The Role of Fresh versus Old Leaf Damage in the Attraction of Parasitic Wasps to Herbivore-Induced Maize Volatiles

The odor produced by a plant under herbivore attack is often used by parasitic wasps to locate hosts. Any type of surface damage commonly causes plant leaves to release so-called green leaf volatiles, whereas blends of inducible compounds are more specific for herbivore attack and can vary considerably among plant genotypes. We compared the responses of naïve and experienced parasitoids of the species Cotesia marginiventris and Microplitis rufiventris to volatiles from maize leaves with fresh damage (mainly green leaf volatiles) vs. old damage (mainly terpenoids) in a six-arm olfactometer. These braconid wasps are both solitary endoparasitoids of lepidopteran larvae, but differ in geographical origin and host range. In choice experiments with odor blends from maize plants with fresh damage vs. blends from plants with old damage, inexperienced C. marginiventris showed a preference for the volatiles from freshly damaged leaves. No such preference was observed for inexperienced M. rufiventris. After an oviposition experience in hosts feeding on maize plants, C. marginiventris females were more attracted by a mixture of volatiles from fresh and old damage. Apparently, C. marginiventris has an innate preference for the odor of freshly damaged leaves, and this preference shifts in favor of a blend containing a mixture of green leaf volatiles plus terpenoids, after experiencing the latter blend in association with hosts. M. rufiventris responded poorly after experience and preferred fresh damage odors. Possibly, after associative learning, this species uses cues that are more directly related with the host presence, such as volatiles from host feces, which were not present in the odor sources offered in the olfactometer. The results demonstrate the complexity of the use of plant volatiles by parasitoids and show that different parasitoid species have evolved different strategies to exploit these signals.

The venom composition of the parasitic wasp Chelonus inanitus resolved by combined expressed sequence tags analysis and proteomic approach

Background Parasitic wasps constitute one of the largest group of venomous animals. Although some physiological effects of their venoms are well documented, relatively little is known at the molecular level on the protein composition of these secretions. To identify the majority of the venom proteins of the endoparasitoid wasp Chelonus inanitus (Hymenoptera: Braconidae), we have randomly sequenced 2111 expressed sequence tags (ESTs) from a cDNA library of venom gland. In parallel, proteins from pure venom were separated by gel electrophoresis and individually submitted to a nano-LC-MS/MS analysis allowing comparison of peptides and ESTs sequences. Results About 60% of sequenced ESTs encoded proteins whose presence in venom was attested by mass spectrometry. Most of the remaining ESTs corresponded to gene products likely involved in the transcriptional and translational machinery of venom gland cells. In addition, a small number of transcripts were found to encode proteins that share sequence similarity with well-known venom constituents of social hymenopteran species, such as hyaluronidase-like proteins and an Allergen-5 protein. An overall number of 29 venom proteins could be identified through the combination of ESTs sequencing and proteomic analyses. The most highly redundant set of ESTs encoded a protein that shared sequence similarity with a venom protein of unknown function potentially specific of the Chelonus lineage. Venom components specific to C. inanitus included a C-type lectin domain containing protein, a chemosensory protein-like protein, a protein related to yellow-e3 and ten new proteins which shared no significant sequence similarity with known sequences. In addition, several venom proteins potentially able to interact with chitin were also identified including a chitinase, an imaginal disc growth factor-like protein and two putative mucin-like peritrophins. Conclusions The use of the combined approaches has allowed to discriminate between cellular and truly venom proteins. The venom of C. inanitus appears as a mixture of conserved venom components and of potentially lineage-specific proteins. These new molecular data enrich our knowledge on parasitoid venoms and more generally, might contribute to a better understanding of the evolution and functional diversity of venom proteins within Hymenoptera.

The prospect of applying chemical elicitors and plant strengtheners to enhance the biological control of crop pests

An imminent food crisis reinforces the need for novel strategies to increase crop yields worldwide. Effective control of pest insects should be part of such strategies, preferentially with reduced negative impact on the environment and optimal protection and utilization of existing biodiversity. Enhancing the presence and efficacy of native biological control agents could be one such strategy. Plant strengthener is a generic term for several commercially available compounds or mixtures of compounds that can be applied to cultivated plants in order to ‘boost their vigour, resilience and performance’. Studies into the consequences of boosting plant resistance against pests and diseases on plant volatiles have found a surprising and dramatic increase in the plants' attractiveness to parasitic wasps. Here, we summarize the results from these studies and present new results from assays that illustrate the great potential of two commercially available resistance elicitors. We argue that plant strengtheners may currently be the best option to enhance the attractiveness of cultivated plants to biological control agents. Other options, such as the genetic manipulation of the release of specific volatiles may offer future solutions, but in most systems, we still miss fundamental knowledge on which key attractants should be targeted for this approach.

Less is More: Treatment with BTH and Laminarin Reduces Herbivore-Induced Volatile Emissions in Maize but Increases Parasitoid Attraction

Chemical plant strengtheners find increasing use in agriculture to enhance resistance against pathogens. In an earlier study, it was found that treatment with one such resistance elicitor, BTH (benzo-(1, 2, 3)-thiadiazole-7-carbothioic acid S-methyl ester), increases the attractiveness of maize plants to a parasitic wasp. This surprising additional benefit of treating plants with BTH prompted us to conduct a series of olfactometer tests to find out if BTH and another commercially available plant strengthener, Laminarin, increase the attractiveness of maize to three important parasitic wasps, Cotesia marginventris, Campoletis sonorensis, and Microplitis rufiventris. In each case, plants that were sprayed with the plant strengtheners and subsequently induced to release volatiles by real or mimicked attack by Spodoptera littoralis caterpillars became more attractive to the parasitoids than water treated plants. The elicitors alone or in combination with plants that were not induced by herbivory were not attractive to the wasps. Interestingly, plants treated with the plant strengtheners did not show any consistent increase in volatile emissions. On the contrary, treated plants released less herbivore-induced volatiles, most notably indole, which has been reported to interfere with parasitoid attraction. The emission of the sesquiterpenes (E)-β-caryophyllene, β-bergamotene, and (E)-β-farnesene was similarly reduced by the treatment. Expression profiles of marker genes showed that BTH and Laminarin induced several pathogenesis related (PR) genes. The results support the notion that, as yet undetectable and unidentified compounds, are of major importance for parasitoid attraction, and that these attractants may be masked by some of the major compounds in the volatile blends. This study confirms that elicitors of pathogen resistance are compatible with the biological control of insect pests and may even help to improve it.

Characterization of Chelonus inanitus polydnavirus segments: sequences and analysis, excision site and demonstration of clustering

Polydnaviruses (genera Ichnovirus and Bracovirus) have a segmented genome of circular double-stranded DNA molecules, replicate in the ovary of parasitic wasps and are essential for successful parasitism of the host. Here we show the first detailed analysis of various segments of a bracovirus, the Chelonus inanitus virus (CiV). Four segments were sequenced and two of them, CiV12 and CiV14, were found to be closely related while CiV14.5 and CiV16.8 were unrelated. CiV12, CiV14.5 and CiV16.8 are unique while CiV14 occurs also nested in another larger segment. All four segments are predicted to contain genes and predictions could be substantiated in most cases. Comparison with databases revealed no significant similarities at either the nucleotide or amino acid level. Inverted repeats with identities between 77% and 92% and lengths between 26 bp and 100 bp were found on all segments outside of predicted genes. Hybridization experiments indicate that CiV12 and CiV14 are both flanked by other virus segments, suggesting that proviral CiV segments are clustered in the genome of the wasp. The integration/excision site of CiV14 was analysed and compared to that of CiV12. On both termini of proviral CiV12 and CiV14 as well as in the excised circular molecule and the rejoined DNA a very similar repeat of 14 bp was found. A model to illustrate where the terminal repeats might recombine to yield the circular molecule is presented. Excision of CiV12 and CiV14 is restricted to the female and sets in at a very specific time-point in pupal-adult development.

Comparative genomics of metabolic networks of free-living and parasitic eukaryotes

Background Obligate endoparasites often lack particular metabolic pathways as compared to free-living organisms. This phenomenon comprises anabolic as well as catabolic reactions. Presumably, the corresponding enzymes were lost in adaptation to parasitism. Here we compare the predicted core metabolic graphs of obligate endoparasites and non-parasites (free living organisms and facultative parasites) in order to analyze how the parasites' metabolic networks shrunk in the course of evolution. Results Core metabolic graphs comprising biochemical reactions present in the presumed ancestor of parasites and non-parasites were reconstructed from the Kyoto Encyclopedia of Genes and Genomes. While the parasites' networks had fewer nodes (metabolites) and edges (reactions), other parameters such as average connectivity, network diameter and number of isolated edges were similar in parasites and non-parasites. The parasites' networks contained a higher percentage of ATP-consuming reactions and a lower percentage of NAD-requiring reactions. Control networks, shrunk to the size of the parasites' by random deletion of edges, were scale-free but exhibited smaller diameters and more isolated edges. Conclusions The parasites' networks were smaller than those of the non-parasites regarding number of nodes or edges, but not regarding network diameters. Network integrity but not scale-freeness has acted as a selective principle during the evolutionary reduction of parasite metabolism. ATP-requiring reactions in particular have been retained in the parasites' core metabolism while NADH- or NADPH-requiring reactions were lost preferentially.

The evolution of antennal courtship in diplazontine parasitoid wasps (Hymenoptera, Ichneumonidae, Diplazontinae)

Background As predicted by theory, traits associated with reproduction often evolve at a comparatively high speed. This is especially the case for courtship behaviour which plays a central role in reproductive isolation. On the other hand, courtship behavioural traits often involve morphological and behavioural adaptations in both sexes; this suggests that their evolution might be under severe constraints, for instance irreversibility of character loss. Here, we use a recently proposed method to retrieve data on a peculiar courtship behavioural trait, i.e. antennal coiling, for 56 species of diplazontine parasitoid wasps. On the basis of a well-resolved phylogeny, we reconstruct the evolutionary history of antennal coiling and associated morphological modifications to study the mode of evolution of this complex character system. Results Our study reveals a large variation in shape, location and ultra-structure of male-specific modifications on the antennae. As for antennal coiling, we find either single-coiling, double-coiling or the absence of coiling; each state is present in multiple genera. Using a model comparison approach, we show that the possession of antennal modifications is highly correlated with antennal coiling behaviour. Ancestral state reconstruction shows that both antennal modifications and antennal coiling are highly congruent with the molecular phylogeny, implying low levels of homoplasy and a comparatively low speed of evolution. Antennal coiling is lost on two independent occasions, and never reacquired. A zero rate of regaining antennal coiling is supported by maximum parsimony, maximum likelihood and Bayesian approaches. Conclusions Our study provides the first comparative evidence for a tight correlation between male-specific antennal modifications and the use of the antennae during courtship. Antennal coiling in Diplazontinae evolved at a comparatively low rate, and was never reacquired in any of the studied taxa. This suggests that the loss of antennal coiling is irreversible on the timescale examined here, and therefore that evolutionary constraints have greatly influenced the evolution of antennal courtship in this group of parasitoid wasps. Further studies are needed to ascertain whether the loss of antennal coiling is irreversible on larger timescales, and whether evolutionary constraints have influenced courtship behavioural traits in a similar way in other groups.

Identification of a host cell target for the thiazolide class of broad-spectrum anti-parasitic drugs

The thiazolide nitazoxanide (NTZ) and some derivatives exhibit considerable in vitro activities against a broad range of parasites, including the apicomplexans Neospora caninum and Toxoplasma gondii tachyzoites. In order to identify potential molecular targets for this compound in both parasites, RM4847 was coupled to epoxy-agarose and affinity chromatography was performed. A protein of approximately 35 kDa was eluted upon RM4847-affinity-chromatography from extracts of N. caninum-infected human foreskin fibroblasts (HFF) and non-infected HFF, but no protein was eluted when affinity chromatography was performed with T. gondii or N. caninum tachyzoite extracts. Mass spectrometry analysis identified the 35 kDa protein as human quinone reductase NQO1 (P15559; QR). Within 8h after infection of HFF with N. caninum tachyzoites, QR transcript expression levels were notably increased, but no such increase was observed upon infection with T. gondii tachyzoites. Treatment of non-infected HFF with RM4847 did also lead to an increase of QR transcript levels. The enzymatic activity of 6-histidine-tagged recombinant QR (recQR) was assayed using menadione as a substrate. The thiazolides NTZ, tizoxanide and RM4847 inhibited recQR activity on menadione in a concentration-dependent manner. Moreover, a small residual reducing activity was observed when these thiazolides were offered as substrates.

The use of light- and electron microscopy for studies on the cell- and molecular biology of parasites and parasitic diseases

Lightmicroscopical (LM) and electron microscopi cal (EM) techniques, have had a major influence on the development and direction of cell biology, and particularly also on the investigation of complex host-parasite relationships. Earlier, microscopy has been rather descriptive, but new technical and scientific advances have changed the situation. Microscopy has now become analytical, quantitative and three-dimensional, with greater emphasis on analysis of live cells with fluorescent markers. The new or improved techniques that have become available include immunocytochemistry using immunogold labeling techniques or fluorescent probes, cryopreservation and cryosectioning, in situ hybridization, fluorescent reporters for subcellular localization, micro-analytical methods for elemental distribution, confocal laser scanning microscopy, scanning tunneling microscopy and live-imaging. Taken together, these tools are providing both researchers and students with a novel and multidimensional view of the intricate biological processes during parasite development in the host.

Travel medicine of parasitic diseases in the dog

Companion animals are increasingly brought along by their owners to foreign countries. Thus, small animal travel medicine is becoming more important. The field includes both prophylaxis and metaphylaxis against various infectious diseases, as well as their diagnosis and treatment. Dogs returning from Southern Europe, but also from more tropical regions, may be infected with exotic pathogens. In addition, imported pedigree or working dogs, and especially stray dogs imported through welfare organisations, are at high risk.The present overview summarises the clinical and practical aspects of exotic parasitic diseases that may affect such dogs, and the risk of such diseases becoming autochthonously transmitted in Switzerland. Furthermore, the zoonotic potential of these infections will be considered.

Neospora caninum: functional inhibition of protein disulfide isomerase by the broad-spectrum anti-parasitic drug nitazoxanide and other thiazolides

Nitazoxanide (NTZ) and several NTZ-derivatives (thiazolides) have been shown to exhibit considerable anti-Neospora caninum tachyzoite activity in vitro. We coupled tizoxanide (TIZ), the deacetylated metabolite, to epoxy-agarose-resin and performed affinity chromatography with N. caninum tachyzoite extracts. Two main protein bands of 52 and 43kDa were isolated. The 52kDa protein was readily recognized by antibodies directed against NcPDI, and mass spectrometry confirmed its identity. Poly-histidine-tagged NcPDI-cDNA was expressed in Escherichia coli and recombinant NcPDI (recNcPDI) was purified by Co2+-affinity chromatography. By applying an enzyme assay based on the measurement of insulin crosslinking activity, recNcPDI exhibited properties reminiscent for PDIs, and its activity was impaired upon the addition of classical PDI inhibitors such as bacitracin (1-2mM), para-chloromercuribenzoic acid (0.1-1mM) and tocinoic acid (0.1-1mM). RecNcPDI-mediated insulin crosslinking was inhibited by NTZ (5-100 microM) in a dose-dependent manner. In addition, the enzymatic activity of recNcPDI was inhibited by those thiazolides that also affected parasite proliferation. Thus, thiazolides readily interfere with NcPDI, and possibly also with PDIs from other microorganisms susceptible to thiazolides.