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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.

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.

Lepidoptera sex pheromone biosynthesis is regulated by pheromone biosynthesis activating neuropeptide (PBAN). PBAN regulates not only female sex pheromone biosynthesis but also male sex pheromone biosynthesis. Previous research has confirmed that PBAN regulates sex pheromone biosynthesis using Ca2+ as a secondary messenger in all examined species to date. However, the downstream signal of Ca2+ has remained elusive. In the present study, calcineurin A (CNA), a downstream signal of Ca2+ , was discovered in Helicoverpa armigera male hairpencil and named HaCNA. Sequence analysis demonstrated that the open reading frame of HaCNA contains 1488 nucleotides encoding 495 amino acid residues. A homology search revealed that HaCNA shares a high amino acid identity with the CNA of other insects. Developmental and spatial expression analyses revealed that the mRNA levels of HaCNA peaked at 24 h after emergence and that HaCNA expression was ubiquitous in all examined tissues. Activity analysis revealed that PBAN activates HaCNA, and a Ca2+ inhibitor, Lacl3 , attenuated the effect of PBAN by decreasing HaCNA activity. Pharmacological inhibitor and RNA interference-mediated knockdown assays revealed that both activity inhibition and decreased mRNA levels of HaCNA led to a significant decrease in the production of the male sex pheromone components [octadecanol and (Z)-11 hexadecanol)] and in the efficacy of female mating acceptance. Our results demonstrate that HaCNA acts as downstream signal of PBAN/Ca2+ and plays an important role in PBAN-induced male sex pheromone biosynthesis and female mating acceptance.

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 (LC50 ) was midgut (31.0 μg/mL) > fat body (59.0 μg/mL) and SF9 cell (99.6 μg/mL). By contrast, the fat body cell line (LC50 = 7.55 μg/mL) was about twice more susceptible to Cry2Ab than the midgut cell line (16.0 μg/mL), the susceptibility of which was not significantly greater than that of SF9 cells (27.0 μg/mL). Further, ligand blot showed the binding differences between Cry1Ac and Cry2Ab in the three cell lines. These results indicated that the receptors of Cry2Ab were enriched in fat body cells and thus largely different from the receptors of Cry1Ac, which were enriched in midgut cells.

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.