Genetic variation in the triosephosphate isomerase gene of the fall armyworm and its distribution across China.

The fall armyworm (FAW), Spodoptera frugiperda, has colonized and caused consistent damage in the Eastern hemisphere. The identification of various FAW strains is essential for developing precise prevention and control measures. The triosephosphate isomerase (Tpi) gene is recognized as an effective marker closely linked to FAW subpopulations. However, most current studies primarily focus on the comparison of variations in specific gene sites of this gene. In this study, we conducted full-length sequencing of the Tpi genes from 5 representative FAW groups. Our findings revealed that the Tpi genes varied in length from 1220 to 1420 bp, with the primary variation occurring within 4 introns. Notably, the exon lengths remained consistent, at 747 bp, with 37 observed base variations; however, no amino acid variations were detected. Through sequence alignment, we identified 8 stable variation sites that can be used to distinguish FAW strains in the Eastern hemisphere. Additionally, we performed strain identification on 1569 FAW samples collected from 19 provinces in China between 2020 and 2021. The extensive analysis indicated the absence of the rice strain in the samples. Instead, we only detected the presence of the corn strain and the Zambia strain, with the Zambia strain being distributed in a very low proportion (3.44%). Furthermore, the corn strain could be further categorized into 2 subgroups. This comprehensive study provides a valuable reference for enhancing our understanding of FAW population differentiation and for improving monitoring and early warning efforts.

Rapid test to detect insecticide resistance in field populations of Spodoptera frugiperda (Lepidoptera: Noctuidae).

Introduction: The development of insecticide resistance in Spodoptera frugiperda populations is a serious threat to the crop industry. Given the spread of invasive resistant populations, prospective monitoring should be accelerated, and the development of diagnostic tools for rapid and accurate assessments of insecticide resistance is essential. Methods: First, the discriminating dose and diagnostic time of the kit were determined by the glass vial method based on a susceptible strain. Then, pests that were collected from field populations were used to determine their susceptibility to seven insecticides by using the diagnostic kit. Finally, the accuracy of the kit was verified based on correlation analyses and the likelihood of insecticide control failure was assessed. Results: Here, we describe a diagnostic kit that enables the rapid detection of resistance to chlorpyrifos, bifenthrin, deltamethrin, lambda-cyhalothrin, phoxim, chlorantraniliprole and chlorfenapyr within 1-2 h in S. frugiperda at diagnostic doses of 0.98, 0.84, 0.38, 1.64, 0.0082, 1.75 and 0.65 µg/cm2, respectively. The linear equation between mortalities under diagnostic doses and actual resistance ratios measured by the diet-overlay bioassay was determined. The high correlation indicates that the insecticide resistance levels diagnosed by the kit were consistent with the results of the diet-overlay bioassay. Moreover, we found a significant negative correlation between diagnostic mortality and the likelihood of control failure for bifenthrin (r = -0.899, p = 0.001), deltamethrin (r = -0.737, p = 0.024) and lambda-cyhalothrin (r = -0.871, p = 0.002). Discussion: The insecticide resistance diagnostic kit for S. frugiperda is a user-friendly tool (portable, short detection time). Its excellent performance qualifies the kit as a reliable screening tool for identifying effective insecticides in sustainable resistance management.

Chromosomal deletions mediated by CRISPR/Cas9 in Helicoverpa armigera.

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 terpene synthase gene family in Gossypium hirsutum harbors a linalool synthase GhTPS12 implicated in direct defence responses against herbivores.

Herbivore-induced terpenes have been reported to function as ecological signals in plant-insect interactions. Here, we showed that insect-induced cotton volatile blends contained 16 terpenoid compounds with a relatively high level of linalool. The high diversity of terpene production is derived from a large terpene synthase (TPS) gene family. The TPS gene family of Gossypium hirsutum and Gossypium raimondii consist of 46 and 41 members, respectively. Twelve TPS genes (GhTPS4-15) could be isolated, and protein expression in Escherichia coli revealed catalytic activity for eight GhTPS. The upregulation of the majority of these eight genes additionally supports the function of these genes in herbivore-induced volatile biosynthesis. Furthermore, transgenic Nicotiana tabacum plants overexpressing GhTPS12 were generated, which produced relatively large amounts of (3S)-linalool. In choice tests, female adults of Helicoverpa armigera laid fewer eggs on transgenic plants compared with non-transformed controls. Meanwhile, Myzus persicae preferred feeding on wild-type leaves over leaves of transgenic plants. Our findings demonstrate that transcript accumulation of multiple TPS genes is mainly responsible for the production and diversity of herbivore-induced volatile terpenes in cotton. Also, these genes might play roles in plant defence, in particular, direct defence responses against herbivores.

Transgenic cotton co-expressing chimeric Vip3AcAa and Cry1Ac confers effective protection against Cry1Ac-resistant cotton bollworm.

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.

Transgenic cotton coexpressing Vip3A and Cry1Ac has a broad insecticidal spectrum against lepidopteran pests.

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.

Dynamic transcriptome analysis and volatile profiling of Gossypium hirsutum in response to the cotton bollworm Helicoverpa armigera.

In response to insect herbivory, plants emit elevated levels of volatile organic compounds for direct and indirect resistance. However, little is known about the molecular and genomic basis of defense response that insect herbivory trigger in cotton plants and how defense mechanisms are orchestrated in the context of other biological processes. Here we monitored the transcriptome changes and volatile characteristics of cotton plants in response to cotton bollworm (CBW; Helicoverpa armigera) larvae infestation. Analysis of samples revealed that 1,969 transcripts were differentially expressed (log2|Ratio| ≥ 2; q ≤ 0.05) after CBW infestation. Cluster analysis identified several distinct temporal patterns of transcriptome changes. Among CBW-induced genes, those associated with indirect defense and jasmonic acid pathway were clearly over-represented, indicating that these genes play important roles in CBW-induced defenses. The gas chromatography-mass spectrometry (GC-MS) analyses revealed that CBW infestation could induce cotton plants to release volatile compounds comprised lipoxygenase-derived green leaf volatiles and a number of terpenoid volatiles. Responding to CBW larvae infestation, cotton plants undergo drastic reprogramming of the transcriptome and the volatile profile. The present results increase our knowledge about insect herbivory-induced metabolic and biochemical processes in plants, which may help improve future studies on genes governing processes.