Abstract: 【Aim】To identify gene groups with the most significant changes in the expression in integuments of Anopheles sinensis pupae under melanization-inducing conditions, and explore their correlations with melanin precursor accumulation and cuticular structural features. 【Methods】Grace’s insect cell medium was supplemented with the melanin precursors dopa and dopamine. An. sinensis pupal integuments were subjected to in vitro cultivation within the first 20 min of pupation, and a control group (CK) was not treated with melanin precursors. At 16 h after cultivation, pupal integuments from the melanin precursor-treated groups and control group were observed to assess their coloration and cuticular cross-sectional features using a stereomicroscope. The integument transcriptome sequencing of the melanin precursor-treated and control groups was performed using the Illumina platform. GO functional classification and KEGG pathway enrichment analysis were performed on the differentially expressed genes (DEGs). The gene annotations and chromosome distributions of significantly enriched DEGs were analyzed. qRT-PCR was conducted to validate the transcriptome data. 【Results】The integuments and cuticular cross-sections of pupae of An. sinensis treated with dopa and dopamine exhibited noticeable melanization compared with those of CK, and dopa treatment resulted in the deepest pigmentation. The cuticle exhibited significant thickening in the dopa and dopamine treatment groups compared with that of CK, and the degree of thickness was correlated with the degree of melanization. There were 2 952 and 697 DEGs were identified from dopa treatment group vs CK and dopamine treatment group vs CK comparison groups, respectively, and the down-regulated genes predominated. The numbers of the shared up-regulated DEGs and down-regulated DEGs between these two comparison groups were 223 and 347, respectively. GO functional classification result showed that the shared up-regulated DEGs in the above comparison groups and the shared up-regulated DEGs in both comparison groups were significantly enriched to structural constituent of cuticle (GO: 0042302). KEGG pathway enrichment analysis result indicated that the up-regulated DEGs in the dopa treatment group vs CK comparison group were significantly enriched to spliceosome pathway, and the down-regulated DEGs were significantly enriched to Toll-Imd and Hippo signaling pathways. The down-regulated DEGs in the dopamine treatment group vs CK comparison group were significantly enriched to autophagy pathway. No DEGs shared between these two comparison groups were significantly enriched to any pathways. In addition, 65 significantly enriched up-regulated cuticular protein genes belonged to the CPR, CPF, TWDL, CPLC, and CPAP five families and distributed on three chromosomes, and some members were clustered. The expression levels detected by qPCR-PCR for 12 selected shared up-regulated cuticular protein genes in both comparison groups were consistent with the transcriptome data. 【Conclusion】This omics-level study showed that the excessive accumulation of melanin in mosquito pupal integuments led to the significant up-regulation of a large number of cuticular protein genes and changes in cuticular structural features. These findings facilitate subsequent studies of the mechanisms that allow melanin precursors to regulate cuticular structural genes, and to understand the impacts of interactions between important components of the cuticle on the adaptive traits in insects, supplying a new perspective on the impact of insect adaptive traits.

Key words: Anopheles sinensis, melanization, melanin precursors, integument, transcriptome, cuticular protein genes