Deep learning-based quantification and transcriptomic profiling reveal a methyl jasmonate-mediated glandular trichome formation pathway in Cannabis sativa.

Cannabis glandular trichomes (GTs) are economically and biotechnologically important structures that have a remarkable morphology and capacity to produce, store, and secrete diverse classes of secondary metabolites. However, our understanding of the developmental changes and the underlying molecular processes involved in cannabis GT development is limited. In this study, we developed Cannabis Glandular Trichome Detection Model (CGTDM), a deep learning-based model capable of differentiating and quantifying three types of cannabis GTs with a high degree of efficiency and accuracy. By profiling at eight different time points, we captured dynamic changes in gene expression, phenotypes, and metabolic processes associated with GT development. By integrating weighted gene co-expression network analysis with CGTDM measurements, we established correlations between phenotypic variations in GT traits and the global transcriptome profiles across the developmental gradient. Notably, we identified a module containing methyl jasmonate (MeJA)-responsive genes that significantly correlated with stalked GT density and cannabinoid content during development, suggesting the existence of a MeJA-mediated GT formation pathway. Our findings were further supported by the successful promotion of GT development in cannabis through exogenous MeJA treatment. Importantly, we have identified CsMYC4 as a key transcription factor that positively regulates GT formation via MeJA signaling in cannabis. These findings provide novel tools for GT detection and counting, as well as valuable information for understanding the molecular regulatory mechanism of GT formation, which has the potential to facilitate the molecular breeding, targeted engineering, informed harvest timing, and manipulation of cannabinoid production.

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.

Resistance to Bacillus thuringiensis Cry1Ac toxin requires mutations in two Plutella xylostella ATP-binding cassette transporter paralogs.

The diamondback moth, Plutella xylostella, is a cosmopolitan pest and the first species to develop field resistance to toxins from the gram-positive bacterium Bacillus thuringiensis (Bt). Although previous work has suggested that mutations of ATP-binding cassette transporter subfamily C2 (ABCC2) or C3 (ABCC3) genes can confer Cry1Ac resistance, here we reveal that P. xylostella requires combined mutations in both PxABCC2 and PxABCC3 to achieve high-level Cry1Ac resistance, rather than simply a mutation of either gene. We identified natural mutations of PxABCC2 and PxABCC3 that concurrently occurred in a Cry1Ac-resistant strain (Cry1S1000) of P. xylostella, with a mutation (RA2) causing the mis-splicing of PxABCC2 and another mutation (RA3) leading to the premature termination of PxABCC3. Genetic linkage analysis showed that RA2 and RA3 were tightly linked to Cry1Ac resistance. Introgression of RA2 and RA3 enabled a susceptible strain (G88) of P. xylostella to obtain high resistance to Cry1Ac, confirming that these genes confer resistance. To further support the role of PxABCC2 and PxABCC3 in Cry1Ac resistance, frameshift mutations were introduced into PxABCC2 and PxABCC3 singly and in combination in the G88 strain with CRISPR/Cas9 mediated mutagenesis. Bioassays of CRISPR-based mutant strains, plus genetic complementation tests, demonstrated that the deletion of PxABCC2 or PxABCC3 alone provided < 4-fold tolerance to Cry1Ac, while disruption of both genes together conferred >8,000-fold resistance to Cry1Ac, suggesting the redundant/complementary roles of PxABCC2 and PxABCC3. This work advances our understanding of Bt resistance in P. xylostella by demonstrating mutations within both PxABCC2 and PxABCC3 genes are required for high-level Cry1Ac resistance.

Gene flow, linked selection, and divergent sorting of ancient polymorphism shape genomic divergence landscape in a group of edaphic specialists.

Interpreting the formation of genomic variation landscape, especially genomic regions with elevated differentiation (i.e. islands), is fundamental to a better understanding of the genomic consequences of adaptation and speciation. Edaphic islands provide excellent systems for understanding the interplay of gene flow and selection in driving population divergence and speciation. However, discerning the relative contribution of these factors that modify patterns of genomic variation remains difficult. We analysed 132 genomes from five recently divergent species in Primulina genus, with four species distributed in Karst limestone habitats and the fifth one growing in Danxia habitats. We demonstrated that both gene flow and linked selection have contributed to genome-wide variation landscape, where genomic regions with elevated differentiation (i.e., islands) were largely derived by divergent sorting of ancient polymorphism. Specifically, we identified several lineage-specific genomic islands that might have facilitated adaptation of P. suichuanensis to Danxia habitats. Our study is amongst the first cases disentangling evolutionary processes that shape genomic variation of plant specialists, and demonstrates the important role of ancient polymorphism in the formation of genomic islands that potentially mediate adaptation and speciation of endemic plants in special soil habitats.

Vitelline Membrane Protein 26 Mutagenesis, Using CRISPR/Cas9, Results in Egg Collapse in Plutella xylostella.

Vitelline membrane proteins (VMPs) are the main proteins that form the inner shell (vitelline membrane layer) of insect eggs and are an integral part of egg formation and embryo development. Here, we characterized the molecular structure and expression patterns of the VMP26 gene and analyzed its reproductive functions in diamondback moth, Plutella xylostella (L.), a worldwide migratory pest of cruciferous plants. The PxVMP26 gene was shown to be a single exon gene that contained an open reading frame of 852 base pairs (bp) encoding 283 amino acids. Both qPCR and western blot analyses showed that PxVMP26 was specifically expressed in female adults and was significantly highly expressed in the ovary. Further anatomical analysis indicated that the expression level of PxVMP26 in the ovarian tube with an incomplete yolk was significantly higher than that in the ovarian tube with a complete yolk. CRISPR/Cas9-induced PxVMP26 knockout successfully created two homozygous strains with 8- and 46-bp frameshift mutations. The expression deficiency of the PxVMP26 protein was detected in the mutant strains using immunofluorescence and western blot. No significant difference was found in the number of eggs laid within three days between wild and mutant individuals, but there was a lower egg hatchability. The loss of the PxVMP26 gene changed the mean egg size, damaged the structure of the vitelline membrane, and increased the proportion of abnormal eggs due to water loss, resulting in egg collapse. This first analysis of the roles of the VMP gene in the oocyte formation and embryonic development of P. xylostella, using CRISPR/Cas9 technology, provides a basis for screening new genetic control targets of P. xylostella.

Differential Profiles of Gut Microbiota and Metabolites Associated with Host Shift of Plutella xylostella.

Evolutionary and ecological forces are important factors that shape gut microbial profiles in hosts, which can help insects adapt to different environments through modulating their metabolites. However, little is known about how gut microbes and metabolites are altered when lepidopteran pest species switch hosts. In the present study, using 16S-rDNA sequencing and mass spectrometry-based metabolomics, we analyzed the gut microbiota and metabolites of three populations of Plutella xylostella: one feeding on radish (PxR) and two feeding on peas (PxP; with PxP-1 and PxP-17 being the first and 17th generations after host shift from radish to peas, respectively). We found that the diversity of gut microbes in PxP-17 was significantly lower than those in PxR and PxP-1, which indicates a distinct change in gut microbiota after host shift. Kyoto Encyclopedia of Genes and Genomes analysis revealed that the functions of energy metabolism, signal transduction, and xenobiotics biodegradation and metabolism were increased in PxP-17, suggesting their potential roles in host adaptation. Metabolic profiling showed a significant difference in the abundance of gut metabolites between PxR and PxP-17, and significant correlations of gut bacteria with gut metabolites. These findings shed light on the interaction among plants, herbivores, and symbionts, and advance our understanding of host adaptation associated with gut bacteria and metabolic activities in P. xylostella.

Genome-wide profiling of the alternative splicing provides insights into development in Plutella xylostella.

BACKGROUND: The diamondback moth (DBM), Plutella xylostella (L.), is a major pest of cruciferous crops worldwide. While the species has become a model for genomics, post-transcriptional mechanisms associated with development and sex determination have not been comprehensively studied and the lack of complete structure of mRNA transcripts limits further research. RESULTS: Here, we combined the methods of single-molecule long-read sequencing technology (IsoSeq) and RNA-seq to re-annotate the published DBM genome and present the genome-wide identification of alternative splicing (AS) associated with development and sex determination of DBM. In total, we identified ~ 13,900 genes (~ 77%) annotated in the DBM genome (version-2), resulting in the correction of 1586 wrongly annotated genes and identification of 78,000 previously unannotated transcripts. We also identified 1804 genes showing alternative splicing (AS) in each of the developmental stages and sexes, suggesting that AS events are ubiquitous in DBM. Comparative analyses showed that these AS events were rarely shared among developmental stages, indicating that they may play key specific roles in regulation of insect development. Further, we found 156 genes showing different AS events and expression patterns between males and females, linking them to potential functions in sex determination. CONCLUSION: Overall, the P. xylostella transcriptome provides the significant information about regulatory alternative splicing events, which are shown to be involved in development and sex determination. Our work presents a solid foundation to better understand the mechanism of post-transcriptional regulation, and offers wider insights into insect development and sex determination.

Implication for DNA methylation involved in the host transfer of diamondback moth, Plutella xylostella (L.).

DNA methylation exerts extensive impacts on gene expression of various living organisms exposed to environmental variation. However, little is known whether DNA methylation is involved in the host transfer of diamondback moth, Plutella xylostella (L.), a worldwide destructive pest of crucifers. In this study, we found that P. xylostella genome exhibited a relatively low level of DNA methylation on the basis of the CpG O/E prediction and experimental validation. A significant positive linear correlation was observed between the stage-specific expressions of PxDNMT1 and DNA methylation levels (5mC content). Particularly, high levels of DNA methylation and gene expression of PxDNMT1 were observed in eggs and mature females of P. xylostella. After host transfer of P. xylostella from Raphanus sativus to Arabidopsis thaliana, we identified some potential genomic loci that might have changed methylation levels. Using the method of fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP), we also found the corresponding genes primarily involved in neural system and signaling. The expressions of six candidate genes were verified by qRT-PCR. One of the genes, Px009600, might be regulated by a DNA methylation-mediated mechanism in response to host transfer. Our study provides evidence for a functional system of DNA methylation in P. xylostella and its possible role in adaptation during host transfer. Further studies should examine methylation as responsive factors to different host plants and environmental cues in insect pests.

Genome-wide investigation of transcription factors provides insights into transcriptional regulation in Plutella xylostella.

Transcription factors (TFs), which play a vital role in regulating gene expression, are prevalent in all organisms and characterization of them may provide important clues for understanding regulation in vivo. The present study reports a genome-wide investigation of TFs in the diamondback moth, Plutella xylostella (L.), a worldwide pest of crucifers. A total of 940 TFs distributed among 133 families were identified. Phylogenetic analysis of insect species showed that some of these families were found to have expanded during the evolution of P. xylostella or Lepidoptera. RNA-seq analysis showed that some of the TF families, such as zinc fingers, homeobox, bZIP, bHLH, and MADF_DNA_bdg genes, were highly expressed in certain tissues including midgut, salivary glands, fat body, and hemocytes, with an obvious sex-biased expression pattern. In addition, a number of TFs showed significant differences in expression between insecticide susceptible and resistant strains, suggesting that these TFs play a role in regulating genes related to insecticide resistance. Finally, we identified an expansion of the HOX cluster in Lepidoptera, which might be related to Lepidoptera-specific evolution. Knockout of this cluster using CRISPR/Cas9 showed that the egg cannot hatch, indicating that this cluster may be related to egg development and maturation. This is the first comprehensive study on identifying and characterizing TFs in P. xylostella. Our results suggest that some TF families are expanded in the P. xylostella genome, and these TFs may have important biological roles in growth, development, sexual dimorphism, and resistance to insecticides. The present work provides a solid foundation for understanding regulation via TFs in P. xylostella and insights into the evolution of the P. xylostella genome.

Selecting and validating reference genes for quantitative real-time PCR in Plutella xylostella (L.).

Gene expression analysis provides important clues regarding gene functions, and quantitative real-time PCR (qRT-PCR) is a widely used method in gene expression studies. Reference genes are essential for normalizing and accurately assessing gene expression. In the present study, 16 candidate reference genes (ACTB, CyPA, EF1-α, GAPDH, HSP90, NDPk, RPL13a, RPL18, RPL19, RPL32, RPL4, RPL8, RPS13, RPS4, α-TUB, and ß-TUB) from Plutella xylostella were selected to evaluate gene expression stability across different experimental conditions using five statistical algorithms (geNorm, NormFinder, Delta Ct, BestKeeper, and RefFinder). The results suggest that different reference genes or combinations of reference genes are suitable for normalization in gene expression studies of P. xylostella according to the different developmental stages, strains, tissues, and insecticide treatments. Based on the given experimental sets, the most stable reference genes were RPS4 across different developmental stages, RPL8 across different strains and tissues, and EF1-α across different insecticide treatments. A comprehensive and systematic assessment of potential reference genes for gene expression normalization is essential for post-genomic functional research in P. xylostella, a notorious pest with worldwide distribution and a high capacity to adapt and develop resistance to insecticides.

Molecular Characterization and the Function of Argonaute3 in RNAi Pathway of Plutella xylostella.

Argonaute (Ago) protein family plays a key role in the RNA interference (RNAi) process in different insects including Lepidopteran. However, the role of Ago proteins in the RNAi pathway of Plutella xylostella is still unknown. We cloned an Argonaute3 gene in P. xylostella (PxAgo3) with the complete coding sequence of 2832 bp. The encoded protein had 935 amino acids with an expected molecular weight of 108.9 kDa and an isoelectric point of 9.29. It contained a PAZ (PIWI/Argonaute/Zwile) domain and PIWI (P-element-induced whimpy testes) domain. PxAgo3 was classified into the Piwi subfamily of Ago proteins with a high similarity of 93.0% with Bombyx mori Ago3 (BmAgo3). The suppression of PxAgo3 by dsPxAgo3 was observed 3 h after treatment and was maintained until 24 h. Knockdown of PxAgo3 decreased the suppression level of PxActin by dsPxActin in P. xylostella cells, while overexpression of PxAgo3 increased the RNAi efficiency. Our results suggest that PxAgo3 play a key role in the double stranded RNA (dsRNA)-regulated RNAi pathway in P. xylostella.

Segmental duplications: evolution and impact among the current Lepidoptera genomes.

BACKGROUND: Structural variation among genomes is now viewed to be as important as single nucleoid polymorphisms in influencing the phenotype and evolution of a species. Segmental duplication (SD) is defined as segments of DNA with homologous sequence. RESULTS: Here, we performed a systematic analysis of segmental duplications (SDs) among five lepidopteran reference genomes (Plutella xylostella, Danaus plexippus, Bombyx mori, Manduca sexta and Heliconius melpomene) to understand their potential impact on the evolution of these species. We find that the SDs content differed substantially among species, ranging from 1.2% of the genome in B. mori to 15.2% in H. melpomene. Most SDs formed very high identity (similarity higher than 90%) blocks but had very few large blocks. Comparative analysis showed that most of the SDs arose after the divergence of each linage and we found that P. xylostella and H. melpomene showed more duplications than other species, suggesting they might be able to tolerate extensive levels of variation in their genomes. Conserved ancestral and species specific SD events were assessed, revealing multiple examples of the gain, loss or maintenance of SDs over time. SDs content analysis showed that most of the genes embedded in SDs regions belonged to species-specific SDs ("Unique" SDs). Functional analysis of these genes suggested their potential roles in the lineage-specific evolution. SDs and flanking regions often contained transposable elements (TEs) and this association suggested some involvement in SDs formation. Further studies on comparison of gene expression level between SDs and non-SDs showed that the expression level of genes embedded in SDs was significantly lower, suggesting that structure changes in the genomes are involved in gene expression differences in species. CONCLUSIONS: The results showed that most of the SDs were "unique SDs", which originated after species formation. Functional analysis suggested that SDs might play different roles in different species. Our results provide a valuable resource beyond the genetic mutation to explore the genome structure for future Lepidoptera research.

Characterization and expression profiling of ATP-binding cassette transporter genes in the diamondback moth, Plutella xylostella (L.).

BACKGROUND: ATP-binding cassette (ABC) transporters are one of the major transmembrane protein families found in all organisms and play important roles in transporting a variety of compounds across intra and extra cellular membranes. In some species, ABC transporters may be involved in the detoxification of substances such as insecticides. The diamondback moth, Plutella xylostella (L.), a destructive pest of cruciferous crops worldwide, is an important species to study as it is resistant to many types of insecticides as well as biological control Bacillus thuringiensis toxins. RESULTS: A total of 82 ABC genes were identified from our published P. xylostella genome, and grouped into eight subfamilies (ABCA-H) based on phylogenetic analysis. Genes of subfamilies ABCA, ABCC and ABCH were found to be expanded in P. xylostella compared with those in Bombyx mori, Manduca sexta, Heliconius melpomene, Danaus plexippus, Drosophila melanogaster, Tetranychus urticae and Homo sapiens. Phylogenetic analysis indicated that many of the ABC transporters in P. xylostella are orthologous to the well-studied ABC transporter genes in the seven other species. Transcriptome- and qRT-PCR-based analysis elucidated physiological effects of ABC gene expressions of P. xylostella which were developmental stage- and tissue-specific as well as being affected by whether or not the insects were from an insecticide-resistant strain. Two ABCC and one ABCA genes were preferentially expressed in midgut of the 4th-instar larvae of a susceptible strain (Fuzhou-S) suggesting their potential roles in metabolizing plant defensive chemicals. Most of the highly expressed genes in insecticide-resistant strains were also predominantly expressed in the tissues of Malpighian tubules and midgut. CONCLUSIONS: This is the most comprehensive study on identification, characterization and expression profiling of ABC transporter genes in P. xylostella to date. The diversified features and expression patterns of this gene family may be associated with the evolutionary capacity of this species to develop resistance to a wide range of insecticides and biological toxins. Our findings provide a solid foundation for future functional studies on specific ABC transporter genes in P. xylostella, and for further understanding of their physiological roles and regulatory pathways in insecticide resistance.

Genome-wide identification and expression profiling of serine proteases and homologs in the diamondback moth, Plutella xylostella (L.).

BACKGROUND: Serine proteases (SPs) are crucial proteolytic enzymes responsible for digestion and other processes including signal transduction and immune responses in insects. Serine protease homologs (SPHs) lack catalytic activity but are involved in innate immunity. This study presents a genome-wide investigation of SPs and SPHs in the diamondback moth, Plutella xylostella (L.), a globally-distributed destructive pest of cruciferous crops. RESULTS: A total of 120 putative SPs and 101 putative SPHs were identified in the P. xylostella genome by bioinformatics analysis. Based on the features of trypsin, 38 SPs were putatively designated as trypsin genes. The distribution, transcription orientation, exon-intron structure and sequence alignments suggested that the majority of trypsin genes evolved from tandem duplications. Among the 221 SP/SPH genes, ten SP and three SPH genes with one or more clip domains were predicted and designated as PxCLIPs. Phylogenetic analysis of CLIPs in P. xylostella, two other Lepidoptera species (Bombyx mori and Manduca sexta), and two more distantly related insects (Drosophila melanogaster and Apis mellifera) showed that seven of the 13 PxCLIPs were clustered with homologs of the Lepidoptera rather than other species. Expression profiling of the P. xylostella SP and SPH genes in different developmental stages and tissues showed diverse expression patterns, suggesting high functional diversity with roles in digestion and development. CONCLUSIONS: This is the first genome-wide investigation on the SP and SPH genes in P. xylostella. The characterized features and profiled expression patterns of the P. xylostella SPs and SPHs suggest their involvement in digestion, development and immunity of this species. Our findings provide a foundation for further research on the functions of this gene family in P. xylostella, and a better understanding of its capacity to rapidly adapt to a wide range of environmental variables including host plants and insecticides.

Characterization and expression profiling of glutathione S-transferases in the diamondback moth, Plutella xylostella (L.).

BACKGROUND: Glutathione S-transferases (GSTs) are multifunctional detoxification enzymes that play important roles in insects. The completion of several insect genome projects has enabled the identification and characterization of GST genes over recent years. This study presents a genome-wide investigation of the diamondback moth (DBM), Plutella xylostella, a species in which the GSTs are of special importance because this pest is highly resistant to many insecticides. RESULTS: A total of 22 putative cytosolic GSTs were identified from a published P. xylostella genome and grouped into 6 subclasses (with two unclassified). Delta, Epsilon and Omega GSTs were numerically superior with 5 genes for each of the subclasses. The resulting phylogenetic tree showed that the P. xylostella GSTs were all clustered into Lepidoptera-specific branches. Intron sites and phases as well as GSH binding sites were strongly conserved within each of the subclasses in the GSTs of P. xylostella. Transcriptome-, RNA-seq- and qRT-PCR-based analyses showed that the GST genes were developmental stage- and strain-specifically expressed. Most of the highly expressed genes in insecticide resistant strains were also predominantly expressed in the Malpighian tubules, midgut or epidermis. CONCLUSIONS: To date, this is the most comprehensive study on genome-wide identification, characterization and expression profiling of the GST family in P. xylostella. The diversified features and expression patterns of the GSTs are inferred to be associated with the capacity of this species to develop resistance to a wide range of pesticides and biological toxins. Our findings provide a base for functional research on specific GST genes, a better understanding of the evolution of insecticide resistance, and strategies for more sustainable management of the pest.

Identification of Empoasca onukii (Hemiptera: Cicadellidae) and Monitoring of its Populations in the Tea Plantations of South China.

Tea green leafhoppers (Empoasca spp.) are considered one of the major pests in tea plantations in Asia. They are, however, difficult to monitor due to their size and flying and jumping abilities. In this study, we clarified the identification of the leafhopper species encountered in our study plantations and examined the impacts of sampling methods in estimating population abundance and sex ratio. The natural sex ratio of eggs, nymphs, and adults of tea green leafhopper and the differences between male and female were tested. Despite previous reports that Empoasca vitis (Goethe) was the major leafhopper present in our study area, our results showed that only Empoasca onukii Matsuda was found. Variation in population size over time and bias in sex ratio depending on the sampling methods were found in our monitoring experiments. In general, adult males were more attracted to yellow sticky cards than females. We believe that because female leafhoppers should be the target in pest control, yellow sticky cards may not be the most suitable monitoring or effective control of tea green leafhopper. We demonstrate the importance of understanding the implications of sampling techniques for population estimation and sex ratio bias as well as how temporal variation may affect monitoring results. Precise monitoring should take into consideration the different life histories of male and female.

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 and insecticide-resistant insights from the de novo assembled transcriptome of the diamondback moth, Plutella xylostella.

We present here the de novo assembly and annotation of the transcriptome of Plutella xylostella (diamondback moth (DBM)), a widespread destructive pest of cruciferous plants, using short reads generated by Illumina sequencing from different developmental stages and insecticide-resistant strains. A total of 171,262 non-redundant sequences, denoted as unigenes, were obtained. They represented approximately 100-fold of all DBM mRNA and EST sequences in GenBank thus far. We identified 38,255 unigenes highly similar to the known functional protein-coding genes, most of which were annotated using gene ontology (GO) and orthologous groups of proteins (COG). Global profiling of differentially expressed unigenes revealed enriched GOs and biological pathways that were related to specific developmental stages and insecticide resistance. We also evaluated the resistance-related single nucleotide polymorphism (SNP) using this high-throughput genotyping method. The newly developed transcriptome will facilitate researches on the DBM developmental biology and insecticide resistance evolution, and ultimately provide better pest management systems.

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.

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.