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.

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.

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.

Bowen's disease of the vulva and perianal area in a SLE patient successfully treated with Photodynamic therapy: A case report.

Photodynamic therapy (PDT) has emerged as a non-invasive treatment modality for superficial skin cancers. It has the advantage of greater tolerance and providing better cosmetic outcomes than conventional treatment methods. Because of the rarity of extensive Bowen's disease located in the genital area, evidence of efficacy for therapies is mainly based on case reports and clinical experience. This report presents a case of a 32-year-old female with Bowen's disease of the vulva and perianal area with systemic lupus erythematosus successfully treated by 5-aminolaevulinic acid PDT. There was no evidence of recurrence after five-years of follow-up.

UDP-Glycosyltransferases UGT350C3 and UGT344L7 Confer Tolerance to Neonicotinoids in Field Populations of Aphis gossypii.

The cotton aphid, Aphis gossypii, is a polyphagous pest that stunts host plant growth via direct feeding or transmitting plant virus. Due to the long-term application of insecticides, A. gossypii has developed different levels of resistance to numerous insecticides. We found that five field populations had evolved multiple resistances to neonicotinoids. To explore the resistance mechanism mediated by uridine diphosphate glycosyltransferases (UGTs), two upregulated UGT genes in these five strains, UGT350C3 and UGT344L7, were selected for functional analysis of their roles in neonicotinoid detoxification. Transgenic Drosophila bioassay results indicated that compared with the control lines, the UGT350C3 and UGT344L7 overexpression lines were more tolerant to thiamethoxam, imidacloprid, and dinotefuran. Knockdown of UGT350C3 and UGT344L7 significantly increased A. gossypii sensitivity to thiamethoxam, imidacloprid, and dinotefuran. Molecular docking analysis demonstrated that these neonicotinoids could bind to the active pockets of UGT350C3 and UGT344L7. This study provides functional evidence of neonicotinoid detoxification mediated by UGTs and will facilitate further work to identify strategies for preventing the development of neonicotinoid resistance in insects.

Stability, Inheritance, Cross-Resistance, and Fitness Cost of Resistance to λ-Cyhalothrin in Cydia pomonella.

Insecticides are commonly utilized in agriculture and forestry for pest control, but their dispersal can pose hazards to humans and environment. Understanding resistance, inheritance patterns, and fitness costs can help manage resistance. A λ-cyhalothrin-resistant population (LCR) of Cydia pomonella, a global pest of pome fruits and walnuts, was obtained through selective insecticide breeding for 15 generations, showing stable moderate resistance (23.85-fold). This population was cross-resistant to deltamethrin (4.26-fold) but not to ß-cypermethrin, chlorantraniliprole, chlorpyrifos, and avermectin. Genetic analysis revealed the resistance was autosomal, incompletely dominant, and controlled by multiple genes. Increased activity of glutathione S-transferases and cytochrome P450 monooxygenases (P450s) played a primary role in resistance, with specific genes up-regulated in LCR, and exhibited significant expression in midgut. LCR also exhibited fitness costs, including delays in development, reduced fecundity, and slower population growth. These findings contribute to understanding λ-cyhalothrin resistance in C. pomonella and can guide resistance management strategies.

Inducible Gut-Specific Carboxylesterase SlCOE030 in Polyphagous Pests of Spodoptera litura Conferring Tolerance between Nicotine and Cyantraniliprole.

Insecticides tolerance in herbivorous arthropods is associated with preadaptation to host plant allelochemicals. However, how plant secondary metabolites activate detoxifying metabolic genes to develop tolerance remains unclear. Herein, the tolerance of Spodoptera litura larvae to cyantraniliprole was increased after nicotine exposure. An S. litura α esterase, SlCOE030, was predominantly expressed in the midgut and induced after exposure to cyantraniliprole, nicotine, and cyantraniliprole plus nicotine. Drosophila melanogaster with ectopically overexpressed SlCOE030 enhanced cyantraniliprole and nicotine tolerance by 4.91- and 2.12-fold, respectively. Compared to UAS-SlCOE030 and Esg-GAL4 lines, the Esg > SlCOE030 line laid more eggs after nicotine exposure. SlCOE030 knockdown decreased the sensitivity of nicotine-treated S. litura larvae to cyantraniliprole. Metabolism assays indicated that recombinant SlCOE030 protein metabolizes cyantraniliprole. Homology modeling and molecular docking analysis demonstrated that SlCOE030 exhibits effective affinities for cyantraniliprole and nicotine. Thus, insect CarEs may result in the development of cross-tolerance between synthetic insecticides and plant secondary metabolites.

Inducible Cytochrome P450s in the Fat Body and Malpighian Tubules of the Polyphagous Pests of Spodoptera litura Confer Xenobiotic Tolerance.

Cytochrome P450 plays vital roles in detoxifying xenobiotics. In this study, SlCYP340A and SlCYP340L expression in the Spodoptera litura fat body and SlCYP332A1, SlCYP6AB12, SlCYP6AB58, SlCYP6AB59, and SlCYP6AN4 expression in the Malpighian tubules were significantly upregulated after cyantraniliprole exposure, and SlCYP6AB58 and SlCYP6AB59 expression levels were simultaneously increased in the Malpighian tubules after gossypol treatment. Drosophila ectopically expressing candidate P450 genes showed that SlCYP332A1, SlCYP6AB12, SlCYP6AB59, SlCYP6AN4, and SlCYP340A conferred cyantraniliprole tolerance. The overexpression of SlCYP6AB58 and SlCYP6AB59 in Drosophila increased the number of eggs laid under the gossypol treatment. Moreover, the knockdown of SlCYP332A1, SlCYP6AB12, SlCYP6AB59, SlCYP6AN4, and SlCYP340A increased S. litura mortality under the cyantraniliprole treatment. Homology modeling and molecular docking results suggested that candidate P450 has the potential to bind with cyantraniliprole. These results indicate that the CYP3 and CYP4 genes participate in cyantraniliprole detoxification and that SlCYP6AB59 may be simultaneously involved in the gossypol tolerance of S. litura.

The C2H2 zinc finger transcription factor CF2-II regulates multi-insecticide resistance-related gut-predominant ABC transporters in Aphis gossypii Glover.

Clarifying the molecular mechanisms of cotton aphid resistance to various insecticides is crucial for the long-term safe application of insecticides in chemical control. ATP-binding cassette (ABC) transporters mediate the membrane transport of various substrates (including exogenous substances). Experiments confirmed that ABCB5, ABCF2, and MRP12 contributed to high levels of resistance to spirotetramat, cyantraniliprole, thiamethoxam or imidacloprid. Binding sites of the C2H2 zinc finger transcription factor CF2-II was predicted to be located in the promoters of ABCB5, ABCF2, and MRP12. The expression levels of ABCB5, ABCF2, and MRP12 were significantly upregulated after silencing CF2-II. The results of dual-luciferase reporter assays demonstrated a negative regulatory relationship between CF2-II and ABC transporter promoters. Furthermore, yeast one-hybrid (Y1H) and electrophoresis mobility shift assays (EMSAs) revealed that CF2-II inhibited the expression of ABC transporter genes through interaction with binding sites [ABCF2.p (-1149/-1140) or MRP12.p (-1189/-1181)]. The above results indicated that ABCB5, ABCF2, and MRP12 were negatively regulated by the transcription factor CF2-II, which will help us further understand the mechanism of transcriptional adaption of multi-insecticides resistant related ABC transporters in response to xenobiotics.

A masked gene concealed hand in glove in the forkhead protein crocodile regulates the predominant detoxification CYP6DA1 in Aphis gossypii Glover.

Cytochrome P450-mediated metabolism is an important mechanism of insecticide resistance, most studies show upregulated transcript levels of P450s in resistant insect strains. Our previous studies illustrated that some upregulated P450s were associated with cyantraniliprole resistance, and it is more comprehensive to use the tissue specificity of transcriptomes to compare resistant (CyR) and susceptible (SS) strains. In this study, the expression profiles of P450s in a CyR strain compared with a SS strain in remaining carcass or midgut were investigated by RNA sequencing, and candidate genes were selected for functional study. Drosophila melanogaster bioassays suggested that ectopic overexpression of CYP4CK1, CYP6CY5, CYP6CY9, CYP6CY19, CYP6CZ1 and CYP6DA1 in flies was sufficient to confer cyantraniliprole resistance, among which CYP6DA1 was the predominant contributor to resistance (12.24-fold). RNAi suppression of CYP4CK1, CYP6CY5, CYP6CY9 and CYP6DA1 significantly increased CyR aphid sensitivity to cyantraniliprole. The CYP6DA1 promoter had two predicted binding sites for crocodile (CROC), an intron-free ORF with bidirectional transcription yielding CROC (+) and CROC (-). Y1H, RNAi and EMSA found that CROC (-) was a transcription factor directly regulating CYP6DA1 expression. In conclusion, P450 genes contribute to cyantraniliprole resistance, and the transcription factor CROC (-) regulates the expression of CYP6DA1 in A. gossypii.

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.

Functional Validation of the Roles of Cytochrome P450s in Tolerance to Thiamethoxam and Imidacloprid in a Field Population of Aphis gossypii.

Field populations of Aphis gossypii (SDR) have evolved high resistance to neonicotinoids, including thiamethoxam and imidacloprid. Synergism bioassays and transcriptomic comparison of the SDR and susceptible (SS) strains revealed that the cytochrome P450s may contribute to the neonicotinoid resistance evolution. The transcripts of some P450s were constitutively overexpressed in the SDR strain, and many genes showed expression plasticity under insecticide exposure. Drosophila that ectopically expressed CYPC6Y9, CYP4CK1, CYP6DB1, and CYP6CZ1 showed greater resistance (>8.0-fold) to thiamethoxam, and Drosophila that expressed CYPC6Y9, CYP6CY22, CYP6CY18, and CYP6D subfamily genes showed greater resistance (>5-fold) to imidacloprid. Five P450 genes that caused thiamethoxam resistance also conferred resistance to α-cypermethrin. Furthermore, the knockdown of CYP4CK1, CYP6CY9, CYP6CY18, CYPC6Y22, CYP6CZ1, and CYP6DB1 dramatically increased the sensitivity of the SDR strain to thiamethoxam or imidacloprid. These results indicate the involvement of multiple P450 genes, rather than one key gene, in neonicotinoid resistance in field populations.

Functional Inquiry into ATP-Binding Cassette Transporter Genes Contributing to Spirotetramat Resistance in Aphis gossypii Glover.

ATP-binding cassette (ABC) transporters regulate the efflux of a broad spectrum of substrates to extracellular transporting, which play an important role in the detoxification process in arthropods. Here, we described a comprehensive approach to explore the involvement of ABC transporters in spirotetramat resistance in cotton aphids. In this study, synergism bioassays showed 17.05% and 35.42% increases in the toxicity to spirotetramat with the ABC inhibitor verapamil in adult and 3rd instar nymph aphids of the SR strain, respectively. In a competitive assay based on the microinjection of a fluorescent ABC transporter substrate, verapamil (a general ABC inhibitor) and spirotetramat significantly inhibited the elimination of Texas Red. Based on transcriptome data of midguts of spirotetramat-susceptible (SS) and -resistant (SR) strains, the expression levels of ABCB4, ABCB5, ABCF2, MRP11, and MRP12 were significantly upregulated in the SR strain midgut compared to that of the SS strain. Gene functional analysis based on ectopic expression and RNA interference (RNAi) proved that ABCB4, ABCB5, ABCF2, MRP11, and MRP12 were involved in the tolerance of cotton aphids to spirotetramat. Moreover, the upregulated ABCF2, ABCB4, and ABCB5 in the midgut of the SR strain contributed more to the resistance of spirotetramat in in vitro functional analysis. In summary, these results demonstrate that candidate ABC transporter genes in the midgut tissue were involved in spirotetramat resistance, which will help reveal the relationship between ABC transporters and the development of spirotetramat resistance in field populations.

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.

The dynamic structure of Spitzenkörpers of Trichosporon asahii examined by the fluorescent probe FM4-64.

The Spitzenkörper is a dynamic and specialized multicomponent cell complex present in the tips of hyphal cells. The amphiphilic styryl dye FM4-64 was found to be ideal for imaging the dynamic changes of the apical vesicle cluster within growing hyphal tips. It is widely used as a marker of endocytosis and to visualize vacuolar membranes. Here we performed uptake experiments using FM4-64 to study the dynamic of the Spitzenkörper in Trichosporon asahii. We observed that Spitzenkörpers were present at the tip of the budding site of the spore, blastospore, and the germ tube of T. asahii. We also found that Spitzenkörpers were present at the tip of the hyphae as well as the subapical regions. Cytochalasin D, an inhibitor of actin polymerization, leads to abnormal Spitzenkörper formation and loss of cell polarity.

The dynamic structure of Spitzenkörpers of Trichosporon asahii examined by the fluorescent probe FM4-64

Abstract The Spitzenkörper is a dynamic and specialized multicomponent cell complex present in the tips of hyphal cells. The amphiphilic styryl dye FM4-64 was found to be ideal for imaging the dynamic changes of the apical vesicle cluster within growing hyphal tips. It is widely used as a marker of endocytosis and to visualize vacuolar membranes. Here we performed uptake experiments using FM4-64 to study the dynamic of the Spitzenkörper in Trichosporon asahii. We observed that Spitzenkörpers were present at the tip of the budding site of the spore, blastospore, and the germ tube of T. asahii. We also found that Spitzenkörpers were present at the tip of the hyphae as well as the subapical regions. Cytochalasin D, an inhibitor of actin polymerization, leads to abnormal Spitzenkörper formation and loss of cell polarity.