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.     

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.     

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.     

The diet of the Harlequin crab Lissocarcinus orbicularis,an obligate symbiont of sea cucumbers (holothuroids) belonging to the genera Thelenota,Bohadschia and Holothuria

The present paper characterizes, for the first time, the diet of the Harlequin crab Lissocarcinus orbicularis, an obligate symbiotic crab that associates with sea cucumbers (holothuroids) belonging to the genera Thelenota, Bohadschia and Holothuria. These tropical holothuroids host a rich symbiotic community in the Indo-West Pacific Ocean of which the Harlequin crab is the best known. The diet of L. orbicularis was characterized by analyzing the microscopic, molecular and isotopic signatures obtained from its gastric content. The presence of sea cucumber ossicles in the gastric mills of the crabs suggests that symbionts eat the superficial integument of their host and this was supported by the fact that Holothuroid DNA was detected in the stomach of L. orbicularis after DGGE and sequencing of the 18S rDNA gene. The stable isotopic δ¹³C and δ¹⁵N values of crab tissues were compared with diverse potential food sources including three holothuroids, three algae, one sea grass as well as the organic matter contained in the water column, in the sediment, and the second most abundant symbiont, the polychaete Gastrolepidia clavigera. The low δ¹⁵N values of crabs suggests that the crabs do not exclusively feed on sea cucumber tissue but assimilate diverse food sources such as sea grasses and organic matter contained in sediment that have similar δ¹³C values. There were no differences between the feeding of males and females but there was a positive correlation between the carapace length and the stable isotopic values indicating a shift of the food source as crabs grow larger.     

Endosymbiotic Bacteroidales Bacteria of the Flagellated Protist Pseudotrichonympha grassii in the Gut of the Termite Coptotermes formosanus

A unique lineage of bacteria belonging to the order Bacteroidales was identified as an intracellular endosymbiont of the protist Pseudotrichonympha grassii (Parabasalia, Hypermastigea) in the gut of the termite Coptotermes formosanus. We identified the 16S rRNA, gyrB, elongation factor Tu, and groEL gene sequences in the endosymbiont and detected a very low level of sequence divergence (<0.9% of the nucleotides) in the endosymbiont population within and among protist cells. The Bacteroidales endosymbiont sequence was affiliated with a cluster comprising only sequences from termite gut bacteria and was not closely related to sequences identified for members of the Bacteroidales attached to the cell surfaces of other gut protists. Transmission electron microscopy showed that there were numerous rod-shaped bacteria in the cytoplasm of the host protist, and we detected the endosymbiont by fluorescence in situ hybridization (FISH) with an oligonucleotide probe specific for the 16S rRNA gene identified. Quantification of the abundance of the Bacteroidales endosymbiont by sequence-specific cleavage of rRNA with RNase H and FISH cell counting revealed, surprisingly, that the endosymbiont accounted for 82% of the total bacterial rRNA and 71% of the total bacterial cells in the gut community. The genetically nearly homogeneous endosymbionts of Pseudotrichonympha were very abundant in the gut symbiotic community of the termite.     

Vertical Distribution of Methanogens in the Anoxic Sediment of Rotsee (Switzerland)

Anoxic sediments from Rotsee (Switzerland) were analyzed for the presence and diversity of methanogens by using molecular tools and for methanogenic activity by using radiotracer techniques, in addition to the measurement of chemical profiles. After PCR-assisted sequence retrieval of the 16S rRNA genes (16S rDNA) from the anoxic sediment of Rotsee, cloning, and sequencing, a phylogenetic analysis identified two clusters of sequences and four separated clones. The sequences in cluster 1 grouped with those of Methanosaeta spp., whereas the sequences in cluster 2 comprised the methanogenic endosymbiont of Plagiopyla nasuta. Discriminative oligonucleotide probes were constructed against both clusters and two of the separated clones. These probes were used subsequently for the analysis of indigenous methanogens in a core of the sediment, in addition to domain-specific probes against members of the domains Bacteria and Archaea and the fluorescent stain 4′,6-diamidino-2-phenylindole (DAPI), by fluorescent in situ hybridization. After DAPI staining, the highest microbial density was obtained in the upper sediment layer; this density decreased with depth from (1.01 ± 0.25) × 10¹⁰ to (2.62 ± 0.58) × 10¹⁰ cells per g of sediment (dry weight). This zone corresponded to that of highest metabolic activity, as indicated by the ammonia, alkalinity, and pH profiles, whereas the methane profile was constant. Probes Eub338 and Arch915 detected on average 16 and 6% of the DAPI-stained cells as members of the domains Bacteria and Archaea, respectively. Probe Rotcl1 identified on average 4% of the DAPI-stained cells as Methanosaeta spp., which were present throughout the whole core. In contrast, probe Rotcl2 identified only 0.7% of the DAPI-stained cells as relatives of the methanogenic endosymbiont of P. nasuta, which was present exclusively in the upper 2 cm of the sediment. Probes Rotp13 and Rotp17 did not detect any cells. The spatial distribution of the two methanogenic populations corresponded well to the methane production rates determined by incubation with either [¹⁴C]acetate or [¹⁴C]bicarbonate. Methanogenesis from acetate accounted for almost all of the total methane production, which concurs with the predominance of acetoclastic Methanosaeta spp. that represented on average 91% of the archaeal population. Significant hydrogenotrophic methanogenesis was found only in the organically enriched upper 2 cm of the sediment, where the probably hydrogenotrophic relatives of the methanogenic endosymbiont of P. nasuta, accounting on average for 7% of the archaeal population, were also detected.     

Novel Chemoautotrophic Endosymbiosis between a Member of the Epsilonproteobacteria and the Hydrothermal-Vent Gastropod Alviniconcha aff. hessleri (Gastropoda: Provannidae) from the Indian Ocean

The hydrothermal-vent gastropod Alviniconcha aff. hessleri from the Kairei hydrothermal field on the Central Indian Ridge houses bacterium-like cells internally in its greatly enlarged gill. A single 16S rRNA gene sequence was obtained from the DNA extract of the gill, and phylogenetic analysis placed the source organism within a lineage of the epsilon subdivision of the Proteobacteria. Fluorescence in situ hybridization analysis with an oligonucleotide probe targeting the specific epsilonproteobacterial subgroup showed the bacterium densely colonizing the gill filaments. Carbon isotopic homogeneity among the gastropod tissue parts, regardless of the abundance of the endosymbiont cells, suggests that the carbon isotopic composition of the endosymbiont biomass is approximately the same as that of the gastropod. Compound-specific carbon isotopic analysis revealed that fatty acids from the gastropod tissues are all ¹³C enriched relative to the gastropod biomass and that the monounsaturated C₁₆ fatty acid that originates from the endosymbiont is as ¹³C enriched relative to the gastropod biomass as that of the epsilonproteobacterial cultures grown under chemoautotrophic conditions. This fractionation pattern is most likely due to chemoautotrophy based on the reductive tricarboxylic-acid (rTCA) cycle and subsequent fatty acid biosynthesis from ¹³C-enriched acetyl coenzyme A. Enzymatic characterization revealed evident activity of several key enzymes of the rTCA cycle, as well as the absence of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in the gill tissue. The results from anatomic, molecular phylogenetic, bulk and compound-specific carbon isotopic, and enzymatic analyses all support the inference that a novel nutritional strategy relying on chemoautotrophy in the epsilonproteobacterial endosymbiont is utilized by the hydrothermal-vent gastropod from the Indian Ocean. The discrepancies between the data of the present study and those of previous ones for Alviniconcha gastropods from the Pacific Ocean imply that at least two lineages of chemoautotrophic bacteria, phylogenetically distinct at the subdivision level, occur as the primary endosymbiont in one host animal type.     

Comparative Allometric Growth of the Mimetic Ephippid Reef Fishes Chaetodipterus faber and Platax orbicularis

Mimesis is a relatively widespread phenomenon among reef fish, but the ontogenetic processes relevant for mimetic associations in fish are still poorly understood. In the present study, the allometric growth of two allopatric leaf-mimetic species of ephippid fishes, Chaetodipterus faber from the Atlantic and Platax orbicularis from the Indo-Pacific, was analyzed using ten morphological variables. The development of fins was considered owing to the importance of these structures for mimetic behaviors during early life stages. Despite the anatomical and behavioral similarities in both juvenile and adult stages, C. faber and P. orbicularis showed distinct patterns of growth. The overall shape of C. faber transforms from a rounded-shape in mimetic juveniles to a lengthened profile in adults, while in P. orbicularis, juveniles present an oblong profile including dorsal and anal fins, with relative fin size diminishing while the overall profile grows rounder in adults. Although the two species are closely-related, the present results suggest that growth patterns in C. faber and P. orbicularis are different, and are probably independent events in ephippids that have resulted from similar selective processes.     

Cospeciation of Psyllids and Their Primary Prokaryotic Endosymbionts

Psyllids are plant sap-feeding insects that harbor prokaryotic endosymbionts in specialized cells within the body cavity. Four-kilobase DNA fragments containing 16S and 23S ribosomal DNA (rDNA) were amplified from the primary (P) endosymbiont of 32 species of psyllids representing three psyllid families and eight subfamilies. In addition, 0.54-kb fragments of the psyllid nuclear gene wingless were also amplified from 26 species. Phylogenetic trees derived from 16S-23S rDNA and from the host wingless gene are very similar, and tests of compatibility of the data sets show no significant conflict between host and endosymbiont phylogenies. This result is consistent with a single infection of a shared psyllid ancestor and subsequent cospeciation of the host and the endosymbiont. In addition, the phylogenies based on DNA sequences generally agreed with psyllid taxonomy based on morphology. The 3′ end of the 16S rDNA of the P endosymbionts differs from that of other members of the domain Bacteria in the lack of a sequence complementary to the mRNA ribosome binding site. The rate of sequence change in the 16S-23S rDNA of the psyllid P endosymbiont was considerably higher than that of other bacteria, including other fast-evolving insect endosymbionts. The lineage consisting of the P endosymbionts of psyllids was given the designation Candidatus Carsonella (gen. nov.) with a single species, Candidatus Carsonella ruddii (sp. nov.).     

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.     

Evolution of symbiotic organs and endosymbionts in lygaeid stinkbugs

We investigated seed bugs of the genus Nysius (Insecta: Hemiptera: Lygaeidae) for their symbiotic bacteria. From all the samples representing 4 species, 18 populations and 281 individuals, specific bacterial 16S rRNA gene sequences were consistently identified, which formed a distinct clade in the Gammaproteobacteria. In situ hybridization showed that the bacterium was endocellularly localized in a pair of large bacteriomes that were amorphous in shape, deep red in color, and in association with gonads. In the ovary of adult females, the endosymbiont was also localized in the ‘infection zone'' in the middle of each germarium and in the ‘symbiont ball'' at the anterior pole of each oocyte, indicating vertical transmission of the endosymbiont through the ovarial passage. Phylogenetic analyses based on bacterial 16S rRNA, groEL and gyrB genes consistently supported a coherent monophyly of the Nysius endosymbionts. The possibility of a sister relationship to ‘Candidatus Kleidoceria schneideri'', the bacteriome-associated endosymbiont of a lygaeid bug Kleidocerys resedae, was statistically rejected, indicating independent evolutionary origins of the endosymbionts in the Lygaeidae. The endosymbiont genes consistently exhibited AT-biased nucleotide compositions and accelerated rates of molecular evolution, and the endosymbiont genome was only 0.6 Mb in size. The endosymbiont phylogeny was congruent with the host insect phylogeny, suggesting strict vertical transmission and host–symbiont co-speciation over evolutionary time. Based on these results, we discuss the evolution of bacteriomes and endosymbionts in the Heteroptera, most members of which are associated with gut symbiotic bacteria. The designation ‘Candidatus Schneideria nysicola'' is proposed for the endosymbiont clade.     

A simple protocol to obtain highly pure Wolbachia endosymbiont DNA for genome sequencing

Most genome sequencing projects using intracellular bacteria face difficulties in obtaining sufficient bacterial DNA free of host contamination. We have developed a simple and rapid protocol to isolate endosymbiont DNA virtually free from fly and mosquito host DNA. We purified DNA from six Wolbachia strains in preparation for genome sequencing using this method, and achieved up to 97% pure Wolbachia sequence, even after using frozen insects. This is a significant improvement for future Wolbachia and other endosymbiont genome projects.     

Spatial distribution and species composition of prosthecate bacteria in Lake Baikal

From the water column of Lake Baikal, several strains of prosthecate bacteria belonging to the genera Caulobacter and Brevundimonas were isolated. In this article, the methods applied for their isolation and cell number determination are described; the occurrence frequency and spatial distribution of these microorganisms in the lake are demonstrated. Characterization of the species composition of cultivable and uncultivable prosthecate bacteria was carried out using the methods of traditional and molecular microbiology, respectively. A comparative phylogenetic analysis of the DNA sequences of uncultivable bacteria, which showed homology to the members of the alpha subclass of proteobacteria, was carried out. It was demonstrated that the lake water column is inhabited by uncultivable alpha-proteobacteria of uncertain phylogenetic affinity, in addition to representatives of the species Caulobacter vibrioides and C. leidyi, which were detected by traditional microbiological methods.     

Comparative Genomics of Wolbachia and the Bacterial Species Concept

The importance of host-specialization to speciation processes in obligate host-associated bacteria is well known, as is also the ability of recombination to generate cohesion in bacterial populations. However, whether divergent strains of highly recombining intracellular bacteria, such as Wolbachia, can maintain their genetic distinctness when infecting the same host is not known. We first developed a protocol for the genome sequencing of uncultivable endosymbionts. Using this method, we have sequenced the complete genomes of the Wolbachia strains wHa and wNo, which occur as natural double infections in Drosophila simulans populations on the Seychelles and in New Caledonia. Taxonomically, wHa belong to supergroup A and wNo to supergroup B. A comparative genomics study including additional strains supported the supergroup classification scheme and revealed 24 and 33 group-specific genes, putatively involved in host-adaptation processes. Recombination frequencies were high for strains of the same supergroup despite different host-preference patterns, leading to genomic cohesion. The inferred recombination fragments for strains of different supergroups were of short sizes, and the genomes of the co-infecting Wolbachia strains wHa and wNo were not more similar to each other and did not share more genes than other A- and B-group strains that infect different hosts. We conclude that Wolbachia strains of supergroup A and B represent genetically distinct clades, and that strains of different supergroups can co-exist in the same arthropod host without converging into the same species. This suggests that the supergroups are irreversibly separated and that barriers other than host-specialization are able to maintain distinct clades in recombining endosymbiont populations. Acquiring a good knowledge of the barriers to genetic exchange in Wolbachia will advance our understanding of how endosymbiont communities are constructed from vertically and horizontally transmitted genes.     

Analysis of Subfossil Molecular Remains of Purple Sulfur Bacteria in a Lake Sediment

Molecular remains of purple sulfur bacteria (Chromatiaceae) were detected in Holocene sediment layers of a meromictic salt lake (Mahoney Lake, British Columbia, Canada). The carotenoid okenone and bacteriophaeophytin a were present in sediments up to 11,000 years old. Okenone is specific for only a few species of Chromatiaceae, including Amoebobacter purpureus, which presently predominates in the chemocline bacterial community of the lake. With a primer set specific for Chromatiaceae in combination with denaturing gradient gel electrophoresis, 16S rRNA gene sequences of four different Chromatiaceae species were retrieved from different depths of the sediment. One of the sequences, which originated from a 9,100-year-old sample, was 99.2% identical to the 16S rRNA gene sequence of A. purpureus ML1 isolated from the chemocline. Employing primers specific for A. purpureus ML1 and dot blot hybridization of the PCR products, the detection limit for A. purpureus ML1 DNA could be lowered to 0.004% of the total community DNA. With this approach the DNA of the isolate was detected in 7 of 10 sediment layers, indicating that A. purpureus ML1 constituted at least a part of the ancient purple sulfur bacterial community. The concentrations of A. purpureus DNA and okenone in the sediment were not correlated, and the ratio of DNA to okenone was much lower in the subfossil sediment layers (2.7 · 10⁻⁶) than in intact cells (1.4). This indicates that degradation rates are significantly higher for genomic DNA than for hydrocarbon cell constituents, even under anoxic conditions and at the very high sulfide concentrations present in Mahoney Lake.     

Development and Evaluation of Functional Gene Arrays for Detection of Selected Genes in the Environment

To determine the potential of DNA array technology for assessing functional gene diversity and distribution, a prototype microarray was constructed with genes involved in nitrogen cycling: nitrite reductase (nirS and nirK) genes, ammonia mono-oxygenase (amoA) genes, and methane mono-oxygenase (pmoA) genes from pure cultures and those cloned from marine sediments. In experiments using glass slide microarrays, genes possessing less than 80 to 85% sequence identity were differentiated under hybridization conditions of high stringency (65°C). The detection limit for nirS genes was approximately 1 ng of pure genomic DNA and 25 ng of soil community DNA using our optimized protocol. A linear quantitative relationship (r² = 0.89 to 0.94) was observed between signal intensity and target DNA concentration over a range of 1 to 100 ng for genomic DNA (or genomic DNA equivalent) from both pure cultures and mixed communities. However, the quantitative capacity of microarrays for measuring the relative abundance of targeted genes in complex environmental samples is less clear due to divergent target sequences. Sequence divergence and probe length affected hybridization signal intensity within a certain range of sequence identity and size, respectively. This prototype functional gene array did reveal differences in the apparent distribution of nir and amoA and pmoA gene families in sediment and soil samples. Our results indicate that glass-based microarray hybridization has potential as a tool for revealing functional gene composition in natural microbial communities; however, more work is needed to improve sensitivity and quantitation and to understand the associated issue of specificity.     

A Polyphasic Approach To Study the Diversity and Vertical Distribution of Sulfur-Oxidizing Thiomicrospira Species in Coastal Sediments of the German Wadden Sea

Recently, four Thiomicrospira strains were isolated from a coastal mud flat of the German Wadden Sea (T. Brinkhoff and G. Muyzer, Appl. Environ. Microbiol. 63:3789–3796, 1997). Here we describe the use of a polyphasic approach to investigate the functional role of these closely related bacteria. Microsensor measurements showed that there was oxygen penetration into the sediment to a depth of about 2.0 mm. The pH decreased from 8.15 in the overlaying water to a minimum value of 7.3 at a depth of 1.2 mm. Further down in the sediment the pH increased to about 7.8 and remained constant. Most-probable-number (MPN) counts of chemolithoautotrophic sulfur-oxidizing bacteria revealed nearly constant numbers along the vertical profile; the cell concentration ranged from 0.93 × 10⁵ to 9.3 × 10⁵ cells per g of sediment. A specific PCR was used to detect the presence of Thiomicrospira cells in the MPN count preparations and to determine their 16S rRNA sequences. The concentration of Thiomicrospira cells did not decrease with depth. It was found that Thiomicrospira strains were not dominant sulfur-oxidizing bacteria in this habitat. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA fragments followed by hybridization analysis with a genus-specific oligonucleotide probe revealed the diversity of Thiomicrospira strains in the MPN cultures. Sequence analysis of the highest MPN dilutions in which the genus Thiomicrospira was detected revealed that there were four clusters of several closely related sequences. Only one of the 10 Thiomicrospira sequences retrieved was related to sequences of known isolates from the same habitat. Slot blot hybridization of rRNA isolated from different sediment layers showed that, in contrast to the concentration of Thiomicrospira cells, the concentration of Thiomicrospira-specific rRNA decreased rapidly in the region below the oxic layer of the sediment. This study revealed the enormous sequence diversity of closely related microorganisms present in one habitat, which so far has been found only by sequencing molecular isolates. In addition, it showed that most of the Thiomicrospira populations in the sediment studied were quiescent.     

Big data can cause big mistakes: using the Societas Europaea Herpetologica atlas by Sillero et al. (2014), the distribution of <Emphasis Type="Italic">Emys orbicularis</Emphasis> will be misunderstood

Problems with the reliability of the distribution map for Emys orbicularis in the online atlas of European amphibians and reptiles (NA2RE) are highlighted. Two distinct species (E. orbicularis, E. trinacris) are lumped together there. For E. orbicularis, many regions are included into the native distribution range where the species does not occur naturally. On the contrary, records are missing for vast parts of the native range. Using data from the NA2RE atlas cannot be recommended presently, especially not for species distribution modelling.