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Endozoicomonas Are Specific, Facultative Symbionts of Sea Squirts.

Schreiber L, Kjeldsen KU, Funch P, Jensen J, Obst M, López-Legentil S, Schramm A - Front Microbiol (2016)

Bottom Line: The strains tested negative for cytotoxic or antibacterial activity.Based on these observations, we propose ascidian-associated Endozoicomonas to be commensals, living off the mucus continuously secreted into the pharynx.The combined results indicate a host-specific, yet facultative symbiosis between ascidians and Endozoicomonas.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience, Center for Geomicrobiology and Section for Microbiology, Aarhus University Aarhus, Denmark.

ABSTRACT
Ascidians are marine filter feeders and harbor diverse microbiota that can exhibit a high degree of host-specificity. Pharyngeal samples of Scandinavian and Mediterranean ascidians were screened for consistently associated bacteria by culture-dependent and -independent approaches. Representatives of the Endozoicomonas (Gammaproteobacteria, Hahellaceae) clade were detected in the ascidian species Ascidiella aspersa, Ascidiella scabra, Botryllus schlosseri, Ciona intestinalis, Styela clava, and multiple Ascidia/Ascidiella spp. In total, Endozoicomonas was detected in more than half of all specimens screened, and in 25-100% of the specimens for each species. The retrieved Endozoicomonas 16S rRNA gene sequences formed an ascidian-specific subclade, whose members were detected by fluorescence in situ hybridization (FISH) as extracellular microcolonies in the pharynx. Two strains of the ascidian-specific Endozoicomonas subclade were isolated in pure culture and characterized. Both strains are chemoorganoheterotrophs and grow on mucin (a mucus glycoprotein). The strains tested negative for cytotoxic or antibacterial activity. Based on these observations, we propose ascidian-associated Endozoicomonas to be commensals, living off the mucus continuously secreted into the pharynx. Members of the ascidian-specific Endozoicomonas subclade were also detected in seawater from the Scandinavian sampling site, which suggests acquisition of the symbionts by horizontal transmission. The combined results indicate a host-specific, yet facultative symbiosis between ascidians and Endozoicomonas.

No MeSH data available.


Related in: MedlinePlus

Ascidian anatomy and FISH detection of Endozoicomonas in the pharynx tissue of A. aspersa. (A) Anatomical sketch of a solitary ascidian (redrawn from multiple sources). (B) Micrograph of Endozoicomonas microcolonies detected by probe ENDO-1240 (green) and probe mix EUB338 I-III (red). Overlay of the two probes produced the yellow-colored colonies observed in the micrograph. Autofluorescent pharynx tissue is shown in green. (C) Confocal micrograph of pharynx-associated Endozoicomonas microcolonies detected by probe ENDO-580 (red) and probe mix EUB338 I-III (pink). Overlay of the two probes produced the magenta-colored colonies shown in the micrograph. Pharynx nuclei were stained with DAPI (cyan). (D) Confocal micrograph of pharynx-associated Endozoicomonas microcolonies detected by probe ENDO-580 (red). Pharynx nuclei were stained with DAPI (blue). All scale bars, 10 5m. As, atrial siphon; Bs, buccal siphon; En, Endozoicomonas microcolonies; Es, endostyle; Gd, gonads; Gt, gut; Nc, nuclei of pharynx tissue; Pb, pharyngeal bars; Ph, pharynx; Ps, pharyngeal stigmata; Tn, tunic; Vc, visceral cavity.
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Figure 3: Ascidian anatomy and FISH detection of Endozoicomonas in the pharynx tissue of A. aspersa. (A) Anatomical sketch of a solitary ascidian (redrawn from multiple sources). (B) Micrograph of Endozoicomonas microcolonies detected by probe ENDO-1240 (green) and probe mix EUB338 I-III (red). Overlay of the two probes produced the yellow-colored colonies observed in the micrograph. Autofluorescent pharynx tissue is shown in green. (C) Confocal micrograph of pharynx-associated Endozoicomonas microcolonies detected by probe ENDO-580 (red) and probe mix EUB338 I-III (pink). Overlay of the two probes produced the magenta-colored colonies shown in the micrograph. Pharynx nuclei were stained with DAPI (cyan). (D) Confocal micrograph of pharynx-associated Endozoicomonas microcolonies detected by probe ENDO-580 (red). Pharynx nuclei were stained with DAPI (blue). All scale bars, 10 5m. As, atrial siphon; Bs, buccal siphon; En, Endozoicomonas microcolonies; Es, endostyle; Gd, gonads; Gt, gut; Nc, nuclei of pharynx tissue; Pb, pharyngeal bars; Ph, pharynx; Ps, pharyngeal stigmata; Tn, tunic; Vc, visceral cavity.

Mentions: Endozoicomonas cells were detected in pharynx samples of A. aspersa (Figure 3), A. scabra, and Ascidia sp. (one specimen each) by FISH. Endozoicomonas formed microcolonies on the pharyngal epithelium outside of the host cells. However, due to high background fluorescence of the pharynx tissue, an additional intracellular localization within the host's pharynx cells cannot be excluded. Host cell nuclei and Endozoicomonas microcolonies did never co-localize (Figures 3C,D), rendering an intranuclear localization of Endozoicomonas (as in bathymodiolin mussels) unlikely. These FISH results suggest that Endozoicomonas are not just enriched by the ascidians from seawater by filtration but are actually able to grow in situ in their host, where they occupy a protected niche in crevices and grooves of the pharynx. Currently no data exist on the localization of Endozoicomonas in other ascidian species. However, similar bacterial microcolonies, albeit of unknown identity, have previously been reported in association with the pharynx of larvae of the ascidian Ecteinascidia turbinata (Moss et al., 2003). Endozoicomonas associated with the Red Sea coral Stylophora pistillata also grow as microcolonies in the coral endoderm (Bayer et al., 2013), while Endozoicomonas associated with bathymodiolin mussels grow inside cell nuclei of the host (Zielinski et al., 2009). Interestingly, in both ascidians and the coral, Endozoicomonas assumes a smaller cell size (diameter, 1 μm) and coccoid morphology in situ, compared to the larger rod-shaped cells (cell size up to 0.5 × 10 μm) of laboratory pure cultures (Kurahashi and Yokota, 2007; Yang et al., 2010; Nishijima et al., 2013; Pike et al., 2013; Hyun et al., 2014; Figure S7, this study), indicating morphological adaptation to the host environment.


Endozoicomonas Are Specific, Facultative Symbionts of Sea Squirts.

Schreiber L, Kjeldsen KU, Funch P, Jensen J, Obst M, López-Legentil S, Schramm A - Front Microbiol (2016)

Ascidian anatomy and FISH detection of Endozoicomonas in the pharynx tissue of A. aspersa. (A) Anatomical sketch of a solitary ascidian (redrawn from multiple sources). (B) Micrograph of Endozoicomonas microcolonies detected by probe ENDO-1240 (green) and probe mix EUB338 I-III (red). Overlay of the two probes produced the yellow-colored colonies observed in the micrograph. Autofluorescent pharynx tissue is shown in green. (C) Confocal micrograph of pharynx-associated Endozoicomonas microcolonies detected by probe ENDO-580 (red) and probe mix EUB338 I-III (pink). Overlay of the two probes produced the magenta-colored colonies shown in the micrograph. Pharynx nuclei were stained with DAPI (cyan). (D) Confocal micrograph of pharynx-associated Endozoicomonas microcolonies detected by probe ENDO-580 (red). Pharynx nuclei were stained with DAPI (blue). All scale bars, 10 5m. As, atrial siphon; Bs, buccal siphon; En, Endozoicomonas microcolonies; Es, endostyle; Gd, gonads; Gt, gut; Nc, nuclei of pharynx tissue; Pb, pharyngeal bars; Ph, pharynx; Ps, pharyngeal stigmata; Tn, tunic; Vc, visceral cavity.
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Figure 3: Ascidian anatomy and FISH detection of Endozoicomonas in the pharynx tissue of A. aspersa. (A) Anatomical sketch of a solitary ascidian (redrawn from multiple sources). (B) Micrograph of Endozoicomonas microcolonies detected by probe ENDO-1240 (green) and probe mix EUB338 I-III (red). Overlay of the two probes produced the yellow-colored colonies observed in the micrograph. Autofluorescent pharynx tissue is shown in green. (C) Confocal micrograph of pharynx-associated Endozoicomonas microcolonies detected by probe ENDO-580 (red) and probe mix EUB338 I-III (pink). Overlay of the two probes produced the magenta-colored colonies shown in the micrograph. Pharynx nuclei were stained with DAPI (cyan). (D) Confocal micrograph of pharynx-associated Endozoicomonas microcolonies detected by probe ENDO-580 (red). Pharynx nuclei were stained with DAPI (blue). All scale bars, 10 5m. As, atrial siphon; Bs, buccal siphon; En, Endozoicomonas microcolonies; Es, endostyle; Gd, gonads; Gt, gut; Nc, nuclei of pharynx tissue; Pb, pharyngeal bars; Ph, pharynx; Ps, pharyngeal stigmata; Tn, tunic; Vc, visceral cavity.
Mentions: Endozoicomonas cells were detected in pharynx samples of A. aspersa (Figure 3), A. scabra, and Ascidia sp. (one specimen each) by FISH. Endozoicomonas formed microcolonies on the pharyngal epithelium outside of the host cells. However, due to high background fluorescence of the pharynx tissue, an additional intracellular localization within the host's pharynx cells cannot be excluded. Host cell nuclei and Endozoicomonas microcolonies did never co-localize (Figures 3C,D), rendering an intranuclear localization of Endozoicomonas (as in bathymodiolin mussels) unlikely. These FISH results suggest that Endozoicomonas are not just enriched by the ascidians from seawater by filtration but are actually able to grow in situ in their host, where they occupy a protected niche in crevices and grooves of the pharynx. Currently no data exist on the localization of Endozoicomonas in other ascidian species. However, similar bacterial microcolonies, albeit of unknown identity, have previously been reported in association with the pharynx of larvae of the ascidian Ecteinascidia turbinata (Moss et al., 2003). Endozoicomonas associated with the Red Sea coral Stylophora pistillata also grow as microcolonies in the coral endoderm (Bayer et al., 2013), while Endozoicomonas associated with bathymodiolin mussels grow inside cell nuclei of the host (Zielinski et al., 2009). Interestingly, in both ascidians and the coral, Endozoicomonas assumes a smaller cell size (diameter, 1 μm) and coccoid morphology in situ, compared to the larger rod-shaped cells (cell size up to 0.5 × 10 μm) of laboratory pure cultures (Kurahashi and Yokota, 2007; Yang et al., 2010; Nishijima et al., 2013; Pike et al., 2013; Hyun et al., 2014; Figure S7, this study), indicating morphological adaptation to the host environment.

Bottom Line: The strains tested negative for cytotoxic or antibacterial activity.Based on these observations, we propose ascidian-associated Endozoicomonas to be commensals, living off the mucus continuously secreted into the pharynx.The combined results indicate a host-specific, yet facultative symbiosis between ascidians and Endozoicomonas.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience, Center for Geomicrobiology and Section for Microbiology, Aarhus University Aarhus, Denmark.

ABSTRACT
Ascidians are marine filter feeders and harbor diverse microbiota that can exhibit a high degree of host-specificity. Pharyngeal samples of Scandinavian and Mediterranean ascidians were screened for consistently associated bacteria by culture-dependent and -independent approaches. Representatives of the Endozoicomonas (Gammaproteobacteria, Hahellaceae) clade were detected in the ascidian species Ascidiella aspersa, Ascidiella scabra, Botryllus schlosseri, Ciona intestinalis, Styela clava, and multiple Ascidia/Ascidiella spp. In total, Endozoicomonas was detected in more than half of all specimens screened, and in 25-100% of the specimens for each species. The retrieved Endozoicomonas 16S rRNA gene sequences formed an ascidian-specific subclade, whose members were detected by fluorescence in situ hybridization (FISH) as extracellular microcolonies in the pharynx. Two strains of the ascidian-specific Endozoicomonas subclade were isolated in pure culture and characterized. Both strains are chemoorganoheterotrophs and grow on mucin (a mucus glycoprotein). The strains tested negative for cytotoxic or antibacterial activity. Based on these observations, we propose ascidian-associated Endozoicomonas to be commensals, living off the mucus continuously secreted into the pharynx. Members of the ascidian-specific Endozoicomonas subclade were also detected in seawater from the Scandinavian sampling site, which suggests acquisition of the symbionts by horizontal transmission. The combined results indicate a host-specific, yet facultative symbiosis between ascidians and Endozoicomonas.

No MeSH data available.


Related in: MedlinePlus