Yvan Rahbé

Immunity and other defenses in pea aphids, Acyrthosiphon pisum

BackgroundRecent genomic analyses of arthropod defense mechanisms suggest conservation of key elements underlying responses to pathogens, parasites and stresses. At the center of pathogen-induced immune responses are signaling pathways triggered by the recognition of fungal, bacterial and viral signatures. These pathways result in the production of response molecules, such as antimicrobial peptides and lysozymes, which degrade or destroy invaders. Using the recently sequenced genome of the pea aphid (Acyrthosiphon pisum), we conducted the first extensive annotation of the immune and stress gene repertoire of a hemipterous insect, which is phylogenetically distantly related to previously characterized insects models.ResultsStrikingly, pea aphids appear to be missing genes present in insect genomes characterized to date and thought critical for recognition, signaling and killing of microbes. In line with results of gene annotation, experimental analyses designed to characterize immune response through the isolation of RNA transcripts and proteins from immune-challenged pea aphids uncovered few immune-related products. Gene expression studies, however, indicated some expression of immune and stress-related genes.ConclusionsThe absence of genes suspected to be essential for the insect immune response suggests that the traditional view of insect immunity may not be as broadly applicable as once thought. The limitations of the aphid immune system may be representative of a broad range of insects, or may be aphid specific. We suggest that several aspects of the aphid life style, such as their association with microbial symbionts, could facilitate survival without strong immune protection.

Transgenic potato plants with enhanced resistance to the peach-potato aphid Myzus persicae

Potato plants (Solanum tuberosum) cv. Desireé were transformed with the genes encoding the proteins bean chitinase (BCH), snowdrop lectin (GNA) and wheat α‐amylase inhibitor (WAI) under the control of the constitutive CaMV 35S promoter. Transgenic plants with detectable levels of foreign RNA were then selected for further characterisation with respect to protein expression levels by immunodot blot analysis using polyclonal antibodies raised against the respective protein. With the exception of WAI, plants expressing high levels of RNA, expressed correspondingly high levels of the foreign protein (1.5–2.0% of the total soluble protein). Although high levels of WAI mRNA were detected in some of the transformants, the protein could not be detected. On the bases of expression levels, two lines, designated PWG6#85 (transformed with the double construct WAI/GNA) and PBG6#47 (transformed with the double construct BCH/GNA), were selected for testing in aphid trials for enhanced levels of resistance.

Toxicity of lectins and processing of ingested proteins in the pea aphid Acyrthosiphon pisum

Acute toxicity of thirty lectins was tested against the pea aphid Acyrthosiphon pisum (Harris) (Homoptera, Aphididae: Macrosiphini). Activity was measured on artificial diets containing moderate concentrations of lectins (10–250 μg/ml) by scoring mortality and growth inhibition over the whole nymphal period (7 days at 20°C). Most of the proteins tested exhibited low toxicity, but some induced significant mortality; these included the lectins from jackbean (Concanavalin A), amaranth, lentil and snowdrop. There was no direct correlation between toxicity and sugar specificity of the lectin; however, many mannose‐binding lectins were toxic towards A. pisum. Concanavalin A was also tested on five other aphid species (Aphis gossypii, Aulacortum solani, Macrosiphum euphorbiae, Macrosiphum albifrons and Myzus persicae) at concentrations between 10–1500 μg/ml. Mortality was very variable from one species to another. Strong growth inhibition invariably occurred within this concentration range, although dose‐response curves differed substantially between aphid species. The peptidase complement of A. pisums digestive tract was also investigated, as well as the oral toxicity of some protease inhibitors (PIs) to this aphid. Most protein PIs were inactive, and no part of the digestive tract contained detectable amounts of endo‐protease activity. This is in contrast to the strong amino‐peptidase activity which was shown to occur predominantly in the midgut and crop portions of the digestive tract. The potential of lectins in transgenic crops to confer Host‐Plant Resistance to aphids is discussed.

Midgut adaptation and digestive enzyme distribution in a phloem feeding insect, the pea aphid Acyrthosiphon pisum.

Transmission electron micrographs of the pea aphid midgut revealed that its anterior region has cells with an apical complex network of lamellae (apical lamellae) instead of the usual regularly-arranged microvilli. These apical lamellae are linked to one another by trabeculae. Modified perimicrovillar membranes (MPM) are associated with the lamellae and project into the lumen. Trabeculae and MPM become less conspicuous along the midgut. The most active A. pisum digestive enzymes are membrane-bound. An aminopeptidase (APN) is described elsewhere. An alpha-glucosidase (alpha-Glu) has a molecular mass of 72 kDa, pH optimum 6.0 and catalyzes in vitro transglycosylations in the presence of an excess of the substrate sucrose. There is a major cysteine proteinase activity (CP) on protein substrates that has a molecular mass of 40 kDa, pH optimum 5.5, is inhibited by E-64 and chymostatin and is activated by EDTA+cysteine. The enzyme is more active against carbobenzoxy-Phe-Arg-4-methylcoumarin-7-amide (ZFRMCA) than against ZRRMCA. These features identify the purified CP as a cathepsin-L-like cysteine proteinase. Most CP is found in the anterior midgut, whereas alpha-Glu and APN predominate in the posterior midgut. With the aid of antibodies, alpha-Glu and CP were immunolocalized in cell vesicles and MPM, whereas APN was localized in vesicles, apical lamellae and MPM. The data suggest that the anterior midgut is structurally reinforced to resist osmotic pressures and that the transglycosylating alpha-Glu, together with CP and APN are bound to MPM, thus being both distributed over a large surface and prevented from excretion with honeydew. alpha-Glu frees glucose from sucrose without increasing the osmolarity, and CP and APN may process toxins or other proteins occasionally present in phloem.

Effects of the cysteine protease inhibitor oryzacystatin (OC-I) on different aphids and reduced performance of Myzus persicae on OC-I expressing transgenic oilseed rape

Abstract When administered in artificial diets, the cysteine protease inhibitor (PI) oryzacystatin I (OC-I) induced moderate but significant growth inhibition on the pea aphid (Acyrthosiphon pisum Harris), the cotton/melon aphid (Aphis Gossypii Glover) and the peach potato aphid (Myzus persicae Sulzer). In transgenic oilseed rape plants (Brassica napus L. cv. Drakkar) expressing OC-I under the control of the CaMV-35S RNA promoter, oryzacystatin was detected in both leaves and phloem sap. Transgenic plants from three independent homozygous lines were used to test the effect of in planta-expression of OC-I on M. persicae. Mean adult weight and fecundity were significantly reduced, and aphid biomass produced in 2 weeks was decreased by 25–40% for aphids fed transgenic plants, when compared to those fed control plants. The effects of OC-I on M. persicae were correlated with the decrease of a major cathepsin L/H-type cysteine protease activity, detected in whole insect extracts. Immuno-histological analysis showed OC-I labeling along the gut epithelium, but also its association with aphid bacteriocytes and oenocytes. These results suggest that OC-I affects M. persicae through digestive tract targets, but also by reaching the haemolymph, thereby inhibiting extra-digestive proteolytic activities and interacting with functions related to aphid reproduction. Overall, it appears that PIs can display deleterious effects against phloem-feeding insects in addition to their activity on leaf-feeding insects.

Effects of GNA and other mannose binding lectins on development and fecundity of the peach‐potato aphid Myzus persicae

Three mannose‐binding lectins were assayed in artificial diets for their toxic and growth‐inhibitory effects on nymphal development of the peach‐potato aphid Myzus persicae. The snowdrop (Galanthus nivalis) lectin GNA was the most toxic, with an induced nymphal mortality of 42% at 1500 μg ml−1 (30 μM) and an IC50 (50% growth inhibition) of 630 μg ml−1 (13 μM). The daffodil (Narcissus pseudonarcissus) lectin NPA and a garlic (Allium sativum) lectin ASA induced no significant mortality in the range 10–1500 μg ml−1, but did result in growth inhibition of 59% (NPA) and 26% (ASA) at 1500 μg ml−1 (40 μM for NPA, 63 μM for ASA). All three lectins were responsible for a slight but significant growth stimulation when ingested at 10 μg ml−1, reaching + 26%, + 18% and + 11% over the control values for the garlic lectin, the daffodil lectin and the snowdrop lectin, respectively. GNA, as well as the glucose/mannose binding lectin Concanavalin A, were also provided at sublethal doses throughout the life cycle of the aphids, and effects on adult performance were monitored. Adult survival was not significantly altered, but both lectins adversely affected total fecundity and the dynamics of reproduction, resulting in significant reduction in calculated rms (population intrinsic rate of natural increase) on lectin‐containing diets. These effects are discussed in relation to the use of transgenic plants expressing these toxic lectins for potential control of aphid populations.

Large-scale gene discovery in the pea aphid Acyrthosiphon pisum (Hemiptera)

Aphids are the leading pests in agricultural crops. A large-scale sequencing of 40,904 ESTs from the pea aphid Acyrthosiphon pisum was carried out to define a catalog of 12,082 unique transcripts. A strong AT bias was found, indicating a compositional shift between Drosophila melanogaster and A. pisum. An in silico profiling analysis characterized 135 transcripts specific to pea-aphid tissues (relating to bacteriocytes and parthenogenetic embryos). This project is the first to address the genetics of the Hemiptera and of a hemimetabolous insect.

Protein toxicity to aphids: an in vitro test on Acyrthosiphon pisum

Recent progress in plant transformation for insect resistance has increased the interest in the potential toxicity of proteins towards insect pests. While studies have been targeted to a large array of insect species, phloem‐feeding Homoptera have not been investigated yet. The paper describes a routine test for screening toxicity and growth inhibition of purified proteins in artificial diets on Acyrthosiphon pisum (Harris). Twenty‐five commercially available proteins of different classes were tested and compared to some non‐protein chemicals (an insecticide, an antibiotic …). A. pisum proved to be very sensitive to all proteases tested and to some venoms with general cytolytic properties. A plant lectin, concanavalin A, displayed significant toxicity and growth inhibition, while various proteins such as a soybean proteinase inhibitor, a chitinase, and bovine serum albumin showed measurable impairments of growth only at higher dose (≥250 μg.ml−1). Some proteins were without short‐term effect on A. pisum physiology. The influence of these results on aphid‐plant interactions are discussed.

The Phytopathogen Dickeya dadantii (Erwinia chrysanthemi 3937) Is a Pathogen of the Pea Aphid

ABSTRACT Dickeya dadantii (Erwinia chrysanthemi) is a phytopathogenic bacterium causing soft rot diseases on many crops. The sequencing of its genome identified four genes encoding homologues of the Cyt family of insecticidal toxins from Bacillus thuringiensis, which are not present in the close relative Pectobacterium carotovorum subsp. atrosepticum. The pathogenicity of D. dadantii was tested on the pea aphid Acyrthosiphon pisum, and the bacterium was shown to be highly virulent for this insect, either by septic injury or by oral infection. The lethal inoculum dose was calculated to be as low as 10 ingested bacterial cells. A D. dadantii mutant with the four cytotoxin genes deleted showed a reduced per os virulence for A. pisum, highlighting the potential role of at least one of these genes in pathogenicity. Since only one bacterial pathogen of aphids has been previously described (Erwinia aphidicola), other species from the same bacterial group were tested. The pathogenic trait for aphids was shown to be widespread, albeit variable, within the phytopathogens, with no link to phylogenetic positioning in the Enterobacteriaceae. Previously characterized gut symbionts from thrips (Erwinia/Pantoea group) were also highly pathogenic to the aphid, whereas the potent entomopathogen Photorhabdus luminescens was not. D. dadantii is not a generalist insect pathogen, since it has low pathogenicity for three other insect species (Drosophila melanogaster, Sitophilus oryzae, and Spodoptera littoralis). D. dadantii was one of the most virulent aphid pathogens in our screening, and it was active on most aphid instars, except for the first one, probably due to anatomical filtering. The observed difference in virulence toward apterous and winged aphids may have an ecological impact, and this deserves specific attention in future research.

Xerocomus chrysenteron lectin: identification of a new pesticidal protein.

Xerocomus chrysenteron is an edible mushroom with insecticidal properties. In an earlier work, we found that proteins are responsible for this toxicity. Here we describe the purification of a approximately 15 kDa lectin, named XCL, from the mushroom. Its cDNA and gDNA were cloned by PCR strategies and a recombinant form was expressed in Escherichia coli. Sequence alignments and sugar specificity showed that this protein is the third member of a new saline-soluble lectin family present in fungi. This protein, either purified from mushroom or expressed in vitro in E. coli, was found to be toxic to some insects, such as the dipteran Drosophila melanogaster and the hemipteran, Acyrthosiphon pisum. The lectin possesses a high insecticidal activity compared to lectin isolated from leguminosae (Lathyrus ochrus) or from the snowdrop (Galanthus nivalis).