Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit-fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The doublesex gene of S. litura (Sldsx) has male-specific SldsxM and female-specific SldsF isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/μL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at Go generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild-type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of doublesex on the male are similar to other insects.
Qian DuLiang WenSi-Chun ZhengHong-Lun BiYong-Ping HuangQi-Li FengLin Liu
Kriippel homolog 1(Kr-hl),a zinc finger transcription factor,is involved in the metamorphosis and adult reproduction of insects.However,the role of Kr-hl in reproduction of holometabolic insects remains to be elucidated.The regulation network of 7-associated genes in the reproduction in Bombyx mori was investigated in this study.The higher expression level of BmKr-hl in the ovaries was detected during the late pupal stage and adults.RNA interference(RNAi)-mediated depletion of BmKr-hl in the female at day 6 of pupae resulted in abnormal oocytes at 48 h post-double-stranded RNA treatment,which showed less yolk protein deposition and partially transparent chorion.RNA-seq and subsequent differentially expressed transcripts analysis showed that knockdown of BmKr-hl caused a decrease in the expression of 2882 genes and an increase in the expression of 2565 genes in the oocytes at day 8 of pupae.Totally,27 genes coding for transcription factors were down-regulated,while six genes coding for other transcription factors were up-regulated.BmKr-hl bound to the Kr-hl binding site of the transcription factors AP-1(activating protein-1)and FOXG1 to increase their messenger RNA tran・scripts in the BmN cells,respectively.Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses of that positively co-expressed with AP-1 and FOXG1 transcripts showed mainly enrichment in the metabolic-related pathways,the nu・tri ent absorption and the yolk protein absorption processes.These data suggested that BmKr-h 1 might directly regulate the metabolic-related pathways,the nutrient absorption and the yolk protein absorption processes or probably through AP-1 and/or FOXG1 to regulate oocyte development.
Zi-Dan ZhuQi-Hao HuChun-Mei TongHong-Guang YangSi-Chun ZhengQi-Li FengHui-Min Deng
