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This study aimed to investigate the community diversity and functional characteristics of spider-associated bacteria in four tissues of <jats:italic>Heteropoda venatoria<\/jats:italic> using HTS of the 16S rRNA gene and culturomics technologies, as well as the functional verification of the isolated strains. The results of HTS showed that the spider-associated bacteria in different tissues belonged to 34 phyla, 72 classes, 170 orders, 277 families, and 458 genera. <jats:italic>Bacillus<\/jats:italic> was found to be the most abundant bacteria in the venom gland, silk gland, and ovary, while <jats:italic>Stenotrophomonas<\/jats:italic>, <jats:italic>Acinetobacter<\/jats:italic>, and <jats:italic>Sphingomonas<\/jats:italic> were dominant in the gut microbiota<jats:italic>.<\/jats:italic> Based on the amplicon sequencing results, 21 distinct cultivation conditions were developed using culturomics to isolate bacteria from the ovary, gut, venom gland, and silk gland. A total of 119 bacterial strains, representing 4 phyla and 25 genera, with <jats:italic>Bacillus<\/jats:italic> and <jats:italic>Serratia<\/jats:italic> as the dominant genera, were isolated. Five strains exhibited high efficiency in degrading pesticides in the <jats:italic>in vitro<\/jats:italic> experiments. Out of the 119 isolates, 28 exhibited antibacterial activity against at least one of the tested bacterial strains, including the pathogenic bacteria <jats:italic>Staphylococcus aureus<\/jats:italic>, <jats:italic>Acinetobacter baumanii<\/jats:italic>, and <jats:italic>Enterococcus faecalis<\/jats:italic>. The study also identified three strains, GL312, PL211, and PL316, which exhibited significant cytotoxicity against MGC-803. The crude extract from the fermentation broth of strain PL316 was found to effectively induce apoptosis in MGC-803 cells. Overall, this study offers a comprehensive understanding of the bacterial community structure associated with <jats:italic>H. venatoria<\/jats:italic>. It also provides valuable insights into discovering novel antitumor natural products for gastric cancer and xenobiotic-degrading bacteria of spiders.<\/jats:p>","DOI":"10.1007\/s00248-024-02383-2","type":"journal-article","created":{"date-parts":[[2024,5,4]],"date-time":"2024-05-04T13:01:37Z","timestamp":1714827697000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Diversity and Functionality of Bacteria Associated with Different Tissues of Spider Heteropoda venatoria Revealed through Integration of High-Throughput Sequencing and Culturomics Approaches"],"prefix":"10.1007","volume":"87","author":[{"given":"Likun","family":"Zhao","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shanfeng","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruoyi","family":"Xiao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chao","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhitang","family":"Lyu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Feng","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,5,4]]},"reference":[{"key":"2383_CR1","unstructured":"World Spider Catalog (2023) World Spider Catalog Version 23.5.,. http:\/\/wsc.nmbe.ch, accessed on (Jan 18, 2023)"},{"key":"2383_CR2","doi-asserted-by":"publisher","first-page":"104","DOI":"10.1186\/s12862-020-01664-x","volume":"20","author":"SJ Dunaj","year":"2020","unstructured":"Dunaj SJ, Bettencourt BR, Garb JE, Brucker RM (2020) Spider phylosymbiosis: divergence of widow spider species and their tissues\u2019 microbiomes. 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