Insect neurobiology: Oviposition crowd control.

A new study examines how Helicoverpa armigera females detect chemicals released by conspecific eggs in order to avoid laying more eggs on the same substrate. This work opens new avenues for basic research inquiries and offers a potential strategy for controlling insect pests.

Symbiotic bacteria confer insecticide resistance by metabolizing buprofezin in the brown planthopper, Nilaparvata lugens (Stål).

Buprofezin, a chitin synthesis inhibitor, is widely used to control several economically important insect crop pests. However, the overuse of buprofezin has led to the evolution of resistance and exposed off-target organisms present in agri-environments to this compound. As many as six different strains of bacteria isolated from these environments have been shown to degrade buprofezin. However, whether insects can acquire these buprofezin-degrading bacteria from soil and enhance their own resistance to buprofezin remains unknown. Here we show that field strains of the brown planthopper, Nilaparvata lugens, have acquired a symbiotic bacteria, occurring naturally in soil and water, that provides them with resistance to buprofezin. We isolated a symbiotic bacterium, Serratia marcescens (Bup_Serratia), from buprofezin-resistant N. lugens and showed it has the capacity to degrade buprofezin. Buprofezin-susceptible N. lugens inoculated with Bup_Serratia became resistant to buprofezin, while antibiotic-treated N. lugens became susceptible to this insecticide, confirming the important role of Bup_Serratia in resistance. Sequencing of the Bup_Serratia genome identified a suite of candidate genes involved in the degradation of buprofezin, that were upregulated upon exposure to buprofezin. Our findings demonstrate that S. marcescens, an opportunistic pathogen of humans, can metabolize the insecticide buprofezin and form a mutualistic relationship with N. lugens to enhance host resistance to buprofezin. These results provide new insight into the mechanisms underlying insecticide resistance and the interactions between bacteria, insects and insecticides in the environment. From an applied perspective they also have implications for the control of highly damaging crop pests.

Reverse genetic study reveals the molecular targets of chordotonal organ TRPV channel modulators.

Insecticides have been widely used for the control of insect pests that have a significant impact on agriculture and human health. A better understanding of insecticide targets is needed for effective insecticide design and resistance management. Pymetrozine, afidopyropen and flonicamid are reported to target on proteins that located on insect chordotonal organs, resulting in the disruption of insect coordination and the inhibition of feeding. In this study, we systematically examined the susceptibility of six Drosophila melanogaster mutants (five transient receptor potential channels and one mechanoreceptor) to three commercially used insecticides, in order to identify the receptor subunits critical to the insect's response to insecticides. Our results showed that iav1, nan36aand wtrw1 mutants exhibited significantly reduced susceptibility to pymetrozine and afidopyropen, but not to flonicamid. The number of eggs produced by the three mutant females were significantly less than that of the w1118 strain. Meanwhile, the longevity of all male mutants and females of nan36a and wtrw1 mutants was significantly shorter than that of the w1118 strain as the control. However, we observed no gravitaxis defects in wtrw1 mutants and the anti-gravitaxis of wtrw1 mutants was abolished by pymetrozine. Behavioral assays using thermogenetic tools further confirmed the bioassay results and supported the idea that Nan as a TRPV subfamily member located in Drosophila chordotonal neurons, acting as a target of pymetrozine, which interferes with Drosophila and causes motor deficits with gravitaxis defects. Taken together, this study elucidates the interactions of pymetrozine and afidopyropen with TRPV channels, Nan and Iav, and TRPA channel, Wtrw. Our research provides another evidence that pymetrozine and afidopyropen might target on nan, iav and wtrw channels and provides insights into the development of sustainable pest management strategies.

Risk assessment, fitness cost, cross-resistance, and mechanism of tetraniliprole resistance in the rice stem borer, Chilo suppressalis.

The rice stem borer (RSB), Chilo suppressalis, a notorious rice pest in China, has evolved a high resistance level to commonly used insecticides. Tetraniliprole, a new anthranilic diamide insecticide, effectively controls multiple pests, including RSB. However, the potential resistance risk of RSB to tetraniliprole is still unknown. In this study, the tetraniliprole-selection (Tet-R) strain was obtained through 10 continuous generations of selection with tetraniliprole 30% lethal concentration (LC30 ). The realized heritability (h2 ) of the Tet-R strain was 0.387, indicating that resistance of RSB to tetraniliprole developed rapidly under the continuous selection of tetraniliprole. The Tet-R strain had a high fitness cost (relative fitness = 0.53). We established the susceptibility baseline of RSB to tetraniliprole (lethal concentration at LC50  = 0.727 mg/L) and investigated the resistance level of 6 field populations to tetraniliprole. All tested strains that had resistance to chlorantraniliprole exhibited moderate- to high-level resistance to tetraniliprole (resistance ratio = 27.7-806.8). Detection of ryanodine receptor (RyR) mutations showed that the Y4667C, Y4667D, I4758M, and Y4891F mutations were present in tested RSB field populations. RyR mutations were responsible for the cross-resistance between tetraniliprole and chlorantraniliprole. Further, the clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9-mediated genome-modified flies were used to study the contribution of RyR mutations to tetraniliprole resistance. The order of contribution of a single RyR mutation to tetraniliprole resistance was Y4667D > G4915E > Y4667C ≈ I4758M > Y4891F. In addition, the I4758M and Y4667C double mutations conferred higher tetraniliprole resistance than single Y4667C mutations. These results can guide resistance management practices for diamides in RSB and other arthropods.

Resistance Monitoring of Nilaparvata lugens to Pymetrozine Based on Reproductive Behavior.

On the basis of the inhibition effects of pymetrozine on the reproductive behavior of N. lugens, we established a bioassay method to accurately evaluate the toxicity of pymetrozine in N. lugens and clarified the level of pymetrozine resistance of N. lugens in the field. In this study, pymetrozine's effects on the fecundity of N. lugens were evaluated using the topical application method and rice-seedling-dipping method. Moreover, the resistance of N. lugens to pymetrozine in a pymetrozine-resistant strain (Pym-R) and two field populations (YZ21 and QS21) was determined using the rice-seedling-dipping method and fecundity assay methods. The results showed that treatment of N. lugens third-instar nymphs with LC15, LC50, and LC85 doses of pymetrozine resulted in a significantly reduced fecundity of N. lugens. In addition, N. lugens adults treated with pymetrozine, using the rice-seedling-dipping and topical application method, also exhibited a significantly inhibited fecundity. Using the rice-stem-dipping method, pymetrozine resistance levels were shown to be high in Pym-R (194.6-fold), YZ21 (205.9-fold), and QS21 (212.8-fold), with LC50 values of 522.520 mg/L (Pym-R), 552.962 mg/L (YZ21), and 571.315 (QS21) mg/L. However, when using the rice-seedling-dipping or topical application fecundity assay method, Pym-R (EC50: 14.370 mg/L, RR = 12.4-fold; ED50: 0.560 ng/adult, RR = 10.8-fold), YZ21 (EC50: 12.890 mg/L, RR = 11.2-fold; ED50: 0.280 ng/adult; RR = 5.4-fold), and QS21 (EC50: 13.700 mg/L, RR = 11.9-fold) exhibited moderate or low levels of resistance to pymetrozine. Our studies show that pymetrozine can significantly inhibit the fecundity of N. lugens. The fecundity assay results showed that N. lugens only developed low to moderate levels of resistance to pymetrozine, indicating that pymetrozine can still achieve effective control on the next generation of N. lugens populations.

Insecticide resistance monitoring of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae) and its mechanism to chlorantraniliprole.

BACKGROUND: The rice leaffolder, Cnaphalocrocis medinalis (Guenée), has become an increasingly occurring pest in Asia in recent years. Chemical control remains the most efficient and primary tool for controlling this pest. In this study, we report the resistance status of C. medinalis in China to multiple insecticides including chlorantraniliprole and the main resistance mechanism. RESULTS: Significant variations among field populations of C. medinalis in their resistance to 10 insecticides were observed during 2019-2022. Most of the tested field populations have developed low-to-moderate levels of resistance to abamectin (RR = 2.4-22.2), emamectin benzoate (RR = 1.9-40.3) and spinetoram (RR = 4.2-24.8). Some field populations have developed low resistance to chlorpyrifos (RR = 0.9-6.8). Indoxacarb, metaflumizone, methoxenozide and Bacillus thuringiensis (Bt) potency against all tested populations remained similar. For diamides, significantly higher levels of resistance to chlorantraniliprole (RR = 64.9-113.7) were observed in 2022, whereas all tested field populations in 2019-2021 exhibited susceptible or moderate resistance level to chlorantraniliprole (RR = 1.3-22.1). Cross-resistance between chlorantraniliprole and tetraniliprole was significant. Analysis of ryanodine receptor (RyR) mutations showed that mutation of I4712M was present in resistant populations of C. medinalis with different levels of chlorantraniliprole resistance and was the main mechanism conferring diamide resistance. Mutation of Y4621D also was detected in one tested population. Resistance management strategies for the control of C. medinalis are discussed. CONCLUSION: C. medinalis has developed high level of resistance to chlorantraniliprole. RyR mutations were deemed as the mechanism. © 2023 Society of Chemical Industry.

Molecular and functional analysis of chitin synthase genes in Chilo suppressalis (Lepidoptera: Crambidae).

The rice stem borer, Chilo suppressalis, has developed a high level of resistance to many of the compounds currently used for control. There is therefore an urgent need to develop novel control methods for C. suppressalis. Insect chitin synthases (CHS) have attracted interest as a potential target for insect pest management. However, to date, CHS have not been characterized in C. suppressalis. Two CHS genes (CsCHS1 and CsCHS2) were identified and cloned from C. suppressalis. Two transcript variants were identified for CsCHS1, CsCHS1a and CsCHS1b. Spatiotemporal expression profiling showed that both transcripts of CsCHS1 are most highly expressed on the last day of each larval instar stage and show the highest expression levels in the integument. In contrast, CsCHS2 is predominantly expressed during the larval feeding stages and shows the highest expression levels in the midgut. Knockdown of CsCHS1 by RNA interference significantly inhibited the molting and pupation of C. suppressalis, and knockdown of CsCHS2 significantly affected growth during the larval stage, but had no significant effect on the pupation. Moreover, knockout of CsCHS1 by CRISPR/Cas9 genome editing severely lowered the hatching rate, larval survivorship, pupation rate, and eclosion rate, but only larval survivorship at the G0 generation was lowered after the knockout of CsCHS2. These results demonstrate that CsCHS1 and CsCHS2 play vital roles in the growth and development of C. suppressalis, and so have potential as insecticidal targets for the control of this highly damaging pest.

Knockout of the odorant receptor co-receptor, orco, impairs feeding, mating and egg-laying behavior in the fall armyworm Spodoptera frugiperda.

The olfactory transduction system of insects is involved in multiple behavioral processes such as foraging, mating, and egg-laying behavior. In the insect olfactory receptor neurons (ORNs), the odorant receptor co-receptor (Orco) is an obligatory component that is required for dimerization with odorant receptors (ORs) to form a ligand-gated ion channel complex. The ORs/Orco heteromeric complex plays a crucial role in insect olfaction. To explore the function of OR-mediated olfaction in the physiological behavior of the fall armyworm, Spodoptera frugiperda, we applied CRISPR/Cas9 genome editing to mutate its Orco gene and constructed a homozygous mutant strain of Orco (Orco-/-) by genetic crosses. Electroantennogram (EAG) analysis showed that the responses of Orco-/- male moths to two universal sex pheromones, Z9-14: Ac and Z7-12: Ac, were abolished. We found that Orco-/- males cannot successfully mate with female moths. An oviposition preference assay confirmed that Orco-/- female moths had a reduced preference for the optimal host plant maize. A larval feeding assay revealed that the time for Orco-/- larvae to locate the food source was significantly longer than in the wild-type. Overall, in the absence of Orco, the OR-dependent olfactory behavior was impaired in both larval and adult stages. Our results confirm that Orco is essential for multiple behavioral processes related to olfaction in the fall armyworm.

The overexpression of cytochrome P450 genes confers buprofezin resistance in the brown planthopper, Nilaparvata lugens (Stål).

BACKGROUND: Buprofezin, an insect growth regulator, has been widely used to control brown planthopper (BPH), Nilaparvata lugens, one of the most destructive pests of rice crops in Asia. The intensive use of this compound has resulted in very high levels of resistance to buprofezin in the field, however, the underpinning mechanisms of resistance have not been fully resolved. RESULTS: Insecticide bioassays using the P450 inhibitor piperonyl butoxide significantly synergized the toxicity of buprofezin in two resistant strains of BPH (BPR and YC2017) compared to a susceptible strain (Sus), suggesting P450s play a role in resistance to this compound. Whole transcriptome profiling identified 1110 genes that were upregulated in the BPR strain compared to the Sus strain, including 13 cytochrome P450 genes, eight esterases and one glutathione S-transferase. Subsequently, qPCR validation revealed that four of the P450 genes, CYP6ER1vA, CYP6CW1, CYP4C77, and CYP439A1 were significantly overexpressed in both the BRP and YC2017 strains compared with the Sus strain. Further functional analyses showed that only suppression of CYP6ER1vA, CYP6CW1, and CYP439A1 gene expression by RNA interference significantly increased the toxicity of buprofezin against BPH. However, only transgenic Drosophila melanogaster expressing CYP6ER1vA and CYP439A1 exhibited significant resistance to buprofezin. Finally, the BPR strain was found to exhibit modest but significant levels of resistance to acetamiprid, dinotefuran and pymetrozine. CONCLUSIONS: Our findings provide strong evidence that the overexpression of CYP6ER1vA and CYP439A1 contribute to buprofezin resistance in BPH, and that resistance to this compound is associated with low-level resistance to acetamiprid, dinotefuran and pymetrozine. These results advance understanding of the molecular basis of BPH resistance to buprofezin and will inform the development of management strategies for the control of this highly damaging pest. © 2022 Society of Chemical Industry.

Function of Transient Receptor Potential-Like Channel in Insect Egg Laying.

The transient receptor potential-like channel (TRPL) is a member of the transient receptor potential (TRP) channel family involved in regulating many fundamental senses, such as vision, pain, taste, and touch, in both invertebrates and vertebrates. Yet, the function of TRPL in other important biological processes remains unclear. We discover that TRPL regulates egg laying in two insect species, the brown planthopper, Nilaparvata lugens, and the fruit fly, Drosophila melanogaster. In both insects, trpl is expressed in the female reproductive organ. Loss of trpl leads to significantly defects in egg laying. In addition, TRPL is functionally interchangeable between the brown planthoppers and flies in egg laying. Altogether, our work uncovers a novel role played by TRPL in regulating egg laying and indicates TRPL as a potential pesticide target in brown planthoppers.

Monitoring, cross-resistance, inheritance, and fitness costs of brown planthoppers, Nilaparvata lugens, resistance to pymetrozine in China.

BACKGROUND: The brown planthopper, Nilaparvata lugens, is considered the most destructive pest of rice in many Asian countries including China. Use of pymetrozine in insect resistance management (IRM) has been one strategy to control this pest. In this study, we reported the status of pymetrozine resistance in Nilaparvata lugens (Stål) collected from China over the period 2017-2021 and selected a strain of N. lugens resistant to pymetrozine and evaluated the cross-resistance, inheritance and fitness costs of the resistance. RESULTS: Monitoring data (2017-2021) showed that field populations of N. lugens in China developed moderate- to high-level pymetrozine resistance during these 5 years. By continuous selection with pymetrozine in the lab, the pymetrozine selected N. lugens strain (Pym-R98 ) developed a 225.2-fold resistance compared to a susceptible strain. The Pym-R98 strain showed high cross-resistance to dinotefuran (66.6-fold) and low cross-resistance to nitenpyram (5.2-fold) and sulfoxaflor (5.8-fold). Inheritance pattern analysis of Pym-R93 revealed that resistance to pymetrozine was polygenic, autosomal and incompletely dominant. Fitness costs of pymetrozine resistance were present in Pym-R90 and WA2020 strains with a relative fitness of 0.72 and 0.60, respectively. The developmental duration of Pym-R90 and WA2020 was significantly longer and hatchability was significantly lower compared to pymetrozine-susceptible strain (Pym-S). CONCLUSIONS: N. lugens has developed high level of resistance to pymetrozine. Pymetrozine-resistance brown planthopper had cross-resistance with some of neonicotinoids such as dinotefuran, nitenpyram and sulfoxaflor. The autosomal, incompletely dominant and polygenic resistance to pymetrozine in N. lugens and the fitness costs associated with this resistance can be exploited in IRM strategies to preserve the lifetime of pymetrozine for control of N. lugens in China. © 2022 Society of Chemical Industry.

Effects of nicotinic acetylcholine receptor subunit deletion mutants on insecticide susceptibility and fitness in Drosophila melanogaster.

BACKGROUND: Nicotinic acetylcholine receptors (nAChRs) are major excitatory neurotransmitter receptors in insects and also the target site for many insecticides. Unfortunately, the effectiveness of these insecticides is diminishing as a consequence of the evolution of insecticide resistance. Further exploration of insecticide targets is important to sustainable pest management. RESULTS: In order to validate the role of nAChR subunits in insecticide susceptibility and test whether the subunit's absence imposes the fitness cost on insects, we determined the susceptibility of eight nAChR subunit deletion mutants of Drosophila melanogaster to nine insecticides. These findings highlighted the specific resistance of the Dα6 deletion mutant to spinosyns. Although triflumezopyrim, dinotefuran and imidacloprid are competitive modulators of nAChRs, differences in susceptibility of the insect with different deletion mutants suggested that the target sites of these three insecticides do not overlap completely. Mutants showed decreased susceptibility to insecticides, accompanied by a reduction in fitness. The number of eggs produced by Dα1attP , Dα2attP , Dß2attP and Dß3attP females was significantly lesser than that of the vas-Cas9 strain as the control. In addition, adults of Dα2attP , Dα3attP and Dα7attP strains showed lower climbing performance. Meanwhile, males of Dα3attP , Dα5attP , Dß2attP and Dß3attP , and females of Dß2attP showed significantly shorter longevity than those of the vas-Cas9 strain. CONCLUSION: This study provides new insights into the interactions of different insecticides with different nAChRs subunit in D. melanogaster as a research model, it could help better understand such interaction in agricultural pests whose genetic manipulations for toxicological research are often challenging. © 2022 Society of Chemical Industry.

Cholecystokinin/sulfakinin peptide signaling: conserved roles at the intersection between feeding, mating and aggression.

Neuropeptides are the most diverse messenger molecules in metazoans and are involved in regulation of daily physiology and a wide array of behaviors. Some neuropeptides and their cognate receptors are structurally and functionally well conserved over evolution in bilaterian animals. Among these are peptides related to gastrin and cholecystokinin (CCK). In mammals, CCK is produced by intestinal endocrine cells and brain neurons, and regulates gall bladder contractions, pancreatic enzyme secretion, gut functions, satiety and food intake. Additionally, CCK plays important roles in neuromodulation in several brain circuits that regulate reward, anxiety, aggression and sexual behavior. In invertebrates, CCK-type peptides (sulfakinins, SKs) are, with a few exceptions, produced by brain neurons only. Common among invertebrates is that SKs mediate satiety and regulate food ingestion by a variety of mechanisms. Also regulation of secretion of digestive enzymes has been reported. Studies of the genetically tractable fly Drosophila have advanced our understanding of SK signaling mechanisms in regulation of satiety and feeding, but also in gustatory sensitivity, locomotor activity, aggression and reproductive behavior. A set of eight SK-expressing brain neurons plays important roles in regulation of these competing behaviors. In males, they integrate internal state and external stimuli to diminish sex drive and increase aggression. The same neurons also diminish sugar gustation, induce satiety and reduce feeding. Although several functional roles of CCK/SK signaling appear conserved between Drosophila and mammals, available data suggest that the underlying mechanisms differ.

Inheritance and fitness cost of buprofezin resistance in a near-isogenic, field-derived strain and insecticide resistance monitoring of Laodelphax striatellus in China.

BACKGROUND: Laodelphax striatellus is one of the most destructive pests of rice and other cereal crops. Chemical control is still the most efficient way to control this pest, but insecticide resistance always threatens this approach. RESULTS: Monitoring data (2003-2020) showed that Chinese field populations of L. striatellus developed high-level buprofezin resistance within the first four years. This high-level resistance to buprofezin was stable for about ten years and persisted even when buprofezin selection pressure was absent. An established near-isogenic strain (YN-NIS) with 90.8-fold resistance to buprofezin had resistance inheritance of autosomal and incomplete dominance, and the resistance was controlled by multiple genes with no obvious fitness costs (relative fitness of 0.8707). Furthermore, the susceptibility of 29 field populations to another seven insecticides (2014-2020) showed that: (i) low-level resistance to pymetrozine, dinotefuran, sulfoxaflor and thiamethoxam was first detected in 2014 (eight years after introduction), 2016 (three years after), 2017 (four years after) and 2019 (19 years after), respectively, (ii) moderate resistance levels to chlorpyrifos were found for all populations across multiple years, and (iii) no resistance was detected for nitenpyram and triflumezopyrim. CONCLUSION: The fast buprofezin resistance development in L. striatellus would be caused by incomplete dominant resistance with almost no fitness cost in the resistant strain. Nitenpyram and triflumezopyrim showed no resistance and can be used as the main insecticide for the control of L. striatellus. These findings provide key fundamental information for controlling L. striatellus.

Cholecystokinin-like peptide mediates satiety by inhibiting sugar attraction.

Feeding is essential for animal survival and reproduction and is regulated by both internal states and external stimuli. However, little is known about how internal states influence the perception of external sensory cues that regulate feeding behavior. Here, we investigated the neuronal and molecular mechanisms behind nutritional state-mediated regulation of gustatory perception in control of feeding behavior in the brown planthopper and Drosophila. We found that feeding increases the expression of the cholecystokinin-like peptide, sulfakinin (SK), and the activity of a set of SK-expressing neurons. Starvation elevates the transcription of the sugar receptor Gr64f and SK negatively regulates the expression of Gr64f in both insects. Interestingly, we found that one of the two known SK receptors, CCKLR-17D3, is expressed by some of Gr64f-expressing neurons in the proboscis and proleg tarsi. Thus, we have identified SK as a neuropeptide signal in a neuronal circuitry that responds to food intake, and regulates feeding behavior by diminishing gustatory receptor gene expression and activity of sweet sensing GRNs. Our findings demonstrate one nutritional state-dependent pathway that modulates sweet perception and thereby feeding behavior, but our experiments cannot exclude further parallel pathways. Importantly, we show that the underlying mechanisms are conserved in the two distantly related insect species.

Double ryanodine receptor mutations confer higher diamide resistance in rice stem borer, Chilo suppressalis.

BACKGROUND: The striped rice stem borer, Chilo suppressalis (Lepidoptera: Pyraidae), is one of the most serious rice pests in China. Chlorantraniliprole was used extensively for C. suppressalis control over the past ten years, and some field populations have developed high resistance. In this study, we report the chlorantraniliprole resistance status of C. suppressalis in China and the resistance mechanism. RESULTS: Significant geographical variations of chlorantraniliprole susceptibility were observed among 28 C. suppressalis field populations in 2019-2020. The LC50 values varied from 2907.874 mg L-1 (XS19) to 1.524 mg L-1 (QW19). Most tested field populations collected from Zhejiang, Jiangxi, Hunan and Anhui provinces in 2020 showed a high level of resistance to chlorantraniliprole (RR = 311.9-2060.1), whereas Jiangsu and Sichuan province populations remained susceptible. Analysis of RyR mutations showed that mutations of I4758M, Y4667D, Y4667C and Y4891F were present in resistant populations of C. suppressalis with different levels of chlorantraniliprole resistance. The frequency of the Y4667C mutation was correlated with chlorantraniliprole resistance in YY19 (RR = 702.6) and YY20 (RR = 1426.8) populations, with the homozygous mutation frequencies of 15.6% and 29.4%, respectively. High contributions of the I4758M and Y4667C double mutation to diamide resistance was demonstrated with CRISPR/Cas9-modified D. melanogaster. Flies bearing the Y4667C mutation (I4758M and Y4667C double mutation in C. suppressalis) exhibited high resistance to chlorantraniliprole (RR = 172.1), and moderate resistance to cyantraniliprole (RR = 79.2) and tetra chlorantraniliprole (RR = 43.6), which were higher than that of single mutations. CONCLUSIONS: Chlorantraniliprole resistance in C. suppressalis is intensifying in China. RyR double mutations (i.e. I4758M and Y4667C) confer higher diamide resistance than single mutations.

Mechanism of metabolic resistance to pymetrozine in Nilaparvata lugens: over-expression of cytochrome P450 CYP6CS1 confers pymetrozine resistance.

BACKGROUND: Pymetrozine is commonly used for the control of Nilaparvata lugens, and resistance to pymetrozine has been frequently reported in the field populations in recent years. However, the mechanism of brown planthopper resistance to pymetrozine is still unknown. RESULTS: In this study, a pymetrozine-resistant strain (PMR) was established, and the potential biochemical resistance mechanism of N. lugens to pymetrozine was investigated. Pymetrozine was synergized by the inhibitor piperonyl butoxide (PBO) in the PMR with 2.83-fold relative synergistic ratios compared with the susceptible strain (Sus). Compared with the Sus, the cytochrome P450 monooxygenase activity of PMR was increased by 1.7 times, and two P450 genes (NlCYP6CS1 and NlCYP301B1) were found to be significantly overexpressed more than 6.0-fold in the PMR. Pymetrozine exposure induced upregulation of NlCYP6CS1 expression in the Sus, but the expression of NlCYP301B1 did not change significantly. In addition, RNA interference (RNAi)-mediated suppression of NlCYP6CS1 gene expression dramatically increased the toxicity of pymetrozine against N. lugens. Moreover, transgenic lines of Drosophila melanogaster expressing NlCYP6CS1 were less susceptible to pymetrozine, and had a stronger ability to metabolize pymetrozine. CONCLUSIONS: Taken together, our findings indicate that the overexpression of NlCYP6CS1 is one of the key factors contributing to pymetrozine resistance in N. lugens. And this result is helpful in proposing a management strategy for pymetrozine resistance.

Monitoring and mechanisms of insecticide resistance in Spodoptera exigua (Lepidoptera: Noctuidae), with special reference to diamides.

The beet armyworm, Spodoptera exigua, is a major lepidopteran pest of global importance in cultivation of numerous crops including cotton, maize, soybean, onion, cabbage, and ornamentals. It has evolved resistance to different insecticides. However, the current status of insecticide resistance in S. exigua has not been well examined in China. In this study, concentration-mortality responses of S. exigua to seven insecticides, including chlorantraniliprole, tetraniliprole, methoxyfenozide, indoxacarb, chlorfenapyr, emamectin benzoate and beta-cypermethrin were evaluated. The results showed that most of the tested populations had developed moderate to high resistance to chlorantraniliprole, with resistance ratios ranging from 6.3 to 2477.3-fold. Our results also showed that chlorantraniliprole have cross-resistance with tetraniliprole in S. exigua. The AY19 population collected from Anyang in Henan Province in 2019 exhibited a high resistance level to beta-cypermethrin (RR = 277.5). Methoxyfenozide and chlorfenapyr were highly effective against all of the tested populations with resistance ratios (RR) ranging from 0.1 to 2.2-fold. One of the tested populations showed moderate resistance to indoxacarb and emamectin benzoate. We detected the known ryanodine receptor target site resistance mutation, I4743M, in the field populations of S. exigua with different levels of diamide resistance.

Molecular characterizations and expression profiles of transient receptor potential channels in the brown planthopper, Nilaparvata lugens.

Transient receptor potential (TRP) is a superfamily of important cation channels located on the cell membrane. It can regulate almost all sensory modality and control a series of behaviors, including hearing, locomotion, gentle touch, temperature sensation, dry air and food texture detection. The expression profiles of TRP channels have been well documented in the model insect Drosophila melanogaster. However, little is known about the TRP channels of agricultural pests. In this study, we cloned 9 TRP ion channel genes from brown planthopper. Their amino acid sequences are highly conserved with homologues of other insects and have typical TRP channel characteristics: six transmembrane domains (TM1 - TM6) and a pore region between TM5 and TM6. These TRP channels of N. lugens were expressed in all developmental stages and various body parts. The expression levels of almost all TRP channels were relatively higher in adults than nymph stages, and lowest in the eggs. Antenna and abdomen were the main body parts with high expression of these genes. Furthermore, the mRNA levels of these TRP genes were significantly decreased in the third-instar nymphs injected with double-stranded RNA (dsRNA). The survival rate of different TRP dsRNA injected nymphs all exceeded 81%, which was no significant difference compared with the control group. These results suggested that these 9 TRP channels are expressed throughout the body and all ages of the brown planthopper, and are involved in regulating multiple physiological and behavioral processes. The identification of TRP channel genes in this study not only provides a foundation for further exploring the potential roles of TRP channels, but also serves as targets to develop new insecticides for the control of agricultural pests.

Leucokinins: Multifunctional Neuropeptides and Hormones in Insects and Other Invertebrates.

Leucokinins (LKs) constitute a neuropeptide family first discovered in a cockroach and later identified in numerous insects and several other invertebrates. The LK receptors are only distantly related to other known receptors. Among insects, there are many examples of species where genes encoding LKs and their receptors are absent. Furthermore, genomics has revealed that LK signaling is lacking in several of the invertebrate phyla and in vertebrates. In insects, the number and complexity of LK-expressing neurons vary, from the simple pattern in the Drosophila larva where the entire CNS has 20 neurons of 3 main types, to cockroaches with about 250 neurons of many different types. Common to all studied insects is the presence or 1-3 pairs of LK-expressing neurosecretory cells in each abdominal neuromere of the ventral nerve cord, that, at least in some insects, regulate secretion in Malpighian tubules. This review summarizes the diverse functional roles of LK signaling in insects, as well as other arthropods and mollusks. These functions include regulation of ion and water homeostasis, feeding, sleep-metabolism interactions, state-dependent memory formation, as well as modulation of gustatory sensitivity and nociception. Other functions are implied by the neuronal distribution of LK, but remain to be investigated.