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Spodoptera litura is a serious polyphagous pest in the whole world, which has developed resistance to most conventional insecticides and even some Bacillus thuringiensis (Bt) toxins. Cry1Ca has excellent insecticide activity against S. litura with potential application to control S. litura and delay the development of insect resistance. However, the mode of action of Cry1Ca in S. litura is poorly understood. Here, Cry1Ca-binding proteins were identified from S. litura by using pull down assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicated that aminopeptidase-N (APN), ATP binding cassette subfamily C member 2 (ABCC2), polycalin, actin and V-type proton ATPase subunit A may bind with Cry1Ca. Further study confirmed that ABCC2 fragment expressed in vitro can bind to Cry1Ca as demonstrated by Ligand blot and homologous competition experiments. The over-expression of endogenous SlABCC2 in Sf9 cells increased Cry1Ca cytotoxicity. Correspondingly, the vivo loss of function analyses by SlABCC2 small interfering RNAs (siRNAs) in S. litura larvae decreased the toxicity of Cry1Ca to larvae. Altogether, these results show that ABCC2 of S. litura is a functional receptor that is involved in the action mode of Cry1Ca.

Background The extensive planting of transgenetic Bacillus thuringiensis crops has driven the evolution of pest resistance to Cry1Ac. The adjustment of cropping structures has promoted the re-outbreak of Helicoverpa armigera in China. To control this pest, a combination of pyramiding RNA interference (RNAi) and Cry2Ab is considered as a promising strategy for countering the cross-resistance and enhancing the toxicity of Cry2Ab to cotton bollworm. We explored the possibility of using calcineurin (CAN) as a target RNAi gene, because it is involved in the cotton bollworm's responses to the toxicity of Cry2Ab. Results Cry2Ab treatment led to a significant increase in HaCAN mRNA level and HaCAN activity. The suppression of HaCAN activity due to RNAi-mediated knockdown of HaCAN increased the susceptibility of midgut cells to Cry2Ab. The increase in HaCAN activity shown by heterologous expression of HaCAN, reduced the cytotoxicity of Cry2Ab to Sf9 cells. Moreover, the ingestion of HaCAN-specific inhibitor FK506 increased the toxicity of Cry2Ab in the larvae. Interestingly, HaCAN does not function as a Cry2Ab direct binding protein that participates in Cry2Ab toxicity. Conclusions The results in present study provide evidence that suppression of HaCAN did not only affect the development of the cotton bollworm, but also enhanced the toxicity of Cry2Ab to the pest. HaCAN is therefore an important candidate gene in the cotton bollworm which can be targeted for pest control, when the pest infests RNAi+Cry2Ab crops. Meanwhile, the mechanism of action of HaCAN in Cry2Ab toxicity suggested that protein dephosphorylation was involved in Cry2Ab toxicity. This article is protected by copyright. All rights reserved.

Insect resistance to Bacillus thuringiensis (Bt) is a critical limiting factor for applying the Bt crops. Some studies indicated that decreased protoxin activation because of lower enzymatic activities of trypsin and chymotrypsin and increased expression of serpin might involve in Bt resistance. Our previous study identified an endogenous serpin could inhibit the midgut proteases to activate Cry1Ac and reduce the insecticide activity to Helicoverpa armigera. We hypothesis that up-regulated serpin involve in resistance via inhibiting enzymatic activities of trypsin and chymotrypsin to decrease protoxin activation. Herein, we found the serpin-e gene relative expression in midgut was significantly higher in the LF30 resistant strain than that in the susceptible strain during all developmental stages. Importantly, RNAi-mediated silencing of serpin-e gene expression caused 4.46-fold mortality changes in LF30 strain, but the trypsin and chymotrypsin proteases activities were only changed 0.79-fold and 2.22-fold. In addition, although proteases activities were significantly lower in LF30 strain than that in the susceptible strain, the resistance ratios of LF30 to Cry1Ac protoxin and to activated Cry1Ac toxin were no difference. The results indicated serpins caused insect resistance to Cry1Ac protoxins partly through inhibiting the trypsin and chymotrypsin proteases activities, but it also existed other mechanisms in LF30.

Receptor proteins on the brush border membrane of the insect midgut epithelium are involved in the mode of action of insecticidal Cry proteins from Bacillus thuringiensis (Bt). Polycalin has been identified as a binding protein of the Bt Cry1Ac toxin in several Lepidoptera including Helicoverpa armigera, but its role in the action mechanism of Cry2Aa is still unclear. In this study, we investigated the binding characteristics of polycalin from the midgut of H. armigera with Cry2Aa and its role in the toxicity of Cry2Aa. The results demonstrated that heterologously expressed H. armigera polycalin peptide could bind with Cry2Aa with high affinity (Kd=32 nmol L–1). The toxicity of Cry2Aa decreased by 27% after H. armigera larvae ingested polycalin antisera. These results suggested that polycalin could be a potential functional receptor for Cry2Aa, and it plays an important role in the susceptibility of H. armigera to Cry2Aa.

Although membrane-bound alkaline phosphatases (ALPs) have been proposed as a receptor for Cry1Ac in a few lepidopteran species, their potential functions as a Cry2Ab receptor are yet to be verified. To determine if ALP2 also serves as a receptor for Cry1Ac and even for Cry2Ab in Helicoverpa zea, we measured the potency of activated Cry1Ac and Cry2Ab against midgut and fat body cell lines of H. zea and the ovarian cell line of Spodoptera frugiperda (Sf9) expressing H. zea ALP2 (HzALP2) or transfected with HzALP2 double-stranded RNA (dsRNA). Relative to the control cells, the three cell lines expressing HzALP2 were more susceptible to Cry1Ac but there was no difference for Cry2Ab. By contrast, the two H. zea cell lines transfected with HzALP2 dsRNA were resistant to Cry1Ac while kept susceptible to Cry2Ab. Furthermore, RNA interference knockdown of HzALP2 in H. zea larvae enhanced larval survival on Cry1Ac-containing diets. These findings indicate that HzALP2 functions as a receptor of Cry1Ac but not Cry2Ab.

Helicoverpa armigera (cotton bollworm) is one of the most destructive pests worldwide. Due to resistance to Bacillus thuringiensis and conventional insecticides, an effective management strategy to control this pest is urgently needed. Spinosad, a natural pesticide, is considered an alternative; however, the mechanism underlying the developmental effects of sublethal spinosad exposure remains elusive. In this study, the mechanism was examined using an insect model of H. armigera. Results confirmed that exposure to sublethal spinosad led to reduced larval wet weight, delayed larval developmental period, caused difficulty in molting, and deformed pupae. Further investigation demonstrated that exposure to sublethal spinosad caused a significant decrease in 20E titer and increase in JH titer, thereby leading to the discordance between 20E and JH titers, and consequently alteration in the expression levels of HR3 and Kr-h1. These results suggested that sublethal spinosad caused hormonal disorders in larvae, which directly affect insect development. Our study serves as a reference and basis for the toxicity evaluation of spinosad on molting and pupation in insect metamorphosis, which may contribute to identifying targets for effective control of cotton bollworm.

Bt protoxins are required to convert to a smaller activated form by insect midgut proteases to exert toxicity against insect pests. Serine protease inhibitors (serpins) play a valuable part in gut protease of insect that hamper digestive proteases activity of insects. Whether the insect serpins induced by Bt protoxin affect the insecticidal activity were rare studied. Here, we identified a serpin-e gene from Helicoverpa armigera, which had potential RCL (Reactive Center Loop) region near the C-terminus like other serpin proteins. It widely expressed in different development stages and in various tissues, but highest expressed in fourth-instar larvae and in larval hemolymph. This Haserpin-e could be induced by Cry1Ac protoxin in vivo and inhibit the midgut proteases to activate Cry1Ac in vitro. Importantly, the functional study indicated it could inhibit the process from Cry1Ac protoxin to activated toxin, and led to the reduction of Cry1Ac insecticide activity to cotton bollworm. Based on our results, we proposed that Haserpin-e involved in the toxicity of Cry1Ac to cotton bollworm by blocking the serine protease to activate the protoxin.

Bt crop pyramids produce two or more Bt proteins active to broaden the spectrum of action and to delay the development of resistance in exposed insect populations. The cross-resistance between Bt toxins is a vital restriction factor for Bt crop pyramids, which may reduce the effect of pyramid strategy. In this review, the status of the cross-resistance among more than 20 Bt toxins that are most commonly used against 13 insect pests was analyzed. The potential mechanisms of cross-resistance are discussed. The corresponding measures, including pyramid RNA interference and Bt toxin, "high dose/refuge," and so on are advised to be taken for adopting the pyramided strategy to delay the Bt evolution of resistance and control the target pest insect.

Helicoverpa armigera can develop resistance to Bacillus thuringiensis (Bt), which threaten the long-term success of Bt crops. In the present study, RNAseq was employed to investigate the midgut genes response to strains with different levels of resistance (LF5, LF10, LF20, LF30, LF60, and LF120) in H. armigera. Results revealed that a series of differentially expressed unigenes (DEGs) were expressed significantly in resistant strains compared with the LF-susceptible strain. Nine trypsin genes, ALP2, were downregulated significantly in all the six resistant strains and further verified by qRT-PCR, indicating that these genes may be used as markers to monitor and manage pest resistance in transgenic crops. Most importantly, the differences in DEG functions in the different resistant strains revealed that different resistance mechanisms may develop during the evolution of resistance. The immune and detoxification processes appear to be associated with the low-level resistance (LF5 strain). Metabolic process-related macromolecules possibly lead to resistance to Cry1Ac in the LF10 and LF20 strains. The DEGs involved in the “proton-transporting V-type ATPase complex” and the “proton-transporting two-sector ATPase complex” were significantly expressed in the LF30 strain, probably causing resistance to Cry1Ac in the LF30 strain. The DEGs involved in binding and iron ion homeostasis appear to lead to high-level resistance in the LF60 and LF120 strains, respectively. The multiple genes and different pathways seem to be involved in Cry1Ac resistance depending on the levels of resistance. Although the mechanisms of resistance are very complex in H. armigera, a main pathway seemingly exists, which contributes to resistance in each level of resistant strain. Altogether, the findings in the current study provide a transcriptome-based foundation for identifying the functional genes involved in Cry1Ac resistance in H. armigera.

To delay evolution of resistance by insect pests, farmers are rapidly increasing their use of transgenic crops producing two or more Bacillus thuringiensis (Bt) toxins that kill the same pest. A key condition favoring durability of these "pyramided" crops is the absence of cross-resistance between toxins. Here we evaluated cross-resistance in the major lepidopteran pest Helicoverpa zea (Boddie) to Bt toxins used in pyramids. In the laboratory, we selected a strain of this pest with Bt toxin Cry1Ac followed by selection with MVP II, a formulation containing a hybrid protoxin that is identical to Cry1Ac in the active portion of the toxin and 98.5% identical overall. We calculated the resistance ratio as the EC50 (concentration causing mortality or failure to develop beyond the first instar of 50% of larvae) for the laboratory-selected strain divided by the EC50 for its field-derived parent strain that was not selected in the laboratory. The resistance ratio was 20.0-33.9 (mean=27.0) for MVP II, 57.0 for Cry1Ac, 51.3 for Cry1A.105, 22.4 for Cry1Ab, 3.3 for Cry2Ab, 1.8 for Cry1Fa, and 1.6 for Vip3Aa. Resistance ratios were 2.9 for DiPel ES and 2.0 for Agree VG, which are commercial Bt spray formulations containing Cry1Ac, other Bt toxins, and Bt spores. By the conservative criterion of non-overlap of 95% fiducial limits, the EC50 was significantly higher for the selected strain than its parent strain for MVP II, Cry1Ac, Cry1A.105, Cry1Ab, Cry2Ab and DiPel ES. For Cry1Fa, Vip3Aa, and Agree VG, significantly lower susceptibility to a high concentration indicated low cross-resistance. The resistance ratio for toxins other than Cry1Ac was associated with their amino acid sequence similarity to Cry1Ac in domain II. Resistance to Cry1Ac and the observed cross-resistance to other Bt toxins could accelerate evolution of H. zea resistance to currently registered Bt sprays and pyramided Bt crops.

Helicoverpa armigera is an universal pest around the world, which has recovered again in recent years because of the adjustment of cropping structure and resistance to Bacillus thuringiensis (Bt) in China. As a new insecticide spinetoram is extensively used to control many pest insects, including H. armigera. However the lethal and sublethal effects of spinetoram on cotton bollworm have not been assessed. In the present study, the toxicity of spinetoram against cotton bollworm was tested under laboratory conditions. Results demonstrated spinetoram showed an excellent activity against H. armigera, especially, against Bt (Cry1Ac) resistant H. armigera. Treatment with spinetoram at the doses of 0.19 mg/kg and 0.36 mg/kg (LC8 and LC20 after 24h oral exposure) significantly arrested the development of surviving larvae and caused significant decrease in larvae wet weight. Besides, the survivors after spinetoram treatments showed significant reduction of pupation ratio, pupal weight, emergence ratio, longevity and fecundity of adults. At same time, spinetoram treatments resulted in significant increase in the prepupal and pupal periods of survivors. In summary, these results showed that spinetoram could be used as an effective pesticide to control H. armigera, especially Cry1Ac-ressitacne, consequently to take both lethal and sublethal effects to cotton bollworm into consideration in cotton bollworm control strategy.

BACKGROUND Field-evolved resistance of Helicoverpa zea to Bacillus thuringiensis (Bt) toxin Cry1Ac was first reported more than a decade ago, yet the underlying mechanisms remain elusive. Towards understanding the mechanisms of resistance to Cry1Ac, we analyzed a susceptible (LAB-S) and two resistant (GA and GA-R) strains of H. zea. The GA strain was derived from Georgia and exposed to Bt toxins only in the field. The GA-R strain was derived from the GA strain and selected for increased resistance to Cry1Ac in the laboratory. RESULTS Resistance to MVPII, a liquid formulation containing a hybrid protoxin similar to Cry1Ac, was 110-fold for GA-R and 7.8-fold for GA relative to LAB-S. In midgut brush border membrane vesicles, activity of alkaline phosphatase and aminopeptidase N did not vary significantly among strains. The activity of total proteases, trypsin-like proteases and chymotrypsin-like proteases was significantly lower for GA-R and GA than LAB-S, but did not differ between GA-R and GA. When H. zea midgut cells were exposed to Cry1Ac protoxin that had been digested with midgut extracts, toxicity was significantly lower for extracts from GA-R and GA relative to extracts from LAB-S, but did not differ between GA-R and GA. Transcriptional analysis showed that none of the five protease genes examined was associated with the decline in Cry1Ac activation in GA-R and GA relative to LAB-S. CONCLUSION The results suggest that decreased Cry1Ac activation is a contributing field-selected mechanism of resistance that helps explain the reduced susceptibility of the GA-R and GA strains. Relative to the LAB-S strain, the two Cry1Ac-resistant strains had lower total protease, trypsin and chymotrypsin activities, a lower Cry1Ac activation rate, and Cry1Ac protoxin incubated with their midgut extracts was less toxic to H. zea midgut cells. © 2018 Society of Chemical Industry.

In this study, the results showed that the population of midgut bacteria and larval mortality due to Cry1Ac are significantly reduced in antibiotic-treated larvae from Bt-susceptible, -resistant and field-collected strains (96S, BtR, FS respectively) of Helicoverpa armigera. The percentage reduction of larval mortality with increasing concentrations of antibiotics was significantly different among strains with the smallest effect observed in FS. It has been suggested that antibiotics could influence the toxicity of Cry1Ac, possibly by eliminating gut bacteria, hence gut bacteria might be playing essential roles in Bt-induced killing of H. armigera. But elimination of midgut microflora with antibiotics had no effect on resistance level.

A pyramid strategy combining the Cry1A and Cry2A toxins in Bt crops has been widely used throughout the world to delay pest adaption to transgenic crops and broaden the insecticidal spectrum. Midgut membrane-bound cadherin (CAD), aminopeptidase-N (APN) and alkaline phosphatase (ALP) are important for Cry1A toxicity in some lepidopteran larvae, but the proteins that bind Cry2A in the midgut of target insects and their role in the Cry2A mechanism of action are still unclear. In this study, we found that heterologously expressed CAD, APN4 and ALP2 peptides from the midgut of Helicoverpa armigera could bind to the Cry2Aa toxin with a high affinity. Additionally, the efficiency of Cry2Aa insecticidal activity against H. armigera larvae was obviously reduced after the genes encoding these proteins were silenced with specific siRNAs: CAD- and ALP2-silenced larvae showed significantly similar reductions in mortality due to the Cry2Aa toxin (41.67% and 43.06%, respectively), whereas a larger reduction in mortality was observed in APN4-silenced larvae (61.11%) than in controls. These results suggest that CAD, APN4 and ALP2 are involved in the mechanism of action of Cry2Aa in H. armigera and may play important functional roles in the toxicity of the Cry2Aa toxin.

While Cry1Ac has been known to bind with larval midgut proteins cadherin, APN (amino peptidase N), ALP (alkaline phosphatase) and ABCC2 (adenosine triphosphate-binding cassette transporter subfamily C2), little is known about the receptors of Cry2Ab. To provide a clue to the receptors of Cry2Ab, we tested the baseline cytotoxicity of activated Cry1Ac and Cry2Ab against the midgut and fat body cell lines of Helicoverpa zea and the ovary cell line of Spodoptera frugiperda (SF9). As expected, the descending order of cytotoxicity of Cry1Ac against the three cell lines in terms of 50% lethal concetration (LC

Crystalline (Cry) proteins from Bacillus thuringiensis (Bt) are used extensively for insect control in sprays and transgenic plants, but their efficacy is reduced by evolution of resistance in pests. Here we evaluated reduced activation of Cry1Ac protoxin as a potential mechanism of resistance in the invasive pest Helicoverpa armigera. Based on the concentration killing 50% of larvae (LC50) for a laboratory-selected resistant strain (LF120) divided by the LC50 for its susceptible parent strain (LF), the resistance ratio was 1600 for Cry1Ac protoxin and 1200 for trypsin-activated Cry1Ac toxin. The high level of resistance to activated toxin as well as to protoxin indicates reduced activation of protoxin is not a major mechanism of resistance to Cry1Ac in LF120. For both insect strains, treatment with either the trypsin inhibitor N-a-tosyl-L-lysine chloromethyl ketone (TLCK) or the chymotrypsin inhibitor N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not significantly affect the LC50 of Cry1Ac protoxin. Enzyme activity was higher for LF than LF120 for trypsin-like proteases, but did not differ between strains for chymotrypsin-like proteases. The results here are consistent with previous reports indicating that reduced activation of protoxin is generally not a major mechanism of resistance to Bt proteins.

Transgenic crops that produce Bacillus thuringiensis (Bt) proteins for pest control are grown extensively, but insect adaptation can reduce their effectiveness. Established mode of action models assert that Bt proteins Cry1Ab and Cry1Ac are produced as inactive protoxins that require conversion to a smaller activated form to exert toxicity. However, contrary to this widely accepted paradigm, we report evidence from seven resistant strains of three major crop pests showing that Cry1Ab and Cry1Ac protoxins were generally more potent than the corresponding activated toxins. Moreover, resistance was higher to activated toxins than protoxins in eight of nine cases evaluated in this study. These data and previously reported results support a new model in which protoxins and activated toxins kill insects via different pathways. Recognizing that protoxins can be more potent than activated toxins against resistant insects may help to enhance and sustain the efficacy of transgenic Bt crops.

To delay evolution of pest resistance to transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt), the "pyramid" strategy uses plants that produce two or more toxins that kill the same pest. We conducted laboratory diet experiments with the cotton bollworm, Helicoverpa armigera, to evaluate cross-resistance and interactions between two toxins in pyramided Bt cotton (Cry1Ac and Cry2Ab). Selection with Cry1Ac for 125 generations produced 1000-fold resistance to Cry1Ac and 6.8-fold cross-resistance to Cry2Ab. Selection with Cry2Ab for 29 generations caused 5.6-fold resistance to Cry2Ab and 61-fold cross-resistance to Cry1Ac. Without exposure to Bt toxins, resistance to both toxins decreased. For each of the four resistant strains examined, 67 to 100% of the combinations of Cry1Ac and Cry2Ab tested yielded higher than expected mortality, reflecting synergism between these two toxins. Results showing minor cross-resistance to Cry2Ab caused by selection with Cry1Ac and synergism between these two toxins against resistant insects suggest that plants producing both toxins could prolong the efficacy of Bt cotton against this pest in China. Including toxins against which no cross-resistance occurs and integrating Bt cotton with other control tactics could also increase the sustainability of management strategies.