Anterior foregut microbiota of the glassy-winged sharpshooter explored using deep 16S rRNA gene sequencing from individual insects.

Elizabeth E Rogers,Elaine A Backus

doi:10.1371/journal.pone.0106215

Abstract

The glassy-winged sharpshooter (GWSS) is an invasive insect species that transmits Xylella fastidiosa, the bacterium causing Pierce's disease of grapevine and other leaf scorch diseases. X. fastidiosa has been shown to colonize the anterior foregut (cibarium and precibarium) of sharpshooters, where it may interact with other naturally-occurring bacterial species. To evaluate such interactions, a comprehensive list of bacterial species associated with the sharpshooter cibarium and precibarium is needed. Here, a survey of microbiota associated with the GWSS anterior foregut was conducted. Ninety-six individual GWSS, 24 from each of 4 locations (Bakersfield, CA; Ojai, CA; Quincy, FL; and a laboratory colony), were characterized for bacteria in dissected sharpshooter cibaria and precibaria by amplification and sequencing of a portion of the 16S rRNA gene using Illumina MiSeq technology. An average of approximately 150,000 sequence reads were obtained per insect. The most common genus detected was Wolbachia; sequencing of the Wolbachia ftsZ gene placed this strain in supergroup B, one of two Wolbachia supergroups most commonly associated with arthropods. X. fastidiosa was detected in all 96 individuals examined. By multilocus sequence typing, both X. fastidiosa subspecies fastidiosa and subspecies sandyi were present in GWSS from California and the colony; only subspecies fastidiosa was detected in GWSS from Florida. In addition to Wolbachia and X. fastidiosa, 23 other bacterial genera were detected at or above an average incidence of 0.1%; these included plant-associated microbes (Methylobacterium, Sphingomonas, Agrobacterium, and Ralstonia) and soil- or water-associated microbes (Anoxybacillus, Novosphingobium, Caulobacter, and Luteimonas). Sequences belonging to species of the family Enterobacteriaceae also were detected but it was not possible to assign these to individual genera. Many of these species likely interact with X. fastidiosa in the cibarium and precibarium.

Highlights

Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae: Cicadellinae) or glassy-winged sharpshooter (GWSS) is a leafhopper that exclusively ingests plant xylem fluid from a wide range of host plants [1]
FastQ files were exported from MiSeq Reporter and Genomics Workbench was used to align paired ends, remove primer sequence, and group reads into operational taxonomic units (OTUs) of at least 97% identity
Manual BLAST searches revealed only 93% identity between the OTU and published Solibacter usitatus Ellin6076 16S sequence. Since this is below the 97% identity threshold for OTU formation, this group was relabeled ‘‘novel acidobacteria.’’ Its consensus 16S sequence is 99% identical to several uncultured bacterium clones from two separate unpublished studies, one of a constructed wetland in France and the other from Tibetan permafrost

Summary

Introduction

Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae: Cicadellinae) or glassy-winged sharpshooter (GWSS) is a leafhopper that exclusively ingests plant xylem fluid from a wide range of host plants [1]. Studies of the cibarium and precibarium, using both light and electron microscopy, have shown patch-like colonies of bacteria interspersed with naked cuticle. The earliest microscopy study used both light and scanning electron microscopy (SEM) to show rod-shaped bacteria embedded in a gum-like matrix in inoculative, i.e. competent to inoculate pathogen, but not in non-inoculative, Graphocephala atropunctata (Signoret) (blue-green sharpshooter; BGSS) [4]; these bacteria were assumed to be the PD bacterium ( known as X. fastidiosa). Three confocal light microscopy studies introducing and visualizing fluorescent protein-expressing bacteria (either X. fastidiosa or an Alcaligenes sp.) have shown single bacterial cells and small clumps or colonies of cells in the anterior foregut of inoculative sharpshooters [3,7,8]

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Conclusion