Identification of carboxylesterase genes implicated in temephos resistance in the dengue vector Aedes aegypti

Abstract

Background: Thailand is currently experiencing one of its worst dengue outbreaks in decades. As in most countries where this disease is endemic, dengue control in Thailand is largely reliant on the use of insecticides targeting both immature and adult stages of the Aedes mosquito, with the organophosphate insecticide, temephos, being the insecticide of choice for attacking the mosquito larvae. Resistance to temephos was first detected in Aedes aegypti larvae in Thailand approximately 25 years ago but the mechanism responsible for this resistance has not been determined.

Principal findings: Bioassays on Ae. aegypti larvae from Thailand detected temephos resistance ratios ranging from 3.5 fold in Chiang Mai to nearly 10 fold in Nakhon Sawan (NS) province. Synergist and biochemical assays suggested a role for increased carboxylesterase (CCE) activities in conferring temephos resistance in the NS population and microarray analysis revealed that the CCE gene, CCEae3a, was upregulated more than 60 fold in the NS population compared to the susceptible population. Upregulation of CCEae3a was shown to be partially due to gene duplication. Another CCE gene, CCEae6a, was also highly regulated in both comparisons. Sequencing and in silico structure prediction of CCEae3a showed that several amino acid polymorphisms in the NS population may also play a role in the increased resistance phenotype.

Significance: Carboxylesterases have previously been implicated in conferring temephos resistance in Ae aegypti but the specific member(s) of this family responsible for this phenotype have not been identified. The identification of a strong candidate is an important step in the development of new molecular diagnostic tools for management of temephos resistant populations and thus improved control of dengue.

Figure 1. Comparison of esterase activities between Phatthalung and Nakhon Sawan 2 populations.

Absorbance values were measured after 4(*** P-val<0.05, Mann-Whitney).

Figure 2. Summary of the genes differentially transcribed in the comparisons Nakhon Sawan 2 Unexp and Exp vs Phatthalung.

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 comparisons. Upward arrows indicate over- transcribed in Nakhon Sawan 2 compared to Phatthalung, downward represent under-transcribed.

Figure 3. GO term enrichment analysis conducted on transcripts found upregulated in both comparisons “NS 2 Exp vs P” and NS 2 Unexp vs P”.

GO-term categories represented were found significantly enriched compared to the reference set (all transcripts present on the microarray) after a Fisher's exact (Pval<0.01) with multiple testing correction. 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 4. Significant up-regulated probes commonly found in NS2 Exp vs P and NS2 Unexp vs P.

Colored probes correspond to the transcripts with fold changes 1.25 fold higher in NS 2 (exposed) vs Phatthalung compared to NS 2 (Unexposed) vs Phatthalung comparison.

Figure 5. Partial alignment between three translated CCEae3a sequences: Two sequences from the susceptible strain Phatthalung and Vectorbase and one from the resistant strain Nakhon Sawan 2.

Grey highlight shows the amino acid changes between the resistant and susceptible populations. Catalytic triad is highlighted by the symbol▾.

Figure 6. Analysis of CCEae3a 3D models.

(A) CCEae3a_Nakhon Sawan (green) and CCEae3a_Phatthalung (blue) model superimposition shows no significant differences in the protein overall fold between resistant and susceptible alleles due to the polymorphic residues despite variations on the substrate binding site. (B) Close-up view of conformational differences at the substrate binding site between CCEae3a_Nakhon Sawan (green) and CCEae3a_Phatthalung (blue) revealed that F286, involved in the substrate recognition by homology with LcαE7 (orange), could be the key to explain both CCEae3a_Phatthalung and CCEae3a_vectorbase (cyan) susceptibility.