Bee-safe peptidomimetic acaricides achieved by comparative genomics

Abstract

The devastating Varroa mite (Varroa destructor Anderson and Trueman) is an obligatory ectoparasite of the honey bee, contributing to significant colony losses in North America and throughout the world. The limited number of conventional acaricides to reduce Varroa mites and prevent disease in honey bee colonies is challenged with wide-spread resistance and low target-site selectivity. Here, we propose a biorational approach using comparative genomics for the development of honey bee-safe and selective acaricides targeting the Varroa mite-specific neuropeptidergic system regulated by proctolin, which is lacking in the honey bee. Proctolin is a highly conserved pentapeptide RYLPT (Arg-Tyr-Leu-Pro-Thr) known to act through a G protein-coupled receptor to elicit myotropic activity in arthropod species. A total of 33 different peptidomimetic and peptide variants were tested on the Varroa mite proctolin receptor. Ligand docking model and mutagenesis studies revealed the importance of the core aromatic residue Tyr2 in the proctolin ligand. Peptidomimetics were observed to have significant oral toxicity leading to the paralysis and death of Varroa mites, while there were no negative effects observed for honey bees. We have demonstrated that a taxon-specific physiological target identified by advanced genomics information offers an opportunity to develop Varroa mite-selective acaricides, hence, expedited translational processes.

Figure 1

Taxonomic distribution of proctolin signaling system. The sequence of the proctolin of Varroa desructor (A), distribution of proctolin orthologous genes in arthropods (B) and phylogeny of the proctolin receptors rooted by FMRFamide receptors and sex peptide/myoinhibitory peptide receptors (C). The footnotes 1, 2, and 3, for Blattodea and Orthoptera proctolin is described in Supplementary data 1. Highlighted raw in B is for the taxonomic groups that is relevant to this study. Solid and empty circles in C are for bootstrapping values > 85% and > 50%, respectively.

Figure 2

In silico docking model. The importance of cation-π interactions of Arg1 and Tyr2 of the proctolin (A), the charges of receptor and ligands on the docking model (B), and the mutated ligands and receptor activities tested on the receptors expressed in the CHO cell (C). In B, blue is for positive and the red is for negative charges. In C, the values are normalized values for the log10-based scale activities relative to the 5 as the standard for wild-type receptor with the proctolin.

Figure 3

Activities of proctolin peptidomimetics. Log-dose response curves showing the activities of proctolin analogs and peptidomimetics on the Varroa destructor proctolin receptor (A to E), and Varroa mite showing excretion induced by injection of proctolin solution (F). In F, the white arrow is for the location of microinjection needle and the black arrow shows the excretion from the anus of Varroa mite.

Figure 4

Proctolin peptidomimetics activities. Time-dependent efficacy of peptide mimics to Varroa mites. Immobilized Female Varroa mites were topically treated on the gnathosoma; 2326 at 3 nM and 2334, 2336, and 2442 at 0.01 nM and the number of mites affected (i.e., paralyzed or dead) was recorded for 1 to 48 h post-treatment. Vertical bars represent the mean ± standard error. Different letters above the bars indicate a significant difference between the mean percent effect at each time point using a one-way ANOVA with a Tukey’s post-hoc test (p < 0.05). The mortalities in water controls were ~  < 10% at 48 h exposure.