The first chromosome-level genome of the stag beetle Dorcus hopei Saunders, 1854 (Coleoptera: Lucanidae).

Stag beetles (Coleoptera: Lucanidae) represent a significant saproxylic assemblage in forest ecosystems and are noted for their enlarged mandibles and male polymorphism. Despite their relevance as ideal models for the study of exaggerated mandibles that aid in attracting mates, the regulatory mechanisms associated with these traits remain understudied, and restricted by the lack of high-quality reference genomes for stag beetles. To address this limitation, we successfully assembled the first chromosome-level genome of a representative species Dorcus hopei. The genome was 496.58 Mb in length, with a scaffold N50 size of 54.61 Mb, BUSCO values of 99.8%, and 96.8% of scaffolds anchored to nine pairs of chromosomes. We identified 285.27 Mb (57.45%) of repeat sequences and annotated 11,231 protein-coding genes. This genome will be a valuable resource for further understanding the evolution and ecology of stag beetles, and provides a basis for studying the mechanisms of exaggerated mandibles through comparative analysis.

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.

Cloning and characterization of luciferase from an Asian firefly Pygoluciola qingyu and its comparison with other beetle luciferases.

The bioluminescence system of luminescent beetles has extensive applications in biological imaging, protein labeling and drug screening. To explore wild luciferases with excellent catalytic activity and thermal stability, we cloned the luciferase of Pygoluciola qingyu, one species living in areas of high temperature and with strong bioluminescence, by combining transcriptomic sequencing and reverse transcription polymerase chain reaction (RT-PCR). The total length of luciferase gene is 1638 bp and the luciferase consists 544 amino acids. The recombinant P. qingyu luciferase was produced in vitro and its characteristics were compared with those of eight luciferases from China firefly species and two commercial luciferases. Compared with these luciferases, the P. qingyu luciferase shows the highest luminescence activity at room temperature (about 25-28 â„ƒ) with similar KM value for D-luciferin and ATP to the Photinus pyralis luciferase. The P. qingyu luciferase activity was highest at 35 â„ƒ and can keep high activity at 30-40 â„ƒ, which suggests the potential of P. qingyu luciferase for in vivo and cell application. Our results provide new insights into P. qingyu luciferase and give a new resource for the application of luciferases.

Multiple Origins of Bioluminescence in Beetles and Evolution of Luciferase Function.

Bioluminescence in beetles has long fascinated biologists, with diverse applications in biotechnology. To date, however, our understanding of its evolutionary origin and functional variation mechanisms remains poor. To address these questions, we obtained high-quality reference genomes of luminous and nonluminous beetles in 6 Elateroidea families. We then reconstructed a robust phylogenetic relationship for all luminous families and related nonluminous families. Comparative genomic analyses and biochemical functional experiments suggested that gene evolution within Elateroidea played a crucial role in the origin of bioluminescence, with multiple parallel origins observed in the luminous beetle families. While most luciferase-like proteins exhibited a conserved nonluminous amino acid pattern (TLA346 to 348) in the luciferin-binding sites, luciferases in the different luminous beetle families showed divergent luminous patterns at these sites (TSA/CCA/CSA/LVA). Comparisons of the structural and enzymatic properties of ancestral, extant, and site-directed mutant luciferases further reinforced the important role of these sites in the trade-off between acyl-CoA synthetase and luciferase activities. Furthermore, the evolution of bioluminescent color demonstrated a tendency toward hypsochromic shifts and variations among the luminous families. Taken together, our results revealed multiple parallel origins of bioluminescence and functional divergence within the beetle bioluminescent system.

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.

Complete mitochondrial genome of Pectocera sp. (Elateridae: Dendrometrinae: Oxynopterini) and its phylogenetic implications.

The Elateridae family (click beetles) represents a highly diverse lineage that possesses a specialized clicking mechanism to startle predators. At present, however, phylogenetic relationships, especially among recognized subfamilies, remain contentious. Mitochondrial genomes (mitogenomes) can help resolve previously intractable phylogenetic relationships using morphological or limited molecular data. Here, we report the complete mitogenome of Pectocera sp. (Elateridae: Dendrometrinae: Oxynopterini), which was 15,962 bp in length and showed a typical gene number and order as most beetle mitogenomes, including 13 protein-coding genes (PCGs), 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 noncoding control region (AT-rich region). Comparative genomic analyses showed a high degree of feature similarity among Pectocera sp. and other click beetles. Evolutionary analysis of all PCGs based on the nonsynonymous/synonymous substitution rate ratio (ω) indicated that cox1 and atp8 exhibited the lowest and highest evolutionary rates, respectively, and that the evolutionary rates of all PCGs, except for cox3, nad2, and nad3, were lower than the average ω of click beetles. Phylogenetic analyses based on concatenated and coalescent approaches indicated that Pectocera sp. was sister to Campsosternus auratus in the same tribe (Oxynopterini) with high support. This study offers insight into the mitogenomic basis of Pectocera sp. and provides an important data resource for exploring the taxonomy, phylogeny, and evolution of click beetles.

Genomic insights into the secondary aquatic transition of penguins.

Penguins lost the ability to fly more than 60 million years ago, subsequently evolving a hyper-specialized marine body plan. Within the framework of a genome-scale, fossil-inclusive phylogeny, we identify key geological events that shaped penguin diversification and genomic signatures consistent with widespread refugia/recolonization during major climate oscillations. We further identify a suite of genes potentially underpinning adaptations related to thermoregulation, oxygenation, diving, vision, diet, immunity and body size, which might have facilitated their remarkable secondary transition to an aquatic ecology. Our analyses indicate that penguins and their sister group (Procellariiformes) have the lowest evolutionary rates yet detected in birds. Together, these findings help improve our understanding of how penguins have transitioned to the marine environment, successfully colonizing some of the most extreme environments on Earth.

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.

Complete mitogenome and phylogenetic significance of Metoecus javanus (Pic, 1913) (Coleoptera: Ripiphoridae) from Southwest China, with notes on morphological traits of adult and immature stages.

Wedge-shaped beetles (Ripiphoridae) not only exhibit enigmatic morphological and biological traits but also disputable phylogenetic positions. At present, however, genetic information regarding this family remains limited. In this study, we report on the complete mitogenome of one ripiphorid beetle, Metoecus javanus (Pic, 1913), from Southwest China, as well as its different developmental stages, populations, and morphological variability. The complete mitogenome of M. javanus was 16 109 bp in length, containing 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and a noncoding control region. Of the 37 genes, 23 were located on the majority strand (J-strand) and 14 were located on the minority strand (N-strand). All PCGs started with "ATN" (N represents A, T, G, and C), and terminated with "TAA", except for NAD1 with "TAG" and COX2 with a single "T". The five most used codons in the PCGs were UUA(L), UCU(S2), CCU(P), UCA(S2), and GGA(G), indicating a strong bias toward A + T-rich codons. All 22 tRNAs showed typical cloverleaf structures, except trnS1, which lacked a dihydrouridine (DHU) stem. The control region contained five types of tandem repeats, with the repeat units ranging from 17 to 24 bp. Phylogenetic analysis of the concatenated set of 13 PCGs and two rRNAs (rrnL and rrnS) of M. javanus and 17 other Tenebrionoidea species indicated that M. javanus did not cluster with Pelecotoma fennica (Ripiphoridae: Pelecotominae), another wedge-shaped beetle, but was located at the base of the Mordellidae + P. fennica clade. This reconstruction supported the paraphyly of Ripiphoridae with respect to Mordellidae. Using the mitogenome COX1 data, wedge-shaped beetles from different stages (male adult, female adult, and pupa), different geographical populations (Nujiang and Lincang), and different wasp hosts (Vespidae: Vespa velutina Lepeletier, 1836 and Vespa bicolor Fabricius, 1787) were identified as a same species (i.e., M. javanus). Based on morphological observations of all specimens, we identified and described variability in the adult pronotum, male genitalia, and pupa of M. javanus. The present results provide important genetic and morphological information for further investigations on the phylogenetic position of Ripiphoridae and its evolutionary diversity.

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.

The mitochondrial genome of a leaf insect Phyllium westwoodii (Phasmatodea: Phylliidae) in Southeast Asia.

The nearly complete mitochondrial genome (mitogenome) of Phyllium westwoodii, a typical leaf mimic insect in Phasmatodea, was obtained in this study. This mitogenome is 17,222 bp in length and contains 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and almost complete control regions. All PCGs initiate with 'ATN' except for NAD4L that uses 'TTG' as the start codon, and terminate with 'TAA' except for COX2 that uses a single 'T' residue as the stop codon. The phylogenetic analysis based on the concatenated sequences of 13 PCGs and two rRNAs shows that P. westwoodii is closer to Phyllium tibetense than Phyllium giganteum.

Three new species of emLamprigera/em Motschulsky (Coleoptera, Lampyridae) from China, with notes on known species.

Lamprigera is found only in those countries from the Himalaya-Karakoran -Tibet region to SE Asia where 17 species have been previously recorded. These 17 include four species from China. In this work, combined molecular data (COI) and morphological traits identified eight species in our collections. Among these, we found three Chinese species (Lamprigera alticola Dong Li, sp. nov., Lamprigera luquanensis Dong Li, sp. nov. and Lamprigera magnapronotum Dong Li, sp. nov.) that are new to science, bringing the total number of species of Lamprigera to 20 (17+3), and four other known species that are herein newly recorded for the first time in China. These four new records, the three new species, and the four previously known records bring the total number of Chinese species to 11. The morphological traits, especially the male genitalia and pronotum, are described for all eight species. We conclude that male genitalia and pronotum are the most important diagnostic traits for separating species of Lamprigera, and this is confirmed by COI data.

The mitochondrial genome of one 'twisted-wing parasite' Xenos cf. moutoni (Insecta, Strepsiptera, Xenidae) from Gaoligong Mountains, Southwest of China.

The nearly complete mitochondrial genome (mitogenome) of Xenos cf. moutoni, one twisted-wing parasite on wasp Vespa velutina from Southwest of China, is described in this study. The total length of this mitogenome is 16,717 bp, containing 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and an incomplete A + T-rich control region . All of the 13 PCGs are initiated with canonical ATN (N represents A, T, G, C) as start codons; 8 PCGs are terminated with a complete typical stop codon TAA, and the remaining five PCGs (cox2, cox3, nad3, nad4 and nad5) have an incomplete stop codon with just a T. The phylogenetic analysis based on the nucleotide sequences of PCGs and rRNAs indicates that Xenos cf. moutoni has a close relationship with Xenos vesparum, confirming its placement in the family Xenidae.

Genome-wide identification and gene-editing of pigment transporter genes in the swallowtail butterfly Papilio xuthus.

BACKGROUND: Insect body coloration often functions as camouflage to survive from predators or mate selection. Transportation of pigment precursors or related metabolites from cytoplasm to subcellular pigment granules is one of the key steps in insect pigmentation and usually executed via such transporter proteins as the ATP-binding cassette (ABC) transmembrane transporters and small G-proteins (e.g. Rab protein). However, little is known about the copy numbers of pigment transporter genes in the butterfly genomes and about the roles of pigment transporters in the development of swallowtail butterflies. RESULTS: Here, we have identified 56 ABC transporters and 58 Rab members in the genome of swallowtail butterfly Papilio xuthus. This is the first case of genome-wide gene copy number identification of ABC transporters in swallowtail butterflies and Rab family in lepidopteran insects. Aiming to investigate the contribution of the five genes which are orthologous to well-studied pigment transporters (ABCG: white, scarlet, brown and ok; Rab: lightoid) of fruit fly or silkworm during the development of swallowtail butterflies, we performed CRISPR/Cas9 gene-editing of these genes using P. xuthus as a model and sequenced the transcriptomes of their morphological mutants. Our results indicate that the disruption of each gene produced mutated phenotypes in the colors of larvae (cuticle, testis) and/or adult eyes in G0 individuals but have no effect on wing color. The transcriptomic data demonstrated that mutations induced by CRISPR/Cas9 can lead to the accumulation of abnormal transcripts and the decrease or dosage compensation of normal transcripts at gene expression level. Comparative transcriptomes revealed 606 ~ 772 differentially expressed genes (DEGs) in the mutants of four ABCG transporters and 1443 DEGs in the mutants of lightoid. GO and KEGG enrichment analysis showed that DEGs in ABCG transporter mutants enriched to the oxidoreductase activity, heme binding, iron ion binding process possibly related to the color display, and DEGs in lightoid mutants are enriched in glycoprotein binding and protein kinases. CONCLUSIONS: Our data indicated these transporter proteins play an important role in body color of P. xuthus. Our study provides new insights into the function of ABC transporters and small G-proteins in the morphological development of butterflies.

African lungfish genome sheds light on the vertebrate water-to-land transition.

Lungfishes are the closest extant relatives of tetrapods and preserve ancestral traits linked with the water-to-land transition. However, their huge genome sizes have hindered understanding of this key transition in evolution. Here, we report a 40-Gb chromosome-level assembly of the African lungfish (Protopterus annectens) genome, which is the largest genome assembly ever reported and has a contig and chromosome N50 of 1.60 Mb and 2.81 Gb, respectively. The large size of the lungfish genome is due mainly to retrotransposons. Genes with ultra-long length show similar expression levels to other genes, indicating that lungfishes have evolved high transcription efficacy to keep gene expression balanced. Together with transcriptome and experimental data, we identified potential genes and regulatory elements related to such terrestrial adaptation traits as pulmonary surfactant, anxiolytic ability, pentadactyl limbs, and pharyngeal remodeling. Our results provide insights and key resources for understanding the evolutionary pathway leading from fishes to humans.

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.

High-quality chromosome-level genome assembly and full-length transcriptome analysis of the pharaoh ant Monomorium pharaonis.

BACKGROUND: Ants with complex societies have fascinated scientists for centuries. Comparative genomic and transcriptomic analyses across ant species and castes have revealed important insights into the molecular mechanisms underlying ant caste differentiation. However, most current ant genomes and transcriptomes are highly fragmented and incomplete, which hinders our understanding of the molecular basis for complex ant societies. FINDINGS: By hybridizing Illumina, Pacific Biosciences, and Hi-C sequencing technologies, we de novo assembled a chromosome-level genome for Monomorium pharaonis, with a scaffold N50 of 27.2 Mb. Our new assembly provides better resolution for the discovery of genome rearrangement events at the chromosome level. Analysis of full-length isoform sequencing (ISO-seq) suggested that ∼15 Gb of ISO-seq data were sufficient to cover most expressed genes, but the number of transcript isoforms steadily increased with sequencing data coverage. Our high-depth ISO-seq data greatly improved the quality of gene annotation and enabled the accurate detection of alternative splicing isoforms in different castes of M. pharaonis. Comparative transcriptome analysis across castes based on the ISO-seq data revealed an unprecedented number of transcript isoforms, including many caste-specific isoforms. We also identified a number of conserved long non-coding RNAs that evolved specifically in ant lineages and several that were conserved across insect lineages. CONCLUSIONS: We produced a high-quality chromosome-level genome for M. pharaonis, which significantly improved previous short-read assemblies. Together with full-length transcriptomes for all castes, we generated a highly accurate annotation for this ant species. These long-read sequencing results provide a useful resource for future functional studies on the genetic mechanisms underlying the evolution of social behaviors and organization in ants.