Knockout of the delta11-desaturase SfruDES1 disrupts sex pheromone biosynthesis, mating and oviposition in the fall armyworm, Spodoptera frugiperda.

Moth insects rely on sex pheromones for long distance attraction and searching for sex partners. The biosynthesis of moth sex pheromones involves the catalytic action of multiple enzymes, with desaturases playing a crucial role in the process of carbon chain desaturation. However, the specific desaturases involved in sex pheromone biosynthesis in fall armyworm (FAW), Spodoptera frugiperda, have not been clarified. In this study, a Δ11 desaturase (SfruDES1) gene in FAW was knocked out using the CRISPR/Cas9 genome editing system. A homozygous mutant of SfruDES1 was obtained through genetic crosses. The gas chromatography-mass spectrometry (GC-MS) analysis results showed that the three main sex pheromone components (Z7-12:Ac, Z9-14:Ac, and Z11-16:Ac) and the three minor components (Z9-14:Ald, E11-14:Ac and Z11-14:Ac) of FAW were not detected in homozygous mutant females compared to the wild type. Furthermore, behavioral assay demonstrated that the loss of SfruDES1 resulted in a significant reduction in the attractiveness of females to males, along with disruptions in mating behavior and oviposition. Additionally, in a heterologous expression system, recombinant SfruDES1 could introduce a cis double bond at the Δ11 position in palmitic acid, which resulted in the changes in components of the synthesized products. These findings suggest desaturase plays a key role in the biosynthesis of sex pheromones, and knockout of the SfruDES1 disrupts sex pheromone biosynthesis and mating behavior in FAW. The SfruDES1 could serve as tool to develop a control method for S. frugiperda.

CRISPR-Cas9 mediated dsRNase knockout improves RNAi efficiency in the fall armyworm.

Lepidopteran insects are refractory to RNA interference (RNAi) response, especially to orally delivered double-stranded RNA (dsRNA). High nuclease activity in the midgut lumen is proposed as one of the major reasons for RNAi insensitivity. We identified three dsRNase genes highly expressed in the midgut of fall armyworm (FAW), Spodoptera frugiperda. The genomic region harboring those three dsRNase genes was deleted using the CRISPR-Cas9-mediated genome editing method. A homozygous line with deletion of three dsRNase genes was produced. dsRNA degradation by midgut lumen contents of mutant larvae was lower than in wild-type larvae. Feeding dsRNA targeting the inhibitor of apoptosis (IAP) gene increased knockdown of the target gene and mortality in mutants compared to wild-type larvae. These results suggest that dsRNases in the midgut contribute to RNAi inefficiency in FAW. Formulations that protect dsRNA from dsRNase degradation may improve RNAi efficiency in FAW and other lepidopteran insects.

Comparative analysis of Spodoptera frugiperda (J. E. Smith) (Lepidoptera, Noctuidae) corn and rice strains microbiota revealed minor changes across life cycle and strain endosymbiont association.

Background: Spodoptera frugiperda (FAW) is a pest that poses a significant threat to corn production worldwide, causing millions of dollars in losses. The species has evolved into two strains (corn and rice) that differ in their genetics, reproductive isolation, and resistance to insecticides and Bacillus thuringiensis endotoxins. The microbiota plays an important role in insects' physiology, nutrient acquisition, and response to chemical and biological controls. Several studies have been carried out on FAW microbiota from larvae guts using laboratory or field samples and a couple of studies have analyzed the corn strain microbiota across its life cycle. This investigation reveals the first comparison between corn strain (CS) and rice strain (RS) of FAW during different developmental insect stages and, more importantly, endosymbiont detection in both strains, highlighting the importance of studying both FAW populations and samples from different stages. Methods: The composition of microbiota during the life cycle of the FAW corn and rice strains was analyzed through high-throughput sequencing of the bacterial 16S rRNA gene using the MiSeq system. Additionally, culture-dependent techniques were used to isolate gut bacteria and the Transcribed Internal Spacer-ITS, 16S rRNA, and gyrB genes were examined to enhance bacterial identification. Results: Richness, diversity, and bacterial composition changed significantly across the life cycle of FAW. Most diversity was observed in eggs and males. Differences in gut microbiota diversity between CS and RS were minor. However, Leuconostoc, A2, Klebsiella, Lachnoclostridium, Spiroplasma, and Mucispirilum were mainly associated with RS and Colidextribacter, Pelomonas, Weissella, and Arsenophonus to CS, suggesting that FAW strains differ in several genera according to the host plant. Firmicutes and Proteobacteria were the dominant phyla during FAW metamorphosis. Illeobacterium, Ralstonia, and Burkholderia exhibited similar abundancies in both strains. Enterococcus was identified as a conserved taxon across the entire FAW life cycle. Microbiota core communities mainly consisted of Enterococcus and Illeobacterium. A positive correlation was found between Spiroplasma with RS (sampled from eggs, larvae, pupae, and adults) and Arsenophonus (sampled from eggs, larvae, and adults) with CS. Enterococcus mundtii was predominant in all developmental stages. Previous studies have suggested its importance in FAW response to B. thuringensis. Our results are relevant for the characterization of FAW corn and rice strains microbiota to develop new strategies for their control. Detection of Arsenophonus in CS and Spiroplasma in RS are promising for the improvement of this pest management, as these bacteria induce male killing and larvae fitness reduction in other Lepidoptera species.

miR-317-3p and miR-283-5p Play a Crucial Role in Regulating the Resistance to Indoxacarb in Spodoptera frugiperda by Targeting GSTs4.

Spodoptera frugiperda is primarily controlled through chemical insecticides. Our RNA-seq data highlight the overexpression of GSTs4 in indoxacarb-resistant S. frugiperda. However, the exact role of GSTs4 in indoxacarb resistance and its regulatory mechanisms remains elusive. Therefore, we investigated the functional role of GSTs4 in S. frugiperda and explored the underlying post-transcriptional regulatory mechanisms. GSTs4 was highly overexpressed (27.6-fold) in the indoxacarb-resistant strain, and GSTs4 silencing significantly increases the susceptibility of S. frugiperda to indoxacarb, increasing mortality by 27.3%. miR-317-3p and miR-283-5p can bind to the 3'UTR of GSTs4, and the targeting relationship was confirmed by dual-luciferase reporter assays. Injecting miR-317-3p and miR-283-5p agomirs reduces GSTs4 levels by 64.8 and 42.3%, respectively, resulting in an increased susceptibility of S. frugiperda to indoxacarb. Conversely, the administration of miR-317-3p and miR-283-5pantagomirs increases GSTs4 expression and reduces larval susceptibility to indoxacarb. These findings demonstrate that miR-317-3p and miR-283-5p contribute to indoxacarb resistance in S. frugiperda by regulating the overexpression of GSTs4.

Integrated transcriptomic and proteomic analyses reveal the molecular mechanism underlying the thermotolerant response of Spodoptera frugiperda.

Spodoptera frugiperda (Lepidoptera: Noctuidae) is a highly destructive invasive pest with remarkable adaptability to extreme climatic conditions, posing a substantial global threat. Although the effects of temperature stress on the biological and ecological properties of S. frugiperda have been elucidated, the molecular mechanisms underlying its responses remain unclear. Herein, we combined transcriptomic and proteomic analyses to explore the key genes and proteins involved in thermotolerance regulation in S. frugiperda larvae at 42 °C. Overall, 1528 differentially expressed genes (DEGs) and 154 differentially expressed proteins (DEPs) were identified in S. frugiperda larvae under heat stress, including antioxidant enzymes, heat shock proteins (Hsps), cytochrome P450s, starch and sucrose metabolism genes, and insulin signaling pathway genes, indicating their involvement in heat tolerance regulation. Correlation analysis of DEGs and DEPs revealed that seven and eight had the same and opposite expression profiles, respectively. After nanocarrier-mediated RNA interference knockdown of SfHsp29, SfHsp20.4, SfCAT, and SfGST, the body weight and mortality of S. frugiperda larvae significantly decreased and increased under heat stress, respectively. This indicates that SfHsp29, SfHsp20.4, SfCAT, and SfGST play a crucial role in the thermotolerance of S. frugiperda larvae. These results provide insight into the mechanism of heat tolerance in S. frugiperda.

Determination of internal controls for quantitative gene expression of Spodoptera litura under microbial pesticide stress.

Quantitative real-time polymerase chain reaction (qRT-PCR) has become a commonly used method for the quantification of gene expression. However, accurate qRT-PCR analysis requires a valid internal reference for data normalization. To determine the valid reference characterized with low expression variability among Spodoptera litura samples after microbial pesticide treatments, nine housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), arginine kinase, ubiquitin C, actin-5C (ACT5C), actin, ribosomal protein S13 (RPS13), tubulin, acidic ribosomal protein P0 (RPLP0) and ubiquinol-cytochrome c reductase, were evaluated for their suitability using geNorm, Normfinder, BestKeeper, RefFinder and the comparative delta CT methods in this study. S. litura larvae after direct treatment (larvae were immersed in biopesticides), indirect treatment (larvae were fed with biopesticide immersed artificial diets) and comprehensive treatment (larvae were treated with the first two treatments in sequence), respectively with Metarhizium anisopliae, Empedobacter brevis and Bacillus thuringiensis, were investigated. The results indicated that the best sets of internal references were as follows: RPLP0 and ACT5C for direct treatment conditions; RPLP0 and RPS13 for indirect treatment conditions; RPS13 and GAPDH for comprehensive treatment conditions; RPS13 and RPLP0 for all the samples. These results provide valuable bases for further genetic researches in S. litura.

The effect of diet composition on the diversity of active gut bacteria and on the growth of Spodoptera exigua (Lepidoptera: Noctuidae).

Gut microbiota plays a functional role in nutrition among several insects. However, the situation is unclear in Lepidoptera. Field studies suggest the microbiome may not be stable and is determined by diet, while in the laboratory, Lepidoptera are routinely reared on diet containing antibiotics with unknown effects on microbial communities. Furthermore, molecular approaches for the characterization of lepidopteran microbiomes rarely describe the metabolically active gut bacteria. The aim of this study was to evaluate how diet and antibiotics affect Spodoptera exigua (Hübner) growth and the diversity and activity of the gut bacteria community. We assessed how alfalfa and wheat germ-based diets affected larval growth, in the presence and absence of streptomycin. Alfalfa diet improved larval growth, pupal mass, and survival, but antibiotic was only beneficial in the wheat germ diet. We observed diet-driven changes in the gut bacterial communities. In the active community, the alfalfa colony was dominated by Enterococcus and Rhodococcus whereas in the wheat germ colony, only Enterococcus was present. In contrast, spore-forming Bacilli species were very common members of the DNA community. In both cases, streptomycin had a selective effect on the relative abundance of the taxa present. Our study highlights the importance of characterizing both the diversity and activity of the gut microbiota community. DNA-derived communities may include environmental DNA, spores, or non-viable bacteria, while RNA-derived communities are more likely to give an accurate representation of the diversity of active members that are potentially directly involved in the metabolic processes of the host.

Invasive fall armyworms are corn strain.

The fall armyworm (Spodoptera frugiperda) is one of the major pest insects in diverse crop plants, including maize, rice, and cotton. While the fall armyworm is native to North and South America, its invasion was first reported in West Africa in 2016. Since then, this species has rapidly spread across Sub-Saharan Africa, Asia, and Oceania, as well as Egypt and Cyprus. The fall armyworm is composed of two sympatric strains, the corn and rice strains, designated to their preferred host plants, in native areas. It remains surprisingly unclear whether invasive fall armyworms belong to the corn strain, rice strain, or hybrids of the two, despite a large number of population genetics studies. In this study, we performed population genomics analyses using globally collected 116 samples to identify the strains of invasive fall armyworms. We observed that invasive fall armyworms are genomically most similar to the corn strain. The reconstructed phylogenetic tree supports the hypothesis that invasive fall armyworms originated from the corn strain. All genomic loci of invasive populations exhibit higher genetic similarity to the corn strains compared to the rice strains. Furthermore, we found no evidence of gene flow from rice strains to invasive populations at any genomic locus. These results demonstrate that invasive fall armyworms belong to the corn strain. These results suggest that invasive fall armyworms likely have very limited potential to infest rice. Therefore, the management plan should primarily focus on crops preferred by the corn strain.

Transcriptomic and biochemical insights into fall armyworm (Spodoptera frugiperda) responses on silicon-treated maize.

Background: The fall armyworm, Spodoptera frugiperda, is an agricultural pest of significant economic concern globally, known for its adaptability, pesticide resistance, and damage to key crops such as maize. Conventional chemical pesticides pose challenges, including the development of resistance and environmental pollution. The study aims to investigate an alternative solution: the application of soluble silicon (Si) sources to enhance plant resistance against the fall armyworm. Methods: Silicon dioxide (SiO2) and potassium silicate (K2SiO3) were applied to maize plants via foliar spray. Transcriptomic and biochemical analyses were performed to study the gene expression changes in the fall armyworm feeding on Si-treated maize. Results: Results indicated a significant impact on gene expression, with a large number of differentially expressed genes (DEGs) identified in both SiO2 and K2SiO3 treatments. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis identified critical DEGs involved in specific pathways, including amino acid, carbohydrate, lipid, energy, xenobiotics metabolisms, signal transduction, and posttranslational modification, significantly altered at both Si sources. Biochemical analyses further revealed that Si treatments inhibited several enzyme activities (glutamate dehydrogenase, trehalase, glucose-6-phosphate dehydrogenase, chitinase, juvenile hormone esterase, and cyclooxygenase while simultaneously inducing others (total protein, lipopolysaccharide, fatty acid synthase, ATPase, and cytochrome P450), thus suggesting a toxic effect on the fall armyworm. In conclusion, Si applications on maize influence the gene expression and biochemical activities of the fall armyworm, potentially offering a sustainable pest management strategy.

Characterization of the inheritance of field-evolved resistance to diamides in the fall armyworm (Spodoptera frugiperda) (Lepidoptera: Noctuidae) population from Puerto Rico.

The fall armyworm (Spodoptera frugiperda) is one of the most destructive pests of corn. New infestations have been reported in the East Hemisphere, reaching India, China, Malaysia, and Australia, causing severe destruction to corn and other crops. In Puerto Rico, practical resistance to different mode of action compounds has been reported in cornfields. In this study, we characterized the inheritance of resistance to chlorantraniliprole and flubendiamide and identified the possible cross-resistance to cyantraniliprole and cyclaniliprole. The Puerto Rican (PR) strain showed high levels of resistance to flubendiamide (RR50 = 2,762-fold) and chlorantraniliprole (RR50 = 96-fold). The inheritance of resistance showed an autosomal inheritance for chlorantraniliprole and an X-linked inheritance for flubendiamide. The trend of the dominance of resistance demonstrated an incompletely recessive trait for H1 (♂ SUS × â™€ PR) × and an incompletely dominant trait for H2 (♀ SUS × â™‚ PR) × for flubendiamide and chlorantraniliprole. The PR strain showed no significant presence of detoxification enzymes (using synergists: PBO, DEF, DEM, and VER) to chlorantraniliprole; however, for flubendiamide the SR = 2.7 (DEM), SR = 3.2 (DEF) and SR = 7.6 (VER) indicated the role of esterases, glutathione S- transferases and ABC transporters in the metabolism of flubendiamide. The PR strain showed high and low cross-resistance to cyantraniliprole (74-fold) and cyclaniliprole (11-fold), respectively. Incomplete recessiveness might lead to the survival of heterozygous individuals when the decay of diamide residue occurs in plant tissues. These results highlight the importance of adopting diverse pest management strategies, including insecticide rotating to manage FAW populations in Puerto Rico and other continents.

Characterization of Spodoptera littoralis (Lepidoptera: Noctuidae) resistance to indoxacarb: inheritance mode, realized heritability, and fitness costs.

Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) is a major economic pest attacking a variety of crops in Egypt and other Mediterranean countries. S. littoralis has developed resistance to both traditional and novel insecticides. The current study investigated S. littoralis resistance to indoxacarb regarding inheritance mode, realized heritability (h2), and fitness costs. An indoxacarb-resistant strain (Indoxa-SEL) was obtained by selecting a field strain with indoxacarb. Indoxa-SEL strain outperformed the susceptible one (Indoxa-S) by 29.77-fold after 16 consecutive generations of selection. Based on the LC50 values of the progenies of reciprocal crosses F1 (R♂ × S♀) and F1' (R♀ × S♂), S. littoralis resistance to indoxacarb was found to be autosomal and partially recessive. Chi-square tests for goodness-of-fit between observed and expected mortalities of self-bred F1 and resistant strain reciprocal crosses revealed that the resistance was controlled by multiple genes. The resistant strain had a relative fitness of 0.80, with significantly increased total preovipositional period of females, egg, larvae, pupae, preadult, adult, and total longevity period. The estimated realized heritability value in the Indoxa-SEL strain was 0.21. The current study will contribute to sustaining indoxacarb efficacy and designing effective resistance management programs against S. littoralis.

Identification of differentially expressed miRNAs associated with diamide detoxification pathways in Spodoptera frugiperda.

The fall armyworm (FAW) Spodoptera frugiperda is a severe economic pest of multiple crops globally. Control of this pest is often achieved using insecticides; however, over time, S. frugiperda has developed resistance to new mode of action compounds, including diamides. Previous studies have indicated diamide resistance is a complex developmental process involving multiple detoxification genes. Still, the mechanism underlying the possible involvement of microRNAs in post-transcriptional regulation of resistance has not yet been elucidated. In this study, a global screen of microRNAs (miRNAs) revealed 109 known and 63 novel miRNAs. Nine miRNAs (four known and five novel) were differentially expressed between insecticide-resistant and -susceptible strains. Gene Ontology analysis predicted putative target transcripts of the differentially expressed miRNAs encoding significant genes belonging to detoxification pathways. Additionally, miRNAs are involved in response to diamide exposure, indicating they are probably associated with the detoxification pathway. Thus, this study provides comprehensive evidence for the link between repressed miRNA expression and induced target transcripts that possibly mediate diamide resistance through post-transcriptional regulation. These findings highlight important clues for further research to unravel the roles and mechanisms of miRNAs in conferring diamide resistance.

Early transcriptomic responses of rice leaves to herbivory by Spodoptera frugiperda.

During herbivory, chewing insects deposit complex oral secretions (OS) onto the plant wound. Understanding how plants respond to the different cues of herbivory remains an active area of research. In this study, we used an herbivory-mimick experiment to investigate the early transcriptional response of rice plants leaves to wounding, OS, and OS microbiota from Spodoptera frugiperda larvae. Wounding induced a massive early response associated to hormones such as jasmonates. This response switched drastically upon OS treatment indicating the activation of OS specific pathways. When comparing native and dysbiotic OS treatments, we observed few gene regulation. This suggests that in addition to wounding the early response in rice is mainly driven by the insect compounds of the OS rather than microbial. However, microbiota affected genes encoding key phytohormone synthesis enzymes, suggesting an additional modulation of plant response by OS microbiota.

Pheromonal variation and mating between two mitotypes of fall armyworm (Spodoptera frugiperda) in Africa.

In the Americas, the fall armyworm (Spodoptera frugiperda) exists in two genetically distinct strains, the corn (C) and rice (R) strains. Despite their names, these strains are not associated with host plant preferences but have been shown to vary in pheromone composition and male responses. Recently, S. frugiperda was detected in Africa as an invasive species, but knowledge about variation in strain types, pheromone composition and inter-strain mating of populations of the pest in the continent has not been fully examined. Therefore, this study aimed to investigate variations, if any in the pheromone composition of female moths, male moth responses, and mating between C and R mitotypes of S. frugiperda populations in Kenya, as well as their geographic distribution. Strains (mitotypes) of S. frugiperda were identified using mitochondrial DNA (mtDNA) markers, and their pheromonal composition determined by coupled gas chromatography-mass spectrometric (GC-MS) analysis. Male moth responses to these compounds were evaluated using GC-electroantennographic detection (EAD), electroantennogram (EAG), and wind tunnel assays. Oviposition assays were used to determine whether R and C mitotype moths could mate and produce eggs. The results showed that both the R and C mitotypes were present, and there were no statistically significant differences in their distribution across all sampled locations. Five pheromone compounds including (Z)-7-dodecenyl acetate (Z7-12:OAc), (Z)-7-tetradecenyl acetate (Z7-14:OAc), (Z)-9-tetradecenyl acetate (Z9-14:OAc), (Z)-11-tetradecenyl acetate (Z11-14:OAc) and (Z)-11-hexadecenyl acetate (Z11-16:OAc), were detected in the pheromone glands of female moths of both mitotypes, with Z9-14:OAc being the most abundant. The relative percentage composition of Z9-14:OAc was similar in both mitotypes. However, the R mitotype had a 2.7 times higher relative percentage composition of Z7-12:OAc compared to the C mitotype moth, while the C mitotype moth had a 2.4 times higher relative percentage composition of Z11-16:OAc than the R mitotype moth. Male moths of both mitotypes exhibited similar responses to the pheromone compounds, showing the strongest responses to Z9-14:OAc and Z7-12:OAc in electrophysiological and behavioural assays. There was mating between R and C mitotypes with egg production comparable to mating within the same mitotype. Our results revealed that differences between the two S. frugiperda mitotypes are characterized by female moth pheromone composition rather than male moth responses to the pheromones, and that this does not prevent hybridisation between the mitotypes, which may have implications for their management.

Rice responds to Spodoptera frugiperda infestation via epigenetic regulation of H3K9ac in the jasmonic acid signaling and phenylpropanoid biosynthesis pathways.

KEY MESSAGE: This study provided important insights into the complex epigenetic regulatory of H3K9ac-modified genes involved in the jasmonic acid signaling and phenylpropanoid biosynthesis pathways of rice in response to Spodoptera frugiperda infestation. Physiological and molecular mechanisms underlying plant responses to insect herbivores have been well studied, while epigenetic modifications such as histone acetylation and their potential regulation at the genomic level of hidden genes remain largely unknown. Histone 3 lysine 9 acetylation (H3K9ac) is an epigenetic marker widely distributed in plants that can activate gene transcription. In this study, we provided the genome-wide profiles of H3K9ac in rice (Oryza sativa) infested by fall armyworm (Spodoptera frugiperda, FAW) using CUT&Tag-seq and RNA-seq. There were 3269 and 4609 up-regulated genes identified in plants infested by FAW larvae for 3 h and 12 h, respectively, which were mainly enriched in alpha-linolenic acid and phenylpropanoid pathways according to transcriptomic analysis. In addition, CUT&Tag-seq analysis revealed increased H3K9ac in FAW-infested plants, and there were 422 and 543 up-regulated genes enriched with H3K9ac observed at 3 h and 12 h after FAW feeding, respectively. Genes with increased H3K9ac were mainly enriched in the transcription start site (TSS), suggesting that H3K9ac is related to gene transcription. Integrative analysis of both RNA-seq and CUT&Tag-seq data showed that up-expressed genes with H3K9ac enrichment were mainly involved in the jasmonic acid (JA) and phenylpropanoid pathways. Particularly, two spermidine hydroxycinnamoyl transferase genes SHT1 and SHT2 involved in phenolamide biosynthesis were highly modified by H3K9ac in FAW-infested plants. Furthermore, the Ossht1 and Ossht2 transgenic lines exhibited decreased resistance against FAW larvae. Our findings suggest that rice responds to insect herbivory via H3K9ac epigenetic regulation in the JA signaling and phenolamide biosynthesis pathways.

Identification of inducible CYP3 and CYP4 genes associated with abamectin tolerance in the fat body and Malpighian tubules of Spodoptera litura.

Abamectin, as a broad-spectrum bioinsecticide, has been widely used for the control of Lepidoptera insects, resulting in different levels of resistance to abamectin in Spodoptera litura. Cytochrome P450 monooxygenases (P450s) are known for their important roles in insecticide detoxification. In this study, the expression of SlCYP6B40, SlCYP4L12 and SlCYP9A32 in the fat body, and SlCYP4S9, SlCYP6AB12, SlCYP6AB58, SlCYP9A75a and SlCYP9A75b in Malpighian tubules was found to be significantly upregulated after abamectin exposure. SlCYP6AE44 and SlCYP6AN4 were simultaneously upregulated in these two tissues after abamectin exposure. Ectopically overexpressed SlCYP6AE44, SlCYP9A32 and SlCYP4S9 in transgenic Drosophila conferred tolerance to abamectin. In addition, homology modeling and molecular docking results suggested that SlCYP6AE44, SlCYP9A32 and SlCYP4S9 may be capable of binding with abamectin. These results demonstrate that upregulation of CYP3 and CYP4 genes may contribute to abamectin detoxification in S. litura and provide information for evidence-based insecticide resistance management strategies.

JAK/STAT signaling regulated intestinal regeneration defends insect pests against pore-forming toxins produced by Bacillus thuringiensis.

A variety of coordinated host-cell responses are activated as defense mechanisms against pore-forming toxins (PFTs). Bacillus thuringiensis (Bt) is a worldwide used biopesticide whose efficacy and precise application methods limits its use to replace synthetic pesticides in agricultural settings. Here, we analyzed the intestinal defense mechanisms of two lepidopteran insect pests after intoxication with sublethal dose of Bt PFTs to find out potential functional genes. We show that larval intestinal epithelium was initially damaged by the PFTs and that larval survival was observed after intestinal epithelium regeneration. Further analyses showed that the intestinal regeneration caused by Cry9A protein is regulated through c-Jun NH (2) terminal kinase (JNK) and Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways. JAK/STAT signaling regulates intestinal regeneration through proliferation and differentiation of intestinal stem cells to defend three different Bt proteins including Cry9A, Cry1F or Vip3A in both insect pests, Chilo suppressalis and Spodoptera frugiperda. Consequently, a nano-biopesticide was designed to improve pesticidal efficacy based on the combination of Stat double stranded RNA (dsRNA)-nanoparticles and Bt strain. This formulation controlled insect pests with better effect suggesting its potential use to reduce the use of synthetic pesticides in agricultural settings for pest control.

Different development and fecundity between Spodoptera frugiperda USA and China populations, influenced by ecdysone-related genes.

The fall armyworm (FAW), Spodoptera frugiperda, is one of the most harmful plant pests in the world and is globally distributed from the American continent to the Asian region. The FAW USA population (Sf-USA) and China population (Sf-CHN), which belong to corn strain, showed different developmental periods and fecundity rates in lab conditions. Sf-USA had faster development and higher fecundity compared with Sf-CHN. To examine these differences, transcriptomic data from two FAW populations were analyzed and compared. Twelve gigabytes of transcripts were read from each sample and 21,258 differentially expressed genes (DEGs) were detected. DEGs with log2 fold change ≥ 2 were identified and compared in two populations. In comparison to the Sf-CHN, we discovered that 3471 and 3851 individual DEGs upregulated and downregulated, respectively. Comparing transcriptome profiles for differential gene expression revealed several DEGs, including 39 of ecdysone (E)-, 25 of juvenile hormone-, and 15 of insulin-related genes. We selected six of E-related genes, such as Neverland, Shade, Ecdysone receptor, Ecdysone-inducible protein 74 (E74), E75, and E78 from DEGs. Gene expressions were suppressed by RNA interference to confirm the physiological functions of the selected genes from Sf-USA. The Sf-USA showed developmental retardation and a decrease in fecundity rate by suppression of E-related genes. These findings show that biological characteristics between Sf-USA and Sf-CHN are influenced by E-related genes.

Characterization and functional analysis of UDP-glycosyltransferases reveal their contribution to phytochemical flavone tolerance in Spodoptera litura.

Efficient detoxification is the key factor for phytophagous insect to adapt to phytochemicals. However, the role of uridine diphosphate (UDP)-glycosyltransferases (UGTs) in insect anti-defense to phytochemical flavone is largely unknown. In this study, 52 UGT genes were identified in Spodoptera litura and they presented evident gene duplication. UGT played a crucial part in larval tolerance to flavone because the enzyme activity and transcriptional level of 77 % UGT members were remarkably upregulated by flavone administration and suppression of UGT enzyme activity and gene expressions significantly increased larval susceptibility to flavone. Bacteria coexpressing UGTs had high survival rates under flavone treatment and flavone was dramatically metabolized by UGT recombinant cells, which indicated the involvement of UGTs in flavone detoxification. What's more, ecdysone pathway was activated by flavone. Topical application of 20-hydroxyecdysone highly upregulated UGT enzyme activity and more than half of UGT expressions. The effects were opposite when ecdysone receptor (EcR) and ultraspiracle (USP)-mediated ecdysone signaling pathway was inhibited. Furtherly, promoter reporter assays of 5 UGT genes showed that their transcription activities were notably increased by cotransfection with EcR and USP. In consequence, this study suggested that UGTs were involved in flavone detoxification and their transcriptional expressions were regulated by ecdysone pathway.

An OBP gene highly expressed in non-chemosensory tissues affects the phototaxis and reproduction of Spodoptera frugiperda.

Insect odorant binding proteins (OBPs) were initially regarded as carriers of the odorants involved in chemosensation. However, it had been observed that a growing number of OBP genes exhibited broad expression patterns beyond chemosensory tissues. Here, an OBP gene (OBP31) was found to be highly expressed in the larval ventral nerve cord, adult brain and male reproductive organ of Spodoptera frugiperda. An OBP31 knockout strain (OBP31-/- ) was generated by CRISPR/Cas9 mutagenesis. For OBP31-/- , the larvae needed longer time to pupate, but there was no difference in the pupal weight between OBP31-/- and wild type (WT). OBP31-/- larvae showed stronger phototaxis than the WT larvae, indicating the importance of OBP31 in light perception. For mating rhythm of adults, OBP31-/- moths displayed an earlier second mating peak. In the cross-pairing of OBP31-/- and WT moths, the mating duration was longer, and hatchability was lower in OBP31-/- group and OBP31+/- ♂ group than that in the WT group. These results suggested that OBP31 played a vital role in larval light perception and male reproductive process and could provide valuable insights into understanding the biological functions of OBPs that were not specific in chemosensory tissues.