Baculoviral p94 homologs encoded in Cotesia plutellae bracovirus suppress both immunity and development of the diamondback moth,Plutellae xylostella

Polydnaviruses (PDVs) are a group of insect DNA viruses, which exhibit a mutual symbiotic relationship with their specific host wasps. Moreover, most encapsidated genes identified so far in PDVs share homologies with insect‐originated genes, but not with virus‐originated genes. In the meantime, PDVs associated with 2 wasp genera Cotesia and Glytapanteles encode some genes presumably originated from other viruses. Cotesia plutellae bracovirus (CpBV) encodes 4 genes homologous to baculoviral p94: CpBV‐E94k1, CpBV‐E94k2, CpBV‐E94k3, and CpBV‐E94k4. This study was conducted to predict the origin of CpBV‐E94ks by comparing their sequences with those of baculoviral orthologs and to determine the physiological functions by their transient expressions in nonparasitized larvae and subsequent specific RNA interference. Our phylogenetic analysis indicated that CpBV‐E94ks were clustered with other E94ks originated from different PDVs and shared high similarity with betabaculoviral p94s. These 4 CpBV genes were expressed during most developmental stages of the larvae of Plutella xylostella parasitized by C. plutellae. Expression of these 4 E94ks was mainly detected in hemocytes and fat body. Subsequent functional analysis by in vivo transient expression showed that all 4 viral genes significantly inhibited both host immune and developmental processes. These results suggest that CpBV‐E94ks share an origin with betabaculoviral p94s and play parasitic roles in suppressing host immune and developmental processes.     

Transient expression of specific Cotesia plutellae bracoviral segments induces prolonged larval development of the diamondback moth, Plutella xylostella

A polydnavirus, Cotesia plutellae bracovirus (CpBV), possesses a segmented and dispersed genome that is located on chromosome(s) of its symbiotic endoparasitic wasp, C. plutellae. When the host wasp parasitizes larvae of the diamondback moth, Plutella xylostella, at least 27 viral genome segments are delivered to the parasitized host along with the wasp egg. The parasitized P. xylostella exhibits significant immunosuppression and a prolonged larval development. Parasitized larvae take about 2 days longer than nonparasitized larvae to develop until the wandering stage of the final larval instar, and die after egress of the full grown wasp larvae. Developmental analysis using juvenile hormone and ecdysteroid analogs suggests that altering endocrine signals could induce the retardation of larval developmental rate in P. xylostella. In this study we used a transient expression technique to micro-inject individual CpBV genome segments, and tested their ability to induce delayed larval development of P. xylostella. We demonstrated that a CpBV segment was able to express its own encoded genes when it was injected into nonparasitized larvae, in which the expression patterns of the segment genes were similar to those in the larvae parasitized by C. plutellae. Twenty three CpBV genome segments were individually cloned and injected into the second instar larvae of P. xylostella and their effects assessed by measuring the time taken for host development to the cocooning stage. Three CpBV genome segments markedly interfered with the host larval development. When the putative genes of these segments were analyzed, it was found that they did not share any common genes. Among these segments able to delay host development, segment S27 was predicted to encode seven protein tyrosine phosphatases (CpBV-PTPs), some of which were mutated by insertional inactivation with transposons, while other encoded gene expressions were unaffected. The mutant segments were unable to induce prolonged larval development of P. xylostella. These results suggest that CpBV can induce prolonged larval development of P. xylostella, and that at least some CpBV-PTPs may contribute to the parasitic role probably by altering titers of developmental hormones.     

Transient transcription of a putative RNase containing BEN domain encoded in Cotesia plutellae bracovirus induces an immunosuppression of the diamondback moth, Plutella xylostella

A polydnavirus, Cotesia plutellae bracovirus (CpBV), possesses segmented genome located on chromosome(s) of an endoparasitoid wasp, C. plutellae. An episomal viral segment (CpBV-S3) consists of 11,017 bp and encodes two putative open reading frames (ORFs). ORF301 shows amino acid sequence homologies (28-50%) with RNase T2s of various organisms. It also contains BEN domain in C-terminal region. ORF302 is a hypothetical gene, which is also found in other bracoviruses. Both genes were expressed in larvae of Plutella xylostella parasitized by C. plutellae. Their expressions were detected in all tested tissues including hemocyte, fat body, gut, and epidermis. To analyze effects of these genes on the parasitism, the segment of CpBV-S3 was injected to nonparasitized larvae of P. xylostella, in which the two genes were expressed at least for 4 days post-injection. The larvae injected with CpBV-S3 exhibited significant immunosuppression, such as reduction in total hemocyte population and impairment in nodule formation behavior of hemocytes in response to bacterial challenge. Each gene expression in the treated larvae was inhibited by co-injecting respective double strand RNA (dsRNA) specific to each ORF. Injection of dsRNA of ORF301 could rescue the immunosuppression of the viral segment-treated larvae, while dsRNA specific to ORF302 did not. These results suggest that a putative RNase fused with a BEN domain encoded in CpBV-S3 plays a parasitic role in inducing host immunosuppression in the parasitism.     

IkB genes encoded in Cotesia plutellae bracovirus suppress an antiviral response and enhance baculovirus pathogenicity against the diamondback moth, Plutella xylostella

An endoparasitoid wasp, Cotesia plutellae, parasitizes larvae of the diamondback moth, Plutella xylostella, with its symbiotic polydnavirus, C. plutellae bracovirus (CpBV). This study analyzed the role of Inhibitor-kB (IkB)-like genes encoded in CpBV in suppressing host antiviral response. Identified eight CpBV-IkBs are scattered on different viral genome segments and showed high homologies with other bracoviral IkBs in their amino acid sequences. Compared to an insect ortholog (e.g., Cactus of Drosophila melanogaster), they possessed a shorter ankyrin repeat domain without any regulatory domains. The eight CpBV-IkBs are, however, different in their promoter components and expression patterns in the parasitized host. To test their inhibitory activity on host antiviral response, a midgut response of P. xylostella against baculovirus infection was used as a model reaction. When the larvae were orally fed the virus, they exhibited melanotic responses of midgut epithelium, which increased with baculovirus dose and incubation time. Parasitized larvae exhibited a significant reduction in the midgut melanotic response, compared to nonparasitized larvae. Micro-injection of each of the four CpBV genome segments containing CpBV-IkBs into the hemocoel of nonparasitized larvae showed the gene expressions of the encoded IkBs and suppressed the midgut melanotic response in response to the baculovirus treatment. When nonparasitized larvae were orally administered with a recombinant baculovirus containing CpBV-IkB, they showed a significant reduction in midgut melanotic response and an enhanced susceptibility to the baculovirus infectivity.     

Transient expression of a polydnaviral gene, CpBV15beta, induces immune and developmental alterations of the diamondback moth, Plutella xylostella

The diamondback moth, Plutella xylostella, parasitized by its endoparasitoid wasp, Cotesia plutellae, undergoes various physiological alterations which include immunosuppression and an extended larval development. Its symbiotic virus, C. plutellae bracovirus (CpBV), is essential for their successful parasitization with more than 136 putative genes encoded in the viral genome. CpBV15β, a CpBV gene, has been known to play significant role in altering host physiological processes including hemocyte-spreading behavior through inhibition of protein synthesis under in vitro conditions. In the current study, we investigated its specific involvement in physiological processes of the host by transient expression and RNA interference techniques. The open reading frame of CpBV15β was cloned into a eukaryotic expression vector and this recombinant CpBV15β was transfected into nonparasitized 3rd instar P. xylostella by microinjection. CpBV15β was expressed as early as 24 h and was consistent up to 72 h. Due to the expression of this gene, plasma protein levels were significantly reduced and the ability of the hemocytes to adhere and spread on extracellular matrix was inhibited, wherein CpBV15β was detectable in the cytoplasm of hemocytes based on an indirect immunofluorescence assay. To confirm the role of CpBV15β, its double stranded RNA could efficiently recover the hemocyte-spreading behavior and synthesis of plasma proteins suppressed by the transient expression of CpBV15β. In addition, the larvae transfected with CpBV15β significantly suffered poor adult development probably due to lack of storage proteins. Thus these results demonstrate the role of CpBV15β in altering the host physiological processes involving cellular immune response and metamorphic development, which are usually induced by wasp parasitization.     

Altered actin polymerization of Plutella xylostella (L.) in response to ovarian calyx components of an endoparasitoid Cotesia plutellae (Kurdjumov)

Abstract Cotesia plutellae (Kurdjumov) (Hymenoptera: Braconidae), a solitary braconid endoparasitoid wasp, parasitizes the diamondback moth Plutella xylostella (L.) (Lepidoptera: Yponomeutidae) by suppressing the host defense response, thereby resulting in successful parasitization. During parasitization, ovarian calyx fluid is also delivered into the haemocoel of the host along with the wasp egg. The effect of calyx fluid constituents on haemocyte‐spreading behaviour of P. xylostella is analysed by measuring F‐actin development in the haemocytes. For this purpose, the calyx fluid of C. plutellae is separated into ovarian protein and C. plutellae bracovirus (CpBV). The ovarian protein consists of a wide range of molecular weight proteins, which are apparently different from those of CpBV. When nonparasitized P. xylostella haemocytes are incubated with either ovarian protein or CpBV for 1 or 2 h, haemocytes lose their responsiveness to a cytokine, plasmatocyte‐spreading peptide, in a dose‐dependent manner for each calyx component and fail to exhibit haemocyte‐spreading behaviour. Some CpBV genes are expressed within 1 h of parasitization. The inhibition of haemocyte‐spreading could be explained by measuring F‐actin contents, in which parasitization by C. plutellae inhibits F‐actin development in the haemocytes of P. xylostella. Either ovarian protein or CpBV could inhibit F‐actin development in the nonparasitized haemocytes. In addition, co‐incubation of ovarian protein and CpBV results in significant additive inhibition of both haemocyte‐spreading and F‐actin development in the haemocytes in response to cytokine. These results suggest that both components of C. plutellae calyx fluid function in a synergistic manner, leading to immunosuppression during the early stage of parasitization.     

Transient expression of a viral histone H4, CpBV-H4, suppresses immune-associated genes of Plutella xylostella and Spodoptera exigua

A viral histone H4, CpBV-H4, is encoded in the Cotesia plutellae bracovirus (CpBV) genome. This polydnavirus is symbiotic with C. plutellae, an endoparasitoid wasp. When the wasp parasitizes its host, Plutella xylostella, the symbiotic CpBV is delivered to host hemocoel and infects different internal tissues. CpBV-H4 encoded in the virus exhibits high sequence similarity to host histone H4, except for an extended N-terminal tail (38 amino acids long). When the CpBV-H4 cloned in a eukaryotic expression vector was transiently expressed in P. xylostella and a nonhost, Spodoptera exigua, it clearly inhibited several immune-associated genes, including cecropin, gloverin, serpin, apolipophorin III, and transferrin. However, its truncated construct, prepared by deleting 38 amino acids at the N-terminal tail, lost its inhibitory activity against immune-associated genes of the both species. This study has verified an inhibitory activity of CpBV-H4 against host immune-associated genes and has provided a possibility to expand its activity spectrum to the genes of other insect species.     

<Emphasis Type="Italic">Cotesia plutellae</Emphasis> bracovirus suppresses expression of an antimicrobial peptide,cecropin, in the diamondback Moth, <Emphasis Type="Italic">Plutella xylostella</Emphasis>, challenged by bacteria

An endoparasitoid wasp, Cotesia plutellae, induces significant immunosuppression of host insect, Plutella xylostella. This study was focused on suppression in humoral immune response of P. xylostella parasitized by C. plutellae. An EST database of P. xylostella provided a putative cecropin gene (PxCec) which is 627 bp long and encodes 66 amino acids. A signal peptide (22 amino acids) is predicted and two putative O-glycosylation sites in threonine are located at positions 58 and 64. Without bacterial infection, PxCec was expressed in pupa and adult stages but not in the egg and larval stages. Upon bacterial challenge, however, the larvae expressed PxCec as early as 3 h post infection (PI) and maintained high expression levels at 12–24 h PI. By 48 h PI, its expression noticeably diminished. All tested tissues of bacteria-infected P. xylostella showed PxCec expression. However, other microbes, such as virus and fungus, did not induce the PxCec expression. Parasitization by C. plutellae suppressed the expression of PxCec in response to bacterial challenge. Among the parasitic factors of C. plutellae, its symbiotic virus (C. plutellae bracovirus: CpBV) alone was able to inhibit the expression of PxCec of P. xylostella challenged by bacteria. These results indicate that PxCec expression is regulated by both immune and developmental processes in P. xylostella. The parasitization by C. plutellae inhibited the expression of PxCec by the wasp’s symbiotic virus.     

Transient expression of a viral histone H4 inhibits expression of cellular and humoral immune-associated genes in Tribolium castaneum

A viral histone H4 is encoded in a polydnavirus called Cotesia plutellae bracovirus (CpBV), which is symbiotic to an endoparasitoid wasp, C. plutellae. Compared to general histone H4s, the viral H4 possesses an extra N-terminal tail containing 38 amino acid residues, which has been presumed to control host gene expression in an epigenetic mode. To analyze the epigenetic control activity of CpBV-H4 on expression of immune-associated genes, it was transiently expressed in larvae of Tribolium castaneum that had been annotated in the immune genes from a full genome sequence. Subsequent alteration of gene expression pattern was compared with that of its mutant form deleting N-terminal tail (truncated CpBV-H4). In response to bacterial challenge, T. castaneum induces expression of 13 antimicrobial peptide (AMP) genes. When CpBV-H4 was expressed, the larvae failed to express 12 inducible AMP genes. By contrast, when truncated CpBV-H4 was transiently expressed, all AMP genes were expressed. Hemocyte nodule formation was significantly impaired by expression of CpBV-H4, in which expressions of tyrosine hydroxylase and dihydroxyphenylalanine decarboxylase were suppressed. However, expression of truncated CpBV-H4 did not give any significant adverse effect on the cellular immunity. The immunosuppression of CpBV-H4 was further supported by its activity of enhancing bacterial pathogenicity of an entomopathogenic bacterium, Xenorhabdus nematophila, against larvae transiently expressing CpBV-H4. These results suggest that CpBV-H4 suppresses both humoral and cellular immune responses of T. castaneum by altering a normal epigenetic control of immune-associated gene expression.     

Parasitism of Cotesia spp. Enhances Susceptibility of Plutella xylostella to Other Pathogens

Two endoparasitoids, Cotesia plutellae and C. glomerata, parasitize the diamondback moth, Plutella xylostella, and induce significant host immunosuppression. This study analyzed the susceptibility changes of the parasitized P. xylostella against other pathogens using an entomopathogenic bacterium, Xenorhabdus nematophila (Xn), and a viral pathogen, Autographa californica nucleopolyhedrosis virus (AcNPV). The P. xylostella parasitized by either C. plutellae or C. glomerata exhibited higher susceptibilities to both microbial pathogens than the nonpara-sitized. To determine the parasitism factors inducing the enhanced susceptibility, three polydnaviral genes so far successfully cloned were selected from C. plutellae bracovirus (CpBV). CpBV-lectin and CpBV15 α/β were inserted into AcNPV under a CpBV promote and analyzed in their pathogenicities against P. xylostella larvae. Two AcNPVs recombined with CpBV15α/β were more potent than the control AcNPV recombined with an enhanced green fluorescent protein gene or the AcNPV recombined with CpBV-lectin. These results suggest that the wasp parasitization enhances other pathogen susceptibilities by inducing host immunosuppression, in which the symbiotic polydnavirus can play significant role in the enhanced susceptibility.     

A copy of cystatin from the diamondback moth Plutella xylostella is encoded in the polydnavirus Cotesia plutellae bracovirus

Cystatins (CSTs) are reversible and competitive inhibitors of cysteine proteases. Some polydnaviruses encode viral CSTs that have been speculated to play a crucial role in viral pathology. Four CSTs have been reported in the episomal genome of a polydnavirus, Cotesia plutellae (synonymous with C. vestalis) bracovirus (CpBV). These 4 CSTs share high sequence homologies with other bracoviral CSTs. Further sequence analysis showed that 2 of the CpBV-CSTs are identical. The remaining 3 CSTs have been designated CpBV-CST1, CpBV-CST2, and CpBV-CST3. Expression analysis indicated that CpBV-CST2 was not expressed in any stage of Plutella xylostella, either parasitized or non-parasitized by C. plutellae. However, both CpBV-CST1 and CpBV-CST3 were expressed in all stages of P. xylostella. Interestingly, these 2 genes were also expressed in non-parasitized P. xylostella in all developmental stages. A CST sequence from the non-parasitized larva was 100% identical with that of CpBV-CST1 for the entire open reading frame (ORF). To understand the role of CpBV-CST1 in viral pathology, the ORF was cloned into a eukaryotic expression vector and transiently expressed in non-parasitized larvae. The in vivo transient expression lasted for at least 4 days. Under this condition, the treated larvae suffered significant suppression in immune responses and in development. These results suggest that CpBV-CSTs play a crucial role in parasitism, altering host immune and developmental processes by interrupting normal interactions between CSTs and cysteine proteases in P. xylostella.     

Combined transcriptomic and proteomic analysis of developmental features in the immune system of Plutella xylostella during larva-to-adult metamorphosis

Diamondback moth (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae) is considered one of the most destructive worldwide agricultural pests and has developed various defence mechanisms to fight against the available pesticides. Understanding the host-defence system of P. xylostella is vital for developing biocontrol-based pest management strategies. Although there are several studies on P. xylostella, little is known about the changes in the immune system during the larva-to-adult metamorphosis. RNA-seq and iTRAQ investigations of P. xylostella from 2-day-old fourth instar larvae (L4D2), pupa (P0), and adult (A0) were done to understand these alterations at a molecular level. A total of 412/ 584 up-regulated and 1430/ 757 down-regulated genes/proteins between larva and pupa, 813/ 589 up-regulated and 1206/ 846 down-regulated genes/proteins between pupa and adult were identified. It was shown that the differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) expression were up-regulated during the pupation and emergence of metamorphosis. The pathway enrichment analysis demonstrated that DEGs and DEPs were mainly associated with the energy generation and metabolism and innate immunity of the insect. The expression of immune-related and developmental-related genes were significantly different during the developmental process of P. xylostella. Moreover, the expression of four focused genes, i.e., serine proteinase inhibitor (Serpin-15), prophenoloxidase activating proteinase 1 (PAP-1) and 3a (PAP-3a), Gram-negative bacteria-binding protein (GNBP-6), was different in developmental stages and after Bacillus thuringiensis HD73 and Metarhizium anisopliae infection. The phenoloxidase (PO) activity in plasma was also significantly up-regulated during the pathogen infection. Recombinant proteins PAP-1, PAP-3a, GNBP-6 could significantly trigger the PO activity in vitro, Serpin-15 could suppress the PO activity. Taken together, these results indicate that Serpin-15, PAP-1, PAP-3a, and GNBP-6 might have the potential for co-regulation of immunity and development in P. xylostella. In conclusion, this study provided the immune system dynamics in the developmental process of P. xylostella and identified four candidate genes that can serve as potential targets for pest control strategies.     

Implication for DNA methylation involved in the host transfer of diamondback moth,Plutella xylostella (L.)

DNA methylation exerts extensive impacts on gene expression of various living organisms exposed to environmental variation. However, little is known whether DNA methylation is involved in the host transfer of diamondback moth, Plutella xylostella (L.), a worldwide destructive pest of crucifers. In this study, we found that P. xylostella genome exhibited a relatively low level of DNA methylation on the basis of the CpG O/E prediction and experimental validation. A significant positive linear correlation was observed between the stage‐specific expressions of PxDNMT1 and DNA methylation levels (5mC content). Particularly, high levels of DNA methylation and gene expression of PxDNMT1 were observed in eggs and mature females of P. xylostella. After host transfer of P. xylostella from Raphanus sativus to Arabidopsis thaliana, we identified some potential genomic loci that might have changed methylation levels. Using the method of fluorescence‐labeled methylation‐sensitive amplified polymorphism (F‐MSAP), we also found the corresponding genes primarily involved in neural system and signaling. The expressions of six candidate genes were verified by qRT‐PCR. One of the genes, Px009600, might be regulated by a DNA methylation‐mediated mechanism in response to host transfer. Our study provides evidence for a functional system of DNA methylation in P. xylostella and its possible role in adaptation during host transfer. Further studies should examine methylation as responsive factors to different host plants and environmental cues in insect pests.     

小菜蛾幼虫偏好表达的microRNA鉴定及功能研究

[目的]microRNA(miRNA)在昆虫生长发育中发挥重要功能,本研究拟通过鉴定小菜蛾不同发育阶段的miRNA,挖掘幼虫偏好表达的miRNA及其潜在功能.[方法]对小菜蛾卵、3龄幼虫、蛹和成虫的miRNA开展高通量测序,结合生物信息学分析方法,筛选在幼虫期偏好表达的miRNA;借助实时荧光定量PCR技术,验证候选m...