Development of resistance monitoring for Helicoverpa armigera (Lepidoptera: Noctuidae) resistance to pyramided Bt cotton in China.

The cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), is a significant cotton pest worldwide. Bacillus thuringiensis (Bt) cotton producing Cry1Ac has been used since 1997 for the control of this pest in China and a significant increase in H. armigera resistance to Cry1Ac has occurred in northern China. To mitigate resistance evolution, it is necessary to develop and plant pyramided 2- and 3-toxin Bt cotton to replace Cry1Ac cotton. For sustainable use of pyramided Bt cotton, we used diet overlay bioassays to measure the baseline susceptibility of H. armigera to Cry2Ab in 33 populations collected in 2017, 2018, and 2021 in 12 locations from major cotton-producing areas of China. The lethal concentration killing 50% (LC50) or 99% (LC99) of individuals from the populations ranged from 0.030 to 0.138 µg/cm2 and 0.365 to 2.964 µg/cm2, respectively. The ratio of the LC50 for the most resistant and susceptible population was 4.6, indicating moderate among-population variability in resistance. The susceptibility of H. armigera to Cry2Ab did not vary significantly over years. A diagnostic concentration of 2 µg/cm2 was calculated as twice the LC99 from an analysis of pooled data for the field-collected populations. This concentration discriminated well between susceptible and resistant individuals, as it killed all larvae from a susceptible laboratory strain and 0%, 0%, and 23% of larvae from 3 laboratory strains with > 100-fold resistance to Cry2Ab. These baseline susceptibility data and diagnostic concentration for Cry2Ab will be useful for monitoring the evolution of H. armigera resistance to pyramided Bt cotton in China.

Differentiation in detoxification gene complements, including neofunctionalization of duplicated cytochrome P450 genes, between lineages of cotton bollworm, Helicoverpa armigera.

Here we investigate the evolutionary dynamics of five enzyme superfamilies (CYPs, GSTs, UGTs, CCEs and ABCs) involved in detoxification in Helicoverpa armigera. The reference assembly for an African isolate of the major lineages, H. a. armigera, has 373 genes in the five superfamilies. Most of its CYPs, GSTs, UGTs and CCEs and a few of its ABCs occur in blocks and most of the clustered genes are in subfamilies specifically implicated in detoxification. Most of the genes have orthologues in the reference genome for the Oceania lineage, H. a. conferta. However, clustered orthologues and subfamilies specifically implicated in detoxification show greater sequence divergence and less constraint on non-synonymous differences between the two assemblies than do other members of the five superfamilies. Two duplicated CYPs, which were found in the H. a. armigera but not H. a. conferta reference genome, were also missing in 16 Chinese populations spanning two different lineages of H. a. armigera. The enzyme produced by one of these duplicates has higher activity against esfenvalerate than a previously described chimeric CYP mutant conferring pyrethroid resistance. Various transposable elements were found in the introns of most detoxification genes, generating diverse gene structures. Extensive resequencing data for the Chinese H. a. armigera and H. a. conferta lineages also revealed complex copy number polymorphisms in 17 CCE001s in a cluster also implicated in pyrethroid metabolism, with substantial haplotype differences between all three lineages. Our results suggest that cotton bollworm has a versatile complement of detoxification genes which are evolving in diverse ways across its range.

Historical museum samples reveal signals of selection and drift in response to changing insecticide use in an agricultural pest moth.

In response to environmental and human-imposed selective pressures, agroecosystem pests frequently undergo rapid evolution, with some species having a remarkable capacity to rapidly develop pesticide resistance. Temporal sampling of genomic data can comprehensively capture such adaptive changes over time, for example, by elucidating allele frequency shifts in pesticide resistance loci in response to different pesticides. Here, we leveraged museum specimens spanning over a century of collections to generate temporal contrasts between pre- and post-insecticide populations of an agricultural pest moth, Helicoverpa armigera. We used targeted exon sequencing of 254 samples collected across Australia from the pre-1950s (prior to insecticide introduction) to the 1990s, encompassing decades of changing insecticide use. Our sequencing approach focused on genes that are known to be involved in insecticide resistance, environmental sensation, and stress tolerance. We found an overall lack of spatial and temporal population structure change across Australia. In some decades (e.g., 1960s and 1970s), we found a moderate reduction of genetic diversity, implying stochasticity in evolutionary trajectories due to genetic drift. Temporal genome scans showed extensive evidence of selection following insecticide use, although the majority of selected variants were low impact. Finally, alternating trajectories of allele frequency change were suggestive of potential antagonistic pleiotropy. Our results provide new insights into recent evolutionary responses in an agricultural pest and show how temporal contrasts using museum specimens can improve mechanistic understanding of rapid evolution.

The larva and adult of Helicoverpa armigera use differential gustatory receptors to sense sucrose.

Almost all herbivorous insects feed on plants and use sucrose as a feeding stimulant, but the molecular basis of their sucrose reception remains unclear. Helicoverpa armigera as a notorious crop pest worldwide mainly feeds on reproductive organs of many plant species in the larval stage, and its adult draws nectar. In this study, we determined that the sucrose sensory neurons located in the contact chemosensilla on larval maxillary galea were 100-1000 times more sensitive to sucrose than those on adult antennae, tarsi, and proboscis. Using the Xenopus expression system, we discovered that Gr10 highly expressed in the larval sensilla was specifically tuned to sucrose, while Gr6 highly expressed in the adult sensilla responded to fucose, sucrose and fructose. Moreover, using CRISPR/Cas9, we revealed that Gr10 was mainly used by larvae to detect lower sucrose, while Gr6 was primarily used by adults to detect higher sucrose and other saccharides, which results in differences in selectivity and sensitivity between larval and adult sugar sensory neurons. Our results demonstrate the sugar receptors in this moth are evolved to adapt toward the larval and adult foods with different types and amounts of sugar, and fill in a gap in sweet taste of animals.

Improved effects of Helicoverpa armigera nucleopolyhedrovirus integrated with Campoletis chlorideae against H. armigera and impact of the virus on the parasitoid.

BACKGROUND: Combined use can be an effective measure to improve pest control by viruses and parasitic wasps. However, not all combinations of natural enemies show improved effects. Helicoverpa armigera nucleopolyhedrovirus (HearNPV) and Campoletis chlorideae Uchida are two important natural enemies of Helicoverpa armigera. This study aimed to investigate the combined effects of C. chlorideae and HearNPV against H. armigera larvae and the impact of HearNPV on C. chlorideae. RESULTS: The combination of HearNPV and C. chlorideae exerted increased mortality on H. armigera when C. chlorideae parasitized larvae one day after infection with HearNPV. C. chlorideae could distinguish between HearNPV-infected and noninfected larvae. Besides influencing host selection of C. chlorideae, HearNPV infection had negative effects on the development and reproduction of C. chlorideae. The developmental time of C. chlorideae was significantly prolonged and the percentage of emergence and adult eclosion of C. chlorideae was lower in infected hosts. The adult wasps were also smaller in body size, and female adults had fewer eggs when they developed in virus-infected hosts. CONCLUSIONS: HearNPV combined with C. chlorideae could improve the efficacy of biological control against H. armigera. The results provided valuable information on the importance of timing in the combined use of HearNPV and C. chlorideae for the biological control of H. armigera. © 2023 Society of Chemical Industry.

Frequency and diversity of indoxacarb resistance in Australian Helicoverpa armigera (Lepidoptera: Noctuidae).

Annual indoxacarb resistance in Helicoverpa armigera (Hübner) populations collected from various crops in Australia was monitored between 2013 and 2023. Resistance frequency determined by F2 screening using a predetermined discriminating dose of indoxacarb, was lowest in the 2013-2014 and 2015-2016 seasons at 0.0164 and 0.0246, respectively. Resistance then increased significantly to a ten-year high of 0.0869 in 2018-2019 but declined to 0.0557 in 2019-2020 during a severe drought, remaining relatively stable thereafter to 2023. Indoxacarb resistance was first detected in H. armigera collected from maize in the Gwydir valley, New South Wales, in 2013 (strain GY7-39). In 2017, a second indoxacarb resistant H. armigera strain (UN1U3-10) was isolated from a population collected in chickpeas in the Liverpool Plains, New South Wales. Indoxacarb resistance of this strain was characterized to evaluate its potential to compromise the ongoing effectiveness of insecticide resistance management strategies in Australian farming systems. Survival at the discriminating dose of indoxacarb in UN1U3-10 was 28.9, 52.6, 86.7, and 92.9% in the F2, F3, F4, and F5, respectively. Following introgression with a susceptible strain and reselection with the discriminating dose of indoxacarb, the resistance ratio of UN1U3-10 was approximately 800-fold. Resistance was autosomal, incompletely dominant and conferred by more than 1 locus. While indoxacarb resistance in UN1U3-10 did not confer to emamectin benzoate or spinetoram and there was no evidence of major cross-resistance to the Bt toxins Cry1A, Cry2A or Vip3A, there was 5-fold reduced sensitivity to chlorantraniliprole. Indoxacarb resistance was suppressed by approximately 10-fold by PBO with no synergism by TPP or DEM, suggesting the involvement of cytochrome P450 enzymes. A stability analysis indicated a fitness cost may be associated with the genes that confer resistance in the UN1U3-10 strain. The potential risk for diverse indoxacarb resistance in the Australian H. armigera population is discussed.

Large-area field application confirms the effectiveness of toxicant-infused bait for managing Helicoverpa armigera (Hübner) in maize fields.

BACKGROUND: Maize is one of the world's most important crops, so its stable production and supply is crucial for food security and socioeconomic development. The cotton bollworm, Helicoverpa armigera (Hübner), is one of the major pests in maize. We evaluated the control effect of a bio-bait, an adult attractant, combined with insecticide, a 'toxicant-infused bait', on H. armigera populations in maize fields, as well as the impact on crop yield and quality through large-scale field applications in Hebei Province, China over a period spanning 2019 to 2021. RESULT: The number of male and female H. armigera adults killed by strip application ranged from 1 to 37 and 4 to 36 per strip, respectively, of which female moths were 53%. Following the application of toxicant-infused bait, we observed a significant reduction in the populations of eggs and larvae, with the average adjusted decrease range from 58% to 63% for eggs and from 34% to 62% for larvae. The application of toxicant-infused bait also resulted in a notable reduction in the proportion of damaged maize plants, with an adjusted decline rate ranging from 59% to 69%. Concurrently, we observed an increase in yield by 4% to 8%. The concentration of aflatoxin in harvested maize grains was significantly reduced from an initial level of 1.24 to 0.1 ug/kg. CONCLUSION: By applying toxicant-infused bait, there was a significant reduction in the population of H. armigera adults and their offspring, resulting in an improved yield and quality of maize. Toxicant-infused bait has great application potential in the integrated pest management of H. armigera. © 2023 Society of Chemical Industry.

Variation of Helicoverpa armigera symbionts across developmental stages and geographic locations.

Cotton bollworm (Helicoverpa armigera) poses a global problem, causing substantial economic and ecological losses. Endosymbionts in insects play crucial roles in multiple insect biological processes. However, the interactions between H. armigera and its symbionts have not been well characterized to date. We investigated the symbionts of H. armigera in the whole life cycle from different geographical locations. In the whole life cycle of H. armigera, Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria were the dominant bacteria at the phylum level, while Enterococcus, Enterobacter, Glutamicibacter, and Bacillus were the four dominant bacteria at the genus level. Furthermore, high similarity in symbiotic bacterial community was observed in different stages of H. armigera, which were dominated by Enterococcus and Enterobacter. In fields, the dominant bacteria were Proteobacteria and Bacteroidetes, whereas, in the laboratory, the dominant bacteria were Proteobacteria. At the genus level, the dominant bacteria in cotton bollworm eggs of wild populations were Enterobacter, Morganella, Lactococcus, Asaia, Apibacter, and Enterococcus, and the subdominant bacteria were Bartonella, Pseudomonas, and Orbus. Moreover, the symbionts varied with geographical locations, and the closer the geographical distance, the more similar the microbial composition. Taken together, our study identifies and compares the symbiont variation along with geographical gradients and host development dynamic and reveals the high flexibility of microbiome communities in H. armigera, which probably benefits for the successful survival in a complicated changing environment.

Factors Affecting Population Dynamics of Helicoverpa zea (Lepidoptera: Noctuidae) in a Mixed Landscape with Bt Cotton and Peanut.

In North America, weather and host-plant abundance drive the population dynamics of the migratory pest Helicoverpa zea. The objectives of this study were to (i) estimate monthly abundance of H. zea moths in Bt cotton and peanut fields, (ii) document the effects of weather on H. zea trap catches, and (iii) determine larval hosts supporting H. zea populations from 2017 to 2019. Year-round trapping of H. zea moths was conducted in 16 commercial fields in two regions of the Florida Panhandle using delta traps. H. zea moth catches were associated with temperature, rainfall, and relative humidity. Larval hosts were determined by isotopic carbon analysis. Our results showed year-round H. zea flights in both regions across two years, with the highest and lowest moth catches occurring from July to September and November to March, respectively. There was no difference in catches between traps set on Bt cotton and peanut. In the Santa Rosa/Escambia counties, weather explained 59% of the variance in H. zea catches, with significant effects of temperature, relative humidity, and rainfall. In Jackson County, weather explained 38% of H. zea catches, with significant effects of temperature and relative humidity. Carbon isotopic data showed that feeding on C3 plants, including Bt cotton, occurred over most of the year, although feeding on C4 hosts, including Bt corn, occurred during the summer months. Hence overwintering and resident populations of H. zea in the Florida Panhandle may be continually exposed to Bt crops, increasing the risk for the evolution of resistance.

Population Genomics of Nonrecessive Resistance to Bt Toxin Cry1Ac in Helicoverpa armigera From Northern China.

Transgenic crops that produce insecticidal proteins from Bacillus thuringiensis (Bt) have provided control of some key pests since 1996. However, the evolution of resistance by pests reduces the benefits of Bt crops. Resistance to Bt crops that is not recessively inherited is especially challenging to manage. Here we analyzed nonrecessive resistance to Bt toxin Cry1Ac in eight field populations of Helicoverpa armigera sampled in 2018 from northern China, where this global pest has been exposed to Cry1Ac in Bt cotton since 1997. Bioassays revealed 7.5% of field-derived larvae were resistant to Cry1Ac of which 87% had at least one allele conferring nonrecessive resistance. To analyze this nonrecessive resistance, we developed and applied a variant of a genomic mapping approach called quantitative trait locus (QTL)-seq. This analysis identified a region on chromosome 10 associated with nonrecessive resistance to Cry1Ac in all 21 backcross families derived from field-collected moths. Individual sequencing revealed that all 21 field-collected resistant grandparents of the backcross families had a previously identified dominant point mutation in the tetraspanin gene HaTSPAN1 that occurs in the region of chromosome 10 identified by QTL-seq. QTL-seq also revealed a region on chromosome 26 associated with nonrecessive resistance in at most 14% of the backcross families. Overall, the results imply the point mutation in HaTSPAN1 is the primary genetic basis of nonrecessive resistance to Cry1Ac in field populations of H. armigera from northern China. Moreover, because nonrecessive resistance is predominant, tracking the frequency of this point mutation could facilitate resistance monitoring in the region.

A Chromosome-Scale Genome Assembly of a Helicoverpa zea Strain Resistant to Bacillus thuringiensis Cry1Ac Insecticidal Protein.

Helicoverpa zea (Lepidoptera: Noctuidae) is an insect pest of major cultivated crops in North and South America. The species has adapted to different host plants and developed resistance to several insecticidal agents, including Bacillus thuringiensis (Bt) insecticidal proteins in transgenic cotton and maize. Helicoverpa zea populations persist year-round in tropical and subtropical regions, but seasonal migrations into temperate zones increase the geographic range of associated crop damage. To better understand the genetic basis of these physiological and ecological characteristics, we generated a high-quality chromosome-level assembly for a single H. zea male from Bt-resistant strain, HzStark_Cry1AcR. Hi-C data were used to scaffold an initial 375.2 Mb contig assembly into 30 autosomes and the Z sex chromosome (scaffold N50 = 12.8 Mb and L50 = 14). The scaffolded assembly was error-corrected with a novel pipeline, polishCLR. The mitochondrial genome was assembled through an improved pipeline and annotated. Assessment of this genome assembly indicated 98.8% of the Lepidopteran Benchmark Universal Single-Copy Ortholog set were complete (98.5% as complete single copy). Repetitive elements comprised approximately 29.5% of the assembly with the plurality (11.2%) classified as retroelements. This chromosome-scale reference assembly for H. zea, ilHelZeax1.1, will facilitate future research to evaluate and enhance sustainable crop production practices.

Evaluation of artificial diets based on different legume seeds on the nutritional physiology and digestive function of Helicoverpa armigera (Hübner).

Helicoverpa armigera (Hübner) is considered a serious agricultural pest worldwide. We explored the effects of artificial diets containing ten legumes, including broad beans (Shadan, Feyz, Saraziri, Barekat, and Mahta cultivars), white kidney beans (Dehghan cultivar), red kidney beans (Goli cultivar), common beans (Khomein cultivar), cowpeas (Mashhad and Arabi cultivars) on the feeding responses of H. armigera by quantifying specific primary and secondary metabolites in the studied legumes and determining larval nutritional indices and digestive enzyme activities. The results showed that the highest efficiency of conversion of digested food (ECD) and relative growth rate values (RGR) of whole larval instars were obtained in the Dehghan and Goli cultivars. However, the lowest values of ECD and RGR were observed in the larvae fed on the Khomein and Mahta cultivars. The highest proteolytic and amylolytic activities of larvae were found on the Dehghan and Mashhad cultivars. The highest and lowest values of standardized insect-growth index and index of plant quality were observed in larvae feeding on the Dehghan and Khomein cultivars, respectively. Additionally, significant variations in phytochemical metabolites were recorded among the studied legume cultivars. Significant negative or positive correlations were also found between feeding characteristics and enzymatic activities of H. armigera with the biochemical composition of the studied legumes. The cluster analysis results revealed that artificial diets containing Mahta and Khomein cultivars were unsuitable for H. armigera, and can be used as candidates for integrated pest management programs or for screening insect inhibitors to produce genetically modified pest-resistant plants.

A highly accumulated secretory protein from cotton bollworm interacts with basic helix-loop-helix transcription factors to dampen plant defense.

Caterpillar oral secretion (OS) contains active molecules that modulate plant defense signaling. We isolated an effector-like protein (Highly Accumulated Secretory Protein 1, HAS1) from cotton bollworm (Helicoverpa armigera) that is the most highly accumulated secretory protein of the nondigestive components in OS and belongs to venom R-like protein. Elimination of HAS1 by plant-mediated RNA interference reduced the suppression of OS on the defense response in plants. Plants expressing HAS1 are more susceptible to insect herbivory accompanied by the reduced expressions of multiple defense genes. HAS1 binds to the basic helix-loop-helix (bHLH) transcription factors, including GoPGF involved in pigmented gland formation and defense compounds biosynthesis in cotton and MYC3/MYC4 the main regulators in jasmonate (JA) signaling in Arabidopsis. The binding activity is required for HAS1 to inhibit the activation of bHLHs on plant defense gene expressions. Together with our previous study that another venom R-like protein HARP1 in cotton bollworm OS blocks JA signaling by interacting with JASMONATE-ZIM-domain repressors, we conclude that the venom R-like proteins in OS interfere with plant defense in a dual suppression manner. Considering the venom proteins in parasitic wasp assault the immune system of its host animal, our investigation reveals their conserved function in carnivorous and herbivorous insects.

Baseline susceptibility of Helicoverpa armigera, Plutella xylostella, and Spodoptera frugiperda to the meta-diamide insecticide broflanilide.

Broflanilide is a novel meta-diamide insecticide that acts as a γ-aminobutyric acid-gated chloride channel allosteric modulator. With its unique mode of action, broflanilide has no known cross-resistance with existing insecticides and is expected to be an effective tool for the management of insecticide resistance. Establishing the baseline susceptibility to this insecticide is an essential step for developing and implementing effective resistance management strategies. Here we evaluated the baseline susceptibility to broflanilide for 3 cosmopolitan lepidopteran pest species, Helicoverpa armigera, Plutella xylostella, and Spodoptera frugiperda. Broflanilide exhibited high activity against populations sampled in the major distribution range of these pests in China, with median lethal concentrations (LC50 ) ranging between 0.209 and 0.684, 0.076 and 0.336, and 0.075 and 0.219 mg/L for H. armigera, P. xylostella, and S. frugiperda, respectively. Among-population variability in susceptibility to broflanilide was moderate for H. armigera (3.3-fold), P. xylostella (4.4-fold), and S. frugiperda (2.9-fold). The recommended diagnostic concentrations for H. armigera, P. xylostella, and S. frugiperda were 8, 4, and 2 mg/L, respectively. Little or no cross-resistance to broflanilide was detected in 3 diamide-resistant strains of P. xylostella and 1 spinosyns-resistant strain of S. frugiperda. Our results provide critical information for the development of effective resistance management programs to sustain efficacy of broflanilide against these key lepidopteran pests.

Effect of Transgenic Cotton Expressing Bt Cry1Ac or Cry1Ab/Ac Toxins on Lacewing Larvae Mediated by Herbivorous Insect Pests.

A simple food chain (plant, insect pests, and predatory arthropods) in an agro-ecosystem was set up here as a model system to elucidate the potential effect of transgenic Bacillus thuringiensis (Bt) cotton on non-target organisms. The system included transgenic/non-transgenic cotton, neonate larvae of three herbivorous insects (Spodoptera exigua, Helicoverpa armigera, and S. litura), and predatory lacewing larvae (Chrysopa spp.), which represent the first, second, and third trophic levels, respectively. The results showed that transgenic treatments and different densities of prey had significant effects on both body-weight gain of neonate herbivorous larvae and the number of prey captured by lacewing larvae, respectively. It was found that Bt toxin could persist at the third trophic level in lacewing larvae. The diet mixture bioassay showed that body-weight gain of lacewing larvae was significantly affected by various treatments, especially at lower concentrations of plant-expressed Bt toxin in the diet mixture, which caused significant decreases in body-weight gain. In contrast, synthetic Bt toxin at higher concentrations in the diet did not show this effect. Thus, we inferred that Bt toxin indirectly affected the growth of the lacewings and the lacewings may not be susceptible to Bt toxin or are able to metabolize it.

Baseline Susceptibility and Cross-Resistance of HearNPV in Helicoverpa armigera (Lepidoptera: Noctuidae) in Brazil.

The marked adoption of bioinsecticides in Brazilian agriculture in recent years is, at least partially, explained by the increasingly higher levels of insect pest resistance to synthetic insecticides. In particular, several baculovirus-based products have been registered in the last 5 years, including Helicoverpa armigera nucleopolyhedrovirus (HearNPV: Baculoviridae: Alphabaculovirus (Armigen®)). Understanding the susceptibility of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) to HearNPV is an important step toward development of robust Integrated Pest Management (IPM) and Insect Resistance Management programs (IRM) aimed at managing this serious insect pest. In this study, droplet feeding bioassays were used to characterize the baseline susceptibility to HearNPV (Armigen®) in H. armigera populations collected from major soybean and cotton-growing regions in Brazil. We defined and validated a diagnostic concentration for susceptibility monitoring of H. armigera populations to HearNPV. Additionally, cross-resistance between HearNPV and the insecticides flubendiamide and indoxacarb was evaluated by testing HearNPV in a susceptible strain and in resistant strains of H. armigera to these insecticides. A low interpopulation variation of H. armigera to HearNPV was detected. The LC50 values ranged from 1.5 × 105 to 1.1 × 106 occlusion bodies (OBs) per mL (7.3-fold variation). The mortality rate at the identified diagnostic concentration of 6.3 × 108 OBs/mL, based on the calculated LC99, ranged from 98.6 to 100% in populations of H. armigera collected from 2018 to 2020. No cross-resistance was detected between HearNPV and flubendiamide or indoxacarb. These results suggest that HearNPV (Armigen®) can be an effective tool in IPM and IRM programs to control H. armigera in Brazil.

The Micrococcus luteus infection activates a novel melanization pathway of cSP10, cSP4, and cSP8 in Helicoverpa armigera.

Melanization is a key immune response mediated by serine protease (SP) cascade in insects. Multiple SP pathways exist in different species and it is unclear how conserved these cascades are. The cotton bollworm Helicoverpa armigera is a major worldwide agricultural pest. We reported a conserved melanization pathway in this species, which consists of SP41, cSP1, and cSP6. In this study, we attempted to identify an insect pathogen that elicits the cascade and test whether or not there are other SP cascades in H. armigera. After Micrococcus luteus, Enterobacter cloacae, Beauveria bassiana, or Helicoverpa armigera nucleopolyhedrovirus were injected into larvae, pathogen-induced hemolymph samples were collected for in vitro biochemical assays, which failed to detect proSP41 or procSP1 activation. In contrast, we found that procSP4, a protein proposed to participate in H. armigera melanization, was activated in M. luteus infected hemolymph. We further revealed that cSP8 was a prophenoloxidase (PPO) activating protease downstream of cSP4, and cSP4 was activated by cSP10. The pathway of cSP10-cSP4-cSP8 activated PPO in vitro. Efficiently cleaved procSPH11 and procSPH50 by cSP8 substantially enhanced phenoloxidase activity, suggesting they work together as a cofactor for cSP8 mediated PPO activation. Hemolymph from larvae challenged with M. luteus or its peptidoglycan effectively activated procSP10. Collectively, these results revealed a new PPO activation cascade specifically triggered by the bacterium. In addition, we found that the PPO activation cascades in H. armigera and Manduca sexta are conserved.

Coocclusion of Helicoverpa armigera Single Nucleopolyhedrovirus (HearSNPV) and Helicoverpa armigera Multiple Nucleopolyhedrovirus (HearMNPV): Pathogenicity and Stability in Homologous and Heterologous Hosts.

Helicoverpa armigera single nucleopolyhedrovirus (HearSNPV) is a virulent pathogen of lepidopterans in the genera Heliothis and Helicoverpa, whereas Helicoverpa armigera multiple nucleopolyhedrovirus (HearSNPV) is a different virus species with a broader host range. This study aimed to examine the consequences of coocclusion of HearSNPV and HearMNPV on the pathogenicity, stability and host range of mixed-virus occlusion bodies (OBs). HearSNPV OBs were approximately 6-fold more pathogenic than HearMNPV OBs, showed faster killing by approximately 13 h, and were approximately 45% more productive in terms of OB production per larva. For coocclusion, H. armigera larvae were first inoculated with HearMNPV OBs and subsequently inoculated with HearSNPV OBs at intervals of 0-72 h after the initial inoculation. When the interval between inoculations was 12-24 h, OBs collected from virus-killed insects were found to comprise 41-57% of HearSNPV genomes, but the prevalence of HearSNPV genomes was greatly reduced (3-4%) at later time points. Quantitative PCR (qPCR) analysis revealed the presence of HearSNPV genomes in a small fraction of multinucleocapsid ODVs representing 0.47-0.88% of the genomes quantified in ODV samples, indicating that both viruses had replicated in coinfected host cells. End-point dilution assays on ODVs from cooccluded mixed-virus OBs confirmed the presence of both viruses in 41.9-55.6% of wells that were predicted to have been infected by a single ODV. A control experiment indicated that this result was unlikely to be due to the adhesion of HearSNPV ODVs to HearMNPV ODVs or accidental contamination during ODV band extraction. Therefore, the disparity between the qPCR and end-point dilution estimates of the prevalence of mixed-virus ODVs likely reflected virus-specific differences in replication efficiency in cell culture and the higher infectivity of pseudotyped ODVs that were produced in coinfected parental cells. Bioassays on H. armigera, Spodoptera frugiperda and Mamestra brassicae larvae revealed that mixed-virus OBs were capable of infecting heterologous hosts, but relative potency values largely reflected the proportion of HearMNPV present in each mixed-virus preparation. The cooccluded mixtures were unstable in serial passage; HearSNPV rapidly dominated during passage in H. armigera whereas HearMNPV rapidly dominated during passage in the heterologous hosts. We conclude that mixed-virus coocclusion technology may be useful for producing precise mixtures of viruses with host range properties suitable for the control of complexes of lepidopteran pests in particular crops, although this requires validation by field testing.

Resistance to RNA interference by plant-derived double-stranded RNAs but not plant-derived short interfering RNAs in Helicoverpa armigera.

Plant-mediated RNA interference (RNAi) has emerged as a promising technology for pest control through expression of double-stranded RNAs (dsRNAs) targeted against essential insect genes. However, little is known about the underlying molecular mechanisms and whether long dsRNA or short interfering RNAs (siRNAs) are the effective triggers of the RNAi response. Here we generated transplastomic and nuclear transgenic tobacco plants expressing dsRNA against the Helicoverpa armigera ATPaseH gene. We showed that expression of long dsRNA of HaATPaseH was at least three orders of magnitude higher in transplastomic plants than in transgenic plants. HaATPaseH-derived siRNAs are absent from transplastomic plants, while they are abundant in transgenic plants. Feeding transgenic plants to H. armigera larvae reduced gene expression of HaATPaseH and delayed growth. Surprisingly, no effect of transplastomic plants on insect growth was observed, despite efficient dsRNA expression in plastids. Furthermore, we found that dsRNA ingested by H. armigera feeding on transplastomic plants was rapidly degraded in the intestinal fluid. In contrast, siRNAs are relatively stable in the digestive system. These results suggest that plant-derived siRNAs may be more effective triggers of RNAi in Lepidoptera than dsRNAs, which will aid the optimization of the strategies for plant-mediated RNAi to pest control.

Bacillus thuringiensis Cry1Ac Protoxin and Activated Toxin Exert Differential Toxicity Due to a Synergistic Interplay of Cadherin with ABCC Transporters in the Cotton Bollworm.

Bacillus thuringiensis Cry proteins are used worldwide for insect control. It was proposed that Cry-protoxins must be converted into activated toxin by proteases to bind midgut cell proteins to kill insects. However, Cry-protoxins also bind to midgut proteins and kill insects that have evolved resistance to activated toxins suggesting an independent toxicity pathway. Cadherin (CAD) and ABCC transporters are recognized as important receptors for Cry proteins. Here we constructed different Helicoverpa armigera mutations in these receptors by CRISPR/Cas9. HaCAD-KO mutant showed much higher resistance to Cry1Ac activated toxin than to Cry1Ac protoxin. In contrast, the HaABCC2-M and HaABCC3-M mutants showed higher resistance to Cry1Ac-protoxin than to activated toxin. However, in the double HaABCC2/3-KO mutant, very high levels of resistance were observed to both Cry1Ac protoxin and activated toxin, supporting that both ABC transporters have redundant functions for these two proteins. In addition, Hi5 cells transfected with HaCAD were susceptible only to the activated toxin but not to protoxin. In contrast, both forms of Cry1Ac were similarly toxic to Hi5 cells expressing HaABCC2 or HaABCC3. Co-expression of HaCAD with HaABCC2 or HaABCC3 revealed a more important synergistic effect for activated toxin compared to protoxin. Overall, our results show that toxicity of Cry1Ac activated toxin involves synergistic interplay of HaCAD with ABCC transporters, while the Cry1Ac protoxin toxicity is mainly mediated by ABCC transporters with little participation of HaCAD. These data help to understand the mode of action of Cry proteins that will be relevant to enhance efficacy and durability of Bt-crops. IMPORTANCE Better understanding of the mode of action of Bacillus thuringiensis toxins is beneficial for the sustainable application of Bt crops. It is generally accepted that Cry-protoxins need to be activated by proteases to bind with midgut cell proteins and exert toxicity against insects. Here, we provide new insights into the toxic pathway of Cry proteins in the cotton bollworm. First, our results demonstrate that Cry1Ac protoxin is able to exert cytotoxicity against the insect cells expressing ABCC transporters. Second, we reveal that CAD plays a critical role in the different toxicity of protoxin and toxin by facilitating a synergistic interplay with ABCC transporters. Our results provide in vivo and in vitro experimental evidence supporting that Cry1Ac protoxin exerts toxicity against H. armigera via different steps from that of toxin. These new findings on the mode of action of Cry proteins could be beneficial for efficacy enhancement and durability of Bt-crops.