Streamlined phage display library protocols for identification of insect gut binding peptides highlight peptide specificity.

Phage display libraries have been used to isolate insect gut binding peptides for use as pathogen transmission blocking agents, and to provide artificial anchors for increased toxicity of bacteria-derived pesticidal proteins. Previously, phage clones displaying enriched peptides were sequenced by Sanger sequencing. Here we present a streamlined protocol for identification of insect gut binding peptides, using insect-appropriate feeding strategies, with next generation sequencing and tailored bioinformatics analyses. The bioinformatics pipeline is designed to eliminate poorly enriched and false positive peptides, and to identify peptides predicted to be stable and hydrophilic. In addition to developing streamlined protocols, we also sought to address whether candidate gut binding peptides can bind to insects from more than one order, which is an important consideration for safe, practical use of peptide-modified pesticidal proteins. To this end, we screened phage display libraries for peptides that bind to the gut epithelia of two pest insects, the Asian citrus psyllid, (Hemiptera) and beet armyworm, (Lepidoptera), and one beneficial insect, the western honey bee, (Hymenoptera). While unique peptide sequences totaling 13,427 for , 89,561 for and 69,053 for were identified from phage eluted from the surface of the insect guts, final candidate pools were comprised of 53, 107 and 1423 peptides respectively. The benefits of multiple rounds of biopanning, along with peptide binding properties in relation to practical use of peptide-modified pesticidal proteins for insect pest control are discussed.