Insights from different reproductive gene knockdowns via RNA interference in the lady beetle Eriopis connexa: Establishing a new model for molecular studies on natural enemies.

Insect pest control can be achieved by the application of RNA interference (RNAi), a key molecular tool in functional genomics. Whereas most RNAi research has focused on insect pests, few studies have been performed on natural enemies. Validating the efficacy of RNAi in natural enemies is crucial for assessing its safety and enabling molecular research on these organisms. Here, we assessed the efficacy of RNAi in the ladybird beetle Eriopis connexa Germar (Coleoptera: Coccinellidae), focusing on genes related to reproduction, such as vitellogenin (Vg) and its receptor (VgR). In the transcriptome of E. connexa, we found one VgR (EcVgR) and two Vg genes (EcVg1 and EcVg2). These genes have been validated by in silico analyses of functional domains and evolutionary relationships. Five-day-old females were injected with 500 ng/µL of a specific double-stranded RNA (dsRNA) (dsEcVg1, dsEcVg2, or dsEcVgR) for RNAi tests, while nonspecific dsRNA (dsGFP or dsAgCE8.1) was used as a control. Interestingly, dsEcVg2 was able to knockdown both Vg genes, while dsEcVg1 could silence only EcVg1. Additionally, the viability of the eggs was significantly reduced when both Vg genes were knocked down at the same time (after treatment with dsEcVg2 or "dsEcVg1+dsEcVg2"). Ultimately, malformed, nonviable eggs were produced when EcVgR was silenced. Interestingly, no dsRNA treatment had an impact on the quantity of eggs laid. Therefore, the feasibility of RNAi in E. connexa has been confirmed, suggesting that this coccinellid is an excellent Neotropical model for molecular research on natural enemies and for studying RNAi nontarget effects.

Functional studies of McSTE24, McCYP305a1, and McJHEH, three essential genes act in cantharidin biosynthesis in the blister beetle (Coleoptera: Meloidae).

Cantharidin is a toxic defensive substance secreted by most blister beetles when attacked. It has been used to treat many complex diseases since ancient times and has recently regained popularity as an anticancer agent. However, the detailed mechanism of the cantharidin biosynthesis has not been completely addressed. In this study, we cloned McSTE24 (encoding STE24 endopeptidase) from terpenoid backbone pathway, McCYP305a1 (encoding cytochrome P450, family 305) and McJHEH [encoding subfamily A, polypeptide 1 and juvenile hormone (JH) epoxide hydrolase] associated to JH synthesis/degradation in the blister beetle Mylabris cichorii (Linnaeus, 1758, Coleoptera: Meloidae). Expression pattern analyses across developmental stages in adult males revealed that the expressions of 3 transcripts were closely linked to cantharidin titer exclusively during the peak period of cantharidin synthesis (20-25 days old). In contrast, at other stages, these genes may primarily regulate different biological processes. When RNA interference with double-stranded RNA suppressed the expressions of the 3 genes individually, significant reductions in cantharidin production were observed in males and also in females following McJHEH knockdown, indicating that these 3 genes might primarily contribute to cantharidin biosynthesis in males, but not in females, while females could self-synthesis a small amount of cantharidin. These findings support the previously hypothesized sexual dimorphism in cantharidin biosynthesis during the adult phase. McCYP305a1 collaborates with its upstream gene McSTE24 in cantharidin biosynthesis, while McJHEH independently regulates cantharidin biosynthesis in males.

Establishing Silphids in the invertebrate DNA toolbox: a proof of concept.

Environmental DNA (eDNA) analyses are an increasingly popular tool for assessing biodiversity. eDNA sampling that uses invertebrates, or invertebrate DNA (iDNA), has become a more common method in mammal biodiversity studies where biodiversity is assessed via diet analysis of different coprophagous or hematophagous invertebrates. The carrion feeding family of beetles (Silphidae: Coleoptera, Latreille (1807)), have not yet been established as a viable iDNA source in primary scientific literature, yet could be useful indicators for tracking biodiversity in forested ecosystems. Silphids find carcasses of varying size for both food and reproduction, with some species having host preference for small mammals; therefore, iDNA Silphid studies could potentially target small mammal communities. To establish the first valid use of iDNA methods to detect Silphid diets, we conducted a study with the objective of testing the validity of iDNA methods applied to Silphids using both Sanger sequencing and high throughput Illumina sequencing. Beetles were collected using inexpensive pitfall traps in Alberta, Michigan in 2019 and 2022. We successfully sequenced diet DNA and environmental DNA from externally swabbed Silphid samples and diet DNA from gut dissections, confirming their potential as an iDNA tool in mammalian studies. Our results demonstrate the usefulness of Silphids for iDNA research where we detected species from the genera Anaxyrus, Blarina, Procyon, Condylura, Peromyscus, Canis, and Bos. Our results highlight the potential for Silphid iDNA to be used in future wildlife surveys.

A chromosome-level genome assembly of Prosopocoilus inquinatus Westwood, 1848 (Coleoptera: Lucanidae).

Lucanidae (Coleoptera: Scarabaeidae) are fascinating beetles exhibiting significant dimorphism and are widely used as beetle evolutionary study models. However, lacking high-quality genomes prohibits our understanding of Lucanidae. Herein, we proposed a chromosome-level genome assembly of a widespread species, Prosopocoilus inquinatus, combining PacBio HiFi, Illumina, and Hi-C data. The genome size reaches 649.73 Mb, having the scaffold N50 size of 59.50 Mb, and 99.6% (647.13 Mb) of the assembly successfully anchored on 12 chromosomes. The BUSCO analysis of the genome exhibits a completeness of 99.6% (n = 1,367), including 1,362 (98.5%) single-copy BUSCOs and 15 (1.1%) duplicated BUSCOs. The genome annotation identifies that the genome contains 61.41% repeat elements and 13,452 predicted protein-coding genes. This high-quality Lucanidae genome provides treasured genomic information to our knowledge of stag beetles.

Cloning and functional analysis of the juvenile hormone receptor gene CsMet in Coccinella septempunctata.

The potential role of the juvenile hormone receptor gene (methoprene-tolerant, Met) in reproduction of Coccinella septempunctata L. (Coleoptera: Coccinellidae)(Coleoptera: Coccinellidae), was investigated by cloning, analyzing expression profiles by quantitative real-time PCR, and via RNA interference (RNAi). CsMet encoded a 1518-bp open reading frames with a predicted protein product of 505 amino acids; the latter contained 2 Per-Arnt-Sim repeat profile at amino acid residues 30-83 and 102-175. CsMet was expressed in different C. septempunctata larvae developmental stages and was most highly expressed in third instar. CsMet expression in female adults gradually increased from 20 to 30 d, and expression levels at 25 and 30 d were significantly higher than levels at 1-15 d. CsMet expression in 20-d-old male adults was significantly higher than in males aged 1-15 d. CsMet expression levels in fat body tissues of male and female adults were significantly higher than expression in the head, thorax, and reproductive system. At 5 and 10 d after CsMet-dsRNA injection, CsMet expression was significantly lower than the controls by 75.05% and 58.38%, respectively. Ovary development and vitellogenesis in C. septempunctata injected with CsMet-dsRNA were significantly delayed and fewer mature eggs were produced. This study provides valuable information for the large-scale rearing of C. septempunctata.

The integral role of de novo lipogenesis in the preparation for seasonal dormancy.

Animals can alter their body compositions in anticipation of dormancy to endure seasons with limited food availability. Accumulation of lipid reserves, mostly in the form of triglycerides (TAGs), is observed during the preparation for dormancy in diverse animals, including insects (diapause) and mammals (hibernation). However, the mechanisms involved in the regulation of lipid accumulation and the ecological consequences of failure to accumulate adequate lipid stores in preparation for animal dormancy remain understudied. In the broadest sense, lipid reserves can be accumulated in two ways: the animal either receives lipids directly from the environment or converts the sugars and amino acids present in food to fatty acids through de novo lipogenesis and then to TAGs. Here, we show that preparation for diapause in the Colorado potato beetle (Leptinotarsa decemlineata) involves orchestrated upregulation of genes involved in lipid metabolism with a transcript peak in 8- and 10-d-old diapause-destined insects. Regulation at the transcript abundance level was associated with the accumulation of substantial fat stores. Furthermore, the knockdown of de novo lipogenesis enzymes (ACCase and FAS-1) prolonged the preparatory phase, while the knockdown of fatty acid transportation genes shortened the preparatory phase. Our findings suggest a model in which the insects dynamically decide when to transition from the preparation phase into diapause, depending on the progress in lipid accumulation through de novo lipogenesis.

Chromosome-level genome assembly of an oligophagous leaf beetle Ophraella communa (Coleoptera: Chrysomelidae).

The leaf beetle Ophraella communa LeSage (Coleoptera: Chrysomelidae) is an effective biological control agent of the common ragweed. Here, we assembled a chromosome-level genome of the O. communa by combining Illumina, Nanopore, and Hi-C sequencing technologies. The genome size of the final genome assembly is 733.1 Mb, encompassing 17 chromosomes, with an improved contig N50 of 7.05 Mb compared to the original version. Genome annotation reveals 25,873 protein-coding genes, with functional annotations available for 22,084 genes (85.35%). Non-coding sequence annotation identified 204 rRNAs, 626 tRNAs, and 1791 small RNAs. Repetitive elements occupy 414.41 Mb, constituting 57.76% of the genome. This high-quality genome is fundamental for advancing biological control strategies employing O. communa.

Climate change will lead to range shifts and genetic diversity losses of dung beetles in the Gobi Desert and Mongolian Steppe.

Desertification is known to be a major threat to biodiversity, yet our understanding of the consequent decline in biodiversity remains insufficient. Here, we predicted climate change-induced range shifts and genetic diversity losses in three model dung beetles: Colobopterus erraticus, Cheironitis eumenes, and Gymnopleurus mopsus, distributed across the Gobi Desert and Mongolian Steppe, areas known for desertification. Phylogeographic analyses of mitochondrial COI sequences and species distribution modeling, based on extensive field investigations spanning 14 years, were performed. Species confined to a single biome were predicted to contract and shift their distribution in response to climate change, whereas widespread species was predicted to expand even if affected by range shifts. We indicated that all species are expected to experience significant haplotype losses, yet the presence of high singleton frequencies and low genetic divergence across geographic configurations and lineages mitigate loss of genetic diversity. Notably, Cheironitis eumenes, a desert species with low genetic diversity, appears to be the most vulnerable to climate change due to the extensive degradation in the Gobi Desert. This is the first study to predict the response of insects to desertification in the Gobi Desert. Our findings highlight that dung beetles in the Gobi Desert and Mongolian Steppe might experience high rates of occupancy turnover and genetic loss, which could reshuffle the species composition.

High-quality reference genome of cowpea beetle Callosobruchus maculatus.

Callosobruchus maculatus is one of the most competitive stored grain pests, which causes a great loss to agricultural economy. However, due to an inadequacy of high-quality reference genome, the molecular mechanisms for olfactory and hypoxic adaptations to stored environments are unknown and require to be revealed urgently, which will contribute to the detection and prevention of the invasive pests C. maculatus. Here, we presented a high-quality chromosome-level genome of C. maculatus based on Illumina, Nanopore and Hi-C sequencing data. The total size was 1.2 Gb, and 65.17% (797.47 Mb) of it was identified to be repeat sequences. Among assembled chromosomes, chromosome 10 was considered the X chromosome according to the evidence of reads coverage and homologous genes among species. The current version of high-quality genome provides preferable data resources for the adaptive evolution research of C. maculatus.

The genomic and cellular basis of biosynthetic innovation in rove beetles.

How evolution at the cellular level potentiates macroevolutionary change is central to understanding biological diversification. The >66,000 rove beetle species (Staphylinidae) form the largest metazoan family. Combining genomic and cell type transcriptomic insights spanning the largest clade, Aleocharinae, we retrace evolution of two cell types comprising a defensive gland-a putative catalyst behind staphylinid megadiversity. We identify molecular evolutionary steps leading to benzoquinone production by one cell type via a mechanism convergent with plant toxin release systems, and synthesis by the second cell type of a solvent that weaponizes the total secretion. This cooperative system has been conserved since the Early Cretaceous as Aleocharinae radiated into tens of thousands of lineages. Reprogramming each cell type yielded biochemical novelties enabling ecological specialization-most dramatically in symbionts that infiltrate social insect colonies via host-manipulating secretions. Our findings uncover cell type evolutionary processes underlying the origin and evolvability of a beetle chemical innovation.

Comparative Genomic Analysis of the Pattern of Evolution of Male and Female Reproductive Proteins in Seed Beetles.

Male seminal fluid proteins often show signs of positive selection and divergent evolution, believed to reflect male-female coevolution. Yet, our understanding of the predicted concerted evolution of seminal fluid proteins and female reproductive proteins is limited. We sequenced, assembled, and annotated the genome of two species of seed beetles allowing a comparative analysis of four closely related species of these herbivorous insects. We compare the general pattern of evolution in genes encoding seminal fluid proteins and female reproductive proteins with those in digestive protein genes and well-conserved reference genes. We found that female reproductive proteins showed an overall ratio of nonsynonymous to synonymous substitutions (ω) similar to that of conserved genes, while seminal fluid proteins and digestive proteins exhibited higher overall ω values. Further, seminal fluid proteins and digestive proteins showed a higher proportion of sites putatively under positive selection, and explicit tests showed no difference in relaxed selection between protein types. Evolutionary rate covariation analyses showed that evolutionary rates among seminal fluid proteins were on average more closely correlated with those in female reproductive proteins than with either digestive or conserved genes. Gene expression showed the expected negative covariation with ω values, except for male-biased genes where this negative relationship was reversed. In conclusion, seminal fluid proteins showed relatively rapid evolution and signs of positive selection. In contrast, female reproductive proteins evolved at a lower rate under selective constraints, on par with genes known to be well conserved. Although our findings provide support for concerted evolution of seminal fluid proteins and female reproductive proteins, they also suggest that these two classes of proteins evolve under partly distinct selective regimes.

Coevolution of longevity and female germline maintenance.

An often-overlooked aspect of life-history optimization is the allocation of resources to protect the germline and secure safe transmission of genetic information. While failure to do so renders significant fitness consequences in future generations, germline maintenance comes with substantial costs. Thus, germline allocation should trade off with other life-history decisions and be optimized in accordance with an organism's reproductive schedule. Here, we tested this hypothesis by studying germline maintenance in lines of seed beetle, selected for early (E) or late (L) reproduction for 350 and 240 generations, respectively. Female animals provide maintenance and screening of male gametes in their reproductive tract and oocytes. Here, we reveal the ability of young and aged E- and L-females to provide this form of germline maintenance by mating them to males with ejaculates with artificially elevated levels of protein and DNA damage. We find that germline maintenance in E-females peaks at young age and then declines, while the opposite is true for L-females, in accordance with the age of reproduction in the respective regime. These findings identify the central role of allocation to secure germline integrity in life-history evolution and highlight how females can play a crucial role in mitigating the effects of male germline decisions on mutation rate and offspring quality.

Evolutionary radiation strategy revealed in the Scarabaeidae with evidence of continuous spatiotemporal morphology and phylogenesis.

Evolutionary biology faces the important challenge of determining how to interpret the relationship between selection pressures and evolutionary radiation. The lack of morphological evidence on cross-species research adds to difficulty of this challenge. We proposed a new paradigm for evaluating the evolution of branches through changes in characters on continuous spatiotemporal scales, for better interpreting the impact of biotic/abiotic drivers on the evolutionary radiation. It reveals a causal link between morphological changes and selective pressures: consistent deformation signals for all tested characters on timeline, which provided strong support for the evolutionary hypothesis of relationship between scarabs and biotic/abiotic drivers; the evolutionary strategies under niche differentiation, which were manifested in the responsiveness degree of functional morphological characters with different selection pressure. This morphological information-driven integrative approach sheds light on the mechanism of macroevolution under different selection pressures and is applicable to more biodiversity research.

Identification of candidate chemosensory genes in the antennal transcriptome of Monolepta signata.

In the polyphagous insect Monolepta signata (M. signata) (Coleoptera: Chrysomelidae), antennae are important for olfactory reception used during feeding, mating, and finding a suitable oviposition site. Based on NextSeq 6000 Illumina sequencing, we assembled the antennal transcriptome of mated M. signata and described the first chemosensory gene repertoire expressed in this species. The relative expression levels of some significant chemosensory genes were conducted by quantitative real-time PCR. We identified 114 olfactory-related genes based on the antennal transcriptome database of M. signata, including 21 odorant binding proteins (OBPs), six chemosensory proteins (CSPs), 46 odorant receptors (ORs), 15 ionotropic receptors (IRs), 23 gustatory receptors (GRs) and three sensory neuron membrane proteins (SNMPs). Blastp best hit and phylogenetic analyses showed that most of the chemosensory genes had a close relationship with orthologs from other Coleoptera species. Overall, this study provides a foundation for elucidating the molecular mechanism of olfactory recognition in M. signata as well as a reference for the study of chemosensory genes in other species of Coleoptera.

StaufenC facilitates utilization of the ERAD pathway to transport dsRNA through the endoplasmic reticulum to the cytosol.

RNA interference (RNAi) is more efficient in coleopteran insects than other insects. StaufenC (StauC), a coleopteran-specific double-stranded RNA (dsRNA)-binding protein, is required for efficient RNAi in coleopterans. We investigated the function of StauC in the intracellular transport of dsRNA into the cytosol, where dsRNA is digested by Dicer enzymes and recruited by Argonauts to RNA-induced silencing complexes. Confocal microscopy and cellular organelle fractionation studies have shown that dsRNA is trafficked through the endoplasmic reticulum (ER) in coleopteran Colorado potato beetle (CPB) cells. StauC is localized to the ER in CPB cells, and StauC-knockdown caused the accumulation of dsRNA in the ER and a decrease in the cytosol, suggesting that StauC plays a key role in the intracellular transport of dsRNA through the ER. Using immunoprecipitation, we showed that StauC is required for dsRNA interaction with ER proteins in the ER-associated protein degradation (ERAD) pathway, and these interactions are required for RNAi in CPB cells. These results suggest that StauC works with the ERAD pathway to transport dsRNA through the ER to the cytosol. This information could be used to develop dsRNA delivery methods aimed at improving RNAi.

The South American genus Cnemalobus (Coleoptera: Carabidae: Cnemalobini): phylogeny and biogeographic analysis with the description of four new species from extra-Andean Patagonian mountains.

The carabid beetle Cnemalobus Guérin-Ménéville, 1838 inhabits high- and lowland grasslands of southern South America. The highest diversity is found in the Patagonian Steppe, where distribution patterns are associated with latitude and elevation. Northern Patagonia, a large volcanic region with a complex geoclimatic history, exhibits elevated grades of endemism. However, a great deal remains unknown regarding diversification and biogeographical patterns for most of the endemic groups. We describe new Cnemalobus species restricted to isolated volcanoes from these extra-Andean mountain systems. We assess the phylogenetic relationships by updating the phylogeny of the genus and conduct a Bayesian binary Markov chain-Monte Carlo (MCMC) analysis on the resulting phylogenetic tree to discuss the biogeographical distribution patterns. We also provide a taxonomic key to all currently known species of Cnemalobus from the Patagonian Steppe. Our phylogenetic analysis supports the monophyly of the new species Cnemalobus tromen sp. nov., Cnemalobus silviae sp. nov., Cnemalobus aucamahuida sp. nov. and Cnemalobus domuyo sp. nov. grouped with C. diamante and C. nevado , referred to as the 'Extra-Andean' mountain lineage. Biogeographical analysis recognises vicariant events as the most plausible explanation for the allopatric distributions of the new species. We hypothesise that these vicariant events could be related to climatic barriers that likely promoted speciation processes by generating geographical isolation in ancestral populations. Our findings contribute significantly to the biogeographical understanding of the Patagonian volcanic region, prompting new inquiries to unravel the speciation processes of the endemic biota in extra-Andean mountain systems. ZooBank: urn:lsid:zoobank.org:pub:6A7585E8-5006-45BC-A1A3-F874F18A6049.

Odd-Paired is Involved in Morphological Divergence of Snail-Feeding Beetles.

Body shape and size diversity and their evolutionary rates correlate with species richness at the macroevolutionary scale. However, the molecular genetic mechanisms underlying the morphological diversification across related species are poorly understood. In beetles, which account for one-fourth of the known species, adaptation to different trophic niches through morphological diversification appears to have contributed to species radiation. Here, we explored the key genes for the morphological divergence of the slender to stout body shape related to divergent feeding methods on large to small snails within the genus Carabus. We show that the zinc-finger transcription factor encoded by odd-paired (opa) controls morphological variation in the snail-feeding ground beetle Carabus blaptoides. Specifically, opa was identified as the gene underlying the slender to stout morphological difference between subspecies through genetic mapping and functional analysis via gene knockdown. Further analyses revealed that changes in opa cis-regulatory sequences likely contributed to the differences in body shape and size between C. blaptoides subspecies. Among opa cis-regulatory sequences, single nucleotide polymorphisms on the transcription factor binding sites may be associated with the morphological differences between C. blaptoides subspecies. opa was highly conserved in a wide range of taxa, especially in beetles. Therefore, opa may play an important role in adaptive morphological divergence in beetles.

Evidence for Admixture and Rapid Evolution During Glacial Climate Change in an Alpine Specialist.

The pace of current climate change is expected to be problematic for alpine flora and fauna, as their adaptive capacity may be limited by small population size. Yet, despite substantial genetic drift following post-glacial recolonization of alpine habitats, alpine species are notable for their success surviving in highly heterogeneous environments. Population genomic analyses demonstrating how alpine species have adapted to novel environments with limited genetic diversity remain rare, yet are important in understanding the potential for species to respond to contemporary climate change. In this study, we explored the evolutionary history of alpine ground beetles in the Nebria ingens complex, including the demographic and adaptive changes that followed the last glacier retreat. We first tested alternative models of evolutionary divergence in the species complex. Using millions of genome-wide SNP markers from hundreds of beetles, we found evidence that the N. ingens complex has been formed by past admixture of lineages responding to glacial cycles. Recolonization of alpine sites involved a distributional range shift to higher elevation, which was accompanied by a reduction in suitable habitat and the emergence of complex spatial genetic structure. We tested several possible genetic pathways involved in adaptation to heterogeneous local environments using genome scan and genotype-environment association approaches. From the identified genes, we found enriched functions associated with abiotic stress responses, with strong evidence for adaptation to hypoxia-related pathways. The results demonstrate that despite rapid demographic change, alpine beetles in the N. ingens complex underwent rapid physiological evolution.

A Farnesyl Pyrophosphate Synthase Gene Is Expressed in Fat Body Regulates Cantharidin Synthesis in Male Epicauta impressicornis Blister Beetle.

Blister beetles of Epicauta impressicornis have attracted attention because they contain a large amount of cantharidin (CTD). To date, however, the synthesis and transfer of CTD in adults of E. impressicornis are largely unknown. Here, we showed that the larvae E. impressicornis are capable of synthesizing CTD and they consume CTD during pupation. Before sexual maturity, both male and female adults synthesized a small amount of CTD, while after sexual maturity, males produced larger amounts of CTD, but females did not. The newly synthesized CTD in males first appeared in the hemolymph and then accumulated in the reproductive system. During the mating, the males transferred CTD to the reproductive system of females. In addition, a farnesyl pyrophosphate synthase (FPPS) gene was identified in male E. impressicornis. RNA-seq analysis, quantitative RT-PCR, and RNA interference analyses were conducted to investigate expression patterns and the functional roles of E. impressicornis FPPS (EiFPPS). Our results indicate that EiFPPS is highly expressed in the fat body of males. Moreover, the knock-down of EiFPPS led to a significant decrease in CTD synthesis. The current study indicates that EiFPPS is expressed in the fat body to regulate CTD synthesis in male E. impressicornis blister beetles.

Candidate membrane protein gene families related to chemoreception in a wood-boring beetle, Pharsalia antennata Gahan (Coleoptera: Cerambycidae).

The longhorned beetles are key players for the maintenance of biodiversity in the terrestrial ecosystem. As xylophagous cerambycid insects in Coleoptera, the beetles have evolved specialized olfactory and gustatory systems to recognize chemical cues in the surrounding habitats. Despite over 36,000 described species in the Cerambycidae family including a wood-boring pest Pharsalia antennata, only a limited number of them (<1 %) have been characterized regarding their chemical ecology at the molecular level. Here, we surveyed four membrane protein gene families in P. antennata related to chemoreception through transcriptomics, phylogenetics and expression profiling analyses. In total, 144 genes encoding 72 odorant receptors (ORs), 33 gustatory receptors (GRs), 23 ionotropic receptors (IRs), four sensory neuron membrane proteins (SNMPs) and 12 ionotropic glutamate receptors (iGluRs) were harvested from the transcriptome of multiple tissues including antennae and legs of both sexes. The lineage-specific expansion of PantORs possibly implied a diverse range of host plants in this beetle, supporting this correlation between the host range and olfactory receptor repertoire sizes across cerambycid species. Further phylogenetic analysis revealed that Group 2 was contributed mainly to the large OR gene repertoire in P. antennata, representing 18 genes in Group 2A and eight in Group 2B. On the other hand, some key chemosensory genes were identified by applying a phylogenetics approach, such as PantOR21 close to the 2-phenylethanol receptor in Megacyllene caryae, three carbon dioxide GRs and seven Antennal IRs (A-IRs) clades. We also determined sex- and tissue-specific expression profiles of 69 chemosensory genes, revealing the high expression of most PantORs in antennae. Noticeably, 10 sex-biased genes (six PantORs, three PantIRs and PantSNMP1a) were presented in antennae, five sex-biased PantGRs in legs and 39 sex-biased genes (15 PantORs, 13 PantGRs, eight PantIRs and three PantSNMPs) in abdomens. These findings have greatly enhanced our knowledge about the chemical ecology of P. antennata and identify candidate molecular targets for mediating smell and taste of this beetle.