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.     

Location of glycogen in spermatids and spermatozoa of the shipworm,Bankia australis (Teredinidae,Bivalvia, Mollusca)

The periodic acid-thiocarbohydrazide-silver proteinate technique and alpha-amylase digestion were used to locate glycogen in the spermatids and sperms of the bivalve Bankia australis. Glycogen was found in the middle piece and around the acrosome in spermatozoa, apparently randomly scattered throughout the cytoplasm of young spermatids, and in a cytoplasmic bead in old spermatids.     

Extensive variation in intracellular symbiont community composition among members of a single population of the wood-boring bivalve Lyrodus pedicellatus (Bivalvia: Teredinidae)

Shipworms (wood-boring bivalves of the family Teredinidae) harbor in their gills intracellular bacterial symbionts thought to produce enzymes that enable the host to consume cellulose as its primary carbon source. Recently, it was demonstrated that multiple genetically distinct symbiont populations coexist within one shipworm species, Lyrodus pedicellatus. Here we explore the extent to which symbiont communities vary among individuals of this species by quantitatively examining the diversity, abundance, and pattern of occurrence of symbiont ribotypes (unique 16S rRNA sequence types) among specimens drawn from a single laboratory-reared population. A total of 18 ribotypes were identified in two clone libraries generated from gill tissue of (i) a single specimen and (ii) four pooled specimens. Phylogenetic analysis assigned all of the ribotypes to a unique clade within the gamma subgroup of proteobacteria which contained at least five well-supported internal clades (phylotypes). By competitive quantitative PCR and constant denaturant capillary electrophoresis, we estimated the number and abundance of symbiont phylotypes in gill samples of 13 individual shipworm specimens. Phylotype composition varied greatly; however, in all specimens the numerically dominant symbiont belonged to one of two nearly mutually exclusive phylotypes, each of which was detected with similar frequencies among specimens. A third phylotype, containing the culturable symbiont Teredinibacter turnerae, was identified in nearly all specimens, and two additional phylotypes were observed more sporadically. Such extensive variation in ribotype and phylotype composition among host specimens adds to a growing body of evidence that microbial endosymbiont populations may be both complex and dynamic and suggests that such genetic variation should be evaluated with regard to physiological and ecological differentiation.     

The fine structure of the oocyte of Bankia australis (Teredinidae,Bivalvia) before and after fertilization

The fine structure of the oocyte of Bankia australis is compared with that of other bivalve oocytes. It was observed that following fertilization, the microvilli changed their spatial organisation and behaviour towards sperm, the cortical granules disappeared in regions of high concentrations of supernumerary sperm, and the mitochondria apparently stated to divide.     

Occurrence and settlement of the common shipworm Teredo navalis (Bivalvia: Teredinidae) in Bremerhaven harbours, northern Germany

The shipworm Teredo navalis L. is a xylophagous bivalve mollusc (Bivalvia: Teredinidae) with a long record of being very destructive to wooden ships and harbour buildings. It has been reported from numerous sites at the coasts of both the North and Baltic Seas since the eighteenth century. Here, we document for the first time the occurrence of live adult T. navalis in the harbours of Bremerhaven (Weser estuary, northern Germany). From August to December 1998, various wooden structures (fir floating fenders and pier posts, oak piles) from seven stations in different docks of two harbours (überseehafen, Fischereihafen) were investigated for the presence and density of live specimens and burrows of T. navalis. The settlement of larval shipworms was studied by exposing experimental fir panels 0.06 m² in size at 20 stations at water depths between 1 and 2 m for periods of 4 months between July and November. In addition, hydrographic profiles (0–8 m water depth) were obtained at 17 stations in five docks once every month from August to December. Live adult shipworms were found in both fir floating fenders and oak piles at four stations. The largest specimen found was 250 mm long. Shipworm burrows were detected at five stations in almost every wooden structure investigated but their abundances differed significantly: Maximum values were >10,000 m^–2 in fir floating fenders, 4,600 m^–2 in oak piles and 200 m^–2 in fir pier posts. Actual shipworm infestation was detected at three of 16 stations in the exposed fir panels (1–3 burrow holes per panel). Water temperatures and salinities varied considerably during the 4-month investigation period. Temperatures decreased from 19.9°C in August to 0.7°C in December. Salinities ranged from 17.6 in August to 1.1 in November, but only at two lock stations during November and December did value drop below 5, which is regarded as the lethal limit for the larvae of this euryhaline teredinid species. We conclude that T. navalis encounters favourable conditions for growth and reproduction in the harbours of Bremerhaven, at least during summer and autumn, and is a common element of the harbour ecosystem. Therefore, a persistent infestation of all wooden structures after a relatively short period of time seems to be highly probable. Electronic Publication     

Physiological and biochemical energetics of larvae of Teredo navalis L. and Bankia gouldi (Bartsch) (Bivalvia : Teredinidae)

The biochemical composition of larvae of Teredo navalis L. and Bankia gouldi (Bartsch) (Bivalvia: Teredinidae) was examined throughout larval development at 23°C and 30–32%. salinity in the presence of the phytoplankton food Isochrysis aff. galbana (clone T-ISO), during a delay of metamorphosis in the presence of food but absence of a wood substratum and during periods of enforced starvation. Newly released Teredo navalis larvae had a mean length (L) and height (H) of 89.3 and 76.1 μm respectively. The first appearance of pediveliger larvae at 212.1 μm L and 230.0 μm H occurred 27 days after release. Larval dry weight increased from 0.29 μg to 1.96 μg during this period. Newly formed straight hinge larvae of Bankiagouldi had dimensions of 62.8 μm L and 49.8 μm H. Metamorphically competent B. gouldi larvae had dimensions of 230.0 μm L and 282.9 μm H and were first observed 20 days after fertilization. Larval dry weight increased from 0.06 μg to 2.20 μg during this period. During enforced delay of metamorphosis the ash-free dry weight of Teredo navalis larvae decreased whereas the ash free dry weight of Bankia gouldi larvae increased. During the early period of shelled larval development both species showed similar decreases in lipid, protein and carbohydrate levels (μg·mg dry weight^?1); however, this was reflected in a decrease in biochemical content (μg·larva^?1) only in Teredo navalis. During enforced starvation the major proportion of both the weight and caloric losses were due to protein. Lipid also contributes significantly to these losses whereas the contribution of carbohydrate was small. Larval oxygen consumption rates were determined directly by manometry and indirectly by estimates of decrease in caloric content during periods of enforced starvation. Direct and indirect determinations for T. navalis are described by the relationships R = 1.16 W^1.05 and R = 0.98 W^1.24 respectively where R is the respiration rate in nl O₂ · larva^?1 · h^?1 and W is dry weight inclusive of shell in μg. Direct and indirect determinations for Bankia gouldi are described by the relationships R = 1.37 W^1.25 and R = 1.81 W^1.25 respectively. When data for both assay procedures are combined for each species the relationships R = 1.10 W^1.07 and R = 1.44 W^1.18 are obtained for Teredonavalis and Bankia gouldi respectively.     

The broadcast spawning Caribbean shipworm,Teredothyra dominicensis (Bivalvia,Teredinidae), has invaded and become established in the eastern Mediterranean Sea

Teredinids, commonly referred to as shipworms, are wood-boring bivalves estimated to cause over one billion dollars’ worth of damage to submerged wooden structures per annum. This paper reports the detection and establishment of the Caribbean shipworm Teredothyra dominicensis (Bivalvia, Teredinidae) in the eastern Mediterranean Sea. Identification was confirmed using an integrative taxonomical approach combining morphology, morphometry and molecular markers (COI-5P and 18S), thus improving both the taxonomic resolution and tractability of this invasive species. Sequence comparisons between indigenous Caribbean and Mediterranean specimens were at least 99 % identical. Wooden panels placed at the site of discovery were infested exclusively by T. dominicensis with specimens of varying size and age, indicating multiple settlement events and the presence of breeding populations in the region. Anatomical and behavioural observations confirm the species as a broadcast spawner with larvae undergoing planktotrophic development, thus distribution range is potentially extensive. Of the possible introduction vectors, transport via ballast water is proposed as the most likely. The establishment of breeding populations of a tropical teredinid in the Mediterranean is of considerable concern as tropical species are particularly destructive and degrade wood more rapidly than the species currently found in the region. This threat is likely to increase in severity due to global warming, as increases in temperature and salinity may lead to an increase in the distribution range, development rate and boring activity of teredinids.     

Distribution, settlement, and growth of first-year individuals of the shipworm Teredo navalis L. (Bivalvia: Teredinidae) in the Port of Rotterdam area, the Netherlands

During the period 2004-2008 the distribution, settlement, and growth of first-year shipworms (Teredo navalis L., 1758) was studied by exposing fir and oak panels in the Port of Rotterdam area, which is situated in the Rhine-Meuse estuary in the Netherlands and covers the complete salinity gradient. Shipworms were found yearly in the western large polyhaline harbours. On only a few occasions were they were found in harbours that showed large seasonal and daily fluctuations in salinity. In 2006 the shipworm was found in fir panels 20 km upstream from the polyhaline harbours, demonstrating their ability to travel with the tidal currents over considerable distances and to settle once the abiotic conditions become favourable. Although the water temperatures allowed them to breed from April until November, infestations were not found before September, and from the size of the animals in the panels it was concluded that in the Port of Rotterdam area they spawned from August until the end of November. The settlement height was negatively correlated with the distance of the panels to the sea floor. In the first season after settlement they showed a substantial growth rate of 0.18 cm day⁻¹. The longest shipworm found measured 36.8 cm after 4-5 months of growth after settlement. Infestations and growth were lower in oak than in fir wood. In 2006 the maximum consumption of wood by individuals settled in the same year in panels at the bottom accounted for 12.4%. Shell size and body length of the animal after the first season of growth showed a significant positive logarithmic relation. In both 2006 and 2007 a similar relation between the average boring tube diameter and the length of the animals was found. Lower river discharges leading to salinisation of the eastern part of the Port of Rotterdam area create conditions favourable for the shipworm, with serious consequences for the piles upon which the quays are built.     

Microbial Distribution and Abundance in the Digestive System of Five Shipworm Species (Bivalvia: Teredinidae)

Marine bivalves of the family Teredinidae (shipworms) are voracious consumers of wood in marine environments. In several shipworm species, dense communities of intracellular bacterial endosymbionts have been observed within specialized cells (bacteriocytes) of the gills (ctenidia). These bacteria are proposed to contribute to digestion of wood by the host. While the microbes of shipworm gills have been studied extensively in several species, the abundance and distribution of microbes in the digestive system have not been adequately addressed. Here we use Fluorescence In-Situ Hybridization (FISH) and laser scanning confocal microscopy with 16S rRNA directed oligonucleotide probes targeting all domains, domains Bacteria and Archaea, and other taxonomic groups to examine the digestive microbiota of 17 specimens from 5 shipworm species (Bankia setacea, Lyrodus pedicellatus, Lyrodus massa, Lyrodus sp. and Teredo aff. triangularis). These data reveal that the caecum, a large sac-like appendage of the stomach that typically contains large quantities of wood particles and is considered the primary site of wood digestion, harbors only very sparse microbial populations. However, a significant number of bacterial cells were observed in fecal pellets within the intestines. These results suggest that due to low abundance, bacteria in the caecum may contribute little to lignocellulose degradation. In contrast, the comparatively high population density of bacteria in the intestine suggests a possible role for intestinal bacteria in the degradation of lignocellulose.     

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.     

Extensive Variation in Intracellular Symbiont Community Composition among Members of a Single Population of the Wood-Boring Bivalve Lyrodus pedicellatus (Bivalvia: Teredinidae)

Shipworms (wood-boring bivalves of the family Teredinidae) harbor in their gills intracellular bacterial symbionts thought to produce enzymes that enable the host to consume cellulose as its primary carbon source. Recently, it was demonstrated that multiple genetically distinct symbiont populations coexist within one shipworm species, Lyrodus pedicellatus. Here we explore the extent to which symbiont communities vary among individuals of this species by quantitatively examining the diversity, abundance, and pattern of occurrence of symbiont ribotypes (unique 16S rRNA sequence types) among specimens drawn from a single laboratory-reared population. A total of 18 ribotypes were identified in two clone libraries generated from gill tissue of (i) a single specimen and (ii) four pooled specimens. Phylogenetic analysis assigned all of the ribotypes to a unique clade within the γ subgroup of proteobacteria which contained at least five well-supported internal clades (phylotypes). By competitive quantitative PCR and constant denaturant capillary electrophoresis, we estimated the number and abundance of symbiont phylotypes in gill samples of 13 individual shipworm specimens. Phylotype composition varied greatly; however, in all specimens the numerically dominant symbiont belonged to one of two nearly mutually exclusive phylotypes, each of which was detected with similar frequencies among specimens. A third phylotype, containing the culturable symbiont Teredinibacter turnerae, was identified in nearly all specimens, and two additional phylotypes were observed more sporadically. Such extensive variation in ribotype and phylotype composition among host specimens adds to a growing body of evidence that microbial endosymbiont populations may be both complex and dynamic and suggests that such genetic variation should be evaluated with regard to physiological and ecological differentiation.     

Developmental dynamics of myogenesis in the shipworm <Emphasis Type="Italic">Lyrodus pedicellatus</Emphasis> (Mollusca: Bivalvia)

Background

The shipworm Lyrodus pedicellatus is a wood-boring bivalve with an unusual vermiform body. Although its larvae are brooded, they retain the general appearance of a typical bivalve veliger-type larva. Here, we describe myogenesis of L. pedicellatus revealed by filamentous actin labelling and discuss the data in a comparative framework in order to test for homologous structures that might be part of the bivalve (larval) muscular ground pattern.

Results

Five major muscle systems were identified: a velum retractor, foot retractor, larval retractor, a distinct mantle musculature and an adductor system. For a short period of larval life, an additional ventral larval retractor is present. Early in development, a velum muscle ring and an oral velum musculature emerge. In late stages the lateral and dorsal mantle musculature, paired finger-shaped muscles, an accessory adductor and a pedal plexus are formed. Similar to other bivalve larvae, L. pedicellatus exhibits three velum retractor muscles, but in contrast to other species, one of them disappears in early stages of L. pedicellatus. The remaining two velum retractors are considerably remodelled during late larval development and are most likely incorporated into the elaborate mantle musculature of the adult.

Conclusions

To our knowledge, this is the first account of any larval retractor system that might contribute to the adult bodyplan of a (conchiferan) mollusk. A comparative analysis shows that a pedal plexus, adductors, a larval velum ring, velum retractors and a ventral larval retractor are commonly found among bivalve larvae, and thus most likely belong to the ground pattern of the bivalve larval musculature.

     

Characterization of the population of the sulfur-oxidizing symbiont of Codakia orbicularis (Bivalvia, Lucinidae) by single-cell analyses

We investigated the characteristics of the sulfur-oxidizing symbiont hosted in the gills of Codakia orbicularis, a bivalve living in shallow marine tropical environments. Special attention was paid to describing the heterogeneity of the population by using single-cell approaches including flow cytometry (FCM) and different microscopic techniques and by analyzing a cell size fractionation experiment. Up to seven different subpopulations were distinguished by FCM based on nucleic acid content and light side scattering of the cells. The cell size analysis of symbionts showed that the symbiotic population was very heterogeneous in size, i.e., ranging from 0.5 to 5 mum in length, with variable amounts of intracellular sulfur. The side-scatter signal analyzed by FCM, which is often taken as a proxy of cell size, was greatly influenced by the sulfur content of the symbionts. FCM revealed an important heterogeneity in the relative nucleic acid content among the subclasses. The larger cells contained exceptionally high levels of nucleic acids, suggesting that these cells contained multiple copies of their genome, i.e., ranging from one copy for the smaller cells to more than four copies for the larger cells. The proportion of respiring symbionts (5-cyano-2,3-ditolyl-terazolium chloride positive) in the bacteriocytes of Codakia revealed that around 80% of the symbionts hosted by Codakia maintain respiratory activity throughout the year. These data allowed us to gain insight into the functioning of the symbionts within the host and to propose some hypotheses on how the growth of the symbionts is controlled by the host.     

The rectal glands of Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae) hermaphrodites and their role in symbiont transmission

Differential interference contrast, transmission electron and epifluorescence microscopy techniques were employed to examine the ultrastructure of the rectal glands in Heterorhabditis bacteriophora hermaphrodites, with special attention to the location of Photorhabdus bacteria symbionts within these structures. Three rectal glands were clearly visualized in all examined specimens, with two glands positioned sub-ventrally and another gland located dorsally. The dorsal rectal gland in all examined specimens is larger than the subventral ones. Our observations indicate that Photorhabdus bacteria do not colonize the rectal glands of H. bacteriophora hermaphrodites, but rather are present in the most posterior-intestinal cells.     

Presumptive horizontal symbiont transmission in the fungus-growing termite Macrotermes natalensis

All colonies of the fungus-growing termite Macrotermes natalensis studied so far are associated with a single genetically variable lineage of Termitomyces symbionts. Such limited genetic variation of symbionts and the absence of sexual fruiting bodies (mushrooms) on M. natalensis mounds would be compatible with clonal vertical transmission, as is known to occur in Macrotermes bellicosus. We investigated this hypothesis by analysing DNA sequence polymorphisms as codominant SNP markers of four single-copy gene fragments of Termitomyces isolates from 31 colonies of M. natalensis. A signature of free recombination was found, indicative of frequent sexual horizontal transmission. First, all 31 strains had unique multilocus genotypes. Second, SNP markers (n = 55) were largely in Hardy-Weinberg equilibrium (90.9%) and almost all possible pairs of SNPs between genetically unlinked loci were in linkage equilibrium (96.7%). Finally, extensive intragenic recombination was found, especially in the EF1alpha fragment. Substantial genetic variation and a freely recombining population structure can only be explained by frequent horizontal and sexual transmission of Termitomyces. The apparent variation in symbiont transmission mode among Macrotermes species implies that vertical symbiont transmission can evolve rapidly. The unexpected finding of horizontal transmission makes the apparent absence of Termitomyces mushrooms on M. natalensis mounds puzzling. To our knowledge, this is the first detailed study of the genetic population structure of a single lineage of Termitomyces.