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Carotenoids are involved in many essential physiological functions and are produced from geranylgeranyl pyrophosphate through synthase, desaturase, and cyclase activities. In the pea aphid (Acyrthosiphon pisum), the duplication of carotenoid biosynthetic genes, including carotenoid synthases/cyclases (ApCscA-C) and desaturases (ApCdeA-D), through horizontal gene transfer from fungi has been detected, and ApCdeB has known dehydrogenation functions. However, whether other genes contribute to aphid carotenoid biosynthesis, and its specific regulatory pathway, remains unclear. In the current study, functional analyses of seven genes were performed using heterologous complementation and RNA interference assays. The bifunctional enzymes ApCscA-C were responsible for the synthase of phytoene, and ApCscC may also have a cyclase activity. ApCdeA, ApCdeC, and ApCdeD had diverse dehydrogenation functions. ApCdeA catalyzed the enzymatic conversion of phytoene to neurosporene (three-step product), ApCdeC catalyzed the enzymatic conversion of phytoene to ζ-carotene (two-step product), and ApCdeD catalyzed the enzymatic conversion of phytoene to lycopene (four-step product). Silencing of ApCscs reduced the expression levels of ApCdes, and silencing these carotenoid biosynthetic genes reduced the α-, β-, and γ-carotene levels, as well as the total carotenoid level. The results suggest that these genes were activated and led to carotenoid biosynthesis in the pea aphid.

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

At the post-transcriptional level, microRNAs (miRNAs) play an important role in the regulation of gene expression, thereby influencing the outcome of many biological processes in insects, such as development, reproduction, metamorphosis, immunity, and insecticide resistance. The alteration of miRNA expression by mimic/agomir or inhibitor/antagomir via injection/feeding can lead to pest developmental abnormalities, death, or reduced pesticide resistance, indicating that miRNAs are potential targets for pest control. This review provides an overview of recent advances in understanding the regulatory roles of miRNA in agricultural and public health insect pest, and further highlights the potential of miRNAs as prospective targets in pest control.

The oriental fruit fly, Bactrocera dorsalis Hendel (Diptera: Tephretidae), is a serious pest of fruits and vegetables and has developed high levels of insecticide resistance. ATP-binding cassette transporter genes (ABC transporters) are involved in mediating the energy-driven transport of many substances across membranes and are closely associated with development and insecticide detoxification. In this study, three ABC transporters in the H subfamily were identified, and the possible roles of these genes in B. dorsalis are discussed. Bioinformatics analysis revealed that those genes are conserved, typical of half-transporters. The expression profiles of BdABCH genes (BdABCHs) in the developmental stages, tissues, and following insecticide exposure, extreme temperature, warm- and cold-acclimated strain, starvation, and desiccation stress were determined by quantitative real-time PCR. Expression of BdABCHs can be detected in various tissues and in different developmental stages. They were most highly expressed in the hindgut and in newly emerged adults. The mRNA levels of BdABCHs in males (including most tissues and body segments) were higher than in females. The expression of BdABCH1 was significantly upregulated 3.8-fold in the cold-acclimated strain, and was significantly upregulated by 1.9-, 3.8- and 4.1-fold in the 0°C, starvation, and desiccation treatments, respectively. Treatment with malathion and avermectin at LD20 and LD30 concentrations produced no obvious changes in the levels of BdABCHs. BdABCHs may be involved in the transport of related hormones during eclosion, as well as water and inorganic salts. BdABCH1 also demonstrated that it is related to the ability to cope with adverse environments.

BACKGROUND: Odorant‐binding proteins (OBPs) in insects contribute to the sensitivity of the olfactory system and connect external odorants to olfactory receptor neurons. Determination of the chemosensory functions in Diaphorina citri, a vector of the citrus Huanglongbing pathogen, may help in developing a potential target for pest management. RESULTS: Diaphorina citri showed dose‐dependent electroantennogram recording (EAG) responses to 12 host plant volatiles. A two‐choice behavioral trap experiment showed that four compounds (methyl salicylate, linalool, citral and R‐(+)‐limonene) that elicited high EAG responses also had significant attraction to adults. The expression profiles induced by these compounds were detected in nine OBP genes, DcitOBP1–9. DcitOBP3, DcitOBP6 and DcitOBP7 commonly showed significant upregulation or downregulation compared with the control. Microscale thermophoresis (MST) showed that the recombinant protein DcitOBP7 had high in vitro binding affinities (Kd

Background Olfactory systems take on important tasks to distinguish salient information from a complex olfactory environment, such as locating hosts, mating, aggression, selecting oviposition sites, and avoiding predators. The olfactory system of an adult insect consists of two pairs of main olfactory appendages on the head, the antennae, and the palps, which are covered with sensilla. Benzothiazole and 1-octen-3-ol could elicit oviposition behavior in gravid B. dorsalis are regarded as oviposition stimulants. However, the mechanism for how B. dorsalis percepts benzothiazole and 1-octen-3-ol still remains unknown. Results We conducted a comparative analysis of the antennal transcriptomes in different genders of B. dorsalis using Illumina RNA sequencing (RNA-seq). We identified a total of 1571 differentially expressed genes (DEGs) among the two sexes, including 450 female-biased genes and 1121 male-biased genes. Among these DEGs, we screened out 24 olfaction-related genes and validated them by qRT-PCR. The expression patterns of these genes in different body parts were further determined. In addition, we detected the expression profiles of the screened female-biased chemosensory genes in virgin and mated female flies. Furthermore, the oviposition stimulants-induced expression profilings were used to identify chemosensory genes potentially responsible for benzothiazole and 1-octen-3-ol perception in this fly. Conclusions The results from this study provided fundamental data of chemosensory DEGs in the B. dorsalis antenna. The odorant exposure assays we employed lay a solid foundation for the further research regarding the molecular mechanism of benzothiazole and 1-octen-3-ol mediated oviposition behavior in B. dorsalis.

The innate immune system of insects is the front line of self-defense against pathogen invasion. Peptidoglycan recognition proteins (PGRPs) are important components and play key roles in insect immune systems by recognizing peptidoglycan (PGN) in bacterial cell walls. We characterized two isoforms of the PGRP-LC gene, BdPGRP-LCa and BdPGRP-LCb, from Bactrocera dorsalis (Hendel), an important fruit and vegetable pest worldwide. These two isoforms contain an open reading frames of 1 668 bp and 1 731 bp, encoding a protein of 555 and 576 amino acids, respectively. Quantitative real-time PCR results showed that both transcripts were prominently expressed in midgut and fat body of B. dorsalis adult. Inoculation of pathogens showed that both isoforms actively responded to Escherichia coli PGN. We also observed a light response to Staphylococcus aureus PGN. Upon Beauveria bassiana inoculation, the expression of BdPGRP-LCa was enhanced, but the expression of BdPGRP-LCb was suppressed. Suppression of both transcripts by RNA interference led to increased mortality of flies challenged by E. coli, indicating that the two isoforms are involved in sensing Gram-negative bacterial infections.

Cuticular proteins (CPs) are critical components of the insect cuticle and play important roles in maintaining normal insect development and defense against various environmental stresses. The oriental fruit fly (Bactrocera dorsalis) is one of the most destructive pests worldwide, and its eight CPs analogous to peritrophin 3 (BdCPAP3) family genes have been identified in our previous study. In the present study, we further explored the possible roles of CPAP3 genes in B. dorsalis development. Each sequence of BdCPAP3 genes contained three conserved ChtBD2 (chitin-binding) domains. Spatial and temporal expression patterns revealed that the four BdCPAP3 genes (BdCPAP3-A1, B, E, and E2) might play important roles in larval pupariation of B. dorsalis. Moreover, treatment with a juvenile hormone analog (methoprene) significantly restricted expression of these four CPAP3 genes, whereas treatment with 20-hydroxy-ecdysone induced expression. The RNA interference (RNAi) results revealed that down-regulated CPAP3 genes led to significant delay of pupariation, and injection of dsBdCPAP3-E into 5-d-old B. dorsalis larvae caused approximately 40% mortality. Interestingly, we also confirmed that BdCPAP3-D2 was involved in B. dorsalis ovarian development. This study showed that some specific CPAP3 genes had crucial roles in B. dorsalis development, and these CP genes could be used as potential targets to control this pest via RNAi.

Background Bactrocera dorsalis (Hendel) is a notorious agricultural pest worldwide, and its resistance to insecticides is a major obstacle in successful control. Cytochrome P450s (P450s) are major metabolic enzymes associated with insecticide resistance. The genome of B. dorsalis was sequenced recently, allowing an integrated genome-wide analysis of P450 genes (P450s) and the analysis of correlations between these genes and insecticide resistance in this pest. Results Totally, 101 P450s were identified in the B. dorsalis genome and classified into four clans, 25 families and 57 subfamilies. Quantitative reverse transcription polymerase chain reaction results showed that most of these genes were highly expressed in adults (46) and in metabolic tissues, including the fatbody (63), midgut (61) and Malphagian tubules (66). In a malathion-resistant strain, 13 and 9 genes were significantly upregulated and downregulated, respectively, compared with a susceptible strain, and these genes were screened as candidate genes associated with malathion resistance. Conclusion This study provides useful information for understanding the evolution and potential functions of P450s in B. dorsalis, and the results lay the foundation for further studies on the correlations between P450s and malathion resistance in B. dorsalis. © 2020 Society of Chemical Industry.

The neurohormone octopamine regulates many crucial physiological processes in insects and exerts its activity via typical G-protein coupled receptors. The roles of octopamine receptors in regulating behavior and physiology in Coleoptera (beetles) need better understanding. We used the red flour beetle, Tribolium castaneum, as a model species to study the contribution of the octopamine receptor to behavior and physiology. We cloned the cDNA of a β-adrenergic-like octopamine receptor (TcOctβ2R). This was heterologously expressed in human embryonic kidney (HEK) 293 cells and was demonstrated to be functional using an in vitro cyclic AMP assay. In an RNAi assay, injection of dsRNA demonstrated that TcOctβ2R modulates beetle locomotion, mating duration, and fertility. These data present some roles of the octopaminergic signaling system in T. castaneum. Our findings will also help to elucidate the potential functions of individual octopamine receptors in other insects.

In insects, the ecdysteroid 20-hydroxyecdysone coordinates with juvenile hormone (JH) to regulate the process of molting, development and metamorphosis; however, this interaction is still unclear in the mites. In this study, we investigated the gene related to ecdysteroid and JH biosynthesis pathways, including four ecdysteroid and 11 JH biosynthesis genes. We examined their expression patterns during molting of different developmental stages of the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), an important agricultural pest that feeds on more than 1100 plant species. The expression of ecdysteroid biosynthesis Halloween genes exhibited a positive zigzag-like pattern, with a peak after 8 h of molting and a drop 8 h after entering each quiescent stage. In contrast, JH biosynthesis genes expression displayed a negative zigzag-like pattern, with a peak at 8 h after entering each quiescent stage and a drop after 8 h of each molting. These opposite patterns imply that ecdysteroid and JH expression is coordinated during the developmental transition. Our data provide an initial perspective on the co-expression of ecdysteroid and JH biosynthesis genes to regulate this important developmental process in the two-spotted spider mite.

Olfaction underpins many insect behaviors, such as foraging, host location, mating, and predator avoidance . In the first step of insect olfaction, odorant-binding proteins (OBPs) bind hydrophobic odorants and transport them to odorant receptors . Methyl eugenol (ME) is a powerful attractant for mature males of the oriental fruit fly Bactrocera dorsalis (Hendel), one of the most destructive fruit pests. The underlying molecular mechanism is unclear, but there is in vitro evidence that BdorOBP56f-2 is involved in ME perception. We used microscale thermophoresis to confirm that BdorOBP56f-2 directly binds ME with strong affinity in vitro. We then used CRISPR/Cas9 to knock out the BdorOBP56f-2 gene, allowing us to establish a homozygous mutant B. dorsalis line. The electroantennogram response and behavioral attraction to ME were significantly reduced in the mutant, providing in vivo evidence that BdorOBP56f-2 is necessary for efficient ME perception. Our results offer insight into the molecular mechanism of ME perception in B. dorsalis and provide a theoretical basis for the functional analysis of other OBPs.

Spider mites have one ecdysone receptor (EcR) and multiple retinoid X receptors (RXRs). However, the function of these RXRs in spider mite development is unknown. Here, we screened the expression dynamics of two PcRXR isoforms at 4 h intervals in the deutonymphal stage of Panonychus citri. The results showed that PcEcR had an expression pattern similar to that of PcRXR2. For PcRXR1, its expression remained at a certain high level, when there was a decrease of both PcEcR and PcRXR2. In situ hybridization showed that PcRXR2 was detected in the central nervous mass, while the ecdysteroid biosynthesis gene PcSpo was mainly expressed at the edge of the central nervous mass. RNAi-based silencing of PcRXR1 or PcRXR2 showed the same phenotype as in mites with that of silencing PcEcR. Furthermore, RNA-seq was used to mine the genes associated with the expression dynamics of PcRXR1 or PcRXR2, which revealed that the heterodimer of EcR-RXR2 in spider mites might be linked with the cell autophagy and tissue remodeling during apolysis, and RXR1 might be linked with new epicuticle and exocuticle secretion during ecdysis. Taken together, these results increase our understanding of the regulation mechanism of ecdysteroid signal pathway in spider mite development.

Simple Summary The melon fly, Zeugodacus cucurbitae, is an important agricultural pest. At present, chemical pesticide treatment is the main method for field control, but this promotes pesticide resistance by Z. cucurbitae, because of its frequent use. ABC transporters are involved in detoxification metabolism, but few studies have yet considered their expression in melon fly. In this study, we identified the ABC transporters genes at a genome-wide level in melon fly, and analysed their spatiotemporal expression patterns, as well as changes in expression after insecticides treatments. A total of 49 ABC transporters were identified, and their expression levels varied at different developmental stages and between tissues. After three insecticides treatment, ZcABCB7 and ZcABCC2 were up-regulated. After β-cypermethrin induction, tissues were dissected at 12, 24 and 48 h, and the expression levels of a number of ABC genes were highly expressed within the fat body. From these results, we conclude that ZcABCB7 and ZcABCC2 may be involved in detoxification metabolism, and that the fat body is the main tissue that plays this role. Abstract The ATP-binding cassette (ABC) transporter is a protein superfamily that transports specific substrate molecules across lipid membranes in all living species. In insects, ABC transporter is one of the major transmembrane protein families involved in the development of xenobiotic resistance. Here, we report 49 ABC transporter genes divided into eight subfamilies (ABCA-ABCH), including seven ABCAs, seven ABCBs, 10 ABCCs, two ABCDs, one ABCE, three ABCFs, 16 ABCGs, and three ABCHs according to phylogenetic analysis in Zeugodacus cucurbitae, a highly destructive insect pest of cucurbitaceous and other related crops. The expressions level of 49 ABC transporters throughout various developmental stages and within different tissues were evaluated by quantitative transcriptomic analysis, and their expressions in response to three different insecticides were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). These ABC transporter genes were widely expressed at developmental stages but most highly expressed in tissues of the midgut, fat body and Malpighian tube. When challenged by exposure to three insecticides, abamectin, β-cypermethrin, and dinotefuran, the expressions of ZcABCB7 and ZcABCC2 were significantly up-regulated. ZcABCB1, ZcABCB6, ZcABCB7, ZcABCC2, ZcABCC3, ZcABCC4, ZcABCC5, and ZcABCC7 were significantly up-regulated in the fat body at 24 h after β-cypermethrin exposure. These data suggest that ZcABCB7 and ZcABCC2 might play key roles in xenobiotic metabolism in Z. cucurbitae. Collectively, these data provide a foundation for further analysis of ABCs in Z. cucurbitae.

BACKGROUND Long non-coding RNAs (lncRNAs) play important roles in the regulation of biological processes and have been identified in many species including insects. However, the association between lncRNAs and pesticide resistance in insect species such as Bactrocera dorsalis is unknown. RESULTS RNA-seq was performed on malathion resistant (MR1) and susceptible (MS) strains of B. dorsalis and a total of 6171 lncRNAs transcripts were identified. These included 3728 lincRNAs, 653 antisense lncRNAs, 1402 intronic lncRNAs, and 388 sense lncRNAs. A total of 40 and 52 upregulated lncRNAs were found in females and males of the MR1 strain compared to 54 and 49 in the same sexes of the MS strain, respectively. Twenty-seven of these lncRNAs showed the same trend of expression in both females and males in the MR1 strain, in which 15 lncRNAs were upregulated and 12 were downregulated. RT-qPCR results indicated that the differentially expressed lncRNAs were associated with malathion resistance. The lnc15010.10 and lnc3774.2 were highly expressed in the cuticle of the MR1 strain, indicating that these two lncRNAs may be related to malathion resistance. RNAi of lnc3774.2 and a bioassay showed that malathion resistance was possibly influenced by changes in the B. dorsalis cuticle. CONCLUSION LncRNAs of B. dorsalis potentially related to the malathion resistance were identified. Two lncRNAs appear to influence malathion resistance via modulating the structure, or components, of the cuticle. © 2021 Society of Chemical Industry.

Psocids are a new risk for global food security and safety because they are significant worldwide pests of stored products. Among these psocids, Liposcelis bostrychophila has developed high levels of resistance or tolerance to heat treatment in grain storage systems, and thus has led to investigation of molecular mechanisms underlying heat tolerance in this pest. In this study, the time-related effects of thermal stress treatments at relatively high temperatures on the activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidases (POD), glutathione-S-transferases (GST) and malondialdehyde (MDA), of L. bostrychophila were determined. Thermal stress resulted that L. bostrychophila had a significantly higher MDA concentration at 42.5 &deg, C, which indicated that the heat stress increased lipid peroxidation (LPO) contents and oxidative stress in this psocid pest. Heat stress also resulted in significant elevation of SOD, CAT and GST activities but decreased POD activity. Our data indicates that different antioxidant enzymes contribute to defense mechanisms, counteracting oxidative damage in varying levels. POD play minor roles in scavenging deleterious LPO, while enhanced SOD, CAT and GST activities in response to thermal stress likely play a more important role against oxidative damage. Here, we firstly identified five LbHsps (four LbHsp70s and one LbHsp110) from psocids, and most of these LbHsps (except LbHsp70-1) are highly expressed at fourth instar nymph and adults, and LbHsp70-1 likely presents as a cognate form of HSP due to its non-significant changes of expression. Most LbHsp70s (except LbHsp70-4) are significantly induced at moderate high temperatures (&lt, 40 &deg, C) and decreased at extreme high temperatures (40&ndash, 45 &deg, C), but LbHsp110-1 can be significantly induced at all high temperatures. Results of this study suggest that the LbHsp70s and LbHsp110 genes are involved in tolerance to thermal stress in L. bostrychophila, and antioxidant enzymes and heat shock proteins may be coordinately involved in the tolerance to thermal stress in psocids.

The neuropeptide adipokinetic hormone (AKH) binds to the AKH receptor (AKHR) to regulate carbohydrate and lipid metabolism. It also participates in the insect anti-stress response. We used RT-qPCR to detect the expression levels of 39 neuropeptides in malathion-susceptible (MS) and malathion-resistant (MR) strains of Bactrocera dorsalis. AKH and AKHR were highly expressed in the MR strain. Using a malathion bioassay and RNA interference (RNAi), we demonstrated that AKHR is involved in the susceptibility of B. dorsalis to malathion. We found significantly reduced expression of two detoxification enzyme genes (glutathione-S-transferase, GST and α-esterase, CarE) after AKHR RNAi. Based on our previous data, GSTd10 and CarE6 participate the direct metabolism of malathion in this fly, which is also verified by a malathion metabolism assay by HPLC using the crude enzymes in the current study. These results suggest that AKHR plays an important role in affecting malathion susceptibility via detoxification enzyme genes.

Eotetranychus kankitus is an important mite pest in citrus, but molecular data on the developmental processes of E. kankitus are lacking. The different development stages mix of E. kankitus was used to sequence for transcriptome and small RNAs to identify genes and predict miRNAs associated with sesquiterpenoid and ecdysteroid biosynthesis and signaling pathways. More than 36 million clean reads were assembled and 67,927 unigenes were generated. Of the unigenes, 19,300 were successfully annotated through annotation databases NR, SwissProt, COG, GO, KEGG, PFAM, and KOG. The transcripts were involved in sesquiterpenoid biosynthesis (11 genes) and ecdysteroid biosynthesis and signaling pathway (13 genes). Another, small RNA library was obtained and 31 conserved miRNAs were identified. Five most abundant miRNAs were Ek-miR-5735, Ek-miR-1, Ek-miR-263a, Ek-miR-184, and Ek-miR-8. The target genes related to sesquiterpenoid and ecdysteroid showed that 10 of the conserved miRNAs could potentially target the sesquiterpenoid and ecdysteroid pathway according to four-prediction software, sRNAT, miRanda, RNAhybrid, and Risearch2. Thus, the results of this study will provide bioinformatics information for further molecular studies of E. kankitus which may facilitate improved pest control strategies.

Background The oriental fruit fly, Bactrocera dorsalis (Hendel), is a widespread agricultural pest that has evolved resistance to many commonly used insecticides including malathion. Glutathione S-transferases (GSTs) are multifunctional enzymes that metabolize insecticides directly or indirectly. The specific mechanism used by GSTs to confer malathion resistance in B. dorsalis is unclear. Results BdGSTd9 was identified from B. dorsalis and was expressed at twice the level in a malathion-resistant strain (MR) than in a susceptible strain (MS). By using RNAi of BdGSTd9, the toxicity of malathion against MR was increased. Protein modelling and docking of BdGSTd9 with malathion and malaoxon indicated key amino acid residues for direct binding in the active site. In vitro assays with engineered Sf9 cells overexpressing BdGSTd9 demonstrated lower cytotoxicity of malathion. High performance liquid chromatography (HPLC) analysis indicated that malathion could be broken down significantly by BdGSTd9, and it also could deplete the malathion metabolite malaoxon, which possesses a higher toxicity to B. dorsalis. Taken together, the BdGSTd9 of B. dorsalis could not only deplete malathion, but also react with malaoxon and therefore enhance malathion resistance. Conclusion BdGSTd9 is a component of malathion resistance in B. dorsalis. It acts by depleting both malathion and malaoxon. © 2020 Society of Chemical Industry.

Esterases (ESTs) play important roles in metabolizing various physiologically endogenous and exogenous compounds, and various environmental xenobiotics in insects. The psocid, Liposcelis bostrychophila is a major pest of stored products worldwide and rapidly develops resistance to commonly insecticides. However, the involvement of ESTs in insecticide metabolization and the application of RNAi approach in psocids have not been well elucidated. In this study, we characterized four LbEST genes and investigated the transcriptional levels of these genes at different developmental stages and under different insecticides exposures to assess their potential roles in response to insecticides. The four LbESTs contain a catalytic triad (Ser-His-Glu) linked to an oxyanion hole and acyl pocket involved in substrate stabilization during its hydrolysis. Synergism observed with the esterase-inhibitor DEF suggests the involvement of esterases in malathion detoxification. LbESTs were expressed during the whole of developmental stages, but predominant abundance in the first nymphal instar and adult stage. The mRNA level of three LbEST genes (except for LbEST4) was induced (1.29- to 5.60 fold) in response to malathion or deltamethrin exposures, indicating that these esterases are involved in the detoxification process. Silencing of LbEST1, LbEST2 or LbEST3 through dsRNA feeding led to a higher mortality of psocids upon the malathion treatment compared to controls (1.83 to 2.69-fold), demonstrating that these esterase genes play roles in malathion detoxification in L. bostrychophila. Our study provides new evidence for understanding of the function and regulation mechanism of esterases in L. bostrychophila in insecticide detoxification. The current study also suggests that the present RNAi method could be applied for gene functional studies in psocids.

Regioselective reactions of methyl pyropheophorbide a (MPPa) with formaldehyde based on hydroxymethylation have been studied. It was found that MPPa can react regioselectively with formaldehyde under different conditions to produce a series of 3-dioxane, 12-, 132- or 20-hydroxymethyl and 12-/132-alkenyl-substituted chlorins via Prins reaction, Blanc chloromethylation and aldol reaction, respectively. The first examples of direct C–C bond formation at 12-position of chlorophyll derivatives were also reported to give a series of 12-vinyl-substituted chlorins. These chlorins showed extend Qy absorptions and efficient singlet oxygen generation property, indicating their potential as photosensitizers for application in photodynamic therapy.

The present study investigated the protective effect of phytoecdysteroids extracted from the Ajuga iva plant on body weight changes, blood glucose, insulin total protein, blood urea nitrogen (BUN), creatinine, triglycerides (TG), cholesterol, lipid peroxidation, antioxidant enzymes, pancreatic histopathology and hexokinase-I expression in the alloxan-induced diabetic rats. Experimental diabetes was induced following 15day intraperitoneal administration of alloxan. The rats were divided into four groups. Group I served as a sham group, and group II served as the diabetic control. Group III served as a treatment for phytoecdysteroids (10mg/kg), and group IV served as a treatment for phytoecdysteroids (20mg/kg). Phytoecdysteroids restored body weight loss to its antihyperglycemic effect. Blood glucose was reduced 19.2 and 52.9% in group III and IV respectively. Blood insulin (54.9 and 105.88%) and total protein (25 and 72.2%) was increased in group III and IV respectively. BUN, creatinine, TG, cholesterol and lipid peroxidation was significantly reduced following treatment. Catalase, superoxide dismutase (SOD), and glutathione peroxidase activity were significantly increased following treatment. Islet β-cells are lost in alloxan-induced diabetic rats. Regeneration of islets and reduced atrophy of acinar cells were noted. The number of insulin-secreting cells was tremendously reduced in alloxan-induced diabetic rats. Insulin-secreting cells were increased 48 and 61% in group III and IV respectively. Hexokinase-I mRNA (28.3 & 93.5%) and protein (27.9 and 55.3%) expression were significantly increased following treatment. Taking all these data together, it is suggested that the phytoecdysteroid could be a potential therapeutic agent against experimental diabetes.

Molting is essential for arthropods to grow. As one of the important arthropod pests in agriculture, key spider mite species (Tetranychus and Panonychus) can normally molt three times from the larva to adult stage within a week. This physiological strategy results in the short lifecycle of spider mites and difficulties in their control in the field. Long non-coding RNAs (lncRNAs) regulate transcriptional editing, cellular function, and biological processes. Thus, analysis of the lncRNAs in the spider mite molting process may provide new insights into their roles in the molting mechanism. For this purpose, we used high-throughput RNA-seq to examine the expression dynamics of lncRNAs and mRNAs in the molting process of different development stages in Panonychus citri. We identified 9199 lncRNAs from 18 transcriptomes. Analysis of the lncRNAs suggested that they were shorter and had fewer exons and transcripts than mRNAs. Among these, 356 lncRNAs were differentially expressed during three molting processes: late larva to early protonymph, late protonymph to early deutonymph, and late deutonymph to early adult. A time series profile analysis of differentially expressed lncRNAs showed that 77 lncRNAs were clustered into two dynamic expression profiles (Pattern a and Pattern c), implying that lncRNAs were involved in the molting process of spider mites. Furthermore, the lncRNA–mRNA co-expression networks showed that several differentially expressed hub lncRNAs were predicted to be functionally associated with typical molting-related proteins, such as cuticle protein and chitin biosynthesis. These data reveal the potential regulatory function of lncRNAs in the molting process and provide datasets for further analysis of lncRNAs and mRNAs in spider mites.

Tandem reactions of methyl pyropheophorbide a with paraformaldehyde, reactive methylene compounds, and/or ammonium acetate provided novel pyrido[2,3-n]chlorophylls in good yields as the first examples of pyridine fused chlorophyll a derivatives, which displayed near-infrared absorptions (748–766 nm) with unusually broadened Q bands and small electrochemical HOMO-LUMO gaps (1.89–2.02 eV).

Acetylcholinesterase (AChE) is the primary target of organophosphate- and carbamate-based insecticides. We sequenced the full-length cDNAs of two AChE genes from the brown citrus aphid Aphis (Toxoptera) citricidus (Kirkaldy). These two genes, Tcace1 and Tcace2, which encode TcAChE1 and TcAChE2, respectively, had a shared amino acid identity of 29% and were highly similar to other insect ace1 and ace2 genes, respectively, having specific functional motifs. Potential differences in enzymatic function were characterized by the heterologous expression of the two genes using a baculovirus system in Sf9 insect cells. Both of the recombinant AChEs had high specific activities for three typical substrates, acetylthiocholine iodide, butyrylthiocholine iodide, and propinylthiocholine iodide. TcAChE1 had a lower Michaelis-Menten constant value and a higher maximal reaction velocity than recombinant TcAChE2, indicating a higher affinity for substrates and greater catalytic efficiency, respectively. Bioassays showed a greater sensitivity of recombinant TcAChE1 to the 10 tested insecticides. Silencing of Tcace1 and Tcace2 by RNA interference significantly increased the susceptibility of A. citricidus to malathion and carbaryl; however, silencing Tcace1 resulted in a higher mortality rate than silencing Tcace2. Additionally, the specific enzyme activity decreased more after silencing Tcace1 than after silencing Tcace2. Thus, TcAChE1 plays a major role in postsynaptic neurotransmission in A. citricidus.

Cecropins and defensins are important antimicrobial peptides in insects and are inducible after injection of immune triggers. In this study, we cloned the cDNAs of two antimicrobial peptides (AMPs), cecropin-2 (BdCec-2) and defensin (BdDef) from Bactrocera dorsalis (Hendel), a serious pest causing great economic losses to fruits and vegetables. The BdCec-2 sequence of 192 bp encodes a protein of 63 amino acids residues with a predicted molecular weight of 6.78 kD. The 282 bp cDNA of BdDef encodes a protein of 93 residues with a predicted molecular weight of 9.81 kD. Quantitative real-time PCR analyses showed that BdCec-2 and BdDef had similar expression profiles among development stages, the highest mRNA levels of these two AMP genes were observed in the adult stage. Among different adult body segments and tissues, both genes had similar transcriptional profiles, the highest mRNA levels appeared in abdomen and fat body, which was consistent with the reported fact that fat body was the main organ synthesizing AMPs in insects. The expression of BdCec-2 and BdDef were up-regulated after challenge with peptidoglycans from Escherichia coli (PGN-EB) and Staphylococcus aureus (PGN-SA), respectively, suggesting their antimicrobial activity against Gram-negative and Gram-positive microorganisms. These results describe for the first time the basic properties of the cecropin-2 and defensin genes from B. dorsalis that probably play an important role in the defense response against invading microbes.

Carotenoids play many crucial roles in organisms. Recently, the de novo synthesis of carotenoids has been reported in pea aphid (Acyrthosiphon pisum) through horizontally transferred genes. However, their upstream pathway in the pea aphid is poorly understood. Geranylgeranyl diphosphate synthase (GGPPS) is the functional enzyme in the synthesis of geranylgeranyl diphosphate (GGPP) which is a precursor for the biosynthesis of many biological metabolites, including carotenoid synthesis. In this study, we performed a series of experiments to characterize GGPPS gene and its association with carotenoid biosynthesis. (1) determining the transcript abundance and carotenoid content in two geographical strain with red and green morphs, and (2) examining the abundance of carotenoid related genes and carotenoid levels after silencing of GGPPS in both red and green morphs. We observed that GGPPS was more highly expressed in the green morph than in the red morph of two strains of the pea aphid. The total level of carotenoids was also higher in green morphs than in red morphs in both strains. In addition to the total carotenoid difference, the carotenoids found in the two morphs also differed. There were α-carotene, β-carotene, and γ-carotene in the green morphs, but three additional carotenoids, including cis-torulene∗, trans-torulene∗, and 3,4-didehydrolycopene∗, were present in the red morphs. Silencing the GGPPS by RNAi in both the red and green morphs decreased the expression of some carotenoid biosynthesis-related genes, including carotenoid synthase/cyclase genes and carotenoid desaturase genes in green morphs. Carotenoid levels were decreased in both green and red morphs. However, the specific carotenoids present were not changed after silencing GGPPS. These results demonstrated that GGPPS may act as the upstream enzyme to influence the synthesis of the total amount of carotenoids. The present study provided important molecular evidence for the conserved roles of GGPPS associated with carotenoids biosynthesis and will enhance further investigation on the mechanisms of carotenoid biosynthesis in pea aphid.

BACKGROUND: Aphids obtained carotenoid biosynthesis genes via horizontal gene transfers from fungi. However, the roles of these genes in the contributions of in aphids'adaptation and whether these genes could be used as RNAi-based pest control targets are not yet clear. Thus, in this study we used parental RNAi to analyze the potential function of a carotenoid desaturase gene (CdeB) by combined molecular and chemical approaches in the pea aphid (Acyrthosiphon pisum). RESULTS: Transcriptional analyses showed that CdeB was significantly more highly expressed in the red morphs compared to the green ones and was associated with the production of red carotenoid. Co-transferring of pET28a-CdeB (the CdeB gene was cloned into pET28a) and pACCRT-EIB (produced lycopene) showed a deep red color in the bacterial precipitate and produced more of a red pigment, lycopene, in vitro. Parental gene-silencing of CdeB resulted in a lower body color intensity in the treated aphids and following generations in vivo. Interestingly, the dsCdeB treatment also reduced aphid performance as reflected by a delay in nymphal developmental duration, lower weight, smaller number, and altered age structure of the population. CONCLUSION: Our results demonstrate that CdeB is involved in red color formation and the silencing of this gene by parental RNAi reduced fitness in the pea aphid. The results enhance our understanding of the biosynthesis of carotenoid in aphids and provide insights into the potential ecological significance of carotenoids in the adaptation of the aphid's biology to the environment and developing environmentally friendly control strategies for this pest.

Pesticide resistance is a serious problem that poses a major challenge to pest control. One of the most potent resistance mechanisms is the overexpression of genes coding for detoxification enzymes. The expression of detoxification genes is regulated by a series of transcription factors. Previous studies have revealed that the increased expression of detoxification genes contributes to the insecticide tolerance of Bactrocera dorsalis. Our objective was thus to identify the transcription factors involved in this process. Temporal expression profiles showed that the transcription factor MafB and detoxification genes were expressed highly in the fat body. Further analysis showed that the expression of MafB, GSTz2, and CYP473A3 was induced by abamectin. Disruption of the MafB transcription factor through RNA interference decreased the transcript levels of GSTz2 and CYP473A3 and increased the susceptibility to abamectin significantly. Direct silencing of the expression of GSTz2 also increased susceptibility to abamectin, while CYP473A3 did not. In conclusion, these results suggest that the expression of GSTz2 and CYP473A3 was regulated by the transcription factor MafB, and the up-regulation of GSTz2 via MafB decreased the susceptibility of B. dorsalis to abamectin.

Bactrocera dorsalis (Hendel) is a notorious insect pest that attacks diverse vegetables and fruits worldwide. The sterile insect technique has been developed as an environmentally friendly and effective control method that depends on the mass production of target flies. Because dietary yeast (protein) and sucrose (carbohydrate) are important in adult diets, yeast:sucrose (Y:S) mixtures are crucial for the mass-rearing of B. dorsalis. In this study, we found adult diets with different ratios of yeast to sucrose-influenced fecundity, and an extremely high or low Y:S ratios significantly decreased egg production of B. dorsalis. Additionally, the maximum oviposition efficiency was realized at dietary yeast to sucrose ratios of 1:1 and 1:3, suggesting their potential use to produce more eggs for the mass production of B. dorsalis. Here, new gel diets having different yeast concentrations (g/L water) were also assessed for rearing B. dorsalis larvae. Gel diets containing 20 g/L yeast led to a higher pupation, pupal weight and adult eclosion rate, and a shorter developmental time than other yeast concentrations. Moreover, the present gel diet also resulted in greater pupal production and adult emergence rates than previously used liquid and solid artificial diets, revealing that it is suitable for rearing B. dorsalis larvae. This research provides a useful reference on artificial diets mixtures for mass rearing B. dorsalis, which is critical for employing the sterile insect technique.

The citrus red mite, Panonychus citri (McGregor), is an important spider mite pest in citrus producing areas. Owing to long-term acaricide exposure, resistance has evolved rapidly in recent years. To evaluate the extent of resistance, seven field mite populations sampled from various geographical locations in China during 2015–2018 were tested using the leaf-dip bioassay method to determine their susceptibilities to four acaricides. In comparison with the susceptible strain maintained in the laboratory, low or moderate levels of fenpropathrin resistance, while no resistance to abamectin or cyflumetofen, were found among populations sampled from Liangping, Wanzhou, Daying, and Anyue in Southwestern China during the test period. High levels (>1,000-fold, with LC50 values that were greater than the recommended concentration) of resistance to fenpropathrin had evolved in field populations from Southern China, including Guilin, Nanning, and Yuxi, when compared with that of the susceptible strain. Populations from Guilin and Nanning also evolved high resistance levels to abamectin (1,088-fold and 1,401-fold) and cyflumetofen (2,112-fold and 9,093-fold). All the populations sampled in 2018 showed a moderate or high resistance to bifenazate. Generally, field populations of citrus red mites from Southwestern China were more sensitive to the tested acaricides than those of Southern China. The data provide a foundation for developing acaricide resistance management strategies in these regions.

Peptidoglycans (PGNs) are major bacterial components recognized by the immune systems of insects and mammals. PGN recognition proteins (PGRPs) are widely distributed and highly conserved in vertebrates and invertebrates. PGRPs are a family of pattern recognition receptors that recognize peptidoglycan and regulate immune responses. In this study, we cloned two PGRP genes (BdPGRP-SA and BdPGRP-SD) from Bactrocera dorsalis (Hendel), which encode 192 and 196 amino acid residues, respectively. Both genes were highly expressed in adults, especially in the fat body and midgut. These two genes were up-regulated when challenged by the immune triggers, PGN-EB (Escherichia coli O111:B4) and PGN-SA (Staphylococcus aureus). The suppression of transcriptional expression of either gene by RNA interference (RNAi) resulted in increased sensitivities to Gram-negative E. coli and Gram-positive S. aureus PGNs. Suppression of BdPGRP-SA and -SD expression by RNAi resulted in weak expressions of four antimicrobial peptides (AMPs) upon injected with E. coli or S. aureus. BdPGRP-SA and -SD are involved in recognizing both Gram-negative and Gram-positive bacteria independently to activate the downstream AMP&rsquo, s response to bacterial infection.

Ecdysis triggering hormone (ETH), released by the Inka cells, is a master hormone in regulating the ecdysis process in insect. Here we investigated the presence and role of the ETH signaling in the female adult of the oriental fruit fly, Bactrocera dorsalis (Hendel) that is one of the most important invasive pest insects in agriculture worldwide. In the female adult, ETH was confirmed in the Inka cells at the tracheae by immunostaining and also in vitro exposure to ETH stimulated the isolated corpora allata of adult in activity. Then we prepared cDNA of females at 0, 5, 10, 15, and 20 days after adult eclosion, and RT-qPCR showed that the expression pattern of ETH and its receptor ETHR-B started from a peak at the day of adult eclosion (day 0), then dropped to basal levels and increased again between day 10 and 15 which is also the period corresponding to ovary growth. In contrast, ETHR-A was absent with Ct values of >33. The expression patterns of the ecdysteroid-producing Halloween genes Spook and Shade, and the vitellogenin genes Vg1, Vg2, and Vg3 co-occurred with peak levels at days 10–15, and also juvenile hormone acid methyltransferase (JHAMT) showed increased levels on day 15. Further in RNAi assays to better understand the role of ETH and ETHR, dsRNA was injected to adult and this led to a respective decrease in expression of 62 and 56% for ETH and ETHR-B, while ETHR-A stayed absent with Ct values of 33. In these RNAi-females, there was an apparently decreased expression for JHAMT and Vg2, together with a significant decrease of the JH titer and egg production. Injection of the JH mimetic methoprene could rescue Vg2 expression and egg production. Upstream, in dsETH/dsETHR-injected females, 20-hydroxyecdysone (20E) injection rescued the transcriptions of ETH and ETHR and also egg production. In summary, our results shed more light on the pivotal role that the ETH peptide hormone and its receptor ETHR-B play an essential role in the reproduction of the female adult of B. dorsalis, via the regulation of JH and vitellogenin, which are controlled by a pulse of 20E.

The oriental fruit fly, Bactrocera dorsalis, is one of the most destructive pests worldwide. The frequent use of chemical insecticides has led B. dorsalis to develop resistance to many insecticides in recent decades. New high-throughput-sequenced transcriptomes, as well as genomes, have revealed a large number of reference genes for functional target identification. Here, we performed digital gene expression profiling of ovary and testis of B. dorsalis adults. Various genes were identified to be highly expressed in B. dorsalis ovary. The genes encoding components of eggshell, vitelline membrane proteins (Vmps) and chorion-related proteins, were identified to be tissue-specifically expressed in ovary. Five cytochrome P450 genes were also identified to be highly expressed in ovary. Three of them were ecdysone synthesis pathway genes indicating the ovary as a potential synthesis site of female. The up-regulated expression of Vmps by exogenous 20-hydroxyecdysone implied the hormonal regulation of eggshell formation during ovarian development. Many other genes with potential functions in ovarian development were also identified, including vitellogenin receptor, insulin receptor, NASP protein, and odorant binding protein. These findings should promote our understanding of the regulation of vitellogenesis and eggshell formation and enable exploration of potentially novel pest control targets.

The citrus red mite, Panonychus citri (McGregor), a major citrus pest distributed worldwide, has been found to be resistant to various insecticides and acaricides used in China. However, the molecular mechanisms associated with the abamectin resistance in this species have not yet been reported. In this study, results showed over-expression of a novel glutathione S-transferases (GSTs) gene (PcGSTm5) in abamectin-resistant P. citri. Quantitative real-time PCR analysis showed that the transcripts of PcGSTm5 were also significantly up-regulated after exposure to abamectin and the maximum mRNA expression level at nymphal stage. The recombinant protein of PcGSTm5-pET-28a produced by Escherichia coli showed a pronounced activity toward the conjugates of 1-chloro-2,4 dinitrobenzene (CDNB) and glutathione (GSH). The kinetics of CDNB and GSH and its optimal pH and thermal stability were also determined. Reverse genetic study through a new method of leaf-mediated dsRNA feeding further support a link between the expression of PcGSTm5 and abamectin resistance. However, no direct evidence was found in metabolism or inhibition assays to confirm the hypothesis that PcGSTm5 can metabolize abamectin. Finally, it is here speculated that PcGSTm5 may play a role in abamectin detoxification through other pathway such as the antioxidant protection.

Chitinases are hydrolytic enzymes that are required for chitin degradation and reconstruction in arthropods. In this study, we report a cDNA sequence encoding a putative chitinase (PcCht1) from the citrus red mite, Panonychus citri. The PcCht1 (564 aa) possessed a signal peptide, a conserver domain, and a chitin-binding domain. Structural and phylogenetic analyses found that PcCht1 had high sequence similarity to chitinases in Tetranychus urticae. Real-time quantitative PCR analyses showed that the transcript levels of PcCht1 peaked periodically in larval and nymph stages. Moreover, significant increase of PcCht1 transcript level in the larvae was observed upon the exposure of diflubenzuron. In contrast, exposures of the larvae to diflubenzuron resulted in the decreased chitin content. Furthermore, through a feeding-based RNA interference approach, we were able to reduce the PcCht1 transcript level by 59.7 % in the larvae, and consequently the treated larvae showed a very low molting rate compared with the control. Our results expanded the understanding of the important role of PcCht1 in the growth and development of P. citri.