Anopheles mosquito species composition
A total of 3, 675 mosquitoes were collected during the study. Of these, 1,122 (31%) were female An. gambiae s.l., 2,358 (64%) An. funestus complex and 195 (5%) Culicine mosquitoes. The majority (58%, 652) of An. gambiae s.l. mosquitoes were found resting outdoors than indoors (42%, 470). Anopheles coluzzii was the predominant species, both indoors (36%, 375) and outdoors (39%, 413), followed by An. arabiensis (3%, 33) indoors and outdoors (12%, 125), and An. gambiae s.s. indoors (2%, 27) and outdoors (8%, 83). Five (5) hybrids of An. coluzzii/gambiae s.s. were also identified.
Phenotypic resistance in F1 Anopheles coluzzii populations.
Overall, a total of 780 mosquitoes (indoor: 380, outdoor: 400) were exposed to insecticides (Additional file 1: Table S1) from about 160 (indoor) and 320 (outdoor) F0 adults that successfully laid eggs. Species identification of all phenotyped samples revealed 98% of F1 progeny were An. coluzzii both indoors and outdoors while the remaining species (An. arabiensis and An. gambiae) represented 2%. Mortality was generally higher in outdoor mosquitoes than the indoor populations. A 24-hour post-exposure mortality of 0% and 9% (95%CI: 3-12%) was observed for DDT with progeny of mosquitoes from indoor and outdoor respectively (Fig. 2) and this difference was statistically significant (Pearson X2=7.58, df=1, P=0.006). Progeny of mosquitoes exposed to deltamethrin showed an overall mortality of 5% (95% CI: 1-12%) for indoor mosquitoes and 2.5% (95%CI: 8-34%) for outdoor-resting mosquitoes (Pearson X2=5.44, df=1, P=0.02).
The indoor and outdoor mosquitoes exposed to bendiocarb showed suspected resistance with mortality of 90% (95%CI: 64-95%) in the indoor population and 95% (95%CI: 87-100%) in the outdoor population (Pearson X2=1.07, df=1, P=0.30). Both the indoor and outdoor populations were fully susceptible to malathion, with 98% and 100% (95%CI: 87-100%) mortality for indoor and outdoor mosquitoes, respectively (Pearson X2=2.02, df=1, P=0.16). There was no observed mortality (0%) in the controls for all insecticides tested.
Detection of resistance alleles in F1 An. coluzzii populations
Resistance-associated allele frequencies were higher in outdoor-resting mosquitoes than the indoor population (Table 1). Vgsc-1014F and GSTe2-114T alleles were the most common in both phenotypically resistant and susceptible indoor and outdoor mosquitoes. In the deltamethrin-resistant mosquitoes, Vgsc-1014F frequency was 0.65 (indoor) and 0.67 (outdoor). However in the DDT-resistant mosquitoes, Vgsc-1014F frequency was 0.65 (indoor) and 0.73 (outdoor). These observed differences were not statistically significant between the indoor and outdoor mosquito populations (Deltamethrin: Pearson X2=0.22, df=1, P=0.64. DDT: Pearson X2=0.41, df=1, P=0.52). The carriage of Vgsc-1014F mutation was strongly associated with resistance to deltamethrin (OR =5.46, P=0.001, 95% CI: 1.94-15.41) but not with DDT resistance (OR=0.69, P=0.75, 95% CI: 0.066-7.14). No Vgsc-1014S allele was detected in any of the mosquitoes.
Vgsc-1575Y mutation was detected mainly in the deltamethrin-resistant outdoor An. coluzzii populations (frequency= 0.27). GSTe2-114T mutation was significantly higher in outdoor-resting (0.85) mosquitoes than the indoor (0.56) DDT-resistant mosquitoes (Pearson X2 = 5.73, df=1, P= 0.02). This mutation was also identified in mosquitoes resistant to deltamethrin (indoor=0.62, outdoor=0.84).
Ace1-119S was detected in a single indoor and an outdoor An. coluzzii specimens that survived bendiocarb exposure. It was also found in a single bendiocarb-resistant outdoor mosquito. The allele was detected only in malathion-susceptible mosquitoes at frequency of 0.08 (indoor) and 0.12(outdoor) with no significant difference (Pearson X2 = 0.003, df=1, P=0.96).
Detection of resistance alleles in F0 An. gambiae populations
The frequency of resistance alleles between the indoor and outdoor mosquitoes varied by mosquito species. Whereas Vgsc-1014S was not detected in the F1 An. coluzzii, it was observed mainly in the F0 An. arabiensis resting outdoors. Vgsc-1014F mutation was significantly higher in outdoor (0.99) resting mosquitoes compared to those indoors (0.77) in An. gambiae s.s. (Pearson X2=31.6, df=2, P=0.001) (Table 2). There was an indication of association of outdoor-resting behaviour with resistance in An. gambiae s.s. population carrying the Vgsc-1014F mutation (OR =0.05, P=0.01, 95% CI: .005-0.419). Although, An. coluzzii was the predominant species collected both indoors and outdoors, the difference in the frequency of this mutation in indoor (0.65) and outdoor (0.70) populations was not statistically significant (Pearson X2=0.7, df=2, P=0.4). However, the higher prevalence of the mutation in indoor (0.48) than the outdoor (0.21) An. arabiensis population was significant (Pearson X2= 6.42, df=2, P = 0.04). Vgsc-1014S was mainly found in indoor An. arabiensis (0.42).
Vgsc-1575Y was detected at an almost similar level (frequencies: Indoor=0.21, outdoor=0.2) in An. coluzii populations. Further, no significant difference was observed in indoor (0.30) and outdoor (0.18) An. gambiae s.s. (Pearson X2=1.2, df=1, P=0.27).
Ace1-119S mutation was most frequent in An. gambiae s.s., although there was no statistically significant difference in the frequencies between indoor (0.25) and outdoor (0.31) populations (Pearson X2=0.2, df=1, P=0. 65). The prevalence was 0.1 in indoor and outdoor An. coluzzii.
Metabolic enzyme activities in F1 An. coluzzii populations
An overall highly significant elevated levels of AChE (F2, 237= 55.93, P<0.0001) and β-esterase (F2, 237= 159.0, P<0.0001) activities were observed in both indoor and outdoor mosquito populations compared to the susceptible reference strain, Kisumu (Figures 3A-D). Conversely, in both mosquito populations, the activities of the GSTs and monooxygenases were less relative to Kisumu but this was not significant in monooxygenase activity (F2, 237 = 0.6589, P=0.52).
AChE activity was not significantly higher in the outdoor (0.62/mg protein) than the indoor (0.57/mg protein) population (Mann–Whitney U=5037, Z=-1.73, P=0.08). The elevation in enzyme activity was found (Table 3) to be 2.48 fold (indoor) and 2.7 fold (outdoor) significantly higher than in Kisumu (P<0.0001). Similarly, non-specific β-esterase activity in the outdoor-resting mosquitoes (1.70/mg protein) was significantly more than the indoor mosquitoes (1.35) (Mann–Whitney U=0.5, Z=-8.33, P<0.0001); with 1.69 (indoor) and 2.13 (outdoor) significant fold changes (P<0.0001). No significant difference was detected in the level of GST activity between the two mosquito populations (indoor: 0.01/mg protein, outdoor: 0.02/mg protein, (Mann–Whitney U=5709, Z= -0.29, P=0.78). Monooxygenase activities also showed a similar level in both indoor and outdoor mosquitoes (mean activity = 0.21/mg protein, (Mann–Whitney U =4989, Z= -1.84, P=0.07). The fold difference in monooxygenase activities in Kisumu population (1.11) was not statistically significant (P=0.59).
Host blood meal sources of wild F0 mosquitoes
A total of 165 out of 214 blood-fed mosquitoes were successfully identified to have fed either on human or animal hosts. The overall vertebrate positivity rate was higher in indoor-resting mosquitoes (Table 4), predominantly in An. coluzzii, which was the most abundant species in the study sites. Overall human blood index (HBI) was 21% and again more prominent in indoor (18%) than outdoor (3%) mosquitoes. 69% of HBI was detected from indoor An. coluzzii, followed by 17% in outdoor An. coluzzii. Anopheles arabiensis was found with only 9% (indoor) and 1% (outdoor) HBI, while 1% HBI was identified in indoor An. gambiae only.
The principal animal blood meal source was from goat, representing 36% of indoor against 8% of outdoor-resting An. coluzzii. The other animal blood sources included cows, dogs, donkeys, horses and pigs and were mainly detected in indoor An. coluzzii specimens. Fewer (4%) An. arabiensis resting indoors fed on animal blood compared to the outdoor population (10%), similar to An. gambiae s.s. indoor (0.6%) and outdoor (1.2%) population. Mixed human and goat blood meal was identified from a single indoor An. coluzzii specimen. Also, mixed cow and goat meal were found in three outdoor An. coluzzii specimens and one outdoor An. gambiae s.s. specimen.