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.

Genome of the parasitoid wasp Cotesia chilonis sheds light on amino acid resource exploitation.

BACKGROUND: A fundamental feature of parasitism is the nutritional exploitation of host organisms by their parasites. Parasitoid wasps lay eggs on arthropod hosts, exploiting them for nutrition to support larval development by using diverse effectors aimed at regulating host metabolism. However, the genetic components and molecular mechanisms at the basis of such exploitation, especially the utilization of host amino acid resources, remain largely unknown. To address this question, here, we present a chromosome-level genome assembly of the parasitoid wasp Cotesia chilonis and reconstruct its amino acid biosynthetic pathway. RESULTS: Analyses of the amino acid synthetic pathway indicate that C. chilonis lost the ability to synthesize ten amino acids, which was confirmed by feeding experiments with amino acid-depleted media. Of the ten pathways, nine are known to have been lost in the common ancestor of animals. We find that the ability to synthesize arginine was also lost in C. chilonis because of the absence of two key genes in the arginine synthesis pathway. Further analyses of the genomes of 72 arthropods species show that the loss of arginine synthesis is common in arthropods. Metabolomic analyses by UPLC-MS/MS reveal that the temporal concentrations of arginine, serine, tyrosine, and alanine are significantly higher in host (Chilo suppressalis) hemolymph at 3 days after parasitism, whereas the temporal levels of 5-hydroxylysine, glutamic acid, methionine, and lysine are significantly lower. We sequence the transcriptomes of a parasitized host and non-parasitized control. Differential gene expression analyses using these transcriptomes indicate that parasitoid wasps inhibit amino acid utilization and activate protein degradation in the host, likely resulting in the increase of amino acid content in host hemolymph. CONCLUSIONS: We sequenced the genome of a parasitoid wasp, C. chilonis, and revealed the features of trait loss in amino acid biosynthesis. Our work provides new insights into amino acid exploitation by parasitoid wasps, and this knowledge can specifically be used to design parasitoid artificial diets that potentially benefit mass rearing of parasitoids for pest control.

Altered Gene Expression of the Parasitoid Pteromalus puparum after Entomopathogenic Fungus Beauveria bassiana Infection.

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.

Comparative Genomics Sheds Light on the Convergent Evolution of Miniaturized Wasps.

Miniaturization has occurred in many animal lineages, including insects and vertebrates, as a widespread trend during animal evolution. Among Hymenoptera, miniaturization has taken place in some parasitoid wasp lineages independently, and may have contributed to the diversity of species. However, the genomic basis of miniaturization is little understood. Diverged approximately 200 Ma, Telenomus wasps (Platygastroidea) and Trichogramma wasps (Chalcidoidea) have both evolved to a highly reduced body size independently, representing a paradigmatic example of convergent evolution. Here, we report a high-quality chromosomal genome of Telenomus remus, a promising candidate for controlling Spodoptera frugiperda, a notorious pest that has recently caused severe crop damage. The T. remus genome (129 Mb) is characterized by a low density of repetitive sequence and a reduction of intron length, resulting in the shrinkage of genome size. We show that hundreds of genes evolved faster in two miniaturized parasitoids Trichogramma pretiosum and T. remus. Among them, 38 genes exhibit extremely accelerated evolutionary rates in these miniaturized wasps, possessing diverse functions in eye and wing development as well as cell size control. These genes also highlight potential roles in body size regulation. In sum, our analyses uncover a set of genes with accelerated evolutionary rates in Tri. pretiosum and T. remus, which might be responsible for their convergent adaptations to miniaturization, and thus expand our understanding on the evolutionary basis of miniaturization. Additionally, the genome of T. remus represents the first genome resource of superfamily Platygastroidea, and will facilitate future studies of Hymenoptera evolution and pest control.

Genes acting in longevity-related pathways in the endoparasitoid, Pteromalus puparum.

Among insects, lifespans vary over a broad range, from the short-lived mayflies to the 17-year periodical cicadas. Generally, lifespans are determined by a phase in life, the reproductive lifespan, which varies among species. Numerous pathways, such as the insulin/insulin-like growth factor signaling pathway, the target of rapamycin pathway and the mitogen-activated protein kinase/extracellular signal-regulated kinases pathways, influence aging and lifespan. Components of these pathways were identified as lifespan-related genes, including genes mediating growth, metabolism, development, resistance, and other processes. Many age-related genes have been discovered in fruit flies, honeybees, and ants among other insect species. Studies of insect aging and longevity can help understand insect biology and develop new pest management technologies. In this paper, we interrogated the new Pteromalus puparum genome, from which we predicted 133 putative lifespan-related genes based on their homology with known lifespan-related genes of Drosophila melanogaster. These genes function in five signaling pathways and three physiological processes. The conserved domain structures of these genes were predicted and their expression patterns were analyzed. Amino acid sequence alignments and domain structure analysis indicate that most components remain conserved across at least six insect orders. The data in this paper will facilitate future work on parasitoid lifespans, which may have economic value in biocontrol programs.

Identification and characterization of miRNAs in an endoparasitoid wasp, Pteromalus puparum.

MicroRNAs (miRNAs) are a form of endogenous small noncoding RNAs that regulate protein-coding gene expression at the posttranscriptional level. So far, knowledge of miRNAs in parasitoids remains rudimentary. We investigated miRNAs in Pteromalus puparum, a pupal endoparasitoid wasp with genome and transcriptome sequences completed. In this study, we constructed eight small RNA libraries from selected developmental stages and genders: male embryos, male larvae, male pupae, male adults, mixed-sex embryos, mixed-sex larvae, mixed-sex pupae, and female adults. We identified 254 mature miRNAs with 5p/3p arm features originated from 75 known and 119 novel miRNA genes in P. puparum, 88 of which reside in 26 clusters. The miRNAs in more than half of the clusters exhibit a consistent expression pattern, indicating they were co-transcribed from a long transcript. Comparing miRNA expression in the eight libraries, we found that 84 mature miRNAs were differentially expressed between embryos and larvae, 20 between larvae and pupae, and 26 between pupae and adults. We found some miRNAs were differentially expressed between sexes in embryos (10), larvae (29), pupae (8), and adults (14). Target predictions resulted in 211,571 miRNA-mRNA interactions for 254 different mature miRNAs. These miRNAs may be involved in sexual and developmental regulation of gene expression.

An Ovarian Protein Involved in Passive Avoidance of an Endoparasitoid To Evade Its Host Immune Response.

Through a combination of transcriptomic and proteomic analyses, we identified 817 secreted ovarian proteins from an endoparasitoid wasp, Cotesia chilonis, of which five proteins are probably involved in passive evasion. The results of an encapsulation assay revealed that one of these passive evasion-associated proteins (Crp32B), a homologue of a 32-kDa protein (Crp32) from C. rubecula, could protect resin beads from being encapsulated by host hemocytes in a dose-dependent manner. Crp32B is transcribed in ovarian cells, nurse cells, follicular cells, and oocytes, and the protein is located throughout the ovary and on the egg surface. Moreover, Crp32B has antigenic similarity to several host components. These results indicate that C. chilonis may use molecular mimicry as a mechanism to avoid host cellular immune response.

CRISPR/Cas9-mediated mutagenesis of the white gene in an ectoparasitic wasp, Habrobracon hebetor.

BACKGROUND: The ectoparasitic wasp Habrobracon hebetor (Hymenoptera, Braconidae) can parasitize various species of lepidopteran pests. To maximize its potential for biological control, it is necessary to investigate its gene function through genome engineering. RESULTS: To test the effectiveness of genome engineering system in H. hebetor, we injected the mixture of clustered regularly interspaced short palindromic repeats (CRISPR) -associated (Cas) 9 protein and single guide RNA(s) targeting gene white into embryos. The resulting mutants display a phenotype of eye pigment loss. The phenotype was caused by small indel and is heritable. Then, we compared some biological parameters between wildtype and mutant, and found there were no significant differences in other parameters except for the offspring female rate and adult longevity. In addition, cocoons could be used to extract genomic DNA for genotype during the gene editing process without causing unnecessary harm to H. hebetor. CONCLUSION: Our results demonstrate that the CRISPR/Cas9 system can be used for H. hebetor genome editing and it does not adversely affect biological parameters of the parasitoid wasps. We also provide a feasible non-invasive genotype detection method using genomic DNA extracted from cocoons. Our study introduces a novel tool and method for studying gene function in H. hebetor, and may contribute to better application of H. hebetor in biocontrol. © 2023 Society of Chemical Industry.

Regulatory network in heat stress response in parasitoid wasp focusing on Xap5 heat stress regulator.

Insects are susceptible to elevated temperatures, resulting in impaired fertility, and shortened lifespan. This study investigated the genetic mechanisms underlying heat stress effects. We conducted RNA sequencing on Pteromalus puparum exposed to 25°C and 35°C, revealing transcriptional signatures. Weighted Gene Co-expression Network Analysis uncovered heat stress-associated modules, forming a regulatory network of 113 genes. The network is naturally divided into two subgroups, one linked to acute heat stress, including heat shock proteins (HSPs), and the other to chronic heat stress, involving lipogenesis genes. We identified an Xap5 Heat Shock Regulator (XHSR) gene as a crucial network component, validated through RNA interference and quantitative PCR assays. XHSR knockdown reduced wasps' lifespan while directly inducing HSPs and mediating lipogenesis gene induction. CRISPR/Cas9-mediated knockout of the Drosophila XHSR homolog reduced mutants' survival, highlighting its conserved role. This research sheds light on thermal tolerance mechanisms, offering potential applications in pest control amid global warming.

Identification and functional analysis of αB-crystallins in Pteromalus puparum.

Heat shock proteins, including αB-crystallins (CRYAB), are pivotal in cellular defense mechanisms and stress response. This study presents a comprehensive investigation of heat shock proteins (HSPs), with a specific focus on the CRYAB family, within the genome of Pteromalus puparum. The analysis encompasses the identification of these proteins, exploration of their phylogenetic relationships, examination of conserved domains, and evaluation of their response to high temperature conditions. A total of 46 HSPs were identified in the P. puparum genome, and the differential expression of mRNA at 35°C and 25°C drew attention to five genes belonging to the CRYAB family, namely, PpCRYAB-1 to PpCRYAB-5. The conservation level of CRYAB family genes across different species was observed to be relatively modest. Through genome-wide screening of 22 species representing six insect orders, a total of 235 CRYAB proteins were identified, with P. puparum harboring eight CRYAB proteins, indicative of a moderate abundance compared to other species. Intriguingly, evolutionary analysis highlighted PpCRYAB-4 with potentially intricate differentiation in comparison to other members of the CRYAB family. Furthermore, RNA interference (RNAi) results demonstrated significant regulatory effects on adult lifespan under heat stress at 35°C for PpCRYAB-4 and PpCRYAB-5. These findings lay a groundwork for future investigations into stress resistance mechanisms in parasitic wasps, providing fresh insights for the study of insect resilience amidst the backdrop of global climate change.

Multi-Omic Identification of Venom Proteins Collected from Artificial Hosts of a Parasitoid Wasp.

Habrobracon hebetor is a parasitoid wasp capable of infesting many lepidopteran larvae. It uses venom proteins to immobilize host larvae and prevent host larval development, thus playing an important role in the biocontrol of lepidopteran pests. To identify and characterize its venom proteins, we developed a novel venom collection method using an artificial host (ACV), i.e., encapsulated amino acid solution in paraffin membrane, allowing parasitoid wasps to inject venom. We performed protein full mass spectrometry analysis of putative venom proteins collected from ACV and venom reservoirs (VRs) (control). To verify the accuracy of proteomic data, we also collected venom glands (VGs), Dufour's glands (DGs) and ovaries (OVs), and performed transcriptome analysis. In this paper, we identified 204 proteins in ACV via proteomic analysis; compared ACV putative venom proteins with those identified in VG, VR, and DG via proteome and transcriptome approaches; and verified a set of them using quantitative real-time polymerase chain reaction. Finally, 201 ACV proteins were identified as potential venom proteins. In addition, we screened 152 and 148 putative venom proteins identified in the VG transcriptome and the VR proteome against those in ACV, and found only 26 and 25 putative venom proteins, respectively, were overlapped with those in ACV. Altogether, our data suggest proteome analysis of ACV in combination with proteome-transcriptome analysis of other organs/tissues will provide the most comprehensive identification of true venom proteins in parasitoid wasps.

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.

Effects of sugar sources on adult longevity, survival and related gene expression in an endoparasitoid, Pteromalus puparum (Hymenoptera: Pteromalidae).

BACKGROUND: Adult parasitic wasps take sugars to meet their energy needs and display different lifespans and fertility in response to different sugar sources. Pteromalus puparum is an endoparasitoid with a wide range of hosts, including many lepidopteran pests. As a potential natural enemy resource, the availability of sugar sources has profound effects for wasp applications and host populations dynamics. RESULTS: We assessed the effect of feeding sucrose and honey on the lifespan of P. puparum in the range 0-40% (w/v). The results indicated a statistically significant positive effect of sucrose and honey solutions on the lifespan of P. puparum female adults. Correlation analyses confirmed a strong positive correlation between high concentrations of sugar and extended lifespan. The optimum concentration of sucrose solution for wasps was 20%, while 10% for honey. Then, we examined the expression patterns of 15 lifespan-related genes. The results showed that the relative expression levels of 14 genes were significantly correlated with the mean lifespan of sucrose-fed wasps, and six genes correlated with the mean lifespan of honey-fed wasps. In addition, the models for lifespan prediction were constructed. CONCLUSION: We elaborated the quantitative effects of two sugar sources (sucrose and honey) on P. puparum lifespan, investigated the expression patterns of lifespan-related genes when fed different sugar sources, and developed round lifespan prediction models accordingly. This study provides a novel tool for studying the longevity regulating mechanisms of parasitic wasps, and may be instructive for mass-production of parasitoids as biological control agents. © 2020 Society of Chemical Industry.

Identification of Neuropeptides and Their Receptors in the Ectoparasitoid, Habrobracon hebetor.

Neuropeptides are a group of signal molecules that regulate many physiological and behavioral processes by binding to corresponding receptors, most of which are G-protein-coupled receptors (GPCRs). Using bioinformatic methods, we screened genomic and transcriptomic data of the ectoparasitoid wasp, Habrobracon hebetor, and annotated 34 neuropeptide candidate precursor genes and 44 neuropeptide receptor candidate genes. The candidate neuropeptide genes were found to encode all known insect neuropeptides except allatotropin, neuropeptide F, pigment dispersing factor, and CCHamides. When compared with the endoparasitic wasp Pteromalus puparum and the ectoparasitic wasp Nasonia vitripennis, trissin and FMRFamide were found only in H. hebetor. A similar result held for the neuropeptide receptor genes, for the receptors were found in H. hebetor except the receptors of CCHamides and neuroparsin. Furthermore, we compared and analyzed the differences in neuropeptides in eight Braconidae wasps and identified natalisin in H. hebetor, Diachasma alloeum, Fopius arisanus and Microplitis demolitor, but not in the other wasps. We also analyzed the transcriptome data and qRT-PCR data from different developmental stages and tissues to reveal the expression patterns of the neuropeptides and their receptors. In this study, we revealed composition of neuropeptides and neuropeptide receptors in H. hebetor, which may contribute to future neurobiological studies.

A chromosome-level genome assembly of the parasitoid wasp Pteromalus puparum.

Parasitoid wasps represent a large proportion of hymenopteran species. They have complex evolutionary histories and are important biocontrol agents. To advance parasitoid research, a combination of Illumina short-read, PacBio long-read and Hi-C scaffolding technologies was used to develop a high-quality chromosome-level genome assembly for Pteromalus puparum, which is an important pupal endoparasitoid of caterpillar pests. The chromosome-level assembly has aided in studies of venom and detoxification genes. The assembled genome size is 338 Mb with a contig N50 of 38.7 kb and a scaffold N50 of 1.16 Mb. Hi-C analysis assembled scaffolds onto five chromosomes and raised the scaffold N50 to 65.8 Mb, with more than 96% of assembled bases located on chromosomes. Gene annotation was assisted by RNA sequencing for the two sexes and four different life stages. Analysis detected 98% of the BUSCO (Benchmarking Universal Single-Copy Orthologs) gene set, supporting a high-quality assembly and annotation. In total, 40.1% (135.6 Mb) of the assembly is composed of repetitive sequences, and 14,946 protein-coding genes were identified. Although venom genes play important roles in parasitoid biology, their spatial distribution on chromosomes was poorly understood. Mapping has revealed venom gene tandem arrays for serine proteases, pancreatic lipase-related proteins and kynurenine-oxoglutarate transaminases, which have amplified in the P. puparum lineage after divergence from its common ancestor with Nasonia vitripennis. In addition, there is a large expansion of P450 genes in P. puparum. These examples illustrate how chromosome-level genome assembly can provide a valuable resource for molecular, evolutionary and biocontrol studies of parasitoid wasps.

Amino acid synthesis loss in parasitoid wasps and other hymenopterans.

Insects utilize diverse food resources which can affect the evolution of their genomic repertoire, including leading to gene losses in different nutrient pathways. Here, we investigate gene loss in amino acid synthesis pathways, with special attention to hymenopterans and parasitoid wasps. Using comparative genomics, we find that synthesis capability for tryptophan, phenylalanine, tyrosine, and histidine was lost in holometabolous insects prior to hymenopteran divergence, while valine, leucine, and isoleucine were lost in the common ancestor of Hymenoptera. Subsequently, multiple loss events of lysine synthesis occurred independently in the Parasitoida and Aculeata. Experiments in the parasitoid Cotesia chilonis confirm that it has lost the ability to synthesize eight amino acids. Our findings provide insights into amino acid synthesis evolution, and specifically can be used to inform the design of parasitoid artificial diets for pest control.

Protein Discovery: Combined Transcriptomic and Proteomic Analyses of Venom from the Endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae).

Many species of endoparasitoid wasps provide biological control services in agroecosystems. Although there is a great deal of information on the ecology and physiology of host/parasitoid interactions, relatively little is known about the protein composition of venom and how specific venom proteins influence physiological systems within host insects. This is a crucial gap in our knowledge because venom proteins act in modulating host physiology in ways that favor parasitoid development. Here, we identified 37 possible venom proteins from the polydnavirus-carrying endoparasitoid Cotesia chilonis by combining transcriptomic and proteomic analyses. The most abundant proteins were hydrolases, such as proteases, peptidases, esterases, glycosyl hydrolase, and endonucleases. Some components are classical parasitoid venom proteins with known functions, including extracellular superoxide dismutase 3, serine protease inhibitor and calreticulin. The venom contains novel proteins, not recorded from any other parasitoid species, including tolloid-like proteins, chitooligosaccharidolytic ß-N-acetylglucosaminidase, FK506-binding protein 14, corticotropin-releasing factor-binding protein and vascular endothelial growth factor receptor 2. These new data generate hypotheses and provide a platform for functional analysis of venom components.