Population genetics and bacterial endosymbionts of Aphrophoridae putative vectors of Xylella fastidiosa and other Auchenorrhyncha species in Greece

Abstract: Xylella fastidiosa is a Gram-negative bacterium which is responsible for many diseases in economically important crops such as olives. Its vectors are xylem fluid-feeding insects of the suborder Auchenorrhyncha (Order: Hemiptera). Philaenus spumarius is the main vector of the bacterium in Europe, responsible for the spread of X. fastidiosa in Apulia region of South-East Italy causing the olive quick decline syndrome in olive orchards. The knowledge of the distribution and the genetic structure of the potential insect vectors of X. fastidiosa or other pathogens is important in preventing such epidemics. Here, we investigated the genetic diversity and the infection status by five secondary symbionts of three Aphrophoridae (P. spumarius, Neophilaenus campestris, P. signatus) putative vectors of Xylella collected from olive orchards in 19 regions of Greece. Additionally, the polymorphism of two Deltocephalinae species (Euscelidius variegatus and Euscelis lineolatus) collected from ground vegetation of the olive orchards was also investigated. The genetic polymorphism of the sampled populations was determined based on the mitochondrial (mt) cytochrome c oxidase subunit I (COI) and cytochrome b (cytb) genes. In total, 238 COI and cytb sequences obtained in the present study were analyzed together with 91 additional from GenBank. Analysis of the mtDNA sequences revealed high polymorphism Greek P. spumarius, (26 haplotypes for COI and 52 for cytb) and N. campestris (13 and 7 haplotypes for COI and cytb, respectively). In addition, the presence of secondary symbionts known for their interference in shaping the genetic structure of insect species was determined by PCR using species-specific primers for Hamiltonella, Rickettsia, Arsenophonus, Cardinium and Wolbachia. The infection statuses ranged from 0 to 100 % depending on the sampled species and the secondary symbiont detected. Every species tested, harbored at least one secondary endosymbiont with Arsenophonus being found as the most prevalent one (overall 9.2 % infection frequency). In P. spumarius, only Hamiltonella, Rickettsia and Wolbachia were present in equally low frequencies (4 %). Regarding N. campestris, Arsenophonus had the highest frequency (35 %) whereas Cardinium was not found in any individual of N. campestris. Moreover, to determine the overall bacterial composition of P. spumarius, we obtained Next Generation Sequencing data of 16S metagenome by examining different parts of the insect body. The comparative analysis of populations of P. spumarius from several regions in Greece revealed constant differences in bacterial composition between the head and the rest of the insect body.