Self-assembled co-delivery nanoplatform for increasing the broad-spectrum susceptibility of fall armyworm toward insecticides.

INTRODUCTION: Unscientific application of insecticides has led to severe resistance of pests to almost all classes of insecticides. Enhanced detoxification is the most common mechanism for this kind of resistance. OBJECT: Fall armyworm (FAW) has developed insecticide resistance, which is often linked to the overexpression of detoxification genes. Herein, a multicomponent nano-pesticide is designed to increase its broad-spectrum susceptibility toward insecticides. METHOD: Regulatory function of nuclear factor erythroid 2-related factor 2 (Nrf2) in detoxification was confirmed using transcriptome sequencing, quantitative real-time PCR and enzyme activity measurement. A star polycation (SPc) was adopted to construct the pesticide/SPc/complex, whose self-assembly mechanism and characterization were examined using isothermal titration calorimetry, dynamic light scattering and transmission electron microscope. The delivery efficiency of SPc-loaded dsRNA was examined in vitro and in vivo using fluorescent tracer technique. A multicomponent nano-pesticide was created through the integration of bacterial expression system and nano-delivery system, and its bioactivity was tested in laboratory and field. RESULTS: We confirmed the crucial role of Nrf2 in regulating the detoxification in FAW, and silencing Nrf2 could decrease detoxification gene expression and increase insecticide susceptibility. We then applied the SPc to self-assemble a nanoplatform for delivering Nrf2 double-stranded RNA (dsRNA) and pesticide simultaneously. Nano-sized pesticide/SPc/dsRNA complex exhibited high delivery efficiency in vitro and in vivo. Excitingly, the insecticidal activities of pesticide/SPc/dsNrf2 complexes were remarkably improved with the normalized synergistic ratios of 5.43-6.25 for chlorantraniliprole, 4.45-15.00 for emamectin benzoate, and 6.75-15.00 for spinetoram. Finally, we developed a multicomponent nano-pesticide (pesticide/SPc/dsNrf2 complex) using a bacterial expression system and nano-delivery system. This approach exhibited excellent leaf protection and pest control efficacy. CONCLUSION: The integration between the pesticide nanometerization and insecticide susceptibility improvement offers a promising strategy to increase insecticidal activity. Our study provides a revolutionary and universal strategy to increase insecticidal activity and decease application doses.

The gut symbiont Sphingomonas mediates imidacloprid resistance in the important agricultural insect pest Aphis gossypii Glover.

BACKGROUND: Neonicotinoid insecticides are applied worldwide for the control of agricultural insect pests. The evolution of neonicotinoid resistance has led to the failure of pest control in the field. The enhanced detoxifying enzyme activity and target mutations play important roles in the resistance of insects to neonicotinoid resistance. Emerging evidence indicates a central role of the gut symbiont in insect pest resistance to pesticides. Existing reports suggest that symbiotic microorganisms could mediate pesticide resistance by degrading pesticides in insect pests. RESULTS: The 16S rDNA sequencing results showed that the richness and diversity of the gut community between the imidacloprid-resistant (IMI-R) and imidacloprid-susceptible (IMI-S) strains of the cotton aphid Aphis gossypii showed no significant difference, while the abundance of the gut symbiont Sphingomonas was significantly higher in the IMI-R strain. Antibiotic treatment deprived Sphingomonas of the gut, followed by an increase in susceptibility to imidacloprid in the IMI-R strain. The susceptibility of the IMI-S strain to imidacloprid was significantly decreased as expected after supplementation with Sphingomonas. In addition, the imidacloprid susceptibility in nine field populations, which were all infected with Sphingomonas, increased to different degrees after treatment with antibiotics. Then, we demonstrated that Sphingomonas isolated from the gut of the IMI-R strain could subsist only with imidacloprid as a carbon source. The metabolic efficiency of imidacloprid by Sphingomonas reached 56% by HPLC detection. This further proved that Sphingomonas could mediate A. gossypii resistance to imidacloprid by hydroxylation and nitroreduction. CONCLUSIONS: Our findings suggest that the gut symbiont Sphingomonas, with detoxification properties, could offer an opportunity for insect pests to metabolize imidacloprid. These findings enriched our knowledge of mechanisms of insecticide resistance and provided new symbiont-based strategies for control of insecticide-resistant insect pests with high Sphingomonas abundance.

Persistent toxicity and dissipation dynamics of afidopyropen against the green peach aphid Myzus persicae (Sulzer) in cabbage and chili.

The green peach aphid, Myzus persicae (Sulzer), is a significant global pest in horticultural and field crops. Afidopyropen is a novel systemic insecticide with high efficacy against sucking pests, and it is suitable for the management of M. persicae. However, the persistent toxicity and dissipation dynamics of afidopyropen in vegetables remain unknown. In this study, we determined the residual activity and dissipation dynamics of afidopyropen against M. persicae on cabbage and chili. The data showed that the toxicity of afidopyropen against M. persicae lasted more than 30 days; the corrected mortality was greater than 80% 10 days after application and was 50-60% 30 days post-application. The afidopyropen residues on cabbage and chili plants were quantified using ultrahigh-pressure liquid chromatography-tandem mass spectrometry. The dissipation half-lives of afidopyropen on cabbage and chili plants ranged from 1.45 to 2.34 days and 3.98-5.98 days at different recommended dosages, respectively. Our findings provide valuable data for the maximum residue limits of afidopyropen on vegetables and will help growers determine the frequency and timing of its application on cabbage and chili.

The variation of a cytochrome P450 gene, CYP6G4, drives the evolution of Musca domestica L. (Diptera: Muscidae) resistance to insecticides in China.

Long term and excessive insecticide use has resulted in some environmental problems and especially, insecticide resistance evolution in insect pests. The variation of cytochrome P450 monooxygenases (P450s), associated with the metabolic detoxification of toxic xenobiotics, is often involved in insecticide resistance. Here, we found that the variation in a P450 gene, CYP6G4, is the most important driver of carbamates resistance in the house fly (Musca domestica). Deciphering the detailed molecular mechanisms of the insecticide resistance is critical for performing suitable insecticide resistance management strategies. Our research results revealed that the combination of amino acid mutations (110C-330E-360N/S, 110C-330E-360S) of CYP6G4 could improve the resistance to propoxur. The nucleotide variations in the promoter region of CYP6G4 significantly increased the luciferase activity by the reporter gene assays. Additionally, miR-281-1-5p was confirmed to post-transcriptionally down-regulate the expression of CYP6G4. These findings suggest that three independent mechanisms; amino acid mutations of the P450 protein, mutations in the promoter region and low expression of post-trans-regulatory factors, as the powerful strategies for the insect resistance to toxic compounds, play a crucial role in the evolutionary processes of insecticide resistance.

Cap 'n' Collar C and Aryl Hydrocarbon Receptor Nuclear Translocator Facilitate the Expression of Glutathione S-Transferases Conferring Adaptation to Tannic Acid and Quercetin in Micromelalopha troglodyta (Graeser) (Lepidoptera: Notodontidae).

Micromelalopha troglodyta (Graeser) (Lepidoptera: Notodontidae) is a notorious pest of poplar. Coevolution with poplars rich in plant secondary metabolites prompts M. troglodyta to expand effective detoxification mechanisms against toxic plant secondary metabolites. Although glutathione S-transferases (GSTs) play an important role in xenobiotic detoxification in M. troglodyta, it is unclear how GSTs act in response to toxic secondary metabolites in poplar. In this study, five GST gene core promoters were accurately identified by a 5' loss luciferase reporter assay, and the core promoters were significantly induced by two plant secondary metabolites in vitro. Two transcription factors, cap 'n' collar C (CncC) and aryl hydrocarbon receptor nuclear translocator (ARNT), were cloned in M. troglodyta. MtCncC and MtARNT clustered well with other insect CncCs and ARNTs, respectively. In addition, MtCncC and MtARNT could bind the MtGSTt1 promoter and strongly improve transcriptional activity, respectively. However, MtCncC and MtARNT had no regulatory function on the MtGSTz1 promoter. Our findings revealed the molecular mechanisms of the transcription factors MtCncC and MtARNT in regulating the GST genes of M. troglodyta. These results provide useful information for the control of M. troglodyta.

Characterization, expression, and functional analysis of TRPV genes in cotton aphid, Aphis gossypii Glover.

Transient receptor potential vanilloid (TRPV) channels have been found to be the molecular target of afidopyropen, a novel insecticide that is highly effective in controlling Aphis gossypii Glover in the field. However, the TRPV genes of A. gossypii has not yet been characterized. In this study, two TRPV genes of A. gossypii (AgNan and AgIav) were cloned and their expression levels were determined by quantitative real-time PCR (RT-qPCR). The deduced amino acids of AgNan and AgIav contain all conserved domains of TRPV and share very high amino acid identity with other insect TRPVs. AgNan and AgIav expressed in all developmental stages and their expression can be induced by afidopyropen in a dose- and time-dependent manner. Moreover, we found that silencing of AgNan and AgIav by RNA interference resulted in a significant mortality increase of adult A. gossypii compared to the control, which was even higher than 93 % at five days after feeding with dsAgIav, suggesting that knockdown of AgNan and AgIav have great effects on the survival of A. gossypii. The results of this study would be helpful for determining the reasonable use of afidopyropen in the integrated pest management programs of A. gossypii and provide useful information for further functional study of TRPVs in insects.

Clathrin-dependent endocytosis plays a critical role in larval and pupal development, and female oocyte production in the red flour beetle (Tribolium castaneum).

BACKGROUND: Clathrin-dependent endocytosis is a vesicular transport process by which cells take macromolecules from the extracellular space to the intracellular space. It plays important roles in various cellular functions, but its biological significance in insect development and reproduction has not been well studied. RESULTS: We characterized and functionally analyzed four major clathrin-dependent endocytic pathway genes (TcChc, TcAP50, TcVhaSFD, TcRab7) in Tribolium castaneum. RNA interference (RNAi) by injecting double-stranded RNA (dsRNA) targeting each gene at three doses (50, 100, or 200 ng per insect) in 20-day-old larvae led to 100% larval mortality. When the expressions of TcChc, TcVhaSFD, and TcRab7 were suppressed by injecting their respective dsRNAs at each dose in 1-day-old pupae, the adults that emerged from the dsRNA-injected pupae were deformed, with the absence of wing development. The deformed adults died within 2 days after eclosion. When the expression of TcAP50 was suppressed by injecting its dsRNA into 1-day-old pupae, although no apparent deformed adults were observed, all the adults died within 35 days after eclosion. In addition, when the expressions of TcChc and TcVhaSFD were suppressed by injecting their respective dsRNAs at a reduced dose (10 ng per insect) in 5-day-old pupae, the ovarian development and oocyte production in the resultant females were completely inhibited. CONCLUSION: Our results indicate that clathrin-dependent endocytosis is essential for insect development and reproduction. The results from this study can help researchers identify potential molecular targets for developing novel strategies for insect pest management. © 2023 Society of Chemical Industry.

A novel V263I mutation in the glutamate-gated chloride channel of Plutella xylostella (L.) confers a high level of resistance to abamectin.

The frequent and extensive use of insecticides leads to the evolution of insecticide resistance, which has become one of the constraints on global agricultural production. Avermectins are microbial-derived insecticides that target a wide number of insect pests, including the diamondback moth Plutella xylostella, an important global pest of brassicaceous vegetables. However, field populations of P. xylostella have evolved serious resistance to avermectins, including abamectin, thereby threatening the efficiency of these insecticides. In this study, a novel valine to isoleucine mutation (V263I) was identified in the glutamate-gated chloride channel (GluCl) of field P. xylostella populations, which showed different levels of resistance to abamectin. Electrophysiological analysis revealed that the V263I mutation significantly reduced the sensitivity of PxGluCl to abamectin by 6.9-fold. Genome-modified Drosophila melanogaster carrying the V263I mutation exhibited 27.1-fold resistance to abamectin. Then, a knockin strain (V263I-KI) of P. xylostella expressing the homozygous V263I mutation was successfully constructed using the CRISPR/Cas9. The V263I-KI had high resistance to abamectin (106.3-fold), but significantly reduced fecundity. In this study, the function of V263I mutation in PxGluCl was verified for the first time. These findings provide a more comprehensive understanding of abamectin resistance mechanisms and lay the foundation for providing a new molecular detection method for abamectin resistance monitoring.

The sulfoximine insecticide sulfoxaflor exposure reduces the survival status and disrupts the intestinal metabolism of the honeybee Apis mellifera.

Honeybees (Apis mellifera) are indispensable pollinators in agricultural production, biodiversity conservation, and nutrients provision. The abundance and diversity of honeybees have been rapidly diminishing, possibly related to the extensive use of insecticides in ecosystems. Sulfoxaflor is a novel sulfoximine insecticide that, like neonicotinoids, acts as a competitive modulator of nicotinic acetylcholine receptors (nAChR) in insects. However, few studies have addressed the negative effects of sulfoxaflor on honeybees at environmentally relevant concentrations. In the present study, adult workers were fed a 50% (w/v) of sugar solution containing different concentrations (0, 0.05, 0.5 and 2.0 mg/L) of sulfoxaflor for two weeks consecutively. The survival rates, food intake, and body weight of the honeybees significantly decreased after continuous exposure at higher doses (0.5 and 2.0 mg/L) of sulfoxaflor when compared with the control. The change in the metabolites in the honeybee gut was determined using high-throughput non-targeted metabolomics on day 14 after sulfoxaflor treatment. The results revealed that 24 and 105 metabolites changed after exposure to 0.5 and 2.0 mg/L sulfoxaflor, respectively, compared with that of the control groups. A total of 12 changed compounds including pregenolone and glutathione were detected as potential biomarkers, which were eventually found to be enriched in pathways of the steroid hormone biosynthesis (p = 0.0001) and glutathione metabolism (p = 0.021). These findings provide a new perspective on the physiological influence of sulfoxaflor stress in honeybees.

CRISPR/Cas9-mediated D472N substitution in the Rdl1 of Plutella xylostella confers low resistance to abamectin.

BACKGROUND: Abamectin is one of the main insecticides used for the control of Plutella xylostella, a destructive pest of cruciferous crops. Target-site mutation plays an important role in insecticide resistance. A point mutation (D472N) has been reported in the Rdl1 γ-aminobutyric acid receptor (GABAR) in P. xylostella, but its roles in insecticide resistance remain unknown. RESULTS: In this study, the D472N mutation of the Rdl1 GABAR was detected in several field populations of P. xylostella and showed a positive correlation with abamectin resistance. A knock-in homozygous mutation strain (D472N-KI) of P. xylostella was successfully constructed using CRISPR/Cas9 coupled with homology-directed repair, and the bioassay results demonstrated that compared with the susceptible strain, the D472N-KI strain had 11.1- and 3.7-fold increased resistance to abamectin and endosulfan, respectively. There was no difference in resistance to fipronil, broflanilide or isocycloseram, which also target the GABAR. In addition, the total fecundity of the D472N-KI strain was significantly reduced by 50.0%. CONCLUSION: Our results suggest that the homozygous D472N mutation in Rdl1 confers a low level of resistance to abamectin in P. xylostella but causes significant fecundity disadvantages, which may delay the development of resistance to some extent. These results lay a foundation for further understanding the mechanisms of abamectin resistance in insect pests. © 2022 Society of Chemical Industry.

Functional characterization of ABC transporters mediates multiple neonicotinoid resistance in a field population of Aphis gossypii Glover.

The ATP-binding cassette (ABC) transporters C and G subfamilies have been reported to be involved in insecticide detoxification, with most studies showing increased gene transcript levels in response to insecticide exposure. Our previous studies have suggested that ABCC and G transporters participate in cyantraniliprole and thiamethoxam resistance of Aphis gossypii. In this study, we focused on the potential roles of the ABCC and G transporters of an A. gossypii field population (SDR) in neonicotinoid detoxification. The results of leaf dip bioassays showed 629.17- and 346.82-fold greater resistance to thiamethoxam and imidacloprid in the SDR strain, respectively, than in the susceptible strain (SS). Verapamil, an ABC inhibitor, was used for synergism bioassays, and the results showed synergistic effects with thiamethoxam, with synergistic ratios (SRs) of 2.07 and 6.68 in the SS and SDR strains, respectively. In addition to thiamethoxam, verapamil increased imidacloprid toxicity by 1.68- and 1.62-fold in the SS and SDR strains respectively. Then, the expression levels of several ABCC and G transporters were analyzed in different treatments. We found that the transcript levels of AgABCG4, AgABCG17, AgABCG26, AgMRP8 and AgMRP12 were higher in the SDR strain than in the SS strain. The mRNA expression of AgABCG4, AgABCG7, AgABCG13, AgABCG17, AgABCG26, AgMRP8 and AgMRP12 in the SDR strain was increased after thiamethoxam and imidacloprid exposure. The results of transgenic Drosophila melanogaster bioassays suggested that overexpression of AgABCG4, AgABCG7, AgABCG13, AgABCG17, AgABCG26, AgMRP8 and AgMRP12 in transgenic flies was sufficient to confer thiamethoxam and imidacloprid resistance, and AgABCG4, AgABCG7, AgABCG13, AgABCG26 and AgMRP12 may be related to α-cypermethrin cross-resistance with weak effects. In addition, the knockdown of AgABCG4, AgABCG13, AgABCG26, AgMRP8 and AgMRP12, and the knockdown of AgABCG7 and AgABCG26 increased thiamethoxam and imidacloprid mortality in the SDR strain, respectively. Our results suggest that changes in the expression levels of ABCC and G transporters may contribute to neonicotinoid detoxification in the SDR strain, and provide a foundation for clarify the potential roles of ABCC and G transporters in insecticide resistance.

A General Signal Pathway to Regulate Multiple Detoxification Genes Drives the Evolution of Helicoverpa armigera Adaptation to Xenobiotics.

The study of insect adaptation to the defensive metabolites of host plants and various kinds of insecticides in order to acquire resistance is a hot topic in the pest-control field, but the mechanism is still unclear. In our study, we found that a general signal pathway exists in H. armigera which can regulate multiple P450s, GSTs and UGTs genes to help insects decrease their susceptibility to xenobiotics. Knockdown of HaNrf2 and HaAhR expression could significantly increase the toxicity of xenobiotics to H. armigera, and simultaneously decrease the gene expression of P450s, GSTs and UGTs which are related to the xenobiotic metabolism and synthesis of insect hormone pathways. Then, we used EMSA and dual luciferase assay to verify that a crosstalk exists between AhR and Nrf2 to regulate multiple P450s, GSTs and UGTs genes to mediate H. armigera susceptibility to plant allelochemicals and insecticides. The detoxification genes' expression network which can be regulated by Nrf2 and AhR is still unknown, and there were also no reports about the crosstalk between AhR and Nrf2 that exist in insects and can regulate multiple detoxification genes' expression. Our results provide a new general signaling pathway to reveal the adaptive mechanism of insects to xenobiotics and provides further insight into designing effective pest-management strategies to avoid the overuse of insecticides.

Establishment of Toxicity and Susceptibility Baseline of Broflanilide for Aphis gossypii Glove.

The Aphis gossypii is an important pest that can damage cotton plants and can cause a huge economic loss worldwide. Chemical control is a main method to manage this pest, but the cotton aphid resistance to insecticides has become a severe problem in the management of the cotton aphid. It is important to introduce a novel insecticide for rotational application with other insecticides. Broflanilide, as a meta-diamide insecticide with a special mode of action, showed high efficiency against lepidopterous larvae. However, we found that broflanilide possessed high insecticidal activity against the sap-sucking pest A. gossypii. The susceptibility of A. gossypii to broflanilide from 20 field populations in main cotton planting areas of China in 2021 was determined by the leaf-dipping method. LC50 values of broflanilide to A. gossypii ranged from 0.20 µg mL-1 to 1.48 µg mL-1. The susceptible baseline of A. gossypii to broflanilide was established with the LC50 value of 0.41 µg mL-1 and might be used to calculate the resistance ratio (RR) of cotton aphid population in broflanilide resistance monitoring. The RR value of field populations in China was from 0.49 to 3.61 in 2021. It suggested that the broflanilide may be a potential agent in the resistance management of A. gossypii to insecticides. These results are significantly useful for the rational chemical control of cotton aphids.

Resistance Mechanisms of Sitobion miscanthi (Hemiptera: Aphididae) to Malathion Revealed by Synergist Assay.

A resistant strain (MRS) of Sitobion miscanthi was cultured by continuous selection with malathion for over 40 generations. The MRS exhibited 32.7-fold resistance to malathion compared to the susceptible strain (MSS) and 13.5-fold, 2.9-fold and 4.8-fold cross-resistance for omethoate, methomyl and beta-cypermethrin, respectively. However, no cross-resistance was found to imidacloprid in this resistant strain. The realized heritability for malathion resistance was 0.02. Inhibitors of esterase activity, both triphenyl phosphate (TPP) and S,S,S,-tributyl phosphorotrithioate (DEF) as synergists, exhibited significant synergism to malathion in the MRS strain, with 11.77-fold and 5.12-fold synergistic ratios, respectively, while piperonyl butoxide (PBO) and diethyl maleate (DEM) showed no significant synergism in the MRS strain. The biochemical assay indicated that carboxylesterase activity was higher in MRS than in MSS. These results suggest that the increase in esterase activity might play an important role in S. miscanthi resistance to malathion. Imidacloprid could be used as an alternative for malathion in the management of wheat aphid resistance.

Impact of Imidacloprid Resistance on the Demographic Traits and Expressions of Associated Genes in Aphis gossypii Glover.

Imidacloprid is one of the most widely used neonicotinoid insecticides to control sap-sucking insect pests, including Aphis gossypii. The intensive application of chemical insecticides to A. gossypii led to the development of resistance against several insecticides, including imidacloprid. Therefore, it is crucial to understand the association between imidacloprid resistance and the fitness of A. gossypii to limit the spread of the resistant population under field contexts. In this study, we used the age-stage, two-sex life table method to comprehensively investigate the fitness of imidacloprid resistant (ImR) and susceptible strains (SS) of melon aphids. Results showed that ImR aphids have prolonged developmental stages and decreased longevity, fecundity, and reproductive days. The key demographic parameters (r, λ, and R0) were significantly reduced in ImR strain compared to SS aphids. Additionally, the molecular mechanism for fitness costs was investigated by comparing the expression profile of juvenile hormone-binding protein (JHBP), juvenile hormone epoxide hydrolase (JHEH), juvenile hormone acid O-methyltransferase (JHAMT), Vitellogenin (Vg), ecdysone receptor (EcR), and ultraspiracle protein (USP) supposed to be associated with development and reproduction in insects. The results of RT-qPCR showed that EcR, JHBP, JHAMT, JHEH, and Vg genes were downregulated, while USP was statistically the same in ImR A. gossypii compared to the SS strain. Together, these results provide in-depth information about the occurrence and magnitude of fitness costs against imidacloprid resistance that could help manage the evolution and spread of A. gossypii resistance in field populations.

Status of the Resistance of Aphis gossypii Glover, 1877 (Hemiptera: Aphididae) to Afidopyropen Originating from Microbial Secondary Metabolites in China.

The resistance of cotton aphids to various forms of commonly used pesticides has seriously threatened the safety of the cotton production. Afidopyropen is a derivative of microbial metabolites with pyropene insecticide, which has been shown to be effective in the management of Aphis gossypii. Several field populations of Aphis gossypii were collected from the major cotton-producing regions of China from 2019 to 2021. The resistance of these populations to afidopyropen was estimated using the leaf-dipping method. The LC50 values of these field populations ranged from 0.005 to 0.591 mg a.i. L-1 in 2019, from 0.174 to 4.963 mg a.i. L-1 in 2020 and from 0.517 to 14.16 mg a.i. L-1 in 2021. The resistance ratios for all A. gossypii populations ranged from 0.03 to 3.97 in 2019, from 1.17 to 33.3 in 2020 and from 3.47 to 95.06 in 2021. The afidopyropen resistance exhibited an increasing trend in the field populations of Cangzhou, Binzhou, Yuncheng, Kuerle, Kuitun, Changji and Shawan from 2019 to 2021. This suggests that the resistance development of the cotton aphid to afidopyropen is inevitable. Therefore, it is necessary to rotate or mix afidopyropen with other insecticides in order to inhibit the development of afidopyropen resistance in field populations.

Functional Inquiry into ATP-Binding Cassette Transporter Genes Contributing to Spirotetramat Resistance in Aphis gossypii Glover.

ATP-binding cassette (ABC) transporters regulate the efflux of a broad spectrum of substrates to extracellular transporting, which play an important role in the detoxification process in arthropods. Here, we described a comprehensive approach to explore the involvement of ABC transporters in spirotetramat resistance in cotton aphids. In this study, synergism bioassays showed 17.05% and 35.42% increases in the toxicity to spirotetramat with the ABC inhibitor verapamil in adult and 3rd instar nymph aphids of the SR strain, respectively. In a competitive assay based on the microinjection of a fluorescent ABC transporter substrate, verapamil (a general ABC inhibitor) and spirotetramat significantly inhibited the elimination of Texas Red. Based on transcriptome data of midguts of spirotetramat-susceptible (SS) and -resistant (SR) strains, the expression levels of ABCB4, ABCB5, ABCF2, MRP11, and MRP12 were significantly upregulated in the SR strain midgut compared to that of the SS strain. Gene functional analysis based on ectopic expression and RNA interference (RNAi) proved that ABCB4, ABCB5, ABCF2, MRP11, and MRP12 were involved in the tolerance of cotton aphids to spirotetramat. Moreover, the upregulated ABCF2, ABCB4, and ABCB5 in the midgut of the SR strain contributed more to the resistance of spirotetramat in in vitro functional analysis. In summary, these results demonstrate that candidate ABC transporter genes in the midgut tissue were involved in spirotetramat resistance, which will help reveal the relationship between ABC transporters and the development of spirotetramat resistance in field populations.

Detection of Chitin Synthase Mutations in Lufenuron-Resistant Spodoptera frugiperda in China.

Spodoptera frugiperda (J. E. Smith), is commonly known as fall armyworm, native to tropical and subtropical regions of America, is an important migratory agricultural pest. It is important to understand the resistance and internal mechanism of action of S. frugiperda against lufenuron in China. Lufenuron is one of the main insecticides recommended for field use in China and has a broad prospect in the future. We conducted a bioassay using the diet-overlay method and found that the current S. frugiperda in China are still at a low level of resistance to lufenuron. Secondly, we examined whether the mutation I1040M (I1042M in Plutella xylostella), associated with lufenuron resistance, was produced in the field. And then we tested the expression of chitin synthase SfCHSA and SfCHSB in different tissues, and the changes of these two genes after lufenuron induction. The results showed that there is still no mutation generation in China and there is a significant change in the expression of SfCHSA under the effect of lufenuron. In conclusion, our study suggests that field S. frugiperda populations in 2019 and 2020 were less resistant to lufenuron. In fall armyworm, chitin synthases included SfCHSA and SfCHSB genes, and after induction treatment with lufenuron, the expression of the SfCHSA gene was significantly increased. In SfCHSA, no mutation has been detected in the site associated with lufenuron resistance. Secondly, in S. frugiperda larvae, the SfCHSA gene was the highest in the head of the larvae, followed by the integument; while the SfCHSB gene was mainly concentrated in the midgut. Therefore, we believe that the SfCHSA gene plays a greater role in the resistance of S. frugiperda to lufenuron than the SfCHSB gene. It is worth noting that understanding the level of resistance to lufenuron in China, the main mechanism of action of lufenuron on larvae, and the mechanism of resistance to lufenuron in S. frugiperda will help in crop protection as well as in extending the life span of this insecticide.

Corynebacterium sp. 2-TD Mediated Toxicity of 2-Tridecanone to Helicoverpa armigera.

Cotton bollworm (Helicoverpa armigera) is a Lepidopteran noctuid pest with a global distribution. It has a wide range of host plants and can harm cotton, tomato, tobacco, and corn, as well as other crops. H. armigera larvae damage the flower buds, flowers, and fruits of tomato and cause serious losses to tomato production. Tomato uses the allelochemical 2-tridecanone to defend against this damage. So far, there have been no reports on whether the adaptation of H. armigera to 2-tridecanone is related to its symbiotic microorganisms. Our study found that Corynebacterium sp. 2-TD, symbiotic bacteria in H. armigera, mediates the toxicity of the 2-tridecanone to H. armigera. Corynebacterium sp. 2-TD, which was identified by 16S rDNA gene sequence analysis, was screened out using a basal salt medium containing a unique carbon source of 2-tridecanone. Then, Corynebacterium sp. 2-TD was confirmed to be distributed in the gut of H. armigera by quantitative PCR (qPCR) and fluorescence in situ hybridization (FISH). The survival rate of H. armigera increased by 38.3% under 2-tridecanone stress after inoculation with Corynebacterium sp. 2-TD. The degradation effect of Corynebacterium sp. 2-TD on 2-tridecanone was verified by ultra-high-performance liquid chromatography (UPLC). Our study is the first to report the isolation of gut bacteria that degrade 2-tridecanone from the important agricultural pest H. armigera and to confirm bacterial involvement in host adaptation to 2-tridecanone, which provides new insights into the adaptive mechanism of agricultural pests to host plants.

Plant volatile compound methyl benzoate is highly effective against Spodoptera frugiperda and safe to non-target organisms as an eco-friendly botanical-insecticide.

Recent studies have indicated that the plant volatile methyl benzoate (MB) exhibits significant insecticidal bioactivity against several common insects. However, the potential environmental hazards of MB and its safety to non-target organisms is poorly understood. In the present study, these characteristics were investigated through laboratory experiments and field investigations. The results revealed that MB was highly toxic to the agricultural pest, fall armyworm Spodoptera frugiperda. Compared with the commercial pesticide lambda-cyhalothrin, the toxicities of MB against S. frugiperda larvae and adults were comparable and 3.41 times higher, respectively. Behavioral bioassays showed that the percentage repellency of MB to S. frugiperda larvae was 56.72 %, and MB induced 69.40 % oviposition deterrence rate in S. frugiperda female adults. Furthermore, in terms of median lethal concentration (LC50) and median lethal doses (LD50), MB exhibited non-toxic effects on non-target animals with 3-d LC50 of > 1 % to natural predators (Coccinella septempunctata and Harmonia axyridis), 3-d LD50 of 467.86 µg/bee to the bumblebee Bombus terrestris, 14-d LC50 of 971.09 mg/kg to the earthworm Eisenia fetida, and 4-d LC50 of 47.30 mg/L to the zebrafish Brachydanio rerio. The accumulation of MB in the soil and earthworms was found to be extremely limited. Our comparative study clearly demonstrated that MB is effective as a selective botanical pesticide against S. frugiperda and it is safe to use in the tested environment, with no toxic effects on non-target animals and natural predators.