Identification and functional analysis of the doublesex gene in the mud crab Scylla paramamosain

Jan 12, 2022Comparative biochemistry and physiology. Part A, Molecular & integrative physiology

Role of the doublesex gene in the mud crab Scylla paramamosain

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Abstract

The Spdsx gene in the mud crab, Scylla paramamosain, encodes a protein of 266 amino acids with a conserved DM domain.

The Spdsx gene is present in various analyzed crustacean species, showing low conservation except for the DM domain.
Only one dsx gene was found in the analyzed crab and lobster species, while two dsx genes were identified in shrimps.
Spdsx is widely expressed in various tissues, with significantly higher levels in the androgenic gland compared to other tissues.
Expression levels of Spdsx in the ovary are significantly higher than in the testis (p < 0.05).
During embryo development, Spdsx expression remains stable until the 5 pairs of appendages stage, after which it increases until hatching.
Silencing Spdsx resulted in significant changes in the expression levels of reproduction-related marker genes in the testis, ovary, and androgenic gland.

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Doublesex (Dsx) is a crucial member of the Dmrt gene family and plays a vital role in sex determination and differentiation among the animal kingdom. In the present study, a doublesex (designated as Spdsx) gene was identified and characterized for the first time in the mud crab, Scylla paramamosain. The Spdsx cDNA contains an 801 bp open reading frame (ORF) encoding 266 amino acids with a conserved DM domain. Meanwhile, to elucidate the conservation of Dsx, its orthologus were identified in several crustacean species as well. In addition, the expression pattern of Spdsx in various adult tissues and during embryo development was analyzed with qRT-PCR technology. Finally, the roles of Spdsx might play in the testis, androgenic gland, and ovary were analyzed by RNAi technology. The main results are as follows: (1) the Spdsx gene widely existed in analyzed crustacean species, and the multiple sequences alignment result indicated the conservation of Dsx was low except for the DM domain; (2) only one dsx gene was identified in analyzed crab and lobster, while 2 dsx genes (dsx-1 and dsx-2) were identified in shrimps; (3) the Spdsx gene was widely expressed in analyzed tissues, and the expression level in androgenic gland was obviously higher than that in other tissues. Interestingly, the expression level of Spdsx in the ovary was significantly higher than that in testis (p < 0.05); (4) The expression pattern of Spdsx during embryo development was divided into two groups: remained stable from blastula stage to 5 pairs of appendages stage; after 5 pairs of appendages stage, the expression level increased and remained stable from 7 pairs of appendages stage to hatching stage; (5) After the silencing of Spdsx, the expression level of marker genes in testis, ovary, and androgenic gland significantly changed, among which the expression level of vtg and vtgR in ovary down-regulated, the dmrt-like and dmrt-1a (exclusively expressed in testis) in testis up-regulated and the IAG in androgenic gland down-regulated. All the results above demonstrated that the Spdsx play crucial roles in regulating the reproduction system development of mud crab.

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Identification and functional analysis of the doublesex gene in the mud crab Scylla paramamosain

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