Proc. Natl. Acad. Sci. USAVol. 90, pp. 5695-5699, June 1993Microbiology

Transovarial inheritance of endosymbiotic bacteria in clamsinhabiting deep-sea hydrothermal vents and cold seeps

(chemoautotroph/bacterial/endosymbiont/vesicomids/transmission)

S. CRAIG CARY* AND STEPHEN J. GIOVANNONIDepartment of Microbiology, Oregon State University, Corvallis, OR 97331-3804

Communicated by George N. Somero, January 25, 1993 (received for review November 24, 1992)

ABSTRACT Vesicomyid clams are conspicuous fauna atmany deep-sea hydrothermal-vent and cold-seep habitats. AUspecies examined have specialized gill tissue harboring endo-symbiotic bacteria, which are thought to provide the hosts' solenutritional support. In these species mechanisms of symbiontinheritance are likely to be key elements of dispersal strategies.These mechanisms have remained unresolved because the earlylife stages are not available for developmental studies. Aspecific 16S rRNA-directed oligodeoxynucleotide probe(CG1255R) for the vesocomyid endosymbionts was used in acombination of sensitive hybridization techniques to detect andlocalize the endosymbionts in host germ tissues. Symbiont-specific polymerase chain reaction amplifications, comparativegene sequencing, and restriction fragment length polymor-phisms were used to detect and confirm the presence ofsymbiont target in tissue nucleic acid extracts. Nonradioactivein situ hybridizations were used to resolve the position of thebacterial endosymbionts in host cells. Symbiont 16S rRNAgenes were consistently amplified from the ovarial tissue ofthree species of vesicomyid clams: Calyptogena magnifica, C.phaseolaformis, and C. pacifica. The nucleotide sequences of thegenes amplified from ovaries were identical to those from therespective host symbionts. In situ hybridizations to CG1255Rlabeled with digoxigenin-11-dUTP were performed on ovarialtissue from each of the vesicomyid clams. Detection of hybridslocalized the symbionts to follicle cells surrounding the primaryoocytes. These results suggest that vesicomyid clams assuresuccessful, host-specific inoculation of all progeny by using atransovarial mechanism of symbiont transmission.

Determining the mechanisms by which symbionts are inher-ited has been an elusive problem in marine symbiosis re-search. This is particularly the case in the associationsinvolving endosymbiotic bacteria and invertebrate hosts,where successful transmission may depend on an initialinoculation of only a single cell.

Several pathways by which the transmission of bacterialendosymbionts might occur are (i) horizontally (spread ofsymbionts between contemporary hosts), (ii) vertically(transfer from parent to offspring), and (iii) environmentally(reinfection of the new host generation from an environmen-tal stock of microorganisms). Many terrestrial arthropodshave evolved elegant transmission mechanisms to ensure thatprogeny are inoculated with a precise complement of bacte-ria. Similar mechanisms are not defined so clearly in marinesystems. The vertical process of bacterial endosymbionttransmission and incorporation has been studied only in alimited number of marine invertebrates, where the host caneasily be cultured (1-5).

Following the discovery of deep-sea hydrothermal ventscame the introduction to a novel symbiosis between

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chemoautotrophic sulfur-oxidizing bacteria and several un-described invertebrate hosts (6, 7). Similar associations arenow known to occur in a wider variety of taxa occupying abroad range of reducing habitats in the marine environment(8). An understanding of transmission mechanisms in deep-sea invertebrate-bacterial associations, in particular thoseinhabiting hydrothermal-vent and cold-seep environments,has been problematic, primarily because the early life stagesof the host have not been found. In one electron microscopicstudy, bacteria-like inclusions were observed in the ovariesof the vesicomyid Calyptogena soyoae. However, thesestructures could not be conclusively identified (9).Vesicomyid clams (Calyptogena spp.) are one of the most

ubiquitous symbiont-containing groups found occupyingdeep-sea reducing environments. Although vesicomyids re-tain the usual complement of digestive organs, a generalreduction in the size of structures associated with particulatenutrition suggests that these structures are virtually nonfunc-tional and that the hosts must rely primarily on symbiont-mediated autotrophic nutrition (10). Stable carbon isotopicanalyses have revealed the host tissue and symbionts to beidentically depleted in 8-13C (11), verifying the critical role ofchemoautotrophy in host nutrition. This redox dependencyappears to restrict these clams to the sub-oxic interfacebetween ambient oxygenated sea water and hydrothermalfluids or sediment pore waters where hydrogen sulfide isreadily available.

Little is known about the reproductive and dispersal strat-egies of this deep-sea family. In one species, Calyptogenamagnifica, oogenesis appears continuous (12), producinglarge yolk-fflled eggs suggestive of a lecithotrophic larvalstrategy. These eggs are bouyant at physiological tempera-ture (2°C) and pressure (260 bars or 26 MPa) and presumablyare capable ofleaving the spawning site (S.C.C., unpublisheddata). C. magnifica has been collected at the Galapagos Riftsites and as far as 21° N on the East Pacific Rise, a distanceof >3500 km (13, 14). While the genetic similarity of thesedistant populations has not been examined, the bouyancy ofthe eggs suggests a broadcast-type dispersal strategy.A knowledge of the mechanism of symbiont inheritance is

critical to our understanding of dispersal and the biologicalconstraints on settlement and colonization of the vesicomy-ids. The island-like and ephemeral nature of these deep-seareducing habitats suggests that vesicomyids may have long-distance dispersal ability. Furthermore, the monospecificity(15) and obligatory nature of the symbioses strongly suggestselective pressures for the evolution of mechanisms thatensure successful symbiont inheritance by the hosts.

Conceivably, only a single bacterium would be required toinitiate the symbiosis in a developing larva. Detection at thislevel ofresolution is not possible with conventional biochem-ical assays. Nucleic acid probe technology based on 16SrRNA sequences provides the high resolution necessary to

Abbreviation: rDNA, DNA encoding rRNA.*To whom reprint requests should be addressed.

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identify the presence and location of extremely low copynumbers ofa specific target sequence. Ribosomal genes offerideal targets for hybridization probes because they are pre-sent in multiple copies and offer a mosaic of conserved andvariable regions. Specific regions may be invariant among allliving organisms or unique to particular organisms or torelated groups of organisms. 16S rRNA-directed oligodeox-ynucleotide probes have been successfully used to examinenucleic acid extracts and intact cells of native bacterialpopulations with fine levels of phylogenetic specificity (16,17). Several recent studies have used similar oligodeoxynu-cleotides as probes in in situ hybridizations to identify andlocalize symbiotic bacteria within eukaryotic tissue (18-20).

In this study an oligonucleotide probe (CG-1255R) com-plementary to a unique domain of the vesicomyid clamsymbiont 16S rDNA (DNA encoding rRNA) (19) was used asa primer in a polymerase chain reaction (PCR) assay capableof detecting extremely low copy numbers of symbiont rDNAfrom mixed nucleic acid samples. CG1255R was also used asa probe in tissue in situ hybridizations to localize symbionttarget within host cells.

MATERIALS AND METHODSSpecimen Collection. Vesicomyid clams were collected by

the deep-sea research vessel (DSRV) Alvin from three sitesin the Eastern Pacific: C. magnifica, from the East PacificRise at a depth of 2615 m (200 50' N 1090 6' W); Calyptogenaphaseoliformis, from the Monterey Canyon at a depth of 3399m (360 23' N 1220 53' W); and Calyptogena pacifica, from theAxial site on the Juan de Fuca Ridge at a depth of 1547 m (450N 56.1', 1300 0.8' W). Bulk nucleic acids were extracted fromthe gill, ovary, foot, and mantle tissues from three specimensof each species. To eliminate the possibility of external

contamination of the gonad samples during the dissections,the entire ovary was removed from each specimen, washedin filtered sea water, placed in a 5% hypochlorite wash for 3min, and finally rinsed with 100% ethanol. Stringent aseptictechniques were maintained throughout the dissections. Theventral portion of the foot was removed to expose the ovaryand allow the sampling of tissue. Mantle, foot, and outergonadal epithelial tissue samples were processed in the samemanner as the ovarian tissue and were used as controls tonegate contamination as a source of symbiont signal in thePCR reactions.

Nucleic Acid Extraction and Sequencing. Nucleic acids wereextracted and recovered from the tissues by using guanidiniumisothiocyanate (19). 16S rRNA genes were amplified from thebulk nucleic acids by the PCR. Three independent primer setswere used in the amplifications: a eukaryotic set (21), aeubacterial set (22), and a symbiont-specific set for the vesi-comyid clams (Calyptogena spp.) (19). The reaction mixturecontained 50 mM KCl, 10 mM Tris HCl (pH 8.4 at 25°C), 2.5mM MgCl2, 0.2mM each dNTP, 0.2 uM ofeach amplificationprimer, 0.1 Mg of template DNA, and 2.5 units of Taq poly-merase (Promega)-all in a total volume of 100 ,ul. Amplifi-cation profile conditions were 1 min at 95°C, 2 min at 630C, and3 min at 720C for 40 cycles. Gene amplifications were per-formed by using one unmodified amplification primer and oneprimer with a biotin moiety covalently linked to the 5' termi-nus. Magnetic beads coupled to streptavidin were used toseparate the strands of PCR amplification products (23) fordirect dideoxy-terminated sequencing (24). Eubacterial andsymbiont-specific polymerase chain reaction products fromthe gill and ovary DNA ofboth species were digested with theendonuclease Hae III and examined by agarose gel electro-phoresis to verify similarity.

CalyptogenamagnificaI 'I

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bB CG/EubBI X I I I I I I X a

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FIG. 1. PCR detection of symbiont rDNA in ovary and gill tissue from three species ofvesicomyid clams. Primer sets specific for eukaryotes(Euk A/B) (21), eubacteria (Eub A/B) (19), and the symbionts of Calyptogena spp. (CG/EubB) (19) were used in amplifications ofbulk genomicDNA extracted from the ovaries and symbiont-containing gill tissue. (A) Successful amplification from ovarial tissue occurred with mosteubacterial and all symbiont-specific primer sets, indicating the presence of symbiont targetrDNA in the ovaries. (B) All amplifications attemptedfrom gill DNA with the same primer sets were successful.

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In Situ Hybridization. The symbiont-specific oligonucleo-tide (CG-1255R) was enzymatically labeled with digoxigenin-11-dUTP by terminal deoxynucleotidyltransferase (Boeh-ringer-Mannheim). Tissue samples were fixed in Mops fixative(1x Mops/0.5 M sodium chloride/4% paraformaldehyde) for6 hr, dehydrated in ethanol, and embedded in paraffin onshipboard. Serial sections (6 ,um) were cut, deparaffinized inxylene, rehydrated in ethanol, and equilibrated in 2x SSPE(0.45 M NaCl/20 mM NaH2PO4/2 mM EDTA) for 2 min. Thesections were then treated with 0.25% acetic anhydride in 0.1M triethanolamine (pH 8.0 with HCI) for 10 min, re-equilibrated in 2x SSPE, and prehybridized for 60 min at roomtemperature in a hybridization buffer [2x SSPE containing 0.5mg of salmon sperm DNA per ml, 0.25 mg of yeast tRNA perml, and 5x Denhardt's reagent (lx = 0.02% polyvinylpyr-rolidone/0.02% Picoll/0.02% bovine serum albumin)]. Thesections were hybridized with the probe (0.85 ,ug/ml) under aParafim coverslip in a sealed chamber at 43°C for 16 hr. Thesections were washed for 1 hr at room temperature consecu-tively in 2x, lx, and 0.2x SSPE, followed by a final highstringency wash in 0.2x SSPE at 55°C for 60 min. Hybridswere subsequently detected with an alkaline phosphatase-

conjugated IgG specific for the digoxigenin moiety (Boehring-er-Mannheim) and were resolved with an enzyme-catalyzedcolor reaction with 5-bromo-4-chloro-3-indoyl ,B3D-galactosidephosphate and nitro blue tetrazolium salt. The sections werecounterstained with pyronin. Probes specific for the symbiontof the vestimentiferan tubeworm Riftia pachyptila (19) and forLactococcus cremoris (25), a gram negative bacterium, servedas negative controls. Sections incubated without a probeserved as controls for nonspecific binding of the IgG.

RESULTS AND DISCUSSIONBulk DNA purified from the various tissues was amplified byPCR using three primer sets specific for (i) all eukaryotes, (ii)all eubacteria; and (iii) the vesicomyid symbionts (Fig. 1).Each of the three primer sets clearly amplified its specifictarget from the gill and ovary of C. pacifica and C. phae-soliformis. Only the primer sets specific for eukaryotes andvesicomyid symbiont ribosomal DNA (rDNA; DNA encod-ing rRNA) amplified symbiont rDNA from the ovaries of C.magnifica. We do not know the cause of the failure of theeubacterial primers to amplify eubacterial rDNA in this case.Mismatches to these primers are rare among eubacteria, but

EubA/B CG-1255/EubB-Gill- -Ovary-

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FIG. 2. Restriction analysis of ovarial amplification products. Agarose gel showing amplified ovarial and gill eubacterial 16S rDNA (lanesA) and the same product cut with the restriction endonuclease Hae III (Promega) (lanes B). Two independent primer sets were used in theamplifications: a eubacterial set and a symbiont-specific set for the vesicomyid clams (Calyptogena spp.). All amplifications of ovarial rDNAhad restriction fragments identical to the symbiont amplification products out of the gills from respective hosts.

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possible. Such a mismatch in the eubacterial B (Eub B)primer, coupled with the low template concentrations ex-pected in ovary extracts, could explain the failure of thesePCR reactions. The control tissues were successfully ampli-fied only by the eukaryote-specific primers, indicating theabsence of contaminating eubacterial DNA.To verify that the amplification products from ovaries were

from symbiont 16S rDNA, each of the amplified productsfrom the gills and ovaries was digested with a restrictionendonuclease and characterized on agarose gels. Addition-ally, the PCR products were directly sequenced. Restrictionfragment length polymorphisms (RFLPs) indicated that thegenes amplified from the ovaries were identical and unique tothe symbiont genes amplified from each of the respective gilltissues (Fig. 2). Partial 16S rRNA sequence comparisons of>300 bases encompassing two hypervariable regions of thegene demonstrated that the rDNA amplified from the ovarieswas identical to the symbiont rDNA amplified from the gilltissues of the respective hosts.

Although PCR assays indicated the presence of specificsymbiont 16S rDNAs in the ovary ofeach ofthe host species,a further experiment utilizing in situ hybridization was usedto localize the position of the symbionts in tissue sections.Nonradioactive in situ hybridizations to ovarial tissues fromadult C. pac fica and C. magniflca with the symbiont-specificoligonucleotide revealed hybrid formation coincident withthe follicle cells associated with primary oocytes (Fig. 3).Similar sections did not hybridize to either of the nonhomol-ogous probes. Multiple sections demonstrated that, regard-less of oocyte maturity, >90% of the associated follicle cellscontained high concentrations ofhybrids, with little variationobserved between animal specimens. No attempt was madeto quantify endosymbiont distribution in the follicle cells dueto poor cellular resolution at higher magnifications. Hybrid-ization of the probes within oocytes was not observed.However, in several instances, sections revealed the folliclecells invaginate the developing oocytes. The infection ofthese nutritive cells would provide the ideal mechanism for

FiG. 3. Light micrographs of thick sections of ovarial tissue from the vesicomyid clam, C. pacifica, after an in situ hybridization with thesymbiont-specific probe CG-1255R (A) and with a nonhomologous probe specific to L. cremoris (25) (B), which served as a negative control.The follicle cells (F) surrounding the primary oocytes (0) clearly hybridized to the symbiont-specific probe (dark blue), identifying the locationof symbiont target 16S rRNA. No hybrids were detected in the control sections. (Bar = 100 ,um.)

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inoculation of the developing eggs. This strategy is similar tothat observed for the transmission of luminescent bacteria intunicates (1) and for the symbionts of certain sponges, wherebacteria-containing follicle cells are shed with the eggs (2).These analyses have identified and localized symbiont 16S

rDNAs in the ovarial tissue of three species of vesicomyidclams. The evidence strongly suggests a vertical mechanismof symbiont transmission. This is in contrast to the hydro-thermal vent tubeworm Riftia pachyptila, whose progenynow are thought to acquire their symbionts by reinfectionfrom a free-living stock of microorganisms (19). Past studieshave demonstrated that certain vent megafauna, includingthe vesicomyid C. magnifica, are broadly distributed on theEast Pacific Rise. Electrophoretic studies have failed toresolve gene flow between isolated populations of the vesti-mentiferans and mytilid bivalves in the same region (26, 27).The archipelago geography of venting sites along spreadingridges (28) may provide a temporal mechanism for dispersalof species constrained by the autotrophic requirements oftheir symbionts. The vertical transmission of the symbiontsin these vesicomyids maintains the continuity of the associ-ation in the progeny, perhaps providing them with the meansto freely settle in isolated areas where sulfide and oxygenco-occur.

Resolving the transmission processes in these obligatesymbiotic associations is essential to understanding howcolonists successfully traverse hundreds of kilometers fromestablished vent sites to nascent ones. Recent preliminarystudies have revealed a similar transovarial transmissionmechanism in the symbiont of the mytilid bivalve Bathymo-diolus thermophilus, also found inhabiting hydrothermal-vent systems, and in the shallow water protobranch bivalveSolemya reidi (S.C.C., unpublished data). Further applica-tion of in situ probing technologies will continue to providevaluable insight into ontogenetic and biochemical processesof symbiont incorporation in these specialized marine bi-valves.

We especially thank chief scientists R. Lutz and R. Vrijenhoek forproviding space and assistance on numerous cruises. We also thankW. Warren for assistance in data collection; K. Field, A. Hacker, andP. Chevaldonnt for critically reviewing the manuscript; and the pilotsand crew of the Alvin and Atlantis II for their efforts in collectingspecimens. This research was supported through National ScienceFoundation Marine Biotechnology Postdoctoral Fellowship OCE-8915305 to S.C.C. and by National Science Foundation GrantOCE-9016373 to S.J.G.

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