In order to unveil the microbiome signature and diversity in the gut and flesh samples of hilsa fish collected from three habitats (e.g., FW, BW and MW) of Bangladesh, we analysed 15 gut samples (e.g., intestinal contents) and three flesh samples through 16S rRNA amplicon sequencing. The study sampling information, demographics, amplicon sequence related data, assigned operational taxonomic units (OTUs) per sample and SRA (sequence read archives) accession numbers of the study subjects are summarized in Table 1. The mean length, girth and weight of the hilsa fishes were 38.83 cm, 24.31 cm and 786.17 gm (Table S1). Among these hilsa fishes, 15 samples were from gut (83.33%) and three were flesh (16.67%). The 16S rRNA gene amplicon sequencing of the 18 hilsa fish samples generated 3,696,608 raw reads (average: 205,367 reads/sample), of which 390,289 quality reads (10.56%) mapped to 325 OTUs of bacteria. Among the observed OTUs, 67 and 258 OTUs were identified in flesh and gut samples, respectively (Table 1).
To elucidate whether diversity of the hilsa fish varies according to sample categories (e.g., gut and flesh) and habitats (e.g., FW, BW and MW), we examined both within sample (alpha) and across the samples (beta) diversities of the detected bacterial communities (Fig. 1). The alpha diversity measured using Observed species, Chao1, Shannon, Simpson, InvSimpson and Fisher indices showed significant differences in bacterial community richness, keeping substantially higher diversity in gut samples associated bacteriomes (p = 0.05; Wilcoxon test) than the flesh samples (Fig. 1A). The within sample diversities were more distinct according to the habitats of the hilsa fishes where higher diversity was estimated in FW followed by BW and MW (p = 0.05; Wilcoxon test) (Fig. 1B). The Bray–Curtis dissimilarity distance estimated principal coordinate analysis (PCoA) plot showed that bacteriome composition in hilsa fishes differed significantly according to sample categories (gut versus flesh, p = 0.03, R2 = 0.076, PERMANOVA test) (Fig. 1C). Moreover, the beta diversity of the bacteriomes also varied significantly according to the host habitats (p = 0.05, R2 = 0.246, PERMANOVA test) (Fig. 1D). The observed OTUs were represented by six phyla, nine classes, 19 orders, 26 families and 40 genera of bacteria (Table S2). At phylum level, the hilsa fish bacteriome was dominated by Firmicutes, Proteobacteria, and Planctomycetes, comprising > 97.5% of the total abundances (Data S1). Among these phyla, Firmicutes was the most abundant phylum with a relative abundance of 62.71% and 88.0% in gut and flesh samples, respectively. Proteobacteria was found as the second top abundant bacterial phylum with 23.11% and 7.66% relative abundances in gut and flesh, respectively (Data S1). By comparing the relative abundances of the detected phyla across the habitats of the hilsa fish, we found that Firmicutes was predominant bacterial phylum in FW, BW and MW (> 65.0% relative abundances). Likewise, Proteobacteria was the second most predominating phylum in FW (16.97%) and BW (24.52%) while Planctomycetes remained as the second most predominant phylum (15.39%) in MW. The rest of phyla also differed significantly between gut and flesh samples and across the habitats (Fig. 2, Data S1). By comparing the bacterial taxa at order level, we found that Lactobacillales was the top most abundant (76.98%) bacterial order in gut whereas Enterobacteriales was the predominant order in flesh samples with a relative abundance of 86.83% (Fig. 3, Data S1). The relative abundance of these two bacterial orders also varied across the habitats of the hilsa fish. For instance, Lactobacillales and Enterobacteriales were detected with relative abundances of 82.63% and 15.36%, respectively in FW, while Enterobacteriales and Lactobacillales had 92.45% and 2.20% abundances, respectively in BW, and 79.67% and 13.39% abundances, respectively in MW (Fig. 3). Enterobacteriaceae (89.64%), Aeromonadaceae (4.84%) and Moraxellaceae (1.31%) were the top abundant families in the gut of hilsa fishes whereas Enterococcaceae (82.91%) and Enterobacteriaceae (15.44%) were the most predominating bacterial families in the flesh (Fig. S1, Data S1). Despite, having had relatively lower abundances (< 2.0%), rest of the bacterial orders and families also showed discriminations according to sample groups and habitats (Data S1).
We also demonstrated noteworthy differences in both composition and the relative abundances of bacterial taxa at genus-level according to sample categories (gut and flesh; p = 0.0127; Kruskal Wallis test) and habitats (FW, BW and MW; p = 0.007; Kruskal Wallis test) of the hilsa fishes. In this study, we detected 40 bacterial genera in gut and 30 genera in flesh samples of the hilsa fishes, of which 30 (75%) were identified as the shared genera between the gut and flesh (Fig. S2a). Likewise, 40, 35 and 22 bacterial genera were detected in FW, BW and MW, and of them, 18 (45%) genera were found to be shared in all of the three habitats (Fig. S2b, Table S2). By studying the phylogenetic relationship of the identified bacterial genera, we found that majority of these genera (n = 18) belonged to Gammaproteobacteria followed by Bacilli (n = 15), Actinobacteria (n = 2), Planctomycetia (n = 2) and others (n = 3) (Fig. S3).
To examine whether genus level composition and relative abundance of the bacteria vary between the sample categories and across the host habitats, we performed pairwise Kruskal–Wallis test of the relative abundances of all genera identified (Figs. 4 and 5). Although, 40 bacterial genera were detected, twenty-five genera had differentially abundant OTUs in the study samples and host habitats (p < 0.05, Kruskal–Wallis test) (Fig. 5). Of the identified genera, Vagococcus (68.67%), Morganella (13.57%), Enterobacter (5.37%), Plesiomonas (3.56%), Shigella (1.75%), Clostridium (1.58%) were the top abundant genera in the gut of hilsa fishes. Conversely, Enterobacter (19.87%), Serratia (19.85%), Aeromonas (18.56), Klebsiella (14.33%), Acinetobacter (4.85%), Vagococcus (4.21%), Macrococcus (2.84%), Clostridium (2.55%), Pseudomonas (1.97%), Shigella (1.69%), Hafnia (1.59%), Plesiomonas (1.51%), Weissella (1.1%), and Morganella (1.0%) were the predominating genera in flesh of hilsa fishes (Fig. 4A). Despite having lower relative abundances, the gut samples had sole association of ten genera such as Proteus, Cronobacter, Synechococcus, Streptococcus, Photobacterium, Lactobacillus, Peptoniphilus, Gemmata, Serinicoccus, and Saccharopolyspora (Data S1).
The FW samples were dominated by Vagococcus (69.54%), Morganella (13.81%), Enterobacter (4.98%), Plesiomonas (3.59%), Shigella (1.68%) and Clostridium (1.45%). In addition, BW samples were mostly dominated by Enterobacter (23.13%), Proteus (14.59%), Vagococcus (12.68%), Aeromonas (11.14%), Serratia (7.55%), Cronobacter (6.98%), Klebsiella (6.31%), Clostridium (5.18%), Shigella (3.36%), Acinetobacter (3.30%), and Plesiomonas (1.79%). On the other hand, Serratia (35.11%), Morganella (20.68%), Vagococcus (17.69%), Erwinia (7.02%), Klebsiella (4.81%), Shigella (3.90%), Staphylococcus (3.77%), Sinobaca (2.60%), and Clostridium (1.04%) were the predominating bacterial genera detected in the MW samples of the hilsa fishes (Fig. 4B). Besides, Vagococcus, Shigella and Morganella had significant association with gut (p < 0.05, Kruskal Wallis test) while Acinetobacter, Aeromonas, Hafnia, Klebsiella, Pseudomonas, Enterobacter, Weissella, Salmonella and Serratia had substantial association with flesh samples (p < 0.05, Kruskal Wallis test) of the hilsa fishes (Fig. 5A). Likewise, Acinetobacter, Klebsiella, Pseudomonas and Planctomyces in FW, Enterobacter, Morganella, Klebsiella and Shigella in BW, and Proteus in MW had significant correlations (p < 0.05, Kruskal Wallis test). Remarkably, Lactococcus, Macrococcus and Vagococcus had stronger correlation (p < 0.05, Kruskal Wallis test) with all of the samples from three habitats (i.e., FW, BW and MW) (Fig. 5B). Interestingly, of the identified 40 bacterial genera, six genera (e.g., Sinobaca, Synechococcus, Gemmata, Serinicoccus, Saccharopolyspora, and Paulinella) have not been identified in any aquatic and marine fish species. Though, rest of the genera had relatively lower abundances (< 1.0%), but their relative abundances differed in two sample groups (gut vs flesh), and across three habitats (e.g., FW, BW and MW) of the hilsa fishes (Fig. 4, Data S1).