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.     

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.     

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.     

Molecular Identification of Algal Endosymbionts in Large Miliolid Foraminifera: 1. Chlorophytes

Large miliolid foraminifers bear various types of algal endosymbionts including chlorophytes, dinoflagellates, rhodophytes, and diatoms. Symbiosis plays a key role in the adaptation of large foraminifera to survival and growth in oligotrophic seas. The identity and diversity of foraminiferal symbionts, however, remain largely unknown. In the present work we use ribosomal DNA (rDNA) sequences to identify chlorophyte endosymbionts in large miliolid foraminifera of the superfamily Soritacea. Partial 18S and complete Internal Transcribed Spacer (ITS) rDNA sequences were obtained from symbionts of eight species representing all genera of extant chlorophyte-bearing Soritacea. Phylogenetic analysis of the sequences confirms the previous fine structure-based identification of these endosymbionts as belonging to the genus Chlamydomonas. All foraminiferal symbionts form a monophyletic group closely related to Chlamydomonas noctigama. The group is composed of seven types identified in this study, including one previously morphologically described species, Chlamydomonas hedleyi. Each of these types can be considered as a separate species, based on the comparison of genetic differences observed between other established Chlamydomonas species. Several foraminiferal species share the same symbiont type, but only one species, Archaias angulatus, was found to bear more than one type.     

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.     

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     

Characterization of chemoautotrophic bacterial symbionts in a gutless marine worm Oligochaeta, Annelida) by phylogenetic 16S rRNA sequence analysis and in situ hybridization.

The phylogenetic relationships of chemoautotrophic endosymbionts in the gutless marine oligochaete Inanidrilus leukodermatus to chemoautotrophic ecto- and endosymbionts from other host phyla and to free-living bacteria were determined by comparative 16S rRNA sequence analysis. Fluorescent in situ hybridization confirmed that the 16S rRNA sequence obtained from these worms originated from the symbionts. The symbiont sequence is unique to I. leukodermatus. In phylogenetic trees inferred by both distance and parsimony methods, the oligochaete symbiont is peripherally associated with one of two clusters of chemoautotrophic symbionts that belong to the gamma subdivision of the Proteobacteria. The endosymbionts of this oligochaete form a monophyletic group with chemoautotrophic ectosymbionts of a marine nematode. The oligochaete and nematode symbionts are very closely related, although their hosts belong to separate, unrelated animal phyla. Thus, cospeciation between the nematode and oligochaete hosts and their symbionts could not have occurred. Instead, the similar geographic locations and habitats of the hosts may have influenced the establishment of these symbioses.     

Molecular and ecological evidence for species specificity and coevolution in a group of marine algal-bacterial symbioses

The phylogenetic relationships of bacterial symbionts from three gall-bearing species in the marine red algal genus Prionitis (Rhodophyta) were inferred from 16S rDNA sequence analysis and compared to host phylogeny also inferred from sequence comparisons (nuclear ribosomal internal-transcribed-spacer region). Gall formation has been described previously on two species of Prionitis, P. lanceolata (from central California) and P. decipiens (from Peru). This investigation reports gall formation on a third related host, Prionitis filiformis. Phylogenetic analyses based on sequence comparisons place the bacteria as a single lineage within the Roseobacter grouping of the alpha subclass of the division Proteobacteria (99.4 to 98.25% sequence identity among phylotypes). Comparison of symbiont and host molecular phylogenies confirms the presence of three gall-bearing algal lineages and is consistent with the hypothesis that these red seaweeds and their bacterial symbionts are coevolving. The species specificity of these associations was investigated in nature by whole-cell hybridization of gall bacteria and in the laboratory by using cross-inoculation trials. Whole-cell in situ hybridization confirmed that a single bacterial symbiont phylotype is present in galls on each host. In laboratory trials, bacterial symbionts were incapable of inducing galls on alternate hosts (including two non-gall-bearing species). Symbiont-host specificity in Prionitis gall formation indicates an effective ecological separation between these closely related symbiont phylotypes and provides an example of a biological context in which to consider the organismic significance of 16S rDNA sequence variation.     

Independent origins and horizontal transfer of bacterial symbionts of aphids

Many insect groups have obligate associations with primary endosymbionts: mutualistic bacteria that are maternally transmitted and derived from an ancient infection. Often, the same insects are hosts to 'secondary' bacterial symbionts which are maternally transmitted but relatively labile within host lineages. To explore the dynamics of secondary symbiont associations in aphids, we characterized bacteria infecting 15 species of macrosiphine aphids using DNA sequencing, diagnostic polymerase chain reaction (PCR), diagnostic restriction digests, phylogenetic analyses, and electron microscopy to examine aphids from nature and from laboratory colonies. Three types of bacteria besides Buchnera were found repeatedly; all three fall within the Enterobacteriaceae. The R-type has a 16S rDNA less than 0.1% different from that of the secondary symbiont previously reported from Acyrthosiphon pisum and is related to Serratia species. The T-type includes a symbiont previously reported from a whitefly; the U-type comprises a new cluster near the T-type. The T-type was found in every one of 40 Uroleucon ambrosiae clones collected throughout the United States. In contrast, A. pisum individuals were infected by any combination of the three symbiont types. Secondary symbionts were maternally transmitted for 11 months within laboratory-reared A. pisum clones and were present in sexually produced eggs. PCR screens for a bacteriophage, APSE-1, indicated its presence in both A. pisum and U. ambrosiae containing secondary symbionts. Electron microscopy of R-type and T-type bacteria in A. pisum and in U. ambrosiae revealed rod-shaped organisms that attain extremely high densities within a few bacteriocytes.     

Phylogenetic characterization of endosymbionts of the hydrothermal vent mussel Bathymodiolus azoricus by analysis of the 16S rRNA, pmoL, and cbbA genes

In order to assess the phylogenetic diversity of the endosymbiotic microbial community of the gills of marine shellfish Bathymodiolus azoricus, total DNA was extracted from the gills. The PCR fragments corresponding to the genes encoding 16S rRNA, ribulose-bisphosphate carboxylase (cbbL), and particulate methane monooxygenase (pmoA) were amplified, cloned, and sequenced. For the 16S rDNA genes, only one phylotype was revealed; it belonged to the cluster of Mytilidae thiotrophic symbionts within the Gammaproteobacteria. For the RuBisCO genes, two phylotypes were found, both belonging to Gammaproteobacteria. One of them was closely related to the previously known mytilid symbiont, the other, to a pogonophore symbiont, presumably a methanotrophic bacterium. One phylotype of particulate methane oxygenase genes was also revealed; this finding indicated the presence of a methanotrophic symbiont. Phylogenetic analysis of the pmoA placed this endosymbiont within the Gammaproteobacteria, in a cluster including the methanotrophic bacterial genus Methylobacter and other methanotrophic Bathymodiolus gill symbionts. These results provide evidence for the existence of two types of endosymbionts (thioautotrophic and methanotrophic) in the gills of B. azoricus and demonstrate that, apart from the phylogenetic analysis of 16S rRNA genes, parallel analysis of functional genes is essential.     

Coexistence of Wolbachia with Buchnera aphidicola and a secondary symbiont in the aphid Cinara cedri

Intracellular symbiosis is very common in the insect world. For the aphid Cinara cedri, we have identified by electron microscopy three symbiotic bacteria that can be characterized by their different sizes, morphologies, and electrodensities. PCR amplification and sequencing of the 16S ribosomal DNA (rDNA) genes showed that, in addition to harboring Buchnera aphidicola, the primary endosymbiont of aphids, C. cedri harbors a secondary symbiont (S symbiont) that was previously found to be associated with aphids (PASS, or R type) and an alpha-proteobacterium that belongs to the Wolbachia genus. Using in situ hybridization with specific bacterial probes designed for symbiont 16S rDNA sequences, we have shown that Wolbachia was represented by only a few minute bacteria surrounding the S symbionts. Moreover, the observed B. aphidicola and the S symbionts had similar sizes and were housed in separate specific bacterial cells, the bacteriocytes. Interestingly, in contrast to the case for all aphids examined thus far, the S symbionts were shown to occupy a similarly sized or even larger bacteriocyte space than B. aphidicola. These findings, along with the facts that C. cedri harbors the B. aphidicola strain with the smallest bacterial genome and that the S symbionts infect all Cinara spp. analyzed so far, suggest the possibility of bacterial replacement in these species.     

Endosymbiotic microbiota of the bamboo pseudococcid Antonina crawii (Insecta, Homoptera)

We characterized the intracellular symbiotic microbiota of the bamboo pseudococcid Antonina crawii by performing a molecular phylogenetic analysis in combination with in situ hybridization. Almost the entire length of the bacterial 16S rRNA gene was amplified and cloned from A. crawii whole DNA. Restriction fragment length polymorphism analysis revealed that the clones obtained included three distinct types of sequences. Nucleotide sequences of the three types were determined and subjected to a molecular phylogenetic analysis. The first sequence was a member of the gamma subdivision of the division Proteobacteria (gamma-Proteobacteria) to which no sequences in the database were closely related, although the sequences of endosymbionts of other homopterans, such as psyllids and aphids, were distantly related. The second sequence was a beta-Proteobacteria sequence and formed a monophyletic group with the sequences of endosymbionts from other pseudococcids. The third sequence exhibited a high level of similarity to sequences of Spiroplasma spp. from ladybird beetles and a tick. Localization of the endosymbionts was determined by using tissue sections of A. crawii and in situ hybridization with specific oligonucleotide probes. The gamma- and beta-Proteobacteria symbionts were packed in the cytoplasm of the same mycetocytes (or bacteriocytes) and formed a large mycetome (or bacteriome) in the abdomen. The spiroplasma symbionts were also present intracellularly in various tissues at a low density. We observed that the anterior poles of developing eggs in the ovaries were infected by the gamma- and beta-Proteobacteria symbionts in a systematic way, which ensured vertical transmission. Five representative pseudococcids were examined by performing diagnostic PCR experiments with specific primers; the beta-Proteobacteria symbiont was detected in all five pseudococcids, the gamma-Proteobacteria symbiont was found in three, and the spiroplasma symbiont was detected only in A. crawii.     

Phylogenetic diversity of bacterial symbionts of Solemya hosts based on comparative sequence analysis of 16S rRNA genes.

The bacterial endosymbionts of two species of the bivalve genus Solemya from the Pacific Ocean, Solemya terraeregina and Solemya pusilla, were characterized. Prokaryotic cells resembling gram-negative bacteria were observed in the gills of both host species by transmission electron microscopy. The ultrastructure of the symbiosis in both host species is remarkably similar to that of all previously described Solemya spp. By using sequence data from 16S rRNA, the identity and evolutionary origins of the S. terraeregina and S. pusilla symbionts were also determined. Direct sequencing of PCR-amplified products from host gill DNA with primers specific for Bacteria 16S rRNA genes gave a single, unambiguous sequence for each of the two symbiont species. In situ hybridization with symbiont-specific oligonucleotide probes confirmed that these gene sequences belong to the bacteria residing in the hosts gills. Phylogenetic analyses of the 16S rRNA gene sequences by both distance and parsimony methods identify the S. terraeregina and S. pusilla symbionts as members of the gamma subdivision of the Proteobacteria. In contrast to symbionts of other bivalve families, which appear to be monophyletic, the S. terraeregina and S. pusilla symbionts share a more recent common ancestry with bacteria associating endosymbiotically with bivalves of the superfamily Lucinacea than with other Solemya symbionts (host species S. velum, S. occidentalis, and S. reidi). Overall, the 16S rRNA gene sequence data suggest that the symbionts of Solemya hosts represent at least two distinct bacterial lineages within the gamma-Proteobacteria. While it is increasingly clear that all extant species of Solemya live in symbiosis with specific bacteria, the associations appear to have multiple evolutionary origins.     

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.     

Independent phylogenetic origins of methanotrophic and chemoautotrophic bacterial endosymbioses in marine bivalves.

The discovery of bacterium-bivalve symbioses capable of utilizing methane as a carbon and energy source indicates that the endosymbionts of hydrothermal vent and cold seep bivalves are not restricted to sulfur-oxidizing chemoautotrophic bacteria but also include methanotrophic bacteria. The phylogenetic origin of methanotrophic endosymbionts and their relationship to known symbiotic and free-living bacteria, however, have remained unexplored. In situ localization and phylogenetic analysis of a symbiont 16S rRNA gene cloned from the gills of a recently described deep-sea mussel species demonstrate that this symbiont represents a new taxon which is closely related to free-living, cultivable Type I methanotrophic bacteria. This symbiont is distinct from known chemoautotrophic symbionts. Thus, despite compelling similarities between the symbioses, chemoautotrophic and methanotrophic symbionts of marine bivalves have independent phylogenetic origins.     

The genus Caedibacter comprises endosymbionts of Paramecium spp. related to the Rickettsiales (Alphaproteobacteria) and to Francisella tularensis (Gammaproteobacteria)

Obligate bacterial endosymbionts of paramecia able to form refractile inclusion bodies (R bodies), thereby conferring a killer trait upon their ciliate hosts, have traditionally been grouped into the genus CAEDIBACTER: Of the six species described to date, only the Paramecium caudatum symbiont Caedibacter caryophilus has been phylogenetically characterized by its 16S rRNA gene sequence, and it was found to be a member of the Alphaproteobacteria related to the RICKETTSIALES: In this study, the Caedibacter taeniospiralis type strain, an R-body-producing cytoplasmatic symbiont of Paramecium tetraurelia strain 51k, was investigated by comparative 16S rRNA sequence analysis and fluorescence in situ hybridization with specific oligonucleotide probes. C. taeniospiralis is not closely related to C. caryophilus (80% 16S rRNA sequence similarity) but forms a novel evolutionary lineage within the Gammaproteobacteria with the family Francisellaceae as a sister group (87% 16S rRNA sequence similarity). These findings demonstrate that the genus Caedibacter is polyphyletic and comprises at least two phylogenetically different bacterial species belonging to two different classes of the PROTEOBACTERIA: Comparative phylogenetic analysis of C. caryophilus, five closely related Acanthamoeba endosymbionts (including one previously uncharacterized amoebal symbiont identified in this study), and their hosts suggests that the progenitor of the alphaproteobacterial C. caryophilus lived within acanthamoebae prior to the infection of paramecia.     

Lucinidae/sulfur-oxidizing bacteria: ancestral heritage or opportunistic association? Further insights from the Bohol Sea (the Philippines)

The first studies of the 16S rRNA gene diversity of the bacterial symbionts found in lucinid clams did not clarify how symbiotic associations had evolved in this group. Indeed, although species-specific associations deriving from a putative ancestral symbiotic association have been described (coevolution scenario), associations between the same bacterial species and various host species (opportunistic scenario) have also been described. Here, we carried out a comparative molecular analysis of hosts, based on 18S and 28S rRNA gene sequences, and of symbionts, based on 16S rRNA gene sequences, to determine as to which evolutionary scenario led to modern lucinid/symbiont associations. For all sequences analyzed, we found only three bacterial symbiont species, two of which are harbored by lucinids colonizing mangrove swamps. The last symbiont is the most common and was found to be independent of biotope or depth. Another interesting feature is the similarity of ctenidial organization of lucinids from the Philippines to those described previously, with the exception that two bacterial morphotypes were observed in two different species (Gloverina rectangularis and Myrtea flabelliformis). Thus, there is apparently no specific association between Lucinidae and their symbionts, the association taking place according to which bacterial species is present in the environment.     

INTRACELLULAR CYANOBACTERIAL SYMBIONTS IN THE MARINE DIATOM CLIMACODIUM FRAUENFELDIANUM (BACILLARIOPHYCEAE)

The diatom Climacodium frauenfeldianum Grunow was collected in the tropical Atlantic and Pacific Oceans. Observations with epifluorescence microscopy revealed that this diatom contained coccoid symbionts (2.5–3.5 μm) with a typical cyanobacterial fluorescence in addition to that of their own chloroplasts. Mean concentration of C. frauenfeldianum for 28 stations in the SW tropical Pacific Ocean was 530 x 10³ (SE = 1372) cells·m ^{− 2}, with highest concentration (mean 17.5 cells·L ^{− 1}) at 40-m depth. The symbiosis was only observed at water temperatures between 26.3 and 28.9° C, with highest concentrations at 27.7° C. Three almost complete 16S rDNA sequences from one sample were determined, and they were identical. The phylogenetic analysis of this 16S rDNA sequence and those from other cyanobacteria and plastids revealed that it was closely related to the 16S rDNA sequence from Cyanothece sp. ATCC 51142. Cyanothece sp. ATCC 51142 is a unicellular nitrogen-fixing cyanobacterium isolated from a coastal marine environment and has ultrastructural features similar to the symbionts of C. frauenfeldianum . The close relationship between Cyanothece sp. and the cyanobacterial symbiont in C. frauenfeldianum suggests the potential for nitrogen fixation in the symbiosis.     

Endosymbioses between bacteria and deep-sea siboglinid tubeworms from an Arctic Cold Seep (Haakon Mosby Mud Volcano, Barents Sea)

Siboglinid tubeworms do not have a mouth or gut and live in obligate associations with bacterial endosymbionts. Little is currently known about the phylogeny of frenulate and moniliferan siboglinids and their symbionts. In this study, we investigated the symbioses of two co-occurring siboglinid species from a methane emitting mud volcano in the Arctic Ocean (Haakon Mosby Mud Volcano, HMMV): Oligobrachia haakonmosbiensis (Frenulata) and Sclerolinum contortum (Monilifera). Comparative sequence analysis of the host-specific 18S and the symbiont-specific 16S rRNA genes of S. contortum showed that the close phylogenetic relationship of this host to vestimentiferan siboglinids was mirrored in the close relationship of its symbionts to the sulfur-oxidizing gammaproteobacterial symbionts of vestimentiferans. A similar congruence between host and symbiont phylogeny was observed in O. haakonmosbiensis: both this host and its symbionts were most closely related to the frenulate siboglinid O. mashikoi and its gammaproteobacterial symbiont. The symbiont sequences from O. haakonmosbiensis and O. mashikoi formed a clade unaffiliated with known methane- or sulfur-oxidizing bacteria. Fluorescence in situ hybridization indicated that the dominant bacterial phylotypes originated from endosymbionts residing inside the host trophosome. In both S. contortum and O. haakonmosbiensis, characteristic genes for autotrophy (cbbLM) and sulfur oxidation (aprA) were present, while genes diagnostic for methanotrophy were not detected. The molecular data suggest that both HMMV tubeworm species harbour chemoautotrophic sulfur-oxidizing symbionts. In S. contortum, average stable carbon isotope values of fatty acids and cholesterol of -43 per thousand were highly negative for a sulfur oxidizing symbiosis, but can be explained by a (13)C-depleted CO(2) source at HMMV. In O. haakonmosbiensis, stable carbon isotope values of fatty acids and cholesterol of -70 per thousand are difficult to reconcile with our current knowledge of isotope signatures for chemoautotrophic processes.     

Endosymbionts of ticks and their relationship to Wolbachia spp. and tick-borne pathogens of humans and animals.

The presence, internal distribution, and phylogenetic position of endosymbiotic bacteria from four species of specific-pathogen-free ticks were studied. These included the hard ticks Ixodes scapularis (the black-legged tick), Rhipicephalus sanguineus (the brown dog tick), and Haemaphysalis longicornis and the African soft tick Ornithodoros moubata. PCR assays for bacteria, using two sets of general primers for eubacterial 16S and 23S rRNA genes (rDNAs) and seven sets of specific primers for wolbachial, rickettsial, or Francisella genes, indicated that I. scapularis possessed symbiotic rickettsiae in the ovaries and that the other species harbored eubacteria in both the ovaries and Malpighian tubules. Phylogenetic analysis based on the sequence of 16S rDNA indicated that the symbiont of I. scapularis belonged to the alpha subgroup of proteobacteria and was closely related to the members of the genus Rickettsia. The other species had similar microorganisms in the ovaries and Malpighian tubules, which belonged to the gamma subgroup of proteobacteria, and formed a monophyletic group with the Q-fever pathogen, Coxiella burnetii. O. moubata harbored another symbiont, which formed a monophyletic group with Francisella tularensis and Wolbachia persica, the latter a symbiont previously isolated from Malpighian tubules of the soft tick Argas (Persicargas) arboreus. Thus, the symbionts of these four tick species were not related to the Wolbachia species found in insects. The two symbionts that live in the Malpighian tubules, one closely related to C. burnetii and the other closely related to F. tularensis, appear to be of ancient origin and be widely distributed in ticks.