Functional investigation of lncRNAs and target cytochrome P450 genes related to spirotetramat resistance in Aphis gossypii Glover.

BACKGROUND: Spirotetramat is a tetramic acid derivative insecticide with novel modes of action for controlling Aphis gossypii Glover in the field. Previous studies have shown that long noncoding RNAs (lncRNAs) and cytochrome P450 monooxygenases (P450s) are involved in the detoxification process. However, the functions of lncRNAs in regulating P450 gene expression in spirotetramat resistance in A. gossypii are unknown. RESULTS: In this study, we found CYP4CJ1, CYP6CY7 and CYP6CY21 expression levels to be significantly upregulated in a spirotetramat-resistant (SR) strain compared with a susceptible (SS) strain. Furthermore, knockdown of CYP4CJ1, CYP6CY7 and CYP6CY21 increased nymph and adult mortality in the SR strain following exposure to spirotetramat. Drosophila ectopically expressing CYP380C6, CYP4CJ1, CYP6DA2, CYP6CY7 and CYP6CY21 showed significantly decreased mortality after spirotetramat exposure, and CYP380C6, CYP4CJ1 and CYP6CY21 are putative targets of six lncRNAs. Silencing of lncRNAs MSTRG.36649.2/5 and MSTRG.71880.1 changed CYP6CY21 and CYP380C6 expression, altering the sensitivity of the SR strain to spirotetramat. Moreover, MSTRG.36649.2/5 did not compete for microRNA (miRNA) binding to regulate CYP6CY21 expression. CONCLUSION: Our results confirm that CYP380C6, CYP4CJ1, CYP6DA2, CYP6CY7 and CYP6CY21 are potentially involved in the development of spirotetramat resistance in A. gossypii, and MSTRG.36649.2/5 and MSTRG.71880.1 probably regulate CYP6CY21 and CYP380C6 expression other than through the "sponge effect" of competing for miRNA binding. Our results provide a favorable molecular basis for studying cotton aphid P450 genes and lncRNA functions in spirotetramat resistance development.

Identification and the potential roles of long non-coding RNAs in regulating acetyl-CoA carboxylase ACC transcription in spirotetramat-resistant Aphis gossypii.

Long non-coding RNAs (lncRNAs) represent the largest class of non-coding transcripts. They act a pivotal part in various insect developmental processes and stress responses. However, the investigation of lncRNA functions in insecticide resistant remains at an early phase. Herein, we conducted whole-transcriptome RNA sequencing for two cotton aphid (Aphis gossypii Glover) strains, i.e., insecticide-susceptible (SS) and spirotetramat-resistant (SR). We discovered 6059 lncRNAs in the RNA-Seq data, and 874 lncRNAs showed differential expression. In addition, 5 lncRNAs among 874 lncRNAs were predicted as targets of acetyl-CoA carboxylase (ACC). Reverse transcription real-time quantitative PCR (RT-qPCR) combined with RNA interference (RNAi) confirmed that selected ACC lncRNA was related to the expression of ACC. Moreover, we also identified two transcription factors, i.e., C/EBP and C/EBPzeta, that regulate the transcription level of ACC lncRNA. These results provide a good basis for the study of cotton aphid lncRNA functions in insecticide resistance development.

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.

Resistance Risk Assessment of the Ryanoid Anthranilic Diamide Insecticide Cyantraniliprole in Aphis gossypii Glover.

Cyantraniliprole targets the ryanodine receptor and shows cross-spectrum activity against a broad range of chewing and sucking pests. In this study, a cyantraniliprole-resistant cotton aphid strain (CyR) developed resistance 17.30-fold higher than that of a susceptible (SS) strain. Bioassay results indicated that CyR developed increased cross-resistance to cyfluthrin, α-cypermethrin, imidacloprid, and acephate. In CyR, piperonyl butoxide synergistically increased the toxicity of cyantraniliprole, α-cypermethrin, and cyfluthrin. The cytochrome P450 activities in the CyR strain were significantly higher than those in the SS strain. The mRNA expression of CYP6CY7, CYP6CY12, CYP6CY21, CYP6CZ1, CYP6DA1, and CYP6DC1 in the CYP3 clade, and CYP380C6, CYP380C12, CYP380C44, CYP4CJ1, and CYP4CJ5 in the CYP4 clade, was significantly higher in CyR than in SS. The depletion of the most abundant CYP380C6 transcript by RNAi also significantly increased the sensitivity of CyR to cyantraniliprole. Transgenic expression of CYP380C6, CYP6CY7, CYP6CY21, and CYP4CJ1 in Drosophila melanogaster suggested that the expression of CYP380C6 and CYP4CJ1 was sufficient to confer cyantraniliprole resistance, with CYP380C6 being the most effective, and that CYP380C6, CYP6CY7, and CYP6CY21 were related to α-cypermethrin cross-resistance. These results indicate the involvement of P450 genes in cyantraniliprole resistance and pyrethroid cross-resistance and provide an overall view of the metabolic factors involved in resistance development.

Multiple ATP-binding cassette transporters genes are involved in thiamethoxam resistance in Aphis gossypii glover.

ATP-binding cassette (ABC) transporters represent the largest known group of efflux pumps, utilizing ATP to translocate a broad spectrum of substrates across lipid membranes, which play an important role in phase III of the detoxification process. The presence of ABC transporters and their potential association with insecticide resistance have not been investigated in Aphis gossypii, one of the most economically important agricultural pests worldwide. In this study, the ABC transporter inhibitor-verapamil significantly increased thiamethoxam toxicity against resistant cotton aphids, suggesting that ABCs are involved in thiamethoxam resistance. ABC transporter genes were identified using the A. gossypii genome database and transcriptome data. A total of 69 ABC transporters were identified and grouped into seven subfamilies (A-G), including 4 ABCAs, 5 ABCBs, 25 ABCCs, 2 ABCDs, 1 ABCE, 4 ABCFs and 30 ABCGs. Of these ABC transporters, 53 were predicted to be functional, 19 were full transporters, 30 were half-transporters and 4 had two NBDs. Subfamilies C and G accounted for 77% (32 and 45%, respectively) of the genes. The transcripts of 20 of 26 ABCs based on the transcriptome were upregulated, and ABCA1, ABCA2, ABCB1, ABCB4, ABCB8, ABCD1, ABCD2, ABCE1, ABCF1, ABCF3, ABCG7, ABCG15, ABCG17, ABCG24, ABCG27, ABCG30, MRP1, MRP7, MRP14 and MRP21 transcripts were significantly increased in the thiamethoxan resistant strain compared to the susceptible strain with qRT-PCR. The suppression of overexpressed ABCs (ABCA2, ABCD1, ABCD2, ABCE1 and ABCG15) significantly increased the thiamethoxam sensitivity of resistant aphids. These results suggest that ABC transporters might be involved in thiamethoxam resistance in A. gossypii and will facilitate further work to validate the functional roles of these ABCs in thiamethoxam resistance. These results are useful for understanding the multiple resistance mechanisms of thiamethoxam and the management of insecticide-resistant cotton aphids.

UDP-glycosyltransferases contribute to spirotetramat resistance in Aphis gossypii Glover.

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyze the conjugation of small lipophilic endogenous and exogenous compounds with sugars to produce water-soluble glycosides, playing an important role in insect endobiotic regulation and xenobiotic detoxification. In this study, two UGT-inhibitors, sulfinpyrazone and 5-nitrouracil, significantly increased spirotetramat toxicity against third instar nymphs of resistant Aphis gossypii, whereas there were no synergistic effects in apterous adult aphids, suggesting UGT involvement in spirotetramat resistance in cotton aphids. Furthermore, the UHPLC-MS/MS was employed to determine the content of spirotetramat and its four metabolites (S-enol, S-glu, S-mono, S-keto) in the honeydew of resistant cotton aphids under spirotetramat treatment. No residual spirotetramat was detected in the honeydew, while its four metabolites were detected at a S-enol: S-glu: S-mono: S-keto ratio of 69.30: 6.54: 1.44: 1.00. Therefore, glycoxidation plays a major role in spirotetramat inactivation and excretion in resistant aphids. Compared with the susceptible strain, the transcriptional levels of UGT344M2 were significantly upregulated in nymphs and adults of the resistant strain. RNA interference of UGT344M2 dramatically increased spirotetramat toxicity in nymphs, but no such effect were found in the resistant adult aphids. Overall, UGT-mediated glycoxidation were found to be involved in spirotetramat resistance. The suppression of UGT344M2 significantly increased the sensitivity of resistant nymphs to spirotetramat, suggesting that UGT344M2 upregulation might be associated with spirotetramat detoxification. This study provides an overview of the involvement of metabolic factors, UGTs, in the development of spirotetramat resistance.

Transcription Factors AhR/ARNT Regulate the Expression of CYP6CY3 and CYP6CY4 Switch Conferring Nicotine Adaptation.

Nicotine is one of the most toxic secondary plant metabolites in nature and it is highly toxic to herbivorous insects. The overexpression of CYP6CY3 and its homologous isozyme CYP6CY4 in Myzus persicae nicotianae is correlated with nicotine tolerance. The expanded (AC)n repeat in promoter is the cis element for CYP6CY3 transcription. These repeat sequences are conserved in the CYP6CY3 gene from Aphis gossypii and the homologous P450 genes in Acyrthosiphon pisum. The potential transcriptional factors that may regulate CYP6CY3 were isolated by DNA pulldown and sequenced in order to investigate the underlying transcriptional regulation mechanism of CYP6CY3. These identified transcriptional factors, AhR and ARNT, whose abundance was highly correlated with an abundance of the CYP6CY3 gene, were validated. RNAi and co-transfection results further confirm that AhR and ARNT play a major role in the transcriptional regulation of the CYP6CY3 gene. When the CYP6CY3 transcript is destabilized by AhR/ARNT RNAi, the transcription of the CYP6CY4 is dramatically up-regulated, indicating a compensatory mechanism between the CYP6CY3 and CYP6CY4 genes. Our present study sheds light on the CYP6CY3 and CYP6CY4 mediated nicotine adaption of M. persicae nicotianae to tobacco. The current studies shed light on the molecular mechanisms that underlie the genotypic and phenotypic changes that are involved in insect host shifts and we conclude that AhR/ARNT regulate the expression of CYP6CY3 and CYP6CY4 cooperatively, conferring the nicotine adaption of M. persicae nicotianae to tobacco.

Thiamethoxam Resistance in Aphis gossypii Glover Relies on Multiple UDP-Glucuronosyltransferases.

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