High-throughput sequencing reveals the dominance of Shewanella species in the intestinal microbiota of barbour’s seahorses (Hippocampus barbouri)

In this study, taxonomic classification of intestinal microbiota from healthy Barbour’s seahorses (Hippocampus barbouri) was determined as it plays an important role in host nutrition and immunity. Genomic DNA was extracted from the intestinal mucus samples of eleven Barbour’s seahorses, which were subjected to high-throughput sequencing of bacterial 16 S rRNA genes targeting V3-V4 regions. The results showed that there were 302,891 amplicon sequence reads representing all samples, with 46 total operational taxonomic units (OTU). Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria were the most predominant phyla observed, with 13 classes, 22 orders, 28 families, and 35 genera, respectively. The results also revealed that the most abundant OTUs were affiliated with the genus Shewanella. Findings in this study may shed light on further studies in exploring the potential implications of intestinal microbiota to seahorses in terms of health status, development, growth, and survival. This could also be used as baseline data for preliminary screening of bacterial species as potential probiotics.


OTU
(Operational Taxonomic Unit) is used to classify closely related organisms by grouping them based on their DNA sequence similarity of a specific taxonomic marker gene (e.g., 16 S rRNA genes) DNA Sequence similarity of a specific taxonomic marker gene (e.g., 16S rRNA genes) BFAR (Bureau of Fisheries and Aquatic Resources) is the government agency responsible for the development, improvement, management, and conservation of the country's fisheries and aquatic resources.It was reconstituted as a line bureau by virtue of Republic Act No. 8550 (Philippine Fisheries Code of 1998) QIIME (Quantitative Insights into Microbial Ecology) is a bioinformatic pipeline that was created with the goal of analyzing microbial communities that are sampled using marker genes (e.g., 16 or 18 S rRNA genes) DNA (Deoxyribonucleic Acid) is a molecule that

Introduction
There has been considerable attention paid to the intestinal microbiota because of its importance to host metabolism, immunity, and infection response (Pérez et al. 2010;Ni et al. 2014).Intestinal microbiota has been found to have a significant influence on the health of humans and mammals, but they also have a major impact on fish health (Butt and Volkoff 2019).However, conventional characterization of intestinal microbiota has depended on cultivation-based techniques, thereby limiting our understanding of its importance to only those microorganisms that can be cultured (Ito et al. 2019).In fact, due to the low culturability (often < 2%) of bacteria from different environments, culture-independent techniques have been used to provide a more comprehensive picture of fish microbiota (Huber et al. 2004).For instance, the advent of high-throughput sequencing technologies, such as Illumina or Oxford Nanopore, has led to significant advances in understanding bacterial community composition and structure in diverse environments (Straub et al. 2020).This has paved the way to reducing the cost, time, and complexity to explore the taxonomic diversity and abundance of bacterial communities within the intestinal ecosystem of fish species.We therefore used this technology to explore the bacterial composition within the intestinal mucus of Barbour's seahorse (Hippocampus barbouri), with particular emphasis on the most dominant species.

Materials and methods
Under the project "Seahorses and Pipefishes with Pharmaceutical Potentials from selected areas in Mindanao, Philippines" with No. 0184 − 19, a Gratuitous Permit from the national office of the Bureau of Fisheries and Aquatic Resources (BFAR) was requested and granted.From coral reefs off the Coast of Cantiasay Island in northeastern Mindanao, the Philippines, eleven healthy Hippocampus barbouri were collected.Samples were immediately transported to our laboratory, where they were kept in an aquarium previously disinfected and under controlled aeration.
Seahorses were starved within 24 h prior to dissection to remove dietary contents (Ortega et al. 2021).The abdominal region was incised from the lateral side using sterile materials (Tanu et al. 2011).Intestinal mucus was collected and homogenized in a sterile saline solution.Genomic DNA was then extracted from the intestinal mucus of six female Barbour's seahorses and five male Barbour's seahorses.Samples were pooled according to their origin and HiPurA™ DNA Purification Kit (HIMEDIA; Mumbai, India) was used for DNA extraction, according to the manufacturer's instructions with slight modifications.Universal primers targeting the V3-V4 regions were used for PCR amplification of 16 S rRNA genes, whose purified products were sent to Macrogen Inc. (Seoul, South Korea) for high-throughput sequencing on the Illumina MiSeq platform.Raw data were processed using the Quantitative Insights into Microbial Ecology (QIIME 2) pipeline described by Bolyen et al. in 2019.At 97% sequence similarity of 16 S rRNA genes, operational taxonomic units (OTUs) were initially defined.The weighted UniFrac test was then applied to determine whether two or more communities have the same structure (Lozupone and Knight 2005).Subsequently, OTUs were clustered at 99% sequence similarity to define a core set of representative sequences for phylogenetic analyses.A heatmap was generated showing the relative abundance of OTUs assigned to the Shewanellaceae family across the samples, which were classified using the EzBioCloud database (Yoon et al. 2017).Phylogenetic analysis was performed by using MEGA version 6.0, as previously described (Balcázar et al. 2010).

Results and discussion
A total of 66,547 sequence reads were collected from the eleven gut samples of seahorses' intestinal tissue.After quality filtering, random subsampling was performed to ensure that all sequence libraries contained the same number of sequences for α-and β-diversity comparisons.The number of OTUs was estimated at a level of 97% 16 S rRNA gene sequence similarity, which resulted in 45 and 41 OTUs in intestinal samples from female and male seahorses, respectively.The species diversity and richness estimators (Shannon and Chao1) showed relatively similar values; however, the weighted UniFrac test (sensitive to abundances of taxa) revealed that while these intestinal samples have similar memberships, the relative abundances of each OTU are different.
Although previous studies have demonstrated that bacterial communities within the fish intestinal ecosystem are dominated by different families belonging to the phylum Proteobacteria (Egerton et al. 2018;Huang et al. 2020), sequences affiliated with the family Shewanellaceae were found to be highly dominant in the intestinal samples from both groups.Several factors may affect the diversity and abundance of the intestinal microbiota, such as diet, trophic level, season, habitat, and sex (Clements et al. 2007;Dhanasiri et al. 2011;Hovda et al. 2012;Cordero et al. 2015;Etyemez and Balcázar 2015;Etyemez-Büyükdeveci et al. 2018).In this study, we also took into account the variations in the relative abundance of bacteria present in the male and female H. barbouri.Factors affecting such presumption are the different physiological exposure of the male and female seahorses (Vincent and Sadler 1995).Our study was supported by the findings of Org et al. (2016), who studied gut microbiota in mice and showed that hormonal effects may be a reason for the differences in microbiota between genders.Our results were also supported by Li et al. (2022), who reported significant differences in gut microbial composition between male and female Nibea albiflora.However, Jiang et al. (2020) excluded the sex difference in terms of the factors affecting the gut microbiota as they found no significant differences between males and females.They however verified that body size and habitat may have greatly contributed to the intestinal microbiota of their Coilia ectenes taihuensis samples.
Because the sequences belonging to the family Shewanellaceae were abundant, a phylogenetic analysis was carried out to establish their taxonomic affiliation.Sequences clustered at 99% sequence similarity and represented by OTU 1, OTU 2, OTU 96, and OTU 126 were found in the intestinal samples from both groups, with OTU 1 being the most abundant in female Barbour's seahorses and OTU 1 and OTU 2 in male Barbour's seahorses (Fig. 1b).Sequence similarity calculations based on a neighbor-joining analysis revealed that these OTUs grouped with known Shewanella species (Fig. 1c).The closest described relative of OTU 1 was S. baltica NCTC 10,735 T (98.5%), whereas OTU 2 was closely related to S. basaltis J83 T (99.6%) and S. ulleungensis MMS16-UL253 T (99.1%).Likewise, OTU 126 was related to S. denitrificans OS217 T (96.5%) and S. maritima D4-2 T (96.2%), whereas OTU 96 was closely related to unclassified Shewanella sp.MR-7 (99.5%).
Shewanella species are ubiquitously distributed in nature and have been usually isolated from surface freshwater and the deepest marine trenches (Satomi 2014;Lemaire et al. 2020).As they are natural inhabitants of marine waters, the presence of Shewanella species within the seahorse intestinal ecosystem may be influenced by their surrounding environment, in which they obtain their food.The wide distribution of Shewanella species in the aquatic environment is due to their unparalleled capacity to respire compounds found in nature and their capacity to thrive at low temperatures (Hau and Garlnick 2007).Moreover, bioactive compounds have been found in Shewanella spp., which may help increase the cytotoxic activity of the host immune response.Some strains have also been proposed as candidates for antibiotic synthesis (Leonardo et al. 1999;Kizhakkekalam et al. 2020).As a consequence, the availability of cutting-edge technologies and tools, sequenced genomes, rapid growth, and robust metabolism make them ideal candidates for biotechnological applications (Amiri-Jami et al. 2006;Lemaire et al. 2020;Chakraborty et al. 2021).
In conclusion, the present study shows that high-throughput sequencing of 16 S rRNA genes provides a reliable method for identifying the composition and structure of bacterial communities within the seahorse intestinal ecosystem.It should be noted that normal intestinal microbiota may improve biological processes that benefit the host, including nutrition and immune system modulation (Maynard et al. 2012;Xing et al. 2013).Although these Shewanella species were found in apparently healthy seahorses, further studies are needed to isolate and characterize these species.Moreover, the information at hand may provide baseline data for further research in order to determine the potential implications to health status, development, growth, and survival of other seahorse species.

Fig. 1
Fig. 1 (a) Relative abundance of dominant bacterial families found in the intestinal samples from female and male Barbour's seahorses.(b) Heatmap of OTUs assigned to the Shewanellaceae family, which were compared with their abundances in each sample.The color intensity (log2 scale) in each panel shows the relative abundance of each OTU.