Endozoicomonas Are Specific, Facultative Symbionts of Sea Squirts

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.

Keywords: Endozoicomonas; ascidians; marine; mucin; sea squirts; symbiosis; tunicates.

Figure 1

Dendrogram of 16S rRNA gene sequences of the Endozoicomonas clade. Strict consensus representation (i.e., the shown branching patterns and subclades were present in all source trees) of phylogenetic trees calculated by MP, ML, and BI analyses using only nearly full-length (≥1400 bp) 16S rRNA gene sequences. The dendrogram was rooted with sequences of the genera Zooshikella and Hahella (not shown). The dendrogram was truncated (indicated with dotted line and arrow) and does not show an additional 80 sequences of bacteria associated with coral hosts. Host species and accession numbers of single sequences are shown in brackets. For subclades, numbers of containing sequences are shown in brackets. Major host groups are indicated with silhouette symbols. Sequences of ascidian origin are additionally marked with green. Branch lengths do not represent phylogenetic distances.

Figure 2

Dendrogram of 16S rRNA gene sequences of Endozoicomonas affiliating with the ascidian-specific subclade depicted in Figure 1. Host species and geographic origin are given for each sequence, followed by the corresponding accession number in brackets. Sequences generated in the present study are shown in bold face. Sequences originating from Endozoicomonas isolates are marked with an asterisk. The dendrogram was truncated and does not show an additional 77 sequences of bacteria associated with Atlantic and Pacific specimens of the ascidian Ciona intestinalis (all from Dishaw et al., 2014). A summary of partial Endozoicomonas sequences generated in this study and confidently assigned to the ascidian-specific subclade was added manually (indicated with dotted lines) to illustrate host and geographic distribution. For these, the number of specimens harboring representatives of the ascidian-specific subclade as well as the total number of positively assigned sequences is shown in brackets (see also Table S6). Branch lengths do not represent phylogenetic distances.

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.