Cloning and effect of RXR on molting-related genes expression in Macrobrachium rosenbergii

Like all arthropods, crustaceans shed the exoskeleton periodically through their life cycle, which is called ecdysis. Molting occurs throughout the life cycle of crustaceans and significantly influences their growth, reproduction, and survival. This physiological event is regulated by several hormones, in which ecdysteroids are the important part. The effect of ecdysteroids is mediated by a heterodimer of ecdysone receptor (EcR) and retinoid X receptor (RXR) homolog, ultraspiracle (USP) in insects or RXR in crustaceans. EcR/RXR or EcR/USP complex could bind to response elements to elicit expression of early ecdysone responsive genes, such as E75, and eventually to regulate downstream transcriptional ecdysteroid cascade reaction. RXR, belonging to a nuclear receptor family, plays an important role in the regulation of molting. In recent years, RXR has been identified in several crustaceans, such as Eriocheir sinensis, Callinectes sapidu, Gecarcinus lateralis, Leptuca pugilator, Macrobrachium nipponense and so on. However, most of the studies focused on the cloning and specific expression analysis among different tissues or different molting stages, its regulation of other molting-related genes remains unclear. Therefore, this study identified a retinoid X receptor (RXR) in M. rosenbergii termed Mr-RXR and further investigated its regulation function through its mRNA expression analysis at different molting stages and RNA interference (RNAi). The full length of the Mr-RXR was 2241 bp, consisting of a 36 bp 5′ UTR (untranslated regions), a 719 bp 3′-UTR, and a 1 386 bp ORF (open reading frame). It encoded 461 amino acids with a predicted molecular weight of 50.6 ku and the theoretical isoelectric point of 7.02. Mr-RXR showed high similarity to other crustaceans with conserved DNA binding domain (DBD) and ligand binding domain (LBD). Phylogenetic analyses showed that the RXR of crustaceans clustered in a group, with the closest relationship between M. rosenbergii and M. nipponense. The expression of Mr-RXR was detected in 11 tissues of M. rosenbergii, including the stomach, gill, brain, abdominal ganglion, intestine, muscle, hepatopancreas, eyestalk, heart, testis, and ovary. The result showed that Mr-RXR was expressed in all tissues with relatively high expression level in the testis, eyestalk, heart, brain, muscle, and relatively low expression in the gill, hepatopancreas and stomach. The expression level of Mr-RXR changes at different molting stages, showing the highest level at premolt stages in both eyestalk and muscle tissues. Besides, the temporal expression of four molting-related genes, including EcR, E75, CHIT and MIH, were also examined. The expression pattern of EcR followed the same trend as Mr-RXR, while E75 and CHIT showed similar mRNA expression patterns with the highest level at premolt and postmolt in the eyestalk and muscle tissues, respectively. MIH was only expressed in eyestalk, reaching its peak at intermolt. To further investigate the function of Mr-RXR, RNAi was conducted for 12 days, which caused the death of 63% of individuals. The expression of Mr-RXR was extremely reduced and the efficiency of silence was 79% and 55% in the eyestalk and muscle, respectively. In the eyestalk, Mr-RXR dsRNA injection did not alter the expression of MIH, but significantly reduced the expression of EcR, E75and CHIT. In the muscle, however, RNAi of Mr-RXR only induced significantly declined expression of CHIT. The correlation between Mr-RXR and other molting-related genes indicated that Mr-RXR might be involved in the regulation of EcR, E75 and CHIT transcription and further mediate the molting process in M. rosenbergii. Results in this study contribute to better understanding of the mechanism of molting in crustaceans.