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.

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.

The G932C mutation of chitin synthase 1 gene (CHS1) mediates buprofezin resistance as confirmed by CRISPR/Cas9-mediated knock-in approach in the brown planthopper, Nilaparvata lugens.

The brown planthopper (Nilaparvata lugens) is a major destructive rice pest in Asia. High levels of insecticide resistance have been frequently reported, and the G932C mutation in the chitin synthase 1 (CHS1) gene has been found to mediate buprofezin resistance. However, there has been no direct evidence to confirm the functional significance of the single G932C substitution mutation leading to buprofezin resistance in N. lugens. Here, we successfully constructed a knock-in homozygous strain (Nl-G932C) of N. lugens using CRISPR/Cas9 coupled with homology-directed repair (HDR). Compared with the background strain susceptible to buprofezin (Nl-SS), the knock-in strain (Nl-G932C) showed a 94.9-fold resistance to buprofezin. Furthermore, resistant strains (Nl-932C) isolated from the field exhibited a 2078.8-fold resistance to buprofezin, indicating that there are other mechanisms contributing to buprofezin resistance in the field. Inheritance analysis showed that the resistance trait is incomplete dominance. In addition, the Nl-G932C strain had a relative fitness of 0.33 with a substantially decreased survival rate, emergence rate, and fecundity. This study provided in vivo functional evidence for the causality of G932C substitution mutation of CHS1 with buprofezin resistance and valuable information for facilitating the development of resistance management strategies in N. lugens. This is the first example of using CRISPR/Cas9 gene-editing technology in a hemipteran insect to directly confirm the role of a candidate target site mutation in insecticide resistance.

The role of the Bemisia tabaci and Trialeurodes vaporariorum cytochrome-P450 clade CYP6DPx in resistance to nicotine and neonicotinoids.

The alkaloid, nicotine, produced by tobacco and other Solanaceae as an anti-herbivore defence chemical is one of the most toxic natural insecticides in nature. However, some insects, such as the whitefly species, Trialeurodes vaporariorum and Bemisia tabaci show strong tolerance to this allelochemical and can utilise tobacco as a host. Here, we used biological, molecular and functional approaches to investigate the role of cytochrome P450 enzymes in nicotine tolerance in T. vaporariorum and B. tabaci. Insecticide bioassays revealed that feeding on tobacco resulted in strong induced tolerance to nicotine in both species. Transcriptome profiling of both species reared on tobacco and bean hosts revealed profound differences in the transcriptional response these host plants. Interrogation of the expression of P450 genes in the host-adapted lines revealed that P450 genes belonging to the CYP6DP subfamily are strongly upregulated in lines reared on tobacco. Functional characterisation of these P450s revealed that CYP6DP1 and CYP6DP2 of T. vaporariorum and CYP6DP3 of B. tabaci confer resistance to nicotine in vivo. These three genes, in addition to the B. tabaci P450 CYP6DP5, were also found to confer resistance to the neonicotinoid imidacloprid. Our data provide new insight into the molecular basis of nicotine resistance in insects and illustrates how divergence in the evolution of P450 genes in this subfamily in whiteflies may have impacted the extent to which different species can tolerate a potent natural insecticide.

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.

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.

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.

Susceptibility of fall armyworm, Spodoptera frugiperda (J.E.Smmith), to eight insecticides in China, with special reference to lambda-cyhalothrin.

Fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), is the main destructive insect pest of grain crops that occurs in all maize growing regions of the Americas. It has rapidly invaded the Southern China since January 2019. However, the current status of insecticide resistance in S. frugiperda has not been reported in China. In this study, we determined the susceptibility of eight populations of FAW to eight insecticides by an artificial diet incorporation method. The results showed that among eight insecticides, emamectin benzoate, spinetoram, chlorantraniliprole, chlorfenapyr, and lufenuron showed higher toxicity to this pest, while lambda-cyhalothrin and azadirachtin exhibited lower toxicity. Susceptibility of S. frugiperda to indoxacarb was significantly different (10.0-fold for LC50) across the various geographic populations. To investigate the biochemical mechanism of FAW to lambda-cyhalothrin, we performed the synergism tests and the results showed that piperonyl butoxide (PBO) and triphenyl phosphate (TPP) produced a high synergism of lambda-cyhalothrin effects in the two field populations. Sequencing of the gene encoding the acetylcholinesterase (AChE) gene in the two field populations identified two amino acid mutations, all of which have been shown previously to confer resistance to organophosphates (OPs) in several arthropod species. The results of this study provided valuable information for choosing alternative insecticides and for insecticide resistance management of S. frugiperda.

Baseline determination, susceptibility monitoring and risk assessment to triflumezopyrim in Nilaparvata lugens (Stål).

Triflumezopyrim, a novel mesoionic chemical insecticide, is promoted as a powerful tool for control of susceptible and resistant hopper species in rice throughout Asia. For a newly commercialized insecticide it is important to establish susceptibility baseline, conduct susceptibility monitoring, and assess the risk of resistance via artificial selection to provide foundational information on designing resistance management strategy. The susceptibility baseline of triflumezopyrim was established for three rice planthopper species, Nilarpavata lugens (Stål), Sogatella furcifera (Horváth) and Laodelphax striatellus (Fallén). The LD50 of triflumezopyrim was 0.026, 0.032 and 0.094 ng/individual for the adults of the susceptible strains of S. furcifera, L. striatellus and N. lugens, respectively, determined by a topical application method. Using a rice stem (seedling) dipping method, the LC50 was determined as 0.042, 0.024 and 0.150 mg/L for the nymphs (3rd instar) of the three hopper species, respectively. In the meanwhile, the LC50 of Pyraxalt™ (triflumezopyrim 10% SC) was 0.064 mg/L for the N. lugens susceptible strain. Furthermore, the susceptibility of triflumezopyrim and other five neonicotinoid insecticides were monitored for N. lugens field populations collected from major rice production areas in China in 2015-2019. All monitored populations were susceptible to triflumezopyrim (0.5 to 3.9-fold resistance ratio), and showed no cross-resistance to the other five neonicotinoids. These results suggested that triflumezopyrim is a good option to control resistant N. lugens. In addition, a field-collected population of N. lugens was artificially selected with triflumezopyrim for 20 generations and resulted in 3.5-fold increase in LC50 from F0 and 6.0-fold increase from that of the susceptible strain. The realized heritability (h2) of resistance was estimated as 0.0451 by using threshold trait analysis. With this h2 value, the projected triflumezopyrim resistance development (a 10-fold increase in LC50) would be expected after 30.3 or 24.0 generations if 80% or 90% of the population was killed at each generation.

Pymetrozine inhibits reproductive behavior of brown planthopper Nilaparvata lugens and fruit fly Drosophila melanogaster.

Pymetrozine is a promising chemical used to control brown planthopper, which developed resistance to imidacloprid and buprofezin in the past decade. Field efficacy indicates that pymetrozine can reduce the number of offsprings of brown planthopper, but the specific physiological mechanism is unknown. In this study, we systematically described the mating process of brown planthopper including 8 steps (abdominal vibration, following, positioning, wing extension, attempted copulation, copulation, terminated copulation and leaving) and explored the optimal mating time after adult eclosion (3-5 days) and observation time (30 mins). Also, behavioral data showed that pymetrozine can affect the mating behavior and female fecundity of brown planthopper and fruit fly. As one of the target genes for pymetrozine, Nanchung (Nan), the nan36a mutant male courtship index, female receptivity and the number of offsprings were significantly decreased. Behavioral defects in nan36a mutant flies can be rescued by expressed NlNan. Our results indicated that Nan plays essential roles in the mating behavior and female fecundity. These findings provide useful information for demonstrating that pymetrozine effectively reduce the reproduction of brown planthopper and contribute to our understanding of reproductive strategies controlled by pymetrozine in insects.

Pymetrozine activates TRPV channels of brown planthopper Nilaparvata lugens.

The commercial insecticide pymetrozine has been extensively used for brown planthopper control in East Asia. The transient receptor potential vanilloid (TRPV) channel, which consists of two proteins, Nanchung (Nan) and Inactive (Iav), has recently been shown to be the molecular target of pymetrozine in the fruit fly (Drosophila melanogaster) and pea aphid (Acyrthosiphon pisum). In this study, we characterized the Nan and Iav TRPV channel subunits of N. lugens and measured the action of pymetrozine on them. NlNan and NlIav are structurally similar to homologs from other insects. The expression pattern analysis of various body parts showed that NlNan and NlIav were both more abundantly expressed in antennae. When NlNan and NlIav were co-expressed in Xenopus laevis oocytes, they formed channels with high sensitivity to pymetrozine (EC50 = 5.5 × 10-8 M). Behavioral observation revealed that the gravitaxis defect in the fruit fly nan36a mutant was rescued by ectopically expressed NlNan and the rescued behavior could be abolished by pymetrozine. Our results confirm that NlNan and NlIav co-expressed complexes can be activated by pymetrozine both in vitro and in vivo and provide useful information for future resistance mechanism studies.

Molecular characterization and expression profiling of ryanodine receptor gene in the pink stem borer, Sesamia inferens (Walker).

The susceptibilities of three field populations of pink stem borer (PSB), Sesamia inferens (walker) to diamide insecticides, chlorantraniliprole and flubendiamide, were evaluated in this study. The results showed that these PSB field populations were still sensitive to the two diamide insecticides after many years of exposure. To further understand PSB and diamide insecticide, the full-length ryanodine receptor (RyR) cDNA (named as SiRyR), the molecular target of diamide insecticides was cloned from PSB and characterized. The SiRyR gene contains an open reading frame of 15,420 nucleotides, encoding 5140 amino acid residues, which shares 77% to 98% sequence identity with RyR homologous of other insects. All hallmarks of RyR proteins are conserved in the SiRyR protein, including the conserved C-terminal domain with the consensus calcium-biding EF-hands (calcium-binding motif), the six transmembrane domains, as well as mannosyltransferase, IP3R and RyR (pfam02815) (MIR) domains. Real-time qPCR analysis revealed that the highest mRNA expression levels of SiRyR were observed in pupa and adults, especially in males. SiRyR was expressed at the highest level in thorax, and the lowest level in wing. The full genetic characterization of SiRyR could provide useful information for future functional expression studies and for discovery of new insecticides with selective insecticidal activity.

Molecular characterization and expression pattern of three GABA receptor-like subunits in the small brown planthopper Laodelphax striatellus (Hemiptera: Delphacidae).

Ionotropic γ-aminobutyric acid (GABA)-gated chloride channel receptors mediate rapid inhibitory neurotransmission in vertebrates and invertebrates. GABA receptors are well known to be the molecular targets of synthetic insecticides or parasiticides. Three GABA receptor-like subunits, LsLCCH3, LsGRD and LS8916, of the small brown planthopper, Laodelphax striatellus (Fallén), a major insect pest of crop systems in East Asia, had been identified and characterized in this study. All three genes were cloned using the reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). They shared common structural features with known Cys-loop ligand-gated ion channels (LGICs): the well-conserved dicysteine-loop structures, an extracellular N-terminal domain containing six distinct regions (loops A-F) that form the ligand binding sites and four transmembrane regions (TM1-4). Additionally, temporal and spatial transcriptional profiling analysis indicated that Lslcch3 was significantly higher than the other two genes. All of them were expressed at higher levels in fifth-instar nymph and adults than in eggs and from first- to fourth-instar nymph. They were predominantly expressed in the heads of 2-d old female adults. These findings enhanced our understanding of cys-loop LGIC functional characterization in Hemiptera and provided a useful basis for the development of improved insecticides that targeting this important agricultural pest.

Resistance monitoring and cross-resistance patterns of three rice planthoppers, Nilaparvata lugens, Sogatella furcifera and Laodelphax striatellus to dinotefuran in China.

Three rice planthoppers, brown planthopper, Nilaparvata lugens, white-backed planthopper, Sogatella furcifera and small brown planthopper, Laodelphax striatellus, are important pests of cultivated rice in tropical and temperate Asia. They have caused severe economic loss and developed resistance to insecticides from most chemical classes. Dinotefuran is the third neonicotinoid which possesses a broad spectrum and systemic insecticidal activity. We determined the susceptibility of dinotefuran to field populations from major rice production areas in China from 2013 to 2015. All the populations of S. furcifera and L. striatellus were kept susceptible to dinotefuran (0.7 to 1.4-fold of S. furcifera and 1.1-to 3.4-fold of L. striatellus) However, most strains of N. lugens (except FQ15) collected in 2015 had developed moderate resistance to dinotefuran, with resistance ratios (RR) ranging from 23.1 to 100.0 folds. Cross-resistance studies showed that chlorpyrifos-resistant and buprofezin-resistant Sogatella furcifera, chlorpyrifos-resistant and fipronil-resistant L. striatellus, imidacloprid-resistant and buprofezin-resistant Nilaparvata lugens exhibited negligible or no cross-resistance to dinotefuran. Synergism tests showed that piperonyl butoxide (PBO) produced a high synergism of dinotefuran effects in the DY15 and JS15 populations (2.14 and 2.52-fold, respectively). The obvious increase in resistance to dinotefuran in N. lugens indicates that insecticide resistance management strategies are urgently needed to prevent or delay further increase of insecticide resistance in N. lugens.

Knockdown of the ionotropic γ-aminobutyric acid receptor (GABAR) RDL gene decreases fipronil susceptibility of the small brown planthopper, Laodelphax striatellus (Hemiptera: Delphacidae).

Insect γ-aminobutyric acid receptors (GABARs) are important molecular targets of cyclodiene and phenylpyrazole insecticides. Previously GABARs encoding rdl (resistant to dieldrin) genes responsible for dieldrin and fipronil resistance were identified in various economically important insect pests. In this study, we cloned the open reading frame cDNA sequence of rdl gene from fipronil-susceptible and fipronil-resistant strains of Laodelphax striatellus (Lsrdl). Sequence analysis confirmed the presence of a previously identified resistance-conferring mutation. Different alternative splicing variants of Lsrdl were noted. Injection of dsLsrdl reduced the mRNA abundance of Lsrdl by 27-82%, and greatly decreased fipronil-induced mortality of individuals from both susceptible and resistant strains. These data indicate that Lsrdl encodes a functional RDL subunit that mediates susceptibility to fipronil. Additionally, temporal and spatial expression analysis showed that Lsrdl was expressed at higher levels in eggs, fifth-instar nymphs, and female adults than in third-instar and fourth-instar nymphs. Lsrdl was predominantly expressed in the heads of 2-day-old female adults. All these results provide useful background knowledge for better understanding of fipronil resistance related ionotropic GABA receptor rdl gene expressed variants and potential functional differences in insects.

Comparison of Insecticide Susceptibilities of Empoasca vitis (Hemiptera: Cicadellidae) from Three Main Tea-Growing Regions in China.

Empoasca vitis (Göthe) is an important insect pest in tea-growing areas of China, and chemical control is the main tactic for the management of this pest. Due to the pressure of increasing insecticide resistance and more stringent food safety regulations, development of sound IPM strategies for E. vitis is an urgent matter. This study comparatively evaluated four field populations of E. vitis from three different tea-growing regions in China for their susceptibilities to eight insecticides using a simple leaf-dip methodology. E. vitis was found to be most sensitive to indoxacarb (LC50<0.5 mg/liter) and least sensitive to isoprocarb (LC50>5 mg/liter) and sophocarpidine (LC50>95 mg/liter, a botanical pesticide) regardless of populations. Population (geographical) variations were higher for indoxacarb and imidacloprid than other compounds. Judging by the 95% fiducial limits of LC50 values, all populations had similar susceptibilities to chlorfenapyr, bifenthrin, and acetamiprid or imidacloprid. Correlation analysis suggested that chlorfenapyr and indoxacarb or isoprocarb may have a high risk of cross resistance. Considering potency (LC50) and maximum residual levels, chlorfenapyr and bifenthrin are good insecticide options followed by acetamiprid and indoxacarb. These results provide valuable information to intelligently select insecticides for IPM programs that are efficacious against E. vitis while also managing insecticide resistance and maximum residual levels for tea production in China.

Thiamethoxam resistance selected in the western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae): cross-resistance patterns, possible biochemical mechanisms and fitness costs analysis.

The western flower thrips (WFT) Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), an important pest of various crops in the world, has invaded China since 2003. To understand the risks and to determine possible mechanisms of resistance to thiamethoxam in WFT, a resistant strain was selected under the laboratory conditions. Cross-resistance and the possible biochemical resistance mechanisms were investigated in this study. A 15.1-fold thiamethoxam-resistant WFT strain (TH-R) was established after selection for 55 generations. Compared with the susceptible strain (TH-S), the selected TH-R strain showed extremely high level cross-resistance to imidaclothiz (392.1-fold) and low level cross-resistance to dinotefuran (5.7-fold), acetamiprid (2.9-fold) and emamectin benzoate (2.1-fold), respectively. No cross-resistance to other fourteen insecticides was detected. Synergism tests showed that piperonyl butoxide (PBO) and triphenyl phosphate (TPP) produced a high synergism of thiamethoxam effects in the TH-R strain (2.6- and 2.6-fold respectively). However, diethyl maleate (DEM) did not act synergistically with thiamethoxam. Biochemical assays showed that mixed function oxidase (MFO) activities and carboxylesterase (CarE) activity of the TH-R strain were 2.8- and 1.5-fold higher than that of the TH-S strain, respectively. When compared with the TH-S strain, the TH-R strain had a relative fitness of 0.64. The results show that WFT develops resistance to thiamethoxam after continuous application and thiamethoxam resistance had considerable fitness costs in the WFT. It appears that enhanced metabolism mediated by cytochrome P450 monooxygenases and CarE was a major mechanism for thiamethoxam resistance in the WFT. The use of cross-resistance insecticides, including imidaclothiz and dinotefuran, should be avoided for sustainable resistance management.

Susceptibility of Sogatella furcifera and Laodelphax striatellus (Hemiptera: Delphacidae) to Six Insecticides in China.

The whitebacked planthopper, Sogatella furcifera (Horváth), and small brown planthopper, Laodelphax striatellus (Fallén), both are important crop pests throughout China, especially in rice. Application of chemical insecticides is the major control practice. Consequently, insecticide resistance has become an urgent issue. In this study, resistance levels to six conventional insecticides were evaluated for these two species collected from major occurring areas of China. Additionally, imidacloprid- (resistance ratio [RR] = 10.4-fold) and buprofezin (RR = 15.1-fold)-resistant strains of whitebacked planthopper were obtained through laboratory selections for cross-resistance profiling and synergism assessment to understand resistance mechanisms. The results showed that all tested populations of both species exhibited low to high levels of resistance to chlorpyrifos, while remaining susceptible to thiamethoxam. Three of the 14 whitebacked planthopper populations showed low to moderate resistance to imidacloprid, while all small brown planthopper populations reminded susceptible. All small brown planthopper and whitebacked planthopper (except one) populations showed at least moderate resistance (RR = 10.1-271.1) to buprofezin. All small brown planthopper populations remained susceptible to pymetrozine and nitenpyram, and all whitebacked planthopper populations remained susceptible to isoprocarb. The imidacloprid-resistant whitebacked planthopper strain showed no significant cross-resistance to other tested insecticides. However, the buprofezin-resistant strain exhibited a low-level cross-resistance (CR = 3.1) to imidacloprid. Piperonyl butoxide, triphenyl phosphate, and diethylmaleate displayed no synergism effect on the resistant whitebacked planthopper strains.

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.

CRISPR/Cas9-mediated knockout of NlCYP6CS1 gene reveals its role in detoxification of insecticides in Nilaparvata lugens (Hemiptera: Delphacidae).

BACKGROUND: The brown planthopper (Nilaparvata lugens) is one of the major rice insect pests in Asia. Recently, high levels of insecticide resistance have been frequently reported and cytochrome P450 monooxygenase (P450)-mediated metabolic detoxification is a common resistance mechanism in N. lugens. However, there has been no persuasive genetic method to prove the role of P450s in insecticide resistance in N. lugens. RESULTS: Here, CRISPR/Cas9 system was used to disrupt the P450 gene NlCYP6CS1 to elucidate its role in insecticide resistance in field populations of N. lugens. We successfully constructed a homozygous strain (Nl6CS1-KO) with a 5-bp deletion and 1-bp insertion mutation of NlCYP6CS1. Compared with a background resistant strain (Nl-R), the susceptibility of knockout strain Nl6CS1-KO to imidacloprid, nitenpyram, thiamethoxam, dinotefuran, and pymetrozine was increased by 2.3-, 3.4-, 7.0-, 4.2- and 3.9-fold, respectively, but not significantly changed to triflumezopyrim, chlorpyrifos and buprofezin. Life table analysis demonstrated that the Nl6CS1-KO strain resembled the Nl-R strain in terms of egg and nymph developmental duration and adult lifespan, but differed from the Nl-R strain in the survival rate of eggs and nymphs, reproduction, and body weight. CONCLUSIONS: Our study demonstrates the effect of functional deletion of NlCYP6CS1 on multiple insecticide resistance in N. lugens. For the first time, we applied CRISPR/Cas9 system to reveal the mechanism of insecticide resistance in N. lugens, which may shed light on similar studies in other hemipteran insects. © 2023 Society of Chemical Industry.