Comprehensive isoform-level analysis reveals the contribution of alternative isoforms to venom evolution and repertoire diversity.

Animal venom systems have emerged as valuable models for investigating how novel polygenic phenotypes may arise from gene evolution by varying molecular mechanisms. However, a significant portion of venom genes produce alternative mRNA isoforms that have not been extensively characterized, hindering a comprehensive understanding of venom biology. In this study, we present a full-length isoform-level profiling workflow integrating multiple RNA sequencing technologies, allowing us to reconstruct a high-resolution transcriptome landscape of venom genes in the parasitoid wasp Pteromalus puparum Our findings demonstrate that more than half of the venom genes generate multiple isoforms within the venom gland. Through mass spectrometry analysis, we confirm that alternative splicing contributes to the diversity of venom proteins, acting as a mechanism for expanding the venom repertoire. Notably, we identified seven venom genes that exhibit distinct isoform usages between the venom gland and other tissues. Furthermore, evolutionary analyses of venom serpin3 and orcokinin further reveal that the co-option of an ancient isoform and a newly evolved isoform, respectively, contributes to venom recruitment, providing valuable insights into the genetic mechanisms driving venom evolution in parasitoid wasps. Overall, our study presents a comprehensive investigation of venom genes at the isoform level, significantly advancing our understanding of alternative isoforms in venom diversity and evolution and setting the stage for further in-depth research on venoms.

Chloride intracellular channel gene knockdown induces insect cell lines death and level increases of intracellular calcium ions.

Chloride intracellular channel (CLIC) is a member of the chloride channel protein family for which growing evidence supports a pivotal role in fundamental cellular events. However, the physiological function of CLIC in insects is still rarely uncovered. The ovary-derived High Five (Hi-5) cell line isolated from the cabbage looper (Trichoplusia ni) is widely used in laboratories. Here, we studied both characteristics and functions of CLIC in Hi-5 cells (TnCLIC). We identified the TnCLIC gene in Hi-5 cells and annotated highly conserved CLIC proteins in most insect species. After RNA interference of TnCLIC, the phenomenon of significantly increased cell death suggests that the TnCLIC protein is essential for the survival of Hi-5 cells. The same lethal effect was also observed in Spodoptera frugiperda 9 and Drosophila melanogaster Schneider 2 cells after CLIC knockdown. Furthermore, we found that this kind of cell death was accompanied by increases in intracellular calcium ions after TnCLIC knockdown with the transcriptomic analyses and the detection of calcium levels. Our results provide insights into insect CLIC as a key factor for cell survival and lay the foundation for the cell death mechanism.

DeepAlgPro: an interpretable deep neural network model for predicting allergenic proteins.

Allergies have become an emerging public health problem worldwide. The most effective way to prevent allergies is to find the causative allergen at the source and avoid re-exposure. However, most of the current computational methods used to identify allergens were based on homology or conventional machine learning methods, which were inefficient and still had room to be improved for the detection of allergens with low homology. In addition, few methods based on deep learning were reported, although deep learning has been successfully applied to several tasks in protein sequence analysis. In the present work, a deep neural network-based model, called DeepAlgPro, was proposed to identify allergens. We showed its great accuracy and applicability to large-scale forecasts by comparing it to other available tools. Additionally, we used ablation experiments to demonstrate the critical importance of the convolutional module in our model. Moreover, further analyses showed that epitope features contributed to model decision-making, thus improving the model's interpretability. Finally, we found that DeepAlgPro was capable of detecting potential new allergens. Overall, DeepAlgPro can serve as powerful software for identifying allergens.

Genomic signatures associated with maintenance of genome stability and venom turnover in two parasitoid wasps.

Parasitoid wasps are rapidly developing as a model for evolutionary biology. Here we present chromosomal genomes of two Anastatus wasps, A. japonicus and A. fulloi, and leverage these genomes to study two fundamental questions-genome size evolution and venom evolution. Anastatus shows a much larger genome than is known among other wasps, with unexpectedly recent bursts of LTR retrotransposons. Importantly, several genomic innovations, including Piwi gene family expansion, ubiquitous Piwi expression profiles, as well as transposable element-piRNA coevolution, have likely emerged for transposable element silencing to maintain genomic stability. Additionally, we show that the co-option evolution arose by expression shifts in the venom gland plays a dominant role in venom turnover. We also highlight the potential importance of non-venom genes that are coexpressed with venom genes during venom evolution. Our findings greatly advance the current understanding of genome size evolution and venom evolution, and these genomic resources will facilitate comparative genomics studies of insects in the future.

Chromosome-level genome assembly of an agricultural pest, the rice leaffolder Cnaphalocrocis exigua (Crambidae, Lepidoptera).

The rice leaffolder Cnaphalocrocis exigua (Crambidae, Lepidoptera) is an important agricultural pest that damages rice crops and other members of related grass families. C. exigua exhibits a very similar morphological phenotype and feeding behaviour to C. medinalis, another species of rice leaffolder whose genome was recently reported. However, genomic information for C. exigua remains extremely limited. Here, we used a hybrid strategy combining different sequencing technologies, including Illumina, PacBio, 10× Genomics, and Hi-C scaffolding, to generate a high-quality chromosome-level genome assembly of C. exigua. We initially obtained a 798.8 Mb assembly with a contig N50 size of 2.9 Mb, and the N50 size was subsequently increased to 25.7 Mb using Hi-C technology to anchor 1413 scaffolds to 32 chromosomes. We detected a total of 97.7% Benchmarking Universal Single-Copy Orthologues (BUSCO) in the genome assembly, which was comprised of ~52% repetitive sequence and annotated 14,922 protein-coding genes. Of note, the Z and W sex chromosomes were assembled and identified. A comparative genomic analysis demonstrated that despite the high synteny observed between the two rice leaffolders, the species have distinct genomic features associated with expansion and contraction of gene families and selection pressure. In summary, our chromosome-level genome assembly and comparative genomic analysis of C. exigua provide novel insights into the evolution and ecology of this rice insect pests and offer useful information for pest control.

A chromosome-level genome assembly of rice leaffolder, Cnaphalocrocis medinalis.

The rice leaffolder, Cnaphalocrocis medinalis Guenée (Crambidae, Lepidoptera), is an important agricultural pest that causes serious losses to rice production in rice-growing regions with high humidity and temperature. However, a lack of genomic resources limits in-depth understanding of its biological characteristics and ecological adaptation. Here, we sequenced the genome of rice leaffolder using the Illumina and PacBio platforms, yielding a genome assembly of 528.3 Mb with a contig N50 of 524.6 kb. A high percentage (96.4%) of Benchmarking Universal Single-Copy Orthologs (BUSCOs) were successfully detected, suggesting high-level completeness of the genome assembly. In total, 39.5% of the genome consists of repeat sequences and 15,045 protein-coding genes were annotated. Comparative phylogenomic analysis showed that some gene families associated with hormone biosynthesis expanded in rice leaffolder. Next, we used the Hi-C technique to produce a chromosome-level genome assembly with a scaffold N50 of 16.1 Mb by anchoring 3,248 scaffolds to 31 chromosomes. The rice leaffolder genome showed high chromosomal synteny with the genome of four other lepidopteran insects. By comparing coverage ratios from the genome resequencing of male and female pupae, we identified near intact Z and W chromosomes. The W chromosome is estimated as 20.75 Mb, which is the most complete known W chromosome in Lepidoptera. The protein-coding genes on the W chromosome were significantly enriched in metabolic pathways. In all, the high-quality genome assembly and the near-intact W chromosome of rice leaffolder should be a useful resource for the fields of insect migration, chromosome evolution and pest control.

A chromosome-level genome assembly provides new insights into paternal genome elimination in the cotton mealybug Phenacoccus solenopsis.

Mealybugs (Hemiptera: Pseudococcidae) are economically important agricultural pests with several compelling biological phenomena including paternal genome elimination (PGE). However, limited high-quality genome assemblies of mealybugs hinder a full understanding of this striking and unusual biological phenomenon. Here, we generated a chromosome-level genome assembly of cotton mealybug, Phenacoccus solenopsis, by combining Illumina short reads, PacBio long reads and Hi-C scaffolding. The assembled genome was 292.54 Mb with a contig N50 of 489.8 kb and a scaffold N50 of 49.0 Mb. Hi-C scaffolding assigned 84.42% of the bases to five chromosomes. A total of 110.75 Mb (37.9%) repeat sequences and 11,880 protein-coding genes were predicted. The completeness of the genome assembly was estimated to be 95.5% based on BUSCO genes. In addition, 27,086 (95.3%) full-length PacBio transcripts were uniquely mapped to the assembled scaffolds, suggesting the high quality of the genome assembly. We showed that cotton mealybugs lack differentiated sex chromosomes by analysing genome resequencing data of males and females. DAPI staining confirmed that one chromosome set in males becomes heterochromatin at an early embryo stage. Chromatin immunoprecipitation assays with sequencing analysis demonstrated that the epigenetic modifications H3K9me3 and H3K27me3 are distributed across the whole genome in males, suggesting that these two modifications might be involved in maintaining heterochromatin status. Both markers were more likely to be distributed in repeat regions, while H3K27me3 had higher overall enrichment. Our results provide a valuable genomic resource and shed new light on the genomic and epigenetic basis of PGE in cotton mealybugs.

A chromosome-level genome assembly reveals the genetic basis of cold tolerance in a notorious rice insect pest, Chilo suppressalis.

The rice stem borer, Chilo suppressalis, is one of the most damaging insect pests to rice production worldwide. Although C. suppressalis has been the focus of numerous studies examining cold tolerance and diapause, plant-insect interactions, pesticide targets and resistance, and the development of RNAi-mediated pest management, the absence of a high-quality genome has limited deeper insights. To address this limitation, we generated a draft C. suppressalis genome constructed from both Illumina and PacBio sequences. The assembled genome size was 824.35 Mb with a contig N50 of 307 kb and a scaffold N50 of 1.75 Mb. Hi-C scaffolding assigned 99.2% of the bases to one of 29 chromosomes. Based on universal single-copy orthologues (BUSCO), the draft genome assembly was estimated to be 97% complete and is predicted to encompass 15,653 protein-coding genes. Cold tolerance is an extreme survival strategy found in animals. However, little is known regarding the genetic basis of the winter ecology of C. suppressalis. Here, we focused our orthologous analysis on those gene families associated with animal cold tolerance. Our finding provided the first genomic evidence revealing specific cold-tolerant strategies in C. suppressalis, including those involved in glucose-originated glycerol biosynthesis, triacylglycerol-originated glycerol biosynthesis, fatty acid synthesis and trehalose transport-intermediate cold tolerance. The high-quality C. suppressalis genome provides a valuable resource for research into a broad range of areas in molecular ecology, and subsequently benefits developing modern pest control strategies.

A chromosome-level genome assembly of Cydia pomonella provides insights into chemical ecology and insecticide resistance.

The codling moth Cydia pomonella, a major invasive pest of pome fruit, has spread around the globe in the last half century. We generated a chromosome-level scaffold assembly including the Z chromosome and a portion of the W chromosome. This assembly reveals the duplication of an olfactory receptor gene (OR3), which we demonstrate enhances the ability of C. pomonella to exploit kairomones and pheromones in locating both host plants and mates. Genome-wide association studies contrasting insecticide-resistant and susceptible strains identify hundreds of single nucleotide polymorphisms (SNPs) potentially associated with insecticide resistance, including three SNPs found in the promoter of CYP6B2. RNAi knockdown of CYP6B2 increases C. pomonella sensitivity to two insecticides, deltamethrin and azinphos methyl. The high-quality genome assembly of C. pomonella informs the genetic basis of its invasiveness, suggesting the codling moth has distinctive capabilities and adaptive potential that may explain its worldwide expansion.