Table Of ContentReference: Biol. Bull. 197: 1-6. (August 1999)
Annual Viral Expression in a Sea Slug Population:
Life Cycle Control and Symbiotic Chloroplast
Maintenance
SIDNEY K. PIERCE1 *, TIMOTHY K. MAUGEL1. MARY E. RUMPHO2.
JEFFREY J. HANTEN1, AND WILLIAM L. MONDY1
Department ofBiologv. University ofMan-land, College Park, Maryland 20742: and 2Department of
Horticultural Sciences, Texas A & M University, College Station, Texas 77843
In afew well-known cases, animal population dynamics juvenile slug while still attached to the algal filament. Ifthe
are regulated by cyclical infections ofprotists, bacteria, or algal filaments are not present, metamorphosis rarely oc-
viruses. In most ofthese cases, thepathogenpersists in the curs, at least not in laboratory cultures (5. 6). Vaucheria is
environment, where it continues to infect some percentage the only alga that E. chlorotica eats, and it is the source of
ofsuccessive generations ofthe host organism. Thispersis- the symbiotic chloroplasts that are acquired during feeding.
tent re-infection causes a long-lived decline, in eitherpop- The juvenile slug immediately begins eating the algal fila-
ulation size orcycle, to a level thatdepends upon pathogen ments and taking on its first load ofchloroplasts, which are
density and infection level (1-4). We have discovered, on sequestered by specialized cells in the epithelium lining the
the basis of9years ofobservation, an annual viral expres- digestive diverticula (5, 6). During the next several months.
sion in Elysia chlorotica, an ascoglossan sea slug, that the slugs continue to eat Vaucheria and grow, until winter
coincides with the yearly, synchronized death of all the temperatures cause them to become inactive. As the salt
adults in the population. This coincidence ofviral expres- marshes warm in the spring, the slugs become active again,
sion and mass death is ubiquitous, and it occurs in the begin laying egg masses, and then die. By the time the egg
laboratory as well as in thefield. Our evidence also sug- masses have hatched in May. all the adults are gone. This
gests that the viruses do not re-infect subsequent genera- mass mortality occurs synchronously in the laboratory as
tionsfrom an external pathogen pool, but are endogenous well as in the field and regardless ofthe time ofyearthat the
to the slug. We are led, finally, to the hypothesis that the slugs were collected.
viruses may be involved in the maintenance ofsymbiotic Symbioses in which chloroplasts usually from a partic-
chloroplasts within the molluscan cells. ular species of alga are taken up and retained within the
Populations ofthe ascoglossan sea slug Elysia chlorotica cytoplasm ofan animal cell occur in several phyla, but they
occur in salt marshes from the Chesapeake Bay to Nova are most commonly encountered in molluscan sea slugs,
Scotia. The life cycle ofthe slug lasts about 10 months. The particularly in the order Ascoglossa (=Sacoglossa) (Opis-
hermaphroditic adults lay egg masses in the spring of each thobranchia). Certain of the molluscan cells can capture
year, and all of the adults die shortly afterward (5, 6). A chloroplasts from algal food (usually fromaspecific species
week or so after the egg deposition, veliger larvae hatch. of either Rhodophyceae or Chlorophyceae), and these or-
These larvae spend a few weeks in the plankton and, it ganelles retain some degree ofphotosynthetic function fora
filaments of the chromophytic alga Vaucheria litorea are time (e.g.. 7, 8, 9). Whether this intracellular association is
present, each veliger homes in on one ofthem and attaches a symbiosis in the strict sense is debatable; some authors
to it. During the next 24 h, the larva metamorphoses into a prefer terms like chloroplast symbiosis, chloroplast reten-
tion, or kleptoplasty (7. 9, 11, 12, 13), which indicate that
R*eTcoeivwehdo1mJucnoerr1e9s9p9o;nadcecnecpetedsho1u7lJdunbee19a9d9d.ressed: E-mail: sp30@ theThbeenedfuirtatoifotnheofastshoeciaastsioocniaitsieonntibreeltyweteonthteheanmiomalll.uscan
umail.umd.edu cell and the algal plastid varies from species to species.
S. K. PIERCE ET AL
Some associations last less than a week [e.g., Hermaea In 1990, as part of an ongoing ultrastructural study of
hifida and Elysia hedgpethi (see 14)]; and although the morphological changes in the fine structure of the dying
plastids are initially functional, photosynthesis stops or is slugs from a population on Martha's Vineyard, Massachu-
greatly reduced after a week of starvation. In contrast, setts, we discovered viruses in digestive cells and hemo-
plastid function continues for more than a week in starved cytes (29; also Fig. 1). Every year since then, every animal
specimens of several species, most belonging to the asco- examined near the end of the life cycle has had viruses
glossan family Elysiidae (e.g., 7, 10, 15, 16). In the species present, whetherit was fixedwithin hours ofcollection from
with the longer duration symbioses, the algal chloroplasts the field or maintained for months in our aquarium. No
within the molluscan cells fix I4CO:, and the I4C appears in evidence ofviruses has been found in any slug earlier in the
a variety ofcompounds in the animal tissues ( 15, 17. 18, 19, year, except occasionally within the confines of the plastid
20, 21). Thus, there is no doubt that the captured chloro- (Fig. IE, see below).
plasts are photosynthetically active within the molluscan Viruses in the spring slugs are present in the nucleus and
cell cytoplasm, and that the products of the synthesis are cytoplasm of several cell types. They appear to be assem-
utilized by the host animal. Indeed, once the symbiosis is bled in the nucleus, then move into the cytoplasm, and
established, the species with the longer-lived chloroplast finally bud out either into the extracellular space or into a
associations can be starved and, as long as light is provided, vacuole (Fig. 1A). The diameters of the icosahedral viral
will maintain or actually gain body weight until the chlo- capsids in the nuclei average 89 nm (1.0); but in the
roplast function finally fails (7). cytoplasm, after they have picked up an envelope, they are
In plant cells, continued photosynthetic activity requires 109 nm (1.6) (Fig. IB). The shapes and sizes of the
continuous synthesis ofchloroplast proteins because several capsids and envelopes are very similar to those in the
proteins, including those used in light harvesting, are rap- Retroviridae, but other viral types have similar dimensions,
idly turned-over during the process and must be replaced and known retroviruses are not assembled in the nucleus
(22, 23, 24). But, several of these photosynthetic proteins (30, 31). In addition to these larger viruses, some chloro-
(or their subunits) are encoded in the nucleus, so plastid plasts within the cells of spring slugs contain structures
protein synthesis requires the integration of two distinct (diameter = 20 nm 0.4) that could either be smaller
genomes, that of the chloroplast and that of the plant cell viruses or viral cores. These particles occur loosely col-
nucleus (reviewed in 25). Because normal plastid photosyn- lected in areas between the plastid membranes (Fig. 1C).
thetic function is dependent upon major nuclear and cyto- They also occur as particle clumps in the cytoplasm (Fig.
plasmic support, the ultimate failure of photosynthesis by ID), sometimes near material that could be the remnants of
the symbiotic chloroplasts within the molluscan cells is not chloroplasts. In some instances, in a few fall animals, these
surprising. But the symbiotic plastids of E. chlorotica stay particles are present in crystalline arrays (Fig. IE). Ribu-
photosynthetically functional for 8-9 months (5, 26) lose-l,5-bisphosphate-carboxylase oxygenase (RuBisCO)
many months longer than those of any other species yet occurs in crystalline form in some plant plastids fixed under
described. This remarkable persistence ofchloroplast func- hyperosmotic conditions (32, 33). However, the particles in
tion during starvation indicates that replacement of at least such RuBisCO crystals are much smaller and usually lack a
the essential photosynthetic proteins must be occurring visual substructure (32, 33), whereas the array particles in
within the plastid while it is housed in the molluscan cyto- the symbiotic plastids in Elysia clearly have a substructure
plasm; and indeed, synthesis of plastid proteins, primarily (Fig. IE). The particle arrays in the E. chlorotica plastids
those associated with photosynthesis, occurs in the E. chlo- are more reminiscent of mosaic viruses in plants (34),
rotica plastids (26. 27. 28). although almost nothing is known about such crystals in the
The plastid proteins synthesized within the cells of E. viruses and plastids of algae. In summary, the morphology
chlorotica are of two pharmacologically distinguishable suggests thateithermore than one viral type is present in the
sorts: those whose synthesis is blocked by chloramphenicol, slug cell and captured plastids, or we have found several
and those blocked by cycloheximide (26). Protein synthesis stages of a single viral type.
on pltistul ribosomes is blocked by chloramphenicol, In addition to the morphology, we have some biochemi-
whereas synthesis on cytoplasmic ribosomes, usually di- cal information about the identity of the viruses. Using
rected by nuclear genes, is blocked by cycloheximide. differential and sucrose-gradient centrifugation, we have
These results suggest strongly that some plastid proteins are isolated a fraction from slug homogenates at a density of
synthesized upon slug cell ribosomes (26). Ifthis is thecase, 1.18 g/ml that has reverse transcriptase (RT) activity. This
the genetic information coding for these proteins must activity, which is considered diagnostic for retroviruses, is
somehow be present in the molluscan DNA or must be two orders ofmagnitude higher in spring slugs than in slugs
acquired by all the slugs from the alga in every generation. tested in the fall (Fig. 2). In addition, the RT activity offall
Our findings presented below suggest a vehicle for this animals, but not spring animals, is inhibited by rifampicin
transfer of genetic information from the alga to the slug. (Fig. 3). This inhibition indicates that the activity measured
SEA SLUGS, PLASTIDS. AND VIRUSES
Figure 1. Electron micrographs of viruses in Elysiu chlorulica. (A) Viral particles in various stages of
maturation are present in the nucleus (n) and cytoplasm of a hemocyte from a dying slug (magnification =
33,750X, scalebar = 1 /urn). (B)Highermagnification (85.000x, scalebar = 0.? /j.m)ofvirusesbudding into
cytoplasmic vacuoles. Icosahedral shape and double envelopes of the mature virus are apparent. (C) Viral
= =
aggregates(arrows)withinthesymbioticchloroplastofaspringElysiu(magnification 16,880,scalebar 1.0
fim). (D) Viral particles very similar in appearance to those in (C) located in the cytoplasm ofa chloroplast
(cp)-containingdigestivecellofaspringslug.Thediffuse,grayareasinthecytoplasmarelipidproducedbythe
plastid(magnification = 27,000,scalebar= 1.0/j.m).(E)Viralcrystalcontainedinasymbioticchloroplastfrom
= =
a fall slug (magnification 54,000, scale bar 0.5 jim).
S. K. PIERCE ET AL.
15(1(1(111-1 150000-
A-fallanimals B-springanimals
100000- 100000-
3.
E
50000- 50000-
1.1i2 1 1i2 I 1i4 11I8 I 1i8 1 1i8 I 1i9 1 1i9 1.1i9 I2i0 1.2i0 1.21i 121i 122i 1~106 1.I10 II12 II14 II15 1I17 1117 1I18 II18 1IIS II19 1.I19 1.119 1I20 1.I20
Gradientfractions(gm/ml)
Figure2. Comparisonofreversetranscriptaseactivity insucrose-gradientfractionsfromatypicalextractof
fall slugs (A) and spring slugs (B). The results are essentially the same whether the animals were freshly
collected in the spring orcollected in the fall and overwintered in aquaria.
in the fall animals is due to DNA dependent-RNA polymer- The relationships between the nuclear, cytoplasmic, and
ase (which utilizes the same substrates in the RT assay) plastid viruses are not known at present. However, for the
rather than RT, and confirms the absence of viruses in the last9 years, the viruses have been found inevery dying slug
fall animals. Taken together, the morphology, thebuoyancy, examined. Since some of the slugs had been maintained in
and the presence ofRT all suggest that a retro-like virus is the laboratory, in aquaria containing artificial seawater and
present in the cells of the dying slugs. with no access to Vauclierui for 8 months before the viruses
appeared, the infection is unlikely to have been opportunis-
tic. Instead, the results suggest either that the demise ofthe
150-1 entire population is caused by an endogenous virus or that
the virus can be expressed only as the defense systems of
the aging animals begin to fail. Furthermore, ifthe effecton
the life cycle is due to a retrovirus, as ourdata suggest, then
the viral genome is probably transmitted to the next gener-
100- ation ofslugs in the molluscan DNA. Infection ofgermcells
by retroviruses produces endogenous proviruses that are
inheritedas Mendelian genes (33). Alternatively, the viruses
3 might enter all ofthe slugs via the sequestered chloroplasts.
either as part of the plastid genome or as constituted viral
50- particles. In either case, viral expression is coincident with
increases in environmental temperature, at least in the field
slugs. Both the onset of egg laying and the death of the
population are associated with the rise of water tempera-
tures in the spring. We can delay the demise ofthe slugs by
maintaining them at very cold temperatures (2-5C), but
O-1 eventually 6-8 weeks after the warmer-maintained slugs
Fall Spring have died the cooled slugs also die with viruses in their
cells, indicating that temperature is not the only expression
Figure 3. Rilampicin inhibits reverse transcriptase activity. Enzyme stimulus.
atcoti1v.i1t8ygw/amsl parsespaayreeddifnrpoomolfaeldl agrnaddisepnrtinfgrascltuigso.nsTwhiethefdfeecntsiotfietshefrionhmib1i.t1o6r It will take some time to sort out both the types ofviruses
is expressed as a percentage ofcontrol values. involved here and the molecular relationships between the
SEA SLUGS. PLASTIDS. AND VIRUSES
slugs, the algae, the plastids. and the viruses. Nevertheless, The gradients were then fractionated by piercing the
the nine-year, annual occurrence ofthe association between bottom ofthe centrifuge tube and raising the gradient out of
population demise and viral expression at the end ofthe E. the tube with 65% sucrose. Twenty 600-/xl fractions were
chlorotica life cycle strongly suggests that the virus has a collected, and the density ofeach was determined by weigh-
role in regulating thiscoincidence. Indeed, we speculate that ing 50 /xl with an analytical balance. The sucrose in each
the viral infection may have caused the transfer, from the fraction was then diluted with homogenization buffer (with-
algatothe slug, ofalgal genes that allow the molluscan cells out /i-acetyl cysteine), and each fraction was centrifuged a
to assist in plastid maintenance. Although the transfer, in- final time at 180,000 X g for2 h. The supernatants from this
tegration, and expression ofsuch a group ofgenes between last spin were discarded, and RT assays (see below) and
species should succeed only rarely, those successes that did protein assays (37) were run on the pellets.
occurshould have profound, immediate, heritable effects on
the phenotype ofthe infected species. Such heritable effects Reverse transcriptase assav
must certainly be associated with the mechanism of the
widely accepted endosymbiotic origin of intracellular or- The final pellets (above) were treated with a detergent
mM mM mM
ganelles such as mitochondria and chloroplasts; a variety of buffer (50 Tris-HCl, 5 KC1, 0.2 EDTA. and
genes musthave been transferredfromthe symbiont into the 0.02% Triton X-100, pH 8.2). Fifteen-microliter aliquots of
mM
host cell nucleus to consummate such a relationship. In this digest were added last to 50 /xl of buffer (100
mM mM
addition, a retrovirus as a gene transmission vehicle might TmrMis-HCl, 200 KC1.m1M0 MgCl2, pH 8.2), ft /xl 100
have merit as a hypothesis to explain genetic similarities DTT, 9.75 LI! 100 thymidine triphosphate (TTP),
between distantly related or unrelated species (e.g., 35) and 1.25 /xl RNA-guard (Pharmacia), 1.0 /xl poly(rA)-p(dT)
is the basis of some genetic therapies (36). If a successful (Pharmacia), and 10 /xCi of32P-TTP (ICN, 10 /xCi//xl). The
interspecies gene transfer between an alga and a slug me- final volume was adjusted to 100 /xl with buffer, as neces-
diated by an endogenous virus could be demonstrated in the sary, to compensate for 12P half life. This solution was
case ofE. chlorotica. then an exemplary mechanism forthis mixed and incubated at 37C for 65 min on a shaker table.
mM
process would have been provided. The reaction was terminated by adding 30 /xl of 10
EDTA in 5% TCA and placing the reaction mixture on ice
Methods for 20 min. DNA was then precipitated by the addition of9
Viral isolation /xl of 72% TCA, and the precipitate was pelleted by cen-
trifugation at 6610 X g for 10 min. The pellet was washed
The fractionation procedure was carried out using au- three times in 5% TCA, the final pellet dissolved in 0.1 N
toclaved equipment. All reagents were molecular biolog- NaOH. and the radioactivity determined by scintillation
ical grade (DNAase-, RNAase-. and protease-free; from counting. The protein concentration of a separate aliquot
Sigma Chemicals, unless otherwise noted) and filtered was determined, and RT activity was expressed as counts
(0.2 /xm pore). Approximately 3.0 g ofE. chlorotica was per minute per microgram (cpm//xg) protein (38).
mM
homogenized in an ice-cold buffer (450 NaCl, 1.0
mM mM
EDTA, 5.0 3-[/V-morpholinom]pMropane-sulfonic Electron microscopy
acid (MOPS), 2.3 yuM leupeptin. 1.0 dithiothreitol
(DTT), 500 /xM phenylmethylsulfonyl fluoride (PMSF), Small pieces of tissue were prepared for microscopy by
M M
pH 7.5) containing the mucolytic agent H-acetyl cysteine fixation in 2% glutaraldehyde in 0.15 cacodylate-0.58
(500 mM), which is necessary to disperse the copious sucrose buffer(pH 7.3) at 900 mosm. The tissuepieces were
mucus produced by the slug (26). The homogenate was post-fixed in 1.0% OsO4 in the same buffer followed by
filtered through six layers of cheesecloth, then through 2.0% aqueous uranyl acetate. The fixed tissue was dehy-
one layer of Miracloth (Calbiochem), and finally through drated in an ethanol series, infiltrated with propylene oxide,
two layers of Miracloth. The filtrate was centrifuged for and embedded in Spurr's medium. Silver sections were cut
5 min at 4300 x g (4C), and the supernatant was with an ultramicrotome (Reichert), mounted on 75 X 300
centrifuged at 20,000 X g for 30 min (4C). The super- mesh coppergrids, and stained with uranyl acetate and lead
natant from the second spin was layered over a 20% citrate. The sections were viewed and photographed with a
sucrose cushion and centrifuged at 180,000 X g for 2 h transmission electron microscope (Zeiss EM 10).
(4C) in a swinging bucket rotor. The supernatant was
discarded, and the pellet was resuspended in ice-cold Acknowledgments
homogenization buffer (without H-acetyl cysteine). This
suspension was layered on the top of a 15%-50% con- This work was supported by an NSF grant to SKP and
tinuous sucrose gradient and centrifuged at 180,000 X g MER. We thank Margaret Palmer, Ulrich Mueller, and
for 43 h (4C). Jeffery DeStefano for critically reading early versions of
6 S. K. PIERCE ET AL
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