Trophic microRNA: Post-transcriptional regulation of target genes and larval development impairment in Plutella xylostella upon precursor and mature microRNA ingestion.

MicroRNAs (miRNAs) are post-transcriptional gene regulators. In the miRNA pathway's cytoplasmic part, the miRNA is processed from a hairpin-structured precursor to a double-stranded (ds) mature RNA and ultimately to a single-stranded mature miRNA. In insects, ingesting these two ds forms can regulate the target gene expression; this inspired the trophic miRNA's use as a functional genomics and pest management tool. However, systematic studies enabling comparisons of pre- and mature forms, dosages, administration times and instar-wise effects on target transcripts and phenotypes, which can help develop a miRNA administration method, are unavailable due to the different focuses of the previous investigations. We investigated the impact of trophically delivered Px-let-7 miRNA on the lepidopteran pest Plutella xylostella, to compare the efficacies of its pre- and ds-mature forms. Continuous feeding on the miRNA-supplemented diet suppressed expressions of FTZ-F1 and E74, the target ecdysone pathway genes. Both the pre-let-7 and mature let-7 miRNA forms similarly downregulated the target transcripts in all four larval instars. Pre-let-7 and let-7 ingestions decreased larval mass and instar duration and increased mortality in all instars, exhibiting adverse effects on larval growth and development. miRNA processing Dicer-1 and AGO-1's upregulations upon miRNA ingestion denoted the systemic miRNA spread in larval tissues. The scrambled sequence controls did not affect the target transcripts, suggesting the sequence-specific targeting by the mature miRNA and hairpin cassette's non-involvement in the target downregulation. This work provides a framework for miRNA and target gene function analyses and potentiates the trophic miRNA's utility in pest management.

Silence of Aminopeptidase N 2 gene reveals the trade-offs for acquiring Cry1Ac resistance in Plutella xylostella.

Bacillus thuringiensis (Bt) is widely used as a biopesticide worldwide. To date, at least eight pest species have been found to be resistant to Bt in the field. As the first pest that was reported having resistance to Bt in the field, considerable research has been done on the mechanisms of Bt resistance in Plutella xylostella. However, whether the acquisition of Bt resistance by P. xylostella comes at a fitness cost is also a valuable question. In this study, Aminopeptidase-N 2 (APN2), a Cry toxin receptor gene of P. xylostella, was knocked down by RNA interference, resulting in improved resistance to Cry1Ac. It was also found that larval mortality of APN2 knockdown P. xylostella was significantly higher than that of the control, while the pupation rate, pupal weight, eclosion rate, fecundity (egg/female), hatchability, and female adult longevity were significantly lower in APN2 knockdown P. xylostella than in the control. These results illustrate that if Cry1Ac resistance was obtained only through the reduction of APN2 expression, P. xylostella would need to incur some fitness costs for it.

Comparative transcriptome analysis provides comprehensive insight into the molecular mechanisms of heat adaption in Plutella xylostella.

Plutella xylostella is one of the most destructive pests for cruciferous vegetables, and is adaptability to different environmental stressors. However, we still know little about the molecular mechanisms of how P. xylostella adapt to thermal stress. Here, the comparative transcriptome analysis was conducted from the samples of control (27 °C, CK) and heat treatment (40 °C, 40 T) P. xylostella. The results showed 1253 genes were differentially expressed, with 624 and 629 genes up- and down-regulated respectively. The annotation analysis demonstrated that "Energy production and conversion", "Protein processing in endoplasmic reticulum", "Peroxisome" and "Tyrosine metabolism" pathways were significantly enriched. Additionally, we found the expression levels of heat shock protein genes (Hsps), cuticle related genes and mitochondrial genes were significantly up-regulated in 40 T insects, suggesting their vital roles in improving adaption to heat stress. Importantly, the SOD activity and MDA content of P. xylostella were both identified to be increased under high temperature stress, indicating the elevated antioxidant reactions might be involved in response to heat stress. In conclusion, the present study offered us an overview of gene expression changes after 40 °C treatments, and found some critical pathways and genes of P. xylostella might play the critical roles in resisting heat stress.

Insecticide resistance and characteristics of mutations related to target site insensitivity of diamondback moths in Taiwan.

Diamondback moth (DBM, Plutella xylostella) is the most significant pest of cruciferous vegetables as they rapidly develop high-level resistance to many insecticides. Monitoring DBM susceptibility and target-site mutation frequency is essential for pest control. In this study, 10 insecticides were tested on 11 field populations. Frequencies of target-site mutations (including para, ace1, Rdl1, RyR1, and nAChRα6 genes) were estimated by pyrosequencing. Insecticides registered after 2007 for DBM control in Taiwan, i.e., spinetoram, chlorantraniliprole, chlorfenapyr, metaflumizone, and flubendiamide, showed >80% mortality toward several populations; Bacillus thurigiensis, emamectin benzoate, and chlorfluazuron showed medium to low efficacy in all populations; and tolfenpyrad and mevinphos were highly ineffective. Susceptibility to insecticides varied substantially among populations: eight out of nine populations were highly susceptible to spinetoram, but only one was susceptible to flubendiamide. Target-site mutations related to organophosphates, pyrethroids, fipronil, and diamides were detected in all populations, but there were few spinosad and spinetoram mutations. Our three-year field study demonstrated rapid efficacy loss for all insecticides tested, particularly for more toxic insecticides. Skipped-generation selection of a field DBM strain to emamectin benzoate, metaflumizone, chlorantraniliprole, and flubendiamide revealed that mortality rates dropped from 60 to 80% to <10% after 6 generations. Next-generation sequencing was performed to identify possible target gene mutations. A resistance management program that considers the instability of resistance to some chemicals and pertinent data on resistance mechanisms should be established. Identifying compounds to overcome high-frequency field DBM point mutations could be beneficial for pest control.

microRNA-8514-5p regulates adipokinetic hormone/corazonin-related peptide receptor to affect development and reproduction of Plutella xylostella.

BACKGROUND: Dicer1 plays a crucial role in regulating the development and reproduction of insects. Knockout of Dicer1 causes pupal deformity, low eclosion and low fecundity in Plutella xylostella, but the mechanism behind this phenomenon is not clear. This study aims to identify differentially-expressed genes and miRNAs in the Dicer1-knockout strain (ΔPxDcr-1) and assess their impact on the reproduction and development of P. xylostella. RESULTS: The knockout of Dicer1 affected the expression of genes including the adipokinetic hormone/corazonin-related peptide receptor (PxACPR). The expression of PxACPR was upregulated, and the expression of miR-8514-5p was downregulated in ΔPxDcr-1 of P. xylostella. The dual luciferase reporter assay and pull-down assay showed that miR-8514-5p bound to PxACPR in vitro and in vivo. The expression profiles demonstrated a negative correlation between PxACPR mRNA and miR-8514-5p in different developmental stages of the wild-type strain. Both the miR-8514-5p agomir and double-stranded RNA of ACPR (dsPxACPR) injected into the pre-pupae inhibited the mRNA level of PxACPR, causing high mortality and deformity of pupae, and low fecundity and hatching rate, which were consistent with the phenotype of ΔPxDcr-1. The injection of miR-8514-5p antagomir caused a similar phenotype to the injection of miR-8514-5p agomir. Additionally, the injection of miR-8514-5p antagomir significantly rescued the phenotype caused by dsPxACPR. CONCLUSION: These results indicate that miR-8514-5p affects the development and reproduction of P. xylostella by regulating PxACPR, and the homeostasis of PxACPR expression is essential for the development and reproduction of P. xylostella. © 2024 Society of Chemical Industry.

Verification of AKT and CDK5 Gene and RNA Interference Combined with Irradiation to Mediate Fertility Changes in Plutella xylostella (Linnaeus).

Plutella xylostella (Linnaeus) mainly damages cruciferous crops and causes huge economic losses. Presently, chemical pesticides dominate its control, but prolonged use has led to the development of high resistance. In contrast, the sterile insect technique provides a preventive and control method to avoid the development of resistance. We discovered two genes related to the reproduction of Plutella xylostella and investigated the efficacy of combining irradiation with RNA interference for pest management. The results demonstrate that after injecting PxAKT and PxCDK5, there was a significant decrease of 28.06% and 25.64% in egg production, and a decrease of 19.09% and 15.35% in the hatching rate compared to the control. The ratio of eupyrene sperm bundles to apyrene sperm bundles also decreased. PxAKT and PxCDK5 were identified as pivotal genes influencing male reproductive processes. We established a dose-response relationship for irradiation (0-200 Gy and 200-400 Gy) and derived the irradiation dose equivalent to RNA interference targeting PxAKT and PxCDK5. Combining RNA interference with low-dose irradiation achieved a sub-sterile effect on Plutella xylostella, surpassing either irradiation or RNA interference alone. This study enhances our understanding of the genes associated with the reproduction of Plutella xylostella and proposes a novel approach for pest management by combining irradiation and RNA interference.

Frequencies of insecticide resistance mutations detected by the amplicon sequencing in Plutella xylostella (Lepidoptera: Plutellidae) and Spodoptera exigua (Lepidoptera: Noctuidae) from China.

The globally prevalent pests, Diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) and Beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), pose significant threats to cruciferous vegetables. They have rapidly developed resistance to a wide range of insecticides, leading to significant yield losses and increased control expenses. In this study, we have established an efficient approach utilizing amplicon sequencing to detect the frequency of 15 target resistance mutant sites in 6 molecular targets, acetylcholinesterase 1 (ACE1), chitin synthase 1 (CHS1), the γ-aminobutyric acid receptor (GABAR), glutamate-gated chloride channel (GluCl), voltage-gated sodium channels (NaV), and ryanodine receptor (RyR) in P. xylostella and the frequency of 11 mutations in 5 molecular targets (except GluCl) in S. exigua in China. Our findings indicate that P. xylostella exhibits remarkably high frequency (over 88.67%) in pyrethroid resistance-related mutations T929I and L1014F of NaV. In S. exigua, the frequencies of L659F mutation were ranging from 41.92% to 74.89%. In addition, the organophosphorus resistance-related mutations A298S and G324A of ACE1 were detected at frequencies ranging from 34.29% to 75.66%, and these 2 mutations occurred simultaneously (from 29.22% to 65.79%) in P. xylostella. An interannual variation in mutation frequency from 2019 to 2021 was found for P. xylostella in HNCS. The frequency of A298S and G324A mutations steadily increased while the frequency of G4946E and I4790M mutations continuously decreased. These results unveil a worrisome scenario of multiple resistance sites in these 2 pests in China and provide valuable insights for the practical application of pesticides in the field.

Functional analysis of three odorant receptors in Plutella xylostella response to repellent activity of 2,3-dimethyl-6-(1-hydroxy)-pyrazine.

Plutella xylostella is an important pest showing resistance to various chemical pesticides, development of botanical pesticides is an effective strategy to resolve above problem and decrease utilization of chemical pesticides. Previous study showed that 2,3-dimethyl-6-(1-hydroxy)-pyrazine has significant repellent activity to P. xylostella adult which mainly effect to the olfactory system, however the molecular targets and mechanism are still unclear. Based on the RNA-Seq and RT-qPCR data, eight ORs (Odorant receptor) in P. xylostella were selected as candidate targets response to repellent activity of 2,3-dimethyl-6-(1-hydroxy)-pyrazine. Here, most of the ORs in P. xylostella were clustered into three branches, which showed similar functions such as recognition, feeding, and oviposition. PxylOR29, PxylOR31, and PxylOR46 were identified as the potential molecular targets based on the results of repellent activity and EAG response tests to the adults which have been injected with dsRNA, respectively. Additionally, the three ORs were higher expressed in antenna of P. xylostella, followed by those in the head segment. Furthermore, it was found that the bindings between these three ORs and 2,3-dimethyl-6-(1-hydroxy)-pyrazine mainly depend on the hydrophobic effect of active cavities, and the binding to PxylOR31 was more stabler and easier with an energy of -16.34 kcal/mol, together with the π-π T-shaped interaction at PHE195 site. These findings pave the way for the complete understanding of pyrazine repellent mechanisms.

Functional types of long trichoid sensilla responding to sex pheromone components in Plutella xylostella.

Sex pheromones, which consist of multiple components in specific ratios promote intraspecific sexual communications of insects. Plutella xylostella (L.) is a worldwide pest of cruciferous vegetables, the mating behavior of which is highly dependent on its olfactory system. Long trichoid sensilla on male antennae are the main olfactory sensilla that can sense sex pheromones. However, the underlying mechanisms remain unclear. In this study, 3 sex pheromone components from sex pheromone gland secretions of P. xylostella female adults were identified as Z11-16:Ald, Z11-16:Ac, and Z11-16:OH in a ratio of 9.4 : 100 : 17 using gas chromatography - mass spectrometry and gas chromatography with electroantennographic detection. Electrophysiological responses of 581 and 385 long trichoid sensilla of male adults and female adults, respectively, to the 3 components were measured by single sensillum recording. Hierarchical clustering analysis showed that the long trichoid sensilla were of 6 different types. In the male antennae, 52.32%, 5.51%, and 1.89% of the sensilla responded to Z11-16:Ald, Z11-16:Ac, and Z11-16:OH, which are named as A type, B type, and C type sensilla, respectively; 2.93% named as D type sensilla responded to both Z11-16:Ald and Z11-16:Ac, and 0.34% named as E type sensilla were sensitive to both Z11-16:Ald and Z11-16:OH. In the female antennae, only 7.53% of long trichoid sensilla responded to the sex pheromone components, A type sensilla were 3.64%, B type and C type sensilla were both 0.52%, D type sensilla were 1.30%, and 1.56% of the sensilla responded to all 3 components, which were named as F type sensilla. The responding long trichoid sensilla were located from the base to the terminal of the male antennae and from the base to the middle of the female antennae. The pheromone mixture (Z11-16:Ald : Z11-16:Ac : Z11-16:OH = 9.4 : 100 : 17) had a weakly repellent effect on female adults of P. xylostella. Our results lay the foundation for further studies on sex pheromone communications in P. xylostella.

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.

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.

Toxicity, biochemical and molecular docking studies of Acacia nilotica L., essential oils against insect pests.

Botanical essential oils are natural insecticides derived from plants, offering eco-friendly alternatives to synthetic chemicals for pest control. In this study, the essential oils were extracted from Acacia nilotica seed cotyledons, and their toxicity was tested against insect pests. Furthermore, the chemical components of the essential oils were identified through gas chromatography-mass spectrometry (GC-MS) analysis. The essential oil extracted from A. nilotica seeds exhibited the highest mortality rates of 60% and 98% in Culex quinquefasciatus, and 60% and 96.66% mortality in Plutella xylostella at 24 and 48 h after treatment, respectively. The essential oils resulted in a lower LC50 of 159.263 ppm/mL, and LC90 of 320.930 ppm/mL within 24 h. In 48 h, the LC50 was 52.070 ppm/mL and the LC90 was 195.123 ppm/mL for C. quinquefasciatus. In the essential oil treatment of P. xylostella, the lower LC50 was 165.900 ppm/mL, and the LC90 was 343.840 ppm/mL 24 h after the treatment. At 48 h post-treatment, the LC50 decreased to 62.965 ppm/mL, and the LC90 decreased to 236.795 ppm/mL in P. xylostella. The study investigated the impact of essential oils on insect enzymes 24 h after treatment. The study revealed significant changes in the levels of insect enzymes, including a decrease in acetylcholinesterase enzymes and an increase in glutathione S-transferase compared to the control group. Essential oils had minimal effects, resulting in mortality rates of 30.66% and 46% at 24 and 48 h after treatment on Artemia salina. After 48 h, minimal toxic effects of essential oils were observed on E. eugeniae, with a mortality rate of 11.33%. The GC-MS analysis of A. nilotica seed-derived essential oils revealed ten major chemical constituents, including 6-hydroxymellein, phthalic acid, trichloroacetic acid, hexadecane, acetamide, heptacosane, eicosane, pentadecane, 1,3,4-eugenol, and chrodrimanin B. Among these constituents, Heptacosane is the major chemical component, and this molecule has a high potential for involvement in insecticidal activity.

Inhibition Effect of Non-Host Plant Volatile Extracts on Reproductive Behaviors in the Diamondback Moth Plutella xylostella (Linnaeus).

The pest management of Plutella xylostella, the global pest of cruciferous plants, is primarily dependent upon continued applications of insecticides, which has led to severe insecticide resistance and a series of ecological concerns. The essential oils (EOs) of non-host plants are considered to have a high application potential in pest behavioral control. In P. xylostella, the insecticidal properties, antifeeding activities, and oviposition inhibition effects of many EOs have been studied in larvae and female moths. However, less focus has been placed on the inhibitory effect on sex pheromone communication during courtship, which is vital for the reproduction of the offspring. In this study, by combining electrophysiological studies, laboratory behavioral assays, and field traps, we demonstrated that non-host plant EOs significantly inhibited the reproductive behaviors of both sexes. Notably, the calamus (Acorus gramineus) EO inhibited the preference of male moths for synthetic sex pheromone blends and reduced the egg-laying number of female moths on host plants, with the highest inhibition rates of 72% and 100%, respectively, suggesting a great application prospect of calamus and its EO on the behavioral control strategies of P. xylostella.

Insecticidal action of mammalian galectin-1-transfected Arabidopsis thaliana.

BACKGROUND: Galectins (GALs) are a family of mammalian sugar-binding proteins specific for ß-galactosides. Our previous studies have shown that the larval development of the diamondback moth (Plutella xylostella) is significantly disturbed when fed with recombinant mammalian galectin 1 (GAL1) derived from Escherichia coli. To further explore its applicability, two GAL1-overexpressed Arabidopsis [GAL1-Arabidopsis (whole plant) and GAL1-Arabidopsis-vas (vascular bundle-specific)] lines were established for insecticidal activity and mechanism studies. RESULTS: The expression level of GAL1 in transgenic Arabidopsis is 1-0.5% (GAL1-Arabidopsis) and 0.08-0.01% (GAL1-Arabidopsis-vas) of total leaf soluble protein. Survival, body weight, and food consumption significantly decreased in a time-dependent manner in P. xylostella larvae (with chewing mouthparts) fed on GAL1-Arabidopsis. The mortality of Kolla paulula (with piercing-sucking mouthparts and xylem feeder) fed on GAL1-Arabidopsis-vas was also significantly higher than that fed on wild-type Arabidopsis (WT-Arabidopsis), but was lower than that fed on GAL1-Arabidopsis. The histochemical structure and results of immunostaining suggested that the binding of GAL1 to the midgut epithelium of P. xylostella fed on GAL1-Arabidopsis was dose- and time-dependent. Ultrastructural studies further showed the disruption of microvilli, abnormalities in epithelial cells, and fragments of the peritrophic membrane (PM) in P. xylostella larvae fed on GAL1-Arabidopsis. CONCLUSION: The insecticidal mechanism of GAL1 involves interference with PM integrity and suggests that GAL1 is a potential candidate for bioinsecticide development. © 2024 Society of Chemical Industry.

Polygenic adaptation of a cosmopolitan pest to a novel thermal environment.

The fluctuation in temperature poses a significant challenge for poikilothermic organisms, notably insects, particularly in the context of changing climatic conditions. In insects, temperature adaptation has been driven by polygenes. In addition to genes that directly affect traits (core genes), other genes (peripheral genes) may also play a role in insect temperature adaptation. This study focuses on two peripheral genes, the GRIP and coiled-coil domain containing 2 (GCC2) and karyopherin subunit beta 1 (KPNB1). These genes are differentially expressed at different temperatures in the cosmopolitan pest, Plutella xylostella. GCC2 and KPNB1 in P. xylostella were cloned, and their relative expression patterns were identified. Reduced capacity for thermal adaptation (development, reproduction and response to temperature extremes) in the GCC2-deficient and KPNB1-deficient P. xylostella strains, which were constructed by CRISPR/Cas9 technique. Deletion of the PxGCC2 or PxKPNB1 genes in P. xylostella also had a differential effect on gene expression for many traits including stress resistance, resistance to pesticides, involved in immunity, trehalose metabolism, fatty acid metabolism and so forth. The ability of the moth to adapt to temperature via different pathways is likely to be key to its ability to remain an important pest species under predicted climate change conditions.

Does fungal infection increase the palatability of oilseed rape to insects?

BACKGROUND: Multiple and simultaneous attacks by pathogens and insect pests frequently occur in nature. Plants respond to biotic stresses by activating distinct defense mechanisms, but little is known about how plants cope with multiple stresses. The focus of this study was the combined interaction of fungal infection caused by Leptosphaeria maculans (synonym Plenodomus lingam) and arthropod infestation by the diamondback moth (Plutella xylostella) in oilseed rape (Brassica napus). We hypothesized that infection by the fungal pathogen L. maculans could alter oilseed rape palatability to P. xylostella-chewing caterpillars. Feeding preference tests were complemented with analyses of defense gene transcription, and levels of glucosinolates (GLSs) and volatile organic compounds (VOCs) in L. maculans-inoculated and non-inoculated (control) leaves to determine possible causes of larval choice. RESULTS: Caterpillars preferred true leaves to cotyledons, hence true leaves were used for further experiments. True leaves inoculated with L. maculans were more palatable to caterpillars over control leaves during the early stage of infection at 3 days post inoculation (dpi), but this preference disappeared in the later stages of infection at 7 dpi. In parallel, genes involved in the salicylic acid and ethylene pathways were up-regulated in L. maculans-inoculated leaves at 3 and 7 dpi; L. maculans increased the level of total aliphatic GLSs, specifically glucobrassicanapin, and decreased the level of glucoiberin at 3 dpi and altered the content of specific VOCs. A group of 55 VOCs with the highest variability between treatments was identified. CONCLUSION: We suggest that the P. xylostella preference for L. maculans-inoculated leaves in the early stage of disease development could be caused by the underlying mechanisms leading to changes in metabolic composition. Further research should pinpoint the compounds responsible for driving larval preference and evaluate whether the behavior of the adult moths, i.e. the stage that makes the first choice regarding host plant selection in field conditions, correlates with our results on larval host acceptance. © 2024 Society of Chemical Industry.

Ligands of HMG-like dorsal switch protein 1 of Spodoptera exigua leads to mortality in diamondback moth, Plutellaxylostella.

HMG-like dorsal switch protein 1 (DSP1) is the insect homolog of the high mobility group box 1 (HMGB1) protein of the vertebrates. Previous studies confirmed DSP1 in Spodoptera exigua, Tenebrio molitor, and Aedes albopictus, and were analyzed for their immune roles, survivability, and binding affinity with entomopathogenic bacterial metabolites. The present study aimed to predict, and confirm DSP1 in diamondback moth, Plutella xylostella along with the effect of Spodoptera exigua DSP1 ligands in the survivability of this insect. DSP1 of Plutella xylostella (Px-DSP1) consists of 465 amino acids (AA). Phylogeny analysis showed that Px-DSP1 clustered with other Lepidopteran insects where each insect order clustered separately. Domain analysis showed that like other insects, Px-DSP1 contains two HMG boxes (Box A and Box B), one coiled-coil (CC), five Q-rich low complexity (LC), and an acidic tail (AT). Px-DSP1 was expressed in each developmental stage and tissue. The highest expression was in L4 larvae and fat body tissues. Thermal shift assay (TSA) showed the binding affinity of 3-Ethoxy-4-Methoxyphenol (EMP), Phthalimide (PM), and o-Cyanobenzoic acid (CBA) to rDSP1 of Spodoptera exigua. Mortality bioassay showed that all these metabolites were toxic against P. xylostella larvae. Among these, EMP was more toxic providing more than 65% mortality at 500 ppm concentration. However, PM and CBA also showed more than 60 and 50% mortality, respectively at 500 ppm concentration. We assume that like Se-DSP1, these compounds also bind with Px-DSP1 which leads to the inhibition of DSP1-mediated immunity and impose the mortality of Plutella xylostella larvae.

Eco-Geographical and Botanical Patterns of Resistance to Lepidoptera Insects in Brassica rapa L.

In the context of the widespread expansion of damage by herbivorous pests of Brassica crops, taking into account the requirements for minimizing pesticide pollution of the environment, it is important to have fundamental knowledge of the geographical features of the distribution of pests and about the botanical confinement of plant resistance in order to develop a strategy for creating new Brassica cultivars with complex resistance to insects. The relevance of our work is related to the study of the variability in the degree of resistance of the extensive genetic diversity of Brassica rapa accessions to the main herbivorous pests of Brassica crops in contrasting ecological and geographical zones of the Russian Federation (Arctic, northwestern, and southern zones). We have studied the distribution and food preferences of Lepidoptera insects (diamondback moth Plutella xylostella and cabbage moth Mamestra brassicae) on a set of 100 accessions from the VIR B. rapa collection (Chinese cabbage, pakchoi, wutacai, zicaitai, mizuna, and leaf and root turnips) in the field in three zones of the Russian Federation. We have found that the diamondback moth and cabbage moth are largely harmful in three zones of the European part of the Russian Federation, although the degree of damage to plants by these insects varies by year of cultivation. On average, for the set studied during the two years of the experiment, the degree of plant damage by both pests in the Arctic zone was low and almost low, and in the northwestern and southern zones, it was medium. It was noted that diamondback moth damage was greater in the northwestern zone in both years and in the southern and Arctic zones in 2021, while in 2022, the degree of cabbage moth damage was slightly higher in the southern and Arctic zones. Under the conditions of field diamondback moth damage, the accessions of Chinese cabbage, wutacai, and mizuna turned out to be the most resistant (the damage score was 1.92-1.99), whereas the accessions of wutacai and pakchoi were the most resistant to the cabbage moth (the damage score was 1.62-1.78). A high variability in the degree of resistance of Brassica crops to Lepidoptera insects from complete resistance to susceptibility was revealed. We have identified sources of resistance to insects, including complex resistance in all study areas, among landraces and some modern cultivars of Chinese cabbage, pakchoi, wutacai, and mizuna from Japan and China, as well as European turnips. The highest susceptibility to pests in the studied set was noted in the accession of root turnip "Hinona" (k-1422, USA) (average damage score of 3.24-3.53 points). We were not able to establish the morphological features of resistant plants or the geographical confinement of the origin of resistance of B. rapa crop accessions.

An odorant receptor mediates the avoidance of Plutella xylostella against parasitoid.

BACKGROUND: Ecosystems are brimming with myriad compounds, including some at very low concentrations that are indispensable for insect survival and reproduction. Screening strategies for identifying active compounds are typically based on bioassay-guided approaches. RESULTS: Here, we selected two candidate odorant receptors from a major pest of cruciferous plants-the diamondback moth Plutella xylostella-as targets to screen for active semiochemicals. One of these ORs, PxylOR16, exhibited a specific, sensitive response to heptanal, with both larvae and adult P. xylostella displaying heptanal avoidance behavior. Gene knockout studies based on CRISPR/Cas9 experimentally confirmed that PxylOR16 mediates this avoidance. Intriguingly, rather than being involved in P. xylostella-host plant interaction, we discovered that P. xylostella recognizes heptanal from the cuticular volatiles of the parasitoid wasp Cotesia vestalis, possibly to avoid parasitization. CONCLUSIONS: Our study thus showcases how the deorphanization of odorant receptors can drive discoveries about their complex functions in mediating insect survival. We also demonstrate that the use of odorant receptors as a screening platform could be efficient in identifying new behavioral regulators for application in pest management.

Exploring the role of the ovary-serine protease gene in the female fertility of the diamondback moth using CRISPR/Cas9.

BACKGROUND: Oogenesis is a complex pathway necessary for proper female reproduction in insects. Ovary-serine protease (Osp) is a homologous gene of serine protease Nudel (SpNudel) and plays an essential role in the oogenesis and ovary development of Drosophila melanogaster. However, the function of Osp is not determined in Plutella xylostella, a highly destructive pest of cruciferous crops. RESULTS: The PxOsp gene comprises a 5883-bp open-reading frame that encodes a protein consisting of 1994 amino acids, which contain four conserved domains. PxOsp exhibited a high relative expression in adult females with a specific expression in the ovary. Through the utilization of CRISPR/Cas9 technology, homozygous mutants of PxOsp were generated. These homozygous mutant females produced fewer eggs (average of 56 eggs/female) than wild-type (WT) females (average of 97 eggs/female) when crossed with WT males, and these eggs failed to hatch. Conversely, mutant males produced normal progeny when crossed with WT females. The ovarioles in homozygous mutant females were significantly shorter (5.02 mm in length) and contained fewer eggs (average of 3 eggs/ovariole) than WT ovarioles (8.09 mm in length with an average of 8 eggs/ovariole). Moreover, eggs laid by homozygous mutant females were fragile, with irregular shapes, and were unable to maintain structural integrity due to eggshell ruptures. However, no significant differences were observed between WT and mutant individuals regarding developmental duration, pupal weight, and mating behavior. CONCLUSION: Our study suggesteds that PxOsp plays a vital role in female reproduction, particularly in ovary and egg development. Disrupting PxOsp results in recessive female sterility while leaving the male reproductive capability unaffected. This report represents the first study of a haplosufficient gene responsible for female fertility in lepidopteran insects. Additionally, these findings emphasize PxOsp as a potential target for genetically-based pest management of P. xylostella. © 2024 Society of Chemical Industry.