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Developmental processes in animals are influenced by colonization and/or signaling from microbial symbionts. Here, we show that bacteria from the environment are linked to development of a symbiotic organ that houses a bacterial consortium in female Hawaiian bobtail squid, Euprymna scolopes . In addition to the well-characterized light organ association with the bioluminescent bacterium Vibrio fischeri, female E. scolopes house a simple bacterial community in a reproductive organ, the accessory nidamental gland (ANG). In order to understand the influences of bacteria on ANG development, squid were raised in the laboratory under conditions where exposure to environmental microorganisms was experimentally manipulated. Under conditions where hosts were exposed to depleted environmental bacteria, ANGs were completely absent or stunted, a result independent of the presence of the light organ symbiont V. fischeri. When squid were raised in the laboratory with substrate from the host's natural environment containing the native microbiota, normal ANG development was observed, and the bacterial communities were similar to wild-caught animals. Analysis of the bacterial communities from ANGs and substrates of wild-caught and laboratory-raised animals suggests that certain bacterial groups, namely, the Verrucomicrobia , are linked to ANG development. The ANG community composition was also experimentally manipulated. Squid raised with natural substrate supplemented with a specific ANG bacterial strain, Leisingera sp. JC1, had high proportions of this strain in the ANG, suggesting that once ANG development is initiated, specific strains can be introduced and subsequently colonize the organ. Overall, these data suggest that environmental bacteria are required for development of the ANG in E. scolopes . IMPORTANCE Microbiota have profound effects on animal and plant development. Hosts raised axenically or without symbionts often suffer negative outcomes resulting in developmental defects or reduced organ function. Using defined experimental conditions, we demonstrate that environmental bacteria are required for the formation of a female-specific symbiotic organ in the Hawaiian bobtail squid, Euprymna scolopes . Although nascent tissues from this organ that are involved with bacterial recruitment formed initially, the mature organ failed to develop and was absent or severely reduced in sexually mature animals that were not exposed to microbiota from the host's natural environment. This is the first example of complete organ development relying on exposure to symbiotic bacteria in an animal host. This study broadens the use of E. scolopes as a model organism for studying the influence of beneficial bacteria on animal development.

AbstractThe Hawaiian bobtail squid, Euprymna scolopes , has a female reproductive organ called the accessory nidamental gland that contains a symbiotic bacterial consortium. These bacteria are deposited from the accessory nidamental gland into the squid's egg cases, where the consortium prevents microbial fouling. The symbiont community is environmentally transmitted and conserved across host populations, yet little is known about how the organ develops and is colonized by bacteria. In order to understand accessory nidamental gland development in E. scolopes , we characterized the gland during maturation by using histology and confocal and transmission electron microscopy. We found that an epithelial field formed first about four weeks after hatching, followed by the proliferation of numerous pores during what we hypothesize to be the initiation of bacterial recruitment (early development). Microscopy revealed that these pores were connected to ciliated invaginations that occasionally contained bacteria. During mid development, these epithelial fields expanded, and separate colonized tubules were observed below the epithelial layer that contained the pores and invaginations. During late development, the superficial epithelial fields appeared to regress as animals approached sexual maturity and were never observed in fully mature adults (about 2-3 months post-hatching), suggesting that they help facilitate bacterial colonization of the accessory nidamental gland. An analysis of 16S rRNA gene diversity in accessory nidamental glands from females of varying size showed that the bacterial community changed as the host approached sexual maturity, increasing in community evenness and shifting from a Verrucomicrobia-dominated to an Alphaproteobacteria-dominated consortium. Given the host's relationship with the well-characterized light organ symbiont Vibrio fischeri , our work suggests that the accessory nidamental gland of E. scolopes may have similar mechanisms to recruit bacteria from the environment. Understanding the developmental and colonization processes of the accessory nidamental gland will expand the use of E. scolopes as a model organism for studying bacterial consortia in marine symbioses.