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Background: Hylurgus ligniperda (Coleoptera: Curculionidae) is a worldwide forest quarantine pest. It is widely distributed, has many host tree species, and possesses strong adaptability. To explore its environmental adaptability and the related molecular mechanisms, we conducted chromosome-level genome sequencing and analyzed the transcriptome under different environmental factors, identifying key expressed genes., Results: We employed PacBio, Illumina, and Hi-C sequencing techniques to assemble a 520 Mb chromosomal-level genome of H. ligniperda, obtaining an N50 of 39.97 Mb across 138 scaffolds. A total of 10,765 protein-coding genes were annotated after repeat masking. Fourteen chromosomes were identified, among which Hyli14 was determined to be the sex chromosome. Survival statistics were tested over various growth periods under high temperature and low humidity conditions. The maximum survival period of adults reached 292 days at 25 °C, 65% relative humidity. In comparison, the maximum survival period was 14 days under 35 °C, 65% relative humidity, and 106 days under 25°C, 40% relative humidity. This indicated that environmental stress conditions significantly reduced adults' survival period. We further conducted transcriptome analysis to screen for potentially influential differentially expressed genes, such as CYP450 and Histone. Subsequently, we performed gene family analysis to gain insights into their functions and interactions, such as CYP450 and Histone. CYP450 genes affected the detoxification metabolism of enzymes in the Cytochrome P450 pathway to adapt to different environments. Histone genes are involved in insect hormone biosynthesis and longevity-regulating pathways in H. ligniperda to adapt to environmental stress., Conclusions: The genome at the chromosome level of H. ligniperda was assembled for the first time. The mortality of H. ligniperda increased significantly at 35 ℃, 65% RH, and 25 ℃, 40% RH. CYP450 and Histone genes played an important role in response to environmental stress. This genome offers a substantial genetic resource for investigating the molecular mechanisms behind beetle invasion and spread., (© 2024. The Author(s).)

Background: The Argentine stem weevil (ASW, Listronotus bonariensis) is a significant pasture pest in Aotearoa New Zealand, primarily controlled by the parasitoid biocontrol agent Microctonus hyperodae. Despite providing effective control of ASW soon after release, M. hyperodae parasitism rates have since declined significantly, with ASW hypothesised to have evolved resistance to its biocontrol agent. While the parasitism arsenal of M. hyperodae has previously been investigated, revealing many venom components and an exogenous novel DNA virus Microctonus hyperodae filamentous virus (MhFV), the effects of said arsenal on gene expression in ASW during parasitism have not been examined. In this study, we performed a multi-species transcriptomic analysis to investigate the biology of ASW parasitism by M. hyperodae, as well as the decline in efficacy of this biocontrol system., Results: The transcriptomic response of ASW to parasitism by M. hyperodae involves modulation of the weevil's innate immune system, flight muscle components, and lipid and glucose metabolism. The multispecies approach also revealed continued expression of venom components in parasitised ASW, as well as the transmission of MhFV to weevils during parasitism and some interrupted parasitism attempts. Transcriptomics did not detect a clear indication of parasitoid avoidance or other mechanisms to explain biocontrol decline., Conclusions: This study has expanded our understanding of interactions between M. hyperodae and ASW in a biocontrol system of critical importance to Aotearoa-New Zealand's agricultural economy. Transmission of MhFV to ASW during successful and interrupted parasitism attempts may link to a premature mortality phenomenon in ASW, hypothesised to be a result of a toxin-antitoxin system. Further research into MhFV and its potential role in ASW premature mortality is required to explore whether manipulation of this viral infection has the potential to increase biocontrol efficacy in future., (© 2024. The Author(s).)

Background: Biocontrol is a key technology for the control of pest species. Microctonus parasitoid wasps (Hymenoptera: Braconidae) have been released in Aotearoa New Zealand as biocontrol agents, targeting three different pest weevil species. Despite their value as biocontrol agents, no genome assemblies are currently available for these Microctonus wasps, limiting investigations into key biological differences between the different species and strains., Methods and Findings: Here we present high-quality genomes for Microctonus hyperodae and Microctonus aethiopoides, assembled with short read sequencing and Hi-C scaffolding. These assemblies have total lengths of 106.7 Mb for M. hyperodae and 129.2 Mb for M. aethiopoides, with scaffold N50 values of 9 Mb and 23 Mb respectively. With these assemblies we investigated differences in reproductive mechanisms, and association with viruses between Microctonus wasps. Meiosis-specific genes are conserved in asexual Microctonus, with in-situ hybridisation validating expression of one of these genes in the ovaries of asexual Microctonus aethiopoides. This implies asexual reproduction in these Microctonus wasps involves meiosis, with the potential for sexual reproduction maintained. Investigation of viral gene content revealed candidate genes that may be involved in virus-like particle production in M. aethiopoides, as well as a novel virus infecting M. hyperodae, for which a complete genome was assembled., Conclusion and Significance: These are the first published genomes for Microctonus wasps which have been deployed as biocontrol agents, in Aotearoa New Zealand. These assemblies will be valuable resources for continued investigation and monitoring of these biocontrol systems. Understanding the biology underpinning Microctonus biocontrol is crucial if we are to maintain its efficacy, or in the case of M. hyperodae to understand what may have influenced the significant decline of biocontrol efficacy. The potential for sexual reproduction in asexual Microctonus is significant given that empirical modelling suggests this asexual reproduction is likely to have contributed to biocontrol decline. Furthermore the identification of a novel virus in M. hyperodae highlights a previously unknown aspect of this biocontrol system, which may contribute to premature mortality of the host pest. These findings have potential to be exploited in future in attempt to increase the effectiveness of M. hyperodae biocontrol., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Pagiophloeus tsushimanus (Coleoptera: Curculionidae), an emerging forest pest exclusively infesting camphor trees, has recently caused severe ecological and economic damage in localized areas in China. Its population outbreak depends largely on the capacity to overcome the pressure of terpenoid-derived metabolites (e.g. linalool) from camphor trees. At present, the molecular basis of physiological adaptation of P. tsushimanus to dietary linalool is poorly understood, and there is no available reference genome or transcriptome., Results: Herein, we constructed the transcriptome profiling of P. tsushimanus larvae reared on linalool-infused diets using RNA sequencing and single-molecule real-time sequencing. A total of 20,325 high-quality full-length transcripts were identified as a reference transcriptome, of which 14,492 protein-coding transcripts including 130 transcription factors (TFs), and 5561 long non-coding RNAs (lncRNAs) were detected. Also, 30 alternative splicing events and 8049 simple sequence repeats were captured. Gene ontology enrichment of differential expressed transcripts revealed that overall up-regulation of both cytochrome P450s (CYP450s) and cuticular proteins (CPs), was the primary response characteristic against dietary linalool. Other physiological effects possibly caused by linalool exposure, such as increase in Reactive Oxygen Species (ROS) and hormetic stimulation, were compensated by a handful of induced genes encoding antioxidases, heat shock proteins (HSPs), juvenile hormone (JH) epoxide hydrolases, and digestive enzymes. Additionally, based on co-expression networks analysis, a diverse array of hub lncRNAs and TFs co-expressed with CYP450s and CPs were screened as the potential gene regulators. Temporal expression of candidate transcripts determined by quantitative real-time PCR also indicated a cooperative relationship between the inductions of CYP450s and CPs upon exposure to linalool., Conclusions: Our present study provides an important transcriptome resource of P. tsushimanus, and lays a valuable foundation for understanding how this specialist pest copes with chemical challenges in its specific host environments., (© 2023. The Author(s).)

Background: The first metazoan genome sequenced, that of Caenorhabditis elegans, has motivated animal genome evolution studies. To date > 50 species from the genus Caenorhabditis have been sequenced, allowing research on genome variation., Results: In the present study, we describe a new gonochoristic species, Caenorhabditis niphades n. sp., previously referred as C. sp. 36, isolated from adult weevils (Niphades variegatus), with whom they appear to be tightly associated during its life cycle. Along with a species description, we sequenced the genome of C. niphades n. sp. and produced a chromosome-level assembly. A genome comparison highlighted that C. niphades n. sp. has the smallest genome (59 Mbp) so far sequenced in the Elegans supergroup, despite being closely related to a species with an exceptionally large genome, C. japonica., Conclusions: The compact genome of C. niphades n. sp. can serve as a key resource for comparative evolutionary studies of genome and gene number expansions in Caenorhabditis species., (© 2022. The Author(s).)

Background: Olfaction and gustation underlie behaviors that are crucial for insect fitness, such as host and mate selection. The detection of semiochemicals is mediated via proteins from large and rapidly evolving chemosensory gene families; however, the links between a species' ecology and the diversification of these genes remain poorly understood. Hence, we annotated the chemosensory genes from genomes of select wood-boring coleopterans, and compared the gene repertoires from stenophagous species with those from polyphagous species., Results: We annotated 86 odorant receptors (ORs), 60 gustatory receptors (GRs), 57 ionotropic receptors (IRs), 4 sensory neuron membrane proteins (SNMPs), 36 odorant binding proteins (OBPs), and 11 chemosensory proteins (CSPs) in the mountain pine beetle (Dendroctonus ponderosae), and 47 ORs, 30 GRs, 31 IRs, 4 SNMPs, 12 OBPs, and 14 CSPs in the emerald ash borer (Agrilus planipennis). Four SNMPs and 17 CSPs were annotated in the polyphagous wood-borer Anoplophora glabripennis. The gene repertoires in the stenophagous D. ponderosae and A. planipennis are reduced compared with those in the polyphagous A. glabripennis and T. castaneum, which is largely manifested through small gene lineage expansions and entire lineage losses. Alternative splicing of GR genes was limited in D. ponderosae and apparently absent in A. planipennis, which also seems to have lost one carbon dioxide receptor (GR1). A. planipennis has two SNMPs, which are related to SNMP3 in T. castaneum. D. ponderosae has two alternatively spliced OBP genes, a novel OBP "tetramer", and as many as eleven IR75 members. Simple orthology was generally rare in beetles; however, we found one clade with orthologues of putative bitter-taste GRs (named the "GR215 clade"), and conservation of IR60a from Drosophila melanogaster., Conclusions: Our genome annotations represent important quantitative and qualitative improvements of the original datasets derived from transcriptomes of D. ponderosae and A. planipennis, facilitating evolutionary analysis of chemosensory genes in the Coleoptera where only a few genomes were previously annotated. Our analysis suggests a correlation between chemosensory gene content and host specificity in beetles. Future studies should include additional species to consolidate this correlation, and functionally characterize identified proteins as an important step towards improved control of these pests.

Background: Cytochrome P450-dependent monooxygenases (P450s), constituting one of the largest and oldest gene superfamilies found in many organisms from bacteria to humans, play a vital role in the detoxification and inactivation of endogenous toxic compounds. The use of various insecticides has increased over the last two decades, and insects have developed resistance to most of these compounds through the detoxifying function of P450s. In this study, we focused on the red palm weevil (RPW), Rhynchophorus ferrugineus, the most devastating pest of palm trees worldwide, and demonstrated through functional analysis that upregulation of P450 gene expression has evolved as an adaptation to insecticide stress arising from exposure to the neonicotinoid-class systematic insecticide imidacloprid., Results: Based on the RPW global transcriptome analysis, we identified 101 putative P450 genes, including 77 likely encoding protein coding genes with ubiquitous expression. A phylogenetic analysis revealed extensive functional and species-specific diversification of RPW P450s, indicating that multiple CYPs actively participated in the detoxification process. We identified highly conserved paralogs of insect P450s that likely play a role in the development of resistance to imidacloprid: Drosophila Cyp6g1 (CYP6345J1) and Bemisia tabaci CYP4C64 (CYP4LE1). We performed a toxicity bioassay and evaluated the induction of P450s, followed by the identification of overexpressed P450s, including CYP9Z82, CYP6fra5, CYP6NR1, CYP6345J1 and CYP4BD4, which confer cross-resistance to imidacloprid. In addition, under imidacloprid insecticide stress in a date palm field, we observed increased expression of various P450 genes, with CYP9Z82, CYP4BD4, CYP6NR1 and CYP6345J1 being the most upregulated detoxification genes in RPWs. Expression profiling and cluster analysis revealed P450 genes with multiple patterns of induction and differential expression. Furthermore, we used RNA interference to knock down the overexpressed P450s, after which a toxicity bioassay and quantitative expression analysis revealed likely candidates involved in metabolic resistance against imidacloprid in RPW. Ingestion of double-stranded RNA (dsRNA) successfully knocked down the expression of CYP9Z82, CYP6NR1 and CYP345J1 and demonstrated that silencing of CYP345J1 and CYP6NR1 significantly decreased the survival rate of adult RPWs treated with imidacloprid, indicating that overexpression of these two P450s may play an important role in developing tolerance to imidacloprid in a date palm field., Conclusion: Our study provides useful background information on imidacloprid-specific induction and overexpression of P450s, which may enable the development of diagnostic tools/markers for monitoring the spread of insecticide resistant RPWs. The observed trend of increasing tolerance to imidacloprid in the date palm field therefore indicated that strategies for resistance management are urgently needed.

Background: The Red Palm Weevil (RPW) Rhynchophorus ferrugineus (Oliver) is one of the most damaging invasive insect species in the world. This weevil is highly specialized to thrive in adverse desert climates, and it causes major economic losses due to its effects on palm trees around the world. RPWs locate palm trees by means of plant volatile cues and use an aggregation pheromone to coordinate a mass-attack. Here we report on the high throughput sequencing of the RPW antennal transcriptome and present a description of the highly expressed chemosensory gene families., Results: Deep sequencing and assembly of the RPW antennal transcriptome yielded 35,667 transcripts with an average length of 857 bp and identified a large number of highly expressed transcripts of odorant binding proteins (OBPs), chemosensory proteins (CSPs), odorant receptors/co-receptors (ORs/Orcos), sensory neuron membrane proteins (SNMPs), gustatory receptors (GRs) and ionotropic receptors (IRs). In total, 38 OBPs, 12 CSPs, 76 ORs, 1 Orco, 6 SNMPs, 15 GRs and 10 IRs were annotated in the R. ferrugineus antennal transcriptome. A comparative transcriptome analysis with the bark beetle showed that 25% of the blast hits were unique to R. ferrugineus, indicating a higher, more complete transcript coverage for R. ferrugineus. We categorized the RPW ORs into seven subfamilies of coleopteran ORs and predicted two new subfamilies of ORs. The OR protein sequences were compared with those of the flour beetle, the cerambycid beetle and the bark beetle, and we identified coleopteran-specific, highly conserved ORs as well as unique ORs that are putatively involved in RPW aggregation pheromone detection. We identified 26 Minus-C OBPs and 8 Plus-C OBPs and grouped R. ferrugineus OBPs into different OBP-subfamilies according to phylogeny, which indicated significant species-specific expansion and divergence in R. ferrugineus. We also identified a diverse family of CSP proteins, as well as a coleopteran-specific CSP lineage that diverged from Diptera and Lepidoptera. We identified several extremely diverged IR orthologues as well as highly conserved insect IR co-receptor orthologous transcripts in R. ferrugineus. Notably, GR orthologous transcripts for CO2-sensing and sweet tastants were identified in R. ferrugineus, and we found a great diversity of GRs within the coleopteran family. With respect to SNMP-1 and SNMP-2 orthologous transcripts, one SNMP-1 orthologue was found to be strikingly highly expressed in the R. ferrugineus antennal transcriptome., Conclusion: Our study presents the first comprehensive catalogue of olfactory gene families involved in pheromone and general odorant detection in R. ferrugineus, which are potential novel targets for pest control strategies.

Background: Insects subsisting on nutritionally unbalanced diets have evolved long-term mutualistic relationships with intracellular symbiotic bacteria (endosymbionts). The endosymbiont population load undergoes changes along with insect development. In the cereal weevil Sitophilus oryzae, the midgut endosymbionts Sodalis pierantonius drastically multiply following adult metamorphosis and rapidly decline until total elimination when the insect achieves its cuticle synthesis. Whilst symbiont load was shown to timely meet insect metabolic needs, little is known about the host molecular and immune processes underlying this dynamics., Methods: We performed RNA sequencing analysis on weevil midguts at three representative phases of the endosymbiont dynamics (i.e. increase, climax and decrease). To screen genes which transcriptional changes are specifically related to symbiont dynamics and not to the intrinsic development of the midgut, we further have monitored by RT-qPCR sixteen gene transcript levels in symbiotic and artificially non-symbiotic (aposymbiotic) weevils. We also localized the endosymbionts during the elimination process by fluorescence microscopy., Results: Functional analysis of the host differentially expressed genes by RNA sequencing showed that the main transcriptional changes occur during endosymbiont growth phase and affect cell proliferation, apoptosis, autophagy, phagocytosis, and metabolism of fatty acids and nucleic acids. We also showed that symbiont dynamics alters the expression of several genes involved in insect development. Our results strengthened the implication of apoptosis and autophagy processes in symbiont elimination and recycling. Remarkably, apart from the coleoptericin A that is known to target endosymbionts and controls their division and location, no gene coding antimicrobial peptide was upregulated during the symbiont growth and elimination phases., Conclusion: We show that endosymbiont dynamics parallels numerous transcriptional changes in weevil developing adults and affects several biological processes, including metabolism and development. It also triggers cell apoptosis, autophagy and gut epithelial cell swelling and delamination. Strikingly, immunity is repressed during the whole process, presumably avoiding tissue inflammation and allowing insects to optimize nutrient recovery from recycled endosymbiont.