Symbiotic bracovirus of a parasite manipulates host lipid metabolism via tachykinin signaling

Parasites alter host energy homeostasis for their own development, but the mechanisms underlying this phenomenon remain largely unknown. Here, we show that Cotesia vestalis, an endoparasitic wasp of Plutella xylostella larvae, stimulates a reduction of host lipid levels. This process requires excess secretion of P. xylostella tachykinin (PxTK) peptides from enteroendocrine cells (EEs) in the midgut of the parasitized host larvae. We found that parasitization upregulates PxTK signaling to suppress lipogenesis in midgut enterocytes (ECs) in a non-cell-autonomous manner, and the reduced host lipid level benefits the development of wasp offspring and their subsequent parasitic ability. We further found that a C. vestalis bracovirus (CvBV) gene, CvBV 9–2, is responsible for PxTK induction, which in turn reduces the systemic lipid level of the host. Taken together, these findings illustrate a novel mechanism for parasite manipulation of host energy homeostasis by a symbiotic bracovirus gene to promote the development and increase the parasitic efficiency of an agriculturally important wasp species.     

A viral lectin encoded in Cotesia plutellae bracovirus and its immunosuppressive effect on host hemocytes

An endoparasitoid wasp, Cotesia plutellae, induces immunosuppression of the host diamondback moth, Plutella xylostella. To identify an immunosuppressive factor, the parasitized hemolymph of P. xylostella was separated into plasma and hemocyte fractions. When nonparasitized hemocytes were overlaid with parasitized plasma, they showed significant reduction in bacterial binding efficacy. Here, we considered a viral lectin previously known in other Cotesia species as a humoral immunosuppressive candidate in C. plutellae parasitization. Based on consensus regions of the viral lectins, the corresponding lectin gene was cloned from P. xylostella parasitized by C. plutellae. Its cDNA is 674 bp long and encodes 157 amino acid residues containing a signal peptide (15 residues) and one carbohydrate recognition domain. Open reading frame is divided by one intron (156 bp) in its genomic DNA. Amino acid sequence shares 80% homology with that of C. ruficrus bracovirus lectin and is classified into C-type lectin. Southern hybridization analysis indicated that the cloned lectin gene was located at C. plutellae bracovirus (CpBV) genome. Both real-time quantitative RT-PCR and immunoblotting assays indicated that CpBV-lectin showed early expression during the parasitization. A recombinant CpBV-lectin was expressed in a bacterial system and the purified protein significantly inhibited the association between bacteria and hemocytes of nonparasitized P. xylostella. In the parasitized P. xylostella, CpBV-lectin was detected on the surface of parasitoid eggs after 24 h parasitization by its specific immunostaining. The 24 h old eggs were not encapsulated in vitro by hemocytes of P. xylostella, compared to newly laid parasitoid eggs showing no CpBV-lectin detectable and easily encapsulated. These results support an existence of a polydnaviral lectin family among Cotesia-associated bracovirus and propose its immunosuppressive function.     

椰扁甲啮小蜂寄生对椰心叶甲蛹免疫反应的影响

为了测明椰扁甲啮小蜂Tetrastichus brontispae寄生对寄主椰心叶甲Brontispa longissima蛹的血细胞和体液免疫反应的影响,开展了椰扁甲啮小蜂寄生对椰心叶甲蛹血细胞数量和延展性、血淋巴酚氧化酶活性、血淋巴黑化百分率和血细胞凝集素活性等影响的研究。结果表明：与同期未被寄生蛹相比,寄生蛹血细胞总量在寄生后2 d显著降低,但寄生后4 d显著升高; 寄生蛹的浆血细胞延展率在寄生后2 d显著降低,寄生后4 d显著升高;寄生蛹的血淋巴黑化百分率在寄生后0.5～2 d较高,寄生后3～4 d降低直至为0;寄生蛹的血淋巴酚氧化酶活性在寄生后0.5 d,1 d和4 d时显著升高;寄生蛹的血凝素活性在寄生后2 d较高,寄生后1 d和4 d较低。结果说明椰扁甲啮小蜂寄生使寄主椰心叶甲蛹血细胞和体液免疫反应呈现不规律的变化。     

Bracoviruses recruit host integrases for their integration into caterpillar’s genome

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.     

Characterization of a novel Cotesia vestalis polydnavirus (CvBV) gene containing a ser-rich motif expressed in Plutella xylostella larvae

Cotesia vestalis is an endoparasitoid of Plutella xylostella larvae and injects a polydnavirus (CvBV) into its host during oviposition. In this report we characterize the gene, CvBV3307, and its products. CvBV3307 is located on segment S33 of the CvBV genome, is 517 bp, and encodes a putative protein of 122 amino acids, including a serine-rich region. The expression pattern of CvBV3307 in parasitized larvae and the subcellular localization of CvBV3307 only in granulocytes indicated that it might be involved in early protection of parasitoid eggs from host cellular encapsulation and in manipulating the hormone titer and developmental rhythm of host larvae. Western blot analysis showed that the size of the immunoreactive protein (about 55 kDa) in parasitized hosts at 48 hours post parasitization (h p.p.) is much larger than the predicted molecular weight of 13.6 kDa, which suggests that CvBV3307 undergoes extensive post-translational modification in hosts.     

Does the solitary parasitoid Microplitis pennatulae use a combinatorial approach to manipulate its host?

Host manipulation is a strategy used by some parasites to enhance their transmission. These parasites use a combination of neuropharmacological, psychoneuroimmunological, genomic/proteomic, or symbiont-mediated mechanisms to manipulate their hosts. Bodyguard manipulation occurs when parasitized hosts guard parasitoid pupae to protect them from their natural enemies. Bodyguard-manipulated hosts exhibit altered behaviours only after the egression of parasitoid prepupae. Behavioural changes in post-parasitoid egressed hosts could have resulted from their altered physiology. Previous studies have shown that gregarious manipulative parasitoids induce multiple physiological changes in their host, but the physiological changes induced by solitary manipulative parasitoids are unknown. Microplitis pennatulae Ranjith & Rajesh (Hymenoptera: Braconidae) is a larval parasitoid of Psalis pennatula Fabricius (Lepidoptera: Erebidae). After the egression of parasitoid prepupae, P. pennatula stops its routine activities and protects the parasitoid pupa from hyperparasitoids by body thrashes. In this study, we looked into the physiological changes induced by the solitary manipulative parasitoid, M. pennatulae, in its host, P. pennatula, during various stages of parasitization. We considered octopamine concentration and phenoloxidase (PO) activity as biomarkers of physiological change. We also examined whether M. pennatulae has a symbiotic virus and whether the wasp transfers it to the host during parasitization. We found that octopamine concentration was low in the pre-parasitoid egressed host, but it was elevated after the parasitoid egressed. Phenoloxidase activity was lower in the pre- and post-parasitoid egressed host than in the unparasitized host. We also detected symbiotic bracovirus (BV) in the wasp ovaries and isolated the BV virulence gene from the parasitised host. Our study suggests that solitary parasitoids also induce multiple physiological changes to influence the host behaviour to their advantage, as is the case with the gregarious parasitoids.     

Parasitism-induced effects on host growth and metabolic efficiency in Plutella xylostella larvae parasitized by Cotesia vestalis or Diadegma semiclausum

The nutritional physiology of the diamondback moth, Plutella xylostella, larvae was examined after parasitization by the solitary endoparasitoids Cotesia vestalis or Diadegma semiclausum. Examinations were performed in two phases, one was examined at the time point of 24 h post‐parasitization, and the other was done at the end of the 4th instar larvae of host. Rates of growth, food consumption, assimilation, excretion, and respiration were calculated as well as approximate digestibility and the rate ratios ECI (percent efficiency of conversion of ingested food to body substance), and ECD (percent efficiency of conversion of digested food to body substance). Parasitization by C. vestalis resulted in significant decrease in the rates of growth, feeding, excretion, assimilation, and respiration, but the final dry rate of respiration at the end of last larval stadium was elevated. The ECI and ECD were also reduced as the result of parasitization, but digestibility was increased. All these parameters in the larvae parasitized by D. semiclausum at 24 h post‐parasitization were also significantly changed compared to the control; however, these differences were quantitatively, but not qualitatively before pupation, similar to those resulted from parasitization by C. vestalis. In spite of the similarities of the parasitism‐induced effects caused by these endoparasitoids, the final metabolic rate, that is, the rate of intake of nutrients required to compensate for metabolism, was much lower in the larvae parasitized by C. vestalis than that of the larvae parasitized by D. semiclausum. All of the results discussed here will contribute toward explaining the different ways these two wasps regulate the parasitoid‐host relationship.     

Effects of the endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae) parasitism,venom, and calyx fluid on cellular and humoral immunity of its host Chilo suppressalis (Lepidoptera: Crambidae) larvae

The larval endoparasitoid Cotesia chilonis injects venom and bracoviruses into its host Chilo suppressalis during oviposition. Here we study the effects of the polydnavirus (PDV)-carrying endoparasitoid C. chilonis (Hymenoptera: Braconidae) parasitism, venom and calyx fluid on host cellular and humoral immunity, specifically hemocyte composition, cellular spreading, encapsulation and melanization. Total hemocyte counts (THCs) were higher in parasitized larvae than in unparasitized larvae in the late stages following parasitization. While both plasmatocyte and granulocyte fractions and hemocyte mortality did not differ between parasitized and unparasitized hosts, in vitro spreading behavior of hemocytes was inhibited significantly by parasitism throughout the course of parasitoid development. C. chilonis parasitism suppressed the encapsulation response and melanization in the early stages. Venom alone did not alter cellular immune responses, including effects on THCs, mortality, hemocyte composition, cell spreading and encapsulation, but venom did inhibit humoral immunity by reducing melanization within 6 h after injection. In contrast to venom, calyx fluid had a significant effect on cell spreading, encapsulation and melanization from 6 h after injection. Dose–response injection studies indicated the effects of venom and calyx fluid synergized, showing a stronger and more persistent reduction in immune system responses than the effect of either injected alone.     

Transfer of a chromosomal <Emphasis Type="Italic">Maverick</Emphasis> to endogenous bracovirus in a parasitoid wasp

Bracoviruses are used by parasitoid wasps to allow development of their progeny within the body of lepidopteran hosts. In parasitoid wasps, the bracovirus exists as a provirus, integrated in a wasp chromosome. Viral replication occurs in wasp ovaries and leads to formation of particles containing dsDNA circles (segments) that are injected into the host body during wasp oviposition. We identified a large DNA transposon Maverick in a parasitoid wasp bracovirus. Closely related elements are present in parasitoid wasp genomes indicating that the element in CcBV corresponds to the insertion of an endogenous wasp Maverick in CcBV provirus. The presence of the Maverick in a bracovirus genome suggests the possibility of transposon transfers from parasitoids to lepidoptera via bracoviruses.     

菜蛾盘绒茧蜂卵携带的免疫抑制因子

抑制寄主昆虫的免疫反应是内寄生蜂存活的关键。菜蛾盘绒茧蜂Cotesia vestalis（Haliday）寄生小菜蛾Plutella xylostella （L.）幼虫后,蜂卵如何逃避和抑制寄主的免疫攻击,尚未得到全面揭示。本文采用电镜技术系统观察了菜蛾盘绒茧蜂卵表面的超微结构。结果显示：蜂卵表面覆盖有纤维层和絮状的类病毒样纤丝（VLFs）,同时携带了含多分DNA病毒粒子（PDV）的萼液。在寄生初期,包裹在蜂卵表面的纤维层和VLFs首先起到保护蜂卵不被小菜蛾血细胞包囊的被动防御作用。随后,PDV发挥主动的免疫抑制作用。通过假寄生手段,证明了菜蛾盘绒茧蜂PDV (CvBV) 具有较持久的克服寄主免疫攻击的能力,是主要的免疫抑制因子。在假寄生后连续8 d的观察时间内,菜蛾盘绒茧蜂的蜂卵均未被包囊。结果提示,在菜蛾盘绒茧蜂-小菜蛾寄生体系中,菜蛾盘绒茧蜂采取被动防御和主动攻击两种方式应对寄主小菜蛾的免疫攻击。     

Venom of the egg-larval parasitoid Chelonus inanitus is a complex mixture and has multiple biological effects

The egg-larval parasitoid Chelonus inanitus injects bracoviruses (BVs) and venom along with the egg into the host egg; both components are essential for successful parasitoid development. All stages of eggs of its natural host, Spodoptera littoralis, can be successfully parasitized, i.e. from mainly a yolk sphere to a fully developed embryo. Here, we show that the venom contains at least 25 proteins with masses from 14 kDa to over 300 kDa ranging from acidic to basic. The majority is glycosylated and their persistence in the host is short when old eggs are parasitized and much longer when young eggs are parasitized. Physiological experiments indicated three different functions. (1) Venom synergized the effect of BVs in disrupting host development when injected into third instar larvae. (2) Venom had a transient paralytic effect when injected into sixth instar larvae. (3) In vitro experiments with haemocytes of fourth instar larvae suggested that venom alters cell membrane permeability. We propose that venom promotes entry of BVs into host cells and facilitates placement of the egg in the embryo's haemocoel when old eggs are parasitized. The multifunctionality of the venom might thus be essential in enabling parasitization of all stages of host eggs.     

Bracoviruses contain a large multigene family coding for protein tyrosine phosphatases

The relationship between parasitic wasps and bracoviruses constitutes one of the few known mutualisms between viruses and eukaryotes. The virions produced in the wasp ovaries are injected into host lepidopteran larvae, where virus genes are expressed, allowing successful development of the parasite by inducing host immune suppression and developmental arrest. Bracovirus-bearing wasps have a common phylogenetic origin, and contemporary bracoviruses are hypothesized to have been inherited by chromosomal transmission from a virus that originally integrated into the genome of the common ancestor wasp living 73.7 +/- 10 million years ago. However, so far no conserved genes have been described among different braconid wasp subfamilies. Here we show that a gene family is present in bracoviruses of different braconid wasp subfamilies (Cotesia congregata, Microgastrinae, and Toxoneuron nigriceps, Cardiochilinae) which likely corresponds to an ancient component of the bracovirus genome that might have been present in the ancestral virus. The genes encode proteins belonging to the protein tyrosine phosphatase family, known to play a key role in the control of signal transduction pathways. Bracovirus protein tyrosine phosphatase genes were shown to be expressed in different tissues of parasitized hosts, and two protein tyrosine phosphatases were produced with recombinant baculoviruses and tested for their biochemical activity. One protein tyrosine phosphatase is a functional phosphatase. These results strengthen the hypothesis that protein tyrosine phosphatases are involved in virally induced alterations of host physiology during parasitism.     

The Bracovirus Genome of the Parasitoid Wasp Cotesia congregata Is Amplified within 13 Replication Units,Including Sequences Not Packaged in the Particles

The relationship between parasitoid wasps and polydnaviruses constitutes one of the few known mutualisms between viruses and eukaryotes. Viral particles are injected with the wasp eggs into parasitized larvae, and the viral genes thus introduced are used to manipulate lepidopteran host physiology. The genome packaged in the particles is composed of 35 double-stranded DNA (dsDNA) circles produced in wasp ovaries by amplification of viral sequences from proviral segments integrated in tandem arrays in the wasp genome. These segments and their flanking regions within the genome of the wasp Cotesia congregata were recently isolated, allowing extensive mapping of amplified sequences. The bracovirus DNAs packaged in the particles were found to be amplified within more than 12 replication units. Strikingly, the nudiviral cluster, the genes of which encode particle structural components, was also amplified, although not encapsidated. Amplification of bracoviral sequences was shown to involve successive head-to-head and tail-to-tail concatemers, which was not expected given the nudiviral origin of bracoviruses.     

Cover Caption

Cotesia vestalis has been used as a good biological control agent for management the pest of global cruciferous plants, Plutella xylostella. The successful parasitization strictly relies on C. vestalis olfactory perception, and odorant binding protein (OBP) play a key role in searching for hosts (see pages 1354-1368). The cover photo shows C. vestalis is parasitizing the host with the help of three OBP genes expressed in C. vestalis antennae for locating P. xylostalla larva. Photo provided by Xi-Qian Ye.     

Survival of the parasitoidEncarsia formosa after treatment of parasitized greenhouse whitefly larvae with fungal spores ofAschersonia aleyrodis

The interaction between the entomopathogenic fungusAschersonia aleyrodis and the parasitoidEncarsia formosa on greenhouse whitefly as a host organism was studied, in particular, the survival of the parasitoid after treatment of parasitized hosts with fungal spores. The mean number of parasitized black pupae per parasitoid produced at 25°C was significantly reduced after spore treatment in the first three days following parasitization. Spore treatment four, seven or ten days after parasitization resulted in a mean number of parasitized pupae not significantly different from the number of black pupae in the control. The rather sudden change from low to high survival of parasitized hosts when treated with spores four days after parasitization in spite of high numbers of infected unparasitized larvae, coincided with the hatching of the parasitoid larva from the egg inside the host. Possible reasons for this decrease in susceptibility to infection after parasitoid egg hatch, such as induced changes in host cuticle or haemolymph, are discussed. Parasitoids emerged from treated hosts did not show differences in reproduction compared with parasitoids emerging from untreated hosts. Both natural enemeies of whitefly are compatible to a great extent.     

Cotesia plutellae Bracovirus Genome and Its Function in Altering Insect Physiology

Polydnavirus is a group of animal DNA virus mutually associated with some ichneumonoid wasp. Its relatively large size of genome has been considered as a major source of the parasitoid function to manipulate developmental and immunological processes of target parasitized insects. Cotesia plutellae bracovirus (CpBV) is a polydnavirus derived from C. plutellae, which parasitizes the diamondback moth, Plutella xylostella. Parasitized P. xylostella exhibits altered physiological symptoms in development and immune reactions. Though several other parasitic factors such as ovarian proteins, venom, and teratocytes are identified, CpBV has been more focused on elucidating various host physiological alterations occurring due to the parasitism, which has driven the CpBV genome project. CpBV attains a typical bracovirus structure by its single unit membrane envelope, in which multiple nucleocapsids are enclosed. Its genome DNAs are segmented and located on the genome of C. plutellae. Its replication begins at adult tissue development during pupal stage. An apparent genome size is 471 kb estimated from 27 segments separated on 5% agarose gel. A current work on the genome has been completely sequenced 24 genomic segments and analyzed their genomic structure. The aggregated genome size is 351, 299 bp long and exhibits an average GC content of approximately 34.6%. Average coding density is about 32.3% and 125 putative open reading frames are predicted. Though more than half (52.5%) of predicted genes are annotated as hypothetical, the annotated CpBV genes share amino acid sequence homologies with those of other bracoviral genomes. The annotated genes are classified into the known bracoviral families, in which a family of protein tyrosine phosphatase is the largest including 36 ORFs, suggesting a significant role during parasitization. In addition, 8 and 7 ORFs encode Iκβ-like and EP1-like, respectively. Some predicted genes are known only in Cotesia-associated bracoviral genomes. Finally, two homologous genes, CpBV15α/β, are unique in CpBV genome, which are not matched to any other known polydnaviral genes. Their homology with malarian circumsporozoite toxin and eukaryotic translation inhibition factors suggests their function in host translation inhibitory factor. This review discusses CpBV genes on their putative physiological functions based on the molecular interactions between the host-parasite.