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.

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.

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.

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.

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.

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.

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.

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.

The evolution of opsin genes in five species of mirid bugs: duplication of long-wavelength opsins and loss of blue-sensitive opsins.

BACKGROUND: Color vision and phototactic behavior based on opsins are important for the fitness of insects because of their roles in foraging and mate choice. Related topics, including the duplication and loss of opsin genes, have been well investigated in insect orders such as Coleoptera, Lepidoptera, Hymenoptera, Odonata and Orthoptera, and the findings have been used to develop pest management strategies involving light trapping. Mirid bugs of Hemiptera, which are pests that cause heavy economic losses, show capacity for color discrimination and phototaxis. However, the opsins in mirid bugs remain uncharacterized. Herein, we examined five species to investigate the evolution of opsins in the family Miridae. RESULTS: Using RNA-seq, we identified several contigs showing high identity with opsins, including four contigs in Apolygus lucorum and three contigs each in Adelphocoris suturalis, Adelphocoris fasciaticollis, Adelphocoris lineolatus and Nesidiocoris tenuis. Phylogenetic analyses indicated that one of these genes clustered with ultraviolet-sensitive (UV) opsins and that the others clustered with long-wavelength (LW) opsins, suggesting that duplication of LW opsins and loss of blue light-sensitive (B) opsins occurred in mirid bugs. The existence of introns in the LW opsins of mirid bugs suggested that the duplication events were DNA based. Both LW1 and LW2 opsins of mirid bugs were found to be under strong purifying selection. The LW1 opsins were significantly more highly expressed than the LW2 and UV opsins. CONCLUSIONS: We identified the opsins of mirid bugs using five selected mirid species as a representative sample. Phylogenetic analyses clustered one of the genes with UV opsins and the others with LW opsins, suggesting the occurrence of LW opsin duplication and B opsin loss during the evolution of mirid bugs. Intron detection suggested that the identified duplication event was DNA based. The evidence of strong purifying selection and the relatively high expression levels suggested that these opsins exhibit fundamental functions in mirid bugs.

Dissecting the roles of FTZ-F1 in larval molting and pupation, and the sublethal effects of methoxyfenozide on Helicoverpa armigera.

BACKGROUND: In holometabolous insects, the major developmental transitions - larval molting and pupation - are triggered by a pulse of 20-hydroxyecdysone (20E) and coordinated by juvenile hormone. Methoxyfenozide (MF), an ecdysteroid agonist, represents a new class of insect growth regulators and is effective against lepidopteran pests. Fushi-tarazu factor 1 (FTZ-F1) is an ecdysone-inducible transcription factor. To date, the effect of MF on 20E-response genes remains unclear, and we speculate the involvement of FTZ-F1 in MF's growth regulating effect. RESULTS: MF at LC25 and LC10 caused severe ecdysis failure in Helicoverpa armigera, extended their larval duration, lowered their pupal weight, and reduced the respiratory, pupation and emergence rates. Furthermore, sublethal doses of MF inhibited ecdysteroidogenesis and lowered the intrinsic 20E titer, but showed an inductive effect on 20E-response genes including HaFTZ-F1. HaFTZ-F1, predominantly expressed in larval epidermis, was markedly upregulated before or right after larval ecdysis, and maintained a high level in prepupal stage. Knockdown of HaFTZ-F1 in 4th-instar larvae severely impaired larval ecdysis, whereas its knockdown in final-instar larvae caused abnormal pupation. Moreover, knocking down HaFTZ-F1 downregulated three critical ecdysteroidogenesis genes, lowered 20E titer, and suppressed the expression of 20E receptors and 20E-response genes. The introduction of 20E into HaFTZ-F1-RNAi larvae partly relieved the negative effects on the 20E-induced signaling cascade. CONCLUSION: Our findings reveal the adverse effects of sublethal doses of MF on the development of H. armigera and elucidate the resulting perturbations on the 20E-induced signaling cascade; we propose that HaFTZ-F1 regulates ecdysis and pupation by mediating 20E titer and its signaling pathway. © 2020 Society of Chemical Industry.

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.

Dissecting the Role of Juvenile Hormone Binding Protein in Response to Hormone and Starvation in the Cotton Bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae).

Juvenile hormone (JH) regulates many physiological processes in insect development, diapause, and reproduction. Juvenile hormone binding protein (JHBP), the carrier partner protein of JH, is essential for the balance of JH titer to regulate the metamorphosis and development of insect. In this study, two JHBP genes were identified from Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), namely HaJHBP1 and HaJHBP2. The tissue and temporal expression pattern revealed that both HaJHBP1 and HaJHBP2 were dominantly expressed in larval fat body, and their high transcription stages were detected in fourth and fifth instars. The ingestion of methoprene, a JH analogue, significantly induced the expression of HaJHBP1 and HaJHBP2. However, both HaJHBP1 and HaJHBP2 mRNA levels were significantly downregulated after treated with a JH antagonist, precocene. When subject to starvation, larvae showed a marked suppressive effect in the expression of HaJHBP1 and HaJHBP2. These results indicate that JHBP plays a part in the JH-regulated metabolism, growth, or development in reaction to different nutritional conditions.

Transcriptomic Responses to Different Cry1Ac Selection Stresses in Helicoverpa armigera.

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.

Methoprene-Tolerant (Met) Is Indispensable for Larval Metamorphosis and Female Reproduction in the Cotton Bollworm Helicoverpa armigera.

Juvenile hormone (JH) represses larval metamorphosis and induces adult reproduction in insects. Methoprene-tolerant (Met) is identified as an intranuclear receptor that mediates JH actions. In the present study, we characterized a Met from the severe agricultural pest, Helicoverpa armigera, namely HaMet. In the larval stage, HaMet is predominantly expressed in the epidermis and midgut, and is upregulated before each molting, whereas in adults HaMet is maximally expressed in the ovary, testis, and fat body. The immunofluorescence assay revealed that HaMet was distributed in the longitudinal and circular muscle layers of midgut in larvae, whereas in the ovary of female adults, HaMet was localized in the nucleus of the oolemma. Knockdown of HaMet in final-instar larvae shortened the time of pupation, induced abnormal pupation, and dampened pupation rate. In female adults, HaMet depletion severely suppressed the transcription of Vitellogenin (Vg) and Vitellogenin Receptor (VgR), disrupted the Vg accumulation in fat body and the yolk protein uptake in oocytes, and finally led to an impaired fecundity. Our findings therefore confirmed that HaMet acted as a nuclear receptor of JH and played an essential role in larval metamorphosis, vitellogenesis, and oocyte maturation.

Assessment of the lethal and sublethal effects by spinetoram on cotton bollworm.

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.

Specific Binding Protein ABCC1 Is Associated With Cry2Ab Toxicity in Helicoverpa armigera.

A pyramid strategy combining the crystal (Cry) 1A and 2A toxins in Bacillus thuringiensis (Bt) crops are active against many species of insects and nematode larvae. It has been widely used to delay pest adaption to genetically modified plants and broaden the insecticidal spectrum in many countries. Unfortunately, Cry2A can also bind with the specific receptor proteins of Cry1A. ATP-binding cassette (ABC) transporters can interact with Cry1A toxins as receptors in the insect midgut, and ABC transporter mutations result in resistance to Bt proteins. However, there is limited knowledge of the ABC transporters that specifically bind to Cry2Ab. Here, we cloned the ABCC1 gene in Helicoverpa armigera, which expressed at all larval stages and in nine different tissues. Expression levels were particularly high in fifth-instar larvae and Malpighian tubules. The two heterologously expressed HaABCC1 transmembrane domain peptides could specifically bind to Cry2Ab with high affinity levels. Moreover, transfecting HaABCC1 into the Spodoptera frugiperda nine insect cell significantly increased its mortality when exposed to Cry2Ab in vitro, and silencing HaABCC1 in H. armigera by RNA interference significantly reduced the mortality of larvae exposed to Cry2Ab in vivo. Altogether current results suggest that HaABCC1 serves as a functional receptor for Cry2Ab.

Establishment of a dietary exposure assay for evaluating the toxicity of insecticidal compounds to Apolygus lucorum (Hemiptera: Miridae).

With the commercialization of transgenic cotton that expresses Bt (Bacillus thuringiensis) insecticidal proteins, mirid bugs have become key pests in cotton and maize fields in China. Genetically engineered (GE) crops for controlling mirids are unavailable owing to a lack of suitable insecticidal genes. In this study, we developed and validated a dietary exposure assay for screening insecticidal compounds and for assessing the potential effects of insecticidal proteins produced by GE plants on Apolygus lucorum, one of the main mirid pests of Bt cotton and Bt maize. Diets containing potassium arsenate (PA) or the cysteine protease inhibitor E-64 were used as positive controls for validating the efficacy of the dietary exposure assay. The results showed that with increasing concentrations of PA or E-64, A. lucorum larval development time was prolonged and adult weight and fecundity were decreased, suggesting that the dietary exposure assay was useful for detecting the toxicity of insecticidal compounds to A. lucorum. This assay was then used to assess the toxicity of Cry1Ab, Cry1Ac, Cry1F, Cry2Aa, and Cry2Ab proteins, which have been transformed into several crops, against A. lucorum. The results showed that A. lucorum did not show a negative effect by feeding on an artificial diet containing any of the purified Cry proteins. No significant changes in the activities of digestive, detoxifying, or antioxidant enzymes were detected in A. lucorum that fed on a diet containing Cry proteins, but A. lucorum fitness was reduced when the insect fed on a diet containing E-64 or PA. These results demonstrate that A. lucorum is not sensitive to the tested Cry proteins and that the dietary exposure assay is useful for evaluating the toxicity of insecticidal compounds to this species.