Transcriptome analysis and functional study of phospholipase A2 in Galleria mellonella larvae lipid metabolism in response to envenomation by an ectoparasitoid, Iseropus kuwanae.

There is abundant evidence that parasitoids manipulate their hosts by envenomation to support the development and survival of their progeny before oviposition. However, the specific mechanism underlying host nutritional manipulation remains largely unclear. To gain a more comprehensive insight into the effects induced by the gregarious ectoparasitoid Iseropus kuwanae (Hymenoptera: Ichneumonidae) on the greater wax moth Galleria mellonella (Lepidoptera: Pyralidae) larvae, we sequenced the transcriptome of both non-envenomed and envenomed G. mellonella larvae, specifically targeting genes related to lipid metabolism. The present study revealed that 202 differentially expressed genes (DEGs) were identified and 9 DEGs were involved in lipid metabolism. The expression levels of these 9 DEGs relied on envenomation and the duration post-envenomation. Further, envenomation by I. kuwanae induced an increase in triglyceride (TG) level in the hemolymph of G. mellonella larvae. Furthermore, silencing GmPLA2 in G. mellonella larvae 24 h post-envenomation significantly decreased the content of 4 unsaturated fatty acids and TG levels in the hemolymph. The content of linoleic acid and α-linoleic acid were significantly decreased and the content of oleic acid was significantly increased by exogenous supplement of arachidonic acid. Meanwhile, the reduction in host lipid levels impairs the growth and development of wasp offspring. The present study provides valuable knowledge about the molecular mechanism of the nutritional interaction between parasitoids and their hosts and sheds light on the coevolution between parasitoids and host insects.

Binding characteristics of pheromone-binding protein 1 in Glyphodes pyloalis to organophosphorus insecticides: Insights from computational and experimental approaches.

Glyphodes pyloalis (Lepidoptera: Pyralidae) is one of the major pests in mulberry production in China, which has developed resistance to various insecticides. Chemoreception is one of the most crucial physiological tactics in insects, playing a pivotal role in recognizing chemical stimuli in the environment, including noxious stimuli such as insecticides. Herein, we obtained recombinant pheromone-binding protein 1 (GpylPBP1) that exhibited antennae-biased expression in G. pyloalis. Ligand-binding assays indicated that GpylPBP1 had the binding affinities to two organophosphorus insecticides, with a higher binding affinity to chlorpyrifos than to phoxim. Computational simulations showed that a mass of nonpolar amino acid residues formed the binding pocket of GpylPBP1 and contributed to the hydrophobic interactions in the bindings of GpylPBP1 to both insecticides. Furthermore, the binding affinities of three GpylPBP1 mutants (F12A, I52A, and F118A) to both insecticides were all significantly reduced compared to those of the GpylPBP1-wild type, suggesting that Phe12, Ile52, and Phe118 residues were crucial binding sites and played crucial roles in the bindings of GpylPBP1 to both insecticides. Our findings can be instrumental in elucidating the effects of insecticides on olfactory recognition in moths and facilitating the development of novel pest management strategies using PBPs as targets based on insect olfaction.

Host trehalose metabolism disruption by validamycin A results in reduced fitness of parasitoid offspring.

The general cutworm, Spodoptera litura (Lepidoptera: Noctuidae) is a worldwide destructive omnivorous pest and the endoparasitoid wasp Meteorus pulchricornis (Hymenoptera: Braconidae) is the dominant endoparasitoid of S. litura larvae. Trehalase is a key enzyme in insect trehalose metabolism and plays an important role in the growth and development of insects. However, the specific function of trehalase in parasitoid and host associations has been less reported. In this study, we obtained two trehalase genes (SlTre1 and SlTre2) from our previously constructed S. litura transcriptome database; they were highly expressed in 3rd instar larvae. SlTre1 was mainly expressed in the midgut, and SlTre2 was expressed highest in the head. SlTre1 and SlTre2 were highly expressed 5 days after parasitization by M. pulchricornis. Treatment with the trehalase inhibitor validamycin A significantly inhibited the expression levels of SlTre1 and SlTre2, and the trehalase activity. Besides, the content of trehalose was increased but the content of glucose was decreased 24 h after validamycin A treatment in parasitized S. litura larvae. In addition, the immune-related genes in phenoloxidase (PO) pathway and fatty acid synthesis-related genes in lipid metabolism were upregulated in parasitized host larvae after validamycin A treatment. Importantly, the emergence rate, proportion of normal adults, and body size of parasitoid offspring was decreased in parasitized S. litura larvae after validamycin A treatment, indicating that validamycin A disrupts the trehalose metabolism of parasitized host and thus reduces the fitness of parasitoid offspring. The present study provides a novel perspective for coordinating the application of biocontrol and antibiotics in agroecosystem.

Identification and functional study of detoxification-related genes in response to tolfenpyrad stress in Glyphodes pyloalis Walker (Lepidoptera: Pyralidae).

Glyphodes pyloalis Walker (G. pyloalis) is a common destructive mulberry pest. Due to the long-term and frequent use of insecticides, it has developed tolerance to commonly used insecticides. Tolfenpyrad (TFP) is a novel pyrazole heterocyclic insecticide. In order to understand the TFP detoxification mechanism of G. pyloalis larvae, we first estimated the LC30 dose of TFP for 3rd instar G. pyloalis larvae. Next, we identified genes that were differentially expressed in 3rd instar G. pyloalis larvae treated with TFP compared to the control group by transcriptome sequencing. In total, 86,949,569 and 67,442,028 clean reads were obtained from TFP-treated and control G. pyloalis larvae, respectively. A total of 5588 differentially expressed genes (DEGs) were identified in TFP-treated and control G. pyloalis larvae, of which 3084 genes were upregulated and 2504 genes were downregulated. We analyzed the expression of 43 candidate detoxification enzyme genes associated with insecticide tolerance using qPCR. According to the spatiotemporal expression pattern of DEGs, we found that CYP6ABE1, CYP333A36 and GST-epsilon8 were highly expressed in the midgut, while CarEs14 was strongly expressed in haemolymph. Furthermore, we successfully knocked down these genes by RNA interference. After silencing CYP6ABE1 and CYP333A36, bioassay showed that the mortality rate of TFP-treated G. pyloalis larvae was significantly higher compared to the control group. This study provides a theoretical foundation for understanding the sensitivity of G. pyloalis to TFP and establish the basis for the effective and green management of this pest.

Odorant-Binding Protein 6 Contributes High Binding Affinity to Insecticides in a Parasitic Wasp Meteorus pulchricornis (Hymenoptera: Braconidae).

Meteorus pulchricornis is a preponderant parasitic wasp of various lepidopteran pests. The extensive application of broad-spectrum insecticides usually causes serious threats to the olfactory recognition of nontarget insects such as parasitoid wasps. However, the binding mechanism of odorant-binding proteins (OBPs) to insecticides in parasitoid wasps remains unknown. Herein, we find that the MpulOBP6 protein had a strong binding affinity to three insecticides (phoxim, chlorpyrifos, and chlorfenapyr). Results of computational simulations revealed that the hydrophobic interaction contributed by a mass of nonpolar amino acid residues was the primary driving force in the formation and stabilization of MpulOBP6-insecticide complexes. Among them, four residues (Met75, Val84, Phe121, and Pro122) and two residues (Val84 and Phe111) play an essential role in the binding of MpulOBP6 to phoxim and chlorfenapyr, respectively. Our findings could be instrumental to elucidate the effects of insecticide application toward the olfactory recognition of nontarget insects in the processes of agricultural production.

Efficient acquisition of high-purity cyanidin-3-O-glucoside from mulberry fruits: An integrated process of ATPS whole-cell transformation and semi-preparative HPLC purification.

As a nutritious fruit, mulberry is an ideal source of high-quality cyanidin-3-O-glucoside (C3G) with various biological activities. However, the difficult separation process of high-purity C3G leads to its high price. To rapidly prepare high-purity C3G, cyanidin-3-O-rutinoside is converted to C3G by direct hydrolysis of rhamnose bond using a whole-cell catalyst containing α-rhamnosidase. Combined with an aqueous two-phase system, a coupling reaction separation system was established. Two monomers were successfully separated by semi-preparative high performance liquid chromatography (semi-preparative HPLC). The conversion of C3G catalyzed by whole-cells in the PEG/Na2SO4 system increased from 47.11 % to 66.56 %, compared with the EtOH/(NH4)2SO4 system, and the whole-cell activity remained above 50 % after five rounds of reuse. Meanwhile, the purity of C3G was increased to 99 % via the semi-preparative HPLC purification and identified by MS. Thus, an integrated process of whole-cell-catalyzed conversion and product peak cutting partition collection provides a novel strategy for efficient biomanufacturing of high-purity C3G.

The Key Role of Fatty Acid Synthase in Lipid Metabolism and Metamorphic Development in a Destructive Insect Pest, Spodoptera litura (Lepidoptera: Noctuidae).

Fatty acid synthase (FAS) is a key enzyme in the lipid synthesis pathway, however, its roles in insects remain largely unknown. Here, we firstly identified two FAS genes from the transcriptome dataset of the general cutworm Spodoptera litura, which is a destructive insect pest of many crops. Both SlFAS1 and SlFAS2 were highly expressed in third instar larvae and in their fat bodies. Then, we successfully silenced SlFAS1 in third instar larvae and the content of α-linolenic acid and triglyceride was significantly decreased. Besides that, the effect of FAS on the metamorphic development in S. litura was evaluated. The results indicate that after silencing SlFAS1, the survival rates of S. litura larvae decreased significantly compared to the control groups. Silencing SlFAS1 in fifth instar larvae resulted in more malformed pupae and adults, and the emergence rates were significantly reduced. Furthermore, the ecdysone content in the haemolymph of fifth instar larvae was significantly decreased after silencing SlFAS1. In addition, knocking down SlFAS1 significantly alters the expression of other key genes in the lipogenesis pathway, implying that FAS has an impact on the lipogenesis pathway. The present study deepens the understanding of FAS in insects and provides novel potential targets for managing insect pests.

Microfluidic preparation of a novel phoxim nanoemulsion pesticide against Spodoptera litura.

With continuous development of pesticide dosage forms, emulsifiable concentrates using large amounts of organic solvents are gradually obsoleted. Nanoemulsions with high water content have been developed and the preparation processes also evolved, but these processes still exist some problems, such as poor controllability and high energy consumption. Microfluidic is a controllable nanoemulsion preparation system which mainly applied to pharmaceutical synthesis. In this study, the pesticide phoxim nanoemulsion was prepared by microfluidic technology. The optimized formulation of phoxim nanoemulsion was composed of Tween 80 and pesticide emulsifier 500 as surfactant, hexyl acetate as oil, and n-propanol as co-surfactant. Moreover, when the flow rates of water and oil in the microfluidic system were adjusted to 5 µL/min and 20 µL/min, phoxim nanoemulsion was obtained with a cloud point/boiling point of 109 °C, a particle size of 21.5 ± 0.8 nm and a potential value of - 18.7 ± 0.6 mV. Furthermore, the nanoemulsion had a rapid release effect in vitro which could be fitted by the Ritger-Peppas model. The feeding toxicity of the phoxim nanoemulsion was higher than that of commercial formulation while the contact killing effect was higher than that of the active ingredient. Therefore, pesticide dosage was reduced and the insecticidal effect was enhanced by using phoxim nanoemulsions. These results also confirm the potential of microfluidics as a green process to produce pesticide nanoemulsions.

Fatty acid synthases and desaturases are essential for the biosynthesis of α-linolenic acid and metamorphosis in a major mulberry pest, Glyphodes pyloalis walker (Lepidoptera: Pyralidae).

BACKGROUND: α-linolenic acid is an essential unsaturated fatty acid in organisms. However, there is a large gap between α-linolenic acid accumulation and its synthesis mechanism in insects. Fatty acid synthases (FASs) and desaturases (Desats) are vital enzymes required for the synthesis of unsaturated fatty acids. RESULTS: The pupae of Glyphodes pyloalis (Lepidoptera: Pyralidae), which is a destructive pest of mulberry trees, contain the highest level of α-linolenic acid compared to other life-history stages. To further explore the synthesis mechanism of α-linolenic acid in G. pyloalis pupae, we constructed a pupal transcriptome dataset and identified 106 genes related to fatty acid metabolism from it. Among these, two fatty acid synthases (GpylFAS) and five desaturases (GpylDesat) were identified. A qRT-PCR validation revealed that GpylFAS1 and GpylDesat1, 2, 3, 5 were expressed highest at pupal stages. Furthermore, the content of α-linolenic acid decreased significantly after silencing GpylFAS1 and GpylDesat5, respectively. Besides, knocking down GpylFAS1 or GpylDesat5 resulted in more malformed pupae and adults, as well as lower emergence rates. Meanwhile, silencing GpylFAS1 or GpylDesat5 affected the expressions of the other GpylFASs and GpylDesats. CONCLUSION: The present results illustrate the pivotal function of FASs and Desats in α-linolenic acid biosynthesis and metamorphosis in insects. Our research also broadens the sources of unsaturated fatty acids, especially for α-linolenic acid from insects, and provides novel insights for the management of mulberry insect pests from the perspective of utilization rather than control. © 2022 Society of Chemical Industry.

A double-population chaotic self-adaptive evolutionary dynamics model for the prediction of supercritical carbon dioxide solubility in polymers.

Solubility of gas in polymers is an important physico-chemical property of foam materials and widely used in the preparation and modification of new materials. Under the conditions of high temperature and high pressure, the dissolution process is a nonlinear, non-equilibrium and dynamic process, so it is difficult to establish an accurate solubility calculation model. Inspired by particle dynamics and evolutionary algorithm, this paper proposes a hybrid model based on chaotic self-adaptive particle dynamics evolutionary algorithm (CSA-PD-EA), which can use the iterative process of particles in evolutionary algorithms at the dynamic level to simulate the mutual diffusion process of molecules during dissolution. The predicted solubility of supercritical CO2 in poly(d,l-lactide-co-glycolide), poly(l-lactide) and poly(vinyl acetate) indicated that the comprehensive prediction performance of the CSA-PD-EA model was high. The calculation error and correlation coefficient were, respectively, 0.3842 and 0.9187. The CSA-PD-EA model showed prominent advantages in accuracy, efficiency and correlation over other computational models, and its calculation time was 4.144-15.012% of that of other dynamic models. The CSA-PD-EA model has wide application prospects in the computation of physical and chemical properties and can provide the basis for the theoretical calculation of multi-scale complex systems in chemistry, materials, biology and physics.

The role of Glutathione-S-transferases in phoxim and chlorfenapyr tolerance in a major mulberry pest, Glyphodes pyloalis walker (Lepidoptera: Pyralidae).

Glyphodes pyloalis Walker is a destructive pest on mulberry trees and poses a significant threat to the sericultural industry in China. Phoxim and chlorfenapyr are two commonly used insecticides in mulberry fields. Glutathione-S-transferases (GSTs) comprise a multifunctional protein superfamily that plays important roles in the detoxification of insecticides and xenobiotic compounds in insects. However, whether GSTs participate in the tolerance of phoxim and chlorfenapyr in G. pyloalis is still unknown. To better understand the mechanism of insecticide tolerance in G. pyloalis, the enzymatic activity of GSTs was evaluated under phoxim and chlorfenapyr exposure, respectively. GST enzyme activity was significantly increased after 12, 36 and 48 h of phoxim treatment and 12, 24, 36 and 48 h of chlorfenapyr treatment. Subsequently, eighteen GST genes were identified from the larvae transcriptome of G. pyloalis. Among these, ten GpGSTs had GSH-binding sites and fifteen GpGSTs had variable hydrophobic substrate-binding sites. The expression levels of Delta-GpGST and Epsilon-GpGST genes were significantly influenced by phoxim and chlorfenapyr treatment, and by the time post insecticide application. Furthermore, after silencing GpGST-E4, the mortality rate of G. pyloalis larvae was increased when they were exposed to chlorfenapyr, but it did not significantly alter when the larvae were exposed to phoxim. Our results indicated the vital roles of GpGSTs in the tolerance of insecticides and this action depends on the categories of insecticides. The present study provides a theoretical basis for elucidating insecticide susceptibility and promotes functional research on GST genes in G. pyloalis.

Sweet-flavored peptides with biological activities from mulberry seed protein treated by multifrequency countercurrent ultrasonic technology.

To increase the reuse of food residues, multifrequency countercurrent ultrasonic treatment was used to enhance the extraction yield of defatted mulberry seed protein (DMSP), and sweet-flavored peptides from DMSP hydrolysates (DMSPHs) were obtained for the first time. Here, the DMSP yield was increased by 16.2% (p < 0.05) while the power density was halved compared with single-frequency ultrasonic treatment. According to Fick's second law, a molecular diffusion dynamics model was developed to be suitable for predicting the pretreatment conditions (R2 = 0.9785). After that, the sweet-flavored peptides were purified and the main amino acid sequences were identified, i.e., FEGGSIE, KDFPEAHSQAT, and GSQPAEGAK. Moreover, the antioxidant activities of DMSPHs prepared with tri-frequency treatment was higher than 60%. The DMSPHs retarded the growth of HepG2 cells in vitro, increased the necrotic quadrant (Q1-UL), and extended the S phase. Therefore, the sweet-flavored peptides prepared from DMSPHs using the multifrequency-ultrasonic treatment have significant biological activities.

Sublethal effects of organophosphorus insecticide phoxim on patch time allocation and oviposition behavior in a parasitoid wasp Meteorus pulchricornis.

Parasitoid wasps are key agents for controlling insect pests in integrated pest management programs. Although many studies have revealed that the behavior of parasitic wasps can be influenced by insecticides, the strategies of patch time allocation and oviposition have received less attention. In the present study, we forced the endoparasitoid Meteorus pulchricornis to phoxim exposure at the LC30 and tested the foraging behavior within patches with different densities of the host, the larvae of the tobacco cutworm Spodoptera litura. The results showed that phoxim treatment can significantly increase the patch-leaving tendency of female wasps, while host density had no impact. The number of oviposition and the number of previous patch visits also significantly influenced the patch time allocation decisions. The occurrence of oviposition behavior was negatively affected by phoxim exposure; however, progeny production was similar among patches with different host densities. Phoxim exposure shaped the offspring fitness correlates, including longer durations from cocoon to adult wasps, smaller body size, and shorter longevity. The findings of the present study highlight the sublethal effects that reduce the patch residence time and the fitness of parasitoid offspring, suggesting that the application of phoxim in association with M. pulchricornis should be carefully schemed in agroecosystems.

Correction: Nutritional targeting modification of silkworm pupae oil catalyzed by a smart hydrogel immobilized lipase.

Correction for 'Nutritional targeting modification of silkworm pupae oil catalyzed by a smart hydrogel immobilized lipase' by Jin-Zheng Wang et al., Food Funct., 2021, 12, 6240-6253, DOI: 10.1039/D1FO00913C.

Detraining Effect on Pulmonary and Cardiovascular Autonomic Function and Functional Outcomes in Patients With Parkinson's Disease After Respiratory Muscle Training: An 18-Month Follow-Up Study.

Background: The effect of 3-month respiratory muscle training (RMT) on pulmonary and autonomic function and functional outcomes has been demonstrated in patients with Parkinson's disease (PD); however, there is a paucity of information on the durability of the training effect. In this study, we monitored the pulmonary and cardiovascular autonomic function and clinical severity scales until 18 months after the cessation of RMT to elucidate the detraining effect after RMT. Methods: All patients with PD receiving RMT were assessed with clinical severity scales as well as pulmonary and autonomic function tests at four different stages (baseline on enrollment, immediately after 3 months of RMT, and 6 and 18 months after cessation of RMT). A control group of PD patients who did not receive RMT was also recruited for comparison. Pulmonary function parameters, including forced vital capacity (FVC), forced expiratory volume in one second (FEV1), maximum inspiratory pressure (MIP), and maximum expiratory pressure (MEP), were assessed. Cardiovascular autonomic function was assessed using measures including heart rate response to deep breathing (HRDB), Valsalva ratio, and baroreflex sensitivity. Clinical severity scores were also measured using the Hoehn and Yahr staging and the Unified Parkinson's Disease Rating Scale (UPDRS). Results: The results showed significant improvements in MIP, MEP, HRDB, and UPDRS immediately after RMT. Despite some decay, the improvements in pulmonary function (MIP and MEP) and functional outcomes (UPDRS) remained significant until 6 months of detraining (9 months after enrollment). However, the improvement in cardiovascular autonomic function (HRDB) was reversed after 6 months of detraining. Conclusions: Based on these findings, we recommend that RMT may be repeated after at least 6 months after previous session (9 months after enrollment) for patients with PD to maintain optimal therapeutic effects.

Characterization, and Functional Analysis of Hsp70 and Hsp90 Gene Families in Glyphodes pyloalis Walker (Lepidoptera: Pyralidae).

Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) is a destructive mulberry pest, causing great damage to mulberry in China. Heat shock proteins (Hsps) are involved in various signal pathways and regulate lots of physiological processes in insects. The function of Hsps in G. pyloalis, however, has still received less attention. Here, we identified five Hsp genes from G. pyloalis transcriptome dataset including two Hsp70 family genes (GpHsp71.3 and GpHsp74.9) and three Hsp90 family genes (GpHsp82.4, GpHsp89, and GpHsp93.4). Quantitative Real-time PCR validation revealed that all Hsps of G. pyloalis have significant expression in pupal and diapause stage, at which the larvae arrest the development. Expressions of GpHsp71.3 and GpHsp82.4 were increased significantly after thermal treatment at 40°C, and this upregulation depended on heat treatment duration. Furthermore, silencing GpHsp82.4 by RNA interference led to a significant increase in mortality of G. pyloalis larvae under the heat stress compared to the control group. After starvation stress, the expression levels of GpHsp82.4 and GpHsp93.4 were significantly increased. At last, after being parasitized by the parasitoid wasp Aulacocentrum confusum, Hsp70 and Hsp90 genes of G. pyloalis were decreased significantly in the early stage of parasitization and this moderation was affected by time post-parasitization. This study highlights the function of G. pyloalis Hsps in response to environmental stress and provides a perspective for the control of this pest.

UDP-glycosyltransferases contribute to the tolerance of parasitoid wasps towards insecticides.

Meteorus pulchricornis (Wesmael) (Hymenoptera: Braconidae) is a predominant endoparasitoid of lepidopteran pests in mulberry fields. Extensive application of insecticides puts natural enemies under threat. UDP-glycosyltransferases (UGTs), as important detoxification enzymes, potentially contribute to the detoxification of pesticides in insects. To investigate the roles of UGTs in the process of tolerance towards commonly used insecticides in M. pulchricornis, ten UGT genes were identified from the transcriptome database of M. pulchricornis. Seven UGT genes contained full-length ORFs and shared 47.12-78.28% identity with other homologous hymenopteran insects. qRT-PCR validation revealed that UGT genes can be induced by treatment of sublethal doses of phoxim, cypermethrin and chlorfenapyr, respectively, and these upregulations were depending on the time post insecticide treatments. To further explore the functions of UGT genes, three MpulUGT genes were singly knocked down, which resulted in the decline of UGT expression and significantly increased mortality of parasitoids under sublethal doses of insecticides exposure. This study revealed that UGTs in M. pulchricornis contributed to the tolerance towards insecticides and provided basic insight into the insecticide detoxification mechanism in parasitoid wasps.

A role of peptidoglycan recognition protein in mediating insecticide detoxification in Glyphodes pyloalis.

Glyphodes pyloalis Walker has become one of the most significant mulberry pests, and it has caused serious economic losses in major mulberry growing regions in China. Peptidoglycan recognition proteins (PGRPs) are responsible for initiating and regulating immune signalling pathways in insects. However, their roles responding to chemical pesticides is still less known. This study aimed to investigate the possible detoxication function of GpPGRP-S2 and GpPGRP-S3 in G. pyloalis in response to chlorfenapyr and phoxim. The chlorfenapyr and phoxim treatment significantly induced the expression level of GpPGRP-S3 at 48 h. In addition, the expression levels of GpPGRP-S2 and GpPGRP-S3 in the chlorfenapyr/phoxim treatment group were significantly higher in midgut than those in the control group at 48 h. The results of the survival experiment showed that silencing either GpPGRP-S2 or GpPGRP-S3 would not influence the survival rate of G. pyloalis which treated with phoxim, however, silencing GpPGRP-S2 or GpPGRP-S3 would cause G. pyloalis to be more easily killed by chlorfenapyr. The expression of carboxylesterase GpCXE1 was significantly induced by chlorfenapyr/phoxim treatment, while it was suppressed once silenced GpPGRP-S2 followed with chlorfenapyr treatment or silenced GpPGRP-S3 followed with phoxim treatment. These results might suggest that under the chlorfenapyr/phoxim treatment condition, the connection between GpPGRPs and detoxification genes in insect was induced to maintain physiological homeostasis; and these results may further enrich the mechanisms of insects challenged by insecticides.

Cytochrome P450s Are Essential for Insecticide Tolerance in the Endoparasitoid Wasp Meteorus pulchricornis (Hymenoptera: Braconidae).

With the widespread application of insecticides, parasitoid wasps may also be under risk when exposed to insecticides directly at their free-living stages. The endoparasitoid wasp Meteorus pulchricornis is the predominant natural enemy of many lepidopteran pests, such as Spodoptera litura and Helicoverpa armigera. The cytochrome P450 monooxygenases constitute a ubiquitous and complex superfamily of hydrophobic, haem-containing enzymes. P450s are involved in the detoxification of many xenobiotics. However, their exact roles in the tolerance mechanism in parasitoids toward insecticides has received less attention. Here, 28 P450 genes in M. pulchricornis were identified from a previously constructed transcriptome dataset. These P450 genes belonged to CYP2, -3, and -4, and mitochondrial clans. Subsequently, eight candidate MpulCYPs were selected from four CYP clans to validate their expression patterns under phoxim, cypermethrin, and chlorfenapyr exposure by qRT-PCR. The results showed that all three insecticides had significant effects on the expression of MpulCYPs. To further study the function of P450s, CYP369B3 was silenced, and its expression levels of CYP369B3 were significantly decreased. Survival analysis indicated that after dsRNA injection, the mortality rate of wasps was significantly increased when M. pulchricornis females were exposed to insecticides compared to control groups. Our findings provide a theoretical base for elucidating the mechanism of insecticide tolerance and promote functional research on P450 genes in parasitoid wasps.

Identification of chemosensory genes by antennal transcriptome analysis and expression profiles of odorant-binding proteins in parasitoid wasp Aulacocentrum confusum.

The endoparasitoid wasp, Aulacocentrum confusum (Hymenoptera: Braconidae), is a preponderant natural enemy of the larvae of Glyphodes pyloalis Walker (Lepidoptera: Pyralidae), which is a destructive pest of mulberry trees. We first constructed the antennal transcriptome database of A. confusum. In total, we obtained 48,262,304 clean reads from the dataset and assembled 24,324 unigenes. A total of 12,690 (52.17%) unigenes indicated significant similarity (E-value < 10-5) compared to known protein sequences of other species from the NCBI non-redundant protein database. Gene ontology (GO) and cluster of orthologous groups (COG) analyses were used to determine the functional categories of these genes. A total of 84 putative chemosensory genes were identified from the antennal transcriptome of A. confusum, including 11 putative odorant-binding protein (OBP) genes, six chemosensory protein (CSP) genes, 44 olfactory receptor (OR) genes (including one olfactory co-receptor, Orco), 19 ionotropic receptor (IR) genes, and four sensory neuron membrane protein (SNMP) genes. Results of qPCR assays indicated that among of 11 AconOBPs, nine AconOBP genes were significantly expressed in the antennae of A. confusum adults. AconOBP8 was significantly expressed in the abdomen and AconOBP10 was highly expressed in the thorax. These findings can build a basis for further study on the processes of chemosensory perception in A. confusum at the molecular level.