RNAi efficiency is enhanced through knockdown of double-stranded RNA-degrading enzymes in butterfly Papilio xuthus.

The efficiency of RNA interference (RNAi) has always limited the research on the phenotype innovation of Lepidoptera insects. Previous studies have found that double-stranded RNA-degrading enzyme (dsRNase) is an important factor in RNAi efficiency, but there have been no relevant reports in butterflies (Papilionoidea). Papilio xuthus is one of the important models in butterflies with an extensive experimental application value. To explore the effect of dsRNase in the RNAi efficiency on butterflies, six dsRNase genes (PxdsRNase 1-6) were identified in P. xuthus genome, and their dsRNA-degrading activities were subsequently detected by ex vivo assays. The result shows that the dsRNA-degrading ability of gut content (<1 h) was higher than hemolymph content (>12 h). We then investigated the expression patterns of these PxdsRNase genes during different tissues and developmental stages, and related RNAi experiments were carried out. Our results show that different PxdsRNase genes had different expression levels at different developmental stages and tissues. The expression of PxdsRNase2, PxdsRNase3, and PxdsRNase6 were upregulated significantly through dsGFP injection, and PxdsRNase genes can be silenced effectively by injecting their corresponding dsRNA. RNAi-of-RNAi studies with PxEbony, which acts as a reporter gene, observed that silencing PxdsRNase genes can increase RNAi efficiency significantly. These results confirm that silencing dsRNase genes can improve RNAi efficiency in P. xuthus significantly, providing a reference for the functional study of insects such as butterflies with low RNAi efficiency.

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.

Comprehensive silk gland multi-omics comparison illuminates two alternative mechanisms in silkworm heterosis.

Heterosis is a common phenomenon in plants and animals with diverse underlying mechanisms. Here, we applied two widely used silkworm hybrid systems and performed multi-omics analysis to identify possible intrinsic associations between different hybrid strategies and epigenetic mechanisms with silkworm heterosis. We found significant differences in the silk gland transcriptomic landscape between the two systems, including differentially expressed genes and expression patterns in the hybrid offspring compared to their parents. In the quaternary hybrid system, hybrid vigor was primarily due to up-regulated genes and the parent-dominant up-regulated expression pattern, involving multiple transport processes, cellular nitrogen compound catabolism, glucose metabolism, and tricarboxylic acid cycle. In the binary system, hybrid vigor was mainly due to the down-regulated genes and transgressively down-regulated expression pattern, mainly involving basic nitrogen synthesis metabolism and body function. We also demonstrated that DNA methylation may affect hybrid vigor by regulating the expression of several heterosis-related genes. Thus, this study revealed two alternative mechanisms that may contribute to silkworm heterosis, both of which facilitate the efficient utilization of energy and nitrogen for silk production.

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.

Molecular cloning, characterization, and evolution analysis of the luciferase genes from three sympatric sibling fireflies (Lampyridae: Lampyrinae, Diaphanes).

Firefly adult bioluminescence functions as signal communication between sexes. How sympatric sibling species with similar glow pattern recognize their conspecific mates remains largely unknown. To better understand the role of the luciferases of sympatric fireflies in recognizing mates, we cloned the luciferase genes of three sympatric forest dwelling fireflies (Diaphanes nubilus, Diaphanes pectinealis, and Diaphanes sp2) and evaluated their enzyme characteristics. Our data show that the amino acid (AA) sequences of all three luciferases are highly conserved, including the identities (D. nubilus vs D. pectinealis: 99%; D. nubilus vs Diaphanes sp2: 98.5%; D. pectinealis vs Diaphanes sp2: 99.4%) and the protein structures. Three recombinant luciferases produced in vitro all possess significant luminescence activity at pH 7.8, and similar maximum emission spectrum (D. nubilus: 562 nm; D. pectinealis and Diaphanes sp2: 564 nm). They show the highest activity at 10 °C (D. pectinealis, Diaphanes sp2) and 15 °C (D. nubilus), and completely inactivation at 45 °C. Their KM for D-luciferin and ATP were 2.7 µM and 92 µM (D. nubilus), 3.7 µM and 49 µM (D. pectinealis), 3.5 µM and 46 µM (Diaphanes sp2). Phylogenetic analyses support that D. nubilus is sister to D. pectinealis with Diaphanes sp2 at their base, which further cluster with Pyrocoelia. All combined data indicate that sympatric Diaphanes species have similar luciferase characteristics, suggesting that other strategies (e.g., pheromone, active time, etc.) may be adopted to recognize mates. Our data provide new insights into Diaphanes luciferases and their evolution.

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.

Integrated Analysis of Transcriptome and Proteome to Reveal Pupal Color Switch in Papilio xuthus Butterflies.

Pupal color polyphenism in Papilio butterflies, including green, intermediate, or brown, is an excellent study system for understanding phenotypic plasticity. Previous studies suggested that development of brown pupae may be controlled by a hormone called pupal-cuticle-melanizing-hormone (PCMH) which is synthesized and secreted from brain-suboesophageal ganglion and prothoracic ganglion complexes (Br-SG-TG1) during the pre-pupa stage. However, detailed molecular mechanisms of neuroendocrine regulation in pupal color development remain unknown. In this study, we integrated the expression profiles of transcriptome and proteome at pre-pupa stages [2 h after gut purge (T1) and 3 h after forming the garter around the body (T2)] and pigmentation stages [10 h after ecdysis (T3) and 24 h after ecdysis (T4)] to identify important genes and pathways underlying the development of green and brown pupa in the swallowtail butterfly Papilio xuthus. Combined comparisons of each developmental stage and each tissue under green and brown conditions, a total of 1042 differentially expressed genes (DEGs) and 430 different abundance proteins (DAPs) were identified. Weighted gene co-expression network analysis (WGCNA) and enrichment analysis indicate that these DEGs were mainly related to oxidation-reduction, structural constituent of cuticle, and pigment binding. Soft clustering by Mfuzz and enrichment analysis indicate that these DAPs are mainly involved in tyrosine metabolism, insect hormone biosynthesis, and melanogenesis. By homologous alignment, we further identified those genes encoding neuropeptides (51), GPCRs (116), G-proteins (8), cuticular proteins (226), chitinases (16), and chitin deacetylases (8) in the whole genome of P. xuthus and analyzed their expression profiles. Although we identified no gene satisfying with hypothesized expression profile of PCMH, we found some genes in the neuropeptide cascade showed differentially expressed under two pupal color conditions. We also found that Toll signaling pathway genes, juvenile hormone (JH) related genes, and multiple cuticular proteins play important roles in the formation of selective pupal colors during the prepupal-pupal transition. Our data also suggest that both green and brown pupa include complex pigment system that is regulated by genes involved in black, blue, and yellow pigments. Our results provide important insights into the evolution of pupal protective colors among swallowtail butterflies.

Second Rhagophthalmid Luciferase Cloned from Chinese Glow-worm Menghuoius giganteus (Rhagophthalmidae: Elateroidea).

The pH-insensitive beetle luciferases cloned from Rhagophthalmidae, Phengodidae, and Elateridae exhibit great potential application as reporter assays for monitoring gene expression. At present, however, only one luciferase has been reported from the enigmatic and predominantly Asian distributed luminous family Rhagophthalmidae. Here, we cloned the second rhagophthalmid luciferase from the Chinese glow-worm Menghuoius giganteus (Rhagophthalmidae: Elateroidea) by combining reverse transcription polymerase chain reaction (RT-PCR) with rapid amplification of complementary DNA ends (RACE). The luciferase consisted of 546 amino acids and showed high identity to that of Rhagophthalmus ohbai (90.4%). The recombinant M. giganteus luciferase was produced in vitro and exhibited significant bioluminescent activity under neutral conditions (pH 7.8), with low KM for D-luciferin (2.2 µm) and ATP (53 µm). Activity was highest at 10°C and inactivation occurred at 45°C. This luciferase showed pH-insensitivity and maximum emission spectrum at 560 nm. Phylogenetic analyses based on the deduced amino acids indicated a close relationship between the M. giganteus luciferase and that of R. ohbai. These results increase our understanding of rhagophthalmid luciferases and provide a new resource for the application of luciferases.

Cloning and Characterization of Luciferase from the Chinese Firefly Lamprigera yunnana.

Lamprigera (Lampyridae) is a small genus with only 17 species distributing in Asian countries. Its larviform females and alate males can produce continuously strong yellow-green light at night. However, no luciferase gene was reported for this genus and its subfamily-level phylogenetic position still remains uncertain. Here, we cloned the luciferase gene from one Chinese species, Lamprigera yunnana, by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). This luciferase includes 551 deduced amino acids (AA) with the sequence identity of 71.8-76.8%, 67.5-70.7%, 68.4-75.3%, 77.8% and 59.5% to those from Lampyrinae, Luciolinae, Ototretinae, Cyphonocerinae and Photurinae, respectively. Phylogenetic analyses of deduced AA of luciferases suggest that Lamprigera locates outside Lampyrinae, in which it was originally placed in traditional taxonomy. The luciferase was produced in vitro as recombinant protein, and its biochemical properties were characterized. It possesses significant luminescence activity at pH 7.8, and its KM for D-luciferin and ATP are 61 µm and 122 µm, respectively. It shows the highest activity at 37°C and is completely inactivated at 55°C. It is pH-sensitive with the maximum emission spectrum of 566 nm at pH 7.8. Our data provide new insights into Lamprigera luciferase and its phylogenetic position.

Genome size of 14 species of fireflies (Insecta, Coleoptera, Lampyridae).

Eukaryotic genome size data are important both as the basis for comparative research into genome evolution and as estimators of the cost and difficulty of genome sequencing programs for non-model organisms. In this study, the genome size of 14 species of fireflies (Lampyridae) (two genera in Lampyrinae, three genera in Luciolinae, and one genus in subfamily incertae sedis) were estimated by propidium iodide (PI)-based flow cytometry. The haploid genome sizes of Lampyridae ranged from 0. 42 to 1. 31 pg, a 3. 1-fold span. Genome sizes of the fireflies varied within the tested subfamilies and genera. Lamprigera and Pyrocoelia species had large and small genome sizes, respectively. No correlation was found between genome size and morphological traits such as body length, body width, eye width, and antennal length. Our data provide additional information on genome size estimation of the firefly family Lampyridae. Furthermore, this study will help clarify the cost and difficulty of genome sequencing programs for non-model organisms and will help promote studies on firefly genome evolution.