GPCR-MAPK signaling pathways underpin fitness trade-offs in whitefly.

Trade-offs between evolutionary gain and loss are prevalent in nature, yet their genetic basis is not well resolved. The evolution of insect resistance to insecticide is often associated with strong fitness costs; however, how the fitness trade-offs operates remains poorly understood. Here, we show that the mitogen-activated protein kinase (MAPK) pathway and its upstream and downstream actors underlie the fitness trade-offs associated with insecticide resistance in the whitefly Bemisia tabaci. Specifically, we find a key cytochrome P450 gene CYP6CM1, that confers neonicotinoids resistance to in B. tabaci, is regulated by the MAPKs p38 and ERK through their activation of the transcription factor cAMP-response element binding protein. However, phosphorylation of p38 and ERK also leads to the activation of the transcription repressor Cap "n" collar isoform C (CncC) that negatively regulates exuperantia (Ex), vasa (Va), and benign gonial cell neoplasm (Bg), key genes involved in oogenesis, leading to abnormal ovary growth and a reduction in female fecundity. We further demonstrate that the transmembrane G protein-coupled receptor (GPCR) neuropeptide FF receptor 2 (NPFF2) triggers the p38 and ERK pathways via phosphorylation. Additionally, a positive feedback loop between p38 and NPFF2 leads to the continuous activation of the MAPK pathways, thereby constitutively promoting neonicotinoids resistance but with a significant reproductive cost. Collectively, these findings provide fundamental insights into the role of cis-trans regulatory networks incurred by GPCR-MAPK signaling pathways in evolutionary trade-offs and applied knowledge that can inform the development of strategies for the sustainable pest control.

Cytochrome P450 CYP6EM1 Underpins Dinotefuran Resistance in the Whitefly Bemisia tabaci.

Being a destructive pest worldwide, the whitefly Bemisia tabaci has evolved resistance to neonicotinoid insecticides. The third-generation neonicotinoid dinotefuran has commonly been applied to the control of the whitefly, but its underlying mechanism is currently unknown. On the base of our transcriptome data, here we aim to investigate whether the cytochrome P450 CYP6EM1 underlies dinotefuran resistance in the whitefly. Compared to the susceptible strain, the CYP6EM1 gene was found to be highly expressed in both laboratory and field dinotefuran-resistant populations. Upon exposure to dinotefuran, the mRNA levels of CYP6EM1 were increased. These results demonstrate the involvement of this gene in dinotefuran resistance. Loss and gain of functional studies in vivo were conducted through RNAi and transgenic Drosophila melanogaster assays, confirming the role of CYP6EM1 in conferring such resistance. In a metabolism assay in vitro, the CYP6EM1 protein could metabolize 28.11% of dinotefuran with a possible dinotefuran-dm-NNO metabolite via UPLC-QTOF/MS. Docking of dinotefuran to the CYP6EM1 protein showed a good binding affinity, with an energy of less than -6.0 kcal/mol. Overall, these results provide compelling evidence that CYP6EM1 plays a crucial role in the metabolic resistance of B. tabaci to dinotefuran. Our work provides new insights into the mechanism underlying neonicotinoid resistance and applied knowledge that can contribute to sustainable control of a global pest such as whitefly.

Dual mutations in the whitefly nicotinic acetylcholine receptor ß1 subunit confer target-site resistance to multiple neonicotinoid insecticides.

Neonicotinoid insecticides, which target insect nicotinic acetylcholine receptors (nAChRs), have been widely and intensively used to control the whitefly, Bemisia tabaci, a highly damaging, globally distributed, crop pest. This has inevitably led to the emergence of populations with resistance to neonicotinoids. However, to date, there have been no reports of target-site resistance involving mutation of B. tabaci nAChR genes. Here we characterize the nAChR subunit gene family of B. tabaci and identify dual mutations (A58T&R79E) in one of these genes (BTß1) that confer resistance to multiple neonicotinoids. Transgenic D. melanogaster, where the native nAChR Dß1 was replaced with BTß1A58T&R79E, were significantly more resistant to neonicotinoids than flies where Dß1 were replaced with the wildtype BTß1 sequence, demonstrating the causal role of the mutations in resistance. The two mutations identified in this study replace two amino acids that are highly conserved in >200 insect species. Three-dimensional modelling suggests a molecular mechanism for this resistance, whereby A58T forms a hydrogen bond with the R79E side chain, which positions its negatively-charged carboxylate group to electrostatically repulse a neonicotinoid at the orthosteric site. Together these findings describe the first case of target-site resistance to neonicotinoids in B. tabaci and provide insight into the molecular determinants of neonicotinoid binding and selectivity.

Dynamic monitoring of the insecticide resistance status of Bemisia tabaci across China from 2019-2021.

BACKGROUND: Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a major agricultural insect pest that causes severe economic losses worldwide. Several insecticides have been applied to effectively control this key pest. However, owing to the indiscriminate use of chemical insecticides, B. tabaci has developed resistance against these chemical compounds over the past several years. RESULTS: From 2019 to 2021, 23 field samples of B. tabaci were collected across China. Twenty species were identified as the Mediterranean 'Q' type (MED) and three were identified as MED/ Middle East-Asia Minor 1 mixtures. Subsequently, resistance of the selected populations to different insecticides was evaluated. The results showed that 13 populations developed low levels of resistance to abamectin. An overall upward trend in B. tabaci resistance toward spirotetramat, cyantraniliprole and pyriproxyfen was observed. In addition, resistance to thiamethoxam remained low-to-moderate in the 23 field populations. CONCLUSION: These findings suggest that the overall resistance of the field-collected B. tabaci populations has shown an upward trend over the years in China. We believe our study can provide basic data to support integrated pest management and insecticide resistance management of field B. tabaci in China. © 2023 Society of Chemical Industry.

CYP4CS5-mediated thiamethoxam and clothianidin resistance is accompanied by fitness cost in the whitefly Bemisia tabaci.

BACKGROUND: Understanding the trade-offs between insecticide resistance and the associated fitness is of particular importance to sustainable pest control. One of the most devastating pest worldwide, the whitefly Bemisia tabaci, has developed resistance to various insecticides, especially the neonicotinoid group. Although neonicotinoid resistance often is conferred by P450s-mediated metabolic resistance, the relationship between such resistance and the associated fitness phenotype remains largely elusive. By gene cloning, quantitative reverse transcription (qRT)-PCR, RNA interference (RNAi), transgenic Drosophila melanogaster, metabolism capacity in vitro and 'two sex-age stage' life table study, this study aims to explore the molecular role of a P450 gene CYP4CS5 in neonicotinoid resistance and to investigate whether such resistance mechanism carries fitness costs in the whitefly. RESULTS: Our bioassay tests showed that a total of 13 field-collected populations of B. tabaci MED biotype displayed low-to-moderate resistance to thiamethoxam and clothianidin. Compared to the laboratory susceptible strain, we then found that an important P450 CYP4CS5 was remarkably upregulated in the field resistant populations. Such overexpression of CYP4CS5 had a good match with the resistance level among the whitefly samples. Further exposure to the two neonicotinoids resulted in an increase in CYP4CS5 expression. These results implicate that overexpression of CYP4CS5 is closely correlated with thiamethoxam and clothianidin resistance. RNAi knockdown of CYP4CS5 increased mortality of the resistant and susceptible populations after treatment with thiamethoxam and clothianidin in bioassay, but obtained an opposite result when using a transgenic line of D. melanogaster expressing CYP4CS5. Metabolic assays in vitro revealed that CYP4CS5 exhibited certain capacity of metabolizing thiamethoxam and clothianidin. These in vivo and in vitro assays indicate an essential role of CYP4CS5 in conferring thiamethoxam and clothianidin resistance in whitefly. Additionally, our life-table analysis demonstrate that the field resistant whitefly exhibited a prolonged development time, shortened longevity and reduced fecundity compared to the susceptible, suggesting an existing fitness cost as a result of the resistance. CONCLUSION: Collectively, in addition to the important role of CYP4CS5 in conferring thiamethoxam and clothianidin resistance, this resistance mechanism is associated with fitness costs in the whitefly. These findings not only contribute to the development of neonicotinoids resistance management strategies, but also provide a new target for sustainable whitefly control. © 2023 Society of Chemical Industry.

Over-expression of UDP-glycosyltransferase UGT353G2 confers resistance to neonicotinoids in whitefly (Bemisia tabaci).

The whitefly, Bemisia tabaci, comes up high metabolic resistance to most neonicotinoids in long-term evolution, which is the key problem of pest control. UGT glycosyltransferase, as a secondary detoxification enzyme, plays an indispensable role in detoxification metabolism. In this study, UGT inhibitors, 5-nitrouracil and sulfinpyrazone, dramatically augmented the toxic damage of neonicotinoids to B. tabaci. A UGT named UGT353G2 was identified in whitefly, which was notably up-regulated in resistant strain (3.92 folds), and could be induced by most neonicotinoids. Additionally, the using of RNA interference (RNAi) suppresses UGT353G2 substantially increased sensitivity to neonicotinoids in resistant strain. Our results support that UGT353G2 may be involved in the neonicotinoids resistance of whitefly. These findings will help further verify the functional role of UGTs in neonicotinoid resistance.

Cytochrome P450 CYP6DB3 was involved in thiamethoxam and imidacloprid resistance in Bemisia tabaci Q (Hemiptera: Aleyrodidae).

High level resistance for a variety of insecticides has emerged in Bemisia tabaci, a globally notorious insect. Neonicotinoid insecticides have been applied widely to control B. tabaci. Whether a differentially expressed gene CYP6DB3 discovered from transcriptome data of B. tabaci is involved in the resistance to neonicotinoid insecticides remains unclear. In the study, CYP6DB3 expression was significantly up-regulated in both thiamethoxam- and imidacloprid-resistant strains relative to the susceptive strains. We also found that CYP6DB3 expression was up-regulated after B. tabaci adults were exposed to thiamethoxam and imidacloprid. Moreover, knocking down CYP6DB3 expression via feeding corresponding dsRNA significantly reduced CYP6DB3 mRNA levels by 34.1%. Silencing CYP6DB3 expression increased the sensitivity of B. tabaci Q adults against both thiamethoxam and imidacloprid. Overexpression of CYP6DB3 gene reduced the toxicity of imidacloprid and thiamethoxam to transgenic D. melanogaster. In addition, metabolic studies showed that CYP6DB3 can metabolize 24.41% imidacloprid in vitro. Collectively, these results strongly support that CYP6DB3 plays an important role in the resistance of B. tabaci Q to imidacloprid and thiamethoxam. This work will facilitate a deeper insight into the part of cytochrome P450s in the evolution of insecticide resistance and provide a theoretical basis for the development of new integrated pest resistance management.

Novel_miR-1517 mediates CYP6CM1 to regulate imidacloprid resistance in Bemisia tabaci (Hemiptera: Gennadius).

Bemisia tabaci (Hemiptera: Gennadius) is a notorious pest that is capable of feeding on >600 kinds of agricultural crops. Imidacloprid is critical in managing pest with sucking mouthparts, such as B. tabaci. However, the field population of B. tabaci has evolved resistance because of insecticide overuse. The overexpression of the detoxification enzyme cytochrome P450 monooxygenase is considered the main mechanism of imidacloprid resistance, but the mechanism underlying gene regulation remains unclear. MicroRNAs are a type of endogenous small molecule compounds that is fundamental in regulating gene expression at the post-transcriptional level. Whether miRNAs are related to the imidacloprid resistance of B. tabaci remains unknown. To gain deep insight into imidacloprid resistance, we conducted on miRNAs expression profiling of two B. tabaci Mediterranean (MED) strains with 19-fold resistance through deep sequencing of small RNAs. A total of 8 known and 1591 novel miRNAs were identified. In addition, 16 miRNAs showed significant difference in expression levels between the two strains, as verified by quantitative reverse transcription PCR. Among these, novel_miR-376, 1517, and 1136 significantly expressed at low levels in resistant samples, decreasing by 36.9%, 60.2%, and 15.6%, respectively. Moreover, modulating novel_miR-1517 expression by feeding with 1517 inhibitor and 1517 mimic significantly affected B. tabaci imidacloprid susceptibility by regulating CYP6CM1 expression. In this article, miRNAs related to imidacloprid resistance of B. tabaci were systematically screened and identified, providing important information for the miRNA-based technological innovation for this pest management.

20E biosynthesis gene CYP306A1 confers resistance to imidacloprid in the nymph stage of Bemisia tabaci by detoxification metabolism.

BACKGROUND: Difference in physiology level between the immature and mature stages of insects likely contribute to different mechanisms of insecticide resistance. It is well acknowledged that insect 20-hydroxyecdysone (20E) plays an important role in many biological processes in the immature stage, whether 20E confers insecticide resistance at this specific stage is still poorly understood. By gene cloning, reverse transcription quantitative real-time PCR, RNA interference (RNAi) and in vitro metabolism experiments, this study aimed to investigate the potential role of 20E-related genes in conferring imidacloprid (IMD) resistance in the immature stage of the whitefly Bemisia tabaci Mediterranean. RESULTS: After identification of low to moderate IMD resistance in the whitefly, we found CYP306A1 of the six 20E-related genes was overexpressed in the nymph stage of the three resistant strains compared to a laboratory reference susceptible strain, but not in the adult stage. Further exposure to IMD resulted in an increase in CYP306A1 expression in the nymph stage. These results together imply that CYP306A1 may be implicated in IMD resistance in the nymph stage of the whitefly. RNAi knockdown of CYP306A1 increased the mortality of nymphs after treatment with IMD in bioassay, suggesting a pivotal role of CYP306A1 in conferring IMD resistance in the nymph stage. Additionally, our metabolism experiments in vivo showed that the content of IMD reduced by 20% along with cytochrome P450 reductase and heterologously expressed CYP306A1, which provides additional evidence for the important function of CYP306A1 in metabolizing IMD that leads to the resistance. CONCLUSION: This study uncovers a novel function of the 20E biosynthesis gene CYP306A1 in metabolizing imidacloprid, thus contributing to such resistance in the immature stage of the insect. These findings not only advance our understanding of 20E-mediated insecticide resistance, but also provide a new target for sustainable pest control of global insect pests such as whitefly. © 2023 Society of Chemical Industry.

CYP6CX2 and CYP6CX3 mediate thiamethoxam resistance in field whitefly, Bemisia tabaci (Hemiptera:Aleyrodidae).

Cytochrome P450 monooxygenases (P450s) are well-known for their crucial roles in the detoxification of xenobiotics. However, whether CYP6CX2 and CYP6CX3, 2 genes from our Bemisia tabaci (B. tabaci) MED/Q genome data were associated with detoxification metabolism and confer resistance to thiamethoxam is unclear. In this study, we investigated the role of CYP6CX2 and CYP6CX3 in mediating whitefly thiamethoxam resistance. Our results showed that mRNA levels of CYP6CX2 and CYP6CX3 were up-regulated after exposure to thiamethoxam. Transcriptional levels of 2 genes were overexpressed in laboratory and field thiamethoxam resistant strains by RT-qPCR. These results indicate that the enhanced expression of CYP6CX2 and CYP6CX3 appears to confer thiamethoxam resistance in B. tabaci. Moreover, linear regression analysis showed that the expression levels of CYP6CX2 and CYP6CX3 were positively correlated with thiamethoxam resistance levels among populations. The susceptibility of whitefly adults was markedly increased after silencing 2 genes by RNA interference (RNAi) which further confirming their major role in thiamethoxam resistance. Our findings provide information to better understand the roles of P450s in resistance to neonicotinoids and suggest that these genes may be applied to develop target genes for sustainable management tactic of agricultural pests such as B. tabaci.

Knockdown of the Nicotinic Acetylcholine Receptor ß1 Subunit Decreases the Susceptibility to Five Neonicotinoid Insecticides in Whitefly (Bemisia tabaci).

The sweet potato whitefly, Bemisia tabaci, (Gennadius) (Hemiptera:Aleyrodidae) is a global pest of crops. Neonicotinoids are efficient insecticides used for control of this pest. Insecticidal targets of neonicotinoids are insect nicotinic acetylcholine receptors (nAChRs). Here, we characterized and cloned the full length of the nAChR ß1 subunit (BTß1) in B. tabaci and confirmed the consistency of BTß1 in B. tabaci MEAM1 and MED. Expression levels of BTß1 in different developmental stages and body parts of adults were investigated and compared in B. tabaci MED. dsRNA was prepared to knock down BTß1 in adult B. tabaci and significantly decreases the susceptibility to five neonicotinoid insecticides, including imidacloprid, clothianidin, thiacloprid, nitenpyram, and dinotefuran. This study indicated BTß1 as a notable site influencing the susceptibility of B. tabaci to neonicotinoids.

Over-expression of CP9 and CP83 increases whitefly cell cuticle thickness leading to imidacloprid resistance.

Cuticular proteins (CPs) play an important role in protecting insects from adverse environmental conditions, like neonicotinoid insecticides, which are heavily used for numerous pests and caused environmental problems and public health concerns worldwide. However, the relationship between CPs and insecticides resistance in Bemisia tabaci, a serious and developed high insecticide resistance, is lacking. In this study, 125 CPs genes were identified in B. tabaci. Further phylogenetic tree showed the RR-2-type genes formed large gene groups in B. tabaci. Transcriptional expression levels of CPs genes at different developmental stages revealed that some CPs genes may play a specific role in insect development. The TEM results indicated that the cuticle thickness of susceptible strain was thinner than imidacloprid-resistance strain. Furthermore, 16 CPs genes (5 in RR-1 subfamily, 7 in RR-2 subfamily, 3 in CPAP3 subfamily and 1 in CPCFC subfamily) were activated in response to imidacloprid. And RNAi results indicated that CP9 and CP83 involved in imidacloprid resistance. In conclusion, this study was the first time to establish a basic information framework and evolutionary relationship between CPs and imidacloprid resistance in B. tabaci, which provides a basis for proposing integrated pest management strategies.

CYP6DW3 Metabolizes Imidacloprid to Imidacloprid-urea in Whitefly (Bemisia tabaci).

Bemisia tabaci has developed high resistance to many insecticides and causes substantial agricultural and economic losses annually. The insecticide resistance of whitefly has been widely reported in previous studies; however, the underlying mechanism remains little known. In this study, we cloned two P450 genes: CYP6DW3 and CYP6DW5v1; these genes were markedly overexpressed in imidacloprid-resistant whitefly populations compared with susceptible populations, and knockdown of these genes decreased the imidacloprid resistance of whitefly. Moreover, heterologous expression of whitefly P450 genes in SF9 cells and metabolic studies showed that the CYP6DW3 protein could metabolize 14.11% imidacloprid and produced imidacloprid-urea in vitro. Collectively, the expression levels of CYP6DW3 and CYP6DW5v1 are positively correlated with imidacloprid resistance in B. tabaci. Our study further reveals that cytochrome P450 enzymes affect the physiological activities related to resistance in insects, which helps scholars more deeply understand the resistance mechanism, and contributes to the development of integrated pest management framework.

Implications of environmentally shaped microbial communities for insecticide resistance in Sitobion miscanthi.

Insect-associated bacteria play an important role in the resistance to pesticides, yet bacterial community compositions in wild insect host populations and the environmental factors that shape them are mostly elusive. In this study, Sitobion miscanthi (Takahashi) populations were collected from major wheat growing regions in China. Following high-throughput sequencing of 16S rRNA gene fragments, association analyses were performed within the bacterial community associated with S. miscanthi, as well as with population resistance levels to four commonly used pesticides and different environmental factors. We found that bacterial community structures differed in various regions, and that the abundances of dominant bacteria such as Buchnera, Candidatus Regiella, Candidatus Hamiltonella showed high variations. The resistance of S. miscanthi to avermectin and bifenthrin was shown to decline with increasing bacterial diversity. Meanwhile, with the increase of bacterial network modularity, the resistance of S. miscanthi populations to imidacloprid, avermectin and bifenthrin also increased correspondingly. In addition, correlation analysis indicated that altitude and air pressure had the strongest impact on bacterial community diversity and relative abundance, followed by humidity, rainfall and temperature. Overall, insights into such complex interactions between bacteria and their insect hosts offer new directions for biological pest control.

Knockdown of UGT352A5 decreases the thiamethoxam resistance in Bemisia tabaci (Hemiptera: Gennadius).

Uridine diphosphate (UDP)-glycosyltransferases (UGTs), which are major phase II detoxification enzymes, have been implicated in the glycosylation of lipophilic endobiotics and xenobiotics and thus potentially lead to the evolution of insecticide resistance. In this study, we identified and cloned two putative UGT genes from transcriptome data which are named UGT352A4 and UGT352A5. As demonstrated by qRT-PCR, two UGT genes were over-expressed in the thiamethoxam-resistant (THQR) strain relative to the susceptible (THQS) strain. Moreover, the induction experiment revealed that the expression of the UGT352A5 gene was significantly increased following exposure to thiamethoxam in the THQR strain. Furthermore, the expression of both UGT352A4 and UGT352A5 was downregulated after RNA interference, whereas only the silencing of UGT352A5 resulted in a noticeable increase in the mortality of THQR adults. Our results represent the first line of evidence showing that UGT352A5 might be responsible for conferring thiamethoxam resistance in B. tabaci. The results will be shed new insights for obtaining a better understanding of the role of UGTs in the evolution of insecticide resistance and developing new insect resistance management tactics within the sustainable integrated pest management framework.

Epitranscriptomic regulation of insecticide resistance.

N 6-methyladenosine (m6A) is the most prevalent messenger RNA modification in eukaryotes and an important posttranscriptional regulator of gene expression. However, the biological roles of m6A in most insects remain largely unknown. Here, we show that m6A regulates a cytochrome P450 gene (CYP4C64) in the global whitefly pest, Bemisia tabaci, leading to insecticide resistance. Investigation of the regulation of CYP4C64, which confers resistance to the insecticide thiamethoxam, revealed a mutation in the 5' untranslated region of this gene in resistant B. tabaci strains that introduces a predicted m6A site. We provide several lines of evidence that mRNA methylation of the adenine at this position, in combination with modified expression of m6A writers, acts to increase expression of CYP4C64 and resistance. Collectively, these results provide an example of the epitranscriptomic regulation of the xenobiotic response in insects and implicate the m6A regulatory axis in the development of insecticide resistance.

Current susceptibilities of brown planthopper Nilaparvata lugens to triflumezopyrim and other frequently used insecticides in China.

The brown planthopper is a notorious rice pest in many areas of Asia. The evolution of insecticide resistance in Nilaparvata lugens has become a serious problem in the effective control of this pest in the paddy field. In this article, the current susceptibility of N. lugens field populations to novel mesoionic insecticide triflumezopyrim and major classes of chemical insecticides was determined and compared. The monitoring results indicated that field populations of N. lugens had developed low resistance to triflumezopyrim (resistance ratio, RR: 1.3-7.3-fold) during 2015-2018 in China, and the median lethal concentration values varied from 0.05 to 0.29 mg/L. Additionally, during 2017 to 2018, field populations of N. lugens showed high resistance levels to thiamethoxam (RR: 456.1-1025.6-fold), imidacloprid (RR: 2195.3-6899.0-fold) and buprofezin (RR: 1241.5-4521.7-fold), moderate to high resistance levels to dinotefuran (RR: 97.6-320.1-fold), clothianidin (RR: 69.4-230.1-fold) and isoprocarb (RR: 44.1-108.0-fold), and low to moderate levels of resistance to chlorpyrifos (RR: 12.0-29.7-fold) and nitenpyram (RR: 6.9-24.1-fold). In contrast, N. lugens just showed low resistance to sulfoxaflor (RR: 3.3-8.5-fold) and etofenprox (RR: 5.0-9.1-fold) in the field. Additionally, the P450 gene CYP6ER1 was found to be significantly overexpressed in all five field populations of N. lugens collected in 2018 when compared with a laboratory susceptible strain. Our findings will provide useful information to delay the evolution of insecticide resistance in N. lugens.

Field evolved resistance to pyrethroids, neonicotinoids, organophosphates and macrolides in Rhopalosiphum padi (Linnaeus) and Sitobion avenae (Fabricius) from China.

Rhopalosiphum padi (Linnaeus) and Sitobion avenae (Fabricius) are the predominant pests coexisting on wheat plants. In this study, the susceptibilities of 29 R. padi and 30 S. avenae populations from 15 provinces in China to pyrethroids (beta-cypermethrin and bifenthrin), neonicotinoids (imidacloprid and thiamethoxam), organophosphates (omethoate and chlorpyrifos) and macrolides (avermectin) were determined during 2018-2019. The median lethal insecticide concentrations (LC50) indicated that R. padi was more sensitive than S. avenae to most of the insecticides. Monitor results showed that most wheat aphid populations were moderately resistant to pyrethroids. Two R. padi populations were highly resistant to beta-cypermethrin with 127.3-fold and 442.8-fold resistance ratio (RR), and two were highly resistant to bifenthrin (RR of 293.9 and 320.6, respectively). One S. avenae population was highly resistant to beta-cypermethrin (RR of 136.8) and one was highly resistant to bifenthrin (RR of 313.4). All populations of two wheat aphids exhibited low to moderate resistance to neonicotinoids (RR < 100). But over half populations were sensitive or exhibited low resistance to organophosphates and macrolides. The pair-wise correlation coefficients for the insecticide LC50 revealed a positive correlation between beta-cypermethrin and bifenthrin resistance, as well as between the resistance to bifenthrin and omethoate for the two-aphid species. Similarly, significant correlations were detected between the resistance to beta-cypermethrin and avermectin for R. padi. These results may be relevant for developing effective insecticide management strategies that prevent or delay the development of resistance among wheat aphids.

Selection and validation of experimental condition-specific reference genes for qRT-PCR in Metopolophium dirhodum (Walker) (Hemiptera: Aphididae).

Metopolophium dirhodum (Walker) (Hemiptera: Aphididae) is one of the most common aphid pests of winter cereals. To facilitate accurate gene expression analyses with qRT-PCR assays, the expression stability of candidate reference genes under specific experimental conditions must be verified before they can be used to normalize target gene expression levels. In this study, 10 candidate reference genes in M. dirhodum were analyzed by qRT-PCR under various experimental conditions. Their expression stability was evaluated with delta Ct, BestKeeper, geNorm, and NormFinder methods, and the final stability ranking was determined with RefFinder. The results indicate that the most appropriate sets of internal controls were SDHB and RPL8 across geographic population; RPL8, Actin, and GAPDH across developmental stage; SDHB and NADH across body part; RPL8 and Actin across wing dimorphism and temperature; RPL4 and EF1A across starvation stress; AK and RPL4 across insecticide treatments; RPL8 and NADH across antibiotic treatments; RPL8, RPL4, Actin, and NADH across all samples. The results of this study provide useful insights for establishing a standardized qRT-PCR procedure for M. dirhodum and may be relevant for identifying appropriate reference genes for molecular analyses of related insects.

Susceptibility of Four Species of Aphids in Wheat to Seven Insecticides and Its Relationship to Detoxifying Enzymes.

Sitobion avenae (Fabricius), Rhopalosiphum padi (Linnaeus), Schizaphis graminum (Rondani), and Metopolophium dirhodum (Walker) (Hemiptera: Aphididae) are important pests of wheat and other cereals worldwide. In this study, the susceptibilities of four wheat aphid species to seven insecticides were assessed. Furthermore, the activities of carboxylesterase (CarE), glutathione S-transferase (GSTs), and cytochrome P450 monooxygenase (P450s) were determined in imidacloprid treated and untreated aphids. The results showed that the susceptibilities of four wheat aphid species to tested insecticides are different and M. dirhodum has shown higher tolerance to most insecticides. Relatively higher CarE and GST activities were observed in M. dirhodum, and P450s activities increased significantly in response to imidacloprid treatment. Moreover, susceptibility to imidacloprid were increased by the oxidase inhibitor piperonyl butoxide in M. dirhodum (20-fold). The results we have obtained imply that P450s may play an important role in imidacloprid metabolic process in M. dirhodum. We suggest that a highly species-specific approach is essential for managing M. dirhodum.