Gene amplification, ABC transporters and cytochrome P450s: unraveling the molecular basis of pyrethroid resistance in the dengue vector, Aedes aegypti

Abstract

Background: Pyrethroid insecticides are widely utilized in dengue control. However, the major vector, Aedes aegypti, is becoming increasingly resistant to these insecticides and this is impacting on the efficacy of control measures. The near complete transcriptome of two pyrethroid resistant populations from the Caribbean was examined to explore the molecular basis of this resistance.

Principal findings: Two previously described target site mutations, 1016I and 1534C were detected in pyrethroid resistant populations from Grand Cayman and Cuba. In addition between two and five per cent of the Ae. aegypti transcriptome was differentially expressed in the resistant populations compared to a laboratory susceptible population. Approximately 20 per cent of the genes over-expressed in resistant mosquitoes were up-regulated in both Caribbean populations (107 genes). Genes with putative monooxygenase activity were significantly over represented in the up-regulated subset, including five CYP9 P450 genes. Quantitative PCR was used to confirm the higher transcript levels of multiple cytochrome P450 genes from the CYP9J family and an ATP binding cassette transporter. Over expression of two genes, CYP9J26 and ABCB4, is due, at least in part, to gene amplification.

Significance: These results, and those from other studies, strongly suggest that increases in the amount of the CYP9J cytochrome P450s are an important mechanism of pyrethroid resistance in Ae. aegypti. The genetic redundancy resulting from the expansion of this gene family makes it unlikely that a single gene or mutation responsible for pyrethroid resistance will be identified in this mosquito species. However, the results from this study do pave the way for the development of new pyrethroid synergists and improved resistance diagnostics. The role of copy number polymorphisms in detoxification and transporter genes in providing protection against insecticide exposure requires further investigation.

Figure 1. Summary of the genes differentially transcribed between resistant and susceptible strains.

The Venn diagram shows the number of genes found significantly (P value<0.01) over- or under-transcribed (>2 fold in either direction) in one or both resistant strains compared to the susceptible New Orleans strain. Upward arrows indicate over- transcribed in resistant strains, downward represent under-transcribed.

Figure 2. GO term enrichment analysis.

The analysis was performed on the significantly up-regulated genes found in CAYMAN and CUBA-DELTA compared to NO. The BLAST2GO software was used for the annotation, mapping and enrichment analysis. The figure represents all the significant GO-term categories found significantly enriched compared to the reference set (all transcripts present on the microarray) after a Fisher's exact test and Benjamini and Hochberg multiple testing correction (Pval<0.05). The test set percentage indicates the percentage of up regulated genes belonging to a GO term category compared to all up-regulated genes used in the GO-term analysis while the reference set percentage indicates the percentage of a particular GO-term category compared to all genes with GO-terms on the microarray.

Figure 3. Quantitative PCR analysis of selected genes from the microarray experiments.

Relative-fold change in transcript and gene copy number normalised to two ribosomal genes was compared between the resistant CAYMAN (A) and CUBA-DELTA (B) strains against the NEW ORLEANS susceptible strain. Transcript levels are shown by the white columns and gene copy numbers by the grey columns. Error bars represent 95% confidence intervals.

Figure 4. Unrooted distance neighbour joining tree showing phylogeny of Aedes aegypti CYP9 genes.

Nodes with >70% bootstrap support (500 pseudoreplicates) are indicated. Sequences in blue are up-regulated in both CUBA-DELTA and CAYMAN strains from the current study. Sequences underlined are over expressed in ≥2 populations versus laboratory susceptible strains from previously published studies (see text for further details). Sequences marked with * have proven ability to metabolise pyrethroids (Stevenson et al, submitted).