Effects of the kdr resistance mutation on the susceptibility of wild Anopheles gambiae populations to Plasmodium falciparum: a hindrance for vector control

Anopheles gambiae s.s. (molecular form S) and An. coluzzii larvae were collected at ten breeding sites in the village of Dielmo[32] (13°43’N, 16°24’W) between August and September 2012. Larvae were raised until emergence; adults were fed on rabbit blood, and 200 females (F0) were randomly selected (20 from each collection site). Each F0 female was allowed to lay its eggs individually before it was genotyped for species and molecular form by PCR-RFLP[14].

The frequency of kdr was determined before (in the F0 population) and after infection (in F4 population) but not in the rearing females (F1 to F3 populations), for which only the molecular form was determined. Previous studies have shown that the kdr frequency in Anopheles populations in Dielmo can reach 47%[12, 33] and may increase significantly after inter-generational crosses.

The offspring of F0 females of the same taxa were then pooled and bred under the same conditions. Larvae were fed Tetramin fish food. Pupae were collected and placed in 10-L plastic buckets, which were covered with mosquito gauze with a cotton sleeve for introducing 10% glucose on filter paper. Adults were maintained at 28°C, 80% relative humidity and 12:12 hr light:dark cycle. In order to increase the proportion of mosquitoes accustomed to feeding on membrane, aggressive F1, F2 and F3 females were selected.

F4 females used for infection were genotyped, and species and molecular forms were confirmed by PCR RFLP[14]. L1014F and L1014S kdr mutations (hereafter referred to as kdr-w and kdr-e, respectively) were detected by PCR[34, 35].

Gametocyte carriers were detected in cross-sectional surveys in villages and schools during the high transmission period (October–November) in Anene (14°47'N, 16°55'W Thies region). Finger-prick blood was taken from each volunteer. Thick blood smears were stained with 10% Giemsa and examined microscopically under a (100×) oil immersion lens for the presence of sexual and asexual parasites. Parasite density was estimated by counting against 1,000 white blood cells and converted to numbers of parasites per microlitre by assuming a standard white blood cell count of 8,000/μL. Symptomatic and non-symptomatic individuals with asexual parasites were treated with artemisinin-based combination therapy according to national recommendations. The inclusion criteria for gametocyte carriers were: age over ten years, a P. falciparum gametocyte density over 20/mm³ of blood and no anti-malarial treatment in the previous month. Each gametocyte carrier provided 6 mL of blood drawn into a heparinized vacutainer tube, and each was given an insecticide-impregnated bed net in compensation.

Experimental infections were carried out in the direct membrane feeding assay as described by Mulder et al.[36]. Blood was rapidly distributed to two pools of three-day-old females of each taxon through a serially connected, warm water (37°C), jacketed membrane feeder, and the mosquitoes were allowed to feed for 15 min; then, partially fed and non-fed specimens were removed. Two batches of 50 mosquitoes of each taxon were randomly selected from among fed females and maintained in the insectary on a 10% sucrose diet for further analyses. The first batch of mosquitoes was dissected seven days later, and their midguts were stained with 3% mercurochrome in PBS and examined under a light microscope (40× objective) for detection and quantification of oocysts. The presence or absence of oocysts (status of infection by oocyst) and their number (intensity of infection) were recorded. The heads and thoraxes of the second batch of mosquitoes were used 14 days after feeding to evaluate the presence of the circumsporozoite protein of P. falciparum in an enzyme-linked immunosorbent assay (ELISA)[37]. A mosquito was considered to have sporozoites when the optical density was >0.6, which is that of the control strain. The status of infection by sporozoites and the optical density (proxy for the intensity of infection) were recorded. PCR RFLP[14] was performed on the carcasses of dissected mosquitoes, and the identity of the molecular forms was confirmed.

Experiments were repeated five times on different days with different samples of gametocyte-containing blood. Gametocytaemia was 78, 92, 113, 136, and 218 gametocytes/μL in the five assays.

Susceptibility to oocysts and sporozoites were assessed separately. The first batch of mosquitoes was used to study infection by oocysts (N = 445, 358 infected), while the second batch was used to study infection by sporozoites (N = 303, 244 infected).

The fixed effects of strain (An. coluzzii and An. gambiae S form), genotype at the kdr locus (RR, RS and SS) and the random effect of assay (a five-level categorical control variable accounting for the day of dissection and the donor) were tested.

Statistical analyses were performed with R software v3.0.2 . The overall method was the same for all four analyses (i.e., of each of the two responses variables in each of the two datasets). First, a model containing all explanatory variables and the strain-genotype interaction was adjusted with a linear mixed-effect model. The glm (fixed-effect generalized linear model) and glmer (mixed generalized linear model) procedures with binomial error distribution were used to analyse the status of infection, the glm and glmer procedures with negative binomial error distribution to analyse oocyst numbers in infected individuals, and the lm (fixed-effect linear model) and lmer (mixed linear model) procedures with Gaussian error distribution to analyse sporozoite density in infected individuals (because absorbance exhibited a Gaussian distribution). Secondly, the significance of the assay effect was assessed. Fixed-effect and mixed models where compared with the Akaike information criterion (AIC): the model with the lowest AIC was chosen. Thirdly, the best model was selected step by step with the drop1 procedure, which performs a Chi-square test for linear models and a likelihood ratio test (approximating a Chi-square distribution) for generalized linear models. If a variable was not significant, it was removed from the model. A p value of ≤0.05 was considered significant.

To analyse infection rates, odds ratios (ORs) were obtained from the model estimates, which are logarithms of ORs (OR = exp^estimates), and their 95% confidence intervals (CIs) were computed with the confint procedure. For oocyst density analysis, the number of oocysts in a mosquito when infection occurred was calculated from the model estimates. For sporozoite density analysis, estimates are meaningless, as the absorbance is not expected to vary linearly with sporozoite density, therefore the focus will be put on variables significativity and the trend given by these estimates.

Pairwise comparisons were then done to determine differences with the difflsmeans procedure (lmerTest library, applicable to mixed-effect models), the wald.test procedure (aod library, applicable to glm) and individual Welsh-corrected t-tests with the Bonferonni correction (linear model).

The strain-genotype interaction, the strain and the assay effects were not significant in either the oocyst model nor the sporozoite infection model, while the effect of genotype (SS, RS and RR) was significant (Table 1). Therefore this is the output of fixed-effect models where only the genotype variable is kept as an explanatory variable that is presented.

The odds of being infected with oocysts were signicantly greater in RS than in SS groups (χ² = 42.8, df = 1, P(>χ²) = 6.1e-11). As all RR individuals were infected (no variability), it was not possble to test how significantly different these individuals are from others, but it appears that homozygote-resistant individuals are much more sensitive to oocyst infection than SS (and probably RS) individuals (Figure 1A).

The results for sporozoite infection were qualitatively similar. The odds of being infected were greater in RS (χ² = 39.8, df = 1, P > χ² = 2.8e-10) and RR (χ² = 38.4, df = 1, P > χ² = 5.8e-10) than in SS genotypes (Figure 1B). The odds of being infected were also significantly higher in RR than in RS groups (χ² = 4.5, df = 1, P > χ² = 0.034.

In the oocyst number model, assay, strain-genotype interaction, strain and genotype were all significant. Hence a mixed-model involving all these variableswas used to test significances and calculate estimates. In the model of sporozoite density (approximated by absorbance), the effects of assay and strain-genotype interaction were not significant, but the effects of strain and genotype were significant. Hence a fixed-effect model involving significances variables was used to test significances and calculate estimates (Table 1).

Pairwise comparisons of oocyst number and absorbance (Tables 2 and3) indicated a small effect of species (small differences in oocyst numbers, barely significant p values) but a strong effect of genotype (larger differences in oocyst numbers, highly significant p values). Thus, the effect of species on the intensity of infection is much smaller than that of genotype.

More specifically, oocyst density is significantly more important in RS and RR than in SS, and significantly more important in RR than in RS. This is true both for An. gambiae S form and An. coluzzii (Figure 1C, Table 2).

Sporozoite density was greater in RS and RR than in SS individuals and greater in An. gambiae S form than in An. coluzzii individuals (Figure 1D). Pairwise comparison indicated that differences are all significant, e.g., there is both an effect of genotype and species on sporozoite density (Table 3).