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.

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.

Thermal acclimation uncovers a simple genetic basis of adaptation to high temperature in a cosmopolitan pest.

Understanding a population's fitness heterogeneity and genetic basis of thermal adaptation is essential for predicting the responses to global warming. We examined the thermotolerance and genetic adaptation of Plutella xylostella to exposure to hot temperatures. The population fitness parameters of the hot-acclimated DBM strains varied in the thermal environments. Using genome scanning and transcription profiling, we find a number of genes potentially involved in thermal adaptation of DBM. Editing two ABCG transporter genes, PxWhite and PxABCG, confirmed their role in altering cuticle permeability and influencing thermal responses. Our results demonstrate that SNP mutations in genes and changes in gene expression can allow DBM to rapidly adapt to thermal environment. ABCG transporter genes play an important role in thermal adaptation of DBM. This work improves our understanding of genetic adaptation mechanisms of insects to thermal stress and our capacity to predict the effects of rising global temperatures on ectotherms.

Developmental Shifts in the Microbiome of a Cosmopolitan Pest: Unraveling the Role of Wolbachia and Dominant Bacteria.

Wolbachia bacteria (phylum Proteobacteria) are ubiquitous intracellular parasites of diverse invertebrates. In insects, coevolution has forged mutualistic associations with Wolbachia species, influencing reproduction, immunity, development, pathogen resistance, and overall fitness. However, the impact of Wolbachia on other microbial associates within the insect microbiome, which are crucial for host fitness, remains less explored. The diamondback moth (Plutella xylostella), a major pest of cruciferous vegetables worldwide, harbors the dominant Wolbachia strain plutWB1, known to distort its sex ratio. This study investigated the bacterial community diversity and dynamics across different developmental life stages and Wolbachia infection states in P. xylostella using high-throughput 16S rDNA amplicon sequencing. Proteobacteria and Firmicutes dominated the P. xylostella microbiome regardless of life stage or Wolbachia infection. However, the relative abundance of dominant genera, including an unclassified genus of Enterobacteriaceae, Wolbachia, Carnobacterium, and Delftia tsuruhatensis, displayed significant stage-specific variations. While significant differences in bacterial diversity and composition were observed across life stages, Wolbachia infection had no substantial impact on overall diversity. Nonetheless, relative abundances of specific genera differed between infection states. Notably, Wolbachia exhibited a stable, high relative abundance across all stages and negatively correlated with an unclassified genus of Enterobacteriaceae, Delftia tsuruhatensis, and Carnobacterium. Our findings provide a foundational understanding of the complex interplay between the host, Wolbachia, and the associated microbiome in P. xylostella, paving the way for a deeper understanding of their complex interactions and potential implications for pest control strategies.

Biology, Ecology, and Management of Flea Beetles in Brassica Crops.

Brassica vegetable and oilseed crops are attacked by several different flea beetle species (Chrysomelidae: Alticini). Over the past decades, most research has focused on two Phyllotreta species, Phyllotreta striolata and Phyllotreta cruciferae, which are major pests of oilseed rape in North America. More recently, and especially after the ban of neonicotinoids in the European Union, the cabbage stem flea beetle, Psylliodes chrysocephala, has become greatly important and is now considered to be the major pest of winter oilseed rape in Europe. The major challenges to flea beetle control are the prediction of population dynamics in the field, differential susceptibility to insecticides, and the lack of resistant plant cultivars and other economically viable alternative management strategies. At the same time, many fundamental aspects of flea beetle biology and ecology, which may be relevant for the development of sustainable control strategies, are not well understood. This review focuses on the interactions between flea beetles and plants and summarizes the literature on current management strategies with an emphasis on the potential for biological control in flea beetle management.

Identification of floral volatiles from Fagopyrum esculentum that attract Cotesia vestalis with potentially better biocontrol efficacy against Plutella xylostella.

BACKGROUND: Nectar plants provide extra nourishment for parasitoids, which can utilize floral volatiles to locate nectar-rich flowers. A promising strategy is to screen potential floral species based on the wasps' olfactory preferences for nectar sources, and to ensure their suitability for both natural enemies and targeted pests. Cotesia vestalis (Haliday) is a dominant parasitoid of the oligophagous pest Plutella xylostella, which poses a significant threat to cruciferous vegetables globally. However, the chemical cues in plant-parasitoid complexes mediating Cotesia vestalis to locate nectar food resources and the positive effect of nectar plants on the Cotesia vestalis population are poorly understood. RESULTS: The results showed that Fagopyrum esculentum was the most attractive plant that attracted Cotesia vestalis, not Plutella xylostella in 44 flowering plants from 19 families. 1,2-Diethyl benzene and 1,4-diethyl benzene, identified from the floral volatiles from F. esculentum in full bloom, were found to elicit dose-dependent electrophysiological responses and attract Cotesia vestalis adults, demonstrating their potential as semiochemicals. Moreover, the age-stage, two-sex life table revealed that feeding on nectar food increased the efficacy of Cotesia vestalis adults against Plutella xylostella. CONCLUSION: In summary, the findings provide insights into the chemical ecology of plant-parasitoid complexes and support the potential use of F. esculentum as insectary plants in habitat manipulation against Plutella xylostella by supplying natural nectar food for the Cotesia vestalis population. Our results suggest an attract and reward strategy based on an attractant for Cotesia vestalis to control Plutella xylostella, or the development of volatile-based artificial food for Cotesia vestalis. © 2023 Society of Chemical Industry.

Gut bacteria mediated adaptation of diamondback moth, Plutella xylostella, to secondary metabolites of host plants.

IMPORTANCE: In this study, we identify an important role of gut bacteria in mediating the adaptation of diamondback moth (DBM) to plant secondary metabolites. We demonstrate that kaempferol's presence in radish seedlings greatly reduces the fitness of DBM with depleted gut biota. Reinstatement of gut biota, particularly Enterobacter sp. EbPXG5, improved insect performance by degrading kaempferol. This bacterium was common in the larval gut of DBM, lining the epithelium as a protective film. Our work highlights the role of symbiotic bacteria in insect herbivore adaptation to plant defenses and provides a practical and mechanistic framework for developing a more comprehensive understanding of insect-gut microbe-host plant co-evolution.

Glucose Oxidase of a Crucifer-Specialized Insect: A Potential Role in Suppressing Plant Defense via Modulating Antagonistic Plant Hormones.

Glucose oxidase (GOX) is a representative compound found in most insect saliva that can suppress plant-defensive responses. However, little is known about the origin and role of GOX in the crucifer-specialized pest Plutella xylostella. In this study, we showed obvious regurgitation from the larval gut of P. xylostella and identified abundant peptides highly similar to known GOX. Three PxGOX genes were verified with PxGOX2 preferentially expressed in the gut. The heterologously expressed PxGOX2 confirmed its function to be a GOX, and it was detected in plant wounds together with the gut regurgitant. Further experiments revealed that PxGOX2 functioned as an effector and may suppress defensive responses in plant through the production of H2O2, which modulates levels of antagonistic salicylic acid and jasmonic acid. However, excessive H2O2 in the host plant may be neutralized by peroxidase, thus forming defensive feedback. Our findings provided new insights into understanding the GOX-mediated insect-plant interactions.

Effects of Antibiotic Treatment on the Development and Bacterial Community of the Wolbachia-Infected Diamondback Moth.

Based on the important role of antibiotic treatment in the research of the interaction between Wolbachia and insect hosts, this study aimed to identify the most suitable antibiotic and concentration for Wolbachia elimination in the P. xylostella, and to investigate the effect of Wolbachia and antibiotic treatment on the bacterial community of P. xylostella. Our results showed that the Wolbachia-infected strain was plutWB1 of supergroup B in the P. xylostella population collected in Nepal in this study; 1 mg/mL rifampicin could remove Wolbachia infection in P. xylostella after 1 generation of feeding treatment and the toxic effect was relatively low; among the 29 samples of adult P. xylostella in our study (10 WU samples, 10 WA samples, and 9 WI samples), 52.5% of the sequences were of Firmicutes and 47.5% were of Proteobacteria, with the dominant genera being mainly Carnobacterium (46.2%), Enterobacter (10.1%), and Enterococcus (6.2%); Moreover, antibiotic removal of Wolbachia infection in P. xylostella and transfer to normal conditions for 10 generations no longer significantly affected the bacterial community of P. xylostella. This study provides a theoretical basis for the elimination method of Wolbachia in the P. xylostella, as well as a reference for the elimination method of Wolbachia in other Wolbachia-infected insect species, and a basis for the study of the extent and duration of the effect of antibiotic treatment on the bacterial community of the P. xylostella.

Gut microbiota assemblages of generalist predators are driven by local- and landscape-scale factors.

The gut microbiomes of arthropods have significant impact on key physiological functions such as nutrition, reproduction, behavior, and health. Spiders are diverse and numerically dominant predators in crop fields where they are potentially important regulators of pests. Harnessing spiders to control agricultural pests is likely to be supported by an understanding of their gut microbiomes, and the environmental drivers shaping microbiome assemblages. This study aimed to deciphering the gut microbiome assembly of these invertebrate predators and elucidating potential implications of key environmental constraints in this process. Here, we used high-throughput sequencing to examine for the first time how the assemblages of bacteria in the gut of spiders are shaped by environmental variables. Local drivers of microbiome composition were globally-relevant input use system (organic production vs. conventional practice), and crop identity (Chinese cabbage vs. cauliflower). Landscape-scale factors, proportion of forest and grassland, compositional diversity, and habitat edge density, also strongly affected gut microbiota. Specific bacterial taxa were enriched in gut of spiders sampled from different settings and seasons. These findings provide a comprehensive insight into composition and plasticity of spider gut microbiota. Understanding the temporal responses of specific microbiota could lead to innovative strategies development for boosting biological control services of predators.

Clip-SP1 cleavage activates downstream prophenoloxidase activating protease (PAP) in Plutella xylostella.

Melanization is a component of the humoral immune defense of insects and is induced by serine protease-mediated phenoloxidase (PO) catalysis. Prophenoloxidase (PPO) in the midgut of Plutella xylostella is activated by the CLIP domain serine protease (clip-SP) in response to Bacillus thuringiensis (Bt) infection, but the detailed signaling cascade following this activation is unknown. Here, we report that activation of clip-SP enhances PO activity in the P. xylostella midgut by cleaving three downstream PPO-activating proteases (PAPs). First, the expression level of clip-SP1 was increased in the midgut after Bt8010 infection of P. xylostella. Then, purified recombinant clip-SP1 was able to activate three PAPs - PAPa, PAPb and PAP3 - which in turn enhanced their PO activity in the hemolymph. Furthermore, clip-SP1 showed a dominant effect on PO activity compared to the individual PAPs. Our results indicate that Bt infection induces the expression of clip-SP1, which is upstream of a signaling cascade, to efficiently activate PO catalysis and mediate melanization in the midgut of P. xylostella. And it provides a basis for studying the complex PPO regulatory system in the midgut during Bt infection.

A very long-chain fatty acid enzyme gene, PxHacd2 affects the temperature adaptability of a cosmopolitan insect by altering epidermal permeability.

Temperature fluctuations pose challenges to poikilotherms, such as insects, especially under climate change conditions. Very long-chain fatty acids (VLCFAs) form important structural components of membranes and epidermal surfaces, so play important roles in adaptation to temperature stress in plants. It has been unclear whether VLCFAs are involved in epidermis formation and thermal resistance in insects. In this study, we focused on the 3-hydroxy acyl-CoA dehydratase 2 (Hacd2), an important enzyme in the synthesis pathway of VLCFAs, in a cosmopolitan pest, the diamondback moth, Plutella xylostella. Hacd2 was cloned from P. xylostella and the relative expression pattern was identified. Epidermal permeability increased with the decreased VLCFAs in the Hacd2-deficient P. xylostella strain, which was constructed by using the CRISPR/Cas9 system. Survival and fecundity of the Hacd2-deficient strain was significantly lower than that of the wildtype strain when subject to desiccating environmental stress. Hacd2 mediates thermal adaptability in P. xylostella by changing epidermal permeability so is likely to be key to its remaining a major pest species under predicted climate change conditions.

Enhanced resistance to Bacillus thuringiensis Cry1Ac toxin mediated by the activation of prophenoloxidase in a cosmopolitan pest.

Plutella xylostella has evolved resistance to Bacillus thuringiensis Cry1Ac toxin over a long evolutionary period. Enhanced immune response is an important factor in insect resistance to a variety of insecticides, and whether phenoloxidase (PO), an immune protein, is involved in resistance to Cry1Ac toxin in P. xylostella remains unclear. Here, spatial and temporal expression patterns showed that prophenoloxidase (PxPPO1 and PxPPO2) in the Cry1S1000-resistant strain was more highly expressed in eggs, 4th instar, head, and hemolymph than those in G88-susceptible strain. The results of PO activity analysis showed that after treatment with Cry1Ac toxin PO activity was about 3 times higher than that before treatment. Furthermore, knockout of PxPPO1 and PxPPO2 significantly increased the susceptibility to Cry1Ac toxin. These findings were further supported by the knockdown of Clip-SPH2, a negative regulator of PO, which resulted in increased PxPPO1 and PxPPO2 expression and Cry1Ac susceptibility in the Cry1S1000-resistant strain. Finally, the synergistic effect of quercetin showed that larval survival decreased from 100 % to <20 % compared to the control group. This study will provide a theoretical basis for the analysis of immune-related genes (PO) genes involved in the resistance mechanism and pest control of P. xylostella.

Analysis of the Effect of Plutella xylostella Polycalin and ABCC2 Transporter on Cry1Ac Susceptibility by CRISPR/Cas9-Mediated Knockout.

Many insects, including the Plutella xylostella (L.), have developed varying degrees of resistance to many insecticides, including Bacillus thuringiensis (Bt) toxins, the bioinsecticides derived from Bt. The polycalin protein is one of the potential receptors for Bt toxins, and previous studies have confirmed that the Cry1Ac toxin can bind to the polycalin protein of P. xylostella, but whether polycalin is associated with the resistance of Bt toxins remains controversial. In this study, we compared the midgut of larvae from Cry1Ac-susceptible and -resistant strains, and found that the expression of the Pxpolycalin gene was largely reduced in the midgut of the resistant strains. Moreover, the spatial and temporal expression patterns of Pxpolycalin showed that it was mainly expressed in the larval stage and midgut tissue. However, genetic linkage experiments showed that the Pxpolycalin gene and its transcript level were not linked to Cry1Ac resistance, whereas both the PxABCC2 gene and its transcript levels were linked to Cry1Ac resistance. The larvae fed on a diet containing the Cry1Ac toxin showed no significant change in the expression of the Pxpolycalin gene in a short term. Furthermore, the knockout of polycalin and ATP-binding cassette transporter subfamily C2 (ABCC2) genes separately by CRISPR/Cas9 technology resulted in resistance to decreased susceptibility to Cry1Ac toxin. Our results provide new insights into the potential role of polycalin and ABCC2 proteins in Cry1Ac resistance and the mechanism underlying the resistance of insects to Bt toxins.

Diversity of Wolbachia infection and its influence on mitochondrial DNA variation in the diamondback moth, Plutella xylostella.

Plutella xylostella is a pest that severely damages cruciferous vegetables worldwide and has been shown to be infected with the maternally inherited bacteria Wolbachia, with the main infected strain was plutWB1. In this study, we performed a large-scale global sampling of P. xylostella and amplified 3 mtDNA genes of P. xylostella and 6 Wolbachia genes to analyze the infection status, diversity of Wolbachia in P. xylostella, and its effect on mtDNA variation in P. xylostella. This study provides a conservative estimate of Wolbachia infection rates in P. xylostella, which was found to be 7% (104/1440). The ST 108 (plutWB1) was shared among butterfly species and the moth species P. xylostella, revealing that Wolbachia strain plutWB1 acquisition in P. xylostella may be through horizontal transmission. The Parafit analyses indicated a significant association between Wolbachia and Wolbachia-infected P. xylostella individuals, and individuals infected with plutWB1 tended to cluster in the basal positions of the phylogenetic tree based on the mtDNA data. Additionally, Wolbachia infections were associated with increased mtDNA polymorphism in the infected P. xylostella population. These data suggest that Wolbachia endosymbionts may have a potential effect on mtDNA variation of P. xylostella.

Oxygen stress on age-stage, two-sex life tables and transcriptomic response of diamondback moth (Plutella xylostella).

Elucidating the genetic basis of local adaption is one of the important tasks in evolutionary biology. The Qinghai-Tibet Plateau has the highest biodiversity for an extreme environment worldwide, and provides an ideal natural laboratory to study adaptive evolution. The diamondback moth (DBM), Plutella xylostella, is one of the most devastating pests of the global Brassica industry. A highly heterozygous genome of this pest has facilitated its adaptation to a variety of complex environments, and so provides an ideal model to study fast adaptation. We conducted a pilot study combining RNA-seq with an age-stage, two-sex life table to study the effects of oxygen deprivation on DBM. The developmental periods of all instars were significantly shorter in the hypoxic environment. We compared the transcriptomes of DBM from Fuzhou, Fujian (low-altitude) and Lhasa, Tibet (high-altitude) under hypoxia treatment in a hypoxic chamber. Some DEGs are enriched in pathways associated with DNA replication, such as DNA repair, nucleotide excision repair, base excision repair, mismatch repair and homologous recombination. The pathways with significant changes were associated with metabolism process and cell development. Thus, we assumed that insects could adapt to different environments by regulating their metabolism. Our findings indicated that although adaptive mechanisms to hypoxia in different DBM strains could be similar, DBM individuals from Tibet had superior tolerance to hypoxia compared with those of Fuzhou. Local adaptation of the Tibetan colony was assumed to be responsible for this difference. Our research suggests novel mechanisms of insect responses to hypoxia stress.

Novel miR-108 and miR-234 target juvenile hormone esterase to regulate the response of Plutella xylostella to Cry1Ac protoxin.

Insect hormones, such as juvenile hormone (JH), precisely regulate insect life-history traits. The regulation of JH is tightly associated with the tolerance or resistance to Bacillus thuringiensis (Bt). JH esterase (JHE) is a primary JH-specific metabolic enzyme which plays a key role in regulating JH titer. Here, we characterized a JHE gene from Plutella xylostella (PxJHE), and found it was differentially expressed in the Bt Cry1Ac resistant and susceptible strains. Suppression of PxJHE expression with RNAi increased the tolerance of P. xylostella to Cry1Ac protoxin. To investigate the regulatory mechanism of PxJHE, two target site prediction algorithms were applied to predict the putative miRNAs targeting PxJHE, and the resulting putative miRNAs were subsequently verified for their function targeting PxJHE using luciferase reporter assay and RNA immunoprecipitation. MiR-108 or miR-234 agomir delivery dramatically reduced PxJHE expression in vivo, whilst only miR-108 overexpression consequently increased the tolerance of P. xylostella larvae to Cry1Ac protoxin. By contrast, reduction of miR-108 or miR-234 dramatically increased PxJHE expression, accompanied by the decreased tolerance to Cry1Ac protoxin. Furthermore, injection of miR-108 or miR-234 led to developmental defects in P. xylostella, whilst injection of antagomir did not cause any obvious abnormal phenotypes. Our results indicated that miR-108 or miR-234 can be applied as potential molecular targets to combat P. xylostella and perhaps other lepidopteran pests, providing novel insights into miRNA-based integrated pest management.

Immune Responses and Transcriptomic Analysis of Nilaparvata lugens against Metarhizium anisopliae YTTR Mediated by Rice Ragged Stunt Virus.

Plant viruses and entomopathogenic fungi (EPF) can both elicit immune responses in insects. This study was designed to clarify whether plant viruses could affect the efficacy of EPF and explore the immune responses of brown planthopper (BPH), Nilaparvata lugens, in response to different pathogen infections. In this study, a strain of Metarhizium anisopliae YTTR with high pathogenicity against BPH was selected and explored whether rice ragged stunt virus (RRSV) could affect its lethality against BPH. RNA-seq was used to detect the inner responses of BPH in response to RRSV and M. anisopliae YTTR infection. Results showed that M. anisopliae YTTR has strong lethality against BPH (RRSV-carrying and RRSV-free). RRSV invasion did not affect the susceptibility of BPH against M. anisopliae YTTR at all concentrations. At 1 × 108 spores/mL, M. anisopliae YTTR caused a cumulative mortality of 80% to BPH at 7 days post-treatment. The largest numbers of differentially expressed genes (DEGs) was obtained in BPH treated with the two pathogens than in other single pathogen treatment. In addition, KEGG enrichment analysis showed that the DEGs were mostly enriched in immune and physiological mechanisms-related pathways. Both RRSV and M. anisopliae YTTR could induce the expression changes of immune-related genes. However, most of the immune genes had varying expression patterns in different treatment. Our findings demonstrated that RRSV invasion did not have any significant effect on the pathogenicity of M. anisopliae YTTR, while the co-infection of M. anisopliae YTTR and RRSV induced more immune and physiological mechanisms -related genes' responses. In addition, the presence of RRSV could render the interplay between BPH and M. anisopliae YTTR more intricate. These findings laid a basis for further elucidating the immune response mechanisms of RRSV-mediated BPH to M. anisopliae infection.

Silencing RNAs expressed from W-linked PxyMasc "retrocopies" target that gene during female sex determination in Plutella xylostella.

The Lepidoptera are an insect order of cultural, economic, and environmental importance, representing ∼10% of all described living species. Yet, for all but one of these species (silkmoth, Bombyx mori), the molecular genetics of how sexual fate is determined remains unknown. We investigated this in the diamondback moth (Plutella xylostella), a globally important, highly invasive, and economically damaging pest of cruciferous crops. Our previous work uncovered a regulator of male sex determination in P. xylostella-PxyMasc, a homolog of B. mori Masculinizer-which, although initially expressed in embryos of both sexes, is then reduced in female embryos, leading to female-specific splicing of doublesex. Here, through sequencing small RNA libraries generated from early embryos and sexed larval pools, we identified a variety of small silencing RNAs (predominantly Piwi-interacting RNAs [piRNAs]) complementary to PxyMasc, whose temporal expression correlated with the reduction in PxyMasc transcript observed previously in females. Analysis of these small RNAs showed that they are expressed from tandemly arranged, multicopy arrays found exclusively on the W (female-specific) chromosome, which we term "Pxyfem". Analysis of the Pxyfem sequences showed that they are partial complementary DNAs (cDNAs) of PxyMasc messenger RNA (mRNA) transcripts, likely integrated into transposable element graveyards by the noncanonical action of retrotransposons (retrocopies), and that their apparent similarity to B. mori feminizer more probably represents convergent evolution. Our study helps elucidate the sex determination cascade in this globally important pest and highlights the "shortcuts" that retrotransposition events can facilitate in the evolution of complex molecular cascades, including sex determination.

An inducible gene from glycoside hydrolase one family of Plutella xylostella decreases larval survival when feeding on host plant.

Glycoside hydrolase family 1 (GH1) members exhibit a broad substrate spectrum and play important roles in insect-plant interactions, such as the defensive ß-glucosidase and ß-thioglucosidase (so-called myrosinase). However, knowledge about the expression profiling and function of glycoside hydrolase family 1 members in a specialist pest of crucifers Plutella xylostella is still limited. In this study, 13 putative glycoside hydrolase family 1 members of P. xylostella were identified based on the sequence characteristics, while no myrosinase activity was detectable in P. xylostella using gas chromatography-mass spectrometry (GC-MS). Expression profiling of these glycoside hydrolase family 1 members identified the midgut-specific gene Px008848 that is induced by host plant. Further experiments revealed that the in vitro expressed Px008848 protein had ß-glucosidase activity and the survival rate of the larvae feeding on wounded Arabidopsis thaliana leaves declined when leaves were treated with purified Px008848 protein. When CRISPR/Cas9-based homozygous mutant larvae of Px008848 and wild-type larvae were respectively transferred onto the A. thaliana, the larval survival rate of the mutant larvae was significantly higher than that of the wild-type individuals. Our work showed that certain insect glycoside hydrolase family 1 gene may have negative effect on the development of larvae feeding on the host plant, which broadened our understandings on the evolutionary function of this gene family in the insect-plant interaction.