The widespread use of neonicotinoid insecticides has resulted in extensive resistance in field populations of the whitefly Bemisia tabaci, posing a major challenge to sustainable pest management. However, the molecular basis of cross-resistance to multiple neonicotinoids remains to be fully elucidated. In this study, resistance levels to thiamethoxam, thiacloprid, and nitenpyram were evaluated in eleven field populations of B. tabaci MED collected over a two-year period from vegetable fields in multiple provinces of China. Comparative transcriptomic analysis identified CYP402C5 as a candidate cytochrome P450 gene consistently overexpressed in resistant populations. RNA interference-mediated silencing of CYP402C5 significantly increased the susceptibility of resistant populations to all three neonicotinoids but had little effect on the susceptible strain, indicating its important role in neonicotinoid resistance. Structural prediction and molecular docking suggested that CYP402C5 possesses a conserved P450 catalytic pocket capable of accommodating multiple neonicotinoids. Functional characterization using a baculovirus expression system, coupled with UPLC-MS/MS analysis, demonstrated NADPH-dependent metabolism of thiamethoxam, thiacloprid, and nitenpyram by recombinant CYP402C5. Moreover, the identification of a nitenpyram-derived metabolite (NIT-IM) provided direct evidence for CYP402C5-mediated biotransformation. Collectively, these results demonstrate that CYP402C5 functions as a neonicotinoid-detoxifying P450 enzyme and contributes to cross-resistance in B. tabaci. This study expands the repertoire of resistance-associated P450 genes in B. tabaci and provides valuable insights for resistance monitoring and neonicotinoid resistance management.

Abstract Horizontal gene transfer (HGT) has enabled insects to acquire novel genetic material that can fuel adaptation to environmental change. However, the role of HGT in the evolution of insecticide resistance remains poorly characterised. Here, we identify BtUCH19 , a fungal gene that has integrated into the genome of the global pest Bemisia tabaci and functions as a deubiquitinating enzyme (DUB). We show that compared to endogenous DUB, BtUCH19 specifically removes K63-linked ubiquitin chains from a cytochrome P450, CYP4C64, thereby stabilizing this key detoxification enzyme in vivo . Sustained CYP4C64 abundance enhances metabolic detoxification of two commonly used insecticides thiamethoxam (TMX) and clothianidin (CLO) to their low-toxicity products TMX-Urea and CLO-Urea. Consequently, BtUCH19 drives insecticide resistance through a novel "HGT-PTM-Metab" axis. Our work reveals the instrumental role of a HGT in orchestrating ubiquitin-proteasome system (UPS)-mediated protein level regulation of a key detoxification pathway. These findings offer new insights into the intricate mechanisms underlying co-evolutionary biology, and provide a new molecular target for Integrated Pest Management (IPM).

Abstract N6-methyladenosine (m 6 A) modification is the most predominant and ubiquitous internal modification of RNA in eukaryotes, serving as a key post-transcriptional regulator of gene expression that is dynamically modulated by methyltransferases (writers) and demethylases (erasers). However, while the functions of m 6 A methylases have been partially elucidated in insects, the identity of m 6 A erasers in arthropods and their chemical catalytic mechanisms, as well as biological functions, remains largely enigmatic. Here, we uncovered 2499 putative methylase genes and 1148 putative demethylase genes in 266 insect genomes, and demonstrated that ALKBH4 functions as an m 6 A demethylase in the whitefly, Bemisia tabaci , catalyzing the oxidative reversal of mRNA m 6 A modifications both in vitro and in vivo. Furthermore, we established that ALKBH4, in coordination with other core components of the m 6 A pathway, fulfills an essential function in regulating the transcript stability of Imaginal Disk Growth Factor 1 (IDGF1) during whitefly development. Collectively, our findings expand the evolutionary scope of the eukaryotic m 6 A modification system, and reveal a conserved yet insect-specific epitranscriptomic regulatory mechanism governing fundamental physiological processes and adaptive phenotypes. Significance statement The addition of a methyl group to the N6-position of adenosine (m 6 A) is a highly abundant chemical modification of RNA. However, the functional role of m 6 A in insects and the key enzymes that regulate its levels remains poorly understood. In this study, we explored putative methylase genes and demethylase genes in hundreds insect genomes, and identified an m 6 A RNA demethylase, ALKBH4, in the whitefly, Bemisia tabaci . We demonstrate that ALKBH4 oxidatively reverses mRNA methylation in vivo and in vitro, in combination with other components of the m 6 A pathway, plays an important role in whitefly development. These findings provide new insight into m 6 A methylation system of insect.

INTRODUCTION:The evolution of insecticide resistance is often accompanied by fitness costs on life-history traits; however, the genetic mechanisms underlying this 'benefit-cost' trade-offs remain limited. OBJECTIVES:Here, we aim to elucidate the molecular basis of fitness trade-offs associated with P450-mediated insecticide resistance. METHODS:Insecticide bioassay and life-table methodology were used to determine thiacloprid resistance and its associated fitness cost in the whitefly Bemisia tabaci MED, a notorious pest of crops worldwide. Reverse genetics, western blots, immunofluorescence, molecular docking and dynamics simulation, recombinant P450 enzymes and UPLC-MS/MS were performed to demonstrate the role of the P450 CYP6EM1 in thiacloprid resistance. Dual-luciferase reporter assays, Phos-tag, EMSA, yeast one-hybrid and immunoprecipitation assay were conducted to investigate the role of a transcription factor in regulating the fitness trade-offs between thiacloprid resistance and the associated fitness cost. RESULTS:Here, we uncover that a single transcription factor, Ecdysone-induced protein 75 (E75), underpins trade-offs associated with thiacloprid resistance in the whitefly. We demonstrate that the transmembrane G protein-coupled receptor (GPCR) Neuropeptide FF receptor 2 (NPFF2) triggers the mitogen-activated protein kinase (MAPK) p38 and ERK signaling pathways, which then promote phosphorylation of E75. This GPCR-E75 regulatory axis not only activates the P450 gene CYP6EM1 but also represses a key oogenesis gene Bg, thereby contributing to the evolution of thiacloprid resistance at the expense of reproductive fitness in the whitefly. CONCLUSION:Our findings demonstrate a pivotal role of cis-trans regulatory networks in fitness trade-offs mediated by GPCR-MAPK-E75 signaling, providing in-depth insights into developing effective measures for sustainable pest control.

Thiacloprid is widely used to manage Bemisia tabaci, a major agricultural pest, yet field-evolved resistance and its fitness consequences remain poorly characterized. In this study, we surveyed 63 field populations across China from 2019 to 2022 to assess thiacloprid resistance, and conducted life-table experiments to evaluate associated fitness costs. Of these populations, 7 had resistance ratios below 10-fold and were classified as susceptible, whereas 45 populations exhibited moderate or higher resistance (>20-fold), including 33 populations with high or extremely high resistance (>50-fold), indicating that high-level resistance to thiacloprid is widespread. Using a two-sex, age-stage life table, we found that the resistant population developed faster but had lower fecundity compared with a susceptible population, suggesting that accelerated immature development may be accompanied by reduced reproductive investment and reflecting a trade-off between development and fecundity. The resistant population’s net reproductive rate (R₀) and intrinsic rate of increase (r) were 6.46 offspring and 0.06 d⁻¹, while for the susceptible population they were 27.84 offspring and 0.10 d⁻¹. Based on those parameters, the relative fitness of resistant populations was estimated at 0.23 (based on R₀) and 0.60 (based on r), supporting a measurable fitness penalty. These results imply that thiacloprid use must be more judicious in view of widespread resistance, but the detectable fitness cost also provide an opportunity for resistance management. Adoption of strategies such as rotating insecticides or combining insecticides with distinct modes of action may enhance sustainable control of Bemisia. tabaci.

Protein phosphorylation plays a key role in regulating cellular function. However, the role of this post-translational modification in the evolution of adaptive traits such as insecticide resistance remains poorly resolved. Here, we show that two protein kinases, belonging to components of different signaling pathways, act antagonistically via phosphorylation to regulate a single transcription factor, which in turn trans-regulates a resistance gene in the global pest Bemisia tabaci. We reveal that the transcription factor Eagle is constitutively overexpressed and trans-activates the 20E-related gene CYP306A1, enhancing imidacloprid detoxification and metabolic resistance. Eagle activity is antagonistically regulated by the protein kinase mitogen-activated protein kinase p38 and the cyclic adenosine monophosphate-dependent protein kinase A PKA_C1 via phosphorylation at two distinct phosphoacceptor sites, with p38 upregulation and PKA_C1 downregulation sustaining resistance. These findings reveal the role of antagonistic phosphorylation in the evolution of adaptive phenotypes and uncover a complex trans-regulatory network underpinning insecticide resistance.

The whitefly Bemisia tabaci (Hemiptera: Gennadius) is a notorious and highly polyphagous agricultural pest that is well known for its ability to transmit a wide range of serious plant pathogenic viruses. The field populations of B. tabaci in some areas have developed resistance to thiamethoxam. We found that high expression of CYP6EM1 can enhance the resistance of B. tabaci to dinotefuran. It is unclear whether CYP6EM1 is involved in the resistance of B. tabaci to the same neonicotinoid pesticide, thiamethoxam. The results of the present study demonstrated that the expression of CYP6EM1 could be induced within 9 h after the exposure of B. tabaci adults to thiamethoxam. Molecular docking analyses, with a binding energy of-6.13 cal/mol, revealed a strong binding affinity between thiamethoxam and the CYP6EM1 protein, implying that CYP6EM1 may be involved in thiamethoxam resistance. Compared with that in the susceptible strain, the mRNA expression level of the CYP6EM1 gene was significantly greater in thiamethoxam-resistant strains (R#1, 9.93-fold, P = 0.0008; R#2, 40.43-fold, P = 0.0013; R#3, 27.40-fold, P = 0.0002; R#4, 21.63-fold, P = 0.0003 and R#5, 28.65-fold, P = 0.0006). Loss and gain of function studies in vivo were performed via RNA interference and transgenic expression in Drosophila melanogaster, and the results confirmed the role of CYP6EM1 in conferring such resistance. An in vitro metabolism assay revealed that CYP6EM1 directly metabolized 15.60 % of thiamethoxam. This study provides solid evidence for the critical role of CYP6EM1 in the metabolism of thiamethoxam, which contributes to resistance. Our work provides a deeper understanding of the mechanism underlying neonicotinoid resistance and contributes valuable insights for the sustainable management of global pests such as whiteflies.

N6-methyladenosine (m6A) is the most prevalent and evolutionarily conserved internal RNA modification; however, its role in insect biology and insecticide resistance remains largely unexplored. Here, we investigate the involvement of m6A methylation in imidacloprid resistance in the whitefly Bemisia tabaci. Our results identify five core m6A methyltransferases linked to imidacloprid resistance. Specifically, we highlight CYP417B1, a key detoxification enzyme, as a critical contributor to metabolic resistance against imidacloprid. Furthermore, we demonstrate that m6A modification regulates CYP417B1 expression by modulating its 3' untranslated region (3' UTR). These findings uncover a novel mechanism by which m6A modification influences insecticide resistance through the regulation of detoxification gene expression. This study enhances our understanding of the biological functions of m6A methyltransferases in insecticide resistance and offers valuable insights for developing sustainable pest management strategies.

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.

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 (BTβ1) that confer resistance to multiple neonicotinoids. Transgenic D. melanogaster, where the native nAChR Dβ1 was replaced with BTβ1A58T&R79E, were significantly more resistant to neonicotinoids than flies where Dβ1 were replaced with the wildtype BTβ1 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.

BACKGROUND:Elucidating fitness cost associated with field-evolved insect resistance to insecticide is of particular importance to current sustainable pest control. The global pest whitefly Bemisia tabaci has developed resistance to many members of neonicotinoids, but little is known about whitefly resistance to neonicotinoid nitenpyram and its associated fitness cost. Using insecticide bioassay and life-table approach, this study aims to investigate nitenpyram resistance status in field-collected whitefly populations, and to explore whether such resistance is accompanied by a fitness cost. RESULTS:The bioassay results revealed that 14 of 29 whitefly populations displayed moderate to extremely high resistance to nitenpyram, demonstrating a widespread field-evolved resistance to nitenpyram. This field-evolved resistance in the whitefly has increased gradually over the past 3 years from 2021 to 2023. Further life-table study showed that two resistant whitefly populations exhibited longer developmental time, shorter lifespans of adult, and lower fecundity compared with the most susceptible population. The relative fitness cost of the two resistant populations was calculated as 0.69 and 0.56 by using net productive rate R0, which suggests that nitenpyram resistance comes with fitness cost in the whitefly, especially on reproduction. CONCLUSION:Overall, these results represent field-evolved high resistance to nitenpyram in the whitefly. The existing fitness costs associated with nitenpyram resistance are helpful to propose a suitable strategy for sustainable control of whiteflies by rotation or mixture of insecticide with different modes of action. © 2024 Society of Chemical Industry.

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.(c) 2023 Society of Chemical Industry.

The whitefly Bemisia tabaci poses a significant threat to various crops and ornamental plants and causes severe damage to the agricultural industry. Over the past few decades, B. tabaci has developed resistance to several pesticides, including imidacloprid. Therefore, elucidating the mechanism that leads to insecticide detoxification is very important for controlling B. tabaci and managing whitefly resistance to neonicotinoid insecticides. Among insect detoxification enzymes, glutathione S-transferase (GST) is an important phase II detoxification enzyme that helps detoxify exogenous toxic substances. In this study, we cloned the BtGSTz1 gene and observed that its expression level was greater in imidacloprid-resistant populations than sensitive populations of B. tabaci. By silencing BtGSTz1 via RNA interference, we found a significant increase in the mortality of imidacloprid-resistant B. tabaci. Additionally, prokaryotic expression and in vitro metabolism studies revealed that the recombinant BtGSTz1 protein could metabolize 36.36% of the total imidacloprid, providing direct evidence that BtGSTz1 plays a crucial role in the detoxification of imidacloprid. Overall, our study elucidated the role of GSTs in physiological activities related to insecticide resistance, which helps clarify the resistance mechanisms conferred by GSTs and provides useful insights for sustainable integrated pest management.

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. (c) 2023 Society of Chemical Industry.

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.

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.

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.

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.

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 β1 subunit (BTβ1) in B. tabaci and confirmed the consistency of BTβ1 in B. tabaci MEAM1 and MED. Expression levels of BTβ1 in different developmental stages and body parts of adults were investigated and compared in B. tabaci MED. dsRNA was prepared to knock down BTβ1 in adult B. tabaci and significantly decreases the susceptibility to five neonicotinoid insecticides, including imidacloprid, clothianidin, thiacloprid, nitenpyram, and dinotefuran. This study indicated BTβ1 as a notable site influencing the susceptibility of B. tabaci to neonicotinoids.