Diversity and Functionality of Bacterial Symbionts in Different Tissues of Spider Heteropoda venatoria Revealed through Integration of High-throughput Sequencing and Culturomic Approaches

Spiders host a diverse range of bacteria in their guts and other tissues, which have been found to play a significant role in their fitness. This study aimed to investigate the community diversity and functional charcateristics of four tissue-associated symbiotic bacteria in Heteropoda venatoria using high-throughput sequencing (HTS) of 16S ribosomal RNA (rRNA) gene and culturomic technologies. The results of HTS showed that the symbiotic bacteria in different tissues belonged to 34 phyla, 72 classes, 170 orders, 277 families, and 458 genera. Bacillus was found to be the most abundant bacteria in the venom gland, silk gland, and ovary, while Stenotrophomonas , Acinetobacter and Sphingomonas were dominant in the gut microbiota. Based on the amplicon sequencing results, 21 different cultivation conditions were disigned using culturomics to isolate bacteria from the ovary, gut, venom gland, and silk gland. A total of 119 bacterial strains belonging to 4 phyla and 25 genera, with Bacillus and Serratia as the main genera, were isolated. Functional predictions revealed that metabolism was the most abundant, and in vitro validation experiments showed that five strains had high efficiency in pesticide degrading. Furthermore, 28 of the 119 isolates exhibited antibacterial activity against at least one of the tested bacterial strains, including pathogenic bacteria Staphylococcus aureus , Acinetobacter baumanii and Enterococcus faecalis . The study also identified three strains, GL312, PL211and PL316, which had significant cytotoxicity against MGC-803, with the fermentation crude extract of strain PL316 found to be an effective inducer of MGC-803 cell apoptosis. Overall, this study provides a comprehensive understanding of the community structure of symbiotic bacteria in H. venatoria and offers valuable insights into the discovery of novel antitumor natural products for gastric cancer and xenobiotic degrading bacteria of spiders.