Multiple resistances and complex mechanisms of Anopheles sinensis mosquito: a major obstacle to mosquito-borne diseases control and elimination in China

Abstract

Malaria, dengue fever, and filariasis are three of the most common mosquito-borne diseases worldwide. Malaria and lymphatic filariasis can occur as concomitant human infections while also sharing common mosquito vectors. The overall prevalence and health significance of malaria and filariasis have made them top priorities for global elimination and control programmes. Pyrethroid resistance in anopheline mosquito vectors represents a highly significant problem to malaria control worldwide. Several methods have been proposed to mitigate insecticide resistance, including rotational use of insecticides with different modes of action. Anopheles sinensis, an important malaria and filariasis vector in Southeast Asia, represents an interesting mosquito species for examining the consequences of long-term insecticide rotation use on resistance. We examined insecticide resistance in two An. Sinensis populations from central and southern China against pyrethroids, organochlorines, organophosphates, and carbamates, which are the major classes of insecticides recommended for indoor residual spray. We found that the mosquito populations were highly resistant to the four classes of insecticides. High frequency of kdr mutation was revealed in the central population, whereas no kdr mutation was detected in the southern population. The frequency of G119S mutation in the ace-1 gene was moderate in both populations. The classification and regression trees (CART) statistical analysis found that metabolic detoxification was the most important resistance mechanism, whereas target site insensitivity of L1014 kdr mutation played a less important role. Our results indicate that metabolic detoxification was the dominant mechanism of resistance compared to target site insensitivity, and suggests that long-term rotational use of various insecticides has led An. sinensis to evolve a high insecticide resistance. This study highlights the complex network of mechanisms conferring multiple resistances to chemical insecticides in mosquito vectors and it has important implication for designing and implementing vector resistance management strategies.

Figure 1. Sampling sites of Anopheles sinensis mosquitoes in southern (Yunnan) and central (Anhui) China.

Figure 2. Anopheles sinensis mosquito mortality rates to multiple classes of insecticides in the standard WHO tube resistance bioassay.

Two mosquito populations (Anhui in central China and Yunnan in southern China) were tested. Insecticides tested and concentrations were: DE (0.05% deltamethrin), PE (0.75% permethrin), DT (4% DDT), MA (5% malathion) and BE (0.1% bendiocarb). Insecticide resistance classification based on 2013 WHO criteria: susceptible if mortality rate >98%, probable resistant if mortality rate ranges 90–98%, resistant if mortality rate <90%.

Figure 3. Anopheles sinensis G119S mutation detection in ace-1 by PCR-RFLP assay.

Lane 1: 50 bp ladder; lanes 2 and 4: homozygous for ace-1 wildtype; lanes 3 and 5: homozygous for ace-1 resistance mutation; lanes 6 and 7: heterozygous for ace-1 mutation.

Figure 4. Boxplots of metabolic detoxification enzyme activities in Anopheles sinensis populations from laboratory susceptible strain, Anhui and Yunnan populations.

The median activity is shown by a horizontal bar; the box denotes the upper and lower quartiles. The vertical lines show the full range of the data set. *, P<0.05 and **, P<0.001 represent variance significantly different from lab strain with pairwise comparison after the analysis of variance. A: P450 monooxygenases; B: glutathione S-transferases; and C: carboxylesterases.

Figure 5. Ratio of metabolic enzyme activities of resistant mosquitoes to susceptible mosquitoes.

Resistant individuals were those that survived the standard WHO resistance tube bioassay; susceptible individuals were those that died in the bioassay with the discriminating insecticide dosage. Insecticides and their discriminating dosages tested were: deltamethrin 0.05% (DE), permethrin 0.75% (PE), DDT 4% (DDT), malathion 5% (MA) and bendiocarb 0.1% (BE). A: P450 monooxygenases (P450); B: glutathione S-transferases (GST); and C: carboxylesterases (COE). *, P<0.05; **, P<0.01; and ***, P<0.001.

Figure 6. Schematic representation of the relative importance of target-site insensitivity and metabolic detoxification enzymes in resistance to multiple classes of insecticides in two populations of Anopheles sinensis, Anhui and Yunnan.

Circle size reflects the relative impact of the mechanism. Variable importance for a particular predictor is the sum, across all nodes, of the improvement scores the predictor has when it acts as a splitter. The most important variable is always expressed as 100%. P450s: P450 monooxygenases; GSTs: glutathione S-transferases; COEs: carboxylesterases; kdr: knockdown resistance; ace-1: acetylcholinesterase gene 1; and “-”: genotype not examined.