Gonochorism is the most common reproductive strategy in decapod crustacean species (Juchault, 1999). Males and females have significantly different reproductive systems, as well as external features (Nagaraju, 2011). For instance, the reproductive system of male American lobster Homarus americanus consists of the paired testes, vas deferens, spermiducts, and gonopores located on the fifth pair of walking legs. Besides, the specialized first pair of pleopods serves as a gonopod during mating (Comeau and Benhalima, 2018b). However, the female reproductive system of H. americanus consists of the paired ovaries, oviducts, and gonopores located on the third pair of walking legs. The specialized organ, seminal receptacle, arises on the thoracic ventral side between the fourth and the fifth pairs of walking legs (Comeau and Benhalima, 2018a). Interestingly, a review of the literature shows that Caridean shrimps exhibit several other protandry sexual systems apart from gonochorism (Bauer, 2000). Among them, strictly sequential protandric hermaphroditism (SPH) and protandric simultaneous hermaphroditism (PSH) are well-studied. For example, Northern spot shrimp Pandalus platyceros, a SPH species, first mature as functional males with hermaphrodite gonads, and then go to a transitional phase followed by a functional female phase (Levy et al., 2020b). Meanwhile, all the Lysmata species with the PSH sexual system, eventually acquire reproductive functions for both males and females following the male phase and a short transitional phase (Bauer, 2000; Chen et al., 2019).

As with insect, sexual differentiation is controlled by key reproductive hormones in decapods. It is generally believed that insulin-like androgenic gland hormone (IAG) regulates male sexual differentiation in dioecious decapods (Ventura et al., 2009). 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). For instance, in the giant freshwater prawn, Macrobrachium rosenbergii, IAG gene silencing induced degeneration of male primary and secondary characteristics (Ventura et al., 2009), and feminization in young males (Ventura et al., 2012). Silencing IAG gene could also feminize male-related phenotypes in the intersex red-claw crayfish C. quadricarinatus (Rosen et al., 2010) and the male Chinese mitten crab Eriocheir sinensis (Fu et al., 2020). Moreover, augmentation of IAG hormone transcripts in adult M. rosenbergii males resulted in significantly higher (orange claw) OC to (blue claw) BC transformations and vice versa (Priyadarshi et al., 2017). Considering its universal and pivotal role as a master regulator of crustacean sexual development, IAG is regard as the sexual “IAG-switch” in decapod crustacean (Levy and Sagi, 2020).

Crustacean female sex hormone (CFSH) is a neurohormone that is closely related to female sexual differentiation in decapods. It was first isolated from the eyestalk ganglion in the Atlantic blue crab Callinectes sapidus, and was found to be pivotal in the development of the female’s mating and egg brooding systems, including the gonopores and ovigerous setae (Zmora and Chung, 2014). Similar functions have also been established in the mud crab Scylla paramamosain (Jiang et al., 2020). Besides, CFSH was also reported as an inhibitor of IAG, as recombinant Sp-CFSH protein significantly suppressed Sp-IAG expression in the AG explants of the mud crab S. paramamosain in vitro (Liu et al., 2017).

In crustacean males, previous studies have revealed that the X-organ-sinus gland complex (XO-SG) in the eyestalk ganglion, a major source of neuropeptides, modulates reproductive functions via the eyestalk-AG-testicular axis (Khalaila et al., 2002; Nagaraju, 2011). Besides, its inhibitory effects on AG development and IAG expression have been intensively explored through eyestalk ablation. The active components suppressing AG development have been identified in XO-SG. Among them, researchers have comprehensively characterized the crustacean hyperglycemic hormone (CHH) superfamily neuropeptides as well as CFSH mentioned above. Most members of the CHH superfamily exerted inhibitory effects on either AG development or IAG expression (Das et al., 2015; Li et al., 2015; Guo et al., 2019). Following the silencing of gonad-inhibiting hormone (GIH), molt-inhibiting hormone (MIH), and CHH gene, IAG transcription level increased significantly in the black tiger prawn Penaeus monodon (Das et al., 2015), the oriental river prawn Macrobrachium nipponense (Li et al., 2015), and the Pacific white shrimp Penaeus (Litopenaeus) vannamei (Guo et al., 2019).

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. The very low but detectable expression of Lw-IAG during the euhermaphrodite phases suggested that Lw-IAG was possibly responsible for the maintenance of the male reproductive activity in euhermaphrodite phase. Recently, a transcriptomic study identified two insulin-like peptide (ILP) transcripts (Lvit-IAG1 and Lvit-IAG2) in the peppermint shrimp Lysmata vittata (Bao et al., 2020), and biological functions of Lvit-IAG1 have been explored in detail (Liu et al., 2020). In L.vittata, silencing Lvit-IAG1 impeded development of male-related phenotypes (appendices masculinae (AM) and male gonopores) while suppressing the germ cells at the primary spermatocyte stage, demonstrating that Lvit-IAG1 was indeed a functional IAG. Meanwhile, Lvit-IAG1 was also suggested to regulate the ovarian development by inhibiting Lvit-GIHs and Lvit-CFSHs expression in the eyestalk ganglion (Liu et al., 2020). In decapods, four types of insulin and related peptides were identified: insulin-like androgenic gland hormone (IAG), insulin, relaxins, and gonadulins (Veenstra, 2020). Besides, there is usually only one IAG gene identified in a species (Li et al., 2012; Huang et al., 2017b). Therefore, further in-depth studies are warranted to explore whether Lvit-IAG2 is another functional IAG or other insulin and related peptides.

The peppermint shrimp L. vittata, a relatively small caridean species (maximum carapace length < 1 cm), like other species, displays a unique sexual system, PSH, whereby individuals first mature as males; however, with increasing age and size, they acquire reproductive functions for both males and females (Bauer, 2000; Alves et al., 2019). Given its impressive reproductive fecundity, short generation time, and relatively clear ontogenetic gonad development staging system, L. vittata is a good model organism for exploring the molecular mechanisms for endocrine regulation of the unique PSH sexual system in crustaceans (Chen et al., 2019).

In this study, we continuously explored the putative function of Lvit-IAG2 in L. vittata. Following its similar spatial and temporal expression profiles with Lvit-IAG1, we hypothesized that Lvit-IAG2 and Lvit-IAG1 exert similar biological functions and they coordinatively regulate sexual differentiation of the species. To validate this hypothesis, we performed both short-term and long-term gene knockdown via RNA interference (RNAi). In addition to morphological characteristics, expression levels of gonadal development-associated genes [GIH, vitellogenin (Vg), and vitellogenin Receptor (VgR)], carbohydrate metabolism (CHH), and sexual differentiation (CFSH) were assessed via qRT-PCR.

Following a previous transcriptome study, two ILP transcripts, Lvit-IAG1 and Lvit-IAG2, exist in the PSH shrimp, L. vittata (Bao et al., 2020). Lvit-IAG1 has also been identified as an IAG via functional study (Liu et al., 2020). In this study, we expounded on the biological functions of another ILP, Lvit-IAG2. Lvit-IAG2 contained features that were structurally conserved in IAGs: a preproprotein comprised of a signal peptide, B chain, C peptide, and A chain; six strictly conserved cysteine residues forming three disulfide bonds in the B chain and A chain among predicted mature peptides. Besides, based on phylogenetic tree analysis, Lvit-IAG2 was classified into the IAG clade and subclade of the family Caridea. These results confirmed that Lvit-IAG2 was identified structurally as an IAG. Meanwhile, we noticed that some studies reported more than one transcript of IAG in a decapod species (Li et al., 2012; Huang et al., 2017b). However, these transcripts were generated by alternative splicing of one IAG gene, and deduced mature amino acid sequences of these encoded IAG proteins were nearly identical (Li et al., 2012; Huang et al., 2017b). Herein, mature peptides of Lvit-IAG2 and Lvit-IAG1 shared only 58.82% similarity for B chain and 44.12% similarity for A chain, respectively. These results might suggest that two different IAG genes exist in the species, which has never been reported.

Earlier experiments demonstrated that IAG genes were mainly expressed in the AG in decapod crustaceans (Manor et al., 2007). More recently, studies detected IAG mRNA expression in the hepatopancreas and ovary (Chung, 2014; Huang et al., 2014). Herein, the Lvit-IAG2 gene was exclusively expressed in the AG; its expression level was significantly higher in the male phase (Stage I-III) than that in the euhermaphrodite phase (Stage IV) during the life cycle of the PSH species. These reports implied that Lvit-IAG2 potentially can initiate male sexual differentiation in the male phase and maintain male reproductive activity in the euhermaphrodite phase. Notably, Lvit-IAG2 and Lvit-IAG1 displayed slightly different expression trends during gonadal development. The expression levels of Lvit-IAG2 decreased sharply at stage II, while significant downregulation of Lvit-IAG1 expression appeared at stage III. The distinction suggested different functions of the two IAGs.

The present results demonstrated that gene silencing via RNAi successfully induced a specific knockdown of Lvit-IAG2 transcripts levels by 77 and 95% in short-term and long-term silencing experiments, respectively. Furthermore, we assessed the influence of Lvit-IAG2 on the development of male features by comparing male external features and germ cell type composition in the testicular region. Notably, previous studies revealed that IAG silencing delayed the appearance of male sexual characteristics and induced testicular spermatogenesis arrest in the freshwater prawn, M. rosenbergii (Ventura et al., 2009) and the red swamp crayfish Procambarus clarkii (Shi et al., 2019). Similar results were observed for Lvit-IAG1 in L. vittata. Lvit-IAG1 knockdown induced abnormal development of both AM and male gonopores, and suppressed germ cells at the primary spermatocytes (Liu et al., 2020). However, we found that Lvit-IAG2 and Lvit-IAG1 exerted distinct functions in the male sexual differentiation process. Lvit-IAG2 knockdown solely suppressed AM development and germ cells arrested at the secondary spermatocytes, implying its crucial contribution to secondary spermatocyte-to-spermatid transition. These results demonstrated that Lvit-IAG2 was indeed another functional IAG of L.vittata. On the other hand, either primary-to-secondary spermatocyte transition or secondary spermatocyte-to-spermatid transition was essential for the normal development of testicular region. Meanwhile, both of the two Lvit-IAGs might be involved in developmental processes specific to AM. Thus, Lvit-IAG2 and Lvit-IAG1 acted in concert, and both of them were indispensable in the male sexual differentiation process of the PSH species.

Simultaneously, we evaluated the effects of Lvit-IAG2 on female development; notably, no significant difference in Lvit-CFSHs gene expression among the three treatments in both short-term and long-term silencing experiments was reported. Further morphological characterization revealed similar results as Lvit-IAG2 knockdown did not impact the development of female gonopores. Nonetheless, a significant inhibitory effect on the development of ovarian region was observed. 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 induced by the difference between SPH and PSH. For SPH shirimp P. platyceros, individuals first mature as male and eventually acquire reproductive function of female, indicating that functional males and females have different reproductive systems (Levy et al., 2020b). This phase-specific gender pattern is similar, to some extent, with dioecious species, and so it is with reproductive endocrine system. However, For SPH shrimp L. vitata, individuals finally acquire the same euhermaphrodite reproductive system with both male and female functions. It is therefore reasonable to speculate that different reproductive endocrine system presents in PSH species, and IAG is likely to possess different functions in ovarian development. Following reports from previous experiments, Lvit-IAG1 could regulate ovarian development by inhibiting Lvit-GIHs expression (Liu et al., 2020). However, in the present experiment, we reported no significant change in Lvit-GIHs transcript levels. Remarkably, knockdown of Lvit-IAG1 and Lvit-IAG2 suppressed ovarian development. Thus, we could not eliminate the possibility that insulin and related peptides could directly regulate Vg expression in the hepatopancreas, and promote oocyte growth and development. Following a review by Das and Arur (2017), insulin signaling played a pivotal role in the regulation of oocyte growth, development, and maturation in both vertebrates and invertebrates. For instance, in the rainbow trout Oncorhynchus mykiss, insulin was reported to induce the accumulation of vitellogenin in cultured oocytes (Shibata et al., 1993). Besides, ILPs could also control germline cyst growth and vitellogenesis in Drosophila (LaFever and Drummond-Barbosa, 2005). In the silkmoth Bombyx mori, knock-out of an insulin-like growth factor (IGF)-like peptide gene induced drastically lower ovary weights and number of eggs in female individuals (Fujinaga et al., 2019). In the kuruma shrimp Marsupenaeus japonicas, Mj-IAG was proved to have insulin-type disulfide, rather than AGH-type (Katayama et al., 2014), indicating that IAG potentially possessed similar functions as IGF or insulin. Moreover, a recent in vitro experiment revealed that bovine insulin induced the Vg expression in the hepatopancreas of the mud crab S. paramamosain, implying that insulin-like peptide could directly modulate vitellogenesis in the hepatopancreas (Huang et al., 2017a). Thus, Lvit-IAG2 potentially possessed similar functions as IGF or insulin in the PSH L. vittata, which might be explained by their structural similarities (Martin et al., 1998, 1999; Manor et al., 2007). On the other hand, growth and reproduction are closely related (Michalakis et al., 2013). Herein, we noted that Lvit-IAG2 silencing induced significantly slower growth in the treated shrimp, implying that Lvit-IAG2 is potentially a growth regulator. Similar results have been reported in the freshwater prawn M. rosenbergii (Ventura et al., 2009) and the mud crab S. paramamosain (Huang et al., 2014), and with the function of insulin and related peptide in mammals (Bruning et al., 2000; Neirijnck et al., 2019), fishes (Reinecke, 2010), and insects (Wu and Brown, 2006). Moreover, transcript levels of Lvit-CHH significantly decreased following knock down of Lvit-IAG2 in both short-term and long-term experiment. CHH is defined as a metabolism associated hormone as its participation in multiple metabolic processes, including glycometabolism (Webster et al., 2012), lipid metabolism (Montiel-Arzate et al., 2020) and ammonia metabolism (Zhang et al., 2020). Of note, the low expression of Lvit-CHH implied the growth and developmental abnormalities of individuals. Hence, it could also be reasonably hypothesized that retardation in the development of the ovarian region in Lvit-IAG2 silencing shrimp potentially arose from the abnormity in the growth and development of the shrimp. Further in vitro experiments are required to test the two hypotheses.

Taken together, the present work characterized an IAG gene, Lvit-IAG2, from the AG of the peppermint shrimp L. vittata and explored its crucial functions in regulating primary and secondary sexual characteristics of the PSH species. Moreover, this study implicated Lvit-IAG2 and Lvit-IAG1 to act in concert, aimed at regulating male sexual differentiation in the PSH shrimp. Also, Lvit-IAG2 may directly contribute to ovarian development, or via the promotion of growth in L. vittata. These findings provide new insights into molecular mechanisms of sexual differentiation in PSH crustaceans, expounding our current knowledge on crustacean reproductive endocrinology.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

FL contributed to conceptualization, methodology, software, validation, formal analysis, investigation, data curation, visualization and writing—original draft preparation of the study. HY contributed to conceptualization, methodology, validation, data curation, writing—review and editing, supervision, project administration and funding acquisition. WS contributed to investigation. AL contributed to validation and writing—review and editing of the study. ZZ contributed to funding acquisition and provided resources. All authors contributed to manuscript revision, read, and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.