glover & Morris, 1979 Falkowski & Owens, 1980 Glover, 1980 Taguchi, 1981 Rivkin et al., 1982 Bienfang & Takahashi, 1983 Furnas, 1983 Smith et al., 1983 Williams, Heinemann, Marra & Purdie, 1983 Gieskes & Kraay, 1984 Laws et al., 1984 Barlow & Alberte, 1985 Glibert, Dennett & Goldman, 1985 Hobson, Morris & Guest, 1985 Holdsworth, 1985 Thinh & Griffiths, 1985 Forbes, Denman & Mackas, 1986 Furuya, Hasumoto, Nakai & Nemoto, 1986 Joint & Pomroy, 1986 King, 1986 Mills & Wilkinson, 1986 Palmisano et al., 1986 Prézelin et al., 1986 Rochet, Legendre & Demers, 1986 Sakshaug & Holm-Hansen, 1986 Furnas & Mitchell, 1987 Kana & Glibert, 1987a Laws et al., 1987 Mortain-Bertrand, Descolas-Gros & Jupin, 1987 Plante-Cuny & Bodoy, 1987

PHOTOSYNTHESIS: A BIOCHEMICAL PERSPECTIVE

Alga or locality

Assimilation numbers mg C·mg chl a −1·h−1

Reference

McMurdo Sound Brittany Coast Prorocentrum mariae-lebouriae Gulf of Maine Mediterranean Sea

0.1–8.1 3.4 0.5–5.8 5 5.1

Rivkin & Putt, 1987 Videau, 1987 Coats & Harding, 1988 Legendre et al., 1988 Lohrenz et al., 1988

5

plants (Delaney & Walker, 1978). Marine phytoplankton, however, have been credited with assimilation numbers two or three times this value (see Table II). Such high assimilation numbers could be the consequence of either (1) errors in the estimate of either photosynthetic CO2 fixation rate or chlorophyll content, or (2) a photosynthetic biochemistry that has the capacity for a several-fold greater rate of CO2 fixation than that in the leaves of land plants. These possibilities are discussed in this review. Attention is being given to these points, not so much because the values appear high (although in most cases not inconceivably high), but because concern has been expressed in the oceanographic literature that the 14C technique may have often led to photosynthesis—and hence the primary productivity of the oligotrophic open ocean—being under-estimated (Sheldon & Sutcliffe, 1978; Shulenberger & Reid, 1981; Dring, 1982, p. 89; Jenkins, 1982; Gieskes & Kraay, 1984; Joiris, 1985; Cullen, Zhu & Pierson, 1986). TABLE III Comparison of oceanic photosynthesis with terrestrial photosynthesis Land

Ocean

White light Up to full sunlight (1700 µE·m−2·s−1) but many leaves shaded Photosynthetic cells support many non-photosynthetic cells and skeletal material Often water-restricted Often nutrient-restricted Limited by supply of inorganic carbon (CO2)

Blue-green light Lower intensity (10 to several hundred µE·m−2·s−1

a

Little to support Never water-restricted Adequate nutrients?a Probably not limited (sea water at pH 8·2 contains 2 mM )

The relationship between phytoplankton growth and nutrient supply is largely beyond the scope of this review. Suffice to mention that certain nutrients (particularly nitrogen, phosphorus, and iron) are often at very low levels in the oligotrophic open ocean, such that any long-term expansion of the phytoplankton population as a whole is prevented, while grazing of phytoplankton and subsequent nutrient regeneration by heterotrophs are tightly coupled, so that the growth of individual phytoplankton cells is seldom nutrientlimited (deficiency symptoms have seldom been observed in natural populations) (Goldman, McCarthy & Peavey, 1979; Rivkin et al., 1982; Goldman, 1986; Balch, Garside & Renger, 1987; Martin & Fitzwater, 1988). In coastal and highlatitude regions of the oceans, physical mixing and advection, as well as regeneration, become, however, major processes for supplying nutrients, contributing to greater and seasonally more variable primary productivity in these regions (Lorenzen, 1976; Kirk, 1983, p. 277).

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THE 14C TECHNIQUE The most popular method for estimating phytoplankton CO2 fixation used at present was introduced by Steemann Nielsen (1952) some 35 years ago. The procedure is described in detail by Strickland & Parsons (1972), but in essence, 14C-labelled bicarbonate is added to a sample of sea water containing its natural population of phytoplankton. Photosynthesis is allowed to proceed for several hours, after which the sample is filtered, washed (to remove unfixed ), and the remaining radioactivity is measured. Assimilation numbers are calculated from this measurement and an estimate of chlorophyll. In general, the procedure has yielded acceptably high assimilation numbers for Pmax (Table II). Nevertheless, there is a widely held suspicion that it gives greatly under-estimated values (Round, 1981, p. 259; Gieskes & Kraay, 1984) because it does not allow for the presence of toxic metal ions on bottle walls, the depletion of nutrients, the rupture of cells on filters, respiration, and grazing by microzooplankton. It is, however, unlikely that the underestimates would be large given that the assimilation numbers obtained are of the same order of magnitude as the accepted Pmax of land plants. Instead, the likelihood of over-estimation needs to be considered. This could result from either erroneously high values of 14C incorporation or erroneously low values for chlorophyll (chlorophyll is discussed below). Admittedly it is, at present, difficult to see how either of these possibilities could arise frequently. Evidence for some over-estimation of net CO2 fixation has been provided from 14C experiments with cultures; it has been attributed to respiration of carbohydrate reserves not labelled with 14C during the experiments (Li & Goldman, 1981; Richardson, Samuelsson & Hällgren, 1984). In what is known of the carbon metabolism of photosynthetic cells, there is, however, no major pathway whereby a separate pool of carbohydrate reserves are respired during photosynthesis. Alternatively, in the field, over-estimates might result from non-photosynthetic fixation of Bacteria (including, presumably, marine bacteria) contain enzymes that catalyse the carboxylation of phosphoenolpyruvate (PEP) (Gieskes & Kraay, 1984) and/or pyruvate, and phytoplankton are also able to carboxylate PEP. These carboxylations are not connected to photosynthesis in bacteria, and cannot contribute more than 10% of the net photosynthetic carbon fixation in phytoplankton (see Fig 4, p. 30)—or in any other photosynthetic cell for that matter, because the only carboxylase connected to a series of reactions that permits plant growth is ribulose-l,5-bisphosphate carboxylase (RuBP carboxylase, see p. 26). PEP carboxylation may, however, occur temporarily at high rates, using PEP generated from carbohydrate reserves accumulated during active photosynthesis at some other time or place. In attempts to correct for PEP carboxylations, researchers usually include control incubations in the dark. These have often given positive values (so-called “dark fixation”), not infrequently equal to 50% or more of the values obtained with incubations in the light (Yentsch, 1974). This dark fixation may, however, not all continue in the light; for example, the phytoplankton may carboxylate more PEP in the dark than in the light. Attempts to control this unknown factor by replacing dark controls with light-plus-photosyntheticpoison controls (Legendre, Demers, Yentsch & Yentsch, 1983) are of questionable value, because poisoned photosynthesis can lead to metabolic perturbations that re-establish “dark metabolism”.

PHOTOSYNTHESIS: A BIOCHEMICAL PERSPECTIVE

7

CHLOROPHYLL ESTIMATION OF CHLOROPHYLL Assimilation numbers could just as easily be wrong because of chlorophyll estimates as carbon-fixation estimates. This possibility will not be exhaustively discussed here, but four points may be mentioned. First, chlorophyll in small picoplanktonic algae may have escaped detection in the past because the pores of filters were too large to retain these cells. Secondly, acetone does not extract any chlorophyll from intact cells of some algae (e.g. Pelagococcus: Vesk & Jeffrey, 1987); thus the proportion of tough cells such as these actually broken by preliminary homogenisation or sonication should be considered. Methanol will extract chlorophyll from these cells (Holm-Hansen & Riemann, 1978; Wood, 1985), but the subsequent estimation is less exact because methanol causes allomerisation of chlorophyll. Thirdly, the accuracy of chlorophyll estimations from in vivo fluorescence is still being debated (Harris, 1978; Leftley, Bonin & Maestrini, 1983; Falkowski & Kiefer, 1985; Keller, 1987). Finally, some microalgae are reported to have chloroplasts so fragile that enzymic degradation of chlorophyll a occurs during preparation of the sample for estimation. Jeffrey & Hallegraeff (1987) found that degradation to chlorophyllide a occurred to a significant extent in 27 of 93 examined species. The chlorophyllide a would, however, be estimated as chlorophyll a in spectrophotometric and fluorimetric procedures (but not using HPLC), so prob ably did not contribute to under-estimates in the past. Suzuki & Fujita (1986), however, reported that degradation to phaeophorbide a occurred in the diatom Skeletonema costatum; if this is a widespread occurrence, then it is likely that chlorophyll a would have been under-estimated by spectrophotometry and fluorimetry. CELLULAR CHLOROPHYLL a CONTENT Another reason for phytoplankton having relatively higher assimilation numbers than terrestrial plants might be because phytoplankton cells contain relatively less chlorophyll a. This would be possible if a greater share of the light-harvesting task in marine algal cells were taken over by pigments other than chlorophyll a, such as chlorophylls b and c, carotenoids, and biliproteins (Hiller & Goodchild, 1981; Prézelin, 1981; Siefermann-Harms, 1987). This would result in high assimilation numbers (i.e. CO2 fixed per unit of chlorophyll a). Most available information argues, however, against this explanation. First, the limiting step that determines photosynthetic capacity (at light-saturation) is believed to be a reaction of photosynthetic electron transport or of CO2 fixation rather than light-harvesting (see p. 20). Thus, the contents of accessory pigments would be irrelevant to the light-saturated rate of photosynthesis. Secondly, even under light-limited conditions where the photosynthetic rate is determined by lightharvesting, high values for carbon fixed per unit of chlorophyll a are unlikely to result from low chlorophyll a contents, because reported values for the latter are similar to those of the leaves of terrestrial plants. The mean chlorophyll a content of the leaves of 49 C3 land plants calculated from values given by Björkman (1981), is about 0.9% of dry weight. Similarly, values of 0.8% and 1.3% are obtained from values given by Boardman (1977) for five sun species and five shade species, respectively (assuming dry weight to be onesixth of fresh weight). From data on marine microalgae, I estimate the values also to be about 1 % of dry weight (Table IV). In addition, 1 % is the global estimate for the open ocean, from 3g dry matter (biomass) ·m−2 and 0.03g chl.·m−2 (Whittaker & Likens, 1975). Thus, TABLE IV

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GRAHAME J.KELLY

Chlorophyll a content of marine phytoplankton Alga(e)

Chlorophyll a % dry weight

Reference

Skeletonema costatum Six species Two dinoflagellates Dunaliella and Skeletonema 11 diatoms and 8 dinoflagellates Five field samples Two psychrophilic diatoms 15 species 11 species Two Thalassiosira species Pelagococcus subviridis Prorocentrum mariae-lebouriae

3.0a

Jørgensen, 1970 Vesk & Jeffrey, 1977 Prézelin & Alberte, 1978 Falkowski, 1981 Hitchcock, 1982 Kirk, 1983, p. 181 Van Baalen, 1985 Geider, 1987 Moal et al., 1987 Sakshaug et al., 1987 Vesk & Jeffrey, 1987 Coats & Harding, 1988

0.40–3.7a 0.8a 1–3 0.6a 0.9b 0.47–2.2 1.2b,c 1.5a,b 2.9a,b 1.5a 1.1–2.9a

a

Calculated from cell volume, and assuming dry weight to be one sixth of fresh weight. Mean value. c Calculated from carbon: chlorophyll a ratios corrected to growth at 50 µE·m−2·s−1, and taking dry matter to be 45% carbon. b

most available measurements do not indicate any significant difference between the chlorophyll a contents of land plants and marine plants. Nevertheless, some scattered information does suggest that certain microalgae may contain relatively little chlorophyll a. Cells that are nutrient-depleted, and thus growing slowly, have low chlorophyll a contents, as evidenced by carbon: chlorophyll a ratios in excess of 100 (Eppley, 1972). Nevertheless, due to their slow growth these cells would not be expected to have high assimilation numbers. The low chlorophyll contents may represent a physiological response of the cells to avoid harvesting more light energy than can be used for growth, thus avoiding photoinhibition (see p. 34). The low contents are unlikely to be due to a lack of nitrogen for incorporation into additional chlorophyll molecules, because the nitrogen demand for chlorophyll is quite low—even in healthy cells it is only about 2% of the nitrogen demand for protein. Cyanobacteria, in which phycobilipigments perform much of the light-harvesting, and most of the chlorophyll a is restricted to photosystem I (Mimuro & Fujita, 1977; Hiller & Goodchild, 1981), might also be thought to have relatively low chlorophyll a levels. But this is not necessarily so: in white-light grown Anacystis nidulans (a freshwater species) it was 0.68% of dry weight (Evans & Alien, 1973), and from studies of marine Synechococcus (Barlow & Alberte, 1985; Kana & Glibert, 1987b; Prézelin, Glover & Campbell, 1987) chlorophyll contents of between 0.3 and 3.3% of dry weight are obtained on the basis of dry matter being 45% carbon, dry weight being one-sixth of fresh weight, and cells being spheres of 1.0 µm diameter. Thus, it is difficult to propose that marine cyanobacteria have notably less chlorophyll a than do eukaryotic photosynthetic cells. This conclusion is relevant because cyanobacteria, together with small eukaryotes, comprise the “picoplankton” that make up a significant portion of the biomass of autotrophic cells in deep regions of the tropical oceans (Platt, Subba Rao & Irwin, 1983; Glover, Keller & Guillard, 1986; Iturriaga & Mitchell, 1986). In an extensive review of algal picoplankton, Stockner & Antia (1986) tabulated eight marine assimilation numbers, the approximate mean of which was 2.3, which is lower than the mean (6.4) of the values in Table II.

PHOTOSYNTHESIS: A BIOCHEMICAL PERSPECTIVE

9

A final point is that the chlorophyll content of a sea-water sample may not be taken as an indicator of the photo synthetic capacity of the microalgae in that water. Work with terrestrial plants has shown that lightsaturated photosynthesis in air is only poorly correlated with the chlorophyll content of leaves, but is highly correlated with the leaf’s content of ribulose-1,5-bisphosphate (RuBP) carboxylase, which is the enzyme responsible for at least 90% of the photosynthetic CO2 assimilation (Björkman, 1981; Usuda, Ku & Edwards, 1984; Makino, Mae & Ohira, 1984; Evans, 1986). A similar conclusion was recently made for the marine alga Dunaliella tertiolecta (Sukenik, Bennett & Falkowski, 1987). CELL GROWTH RATES The values for open-ocean phytoplankton listed in Table I, show annual production to be 42-times the standing stock. This implies that the mean turnover time of the phytoplankton carbon pool in surface water is days, from which it can be inferred that the growth rate of cells varies between low values (growth effectively ceased) to values typical of rapidly growing cells (e.g. 1·day−1). In cells with a carbon content of 45% of the dry weight and a chlorophyll content of 1% (i.e. carbon: chlorophyll a ratio of 45), a growth rate of 1·day−1 corresponds to a photosynthetic rate of 45 mg carbon fixed·mg chl. a−1·day−1, or assimilation number (for 12 h of daylight) of 3.8 (see also Eppley, 1972; Falkowski, 1981). This is 60% of the mean of the values in Table II, and close to the maximum values observed in land plants. Very high growth rates, such as the 3 h generation time estimated by Sheldon & Sutcliffe (1978), would give assimilation numbers greater than those usually observed for land plants. Do such values represent the upper limits for Pmax? It would seem so: the maximum growth rate of Chlorella “fully charged with reducing equivalents and energy” was reported as a doubling time of 3 h (Pirt, 1986). Other data are consistent with doubling times being considerably greater than 3 h. Eppley (1972) reviewed the growth rates for 160 laboratory cultures of both freshwater and marine microalgae grown at temperatures up to 33°C “largely in continuous light”, and all were below 4.4·day−1. This maximum corresponds to a doubling time of 5.5 h, and to an assimilation number of 8.4 if carbon and chlorophyll a contents were as above, and lighting was continuous. Geider (1987) concludes from theoretical considerations that a growth rate of 2.6·day−1 at 20°C is the maximum attainable, but requires a carbon: chlorophyll a ratio of 27, in which case the chlorophyll a content would approach 1.7% of dry weight (once again assuming dry matter to be 45% carbon) and the corresponding maximum assimilation number would be 5.7, which is close to the mean of the values in Table II. Langdon (1987) found that the photosynthetic efficiencies of several species were very similar, at about 0.02 µg carbon·µg chl. a−1·h−1. This value predicts that a potential assimilation number of 6.4 (i.e. the mean of the values in Table II) will occur if light saturation of photosynthesis is reached at 320 Most species would be almost or completely light-saturated at this irradiance. TEMPERATURE As most values of marine and terrestrial photosynthesis have been obtained from measurements at ambient temperatures, temperature is not discussed in detail in this review. The reader is referred to Eppley (1972) for an account of the effect of temperature on phytoplankton production, including observations of temperature coefficient (Q10) values close to 2.0. This author briefly referred to the Bělehrádek equation, and it may be worth noting that the response of photosynthesis to change in temperature possibly follows the Bělehrádek equation (McMeekin et al., 1987) more closely than the Arrhenius equation. Re-analysis of data presented by Fu & Gibbs (1987) on the effect of temperature on photosynthesis by spinach, pea, and

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Chlorella indicates that, in each case, the observed responses obey the Bělehrádek equation, with an exponent of 1, quite closely (D.A.Ratkowsky, pers. comm.). Tmin values of between +1°C and −2°C were obtained. This parameter Tmin specifies a lower temperature where growth (or, in this case, photosynthesis) theoretically becomes zero due exclusively to the lowering of temperature. (In practice, other factors such as freezing may stop growth before Tmin is reached). In a study of the response of phytoplankton photosynthesis to temperature, Li (1985) found that the Bělehrádek equation, with an exponent of 2, fitted the data quite well. Tmin values between 14.4°C and −23.4°C were obtained, the lower values being for phytoplankton from colder waters. Optimum temperatures varied from 11.8°C (for samples collected from −1.1°C water) to 27.3°C (for samples from 27.5°C water). BIOCHEMICAL DETERMINANTS OF Pmax A comparison of the reported photosynthetic capacities of leaves of terrestrial plants and marine phytoplankton raises the question of which biochemical step determines the Pmax. Boardman (1977) noted that photosynthetic capacity will be influenced by one or more of the “dark” steps of photosynthesis, such as the rate of diffusion of CO2 from the cell wall to the chloroplast, the rate of photosynthetic electron transport, and the carboxylation reaction, but is expected to be independent of the efficiency of light absorption and the primary photochemistry. Leaves of plants grown under high light intensities are capable of higher rates of light-saturated photosynthesis (measured with air-levels of CO2) than are leaves of plants grown under low light intensities; this applies whether the rates are expressed on a chlorophyll basis or a leaf area basis, since chlorophyll per unit leaf area varies little between the two groups of plants. The increased photosynthesis by the high-light plants correlates with increased activities of RuBP carboxylase and photosynthetic electron transport, and increased amounts in the leaf of the electron transport component cytochrome f (Boardman, 1977; Björkman, 1981; Chow & Anderson, 1987a; Evans, 1987). Thus, in leaves of terrestrial plants, Pmax appears to be primarily determined by the contents of electron transport chains and RuBP carboxylase molecules (von Caemmerer & Farquhar, 1981; Dietz, Neimanis & Heber, 1984; Heber, Neimanis & Dietz, 1988). A comparable study of a marine microalga (Dunaliella tertiolecta), grown at five irradiances between 80 and 1900 has been reported recently by Sukenik et al. (1987). They also obtained evidence that the cell’s content of RuBP carboxylase determined its Pmax, except at very high light intensities where the capacity for photosynthetic electron transport would determine its Pmax. The response of the microalga to low or high light was, however, rather different to that of plant leaves: neither the cell’s content of RuBP carboxylase nor the photosynthetic capacity per cell changed, but the chlorophyll a content in the low-light cells was about six times greater than in the high-light cells; their Pmax was consequently one-sixth that of highlight cells. The cytochrome fcontent of Dunaliella cells also increased fourfold in response to low light (rather than decreasing, as happens in plant leaves). Another electron transport component, plastoquinone (which is implicated in the rate-limiting reaction of photosynthetic electron transport: Harris, 1978; Richardson, Beardall & Raven, 1983), also increased in low-light grown Dunaliella. In pea leaves (Chow & Anderson, 1987b) and in the freshwater microalga Scenedesmus obliquus (Fleischhacker & Senger, 1978), the levels of both cytochrome f and plastoquinone were, however, lower in low-light grown plants than in high-light grown. The responses of Dunaliella also differed from those reported for diatoms. Some diatoms (but not all) contain similar amounts of chlorophyll a, whether grown at low or high light intensities (see Kirk, 1983, p. 305); and with respect to RuBP carboxylase, the activity per cell in Phaeodactylum tricornutum increased over sixfold between about 10 and 200 (Beardall & Morris, 1976). Thus it appears

PHOTOSYNTHESIS: A BIOCHEMICAL PERSPECTIVE

11

Fig 1.—A stoichiometric representation of the generation of NADPH and ATP during non-cyclic photosynthetic electron transport in the pigment-containing membranes of chloroplasts.

that, while photosynthetic capacity is determined by the capacities for photosynthetic transport and RuBP carboxylation, the mechanism(s) by which this is achieved may not be the same in all species. As the efficiency of light absorption is not expected to influence photosynthetic capacity, the variations in chlorophyll a content that are normally observed should not directly influence this parameter. These changes in chlorophyll a level will, however, influence Pmax, because Pmax is an expression of photosynthetic capacity per unit of chlorophyll a. The independence of photosynthetic capacity—and the dependence on Pmax—on chlorophyll a level needs to be kept in mind when chlorophyll a levels vary considerably. Variation appears to be more common in phytoplankton than in leaves of terrestrial plants, but the tendency in phytoplankton appears to be in the direction of high chlorophyll levels in low-light adapted cells. There is no strong indication that the opposite trend (low chlorophyll levels in highlight adapted cells) extends to the point where chlorophyll levels are markedly lower (and thus Pmax values are markedly higher) than for the leaves of sun plants on land. PHOTOSYNTHETIC ELECTRON TRANSPORT AND LIGHT-HARVESTING POTENTIAL OF PHYTOPLANKTON PHOTOSYNTHETIC ELECTRON TRANSPORT Theoretically, it should be possible to demonstrate that chloroplast membranes isolated from marine algae are able to evolve oxygen during photosynthetic electron transport at a rate comparable with an assimilation number of 6.4 (Table II). This is because the photo-assimilation of one molecule of CO2 utilises 2 NADPH, and 2 NADPH are produced by the photosynthetic electron transport that releases one molecule of O2, as shown in Figure 1. The oxidation of two molecules of water by a poorly understood (Avron, 1981), Mndependent system (“black box”—“s” represents oxidation state) releases one molecule of oxygen, four protons (into the thylakoid lumen) and four electrons. Each electron is moved through the electron transport chain, receiving as it goes two boosts of light energy (from captured photons) to move it from the reaction centre chlorophylls P-680 and P-700 to (respectively) the relatively electronegative components Qox (a special plastoquinone) and the Fe3+ of bound iron-sulphur centres. Thus, the complete transit of the four electrons requires eight photons. During this electron transport, four more protons are translocated from the stroma to the thylakoid lumen by the plastoquinone (PQ) oxidation/reduction cycle. The total of eight accumulated protons subsequently moves out through a coupling factor that uses the energy in this proton gradient to synthesise 2.67 ATP. Four of the protons return to the plastoquinone cycle, while the other four (conceivably those released from the water-splitting step) rejoin, in effect, their four electrons when the latter arrive at NADP+, to form NADPH. This NADPH may then be used in CO2 fixation (see Fig.3, p. 28). As mentioned above, the processes are balanced so that, during carbohydrate synthesis, one CO2 is fixed for each O2 evolved and each eight photons captured: (a) , (b) as shown in Figure 3, exactly are used during the conversion of one CO2 to carbohydrate. Excellent representations of the dynamics of photosynthetic electron transport have recently been presented by Gounaris, Barber & Harwood (1986) and Murphy (1986). These authors describe how plastoquinone and (probably over shorter distances) plastocyanin, may diffuse along thylakoid membranes, moving reducing equivalents between three intrinsic supermolecular complexes: (1) photosystem II with

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GRAHAME J.KELLY

associated light-harvesting pigment-proteins and proteins involved in water-splitting, (2) cytochromes b6 and f and iron-sulphur protein, and (3) photosystem I with associated light-harvesting pigment-proteins and proteins involved in ferredoxin reduction. The light-harvesting pigment-proteins of plant leaves, green algae and phycobiliprotein-containing algae have been rather well characterised (Hiller & Goodchild, 1981; Gounaris et al., 1986; Murphy, 1986), and descriptions of the comparable pigment-proteins from diatoms and other algae containing chlorophyll c are now becoming available (Brown, 1988; Hiller, Larkum & Wrench, 1988; and references therein). In contrast, studies of the function of chloroplast membranes from these algae are few, and the highest measured rates of O2 evolution are equivalent to an assimilation number of only 1.4 (Popovic, Colbow, Vidaver & Bruce, 1983; Samuelsson & Prezélin, 1985; Sandmann, 1985), well below the average of 6.4 obtained for the Pmax of intact phytoplankton cells (Table II). If Pmax is indeed limited by the rate of photosynthetic electron transport (specifically, by the rate of re-oxidation of reduced plastoquinone: Harris 1978; Richardson et al., 1983) then, in order to support the high rates of photosynthesis reported for phytoplankton, evidence must be found of higher rates of O2 evolution by algal photosynthetic membranes. LIGHT-HARVESTING POTENTIAL A second aspect of the conversion of light energy to chemical energy is to consider, from first principles, how it is possible that open-ocean phytoplankton, which contain only 4% of the world’s chlorophyll, are capable of carrying out 24% of the production (Table I) using the low irradiances available to them at the depths where most live. The average chlorophyll content of the open ocean is around 30 mg·m−2 (Whittaker & Likens, 1975). From calculations based on Falkowski (1981), it is estimated that the cells containing this 30 mg chlorophyll could process mg chlorophyll a (molecular weight 894) contains molecules, and therefore would be incorporated into 1016 oxygen-evolving units (OEU’s), each involving 2000 molecules chlorophyll (plus other machinery; the OEU is the ratio of the chlorophyll molecules to the evolved O2 molecules under saturating flashes of light; it is not necessarily a structural entity). The OEU’s process eight photons per turnover (Fig 1). With turnover time of s (mean of the three fastest marine microalgal values in Fig 1 of Falkowski, 1981), 1016 OEU’s would process photons·s−1, or (because 1 µE contains [i.e. 1 µmole] of photons). A value higher than would be obtained if faster turnover times and/ or smaller units were entered into the calculation, but Falkowski (1981) and Sukenik, Bennett & Falkowski (1987) present minimum values of about 2.4 ms and 3.5 ms, respectively, for turnover time, and a minimum of 2000 for OEU size. Higher values were obtained for cells grown under low light intensities, which suggests the calculated capacity of would be realistic for field populations of phytoplankton. This capacity of is equivalent to only 2% of the photosynthetically active radiation that arrives at the sea surface during full sunshine (i.e. : Harris 1978; Richardson et al., 1983), but of course it is equivalent to a higher percentage of the lower irradiances available at the depths where most phytoplankton occur (see Lorenzen, 1976; Kirk, 1983, pp. 237 and 242). Nevertheless, this light-harvesting capacity of is ample to provide the assimilatory power to perform the estimated in situ photosynthesis of 69 g carbon·m−1.yr−1 (from Table I, and ocean area of ). Because 69 g carbon is 5.75 moles, and because the photo-assimilation of each mole of carbon requires at least 8 moles photons (i.e. 8 E), then a total of 46 E·yr−1 must be captured, or Admittedly, this is a theoretical minimum, based on a quantum yield of 1/8 (1 CO2 fixed per 8 photons used). It is equivalent to 9% of the potential light capture of . This calculation is, in effect, the photochemical equivalent of the earlier calculation that

PHOTOSYNTHESIS: A BIOCHEMICAL PERSPECTIVE

13

gave the actual assimilation number for open ocean productivity (from Table I) as 7% of the mean potential value (from Table II). Both this calculation and the earlier one, however, represent theoretical extremes. They are based on a quantum yield of 1/8, whereas actual quantum yields seldom exceed 1/10 (Myers, 1980; Priscu, 1984; Dubinsky, Falkowski & Wyman, 1986; but see Osborne & Geider, 1987). This raises the light requirement to Furthermore, to compensate for dark respiratory losses, and to accommodate the extra photochemical energy needed to assimilate nitrogen (including the substantial energy investment of 16 photons to reduce ), produce lipids, and power energy-dependent membrane transporters, a value of (say) would be more realistic. Thus, the cells under the average m2 of ocean actually use about one-seventh of the light energy they are capable of using This relatively low value presumably reflects imperfect light capture because of such factors as uneven packaging of chlorophyll within cells, reaction centres being temporarily closed when photons arrive, optical properties of cell walls and, of course, the cells sometimes being at depths where available irradiance is low. If these estimates are correct, the capacity of algae to convert light energy into biomass restricts ocean photosynthesis to a ceiling of not more than seven times the assimilation number of 0.43 (the estimate calculated from Table I), and this ceiling would only be reached if all the phytoplankton were saturated with light every day for 12 h. The assimilation number would then increase to 3.0 which, perhaps not incidentally, is close to the maximum considered possible for land plants (Delaney & Walker, 1978). Thus, it must be concluded that if ocean photosynthesis is greater than estimated at present, it must be a consequence of one or more of the following: (1) the phytoplankton being more saturated with light than previously believed, (2) the ocean’s phytoplankton biomass being under-estimated, (3) photochemical reactions being unexpectedly efficient. EFFICIENCY OF CONVERSION OF RADIANT ENERGY TO BIOMASS The use of 46 Einsteins of light to fix 69 g carbon does not mean that all the energy of this light is converted to chemical energy. The maximum conversion theoretically possible is 34% with 680 nm light. This value is obtained from the following. The eneregy content of a Thus, the energy in 46 Einsteins of light The energy released by burning an amount of glucose containing 69 g carbon (i.e. 0.96 mole) is, however, only 34% of this, i.e. When light of shorter wavelengths—and therefore consisting of higher-energy photons—is harvested (as is common in the marine environment where accessory pigments such as fucoxanthin and peridinin collect blue-green light), the theoretical maximum efficiency of conversion is lower, because the extra energy content of the blue-green photons, as compared with 680 nm photons, is lost as radiation-less (thermal) deexcitation before any conversion of light energy to chemical energy begins (Walker, 1979). In addition, the conversion efficiency is further reduced if the more realistic quantum yield of 1 CO2 fixed per. 10 photons is adopted. Thus, with this quantum yield and 500 nm light, the maximum possible conversion reduces to 19%. In practice, actual energy conversion is well below the theoretical maximum because only a fraction of the available photons are captured and used (see Kirk, 1983, p. 237). For naturally occurring phytoplankton, one of the higher measurements of photosynthesis—4 g carbon fixed·m−2·day−1— was obtained off the Coast of Peru (Strickland, Eppley & Rojas de Mendiola, 1969). If these algae were from a depth where average irradiance was 300 then the efficiency of energy conversion would have been 6%. By contrast, a coastal pond culture of Phaeodactylum tricornutum converted 13% of the photosynthetically active radiant energy incident on the pond surface (Thomas et al., 1984). On a global scale, only about 0.

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Fig 2.—Tenfold elevation of the intracellular CO2 concentration in marine algae by movement of 2 mM from outside at pH 8.2 to inside at pH 7.2.

12% of the energy in photosynthetically active radiation reaching the ocean surface is, however, assimilated in phytoplankton production (based on a fixation rate of 69 g carbon fixed·m−2·yr−1, and an average irradiance each 12-h day of see Fig. 7, p. 38). Lieth (1973) gives an even lower estimate of 0.07%. These values appear small. The radiant energy available at the depths where phytoplankton grow is, however, much lower than at the surface, and if all cells are, on average, at the 5% light level (i.e. ) then the efficiency of conversion of the light energy actually available is a more respectable 2.4%. This value agrees closely with that recently obtained in the field by Kishino, Okami, Takahashi & Ichimura (1986). Using sophisticated instrumentation, these authors determined that the efficiency was 5% at the 1.5% light level.

INORGANIC CARBON UPTAKE Inorganic carbon exists in water as CO2, H2CO3, and (Raven, 1970; Kremer 1981a). Water in equilibrium with the atmosphere contains dissolved CO2 at a concentration of 10 µM. This concentration is little influenced by either pH or salinity. Thus, the oceans contain But CO2 dissolved in water is in equilibrium with H2CO3 and according to: From this equation, it is clear that the concentration of HCO3 will vary with pH. In salt water, H2CO3 is half-dissociated at pH 5.93 (Raven, 1970), at which pH the concentrations of CO2 and will both be 10 µM; as pH increases, the concentration of increases. Consequently, the ocean, which is at a slightly alkaline pH of 8.2, contains at close to 2mM, 200 times the concentration of CO2. The species of inorganic carbon used for photosynthesis is CO2, which is the substrate for RuBP carboxylase. A low level of CO2 in the environment can limit photosynthesis (the growth of wellilluminated C3 plants on land is CO2-limited). In water, the problem becomes greater, because the rate of diffusion of CO2 in water is less by a factor of 105 than that in air (Raven 1970). It may not be surprising, then, that current evidence points to as the species of inorganic carbon taken up by marine microalgae (Colman & Gehl, 1983; Aizawa, Tsuzuki & Miyachi, 1986; Burns & Beardall, 1987; Dixon & Merrett, 1988) and seaweeds (Beer & Eshel, 1983; Brechignac & Andre, 1984; Bidwell & McLachlan, 1985; Cook, Lanaras & Colman, 1986). Experiments with Dunaliella, Synechococcus, and Ulva indicate that the uptake is not via simple diffusion into the cell, but rather that a carrier (presumably a protein) actively pumps into the cell, using energy (probably ATP) generated during photosynthesis. In some instances, accumulation of inorganic carbon in the cell to concentrations several- to many-fold that of the surrounding medium was detected (Zenvirth & Kaplan, 1981 ; Badger & Andrews, 1982; Colman, 1984). The taken up by cells must be protonated to form H2CO3, and this must then be dehydrated to CO2 for use by RuBP carboxylase. The slow dehydration step is catalysed by carbonic anhydrase, a common enzyme in photosynthetic cells, including marine microalgae (Aizawa & Miyachi, 1984; Lucas & Berry, 1985; Burns & Beardall, 1987; Dixon & Merrett, 1988). If the pH of the cytoplasm of phytoplankton cells is close to that of leaf cells (about 7.2) then the instantaneous effect of moving 2 mM into the cytoplasm at this pH would be to increase the intracellular CO2 concentration to over 100 µM (Fig 2), or even higher if active uptake raises the intracellular concentration about 2 mM. This would have a profound influence on the likelihood of photorespiration occurring in marine phytoplankton (see p. 31).

PHOTOSYNTHESIS: A BIOCHEMICAL PERSPECTIVE

15

Fig 3.—The Calvin cycle of CO2 fixation. Lower: the cycle drawn to show net conversion of 3 CO2 to a 3-carbon sugar (triose-P) ; note that the three CO2-accepting molecules (3 RuBP) are fully regenerated by the cycle. Upper: details of the reactions in which CO2 is fixed, and the products (two glycerate-3-P) converted to two triose-P in reactions that use ATP and i.e. it is in these two reactions that most of the solar energy captured by the photochemistry (Fig 1) is incorporated into carbohydrate.

CARBOXYLATION RuBP CARBOXYLATION At least half a dozen enzymes can fix CO2 into organic compounds, but only one of these is connected to a series of reactions that permits the net fixation of CO2, and therefore plant growth (Walker, 1974; Kelly, Latzko & Gibbs, 1976). This enzyme is RuBP carboxylase and the sequence of reactions (popularly termed the Calvin cycle) permits plant growth because it is able to reduce CO2 to carbohydrate while at the same time regenerating the original amount of the CO2-acceptor (RuBP). For example, in Figure 3 the net reduction of three CO2 to one triose-sugar occurs concomitantly with the re-synthesis of the three RuBP (ready, as it were, to fix three more CO2). The activity of RuBP carboxylase in phytoplankton must therefore be comparable with observed photosynthesis rates, i.e. able to accommodate the measured assimilation number (average 6.4 in Table II). Relative to land plants, few measurements of the carboxylase activity have been made; the average of those available is equivalent to an assimilation number of only 2.0 (Table V). In contrast, numbers averaging 7.6 are reported for the activities in the leaves of land plants (Lilley & Walker, 1975; Delaney & Walker, 1978; Yokota & Canvin, 1985; Kobza & Edwards, 1987; Ramachandra Reddy & Das, 1987; Terashima & Evans, 1988) which are not claimed to have such high photosynthetic rates as are claimed for phytoplankton. Higher activities will need to be demonstrated, especially given the conclusion of Sukenik, Bennett & Falkowski (1987) that Pmax can be limited by carbon fixation rather than electron transport. Several reports present values for RuBP carboxylase activity in units that cannot be converted to assimilation numbers. Glover & Morris (1979) compared the activity per cell with maximum photosynthesis rates in seven cultured microalgae; the average activity was only 34% of the average photosynthetic rate (Table V). Other workers have presented activity in unwieldy units that the reader must translate, e.g. Smith & Platt (1985) use “untransformed units, d.p.m. L−1 hr−1”. Taken together, these results imply that either photosynthesis rates have been over-estimated, or RuBP carboxylase activities have been under-estimated. Given that the extraction of active enzymes from algae, particularly diatoms and dinoflagellates, is not easy (Everest, Hipkin & Syrett, 1986), and neither is the assay of RuBP carboxylase, the second possibility seems to be the more likely, although some contribution from the other is not ruled out. PEP CARBOXYLATION Most scientists do not, however, favour either of the above explanations. Rather, they suggest that the deficit in measured RuBP carboxylase activity may be made up by the carboxylation of phosphoenolpyruvate (PEP). Two enzymes can carboxylate PEP: PEP carboxylase e.g., in dinoflagellates, and PEP carboxykinase e.g., in diatoms and macrophytic brown algae (Akagawa, Ikawa & Nisizawa, 1972; Holdsworth & Bruck, 1977; Kerby & Evans, 1983; Descolas-Gros & Fontugne, 1985). The finding that marine algae have a significant potential for PEP carboxylation (Karekar & Joshi, 1973; Beardall, Mukerji,

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Glover & Morris, 1976) originally resulted in suggestions that their photosynthetic mechanisms were akin to those of terrestrial C4 plants and CAM plants (see below), which use PEP carboxylation extensively for cap TABLE V Laboratory and field measurements of RuBP carboxylase activity and photosynthesis Assimilation number Alga or locality

Carboxylase mg carbon.mg

Photo-synthesis chl a Carboxylase/ −1·h−1 Photosynthesis %

Reference

Phaeodactylum tricornutum Boothbay Harbor Phaeodactylum tricornutum Gulf of Maine

3.0

5.9

51

4.0 1.1

? 0.56

– >100

0.4a

8a

5

Seven cultures

–

–

34a

Amphidinium carterae

1.1

?

–

Pyrocystis noctiluca Bedford Basin Dunaliella tertiolecta Skeletonema costatum Synechococcus spp. in eddy surface waters

1.2 ? 5.4 0.5

4.0 1.6 6.8 3.3

30 14 79 15

1.0a

1.0a

100

Beardall & Morris, 1976 Beardall et al., 1976 Holdsworth & Colbeck, 1976 Glover & Morris, 1979 Glover & Morris, 1979 Appleby, Colbeck, Holdsworth & Wadman, 1980 Rivkin et al., 1982 Smith et al., 1983 Sukenik et al., 1987 Mortain-Bertrand et al., 1987 Prézelin et al., 1987

a

Approximate mean values.

turing CO2, but this idea lost popularity when it was found that algal photosynthetic rates correlated with RuBP carboxylase activity, but not with PEP carboxylase activity (Mukerji, Glover & Morris, 1978; Glover & Morris, 1979; Smith, Platt & Harrison, 1983). Terrestrial C4 plants and CAM plants utilize PEP carboxylase to capture CO2 in, respectively, the C4 pathway and crassulacean acid metabolism (CAM); hence the terms “C4 plants” and “CAM plants”. These mechanisms are not alternatives to the Calvin cycle because, by themselves, they would not permit plant growth. In any event, it is quite unlikely that any unicellular marine alga could be classed as a C4 plant or CAM plant, because the former requires co-operation between two types of cells in the one plant, while the latter is an adaptation for survival in arid environments. Consequently, marine algae are almost certainly “C3 plants”. The origin of the terms “C3” and “C4” have, however, caused some confusion. C3 refers to the fact that a three-carbon compound (glycerate-3-P, see Fig 3) is the first product of CO2 fixation, whereas C4 denotes that a four-carbon compound (oxaloacetate, see Fig 4) is initially observed. As phytoplankton synthesise more protein and less carbohydrates than do multicellular plants, much triose-P produced by the Calvin cycle must move via PEP to produce the carbon skeletons needed for amino-acid biosynthesis. A good portion of this PEP is carboxylated with CO2 to produce oxaloacetate (which is, itself, a carbon

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Fig 4.—The Calvin cycle as the producer of PEP for PEP carboxylation. The maximum long-term ratio of PEP carboxylation: RuBP carboxylation during growth is 1:3. If two thirds of the triose-P (representing the product of CO2 fixation) is used for the synthesis of carbohydrates, lipids, and amino acids not derived from oxaloacetate (as is usual), then the ratio reduces to 1:9. Consequently, attempts to credit PEP carboxylation with more than 10% of the total photosynthetic carbon fixation are fundamentally in error.

skeleton, and is a precursor of several others). Although this gives a “C4 flavour” to algal CO2 fixation, the principal purpose of oxaloacetate synthesis in marine algae (amino-acid synthesis) is entirely unrelated to that of C4 and CAM plants (a CO2-capturing mechanism). Nevertheless, the concept that PEP carboxylation may make up for a perceived insufficiency of RuBP carboxylation has persisted, and has supported a considerable amount of theoretical discussion (Morris, 1980; Priscu & Goldman, 1983; Smith et al., 1983). But, as emphasised by Figure 4, the concept itself is wrong. In growing photosynthetic cells, three CO2 must be fixed by RuBP carboxylase in order to generate a PEP before a CO2 can be fixed by PEP carboxylase or PEP carboxykinase. This is because there is no known biochemical pathway whereby the product of PEP carboxylation (oxaloacetate) can be metabolised so that three of its four carbons are used to regenerate CO2-acceptor (PEP) while the other is kept in the reduced form and used for plant growth (Kelly et al., 1976). Consequently, since one net PEP carboxylation requires three prior RuBP carboxylations, the maximum contribution of PEP carboxylation to total photosynthetic CO2 fixation is 25%. This theoretical limit reduces to about 10% in practice because not all amino-acid skeletons can be derived from oxaloacetate, and because protein is only one of the major products of cell growth; others, for example sugars, polysaccharides and lipids, cannot be synthesised from oxaloacetate without the loss of the CO2 that was fixed during PEP carboxylation. The practical value of about 10% for the contribution of PEP carboxylation is in good agreement with experimental values of 6.7 to 12.7% recently obtained for two diatoms (Mortain-Bertrand, Descolas-Gros & Jupin, 1988). Finally, there may be stages in the growth cycle of an alga where PEP carboxylation equals or exceeds RuBP carboxylation. Microalgal cultures in the stationary phase appear to have this attribute (Mukerji et al., 1978; Glover & Morris, 1979), but these cells are obtaining PEP from a carbohydrate reserve (e.g. mannitol in brown algae) previously synthesised entirely through RuBP carboxylation (Kremer, 1981b). PHOTORESPIRATION AND MARINE ALGAE DOES PHOTORESPIRATION OCCUR IN MARINE ALGAE? Photosynthetic cells of terrestrial C3 plants exhibit a major light-dependent set of reactions that results in O2 uptake, CO2 release, and the consumption of energy (Fig 5). Due to its light dependence and gas exchanges, it has been called photorespiration, but it is otherwise not at all like dark (mitochondrial) respiration. Photorespiration occurs when O2 replaces CO2 in the RuBP carboxylase (now called “carboxylase/ oxygenase”) reaction, leading to the formation of one glycerate-3-P, plus a smaller (2-carbon) compound called glycolate (not two glycerate-3-P, as in Fig 3). A series of six reactions then converts two glycolate molecules to one glycerate-3-P and one CO2 (Fig 5). If all molecules of RuBP were oxygenated, plant growth would cease. The combination of the enzyme’s relative affinities for CO2 and O2 and the relative concentrations of these two gases in air results, however, in a situation somewhat akin to Figure 5b where carboxylation exceeds oxygenation sufficiently for plant growth to proceed. The real function of photorespiration is not yet known. It may act as a “release valve” to dissipate photosynthetically generated energy, as in Figure 5a, when the CO2 supply is restricted (e.g. during periods

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Fig 5.—Photorespiration. a, at the CO2 compensation point, where the amount of CO2 lost by photorespiration is exactly balanced by the amount fixed in the Calvin cycle; at this state, where the ratio of RuBP carboxylated: oxygenated is 1:2, 9 ATP and 6 NAD(P)H are ‘wasted’, while plant growth is zero. b, the situation with terrestrial C3 plants in normal air, where carboxylation to oxygenation is approximately 1:0.7, and growth (represented by the synthesis of a molecule of sucrose from 12 CO2) in the presence of photorespiration is possible.

of water stress when stomates close), thereby preventing “over-reduction” of the photochemical apparatus and subsequent photoinhibition (see below; Osmond, 1981; but see Powles, Comic & Louason, 1984). Nevertheless, it still proceeds in C3 plants when stomates are open, reducing productivity by 30 to 50%. In C4 plants photorespiration is, however, inhibited because the C4 pathway concentrates CO2 in the vicinity of RuBP carboxylase, and because O2 and CO2 are straightforward competitors for entry onto the enzyme’s active site, the increased CO2 concentration overwhelms the oxygenase reaction so that it is effectively eliminated. A different mechanism appears to produce a similar consequence in marine algae. As mentioned on p. 26, the effects of pH and/or the active uptake of inorganic carbon lead to intracellular CO2 concentrations of 100 µM or more in marine algae. Consequently, the CO2:O2 ratio would be expected to increase such that, in these cells too, the oxygenase reaction would be overwhelmed. This does not mean that marine algae may not be capable of photo-respiration. They can synthesise glycolate (Lloyd, Canvin & Culver, 1977; Glover & Morris, 1981; Fogg, 1983) and they possess enzymes for converting it to glycerate (Paul & Volcani, 1976). But despite claims that photorespiration may occur (Burris, 1977; Morris & Glover, 1981), the likelihood of it occurring under natural conditions is small (Lloyd et al., 1977; Birmingham, Coleman & Colman, 1982; Burns & Beardall, 1987), and most recent work showing that O2 has little or no effect on the rates of photosynthesis by marine algae argues against it (Colman & Gehl, 1983; Colman, 1984; Bidwell & McLachlan, 1985; Beer & Israel, 1986; Cook & Colman 1987; Johnston & Raven, 1987). A study of coccoid marine cyanobacteria by Glover & Morris (1981) indicated that these marine photosynthetic cells have a greater potential for photorespiration than have eukaryotic marine algae, but even in this case the magnitude of photorespiration in the presence of 2 mM was near zero. One further consideration, however, counteracts these arguments. Build-up of CO2 in cells (due to effects of pH and/or active uptake) will lead to increased photosynthesis and, consequently, increased O2 evolution. This may produce locally high concentrations of O2 in chloroplasts, favouring RuBP oxygenase activity and photorespiration. Evidence for such an event in three freshwater green microalgae provided with inorganic carbon (CO2 plus HCO3) at concentrations up to 0.6 mM has recently been obtained (Yokota & Kitaoka, 1987). Similar experiments with marine microalgae growing in 2 mM inorganic carbon are required. PHOTORESPIRATION AND PRODUCTIVITY ESTIMATES If photorespiration in phytoplankton does occur, it would not affect productivity estimations based on measurements of O2 evolution (using the Winkler technique), as suspected by some workers (see Round, 1981, p. 259). Reference to Figure 5a shows that a zero change in CO2 (one CO2 taken up, and one released) correlates with a zero change in O2: three O2 are used— two during oxidation of two RuBP, and net of one during oxidation of two glycolate—while three O2 are liberated from water (see Fig. 1, p. 22) during the photosynthetic electron transport that generates the required reducing power (five NADPH and one NADH; Fig 5a). The amount of O2 used during glycolate oxidation is the same whether the glycolate is oxidised by an oxidase, as shown in Figure 5, or by a mitochondrial dehydrogenase linked to respiratory O2

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consumption; both types of oxidation have been detected in algae. With photorespiration proportionately less than photosynthesis, as when net CO2 fixation proceeds (Fig 5b), the net O2 evolution will equal the net CO2 assimilation, as if this CO2 fixation were occurring in the absence of photorespiration. Photorespiration would affect productivity estimations using the 14C-technique, but not dramatically. It is most probable that “total CO2 fixation minus photorespiratory CO2 evolution” would be estimated, because photorespiratory and photosynthetic metabolisms share common intermediates and are obligatorily interconnected (Fig 5), so that 14C entering the Calvin cycle will also enter photorespiration after a short delay. In a sense, the result would be the same as for O2 measurements, i.e. (net) CO2 fixation would be measured as if it were occurring in the absence of photorespiration.

PHOTOINHIBITION If photorespiration is absent, the question arises as to how phytoplankton dispose of excess photosynthetically generated energy. Failure to dispose of this excess energy is thought to be one cause of photoinhibition, whereby over-reduction of electron transport components causes a chain of events that results in damage to one or more of the components that make up the reaction centre of photosystem II (Allakhverdiev, Šetlíková, Klimov & Šetlik, 1987; Demeter, Neale & Melis, 1987; Lidholm, Gustafsson & Öquist, 1987; Kirilovsky, Vernotte, Astier & Étienne, 1988; Ohad, Koike, Shochat & Inoue, 1988). The damage can become so extensive as to be irreversible, and cell death follows. Other possible mechanisms for protecting against photoinhibition implicate (1) carotenoids; (2) use of energy to reduce O2 to the superoxide anion (Mehler reaction) which, however, must subsequently be dealt with; (3) spillover of energy from photosystem II to photosystem I; (4) cyclic electron flow around photosystem II; and (5) reduction in the level of light-harvesting pigments (Samuelsson et al., 1985; Ben-Amotz, Gressel & Avron, 1987; Demmig, Winter, Krüger & Czygan, 1987; Geider, 1987; Siefermann-Harms, 1987; Falkowski, Kolber & Fujita, 1988). In addition, as Harris (1978) has argued, phytoplankton populations may normally largely avoid photoinhibition, either passively (vertical mixing; sinking), or actively (motile microalgae may swim away form excessive light). Support for this viewpoint has come from Vincent, Neale & Richerson (1984), who describe an extensive (but still reversible) photoinhibition of phytoplankton each morning in the high-altitude Lake Titicaca (Peru-Bolivia) when the phytoplankton became temporarily trapped under extreme irradiance by a near-surface thermocline. On the other hand, algae that are continually exposed to high light intensities are apparently more resistant to photoinhibition. Seaweeds, Synechococcus, Thalassiosira pseudonana, and a population of benthic microalgae dominated by Euglena obtusa and Pleurosigma angulatum all showed no evidence of photoinhibition under almost full sea-surface irradiance (Penniman & Mathieson, 1985; Mills & Wilkinson, 1986; Prézelin, Putt & Glover, 1986; Kana & Glibert, 1987a; Sakshaug, Demers & Yentsch, 1987). Thus, there appears to be a broad spectrum of tolerance to photoinhibition in algae. At least some of this variation may be an adaptation, in that photosynthetic cells exposed daily to high irradiance contain less lightharvesting pigments per electron-transport chain (compared with “shade-adapted” cells), and are thus less likely to overload their light processing machinery (Osmond, 1981). To a certain degree, microalgae may avoid photoinhibition by using excess photochemical energy rather than allowing it to accumulate to the point where all reaction centres become occupied. Such utilisation could involve the assimilation of more CO2 than is required for growth. This may help to explain why stressed (e.g. nitrogen-deficient) cells of some species excrete large quantities of carbohydrates, as polysaccharides or glycolate, during photosynthesis (Jensen, 1984; Vieira & Myklestad, 1986; Al-Hasan &

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Fogg, 1987); these excretions would be the end-product of energy harvested in a process not linked to cell growth (see also Dring, 1982, p. 75). Exudation of low molecular weight organic compounds by nonstressed cells is, however, believed by Bjørnsen (1988) to be a consequence of passive diffusion through the cell membrane. Not only high intensities of photosynthetically active radiation, but also the much lower intensities of UVB light (290–320 nm) normally encountered in the solar spectrum cause photoinhibition of algae that occur high in water columns (Harris, 1978; Richardson, Beardall & Raven, 1983, Döhler, 1984; Jokiel & York, 1984; Vincent et al., 1984, Wood, 1987). The mechanism of this inhibition is not known. Likely explanations are that the nucleic acids or components of the photosynthetic electron transport chain are damaged. The photosynthetic cells of leaves of terrestrial plants are protected from UV-B-induced photoinhibition by flavonoid compounds in the non-photosynthetic cells of the leaf epidermis (Caldwell, 1981). DARK (MITOCHONDRIAL) RESPIRATION DOES MITOCHONDRIAL RESPIRATION PROCEED DURING PHOTOSYNTHESIS? In productivity experiments using the question arises whether gross photosynthesis (total CO2 fixation itself) or net photosynthesis (total CO2 fixation minus CO2 loss through respiration and/or photorespiration) is measured. Investigations of this question have reached differing conclusions: that it measures gross photosynthesis (Dring & Jewson, 1982), net photosynthesis (Smith & Platt, 1984), or some value in between, depending on the growth rate (Harris & Piccinin, 1983). This problem will not be solved until the extents of mitochondrial respiration (in the dark and in the light) and photorespiration are known. There are, however, some indications. First, as outlined above, photorespiration may not occur in phytoplankton living in natural conditions, and even if it did, it would not be likely to affect productivity measurements. Thus, only dark (mitochondrial) respiration need be considered. It is not known whether the dark (mitochondrial) respiration of photosynthetic cells is suppressed during photosynthesis, and if so, to what extent (Kelly, 1983). In one sense, respiration may be unnecessary in that its prime purpose (complete oxidation of carbohydrate to CO2 in order to generate energy) would seem inappropriate in a cell harvesting light energy and reducing CO2 to carbohydrate. A portion of the Krebs cycle (tricarboxylic acid cycle) would, however, need to proceed at a low rate in order to generate α-ketoglutarate, which is the precursor of four amino acids and of chlorophyll. This could proceed as shown in Figure 6, in which a net evolution of one CO2 per α-ketoglutarate synthesised would occur. But note that, for the growth of cells containing 50% protein and 1% chlorophyll (dry weight basis), the required rate of this partial Krebs cycle would lead to a CO2 evolution equivalent to only 2% of the photosynthetic CO2 fixation. Despite this requirement for some Krebs cycle activity for growth, a concomitant respiratory O2 uptake would not necessarily occur because the reducing power (NADH) generated during α-ketoglutarate synthesis could be delivered to the cytosol and used there, as shown in Figure 6. The respiratory electron transport chain would then remain inactive. Some recent experiments with the freshwater green microalgae Chlamydomonas reinhardtii and Selenastrum minutum suggest, however, that both the Krebs cycle and respiratory electron transport function during photosynthesis in these algae (Peltier & Thibault, 1985; Husic & Tolbert, 1987; Weger, Birch, Elrifi & Turpin, 1988), but the extent to which this is so in other algae is not known, and once again

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Fig 6.—Limited operation of the mitochondrial Krebs cycle during photosynthesis in order to generate α-ketoglutarate. Two molecules of glycerate-3-P (from photosynthetic CO2 fixation) are converted to one α-ketoglutarate and one CO2, and two NAD+ are reduced in the process. A metabolic shuttle may dispose of this reducing power in the cytosol (as shown) or respiratory electron transport may oxidise it, with consumption of one O2.

the question of function arises. With respect to the function, Husic & Tolbert (1987) have alluded to the alternative (cyanide-resistant) pathway of respiratory electron transport in plant mitochondria. This pathway is not coupled to ATP synthesis and is therefore a means of “burning off” excess carbohydrate (Lambers, 1982), an intriguing possibility in view of the suggestion that marine algae may not be able to dispose of excess energy by photorespiring as land plants do. The alternative pathway, however, generates superoxide anions and peroxide, thus mechanisms for the removal of these toxic substances would need to accompany any alternative pathway activity. All of these considerations have been subsequently disturbed by an experiment, elegant in its simplicity, in which an inhibitor of mitochondrial electron transport was shown to inhibit photosynthesis by barley leaf protoplasts, but to have no effect on the photosynthesis of chloroplasts isolated from these protoplasts (Krömer, Stitt & Heldt, 1988). It was concluded that mitochondrial electron transport is required during photosynthesis to generate ATP for cytosolic metabolism, and that the ultimate electron donor is photosynthetically-generated NADPH shuttled from chloroplasts to mitochondria, more or less by a reversal (and extension) of the malateoxaloacetate shuttle shown in Figure 6. The advantage of this system is that it is energetically efficient (3.8 ATP per 4 photons, compared with 1.3 ATP per 4 photons when the ATP is generated during either cyclic or pseudocyclic electron transport in chloroplasts). It could therefore be useful to phytoplankton cells growing in a low light environment, especially because these cells require cytosolic ATP for protein synthesis (an ATP supply rate of about 7% of the rate of CO2 fixation is needed only for the activation and attachment of amino acids to growing polypeptide chains), and to pump inorganic carbon into cells (possibly an even more ATP-demanding process; Yokota et al., 1987). Note that this system of producing ATP by oxidising NADPH of chloroplast origin involves mitochondrial electron transport only; Krebs cycle activity would not be required. Thus, respiratory oxygen uptake would occur, but respiratory CO2 evolution would not (other than that noted above for αketoglutarate synthesis). Phytoplankton appear to possess ample respiratory electron transport activity (Kenner & Ahmed, 1975). MITOCHONDRIAL RESPIRATION AND PRODUCTIVITY ESTIMATES Of course, the extent of mitochondrial respiration during the night also needs to be evaluated for productivity estimates. Most data indicate that the rate at which marine algae respire in darkness is equivalent to about 10% of the light-saturated rate of photosynthesis (Harris, 1978; Falkowski & Owens, 1980; Taguchi, 1981; Richardson et al., 1983; Geider, Osborne & Raven, 1985; Smith & Geider, 1985; Gerard, 1986). Assuming respiration occurs both day and night, then at maximum growth rates the net photosynthesis would equal 80% of gross photosynthesis. This percentage may, however, be higher if respiration is partially or completely inhibited during photosynthesis, as discussed above, and there is other evidence for this (Burris, 1980; Scherer, Stürzl & Böger, 1984). There is also evidence that any respiratory CO2 released in illuminated cells is quickly re-assimilated by photosynthesis before it can leave the cell (Scherer et al., 1984). This possibility, which was denied by Bidwell (1977), was proposed in the 1950s for Dunaliella euchlora (Ryther, 1956).

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Fig 7.—Efficiencies in marine production. Mean daytime surface irradiance of is equal to (Richardson et al., 1983), and thus to and to Average photosynthesis (Table I) of 69 g carbon·m−2·yr−1 is converted to its energy equivalent as described on p. 25. Average fish production is calculated by dividing total production at the end of food chains ( tonnes fresh weight [Ryther, 1969], equivalent to approximately tonnes carbon) by total ocean area and then converting to energy units as for the phytoplankton.

Although respiration appears to use only a small fraction of carbohydrate produced under light saturation, it may become more significant to the balance of carbon metabolism under light limitation. Phytoplankton commonly occur at depths where irradiance is low, so respiration could be important in assessing net photosynthesis in the ocean, but to what extent is not clear; it has been estimated that 40% of gross photosynthesis in the ocean is lost in dark respiration (see Burris, 1980) but some species, when growing slowly in dim light, appear able to adjust their respiration to a correspondingly low level (Falkowski & Owens, 1980; Geider, 1987; Langdon, 1987). Of course, if irradiance falls so low that photosynthesis and respiration are equal, then growth will cease. At this point, the irradiance is termed the “light compensation point”. Experiments indicate this point to range between low values near for diatoms grown in low light, to high values of over for dinoflagellates (Falkowski & Owens, 1980; Rivkin, Seliger, Swift & Biggley, 1982; Hobson & Guest, 1983; Geider & Osborne, 1986; Langdon, 1987). CONCLUDING COMMENT The efficiency with which solar energy is used to convert inorganic precursors into fish in the ocean is 0. 00014%, based on an average sea-surface irradiance of and an annual total fish production of tonnes carbon (Fig. 7). Taking the value for marine algal photosynthesis to be 69 g carbon fixed·m−2·yr−1 (Table I, see p. 13), then the global efficiencies for the two sections of this conversion (i.e. sunlight to algae, and algae to fish) are a little above 0.1 % each. For sunlight to algae, the value varies regionally from 0.02 to 5%; the overall mean is relatively low (0.12%) because much light energy is atenuated by the overlying water column before it reaches the algae (Kirk, 1983, p. 244), resulting in marine photosynthesis being, on average, markedly light-limited. For algae to fish, the low value is a consequence of having about three links, on average, in the marine food chain from the microscopic phytoplankton to the fish at the end of the food chains (Ryther, 1969). In time, the value for marine algal production, and thus the efficiency values shown in Figure 7, may be revised. From the biochemical viewpoint, revisions may be needed when the extents of respiration and photorespiration in marine phytoplankton are better understood. As emphasised in this review, upward revisions cannot, however, be confidently based on high estimates of Pmax values for marine photosynthetic cells unless it is demonstrated that these cells have a photosynthetic machinery with a significantly greater capacity for CO2 fixation than that of the photosynthetic cells of terrestrial plants. ACKNOWLEDGEMENTS I am grateful for many helpful discussions with S.W.Jeffrey who introduced me to the marvellous minute plants of the ocean, and with J.S.Parslow, who taught me much about their growth. REFERENCES Aizawa, K. & Miyachi, S., 1984. FEBS Lett., 173, 41–44.

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Oceanogr. Mar. Biol. Annu. Rev., 1989, 27, 45–90 Margaret Barnes, Ed. Aberdeen University Press

THE ECOLOGY AND BEHAVIOUR OF ASCIDIAN LARVAE* IB SVANE Kristineberg Marine Biological Station, Kristineberg 2130, S-450 34 Fiskebäckstil, Sweden and CRAIG M.YOUNG† Harbor Branch Oceanographie Institution, 5600 Old Dixie Highway, Fort Pierce, Florida 34946, USA

ABSTRACT Recent studies of ascidian larval biology reveal a diversity of structure and behaviour not previously recognised. The introduction of direct methods for observing ascidian larvae in situ has provided important insights on larval behaviour, mortality, and dispersal not possible with the microscopic larvae of most other marine invertebrates. In the context of these recent advances, this review considers the ecology of pelagic phases (egg, embryo, and larva) of the ascidian life cycle and relates aspects of reproduction and larval biology to the recruitment, abundance, and distribution of adult populations. INTRODUCTION The ascidian larva functions in site selection and dispersal while transporting adult rudiments between generations (Berrill, 1955, 1957; Millar, 1971; Cloney, 1987). Interest in the ecology of ascidian larvae has recently been stimulated by the introduction of new ideas and methods, most notably in situ observations of living larvae. The resulting advances justify a reconsideration of ascidian larval ecology. From an ecological standpoint, all pelagic phases of the ascidian life cycle (which may include eggs, embryos and larvae) influence adult populations. Thus, our definition of larval ecology is broad; it includes reproductive processes leading to the production of gametes and larvae, as well as post-settlement consequences of larval processes. The literature on larval evolution as it relates to ascidian and chordate phylogeny is large, and will not be considered in the present paper. Reviews on ascidian biology dealing with aspects of reproduction and larval ecology have been published by Millar (1971), Berrill (1975), and Cloney (1987), and detailed reviews on

* Harbor Branch Contribution No. 678.

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Fig 1.—Generalised life cycles of typical solitary ascidians (top) and compound ascidians (bottom). The diagrams incorporate characteristics of many species; not all aspects (e.g., fusion of colonies) are present in the life cycle of any given species.

ascidian metamorphosis have been presented by Cloney (1978, 1982). More recently, ascidian metamorphosis and larval locomotion have been treated as parts of general comprehensive reviews by Burke (1983) and Chia, Buckland-Nicks & Young (1984), respectively. It is our goal to bring together a scattered literature that comprises ascidian larval ecology. While emphasising ecological aspects of larval release, development, dispersal, behaviour, and settlement in relation to the distribution and abundance of adult populations, we also deal with reproductive, morphological, and developmental problems that help to elucidate aspects of ascidian larval ecology. REPRODUCTION, FERTILISATION, AND LARVAL ANATOMY MODES OF REPRODUCTION Ascidians may be either oviparous (typical of most solitary ascidians), ovoviviparous (typical of most compound ascidians), or viviparous (Fig 1). True viviparity in which nutrients are exchanged between parent and embryo across a placenta-like structure is known to occur in only a single species, Hypsistozoa fasmeriana (Brewin, 1959). Much of the earlier literature, including the classic works of Berrill (e.g. 1929, 1935, 1950) uses the term in reference to ovoviviparous species that retain embryos to hatching, but without apparent transfer of nutrients after ovulation. In oviparous forms, fertilisation and all subsequent developmental stages occur externally. Ascidian embryology and developmental biology have been the subjects of numerous important studies which have been reviewed by Cloney (1987) and Berrill (1975), and provided the theme for a recent symposium (Lambert, 1982). Conklin’s (1905) study is still the classic description of cell lineage, although there has been a late resurgence of cell lineage studies using modern fluorescent markers and immunological techniques (Whittaker, 1987). The most common developmental pattern (urodele development) terminates in the production of a swimming tadpole larva. Many molgulids and a few species in the family Styelidae (Pelonia corrugata and Polycarpa tinctor) undergo direct development, bypassing the larval stage completely (reviewed by Berrill, 1931; Millar, 1971; Young et al., 1988). In ascidians, such direct development is known as anural development (Fig 2). As in many other kinds of animals, larval size is correlated with the degree of parental protection. Compound ascidians releasing fully developed larvae generally produce relatively few large larvae, whereas the eggs released by oviparous solitary ascidians are relatively more numerous and result in the production of much smaller, less differentiated tadpoles. In many invertebrates, there appears to be a correlation between adult size and brooding (Menge, 1975; Strathmann & Strathmann, 1982); within a given taxon, species with smaller individuals are more likely to brood than those with larger individuals. If we define an individual as a single zooid, not a colony, the relationship also seems to hold in ascidians. Accordingly, most colonial ascidians, having small zooids, brood relatively large complex larvae which develop from large eggs. Among solitary ascidians, brooders tend to have small bodies and produce relatively large eggs and larvae, whereas oviparous species often

† Order of authorship is alphabetical.

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Fig 2.—Generalised life cycle of a molgulid ascidian with anural development.

have relatively larger bodies and produce small eggs. Exceptions to this pattern are found in Boltenia echinata (Berrill, 1948b; Svane, 1983) and Corella inflata (Child, 1927; Lambert, Lambert & Abbott, 1981), both of which brood small eggs and produce small, simple tadpole larvae. At least one solitary ascidian, Polycarpa pomaria, is observed in the laboratory to brood facultatively. It is normally oviparous and produces small eggs, but developing larvae have not been found in the atrial cavity in nature (Berrill, 1950). One explanation for the relationship between brooding and zooid size is that cost of reproduction prohibits formation of numerous eggs in small individuals (Vance, 1973; Chia, 1974; Menge, 1976; Heath, 1977; Strathmann & Strathmann, 1982). This explanation, however, begs the question of why very large colonies of genetically identical zooids invest their resources for sexual reproduction in few large tadpoles rather than numerous small ones. Strathmann, Strathmann & Emson (1984) have suggested that selffertilisation can lead to brooding in echinoderms. The same argument could be applied to ascidians if we knew the extent of self-fertilisation. Botryllus schlosseri, which brood their embryos in the atrial cavity, can be either semelparous or iteroparous (R.Grosberg, pers. comm.). When fully developed larvae are removed surgically from the atrial cavities of a semelparous colony, the brood is replaced, suggesting that reproduction is not constrained by energy limitations. When a brood is replaced with inert glass beads, the colony dies shortly thereafter, just as in a typical semelparous colony (R.Grosberg, pers. comm.). Based on these findings, Grosberg has suggested that large broods prevent water flow through the atrium, killing the parent colony before a second brood is formed. FERTILISATION Sperm morphology and the physiology of ascidian fertilisation have been reviewed by Lambert (1982). Ascidian sperm are among the simplest in the animal kingdom in that they lack a midpiece. The excentric mitochondrion lies next to the nucleus in the head region. Paradoxically, this simple sperm must penetrate a complex egg covering consisting of two layers of somatic cells and a chorion. Ascidian eggs have an effective block to polyspermy that appears to involve the egg membrane rather than the accessory cells or chorion (Lambert & Lambert, 1981). Moreover, many species demonstrate complete or partial blocks to self-fertilisation (Morgan, 1938, 1942). Stolidobranchs tend to have a better block to self-fertility than phlebobranchs (Cloney, 1987). Many ascidiids and corellids are completely self-fertile. Several species in the genus Ascidia demonstrate a partial block to self-fertility when first spawned, but this block wears off during the first few hours, ultimately permitting virtually 100% self-fertilisation (Scofield, Schlumpberger, West & Weismann, 1982 for A. ceratodes; Young, unpubl. data on A. callosa and A. mentula). Although self-sterility clearly implies the absence of self-fertilisation in nature, the converse (absence of a block to self-fertility) does not necessarily indicate that eggs are routinely self-fertilised in the field. Indeed, the extent to which self-fertilisation occurs in nature is unknown. Ovulation and subsequent development have been observed in isolated zooids of Ecteinascidia turbinata, suggesting that selffertilisation is likely (Lambert, 1982). Some species release sperm seconds to minutes before eggs are spawned, providing an opportunity for sperm to disperse and mix with that of other individuals before fertilisation (Lambert & Brandt, 1967; Lambert et al., 1981). Recent electrophoretic evidence for two selffertile species, Chelyosoma production and Corella inflata, indicates that populations are not inbred; both populations have high levels of heterozygosity (S.Cohen, pers. comm.). Although the observed

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Fig 3.—Representative tadpole larvae of compound ascidians, including Diplosoma macdonaldi (A), Botryllus planus (B), Distaplia occidentalis (C), Ecteinascidia turbinata (D) and Eudistoma capsulatum (E). Only the styelid Botryllus has a modified sensory vesicle. BB: branchial basket. BS: oral siphon. AS: atrial siphon. Other labels as in Fig 4. Fig 4.—Representative tadpole larvae of solitary ascidians. Boltenia villosa (A), Corella inflata (B), and Halocynthia igaboja (C) have both a statocyte and an ocellus in the sensory vesicle. Styela coriacea (D) has a compound sensory organ known as a photolith, whereas Molgula occidentalis (E) has a statocyte but no ocellus. N: notochord; O: ocellus; S: statocyte; P: adhesive papilla ; PL: photolith; A: ampulla; TF: tail fin; F: tail tunic.

heterozygosity is not given, all alleles of Ciona intestinalis reported by Schmidtke, Weiler, Kunz & Engel (1977) were highly polymorphic. Lyerla & Lyerla (1978), however, found that the colonial species Clavelina picta and C. oblonga were monomorphic and may experience high levels of inbreeding. Whether this is due to obligate self-fertilisation or almost total asexual reproduction, or a combination of both, is unknown (Lyerla & Lyerla, 1978). Ascidian sperm demonstrate chemotactic attraction to eggs under laboratory conditions (Miller, 1975, 1982). Although there is some species specificity in these interactions, many stolidobranchs demonstrate low levels of specificity (Miller, 1982). LARVAL ANATOMY Ever since the chordate nature of the ascidian tadpole larva was recognised by Kowalevsky (1866), phylogenetic questions have motivated detailed studies of tadpole structure and development. Because details of tadpole morphology have been reviewed in depth several times during the past two decades (Millar, 1971; Berrill, 1975; Katz, 1983; Cloney, 1978, 1987), we shall deal only with structures that control larval habitat selection and swimming behaviour that are directly relevant to the ecology of larvae themselves. Those portions of the larva that have been called the “adult action system” (developing and fully developed adult structures present in the larva) will not be reviewed here. The overall sizes of ascidian larvae range up to 4.5 mm, with the largest being found among colonial ascidians in the order Aplousobranchiata: Hypsistozoa fasmeriana (Brewin, 1959), Polycitor circes (Millar, 1963), and Eudistoma digitatum (Millar, 1964). The largest tadpoles are also among the most complex, as they contain well-developed rudiments of adult structures (Fig 3). By contrast, the simple larvae typically produced by oviparous species are generally about 1 mm long (see Fig 4). Cloney (1978) has calculated that the large complex larva of Hypsistozoa fasmeriana is approximately 3500 times greater in volume than Molgula occidentalis which has a simple tadpole larva. The typical tadpole of a solitary ascidian consists of a trunk (also called the “body” or “head” in the earlier literature), which is 150–250 µm long and 100–125 µm wide, and a tail about 750 µm in length, though this may vary. A detailed review of larval morphology in a simple tadpole is given for ciona intestinalis by Katz (1983). Katz, following earlier workers (Grave, 1944; Scott, 1946) classified the larval tissues into two categories. The six organ systems that comprise the larval action system are the tunic, epidermis, notochord, tail musculature, adhesive organs and nervous system. Four additional organ systems carried by the larva will ultimately become organs of the adult stage, but without immediate ecological relevance during the larval period (Grave, 1944; Scott, 1946). The simple type of larva is found in the phlebobranchiate familes Cionidae, Ascidiidae, Corellidae, and Diazonidae, but also has been retained in the phylogenetically more derived stolidobranchiate families Pyuridae, Styelidae, and Molgulidae. In colonial ascidians (Aplousobranchiata and the stolidobranchiate subfamily Botryllinae), a more elaborate larva is found wherein oral papillae occur in great variety, and numerous adult structures, non-functional in

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the larvae, may be present (Barnes, 1971; Millar, 1971; Cloney, 1977). Larvae of some colonial ascidians may even have a functional heart (Cloney, 1982). Although the general complement of sensory and locomotory structures is similar in the larvae of solitary and compound ascidians, many of the subtle differences could have ecological or behavioural significance. The surface of an ascidian larva is covered with a secreted, non-cellular tunic layer. This transparent tunic, also called the test or cuticle, is elaborated into two fins which, in tadpoles of solitary ascidians, are situated dorsally and ventrally on the trunk and tail. The fins are formed by extra-embryonic test cells present between the egg chorion and the embryo (Cloney & Cavey, 1982; Robinson, Kusten & Cloney, 1986). At hatching, the test cells often persist on the outside of the tunic fin for a period of time (Grave, 1921; Cloney & Cavey, 1982; Katz, 1983), although it is not known whether their persistence into the posthatching stage has any ecological significance. In any case, the test cells generally fall off before metamorphosis. Tadpole larvae of most solitary ascidians are laterally flattened for their entire length. In addition to the large dorsal and ventral fins, other smaller fins may be arranged in various positions on the lateral sides of the tail (Katz, 1983). Some colonial ascidians, but not all (see Grave & Woodbridge, 1924), have tails which are rotated through 90 degrees by a quarter twist at the base of the tail (Damas, 1904; Grave, 1921; Grave & McCosh, 1924). Berrill (1950) hypothesised that the twist results from limited space for development in the chorion. Although this appears to be an adequate proximate explanation, there has been no speculation as to why chorion size should be constrained in larger embryos. Perhaps it is limited in small zooids by the space available for brooding. If this is the case, one might expect larger chorionic spaces (and perhaps untwisted tails) in the embryos of atrial brooders than oviducal brooders. One consequence of the twisted tail is a more erratic, spiral swimming pattern. All ascidian larvae move by flexing muscles in the tail. These muscles are arranged in three longitudinal bands situated laterally (or, in the case of compound ascidians with twisted tails, dorsally and ventrally) and bounding the notochord. The notochord consists of about 40 squamous, matrix-filled cells surrounded by a fibrous sheath (Cloney, 1964; Mancuso & Dolcemascolo, 1977). The tail is innervated not only by the neuromuscular junctions that stimulate the muscle blocks (reviewed by Katz, 1983), but also by axons connected to pairs of ciliary sensory cells lying along the midline of the tail epidermis (Torrence & Cloney, 1982). It is not known whether these are proprioceptors that signal tail posture or if the receptors provide the larva with sensory input concerning some aspect of the outside environment. Most tadpoles respond quickly to tactile stimulation of the tail tunic (R.A.Cloney, pers. comm.). The anterior end of the trunk is equipped with three adhesive papillae. Cloney (1978) has identified at least nine different types of papillae, which he has classified into three major groups: non-everting glandular papillae, everting glandular papillae, and non-glandular papillae. The papilla structure may play a role in determining how well larvae select substrata. For example, some tadpoles with simple coniform papillae secrete adhesive even before contacting the substratum (Cloney, 1978). Although one might expect that this would reduce their capability for habitat selection, several species with this sort of papillae are known to discriminate among substrata at settlement (Young & Braithwaite, 1980a; Young, 1982). Also counter-intuitive is the observation that some tadpoles which can expose their adhesive abruptly by everting the papillae (reviewed by Cloney, 1978) seem to be quite indiscriminate with respect to attachment surface. The larvae of Botryllus schlosseri and Symplegma viride have a sucker-like holdfast mechanism, under nervous control, which reportedly allows the larva to attach and release its hold several times before selecting a final site for metamorphosis (Grave, 1934). At the opposite extreme, larvae of some urodele molgulids (e.g., Molgula citrina, M. manhattensis, M. occidentalis) either lack papillae (Cloney, 1978) or have tiny oral papillae that do not function in attachment. In these species, either the entire larval body becomes adhesive (Kingsley, 1882; Grave, 1944) or attachment is by means of a large primary ampulla near

THE ECOLOGY AND BEHAVIOUR OF ASCIDIAN LARVAE

33

the anterior end. The larvae of M. occidentalis, which fall into this latter category, are capable of discriminating among substratum types (Young, in press), presumably by delaying the release of ampullar adhesive until after encountering an appropriate habitat. It is not surprising, therefore, that Torrence & Cloney (1983, 1988) have reported sensory neurons in the anterior epidermis of this species. Attachment in some anural species occurs shortly after the egg contacts sea water (Young et al., 1988). Follicle cells surrounding the egg chorion of M. pacifica undergo a holocrine secretion of adhesive shortly after spawning. The embryo develops to hatching on the first substratum contacted by the egg, after which the juvenile moves away a short distance (Young et al., 1988). The adhesive papillae of most tadpoles are probably sensory as well as adhesive in function. Torrence & Cloney (1983,1988) have demonstrated the presence of sensory neurons in the oral papillae of Diplosoma macdonaldi and many other ascidians in several families. Similar neurons, as well as an associated papillar ganglion, have been reported at the light microscope level for the larvae of Botryllus schlosseri (Grave, 1934; Grave & Riley, 1935). Although the ultrastructural studies provide few cues as to the functions of the papillar receptors, behavioural evidence suggests that they may function both as mechanoreceptors and chemoreceptors (Cloney, 1978; Young & Braithwaite, 1980a; Young, 1982; Davis, 1987; Torrence & Cloney, 1988). The cerebral vesicle of an ascidian larva generally contains at least two sensory structures that control swimming behaviour and orientation: a multicellular ocellus for the reception of light, and a statocyte containing a statolith for the reception of gravity (Fig 4). A third type of possible sensory structure has been found in both phlebobranchs and stolidobranchs (Dilly, 1969; Eakin & Kuda, 1971; Reverberi, 1979; Svane, 1982). This structure is located in the dorso-posterior wall of the cerebral vesicle, imbedded in the tissue lateral to the cerebral vesicle, or confined to an auxiliary vesicle which communicates with the cerebral vesicle at the level of the statocyte (Svane, 1982; Vorontosova & Malakhov, 1984; Svane & Young, unpubl.). The cells carry 2 µm globular ciliary structures and resemble coronet cells (although more simple) typical for the saccus vasculosus found in elasmobranchs and many ganoid and teleost fishes. The globular cilia possess a modified (9+0) arrangement with tubules that open into the lumen of the cerebral vesicle (Eakin & Kuda, 1971). These structures have been variously interpreted as a second ocellus (Dilly, 1969; Torrence & Cloney, 1988) or hydrostatic pressure receptors (Eakin & Kuda, 1971). The only species of tadpole larva that has, however, been tested for a pressure sensitivity demonstrated no such response (Crisp & Ghobashy, 1971). Alternatively, if the ascidian “coronet cells” are homologous to those of fishes, they could have a secretory function as suggested by Lanzing & Lennep (1971) and Emanuelsson & Mecklenburg (1974) or they could function as a receptor for low molecular weight materials (Jansen & Flight, 1969). The actual function or functions, however, remain unknown. The morphology and ultrastructure of the ocellus and statocyte have been studied in detail (e.g. Dilly, 1961, 1962, 1964; Barnes, 1971; Eakin & Kuda, 1971; Katz, 1983; Vorontosova & Malakhov, 1984). Structural details of these organs vary substantially among species, and these differences have often been assumed to produce behavioural differences (Berrill, 1975). A typical ocellus, as exemplified by Ciona intestinalis, consists of three lens cells, a number of neurosensory retinal cells and a unicellular pigmented cup (Dilly, 1964; Eakin & Kuda, 1971; Turon, 1988). Similar ocelli are found in colonial aplousobranchs (Grave, 1921; Barnes, 1971, 1974). The larvae of most styelids have either simplified ocelli or compound sensory structures that incorporate elements of both the ocellus and the statocyte into a single structure. For example, in larvae of Styela partita, the ocellus is reduced to a small melanic granule surrounded by a thin layer of optically clear material (Grave, 1944; Whittaker, 1966), whereas colonial styelids such as Botryllus have the statocyte and ocellus combined into a “photolith” (Garstang & Garstang, 1928; Grave, 1934; Berrill, 1949; Torrence & Cloney, 1988). Larvae of the solitary styelid Cnemidocarpa finmarkiensis are

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reported to possess both an ocellus and a photolith (Vorontosova & Malakhov, 1984). The styelid genera Dendrodoa and Polycarpa (Millar, 1971), as well as the pyurid Herdmania momus (Svane & Young, unpubl.), and most urodele molgulids (Grave, 1926; Berrill, 1931) have statocytes, but no pigmented ocellus. The behavioural and ecological implications of these structural variations remain largely undocumented. TIMING AND SYNCHRONY OF REPRODUCTION SEASONAL PATTERNS OF REPRODUCTION Successful reproduction in cross-fertilising organisms depends on synchrony among members of the population. Although a few species, including both tropical (Goodbody, 1961, 1963) and temperature (Lambert, 1968; Berrill, 1957; Svane & Lundälv, 1981) ones, reproduce throughout the year, most ascidians reproduce in one or two discrete peaks during the annual cycle (reviewed by Millar, 1971; Berrill, 1975). Sexual and asexual portions of the reproductive cycle occur simultaneously in some compound ascidians and alternate in regular cycles in others (Millar, 1971). Previous workers have assumed that because reproductive cycles are correlated with temperature, temperature is the causative factor that entrains the cycles. Other factors that covary with temperature on an annual basis, including day length, phytoplankton concentrations, lunar periods, and tide levels, have not, however, been investigated. The important work of Pearse and his colleagues (Pearse & Eernisse, 1982; Pearse & Beauchamp, 1986; Pearse & Walker, 1986; and others) on the role of light in entraining circannual reproductive cycles in echinoderms should inspire similar studies in ascidians, particularly because many solitary ascidians are easily reared in laboratory seawater systems. DIEL PATTERNS OF LARVAL RELEASE AND SPAWNING In colonial ascidians, fully developed larvae may be incubated in the oviduct, in the atrial chamber of a zooid, in a specialised brood pouch (e.g. Distaplia spp.), or in the common cloacal chambers of the colony. Larval release may involve expulsion of the larva from an existing opening or directly through the tunic of the colony. The timing of larval release is controlled by light in every species that has been experimentally investigated (Table I). In general, spawning occurs in response to light following a period of dark adaptation. The duration of light exposure required, which has been called the dormant period (Huus, 1939) or the latency period (Lambert, Lambert & Abbott, 1981), varies somewhat among species. Olson (1983) tabulated the latency period for nine species of compound ascidians. We have expanded his table to include many additional species, including solitary ascidians and several colonial species not reported in the primary literature (Table I). Of 27 species for which observations or data are available, 16 release larvae in the early morning or within a short time after exposure to light in the laboratory. Five species release larvae all day (one of which continues to produce larvae throughout the night; Grave, 1936), five release between the late morning and early afternoon hours, and one releases in the afternoon (Table I). With a single exception, Podoclavella moluccensis (A.Davis, pers. comm.), the species releasing larvae at midday are didemnids which contain symbiotic algae, and which transfer the symbionts between generations during the larval stage (Duyl, Bak & Sybesma, 1981; Olson, 1983). The actual mechanism of larval release remains poorly understood for most colonial ascidians. Metandrocarpa taylori has been observed to eject larvae forcefully by means of a general contraction of body wall musculature (Abbott, 1955). Euherdmania claviformis and Pycnoclavella stanleyi move larvae through the atrium by a series of contractions in the thoracic region according to Trason (1957, 1963).

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35

Larvae of Eudistoma ritteri have been observed to swim actively from the atrial chamber of the adult zooid (Levine, 1962). We do not, however, know whether release is under control of the adult nervous system or if, alternatively, larvae leave spontaneously following stimulation of their photoreceptors. In Distaplia occidentalis, adult control TABLE I Spawning or larval release in relation to light-dark cycles Species Polyclinidae Aplidium (Amaroucium) constallatum A. stellatum A. antillense Polycitoridae Distaplia occidentalis D. corolla Polycitor mutabilis Cystodytes lobatus Eudistoma hepaticum E. olivaceum E. capsulatum Clavelina oblonga Didemnidae Diplosoma listerianum D. similis Didemnum molle Leptoclinum mitsukurii Trididemnum solidum Lissoclinum voeltzkowi L. patella Perophoridae Perophora viridis Ecteinascidia turbinata Cionidae Ciona intestinalis

Corellidae Corella paralellogramma C. willmeriana

Average spawning latency Spawning time Settlement time References 20 min

morning

morning

Scott, 1954; Mast, 1921

2–3 h

morning morning

morning morning

Gotelli, 1987 Bingham, pers. comm.

15 min

morning

morning

late morning continuous all day morning morning morning all day

midday continuous all day morning morning morning all day

Watanabe & Lambert, 1973 Bingham, pers. comm. Oka, 1943 Lambert, 1979 Bingham, pers. comm. Bingham, pers. comm. Bingham, pers. comm. Bingham, pers. comm.

morning midday 1100–1400 morning midday midday 1100–1800

morning midday midday morning midday midday afternoon

Crisp & Ghobashy, 1971 Olson, 1983 Olson, 1983 Yamaguchi, 1975 Duyl et al., 1981 Olson, 1983 Olson & McPherson, 1987

morning morning

morning morning

Grave & McCosh, 1924 Young, unpubl.

3–4 h

min

0–3 h

27 min

morning

morning

Lambert & Brandt, 1967; Conklin, 1905 ; Whittingham, 1967

11–17 min

morning

morning

Huus, 1939, 1941

15.1 min (sperm)

morning

Lambert et al., 1981

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C. inflata Chelyosoma productum Styelidae Styela partita S. plicata (Japan) S. plicata (Calif.) Botryllus schlosseri

12.6 min (sperm)

20.8 min (eggs) Lambert et al., 1981 18.9 min (eggs) Young & Braithwaite, 1980a

morning morning

11–12 h

afternoon

10–12 h

evening afternoon all day

all day morning morning morning morning

B. planus Botrylloides mutabilis B. nigrum Metandrocarpa taylori

15 min

morning morning morning morning

Polyandrocarpa tincta P. misakiensis

morning afternoon (16.30)

morning

11.5h

24 h

continuous

Symplegma viride Pyuridae Halocynthia roretzi

11–12pm twice a day (2 types)

Molgulidae Molgula manhattensis

M. citrina M. pacifica

few min

Conklin, 1905; Rose, 1939 Yamaguchi, 1975 West & Lambert, 1976 Grave, 1934; Grave & Woodbridge, 1924 Bingham, pers. comm. Yamaguchi, 1975 Morgan, 1977 Watanabe & Lambert, 1973 Grave, 1936 Hashimoto & Watanabe, 1982 Grave, 1936 Hirai & Tsubata, 1956

Watanabe & Tokioka, 1972 morning

all day night

Berrill, 1931; Whittingham, 1967; Conklin, 1905 Grave, 1926 Young et al., 1988

seems unlikely, since the brood pouches (which originate from the adult oviducts; Berrill, 1948a) often occur in the tunic as isolated entities following the regression of the zooids (Watanabe & Lambert, 1973). Reese (1967) and Woollacott (1979) have shown that isolated sperm ducts of Ciona intestinalis can be induced to spawn by exposure to light. Whether Distaplia brood pouches release larvae by a similar mechanism is not known. Because larvae of D. occidentalis are not active at the moment of release, larval activity seems, however, an unlikely explanation in this species (Watanabe & Lambert, 1973). The physiology of larval release has been investigated in greatest detail for two temperate species, D. occidentalis and Metandrocarpa taylori (Watanabe & Lambert, 1973). Under natural illumination, larvae were released about 1 h after sunrise, but with higher intensities of artificial illumination, the average latency period was reduced to 15 min. Because colonies could be stimulated to release larvae at any time of the day (following adequate dark adaptation), it was concluded that endogenous rhythms play no role in larval release. The rate of larval release was correlated positively with the length of the dark adaptation period. Colonies kept in continuous darkness released relatively fewer larvae than colonies maintained in

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continuous light. Larval release rate has also been correlated with temperature in Podoclavella moluccensis (Davis, in press). Among solitary ascidians, many phlebobranchs have large gonoducts in which masses of eggs and sperm are stored before spawning, whereas most stolidobranchs have no such storage gonoducts. In the latter group, eggs and sperm are released directly from the gonads. Ciona intestinalis has been reported to spawn in approximately three-day cycles in which gametes are shed into the gonoduct for several successive days, then spawned when the oviduct is full (Yamaguchi, 1975). Although few case studies are available, the mechanisms of spawning in solitary ascidians are understood better than the mechanisms of larval release in colonial ascidians. C. intestinalis, which spawns shortly after exposure to light, has been investigated most intensively (Lambert & Brandt, 1967; Whittingham, 1967). Because the photoreceptor that mediates spawning is located at the distal tip of the spermoduct (Reese, 1967), and the spermoduct possesses an extensive network of microtubules (Woollacott, 1979) the spermoduct acts as an independent effector system. The isolated spermoduct can be stimulated to spawn in the same manner as the intact animal (Reese, 1967; Woollacott, 1979). The action spectrum for spawning peaks at wavelengths that implicate cytochrome c as the photoreceptor pigment (Lambert & Brandt, 1967). Other solitary ascidians that spawn in the early morning include several phlebobranchs in the family Corellidae (Huus, 1939; Lambert et al., 1981), as well as the molgulid Molgula manhattensis. It may be significant that every one of these, irrespective of taxonomic affinity, has transparent or transluscent tunic. The reverse side of this correlation also holds; every solitary stolidobranch species with opaque tunic that has been examined spawns many house after the onset of light stimulation (as in Styela plicata; Yamaguchi, 1975; West & Lambert, 1976 and Halocynthia roretzi; Hirai & Tsubata, 1956). Only one solitary ascidian, Molgula pacifica, has been reported to spawn in the dark (Young et al., 1988). As gametes of the latter species are entirely self-fertile, its reproductive pattern may not require diel synchrony among individuals in a population. Likewise, Grave (1926) observed no pattern of release in response to light in Molgula citrina. Yamaguchi (1975) reports that Styela plicata and Ciona intestinalis both spawn after dark in Japan. Both of these species spawn, however, earlier in the day in other parts of the world. The ecological significance of morning larval release by most ovoviviparous ascidians has not been investigated. Thorson (1964) speculated that early morning release allows the larvae maximal time to distinguish between light and dark substrata at settlement. Most compound ascidian larvae swim for no more than a few hours; presumably they select habitats long before the sun has set. With only a few exceptions, all reported data on ascidian spawning times come from laboratory settings in which the adults were exposed to relatively high light intensities. Watanabe & Lambert (1973) have shown with at least one species that the latency period is a function of the cumulative quanta of energy with which the ascidian is illuminated. Thus, spawning can be delayed by illuminating with lower light intensities. Although diel changes in underwater light regimes have not been related quantitatively to any of the species with “morning release”, it seems possible that release could actually occur long after sunrise. Water turbidity, depth, cloud cover, season, and other factors influencing underwater irradiance should all play a role in determining when compound ascidians release larvae. Larval release has been observed in situ by divers for several tropical didemnids, all of which support extracellular algal symbionts of the genus Prochloron or Synechocystis (Duyl et al., 1981; Olson, 1983; Olson & McPherson, 1987) and all of which release larvae at midday. The algae are transferred between generations by the larvae (Kott, 1981; Lafargue & Duclaux, 1979; Olson, 1980). When a larva is released into the common cloacal cavity, algae residing in the cavity become entangled in filaments of the rastrum, a specialised structure located at the postero-dorsal end of the larval trunk (Kott, 1981). Olson (1983) has provided evidence that juveniles, which lack adult pigmentation, require lower light levels than adult colonies can tolerate, but also must select habitats where there is

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adequate light for photosynthesis of the symbionts. He argues that selection of such habitats is best done when conditions for ultraviolet irradiation are harshest, in the middle of the day. Didemnum molle and other didemnids with short (about 15 min) larval lives accomplish this by releasing brooded larvae between late morning and early afternoon (Duyl et al., 1981; Olson, 1983). Because light is an important settlement cue for the tadpole larvae of many solitary ascidians (Young, 1982; Young & Chia, 1984; Svane, 1987), one might expect that spawning of these oviparous species would be timed so that larvae would attain metamorphic competency during daylight hours (Thorson, 1964). Data are insufficient to support strong conclusions in this regard, but it is interesting to note that most solitary ascidians that reportedly spawn in the morning hatch after about 24 h, but that Styela plicata which has a 12h latency period for spawning (West & Lambert, 1976), develops in half that time (Yamaguchi, 1975). Thus, many solitary ascidians attain metamorphic competency at about the same time of day, but accomplish it by different means. This observation suggests that spawning times and developmental periods could have evolved jointly to increase opportunities for habitat selection. Additional work is needed on the interactions linking these various factors.

THE PELAGIC PERIOD DISPERSAL POTENTIAL AND DURATION OF THE PLANKTONIC PERIOD The potential for ascidian dispersal in the larval stage is correlated with the kind of development they exhibit. Direct developers, viviparous, and ovoviviparous species generally spend no more than a few hours in the plankton (Berrill, 1935), whereas oviparous species may sometimes prolong larval life for as much as a week and still undergo successful metamorphosis (Svane, 1984; Young; unpubl. data on Ascidia callosa). In one respect this correlation between the length of larval life and developmental period is unexpected. The large larvae of compound ascidians, being orders of magnitude greater in volume than those of solitary ascidians, presumably have energy stores sufficient to support a longer larval life. Variability in the total length of pelagic life can originate from processes and adaptations occurring in any of the pre-settlement stages (egg, embryo, or larva). In oviparous ascidians, a portion of the dispersal period is passed in the egg and embryonic stages. Although these life-history stages cannot swim; they demonstrate several possible adaptations that may affect dispersal (Fig 5). At a given sea-water density and viscosity, the sinking rate of a sphere at low Reynolds number is determined by the balance between its density and its drag, the latter being a function of cross-sectional area. If an organism can increase its drag without a proportional increase in density, it will sink more slowly (Vogel, 1981; Chia, Buckland-Nicks & Young, 1984). Ascidians seem to have accomplished this in three different ways. First, many ascidian eggs undergo an osmotic expansion of the perivitelline space shortly after contacting sea water (Berrill, 1929, 1975). Secondly, additional diameter is conferred by the follicle cells, which are heavily vacuolated (hence, light) cells, extraembryonic in origin, that cover the outside of the chorion. Finally, the follicle cells of a few phlebobrachs (Ascidiella aspersa, Corella inflata, C. willmeriana, C. parallelogramma) are lighter than sea water and cause the eggs to float. Lambert & Lambert (1978) have demonstrated that the follicle cells of C. inflata float because their vacuoles contain ammonia, which is lighter than sea water. In the latter species, however, egg flotation facilitates brooding rather than dispersal (Child, 1927; Young, 1988). The duration of the embryonic (i.e. pre-hatching) period in ascidians is partly a function of egg-size and water temperature (Figs 6 and 7), so temperate and cold-water species with oviparous development

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Fig 5.—Ascidian egg adaptations. Ova of Styela montereyensis (A and B), an ascidian with typical extraembryonic cell layers. Newly spawned eggs (C) and 16-celled embryos (D) of Halocynthia aurantium. Note that the chorion swells after spawning, increasing the effective surface area and drag of the egg without a commensurate increase in mass. Eggs of Corella inflata (E) showing floating, ammonia-filled follicle cells that facilitate brooding. Eggs of Molgula pacifica (F) attached to the substratum by a sticky adhesive coat secreted by follicle cells. FC: follicle cells; CH: chorion; TC: test cell; OV: ova; EM: embryo; AC: adhesive coat. Fig 6.—Relationship between temperature and duration of embryonic development (to completion of tadpole). Species: Ascidia atra, A.curvata, A.mentula, A.prunum, A.mammillata, Ascidiella aspersa. Redrawn from Berrill (1935). Fig 7.—Relationship between egg-size and duration of embryonic development (up to completion of tadpole) at 16°C. Species: Molgula, Styela, Ascidia, Ciona, Diazona, Corella, Boltenia, Polycarpa, Tethyum, Syplegma, Dendrodoa, Distomus, Stolonica. Redrawn from Berrill (1935).

probably spend more time in the plankton than similar species in the tropics. As an example, Styela gibbsii takes 30 h to hatch as a competent larva at 10°C in Washington (Cloney, 1987), whereas the congeneric S. plicata spends only 12 h in the embryonic stage at temperatures between 18 and 26°C (Yamaguchi, 1975). The same relationship applies within single species that occur over wide geographic ranges or in areas where temperatures fluctuate over a wide range. For example, the time from fertilisation to hatching in Ascidiella aspersa may be as short as 15 hours at 23°C or as long as 39 hours at 8°C (Knaben, 1952). Within a species, there may be substantial variability in the duration of the free-swimming period (Grave, 1920, 1928; Grave & McCosh, 1924; Grave & Woodbridge, 1924; Levine, 1962; Olson & McPherson, 1987, and many others). At least three major monographs (Berrill, 1935; Grave, 1935; Grave & Nicoll, 1939), attempted to explain this variability over a half century ago; nevertheless, many of the variables influencing length of larval life remain undetermined. Habitat selection may play a major role in determining the length of larval life. Some species with specific habitat requirements delay metamorphosis in the absence of suitable cues (Young, 1982); others settle faster in the presence of conspecifics (or extracts of conspecifics; Grave, 1935; Svane, Havenhand & Jørgensen, 1987) or under particular light conditions (Crisp & Ghobashy, 1971) than they otherwise might. Larval life may be shortened or prolonged artificially by a wide array of chemical reagents, hormones, and other artificial treatments (Grave, 1935; Grave & Nicoll, 1939; Lynch, 1961), but it is doubtful if these have any ecological relevance. In some oviparous species, only a fraction of larvae delay settlement for a prolonged period (e.g., up to 10 days in Ascidia mentula and 6 days in Ciona intestinalis; Svane, 1984, and unpubl.). The remainder settle much earlier. Indeed, settling curves for many species plotted from the literature demonstrate a marked negative skew (long tails on the upper end of the curve) (Millar, 1971). Grave & Nicoll (1939) observed that larval longevity of Ascidia nigra decreased as the breeding season advanced but did not discover the factor controlling this phenomenon. Although they hypothesised the existence of an hormonal “aging factor” that controlled the onset of metamorphosis, the fact that many larvae settled shortly after hatching indicated that such a factor was not necessary (Grave & Nicoll, 1939). Larvae of some colonial ascidians are incapable of settlement immediately upon release, whereas others (e.g., Didemnum molle; Olson, 1985) apparently have no pre-competent period. Within a given species, there is some variation in the time to settlement. For example, Olson (1985) reported that some larvae of D. molle settled immediately, whereas others swam for as long as 20 min. It is not known whether the latter individuals were competent to settle upon release. It seems likely that ascidians demonstrate the same inverse relationship between larval survival and time spent in the plankton as other invertebrates (Thorson, 1950; Menge, 1972; Vance, 1973; Chia, 1974; Emlet, McEdward & Strathmann, 1987), although this question has not been specifically addressed for ascidians. Ascidians in general may have short larval life because there is greater advantage to survival than dispersal.

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Certainly the advantages of dispersal to benthic invertebrates have been difficult to demonstrate (see Jackson & Strathmann, 1981; Palmer & Strathmann, 1981; Svane, 1984). Because the larvae of many compound ascidians are large enough to observe underwater, dispersal of several such species has been studied in situ. Olson (1985) followed numerous larvae of D. molle at Lizard Island on the Great Barrier Reef. Direction and speed of dispersal were related directly to current speed. Although the larvae swam during the dispersal phase, their swimming appeared virtually ineffectual because of the dominant influence of currents. Most recruitment occurred on the upstream edges of patch reefs downstream from source populations. Active swimming against the current was only observed close to the bottom or in the lee of coral heads, where the current moved very slowly (Olson, 1985). This observation suggests that swimming may function primarily in site selection rather than dispersal. Young (1985), noted that the larvae of Ecteinascidia turbinata spent a large portion of their pelagic period drifting passively without any spontaneous movements whatsoever, but Davis & Butler (in press) have found that larvae of Podoclavella moluccensis swim almost continuously, with only brief resting periods. In both species, dispersal distance seemed, however, to correlate with current speed. Observations made by Olson & McPherson (1987) confirm that dispersal depends mainly on currents, not swimming. There is evidence that larval swimming time is severely over-estimated by experiments conducted in closed containers of sea water. Olson (1985) found that swimming times of larvae released naturally on the reef ranged from 40 to 370 s, whereas larvae captured and retained underwater in closed Plexiglass boxes had settlement times distributed around a mean of 15 min, even when a dark surface was present in the box. Likewise, Duyl et al. (1981) noted that although some beaker-reared larvae of Trididemnum solidum settled in about 15 min, others delayed settlement as long as 3 h. More field observations are needed before we completely understand the nature and extent of the laboratory bias. In quiet harbours, dispersal of ovoviviparous ascidians may be very short indeed. Grosberg & Quinn (1986) investigated recruitment of Botryllus schlosseri on a 1-m square settling panel in the Eel Pond at Woods Hole, Massachusetts. A genetically characterised adult having a rare allele in the population was placed in the middle of the panel, and positions of recruits having the same allele were plotted. Settlement was found to be a function of distance from the adult colony, with most individuals settling within a few centimetres. Berrill (1955) has attributed aggregation in the brooding styelid Dendrodoa grossularia to similar short distance dispersal. Lambert (1968) also noted consistently high local recruitment near parents in Corella inflata, a solitary corellid that retains larvae until they are competent to settle. Although maximum dispersal potential can be estimated by knowing something about the combined lengths of the pre-competent and competent periods (Jackson & Strathmann, 1981) and the speeds of currents larvae are likely to encounter, such estimates may not be representative of the actual dispersal distances achieved by the larvae. Dispersal can be shortened by mortality, early settlement, or entrapment in areas with little or no current. Olson & McPherson (1987) have provided a convincing in situ demonstration of this point for the larvae of Lissoclinum patella on the Great Barrier Reef. Based on in vitro field measurements of the length of larval life, it was predicted that a larva should be able to disperse several hundred metres over a period of about two hours. In observing free-swimming larvae in the field, it was, however, discovered that the majority were consumed by predatory fishes and cnidarians, and the remainder settled less than 10 m from the parent colonies (Olson & McPherson, 1987). Thus, the potential dispersal was approximately an order of magnitude greater than the realised dispersal. Many colonial marine animals, including bryozoans, sponges, and some cnidarians demonstrate very short dispersal distances as a result of brooding (reviewed by Jackson, 1986). Philopatry (as short dispersal distance is sometimes called) may result in greater temporal stability of local populations than is found in

THE ECOLOGY AND BEHAVIOUR OF ASCIDIAN LARVAE

41

species with longer dispersal (Chernoff, 1985). Other ecological and evolutionary consequences of philopatry have been discussed by Shields (1982) and Jackson (1986). DYNAMICS OF LOCOMOTION From the standpoint of anatomy and ultrastructure, the tail is the best understood portion of a tadpole (reviewed by Katz, 1983). It is, therefore, surprising that there have been no careful studies of the mechanics of larval swimming in ascidians. In general terms, tadpoles move by alternating contractions and relaxations of the lateral muscle blocks that run alongside the tail. The elastic notochord antagonises the contractions of muscle blocks, translating the longitudinal forces into lateral (or sometimes dorso-ventral) flexions of the tail. Tadpole larvae, because they are small in size, function at low Reynolds numbers where inertial forces are overshadowed by the effects of viscosity. Consequently, tadpoles only move when their tails beat; there is virtually no glide stroke. The trunk of a tadpole does not remain stationary as the head of a fish does. Instead, it moves from side to side, with a displacement from the midline that may be either equal to or slightly less than the amplitude of the tail flexions. Cloney (1959) has noted that a swimming larva of Boltenia villosa flexes around two stationary nodes in the tail region, one just behind the trunk and the other about one-third of the way forward from the end of the tail fin. That portion of the tail between the nodes moves with about the same amplitude as the tip of the fin and the anteriormost portion of the trunk. In addition, larvae of many colonial ascidians have a 90-degree twist in the tail, causing the tail to beat dorsally and ventrally rather than laterally. Grave (1920) was apparently the first to note that larvae of ascidians spin on their longitudinal axis while swimming. He was not able to ascertain experimentally the cause of this spin, but proposed several possible mechanisms. In Aplidium constellation, Grave (1920) attributed the spin to an interaction among three mechanisms: a horizontal tail fin, lateral asymmetry in the trunk, and oblique alignment of the myofibrils in the muscle blocks of the tail (Grave, 1920). Both the horizontal tail fin and the lateral trunk asymmetry apparently resulted from development of the tail within the narrow confines of a small chorionic space. The manner in which the tail folds against the trunk during development molds the trunk tunic into a sort of short spiral (Grave, 1920, 1921). Because of this corkscrew shape, the tadpoles of Aplidium constellatum rotate even as they sink passively through the water column (Grave, 1920). In Botryllus schlosseri (which has a vertical tail fin; Grave & Woodbridge, 1924) and Perophora viridis (horizontal fin; Grave & McCosh, 1924), clockwise rotation during swimming was attributed in part to asymmetrical insertion of the tail at the posterior end of the trunk (Grave & McCosh, 1924; Grave & Woodbridge, 1924) and in part to the axial torsion imparted to the tail movements by a spiral arrangement of myofibrils. Now that methods are available for analysis of movements and flow effects at low Reynolds numbers (Vogel, 1981), the time is right for a re-analysis of locomotory methods in ascidian tadpoles. Ascidian tadpoles move faster than invertebrate larvae which employ ciliary locomotion, but slower than some crustacean larvae (reviewed by Chia et al., 1984). Berrill (1931) demonstrated a positive correlation between swimming speed and larval size. Swimming speeds of tadpoles, as measured in the laboratory, range from 0.2–2.5 cm/s (Berrill, 1931; Chia et al., 1984; Olson, 1985). LARVAL ORIENTATION WITH RESPECT TO PHYSICAL CUES The sensory structures in the tadpole cerebral vesicle (ocelli, statocysts or modifications thereof) mediate both kinetic and tactic (sensu Fraenkel & Gunn, 1940) responses. Although some workers (e.g., Millar, 1971) have referred to ascidian phototaxis as a phototropism, the term is incorrect. Tropisms (orientation of new

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growth with respect to a physical scalar or vector) may occur in adult ascidians, but not in ascidian larvae. Photokinesis, which involves a change in activity level resulting from a light stimulus, is common in ascidian tadpoles, and there is also evidence for directional responses to the physical vectors of light and gravity. Such directional responses are correctly termed phototaxis and geotaxis, respectively. Following Thorson’s (1964) review in which generalised behaviours of invertebrate photoresponse were suggested, reviews on ascidian biology have adopted the generalisation that larvae are photopositive at release or hatching, then become progressively more photonegative before settlement (Millar, 1971; Berrill, 1975). While this paradigm seems to fit a few species, careful examination of the available data on ascidian phototaxis reveals so much variability within and among species that broad generalisations seem unwarranted. Even different broods of larvae from the same individual or population often show statistical differences in the proportions of larvae demonstrating particular behaviours. In this section, we will review the nature of this behavioural variability and discuss the ecological ramifications thereof. The light wavelengths and intensities that ascidian larvae can detect have been considered by Mast (1921), Grave (1935) and Young (1982). Mast (1921), found that the larvae of Aplidium constellation were unable to detect very low (30 m.c.) absolute intensities of white light, but demonstrated a shadow response when higher intensities were reduced abruptly by even 10 m.c. of energy. Tadpoles also responded to red light, though the spectral distribution of this light was not reported (Mast, 1921). The wavelength and intensity thresholds for the shadow response were studied by Young (1982) for 12 species of solitary ascidians. All species were able to sense light intensities as low as and tadpoles of one species, Ascidia callosa, responded at the lowest intensity tested, (Young, 1982). Using a monochromator to produce 10 nm band-width beams of light, it was shown that all 12 species were most sensitive to wavelengths in the blue and green regions of the spectrum. The upper visual threshold was between 575 and 650 nm (red-orange light), and none of the tadpoles tested responded to light below 425 nm (Young, 1982). Grave (1935) reported that the larvae of several colonial ascidians responded equally to all wavelengths in the human visible spectrum, although he did not report the bandpass data on the filters used for making these observations. Photokinesis In ascidian tadpoles, two kinds of photokinetic responses have been documented. In the first, tadpoles accumulate in regions of low light intensity just before settlement. Crisp & Ghobashy (1971) have demonstrated the kinetic nature of this phenomenon by studying the settlement distribution of Diplosoma listerianum in a chamber illuminated by horizontal light passing through a graded neutral density filter. Because the light source was diffuse and at right angles to the chamber, the authors interpreted the responses as a photokinesis. Larvae did not show any preference for a particular region of the light gradient while swimming, but accumulated at an intensity of 300– 500 lux at settlement (Crisp & Ghobashy, 1971). Dim light was preferred over complete darkness. The second manifestation of photokinesis, commonly called the shadow response, seems to be present in virtually all tadpoles with functional ocelli (Young & Chia, 1985) and at least one species, Molgula occidentalis, which lacks an ocellus (Torrence & Cloney, 1988). The only exception reported in the literature is Metandrocarpa taylori (Abbott, 1955). In the laboratory, ascidian larvae resting on the bottom of the culture vessel or drifting passively in the water column are stimulated to swim by an abrupt reduction in light intensity. This response was discovered independently by Mast (1921) and Grave (1920) in Aplidium constellatum. Mast (1921) also reported that swimming tadpoles respond to shadows by changing the position of the tail, a process which he used to explain phototactic orientation. Sudden increases in light

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43

intensity do not elicit kinetic responses in resting ascidian tadpoles, though they may elicit changes in tail position in active ones (Mast, 1921). Only a few hypotheses have been advanced to explain the function of the shadow response. Crisp & Ghobashy (1971) and Grave (1935) noted that ascidian larvae settle sooner when stimulated to swim by frequent shadows than when held under constant illumination, but Young & Chia (1985) found this same relationship with only one of eight solitary ascidian species tested. For a species that needs shaded areas for survival (Olson, 1983; Young & Chia, 1984), it would seem reasonable that a tadpole should quickly undergo metamorphosis in a region with many shadows. Woodbridge (1924) extended this idea to explain how Botryllus schlosseri tadpoles locate seagrass blades, which is their normal habitat. She noted that larvae drifting passively in the water column would be stimulated to swim when passing through the shadow cast by a blade of seagrass. Larvae so stimulated swam upward 64% of the time and often contacted a seagrass blade. Because overhangs, rock crevices, and other shaded habitats tend to be excellent habitats for many ascidians (Millar, 1971; Berrill, 1975; Olson, 1983; Young & Chia, 1984; and others), one might suppose that the almost universally occurring shadow response could function in habitat location for many species of ascidians. This was tested experimentally in the laboratory for eight species by Young & Chia (1985). Tadpoles were offered choices between shaded overhangs and unshaded habitats under conditions of constant and fluctuating illumination. It was expected that larvae in the latter treatments would be stimulated to swim more often than larvae in the former, and would thereby be more likely to locate the optimal habitats. Only one species, Styela gibbsii, located the downward-facing surface significantly more often in fluctuating light than in constant light. None of the species located shaded habitats more often as a result of the shadow response. It was concluded that the shadow response does not facilitate selection of an optimal light regime at settlement (Young & Chia, 1985). Geotaxis and phototaxis A generalised description of ontogenetic changes in ascidian orientation behaviour has persisted in the literature for nearly 60 years. Millar (1971) has summarised the stereotyped behaviour as follows: “The ascidian larva has a characteristic pattern of behaviour consisting of an initial period when it swims upward (positive phototropism and negative geotropism) followed by a period when it swims or sinks downwards. The initial phase serves to distribute larvae, and it is in the second phase that the critical reaction is elicited in response to a decrease in light. The larva then swims towards dark areas, which in nature tend to be the vertical or lower surfaces of rocks etc.” This idea of ontogenetic change in orientation behaviour holds best for the compound ascidians that have been investigated; solitary ascidians seem to exhibit much more variability in their behaviour (Table II, Young & Braithwaite, 1980a; Young, 1982; Svane, 1987). Most data reporting the phototactic behaviour in ascidian larvae are casual observations made during studies of settlement and metamorphosis (Thorson, 1964). Many authors have, however, noted that all tadpoles do not behave the same way. There is often marked variability in behaviour among tadpoles of the same age and parentage, and also among individuals of different parentage within a given species. Thus, phototaxis in ascidians is best considered not as a stereotyped behaviour, but as a phenomenon with a statistical distribution. Ontogenetic changes in phototaxis and geotaxis have been documented convincingly in several ovoviviparous colonial ascidians which have a comparatively short planktonic life (Table II). The most detailed study, that of Crisp & Ghobashy (1971), indicates that Diplosoma listerianum conforms nicely to the classical generalisation. Larvae of this species remain strongly geonegative and slightly photopositive

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throughout the swimming period then switch to the opposite responses just before settlement (Crisp & Ghobashy, 1971). Swimming behaviour is modified by suboptimal temperatures and strong light, but is not influenced by hydrostatic pressures as high as two atmospheres (Crisp & Ghobashy, 1971). Botryllus schlosseri, like Diplosoma listerianum, remains weakly photopositive throughout most of its larval life (Grave & Woodbridge, 1924). Approximately two thirds of the larvae of Perophora viridis retain their positive phototaxis for their entire larval period; the remaining one third reportedly become photonegative towards the end of larval life (Grave & McCosh, 1924). In Aplidium constellatum, the photopositive period lasts for only a few seconds and the larvae spend most of their free-swimming period alternating periods of rest and activity in less illuminated regions (Grave, 1920). Several workers have made observations on behavioural changes of colonial ascidians in the field. Duyl, Bak & Sybesma (1981), Olson (1983, 1985) and Olson & McPherson (1987) all noted initial upwardswimming periods following release of didemnids on coral reefs. In Lissoclinum patella, the larvae moved upward for only about one minute (over a distance of about a metre) before they began swimming downward. In situ settlement experiments in a sealed chamber with a window at one end, however, indicated that larvae settle near the light (Olson & McPherson, 1987). Young (1986) made continuous in situ observations of behavioural changes in Ecteinascidia turbinata larvae of different ages. Contrary to the work with other compound ascidians, he found no evidence for an initial upward swimming period. Newly released larvae drifted passively more often than they swam. Throughout larval life, there were no significant differences in the amounts of time spent swimming upward and swimming downward. Swimming behaviour was extremely vari TABLE II Summary of ascidian tadpole photoresponses investigated to date. C: compound, S: solitary, X: response present, 0: response absent, +: responcses positive, −: respo onse negative, V: response variable, ?: not reported Shadow Phototaxis Family

Species

C/S response early late

Polyclinidae pellucidum

Aplidium (Amaroucium) C

X

+

–

A. constellatum

C

X

+

–

Didemnidae

C

X

+

Trididemnum solidum Polycitoridae Cionidae

Diplosoma listerianum C Distaplia sp. Ciona intestinalis

Grave, 1920; Mast, 1921 Grave, 1936; Mast, 1921 V

? C S

+ ? ?

– 0 +

Duyl et al., 1981 0 –

Perophoridae

Perophora viridis

C

?

+

V

Corellidae C. willmeriana Chelyosoma productum

Corella inflata S S

S X X

X 0 0

0 – V

– Young, 1982 Young & Braithwaite, 1980a

Reference

Crisp & Ghobashy, 1971 Berrill, 1948a Berrill, 1947; Castle, 1896; Dilly, 1964; Dybern, 1963 Grave & McCosh, 1924 Young, 1982

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Shadow Phototaxis Family

Species

C/S response early late

Reference

Ascidiidae

Ascidia nigra

S

X

+

_

Goodbody, 1963; Grave, 1936

A. callosa A. paratropa A. mentula Styelidae

S S S Dendrodoa grossularia C

X X X S

0 0 – ?

– – – 0

Young, 1982 Young, 1982 Svane, 1987 0

X

+

–

Symplegma viride Polyandrocarpa tincta P. gravel Styela montereyensis

C C

X X

+ +

_ _

Grave & Woodbridge, 1924; Woodbridge, 1924 Grave, 1936 Grave, 1936

C S

X X

+ 0

_ V

S. coriacea S. gibbsii S. partita Cnemidocarpa finmarkiensis Metandrocarpa taylori Pyuridae Boltenia villosa Halocynthia igaboja Molgulidae

S S S S

X X X X

0 0 0 0

_ _ _ –

Grave, 1936 Young & Braithwaite, 1980b Young, 1982 Young, 1982 Grave, 1941, 1944 Young, 1982

C

0

–

_

Abbott, 1955

Pyura haustor S S Molgula citrina

S X X S

X 0 0 0

0 _ V 0

– Young, 1982 Young, 1982 0

Botryllus schlosseri

Berrill, 1950

Young, 1982

Grave, 1926

able among individual tadpoles, and the ontogenetic changes in swimming behaviours predicted by the standard paradigm (Thorson, 1964; Millar, 1971; Berrill, 1975) did not occur (Young, 1986). In solitary ascidians, behavioural responses to light and gravity are extremely variable. Two species, Ascidia nigra (Grave, 1935; Goodbody, 1963) and Ciona intestinalis (Berrill, 1947; Millar, 1953; Dybern, 1963), have been reported to pass through photopositive and photonegative phases, but this conclusion is based on casual observations and field distributions (Dybern, 1963) rather than careful behavioural experiments. Svane (1987, and unpubl.) has demonstrated experimentally that both C. intestinalis and Ascidia mentula remain photonegative during their entire free-swimming period. Styela partita is negatively geotactic under all light conditions, and demonstrates negative phototaxis early in larval life (Grave, 1941, 1944). Late-stage larvae cease swimming and sink passively to the bottom (Grave, 1941, 1944). Although Grave (1944) attributed this behaviour to a reduced ocellus in styelid larvae, similar behaviour is demonstrated by Ascidia mentula, a species with a complete 3-lens ocellus (Svane, 1987). The larva of Molgula citrina, which lacks an ocellus, displays short bouts of negative geotaxis followed by random swimming and periods of rest (Grave, 1926). No response to light was observed (Grave, 1926).

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Fig 8.—Phototactic orientation mechanism of Aplidium constellation, as explained by Mast (1921). The mechanism relies on the orientation of the pigment cup, which allows light to strike the retinal cells only from the anterior or right sides.

The larval ocellus of Ciona savignyi differentiates after hatching, and the larvae are reported to pass through at least four different behavioural phases that correspond to the ocellar changes (Kajiwara & Yoshida, 1985). Newly hatched larvae are strongly geonegative but do not respond to light. Within 30 min of hatching, larvae in culture vessels swarm at the surface in dense aggregations (Kajiwara & Yoshida, 1985). Larvae begin to exhibit the shadow response 1.5 h after hatching, and the response becomes stronger as larval life proceeds. Finally, larvae developed a strong negative phototaxis after 3.5 h according to Kajiwara & Yoshida (1985). In the San Juan Islands of Washington, photoresponses of 12 species of solitary ascidians have been studied (Young & Braithwaite, 1980a,b; Young, 1982). None of the species showed predictable positive phototaxis at hatching, although most demonstrated an intermittent negative geotaxis early in larval life. Photoresponses at settlement were extremely variable in some species (e.g., Chelyosoma productum, Styela montereyensis), while other species (e.g., Pyura haustor, Corella willmeriana, Cnemidocarpa finmarkiensis) chose shaded substrata more often than expected by chance. The settlement distributions indicative of geotactic responses (choices of upward-facing and downward-facing surfaces) were similarly variable within and among species (Young, 1982). The species differences in phototaxis and geotaxis correlated well with the distributions of adults in the field. Taken together, the data on taxes in solitary ascidians suggest that photoresponse is variable among species and that broad generalisations cannot be made. There is more consistency among the short-lived larvae of colonial ascidians, but even the latter differ in many important details. Mast (1921) is the only worker to have considered in detail the mechanism of phototactic orientation in ascidian tadpoles. Many invertebrate larvae that respond to directional light stimuli (e.g., crustacean zoeae, polychaete setigers) have two distantly positioned photoreceptors. The mechanism of orientation seems to involve moving back and forth until an equal amount of energy enters each eye. Using simple but elegant observational techniques with larvae of Aplidium constellatum, Mast postulated a credible mechanism by which tadpole larvae orientate with a single photoreceptor (Fig. 8). The pigment cup in the ocellus of A. constellatum is directed anterolaterally on the right side (which Mast termed the ocular side) of the trunk. Thus, the retinal cells are stimulated only by light directed from the front or from the ocular side; light directed from the rear of the animal or from the left side does not stimulate the ocellus. In a tadpole larva moving perpendicular to a beam of light, the ocellus is alternately illuminated and shaded by the trunk as the animal spins on its own longitudinal axis. Each time the ocellus is shaded, it stimulates the tail to bend slightly and briefly (while still vibrating) toward the ocular side. This causes the animal to turn slightly away from the light. With another half rotation, the retinal cells receive full illumination, causing the tail to bend toward the abocular side and producing the same effect as before. After this process has been repeated during several revolutions, the animal becomes situated such that its pigment cup shades the retinal cells continuously. In this position, no tail bending occurs, and the direction of movement is away from the light in a straight line. Photopositive animals orientate exactly the same way, except that they bend their tails toward the abocular side when the ocellus is shaded and toward the ocular side when it is abruptly illuminated (Mast, 1921). Both the shadow response and the phototactic response described by Mast (1921) depend on reflexes stimulated by abrupt changes in light intensity. There are, however, several subtle differences in addition to the net behavioural effect. First, the shadow response occurs only when an animal is at rest, whereas the phototactic stimulation occurs only in actively swimming animals. Secondly, the

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47

shadow response occurs only when light intensity is decreased. An abrupt increase in intensity has no effect on resting tadpole larvae. Swimming animals, on the other hand, respond both to increases and decreases in illumination. Finally, the shadow response stimulates vibration of the tail, whereas phototaxis stimulates a bending of the tail while the latter continues to vibrate (Mast, 1921). Mast’s (1921) hypothesis on the mechanism of phototactic orientation is supported by the work of Kajiwara & Yoshida (1985) on Ciona savignyi. In this species, the pigment of the ocellus is diffuse and irregular in shape at hatching. It passes through a series of changes and increases in pigment density for the first 3.5 h of larval life until it finally takes on the characteristic cup shape. Behavioural changes correspond remarkably well to the changes in ocellus morphology. The shadow response begins when the ocellus becomes packed with dense pigmented tubules, 1.5 h after hatching, but directed swimming (negative phototaxis) does not occur until the pigment cup is completely formed (Kajiwara & Yoshida, 1985). LARVAL MORTALITY As with most invertebrate larvae, relatively little is known about the sources of mortality occurring during the pelagic phase. Most work to date has been on predators. Encounters between predatory fishes and three different species of larvae have been observed in the field (Olson, 1983; Olson & McPherson, 1987; Davis & Butler, in press). 87% of 133 larvae of Lissoclinum patella followed in the field were consumed by predatory fishes, zoanthids, or corals (Olson & McPherson, 1987). The major predators were territorial pomacentrids. Although the fish were equally abundant at all depths between 5 and 25 m, a higher percentage of tadpoles were consumed at shallow depths than at greater depths. Olson & McPherson (1987) attributed this difference to the fact that tadpoles passed pomacentrid territories repeatedly in shallow water as they were buffeted by the surge. One species of fish with a small mouth, Pomacentrus lepidogenys rejected larvae that they ingested. However, the egested larvae were always damaged too badly to continue swimming (Olson & McPherson, 1987). Predation rates on Podoclavella moluccensis in Southern Australia were much lower; of 270 larvae followed in the field, only two were mouthed by fishes, and both lived to settle successfully (Davis, unpubl.). Similarly, larvae of the didemnid, Didemnum molle were always rejected by fishes and always survived the attacks (Olson, 1983). Unidentified ascidian tadpoles have been taken in the guts of the pinfish Lagodon rhomboides in the Gulf of Mexico (J.Luzcovich, pers. comm.). This same fish species and several others accept tadpoles of many species of compound ascidians in laboratory aquaria (B.Bingham, unpubl.). Larval defences against predators (reviewed by Young & Chia, 1987) may be structural, behavioural, or chemical. The prevalence of both structural (Young, 1985) and chemical (Stoecker, 1978, 1980) defences in adult ascidians suggests that such mechanisms might also be present in larvae. However, besides anecdotal evidence (e.g., rejection of larvae by fishes in the field; Olson, 1983; Davis & Butler, in press), there is only one example of an ascidian larval defence in the literature: distastefulness in larvae of Ecteinascidia turbinata. The bright orange tadpole larvae of this perophorid ascidian are mouthed then rejected by several species of fish, including Lagodon rhomboides (Young & Bingham, 1987). By homogenising the larvae and embedding the homogenate in agar pellets, it was shown that the tadpoles are rendered unpalatable not by a structural defence but by a potent chemical substance (Young & Bingham, 1987). Fish that had experienced the taste of Ecteinascidia turbinata larvae learned quickly to avoid them as food, suggesting the possibility of aposematic (or “warning”) coloration. This hypotheses was tested by creating artifical mimics of palatable ascidian tadpoles (Clavelina oblonga) using orange stain. Fish that had not tasted unpalatable orange tadpoles of Ecteinascidia turbinata consumed the mimics readily, but experienced fish avoided all orange tadpoles regardless of species (Young & Bingham, 1987). The chemistry of the defence mechanism

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IB SVANE AND CRAIG M.YOUNG

remains unknown, but Bingham (pers. comm.) has now found several other ascidians in which orange coloration is correlated with unpalatability to fishes. Several sessile benthic predators are known to prey on ascidian larvae. The temperate octocoral Alcyonium siderium and the sea anemone Metridium senile, both of which are common on subtidal rocks in New England, often contain large numbers of ascidian tadpoles in their guts (Sebens & Koehl, 1984). It has been proposed that these cnidarians reduce competitive interactions with adult compound ascidians (Aplidium stellatum) by consuming the competitors’ larvae (Sebens & Koehl, 1984). Davis & Butler (in press) observed ingestion of larval Podoclavella moluccensis by the hard coral Culicia tenella in the field. Corals also captured a small percentage of Lissoclinum patella larvae on the Great Barrier Reef (Olson & McPherson, 1987). Adult ascidians sometimes consume their own eggs and larvae or the offspring of other species of ascidians (Young, 1988). It has been shown that the tendency toward larval cannibalism is greater in species that occur as solitary individuals in the field than among species that live in aggregations and settle gregariously as larvae (Young, 1988). Rejection of conspecific eggs relative to those of other species has also been observed for Ciona intestinalis (Havenhand, pers. comm.). Species that reject their own tadpoles do so by means of the “crossed reflex” (Hecht, 1918), in which objects stimulating the oral tentacles or the inside of the incurrent siphon epithelium elicit closure of the excurrent siphon followed by a strong contraction of the body wall musculature (Young, 1988). Species with larger siphon diameters tend to reject eggs and larvae less often than species with small siphon diameters. Young (1988) has proposed that an efficient rejection mechanism might be an important prerequisite for gregarious settlement responses in ascidians. There are undoubtedly numerous sources of mortality other than predators that eliminate larvae or embryos during their planktonic period (Thorson, 1950, 1966). However, very few such factors have been investigated for ascidians. Goodbody & Fisher (1974) reared the embryos of Ascidia nigra in sea water collected from three different habitats and demonstrated higher hatching success in water from the open ocean than in water from two inshore habitats. Survival differences were attributed to pH and salinity (Goodbody & Fisher, 1974). Many larvae are probably lost by drifting away from appropriate settlement sites. However, the extent of this loss has not been estimated. SETTLEMENT The transition between pelagic and benthic existence involves two processes, settlement and metamorphosis. In ascidians, settlement may be defined as the process of locating and affixing to the juvenile habitat. It generally, though not always, precedes metamorphosis. Metamorphosis is defined as the sequence of morphological events that transform the larva into a sessile, feeding juvenile (Cloney, 1982). These processes may include (but are not limited to) attachment, resorption of the tail, rotation of the trunk, emigration of blood cells from the hemocoel to the tunic, extension of epidermal ampullae, retraction of the sensory vesicle, and destruction of larval structures (Cloney, 1982). A larva capable of undergoing these metamorphic changes successfully is termed “competent” (Jackson & Strathmann, 1981). The processes by which ascidian tadpoles select habitats may involve numerous characteristic behaviours including phototaxis, thigmotaxis, chemotaxis, kin recognition, and gregariousness. Behaviour only becomes important, however, given the appropriate ecological opportunities (Moore, 1975). Thus, inherent behavioural patterns must interact with the distribution of available habitats, with pre-settlement mortality, and with factors modifying larval distribution in the plankton to produce the pattern of settlement.

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Settlement distributions are modified in turn by post-settlement mortality and migration (the latter in didemnids and some molgulids) to produce the distributional patterns of the juveniles and adults. Because microscopic juveniles are generally difficult to monitor in the field, there are few data on the settlement distributions of invertebrate larvae in general. Just as the number of recruits (where “recruit” is defined as an individual large enough to observe in the field) is not necessarily representative of the number of settlers because of mortality occurring between the two stages (Keough & Downes, 1982; Connell, 1985; Butler, 1986; Davis, 1987), so the distribution of recruits does not always reflect the settlement distribution. Nevertheless, the positive information contained in recruitment or distributional data should not be ignored; often, the only sites where we know with certainty that settlement occurred are those occupied by juveniles or adults. Colonial ascidians are often large enough to observe from the very moment of settlement, so the problem of distinguishing recruits from settlers is less acute in some members of the Ascidiacea than in many other invertebrate groups. Most behavioural studies have considered responses to single factors in isolation, whereas tadpoles are confronted in the field with interactive, complex suites of potential settlement cues. Whether settlement cues are ranked consistently or change in relative importance under different environmental conditions is not known. Some larvae, including those of soft-sediment molgulids, have been described as non-discriminating at settlement. Such are presumed to settle randomly within whatever regions competent larvae arrive. In the literature, three major spatial patterns have been attributed to larval behaviour: (1) small-scale singlespecies aggregations that result from gregarious settlement behaviour, (2) epibiosis and multiple-species aggregations, and (3) occurrence in shaded or cryptic habitats, generally attributed to negative phototaxis. LARVAL RESPONSES TO CONSPECIFICS Many solitary ascidians form clumps (Fig 9). Dense aggregations may occur on virtually any scale, only the smallest of which are likely to be influenced by settlement choices of the larvae (Butman, 1987). Aggregation is common on soft bottoms where other ascidians may be among the only available hard substrata for settlement (Young, 1985), but also occurs on rock surfaces where ascidian tunic is much less common than other available substrata (Young, 1982). Members of the genus Pyura, particularly in the southern hemisphere, commonly form dense beds in the lower intertidal zone. These include P. praeputialis in New South Wales, Australia (Dakin, Bennett & Pope, 1948; Underwood & Fairweather, 1986), P. chilensis and P. stolonifera in Chile (Gutierrez & Lay, 1965; Paine & Suchanek, 1983), P. pachydermatina in New Zealand (Batham, 1956), P. stolonifera in South Africa (Stephenson, 1942; Day, 1974) and P. haustor in Washington, USA (Young, 1982). Subtidal styelids and pyurids that aggregate include Bolteniopsis prenenti (Monniot, 1965), Microcosmus vulgaris (Monniot, 1965), Styela gibbsii (Young, 1985), Dendrodoagrossularia (Berrill, 1955) and Pyura haustor (Young, 1982, 1985). Molgula occidentalis form aggregations on virtually all scales from fourths of metres up to tens of metres on the relatively flat terrain of intertidal sandbars (Young, in press). Molgulids occurring in small-scale aggregations on hard substratum include M. complanata (Schmidt, 1982a, b), M.manhattensis (Monniot, 1965), M. occulata (Monniot, 1965) and the anural M. pacifica (Young et al., 1988). Subtidal aggregations have been reported in numerous phlebobranchs including Chelyosoma productum (Young & Braithwaite, 1980a), Corella inflata (Lambert, 1968), and Ascidia mentula (Havenhand & Svane, in press). Aggregations of some species tend to have unimodal size distributions, suggesting that they are formed by the nearly simultaneous settlement of larvae on a common substratum, whereas aggregations of other species are polymodal, consisting of numerous small individuals attached to the tunic of larger ones. These patterns suggest two different kinds

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Fig 9.—Small-scale aggregations of ascidians. A: subtidal clump of Ciona intestinalis in Gullmarsfjorden, Sweden, B: intertidal aggregation of Dendrodoa grossularia, probably formed by philopatric dispersal. Note two large adults (A) surrounded by numerous younger recruits (R). C: aggregation of Pyura haustor, a species with strongly gregarious larvae at 20 m depth in the San Juan Islands, Washington, USA. D: adult and juvenile Ascidia mentula in Gullmarsfjorden, Sweden. This species settles gregariously. E: “social” ascidian Clavelina picta at 15 m depth in the Bahamas. Apparent aggregations are formed by asexual budding, not larval processes. F: high-density clump of Styela montereyensis on an intertidal piling in Neah Bay, Washington. Larvae of this species are not gregarious (Young, 1988).

of behaviour, both of which have been documented in ascidians: aggregation among siblings or other individuals of the same age, and selection of adult conspecifics as a settlement site. Chemical cues associated with adults and juveniles stimulate metamorphosis and settlement in many ascidian species. Grave (1935) and Grave & Nicoll (1939) discovered that larvae of Ascidia nigra and Polyandrocarpa sp. were induced to settle sooner in adult or larval tissue extracts than in plain sea water. The same phenomenon has now been shown to occur in Ascidia mentula, Ascidiella scabra, (Svane, Havenhand & Jørgensen, 1987), and Pyura haustor (Young, unpubl. data). In all of these species, the time to settlement is related to the concentration of the adult extracts to which larvae are exposed. Significantly, extracts of tunic, the tissue larvae are most likely to contact, are more potent inducers than visceral extracts (Svane et al., 1987). Ascidiella scabra can be stimulated to undergo metamorphosis while still enclosed in their egg envelope (Svane et al., 1987), suggesting that larvae are competent to settle immediately after hatching. Although Grave & Nicoll (1939) reported a species specific effect (tissue extracts of Polyandrocarpa do not stimulate metamorphosis in Ascidia nigra), Svane et al. (1987) found little evidence for specificity between the two closely related ascidiids, Ascidiella scabra and Ascidia mentula. Larvae of Pyura haustor can be stimulated to undergo metamorphosis by exposure to sea water that has passed through the branchial sac and atrium of conspecific adults (Young, 1978). All of these phenomena are known only from the laboratory. Although their ecological significance remains unproven, the observations suggest not only that adult-associated cues could be important inducers of metamorphosis, but also that larvae may be able to detect water-borne (as opposed to surface-bound) cues. Larvae of several ascidians respond to newly settled juveniles. In dense cultures of Chelyosoma productum, larvae settle in a distinctly non-random manner (Young & Braithwaite, 1980a) and select areas with established juveniles much more often than regions colonised only by algae and bacteria (Young & Braithwaite, 1980a). Duyl, Bak & Sybesma (1981) showed that larvae of Trididemnum solidum settle sooner when even a single juvenile is present in a beaker than when no established juvenile is present. In related studies with Ascidia nigra, it was shown that curves of settlement versus time have steeper slopes when large numbers of larvae are present in the vials (Grave & Nicoll, 1939). Grosberg (1981) compared settlement density of Botryllus schlosseri among plates with varying densities of established juvenile colonies present. Although juveniles were avoided by many species of invertebrates, the presence of juveniles had no apparent effect on the settlement rates of conspecifics or any other species of ascidian. These data are supported by Schmidt (1982b) showing random spatial distributions among B. schlosseri settlers in Britain. Schmidt (1982b) also recorded the distribution of two other species of ascidians, Diplosoma listerianum, and Molgula complanata, on Perspex panels after submergence for four weeks; M. complanata was found to be aggregatively distributed while Diplosoma listerianum were distributed randomly. Schmidt (1982a,b) concluded that the observed distribution pattern resulted from tadpole choices or the absence thereof. In D. listerianum this conclusion was supported by nearest neighbour distances in laboratory settlement experiments, but the evidence for larval behaviour as a determinant of juvenile spatial pattern in Molgula complanata is equivocal, since post-settlement processes operating during the four week

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submergence period were not addressed. Havenhand & Svane (in press) demonstrated that distributions of newly settled Ascidia mentula in Petri dishes were significantly different from random at densities higher than 5 per cm2, but not at lower densities. The second pattern of single-species aggregation, characterised by a polymodal age distribution within a given clump, implies that larvae attach directly to the surfaces of established adults. Because the same pattern can, however, result from differential mortality in which survival is greater in that portion of the population settled on adults, careful settlement experiments are required before behavioural preferences are implicated as the cause. Such choice experiments have been run with several species. Larvae of Pyura haustor, a species that forms aggregations in the rocky intertidal zone, and in both soft and hard subtidal habitats, demonstrate a strong preference for the tunic of adult conspecifics as a settlement site (Young, 1982). Other substrata, including rocks, mollusc shell, and the tunic from various other ascidians are selected much less often than P. haustor tunic. Experiments in which the grooves and crevices of adult tunic are removed by a clean cut indicate that the attraction is not to structural or textural features, but rather to some chemical aspect of the adult. The possible selective advantage of this behaviour has been investigated by allowing larvae to settle on living adults and on rocks in the laboratory, then outplanting the juveniles to intertidal habitats where the adults naturally occur (Young, 1983). Over a two-week period, survival on adults was significantly higher than survival on adjacent rocks. Young (1984) attributed this difference to higher moisture and lower temperatures in the ascidian clumps during periods of tidal exposure. Havenhand & Svane (in press) have investigated gregarious settlement of Ascidia mentula, a species that consistently aggregates in the fjords of western Sweden. Over an 11-year period, recruitment of juveniles at several subtidal sites was correlated with adult density. In the laboratory, three pieces of evidence were indicative of gregarious settlement responses: aggregation of juveniles, acceleration of metamorphosis in response to adult tunic extracts, and apparent attraction to adult tunic. In the latter experiments, larval distribution was examined after 10 min of incubation in a submerged tube with an adult affixed to one end. There were consistently more larvae at the adult end than the opposite end. Although this experiment is indicative of gregarious larval behaviour, it does not demonstrate whether distant chemotaxis is involved or if larvae simply remain in the vicinity of an adult following random encounter. Adults and juveniles of Molgula occidentalis cover themselves completely with sand by means of fine tunic filaments all over the body. Larvae offered a choice of sand collected from the environment, sand removed from adults, or adult tunic selected the adult sand significantly more often than the other substrata (Young, in press). This ability to select a habitat may seem surprising in that these molgulid larvae have very reduced oral papillae (Cloney, 1978), but sensory neurons have been located in the anterior epidermis of the apapillate larva (Torrence & Cloney, 1988) (Fig 10). Young (1988) tested eight ascidian species for gregarious settlement by offering natural rocks and adult tunic to larvae as alternative substratum choices. Besides those species already discussed (Pyura haustor, Chelyosoma productum), only one, Styela gibbsii, was gregarious in laboratory experiments. Four other species that sometimes attach to adults under field conditions (Styela montereyensis, Boltenia villosa, Ascidia callosa, and Halocynthia igabojd) did not demonstrate strong preferences for adult tunic in the laboratory. The remaining five species tested did not select adult tunic as a preferred substratum. This pattern was expected, as none of them are found attached to conspecifics in the field (Young, 1988). Young

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Fig 10.—Attachment process of the apapillate larvae of Molgula occidentalis (A). Newly metamorphosed juvenile (B) showing sticky primary ampulla which provides initial attachment to the substratum. Juvenile several days old (C) attached to adjacent sand grains with secondary ampullae. Sediment-covered adults (D) on an intertidal sandflat.

(1985) provided field evidence that aggregation in Styela gibbsii reduces predation by the subtidal asteroid Evasterias troschelii by limiting the predator’s access to a region of soft tunic on the posterior end. SETTLEMENT RESPONSES TO OTHER ORGANISMS On both hard and soft bottoms, many species of solitary ascidians occur in multiple species aggregations where individuals are attached directly to individuals of other species. Monniot (1965) described the ecological relationships in such aggregations, termed “blocs de Microcosmus” in which large individuals of Microcosmus vulgaris provide attachment surfaces for many species of ascidians and other invertebrates. In Washington, aggregations dredged from deep, muddy bottoms often include as many as eight different species of solitary ascidians. Boltenia villosa and Styela gibbsii live attached to the tunic of Halocynthia igaboja and Pyura haustor, where they obtain protection from the predatory gastropod Fusitriton oregonensis (Young, 1985, 1986). In laboratory experiments, it has been demonstrated that larvae of both these epizooitic species prefer the tunic of the host species over the tunics of other species, as well as common inorganic substrata from their environment including rock and shell (Young, 1982). Moreover, the larvae of both Boltenia villosa and Styela gibbsii delay metamorphosis in the absence of suitable substratum (Young, 1982). Davis (1987) documented the settlement choices of larvae of Podoclavella moluccensis by following, larvae underwater and noting the outcome (settlement or rejection) of their first encounter with a substratum. Besides wooden pier pilings, most of the available substrata were sponges of several species. When a substratum was acceptable, a larva beat its tail vigorously for 3–5 min after attachment. Attached larvae were capable of rejecting a substratum by detaching with a short flick of the tail. Larvae generally rejected sponges, particularly those of the genera Dendrocia and Mycale. During the first month after settlement, survival was significantly higher on bare space and on the preferred sponges than on those sponges most often rejected by the larvae, suggesting that the settlement choices had an adaptive component (Davis, 1987). Moreover, larval choices were good predictors of the relative proportions of recruits appearing on the various available substrata in the system. SETTLEMENT RESPONSES TO PHYSICAL CUES Ascidians often occur in cryptic habitats such as cracks, crevices, and the dark undersides of rocks or overhangs. It has often been assumed that these habitats are located by negtive photo taxis (Dybern, 1963; Thorson, 1964; Crisp & Ghobashy, 1971; Millar, 1971; Berrill, 1975). The same pattern could, however, result from other behaviours including the shadow response (Woodbridge, 1924; Young & Chia, 1984), rugophilia, and negative geotaxis. Many selective pressures operate more intensely in open than cryptic sites, particularly during the juvenile stage. These include predation (Keough & Downes, 1986), silt (Young & Chia, 1984; Svane, 1987), competition with diatoms or filamentous algae (Goodbody, 1963; Young & Chia, 1984), grazing by herbivorous snails (Young & Chia, 1984) and ultraviolet light (Olson, 1983). Thus, both behaviour and selective mortality must be considered as important determinants of a cryptophilic distribution. Indeed, photonegative behaviour probably evolved in response to the predictable differences in mortality between open and shaded habitats.

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By integrating field recruitment studies with laboratory experiments on larval behaviour, Dybern (1963) demonstrated that negative phototaxis in the larval stage can explain the crytophilic distribution of Ciona intestinalis in Gullmarsfjorden, Sweden. Although he did not consider the possibility that post-settlement mortality on upward-facing surfaces could enhance the distributional pattern, Dybern’s (1963) observation that deep-water (low light) populations often occur on such surfaces supports his hypothesis that larval behaviour sets the pattern of distribution. On subtidal limestone outcroppings in the Gulf of Mexico, the compound ascidian Aplidium stellatum recruits primarily on vertical surfaces, which are much less common than horizontal ones (Gotelli, 1987). Laboratory experiments with the larvae demonstrated that vertical surfaces were chosen over horizontal ones in a consistent 2:1 ratio regardless of the relative proportions of the two kinds of surfaces. The nature of the behaviour producing this pattern has not been investigated. While following larvae of Didemnum molle underwater on the Great Barrier Reef, Olson (1983) observed that nearly all individuals settled on the undersurfaces of coral rubble and that recruits on horizontal settlement panels occur mostly on the undersides, within a few centimetres from the edge. In vitro experiments demonstrated, in agreement with the field observations, that larvae select shaded over unshaded surfaces. The advantage of photonegative settlement behaviour becomes apparent in the juvenile stage. Individuals reared in exposed reef habitats died within four days of settlement, possibly of exposure to ultraviolet light (Olson, 1983). The behaviour of D. molle larvae contrasts markedly with that of Lissoclinum patella, another didemnid containing symbiotic Prochloron algae (Olson & McPherson, 1987). Larvae of the latter species settled exclusively near the light end of a 1-m long light gradient. Olson & McPherson (1987) used this photopositive settlement behaviour to explain the low density of adult colonies at depths greater than 25 m. Alternative hypotheses (e.g., low survival of symbionts due to lower light levels) were, however, not tested. A comparative study of larval settlement behaviour in 12 species of ascidians in the Puget Sound region of Washington, USA, showed that some species were strongly photonegative at settlement, whereas others did not discriminate between illuminated and shaded substrata (Young, 1982). In most cases, larval behaviour was a good predictor of adult distribution in rocky subtidal habitats. Photonegative larval behaviour has also been implicated as a determinant of distribution in Diplosoma listerianum (Crisp & Ghobashy, 1971) and Botryllus schlosseri (Woodbridge, 1924; Dybern, 1963). Young & Svane (unpubl.) compared larval behaviour and field distribution of two solitary ascidians in Florida, one (Microcosmus exasperatus) of which lacks a pigmented ocellus, and one (Ascidia nigra) of which discriminates between light and dark regions at settlement. Recruitment of the two species on half-shaded Plexiglas plates in the field followed the laboratory predictions; Microcosmus exasperatus settled randomly, whereas Ascidia nigra demonstrated a preference for the dark portions of the plates (Young & Svane, unpubl.). ROLE OF REPRODUCTIVE AND LARVAL PROCESSES IN RECRUITMENT In recent years, population biologists working in the marine environment have placed an increasing emphasis on processes that control the abundance (or “supply”) of new recruits (Underwood & Denley, 1984; Connell, 1985; Roughgarden, Iwasa & Baxter, 1985). In ascidians, as in other benthic marine invertebrates with open populations, both temporal patterns of abundance (i.e. population dynamics) and spatial patterns of distribution are influenced by developmental mode, mortality occurring during the larval stage, advection and diffusion of larvae, and larval behaviour, as well as the distribution, fecundity and mortality of adult populations. We have already discussed many of these factors as isolated topics. In this

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section, we will consider the relative contributions of these factors in producing spatial and temporal patterns of ascidian recruitment. For our purposes, recruitment may be defined as appearance of a new generation of ascidians in a benthic population. The definition is operational rather than absolute, since different sampling protocols cause recruits to be first documented at different ages after settlement (Keough & Downes, 1982; Connell, 1985). Thus, a settler is the product of larval processes alone, but a recruit is the composite product of larval processes and events occurring during that portion of the post-settlement stage before the animal is first noticed by the ecologists. It is axiomatic that recruitment varies from place to place and from time to time. One might expect that recruitment events would be tied closely to reproductive seasonality (reviewed by Millar, 1971), but local patterns of mortality can result in no recruitment even when many larvae are produced. In a discrete population, it should be possible to estimate pre-recruitment mortality by comparing fecundity and recruitment. If recruitment were determined entirely by larval production, then one would expect a perfect correlation between patterns of fecundity and patterns of recruitment (Gotelli, 1987); lower levels of correlation should reflect variability in pre-recruitment losses. Most benthic invertebrates have, however, open populations where immigration and emigration are difficult or impossible to assess (Roughgarden et al., 1985). Quantitative comparisons of fecundity and recruitment become meaningful only where populations are closed or discrete, where dispersal time is very short, or where larval losses can be measured by direct obervations. Fortunately, some ascidian populations meet these criteria. Davis & Butler (in press) have presented evidence that the ascidian Podoclavella moluccensis has closed populations in the gulfs of South Australia. Using underwater observation of larvae, Davis (1988) estimated the number of individuals surviving to each major life history stage. Of 6430 larvae produced per m2, only 6.1 % settled. This estimate of larval mortality is about the same order of magnitude as estimates for other benthic invertebrates, including bivalves with much longer-lived larvae (reviewed by Strathmann, 1985; Young & Chia, 1987). About 63% of the settlers survived the first month to become “recruits” and 14% of recruits survived to become reproductive adults. Thus, only 0.56% of all larvae survived to sexual maturity (Davis, 1987). Gotelli (1987) studied a shallow subtidal population of Aplidium stellatum on a discrete limestone outcropping. Just over 50% of the temporal variation in recruitment could be explained by variation in the number of zooids brooding larvae in the previous month (Gotelli, 1987). The rest of the variability in recruitment was attributed to unstudied and variable losses in the larval and juvenile stages. Life table and survivorship curves have been calculated for many ascidians (Goodbody, 1962, 1963; Lambert, 1968; Goodbody & Gibson, 1974; Nomaguchi, 1974; Svane & Lundälv, 1981; Svane, 1983, 1987; Young & Chia, 1984; Young, 1985; Keough & Downes, 1986; Davis, 1987), but almost none of these consider mortality (“wastage”, in the terminology of Thorson, 1950, 1966) in the plankton, and only a few have characterised mortality immediately after settlement. The compound ascidian Podoclavella moluccensis is the only species for which mortality in all life history stages has been estimated (Davis, 1988). Olson & McPherson (1987) have estimated larval mortality of one additional species by direct observation, and several workers have studied the contribution of early post-settlement mortality. One method of documenting the mortality occurring during these early (and often microscopic) benthic stages is to transplant laboratory-settled juveniles on artificial substrata to the field habitats of interest. This method has been used for eight species of solitary ascidians in Washington (Young & Chia, 1984), for Ascidia mentula in Sweden (Svane, 1987), for Ascidia nigra in the Caribbean (Goodbody, 1963), and for Ciona intestinalis in Japan (Nomaguchi, 1974; Yamaguchi, 1975). High resolution monitoring of larger juveniles in the field has yielded similar data for several compound ascidians (Olson, 1983; Keough & Downes, 1986; Davis, 1987). Most of these studies indicated high habitat specific early mortality. All of those studies in which

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Fig 11.—Cluster dendrogram of correlation coefficients of recruitment over a 12-year period for Ascidia mentula at three stations each at two depth levels in Gullmarsfjorden on the Swedish west coast. Redrawn from Svane (1984).

mortality was measured in different seasons or in different years showed high temporal variation. The causes of this temporal variation in survival have not been explored adequately in any system. Because temperature is correlated with the timing of reproduction in many ascidian species (Millar, 1971), populations of the same species living under different temperature regimes might be expected to either recruit at different times or demonstrate differences in the duration of the reproductive period. This is reflected in the size-frequency distributions of Scandinavian populations of Ciona intestinalis. Shallowwater populations which experience relatively high summer temperatures consist of only a single generation at a time, whereas deeper populations living where temperatures are relatively low and stable may have up to three generations represented in the age structure, because of multiple recruitment events (Dybern, 1963; Millar, 1971; Svane, 1983). Synchronous recruitment of Podoclavella moluccensis has been noted by Davis & Butler (in press) at several sites in South Australia. However, Keough (1983) compared recruitment of Ciona intestinalis, Botrylloides leachii, Didemnum sp. and other sessile animals at two sites in Southern Australia separated by a distance of approximately 100 km and found no synchrony between sites. Svane (1983, 1984, 1988) found good synchrony in recruitment of Ascidia mentula, Ciona intestinalis, and Ascidiella sp. between sites separated by distances of 110 km and 9 km in Sweden. When comparing 12 years of recruitment patterns from stations in Gullmarsfjorden and in the archipelago off the Swedish west coast, Svane (1984) showed that recruitment correlated significantly between stations in the inner fjord and that portion of the central fjord greater than 20 m deep, while the station in the exposed archipelago correlated significantly with the shallow (15 m) station in the central portion of the fjord (Fig 11). This pattern was explained by the hydrographic properties of the area which may cause entrapment of eggs and larvae for long periods of time. Although temperature or other factors may help to explain the absence of recruitment synchrony between widely separated sites, many other factors (e.g., larval mortality; aggregation, local physical factors) are likely to be important; as always, correlation is fraught with the danger of misinterpretation! The solitary ascidians on subtidal rock walls of the Swedish west coast (Svane & Lundälv, 1981, 1982; Svane, 1983) have been monitored more intensively than those at any other site. Over a 12-year period, repeated sampling revealed major differences in the longevity and recruitment dynamics of four major species, Ciona intestinalis, Boltenia echinata, Ascidia mentula, and Pyura tessellata. The most common annual species in the system, Ciona intestinalis, fluctuated widely from year to year (Svane, 1983), whereas the longest-lived species, Pyura tessellata, had a constant population size over the entire study (Svane & Lundälv, 1982; Svane, 1983). Those species with the most stable populations also had low fecundity and recruitment. Solitary ascidians with relatively short (less than two years) generation time such as Corella inflata (Lambert, 1968), Ascidia nigra (Goodbody, 1962), and Ciona intestinalis (Dybern, 1963; Gulliksen, 1972; Svane, 1983) often demonstrate large local variations in population size from year to year. Current paradigms would predict that such species should be excellent colonisers, having high fecundities and fast growth to reproductive maturity (Jackson, 1977). These species are “weedy”; by producing many gametes, they are likely to have larvae present whenever an ecological opportunity (e.g. in the form of a new substratum) becomes available for settlement. Many short-lived species are poor competitors for space (Goodbody, 1965; Lambert, 1968) or are readily preyed upon due to their thin, gelatinous tunic (Lambert, 1968; Gulliksen & Skjaeveland, 1973; Young, 1986). Lambert (1968) has suggested that the brooding mechanism of Corella inflata is a method for producing multiple generations at a good site before individuals

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begin to lose ground to superior competitors such as compound ascidians (but see Young, 1988, for an alternative hypothesis). Annual, or short lived, ascidians are commonly found in fouling communities occurring on submerged hard substrata such as experimental panels (e.g. Sutherland, 1974; Field, 1982; Kay & Butler, 1983; Todd & Turner, 1986, and many others). Recruitment onto these hard substrata is highly unpredictable and the communities themselves are highly unstable both temporally and spatially (Dayton, 1984). Longer lived ascidians may, however, stabilise these communities for a period of time (Sutherland, 1981). Recruitment onto fouling panels and subsequent community development is influenced strongly by substratum size, quality, and the orientation of the panels (e.g., Keough, 1983; Todd & Turner, 1986). The developing communities are rarely comparable to those found on natural hard substrata since different organising forces operate (Svane, 1988). Kay & Keough (1981) found that relatively small isolated patches (the pen shell Pinna bicolor) were colonised mainly by larval recruitment while cleared patches on pilings nearby were colonised by the vegetative extension of adjacent sponges and colonial ascidians. Submerged artificial hard substrata may therefore be regarded as larval filters in which the effective pore size is determined by the physical properties of the material and by the surrounding environment, on both small and large scales. Fecundity is related inversely to tunic dry weight (Svane, 1983). Thus, species with low fecundity (e.g. Pyura tessellata) compensate by increasing longevity (more than 11 years) with a tough, protective adult tunic. Although these species seem to recruit rarely because of their low fecundity, their populations are often more stable than populations of species with higher recruitment potential that risk more of their available energy by sending larvae into the plankton. EPILOGUE Over the past half century, studies of ascidian tadpole larvae have progressed along many fronts. In recent years, ultrastructural studies have shed light on attachment, locomotory, and behavioural mechanisms, and behavioural studies indicate functions for known structures. Studies of long-term recruitment dynamics integrated with laboratory studies of larvae show how various life-history stages integrate to produce temporal and spatial patterns of variation which are of interest to the ecologist. Ascidians have proved ideal for general studies of larvae for many reasons, among which are short larval life, lecithotrophy (larvae need not be fed in culture), diversity of behaviours and structures, and ease of obtaining material for experiments. The large size of some colonial ascidian larvae allows us to follow, observe, and manipulate larvae in their natural environment. Logistic problems prohibit the use of such direct methods in most other invertebrate groups. Although in the past five years a rapid development in the use of in situ techniques has been seen, there remains a variety of important and unresolved questions in ascidian larval ecology specifically and invertebrate larval ecology in general that could be addressed using ascidian tadpole larvae. ACKNOWLEDGEMENTS We wish to thank our colleagues for valuable comments and criticism. We are particularly indebted to Brian Bingham, Lane Cameron, Andy Davis, Jon Havenhand, and Randy Olson for providing assistance and valuable information. One of us (I.Svane) is grateful to Dr T.Brattegård and the Marine Biological Station, University of Bergen, for providing excellent working facilities during the preparation of this work. The study was supported by the Swedish Natural Science Research Council, contract no. B-BU 8526–300 to I.Svane and in part by National Science Foundation grant no. OCE-8400406 to C.M.Young. The project was conceived and completed during reciprocal transatlantic visits supported by our respective institutions.

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Oceanogr. Mar. Biol. Annu. Rev., 1989, 27, 91–166 Margaret Barnes, Ed. Aberdeen University Press

EGG PRODUCTION IN CIRRIPEDES MARGARET BARNES The Scottish Marine Biological Association, The Dunstaffnage Marine Research Laboratory, Oban, Argyll, PA34 4AD, Scotland

ABSTRACT gE g production in three orders, Acrothoracica, Rhizoce phala, and Thoracica has been dealt with in this review. The Acrothoracica are burrowing forms, the Rhizocephala are parasitic, and the Thoracica the true barnacles. uM ch of the information on egg production concerns the Thoracica. There are, however, scattered references to egg production in the other two orders. Cirripedes may be hermaphroditic or have separate sexes in which case dwarf males are needed for fertilisation. In some cases hermaphrodites have complemental or apertural males. Transfer of spermatozoa, ac tivated by secretions of the oviducal gland, penetrate the oviducal sac and fertilisation results. gE gs a re developed outside the body but within the mantle cavity of the adult. The relative virtues of self- and cross-fertilisation have been discussed. Animals producing lecithotrophic nauplii usually have broods of fewer than usual eggs of a larger siz e than normal. The rhiz ocephalans are an exception; their eggs are small and numerous. Acrothoracicans also have lecithotrophic nauplii but the eggs are bigger and fewer than in the rhizoce phalans. Some lepadomorphs have lecithotrophic larvae and within the balanomorphs the teT racl ita species produce some anomalies; three species are lecithotrophic and the rest, as far as is known, are planktotrophic. At high latitudes and low temperature the fertilisation of eggs and release of nauplii has to be carefully controlled with probably only one brood a year. At lower latitudes, where temperature is higher and environmental conditions remain favourable longer, many broods can be produced in quick succe ssion. INTRODUCTION As members of the Crustacea, cirripedes have an exclusively sessile habit either as free-living filter-feeders or as highly specialised parasites. They may be estuarine or marine and are found at all depths of the ocean in a wide variety of habitats. The sub-class Cirripedia (a member of the Maxillopoda, see e.g. Newman,

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Zullo & Withers, 1969) was traditionally regarded to comprise the orders Apoda, Ascothoracica, Thoracica, Acrothoracica, and Rhizocephala. The Apoda, represented by the parasite Protolepas bivincta, was removed from the Cirripedia after Bocquet-Védrine (1972a, 1979) showed that the parasite was an epicaridean. The Ascothoracica encompasses primitive crustaceans living as parasites of echinoderms and coelenterates and are generally found in deep water. They are suctorial rather than cirral feeding, the nauplii lack frontal horns (Zullo, 1979) and all stages are capable of feeding; this is not true of the other orders. The sperm morphology differs from that of the remainder of the cirripedes (Grygier, 1982, 1983a). There has also been some discussion about the position of the gonopores and whether carapace pores could be homologous to frontal horn glands (Grygier, 1983b). Hallberg, Elofsson & Grygier (1985) have now found that the ascothoracid larva described by Grygier (1983c) does indeed have functional compound eyes suggesting a close relationship to the Cirripedia. There is still, however, only a limited amount of material available for such comparisons. In view of the controversy that has been apparent for some time about the position of the Ascothoracica—the most recent account being that of Grygier (1987)—it seems reasonable at present to keep it separate from the Cirripedia but as a sub-class of the Maxillopoda (Newman, 1982). This account of egg production in cirripedes will, therefore, only be concerned with the orders Thoracica, Acrothoracica, and Rhizocephala. The Acrothoracica represent burrowing forms, the Rhizocephala parasitic forms without appendages or digestive tract, and the Thoracica the true barnacles. Most of the information regarding egg production concerns the last of these three orders. There are, however, numerous references, many of them anecdotal, to egg production in the other orders and an attempt will be made to summarise these. Systematics and nomenclature are not the concern of this article and it is, therefore, the intention to use the names given by the various workers in their published papers. GENERAL Cirripedes may be either hermaphroditic or have separate sexes (Darwin, 1851, 1873; Batham, 1945a; Henry & McLaughlin, 1965, 1967; McLaughlin & Henry, 1972; Gomez, 1974; Foster, 1978, 1983; Newman, 1980; Dayton, Newman & Oliver, 1982; Crisp, 1983; Hui & Moyse, 1984). When the sexes are separate the males are referred to as dwarf males. In some cases hermaphrodites may also have one or more males attached in which case they are called complemental males. Crisp (1983) introduced the term apertural males for individuals he found settled on the operculum of Chelonobia patula. Similar males have been found in Chirona tennis (Zevina & Poliakova, 1986). These apertural males are capable of feeding and appear to be potential hermaphrodites in which development has been arrested. The determination of sex in cyprids of barnacles carrying dwarf or complemental males has received some attention over the years as can be seen from the work of the following authors: Kühnert (1934), Callan (1941), Veillet (1956, 1961), Yanagimachi (1961a), Gomez, Faulkner, Newman & Ireland (1973), Gomez (1974, 1975), Høeg (1984), and Walker (1985, 1987). Complemental and dwarf males are greatly reduced and consist of little more than the reproductive structures; their comparative anatomy has been reviewed recently by Klepal (1987). They may be attached in or about the external aperture of a normal hermaphrodite or female. At maturity spermatozoa are introduced into the visceral tissue or mantle cavity (body chamber) of the mature hermaphrodite or female. The transfer of spermatozoa may be by an injection of cells from the male in the Rhizocerphala (see e.g. Ichikawa & Yanagimachi, 1958; Reischman, 1959) or via a penis in the Acrothoracica and Thoracica (first seen by R. Bishop and recorded by Bate, 1869). In some cases the penis is capable of great extension in length for this purpose. Because semen is introduced into a functional female near the oviducal opening but

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not into the female genital tract exception may be taken to the use of the word copulation; it has, however, been widely used although some people prefer the term pseudo-copulation. In the Acrothoracica and Thoracica oviducts running from the ovaries enter the mantle cavity at the base of the first cirri. Movement of ova from the ovaries via the oviducts is stimulated by semen covering the exit of the oviducts in the mantle cavity. Spermatozoa, if not already motile, are activated by secretions from the oviducal gland and the oviducal sac containing the ova is penetrated (Walley, 1965; Barnes, Barnes & Klepal, 1977; Klepal & Barnes, 1977; Klepal, Barnes & Barnes, 1977; Walker, 1977a, 1980). Fertilisation takes place at this time and in all cirripedes the eggs develop external to the body but within the mantle cavity of the adult. In what follows the term egg will be used for the fertilised ovum plus its nutritive and protective tissues and from which, in the case of cirripedes, one of the planktonic stages emerges. The embryo will be regarded as the young organism in its stages of development within the egg. Within the eggs of cirripedes the embryos develop, eventually hatch and the young leave the mantle cavity. At this time it may be as a Stage I nauplius or, in some cases, as a cyprid. There are several nauplius stages (up to six) followed by a cyprid stage which eventually settles and becomes the adult animal. In some Rhizocephala the cyprid stage is followed by a kentrogon stage (which produces the reproductive adult) or a trichogon stage (Høeg, 1987). FERTILISATION Cross-fertilisation is obligatory in most cirripedes although several instances of self-fertilisation are known. Self-fertilisation may occur in some species when individuals are isolated by distances greater than the length of the extended penis (see e.g. Barnes & Crisp, 1956; Barnes & Barnes, 1958). In such cases parthenogenesis or activation by water-borne spermatozoa must be shown to be absent. The former could be eliminated by detailed cytological investigations. This has never been done but because male and female gonads are ripe simultaneously and cross-fertilisation is normal in the same species, parthenogenesis is unlikely. Fertilisation by water-borne spermatozoa also appears improbable (Barnes & Crisp, 1956). First, there is no decrease in the incidence of egg masses in isolated animals with increasing distance of isolation. Secondly, seminal fluid is rarely seen to be emitted except as a result of the stimulus of copulation and then takes place within the mantle cavity when the semen at first coagulates on contact with sea water. Thirdly, oviposition itself normally only occurs in association with the copulatory stimulus. Self-fertilisation, therefore, remains the only possible mechanism. The percentage of a population whose egg masses have reached a given stage of development is always greater in contiguous compared with isolated individuals, indicating that in the latter oviposition may be delayed. This delay is almost certainly dependent on the absence of the appropriate stimulus to oviposition associated with copulation. Eventually the threshold value of this stimulus must be reached and perhaps under these conditions movements of the penis within the mantle cavity are sufficient to initiate discharge of semen and to induce oviposition. Although many apparently viable nauplii may be obtained from the ripe egg masses contained in isolated, and presumably self-fertilised, individuals there are often many unsegmented and abnormal eggs. This suggests that self-fertilised eggs are less viable. The fact that the number of normal, viable nauplii from an animal of a given size is less when it is growing isolated than when it is in a position to be cross-fertilised also suggests that the latter is a more effective form of reproduction (Barnes & Crisp, 1956). Instances of self-fertilisation have been found in Verruca stroemia (Barnes & Crisp, 1956), Balanus amphitrite (Patel & Crisp, 1961), B. amphitrite communis (Pillay & Nair, 1972), B. balanus (Barnes & Barnes, 1954; Crisp, 1954), B. eburneus (Cheung & Nigrelli, 1969; Landau, 1976), B. improvisus

EGG PRODUCTION IN CIRRIPEDES

65

(E.Furman, pers. comm.), B. perforatus (Barnes & Crisp, 1956), B. trigonus (Werner, 1967), Octolasmis warwickii (Harker, 1975), and possibly in Platylepas ophiophilus (Zann, 1975) although this is not yet confirmed. There is evidence that several species of Chthamalus can self-fertilise (Barnes & Crisp, 1956; Barnes & Barnes, 1958; Tenerelli, 1958; Klepal & Barnes, 1975). In this genus many of the species extend to high intertidal levels and the capacity to resort ultimately to self-fertilisation may have a survival value. The population density at such levels may not always be sufficient to ensure proximity of individuals for mutual copulation. The advantages of hermaphrodites which normally cross-fertilise being able, in some circumstances, to self-fertilise have been discussed by Tomlinson (1966), Ghiselin (1969), and Sastry (1983). Ghiselin (1984) has discussed the use of dwarf or complemental males which can be regarded as a form of self-fertilisation when they originate from the adult to which they eventually become attached. In many instances it is assumed that this is not the case. REPRODUCTIVE GONADS The processes of spermatogenesis and oogenesis are outwith the scope of this review albeit spermatozoa and ova are necessary for egg production and deserve a mention. The most detailed experimental work on these processes has been done on members of the Thoracica. Early workers were aware that the spermatozoa of some cirripedes were finely filiform and capable of movement at some time in their life. In more recent times modern methods of study including scanning and electron microscopy have been used to extend our knowledge of cirripede spermatozoa and spermatogenesis. Rigo (1941) worked on Balanus amphitrite communis; Bocquet-Védrine & Pochon-Masson (1969) on B. perforatus; Munn & Barnes (1970a,b) and Barnes, Klepal & Munn (1971) on B. amphitrite amphitrite, B. balanoides, B. balanus, B. crenatus, B. eburneus, and B. perforatus, also Chthamalus stellatus, Verruca stroemia, and Elminius modestus; and Honma & Nakajima (1973) on Balanus eburneus. The relative inactivity of the spermatozoa of B. balanoides was commented on by several of these authors and also by Walley, White & Brander (1971) and Walker (1977b) who found that fluid from the oviducal gland stimulated activation at the time of oviposition. Turquier & Pochon-Masson (1969) worked on the spermatozoa of the acrothoracid Trypetesa (=Alcippe) nassarioides. References to the early work can be found in many of these recent papers. Gonad production involves biochemical synthesis with the formation of nucleic acids for spermatozoa and the mobilization of lipid and protein for ova. Food reserves may or may not be stored by the adult animal prior to gonad development. In boreo-arctic barnacles such as Balanus balanoides and B. balanus assimilation and storage of reserves begins in March to April at the time of the diatom increase (Barnes, Barnes & Finlayson, 1963). Gonad production in these species is relatively slow, beginning in early May and lasting until October in B. balanoides and early February in B. balanus. Final maturation takes place immediately before fertilisation (Barnes, Barnes & Klepal, 1977) in late October to November in the former species and February in the latter. The precise time of fertilisation varies by a few weeks depending on latitude. After oviposition and fertilisation these animals are devoid of spermatozoa and ova until feeding begins again. After fertilisation the penis of B. balanoides is shed in one or two moults (Klepal, Barnes & Barnes, 1975) and is gradually replaced, the length rapidly increasing from August to reach a maximum in October immediately prior to copulation (Barnes & Stone, 1972). In warm-water genera such as Chthamalus there are several cycles of gonad production in a year allowing for several broods of eggs (Crisp & Patel, 1969) in contrast to the one annual brood in boreo-arctic species. In general the behaviour of C. stellatus seems to be markedly independent of environmental conditions as far as food is concerned (Barnes, 1972). In some species the male gonads may remain active throughout the

66

MARGARET BARNES

year and ovaries may or may not carry mature ova or ova available for final maturation as soon as environmental conditions are favourable. In Balanus improvisus Blom (1965) reports large amounts of spermatozoa in winter but practically no oocytes— they begin to increase when sea-water temperature rises in April. In B. rostratus, however, oocytes are produced throughout the year according to Korn (1985) while male gonads only regenerate from June onwards and the animals fertilise in late September to October. A large number of oocytes degenerate before fertilisation; resorption of oocytes can also be seen in figures given for B. amphitrite and B. eburneus by Fyhn & Costlow (1975). Other species, although not breeding continuously, retain mature germinal cells throughout the year, reproduction being initiated as soon as conditions allow it. This appears to be so in B. perforatus and B. eburneus near Tarante, Italy (Lepore, Sciscioli & Gherardi, 1979) and in B. algicola in South Africa (Sandison, 1954). RHIZOCEPHALA The Rhizocephala are parasites of crustaceans, particularly decapods. The adult has a modified form and bears no resemblance to other cirripedes; the affinities are only shown by the larval stages and the spermatozoa. An up-to-date account of rhizocephalans has been given by Høeg & Lützen (1985). There are usually four nauplius stages, with the characteristic frontal horns but no alimentary canal, followed by a cyprid (Codreanu, 1959; Høeg, 1982). In some cases the hatching embryo is a nauplius and sometimes a cyprid. The more primitive rhizocephalans are ectoparasitic and the adult (reproductive) body develops in situ at the site of attachment of the cyprid to the host; there is no kentrogon stage. Such is the case in Duplorbis (Smith, 1906), Chthamalophilus delagei (Bocquet-Védrine, 1961), and Boschmaella balani (BocquetVédrine, 1968). The majority of rhizocephalans, however, develop from a kentrogon formed from the settled cyprid. The kentrogon penetrates the host and develops endoparasitically for several months, depending on the species. An adult reproductive body eventually emerges and becomes the externa while the internal nutritive structure (the interna) remains within the host’s body. This process has been known since the time of Delage (1884) but has recently been studied in detail by Høeg (1985a) for Lernaeodiscus porcellanae. The complete life history of this species has been determined by Ritchie & Høeg (1981) and Høeg & Ritchie (1985). The externa has a visceral mass, containing the ovaries, and is surrounded by a mantle. This is separated ventrolaterally from the visceral mass by a mantle cavity which develops as the externa matures. There is usually one mantle aperture at an extremity of the externa. Sylon, the sole genus of the Sylonidae, differs from most other rhizocephalans in having two mantle apertures (Lützen, 1981). In most rhizocephalans the aperture is open soon after the emergence of the externa but in some it may only open very late in the development of the externa or even when the embryos are ready for release. Such is the case in Clistosaccus paguri (Høeg, 1982, 1985b). In Thompsonia there is no mantle aperture (Reinhard & Stewart, 1956). Development of the externae depends on exposure to male cyprids. If virgin externae are kept in isolation and out of contact of male cyprids, they neither moult nor begin production of ova. In Lernaeodiscus porcellanae they can remain in this state almost indefinitely (Ritchie & Høeg, 1981). In Sacculina carcini virgin externae quickly die and drop off the host if no contact with male cyprids is made. Lützen (1981) has, however, reported ovulation in two full-grown Sylon hippolytes in which implantation of male cells had failed; the ova, however, did not develop. Rhizocephalans are now known to have separate sexes (see e.g. Reinhard, 1942a; Ichikawa &. Yanagimachi, 1958, 1960; Yanagimachi, 1961a,b; Yanagimachi & Fujimaki, 1967) although originally (Delage, 1884; Smith, 1906) they were thought to be hermaphroditic. Bocquet-Védrine (1961) described the

EGG PRODUCTION IN CIRRIPEDES

67

Chthamalophilidae as self-fertilising hermaphrodites and she later (Bocquet-Védrine, 1972b) expressed doubts about the function of male cyprids in Sacculina carcini. This has, however, now been settled by the work of Høeg (1984, 1987). He found that there are small (female) and large (male) cyprids in S. carcini. The female cyprids settle on the host and the males on the externae; this is in agreement with other rhizocephalans. Two sizes of cyprids were recognised by Veillet (1943a, 1945) in Triangulus galatheae and their separate roles were clarified by Yanagimachi (1961a,b) during experiments with Peltogasterella gracilis. A size difference (greater in some cases than others) between male and female cyprids is now established in the Peltogastridae and the Lernaeodiscidae (Ritchie & Høeg, 1981), the Sylonidae and the Clistosaccidae (Lùtzen, 1981; Høeg, 1982), as well as in the Sacculinidae (Høeg, 1984). Invasion by cells from male cyprids is necessary for further development of externae (see above). This invasion is generally thought to be through .the mantle aperture into one or two male cell receptacles, the testes (Ichikawa & Yanagimachi, 1960; Høeg, 1985b; Høeg & Ritchie, 1985). Høeg (1987) found a trichogon stage produced by male cyprids and has described what happens between settlement of the cyprid on an externa and the arrival of male cells in the receptacle of Sacculina carcini. A trichogon stage is present in the Sacculinidae, the Lernaeodiscidae and possibly in the Peltogastridae. A few genera lack receptacles, e.g. Mycetomorpha, Thompsonia, and Sylon. In those species that lack a mantle opening, or it only opens after spermatogenesis, the male cells must be injected through the integument and mantle of the virgin externa. Ichikawa & Yanagimachi (1958, 1960) do not regard the male “testis” in Sylonidae as a testis in the true sense of the word but rather that there is a female organ serving to hold a mass of male cells from the transformed male cyprid. There is no special receptacle for these cells and where they are found varies according to the genus. Spermatogenesis may, therefore, take place in the mantle cavity as in Duplorbis (Smith, 1906), in the colleteric gland in Sylon (Veillet, 1962; Lützen, 1981) or in the mantle of Mycetomorpha (Reinhard & Evans, 1951) and Thompsonia (Yanagimachi & Fujimaki, 1967). In the Peltogasteridae (Reinhard, 1942a; Ichikawa & Yanagimachi, 1958), Sacculinidae (Ichikawa & Yanagimachi, 1960), and Lernaeodiscidae (Ritchie & Høeg, 1981) spermatogenesis begins in the male cell receptacles and they become the only source of spermatozoa. This process begins in Clistosaccus paguri, which only has one receptacle, when ova within the ovary reach about 60 µm diameter. When the ova are about 100 µm spermatogonia differentiate and when 165 µm the receptacle is filled with spermatozoa. Ovulation and release of spermatozoa into the mantle cavity are simultaneous and in this species the mantle aperture opens at the same time (Høeg, 1982). A similar synchrony is recorded for other species although the mantle aperture may be already open (Lützen, 1981; Ritchie & Høeg, 1981). Ova pass through the atrium of the colleteric gland and are fertilised in the mantle cavity. After ovulation the externa exhibits peristaltic movements which serve to mix spermatozoa and ova together and to ventilate the mantle cavity. Although the mantle aperture is technically open it remains compressed during embryogenesis. During ovulation in Peltogaster paguri the aperture is tightly closed (Reinhard, 1942b). In Lernaeodiscus porcellanae ventilation may also be assisted by the grooming action of the castrated host which treats the externae as its own brood. When this is prevented the externae soon become fouled. In particular, the grooming assists during the moult which occurs after the release of each batch of nauplii (or cyprids). The nauplii are also released into the current generated by the host during grooming; this aids dispersion of the cirripede young (Ritchie & Høeg, 1981). When embryogenesis is complete the embryos hatch and are expelled from the mantle cavity through the mantle aperture; Reischman (1959) has described this for Peltogasterella. Information on the sizes of ova and eggs produced by rhizocephalans is scattered throughout the literature from as early as Delage (1884) to the present day. Even Leuckart (1859) stated of Sacculina inflata “… ovarian eggs were usually smaller than those of the egg-tubes…” but gave no sizes. Descriptions of the rhizocephalans collected during the Siboga Expedition (Van Kampen & Boschma, 1925; Boschma, 1931)

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mention the presence of eggs in the mantle cavity of many species but only give sizes in a few cases. These are given in Table I; sometimes sizes have been estimated from scaled drawings. Other records from the literature are also shown in Table I. Sometimes the size of nauplius stage I is given if no information about the egg is TABLE I Rhizocephala: summary of relevant literature on breeding seasons, sizes and numbers of young, cyp=cyprid ; St I=stage I nauplius ; +=or more ; *=embryo released as cyprid from mantle cavity of adult, if known Species

Place

Breeding season

Incubation time, days

Sizes (L×B), Number of µm eggs per brood

Number of broods per year

References

Chthamalo philus delagei* Clistosaccu s paguri*

–

–

–

cyp 60–70

–

–

Sweden

About 30

ova 165 cyp 184×90

–

1 or 1+ per life

Drepanorch is neglecta

–

All year, summer peak –

BocquetVédrine, 1961 Høeg, 1982, 1985b

–

cyp 140

–

–

Drepanorch is villosa Heterosacc us ruginosus Lernaeodis cus cornutus Lernaeodis cus galatheae

Siboga, Exped. –

–

–

egg 100

–

–

–

–

–

–

–

–

–

egg 135×108 St I 200 egg 94

–

–

Krüger, 1940

–

–

–

–

–

Veillet, 1943a,b

Lernaeodis cus porcellanae –

–

–

–

small cyp 130 large cyp 180 egg 160×126

–

–

Krüger, 1940

–

–

–

–

–

All year

10 to 14

100+ to 20 000

About every 15 days

Müller, 1862 Ritchie & Høeg, 1981

Peltogaster paguri

–

–

egg 200×120 small cyp 227×202 large cyp 255×232 30 to 40

St I 210 to 280

9800 to 28000

6

BocquetVédrine, 1961 Boschma, 1931 George, 1959

McMumch, 1917; NilssonCantell, 1921; Reinhard, 1942b, 1946

EGG PRODUCTION IN CIRRIPEDES

Species

Place

Breeding season

Incubation time, days

Sizes (L×B), Number of µm eggs per brood

Number of broods per year

References

Peltogaster sulcatus

–

–

–

St I 250

–

–

Peltogaster ella gracilis Hokkaido, Japan

Bering Sea

–

–

St I L=B

–

–

NilssonCantell, 1921 Reischman, 1959

–

–

Small ova 140 to 150 Large ova 158 to 160 Small egg 140 to 150 Large egg 160 to 170 Small St I 220 to 245 Large St I 255 to 275

–

–

Yanagimach i, 1961a

Peltogastere lla socialis

–

–

St I 207 to 247

–

–

Japan

N.W. Pacific, Alaska, Bering Sea –

About 14

–

–

–

Peltogastere lla subterminali s Sacculina carcini

N.W. Pacific, Alaska California Millport, Scotland

–

–

St I (190 to 216) ×(126 to 148)

–

Shirase & Yanagimac hi, 1957 –

–

–

–

–

R.Mersey, England Plymouth, England Roscoff, France

Mar–May

Max Aug– Dec Min Jan– Mar –

–

–

–

Day, 1935

Mar–Oct

–

–

–

–

Orton, 1936

June–Aug

28 to 35

–

2+

Delage, 1884

France

–

–

ova 80×60 egg 180×150 egg 120×100

–

–

Isefjord, Denmark

July–Oct

12 to 18

100 000 to 300 000

6

BocquetVédrine, 1964 Lützen, 1984

–

Reinhard, 1944

Reinhard, 1944

Foxon, 1940; Heath, 1971

69

70

MARGARET BARNES

–

–

–

egg 70×54

–

–

–

–

–

–

–

Sacculina micracantha Sacculina papposa Sacculina rotundata Madras, India Sacculina setosa Sacculina sulcata Sacculina sp.

Siboga Exped. Siboga Exped. Siboga Exped. –

–

St I 165×120 –

Krüger, 1940 Hoek, 1909

egg 134

–

–

–

–

egg 100

–

–

–

–

egg 100

–

–

–

–

–

–

St I 215×135 –

egg 100

–

George, 1949 –

–

–

egg 110

–

–

July–Jan

–

–

(75 ova in 2 mm adult)

–

–

–

–

–

–

Black Sea

–

9

Sylon challengir* Sylon hippolytes* Sylon schneideri Thompsonia cubensis

–

–

–

egg 182×129 egg 170×125 St I 190×135 ova 60

–

–

Hoek, 1888

–

–

–

–

1 per life

–

–

–

ova 80×60

20 000 to 200 000 –

Lützen, 1981 Hoek, 1888

Cuba

–

–

ova 95×80 egg 85+

–

–

Thompsonia sp.* Thompsonia sp.*

Great Barrier Reef –

–

–

egg 34

–

–

–

–

cyp 200

–

–

Triangulus galatheae Bocquetia rosea

–

–

–

–

–

S California

–

–

St I 257×100 cyp 88.9±1. 89

–

–

Septodiscus flabellum Septosaccus cuenoti

Siboga Exped. Siboga Exped. Mutsu Bay, Japan

–

Boschma, 1931 Boschma, 1931 Boschma, 1931

Boschma, 1931 Boschma, 1931 Elston, Wilkinson & Burge, 1985; Krüger, 1940 Codreanu, 1959

Reinhard & Stewart, 1956 Potts, 1915 BocquetVédrine, 1961 Veillet, 1945 Pawlik, 1987

EGG PRODUCTION IN CIRRIPEDES

71

available. The difference in size between the mature egg and nauplius stage I is very little. Egg size may also increase during embryogenesis and in many cases the stage of development is not recorded. Veillet’s (1943a, 1945) suggestion that the sex of a cyprid is determined at the egg stage was confirmed by Yanagimachi (1961a,b) who went further and said it was determined in the ovary. Yanagimachi (1960) also considered the chromosome numbers of large and small eggs. Ritchie & Høeg (1981) confirmed the earlier work on Peltogaster paguri and Sacculina senta (Ichikawa & Yanagimachi, 1960) and Peltogasterella gracilis (Yanagimachi, 1961b) that two sizes of eggs may be produced but added that Lernaeodiscus porcellanae does not always produce broods of one sex. They found that the sex of the broods varied with season and that during the transition broods of mixed sex could be produced. Broods were predominantly female in summer and male in winter—these males being necessary for virgin externae that would emerge in the following spring. In Sacculina carcini (Høeg, 1984) some broods contain only one sex while others may be mixed. Høeg suggests that this may reflect the nutritional state of the externa which may itself be determined by the condition of the host. It may also be that in summer broods are laid in quick succession thus tending to deplete the nutritional reserves of the externa. Walker (1985, 1987) has examined nauplii from broods of S. carcini taken at different times of the year; he also found a progressive change from male to female cyprids from spring to summer reverting to males again from autumn to winter. The sizes of externae are difficult to measure because of their shape and because they may be distended when carrying eggs in the mantle cavity—the amount of distension depending on the stage of development of the embryos (Reinhard, 1942b). The size of the externa at maturity may vary according to species or the number of externae per host. According to Boschma (1931) Peltogasterella socialis externae 5–7 mm long have eggs in the mantle cavity whereas Sacculina carcini externae do not carry eggs until they are between 10 and 12 mm long (Foxon, 1940; Heath, 1971). In Sylon hippolytes size at maturity may be 10.4, 8.4 or 7.2 mm long depending on whether the host is carrying 1, 2 or 4 externae (Lützen, 1981). In Peltogaster paguri (Reinhard, 1942b) externae of 6 mm length may be mature on small hosts; on larger hosts they are not mature until 9 mm long. Season of maturity can also vary depending on temperature or even the life cycle of the host (Foxon, 1940). Some species have peak periods of reproduction during the summer and a rest period in the winter, such as Sacculina carcini in Denmark (Lützen, 1984), although further south the reproductive period of this species may be longer (Delage, 1884; Day, 1935; Orton, 1936; Foxon, 1940; Heath, 1971; Walker, 1987). Some species may be continuously reproductive, such as Lernaeodiscus porcellanae (Ritchie & Høeg, 1981), with incubation time depending on temperature and the time of year. In some species the externa may produce only one brood and then die and fall off the host, such as in Sylon hippolytes (Lützen, 1981). Results are not expressed in the same way in the literature (see Table I) which often makes comparisons difficult. Day (1935) suggested that reproduction in Sacculina carcini is synchronised with that of its host so that food reserves normally used by the host can be appropriated by the parasite. While this is possible it is known (Heath & Barnes, 1970) that the ovaries of the host, Carcinus maenas, show their greatest increase in size in the summer and yet nauplii and cyprids of Sacculina carcini are most abundant in winter (Pyefinch, 1948). It is suggested, therefore, that it is the developing interna, rather than the externa, which requires optimal conditions for growth. Furthermore, as rhizocephalan nauplii are lecithotrophic their release at a time when there is little plankton in the winter, far from being disadvantageous, may be an advantage in reduced losses due to predation (Heath, 1971). The number of eggs found in the mantle cavity after each ovulation depends on the size of the externa and some authors are careful to give this. The most comprehensive account is that of Lützen (1981; see Table II) for Sylon hippolytes. Numbers of eggs found in other species are given in Table I. It is of interest

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MARGARET BARNES

that Peltogaster paguri of externa size 12 mm produces 28 000 eggs per brood which, allowing say 6 broods per summer (Reinhard, 1942b) would be 168 000 eggs per year (life?). From Table II it can be seen that Sylon hippolytes of the same size externa produces 120 000–152 700 eggs in its one brood per life. The former species releases the embryo as a nauplius and the latter as a cyprid. It has been said that species releasing as cyprids may not produce as many offspring as those releasing as nauplii (Høeg, 1982). This is hardly borne out by this result. A better comparison may be with Lernaeodiscus porcellanae which releases as a nauplius and may produce several hundred to 20 000 eggs per brood (Ritchie & Høeg, 1981). In summer this can be every 10–14 days but will obviously be less frequent at lower winter temperatures. If one assumes a brood say every month on the average (it breeds continuously according to Ritchie & Høeg, 1981) then it will produce up to 240 000 eggs per year which is more than in Sylon hippolytes and may be an under-estimate. At two more broods per year the number would increase to 280 000; in this case there are many more nauplii produced than cyprids. The merits of releasing young as nauplii or cyprids is discussed by Høeg (1982). Dispersion of the species is ensured by nauplii which remain swimming in the water for several days. Reduction of the planktonic stages, and consequently of the length of planktonic life, reduces the risk of predation and increases the chance of survival. In species such as Clistosaccus paguri, TABLE II Number of eggs in six externae of Sylon hippolytes calculated from serial sections (Lützen, 1981) Size of externae (mm)

Approximate

Length

Width

Height

number eggs

5.9 7.6 8.1 10.0 14.0 15.2

3.4 5.2 6.1 8.3 10.0 9.6

4.1 4.8 4.9 7.0 5.0 9.0

18 900 75 500 105 900 120 000 152 700 203 300

producing only one or very few broods during the lifetime of an externa, the lack of dispersion and greater chance of survival ensures that the cyprids remain in the vicinity of the hermit crab (host) population. ACROTHORACICA Cirripedes of the order Acrothoracica are burrowing and non-parasitic. They have a soft mantle without calcareous plates and are dioecious with dwarf males. The order is predominantly found in warm-temperate regions and generally in shallow (30 m) water (Ross & Newman, 1969; Zullo, 1979). Newman & Ross (1971) have, however, reported a species, Australophialus tomlinsoni, from depths of 300–600 m south of the Antarctic Convergence and two truly deep-water species have been found: Weltneria hessleri at 1000 m depth off Bermuda (Newman, 1971) and W. exargilla in the Bay of Biscay at a depth of 1500 m (Newman, 1974). The females live in self-excavated burrows usually in limestone, coral skeletons or mollusc shells. W. exargilla is different in that it burrows into soft clayey siliceous substrata which are fairly low in calcium carbonate. The tiny males are generally found near the aperture of the females in a pocket of the mantle tissue near the area of the ovary. In some species the males are, however, found attached to the wall of the

EGG PRODUCTION IN CIRRIPEDES

73

burrow. The acrothoracids so far described are small usually being only a few millimetres long (see Table III). The females are about ten times longer than the males. The burrows of these soft bodied barnacles provide them with the protection necessary because of the absence of a calcareous shell. The burrow is largely formed by abrasion caused by chitinous teeth on the mantle surface of the female although a cyprid larva settling on a suitable substratum has no mantle teeth suggesting that the initial penetration may be by chemical dissolution (Tomlinson, 1969). Indeed Turquier (1968) mentioned the use of carbonic anhydrase in burrowing by Trypetesa nassarioides and Tomlinson (1973) found papillae on the external surface of the mantle. In view of Turquier’s work Tomlinson suggested that these papillae might be secreting carbonic anhydrase or other alkaline phosphatase and that teeth, developed later, act only after enzymatic softening of the substratum. A chemical action combined with abrasion was also mentioned by Kamens (1981). The attachment area of the female is a major structural element within the burrow and allows movement of the mantle. As the mantle is cemented to the burrow wall at this point it cannot moult in this area and remains of exuxiae build up forming a horny disk (see Grygier & Newman, 1985, for a discussion about this disk). Dwarf males may be attached to the margins of this disk embedded in the mantle of the female, e.g. in Alcippe lampas (=Trypetesa lampas) (Berndt, 1903), Lithoglyptes indiens and Trypetesa lateralis (Tomlinson, 1969), and Cryptophialus melampygos (Batham & Tomlinson, 1965). In some cases the males may be attached to the burrow walls, e.g. in Berndtia purpurea (Utinomi, 1950). There are also cases where males have been found attached to either the mantle of the female or the burrow wall, such as in Kochlorine floridana (Wells & Tomlinson, 1966), Berndtia nodosa (Tomlinson, 1967), and Cryptophialus coronophorus (Smyth, 1986). Utinomi (1964) failed to find any males in his study of Tryptesa habei; they TABLE III Acrothoracica: comparison of size of adult female and size and number of young, cyp=cyprid;? stage doubtful Species Trypetesidae Trypetesa lateralis Cryptophialidae Australophialus pecorus Cryptophialus melampygos 2.5 2.6 2.7 Cryptophialus minutus Lithoglyptidae Berndtia purpurea 2.2 2.4 2.5

Size of young (L×B), Size of female (L×B), Number of young per µm mm brood

References

1.2–1.3 also 2.0

egg 250×180 cyp 500×200

180

Tomlinson, 1953, 1955

largest 1.28×0.82

egg 220 to 270 cyp 350 to 370 egg 260×210

1 to 8

Turquier, 1985a

14

Batham & Tomlinson, 1965

19 to 60

Darwin, 1854

14

Utinomi, 1961

1.9 to 2.1 cyp 530×230

largest 2.54

1.7 cyp 470×260

33 44 31 egg 254 cyp 229 to 406 egg 320 65 21 85

74

MARGARET BARNES

Species 2.6 2.6 2.7 Lithoglyptes tectoscrobis Weltneria exargilla

Size of young (L×B), Size of female (L×B), Number of young per µm mm brood

5.7×(1.7 to 1.0) (6.2 to 11.4)× (1.5 to 3.9)

64 57 32 egg 180×130 St? 510×370

1200 estimated by volume About 150

References

Grygier & Newman, 1985 Newman, 1974

may have been lost during the preparation of the material for examination. The size of the adult male is less than that of the cyprid from which it develops. The adult male lacks feeding appendages and the maturation process is at the expense of larval food reserves. Mature males consist mainly of reproductive material and its maturation must be synchronised in some way with that of the female if fertilisation is to be effective. Kühnert (1934) raised larvae of Alcippe lampas (=Trypetesa lampas) in separate vessels and found that males would differentiate without the presence of females but would not become fully mature. Tomlinson (1969) suggests that part of the maturation of the male may be under the chemical influence of the female. The partial burial of the male in the female’s mantle tissue near the ovary may be significant in this respect. The dwarf males in acrothoracids usually have a penis although it has not been seen in some species and is even reported as absent in others. In some species of Lithoglyptes the presence or absence has not been resolved but according to Tomlinson (1969) it is usually present. He was, however, not sure of it in L. hirsutus (Turquier, 1963). Newman (1971) did not find a penis in Weltneria hessleri; this may have been due to the immaturity of the males examined as Tomlinson (1969) suspected in Cryptophialus heterodontus. In Kochlorine ulula and Cryptophialus rossi a penis could not be positively identified by Tomlinson (1973), but he did find a penis sheath in the latter species. In C. epacrus a penis was found. In Kochlorine floridana the penis matures late in life (Wells & Tomlinson, 1966) whereas in K. bocqueti Turquier (1977) found none. In Trypetesa lateralis (Turquier, 1967c) the male is greatly reduced and there is no penis sheath. There is only a short penis in T. nassarioides (Turquier, 1967c, 1971) and Utinomi (1964) records its presence as doubtful in T. habei but a penis sheath is present. The sheath is formed from the mantle of the male (Smyth, 1986). The spermatozoa of T. lateralis can be activated by sea water, the length of time depending on the salinity of the water (Tomlinson, 1969). Turquier & Pochon-Masson (1969) made an ultrastructural study of spermiogenesis in T. nassarioides and showed it to be normal for cirripedes; dwarf males have a true fertilising role. The method of insemination needs further study as details of copulation and fertilisation have not been described in detail for any acrothoracid (Tomlinson, 1960, 1969; but see also Turquier, 1977). By whatever method involved the mature ova and sperm come into contact and fertilised egg masses are found in the mantle cavity of the females. During its life a female may be served by a few or many males. In Kochlorine floridana Wells & Tomlinson (1966) found up to five relatively short-lived dwarf males per female. In Australophialus turbonis there may be about 12 per female; up to 17 have been seen (Tomlinson, 1969). In Cryptophialus zulloi Tomlinson (1973) found remains of at least 27 males in the area of the attachment disk of the female. According to Turquier (1972a) the dwarf males of Trypetesa, in spite of their short life, are able to fertilise several egg masses. When their testes are empty they may be replaced by other males. In Cryptophialus melampygos old spent males are replaced during the life of the female (Batham & Tomlinson, 1965).

EGG PRODUCTION IN CIRRIPEDES

75

The ovaries lie on the dorsal side of the female under the thickened layer of the disk and the oviducts each end in an atrium which opens into the mantle cavity. Ovaries and oviducts have been described in Alcippe lampas by Berndt (1903) and in Berndtia purpurea by Utinomi (1950, 1960). The fertilised ova (eggs) remain in the mantle cavity of the female as two separate egg lamellae until hatching takes place (Tomlinson, 1969). Embryonic development takes place within the egg case. As early as 1849 Hancock reported seeing eggs in the mantle cavity of Alcippe lampas and watched the hatching of swimming nauplii. Whether the embryos eventually hatch as nauplii or cyprid larvae varies in different genera. In the Crytophialidae where hatching has been described it is usually as a cyprid. Although in Australophialus turbonis (Tomlinson, 1969) whether the nauplius is free-swimming or retained to the cyprid stage is not known; in A. pecorus the larvae is liberated as a cyprid (Turquier, 1985a). In the Lithoglyptidae species of the genus Weltneria are released as cyprids (Tomlinson, 1969: W. spinosa, W. reticulata; Turquier, 1985a: W. zibrowii; Newman, 1974: W. exargilla). Balanodytes taiwanus (Tomlinson, 1969) also hatches as a cyprid. In species of the genera Berndtia and Lithoglyptes so far described the embryos, however, hatch as nauplii (Tomlinson, 1969: L. indicuns, L. mitis, L. scamborachis; Utinomi, 1950, 1960: Berndtia purpurea). In the Trypetesidae some members of the same genus may hatch as nauplii (Berndt, 1903; Genthe, 1905; Kühnert, 1934: Alcippe (=Trypetesa) lampas; Turquier, 1976b: Trypetesa nassarioides) whereas T. lateralis hatches as a cyprid (Tomlinson, 1955). Whether hatched as nauplii, which develop through four stages (Visscher, 1938, says six in Alcippe lampas but this has never been confirmed by other workers) or cyprids the larvae are expelled from the female by contractions of the mantle cavity as seen by Genthe (1905) in Alcippe (=Trypetesa) lampas. The cyprids may be free swimming or have reduced swimming appendages as, for example in Cryptophialus melampygos (Batham & Tomlinson, 1965). In this species the cyprids can only crawl and so are splashed about in the sea water and as