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Androgen administration has been widely used for masculinization in fish. The mechanism of the sex change in sexual fate regulation is not clear. Oral administration or pellet implantation was applied. We orally applied an aromatase inhibitor (AI, to decrease estrogen levels) and 17α-methyltestosterone (MT, to increase androgen levels) to induce masculinization to clarify the mechanism of the sex change in the protogynous orange-spotted grouper. After 3 mo of AI/MT administration, male characteristics were observed in the female-to-male sex change fish. These male characteristics included increased plasma 11-ketotestosterone (11-KT), decreased estradiol (E2) levels, increased male-related gene (dmrt1, sox9, and cyp11b2) expression, and decreased female-related gene (figla, foxl2, and cyp19a1a) expression. However, the reduced male characteristics and male-to-female sex change occurred after AI/MT-termination in the AI- and MT-induced maleness. Furthermore, the MT-induced oocyte-depleted follicle cells (from MT-implantation) had increased proliferating activity, and the sexual fate in a portion of female gonadal soma cells was altered to male function during the female-to-male sex change. In contrast, the gonadal soma cells were not proliferative during the early process of the male-to-female sex change. Additionally, the male gonadal soma cells did not alter to female function during the male-to-female sex change in the AI/MT-terminated fish. After MT termination in the male-to-female sex-changed fish, the differentiated male germ cells showed increased proliferating activities together with dormancy and did not show characteristics of both sexes in the early germ cells. In conclusion, these findings indicate for the first time in a single species that the mechanism involved in the replacement of soma cells is different between the female-to-male and male-to-female sex change processes in grouper. These results also demonstrate that sexual fate determination (secondary sex determination) is regulated by endogenous sex steroid levels.

Since females grow faster in penaeid shrimp, all-female aquaculture was proposed. Environmental conditions in the Pacific white shrimp were not found to affect genetic sex determination (ZZ/ZW system). The androgenic gland–secreting insulin-like androgenic gland hormone is a key controlling factor in crustacean male differentiation. However, functional sex reversal (neo-male) in penaeid shrimp has not yet been achieved by manipulating the insulin-like androgenic gland hormone–sexual switch. Therefore, understanding the molecular mechanisms of gonadal differentiation may help build appropriate tools to generate neo-male for all-female breeding. This study describes the potential role of the novel penaeid-specific testicular zinc finger protein (pTZFP) in the gonads of Pacific white shrimp. First, pTZFP transcripts show a male-bias expression pattern in undifferentiated gonads, which is then exclusively expressed in the testis and absent or slightly expressed in the ovary and other tissues. Besides, the knockdown of pTZFP in undifferentiated males results in smaller testes but no sex reversal. Immunohistochemical staining of proliferating cell nuclear antigen further confirmed that the smaller testes in pTZFP-deficient males are due to the lower proliferating activity of spermatogonia. These data reveal that pTZFP may be involved in testicular development but have fewer effects on gonadal differentiation. Moreover, testicular pTZFP transcription levels were not reduced with estradiol-17β (E2) administration or AG excision. Therefore, our data suggest that pTZFP may regulate testicular development through downstream genes regulating spermatogonia proliferation. Moreover, our data provide an appropriate molecular marker for identifying the sex of undifferentiated gonads. Summary Sentence pTZFP participates in testicular development by regulating spermatogonia proliferation, and its male-biased expression profile provides an appropriate molecular marker for identifying the critical window of gonadal sex differentiation in penaeid shrimp. Graphical Abstract

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.

In most animals, excess dietary energy is stored as lipids in specialized tissues, such as the liver in vertebrates or the hepatopancreas and fat body in invertebrates, which function as energy reservoirs for reproduction. In cephalopods, however, dietary energy is rapidly mobilized from the digestive gland for growth rather than stored for reproduction. How excess energy is allocated for reproduction activity in cephalopods remains largely unclear. Lipogenesis is initiated by acetyl-CoA carboxylase (ACC), which converts acetyl-CoA derived from dietary carbon sources into malonyl-CoA; subsequent synthesis of saturated fatty acids is catalyzed by fatty acid synthase (FAS). Using the bigfin reef squid as a model, we investigate the role of fas in female development. fas mRNA was highly expressed in ovaries but weak in other tissues, including the lipid-rich digestive gland. fas showed female-biased expression in gonads, with level highest in juvenile ovaries and progressively decreasing to their lowest in mature ovaries. Expression was also high in primary and multiple follicular oocytes but declined in later stages. In situ hybridization and immunohistochemistry confirmed fas mRNA and protein localization in oocytes, particularly in primary and multiple follicular oocytes. In vitro ovarian culture further showed that inhibiting FAS activity enhanced somatic cell proliferation. Together, these findings suggest that squid ovary is a primary site of fatty acid synthesis, supporting early oocyte growth and membrane biogenesis in the absence of dedicated lipid storage tissues. The decline of FAS activity during oogenesis, and the associated reproduction in fatty acid synthesis, may act as a regulatory signal to promote somatic cell proliferation.

More than 1,500 fish species are hermaphroditic, but no hermaphroditic lineage appears to be evolutionarily ancient in fishes. Thus, whether more than one sex at a time was present during the evolutionary shift from gonochorism to hermaphroditism in fishes is an intriguing question. Ectopic oocytes were created in the ovotestes of protandrous black porgy via the withdrawal of estradiol (E2) administration. These ectopic oocytes reprogrammed the surrounding cells, which changed from Sertoli cells to follicle-like cells. We observed that gdf9 and bmp15 expression was localized in the primary oocytes and gradually decreased after oocytes entered a secondary oocyte stage. Robust expression of gdf9 and bmp15 in ectopic oocytes was associated with the surrounding Sertoli cells. However, blocking Cyp19a1a activity and increasing androgen levels did not stimulate the expression of gdf9 and bmp15. Thus, the robust gdf9 and bmp15 expression was not related to the inappropriate male microenvironment. Furthermore, in vitro data demonstrated that gdf9 and bmp15 were not downstream genes of Figla signaling. Therefore, our results suggest that there are two independent mechanisms, a Figla-dependent pathway and a Figla-independent pathway, by which oocyte-surrounding cells are altered from a male somatic fate to a female somatic fate. This functional switch might clarify how oocytes created an appropriate microenvironment during the transition from the ancient gonochorism to the present hermaphroditism.

Unlike vitellogenin, which is the sole major precursor of yolk protein in all oviparous vertebrates, a variety of major precursor of yolk proteins are found among oviparous invertebrates. Sea urchins have a transferrin-like yolk protein, while all other major precursors of yolk proteins in oviparous invertebrates belong to the superfamily of large lipid transfer proteins (LLTPs). However, a comprehensive understanding of vitellogenesis is absent in cephalopods. To understand control of vitellogenesis by the LLTPs gene, two vitellogenins (VTG1 and VTG2), two apolipophorins (APOLP2A and APOLP2B), and a cytosolic large subunit of microsomal triglyceride transfer protein (MTTP) found in the bigfin reef squid. Only the two VTGs showed high levels of expression in mature females compared to males.We further analyzed the expression profile and localization of both VTGs/VTGs during ovarian development. Our data showed that VTGs/VTGs expressions were correlated to the female reproductive cycle. Ovarian VTG1 and VTG2 were localized in the follicle cells but not in oocytes. In addition, VTG1 and VTG2 were represented in follicle cells and oocytes. Thus, our results showed that both VTGs were synthesized by follicle cells and are then delivered to oocytes. In addition, we demonstrated that VTGs were the major precursor of yolk protein in bigfin reef squid. We also found differential proteolytic cleavage processes of VTG1 and VTG2 during VTGs accumulation in oocytes. Therefore, our data shed light on the molecular mechanism of the yolk accumulation pathway in cephalopods. Summary Sentence Two distinct vitellogenins, which are synthesized by the follicle cells, are the major precursor of yolk protein of oocytes in bigfin reef squid.

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.

The bigfin reef squid, Sepioteuthis lessoniana , are a valuable commercial species in East Asian regions such as Taiwan and Japan. A lack of genomic information limits the application of potential aquaculture techniques, especially in breeding when considering the hatching rate of offspring. In some squids and cuttlefishes, symbiotic bacteria are transmitted from the accessory nidamental gland (ANG) to the jelly coat of eggs. In Hawaiian bobtail squid, these parent-delivered mutualistic bacteria play an important role in preventing lethal biofouling of the eggs and accelerating the hatch rate of offspring. The bacterial consortium, which is housed in the female squids ANG, are governed by host selection during female maturation. Immune functions are typically used to explain the regulatory mechanism of symbioses by host selection. In this study, we evaluated the transcripts featured in bacterial selection and maintenance during ANG development using RNA-seq. Different developmental stages of ANGs (stages 1–4) were sequenced. The de novo transcriptome assembly resulted in 524,918 unigenes. Two groups, non-pigmentation group (stage 1 and stage 3) and pigmentation group (stage 4), were clustered by transcriptome-wide expression profile analysis. The gene expression analyses indicated that 9,475 differential expression genes (DEGs) in three different phases and 1,363 (14.3%) DEGs were matched in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Furthermore, KEGG-enriched analysis results suggested that immune responses are a dominant pathway in the non-pigmentation group (stage 1 and stage 3) whereas lipid metabolism and metabolism of flora fermentation are dominant in the pigmentation group (stage 4). Although the host immunity plays an important role during bacterial colonization of the ANG in bigfin reef squid, our results showed that most immune-related genes had a reduced transcriptomic level in the pigmentation group compared with the non-pigmentation group. Therefore, our results provide new insight to understand the regulatory mechanisms of initial bacterial colonization and later bacterial pigmentation in the bigfin reef squid.

Gonadal differentiation is tightly regulated by the initial sex determining gene and the downstream sex-related genes in vertebrates. However, sex change in fish can alter the sexual fate from one sex to the other. Chemical-induced maleness in the protogynous orange-spotted grouper is transient, and a reversible sex change occurs after the chemical treatment is withdrawn. We used these characteristics to study Amh signaling during bi-directional sex change in the grouper. We successfully induced the female-to-male sex change by chemical (aromatase inhibitor, AI, or methyltestosterone, MT) treatment. A dormant gonad (a low proliferation rate of early germ cells and no characteristics of both sexes) was found during the transient phase of reversible male-to-female sex change after the withdrawal of chemical administration. Our results showed that amh (anti-mullerian hormone) and its receptor amhr2 (anti-mullerian hormone receptor type 2) were significantly increased in the gonads during the process of female-to-male sex change. Amh is expressed in the Sertoli cells surrounding the type A spermatogonia in the female-to-male grouper. Male-related gene (dmrt1 and sox9) expression was immediately decreased in MT-terminated males during the reversible male-to-female sex change. However, Amh expression was found in the surrounding cells of type A spermatogonia-like cells during the transient phase of reversible male-to-female sex change. This phenomenon is correlated with the dormancy of type A spermatogonia-like cells. Thus, Amh signaling is suggested to play roles in regulating male differentiation during the female-to-male sex change and in inhibiting type-A spermatogonia-like cell proliferation/differentiation during the reversible male-to-female sex change. We suggest that Amh signaling might play dual roles during bi-directional sex change in grouper.

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 ( sl PGRP2-5), 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 sl PGRP2 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.