Regulation of GSTu1-mediated insecticide resistance in Plutella xylostella by miRNA and lncRNA.

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.

The gut symbiont Sphingomonas mediates imidacloprid resistance in the important agricultural insect pest Aphis gossypii Glover.

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.

Persistent toxicity and dissipation dynamics of afidopyropen against the green peach aphid Myzus persicae (Sulzer) in cabbage and chili.

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.

Cap 'n' Collar C and Aryl Hydrocarbon Receptor Nuclear Translocator Facilitate the Expression of Glutathione S-Transferases Conferring Adaptation to Tannic Acid and Quercetin in Micromelalopha troglodyta (Graeser) (Lepidoptera: Notodontidae).

Micromelalopha troglodyta (Graeser) (Lepidoptera: Notodontidae) is a notorious pest of poplar. Coevolution with poplars rich in plant secondary metabolites prompts M. troglodyta to expand effective detoxification mechanisms against toxic plant secondary metabolites. Although glutathione S-transferases (GSTs) play an important role in xenobiotic detoxification in M. troglodyta, it is unclear how GSTs act in response to toxic secondary metabolites in poplar. In this study, five GST gene core promoters were accurately identified by a 5' loss luciferase reporter assay, and the core promoters were significantly induced by two plant secondary metabolites in vitro. Two transcription factors, cap 'n' collar C (CncC) and aryl hydrocarbon receptor nuclear translocator (ARNT), were cloned in M. troglodyta. MtCncC and MtARNT clustered well with other insect CncCs and ARNTs, respectively. In addition, MtCncC and MtARNT could bind the MtGSTt1 promoter and strongly improve transcriptional activity, respectively. However, MtCncC and MtARNT had no regulatory function on the MtGSTz1 promoter. Our findings revealed the molecular mechanisms of the transcription factors MtCncC and MtARNT in regulating the GST genes of M. troglodyta. These results provide useful information for the control of M. troglodyta.

Silencing of CYP6AS160 in Solenopsis invicta Buren by RNA interference enhances worker susceptibility to fipronil.

Cytochrome P450 monooxygenases play a key role in pest resistance to insecticides by detoxification. Four new P450 genes, CYP6AS160, CYP6AS161, CYP4AB73 and CYP4G232 were identified from Solenopsis invicta. CYP6AS160 was highly expressed in the abdomen and its expression could be induced significantly with exposure to fipronil, whereas CYP4AB73 was not highly expressed in the abdomen and its expression could not be significantly induced following exposure to fipronil. Expression levels of CYP6AS160 and CYP4AB73 in workers were significantly higher than that in queens. RNA interference-mediated gene silencing by feeding on double-stranded RNA (dsRNA) found that the levels of this transcript decreased (by maximum to 64.6%) when they fed on CYP6AS160-specific dsRNA. Workers fed dsCYP6AS160 had significantly higher mortality after 24 h of exposure to fipronil compared to controls. Workers fed dsCYP6AS160 had reduced total P450 activity of microsomal preparations toward model substrates p-nitroanisole. However, the knockdown of a non-overexpressed P450 gene, CYP4AB73 did not lead to an increase of mortality or a decrease of total P450 activity. The knockdown effects of CYP6AS160 on worker susceptibility to fipronil, combined with our other findings, indicate that CYP6AS160 is responsible for detoxification of fipronil. Feeding insects dsRNA may be a general strategy to trigger RNA interference and may find applications in entomological research and in the control of insect pests in the field.

The influence of Bt cotton cultivation on the structure and functions of the soil bacterial community by soil metagenomics.

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.

Plant volatile compound methyl benzoate is highly effective against Spodoptera frugiperda and safe to non-target organisms as an eco-friendly botanical-insecticide.

Recent studies have indicated that the plant volatile methyl benzoate (MB) exhibits significant insecticidal bioactivity against several common insects. However, the potential environmental hazards of MB and its safety to non-target organisms is poorly understood. In the present study, these characteristics were investigated through laboratory experiments and field investigations. The results revealed that MB was highly toxic to the agricultural pest, fall armyworm Spodoptera frugiperda. Compared with the commercial pesticide lambda-cyhalothrin, the toxicities of MB against S. frugiperda larvae and adults were comparable and 3.41 times higher, respectively. Behavioral bioassays showed that the percentage repellency of MB to S. frugiperda larvae was 56.72 %, and MB induced 69.40 % oviposition deterrence rate in S. frugiperda female adults. Furthermore, in terms of median lethal concentration (LC50) and median lethal doses (LD50), MB exhibited non-toxic effects on non-target animals with 3-d LC50 of > 1 % to natural predators (Coccinella septempunctata and Harmonia axyridis), 3-d LD50 of 467.86 µg/bee to the bumblebee Bombus terrestris, 14-d LC50 of 971.09 mg/kg to the earthworm Eisenia fetida, and 4-d LC50 of 47.30 mg/L to the zebrafish Brachydanio rerio. The accumulation of MB in the soil and earthworms was found to be extremely limited. Our comparative study clearly demonstrated that MB is effective as a selective botanical pesticide against S. frugiperda and it is safe to use in the tested environment, with no toxic effects on non-target animals and natural predators.

Sublethal and transgenerational effects of afidopyropen on biological traits of the green peach aphid Myzus persicae (Sluzer).

The green peach aphid, Myzus persicae (Sulzer), is a cosmopolitan agricultural pest and causes great damages each year. Afidopyropen is a novel insecticide with high efficacy against even the insecticides resistant M. persicae. However, the sublethal and transgenerational effects of afidopyropen on M. persicae is not clear. In the present paper, sublethal and transgenerational effects of afidopyropen on biological traits of M. persicae were determined based on the age-stage, two-sex life table theory. The afidopyropen was more toxic against M. persicae than other widely used insecticides, with LC50 of 0.086 mg/L. The treatment with LC5, LC15 and LC25 concentrations of afidopyropen remarkably reduced the longevity and fecundity of F0M. persicae by 15.9-64.4% and 24.3-76.7%, respectively, compared with those of the control. The life history traits of F1 generation including the pre-adult development time, mean total longevity, pre-adult survival rate, total pre-oviposition period and fecundity were significantly affected after treatment of the F0 with afidopyropen, and the population parameters, including the net reproductive rate (R0), intrinsic rate of increase (r) and finite rate of increase (λ) were also remarkably decreased, while the mean generation time (T) was extended by 6.94%. Among four development and reproduction related genes investigated, JHEH was downregulated by 31.8-38.0% in the afidopyropen treated F0 generation, while the EcR and JHAMT were overexpressed and the Vg was significantly downregulated in F1 generation compared to the control group. All these data indicated that the afidopyropen had significant sublethal and transgenerational effects on M. persicae. These results provide insights into comprehensively understanding of the insecticidal effects of afidopyropen on M. persicae as well as the management of resistant M. persicae.

Integration analysis of PacBio SMRT- and Illumina RNA-seq reveals P450 genes involved in thiamethoxam detoxification in Bradysia odoriphaga.

The sciarid fly Bradysia odoriphaga is a serious pest of Chinese chive (Liliaceae). Neonicotinoid insecticides including thiamethoxam have been used for B. odoriphaga control. However, thiamethoxam resistance in B. odoriphaga has developed in recent years. To identify potential genes involved in detoxification metabolism of thiamethoxam in B. odoriphaga, a PacBio single-molecule real-time (SMRT) transcriptome sequencing and Illumina RNA-seq analysis on thiamethoxam treated B. odoriphaga were performed to explore differentially expressed genes in B. odoriphaga. After SMRT sequencing, analysis of Illumina RNA-Seq data showed a total of 172 differentially expressed genes (DEGs) after thiamethoxam treatment, among which eight upregulated DEGs were P450 genes that may be related to thiamethoxam metabolism. The qRT-PCR results of the eight up-regulated P450 unigenes after thiamethoxam treatment were consistent with RNA-Seq data. Furthermore, oral delivery mediated RNA interference of the eight upregulated P450 transcripts followed by insecticide bioassay was conducted, and three P450 unigenes were verified to be related to thiamethoxam detoxification in B. odoriphaga. This study provides new information about the P450 genes involved in thiamethoxam detoxification in B. odoriphaga.

Chemosensory proteins confer adaptation to the ryanoid anthranilic diamide insecticide cyantraniliprole in Aphis gossypii glover.

Chemosensory proteins (CSPs) are a class of small transporter proteins expressed only in arthropods with various functions beyond chemoreception. Previous studies have been reported that CSPs are involved in the insecticide resistance. In this study, we found that AgoCSP1, AgoCSP4, and AgoCSP5 were constitutively overexpressed in an insecticide-resistant strain of Aphis gossypii and showed higher expression in broad body tissue (including fat bodies) than in the midgut but without tissue specificity. However, the function of these three upregulated AgoCSPs remains unknown. Here, we investigated the function of AgoCSPs in resistance to the diamide insecticide cyantraniliprole. Suppression of AgoCSP1, AgoCSP4 and AgoCSP5 transcription by RNAi significantly increased the sensitivity of resistant aphids to cyantraniliprole. Molecular docking and competitive binding assays indicated that these AgoCSPs bind moderate with cyantraniliprole. Transgenic Drosophila melanogaster expressing these AgoCSPs in the broad body or midgut showed higher tolerance to cyantraniliprole than control flies with the same genetic background; AgoCSP4 was more effective in broad body tissue, and AgoCSP1 and AgoCSP5 were more effective in the midgut, indicating that broad body and midgut tissues may be involved in the insecticide resistance mediated by the AgoCSPs examined. The present results strongly indicate that AgoCSPs participate in xenobiotic detoxification by sequestering and masking toxic insecticide molecules, providing insights into new factors involved in resistance development in A. gossypii.

Functional analysis of cyantraniliprole tolerance ability mediated by ATP-binding cassette transporters in Aphis gossypii glover.

Cyantraniliprole, a second-generation anthranilic diamide insecticide, is widely used to control chewing and sucking pests. ATP-binding cassette transporters (ABCs) are a ubiquitous family of membrane proteins that play important roles in insect detoxification mechanisms. However, the potential effects of ABCs on cyantraniliprole-resistance remain unclear. In the present study, synergism bioassays revealed that verapamil, an ABC inhibitor, increased the toxicity of cyantraniliprole by 2.00- and 12.25-fold in the susceptible and cyantraniliprole-resistant strains of Aphis gossypii. Based on transcriptome data, the expression levels of ABCB4, ABCB5, ABCD1, ABCG4, ABCG7, ABCG13, ABCG16, ABCG17, ABCG26 and MRP12 were upregulated 1.56-, 1.32-, 1.51-, 2.03-, 1.65-, 1.50-, 4.18-, 6.07-, 4.68- and 4.69-fold, respectively, in the cyantraniliprole-resistant strain (CyR) compared to the susceptible strain (SS), as determined using RT-qPCR. Drosophila melanogaster ectopically overexpressing ABCB5, ABCG4, ABCG7, ABCG16, ABCG17, ABCG26 and MRP12 exhibited significantly increased tolerance to cyantraniliprole by 11.71-, 2.39-, 4.85-, 2.06-, 3.75-, 4.20- and 3.50-fold, respectively, with ABCB5 and ABCG family members being the most effective. Furthermore, the suppression of ABCB5, ABCG4, ABCG7, ABCG16, ABCG17, ABCG26 and MRP12 significantly increased the sensitivity of the CyR strain to cyantraniliprole. These results indicate that ABCs may play crucial roles in cyantraniliprole resistance and may provide information for shaping resistance management strategies.

Functional characterization of ABC transporters mediates multiple neonicotinoid resistance in a field population of Aphis gossypii Glover.

The ATP-binding cassette (ABC) transporters C and G subfamilies have been reported to be involved in insecticide detoxification, with most studies showing increased gene transcript levels in response to insecticide exposure. Our previous studies have suggested that ABCC and G transporters participate in cyantraniliprole and thiamethoxam resistance of Aphis gossypii. In this study, we focused on the potential roles of the ABCC and G transporters of an A. gossypii field population (SDR) in neonicotinoid detoxification. The results of leaf dip bioassays showed 629.17- and 346.82-fold greater resistance to thiamethoxam and imidacloprid in the SDR strain, respectively, than in the susceptible strain (SS). Verapamil, an ABC inhibitor, was used for synergism bioassays, and the results showed synergistic effects with thiamethoxam, with synergistic ratios (SRs) of 2.07 and 6.68 in the SS and SDR strains, respectively. In addition to thiamethoxam, verapamil increased imidacloprid toxicity by 1.68- and 1.62-fold in the SS and SDR strains respectively. Then, the expression levels of several ABCC and G transporters were analyzed in different treatments. We found that the transcript levels of AgABCG4, AgABCG17, AgABCG26, AgMRP8 and AgMRP12 were higher in the SDR strain than in the SS strain. The mRNA expression of AgABCG4, AgABCG7, AgABCG13, AgABCG17, AgABCG26, AgMRP8 and AgMRP12 in the SDR strain was increased after thiamethoxam and imidacloprid exposure. The results of transgenic Drosophila melanogaster bioassays suggested that overexpression of AgABCG4, AgABCG7, AgABCG13, AgABCG17, AgABCG26, AgMRP8 and AgMRP12 in transgenic flies was sufficient to confer thiamethoxam and imidacloprid resistance, and AgABCG4, AgABCG7, AgABCG13, AgABCG26 and AgMRP12 may be related to α-cypermethrin cross-resistance with weak effects. In addition, the knockdown of AgABCG4, AgABCG13, AgABCG26, AgMRP8 and AgMRP12, and the knockdown of AgABCG7 and AgABCG26 increased thiamethoxam and imidacloprid mortality in the SDR strain, respectively. Our results suggest that changes in the expression levels of ABCC and G transporters may contribute to neonicotinoid detoxification in the SDR strain, and provide a foundation for clarify the potential roles of ABCC and G transporters in insecticide resistance.

MicroRNA-263b confers imidacloprid resistance in Sitobion miscanthi (Takahashi) by regulating the expression of the nAChRß1 subunit.

The Chinese wheat aphid Sitobion miscanthi (CWA) is an important harmful pest in wheat fields. Imidacloprid plays a critical role in controlling pests with sucking mouthparts. However, imidacloprid-resistant pests have been observed after insecticide overuse. Point mutations and low expression levels of the nicotinic acetylcholine receptor ß1 (nAchRß1) subunit are the main imidacloprid-resistant mechanisms. However, the regulatory mechanism underlying nAChRß1 subunit expression is poorly understood. In this study, a target of miR-263b was isolated from the 5'UTR of the nAchRß1 subunit in the CWA. Low expression levels were found in the imidacloprid-resistant strain CWA. Luciferase reporter assays showed that miR-263b could combine with the 5'UTR of the nAChRß1 subunit and suppress its expression by binding to a site in the CWA. Aphids treated with the miR-263b agomir exhibited a significantly reduced abundance of the nAchRß1 subunit and increased imidacloprid resistance. In contrast, aphids treated with the miR-263b antagomir exhibited significantly increased nAchRß1 subunit abundance and decreased imidacloprid resistance. These results provide a basis for an improved understanding of the posttranscriptional regulatory mechanism of the nAChRß1 subunit and further elucidate the function of miRNAs in regulating susceptibility to imidacloprid in the CWA. These results provide a better understanding of the mechanisms of posttranscriptional regulation of nAChRß1 and will be helpful for further studies on the role of miRNAs in the regulation of nAChRß1 subunit resistance in homopteran pests.

A General Signal Pathway to Regulate Multiple Detoxification Genes Drives the Evolution of Helicoverpa armigera Adaptation to Xenobiotics.

The study of insect adaptation to the defensive metabolites of host plants and various kinds of insecticides in order to acquire resistance is a hot topic in the pest-control field, but the mechanism is still unclear. In our study, we found that a general signal pathway exists in H. armigera which can regulate multiple P450s, GSTs and UGTs genes to help insects decrease their susceptibility to xenobiotics. Knockdown of HaNrf2 and HaAhR expression could significantly increase the toxicity of xenobiotics to H. armigera, and simultaneously decrease the gene expression of P450s, GSTs and UGTs which are related to the xenobiotic metabolism and synthesis of insect hormone pathways. Then, we used EMSA and dual luciferase assay to verify that a crosstalk exists between AhR and Nrf2 to regulate multiple P450s, GSTs and UGTs genes to mediate H. armigera susceptibility to plant allelochemicals and insecticides. The detoxification genes' expression network which can be regulated by Nrf2 and AhR is still unknown, and there were also no reports about the crosstalk between AhR and Nrf2 that exist in insects and can regulate multiple detoxification genes' expression. Our results provide a new general signaling pathway to reveal the adaptive mechanism of insects to xenobiotics and provides further insight into designing effective pest-management strategies to avoid the overuse of insecticides.

Chemosensory Proteins Are Associated with Thiamethoxam and Spirotetramat Tolerance in Aphis gossypii Glover.

Chemosensory proteins (CSPs) are a class of transporters in arthropods. Deeper research on CSPs showed that CSPs may be involved in some physiological processes beyond chemoreception, such as insect resistance to pesticides. We identified two upregulated CSPs in two resistant strains of Aphis gossypii Glover. To understand their role in the resistance of aphids to pesticides, we performed the functional verification of CSP1 and CSP4 in vivo and in vitro. Results showed that the sensitivity of the thiamethoxam-resistant strain to thiamethoxam increased significantly with the silencing of CSP1 and CSP4 by RNAi (RNA interference), and the sensitivity of the spirotetramat-resistant strain to spirotetramat increased significantly with the silencing of CSP4. Transgenic Drosophila melanogaster expressing CSPs exhibited stronger resistance to thiamethoxam, spirotetramat, and alpha-cypermethrin than the control did. In the bioassay of transgenic Drosophila, CSPs showed different tolerance mechanisms for different pesticides, and the overexpressed CSPs may play a role in processes other than resistance to pesticides. In brief, the present results prove that CSPs are related to the resistance of cotton aphids to insecticides.

Sublethal and lethal effects of the imidacloprid on the metabolic characteristics based on high-throughput non-targeted metabolomics in Aphis gossypii Glover.

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.

Sublethal concentrations of clothianidin affect fecundity and key demographic parameters of the chive maggot, Bradysia odoriphaga.

Bradysia odoriphaga is a major insect pest that infests Chinese chive in northern China. Clothianidin is a second-generation neonicotinoid insecticide that is commonly used against B. odoriphaga. In this study, the effect of sublethal clothianidin concentrations (LC5 and LC10) on key biological characteristics of B. odoriphaga was investigated using an age-stage, two-sex life table method. Bioassays results showed that clothianidin exhibited high toxicity against B. odoriphaga with LC50 of 1.898 mg L-1 following 24 h exposure. The developmental duration of larvae was significantly increased when exposed to the LC5 (0.209 mg L-1) and LC10 (0.340 mg L-1) of clothianidin. No significant effects were observed on the pupal stage, adult pre-oviposition period (APOP), total pre-oviposition period (TPOP), and mean longevities of male and female. The oviposition period and fecundity of B. odoriphaga were reduced in clothianidin-treated groups. Moreover, key demographic parameters, including the intrinsic rate of increase (r), finite rate of increase (λ), and net reproductive rate (R0), were significantly decreased by the LC5 and LC10 of clothianidin, while no effects were noted on mean generation time (T). Overall, this study showed that sublethal concentrations of clothianidin have a detrimental effect on B. odoriphaga developmental period, fecundity, and life table parameters. Therefore, clothianidin has the potential to suppress the population of B. odoriphaga even at sublethal concentrations.

Acetamiprid resistance and fitness costs of melon aphid, Aphis gossypii: An age-stage, two-sex life table study.

The melon aphid, Aphis gossypii is a globally distributed crop pest with a wide host range. The intensive use of insecticides against this insect over several years has led to develop resistance against many insecticides including acetamiprid. Understanding the relationship between acetamiprid resistance and fitness of A. gossypii is essential to limit the spread of the resistant population in the field. In this study, age-stage, two-sex life table approach was used to investigate these relationships in the lab. Results showed that resistant strain (Ace-R) had a reduced fitness (relative fitness = 0.909) along with significantly decreased adult longevity, fecundity, net reproductive (R0), mean generation time (T) and gross reproductive rate (GRR). Compared to the susceptible strain (Ace-S), the pre-adult period and total pre-oviposition period (TPOP) were also significantly shorter in Ace-R strain. Moreover, the expression profiles of EcR, JHBP, JHAMT, JHEH, USP and Vg genes supposed to be involved in insect reproduction and development were analyzed using Quantitative Real Time PCR. The EcR, JHBP, JHAMT and USP genes were up-regulated, Vg gene was down-regulated while the mRNA level of JHEH gene was statistically same in the Ace-R strain compared to the Ace-S strain. Collectively, this study provides the occurrence and magnitude of fitness costs of A. gossypii against acetamiprid resistance and could be helpful to manage the resistance evolution in field populations.

Characterization and functional analysis of two acetylcholinesterase genes in Bradysia odoriphaga Yang et Zhang (Diptera: Sciaridae).

Two acetylcholinesterase genes (Boace1 and Boace2) were cloned from Bradysia odoriphaga, a devastating soil pest that mainly damages Chinese chives. The Boace1 encodes BoAChE1 protein consisting of 696 amino acid residues, while Boace2 encodes BoAChE2 containing 638 amino acids. Phylogenetic analysis showed that Boace1 and Boace2 are appeared to be distinct clusters. The gene expression patterns at different development stages and various body parts tissues were examined, and their biological functions were characterized by RNA interference and analog docking prediction. The results showed that both Boace genes were expressed in all developmental stages and examined tissues. The transcript level of Boace2 was significantly higher than Boace1 in all tested samples, and Boace1 was found most abundant in the head while Boace2 was highly expressed in the fat body of B. odoriphaga. The silencing of Boace1 and Boace2 significantly decreased the AChE activity of 36.6% and 14.8% separately, and increased the susceptibility of B. odoriphaga to phoxim, with 60.8% and 44.7% mortality. Besides, overexpression and gene duplication of Boace1 were found in two field resistant populations, and two major mutations, A319S and G400V, were detected in Boace1. Moreover, the docking results revealed that BoAChE1 had a higher affinity towards organophosphorus than BoAChE2. It is concluded that Boace2 is the most abundant ace type in B. odoriphaga, while both Boace play vital roles. Boace1 might play a major neurological function and more likely be the prime target for insecticides, while Boace2 might play some important unidentified roles.

Functional validation of key cytochrome P450 monooxygenase and UDP-glycosyltransferase genes conferring cyantraniliprole resistance in Aphis gossypii Glover.

Cytochrome P450 monooxygenases (P450s) and UDP-glycosyltransferases (UGTs) are major detoxifying enzymes that metabolize plant toxins and insecticides. In the present study, the synergists of piperonyl butoxide, sulfinpyrazone and 5-nitrouracil significantly increased cyantraniliprole and α-cypermethrin toxicity against the resistant strain. The transcripts of UGT341A4, UGT344B4, UGT344D6, UGT344J2 and UGT344M2 increased significantly in the CyR strain compared with the susceptible strain. Among these upregulated genes (including P450s), CYP6CY7 and UGT344B4 were highly expressed in the midgut. Transgenic expression of the P450 and UGT genes in broad body tissues in Drosophila melanogaster indicated that the expression of CYP380C6, CYP4CJ1, UGT341A4, UGT344B4 and UGT344M2 is sufficient to confer cyantraniliprole resistance, and CYP380C6, CYP6CY7, CYP6CY21, UGT341A4 and UGT344M2 are related to α-cypermethrin cross-resistance. The midgut-specific overexpression of CYP380C6, CYP6CY7, CYP6CY21, CYP4CJ1, UGT341A4, UGT344B4 and UGT344M2 significantly increased insensitivity to cyantraniliprole, and CYP380C6, CYP6CY7, CYP6CY21, UGT344B4 and UGT344M2 confer α-cypermethrin cross-resistance. The expression of CYP380C6, CYP4CJ1, UGT341A4 and UGT344M2 in broad tissues or in midgut has similar effects on insensitivity to insecticides; however, CYP6CY7, CYP6CY21 and UGT344B4 are more effective in the midgut. This result indicates that broad body tissues and midgut tissue are involved in insecticide resistance mediated by the candidate P450s and UGTs examined.