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Cotesia ruficrus presents a promising local natural enemy for controlling the invasive fall armyworm Spodoptera frugiperda in China. However, the mechanisms underlying how C. ruficrus locates its target pest remain unclear. In this study, we analyzed the expression patterns of 18 CrufOBPs across different developmental stages of C. ruficrus, and found that CrufOBP1 exhibited consistent and high expression levels in female adults. CrufOBP1 transcript was predominantly localized in sensilla placodea and sensilla trichodea on the antennae. Additionally, we confirmed the binding properties of CrufOBP1 protein to various cuticular compounds of S. frugiperda larvae. Subsequent electroantennogram and behavioral assays revealed that 1-(2-hydroxy-5-methylphenyl)-ethanone attracted female C. ruficrus, consequently increased the parasitism rate. However, upon silencing CrufOBP1, females exhibited reduced attraction towards 1-(2-hydroxy-5-methylphenyl)-ethanone, indicating the crucial role of CrufOBP1 in the chemoreception of C. ruficrus. These findings shed light on the kairomone-based mechanism employed by C. ruficrus to locate S. frugiperda larvae and hold a promise for the development of environmentally friendly pest management strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In vertebrates dysregulation of the antioxidant defense system has a detrimental impact on male fertility and reproductive physiology. However, in insects, especially mosquitoes the importance of sperm quality has been poorly studied. Since long-term storage of healthy and viable sperm earmarks male reproductive competency, we tested whether the heme peroxidase, a member of antioxidant enzyme family proteins, and abundantly expressed in the testis, also influence male fertility in the mosquito An. stephensi. Here, we show that a heme peroxidase 12 (HPX12), is an important cellular factor to protect the sperms from oxidative stress, and maintains semen quality in the male mosquito reproductive organ. We demonstrate that knockdown of the HPX12 not only impairs the sperm parameters such as motility, viability but also causes a significant down-regulation of MAG expressing transcripts such as ASTEI02706, ASTEI00744, ASTEI10266, likely encoding putative Accessory gland proteins. Mating with HPX12 knockdown male mosquitoes, resulted in ~ 50% reduction in egg-laying, coupled with diminished larval hatchability of a gravid female mosquito. Our data further outlines that increased ROS in the HPX12 mRNA depleted mosquitoes is the ultimate cause of sperm disabilities both qualitatively as well as quantitatively. Our data provide evidence that testis expressing AsHPX12 is crucial for maintaining optimal homeostasis for storing and protecting healthy sperms in the male mosquito's reproductive organs. Since, high reproductive capacity directly influences the mosquito population, manipulating male mosquito reproductive physiology could be an attractive tool to combat vector-borne diseases., (© 2022. The Author(s).)

Baculoviruses are double-stranded DNA entomopathogenic viruses that infect predominantly insects of the order Lepidoptera. Research in the last decade has started to disentangle the mechanisms underlying the insect-virus interaction, particularly focusing on the effects of the baculovirus infection in the host's physiology. Among crucial physiological functions, olfaction has a key role in reproductive tasks, food source detection and enemy avoidance. In this work, we describe that Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) induces expression changes in some odorant receptors (ORs) - the centrepiece of insect's olfaction - when infecting larvae from its natural host Spodoptera exigua (Lepidoptera: Noctuidae). Different ORs are up-regulated in larvae after SeMNPV infection, and two of them, SexiOR35 and SexiOR23, were selected for further functional characterization by heterologous expression in empty neurons of Drosophila melanogaster coupled to single-sensillum recordings. SexiOR35 appears to be a broadly tuned receptor able to recognise multiple and different chemical compounds. SexiOR23, although correctly expressed in Drosophila neurons, did not display any significant response to a panel of 58 stimuli. Behavioural experiments revealed that larvae infected by SeMNPV exhibit altered olfactory-driven behaviour to diet when it is supplemented with the plant volatiles linalool or estragole, two of the main SexiOR35 ligands, supporting the hypothesis that viral infection triggers changes in host perception through changes in the expression level of specific ORs., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Recent identification of a Piwi-interacting RNA (piRNA)-initiated sex determination cascade in the silkworm, Bombyx mori, provides novel insights into high diversity of insect sex determination pathways. In this system, the W-chromosome-derived Fem piRNA is the primary sex determination signal. A CCCH-type zinc finger gene Masculinizer (Masc), which is targeted by Fem piRNA-PIWI complex in female animals, is indispensable for male-specific splicing of B. mori doublesex (Bmdsx). Although many genes involved in this cascade have been identified, the regulatory mechanisms of silkworm sex determination remain to be elucidated. Here we show that another CCCH-type zinc finger gene, Bmznf-2, is a masculinization factor in B. mori. Bmznf-2 shows testis-abundant expression and loss of Bmznf-2 function via clustered regularly interspaced short palindromic repeats / single-guide RNA-mediated mutagenesis results in feminized differentiation and appearance of the female-specific splicing variants of Bmdsx transcripts in males. In contrast, there is no phenotypic consequence in mutant females. In mutant males, relative messenger RNA expression levels of female-dominant genes such as vitellogenin and sex-specific storage protein 1 are significantly elevated while several male-dominant genes are significantly down-regulated. Furthermore, male mutants show delayed developmental timing, smaller body sizes of larvae and malformation of moth wings. Our data thus reveal that Bmznf-2 plays an indispensable role in silkworm male sexual differentiation., (© 2020 Institute of Zoology, Chinese Academy of Sciences.)

Chemosensory systems are considered to play an important role in host plant selection in herbivorous insects. However, few studies have focused on chemosensory proteins (CSPs) for aphid host-location mechanisms. The roles of CSPs in searching for different Poaceae species (wheat, barley, triticale, maize and sorghum) were tested in Rhopalosiphum padi, an important cereal pest. The olfactometer assays showed that R. padi responds to plant odors. Seven R. padi CSP genes were identified. Influence of aphid morph, tissue and starvation state on expression patterns of CSPs was evaluated. Expression levels of CSP1, CSP4, CSP5 and CSP6 in winged aphids were significantly higher than those in wingless ones. Transcription levels of four genes (CSP1, CSP4, CSP5 and CSP6) were relatively higher in the head with antennae, and the four genes tended to be upregulated following starvation. Silencing of three CSPs (CSP4, CSP5 and CSP6) altered aphid host-location behavior in response to the five different host plants tested. Three volatile compounds of host plants (octanal, [E]-2-hexenol and linalool) have significant attraction to winged R. padi according to the four-arm olfactometer tests. Molecular docking predicted hydrogen bonding sites which played key roles in the binding of CSP4, CSP5 and CSP6 with volatile compounds. Knockdown of CSP4 or CSP5 significantly decreased the staying time of R. padi in the arms with octanal. However, knockdown of CSP6 could not affect the response of R. padi to octanal. These results bring evidence for the involvement of three CSPs in R. padi host-location behavior., (© 2020 Institute of Zoology, Chinese Academy of Sciences.)

Animal development relies on a sequence of specific stages that allow the formation of adult structures with a determined size. In general, juvenile stages are dedicated mainly to growth, whereas last stages are devoted predominantly to the maturation of adult structures. In holometabolous insects, metamorphosis marks the end of the growth period as the animals stops feeding and initiate the final differentiation of the tissues. This transition is controlled by the steroid hormone ecdysone produced in the prothoracic gland. In Drosophila melanogaster different signals have been shown to regulate the production of ecdysone, such as PTTH/Torso, TGFß and Egfr signaling. However, to which extent the roles of these signals are conserved remains unknown. Here, we study the role of Egfr signaling in post-embryonic development of the basal holometabolous beetle Tribolium castaneum. We show that Tc-Egfr and Tc-pointed are required to induced a proper larval-pupal transition through the control of the expression of ecdysone biosynthetic genes. Furthermore, we identified an additional Tc-Egfr ligand in the Tribolium genome, the neuregulin-like protein Tc-Vein (Tc-Vn), which contributes to induce larval-pupal transition together with Tc-Spitz (Tc-Spi). Interestingly, we found that in addition to the redundant role in the control of pupa formation, each ligand possesses different functions in organ morphogenesis. Whereas Tc-Spi acts as the main ligand in urogomphi and gin traps, Tc-Vn is required in wings and elytra. Altogether, our findings show that in Tribolium, post-embryonic Tc-Egfr signaling activation depends on the presence of two ligands and that its role in metamorphic transition is conserved in holometabolous insects., (© 2021. The Author(s).)

Neuropeptide CCHamide-2 is a recently discovered peptide presents in many arthropods and it is the ligand of the CCHamide-2 receptor (CCHa2-R), which is playing a regulatory role in diverse physiological processes, such as feeding, insulin production, lipid metabolism, growth and reproduction, however, the function of this gene in aphids is still unknown. Here, we characterized and determined the presumed role of CCHa2-R signaling in the wingless pea aphid, Acyrthosiphon pisum. Quantitative real-time reverse transcription-PCR (qRT-PCR) revealed the expression levels of CCHa2-R transcripts in different development stages and different tissues, which indicates that the CCHa2-R expression was high in the first instar as compared to the upcoming nymphal instars and adult and was predominantly high in the brain. The CCHa2-R transcript levels were significantly up-regulated in starved aphids as compared to fed aphids. Moreover, RNAi knockdown by the injection of dsRNA-CCHa2-R significantly reduced the expression and also reduced their food intake in adult aphids, as revealed by electrical penetration graphs (EPGs) results. CCHa2-R-silencing was also shown to reduce reproduction but not survival in A. pisum. These observations suggest a role for the CCHa2-R pathway in the response of wingless parthenogenetic aphids to their nutritional status, and this role involves the regulation of the expression of CCHa2-R. Overall, the results of these experiments indicate that CCHa2-R plays a dominant role in the regulation of feeding in A. pisum., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The Masculinizer ( Masc ) gene has been known to control sex development and dosage compensation in lepidopterans. However, it remains unclear whether its ortholog exists and plays the same roles in distantly related lepidopterans such as Helicoverpa armigera . To address this question, we cloned Masc from H. armigera ( HaMasc ), which contains all essential functional domains of BmMasc, albeit with less than 30% amino acid sequence identity with BmMasc. Genomic PCR and qPCR analyses showed that HaMasc is a Z chromosome-linked gene since its genomic content in males (ZZ) was two times greater than that in females (ZW). RT-PCR and RT-qPCR analyses revealed that HaMasc expression was sex- and stage-biased, with significantly more transcripts in males and eggs than in females and other stages. Transfection of a mixture of three siRNAs of HaMasc into a male embryonic cell line of H. armigera led to the appearance of female-specific mRNA splicing isoforms of H. armigera doublesex ( Hadsx ), a downstream target gene of HaMasc in the H. armigera sex determination pathway. The knockdown of HaMasc, starting from the third instar larvae resulted in a shift of Hadsx splicing from male to female isoforms, smaller male pupa and testes, fewer but larger/longer spermatocytes and sperm bundles, delayed pupation and internal fusion of the testes and follicles. These data demonstrate that HaMasc functions as a masculinizing gene in the H. armigera sex-determination cascade.

Chemical communication via pheromones is an integral component in insect behavior, particularly for mate searching and reproduction. Aggregation pheromones, that attract conspecifics of both sexes, are particularly common and have been identified for hundreds of species. These pheromones are among the most ecologically selective pest suppression agents. In this study, we identified an activating effect of the aggregation pheromone of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenibroidae) on a highly conserved circadian clock gene (Tctimeless). Tribolium castaneum is one of the most damaging cosmopolitan pest of flour and other stored food products. Its male produced aggregation pheromone, 4,8-dimethyldecanal (DMD), attracts both conspecific males and females and is used for pest management via monitoring and mating disruption. The Tctimeless gene is an essential component for daily expression patterns of the circadian clock and plays vital roles in eclosion, egg production, and embryonic development. In this study, we demonstrate that constant exposure to the species-specific aggregation pheromone led to Tctimeless up-regulation and a different pattern of rhythmic locomotive behavior. We propose that changing the well-adapted "alarm clock", using DMD is liable to reduce fitness and can be highly useful for pest management., (© 2021. The Author(s).)

Many insect species have several genes coding for insulin-related peptides (IRPs), but so far only a single IRP gene has been identified in migratory locusts. Here, we report and characterize two other genes coding for peptides that are related to insulin, namely gonadulin and arthropod insulin-like growth factor (aIGF); peptides postulated to be orthologs of Drosophila melanogaster insulin-like peptides 8 and 6 respectively. In Locusta migratoria the aIGF transcript is expressed in multiple tissues as was previously reported for IRP in both L. migratoria and Schistocerca gregaria , but there are significant differences in expression patterns between the two species. The gonadulin transcript, however, seems specific to the ovary, whereas its putative receptor transcript is expressed most abundantly in the ovary, fat body and the central nervous system. Since the central nervous system-fat body-ovary axis is essential for successful reproduction, we studied the influence of gonadulin on vitellogenesis and oocyte growth. A reduction in the gonadulin transcript ( via RNA interference) led to a significant reduction in vitellogenin mRNA levels in the fat body and a strong oocyte growth inhibition, thus suggesting an important role for gonadulin in reproduction in this species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Veenstra, Leyria, Orchard and Lange.)

Density-dependent phase polyphenism in locusts is one of the most extreme forms of phenotypic plasticity. Locusts exist along the continuum between two density-dependent phenotypes that differ in nymphal coloration, behavior, morphology, physiology, and reproduction among others. Nymphs of the solitarious phase, found in low population densities, are usually green, relatively inactive, and avoid each other, while gregarious nymphs, found in high density, exhibit a very obvious yellow/orange background with black patterning, and are highly active and attracted to each other. The multifunctional neuropeptide [His 7 ]-corazonin has been shown to strongly affect black coloration and several other phase-related characteristics in at least two locust species, even though no effect on phase-related behavioral traits has been found. In this study, we investigate the role of [His 7 ]-corazonin in the Central American locust Schistocerca piceifrons (Walker), which evolved density-dependent phase polyphenism independently from the two previously studied locust species. After successfully knocking down the transcript encoding [His 7 ]-corazonin (CRZ) using RNA interference, we show that such a knockdown influences both color and morphometrics in this species, but does not influence phase-related behavioral traits. Our results suggest that the role of [His 7 ]-corazonin is conserved in different locust species. Finally, our study represents the first controlled study of behavioral solitarization in S. piceifrons., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Cytochrome P450 monooxygenases (P450s) play significant roles in protecting organisms from abiotic stress damage. Here, we report the sequence and characterization of a P450s gene (AccCYP4AV1), isolated from Apis cerana cerana Fabricius. The open reading frame of AccCYP4AV1 is 1506 base pairs long and encodes a predicted protein of 501 amino acids and 57.84 kDa, with an isoelectric point of 8.67. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis indicated that AccCYP4AV1 is more highly expressed in the midgut than in other tissues. In addition, the highest expression occurs in newly emerged adult workers, followed by the first instar of the larval stage. In addition, the expression of the AccCYP4AV1 was upregulated by low temperature (4 °C), ultraviolet radiation, hydrogen peroxide, paraquat, and dichlorvos treatments. In contrast, AccCYP4AV1 transcription was downregulated by other abiotic stress conditions: exposure to increased temperature (44 °C), deltamethrin, cadmium chloride, and mercury (II) chloride. Moreover, when AccCYP4AV1 was knocked-down by RNA interference, the results suggested that multiple antioxidant genes (AccsHSP22.6, AccSOD2, AccTpx1, and AccTpx4) were downregulated and antioxidant genes AccGSTO1 and AccTrx1 were upregulated. The activity levels of peroxidase and catalase were upregulated in the AccCYP4AV1-knocked-down samples, compared with those in the control groups. These findings suggest that the AccCYP4AV1 protein might be involved in the defense against abiotic stress damage.

DMRT (Doublesex and Mab-3-related transcription factor) is a highly conserved transcription factor family involved in sex determination in numerous animal species. One DMRT, dmrt2/dmrt11E, has entirely different functions in invertebrate and vertebrate species, indicating unpredicted functions. Here, we performed functional analysis of the dmrt11E gene in the domesticated silkworm, Bombyx mori. This gene was preferentially expressed in ovarioles at the last larval instar stage. Its mRNA accumulated in ovarian eggs during the adult stage. CRISPR/Cas9-mediated knockout of Bombyx dmrt11E (Bmdmrt11E) caused defects in oogenesis, resulting in the production of abnormal eggs with transparent liquids. These eggs had significantly reduced fertility and lipid levels. Transcriptomic comparisons between ovaries of control and mutant insects at two developmental stages identified six genes that may be under the control of Bmdmrt11E. Finally, we provide a possible model for lipid uptake and storage in eggs of Bombyx mori., (Copyright © 2020. Published by Elsevier Ltd.)

Hymenoptera are characterised by the presence of one forewing pair and one hindwing pair. The two wings of each body side are coupled to each other during flight making the morphologically four-winged insects functionally two-winged. This coupling is formed by a row of hook-like structures, called hamuli, that are located at the leading edge of the hindwing and interlock with a thickened and recurved margin present at the trailing edge of the forewing. In this study, autofluorescence analyses performed with confocal laser scanning microscopy revealed differences in the exoskeleton material composition of the interlocking structures. While the wing veins and the recurved margin are strongly sclerotised and chitinous, the wing membranes mainly contain the elastomeric protein resilin. The hamuli are composed of sclerotised chitinous material, and each hamulus base is surrounded by and embedded in material that features large proportions of resilin and is located in strongly sclerotised socket-like wing vein structures. This exoskeleton organisation likely allows movements of the hamuli and, in combination with the exoskeleton material gradients visualized in the other interlocking structures, is assumed to guarantee an effective wing coupling and to simultaneously decrease the risk of wear and damage under mechanical loads occurring in flight, coupling and decoupling situations., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The CP2 transcription factors are highly conserved in metazoans, where they are divided into two groups: grainyhead and late SV40 factor (LSF). We traced their evolutionary history in the Hexapoda using over 500 insect transcriptomes, to test the hypothesis that the evolution of holometaboly involved novel isoforms of these genes. All insects appear to express at least one grainyhead and one LSFlike gene, regardless of life cycle, as in most known metazoa. No major evolutionary events in these gene families occurred during the evolution of insects.

The Chinese citrus fly, Bactrocera minax, is a notorious univoltine pest that causes damage to citrus. B. minax enters obligatory pupal diapause in each generation to resist harsh environmental conditions in winter. Despite the enormous efforts that have been made in the past decade, the understanding of pupal diapause of B. minax is currently still fragmentary. In this study, the 20-hydroxyecdysone solution and ethanol solvent was injected into newly-formed pupae to obtain non-diapause- (ND) and diapause-destined (D) pupae, respectively, and a comparative proteomics analysis between ND and D pupae was performed 1 and 15 d after injection. A total of 3,255 proteins were identified, of which 190 and 463 were found to be differentially abundant proteins (DAPs) in ND1 vs D1 and ND15 vs D15 comparisons, respectively. The reliability and accuracy of LFQ method was validated by qRT-PCR. Functional analyses of DAPs, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction (PPI) network construction, were conducted. The results revealed that the diapause program of B. minax is closely associated with several physiological activities, such as phosphorylation, chitin biosynthesis, autophagy, signaling pathways, endocytosis, skeletal muscle formation, protein metabolism, and core metabolic pathways of carbohydrate, amino acid, and lipid conversion. The findings of this study provide insights into diapause program of B. minax and lay a basis for further investigation into its underlying molecular mechanisms., Competing Interests: The authors have declared that no competing interests exist.

Background: Elasticity prevents fatigue of tissues that are extensively and repeatedly deformed. Resilin is a resilient and elastic extracellular protein matrix in joints and hinges of insects. For its mechanical properties, Resilin is extensively analysed and applied in biomaterial and biomedical sciences. However, there is only indirect evidence for Resilin distribution and function in an insect. Commonly, the presence of dityrosines that covalently link Resilin protein monomers (Pro-Resilin), which are responsible for its mechanical properties and fluoresce upon UV excitation, has been considered to reflect Resilin incidence., Results: Using a GFP-tagged Resilin version, we directly identify Resilin in pliable regions of the Drosophila body, some of which were not described before. Interestingly, the amounts of dityrosines are not proportional to the amounts of Resilin in different areas of the fly body, arguing that the mechanical properties of Resilin matrices vary according to their need. For a functional analysis of Resilin matrices, applying the RNA interference and Crispr/Cas9 techniques, we generated flies with reduced or eliminated Resilin function, respectively. We find that these flies are flightless but capable of locomotion and viable suggesting that other proteins may partially compensate for Resilin function. Indeed, localizations of the potentially elastic protein Cpr56F and Resilin occasionally coincide., Conclusions: Thus, Resilin-matrices are composite in the way that varying amounts of different elastic proteins and dityrosinylation define material properties. Understanding the biology of Resilin will have an impact on Resilin-based biomaterial and biomedical sciences.

Postcopulatory sexual selection (PCSS), comprised of sperm competition and cryptic female choice, has emerged as a widespread evolutionary force among polyandrous animals. There is abundant evidence that PCSS can shape the evolution of sperm. However, sperm are not the whole story: they are accompanied by seminal fluid substances that play many roles, including influencing PCSS. Foremost among seminal fluid models is Drosophila melanogaster , which displays ubiquitous polyandry, and exhibits intraspecific variation in a number of seminal fluid proteins (Sfps) that appear to modulate paternity share. Here, we first consolidate current information on the identities of D. melanogaster Sfps. Comparing between D. melanogaster and human seminal proteomes, we find evidence of similarities between many protein classes and individual proteins, including some D. melanogaster Sfp genes linked to PCSS, suggesting evolutionary conservation of broad-scale functions. We then review experimental evidence for the functions of D. melanogaster Sfps in PCSS and sexual conflict. We identify gaps in our current knowledge and areas for future research, including an enhanced identification of PCSS-related Sfps, their interactions with rival sperm and with females, the role of qualitative changes in Sfps and mechanisms of ejaculate tailoring. This article is part of the theme issue 'Fifty years of sperm competition'.

Hepatocyte nuclear factor 4 (HNF4) plays essential roles in regulating lipid metabolism and glucose homeostasis in female insects. However, little is known about the role of HNF4 in insect fecundity. Here we demonstrate that HNF4 regulates female fecundity by affecting egg hatching in the brown planthopper (BPH) Nilaparvata lugens. HNF4 was highly expressed in the ovary and fat body of female adult. RNA interference-mediated HNF4 knockdown resulted in a dramatic reduction in egg hatchability and caused a severe block in embryonic development, while showed no significant effects on ovary development and egg laying. Transcriptome sequencing analysis showed that 72 genes encoding ribosome proteins were significantly down-regulated in the HNF4-silenced BPH and "ribosome" was the most-enriched pathway for the down-regulated genes. These results suggest that HNF4 controls the dynamics of egg structure, likely through its regulation of ribosome protein genes, which in turn affects the embryonic development and egg hatching., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Drosophila suzukii (spotted wing drosophila) has become a major invasive insect pest of soft fruits in the America and Europe, causing severe yield losses every year. The female D. suzukii shows the oviposition preference for ripening or ripe fruit by cutting the hard skin with its serrated ovipositor. A recent study reported that mechanosensation is involved in the texture discrimination during egg-laying behaviour in D. suzukii. However, the underlying mechanism and molecular entity that control this behaviour are not known. The transient receptor potential (TRP) channels and degenerin/epithelial sodium channels (DEG/ENaC) are two candidate gene families of mechanically activated ion channels. Thus, we first identified TRP and DEG/ENaC genes in D. suzukii by bioinformatic analysis. Using transcriptome sequencing, we found that many TRP genes were expressed in the ovipositor in both D. suzukii and D. melanogaster, while some DEG/ENaCs showed species-specific expression patterns. Exposure to drugs targeting TRP and DEG/ENaC channels abolished the oviposition preference for harder texture in female D. suzukii. Therefore, mechanosensitive ion channels may play significant roles in the texture assessment of egg-laying behaviour in D. suzukii, which has promising implications to further research on the development of novel control measures., (© 2020 The Royal Entomological Society.)

In insects, synthesis and deposition of the chorion (eggshell) are performed by the professional secretory follicle cells (FCs) that surround the oocytes in the course of oogenesis. Here, we found that ULK1/ATG1, an autophagy-related protein, is highly expressed in the FCs of the Chagas-Disease vector Rhodnius prolixus, and that parental RNAi silencing of ULK1/ATG1 results in oocytes with abnormal chorion ultrastructure and FCs presenting expanded rough ER membranes as well as increased expression of the ER chaperone BiP3, both indicatives of ER stress. Silencing of LC3/ATG8, another essential autophagy protein, did not replicate the ULK1/ATG1 phenotypes, whereas silencing of SEC16A, a known partner of the noncanonical ULK1/ATG1 function in the ER exit sites phenocopied the silencing of ULK1/ATG1. Our findings point to a cooperated function of ULK1/ATG1 and SEC16A in the FCs to complete choriogenesis and provide additional in vivo phenotype-based evidence to the literature of the role of ULK1/ATG1 in the ER in a professional secretory cell., (© 2020 Federation of American Societies for Experimental Biology.)

Deducing impacts of environmental change on species and the populations they form in nature is an important goal in contemporary ecology. Achieving this goal is hampered by our limited understanding of the influence of naturally occurring environmental variation on the molecular systems of ecologically relevant species, as the pathways underlying fitness-affecting plastic responses have primarily been studied in model organisms and under controlled laboratory conditions. Here, to test the hypothesis that proteome variation systematically relates to variation in abiotic conditions, we establish such relationships by profiling the proteomes of 24 natural populations of the spring-dwelling caddisfly Crunoecia irrorata. We identified protein networks whose abundances correlated with environmental (abiotic) gradients such as in situ pH, oxygen- and nitrate concentrations but also climatic data such as past thermal minima and temperature seasonality. Our analyses suggest that variations in abiotic conditions induce discrete proteome responses such as the differential abundance of proteins associated with cytoskeletal function, heat-shock proteins and proteins related to post-translational modification. Identifying these drivers of proteome divergence characterizes molecular "noise", and positions it as a background against which molecular signatures of species' adaptive responses to stressful conditions can be identified.

Neuropeptides of short neuropeptides F family (sNPF) have been identified in various arthropods. They are pleiotropic neuromolecules which so far have been mainly associated with regulation of feeding and metabolism, as well as growth and development, locomotion, circadian rhythm or learning and memory. Here, we describe the effects of Tenebrionid sNPF peptide (SGRSPSLRLRFa) on various aspects of the male reproductive physiology in the Tenebrio molitor beetle. We identified in silico the putative sNPF receptor Tenmo-sNPFR. Based on RT-PCR technique, it was shown that the receptor might be present in the male reproductive tissues of this beetle. The analysis of receptor amino acid sequence showed that it is similar to other beetle sNPFRs, as well as other insect species, and belongs rhodopsin-like G-protein-coupled receptors (GPCRs). Injections of Trica-sNPF and its shorter form Trica-sNPF (4-11) caused differentiated effects in T. molitor male reproductive tissues. After 24 h post injections, the peptides decreased the concentration of the soluble protein fraction in testes of 4- and 8-day-old beetles as well as the dry mass of these organs but only in 8-day-old individuals. The same effects were shown with regard to accessory glands. Both peptides decrease the concentration of the soluble protein fraction but do not affect the dry mass of this organ. Furthermore, injections of Trica-sNPF at the 10 -7  M concentration decrease the total sperm number in the reproductive system. Surprisingly, the same concentration of the shorter form, Trica-sNPF (4-11) increased the sperm number. It was also shown that both peptides in different manner influence contractions of ejaculatory duct. The data presented in this article give new evidence that sNPFs are involved in the regulation of reproductive events in beetles, which might be the part of a larger neuropeptide network combining feeding, growth and development with the physiology of reproduction.

In insects, nuclear receptors (NRs) including EcR (NR1H1), USP (NR2B4), E75 (NR1D3), HR3 (NR1F), HR4 (NR6) and FTZ-F1 (NR5A3) mediate the 20-hydroxyecdysone (20E) signaling cascade to play a critical role during larval metamorphosis. In this present paper, we focused on hormone receptor 38 (HR38) in Leptinotarsa decemlineata, the only insect homolog of the NR4A subclass. RNA interference (RNAi) of LdHR38 in the penultimate (third) instar larvae reduced the expression of an ecdysteroidogenesis gene and declined the titer of 20E. Knockdown of LdHR38 intensified the expression of LdUSP, LdE75, LdE74, LdE93, LdBroad and LdHR3, whereas repressed the transcription of LdFTZ-F1. Disruption of 20E signaling inhibited chitin biosynthesis in the larval cuticle. Approximately 25% of the LdHR38 RNAi larvae died, around 40% of the resultant larvae remained as prepupae or become deformed pupae. The body surface of the HR38 depleted abnormal prepupae and pupae looked wet, just like the cuticle being covered with a layer of liquid. Moreover, the increase of larval mortality, and the impairment of pupation and emergence exhibited dose-dependent manners. Furthermore, silencing LdHR38 at the final (fourth) instar caused similar but less severe impairment of pupation. Dietary supplement with 20E for the third instar larvae did not rescue the high larval death and only slightly alleviated the low pupation rate in the LdHR38 RNAi hypomorphs. Accordingly, we propose that HR38 is necessary for tune of ecdysteroidogenesis and for mediation of 20E signaling during metamorphosis in L. decemlineata., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Methyl-CpG binding domain proteins (MBD) can specifically bind to methylated CpG sites and play important roles in epigenetic gene regulation. Here, we identified and functionally characterized the MBD protein in Tribolium castaneum. T. castaneum genome encodes only one MBD protein: TcMBD2/3. RNA interference targeting this gene at different developmental stages caused lethal phenotypes including metamorphosis deficiency in larvae and pupae, gastrointestinal system problems and fecundity deficiency in adult. Moreover, Tcmbd2/3 knockdown adult showed progressive reduced locomoter activity, a typical neurodegeneration phenotype. This is a common feature of DNA methylation in mammals and has not been found in other insects. However, band shift assays demonstrated that TcMBD2/3 could not bind to methylated DNA, indicating the essential roles of TcMBD2/3 is independent of DNA methylation. Our study provides Tcmbd2/3 plays important roles in T. castaneum and gives new insights into the potential mechanism of action of MBD proteins in insect., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Sex determination and differentiation are nearly universal to all eukaryotic organisms, encompassing diverse systems and mechanisms. Here, we identified a spliceosomal protein gene BmSPX involved in sex determination of the lepidopeteran insect, Bombyx mori . In a transgenic silkworm line that overexpressed the BmSPX gene, transgenic silkworm males exhibited differences in their external genitalia compared to wild-type males, but normal internal genitalia. Additionally, transgenic silkworm females exhibited a developmental disorder of the reproductive organs. Upregulation of BmSPX significantly increased the expression levels of sex-determining genes ( BmMasc and BmIMP ) and reduced the female-type splice isoform of Bmdsx , which is a key switch gene downstream of the sex-determination pathway. Additionally, co-immunoprecipitation assays confirmed an interaction between the BmSPX protein and BmPSI, an upstream regulatory factor of Bmdsx . Quantitative real-time PCR showed that BmSPX over-expression upregulated the expression of the Hox gene abdominal-B ( Adb-B ), which is required for specification of the posterior abdomen, external genitalia, and gonads of insects, as well as the genes in the Receptor Tyrosine Kinase (RTK) signaling pathway. In conclusion, our study suggested the involvement of BmSPX, identified as a novel regulatory factor, in the sex-determination pathway and regulation of reproductive organ development in silkworms.

Background: Lepidoptera is one group of the largest plant-feeding insects and Spodoptera litura (Lepidoptera: Noctuidae) is one of the most serious agricultural pests in Asia countries. An interesting and unique phenomenon for gonad development of Lepidoptera is the testicular fusion. Two separated testes fused into a single one during the larva-to-pupa metamorphosis, which is believed to contribute to sperm production and the prevalence in field. To study the molecular mechanism of the testicular fusion, RNA sequencing (RNA-seq) experiments of the testes from 4-day-old sixth instar larvae (L6D4) (before fusion), 6-day-old sixth instar larvae (L6D6, prepupae) (on fusing) and 4-day-old pupae (P4D) (after fusion) of S. litura were performed., Results: RNA-seq data of the testes showed that totally 12,339 transcripts were expressed at L6D4, L6D6 and P4D stages. A large number of differentially expressed genes (DEGs) were up-regulated from L6D4 to L6D6, and then more genes were down-regulated from L6D6 to P4D. The DEGs mainly belongs to the genes related to the 20E signal transduction pathway, transcription factors, chitin metabolism related enzymes, the families of cytoskeleton proteins, extracellular matrix (ECM) components, ECM-related protein, its receptor integrins and ECM-remodeling enzymes. The expression levels of these genes that were up-regulated significantly during the testicular fusion were verified by qRT-PCR. The matrix metalloproteinases (MMPs) were found to be the main enzymes related to the ECM degradation and contribute to the testicular fusion. The testis was not able to fuse if MMPs inhibitor GM6001 was injected into the 5th abdomen region at L6D6 early stage., Conclusions: The transcriptome and DEGs analysis of the testes at L6D4, L6D6 and P4D stages provided genes expression information related to the testicular fusion in S. litura. These results indicated that cytoskeleton proteins, ECM-integrin interaction genes and ECM-related proteins were involved in cell migration, adhesion and fusion during the testicular fusion. The ECM degradation enzymes MMPs probably play a critical role in the fusion of testis.

Phospholipase A 2 (PLA 2 ) catalyzes release of free fatty acids linked to phospholipids at sn-2 position. Some of these released free fatty acids are used to synthesize eicosanoids that mediate various physiological processes in insects. Although a large number of PLA 2 s form a superfamily consisting of at least 16 groups, few PLA 2 s have been identified and characterized in insects. Furthermore, physiological functions of insect PLA 2 s remain unclear. Clustered regularly interspaced short parlindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has been a useful research tool to validate gene function. This study identified and characterized a secretory PLA 2 (sPLA 2 ) from legume pod borer, Maruca vitrata (Lepidoptera: Crambidae), and validated its physiological functions using CRISPR/Cas9. An open reading frame of M. vitrata sPLA 2 (Mv-sPLA 2 ) encoding 192 amino acids contained signal peptide, calcium-binding domain, and catalytic site. Phylogenetic analysis indicated that Mv-sPLA 2 was related to other Group III sPLA 2 s. Mv-sPLA 2 was expressed in both larval and adult stages. It was inducible by immune challenge. RNA interference (RNAi) of Mv-sPLA 2 significantly suppressed cellular immunity and impaired larval development. Furthermore, RNAi treatment in female adults prevented oocyte development. These physiological alterations were also observed in a mutant line of M. vitrata with Mv-sPLA 2 deleted by using CRISPR/Cas9. Mv-sPLA 2 was not detected in the mutant line from western blot analysis. Addition of an eicosanoid, PGE 2 , significantly rescued oocyte development of females of the mutant line. These results suggest that Mv-sPLA 2 plays crucial role in immune, developmental, and reproductive processes of M. vitrata., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A second intracellular copper/zinc superoxide dismutase (icCuZnSOD2) and manganese SOD (MnSOD) were cloned and characterized in Oxya chinensis. The open reading frame (ORF) of OcicCuZnSOD2 and OcMnSOD are 462 and 672 bp encoding 153 and 223 amino acids, respectively. OcicCuZnSOD2 contains two signature sequences, one potential N-glycosylation site, and seven copper/zinc binding sites. OcMnSOD includes a mitochondria targeting sequence of 7 amino acids at N-terminal, one signature sequence, two N-glycosylation sites, and four manganese binding sites. The secondary structure and homology model of OcicCuZnSOD2 include nine β sheets, two Greek-key motifs, and one electrostatic loop. OcMnSOD contains nine α-helices and three β-sheets. Phylogenetic analysis shows that OcMnSOD is evolutionarily conserved while OcicCuZnSOD2 may be gene duplication and is paralogous to OcicCuZnSOD1. OcMnSOD expressed widely in all tissues and developmental stages. OcicCuZnSOD2 showed testis-specific expression and expressed highest in the 5th-instar nymph and the adult. The optimum temperatures and pH values of the recombinant OcicCuZnSOD2 and OcMnSOD were 40 °C and 8.0. They were stable at 25-55 °C and at pH 5.0-12.0 and pH 6.0-12.0, respectively. The activity and mRNA expression of each OcSOD were assayed after chlorpyrifos treatments. Total SOD and CuZnSOD activities first increased then declined under chlorpyrifos stress. Chlorpyrifos induced the mRNA expression and activity of OcMnSOD as a dose-dependent manner and inhibited OcicCuZnSOD2 transcription. The role of each OcSOD gene in chlorpyrifos stress was investigated using RNAi and disc diffusion assay with Escherichia coli overexpressing OcSOD proteins. Silencing of OcMnSOD significantly increased ROS content in chlorpyrifos-exposed grasshoppers. Disc diffusion assay showed that the plates with E. coli overexpressing OcMnSOD had the smaller inhibition zones around the chlorpyrifos-soaked filter discs. These results implied that OcMnSOD played a significant role in defense chlorpyrifos-induced oxidative stress., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Sex determination has been studied in the model lepidopteran species Bombyx mori, but it remains poorly understood in lepidopteran pests. In the present study, we identified and characterized the Masculinizer (Masc) gene in a Noctuidae pest species, Agrotis ipsilon. Sequence analysis revealed that AiMasc encodes a protein of 658 amino acids that has two CCCH-type zinc finger domains and two conserved cysteine residues (Cys-277 and Cys-280). We assessed the masculinizing activity of AiMasc in BmN cells and found that AiMasc induced expression of the male-specific doublesex isoform. Disruption of Masc via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) in A. ipsilon caused abnormalities in abdominal segments and external genitalia, resulting in male-specific sterility. These results suggest that Masc participates in the process of sex determination in A. ipsilon. Successful identification of sex-determination gene in a pest species may enable the development of novel genetic approaches for pest control., (© 2018 Institute of Zoology, Chinese Academy of Sciences.)

Three different pheromone binding proteins (PBPs) can typically be found in the sensilla lymph of noctuid moth antennae, but their relative contributions in perception of the sex pheromone is rarely verified in vivo. Previously, we demonstrated that SlitPBP3 plays a minor role in the sex pheromone detection in Spodoptera litura using the CRISPR/Cas9 system. In the present study, the roles of two other SlitPBPs (SlitPBP1 and SlitPBP2) are further verified using the same system. First, by co-injection of Cas9 mRNA/sgRNA into newly laid eggs, a high rate of target mutagenesis was induced, 51.5% for SlitPBP1 and 46.8% for SlitPBP2 as determined by restriction enzyme assay. Then, the homozygous SlitPBP1 and SlitPBP2 knockout lines were obtained by cross-breeding. Finally, using homozygous knockout male moths, we performed electrophysiological (EAG recording) and behavioral analyses. Results showed that knockout of either SlitPBP1 or SlitPBP2 in males decreased EAG response to each of the 3 sex pheromone components (Z9,E11-14:Ac, Z9,E12-14:Ac and Z9-14:Ac) by 53%, 60% and 63% (for SlitPBP1 knockout) and 40%, 43% and 46% (for SlitPBP2 knockout), respectively. These decreases in EAG responses were similar among 3 pheromone components, but were more pronounced in SlitPBP1 knockout males than in SlitPBP2 knockout males. Consistently, behavioral assays with the major component (Z9,E11-14:Ac) showed that SlitPBP1 knockout males responded in much lower percentages than SlitPBP2 knockout males in terms of orientation to the pheromone, along with reduction in close range behaviors such as hairpencil display and mating attempt. Taken together, this study provides direct functional evidence for the roles of SlitPBP1 and SlitPBP2, as well as their relative importance (SlitPBP1 > SlitPBP2) in the sex pheromone perception. This information is valuable in understanding mechanisms of sex pheromone perception and may facilitate the development of PBP-targeted pest control techniques., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

MAP kinase phosphatase 3 (MKP3), a member of the dual-specificity protein phosphatase (DUSP) superfamily, has been widely studied for its role in development, cancer, and environmental stress in many organisms. However, the functions of MKP3 in various insects have not been well studied, including honeybees. In this study, we isolated an MKP3 gene from Apis cerana cerana and explored the role of this gene in the resistance to oxidation. We found that AccMKP3 is highly conserved in different species and shares the closest evolutionary relationship with AmMKP3. We determined the expression patterns of AccMKP3 under various stresses. qRT-PCR results showed that AccMKP3 was highly expressed during the pupal stages and in adult muscles. We further found that AccMKP3 was induced in all the stress treatments. Moreover, we discovered that the enzymatic activities of peroxidase, superoxide dismutase, and catalase increased and that the expression levels of several antioxidant genes were affected after AccMKP3 was knocked down. Collectively, these results suggest that AccMKP3 may be associated with antioxidant processes involved in response to various environmental stresses.

Inositol phosphate synthase (IPS) is a rate-limiting enzyme in myo-inositol biosynthesis, which can regulate stress responses in plants and animals. However, there are few studies on the function of IPS in insects, especially in Apis cerana cerana. In this study, the inositol-3-phosphate synthase 1-B gene (AccIPS1-B) was isolated from Apis cerana cerana, and its connection to antioxidant defence was investigated. The open reading frame of AccIPS1-B was 1542 bp, encoding a 513 amino acid polypeptide. Quantitative real-time PCR analysis revealed that the expression level of AccIPS1-B was highest in pupae of Apis cerana cerana, and it was expressed at higher levels in the thorax than in other tissues tested. Moreover, the expression of AccIPS1-B was significantly upregulated by abiotic stresses. The recombinant AccIPS1-B also displayed significant tolerance to cumene hydroperoxide and HgCl 2 . In addition, knockdown of AccIPS1-B significantly suppressed the expression of most of the antioxidant genes and decreased the antioxidant enzymatic activities of SOD, POD, and GST. Taken together, these findings indicate that AccIPS1-B may be involved in the response to antioxidant defence and development in Apis cerana cerana.

The AMP-activated protein kinase (AMPK) has important roles in the regulation of energy metabolism, and AMPK activity and its regulation have been the focus of relevant investigations. However, functional characterization of AMPK is still limited in insects. In this study, the full-length cDNA coding AMPKα (TcAMPKα) was isolated from the red flour beetle, Tribolium castaneum. The TcAMPKα gene contains an ORF of 1581 bp encoding a protein of 526 amino acid residues, which shared conserved domain structure with Drosophila melanogaster and mammalian orthologs. Exposure of female adults to oxidative, heat, and cold stresses caused an increase in TcAMPKα mRNA expression levels and phosphorylation of Thr-173 in the activation loop. The RNAi-mediated knockdown of TcAMPKα resulted in the increased sensitivity of T. castaneum to oxidative, heat, and cold stresses. These results suggest that stress signals regulate TcAMPKα activity, and TcAMPKα plays an important role in enabling protective mechanisms and processes that confer resistance to environmental stress.

Elucidating the genetic basis of metabolic resistance to insecticides in malaria vectors is crucial to prolonging the effectiveness of insecticide-based control tools including long lasting insecticidal nets (LLINs). Here, we show that cis-regulatory variants of the cytochrome P450 gene, CYP6P9b, are associated with pyrethroid resistance in the African malaria vector Anopheles funestus. A DNA-based assay is designed to track this resistance that occurs near fixation in southern Africa but not in West/Central Africa. Applying this assay we demonstrate, using semi-field experimental huts, that CYP6P9b-mediated resistance associates with reduced effectiveness of LLINs. Furthermore, we establish that CYP6P9b combines with another P450, CYP6P9a, to additively exacerbate the reduced efficacy of insecticide-treated nets. Double homozygote resistant mosquitoes (RR/RR) significantly survive exposure to insecticide-treated nets and successfully blood feed more than other genotypes. This study provides tools to track and assess the impact of multi-gene driven metabolic resistance to pyrethroids, helping improve resistance management.

The number of cells in tissues is under strict genetic control, and research on the determination of cell number is of great importance to understand the growth and development of organs. Bmsage, a bHLH transcription factor, is involved in the development of the silk gland during the embryonic stage in Bombyx mori. However, the mechanism by which it influences silk gland development is unclear. In the present study, we determined via immunofluorescence staining during the embryonic stage of Bombyx mori that Bmsage is expressed in silk gland cells from the beginning of development of the silk gland until its complete formation. By comparing different silkworm strains, we found that Bmsage expression is positively correlated with the number of silk gland cells. Bmsage knockdown by RNAi resulted in shorter silk glands and lower cell numbers, especially in the posterior silk gland. The silk gland lumen also shriveled, and the silk protein content was significantly lower than that in the control. Further investigation revealed that all cyclins decreased after knock down of Bmsage, and cyclin B and cyclin 3 were significantly down-regulated. Bmsage may be involved in the regulation of the cyclin pathway to control silk gland development. Taken together, it can be concluded from our results that Bmsage is involved in the determination of cell number in silk glands. Our results help clarify the process of cell number determination in silk gland and identify a potential target for silkworm breeding., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Insect G protein coupled receptors (GPCRs) have been identified as a highly attractive target for new generation insecticides discovery due to their critical physiological functions. However, few insect GPCRs have been functionally characterized. Here, we cloned the full length of a methuselah-like GPCR gene (Ldmthl1) from the Asian gypsy moth, Lymantria dispar. We then characterized the secondary and tertiary structures of Ldmthl1. We also predicted the global structure of this insect GPCR protein which is composed of three major domains. RNA interference of Ldmthl1 resulted in a reduction of gypsy moths' resistance to deltamethrin and suppressed expression of downstream stress-associated genes, such as P450s, glutathione S transferases, and heat shock proteins. The function of Ldmthl1 was further investigated using transgenic lines of Drosophila melanogaster. Drosophila with overexpression of Ldmthl1 showed significantly longer lifespan than control flies. Taken together, our studies revealed that the physiological functions of Ldmthl1 in L. dispar are associated with longevity and resistance to insecticide stresses. Potentially, Ldmthl1 can be used as a target for new insecticide discovery in order to manage this notorious forest pest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Venoms are comprised of diverse mixtures of proteins, peptides, and small molecules. Identifying individual venom components and their target(s) with mechanism of action is now attainable to understand comprehensively the effectiveness of venom cocktails and how they collectively function in the defense and predation of an organism. Here, structure-based computational methods were used with bioinformatics tools to screen and identify potential biological targets of tertiapin (TPN), a venom peptide from Apis mellifera (European honey bee). The small hive beetle ( Aethina tumida ( A. tumida )) is a natural predator of the honey bee colony and was found to possess multiple inwardly rectifying K + (Kir) channel subunit genes from a genomic BLAST search analysis. Structure-based virtual screening of homology modelled A. tumida Kir ( at Kir) channels found TPN to interact with a docking profile and interface "footprint" equivalent to known TPN-sensitive mammalian Kir channels. The results support the hypothesis that at Kir channels, and perhaps other insect Kir channels, are natural biological targets of TPN that help defend the bee colony from infestations by blocking K + transport via at Kir channels. From these in silico findings, this hypothesis can now be subsequently tested in vitro by validating at Kir channel block as well as in vivo TPN toxicity towards A. tumida . This study highlights the utility and potential benefits of screening in virtual space for venom peptide interactions and their biological targets, which otherwise would not be feasible.

Umami taste perception indicates the presence of amino acids, which are essential nutrients. Although the physiology of umami perception has been described in mammals, how insects detect amino acids remains unknown except in Drosophila melanogaster. We functionally characterized a gustatory receptor responding to L-amino acids in the western honey bee, Apis mellifera. Using a calcium-imaging assay and two-voltage clamp recording, we found that one of the honey bee's gustatory receptors, AmGr10, functions as a broadly tuned amino acid receptor responding to glutamate, aspartate, asparagine, arginine, lysine, and glutamine, but not to other sweet or bitter compounds. Furthermore, the sensitivity of AmGr10 to these L-amino acids was dramatically enhanced by purine ribonucleotides, like inosine-5'-monophosphate (IMP). Contact sensory hairs in the mouthpart of the honey bee responded strongly to glutamate and aspartate, which house gustatory receptor neurons expressing AmGr10. Interestingly, AmGr10 protein is highly conserved among hymenopterans but not other insects, implying unique functions in eusocial insects.

Aedes (Stegomyia) aegypti, the principal global vector of dengue viruses, has differences in its susceptibility to dengue virus infection. We compared the global expression of genes in the midguts of Colombian Ae. aegypti dengue-susceptible (Cali-S) and dengue-refractory (Cali-MIB) field derived strains after ingesting either a sugarmeal, a bloodmeal, or a bloodmeal containing dengue virus serotype 2 (DENV-2). Microarray-based transcriptome analysis among treatments indicated a total of 4725 transcripts with differential expression between the two strains. Eleven genes were selected from different functional groups based on their significant up or down expression levels as well as reports in the literature suggesting they are associated with dengue virus elimination. We measured mRNA abundance of these 11 genes at 0, 8, 24, and 36 h postinfection using quantitative real time PCR (qPCR) to confirm the microarray results and assess any temporal patterns. Four genes were selected (Gram-negative binding protein-GNBP [AAEL009176], Niemann Pick Type-C2-NPC2 [AAEL015136], Keratinocyte lectin [AAEL009842], and Cathepsin-b [AAEL007585]) for knockdown experiments using RNA interference (RNAi) methodology to determine the phenotype (DENV-2 susceptible or refractory). Silencing GNBP, Cathepsin-b and Keratinocyte lectin reduced the percentage of mosquitoes with disseminated virus in the Cali-S strain to 8%, 20%, and 12% respectively compared with 96% in the controls. Silencing of NPC2 increased the percentage of mosquitos with disseminated virus infections in Cali-MIB to 66% compared with 35% in the controls. This study provides insight into genes that may contribute to the Cali-S susceptible and Cali-MIB refractory phenotypes in Ae. aegypti., (© 2018 Institute of Zoology, Chinese Academy of Sciences.)

Darwin's bark spider ( Caerostris darwini ) produces giant orb webs from dragline silk that can be twice as tough as other silks, making it the toughest biological material. This extreme toughness comes from increased extensibility relative to other draglines. We show C. darwini dragline-producing major ampullate (MA) glands highly express a novel silk gene transcript (MaSp4) encoding a protein that diverges markedly from closely related proteins and contains abundant proline, known to confer silk extensibility, in a unique GPGPQ amino acid motif. This suggests C. darwini evolved distinct proteins that may have increased its dragline's toughness, enabling giant webs. Caerostris darwini's MA spinning ducts also appear unusually long, potentially facilitating alignment of silk proteins into extremely tough fibers. Thus, a suite of novel traits from the level of genes to spinning physiology to silk biomechanics are associated with the unique ecology of Darwin's bark spider, presenting innovative designs for engineering biomaterials., Competing Interests: Competing interestsThe authors declare no competing interests.

Through a combination of transcriptomic and proteomic analyses, we identified 817 secreted ovarian proteins from an endoparasitoid wasp, Cotesia chilonis, of which five proteins are probably involved in passive evasion. The results of an encapsulation assay revealed that one of these passive evasion-associated proteins (Crp32B), a homologue of a 32-kDa protein (Crp32) from C. rubecula, could protect resin beads from being encapsulated by host hemocytes in a dose-dependent manner. Crp32B is transcribed in ovarian cells, nurse cells, follicular cells, and oocytes, and the protein is located throughout the ovary and on the egg surface. Moreover, Crp32B has antigenic similarity to several host components. These results indicate that C. chilonis may use molecular mimicry as a mechanism to avoid host cellular immune response.

Our previous study demonstrated that predominant feeding inhibitory effects were found in the crude extracts of foregut and midgut of the silkworm Bombyx mori larvae. To address the entero-intestinal control crucial for the regulation of insect feeding behavior, the present study identified and functionally characterized feeding inhibitory peptides from the midgut of B. mori larvae. Purification and structural analyses revealed that the predominant inhibitory factors in the crude extracts were allatotropin (AT) and GSRYamide after its C-terminal sequence. In situ hybridization revealed that AT and GSRYamide were expressed in enteroendocrine cells in the posterior and anterior midgut, respectively. Receptor screening using Ca2+-imaging technique showed that the B. mori neuropeptide G protein-coupled receptor (BNGR)-A19 and -A22 acted as GSRYamide receptors and BNGR-A5 acted as an additional AT receptor. Expression analyses of these receptors and the results of the peristaltic motion assay indicated that these peptides participated in the regulation of intestinal contraction. Exposure of pharynx and ileum to AT and GSRYamide inhibited spontaneous contraction in ad libitum-fed larvae, while exposure of pharynx to GSRYamide did not inhibit contraction in non-fed larvae, indicating that the feeding state changed their sensitivity to inhibitory peptides. These different responses corresponded to different expression levels of their receptors in the pharynx. In addition, injection of AT and GSRYamide decreased esophageal contraction frequencies in the melamine-treated transparent larvae. These findings strongly suggest that these peptides exert feeding inhibitory effects by modulating intestinal contraction in response to their feeding state transition, eventually causing feeding termination., Competing Interests: NK is employed by LPixel Inc. There are no other competing interests to declare. This does not affect our adherence to PLOS ONE policies on data or materials sharing.

As detoxification enzymes, proteins in the glutathione S-transferase (GST) superfamily are reported to participate in oxidative stress resistance. Nevertheless, microsomal GSTs (MGSTs), a unique subclass of the GST superfamily associated with membranes, are rarely studied in insects. Here, we isolated an MGST gene in Apis cerana cerana (AccMGST1) and verified its role in oxidative stress response. We found higher expression of AccMGST1 in protective or defensive tissue, that is, the epidermis, which indicated its role in stress resistance. Real-time quantitative PCR (qRT-PCR) analysis indicated that AccMGST1 was upregulated by oxidative stresses at the transcriptional level. In contrast, AccMGST1 expression was inhibited when the antioxidant vitamin C (VC) was fed to experimental bees. Through western blotting, we found that the protein level of AccMGST1 under oxidative stress corresponded to the transcript level. Disc diffusion and mixed-function oxidation (MFO) assays suggested that AccMGST1 can protect not only cells but also DNA against oxidative damage. Furthermore, we discovered that the expression patterns of known antioxidant genes were changed in A. cerana cerana after AccMGST1 was silenced by RNA interference (RNAi). Thus, we concluded that the gene AccMGST1 exerts a significant role in the antioxidant mechanism.

Pheromone-binding proteins (PBPs) are thought to bind and transport sex pheromones onto the olfactory receptors on the dendrite membrane of olfactory neurons, and thus play a vital role in sex pheromone perception. However, the function of PBPs has rarely been demonstrated in vivo. In this study, two PBPs (PBP1 and PBP3) of Chilo suppressalis, one of the most notorious pyralid pests, were in vivo functionally characterized using insects with the PBP gene knocked out by the CRISPR/Cas9 system. First, through direct injection of PBP-single guide RNA (sgRNA)/Cas9 messenger RNA into newly laid eggs, a high rate of target-gene editing (checked with polled eggs) was induced at 24 h after injection, 21.3% for PBP1-sgRNA injected eggs and 19.5% for PBP3-sgRNA injected eggs. Second, by an in-crossing strategy, insects with mutant PBP1 or PBP3 (both with a premature stop codon) were screened, and homozygous mutants were obtained in the G3 generation. Third, the mutant insects were measured for electroantennogram (EAG) response to female sex pheromones. As a result, both PBP mutant males displayed significant reduction in EAG response, and this reduction in PBP1 mutants was higher than that in PBP3 mutants, indicating a more important role of PBP1. Finally, the relative importance of two PBPs and the possible off target effect induced by sgRNA-injection are discussed. Taken together, our study provides a deeper insight into the function of and interaction between different PBP genes in sex pheromone perception of C. suppressalis, as well as a valuable reference in methodology for gene functional study in other genes and other moth species., (© 2017 The Authors. Insect Science published by John Wiley & Sons Australia, Ltd on behalf of Institute of Zoology, Chinese Academy of Sciences.)

Hematological and neurological expressed 1-like (HN1L) protein is an evolutionarily conserved protein that plays an important role in embryonic development. It has been reported that HN1L is involved in the process of cell growth and cancer formation and that cell cycle arrest occurs during suppression of HN1L expression. Previous studies have demonstrated that the expression levels of the Bombyx mori HN1L protein were significantly downregulated in Bombyx mori Nucleopolyhedrovirus (BmNPV) infected silkworm cells. Transient transfections were performed with plasmids for pIEX-1-HN1L expression in Bombyx mori ovarian cells (BmN) in order to explore the effect of the HN1L protein on the growth of silkworm cells and its regulatory role in the process of viral infection. Cellular localization analysis revealed that HN1L was localized in the cytoplasm and that its upregulation could significantly enhance cellular activity. Furthermore, HN1L could promote G1/S phase conversion, thereby contributing to cell proliferation. Upon infection of BmN cells with BmNPV, the induction of apoptosis increased, although HN1L overexpression could inhibit DNA fragmentation, suggesting that the HN1L protein could inhibit cell apoptosis induced by viral invasion. In addition, Western blotting indicated that the HN1L protein inhibited the activation of caspase-9 zymogen and the expression of Bax protein, although it promoted Bcl-2 expression. Flow cytometry analysis further confirmed that overexpression of HN1L significantly inhibited apoptosis induced by BmNPV infection. Consequently, we demonstrated that BmN HN1L is a protein with multiple functions, which enhanced cell activity, regulated the cell cycle and induced an anti-apoptotic response by BmNPV infection., Competing Interests: The authors have declared that no competing interests exist.

Cuticle tanning occurs in insects immediately after hatching or molting. During this process, the cuticle becomes dark and rigid due to melanin deposition and protein crosslinking. In insects, different from mammals, melanin is synthesized mainly from dopamine, which is produced from DOPA by the enzyme DOPA decarboxylase. In this work, we report that the silencing of the RpAadc-2 gene, which encodes the putative Rhodnius prolixus DOPA decarboxylase enzyme, resulted in a reduction in nymph survival, with a high percentage of treated insects dying during the ecdysis process or in the expected ecdysis period. Those treated insects that could complete ecdysis presented a decrease in cuticle pigmentation and hardness after molting. In adult females, the knockdown of AADC-2 resulted in a reduction in the hatching of eggs; the nymphs that managed to hatch failed to tan the cuticle and were unable to feed. Despite the failure in cuticle tanning, knockdown of the AADC-2 did not increase the susceptibility to topically applied deltamethrin, a pyrethroid insecticide. Additionally, our results showed that the melanin synthesis pathway did not play a major role in the detoxification of the excess (potentially toxic) tyrosine from the diet, an essential trait for hematophagous arthropod survival after a blood meal., (Copyright © 2019. Published by Elsevier Ltd.)

Development requires highly coordinated changes in chromatin accessibility in order for proper gene regulation to occur. Here, we identify factors associated with major, discrete changes in chromatin accessibility during butterfly wing metamorphosis. By combining mRNA sequencing (mRNA-seq), assay for transposase-accessible chromatin using sequencing (ATAC-seq), and machine learning analysis of motifs, we show that distinct sets of transcription factors are predictive of chromatin opening at different developmental stages. Our data suggest an important role for nuclear hormone receptors early in metamorphosis, whereas PAS-domain transcription factors are strongly associated with later chromatin opening. Chromatin immunoprecipitation sequencing (ChIP-seq) validation of select candidate factors showed spineless binding to be a major predictor of opening chromatin. Surprisingly, binding of ecdysone receptor (EcR), a candidate accessibility factor in Drosophila, was not predictive of opening but instead marked persistent sites. This work characterizes the chromatin dynamics of insect wing metamorphosis, identifies candidate chromatin remodeling factors in insects, and presents a genome assembly of the model butterfly Junonia coenia., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Body size in holometabolous insects is determined by the size at which the juvenile larva undergoes metamorphosis to the pupal stage. To undergo larva-pupa transition, larva must reach a critical developmental checkpoint, the threshold size (TS); however, the molecular mechanisms through which the TS cues this transition remain to be fully characterized. Here, we use the flour beetle Tribolium castaneum to characterize the molecular mechanisms underlying entry into metamorphosis. We found that T. castaneum reaches a TS at the beginning of the last larval instar, which is associated with the downregulation of TcKr-h1 and the upregulation of TcE93 and TcBr-C. Unexpectedly, we found that while there is an association between TS and TcE93 upregulation, it is the latter that constitutes the molecular trigger for metamorphosis initiation. In light of our results, we evaluate the interactions that control the larva-pupa transition and suggest alternative models., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)