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. 2021 Mar 1;16(3):e0240771. doi: 10.1371/journal.pone.0240771

Insecticide resistance status of indoor and outdoor resting malaria vectors in a highland and lowland site in Western Kenya

Kevin O Owuor 1,2, Maxwell G Machani 1, Wolfgang R Mukabana 2,3, Stephen O Munga 1, Guiyun Yan 4, Eric Ochomo 1, Yaw A Afrane 5,*
Editor: Basil Brooke6
PMCID: PMC7920366  PMID: 33647049

Abstract

Background

Long Lasting Insecticidal Nets (LLINs) and indoor residual spraying (IRS) represent powerful tools for controlling malaria vectors in sub-Saharan Africa. The success of these interventions relies on their capability to inhibit indoor feeding and resting of malaria mosquitoes. This study sought to understand the interaction of insecticide resistance with indoor and outdoor resting behavioral responses of malaria vectors from Western Kenya.

Methods

The status of insecticide resistance among indoor and outdoor resting anopheline mosquitoes was compared in Anopheles mosquitoes collected from Kisumu and Bungoma counties in Western Kenya. The level and intensity of resistance were measured using WHO-tube and CDC-bottle bioassays, respectively. The synergist piperonyl butoxide (PBO) was used to determine if metabolic activity (monooxygenase enzymes) explained the resistance observed. The mutations at the voltage-gated sodium channel (Vgsc) gene and Ace 1 gene were characterized using PCR methods. Microplate assays were used to measure levels of detoxification enzymes if present.

Results

A total of 1094 samples were discriminated within Anopheles gambiae s.l. and 289 within An. funestus s.l. In Kisian (Kisumu county), the dominant species was Anopheles arabiensis 75.2% (391/520) while in Kimaeti (Bungoma county) collections the dominant sibling species was Anopheles gambiae s.s 96.5% (554/574). The An. funestus s.l samples analysed were all An. funestus s.s from both sites. Pyrethroid resistance of An.gambiae s.l F1 progeny was observed in all sites. Lower mortality was observed against deltamethrin for the progeny of indoor resting mosquitoes compared to outdoor resting mosquitoes (Mortality rate: 37% vs 51%, P = 0.044). The intensity assays showed moderate-intensity resistance to deltamethrin in the progeny of mosquitoes collected from indoors and outdoors in both study sites. In Kisian, the frequency of vgsc-L1014S and vgsc-L1014F mutation was 0.14 and 0.19 respectively in indoor resting malaria mosquitoes while those of the outdoor resting mosquitoes were 0.12 and 0.12 respectively. The ace 1 mutation was present in higher frequency in the F1 of mosquitoes resting indoors (0.23) compared to those of mosquitoes resting outdoors (0.12). In Kimaeti, the frequencies of vgsc-L1014S and vgsc-L1014F were 0.75 and 0.05 respectively for the F1 of mosquitoes collected indoors whereas those of outdoor resting ones were 0.67 and 0.03 respectively. The ace 1 G119S mutation was present in progeny of mosquitoes from Kimaeti resting indoors (0.05) whereas it was absent in those resting outdoors. Monooxygenase activity was elevated by 1.83 folds in Kisian and by 1.33 folds in Kimaeti for mosquitoes resting indoors than those resting outdoors respectively.

Conclusion

The study recorded high phenotypic, metabolic and genotypic insecticide resistance in indoor resting populations of malaria vectors compared to their outdoor resting counterparts. The indication of moderate resistance intensity for the indoor resting mosquitoes is alarming as it could have an operational impact on the efficacy of the existing pyrethroid based vector control tools. The use of synergist (PBO) in LLINs may be a better alternative for widespread use in these regions recording high insecticide resistance.

Introduction

The decline in malaria incidence and prevalence have been achieved in sub-Saharan Africa through the widespread use of anti-malarial drug therapies and scaling up of vector control interventions that primarily target malaria vectors feeding and resting indoor [1]. Despite the observed achievements in malaria reduction, many parts of sub-Saharan Africa still suffer greatly from the disease [2, 3]. The recent increases in malaria transmission in many parts of sub-Saharan Africa has been partly attributed to the shifts in the mosquito biting and resting behaviours [4–7] and increasing insecticide resistance in the mosquitoes [8–10].

Insecticide resistance in malaria mosquitoes has been linked to target-site insensitivity, elevated levels of metabolic detoxifying enzymes, and behavioural resistance mechanisms [11]. Metabolic enzyme detoxification [12] and target site insensitivity [13] are responsible for higher levels of insecticide resistance [14]. Detoxification enzyme systems that have been reported to confer resistance include three major families of enzymes; the cytochrome P450 monooxygenases, esterases, and the Glutathione S-transferases. In western Kenya, about 80% of reported resistance genotypes are Vgsc-1014S kdr mutation, Vgsc-1014F mutations in the major vectors Anopheles gambiae s.l. mainly in An. gambiae and Anopheles arabiensis [15–18]. The malaria vector Anopheles arabiensis has been reported with increasing levels of kdr mutations [19]. There are no reports of kdr mutation at the locus 1014 in Anopheles funestus, also an important vector in western Kenya and many parts of Africa despite having several reports of metabolic resistance [20–22]. The increasing levels of insecticide resistance in malaria mosquitoes is believed to be mainly caused by scaling up of insecticidal treated nets (ITNs) [23, 24] and indiscriminate use of agro-chemicals for controlling crop pests in agriculture [25–27].

Field studies in East Africa have reported increased zoophagy [23, 24, 28], feeding outdoors or early evening biting [29] and changes in resting behaviour from indoor to outdoor [28, 30, 31]. These behavioural changes might have been due to selection pressure from increased coverage of LLINs [32–35]. The scale-up of LLINs in Africa has been associated with a species shift from the highly endophilic An. gambiae to the more exophilic An. arabiensis in Kenya [3, 36, 37]. The intervention pressure may selectively eliminate the most susceptible species from a population leaving the less vulnerable species able to adapt to the new environment [38]. While the majority of studies have reported the existence of insecticide resistance and the mechanisms involved, there is a paucity of detailed information on the association of insecticide resistance in malaria vectors with the observed resting behavior in the field.

Malaria transmission is dependent on the propensity of malaria vectors to feed on human hosts and preference to live in close proximity to human dwellings [7]. Given the importance of mosquito feeding and resting behaviour to the successes of malaria vector control and transmission, it is important to understand the influence of physiological resistance on the resting behaviour of malaria vectors and how the observed behaviours could impact the effectiveness of the existing frontline interventions. Currently, the mechanisms underlying the observed behavioural shifts in malaria vectors are poorly known, and it may have an epidemiological consequence. In order to maintain the efficacy of insecticide-based vector control, insecticide resistance should be constantly monitored and management strategies developed and deployed [8, 39–43]. The present study attempts to answer how insecticide use and resistance influences resting behaviours and reports on the status of insecticide resistance and mechanisms involved in indoor and outdoor resting malaria vectors.

Methods

Study sites

The study was carried out in the lowland site of Kisian (00.0749° S, 034.6663° E, altitude 1,137–1,330 m above sea level) in Kisumu county and the highland site of Kimaeti (00.6029° N, 034.4073° E, altitude 1,430–1545 m above sea level) in Bungoma county all in Western Kenya. These sites have high abundance of malaria mosquitoes (An. gambiae s.l. and An. funestus s.l.) and high level of insecticide resistance [15, 17]. Kimaeti (Bungoma county) has extensive tobacco cultivation visible by large farms with numerous curing kilns observed within the village in the region. In Kisian (Kisumu county), there is sand harvesting from river beds, fishing, rice and maize farming most of which enhance mosquito breeding habitats. There is extensive use of agrochemicals on these farms which could have a potential role in the mediation of resistance to insecticides [44]. Western Kenya experiences long rainy seasons between the months of March to June and the short rainy seasons between the months of October and November [45].

Mosquito sampling

Resting Anopheles mosquitoes were sampled indoors and outdoors from household units. Mosquito collections were made during the long rainy season (May-July) and the short rainy season (October-November) of 2019. Thirty (30) houses were randomly selected per site and resting mosquitoes collected from 06:00 to 09:00 h both indoor and outdoor resting points. For indoor resting mosquitoes, a Prokopack aspirator (JohnWHock, Gainesville, FL, USA) and mouth aspirator were employed to collect mosquitoes indoors. Briefly, collections were done by hovering the aspirator systematically over the walls up and down, under the furniture and on hanged clothing for about 1 minute per second [46, 47]. Outdoor collections were sampled from pit shelters dug (1.5M×1.5M×1.5M) in the ground constructed according to Muirhead-Thomson’s method [48], from clay pots or containers placed at least 10 meters outside of houses and from any proximal human outdoor resting points such as granaries, outdoor kitchen, under shaded places and evening outdoor human resting points. Sampled anophelines were first discriminated using morphological keys [49]. Further species-specific identification within the An. gambiae s.l. and An. funestus s.l. was conducted using PCR. Mosquito collections were done at the beginning and at the end of the dry and rainy seasons. The samples collected were taken to the entomology laboratories at the Kenya Medical Research Institute (KEMRI), Center for Global Health Research (CGHR) for subsequent rearing, phenotypic, biochemical and molecular analyses.

Rearing of mosquitoes

Blood-fed and half-gravid female Anopheles mosquitoes from both the indoor and outdoor collections were aspirated into separate labeled netted mosquito holding cages measuring 30cm × 30cm × 30cm where they were maintained at 25 ± 2°C and relative humidity of 80 ± 4% with 12:12 hours of light and dark. They were provided with 10% sucrose solution imbibed in cotton wool. Oviposition cups were introduced into the cages for egg collection. Since all collections made were put together in similar cages, the number of mosquitoes that laid eggs was not determined. Eggs collected were transferred into larval rearing trays containing spring water where they hatched. The aquatic larval stages were maintained in water 26–27°C and were fed on a mixture of Tetramin™ fish food and brewer’s yeast. After the four larval stages, pupae were picked and transferred into netted holding cages in small cups where the emergent adults were provided with 10% sucrose solution [50].

Testing phenotypic resistance in the F1 progeny of indoor and outdoor resting mosquitoes

First filial generation (F1) females raised from field-collected adults that were resting either indoors or outdoors, that were 3–5 -day old, were tested for susceptibility using the standard WHO tube bioassays (WHO, 2016) against discriminating doses of four insecticides selected from two classes: (i) Pyrethroids—(0.05% deltamethrin, 0.75% permethrin and (0.05% Alphacypermethrin); and (ii) organophosphate—(5% malathion). For each test about 100–150 mosquitoes were used for the assay comprising 20–25 mosquitoes for each of four replicates for each of the insecticides and controls. Silicone oil-treated papers were used as a control for pyrethroid assays while olive oil was used for the malathion (organophosphate) test. Mosquitoes were exposed for 1hour for each insecticide and the number that were knocked down recorded after every 10 mins within the 1-hour exposure period. After 1-hour exposure to the diagnostic concentrations, mosquitoes were transferred to recovery cups and maintained on 10% sucrose solution for 24 hrs. Mosquito survival status was examined at 24-hour post-exposure, where the survived and dead mosquitoes were collected and preserved at -20°C prior to molecular analysis. Percentage mortality was calculated for both indoor and outdoor F1 mosquitoes.

Piperonyl butoxide (PBO) synergist bioassays

The involvement of oxidase (P450) resistance mechanism in pyrethroid resistance was determined by pre-exposing test populations to the oxidase inhibitor; Piperonyl butoxide synergist (PBO). Briefly, unfed females aged 3–5 days were pre-exposed to 4% PBO impregnated test papers for one hour. After pre-exposure to PBO, the mosquitoes were immediately exposed to each of the three pyrethroids (deltamethrin, permethrin and alphacypermethrin) separately for another hour. One batch of 25 females was only exposed to 4% PBO without insecticide as a control. Mosquitoes were transferred to holding tubes and supplied with 10% sugar solution. Mortality was recorded after 24 hour recovery period.

Measurement of insecticide resistance intensity in the F1 progeny

Insecticide resistance intensity testing to deltamethrin was determined by using CDC bottle bioassay with serial dosages. Serial concentrations (1×, 5× and 10×) of deltamethrin were prepared and used for the CDC bottle assays. The bottles were coated in batches for each working concentration, to which mosquitoes were exposed as per the CDC procedure guide MR4 [50, 51]. The number of knocked-down mosquitoes was recorded every 10 minutes until either all mosquitoes in the test bottles were dead or it reached 1 hour after the start of the experiment. Mosquitoes were transferred to holding cups and fed on 10% sucrose solution. Mortality was recorded after 24-hours.

Molecular identification and genotyping of resistance alleles

Genomic DNA was extracted by the alcohol precipitation method and conventional PCR was used to speciate the samples [50, 52, 53]. The taqMan assay was used to detect the mutations (Vgsc-1014S, Vgsc-1014F and N1575Y) at the voltage-gated sodium channel [54, 55] and the same set of samples were used to detect the G119S mutation in Ace 1 [56].

Biochemical enzyme levels in F1 progeny of indoor and outdoor resting An. gambiae s.l.

From both sites, indoor and outdoor, 100-three-day old female mosquitoes, were killed by freezing for 10 minutes and homogenized individually in 0.1 M potassium Phosphate (KPO4) buffer as described by Benedict, (2014). The levels of metabolic enzymes; β-esterases, Glutathione S-transferase (GST) and Oxidases were measured using microplate enzyme assays. To correct for variations in mosquito sizes, the protein content of each mosquito was measured by adding 20μl of mosquito homogenate to the microtiter plates in triplicates and 80μl of KPO4 to each well after which 200μl of protein-dye reagent was toped up. A standard curve was used to relate amount of protein used. The absorbances were taken using a microplate reader [50, 57, 58].

Ethical considerations

Ethical approval for the study was obtained from Ethical Review Board of Kenya Medical Research Institute under number SERU 3613. Permission was sought from community leaders of each study site. Informed consent was obtained from the household heads. For mosquito larvae collection, oral consent was obtained from field owners in each location. These locations were not protected land, and the field studies did not involve endangered or protected species.

Data analysis

The phenotypic resistance assays were expressed as proportions of mortality around 95% confidence interval and classified by WHO (2016) as a guide. Genotypic data for species identification was weighted as proportions of the samples assessed. The allele frequencies for resistant genotypes were calculated using the Hardy-Weinberg equilibrium equation. Metabolic resistance enzymes were analyzed by ANOVA after which the source of variation between the fold changes was determined by the Turkey-Kramer HSD test. All statistical analyses were done in R software version 3.6.3.

Results

Species discrimination of An. gambiae s.l. and An. funestus s.l.

A total of 1074 samples were identified to species within the An. gambiae s.l. and 289 from the An. funestus s.l. from the two sites. In the lowland site of Kisian (Kisumu county), out of 500 An. gambiae s.l. samples analysed, An. arabiensis composition was 74.2% (95% CI; 71.5–78.9%) while An. gambiae s.s. was 25.8% (95% CI; 21.1–28.5%). All 122 An. funestus s.l samples analysed from indoors were An. funestus s.s. (Table 1). In the highland site of Kimaeti (Bungoma county) out of 574 An. gambiae s.s. composition was 96.5% (95% CI; 95.0–98.0%) while An. arabiensis was 3.5% (95% CI; 2.0–5.0%). The 167 An. funestus s.l. analysed were all An. funestus s.s. (Table 1).

Table 1. An. gambiae s.l. and An. funestus s.l. species composition from indoor and outdoor resting collections from Western Kenya.

Site Anopheles gambiae s.l Anopheles funestus s.l.
Location An. gambiae s.s (%) An. arabiensis(%) Total An. funestus s.s
Kisian Indoor 83(33.2) 167(66.8) 250 122(100)
Outdoor 46(18.4) 204(81.6) 250 0
Total 129(25.8) 371(74.2) 500 122(100)
Kimaeti Indoor 304(99.1) 3(0.9) 307 167(100)
Outdoor 250(93.6) 17(6.4) 267 0
Total 554(96.5) 20(3.5) 574 167(100)
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Phenotypic resistance in the F1 progeny of indoor and outdoor mosquitoes

A total of 2,800 female An. gambiae s.l. (Kisian = 1,400 and Kimaeti = 1,400) and 1,600 female An. funestus s.l. (Kisian = 800 and Kimaeti = 800) were used in the WHO tube assays. In the lowland site of Kisian, the mortality rate of the indoor resting An. gambiae s.l. mosquitoes exposed to deltamethrin was significantly lower than outdoors resting ones (37%, 95% [CI; 28–46%]) vs 51% [95% CI; 41–61%] respectively; t = 2.035, df = 6, P = 0.044). The indoor resting An. gambiae s.l. had significantly lower mortality rate to permethrin than those resting outdoors (31% [95% CI; 22–40%] vs 51% [95% CI; 41–61%], t = 2.078, df = 6, P = 0.042). Following exposure to alphacypermethrin, the mortality rate for indoor resting An. gambiae s.l. was 30% (95% CI; 21–39%) compared to their outdoor counterparts with 60% (95% CI; 50–70%) (t = 4.392, df = 6, P<0.05). There was 100% mortality for both the indoor resting and outdoor resting Anopheles gambiae s.l. when exposed to malathion (Fig 1A).

Fig 1. Percentage mortality rates for indoor and outdoor resting A.) An gambiae s.l B.) An funestus F1 progeny from Kisian (lowland) and Kimaeti (Highland) using WHO tube bioassays.

Fig 1

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Error bars indicate 95% confidence intervals. The 90% mortality threshold for declaring suspected resistance and 98% mortality threshold for calling full susceptibility based on the WHO criteria are indicated.

Indoor resting F1 progeny raised from Anopheles gambiae s.l. collected from the highland site of Kimaeti had a mortality rate of 49% (95% CI; 39–59%) compared to those resting outdoors 53% (95% CI; 43–63%) when exposed to deltamethrin. Although the indoor resting mosquitoes showed a slightly lower mortality rate compared to outdoors, this was not statistically significant (t = 0.474, df = 6, P>0.05). Exposure of mosquitoes to permethrin showed for indoor resting mosquitoes had a significantly lower mortality 7% (95% CI; 1–12%) compared to those resting outdoors 51% (95% CI; 41–61%), (t = 6.063, df = 6, P<0.001). Mosquitoes exposed to alphacypermethrin on the other hand showed a mortality rate of 70% (95% CI; 61–79%) for indoor resting mosquitoes compared to those resting outdoors outdoors80% (95% CI; 72–88%), though this was not significantly different (t = 1.058, df = 6, P>0.05). Exposure of mosquitoes from the indoor or outdoor location in showed that An gambiae s.l. were fully susceptible to malathion with 100% mortality (Fig 1A).

Addition of PBO synergist to the test, partially restored the resistance of indoor resting mosquitoes from 37% to 96% for deltamethrin (t = 9.0, df = 6, P<0.001), 31% to 79% permethrin (t = 5.908, df = 6 P = 0.005) and 30% to 92% for alphacypermethrin (t = 8.598, df = 6, P<0.001) in Kisian. The effects of the PBO synergist was evident in the outdoor resting mosquitoes with mortality rate range; 98%-100% for the three pyrethroids used, confirming the full involvement of monooxygenase enzyme activity in the pyrethroid detoxification (Fig 1A).

In Kimaeti, the addition of PBO to tests involving indoor resting An. gambiae s.l. showed significantly increased mortality rate from 49% to 100% (t = 7.095, df = 6, P<0.001) for deltamethrin, 7% to 95% (t = 16.436, df = 6, P<0.001) for permethrin and 70% to 99% (t = 5.385, df = 6, P = 0.001) for alphacypermethrin. The effects of the PBO synergist was also seen in outdoor resting mosquitoes with the mortality rate ranging between 94% and 100% (Fig 1A).

Due to the small number collected outdoors and the general difficulty in raising the F1, only indoor An. funestus s.l. from both study sites were assayed. In Kisian, the mortality rate of An. funestus was 68% (95% CI; 59–77%) to deltamethrin, 74% (95% CI; 65–83%) to permethrin and 77% (95% CI; 69–85%) to alphacypermethrin (Fig 1B). In Kimaeti, the F1 of An. funestus showed mortality rates of 62% (95% CI; 52–72%) when exposed to deltamethrin, 89% (95% CI; 83–95%) to permethrin and 61% (95% CI; 51–71%) following alphacypermethrin exposure. There was 100% mortality across both sites with PBO pre-exposure (Fig 1B).

Intensity of insecticide resistance in F1 of An. gambiae s.l. resting indoors and outdoors

The mortality rate for indoor An. gambiae s.l. from Kisian that were exposed to 1×, 5× and 10× of the diagnostic doses of deltamethrin was 42% (95% CI; 32–52%), 78% and 100% respectively whilst for outdoors was 51% (95% CI; 41–61%), 83% (95% CI; 76–90%) and 100%, indicating moderate-intensity resistance across both locations according to the WHO 2016 criteria [59] (Fig 2). Although there was lower mortality among the indoor resting mosquitoes compared to their outdoor counterparts at 1× (t = 1.269, df = 6, P = 0.130) and at 5× (t = 0.823, df = 6, P = 0.221), this was not statistically significant (Fig 2).

Fig 2. Mortality rates of An. gambiae s.l F1 progeny from indoor and outdoor resting collections recorded using CDC bottle intensity assays.

Fig 2

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Error bars indicate 95% confidence intervals. The 90% mortality threshold for declaring suspected resistance and 98% mortality threshold for calling full susceptibility based on the WHO criteria are indicated.

The mortality rate of indoor resting population from Kimaeti exposed to 1×, 5× and 10× concentration of deltamethrin were 31% (95% CI; 22–40%), 75% (95% CI; 67–83%) and 100% respectively while the outdoors were 48% (95% CI; 38–58%), 80% (95% CI; 72–88%) and 100% respectively indicating moderate-intensity resistance in both locations according to the WHO 2016 criteria [59]. Similarly, even though the mortality rates were lower indoors than outdoors, there was no significant statistical difference between the two populations at 1× (t = 1.512, df = 6, P>0.05) and at 5× (t = 0.808, df = 6, P>0.05) (Fig 2).

Target site genotyping for resistance alleles in the F1 of indoor and outdoor resting An. gambiae s.l.

In Kisian, the frequency of the vgsc L1014S and L1014F in the progeny of mosquitoes resting indoors were present with frequencies of 0.14 and 0.19 respectively for the F1of indoor resting mosquitoes whereas those raised from mosquitoes resting outdoors were 0.14 and 0.12 respectively. The ace 1 mutation was present by higher frequency in the F1 of mosquitoes resting indoors (0.23) compared to those of the ones resting outdoors (0.12). The vgsc-1014S and ace 1 mutations were not observed in An. gambiae from Kisian due to the small sample size.

The frequency of L1014S and L1014F present in mosquitoes collected indoors were 0.75 and 0.05 respectively in Kimaeti compared to those raised from mosquitoes collected outdoors (0.67 and 0.03 respectively). The ace 1 G119S mutation was observed in the F1 of mosquitoes resting indoors with a frequency of 0.05 and was not present in those of mosquitoes resting outdoors. The kdr point mutation at locus 1575Y was not present in both study sites (Table 2).

Table 2. Frequency of resistant alleles (Kdr and Ace1-G119S) in indoor and outdoor-resting An. gambiae s.s and An. arabiensis populations from Western Kenya.

Site Location Species n Vgsc (kdr) Ace 1
Locus 1014 Locus 1575 Locus 119
L1014S L1014F 1575Y G119S
Kisian Indoor An.gambiae 8 0 0.25 0 0
An.arabiensis 36 0.14 0.19 0 0.23
Outdoor An.gambiae 1 0 0 0 0
An.arabiensis 43 0.14 0.12 0 0.12
Total An. gambiae 9 0 0.33 0 0
An.arabiensis 79 0.08 0.06 0 0.19
Kimaeti Indoor An.gambiae 43 0.75 0.05 0 0.05
An.arabiensis 1 0.01 0 0 0
Outdoor An.gambiae 39 0.67 0.03 0 0
An.arabiensis 5 0.60 0 0 0
Total An.gambiae 82 0.72 0.06 0 0.02
An.arabiensis 6 0.07 0 0 0
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Biochemical enzyme levels in F1 progeny of indoor and outdoor resting An. gambiae s.l.

The monooxygenases, β-Esterase and Glutathione S-transferases activities were analyzed to determine the level of involvement in the F1 of An. gambiae s.l. insecticide resistance. In Kisian, the monooxygenase activity was increased by 1.83 folds in the progeny of An. gambiae s.l. resting indoors and by 1.66-folds for those resting outdoors when compared to the insectary reference Kisumu strain (F2,134 = 105.20, P<0.05, Fig 3A). The β-Esterases fold change was not significantly different between F1 progeny raised from indoor and outdoor resting An. gambiae s.l. mosquitoes (F2,134 = 188.50, P<0.05, Fig 3B). In Kisian, the elevation of GSTs was by a 2.3-fold change in the F1 of indoor-resting mosquitoes which was significantly higher than that of the F1 of those resting outdoors (F2,134 = 95.14, P<0.05, Fig 3C).

Fig 3. Metabolic enzyme activity for indoor and outdoor resting F1 progeny of An. gambiae from Kisian and Kimaeti in Western Kenya.

Fig 3

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A: monooxygenases; B: β-esterases; and C: Glutathione S-transferase. Enzyme activities were expressed as the ratio of a population of interest to the Kisumu reference strain. Error bars indicates 95% confidence intervals. *, P < 0.05; ***, P < 0.001; NS; not significant.

The enzyme activity of monooxygenases was higher by 1.3-fold in the indoor population from Kimaeti compared to the outdoor population (F2,134 = 51.43, P<0.05, Fig 3A). The activity of β-esterases from Kimaeti was elevated by 1.2 folds for the indoor-resting population which was significantly different compared to that of the outdoor resting mosquitoes (F2,134 = 36.66, P<0.001, Fig 3B). The activity of Glutathione S-transferase was elevated by a 3.0-fold change in the progeny of mosquitoes found resting indoors than those found resting outdoors (F2,134 = 119.9, P<0.05) (Fig 3C).

Discussion

This study set out to determine the level of insecticide resistance of Anopheles mosquito species between populations found resting indoors and those resting outdoors. Generally, high phenotypic, physiological (genotypic and metabolic) resistance was observed in the progeny of indoor resting malaria mosquitoes than the outdoor resting vectors.

In the lowland sites of Kisian (Kisumu county), An. arabiensis was the most abundant malaria vector compared to its sibling species An. gambiae s.s. whereas in Kimaeti (Bungoma county), the dominant species was An. gambiae s.s. similar to earlier reports [17, 21, 28, 60, 61]. The lowlands tend to have high temperatures and low humidity which favour the more resilient An arabiensis whereas in the highlands, there are low temperatures and high relative humidity which favour An gambiae [62].

The indoor population recorded high phenotypic resistance to pyrethroids than outdoors. The phenotypic insecticide resistance to pyrethroids in An. gambiae s.l. is widespread in Western Kenya evident in previous studies [15, 17, 19]. The resistance to pyrethroids by An. funestus was observed and has as well been reported before [63]. These regions of Western Kenya have been reported to have increasing resistance to pyrethroids which are the public health approved insecticides for use in LLINs [15, 17, 20, 42]. There was 100% susceptibility to malathion of mosquitoes just as similar studies have shown in Ghana [64]. Synergist PBO pre-exposure restored susceptibility for both indoor and outdoor resting mosquitoes, revealing the role of detoxifying metabolic enzymes in the insecticide resistance in these regions. This means, therefore, that there are more factors at play contributing to the insecticide resistance present in Western Kenya similar to studies before [12, 65, 66]. Increasing the concentration of the deltamethrin in CDC bottle assays restored susceptibility to 100% suggesting that the continuous exposure to the current dosage in LLINs and possible interaction with non-lethal doses in agricultural chemicals could have been at play to contribute to the development of resistance to pyrethroids as previously demonstrated [38] in indoor resting and outdoor resting malaria mosquitoes. The result showed moderate intensity insecticide resistance since the mosquitoes succumbed to the highest concentration according to the WHO test procedures for insecticide resistance monitoring in malaria vectors [59]. The buildup of the phenotypic resistance which was higher in indoor resting mosquitoes compared to the outdoor resting counterparts might be threatening current insecticide-based malaria control interventions as suggested by prior studies [67, 68].

The presence of resistance-associated point mutations was more in indoor resting mosquitoes than their outdoor resting counterparts. This can be attributed to the adaptations from selection pressures due to constant exposure to insecticide-based interventions such as LLINs [17, 23, 39, 69] and the extensive chemicals used in the tobacco farms in Kimaeti. The study also detected, even though in lower frequencies, a significant proportion of the vgsc-1014S and 1014F in An. arabiensis a phenomenon that has been previously reported [17, 19, 66]. This is in line with studies that have shown the occurrence of more than one kdr associated point mutation within a population of An. gambiae s.l. already reported previously [17, 20, 61, 66, 70]. The significant vgsc mutations observed could be a result of selection pressure build-up that is due to more contact with insecticides in indoor-based interventions [17, 39, 42, 61, 66]. From Kisian, the G119S mutation was present at low frequencies even though it was higher in the progeny of mosquitoes resting indoors compared to those resting outdoors. This was more in Kisian, where the vgsc mutations were at lower frequencies than in Kimaeti. These findings suggest that these mutations could be arising from different pressures that could be present in the lowland and absent in the highland.

The metabolic enzymes, associated with insecticide resistance (monooxygenases, β-esterases, and glutathione S-transferases) activities were found to be elevated, more in indoor resting malaria mosquitoes compared to the outdoor counterparts from both sites. From the phenotypic assays, pre-exposure to PBO synergist restored the susceptibility of the malaria vectors to the pyrethroids commonly used in LLINs by public health. Phenotypic exposures with prior PBO contact demonstrated more activity of monooxygenases in aiding metabolic resistance. The involvement of monooxygenases in pyrethroid resistance has been reported in Western Kenya [17]. In Kimaeti, there was increased levels β-esterases, higher indoors than outdoors. Kisian, on the other hand, did not show involvement of β-esterases in contributing to resistance as shown by similar levels in indoor and outdoor resting mosquitoes. The glutathione-S-transferase possibly played a part in the resistance levels as a previous study reported [71] since it was higher in mosquitoes resting indoors than those resting outdoors from both Kisian and Kimaeti. These levels, therefore, suggest that monooxygenases were the main mechanism of insecticide resistance in Kisian, especially with the low frequency of resistant alleles, whereas in Kimaeti, the case pointed be a combination of genotypic and metabolic mechanisms.

The expression of phenotypic, genotypic and metabolic resistance appears to be higher in indoor than outdoor resting malaria mosquitoes in these regions. The widespread use of LLINs in attempts to controlling these vectors and the extensive agrochemical use could be strengthening the increase of insecticide resistance in the sites [21, 61]. The higher levels indoors suggest that these mosquitoes could be resting indoors because they are adequately resistant to the insecticides used in LLINs, posing a threat to the wide coverage LLINs [21]. On the other hand, outdoors, the resistance mechanisms were present as well pointing to exposure to these insecticide-based interventions in just enough pressure to elicit expression of the resistance traits. The levels of resistance could be enough to elicit an increase in malaria incidence due to the reduced mortality of resistant malaria vectors that could hinder current vector control interventions [67].

Conclusion

In this study there was high phenotypic, genotypic and metabolic insecticide resistance in indoor resting malaria vectors (An. gambiae s.l and An. funestus) compared to outdoor-resting mosquitoes. Indoor-based insecticide control interventions are potentially at the verge of becoming obsolete due to the reduced efficacy in controlling resistant malaria vectors which in turn might lead to rise in malaria incidence. This calls for urgent improvement of these interventions and development of alternative tools for indoor malaria control coupled with strengthening of insecticide resistance monitoring. The use of synergist (PBO) in LLINs may be a better alternative for widespread use in these regions recording high insecticide resistance.

Acknowledgments

The authors wish to thank the villagers and community leaders for their permission to collect mosquitoes in their houses. In particular, we thank the community leaders. We acknowledge the Entomology Laboratory at Kenya Medical Research Institute, Kisumu, more specific Joyce K. Osoro, Mathew Kipsum, Christabel A. Winter, Duncan O. Tindi and Benard O. Alele for providing technical support and laboratory space for the study. This paper is published with permission from the director of Kenya Medical Research Institute.

Data Availability

All relevant data are within the paper.

Funding Statement

This study was supported by grants from the National Institute of Health (R01 A1123074, U19 AI129326, R01 AI050243, D43 TW001505). There was no additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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10 Dec 2020

PONE-D-20-30800

Insecticide resistance status of indoor and outdoor resting malaria vectors in a highland and lowland site in Western Kenya

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: My comments are below

Introduction

Line 73 to 75 « Despite the observed improvement in malaria incidence and prevalence in many parts of sub-Saharan Africa, transmission is increasing in several countries [2,3]. » I don’t think the term « improvement » is appropriate and the sentence doesn’t make sense

Line 80 to 81 « Insecticide resistance in malaria mosquitoes is linked to presence and increase in metabolic detoxification enzymes, target site insensitivity and behavioural resistance [11]. » the sentence is not clear can the author rephrase the sentence to improve understanding?

Line 84 to 86 « Detoxification enzyme systems that have been reported to confer resistance include three major families of enzymes; the cytochrome P450 monooxygenases, β-esterases, and the Glutathione S-transferases. » please write esterase as you are refering to enzymes families

Line 92 to 95 « The increasing levels of insecticide resistance in malaria mosquitoes have been associated with continuous exposure to insecticides in Long Lasting Insecticide Nets( LLINs) [23,24] and agro-chemicals such as pesticides due to the creation of selection pressures [25-27]. » The phrase is not clear and need to be rewritten to improve understanding

The introduction is not straight forward and is poorly written the authors need to revise this section carefully to improve understanding

Methods

Study sites

Line 120 « The study was carried out in the lowland site of Kisian (0.0749° S, 34.6663° E, 1,137m) in Kisumu county … » The geographical coordinates is not appropriate can the authors check for the correct coordinates ?

Line 127 to 128 « There is extensive use of agrochemicals on these farms which could have a potential role in the mediation of resistance to insecticides. » Are anopheline breeding habitats found in tobacco farms and what type of insecticides are used in these farms please provide more information. Please also say how frequent insecticides are sprayed in these farms. How distant are these farms from the village.

Mosquito sampling

Mosquitoes were collected in how many houses per site and outdoor places please include information

Line 156 to 160 « First filial generation (F1) females raised from field-collected adults that were resting either indoors or outdoors, that were 3-5 -day old, were tested for susceptibility using the standard WHO tube bioassays (WHO, 2016) against discriminating doses of five insecticides selected from three classes: (i) Pyrethroids - (0.05% deltamethrin, 0.75% permethrin and (0.05% Alphacypermethrin); and (ii) organophosphate - (5% malathion). » The author say they tested 5 insecticides only a list of four insecticide is provided please correct.

Line 166 to 167 « Mortality was defined as the inability of the mosquitoes to stand or to fly in a coordinated manner. » is this mortality or knock down effect please correct

Line 175 to 176 « After pre-exposure to PBO, the mosquitoes were immediately exposed to the three pyrethroids (deltamethrin, permethrin and alphacypermethrin) for an additional hour. » Where mosquitoes exposed to all three insecticides at once or seperately to each insecticide please clarify

Results

There are data missen in this part the author need to give the number of gravid and non gravid females collected outdoor and indoor in each site. They could also indicate the number collected during each season.

The number of mosquitoes tested for each insecticide need to be provided

Table 1

The authors say that the percentage is in bracket but it is not clear how they did the calculations of the percentage of An. funestus in Kisian 122(19%) in Kimaeti they also wrote 167 (23.16) can they provide explanation for their calculations or correct.

The author also need to check their calculations the row on An. arabiensis in Kisian the total in the column and line also need to be corrected.

Reviewer #2: Comments:

This study intends to show the insecticides resistance between indoors and outdoors resting vectors in highlands and lowlands

This study has a major drawback in methodology.

To start with the ecological behavior of An. gambiae s.l is to feed indoor and rest indoor for An. gambiae s.s. while for An. arabienses is feeding indoor and resting outdoor or feed outdoor and rest outdoors. Due to the wide coverage of the insecticide treated surfaces (LLINs and RS), mosquitoes have changed the feeding and resting behavior to forfeit the effect of the insecticides treated surfaces. Due to this, higher proportions have found to feed and rest outdoors regardless of the vector species.

Sampling:

The samplings of vectors were done indoor and outdoor. The vector after feeding indoor they get out to rest and search for oviposition site. This is indeed a challenge to mark that this mosquito was found resting outdoor while it fed indoor. The physiological and enzymatic dynamics in mosquitoes is mostly reflected by blood meal source. There is no barrier between indoor and outdoor resting populations hence free mating is possible. In first place, authors should be aware that resistance is genetical and is clearly related age of the mosquito. These mosquitoes share breeding sited and feeding sources. The emerging young mosquitoes resting outdoor before searching for host. The outdoor collection is probably that it collected emerged and gravid mosquitoes searching for oviposition site. The methods were weak not to show the separation of population fed outdoor or indoor. So collection alone cannot provide a guarantee of the biochemical processes to be elevated by resting position.

I personally reject this paper as the basis used of outdoor and indoor resting have no barrier separation between the populations collected. Since resistance is genetical and biochemical process is influenced by blood meal sources. No evidence of where the mosquitoes fed (i.e. blood meal source). That was not shown so as to give the=at strong evidence.

This study can compare the enzyme changes and resistance and enzyme presentation between lowland and highland but not between indoor and outdoor resting mosquitoes

More specific comments are uploaded in attached document.

**********

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Reviewer #1: No

Reviewer #2: No

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Attachment

Submitted filename: comments.docx

Click here for additional data file. (12.5KB, docx)
PLoS One. 2021 Mar 1;16(3):e0240771. doi: 10.1371/journal.pone.0240771.r002

Author response to Decision Letter 0

13 Jan 2021

Response to reviewer comments

The following are our point-by-point responses to the comments raised by the reviewers. We really thank the reviewers for their constructive criticism and insights that have helped to improve this paper.

Reviewer Comments:

Reviewer #1

Reviewer: Line 73 to 75 « Despite the observed improvement in malaria incidence and prevalence in many parts of sub-Saharan Africa, transmission is increasing in several countries [2,3]. » I don’t think the term « improvement » is appropriate and the sentence doesn’t make sense

Response: The sentence has been revised and now reads” Despite the observed achievements in malaria reduction, many parts of sub-Saharan Africa still suffer greatly from the disease”….Line 72-74

Reviewer: Line 80 to 81 « Insecticide resistance in malaria mosquitoes is linked to presence and increase in metabolic detoxification enzymes, target site insensitivity and behavioural resistance [11]. » the sentence is not clear can the author rephrase the sentence to improve understanding?

Response: The sentence has been revised to read “Insecticide resistance in malaria mosquitoes has been linked to target-site insensitivity, elevated levels of metabolic detoxifying enzymes”……Line 77-78

Reviewer: Line 84 to 86 « Detoxification enzyme systems that have been reported to confer resistance include three major families of enzymes; the cytochrome P450monooxygenases, β-esterases, and the Glutathione S-transferases. » please write esterase as you are refering to enzymes families

Response: This has been addressed …Line 82.

Reviewer: Line 92 to 95 « The increasing levels of insecticide resistance in malaria mosquitoes have been associated with continuous exposure to insecticides in LongLasting Insecticide Nets( LLINs) [23,24] and agro-chemicals such as pesticides due to the creation of selection pressures [25-27]. » The phrase is not clear and need to be rewritten to improve understanding

Response: The statement has been rewritten and now it reads “ The increasing levels of insecticide resistance in malaria mosquitoes is believed to be mainly caused by scaling up of insecticidal treated nets (ITNs) [23,24] and indiscriminate use of agro-chemicals for controlling crop pests in agriculture[25-27] ..Line 88-90

Reviewer: The introduction is not straight forward and is poorly written the authors need to revise this section carefully to improve understanding

Response: The introduction section has been revised to improve understanding.

Reviewer: Line 120 « The study was carried out in the lowland site of Kisian (0.0749° S, 34.6663° E, 1,137m) in Kisumu county … » The geographical coordinates is not appropriate can the authors check for the correct coordinates ?

Response: The geographical coordinates provided for Kisian are the correct one “00.0749° S, 034.6663° E, altitude 1,137-1,330 m above sea level)”

Reviewer: Line 127 to 128 « There is extensive use of agrochemicals on these farms which could have a potential role in the mediation of resistance to insecticides. » Are anopheline breeding habitats found in tobacco farms and what type of insecticides are used in these farms please provide more information. Please also say how frequent insecticides are sprayed in these farms. How distant are these farms from the village.

Response: The sentence has been modified from line 120 -125 and also cited Wanjala et al 2015 which has detailed information on the agrochemicals used in the study area.

Reviewer: Mosquitoes were collected in how many houses per site and outdoor places please include information.

Response: The information on the number of houses sampled and outdoor places has been included under mosquito sampling section …. ‘Thirty (30) houses were randomly selected per site and resting mosquitoes collected from 06:00 to 09:00 h……” Line 130-139

Reviewer: Line 156 to 160 « First filial generation (F1) females raised from field-collected adults that were resting either indoors or outdoors, that were 3-5 -day old, were tested for susceptibility using the standard WHO tube bioassays (WHO, 2016) against discriminating doses of five insecticides selected from three classes: (i)Pyrethroids - (0.05% deltamethrin, 0.75% permethrin and (0.05% Alphacypermethrin); and (ii) organophosphate - (5% malathion). » The author say they tested 5 insecticides only a list of four insecticide is provided please correct.

Response: This has been corrected in the text, four insecticides from 2 classes of insecticides were used Line161.

Reviewer: Line 166 to 167 « Mortality was defined as the inability of the mosquitoes to stand or to fly in a coordinated manner. » is this mortality or knock down effect please correct.

Response: The statement has been deleted from the text.

Reviewer: Line 175 to 176 « After pre-exposure to PBO, the mosquitoes were immediately exposed to the three pyrethroids (deltamethrin, permethrin and alphacypermethrin) for an additional hour. » Where mosquitoes exposed to all three insecticides at once or seperately to each insecticide please clarify

Response: The sentence has been corrected and now reads “After pre-exposure to PBO, the mosquitoes were immediately exposed to each of the three pyrethroids (deltamethrin, permethrin and alphacypermethrin) separately for another hour….Line 177-178

Reviewer: There are data missen in this part the author need to give the number of gravid and non gravid females collected outdoor and indoor in each site. They could alsoindicate the number collected during each season.

Response: We have included the data for the gravid females that were used for the study from line 155-157.

Reviewer: The number of mosquitoes tested for each insecticide need to be provided

Responses: The number of mosquitoes tested for each insecticide was 150 according to the WHO 2016 Insecticide resistance testing procedures the information is already provide in the text.. Line 163

Reviewer: The authors say that the percentage is in bracket but it is not clear how they did the calculations of the percentage of An. funestus in Kisian 122(19%) in Kimaeti they also wrote 167 (23.16) can they provide explanation for their calculations or correct. The author also need to check their calculations the row on An. arabiensis in Kisian the total in the column and line also need to be corrected.

Response: The figures in table 1 has been corrected

Reviewer #2

This study intends to show the insecticides resistance between indoors and outdoors resting vectors in highlands and lowlands This study has a major drawback in methodology. To start with the ecological behavior of An. gambiae s.l is to feed indoor and rest indoor for An. gambiae s.s. while for An. arabienses is feeding indoor and resting outdoor or feed outdoor and rest outdoors. Due to the wide coverage of the insecticide treated surfaces (LLINs and RS), mosquitoes have changed the feeding and resting behavior to forfeit the effect of the insecticides treated surfaces. Due to this, higher proportions have found to feed and rest outdoors regardless of the vector species.

Sampling: The samplings of vectors were done indoor and outdoor. The vector after feeding indoor they get out to rest and search for oviposition site. This is indeed achallenge to mark that this mosquito was found resting outdoor while it fed indoor. The physiological and enzymatic dynamics in mosquitoes is mostly reflected byblood meal source. There is no barrier between indoor and outdoor resting populations hence free mating is possible. In first place, authors should be aware thatresistance is genetical and is clearly related age of the mosquito. These mosquitoes share breeding sited and feeding sources. The emerging young mosquitoesresting outdoor before searching for host. The outdoor collection is probably that it collected emerged and gravid mosquitoes searching for oviposition site. Themethods were weak not to show the separation of population fed outdoor or indoor. So collection alone cannot provide a guarantee of the biochemical processes tobe elevated by resting position. I personally reject this paper as the basis used of outdoor and indoor resting have no barrier separation between the populations collected. Since resistance isgenetical and biochemical process is influenced by blood meal sources. No evidence of where the mosquitoes fed (i.e. blood meal source). That was not shown so as to give the=at strong evidence.

This study can compare the enzyme changes and resistance and enzyme presentation between lowland and highland but not between indoor and outdoor resting mosquitoes More specific comments are uploaded in attached document.

Response: Our study looked at the differences in insecticide resistance between indoor and outdoor resting mosquitoes. The focus was not on biting mosquitoes as mosquitoes are likely to be influenced by where the host might be, whether indoor or outdoor, to take a blood meal. They are likely to make a choice, after taking a blood meal, between resting indoors or outdoors based on their preferences as exophilic or endophilic vectors. We acknowledge that the taking of a blood meal by mosquitoes might influence their insecticide resistance status through the raising of biochemical enzymes to tolerate insecticides. This made us not to use the female mosquitoes collected but their F1 progeny so that we can understand whether resting behaviour is a consistent behaviour. We have clarified our objective and some parts of the methodology, which should make our methodology clear to answer the points raised by the reviewer 2. We strongly disagree with the reviewer that we should have shown where the mosquitoes took their blood meals. We also reject the suggestion that we “can compare the enzyme changes and resistance and enzyme presentation between lowland and highland”. This has no scientific basis. The methodology in our study was not weak as alleged by the reviewer but probably he may not have clearly understood it.

Editorial comments

To the editor:

Figure 1 image has been removed from the manuscript.

We have also taken the ethics statement to the methods section

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file. (27.2KB, docx)

Decision Letter 1

Basil Brooke

16 Feb 2021

Insecticide resistance status of indoor and outdoor resting malaria vectors in a highland and lowland site in Western Kenya

PONE-D-20-30800R1

Dear Dr. Afrane,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Basil Brooke, PhD

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

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Reviewer #2: Yes

**********

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Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: I have realized that, all comments I raised in first place were all addressed in this revised version.

**********

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Acceptance letter

Basil Brooke

19 Feb 2021

PONE-D-20-30800R1

Insecticide resistance status of indoor and outdoor resting malaria vectors in a highland and lowland site in Western Kenya

Dear Dr. Afrane:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

Dr Basil Brooke

Academic Editor

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