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Beetles, representing the majority of the insect species diversity, are characterized by thick and hard cuticle, which plays important roles for their environmental adaptation and underpins their inordinate diversity and prosperity. Here, we report a bacterial endosymbiont extremely specialized for sustaining beetle’s cuticle formation. Many weevils are associated with a γ-proteobacterial endosymbiont lineage Nardonella, whose evolutionary origin is estimated as older than 100 million years, but its functional aspect has been elusive. Sequencing of Nardonella genomes from diverse weevils unveiled drastic size reduction to 0.2 Mb, in which minimal complete gene sets for bacterial replication, transcription, and translation were present but almost all of the other metabolic pathway genes were missing. Notably, the only metabolic pathway retained in the Nardonella genomes was the tyrosine synthesis pathway, identifying tyrosine provisioning as Nardonella’s sole biological role. Weevils are armored with hard cuticle, tyrosine is the principal precursor for cuticle formation, and experimental suppression of Nardonella resulted in emergence of reddish and soft weevils with low tyrosine titer, confirming the importance of Nardonella-mediated tyrosine production for host’s cuticle formation and hardening. Notably, Nardonella’s tyrosine synthesis pathway was incomplete, lacking the final step transaminase gene. RNA sequencing identified host’s aminotransferase genes up-regulated in the bacteriome. RNA interference targeting the aminotransferase genes induced reddish and soft weevils with low tyrosine titer, verifying host’s final step regulation of the tyrosine synthesis pathway. Our finding highlights an impressively intimate and focused aspect of the host–symbiont metabolic integrity via streamlined evolution for a single biological function of ecological relevance.

Background Climatic changes during glacial periods have had a major influence on the recent evolutionary history of living organisms, even in temperate forests on islands, where the land was not covered with ice sheets. We investigated the phylogeographical patterns of the weevil Curculio sikkimensis (Curculionidae), a generalist seed predator of Fagaceae plants living in both deciduous oak and evergreen forests of Japan. Its genetic structure was compared to that of another host-specific seed predator, C. hilgendorfi , inhabiting only evergreen forests. Results We examined 921 bp of mitochondrial DNA for 115 individuals collected from 33 populations of C. sikkimensis from 11 plant species of three genera, Quercus , Lithocarpus , and Castanopsis . An analysis of molecular variance revealed that a large proportion (almost 50%, P < 0.001) of the total genetic variance could be explained by differences between two geographical regions, the southwestern and northeastern parts of the main islands of Japan. In contrast, no significant genetic differentiation of the weevil was observed among vegetation types of their utilized host plant species. The phylogeographical patterns of the generalist and the host-specific seed predator exhibited a congruent genetic boundary in the Chugoku-Shikoku region. Conclusion Our results suggest that geology and historical environment have contributed to shaping the present genetic structure of C. sikkimensis . The geographical patterns of genetic differentiation in the Chugoku-Shikoku region observed in the two types of Fagaceae-associated Curculio in this study have also been observed in several plant species growing in warm and cool temperate zones of Japan. The occurrence of this common pattern suggests that deciduous oak and evergreen forests of Japan survived together, or adjacent to each other, in small refugia during glacial ages, in the southwestern and northeastern parts of the main islands, although these two types of forests are presently distributed in cool and warm temperate zones of Japan, respectively.

Complete mitochondrial genomes have been shown to be reliable markers for phylogeny reconstruction among diverse animal groups. However, the relative difficulty and high cost associated with obtaining de novo full mitogenomes have frequently led to conspicuously low taxon sampling in ensuing studies. Here, we report the successful use of an economical and accessible method for assembling complete or near-complete mitogenomes through shot-gun next-generation sequencing of a single library made from pooled total DNA extracts of numerous target species. To avoid the use of separate indexed libraries for each specimen, and an associated increase in cost, we incorporate standard polymerase chain reaction-based “bait” sequences to identify the assembled mitogenomes. The method was applied to study the higher level phylogenetic relationships in the weevils (Coleoptera: Curculionoidea), producing 92 newly assembled mitogenomes obtained in a single Illumina MiSeq run. The analysis supported a separate origin of wood-boring behavior by the subfamilies Scolytinae, Platypodinae, and Cossoninae. This finding contradicts morphological hypotheses proposing a close relationship between the first two of these but is congruent with previous molecular studies, reinforcing the utility of mitogenomes in phylogeny reconstruction. Our methodology provides a technically simple procedure for generating densely sampled trees from whole mitogenomes and is widely applicable to groups of animals for which bait sequences are the only required prior genome knowledge.

The cereal weevil, Sitophilus zeamais is one of the most destructive pests of stored cereals worldwide. Frequent use of fumigants for managing stored-product insects has led to the development of resistance in insects. Essential oils from aromatic plants including the tea oil plant, Melaleuca alternifolia may provide environmentally friendly alternatives to currently used pest control agents. However, little is known about molecular events involved in stored-product insects in response to plant essential oil fumigation. M. alternifolia essential oil was shown to possess the fumigant toxicity against S. zeamais. The constituent, terpinen-4-ol was the most effective compound for fumigant toxicity. M. alternifolia essential oil significantly inhibited the activity of three enzymes in S. zeamais, including two detoxifying enzymes, glutathione S-transferase (GST), and carboxylesterase (CarE), as well as a nerve conduction enzyme, acetylcholinesterase (AChE). Comparative transcriptome analysis of S. zeamais through RNA-Seq identified a total of 3,562 differentially expressed genes (DEGs), of which 2,836 and 726 were up-regulated and down-regulated in response to M. alternifolia essential oil fumigation, respectively. Based on gene ontology (GO) analysis, the majority of DEGs were involved in insecticide detoxification and mitochondrial function. Furthermore, an abundance of DEGs mapped into the metabolism pathway in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database were associated with respiration and metabolism of xenobiotics, including cytochrome P450s, CarEs, GSTs, and ATP-binding cassette transporters (ABC transporters). Some DEGs mapped into the proteasome and phagosome pathway were found to be significantly enriched. These results led us to propose a model of insecticide action that M. alternifolia essential oil likely directly affects the hydrogen carrier to block the electron flow and interfere energy synthesis in mitochondrial respiratory chain. This is the first study to perform a comparative transcriptome analysis of S. zeamais in response to M. alternifolia essential oil fumigation. Our results provide new insights into the insecticidal mechanism of M. alternifolia essential oil fumigation against S. zeamais and eventually contribute to the management of this important agricultural pest.

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 CO 2 -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 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.

The phylogeny and evolution of weevils (the beetle superfamily Curculionoidea) has been extensively studied, but many relationships, especially in the large family Curculionidae (true weevils; > 50,000 species), remain uncertain. We used phylogenomic methods to obtain DNA sequences from 522 protein-coding genes for representatives of all families of weevils and all subfamilies of Curculionidae. Most of our phylogenomic results had strong statistical support, and the inferred relationships were generally congruent with those reported in previous studies, but with some interesting exceptions. Notably, the backbone relationships of the weevil phylogeny were consistently strongly supported, and the former Nemonychidae (pine flower snout beetles) were polyphyletic, with the subfamily Cimberidinae (here elevated to Cimberididae) placed as sister group of all other weevils. The clade comprising the sister families Brentidae (straight-snouted weevils) and Curculionidae was maximally supported and the composition of both families was firmly established. The contributions of substitution modeling, codon usage and/or mutational bias to differences between trees reconstructed from amino acid and nucleotide sequences were explored. A reconstructed timetree for weevils is consistent with a Mesozoic radiation of gymnosperm-associated taxa to form most extant families and diversification of Curculionidae alongside flowering plants—first monocots, then other groups—beginning in the Cretaceous.

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.

Many insects sustain long-term relationships with intracellular symbiotic bacteria that provide them with essential nutrients. Such endosymbiotic relationships likely emerged from ancestral infections of the host by free-living bacteria, the genomes of which experience drastic gene losses and rearrangements during the host–symbiont coevolution. While it is well documented that endosymbiont genome shrinkage results in the loss of bacterial virulence genes, whether and how the host immune system evolves towards the tolerance and control of bacterial partners remains elusive. Remarkably, many insects rely on a ‘compartmentalization strategy’ that consists in secluding endosymbionts within specialized host cells, the bacteriocytes, thus preventing direct symbiont contact with the host systemic immune system. In this review, we compile recent advances in the understanding of the bacteriocyte immune and cellular regulators involved in endosymbiont maintenance and control. We focus on the cereal weevils Sitophilus spp., in which bacteriocytes form bacteriome organs that strikingly evolve in structure and number according to insect development and physiological needs. We discuss how weevils track endosymbiont dynamics through at least two mechanisms: (i) a bacteriome local antimicrobial peptide synthesis that regulates endosymbiont cell cytokinesis and helps to maintain a homeostatic state within bacteriocytes and (ii) some cellular processes such as apoptosis and autophagy which adjust endosymbiont load to the host developmental requirements, hence ensuring a fine-tuned integration of symbiosis costs and benefits. This article is part of the themed issue ‘Evolutionary ecology of arthropod antimicrobial peptides’.

Background Bark beetles are major pests of conifer forests, and their behavior is primarily mediated via olfaction. Targeting the odorant receptors (ORs) may thus provide avenues towards improved pest control. Such an approach requires information on the function of ORs and their interactions with ligands, which is also essential for understanding the functional evolution of these receptors. Hence, we aimed to identify a high-quality complement of ORs from the destructive spruce bark beetle Ips typographus (Coleoptera, Curculionidae, Scolytinae) and analyze their antennal expression and phylogenetic relationships with ORs from other beetles. Using 68 biologically relevant test compounds, we next aimed to functionally characterize ecologically important ORs, using two systems for heterologous expression. Our final aim was to gain insight into the ligand-OR interaction of the functionally characterized ORs, using a combination of computational and experimental methods. Results We annotated 73 ORs from an antennal transcriptome of I. typographus and report the functional characterization of two ORs (ItypOR46 and ItypOR49), which are responsive to single enantiomers of the common bark beetle pheromone compounds ipsenol and ipsdienol, respectively. Their responses and antennal expression correlate with the specificities, localizations, and/or abundances of olfactory sensory neurons detecting these enantiomers. We use homology modeling and molecular docking to predict their binding sites. Our models reveal a likely binding cleft lined with residues that previously have been shown to affect the responses of insect ORs. Within this cleft, the active ligands are predicted to specifically interact with residues Tyr84 and Thr205 in ItypOR46. The suggested importance of these residues in the activation by ipsenol is experimentally supported through site-directed mutagenesis and functional testing, and hydrogen bonding appears key in pheromone binding. Conclusions The emerging insight into ligand binding in the two characterized ItypORs has a general importance for our understanding of the molecular and functional evolution of the insect OR gene family. Due to the ecological importance of the characterized receptors and widespread use of ipsenol and ipsdienol in bark beetle chemical communication, these ORs should be evaluated for their potential use in pest control and biosensors to detect bark beetle infestations.