Comparing the spatial genetic structures of the Flavescence dorée phytoplasma and its leafhopper vector Scaphoideus titanus.

The Nearctic leafhopper Scaphoideus titanus Ball is the vector of "Flavescence dorée" phytoplasma (FDp) in European vineyards. We studied the genetic diversity and structure of S. titanus populations in France and of the FDp they carried. A total of 621 S. titanus individuals, sampled in 24 FDp-infected and uninfected vineyards, were genotyped using seven polymorphic microsatellite loci. The mean observed heterozygosity in S. titanus populations was between 0.364 and 0.548. There was evidence of only a low level of population genetic differentiation (mean F(ST)=0.027) suggesting that there is long-distance gene flow between S. titanus populations. This may be a consequence of the high migration capacity of the vector associated with large effective population size and, at least in part, of passive dispersion over long distances by the transport of grapevine-planting material carrying eggs. For each insect, FDp was detected and typed by nested-PCR followed by RFLP and sequencing of a 674 bp fragment of the FDp map gene. Twelve of the 24 populations were found to be infected by FDp, with the percentage of infected individuals varying from 3% to 29%. FDp isolates were classified into two FDp genetic clusters (FD1 and FD2), which differed by 12-13 SNPs. FD1 genotypes were detected in the insect populations at two sites and the FD2 genotypes in the other ten populations. Both FD1 and FD2 genotypes were found to be transmitted by the insect. No significant relationship was found between the genetic structure of these French S. titanus populations and the distribution of the various FDp strain types they carried. Nevertheless, overall genetic differentiation between FDp-infected and healthy S. titanus "subsamples" was found to be significantly higher than zero. These results suggest that FDp-infected S. titanus individuals are more philopatric (disperse less) than healthy S. titanus.

Isolation and characterization of polymorphic microsatellite loci in the green leafhopper Empoasca vitis Goethe (Homoptera).

Eight dinucleotide microsatellite loci were isolated and characterized within the green leafhopper Empoasca vitis (Goethe) using an enrichment cloning procedure. Primers were tested on 171 individuals collected in the southwest of France from the vine plants. The identified loci were polymorphic, with allelic diversity ranging from two to 18 alleles per locus. Observed heterozygosities were from 0.021 to 0.760. These microsatellite markers should prove to be a useful tool for estimating the population genetic structure, host-plant specialization and migration capacity of this insect.

Predominance of sexual reproduction in Romanian populations of the aphid Sitobion avenae inferred from phenotypic and genetic structure.

Models of coexistence of sexual and asexual lineages in aphids assume that obligate parthenogenetic lineages predominate in areas with mild winter climate because of their high reproductive output, while sexual lineages predominate in areas with severe winter because they produce eggs resistant to frost. To validate this hypothesis in natural conditions, the reproductive mode of populations of the aphid Sitobion avenae was assessed in two very contrasting climatic situations, Romania (severe winter) and Western France (mild winter). To achieve this, reproductive modes were inferred from both (1) the population composition in sexual and asexual forms in autumn, and (2) the genetic structure of Romanian and French populations of S. avenae using microsatellite markers. Romanian populations encompassed a high proportion of sexual forms and were characterised by a very high genotypic diversity and low linkage disequilibrium. In constrast, the French population showed frequent linkage disequilibria, low genetic diversity, and high level of clonal amplification with two asexual genotypes representing over 60% of the sample. In agreement with the model's predictions, these results clearly indicate that sexual reproduction in S. avenae is predominant under the continental climate of Romania, while asexual lineages prevail under the oceanic climate of Western France.

Two-dimensional electrophoresis of proteins discriminates aphid clones of Sitobion avenae differing in BYDV-PAV transmission.

Aset of 39 F1 Sitobion avenae clones was obtained by selfing a poorly efficient BYDV-PAV vector clone. These clones were genetically typed by 11 microsatellite loci, and tested for BYDV-PAV4 transmission to barley. The 39 clones displayed a continuum in transmission percentages, from 0% to 88% with a significant clone effect. From this set, two highly efficient (HEV) and two poorly efficient (PEV) vectoring clones were more precisely characterized for transmission of two other PAV isolates. The molecular bases of the lower transmissibility of BYDV-PAV4 by PEV clones and of the aphid vectoring properties were investigated respectively by comparing the sequences corresponding to structural proteins (CP and RTD) of BYDV, and by using proteomic analysis of aphids in two dimensional electrophoresis (2-DE) with immobilized pH gradients (IPG) after an improved protein extraction. Four residues specific to BYDV-PAV4 located in the CP sequence (A(24) and L(130)) or in the RTD region (M(334) and S(456)) could be responsible for the lower transmissibility of this isolate by PEV clones. Among a total of 2150 well-resoluted spots scored on S. avenae proteinic pattern, only twelve proteins were qualitatively or quantitatively different between clones. Four out of them discriminated HEV and PEV groups.