Gill filament differentiation and experimental colonization by symbiotic bacteria in aposymbiotic juveniles of Codakia orbicularis (Bivalvia: Lucinidae)

Summary

Codakia orbicularis is a tropical lucinid harboring gill endosymbionts which are environmentally transmitted from a free living-symbiont form to the new host generation after metamorphosis. Structural changes occurring in the cellular organization from incomplete gill filaments in young aposymbiotic juveniles to full differentiated gill filaments containing bacterial endosymbionts in reared symbiotic juveniles, were analyzed for juveniles from 250 μm to 2 μm shell-length. Aposymbiotic juveniles possess differentiated gill filaments with ciliated, intermediary, and lateral zones similar to those described in wild juveniles, except for the bacteriocytes which are lacking. Granule cells, which progressively differentiate during the morphogenesis of the gill filament, do not appear as a consequence of symbiosis. Experimental colonization of aposymbiotic juveniles by the free-living symbiont form has been obtained through the addition of unsterilized sand collected from the natural habitat of C. orbicularis. Two days after exposure to crude sand, symbiosis-competent bacteria enter by endocytosis at the apical pole of undifferentiated cells which progressively differentiate into classical bacteriocytes similar to those found in the adult gill filaments. Undifferentiated cells of aposymbiotic gill filaments remain receptive to bacteria several months after metamorphosis, and become bacteriocytes when aposymbiotic juveniles get contact with the symbiont free-living form. Therefore, the environmental transmission of symbionts does not appear to be restrained to a defined period of time during post-larval development in C. orbicularis.     

Environmental transmission of a sulfur-oxidizing bacterial gill endosymbiont in the tropical lucinid bivalve Codakia orbicularis.

Codakia orbicularis is a large tropical member of the bivalve mollusk family Lucinidae which inhabits shallow-water sea-grass beds (Thalassia testudinum environment) and harbors sulfur-oxidixing endosymbiotic bacteria within bacteriocytes of its gill filaments. When a C. orbicularis-specific 16S rDNA (DNA encoding rRNA) primer is used with a bacterium-specific 16S rDNA reverse primer in amplifications by PCR, the primer set was unsuccessful in amplifying symbiont DNA targets from ovaries, eggs, veligers, and metamorphosed juveniles (600 microns to 1 mm in shell length) cultivated in sterile sand, whereas successful amplifications were obtained from gill tissue of adult specimens and from metamorphosed juveniles (600 microns to 1 mm in shell length) cultivated in unsterilized sea-grass bed sand. To ascertain the presence of the symbiont target in juveniles, restriction fragment length polymorphism analysis, Southern blotting, and transmission electron microscopy were used. Specific hybridizations and observation of endosymbiotic bacteria in the gills of numerous juveniles cultivated in unsterilized sea-grass bed sand showed that the sulfur-oxidizing endosymbionts of C. orbicularis are environmentally transmitted to the new generation after larval metamorphosis.     

Respiration Strategies Utilized by the Gill Endosymbiont from the Host Lucinid Codakia orbicularis (Bivalvia: Lucinidae)

The large tropical lucinid clam Codakia orbicularis has a symbiotic relationship with intracellular, sulfide-oxidizing chemoautotrophic bacteria. The respiration strategies utilized by the symbiont were explored using integrative techniques on mechanically purified symbionts and intact clam-symbiont associations along with habitat analysis. Previous work on a related symbiont species found in the host lucinid Lucinoma aequizonata showed that the symbionts obligately used nitrate as an electron acceptor, even under oxygenated conditions. In contrast, the symbionts of C. orbicularis use oxygen as the primary electron acceptor while evidence for nitrate respiration was lacking. Direct measurements obtained by using microelectrodes in purified symbiont suspensions showed that the symbionts consumed oxygen; this intracellular respiration was confirmed by using the redox dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride). In the few intact chemosymbioses tested in previous studies, hydrogen sulfide production was shown to occur when the animal-symbiont association was exposed to anoxia and elemental sulfur stored in the thioautotrophic symbionts was proposed to serve as an electron sink in the absence of oxygen and nitrate. However, this is the first study to show by direct measurements using sulfide microelectrodes in enriched symbiont suspensions that the symbionts are the actual source of sulfide under anoxic conditions.     

Embryonic development and endosymbiont transmission mode in the symbiotic clam Lucinoma aequizonata (Bivalvia: Lucinidae)

Summary

Lucinoma aequizonata is a large lucinid clam which lives in reducing mud around 500 m deep. Adults harbor intracellular chemoautotrophic sulfur-oxidizing bacteria in specialized gill cells called bacteriocytes. The embryonic and early larval development of L. aequizonata is described by using light and scanning electron microscopy. Gametes were obtained by injection of 0.2ml of 4 mM serotonin solution in seawater into the posterior adductor muscle. The oocytes, 200 μm in diameter, are surrounded by a glycoprotein capsule which gives to the egg a total diameter of 500μm. The development which occurs at 10°C is slow. The first polar body is detected 2.5 h after contact between sperm and oocytes (T_o+2.5 h), and the first cleavage begins 10 h later (T_o+12.5 h). The following successive cleavages produce a nonciliated morula, then a ciliated gastrula which begins to rotate within the egg-capsule at T_o+4.5 days. At this stage, the first shell pellicle appears on the dorsal side of the embryo. At T₀+8 days, the trochophore larvae develop discrete ciliary bands which constitute the prototroch. Typical straight-hinge veligers, D-shaped larvae, hatch from the egg-capsule 12 days after fertilization. The newly hatched larvae are 240 μm in length and 200μm in height, and the straight hinge 150μm long. To elucidate the symbiont transmission mode, two symbiont-specific primers were designed and used in amplifications by PCR. This primer set was unsuccessful in amplifying symbiont DNA targets from mature gonads, spawned oocytes, eggs, and veligers whereas successful amplifications were obtained from symbiont-containing gill tissues. These data rule out the vertical transmission mode and strongly suggest that the symbionts are environmentally transmitted to the new host generation in L. aequizonata as for all tropical lucinids examined to date.     

Detection of the Free-Living Forms of Sulfide-Oxidizing Gill Endosymbionts in the Lucinid Habitat (Thalassia testudinum Environment)

Target DNA from the uncultivable Codakia orbicularis endosymbiont was PCR amplified from sea-grass sediment. To confirm that such amplifications originated from intact bacterial cells rather than free DNA, whole-cell hybridization (fluorescence in situ hybridization technique) with the specific probe Symco2 was performed along with experimental infection of aposymbiotic juveniles placed in contact with the same sediment. Taken together, the data demonstrate that the sulfide-oxidizing gill endosymbiont of Codakia orbicularis is present in the environment as a free-living uncultivable form.     

Bacterial host specificity of Lucinacea endosymbionts: Interspecific variation in 16S rRNA sequences

Abstract Three tropical lucinid clams ( Codakia orbiculata, Codakia pectinella and Lucina nassula ) from a shallow coastal environment have been studied regarding to their thioautotrophic bacterial endosymbionts. The 16S rRNA genes (rDNA) from these three endosymbionts were amplified using PCR. Phylogenetic analysis by distance matrix and parsimony methods always placed the newly examined symbionts within the monophyletic group composed of symbionts of the bivalve superfamily Lucinacea. A same single 16S rRNA sequence was found in C. orbiculata and C. pectinella and was identical to that found in C. orbicularis and Linga pensylvanica , two other lucinids living in the same type of environment. These data indicate that a same symbiont species may be associated with different host species. Lucina nassula hosts a symbiont with a distinct 16S rDNA sequence, but very closely related to the former.     

Plasticity of symbiont acquisition throughout the life cycle of the shallow-water tropical lucinid Codakia orbiculata (Mollusca: Bivalvia)

In marine invertebrates that acquire their symbionts from the environment, these are generally only taken up during early developmental stages. In the symbiosis between lucinid clams and their intracellular sulfur-oxidizing bacteria, it has been shown that the juveniles acquire their symbionts from an environmental stock of free-living symbiont forms, but it is not known if adult clams are still competent to take up symbiotic bacteria from the environment. In this study, we investigated symbiont acquisition in adult specimens of the lucinid clam Codakia orbiculata, using transmission electron microscopy, fluorescence in situ hybridization, immunohistochemistry and PCR. We show here that adults that had no detectable symbionts after starvation in aquaria for 6 months, rapidly reacquired symbionts within days after being returned to their natural environments in the field. Control specimens that were starved and then exposed to seawater aquaria with sulfide did not reacquire symbionts. This indicates that the reacquisition of symbionts in the starved clams returned to the field was not caused by high division rates of a small pool of remaining symbionts that we were not able to detect with the methods used here. Immunohistochemistry with an antibody against actin, a protein involved in the phagocytosis of intracellular bacteria, showed that actin was expressed at the apical ends of the gill cells that took up symbionts, providing further evidence that the symbionts were acquired from the environment. Interestingly, actin expression was also observed in symbiont-containing cells of untreated lucinids freshly collected from the environment, indicating that symbiont acquisition from the environment occurs continuously in these clams throughout their lifetime.     

Respiration strategies utilized by the gill endosymbiont from the host lucinid Codakia orbicularis (Bivalvia: Lucinidae)

The large tropical lucinid clam Codakia orbicularis has a symbiotic relationship with intracellular, sulfide-oxidizing chemoautotrophic bacteria. The respiration strategies utilized by the symbiont were explored using integrative techniques on mechanically purified symbionts and intact clam-symbiont associations along with habitat analysis. Previous work on a related symbiont species found in the host lucinid Lucinoma aequizonata showed that the symbionts obligately used nitrate as an electron acceptor, even under oxygenated conditions. In contrast, the symbionts of C. orbicularis use oxygen as the primary electron acceptor while evidence for nitrate respiration was lacking. Direct measurements obtained by using microelectrodes in purified symbiont suspensions showed that the symbionts consumed oxygen; this intracellular respiration was confirmed by using the redox dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride). In the few intact chemosymbioses tested in previous studies, hydrogen sulfide production was shown to occur when the animal-symbiont association was exposed to anoxia and elemental sulfur stored in the thioautotrophic symbionts was proposed to serve as an electron sink in the absence of oxygen and nitrate. However, this is the first study to show by direct measurements using sulfide microelectrodes in enriched symbiont suspensions that the symbionts are the actual source of sulfide under anoxic conditions.     

Bacterial Symbiont Transmission in the Wood-Boring Shipworm Bankia setacea (Bivalvia: Teredinidae)

The Teredinidae (shipworms) are a morphologically diverse group of marine wood-boring bivalves that are responsible each year for millions of dollars of damage to wooden structures in estuarine and marine habitats worldwide. They exist in a symbiosis with cellulolytic nitrogen-fixing bacteria that provide the host with the necessary enzymes for survival on a diet of wood cellulose. These symbiotic bacteria reside in distinct structures lining the interlamellar junctions of the gill. This study investigated the mode by which these nutritionally essential bacterial symbionts are acquired in the teredinid Bankia setacea. Through 16S ribosomal DNA (rDNA) sequencing, the symbiont residing within the B. setacea gill was phylogenetically characterized and shown to be distinct from previously described shipworm symbionts. In situ hybridization using symbiont-specific 16S rRNA-directed probes bound to bacterial ribosome targets located within the host gill coincident with the known location of the gill symbionts. These specific probes were then used as primers in a PCR-based assay which consistently detected bacterial rDNA in host gill (symbiont containing), gonad tissue, and recently spawned eggs, demonstrating the presence of symbiont cells in host ovary and offspring. These results suggest that B. setacea ensures successful inoculation of offspring through a vertical mode of symbiont transmission and thereby enables a broad distribution of larval settlement.     

Lack of endosymbiont release by two Lucinidae (Bivalvia) of the genus Codakia: consequences for symbiotic relationships

Associations between marine invertebrates and chemoautotrophic bacteria constitute a wide field for the study of symbiotic associations. In these interactions, symbiont transmission must represent the cornerstone allowing the persistence of the association throughout generations. Within Bivalvia , in families such as Solemyidae or Vesicomyidae , symbiont transmission is undoubtedly vertical. However, in Lucinidae , symbiont transmission is described in the literature as 'environmental', symbionts being acquired from the environment by the new host generations. Hence, if there is transmission, symbionts should be transmitted from adults to juveniles via the environment. Consequently, we should observe a release of the symbiont by adults. We attempted to detect such a release within two Lucinidae species of the genus Codakia . We sampled 10 Codakia orbicularis and 20 Codakia orbiculata distributed in 10 crystallizing dishes containing filtered seawater. During 1 month of investigation, we analyzed water of the dishes in order to detect any release of a symbiont using catalyzed report deposition-FISH techniques. For 140 observations realized during this period, we did not observe any release of symbionts. This suggests that the idea of host-to-host passage in Lucinidae is inaccurate. We could therefore consider that the transmission mode from generation to generation does not occur within Lucinidae , symbiosis appearing to be advantageous in this case only for the host, and constitutes an evolutionary dead-end for the bacteria.     

In situ localization of sulphur in the thioautotrophic symbiotic model Lucina pectinata (Gmelin, 1791) by cryo-EFTEM microanalysis

BACKGROUND INFORMATION: Lucina pectinata is a large tropical lucinid known to harbour sulphide-oxidizing bacteria in specialized gill cells. Conventional TEM (transmission electron microscopy) has shown that bacteriocytes also harbour visibly 'empty' vesicles whose chemical content remains, to date, only roughly determined. RESULTS: In the present study, L. pectinata gill tissues were cryo-fixed as fast as possible by performing high-pressure freezing before a freeze-substitution process and finally performing a cryo-embedding in Lowicryl. Ultrathin sections were then used for a cryo-EFTEM (where EFTEM stands for energy-filtered TEM) microanalysis. Results show that bacteriocytes within the gill tissues contain elemental sulphur in small vesicles produced by the host itself. In instances of sporadic depletion of sulphur in the environment, such structures may act as energy sources for bacterial endosymbionts. CONCLUSIONS: The cryo-EFTEM techniques represent (i) the only method used to date to locate and preserve sulphur at the cellular level and (ii) a powerful tool for sulphur metabolism analysis in thioautotrophic symbiont relationships.     

Environmental Acquisition of Thiotrophic Endosymbionts by Deep-Sea Mussels of the Genus Bathymodiolus

Deep-sea Bathymodiolus mussels, depending on species and location, have the capacity to host sulfur-oxidizing (thiotrophic) and methanotrophic eubacteria in gill bacteriocytes, although little is known about the mussels' mode of symbiont acquisition. Previous studies of Bathymodiolus host and symbiont relationships have been based on collections of nonoverlapping species across wide-ranging geographic settings, creating an apparent model for vertical transmission. We present genetic and cytological evidence for the environmental acquisition of thiotrophic endosymbionts by vent mussels from the Mid-Atlantic Ridge. Open pit structures in cell membranes of the gill surface revealed likely sites for endocytosis of free-living bacteria. A population genetic analysis of the thiotrophic symbionts exploited a hybrid zone where two Bathymodiolus species intergrade. Northern Bathymodiolus azoricus and southern Bathymodiolus puteoserpentis possess species-specific DNA sequences that identify both their symbiont strains (internal transcribed spacer regions) and their mitochondria (ND4). However, the northern and southern symbiont-mitochondrial pairs were decoupled in the hybrid zone. Such decoupling of symbiont-mitochondrial pairs would not occur if the two elements were transmitted strictly vertically through the germ line. Taken together, these findings are consistent with an environmental source of thiotrophic symbionts in Bathymodiolus mussels, although an environmentally “leaky” system of vertical transmission could not be excluded.     

Effects of Long-Term Starvation on a Host Bivalve (Codakia orbicularis,Lucinidae) and Its Symbiont Population

The bivalve Codakia orbicularis, hosting sulfur-oxidizing gill endosymbionts, was starved (in artificial seawater filtered through a 0.22-μm-pore-size membrane) for a long-term experiment (4 months). The effects of starvation were observed using transmission electron microscopy, fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH), and flow cytometry to monitor the anatomical and physiological modifications in the gill organization of the host and in the symbiotic population housed in bacteriocytes. The abundance of the symbiotic population decreased through starvation, with a loss of one-third of the bacterial population each month, as shown by CARD-FISH. At the same time, flow cytometry revealed significant changes in the physiology of symbiotic cells, with a decrease in cell size and modifications to the nucleic acid content, while most of the symbionts maintained a high respiratory activity (measured using the 5-cyano-2,3-ditolyl tetrazolium chloride method). Progressively, the number of symbiont subpopulations was reduced, and the subsequent multigenomic state, characteristic of this symbiont in freshly collected clams, turned into one and five equivalent genome copies for the two remaining subpopulations after 3 months. Concomitant structural modifications appeared in the gill organization. Lysosymes became visible in the bacteriocytes, while large symbionts disappeared, and bacteriocytes were gradually replaced by granule cells throughout the entire lateral zone. Those data suggested that host survival under these starvation conditions was linked to symbiont digestion as the main nutritional source.The entire marine Lucinidae family, found in a wide range of sulfidic habitats, lives in association with chemoautotrophic sulfide-oxidizing bacterial symbionts, generally hosted in the gills of the bivalve. Lucinids are usually found in shallow water, such as intertidal mud or seagrasses (4, 53), in deeper water, e.g., Bathyaustriella thionipta (30), and in deep oceans at a 2,000-m depth, i.e., Lucinoma kazani (21, 55). The chemoautotrophic endosymbionts involved in such relationships are always localized inside specialized cells called bacteriocytes, and they have been found in several genera of the Lucinidae family, such as Codakia (4, 28), Loripes (39, 43), Lucina, and Lucinoma (17). Sulfur granules inside the symbiont cytoplasm have been demonstrated in most of the investigated species. The intracellular symbionts take energy from the oxidation of reduced sulfur compounds (27, 56, 59) and synthesize organic molecules by CO₂ fixation in a Calvin-Benson cycle, translocated to the host (18). This relationship between the host and its symbionts represents the autotrophic pathway for host nutrition (27). It has also been suggested that in symbiotic bivalves, intracellular digestion of the symbionts may be a nutrient source for the host, based on studies of hydrothermal vent and shallow water bivalves (6, 26, 39).The relative importance of the autotrophic versus heterotrophic nutritional pathway can be estimated by measuring the carbon isotope (δ-¹³C) ratios in the host tissue. Measuring this δ-¹³C ratio on a wide range of invertebrates suggested that bivalves, including members of the Lucinidae, that live in reduced sediment may obtain a significant proportion of their organic carbon from chemoautotrophic endosymbionts (4, 10, 51, 52, 57). This suggestion was in agreement with the reduced functional digestive system previously described for the Lucinidae family (2, 53). Structural and morphological studies of gills of a few lucinids (belonging to the genera Lucina and Lucinoma) strongly suggested that symbionts play an important role in host nutrition since they occupy about 30% of the gill tissue and produce most of the host energy (17). Nevertheless, an alternative pathway for feeding, i.e., heterotrophic particulate feeding, could occur in some of the lucinid bivalves, since diatoms were found in the stomach of some lucinids (57). Duplessis et al. (22) showed that particulate feeding could be an important part of the nutritional strategy in symbiont-bearing Lucinoma, as opposed to the anatomical features that gave the impression that this bivalve relied only on symbiont nutrition.In natural habitats, chemoautotrophic bivalves live at the interface between an anoxic sulfide-generating zone and water column oxygenated sediment. However, even if they are not close to a vent, these symbiotic organisms often have to deal with environments that are periodically depleted of oxygen (5, 12) and with extremely low sulfide concentration (13, 15, 16). These natural environmental variations lead to annual and seasonal changes in the δ-¹³C ratio, as observed for some thyasirid species (13, 14). This δ-¹³C ratio variation may be assumed to correspond to the capability of the host to rely on both autotrophic and heterotrophic pathways, and the preponderance of one pathway versus the other in the mixotrophic diet has been considered to be the way in which these organisms deal with changes in the chemical composition of their environment.Apart from the decrease in symbiont abundance suggested by transmission electron microscopy (TEM) analysis and a decrease in sulfur and protein content in the gill tissue of thyasirids (20, 38, 40), little is known about the physiological status of these symbionts and the changes undergone by the symbiotic population of starved bivalves. A previous study of the population was carried out under natural conditions with Codakia orbicularis, a chemoautotrophic bivalve. This tropical bivalve lives in shallow-water sediment among the roots of seagrasses (Thalassia testudinum) (1). Like all lucinids studied so far, it is associated with sulfur-oxidizing symbionts (4, 27, 28) containing elemental sulfur in their cytoplasm (42). The bacterial symbiont of C. orbicularis, environmentally transmitted to the host (31), belongs to a single taxonomic group (Gammaproteobacteria) (25) and is shared by several other tropical lucinids (24, 25, 34, 35). Only a few data are available on the physiology of this symbiont. It was characterized by the presence of Rubisco and ATP sulfurylase enzymes and a δ-¹³C ratio typical of chemoautotrophic bivalves (4). Unlike other related thyasirids tested for nitrate respiration even under oxygenated conditions (37), the symbionts of C. orbicularis use oxygen as the primary electron acceptor (23). Initial investigations of the population of Codakia orbicularis'' symbiont revealed that the symbiotic population hosted by freshly collected individuals contained a high proportion of large bacterial cells containing multiple copies of their genome, typical of actively growing cells, despite the absence of dividing cells (11). It was assumed that the host could maintain a pure culture of the symbiont inside the bacteriocytes by regulating the entry and growth of newly recruited symbionts from sediment and probably regulating symbiont densities by host digestion (11).This study was undertaken to investigate the dynamics of the symbiotic population hosted by C. orbicularis under experimental conditions based on long-term starvation of bivalves, i.e., incubated without planktonic food. We set out the ultrastructural, structural, and physiological changes that occurred in the symbiont population by examining the host gill sections using TEM and fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH). A purified fraction of gill endosymbionts was analyzed by flow cytometry (FCM) to investigate the nucleic acid content and cell size of symbionts and by using the 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) method and epifluorescence microscopy to detect the respiratory activity of symbionts. The modifications induced by host starvation in the symbiotic population are described for the period of long-term starvation.     

Transitions in symbiosis: evidence for environmental acquisition and social transmission within a clade of heritable symbionts

A dynamic continuum exists from free-living environmental microbes to strict host-associated symbionts that are vertically inherited. However, knowledge of the forces that drive transitions in symbiotic lifestyle and transmission mode is lacking. Arsenophonus is a diverse clade of bacterial symbionts, comprising reproductive parasites to coevolving obligate mutualists, in which the predominant mode of transmission is vertical. We describe a symbiosis between a member of the genus Arsenophonus and the Western honey bee. The symbiont shares common genomic and predicted metabolic properties with the male-killing symbiont Arsenophonus nasoniae, however we present multiple lines of evidence that the bee Arsenophonus deviates from a heritable model of transmission. Field sampling uncovered spatial and seasonal dynamics in symbiont prevalence, and rapid infection loss events were observed in field colonies and laboratory individuals. Fluorescent in situ hybridisation showed Arsenophonus localised in the gut, and detection was rare in screens of early honey bee life stages. We directly show horizontal transmission of Arsenophonus between bees under varying social conditions. We conclude that honey bees acquire Arsenophonus through a combination of environmental exposure and social contacts. These findings uncover a key link in the Arsenophonus clades trajectory from free-living ancestral life to obligate mutualism, and provide a foundation for studying transitions in symbiotic lifestyle.Subject terms: Microbial ecology, Molecular evolution, Bacterial evolution, Bacterial genetics, Phylogenetics     

Diversity and characterization of bacterial communities of five co‐occurring species at a hydrothermal vent on the Tonga Arc

Host–symbiont relationships in hydrothermal vent ecosystems, supported by chemoautotrophic bacteria as primary producers, have been extensively studied. However, the process by which densely populated co‐occurring invertebrate hosts form symbiotic relationships with bacterial symbionts remains unclear. Here, we analyzed gill‐associated symbiotic bacteria (gill symbionts) of five co‐occurring hosts, three mollusks (“Bathymodiolus” manusensis, B. brevior, and Alviniconcha strummeri) and two crustaceans (Rimicaris variabilis and Austinograea alayseae), collected together at a single vent site in the Tonga Arc. We observed both different compositions of gill symbionts and the presence of unshared operational taxonomic units (OTUs). In addition, the total number of OTUs was greater for crustacean hosts than for mollusks. The phylogenetic relationship trees of gill symbionts suggest that γ‐proteobacterial gill symbionts have coevolved with their hosts toward reinforcement of host specificity, while campylobacterial Sulfurovum species found across various hosts and habitats are opportunistic associates. Our results confirm that gill symbiont communities differ among co‐occurring vent invertebrates and indicate that hosts are closely related with their gill symbiont communities. Considering the given resources available at a single site, differentiation of gill symbionts seems to be a useful strategy for obtaining nutrition and energy while avoiding competition among both hosts and gill symbionts.     

Cenozoic Methane-Seep Faunas of the Caribbean Region

We report new examples of Cenozoic cold-seep communities from Colombia, Cuba, the Dominican Republic, Trinidad, and Venezuela, and attempt to improve the stratigraphic dating of Cenozoic Caribbean seep communities using strontium isotope stratigraphy. Two seep faunas are distinguished in Barbados: the late Eocene mudstone-hosted ‘Joes River fauna’ consists mainly of large lucinid bivalves and tall abyssochrysoid gastropods, and the early Miocene carbonate-hosted ‘Bath Cliffs fauna’ containing the vesicomyid Pleurophopsis, the mytilid Bathymodiolus and small gastropods. Two new Oligocene seep communities from the Sinú River basin in Colombia consist of lucinid bivalves including Elongatolucina, thyasirid and solemyid bivalves, and Pleurophopsis. A new early Miocene seep community from Cuba includes Pleurophopsis and the large lucinid Meganodontia. Strontium isotope stratigraphy suggests an Eocene age for the Cuban Elmira asphalt mine seep community, making it the oldest in the Caribbean region. A new basal Pliocene seep fauna from the Dominican Republic is characterized by the large lucinid Anodontia (Pegophysema). In Trinidad we distinguish two types of seep faunas: the mudstone-hosted Godineau River fauna consisting mainly of lucinid bivalves, and the limestone-hosted Freeman’s Bay fauna consisting chiefly of Pleurophopsis, Bathymodiolus, and small gastropods; they are all dated as late Miocene. Four new seep communities of Oligocene to Miocene age are reported from Venezuela. They consist mainly of large globular lucinid bivalves including Meganodontia, and moderately sized vesicomyid bivalves. After the late Miocene many large and typical ‘Cenozoic’ lucinid genera disappeared from the Caribbean seeps and are today known only from the central Indo-Pacific Ocean. We speculate that the increasingly oligotrophic conditions in the Caribbean Sea after the closure of the Isthmus of Panama in the Pliocene may have been unfavorable for such large lucinids because they are only facultative chemosymbiotic and need to derive a significant proportion of their nutrition from suspended organic matter.     

Transmission Mode Predicts Specificity and Interaction Patterns in Coral-Symbiodinium Networks

Most reef-building corals in the order Scleractinia depend on endosymbiotic algae in the genus Symbiodinium for energy and survival. Significant levels of taxonomic diversity in both partners result in numerous possible combinations of coral-Symbiodinium associations with unique functional characteristics. We created and analyzed the first coral-Symbiodinium networks utilizing a global dataset of interaction records from coral reefs in the tropical Indo-Pacific and Atlantic Oceans for 1991 to 2010. Our meta-analysis reveals that the majority of coral species and Symbiodinium types are specialists, but failed to detect any one-to-one obligate relationships. Symbiont specificity is correlated with a host’s transmission mode, with horizontally transmitting corals being more likely to interact with generalist symbionts. Globally, Symbiodinium types tend to interact with only vertically or horizontally transmitting corals, and only a few generalist types are found with both. Our results demonstrate a strong correlation between symbiont specificity, symbiont transmission mode, and community partitioning. The structure and dynamics of these network interactions underlie the fundamental biological partnership that determines the condition and resilience of coral reef ecosystems.     

The three-dimensional structure of codakine and related marine C-type lectins

Codakine is a new Ca(2+)-dependent mannose-binding C-type lectin (MBL) isolated from the gill tissue of the tropical clam, Codakia orbicularis. Bioinformatic analyses with the BLAST program have revealed similarities with marine lectins involved in immunity whose three-dimensional (3D) structures were unknown up until recently. In this article, we present bioinformatic analyses of marine lectins that are homologous to codakine, in particular lectins from the sea worm Laxus oneistus, named mermaid. These lectins are involved in the symbiotic association with sulphur-oxidizing bacteria which are closely related to the C. orbicularis gill symbiont. Using homology modelling, folding that is characteristic of C-type lectins was observed in all the marine Ca(2+)-dependent lectins studied, with conservation of random coiled structures of the carbohydrate recognition domain (CRD) and Ca(2+)-binding sites. Like codakine, the marine lectins analysed contain a signal peptide commonly found in secreted and transmembrane proteins. The majority of the predictive 3D models established from the lectins exhibit a common feature, namely the involvement in invertebrate and vertebrate immunity (dendritic cell receptor, macrophage receptor, etc.). These bioinformatic analyses and the literature data support the hypothesis that codakine, like the L. oneistus mermaids, is probably involved in the cellular mediation of symbiosis and defence against pathogenic microorganisms.     

Detection of the free-living forms of sulfide-oxidizing gill endosymbionts in the lucinid habitat (Thalassia testudinum environment)

Target DNA from the uncultivable Codakia orbicularis endosymbiont was PCR amplified from sea-grass sediment. To confirm that such amplifications originated from intact bacterial cells rather than free DNA, whole-cell hybridization (fluorescence in situ hybridization technique) with the specific probe Symco2 was performed along with experimental infection of aposymbiotic juveniles placed in contact with the same sediment. Taken together, the data demonstrate that the sulfide-oxidizing gill endosymbiont of Codakia orbicularis is present in the environment as a free-living uncultivable form.     

Sulfur-oxidizing bacterial endosymbionts: analysis of phylogeny and specificity by 16S rRNA sequences.

The 16S rRNAs from the bacterial endosymbionts of six marine invertebrates from diverse environments were isolated and partially sequenced. These symbionts included the trophosome symbiont of Riftia pachyptila, the gill symbionts of Calyptogena magnifica and Bathymodiolus thermophilus (from deep-sea hydrothermal vents), and the gill symbionts of Lucinoma annulata, Lucinoma aequizonata, and Codakia orbicularis (from relatively shallow coastal environments). Only one type of bacterial 16S rRNA was detected in each symbiosis. Using nucleotide sequence comparisons, we showed that each of the bacterial symbionts is distinct from the others and that all fall within a limited domain of the gamma subdivision of the purple bacteria (one of the major eubacterial divisions previously defined by 16S rRNA analysis [C. R. Woese, Microbiol. Rev. 51: 221-271, 1987]). Two host specimens were analyzed in five of the symbioses; in each case, identical bacterial rRNA sequences were obtained from conspecific host specimens. These data indicate that the symbioses examined are species specific and that the symbiont species are unique to and invariant within their respective host species.