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.

Molecular and functional characterization of an antenna-enriched glutathione S-transferase BminGSTd3 involved in undecanol degradation in the citrus fruit fly, Bactrocera minax (Enderlein) (Diptera Tephritidae).

Bactrocera minax is a disastrous pest of citrus crops in China. Numerous studies focused on the molecular mechanism of odorant perception of B. minax, but the molecular mechanism of odorant degradation remains unclear. Glutathione S-transferases (GSTs) are considered as a class of odorant-degrading enzymes involved in degrading odorant molecules in insects' olfactory system. Here, we identified a delta-class GST gene, BminGSTd3, from B. minax. It was predominantly expressed in adult's olfactory organ antennae. The bacterially expressed recombinant BminGSTd3 was able to catalyze the conjugation of glutathione (GSH) with 2, 4-dinitrochlorobenzene (CDNB). Spectrophotometric analysis showed that undecanol can inhibit catalytic activities of BminGSTd3. Metabolic assays exhibited that undecanol can be depleted by BminGSTd3. Undecanol is believed to be an important B. minax sex pheromone component. The other components of the pheromone remain unclear. To understand how BminGSTd3 specifically recognizes undecanol, a 3D model of BminGSTd3 was constructed by homology modeling. Molecular docking based on this model revealed that E64 and S65 are the key amino acids recognizing undecanol, and this was proven by site-directed mutagenesis and intrinsic fluorescence assays. We suggest that BminGSTd3 is an undecanol metabolizing GST in B.minax, and E64 and S65 may serve as the key binding sites.

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.

Key Amino Acid Residues Involved in Binding Interactions between Bactrocera minax Odorant-Binding Protein 3 (BminOBP3) and Undecanol.

Insect odorant-binding proteins (OBPs) are significant in binding and transporting odorants to specific receptors. Our previous study demonstrated that BminOBP3 exhibited a strong affinity with undecanol. However, the binding mechanism between them remains unknown. Here, using homology modeling and molecular docking, we found that the C-terminus (I116-P122), especially the hydrogenbonds formed by the last three amino acid residues (V120, F121, and P122) of the C-terminus, is essential for BminOBP3's ligand binding. Mutant binding assays showed that the mutant T-OBP3 that lacks C-terminus (I116-P122) displayed a significant decrease in affinity to undecanol (Ki = 19.57 ± 0.45) compared with that of the wild-type protein BminOBP3 (Ki = 11.59 ± 0.51). In the mutant 3D2a that lacks F121 and P122 and the mutant V120A in which V120 was replaced by alanine, the bindings to undecanol were completely abolished. In conclusion, the C-terminus plays a crucial role in the binding interactions between BminOBP3 and undecanol. Based on the results, we discussed the ligand-binding process of BminOBP3.

Real-Time Recognition and Detection of Bactrocera minax (Diptera: Trypetidae) Grooming Behavior Using Body Region Localization and Improved C3D Network.

Pest management has long been a critical aspect of crop protection. Insect behavior is of great research value as an important indicator for assessing insect characteristics. Currently, insect behavior research is increasingly based on the quantification of behavior. Traditional manual observation and analysis methods can no longer meet the requirements of data volume and observation time. In this paper, we propose a method based on region localization combined with an improved 3D convolutional neural network for six grooming behaviors of Bactrocera minax: head grooming, foreleg grooming, fore-mid leg grooming, mid-hind leg grooming, hind leg grooming, and wing grooming. The overall recognition accuracy reached 93.46%. We compared the results obtained from the detection model with manual observations; the average difference was about 12%. This shows that the model reached a level close to manual observation. Additionally, recognition time using this method is only one-third of that required for manual observation, making it suitable for real-time detection needs. Experimental data demonstrate that this method effectively eliminates the interference caused by the walking behavior of Bactrocera minax, enabling efficient and automated detection of grooming behavior. Consequently, it offers a convenient means of studying pest characteristics in the field of crop protection.

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.

Catalase promotes whitefly adaptation to high temperature by eliminating reactive oxygen species.

Thermal stress usually leads to excessive production of reactive oxygen species (ROS) in all aerobic organisms. Catalases (CAT) are the key antioxidant enzymes, which act as the first line of defense against ROS in the antioxidant pathway. The highly invasive and widely distributed whitefly Bemisia tabaci MED damages plants by feeding as well as by transmitting many plant viruses. Previous studies have shown that strong adaptability to high temperature helps explain the spread of MED around the world. However, the mechanism underlying high temperature adaptation of this pest is not well understood. In this study, 6 CAT genes were identified from the MED genome and transcriptome dataset, among which BtCAT1, BtCAT2, and BtCAT3 were found to be highly expressed in adults. The expression of BtCAT1, BtCAT2, or BtCAT3 increased with induction temperature and induction time. The MED was exposed with mean high temperature (30 °C or 35 °C) and a short-term extremely high temperature (39 °C or 41 °C) after the silencing of BtCAT1, BtCAT2, or BtCAT3 to significantly increased ROS levels by at least 0.5 times and significantly decreased survival rate and fecundity of MED adults. The ROS level in the treated specimens gradually returned to a normal level after 24 h at 25 °C, but the survival rate still declined significantly. Taken together, our results demonstrate that CAT could help B. tabaci adapt to long-term mean high temperatures and short-term extremely high temperatures by eliminating excessive ROS.

Fitness cost of spinosad resistance related to vitellogenin in Frankliniella occidentalis (Pergande).

BACKGROUND: The western flower thrips Frankliniella occidentalis, a worldwide agricultural pest, has developed resistance to an array of insecticides. Spinosad resistance confers an apparent fitness cost in F. occidentalis. In the present study, we compared the reproductive capacities, ovary development, and the expression of the vitellogenin (Vg) gene in spinosad-susceptible (Ivf03) and -resistant (NIL-R) near isogenetic lines of F. occidentalis in order to clarify the reason for the fitness cost in spinosad resistance. RESULTS: The NIL-R strain exhibited a 17.9% decrease in fecundity (eggs laid per female) as compared to the Ivf03 strain, and the ovariole was significantly shortened by 2.8% in the NIL-R strain relative to the Ivf03 strain. Compared to the Ivf03 strain, the expression levels of Vg mRNA and protein were downregulated by 33.7% and 32.9% in the NIL-R strain, respectively. Moreover, interference with the Vg gene significantly reduced the expression levels of Vg mRNA and protein, and decreased ovariole length, survival rates and the fecundity of both strains. CONCLUSION: The results indicate that the downregulated expression of Vg may contribute to the reduction of ovariole length and consequently to a fitness cost in spinosad-resistant F. occidentalis. The results not only increase our understanding of the evolution of insecticide resistance, but also could contribute to the formulation of control strategy of F. occidentalis. © 2022 Society of Chemical Industry.

Spinetoram resistance drives interspecific competition between Megalurothrips usitatus and Frankliniella intonsa.

BACKGROUND: Species displacement by the outcome of interspecific competition is of particular importance to pest management. Over the past decade, spinetoram has been extensively applied in control of the two closely related thrips Megalurothrips usitatus and Frankliniella intonsa worldwide, while whether its resistance is implicated in mediating interspecific interplay of the two thrips remains elusive to date. RESULTS: Field population dynamics (from 2017 to 2019) demonstrated a trend toward displacement of F. intonsa by M. usitatus on cowpea crops, supporting an existing interspecific competition. Following exposure to spinetoram, M. usitatus became the predominate species, which suggests the use of spinetoram appears to be responsible for mediating interspecific interactions of the two thrips. Further annual and seasonal analysis (from 2016 to 2020) of field-evolved resistance dynamics revealed that M. usitatus developed remarkably higher resistance to spinetoram compared to that of F. intonsa, implying a close relationship between evolution of spinetoram resistance and their competitive interactions. After 12 generations of laboratory selection, resistance to spinetoram in M. usitatus and F. intonsa increased up to 64.50-fold and 28.33-fold, and the average realized heritability (h2 ) of resistance was calculated as 0.2550 and 0.1602, respectively. Interestingly, two-sex life table analysis showed that the spinetoram-resistant strain of F. intonsa exhibited existing fitness costs, but not the M. usitatus. These indicate that a rapid development of spinetoram resistance and the lack of associated fitness costs may be the mechanism underlying recent dominance of M. usitatus over F. intonsa. CONCLUSION: Collectively, our results uncover the involvement of insecticide resistance in conferring displacement mechanism behind interspecific competition, providing a framework for understanding the significance of the evolutionary relationships among insects under ongoing changing environments. These findings also can be invaluable in proposing the most appropriate strategies for sustainable thrips control programs. © 2022 Society of Chemical Industry.

Functional Characterization of Two Antenna-Enriched Odorant-Binding Proteins From Bactrocera minax (Diptera: Tephritidae).

Olfaction is of great significance for insect mate-seeking and host-locating behaviors. Insect odorant-binding proteins (OBPs), especially those antenna-enriched OBPs, are thought to discriminate, capture and transport odorant molecules to olfactory receptors, but this has not been fully clarified in Bactrocera minax (Enderlein), an economically important pest of citrus crops. Our previous studies showed that seven OBP genes (BminOBP1-7) were identified from B. minax adults via a head transcriptome analysis, of which only BminOBP3 and 6 were highly expressed in antennae, suggesting an olfactory role. To confirm their functions, here, BminOBP3 and 6 were cloned, expressed in Escherichia coli cells. Binding properties of the recombinant BminOBPs with 13 volatiles, most of which can elicit a significant behavioral response from B. minax adults, were determined by fluorescent competitive binding assays. The results showed that Both BminOBP3 and 6 exhibited a remarkable selectivity towards the 13 ligands tested. BminOBP3 displayed strong binding affinity only with undecanol. BminOBP6 demonstrated strong binding affinity with undecanol and limonene among 13 ligands tested. Undecanol is believed to be main sex pheromone component of B. minax. Limonene is an important volatile compound enriched in citrus fruits. Taken together, we concluded that BminOBP3 and 6 may play a prominent role in the process of B. minax mate-seeking and host-locating behaviors through recognizing and transporting these volatiles. It is conceivable that this study will increase our molecular understanding of B. minax olfaction, facilitating the development of OBP-based behavioral interference that is potentially useful for the integrated management of B. minax.

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.

Characterization and Levels of Expression of Sensory Neuron Membrane Proteins in the Adult Citrus Fruit Fly (Diptera: Tephritidae).

Sensory neuron membrane proteins (SNMPs) play an important role in insect chemoreception; however, the SNMPs for Bactrocera minax (Enderlein) (Diptera: Tephritidae), an economically important pest of citrus, remain uncharacterized. Here, we report on the molecular characterization of SNMPs (BminSNMP1 and BminSNMP2) from adult B. minax. The open-reading frames of BminSNMP1 and BminSNMP2 were 1,608 and 1,647 nucleotides, encoding proteins of 535 and 557 amino acid residues, respectively. Phylogenetic analysis showed that the two BminSNMPs belonged to two distinct subgroups, indicating the possibility of their contrasting function in insect chemoreception. Real-time PCR results showed that BminSNMP1 was expressed primarily in the antennae of males and females, where levels of expression were similar at different developmental stages of females, but lower in 1- and 5-d-old males than in 15- and 20-d-old males. In both sexes, BminSNMP2 was expressed at high levels in antennae and in nonolfactory tissues, especially in legs, where levels were higher than in other nonolfactory tissues. We found highest levels of expression of BminSNMP2 in antennae of both sexes in 30-d-old adults, while in legs of both sexes, highest levels of expression were detected in 1- and 30-d-old adults. We discuss the possible physiological functions of BminSNMPs based on our findings.

[Relationships between eggplant leaf pubescent and its resistance to Polyphagotarsonemus latus].

With 27 eggplant varieties as test objects, this paper studied the effects of their leaf pubescent on the field population density of Polyphagotarsonemus latus, its injury index on eggplant leaf, and population growth rate. The results showed that the density and length of leaf pubescent differed with eggplant varieties. For the same variety, leaf pubescence had a higher density but a shorter length on reverse side than on obverse side. Among the test varieties, 7 resistant varieties had a significantly higher mean pubescence density than 6 susceptive varieties on their reverse side leaf. The higher the pubescence density on reverse side eggplant leaf, the less field population density of P. latus and the lower injury index of eggplant leaf, suggesting a positive correlation between the pubescence density on reverse side eggplant leaf and the resistance of eggplant to P. latus.