Brown planthopper infestation on rice reduces plant susceptibility to Meloidogyne graminicola by reducing root sugar allocation.

Plants are simultaneously attacked by different pests that rely on sugars uptake from plants. An understanding of the role of plant sugar allocation in these multipartite interactions is limited. Here, we characterized the expression patterns of sucrose transporter genes and evaluated the impact of targeted transporter gene mutants and brown planthopper (BPH) phloem-feeding and oviposition on root sugar allocation and BPH-reduced rice susceptibility to Meloidogyne graminicola. We found that the sugar transporter genes OsSUT1 and OsSUT2 are induced at BPH oviposition sites. OsSUT2 mutants showed a higher resistance to gravid BPH than to nymph BPH, and this was correlated with callose deposition, as reflected in a different effect on M. graminicola infection. BPH phloem-feeding caused inhibition of callose deposition that was counteracted by BPH oviposition. Meanwhile, this pivotal role of sugar allocation in BPH-reduced rice susceptibility to M. graminicola was validated on rice cultivar RHT harbouring BPH resistance genes Bph3 and Bph17. In conclusion, we demonstrated that rice susceptibility to M. graminicola is regulated by BPH phloem-feeding and oviposition on rice through differences in plant sugar allocation.

Investigation of lambda-cyhalothrin resistance in Spodoptera frugiperda: Heritability, cross-resistance, and mechanisms.

Lambda-cyhalothrin, a representative pyrethroid insecticide widely used for Spodoptera frugiperda control in China, poses challenges due to the development of resistance. This study investigates the realized heritability, inheritance pattern, cross-resistance, and resistance mechanisms to lambda-cyhalothrin. After 21 generations of selection, the lambda-cyhalothrin-resistant strain (G21) developed a 171.11-fold resistance compared to a relatively susceptible strain (RS-G9), with a realized heritability (h2) of 0.11. Cross-resistance assays revealed that lambda-cyhalothrin-resistant strains showed no significant cross-resistance to the majority of tested insecticides. Genetic analysis indicated that lambda-cyhalothrin resistance in S. frugiperda was autosomal, incompletely dominant, and polygenic inheritance. The P450 enzyme inhibitor PBO significantly enhanced lambda-cyhalothrin toxicity in the resistant strains. Compared with the RS-G9 strain, the P450 enzyme activity was significantly increased and multiple P450 genes were significantly up-regulated in the lambda-cyhalothrin-resistant strains. RNAi targeting the most overexpressed P450 genes (CYP337B5 and CYP321B1) significantly increased the susceptibility of resistant S. frugiperda larvae to lambda-cyhalothrin. This study provides comprehensive insights into lambda-cyhalothrin resistance in S. frugiperda, and the results are helpful for developing effective resistance management strategies of this pest.

The microRNA-7322-5p/p38/Hsp19 axis modulates Chilo suppressalis cell-defences against Cry1Ca: an effective target for a stacked transgenic rice approach.

Bacillus thuringiensis (Bt)-secreted crystal (Cry) toxins form oligomeric pores in host cell membranes and are a common element in generating insect-resistant transgenic crops. Although Cry toxin function has been well documented, cellular defences against pore-formation have not been as well developed. Elucidation of the processes underlying this defence, however, could contribute to the development of enhanced Bt crops. Here, we demonstrate that Cry1Ca-mediated downregulation of microRNA-7322-5p (miR-7322-5p), which binds to the 3' untranslated region of p38, negatively regulates the susceptibility of Chilo suppressalis to Cry1Ca. Moreover, Cry1Ca exposure enhanced phosphorylation of Hsp19, and hsp19 downregulation increased susceptibility to Cry1Ca. Further, Hsp19 phosphorylation occurs downstream of p38, and pull-down assays confirmed the interactions between Hsp19 and Cry1Ca, suggesting that activation of Hsp19 by the miR-7322-5p/p38/Hsp19 pathway promotes Cry1Ca sequestration. To assess the efficacy of targeting this pathway in planta, double-stranded RNA (dsRNA) targeting C. suppressalis p38 (dsp38) was introduced into a previously generated cry1Ca-expressing rice line (1CH1-2) to yield a single-copy cry1Ca/dsp38 rice line (p38-rice). Feeding on this rice line triggered a significant reduction in C. suppressalis p38 expression and the line was more resistant to C. suppressalis than 1CH1-2 in both short term (7-day) and continuous feeding bioassays as well as field trials. These findings provide new insights into invertebrate epithelium cellular defences and demonstrate a potential new pyramiding strategy for Bt crops.

Sublethal effects of acetamiprid and afidopyropen on Harmonia axyridis: insights from transcriptomics analysis.

Evaluating the sublethal effects of insecticide is crucial for protecting and utilizing natural enemies. In this study, we determined the sublethal effects of acetamiprid and afidopyropen on Harmonia axyridis (Pallas) and explored the potential molecular mechanisms underlying these effects through transcriptomics analysis. The results showed that sublethal concentrations of acetamiprid significantly reduced the adult fecundity and longevity of F0H. axyridis and decreased the survival time and survival rate of the F1 generation. Sublethal concentrations of afidopyropen prolonged the developmental time of 4th instar larvae in the F0 generation. Additionally, acetamiprid and afidopyropen treatments significantly decreased the predation of H. axyridis. Furthermore, transcriptome sequencing analysis revealed that several P450 and UGT genes expressed differently when H. axyridis were exposed to sublethal concentrations of acetamiprid and afidopyropen, suggesting that the differential expression of detoxifying genes might be involved in the response and detoxification metabolism of acetamiprid and afidopyropen in H. axyridis. Our findings demonstrate that sublethal concentrations of acetamiprid adversely influences the development and predation of H. axyridis, while afidopyropen has limited effects on H. axyridis. These results are helpful for protecting and utilizing natural enemies and guiding the scientific use of pesticides in the field.

Unintended consequences: Disrupting microbial communities of Nilaparvata lugens with non-target pesticides.

Insects are frequently exposed to a range of insecticides that can alter the structure of the commensal microbiome. However, the effects of exposure to non-target pesticides (including non-target insecticides and fungicides) on insect pest microbiomes are still unclear. In the present study, we exposed Nilaparvata lugens to three target insecticides (nitenpyram, pymetrozine, and avermectin), a non-target insecticide (chlorantraniliprole), and two fungicides (propiconazole and tebuconazole), and observed changes in the microbiome's structure and function. Our results showed that both non-target insecticide and fungicides can disrupt the microbiome's structure. Specifically, symbiotic bacteria of N. lugens were more sensitive to non-target insecticide compared to target insecticide, while the symbiotic fungi were more sensitive to fungicides. We also found that the microbiome in the field strain was more stable under pesticides exposure than the laboratory strain (a susceptible strain), and core microbial species g_Pseudomonas, s_Acinetobacter soli, g_Lactobacillus, s_Metarhizium minus, and s_Penicillium citrinum were significantly affected by specifically pesticides. Furthermore, the functions of symbiotic bacteria in nutrient synthesis were predicted to be significantly reduced by non-target insecticide. Our findings contribute to a better understanding of the impact of non-target pesticides on insect microbial communities and highlight the need for scientific and rational use of pesticides.

The jasmonate-induced bHLH gene SlJIG functions in terpene biosynthesis and resistance to insects and fungus.

Jasmonic acid (JA) is a key regulator of plant defense responses. Although the transcription factor MYC2, the master regulator of the JA signaling pathway, orchestrates a hierarchical transcriptional cascade that regulates the JA responses, only a few transcriptional regulators involved in this cascade have been described. Here, we identified the basic helix-loop-helix (bHLH) transcription factor gene in tomato (Solanum lycopersicum), METHYL JASMONATE (MeJA)-INDUCED GENE (SlJIG), the expression of which was strongly induced by MeJA treatment. Genetic and molecular biology experiments revealed that SlJIG is a direct target of MYC2. SlJIG knockout plants generated by gene editing had lower terpene contents than the wild type from the lower expression of TERPENE SYNTHASE (TPS) genes, rendering them more appealing to cotton bollworm (Helicoverpa armigera). Moreover, SlJIG knockouts exhibited weaker JA-mediated induction of TPSs, suggesting that SlJIG may participate in JA-induced terpene biosynthesis. Knocking out SlJIG also resulted in attenuated expression of JA-responsive defense genes, which may contribute to the observed lower resistance to cotton bollworm and to the fungus Botrytis cinerea. We conclude that SlJIG is a direct target of MYC2, forms a MYC2-SlJIG module, and functions in terpene biosynthesis and resistance against cotton bollworm and B. cinerea.

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.

Fitness costs of sulfoxaflor resistance in the cotton aphid, Aphis gossypii Glover.

Aphis gossypii Glover is an economically important pest of numerous crops throughout the world. Some field populations of A.gossypii in China have developed moderate level of resistance to sulfoxaflor, a newly released sulfoximine insecticide for management of sap-feeding pests. To evaluate the effect of sulfoxaflor resistance on the fitness cost of A. gossypii, the life history traits of sulfoxaflor-resistant strain (SulR) and an isogenic susceptible strain (SS) were compared using the age-stage, two-sex life table approach. The results showed that the resistant strain had a reduction in fitness (relative fitness = 0.917), along with significantly decreases in longevity, fecundity, net reproductive (R0), mean generation time (T) and gross reproductive rate (GRR). Compared to the susceptible strain, SulR strain showing a shorter developmental duration of each nymph instar stage. Moreover, the adult pre-oviposition period (APOP) and total preoviposition period (TPOP) of SulR strain were also significantly shorter than that of the susceptible strain. Investigation of six development and reproduction related genes indicated that EcR, USP and JHBP were overexpressed in the SulR strain, while the mRNA transcript level of Vg was decreased significantly compared to the susceptible strain. These results suggest that there is a fitness cost associated with sulfoxaflor resistance in A. gossypii and the different expression of EcR, USP, JHBP, and Vg may play very important role in this trade-off.

Overexpression of multiple cytochrome P450 genes associated with sulfoxaflor resistance in Aphis gossypii Glover.

Sulfoxaflor is the first commercially available sulfoximine insecticide, which exhibits highly efficacy against many sap-feeding insect pests and has been applied as an alternative insecticide against cotton aphid in China. This study was conducted to investigate the risk of resistance development, the cross-resistance pattern and the potential resistance mechanisms of sulfoxaflor in Aphis gossypii. A colony (SulR strain) of A. gossypii with 245-fold resistance, originated from Xinjiang field population, was established by continuous selection using sulfoxaflor. The SulR strain has developed cross-resistance to imidacloprid (80.8-fold), acetamiprid (19.3-fold), thiamethoxam (10.0-fold), and flupyradifurone (107.5-fold), while no cross-resistance was detected to malathion, omethoate, bifenthrin, methomyl, and carbosulfan. Piperonyl butoxide and S, S, S-tributyl phosphorotrithioate could significantly increase the toxicity of sulfoxaflor to the SulR strain by 5.99- and 4.18-fold, respectively, whereas no synergistic effect with diethyl maleate was observed. The activities of P450s and carboxylesterase were significantly higher in the SulR strain than that in the SS strain. Further gene expression determination demonstrated that nine P450 genes were significantly increased in SulR strain and suppression the expression of CYP6CY13 and CYP6CY19 by RNAi significantly increased the susceptibility of SulR adult aphids to sulfoxaflor. These results demonstrated that the enhancing detoxification by cytochrome P450 monooxygenase may be involved in A.gossypii resistance to sulfoxaflor.

Identification of microRNAs and their response to the stress of plant allelochemicals in Aphis gossypii (Hemiptera: Aphididae).

BACKGROUND: MicroRNAs (miRNAs) are a group of short non-coding RNAs involved in the inhibition of protein translation or in mRNA degradation. Although the regulatory roles of miRNAs in various biological processes have been investigated, there is as yet an absence of studies about the regulatory roles of miRNAs involved in the metabolism of plant allelochemicals in insects. RESULTS: We constructed five small RNA libraries from apterous Aphis gossypii adults that had fed on an artificial diet containing various allelochemicals. Using Illumina sequencing, a total of 73.27 million clean reads was obtained, and 292 miRNAs were identified from A. gossypii. Comparative analysis of read counts indicated that both conserved and novel miRNAs were differently expressed among the five libraries, and the differential expression was validated via qRT-PCR. We found that the transcript levels of several miRNAs were increased or decreased in all of the allelochemical treatment libraries compared to the control. The putative target genes of the miRNAs were predicted using in silico tools, and the target genes of several miRNAs were presumed to be involved in the metabolism of xenobiotic compounds. Furthermore, the target prediction results were confirmed using dual luciferase reporter assay, and Ago-miR-656a-3p was demonstrated to regulate the expression of CYP6J1 post-transcriptionally through binding to the 3' UTR of CYP6J1. CONCLUSION: Our research results indicate that miRNAs may be involved in the metabolism of plant allelochemicals in A. gossypii, and these results also represent an important new small RNA genomics resource for further studies on this topic.

Monitoring insecticide resistance and diagnostics of resistance mechanisms in the green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae) in China.

Myzus persicae (Sulzer) is one of the most serious agricultural pests in China, and management strategies mainly rely on insecticidal treatment. To evaluate the resistance of field populations of M. persicae to seven insecticides, we assessed the susceptibility of 11 field populations collected from eight provinces in China using leaf-dip bioassays. Toxicity assays showed that M. persicae field populations have developed several levels of resistance to each tested insecticide. For pyrethroids, the field populations have developed a high level of resistance to ß-cypermethrin and cypermethrin, while the resistance to bifenthrin is still low. The resistance ratios of field populations to imidacloprid ranged from 1.48 to 52.36, and eight populations have developed moderate to high resistance. Resistance to acetamiprid is low, and only two populations have a moderate level of resistance. Most of the field populations of M. persicae developed moderate to high resistance to methomyl and omethoate. To investigate potential resistance mechanisms, we analyzed the enzyme activity of carboxylesterases, the type of amplified esterase genes, as well as the kdr (L1014F) mutation. All of the field populations exhibited a higher esterase activity compared to the laboratory susceptible strain. An amplified FE4, as well as the L1014F mutation, were also found in all of our experimental field populations. These results provide valuable insight into the current status of insecticide resistance and will prove to be a valuable resource in designing appropriate resistance management strategies for M. persicae in China.

RNA interference of Dicer-1 and Argonaute-1 increasing the sensitivity of Aphis gossypii Glover (Hemiptera: Aphididae) to plant allelochemical.

Plant allelochemicals are a group of important defensive agents of plants, which have been documented to be deleterious to insect herbivores. In the present study, we found that the expression level of Dicer-1 was significantly increased when Aphis gossypii adults fed on plant allelochemicals (tannic acid and gossypol) incorporated artificial diets. Consider that miRNAs play great regulatory roles in various biological processes, this suggested that miRNAs may be related to the regulation of enzymes involved in metabolism of plant allelochemicals in A. gossypii. To further reveal the roles of miRNAs in the response of A. gossypii against plant allelochemicals, both Dicer-1 and Argonaute-1, an important component of the RNA-induced silencing complex (RISC) in miRNA pathway, were silenced using systemic RNA interference (RNAi). The results indicated that silence of Dicer-1 reduced the expression of miRNAs, and resulted in a high mortality of A. gossypii when fed on both tannic acid and gossypol. The silencing of Argonaute-1 resulted in the mortality of A. gossypii by the treatment of tannic acid significantly increased compared with control, however, the sensitivity of A. gossypii to gossypol was not significantly changed. It suggested that miRNAs play potential regulatory roles in the response of A. gossypii to plant allelochemicals. These results should be useful to understand the molecular mechanisms of the cotton aphid adaption to plant allelochemicals.

Both point mutations and low expression levels of the nicotinic acetylcholine receptor ß1 subunit are associated with imidacloprid resistance in an Aphis gossypii (Glover) population from a Bt cotton field in China.

Aphis gossypii Glover is a destructive pest of numerous crops throughout the world. Although the expansion of Bt cotton cultivation has helped to control some insect pests, the damage from cotton aphids has not been mitigated. The evolution of aphid resistance to imidacloprid has made its chemical control more difficult since its introduction in 1991. Field populations of A. gossypii that were collected from different transgenic (Bt) cotton planting areas of China in 2014 developed different levels of resistance to imidacloprid. The IMI_R strain has developed high resistance to imidacloprid with the resistance ratio >1200-fold. Compared with the susceptible IMI_S strain, the IMI_R strain also developed a high level cross resistance to sulfoxaflor and acetamiprid. The limited synergism with either PBO or DEF suggests that resistance may be due to the site mutation of molecular target rather than to enhanced detoxification. Three target-site mutations within the nicotinic acetylcholine receptor (nAChR) ß1 subunit were detected in the IMI_R strain. The R81T mutation has been reported to be responsible for imidacloprid resistance in A. gossypii and M. persicae. Both V62I and K264E were first detected in A. gossypii. These point mutations are also present in field populations, suggesting that they play a role in the resistance to imidacloprid. Furthermore, the expression level of transcripts encoding ß1 subunit was decreased significantly in the IMI_R strain compared with the IMI_S strain, suggesting that both point mutations and the down-regulation of nAChR ß1 subunit expression may be involved in the resistance mechanism for imidacloprid in A. gossypii. These results should be useful for the management of imidacloprid-resistant cotton aphids in Bt cotton fields in China.

Sublethal and transgenerational effects of sulfoxaflor on the biological traits of the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae).

Sulfoxaflor is a novel insecticide belonging to sulfoximine chemical class that can be used to control sap-feeding insects, notably Aphis gossypii Glover. In addition to its acute toxicity, it is also important to consider the possible sublethal effects when establishing a comprehensive understanding of the toxicity of a new insecticide. We assessed the effects of a low lethal concentration (LC25) of sulfoxaflor on biological parameters of A. gossypii adults (F0) and subsequent transgenerational effects, i.e., on the progeny (F1 generation). The data were analyzed using an age-stage life table procedure. The results showed that the longevity and fecundity were not significantly affected by the LC25 of sulfoxaflor in the F0 or F1 generations. In addition, no significant differences were observed on the developmental time of each instar, the adult pre-oviposition period, and on the longevity of F1 individuals. However, the duration of their pre-adult stage and total pre-oviposition period, as well as their mean generation time were significantly increased. These observed effects affected aphid demographic traits; the survival rate, the intrinsic rate of increase (r i ), the finite rate of increase (λ), the net reproductive rate (R0), and the gross reproduction rate (GRR) of the F1 individuals (i.e., from F0 mothers) were significantly lower compared to the control. Our results showed that sublethal effects of sulfoxaflor significantly slowed down A. gossypii population growth; they indicated that effects of sulfoxaflor might be increased (beyond lethal effect) through sublethal effects when concentrations decreased in sulfoxaflor-treated areas after initial application in field.

Identification and Validation of Reference Genes for the Normalization of Gene Expression Data in qRT-PCR Analysis in Aphis gossypii (Hemiptera: Aphididae).

To obtain accurate and reliable results from quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis, it is necessary to select suitable reference genes as standards for normalizing target gene expression data. QRT-PCR is a popular analytical methodology for studying gene expression and it has been used widely in studies of Aphis gossypii Glover in recent years. However, there is absence of study on the stability of the expression of reference genes in A. gossypii. In this study, eight commonly used candidate reference genes, including 18S, 28S, ß-ACT, GAPDH, EF1α, RPL7, α-TUB, and TBP, were evaluated under various experimental conditions to assess their suitability for use in the normalization of qRT-PCR data. The optimal number of reference genes was determined using the geNorm program, and the suitability of particular reference genes was empirically validated by performing normalizations of expression data for the HSP70 gene. The results showed the most suitable combinations of reference genes for the different experimental conditions. For experiments based on divergent developmental stages, EF1α, ß-ACT, and RPL7 are the optimal reference gene combination, both EF1α and ß-ACT are the optimal combination used in the experiments of different geographical populations, whereas for experiments of the temperature changes, the combination of GAPDH and RPL7 is optimal, both 18S and ß-ACT are an optimal combination for feeding assay experiments. These research results should be useful for the selection of the suitable reference genes to obtain reliable qRT-PCR data in the gene expression study of A. gossypii.

Chlorantraniliprole Resistance in Spodoptera frugiperda: Resistance Monitoring, Resistance Risk, and Resistance Mechanisms.

Spodoptera frugiperda is a significant global pest, and chlorantraniliprole (CAP) is extensively used in China for its control. Understanding CAP resistance in S. frugiperda is crucial for effective management of this pest. Field populations exhibited varying degrees of resistance to CAP (RR = 1.74-5.60-fold). After 10 generations of selection, the CAP-resistant strain developed over 10-fold resistance, with a realized heritability (h2) of 0.10. Genetic analysis reveals inheritance patterns as autosomal, incomplete recessive, and monofactorial. The CAP-resistant strain showed limited cross-resistance to lufenuron and tetrachlorantraniliprole, negative cross-resistance to spinetoram, and no observed cross-resistance to other insecticides. Biochemical analysis suggested that P450-mediated detoxification is the primary resistance mechanism, with 26 genes overexpressed in the CAP-resistant strain. Additionally, the knockdown of CYP4L13, CYP6B39, CYP6B40, and CYP4G74 significantly increased the sensitivity of the resistant larvae to CAP. These findings highlight the resistance risk of CAP in S. frugiperda and emphasize the crucial role of P450 enzymes in resistance.

miR-317-3p and miR-283-5p Play a Crucial Role in Regulating the Resistance to Indoxacarb in Spodoptera frugiperda by Targeting GSTs4.

Spodoptera frugiperda is primarily controlled through chemical insecticides. Our RNA-seq data highlight the overexpression of GSTs4 in indoxacarb-resistant S. frugiperda. However, the exact role of GSTs4 in indoxacarb resistance and its regulatory mechanisms remains elusive. Therefore, we investigated the functional role of GSTs4 in S. frugiperda and explored the underlying post-transcriptional regulatory mechanisms. GSTs4 was highly overexpressed (27.6-fold) in the indoxacarb-resistant strain, and GSTs4 silencing significantly increases the susceptibility of S. frugiperda to indoxacarb, increasing mortality by 27.3%. miR-317-3p and miR-283-5p can bind to the 3'UTR of GSTs4, and the targeting relationship was confirmed by dual-luciferase reporter assays. Injecting miR-317-3p and miR-283-5p agomirs reduces GSTs4 levels by 64.8 and 42.3%, respectively, resulting in an increased susceptibility of S. frugiperda to indoxacarb. Conversely, the administration of miR-317-3p and miR-283-5pantagomirs increases GSTs4 expression and reduces larval susceptibility to indoxacarb. These findings demonstrate that miR-317-3p and miR-283-5p contribute to indoxacarb resistance in S. frugiperda by regulating the overexpression of GSTs4.

RNA interference of NADPH-cytochrome P450 reductase increases the susceptibility of Aphis gossypii Glover to sulfoxaflor.

NADPH-cytochrome P450 reductase (CPR) is essential for the detoxification of endogenous and exogenous substances mediated by cytochrome P450. While several insect CPRs have been found to be associated with insecticide resistance, the CPR of Aphis gossypii has not been characterized, and its functional role in insecticide resistance remains undefined. In this study, we cloned and characterized the full-length sequence of A. gossypii CPR (AgCPR). The deduced amino acid sequence of AgCPR contains all conserved domains of CPR, which shows high similarity to other insect CPRs and was clustered into a same branch of aphids according to phylogenetic analysis. The transcript of AgCPR was present in all developmental stages, with the highest expression in the adult stage. Furthermore, the expression of AgCPR could be induced by sulfoxaflor, a commonly used insecticide, in a time- and dose-dependent manner. Further silencing of AgCPR by feeding dsRNA significantly increased the susceptibility of A. gossypii to this insecticide. These findings suggest that AgCPR may play a significant role in the susceptibility of A. gossypii to sulfoxaflor and in the development of P450-mediated resistance to sulfoxaflor.

Characterization, expression, and functional analysis of TRPV genes in cotton aphid, Aphis gossypii Glover.

Transient receptor potential vanilloid (TRPV) channels have been found to be the molecular target of afidopyropen, a novel insecticide that is highly effective in controlling Aphis gossypii Glover in the field. However, the TRPV genes of A. gossypii has not yet been characterized. In this study, two TRPV genes of A. gossypii (AgNan and AgIav) were cloned and their expression levels were determined by quantitative real-time PCR (RT-qPCR). The deduced amino acids of AgNan and AgIav contain all conserved domains of TRPV and share very high amino acid identity with other insect TRPVs. AgNan and AgIav expressed in all developmental stages and their expression can be induced by afidopyropen in a dose- and time-dependent manner. Moreover, we found that silencing of AgNan and AgIav by RNA interference resulted in a significant mortality increase of adult A. gossypii compared to the control, which was even higher than 93 % at five days after feeding with dsAgIav, suggesting that knockdown of AgNan and AgIav have great effects on the survival of A. gossypii. The results of this study would be helpful for determining the reasonable use of afidopyropen in the integrated pest management programs of A. gossypii and provide useful information for further functional study of TRPVs in insects.

Identification and expression analysis of G protein-coupled receptors in the cotton aphid, Aphis gossypii Glover.

G protein-coupled receptors play important roles in mediating signal transformation and physiological processes. As a new type of insecticide target, GPCRs have attracted much attention in recent years. However, GPCRs have not yet been identified in Aphis gossypii. In the present study, a total of 87 GPCRs were identified from A. gossypii, including 65 Family A, 12 Family B, 7 Family C, and 3 Family F receptors. Most of the GPCRs in A. gossypii showed considerable sequence identity, and all of them have conserved transformmembrane domains. Newly identified GPCR genes were differentially expressed in different developmental stages and tissues. Moreover, we found that 34 GPCR genes were highly overexpressed in a sulfoxaflor-resistant strain, 4 and 10 of them were highly induced by LC15 and LC50 of sulfoxaflor, respectively. Furthermore, silencing of two highly overexpressed GPCRs by RNAi indicated that suppression the expression of AgoGPCR48 and AgoGPCR53 significantly increased the susceptibility of A. gossypii to sulfoxaflor, suggesting that these GPCR genes may be associated with sulfoxaflor resistance in A. gossypii. Our results imply that the overexpression of GPCR genes contribute to the sulfoxaflor resistance development in A. gossypii and provide useful targets for developing novel insecticides to manage this pest.