Lepidoptera Research Articles

Nap1 is essential for eupyrene spermatogenesis and migration in Plutella xylostella.

Spermatogenesis is a key process for the sexual reproduction species. In lepidopteran insects, spermatogenesis produces two different types of sperms, in which eupyrene sperm carry genomic DNA and fertilize eggs, whereas apyrene sperm are necessary for eupyrene sperm to enter eggs. However, functional genetic studies of spermatogenesis in Plutella xylostella remain a longstanding puzzle even though the phenomenon in lepidoptera has been widely documented more than a century. In this study, we particularly focus on the gene Nap1 which belongs to the Nucleosome assembly protein family. Our findings revealed that Nap1 was highly expressed in the testes, and the disruption of PxNap1 induced male sterility in P. xylostella, while the fertility of mutant females was comparable to wild-type females. Additionally, through immunofluorescence staining analysis, we found that the eupyrene sperm bundles presented diffusedly scattered nuclei in PxNap1 mutant males, while the nuclei in the wild-type were clustered together presented as needle shape. We also found that PxNap1 deficiency hinders the transfer of eupyrene sperm to the bursa copulatrix and spermatheca of females. However, the apyrene spermatogenesis was not affected in the PxNap1 mutant. RNA-seq analyses indicated that the defects of eupyrene sperm in PxNap1 mutants were related to energy metabolic such as pentose and glucuronate interconversions, biosynthesis of amino acids, and pentose phosphate pathway. Our study demonstrates that PxNap1 plays crucial function in eupyrene spermatogenesis and eupyrene sperm migration. Our research provides valuable insights for the genetic factors underlying reproductive processes in Lepidopteran insects.

Chronologically inappropriate morphogenesis (Chinmo) is required for maintenance of larval stages of fall armyworm

Broad complex (Br-C) and eip93F (E93) transcription factors promote insect metamorphosis from larva to pupa and from pupa to adult, respectively. Recently, chronologically inappropriate morphogenesis (Chinmo) has been proposed as a larval specifier in Drosophila melanogaster. However, whether Chinmo is required for larval maintenance in lepidopteran insects, the underlying mechanisms involved in maintaining the larval stage, and its interactions with the JH signaling pathway are not well understood. Here, we used a binary transgenic CRISPR/Cas9 system to knockout Chinmo and Kr-h1 (primary response gene in the JH signaling pathway) in the fall armyworm (FAW). Kr-h1 knockout induced premature metamorphosis only after L5 (penultimate), whereas Chinmo and Kr-h1 double knockout induced premature metamorphosis in L3. Sequencing and differential gene expression (DEG) analysis of RNA isolated from mutants and single-cell multiome ATAC analysis of Chinmo, Kr-h1, and Chinmo and Kr-h1 double knockout Sf9 cells revealed that Chinmo participates in chromatin modifications that prevent the promoter accessibility and expression of metamorphosis promoting genes. These results suggest that Chinmo is a larval specifier that plays a major role in preventing metamorphosis in early larval stages by controlling chromatin accessibility near the promoters of genes such as Br-C and E93 required for pupal and adult development.

Diet composition and diet choice of Lesser Mouse-tailed Bat Rhinopoma hardwickii (Gray, 1831) (Rhinopomatidae: Chiroptera)

The food composition and food choice of R. hardwickii were assessed through guano analysis at different seasons and geographical locations. Guano samples of R. hardwickii were collected from the roost sites of the Gangetic plains and arid region of Uttar Pradesh. Each intact pellet was dissolved and recognizable insect body parts like legs, antennae, wings, and mouth parts were separated and photographed. Each insect remnant was identified to its lowest rank as much as possible. A total of 10 roost sites of R. hardwickii were observed in the arid region and Gangetic plains of Uttar Pradesh and all of them were found in historical monuments. A total of 61 pellets of 10 sites yielded 1,035 remnants of insects. The highest percentage of remnants belongs to legs, followed by wings, antennae, abdominal segments, and mouthparts. The remnants belong to eight insect orders such as Coleoptera, Hemiptera, Orthoptera, Hymenoptera, Dermaptera, Diptera, Lepidoptera, and Plecoptera. The remnants of order Hemiptera showed the highest frequency of occurrence followed by orders Coleoptera, Orthoptera, and Hymenoptera, and these four orders of insects constitute the major portion of the diet of R. hardwickii. The orders Dermaptera, Diptera, Lepidoptera, and Plecoptera contributed a small proportion to the diet of R. hardwickii. The remnants of orders Lepidoptera and Plecoptera were occasional. The result of the current study shows that the food choice of R. hardwickii did not differ significantly across roost sites, while differed seasonally. Further, it reveals that the Lesser Mouse-tailed Bat acts as a potential and natural insect balancing agent.

RESARCHES REGARDING THE ENTOMOFAUNA FROM THE OSTROVENI - DOLJ AREA

From our observation regarding the entomofauna of Ostroveni area, during the research, 116 species of Arthropods were collected and identified (belonging to the orders: Acari, Orthoptera, Dermaptera, Thysanoptera, Homoptera, Hymenoptera, Neuroptera, Odonata, Coleoptera, Lepidoptera, Diptera) of which 93 species (80%) are harmful and 23 species (20%) are useful species.The most numerous order was Coleoptera (35%), followed by the order Lepidoptera (19%), the order Heteroptera and Hymenoptera (9%) each.

Temperature-Based Extraction, Characterization by Gas Chromatography-Mass Spectrometry and Fourier Transform Infrared Spectroscopy with Prospective Antibacterial Properties of Eri (Philosamia ricini) Pupal Oil.

Lepidopteran insects are novel source of omega-3 fatty acids as revealed by the studies recently. In the current study, we assessed the effects of four different drying temperatures (50, 60, 70, and80 ºC) of eri silkworm (Philosamia ricini) pupal oil (EPO)abbreviated asEPO50, 60, 70, and 80 followed by conducting the characterization and antibacterial efficacy studies. Gas chromatography-mass spectroscopy (GC-MS) and Fourier transform infrared (FTIR) were used for chemical profiling of EPOs. The agar-well diffusion method was performed to evaluate the antibacterial activities. The trend of this study clearly revealed that enhanced drying percentage of temperatures showed raising saturated fats and reduced unsaturated fats. However, the best suitable temperature was found at 60 ºC with high unsaturated fatty acids, including Omega-3. Further, at higher temperature, FTIR analysis revealed an increasing complexity of functional groups with potential bioactive compounds. EPOs were found to have significant antibacterial activity against S. typhi and S. aureus at all tested (10, 20, 30, and 40 µL) volumes used in the study.

Assessment of Bee Diversity and Abundance (Hymenoptera: Apoidea) on Caesalpinia bonduc L. during the Flowering Season in the Southwestern Aravalli Region (Abu Road), Sirohi, Rajasthan, India

The aim of the present study was to document the diversity and abundance of bee species visiting Caesalpinia bonduc (Family: Fabaceae), a plant species found in the foothills of the Aravalli range, in the south-western Rajasthan area of Abu Road Tehsil, Rajasthan, India. The surveys were conducted during the flowering period from July 2021 to September 2021 and again from July 2022 to September 2022. A comprehensive analysis revealed the presence of sixteen bee species (Hymenoptera: Apoidea) visiting Caesalpinia bonduc, belonging to nine different genera and categorized under four families: Apidae, Andrenidae, Halictidae, and Megachilidae. Among these families, Apidae recorded the highest number of bee species, with 11 in total: Amegilla dizona, A. violacea, A. zonata, Apis cerana, A. dorsata, A. florea, Ceratina binghami, Tetragonula iridipennis, Xylocopa aestuans, X. pubescens, and X. fenestrata. The families Megachilidae and Halictidae were each represented by two species: Megachile bicolor, M. cephalotes and Nomia crassipes, Pseudapis oxybeloides respectivily. The highest abundance of all Bee species was recorded between 12.00- 2:00 pm across various time intervals. The highest abundance of Apis florea (13.4 bees/m²/5 minutes) was recorded between 12:00 and 2:00 PM, making it the most abundant species among the 16 bee species observed. At different time intervals, Apis florea consistently recorded the highest number of individuals in each interval. Other entomofauna observed on C. bonduc included Spoladea recurvalis (Fabricius, 1775), Suastus gremius (Fabricius, 1798), Hasora chromus (Cramer, 1780), and Spindasis ictis (Hewitson, 1865), which belong to the order Lepidoptera. Additionally, Poekilocerus pictus (Fabricius, 1775) from the order Orthoptera and Gametis versicolor (Fabricius, 1775) from the order Coleoptera were also recorded.

Physiological and Molecular Mechanisms of Lepidopteran Insects: Genomic Insights and Applications of Genome Editing for Future Research.

Lepidopteran insects are a major threat to global agriculture, causing significant crop losses and economic damage. Traditional pest control methods are becoming less effective due to the rapid evolution of insecticide resistance. This study explores the current status and genomic characteristics of 1315 Lepidopteran records, alongside an overview of relevant research, utilizing advanced functional genomics techniques, including RNA-seq and CRISPR/Cas9 gene-editing technologies to uncover the molecular mechanisms underlying insecticide resistance. Our genomic analysis revealed significant variability in genome size, assembly quality, and chromosome number, which may influence species' biology and resistance mechanisms. We identified key resistance-associated genes and pathways, including detoxification and metabolic pathways, which help these insects evade chemical control. By employing CRISPR/Cas9 gene-editing techniques, we directly manipulated resistance-associated genes to confirm their roles in resistance, demonstrating their potential for targeted interventions in pest management. These findings emphasize the value of integrating genomic data into the development of effective and sustainable pest control strategies, reducing reliance on chemical insecticides and promoting environmentally friendly integrated pest management (IPM) approaches. Our study highlights the critical role of functional genomics in IPM and its potential to provide long-term solutions to the growing challenge of Lepidopteran resistance.

Antifeedant Nanosuspension Formula of <i>Tithonia diversifolia</i> Leaf Extract by Emulsion Inverse Method to Control <i>Crocidolomia pavonana</i> Cabbage Pest Insect

The leaf extract from Tithonia diversifolia is recognized for its ability to deter feeding in various Lepidoptera insect pests, including the larvae of Crocidolomia pavonana. Presently, transformation efforts from conventional formulations into nano-based formulations for biopesticides exhibit enhanced effectiveness and efficiency. Utilizing a low-energy process, an inversion emulsion facilitates the dispersion of the extract suspension in an organic solvent into a water-immiscible solvent using a suitable surfactant. The forming nano-size droplets in water (t1, t2, t3, t4) are influenced by the ratio of surfactant and organic suspension (Water: Tween 80: Organic suspension). The emulsification method successfully formulated T. diversifolia leaf extract, into dispersed nano-size and submicron suspensions in water. The t3 formula exhibits the smallest nano-size dispersed in water (D=23.6 ± 39.6 nm; polydispersity index IP=0.702) and enhanced wettability, evident in the lower contact angle of the droplet on the cabbage leaf surface (49.4°) compare with the control group. The Phytochemicals confirmed by IR-spectra analysis identified the phenols, alkaloids, and steroids constituents of leaf extract, which are known to have antifeedant properties. The enhanced antifeedant properties of T. diversifolia nanosuspension against C. pavonana third-instar larvae demonstrated by the antifeedant test results showing that t3 is the most successful deterrent larvae feeding activity compared to the control (P<0.05), due to the highest total antifeedant coefficient (74.27%) in a category medium antifeedant activity, while the non-emulsification displayed the lowest antifeedant coefficient (25.36%) in a category as low antifeedant activity. T. diversifolia leaf extract with a nano-based formula succeeded resulting in dispersed nano-size and submicron suspension in aqueous media, thereby reducing surface tension and enhancing wettability on the leaf surface during application. The improved dispersion of antifeedant nanosuspension on the leaf surface results in more effective delivery to target insects.

PGE2 Binding Affinity of Hemocyte Membrane Preparations of Manduca sexta and Identification of the Receptor-Associated G Proteins in Two Lepidopteran Species.

Prostaglandin E2 (PGE2) is an eicosanoid that mediates a range of physiological actions in vertebrates and invertebrates, including reproduction and immunity. The PGE2 receptor was identified and functionally assessed in two lepidopteran insects, Manduca sexta and Spodoptera exigua. However, its binding affinity to the receptor has not been reported. The PGE2 receptor is a G-protein coupled receptor (GPCR) although its corresponding G-protein is not identified. PGE2 binding assays were performed with membrane preparations from hemocytes of M. sexta larvae. We recorded an optimal binding in 4 h reactions conducted at pH 7.5 with 12 nM tritium-labeled PGE2. We found that hemocytes express a single population of PGE2 binding sites with a high affinity (Kd = 35 pmol/mg protein), which are specific and saturable. The outcomes of experiments on the influence of purine nucleotides suggested these are functional GPCRs. A bioinformatics analysis led to a proposed trimeric G-protein in the S. exigua transcriptome, in which the Gα subunit is classified into five different types: Gα(o), Gα(q), Gα(s), Gα(12), and Gα(f). After confirming expressions of these five types in S. exigua, individual RNA interference (RNAi) treatments were applied to the larvae using gene-specific double-stranded RNAs. RNAi treatments specific to Gα(s) or Gα(12) gene expression significantly suppressed the cellular immune responses although the RNAi treatments specific to other three Gα components did not. While PGE2 treatments led to elevated hemocyte cAMP or Ca2+ levels, the RNAi treatments specific to Gα(s) or Gα(12) genes led to significantly reduced second messenger levels under PGE2, although the RNAi treatments specific to the other three Gα components did not. These results showed that the PGE2 receptor has high PGE2 affinity in the nanomolar range and binds G-proteins containing a Gα(s) or Gα(12) trimeric component in S. exigua and M. sexta, and likely, all lepidopteran insects.

Molecular Diagnostics for Monitoring Insecticide Resistance in Lepidopteran Pests

Chemical control methods to prevent crop damage have long been directly implicated in the selection of lepidoptera insect populations resistant to insecticides. More recently, new products featuring different modes of action (MoA), developed to mitigate the negative effects of control management on both producers and the environment, are rapidly losing efficacy due to the emergence of insect resistance. Among these, certain resistances are associated with molecular changes in the genomes of pest insects that are valuable for developing molecular markers for diagnostic tools, particularly the point mutations. Molecular diagnosis represents an innovative solution for insecticide resistance management (IRM) practices, allowing for the effective monitoring of insecticide resistance. This approach facilitates decision making by enabling the timely alternation between different modes of action (MoAs). In this context, this review focuses on the major lepidopteran pests that affect globally significant crops, discussing the impacts of insecticide resistance. It gathers literature on diagnostic methods; provides a comparative overview of the advantages of different techniques in terms of efficiency, cost, precision, sensitivity, and applicability; and highlights several novel diagnostic tools. Additionally, this review explores the coffee leaf miner, Leucoptera coffeella, as an applied model to illustrate potential approaches for more effective and sustainable control strategies.

Bacillus thuringiensis strains with high insecticidal activity against insect larvae of the orders Coleoptera and Lepidoptera

Abstract The concept of sustainable agricultural development is aimed at reducing the use of chemical pest control agents. Since the use of chemical pesticides poses a significant environmental threat, one of the possible solutions to overcome this problem is the study of biological approaches, in particular, the use of entomopathogenic microorganisms as biological pest control agents. Insecticidal activity of over 220 strains of Bacillus thuringiensis (Bt) and Lysinibacillus sphaericus from the collection of the Laboratory of Spore-Forming Microorganisms of the Microbial Depository Center of the Scientific and Production Center “Armbiotechnology” and 15 strains isolated from samples of dead insects (imago) of representatives of various genera of the Coleoptera order was studied. The pathogenic effect of the strains was assessed in relation to the insect species Tenebrio molitor, Zophobas morio, Leptinotarsa decemlineata Say and Pieris brassicae, belonging to the Coleoptera and Lepidoptera orders. Among the test objects, the representative of Lepidoptera Pieris brassicae demonstrated the highest degree of sensitivity to B. thuringiensis strains up to 100%, with some Bt strains causing 60–100% mortality of Tenebrio molitor and Zophobas morio larvae of different ages. The larvae of the beetle Leptinotarsa decemlineata Say were also partially susceptible, presenting mortality levels of between 31 and 60%. The article is devoted to the study of Bt bacterial strains that have pronounced insecticidal activity against insect larvae of the orders Coleoptera and Lepidoptera.

A near chromosome-level genome assembly of a ghost moth (Lepidoptera, Hepialidae)

Ghost moths are an unusual family of primitive moths (Lepidoptera: Hepialidae) known for their large body size and crepuscular adult activity. These moths represent an ancient lineage, frequently have soil dwelling larvae, and are adapted to high elevations, deserts, and other extreme environments. Despite being rather speciose with more than 700 species, there is a dearth of genomic resources for the family. Here, we present the first high quality, publicly available hepialid genome, generated from an Andean species of ghost moth, Druceiella hillmani. Our genome assembly has a length of 2,586 Mbp with contig N50 of 28.1 Mb and N50 of 29, and BUSCO completeness of 97.1%, making it one of the largest genomes in the order Lepidoptera. Our assembly is a vital resource for future research on ghost moth genomics.

Host adaptability of Microplitis manilae (Hymenoptera: Braconidae) in Noctuidae.

Biological control is an effective and sustainable method of integrated pest management. Microplitis manilae (Hymenoptera: Braconidae) is a common internal parasitoid wasp of the order Lepidoptera. To determine a suitable host species for the reproduction of parasitoids, it is essential to assess host suitability and parasitic potential of the parasitized pests. Herein, we investigated host selection and exploitation of M. manilae. We evaluated the parasitic efficacy of M. manilae in 4 pest species in the Spodoptera and Mythimna genera of the Noctuidae family. The results indicated that the parasitism rate of M. manilae on 3 species in the Spodoptera genus is higher than on Mythimna separata in the Mythimna genus, with M. manilae exhibiting a higher parasitism rate and shorter development duration on Spodoptera litura compared to other species. The parasitism rate for 1st instars hosts was 86.67 ± 0.04%, while the development duration was 14.1 ± 0.03 days. However, when parasitizing the 3rd instar of Spodoptera frugiperda, parasitoids showed a higher sex ratio, of 0.71 ± 0.05. Additionally, M. manilae had parasitic effect on M. separata, providing a new choice for its parasitism. The results identify the optimal host, which could enhance and reproduction rate and survival rate of M. manilae, thus facilitating their large-scale propagation. Understanding the parasitic effects of M. manilae on pests can further its field application, also plays a major role in promoting the development of biological control technologies and sustainable agricultural production.

Conserved fungal effector NLS1 suppresses Lepidoptera insect immunity by targeting the host defense protein Hdd11.

Entomopathogenic fungi have been widely used as the main mycoinsecticide for controlling agricultural and forest pests. The effector molecules of these mycopathogens have evolved to adapt to their hosts. The role of fungal effectors in evading the host immune system in insects remains mainly unclear. We characterized the widely distributed fungal effector necrosis-inducing-like secreted protein 1 (NLS1) in the entomopathogenic fungus Metarhizium robertsii. Our findings revealed the presence of M. robertsii NLS1 (MrNLS1) in host hemocytes during the early stage of hemocoel infection. MrNLS1 knock down (ΔMrNLS1) reduced fungal pathogenicity during infection and altered the expression of host immune genes. The molecular docking results and the yeast 2-hybrid assay confirmed that MrNLS1 interacts with the host defense protein Hdd11. The phylogenetic analysis indicated that Hdd11 is conserved across a broad range of Lepidoptera species. Knock down of hdd11 in Helicoverpa armigera, Bombyx mori, and Galleria mellonella markedly suppressed their immune responses against M. robertsii. However, no significant difference was observed in the mean lethal time between hdd11-knockdown Lepidoptera species infected with ΔMrNLS1 and those infected with wild-type M. robertsii. Therefore, in Lepidoptera insects, Hdd11 is essential for fungal defense. In conclusion, M. robertsii infects Lepidoptera insects by targeting host Hdd11 through its protein MrNLS1, thereby suppressing the host immune response. Our findings clarify the molecular mechanisms underlying fungal infection pathogenesis.

From Proline to Chlorantraniliprole Mimics: Computer-Aided Design, Simple Preparation, and Excellent Insecticidal Profiles.

Chlorantraniliprole (CHL), a favored agricultural insecticide, is renowned for its high efficiency and broad-spectrum effectiveness against lepidoptera insects. However, the urgency for new insecticide development is underscored by the intricate multistep preparation process and modest overall yields of CHL, along with the escalating challenge of insect resistance. In response, we have crafted CHL mimics from proline employing computer-aided drug design. Molecular docking analysis of CHL's interactions with the ryanodine receptor (RyR) revealed that the nitrogen atom within the pyrazole moiety does not engage in pivotal interactions. Its removal may not abolish bioactivity entirely but could substantially simplify the synthetic process, thereby enhancing atom economy. This revelation prompted the exclusion of nitrogen and the subsequent formation of a pyrrole ring, enabling the meticulous design of synthetic pathways characterized by cost-effective precursors, streamlined synthesis, the avoidance of toxic reagents, minimal instrumentation, and high yields in the pursuit of innovative RyR modulators. Among these modulators, A1 and B1, obtained with yields exceeding 60%, showcased exceptional insecticidal potency, with LC50 values spanning from 0.12 to 1.47 mg L-1 against P. xylostella and M. separate. The inhibitory effects of these two compounds on insect detoxification enzymes imply a reduced likelihood of eliciting resistance in comparison to CHL, a finding further corroborated by their insecticidal potency against resistant pests. Moreover, molecular docking, MD simulations, and DFT calculations provided valuable structural insights, potentially unraveling the superior insecticidal activity of these two molecules, and thus paving the way for developing more potent insecticides.

Unveiling symbiotic bacterial communities in insects feeding on the latex-rich plant Ficus microcarpa.

The diversity and health of insects that feed on plants are closely related to their mutualistic symbionts and host plants. These symbiotic partners significantly influence various metabolic activities in these insects. However, the symbiotic bacterial community of toxic plant feeders still needs further characterisation. This study aims to unravel bacterial communities associated with the different species of insect representing three insect orders: Thysanoptera, Hemiptera, and Lepidoptera, along with their predicted functional role, which exclusively feeds on latex-rich plant species Ficus microcarpa. By using 16S rRNA gene high-throughput sequencing, the analysis was able to define the major alignment of the bacterial population, primarily comprising Proteobacteria, Firmicutes, Bacteroidota, Actinobacteriota, and Acidobacteriota. Significant differences in symbiotic organisms between three insect groups were discovered by the study: hemipterans had Burkholderia and Buchnera, and lepidopterans had Acinetobacter. At the same time, Pseudomonas was detected in high abundance in both lepidopteran and thysanopteran insects. Furthermore, these symbionts exhibit consistent core functions, potentially explaining how different insects can consume the same host plant. The identified core functions of symbionts open avenues for innovative approaches in utilising these relationships to develop environment-friendly solutions for pest control, with broader implications for agriculture and environmental conservation.

Molecular characterization and identification of insect pollinators of Valeriana jatamansi Jones from Shimla Hills, Western Himalaya, India

Valeriana jatamansi Jones a perennial medicinal herb belongs to the family Caprifoliaceae and highly pollinated by wild insect pollinators. In present investigation a total of 51 species of flower visitors were reported on V. jatamansi at altitude range of 1,927 to 2,850 m, in different localities of Shimla Hills, Western Himalaya. The collected insect pollinator species were taxonomically identified and 29 of them were olecularly characterized and identified using the cytochrome c oxidase subunit 1 (COI) sequence from mitochondrial DNA (mtDNA). BLAST analysis revealed 98 to 100 per cent similarity with the existing GenBank sequences. The average AT content was found to be significantly higher (69.8%) compared to the GC content (30.0%), indicating AT bias in all sequences. Phylogenetic analysis of 29 different species of insect ‘pollinator of V. jatamansi revealed two clades, one shows phylogenetic relationship between 28 species which belongs to four orders Diptera, Hymenoptera, Hemiptera and Lepidoptera and the other shows the one species which belongs to order Coleoptera. This study contributes to increasing the number of published accounts of NCBI and helps to accurately distinguish the pollinator fauna of Valeriana Jatamansi.

EpOME mediates the immune resolution and its alkoxide analog enhances the virulence of microbial insecticides against the legume pod borer, Maruca vitrata

Excessive and unnecessary immune responses cause serious adverse effects due to self-tissue damage and energy consumption, particularly at the late stage of infection to terminate the induced immunity. Unlike mammals, which use long-chain fatty acid oxylipins, C18 oxygenated polyunsaturated fatty acids are suggested to act as immune resolvins in insects, including two epoxyoctadecamonoenoic acids (9,10-EpOME and 12,13-EpOME). This study investigated the physiological roles of EpOMEs in immune resolution in the lepidopteran insect, Maruca vitrata. The levels of two EpOMEs in the larvae increased during the late infection stage upon immune challenge. At their peak concentrations at 96 h post-infection (pi), both EpOMEs were found at similar levels: 323.18 pg/mg body weight for 9,10-EpOME and 322.07 pg/mg body weight for 12,13-EpOME. Both EpOMEs inhibited cellular and humoral immune responses, with 12,13-EpOME being more potent than 9,10-EpOME. Genes associated with EpOME synthase and degradation, identified as Mv-CYP1 and Mv-sEH, were detected in various developmental stages and tissues of M. vitrata. RNA interference (RNAi) targeting Mv-CYP1 failed to inhibit the immune response, whereas RNAi targeting Mv-sEH enhanced the immunosuppression. In contrast to the acute (< 12 h pi) immune response involving eicosanoid biosynthesis, the expression of these two genes linked to EpOME metabolism increased significantly at the late infection stage (> 12 h pi). Several alkoxide analogs of EpOME, with the epoxide group replaced by an alkoxide group, were synthesized; one such derivative demonstrated substantially greater efficacy than the natural EpOMEs in inhibiting the immune response. Additionally, using EpOME alkoxide significantly increased the effectiveness of microbial insecticides. Moreover, exposing young larvae to sublethal doses of EpOME alkoxide or sEH inhibitor induced severe developmental delays. These results suggest a novel strategy for insect pest control using insect immune resolvin analogs.

Molecular Functions and Physiological Roles of Gustatory Receptors of the Silkworm Bombyx mori.

Complete elucidation of members of the gustatory receptor (Gr) family in lepidopteran insects began in the silkworm Bombyx mori. Grs of lepidopteran insects were initially classified into four subfamilies based on the results of phylogenetic studies and analyses of a few ligands. However, with further ligand analysis, it has become clear that plant secondary metabolites are important targets not only for Grs in the bitter subfamily but also for the Drosophila melanogaster Gr43a orthologue subfamily and Grs in the sugar subfamily. Gene knockout experiments showed that B. mori Gr6 (BmGr6) and BmGr9 are involved in the recognition of the feeding-promoting compounds chlorogenic acid and isoquercetin in mulberry leaves by the maxillary palps, suggesting that these Grs are responsible for palpation-dependent host recognition without biting. On the other hand, BmGr expression was also confirmed in nonsensory organs. Midgut enteroendocrine cells that produce specific neuropeptides were shown to express specific BmGrs, suggesting that BmGrs are involved in the induction of endocrine secretion in response to changes in the midgut contents. Furthermore, gene knockout experiments indicated that BmGr6 is indeed involved in the secretion of myosuppressin. On the other hand, BmGr9 was shown to induce signal transduction that is not derived from the intracellular signaling cascade mediated by G proteins but from the fructose-regulated cation channel of BmGr9 itself. Cryogenic electron microscopy revealed the mechanism by which the ion channel of the BmGr9 homotetramer opens upon binding of fructose to the ligand-binding pocket. Research on BmGrs has contributed greatly to our understanding of the functions and roles of Grs in insects.

Efficient CRISPR/Cas9-mediated genome editing in the European corn borer, Ostrinia nubilalis.

The European corn borer (Ostrinia nubilalis) is an agricultural pest and burgeoning model for research on speciation, seasonal adaptation and insect resistance management. Although previous work in O. nubilalis has identified genes associated with differences in life cycle, reproduction, and resistance to Bt toxins, the general lack of a robust gene-editing protocol for O. nubilalis has been a barrier to functional validation of candidate genes. Here, we demonstrate an efficient and practical methodology for heritable gene mutagenesis in O. nubilalis using the CRISPR/Cas9 genome editing system. Precise loss-of-function (LOF) mutations were generated at two circadian clock genes, period (per) and pigment-dispersing factor receptor (pdfr), and a developmental gene, prothoracicotropic hormone (ptth). Precluding the need for a visible genetic marker, gene-editing efficiency remained high across different single guide RNAs (sgRNA) and germline transmission of mutations to F1 offspring approached 100%. When single or dual sgRNAs were injected at a high concentration, gene-specific phenotypic differences in behaviour and development were identified in F0 mutants. Specifically, F0 gene mutants demonstrated that PER, but not PDFR, is essential for normal timing of eclosion. PTTH F0 mutants were significantly heavier and exhibited a higher incidence of diapause. This work will accelerate future studies of gene function in O. nubilalis and facilitate the development of similar screens in other Lepidopteran and non-model insects.