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

Abstract

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.

Metabolism Interactions Promote the Overall Functioning of the Episymbiotic Chemosynthetic Community of Shinkaia crosnieri of Cold Seeps

“…Genes involved in the serine pathway were identified in the Methylococcales and Thiotrichales MAGs. However, none of the MAGs encoded the complete pathway, suggesting that the serine pathway was nonfunctional, as previously suggested ( 42 ). Thiotrichales carried complete gene sets for Calvin-Benson-Bassham (CBB) cycles, while most Campylobacteriales could fix carbon through the rTCA cycle with ATP-citrate lyase.…”

“…16S rRNA amplicon analysis and metatranscriptomic analyses have revealed that the episymbionts of S. crosnieri are dominated by bacteria in the orders Thiotrichales , Campylobacterales , and Methylococcales . The symbionts can supply their hosts with carbon through chemosynthetic carbon fixation using methane and sulfide as electron donors ( 36 – 42 ). However, natural stable carbon isotopic compositions of S. crosnieri epibionts differ extensively among habitats, with the mean delta13C‰ values of vent individuals ranging from −26.3 to −37.0 ( 41 ) and up to −54.1 within seep individuals.…”

Comparative transcriptomic analysis of in situ and onboard fixed deep-sea limpets reveals sample preparation-related differences

“…Hence, conventionally obtained faunal samples can hardly reflect their natural physiological and biochemical status as they were inhabiting the deep sea. It is worth mentioning that some researchers realized this issue and tried to obtain in situ fixed deep-sea samples and advocated for the utilization of in situ fixed deep-sea samples in their studies ( Chen et al., 2021 ; Gao et al., 2019 ; Mat et al, 2020 ; Motoki et al., 2020 ; Sanders et al., 2013 ; Sun et al., 2020 ; Wang et al, 2019b ; Watsuji et al., 2014 ; Wei et al., 2020 ). Nevertheless, studies that explicitly compare the biological difference between in situ fixed and conventional sampled deep-sea fauna are still lacking.…”

Full-length 16S rRNA amplicon sequencing reveals the variation of epibiotic microbiota associated with two shrimp species of Alvinocarididae: possibly co-determined by environmental heterogeneity and specific recognition of hosts

“…Given that gill is one of the organs that directly exchange substances between shrimps and the external environment, we believe that the different bacterial communities found on shrimp gills in this study may play important roles in the adaptation to the extreme environments of the hosts. Further research based on metagenomics, metatranscriptomics and proteomics, as well as in situ experiments, are expected to provide deeper and novel insights into the microbe-host system in deep-sea chemosynthetic environments ( Forget & Kim Juniper, 2013 ; Motoki et al, 2020 ).…”