Background
Malaria remains an important global health problem, with 92 % of all deaths occurring in Africa [
1]. In Kenya, more than 70 % of the population is at risk of the disease, with children aged ≤ 5 years and pregnant women being the most vulnerable to infection [
2]. The use of indoor residual spraying (IRS), long-lasting insecticidal nets (LLINs) and other interventions have led to measurable improvements in preventing malaria [
3]. Continued reliance on insecticide-based interventions has also resulted in widespread insecticide resistance in malaria vectors, thus threatening malaria control efforts [
4,
5]. This is the case in western Kenya, where malaria vector control is increasingly being threatened by insecticide resistance due to selection pressure imposed by continued exposure to insecticides [
4,
6].
Although insecticide resistance is increasingly prevalent [
7], its underlying mechanisms are not fully understood. So far, four principal mechanisms of insecticide resistance have been described in mosquitoes, including: (i) metabolic resistance due to elevated activity of detoxification enzymes, (ii) target-site resistance due to genetic alterations at insecticide binding sites, (iii) cuticle modifications that prevent or reduce insecticide penetration, and (iv) behavioral changes resulting in avoidance of, or reduced contact with, insecticides [
8]. Recent studies suggest that the mosquito microbiota may provide a fifth mechanism contributing to insecticide resistance [
9,
10]. Focusing largely on
Anopheles albimanus across different geographical locations including Peru [
9] and Guatemala [
10], these studies have identified significant alterations of the mosquito microbiota associated with insecticide resistance, with enrichment of insecticide-degrading bacteria and enzymes in resistant mosquitoes [
9].
The mosquito microbiota has been shown to affect mosquito physiology [
11]. These microbes, which are predominantly acquired during the aquatic life stage from aquatic habitats, colonize mosquito tissues including the gut, reproductive tracts, exoskeleton, and haemocoel [
12,
13]. Some of these microbes are beneficial to mosquitoes through their role in nutrient provisioning, immunity and development, and subsequent contributions to mosquito fitness [
12]. They also help provide protection against pathogens by modifying the host’s immune system or by synthesizing specific toxins [
12]. The mosquito microbiota can influence and/or be influenced by several mosquito-related factors including mosquito species, developmental stage, genetics, and sex [
11]. In mosquito vectors of malaria, the microbiota play important roles in malaria parasite development, survival, and sporozoite prevalence, thus modulating vector competence [
14‐
17].
Recent studies on the effects of insecticide exposure on microbes associated with mosquitoes and their habitats have so far focused on
Anopheles stephensi, Anopheles albimanus and
Anopheles arabiensis [
9,
10,
18,
19]. The microbiota of
Anopheles gambiae sensu stricto (
s.s.) has, however, largely been unexplored in relation to insecticide resistance. Of particular importance is pyrethroid resistance—a major concern in Kenya, where this class of insecticide is predominantly used in LLINs and IRS [
6,
20,
21]. To address this research gap, this study characterized and compared microbiota between pyrethroid resistant and susceptible
An. gambiae s.s. from an area with intense pyrethroid resistance in Western Kenya. Mosquitoes were also screened for gene mutations that mediate knockdown resistance (
kdr) to pyrethroids, in order to characterize any associations between the mosquito microbiota and
kdr genotype. We discuss these findings on
An. gambiae s.s., and highlight their implications for insecticide resistance monitoring and management.
Discussion
Recently, studies of
An. stephensi, An. arabiensis and
An. albimanus have shown links between mosquito-associated microbiota and resistance to pyrethroids and organophosphates [
9,
10,
18,
19]. In this study the microbiota of pyrethroid resistant and susceptible F
1 progeny of field-derived
An. gambiae s.s. were comparatively characterized. Results showed significant differences in microbiota composition between resistant and susceptible mosquitoes with enrichment of different bacterial taxa between resistant and susceptible mosquitoes.
Intense resistance (at 5× the diagnostic dose) to permethrin was detected, along with high frequency (99.14 %) of the
kdr east allele in the F
1 progeny originating from Tulukuyi, Western Kenya. These findings corroborate earlier reports of high pyrethroid resistance in the same area [
6,
20]. Multiple studies from western Kenya have indicated that the high intensity of insecticide resistance may be contributing to mosquito control failure [
20,
21]. The high frequency of the
kdr east allele suggests that the mutation is fixed in this mosquito population. Other studies conducted in western Kenya have also reported the presence of high
kdr east allele frequencies which is attributed to the continued use of insecticide–based vector control methods [
20,
38‐
40]. However, results showed that the allele was fixed regardless of resistance phenotype, suggesting that additional mechanisms, such as the overexpression of detoxification enzymes (e.g. cytochrome P450s [
41]), are more important than
kdr in conferring the intense permethrin resistance detected in the population. The fixation of the
kdr east mutation in the population also precluded further analysis of any associations between
kdr alleles and the mosquito microbiota. Indeed, a recent study identified no links between the two [
42]. The authors thus hypothesize that any microbe-mediated mechanism of insecticide resistance would be largely distinct from the mosquito host’s genetics, and likely of a metabolic nature.
Results showed diverse bacterial taxa from individual
An. gambiae s.s. samples, a majority of which have previously been identified in
Anopheles and other mosquito genera including
Aedes aegypti [
43‐
46]. However, less than half of the detected microbial taxa were shared between permethrin resistant and susceptible mosquitoes, suggesting insecticide resistance-related physiological differences that favored different bacterial taxa.
Significant differences in microbiota composition and structure between permethrin resistant and susceptible
An. gambiae s.s. were also shown. There is evidence that insecticide detoxifying microbes in agricultural insect pests contribute to insecticide resistance in their hosts [
47,
48]. Recent studies on mosquitoes have also identified insecticide resistance- and/or exposure-driven alterations of the host microbiota. In particular,
An. albimanus microbiota differed by resistance to fenitrothion and was altered by exposure to different pyrethroids, and
Aedes aegypti microbiota differed by resistance to lambda-cyhalothrin [
9,
10,
49]. These findings suggest that insecticide resistance in mosquitoes favour and/or is a consequence of the proliferation of certain bacterial taxa, possibly those that can degrade and metabolize insecticides. Recent studies [
9,
10] identified known insecticide-metabolizing bacterial taxa in
An. albimanus that were exposed or resistant to insecticides. Huang et al. [
50] and Tang et al. [
51] documented that certain microorganisms (considered as potential candidates for bioremediation), including bacteria, degrade pesticides in the soil by breaking them down into smaller compounds, utilizing them as their source of nutrients and making them less toxic to the environment. Some of these microorganisms degrade pesticides to create conducive environments for their survival and not for nutritional requirements [
51]. The different taxa present in the resistant
versus susceptible mosquitoes, particularly those of resistant mosquitoes, is suggestive of this type of adaptation.
Despite significant differences in microbiota composition and structure (beta diversity), there was no significant difference in alpha (Shannon) diversity between the microbiota of resistant and susceptible mosquitoes. This is suggestive of a homeostatic-controlled number of microbial taxa across individual mosquitoes, with an insecticide resistance-associated perturbation of the type and relative abundance of specific microbial taxa. Mosquitoes used in this study were F
1 progeny of wild adult females collected from the same location and reared under identical conditions. Except for their permethrin resistance status, which was determined at 2–3 days post adult eclosion, the mosquitoes had identical physiological characteristics. These identical rearing conditions and subsequent uniform physiological characteristics may explain the homogeneity in alpha diversity across samples. On the other hand, the differences in microbial composition associated with their permethrin resistance status provide further evidence of insecticide selection pressure on the mosquito microbiota. Previous studies have shown that a majority of the mosquito microbiota is obtained from mosquito aquatic habitats at the larval stage, and also from food sources as adults [
13]. Newly emerged adults can also imbibe bacteria along with water from their larval habitats during eclosion or through transstadial transmission [
52]. However, other factors such as mosquito physiological status [
11,
52,
53] affect what microbes persist and colonize the mosquitoes following acquisition, and this could explain the insecticide resistance-associated differences in composition despite similar alpha diversity across all individual samples.
Differential abundance testing identified
Sphingobacterium,
Lysinibacillus,
Streptococcus and
Rubrobacter as significantly more abundant in resistant mosquitoes and
Myxococcus as significantly more abundant in susceptible mosquitoes. The first three genera were only detected in resistant mosquitoes, while
Rubrobacter was at least three-fold more abundant in resistant compared to susceptible mosquitoes. In a study conducted by Hu et al. [
54],
Lysinibacillus sphaericus was identified as a microbe with the ability to degrade up to 83 % of cyfluthrin (a pyrethroid) after 5 days of incubation by utilizing the insecticide as its source of carbon or nitrogen. In the current study,
Lysinibacillus was only detected in resistant mosquitoes, likely as a result of its ability to utilize pyrethroids. Lozano and Dussán [
55] also described the potential of
Lysinibacillus sphaericus to be used in bioremediation of heavy metals.
Sphingobacterium and
Streptococcus, also only detected in resistant mosquitoes in this study, are bacterial genera known to degrade pyrethroid insecticides such as cypermethrin [
56‐
58]. Bacteria belonging to the genera
Streptococcus and
Rubrobacter have been categorized as core microbiota of the digestive system of
Anopheles culicifacies [
59]. Although not documented for pyrethroid degradation or metabolism,
Rubrobacter are known to be thermophilic and extremely resistant to UV thermal and gamma radiations [
60]. Other bacterial genera belonging to
Actinobacteria, the phylum to which
Rubrobacter belongs, have been associated with degradation of insecticides including pyrethroids [
61,
62], and the increased relative abundance of
Rubrobacter in insecticide resistant mosquitoes could suggest their contribution to resistance. On the other hand, the genus
Myxococcus was only detected in susceptible mosquitoes. This bacterial genus is known to be predatory on other bacteria [
63], chitinase-producing [
64], capable of producing various bioactive antifungal agents [
65], and inhibitors of cellular respiration [
66]. However, their association with mosquito physiology or insecticide susceptibility has not yet been described. Given what is known about this bacterial genus as highlighted above, it is possible that they could also be toxic to mosquitoes by directly inhibiting host’s cellular respiration and/or indirectly preying on other members of the mosquito microbiota that are necessary for host’s survival and or insecticide metabolism. Further studies are necessary to elucidate the role of
Myxococcus and their secondary metabolites on mosquito physiology, including insecticide susceptibility.
In an aquatic microcosm, it has been demonstrated that insecticides, if used singly or in combination, can reduce microbial diversity and/or induce shifts in microbial community structure [
67]. Recent studies have also demonstrated shifts in mosquito microbiota and larval water microbiota that were associated with insecticide exposure [
10,
67]. This indicates that insecticide exposure shapes the microbial composition of mosquitoes and their habitats. This is likely due to the toxic effects of insecticides on some microbes, while at the same time favoring the proliferation of other tolerant microbes as described by Johnsen et al. [
68]. It is also possible that in addition to, or rather than selection pressure, the presence of specific insecticide-metabolizing microbes in mosquitoes induce resistance to insecticides and precludes colonization by other microbes. In
Ae. aegypti it has been demonstrated that infections with certain microbes precludes colonization by others [
69], and that microbial interactions within mosquitoes shape their microbial community [
9,
70]. Further research on these microbial networks could shed more light on the role of the mosquito microbiota in insecticide resistance. [
10,
67].
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