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Technologies for gene activation are valuable tools for the study of gene functions and have a wide range of potential applications in bioengineering and medicine. In contrast to existing methods based on recruiting transcriptional modulators via DNA-binding proteins, we developed a strategy termed Narta (nascent RNA-guided transcriptional activation) to achieve gene activation by recruiting artificial transcription factors (aTFs) to transcription sites through nascent RNAs of the target gene. Using Narta, we demonstrate robust activation of a broad range of exogenous and endogenous genes in various cell types, including zebrafish embryos, mouse and human cells. Importantly, the activation is reversible, tunable and specific. Moreover, Narta provides better activation potency of some expressed genes than CRISPRa and, when used in combination with CRISPRa, has an enhancing effect on gene activation. Quantitative imaging illustrated that nascent RNA-directed aTFs could induce the high-density assembly of coactivators at transcription sites, which may explain the larger transcriptional burst size induced by Narta. Overall, our work expands the gene activation toolbox for biomedical research.

Parasitoids are widespread in natural ecosystems and normally equipped with diverse viral factors to defeat host immune responses. On the other hand, parasitoids can enhance the antibacterial abilities and improve the hypoimmunity traits of parasitized hosts that may encounter pathogenic infections. These adaptive strategies guarantee the survival of parasitoid offspring, yet their underlying mechanisms are poorly understood. Here, we focused on Cotesia vestalis, an endoparasitoid of the diamondback moth Plutella xylostella, and found that C. vestalis parasitization decreases the number of host hemocytes, leading to disruption of the encapsulation reaction. We further found that one bracovirus C-type lectin gene, CvBV_28-1, is highly expressed in the hemocytes of parasitized hosts and participates in suppressing the proliferation rate of host hemocytes, which in turn reduces their population and represses the process of encapsulation. Moreover, CvBV_28-1 presents a classical bacterial clearance ability via the agglutination response in a Ca2+-dependent manner in response to gram-positive bacteria. Our study provides insights into the innovative strategy of a parasitoid-derived viral gene that has dual functions to manipulate host immunity for a successful parasitism.

Odorant-binding proteins (OBPs) are crucial in insect's olfactory perception, which participate in the initial step of odorant molecules transporting from the external environment to olfactory receptor neurons. To better understand the roles for OBPs in olfactory perception in Cotesia vestalis, a solitary larval endoparasitoid of diamondback moth, Plutella xylostella, we have comprehensively screened the genome of C. vestalis, and obtained 20 CvesOBPs, including 18 classic OBPs and two minus-C OBPs. Motif-pattern analysis indicates that the motifs of C. vestalis OBPs are highly conserved in Hymenoptera. The results of tissue expression analysis show that five OBPs (CvesOBP1/11/12/14/16) are highly expressed in male antennae, whereas six other OBP genes (CvesOBP7/8/13/17/18/19) are significantly transcriptionally enriched in female antennae. The results of RNA interference experiments for three most highly expressed OBP genes (CvesOBP17/18/19) in female antennae demonstrate that they are likely involved in parasitic processes of female wasps, as the wasps take a longer time to target the hosts when they are knocked down.

Many endoparasitoids adopt several parasitic factors, such as venom, polydnavirus and teratocytes, to suppress the immune response of their associated hosts including melanization for successful parasitism. A teratocyte-specific expressed serpin gene, designated as CvT-serpin6, was identified from the parasitoid Cotesia vestalis. The immunoblot result suggested that CvT-serpin6 was secreted into extracellular space. qPCR results showed that CvT-serpin6 was mainly transcribed at later stages of parasitism, and the transcriptional abundance of CvT-serpin6 in teratocytes was significantly increased in response to the challenge of bacteria. Inhibitory assay indicated that recombinant CvT-serpin6 (rCvT-serpin6) could inhibit the activation of Plutella xylostella prophenoloxidase and ultimately resulted in the inhibition of melanization in P. xylostella haemolymph. Furthermore, we confirmed that rCvT-serpin6 could form SDS-stable complexes with activated PxPAP1 and PxPAP3 in a dose-dependent manner. Altogether, our results further shed insight into the molecular mechanisms that teratocytes involved in controlling host immune response.

Parasitic wasps are an important group of entomophagous insects for pest control. As parasitic wasps often lay eggs on or into their associated hosts, parasitoids evolve to utilize several factors including venom, polydnavirus (PDV) to alter host physiology for successful parasitism. Some taxa of endoparasitoids produce teratocytes, which are a type of cell that is released into host insects when wasp eggs hatch. Teratocytes display multifunction in parasitism such as host nutritional exploration, immune and developmental regulation, by secreting plenty of proteins into host hemocoel.A serpin (CvT-serpin15) secreted by teratocytes was characterized. QPCR results showed the expressional level of CvT-serpin15 was upregulated following bacterial challenges. Enzyme activity experiment indicated the recombinant CvT-serpin15 protein could interfere with the growth of Gram-positive bacteria Staphylococcus aureus. The survival rate assay demonstrated CvT-serpin15 increased survival rate of Plutella xylostella infected by S. aureus.CvT-serpin15 secreted by teratocytes would boost the host immune system when pathogens invade host hemocoel during parasitism, and ultimately protect the development of wasp larva from bacterial infection. © 2021 The Authors. Pest Management Science published by John WileySons Ltd on behalf of Society of Chemical Industry.

The endoparasitic wasp Cotesia vestalis is an important biological agent for controlling the population of Plutella xylostella, a major pest of cruciferous crops worldwide. Though the genome of C. vestalis has recently been reported, molecular mechanisms associated with sexual development have not been comprehensively studied. Here, we combined PacBio Iso-Seq and Illumina RNA-Seq to perform genome-wide profiling of pharate adult and adult development of male and female C. vestalis. Taking advantage of Iso-Seq full-length reads, we identified 14,466 novel transcripts as well as 8770 lncRNAs, with many lncRNAs showing a sex- and stage-specific expression pattern. The differentially expressed gene (DEG) analyses showed 2125 stage-specific and 326 sex-specific expressed genes. We also found that 4819 genes showed 11,856 alternative splicing events through combining the Iso-Seq and RNA-Seq data. The results of comparative analyses showed that most genes were alternatively spliced across developmental stages, and alternative splicing (AS) events were more prevalent in females than in males. Furthermore, we identified six sex-determining genes in this parasitic wasp and verified their sex-specific alternative splicing profiles. Specifically, the characterization of feminizer and doublesex splicing between male and female implies a conserved regulation mechanism of sexual development in parasitic wasps.

Parasitoids are ubiquitous in natural ecosystems. Parasitic strategies are highly diverse among parasitoid species, yet their underlying genetic bases are poorly understood. Here, we focus on the divergent adaptation of a specialist and a generalist drosophilid parasitoids. We find that a novel protein (Lar) enables active immune suppression by lysing the host lymph glands, eventually leading to successful parasitism by the generalist. Meanwhile, another novel protein (Warm) contributes to a passive strategy by attaching the laid eggs to the gut and other organs of the host, leading to incomplete encapsulation and helping the specialist escape the host immune response. We find that these diverse parasitic strategies both originated from lateral gene transfer, followed with duplication and specialization, and that they might contribute to the shift in host ranges between parasitoids. Our results increase our understanding of how novel gene functions originate and how they contribute to host adaptation., Parasitism is a widespread evolutionary strategy. A study that spans functional and evolutionary genomics identifies the molecular basis and history underlying two genes that have mediated divergent parasitic strategies (specialist vs generalist) between two sister species of parasitoid wasp.

Some DNA viruses infect host animals usually by integrating their DNAs into the host genome. However, the mechanisms for integration remain largely unknown. Here, we find that Cotesia vestalis bracovirus (CvBV), a polydnavirus of the parasitic wasp C. vestalis (Haliday), integrates its DNA circles into host Plutella xylostella (L.) genome by two distinct strategies, conservatively and randomly, through high-throughput sequencing analysis. We confirmed that the conservatively integrating circles contain an essential “8+5” nucleotides motif which is required for integration. Then we find CvBV circles are integrated into the caterpillar’s genome in three temporal patterns, the early, mid and late stage-integration. We further identify that three CvBV-encoded integrases are responsible for some, but not all of the virus circle integrations, indeed they mainly participate in the processes of early stage-integration. Strikingly, we find two P. xylostella retroviral integrases (PxIN1 and PxIN2) are highly induced upon wasp parasitism, and PxIN1 is crucial for integration of some other early-integrated CvBV circles, such as CvBV_04, CvBV_12 and CvBV_24, while PxIN2 is important for integration of a late-integrated CvBV circle, CvBV_21. Our data uncover a novel mechanism in which CvBV integrates into the infected host genome, not only by utilizing its own integrases, but also by recruiting host enzymes. These findings will strongly deepen our understanding of how bracoviruses regulate and integrate into their hosts., Author summary Parasitoid wasps are a very large group of animals that live in or on the other arthropods. Polydnaviruses (PDVs) are a special kind of DNA virus associated parasitic wasps (primary hosts), which do not replicate in their infected caterpillar hosts (secondary hosts). During parasitoid oviposition, PDVs enter infected hosts and integrate their DNA into the genome of infected hosts, then their virulence genes begin to express and manipulate multiple biological process of hosts to fulfill all the requirements of parasitoids offspring. However, the mechanisms for integration remain largely unknown. Our research found the integration of PDVs is not only by utilizing its own integrases, but also by recruiting host enzymes. Here, we provide a new concept that host intergrases are involved in the integration of PDVs into host genome, and it greatly enhance our understanding of how PDVs regulate and integrate into their hosts, and may also inspire studies on how some DNA viruses may integrate their DNAs into human genome.

Intraspecific competition is a major force in mediating population dynamics, fuelling adaptation, and potentially leading to evolutionary diversification. Among the evolutionary arms races between parasites, one of the most fundamental and intriguing behavioural adaptations and counter-adaptations are superparasitism and superparasitism avoidance. However, the underlying mechanisms and ecological contexts of these phenomena remain underexplored. Here, we apply the Drosophila parasite Leptopilina boulardi as a study system and find that this solitary endoparasitic wasp provokes a host escape response for superparasitism avoidance. We combine multi-omics and in vivo functional studies to characterize a small set of RhoGAP domain-containing genes that mediate the parasite’s manipulation of host escape behaviour by inducing reactive oxygen species in the host central nervous system. We further uncover an evolutionary scenario in which neofunctionalization and specialization gave rise to the novel role of RhoGAP domain in avoiding superparasitism, with an ancestral origin prior to the divergence between Leptopilina specialist and generalist species. Our study suggests that superparasitism avoidance is adaptive for a parasite and adds to our understanding of how the molecular manipulation of host behaviour has evolved in this system., Evolutionary arms races can drive adaptations in hosts and parasites as well as among competing parasites. A combination of multi-omics and functional tests identifies a set of genes that allow a parasitic wasp to minimize intraspecific competition by inducing hosts to escape before more wasps can parasitize them.

Polydnaviruses (PDVs) are obligatory symbionts of parasitoid wasps and play an important role in suppressing host immune defenses. Although PDV genes that inhibit host melanization are known in Microplitis bracovirus, the functional homologs in Cotesia bracoviruses remain unknown. Here, we find that Cotesia vestalis bracovirus (CvBV) can inhibit hemolymph melanization of its host, Plutella xylostella larvae, during the early stages of parasitization, and that overexpression of highly expressed CvBV genes reduced host phenoloxidase activity. Furthermore, CvBV-7-1 in particular reduced host phenoloxidase activity within 12 h, and the injection of anti-CvBV-7-1 antibody increased the melanization of parasitized host larvae. Further analyses showed that CvBV-7-1 and three homologs from other Cotesia bracoviruses possessed a C-terminal leucine/isoleucine-rich region and had a similar function in inhibiting melanization. Therefore, a new family of bracovirus genes was proposed and named as C-terminal Leucine/isoleucine-rich Protein (CLP). Ectopic expression of CvBV-7-1 in Drosophila hemocytes increased susceptibility to bacterial repression of melanization and reduced the melanotic encapsulation of parasitized D. melanogaster by the parasitoid Leptopilina boulardi. The formation rate of wasp pupae and the eclosion rate of C. vestalis were affected when the function of CvBV-7-1 was blocked. Our findings suggest that CLP genes from Cotesia bracoviruses encoded proteins that contain a C-terminal leucine/isoleucine-rich region and function as melanization inhibitors during the early stage of parasitization, which is important for successful parasitization.

Parasitic wasps produce several factors including venom, polydnaviruses (PDVs) and specialized wasp cells named teratocytes that benefit the survival of offspring by altering the physiology of hosts. However, the underlying molecular mechanisms for the alterations remain unclear. Here we find that the teratocytes of Cotesia vestalis, an endoparasitoid of the diamondback moth Plutella xylostella, and its associated bracovirus (CvBV) can produce miRNAs and deliver the products into the host via different ways. Certain miRNAs in the parasitized host are mainly produced by teratocytes, while the expression level of miRNAs encoded by CvBV can be 100-fold greater in parasitized hosts than non-parasitized ones. We further show that one teratocyte-produced miRNA (Cve-miR-281-3p) and one CvBV-produced miRNA (Cve-miR-novel22-5p-1) arrest host growth by modulating expression of the host ecdysone receptor (EcR). Altogether, our results show the first evidence of cross-species regulation by miRNAs in animal parasitism and their possible function in the alteration of host physiology during parasitism., The moth Plutella xylostella during its larval stage is the host of the endoparasitic wasp Cotesia vestalis. Here the authors show that the parasitoids deliver microRNAs to their hosts through their symbiotic virus and specialized cells leading to induced developmental delay.

Background Insecticide resistance is a substantial problem in controlling agricultural and medical pests. Detecting target site mutations is crucial to manage insecticide resistance. Though PCR-based methods have been widely used in this field, they are time-consuming and inefficient, and typically have a high false positive rate. Acetylcholinesterases (Ace) is the neural target of the widely used organophosphate (OP) and carbamate insecticides. However, there is not any software available to detect insecticide resistance associated mutations in RNA-Seq data at present. Results A computational pipeline ACE was developed to detect resistance mutations of ace in insect RNA-Seq data. Known ace resistance mutations were collected and used as a reference. We constructed a Web server for ACE, and the standalone software in both Linux and Windows versions is available for download. ACE was used to analyse 971 RNA-Seq data from 136 studies in 7 insect pests. The mutation frequency of each RNA-Seq dataset was calculated. The results indicated that the resistance frequency was 30%–44% in an eastern Ugandan Anopheles population, thus suggesting this resistance-conferring mutation has reached high frequency in these mosquitoes in Uganda. Analyses of RNA-Seq data from the diamondback moth Plutella xylostella indicated that the G227A mutation was positively related with resistance levels to organophosphate or carbamate insecticides. The wasp Nasonia vitripennis had a low frequency of resistant reads (

To establish successful infections, endoparasitoid wasps must develop strategies to evade immune responses of the host. Here, we identified and characterized a teratocytes-expressed gene encoding a trypsin inhibitor-like protein containing a cysteine-rich domain from Cotesia vestalis, CvT-TIL. CvT-TIL had a high expression level during the later developmental stage of teratocytes and was secreted into host hemolymph. Further experiments showed CvT-TIL strongly suppressed the prophenoloxidase activation of host hemolymph in a dose-dependent manner by interacting with PxPAP3 of PO cascade. Our results not only provide evidence for an inhibition between CvT-TIL gene and the host's melanization activity, but also expand our knowledge about the mechanisms by which parasitoids regulate humoral immunity of the host.

Background Parasitic insects are well-known biological control agents for arthropod pests worldwide. They are capable of regulating their host’s physiology, development and behaviour. However, many of the molecular mechanisms involved in host-parasitoid interaction remain unknown. Results We sequenced the genomes of two parasitic wasps (Cotesia vestalis, and Diadromus collaris) that parasitize the diamondback moth Plutella xylostella using Illumina and Pacbio sequencing platforms. Genome assembly using SOAPdenovo produced a 178 Mb draft genome for C. vestalis and a 399 Mb draft genome for D. collaris. A total set that contained 11,278 and 15,328 protein-coding genes for C. vestalis and D. collaris, respectively, were predicted using evidence (homology-based and transcriptome-based) and de novo prediction methodology. Phylogenetic analysis showed that the braconid C. vestalis and the ichneumonid D. collaris diverged approximately 124 million years ago. These two wasps exhibit gene gains and losses that in some cases reflect their shared life history as parasitic wasps and in other cases are unique to particular species. Gene families with functions in development, nutrient acquisition from hosts, and metabolism have expanded in each wasp species, while genes required for biosynthesis of some amino acids and steroids have been lost, since these nutrients can be directly obtained from the host. Both wasp species encode a relative higher number of neprilysins (NEPs) thus far reported in arthropod genomes while several genes encoding immune-related proteins and detoxification enzymes were lost in both wasp genomes. Conclusions We present the annotated genome sequence of two parasitic wasps C. vestalis and D. collaris, which parasitize a common host, the diamondback moth, P. xylostella. These data will provide a fundamental source for studying the mechanism of host control and will be used in parasitoid comparative genomics to study the origin and diversification of the parasitic lifestyle.

The pupal endoparasitoid wasp Trichopria drosophilae (Hymenoptera: Diapriidae) plays an important role in biological control of many frugivorous fruit fly species including Drosophila suzukii, a well-known invasive pest. Here, we report the transcriptomes of T. drosophilae among different developmental stages. A total of 601,148,438 high-quality reads were obtained and de novo assembled into 187,704 unigenes with an average length of 1096 bp. Among them, 21,735 unigenes were annotated into 52 Gene Ontology terms and 36,898 were assigned to 25 Cluster of Orthologous Groups categories, whereas 30,585 unigenes were mapped to 270 Kyoto Encyclopedia of Genes and Genomes different pathways. Numbers of differentially expressed genes were found through comparisons between different developmental stages. We further identified 137 cuticular protein genes (CPs) from T. drosophilae transcriptome, including 59 from CPR family, 2 from Tweedle family, 1 from CPF family, 46 from CPAP family, and 29 from other CP families. We analyzed expression patterns of the CPs at different developmental stages of T. drosophilae, and found some stage-specific CPs. Quantitative real-time PCR results confirmed RNA sequencing findings based on the relative expression levels of eight randomly selected CPs. This study provides a valuable transcriptomic resource for a comprehensive understanding of the development and physiology of T. drosophilae, and will help to improve their parasitism efficiency for biological control purposes.