Cadherin Is a Binding Protein but Not a Functional Receptor of Bacillus thuringiensis Cry2Ab in Helicoverpa armigera.

Midgut receptors play a critical role in the specificity of Cry toxins for individual insect species. Cadherin proteins are essential putative receptors of Cry1A toxins in lepidopteran larvae. Cry2A family members share common binding sites in Helicoverpa armigera, and one of them, Cry2Aa, has been widely reported to interact with midgut cadherin. Here, we studied the binding interaction and functional role of H. armigera cadherin in the mechanism of Cry2Ab toxicity. A region spanning from cadherin repeat 6 (CR6) to the membrane-proximal region (MPR) of cadherin protein was produced as six overlapping peptides to identify the specific binding regions of Cry2Ab. Binding assays showed that Cry2Ab binds nonspecifically to peptides containing CR7 and CR11 regions in a denatured state but binds specifically only to CR7-containing peptides in the native state. The peptides CR6-11 and CR6-8 were transiently expressed in Sf9 cells to assess the functional role of cadherin. Cytotoxicity assays showed that Cry2Ab is not toxic to the cells expressing any of the cadherin peptides. However, ABCA2-expressing cells showed high sensitivity to Cry2Ab toxin. Neither increased nor decreased sensitivity to Cry2Ab was observed when the peptide CR6-11 was coexpressed with the ABCA2 gene in Sf9 cells. Instead, treating ABCA2-expressing cells with a mixture of Cry2Ab and CR6-8 peptides resulted in significantly reduced cell death compared with treatment with Cry2Ab alone. Moreover, silencing of the cadherin gene in H. armigera larvae showed no significant effect on Cry2Ab toxicity, in contrast to the reduced mortality in ABCA2-silenced larvae. IMPORTANCE To improve the efficiency of production of a single toxin in crops and to delay the evolution of insect resistance to the toxin, the second generation of Bt cotton, expressing Cry1Ac and Cry2Ab, was introduced. Understanding the mode action of the Cry proteins in the insect midgut and the mechanisms insects use to overcome these toxins plays a crucial role in developing measures to counter them. Extensive studies have been conducted on the receptors of Cry1A toxins, but relatively little has been done about those of Cry2Ab. By showing the nonfunctional binding of cadherin protein with Cry2Ab, we have furthered the understanding of Cry2Ab receptors.

Up-regulated serpin gene involved in Cry1Ac resistance in Helicoverpa armigera.

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.

Cyclosporin A acts as a novel insecticide against Cry1Ac-susceptible and -resistant Helicoverpa armigera.

Cotton bollworm (Helicoverpa armigera) is an economically important pest, which is difficult to manage due to its biological and ecological traits, and resistance to most insecticides. Alternative compounds for the sustainable management of H. armigera are needed. As a fungal metabolite, Cyclosporin A (CsA) has not been applied in agriculture pests. Here, CsA was evaluated as a propective insecticide for H. armigera. The results showed that CsA displayed high insecticidal activity against both Cry1Ac-susceptible and -resistant populations of H. armigera. Moreover, lower concentrations of CsA had clear effects, including significantly reduced pupal weight, pupation rate, emergence rate, ovary size, female fecundity and egg hatchability. Further study confirmed that CsA suppressed calcineurin activity and the subsequent expression of endogenous antimicrobial peptide genes (APMs), leading to impaired immunity, ultimately resulting in delayed development and increased mortality. Thus, CsA treatment could control the cotton bollworm population and even showed efficacy against those with Bt resistance. In addition, the morphological changes observed in insects fed CsA with lower concentrations provide insight into insect immunity, regulation of growth and development, regulation of body color, ovary development and sexual selection under external pressure. Overall, our study provides information on biological control potential of Cry1Ac-susceptible and -resistant populations of H. armigera to develop novel bioinsecticides.

Endogenous serpin reduces toxicity of Bacillus thuringiensis Cry1Ac against Helicoverpa armigera (Hübner).

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.

Polycalin is involved in the toxicity and resistance to Cry1Ac toxin in Helicoverpa armigera (Hübner).

Polycalin has been confirmed as a binding protein of the Cry toxins in a few Lepidoptera insects, but its function in the action mechanism of Cry1Ac and whether it is involved in resistance evolution are still unclear. In this study, Ligand blot and enzyme-linked immunosorbent assays showed that Helicoverpa armigera polycalin could specifically interact with Cry1Ac with a high affinity (Kd = 118.80 nM). Importantly, antisera blocking polycalin in H. armigera larvae decreased the toxicity of Cry1Ac by 31.84%. Furthermore, the relative gene and protein expressions were lower in Cry1Ac-resistant strain (LF60) than that in Cry1Ac-susceptible strain (LF). These findings indicated that H. armigera polycalin was a possible receptor of Cry1Ac and may be contributed to the resistance to Cry1Ac.

A Single Point Mutation Resulting in Cadherin Mislocalization Underpins Resistance against Bacillus thuringiensis Toxin in Cotton Bollworm.

Transgenic plants that produce Bacillus thuringiensis (Bt) crystalline (Cry) toxins are cultivated worldwide to control insect pests. Resistance to B. thuringiensis toxins threatens this technology, and although different resistance mechanisms have been identified, some have not been completely elucidated. To gain new insights into these mechanisms, we performed multiple back-crossing from a 3000-fold Cry1Ac-resistant BtR strain from cotton bollworm (Helicoverpa armigera), isolating a 516-fold Cry1Ac-resistant strain (96CAD). Cry1Ac resistance in 96CAD was tightly linked to a mutant cadherin allele (mHaCad) that contained 35 amino acid substitutions compared with HaCad from a susceptible strain (96S). We observed significantly reduced levels of the mHaCad protein on the surface of the midgut epithelium in 96CAD as compared with 96S. Expression of both cadherin alleles from 96CAD and 96S in insect cells and immunofluorescence localization in insect midgut tissue sections showed that the HaCAD protein from 96S localizes on the cell membrane, whereas the mutant 96CAD-mHaCad was retained in the endoplasmic reticulum (ER). Mapping of the mutations identified a D172G substitution mainly responsible for cadherin mislocalization. Our finding of a mutation affecting membrane receptor trafficking represents an unusual and previously unrecognized B. thuringiensis resistance mechanism.

Effects of antibiotics on biological activity of Cry1Ac in Bt-susceptible and Bt-resistant Helicoverpa armigera strains.

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.

Insecticidal Specificity of Cry1Ah to Helicoverpa armigera Is Determined by Binding of APN1 via Domain II Loops 2 and 3.

Bacillus thuringiensis Cry1Ah protein is highly toxic against Helicoverpa armigera but shows no toxicity against Bombyx mori larvae. In contrast, the closely related Cry1Ai toxin showed the opposite phenotype: high activity against B. mori but no toxicity against H. armigera. Analysis of binding of Cry1Ah to brush border membrane vesicle (BBMV) proteins from H. armigera and B. mori by surface plasmon resonance revealed association of toxin binding with insect specificity. Pulldown experiments identified aminopeptidase N1 (APN1) as a Cry1Ah binding protein that was not observed in the assays using B. mori BBMV proteins. The APN1 Cry1Ah binding region was narrowed to the region from A548 to S798 (fragment H3) by expressing four different APN1 fragments in Escherichia coli and analyzing Cry1Ah binding by ligand blot. Binding competition experiments of Cry1Ah to APN1 fragment H3 using synthetic peptides corresponding to four predicted domain II loop regions showed that loop 2 and loop 3 have additive effects on binding to APN1 fragment H3. Moreover, switching of loop 2 and loop 3 regions from Cry1Ah to Cry1Ai toxins showed that loop 2 and loop 3 are both involved in specificity and toxicity against H. armigera IMPORTANCE: Domain II loop regions have been shown to be involved in binding to larval gut proteins mediating insect specificity. The modification of loop regions is a direct and effective method to construct new Cry toxin variants to increase toxicity or modify specificity. Our results show that the exchange of loop regions from one toxin into another is a successful scheme for modification of B. thuringiensis Cry toxin specificity.

Functional analysis of two polygalacturonase genes in Apolygus lucorum associated with eliciting plant injury using RNA interference.

Salivary enzymes of many piercing-sucking insects lead to host plant injury. The salivary enzymes, polygalacturonase (PGs), act in insect feeding. PG family genes have been cloned from the mirid bug Apolygus lucorum, a pest of cotton and other host crops in China. We investigated the function of two PG genes that are highly expressed in A. lucorum nymphs (PG3-4) and adults (PG3-5), using siRNA injection-based RNA interference (RNAi). Accumulation of mRNA encoding both genes and their cognate proteins was significantly reduced (>60%) in experimental compared control green fluorescent protein (GFP) siRNA-treated mirids at 48 h post injection. Injury levels of cotton buds were also significantly reduced after injecting saliva isolated from PG3-4 and PG3-5 siRNA-treated A. lucorum. These results demonstrate that these two PG act in A. lucorum elicitation of plant injury.

Insecticide Toxicity to Adelphocoris lineolatus (Hemiptera: Miridae) and its Nymphal Parasitoid Peristenus spretus (Hymenoptera: Braconidae).

In China, Adelphocoris lineolatus (Goeze) (Hemiptera: Miridae) is an important pest of alfalfa, cotton, and other crops, while Peristenus spretus (Chen & van Achterberg) (Hymenoptera: Braconidae) is the dominant nymphal parasitoid of this mirid bug. In the present study, the toxicity of 17 common insecticides to A. lineolatus was evaluated, and the susceptibility of P. spretus to the insecticides with high toxicity to A. lineolatus was tested under laboratory conditions. Of the 17 insecticides tested, 12 (beta cypermethrin, deltamethrin, carbosulfan, acetamiprid, emamectin benzoate, imidacloprid, phoxim, chlorpyrifos, acephate, profenophos, hexaflumuron, and abamectin) had a highly toxic effect on second-instar nymphs of A. lineolatus, with LC(50) values ranging from 0.58 to 14.85 mg a.i. (active ingredient) liter(-1). Adults of P. spretus were most sensitive to chlorpyrifos, with LC(50) values of 0.03 mg a.i. liter(-1), followed by phoxim, acetamiprid, profenophos, carbosulfan, acephate, deltamethrin, emamectin benzoate, imidacloprid, beta-cypermethrin, and abamectin, with LC(50) values ranging from 0.06 to 3.09, whereas hexaflumuron exhibited the least toxicity to the parasitoid, with LC(50) values >500 mg a.i. liter(-1). A risk quotient analysis indicated that beta-cypermethrin, emamectin benzoate, abamectin, and hexaflumuron when applied against A. lineolatus were the least toxic to P. spretus.

Mining rare and ubiquitous toxin genes from a large collection of Bacillus thuringiensis strains.

There has been considerable effort made in recent years for research groups and other organizations to build up large collections of strains of Bacillus thuringiensis in the search for genes encoding novel insecticidal toxins, or encoding novel metabolic pathways. Whilst next generation sequencing allows the detailed genetic characterization of a bacterial strain with relative ease it is still not practicable for large strain collections. In this work we assess the practicability of mining a mixture of genomic DNA from a two thousand strain collection for particular genes. Using PCR the collection was screened for both a rare (cry15) toxin gene as well as a more commonly found gene (vip3A). The method was successful in identifying both a cry15 gene and multiple examples of the vip3A gene family including a novel member of this family (vip3Aj). A number of variants of vip3Ag were cloned and expressed, and differences in toxicity observed despite extremely high sequence similarity.

Keap1 Negatively Regulates Transcription of Three Counter-Defense Genes and Susceptibility to Plant Toxin Gossypol in Helicoverpa armigera.

Expressions of a wide range of cytoprotective counter-defense genes are mainly regulated by the Keap1-Nrf2-ARE signaling pathway in response to oxidative stress from xenobiotics. Gossypol is the major antiherbivore secondary metabolite of cotton, but how the polyphagous pest Helicoverpa armigera copes with this phytochemical to utilize its favorite host plant cotton remains largely elusive. In this study, we first suppressed the Keap1 gene in newly hatched larvae of cotton bollworm by feeding them the siRNA diet for 4 days. All of the larvae were subsequently fed the artificial diet supplied with gossypol or the control diet for 5 days. We identified that the knockdown of the Keap1 gene significantly decreased larval mortality and significantly increased the percentages of larval survival, reaching the fourth instar, compared with ncsiRNA when exposed to a diet containing gossypol. Three counter-defense genes CYP9A17, CYP4L11 and UGT41B3, which were related to the induction or metabolism of gossypol according to the report before, were all significantly up-regulated after the knockdown of the Keap1 gene. The Antioxidant Response Elements (AREs) were also detected in the promoter regions of the three counter-defense genes above. These data indicate that the suppression of the Keap1 gene activates the Keap1-Nrf2-ARE signaling pathway, up-regulates the expressions of counter-defense genes involved in the resistance of oxidative stress and finally contributes to reducing the susceptibility of gossypol. Our results provide more knowledge about the transcriptional regulation mechanisms of counter-defense genes that enable the cotton bollworm to adapt to the diversity of host plants including cotton.

Use of a pooled clone method to isolate a novel Bacillus thuringiensis Cry2A toxin with activity against Ostrinia furnacalis.

A pooled clone method was developed to screen for cry2A genes. This metagenomic method avoids the need to analyse isolated Bacillus thuringiensis strains by performing gene specific PCR on plasmid-enriched DNA prepared from a pooled soil sample. Using this approach the novel holotype gene cry2Ah1 was cloned and characterized. The toxin gene was over-expressed in Escherichia coli Rosetta (DE3) and the expressed toxin accumulated in both the soluble and insoluble fractions. The soluble Cry2Ah1 was found to have a weight loss activity against Ostrinia furnacalis, and a growth inhibitory activity to both Cry1Ac-susceptible and resistant Helicoverpa armigera populations.

Involvement of nonbinding site proteinases in the development of resistance of Helicoverpa armigera (Lepidoptera: Noctuidae) to Cry1Ac.

Development of resistance to transgenic crops expressing the Cry toxin from Bacterium thuringiensis (Bt) has been the major concern for the long-term success of Bt crops. Alterations in nonbinding site proteinases and Bt toxin receptors are the two types of mechanisms responsible for Bt resistance in resistant insects. However, little is known about the relative contributions of the two types of mechanisms in the early and late phases of the development of Bt resistance. To address the relative contributions of four nonbinding site proteinases including esterase, total protease, chymotrypsin, and glutathione S-transferase in the early and late phases of the development of Cry1Ac resistance, we analyzed the relationships between nonbinding site proteinases and resistance of three groups of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) strains with different resistance levels because of different geographic origins and selection pressures. Positive correlation (esterase, glutathione-S-tranferases [GST], and chymotrypsin) and negative correlation (total midgut protease) were observed within the low to moderate group II resistant strains. Such correlations were less obvious within the low to moderate group III resistant strains because of only threefold differences in LC50 values. Relative to the unselected susceptible 96S strain, the two highly resistant group I resistant strains BtI and BtR have the same amounts of esterase, GST, and chymotrypsin and disproportionally decreased the amount of total midgut protease. Overall, the low to moderate resistant strains had the lowest amount of the nonbinding site proteinases. The results obtained suggest that alternations in the nonbinding site proteinases probably can only confer low to moderate levels of resistance and thus are enriched in the early phase of the development of Cry1Ac resistance.

Cry51Aa Proteins Are Active against Apolygus lucorum and Show a Mechanism Similar to Pore Formation Model.

Reduced insecticide spray in crop fields due to the widespread adoption of Bacillus thuringiensis (Bt) crops has favored the population increases of mirid bugs. Cry51Aa proteins are new types of Bt proteins that belong to aerolysin-like ß pore-forming proteins with insecticidal activity against hemipteran and coleopteran pests. Here, we studied the activity of Bt Cry51Aa1 and Cry51Aa2 against Apolygus lucorum, an emerging pest in cotton, and their mechanism of action. Cry51Aa1 exhibited almost 5-fold higher toxicity than Cry51Aa2 with LC50 of 11.87 and 61.34 µg/mL, respectively. Protoxins could be activated both in vitro, by trypsin and midgut contents, and in vivo, by A. lucorum midgut. Both Cry51Aa protoxins were processed in two steps, producing pre-activated (∼30 kDa) and final activated (∼25-28 kDa) proteins. Cry51Aa proteins bound to a 25 kDa midgut protein, and Cry51Aa2 showed 2 times higher binding affinity than Cry51Aa1. Incubating Cry51Aa proteins with midgut homogenate resulted in toxin oligomers of 150-200 kDa. Our findings provide a theoretical basis for using Cry51Aa proteins to control A. lucorum and a better understanding of their mode of action.

Reduced expression of the P-glycoprotein gene HaABCB1 is linked to resistance to Bacillus thuringiensis Cry1Ac toxin but not Cry2Ab toxin in Helicoverpa armigera.

Rapid evolution of pest resistance to Bt insecticidal proteins presents a serious threat to the sustainable use of Bt crops. The cotton bollworm has been extensively exposed to Bt cotton worldwide and has evolved resistance in laboratory and field. Previous studies have highlighted the significant roles played by the ABC transporter proteins in Bt resistance. In this study, the ORF of HaABCB1 was cloned and analyzed. The expression of HaABCB1 was detected in all developmental stages and tissues, with the highest expression in third instar larvae stage and hindgut tissue. Compared with susceptible strain, a remarkable decrease of HaABCB1 expression in Cry1Ac resistant strain while no significant change in Cry2Ab resistant strain were found. The HaABCB1 expression reduced after susceptible larvae induced by Cry1Ac, but no obvious expression changes after Cry2Ab exposure. RNAi-mediated down-regulation of HaABCB1 could lead to a significant reduction in larval susceptibility to Cry1Ac, but not to Cry2Ab, in susceptible strain. Genetic linkage analysis confirmed that decreased expression of the HaABCB1 mediates resistance to Cry1Ac, but not Cry2Ab resistance. This knowledge contributes to better understanding of the complex molecular mechanisms underlying Bt resistance and provide theoretical foundation for the development of new strategies for pest resistance management.

Differences in the Sublethal Effects of Sulfoxaflor and Acetamiprid on the Aphis gossypii Glover (Homoptera: Aphididae) Are Related to Its Basic Sensitivity Level.

The cotton aphid, Aphis gossypii, is an important insect pest of many crops around the world, and it has developed resistance to a large number of frequently used insecticides. The sublethal effects of insecticides not only have an environmental risk to arthropods but also have the potential to promote resistance evolution. The sublethal effects (inhibitory or stimulatory) are influenced by many factors, such as the type of insecticide, sublethal concentrations, pest species, and others. In this study, the sublethal effects of sulfoxaflor and acetamiprid on A. gossypii were compared using two field-collected populations. The results show that sulfoxaflor was more toxic than acetamiprid against A. gossypii in both populations, the LC50 concentrations of acetamiprid and sulfoxaflor were 6.35 and 3.26 times higher, respectively, for the Jinghe population than for Yarkant. The LC25 concentration of acetamiprid significantly reduced adult longevity and fecundity in exposed adults (F0) of the Jinghe population, but it had no significant effects on these factors in Yarkant. Similar inhibitory effects were found in the F1 and F2 generations, but the biological traits in the Yarkant population were significantly reduced when the parents (F0) were exposed to LC25 of acetamiprid, whereas the changes in the Jinghe population were not significant. However, sublethal sulfoxaflor showed a stimulatory effect on A. gossypii in the F0 and F1 generation; the adult fecundity and longevity of the F0 generation were significantly higher in Jinghe, while the biological traits of the F1 generation were obviously higher in Yarkant. In the F2 generation, the r and λ were significantly higher in Jinghe; meanwhile, these biological traits were reduced in Yarkant. These results indicate that sulfoxaflor and acetamiprid had different sublethal effects on A. gossypii that varied by generation. In addition, we speculate that the genetic background and the resistance levels of A. gossypii may also influence the sublethal effects. Our findings are useful for assessing the overall effects of sulfoxaflor and acetamiprid on A. gossypii.

Selection and Validation of Reference Genes for Quantitative Real-Time PCR Normalization in Athetis dissimilis (Lepidoptera: Noctuidae) Under Different Conditions.

Reference genes are the key to study gene expression patterns using quantitative real-time PCR (qRT-PCR). No studies on the reference genes of Athetis dissimilis, an important agricultural pest, have been reported. In order to determine the reference genes for qRT-PCR normalization in A. dissimilis under different conditions, 10 candidate genes [18S ribosomal protein (18S), 28S ribosomal protein (28S), arginine kinase (AK), elongation factor 1 alpha (EF1-α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L32 (RPL32), ribosomal protein L40 (RPL40), alpha-tubulin (α-TUB), beta-actin (ß-ACT), and beta-tubulin (ß-TUB)] of A. dissimilis were selected to evaluate their stability as reference genes under different biotic and abiotic conditions by using five tools, geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder. Furthermore, CSP1 and superoxide dismutase (SOD) were used as target genes to validate the candidate reference genes. The results showed that different reference genes were needed under different experimental conditions, among which, EF-1α, RPL40, and 18S are most suitable reference genes for studying genes related development stages of A. dissimilis, RPL40 and α-TUB for larval tissues, α-TUB and 28S for adult tissues, EF-1α and ß-ACT for insecticidal treatments, ß-ACT and 28S for temperature treatments, EF-1α and ß-ACT for starvation treatments, RPL40 and 18S for dietary treatments, and 18S, 28S, and α-TUB for all the samples. These results provide suitable reference genes for studying gene expression in A. dissimilis under different experimental conditions, and also lay the foundation for further research into the function of related genes in A. dissimilis.

ABCC2 is a functional receptor of Bacillus thuringiensis Cry1Ca in Spodoptera litura.

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.

Response of Different Insect Groups to Various Wavelengths of Light under Field Conditions.

Insects in the same taxonomic group generally have similar responses to light at various wavelengths in the laboratory. However, there is lack of direct evidence of between-group differences in insect responses to various light wavelengths under field conditions. During 2014 and 2015, we evaluated the relative attractiveness of LEDs with 19 single wavelengths to three pest orders and four natural predator orders in cotton fields. The average numbers of Lepidoptera, Hemiptera, Coleoptera, and total pests captured by traps with a 395-nm LED wavelength were higher than those for all others, except 440-nm wavelength captured the largest number of Hemiptera in 2015. For natural enemies, the average numbers of Coleoptera, Neuroptera, and total natural enemies were the largest in traps with a 572-nm LED wavelength, except 538-nm wavelength captured the largest number of Coleoptera in 2014. In general, the ratio of pests to natural enemies captured in the 395-nm wavelength LED trap was significantly more than all others. These results demonstrated that insects in different taxonomic groups have significantly different responses to light at various wavelengths under field conditions; these results will provide insights for in-depth studies on insect phototaxis and guide the long-term monitoring of insects in different groups.