Gene redundancy and gene compensation of insulin-like peptides in the oocyte development of bean beetle.

Bean beetle (Callosobruchus maculatus) exhibits clear phenotypic plasticity depending on population density; However, the underlying molecular mechanism remains unknown. Compared to low-density individuals, high-density individuals showed a faster terminal oocyte maturity rate. Four insulin-like peptide (ILP) genes were identified in the bean beetle, which had higher expression levels in the head than in the thorax and abdomen. The population density could regulate the expression levels of CmILP1-3, CmILP2-3, and CmILP1 as well as CmILP3 in the head, thorax, and abdomen, respectively. RNA interference results showed that each CmILP could regulate terminal oocyte maturity rate, indicating that there was functional redundancy among CmILPs. Silencing each CmILP could lead to down-regulation of some other CmILPs, however, CmILP3 was up-regulated in the abdomen after silencing CmILP1 or CmILP2. Compared to single gene silencing, silencing CmILP3 with CmILP1 or CmILP2 at the same time led to more serious retardation in oocyte development, suggesting CmILP3 could be up-regulated to functionally compensate for the down-regulation of CmILP1 and CmILP2. In conclusion, population density-dependent plasticity in terminal oocyte maturity rate of bean beetle was regulated by CmILPs, which exhibited gene redundancy and gene compensation.

Physiological Measurements and Transcriptomics Reveal the Fitness Costs of Monochamus saltuarius to Bursaphelenchus xylophilus.

The pine wood nematode (PWN) uses several Monochamus species as vehicles, through a temporary hitchhiking process known as phoresy, enabling it to access new host plant resources. Monochamus saltuarius acts as a new and major vector of the PWN in Northeastern China, showing lower PWN carrying capacity and a shorter transmission cycle compared to established vectors. The apparently altered symbiotic relationship offers an interesting area for researching the costs and adaptions involved in nematode-beetle, a specialized phoresy. We analyzed the response and fitness costs of M. saltuarius through physiological measurements and transcriptomics. The PWN exerted adverse repercussions on the growth and development of M. saltuarius. The PWN accelerated larval development into pupae, while beetle adults carrying the PWN exhibited an elevated abnormality rate and mortality, and reduced starvation resistance. During the pupal stage, the expression of growth-related genes, including ecdysone-inducible genes (E74EA), cuticle proteins, and chitin genes (CHTs), markedly increased. Meanwhile, the induced immune response, mainly by the IMD and Toll signaling pathways, could be a contributing factor to adult abnormality and mortality. Adult gonads and trachea exhibited enrichment in pathways related to fatty acid elongation, biosynthesis, and metabolism. FASN, ELOVL, and SCD possibly contributed to resistance against PWN. Our research indicated that phoretic interactions between vector beetles and PWN vary throughout the vector's lifespan, particularly before and after entry into the trachea. This study highlighted the fitness costs of immunity and metabolism on the vector beetle, indicating the adaptation mechanisms and evolutionary trade-offs to PWN.

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.

Vertically inherited microbiota and environment modifying behaviours conceal genetic variation in dung beetle life history.

Diverse organisms actively manipulate their (sym)biotic and physical environment in ways that feed back on their own development. However, the degree to which these processes affect microevolution remains poorly understood. The gazelle dung beetle both physically modifies its ontogenetic environment and structures its biotic interactions through vertical symbiont transmission. By experimentally eliminating (i) physical environmental modifications and (ii) the vertical inheritance of microbes, we assess how environment modifying behaviour and microbiome transmission shape heritable variation and evolutionary potential. We found that depriving larvae of symbionts and environment modifying behaviours increased additive genetic variance and heritability for development time but not body size. This suggests that larvae's ability to manipulate their environment has the potential to modify heritable variation and to facilitate the accumulation of cryptic genetic variation. This cryptic variation may become released and selectable when organisms encounter environments that are less amenable to organismal manipulation or restructuring. Our findings also suggest that intact microbiomes, which are commonly thought to increase genetic variation of their hosts, may instead reduce and conceal heritable variation. More broadly, our findings highlight that the ability of organisms to actively manipulate their environment may affect the potential of populations to evolve when encountering novel, stressful conditions.

Molecular characterization of the cytochrome P450 enzyme CYP18A1 in Henosepilachna vigintioctopunctata.

In insects, the expression of 20E response genes that initiate metamorphosis is triggered by a pulse of 20-hydroxyecdysone (20E). The 20E pulse is generated through two processes: synthesis, which increases its level, and inactivation, which decreases its titer. CYP18A1 functions as an ecdysteroid 26-hydroxylase and plays a role in 20E removal in several representative insects. However, applying 20E degradation activity of CYP18A1 to other insects remains a significant challenge. In this study, we discovered high levels of Hvcyp18a1 during the larval and late pupal stages, particularly in the larval epidermis and fat body of Henosepilachna vigintioctopunctata, a damaging Coleopteran pest of potatoes. RNA interference (RNAi) targeting Hvcyp18a1 disrupted the pupation. Approximately 75% of the Hvcyp18a1 RNAi larvae experienced developmental arrest and remained as stunted prepupae. Subsequently, they gradually turned black and eventually died. Among the Hvcyp18a1-depleted animals that successfully pupated, around half became malformed pupae with swollen elytra and hindwings. The emerged adults from these deformed pupae appeared misshapen, with shriveled elytra and hindwings, and were wrapped in the pupal exuviae. Furthermore, RNAi of Hvcyp18a1 increased the expression of a 20E receptor gene (HvEcR) and four 20E response transcripts (HvE75, HvHR3, HvBrC, and HvαFTZ-F1), while decreased the transcription of HvßFTZ-F1. Our findings confirm the vital role of CYP18A1 in the pupation, potentially involved in the degradation of 20E in H. vigintioctopunctata.

Six complete mitochondrial genomes of ground beetles from the Harpalinae and Carabinae (Coleoptera, Carabidae) with phylogenetic analysis based on mitogenomic data.

In this study, we employed high-throughput sequencing technology to determine the complete mitochondrial genomes of six ground beetles, encompassing five Harpalinae species and one Carabinae species. The sizes of mitochondrial genomes ranged from 15,334 to 16,972 bp, encompassing 37 genes, including 13 protein-coding genes, 22 transfer RNA genes, and 2 ribosomal RNA genes. Furthermore, each species was found to possess a putative control region. Combining with 65 published mitochondrial genome sequences of Carabidae as ingroups and four species from Trachypachidae, Gyrinidae and Dytiscidae as outgroups, we conducted phylogenetic analyses utilizing Maximum likelihood and Bayesian inference methods. Moreover, we reconstructed a species tree of Carabidae based on mitochondrial genome data using the coalescent-based species tree method (ASTRAL). The results revealed that the family Carabidae was not a monophyletic group. The subfamily Harpalinae was supported to be a monophyletic group in Maximum likelihood analysis. Although the subfamily Carabinae was found to be nonmonophyletic in the concatenation analyses under both Maximum likelihood and Bayesian inference criteria, it was identified as a monophyletic group in the species tree analysis.

A Measure of the DNA Barcode Gap for Applied and Basic Research.

DNA barcoding has largely established itself as a mainstay for rapid molecular taxonomic identification in both academic and applied research. The use of DNA barcoding as a molecular identification method depends on a "DNA barcode gap"-the separation between the maximum within-species difference and the minimum between-species difference. Previous work indicates the presence of a gap hinges on sampling effort for focal taxa and their close relatives. Furthermore, both theory and empirical work indicate a gap may not occur for related pairs of biological species. Here, we present a novel evaluation approach in the form of an easily calculated set of nonparametric metrics to quantify the extent of proportional overlap in inter- and intraspecific distributions of pairwise differences among target species and their conspecifics. The metrics are based on a simple count of the number of overlapping records for a species falling within the bounds of maximum intraspecific distance and minimum interspecific distance. Our approach takes advantage of the asymmetric directionality inherent in pairwise genetic distance distributions, which has not been previously done in the DNA barcoding literature. We apply the metrics to the predatory diving beetle genus Agabus as a case study because this group poses significant identification challenges due to its morphological uniformity despite both relative sampling ease and well-established taxonomy. Results herein show that target species and their nearest neighbor species were found to be tightly clustered and therefore difficult to distinguish. Such findings demonstrate that DNA barcoding can fail to fully resolve species in certain cases. Moving forward, we suggest the implementation of the proposed metrics be integrated into a common framework to be reported in any study that uses DNA barcoding for identification. In so doing, the importance of the DNA barcode gap and its components for the success of DNA-based identification using DNA barcodes can be better appreciated.

Importance of core microRNA pathway genes and microRNAs associated with the defense of Odontotermes formosanus (Shiraki) against Serratia marcescens infection.

MicroRNAs (miRNAs) are noncoding small regulatory RNAs involved in diverse biological processes. Odontotermes formosanus (Shiraki) is a polyphagous pest that causes economic damage to agroforestry. Serratia marcescens is a bacterium with great potential for controlling this insect. However, knowledge about the miRNA pathway and the role of miRNAs in O. formosanus defense against SM1 is limited. In this study, OfAgo1, OfDicer1 and OfDrosha were differentially expressed in different castes and tissues. SM1 infection affected the expression of all three genes in O. formosanus. Then, we used specific double-stranded RNAs to silence OfAgo1, OfDicer1 and OfDrosha. Knockdown of these genes enhanced the virulence of SM1 to O. formosanus, suggesting that miRNAs were critical in the defense of O. formosanus against SM1. Furthermore, we sequenced miRNAs from SM1-infected and uninfected O. formosanus. 33 differentially expressed (DE) miRNAs were identified, whereby 22 were upregulated and 11 were downregulated. Finally, the miRNA-mRNA networks were constructed, which further suggested the important role of miRNAs in the defense of O. formosanus against SM1. Totally, O. formosanus miRNA core genes defend against SM1 infection by regulating miRNA expression. This study elucidates the interactions between O. formosanus and SM1 and provides new theories for biological control.

The Neurotranscriptome of Monochamus alternatus.

The Japanese pine sawyer Monochamus alternatus serves as the primary vector for pine wilt disease, a devastating pine disease that poses a significant threat to the sustainable development of forestry in the Eurasian region. Currently, trap devices based on informational compounds have played a crucial role in monitoring and controlling the M. alternatus population. However, the specific proteins within M. alternatus involved in recognizing the aforementioned informational compounds remain largely unclear. To elucidate the spatiotemporal distribution of M. alternatus chemosensory-related genes, this study conducted neural transcriptome analyses to investigate gene expression patterns in different body parts during the feeding and mating stages of both male and female beetles. The results revealed that 15 genes in the gustatory receptor (GR) gene family exhibited high expression in the mouthparts, most genes in the odorant binding protein (OBP) gene family exhibited high expression across all body parts, 22 genes in the odorant receptor (OR) gene family exhibited high expression in the antennae, a significant number of genes in the chemosensory protein (CSP) and sensory neuron membrane protein (SNMP) gene families exhibited high expression in both the mouthparts and antennae, and 30 genes in the ionotropic receptors (IR) gene family were expressed in the antennae. Through co-expression analyses, it was observed that 34 genes in the IR gene family were co-expressed across the four developmental stages. The Antenna IR subfamily and IR8a/Ir25a subfamily exhibited relatively high expression levels in the antennae, while the Kainate subfamily, NMDA subfamily, and Divergent subfamily exhibited predominantly high expression in the facial region. MalIR33 is expressed only during the feeding stage of M. alternatus, the MalIR37 gene exhibits specific expression in male beetles, the MalIR34 gene exhibits specific expression during the feeding stage in male beetles, the MalIR8 and MalIR39 genes exhibit specific expression during the feeding stage in female beetles, and MalIR8 is expressed only during two developmental stages in male beetles and during the mating stage in female beetles. The IR gene family exhibits gene-specific expression in different spatiotemporal contexts, laying the foundation for the subsequent selection of functional genes and facilitating the full utilization of host plant volatiles and insect sex pheromones, thereby enabling the development of more efficient attractants.

RNA Interference-Mediated Suppression of Ecdysone Signaling Inhibits Choriogenesis in Two Coleoptera Species.

During choriogenesis in insects, chorion (eggshell) is formed by surrounding follicular epithelial cells in ovarioles. However, the regulatory endocrine factor(s) activating choriogenesis and the effect of chemical components on eggshell deserve further exploration. In two representative coleopterans, a coccinellid Henosepilachna vigintioctopunctata and a chrysomelid Leptinotarsa decemlineata, genes encoding the 20-hydroxyecdysone (20E) receptor heterodimer, ecdysone receptor (EcR) and ultraspiracle (USP), and two chitin biosynthesis enzymes UDP-N-acetylglucosamine pyrophosphorylase (UAP) and chitin synthase (ChS1), were highly expressed in ovaries of the young females. RNA interference (RNAi)-aided knockdown of either HvEcR or Hvusp in H. vigintioctopunctata inhibited oviposition, suppressed the expression of HvChS1, and lessened the positive signal of Calcofluor staining on the chorions, which suggests the reduction of a chitin-like substance (CLS) deposited on eggshells. Similarly, RNAi of LdEcR or Ldusp in L. decemlineata constrained oviposition, decreased the expression of LdUAP1 and LdChS1, and reduced CLS contents in the resultant ovaries. Knockdown of LdUAP1 or LdChS1 caused similar defective phenotypes, i.e., reduced oviposition and CLS contents in the L. decemlineata ovaries. These results, for the first time, indicate that 20E signaling activates choriogenesis in two coleopteran species. Moreover, our findings suggest the deposition of a CLS on the chorions.

Heterochromatin Is Not the Only Place for satDNAs: The High Diversity of satDNAs in the Euchromatin of the Beetle Chrysolina americana (Coleoptera, Chrysomelidae).

The satellitome of the beetle Chrysolina americana Linneo, 1758 has been characterized through chromosomal analysis, genomic sequencing, and bioinformatics tools. C-banding reveals the presence of constitutive heterochromatin blocks enriched in A+T content, primarily located in pericentromeric regions. Furthermore, a comprehensive satellitome analysis unveils the extensive diversity of satellite DNA families within the genome of C. americana. Using fluorescence in situ hybridization techniques and the innovative CHRISMAPP approach, we precisely map the localization of satDNA families on assembled chromosomes, providing insights into their organization and distribution patterns. Among the 165 identified satDNA families, only three of them exhibit a remarkable amplification and accumulation, forming large blocks predominantly in pericentromeric regions. In contrast, the remaining, less abundant satDNA families are dispersed throughout euchromatic regions, challenging the traditional association of satDNA with heterochromatin. Overall, our findings underscore the complexity of repetitive DNA elements in the genome of C. americana and emphasize the need for further exploration to elucidate their functional significance and evolutionary implications.

Species delimitation, discovery and conservation in a tiger beetle species complex despite discordant genetic data.

In an age of species declines, delineating and discovering biodiversity is critical for both taxonomic accuracy and conservation. In recent years, there has been a movement away from using exclusively morphological characters to delineate and describe taxa and an increase in the use of molecular markers to describe diversity or through integrative taxonomy, which employs traditional morphological characters, as well as genetic or other data. Tiger beetles are charismatic, of conservation concern, and much work has been done on the morphological delineation of species and subspecies, but few of these taxa have been tested with genetic analyses. In this study, we tested morphologically based taxonomic hypotheses of polymorphic tiger beetles in the Eunota circumpicta (LaFerté-Sénectère, 1841) species complex using multilocus genomic and mtDNA analyses. We find multiple cryptic species within the previous taxonomic concept of Eunota circumpicta, some of which were historically recognized as subspecies. We found that the mtDNA and genomic datasets did not identify the same taxonomic units and that the mtDNA was most at odds with all other genetic and morphological patterns. Overall, we describe new cryptic diversity, which raises important conservation concerns, and provide a working example for testing species and subspecies validity despite discordant data.

Phylogenomics of Tetraopes longhorn beetles unravels their evolutionary history and biogeographic origins.

Tetraopes longhorn beetles are known for their resistance to milkweed plant toxins and their coevolutionary dynamics with milkweed plants (Asclepias). This association is considered a textbook example of coevolution, in which each species of Tetraopes is specialized to feed on one or a few species of Asclepias. A major challenge to investigating coevolutionary hypotheses and conducting molecular ecology studies lies in the limited understanding of the evolutionary history and biogeographical patterns of Tetraopes. By integrating genomic, morphological, paleontological, and geographical data, we present a robust phylogeny of Tetraopes and their relatives, using three inference methods with varying subsets of data, encompassing 2-12 thousand UCE loci. We elucidate the diversification patterns of Tetraopes species across major biogeographical regions and their colonization of the American continent. Our findings suggest that the genus originated in Central America approximately 21 million years ago during the Miocene and diversified from the Mid-Miocene to the Pleistocene. These events coincided with intense geological activity in Central America. Additionally, independent colonization events in North America occurred from the Late Miocene to the early Pleistocene, potentially contributing to the early diversification of the group. Our data suggest that a common ancestor of Tetraopini migrated into North America, likely facilitated by North Atlantic land bridges, while closely related tribes diverged in Asia and Europe during the Paleocene. Establishing a robust and densely sampled phylogeny of Tetraopes beetles provides a foundation for investigating micro- and macroevolutionary phenomena, including clinal variation, coevolution, and detoxification mechanisms in this ecologically important group.

A near-complete genome assembly of Monochamus alternatus a major vector beetle of pinewood nematode.

The Japanese sawyer beetle, Monochamus alternatus, is not only one of the most important wood boring pest itself, but also a major vector of the invasive pinewood nematode (PWN), which is the causal agent of the devastative pine wilt disease (PWD) and threats the global pine forest. Here, we present a near-complete genome of M. alternatus at the chromosome level. The assembled genome was 792.05 Mb with contig N50 length of 55.99 Mb, which is the largest N50 size among the sequenced Coleoptera insects currently. 99.57% of sequence was anchored onto ten pseudochromosomes (one X-chromosome and nine autosomes), and the final genome harbored only 13 gaps. BUSCO evaluation revealed the presence of 99.0% of complete core genes. Thus, our genome assembly represented the highest-contiguity genome assembly as well as high completeness in insects so far. We identified 20,471 protein-coding genes, of which 20,070 (98.04%) were functionally annotated. The genome assembly of M. alternatus provides a valuable resource for exploring the evolution of the symbiosis between PWN and the vector insects.

The direction, timing and demography of Popillia japonica (Coleoptera) invasion reconstructed using complete mitochondrial genomes.

The Japanese beetle Popillia japonica is a pest insect that feeds on hundreds of species of wild and cultivated plants including important fruit, vegetable, and field crops. Native to Japan, the pest has invaded large areas of the USA, Canada, the Azores (Portugal), Italy, and Ticino (Switzerland), and it is considered a priority for control in the European Union. We determined the complete mitochondrial genome sequence in 86 individuals covering the entire distribution of the species. Phylogenetic analysis supports a major division between South Japan and Central/North Japan, with invasive samples coming from the latter. The origin of invasive USA samples is incompatible, in terms of the timing of the event, with a single introduction, with multiple Japanese lineages having been introduced and one accounting for most of the population expansion locally. The origin of the two invasive European populations is compatible with two different invasions followed by minimal differentiation locally. Population analyses provide the possibility to estimate the rate of sequence change from the data and to date major invasion events. Demographic analysis identifies a population expansion followed by a period of contraction prior to the invasion. The present study adds a time and demographic dimension to available reconstructions.

Mammal dung-dung beetle trophic networks: an improved method based on gut-content DNA.

Background: Dung beetles provide many important ecosystem services, including dung decomposition, pathogen control, soil aeration, and secondary seed dispersal. Yet, the biology of most dung beetles remains unknown. Natural diets are poorly studied, partly because previous research has focused on choice or attraction experiments using few, easily accessible dung types from zoo animals, farm animals, or humans. This way, many links within natural food webs have certainly been missed. In this work, we aimed to establish a protocol to analyze the natural diets of dung beetles using DNA gut barcoding. Methods: First, the feasibility of gut-content DNA extraction and amplification of 12s rDNA from six different mammal dung types was tested in the laboratory. We then applied the method to beetles caught in pitfall traps in Ecuador and Germany by using 12s rDNA primers. For a subset of the dung beetles caught in the Ecuador sampling, we also used 16s rDNA primers to see if these would improve the number of species we could identify. We predicted the likelihood of amplifying DNA using gut fullness, DNA concentration, PCR primer, collection method, and beetle species as predictor variables in a dominance analysis. Based on the gut barcodes, we generated a dung beetle-mammal network for both field sites (Ecuador and Germany) and analyzed the levels of network specificity. Results: We successfully amplified mammal DNA from dung beetle gut contents for 128 specimens, which included such prominent species as Panthera onca (jaguar) and Puma concolor (puma). The overall success rate of DNA amplification was 53%. The best predictors for amplification success were gut fullness and DNA concentration, suggesting the success rate can be increased by focusing on beetles with a full gut. The mammal dung-dung beetle networks differed from purely random network models and showed a moderate degree of network specialization (H2': Ecuador = 0.49; Germany = 0.41). Conclusion: We here present a reliable method of extracting and amplifying gut-content DNA from dung beetles. Identifying mammal dung via DNA reference libraries, we created mammal dung-dung beetle trophic networks. This has benefits over previous methods because we inventoried the natural mammal dung resources of dung beetles instead of using artificial mammal baits. Our results revealed higher levels of specialization than expected and more rodent DNA than expected in Germany, suggesting that the presented method provides more detailed insights into mammal dung-dung beetle networks. In addition, the method could have applications for mammal monitoring in many ecosystems.

scRNA-seq Reveals Novel Genetic Pathways and Sex Chromosome Regulation in Tribolium Spermatogenesis.

Spermatogenesis is critical to sexual reproduction yet evolves rapidly in many organisms. High-throughput single-cell transcriptomics promises unparalleled insight into this important process but understanding can be impeded in nonmodel systems by a lack of known genes that can reliably demarcate biologically meaningful cell populations. Tribolium castaneum, the red flour beetle, lacks known markers for spermatogenesis found in insect species like Drosophila melanogaster. Using single-cell sequencing data collected from adult beetle testes, we implement a strategy for elucidating biologically meaningful cell populations by using transient expression stage identification markers, weighted principal component clustering, and SNP-based haploid/diploid phasing. We identify populations that correspond to observable points in sperm differentiation and find species specific markers for each stage. Our results indicate that molecular pathways underlying spermatogenesis in Coleoptera are substantially diverged from those in Diptera. We also show that most genes on the X chromosome experience meiotic sex chromosome inactivation. Temporal expression of Drosophila MSL complex homologs coupled with spatial analysis of potential chromatin entry sites further suggests that the dosage compensation machinery may mediate escape from meiotic sex chromosome inactivation and postmeiotic reactivation of the X chromosome.

MicroRNA miR-7-5p targets MARK2 to control metamorphosis in Galeruca daurica.

The MARK2 gene, coding microtubule affinity-regulating kinase or serine/threonine protein kinase, is an important modulator in organism microtubule generation and cell polarity. However, its role in the metamorphosis of insects remains unknown. In this study, we found a conserved miRNA, miR-7-5p, which targets MARK2 to participate in the regulation of the larval-pupal metamorphosis in Galeruca daurica. The dual luciferase reporter assay showed that miR-7-5p interacted with the 3' UTR of MARK2 and repressed its expression. The expression profiling of miR-7-5p and MARK2 displayed an opposite trend during the larval-adult development process. In in-vivo experiments, overexpression of miR-7-5p by injecting miR-7-5p agomir in the final instar larvae down-regulated MARK2 and up-regulated main ecdysone signaling pathway genes including E74, E75, ECR, FTZ-F1 and HR3, which was similar to the results from knockdown of MARK2 by RNAi. In contrast, repression of miR-7-5p by injecting miR-7-5p antagomir obtained opposite effects. Notably, both overexpression and repression of miR-7-5p in the final instar larvae caused abnormal molting and high mortality during the larval-pupal transition, and high mortality during the pupal-adult transition. The 20-hydroxyecdysone (20E) injection experiment showed that 20E up-regulated miR-7-5p whereas down-regulated MARK2. This study reveals that the accurate regulation of miRNAs and their target genes is indispensable for insect metamorphosis.

Binding Properties of Odorant Binding Protein 37 in Plagiodera versicolora to Host Volatile, o-Cymene.

The chemosensory system plays an important role in the host plants location. Plagiodera versicolora (Coleoptera: Chrysomelidae) is a worldwide leaf-eating forest pest that feeds exclusively on salicaceous trees. There is no function study of odorant binding proteins (OBPs) in P. versicolora. In the current study, we found that PverOBP37 has a high expression in male and female antennae, heads, and legs by quantitative real-time PCR. The binding properties of PverOBP37 to 18 host plant volatiles were determined by fluorescence competition binding assays. The results showed that PverOBP37 could bind to the host plant volatile, o-cymene. Furthermore, four candidate key amino acid residues (F8, Y50, F103, and R107) of PverOBP37 to o-cymene were identified by molecular docking. The functional assay to confirm Y50, F103, and R107 mutations were key amino acid residues of PverOBP37 involved in the binding to o-cymene. Knockdown of PverOBP37 and Y-tube behavioral bioassays of mated females led to a significantly reduced attraction to o-cymene. This study not only revealed the molecular mechanism of PverOBP37 but also suggested that PverOBP37 is essential to detect host plant volatiles as cues to search for egg-laying sites in P. versicolora.

Comparative transcriptomic and metabolomics analysis of modified atmosphere responses in Tribolium castaneum (Coleoptera: Tenebrionidae).

Modified atmosphere is effective in controlling Tribolium castaneum Herbst, but it has adaptations. Comprehending the potential mechanism of resistance to T. castaneum in a modified atmosphere will help advance related management methods. This study conducted a comparative transcriptomic and metabolomic analysis to understand the physiological mechanism of T. castaneum in adapting to CO2 stress. Results showed that there were a large number of differentially expressed genes (DEGs) in T. castaneum treated with different concentrations of CO2. Gene ontology (GO) analysis revealed significant enrichment of DEGs mainly in binding, catalytic activity, cell, membrane, membrane part, protein-containing complex, biological regulation, and cellular and metabolic process. Kyoto Encyclopedia of Genes and Genomes analysis showed that different treatments had different effects on the metabolic pathways of T. castaneum. DEGs induced by 25% CO2 were involved in arginine and proline metabolism, and 50% air + 50% CO2 treatment affected most kinds of metabolic pathways, mainly the signal transduction pathway, including PI3K-Akt signaling pathway, AMPK signaling pathway, neurotrophin signaling pathway, insulin signaling pathway, and thyroid hormone signaling. Ribosome and DNA replication were enriched under high CO2 stress (75% and 95%). The metabolomics revealed that different concentrations of CO2 treatments might inhibit the growth of T. castaneum through acidosis, or they may adapt to anoxic conditions through histamine and N-acetylhistamine. Multiple analyses have shown significant changes in histamine and N-acetylhistamine levels, as well as their associated genes, with increasing CO2 concentration. In conclusion, this study comprehensively revealed the molecular mechanism of T. castaneum responding to CO2 stress and provided the basis for an effectively modified atmosphere in the T. castaneum.