HIGH-THROUGHPUT SEQUENCING ANALYSIS OF BACTERIAL COMMUNITIES ASSOCIATED WITH TIGER TAIL SEAHORSE, Hippocampus comes FROM HANIGAD ISLAND, SURIGAO DEL NORTE, MINDANAO, PHILIPPINES

[Audio] A pleasant morning to everyone! First, I'd like to thank you for your time and effort to listen to my recorded presentation. I am Chinee S. Padasas, a Ph.D. student from Mindanao State University – Iligan Institute of Technology. And I will be presenting to you a part of my master's thesis entitled high-throughput sequencing analysis of bacterial communities associated with tiger tail seahorse, Hippocampus comes FROM HANIGAD ISLAND, Surigao del Norte, Mindanao, Philippines..

[Audio] I'd like to start my presentation with this strong message from Dr. Amanda Vincent, the director, and co-founder of Project seahorse. She said "Get it right with seahorses and we will help save coastal seas"..

[Audio] So now, let's get to know our seahorses! Seahorses are bony fishes comprising one genus ( Hippocampus) under the family Syngnathidae. They are found in ecosystems like seagrass beds, corals, sponges, and even mangrove areas. Currently, ten species of seahorses are recorded here in the Philippines. The present study focused on the tiger tail seahorse, H. comes found in Surigao del Norte. H. comes is commonly distinguished by a blotchy or striped tail. The figures on your screen now are the male and female seahorses. As you can see, they are strikingly similar in appearance and the only visible, morphological difference is that the male here in figure 1 has a brood stomach pouch, adapted for reproduction purposes. So many would ask, why seahorses? or why study seahorses? Aside from these species being charismatic organisms and possessing unique biology, seahorses are powerful ambassadors of our ocean health. They are considered a popular flagship species for engendering support for marine conservation and sustainable management. This only means that protecting seahorses is saving our coastal seas. In addition to their biological and ecological importance, seahorses display a high economic value globally. Their value as medicinal organisms in traditional Chinese medicine ( TCM) and ornamental display means they provide notable income for subsistence fishers in many parts of the world..

[Audio] Seahorses gained popularity because of their use in traditional Chinese medicine and because of that the seahorse trade now is rapidly growing worldwide; the problem is not with the demand but the supply of seahorses! The pictures on your screen are basically a species of seahorses being sold in the market in some countries. Treatments including seahorses are believed to benefit a range of conditions, including respiratory disorders such as asthma, sexual dysfunctions such as impotence or infertility, and general lethargy and pain. Aside from that, seahorses are also collected and targeted from the wild for aquarium pets and some curios. And because of the increasing market demand, the commercial market value of seahorses has also increased and this provides high notable income to the fisher folks. However, this high demand and popularity have been catastrophic in the population of wild seahorses..

[Audio] In fact, according to the project seahorse organization, it was estimated that the total number of seahorses caught per year is about 70M..

[Audio] And 80 countries are involved in the international trade in seahorses.

[Audio] Where the Philippines is ranked as the second highest exporter of trading seahorses in the international black market. and 95% of this dried seahorse trade is illegal, unmanaged, and unreported..

[Audio] Despite tightening the restrictions of the seahorse trade and strong regulation and implementation of the Convention on International Trade in Endangered Species, the seahorse trade continued in a detrimental manner which resulted in threats and challenges in their wild population. First, it resulted in uncontrolled and overexploitation worldwide..

[Audio] In fact, all 42 seahorse species are now currently included on the IUCN Red List ( IUCN 2021) and the last global assessment of seahorses, conducted in 2016 by Project Seahorse, established that at least one-third of the 42 species that were recognized were now threatened..

[Audio] 2 species are Endangered 12 species are Vulnerable, including my study organism, the Hippocampus comes, and species primarily in East and Southeast Asia, while one is Near Threatened. 17 seahorse species are Data Deficient which basically means that these species are poorly understood and that we couldn't assess their status 10 seahorse species are of Least Concern because (i) reductions in population size are not severe enough to meet thresholds under criterion A despite the presence of known threats, or (ii) no threats are known to affect the species..

[Audio] Second, there is a drastic reduction in the population of Hippocampus comes. The high demand for seahorses far exceeds sustainable levels. In the Philippines, the population of H. comes has been reported to have decreased by up to 70% over the decade based on the available data provided last 1985- 1995 by the FAO Fisheries and Aquaculture Department. Third, there is data deficiency in Hippocampus comes. This suggests that we do not know enough about those species to determine their conservation status and yet it is one of the most heavily exploited species of seahorses worldwide. Fourth, there is only limited success in seahorse aquaculture on a mass scale. Many countries have cultivated different species of seahorses. However, several attempts to culture seahorses on a mass scale in the recent past have met with limited success as a result of severe problems of mass mortality due to inadequate nutrition and infections or diseases. In fact, there is no seahorse aquaculture here in Mindanao despite the fact that Mindanao is heavily involved in exploiting seahorses worldwide..

[Audio] In response to the challenge and as a continuous effort of culturing seahorses in a sustainable manner and addressing the crisis in the black market for TCM, this study was carried out and specifically focused on the following objectives. First is to.

[Audio] Now for the significance of the study, First, with the advancement of high-throughput sequencing, the results of the study will provide us with a much more detailed analysis of the metagenomic data, including the reconstruction of novel microbial genomes and gaining knowledge about the biomedical capacities of the isolated bacteria from the skin mucus and gut content of H. comes and from its surrounding environment. Second, results from this study will reveal the expansive biodiversity of microorganisms, clarify the relationship between host-associated microbial communities, and provide practical significance in their role in the seahorse's overall health and nutrition, which is essential in providing better management programs for culturing seahorses. And lastly, results from this metagenomic research can therefore infer medically or industrially important knowledge by revealing the potential biomedical activities of isolated bacteria using data mining..

[Audio] First is interview and reconnaissance, so I did an initial interview in areas where seahorse sightings were reported. The local fishermen were asked about the features of seahorse species spotted to confirm the presence of Hippocampus comes on their areas. Second, is permit acquisition. A permit to conduct the study was acquired from the local government unit of Surigao City and from the Bureau of Fisheries and Aquatic Resources ( BFAR) office. Third is the collection of samples. So I did an opportunistic sampling and micro-habitats associated with species of seahorses were noted based on underwater surveys. Seahorse samples were temporarily placed in a large aquarium with its carriage water for transportation from the sampling area to the laboratory..

[Audio] Next is DNA Extraction. Now for the DNA extraction, bacterial samples were aseptically collected from the dermal layer of H. comes by conventional direct swabbing method using a sterile swab. Samples were then restored in a 10ml test tube containing 2 ml of sterile phosphate buffer solution ( PBS). DNA was extracted using MolBi HIMEDIA DNA Purification Kit (without enrichment). The entire DNA extraction process followed the manufacturer's suggested protocol and extracted gDNA was sent to Macrogen Inc., Seoul, Korea for metabarcoding analysis. Next is RNA extraction, this was performed at Macrogen Inc. In Seoulm Korea and all samples underwent quality control ( QC). Qualified samples proceeded to library construction. The PCR amplification of the 16S rRNA hypervariable regions ( V1-V3) was amplified from total genomic DNA using general bacterial primers. All samples were then preceded by illumina sequencing. Sequencing data were converted into raw data for analysis. So last is data analysis, from the raw data, pairs of reads were merged and assembled using the program FLASH ( 1. 2. 11). Short reads are filtered out and extra long tails are trimmed. Filtered reads are clustered at 100% identity using CD- HIT-DUP. The diversity analysis and taxonomic composition for each sample from phylum to species levels were identified using QUIME. Obtained sequences were compared to existing gene sequences from NCBI and phylogenetic trees were constructed using Molecular Evolutionary Genetics Analysis ( MEGA) X software ( Kumar and Tamura, 2016). The resulting data were then analyzed using the Neighbor-Joining ( NJ) approach..

[Audio] Overall taxonomic characterization of the bacterial community isolated in this study was analyzed down to the species level. Taxonomic analysis yielded a total of sixteen classifiable phyla of which three phyla were dominant in the entire samples. Proteobacteria ( 49.42- 99.76%) Bacteroidetes ( 0.3- 12.05%) Firmicutes ( 0.01- 4.32%) In most studies, these three phyla are actually the most commonly detected bacterial phyla in marine ecosystems and benthic organisms..

[Audio] In general, the Gammaproteobacteria represented about 86.15%. While other classes of bacteria represented only a total of 13.85% of the total bacteria isolated from all samples. This result is contrary to the study conducted by Balcazar. In their study, Alphaproteobacteria was found to dominate the bacterial population in cutaneous mucus of H. guttulatus, and bacterial isolates belonging to the Gammaproteobacteria were obtained exclusively from the intestinal content. This is the first report of Gammaproteobacteria detected on the skin mucus of H. comes. What's interesting is that the study of Balcazar revealed that 13 bacterial isolates under the class gammaproteobacteria from H. guttulatus exhibited antibacterial properties against at least one of the indicator strains. So since gammaproteobacteria dominated the H. comes skin mucus and gut content, we can say that there is a possibility that the families under these classes will also exhibit antibacterial properties which can be used to combat the common pathogenic agents of seahorses that cause bacterial infections in seahorse aquaculture. As well as explore its possible biomedical potential associated with TCM. Since gammaproteobacteria is the highly dominant class, and the bacterial classes under " others" were about 1% or less of the total bacteria isolated limiting their resolution, only proteobacteria were classified up to species level..

Results & Discussions (Order level). The most prominent order under class gammaproteobacteria :.

[Audio] 143 families were identified in this study. For the skin samples. A total of 27 and 19 families were identified in the skin mucus and gut content, respectively, while 132 and 80 bacteria families were identified in the carriage water and sediment samples. The bacterial families were dominantly represented by the family shewanellacea..

[Audio] Fourteen of these families ( 9.15% of total identified microbes) were present in all samples ( Table 2).

[Audio] The baseline data gathered in this study served as a source of inspiration for biomedical research and drug discovery for treating diseases relevant to the use of seahorses in traditional medicine..

Recommendations. Extract the biomass of the isolated bacteria and screen its antibacterial activity and mechanism of action against pathogenic Halomonas species through microbroth susceptibility assay and minimum inhibitory concentration assay..

Acknowledgements. BIODIVERSITY 2022. “BUILDING RESILIENCE IN TIMES OF PANDEMIC”.

“Small things create big impacts”. Cute Cartoon Seahorse Couple. Male And Female Sea Horses With Hearts. St. Valentines Day Vector Greeting Card Illustration. Royalty Free SVG, Cliparts, Vectors, And Stock Illustration. Image 124243298..