First report of field-evolved resistance to insecticides in Spodoptera frugiperda (Lepidoptera: Noctuidae) from Punjab, Pakistan

Spodoptera frugiperda (Lepidoptera: Noctuidae) is a serious invasive pest, which has attracted concern regarding the effectiveness of environmental bioinsecticide as a substitution for synthetic insecticide in controlling its damage to numerous agricultural crops in recent years. Hence, laboratory and field experiments have investigated insecticidal toxicity in S. frugiperda by 5 insecticides and have determined the activity of acetylcholinesterase (AChE) and detoxifying enzymes, namely mixed function oxidase (MFO), carboxylesterase (CarE), and glutathione S-transferase (GST) on 3rd instar larvae at 25% lethal concentration (LC25) to explore detoxification mechanisms. The results showed that the most effective insecticides were 3.0% emamectin benzoate (0.024 mg/L), 60 g/L spinetoram (0.282 mg/L), and 1.3% matrine (0.380 mg/L) at 48 h depending on LC50 values. The field efficiencies of emamectin benzoate and spinetoram were over 80% at 24 h and 90% at 72 h, indicating that both insecticides had acute and long-lasting toxicity on S. frugiperda; matrine had extended-release toxicity with 90% field efficiency at 72 h. Emamectin benzoate and spinetoram activated the activities of AChE, MFO and GST, and spinetoram decreased in CarE activity; cyantraniliprole induced an increase of 4 detoxifying enzymes; spinosad increased the activities of AChE, MFO, and CarE but not GST. Furthermore, matrine had an inhibiting effect on AChE and acceleration on CarE and GST. Overall results obviously depicted that semi-synthetic insecticide spinetoram and bioinsecticide matrine were recommended to control S. frugiperda with effective and long-lasting toxicity. Moreover, this study will provide basic information for sustainable control of S. frugiperda under field conditions in China.

Toxicity of some insecticides against egg masses and two larval instars of cotton leafworm (CLW), Spodoptera littoralis, field and laboratory strains was determined. The activities of glutathione S-transferases (GST) and alkaline phosphatases (ALP) in the two strains also were compared. Results revealed that, spinosad, spinetoram, emamectin benzoate and lufenuron have low ovicidal activity. Chlorpyrifos and methomyl at 10 ppm achieved 80.4 and 83.6% mortality of treated egg masses, respectively. On the other hand, spinosad, spinetoram and emamectin benzoate at the same concentration caused 18.9, 19.4 and 28.1% mortality of treated egg masses, respectively. While lufenuron at 25 ppm caused 54.9% mortality of treated egg masses, lufenuron at 100 ppm achieved 100% mortality of treated egg masses at 100 ppm. Although spinosad, spinetoram and emamectin benzoate have low ovicidal activity compared with methomyl and chlorpyrifos, they have a comparable residual toxicity with methomyl and chlorpyrifos against the neonates. The residual toxicity of lufenuron against neonates is low at all tested concentrations. The 2nd and 4th instar larvae of the field strain exerts high resistance levels towards methomyl, chlorpyrifos and esfenvalerate. Regarding the 2nd instar, resistance ratios in 2008 and 2009 cotton seasons were 43.9 and 50.8 towards methomyl, 27.6 and 24.7 towards chlorpyrifos and 76.4 and 79.2 towards esfenvalerate. For the 4th instar resistance ratios in 2008 and 2009 cotton seasons were 62.8 and 63.6 towards methomyl, 43.9 and 49.2 towards chlorpyrifos and 112.4 and 114.8 towards esfenvalerate. On the other hand, field strain shows low or no levels of tolerance to spinosad, spinetoram, emamectin benzoate, lufenuron and methoxyfenozide. Activities of GST and ALP in the field strain were higher compared with that in the laboratory strain. In conclusion, the alternation between these insecticides can avoid increasing selection pressure of CLW populations to these insecticides.

Insect glutathione S-transferases (GSTs) serve critical roles in insecticides and other forms of xenobiotic chemical detoxification. The fall armyworm, Spodoptera frugiperda (J. E. Smith), is a major agricultural pest in several countries, especially Egypt. This is the first study to identify and characterize GST genes in S. frugiperda under insecticidal stress. The present work evaluated the toxicity of emamectin benzoate (EBZ) and chlorantraniliprole (CHP) against the third-instar larvae of S. frugiperda using the leaf disk method. The LC50 values of EBZ and CHP were 0.029 and 1.250 mg/L after 24 h of exposure. Moreover, we identified 31 GST genes, including 28 cytosolic and 3 microsomal SfGSTs from a transcriptome analysis and the genome data of S. frugiperda. Depending on the phylogenetic analysis, sfGSTs were divided into six classes (delta, epsilon, omega, sigma, theta, and microsomal). Furthermore, we investigated the mRNA levels of 28 GST genes using qRT-PCR under EBZ and CHP stress in the third-instar larvae of S. frugiperda. Interestingly, SfGSTe10 and SfGSTe13 stood out with the highest expression after the EBZ and CHP treatments. Finally, a molecular docking model was constructed between EBZ and CHP using the most upregulated genes (SfGSTe10 and SfGSTe13) and the least upregulated genes (SfGSTs1 and SfGSTe2) of S. frugiperda larvae. The molecular docking study showed EBZ and CHP have a high binding affinity with SfGSTe10, with docking energy values of -24.41 and -26.72 kcal/mol, respectively, and sfGSTe13, with docking energy values of -26.85 and -26.78 kcal/mol, respectively. Our findings are important for understanding the role of GSTs in S. frugiperda regarding detoxification processes for EBZ and CHP.

Risk assessment, cross-resistance potential, and biochemical mechanism of resistance to emamectin benzoate in a field strain of house fly (Musca domestica Linnaeus)

Simple SummaryThe widespread cultivation of genetically engineered crops producing not only toxic proteins from the bacterium Bacillus thringiensis (Bt) but also plant defensive compounds known as allelochemicals, combined with occasional use of insecticides, is the major tactic to manage some economically important pests. Better understanding of the toxicological interactions of the three types of toxins is needed to rationally deploy them to protect crops from pests. The aim of this study is to examine the sequential and simultaneous interactions of the allelochemical flavone, Bt toxin Vip3A, and insecticide emamectin benzoate in the fall armyworm (Spodoptera frugiperda), a worldwide target pest of Bt crops. Bioassays of S. frugiperda neonates revealed that all interactions of the three toxins, except for 1-day pre-exposure to a sublethal dose (LC5) of flavone followed by 6-day simultaneous exposure to flavone LC5 + emamectin benzoate LC50, are synergistic or additive. The results suggest that the combined use of the three toxins is basically a great strategy to manage S. frugiperda.Target pests of genetically engineered crops producing both defensive allelochemicals and Bacillus thuringiensis (Bt) toxins often sequentially or simultaneously uptake allelochemicals, Bt toxins, and/or insecticides. How the three types of toxins interact to kill pests remains underexplored. Here we investigated the interactions of Bt toxin Vip3A, plant allelochemical flavone, and insecticide emamectin benzoate in Spodoptera frugiperda. Simultaneous administration of flavone LC25 + Vip3A LC25, emamectin benzoate LC25 + Vip3A LC25, and flavone LC15 + emamectin benzoate LC15 + Vip3A LC15 but not flavone LC25 + emamectin LC25 yielded a mortality significantly higher than their expected additive mortality (EAM). One-day pre-exposure to one toxin at LC5 followed by six-day exposure to the same toxin at LC5 plus another toxin at LC50 showed that the mortality of flavone LC5 + Vip3A LC50, emamectin benzoate LC5 + Vip3A LC50, and Vip3A LC5 + emamectin benzoate LC50 were significantly higher than their EAM, while that of flavone LC5 + emamectin benzoate LC50 was significantly lower than their EAM. No significant difference existed among the mortalities of Vip3A LC5 + flavone LC50, emamectin benzoate LC5 + flavone LC50, and their EAMs. The results suggest that the interactions of the three toxins are largely synergistic (inductive) or additive, depending on their combinations and doses.

Maize is affected by more than 30 insect pests and the recent attack by the invasive fall armyworm Spodoptera frugiperda has been a serious concern to maize growers all over the country. Being polyphagous with a wider host range, the pest necessitates the use of insecticides in the hitherto unsprayed maize ecosystems of the country. Evaluation of newer insecticides against fall armyworm revealed that, the combination insecticide, chlorantraniliprole 9.3% + lambda cyhalothrin 4.6%ZC registered lesser infestation after first round of spraying. However after two rounds of spraying, emamectin benzoate 5%SG, spinetoram 11.7%SC, chlorantraniliprole 18.5%SC, flubendiamide 480%SC and chlorantraniliprole 9.3% + lambda cyhalothrin 4.6%ZC were on par in their efficacy in reducing the fall armyworm infestation, though emamectin benzoate and flubendiamide treated plots registered comparatively higher yields. With respect to B:C ratio, emamectin benzoate and chlorantraniliprole 18.5%SC registered comparatively higher BC ratio followed by flubendiamide 480%SC, spinetoram 11.7%SC and chlorantraniliprole 9.3% + lambda cyhalothrin 4.6% ZC. Thus the above insecticides can be used in maize ecosystems to effectively tackle the fall armyworm menace.

The fall armyworm, Spodoptera frugiperda (J. E. Smith), is one of the main pests of corn in many areas of the American continent. The reliance on pesticides to control fall armyworm has led to the development of insecticide resistance in many regions. We determined the resistance levels of fall armyworm to insecticides of different modes of action in fall armyworm populations from Puerto Rico and several Mexican states with different insecticide use patterns. Mexican populations that expressed higher resistance ratios (RR50) were: Sonora (20-fold to chlorpyriphos), Oaxaca (19-fold to permethrin), and Sinaloa (10-fold to flubendamide). The Puerto Rico population exhibited a remarkable field-evolved resistance to many pesticides. The RR50 to the insecticides tested were: flubendiamide (500-fold), chlorantraniliprole (160-fold), methomyl (223-fold), thiodicarb (124-fold), permethrin (48-fold), chlorpyriphos (47-fold), zeta-cypermethrin (35-fold), deltamethrin (25-fold), triflumuron (20-fold), spinetoram (14-fold). Spinosad (eightfold), emamectin benzoate and abamectin (sevenfold) displayed lower resistance ratio. However, these compounds are still effective to manage fall armyworm resistance in Puerto Rico. Fall armyworm populations from Mexico show different levels of susceptibility, which may reflect the heterogeneity of the pest control patterns in this country. The status of insecticide resistance in the fall armyworm from Puerto Rico indicates a challenging situation for the control of this pest with these insecticides in the close future. Lessons learned from this research might be applied in regions with recent invasions of fall armyworm in Africa.

The black inch looper, Hyposidra talaca is a serious defoliator pest of tea and its management relies profoundly on application of insecticides. Here, we investigated the insecticide susceptibility in H. talaca collected from six districts of Assam to five insecticides: bifenthrin and deltamethrin (Pyrethroids), quinalphos (Organophosphates), emamectin benzoate (Avermectins) and flubendiamide (Diamides). Based on relative susceptibility values, all populations showed varied levels of susceptibility to the insecticides evaluated as compared to the susceptible population. Tinsukia and Dibrugarh populations showed high LC50s to most of the insecticides assessed. A moderate to high level of resistance was recorded for deltamethrin [Resistance coefficient (RC) = 19.03–31.75; Resistance ratio (RR) = 8.23–38.90]. Bifenthrin showed moderate level of resistance [RC = 11.80-23.79; RR = 16.52–25.52] while quinalphos demonstrated low-resistance level [RC = 4.35–6.06; RR = 3.22–4.41]. Emamectin benzoate and flubendiamide were effective against H. talaca in all tested populations, except for Tinsukia and Dibrugarh populations where low levels of resistance were recorded. Detoxification enzyme assays indicate that glutathione S-transferases (GST), general esterase (GE) and cytochrome P450 (CYP) activities were high in all field populations as compared to susceptible ones. GE and CYP had significant variation among different population and registered positive correlation with the LC50s of deltamethrin, bifenthrin and quinalphos. Synergistic assays reveal that the use of synergists’ piperonyl butoxide, S.S.S.-Tributylphosphorotrithioate and ethacrynic acid in combination with deltamethrin and quinalphos re-establishes the toxicity of these insecticides. The findings show that detoxification enzymes play important role in the development of resistance to insecticides in H. talaca and will be useful in designing resistance management programmes against the pest.

Biochemical and genetic mechanisms in Pieris rapae (Lepidoptera: Pieridae) resistance under emamectin benzoate stress

Maize is affected by more than 30 insect pests and the recent attack by the invasive fall armyworm Spodoptera frugiperda has been a serious concern to maize growers all over the country. Being polyphagous with a wider host range, the pest necessitates the use of insecticides in the hitherto unsprayed maize ecosystems of the country. Evaluation of newer insecticides against fall armyworm revealed that, the combination insecticide, chlorantraniliprole 9.3% + lambda cyhalothrin 4.6%ZC registered lesser infestation after first round of spraying. However after two rounds of spraying, emamectin benzoate 5%SG, spinetoram 11.7%SC, chlorantraniliprole 18.5%SC, flubendiamide 480%SC and chlorantraniliprole 9.3% + lambda cyhalothrin 4.6%ZC were on par in their efficacy in reducing the fall armyworm infestation, though emamectin benzoate and flubendiamide treated plots registered comparatively higher yields. With respect to B:C ratio, emamectin benzoate and chlorantraniliprole 18.5%SC registered comparatively higher BC ratio followed by flubendiamide 480%SC, spinetoram 11.7%SC and chlorantraniliprole 9.3% + lambda cyhalothrin 4.6% ZC. Thus the above insecticides can be used in maize ecosystems to effectively tackle the fall armyworm menace.

Fall armyworm, Spodoptera frugiperda (J. E. Smith), is a relevant global pest due to severe damage caused on agricultural crops and its capacity to evolve resistance to insecticides. Here, we selected a strain of S.frugiperda resistant to emamectin benzoate under laboratory conditions to understand the inheritance patterns, cross-resistance and synergism involved in the resistance. The emamectin benzoate-resistant (Ben-R) strain was isolated by using F2 screen in a field population collected in Lucas do Rio Verde, Mato Grasso state, Brazil. After ten generations of selection pressure with emamectin benzoate, the estimated LC50 of the Ben-R strain was 678.38 μg a.i. mL-1 whereas that of the susceptible (Sus) strain was 0.29 μg a.i.mL-1 , resulting in a resistance ratio (RR) of 2340-fold. The LC50 values of the offspring from reciprocal crosses of Sus and Ben-R strains were 93.37 and 105.32 μg a.i. mL-1 , suggesting that resistance is an autosomal incompletely dominant trait. The high survival of heterozygous and Ben-R strains (>92%) on non-Bt maize sprayed with the field rate of emamectin benzoate confirmed that resistance is functionally dominant. The minimum number of segregations influencing resistance was 3.55, suggesting a polygenic effect. Low cross-resistance was detected between emamectin benzoate and the insecticides methomyl, chlorpyrifos, lambda-cyhalothrin, spinetoram, indoxacarb and chlorantraniliprole (RR <5.75-fold). There was no effect of synergists piperonyl butoxide, diethyl maleate and S, S, S-tributyl phosphorotrithiotate on the Ben-R strain, suggesting a minor role of metabolic resistance. Our results showed a high risk of resistance evolution of S.frugiperda to emamectin benzoate, based on incompletely dominant inheritance. Rotation of insecticides with different modes of action can be one of the resistance management strategies to be implemented to delay the evolution of resistance of S.frugiperda to emamectin benzoate in Brazil.

The fall armyworm (FAW) Spodoptera frugiperda is a notorious pest, causing severe crop damage worldwide and prompting effective prevention and control. Over-reliance on and intensive use of insecticides are prone to leading to the rapid evolution of insecticide resistance, urging rational insecticide application. One effective way of rational insecticide application is to apply insecticides of different modes of action in combination or supplemented with adjuvants. In this study, we assessed the efficacies of two individual insecticides, emamectin benzoate (EB) and chlorantraniliprole (CT), and their mixture, supplemented with and without the oil adjuvant Jijian® to control FAW in laboratory bioassays and a field trial. Both EB and CT showed high toxicities to FAW. The EB × CT mixture at a mass ratio of 9:1 yielded a remarkable synergistic effect, with the co-toxicity coefficient (CTC) being 239.38 and the median lethal concentration (LC50) being 0.177 mg/L. In leaf-spray bioassays, the addition of the adjuvant reduced the LC50 values of both the individual insecticides and the EB × CT mixture by more than 59%, significantly improving the efficacies. The field trial confirmed the synergistic effects of the adjuvant, which reduced the amount of EB × CT mixture by 80%. This study provides an effective and promising insecticide-adjuvant mixture to control S. frugiperda.

The fall armyworm (Spodoptera frugiperda) is a worldwide maize pest originating from the American continent. It invaded Africa during 2016, causing important economic damages, forcing African countries to take urgent actions to tackle this new invasive pest. In Burkina Faso, several chemical insecticides were promoted, but farmers have quickly and repeatedly reported control failures. In this work, we collected seven fall armyworm populations in as many maize-producing areas of Burkina Faso. Following the approved IRAC leaf bioassay protocol, we evaluated the susceptibility of third instar larvae to seven commercially available insecticide formulations, including various modes of action: methomyl and chlorpyriphos-ethyl (acetylcholinesterase inhibitors), deltamethrin and lambda-cyhalothrin (sodium channel modulators), emamectin benzoate and abamectin (chloride channel activators) and Bacillus thuringiensis (a microbial disruptor of insect midgut membranes). Lethal concentrations (LC50), resistance ratios (RR50) and relative toxicity were calculated for each population and active ingredient. LC50 values for all S. frugiperda populations were, in order of importance: emamectin benzoate (0.33–0.38 µg/l), methomyl (18–73 mg/l), abamectin (58–430 mg/l), chlorpyrifos-ethyl (199–377 mg/l), deltamethrin (70–541 mg/l) and lambda-cyhalothrin (268–895 mg/l). LC50 of the B. thuringiensis formulation ranged from 430 to 614 MIU/l. Lambda-cyhalothrin was the least efficient of the tested chemical pesticides, and emamectin benzoate the most efficient (relative toxicity ×2 712 969). Methomyl (×49), abamectin (×15), deltamethrin (×13), chlorpyriphos-ethyl (×4) were also more toxic than lambda-cyhalothrin. Based on these results, we conclude that emamectin benzoate, methomyl and chlorpyriphos-ethyl insecticides are the most efficient for the control of the fall armyworm in Burkina Faso. We discuss the importance to implement a national-level resistance survey for this major pest, which would allow rapid and efficient adaptation of the control strategy.

Evidence of field-evolved resistance in Spodoptera frugiperda (Lepidoptera: Noctuidae) to emamectin benzoate in Brazil

BackgroundThe indiscriminating and intensive use of insecticides to control the cotton leafworm (CLW), Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), usually induces high levels of resistance. The insecticides are the principal method for controlling this pest because of its critical role in reducing insects when the economic threshold (ETL); therefore, the effectiveness of these insecticides should be maintained. So, the target of the present work was directed to focus on studying the change in the activities of some important enzymes as a result of sublethal treatment concentrations (viz. LC25 values) of tested new insecticides (profenofos, cyfluthrin, emamectin benzoate, lufenuron, and spinetoram). The expected results could offer better understanding and more specific information about the resistance development in field populations of CLW because resistance is a significant challenge to pest control workers and these results may contribute to making the right decision at the right time.Materials and methodsBioassays were performed on fourth instar larvae of S. littoralis field populations compared with the laboratory strain to assess the activity of the emamectin benzoate, lufenuron, and spinetoram by LC25 and study the biochemical activities of some detoxification enzymes, like acetylcholinesterase (AChE), glutathione S-transferases (GST), alkaline phosphatase (ALP), and acid phosphatase (ACP) in fourth larval instar which was treated with LC25; protein content is also determined.ResultsThe emamectin benzoate was the most toxic compound followed by lufenuron, cyfluthrin, spinetoram, and profenofos with the corresponding LC50 values of 0.05, 49.18, 70.99, 130.26, and 156.78 ppm respectively. The results showed that all the tested insecticides at LC25 value gave a slight inhibition of the acetylcholinesterase (AChE) activity, except profenofos was the most potent one. The activity of glutathione S-transferase (GST) of larvae treated with spinetoram has significantly increased, whereas the enzyme activity was significantly inhibited following cyfluthrin and non-significantly inhibited following profenofos, lufenuron, and emamectin benzoate application. There were no significant differences between treated and untreated larvae in ACP activity. In contrast, the alkaline phosphatase (ALP) activity of larvae treated with tested insecticides significantly increased, while the activity was inhibited following cyfluthrin application. On the other hand, spinetoram, emamectin benzoate, and lufenuron exhibited significant increment in the protein content, whereas there was no significant effect following either cyfluthrin or profenofos treatments.ConclusionsIn summary, the present results suggest that not only the lethal effects but also the sublethal effects of newly tested insecticides could have a negative influence on the dynamics of CLW; thus, these sublethal effects can be integrated into pest control to reduce the overuse and misuse of insecticides. This effect appears reduction in the activity of detoxification enzymes, resulting in response to the tested insecticide by the lowest concentration of (sublethal doses). Also, the inhibition of detoxification enzymes, which represents defensive reactions in insects, is playing important role in reducing resistance in S. littoralis, which is one of the most dangerous pests of all agricultural crops in the world. Therefore, these results were valuable for the practical use of these insecticides in IPM programs.