关键词: 大猿叶虫, 促咽侧体素, 抑咽侧体素, 生殖滞育准备, 保幼激素信号

Abstract: 【Aim】 This study aims to clarify the molecular characteristics and expression difference of allatotropin (AT) and allatostatin (AST) genes during the reproduction and diapause of Colaphellus bowringi, so as to further reveal the functions of these genes in the reproductive diapause preparation of C. bowringi. 【Methods】 We identified the sequences of CbAT and CbASTs in C. bowringi based on the previously constructed transcriptome database, cloned their ORFs and carried out the bioinformatics analysis of their sequences. Then, we assayed the expression patterns of CbAT and CbASTs in the heads of the diapause-destined and non-diapause-destined female pupae and female adults via RT-PCR. RNAi experiments of CbAST-B, CbAST-C and CbAST-B+CbAST-C were done in the diapause-destined 2-day-old female pupae, and the expression levels of juvenile hormone (JH) signaling genes and vitellogenin genes in 4-day-old female adults were detected. 【Results】 One allatotropin gene CbAT (GenBank accession no.: MT977128) and two allatostatin genes CbAST-B (GenBank accession no.: MT977126) and CbAST-C (GenBank accession no.: MT977127) of C. bowringi were identified and cloned, with the ORFs of 408, 600 and 303 bp, respectively. By amino acid sequence alignment and phylogenetic analysis, we found that CbAT, CbAST-B and CbAST-C respectively cluster into the same branch with homologous proteins of Coleoptera insects, showing high conservation in the evolution. The qRT-PCR assay indicated that CbAT had no significant expression differences in the female heads between the diapause-destined and non-diapause-destined individuals from the 2-day-old pupal stage to the 4-day-old adult stage. CbAST-B and CbAST-C were significantly highly expressed in the heads of diapause-destined female adult since the 1- and 2-day-old, respectively, and the differential expression state between the diapause-destined and non-diapause-destined individuals lasted until the end of diapause preparation period. After knocking down CbAST-B, CbAST-C and CbAST-B+CbAST-C in the diapause-destined 2-day-old female pupae, the expression of CbAST-B and CbAST-C in the heads of 4-day-old female adults was significantly suppressed with the RNAi efficiency over 70%. Further study revealed that the expression of JH biosynthesis genes AACT, FPPS and JHAMT, in the head without antenna, JH-responsive genes Kr-h1 and JHE1 in the fat body, and vitellogenin genes Vg1 and Vg2 in the fat body was all significantly up-regulated in the treatment groups of dsCbAST-B, dsCbAST-C and dsCbAST-B+dsCbAST-C. 【Conclusion】 CbAT may be not a key regulator mediating the differences of JH signaling between the preoviposition period and diapause preparation phase in C. bowringi. However, CbAST-B and CbAST-C suppress the synthesis of JH and further down-regulate the expression of Vg genes in C. bowringi during the reproductive diapause preparation phase, thereby promoting the occurrence of diapause. This study further reveals an upstream regulation mechanism for the JH signaling in insects during the reproductive diapause preparation phase, being helpful to understand the seasonal adaptation strategy of insects and providing new guidance and reference for potential target genes for pest control.

Key words: Colaphellus bowringi, allatotropin, allatostatin, reproductive diapause preparation, juvenile hormone signaling