The chromosomal-scale genome sequencing and assembly of Athetis lepigone.

Athetis lepigone is an emerging highly polyphagous insect pest reported to cause crop damage in several European and Asian countries. However, our understanding of its genetic adaptation mechanisms has been limited due to lack of high-quality genetic resources. In this study, we present a chromosomal-level genome of A. lepigone, representing the first species in the genus of Athetis. We employed PacBio long-read sequencing and Hi-C technologies to generate 612.49 Mb genome assembly which contains 42.43% repeat sequences with a scaffold N50 of 20.9 Mb. The contigs were successfully clustered into 31 chromosomal-size scaffolds with 37% GC content. BUSCO assessment revealed a genome completeness of 97.4% with 96.3 identified as core Arthropoda single copy orthologs. Among the 17,322 genes that were predicted, 15,965 genes were functionally annotated, representing a coverage of 92.17%. Furthermore, we revealed 106 P450, 37 GST, 27 UGT, and 74 COE gene families in the genome of A. lepigone. This genome provides a significant and invaluable genomic resource for further research across the entire genus of Athetis.

CRISPR/Cas9-Mediated Knockout of the PxJHBP Gene Resulted in Increased Susceptibility to Bt Cry1Ac Protoxin and Reduced Lifespan and Spawning Rates in Plutella xylostella.

Juvenile hormone binding protein (JHBP) is a key regulator of JH signaling, and crosstalk between JH and 20-hydroxyecdysone (20E) can activate and fine-tune the mitogen-activated protein kinase cascade, leading to resistance to insecticidal proteins from Bacillis thuringiensis (Bt). However, the involvement of JHBP in the Bt Cry1Ac resistance of Plutella xylostella remains unclear. Here, we cloned a full-length cDNA encoding JHBP, and quantitative real-time PCR (qPCR) analysis showed that the expression of the PxJHBP gene in the midgut of the Cry1Ac-susceptible strain was significantly higher than that of the Cry1Ac-resistant strain. Furthermore, CRISPR/Cas9-mediated knockout of the PxJHBP gene significantly increased Cry1Ac susceptibility, resulting in a significantly shorter lifespan and reduced fertility. These results demonstrate that PxJHBP plays a critical role in the resistance to Cry1Ac protoxin and in the regulation of physiological metabolic processes associated with reproduction in adult females, providing valuable insights to improve management strategies of P. xylostella.

Biochemistry and transcriptomic analyses of Phthorimaea absoluta (Lepidoptera: Gelechiidae) response to insecticides.

Phthorimaea absoluta is an invasive solanaceous plant pest with highly devastating effects on tomato plant. Heavy reliance on insecticide use to tackle the pest has been linked to insecticide resistance selection in P. absoluta populations. To underline insights on P. absoluta insecticide resistance mechanisms to diamides and avermectins, we evaluated the transcriptomic profile of parental (field-collected) and F8 (lab-reared) populations. Furthermore, to screen for the presence of organophosphate and pyrethroid resistance, we assessed the gene expression levels of acetylcholinesterase (ace1) and para-type voltage-gated sodium channel (VGSG) genes in the F1 to F8 lab-reared progeny of diamide and avermectin exposed P. absoluta field-collected populations. The VGSG gene showed up-regulation in 12.5% and down-regulation in 87.5% of the screened populations, while ace1 gene showed up-regulation in 37.5% and down-regulation in 62.5% of the screened populations. Gene ontology of the differentially expressed genes from both parental and eighth generations of diamide-sprayed P. absoluta populations revealed three genes involved in the metabolic detoxification of diamides in P. absoluta. Therefore, our study showed that the detoxification enzymes found could be responsible for P. absoluta diamide-based resistance, while behavioural resistance, which is stimulus-dependent, could be attributed to P. absoluta avermectin resistance.

CRISPR/Cas9-mediated ebony knockout causes melanin pigmentation and prevents moth Eclosion in Ectropis grisescens.

Ectropis grisescens (Lepidoptera: Geometridae) is a destructive tea pest in China. Mimesis, characterized by changing body color, is an important trait of E. grisescens larvae. Hence, identifying melanin pathway-related genes may contribute to developing new pest control strategies. In the present study, we cloned Egebony, a gene potentially involved in melanin pigmentation in E. grisescens, and subsequently conducted CRISPR/Cas9-mediated targeted mutagenesis of Egebony to analyze its role in pigmentation and development. At the larvae, prepupae, and pupae stages, Egebony-knockout individuals exhibited darker pigmentation than the wild-type. However, Egebony knockout did not impact the colors of sclerotized appendants, including ocelli, setae, and claws. While mutant pupae could successfully develop into moths, they were unable to emerge from the puparium. Notably, embryo hatchability and larval survival of mutants remained normal. Further investigation indicated that mutant pupae exhibited significantly stronger shearing force than the wild-type, with the pigmented layer of mutant pupae appearing darker and thicker. Collectively, these results suggest that the loss of Egebony might increase the rigidity of the puparium and prevent moth eclosion. This study provides new insights into understanding the function and diversification of ebony in insect development and identifies a lethal gene that can be manipulated for developing effective pest control strategies.

Electrophysiological and pharmacological properties of the slowpoke channel in the diamondback moth, Plutella xylostella.

The slowpoke channel responds to the intracellular calcium concentration and the depolarization of the cell membrane. It plays an important role in maintaining the resting potential and regulating the homeostasis of neurons, but it can also regulate circadian rhythm, sperm capacitation, ethanol tolerance, and other physiological processes in insects. This renders it a potentially useful target for the development of pest control strategies. There are relatively few studies on the slowpoke channels in lepidopteran pests, and their pharmacological properties are still unclear. So, in this study, the slowpoke gene of Plutella xylostella (Pxslo) was heterologous expressed in HEK293T cells, and the I-V curve of the slowpoke channel was measured by whole cell patch clamp recordings. Results showed that the slowpoke channel could be activated at -20 mV with 150 µM Ca2+. The subsequent comparison of the electrophysiological characteristics of the alternative splicing site E and G deletions showed that the deletion of the E site enhances the response of the slowpoke channel to depolarization, while the deletion of the G site weakens the response of the slowpoke channel to depolarization. Meanwhile, the nonspecific inhibitors TEA and 4-AP of the Kv channels, and four pesticides were tested and all showed an inhibition effect on the PxSlo channel at 10 or 100 µM, suggesting that these pesticides also target the slowpoke channel. This study enriches our understanding of the slowpoke channel in Lepidopteran insects and can aid in the development of relevant pest management strategies.

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.

Development and characterization of microsatellite markers for population genetics of the cocoa pod borer Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillaridae).

The cocoa pod borer (CPB) Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillaridae) is one of the major constraints for cocoa production in South East Asia. In addition to cultural and chemical control methods, autocidal control tactics such as the Sterile Insect Technique (SIT) could be an efficient addition to the currently control strategy, however SIT implementation will depend on the population genetics of the targeted pest. The aim of the present work was to search for suitable microsatellite loci in the genome of CPB that is partially sequenced. Twelve microsatellites were initially selected and used to analyze moths collected from Indonesia, Malaysia, and the Philippines. A quality control verification process was carried out and seven microsatellites found to be suitable and efficient to distinguish differences between CPB populations from different locations. The selected microsatellites were also tested against a closely related species, i.e. the lychee fruit borer Conopomorpha sinensis (LFB) from Vietnam and eight loci were found to be suitable. The availability of these novel microsatellite loci will provide useful tools for the analysis of the population genetics and gene flow of these pests, to select suitable CPB strains to implement the SIT.

What one genus of showy moths can say about migration, adaptation, and wing pattern.

The Ornate Moth, Utetheisa ornatrix, has served as a model species in chemical ecology studies for decades. Like in the widely publicized stories of the Monarch and other milkweed butterflies, the Ornate Moth and its relatives are tropical insects colonizing whole continents assisted by their chemical defenses. With the recent advances in genomic techniques and evo-devo research, it is becoming a model for studies in other areas, from wing pattern development to phylogeography, from toxicology to epigenetics. We used a genomic approach to learn about Utetheisa's evolution, detoxification, dispersal abilities, and wing pattern diversity. We present an evolutionary genomic analysis of the worldwide genus Utetheisa, then focusing on U. ornatrix. Our reference genome of U. ornatrix reveals gene duplications in the regions possibly associated with detoxification abilities, which allows them to feed on toxic food plants. Finally, comparative genomic analysis of over 100 U. ornatrix specimens from the museum with apparent differences in wing patterns suggest the potential roles of cortex and lim3 genes in wing pattern formation of Lepidoptera and the utility of museum-preserved collection specimens for wing pattern research.

Nuclear and mitochondrial genomes of Polylopha cassiicola: the first assembly in Chlidanotinae (Tortricidae).

Tortricidae is one of the largest families in Lepidoptera, including subfamilies of Tortricinae, Olethreutinae, and Chlidanotinae. Here, we assembled the gap-free genome for the subfamily Chlidanotinae using Illumina, Nanopore, and Hi-C sequencing from Polylopha cassiicola, a pest of camphor trees in southern China. The nuclear genome is 302.03 Mb in size, with 36.82% of repeats and 98.4% of BUCSO completeness. The karyotype is 2n = 44 for males. We identified 15412 protein-coding genes, 1052 tRNAs, and 67 rRNAs. We also determined the mitochondrial genome of this species and annotated 13 protein-coding genes, 22 tRNAs, and one rRNA. These high-quality genomes provide valuable information for studying phylogeny, karyotypic evolution, and adaptive evolution of tortricid moths.

Evaluation of different Kabuli chickpea genotypes against Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in relation to biotic and abiotic factors.

Background: The chickpea pod borer Helicoverpa armigera (Hübner) is a significant insect pest of chickpea crops, causing substantial global losses. Methods: Field experiments were conducted in Central Punjab, Pakistan, to investigate the impact of biotic and abiotic factors on pod borer population dynamics and infestation in nine kabuli chickpea genotypes during two cropping seasons (2020-2021 and 2021-2022). The crops were sown in November in both years, with row-to-row and plant-to-plant distances of 30 and 15 cm, respectively, following a randomized complete block design (RCBD). Results: Results showed a significant difference among the tested genotypes in trichome density, pod wall thickness, and leaf chlorophyll contents. Significantly lower larval population (0.85 and 1.10 larvae per plant) and percent damage (10.65% and 14.25%) were observed in genotype Noor-2019 during 2020-2021 and 2021-2022, respectively. Pod trichome density, pod wall thickness, and chlorophyll content of leaves also showed significant variation among the tested genotypes. Pod trichome density and pod wall thickness correlated negatively with larval infestation, while chlorophyll content in leaves showed a positive correlation. Additionally, the larval population positively correlated with minimum and maximum temperatures, while relative humidity negatively correlated with the larval population. Study results explore natural enemies as potential biological control agents and reduce reliance on chemical pesticides.

The complete mitochondrial genome of Chibiraga houshuaii (Lepidoptera, Limacodidae) and its phylogenetic implications.

Chibiraga is a mall East Asian genus in the family Limacodidae (slug-moths). The latter includes many agricultural pests. Mitochondrial genome analysis is an important tool for studying insect molecular identification and phylogenetics. However, there are very few mitogenome sequences available for Limacodidae species, and none for the genus Chibiraga at all. To explore the mitogenome features of Chibiraga and verify its phylogenetic position, the complete mitogenome of Chibiraga houshuaii was sequenced and annotated. The complete 15,487 bp genome encoded 37 mitochondrial genes, including 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a control region (CR). Most of the PCGs had typical ATN start codons and terminated with TAA or a single T residue. UUA (Leu2), AUU (Ile), UUU (Phe), AUA (Met) and AAU (Asn) were the five most frequently used codons. All tRNAs were folded into cloverleaf secondary structure, except for trnS1, which lacked the DHU arm. Phylogenetic analyses within the superfamily Zygaenoidea were performed based on multiple datasets from mitochondrial genes. The results showed that the families Phaudidae, Limacodidae and Zygaenidae were respectively recovered as monophyly; C. houshuaii was clustered in a clade with nettle type larvae in Limacodidae.

CRISPR/Cas9-mediated knockout of scarlet gene produces eye color mutants in the soybean looper, Chrysodeixis includens.

The CRISPR/Cas9 technology has greatly progressed research on non-model organisms, demonstrating successful applications in genome editing for various insects. However, its utilization in the case of the soybean looper, Chrysodeixis includens, a notable pest affecting soybean crops, has not been explored due to constraints such as limited genomic information and the embryonic microinjection technique. This study presents successful outcomes in generating heritable knockout mutants for a pigment transporter gene, scarlet, in C. includens through CRISPR/Cas9-mediated mutagenesis. The scarlet locus identified in the genome assembly of C. includens consists of 14 exons, with a coding sequence extending for 1,986 bp. Two single guide RNAs (sgRNAs) were designed to target the first exon of scarlet. Microinjection of these two sgRNAs along with the Cas9 protein into fresh embryos resulted in the successful production of variable phenotypes, particularly mutant eyes. The observed mutation rate accounted for about 16%. Genotype analysis revealed diverse indel mutations at the target site, presumably originating from double-strand breaks followed by the nonhomologous end joining repair, leading to a premature stop codon due to frame shift. Single-pair mating of the mutant moths produced G1 offspring, and the establishment of a homozygous mutant strain occurred in G2. The mutant moths exhibited lightly greenish or yellowish compound eyes in both sexes, confirming the involvement of scarlet in pigmentation in C. includens. Notably, the CRISPR/Cas9-mediated genome editing technique serves as a visible phenotypic marker, demonstrating its proof-of-concept applicability in C. includens, as other pigment transporter genes have been utilized as visible markers to establish genetic control for various insects. These results provide the first successful case that the CRISPR/Cas9 method effectively induces mutations in C. includes, an economically important soybean insect pest.

The complete mitochondrial genome of Leucoptera coffeella (Lepidoptera: Lyonetiidae) and phylogenetic relationships within the Yponomeutoidea superfamily.

The coffee leaf miner (Leucoptera coffeella) is one of the major pests of coffee crops in the neotropical regions, and causes major economic losses. Few molecular data are available to identify this pest and advances in the knowledge of the genome of L. coffeella will contribute to improving pest identification and also clarify taxonomy of this microlepidoptera. L. coffeella DNA was extracted and sequenced using PacBio HiFi technology. Here we report the complete L. coffeella circular mitochondrial genome (16,407 bp) assembled using Aladin software. We found a total of 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs) and an A + T rich-region and a D-loop. The L. coffeella mitochondrial gene organization is highly conserved with similarities to lepidopteran mitochondrial gene rearrangements (trnM-trnI-trnQ). We concatenated the 13 PCG to construct a phylogenetic tree and inferred the relationship between L. coffeella and other lepidopteran species. L. coffeella is found in the Lyonetiidae clade together with L. malifoliella and Lyonetia clerkella, both leaf miners. Interestingly, this clade is assigned in the Yponomeutoidea superfamily together with Gracillariidae, and both superfamilies displayed species with leaf-mining feeding habits.

Immunological regulation by Toll-1 and Spätzle-4 in larval density-dependent prophylaxis of the oriental armyworm, Mythimna separata.

High population density has been shown to alter insect prophylactic immunity. Toll-Spätzle pathway performs a key function in insect innate immune response. To determine the role of Toll and Spätzle, two main components of Toll-Spätzle pathway, in the density-dependent prophylaxis of Mythimna separata. We identified full-length cDNA encoding the Toll-1 and Spätzle-4 genes in M. separata (designed MsToll-1 and Ms Spätzle-4). Both MsToll-1 and MsSpätzle-4 were expressed throughout all developmental stages. MsToll-1 expression was highly in fat body and brain and MsSpätzle-4 was highly expressed in brain and Malpighian tubule. With increased larval density, MsToll-1 expression was markedly up-regulated. MsSpätzle-4 expression was found to be raised in larvae that were fed in high density (5 and 10 larvae per jar). Co-immunoprecipitation assays demonstrated that MsToll-1 interacted with MsSpätzle-4. Immune-related genes transcriptions were considerably reduced in high-density larvae MsToll-1 (or MsSpätzle-4) was silenced by dsRNA injection. Meanwhile, a discernible reduction in the survival rate of the larvae exposed to Bacillus thuringiensis infection with silence of MsToll-1 (or MsSpätzle-4) was observed. This study implies that prophylactic immunity was influenced by crowded larvae via modulating the Toll-Spätzle pathway in M. separata and allow for a new understanding of into density-dependent prophylaxis in insects.

Trichoplusia ni Transcriptomic Responses to the Phytosaponin Aglycone Hederagenin: Sex-Related Differences.

Many plant species, particularly legumes, protect themselves with saponins. Previously, a correlation was observed between levels of oleanolic acid-derived saponins, such as hederagenin-derived compounds, in the legume Medicago truncatula and caterpillar deterrence. Using concentrations that reflect the foliar levels of hederagenin-type saponins, the sapogenin hederagenin was not toxic to 4th instar caterpillars of the cabbage looper Trichoplusia ni nor did it act as a feeding deterrent. Female caterpillars consumed more diet than males, presumably to obtain the additional nutrients required for oogenesis, and are, thus, exposed to higher hederagenin levels. When fed the hederagenin diet, male caterpillars expressed genes encoding trypsin-like proteins (LOC113500509, LOC113501951, LOC113501953, LOC113501966, LOC113501965, LOC113499659, LOC113501950, LOC113501948, LOC113501957, LOC113501962, LOC113497819, LOC113501946, LOC113503910) as well as stress-responsive (LOC113503484, LOC113505107) proteins and cytochrome P450 6B2-like (LOC113493761) at higher levels than females. In comparison, female caterpillars expressed higher levels of cytochrome P450 6B7-like (LOC113492289). Bioinformatic tools predict that cytochrome P450s could catalyze the oxygenation of hederagenin which would increase the hydrophilicity of the compound. Expression of a Major Facilitator Subfamily (MFS) transporter (LOC113492899) showed a hederagenin dose-dependent increase in gene expression suggesting that this transporter may be involved in sapogenin efflux. These sex-related differences in feeding and detoxification should be taken into consideration in insecticide evaluations to minimize pesticide resistance.

Characterization of the complete mitochondrial genome of Spirobolus grahami (Diplopoda: Spirobolidae) with phylogenetic analysis.

Diplopoda is one of the most diverse and important groups of soil arthropods, but little research has been done on their phylogenetic relationship and evolution. Here, we sequenced and annotated the complete mitochondrial genomes of Spirobolus grahami. The total mitogenome of S. grahami was typical circular, double-stranded molecules, with 14,875 bp in length, including 13 protein-coding genes, 22 tRNAs, two rRNAs, and one control region. Base composition analysis suggested that the mitochondrial sequences were biased toward A and T, with A + T content of 58.68%. The mitogenomes of S. grahami exhibited negative AT and positive GC skews. Most of the 13 PCGs had ATN as the start codon, except COX1 start with CGA, and most PCGs ended with the T stop codon. The dN/dS values for most PCGs were lower than 1, suggesting that purifying selection was likely the main driver of mitochondrial PCG evolution. Phylogenetic analyses based on 13 PCGs using BI and ML methods support the classification of genus Spirobolus and Tropostreptus. Glomeridesmus spelaeus is distantly related to the other Diplopoda species.

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.

Opsin mutants alter host plant selection by color vision in the nocturnal invasive pest Tuta absoluta.

In insects, vision is crucial in finding host plants, but its role in nocturnal insects is largely unknown. Vision involves responses to specific spectra of photon wavelengths and opsins plays an important role in this process. Long-wavelength sensitive opsin (LW opsin) and blue-sensitive opsin (BL opsin) are main visual opsin proteins and play important in behavior regulation.We used CRISPR/Cas9 technology to mutate the long-wavelength-sensitive and blue wavelength-sensitive genes and explored the role of vision in the nocturnal invasive pest Tuta absoluta. Light wave experiments revealed that LW2(-/-) and BL(-/-) mutants showed abnormal wavelength tropism. Both LW2 and BL mutations affected the preference of T. absoluta for the green environment. Mutations in LW2 and BL are necessary to inhibit visual attraction. The elimination of LW2 and BL affected the preference of leaf moths for green plants, and mutations in both induced a preference in moths for white plants. Behavioral changes resulting from LW2(-/-) and BL(-/-) mutants were not affected by sense of smell, further supporting the regulatory role of vision in insect behavior. To the best of our knowledge, this is the first study to reveal that vision, not smell, plays an important role in the host-seeking behavior of nocturnal insects at night, of which LW2 and BL opsins are key regulatory factors. These study findings will drive the development of the "vision-ecology" theory.

Key role of the ryanodine receptor I4790K mutation in mediating diamide resistance in Plutella xylostella.

The diamondback moth Plutella xylostella, a global insect pest of cruciferous vegetables, has evolved resistance to many classes of insecticides including diamides. Three point mutations (I4790M, I4790K, and G4946E) in the ryanodine receptor of P. xylostella (PxRyR) have been identified to associate with varying levels of resistance. In this study, we generated a knockin strain (I4790K-KI) of P. xylostella, using CRISPR/Cas9 to introduce the I4790K mutation into PxRyR of the susceptible IPP-S strain. Compared to IPP-S, the edited I4790K-KI strain exhibited high levels of resistance to both anthranilic diamides (chlorantraniliprole 1857-fold, cyantraniliprole 1433-fold) and the phthalic acid diamide flubendiamide (>2272-fold). Resistance to chlorantraniliprole in the I4790K-KI strain was inherited in an autosomal and recessive mode, and genetically linked with the I4790K knockin mutation. Computational modeling suggests the I4790K mutation reduces the binding of diamides to PxRyR by disrupting key hydrogen bonding interactions within the binding cavity. The approximate frequencies of the 4790M, 4790K, and 4946E alleles were assessed in ten geographical field populations of P. xylostella collected in China in 2021. The levels of chlorantraniliprole resistance (2.3- to 1444-fold) in these populations were significantly correlated with the frequencies (0.017-0.917) of the 4790K allele, but not with either 4790M (0-0.183) or 4946E (0.017-0.450) alleles. This demonstrates that the PxRyR I4790K mutation is currently the major contributing factor to chlorantraniliprole resistance in P. xylostella field populations within China. Our findings provide in vivo functional evidence for the causality of the I4790K mutation in PxRyR with high levels of diamide resistance in P. xylostella, and suggest that tracking the frequency of the I4790K allele is crucial for optimizing the monitoring and management of diamide resistance in this crop pest.

An epigenetic change in a moth is generated by temperature and transmitted to many subsequent generations mediated by RNA.

Epigenetic changes in sexually reproducing animals may be transmitted usually only through a few generations. Here we discovered a case where epigenetic change lasts 40 generations. This epigenetic phenomenon occurs in the short antennae (sa) mutation of the flour moth (Ephestia kuehniella). We demonstrate that is probably determined by a small RNA (e.g., piRNA, miRNA, tsRNA) and transmitted in this way to subsequent generations through the male and female gametes. The observed epigenetic change cancels sa mutation and creates a wild phenotype (a moth that appears to have no mutation). It persists for many generations (40 recorded). This epigenetic transgenerational effect (suppression homozygous mutation for short antennae) in the flour moth is induced by changes during ontogenetic development, such as increased temperature on pupae development, food, different salts in food, or injection of RNA from the sperm of already affected individuals into the eggs. The epigenetic effect may occasionally disappear in some individuals and/or progeny of a pair in the generation chain in which the effect transfers. We consider that the survival of RNA over many generations has adaptive consequences. It is mainly a response to environmental change that is transmitted to offspring via RNA. In this study, we test an interesting epigenetic effect with an unexpected length after 40 generations and test what is its cause. Such transfer of RNA to subsequent generations may have a greater evolutionary significance than previously thought. Based on some analogies, we also discuss of the connection with the SIR2 gene.