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To harness the diverse industrial applications of cellulase, including its use in the food, pulp, textile, agriculture, and biofuel sectors, this study focused on the high-yield production of a bioactive insect-derived endoglucanase, Monochamus saltuarius glycoside hydrolase family 5 (MsGHF5). MsGHF5 was introduced into the genome of Kluyveromyces lactis to maintain expression stability, and mass production of the enzyme was induced using fed-batch fermentation. After 40 h of cultivation, recombinant MsGHF5 was successfully produced in the culture broth, with a yield of 29,000 U/L, upon galactose induction. The optimal conditions for the activity of purified MsGHF5 were determined to be a pH of 5 and a temperature of 35 °C, with the presence of ferrous ions enhancing the enzymatic activity by up to 1.5-fold. Notably, the activity of MsGHF5 produced in K. lactis was significantly higher than that produced in Escherichia coli, suggesting that glycosylation is crucial for the functional performance of the enzyme. This study highlights the potential use of K. lactis as a host for the production of bioactive MsGHF5, thus paving the way for its application in various industrial sectors., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Phospholipase A2 (PLA2) is widely distributed in animals, plants, and microorganisms, and it plays an important role in many physiological activities. In a previous study, we have identified a secretory PLA2 in Bombyx mori ( BmsPLA2-1-1 ). In this study, we further identified four new sPLA2 genes ( BmsPLA2-1-2 , BmsPLA2-2 , BmsPLA2-3 , and BmsPLA2-4 ) in B . mori genome. All four genes exhibits the characteristic features of sPLA2, including the sPLA2 domain, metal binding sites, and highly conserved catalytic domain. This study completed the cloning, in vitro expression, and expression pattern analysis of the BmsPLA2-4 gene in B . mori . The full length of BmsPLA2-4 is 585 bp, and the recombinant protein obtained through prokaryotic expression has an estimated size of 25 kDa. qRT-PCR analysis revealed that the expression level of BmsPLA2-4 reached its peak on the first day of the fifth instar larval stage. Tissue expression profiling analysis showed that BmsPLA2-4 had the highest expression level in the midgut, followed by the epidermis and fat body. Western blotting analysis results were consistent with those of qRT-PCR. Furthermore, after infecting fifth instar 1-day-old larvae with Escherichia coli and Staphylococcus aureus , the expression level of the BmsPLA2-4 gene significantly increased in 24 h. The findings of this study provides a theoretical basis and valuable experimental data for future related research.

Hemimetabolous insects, such as the two-spotted cricket Gryllus bimaculatus, can recover lost tissues, in contrast to the limited regenerative abilities of human tissues. Following cricket leg amputation, the wound surface is covered by the wound epidermis, and plasmatocytes, which are insect macrophages, accumulate in the wound region. Here, we studied the function of Toll-related molecules identified by comparative RNA sequencing during leg regeneration. Of the 11 Toll genes in the Gryllus genome, expression of Toll2-1, Toll2-2 and Toll2-5 was upregulated during regeneration. RNA interference (RNAi) of Toll, Toll2-1, Toll2-2, Toll2-3 or Toll2-4 produced regeneration defects in more than 50% of crickets. RNAi of Toll2-2 led to a decrease in the ratio of S- and M-phase cells, reduced expression of JAK/STAT signalling genes, and reduced accumulation of plasmatocytes in the blastema. Depletion of plasmatocytes in crickets using clodronate also produced regeneration defects, as well as fewer proliferating cells in the regenerating legs. Plasmatocyte depletion also downregulated the expression of Toll and JAK/STAT signalling genes in the regenerating legs. These results suggest that Spz-Toll-related signalling in plasmatocytes promotes leg regeneration through blastema cell proliferation by regulating the Upd-JAK/STAT signalling pathway., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Lipases play crucial roles in food digestion by degrading dietary lipids into free fatty acids and glycerols. The domesticated silkworm (Bombyx mori) has been widely used as an important Lepidopteran model for decades. However, little is known about the lipase gene family in the silkworm, especially their hydrolytic activities as digestive enzymes. In this study, a total of 38 lipase genes were identified in the silkworm genome. Phylogenetic analysis indicated that they were divided into three major groups. Twelve lipases were confirmed to be expressed in the midgut at both transcriptional and translational levels. They were grouped into the same gene cluster, suggesting that they could have similar physiological functions. Quantitative real-time PCR (qRT-PCR) analyses indicated that lipases were mainly expressed in anterior and middle midgut regions, and their expression levels varied greatly along the length of midgut. A majority of lipases were down-regulated in the midgut when larvae stopped feeding. However, a unique lipase gene (Bmlip10583) showed low expression level during feeding stage, but it was significantly up-regulated during the larvae-pupae transition. These results demonstrated that expression of silkworm lipases was spatially and temporally regulated in the midgut during larval development. Taken together, our results provide a fundamental research of the lipase gene family in the silkworm., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Lytic polysaccharide monooxygenases (LPMOs) are metalloenzymes that cleave structural polysaccharides through an oxidative mechanism. The enzymatic activity of LPMOs relies on the presence of a Cu 2+ histidine-brace motif in their flat catalytic surface. Upon reduction by an external electron donor and in the presence of its co-substrates, O 2 or H 2 O 2 , LPMOs can generate reactive oxygen species to oxidize the substrates. Fungal and bacterial LPMOs are involved in the catabolism of polysaccharides, such as chitin, cellulose, and hemicelluloses, and virulence mechanisms. Based on the reports on the discovery of LPMOs from the family AA15 in termites, firebrats, and flies, the functional role of the LPMO in the biosphere could expand, as these enzymes may be correlated with chitin remodeling and molting in insects. However, there is limited knowledge of AA15 LPMOs due to difficulties in recombinant expression of soluble proteins and purification protocols. In this study, we describe a protocol for the cloning, expression, and purification of insect AA15 LPMOs from Arthropoda, mainly from termites, followed by the expression and purification of an AA15 LPMO from the silkworm Bombyx mori, which contains a relatively high number of disulfide bonds. We also report the recombinant expression and purification of a protein with homology to AA15 family from the western European honeybee Apis mellifera, an LPMO-like enzyme lacking the canonical histidine brace. Therefore, this work can support future studies concerning the role of LPMOs in the biology of insects and inspire molecular entomologists and insect biochemists in conducting activities in this field., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Locusta migratoria apolipophorin III (apoLp-III) possesses the ability to exist as a water soluble amphipathic α-helix bundle and a lipid surface seeking apolipoprotein. The intrinsic ability of apoLp-III to transform phospholipid vesicles into reconstituted discoidal high-density lipoproteins (rHDL) has led to myriad applications. To improve the yield of recombinant apoLp-III, studies were performed in a bioreactor. Induction of apoLp-III expression generated a protein product that is secreted from E. coli into the culture medium. Interaction of apoLp-III with gas and liquid components in media produced large quantities of thick foam. A continuous foam fractionation process yielded a foamate containing apoLp-III as the sole major protein component. The yield of recombinant apoLp-III was ~0.2 g / liter bacterial culture. Mass spectrometry analysis verified the identity of the target protein and indicated no modifications or changes to apoLp-III occurred as a result of foam fractionation. The functional ability of apoLp-III to induce rHDL formation was evaluated by incubating foam fractionated apoLp-III with phosphatidylcholine vesicles. FPLC size exclusion chromatography revealed a single major population of particles in the size range of rHDL. The results described offer a novel approach to bioreactor-based apoLp-III production that takes advantage of its intrinsic biosurfactant properties., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Insect metamorphosis into an adult occurs after the juvenile hormone (JH) titer decreases at the end of the juvenile stage. This generally coincides with decreased transcript levels of JH-response transcription factors Krüppel homolog 1 (Kr-h1) and broad (br), and increased transcript levels of the adult specifier E93. Thrips (Thysanoptera) develop through inactive and non-feeding stages referred to as "propupa" and "pupa", and this type of distinctive metamorphosis is called neometaboly. To understand the mechanisms of hormonal regulation in thrips metamorphosis, we previously analyzed the transcript levels of Kr-h1 and br in two thrips species, Frankliniella occidentalis (Thripidae) and Haplothrips brevitubus (Phlaeothripidae). In both species, the transcript levels of Kr-h1 and br decreased in the "propupal" and "pupal" stages, and their transcription was upregulated by exogenous JH mimic treatment. Here we analyzed the developmental profiles of E93 in these two thrips species. Quantitative RT-PCR revealed that E93 expression started to increase at the end of the larval stage in F. occidentalis and in the "propupal" stage of H. brevitubus, as Kr-h1 and br mRNA levels decreased. Treatment with an exogenous JH mimic at the onset of metamorphosis prevented pupal-adult transition and caused repression of E93. These results indicated that E93 is involved in adult differentiation after JH titer decreases at the end of the larval stage of thrips. By comparing the expression profiles of Kr-h1, br, and E93 among insect species, we propose that the "propupal" and "pupal" stages of thrips have some similarities with the holometabolous prepupal and pupal stages, respectively., Competing Interests: The authors have declared that no competing interests exist.

Dectes texanus is an important coleopteran pest of soybeans and cultivated sunflowers in the Midwestern United States that causes yield losses by girdling stems of their host plants. Although sunflower and giant ragweed are primary hosts of D. texanus, they began colonizing soybeans approximately 50 years ago and no reliable management method has been established to prevent or reduce losses by this pest. To identify genes putatively involved when feeding soybean, we compared gene expression of D. texanus third-instar larvae fed soybean to those fed sunflower, giant ragweed, or artificial diet. Dectes texanus larvae differentially expressed 514 unigenes when fed on soybean compared to those fed the other diet treatments. Enrichment analyses of gene ontology terms from up-regulated unigenes in soybean-fed larvae compared to those fed both primary hosts highlighted unigenes involved in oxidoreductase and polygalacturonase activities. Cytochrome P450s, carboxylesterases, major facilitator superfamily transporters, lipocalins, apolipoproteins, glycoside hydrolases 1 and 28, and lytic monooxygenases were among the most commonly up-regulated unigenes in soybean-fed larvae compared to those fed their primary hosts. These results suggest that D. texanus larvae differentially expressed unigenes involved in biotransformation of allelochemicals, digestion of plant cell walls and transport of small solutes and lipids when feeding in soybean.

Silk gland is an organ that produces and secretes silk proteins. The development of the silk gland is essential for high silk production yield and silk quality. Although Sage reportedly plays a pivotal role in embryonic silk gland development, the mechanism underlying its action remains unclear. Our study aimed to determine the genes downstream of Sage through which it regulates the development of the silk gland. After chromatin immunoprecipitation and sequencing, Dfd was identified as a downstream target gene of Sage and it was confirmed that Sage could inhibit Dfd expression by competing with SGF1. When Dfd was knocked down through RNA interference (RNAi), the number of cells in the middle silk gland decreased, and the posterior silk gland was straightened. Simultaneously, the expression of Ser1 and silk fibroin genes was no longer strictly regional. These changes eventually led to an alteration in the composition of the Dfd RNAi cocoon. In conclusion, our research contributes to a deeper understanding of the development of silk glands., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Odorant binding proteins (OBPs) play an essential role for insect chemosensation in insect peripheral nervous systems of antennae. Each antennal sensilla contains more than one OBP at high concentrations but the interactions and cooperation between co-localized OBPs are rarely reported. In present study, we cloned, expressed and purified eight OBPs of the green peach aphid Myzus persicae. The effects of knocking down the expression of these OBP genes by RNAi on the electrophysiological and behavioural responses of M. persicae to the aphid alarm pheromone, (E)-β-farnesene (EβF) were investigated. The results showed that the aphids could still be repelled by EβF when the expression of each of three OBP genes was individually knocked down. However, the simultaneous knockdown of MperOBP3/7/9 expression significantly reduced the electrophysiological response and the repellent behaviours of M. persicae to EβF than the single OBP gene knockdown (P < 0.05). Rather than a normal saturation binding curve of individual OBP, the binding curve of MperOBP3/7/9 is bell-shaped with a higher affinity for the fluorescent probe N-phenyl-1-naphthylamine (1-NPN). The competitive binding assays confirmed that MperOBP3, MperOBP7, MperOBP9 and MperOBP3/7/9 mixture exhibited a stronger binding affinity for EβF, than for sex pheromones and plant volatiles with a dissociation constant of 2.5 μM, 1.1 μM, 3.9 μM and 1.0 μM, respectively. The competitive binding curve of MperOBP3/7/9 mixture to EβF is shallow without bottom plateau, suggesting a conformational change and a rapid dissociation after the displacement of all 1-NPN (in vivo after the saturation binding of all OBPs by EβF). The interaction between OBPs and formation of a heterogeneous unit may facilitate the delivery EβF to the OR at electrophysiological and behavioural levels during insect odorant signal transduction thus mediate M. persicae response to the alarm pheromone EβF., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In most diploid organisms, mating is a prerequisite for reproduction and, thus, critical to the maintenance of their population and the perpetuation of the species. Besides the importance of understanding the fundamentals of reproduction, targeting the reproductive success of a pest insect is also a promising method for its control, as a possible manipulation of the reproductive system could affect its destructive activity. Here, we used an integrated approach for the elucidation of the reproductive system and mating procedures of the olive fruit fly, Bactrocera oleae . Initially, we performed a RNAseq analysis in reproductive tissues of virgin and mated insects. A comparison of the transcriptomes resulted in the identification of genes that are differentially expressed after mating. Functional annotation of the genes showed an alteration in the metabolic, catalytic, and cellular processes after mating. Moreover, a functional analysis through RNAi silencing of two differentially expressed genes, yellow-g and troponin C , resulted in a significantly reduced oviposition rate. This study provided a foundation for future investigations into the olive fruit fly's reproductive biology to the development of new exploitable tools for its control.

The Chinese white pine beetle, Dendroctonus armandi Tsai and Li, is a serious native pest in the Qinling Mountains of China. exo-Bevicomin, as the main component of bark beetle pheromone, is released by the female D. armandi. In this paper, we identified two genes encoding, (Z)-6-nonen-2-ol dehydrogenase and CYP6CR, that are known to be involved in xo-brevicomin synthesis to improve the understanding of exo-brevicomin biosynthesis in the Chinese white pine beetle. The two protiens had high homology with their orthologs in the exo-brevicomin biosynthesis pathway from D. ponderosae. The expression profiles of CYP6CR12 and DaZnoDH in D. armandi females are closely correlated with exo-brevicomin biosynthesis. The expression levels of CYP6CR12 and DaZnoDH are also regulated by feeding behavior and juvenile hormone levels. Since they are also expressed in males, CYP6CR12 and DaZnoDH are not only important for exo-brevicomin biosynthesis that this might be potential role for the semichemical biosysthesis pathways., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Across the evolutionary history of insects, the shift from nitrogen-rich carnivore/omnivore diets to nitrogen-poor herbivorous diets was made possible through symbiosis with microbes. The herbivorous turtle ants Cephalotes possess a conserved gut microbiome which enriches the nutrient composition by recycling nitrogen-rich metabolic waste to increase the production of amino acids. This enrichment is assumed to benefit the host, but we do not know to what extent. To gain insights into nitrogen assimilation in the ant cuticle we use gut bacterial manipulation, 15 N isotopic enrichment, isotope-ratio mass spectrometry, and 15 N nuclear magnetic resonance spectroscopy to demonstrate that gut bacteria contribute to the formation of proteins, catecholamine cross-linkers, and chitin in the cuticle. This study identifies the cuticular components which are nitrogen-enriched by gut bacteria, highlighting the role of symbionts in insect evolution, and provides a framework for understanding the nitrogen flow from nutrients through bacteria into the insect cuticle.

Rapid adaptive responses were evident from reciprocal host-plant switches on performance, digestive physiology and relative gene expression of gut serine proteases in larvae of crucifer pest P. brassicae transferred from cauliflower (CF, Brassica oleracea var. botrytis, family Brassicaceae) to an alternate host, garden nasturtium, (GN, Tropaeolum majus L., family Tropaeolaceae) and vice-versa under laboratory conditions. Estimation of nutritional indices indicated that larvae of all instars tested consumed the least food and gained less weight on CF-GN diet (significant at p≤0.05) as compared to larvae feeding on CF-CF, GN-GN and GN-CF diets suggesting that the switch to GN was nutritionally less favorable for larval growth. Nevertheless, these larvae, especially fourth instars, were adroit in utilizing and digesting GN as a new host plant type. In vitro protease assays conducted to understand associated physiological responses within twelve hours indicated that levels and properties of gut proteases were significantly influenced by type of natal host-plant consumed, change in diet as well as larval age. Activities of gut trypsins and chymotrypsins in larvae feeding on CF-GN and GN-CF diets were distinct, and represented shifts toward profiles observed in larvae feeding continuously on GN-GN and CF-CF diets respectively. Results with diagnostic protease inhibitors like TLCK, STI and SBBI in these assays and gelatinolytic zymograms indicated complex and contrasting trends in gut serine protease activities in different instars from CF-GN diet versus GN-CF diet, likely due to ingestion of plant protease inhibitors present in the new diet. Cloning and sequencing of serine protease gene fragments expressed in gut tissues of fourth instar P. brassicae revealed diverse transcripts encoding putative trypsins and chymotrypsins belonging to at least ten lineages. Sequences of members of each lineage closely resembled lepidopteran serine protease orthologs including uncharacterized transcripts from Pieris rapae. Differential regulation of serine protease genes (Pbr1-Pbr5) was observed in larval guts of P. brassicae from CF-CF and GN-GN diets while expression of transcripts encoding two putative trypsins (Pbr3 and Pbr5) were significantly different in larvae from CF-GN and GN-CF diets. These results suggested that some gut serine proteases that were differentially expressed in larvae feeding on different species of host plants were also involved in rapid adaptations to dietary switches. A gene encoding nitrile-specifier protein (nsp) likely involved in detoxification of toxic products from interactions of ingested host plant glucosinolates with myrosinases was expressed to similar levels in these larvae. Taken together, these snapshots reflected contrasts in physiological and developmental plasticity of P. brassicae larvae to nutritional challenges from wide dietary switches in the short term and the prominent role of gut serine proteases in rapid dietary adaptations. This study may be useful in designing novel management strategies targeting candidate gut serine proteases of P. brassicae using RNA interference, gene editing or crops with transgenes encoding protease inhibitors from taxonomically-distant host plants., Competing Interests: The authors have declared that no competing interests exist.

The assumption that structural or sequential homology between enzymes implies functional homology is a common misconception. Through in-depth structural and kinetic analysis, we are now beginning to understand the minute differences in primary structure that can alter the function of an enzyme completely. Alternative splicing is one method for which the activity of an enzyme can be controlled, simply by altering its length. Arylalkylamine N-acetyltransferase A (AANATA) in D. melanogaster, which catalyzes the N-acetylation of biogenic amines, has multiple splicoforms - alternatively spliced enzyme isoforms - with differing tissue distribution. As demonstrated here, AANAT1 from Tribolium castaneum is another such enzyme with multiple splicoforms. A screening assay was developed and utilized to determine that, despite only a 35 amino acid truncation, the shortened form of TcAANAT1 is a more active form of the enzyme. This implies regulation of enzyme metabolic activity via alternative splicing., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Small heat shock proteins (sHSPs) are important modulators of insect survival. Previous research revealed that there is only one orthologous cluster of shsps in insects. Here, we identified another novel orthologous cluster of shsps in insects by comparative analysis. Multiple stress experiments and function investigation of Tchsp21.8a belonging to this orthologous cluster and seven species-specific shsps were performed in the stored-grain pest Tribolium castaneum. The results indicated that expression of Tchsp21.8a showed weak responses to different stresses. However, expressions of most species-specific shsps exhibited hyper-responses to heat stress, and expressions of all species-specific shsps displayed diverse responses during other stresses to protect beetles in a cooperative manner. Additionally, Tchsp21.8a and species-specific Tcshsp19.7 played important roles in the development of T. castaneum, and all Tcshsps had a certain impact on the fecundity. Our work created a comprehensive reliable scaffold of insect shsps that can further provide instructive insights to pest bio-control., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Antheraea mylitta arylphorin protein was extracted from the silk gland of fifth instar larvae and purified by ammonium sulphate precipitation, ion-exchange, and gel filtration chromatography. The N-terminal sequencing of ten amino acids (NH 2 -SVVHPPHHEV-COOH) showed similarity with Antheraea pernyi arylphorin. Based on N-terminal and C-terminal A. pernyi arylphorin sequences, primers were designed, and A. mylitta arylphorin cDNA was cloned by RT-PCR from silk gland mRNA. Sequencing of complete cDNA including 25 nucleotides at 5' UTR (obtained by 5' RACE) showed that it consisted of an ORF of 2115 nucleotides which could encode a protein of 704 amino acids (predominantly aromatic residues) having molecular weight 83 kDa. Homology modelling was done using A. pernyi arylphorin as a template. Cloned arylphorin cDNA was expressed in E. coli and recombinant His-tagged protein was purified by Ni-NTA affinity chromatography. Analysis of tissue-specific expression of arylphorin by real-time PCR showed maximum expression in the fat body followed by silk gland and integument. 5' flanking region (759 bp) of arylphorin gene was amplified by inverse PCR and the full length gene (5359 nucleotides) containing five exons and four introns was cloned from the A. mylitta genomic DNA and sequenced. Polyclonal antibody was raised against purified arylphorin and more native arylphorin protein (500 kDa) was purified from the fat body by antibody affinity chromatography. Study of mitogenic effect of native and chymotrypsin hydrolysate of arylphorin on different insect cell lines showed that arylphorin could be used as serum substitute for in vitro cultivation of insect cells., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Chemosensory proteins (CSPs) are soluble carrier proteins typically characterized by a six-helix bundle structure joined by two disulfide bridges and a conserved Cys spacing pattern (C1-X 6-8 -C2-X 16-21 -C3-X 2 -C4). CSPs are functionally diverse with reported roles in chemosensation, immunity, development, and resistance. To expand our molecular understanding of CSP function in plant bugs, we used recently developed transcriptomic resources for Lygus lineolaris and Lygus hesperus to identify 17 and 14 CSP-like sequences, respectively. The Lygus CSPs are orthologous and share significant sequence identity with previously annotated CSPs. Three of the CSPs are predicted to deviate from the typical CSP structure with either five or seven helical segments rather than six. The seven helix CSP is further differentiated by an atypical C3-X 3 -C4 Cys spacing motif. Reverse transcriptase PCR-based profiling of CSP transcript abundance in adult L. lineolaris tissues revealed broad expression for most of the CSPs with antenna specific expression limited to a subset of the CSPs. Comparative sequence analyses and homology modeling suggest that variations in the amino acids that comprise the Lygus CSP binding pockets affect the size and nature of the ligands accommodated., (© 2018 Institute of Zoology, Chinese Academy of Sciences.)

Talisin is a storage protein from Talisia esculenta seeds that presents lectin-like and peptidase inhibitor properties. These characteristics suggest that talisin plays a role in the plant defense process, making it a multifunctional protein. This work aimed to investigate the effects of chronic intake of talisin on fifth instar larvae of Spodoptera frugiperda , considered the main insect pest of maize and the cause of substantial economic losses in several other crops. The chronic intake of talisin presented antinutritional effects on the larvae, reducing their weight and prolonging the total development time of the insects. In addition, talisin-fed larvae also showed a significant reduction in the activity of trypsin-like enzymes. Midgut histology analysis of talisin-fed larvae showed alterations in the intestinal epithelium and rupture of the peritrophic membrane, possibly causing an increase of aminopeptidase activity in the midgut lumen. Talisin also proved to be resistant to degradation by the digestive enzymes of S. frugiperda . The transcription profile of trypsin, chymotrypsin and aminopeptidase genes was also analyzed through qPCR technique. Talisin intake resulted in differential expression of at least two genes from each of these classes of enzymes. Molecular docking studies indicated a higher affinity of talisin for the less expressed enzymes.

Unsuitable pH is a major limiting factor for all organisms, and a low pH can lead to organism death. Late embryogenesis abundant (LEA) peptides confer tolerance to abiotic stresses including salinity, drought, high and low temperature, and ultraviolet radiation same as the LEA proteins from which they originate. In this study, LEA peptides derived from group 3 LEA proteins of Polypedilum vanderplanki were used to enhance low pH tolerance. Recombinant Escherichia coli BL21 (DE3) cells expressing the five designed LEA peptides were grown at pH 4, 3, and 2. The transformants showed higher growth capacity at low pH as compared to control cells. These results indicate that LEA peptide could prevent E. coli cell death under low pH conditions.

To elucidate the functional alteration of the recombinant hybrid chitinases composed of bacterial and insect's domains, we cloned the constitutional domains from chitinase-encoding cDNAs of a bacterial species, Bacillus thuringiensis (BtChi) and a lepidopteran insect species, Mamestra brassicae (MbChi), respectively, swapped one's leading signal peptide (LSP) - catalytic domain (CD) - linker region (LR) (LCL) with the other's chitin binding domain (ChBD) between the two species, and confirmed and analyzed the functional expression of the recombinant hybrid chitinases and their chitinolytic activities in the transformed E. coli strains. Each of the two recombinant cDNAs, MbChi's LCL connected with BtChi's ChBD (MbLCL-BtChBD) and BtChi's LCL connected with MbChi's ChBD (BtLCL-MbChBD), was successfully introduced and expressed in E. coli BL21 strain. Although both of the two hybrid enzymes were found to be expressed by SDS-PAGE and Western blotting, the effects of the introduced genes on the chitin metabolism appear to be dramatically different between the two transformed E. coli strains. BtLCL-MbChBD remarkably increased not only the cell proliferation rate, extracellular and cellular chitinolytic activity, but also cellular glucosamine and N-acetylglucosamine levels, while MbLCL-BtChBD showed about the same profiles in the three tested subjects as those of the strains transformed with each of the two native chitinases, indicating that a combination of the bacterial CD of TIM barrel structure with characteristic six cysteine residues and insect ChBD2 including a conserved six cysteine-rich region (6C) enhances the attachment of the enzyme molecule to chitin compound by MbChBD, and so increases the catalytic efficiency of bacterial CD., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Temperature stress disrupts protein homeostasis in biological cells. High temperature causes death of the silkworm Bombyx mori, while low temperature leads to slow growth of silkworm in sericulture. Small heat shock proteins (sHsps) play a crucial role in maintaining protein homeostasis and protecting cells against various harmful conditions. In this study, the expression patterns of Bmhsp19.9 were analyzed by reverse transcription (RT)-PCR and quantitative PCR. The promoters hsp19.9P1 and hsp19.9P2 of Bmhsp19.9 were cloned, quantitative PCR and western blotting confirmed that the longer promoter of hsp19.9P1 showed higher activity compared to hsp19.9P2. Overexpression of Bmhsp19.9 increased the viability of BmE cells at high temperatures. The transgenic vector pb-H19.9 was constructed, in which Bmhsp19.9 was overexpressed under the control of hsp19.9P1. Two transgenic lines, H19.9A and H19.9B, were generated via microinjection of P50 silkworm embryos. Quantitative PCR and western blotting revealed that the content of Bmhsp19.9 in transgenic silkworms was higher than that in the control. After exposure to 35 °C for 49 h, the survival rate was increased by approximately 40% in transgenic silkworms compared to non-transgenic P50. When P50 and H19.9A were reared at 13 °C, growth was faster and the survival rate and surviving larval weight were significantly higher in H19.9A compared to P50. These results suggest that overexpression of Bmhsp19.9 increased the resistance of transgenic silkworms to extreme temperatures, which may be useful for sericulture in regions and seasons with no optimal average temperatures for silkworm., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Melanization is a common phenomenon in insects, and melanin synthesis is a conserved physiological process that occurs in epidermal cells. Moreover, a comprehensive understanding of the mechanisms of melanin synthesis influencing insect pigmentation are well-suited for investigating phenotype variation. The Asian multi-colored (Harlequin) ladybird beetle, Harmonia axyridis , exhibits intraspecific polymorphism based on relative levels of melanization. However, the specific characteristics of melanin synthesis in H. axyridis remains elusive. In this study, we performed gene-silencing analysis of the pivotal inverting enzyme, tyrosine hydroxylase (TH), and DOPA decarboxylase (DDC) in the tyrosine metabolism pathway to investigate the molecular and regulatory mechanism of melanin synthesis in H. axyridis . Using RNAi of TH and DDC genes in fourth instar larvae, we demonstrated that dopamine melanin was the primary contributor to the overall body melanization of H. axyridis . Furthermore, our study provides the first conclusive evidence that dopamine serves as a melanin precursor for synthesis in the early pupal stage. According to transcription factor Pannier , which is essential for the formation of melanic color on the elytra in H. axyridis , we further demonstrated that suppression of HaPnr can significantly decrease expression levels of HaTH and HaDDC . These results in their entirety lead to the conclusion that transcription factor Pannier can regulate dopamine melanin synthesis in the dorsal elytral epidermis of H. axyridis .

Juvenile hormone binding proteins (JHBPs) serve as the carriers that transport juvenile hormone (JH) to target tissues to activate JH signals. In this study, seven JHBPs from mud crab Scylla paramamosain were obtained. All Sp-JHBPs contained one JHBP domain, and Sp-JHBP4, 5, 6 also contained one Grp7&#95;allergen domain. The predicted secondary and 3D structures are conserved among the seven Sp-JHBP domains, but the complete 3D structure of Sp-JHBP4, 5, 6 were more similar to lipopolysaccharide binding protein. Additionally, the potential ligands for Sp-JHBP1, 2, 3, 7 were ubiquinone 8 (UQ8) and JH II or JH III, and the predicted binding location and sites for JHs were similar to the structured known BmJHBP, suggesting that MF is a potential ligand for these Sp-JHBPs. Furthermore, the expression analysis showed that Sp-JHBP1 expression is relatively low in the 14 detected tissues, and Sp-JHBP2 has the highest expression in the ovary, while the other five JHBPs are highly expressed in the hepatopancreas. During larval development, Sp-JHBP2 is highly expressed during the Z1, M and C1 stages, which are three post-metamorphosis stages, while Sp-JHBP3 and 4 expression levels were significantly lower during the Z5 and M stages, which are two pre-metamorphosis stages. Moreover, the in vivo and in vitro study revealed that Sp-JHBP2 expression was reduced by MF, while the in vitro studies showed that Sp-JHBP3, 6, 7 are induced by MF in a concentration-independent manner. Taken together, we hypothesized Sp-JHBP1, 2, 3, 7 has the potential to bind MF and Sp-JHBP2, 3, 7 play a more important roles in MF signal, while Sp-JHBP4, 5, 6 were more likely to participate in immune response., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Conogethes pinicolalis (Lepidoptera: Crambidae), a major pine pest, possesses a sensitive olfactory system to locate its host. Pheromone binding proteins (PBPs) and general odorant binding proteins (GOBPs) are two types of proteins involved in the process. In this work, we used phylogenetic analysis, gene expression, fluorescence competitive binding assay, and molecular docking to characterize PBPs and GOBPs in C. pinicolalis. The phylogenetic unrooted tree revealed the C. pinicolalis GOBPs and PBPs amino acid sequences showed very close relation with Conogethes punctiferalis (yellow peach moth). Meanwhile, both the PBPs and GOBPs were specifically expressed in the antennae. Binding affinities of PBPs and GOBPs to 19 volatile compounds were tested. PBP2 shows the strongest binding to E10-16: Ald with a K i value of 0.28/1.66 μM; GOBP1 to Z10-16:Ald with a K i value of 3.11/4.24 μM. Furthermore, molecular docking reveals potential active sites in PBP2 and GOBP1 to interact with most of the tested volatiles. Finally, we demonstrate that PBP2 and GOBP1 are the dominant genes in their respective families using several different assays. In conclusion, PBP2 and GOBP1 genes may play similar roles in detecting and transporting sex pheromones and host plant volatiles in C. pinicolalis., (Copyright © 2019. Published by Elsevier B.V.)

This work provides methods for chitosan production with larval exoskeletons from Hermetia illucens and characterizes the produced material. The study aims to provide optimal reaction conditions for the purification of chitin from insect material and evaluates the key characteristics of the insect-based chitosan compared to crab shell chitosan. H. illucens is a sustainable source of high-quality protein, lipids and compost currently applied as aquaculture feed and soil improver. The ecdysis of the H. illucens larvae results in larval exoskeletons (LE) rich in chitin and suitable for chitosan production. Using procedures based on crab shell processing, two steps were performed to purify chitin from the larval exoskeletons. The results showed that formic acid is a suitable demineralization agent, requiring 5 mol per kg larval exoskeletons and thorough washing afterwards for 89 % demineralization effectivity (DME). The effect of the deproteinization variables were explored via design of experiments and analyzed using linear regression resulting in chitin contents of the deproteinized material of 83 % in small-scale and 87 % in 10 L-scale, respectively. Heterogeneous and homogeneous deacetylation was performed to convert chitin in different chitosan fractions. Heterogeneous deacetylation at 120 °C resulted in a deacetylation degree (DD) of 72 % and a yield of 43 % at maximum with regard to the total chitin applied. Homogeneous deacetylation at 4 °C resulted in a low chitosan yield of 13 % and a DD of 34 %. However, chitosan solubilized in acetic acid revealed superior film forming properties and a high viscosity. The results indicated that the chitosan from insects has comparable properties than those produced from crab shells, although the properties are also strongly dependent on the manufacturing conditions., 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 © 2019 Elsevier B.V. All rights reserved.)

Yellow protein of the takeout family (YPT) and albino-related takeout protein (ALTO) are involved in body-color polyphenism in Schistocerca gregaria. YPT has been proposed to bind to β-carotene, whereas the physiological role of ALTO is unclear. Structurally, takeout proteins contain a long continuous tunnel to bind specific ligands. However, the specific ligands of YPT and ALTO have not been fully elucidated. Here, we isolated the full coding cDNAs of these proteins and successfully produced recombinant YPT and ALTO using an Escherichia coli expression system. Absorption spectral analyses of YPT with and without carotenoids revealed that this protein bound to lutein. In contrast, obvious binding of YPT to β-carotene and astaxanthin was not detected. Similar results were obtained for ALTO. The presence of juvenile hormone only weakly affected the protein/carotenoid interactions. These results suggested that YPT and ALTO specifically bound to lutein in a juvenile hormone-independent manner., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Phosphoserine phosphatase (PSP) catalyses the synthesis of l-serine via the phosphorylated pathway by facilitating the dephosphorylation of phosphoserine. A cDNA encoding PSP from the silkworm Bombyx mori (bmPSP) was isolated using reverse transcription-PCR and then sequenced. The resulting clone encoded 236 amino acids with a molecular weight of 26 150, exhibiting 14-60% sequence identity with other PSPs. The recombinant PSP was overexpressed in Escherichia coli and purified. Kinetic studies showed that bmPSP possessed activity toward l-phosphoserine, and Asp20, Asp22 and Asp204 in bmPSP were found to be critical for modulating bmPSP activity. Real-time PCR analysis provided evidence that the amount of bmpsp transcript was reduced in middle silk glands of a sericin-deficient silkworm strain. These findings revealed that bmPSP may play important roles in synthesizing one-carbon donors of l-serine, which is abundant in silk, as well as other cell metabolites in B. mori., (© 2019 The Royal Entomological Society.)

Background: Antibacterial peptides play important roles in the innate immune system of insects and are divided into four categories according to their structures. Although many antibacterial peptides have been reported in lepidopteran insects, the roles of an attacin-like gene in immune response of Antheraea pernyi remain unclear., Objective: In this study, the cloning and immunological functions of an attacin-like gene from Antheraea pernyi were investigated., Methods: The open reading frame of Ap-attacin-like gene was cloned by PCR using the specific primers and then was ligated to the pET-32a vector to construct the recombinant plasmids Ap-attacin- like-pET-32a. The recombinant Ap-attacin-like protein was expressed in E. coli (BL21 DE3) cells and purified by Ni-NTA affinity chromatography. The expression patterns of Ap-attacin-like in different tissues or under microorganism challenges were investigated by real-time PCR and western blotting. Finally, agar well diffusion assay was performed to determine the antimicrobial activity of the recombinant Ap-attacin-like proteins based on the inhibition rate., Results: The expression level of Ap-attacin-like was highest in the fat body compared with the other examined tissues. The expression of Ap-attacin-like in the fat body was significantly elevated after E. coli, Beauveria bassiana, Micrococcus luteus or Nuclear Polyhedrosis Virus challenges. In addition, the recombinant Ap-attacin-like proteins had obvious antibacterial activity against E. coli., Conclusion: Ap-attacin-like was highly expressed in immune-related tissues and its expression level was significantly induced by different microorganism challenges, suggesting that Ap-attacin-like participated in the innate immunity of A. pernyi., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

AcMNPV is the first baculovirus to be sequenced and is considered a model of baculovirus. ac154 is a later expression gene in AcMNPV genome and its function is unknown. In this study, we explored the function of Ac154 in AcMNPV infection process in host Sf9 cells. The results showed that Ac154 was distributed in both nucleus and cytoplasm. Knockout of ac154 did not affect the production of BV, but the yield of progeny virus was reduced, indicating the auxiliary function of Ac154 in virus production. MTT assay showed that Ac154 promoted the proliferation and inhibited apoptosis of Sf9 cells. Overexpression of ac154 gene significantly increased the transcription level of anti-apoptotic gene p35, and delayed the expression of the pro-apoptotic protein SfP53 and reduced its expression level, which indicated its anti-apoptotic role in the host cells. In conclusion, our results demonstrated Ac154 could delay apoptosis process in host cells by regulating the transcription of p35 gene and the expression of SfP53 protein, which provided a more favorable environment for progeny virus replication and packaging, thereby promoting the proliferation of progeny virus. So we provided a potentially improved bac-to-bac eukaryotic protein expression system and biopesticide in this work.

In developing countries, low-cost control and treatment programs that offer combined approaches against diseases and their vectors are certainly needed. Ivermectin (IVM) has been well known for its role in the treatment of parasitic diseases, due to its effect on glutamate-gated chloride channels. These same channels are also present in the mosquito vector, and thus, research has focused on the insecticidal effects of this drug. Possible alternative mechanisms of IVM on the physiology of mosquitoes, however, have not been sufficiently elaborated. We assessed the protease activity, lipid peroxidation, and local expression of STAT, p53, caspase-3, and Bax markers to study the effect of this antibiotic on digestion and immunity in Culex pipiens. Sugar- and blood-feeding assays were employed to investigate the potential influence of blood feeding on the dynamics of these parameters. IVM was found to have an effect on protease activity, lipid peroxidation as well as the expression of different markers investigated in this work. The focus on the detailed effect of this drug certainly opens the gate to broadening the spectrum of IVM and expanding its health and economic benefit, especially that it is relatively more affordable than other antibiotics on the market.

The ATP-binding cassette (ABC) transporters exist in all living organisms and play major roles in various biological functions by transporting a wide variety of substrates across membranes. The functions of ABC transporters in drug resistance have been extensively studied in vertebrates; however, they are rarely characterized in agricultural pests. The Asian citrus psyllid, Diaphorina citri, is one of the most damaging pests of the Citrus genus because of its transmission of Huanglongbing, also known as Yellow Dragon disease. In this study, the next-generation sequencing technique was applied to research the ABC transporters of D. citri. Fifty-three ABC transporter genes were found in the RNA-Seq data, and among these ABC transporters, 4, 4, 5, 2, 1, 4, 18 and 15 ABC proteins belonged to the ABCA-ABCH subfamilies, respectively. Different expression profiles of 52 genes between imidacloprid-resistant and imidacloprid-susceptible strains were studied by qRT-PCR; 5 ABCGs and 4 ABCHs were significantly upregulated in the imidacloprid-resistant strain. In addition, five of the nine upregulated genes were widely expressed in adult tissues in spatial expression analysis. The results suggest that these genes may play key roles in this phenotype. In general, this study contributed to our current understanding of D. citri resistance to insecticides.

Unrelated genes establish head-to-tail polarity in embryos of different fly species, raising the question of how they evolve this function. We show that in moth flies ( Clogmia , Lutzomyia ), a maternal transcript isoform of odd-paired (Zic) is localized in the anterior egg and adopted the role of anterior determinant without essential protein change. Additionally, Clogmia lost maternal germ plasm, which contributes to embryo polarity in fruit flies ( Drosophila ). In culicine ( Culex , Aedes) and anopheline mosquitoes (Anopheles), embryo polarity rests on a previously unnamed zinc finger gene ( cucoid ), or pangolin ( dTcf ), respectively. These genes also localize an alternative transcript isoform at the anterior egg pole. Basal-branching crane flies ( Nephrotoma ) also enrich maternal pangolin transcript at the anterior egg pole, suggesting that pangolin functioned as ancestral axis determinant in flies. In conclusion, flies evolved an unexpected diversity of anterior determinants, and alternative transcript isoforms with distinct expression can adopt fundamentally distinct developmental roles., Competing Interests: YY, JK, IM, FC, EC, JR, US No competing interests declared, (© 2019, Yoon et al.)

Integrating multifunctionality such as stretchability, adhesiveness, and electroconductivity on a single protein hydrogel is highly desirable for various applications, and remains a challenge. Here we present the development of such multifunctional hydrogels based on resilin, a natural rubber-like material with remarkable extensibility and resilience. First, genetically engineered reslin-like proteins (RLPs) with varying molecular weight were biosynthesized to tune mechanical strength and stiffness of the cross-linked RLP hydrogels. Second, glycerol was incorporated into the hydrogels to endow adhesive properties. Next, a graphene-RLP conjugate was synthesized for cross-linking with the unmodified, pristine RLP to form an integrated network. The obtained hybrid hydrogel could be stretched to over four times of its original length, and self-adhered to diverse substrate surfaces due to its high adhesion strength of ∼24 kPa. Furthermore, the hybrid hydrogel showed high sensitivity, with a gauge factor of 3.4 at 200% strain, and was capable of real-time monitoring human activities such as finger bending, swallowing, and phonating. Due to these favorable attributes, the graphene/resilin hybrid hydrogel was a promising material for use in wearable sensors. In addition, the above material design and functionalization strategy may provide intriguing opportunities to generate innovative materials for broad applications.

Insects are the only known animals in which sexual differentiation is controlled by sex-specific splicing. The doublesex transcription factor produces distinct male and female isoforms, which are both essential for sex-specific development. dsx splicing depends on transformer , which is also alternatively spliced such that functional Tra is only present in females. This pathway has evolved from an ancestral mechanism where dsx was independent of tra and expressed and required only in males. To reconstruct this transition, we examined three basal, hemimetabolous insect orders: Hemiptera, Phthiraptera, and Blattodea. We show that tra and dsx have distinct functions in these insects, reflecting different stages in the changeover from a transcription-based to a splicing-based mode of sexual differentiation. We propose that the canonical insect tra-dsx pathway evolved via merger between expanding dsx function (from males to both sexes) and narrowing tra function (from a general splicing factor to dedicated regulator of dsx )., Competing Interests: JW, ED, XB, CS, AW, MA, AK No competing interests declared, (© 2019, Wexler et al.)

Resilin, a protein found in insect cuticles, is renowned for its outstanding elastomeric properties. The authors' laboratory previously developed a recombinant protein, which consisted of consensus resilin-like repeats from Anopheles gambiae, and demonstrated its potential in cartilage and vascular engineering. To broaden the versatility of the resilin-like protein, this study utilizes a cleavable crosslinker, which contains a disulfide bond, to develop smart resilin-like hydrogels that are redox-responsive. The hydrogels exhibit a porous structure and a stable storage modulus (G') of ≈3 kPa. NIH/3T3 fibroblasts cultured on hydrogels for 24 h have a high viability (>95%). In addition, the redox-responsive hydrogels show significant degradation in a reducing environment (10 mm glutathione (GSH)). The release profiles of fluorescently labeled dextrans encapsulated within the hydrogels are assessed in vitro. For dextran that is estimated to be larger than the mesh size of the gel, faster release is observed in the presence of reducing agents due to degradation of the hydrogel networks. These studies thus demonstrate the potential of using these smart hydrogels in a variety of applications ranging from scaffolds for tissue engineering to drug delivery systems that target the intracellular reductive environments of tumors., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the evolutionarily-derived termite subfamily Nasutitermitinae (family Termitidae), soldiers defend their nestmates by discharging polycyclic diterpenes from a head projection called the "nasus." The diterpenes are synthesised in the frontal gland from the precursor geranylgeranyl diphosphate (GGPP), which is generally used for post-translational modification of proteins in animals. In this study, we constructed a comprehensive gene catalogue to search for genes involved in the diterpene biosynthesis by assembling RNA sequencing reads of Nasutitermes takasagoensis, identifying eight gene copies for GGPP synthase (GGPPS). The number of gene copies is much larger in contrast to other related insects. Gene cloning by reverse transcription-PCR and rapid amplification of cDNA ends confirmed that seven GGPPS genes (NtGGPPS1 to NtGGPPS7) have highly variable untranslated regions. Molecular phylogenetic analysis showed that the NtGGPPS7 gene was grouped with homologs obtained from ancestral termites that have only a single copy of the gene, and the NtGGPPS6 gene was grouped with homologs obtained from a basal lineage of termitids, in which soldiers do not synthesise diterpenes. As the sister group to this clade, furthermore, a monophyletic clade included all the other NtGGPPS genes (NtGGPPS1 to NtGGPPS5). Expression analyses revealed that NtGGPPS7 gene was expressed in all the examined castes and tissues, whereas all the other genes were expressed only in the soldier head. These results suggest that gene duplication followed by subfunctionalisation of the GGPPS genes might have accompanied the evolution of chemical defence in the nasute termite lineage., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Juvenile hormones (JH) regulate wide-ranging physiological and developmental processes in insects. However, molecular mechanisms underlying JH signaling remain to be determined. Vitellogenin (Vg) is primarily an egg-yolk protein, but recently proposed to serve many functions in insects. In the female American cockroach (Periplaneta americana), vitellogenin (Vg) genes are activated by JH III and suppressed by 20-hydroxyecdysone (20E) via cis-regulatory elements in a dose-dependent manner. In the present study, the upstream promoter region (935 bp) of Vg1 was cloned to elucidate the action of these hormones. A luciferase reporter assay identified an 81 bp region in the promoter region of Vg1 (-120 to -39 bp) that we found to be critical for JH III activation and 20E suppression. This 81 bp region contains a direct repeat separated by a 2-nucleotide spacer-designated Vg1HRE- that is similar to the Drosophila ecdysone response element direct repeat 4. Moreover, nuclear proteins isolated from nymphs, males, females, and Sf9 cells successfully bound to Vg1HRE, while binding was outcompeted by a 100-fold excess of cold probe or dephosphorylated nuclear protein extracts. In addition, binding was outcompeted by other ecdysone and JH response elements with similar half-site sequences (direct repeats) but to varying extents. Ultimately, we postulate that JH III indirectly activates Vg expression by interfering with or inhibiting the phosphorylation of nuclear proteins bound to Vg1HRE. Involvement of JH III in both induction of Vg1 and control of nuclear proteins binding to Vg1HRE suggest the latter to play an important role in JH signaling., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Baculoviruses are large double-stranded DNA viruses that are virulent pathogens of certain insect species. In a natural host, Trichoplusia ni , infection by the model baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) begins when the occluded form of the virus disassembles in the midgut and virions infect midgut epithelial cells to establish the primary phase of the infection. To better understand the primary phase of the AcMNPV infection cycle, newly molted 5th-instar T. ni larvae were orally infected with AcMNPV occlusion bodies and the transcriptional responses of the T. ni midgut were analyzed at various times from 0 to 72 h postinfection, using transcriptome sequencing analysis and a T. ni reference genome. The numbers of differentially expressed host genes increased as the infection progressed, and we identified a total of 3,372 differentially expressed T. ni transcripts in the AcMNPV-infected midgut. Genes encoding orthologs of HMG176, atlastin, and CPH43 were among the most dramatically upregulated in response to AcMNPV infection. A number of cytochrome P450 genes were downregulated in response to infection. We also identified the effects of AcMNPV infection on a large variety of genes associated with innate immunity. This analysis provides an abundance of new and detailed information on host responses to baculovirus infection during the primary phase of the infection in the midgut and will be important for understanding how baculoviruses establish productive infections in the organism. IMPORTANCE Baculoviruses are virulent pathogens of a number of important insect pest species. In the host Trichoplusia ni , infection begins in the midgut when infectious virions of the occlusion-derived virus (ODV) phenotype enter and subsequently replicate in cells of the midgut epithelium. A second virion phenotype (budded virus [BV]) is produced there, and BV mediates systemic infection of the animal. Most prior detailed studies of baculovirus infections have focused on BV infections of cultured cells. In this study, we examined the transcriptional responses of the T. ni midgut to infection by ODV of the baculovirus AcMNPV and identified a variety of host genes that respond dramatically to viral infection. Understanding the transcriptional responses of the host midgut to viral infection is critically important for understanding the biphasic infection in the animal as a whole., (Copyright © 2019 American Society for Microbiology.)

In insects, the gustatory system has a critical function not only in selecting food and feeding behaviours but also in growth and metabolism. Gustatory receptors play an irreplaceable role in insect gustatory signalling. Trichogramma chilonis is an effective biocontrol agent against agricultural insect pests. However, the molecular mechanism of gustation in T. chilonis remains elusive. In this study, we found that T. chilonis adults had a preference for D-fructose and that D-fructose contributed to prolong longevity and improve fecundity. Then, We also isolated the full-length cDNA encoding candidate gustatory receptor (TchiGR43a) based on the transcriptome data of T. chilonis, and observed that the candidate gustatory receptor gene was expressed from the larval to adult stages. The expression levels of TchiGR43a were similar between female and male. A Xenopus oocyte expression system and two-electrode voltage-clamp recording further verified the function analysis of TchiGR43a. Electrophysiological results showed that TchiGR43a was exclusively tuned to D-fructose. By the studies of behaviour, molecular biology and electrophysiology in T. chilonis, our results lay a basic fundation of further study on the molecular mechanisms of gustatory reception and provide theoretical basis for the nutritional requirement of T. chilonis in biocontrol., Competing Interests: The authors have declared that no competing interests exist.

Many organisms both undergo dramatic morphological changes during post-embryonic development and also regenerate lost structures, but the roles of epigenetic regulators in such processes are only beginning to be understood. In the present study, the functions of two histone modifiers were examined during metamorphosis and larval limb regeneration in the red flour beetle Tribolium castaneum. Polycomb (Pc), a member of Polycomb repressive complex 1 (PRC1), and Enhancer of zeste (E(z)), a member of Polycomb repressive complex 2 (PRC2), were silenced in larvae using RNA interference. In the absence of Pc, the head appendages of adults transformed into a leg-like morphology, and the legs and wings assumed a metathoracic identity, indicating that Pc acts to specify proper segmental identity. Similarly, silencing of E(z) led to homeotic transformation of legs and wings. Additional defects were also observed in limb patterning as well as eye morphogenesis, indicating that PcG proteins play critical roles in imaginal precursor cells. In addition, larval legs and antennae failed to re-differentiate when either Pc or E(z) was knocked down, indicating that histone modification is necessary for proper blastema growth and differentiation. These findings indicate that PcG proteins play extensive roles in postembryonic plasticity of imaginal precursor cells., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In this study, we identified and characterized a phosphoserine aminotransferase (bmPSAT) from Bombyx mori (B. mori) that is responsible for l-serine biosynthesis. A complementary DNA that encodes bmPSAT was cloned by reverse transcriptase polymerase reaction and sequenced. The presumed amino acid sequence revealed 47-87% identity with known PSATs from insects, humans, plants, and bacteria. Through phylogenetic analysis, we found that bmPSAT is evolutionary related to insect PSATs. Recombinant bmPSAT was produced in Escherichia coli by using a cold-shock promotor and purified to homogeneity. This enzyme utilizes phosphohydroxypyruvate and glutamate for transamination. bmPSAT messenger RNA (mRNA) was expressed at higher levels in several tissues of standard strain silkworm including the silk gland, whereas a sericin-deficient silkworm strain exhibited a diminished expression of bmPSAT mRNA in the silk gland. These findings indicate that bmPSAT may play an important role in synthesizing and supplying l-serine in the larva of B. mori., (© 2019 Wiley Periodicals, Inc.)

Insect-derived serine protease inhibitors (serpins) exhibit multiple inhibitory activities. Todate some functional roles for serpins in Hyphantria cunea Drury have been identified. Here, new functional features of the H. cunea serine protease inhibitor (dHC-serpin) were characterized. In this study, the cDNA encoding serpin was amplified from H. cunea (dHC) pupa fat body total RNA using RT-PCR. The full-length dHC-serpin cDNA encoded a protein of 440 amino acids with a predicted 19-amino acid signal peptide and a 421-amino acid functional domain. The functional domain was cloned into a pSUMO vector and transformed into Escherichia coli, resulting in the production of a pSUMO-dHC-serpin fusion protein. The soluble form of this protein was then purified by Ni-IDA chromatography. The SUMO-dHC-serpin fusion protein was then cleaved using a SUMO protease and purified again by Ni-IDA chromatography. dHC-serpin did not inhibit trypsin, elastase, proteinase K or cathepsin B, but strongly inhibited papain. The inhibitor retained its inhibitory activity over a broad range of pH (pH 2-12), temperature (20-50 °C), and DTT concentration (up to 100 mM). A complete loss of inhibitory activity was observed at pH 13 and 70 °C. Serpins generally serve as inhibitors that use a mobile reactive center loop (RCL) as bait to trap protease targets. dHC-serpin, like others serpins, binds papain using the RCL structure., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The reproductive ground plan hypothesis proposes that gene networks regulating foraging behavior and reproductive female physiology in social insects emerged from ancestral gene and endocrine factor networks. Expression of storage proteins such as vitellogenins and hexamerins is an example of this co-option. Hexamerins, through their role modulating juvenile hormone availability, are involved in caste determination in termites. The genome of the fire ant (Solenopsis invicta) encodes four hexamerin genes, hexamerin-like (LOC105192919, hereafter called hexamerin 1), hexamerin (LOC105204474, hereafter called hexamerin 2), arylphorin subunit alpha-like, and arylphorin subunit beta. In this study, a phylogenetic analysis of the S. invicta hexamerins determined that each predicted protein clustered with one of the orthologous Apis mellifera hexamerins. Gene expression analyses by RT-qPCR revealed differential expression of the hexamerins between queens and workers, and between specific task-allocated workers (nurses and foragers). Queens and nurses had significantly higher expression of all genes when compared to foragers. Hexamerin 1 was expressed at higher levels in queens, while hexamerin 2 and arylphorin subunit beta were expressed at significantly higher levels in nurses. Arylphorin subunit alpha-like showed no significant difference in expression between virgin queens and nurses. Additionally, we analyzed the relationship between the expression of hexamerin genes and S-hydroprene, a juvenile hormone analog. Significant changes in hexamerin expression were recorded in nurses, virgin queens, and foragers 12 h after application of the analog. Hexamerin 1 and arylphorin subunit alpha-like expression were significantly lower after analog application in virgin queens. In foragers, hexamerin 2 and arylphorin subunit beta were significantly lower after analog application, while in nurses expression of all genes were significantly lower after analog application. Our results suggest that in S. invicta hexamerin genes could be associated with reproductive division of labor and task-allocation of workers., Competing Interests: The authors have declared that no competing interests exist.

The smaller tea tortrix, Adoxophyes honmai, has developed strong resistance to tebufenozide, a diacylhydrazine-type (DAH) insecticide. Here, we investigated its mechanism by identifying genes responsible for the tebufenozide resistance using various next generation sequencing techniques. First, double-digest restriction site-associated DNA sequencing (ddRAD-seq) identified two candidate loci. Then, synteny analyses using A. honmai draft genome sequences revealed that one locus contained the ecdysone receptor gene (EcR) and the other multiple CYP9A subfamily P450 genes. RNA-seq and direct sequencing of EcR cDNAs found a single nucleotide polymorphism (SNP), which was tightly linked to tebufenozide resistance and generated an amino acid substitution in the ligand-binding domain. The binding affinity to tebufenozide was about 4 times lower in in vitro translated EcR of the resistant strain than in the susceptible strain. RNA-seq analyses identified commonly up-regulated genes in resistant strains, including CYP9A and choline/carboxylesterase (CCE) genes. RT-qPCR analysis and bioassays showed that the expression levels of several CYP9A and CCE genes were moderately correlated with tebufenozide resistance. Collectively, these results suggest that the reduced binding affinity of EcR is the main factor and the enhanced detoxification activity by some CYP9As and CCEs plays a supplementary role in tebufenozide resistance in A. honmai.

In this study, various constructs and hosts were used to produce high levels of cecropin B2 (cecB2). To mitigate cecB2's toxic inhibition of host cells, various cecB2 constructs were built. Results showed that the combination of a chitin-binding domain and an intein self-cleavage motif in front of cecropin B2, without a His-tag, was best for cecB2 expression. E. coli ER2566 was the best host, and 2YT was the best medium for cultivation. Under these conditions, a cecB2 yield of 98.2 mg/L could be obtained after purification. The purified cecB2 expressed a wide antimicrobial effect on most Gram-negative strains, including multidrug-resistant Acinetobactor baumannii and Staphylococcus aureus. This study provides a systematic approach to the efficient production of the antimicrobial peptide (AMP) cecB2 via the recombinant E. coli process, which is expected to be an efficient way for the production of other AMPs., (© 2018 International Union of Biochemistry and Molecular Biology, Inc.)

Arginine kinase (AK) plays a critical role in insect energy metabolism and has been proposed to be a potential insecticide target for commercial exploitation. In this study, the full length cDNA encoding a typical group 1 insect AK (FoAK) was isolated from the western flower thrips (WFT), Frankliniella occidentalis (Pergande). Sequence analysis showed that FoAK contains an open reading frame of 1068 nucleotides, which encods a protein of 355 amino acid residues including the signature sequence pattern of ATP-guanidino kinases. Genomic structure analysis showed that the coding region of FoAK contains five exons connected by four introns. RT-qPCR analysis revealed that the mRNA expression of FoAK was developmentally regulated with the lowest level in prepupal stage. Enzymatic activity analysis of the recombinant enzymes expressed in Escherichia coli showed that FoAK is highly stereo specific for L-arginine versus D-arginine and the apparent Michaelis constant for L-arginine (Km Arg ) is comparable to that of AKs from a variety of species. This research should enable further investigation of the function as well as in vitro screening for inhibitors of FoAK., (Copyright © 2019. Published by Elsevier Inc.)

Periplanetasin-2 is a 15-mer antimicrobial peptide (AMP), derived from the American cockroach Periplaneta americana. This novel AMP exhibits potent antibacterial effect against several pathogenic bacteria including Escherichia coli. Distinct from the targeting cell membrane, which is the general antibacterial mechanism of AMP, periplanetasin-2 exerts its antibacterial activity via apoptosis-like death, which is physiologically and mechanistically similar to eukaryotic apoptosis. E. coli cells treated with periplanetasin-2 showed features of apoptosis in a concentration-dependent manner, such as membrane depolarization, DNA fragmentation, caspase-like protein activation, and phosphatidylserine externalization. These physiological changes were attenuated by pretreatment with the reactive oxygen species (ROS) scavenger, which demonstrates that periplanetasin-2 induced apoptosis-like death in E. coli by generating ROS. In addition, periplantasin-2-induced apoptotic death was affected by SOS response components. In the absence of RecA, an essential protein for SOS response, apoptosis did not occur and the antibacterial activity of periplanetasin-2 was decreased. In contrast, deletion of the SOS gene dinF caused higher ROS accumulation and apoptotic features were detected. Collectively, these results indicate that the antibacterial mechanism of periplanetasin-2 is ROS-induced apoptosis-like death, which requires RecA for proceeding it, and the role of DinF is assumed to contribute to the ROS defense SOS response.

Chitin synthase is responsible for chitin synthesis in the cuticles and cuticular linings of other tissues in insects. We cloned two alternative splicing variants of the chitin synthase 1 gene (SfCHS1) from the white-backed planthopper, Sogatella furcifera. The full-length cDNA of the two variants (SfCHS1a and SfCHS1b) consists of 6408 bp, contains a 4719-bp open reading frame encoding 1572 amino acids, and has 5' and 3' non-coding regions of 283 and 1406 bp, respectively. The two splicing variants occur at the same position in the cDNA sequence between base pairs 4115 and 4291, and consist of 177 nucleotides that encode 59 amino acids but show 74.6% identity at the amino acid level. Analysis in different developmental stages showed that expression of SfCHS1 and SfCHS1a were highest just after molting, whereas SfCHS1b reached its highest expression level 2 days after molting. Further, SfCHS1 and SfCHS1a were mainly expressed in the integument, whereas SfCHS1b was predominately expressed in the gut and fat body. RNAi-based gene silencing inhibited transcript levels of the corresponding mRNAs in S. furcifera nymphs injected with double-stranded RNA of SfCHS1, SfCHS1a, and SfCHS1b, resulted in malformed phenotypes, and killed most of the treated nymphs. Our results indicate that SfCHS1 may be a potential target gene for RNAi-based S. furcifera control.

The mosquito Aedes aegypti is the primary vector for the fastest growing infectious disease in the world, dengue fever. Disease transmission heavily relies on the ability of female mosquitoes to locate their human hosts. Additionally, males may be found in close proximity to humans, where they can find mates. Host seeking behaviour of both sexes is dependent on adult sexual maturation. Identifying the molecular basis for the onset of host seeking may help to determine targets for future vector control. In this study, we investigate modulation of the host seeking behaviour and the transcript abundance of the main chemoreceptor families between sexes and across ages in newly-emerged mosquitoes. Attraction to human odour was assessed using a Y-tube olfactometer, demonstrating that both males and females display age-dependent regulation of host seeking. The largest increase in transcript abundance was identified for select chemosensory genes in the antennae of young adult Ae. aegypti mosquitoes and reflects the increase in attraction to human odour observed between 1 and 3 day(s) post-emergence in both males and females. Future functional characterisation of the identified differentially abundant genes may provide targets for the development of novel control strategies against vector borne diseases.