A survey of Xylella fastidiosa-carrying glassy-winged sharpshooters (Homalodisca vitripennis) across Texas grape vineyards and variations between their internal microbial communities

Because of its role in causing Pierce’s Disease (PD) by the transmission of Xylella fastidiosa spp. fastidiosa to grapevine, the glassy-winged sharpshooter (GWSS; Homalodisca vitripennis) is, economically, one of most important insect pests to the wine and table grape industries in Texas, California, and Florida. Through analysis of bacterial communities living within GWSS samples, an effort is being made to understand the factors influencing the differences between individual insects and groups of individuals in addition to any influence the structure of these communities have on the presence or absence of X. fastidiosa within GWSS. In this work, 25 GWSS were collected from 7 vineyards across Texas, total genomic DNA was extracted from the abdomen and thorax of each sample, and bacterial 16S rRNA sequences of associated bacterial sequences were amplified by PCR and sequenced by 454 pyrosequencing. 16S sequences from each sample were clustered based on similarity, and relationships and trends in bacterial composition within and between samples were analyzed. In addition, total genomic DNA was extracted from the foregut contents of ~200 GWSS samples collected from the same 7 vineyards, and the subspecies of Xylella fastidiosa within the foregut extract of each insect were determined through qrt-PCR amplification with Xylella fastidiosa gyrase B specific primers and high resolution melting temperature analysis. The parameters of analysis of 454 pyrosequencing results were adjusted to determine whether the presence of X. fastidiosa (ssp. fastidiosa or not) or certain environmental variables are correlated with differences in bacterial communities between GWSS samples. Finally, bacterial communities were reanalyzed including only those sequences from primary GWSS endosymbionts.