The IAG-Switch and Further Transcriptomic Insights Into Sexual Differentiation of a Protandric Shrimp

Abstract

The insulin-like androgenic gland hormone (IAG) secreted from the androgenic gland (AG) is a unique endocrine controller (the IAG-switch) of crustacean sexual differentiation. However, while previous studies of the IAG-switch focused mainly on sexual differentiation at early developmental stages of gonochoristic species, this mechanism is yet to be deciphered during naturally occurring sexual shifts in hermaphrodite species. The Northern spot shrimp, Pandalus platyceros, is a protandric hermaphrodite species, native to the North Pacific Ocean. We collected four stages of spot shrimp in Southeast Alaska including: juveniles, adult males, transitionals, and adult females. The AG was dissected from each stage and characterized histologically. Additionally, the IAG mRNA was sequenced. The function of the IAG-switch during the life history of this protandric species was demonstrated through monitoring IAG gene expression. Transcript levels were highest at the juvenile stage, then decreased significantly in mature males and became negligible in the transitional and female stages. Moreover, manipulating the IAG-switch via IAG loss of function in males through RNAi, induced the expected masculine to feminine sexual transformation that naturally occurs in this species. This included reduction in IAG transcript levels in males, elevation of vitellogenin gene expression in hepatopancreas and transformation of the gonad from an ovotestis containing both ovarian and testicular tissue to a true ovary with vitellogenic oocytes. Furthermore, a transcriptomic library from tissues associated with the endocrine axis upstream and downstream the IAG-switch yielded 1,801, 1,707, 1,946, and 182 differentially expressed genes between males, transitionals, and females in the AG, eyestalk, gonad, and hepatopancreas, respectively. Among these genes, the transcriptional pattern of six of them (all of them in the AG), between males, transitionals and females, had similar or inverted transcriptional pattern to that of IAG in P. platyceros. Five of these putatively IAG-switch associated genes are downregulated and one is upregulated throughout the P. platyceros shift from maleness to femaleness. Homolog protein sequences for the above novel genes were found in 17 other decapod species suggesting that they might represent conserved factors associated with the IAG-switch and involved in universal crustacean sexual differentiation mechanisms.

RNAi Reveals Role of Insulin-Like Androgenic Gland Hormone 2 (IAG2) in Sexual Differentiation and Growth in Hermaphrodite Shrimp

“…Contrary to the controls, shrimp in the Lvit-IAG2 silencing treatments exhibited decreased volume of the ovarian region, and germ cells were much smaller and arranged in a disorganized mass. It is interesting to note that silencing IAG gene in another hermaphrodite shrimp P. platyceros induced feminization of individuals, including Vg expression in the hepatopancreas and ovarian development (Levy et al, 2020a), which is consistent with studies in dioecious species. Different outcome might be Ovarian regions of the shrimp from the Lvit-IAG2 silencing treatment were more transparent and smaller in relative size (C).…”

“…In decapod species, IAG was first identified in the androgenic gland (AG) of the red-claw crayfish Cherax quadricarinatus in 2007 (Manor et al, 2007). Since then, numerous studies have found IAG in dioecious (Ventura et al, 2009;Rosen et al, 2010;Ventura et al, 2012;Chung, 2014;Huang et al, 2014), parthenogenetic (Levy et al, 2017), and hermaphroditic (Zhang et al, 2017;Levy et al, 2020a;Liu et al, 2020) species. Furthermore, its critical role in sexual differentiation has been properly studied via loss-of-function experiments (Ventura et al, 2012;Zmora and Chung, 2014;Priyadarshi et al, 2017;Fu et al, 2020).…”

“…To date, however, there are rare reports elaborating sexual differentiation mechanism mediated by IAG in protandric crustaceans. In the SPH shrimp P. platyceros, Pnp-IAG knockdown elevated vitellogenin gene expression in the hepatopancreas and transformation of the gonad from ovotestis to ovary (Levy et al, 2020a). Zhang et al (2017) identified an IAG transcript (Lw-IAG) in a PSH shrimp Lysmata wurdemanni, and examined its expression profile during gonadal development.…”

Protandric Transcriptomes to Uncover Parts of the Crustacean Sex-Differentiation Puzzle

“…In the present study, we collected samples of H. inermis at different stages of the species' protandric life cycle and constructed RNA libraries that yielded both known and novel, yet unannotated, sex-specific genes that are assumed to be associated with sexual differentiation and reproduction. To obtain insight into the function of these novel genes, we also performed a comparative in silico analysis with P. platyceros, taking advantage of RNA libraries of specific tissues previously obtained over the life cycle of P. platyceros (Levy et al, 2020a). P. platyceros is particularly suitable for such an analysis for two reasons: (i) taxonomically, the two species, H. inermis and P. platyceros, belong to families -Pandalidae and Hippolytidae, respectively (Martin et al, 2009) -that are closely related within the infraorder Caridea (Christoffersen, 1990;Wolfe et al, 2019;Levy et al, 2020c), and (ii) technically, the RNA for the H. inermis libraries was extracted from the entire body of the animal (due to its small size that prevented dissection of specific tissues), while the RNA for constructing P. platyceros libraries was obtained by dissecting out specific tissues (Levy et al, 2020a).…”

“…To obtain insight into the function of these novel genes, we also performed a comparative in silico analysis with P. platyceros, taking advantage of RNA libraries of specific tissues previously obtained over the life cycle of P. platyceros (Levy et al, 2020a). P. platyceros is particularly suitable for such an analysis for two reasons: (i) taxonomically, the two species, H. inermis and P. platyceros, belong to families -Pandalidae and Hippolytidae, respectively (Martin et al, 2009) -that are closely related within the infraorder Caridea (Christoffersen, 1990;Wolfe et al, 2019;Levy et al, 2020c), and (ii) technically, the RNA for the H. inermis libraries was extracted from the entire body of the animal (due to its small size that prevented dissection of specific tissues), while the RNA for constructing P. platyceros libraries was obtained by dissecting out specific tissues (Levy et al, 2020a). Therefore, our working hypothesis for the in silico analysis part of this study was that a search for homologs to the newly discovered H. inermis genes in transcriptomic libraries of P. platyceros (and other decapod crustaceans) might reveal the function of those genes in a more general context that includes a wider range of crustaceans.…”