The ABCB Multidrug Resistance Proteins Do Not Contribute to Ivermectin Detoxification in the Colorado Potato Beetle, Leptinotarsa decemlineata (Say)

Abstract

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is a significant agricultural pest that has developed resistance to many insecticides that are used to control it. Investigating the mechanisms of insecticide detoxification in this pest is important for ensuring its continued control, since they may be contributors to such resistance. Multidrug resistance (MDR) genes that code for the ABCB transmembrane efflux transporters are one potential source of insecticide detoxification activity that have not been thoroughly examined in L. decemlineata. In this study, we annotated the ABCB genes found in the L. decemlineata genome and then characterized the expression profiles across midgut, nerve, and Malpighian tubule tissues of the three full transporters identified. To investigate if these genes are involved in defense against the macrocyclic lactone insecticide ivermectin in this insect, each gene was silenced using RNA interference or MDR protein activity was inhibited using a chemical inhibitor, verapamil, before challenging the insects with a dose of ivermectin. Survival of the insects did not significantly change due to gene silencing or protein inhibition, suggesting that MDR transporters do not significantly contribute to defense against ivermectin in L. decemlineata.

Keywords: ATP-binding cassette proteins; RNAi; insecticide defense.

Figure 1

Expression levels of LedMDR1, LedMDR2, and LedMDR3 in the midgut, head, and Malpighian tubules (MT) tissues of adult L. decemlineata. Bars represent the mean normalized fold change of each gene (± SEM, n = 3) relative to the tissue in which each gene is most highly expressed. Different letters represent significantly different expression levels (Tukey’s Honestly Significant Difference (HSD), p < 0.05).

Figure 2

Expression levels of LedMDR1 and LedMDR2 in adult L. decemlineata fed with dsRNA. Expression was measured in combined midgut, head, and MT tissues after three days of feeding on the indicated treatment. Bars represent the mean normalized fold expression (±SEM, n = 6) relative to expression in insects fed with potato. Different letters represent significantly different expression levels within each gene (Tukey’s HSD, p < 0.05).

Figure 3

Proportion of adult L. decemlineata surviving in different dsRNA feeding treatments each day after consuming ivermectin. L. decemlineata adults were fed with the indicated treatment for three days before being given a 1 µL dose of 20 ppm ivermectin. No significant differences were observed between treatments (log-rank test, χ²(4, 24) = 7.5, p = 0.1).

Figure 4

Proportion of adult L. decemlineata surviving in different verapamil and ivermectin feeding treatments. No significant differences were observed between the ivermectin and verapamil + ivermectin groups (log-rank test, χ²(1, 115) = 1.8, p = 0.2).