Microbial diversity and pigment synthesis in the accessory nidamental gland: species-specific and color-associated patterns in bigfin reef squid (Sepioteuthis lessoniana).

Background: In certain cephalopod species, two distinct symbiotic organs host large populations of microorganisms: the light organ, regulated by the daily cycle, and the accessory nidamental gland (ANG), regulated by the female reproductive cycle. While host-microbiota interactions in the light organ of the bobtail squid are well understood, the dynamics within the ANG remain largely unexplored. This study uses the bigfin reef squid, Sepioteuthis lessoniana, as a model to investigate the microbiomes associated with specific regions of the ANG, capitalizing on its relatively large gland size compared to the bobtail squid. Our goal was to characterize species-specific microbiomes in the ANG and explore how pigmented region-dependent microbes contribute to reproductive fitness in bigfin reef squid.

Results: Histological results indicate that four types of epithelial cells were observed in the secondary tubules of inner ANG layer. Using an amplicon-based approach, we found that Alphaproteobacteria were highly abundant in different cephalopod species. Beta diversity analyses revealed significant interspecies differences in microbiomes, while alpha diversity showed that the bigfin reef squid harbored a richer bacterial community than the other two species. Notably, pigmented regions of the ANG exhibited lower microbial diversity compared to whole ANG tissues, with Alphaproteobacteria significantly enriched in these regions. Hyphomicrobiaceae (Alphaproteobacteria) were unique to the orange regions, while Fodinicurvataceae (Alphaproteobacteria) and Flavobacteriaceae (Bacteroidia) were exclusive to the white regions. qPCR results showed higher transcription levels of immune response-associated genes in the orange region compared to other pigmented regions, suggesting localized immune interactions.

Conclusions: These findings suggest that Alphaproteobacteria, particularly the Hyphomicrobiaceae clade, may correlated to the synthesis orange pigmentation in the ANG of the bigfin reef squid. The roles of Hyphomicrobiaceae in ANG symbiosis and reproductive fitness still needs further investigation. With this knowledge, we propose further investigations using in situ hybridization to detect host-expressed genes and pigmented region-dependent bacteria as markers. This approach will facilitate the study of localized host-microbiota interactions in distinct pigmented regions of the ANG, providing deeper insights into the mechanism of host-microbe communication.