SCREENING AND RNA INTERFERENCE STUDY OF TWO GENES RELATED TO REPRODUCTION IN DAPHNIA SINENSIS

Reproductive biology of cladocera has been a research focus among ecologists, however, the molecular mechanism on the reproduction of cladocera remains unclear. In this paper, the genes related to reproduction of Daphnia sinensis were screened. D. sinensis at four growth stages (JF: Juvenile female, BM: the 1^st instar female before maturity, MA: the 1^st adult instar female, and RF: the 4^th adult instar female) were selected for transcriptome sequencing. 6 combinations including BM vs. JF, MA vs. JF, RF vs. JF, RF vs. MA, MA vs. BM, RF vs. BM, were analyzed, and the top 30 upregulated genes from each combination were selected. This resulted in a total of 163 differentially expressed genes. To validate these genes, RT qPCR was performed on the fourth adult females of D. sinensis cultured with fish kairomones, leading to the identification of 19 candidate genes related to reproduction. Among these candidates, two genes (Cluster-13168.32243, Dsussp and Cluster-13168.22885, Dsmapk) were selected for functional analysis. Subsequently, RNAi was conducted, resulting in significantly lower expression levels of Dsussp and Dsmapk genes in E. coli containing their dsRNA compared to the control group (Enhanced Green Fluorescent Protein, EGFP). Compared with the EGFP group, the expression levels of Dsmapk and Dsussp genes decreased by 28.1% and 32.3% in the 2% E. coli concentration, and by 41.1% and 55.6% in the 5% E. coli concentration, respectively. Compared with the control group, the number of eggs and offspring of D. sinensis decreased in the Dsmapk and Dsussp groups after RNAi, particularly in the 5% E. coli concentration compared to the 2% E. coli concentration. The inhibitory effects on the number of eggs and offspring of D. sinensis were strengthened after RNAi. Therefore, the Dsmapk and Dsussp genes have been confirmed to play an important role in the reproductive development of D. sinensis.