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Introduction: Unscientific application of insecticides has led to severe resistance of pests to almost all classes of insecticides. Enhanced detoxification is the most common mechanism for this kind of resistance. Object: Fall armyworm (FAW) has developed insecticide resistance, which is often linked to the overexpression of detoxification genes. Herein, a multicomponent nano-pesticide is designed to increase its broad-spectrum susceptibility toward insecticides. Method: Regulatory function of nuclear factor erythroid 2-related factor 2 (Nrf2) in detoxification was confirmed using transcriptome sequencing, quantitative real-time PCR and enzyme activity measurement. A star polycation (SPc) was adopted to construct the pesticide/SPc/complex, whose self-assembly mechanism and characterization were examined using isothermal titration calorimetry, dynamic light scattering and transmission electron microscope. The delivery efficiency of SPc-loaded dsRNA was examined in vitro and in vivo using fluorescent tracer technique. A multicomponent nano-pesticide was created through the integration of bacterial expression system and nano-delivery system, and its bioactivity was tested in laboratory and field. Results: We confirmed the crucial role of Nrf2 in regulating the detoxification in FAW, and silencing Nrf2 could decrease detoxification gene expression and increase insecticide susceptibility. We then applied the SPc to self-assemble a nanoplatform for delivering Nrf2 double-stranded RNA (dsRNA) and pesticide simultaneously. Nano-sized pesticide/SPc/dsRNA complex exhibited high delivery efficiency in vitro and in vivo. Excitingly, the insecticidal activities of pesticide/SPc/dsNrf2 complexes were remarkably improved with the normalized synergistic ratios of 5.43–6.25 for chlorantraniliprole, 4.45–15.00 for emamectin benzoate, and 6.75–15.00 for spinetoram. Finally, we developed a multicomponent nano-pesticide (pesticide/SPc/dsNrf2 complex) using a bacterial expression system and nano-delivery system. This approach exhibited excellent leaf protection and pest control efficacy. Conclusion: The integration between the pesticide nanometerization and insecticide susceptibility improvement offers a promising strategy to increase insecticidal activity. Our study provides a revolutionary and universal strategy to increase insecticidal activity and decease application doses.

Abstract Background Neonicotinoid insecticides are applied worldwide for the control of agricultural insect pests. The evolution of neonicotinoid resistance has led to the failure of pest control in the field. The enhanced detoxifying enzyme activity and target mutations play important roles in the resistance of insects to neonicotinoid resistance. Emerging evidence indicates a central role of the gut symbiont in insect pest resistance to pesticides. Existing reports suggest that symbiotic microorganisms could mediate pesticide resistance by degrading pesticides in insect pests. Results The 16S rDNA sequencing results showed that the richness and diversity of the gut community between the imidacloprid-resistant (IMI-R) and imidacloprid-susceptible (IMI-S) strains of the cotton aphid Aphis gossypii showed no significant difference, while the abundance of the gut symbiont Sphingomonas was significantly higher in the IMI-R strain. Antibiotic treatment deprived Sphingomonas of the gut, followed by an increase in susceptibility to imidacloprid in the IMI-R strain. The susceptibility of the IMI-S strain to imidacloprid was significantly decreased as expected after supplementation with Sphingomonas. In addition, the imidacloprid susceptibility in nine field populations, which were all infected with Sphingomonas, increased to different degrees after treatment with antibiotics. Then, we demonstrated that Sphingomonas isolated from the gut of the IMI-R strain could subsist only with imidacloprid as a carbon source. The metabolic efficiency of imidacloprid by Sphingomonas reached 56% by HPLC detection. This further proved that Sphingomonas could mediate A. gossypii resistance to imidacloprid by hydroxylation and nitroreduction. Conclusions Our findings suggest that the gut symbiont Sphingomonas, with detoxification properties, could offer an opportunity for insect pests to metabolize imidacloprid. These findings enriched our knowledge of mechanisms of insecticide resistance and provided new symbiont-based strategies for control of insecticide-resistant insect pests with high Sphingomonas abundance.

The green peach aphid, Myzus persicae (Sulzer), is a significant global pest in horticultural and field crops. Afidopyropen is a novel systemic insecticide with high efficacy against sucking pests, and it is suitable for the management of M. persicae. However, the persistent toxicity and dissipation dynamics of afidopyropen in vegetables remain unknown. In this study, we determined the residual activity and dissipation dynamics of afidopyropen against M. persicae on cabbage and chili. The data showed that the toxicity of afidopyropen against M. persicae lasted more than 30 days; the corrected mortality was greater than 80% 10 days after application and was 50–60% 30 days post-application. The afidopyropen residues on cabbage and chili plants were quantified using ultrahigh-pressure liquid chromatography–tandem mass spectrometry. The dissipation half-lives of afidopyropen on cabbage and chili plants ranged from 1.45 to 2.34 days and 3.98–5.98 days at different recommended dosages, respectively. Our findings provide valuable data for the maximum residue limits of afidopyropen on vegetables and will help growers determine the frequency and timing of its application on cabbage and chili.

Abstract Two unique housefly strains, PSS and N-PRS (near-isogenic line with the PSS), were used to clarify the mechanisms associated with propoxur resistance in the housefly, Musca domestica. The propoxur-selected resistant (N-PRS) strain exhibited >1035-fold resistance to propoxur and 1.70-, 12.06-, 4.28-, 57.76-, and 57.54-fold cross-resistance to beta-cypermethrin, deltamethrin, bifenthrin, phoxim, and azamethiphos, respectively, compared to the susceptible (PSS) strain. We purified acetylcholinesterase (AChE) from the N-PRS and PSS strains using a procainamide affinity column and characterized the AChE. The sensitivity of AChE to propoxur based on the bimolecular rate constant (Ki) was approximately 100-fold higher in the PSS strain compared to the N-PRS strain. The cDNA encoding Mdace from both the N-PRS strain and the PSS strain were cloned and sequenced using RT-PCR. The cDNA was 2073 nucleotides long and encoded a protein of 691 amino acids. A total of four single nucleotide polymorphisms (SNPs), I162M, V260L, G342A, and F407Y, were present in the region of the active site of AChE from the N-PRS strain. The transcription level and DNA copy number of Mdace were significantly higher in the resistant strain than in the susceptible strain. These results indicated that mutations combined with the up-regulation of Mdace might be essential in the housefly resistance to propoxur.

Bt cotton successfully controlled major devastating pests in cotton，such as Helicoverpa armigera and Spodoptera exigua, and led to a drastic decrease in insecticide use in cotton fields, and it has been grown commercially worldwide. However, Bt cotton cultivation left Bt toxin residues in the soil, resulting in a response by its microbiome that caused potential environmental risks. In this research, the metagenomics analysis was performed to investigate the structure and functions of the soil bacterial community in the Bt cotton field from the Binzhou, Shandong province of China, where the Bt cotton has been cultivated for over fifteen years. Analysis of the function genes proved that the receptors of Bt toxins were absent in the soil bacteria and Bt toxins failed to target the soil bacteria. The microbiome structure and function were highly influenced by Bt cotton cultivation, however, no significant change in the total abundance of the bacteria was observed. Proteobacteria was the largest taxonomic group in the soil bacterial (42–52%) and its abundance was significantly increased after Bt cotton cultivation. The increase of Proteobacteria abundance resulted in an increase in ABC transporters gene abundance, indicating the improved ability of detoxification metabolism over Bt cotton cultivation. Xanthomonadales could be a biomarker of the Bt cotton group, whose abundance was significantly increased to contribute to the increase of the genes abundance in ABC transporters. The abundance of apoptosis genes was significantly decreased, and it might be related to the increase of Proteobacteria abundance by Bt cotton cultivation. In addition, Myxococcales was responsible for carotenoid biosynthesis, whoes genes abundance was significantly decreased due to the decrease of Myxococcales abundance by Bt cotton cultivation. These changes in soil bacterial community structure and functions indicate the influence by Bt cotton cultivation, leading to an understanding of the bacteria colonization patterns due to successive years of Bt cotton cultivation. These research results should be significant for the rational risk assessment of Bt cotton cultivation.

Both insects and mammals all exhibit a daily fluctuation of susceptibility to chemicals at different times of the day. However, this phenomenon has not been further studied in the house fly (Musca domestica L.) and a better understanding of the house fly on chronobiology should be useful for controlling this widespread disease vector. Here we explored diel time-of-day variations in insecticide susceptibility, enzyme activities, and xenobiotic-metabolizing enzyme gene expressions. The house fly was most tolerant to beta-cypermethrin in the late photophase at Zeitgeber time (ZT) 8 and 12 [i.e., 8 and 12 h after light is present in the light-dark cycle (LD)]. The activities of cytochrome P450, GST, and CarE enzymes were determined in the house flies collected at various time, indicating that rhythms occur in P450 and CarE activities. Subsequently, we observed diel rhythmic expression levels of detoxifying genes, and CYP6D1 and MdαE7 displayed similar expression patterns with enzyme activities in LD conditions, respectively. No diel rhythm was observed for CYP6D3 expression. These data demonstrated a diel rhythm of metabolic detoxification enzymes and insecticide susceptibility in M. domestica. In the future, the time-of-day insecticide efficacy could be considered into the management of the house fly.

The evolution of resistance to insecticides is well known to be closely associated with the overexpression of detoxifying enzymes. Although the role of glutathione S-transferase (GST) genes in insecticide resistance has been widely reported, the underlying regulatory mechanisms are poorly understood. Here, one GST gene (GSTu1) and its antisense transcript (lnc-GSTu1-AS) were identified and cloned, and both of them were upregulated in several chlorantraniliprole-resistant Plutella xylostella populations. GSTu1 was confirmed to be involved in chlorantraniliprole resistance by direct degradation of this insecticide. Furthermore, we demonstrated that lnc-GSTu1-AS interacted with GSTu1 by forming an RNA duplex, which masked the binding site of miR-8525-5p at the GSTu1-3′UTR. In summary, we revealed that lnc-GSTu1-AS maintained the mRNA stability of GSTu1 by preventing its degradation that could have been induced by miR-8525-5p and thus increased the resistance of P. xylostella to chlorantraniliprole. Our findings reveal a new noncoding RNA-mediated pathway that regulates the expression of detoxifying enzymes in insecticide-resistant insects and offer opportunities for the further understanding of the mechanisms of insecticide and drug resistance. Author summary The development of insecticide resistance in insect pests is a worldwide concern and a major problem in agriculture. Understanding the genetics of insecticide resistance is critical for effective crop protection. Plutella. xylostella (L.), a major pest of cruciferous crops, has developed resistance to almost all kinds of insecticide, and has become one of the most resistant pests in the world. Overexpression of detoxification enzymes is closely associated with insecticide resistance, but researches on their regulatory mechanism are still very limited. Here, GSTu1 was identified to be upregulated in several chlorantraniliprole-resistant P. xylostella populations and was confirmed to be involved in chlorantraniliprole resistance by direct degradation of this insecticide. Further, lnc-GSTu1-AS transcribed from the opposite DNA strand to GSTu1 was identified to be able to enhance the mRNA stability of GSTu1 by blocking miRNA activity, and thus increased the resistance of P. xylostella to chlorantraniliprole. The results provide further insights into the mechanisms underlying metabolic resistance.

Abstract Background Most aphids exhibit wing polyphenism in which wingless and winged morphs produce depending on the population density and host plant quality. Although the influence of environmental factors on wing polyphenism of aphids have been extensively investigated, molecular mechanisms underlining morph differentiation (i.e. wing development /degeneration), one downstream aspect of the wing polyphenism, has been poorly understood. Results We examined the expression levels of the twenty genes involved in wing development network, and only vestigial (vg) showed significantly different expression levels in both whole-body and wall-body of third instar nymphs, with 5.4- and 16.14- fold higher expression in winged lines compared to wingless lines, respectively in Rhopalosiphum padi. vg expression was higher in winged lines compared to wingless lines in third, fourth instar nymphs and adults. Larger difference expression was observed in third (21.38-fold) and fourth (20.91-fold) instar nymphs relative to adults (3.12-fold). Suppression of vg using RNAi repressed the wing development of third winged morphs. Furthermore, dual luciferase reporter assay revealed that the miR-147 can target the vg mRNA. Modulation of miR-147b levels by microinjection of its agomir (mimic) decreased vg expression levels and repressed wing development. Conclusions Our findings suggest that vg is essential for wing development in R. padi and that miR-147b modulates its expression.

The Aphis gossypii is an important pest that can damage cotton plants and can cause a huge economic loss worldwide. Chemical control is a main method to manage this pest, but the cotton aphid resistance to insecticides has become a severe problem in the management of the cotton aphid. It is important to introduce a novel insecticide for rotational application with other insecticides. Broflanilide, as a meta-diamide insecticide with a special mode of action, showed high efficiency against lepidopterous larvae. However, we found that broflanilide possessed high insecticidal activity against the sap-sucking pest A. gossypii. The susceptibility of A. gossypii to broflanilide from 20 field populations in main cotton planting areas of China in 2021 was determined by the leaf-dipping method. LC50 values of broflanilide to A. gossypii ranged from 0.20 μg mL−1 to 1.48 μg mL−1. The susceptible baseline of A. gossypii to broflanilide was established with the LC50 value of 0.41 μg mL−1 and might be used to calculate the resistance ratio (RR) of cotton aphid population in broflanilide resistance monitoring. The RR value of field populations in China was from 0.49 to 3.61 in 2021. It suggested that the broflanilide may be a potential agent in the resistance management of A. gossypii to insecticides. These results are significantly useful for the rational chemical control of cotton aphids.

The resistance of cotton aphids to various forms of commonly used pesticides has seriously threatened the safety of the cotton production. Afidopyropen is a derivative of microbial metabolites with pyropene insecticide, which has been shown to be effective in the management of Aphis gossypii. Several field populations of Aphis gossypii were collected from the major cotton-producing regions of China from 2019 to 2021. The resistance of these populations to afidopyropen was estimated using the leaf-dipping method. The LC50 values of these field populations ranged from 0.005 to 0.591 mg a.i. L−1 in 2019, from 0.174 to 4.963 mg a.i. L−1 in 2020 and from 0.517 to 14.16 mg a.i. L−1 in 2021. The resistance ratios for all A. gossypii populations ranged from 0.03 to 3.97 in 2019, from 1.17 to 33.3 in 2020 and from 3.47 to 95.06 in 2021. The afidopyropen resistance exhibited an increasing trend in the field populations of Cangzhou, Binzhou, Yuncheng, Kuerle, Kuitun, Changji and Shawan from 2019 to 2021. This suggests that the resistance development of the cotton aphid to afidopyropen is inevitable. Therefore, it is necessary to rotate or mix afidopyropen with other insecticides in order to inhibit the development of afidopyropen resistance in field populations.

A resistant strain (MRS) of Sitobion miscanthi was cultured by continuous selection with malathion for over 40 generations. The MRS exhibited 32.7-fold resistance to malathion compared to the susceptible strain (MSS) and 13.5-fold, 2.9-fold and 4.8-fold cross-resistance for omethoate, methomyl and beta-cypermethrin, respectively. However, no cross-resistance was found to imidacloprid in this resistant strain. The realized heritability for malathion resistance was 0.02. Inhibitors of esterase activity, both triphenyl phosphate (TPP) and S,S,S,-tributyl phosphorotrithioate (DEF) as synergists, exhibited significant synergism to malathion in the MRS strain, with 11.77-fold and 5.12-fold synergistic ratios, respectively, while piperonyl butoxide (PBO) and diethyl maleate (DEM) showed no significant synergism in the MRS strain. The biochemical assay indicated that carboxylesterase activity was higher in MRS than in MSS. These results suggest that the increase in esterase activity might play an important role in S. miscanthi resistance to malathion. Imidacloprid could be used as an alternative for malathion in the management of wheat aphid resistance.

Cotton bollworm (Helicoverpa armigera) is a Lepidopteran noctuid pest with a global distribution. It has a wide range of host plants and can harm cotton, tomato, tobacco, and corn, as well as other crops. H. armigera larvae damage the flower buds, flowers, and fruits of tomato and cause serious losses to tomato production. Tomato uses the allelochemical 2-tridecanone to defend against this damage. So far, there have been no reports on whether the adaptation of H. armigera to 2-tridecanone is related to its symbiotic microorganisms. Our study found that Corynebacterium sp. 2-TD, symbiotic bacteria in H. armigera, mediates the toxicity of the 2-tridecanone to H. armigera. Corynebacterium sp. 2-TD, which was identified by 16S rDNA gene sequence analysis, was screened out using a basal salt medium containing a unique carbon source of 2-tridecanone. Then, Corynebacterium sp. 2-TD was confirmed to be distributed in the gut of H. armigera by quantitative PCR (qPCR) and fluorescence in situ hybridization (FISH). The survival rate of H. armigera increased by 38.3% under 2-tridecanone stress after inoculation with Corynebacterium sp. 2-TD. The degradation effect of Corynebacterium sp. 2-TD on 2-tridecanone was verified by ultra-high-performance liquid chromatography (UPLC). Our study is the first to report the isolation of gut bacteria that degrade 2-tridecanone from the important agricultural pest H. armigera and to confirm bacterial involvement in host adaptation to 2-tridecanone, which provides new insights into the adaptive mechanism of agricultural pests to host plants.

Imidacloprid is one of the most widely used neonicotinoid insecticides to control sap-sucking insect pests, including Aphis gossypii. The intensive application of chemical insecticides to A. gossypii led to the development of resistance against several insecticides, including imidacloprid. Therefore, it is crucial to understand the association between imidacloprid resistance and the fitness of A. gossypii to limit the spread of the resistant population under field contexts. In this study, we used the age-stage, two-sex life table method to comprehensively investigate the fitness of imidacloprid resistant (ImR) and susceptible strains (SS) of melon aphids. Results showed that ImR aphids have prolonged developmental stages and decreased longevity, fecundity, and reproductive days. The key demographic parameters (r, λ, and R0) were significantly reduced in ImR strain compared to SS aphids. Additionally, the molecular mechanism for fitness costs was investigated by comparing the expression profile of juvenile hormone-binding protein (JHBP), juvenile hormone epoxide hydrolase (JHEH), juvenile hormone acid O-methyltransferase (JHAMT), Vitellogenin (Vg), ecdysone receptor (EcR), and ultraspiracle protein (USP) supposed to be associated with development and reproduction in insects. The results of RT-qPCR showed that EcR, JHBP, JHAMT, JHEH, and Vg genes were downregulated, while USP was statistically the same in ImR A. gossypii compared to the SS strain. Together, these results provide in-depth information about the occurrence and magnitude of fitness costs against imidacloprid resistance that could help manage the evolution and spread of A. gossypii resistance in field populations

Sublethal effect considered as an emerging factor to assess the environmental risk of insecticides, which can impact the insects on both physiology and behavior. Lethal exposure can be causing near immediate mortality. Pests are inevitably exposed to sublethal and lethal dose in the agroecosystem following application of pesticides. Insecticides, widely used for the control of insect pests, are irreplaceable in insect pest management. The effects of imidacloprid by the method of high-throughput non-targeted metabolomics was investigated in Aphis gossypii Glover exposed to LC10 and LC90 doses of the imidacloprid, and the control group was treated with the same condition without imidacloprid. Pairwise comparisons showed that 111 metabolites changed significantly, 60 in the LC10 group, and 66 in the LC90 group compared to the control group, while only 16 changes in the LC10 were same with that in LC90 group. Among the changed metabolites, a total of 16 metabolites were identified as potential biomarkers, which represented the most influential pathways including glycolysis and gluconeogenesis, alanine, aspartate, and glutamate metabolism, ascorbate and aldarate metabolism, glutathione metabolism, phenylalanine metabolism, tyrosine metabolism, caffeine metabolism and parkinson’s disease (PD), which could account for the sublethal and lethal effects on A. gossypii. These modified metabolic pathways demonstrated that high energy consumption, excitotoxicity and oxidative stress (OS) were appeared in both LC10 and LC90 groups, while PD was detected only in the LC90 group. The results of non-targeted metabolomics revealed the effects of neonicotinoid pesticide exposure on A. gossypii successfully, and provided a deep insight into the influenced physiology by the stress of neonicotinoid pesticide in the insect.

The locusts Oedales asiaticus (Bey-Bienko) and Myrmeleotettix palpalis (Zubovski) (Orthoptera Acrididae) and the leaf beetle Galeruca daurica (Joannis) (Coleoptera, Chrysomelidae) are economically devastating insect species in the desert steppes of Northern China. Control is mainly and frequently dependent on highly toxic chemicals. To date, there have been no complete and comprehensive reports of insecticide applications to these key pests. In this study, laboratory bioassays were carried out to determine and compare the toxicity of twelve insecticides to three outbreaking insects, O. asiaticus, M. palpalis, and G. daurica, from three typical desert steppe regions, SZWQ, XHQ and WLTQQ, respectively. The responses of the two locust species and the leaf beetle were evaluated by topical application and leaf dip bioassay techniques across a range of concentrations to develop dosage–mortality regressions. The insecticides tested included six chemical insecticides (β-cypermethrin, imidacloprid, phoxim, λ-cyhalothrin, methomyl, chlorantraniliprole) and six biogenic insecticides (spinosad, avermectin, rotenone, matrine, azadiracthin, and methoxyfenozide). The results showed that phoxim, λ-cyhalothrin, β-cypermethrin and spinosad showed highly toxic activity to O.asiaticus, M. palpalis, and G. daurica, while methonyl, chlorantraniliprole, and rotenone were moderately toxic to both locust species and the leaf beetle. The LC50 values of matrine, azadiractin, and avermectin were more than 1 μg a.i./adult for O. asiaticus and M. palpalis, the LC50 values of which were higher 2 g/L for G. daurica. Our findings complement information from previous similar studies and will inform future studies relating to the control of outbreaking insects, such as O.asiaticus, M. palpalis, and G. daurica in desert steppes of northern China. This study is also expected to provide basic data on the use of chemical and biogenic insecticides for application in desert steppes.

Abstract Cucurbitacin B is a natural triterpene present in plants of Cucurbitaceae family, which are among the host plants for melon aphid, Aphis gossypii. In present study we characterized the effects of two cucurbitacin B concentrations on the biological parameters of adults (F0) and of juveniles and adults of their progeny (F1). The results showed that cucurbitacin B at 25 ppm significantly reduced the adult longevity and fecundity of both F0 and F1 generation. Exposure of F0 generation to 25 ppm though reduced the demographic traits of F1 including the intrinsic rate of increase r (day−1), generation time T (day), finite rate of increase λ (day−1), however, only net reproductive rate R 0 (offspring/individual) decreased significantly. While 100 ppm reduced not only the longevity and fecundity of F0 generation but also the longevity of F1 generation. Fecundity of F1 was not affected by 100 ppm of cucurbitacin B, however, R 0 (offspring/individual) and T (day) of F1 generation were lower than the control population. These results support the hypothesis that high contents of cucurbitacin B caused negative impact on melon aphid and could be used as a lead for classical selection of resistant varieties of plants that are main hosts for the melon aphid.

Abstract Background The oriental armyworm, Mythimna separata, is an economically important and common Lepidopteran pest of cereal crops. Chemoreception plays a key role in insect life, such as foraging, oviposition site selection, and mating partners. To better understand the chemosensory mechanisms in M. separata, transcriptomic analysis of antennae, labial palps, and proboscises were conducted using next-generation sequencing technology to identify members of the major chemosensory related genes. Results In this study, 62 putative odorant receptors (OR), 20 ionotropic receptors (IR), 16 gustatory receptors (GR), 38 odorant binding proteins (OBP), 26 chemosensory proteins (CSP), and 2 sensory neuron membrane proteins (SNMP) were identified in M. separata by bioinformatics analysis. Phylogenetic analysis of these candidate proteins was performed. Differentially expressed genes (DEGs) analysis was used to determine the expressions of all candidate chemosensory genes and then the expression profiles of the three families of receptor genes were confirmed by real-time quantitative RT-PCR (qPCR). Conclusions The important genes for chemoreception have now been identified in M. separata. This study will provide valuable information for further functional studies of chemoreception mechanisms in this important agricultural pest.

Abstract Background Long noncoding RNAs (lncRNAs) are now considered important regulatory factors, with a variety of biological functions in many species including insects. Some lncRNAs have the ability to show rapid responses to diverse stimuli or stress factors and are involved in responses to insecticide. However, there are no reports to date on the characterization of lncRNAs associated with chlorantraniliprole resistance in Plutella xylostella. Results Nine RNA libraries constructed from one susceptible (CHS) and two chlorantraniliprole-resistant P. xylostella strains (CHR, ZZ) were sequenced, and 1309 lncRNAs were identified, including 877 intergenic lncRNAs, 190 intronic lncRNAs, 76 anti-sense lncRNAs and 166 sense-overlapping lncRNAs. Of the identified lncRNAs, 1059 were novel. Furthermore, we found that 64 lncRNAs were differentially expressed between CHR and CHS and 83 were differentially expressed between ZZ and CHS, of which 22 were differentially expressed in both CHR and ZZ. Most of the differentially expressed lncRNAs were hypothesized to be associated with chlorantraniliprole resistance in P. xylostella. The targets of lncRNAs via cis- ( 0.9 or < -0.9, P < 0.05) regulatory effects were also identified; many of the differently expressed lncRNAs were correlated with various important protein-coding genes involved in insecticide resistance, such as the ryanodine receptor, uridine diphosphate glucuronosyltransferase (UGTs), cytochrome P450, esterase and the ATP-binding cassette transporter. Conclusions Our results represent the first global identification of lncRNAs associated with chlorantraniliprole resistance in P. xylostella. These results will facilitate future studies of the regulatory mechanisms of lncRNAs in chlorantraniliprole and other insecticide resistance and in other biological processes in P. xylostella.

Fitness is closely associated with the development of pesticide resistance in insects, which determines the control strategies employed to target species and the risks of toxicity faced by non-target species. After years of selections with beta-cypermethrin in laboratory, a strain of housefly was developed that was 684,521.62-fold resistant (CRR) compared with the susceptible strain (CSS). By constructing ≤ 21 d and ≤ 30 d life tables, the differences in life history parameters between CSS and CRR were analyzed. The total production numbers of all the detected development stages in CRR were lower than in CSS. Except for the lower mortality of larvae, all the other detected mortalities in CRR were higher than in CSS. ♀:♂ and normal females of CRR were also lower than those of CSS. For CRR, the relative fitness was 0.25 in the ≤ 21 d life table and 0.24 in the ≤ 30 d life table, and a lower intrinsic rate of increase (rm) and net reproductive rate (Ro) were detected. Based on phenotype correlation and structural equation model (SEM) analyses, fecundity and viability were the only directly positive fitness components affecting fitness in CRR and CSS, and the other components played indirect roles in fitness. The variations of the relationships among fitness, fecundity and viability seemed to be the core issue resulting in fitness differences between CRR and CSS. The interactions among all the detected fitness components and the mating frequency-time curves appeared to be distinctly different between CRR and CSS. In summary, fecundity and its related factors separately played direct and indirect roles in the fitness costs of a highly beta-cypermethrin-resistant housefly strain.

Clothianidin is a second-generation neonicotinoid insecticide, widely used against sap-sucking insect pest including melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae). This pest causes severe economic damage to Cucurbitaceae plants worldwide. In this study, we investigated clothianidin resistance development under continuous selection pressure. Moreover, the age-stage, two-sex life table approach was used to evaluate the impact of clothianidin resistance on the fitness of A. gossypii. A clothianidin resistant strain (CT-R) with a 23.17-fold resistance level was developed from a susceptible strain (CT-S) after continuous selection for 24 generations. Life table results showed a significant reduction in the relative fitness (0.847) of CT-R strain compared to the CT-S strain of A. gossypii. The developmental duration, oviposition days, total pre-oviposition period (TPOP), longevity, and fecundity of CT-R strain were found to be significantly lower when compared to CT-S strain. The demographic parameters, including the intrinsic rate of increase (r), finite rate of increase (λ), net reproductive rate (R0), and mean generation time (T) were also significantly decreased in CT-R strain compared to the CT-S strain. Both the reproductive and survival rates were affected by clothianidin resistance in CT-R strain compared with the CT-S strain of A. gossypii. Overall, our results demonstrate that in-depth knowledge about the trade-off at play between resistance degree and fitness cost might be useful to design resistance management strategies against A. gossypii.

OBPs play a crucial role in the recognition of ligands and are involved in the initial steps of semiochemical perception. The diverse expression of OBP genes allows them to participate in different physiological functions in insects. In contrast to classic OBPs with typical olfactory roles in A. lineolatus, the physiological functions of Plus-C OBPs remain largely unknown. In addition, detection of the expression of insect OBP genes by conventional methods is difficult in vitro. Here, we focused on AlinOBP14, a Plus-C OBP from A. lineolatus, and we developed a PNA-GO-based mRNA biosensor to detect the expression of AlinOBP14. The results demonstrated that AlinOBP14 plays dual roles in A. lineolatus. The AlinOBP14 is expressed beneath the epidermis of the vertex and gena in heads of A. lineolatus, and it functions as a carrier for three terpenoids, while AlinOBP14 is also expressed in the peripheral antennal lobe and functions as a carrier for endogenous compounds such as precursors for juvenile hormone (JH) and JHⅢ. Our investigation provides a new method to detect the expression of OBP genes in insects, and the technique will facilitate the use of these genes as potential targets for novel insect behavioral regulation strategies against the pest.

UDP-glycosyltransferases (UGTs) are major phase II detoxification enzymes that catalyze the transfer of glycosyl residues from activated nucleotide sugars to acceptor hydrophobic molecules and play very important roles in the biotransformation of various endogenous and exogenous compounds. Our previous studies demonstrated that UGTs participated in the detoxification of insecticides in Aphis gossypii. However, the potential roles of UGTs in A. gossypii resistance to sulfoxaflor are still unclear. In this study, two inhibitors of UGT enzymes, sulfinpyrazone and 5-nitrouracil, significantly increased the toxicity of sulfoxaflor to a resistant strain of A. gossypii, whereas there were no synergistic effects in the susceptible strain. Based on the transcriptome sequencing results, the expression levels of 15 UGTs were analyzed by quantitative real-time PCR, and we found that seven UGT genes were highly over-expressed in a sulfoxaflor-resistant strain compared to the susceptible strain, including UGT344B4, UGT344C5, UGT344A11, UGT344A14, and UGT344L2. Further suppressing the expression of UGT344B4, UGT344C5, and UGT344A11 by RNA interference significantly increased the sensitivity of resistant aphids to sulfoxaflor, indicating that the overexpression of UGT genes is potentially associated with sulfoxaflor resistance. These results could provide valuable information for further understanding the mechanisms of insecticide resistance.

Plants accumulate phenolic compounds such as tannic acid to resist insect herbivores. The survival of insects exposed to toxic secondary metabolites depends on the detoxification metabolism mediated by limited groups of glutathione S-transferases (GSTs). Micromelalopha troglodyta (Graeser) (Lepidoptera: Notodontidae) is an important foliar pest of poplar trees. GSTs play an important role in xenobiotic detoxification in M. troglodyta. Five GST genes were identified in M. troglodyta and were classified into five different cytosolic GST classes, delta, omega, sigma, theta, and zeta. Real-time fluorescent quantitative polymerase chain reaction (qPCR) was used to determine the mRNA expression of the five cloned GSTs in the midguts and fat bodies of M. troglodyta. The mRNA expression of the five GSTs was significantly induced when M. troglodyta was exposed to tannic acid. To further understand the tannic acid regulatory cascade, the 5′-flanking promoter sequences of the five MtGSTs were isolated by genome walking methods, and the promoters were very active and induced by tannic acid. In summary, the induction of GST mRNA expression was due to the response of five MtGST promoters to tannic acid. Therefore, MtGST promoters play an important role in the regulation of GST transcription.

Both inhibitory and stimulatory (known as hormesis) effects of the sublethal flupyradifurone, a butenolide insecticide, on Myzus persicae Sulzer (Hemiptera: Aphididae) were investigated for incorporating it into integrated pest management (IPM). A leaf-dip bioassay showed that flupyradifurone was very toxic against adult M. persicae with a 48 h LC50 of 8.491 mg/L. Using the age-stage two-sex life table approach, we assessed the effects of LC25 of flupyradifurone on adult M. persicae and its progeny (F1 and F2). On the one hand, aphids exposed to flupyradifurone had significantly negative effects on the life history traits acrossing the generations, such as reduced the adult longevity and fecundity of F0, shortened the duration of third instar and fourth instar nymphs, preadult period and the pre-reproductive period of F1, and decreased the reproductive days and adult longevity of F2. On the other hand, stimulatory effects on the duration of pre-adult, adult reproductive days, and reproduction of F1 were observed in the flupyradifurone-treated aphids. Consistently with the stimulation on individual traits, a higher net reproductive rate (R0) of F1 and a shorter mean generation time (T) of F2 were observed in the flupyradifurone-treated aphids, although the other population parameters including the intrinsic rate of increase (r), finite rate of increase (λ) and T of F1 and R0, r and λ of F2 were not significantly affected. These results revealed that adult M. persicae exposed to sublethal concentration of flupyradifurone can induce hormetic effects on F1, and also cause negative effects on F2. Our results would be useful for assessing the overall effects of flupyradifurone on M. persicae and the hormetic effects should take into consideration when use flupyradifurone for control M. persicae.

Plant allelochemicals are a group of secondary metabolites produced by plants to defend against herbivore. The mortality of two plant allelochemicals (tannic acid and gossypol) on the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae), were investigated using feeding assays and the sublethal effects were evaluated using the age-stage, two-sex life table approach. Tannic acid and gossypol have deleterious effects on A. gossypii, and as the concentrations increased, the mortality of cotton aphid increased. The life history traits of A. gossypii including the developmental duration of each nymph stage, the longevity, oviposition days, total preadult survival rate and adult pre-oviposition period were not significantly affected by sublethal concentration of tannic acid (20 mg/L) and gossypol (50 mg/L), while the population parameters (r, λ and R0) were significantly affected by these two plant allelochemicals. Furthermore, tannic acid can increase the pre-adult duration time and TPOP but reduce the fecundity of A. gossypii significantly compared to the control and gossypol treatment groups. These results are helpful for comprehensively understanding the effects of plant allelochemicals on A. gossypii.

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are major phase II enzymes that conjugate a variety of small lipophilic molecules with UDP sugars and alter them into more water-soluble metabolites. Therefore, glucosidation plays a major role in the inactivation and excretion of a great variety of both endogenous and exogenous compounds. In this study, two inhibitors of UGT enzymes, sulfinpyrazone and 5-nitrouracil, significantly increased the toxicity of thiamethoxam against the resistant strain of Aphis gossypii, which indicates that UGTs are involved in thiamethoxam resistance in the cotton aphid. Based on transcriptome data, 31 A. gossypii UGTs belonging to 11 families (UGT329, UGT330, UGT341, UGT342, UGT343, UGT344, UGT345, UGT348, UGT349, UGT350, and UGT351) were identified. Compared with the thiamethoxam-susceptible strain, the transcripts of 23 UGTs were elevated, and the transcripts of 13 UGTs (UGT344J2, UGT348A2, UGT344D4, UGT341A4, UGT343B2, UGT342B2, UGT350C3, UGT344N2, UGT344A14, UGT344B4, UGT351A4, UGT344A11, and UGT349A2) were increased by approximately 2.0-fold in the resistant cotton aphid. The suppression of selected UGTs significantly increased the insensitivity of resistant aphids to thiamethoxam, suggesting that the up-regulated UGTs might be associated with thiamethoxam tolerance. This study provides an overall view of the possible metabolic factor UGTs that are relevant to the development of insecticide resistance. The results might facilitate further work to validate the roles of these UGTs in thiamethoxam resistance.

The mirid bug, Apolygus lucorum Meyer-Dür, has been an important pest of cotton crop in China, and is primarily controlled with insecticides, such as pyrethroids. To elucidate the potential resistant mechanisms of A. lucorum to lambda-cyhalothrin, a series of biological, biochemical, and molecular assays were conducted in the reference (AL-S) and lambda-cyhalothrin-resistant (AL-R) populations. Comparison of the molecular target of pyrethroid insecticides, voltage-gated sodium channel, revealed that there were no mutation sites in the resistant population, indicating target insensitivity is not responsible for increased resistance of AL-R to lambda-cyhalothrin. Furthermore, the synergism assays and the activities of detoxification enzymes were performed to determine detoxification mechanism conferring the lambda-cyhalothrin resistance. In the tested synergists, the piperonyl butoxide had the highest synergism ratio against lambda-cyhalothrin, which was up to five-fold in both populations. In addition, the result also showed that only cytochrome P450 had significantly higher O-deethylase activity with 7-ethoxycoumarin (1.78-fold) in AL-R population compared with AL-S population. Seven cytochrome P450 genes were found to be significantly overexpressed in the resistant AL-R population compared with AL-S population. Taken together, these results demonstrate that multiple over-transcribed cytochrome P450 genes would be involved in the development of lambda-cyhalothrin resistance in AL-R population.

Chitin is a vital part of the insect exoskeleton and peritrophic membrane, synthesized by chitin synthase (CHS) enzymes. Chitin synthase 1 (CHS1) is a crucial enzyme in the final step of chitin biosynthetic pathway and consequently plays essential role towards insect growth and molting. RNA interference (RNAi) is an agent that could be used as an extremely target-specific and ecologically innocuous tactic to control different insect pests associated with economically important crops. The sole purpose of the current study is to use CHS1 as the key target gene against the cotton-melon aphid, Aphis gossypii, via oral feeding on artificial diets mixed with dsRNA-CHS1. Results revealed that the expression level of CHS1 gene significantly decreased after the oral delivery of dsRNA-CHS1. The knockdown of CHS1 gene caused up to 43%, 47%, and 59% mortality in third-instar nymph after feeding of dsCHS1 for 24, 48, and 72 h, respectively, as compared to the control. Consistent with this, significantly lower longevity (approximately 38%) and fecundity (approximately 48%) were also found in adult stage of cotton-melon aphids that were fed with dsCHS1 for 72 h at nymphal stage. The qRT-PCR analysis of gene expression demonstrated that the increased mortality rates and lowered longevity and fecundity of A. gossypii were attributed to the downregulation of CHS1 gene via oral-delivery-mediated RNAi. The results of current study confirm that CHS1 could be an appropriate candidate target gene for the RNAi-based control of cotton-melon aphids.

Quercetin as one of the key plant secondary metabolite flavonol is ubiquitous in terrestrial plants. In this study, the decrease in sensitivity to lambda-cyhalothrin was observed in quercetin-fed Helicoverpa armigera larvae. In order to figure out the mechanisms underlying the decreased sensitivity of H. armigera larvae to lambda-cyhalothrin by quercetin induction, the changes in carboxylesterase activity and in-vitro hydrolytic metabolic capacity to lambda-cyhalothrin were examined. The LC50 value of quercetin-fed H. armigera larvae to lambda-cyhalothrin showed 2.41-fold higher than that of the control. S, S, S-Tributyl phosphorotrithioate (DEF) treatment showed a synergism effect on lambda-cyhalothrin toxicity to quercetin-fed H. armigera. Moreover, the activity of carboxylesterase was significantly higher in quercetin-fed H. armigera larvae after fed on quercetin for 48 h. The in-vitro hydrolytic metabolic capacity to lambda-cyhalothrin in quercetin-fed H. armigera larvae midgut was 289.82 nmol 3-PBA/mg protein/min, which is significant higher than that in the control group (149.60 nmol 3-PBA/mg protein/min). The elevated CarE enzyme activity and corresponding increased hydrolytic metabolic capacity to lambda-cyhalothrin in quercetin-fed H. armigera contributed to the enhanced tolerance to lambda-cyhalothrin.

Imidacloprid is a neonicotinoid insecticide that is effective against house fly, Musca domestica L., which is a major pest with the ability to develop resistance to insecticides. In the present study, we investigated the inheritance mode, the cross-resistance pattern and the mechanisms of resistance to imidacloprid. A near-isogenic house fly line (N-IRS) with 78-fold resistance to imidacloprid was used to demonstrate the mode of inheritance. The overlapping confidence limits of LC50 values and the slopes of the log concentration-probit lines between the reciprocal F1 and F1' progenies suggest that imidacloprid resistance is inherited autosomally in the house fly. There was incomplete dominant inheritance in the F1 and F1' progenies, based on dominance values of 0.77 and 0.75, respectively. A monogenic inheritance model revealed that imidacloprid resistance is governed by more than one factor. Compared to the field strain (CFD), the N-IRS strain developed more cross-resistance to chlorfenapyr and no cross-resistance to chlorpyrifos and acetamiprid, but showed negative cross-resistance to beta-cypermethrin and azamethiphos. Three synergists, diethyl malate (DEM), s,s,s-tributylphosphorotrithioate (DEF), and piperonyl butoxide (PBO), showed significant synergism against to imidacloprid (4.55-, 4.46- and 3.34-fold respectively) in the N-IRS strain. However, both DEM and PBO had no synergism and DEF only exhibited slight synergism in the CSS strain. The activities of carboxylesterase (CarE), glutathione S-transferases (GSTs) and cytochrome P450 in the N-IRS strain were significantly higher than in the CSS strain. But similar synergistic potential of DEF to imidacloprid between the CSS and N-IRS strain suggested that GSTs and cytochrome P450 played much more important role than esterase for the N-IRS strain resistance to imidacloprid. These results should be helpful for developing an improved management strategy to delay the development of imidacloprid resistance in house fly.

Both exogenous and endogenous compounds can induce the expression of cytochrome P450 genes. The insect cytochrome P450 genes related to insecticide resistance are likely to be expressed as the "first line of defense" when challenged with insecticides. In this study, four cytochrome P450 genes, SinvCYP6B1, SinvCYP6A1, SinvCYP4C1, and SinvCYP4G15, were firstly isolated from workers of the red imported fire ant (Solenopsis invicta) through rapid amplification of cDNA ends (RACE) and sequenced. The fipronil induction profiles of the four cytochrome P450 genes and the two previously isolated CYP4AB1 and CYP4AB2 were characterized in workers. The results revealed that the expression of SinvCYP6B1, SinvCYP6A1, CYP4AB2, and SinvCYP4G15, increased 1.4-fold and 1.3-fold more than those of acetone control, respectively, after 24 h exposure to fipronil at concentrations of 0.25 μg mL-1 (median lethal dose) and 0.56 μg mL-1 (90% lethal dose), while no significant induction of the expression of CYP4AB1 and SinvCYP4C1 was detected. Among these genes, SinvCYP6B1 was the most significantly induced, and its maximum expression was 3.6-fold higher than that in acetone control. These results might suggest that multiple cytochrome P450 genes are co-up-regulated in workers of the fire ant through induction mechanism when challenged with fipronil. These findings indicated that cytochrome P450 genes play an important role in the detoxification of insecticides and provide a theoretical basis for the mechanisms of insecticide metabolism in the fire ant.

The wheat aphids, Rhopalosiphum padi (Linnaeus) and Sitobion avenae (Fabricius), are key pests on wheat crops worldwide. Management practices rely primarily on insecticides. The pirimicarb (carbamate) is used extensively as an effective insecticide to control these two aphids. In addition to the mortality caused by pirimicarb, various sublethal effects may occur in aphids when exposed to low lethal or sublethal doses. Understanding the general effect of pirimicarb on aphids could help increasing rational use of this insecticide. Under laboratory conditions, we assessed the sublethal effects of a low lethal concentration of pirimicarb (LC25) on biological traits and acetylcholinesterase (AChE) activity of R. padi and S. avenae. Both direct and transgenerational effects, i.e. on parent and the F1 generations were assessed, respectively. We found that R. padi and S. avenae responded differentially to the LC25 of pirimicarb. The parent generation of R. padi showed a 39% decrease in fecundity and multiple transgenerational effects were observed in the F1 generation; overall juvenile development, reproductive period, adult longevity and lifespan were longer than those of the control group. By contrast, LC25 of pirimicarb showed almost no effects on S. avenae biological traits in both the parent and F1 generations; only the pre-reproductive duration was reduced in F1 generations. Demographic parameter estimates (e.g. rm) showed similar trend, i.e. significant negative effect on R. padi population growth and no effect on S. avenae. However, AChE activity decreased in both R. padi and S. avenae treated by the LC25 of pirimicarb. We demonstrated sublethal and transgenerational effects of pirimicarb in the two wheat aphid species; it hinted at the importance of considering sublethal effects (including hormesis) of pirimicarb for optimizing Integrated Pest Management (IPM) of wheat aphids.

Finding a suitable reference gene is the key for qRT-PCR analysis. However, none of the reference gene discovered thus far can be utilized universally under various biotic and abiotic experimental conditions. In this study, we further examine the stability of candidate reference genes under a single abiotic factor, insecticide treatment. After being exposed to eight commercially available insecticides, which belong to five different classes, the expression profiles of eight housekeeping genes in the sweetpotato whitefly, Bemisia tabaci, one of the most invasive and destructive pests in the world, were investigated using qRT-PCR analysis. In summary, elongation factor 1α (EF1α), α-tubulin (TUB1α) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were identified as the most stable reference genes under the insecticide treatment. The initial assessment of candidate reference genes was further validated with the expression of two target genes, a P450 (Cyp6cm1) and a glutathione S-transferase (GST). However, ranking of reference genes varied substantially among intra- and inter-classes of insecticides. These combined data strongly suggested the necessity of conducting custom reference gene selection designed for each and every experimental condition, even when examining the same abiotic or biotic factor.

BACKGROUND: The whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), is one of the most widely distributed agricultural pests. In recent years, B. tabaci Q has invaded China, and Q has displaced B in many areas now. In a number of regions of the world, invasion by B and/or Q has been followed by outbreaks of tomato yellow leaf curl virus (TYLCV). Our previous study showed TYLCV directly and indirectly modified the feeding behavior of B. tabaci in favor of Q rather than B. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we quantified the salicylic acid (SA) titers and relative gene expression of SA in tomato leaves that were infested with viruliferous or non-viruliferous B and Q. We also measured the impacts of exogenous SA on the performance of B and Q, including the effects on ovary development. SA titer was always higher in leaves that were infested with viruliferous B than with viruliferous Q, whereas the SA titer did not differ between leaves infested with non-viruliferous B and Q. The relative gene expression of SA signaling was increased by feeding of viruliferous B but was not increased by feeding of viruliferous Q. The life history traits of B and Q were adversely affected on SA-treated plants. On SA-treated plants, both B and Q had lower fecundity, shorter longevity, longer developmental time and lower survival rate than on untreated plants. Compared with whiteflies feeding on control plants, those feeding on SA-treated plants had fewer oocytes and slower ovary development. On SA-treated plants, viruliferous B had fewer oocytes than viruliferous Q. CONCLUSIONS/SIGNIFICANCE: These results indicate that TYLCV tends to induce SA-regulated plant defense against B but SA-regulated plant defense against Q was reduced. In other words, Q may have a mutualistic relationship with TYLCV that results in the reduction of the plant's defense response.

Reverse transcription quantitative polymerase chain reaction (qRT-PCR) has rapidly become the most sensitive and accurate method for the quantification of gene expression. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. These housekeeping genes need to show stable expression under the given experimental conditions for the qRT-PCR results to be accurate. Unfortunately, there are no studies on the stability of housekeeping genes used in Spodoptera litura. In this study, eight candidate reference genes, elongation factor 1 alpha (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein S3 (RPS3), beta actin (ACTB), beta FTZ-F1 (FTZF1), ubiquinol-cytochrome c reductase (UCCR), and arginine kinase (AK), were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs, BestKeeper, geNorm and Normfinder, and the comparative ΔCt method. We determined the expression levels of the candidate reference genes for three biotic factors (developmental stage, tissue and population), and four abiotic treatments (temperature, insecticide, food and starvation). The results indicated that the best sets of candidates as reference genes were as follows: GAPDH and UCCR for developmental stages; RPL10, AK and EF1 for different tissues; RPL10 and EF1 for different populations in China; GAPDH and EF1 for temperature-stressed larvae; AK and ACTB for larvae treated with different insecticides; RPL10, GAPDH and UCCR for larvae fed different diets; RPS3 and ACTB for starved larvae. We believe that these results make an important contribution to gene analysis studies in S. litura and form the basis of further research on stable reference genes in S. litura and other organisms.

Phenol is a major pollutant in aquatic ecosystems due to its chemical stability, water solubility and environmental mobility. To date, little is known about the molecular modifications of invertebrates under phenol stress. In the present study, we used Solexa sequencing technology to investigate the transcriptome and differentially expressed genes (DEGs) of midges (Chironomus kiinensis) in response to phenol stress. A total of 51,518,972 and 51,150,832 clean reads in the phenol-treated and control libraries, respectively, were obtained and assembled into 51,014 non-redundant (Nr) consensus sequences. A total of 6,032 unigenes were classified by Gene Ontology (GO), and 18,366 unigenes were categorized into 238 Kyoto Encyclopedia of Genes and Genomes (KEGG) categories. These genes included representatives from almost all functional categories. A total of 10,724 differentially expressed genes (P value

MicroRNAs (miRNAs) are a group of small RNAs involved in various biological processes through negative regulation of mRNAs at the post-transcriptional level. Although miRNA profiles have been documented in over two dozen insect species, few are agricultural pests. In this study, both conserved and novel miRNAs in the diamondback moth, Plutella xylostella L., a devastating insect pest of cruciferous crops worldwide, were documented. High-throughput sequencing of a small RNA library constructed from a mixed life stages of P. xylostella, including eggs, 1st to 4th (last) instar larvae, pupae and adults, identified 384 miRNAs, of which 174 were P. xylostella specific. In addition, temporal expressions of 234 miRNAs at various developmental stages were investigated using a customized microarray analysis. Among the 91 differentially expressed miRNAs, qRT-PCR analysis was used to validate highly expressed miRNAs at each stage. The combined results not only systematically document miRNA profiles in an agriculturally important insect pest, but also provide molecular targets for future functional analysis and, ultimately, genetic-based pest control practice.

Since the report of a paralogous acetylcholinesterase (AChE, EC3.1.1.7) gene in the greenbug (Schizaphis graminum) in 2002, two different AChE genes (Ace1 and Ace2) have been identified in each of at least 27 insect species. However, the gene models of Ace1 and Ace2, and their molecular properties have not yet been comprehensively analyzed in any insect species. In this study, we sequenced the full-length cDNAs, computationally predicted the corresponding three-dimensional protein models, and profiled developmental stage and tissue-specific expression patterns of two Ace genes from the red flour beetle (Tribolium castaneum; TcAce1 and TcAce2), a globally distributed major pest of stored grain products and an emerging model organism. TcAce1 and TcAce2 encode 648 and 604 amino acid residues, respectively, and have conserved motifs including a choline-binding site, a catalytic triad, and an acyl pocket. Phylogenetic analysis show that both TcAce genes are grouped into two insect Ace clusters and TcAce1 is completely diverged from TcAce2, suggesting that these two genes evolve from their corresponding Ace gene lineages in insect species. In addition, TcAce1 is located on chromosome 5, whereas TcAce2 is located on chromosome 2. Reverse transcription polymerase chain reaction (PCR) and quantitative real-time PCR analyses indicate that both genes are virtually transcribed in all the developmental stages and predominately expressed in the insect brain. Our computational analyses suggest that the TcAce1 protein is a robust acetylcholine (ACh) hydrolase and has susceptibility to sulfhydryl agents whereas the TcAce2 protein is not a catalytically efficient ACh hydrolase.