RNA interference validation of detoxification genes involved in ivermectin tolerance in Drosophila melanogaster

Abstract

Previously, we observed increased transcription levels of specific cytochrome P450 monooxygenase (P450) and adenosine triphosphate binding cassette (ABC) transporter genes in human body lice, Pediculus humanus humanus, following exposure to ivermectin using the non-invasive induction assay, which resulted in tolerance. To confirm the roles of these genes in induction and tolerance, the robust genetic model insect Drosophila melanogaster was chosen. Orthologous genes corresponding to the body louse P450 (Cyp9f2, Cyp6g2 and Cyp9h1) and ABC transporter (Mrp1, GC1824 as an ABCB type and CG3327 as an ABCG type) genes were selected for in vivo bioassay. Following a brief treatment with a sublethal dose of ivermectin, the mortality response was significantly slower, indicating the presence of tolerance. Concurrently, the transcription levels of Cyp9f2 and Mrp1 at 3 h and those of Cyp6g2, Cyp9h1, Mrp1, CG1824 and CG3327 at 6 h post-treatment were upregulated, indicating gene induction. In behavioural bioassay using GAL4/UAS-RNA interference transgenic fly lines, increased susceptibility to ivermectin was observed following heat shock in the Cyp9f2 , Cyp6g2 , Cyp9h1 , Mrp1 or CG3327-knockdown flies. Considering that these five genes are orthologous to those which had the largest over-expression level following ivermectin-induced tolerance in the body louse, the current results suggest that they are also associated with ivermectin detoxification in D. melanogaster and that body lice and D. melanogaster are likely to share, in part, similar mechanisms of tolerance to ivermectin.

Functional analysis ofABCGandABCHtransporters from the red flour beetle,Tribolium castaneum

“…ABCG–H genes have been suggested to contribute to insecticide detoxification as well based on the indirect finding of upregulated gene expression in response to exposure to different insecticides 21–30, 54 . Only in the case of ivermectin resistance in Drosophila flies, however, has direct genetic evidence for the contribution of an ABCG transporter to insecticide resistance been provided 31 . Therefore, we focused on TcABCG–H genes and addressed the question of whether they potentially contribute to insecticide detoxification in coleopteran species.…”

“…[21][22][23][24][25][26][27][28][29][30]54 Only in the case of ivermectin resistance in Drosophila flies, however, has direct genetic evidence for the contribution of an ABCG transporter to insecticide resistance been provided. 31 Therefore, we focused on TcABCG-H genes and addressed the question of whether they potentially contribute to insecticide detoxification in coleopteran species.…”

“…In addition, ABCG–H transporters have been suggested to be implicated in the elimination of insecticides mostly based on the observation of increased transcript levels in response to treatments with different insecticides 21–30 . However, only in case of ivermectin tolerance a direct contribution of an ABCG transporter has been demonstrated using a Drosophila GAL4/UAS‐RNAi line 31 …”

“…[21][22][23][24][25][26][27][28][29][30] However, only in case of ivermectin tolerance a direct contribution of an ABCG transporter has been demonstrated using a Drosophila GAL4/UAS-RNAi line. 31 ABCG and ABCH transporters are half transporters which need to dimerize to form functional homo-or heterodimeric transporters. Insect genomes harbour 12-23 ABCG genes and 3-9 ABCH genes.…”

Post-transcriptional modulation of cytochrome P450s, Cyp6g1 and Cyp6g2, by miR-310s cluster is associated with DDT-resistant Drosophila melanogaster strain 91-R

“…Specifically, although the role of Cyp6g1 in DDT resistance is established, neither is any direct role of Cyp6g2 in DDT resistance yet reported from Drosophila, nor is there direct functional evidence for enzymatic products of Cyp6g2 in DDT detoxification. However, a number of previous studies suggested that the overexpression of Cyp6g2 gene was associated with resistance to several insecticides such as imidacloprid, ivermectin, and diazinon in Drosophila 35,38,39 . Moreover, the significant overexpression of Cyp6g2 was associated with the DDT-resistant 91-R compared to susceptible strains Canton-S and 91-C 12,19,31 .…”