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. 2021 May 31:12:675928.
doi: 10.3389/fgene.2021.675928. eCollection 2021.

Transcriptome Profiling Analysis of the Testis After Eyestalk Ablation for Selection of the Candidate Genes Involved in the Male Sexual Development in Macrobrachium nipponense

Shubo Jin  1 , Yin Fu  2 , Yuning Hu  3 , Hongtuo Fu  1   3 , Sufei Jiang  1 , Yiwei Xiong  1 , Hui Qiao  1 , Wenyi Zhang  1 , Yongsheng Gong  1 , Yan Wu  1
Affiliations

Transcriptome Profiling Analysis of the Testis After Eyestalk Ablation for Selection of the Candidate Genes Involved in the Male Sexual Development in Macrobrachium nipponense

Shubo Jin et al. Front Genet. .

Abstract

The eyestalk of crustacean species secretes many hormones, affecting the process of reproduction, molting, metabolism of glucose, and other functions in crustaceans. In this study, important metabolic pathways and candidate genes involved in the male sexual development were identified through performing the transcriptome profiling analysis of the testis after the ablation of eyestalk from Macrobrachium nipponense. The histological observations revealed that the testis development became vigorous after eyestalk ablation, indicating that the hormones secreted by the eyestalk have negative effects on the testis development in M. nipponense. Transcriptome profiling analysis revealed that 1,039, 1,226, and 3,682 differentially expressed genes (DEGs) were identified between normal prawns (CG) vs single-side eyestalk ablation prawns (SS), SS vs double-side eyestalk ablation prawns (DS), and CG vs DS, respectively, indicating that the ablation of double-side eyestalk has more significant regulatory roles on male sexual development than that of single-side ablation, which was consistent with the histological observations. Lysosome, Apoptosis, Glycolysis/Gluconeogenesis, and Insulin signaling pathway were the main enriched metabolic pathways in all of these three comparisons, and the important genes from these metabolic pathways were also selected. The qPCR verifications of 10 DEGs from these metabolic pathways were the same as those of RNA-seq. The qPCR, in situ hybridization, and RNA interference analysis of Mn-NFkBα revealed that NFkBα has a positive regulatory effect on testis development. This study provided new insights on male sexual development in M. nipponense, promoting the studies on male sexual development in other crustaceans as well.

Keywords: Macrobrachium nipponense; NFkBα; eyestalk ablation; male sexual development; testis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
The morphological differences of the testis after the ablation of eyestalk. SG, spermatogonia; SC, spermatocytes; S, sperms; and CT, collected tissue. Scale bars = 20 μm.
FIGURE 2
FIGURE 2
Gene ontology classification of non-redundant transcripts.
FIGURE 3
FIGURE 3
Clusters of orthologous groups of proteins (COG) classification of putative proteins.
FIGURE 4
FIGURE 4
Verification of the expressions of 10 differentially expressed genes (DEGs) by qPCR. The amounts of DEG expression were normalized to the EIF transcript level. Data are shown as mean ± SD (standard deviation) of tissues in three separate individuals. Capital letters indicate expression difference. Control indicates normal prawns; single-side indicates the single-side ablation of eyestalk; double-side indicates the double-side ablation of eyestalk.
FIGURE 5
FIGURE 5
Expression characterization of Mn-NFkBα in different tissues and post-larval developmental stages. The amount of Mn-NFkBα mRNA was normalized to the EIF transcript level. Data are shown as mean ± SD (standard deviation) of tissues from three separate individuals. Capital letters indicate expression difference between different samples. (A) The expression characterization of Mn-NFkBα in different tissues. (B) The expression characterization of Mn-NFkBα in different post-larval developmental stages.
FIGURE 6
FIGURE 6
In situ hybridization analysis of Mn-NFkBα gene in the testis and androgenic gland from reproductive season, and different reproductive cycle of ovary of M. nipponense. SG, spermatogonia; SC, spermatocytes; S, sperms; CT, collected tissue; I, Stage I of androgenic gland cell; II, Stage II of androgenic gland cell; III, Stage III of androgenic gland cell; EB, ejaculatory bulb; OG, oogonium; OC, oocyte; CM, cytoplasmic membrane; N, nucleus; Y, yolk granule; and FC, follicle membrane. Scale bars = 20 μm.
FIGURE 7
FIGURE 7
Expression characterization of Mn-NFkBα and Mn-IAG at different days after Mn-NFkBα dsRNA injection. The amount of Mn-NFkBα and Mn-IAG mRNA was normalized to the EIF transcript level. Data are shown as mean ± SD (standard deviation) of tissues from three separate individuals. Capital letters indicate expression difference between different days after green fluorescent protein (GFP) injection in the control group. Lowercase letters indicate expression difference between different days after Mn-NFkBα dsRNA injection in the RNA interference (RNAi) group. * (p < 0.05) and ** (p < 0.01) indicate significant expression difference between the RNAi group and control group at the sample day. (A) Expression characterization of Mn-NFkBα at different days after Mn-NFkBα dsRNA injection. (B) Expression characterization of Mn-IAG at different days after Mn-NFkBα dsRNA injection.
FIGURE 8
FIGURE 8
The morphological differences of the testis between the RNA interference (RNAi) and control groups. SG, spermatogonia; SC, spermatocytes; S, sperms; and CT, collected tissue. Scale bars = 20 μm.
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