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The diamondback moth (DBM), Plutella xylostella (L.), has evolved resistance to multiple insecticides including Bacillus thuringiensis (Bt). ATP-binding cassette (ABC) transporters are a class of transmembrane protein families, involved in multiple physiological processes and pesticide resistances in insects. However, the role and regulatory mechanism of ABC transporter in mediating the response to Bt Cry1Ac toxin remain unclear. Here, we characterized a MAPK signaling pathway-enriched ABCG subfamily gene PxABCG20 from DBM, and found it was differentially expressed in the Cry1Ac-resistant and Cry1Ac-susceptible strains. RNAi knockdown of PxABCG20 increased the tolerance of DBM to Cry1Ac protoxin. To explore the regulatory mechanism of PxABCG20 expression, we predicted the potential miRNAs targeting PxABCG20 using two target prediction algorithms. Luciferase reporter assay confirmed that novel-miR-310 was able to down-regulate PxABCG20 expression in HEK293T cells. Furthermore, injection of novel-miR-310 agomir markedly inhibited PxABCG20 expression, resulting in increased tolerance to Cry1Ac protoxin in susceptible strain, while injection of novel-miR-310 antagomir markedly induced the expression of PxABCG20, leading to decreased tolerance to Cry1Ac protoxin. Our work provides theoretical basis for exploring novel targets for the DBM response to Cry1Ac toxin and expands the understanding of miRNA role in mediating the susceptibility of insect pest to Cry1Ac toxin.

Odorant-binding protein (OBP) is a potential target for developing insect behavior control agents due to its properties in transporting semiochemicals. In this study, 12 novel jasmonic acid (JA) derivatives were rationally designed and synthesized based on the binding features between

The bean bug Riptortus pedestris is a notorious insect pest that can damage various crops, especially soybean, in East Asia. In insects, the olfactory system plays a crucial role in host finding and feeding behaviour in which the odorant-binding proteins (OBPs) are believed to be involved in initial step in this system. In this study, we produced the R. pedestris adult antennae-expressed RpedOBP4 protein using a recombinant expression system in E. coli. Fluorescence competitive binding confirmed that RpedOBP4 has binding affinities to 7 of 20 soybean volatiles (ligands), and that a neutral condition is the best environment for it. The binding property of RpedOBP4 to these ligands was further revealed by integrating data from molecular docking, site-directed mutagenesis and ligand binding assays. This demonstrated that five amino acid residues (I30, L33, Y47, I57 and Y121) are involved in the binding process of RpedOBP4 to corresponding ligands. These findings will not only help us to more thoroughly explore the olfactory mechanism of R. pedestris during feeding on soybean, but also lead to the identification of key candidate targets for developing environmental and efficient behaviour inhibitors to prevent population expansion of R. pedestris in the future.

The eastern Apis cerana (Ac) and the western Apis mellifera (Am) are two closely related and most economically valuable honeybee species managed extensively worldwide. However, how worker bees of Ac and Am are adapted to their colony organization remains to be uncovered. Here, we found that the expression level of gene encoding antennae-specific proteins 1 (ASP1, a key regulator in recognizing queen mandibular pheromone) was positively correlated with the colony sizes in both bee species, and the expression level in Am was higher than that in Ac, suggesting that ASP1 may play an important role in maintaining colony homeostasis. Using competitive binding assay, molecular docking, and site-directed mutagenesis, we then confirmed the good binding affinities of both Ac-ASP1 and Am-ASP1 to methyl p-hydroxy benzoate (HOB), and Val115 was the key amino acid. However, the affinity of Am-ASP1 was stronger than that of Ac-ASP1. EAG analysis further demonstrated that antennae of Am worker bees had faster depolarization and repolarization in response to HOB stimulation. Taken together, these findings indicate that the differences in expression levels and binding dynamics allow ASP1 recognizing HOB to potentially serve as a specific regulator of colony organization in Ac and Am.

The protein vitellogenin (Vg) plays a central role in lipid transportation in most egg-laying animals. High Vg levels correlate with stress resistance and lifespan potential in honey bees (Apis mellifera). Vg is the primary circulating zinc-carrying protein in honey bees. Zinc is an essential metal ion in numerous biological processes, including the function and structure of many proteins. Measurements of Zn2+ suggest a variable number of ions per Vg molecule in different animal species, but the molecular implications of zinc-binding by this protein are not well-understood. We used inductively coupled plasma mass spectrometry to determine that, on average, each honey bee Vg molecule binds 3 Zn2+-ions. Our full-length protein structure and sequence analysis revealed seven potential zinc-binding sites. These are located in the β-barrel and α-helical subdomains of the N-terminal domain, the lipid binding site, and the cysteine-rich C-terminal region of unknown function. Interestingly, two potential zinc-binding sites in the β-barrel can support a proposed role for this structure in DNA-binding. Overall, our findings suggest that honey bee Vg bind zinc at several functional regions, indicating that Zn2+-ions are important for many of the activities of this protein. In addition to being potentially relevant for other egg-laying species, these insights provide a platform for studies of metal ions in bee health, which is of global interest due to recent declines in pollinator numbers. publishedVersion

Dosage compensation is a process that produces a similar expression of sex-linked and autosomal genes. In the silkworm Bombyx mori with a WZ sex-determination system, the expression from the single Z in WZ females matches that of ZZ males due to the suppression of Z-linked genes in males. A primary maleness determinant gene, Masculinizer (Masc), is also required for dosage compensation. In females, P-element induced wimpy testis (PIWI) is complexed with the W chromosome-derived female-specific Feminizer (Fem) PIWI-interacting RNA (piRNA) and cleaves Masc mRNA. When Fem piRNA-resistant Masc cDNA (Masc-R) is overexpressed in both sexes, only female larvae are dead during the larval stage. In this study, transcriptome analysis was performed in neonate larvae to examine the effects of Masc-R overexpression on a global gene expression profile. Z-linked genes were globally repressed in Masc-R-overexpressing females due to force-driven dosage compensation. In contrast, Masc-R overexpression had little effect on the expression of Z-linked genes and the male-specific isoform of B. mori insulin-like growth factor II mRNA-binding protein in males, indicating that excessive Masc expression strengthens neither dosage compensation nor maleness in males. Fourteen genes were differentially expressed between Masc-R-overexpressing and control neonate larvae in both sexes, suggesting Masc functions other than dosage compensation and masculinization.HighlightsTranscriptome analysis was performed in Masc-R-overexpressing neonate Bombyx mori larvae.Z-linked genes were globally suppressed in Masc-R-overexpressing females.Masc-R overexpression had little effect on BmImpM expression in males.Several genes may be controlled by Masc-R regardless of sex.

Intergenerational effects from fathers to offspring are increasingly reported from diverse organisms, but the underlying mechanisms remain speculative. Paternal trans-generational immune priming (TGIP) was demonstrated in the red flour beetle Tribolium castaneum: non-infectious bacterial exposure of fathers protects their offspring against an infectious challenge for at least two generations. Epigenetic processes, such as cytosine methylation of nucleic acids, have been proposed to enable transfer of information from fathers to offspring. Here we studied a potential role in TGIP of the Dnmt2 gene (renamed as Trdmt1 in humans), which encodes a highly conserved enzyme that methylates different RNAs, including specific cytosines of a set of tRNAs. Dnmt2 has previously been reported to be involved in intergenerational epigenetic inheritance in mice and protection against viruses in fruit flies. We first studied gene expression and found that Dnmt2 is expressed in various life history stages and tissues of T. castaneum, with high expression in the reproductive organs. RNAi-mediated knockdown of Dnmt2 in fathers was systemic, slowed down offspring larval development and increased mortality of the adult offspring upon bacterial infection. However, these effects were independent of bacterial exposure of the fathers. In conclusion, our results point towards a role of Dnmt2 for paternal effects, while elucidation of the mechanisms behind paternal TGIP needs further studies.

Chitin, a major component of insect cuticles, plays a critical role in insect molting and morphogenesis. Thus, coordination of chitin remodeling during insect development requires tight transcriptional control of the chitin metabolism genes involved in chitin synthesis, assembly and degradation. However, the molecular mechanism underlying transcriptional coordination of chitin metabolism genes during beetle development is not yet completely understood.We cloned the full-length cDNA encoding hormone receptor 3 (TcHR3) from Tribolium castaneum and showed a critical role of TcHR3 in modulating chitin metabolism gene expression during molting. Genome-wide transcriptome analysis of HR3-deficient old larvae using RNA sequencing analysis revealed a positive correlation between TcHR3 and transcription of chitin metabolism genes involved in chitin synthesis and degradation. In addition, HR3 overexpression significantly induced the gene promoter activity of N-acetylglucosaminidase 1 (NAG1) involved in chitin degradation and UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1) involved in chitin synthesis. Chromatin immunoprecipitation analysis revealed that HR3 could directly bind to HR3-response element of NAG1 and UAP1 promoters. Finally, HR3-deficient late instar larvae and prepupae exhibited defects in larval-larval and larval-pupal molting, respectively, leading to eventual larval death because developing larvae were trapped inside the old cuticle as a result of abnormal chitin metabolism.TcHR3 is a transcriptional regulator of chitin metabolic genes for molting of T. castaneum. Controlling the molting system by TcHR3 might be a new management strategy for selective control of red flour beetle infestation. © 2022 Society of Chemical Industry.

Cuticular proteins (CPs) are known to play important roles in insect development and defence responses. The loss of CP genes can lead to changes in insect morphology and sensitivity to the external environment. In this study, we identified the AccCPR2 gene, which belongs to the CPR family (including the RR consensus motif) of CPs, and explored its function in the response of Apis cerana cerana to adverse external stresses. Our results demonstrated that AccCPR2 was highly expressed in the late pupal stage and epidermis, and the expression of AccCPR2 may be induced or inhibited under different stressors. RNA interference experiments showed that knockdown of AccCPR2 reduced the activity of antioxidant enzymes, led to the accumulation of oxidative damage and suppressed the expression of several antioxidant genes. In addition, knockdown of AccCPR2 also reduced the pesticide resistance of A. cerana cerana. The overexpression of AccCPR2 in a prokaryotic system further confirmed its role in resistance to various stresses. In summary, AccCPR2 may play pivotal roles in the normal development and environmental stress response of A. cerana cerana. This study also enriched the theoretical knowledge of the resistance biology of bees.

Aphids are hemimetabolous insects that undergo incomplete metamorphosis without pupation. The annual life cycle of most aphids includes both an asexual (viviparous) and a sexual (oviparous) phase. Sexual reproduction only occurs once per year and is followed by many generations of asexual reproduction, during which aphids propagate exponentially with telescopic development. Here, we discuss the potential links between viviparous embryogenesis and derived developmental features in the pea aphidAcyrthosiphon pisum, particularly focusing on germline specification and axis determination, both of which are key events of early development in insects. We also discuss potential evolutionary paths through which both viviparous and oviparous females might have come to utilize maternal germ plasm to drive germline specification. This developmental strategy, as defined by germline markers, has not been reported in other hemimetabolous insects. In viviparous females, furthermore, we discuss whether molecules that in other insects characterize germ plasm, like Vasa, also participate in posterior determination and how the anterior localization of thehunchbackorthologueAp-hbestablishes the anterior-posterior axis. We propose that the linked chain of developing oocytes and embryos within each ovariole and the special morphology of early embryos might have driven the formation of evolutionary novelties in germline specification and axis determination in the viviparous aphids. Moreover, based upon the finding that the endosymbiontBuchnera aphidicolais closely associated with germ cells throughout embryogenesis, we propose presumptive roles forB. aphidicolain aphid development, discussing how it might regulate germline migration in both reproductive modes of pea aphids. In summary, we expect that this review will shed light on viviparous as well as oviparous development in aphids.

The cigarette beetle, Lasioderma serricorne (Fabricius) (Coleoptera: Anobiidae), is a destructive stored product pest worldwide. Adult cigarette beetles are known to rely on host volatiles and pheromones to locate suitable habitats for oviposition and mating, respectively. However, little is known about the chemosensory mechanisms of these pests. Soluble chemoreception proteins are believed to initiate olfactory signal transduction in insects, which play important roles in host searching and mating behaviors. In this study, we sequenced the antennal transcriptome of L. serricorne and identified 14 odorant-binding proteins (OBPs), 5 chemosensory proteins (CSPs), and 2 Niemann-Pick C2 proteins (NPC2). Quantitative realtime PCR (qPCR) results revealed that several genes (LserOBP2, 3, 6, and 14) were predominantly expressed in females, which might be involved in specific functions in this gender. The five LserOBPs (LserOBP1, 4, 8, 10, and 12) that were highly expressed in the male antennae might encode proteins involved in specific functions in males. These findings will contribute to a better understanding of the olfactory system in this stored product pest and will assist in the development of efficient and environmentally friendly strategies for controlling L. serricorne.

Laodelphax striatellus transmits rice stripe virus (RSV) during sap feeding on the rice plant. The insect saliva proteins have direct and indirect roles in mediating RSV transmission; however, the function of most saliva proteins remains unclear.In this study, we sequenced L. striatellus saliva proteins using shotgun liquid chromatography-electrospray ionization-tandem mass spectrometry. We identified 41 secreted saliva proteins, among which a saliva mucin-like protein, designated LssaMP, was the most abundant. In silico analysis revealed the sequence conservation among planthoppers. We revealed that the LssaMP gene is specifically expressed in the salivary glands and the protein is secreted as a component of gel saliva. Using LssaMP-specific double-stranded RNA (dsRNA) to silence gene expression, we revealed that LssaMP is required for formation of the salivary sheath, an important structure for sap feeding. Disrupting LssaMP expression resulted in inefficient formation of the feeding structure, thereby stopping insects from secreting watery saliva and acquiring sufficient nutrients from the phloem sap. We confirmed that RSV is mainly released via the watery saliva, which passes through the salivary sheathes into the plant phloem. An insufficient feeding structure results in decreased release of watery saliva, as well as the arboviruses.This study clarified the function of an insect saliva protein in mediating insect feeding, as well as arbovirus transmission. © 2022 Society of Chemical Industry.

Rearing silkworms (Bombyx mori) using formula feed has revolutionized traditional mulberry feed strategies. However, low silk production efficiencies persist and have caused bottlenecks, hindering the industrial application of formula feed sericulture. Here, we investigated the effects of formula feed amino acid composition on silk yields. We showed that imbalanced amino acids reduced DNA proliferation, decreased Fib-H, Fib-L, and P25 gene expression, and caused mild autophagy in the posterior silk gland, reducing cocoon shell weight and ratio. When compared with mulberry leaves, Gly, Ala, Ser, and Tyr percentages of total amino acids in formula feed were decreased by 5.26%, while Glu and Arg percentages increased by 9.56%. These changes increased uric acid and several amino acids levels in the hemolymph of silkworms on formula feed. Further analyses showed that Gly and Thr (important synthetic Gly sources) increased silk yields, with Gly increasing amino acid conversion efficiencies to silk protein, and reducing urea levels in hemolymph. Also, Gly promoted endomitotic DNA synthesis in silk gland cells via phosphoinositide 3-kinase (PI3K)/Akt/target of rapamycin (TOR) signaling. In this study, we highlighted the important role of Gly in regulating silk yields in silkworms.

The M14 family metal carboxypeptidase genes play an important role in digestion and pathogenic infections in the gut of insects. However, the roles of these genes in Antheraea pernyi (Guérin-Méneville, 1855) remain to be analyzed. In the present study, we cloned a highly expressed M14 metal carboxypeptidase gene (ApMCP1) found in the gut and discovered that it contained a 1,194 bp open reading frame encoding a 397-amino acid protein with a predicted molecular weight of 45 kDa. Furthermore, 14 members of the M14 family metal carboxypeptidases (ApMCP1–ApMCP14) were identified in the A. pernyi genome, with typical Zn_pept domains and two Zn-anchoring motifs, and were further classified into M14A, M14B, and M14D subfamilies. Expression analysis indicated that ApMCP1 and ApMCP9 were mainly expressed in the gut. Additionally, we observed that ApMCP1 and ApMCP9 displayed opposite expression patterns after starvation, highlighting their functional divergence during digestion. Following natural infection with baculovirus NPV, their expression was significantly upregulated in the gut of A. pernyi. Our results suggest that the M14 family metal carboxypeptidase genes are conservatively digestive enzymes and evolutionarily involved in exogenous pathogenic infections.

The black carabid beetle Calosoma maximoviczi is a successful predator that serves as both a beneficial insect and a severe threat to economic herbivores. Its hunting technique relies heavily on olfaction, but the underlying mechanism has not been studied. Here, we report the electrophysiological, ecological and molecular traits of bioactive components identified from a comprehensive panel of natural odorants in the beetle-prey-plant system. The aim of this work was to investigate olfactory perceptions and their influence on the behaviours of C. maximoviczi.Among the 200 identified volatiles, 18 were concentrated in beetle and prey samples, and 14 were concentrated in plants. Insect feeding damage to plants led to a shift in the emission fingerprint. Twelve volatiles were selected using successive electrophysiological tests. Field trials showed that significant sex differences existed when trapping with a single chemical or chemical mixture. Expression profiles indicated that sex-biased catches were related to the expression of 15 annotated CmaxOBPs and 40 CmaxORs across 12 chemosensory organs. In silico evaluations were conducted with 16 CmaxORs using modelling and docking. Better recognition was predicted for the pairs CmaxOR5-(Z)-3-hexenyl acetate, CmaxOR6-β-caryophyllene, CmaxOR18-(E)-β-ocimene and CmaxOR18-tetradecane, with higher binding affinity and a suitable binding pocket. Lastly, 168Y in CmaxOR6 and 142Y in CmaxOR18 were predicted as key amino acid residues for binding β-caryophyllene and tetradecane, respectively.This work provides an example pipeline for de novo investigation in C. maximoviczi baits and the underlying olfactory perceptions. The results will benefit the future development of trapping-based integrated pest management strategies and the deorphanization of odorant receptors in ground beetles. © 2022 Society of Chemical Industry.

The

RNA interference (RNAi) selectively targets genes and silences their expression in vivo, causing developmental defects, mortality and altered behavior. Consequently, RNAi has emerged as a promising research area for insect pest management. However, it is not yet a viable alternative over conventional pesticides despite several theoretical advantages in safety and specificity. As a first step toward a more standardized approach, a machine learning algorithm was used to identify factors that predict trial efficacy. Current research on RNAi for pest management is highly variable and relatively unstandardized. The applied random forest model was able to reliably predict mortality ranges based on bioassay parameters with 72.6% accuracy. Response time and target gene were the most important variables in the model, followed by applied dose, double-stranded RNA (dsRNA) construct size and target species, further supported by generalized linear mixed effect modeling. Our results identified informative trends, supporting the idea that basic principles of toxicology apply to RNAi bioassays and provide initial guidelines standardizing future research similar to studies of traditional insecticides. We advocate for training that integrates genetic, organismal, and toxicological approaches to accelerate the development of RNAi as an effective tool for pest management. © 2022 The Authors. Pest Management Science published by John WileySons Ltd on behalf of Society of Chemical Industry.

Edible insect production relies, among others, on the use of high-quality strains with good productivity characteristics. However, studies on the evaluation of the growth performance of different insect strains are limited. In this framework, the objective of the present study was to evaluate the growth and development under dry conditions of larvae of six Tenebrio molitor strains of different geographical origins, i.e. Greece, Italy, Turkey, Spain and USA, in terms of larval survival and development, as well as feed utilisation efficiency. In light of our results, we report the variable growth performance of different T. molitor strains in the absence of a moisture source. Although no significant differences were shown among the strains tested with regard to the final larval weights and survival rates, there were significant differences in the development time and the growth rate, which may affect the overall efficiency of T. molitor larvae production. Our study aims to highlight the significance of strain for the growth performance and consequently the productivity of T. molitor.

Crystal (Cry) toxins produced from the soil bacterium, Bacillus thuringiensis (Bt), have gained worldwide attention for long due to their insecticidal potential. A number of receptor proteins located on the epithelial cells of the larval midgut were shown to be crucial for Cry intoxication in different insect pests belonging to order Lepidoptera, Diptera and Coleoptera. A beehive pest, Galleria mellonella, serves as an excellent insect model for biochemical research. However, information on the Cry receptor-like genes in G. mellonella is limited. In the present study, the full-length sequences of four putative Cry receptor genes (ABC transporter, alkaline phosphatase, aminopeptidase N and cadherin) were cloned from G. mellonella. All these receptor genes were substantially upregulated in the midgut tissue of fourth-instar G. mellonella larvae upon early exposure (6 h) to a sub-lethal dose of Cry1AcF toxin. Oral and independent delivery of bacterially-expressed dsRNAs corresponding to four receptor genes in G. mellonella suppressed the transcription of target receptors which in turn significantly reduced the larval sensitivity to Cry1AcF toxin. As the laboratory populations of G. mellonella develop Bt resistance in a relatively short time, molecular characterization of Cry receptor genes in G. mellonella performed in the present study may provide some useful information for future research related to the genetic basis of Bt resistance in the model insect.

At each molt of Manduca, the large dermal secretory cells expel the protein contents of their vacuoles into the hemocoel. The constellation of proteins expelled at the last larval-pupal molt, however, differs qualitatively from those proteins released at earlier larval-larval molts. Secretory cells at the two stages not only have different lectin staining properties but also have different proteins that separate on two-dimensional gels. Numerous physiological changes accompany the termination of the last larval instar, including increased chitin synthesis, diminished oxygen delivery, and reduced humoral immunity. Secretion of trehalase that is essential for chitin synthesis and the release of hypoxia up-regulated protein to ameliorate oxygen deprivation help ensure normal transition from larva to pupa. Proteins released by dermal secretory cells at this last molt could supplement the diminished immune defenses mediated by fat body and hemocytes at the end of larval life. Additional immune defenses provided by dermal secretory cells could help ensure a safe transition during a period of increased vulnerability for the newly molted pupa with its soft, thin cuticle and reduced mobility.

Lytic polysaccharide monooxygenases (LPMOs) are important enzymes that boost the hydrolysis of recalcitrant polysaccharides, such as chitin. They are found extensively in different insect species and are classified as auxiliary activities family 15 (AA15) LPMOs (LPMO15). Some of them were identified from the insect midgut and proven to act on chitin. However, knowledge about their physiological roles during insect growth and development remains limited. Here, we found that midgut-specific LPMO15s are widely distributed in different insect orders, such as the orthopteran Locusta migratoria and the lepidopteran Bombyx mori. Using L. migratoria as a model insect, the function of midgut-specific LmLPMO15-3 during development was investigated. Double-stranded RNA-mediated downregulation of LmLPMO15-3 expression at the 4th or 5th instar nymph stage severely decreased the survival rate and resulted in lethal phenotypes. Hematoxylin and eosin staining results indicated that the deficient individuals exhibited incompletely digested peritrophic matrix (PM), which suggested that LmLPMO15-3 is essential for the deconstruction of the PM during molting. This study provides direct evidence of the physiological importance of a midgut-specific LPMO15 during insect development. As L. migratoria is one of the most destructive agricultural pests, LmLPMO15-3 is a potential target for pest management.

Insect midgut plays a central role in food digestion and nutrition absorption. Larval silkworm midgut could be divided into 3 distinct regions based on their morphological colors. However, it remains rudimentary of regional gene expression and physiological function in larval silkworm midgut. Through transcriptome sequencing of 3 midgut compartments, a comprehensive analysis of gene expression atlas along the anterior-posterior axis was conducted. Posterior midgut was found transcriptionally divergent from anterior and middle midgut. Differentially expressed gene analysis revealed the regional specialization of digestive enzyme production, transmembrane transport, chitin metabolism, and hormone regulation in different midgut regions. In addition, gene subsets of pan-midgut and region-specific transcription factors (TFs) along the length of midgut were also identified. The results suggested that homeobox TFs might play an essential role in transcriptional variations across the midgut. Altogether, our study provided the first fundamental resource to investigate physiological function and regulation mechanism in larval midgut compartmentalization.

RNA interference (RNAi) is a breakthrough technology in pest control. It is highly efficient to Coleopteran pests such as the Colorado potato beetle Leptinotarsa decemlineata, a serious pest defoliator mainly attacking potatoes worldwide. The first step for effective pest control by RNAi is the development of effective and reliable target genes.Our results revealed that continuous ingestion of dsLdRan for 3 days successfully silenced the target gene, inhibited larval growth and killed 100% L. decemlineata larvae. When the bioassay began at the second-, third/fourth-instar larval stages, the larval lethality mainly occurred at the fourth larval instar and prepupal stages, respectively. Importantly, consumption of dsLdRan for 3 days by the newly-emerged males and females effectively knocked down the target transcript, reduced fresh weights and caused 100% of lethality within a week. The LdRan females possessed underdeveloped ovaries.Considering that the larvae, adults and eggs are simultaneously sited on the potato plants, bacterially-expressed dsLdRan is a potential RNAi-based strategy for managing L. decemlineata in the potato field. © 2022 Society of Chemical Industry.

Insect wings are subject to strong selective pressure, resulting in the evolution of remarkably diverse wing morphologies that largely determine flight capacity. However, the genetic basis and regulatory mechanisms underlying wing size and shape development are not well understood. The silkworm Bombyx mori micropterous (mp) mutant exhibits shortened wing length and enlarged vein spacings, albeit without changes in total wing area. Thus, the mp mutant comprises a valuable genetic resource for studying wing development. In this study, we used molecular mapping to identify the gene responsible for the mp phenotype and designated it Bmmp. Phenotype-causing mutations were identified as indels and single nucleotide polymorphisms in noncoding regions. These mutations resulted in decreased Bmmp messenger RNA levels and changes in transcript isoform composition. Bmmp null mutants were generated by clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 and exhibited changed wing shape, similar to mp mutants, and significantly smaller total wing area. By examining the expression of genes critical to wing development in wildtype and Bmmp null mutants, we found that Bmmp exerts its function by coordinately modulating anterior-posterior and proximal-distal axes development. We also studied a Drosophila mp mutant and found that Bmmp is functionally conserved in Drosophila. The Drosophila mp mutant strain exhibits curly wings of reduced size and a complete loss of flight capacity. Our results increase our understanding of the mechanisms underpinning insect wing development and reveal potential targets for pest control.

Silkworm pupa (SWP) food anaphylaxis has been described frequently in Asian countries. However, false-positive reactions by skin pricks and serum IgE (sIgE) tests to the extract complicate diagnosis, requiring identification of clinically relevant major allergens.In this study, we characterized a novel SWP allergen, Bomb m 4, a 30-kDa lipoprotein, and evaluated its diagnostic sensitivity.Bomb m 4 was identified by a proteomic analysis. This recombinant (r)Bomb m 4 was overexpressed in Escherichia coli, and the IgE reactivity by ELISA was compared with other reported allergenic proteins: Bomb m 1 (arginine kinase), 27-kDa glycoprotein, Bomb m 3 (tropomyosin) using the serum samples from 17 SWP allergic patients and 11 asymptomatic sensitized subjects.rBomb m 4-specific IgE was recognized by all 17 SWP allergic patients. The 27-kDa glycoprotein and Bomb m 1 sIgE were found in 35.3% and 0%, respectively, in the SWP allergic patients. ELISA sIgE reactivity increased significantly, when 4 M urea was added in serum samples. However, only 16% inhibition of sIgE reactivity to the whole SWP extract was exhibited by rBomb m 4, whereas more than 93% of self-inhibition of rBomb m 4 sIgE was obtained, possibly due to the low abundance of Bomb m 4 in the extract. Three linear epitopes (81-95, 191-205 and 224-238 residues) of rBomb m 4 were identified. These epitopes are shown to be released by pepsin digestion. Receiver operator characteristic (ROC) analysis showed the highest diagnostic value of Bomb m 4 followed by Bomb m 1, 27-kDa glycoprotein and Bomb m 3.Bomb m 4 is the major allergen of SWP allergic patients. It has cryptic epitopes which are exposed to IgE antibodies with digestive enzymes. This recombinant Bomb m 4 allergen permits exact diagnosis of SWP allergy.

Destruxin A (DA) is a mycotoxin secreted by entomogenous fungi, such as Metarhizium anisopliae, which has broad-spectrum insecticidal activity. Insect innate immunity provides resistance against the invasion of entomopathogenic fungi. Previous studies have shown that DA could inhibit the immune response, however, the suppressive mechanism of DA on prophenoloxidase system is still unknown.Based on the transcriptome of Aphis citricola, we screened the scavenger receptor class B(AcSR-B)and identified that it significantly responds to DA. Spatio-temporal expression analysis showed that AcSR-B is highly expressed in adult stage and is mainly distributed in the abdominal region. We further revealed that both M. anisopliae and Escherichia coli could suppress the expression of AcSR-B at 24 h, and that the expressed recombinant protein rAcSR-B possessed agglutination activity to M. anisopliae and E. coli. DA could suppress the protein expression of AcSR-B. In addition, RNA interference of AcSR-B caused death of A. citricola in a dose-dependent manner, and RNA interference of AcSR-B increased mortality in A. citricola under the same lethal concentration of DA. The inhibiting effect of AcSR-B silencing was similar with the DA treatment upon phenol oxidase (PO) activity of A. citricola hemolymph. DA could not decrease PO activity further after AcSR-B silencing.Destruxin A inhibits melanization by suppressing AcSR-B in A. citricola. Our findings are helpful in understanding the underlying molecular mechanism of the DA suppressing immune system, and uncover a potential molecular target for double-stranded RNA (dsRNA) insecticides.

The E2F family of transcription factors is crucial for cell cycle progression and cell fate decisions. Although E2Fs have been widely studied in mammals, there have been few studies performed in insects. Here, we determined the function of E2F4 in the silkworm, Bombyx mori. We demonstrate that E2F proteins are highly conserved among species from lower animals to higher mammals. Overexpression of the BmE2F4 gene led to cell cycle arrest in the G1 phase, whereas interfering with the BmE2F4 mRNA led to accumulation of cells in the S phase. These results indicate that BmE2F4 is important in cell cycle regulation. We also demonstrate that the BmE2F4 gene is involved in DNA replication of BmN-SWU1 cells and DNA synthesis in the silk gland. Furthermore, we identified a protein called Bm14-3-3ζ that can interact with BmE2F4 and allow it to localize in the nucleus. Overexpression of the Bm14-3-3ζ gene led to cell cycle arrest in the G1 phase, while knocking down the gene increased the proportion of cells in S phase. These findings provide important insights into the function of E2F transcription factors and increase our understanding of their involvement in cell cycle regulation.

Juvenile hormone (JH) plays a key role in regulating insect reproductive processes. Methoprene-tolerant (Met), as a putative JH receptor, transduces JH signals by activating the transcription factor krüppel homolog 1 (Kr-h1). To understand the effects of Met and Kr-h1 genes on female reproduction of natural enemy insects, the Met and Kr-h1 were identified and analyzed from Harmonia axyridis Pallas (HmMet and HmKr-h1). The HmMet protein belonged to the bHLH-PAS family with bHLH domain, PAS domains, and PAC domain. HmMet mRNA was detected in all developmental stages, and the highest expression was found in the ovaries of female adults. The HmKr-h1 protein had eight C2H2-type zinc finger domains. HmKr-h1 mRNA was highly expressed from day 7 to day 9 of female adults. The tissue expression showed that HmKr-h1 was highly expressed in its wing, leg, and fat body. Knockdown of HmMet and HmKr-h1 substantially reduced the transcription of HmVg1 and HmVg2, inhibited yolk protein deposition, and reduced fecundity using RNA interference. In addition, the preoviposition period was significantly prolonged after dsMet-injection, but there was no significant difference after dsKr-h1-silencing. However, the effect on hatchability results was the opposite. Therefore, we infer that both HmMet and HmKr-h1 are involved in female reproduction of H. axyridis, and their specific functions are different in certain physiological processes. In several continents, H. axyridis are not only beneficial insects, but also invasive pests. This report will provide basis for applying or controlling the H. axyridis.

The development of novel and safe insecticides remains an important need for a growing world population to protect crops and animal and human health. New chemotypes modulating the insect nicotinic acetylcholine receptors have been recently brought to the agricultural market, yet with limited understanding of their molecular interactions at their target receptor. Herein, we disclose the first crystal structures of these insecticides, namely, sulfoxaflor, flupyradifurone, triflumezopyrim, flupyrimin, and the experimental compound, dicloromezotiaz, in a double-mutated acetylcholine-binding protein which mimics the insect-ion-channel orthosteric site. Enabled by these findings, we discovered novel pharmacophores with a related mode of action, and we describe herein their design, synthesis, and biological evaluation.

Periplaneta americana L. (PA), a type of animal medicine, has been widely used for wound healing in clinical settings. In order to further investigate the bioactive wound healing substances in PA, crude PA protein-polysaccharide complexes were further purified by cellulose DE-52 and Sephadex G100 chromatography in succession. Among these isolated fractions, two fractions eluted by 0.3 M and 0.5 M NaCl with the higher yield, respectively named PaPPc2 and PaPPc3 respectively, were chosen for the wound healing experiments. Mediated by HPGPC, amino acid and monosaccharide composition analysis, circular dichroism spectrum, glycosylation type, FT-IR, and

In Brazil, the major vector of arboviruses is Aedes aegypti, which can transmit several alpha and flaviviruses. In this work, a pacifastin protease inhibitor library was constructed and used to select mutants for Ae. aegypti larvae digestive enzymes. The library contained a total of 3.25 × 10

Apis mellifera major royal jelly protein 2 suppressed leukemia-initiating stem cell (LIC)-related oncogenes, MMP10 and HDAC8 activities, as well as CD34+ LICs in myeloid and lymphoid leukemia cells, resulting in their cell cycle arrest and apoptosis.

Background Pyrethroids are the most widely used insecticides for the control of malaria transmitting Anopheles gambiae mosquitoes and rapid increase in resistance to this insecticide class is of major concern. Pyrethroids target the Voltage Gated Sodium Channels (VGSCs), that have a key role in the normal function of the mosquitoes' nervous system. VGSC mutations L995F and L995S have long been associated with pyrethroid resistance and screening for their presence is routine in insecticide resistance management programs. Recently, a VGSC haplotype containing two amino acid substitutions associated with resistance in other species, V402L and I1527T, was identified. These two VGSC mutations are found in tight linkage and are mutually exclusive to the classical L995F/S mutations. Results We identify the presence of the V402L-I1527 T haplotype in resistant An. coluzzii colonized strains and in field populations from Burkina Faso at frequencies higher than previously reported; in some cases almost reaching fixation. Functional validation of V402L in insecticide resistance using a CRISPR/Cas9 genome modified line showed that it confers reduced mortality after exposure to all tested pyrethroids and DDT, but at lower levels compared to L995F. In contrast however to L995F, no fitness costs were identified for mosquitoes carrying V402L under laboratory conditions. Conclusion The V402L substitution confers pyrethroid resistance in An. gambiae in the absence of any other VGSC substitution and/or alternative resistance mechanisms. The lower fitness cost associated with this kdr mutation may provide a selective advantage over the classical kdr in some settings and genotyping at this locus should be added in the list of resistant alleles for routine screening. This article is protected by copyright. All rights reserved.

The SOCS/CIS is a family of intracellular proteins distributed widely among living organisms. The members of this family have extensively been studied in mammals and have been shown to regulate various physiological processes. In contrast, the functional roles of SOCS/CIS family proteins are unknown in most invertebrates, including insects. Here, we retrieved a full-length open reading frame (ORF) of SOCS-6 from Chines oak silkworm, Antheraea pernyi (Designated as ApSOCS-6), using the RNA-seq database. The predicted ApSOCS-6 amino acid sequence comprised an N-terminal SH2 domain and a C-terminal SOCS-box domain. It shared the highly conserved structures of the SOCS proteins with other lepidopteran species. ApSOCS-6 mRNA transcript was detected in all the tested tissues of the A. pernyi larvae; however, the highest mRNA levels were found in the larval hemocytes, fat bodies, and integuments. The mRNA transcript levels of ApSOCS-6 were increased in the A. pernyi larval hemocytes and fat bodies after a challenge by the Gram-positive bacteria, M. luteus, Gram-negative bacteria, Escherichia coli, Virus, ApNPV, and Fungus, B. bassiana. After the knockdown of ApSOCS-6, we found a significant increase in bacterial clearance and a decrease in the relative replication of bacteria. To evaluate the possible cause of enhanced antibacterial activity, we measured antimicrobial peptides expression in the fat body of A. pernyi larvae. The production of AMPs was strongly increased in the B. cereus infected larval fat bodies following silencing of ApSOCS-6. Our data indicate that ApSOCS-6 negatively regulates the expression of AMPs in immune tissues in response to the B. cereus challenge.

Background In insects, the chemosensory system is crucial in guiding their behaviors for survival. Plagiodera versicolora (Coleoptera: Chrysomelidae), is a worldwide leaf-eating forest pest in salicaceous trees. There is little known about the chemosensory genes in P. versicolora. Here, we conducted a transcriptome analysis of larvae heads in P. versicolora. Results In this study, 29 odorant binding proteins (OBPs), 6 chemosensory proteins (CSPs), 14 odorant receptors (ORs), 13 gustatory receptors (GRs), 8 ionotropic receptors (IRs) and 4 sensory neuron membrane proteins (SNMPs) were identified by transcriptome analysis. Compared to the previous antennae and foreleg transcriptome data in adults, 12 OBPs, 2 CSPs, 5 ORs, 4 IRs, and 7 GRs were newly identified in the larvae. Phylogenetic analyses were conducted and found a new candidate CO2 receptor (PverGR18) and a new sugar receptor (PverGR23) in the tree of GRs. Subsequently, the dynamic expression profiles of various genes were analyzed by quantitative real-time PCR. The results showed that PverOBP31, OBP34, OBP35, OBP38, and OBP40 were highly expressed in larvae, PverOBP33 and OBP37 were highly expressed in pupae, and PverCSP13 was highly expressed in eggs, respectively. Conclusions We identified a total of 74 putative chemosensory genes based on a transcriptome analysis of larvae heads in P. versicolora. This work provides new information for functional studies on the chemoreception mechanism in P. versicolora.

We found the activity of arylsulfatase in the midgut contents of the silkworm, Bombyx mori. We identified a 60-kDa protein that comigrates with the activity on a column chromatography following ammonium sulfate precipitation. Based on its partial amino acid sequence, we searched for its coding gene using Basic Local Alignment Search Tool (BLAST) and identified KWMTBOMO05106. Transcriptional data suggest a specific expression of the gene in middle silk glands. The majority (80%) of arylsulfatase activity was found in the silk glands, concurring the specific transcription in the silk gland. Observing the feeding behaviour of the silkworm, we found that silkworms smear a mucus secretes from the spinneret on the food pellet as they feed on. Arylsulfatase activity was also detected in the food pellet bitten by the silkworm as well as in the gut content. Furthermore, arylsulfatase activity was not detected either in the food pellet and in the gut content when silkworms had obstructed the spinneret. These results suggest that arylsulfatase is secreted from the silk glands and may contribute to digestive function.

Insect trehalases are glycoside hydrolases essential for trehalose metabolism and stress resistance. We here report the extraction and purification of Acyrthosiphon pisum soluble trehalase (ApTreh-1), its biochemical and structural characterization, as well as the determination of its kinetic properties. The protein has been purified by ammonium sulphate precipitation, first followed by an anion-exchange and then by an affinity chromatography. The SDS-PAGE shows a main band at 70 kDa containing two isoforms of ApTreh-1 (X1 and X2), identified by mass spectrometry and slightly contrasting in the C-terminal region. A phylogenetic tree, a multiple sequence alignment, as well as a modelled 3D-structure were constructed and they all reveal the ApTreh-1 similarity to other insect trehalases, i.e. the two signature motifs 179PGGRFRELYYWDTY192 and 479QWDFPNAWPP489, a glycine-rich region 549GGGGEY554, and the catalytic residues Asp336 and Glu538. The optimum enzyme activity occurs at 45 °C and pH 5.0, with Km and Vmax values of ~ 71 mM and ~ 126 µmol/min/mg, respectively. The present structural and functional characterization of soluble A. pisum trehalase enters the development of new strategies to control the aphids pest without significant risk for non-target organisms and human health.

Much research has been directed towards the optimisation of the mass rearing of the yellow mealworm, Tenebrio molitor, in terms of diet and rearing conditions. However, an issue that has not been thoroughly investigated to date is the strain effect on the larval growth and performance of T. molitor. To close this gap, the plasticity in growth and development, as well as the feed conversion efficiency and the nutritional composition of larvae of seven T. molitor strains of different geographic origins (Greece, Italy, Germany, Turkey, Spain and USA) were comparatively evaluated in the present study. Based on the results, there were considerable differences in the performance of the strains tested. Larval weight at harvest was the highest for a strain originated from Germany; however, the higher final body mass was at the cost of longer development time and higher feed intake, the latter having a negative impact in the overall performance and productivity of the strain. No significant differences were detected in the larval nitrogen and lipid content among the strains tested. In overall, the Italian strain II had the best growth performance in terms of survival, larval biomass production, development time and feed utilisation. The present study aims to highlight the need for selection of strains with enhanced performance and production traits for mass-production, which will boost the overall productivity of insect farming systems.

Metallocarboxypeptidases (MCPs) in the mosquito midgut play crucial roles in infection, as well as in mosquito dietary digestion, reproduction, and development. MCPs are also part of the digestive system of plant-feeding insects, representing key targets for inhibitor development against mosquitoes/mosquito-borne pathogens or as antifeedant molecules against plant-feeding insects. Notably, some non-mosquito insect B-type MCPs are primarily insensitive to plant protease inhibitors such as the potato carboxypeptidase inhibitor (PCI; MW 4-kDa), an inhibitor explored for cancer treatment and insecticide design. Here, we report the crystal structure of Ae. aegypti carboxypeptidase-B1 (CPBAe1)-PCI complex and compared the binding with that of PCI-insensitive CPBs. We show that PCI accommodation is determined by key differences in the active-site regions of MCPs. In particular, the loop regions α6-α7 (Leu242 -Ser250 ) and β8-α8 (Pro269 -Pro280 ) of CPBAe1 are replaced by α-helices in PCI-insensitive insect H.zea CPBHz. These α-helices protrude into the active-site pocket of CPBHz, restricting PCI insertion and rendering the enzyme insensitive. We further compared our structure with the only other PCI complex available, bovine CPA1-PCI. The potency of PCI against CPBAe1 (Ki = 14.7 nM) is marginally less than that of bovine CPA1 (Ki = 5 nM). Structurally, the above loop regions that accommodate PCI binding in CPBAe1 are similar to that of bovine CPA1, although observed changes in proteases residues that interact with PCI could account for the differences in affinity. Our findings suggest that PCI sensitivity is largely dictated by structural interference, which broadens our understanding of carboxypeptidase inhibition as a mosquito population/parasite control strategy. This article is protected by copyright. All rights reserved.

Scavenger receptor is pattern-recognition receptor (PRR) that plays a crucial function in host defense against pathogens. Scavenger receptor C (SR-C) is present only in invertebrates and its function has not been studied in detail. In this study, an SR-C homologous gene from the silkworm, Bombyx mori, was identified and characterized. SR-C was largely expressed in hemocytes and Malpighian tubules, with continuous expression in hemocytes. The peak expression was observed in hemocytes during molting and wandering stages both at mRNA and protein levels. Furthermore, immunofluorescence demonstrated it to be mainly distributed in the cell membranes of hemocytes, including oenocytoids and granulocytes. The recombinant SR-C protein (rSR-C) could bind to different types of bacteria and pathogen-associated molecular patterns (PAMPs), with strong binding to gram-positive bacteria and Lys-type peptidoglycans. The overexpression of SR-C induced the expression of genes related to the Toll pathway and antibacterial peptides. While the knockdown of SR-C reduced the expression of AMPs and inhibited the Toll pathway, it impaired the bacterial clearance ability of silkworm larvae, thus decreasing silkworm larvae's survival rate. Altogether, SR-C is a PRR that protect silkworms against bacterial pathogens by enhancing the expression of AMPs expression via the Toll pathway in hemocytes.

The innate immune response of insects provides a robust line of defense against pathogenic microbes and eukaryotic parasites. It consists of two types of overlapping immune responses, named humoral and cellular, which share protective molecules and regulatory mechanisms that closely coordinate to prevent the spread and replication of pathogens within the compromised insect hemocoel. The major feature of the humoral part of the insect immune system involves the production and secretion of antimicrobial peptides from the fat body, which is considered analogous to adipose tissue and liver in vertebrates. Previous research has identified and characterized the nature of antimicrobial peptides that are directed against various targets during the different stages of infection. Here we review this information focusing mostly on the diversity and mode of action of these host defense components, and their critical contribution to maintaining host homeostasis. Extending this knowledge is paramount for understanding the evolution of innate immune function and the physiological balance required to provide sufficient protection to the host against external enemies while avoiding overactivation signaling events that would severely undermine physiological stability.

Zinc finger protein, an important transcription factor, regulates gene expression associated with various physiological and pathological processes. U-shaped, belong to the Friend of GATA (FOG) transcription factor, play a crucial role in hematopoiesis by interacting with the GATA transcription factor as a co-factor. However, little is known about its functions in insects. In the present study, a U-shaped cDNA was identified and characterized from the silkworm Bombyx mori and investigated its potential roles in innate immunity. The predicted silkworm U-shaped amino acid sequence contained a classical nuclear localization signal (NLS) motif "GESSPKRRRR" at position 450-459, and arginine residues at position 456 and 478 are the critical sites of the NLS. U-shaped mRNA was detected in all tested tissues of the B. mori; however, the highest levels were found in the hemocytes. U-shaped mRNA expression levels were up-regulated in the hemocyte after the Escherichia coli and Staphylococcus aureus challenge. Furthermore, U-shaped knockdown significantly reduced the melanization process and suppressed the expression of melanization-associated genes, including PPO1, PPO2, PPAE, and BAEE. In addition, U-shaped interacts with Lozenge protein to regulates the innate immune response of the insect. Our results revealed that U-shaped binds directly to Lozenge protein to modulate the melanization process and innate immune responses in silkworm. This article is protected by copyright. All rights reserved.

Silkworm, as a model organism, has very high economic value due to its silk secretion ability. Although a large number of studies have attempted to elucidate the mechanism of silk secretion, it remains unclear. In this study, the fibroin light chain (Fib-L) gene of silkworm was subjected to CRISPR/Cas9 editing, which yielded premature termination of translation at 135 aa. Compared with those of the wild type, the posterior silk glands (PSGs) of the homozygous mutants on the third day of the fifth instar showed obvious premature degeneration. Comparative transcriptome and proteomic analyses of the PSGs of wild-type individuals, heterozygous mutants and homozygous mutants were performed on the fourth day of the fifth instar. A GO enrichment analysis showed that the differentially expressed genes (DEGs) between homozygous mutants and wild-type individuals were enriched in cytoskeleton-related terms, and a KEGG enrichment analysis showed that the upregulated DEGs between homozygous mutants and wild-type individuals were enriched in the phagosome and apoptosis pathways. These results indicated that apoptosis was activated prematurely in the PSGs of homozygous mutants. Furthermore, autophagy and heat shock response were activated in the PSGs of homozygous mutants, as demonstrated by an analysis of the DEGs related to autophagy and heat shock. A comparative proteomic analysis further confirmed that autophagy, apoptosis and the heat shock response were activated in the PSGs of homozygous mutants, which led to premature degradation of the PSGs. These results provide insights for obtaining a more in-depth understanding of the mechanism of silk secretion in silkworms.

Background Tanning is an important physiological process that play the critical roles in affecting cuticle pigmentation and sclerotization. Previous studies have showed that insect cuticle tanning was closely associated with tyrosine metabolism pathway that consisted of a series of enzymes. Results In this study, a total of 24 tyrosine metabolism pathway genes were identified in the oriental fruit fly Bactrocera dorsalis (Hendel) genome. Then, the gene expression profiles throughout 15 developmental stages of B. dorsalis were established on the basis of our previous RNA-seq data, and we found 13 enzyme genes could be involved in the process of pupariation. Accordingly, a predicted tyrosine-mediated tanning pathway during the pupariation of B. dorsalis was obtained, and a critical enzyme 3,4-dihydroxyphenylalanine (DOPA) decarboxylase (DDC) was selected to explore its possible functions in the formation of puparium. Firstly, the RT-qPCR confirmed that BdDDC had epidermis-specific expression pattern, and it was also highly expressed during the larval metamorphosis of B. dorsalis. Subsequently, the dysfunction of BdDDC by feeding 5-day-old larvae with DDC inhibitor (L-α-Methyl-DOPA) could lead to (i) the significant decreasing of BdDDC enzyme activity and dopamine concentration; (ii) the defects in puparium pigmentation; (iii) the impairment of morphology and less thickness of puparium; (iv) the lower pupal weight and obstacle to eclosion. Conclusion The present study provided us a potential tyrosine metabolic pathway that was responsible for insect tanning during the pupariation, and the BdDDC enzyme has been proved to act the crucial roles in larval-pupal tanning of B. dorsalis. This article is protected by copyright. All rights reserved.

Odorant-binding proteins (OBPs) of vertebrates belong to the lipocalin superfamily and perform a dual function: solubilizing and ferrying volatile pheromones to the olfactory receptors, and complexing the same molecules in specialized glands and assisting their release into the environment. Within vertebrates, to date they have been reported only in mammals, apart from two studies on amphibians. Based on the small number of OBPs expressed in each species, on their sites of production outside the olfactory area and their presence in biological fluids known to be pheromone carriers, such as urine, saliva and sexual secretions, we conclude that OBPs of mammals are specifically dedicated to pheromonal communication. This assumption is further supported by the observation that some OBPs present in biological secretions are endowed with their own pheromonal activity, adding renewed interest to these proteins. Another novel piece of evidence is the recent discovery that glycosylation and phosphorylation can modulate the binding activity of these proteins, improving their affinity to pheromones and narrowing their specificity. A comparison with insects and other arthropods shows a completely different scenario. While mammalian OBPs are specifically tuned to pheromones, those of insects, which are completely different in sequence and structure, include carriers for general odorants in addition to those dedicated to pheromones. Additionally, whereas mammals adopted a single family of carrier proteins for chemical communication, insects and other arthropods are endowed with several families of semiochemical-binding proteins. Here, we review the literature on the structural and functional properties of vertebrate OBPs, summarize the most interesting new findings and suggest possible exciting future developments.

The DNA methyltransferase family contains a conserved set of DNA-modifying enzymatic proteins. They are responsible for epigenetic gene modulation, such as transcriptional silencing, transcription activation, and post-transcriptional modulation. Recent research has revealed that the canonical DNA methyltransferases (DNMTs) biological roles go beyond their traditional functions of establishing and maintaining DNA methylation patterns. Although a complete DNA methylation toolkit is absent in most insect orders, recent evidence indicates the de novo DNA methylation and maintenance function remain conserved. Studies using various molecular approaches provided evidence that DNMTs are multi-functional proteins. However, still in-depth studies on their biological role lack due to the least studied area in insects. Here, we review the DNA methylation toolkit of insects, focusing on recent research on various insect orders, which exhibit DNA methylation at different levels, and for which DNMTs functional studies have become available in recent years. We survey research on the potential roles of DNMTs in the regulation of gene transcription in insect species. DNMTs participate in different physiological processes by interacting with other epigenetic factors. Future studies on insect's DNMTs will benefit to understand developmental processes, responses to various stimuli, and adaptability of insects to different environmental conditions.

Uridine diphosphate (UDP)-glycosyltransferases (UGTs), which are major phase II detoxification enzymes, have been implicated in the glycosylation of lipophilic endobiotics and xenobiotics and thus potentially lead to the evolution of insecticide resistance. In this study, we identified and cloned two putative UGT genes from transcriptome data which are named UGT352A4 and UGT352A5. As demonstrated by qRT-PCR, two UGT genes were over-expressed in the thiamethoxam-resistant (THQR) strain relative to the susceptible (THQS) strain. Moreover, the induction experiment revealed that the expression of the UGT352A5 gene was significantly increased following exposure to thiamethoxam in the THQR strain. Furthermore, the expression of both UGT352A4 and UGT352A5 was downregulated after RNA interference, whereas only the silencing of UGT352A5 resulted in a noticeable increase in the mortality of THQR adults. Our results represent the first line of evidence showing that UGT352A5 might be responsible for conferring thiamethoxam resistance in B. tabaci. The results will be shed new insights for obtaining a better understanding of the role of UGTs in the evolution of insecticide resistance and developing new insect resistance management tactics within the sustainable integrated pest management framework.

Bacterial symbionts, such as Wolbachia species, can manipulate the sexual development and reproduction of their insect hosts. For example, Wolbachia infection induces male-specific death in the Asian corn borer Ostrinia furnacalis by targeting the host factor Masculinizer (Masc), an essential protein for masculinization and dosage compensation in lepidopteran insects. Here we identify a Wolbachia protein, designated Oscar, which interacts with Masc via its ankyrin repeats. Embryonic expression of Oscar inhibits Masc-induced masculinization and leads to male killing in two lepidopteran insects, O. furnacalis and the silkworm Bombyx mori. Our study identifies a mechanism by which Wolbachia induce male killing of host progeny.

Endoparasitoid wasps inject venom proteins into the hemocoel of host insects to ensure survival, growth, and development of their progenies by blocking host immunity. We previously identified ten serine protease inhibitors of the serpin superfamily in venom of the endoparasitoid wasp, Microplitis mediator, but it is unclear how these inhibitors may interact with host immune serine proteases. In this study, we investigated the functions of two serpins, MmvSPN-1 and MmvSPN-2, in the regulation of humoral immune responses in two hosts, the oriental armyworm Pseudaletia separate and the cotton bollworm Helicoverpa armigera, by dsRNA knockdown and biochemical assays using recombinant proteins. Knockdown of the two serpins resulted in increases in prophenoloxidase (PPO) activation and antimicrobial peptide (AMP) production in the hosts. After injection into the host hemocoel, the recombinant serpins inhibited PPO activation and AMP transcription. Mass spectrometry analysis of the pull-downs and in vitro reconstitution experiments revealed that HacSP29, a clip-domain serine protease in H. armigera, is the target of these two serpins. Therefore, these two inhibitors in the wasp venom may protect eggs from attacks by melanization and AMPs in the host insects.