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.

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.

A serpin gene from a parasitoid wasp disrupts host immunity and exhibits adaptive alternative splicing.

Alternative splicing (AS) is a major source of protein diversity in eukaryotes, but less is known about its evolution compared to gene duplication (GD). How AS and GD interact is also largely understudied. By constructing the evolutionary trajectory of the serpin gene PpSerpin-1 (Pteromalus puparum serpin 1) in parasitoids and other insects, we found that both AS and GD jointly contribute to serpin protein diversity. These two processes are negatively correlated and show divergent features in both protein and regulatory sequences. Parasitoid wasps exhibit higher numbers of serpin protein/domains than nonparasitoids, resulting from more GD but less AS in parasitoids. The potential roles of AS and GD in the evolution of parasitoid host-effector genes are discussed. Furthermore, we find that PpSerpin-1 shows an exon expansion of AS compared to other parasitoids, and that several isoforms are involved in the wasp immune response, have been recruited to both wasp venom and larval saliva, and suppress host immunity. Overall, our study provides an example of how a parasitoid serpin gene adapts to parasitism through AS, and sheds light on the differential features of AS and GD in the evolution of insect serpins and their associations with the parasitic life strategy.

ICTV Virus Taxonomy Profile: Lispiviridae 2023.

Members of the family Lispiviridae are viruses with negative-sense RNA genomes of 6.5-15.5 kb that have mainly been found in arthropods and nematodes. The genomes of lispivirids contain several open reading frames, typically encoding a nucleoprotein (N), a glycoprotein (G), and a large protein (L) including an RNA-directed RNA polymerase (RdRP) domain. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Lispiviridae, which is available at ictv.global/report/lispiviridae.

Disruption of serotonin biosynthesis increases resistance to striped stem borer without changing innate defense response in rice.

Striped stem borer (SSB) is one of the most damaging pests in rice production worldwide. Previously, we preliminarily demonstrated that indica rice Jiazhe LM, an OsT5H (encoding tryptamine-5-hydroxylase) knockout mutant deficient in serotonin, had increased resistance to SSB as compared with its wildtype parent Jiazhe B. However, the full scenario of SSB resistance and the underlying mechanism remain unknown. In this study, we first demonstrated that the OsT5H knockout could generally increase rice resistance to SSB and then proved that the OsT5H knockout does not disrupt the innate defense response of rice plants to SSB infestation, that is, OsT5H knockout mutations neither had significant effect on the transcriptional response of defense genes upon SSB infestation, nor the profile of defense related metabolites and plant hormones, such as lignin, salicylic acid, jasmonic acid, and abscisic acid, nor the activity of reactive oxygen species (ROS) scavenging enzymes and the ROS contents. We then demonstrated that supplementation of serotonin promoted SSB growth and performance in artificial diet feeding experiments. We observed that SSB larvae feeding on Jiazhe B had serotonin 1.72- to 2.30-fold that of those feeding on Jiazhe LM at the whole body level, and more than 3.31 and 1.84 times in the hemolymph and head, respectively. Further studies showed that the expression of genes involved in serotonin biosynthesis and transport was ~88.1% greater in SSB larvae feeding on Jiahze LM than those feeding on Jiazhe B. These observations indicated that SSB increases serotonin synthesis when feeding on serotonin deficient rice but is unable to fully compensate the dietary serotonin deficiency. Put together, the present study strongly suggests that it is the deficiency of serotonin, not the secondary effect of OsT5H knockout on innate defense response confers the SSB resistance in rice, which implies that reducing serotonin level, particularly through inhibition of its inductive synthesis upon SSB damage, could be an efficient strategy for breeding SSB resistant varieties.

Transcriptional regulation of host insulin signaling pathway genes controlling larval development by Microplitis manilae parasitization.

Idiobiont parasitoids using other insects as hosts sabotage the host growth and development to ensure their offspring survival. Numerous studies have discovered that insect development is subtly regulated by the conserved insulin signaling pathway. However, little is known about how wasp parasitization disrupts host development controlled by the insulin signaling pathway. Here we address this study to determine the effect of wasp parasitism on host Spodoptera frugiperda development using the idiobiont parasitoid Microplitis manilae as a model. Upon M. manilae parasitization, the body weight, body length, and food consumption of host insect were dramatically reduced compared to the unparasitized S. frugiperda. We next identified the core genes involved in host insulin signaling pathway and further analyzed the domain organizations of these genes. Phylogenetic reconstruction based on the insulin receptors clustered S. frugiperda together with other noctuidae insects. In the latter study, we profiled the expression patterns of host insulin signaling pathway genes in response to M. manilae parasitization at 2, 24, and 48 h, significant decreases in mRNA levels were recorded in S. frugiperda larvae upon 24 and 48 h parasitization. These current findings substantially add to our understanding of the physiological interaction between parasitoid and host insects, thus contributing to revealing the molecular mechanism of parasitic wasps regulating host development.

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.

Virus-Induced Plant Volatiles Promote Virus Acquisition and Transmission by Insect Vectors.

Rice dwarf virus (RDV) is transmitted by insect vectors Nephotettix virescens and Nephotettix cincticeps (Hemiptera: Cicadellidae) that threatens rice yield and results in substantial economic losses. RDV induces two volatiles ((E)-ß-caryophyllene (EBC) and 2-heptanol) to emit from RDV-infected rice plants. However, the effects of the two volatiles on the olfactory behavior of both non-viruliferous and viruliferous N. virescens are unknown, and whether the two volatiles could facilitate the spread and dispersal of RDV remains elusive. Combining the methods of insect behavior, chemical ecology, and molecular biology, we found that EBC and 2-heptanol influenced the olfactory behavior of non-viruliferous and viruliferous N. virescens, independently. EBC attracted non-viruliferous N. virescens towards RDV-infected rice plants, promoting virus acquisition by non-viruliferous vectors. The effect was confirmed by using oscas1 mutant rice plants (repressed EBC synthesis), but EBC had no effects on viruliferous N. virescens. 2-heptanol did not attract or repel non-viruliferous N. virescens. However, spraying experiments showed that 2-heptanol repelled viruliferous N. virescens to prefer RDV-free rice plants, which would be conducive to the transmission of the virus. These novel results reveal that rice plant volatiles modify the behavior of N. virescens vectors to promote RDV acquisition and transmission. They will provide new insights into virus-vector-plant interactions, and promote the development of new prevention and control strategies for disease management.

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.

Toll and IMD Immune Pathways Are Important Antifungal Defense Components in a Pupal Parasitoid, Pteromalus puparum.

Insects employ multifaceted strategies to combat invading fungi, with immunity being a promising mechanism. Immune pathways function in signal transduction and amplification, ultimately leading to the activation of antimicrobial peptides (AMPs). Although several studies have shown that immune pathways are responsible for defending against fungi, the roles of parasitoid immune pathways involved in antifungal responses remain unknown. In this study, we evaluated the roles of the Toll and IMD pathways of a pupal parasitoid, Pteromalus puparum (Hymenoptera: Pteromalidae), in fighting against Beauveria bassiana (Hypocreales: Cordycipitaceae). Successful colonization of B. bassiana on P. puparum adults was confirmed by scanning electron microscopy (SEM). AMPs were induced upon B. bassiana infection. The knockdown of key genes, PpTollA and PpIMD, in Toll and IMD signaling pathways, respectively, significantly compromised insect defense against fungal infection. The knockdown of either PpTollA or PpIMD in P. puparum dramatically promoted the proliferation of B. bassiana, resulting in a decreased survival rate and downregulated expression levels of AMPs against B. bassiana compared to controls. These data indicated that PpTollA and PpIMD participate in Toll and IMD-mediated activation of antifungal responses, respectively. In summary, this study has greatly broadened our knowledge of the parasitoid antifungal immunity against fungi.

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.

Genome of the pincer wasp Gonatopus flavifemur reveals unique venom evolution and a dual adaptation to parasitism and predation.

BACKGROUND: Hymenoptera comprise extremely diverse insect species with extensive variation in their life histories. The Dryinidae, a family of solitary wasps of Hymenoptera, have evolved innovations that allow them to hunt using venom and a pair of chelae developed from the fore legs that can grasp prey. Dryinidae larvae are also parasitoids of Auchenorrhyncha, a group including common pests such as planthoppers and leafhoppers. Both of these traits make them effective and valuable for pest control, but little is yet known about the genetic basis of its dual adaptation to parasitism and predation. RESULTS: We sequenced and assembled a high-quality genome of the dryinid wasp Gonatopus flavifemur, which at 636.5 Mb is larger than most hymenopterans. The expansion of transposable elements, especially DNA transposons, is a major contributor to the genome size enlargement. Our genome-wide screens reveal a number of positively selected genes and rapidly evolving proteins involved in energy production and motor activity, which may contribute to the predatory adaptation of dryinid wasp. We further show that three female-biased, reproductive-associated yellow genes, in response to the prey feeding behavior, are significantly elevated in adult females, which may facilitate the egg production. Venom is a powerful weapon for dryinid wasp during parasitism and predation. We therefore analyze the transcriptomes of venom glands and describe specific expansions in venom Idgf-like genes and neprilysin-like genes. Furthermore, we find the LWS2-opsin gene is exclusively expressed in male G. flavifemur, which may contribute to partner searching and mating. CONCLUSIONS: Our results provide new insights into the genome evolution, predatory adaptation, venom evolution, and sex-biased genes in G. flavifemur, and present genomic resources for future in-depth comparative analyses of hymenopterans that may benefit pest control.

Molecular and Pharmacological Characterization of ß-Adrenergic-like Octopamine Receptors in the Endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae).

Octopamine (OA) is structurally and functionally similar to adrenaline/noradrenaline in vertebrates, and OA modulates diverse physiological and behavioral processes in invertebrates. OA exerts its actions by binding to specific octopamine receptors (OARs). Functional and pharmacological characterization of OARs have been investigated in several insects. However, the literature on OARs is scarce for parasitoids. Here we cloned three ß-adrenergic-like OARs (CcOctßRs) from Cotesia chilonis. CcOctßRs share high similarity with their own orthologous receptors. The transcript levels of CcOctßRs were varied in different tissues. When heterologously expressed in CHO-K1 cells, CcOctßRs induced cAMP production, and were dose-dependently activated by OA, TA and putative octopaminergic agonists. Their activities were inhibited by potential antagonists and were most efficiently blocked by epinastine. Our study offers important information about the molecular and pharmacological properties of ß-adrenergic-like OARs from C. chilonis that will provide the basis to reveal the contribution of individual receptors to the physiological processes and behaviors in parasitoids.

Virus-induced plant volatiles mediate the olfactory behaviour of its insect vectors.

Plant viruses can manipulate their hosts to release odours that are attractive or repellent to their insect vectors. However, the volatile organic compounds (VOCs), either individually or as mixtures, which play a key role in the olfactory behaviour of insect vectors remains largely unknown. Our study focused on green rice leafhoppers (GRLHs) vectoring rice dwarf virus (RDV) revealed that RDV infection significantly induced the emission of (E)-ß-caryophyllene and 2-heptanol by rice plants, which influenced the olfactory behaviour of both non-viruliferous and viruliferous GRLHs. (E)-ß-caryophyllene attracted non-viruliferous GRLHs to settle on RDV-infected plants, but neither attracted nor repelled viruliferous GRLHs. In contrast, 2-heptanol repelled viruliferous GRLHs to settle on RDV-infected plants, but neither repelled nor attracted non-viruliferous GRLHs. Suppression of (E)-ß-caryophyllene synthase OsCAS via CRISPR-Cas9 to generate oscas-1 plants enabled us to confirm the important role played by (E)-ß-caryophyllene in modulating the virus-vector-host plant interaction. These novel results reveal the role of these virus-induced VOCs in modulating the behaviour of its GRLH insect vector and may facilitate the design of new strategies for disease control through manipulation of plant volatile emissions.

Impacts of Bt rice on non-target organisms assessed by the hazard quotient (HQ).

The potential risk of Bt (Bacillus thuringiensis) crops on non-target organisms (NTOs) has drawn a lot of public concerns. Despite a series of risk assessments of Bt crops on NTOs has been conducted, a quantitative approach which could support a precise judgment of their safety is required. In the present work, hazard quotient (HQ) was applied in the safety evaluation of three Bt rice events (Cry1Ab, Cry1C and Cry2Aa rice) on NTOs. Eight NTOs in different functional guilds associated with Bt rice were selected to conduct the tests. The results showed that the HQs of three Bt rice events for eight NTOs were all below the trigger value 1, while the HQ of Cry1Ab rice for one target pest Chilo suppressalis was three times higher than 1. Our results assured the reliability of the HQ and indicated that the three Bt rice events would pose no risks to the eight NTOs. Further testing of three Bt proteins on biological parameters of one NTO Nasonia virtipennis under no observed adverse effect concentration (NOAEC) confirmed the robustness of HQ assessment. We recommend that the HQ could be applied in tier-1 risk assessments of Bt crops on NTOs as a reference data standard, which would provide more clear and credible safety information of transgenic crops for the public and policy makers.

Evolutionary Rate Correlation between Mitochondrial-Encoded and Mitochondria-Associated Nuclear-Encoded Proteins in Insects.

The mitochondrion is a pivotal organelle for energy production, and includes components encoded by both the mitochondrial and nuclear genomes. Functional and evolutionary interactions are expected between the nuclear- and mitochondrial-encoded components. The topic is of broad interest in biology, with implications to genetics, evolution, and medicine. Here, we compare the evolutionary rates of mitochondrial proteins and ribosomal RNAs to rates of mitochondria-associated nuclear-encoded proteins, across the major orders of holometabolous insects. There are significant evolutionary rate correlations (ERCs) between mitochondrial-encoded and mitochondria-associated nuclear-encoded proteins, which are likely driven by different rates of mitochondrial sequence evolution and correlated changes in the interacting nuclear-encoded proteins. The pattern holds after correction for phylogenetic relationships and considering protein conservation levels. Correlations are stronger for both nuclear-encoded OXPHOS proteins that are in contact with mitochondrial OXPHOS proteins and for nuclear-encoded mitochondrial ribosomal amino acids directly contacting the mitochondrial rRNAs. We find that ERC between mitochondrial- and nuclear-encoded proteins is a strong predictor of nuclear-encoded proteins known to interact with mitochondria, and ERC shows promise for identifying new candidate proteins with mitochondrial function. Twenty-three additional candidate nuclear-encoded proteins warrant further study for mitochondrial function based on this approach, including proteins in the minichromosome maintenance helicase complex.

A venom protein, Kazal-type serine protease inhibitor, of ectoparasitoid Pachycrepoideus vindemiae inhibits the hemolymph melanization of host Drosophila melanogaster.

Parasitic wasps inject various virulence factors into the host insects while laying eggs, among which the venom proteins, one of the key players in host insect/parasitoid relationships, act in host cellular and humoral immune regulation to ensure successful development of wasp progeny. Although the investigations into actions of venom proteins are relatively ample in larval parasitoids, their regulatory mechanisms have not been thoroughly understood in pupal parasitoids. Here, we identified a venom protein, Kazal-type serine protease inhibitor, in the pupal ectoparasitoid Pachycrepoideus vindemiae (PvKazal). Sequence analysis revealed that PvKazal is packed by a signal peptide and a highly conserved "Kazal" domain. Quantitative polymerase chain reaction analysis recorded a higher transcript level of PvKazal in the venom apparatus relative to that in the carcass, and the PvKazal messenger RNA level appeared to reach a peak on day 5 posteclosion. Recombinant PvKazal strongly inhibited the hemolymph melanization of host Drosophila melanogaster. Additionally, the heterologous expression of PvKazal in transgenic Drosophila reduced the crystal cell numbers and blocked the melanization of host pupal hemolymph. Our present work underlying the roles of PvKazal undoubtedly increases the understanding of venom-mediated host-parasitoid crosstalk.

Immune signaling pathways in the endoparasitoid, Pteromalus puparum.

Parasitoids serve as effective biocontrol agents for agricultural pests. However, they face constant challenges from host immune defense and numerous pathogens and must develop potent immune defense against these threats. Despite the recent advances in innate immunity, little is known about the immunological mechanisms of parasitoids. Here, we identified and characterized potential immune-related genes of the endoparasitoid, Pteromalus puparum, which act in regulating populations of some members of the Pieridae. We identified 216 immune-related genes based on interrogating the P. puparum genome and transcriptome databases. We categorized the cognate gene products into recognition molecules, signal moieties and effector proteins operating in four pathways, Toll, IMD, JAK/STAT, and JNK. Comparative analyses of immune-related genes from seven insect species indicate that recognition molecules and effector proteins are more expanded and diversified than signaling genes in these signal pathways. There are common 1:1 orthologs between the endoparasitoid P. puparum and its relative, the ectoparasitoid Nasonia vitripennis. The developmental expression profiles of immune genes randomly selected from the transcriptome analysis were verified by a quantitative polymerase chain reaction. Our work provides comprehensive analyses of P. puparum immune genes, some of which may be exploited in advancing parasitoid-based biocontrol technologies.

Genome-wide identification and analysis of genes encoding cuticular proteins in the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae).

The multifunctional insect cuticle serves as the exoskeleton, determines body shape, restricts water loss, provides attachment sites for muscles and internal organs and is a formidable barrier to invaders. It is morphologically divided into three layers, including envelope, epicuticle, and procuticle and is composed of chitin and cuticular proteins (CPs). Annotation of CPs and their cognate genes may help understand the structure and functions of insect cuticles. In this paper, we interrogated the genome of Pteromalus puparum, an endoparasitoid wasp that parasitizes Pieris rapae and Papilio xuthus pupae, and identified 82 genes encoding CPs belonging to six CP families, including 62 in the CPR family, 8 in CPAP3, 5 in CPF/CPFL, 2 low complexity proteins, 2 in TWDL, and 3 in Apidermin. We used six RNA-seq libraries to determine CP gene expression profiles through development and compared the cuticle hydrophobicity between the P. puparum and the ectoparasitoid Nasonia vitripennis based on GRAVY values of CPR sequences. In the Nasonia-Pteromalus comparison, we found in both N. vitripennis and P. puparum, the peak of their CPR hydrophobicity displayed at their pupal stage, whereas their adult stage showed the lowest level. Except at the adult stage, the CPR hydrophobicity in N. vitripennis is always higher than P. puparum. Finally, we identified three novel Apidermin genes, a family found solely in Hymenoptera and revealed a new sequence feature of this family. This new information contributes to a broader understanding of insect CPs generally.

Biogenic amine biosynthetic and transduction genes in the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae).

Biogenic amines (BAs), such as octopamine, tyramine, dopamine, serotonin, and acetylcholine regulate various behaviors and physiological functions in insects. Here, we identified seven genes encoding BA biosynthetic enzymes and 16 genes encoding BA G protein-coupled receptors in the genome of the endoparasitoid wasp, Pteromalus puparum. We compared the genes with their orthologs in its host Pieris rapae and the related ectoparasitic wasp Nasonia vitripennis. All the genes show high (>90%) identity to orthologs in N. vitripennis. P. puparum and N. vitripennis have the smallest number of BA receptor genes among the insect species we investigated. We then analyzed the expression profiles of the genes, finding those acting in BA biosynthesis were highly expressed in adults and larvae and those encoding BA receptors are highly expressed in adults than immatures. Octα1R and 5-HT7 genes were highly expressed in salivary glands, and a high messenger RNA level of 5-HT1A was found in venom apparatuses. We infer that BA signaling is a fundamental component of the organismal organization, homeostasis and operation in parasitoids, some of the smallest insects.