ABSTRACT
The gut and flesh microbiome of the national fish of Bangladesh, Tenualosa ilisha, were analyzed using 16S rRNA gene sequencing. Our findings revealed a significant microbial disparity between sample categories and the habitat of hilsa fish, which will serve as a valuable foundation for further comprehensive studies on the hilsa microbiome.
KEYWORDS: Tenualosa ilisha, Hilsa flesh, gut microbiome, metagenomics
ANNOUNCEMENT
Hilsa (Tenualosa ilisha) is the national fish of Bangladesh. It contributes ~12% of the total fish production and 1.15% GDP of the country (1). It is widely distributed in Southeast and South Asia (2, 3) and found in coastal areas, estuaries, brackish, and freshwater rivers (3, 4). Hilsa is an anadromous marine fish that comes to freshwater in rivers during spawning. Gut microbiota, which modulate host homeostasis, immunity, and fitness (5, 6), have been analyzed in many fish species (7, 8) but rarely in the hilsa fish (1). Nothing is known about the gut and flesh microbiome of hilsa, which is known to be free from diseases. This study aimed to analyze the gut and flesh microbiome of hilsa fish using 16S rRNA gene amplicon sequencing.
Eighteen adult hilsa fishes were collected and frozen from September to October 2020 from five different sites in Bangladesh (Table 1). Sampling of their intestinal contents (n = 15) and flesh (n = 3) was performed within 48 h after the collection. Fish scales from the body of all individual fishes were removed by sterile forceps under aseptic conditions, and fleshes (100 mg) and lower stomach contents (100 mg) from each fish were collected. Genomic DNA was extracted from each specimen after grinding (flesh) and homogenization (gut content) using DNeasy Blood and Tissue Kit (Qiagen, UK) following the manufacturer’s instructions. A NanoDrop 2000c spectrophotometer was used to check the purity and concentration of DNA. The amplicon libraries were prepared using Nextera XT Index Kit (Illumina Inc.) as per the 16S amplicon sequencing library preparation protocol, and V3–V4 regions were amplified through a 40-cycle PCR using 341F and 806R primers (9). Paired-end sequencing (2 × 150 bp, 300 cycles) was performed on the Illumina MiniSeq sequencer. FastQC v0.11.9 (10) was used to check the quality of the reads, while Trimmomatic v0.39 (11) removed Illumina adapters, known Illumina artifacts, and phiX reads. The demultiplexed sequencing data were processed using QIIME 2 (2023.2.0) and associated plugins (12). Sequences showing ≥98% similarity were grouped into operational taxonomic units (OTUs) using the SILVA database v.138 (13). Default parameters were used for bioinformatic analyses, except where otherwise stated.
TABLE 1.
Summary of metadata and SRA accession numbers of the 16S rRNA amplicon sequences of hilsa fish gut and flesh samples, along with OTUs mapped against bacterial taxa
| Sample ID | Collection site | Coordinate | Habitat | Source | No. of raw reads | No. of mapped reads | No. of observed OTUs | SRA accessions |
|---|---|---|---|---|---|---|---|---|
| CG1 | Confluence of Meghna and Padma River, Chandpur | 23.2321° N, 90.6631° E | Freshwater | Gut | 325,812 | 33,295 | 22 | SRR24402593 |
| CG2 | Confluence of Meghna and Padma River, Chandpur | 23.2321° N, 90.6631° E | Freshwater | Gut | 140,124 | 12,418 | 22 | SRR24402592 |
| CG3 | Confluence of Meghna and Padma River, Chandpur | 23.2321° N, 90.6631° E | Freshwater | Gut | 119,676 | 4,738 | 10 | SRR24402608 |
| CG4 | Confluence of Meghna and Padma River, Chandpur | 23.2321° N, 90.6631° E | Freshwater | Gut | 697,544 | 291,731 | 23 | SRR24402607 |
| CG5 | Confluence of Meghna and Padma River, Chandpur | 23.2321° N, 90.6631° E | Freshwater | Gut | 131,276 | 9,514 | 20 | SRR24402606 |
| RG3 | Padma River, Rajshahi | 24.3745° N, 88.6042° E | Freshwater | Gut | 194,464 | 2,256 | 18 | SRR24402605 |
| MG1 | Meghna River, Munshiganj | 23.5422° N, 90.5305° E | Freshwater | Gut | 165,096 | 1,333 | 15 | SRR24402604 |
| MG2 | Meghna River, Munshiganj | 23.5422° N, 90.5305° E | Freshwater | Gut | 126,908 | 7,353 | 15 | SRR24402602 |
| MG4 | Meghna River, Munshiganj | 23.5422° N, 90.5305° E | Freshwater | Gut | 260,420 | 3,077 | 11 | SRR24402601 |
| PG1 | Payra River, Patuakhali | 22.3586° N, 90.3317° E | Brackish water | Gut | 149,988 | 611 | 23 | SRR24402600 |
| PG2 | Payra River, Patuakhali | 22.3586° N, 90.3317° E | Brackish water | Gut | 119,052 | 1,142 | 11 | SRR24402598 |
| PG3 | Payra River, Patuakhali | 22.3586° N, 90.3317° E | Brackish water | Gut | 361,312 | 8,111 | 20 | SRR24402599 |
| PG5 | Payra River, Patuakhali | 22.3586° N, 90.3317° E | Brackish water | Gut | 178,372 | 3,982 | 18 | SRR24402597 |
| XG1 | Bay of Bengal, Cox’s Bazar |
21.4272° N, 92.0058° E | Marine water | Gut | 112,684 | 509 | 16 | SRR24402596 |
| XG3 | Bay of Bengal, Cox’s Bazar |
21.4272° N, 92.0058° E | Marine water | Gut | 151,480 | 260 | 14 | SRR24402595 |
| CF4 | Confluence of Meghna and Padma River, Chandpur | 23.2321° N, 90.6631° E | Freshwater | Flesh | 180,944 | 4,994 | 16 | SRR24402609 |
| CF5 | Confluence of Meghna and Padma River, Chandpur | 23.2321° N, 90.6631° E | Freshwater | Flesh | 106,604 | 2,226 | 14 | SRR24402603 |
| PF4 | Payra River, Patuakhali | 22.3586° N, 90.3317° E | Brackish water | Flesh | 174,852 | 2,739 | 17 | SRR24402594 |
The 16S rRNA amplicon sequencing of 18 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. The rest of the reads (90%) were unassigned to any OTUs and might come from host DNA. Among these, 67 and 258 OTUs were identified in flesh and gut samples, respectively, through the SILVA database v.138 (Table 1). These OTUs were represented by six phyla, nine classes, 19 orders, 26 families, and 40 genera of bacteria. Firmicutes constituted >75.0% of the hilsa bacteriome, with 78.17% and 2.86% relative abundances in the gut and flesh, respectively. Vagococcus (67.02%), Morganella (13.25%), Enterobacter (5.74%), Plesiomonas (3.51%), Shigella (1.75%), Clostridium (1.60%), Klebsiella (1.02%), and Serratia (1.0%) were the top abundant bacterial genera detected in hilsa fish, with distinct variations in their relative abundances according to the sample categories (gut and flesh; P = 0.0127; Kruskal Wallis test). The genomic data of the flesh and gut bacteriome of hilsa fish revealed in this study has laid the foundation for shedding light on the microbiome of this economically important trans-boundary fish.
ACKNOWLEDGMENTS
We are thankful to the "BSMRAU Physical Facility and Research Capacity Strengthening Project" under the Ministry of Education, People’s Republic of Bangladesh, for funding this research. The authors sincerely thank A.Q.M. Robiul Kawser, Assistant Professor, Department of Aquaculture, Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU), Gazipur 1706, Bangladesh, and Nur Uddin Mahmud of the IBGE, BSMRAU, for their support in sampling, technical assistance in genomic DNA extraction, and PCR.
The authors declare no conflicts of interest regarding this paper.
Contributor Information
Tofazzal Islam, Email: tofazzalislam@bsmrau.edu.bd.
Frank J. Stewart, Montana State University, Bozeman, Montana, USA
DATA AVAILABILITY
The 16S rRNA gene amplicon sequencing data are available at the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA964437. The accession numbers for all SRA experiments are listed in Table 1.
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Associated Data
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Data Availability Statement
The 16S rRNA gene amplicon sequencing data are available at the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA964437. The accession numbers for all SRA experiments are listed in Table 1.