The Wiggle Index: An Open Source Bioassay to Assess Sub-Lethal Insecticide Response in Drosophila melanogaster

Abstract

Toxicological assays measuring mortality are routinely used to describe insecticide response, but sub-lethal exposures to insecticides can select for resistance and yield additional biological information describing the ways in which an insecticide impacts the insect. Here we present the Wiggle Index (WI), a high-throughput method to quantify insecticide response by measuring the reduction in motility during sub-lethal exposures in larvae of the vinegar fly Drosophila melanogaster. A susceptible wild type strain was exposed to the insecticides chlorantraniliprole, imidacloprid, spinosad, and ivermectin. Each insecticide reduced larval motility, but response times and profiles differed among insecticides. Two sets of target site mutants previously identified in mortality studies on the basis of imidacloprid or spinosad resistance phenotypes were tested. In each case the resistant mutant responded significantly less than the control. The WI was also able to detect a spinosad response in the absence of the primary spinosad target site. This response was not detected in mortality assays suggesting that spinosad, like many other insecticides, may have secondary targets affecting behaviour. The ability of the WI to detect changes in insecticide metabolism was confirmed by overexpressing the imidacloprid metabolizing Cyp6g1 gene in digestive tissues or the central nervous system. The data presented here validate the WI as an inexpensive, generic, sub-lethal assay that can complement information gained from mortality assays, extending our understanding of the genetic basis of insecticide response in D. melanogaster.

Partitioning the roles of insect and microbial enzymes in the metabolism of the insecticide imidacloprid inDrosophila melanogaster

“…Significantly higher levels of metabolites detected in the HR_Cyp6g1 larvae and their media between 15 and 30 mins suggest that ingested IMI is rapidly metabolised by CYP6G1 in the metabolic tissues and that IMI-5-OH and IMI-Ole are excreted in the media of exposure as they are formed. These data are consistent with a previous report that there is a significant difference in motility between the HR_Cyp6g1 and control strains after only 15 mins of exposure to IMI 49 . The over-expression of Cyp6g1 in the metabolic tissues reduces the levels of IMI in larval bodies reducing the toxicity of this insecticide (Figure 4A).…”

Deletion of the nicotinic acetylcholine receptor subunit gene Dα1 confers insecticide resistance, but at what cost?

“…2). 17 Significant differences in the relative movement are detectable within 15 minutes of exposure to the compound (Fig. 2).…”

“…Following the protocol of Denecke, et al, 2015, 3 rd instar larvae were bathed in (A) 5% sucrose or (B) a 5% sucrose:25 ppm imidacloprid solution, and their movement recorded over time. 17 The differences in movement relative to their starting activity is presented in the graph as a relative movement ratio. No difference is observed between the three genotypes in 5% sucrose solution across the duration of the experiment.…”

Multiple P450s and Variation in Neuronal Genes Underpins the Response to the Insecticide Imidacloprid in a Population ofDrosophila melanogaster

“…The response of D. melanogaster larvae to imidacloprid was measured using the WI assay, which estimates the insecticidal effect by quantifying the reduction in motility during insecticide exposure 31 . Third instar larvae of each genotype were picked, 25 per well, into a NUNC cell culture treated 24 well plate (Thermo-Scientific) preloaded with 200µL 5% w/v sucrose (AR Grade; Chem Supply) in distilled H 2 0.…”