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

Abstract

Shrimps of the family Alvinocarididae, endemic species to deep sea chemosynthetic ecosystems, harbor epibiotic microbes on gills which probably play important roles in the survival of the shrimps. Among them, Alvinocaris longirostris and Shinkaicaris leurokolos occupy different ecological niches within the same hydrothermal vent in Okinawa Trough, and A. longirostris also exists in a methane seep of the South China Sea. In this study, full-length 16S rRNA sequences of the gill associated bacteria of two alvinocaridid species from different chemosynthetically ecological niches were first captured by single-molecule real-time sequencing. Totally, 120,792 optimized circular consensus sequences with ∼1,450 bp in length were obtained and clustered into 578 operational taxonomic units. Alpha diversity analysis showed seep A. longirostris had the highest species richness and evenness (average Chao1 = 213.68, Shannon = 3.39). Beta diversity analysis revealed that all samples were clearly divided into three groups, and microbial community of A. longirostris from seep and vent were more related than the other comparisons. By permutational multivariate analysis of variance, the most significant community compositional variance was detected between seep A. longirostris and vent S. leurokolos (R ² = 0.731, P = 0.001). The taxon tags were further classified into 21 phyla, 40 classes, 89 orders, 124 families and 135 genera. Overall, the microbial communities were dominated by Campylobacteria and Gammaproteobacteria. Alphaproteobacteria, Bacteroidia, Verrucomicrobiae, Bacilli and other minor groups were also detected at lower abundance. Taxonomic groups recovered from the vent S. leurokolos samples were only dominated by Sulfurovaceae (94.06%). In comparison, gill-associated microbiota of vent A. longirostris consisted of more diverse sulfur-oxidizing bacteria, including Sulfurovaceae (69.21%), Thiotrichaceae (6.77%) and a putative novel Gammaproteobacteria group (14.37%), while in seep A. longirostris, Gammaproteobacteria un-group (44.01%) constituted the major component, following the methane-oxidizing bacteria Methylomonadaceae (19.38%), and Sulfurovaceae (18.66%). Therefore, the gill associated bacteria composition and abundance of alvinocaridid shrimps are closely related to the habitat heterogeneity and the selection of microbiota by the host. However, the interaction between these alvinocaridid shrimps and the epibiotic communities requires further study based on metagenome sequencing and fluorescence in situ hybridization.

Keywords: Alvinocarididae; Deep-sea; Hydrothermal vent; Methane seep; Microbial diversity.

Figure 1. Sampling information of alvinocaridid shrimps.

(A) Sampling sites of alvinocaridid shrimps. The map was created by Ocean Data View (ODV) v5.3.0.0 (Schlitzer, 2020). Different colors on the right color bar represent different water depths. ALMS: A. longirostris from Methane Seep; ALHV: A. longirostris from Hydrothermal Vent; SLHV: S. leurokolos from Hydrothermal Vent. (B) The S. leurokolos living environment in the Iheya North hydrothermal vent of Okinawa Trough. The S. leurokolos is marked in the red square. (C) The A. longirostris living environment in the Formosa Ridge methane seep of the South China Sea. The A. longirostris is marked in the blue square. (D) Morphological characteristics of S. leurokolos. (E) Morphological characteristics of A. longirostris. (F) Environmental parameters of the living environment of different sample groups. FR: Formosa Ridge; IN: Iheya North.

Figure 2. Operational taxonomic units (OTUs) of different samples.

(A) Rarefaction curve diagrams of OTUs from samples of ALHV, ALMS, and SLHV groups. (B) OTU number distribution in each sample and groups. (C) Venn diagram of OTUs among the three groups.

Figure 3. Microbiota alpha and beta diversity evaluated based on the Chao1 (A), Shannon (B) indices, and principal coordinates analysis (PCoA) among ALMS, ALHV, and SLHV (C). An asterisk (*) indicates significant differences with P < 0.05; two asterisks (**) indicates extremely significant difference with P < 0.01.

Figure 4. Relative abundances of bacteria at the phylum (A), class (B), family (C) and genus (D) level in gill samples of ALMS, ALHV, and SLHV.

Figure 5. Phylogenetic analysis of the top OTUs.

The phylogenetic tree is constructed based on 16S rRNA full-length sequences with the distance-based maximum-likelihood (ML) method using IQ-TREE. Numbers in the nodes correspond to ML bootstrap proportions (>50). Red, green and blue numbers in brackets represent relative abundances of the OTUs in ALMS, ALHV, and SLHV, respectively.

Figure 6. Bacteria present in A. longirostris core microbiome and their relative abundance in each sample.

The OTUs in red represent bacteria in core microbiome of A. longirostris and S. leurokolos.