Chloroplast symbiosis and the extent to which

Transcription

1 Biol. J. Linn. Sac.. 6: With 1 plate December 1974 Chloroplast symbiosis and the extent to which it occurs in Sacoglossa (Gastropoda: Mollusca) ROSALIND HINDEt AND D. C. SMITHS Department of Agricultural Science, Ox ford Accepted for publication June I974 Criteria for defining chloroplast symbiosis are outlined. Investigations are described into the presence or absence of chloroplast symbiosis in Limapontia capitata, pale and dark forms of L. depressa, Alderia modesta and Elysia viridis. Apart from E. viridis, only pale forms of L. depressa showed signs of the phenomenon. All sacoglossans hitherto shown definitely to possess chloroplast symbiosis have the twin characteristics of being elysioid and having siphonaceous algae as the source of chloroplasts. It is therefore suggested that the phenomenon may not be very widespread in the order, probably occurring in less than half of all species. CONTENTS Introduction 349 Material Limapontia capitata (Miiller) Limapontia depressa (Alder & Hancock) Alderia modesta (Loved Elysia viridis (Montagu) Starvation experiments Methods Measurement of C fixation in light and dark Extraction and analysis of animals Paper chromatography Results Limapontia depressa: pale forms 352 Limapontia depressa: dark forms Limapontia capitata AIden a modesta Elysia viridis Heterotrophic carbon fixation in Limapontia spp,and Alderia modesta Discussion Acknowledgements References INTRODUCTION In certain sacoglossans, the occurrence of chloroplasts which remain functional for appreciable periods of time is now well documented (Greene, 197a, b, c; Taylor, 197; Trench, Boyle & Smith, 1973; Muscatine & Greene, t Present address: School of Biological Sciences, University of Sydney, Australia 26 $ Present address: Dept of Botany, The University, Woodland Rd, Bristol, England. 349

2 3 5 R. HINDE AND D. C. SMITH 1973). There has been consequent speculation that chloroplast symbiosis may be nearly universal in these molluscs (Greene, 197a). This article attempts to clarify the extent to which the phenomenon probably occurs in sacoglossans, and describes relevant observations on some British species. The term chloroplast symbiosis will be defined as the situation where an animal takes up and retains free, undamaged chloroplasts under natural conditions, shows active photosynthesis for at least several days after removal from its host plant, and utilizes products of photosynthesis released from the chloroplasts. Active photosynthesis implies significantly higher rates of carbon fixation in light than in the dark, detectable synthesis of intermediates and products of the photosynthetic carbon fixation pathway, and oxygen evolution. Situations where active photosynthesis does not occur, and where the animal retains only damaged chloroplasts, chloroplast fragments or chloroplast pigments cannot be properly described as symbiotic. Thus defined, there is positive evidence for chloroplast symbiosis in Elysia viridis (Trench et al., 1973), E. hedgpethi (Greene & Muscatine, 19721, Tridachia crispata (Trench, 1969; Trench, Greene 8~ Bystrom, 1969), Placobranchus ianthobapsus (Trench et al., 1969; Greene, 197c) and Tridachiella diomedea (Trench et al., 1969). Probably, it also occurs in Elysia atroviridis (Kawaguti & Yamasu, 1965) and Placobranchus ocellatus (Kawaguti, 1941). The chloroplasts all come from algae of the order Siphonales and occur inside animal cells. Chloroplasts from these algae are remarkably robust; for example, Giles &? Sarafis (1972) found that those of Caulerpa sedoides still showed photosynthetic activity after 27 days incubation in hen s eggs. Chloroplast symbiosis could not be detected in Hermaeina smithi (Greene, 197a), Berthellina chloris and Oxynoe antillarum (Muscatine & Greene, 1974). The evidence is doubtful or confused for Hermaea dendritica (Greene, 197a; Muscatine & Greene, 197 3; Greene & Muscatine, 1972), H. bifida (Taylor, 1967) and Oxynoe panamensis (Muscatine & Greene, 1973). Muscatine & Greene suggested that chloroplast symbiosis is only shown by those Sacoglossa which are considered to be evolutionarily the most advanced, the elysioid forms. Since some eolidiform types feed on Siphonales but do not show chloroplast symbiosis they believed that the animal, rather than the plant, is important in the evolution of the symbiosis. Cerata-bearing forms were considered intermediate since they present some of the examples about which the evidence is equivocal (e.g. Hermaea sp.). Amongst British sacoglossans, chloroplast symbiosis has been conclusively demonstrated hitherto only in Elysia viridis. Observations are presented here on two distinct types of Limapontia depressa, L. capitata and Alderia rnodesta. Lirnapontia sp. are elysioid, and A. modesta bears cerata. For comparison, previously unpublished observations on E. viridis are also given. MATERIAL Limapontia capitata (Muller) This was collected from St. Mary s Island, Northumberland, in June 1973, from Cladophora arcta and C. rupestris, and occasionally from Enterornorpha sp. However, it probably feeds on Cladophora sp. (Gascoigne, 1952). In the

3 CHLOROPLAST SYMBIOSIS 35 1 laboratory, it was kept in beakers of aerated sea water with some Cladophora at 7-8 C with 15 foot candles illumination on a 12-hour light/l2-hour dark cycle. Limapontia depressa (Alder & Hancock): pale and dark forrrls This occurs in nature in two forms (Gascoigne, 1956). The pale form is larger and is transparent creamy yellow with obvious green gut diverticula. The smaller dark form has a superficial layer of dark pigment which usually obscures the diverticula completely. Intermediate forms with uneven pigmentation are sometimes seen. Both forms were collected on saltings at Pagham Harbour, Sussex in April and June They were found in areas where a mat of Vaucheria, often with some Enteromorpha sp., was growing on mud in the shelter of small bushes. Dark forms were also seen at Langstone Harbour, Hampshire, in an area where there was Ulva, Enteromorpha and Cladophora but not Vaucheria. In the laboratory, both forms were kept in getri dishes on filter paper moistened with 5% (v/v) sea water at 7-8 C with 15 foot candles illumination on a 12-hour lightll2-hour dark cycle. Alderia modesta (Loven) This was collected from saltings at Pagham Harbour from the same Vaucheria mats as Limapontia depressa in April 1973, and kept under similar conditions in the laboratory. Elysia viridis (Montagu) This was collected from Bembridge, Isle of Wight in June 1972, from Codium fragile, and maintained in aerated sea water as described by Hinde & Smith (1972), except that the temperature was 7 43 C. Starvation experiments Animals were kept in the same conditions as described above but without the appropriate food plant. Additional techniques for E. viridis are described by Hinde & Smith (1972). METHODS Measurement of I4C fixation in light and dark Samples of animals were incubated in either light (2 foot candles) or dark in one inch diameter glass specimen tubes illuminated from below in media containing NaH 4C,, For Limapontia depressa, 1-25 animals/sample incubated in.2 ml 5% (v/v) sea water containing 1 pci for 2 h at 12.5 ; L. capitata, 3 animals/sample in 1. ml sea water with 1 pci for 2 h at 15.5 ; Alderia modestn, 1 animalshample, conditions otherwise as for L. depressa; Elysia viridis 3 animalshample for 1 h in 3 ml sea water containing 2 pci at 18.

4 352 R. HlNDE AND D. C. SMITH Ex traction and analysis of animals At the end of the incubation period, samples were killed in hot absolute methanol (5 ml per sample) and immediately placed in a refrigerator in the methanol. After 3 h, chlorophyll in the methanol extract was determined by the method of Arnon (1949). Samples were then extracted in 5 ml 5% (NH4),S4 for 1 h at loo, followed by 5 ml M KOH at 1 until they dissolved. Samples of the incubation medium and three extracts were acidified and evaporated down. Scintillant (butyl-pbd,.7 g; toluene, 5 ml; methanol, 5 ml) was added and I4C counted in a Beckman liquid scintillation counter. Paper chromatography The pattern of distribution of fixed I4C in the methanol extract was determined by making autoradiographs of two-dimensional chromatograms developed in phenol-water and butanol-propionic acid-water solvents according to the method of Bassham & Calvin (1957). RESULTS For each species measurements were made of fresh weight, chlorophyll content and the patterns and relative rates of I4C fixation in light and dark. The ratio of chlorophyll a to b was also measured since Vauchen a lacks chlorophyll b whereas it is present in normal amounts in the other food plants. Changes over a period of starvation in pale forms of Limapontia depressa, L. capitata and Elysia viridis were also measured. Results for all species are consolidated into Table 1. Limapontia depressa: pale forms These showed definite evidence of chloroplast symbiosis. Animals had significantly higher rates of 14C fixation in the light than in the dark, and much more I4C was incorporated into glucose in the light (Plate la, B). After 14 days starvation, fixation rates were only 7% higher in light than in dark, but patterns of fixation still differed (Plate lc, D). The very small losses in weight and chlorophyll content during starvation also indicate symbiosis of some duration. The absence of chlorophyll b in the June collection is evidence for the presence of Vaucheria chloroplasts. The presence of some chlorophyll b in the April collection indicates that either the animals had been feeding to a certain extent on algae other than Vaucheria. or that some Vaucheria chloroplasts had been digested and some chlorophyll a degraded to chlorophyll b. Limapontia depressa: dark forms Chlorophyll contents were lower than in pale forms, and some chlorophyll b was always present. Rates of I4C fixation were not significantly higher in light than dark; indeed, the rate of light fixation for the June collection seemed abnormally low, and it is possible that some animals may have died during the

5 Table 1. Presence and absence of chloroplast symbiosis in British sacoglossans Species Limapontia depressa April "pale" June forms "dark" April forms June Intermediates of Chlorophyll ratio I4C fixation ( lo6 counts/min/g photosynthetic 14 Month of Days of Mean wt per content (mg/g Chlorophyll a animal) C fixation collection starvation animal (mg) animal) Chlorophyll b Light Dark detected in light (no - b ) (no b) Limapontia aapitata June Alder& modesta April Elysia viridis June Results in Table adjusted for time and specific activity to be comparable to those for Limapontia depressa and Alderia rnodesta. Intermediates of photosynthetic fixation detected by two-dimensional chromatography of ethanol extracts (cf. Plate 1)

6 354 R. HlNDE AND D. C. SMITH incubation period. No differences could be detected between the pattern of light and dark fixation. The absence of photosynthesis in dark forms could well be due principally to shading of chloroplasts by the animals dark brown pigment (it is rarely possible to see the outline of the gut at all). L imapon t ia capita ta. The chlorophyll content of this species was initially high, but declined rapidly during seven days starvation; there was also a substantial loss of weight during starvation. No differences could be detected in either rates or patterns of 14C fixation, so providing conclusive proof that there was no chloroplast symbiosis. The absence of photosynthesis in L. capitata might be expected since the chloroplasts of Cladophora upon which it feeds are large and reticulate. However, Greene (1 97a) reports that fragments of Cladophora and Chaetornorpha chloroplasts were capable of photosynthetic 14C fixation, and Taylor (1967) observed intact plastids by electron microscopy in L. capitata, implying that the animal might be capable of photosynthesis. Alderia modesta Although A. modesta was collected from Vaucheria mats and appeared to browse on Vaucheria in the laboratory, the chlorophyll content and the ratio a : b was always low. If it fed on Vaucheria rather than on other algae intermingled in the mats, then chlorophyll degradation was presumably rapid. No photosynthesis could be detected. Elysia viridis The results in Table 1 illustrate the-strong photosynthetic ability of this animal. The rise in rate of 14C fixation per g animal tissue after 2 days starvation is probably due to the fact that chloroplast-containing tissues are I depleted less rapidly than others (Hinde & Smith, 1972). Hinde & Smith could even detect photosynthetic activity in an animal starved for three months. Heterotrophic carbon fixation in Limapontia sp. and Alderia modesta After two hours dark fixation, L. depressa (both forms), L. capitata and A. modesta all incorporated 14C into a wide range of compounds, including a number of amino-acids (especially aspartic and glutamic), and organic acids as well as other compounds. The pattern for all three species was similar to that for pale L. depressa shown in Fig. 1B and D. As compared with vertebrates, several molluscan species have shown unusually high rates of heterotrophic fixation of C2 into amino acids, and these rates have been correlated with high levels of phosphoenolpyruvate carboxylase (Simpson & Awapara, 1964)-it may be significant that the highest activities of the enzyme were found in Rungiu cuneatu, a bivalve, which, like L. depressa and A. modesta, lives in brackish water. C2 fixed by molluscs may eventually be incorporated into glycogen by a pathway including several

7 CHLOROPLAST SYMBIOSIS 355 organic acids, PEP and glucose (Goddard & Martin, 1966). Since the products of dark fixation by some molluscs may be much more numerous than for most green plants, the pattern of 14C fixation by a sacoglossan in the light could well give the misleading impression of the presence of photosynthetic activity. Only where there are clear differences between the light and dark pattern can it safely be assumed that photosynthesis occurs. DISCUSSION Apart from Elysia viridis, only pale forms of Limcpontia depressa showed signs of chloroplast symbiosis and even with these, chlorophyll content and ability to maintain high photosynthestic rates during starvation were substantially less than in E. viridis. No direct proof for the utilization of products of photosynthesis by pale L. depressa was obtained, but the negligible loss in weight and chlorophyll during 14 days starvation is presumptive evidence. It is not known if the chloroplasts in pale L. depressa are intra- or extracellular. All other sacoglossans previously shown to exhibit chloroplast symbiosis feed on members of the Siphonales which are known to have robust chloroplasts. Limapontia depressa is different in presumably obtaining chloroplasts from Vaucheria, a member of the Xanthophyta. Gallop (unpubl.) has found that Vaucheria chloroplasts isolated into a simple mineral medium can still fix carbon photosynthetically, but so far there has been no systematic investigation to see if Vaucheria chloroplasts show the same toughness after isolation as do those of Codium and Caulerpa sp. The only other alga shown to have robust chloroplasts is Acetabularia (Shephard, Levin & Bidwell, 1968), a member of the Dasycladiales but like Vaucheria and members of the Siphonales, of siphonaceous habit. The possibility thus arises that robust chloroplasts might be a general characteristic of algae of siphonaceous habit rather than those of a particular taxonomic class. The negative results with other animals investigated raises the question of the true extent of chloroplast symbiosis in Sacoglossa. All species shown to be symbiotic have the twin characteristics of being elysioid and having host plants of siphonaceous habit. In a survey of 38 sacoglossan species, Greene (197b) found just under two thirds fed on siphonaceous algae. Since no eolidiform or cerata-bearing types have been shown unequivocally to be symbiotic even when feeding on siphonaceous algae, the number of sacoglossans with chloroplast symbiosis may well be less than half of all species. The results described here also illustrate the care required to establish the existence of symbiosis: simple electron micrographic observation of the presence of intact plastids is not in itself proof of the occurrence of symbiosis. Because of the very active heterotrophic fixation shown by some molluscs, it is essential to demonstrate differences in both rate and pattern between light and dark fixation. Certainly, sacoglossans which are not elysioid or which do not feed on siphonaceous algae require careful investigation before concluding that they are symbiotic. It is tempting to suggest that Limapontia depressa represents an intermediate form in evolution since pale forms show signs of symbiosis whereas dark ones do not. However, the possibility that the two forms represent developmental stages of the same type cannot yet be excluded.

8 356 R. HINDE AND D. C. SMITH ACKNOWLEDGEMENTS We are most grateful to Dr Tom Gascoigne for his help and advice in collecting Limapontia spp. and Alderia modesta. and to Mrs Northover of Bembridge in collecting EZysia viridis. The assistance of Mrs Angela Gallop in some experiments is gratefully acknowledged. The work was supported by the Science Research Council. REFERENCES ARNON, D. I., Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Eefa vulgaris. PI. Phpiol., 24: BASSHAM, J. A. & CALVIN, M., The path ofcarbon in phorosynrhesis. New Jersey: Prentice-Hall. WSCOIGNE, T., The distribution of Limaponfia capirata (Muller) and of L. depressa (Alder & Hancock) on the coasts of Northumberland and Durham. Proc. malac. SOC. Lond., 29: GASCOIGNE, T., Feeding and reproduction in the Limapontidae. Trans. R. SOC. Edin 43: GODDARD, C. K. & MARTIN, A. W., Carbohydrate metabolism. Chapter 9, In K. M. Wilbur & C. M. Yonge, (Eds), Physiology of Mollusca, II; London: Academic Press. GREENE, R. W., 197a. Symbiosis in sacoglosan opisthobranchs: symbiosis with algal chloroplasts. Malacologia, 1: GREENE, R. W., 197b. Symbiosis in sacoglosan opisthobranchs: translocation of photosynthestic products from chloroplast to host tissuemalacologia, 1: CREENE, R. W., 197c. Symbiosis in sacoglossan opisthobranchs: functional capacity of symbiotic chloroplasts. Mar. Eiol., 7: GREENE, R. W. & MUSCATINE, L., Symbiosis in sacoglosan opisthobranchs: photosynthetic products of animal-chloroplast associations. Mar. Eiol, 14: GILES, K. L. & SARAPIS, V., Chloroplast survival and division in wirro. Nature, New Eiol., 236: HINDE, R. T. & SMITH, D. C., Persistence of functional chloroplasts in Elysia viridis (Opisthobranchia, Sacoglossa). Nafure, New Eiol., 239: 3-1. KAWAGUTI, S., Study on the invertebrates associating unicellular algae. 1. Placobranchus ocellatus von Hasselt, a nudibranch. Palao trop. biol. Stn. Stud. 2: KAWAGUTI, S. & YAMASU, S., Electron microscopy on the symbiosis between an elysioid gastropod and chloroplasts of a green alga. Eiol. J. Okayama Uniw., IZ: MUSCATINE, L. & GREENE, R. W Chloroplasts and algae as symbionts in molluscs. Inf. Rev. Cyfd., 36: SIMPSON, J. W. & AWAPARA, J., Phosphoenolypyruvate carboxykinase activity in invertebrates. Comp. Eiochem. Physiol., 12: SHEPHARD, D. C., LEVIN, W. B. & BIDWELL, R. G. S., Normal photosynthesis in isolated chloroplasts. Biochem. biophys. Res. Commun., 32: TAYLOR, D. L., The occurrence and significance of endosymbiotic chloroplasts in the digestive glands of herbivorous opisthobranchs. J. Phycol, 3: TAYLOR, D. L., 197. Chloroplasts as symbiotic organelles. fnr. Rev. cyror, 27: TRENCH, R. K., Chloroplasts as functional endosymbionts in the mollusc Tridachia crispafa (Bergh), (Opisthobranchia, Sacoglossa). Nature, Lond., 222: TRENCH, R. K., BOYLE, J. ELIZABETH & SMITH, D. C., The association between chloroplasts of Codium jhgile and the mollusc Elysia viridis. I. Characteristics of isolated Codium chloroplasts. Prm. R. SOC. Lond., 184: TRENCH, R. K., GREENE, R. W. & BYSTROM, B. G., Chloroplasts as functional organelles in animal tissues. J. Cell Elol., 42: EXPLANATION OF PLATE PLATE 1 Autoradiogaphs of two-dimensional paper chromatograms of pale forms of Litnaponria depressa after two hours C fixation. Before starvation: A. light furation; B, dark fixation. After 14 days starvation: A, light fixation; B, dark fixation. 1. Alanine; 2, unknown; 3, glucose; 4, serine; 5, glutamic acid; 6, aspanic acid;, origin.

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