Genome-wide survey of open chromatin regions in two swallowtail butterflies Papilio machaon and P. bianor.

Papilio machaon was assigned as the type species for all butterflies by Linnaeus and P. bianor is a congener but exhibits a great difference in morphology (especially larva and adult color pattern) and larval host plants from P. machaon. Thus, they are the ideal models to investigate genetic mechanisms underlying morphology and plasticity between congeners. The reference genomes of both species were dissected in our previous studies, but little is known about their regulatory genome and the epigenetic regulation of gene expression throughout developmental stages. Here, we profiled the chromatin accessibility and gene expression of three developmental stages (the 4th instar larva [L4], the 5th instar larva [L5], and pupa [P]) using transposase accessible chromatin sequencing (ATAC-seq) and RNA-seq. Results showed that many accessible chromatin peaks were identified at three developmental stages (peak number, P. machaon: 44,977 [L4], 36,919 [L5], 47,147 [P]; P. bianor: 20,341 [L4], 44,668 [L5], 62,249 [P]). Moreover, the number of differentially accessible peaks and differentially expressed genes between larval stages of each butterfly species are significantly fewer than that between larval and pupal stages, suggesting a higher similarity within larvae and a significant difference between larvae and pupae. This study added the annotated information of chromatin accessibility genome-wide of the two papilionid species and will promote the investigation of gene regulation in butterfly evolution.

Analysis of gene expression profiles at different stages during preadipocyte differentiation in rabbits.

Excessive accumulation of fat is harmful to human health. The preadipocyte differentiation is a critical process of fat development. Studying the expression profiles of genes related to preadipocyte differentiation contributes to understanding of the mechanism of fat accumulation. Despite being considered an ideal animal model for studying adipogenesis, little is known about the gene expression profiles at different stages during preadipocyte differentiation in rabbits. In the present study, rabbit preadipocytes were cultured in vitro and induced for differentiation, and gene expression profiles of adipocytes collected at days 0, 3, and 9 of differentiation were analyzed by RNA-seq. We identified 1352 differentially expressed genes (DEGs) when comparing day 3 with day 0 and identified 888 DEGs when comparing day 9 with day 3. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the PPAR signaling pathway and PI3K-Akt signaling pathway were significantly enriched by the DEGs that up-regulated within the period of day 0 - day 3, and the GO terms and KEGG pathways that were associated with cell cycle were enriched by the DEGs that up-regulated within the period of day 3 - day 9. The DEGs that specifically up-regulated within the period of day 0 - day 3 might play roles in the cytoplasm, and the DEGs that specifically up-regulated within the period of day 3 - day 9 might act in the nucleus. The protein-protein interaction (PPI) network constructed by DEGs showed that hub node genes might modulate rabbit preadipocyte differentiation via regulating cell cycle.

High-quality reference genomes of swallowtail butterflies provide insights into their coloration evolution.

Swallowtail butterflies (Papilionidae) are a historically significant butterfly group due to their colorful wing patterns, extensive morphological diversity, and phylogenetically important position as a sister group to all other butterflies and have been widely studied regarding ecological adaption, phylogeny, genetics, and evolution. Notably, they contain a unique class of pigments, i.e., papiliochromes, which contribute to their color diversity and various biological functions such as predator avoidance and mate preference. To date, however, the genomic and genetic basis of their color diversity and papiliochrome origin in a phylogenetic and evolutionary context remain largely unknown. Here, we obtained high-quality reference genomes of 11 swallowtail butterfly species covering all tribes of Papilioninae and Parnassiinae using long-read sequencing technology. Combined with previously published butterfly genomes, we obtained robust phylogenetic relationships among tribes, overcoming the challenges of incomplete lineage sorting (ILS) and gene flow. Comprehensive genomic analyses indicated that the evolution of Papilionidae-specific conserved non-exonic elements (PSCNEs) and transcription factor binding sites (TFBSs) of patterning and transporter/cofactor genes, together with the rapid evolution of transporters/cofactors, likely promoted the origin and evolution of papiliochromes. These findings not only provide novel insights into the genomic basis of color diversity, especially papiliochrome origin in swallowtail butterflies, but also provide important data resources for exploring the evolution, ecology, and conservation of butterflies.

Chromatin accessibility profiling provides insights into larval cuticle color and adult longevity in butterflies.

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.