Biol. J. Linn. Sac.. 6: 3 4 9 - 3 5 6 . With 1 plate
December 1 9 7 4
“Chloroplast symbiosis” and the extent to which
it occurs in Sacoglossa (Gastropoda: Mollusca)
ROSALIND HINDEt
AND
D. C. SMITHS
Department of Agricultural Science, Oxford
Accepted for publication J u n e 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. I t is therefore suggested that t h e phenomenon
may not be very widespread in t h e order, probably occurring in less than half of all species.
CONTENTS
Introduction
. . . . . . . . . . . . . . . . . . .
Material
. . . . . . . . . . . . . . . . . . . .
Limapontia capitata (Miiller)
. . . . . . . . . . .
Limapontia depressa (Alder & Hancock)
. . . . . . . .
Alderia modesta ( L o v e d
. . . . . . . . . . . .
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 . . . . . . . . . .
. . . . . . . . .
Limapontia depressa: “dark” forms
Limapontia capitata
. . . . . . . . . . . . .
AIden’a modesta
. . . . . . . . . . . . . . .
Elysia viridis
. . . . . . . . . . . . . . . . .
Heterotrophic carbon fixation in Limapontia spp,and Alderia modesta
Discussion
. . . . . . . . . . . . . . . . . . .
Acknowledgements
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References . . . . . . . . . . . . . . . . . . . .
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INTRODUCTION
In certain sacoglossans, the occurrence of chloroplasts which remain
functional for appreciable periods of time is now well documented (Greene,
1970a, b, c; Taylor, 1970; Trench, Boyle & Smith, 1973; Muscatine & Greene,
t Present address: School of Biological Sciences, University of Sydney, Australia 2006
$ Present address: Dept of Botany, The University, Woodland Rd, Bristol, England.
349
3 50
R. HINDE AND D. C. SMITH
1973). There has been consequent speculation that “chloroplast symbiosis”
may be nearly universal in these molluscs (Greene, 1970a). This article
attempts t o 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, 1970c) 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, 1970a), Berthellina chloris and Oxynoe antillarum
(Muscatine & Greene, 1974). The evidence is doubtful or confused for Hermaea
dendritica (Greene, 1970a; 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
“CHLOROPLAST SYMBIOSIS”
35 1
laboratory, it was kept in beakers of aerated sea water with some Cladophora at
7”-8”C with 150 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 1973. 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 a t 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 50% (v/v) sea water at 7”-8 C with 150 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 (2000 foot candles) or
dark in one inch diameter glass specimen tubes illuminated from below in
media containing NaH’4C0,, For Limapontia depressa, 10-25 animals/sample
incubated in 0.2 ml 50% (v/v) sea water containing 10 pCi for 2 h at 12.5”; L.
capitata, 30 animals/sample in 1.0 ml sea water with 10 pCi for 2 h at 15.5” ;
Alderia modestn, 10 animalshample, conditions otherwise as for L. depressa;
Elysia viridis 3 animalshample for 1 h in 3 ml sea water containing 20 pCi at
18’.
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),S04 for 1 h at loo”, followed by 5 ml M KOH at 100” until they
dissolved. Samples of the incubation medium and three extracts were acidified
and evaporated down. Scintillant (butyl-PBD, 0.7 g; toluene, 50 ml; methanol,
50 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 14Cfixation 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 70% 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
June
Elysia
viridis
20
0
0
0
7
0
0
235.0
178.0
10.12
0.49
0.21
2.82
1.45
4.80
5.25
4.90
Mean wt per
animal (mg)
0.155
-
1 .5
1.92
2.10
0.88
0.77
2.76
1.67
14.5
2.7
(no b )
00
(no b )
7.19
0.055
0.56
0.09
0.13
0.29
0.52
0.59
ratio
Chlorophyll a
Chlorophyll b
57.9
73.5
2.25
3.59
5.7
7.2
1.96
3.55
3.6
3.7
1.3
1.95
2.55
4.6
0.6
2.2
10.9
24.2
+
+
+
+
+
Intermediates of
I4C fixation ( lo6 counts/min/g photosynthetic
14
C fixation
animal)
Light
Dark
detected in light
Results in Table adjusted for time and specific activity t o 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)
April
Alder&
modesta
April
June
14
0
0
April
June
June
forms
"dark"
"pale"
forms
Days of
starvation
Month of
collection
Limapontia
aapitata
Limapontia
depressa
Species
Chlorophyll
content (mg/g
animal)
Table 1. Presence and absence of chloroplast symbiosis in British sacoglossans
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 (1970a) 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 20 days
starvation is probably due to the fact that chloroplast-containing tissues are
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 C 0 2 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. C02 fixed by molluscs may
eventually be incorporated into glycogen by a pathway including several
I
“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. N o 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 (1970b)
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.
R. HINDE AND D. C. SMITH
356
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.
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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; 0, origin.