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Monitoring adult populations of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), a major agricultural pest, provides data useful for its control. Food attractants, considered as adult insect behavior regulators based on the preference of an herbivorous pest for food sources or their volatiles, also have great potential for monitoring populations. To study the feasibility of monitoring the population dynamics and reproductive development of H. armigera in the field using food attractants, we quantitatively analyzed reproductive organ development of adults in a laboratory population as a way to predict the reproductive development of adults trapped using food attractants in the field in 2019 and 2020. The adults trapped using food attractants had obvious generational changes and the same trends in variation for females and males. The extent of ovarian development in trapped females tended to increase within each generation, and the major axis length of testis in trapped males tended to decrease. Reproductive developmental status of trapped adults also differed significantly among months. This study shows that by trapping H. armigera with food attractants, the population dynamics of adults in the field can be monitored, and reproductive anatomy can also be used to monitor adult reproductive status. These approaches are a new way to forecast the population dynamics of this pest.

Background The fall armyworm Spodoptera frugiperda is a major agricultural pest that has invaded the East Hemisphere since 2016, generating a serious threat to food security worldwide including Africa and Asia. The Cry toxins produced by Bacillus thuringiensis (Bt) have been shown to be effective against this insect pest. In different insect ABC transporters (ABCC2 or ABCC3) have been shown to be involved as receptors of some Cry1 toxins. Here we analyzed the role of SfABCC2 and SfABCC3 in the toxicity of Cry1Fa and Cry1Ab toxins in this insect pest. Results Two S. frugiperda SfABCC2 and SfABCC3 knockout strains, coding for potential functional Bt receptors, were created using CRISPR/Cas9 genome editing system. Both knockout strains showed resistance to both Cry1Fa and Cry1Ab toxins compared with the susceptible strain. SfABCC2 knockout strain showed higher resistance to both Cry toxins than SfABCC3 knockout strain, suggesting a major role of SfABCC2 in the mode of action of these Cry toxins. In addition, expression of SfABCC2 and SfABCC3 genes in Trichoplusia ni Hi5 cells also increased the susceptibility to Cry1Ab and Cry1Fa toxins, in agreement with the genome editing results. The double knockout of SfABCC2 and SfABCC3 strain was not viable in contrast to other lepidopteran species. Furthermore, we report here that SfABCC2 or SfABCC3 knockout strains increased their susceptibility to abamectin and spinosad insecticides. Conclusion We provide functional evidence that in S. frugiperda these two ABCC transporters serve as receptors of Bt Cry1Fa and Cry1Ab toxins. © 2020 Society of Chemical Industry.

Background The plant bug, Apolygus lucorum Meyer-Dur, has begun a resurgence and has become a key pest in cotton in northern China, with the wide-scale adoption of transgenic Bt cotton. We attempted to develop a new approach to the control of this plant bug by identifying and utilizing its sex pheromone. Results Extracts from A. lucorum adults and nymphs were analyzed using gas chromatography-mass spectrometry (GC-MS) and gas chromatography-electroantennogram detection (GC-EAD) to identify the sex pheromone components. (E)-4-Oxo-2-hexenal and (E)-2-hexenyl butyrate were the major sex pheromone components from females and were also electrophysiology-active towards male antennae. For males, (E)-4-oxo-2-hexenal and hexyl butyrate were the major sex pheromone components. These three components, however, were not detected in nymphs. Field tests showed that binary blends of (E)-4-oxo-2-hexenal and (E)-2-hexenyl butyrate at a ratio of 3:2 resulted in trapping the greatest numbers of A. lucorum males. A long-term dispenser was developed by loading the pheromone components dissolved in sunflower oil into polyethylene vials. Further field evaluation showed that the polyethylene dispensers attracted significantly more bugs even when deployed in the field for 5 weeks. Conclusion We identified the sex pheromone of A. lucorum and developed a type of high-efficiency and long-term lure. Our results could expand knowledge of the pheromones of plant bugs, and provide novel technologies to monitor and control this pest. © 2019 Society of Chemical Industry.

Summary Genetically‐modified crops expressing Bacillus thuringiensis (Bt) proteins have been widely cultivated, permitting an effective non‐chemical control of major agricultural pests. While their establishment can enable an area‐wide suppression of polyphagous herbivores, no information is available on the impact of Bt crop abandonment in entire landscape matrices. Here, we detail a resurgence of the cosmopolitan bollworm Helicoverpa armigera following a contraction of Bt cotton area in dynamic agro‐landscapes over 2007–2019 in North China Plain. An 80% reduction in Bt cotton was mirrored in a 1.9‐fold increase of ambient H. armigera population levels, culminating in 1.5–2.1‐fold higher yield loss and a 2.0–4.4‐fold increase in pesticide use frequency in non‐Bt crops (i.e. maize, peanut, soybean). Our work unveils the fate of herbivorous insect populations following a progressive dis‐use of insecticidal crop cultivars, and hints at how tactically deployed Bt crops could be paired with agro‐ecological measures to mitigate the environmental footprint of crop production.

Numerous insect species engage in seasonal, trans-latitudinal migration, in response to varying resource availability, climatic conditions and associated opportunities, to maximize fitness and reproductive success. For certain species, the interaction between migrant adults and individual host plants is well-studied under laboratory conditions, but scant knowledge exists on the nutritional ecology of wild (i.e., field-caught) moths. During 2017–2018, we trapped adults of the cotton bollworm Helicoverpa armigera (Hübner) along its migration pathway in northeastern China and used pollen grain analysis to assess its visitation of particular host plants. Next, we assessed life history effects of adult feeding on carbohydrate-rich resources, for migrant individuals. Pollen grain analysis revealed H. armigera visitation of 32 species from 28 families, with the largest carrier ratio for northward migrants. Evening primrose (Oenothera spp.) accounted for 48% of pollen grains, indicating a marked H. armigera feeding preference. Furthermore, feeding on sugar-rich foods benefited adult fitness, enhanced fecundity by 65–82% and increased flight distance by 38–55% as compared to unfed individuals. Also, the degree of enhancement of reproduction and flight performance following sugar feeding varied between different migratory cohorts. Our work combines (polymerase chain reaction [PCR]-assisted) palynology and laboratory-based life history trials to generate novel perspectives on the nutritional ecology of long-distance migratory insects. These findings can aid the development of population monitoring and ‘area-wide’ management strategies for a globally-important agricultural pest.

Parasitic wasps are among the most species‐rich groups on Earth. A major cause of this diversity may be local adaptation to host species. However, little is known about variation in host specificity among populations within parasitoid species. Not only is such knowledge important for understanding host‐driven speciation, but because parasitoids often control pest insects and narrow host ranges are critical for the safety of biological control introductions, understanding variation in specificity and how it arises are crucial applications in evolutionary biology. Here, we report experiments on variation in host specificity among 16 populations of an aphid parasitoid, Aphelinus certus. We addressed several questions about local adaptation: Do parasitoid populations differ in host ranges or in levels of parasitism of aphid species within their host range? Are differences in parasitism among parasitoid populations related to geographical distance, suggesting clinal variation in abundances of aphid species? Or do nearby parasitoid populations differ in host use, as would be expected if differences in aphid abundances, and thus selection, were mosaic? Are differences in parasitism among parasitoid populations related to genetic distances among them? To answer these questions, we measured parasitism of a taxonomically diverse group of aphid species in laboratory experiments. Host range was the same for all the parasitoid populations, but levels of parasitism varied among aphid species, suggesting adaptation to locally abundant aphids. Differences in host specificity did not correlate with geographical distances among parasitoid populations, suggesting that local adaption is mosaic rather than clinal, with a spatial scale of less than 50 kilometers. We sequenced and assembled the genome of A. certus, made reduced‐representation libraries for each population, analyzed for single nucleotide polymorphisms, and used these polymorphisms to estimate genetic differentiation among populations. Differences in host specificity correlated with genetic distances among the parasitoid populations.

Resistance evolution of target pests reduces efficacy of Bacillus thuringiensis Cry toxins used in insect-pest control. Mutations in Cadherin (CAD) or ATP-binding cassette (ABC) transporters genes are linked to Cry resistance in different pests. Also, it has been shown that ABCC2 and CAD have synergistic interaction on Cry toxicity when co-express in cell lines, which we confirmed here by Helicoverpa armigera HaABCC2 and HaCAD expression in Hi5 cells. To confirm that CAD and ABC transporters interact in vivo, we constructed nearly H. armigera isogenic lines such as LFC2 and 96CAD strains, linked to HaABCC2 and HaCAD mutations that showed 512- and 396-fold Cry1Ac resistance-ratios, respectively. Interestingly, Fusion-1 strain linked to both HaABCC2 and HaCAD mutations, showed 6273-fold resistance-ratio, significantly higher than the single mutant strains. To confirm the interaction of HaABCC2 and CAD in Cry1Ac resistance, we analyzed the Cry1Ac susceptibility in CRISPR/Cas9 knockdown strains, C2-KO (ABCC2-gene knockout-strain) and CAD-KO (CAD-gene knockout-strain), that showed 112- and 531-fold Cry1Ac resistance-ratios, respectively. However, the resistance-ratio of Fusion-2 strain obtained from crossing C2-KO and CAD-KO strains, was only 816-fold. The analysis of HaABCC3 gene transcript levels showed nearly 4-fold lower expression in LFC2 and Fusion-1 strains compared to the susceptible strain, suggesting that additional mutations in these strains resulted in low HaABCC3 expression, which contribute to their enhanced Cry1Ac resistance. Our data show that the CAD and ABCC2/ABCC3 interact synergistically to induce high Cry1Ac resistance in H. armigera. These results can be helpful for Bt resistance monitoring and pest management.

Bacillus thuringiensis (Bt) crops have been widely planted and the effects of Bt-crops on populations of the target and non-target insect pests were well studied. However, the effects of Bt-crops exposure on microorganisms that interact with crop pests haven’t previously been quantified. Here, we use laboratory and field data to show that infection of Helicoverpa armigera with a symbiotic densovirus (HaDV2) is associated with its enhanced growth and resistance to Bt-cotton. Moreover, field monitoring showed a much higher incidence of cotton bollworm infection with HaDV2 in regions cultivated with Bt-cotton than in regions without it, with the rate of densovirus infection increasing with increasing use of Bt-cotton. RNA-seq suggested resistance to both baculovirus and Cry1Ac were enhanced via the immune-related pathways. These suggest that the exposure to Bt-crops has selected for beneficial interactions between the target pest and a symbiotic microorganism that enhances its performance on Bt-crops under field conditions.

Bacillus thuringiensis (Bt) crops have been widely planted and the effects of Bt-crops on populations of the target and non-target insect pests have been well studied. However, the effects of Bt-crops exposure on microorganisms that interact with crop pests have not previously been quantified. Here, we use laboratory and field data to show that infection of Helicoverpa armigera with a densovirus (HaDV2) is associated with its enhanced growth and tolerance to Bt-cotton. Moreover, field monitoring showed a much higher incidence of cotton bollworm infection with HaDV2 in regions cultivated with Bt-cotton than in regions without it, with the rate of densovirus infection increasing with increasing use of Bt-cotton. RNA-seq suggested tolerance to both baculovirus and Cry1Ac were enhanced via the immune-related pathways. These findings suggest that exposure to Bt-crops has selected for beneficial interactions between the target pest and a mutualistic microorganism that enhances its performance on Bt-crops under field conditions.

The ability to migrate is an important biological trait of insects, and wingbeat frequency (WBF) is a key factor influencing migratory behavior. The WBF of insects has been shown to be species-specific in previous studies; however, there is scant information on variations in WBF among different taxa of migratory insects. In 2018 and 2019, we investigated the relationship between WBF and 12 morphological variables (e.g., body mass, body length, total wing area, etc.) of the main migratory insects (77 species in 3 orders and 14 families) over the Bohai Sea in China. The WBF of migratory insects was negatively correlated with the 12 morphological variables and varied significantly among orders. In migratory lepidopterans, neuropterans, and odonatans, the ranges of WBF were 6.71–81.28 Hz, 19.17–30.53 Hz, and 18.35–38.01 Hz, respectively. Regression models between WBF and connecting morphological variables were established for these three orders. Our findings revealed the relationship between WBF and morphometrics of migratory insects in Northeast Asia, increased our knowledge on the flight biology of migratory insects, and provided a basis for developing morphological and WBF-based monitoring techniques to identify migrating insects.

Since 2016, the fall armyworm (FAW, Spodoptera frugiperda) has invaded large parts of Africa and Asia, impacting millions of hectares of maize crops and thereby posing a major threat to food security. The rapid geographic spread and outbreak dynamics of S. frugiperda are tied to its unique dispersal ability and long-distance migration capability. Yet, up till present, limited research has been conducted on the physiological determinants of S. frugiperda flight and migration. In this study, we used laboratory experiments to assess whether mating and oviposition affect S. frugiperda flight ability and wingbeat frequency. During 2019-2020, migratory FAW females were trapped in Yunnan (China) and dissected to assess ovarian development. Tethered flight assays showed that gravid S. frugiperda females exhibited strong flight ability at 1-3 days following the onset of oviposition. Flight distance and duration negatively correlated with the number of deposited eggs. Ovarian dissections further showed that over 50% of migrant females were mated and 46-54% had initiated oviposition. Our study shows the complex, yet nuanced effects of reproductive status on flight capacity, with possibly a facultative trade-off between flight and reproduction. These novel insights into S. frugiperda physiology and migration behavior can guide future monitoring and integrated pest management (IPM) programs against this newly-invasive pest in China and abroad.

Evolution of resistance by pests reduces the benefits of transgenic crops that produce insecticidal proteins from Bacillus thuringiensis (Bt). Here we analyzed resistance to Bt toxin Cry1Ac in a field-derived strain of pink bollworm (Pectinophora gossypiella), a global pest of cotton. We discovered that the r14 allele of the pink bollworm cadherin gene (PgCad1) has a 234-bp insertion in exon 12 encoding a mutant PgCad1 protein that lacks 36 amino acids in cadherin repeat 5 (CR5). A strain homozygous for this allele had 237-fold resistance to Cry1Ac, 1.8-fold cross-resistance to Cry2Ab, and developed from neonate to adult on Bt cotton producing Cry1Ac. Inheritance of resistance to Cry1Ac was recessive and tightly linked with r14. PgCad1 transcript abundance in midgut tissues did not differ between resistant and susceptible larvae. Toxicity of Cry1Ac to transformed insect cells was lower for cells expressing r14 than for cells expressing wild-type PgCad1. Wild-type PgCad1 was transported to the cell membrane, whereas PgCad1 produced by r14 was not. In larval midgut tissue, PgCad1 protein occurred primarily on the brush border membrane only in susceptible larvae. The results imply r14 mediates pink bollworm resistance to Cry1Ac by reduced translation, increased degradation, and/or mislocalization of cadherin.

Recent advances in next generation sequencing (NGS) (e.g. metagenomic and transcriptomic sequencing) have facilitated the discovery of a large number of new insect viruses, but the characterization of these viruses is still in its infancy. Here, we report the discovery, using RNA-seq, of three new partiti-like viruses from African armyworm, Spodoptera exempta (Lepidoptera: Noctuidae), which are all vertically-transmitted transovarially from mother to offspring with high efficiency. Experimental studies show that the viruses reduce their host’s growth rate and reproduction, but enhance their resistance to a nucleopolyhedrovirus (NPV). Via microinjection, these partiti-like viruses were transinfected into a novel host, a newly-invasive crop pest in sub-Saharan Africa (SSA), the Fall armyworm, S. frugiperda. This revealed that in this new host, these viruses appear to be deleterious without any detectable benefit; reducing their new host’s reproductive rate and increasing their susceptibility to NPV. Thus, the partiti-like viruses appear to be conditional mutualistic symbionts in their normal host, S. exempta, but parasitic in the novel host, S. frugiperda. Transcriptome analysis of S. exempta and S. frugiperda infected, or not, with the partiti-like viruses indicates that the viruses may regulate pathways related to immunity and reproduction. These findings suggest a possible pest management strategy via the artificial host-shift of novel viruses discovered by NGS., Author summary Modern molecular approaches allow the discovery of new viruses that do not present obvious symptoms of disease in their hosts. Using these methods, we discovered three new partiti-like viruses in a major crop pest, the African armyworm, Spodoptera exempta, which are naturally transmitted vertically from adult females to their offspring in her eggs. We discovered that these viruses decrease their host’s growth rate and reproduction, but they may also help their insect host by protecting them from other lethal viruses, specifically the baculoviruses that are often used as biological pesticides. We transferred these partiti-like viruses into a novel host, the Fall armyworm, S. frugiperda, by microinjection and found that they also decrease S. frugiperda’s growth rate and reproduction. Interestingly, however, it appears that the partiti-like viruses make S. frugiperda more susceptible to baculovirus, potentially making it more vulnerable to control using biopesticides. Our results suggest a possible novel pest management strategy via artificial host-shift of viruses discovered using molecular approaches and offers new insights into host-virus-virus interactions.

Apolygus lucorum (Miridae) is an omnivorous pest that occurs worldwide and is notorious for the serious damage it causes to various crops and substantial economic losses. Although some studies have examined the biological characteristics of the mirid bug, no reference genome is available in Miridae, limiting in-depth studies of this pest. Here, we present a chromosome-scale reference genome of A. lucorum, the first sequenced Miridae species. The assembled genome size was 1.02 Gb with a contig N50 of 785 kb. With Hi-C scaffolding, 1,016 Mb contig sequences were clustered, ordered and assembled into 17 large scaffolds with scaffold N50 length 68 Mb, each corresponding to a natural chromosome. Numerous transposable elements occur in this genome and contribute to the large genome size. Expansions of genes associated with omnivorousness and mesophyll feeding such as those related to digestion, chemosensory perception, and detoxification were observed in A. lucorum, suggesting that gene expansion contributed to its strong environmental adaptability and severe harm to crops. We clarified that a salivary enzyme polygalacturonase is unique in mirid bugs and has significantly expanded in A. lucorum, which may contribute to leaf damage from this pest. The reference genome of A. lucorum not only facilitates biological studies of Hemiptera as well as an understanding of the damage mechanism of mesophyll feeding, but also provides a basis on which to develop efficient control technologies for mirid bugs.

Mamestra brassicae L. is an important, regionally migratory pest of vegetable crops in Europe and Asia. Its migratory activity contributes significantly to population outbreaks, causing severe crop yield losses. Because an in-depth understanding of flight performance is key to revealing migratory patterns, here we used a computer-linked flight mill and stroboscope to study the flight ability and wingbeat frequency (WBF) of M. brassicae in relation to sex, age, temperature, and relative humidity (RH). The results showed that age significantly affected the flight ability and WBF of M. brassicae, and 3-d-old individuals performed the strongest performance (total flight distance: 45.6 ± 2.5 km; total flight duration: 9.3 ± 0.3 h; WBF: 44.0 ± 0.5 Hz at 24°C and 75% RH). The age for optimal flight was considered to be 2–3 d old. Temperature and RH also significantly affected flight ability and WBF; flight was optimal from 23°C to 25°C and 64–75% RH. Because M. brassicae thus has great potential to undertake long-distance migration, better knowledge of its flight behavior and migration will help establish a pest forecasting and early-warning system.

Summary Genetically engineered (GE) rice lines expressing Lepidoptera‐active insecticidal cry genes from the bacterium Bacillus thuringiensis (Bt) have been developed in China. Field surveys indicated that Bt rice harbours fewer rice planthoppers than non‐Bt rice although planthoppers are not sensitive to the produced Bt Cry proteins. The mechanisms underlying this phenomenon remain unknown. Here, we show that the low numbers of planthoppers on Bt rice are associated with reduced caterpillar damage. In laboratory and field‐cage experiments, the rice planthopper Nilapavata lugens had no feeding preference for undamaged Bt or non‐Bt plants but exhibited a strong preference for caterpillar‐damaged plants whether Bt or non‐Bt. Under open‐field conditions, rice planthoppers were more abundant on caterpillar‐damaged non‐Bt rice than on neighbouring healthy Bt rice. GC–MS analyses showed that caterpillar damage induced the release of rice plant volatiles known to be attractive to planthoppers, and metabolome analyses revealed increased amino acid contents and reduced sterol contents known to benefit planthopper development. That Lepidoptera‐resistant Bt rice is less attractive to this important nontarget pest in the field is therefore a first example of ecological resistance of Bt plants to nontarget pests. Our findings suggest that non‐Bt rice refuges established for delaying the development of Bt resistance may also act as a trap crop for N. lugens and possibly other planthoppers.

Evolution of pest resistance reduces the efficacy of insecticidal proteins from the gram-positive bacterium Bacillus thuringiensis (Bt) used widely in sprays and transgenic crops. In some previously studied strains of three major lepidopteran pests, resistance to Bt toxin Cry1Ac is associated with mutations disrupting the extracellular or cytoplasmic domains of cadherin proteins that bind Cry1Ac in the midgut of susceptible larvae. Here we report the first case of a cadherin transmembrane mutation associated with insect resistance to Bt. We discovered this mutation in a strain of the devastating global cotton pest, the pink bollworm (Pectinophora gossypiella), derived from a field population in the Yangtze River Valley of China. The mutant allele analyzed here has a 207 base pair deletion and encodes a cadherin protein lacking its transmembrane domain. Relative to a susceptible strain, a strain homozygous for this allele had 220-fold resistance to Cry1Ac and 2.1-fold cross-resistance to Cry2Ab. On transgenic cotton plants producing Cry1Ac, no susceptible larvae survived, but the resistant strain completed its life cycle. Inheritance of resistance to Cry1Ac was autosomal, recessive and tightly linked with the cadherin gene. Transportation of cadherin protein to the cell membrane and susceptibility to Cry1Ac occurred in transfected insect cells expressing the wild type cadherin allele, but not in transfected insect cells expressing the mutant cadherin allele. The results imply that the mutant allele analyzed here confers resistance to Cry1Ac by disrupting cellular trafficking of cadherin.

Helicoverpa armigera, cotton bollworm, is one of the most disastrous pests worldwide, threatening various food and economic crops. Functional genomic tools may provide efficient approaches for its management. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, dependent on a single guide RNA (sgRNA), has been used to induce indels for targeted mutagenesis in cotton bollworm. However, genomic deletions may be more desirable to disrupt the function of noncoding genes or regulatory sequences. By injecting two sgRNAs with Cas9 protein targeting different exons, we obtained predictable genomic deletions of several hundred bases. We achieved this type of modification with different combinations of sgRNA pairs, including HaCad and HaABCC2. Our finding indicated that CRISPR/Cas9 can be used as an efficient tool to engineer genomes with chromosomal deletion in H. armigera.

The polyphagous mirid bug Apolygus lucorum (Hemiptera: Miridae) is an important pest of cotton and other crops in China. It would be useful to examine the reproductive biology of A. lucorum for the development of management strategies. In this study, the effect of mating on ovarian development, egg maturation, and oviposition of A. lucorum was investigated. The results showed that mating could induce ovary development and oviposition. During the preoviposition period (

Although farmers in China have grown transgenic Bt-Cry1Ac cotton to resist the major pest Helicoverpa armigera since 1997 with great success, many secondary lepidopteran pests that are tolerant to Cry1Ac are now reported to cause considerable economic damage. Vip3AcAa, a chimeric protein with the N-terminal part of Vip3Ac and the C-terminal part of Vip3Aa, has a broad insecticidal spectrum against lepidopteran pests and has no cross resistance to Cry1Ac. In the present study, we tested insecticidal activities of Vip3AcAa against Spodoptera litura, Spodoptera exigua, and Agrotis ipsilon, which are relatively tolerant to Cry1Ac proteins. The bioassay results showed that insecticidal activities of Vip3AcAa against these three pests are superior to Cry1Ac, and after an activation pretreatment, Vip3AcAa retained insecticidal activity against S. litura, S. exigua and A. ipsilon that was similar to the unprocessed protein. The putative receptor for this chimeric protein in the brush border membrane vesicle (BBMV) in the three pests was also identified using biotinylated Vip3AcAa toxin. To broaden Bt cotton activity against a wider spectrum of pests, we introduced the vip3AcAa and cry1Ac genes into cotton. Larval mortality rates for S. litura, A. ipsilon and S. exigua that had fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and Bt-Cry1Ac cotton in a laboratory experiment. These results suggested that the Vip3AcAa protein is an excellent option for a “pyramid” strategy for integrated pest management in China.

While Cry1Ac has been known to bind with larval midgut proteins cadherin, APN (amino peptidase N), ALP (alkaline phosphatase) and ABCC2 (adenosine triphosphate-binding cassette transporter subfamily C2), little is known about the receptors of Cry2Ab. To provide a clue to the receptors of Cry2Ab, we tested the baseline cytotoxicity of activated Cry1Ac and Cry2Ab against the midgut and fat body cell lines of Helicoverpa zea and the ovary cell line of Spodoptera frugiperda (SF9). As expected, the descending order of cytotoxicity of Cry1Ac against the three cell lines in terms of 50% lethal concetration (LC50 ) was midgut (31.0 μg/mL) > fat body (59.0 μg/mL) and SF9 cell (99.6 μg/mL). By contrast, the fat body cell line (LC50 = 7.55 μg/mL) was about twice more susceptible to Cry2Ab than the midgut cell line (16.0 μg/mL), the susceptibility of which was not significantly greater than that of SF9 cells (27.0 μg/mL). Further, ligand blot showed the binding differences between Cry1Ac and Cry2Ab in the three cell lines. These results indicated that the receptors of Cry2Ab were enriched in fat body cells and thus largely different from the receptors of Cry1Ac, which were enriched in midgut cells.

Extensive cultivation of crops genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) has suppressed some major pests, reduced insecticide sprays, enhanced pest control by natural enemies, and increased grower profits. However, these benefits are being eroded by evolution of resistance in pests. We report a strategy for combating resistance by crossing transgenic Bt plants with conventional non-Bt plants and then crossing the resulting first-generation (F1) hybrid progeny and sowing the second-generation (F2) seeds. This strategy yields a random mixture within fields of three-quarters of plants that produce Bt toxin and one-quarter that does not. We hypothesized that the non-Bt plants in this mixture promote survival of susceptible insects, thereby delaying evolution of resistance. To test this hypothesis, we compared predictions from computer modeling with data monitoring pink bollworm (Pectinophora gossypiella) resistance to Bt toxin Cry1Ac produced by transgenic cotton in an 11-y study at 17 field sites in six provinces of China. The frequency of resistant individuals in the field increased before this strategy was widely deployed and then declined after its widespread adoption boosted the percentage of non-Bt cotton plants in the region. The correspondence between the predicted and observed outcomes implies that this strategy countered evolution of resistance. Despite the increased percentage of non-Bt cotton, suppression of pink bollworm was sustained. Unlike other resistance management tactics that require regulatory intervention, growers adopted this strategy voluntarily, apparently because of advantages that may include better performance as well as lower costs for seeds and insecticides.

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.

Previously, we reported a novel iflavirus in Helicoverpa armigera (helicoverpa armigera iflavirus, HaIV) and here we report the effects of HaIV on its host. In a laboratory bioassay, HaIV-positive larvae and pupae developed more slowly and had higher mortality than HaIV-negative larvae, suggesting that the virus is pathogenic. The relative fitness of H. armigera decreased with HaIV infection by a ratio of 0.65. Transcriptional analysis indicated that infection significantly changed the expression levels of host genes, with more genes affected at 72 h after inoculation than at 48 h (138 up- and 229 downregulated at 48 h; 185 up- and 299 downregulated at 72 h). Interestingly, pathways related to digestion and absorption were significantly enriched, e.g., protein digestion and absorption, suggesting developmental regulation of the host by HaIV via these pathways. HaIV-infected H. armigera showed significantly downregulated expression of genes encoding cuticular proteins (CPs), essential for structural and protective functions, at 48 h and 72 h, suggesting that HaIV increased larval mortality by downregulating CP gene expression.

Insecticidal proteins from Bacillus thuringiensis (Bt) are widely used to control insect pests, but their efficacy is reduced when pests evolve resistance. We report on a novel allele (r16) of the cadherin gene (PgCad1) in pink bollworm (Pectinophora gossypiella) associated with resistance to Bt toxin Cry1Ac, which is produced by transgenic cotton. The r16 allele isolated from a field population in China has 1545 base pairs of a degenerate transposon inserted in exon 20 of PgCad1, which generates a mis-spliced transcript containing a premature stop codon. A strain homozygous for r16 had 300-fold resistance to Cry1Ac, 2.6-fold cross-resistance to Cry2Ab, and completed its life cycle on transgenic Bt cotton producing Cry1Ac. Inheritance of Cry1Ac resistance was recessive and tightly linked with r16. Compared with transfected insect cells expressing wild-type PgCad1, cells expressing r16 were less susceptible to Cry1Ac. Recombinant cadherin protein was transported to the cell membrane in cells transfected with the wild-type PgCad1 allele, but not in cells transfected with r16. Cadherin occurred on brush border membrane vesicles (BBMVs) in the midgut of susceptible larvae, but not resistant larvae. These results imply that the r16 allele mediates Cry1Ac resistance in pink bollworm by interfering with the localization of cadherin.

Crystalline (Cry) proteins from Bacillus thuringiensis (Bt) are widely used in sprays and transgenic crops to control insect pests, but the evolution of insect resistance threatens their long-term use. Different resistance mechanisms have been identified, but some have not been completely elucidated. Here, the transcriptome of the midgut and proteome of the peritrophic matrix (PM) were comparatively analyzed to identify potential mechanism of resistance to Cry1Ac in laboratory-selected strain XJ10 of Helicoverpa armigera. This strain had a 146-fold resistance to Cry1Ac protoxin and 45-fold resistance to Cry1Ac activated toxin compared with XJ strain. The mRNA and protein levels for several trypsin genes were downregulated in XJ10 compared to the susceptible strain XJ. Furthermore, 215 proteins of the PM were identified, and nearly all had corresponding mRNAs in the midgut. These results provide new insights that the PM may participate in Bt resistance.

Many insects adopt seasonal, trans-latitudinal migration in response to altering climatic conditions, resource availability or reproductive requirements. Though the migration behavior of the cotton bollworm, Helicoverpa armigera (Hubner)(Lepidoptera: Noctuidae), has been well-studied, little is known about the extent of intra-specific variation between northward- and southward-migrating individuals. In this study, we captured H. armigera adults along the species' migration route during 2017-2018 to determine seasonal variability in their morphology, flight capability, and reproductive performance. Northward migrants have broader, longer bodies and are 1.33 times heavier than southward migrants, hinting at a comparatively higher allocation of resources in the abdomen. Accordingly, the former migrants engaged in longer flights, had greater reproductive capacity and a longer lifespan than southward ones. As northward migrants originate from favorable environmental conditions, their fitness is higher than that of the southward cohorts that develop on less favorable host plants and in perhaps more adverse climatic or ecological conditions. Northward H. armigera migrants thus possess an advantageous morphology that benefits habitat colonization and resource exploitation. Our work offers a novel perspective on the ecological and reproductive benefits of long-distance migration and can aid the development of population monitoring and forecasting methods for this globally important agricultural pest.

Crystalline (Cry) proteins from Bacillus thuringiensis (Bt) are widely used in sprays and transgenic crops to control insect pests, but the evolution of insect resistance threatens their long-term use. Different resistance mechanisms have been identified, but some have not been completely elucidated. Here, the transcriptome of the midgut and proteome of the peritrophic matrix (PM) were comparatively analyzed to identify potential mechanism of resistance to Cry1Ac in laboratory-selected strain XJ10 of Helicoverpa armigera. This strain had a 146-fold resistance to Cry1Ac protoxin and 45-fold resistance to Cry1Ac activated toxin compared with XJ strain. The mRNA and protein levels for several trypsin genes were downregulated in XJ10 compared to the susceptible strain XJ. Furthermore, 215 proteins of the PM were identified, and nearly all had corresponding mRNAs in the midgut. These results provide new insights that the PM may participate in Bt resistance.

The mirid bug Apolygus lucorum Meyer-Dür, 1843, an omnivorous species that feeds on plants and animals, has become a major pest in China as production of Bt-cotton has grown to such a large scale. Its omnivory is likely to be critical for its success, but the digestive mechanism(s) underlying processing and adsorption of such diverse foods is relatively unknown. Here, we examined the activities of digestive enzymes of A. lucorum in the salivary gland complex and midgut and the effect of sex, age, and food source on these activities. Amylase and protease were present in the salivary gland complex and the midgut, but were higher in the salivary gland complex. Trypsin-like enzyme was also present in both organs, but chymotrypsin-like enzyme was present only in the midgut. Sex, age, and food source affected the activities of these digestive enzymes. In general, the activities of these enzymes peaked at 10 d after emergence, and amylase and protease activities were higher in female adults than in males. Of the food sources tested, green bean pods (Gb) induced the highest amylase activity, whereas Helicoverpa armigera Hübner, 1809 eggs (He) and a mixture of Gb and He induced higher activities of the trypsin-like and chymotrypsin-like enzymes. The results from food switching experiments confirmed that amylase activity could be induced by plant sources, and animal sources induced protease activity. Thus, the types and activities of digestive enzymes in A. lucorum provide the physiological basis of the pest’s omnivory.

Glycosphingolipids (GSLs) play important roles in the cellular biology of vertebrate and invertebrate organisms, such as cell differentiation, tumor metastasis, and cell coordination. GSLs also serve as receptors for different bacterial toxins . For example, in the nematode Caenorhabditis elegans , GSLs function as receptors of the insecticidal Cry toxins produced by Bacillus thuringiensis (Bt), and mutations in bre genes involved in GSLs synthesis resulted in resistance to Cry5 toxin in this organism. However, the information of GSLs function in insects is still limited. In this study, three genes for glycosyltransferases, bre 2, bre 3, and bre 4, from Helicoverpa armigera were identified and cloned. The previously reported bre5 gene from H. armigera was also analyzed. Protein sequence alignments revealed that proteins codified by H. armigera Bre shared high identity with homologous proteins from other organisms. Expression profile analysis revealed that the expressions of bre genes varied in the different tissues and also in the different developmental stages of H. armigera. Finally, the heterologous expression of bre genes in Trichoplusia ni Hi5 cell line showed that the corresponding translated proteins were localized in the cytoplasm of Hi5 cells. These results provide the bases for further functional studies of bre genes and analyzing potential roles of GSLs in mode of action of Cry1A toxin in H. armigera.

Vitellogenin proteins (Vgs) are precursors of the major egg yolk proteins in many oviparous vertebrates and invertebrates. Here we cloned the full-length cDNA of the Vg gene of Apolygus lucorum , an omnivorous mirid bug that feeds on various agricultural crops and small insects. The gene has 6,131 base pairs (bp) with an open reading frame (ORF) of 5,988 bp, encoding a protein with 1,996 amino acids, with molecular mass 218.3 kDa and isoelectric point 9.02. Phylogenetic analysis showed that Vg gene has a high similarity with that in Hemiptera insects and was most closely related to the rice leaf bug, Trigonotylus caelestialium . The expression of Vg gene was significantly higher in the fat body than in other tissues in females (ovary, hemolymph, gut, and malpighian tubule). Vg gene expression was extremely low in newly emerged adult females and increased with age. The effect of different food sources on the expression level of Vg in adult females was investigated after they were fed on green bean pods (Gb), H. armigera eggs (He), or a combination of green bean pods and H. armigera eggs (GbHe). The results showed that transcript level of Vg gene in females was significantly higher on the GbHe diet than on Gb or He. Our results demonstrate that A. lucorum can acquire nutrients from both plant and animal origin food sources, and the combined food sources significantly facilitated Vg synthesis.

Cotton bollworm (Helicoverpa armigera) is the major insect herbivore of cotton plants. As its larvae feed and grow on cotton, H. armigera can likely tolerate gossypol, the main defense metabolite produced by cotton plants, through detoxification and sequestration mechanisms. Recent reports have shown that various P450 monooxygenases and UDP-glycosyltransferases in H. armigera are involved in gossypol detoxification, while the roles of ABC transporters, another gene family widely associated with metabolite detoxification, remain to be elucidated. Here, we show that ingestion of gossypol-infused artificial diet and cotton leaves significantly induced the expression of HaABCB6 in H. armigera larvae. Knockdown and knockout of HaABCB6 increased sensitivity of H. armigera larvae to gossypol. Moreover, HaABCB6-GFP fusion protein was localized on lysosomes in Hi5 cells and its overexpression significantly enhanced gossypol tolerance in vitro. These experimental results strongly support that HaABCB6 plays an important role in gossypol detoxification by H. armigera.

We characterize a novel picorna-like virus, named Helicoverpa armigera Nora virus (HaNV), with a genome length of 11,200 nts, the sequence of which was isolated from the lepidopteran host cotton bollworm Helicoverpa armigera, using RNA-Seq. Phylogenetic analysis, using the putative amino acid sequence of the conserved RNA-dependent RNA polymerase (RdRp) domain, indicated that HaNV clustered with Spodoptera exigua Nora virus, Drosophila Nora virus and Nasonia vitripennis virus-3 with a high bootstrap value (100%), which might indicate a new viral family within the order Picornavirales. HaNV was efficiently horizontally transmitted between hosts via contaminated food, and transmission was found to be dose-dependent (up to 100% efficiency with 109 viral copy number/µl). HaNV was also found to be transmitted vertically from parent to offspring, mainly through transovum transmission (virus contamination on the surface of the eggs), but having a lower transmission efficiency (around 43%). Infection distribution within the host was also investigated, with HaNV mainly found in only the gut of both adult moths and larvae (>90%). Moreover, our results showed that HaNV appears not to be an overtly pathogenic virus to its host.

When any living organism is frequently exposed to any drugs or toxic substances, they evolve different detoxification mechanism to confront with toxicants during absorption and metabolism. Likewise, the insects have evolved detoxification mechanisms as they are frequently exposed to different toxic secondary plant metabolites and commercial insecticides. ABC transporter superfamily is one of the largest and ubiquitous group of proteins which play an important role in phase III of the detoxification process. However, knowledge about this gene family remains largely unknown. To help fill this gap, we have identified a total of 54 ABC transporters in the Helicoverpa armigera genome which are classified into eight subfamilies (A-H) by phylogenetic analysis. The temporal and spatial expression profiles of these 54 ABC transporters throughout H. armigera development stages and seven tissues and their responses to five different insecticides, were investigated using RNA-seq analysis. Furthermore, the mRNA expression of eight selected genes in different tissues and six genes responses to insecticides were confirmed by the quantitative real-time PCR (RT-qPCR). Moreover, H. armigera become more sensitive to abamectin and indoxacarb when P-gp was inhibited. These results provide a foundation for further studies of ABCs in H. armigera.

To determine cellular changes caused by the chimeric protein Vip3AcAa against Helicoverpa armigera, we used transmission electron microscopy to examine ultrastructural changes in midgut cells of third-instar larvae of Cry1Ac-susceptible H. armigera after feeding on an artificial diet containing the Vip3AcAa toxin. Midgut epithelial cells of Cry1Ac-resistant H. armigera larvae that had fed on an artificial diet containing Vip3AcAa or on Bt cotton expressing Vip3AcAa+Cry1Ac were also examined using optical microscopy and hematoxylin–eosin staining. In the midgut cells of H. armigera larvae fed with Vip3AcAa, microvilli were swollen and broken; inner cristae of the mitochondria were indistinct and vacuolated; endoplasmic reticulum was swollen, fractured, and disordered; boundaries of karyotheca in the nucleus were indistinct and chromatin underwent pyknosis and was pressed close to the karyotheca. Histopathological changes and the time of onset in midgut tissues of H. armigera larvae fed on Vip3AcAa or Cry1Ac were similar. Vip3AcAa and transgenic cotton expressing Vip3AcAa+Cry1Ac caused the goblet cell cavity and microvilli pathological changes in the midgut epithelial cells of the Cry1Ac-susceptible and Cry1Ac-resistant H. armigera larvae that eventually killed the larvae.

Figure S1: Key volatile compounds emitted by non-Bt rice plants (from 20:00 pm to 4:00 am) that were damaged by Chilo suppressalis larvae for 72 h (A) or remained undamaged (B). IS = internal standard. The peak numbers of all compounds are consistent with those listed in Table 1.; Figure S2: Number of eggs laid by female Chilo suppressalis on undamaged or caterpillar-damaged Bt or non-Bt rice plants (greenhouse cage experiment). Caterpillar symbols indicate damage by two 3rd instar of C. suppressalis. Each choice test was performed with 15 to 20 replicates, each consisting of a group of 10 pairs of C. suppressalis adults. The number of egg masses represents the total egg masses laid on two rice plants. Asterisks indicate significant differences: *P < 0.05; ** P < 0.01; n.s. indicates no significance (P > 0.05) (paired-sample t-test).; Figure S3: Performance of Chilo suppressalis larvae feeding on rice plants that were either undamaged or damaged by 3rd instars of conspecifics. Two-day old larvae of C. suppressalis were individually placed on caterpillar-damaged or undamaged non-Bt rice plants for 7 days. Forty insects were tested in each treatment. Asterisks indicate significant differences between the undamaged and caterpillar-damaged treatments (P

Significance Changes in land use, climate, and agricultural technologies affect pest severity and management. We analyzed long-term longitudinal data (1991–2015) on three major cotton pests for 51 Chinese counties. Bacillus thuringiensis (Bt) insect-resistant cotton had pervasive effects on the whole pest complex and its management. Adoption resulted in major reductions in insecticide use against bollworm. The resulting restoration of aphid biocontrol decreased aphid severity. Mirid bug severity increased, aided by higher May temperatures and reduced insecticide sprays against bollworm. Landscape effects on pest severity varied between species. Farmers sprayed at nondamaging infestation levels. Findings support Bt-based plant resistance as a component of integrated pest management (IPM) but highlight the potential for unintended outcomes for the whole pest complex and the importance of climate change., Long-term changes in land use, climate, and agricultural technologies may affect pest severity and management. The influences of these major drivers can only be identified by analyzing long-term data. This study examines panel data on land use, adoption of genetically modified Bacillus thuringiensis (Bt) insect-resistant cotton, weather, pest severity, and insecticide use on three major cotton pests for 51 counties in China during 1991–2015. Bt cotton had pervasive effects on the whole pest complex in cotton and its management. Adoption resulted in major reductions in insecticide use for bollworm control. The resulting restoration of aphid biological control decreased aphid severity. However, mirid bugs, which have few effective natural enemies in cotton, increased in severity with warming May and reduced insecticide spraying against bollworm. The effects of landscape on pest severity were pest specific. The severity of cotton aphid and mirid bugs decreased with higher land use diversity, but the severity of highly polyphagous cotton bollworm was unrelated to land use diversity. Shares of forest, water body, and unused land area were negatively associated with the severity of mirid bugs, whereas cotton bollworm responded positively to the shares of water body and unused land area. Farmers sprayed insecticides at mild infestation levels and responded aggressively to severe bollworm outbreaks. Findings support the usefulness of Bt-based plant resistance as a component of integrated pest management (IPM) but highlight the potential for unexpected outcomes resulting from agro-ecosystem feedback loops as well as the importance of climate.

Insect migration provides major ecosystem services, and sometimes, migratory pests cause serious crop damage and yield loss. Species identification is critically important in studies of insect migration, for both entomologists and pest managers. Radar is an effective means of detecting insect migrants. Current entomological radars usually operate at X-band, and signal amplitude information is used to estimate body mass and wing-beat frequency, which can then be used to categorize migratory insects into broad taxon classes. To improve the identification performance, this paper presents a novel radar method of measuring insect mass and body length. The multifrequency radar cross sections (RCS) of insects at X-band and Ku-/K-band are fully investigated, and the comprehensive relationship between RCS and insect morphological parameters provides an improvement in the estimation of insect mass. More importantly, estimations of body length can also be realized with an accuracy of 84% based on experimental data acquired by a vector network analyzer in a microwave anechoic chamber. If multifrequency RCS measurements can be obtained by radar in the future, then highly accurate estimations of insect mass and body length will be possible, although it is currently still a challenge to build a radar capable of making the required measurements over such a wide frequency range.

Peristenus spretus Chen et van Achterberg (Hymenoptera: Braconidae), a parasitoid of the plant bug Apolygus lucorum (Hemiptera: Miridae), has been studied for use in augmentative biological control in China. Under laboratory conditions, we explored the development, survival, age-specific and potential lifetime fecundity, oviposition period and progeny sex ratio of P. spretus reared at six constant temperatures (15°C, 19°C, 23°C, 27°C, 31°C, 35°C) on the second instar nymphs of A. lucorum. At 15°C, male and female P. spretus took 48.7 ± 0.3 and 52.5 ± 0.3 days to complete their immature development, while developmental time was reduced by more than half at 23°C and 27°C. The parasitoid can only develop to the larval stage at 31°C and neither larva nor pupa survived at 35°C. The estimated lower developmental threshold of the immature stage was 7.3°C. When parasitoid adults were exposed at 15°C, females laid 90% of their eggs at first 19 days of oviposition and had an extended reproductive life. In contrast,...

Wide planting of transgenic Bt cotton in China since 1997 to control cotton bollworm (Helicoverpa armigera) has increased yields and decreased insecticide use, but the evolution of resistance to Bt cotton by H. armigera remains a challenge. Toward developing a new generation of insect-resistant transgenic crops, a chimeric protein of Vip3Aa1 and Vip3Ac1, named Vip3AcAa, having a broader insecticidal spectrum, was specifically created previously in our laboratory. In this study, we investigated cross resistance and interactions between Vip3AcAa and Cry1Ac with three H. armigera strains, one that is susceptible and two that are Cry1Ac-resistant, to determine if Vip3AcAa is a good candidate for development the pyramid cotton with Cry1Ac toxin. Our results showed that evolution of insect resistance to Cry1Ac toxin did not influence the sensitivity of Cry1Ac-resistant strains to Vip3AcAa. For the strains examined, observed mortality was equivalent to the expected mortality for all the combinations of Vip3AcAa and Cry1Ac tested, reflecting independent activity between these two toxins. When this chimeric vip3AcAa gene and the cry1Ac gene were introduced into cotton, mortality rates of Cry1Ac resistant H. armigera larvae strains that fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and cotton producing only Cry1Ac. These results suggest that the Vip3AcAa protein is an excellent option for a “pyramid” strategy for pest resistance management in China.

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

The cotton bollworm, Helicoverpa armigera, is one of the most important agricultural pests of many economic crops worldwide. Herein, we found a novel single-strand RNA virus by RNA-Seq and Polymerase Chain Reaction (PCR) method in H. armigera named Helicoverpa armigera iflavirus (HaIV), which possessed a genome with 10,017 nucleotides in length and contained a single large open reading frame (ORF) encoding a putative polyprotein of 3,021 amino acids with a predicted molecular mass of 344.16 kDa and a theoretical isoelectric point (pI) of 6.45. The deduced amino acid sequence showed highest similarity (61.0%) with the protein of Lymantria dispar Iflavirus 1. Phylogenetic analysis with putative RdRp amino acid sequences indicated that the virus clustered with members of the genus Iflavirus. The virus was mainly distributed in the fat body of its host and was found to be capable of both horizontal and vertical transmission. The efficiency of perorally horizontal transmission was dose dependent (100% infection rate with a viral dose of 108 copies /μl) while vertical transmission efficiency was found to be relatively low (< 28.57%). These results suggest that we have found a novel member of genus Iflavirus in H. armigera.

Several strategies involving refuge have been proposed for delaying insect resistance to Bt crops. The report was focused on the unstructured 'natural' refuges that contain plants naturally presented as part of the cropping system in the form of non-Bt plants that differ from Bt plant species, or wild host plants of the target pests. The cases of natural refuges applied in different countries were analyzed, and the factors that favor their success are discussed. The results indicate that the effectiveness of a natural refuge strategy depends on the biological characteristics of the target pest, the spatial and temporal distribution and abundance of the host plants in the agricultural system and the quality of the host plants for the pest species.

International audience; Recently, Apolygus lucorum (Hemiptera: Miridae) has become an important insect pest of Bt cotton and fruit trees in China. At present, the application of chemical insecticides is the main control option for this pest. We investigated the lethal effect of seven neonicotinoid insecticides on A. lucorum adults, including six commercialized neonicotinoids and a novel one, cycloxaprid, which is developed in China. In addition, we assessed the sublethal physiological (longevity, fecundity) and behavioral (preoviposition and oviposition periods) effects of LD10 (low lethal dose) and LD40 (moderate lethal dose) of cycloxaprid on the mirid bug. Lethal effect of the insecticides was determined using a topical exposure method; contact toxicity against the adults was ranked as follows: cycloxaprid, nitenpyram > clothianidin > acetamiprid, imidacloprid > thiamethoxam > dinotefuran. Among these insecticides, the toxicity of cycloxaprid against the adults was the highest; its LD50 value was 2.54 ng a.i .(active ingredient)/adult. Both LD10 (0.74 ng a. i./adult) and LD40 (1.98 ng a. i./ adult) of cycloxaprid induced sublethal effects in the adults; we observed a reduced longevity in males and the LD40 also shortened the longevity of females. In addition, the LD40 prolonged the pre-oviposition period and shortened the oviposition period of females, and it decreased their fecundity. The hatching rate of eggs laid by the females exposed to LD10 and LD40 greatly decreased (two-fold lower when exposed to LD40). Our results demonstrate that cycloxaprid is among the best candidate insecticides for controlling A. lucorum, and it showed significant lethal and sublethal effects on this mirid bug.

BACKGROUND Transgenic cotton that expresses a gene derived from the bacterium Bacillus thuringiensis (Bt) has been deployed for combating cotton bollworm in China since 1997. As a follow-up on research started in 2002, the quantitative shifts in larval Cry1Ac resistance of field Helicoverpa armigera populations were monitored from 2009–2013 using bioassays of isofemale lines. RESULTS A total of 2837 lines from Xiajin and 2055 lines from Anci were screened for growth rate on normal artificial diet and on a diet containing 1.0 µg mL−1 of Cry1A(c) toxin. In 2009–2013, the mean relative average development rates (RADRs) of H. armigera larvae in the Xiajin population were 0.62, 0.59, 0.59, 0.58 and 0.62 respectively, and in the Anci population 0.54, 0.58, 0.60, 0.53 and 0.62 respectively. CONCLUSIONS Compared with previous results in 2002, there was an increase in the RADR of H. armigera during 2009–2013, with ratios of 1.53–1.63 and 1.77–2.07 in the respective Xiajin and Anci populations, suggesting that resistance to Cry1Ac has increased in H. armigera populations in northern China. © 2014 Society of Chemical Industry

Evolution of resistance to insecticides usually has fitness tradeoffs associated with adaptation to the stress. The basic regulation mechanism of tradeoff between reproduction and resistance evolution to Bacillus thuringiensis (Bt) toxin in the cotton bollworm, Helicoverpa armigera (Ha), based on the vitellogenin (Vg) gene expression was analyzed here. The full-length cDNA of the Vg gene HaVg (JX504706) was cloned and identified. HaVg has 5704 base pairs (bp) with an open reading frame (ORF) of 5265 bp, which encoded 1756 amino acid protein with a predicted molecular mass of 197.28 kDa and a proposed isoelectric point of 8.74. Sequence alignment analysis indicated that the amino acid sequence of HaVg contained all of the conserved domains detected in the Vgs of the other insects and had a high similarity with the Vgs of the Lepidoptera insects, especially Noctuidae. The resistance level to Cry1Ac Bt toxin and relative HaVg mRNA expression levels among the following four groups: Cry1Ac-susceptible strain (96S), Cry1Ac-resistant strain fed on artificial diet with Bt toxin for 135 generations (BtR stands for the Cry1Ac Bt resistance), progeny of the Cry1Ac-resistant strain with a non-Bt-toxin artificial diet for 38 generations (CK1) and the direct descendants of the 135th-generation resistant larvae which were fed on an artificial diet without the Cry1Ac protein (CK2) were analyzed. Compared with the 96S strain, the resistance ratios of the BtR strain, the CK1 strain and the CK2 strain were 2917.15-, 2.15- and 2037.67-fold, respectively. The maximum relative HaVg mRNA expression levels of the BtR strain were approximately 50% less than that of the 96S strain, and the coming of maximum expression was delayed for approximately 4 days. The overall trend of the HaVg mRNA expression levels in the CK1 strain was similar to that in the 96S strain, and the overall trend of the HaVg mRNA expression levels in the CK2 strain was similar to that in the BtR strain. Our results suggest that the changes in reproduction due to the Bt-toxin resistance evolution in the BtR strain may be regulated by the Vg gene expression. The down-regulation of HaVg at the early stages resulted in a period of delayed reproduction and decreased fecundity in the BtR strain. This performance disappeared when the Bt-toxin selection pressure was lost.

Cotton is one of the most economically important crops in China, while insect pest damage is the major restriction factor for cotton production. The strategy of integrated pest management (IPM), in which bio- logical control plays an important role, has been widely applied. Nearly 500 species of natural enemies have been reported in cotton systems in China, but few species have been examined closely. Seventy- six species, belonging to 53 genera, of major arthropod predators and parasitoids of lepidoptera pests, and 46 species, belonging to 29 genera, of natural enemies of sucking pests have been described. In addi- tion, microsporidia, fungi, bacteria and viruses are also important natural enemies of cotton pests. Tricho- gramma spp., Microplitis mediator, Amblyseius cucumeris, Bacillus thuringiensis and Helicoverpa armigera nuclear polyhedrosis virus (HaNPV) have been mass reared or commercially produced and used in China. IPM strategies for cotton pests comprising of cultural, biological, physical and chemical controls have been developed and implemented in the Yellow River Region (YRR), Changjiang River Region (CRR) and Northwestern Region (NR) of China over the past several decades. In recent years, Bt cotton has been widely planted for selectively combating cotton bollworm, H. armigera, pink bollworm, Pectinophora gos- sypiella, and other lepidopteran pest species. As a result of reduced insecticide sprays, increased abun- dance of natural enemies in Bt cotton fields efficiently prevents outbreaks of other pests such as cotton aphids. In contrast, populations of mirid plant bugs have increased dramatically due to a reduction in the number of foliar insecticide applications for control of the bollworms in Bt cotton, and now pose a key problem in cotton production. In response to this new pest issue in cotton production, control strat- egies including biological control measures are being developed in China.

Peristenus spretus Chen & van Achterberg (Hymenoptera: Braconidae) is a solitary endoparasitoid, which is considered for augmentative biological control of Apolygus lucorum Meyer-Dur (Heteroptera: Miridae) in Chinese cotton fields. Since the association of P. spretus with A. lucorum was only recently discovered, the biology of the parasitoid remains unknown. In order to understand its reproductive biology, the mutual interference and functional response of P. spretus were investigated by altering either the parasitoid or the host density while keeping the other constant. In both experiments, the effects of parasitoid and host densities on parasitism, superparasitism, progeny production and sex ratio were assessed. P. spretus exhibited a Holling type II functional response to changing host densities, indicating that parasitism increases with increasing host density until the parasitoid reaches its maximum reproductive capacity. The model suggested that a single P. spretus female could parasitise a maximum ...

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 Hubner (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.

BACKGROUND Two transgenic lines of the soybean Glycine max, MON87701 expressing the Cry1Ac protein and MON87701RR2Y expressing Cry1Ac + EPSPS proteins, were evaluated for their resistance to four lepidopteran pests in the laboratory using detached-leaf bioassays throughout the soybean growth seasons (before anthesis, during anthesis and after anthesis) in China. Enzyme-linked immunosorbent assays (ELISAs) were used to monitor the Cry1Ac expression in soybean leaves. RESULTS The bioassay results revealed that both transgenic soybean lines exhibited significantly high resistance against Helicoverpa armigera (Hubner) throughout the soybean growing seasons. The survival rates of H. armigera larvae ranged from 5.4 to 24.4% when feeding on the transgenic soybean leaves, significantly lower than the survival rates when feeding on control leaves (71.1–94.9%). Limited resistance was found for both transgenic soybean lines against Spodoptera litura (Fabricius), although the survival rates and weight of S. litura larvae as well as female fecundity were significantly decreased when feeding on Bt soybean leaves compared with feeding on control leaves. In contrast, both transgenic soybean lines provided almost no resistance to Spodoptera exigua (Hubner) and Agrotis ypsilon (Rottemberg). Cry1Ac expression in the leaves of both transgenic soybean lines was relatively stable throughout the soybean growing season, with a peak occurring at V6–8 and V11–12 before anthesis. The ELISA results were positively correlated with the results from the insect bioassays. CONCLUSIONS The results show that, while Cry1Ac-expressing Bt soybeans may provide good protection against H. armigera, alternative control measures are required to manage S. exigua, S. litura and A. ypsilon. © 2013 Society of Chemical Industry