Full Functional Sex Reversal Achieved Through Silencing of MroDmrt11E Gene in Macrobrachium rosenbergii: Production of All-Male Monosex Freshwater Prawn

Abstract

The freshwater prawn Macrobrachium rosenbergii is one kind of important economic aquaculture species and displays remarkable sexual dimorphism. The molecular mechanism of sexual differentiation in M. rosenbergii has been primarily unraveled through the research efforts of the androgenic gland and its related genes. However, the understanding of conserved genes involved in the molecular mechanism underpinning sex determination and sexual differentiation of M. rosenbergii is still fragmentary. MroDmrt11E is a member of the doublesex and mab-3-related transcription factor (Dmrt) gene family and is prominently expressed in the testis. In the present study, in vivo knockdown of MroDmrt11E at the postlarva stage in male prawn induced a complete and functional sex reversal and achieved the production of an all-male monosex population. Furthermore, a great deal of new information of upregulated and downregulated transcriptions involved in sexual differentiation of MroDmrt11E knockdown was enriched by comparative transcriptomic analysis. The effects of RNAi-mediated gene knockdown of MroDmrt11E on the differentially expressed and sex-related candidate genes, such as transformer, fruitless, feminization, insulin-like androgenic gland gene, Dmrt gene family, were primarily focused on, and their possible molecular regulatory relationships in sexual differentiation were analyzed. Meanwhile, the response of primary Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathways was investigated to expound the potential roles of MroDmrt11E in male sexual differentiation, which provided a deeper understanding of the molecular regulatory network underlying sexual differentiation of M. rosenbergii. The finding provided a novel sexual manipulation technique through silencing of Dmrt gene family for achieving a complete and functional sex reversal and offered a new insight regarding the mechanism of the Dmrt gene family in the sexual differentiation of crustaceans.

Keywords: Macrobrachium rosenbergii; MroDmrt11E gene knockdown; all-male monosex population; sex reversal; sexual differentiation.

Figure 1

In vivo knockdown of MroDmrt11E by RNAi in postlarvae. (A) The diagrammatic sketch of in vivo knockdown of MroDmrt11E by RNAi in postlarvae. MroDmrt11E dsRNA injection was performed in postlarvae, and the gene silencing lasted for 3 months. (B) Detection of the relative MroDmrt11E mRNA level in RNAi induced and the control group. (C) The sex identification of differentiated male and female prawn by the appearance of sexual characteristics in M. rosenbergii. There was a pair of male genital pores located at the coxopodite of the fifth pereiopods in males, but a pair of female genital pores located at the coxopodite of the third pereiopods in females as well as neo-females. MGP, male genital pores; PE, pereiopods. (D) The gender of individuals was identified using sex molecular markers in both MroDmrt11E RNAi and the control group. The female-specific sex band indicates female genotype. The female-specific sex bands are indicated by the gray arrows in females. The representative neo-female prawn are shown in a red broken line frame. (E) The neo-female prawn with female appearance features. The MroDmrt11E-silenced genetic males (neo-females) were raised to maturity and induced appearance features of females. Some neo-females spawned and incubated the eggs/embryos in their brood chambers, located ventrally on their abdomens.

Figure 2

The ovarian development of full-functional sex reversal prawn and the breeding of all-male progeny by MroDmrt11E-silenced prawn (neo-females). (A) The ovarian development and maturation of neo-females. The inset photograph indicated the histology of the mature ovary of neo-females. The ovary was full of mature oocytes (Oc4) that were intensely eosinophilic (reddish orange). (B) Ventral view of MroDmrt11E-silenced prawn (neo-females) incubating embryos in their brood chamber. The mature neo-females (B-I) spawned and incubated the embryos in their brood chambers, located ventrally on their enlarged abdomens (B-II, B-III). The color of embryos presented gradual changes from yellow to orange (B-III) then to light-gray color (B-II) during embryonic development. AW, abdomen; E, embryos. (C) Genomic validation of representative embryos from each progeny of neo-females. The first row of PCR products was generated from neo-female embryos, and the second row was from normal females. The presented specific band in gel electrophoresis implied the female embryo.

Figure 3

The significant differentially expressed unigenes enriched KEGG pathway analysis by the comparative transcriptomic analysis of MroDmrt11E knockdown. Four kinds of categories with a higher percentage of upregulated and downregulated transcripts were primarily focused on the pathways of apoptosis and regulation of actin cytoskeleton (A), oocyte meiosis (B), and protein processing in endoplasmic reticulum (C), respectively. The primary upregulated transcripts (red color) and the downregulated transcripts (green color) were briefly profiled in the various signaling pathways.

Figure 4

(A) The sex-related candidate unigenes enriched in putative sexual differentiation pathway by the comparative transcriptomic analysis of MroDmrt11E knockdown. The primary upregulated transcripts (red color) and the downregulated transcripts (green color) were briefly profiled in the signaling pathway. The genes with gray color possibly participated in the sexual differentiation but did not show significant differential expression by MroDmrt11E knockdown. (B) The brief relationship among Dmrt gene family and Mr-IAG was summarized to evaluate their potential influences involved in the sexual differentiation of M. rosenbergii.