Metatranscriptomics by In Situ RNA Stabilization Directly and Comprehensively Revealed Episymbiotic Microbial Communities of Deep-Sea Squat Lobsters.

Shinkaia crosnieri is an invertebrate that inhabits an area around deep-sea hydrothermal vents in the Okinawa Trough in Japan by harboring episymbiotic microbes as the primary nutrition. To reveal physiology and phylogenetic composition of the active episymbiotic populations, metatranscriptomics is expected to be a powerful approach. However, this has been hindered by substantial perturbation (e.g., RNA degradation) during time-consuming retrieval from the deep sea. Here, we conducted direct metatranscriptomic analysis of S. crosnieri episymbionts by applying in situ RNA stabilization equipment. As expected, we obtained RNA expression profiles that were substantially different from those obtained by conventional metatranscriptomics (i.e., stabilization after retrieval). The episymbiotic community members were dominated by three orders, namely, Thiotrichales, Methylococcales, and Campylobacterales, and the Campylobacterales members were mostly dominated by the Sulfurovum genus. At a finer phylogenetic scale, the episymbiotic communities on different host individuals shared many species, indicating that the episymbionts on each host individual are not descendants of a few founder cells but are horizontally exchanged. Furthermore, our analysis revealed the key metabolisms of the community: two carbon fixation pathways, a formaldehyde assimilation pathway, and utilization of five electron donors (sulfide, thiosulfate, sulfur, methane, and ammonia) and two electron accepters (oxygen and nitrate/nitrite). Importantly, it was suggested that Thiotrichales episymbionts can utilize intercellular sulfur globules even when sulfur compounds are not usable, possibly also in a detached and free-living state.IMPORTANCE Deep-sea hydrothermal vent ecosystems remain mysterious. To depict in detail the enigmatic life of chemosynthetic microbes, which are key primary producers in these ecosystems, metatranscriptomic analysis is expected to be a promising approach. However, this has been hindered by substantial perturbation (e.g., RNA degradation) during time-consuming retrieval from the deep sea. In this study, we conducted direct metatranscriptome analysis of microbial episymbionts of deep-sea squat lobsters (Shinkaia crosnieri) by applying in situ RNA stabilization equipment. Compared to conventional metatranscriptomics (i.e., RNA stabilization after retrieval), our method provided substantially different RNA expression profiles. Moreover, we discovered that S. crosnieri and its episymbiotic microbes constitute complex and resilient ecosystems, where closely related but various episymbionts are stably maintained by horizontal exchange and partly by their sulfur storage ability for survival even when sulfur compounds are not usable, likely also in a detached and free-living state.

Compositional and Functional Shifts in the Epibiotic Bacterial Community of Shinkaia crosnieri Baba & Williams (a Squat Lobster from Hydrothermal Vents) during Methane-Fed Rearing.

The hydrothermal vent squat lobster Shinkaia crosnieri Baba & Williams harbors an epibiotic bacterial community, which is numerically and functionally dominated by methanotrophs affiliated with Methylococcaceae and thioautotrophs affiliated with Sulfurovum and Thiotrichaceae. In the present study, shifts in the phylogenetic composition and metabolic function of the epibiont community were investigated using S. crosnieri individuals, which were reared for one year in a tank fed with methane as the energy and carbon source. The results obtained indicated that indigenous predominant thioautotrophic populations, such as Sulfurovum and Thiotrichaceae members, became absent, possibly due to the lack of an energy source, and epibiotic communities were dominated by indigenous Methylococcaceae and betaproteobacterial methylotrophic members that adapted to the conditions present during rearing for 12 months with a supply of methane. Furthermore, the overall phylogenetic composition of the epibiotic community markedly changed from a composition dominated by chemolithotrophs to one enriched with cross-feeding heterotrophs in addition to methanotrophs and methylotrophs. Thus, the composition and function of the S. crosnieri epibiotic bacterial community were strongly affected by the balance between the energy and carbon sources supplied for chemosynthetic production as well as that between the production and consumption of organic compounds.

Molecular evidence of digestion and absorption of epibiotic bacterial community by deep-sea crab Shinkaia crosnieri.

The hydrothermal vent crab Shinkaia crosnieri is considered to obtain nutrition from the epibiotic bacteria found on the setae, but previous studies have not shown how nutrients can be transferred from the epibionts to the host. In this study, microscopic observations of S. crosnieri intestinal components detected autofluorescent setae fragments and pigmentation derived from the digestion of epibionts in a dye-stained epibiont tracer experiment. An in vitro digestion experiment with epibiotic populations using an intestinal extract demonstrated the degradation of epibiotic cells by digestive enzymes. A phylogenetic analysis showed that many of the bacterial 16S ribosomal RNA gene sequences obtained from the intestine were closely related to the sequences of the epibionts, thus they were probably derived from the epibionts. A stable isotope tracer experiment also indicated that (13)C assimilated by the epibionts provided a carbon (nutrition) source for the host. Both activity measurements and isotope studies showed that chemosynthetic metabolism by the gut microbial components were inactive. Together with the feeding behaviour of living S. crosnieri, these results indicate that S. crosnieri ingests the epibionts using maxillipeds and assimilates them via its digestive organs as a nutrient source. The results of this study elucidate the mechanism of nutritional transfer in ectosymbiosis between chemosynthetic bacteria and deep-sea invertebrates.