Abstract
Gonad-inhibiting hormone (GIH) belongs to a family of neuropeptides that are released from the eyestalks of male crustaceans and plays key roles in gonadal maturity, reproduction, and molting. However, the detailed mechanisms underlying the effects of GIH on sexual regulation have yet to be elucidated. In the present study, we aimed to demonstrate how GIH mediate the activity of the androgenic gland (AG) to affect sexual regulation. To do this, we cloned and characterized a GIH sequence from Macrobrachium rosenbergii (MrGIH). The open reading frame (ORF) of MrGIH was 360 bp and codes for a polypeptide of 119 amino acids and a putative protein of 13.56 KDa. Tissue analysis showed that MrGIH is widely expressed in a range of tissues but particularly, the eyestalk, intestine, and nerve cord. Following the dsRNA silencing of MrGIH for 24 h, the expression levels of MrGIH were down-regulated in both the eyestalk and AG when compared with the negative control, but significantly increased the expression of Macrobrachium rosenbergii insulin-like androgenic gland hormone-binding protein (MrIAGBP) in AG, thus suggesting that MrGIH is an inhibitory factor for MrIAGBP. In addition, we found that eyestalk removal on certain days led to increased expression levels of MrIAGBP expression. The expression levels of MrIAGBP peaked at 2 d in the AG after unilateral and bilateral eyestalk ablation, exhibiting a 7.27- and 6.03-fold increase, respectively. Afterward, the expression of GIH protein levels were down-regulated and IAGBP protein levels were up-regulated after GIH silencing using immunohistochemistry method, combined with the increase of IAGBP protein levels after eyestalk ablation, confirming that MrGIH is an inhibitory factor that can moderately regulate AG development and IAGBP expression. Collectively, our findings enriched the mechanisms that control the sexual regulation pathway of male M. rosenbergii, and provided significant information for further explorations of the mechanism underlying sex regulation in other decapod crustaceans.
Highlights
The mechanisms of sex determination and differentiation in Macrobrachium rosenbergii, a commercially important species of giant freshwater prawn, have been the subject of significant research interest in terms of evolutionary biology and endocrinology
Multiple alignment showed that the amino acids of MrGIH shared 98.18% identity with Macrobrachium nipponense (AEJ54622.1), 89.09% identity with Palaemon carinicauda (AIJ49750.1), 83.64% identity with Chorismus antarcticus (ANQ38670.1), 58.93% identity with Homarus gammarus (ABA42181.1), 48.21% identity with Charybdis japonica (ACD11361.1), 47.22% identity with Eriocheir sinensis (AAQ81640.1), 48.05% identity with Cancer pagurus (CAB61424.1), 44.93% identity with Trachypenaeus curvirostris (AAL55259.1), 34.62% identity with Cherax quadricarinatus (AWK57516.1), and 31.67% identity with Litopenaeus vannamei (KF879913.1) (Figure 2)
We found that MrGIH mRNA was expressed at very high levels in the eyestalk both of females and males; Interestingly in the brain, female Gonad-inhibiting hormone (GIH) is highly expressed, while male GIH is low expressed; MrGIH was expressed in a range of other tissues, but with lower levels; for example, in the androgenic gland, ovary, testis, stomach, gill, and heart (Figure 4)
Summary
The mechanisms of sex determination and differentiation in Macrobrachium rosenbergii, a commercially important species of giant freshwater prawn, have been the subject of significant research interest in terms of evolutionary biology and endocrinology. Previous work, based on the pattern of sexual dimorphism in many species of crustaceans, led to the use of monosex culture (all-male or all-female) in the commercial sector as this was the best strategy to achieve the highest yield (Ventura and Sagi, 2012; Levy et al, 2016). Recent research has shown that environmental factors have led to the onset of sexual maturation in some crustaceans at a much earlier time than normal, resulting in slow growth and reduced production (Wilder et al, 2010). It is of great significance to study the mechanisms associated with sex differentiation that involves gonad-related gene expression as this may be advantageous with regards to improving yield. The dynamic processes underlying the regulation of gonadinhibiting factors in the giant freshwater prawn have yet to be elucidated
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