Butterflies are diverse in virtually all aspects of their ontogeny, including morphology, life history, and behavior. However, the developmental regulatory mechanisms underlying the important phenotypic traits of butterflies at different developmental stages remain unknown. Here, we investigated the developmental regulatory profiles of butterflies based on transposase accessible chromatin sequencing (ATAC-seq) at three developmental stages in two representative species ( Papilio xuthus and Kallima inachus). Results indicated that 15%-47% of open chromatin peaks appeared in associated genes located 3 kb upstream (i.e., promoter region) of their transcription start site (TSS). Comparative analysis of the different developmental stages indicated that chromatin accessibility is a dynamic process and associated genes with differentially accessible (DA) peaks show functions corresponding to their phenotypic traits. Interestingly, the black color pattern in P. xuthus 4th instar larvae may be attributed to promoter peak-related genes involved in the melanogenesis pathway. Furthermore, many longevity genes in 5th instar larvae and pupae showed open peaks 3 kb upstream of their TSS, which may contribute to the overwintering diapause observed in K. inachus adults. Combined with RNA-seq analysis, our data demonstrated that several genes enriched in the melanogenesis and longevity pathways also exhibit higher expression, confirming that the expression of genes may be closely related to their phenotypic traits. This study offers new insights into larval cuticle color and adult longevity in butterflies and provides a resource for investigating the developmental regulatory mechanisms underlying butterfly ontogeny.
蝴蝶的多样性体现在其个体发育的各个方面，如形态、生活史、行为等。然而，蝴蝶的许多重要表型性状的发育调控机制尚不清楚。该研究运用转座酶研究染色质可及性 (Assay of transposase accessible chromatin sequencing, ATAC-seq) 技术对两种蝴蝶 (柑橘凤蝶 Papilio xuthus和枯叶蛱蝶 Kallima inachus) 的三个发育时期 (4龄、5龄和蛹期) 的染色质可及性区域进行了研究。在柑橘凤蝶三个发育时期分别鉴定到20 367 (4龄)、9 648 (5龄) 和12 001 (蛹期) 个染色质可及性区域；在枯叶蛱蝶的三个发育时期分别鉴定到28 751 (4龄)、85 433 (5龄) 和105 067 (蛹期) 个染色质可及性区域；有15%–47%的染色质可及性区域位于相关基因转录起始位点 (TSS) 上游3kb (即启动子区域)。另外，对在两个蝴蝶物种三个发育时期中鉴定到的染色质可及性区域进行了Motif预测，再根据已知转录因子 (Transcription Factors, TFs) 数据库搜索匹配，鉴定到了一些与这些Motif结合的TFs。通过对物种内不同发育阶段的比较分析表明，染色质可及性区域是一个动态过程，具有差异可及性区域的相关基因对其表型性状具有相应的功能。我们发现柑橘凤蝶4龄幼虫表皮的黑色模式可能与黑色素生成途径中相关基因启动子区域的染色质可及性区域有关；枯叶蛱蝶5龄幼虫和蛹期的很多长寿基因启动子区域有染色质可及性区域，这些基因可能与其成虫长寿命滞育越冬密切相关。结合RNA-seq分析，这些富集在黑色素生成途径和长寿机制途径的基因均表现出较高的表达水平，进一步证实了这些基因的表达可能与其相应的表型性状密切相关。该研究为蝴蝶幼虫表皮颜色和成虫寿命机制的研究提供了新的思路，也为研究蝴蝶个体发育的调控机制提供了新的数据来源。.
Keywords: ATAC-seq; Kallima inachus; Longevity; Melanogenesis; Papilio xuthus.