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.     

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.     

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.     

Bacterial symbiosis maintenance in the asexually reproducing and regenerating flatworm Paracatenula galateia

Bacteriocytes set the stage for some of the most intimate interactions between animal and bacterial cells. In all bacteriocyte possessing systems studied so far, de novo formation of bacteriocytes occurs only once in the host development, at the time of symbiosis establishment. Here, we present the free-living symbiotic flatworm Paracatenula galateia and its intracellular, sulfur-oxidizing bacteria as a system with previously undescribed strategies of bacteriocyte formation and bacterial symbiont transmission. Using thymidine analogue S-phase labeling and immunohistochemistry, we show that all somatic cells in adult worms - including bacteriocytes - originate exclusively from aposymbiotic stem cells (neoblasts). The continued bacteriocyte formation from aposymbiotic stem cells in adult animals represents a previously undescribed strategy of symbiosis maintenance and makes P. galateia a unique system to study bacteriocyte differentiation and development. We also provide morphological and immunohistochemical evidence that P. galateia reproduces by asexual fragmentation and regeneration (paratomy) and, thereby, vertically transmits numerous symbiont-containing bacteriocytes to its asexual progeny. Our data support the earlier reported hypothesis that the symbiont population is subjected to reduced bottleneck effects. This would justify both the codiversification between Paracatenula hosts and their Candidatus Riegeria symbionts, and the slow evolutionary rates observed for several symbiont genes.     

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.     

Bacteriocyte dynamics during development of a holometabolous insect,the carpenter ant <Emphasis Type="Italic">Camponotus floridanus</Emphasis>

Background  

The carpenter ant Camponotus floridanus harbors obligate intracellular mutualistic bacteria (Blochmannia floridanus) in specialized cells, the bacteriocytes, intercalated in their midgut tissue. The diffuse distribution of bacteriocytes over the midgut tissue is in contrast to many other insects carrying endosymbionts in specialized tissues which are often connected to the midgut but form a distinct organ, the bacteriome. C. floridanus is a holometabolous insect which undergoes a complete metamorphosis. During pupal stages a complete restructuring of the inner organs including the digestive tract takes place. So far, nothing was known about maintenance of endosymbionts during this life stage of a holometabolous insect. It was shown previously that the number of Blochmannia increases strongly during metamorphosis. This implicates an important function of Blochmannia in this developmental phase during which the animals are metabolically very active but do not have access to external food resources. Previous experiments have shown a nutritional contribution of the bacteria to host metabolism by production of essential amino acids and urease-mediated nitrogen recycling. In adult hosts the symbiosis appears to degenerate with increasing age of the animals.     

Gill ultrastructure and symbiotic bacteria in Codakia orbicularis (Bivalvia,Lucinidae)

Summary The cellular organization of the gill, which harbors symbiotic bacteria, is described in juveniles and adults of Codakia orbicularis, a large tropical Lucinidae. The ciliary zone is similar in every species of Lucinidae described and includes the large clear cell which has been previously described as an intermediary cell. The intermediary zone is composed of a few narrow unciliated cells, which bind adjacent filaments together and constitute channels through which sea water circulates along the abfrontal part of the filaments. The lateral zone is more complex in C. orbicularis than in other Lucinidae, being composed of four cell types and differentiated into two distinct regions. The bacteriocytes and intercalary cells occupy the outermost bacteriocyte zone, while mucocytes and numerous cells crowded with proteinic, cystine-rich granules constitute the innermost secretory zone which has not been described in other species. The newly described granule cells are considered to be a key factor in the storage and metabolic conversion of sulfur compounds.     

Putative environmental transmission of sulfur-oxidizing bacterial symbionts in tropical lucinid bivalves inhabiting various environments

Four tropical lucinids, Codakia orbiculata, C. pectinella, Linga pensylvanica, which inhabit sea-grass beds, and Lucina pectinata, which inhabits mangrove swamps, harbor sulfur-oxidizing endosymbiotic bacteria within bacteriocytes of their gill filaments. To elucidate the symbiont transmission mode in these bivalves, symbiont-specific oligonucleotides were designed and used in polymerase chain reaction amplifications (PCR). For all species investigated, each primer set was unsuccessful in amplifying symbiont DNA targets from ovaries and testis, whereas successful amplifications were obtained from symbiont-containing gill tissue. These data suggest that the transmission mode is environmental, independently of the lucinid habitat, as it is in the other tropical lucinid Codakia orbicularis.     

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.     

Ultrastructural Differences Between Species of Trypanosomatids With and Without Endosymbionts1

Species of trypanosomatids without endosymbionts (Leptomonas seymouri, L. collosoma, L. samueli, Crithidia fasciculata, C. luciliae, C. acanthocephali, Herpetomonas megaseliae, H. mariadeanei, H. samuelpessoai, H. muscarum muscarum, Trypanosoma cruzi) and species of trypanosomatids with endosymbionts (Crithidia deanei, C. oncopelti, Blastocrithidia culicis) were comparatively studied by means of electron microscopy. Artificially aposymbiotic strains derived from species with symbiont were also included in the survey. Species with symbiont were found to differ in some ultrastructural aspects from the group of species without symbiont. Paraxial rods of flagella or intraflagellar structure were found exclusively in species without symbiont. Peripheral branching of mitochondria, accompanied by absence of subpellicular microtubules in sites where the mitochondrial branches are appressed to the cell membrane, were found exclusively in species with symbiont. Networks of kinetoplast DNA fibrils were found to be larger and looser in species with symbiont. Symbiont-free strains of species with symbiont retained the same morphological characteristics of their parental species.     

Toxicological effects of biocides on symbiotic and aposymbiotic juveniles of the hermatypic coral Acropora tenuis

Reef-building (or hermatypic) corals harbor the symbiotic dinoflagellates Symbiodinium spp. (Alveolata, Dinophyceae, Gymnodiniales), and contribute to the accretion of coral reefs in tropical and sub-tropical zones. In this study, toxicological effects of three commonly used biocides (dichlorvos (DDVP), a commonly used insecticide; diuron (DCMU), a herbicide; and tributyltin chloride (TBT-Cl), an anti-fouling agent) on the hermatypic coral Acropora tenuis (Anthozoa, Hexacorallia, Scleractinia) were studied using juveniles in both aposymbiotic (symbiont-free) and symbiotic conditions. After exposure to the chemicals, abnormalities such as detachment of soft tissues from the skeleton and/or death were observed, as well as lowered uptake of symbiotic algae (in aposymbiotic juveniles) or reduction of the symbiont population in tentacles (in symbiotic juveniles). Significant reduction of the symbiont population in tentacles of symbiotic juveniles exposed to DDVP, DCMU and TBT-Cl was observed at the concentrations of 100, 10, and 1 μg/l, respectively. Results of this study suggested that symbiotic juveniles of A. tenuis are more sensitive than in the aposymbiotic condition to DDVP and DCMU, but not to TBT-Cl.     

Evidence for phylogenetic congruence among sulfur-oxidizing chemoautotrophic bacterial endosymbionts and their bivalve hosts

Sulfur-oxidizing chemoautotrophic (thioautotrophic) bacteria are now known to occur as endosymbionts in phylogenetically diverse bivalve hosts found in a wide variety of marine environments. The evolutionary origins of these symbioses, however, have remained obscure. Comparative 16S rRNA sequence analysis was used to investigate whether thioautotrophic endosymbionts are monophyletic or polyphyletic in origin and to assess whether phylogenetic relationships inferred among these symbionts reflect those inferred among their hosts. 16S rRNA gene sequences determined for endosymbionts from nine newly examined bivalve species from three families (Vesicomyidae, Lucinidae, and Solemyidae) were compared with previously published 16S rRNA sequences of thioautotrophic symbionts and free-living bacteria. Distance and parsimony methods were used to infer phylogenetic relationships among these bacteria. All newly examined symbionts fall within the gamma subdivision of the Proteobacteria, in clusters containing previously examined symbiotic thioautotrophs. The closest free-living relatives of these symbionts are bacteria of the genus Thiomicrospira. Symbionts of the bivalve superfamily Lucinacea and the family Vesicomyidae each form distinct monophyletic lineages which are strongly supported by bootstrap analysis, demonstrating that host phylogenies inferred from morphological and fossil evidence are congruent with phylogenies inferred for their respective symbionts by molecular sequence analysis. The observed congruence between host and symbiont phylogenies indicates shared evolutionary history of hosts and symbiont lineages and suggests an ancient origin for these symbioses. Correspondence to: D.L. Distel     

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.     

Coral ontogeny affects early symbiont acquisition in laboratory-reared recruits

In most coral species, the critical association with a subset of genetically diverse algal endosymbionts, Symbiodinium, is re-established anew each generation in early coral ontogeny. Yet little is known about the window during which these associations are established or the potential for altering symbiont associations through early exposure to non-native, and/or ecologically beneficial (e.g., stress tolerant), symbiont strains. This study examined the ontogenetic window of symbiont uptake in a restoration target species. Orbicella faveolata recruits, maintained aposymbiotic in laboratory tanks for 4 months, showed a significant decrease in symbiont acquisition upon exposure to natural seawater. Recruits initially inoculated with cultured Symbiodinium readily acquired additional strains from environmental symbiont populations upon exposure, but exogenous uptake also decreased in frequency after 4 months of laboratory rearing. Early exposure to Symbiodinium may benefit laboratory-reared recruits (e.g., enhance growth), but the potential for establishing long-term novel symbiotic associations may be limited.

     

Effects of mass releases of Trichogramma brassicae on predatory insects in maize

Abstract Females of the rove beetle genus Paederus (Coleoptera: Staphylinidae) accumulate large amounts of the defensive compound pederin produced by an endosymbiotic bacterium. Pederin is transferred in the eggs, while the endosymbionts are transmitted via the egg shell. In all three species analyzed (P. melanurus, P. riparius, and P. sabaeus), descendants of aposymbiotic matrilines (lacking both endosymbionts and biosynthetic capabilities) acquire biosynthetic capability by the ingestion of eggs with endosymbionts during larval development. Successful colonization by endosymbionts depends on the number of eggs consumed and the age of the feeding larvae. During the adult stage, however, the females cannot acquire biosynthetic capabilities. Adult females are able to sequester pederin from eggs eaten, and they transfer the substance into their own eggs. Lack of intrinsic biosynthesis after ingestion of endosymbionts indicates that these are not biosynthetically active within the gut and have to reach an unknown internal location before completion of metamorphosis. These results are discussed with regard to the benefit of intraspecific cannibalism.     

Structure and position of bacterial endosymbionts in the gill filaments of lucinidae from bermuda (Mollusca,Bivalvia)

Summary The gills of four lucinid bivalves from fine sediments in shallow reaches of Bermuda, Anodontia philippiana, Lucina multilineata, L. radians, and L. costata, were observed by electron microscope. They harbour intracellular bacterial endosymbionts similar to those in gills of clams from hydrothermal vents and other sulphide biotopes. The bacteria-containing bacteriocytes are found in distinct positions in the gill filaments, alternating with cells not invaded by the prokaryotes but with abundant mitochondria. Referring to the specific environment of the collected bivalves and to literature data from corresponding studies, the significance of this bacteria/animal association is discussed.     

Structural and ultrastructural analysis of the gills in the bacterial-bearing species <Emphasis Type="Italic">Thyasira falklandica</Emphasis> (Bivalvia,Mollusca)

In this study, the cellular organization of the gill that harbors symbiotic bacteria is described in the thyasirid Thyasira falklandica collected from South Shetlands in Antarctic. Sections of the gills revealed that T. falklandica belongs to the gill type 3, as described by Dufour (Biol Bull, 208:200–212, 2005), with an elongated lateral zone along the frontal-abfrontal axis of the gill filaments. The ciliated and intermediary zones looked similar to those described in symbionts-bearing bivalves. The lateral zone is more complex in T. falklandica than in other Thyasiridae already described. Such a zone is composed of four different cell types. Bacteriocytes are abundant in the frontal and abfrontal positions, while the middle part of the lateral zone is occupied mostly by numerous granule cells devoid of bacteria. All along the lateral zone, TEM and SEM observations show some ciliated cells, which are regularly interspersed between bacteriocytes and/or granule cells. Such cells, according to the long double ciliary roots of their cilia, should have a sensory function. Intercalary cells, which have never been observed between bacteriocytes, are restricted to the middle part of the lateral zone where their expansions overlap the adjacent granule cells. Bacterial symbionts occur only extracellularly among long microvilli differentiated by the bacteriocytes. They are abundant, usually spherical in shape (around 0.7 μm length), and covered by the glycocalix from bacteriocyte microvilli. According to TEM views, the empty vesicles located in the periplasmic space should be sulfur storage, as known for other sulfur-oxidizing symbionts.     

Symbiosis in the green leafhopper,Cicadella viridis (Hemiptera,Cicadellidae). Association in statu nascendi?

The green leafhopper, Cicadella viridis lives in symbiotic association with microorganisms. The ultrastructural and molecular analyses have shown that in the body of the C. viridis two types of bacteriocyte endosymbionts are present. An amplification and sequencing of 16S rRNA genes revealed that large, pleomorphic bacteria display a high similarity (94–100%) to the endosymbiont ‘Candidatus Sulcia muelleri’ (phylum Bacteroidetes), whereas long, rod-shaped microorganisms are closely related to the γ-proteobacterial symbiont Sodalis (97–99% similarity). Both endosymbionts may be harbored in their own bacteriocytes as well as may co-reside in the same bacteriocytes. The ultrastructural observations have revealed that the Sodalis-like bacteria harboring the same bacteriocytes as bacterium Sulcia may invade the cells of the latter. Bacteria Sulcia and Sodalis-like endosymbionts are transovarially transmitted from one generation to the next. However, Sodalis-like endosymbionts do not invade the ovaries individually, but only inside Sulcia cells. Apart from bacteriocyte endosymbionts, in the body of C. viridis small, rod-shaped bacteria have been detected, and have been identified as being closely related to γ-proteobacterial microorganism Pectobacterium (98–99% similarity). The latter are present in the sheath cells of the bacteriomes containing bacterium Sulcia as well as in fat body cells.     

Identification and Characterization of a Flagellin Gene from the Endosymbiont of the Hydrothermal Vent Tubeworm Riftia pachyptila

The bacterial endosymbionts of the hydrothermal vent tubeworm Riftia pachyptila play a key role in providing their host with fixed carbon. Results of prior research suggest that the symbionts are selected from an environmental bacterial population, although a free-living form has been neither cultured from nor identified in the hydrothermal vent environment. To begin to assess the free-living potential of the symbiont, we cloned and characterized a flagellin gene from a symbiont fosmid library. The symbiont fliC gene has a high degree of homology with other bacterial flagellin genes in the amino- and carboxy-terminal regions, while the central region was found to be nonconserved. A sequence that was homologous to that of a consensus ς²⁸ RNA polymerase recognition site lay upstream of the proposed translational start site. The symbiont protein was expressed in Escherichia coli, and flagella were observed by electron microscopy. A 30,000-M_r protein subunit was identified in whole-cell extracts by Western blot analysis. These results provide the first direct evidence of a motile free-living stage of a chemoautotrophic symbiont and support the hypothesis that the symbiont of R. pachyptila is acquired with each new host generation.     

Who wins in the weaning process? Juvenile feeding morphology of two freshwater mussel species

The global decline of freshwater mussels can be partially attributed to their complex life cycle. Their survival from glochidium to adulthood is like a long obstacle race, with juvenile mortality as a key critical point. Mass mortality shortly after entering into a juvenile state has been reported in both wild and captive populations, thus weakening the effective bivalve population. A similar phenomenon occurs during metamorphosis in natural and hatchery populations of juvenile marine bivalves. Based on a morphological analysis using scanning electron microscopy of newly formed juveniles of the freshwater species Margaritifera margaritifera (L.) (Margaritiferidae) and Unio mancus Lamarck (Unionidae), we show that a second metamorphosis, consisting of drastic morphological changes, occurs that leads to suspension feeding in place of deposit feeding by the ciliated foot. We hypothesize that suspension feeding in these two species improves due to a gradual development of several morphological features including the contact between cilia of the inner gill posterior filaments, the inner gill reflection, the appearance of the ctenidial ventral groove and the formation of the pedal palps. Regardless of the presence of available food, a suspension feeding mode replaces deposit feeding, and juveniles unable to successfully transition morphologically or adapt to the feeding changes likely perish.