Effects of the polydnavirus of Cotesia congregata on the immune system and development of non-habitual hosts of the parasitoid

Polydnaviruses (PDV) are obligate mutualistic symbionts found in association with some groups of parasitic Hymenoptera. In these groups, they suppress the immune response of the parasitoid’s host and are required for successful parasitoid reproduction. Several PDV effects have been described in different experimental systems, but no clear picture of PDV mode of immunosuppression has emerged. No study to date has directly tested if PDV modes of action are evolutionarily conserved or divergent among parasitoid taxa within the Ichneumonoidea. We hypothesize the divergence in PDV mode of immunosuppression can be detected by identifying points of divergence in the immune response of different host species to PDV from one parasitoid species. This study tests the effects of purified PDV from Cotesia congregata on the immune response of three larval lepidopteran species that naturally are hosts of parasitoid species that differ in taxonomic relatedness to C. congregata. Here we demonstrate that despite associations with distantly related parasitoids (Ichneumonidae and Braconidae), Manduca sexta and Heliothis virescens showed similar patterns of increased glucose dehydrogenase (GLD) activity, suppressed cellular encapsulation in vitro, and increased time to pupation. In contrast, Lymantria dispar showed no response to C. congregata PDV across any of the parameters measured, even though it has an evolutionary association with several parasitoids closely related to C. congregata and within the Microgastrinae. The PDV immunosuppression in H. virescens and M. sexta does not correlate with host molecular phylogeny either. The suborganismal effects shown in M. sexta and H. virescens translated into significantly reduced pupation success in M. sexta only. Results demonstrate that while some PDV modes of immunosuppression in hosts may be divergent, others may be conserved across broad host groups.     

Modulation by eicosanoid biosynthesis inhibitors of immune responses by the insect Manduca sexta to the pathogenic fungus Metarhizium anisopliae

Metarhizium anisopliae conidia (spores) reduced weight gain and caused death when injected into Manduca sexta larvae. When the fungus was co-injected with the eicosanoid biosynthesis inhibitor dexamethasone, larval weight gain was further reduced and mortality increased. These effects were reversed when dexamethasone was given together with the eicosanoid precursor arachidonic acid (AA). Similarly, treatment with other eicosanoid biosynthesis inhibitors (esculetin, phenidone, ibuprofen, and indomethacin) with differing modes of action enhanced the reduction in weight gain caused by mycosis. Injection of M. anisopliae conidia induced nodule formation in vivo; nodule numbers were reduced by dexamethasone, and restored by AA. Incubation of hemocytes with conidia caused microaggregation of hemocytes (indicative of nodule formation) in vitro and this was inhibited by dexamethasone, suggesting that dexamethasone acts directly on hemocytes, although inhibition was only partially reversed by AA. We suggest that the M. sexta immune response to fungal pathogens is normally modulated by physiological systems that include eicosanoid biosynthesis. This is the first demonstration that the virulence of a fungal entomopathogen can be enhanced by compromising the insect host's immune system.     

Parasitization of Manduca sexta larvae by the parasitoid wasp Cotesia congregata induces an impaired host immune response

During oviposition, the parasitoid wasp Cotesia congregata injects polydnavirus, venom, and parasitoid eggs into larvae of its lepidopteran host, the tobacco hornworm, Manduca sexta. Polydnaviruses (PDVs) suppress the immune system of the host and allow the juvenile parasitoids to develop without being encapsulated by host hemocytes mobilized by the immune system. Previous work identified a gene in the Cotesia rubecula PDV (CrV1) that is responsible for depolymerization of actin in hemocytes of the host Pieris rapae during a narrow temporal window from 4 to 8h post-parasitization. Its expression appears temporally correlated with hemocyte dysfunction. After this time, the hemocytes recover, and encapsulation is then inhibited by other mechanism(s). In contrast, in parasitized tobacco hornworm larvae this type of inactivation in hemocytes of parasitized M. sexta larvae leads to irreversible cellular disruption. We have characterized the temporal pattern of expression of the CrV1-homolog from the C. congregata PDV in host fat body and hemocytes using Northern blots, and localized the protein in host hemocytes with polyclonal antibodies to CrV1 protein produced in P. rapae in response to expression of the CrV1 protein. Host hemocytes stained with FITC-labeled phalloidin, which binds to filamentous actin, were used to observe hemocyte disruption in parasitized and virus-injected hosts and a comparison was made to hemocytes of nonparasitized control larvae. At 24h post-parasitization host hemocytes were significantly altered compared to those of nonparasitized larvae. Hemocytes from newly parasitized hosts displayed blebbing, inhibition of spreading and adhesion, and overall cell disruption. A CrV1-homolog gene product was localized in host hemocytes using polyclonal CrV1 antibodies, suggesting that CrV1-like gene products of C. congregata's bracovirus are responsible for the impaired immune response of the host.     

Evidence for an immune function of lepidopteran silk proteins

Hemolymph coagulation stops bleeding and protects against infection. Clotting factors include both proteins that are conserved during evolution as well as more divergent proteins in different species. Here we show that several silk proteins also appear in the clot of the greater wax moth Galleria mellonella. RT-PCR analysis reveals that silk proteins are expressed in immune tissues and induced upon wounding in both Galleria and Ephestia kuehniella, a second pyralid moth. Our results support the idea that silk proteins were co-opted for immunity and coagulation during evolution.     

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.     

A putative protein translation inhibitory factor encoded by Cotesia plutellae bracovirus suppresses host hemocyte-spreading behavior

An endoparasitoid, Cotesia plutellae (Hymenoptera: Braconidae), possesses a mutualistic bracovirus (CpBV), which plays significant roles in the parasitized host, Plutella xylostella (Lepidoptera: Plutellidae). CpBV15beta, a viral gene encoded by CpBV, is expressed at early and late parasitization periods, suggesting that it functions to manipulate the physiology of the parasitized host. This paper reports a physiological function of CpBV15beta as an immunosuppressive agent. The effect of CpBV15beta on cellular immunity was analyzed by assessing hemocyte-spreading behavior. Parasitization by C. plutellae caused altered behavior of hemocytes of P. xylostella, in which the hemocytes were not able to attach and spread on glass slides. CpBV15beta was expressed in Sf9 cells using a baculovirus expression system and purified from the culture media. When hemocytes of nonparasitized P. xylostella were incubated with purified CpBV15beta protein, spreading behavior was impaired in a dose-dependent manner at low micro-molar range. This inhibitory effect of CpBV15beta could also be demonstrated on hemocytes of a non-natural host, Spodoptera exigua. CpBV15beta protein significantly inhibited F-actin growth of hemocytes in response to an insect cytokine. Similarly, cycloheximide, a eukaryotic translation inhibitor, strongly inhibited the spreading behavior and F-actin growth of P. xylostella hemocytes. Under in vitro condition, hemocytes of nonparasitized P. xylostella released proteins into the surrounding medium. Upon incubation of hemocytes with either CpBV15beta or cycloheximide, their ability to release protein molecules was markedly inhibited. This study suggests that CpBV15beta suppresses hemocyte behavior by inhibiting protein translation.     

Antiviral, anti-parasitic, and cytotoxic effects of 5,6-dihydroxyindole (DHI), a reactive compound generated by phenoloxidase during insect immune response

Phenoloxidase (PO) and its activation system are implicated in several defense responses of insects. Upon wounding or infection, inactive prophenoloxidase (proPO) is converted to active PO through a cascade of serine proteases and their homologs. PO generates reactive compounds such as 5,6-dihydroxyindole (DHI), which have a broad-spectrum antibacterial and antifungal activity. Here we report that DHI and its spontaneous oxidation products are also active against viruses and parasitic wasps. Preincubation of a baculovirus stock with 1.25 mM DHI for 3 h near fully disabled recombinant protein production. The LC₅₀ for lambda bacteriophage and eggs of the wasp Microplitis demolitor were 5.6 ± 2.2 and 111.0 ± 1.6 ??M, respectively. The toxicity of DHI and related compounds also extended to cells derived from insects that serve as hosts for several of the aforementioned pathogens. Pretreatment of Sf9 cells with 1.0 mM DHI for 4 h resulted in 97% mortality, and LC₅₀ values of 20.3 ± 1.2 ??M in buffer and 131.8 ± 1.1 ??M in a culture medium. Symptoms of DHI toxicity in Sf9 cells included DNA polymerization, protein crosslinking, and lysis. Taken together, these data showed that proPO activation and DHI production is strongly toxic against various pathogens but can also damage host tissues and cells if not properly controlled.     

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.     

Effects of starvation and parasitism on foregut contraction in larval Manduca sexta

Larvae of Manduca sexta are parasitised by the braconid wasp, Cotesia congregata. In this study we examined whether contraction activity of the semi-isolated foregut was affected by parasitism. Parasitised larvae fed significantly less compared with unparasitised control larvae, therefore starved unparasitised animals were used as controls. Rate and force of foregut contraction in control caterpillars significantly increased with days of starvation. However, only contraction force in foreguts of parasitised larvae increased over time following infection. The presence of food in the foregut of caterpillars starved 7 days suggested that food moved anteriorly from the midgut and that contraction became antiperistaltic, but only normal peristalsis occurred in parasitised caterpillars. Rate and force of gut contractions may be controlled independently and starvation did not truly mimic the effects of the parasitoids. Dissection of caterpillars with emerged wasps indicated that 47% had a single wasp larva wedged between the brain and foregut. Removal of this wasp caused an increased rate of foregut contraction of the caterpillar. Brain removal resulted in an increased rate of foregut contraction only for unparasitised insects. Sectioning of the recurrent nerve temporarily eliminated foregut contraction, but the contraction began again in 250 s in parasitised caterpillars prior to wasp emergence, compared with over 500 s for unparasitised controls and parasitised caterpillars following wasp emergence.     

K<Superscript>+</Superscript> transport in the caterpillar intestine epithelium: role of osmolytes for the K<Superscript>+</Superscript>-secretory capacity of the tobacco hornworm midgut

The midgut of the tobacco hornworm, Manduca sexta, actively secretes potassium ions. This can be measured as short-circuit current (I_sc) with the midgut mounted in an Ussing chamber and superfused with a high-K⁺ saline containing as its major osmolyte 166 mM sucrose. Iso-osmotic substitution of sucrose by non-metabolisable compounds (mannitol, urea, NaCl and the polyethylene glycols 200, 400 and 600) led to a dramatic, though reversible, drop in the current. Acarbose, a specific inhibitor of invertase (sucrase) in vertebrates and insects, had no detectable influence on I_sc. Unexpectedly, after replacing sucrose iso-osmotically with the saccharides glucose, fructose, trehalose or raffinose, the K⁺ current could no longer be supported. However, all osmolytes smaller than sucrose (except for NaCl), metabolisable or not, initiated an immediate, quite uniform but transient, increase in I_sc by about 20%, before its eventual decline far below the control value. Hypo-osmotic treatment by omission of sucrose also transiently increased the K⁺ current. Small osmolytes substituted for sucrose caused no transient I_sc stimulation when the epithelium had been challenged before with hypo-osmolarity; however, the eventual decline in I_sc could not be prevented. Our data seem inconsistent with a role of sucrose as energiser or simple osmolyte. Rather, we discuss here its possible role as analogous to that of sucrose in lower eukaryotes or plants, as an extra- and/or intracellular compatible osmolyte that stabilises structure and/or function of the proteins implicated in K⁺ transport.Communicated by G. Heldmaier     

Expression patterns of odorant-binding proteins in antennae of the moth<Emphasis Type="Italic"> Manduca sexta</Emphasis>

Monoclonal antibodies (MAbs) were generated to six recombinant proteins (odorant-binding proteins; OBPs) of Manduca sexta. The specificity of each MAb was demonstrated by labeling six immunoblots, each of which contained samples of all six recombinant OBPs. The expression patterns of the six OBPs could be grouped into three classes: (1) one (GOBP1) was expressed in sensilla located throughout each annulus; (2) two (ABPX and ABP2) were expressed in the long sensilla trichoidea bordering a zone that was arranged as an arch on the periphery of each annulus; (3) three (PBP2, PBP3, and GOBP2) were expressed in shorter sensilla occupying a wedge-shaped mid-annular zone of each annulus. In female antennae, sensilla expressing these OBPs were intermixed, and the distinct zonation observed in the male antenna was absent. In males, PBP2 was co-expressed in exactly the same cells of the mid-annular zone as those expressing PBP3 and most of the same cells expressing GOBP2, although its expression overlapped with no or only a few sensilla expressing OBPs of class 1 (GOBP1) or class 2 (ABPX, ABP2). This overlap of expression or lack of overlap between PBP2 and the other OBPs for male antennae was mirrored in female antennae. In view of the restricted spatial expression of OBPs within an annulus and the diversity of possible dimeric combinations of OBPs that arises from the co-expression of multiple OBPs in a given sensillum, OBPs could contribute to the specificity of the olfactory responses of insects.This research was supported by grants from the National Science Foundation (IBN-9604095) and the University of Illinois Critical Research Initiatives     

Differential inhibition of Helicoverpa armigera gut proteinases by proteinase inhibitors of pigeonpea (Cajanus cajan) and its wild relatives

The seeds of 36 pigeonpea [Cajanus cajan (L) Millsp.] cultivars, resistant and susceptible to pests and pathogens and 17 of its wild relatives were analysed for inhibitors of trypsin, chymotrypsin, and insect gut proteinases to identify potential inhibitors of insect (Helicoverpa armigera) gut enzymes. Proteinase inhibitors (PIs) of pigeonpea cultivars showed total inhibition of trypsin and chymotrypsin, and moderate inhibition potential towards H. armigera proteinases (HGP). PIs of wild relatives exhibited stronger inhibition of HGP, which was up to 87% by Rhynchosia PIs. Electrophoretic detection of HGPI proteins and inhibition of HGP isoforms by few pigeonpea wild relative PIs supported our enzyme inhibitor assay results. Present results indicate that PIs exhibit wide range of genetic diversity in the wild relatives of pigeonpea whereas pigeonpea cultivars (resistant as well as susceptible to pests and pathogens) are homogeneous. The potent HGPIs identified in this study need further exploration for their use in strengthening pigeonpea defence against H. armigera.     

Trehalose-hydrolysing enzymes of Metarhizium anisopliae and their role in pathogenesis of the tobacco hornworm,Manduca sexta

Trehalose is the main haemolymph sugar in most insects including the tobacco hornworm, Manduca sexta, and is potentially a prime target for an invading pathogenic fungus. There was considerably more trehalose-hydrolysing activity in the haemolymph of caterpillars infected with Metarhizium anisopliae than in controls. This appeared to be due primarily to additional isoforms; one of which could also hydrolyse maltose and was designated an alpha-glucosidase. A comparable isoform was identified in in vitro culture of the fungus, supporting a fungal origin for the in vivo enzyme. The in vitro fungal enzyme, alpha-glucosidase-1 (alpha-gluc-1), was purified to homogeneity and partially characterised. A study with the trehalase inhibitor trehazolin and C14 trehalose suggested that extracellular hydrolysis is important for fungal mobilisation of trehalose. Haemolymph glucose increases significantly during mycosis of tobacco hornworm larvae by M. anisopliae, consistent with the hydrolysis of trehalose by extracellular fungal enzymes. The implications for the host insect are discussed.     

Differential expression of the CrV1 haemocyte inactivation-associated polydnavirus gene in the African maize stem borer Busseola fusca (Fuller) parasitized by two biotypes of the endoparasitoid Cotesia sesamiae (Cameron)

Polydnaviruses are rarely studied for their natural variation in immune suppressive abilities. The polydnavirus harboring braconid Cotesia sesamiae, a widespread endoparasitoid of Busseola fusca and Sesamia calamistis in sub-Saharan Africa exists as two biotypes. In Kenya, the western biotype completes development in B. fusca larvae. However, eggs of the coastal C. sesamiae are encapsulated in this host and ultimately, no parasitoids emerge from parasitized B. fusca larvae. Both biotypes develop successfully in S. calamistis larvae. Encapsulation activity by B. fusca larvae towards eggs of the avirulent C. sesamiae was detectable six hours post-parasitization. The differences in encapsulation of virulent and avirulent strains were associated with differences in nucleotide sequences and expression of a CrV1 polydnavirus (PDV) gene, which is associated with haemocyte inactivation in the Cotesia rubecula/Pieris rapae system. CrV1 expression was faint or absent in fat body and haemolymph samples from B. fusca parasitized by the avirulent C. sesamiae, which exhibited encapsulation of eggs. Expression was high in fat body and haemolymph samples from both B. fusca and S. calamistis larvae parasitized by the virulent C. sesamiae, encapsulation in the former peaking at the same time points as CrV1 expression in the latter. Non synonymous difference in CrV1 gene sequences between virulent and avirulent wasp suggests that variations in B. fusca parasitism by C. sesamiae may be due to qualitative differences in CrV1-haemocyte interactions.     

Expression and in vitro activation of Manduca sexta prophenoloxidase-activating proteinase-2 precursor (proPAP-2) from baculovirus-infected insect cells

Prophenoloxidase activation is a component of the immune system in insects and crustaceans. We recently purified and cloned a new prophenoloxidase-activating proteinase (PAP-2) from hemolymph of the tobacco hornworm Manduca sexta [J. Biol. Chem. 278, 3552-3561]. As the terminal component of a putative serine proteinase cascade, this enzyme activates prophenoloxidase (proPO) via limited proteolysis. To purify and study the activating proteinase for PAP-2 from this insect, we expressed the zymogen of PAP-2 (proPAP-2) in insect cells infected by a recombinant baculovirus that harbors the cDNA. To facilitate the purification of proPAP-2, we modified a commercial vector (pFastBac1) by inserting a synthetic DNA fragment encoding a hexahistidine sequence, allowing fusion of the affinity tag to the carboxyl terminus of a protein. After Spodoptera frugiperda Sf21 cells were infected by the virus, recombinant proPAP-2 was efficiently secreted into the media at a concentration of 5.9 microg/ml under the optimal conditions. After ammonium sulfate precipitation, the proenzyme was purified to near homogeneity by affinity chromatography on Ni(2+)-NTA agarose. Western blot analysis indicated that the recombinant proPAP-2 has a mobility slightly lower than that of the zymogen from M. sexta hemolymph. The molecular mass and isoelectric point of proPAP-2 were determined to be 47,573+/-11Da and 6.6, respectively. After the purified proenzyme was added to hemolymph from induced M. sexta larvae, it was rapidly activated by an unknown proteinase in the presence of peptidoglycan.     

Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus

In insect pathogen interactions, host developmental stage is among several factors that influence the induction of immune responses. Here, we show that the effectiveness of immune reactions to a pathogen can vary markedly within a single larval stage. Pre-wandering fifth-stage (day 5) larvae of the model lepidopteran insect Manduca sexta succumb faster to infection by the insect pathogenic bacterium Photorhabdus luminescens than newly ecdysed fifth-stage (day 0) caterpillars. The decrease in insect survival of the older larvae is associated with a reduction in both humoral and cellular defence reactions compared to less developed larvae. We present evidence that older fifth-stage larvae are less able to over-transcribe microbial pattern recognition protein and antibacterial effector genes in the fat body and hemocytes. Additionally, older larvae show reduced levels of phenoloxidase (PO) activity in the cell-free hemolymph plasma as well as a dramatic decrease in the number of circulating hemocytes, reduced ability to phagocytose bacteria and fewer melanotic nodules in the infected tissues. The decline in overall immune function of older fifth-stage larvae is reflected by higher bacterial growth in the hemolymph and increased colonization of Photorhabdus on the basal surface of the insect gut. We suggest that developmentally programmed variation in immune competence may have important implications for studies of ecological immunity.