Characterization of immunosuppressive surface coat proteins from Steinernema glaseri that selectively kill blood cells in susceptible hosts.

Surface coat proteins (SCPs) of entomopathogenic nematodes are implicated in the suppression/evasion of host immune responses, which is required for successful host colonization. Steinernema glaseri NC strain SCPs suppressed immune responses in oriental beetle larvae (Exomala orientalis), a susceptible host for S. glaseri, in a dosage-dependent manner, thus protecting Heterorhabditis bacteriophora from being killed in the same host. Melanization of H. bacteriophora decreased from 92+/-5% in the untreated check to 1+/-3% when protected by injection of 230ng of S. glaseri SCPs. As the SCPs dosage increased, freely moving H. bacteriophora increased from 3+/-4% in the untreated group to 57+/-15% with an SCPs dose of 940ng. At 2h and in the absence of SCPs, 8% and 11% of hemocytes of E. orientalis were stained by propidium iodide and Hoechst, respectively. When exposed to 300ng/microl SCPs, 70% and 96% were stained, respectively. At 6h, propidium iodide stained 37% and 92% of the hemocytes without and with SCPs, respectively. In contrast, more than 90% of the cells were stained by Hoechst with or without SCPs. As native proteins, two isolated S. glaseri SCPs had an immunosuppressive effect; they were each composed of 38kDa (PI=4.6) and 56kDa (PI=3.6) subunits. SCP peptides were sequenced using LC-MS/MS and the mass fingerprints obtained with MALDI-TOF-MS; there were no significant matches found in peptide databases, which suggests that the SCPs studied are novel proteins. Twelve cDNA sequences were derived based on short peptides and 7 of them had no significant match against the Caenorhabditis elegans protein database. One of the cDNA matched an unknown C. elegans protein and the remaining 4 cDNAs matched proteins of C. elegans and Brugia malayi.

Relationship between the successful infection by entomopathogenic nematodes and the host immune response.

Reproduction of entomopathogenic nematodes requires that they escape recognition by a host's immune system or that they have mechanisms to escape encapsulation and melanization. We investigated the immune responses of larvae for the greater wax moth (Galleria mellonella), tobacco hornworm (Manduca sexta), Japanese beetle (Popillia japonica), northern masked chafer (Cyclocephala borealis), oriental beetle (Exomala orientalis) and adult house crickets (Acheta domesticus), challenged with infective juveniles from different species and strains of entomopathogenic nematodes. The in vivo immune responses of hosts were correlated with nematode specificity and survival found by infection assays. In P. japonica, 45% of injected infective juveniles from Steinernema glaseri NC strain survived; whereas the hemocytes from the beetle strongly encapsulated and melanized the Heterorhabditis bacteriophora HP88 strain, S. glaseri FL strain, Steinernema scarabaei and Steinernema feltiae. Overall, H. bacteriophora was intensively melanized in resistant insect species (E. orientalis, P. japonica and C. borealis) and had the least ability to escape the host immune response. Steinernema glaseri NC strain suppressed the immune responses in susceptible hosts (M. sexta, E. orientalis and P. japonica), whereas S. glaseri FL strain was less successful. Using an in vitro assay, we found that hemocytes from G. mellonella, P. japonica, M. sexta and A. domestica recognized both nematode species quickly. However, many S. glaseri in M. sexta and H. bacteriophora in G. mellonella escaped from hemocyte encapsulation by 24h. These data indicate that, while host recognition underlies some of the differences between resistant and susceptible host species, escape from encapsulation following recognition can also allow successful infection. Co-injected surface-coat proteins from S. glaseri did not protect H. bacteriophora in M. sexta but did protect H. bacteriophora in E. orientalis larva; therefore, surface coat proteins do not universally convey host susceptibility. Comparisons of surface coat proteins by native and SDS-PAGE demonstrated different protein compositions between H. bacteriophora and S. glaseri and between the two strains of S. glaseri.

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.

Alteration in FAD-glucose dehydrogenase activity and hemocyte behavior contribute to initial disruption of Manduca sexta immune response to Cotesia congregata parasitoids.

Cotesia congregata and Manduca sexta were used as a model system to study the mechanism and effect of a polydnavirus (PDV). We hypothesized that (1) FAD-glucose dehydrogenase (GLD) (EC 1.1.99.10) hemolymph titer would increase in response to parasitism, (2) hemocyte targeting behavior would be altered by parasitism, and (3) changes observed in GLD activity and hemocyte behavior immediately post-parasitization would be due to the presence of PDV. GLD specific activity was measured at four time points early during parasitism using a spectrophotometric enzyme assay. Hemocyte behavior was measured using direct observations of hemocyte response to a foreign target in vitro. Results demonstrate that GLD increases immediately post-oviposition and post-injection of purified PDV, indicating that virions elicit nonself recognition. This increase relative to unparasitized controls also occurs in response to trioxsalen-UV inactivated virus, indicating that the initial disruption of the host immune response is not dependent upon viral transcription. Further, we demonstrate that plasmatocytes are actively spreading and aggregating but are not targeting nonself material in both parasitized and polydnavirus treatments. These results indicate that purified PDV is recognized as nonself and is triggering an immediate cellular immune response prior to viral transcription.

Expression of a delta 9 14:0-acyl carrier protein fatty acid desaturase gene is necessary for the production of omega 5 anacardic acids found in pest-resistant geranium (Pelargonium xhortorum).

Anacardic acids, a class of secondary compounds derived from fatty acids, are found in a variety of dicotyledonous families. Pest resistance (e.g., spider mites and aphids) in Pelargonium xhortorum (geranium) is associated with high levels (approximately 81%) of unsaturated 22:1 omega 5 and 24:1 omega 5 anacardic acids in the glandular trichome exudate. A single dominant locus controls the production of these omega 5 anacardic acids, which arise from novel 16:1 delta 11 and 18:1 delta 13 fatty acids. We describe the isolation and characterization of a cDNA encoding a unique delta 9 14:0-acyl carrier protein fatty acid desaturase. Several lines of evidence indicated that expression of this desaturase leads to the production of the omega 5 anacardic acids involved in pest resistance. First, its expression was found in pest-resistant, but not suspectible, plants and its expression followed the production of the omega 5 anacardic acids in segregating populations. Second, its expression and the occurrence of the novel 16:1 delta 11 and 18:1 delta 13 fatty acids and the omega 5 anacardic acids were specific to tall glandular trichomes. Third, assays of the recombinant protein demonstrated that this desaturase produced the 14:1 delta 9 fatty acid precursor to the novel 16:1 delta 11 and 18:1 delta 13 fatty acids. Based on our genetic and biochemical studies, we conclude that expression of this delta 9 14:0-ACP desaturase gene is required for the production of omega 5 anacardic acids that have been shown to be necessary for pest resistance in geranium.

A rapid method for isolating glandular trichomes.

A physical method is described for the rapid isolation of plant trichomes, with emphasis on stalked glandular types. The technique involved breaking frozen trichomes with powdered dry ice and collection of glandular heads by sieving from larger tissue fragments. This method was applied to several plants that bear similar stalked trichomes: geranium (Pelargonium), potato (Solanum tuberosum), tomato (Lycopersicon esculentum), squash (Cucurbita pepo), and velvetleaf (Abutilon theophrasti). The tissue preparation was of sufficient quality without further purification for biochemical and molecular studies. The preparation maintained the biochemical integrity of the trichomes for active enzymes and usable nucleic acids. A large quantity of tissue can be harvested; for example, 351 milligrams dry weight of glandular trichomes were harvested from geranium pedicels in 12 hours. The utility of the technique was demonstrated by examining the fatty acid composition of tall glandular trichomes of geraniums, Pelargonium xhortorum L.H. Bailey. These purified cells contained high concentrations of unusual omega5-unsaturated fatty acids, proportionally 23.4% of total fatty acids in the trichomes. When the trichomes were removed, the supporting tissue contained no omega5-fatty acids, thereby unequivocally localizing omega5-fatty acids to the trichomes. Because omega5-fatty acids are unique precursors for the biosynthesis of omega5-anacardic acids, we conclude that anacardic acid synthesis must occur in the glandular trichomes.

Developmental expression of the glucose dehydrogenase gene in Drosophila melanogaster.

The Gld gene of Drosophila melanogaster is transiently expressed during every stage of development. The temporal pattern of Gld expression is highly correlated with that of ecdysteroids. Exogeneous treatment of third instar larvae with 20-hydroxyecdysone induces the accumulation of Gld mRNA in the hypoderm and anterior spiracular gland cells. During metamorphosis Gld is expressed in a variety of tissues derived from the ectoderm. In the developing reproductive tract, Gld mRNA accumulates in the female spermathecae and oviduct and in the male ejaculatory duct and ejaculatory bulb. These four organs are derived from closely related cell lineages in the genital imaginal disc. Since the expression of Gld is not required for the development of these reproductive structures, this spatial pattern of expression is most likely a fortuitous consequence of a shared regulatory factor in this cell lineage. At the adult stage a high level of the Gld mRNA is only observed in the male ejaculatory duct.