Deciphering taxonomic and functional patterns of microbial communities associated with the tiger tail seahorse (Hippocampus comes)

Deciphering taxonomic and functional patterns of microbial communities associated with the tiger tail seahorse (Hippocampus comes) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Deciphering taxonomic and functional patterns of microbial communities associated with the tiger tail seahorse (Hippocampus comes) Chinee Surita Padasas-Adalla, Rose Chinly Mae Ortega, Rodelyn Dalayap, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3862946/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study aimed to explore the microbial diversity and metabolic functions of the skin and gut of the tiger tail seahorse (Hippocampus comes) and their surrounding environment using shotgun metagenomics and bioinformatics. Members belonging to the Pseudomonadota phylum were dominant on the skin, whereas Bacteroidota was dominant in the gut. Bacillota, Actinomycetota, and Planctomycetota were also detected in the seahorse-associated microbiome. Statistical analysis revealed significant differences ( p < 0.01) in species diversity between skin and gut microbiomes, with members belonging to the Moraxellaceae family being dominant on the skin and the Bacteroidaceae family in the gut. Moreover, the surrounding environment (water or sediment) did not have a direct effect on the seahorse microbiome composition. Functional annotations highlighted the involvement of the skin microbiome in energy, lipid, and amino acid metabolism, as well as terpenoids and polyketides metabolism, xenobiotics biodegradation and metabolism, and cellular processes. Additionally, annotations indicated the presence of quorum sensing and intercellular communication. The relative abundance of bacteriocins was similar in both gut and skin, which is significant in shaping microbial communities due to their antimicrobial properties. Overall, the study highlights the importance of seahorse-microbe relationship for their well-being and holds implications for conservation and sustainable aquaculture. Seahorses Microbiome Functional metabolism Figures Figure 1 Figure 2 Figure 3 Full Text Previous studies have demonstrated that aquatic species have a complex and diverse microbiome, which is crucial for their development and health [1, 2]. Among these species, seahorses stand out as the most emblematic species and are considered ambassadors of marine biodiversity. However, limited information is available regarding the microbial diversity and functional patterns in seahorses, particularly for the vulnerable species known as the tiger tail seahorse ( Hippocampus comes ). This could be of great practical significance for aquaculture initiatives, disease resistance, and overall health of this species [2]. Moreover, exploring their microbiome composition and bioactive compounds holds the potential for enhancing conservation efforts and facilitating biomedical applications. Given these factors, we employed a combination of shotgun metagenomics and bioinformatics to provide a comprehensive view of the microbial communities associated with the tiger tail seahorse, including their metabolic functions. In order to reach our goal, we collected fifteen healthy seahorse specimens ( n = 15), as well as water and sediment samples from Hanigad Island, Surigao del Norte, Philippines. Genomic DNA was extracted from the gut and skin of tiger tail seahorses, as well as from the surrounding water and sediment following previously described methods [3]. Equal amounts of DNA were pooled into three groups, each containing DNA from five specimens. Subsequently, these samples were sequenced on NovaSeq 6000 platform (Illumina Inc., San Diego, CA) using 150-bp paired-end reads. All quality-checked reads were aligned against the non-redundant (nr) protein reference database using the BLASTX command implemented in DIAMOND v2.1.7 [4]. To obtain detailed taxonomic classification, output files were filtered through a lowest common ancestor (LCA) algorithm in MEGAN 6 [5]. The results of this analysis revealed that Pseudomonadota (formerly Proteobacteria) were dominant across all samples except in the gut samples. This supports previous findings in herbivorous fish, where the gut microbiomes are typically characterized by a predominance of Bacteroidetes and Firmicutes, specifically within the order Clostridia [6, 7]. Overall, Pseudomonadota accounted for the largest proportion of bacterial communities in all four environments, followed by Bacillota and Bacteroidota. Moraxellaceae emerged as the predominant family in the skin samples ( Fig. 1 ), particularly represented by members of the Acinetobacter and Psychrobacter genera, a noteworthy finding as it represents the first evidence of this family in healthy seahorses. The gut samples were dominated by Bacteroidaceae , predominantly represented by members of the Bacteroides and Phocaeicola genera, which are commonly found in anaerobic environments. Previous studies have demonstrated that Bacteroidaceae are involved in several bacterial functional gene categories within the active gut microbiome [8]. Although these data should be interpreted with caution due to the low number of water or sediment samples analyzed, the surrounding environment (water or sediment) did not appear to have a direct effect on the seahorse microbiome composition. Clinically relevant families, including Enterobacteriaceae and Enterococcaceae , were identified in skin, gut, and water samples, raising concerns about potential health risks and fecal contamination. Enterobacteriaceae are associated with several infections and are a major cause of foodborne enteritis and zoonotic infections, including outbreaks of human diseases [9]. Some members of the Enterobacteriaceae family have also been identified as pathogens in both wild and farmed fish species [10]. Moreover, the high number of members belonging to the Enterococcaceae family suggests the likelihood of fecal contamination [11]. Furthermore, skin samples revealed the presence of members from the Aeromonadaceae and Shewanellaceae families, emphasizing significant implications and the need for monitoring and assessment. The Shannon diversity index implemented in MEGAN 6 showed a significantly higher diversity ( p <0.01) in the skin samples (3.51±0.31) compared to the gut samples (1.60±0.27), probably influenced by the unique characteristics of the seahorse skin and its exposure to diverse environmental microorganisms. These findings highlight the complex interactions within the tiger tail seahorse microbiome and emphasize the role of environmental factors in shaping microbial populations. Further studies are therefore required to understand the underlying mechanisms and their ecological implications for seahorses and their ecosystems. To elucidate such interactions, we conducted a comprehensive and detailed functional annotation based on the KEGG pathway database [12]. The skin exhibited a higher abundance ( p <0.05) of functional genes related to energy metabolism, lipid metabolism, amino acid metabolism, terpenoids and polyketides metabolism, xenobiotics biodegradation and metabolism compared to the gut ( Fig. 2 ). This suggests that the skin and gut microbiome may be involved in the breakdown, utilization, and detoxification of various compounds, including lipids, amino acids, and xenobiotics [13]. Moreover, the skin microbiome may have the ability to degrade and metabolize foreign substances, such as pollutants or toxins, through xenobiotic metabolism [14]. Functional genes related to xenobiotics biodegradation and metabolism suggest the potential role of the skin and gut microbiome in detoxification processes, resilience to environmental stressors, and adaptation to xenobiotic exposure [15, 16]. The genetic information processing category also showed a higher abundance ( p <0.05) of functional genes related to translation, replication, and repair in the skin compared to the gut. Within the environmental information processing category, the skin displayed a greater abundance of genes related to membrane transport and signal transduction, suggesting its role in interactions with the external environment. Additionally, quorum sensing ability was found in the skin and gut microbiome, whose signaling molecules are secreted to coordinate community behaviors and protect against unfavorable environmental conditions [17]. These findings provide insights into the metabolic and regulatory capacities of the skin and gut microbiome of the tiger tail seahorse, highlighting their ecological and health significance. We also identified bacteriocins using the BAGEL4 database as previously described [18], whose results demonstrated that the relative abundance of bacteriocins was higher in the skin compared to the gut ( Fig. 3 ), but the difference was not significant ( p >0.05). Class III bacteriocins were relatively more abundant compared to class I and class II in both samples. Specifically, Class I bacteriocins, known as lantibiotics, were more prevalent in the skin microbiome, and they have shown promising antimicrobial properties and potential health benefits as probiotics [19]. Both Class II and Class III bacteriocins have also been of significant interest as probiotics, as they can enhance gastrointestinal health and combat pathogens in the gut environment. Our analysis identified a total of 11 bacteriocins, with 5 of them being common to both skin and gut samples. Notably, enterocins were prevalent in both samples and have a wide range of antimicrobial activity against Gram-positive pathogens [20], making them valuable candidates for further investigation. The relatively similar presence and abundance of bacteriocin classes in both skin and gut samples suggest a potential capacity for antimicrobial activity in both microbiomes, offering insights into the dynamics of microbial communities and their potential influence on the tiger tail seahorse’s health. In conclusion, this study reveals the diverse and complex microbiome of the tiger tail seahorse, highlighting its ecological and health importance. The skin and gut microbiomes differ significantly in composition and functional properties, with potential implications for seahorse health and adaptation. The presence of bacteriocins in both microbiomes suggests antimicrobial activity, providing insights into microbial community dynamics. However, further studies are needed to understand the underlying mechanisms and ecological implications, benefiting seahorse conservation and biomedical applications. Overall, this study contributes valuable knowledge to seahorse microbiome research, aiding aquaculture management and disease resistance efforts. Declarations Author contributions CSPA designed the study and performed all experiments with support from JLB. CSPA, COLO, and JLB analyzed the data. The manuscript was written by CSPA and JLB with contributions from SRT, RCMHO, OAA, CSM, JGM, COLO, and RD. Acknowledgments CSPA acknowledges the funding support from the Department of Science and Technology under the Accelerated Science and Technology Human Resource Development Program. The sample processing, including DNA extraction, was conducted at the Mindanao State University – Iligan Institute of Technology and its Premier Research Institute of Science and Mathematics. The bioinformatic analysis was carried out at the Catalan Institute for Water Research (ICRA-CERCA). Moreover, CSPA expresses gratitude to the local government unit of Surigao City and the locals of Hanigad Island, where the sampling took place. The authors also acknowledge funding from Generalitat de Catalunya through Consolidated Research Group 2021 SGR 01282 and from the CERCA program. Data availability The Metagenome sequence data that support the findings of this study are openly available in NCBI’s GenBank under BioProject accession no. PRJNA986485. Funding This work was supported by the Department of Science and Technology – Science Education Institute (DOST-SEI) under the Accelerated Science and Technology Human Resource Development Program (ASTHRDP). The contents of this study are the authors’ sole responsibility and do not necessarily reflect the views of DOST. The funding body were only involved in giving financial support and not involved in either the design of the study or collection, analysis, and interpretation of data, nor were they involved in writing the manuscript. Conflict of Interest Statement The authors declare no competing interests. Ethics Approval The care and use of experimental animals were in accordance with the terms and conditions from the Gratuitous Permit issued by the Bureau of Fisheries and Aquatic Resources (BFAR) under GP No. 0184-19. All experimental protocols were approved including accepted protocols and all methodologies employed in this study were meticulously documented in alignment with accepted practices. References Egerton S, Culloty S, Whooley J, Stanton C, Ross RP. The gut microbiota of marine fish. Front Microbiol. 2018;9:873. Sehnal L, Brammer-Robbins E, Wormington AM, Blaha L, Bisesi J, Larkin I, Martyniuk CJ, Simonin M, Adamovsky O. Microbiome composition and function in aquatic vertebrates: small organisms making big impacts on aquatic animal health. Front Microbiol. 2021;12:567408. Ortega RCMH, Tabugo SRM, Martinez JGT, Padasas CS, Balolong MP, Balcázar JL. High-throughput sequencing-based analysis of bacterial communities associated with Barbour's seahorses ( Hippocampus barbouri ) from Surigao del Norte, Philippines. Lett Appl Microbiol. 2021;73:280–5. Buchfink B, Xie C, Huson DH. Fast and sensitive protein alignment using DIAMOND. Nat Methods. 2015;12:59–60. Huson DH, Beier S, Flade I, Górska A, El-Hadidi M, Mitra S, Ruscheweyh HJ, Tappu R. MEGAN Community Edition - Interactive Exploration and Analysis of Large-Scale Microbiome Sequencing Data. PLoS Comput Biol. 2016;12:e1004957. Sullam KE, Essinger SD, Lozupone CA, O'Connor MP, Rosen GL, Knight R, Kilham SS, Russell JA. Environmental and ecological factors that shape the gut bacterial communities of fish: a meta-analysis. Mol Ecol. 2012;21:3363–78. Miyake S, Ngugi DK, Stingl U. Diet strongly influences the gut microbiota of surgeonfishes. Mol Ecol. 2015;24:656–72. Yap GC, Loo EX, Aw M, Lu Q, Shek LP, Lee BW. Molecular analysis of infant fecal microbiota in an Asian at-risk cohort-correlates with infant and childhood eczema. BMC Res Notes. 2014;7:166. Janda JM, Abbott SL. The changing face of the family Enterobacteriaceae (Order: Enterobacterales): New members, taxonomic issues, geographic expansion, and new diseases and disease syndromes. Clin Microbiol Rev. 2021;34:e00174–20. Sekar VT, Santiago TC, Vijayan KK, Alavandi SV, Raj VS, Rajan JJ, Sanjuktha M, Kalaimani N. Involvement of Enterobacter cloacae in the mortality of the fish, Mugil cephalus . Lett Appl Microbiol. 2008;46:667–72. Sadowsky MJ, Whitman RL, editors. The Fecal Bacteria. Washington, DC: ASM Press; 2011. Kanehisa M, Furumichi M, Sato Y, Kawashima M, Ishiguro-Watanabe M. KEGG for taxonomy-based analysis of pathways and genomes. Nucleic Acids Res. 2023;51:D587–92. Zhang J, Guo Z, Xue Z, Sun Z, Zhang M, Wang L, Wang G, Wang F, Xu J, Cao H, Xu H, Lv Q, Zhong Z, Chen Y, Qimuge S, Menghe B, Zheng Y, Zhao L, Chen W, Zhang H. A phylo-functional core of gut microbiota in healthy young Chinese cohorts across lifestyles, geography and ethnicities. ISME J. 2015;9:1979–90. van der Meer JR, de Vos WM, Harayama S, Zehnder AJ. Molecular mechanisms of genetic adaptation to xenobiotic compounds. Microbiol Rev. 1992;56:677–94. Demain AL, Fang A. The natural functions of secondary metabolites. Adv Biochem Eng Biotechnol. 2000;69:1–39. Kontomina E, Garefalaki V, Fylaktakidou KC, Evmorfidou D, Eleftheraki A, Avramidou M, Udoh K, Panopoulou M, Felföldi T, Márialigeti K, Fakis G, Boukouvala S. A taxonomically representative strain collection to explore xenobiotic and secondary metabolism in bacteria. PLoS ONE. 2022;17:e0271125. Waters CM, Bassler BL. Quorum sensing: cell-to-cell communication in bacteria. Annu Rev Cell Dev Biol. 2005;21:319–46. van Heel AJ, de Jong A, Song C, Viel JH, Kok J, Kuipers OP. BAGEL4: a user-friendly web server to thoroughly mine RiPPs and bacteriocins. Nucleic Acids Res. 2018;46:W278–81. McAuliffe O, Ross RP, Hill C. Lantibiotics: structure, biosynthesis and mode of action. FEMS Microbiol Rev. 2001;25:285–308. Wu Y, Pang X, Wu Y, Liu X, Zhang X. Enterocins: classification, synthesis, antibacterial mechanisms and food applications. Molecules. 2022;27:2258. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3862946","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":267415520,"identity":"3b450a83-d258-4b5e-841b-0f2a1d248539","order_by":0,"name":"Chinee Surita Padasas-Adalla","email":"data:image/png;base64,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","orcid":"","institution":"Mindanao State University, Iligan Institute of Technology","correspondingAuthor":true,"prefix":"","firstName":"Chinee","middleName":"Surita","lastName":"Padasas-Adalla","suffix":""},{"id":267415521,"identity":"e3a708ab-0399-4dc4-8ad6-9129fcc9abaf","order_by":1,"name":"Rose Chinly Mae Ortega","email":"","orcid":"","institution":"Mindanao State University, Iligan Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Rose","middleName":"Chinly Mae","lastName":"Ortega","suffix":""},{"id":267415522,"identity":"a05b1222-35c7-45b1-8161-950f4a3d32b7","order_by":2,"name":"Rodelyn Dalayap","email":"","orcid":"","institution":"Sultan Kudarat State University","correspondingAuthor":false,"prefix":"","firstName":"Rodelyn","middleName":"","lastName":"Dalayap","suffix":""},{"id":267415523,"identity":"6159d973-41e2-440c-b7ac-ae425efd88eb","order_by":3,"name":"Joey Genevieve Martinez","email":"","orcid":"","institution":"Mindanao State University, Iligan Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Joey","middleName":"Genevieve","lastName":"Martinez","suffix":""},{"id":267415524,"identity":"7d82fd25-a13c-47c9-a5c5-3724e518f04e","order_by":4,"name":"Olive Amparado","email":"","orcid":"","institution":"Mindanao State University, Iligan Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Olive","middleName":"","lastName":"Amparado","suffix":""},{"id":267415525,"identity":"4207e2b2-4321-4cdd-b9f2-41d2a48d31f5","order_by":5,"name":"Carlo Stephen Moneva","email":"","orcid":"","institution":"Mindanao State University, Iligan Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Carlo","middleName":"Stephen","lastName":"Moneva","suffix":""},{"id":267415526,"identity":"200ddcb4-6ba7-439e-b540-d314f26193c5","order_by":6,"name":"Carlos O. Lomelí-Ortega","email":"","orcid":"","institution":"Catalan Institute for Water Research (ICRA-CERCA)","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"O.","lastName":"Lomelí-Ortega","suffix":""},{"id":267415527,"identity":"fcdffc25-ba26-44f3-a1b1-7feb07af1d21","order_by":7,"name":"Sharon Rose Tabugo","email":"","orcid":"","institution":"Mindanao State University, Iligan Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Sharon","middleName":"Rose","lastName":"Tabugo","suffix":""},{"id":267415528,"identity":"877c720a-3a66-4197-9024-eee75bea011b","order_by":8,"name":"José Luis Balcázar","email":"","orcid":"","institution":"Catalan Institute for Water Research (ICRA-CERCA)","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"Luis","lastName":"Balcázar","suffix":""}],"badges":[],"createdAt":"2024-01-14 10:14:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3862946/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3862946/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49794798,"identity":"fadeeed1-92a7-413f-9951-3079dfb04111","added_by":"auto","created_at":"2024-01-18 06:47:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":183126,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap describing the relative abundance (log scale 2) of dominant families, normalized to the total number of reads annotated in all metagenomes.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3862946/v1/be03196a4201d02064c99886.png"},{"id":49794083,"identity":"736e67de-d4a5-4fe6-8b40-552d94e726c6","added_by":"auto","created_at":"2024-01-18 06:39:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":360069,"visible":true,"origin":"","legend":"\u003cp\u003eBubble map shows the relative abundance of KEGG pathway of skin and gut microbiomes. Abundance is normalized among different samples based on Z-score. Asterisk (*) denotes a statistically significant difference between skin and gut samples at p\u0026lt;0.05. All data were tested for normality (Shapiro-Wilk’s test) and homoscedasticity (Levene’s test) and then assessed for differences across groups (skin and gut) using Student’s t-test. The resulting p-values were adjusted for multiple comparisons using the False Discovery Rate correction.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3862946/v1/a995349ff41acd7847247a7d.png"},{"id":49794081,"identity":"19e17a61-f22a-4136-8406-d60ee5162c95","added_by":"auto","created_at":"2024-01-18 06:39:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":34451,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance of bacteriocins in skin and gut microbiomes, whose abundances were normalized to the total number of reads annotated as 16S rRNA genes using METAXA2 (https://microbiology.se/software/metaxa2/).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3862946/v1/d89f359dee0aaed6e776d170.png"},{"id":51902307,"identity":"59de7b7e-0ae8-4cad-b682-fdd55ae53c3e","added_by":"auto","created_at":"2024-03-02 16:12:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":667072,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3862946/v1/f919c75c-9cd5-48d1-9026-bf6a75b8566d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Deciphering taxonomic and functional patterns of microbial communities associated with the tiger tail seahorse (Hippocampus comes)","fulltext":[{"header":"Full Text","content":"\u003cp\u003ePrevious studies have demonstrated that aquatic species have a complex and diverse microbiome, which is crucial for their development and health [1, 2]. Among these species, seahorses stand out as the most emblematic species and are considered ambassadors of marine biodiversity. However, limited information is available regarding the microbial diversity and functional patterns in seahorses, particularly for the vulnerable species known as the tiger tail seahorse (\u003cem\u003eHippocampus comes\u003c/em\u003e). This could be of great practical significance for aquaculture initiatives, disease resistance, and overall health of this species [2]. Moreover, exploring their microbiome composition and bioactive compounds holds the potential for enhancing conservation efforts and facilitating biomedical applications. Given these factors, we employed a combination of shotgun metagenomics and bioinformatics to provide a comprehensive view of the microbial communities associated with the tiger tail seahorse, including their metabolic functions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn order to reach our goal, we collected fifteen healthy seahorse specimens\u003cem\u003e\u0026nbsp;\u003c/em\u003e(\u003cem\u003en\u003c/em\u003e= 15), as well as water and sediment samples from Hanigad Island, Surigao del Norte, Philippines. Genomic DNA was extracted from the gut and skin of tiger tail seahorses, as well as from the surrounding water and sediment following previously described methods [3]. Equal amounts of DNA were pooled into three groups, each containing DNA from five specimens. Subsequently, these samples were sequenced on NovaSeq 6000 platform (Illumina Inc., San Diego, CA) using 150-bp paired-end reads. All quality-checked reads were aligned against the non-redundant (nr) protein reference database using the BLASTX command implemented in DIAMOND v2.1.7 [4]. To obtain detailed taxonomic classification, output files were filtered through a lowest common ancestor (LCA) algorithm in MEGAN 6 [5]. The results of this analysis revealed that Pseudomonadota (formerly Proteobacteria) were dominant across all samples except in the gut samples. This supports previous findings in herbivorous fish, where the gut microbiomes are typically characterized by a predominance of Bacteroidetes and Firmicutes, specifically within the order Clostridia [6, 7]. Overall, Pseudomonadota accounted for the largest proportion of bacterial communities in all four environments, followed by Bacillota and Bacteroidota. \u003cem\u003eMoraxellaceae\u003c/em\u003e emerged as the predominant family in the skin samples (\u003cstrong\u003eFig. 1\u003c/strong\u003e), particularly represented by members of the \u003cem\u003eAcinetobacter\u003c/em\u003e and \u003cem\u003ePsychrobacter\u003c/em\u003e genera, a noteworthy finding as it represents the first evidence of this family in healthy seahorses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe gut samples were dominated by \u003cem\u003eBacteroidaceae\u003c/em\u003e, predominantly represented by members of the \u003cem\u003eBacteroides\u003c/em\u003e and \u003cem\u003ePhocaeicola\u003c/em\u003e genera, which are commonly found in anaerobic environments. Previous studies have demonstrated that \u003cem\u003eBacteroidaceae\u003c/em\u003e are involved in several bacterial functional gene categories within the active gut microbiome [8]. Although these data should be interpreted with caution due to the low number of water or sediment samples analyzed, the surrounding environment (water or sediment) did not appear to have a direct effect on the seahorse microbiome composition. Clinically relevant families, including \u003cem\u003eEnterobacteriaceae\u003c/em\u003e and \u003cem\u003eEnterococcaceae\u003c/em\u003e, were identified in skin, gut, and water samples, raising concerns about potential health risks and fecal contamination. \u003cem\u003eEnterobacteriaceae\u003c/em\u003e are associated with several infections and are a major cause of foodborne enteritis and zoonotic infections, including outbreaks of human diseases [9]. Some members of the \u003cem\u003eEnterobacteriaceae\u003c/em\u003e family have also been identified as pathogens in both wild and farmed fish species [10]. Moreover, the high number of members belonging to the \u003cem\u003eEnterococcaceae\u003c/em\u003e family suggests the likelihood of fecal contamination [11]. Furthermore, skin samples revealed the presence of members from the \u003cem\u003eAeromonadaceae\u003c/em\u003e and \u003cem\u003eShewanellaceae\u0026nbsp;\u003c/em\u003efamilies, emphasizing significant implications and the need for monitoring and assessment. The Shannon diversity index implemented in MEGAN 6 showed a significantly higher diversity (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01) in the skin samples (3.51\u0026plusmn;0.31) compared to the gut samples (1.60\u0026plusmn;0.27), probably influenced by the unique characteristics of the seahorse skin and its exposure to diverse environmental microorganisms. These findings highlight the complex interactions within the tiger tail seahorse microbiome and emphasize the role of environmental factors in shaping microbial populations. Further studies are therefore required to understand the underlying mechanisms and their ecological implications for seahorses and their ecosystems.\u003c/p\u003e\n\u003cp\u003eTo elucidate such interactions, we conducted a comprehensive and detailed functional annotation based on the KEGG pathway database [12]. The skin exhibited a higher abundance (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) of functional genes related to energy metabolism, lipid metabolism, amino acid metabolism, terpenoids and polyketides metabolism, xenobiotics biodegradation and metabolism compared to the gut (\u003cstrong\u003eFig. 2\u003c/strong\u003e). This suggests that the skin and gut microbiome may be involved in the breakdown, utilization, and detoxification of various compounds, including lipids, amino acids, and xenobiotics [13].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMoreover, the skin microbiome may have the ability to degrade and metabolize foreign substances, such as pollutants or toxins, through xenobiotic metabolism [14]. Functional genes related to xenobiotics biodegradation and metabolism suggest the potential role of the skin and gut microbiome in detoxification processes, resilience to environmental stressors, and adaptation to xenobiotic exposure [15, 16]. The genetic information processing category also showed a higher abundance (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) of functional genes related to translation, replication, and repair in the skin compared to the gut. Within the environmental information processing category, the skin displayed a greater abundance of genes related to membrane transport and signal transduction, suggesting its role in interactions with the external environment. Additionally, quorum sensing ability was found in the skin and gut microbiome, whose signaling molecules are secreted to coordinate community behaviors and protect against unfavorable environmental conditions [17]. These findings provide insights into the metabolic and regulatory capacities of the skin and gut microbiome of the tiger tail seahorse, highlighting their ecological and health significance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe also identified bacteriocins using the BAGEL4 database as previously described [18], whose results demonstrated that the relative abundance of bacteriocins was higher in the skin compared to the gut (\u003cstrong\u003eFig. 3\u003c/strong\u003e), but the difference was not significant (\u003cem\u003ep\u003c/em\u003e\u0026gt;0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eClass III bacteriocins were relatively more abundant compared to class I and class II in both samples. Specifically, Class I bacteriocins, known as lantibiotics, were more prevalent in the skin microbiome, and they have shown promising antimicrobial properties and potential health benefits as probiotics [19]. Both Class II and Class III bacteriocins have also been of significant interest as probiotics, as they can enhance gastrointestinal health and combat pathogens in the gut environment. Our analysis identified a total of 11 bacteriocins, with 5 of them being common to both skin and gut samples. Notably, enterocins were prevalent in both samples and have a wide range of antimicrobial activity against Gram-positive pathogens [20], making them valuable candidates for further investigation. The relatively similar presence and abundance of bacteriocin classes in both skin and gut samples suggest a potential capacity for antimicrobial activity in both microbiomes, offering insights into the dynamics of microbial communities and their potential influence on the tiger tail seahorse\u0026rsquo;s health.\u003c/p\u003e\n\u003cp\u003eIn conclusion, this study reveals the diverse and complex microbiome of the tiger tail seahorse, highlighting its ecological and health importance. The skin and gut microbiomes differ significantly in composition and functional properties, with potential implications for seahorse health and adaptation. The presence of bacteriocins in both microbiomes suggests antimicrobial activity, providing insights into microbial community dynamics. However, further studies are needed to understand the underlying mechanisms and ecological implications, benefiting seahorse conservation and biomedical applications. Overall, this study contributes valuable knowledge to seahorse microbiome research, aiding aquaculture management and disease resistance efforts.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCSPA designed the study and performed all experiments with support from JLB. CSPA, COLO, and JLB analyzed the data. The manuscript was written by CSPA and JLB with contributions from SRT, RCMHO, OAA, CSM, JGM, COLO, and RD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCSPA acknowledges the funding support from the Department of Science and Technology under the Accelerated Science and Technology Human Resource Development Program. The sample processing, including DNA extraction, was conducted at the Mindanao State University – Iligan Institute of Technology and its Premier Research Institute of Science and Mathematics. The bioinformatic analysis was carried out at the Catalan Institute for Water Research (ICRA-CERCA). Moreover, CSPA expresses gratitude to the local government unit of Surigao City and the locals of Hanigad Island, where the sampling took place. The authors also acknowledge funding from Generalitat de Catalunya through Consolidated Research Group 2021 SGR 01282 and from the CERCA program.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Metagenome sequence data that support the findings of this study are openly available in NCBI’s GenBank under BioProject accession no. PRJNA986485.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Department of Science and Technology – Science Education Institute (DOST-SEI) under the\u0026nbsp;Accelerated Science and Technology Human Resource Development Program (ASTHRDP). The contents of this study are the authors’ sole responsibility and do not necessarily reflect the views of DOST.\u0026nbsp;The funding body were only involved in giving financial support and not involved in either the design of the study or collection, analysis, and interpretation of data, nor were they involved in writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe care and use of experimental animals were in accordance with the terms and conditions from the Gratuitous Permit issued by the Bureau of Fisheries and Aquatic Resources (BFAR) under GP No. 0184-19. All experimental protocols were approved including accepted protocols and all methodologies employed in this study were meticulously documented in alignment with accepted practices.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEgerton S, Culloty S, Whooley J, Stanton C, Ross RP. The gut microbiota of marine fish. Front Microbiol. 2018;9:873.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSehnal L, Brammer-Robbins E, Wormington AM, Blaha L, Bisesi J, Larkin I, Martyniuk CJ, Simonin M, Adamovsky O. Microbiome composition and function in aquatic vertebrates: small organisms making big impacts on aquatic animal health. Front Microbiol. 2021;12:567408.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrtega RCMH, Tabugo SRM, Martinez JGT, Padasas CS, Balolong MP, Balc\u0026aacute;zar JL. 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Molecules. 2022;27:2258.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Seahorses, Microbiome, Functional metabolism","lastPublishedDoi":"10.21203/rs.3.rs-3862946/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3862946/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study aimed to explore the microbial diversity and metabolic functions of the skin and gut of the tiger tail seahorse \u003cem\u003e(Hippocampus comes)\u003c/em\u003e and their surrounding environment using shotgun metagenomics and bioinformatics. Members belonging to the Pseudomonadota phylum were dominant on the skin, whereas Bacteroidota was dominant in the gut. Bacillota, Actinomycetota, and Planctomycetota were also detected in the seahorse-associated microbiome. Statistical analysis revealed significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in species diversity between skin and gut microbiomes, with members belonging to the \u003cem\u003eMoraxellaceae\u003c/em\u003e family being dominant on the skin and the \u003cem\u003eBacteroidaceae\u003c/em\u003e family in the gut. Moreover, the surrounding environment (water or sediment) did not have a direct effect on the seahorse microbiome composition. Functional annotations highlighted the involvement of the skin microbiome in energy, lipid, and amino acid metabolism, as well as terpenoids and polyketides metabolism, xenobiotics biodegradation and metabolism, and cellular processes. Additionally, annotations indicated the presence of quorum sensing and intercellular communication. The relative abundance of bacteriocins was similar in both gut and skin, which is significant in shaping microbial communities due to their antimicrobial properties. Overall, the study highlights the importance of seahorse-microbe relationship for their well-being and holds implications for conservation and sustainable aquaculture.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Deciphering taxonomic and functional patterns of microbial communities associated with the tiger tail seahorse (Hippocampus comes)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-18 06:39:30","doi":"10.21203/rs.3.rs-3862946/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1138d217-d66b-449c-a97a-fe6fee4f87fa","owner":[],"postedDate":"January 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-02T16:04:42+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-18 06:39:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3862946","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3862946","identity":"rs-3862946","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}