Nicolas Sauvion

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.

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.

Melon resistance to the aphid Aphis gossypii: behavioural analysis and chemical correlations with nitrogenous compounds

In the melon, the Vat (monogenic, dominant) resistance gene governs both an antixenotic reaction to the melon aphid Aphis gossypii Glover (Homoptera, Aphididae) and a resistance to non‐persistent virus transmission, restricted to this vector species. We investigated the behavioural features and tissue localisation of the antixenosis resistance by the electrical penetration graph technique (EPG, DC system). We also compared the chemical composition in amino compounds and proteins of the phloem sap collected from two isogenic lines of melon (Cucumis melo L.), carrying the Vat gene or not. All behavioural and chemical data indicated that this resistance is constitutive. EPG analysis clearly showed that access to phloem, although delayed by alterations in pathway activities, was not impaired in terms of frequency of access or initiation of feeding. The most striking feature was, however, a very reduced duration of ingestion from phloem of resistant plants, making this compartment one of the tissues where the effects of the Vat gene are unambiguously expressed. This was confirmed by clear differential activity of phloem extracts in artificial no‐choice bioassays. Chemical analyses have shown that phloem saps from the two isogenic lines were extremely similar in profiles of ninhydrin positive compounds, and contained a low total amount of free amino acids (less than 10 mM). Out of more than 40 distinguishable peaks in the chromatograms (protein and non‐protein amino acids, as well as small peptides), only five differentiated the two genotypes. Two of them were increased in the resistant genotype: glutamic acid and a major unknown peak, probably a non‐protein amino acid (different from β pyrazolyl‐alanine, a Cucumis‐specific amino acid). The three others were depressed in resistant plants, and included the sulphur amino acid cystine and a peptide peak partly composed of the cysteine‐containing peptide glutathione (reduced form). Sap collection also showed that phloem exudation rates, as well as total protein and glutathione levels, were depressed in phloem sap from resistant plants. Such data are all indicative of a modified phloem‐sealing physiology, linked to sulfhydryl oxidation processes, in plants carrying the Vat gene. The originality of the mechanism of Vat resistance to aphids is discussed.

Effects of jackbean lectin (ConA) on the feeding behaviour and kinetics of intoxication of the pea aphid, Acyrthosiphon pisum

Mannose‐binding lectins were shown to be useful in creating transgenic plants resistant to insects, including many phloem‐feeding Hemiptera. Before these plants can be used extensively, it is important to understand how these lectins exert their toxic effects on the target organisms. We investigated the feeding alterations induced by presenting the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae), with a diet containing the lectin from Canavalia ensiformis (ConA). A series of behavioural experiments were carried out to detect potential sensory mediation of lectin activity. Choice tests performed with a 400 µg ml−1 ConA diet (3.7 µm of native tetramer) showed that A. pisum quickly rejected the ConA diet, but that this reaction was not typical of a sensory‐mediated phagodeterrent effect. In addition, the aphids did not develop a conditioned taste aversion to the lectin. Diet uptake was evaluated using a radioactive tracer (14C‐methylated inulin), and showed depression of ingestion only after 16 h at 200 µg ml−1 or after 8 h at 400 µg ml−1 ConA. This effect was reversible under our test conditions. No evidence was obtained for early detection of the lectin, even by intoxicated aphids. An electrical penetration graph technique was adapted to artificial diets and provided short‐term continuous analysis on feeding/probing events. At the 400 µg ml−1 level, adults were affected and had reduced ingestion durations as early as in the first 4 h of contact, but experienced an adaptation to the behavioural alterations induced by lectin feeding. Overall, feeding deterrency following exposure to mannose lectins appeared to be a consequence of intoxication, and not due to a sensory mediated process.

Biological and chemical characteristics of a genetic resistance of melon to the melon aphid

Host plant resistance to pests is an important component of integrated control strategies. Due to the specialised phloem-feeding of aphids and many other hemipteran insect pests, which necessitates specific chemical treatments and control of virus transmission, this genetic strategy seems particularly attractive for this insect group. In the melon Cucumis melo L., a gene labelled vat, for virus aphid transmission (Pitrat & Lecoq, 1982), brings both a resistance to the melon aphid Aphis gossypii Glover and a resistance to the transmission by this vector of many non-persistent viruses. This gene induces both an antixenotic response to adults and an apparent antibiosis towards A. gossypii larvae. The purpose of the experiments reported here was to investigate the mechanisms of this resistance (mechanical, chemical, tissue localisation...) and to characterize any chemical difference between genotypes differing by the presence/absence of the vat gene.

Molecular Test to Assign Individuals within the Cacopsylla pruni Complex

Crop protection requires the accurate identification of disease vectors, a task that can be made difficult when these vectors encompass cryptic species. Here we developed a rapid molecular diagnostic test to identify individuals of Cacopsylla pruni (Scopoli, 1763) (Hemiptera: Psyllidae), the main vector of the European stone fruit yellows phytoplasma. This psyllid encompasses two highly divergent genetic groups that are morphologically similar and that are characterized by genotyping several microsatellite markers, a costly and time-consuming protocol. With the aim of developing species-specific PCR primers, we sequenced the Internal Transcribed Spacer 2 (ITS2) on a collection of C . pruni samples from France and other European countries. ITS2 sequences showed that the two genetic groups represent two highly divergent clades. This enabled us to develop specific primers for the assignment of individuals to either genetic group in a single PCR, based on ITS2 amplicon size. All previously assigned individuals yielded bands of expected sizes, and the PCR proved efficient on a larger sample of 799 individuals. Because none appeared heterozygous at the ITS2 locus (i.e., none produced two bands), we inferred that the genetic groups of C . pruni , whose distribution is partly sympatric, constitute biological species that have not exchanged genes for an extended period of time. Other psyllid species (Cacopsylla, Psylla, Triozidae and Aphalaridae) failed to yield any amplicon. These primers are therefore unlikely to produce false positives and allow rapid assignment of C . pruni individuals to either cryptic species.

Mixed xylem and phloem sap ingestion in sheath-feeders as normal dietary behavior: Evidence from the leafhopper Scaphoideus titanus

In phytophagous piercing-sucking insects, salivary sheath-feeding species are often described as xylem- or phloem-sap feeding specialists. Because these two food sources have very different characteristics, two feeding tactics are often associated with this supposed specialization. Studying the feeding behavior of insects provides substantial information on their biology, ecology, and evolution. Furthermore, study of feeding behavior is of primary importance to elucidate the transmission ability of insects that act as vectors of plant pathogens. In this study, we compared the durations of ingestion performed in xylem versus phloem by a leafhopper species, Scaphoideus titanus Ball, 1932. This was done by characterizing and statistically analyzing electrical signals recorded using the electropenetrography technique, derived from the feeding behaviors of males and females. We identified three groups of S. titanus based on their feeding behavior: 1) a group that reached the phloem quickly and probed for a longer time in phloem tissue than the other groups, 2) a group that reached the xylem quickly and probed for a longer time in xylem tissue than the other groups, and 3) a group where individuals did not ingest much sap. In addition, the numbers and durations of waveforms representing ingestion of xylem and phloem saps differed significantly depending on the sex of the leafhopper, indicating that the two sexes exhibit different feeding behaviors. Males had longer phloem ingestion events than did females, which indicates that males are greater phloem feeders than females. These differences are discussed, specifically in relation to hypotheses about evolution of sap feeding and phytoplasma transmission from plant to plant.

Nine polymorphic microsatellite loci from the psyllid Cacopsylla pruni (Scopoli), the vector of European stone fruit yellows

Cacopsylla pruni is the vector of European stone fruit yellows, a quarantine disease of Prunus trees. Nine polymorphic microsatellite markers were developed from enriched DNA libraries. Allelic variability was assessed in a collection of 149 females obtained from five localities covering a large geographical area in France. The number of detected alleles ranged from 8 to 37. Within the localities, observed and expected heterozygosities averaged across loci ranged from 0.39 to 0.55, and from 0.68 to 0.81, respectively. A heterozygote deficiency was detected for almost all loci, possibly due to a high null allele frequency. Other possible causes of the homozygote excess (mode of reproduction, inbreeding, assortative mating or Wahlund effect) are discussed. These variable microsatellite loci can provide tools to assess overall genetic variation in this important vector species. They will be used to search for population structure and migration patterns of C. pruni.

A framework for estimating the effects of sequential reproductive barriers: implementation using Bayesian models with field data from cryptic species

Determining how reproductive barriers modulate gene flow between populations represents a major step towards understanding the factors shaping the course of speciation. Although many indices quantifying reproductive isolation (RI) have been proposed, they do not permit the quantification of cross direction-specific RI under varying species frequencies and over arbitrary sequences of barriers. Furthermore, techniques quantifying associated uncertainties are lacking, and statistical methods unrelated to biological process are still preferred for obtaining confidence intervals and p-values. To address these shortcomings, we provide new RI indices that model changes in gene flow for both directions of hybridization, and we implement them in a Bayesian model. We use this model to quantify RI between two species of the psyllid Cacopsylla pruni based on field genotypic data for mating individuals, inseminated spermatophores and progeny. The results showed that pre-insemination isolation was strong, mildly asymmetric and undistinguishably different between study sites despite large differences in species frequencies; that post-insemination isolation strongly affected the more common hybrid type; and that cumulative isolation was close to complete. In the light of these results, we discuss how these developments can strengthen comparative RI studies. Author contributions JP and NS initiated the study and obtained biological data. JP and DRJP developed the porosity-based approach. DRJP conceived the Bayesian implementation and code. JP, DRJP and NS wrote the manuscript. Data availability Mitochondrial sequence data will be available at Genbank, source code is available at xxx.