Major Agricultural Pest Research Articles

The Insecticidal Efficacy and Physiological Action Mechanism of a Novel Agent GC16 against Tetranychus pueraricola (Acari: Tetranychidae).

Simple SummarySpider mite is major pest in agriculture and have developed resistance to commonly used pesticides. Therefore, it is urgent to discover new pesticides to control the pest. In order to provide alternatives for its management, we evaluated the effectiveness of a new agent GC16 against the spider mite Tetranychus pueraricola. Then, we preliminarily revealed the its acaricidal mechanism of action based on the damage of cuticle and organelles of mites. We confirmed that GC16 has a good controlling effect on T. pueraricola and it is not harmful to Picromerus lewisi and Harmonia axyridis. Our research provides not only an alternative pesticide for the management of spider mites, but also guidance for the application of GC16 in sustainable agriculture.Chemical control plays a crucial role in pest management but has to face challenges due to insect resistance. It is important to discover alternatives to traditional pesticides. The spider mite Tetranychus pueraricola (Ehara & Gotoh) (Acari: Tetranychidae) is a major agricultural pest that causes severe damage to many crops. GC16 is a new agent that consists of a mixture of Calcium chloride (CaCl2) and lecithin. To explore the acaricidal effects and mode of action of GC16 against T. pueraricola, bioassays, cryogenic scanning electron microscopy (cryo-SEM) and transmission electron microscopy (TEM) were performed. GC16 had lethal effects on the eggs, larvae, nymphs, and adults of T. pueraricola, caused the mites to dehydrate and inactivate, and inhibited the development of eggs. GC16 displayed contact toxicity rather than stomach toxicity through the synergistic effects of CaCl2 with lecithin. Cryo-SEM analysis revealed that GC16 damaged T. pueraricola by disordering the array of the cuticle layer crest. Mitochondrial abnormalities were detected by TEM in mites treated by GC16. Overall, GC16 had the controlling efficacy on T. pueraricola by cuticle penetration and mitochondria dysfunction and had no effects on Picromerus lewisi and Harmonia axyridis, indicating that GC16 is likely a new eco-friendly acaricide.

Modelling the emergence dynamics of the western corn rootworm beetle (Diabrotica virgifera virgifera)

The western corn rootworm is an invasive species to Europe and is a major agricultural pest that causes widespread economic and yield losses to maize producers. The Gompertz curve was originally used to model human population mortality. It is a sigmoidal curve where the beginning and end of a period shows the slowest time for growth, and adequately describes observed dynamics of many phenomena. We propose the use of the Gompertz function in a Bayesian Hierarchical framework to model the emergence dynamics of the western corn rootworm beetle. The proposed model includes the use of climatic variables to assess how weather can influence the observed dynamics. We apply the model to Austrian monitoring data collected in 2004–2015.

Identification of Helicoverpa armigera promoters for biotechnological applications

Helicoverpa armigera and Helicoverpa zea are highly polyphagous major agricultural pests with a global distribution. Their control is based on insecticides, however, new, effective, and environmentally friendly control tools are required to be developed and validated. In an effort to facilitate the development of advanced biotechnological tools in these species that will take advantage of new powerful molecular biology techniques like CRISPR/Cas9, we used available transcriptomic data and literature resources, in order to identify RNA polymerase II and III promoters active in RP-HzGUT-AW1(MG), a midgut derived cell line from Helicoverpa zea. Following functional analysis in insect cell lines, four RNA polymerase II promoters from the genes HaLabial, HaTsp-2A, HaPtx-I and HaCaudal were found to exhibit high transcriptional activity in vitro. The HaTsp-2A promoter did not exhibit any activity in the non-midgut derived cell lines Sf-9 and Hi-5 despite high sequence conservation among Lepidoptera, suggesting that it may function in a gut specific manner. Furthermore, considering the utility of RNA polymerase III U6 promoters in methodologies such as RNAi and CRISPR/Cas9, we identified and evaluated four different U6 promoters of H. armigera. In vitro experiments based on luciferase and GFP reporter assays, as well as in vivo experiments targeting an essential gene of Helicoverpa, indicate that these U6 promoters are functional and can be used to experimentally silence or knockout target genes through the expression of shRNAs and sgRNAs respectively. Taking our findings together, we provide a set of promoters useful for the genetic manipulation of Helicoverpa species, that can be used in various applications in the context of agricultural biotechnology.

Four MicroRNAs, miR-13b-3p, miR-278-5p, miR-10483-5p, and miR-10485-5p, Mediate Insecticide Tolerance in Spodoptera frugiperda.

Spodoptera frugiperda is the world’s major agricultural pest and has the distinctive features of high fecundity, strong migratory capacity, and high resistance to most insecticides. At present, the control of S. frugiperda in China relies mainly on the spraying of chemical insecticides. MicroRNAs (miRNAs) are a class of small, single-stranded, non-coding RNAs and play crucial regulatory roles in various physiological processes, including the insecticide resistance in insects. However, little is known about the regulatory roles of miRNAs on the resistance of S. frugiperda to insecticides. In the present research, the miRNAs that were differentially expressed after cyantraniliprole, spinetoram, and emamectin benzoate treatment were analyzed by RNA-Seq. A total of 504 miRNAs were systematically identified from S. frugiperda, and 24, 22, and 31 miRNAs were differentially expressed after treatments of cyantraniliprole, spinetoram, and emamectin benzoate. GO and KEGG enrichment analyses were used to predict the function of differentially expressed target genes of miRNAs. Importantly, ten miRNAs were significantly differentially expressed among the treatments of three insecticides. miR-278-5p, miR-13b-3p, miR-10485-5p, and miR-10483-5p were significantly downregulated among the treatments of three insecticides by RT-qPCR. Furthermore, the overexpression of miR-278-5p, miR-13b-3p, miR-10485-5p, and miR-10483-5p significantly increased the mortality of S. frugiperda to cyantraniliprole and emamectin benzoate. The mortality was significantly increased with spinetoram treatment after the overexpression of miR-13b-3p, miR-10485-5p, and miR-10483-5p. These results suggest that miRNAs, which are differentially expressed in response to insecticides, may play a key regulatory role in the insecticide tolerance in S. frugiperda.

Cold storage of Spodoptera litura eggs and Telenomus remus adults for improving mass‐rearing efficiency

AbstractTelenomus remus (Nixon) has shown promising potential as a biological control agent for the major invasive agricultural pest, Spodoptera frugiperda (J. E. Smith). Application of biological control programmes in the field requires an efficient mass‐rearing system to provide sufficient numbers of beneficial insects. The short longevity of parasitoid adults and limited period of availability of host eggs are two factors affecting the mass‐rearing efficiency of T. remus. Using Spodoptera litura (Fabricius) eggs as an alternative host, the effects of cold storage of S. litura eggs and T. remus adults at 5°C, 8°C and 11°C for different time durations on the fitness of the parasitoid were investigated to identify the most suitable storage conditions. Results showed that T. remus can successfully exploit stored S. litura eggs, but the number of parasitized eggs decreased with increased egg storage duration and/or reduced storage temperature. For refrigerated adults, the survival rate declined gradually with longer storage duration, and the females' survival rate was higher than the males' at any treatment. Similarly, parasitism capacity of stored adults declined significantly with prolonged storage. Female adult longevity was significantly shorter than the control for all treatments. However, the offspring emergence rate and percentage of females were barely affected by cold storage. In conclusion, S. litura eggs can be stored at 11°C for up to 9 days and adult T. remus can be stored at 8°C or 11°C for up to 7 days without significant losses. Data obtained from this study will help coordinate different aspects of T. remus production, decreasing costs and improving the biocontrol materials' availability.

A Review of the Biology, Ecology, and Management of the South American Locust, Schistocerca cancellata (Serville, 1838), and Future Prospects

In the first half of the twentieth century, the South American Locust (SAL), Schistocerca cancellata (Serville, 1838), was a major pest of agriculture in Argentina, Bolivia, Paraguay, Uruguay, and Brazil. From 1954–2014, a preventive management program appeared to limit SAL populations, with only small- to moderate-scale treatments required, limited to outbreak areas in northwest Argentina. However, the lack of major locust outbreaks led to a gradual reduction in resources, and in 2015, the sudden appearance of swarms marked the beginning of a substantial upsurge, with many swarms reported initially in Argentina in 2015, followed by expansion into neighboring countries over the next few years. The upsurge required a rapid allocation of resources for management of SAL and a detailed examination of the improvements needed for the successful management of this species. This paper provides a review of SAL biology, management history, and perspectives on navigating a plague period after a 60-year recession.

Over-expression in cis of the midgut P450 CYP392A16 contributes to abamectin resistance in Tetranychus urticae

Cytochrome P450 mediated metabolism is a well-known mechanism of insecticide resistance. However, to what extent qualitative or quantitative changes are responsible for increased metabolism, is not well understood. Increased expression of P450 genes is most often reported, but the underlying regulatory mechanisms remain widely unclear. In this study, we investigate CYP392A16, a P450 from the polyphagous and major agricultural pest Tetranychus urticae. High expression levels of CYP392A16 and in vitro metabolism assays have previously associated this P450 with abamectin resistance. Here, we show that CYP392A16 is primarily localized in the midgut epithelial cells, as indicated by immunofluorescence analysis, a finding also supported by a comparison between feeding and contact toxicity bioassays. Silencing via RNAi of CYP392A16 in a highly resistant T. urticae population reduced insecticide resistance levels from 3400- to 1900- fold, compared to the susceptible reference strain. Marker-assisted backcrossing, using a single nucleotide polymorphism (SNP) found in the CYP392A16 allele from the resistant population, was subsequently performed to create congenic lines bearing this gene in a susceptible genetic background. Toxicity assays indicated that the allele derived from the resistant strain confers 3.6-fold abamectin resistance compared to the lines with susceptible genetic background. CYP392A16 is over-expressed at the same levels in these lines, pointing to cis-regulation of gene expression. In support of that, functional analysis of the putative promoter region from the resistant and susceptible parental strains revealed a higher reporter gene expression, confirming the presence of cis-acting regulatory mechanisms.

Ryanodine Receptor as Insecticide Target.

The ryanodine receptor (RyR) is one of the primary targets of commercial insecticides. The diamide insecticide family, including flubendiamide, chlorantraniliprole, cyantraniliprole, etc., targets insect RyRs and can be used to control a wide range of destructive agricultural pests. The diamide insecticides are highly selective against lepidopteran and coleopteran pests with relatively low toxicity for non-target species, such as mammals, fishes, and beneficial insects. However, recently mutations identified on insect RyRs have emerged and caused resistance in several major agricultural pests throughout different continents. This review paper summarizes the recent findings on the structure and function of insect RyRs as insecticide targets. Specifically, we examine the structures of RyRs from target and non-target species, which reveals the molecular basis for insecticide action and selectivity. We also examine the structural and functional changes of RyR caused by the resistance mutations. Finally, we examine the progress in RyR structure-based insecticide design and discuss how this might help the development of a new generation of green insecticides.

Functional Characterization of Silkworm PIWI Proteins by Embryonic RNAi.

The molecular mechanisms of sex-determination systems among insect orders and species are diverse. Therefore, genes involved in sex determination are strong candidates for insect pest management. Even though lepidopterans are major agricultural insect pests that cause widespread economic damage to various crops, their sex-determination systems have not been fully elucidated, even in the silkworm (Bombyx mori), a model lepidopteran insect. In 2014, we found that a female-specific W chromosome-derived PIWI-interacting RNA (piRNA) determines femaleness in silkworms. To analyze the function of two core silkworm piRNA biogenesis pathway genes, Siwi and BmAgo3, in the sex-determination system, we developed a genomic DNA and total RNA extraction strategy for a siRNA-injected single embryo. The siRNA-injected embryo can be molecularly sexed by W chromosome-specific DNA markers. Using complementary DNA (cDNA) reverse transcribed from the sexed RNA, we evaluated the knockdown effect of the PIWI protein-coding genes on a sexual development-related gene, Bombyx mori doublesex.

First Report of Halyomorpha halys (Hemiptera: Pentatomidae) in Montana, USA

Abstract Here, we document the first record for Montana of Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), a major agricultural and nuisance pest in many parts of the world. The insect was found overwintering in a private residence in January 2021 in Billings (Yellowstone Co.). A preliminary survey of sites in Billings from May to October 2021 confirmed the presence of H. halys within a radius of at least 11 km from the January detection. Putatively overwintering H. halys and adult bugs found across the city of Billings in the summer suggest the insect may have been present in the area prior to 2021. Surveys conducted in various regions of the state between 2013 and 2020 reported no detections of H. halys. Yellowstone Co. was previously surveyed in 2017, indicating the species likely arrived between 2018 and 2020 or was present before 2018 at densities too low to be detected. The summary of presence and absence records we provide herein is valuable for future monitoring and management efforts of this economically important species.

Directed Sequencing of Plant Specific DNA Identifies the Dietary History of Four Species of Auchenorrhyncha (Hemiptera)

Abstract Auchenorrhyncha (Hemiptera) includes several families of sap-feeding insects that tend to feed on a wide-range of host plants. Some species within Auchenorrhyncha are major agricultural pests that transmit plant pathogens or cause direct feeding damage. Nearly all pest Auchenorrhyncha are highly polyphagous, have mobile nymphs, and colonize crops from noncrop habitats. We examined whether methods for gut content analysis developed for more specialized Hemiptera identify dietary history of polyphagous Auchenorrhyncha. We used high-throughput sequencing of the plant genes trnF and ITS to examine the dietary history of Circulifer tenellus (Baker) (Cicadellidae), Colladonus geminatus (Van Duzee) (Cicadellidae), Colladonus montanus reductus (Van Duzee) (Cicadellidae), and Lycorma delicatula (White) (Fulgoridae). C. tenellus is a vector of the vegetable pathogens Candidatus Phytoplasma trifolii and beat curly top virus. Both Colladonus species are vectors of Ca. Phytoplasma pruni, the pathogen associated with X-disease of stone fruits. Lycorma delicatula is an invasive pest of grape and ornamentals in the eastern United States. Results showed spring hosts for C. tenellus included Brassicaceae, especially Sisymbrium sp. (tumble mustard), and spring hosts for both Colladonus species included Taraxacum sp. (dandelion). Gut content analysis also detected a decrease in host-breadth by L. delicatula from early to late instars. Results demonstrate that directed sequencing of plant DNA identified the dietary history of leafhopper and planthopper pests. Expanded use of gut content analysis will help identify the noncrop sources of phytoplasma-infected C. tenellus and Colladonus, and to examine seasonal changes in host shifts by L. delicatula.

Diversity and Distribution of Forest Ants (Hymenoptera: Formicidae) in Nepal: Implications for Sustainable Forest Management.

Simple SummaryThere is little information available about the species diversity and distribution patterns of Nepalese ants, as well as their importance in maintaining forest health. A survey of forest ants was conducted in Nepal to learn about their diversity, distribution, and role in forest management. Ants were collected using vegetation beating, sweeping, and hand collection methods in eastern, central, and western Nepal. Seventy ant species from thirty-six genera and six subfamilies were recorded in the study. The research also discovered five genera and nine species new for the country, as well as eight tramp species, four of which are major ecological, agricultural, and/or household pests. The study discovered that western Nepal and the Siwalik region have a relatively high ant diversity. Ant diversity was found to decrease with increasing elevation. The assessment of ant diversity using multiple sampling methods that cover all seasons and forest types may be useful in obtaining complete ant diversity data. Early intervention through sustainable forest management initiatives would aid in preventing invasive ant incursions in the forests of Nepal.The information available on the diversity of ant species and their distribution and interaction with forest health in Nepal remains limited. As part of a nationwide project on forest health, we conducted inventories to assess the diversity and distribution of forest ants and their role in forest management in Nepal. Ants were collected from 187 plots of 10 m × 10 m size along the north–south belt transects in eastern, central, and western Nepal. We used vegetation beating, sweeping, and hand collection methods in selected forest types. In each transect, we designed six plots in each major forest type (Sal, Schima–Castanopsis, and broadleaf mixed forests) and three plots each in deodar, Alnus, riverine, and Cryptomeria forests. We recorded 70 ant species from 36 genera and six subfamilies. This includes five genera and nine species new for the country, as well as eight tramp species, four of which are major ecological, agricultural, and/or household pests. Our study indicates that forest ant species richness is high in western Nepal and the Siwaliks, and it decreases as elevation increases. The high diversity of ant species in the forests of Nepal needs to be assessed with further exploration using multiple sampling methods covering all seasons and forest types. Ants can be useful indicators for ecosystem management and human impacts on forests. Reports of invasive ants in Nepalese forests indicate the relevance of urgent interventions through sustainable forest management initiatives to prevent future incursions.

Characterization of a novel superoxide dismutase in Nilaparvata lugens.

The brown planthopper (Nilaparvata lugens) is a major agricultural pest of rice crops. Analysis of the enzymes produced by N. lugens is important to develop pest-control methods. Superoxide dismutase (SOD) is a detoxification enzyme that catalyzes the conversion of superoxide anions (reactive oxygen species) into oxygen and hydrogen peroxide. As there have been no reports on SOD in N. lugens, in this study, we characterized a new SOD in the brown planthopper, nlSOD1. Amino acid sequence and phylogenetic analyses revealed that nlSOD1 is a member of the Cu/Zn-SOD family. Recombinant nlSOD1, when overexpressed in Escherichia coli, catalyzes the dismutation of superoxide radicals into molecular O2 and H2 O2 . Exposure to various insecticides induced nlSOD1 messenger RNA expression. These results indicate that nlSOD1 may contribute to the insecticide resistance of N. lugens. The findings of this study may assist in the development of novel methods to control the population of N. lugens.

Genome sequence of the English grain aphid, Sitobion avenae and its endosymbiont Buchnera aphidicola.

The English grain aphid, Sitobion avenae, is a major agricultural pest of wheat, barley and oats, and one of the principal vectors of barley yellow dwarf virus leading to significant reductions in grain yield, annually. Emerging resistance to and increasing regulation of insecticides has resulted in limited options for their control. Using PacBio HiFi data, we have produced a high-quality draft assembly of the S. avenae genome; generating a primary assembly with a total assembly size of 475.7 Mb, and an alternate assembly with a total assembly size of 430.8 Mb. Our primary assembly was highly contiguous with only 326 contigs and a contig N50 of 15.95 Mb. Assembly completeness was estimated at 97.7% using BUSCO analysis and 31,007 and 29,037 protein-coding genes were predicted from the primary and alternate assemblies, respectively. This assembly, which is to our knowledge the first for an insecticide resistant clonal lineage of English grain aphid, will provide novel insight into the molecular and mechanistic determinants of resistance and will facilitate future research into mechanisms of viral transmission and aphid behavior.

Cover Caption

The 28‐spotted potato ladybeetle, Henosepilachna vigintioctopunctata (Fabricius) (Coleoptera: Coccinellidae), is a major agricultural pest that primarily causes serious damage to solanaceous plants. The destructive potential of H. vigintioctopunctata is high at the larval stages. The cover photo shows that H. vigintioctopunctata third instar larva eating on the surface of eggplant leaf; it can be easily killed after ingestion of dsHvvATPase A or dsHvvATPase E (see pages xxx‐xxx). Photo provided by Li‐Yuan Liu.

Reciprocal mutations of two multifunctional β-amyrin synthases from Barbarea vulgaris shift α/β-amyrin ratios.

In the wild cruciferous wintercress (Barbarea vulgaris), β-amyrin-derived saponins are involved in resistance against insect herbivores like the major agricultural pest diamondback moth (Plutella xylostella). Enzymes belonging to the 2,3-oxidosqualene cyclase family have been identified and characterized in B. vulgaris G-type and P-type plants that differ in their natural habitat, insect resistance and saponin content. Both G-type and P-type plants possess highly similar 2,3-oxidosqualene cyclase enzymes that mainly produce β-amyrin (Barbarea vulgaris Lupeol synthase 5 G-Type; BvLUP5-G) or α-amyrin (Barbarea vulgaris Lupeol synthase 5 P-Type; BvLUP5-P), respectively. Despite the difference in product formation, the two BvLUP5 enzymes are 98% identical at the amino acid level. This provides a unique opportunity to investigate determinants of product formation, using the B. vulgaris 2,3-oxidosqualene cyclase enzymes as a model for studying amino acid residues that determine differences in product formation. In this study, we identified two amino acid residues at position 121 and 735 that are responsible for the dominant changes in generated product ratios of β-amyrin and α-amyrin in both BvLUP5 enzymes. These amino acid residues have not previously been highlighted as directly involved in 2,3-oxidosqualene cyclase product specificity. Our results highlight the functional diversity and promiscuity of 2,3-oxidosqualene cyclase enzymes. These enzymes serve as important mediators of metabolic plasticity throughout plant evolution.

Bt cotton area contraction drives regional pest resurgence, crop loss, and pesticide use.

SummaryGenetically‐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.

FREQUENT LEPTOSPIRA SPP. DETECTION BUT ABSENCE OF TULA ORTHOHANTAVIRUS IN MICROTUS SPP. VOLES, NORTHWESTERN SPAIN.

The common vole (Microtus arvalis) is a major agricultural pest in Europe and is a reservoir for several zoonotic agents, such as Leptospira spp. and Tula orthohantavirus (TULV). However, little is known about the occurrence of those pathogens in voles from Spain, where the species has largely expanded its distribution range in the past decades, causing agricultural pests and zoonotic diseases. For a molecular survey, 580 common voles and six Lusitanian pine voles (Microtus lusitanicus) were collected in 26 localities from four provinces of northwestern Spain. We assessed the presence of Leptospira spp. DNA in kidney tissue by PCR targeting the lipL32 gene, detecting a prevalence of 7.9% (95% confidence interval, 5.9-10.4) for common voles and of 33.3% (95% confidence interval, 4.3-77.7) for Lusitanian pine voles. We identified Leptospira kirschneri in 24 animals and Leptospira borgpetersenii in two animals, using secY gene-specific PCR. We analyzed environmental and demographic factors (such as age class, weight, and sex) and population dynamics data for their potential effect on the Leptospira spp. prevalence in those voles. The Leptospira spp. DNA detection rate in common voles increased significantly with maximum air temperature, vole weight, and amount of accumulated rainfall during the 90 d before capture and within the peak phase of the population cycle. We assessed the presence of TULV in lung tissue of 389 voles by reverse-transcription PCR, with no positive results. The absence of TULV might be explained by the evolutionary isolation of the common vole in Spain. The detection of two Leptospira genomospecies underlines the necessity for further typing efforts to understand the epidemiology of leptospiral infection in the common vole and the potential risk for human health in Spain.

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

Simple SummaryFour major Spodoptera pests, S. exigua, S. frugiperda, S. litura, and S. littoralis, are widely distributed polyphagous pests affecting various crops. Despite different distribution areas, these four species cause serious damage to agriculture worldwide. As these species are morphologically similar at the larval stage, diagnostic methods have been developed and utilized for their identification. Here, we developed a loop-mediated isothermal amplification (LAMP) assay for rapid and effective species diagnosis, along with PCR, to identify Korean field-collected or overseas samples. The optimal conditions for the LAMP assay were 61 °C for 60 min with four LAMP primers. Additional loop primers increased the amplification efficiency in S. exigua, whereas increased non-specific amplification was found in other species. A broad range of DNA concentrations was observed in the LAMP assay, and the minimum detectable DNA concentration was 1 pg. The DNA release method for LAMP involved incubation of larval or adult tissue samples for 5 min at 95 °C, without a DNA extraction step. Considering the gradual diversification invasive pest incidence, this simple and accurate LAMP assay can be used for intensive field monitoring of invasive pests and integrated management of these species.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.

Functional analysis of small heat shock proteins providing evidence of temperature tolerance in Hyphantria cunea

AbstractA substantial increase in the occurrence of extremely high‐ or low‐temperature events caused by global climate change has imposed pressure on the survival and reproduction of invertebrates. Hyphantria cunea (H. cunea; Lepidoptera: Arctiidae) is a worldwide defoliator and has become a major forestry and agricultural pest during the past three decades in China. The thermal and cold tolerance has directly affected the population and geographical distribution of H. cunea, which remains poorly understood. Here, we examined the thermal tolerance of H. cunea larvae and discovered that they could endure extremely high temperatures, as demonstrated by median lethal times (Ltim50) of 103.3 hr at 37°C, 62.0 hr at 40°C and even 14.5 hr at 43°C. To further explore the molecular evidence of the physiological response to heat and cold, we cloned and characterized six small heat shock proteins (sHSPs). These proteins possessed the conserved α‐domain of the sHSP family at the C‐terminus and various N‐terminal domains. Phylogenetic analysis showed that the HCsHSPs presented a close evolutionary relationship with sHSP genes in noctuidae species. Four sHSPs in H. cunea were sensitive to heat stress, and only HCsHSP28.7 was induced by cold stress. The expression level of HCsHSPs peaked at 40°C and decreased in treatment at 45°C. HCsHSP20.0 transcripts were abundant in the cuticle after heat shock, and HCsHSP28.7 was most enriched in the fat body after cold shock. In addition, an equal molecular weight of the HCsHSP20.0 protein could completely protect malate dehydrogenase (MDH) from thermal aggregation. Recombinant HCsHSP28.7 E. coli cells exhibited tolerance to cold stress. Overall, our results demonstrate that HCsHSPs, especially HCsHSP20.0 and HCsHSP28.7, are vital genes in the adaptation of H. cunea larvae to extreme conditions in the context of global climate change.