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Introduction
It has long been known that photosynthesis takes place in
chlorophyll-containing plants, algae and some bacteria and that it
serves to provide oxygen and energy in the form of biomass to
support heterotrophic life. Some animals (for the purposes of this
review we will address only the metazoans) in the phyla Mollusca
(giant clams, nudibranchs), Porifera (sponges), Cnidaria (corals,
anemones and hydra), Acoelomorpha (flatworms) and Chordata
(ascidians) have evolved mechanisms to capture photosynthetic
products through the formation of symbiotic associations with
intact unicellular algae or cyanobacteria (Fig.1 and supplementary
material TableS1 and references therein). In these cases, the
photobiont (alga or cyanobacterium) acts as an autonomous
photosynthetic factory, providing reduced carbon as a source of
energy to the heterotroph, often receiving nutrients in return.
Whereas initial events leading to primary photosynthetic
eukaryotes involved phagocytosis by a unicellular protist (see
below), the acquisition of photosynthetic symbionts by
multicellular animals poses multiple challenges. Among these are:
localization of the symbiont to differentiated tissues, regulation of
the symbiont environment, protein/metabolite transfer and
turnover, evasion or suppression of the host immune response and,
in order for the symbiosis to become permanent, transmission of
the symbiont to the germline (Venn et al., 2008; Raven et al., 2009).
The ability of animals to acquire photobionts appears limited to
aquatic environments and to the few phyla mentioned above. The
morphology of these multicellular organisms is an important factor
leading to such relationships. Photosynthetic animals tend to
exhibit simple morphologies (in many, only two cell layers)
coupled with a large surface area to volume ratio to accommodate
the organelles and transfer of photosynthates and other nutrients
(Venn et al., 2008) (see examples in Fig.1A–C). Other apparently
adaptive modifications are seen in the morphologically advanced
molluscs (bivalves, nudibranchs and sacoglossans), in which the
finely branching diverticula of the digestive tract achieves a similar
high surface area to volume ratio in the more complex tissue of
these hosts (see examples in Fig.1G and Fig.2) (Graves et al., 1979;
Norton et al., 1992; Venn et al., 2008). Within the host animal,
symbiotic algae and cyanobacteria are either compartmentalized
within a host membrane (e.g. symbiosomes in cnidarians) or
localized to a specific region (e.g. the heamal sinus of tridacnid
clams or the epidermal surface of certain ascidians). The symbionts
are also typically restricted to regions of the body cavity that enable
the most efficient light capture. Often, morphological adaptations
by the host facilitate symbiont light capture, compensating for the
loss of mobility characteristic of the algal symbionts (Trench,
1993). The membrane-bound compartment poses a greater
challenge to the transfer of materials between host and symbiont,
but conversely provides a more controlled environment. Numerous
studies have addressed the dynamics of metabolite and protein
exchange between host and symbiont; in many cases both partners
have evolved adaptations to support the relationship (reviewed by
Trench, 1993; Yellowlees et al., 2008; Venn et al., 2008). Whether
symbionts overcome or avoid innate immune responses of the host
(all photosynthetic animals studied to date have only innate
immunity) is still largely unknown, and the mechanisms are not
The Journal of Experimental Biology 214, 303-311
© 2011. Published by The Company of Biologists Ltd
doi:10.1242/jeb.046540
The making of a photosynthetic animal
Mary E. Rumpho1,*, Karen N. Pelletreau1, Ahmed Moustafa2 and Debashish Bhattacharya3
1Department of Molecular and Biomedical Sciences, 5735 Hitchner Hall, University of Maine, Orono, ME 04469, USA, 2Department
of Biology and Graduate Program in Biotechnology, American University in Cairo, New Cairo 11835, Egypt and 3Department of
Ecology, Evolution and Natural Resources, Institute of Marine and Coastal Sciences, Rutgers University, New Brunswick, NJ 08901,
USA
*Author for correspondence (mrumpho@umit.maine.edu)
Accepted 6 August 2010
Summary
Symbiotic animals containing green photobionts challenge the common perception that only plants are capable of capturing the
sun’s rays and converting them into biological energy through photoautotrophic CO2 fixation (photosynthesis). ‘Solar-powered’
sacoglossan molluscs, or sea slugs, have taken this type of symbiotic association one step further by solely harboring the
photosynthetic organelle, the plastid (chloroplast). One such sea slug, Elysia chlorotica, lives as a ‘plant’ when provided with
only light and air as a result of acquiring plastids during feeding on its algal prey Vaucheria litorea. The captured plastids
(kleptoplasts) are retained intracellularly in cells lining the digestive diverticula of the sea slug, a phenomenon sometimes referred
to as kleptoplasty. Photosynthesis by the plastids provides E. chlorotica with energy and fixed carbon for its entire lifespan of
~10months. The plastids are not transmitted vertically (i.e. are absent in eggs) and do not undergo division in the sea slug.
However, de novo protein synthesis continues, including plastid- and nuclear-encoded plastid-targeted proteins, despite the
apparent absence of algal nuclei. Here we discuss current data and provide hypotheses to explain how long-term photosynthetic
activity is maintained by the kleptoplasts. This fascinating ‘green animal’ provides a unique model to study the evolution of
photosynthesis in a multicellular heterotrophic organism.
Supplementary material available online at http://jeb.biologists.org/cgi/content/full/214/2/303/DC1
Key words: Elysia chlorotica, horizontal gene transfer, mollusc, photosynthesis, plastid, sea slug, symbiosis, Vaucheria litorea.
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understood. However, recent transcriptomic work studying
coral–algal symbioses has shown that gene expression of the host,
when infected by the appropriate symbiont, shows little or no
change over time (Voolstra et al., 2009). By contrast, when the host
is exposed to foreign symbionts (those incapable of establishing a
symbiosis) significant changes in the host transcriptome result and
the immune response is launched. This difference implies that
appropriate symbionts evade detection by the host and fail to elicit
recognition, rejection and immune responses such as apoptosis and
proteolysis (Voolstra et al., 2009). Further studies of the immune
response in other photosynthetic animals are needed to determine
whether similar mechanisms are employed across all of these
organisms. Transmission of photosynthetic symbionts is largely
horizontal, with each generation acquiring its photosynthetic
partner anew from the surrounding environment. Transmission to
the germline remains a final barrier towards a more permanent
photosynthetic animal.
In an even more unusual adaptation leading to photosynthesis by
animals, several sacoglossan molluscs (sea slugs), particularly in
the genus Elysia, have evolved the ability to retain only the
functional plastids from their algal prey (Fig.2 and supplementary
material TableS2 and references therein). The plastids are retained
intracellularly in cells lining the animal’s digestive diverticula and
remain photosynthetically active for varying lengths of time
depending upon the host–symbiont association (reviewed in
Rumpho et al., 2000). Reports on the presence of green pigment
and then green ‘animals’ in sacoglossans were made in the late
1800s (De Negri and De Negri, 1876; Brandt, 1883) [as reported
in Clark et al. (Clark et al., 1990)]. The discovery that the green
‘bodies’ were plastids was made in 1965 after microscopic
observations of Elysia atroviridis (Kawaguti and Yamasu, 1965).
Research publications over the next 10–20years, principally by
Trench (Trench, 1969; Trench et al., 1969; Trench et al., 1972;
Trench et al., 1973a; Trench et al., 1973b; Muscatine et al., 1975;
Trench, 1975), but also by Clark (Clark and Busacca, 1978; Clark
et al., 1990), Jensen (Jensen, 1986), Hinde (Hinde, 1980; Hinde and
Smith, 1972; Hinde and Smith, 1974) and others (Green and
Muscatine, 1972; Graves et al., 1979), greatly advanced our
understanding of functional kleptoplasty in sea slugs, in particular
at the ecological and physiological levels.
Many studies have employed a variety of methods to
characterize the duration of photosynthesis in these sacoglossans,
often generating conflicting results for many species
(supplementary material TableS2). These contradictions warrant
more thorough investigations of photosynthesis in situ.
Phylogenetic studies have been more helpful in elucidating the
evolution and distribution of the photosynthetic abilities of these
animals. Händeler et al. investigated the phylogeny of numerous
sacoglossans coupled with functional photosynthesis (Händeler et
al., 2009). Their work supports the evolution of short-term plastid
retention in sacoglossans once in a common ancestor of the
Plakobranchoidea, and the lack of plastid retention in members of
the other superfamilies (Oxynoacea and Limapontioidea).
Subsequently, at least four species of sacoglossans independently
evolved the ability for long-term plastid retention. Morphology of
these sacoglossans is thought to play a role in long-term plastid
retention; animals with wing-like parapodia are able to regulate
light exposure and have evolved the ability to maintain these
plastids for several months (Händeler et al., 2009) (Fig.2A–C). In
E. chlorotica, one of the four derived sacoglossans with parapodia
(Fig.2A), the captured plastids can serve as the sole energy source
for the animal for over 10months (Green et al., 2000; Rumpho et
al., 2006). These photosynthetic animals are closer to a permanent
symbiosis in that the organelle is intracellular and is often not
bound by a host-derived membrane. Control of the plastid
processes remains enigmatic (discussed below).
Evolution of plastids and photosynthetic organisms
Photosynthetic eukaryotes reflect an evolutionary history of close
physical contact between organisms leading to multiple
endosymbiotic events, gene transfer and the evolution of modern-
day mitochondria and plastids. Mitochondria owe their origin to
the uptake of an -proteobacterium (Gray et al., 2001), and
plastids to the subsequent uptake of a photosynthetic
cyanobacterium (Reyes-Prieto et al., 2007). These two key
primary endosymbioses led to free-living prokaryotes becoming
highly dependent upon their host nucleo-cytosol because genes
M. E. Rumpho and others
Fig.1. Examples of diverse photosynthetic animals with varied symbionts.
(A)Neopetrosia subtriangularis with Synechococcus (photo by Robert
Thacker); (B) Didemnum molle with Prochloron (inset) (photo by Euichi
Hirose); (C) Symsagittifera sp. with Tetraselmis symbionts [modified here
from fig.1 in Hooge and Tyler (Hooge and Tyler, 2008), with permission of
Magnolia Press]; (D) Cassiopea xamachana with Symbiodinium (photo by
Alan Verde); (E) green Hydra with Chlorella (photo by Thomas Bosch);
(F) confocal image of Fungia coral larva (blue) with red autofluorescent
Symbiodinium (photo by Virginia Weis); and (G) Tridacna spp. with
Symbiodinium (photo by Jesús Pineda with permission from Woods Hole
Oceanographic Institution).
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305Mollusc photosynthesis
were lost or transferred to the nucleus in the first examples of
massive intracellular gene transfer. The ancestral photosynthetic
organism gave rise to three primary lineages, the glaucophytes,
the rhodophytes (red algae) and the Chloroplastida or
Viridiplantae (green algae and land plants) (Bhattacharya and
Medlin, 1995; Falkowski et al., 2004; Adl et al., 2005) (see
evolutionary scheme in Fig.3). In turn, a diverse group of
secondary or ‘complex’ algae evolved following the engulfment
of a single-celled green or red alga (or both) by a heterotrophic,
eukaryotic host (McFadden, 2001). Genome modification again
occurred as a result of gene transfer and loss from the symbiont
plastid, but also as a result of the ‘merger’ of the two nuclei (host
and endosymbiont) (reviewed in Lane and Archibald, 2008). The
evolution of photosynthetic organisms did not stop here, however.
Serial secondary endosymbiosis, the replacement of the original
primary plastid by a different primary plastid, and tertiary
endosymbiosis, the engulfment of a secondarily derived plastid
by a heterotrophic or autotrophic eukaryotic host (Bhattacharya
and Nosenko, 2008), gave rise to additional algal diversification
and genome chimerism (Keeling, 2004; Yoon et al., 2005; Lane
and Archibald, 2008; Sanchez-Puerta and Delwiche, 2008). In
organisms that evolved through serial endosymbiosis, previously
transferred plastid genes would have been present in the host
nuclear genome, but could have also been replaced by plastid or
nuclear genes of the new symbiont. In the case of tertiary
endosymbiosis, genes required to support functions of the most
recently acquired plastid may have been present in the host from
a previous endosymbiotic partner, or they may also have been
transferred from the newest endosymbiont’s nuclear and plastid
genomes to the host’s nuclear genome (Lane and Archibald,
2008). Finally, recent data suggest that stramenopiles and other
chromalveolates share a cryptic green algal endosymbiont that
predates the canonical red algal capture that has also contributed
significantly (100s of genes) to their nuclear genome (Moustafa
et al., 2009). Therefore, the genomes of many chromalveolate
taxa (minimally) contain genes of red and green algal origin
derived from eukaryotic endosymbioses (Moustafa et al., 2009).
Evolution of kleptoplasty and photosynthesis in Elysia
To the extent that it has been studied, the sea slug Elysia chlorotica
Gould 1870 is specific for its algal prey, feeding on and acquiring
plastids with any success from only two Vaucheria species (V.
litorea and V. compacta) (West, 1979; West et al., 1984). Not only
is the association specific, but it is also obligate; the sea slug will
not complete metamorphosis and develop into an adult in the
absence of its algal prey and plastid uptake. Vaucheria is a member
of the stramenopiles that currently contain red algal secondary-
derived plastids. Biochemically, the alga is characterized by
chlorophylls a and c, vaucheriaxanthin accessory pigments, lipid
carbon reserves (Anderson, 2004; Lee, 2008) and high mannitol
levels (M.E.R., unpublished data). Its coenocytic (single-celled,
multi-nucleate) filaments are largely vacuolated and surrounded by
a thin cell wall. As a result of secondary plastid evolution, these
organelles are surrounded by four membranes in V. litorea
(Rumpho et al., 2001): the inner and outer envelopes, the periplastid
membrane (a remnant of the plasma membrane of the engulfed
alga) and the outermost plastid endoplasmic reticulum membrane
(Gibbs, 1993; Bhattacharya et al., 2004). Interestingly, the outer
Fig.2. Examples of photosynthetic
sacoglossans with varied times of chloroplast
retention. (A)Elysia chlorotica [reprinted with
permission (Rumpho et al., 2008)], (B) Elysia
crispata, (C) Plakobranchus ocellatus, (D)
Costasiella ocellifera, (E) Thuridilla gracilis, (F)
Costasiella kurishimae, (G) Alderia modesta,
(H) Lobiger viridis and (I) Oxynoe antillarum.
Photos in panels B, D, E, G and I were
provided with permission by Patrick Krug;
photos in panels C, F and H were provided
with permission by Heike Wägele.
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two membranes are not readily observed in the sea slug (Rumpho
et al., 2001) and this has potential implications for protein targeting,
but will not be discussed here.
Because E. chlorotica obtains its plastids from a eukaryote
containing plastids of secondary origin, the evolution of plastid
retention (kleptoplasty) and photosynthesis in the sea slug can be
explained in a parallel fashion to that of other tertiary-evolved
photosynthetic organisms encompassing both endosymbiosis and
potentially horizontal gene transfer (HGT; defined as the non-
sexual exchange of genetic material) as follows. (1) The
heterotrophic eukaryotic host and symbiont establish close,
prolonged physical contact; i.e. the host sea slug (E. chlorotica)
grazes on mats of the algal prey (V. litorea), sucking out the cellular
contents (including intact and broken plastids, mitochondria and
nuclei), which then slowly traverse the digestive gut. (2) The
symbiont is taken up by the host; i.e. algal plastids in the digestive
gut are phagocytosed by the digestive epithelial cells of the host
sea slug. (3) Genome modification occurs; i.e. gene transfer may
have occurred through HGT from the kleptoplasts in the epithelial
cells and from broken algal nuclei releasing DNA in the gut lumen
to the host nucleus. (4) New metabolic properties evolve as a result
of the kleptoplastic association; i.e. an animal (E. chlorotica) is able
to sustain itself solely by photoautotrophic CO2 fixation, as a plant.
There are two major differences to consider in this comparison.
First, the entire V. litorea algal cell, including intact nuclei, is not
intracellularly taken up into the host sea slug. Second, the
kleptoplastic association is not transmitted vertically. In the first
case, the absence of the algal nucleus in the sea slug cell has
significant implications for providing nuclear-encoded plastid
proteins on a long-term basis. In the second case, absence of
heritability is most likely due to a soma–germline barrier that could
prevent the movement of plastids into the invertebrate germline
and, hence, into the progeny. Whether the sea slug is ‘on the path’
to the evolution of permanent photosynthesis is unknown, in large
part because of the lack of genome data.
Laboratory culturing of kleptoplastic sea slugs
To date, most of the studies on E. chlorotica have been carried out
on adult kleptoplastic animals collected from the ocean. The ‘true’
age of the sea slugs, the extent of feeding, the history of the
population and sea slug behavior (e.g. mating) are unknown in
these collections. Furthermore, because the association is already
established we cannot explore the early steps in development,
uptake and retention of the plastids or the initiation of
photosynthesis. Virtually nothing is known at the biochemical or
molecular level about any of these processes. Finally, we also do
not know what contributes to the great disparity in longevity of the
different kleptoplastic associations or, in particular, their
photosynthetic activity.
The successful establishment of a laboratory culture system has
provided us the necessary controls to fully investigate sea slug
development and establishment of kleptoplasty and photosynthesis.
We optimized an artificial saltwater (ASW) culture system using
aposymbiotic eggs produced by E. chlorotica populations from
Martha’s Vineyard Island, MA, USA, and near Halifax, Nova
Scotia (see life cycle in Fig.4). Successful planktotrophic
development was recorded for all developing larvae that were fed
a unicellular algal diet of Isochrysis galbana. Metamorphosis of
larvae to the juvenile stage requires the presence of V. litorea
filaments. Immediately following metamorphosis, the juveniles
begin feeding on the filamentous alga, engulfing plastids and
turning green. A transient nature to the plastid symbiotic
association is observed in recently metamorphosed juvenile sea
slugs if removed from the presence of V. litorea too soon (less than
~6days); this also results in cessation of their morphological
development. Plastid uptake until the establishment of irreversible
kleptoplasty appears to be required for full adult development and
survival, although one report of ‘albino ghost’ E. chlorotica was
documented in 1986 (Gibson et al., 1986). Establishment of the
kleptoplastic association involves specific recognition processes
that comprise at least two steps: (1) planktonic larvae require V.
litorea filaments to be present for settlement and metamorphosis to
the juvenile stage, and (2) adult development requires uptake and
retention of V. litorea plastids by cells lining the digestive
diverticula.
Semi-autonomy of plastids and the need for the nucleus
Endosymbiosis and the associated gene transfer that followed
rendered extant plastid genomes greatly reduced in size
(37–224kb), encoding between 61 and 273 proteins
(http://www.ncbi.nlm.nih.gov/genomes/GenomesGroup.cgi?taxid
2759&optplastid) compared with the cyanobacterial progenitors
[1.6–9.0Mb in free-living taxa, encoding 1717 to 7672 proteins
(Meeks et al., 2001; Rocap et al., 2003)]. These semi-autonomous
organelles encode a small percentage of the predicted 1000 to 5000
M. E. Rumpho and others
Fig.3. Evolutionary scheme for primary, secondary and tertiary plastids.
The secondary endosymbiotic origin of plastids is illustrated in Vaucheria
litorea from the red algal lineage. The subsequent acquisition of V. litorea
plastids by the sea slug Elysia chlorotica in a tertiary endosymbiotic event
imparts photosynthetic activity to this heterotroph.
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307Mollusc photosynthesis
proteins required to sustain the full metabolic capacity of the plastid
(Martin et al., 2002; Richly and Leister, 2004; Bock and Timmis,
2008). This is also true for the 115-kb V. litorea plastid genome,
which we recently sequenced, demonstrating that it contains only
139 protein-encoding genes (Rumpho et al., 2008). If we consider
solely photosynthesis, the V. litorea plastid genome does not
encode all of the components for any of the four multi-subunit
complexes of the photosynthetic electron transport chain
[photosystems I and II (PSI and PSII, respectively), the cytochrome
b6/f complex and ATP synthase] or the reductive pentose phosphate
pathway (RPPP, or the Calvin–Benson cycle) (reviewed in
Raghavendra, 1998; Nelson and Yocum, 2006). Some of the
essential missing genes in the thylakoid-localized electron transport
chain include: the PSI and PSII light-harvesting complex
pigment/protein genes (vcp in V. litorea), the PSII Mn-stabilizing
protein of the oxygen evolution complex (MSP, encoded by psbO),
the Reiske Fe-S protein of the cytochrome b6/f complex and atpC,
which encodes the critical redox-regulated  subunit of ATP
synthase (see Fig.5 schematic).
Only one enzyme of the RPPP is plastid encoded, the essential
carboxylating enzyme ribulose-1,5-bisphosphate carboxylase/
oxygenase (RuBisCO) (Fig.5). Unlike plants and green algae, both
the large (rbcL) and small (rbcS) subunits are plastid-encoded in
V. litorea (Rumpho et al., 2008). The other ten enzymes of the cycle
are nuclear-encoded, and all but phosphoribulokinase (prk) and
sedoheptulose-1,7-bisphosphatase (sbp) are also encoded by the
nuclear genome of animals for glycolysis and/or the oxidative
pentose phosphate pathway [see Rumpho et al. (Rumpho et al.,
2008) for a complete listing of plastid-encoded genes in V. litorea].
As a result, it is possible that the animal could provide substitute
proteins for the majority of the nuclear-encoded RPPP enzymes if
they were properly targeted to the foreign plastids. The remaining
two RPPP enzymes, PRK and SBP, as well as the nuclear-encoded
electron transport proteins discussed above, are excellent targets for
the study of HGT.
Plastids are highly evolved to absorb the intense energy of
sunlight and fuel photosynthetic carbon reduction and, as a result,
plastid proteins are subject to constant photo-oxidative damage by
reactive oxygen species (Aro et al., 1993; Aro et al., 2005). To
maintain homeostasis and uninterrupted function of the plastid,
these damaged proteins must be removed [presumably by proteases
(Adam and Clarke, 2002)] and/or repaired (frequently involving
Fig.4. Life cycle of Elysia chlorotica. After 4days, veliger larvae hatch from
egg ribbons and live planktonically for 3weeks until competent for
metamorphosis. Upon detection of the algal prey Vaucheria litorea, mature
veligers settle out of the water onto the algal filaments and metamorphose
into juvenile sea slugs. Feeding occurs immediately and plastids are
observed inside the animal within 24h of settlement and metamorphosis.
After continual feeding of 5 to 7days, the association becomes permanent
and the plastids are stable within the animal. Additional feeding leads to
growth of the juvenile to the adult stage and further incorporation of
plastids into the animal tissues. Adults live for ~10months in the wild,
senescing often after mating in the spring.
Fig.5. Schematic of the light and dark reactions of photosynthesis showing
plastid- vs nuclear-encoded genes. (A)Adult, kleptoplastic Elysia chlorotica.
(B)Transmission electron micrograph showing numerous algal plastids
within a cell lining the digestive diverticuli of the sea slug. (C,D)Schematic
of the two photosynthetic processes overlaid on a plastid illustrating the
essential proteins required in each pathway. Nuclear-encoded plastid
proteins are shaded blue for both the electron transfer chain (C) and the
Calvin–Bensen cycle (D). In the latter, RuBisCO is shaded green to
indicate a plastid-encoded protein. Two of the enzymes,
phosphoribulokinase and sedoheptulose-1,7-bisphosphatase, are shaded
dark blue to indicate that, although they are nuclear-encoded like the light-
blue-shaded enzymes, these enzymes are unique to phototrophs and are
not typically found in an animal, whereas the light-blue-shaded enzymes all
have homologs in animal metabolism.
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