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.

Interaction between Saikosaponin D, Paeoniflorin, and Human Serum Albumin.

Saikosaponin D (SSD) and paeoniflorin (PF) are the major active constituents of Bupleuri Radix and Paeonia lactiflora Pall, respectively, and have been widely used in China to treat liver and other diseases for many centuries. We explored the binding of SSD/PF to human serum albumin (HSA) by using fluorospectrophotometry, circular dichroism (CD) and molecular docking. Both SSD and PF produced a conformational change in HSA. Fluorescence quenching was accompanied by a blue shift in the fluorescence spectra. Co-binding of PF and SSD also induced quenching and a conformational change in HSA. The Stern-Volmer equation showed that quenching was dominated by static quenching. The binding constant for ternary interaction was below that for binary interaction. Site-competitive experiments demonstrated that SSD/PF bound to site I (subdomain IIA) and site II (subdomain IIIA) in HSA. Analysis of thermodynamic parameters indicated that hydrogen bonding and van der Waals forces were mostly responsible for the binary association. Also, there was energy transfer upon binary interaction. Molecular docking supported the experimental findings in conformation, binding sites and binding forces.

Electrophysiological responses of the rice leaffolder, Cnaphalocrocis medinalis, to rice plant volatiles.

The rice leaffolder, Cnaphalocrocis medinalis Guenée (Lepidoptera: Pyralidae), is one of the most destructive pests of rice. Electrophysiological responses of this species to 38 synthetic volatiles known to be released from rice plants (Poaceae: Oryza spp.) were studied using the electroantennogram (EAG) method. Compounds that elicited the strongest EAG responses for each physiological condition were selected for EAG dose-response tests at five concentrations. These compounds included: methyl salicylate, heptanol, linalool, cyclohexanol, and 2-heptanone for one-day-old male moths; heptanol, hexanal, (Z)-2-hexen-1-ol, and nonadecane for one-day- old females; methyl salicylate, heptanol, (E)-2-hexen-1-ol, and (Z)-2-hexen-1-ol for three-day- old males; linalool, heptanol, (E)-2-hexen-1-ol, 2-heptanone, and hexanal for three-day-old females; 2-heptanone, cyclohexanol, linalool, heptanol, and methyl salicylate for five-day-old virgin females; and methyl benzoate, (Z)-2-hexen-1-ol, heptanol, linalool, and hexanal for five- day-old mated females. Female and male C. medinalis exhibited broad overlap in their EAG responses, and there was no clear difference between male and female EAG responses to different compounds. Statistical analyses revealed that both volatile compound chemical structure and C. medinalis physiological condition (age, sex, and mating condition) had an effect on EAG response.

Transcriptomic Features in a Single Extracellular Vesicle via Single-Cell RNA Sequencing.

Although many studies have investigated functional molecules in extracellular vesicles (EVs), the exact number of ribonucleic acid molecules in a single-EV is unknown. Therefore, it is critical to explore the transcriptomic features and heterogeneity at the level of a single-EV. Here, using the 10x Genomics platform, the RNA cargos are profiled in single EVs derived from human K562 and mesenchymal stem cells. The key steps are labeling intact EVs using calcein-AM, detecting the EV concentration via flow cytometry, and using the CB2 algorithm with adaptive thresholds to effectively distinguish real EVs from background. The gene number in a single-EV varied from 6 to 148, with a mean of 52. Ribosomal genes, mitochondrial genes, and eukaryotic translation elongation factor 1 alpha has a high EV percentage in all EV samples. Hemoglobin genes are uniquely highly expressed in K562-EVs, and cytoskeleton genes are enriched in MSC-EVs. Ten or more clusters with different marker genes in each single-EV dataset demonstrated EV heterogeneity. Moreover, integrating EVs and their parental cells reveal both EVs and cells in each cluster, indicating different cell origins of various EVs. To the best of the author's knowledge, this study provides the first high-throughput transcriptome at the single-EV level and improves the understanding of EVs.

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.