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. 2022 Nov 9;13(11):2079.
doi: 10.3390/genes13112079.

RNA Interference Analysis of the Functions of Cyclin B in Male Reproductive Development of the Oriental River Prawn (Macrobrachium nipponense)

Wenyi Zhang  1 , Pengchao Wang  2 , Yiwei Xiong  1 , Tianyong Chen  3 , Sufei Jiang  1 , Hui Qiao  1 , Yongsheng Gong  1 , Yan Wu  1 , Shubo Jin  1   2 , Hongtuo Fu  1   2
Affiliations

RNA Interference Analysis of the Functions of Cyclin B in Male Reproductive Development of the Oriental River Prawn (Macrobrachium nipponense)

Wenyi Zhang et al. Genes (Basel). .

Abstract

Cyclin B (CycB) plays essential roles in cell proliferation and promotes gonad development in many crustaceans. The goal of this study was to investigate the regulatory roles of this gene in the reproductive development of male oriental river prawns (Macrobrachium nipponense). A phylo-genetic tree analysis revealed that the protein sequence of Mn-CycB was most closely related to those of freshwater prawns, whereas the evolutionary distance from crabs was much longer. A quantitative PCR analysis showed that the expression of Mn-CycB was highest in the gonad of both male and female prawns compared to that in other tissues (p < 0.05), indicating that this gene may play essential roles in the regulation of both testis and ovary development in M. nipponense. In males, Mn-CycB expression in the testis and androgenic gland was higher during the reproductive season than during the non-reproductive season (p < 0.05), implying that CycB plays essential roles in the reproductive development of male M. nipponense. An RNA interference analysis revealed that the Mn-insulin-like androgenic gland hormone expression decreased as the Mn-CycB expression decreased, and that few sperm were detected 14 days after the dsCycB treatment, indicating that CycB positively affects testis development in M. nipponense. The results of this study highlight the functions of CycB in M. nipponense, and they can be applied to studies of male reproductive development in other crustacean species.

Keywords: Macrobrachium nipponense; RNAi analysis; cyclin B; insulin-like androgenic gland hormone; spermatogenesis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Flowchart showing the sample collection procedure.
Figure 2
Figure 2
Nucleotide and deduced amino acid sequence of Mn-CycB. The nucleotide sequence is displayed in the 5′–3′ direction and is numbered on the left. The deduced amino acid sequence is shown as a single capital letter amino acid code. The 3′ and 5′ untranslated regions are shown with lowercase letters. Codons are numbered on the left, the methionine (ATG) initiation codon is shown, and an asterisk denotes the termination codon (TGA). Arrows and line indicated the direction of location of each primer.
Figure 3
Figure 3
Similarities in amino acid sequences of CycB between different species. The black box indicates the cyclin destruction box and the red box indicates the conserved pkA site.
Figure 4
Figure 4
Phylo-genetic tree of CycBs from different organisms. Species names are listed on the right side of the tree. The red asterisk indicates M. nipponense.
Figure 5
Figure 5
Mn-CycB expression in different mature tissues and developmental stages as determined with qPCR. Lowercase letters on the bars indicate expression differences between different tissue samples (analysis of variance (AVONA)). Capital letters on the bars indicate expression differences in the same tissue between male and female prawns (independent sample t-tests). (A) Mn-CycB expression in different mature tissues. (B) Mn-CycB expression in different developmental stages. E: eyestalk; Br: brain; H: heart; He: hepatopancreas; M: muscle; G: gonad; Gi: gill; CS: cleavage stage; BS: blastula stage, GS: gastrula stage; NS: nauplius stage; FS: posterior nauplius stage; PS: protozoa stage; ZS: zoea stage; L: larval developmental stage; PL: post-larval developmental stage.
Figure 6
Figure 6
Mn-CycB expression in the testis and androgenic gland of prawns collected during the reproductive and non-reproductive seasons. Lowercase letters (a, b) on the bars indicate expression differences between different tissue samples (independent sample t-tests). (A) Mn-CycB expression in the testis. (B) Mn-CycB expression in the androgenic gland.
Figure 7
Figure 7
Measurement of Mn-CycB and Mn-IAG expression in the androgenic gland on different days after dsCycB and dsGFP injections. Lowercase letters (a, b) on the bars indicate expression differences between different days after dsGFP injection, and capital letters (X, Y) on the bars indicate expression differences between different days after dsCycB injection (analysis of variance (AVONA)). * p < 0.05 and ** p < 0.01 indicate significant expression differences between the dsGFP- and dsCycB-treated groups on the same day (independent sample t-tests). (A) Mn-CycB expression on different days after dsGFP and dsCycB injections. (B) Measurement of Mn-IAG expression on different days after dsGFP and dsCycB injections.
Figure 8
Figure 8
Histological comparison of testis tissue between dsGFP- and dsCycB-treated prawns. SG: spermatogonia; SC: spermatocyte; S: sperm. Scale bar = 20 μm.
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