The accessory nidamental gland (ANG) is a female reproductive organ found in most squid and cuttlefish that contains a consortium of bacteria. These symbiotic bacteria are transmitted from the marine environment and selected by the host through an unknown mechanism. In animals, a common antimicrobial mechanism of innate immunity is iron sequestration, which is based on the development of transferrin (TF)-like proteins. To understand this mechanism of host-microbe interaction, we attempted to characterize the role of transferrin in bigfin reef squid (Sepioteuthis lessoniana) during bacterial transmission. qPCR analysis showed that Tf was exclusively expressed in the outer layer of ANG,and this was confirmed by in situ hybridization, which showed that Tf was localized in the outer epithelial cell layer of the ANG. Western blot analysis indicated that TF is a soluble glycoprotein. Immunohistochemical staining also showed that TF is localized in the outer epithelial cell layer of the ANG and that it is mainly expressed in the outer layer during ANG growth. These results suggest that robust Tf mRNA and TF protein expression in the outer layer of the ANG plays an important role in microbe selection by the host during bacterial transmission.

Unlike gonochoristic fishes, sex is fixed after gonadal differentiation (primary sex determination), and sex can be altered in adults (secondary sex determination) of hermaphroditic fish species. The secondary sex determination of hermaphroditic fish has focused on the differences between testicular tissue and ovarian tissue during the sex change process. However, comprehensive studies analyzing ovarian tissue or testicular tissue independently have not been performed. Hermaphroditic black porgy shows a digonic gonad (ovarian tissue with testicular tissue separated by connective tissue). Protandrous black porgy has stable maleness during the first two reproductive cycles (<2 years old), and approximately 50% enter femaleness (natural sex change) during the third reproductive cycle. Precocious femaleness is rarely observed in the estradiol-17β (E2)-induced female phase (oocytes maintained at the primary oocyte stage), and a reversible female-to-male sex change is found after E2 is withdrawn in <2-year-old fish. However, precocious femaleness (oocytes entering the vitellogenic oocyte stage) is observed in testis-removed fish in <2-year-old fish. We used this characteristic to study secondary sex determination (femaleness) in ovarian tissue via transcriptomic analysis. Cell proliferation analysis showed that BrdU (5-bromo-2′-deoxyuridine)-incorporated germline cells were significantly increased in the testis-removed fish (female) compared to the control (sham) fish (male) during the nonspawning season (2 months after surgery). qPCR analysis showed that there were no differences in pituitary-releasing hormones (lhb and gtha) in pituitary and ovarian steroidogenesis-related factors (star, cyp11a1, hsd3b1, and cyp19a1a) or female-related genes (wnt4a, bmp15, gdf9, figla, and foxl2) in ovarian tissues between intact and testis-removed fish (2 months after surgery). Low expression of pituitary fshb and ovarian cyp17a1 was found after 2 months of surgery. However, we did find small numbers of genes (289 genes) showing sexual fate dimorphic expression in both groups by transcriptomic analysis (1 month after surgery). The expression profiles of these differentially expressed genes were further examined by qPCR. Our present work identified several candidate genes in ovarian tissue that may be involved in the early period of secondary sex determination (femaleness) in black porgy. The data confirmed our previous suggestion that testicular tissue plays an important role in secondary sex determination in protandrous black porgy.

Some cephalopods carry microorganisms in two specialized organs, the light organ and the accessory nidamental gland (ANG). For the light organ, comprehensive mechanisms have been described for winnowing (bacterial selection) and maintenance of the symbiotic luminescent bacterium Vibrio fischeri (V. fischeri). However, the mechanisms controlling bacterial selection and maintenance during bacterial colonization of the ANG are open biological issues with physiological significance. Our recent study on bigfin reef squid (Sepioteuthis lessoniana) already showed that the ANG bacterial community shifts gradually and exhibits decreased diversity throughout maturation. This study further describes a potential role of an innate immunity-involved molecule, peptidoglycan recognition proteins (PGRPs), in the ANG of bigfin reef squid during bacterial transmission and colonization. First, we found that four homologs of the PGRP family are expressed in the ANG of bigfin reef squid (slPGRP2-5), but only slPgrp2 transcript levels are highly correlated with ANG development and bacterial colonization. Besides, slPgrp2 transcripts are mainly expressed in the epithelial cells of certain secondary tubules of ANG, and the expression levels are varied in the epithelial cells of other secondary tubules. This data reveals that slPgrp2 transcripts may associate with the composition of bacterial consortium and its secretary factors. Moreover, recombinant slPGRP2 had a negative effect of Escherichia coli (E.coli) which inhibited bacterial growth in culture. Therefore, our data suggest slPgrp2 expression in the epithelial cells of secondary tubules in the ANG may have an essential role in the winnowing and maintenance of holobiont homeostasis in bigfin reef squid.