UNASSIGNED: Plumbagin is an important phytochemical and has been reported to exhibit potent larvicidal activity against several insect pests, However, the insecticidal mechanism of plumbagin against pests is still poorly understood. This study aimed to investigate the insecticidal activities of plumbagin and the underlying molecular mechanisms against a devastating agricultural pest, the fall armyworm Spodoptera frugiperda.
UNASSIGNED: The effects of plumbagin on S. frugiperda larval development and the activities of two detoxification enzymes were initially examined. Next, transcriptomic changes in S. frugiperda after plumbagin treatment were investigated. Furthermore, RNA-seq results were validated by qPCR.
UNASSIGNED: Plumbagin exhibited a high larvicidal activity against the second and third instar larvae of S. frugiperda with 72 h LC50 of 0.573 and 2.676 mg/g, respectively. The activities of the two detoxification enzymes carboxylesterase and P450 were significantly increased after 1.5 mg/g plumbagin treatment. Furthermore, RNA-seq analysis provided a comprehensive overview of complex transcriptomic changes in S. frugiperda larvae in response to 1.5 mg/g plumbagin exposure, and revealed that plumbagin treatment led to aberrant expression of a large number of genes related to nutrient and energy metabolism, humoral immune response, insect cuticle protein, chitin-binding proteins, chitin synthesis and degradation, insect hormone, and xenobiotic detoxification. The qPCR results further validated the reproducibility and reliability of the transcriptomic data.
UNASSIGNED: Our findings provide a valuable insight into understanding the insecticidal mechanism of the phytochemical plumbagin.

Dicaffeoyltartaric acid (diCT) and 3,5-dicaffeoylquinic acid (3,5-diCQ) are described for their aphicidal properties on several aphid species. Intending to valorize diCT and 3,5-diCQ as biocontrol products and because of the high adaptive capacities of aphids to xenobiotics, we sought to determine the existence of adaptation first in Myzus persicae (Sulzer) (Hemiptera: Aphididae) and then other aphids. Resistance of aphids to these biopesticides could be promoted by (i) the existence of resistance to synthetic insecticides that may confer cross-resistance and (ii) the presence of these compounds in wild plants likely which may have led to pre-existing adaptation in aphids. We assessed the resistance levels to diCT and 3,5-diCQ in 7 lab strains (including some resistant to synthetic aphicides) and 7 wild populations of M. persicae using biotests. The activities of detoxification enzymes contributing to insecticide resistance were also measured. Additionally, we followed the same method to characterize susceptibility to these caffeic derivatives in wild populations of Nasonovia ribisnigri (Mosley) (Hemiptera: Aphididae), Brevicoryne brassicae(Linnaeus) (Hemiptera: Aphididae) and, Aphis craccivora(Koch) (Hemiptera: Aphididae). Our results show variability in susceptibility to diCT between populations of M. persicae, but resistance ratios (RR) were low (RR = 3.59). We found no cross-resistance between synthetic insecticides and diCT. Carboxylesterase and glutathione-S-transferase did not seem to be involved in its detoxification. A clone of A. craccivora collected from peanut, a species rich in diCT, was not susceptible to either diCT or 3,5-diCQ, suggesting a common molecular target for these 2 molecules and the existence of a high-effect resistance mechanism. These active botanical substances remain good candidates for M. persicae biocontrol in agriculture.

Drought, a major stress for crop plants, is expected to increase in frequency due to climate change. Drought can alter crop growth and levels of secondary plant metabolites, which in turn can affect herbivores, but this latter point is still controversial. This study used three different polyethylene glycol (PEG-6000) levels (0%, 1%, and 3%) to simulate drought stress and evaluated their effects on cotton plants and the impacts on the performance of the cotton aphid Aphis gossypii. Cotton plants under drought stress showed decreased water content, above-ground biomass, and nitrogen content and increased soluble protein, soluble sugar, and tannin contents. Based on analysis of the developmental time and fecundity data from individuals and at the population level, a significantly lower fecundity and population abundance of A. gossypii were detected on cotton plants with drought stress, which supports the \"plant vigor hypothesis\". The poor development of A. gossypii is possibly related to lower xylem sap and phloem ingestion under drought stress. In addition, the increased tannin content of cotton plants induced by drought and lower detoxification enzyme activities of A. gossypii may have affected the responses of aphids to drought-stressed plants. Overall, the results showed that drought stress altered the physiological characteristics of the cotton plants, resulting in adverse bottom-up effects on cotton aphid performances. This implies that the adoption of drip irrigation under plastic film that can help alleviate drought stress may favor the population growth of cotton aphids.

The fall armyworm (FAW), Spodoptera frugiperda, is one of the most devastating invasive polyphagous pests, which has attracted recent global attention by developing resistance to various insecticidal active ingredients with independent mode of action. Fluxametamide, a newly commercialized isoxazoline insecticide, is exceptionally selective towards several lepidopteran pests. The present study aimed to evaluate resistance risk in FAW to fluxametamide and the fitness costs associated with fluxametamide resistance. A field-collected and genetically mixed population of FAW was artificially selected through continuous exposure to fluxametamide. After successive selection of 10 generations, there was no obvious increase in the LC50 (RF: 2.63-fold). The realized heritability (h2) of fluxametamide resistance was estimated as h2 = 0.084 using a quantitative genetic approach. Compared with the susceptible F0 strain, the Flux-SEL (F10) strain of FAW displayed no significant cross-resistance to broflanilide, chlorantraniliprole, fipronil, indoxacarb, lambda cyhalothrin, spinetoram, and tetraniliprole, except emamectin benzoate (RF: 2.08-fold). Increased activity of glutathione S-transferase (ratio 1.94) was observed in the Flux-SEL (F10) strain of FAW, while the cytochrome P450 and carboxylesterase activities were not altered. The fluxametamide-selection significantly affected the development and reproductive traits of FAW with a lower R0, T and relative fitness (Rf = 0.353). The results alluded that the risk of fluxametamide resistance evolution in FAW is relatively lower; however, proactive implementation of resistance management approaches should be done to maintain the field efficacy of fluxametamide against FAW.

The risk assessment and resistance mechanisms of insecticide resistance are critical for resistance management strategy before a new insecticide is widely used. Triflumezopyrim (TFM) is the first commercialized mesoionic insecticide, which can inhibit nicotinic acetylcholine receptor with high-performance against the small brown planthopper (SBPH), Laodelphax striatellus (Fallén). In our study, the resistance of SBPH to TFM increased 26.29-fold, and the actual heritability of resistance was 0.09 after 21 generations of continuous selection by TFM. After five generations of constant feeding under insecticide-free conditions from F16 generation, the resistance level decreased 2.05-fold, and the average resistance decline rate per generation was 0.01, but there were no statistical decline. The TFM resistant strains had no cross-resistance to imidacloprid, nitenpyram, thiamethoxam, dinotefuran, flonicamid, pymetrozine, and chlorfenapyr. The third and fifth nymphal stage duration, pre-adult stage, adult preoviposition period, longevity, emergence rate, and hatchability of the resistant strain were significantly lower than those of the susceptible strain, while the female-male ratio was considerably increased. The fitness cost was 0.89. Further, cytochrome P450 monooxygenase (P450) and carboxylesterase (CarE) activities were markedly increased, but only the enzyme inhibitor piperonyl butoxide (PBO) had a significant synergistic effect on the resistant strain. The expression of CYP303A1, CYP4CE2, and CYP419A1v2 of P450 genes was significantly increased. SBPH has a certain risk of resistance to TFM with continuous application. The TFM resistance may be due to the increased activity of P450 enzyme regulated by the overexpression of P450 genes.

Oedaleus asiaticus (Bey-Bienko) is an economically devastating locust species found in grassland and pastoral areas of the Inner Mongolia region of northern China. In this study, resistance to three frequently used insecticides (beta-cypermethrin, matrine, and azadirachtin) was investigated in six field populations of O. asiaticus using the leaf-dip bioassay method. The inhibitory effects of synergists and the activities of detoxification enzyme activities in the different populations were determined to explore potential biochemical resistance mechanisms. The results showed that the field populations SB (resistance ratio [RR] = 7.85), ZB (RR = 5.64), and DB (RR = 6.75) had developed low levels of resistance to beta-cypermethrin compared with a susceptible control strain. Both the SB (RR = 5.92) and XC (RR = 6.38) populations had also developed low levels of resistance against matrine, with the other populations remaining susceptible to both beta-cypermethrin and matrine. All field populations were susceptible to azadirachtin. Synergism analysis showed that triphenyl phosphate (TPP) and diethyl-maleate (DEM) increased the toxicity of beta-cypermethrin significantly in the SB population, while the synergistic effects of TPP, piperonyl butoxide (PBO), and DEM on the toxicity of matrine were higher in SB (SR 3.86, 4.18, and 3.07, respectively) than in SS (SR 2.24, 2.86, and 2.29, respectively), but no synergistic effects of TPP, PBO, and DEM on azadirachtin were found. Biochemical assays showed that the activities of carboxylesterases (CarEs) and glutathione-S-transferases (GSTs) were significantly raised in all field populations of O. asiaticus, with a significant positive correlation observed between beta-cypermethrin resistance and CarE activity. The activities of cytochrome P450 monooxygenases (P450) and multi-function oxidases (MFO) were elevated in all six field populations, and P450 activity displayed strong positive correlations with the three insecticides. Our findings suggest that resistance to beta-cypermethrin in O. asiaticus may be mainly attributed to elevated CarE and GST activities, while P450 plays an important role in metabolizing matrine and azadirachtin. Our study provides insights that will help improve insecticide resistance management strategies.

The two-spotted spider mite Tetranychus urticate is an important agricultural pest worldwide. It is extremely polyphagous and has developed resistance to many pesticides. Here, we assessed the pesticide resistance of seven field populations of T. urticae in China, their target site mutations and the activities of their detoxification enzymes. The results showed that abamectin and the traditional pesticides pyridaben, profenofos and bifenthrin had higher resistance or lower toxicity than more recently developed pesticides including chlorfenapyr, spinetoram, cyflumetofen, cyenopyrafen, bifenazate and B-azolemiteacrylic. The frequency of point mutations related to abamectin resistance, G314D in the glutamate-gated chloride channel 1 (GluCl1) and G326E in GluCl3, ranged 47%-70% and 0%-97%, respectively. The frequency of point mutations in A1215D and F1538I of the voltage-gated sodium channel gene (VGSC), which may increase resistance to pyrethroids, ranged 88%-100% and 10%-100%, respectively. For target sites related to organophosphate resistance, mutation frequencies ranged 25%-92% for G119S and 0%-23% for A201S in the acetycholinesterase gene (Ace). Mutation G126S in the bifenazate resistance-related cytochrome b gene (Cytb) was observed in three of the seven T. urticae populations. Higher activities of detoxification enzymes (P450, GST, CarEs and UGTs) were observed in two T. urticae populations, with significant difference in the XY-SX population. These results provide useful information on the status of pesticide resistance of T. urticae in China and suggest that T. urticae field populations may have multiple resistance mechanisms.

Tolerance to chemical insecticides can be driven by the necessity of herbivorous insects to defend against host plant-produced phytochemicals. However, how the phytochemicals are sensed and further transduced into a defense response associated with insecticide tolerance is poorly understood. Herein, we show that pre-exposure to flavone, a flavonoid phytochemical, effectively enhanced larval tolerance to multiple synthetic insecticides and elevated detoxification enzyme activities in Spodoptera litura. RNA-Seq analysis revealed that flavone induced a spectrum of genes spanning phase I and II detoxification enzyme families, as well as two transcription factors Cap \"n\" collar isoform C (CncC) and its partner small muscle aponeurosis fibromatosis (MafK). Knocking down of CncC by RNA interference suppressed flavone-induced detoxification gene expression and rendered the larvae more sensitive to the insecticides. Flavone exposure elicited a reactive oxygen species (ROS) burst, while scavenging of ROS inhibited CncC-mediated detoxification gene expression and suppressed flavone-induced detoxification enzyme activation. Metabolome analysis showed that the ingested flavone was mainly converted into three flavonoid metabolites, and only 3-hydroxyflavone was found to affect the ROS/CncC pathway-mediated metabolic detoxification. These results indicate that the ROS/CncC pathway is an important route driving detoxification gene expression responsible for insecticide tolerance after exposure to the phytochemical flavone.

Insecticide resistance is one of the major obstacles for controlling agricultural pests. There have been a lot of studies on insecticides stimulating the development of insect resistance. Herbicides account for the largest sector in the agrochemical market and are often co-applied with insecticides to control insect pests and weeds in the same cropland ecosystem. However, whether and how herbicides exposure will affect insecticide resistance in insect pests is largely unexplored. Here we reported that after exposure to herbicide butachlor, the lepidopteran Spodoptera litura larvae reduced susceptibility to the insecticide chlorpyrifos. Docking simulation studies suggested that general odorant-binding protein 2 (GOBP2) could bind to butachlor with high binding affinity, and silencing SlGOBP2 by RNA interference (RNAi) decreased larval tolerance to chlorpyrifos. Butachlor exposure induced ecdysone biosynthesis, whose function on increasing chlorpyrifos tolerance was supported in synergism experiments and confirmed by silencing the key gene (SlCYP307A1) for ecdysone synthesis. Butachlor exposure also activated the expression of detoxification enzyme genes. Silencing the genes with the highest herbicide-induced expression among the three detoxification enzyme genes led to increased larval susceptibility to chlorpyrifos. Collectively, we proposed a new mechanism that olfactory recognition of herbicides by GOBP2 triggers insect hormone biosynthesis and leads to high metabolic tolerance against insecticides. These findings provide valuable information for the dissection of mechanisms of herbicide-induced resistance to insecticides and also supplements the development of reduced-risk strategies for pest control.

Plants release an array of volatile chemicals into the air to communicate with other organisms in the environment. Insect attack triggers emission of herbivore-induced plant volatiles (HIPVs). How insect herbivores use these odors to plan their detoxification systems is vital for insect adaptation to environmental xenobiotics. Here we show that the larvae of Helicoverpa armigera (Hübner), a broadly polyphagous lepidopteran herbivore, have the capacity to use plant volatiles as cues to upregulate multiple detoxification systems, including cytochrome P450 monooxygenases (P450s), for detoxification of insecticides. Olfactory exposure of the fifth instars to two terpene volatiles limonene and nerolidol, and two green-leaf volatiles 2-heptanone and cis-3-hexenyl acetate significantly reduced larval susceptibility to the insecticide methomyl. However, larval pretreatment with piperonyl butoxide (PBO), a known P450 inhibitor, neutralized the effects of volatile exposure. Furthermore, larval exposure to the four plant volatiles enhanced activities of P450 enzymes in midguts and fatbodies, and upregulated expression of CYP6B2, CYP6B6 and CYP6B7, P450s involved in detoxification of the insecticide. Larval exposure to 2-heptanone and limonene volatiles also enhanced activities of glutathione-s-transferase and carboxylesterase. Our findings suggest that olfactory exposure to HIPVs enhances larval insecticide tolerance via induction of detoxification P450s.