Characterizing the Role of a Bacterial Consortium in a Host Reproductive Organ

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All animals have beneficial relationships with microorganisms. Many aquatic animals lay external eggs that must be protected from fouling by microorganisms to successfully develop. Although beneficial bacteria are thought to contribute to egg defense, the mechanisms of this protection are largely unknown. The Hawaiian bobtail squid is a model animal host for studying relationships with beneficial bacteria. Females of this and many other squid species harbor bacteria in a specialized organ of their reproductive tract. The function of these bacteria is unknown, but they may assist with protecting such eggs from fouling by other microorganisms. This work will use genetic and chemical analyses to determine whether these beneficial bacteria and the compounds that they produce can inhibit the growth of fungi and other microorganisms. The ability of these bacteria to induce development of the squid's reproductive organ will also be studied. The study of defensive symbioses is a growing area of interest in a number of terrestrial and aquatic associations. This project will take a multi-disciplinary approach by investigators with expertise in symbiosis, chemical ecology and drug discovery to shed light on how bacteria and microbial metabolites affect egg protection and development. The proposed study will integrate research with education by contributing to the training of graduate students and a number of undergraduate researchers, focusing on students from underrepresented groups through several outreach programs. Results of the study may lead to the discovery of novel anti-fungal and anti-microbial compounds.Preliminary studies characterized the bacterial consortium found in the accessory nidamental gland (ANG) of the squid Euprymna scolopes, and suggest that ANG bacteria deposited into eggs protect developing embryos from fungal biofilms. Furthermore, female squid raised in the absence of bacteria do not form an ANG by sexual maturity, suggesting that bacteria may be necessary for proper development of the organ. Based on these results, the proposed research has three main objectives. 1) Isolate and identify bioactive metabolites from ANG and egg bacteria that show activity against fungal and other microbial biofilms, and determine if bioactive bacterial strains or isolated compounds can rescue eggs upon fungal challenge. The use of molecular networks would compare secondary metabolite production by these bacterial isolates. 2) Use transcriptomics to characterize bacterial gene expression in active bacterial isolates. This information, along with sequence transcriptomes and metagenomes from whole ANGs, eggs and genomes of active bacterial isolates, would be used to predict the biosynthetic pathways leading to antimicrobial secondary metabolite production that may be involved with egg protection. 3) Characterize the role of bacteria in the development of the ANG, by testing whether ANG bacterial isolates from the consortium are capable of inducing morphogenesis of the ANG. The project would examine whether eggs from females without an ANG are susceptible to fouling, and whether eggs challenged with fungi can be rescued with active bacterial isolates and/or compounds.