Phylogenetic diversity of bacteria in an earth-cave in Guizhou province, southwest of China

The objective of this study was to analyze the phylogenetic composition of bacterial community in the soil of an earth-cave (Niu Cave) using a culture-independent molecular approach. 16S rRNA genes were amplified directly from soil DNA with universally conserved and Bacteria-specific rRNA gene primers and cloned. The clone library was screened by restriction fragment length polymorphism (RFLP), and representative rRNA gene sequences were determined. A total of 115 bacterial sequence types were found in 190 analyzed clones. Phylogenetic sequence analyses revealed novel 16S rRNA gene sequence types and a high diversity of putative bacterial community. Members of these bacteria included Proteobacteria (42.6%), Acidobacteria (18.6%), Planctomycetes (9.0%), Chloroflexi (Green nonsulfur bacteria, 7.5%), Bacteroidetes (2.1%), Gemmatimonadetes (2.7%), Nitrospirae (8.0%), Actinobacteria (High G+C Gram-positive bacteria, 6.4%) and candidate divisions (including the OP3, GN08, and SBR1093, 3.2%). Thirty-five clones were affiliated with bacteria that were related to nitrogen, sulfur, iron or manganese cycles. The comparison of the present data with the data obtained previously from caves based on 16S rRNA gene analysis revealed similarities in the bacterial community components, especially in the high abundance of Proteobacteria and Acidobacteria. Furthermore, this study provided the novel evidence for presence of Gemmatimonadetes, Nitrosomonadales, Oceanospirillales, and Rubrobacterales in a karstic hypogean environment.     

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.     

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.     

Coexistence of multiple proteobacterial endosymbionts in the gills of the wood-boring Bivalve Lyrodus pedicellatus (Bivalvia: Teredinidae)

Wood-boring bivalves of the family Teredinidae (commonly called shipworms) are known to harbor dense populations of gram-negative bacteria within specialized cells (bacteriocytes) in their gills. These symbionts are thought to provide enzymes, e.g., cellulase and dinitrogenase, which assist the host in utilizing wood as a primary food source. A cellulolytic, dinitrogen-fixing bacterium, Teredinibacter turnerae, has been isolated from the gill tissues of numerous teredinid bivalves and has been proposed to constitute the sole or predominant symbiont of this bivalve family. Here we demonstrate that one teredinid species, Lyrodus pedicellatus, contains at least four distinct bacterial 16S rRNA types within its gill bacteriocytes, one of which is identical to that of T. turnerae. Phylogenetic analyses indicate that the three newly detected ribotypes are derived from gamma proteobacteria that are related to but distinct (>6.5% sequence divergence) from T. turnerae. In situ hybridizations with 16S rRNA-directed probes demonstrated that the pattern of occurrence of symbiont ribotypes within bacteriocytes was predictable and specific, with some bacteriocytes containing two symbiont ribotypes. However, only two of the six possible pairwise combinations of the four ribotypes were observed to cooccur within the same host cells. The results presented here are consistent with the existence of a complex multiple symbiosis in this shipworm species.     

A novel betaproteobacterial agent of gill epitheliocystis in seawater farmed Atlantic salmon (Salmo salar)

Epitheliocystis, a disease characterised by cytoplasmic bacterial inclusions (cysts) in the gill and less commonly skin epithelial cells, has been reported in many marine and freshwater fish species and may be associated with mortality. Previously, molecular and ultrastructural analyses have exclusively associated members of the Chlamydiae with such inclusions. Here we investigated a population of farmed Atlantic salmon from the west coast of Norway displaying gill epitheliocystis. Although 'Candidatus Piscichlamydia salmonis', previously reported to be present in such cysts, was detected by PCR in most of the gill samples analysed, this bacterium was found to be a rare member of the gill microbiota, and not associated with the observed cysts as demonstrated by fluorescence in situ hybridization assays. The application of a broad range 16 S rRNA targeted PCR assay instead identified a novel betaproteobacterium as an abundant member of the gill microbiota. Fluorescence in situ hybridization demonstrated that this bacterium, tentatively classified as 'Candidatus Branchiomonas cysticola', was the cyst-forming agent in these samples. While histology and ultrastructure of 'Ca. B. cysticola' cysts revealed forms similar to the reticulate and intermediate bodies described in earlier reports from salmon in seawater, no elementary bodies typical of the chlamydial developmental cycle were observed. In conclusion, this study identified a novel agent of epitheliocystis in sea-farmed Atlantic salmon and demonstrated that these cysts can be caused by bacteria phylogenetically distinct from the Chlamydiae.     

Heterotrophic symbionts of phototrophic consortia: members of a novel diverse cluster of Betaproteobacteria characterized by a tandem rrn operon structure

Phototrophic consortia represent the most highly developed type of interspecific association of bacteria and consist of green sulfur bacterial epibionts attached around a central colourless rod-shaped bacterium. Based on 16S rRNA gene sequencing, the central bacterium of the consortium 'Chlorochromatium aggregatum' was recently shown to represent a novel and phylogenetically isolated lineage of the Comamonadaceae within the beta-subgroup of the Proteobacteria. To date, 19 types of phototrophic consortia are distinguished based on the different 16S rRNA gene sequences of their epibionts, but the diversity and phylogenetic relationships of the heterotrophic partner bacteria are still unknown. We developed an approach based on the specific rrn (ribosomal RNA) operon structure of the central bacterium of 'C. aggregatum' to recover 16S rRNA gene sequences of other central bacteria and their close relatives from natural consortia populations. Genomic DNA of the central bacterium of 'C. aggregatum' was first enriched several hundred-fold by employing a selective method for growth of consortia in a monolayer biofilm followed by a purification of the genome of the central bacterium by cesium chloride-bisbenzimidazole equilibrium density gradient centrifugation. A combination of inverse PCR, cloning and sequencing revealed that two rrn operons of the central bacterium are arranged in a tandem fashion and are separated by an unusually short intergenic region of 195 base pairs. This rare gene order was exploited to screen various natural microbial communities by PCR. We discovered a diverse and previously unknown subgroup of Betaproteobacteria in the chemoclines of freshwater lakes. This group was absent in other freshwater and soil samples. All the 16S rRNA gene sequences recovered are related to that of the central bacterium of 'C. aggregatum'. Fluorescence in situ hybridization indicated that two of these sequences originated from central bacteria of different phototrophic consortia, which, however, were only distantly related to the central bacterium of 'C. aggregatum'. Based on a detailed phylogenetic analysis, these central bacterial symbionts of phototrophic consortia have a polyphyletic origin.     

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

In order to assess the phylogenetic diversity of the endosymbiotic microbial community of the gills of marine bivalve 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 thiotrophic mytilid’s 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.     

Characterization of the endosymbiont of a deep-sea bivalve, Calyptogena soyoae.

We have purified DNA from gill tissue of a marine bivalve, Calyptogena soyoae, collected from the deep-sea cold seep communities in Sagami Bay, Japan. An rRNA gene was amplified, cloned, and sequenced. In situ hybridization revealed that the sequence is that of a bacterial endosymbiont within the gill of C. soyoae.     

Novel approach to quantitative detection of specific rRNA in a microbial community, using catalytic DNA

We developed a novel method for the quantitative detection of the 16S rRNA of a specific bacterial species in the microbial community by using deoxyribozyme (DNAzyme), which possesses the catalytic function to cleave RNA in a sequence-specific manner. A mixture of heterogeneous 16S rRNA containing the target 16S rRNA was incubated with a species-specific DNAzyme. The cleaved target 16S rRNA was separated from the intact 16S rRNA by electrophoresis, and then their amounts were compared for the quantitative detection of target 16S rRNA. This method was used to determine the abundance of the 16S rRNA of a filamentous bacterium, Sphaerotilus natans, in activated sludge, which is a microbial mixture used in wastewater treatment systems. The result indicated that this DNAzyme-based approach would be applicable to actual microbial communities.     

Molecular phylogenetic and isotopic evidence of two lineages of chemoautotrophic endosymbionts distinct at the subdivision level harbored in one host-animal type: the genus Alviniconcha (Gastropoda: Provannidae)

The hydrothermal-vent gastropod Alviniconcha hessleri from the Alice Springs deep-sea hydrothermal field in the Mariana Back-Arc Basin in the Western Pacific houses an intracellular bacterial endosymbiont in its gill. Although enzymatic analysis has revealed that the endosymbiont is a sulfur-oxidizing chemoautotroph using the Calvin-Benson cycle for the fixation of carbon dioxide, the phylogenetic affiliation of, and the trophic relationship of A. hessleri with, the chemoautotrophic endosymbiont remains undetermined. A single 16S rRNA gene sequence was obtained from the DNA extract of the gill, and phylogenetic analysis placed the source organism within the lineage of the gamma subdivision of the Proteobacteria that consists of many chemoautotrophic endosymbionts of marine invertebrates. Fluorescence in situ hybridization analysis showed the bacterium densely colonizing the gill filaments. The fatty acid profile of the symbiont-free mantle contains the high level of the 16:1 fatty acid originating from the endosymbiont, which indicates that the endosymbiont cells are digested by, and incorporated into, the host. Compound-specific carbon isotopic analysis revealed that fatty acids from the gastropod tissues are all (13)C-depleted relative to the gastropod biomass. This fractionation pattern is consistent with chemoautotrophy based on the Calvin-Benson cycle and subsequent fatty-acid biosynthesis from (13)C-depleted acetyl coenzyme A. The results from the present study are clearly different from those from our previous study for A. aff. hessleri from the Indian Ocean that harbors a chemoautotrophic endosymbiont belonging to the epsilon subdivision of the Proteobacteria, which mediates the reductive tricarboxylic acid cycle for carbon fixation. Thus, it is concluded here that two lineages of chemoautotrophic bacteria, phylogenetically distinct at the subdivision level, occur as the primary endosymbiont in one host-animal type, which is unknown for the other metazoans.     

Novel Approach to Quantitative Detection of Specific rRNA in a Microbial Community, Using Catalytic DNA

We developed a novel method for the quantitative detection of the 16S rRNA of a specific bacterial species in the microbial community by using deoxyribozyme (DNAzyme), which possesses the catalytic function to cleave RNA in a sequence-specific manner. A mixture of heterogeneous 16S rRNA containing the target 16S rRNA was incubated with a species-specific DNAzyme. The cleaved target 16S rRNA was separated from the intact 16S rRNA by electrophoresis, and then their amounts were compared for the quantitative detection of target 16S rRNA. This method was used to determine the abundance of the 16S rRNA of a filamentous bacterium, Sphaerotilus natans, in activated sludge, which is a microbial mixture used in wastewater treatment systems. The result indicated that this DNAzyme-based approach would be applicable to actual microbial communities.     

Phylogenetic affiliation of the bacteria that constitute phototrophic consortia

The phylogenetic affiliation of epibionts occurring in three morphologically distinct types of green-colored phototrophic consortia was investigated. Intact consortia of the types "Chlorochromatium aggregatum", "C. glebulum", and a third previously undescribed type, tentatively named "C. magnum" were mechanically separated from accompanying bacteria by either micromanipulation or by chemotactic accumulation in sulfide-containing capillaries. A 540-base-pair-long fragment of the 16S rRNA gene of the epibionts was amplified employing PCR primers specific for green sulfur bacteria. DNA fragments were separated by denaturing gradient gel electrophoresis and subsequently sequenced. The results of this phylogenetic analysis indicated that the symbiotic epibionts, together with only a few free-living strains, form a cluster within the green sulfur bacterial radiation which is only distantly related to the majority of known representatives of this phylum. Consortia with identical morphology but different origin exhibited significant differences in their partial 16S rRNA gene sequences, which could be confirmed by analysis of the 16S rRNA secondary structure. The phylogenetic affiliation of the chemotrophic central rod-shaped bacterium of "C. aggregatum" and "C. magnum" was analyzed by fluorescent in situ hybridization. According to our results and contrary to earlier assumptions, the central bacterium is a member of the beta-subgroup of the Proteobacteria.     

Bacterioplankton community structure and dynamics after large-scale release of nonindigenous bacteria as revealed by low-molecular-weight-RNA analysis.

A set of freshwater mesocosms (1.7 m3 each) was inoculated with large amounts of Escherichia coli, Pseudomonas putida, and their culture medium to substantially disturb the natural microbial community. To monitor microbial community dynamics, low-molecular-weight RNA (5S rRNA and tRNA) obtained directly from bacterioplankton was analyzed by using high-resolution electrophoresis. The introduced bacteria showed no significant effect on the community structure of the natural bacterial assemblage and its dynamics for 16 days. In contrast, the addition of culture medium resulted within 2 days in a reduction of community diversity due to dominance of a single 5S rRNA band from an indigenous bacterium. Partial sequencing of several 5S rRNAs demonstrated the molecular homogeneity of most of the abundant bands and enabled the identification of corresponding bacterial isolates and/or species. The dominating bacterium (around 54% of the total 5S rRNA) in the nutrient-amended mesocosms could be identified by partial sequencing as a member of the Aeromonas hydrophila complex. Another bloom of heterotrophic bacteria belonging to the Cytophaga johnsonae complex was detected in the nutrient-amended mesocosms after 13 days. The dominance of this C. johnsonae-like bacterium could even be seen in the environmental tRNAs of the bacterioplankton, where its specific tRNAs prevailed from day 13 onward. This event was also independent of the introduced nonindigenous bacteria because it occurred at the same time in all nutrient-amended mesocosms. By contrast, in the unamended experiments, a different small 5S rRNA could by observed from day 10 onward with less pronounced dominance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bacterioplankton community structure and dynamics after large-scale release of nonindigenous bacteria as revealed by low-molecular-weight-RNA analysis.

A set of freshwater mesocosms (1.7 m3 each) was inoculated with large amounts of Escherichia coli, Pseudomonas putida, and their culture medium to substantially disturb the natural microbial community. To monitor microbial community dynamics, low-molecular-weight RNA (5S rRNA and tRNA) obtained directly from bacterioplankton was analyzed by using high-resolution electrophoresis. The introduced bacteria showed no significant effect on the community structure of the natural bacterial assemblage and its dynamics for 16 days. In contrast, the addition of culture medium resulted within 2 days in a reduction of community diversity due to dominance of a single 5S rRNA band from an indigenous bacterium. Partial sequencing of several 5S rRNAs demonstrated the molecular homogeneity of most of the abundant bands and enabled the identification of corresponding bacterial isolates and/or species. The dominating bacterium (around 54% of the total 5S rRNA) in the nutrient-amended mesocosms could be identified by partial sequencing as a member of the Aeromonas hydrophila complex. Another bloom of heterotrophic bacteria belonging to the Cytophaga johnsonae complex was detected in the nutrient-amended mesocosms after 13 days. The dominance of this C. johnsonae-like bacterium could even be seen in the environmental tRNAs of the bacterioplankton, where its specific tRNAs prevailed from day 13 onward. This event was also independent of the introduced nonindigenous bacteria because it occurred at the same time in all nutrient-amended mesocosms. By contrast, in the unamended experiments, a different small 5S rRNA could by observed from day 10 onward with less pronounced dominance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence, genetic variability, and potential significance of “Candidatus Midichloria mitochondrii” in the lone star tick Amblyomma americanum

We used next generation sequencing to detect the bacterium "Candidatus Midichloria mitochondrii" for the first time in lone star ticks (Amblyomma americanum) from the eastern United States. 177 individuals and 11 tick pools from seven sites in four states were tested by pyrosequencing with barcoded 16S rRNA gene eubacterial primers targeting variable regions 5-3. Average infection prevalence was 0.15 across all surveyed populations (range 0-0.29) and only the site with the smallest sample size (n?=?5) was negative. Three genotypes differing by 2.6-4.1?% in a 271?bp region of 16S rRNA gene were identified. Two variants co-occurred in sites in North Carolina and New York, but were not observed in the same tick at those sites. The third genotype was found only in Georgia. Phylogenetic analysis of this fragment indicated that the three variants are more closely related to "Candidatus Midichloria mitochondrii" genotypes from other tick species than to each other. This variation suggests that multiple independent introductions occurred in A. americanum which may provide insight into bacterial spread within its ecosystem and parasitism on this tick. Whether the presence of this bacterium affects acquisition or maintenance of other pathogens and symbionts in A. americanum or the survival, biology and evolution of the tick itself is unknown.     

Molecular Characterization of “Candidatus Parilichlamydia carangidicola,” a Novel Chlamydia-Like Epitheliocystis Agent in Yellowtail Kingfish,Seriola lalandi (Valenciennes), and the Proposal of a New Family, “Candidatus Parilichlamydiaceae” fam. nov. (Order Chlamydiales)

Three cohorts of farmed yellowtail kingfish (Seriola lalandi) from South Australia were examined for Chlamydia-like organisms associated with epitheliocystis. To characterize the bacteria, 38 gill samples were processed for histopathology, electron microscopy, and 16S rRNA amplification, sequencing, and phylogenetic analysis. Microscopically, the presence of membrane-enclosed cysts was observed within the gill lamellae. Also observed was hyperplasia of the epithelial cells with cytoplasmic vacuolization and fusion of the gill lamellae. Transmission electron microscopy revealed morphological features of the reticulate and intermediate bodies typical of members of the order Chlamydiales. A novel 1,393-bp 16S chlamydial rRNA sequence was amplified from gill DNA extracted from fish in all cohorts over a 3-year period that corresponded to the 16S rRNA sequence amplified directly from laser-dissected cysts. This sequence was only 87% similar to the reported “Candidatus Piscichlamydia salmonis” ({"type":"entrez-nucleotide","attrs":{"text":"AY462244","term_id":"42525281","term_text":"AY462244"}}AY462244) from Atlantic salmon and Arctic charr. Phylogenetic analysis of this sequence against 35 Chlamydia and Chlamydia-like bacteria revealed that this novel bacterium belongs to an undescribed family lineage in the order Chlamydiales. Based on these observations, we propose this bacterium of yellowtail kingfish be known as “Candidatus Parilichlamydia carangidicola” and that the new family be known as “Candidatus Parilichlamydiaceae.”     

Natural occurrence of Mycobacterium as an endosymbiont of Acanthamoeba isolated from a contact lens storage case

Recent in vitro studies have revealed that a certain Mycobacterium can survive and multiply within free-living amoebae. It is believed that protozoans function as host cells for the intracellular replication and evasion of Mycobacterium spp. under harmful conditions. In this study, we describe the isolation and characterization of a bacterium naturally observed within an amoeba isolate acquired from a contact lens storage case. The bacterium multiplied within Acanthamoeba, but exerted no cytopathic effects on the amoeba during a 6-year amoebic culture. Transmission electron microscopy showed that the bacteria were randomly distributed within the cytoplasm of trophozoites and cysts of Acanthamoeba. On the basis of the results of 18S rRNA gene analysis, the amoeba was identified as A. lugdunensis. A 16S rRNA gene analysis placed this bacterium within the genus Mycobacterium. The bacterium evidenced positive reactivity for acid-fast and fluorescent acid-fast stains. The bacterium was capable of growth on the Middlebrook 7H11-Mycobacterium-specific agar. The identification and characterization of bacterial endosymbionts of free-living protozoa bears significant implications for our understanding of the ecology and the identification of other atypical mycobacterial pathogens.     

Molecular Characterization of “Candidatus Similichlamydia latridicola” gen. nov., sp. nov. (Chlamydiales: “Candidatus Parilichlamydiaceae”), a Novel Chlamydia-Like Epitheliocystis Agent in the Striped Trumpeter,Latris lineata (Forster)

Histological analysis of gill samples taken from individuals of Latris lineata reared in aquaculture in Tasmania, Australia, and those sampled from the wild revealed the presence of epitheliocystis-like basophilic inclusions. Subsequent morphological, in situ hybridization, and molecular analyses were performed to confirm the presence of this disease and discovered a Chlamydia-like organism associated with this condition, and the criteria set by Fredericks and Relman''s postulates were used to establish disease causation. Three distinct 16S rRNA genotypes were sequenced from 16 fish, and phylogenetic analyses of the nearly full-length 16S rRNA sequences generated for this bacterial agent indicated that they were nearly identical novel members of the order Chlamydiales. This new taxon formed a well-supported clade with “Candidatus Parilichlamydia carangidicola” from the yellowtail kingfish (Seriola lalandi). On the basis of sequence divergence over the 16S rRNA region relative to all other members of the order Chlamydiales, a new genus and species are proposed here for the Chlamydia-like bacterium from L. lineata, i.e., “Candidatus Similichlamydia latridicola” gen. nov., sp. nov.     

Molecular evidence for association of chlamydiales bacteria with epitheliocystis in leafy seadragon (Phycodurus eques), silver perch (Bidyanus bidyanus), and barramundi (Lates calcarifer)

Epitheliocystis in leafy seadragon (Phycodurus eques), silver perch (Bidyanus bidyanus), and barramundi (Lates calcarifer), previously associated with chlamydial bacterial infection using ultrastructural analysis, was further investigated by using molecular and immunocytochemical methods. Morphologically, all three species showed epitheliocystis cysts in the gills, and barramundi also showed lymphocystis cysts in the skin. From gill cysts of all three species and from skin cysts of barramundi 16S rRNA gene fragments were amplified by PCR and sequenced, which clustered by phylogenetic analysis together with other chlamydia-like organisms in the order Chlamydiales in a lineage separate from the family Chlamydiaceae. By using in situ RNA hybridization, 16S rRNA Chlamydiales-specific sequences were detected in gill cysts of silver perch and in gill and skin cysts of barramundi. By applying immunocytochemistry, chlamydial antigens (lipopolysaccharide and/or membrane protein) were detected in gill cysts of leafy seadragon and in gill and skin cysts of barramundi, but not in gill cysts of silver perch. In conclusion, this is the first time epitheliocystis agents of leafy seadragon, silver perch and barramundi have been undoubtedly identified as belonging to bacteria of the order Chlamydiales by molecular methods. In addition, the results suggested that lymphocystis cysts, known to be caused by iridovirus infection, could be coinfected with the epitheliocystis agent.