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Cytochrome P450 monooxygenases play a key role in herbivorous pest adaptation to host plants by the detoxification against plant allelochemicals. A new P450 gene (CYP4CJ1) was identified from Aphis gossypii, which displayed a positive response to plant allelochemicals. The transcript levels of CYP4CJ1 could be significantly induced by both gossypol and tannic acid. Knockdown of CYP4CJ1 increased the sensitivity of A. gossypii to these two plant allelochemicals. These results suggest that CYP4CJ1 could be involved in the tolerance of A. gossypii to some plant allelochemicals. Subsequently, we examined the regulatory mechanism of CYP4CJ1 based on the transcriptional and post-transcriptional level. A promoter region from −1422 to −1166 of CYP4CJ1 was identified, which was an essential plant allelochemical responsive region. In addition, miR-4133-3p was found to participate in the regulation of CYP4CJ1 post-transcriptionally. Our results suggest that the transcript abundance of CYP4CJ1, following the exposure of A. gossypii to gossypol and tannic acid can be attributed to both the transcriptional and post-transcriptional regulation mechanisms. These results are important for understanding the roles of P450s in the plant allelochemical tolerance of A. gossypii.

PubMed ID: 30590572, The cosmopolitan pest Aphis gossypii (Glover) causes considerable economic losses on various crops by its feeding damage and transmitting diseases around the world. Flupyradifurone is a novel butenolide pesticide; its toxicity on A. gossypii parent generation (F0) was estimated following treatment with LC 25 concentration for 48 h. The adult longevity and fecundity of the F0 individuals treated by flupyradifurone showed no significant decrease in comparison with the control. Life table method was used to evaluate the sublethal effects on progeny population (F1). Results showed that the development time of the fourth instar and the preadult as well as the total pre-reproductive period were significantly prolonged, while their fecundity was significantly decreased compared with the control. Additionally, the intrinsic rate of increase (r), the finite rate of increase, and the net reproductive rate (R 0) of F1 were all significantly lower in the group treated by LC 25 than in the control group. These results reveal that the sublethal concentration of flupyradifurone could suppress the population growth of A. gossypii and indicate that this novel insecticide may be as a useful tool in pest management. © The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved., National Natural Science Foundation of China, This work was supported by The National Natural Science Foundation of China (no. 31330064).

Aphids transmit CMV (cucumber mosaic virus) in a non-persistent manner. However, little is known about the mechanism of CMV transmission. In this study, an integrated analysis of the mRNA and protein was performed to identify important putative regulators involved in the transmission of CMV by aphids. At the level of transcription, a total of 20,550 genes (≥2-fold expression difference) were identified as being differentially expressed genes (DEGs) 24 h after healthy aphid transfer to infected tobacco plants using the RNA-seq approach. At the protein level, 744 proteins were classified as being differentially abundant between virus-treated and control M. persicae using iTRAQ (isobaric tags for relative and absolute quantitation) analysis. The combined mRNA and protein analysis enabled the identification of some viral putative regulators, such as cuticle proteins, ribosomal proteins, and cytochrome P450 enzymes. The results show that most of the key putative regulators were highly accumulated at the protein level. Based on those findings, we can speculate that the process by which aphids spread CMV is mainly related to post-translational regulation rather than transcription.

Simple Summary The cotton aphid, Aphis gossypii Glover, is a notorious pest in cotton and cucurbit fields. The control of A. gossypii has typically relied on the application of chemical insecticides. Sulfoxaflor is the first commercially available sulfoximine insecticide, which exhibits great efficacy against sap-feeding insect pests and has been applied as an alternative insecticide for controlling of A. gossypii in China. Consequently, A. gossypii quickly developed resistance to this insecticide. Hence, in this study, to clarify the potential detoxifying roles of UGTs (one of the phase II detoxification enzymes) in resistance of A. gossypii against sulfoxaflor, the synergistic effects of two synergists (sulfinpyrazone and 5-nitrouracil) against sulfoxaflor were investigated using the susceptible and laboratory-established sulfoxaflor resistant strain (SulR), and the expression levels of 15 UGT genes were determined by qRT-PCR. Furthermore, the involvement of highly upregulated UGTs in sulfoxaflor-resistant strain was functionally tested by RNA interference (RNAi). Our results suggest that overexpression of UGTs contributes to sulfoxaflor resistance in A. gossypii, which should be useful for understanding sulfoxaflor resistance mechanisms. Abstract 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.

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.

Afidopyropen is a novel pyropene insecticide that has been shown to be effective for the management of Aphis gossypii Glover. Establishing the susceptibility baseline of A. gossypii to this newly commercialized insecticide is important for ensuring its continued efficacy. The aim of this study was to evaluate the susceptibility baseline of field populations of A. gossypii to the novel insecticide afidopyropen. A total of 25 field populations of A. gossypii were collected in China from 2019 to 2020, and the susceptibility of these populations to afidopyropen was determined in the laboratory using a leaf-dipping method. The results indicated that afidopyropen was highly toxic to adult A. gossypii, and the LC50 values ranged from 0.009 mg L−1 to 0.435 mg L−1, suggesting that afidopyropen is effective for controlling A. gossypii. Interestingly, we found that A. gossypii collected from cotton plants showed higher tolerance to afidopyropen compared with individuals collected from the other host plants. The findings of our study will aid future efforts to monitor A. gossypii resistance to afidopyropen.

Insects have developed a good adaptive mechanism in response to environmental stresses in the long-term evolution. They have developed a helpful metabolism system to resist plant allelochemicals. Insects also harbor different kinds of symbiotic bacteria, which provide them a competitive advantage. Here, using cotton aphid as an example, we investigated the effects of four plant allelochemicals on the differential expression of symbiotic bacteria based on transcriptome data. We also studied the composition of symbiotic bacteria and function on pathway level in three kinds of aphids. We found that the bacteria have a significant role in resisting the plant allelochemicals stress and host plant selection by aphids. These results should be useful to investigate the environmental adaption mechanism of aphids in the view of symbiotic bacteria. These results would offer a new insight for improving strategy of aphids and developing new pest control systems.

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) β1 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 β1 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 β1 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.

BACKGROUND The cotton aphid, Aphis gossypii (Glover), is a destructive pest that affects numerous crops throughout the world. Pyrethroid resistance has become endemic in A. gossypii populations in the cotton growing areas of China due to wide- spread application of insecticides. To assess the extent of pyrethroid resistance, bioassays were conducted on field populations collected from several cotton cultivation areas from 2010–2015. The frequency of a known resistance-associated sodium channel mutation (M918L) in A. gossypii was evaluated and the bioassay of bifenthrin with or without the synergist was performed to illuminate the mechanisms underlying resistance to pyrethroids. RESULTS The field populations exhibited very high levels of resistance to both beta-cypermethrin and deltamethrin. Pretreatment with synergists, DEF and PBO, significantly increased the toxicity of bifenthrin to cotton aphid populations collected from Bt cotton fields in China. Further, 96.8%-100% of individuals with the M918L mutation (including both RR and RS individuals) were observed in various populations, and only 2.8%-3.2% of individuals with wild-type homozygotes (SS) were detected. CONCLUSION The mutation M918L in the voltage-gated sodium channel along with detoxifying metabolism was contributed to the pyrethroid resistance in the field populations of Aphis gossypii from cotton growing regions of China. And insecticides with different modes of action should be recommended for the control of A. gossypii in the future.

The joint toxicity of chlorantraniliprole, a novel insecticide that acts on ryanodine receptors, and sinigrin, a natural plant defense compound from brassicaceous vegetables, to the larvae of Spodoptera exigua (Hübner) was determined in this paper. Additionally, the joint effects of the two compounds on cytochrome P450 enzyme activity and on the expression levels of mRNA of three P450 genes (including CYP9A9, CYP6B, and CYP4G37) and an NADPH cytochrome P450 reductase gene (HQ852049) were investigated. The toxicity of the mixture of chlorantraniliprole and sinigrin to fourth-instar S. exigua larvae was 1.60-fold higher than the toxicity of the chlorantraniliprole-only treatment after 24 h. Induced by chlorantraniliprole and sinigrin, the specific activity of the P450 O-deethylase was affected in a time-, dose-, and organ-specific manner in fifth-instar S. exigua larvae. The effects were more pronounced in the midgut than in the fat body. The specific activity of the P450 O-deethylase in almost all treatments increased at 12, 24, and 36 h posttreatment compared with that in the control. Based on real-time PCR analyses, the expression levels of the P450 genes CYP9A9, CYP6B, and CYP4G37 and the NADPH cytochrome P450 reductase gene HQ852049 in fifth-instar S. exigua larvae were induced by chlorantraniliprole and sinigrin, and the trends were similar to the specific activity of the P450 O-deethylase. Therefore, the CYP9A9, CYP6B, and HQ852049 in the tested genes were the most inducible genes that were expressed when the S. exigua larvae were exposed to chlorantraniliprole and sinigrin.

The cotton aphid, Aphis gossypii (Glover), is a destructive pest that affects numerous crops throughout the world. Pyrethroid resistance has become endemic in A. gossypii populations in the cotton growing areas of China due to wide- spread application of insecticides. To assess the extent of pyrethroid resistance, bioassays were conducted on field populations collected from several cotton cultivation areas from 2010 to 2015. The frequency of a known resistance-associated sodium channel mutation (M918 L) in A. gossypii was evaluated and the bioassay of bifenthrin with or without the synergist was performed to illuminate the mechanisms underlying resistance to pyrethroids.The field populations exhibited very high levels of resistance to both beta-cypermethrin and deltamethrin. Pretreatment with synergists, DEF and PBO, significantly increased the toxicity of bifenthrin to cotton aphid populations collected from Bt cotton fields in China. Further, 96.8-100% of individuals with the M918 L mutation (including both RR and RS individuals) were observed in various populations, and only 2.8-3.2% of individuals with wild-type homozygotes (SS) were detected.The mutation M918 L in the voltage-gated sodium channel along with detoxifying metabolism was contributed to the pyrethroid resistance in the field populations of Aphis gossypii from cotton growing regions of China. And insecticides with different modes of action should be recommended for the control of A. gossypii in the future. © 2017 Society of Chemical Industry.

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.

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.

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 (lambda), the net reproductive rate (R-0), 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.

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, β-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, β-ACT, and RPL7 are the optimal reference gene combination, both EF1α and β-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 β-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.

The effects of various insecticides on the fluidity of mitochondrial membranes and cross-resistance were investigated in the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae) using strains that were both resistant and susceptible to avermectin. The resistant strain of P. xylostella, AV-R, developed 1078-fold resistance to avermetins with a high level of cross-resistance to the analogs of avermectins, ivermectin and emamectin benzoate. It had more than 1000 times greater resistance when compared with the avermectin-susceptible strain, XH-S. Mitochondrial membrane fluidity was measured by detecting fluorescence polarization using DPH (1,6-Diphenyl -1,3,5-hexatriene) as the fluorescence probe. Abamectin, emamectin benzoate, ivermectin, cypermethrin and fenvalerate decreased the fluidity of mitochondrial membranes in the XH-S strain at 25 degrees C. However, fipronil and acephate did not change the fluidity of mitochondrial membrane when the concentration of these insecticides was 1x10(-4) mol/L. Membrane fluidity increased as the temperature increased. The thermotropic effect on the polarization value of DPH increased as the insecticide concentration was increased. There was a significant difference of mitochondrial membrane fluidity between both XH-S and AV-R when temperature was less than 25 degrees C and no difference was observed when the temperature was more than 25 degrees C. The low-dose abamectin (0.11 mg/L) in vivo treatment caused a significant change of membrane fluidity in the XH-S strain and no change in the AV-R strain. However, a high-dose abamectin (11.86 mg/L) resulted in 100% mortality of the XH-S strain. In vivo treatment may cause a significant change of membrane fluidity in the AV-R strain.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12867-017-0080-5) contains supplementary material, which is available to authorized users.