Retrotransposon-mediated disruption of a chitin synthase gene confers insect resistance to Bacillus thuringiensis Vip3Aa toxin.

The vegetative insecticidal protein Vip3Aa from Bacillus thuringiensis (Bt) has been produced by transgenic crops to counter pest resistance to the widely used crystalline (Cry) insecticidal proteins from Bt. To proactively manage pest resistance, there is an urgent need to better understand the genetic basis of resistance to Vip3Aa, which has been largely unknown. We discovered that retrotransposon-mediated alternative splicing of a midgut-specific chitin synthase gene was associated with 5,560-fold resistance to Vip3Aa in a laboratory-selected strain of the fall armyworm, a globally important crop pest. The same mutation in this gene was also detected in a field population. Knockout of this gene via CRISPR/Cas9 caused high levels of resistance to Vip3Aa in fall armyworm and 2 other lepidopteran pests. The insights provided by these results could help to advance monitoring and management of pest resistance to Vip3Aa.

Escalation by duplication: Milkweed bug trumps Monarch butterfly.

The iconic Monarch butterfly is probably the best-known example of chemical defence against predation, as pictures of vomiting naive blue jays in countless textbooks vividly illustrate. Larvae of the butterfly take up toxic cardiac glycosides from their milkweed hostplants and carry them over to the adult stage. These compounds (cardiotonic steroids, including cardenolides and bufadienolides) inhibit the animal transmembrane sodium-potassium ATPase (Na,K-ATPase), but the Monarch enzyme resists this inhibition thanks to amino acid substitutions in its catalytic alpha-subunit. Some birds also have substitutions and can feast on cardiac glycoside-sequestering insects with impunity. A flurry of recent work has shown how the alpha-subunit gene has been duplicated multiple times in separate insect lineages specializing in cardiac glycoside-producing plants. In this issue of Molecular Ecology, Herbertz et al. toss the beta-subunit into the mix, by expressing all nine combinations of three alpha- and three beta-subunits of the milkweed bug Na,K-ATPase and testing their response to a cardenolide from the hostplant. The findings suggest that the diversification and subfunctionalization of genes allow milkweed bugs to balance trade-offs between resistance towards sequestered host plant toxins that protect the bugs from predators, and physiological costs in terms of Na,K-ATPase activity.

Peritrophins are involved in the defense against Bacillus thuringiensis and nucleopolyhedrovirus formulations in Spodoptera littoralis (Lepidoptera: Noctuidae).

The peritrophic matrix (or peritrophic membrane, PM) is present in most insects where it acts as a barrier to mechanical insults and pathogens, as well as a facilitator of digestive processes. The PM is formed by the binding of structural PM proteins, referred to as peritrophins, to chitin fibrils and spans the entire midgut in lepidopterans. To investigate the role of peritrophins in a highly polyphagous lepidopteran pest, namely the cotton leafworm (Spodoptera littoralis), we generated Insect Intestinal Mucin (IIM-) and non-mucin Peritrophin (PER-) mutant strains via CRISPR/Cas9 mutagenesis. Both strains exhibited deformed PMs and retarded developmental rates. Bioassays conducted with Bacillus thuringiensis (Bt) and nucleopolyhedrovirus (SpliNPV) formulations showed that both the IIM- and PER- mutant larvae were more susceptible to these bioinsecticides compared to the wild-type (WT) larvae with intact PM. Interestingly, the provision of chitin-binding agent Calcofluor (CF) in the diet lowered the toxicity of Bt formulations in both WT and IIM- larvae and the protective effect of CF was significantly lower in PER- larvae. This suggested that the interaction of CF with PER is responsible for Bt resistance mediated by CF. In contrast, the provision of CF caused increased susceptibility to SpliNPV in both mutants and WT larvae. The study showed the importance of peritrophins in the defense against pathogens in S. littoralis and revealed novel insights into CF-mediated resistance to Cry toxin.

Lipases and carboxylesterases affect moth sex pheromone compounds involved in interspecific mate recognition.

Moth sex pheromones are a classical model for studying sexual selection. Females typically produce a species-specific pheromone blend that attracts males. Revealing the enzymes involved in the interspecific variation in blend composition is key for understanding the evolution of these sexual communication systems. The nature of the enzymes involved in the variation of acetate esters, which are prominent compounds in moth pheromone blends, remains unclear. We identify enzymes involved in acetate degradation using two closely related moth species: Heliothis (Chloridea) subflexa and H. (C.) virescens, which have different quantities of acetate esters in their sex pheromone. Through comparative transcriptomic analyses and CRISPR/Cas9 knockouts, we show that two lipases and two esterases from H. virescens reduce the levels of pheromone acetate esters when expressed in H. subflexa females. Together, our results show that lipases and carboxylesterases are involved in tuning Lepidoptera pheromones composition.

Downregulation of a transcription factor associated with resistance to Bt toxin Vip3Aa in the invasive fall armyworm.

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) have revolutionized control of some major pests. However, more than 25 cases of field-evolved practical resistance have reduced the efficacy of transgenic crops producing crystalline (Cry) Bt proteins, spurring adoption of alternatives including crops producing the Bt vegetative insecticidal protein Vip3Aa. Although practical resistance to Vip3Aa has not been reported yet, better understanding of the genetic basis of resistance to Vip3Aa is urgently needed to proactively monitor, delay, and counter pest resistance. This is especially important for fall armyworm (Spodoptera frugiperda), which has evolved practical resistance to Cry proteins and is one of the world's most damaging pests. Here, we report the identification of an association between downregulation of the transcription factor gene SfMyb and resistance to Vip3Aa in S. frugiperda. Results from a genome-wide association study, fine-scale mapping, and RNA-Seq identified this gene as a compelling candidate for contributing to the 206-fold resistance to Vip3Aa in a laboratory-selected strain. Experimental reduction of SfMyb expression in a susceptible strain using RNA interference (RNAi) or CRISPR/Cas9 gene editing decreased susceptibility to Vip3Aa, confirming that reduced expression of this gene can cause resistance to Vip3Aa. Relative to the wild-type promoter for SfMyb, the promoter in the resistant strain has deletions and lower activity. Data from yeast one-hybrid assays, genomics, RNA-Seq, RNAi, and proteomics identified genes that are strong candidates for mediating the effects of SfMyb on Vip3Aa resistance. The results reported here may facilitate progress in understanding and managing pest resistance to Vip3Aa.

8-HQA adjusts the number and diversity of bacteria in the gut microbiome of Spodoptera littoralis.

Quinolinic carboxylic acids are known for their metal ion chelating properties in insects, plants and bacteria. The larval stages of the lepidopteran pest, Spodoptera littoralis, produce 8-hydroxyquinoline-2-carboxylic acid (8-HQA) in high concentrations from tryptophan in the diet. At the same time, the larval midgut is known to harbor a bacterial population. The motivation behind the work was to investigate whether 8-HQA is controlling the bacterial community in the gut by regulating the concentration of metal ions. Knocking out the gene for kynurenine 3-monooxygenase (KMO) in the insect using CRISPR/Cas9 eliminated production of 8-HQA and significantly increased bacterial numbers and diversity in the larval midgut. Adding 8-HQA to the diet of knockout larvae caused a dose-dependent reduction of bacterial numbers with minimal effects on diversity. Enterococcus mundtii dominates the community in all treatments, probably due to its highly efficient iron uptake system and production of the colicin, mundticin. Thus host factors and bacterial properties interact to determine patterns of diversity and abundance in the insect midgut.

Towards evolutionary predictions: Current promises and challenges.

Evolution has traditionally been a historical and descriptive science, and predicting future evolutionary processes has long been considered impossible. However, evolutionary predictions are increasingly being developed and used in medicine, agriculture, biotechnology and conservation biology. Evolutionary predictions may be used for different purposes, such as to prepare for the future, to try and change the course of evolution or to determine how well we understand evolutionary processes. Similarly, the exact aspect of the evolved population that we want to predict may also differ. For example, we could try to predict which genotype will dominate, the fitness of the population or the extinction probability of a population. In addition, there are many uses of evolutionary predictions that may not always be recognized as such. The main goal of this review is to increase awareness of methods and data in different research fields by showing the breadth of situations in which evolutionary predictions are made. We describe how diverse evolutionary predictions share a common structure described by the predictive scope, time scale and precision. Then, by using examples ranging from SARS-CoV2 and influenza to CRISPR-based gene drives and sustainable product formation in biotechnology, we discuss the methods for predicting evolution, the factors that affect predictability and how predictions can be used to prevent evolution in undesirable directions or to promote beneficial evolution (i.e. evolutionary control). We hope that this review will stimulate collaboration between fields by establishing a common language for evolutionary predictions.

Pesticide resistance in arthropods: Ecology matters too.

Pesticide resistance development is an example of rapid contemporary evolution that poses immense challenges for agriculture. It typically evolves due to the strong directional selection that pesticide treatments exert on herbivorous arthropods. However, recent research suggests that some species are more prone to evolve pesticide resistance than others due to their evolutionary history and standing genetic variation. Generalist species might develop pesticide resistance especially rapidly due to pre-adaptation to handle a wide array of plant allelochemicals. Moreover, research has shown that adaptation to novel host plants could lead to increased pesticide resistance. Exploring such cross-resistance between host plant range evolution and pesticide resistance development from an ecological perspective is needed to understand its causes and consequences better. Much research has, however, been devoted to the molecular mechanisms underlying pesticide resistance while both the ecological contexts that could facilitate resistance evolution and the ecological consequences of cross-resistance have been under-studied. Here, we take an eco-evolutionary approach and discuss circumstances that may facilitate cross-resistance in arthropods and the consequences cross-resistance may have for plant-arthropod interactions in both target and non-target species and species interactions. Furthermore, we suggest future research avenues and practical implications of an increased ecological understanding of pesticide resistance evolution.

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.

Perspectives on gene copy number variation and pesticide resistance.

Although the generation of evolutionary diversity by gene duplication has long been known, the implications for pesticide resistance are just now beginning to be appreciated. A few examples will be cited to illustrate the point that there are many variations on the theme that gene duplication does not follow a set pattern. Transposable elements may facilitate the process but the mechanistic details are obscure and unpredictable. New developments in DNA sequencing technology and genome assembly promise to reveal more examples, yet care must be taken in interpreting the results of transcriptome and genome assemblies and independent means of validation are important. Once a specific gene family is identified, special methods generally must be used to avoid underestimating population polymorphisms and being trapped in preconceptions about the simplicity of the process. © 2021 The Author. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Development of a LAMP-Based Molecular Species Diagnosis Method for Four Major Agricultural Pests in the Genus Spodoptera (Lepidoptera: Noctuidae).

Molecular-based species identification tools are helpful to identify tiny insect and lepidopteran pests that show morphological similarities in the larval stage and are essential for quarantine as well as agricultural research. Here, we focused on four major Spodoptera pests: S. exigua, S. frugiperda, S. litura, and S. littoralis. S. exigua and S. litura mitochondrial genome sequences were newly identified and species-specific sequence regions were identified in the cytochrome c oxidase subunit II and III regions. Species primers were designed and applied in loop-mediated isothermal amplification (LAMP) and PCR to identify Korean field-collected or overseas samples. The optimal incubation conditions for LAMP were 61 °C for 60 min with four LAMP primers. Additional loop primers increased the amplification efficiency for S. exigua, and the nonspecific amplification for other species. The LAMP assay could detect a wide range of DNA concentrations, with the range 1 ng-1 pg in dependence of four LAMP primers. The DNA-releasing technique, without DNA extraction, in the LAMP assay involved larval or adult tissue sample incubation at 95 °C for 5 min. The entire process takes approximately 70 min. This new molecular diagnostic method is simple and accurate, with application in the field and laboratory and for monitoring and ecological studies.

Consumption of gossypol increases fatty acid-amino acid conjugates in the cotton pests Helicoverpa armigera and Heliothis virescens.

Gossypol is a toxic sesquiterpene dimer produced by cotton plants which deters herbivory by insects and vertebrates. Two highly reactive aldehyde groups contribute to gossypol toxicity by cross-linking herbivore proteins. We identified another consequence of consuming gossypol in two insect pests of cotton: increased amounts of fatty acid-amino acid conjugates (FACs). Eight different FACs in the feces of larval Helicoverpa armigera and Heliothis virescens increased when larvae consumed artificial diet containing gossypol, but not a gossypol derivative lacking free aldehyde groups (SB-gossypol). FACs are produced by joining plant-derived fatty acids with amino acids of insect origin in the larval midgut tissue by an unknown conjugase, and translocated into the gut lumen by an unknown transporter. FACs are hydrolyzed back into fatty acids and amino acids by an aminoacylase (L-ACY-1) in the gut lumen. The equilibrium level of FACs in the lumen is determined by a balance between conjugation and hydrolysis, which may differ among species. When heterologously expressed, L-ACY-1 of H. armigera but not H. virescens was inhibited by gossypol; consistent with the excretion of more FACs in the feces by H. armigera. FACs are known to benefit the plant host by inducing anti-herbivore defensive responses, and have been hypothesized to benefit the herbivore by acting as a surfactant and increasing nitrogen uptake efficiency. Thus in addition to its direct toxic effects, gossypol may negatively impact insect nitrogen uptake efficiency and amplify the signal used by the plant to elicit release of volatile compounds that attract parasitoids.

Silencing of an ABC transporter, but not a cadherin, decreases the susceptibility of Colorado potato beetle larvae to Bacillus thuringiensis ssp. tenebrionis Cry3Aa toxin.

The Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), is a major pest of potato plants worldwide and is notorious for its ability to develop resistance to insecticides. Cry3 toxins synthesized by Bacillus thuringiensis ssp. tenebrionis have been used successfully to manage this pest. Resistance to Cry toxins is a concerning problem for many insect pests; therefore, it is important to determine the mechanisms by which insects acquire resistance to these toxins. Cadherin-like and ABC transporter proteins have been implicated in the mode of action of Cry toxins as mutations in these genes render lepidopterans resistant to them; however, clear consensus does not exist on whether these proteins also play a role in Cry3 toxin activity and/or development of resistance in coleopterans. In the current study, we identified the L. decemlineata orthologues of the cadherin (LdCAD) and the ABCB transporter (LdABCB1) that have been implicated in the mode of action of Cry toxins in other coleopterans. Suppression of LdABCB1 via RNA interference reduced toxin-related larval mortality, whereas partial silencing of LdCAD did not. Our results suggest that the ABCB is involved in the mode of action of Cry3Aa toxins; however, no evidence was found to support the role of cadherin as a receptor of Cry3Aa in L. decemlineata.

A Generalist Feeding on Brassicaceae: It Does Not Get Any Better with Selection.

Brassicaceae (Cruciferae) are ostensibly defended in part against generalist insect herbivores by toxic isothiocyanates formed when protoxic glucosinolates are hydrolysed. Based on an analysis of published host records, feeding on Brassicas is widespread by both specialist and generalists in the Lepidoptera. The polyphagous noctuid moth Helicoverpa armigera is recorded as a pest on some Brassicas and we attempted to improve performance by artificial selection to, in part, determine if this contributes to pest status. Assays on cabbage and kale versus an artificial diet showed no difference in larval growth rate, development times and pupal weights between the parental and the selected strain after 2, 21 and 29 rounds of selection, nor in behaviour assays after 50 generations. There were large differences between the two Brassicas: performance was better on kale than cabbage, although both were comparable to records for other crop hosts, on which the species is a major pest. We discuss what determines "pest" status.

So Much for Glucosinolates: A Generalist Does Survive and Develop on Brassicas, but at What Cost?

While plants produce complex cocktails of chemical defences with different targets and efficacies, the biochemical effects of phytotoxin ingestion are often poorly understood. Here, we examine the physiological and metabolic effects of the ingestion of glucosinolates (GSLs), the frontline chemical defenses of brassicas (crucifers), on the generalist herbivore Helicoverpa armigera. We focus on kale and cabbage, two crops with similar foliar GSL concentrations but strikingly different GSL compositions. We observed that larval growth and development were well correlated with the nutritional properties of the insect diets, with low protein contents appearing to exacerbate the negative effects of GSLs on growth, pupation and adult eclosion, parameters that were all delayed upon exposure to GSLs. The different GSLs were metabolized similarly by the insect, indicating that the costs of detoxification via conjugation to glutathione (GSH) were similar on the two plant diets. Nevertheless, larval GSH contents, as well as some major nutritional markers (larval protein, free amino acids, and fat), were differentially affected by the different GSL profiles in the two crops. Therefore, the interplay between GSL and the nitrogen/sulfur nutritional availability of different brassicas strongly influences the effectiveness of these chemical defenses against this generalist herbivore.

Genomic analysis of novel Yarrowia-like yeast symbionts associated with the carrion-feeding burying beetle Nicrophorus vespilloides.

BACKGROUND: Mutualistic interactions with microbes can help insects adapt to extreme environments and unusual diets. An intriguing example is the burying beetle Nicrophorus vespilloides, which feeds and reproduces on small vertebrate carcasses. Its fungal microbiome is dominated by yeasts that potentially facilitate carcass utilization by producing digestive enzymes, eliminating cadaver-associated toxic volatiles (that would otherwise attract competitors), and releasing antimicrobials to sanitize the microenvironment. Some of these yeasts are closely related to the biotechnologically important species Yarrowia lipolytica. RESULTS: To investigate the roles of these Yarrowia-like yeast (YLY) strains in more detail, we selected five strains from two different phylogenetic clades for third-generation sequencing and genome analysis. The first clade, represented by strain B02, has a 20-Mb genome containing ~ 6400 predicted protein-coding genes. The second clade, represented by strain C11, has a 25-Mb genome containing ~ 6300 predicted protein-coding genes, and extensive intraspecific variability within the ITS-D1/D2 rDNA region commonly used for species assignments. Phenotypic microarray analysis revealed that both YLY strains were able to utilize a diverse range of carbon and nitrogen sources (including microbial metabolites associated with putrefaction), and can grow in environments with extreme pH and salt concentrations. CONCLUSIONS: The genomic characterization of five yeast strains isolated from N. vespilloides resulted in the identification of strains potentially representing new YLY species. Given their abundance in the beetle hindgut, and dominant growth on beetle-prepared carcasses, the analysis of these strains has revealed the genetic basis of a potential symbiotic relationship between yeasts and burying beetles that facilitates carcass digestion and preservation.

bric à brac controls sex pheromone choice by male European corn borer moths.

The sex pheromone system of ~160,000 moth species acts as a powerful form of assortative mating whereby females attract conspecific males with a species-specific blend of volatile compounds. Understanding how female pheromone production and male preference coevolve to produce this diversity requires knowledge of the genes underlying change in both traits. In the European corn borer moth, pheromone blend variation is controlled by two alleles of an autosomal fatty-acyl reductase gene expressed in the female pheromone gland (pgFAR). Here we show that asymmetric male preference is controlled by cis-acting variation in a sex-linked transcription factor expressed in the developing male antenna, bric à brac (bab). A genome-wide association study of preference using pheromone-trapped males implicates variation in the 293 kb bab intron 1, rather than the coding sequence. Linkage disequilibrium between bab intron 1 and pgFAR further validates bab as the preference locus, and demonstrates that the two genes interact to contribute to assortative mating. Thus, lack of physical linkage is not a constraint for coevolutionary divergence of female pheromone production and male behavioral response genes, in contrast to what is often predicted by evolutionary theory.

The Essential and Enigmatic Role of ABC Transporters in Bt Resistance of Noctuids and Other Insect Pests of Agriculture.

In the last ten years, ABC transporters have emerged as unexpected yet significant contributors to pest resistance to insecticidal pore-forming proteins from Bacillus thuringiensis (Bt). Evidence includes the presence of mutations in resistant insects, heterologous expression to probe interactions with the three-domain Cry toxins, and CRISPR/Cas9 knockouts. Yet the mechanisms by which ABC transporters facilitate pore formation remain obscure. The three major classes of Cry toxins used in agriculture have been found to target the three major classes of ABC transporters, which requires a mechanistic explanation. Many other families of bacterial pore-forming toxins exhibit conformational changes in their mode of action, which are not yet described for the Cry toxins. Three-dimensional structures of the relevant ABC transporters, the multimeric pore in the membrane, and other proteins that assist in the process are required to test the hypothesis that the ATP-switch mechanism provides a motive force that drives Cry toxins into the membrane. Knowledge of the mechanism of pore insertion will be required to combat the resistance that is now evolving in field populations of insects, including noctuids.

Saltational evolution of a pesticide-metabolizing cytochrome P450 in a global crop pest.

BACKGROUND: The cotton bollworm, Helicoverpa armigera (Hübner), is a damaging insect pest threatening agricultural crops worldwide as a result of its resistance to insecticides. Metabolic resistance to pyrethroid insecticides is conferred by the chimeric P450 enzyme CYP337B3, produced by unequal crossing-over between CYP337B1 and CYP337B2. CYP337B3 is 99.7% similar to CYP337B1 except for the 177 N-terminal amino acids (AAs) containing the substrate recognition site 1 from CYP337B2. Here, we studied the structure-function relationship of CYP337B3 and CYP337B1 to determine the AAs that enable CYP337B3 to efficiently hydroxylate the 4'-carbon position of fenvalerate, which neither CYP337B1 nor CYP337B2 can do. RESULTS: Site-directed mutagenesis showed that the L114F substitution in CYP337B3 reduced its 4'-hydroxylation activity by 89%, but the reciprocal F114L substitution in CYP337B1 increased its 4'-hydroxylation activity to only 49% of the level of CYP337B3. Docking models showed that AA 114 seems to have different functions in CYP337B1 and CYP337B3. Antibodies detected two- to three-fold more CYP337B1 than CYP337B3 in larval cuticle, which along with a 49% 4'-hydroxylation activity increase due to a F114L substitution in vivo might be expected to provide as much protection for the larva against exposure to fenvalerate as CYP337B3. However, CYP337B3 is present at much higher frequencies than CYP337B1-CYP337B2 in most populations, including those recently invading South America. CONCLUSION: The metabolic resistance to pyrethroids in H. armigera has evolved by saltational evolution - by a single mutation, an unequal crossing-over, producing a larger selective advantage than could be attained gradually by stepwise improvement of the parental enzyme. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Development of a simple and accurate molecular tool for Spodoptera frugiperda species identification using LAMP.

BACKGROUND: The fall armyworm, Spodoptera frugiperda is a native species of the Americas. First detected in western and central Africa in early 2016, it has become one of the most serious invasive lepidopteran pests in many African and Asian countries. S. frugiperda has spread very quickly; however, there are no molecular-based, simple and accurate diagnostic tools for identification of this species in the field. Methods to identify invasive S. frugiperda are urgently needed because farmers and agricultural managers have no prior experience with this pest. RESULTS: Based on mitochondrial genome sequence alignment, a S. frugiperda-specific sequence region was identified in the transfer RNA-coding region between NADH dehydrogenase, ND3, and ND5. Using this unique region, species-diagnostic primers were designed and applied in a loop-mediated isothermal amplification (LAMP) assay and a conventional polymerase chain reaction to identify field-collected samples of S. frugiperda. The optimal incubation conditions for the LAMP assay were 61°C for 90 min with four LAMP primers; an additional loop primer increased the amplification efficiency. A response was obtained for a wide range of DNA concentrations in the LAMP assay and the minimum detectable DNA concentration was 10 pg. CONCLUSIONS: We developed a new LAMP-based molecular diagnostic method that it is easy to use and accurate. The LAMP assay was used with a DNA-releasing technique for larval and adult samples, without a DNA extraction step, by incubating the tissue sample at 95°C for 5 min. This method can be applied in intensive field monitoring of S. frugiperda and its ecological studies. © 2021 Society of Chemical Industry.