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.

The ectoparasitoid waspTheocolax elegansis a cosmopolitan and generalist pteromalid parasitoid of several major storage insect pests, and can effectively suppress a host population in warehouses. However, little molecular information about this wasp is currently available. In this study, we assembled the genome ofT. elegansusing PacBio long-read sequencing, Illumina sequencing, and Hi-C methods. The genome assembly is 662.73 Mb in length with contig and scaffold N50 values of 1.15 Mb and 88.8 Mb, respectively. The genome contains 56.4% repeat sequences and 23,212 protein-coding genes were annotated. Phylogenomic analyses revealed thatT. elegansdiverged from the lineage leading to subfamily Pteromalinae (Nasonia vitripennisandPteromalus puparum) approximately 110.5 million years ago. We identified 130 significantly expanded gene families, 34 contracted families, 248 fast-evolving genes, and 365 positively selected genes inT. elegans. Additionally, 260 olfactory receptors and 285 venom proteins were identified. This genome assembly provides valuable genetic bases for future investigations on evolution, molecular biology and application ofT. elegans.

The order Hymenoptera includes a large number of species with diverse lifestyles and is known for its significant contributions to natural ecosystems. To better understand the evolution of this diverse order, we performed large-scale comparative genomics on 131 species from 13 superfamilies, covering most representative groups. We used these genomes to reveal an overall pattern of genomic change in terms of gene content and evolutionary rate throughout hymenopteran history. We identified genes that possibly contributed to the evolution of several key innovations, such as parasitoidism, wasp-waist, stinger, and secondary phytophagy. We also discovered the distinct genomic trajectories between the clade containing major parasitoid wasps (Parasitoida) and stinging species (Aculeata) since their divergence, which are involved in many aspects of genomic change, such as rapidly evolving gene families, gene gain and loss, and metabolic pathway evolution. In addition, we explored the genomic features accompanying the three independent evolution of secondary phytophagy. Our work provides insights for understanding genome evolution and the genomic basis of diversification in Hymenoptera.

Both parasitoids and entomopathogenic fungi are becoming increasingly crucial for managing pest populations. Therefore, it is essential to carefully consider the potential impact of entomopathogenic fungi on parasitoids due to their widespread pathogenicity and the possible overlap between these biological control tools during field applications. However, despite their importance, little research has been conducted on the pathogenicity of entomopathogenic fungi on parasitoids. In our study, we aimed to address this knowledge gap by investigating the interaction between the well-known entomopathogenic fungus Beauveria bassiana, and the pupal endoparasitoid Pteromalus puparum. Our results demonstrated that the presence of B. bassiana significantly affected the survival rates of P. puparum under laboratory conditions. The pathogenicity of B. bassiana on P. puparum was dose- and time-dependent, as determined via through surface spraying or oral ingestion. RNA-Seq analysis revealed that the immune system plays a primary and crucial role in defending against B. bassiana. Notably, several upregulated differentially expressed genes (DEGs) involved in the Toll and IMD pathways, which are key components of the insect immune system, and antimicrobial peptides were rapidly induced during both the early and late stages of infection. In contrast, a majority of genes involved in the activation of prophenoloxidase and antioxidant mechanisms were downregulated. Additionally, we identified downregulated DEGs related to cuticle formation, olfactory mechanisms, and detoxification processes. In summary, our study provides valuable insights into the interactions between P. puparum and B. bassiana, shedding light on the changes in gene expression during fungal infection. These findings have significant implications for the development of more effective and sustainable strategies for pest management in agriculture.

Venoms have repeatedly evolved over 100 occasions throughout the animal tree of life, making them excellent systems for exploring convergent evolutionary novelty. Growing evidence supports that venom evolution is predominantly driven by prey or host-related selection pressures, and the expression patterns of venom glands reflect adaptive evolution. However, it remains elusive whether the evolution of expression patterns in venom glands is likewise a convergent evolution driven by their prey/host species. We utilized parasitoid wasps that had independently adapted to Drosophila hosts as models to investigate the convergent evolution of venom gland transcriptomes in 19 hymenopteran species spanning ~ 200 million years of evolution. Comparative transcriptome analysis reveals that the global expression patterns among the venom glands of Drosophila parasitoid wasps do not achieve higher similarity compared to non-Drosophila parasitoid wasps. Further evolutionary analyses of expression patterns at the single gene, orthogroup, and Gene Ontology (GO) term levels indicate that some orthogroups/GO terms show correlation with the Drosophila parasitoid wasps. However, these groups rarely include genes highly expressed in venom glands or putative venom genes in the Drosophila parasitoid wasps. Our study suggests that convergent evolution may not play a predominant force shaping gene expression levels in the venom gland of the Drosophila parasitoid wasps, offering novel insights into the co-evolution between venom and prey/host.

New genes play a critical role in phenotypic diversity and evolutionary innovation. Parasitoid wasps, a highly abundant and diverse group of insects, parasitize other arthropods and exhibit remarkable evolutionary adaptations, such as evading host immune responses and exploiting host resources. However, the specific contributions of new genes to their unique traits remain poorly understood. Here, we identified 480 new genes that emerged after the Nasonia-Pteromalus divergence. Among these, 272 (56.7%) originated through DNA-mediated duplication, representing the largest proportion, followed by 77 (16.0%) derived from RNA-mediated duplication and 131 (27.3%) that arose de novo. Comparative analysis revealed that these new genes generally have shorter coding sequences and fewer exons compared to single-copy older genes conserved in the seven parasitoid wasps. These new genes are predominantly expressed in the reproductive glands and exhibit venom gland-biased expression. Notably, gene co-expression network analysis further identified that a new gene may act as a hub by interacting with older genes to regulate venom-related networks rather than directly encoding venom proteins. Together, our findings provide novel insights into the role of new genes in driving venom innovation in parasitoid wasps.