Skip to main content

Insecticidal decay effects of long-lasting insecticide nets and indoor residual spraying on Anopheles gambiae and Anopheles arabiensis in Western Kenya



Indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) are the first-line tools for malaria prevention and control in Africa. Vector resistance to insecticides has been extensively studied, however the insecticidal effects of the nets and sprayed walls on pyrethroid resistant mosquitoes has not been studied thoroughly. We evaluated the bioefficacy of LLINs of different ages and lambda-cyhalothrin (ICON 10cs) on the sprayed mud walls for a period of time on malaria vector survivorship.


WHO tube bioassay was performed using diagnostic doses of lambda-cyhalothrin (0.05 %), permethrin (0.75 %) and deltamethrin (0.05 %). Cone bioassays were conducted on netting materials from 0 to 3 years old long-lasting insecticide-impregnated nets. Wall bioassays were performed monthly on mud slabs sprayed with lambdacyhalothrin over a period of seven months. All bioassays used An. gambiae mosquitoes collected from the field and the laboratory susceptible reference Kisumu strain. Concentration of the insecticides on the netting materials was examined using the gas chromatography method. Mosquitoes were identified to species level using PCR and genotyped for the kdr gene mutation frequencies.


WHO bioassays results showed that populations from five sites were highly resistant to the pyrethroids (mortalities ranged from 52.5 to 75.3 %), and two sites were moderately resistant to these insecticides (80.4 – 87.2 %). Homozygote kdr mutations of L1014S ranged from 73 to 88 % in An. gambiae s.s. dominant populations whereas L1014S mutation frequencies were relatively low (7–31 %) in An. arabiensis dominant populations. There was a significant decrease (P < 0.05) in mosquito mortality with time after the spray with both lambda-cyhalothrin (75 % mortality after six months) and with the age of LLINs (60 % mortality after 24 month). Field collected mosquitoes were able to survive exposure to both IRS and LLINs even with newly sprayed walls (86.6–93.5 % mortality) and new LLINs (77.5–85.0 % mortality), Wild mosquitoes collected from the field had significantly lower mortality rates to LLINs (59.6–85.0 %) than laboratory reared susceptible strain (100 %). Insecticide concentration decreased significantly from 0.14 μg/ml in the new nets to 0.077 μg/ml in nets older than 18 months (P < 0.05).


This study confirms that insecticide decay and developing levels of resistance have a negative contribution to reduced efficacy of ITN and IRS in western Kenya. These factors contribute to decreased efficacy of pyrethroid insectides in ongoing malaria control programs. In order to mitigate against the impact of insecticide resistance and decay it is important to follow the WHO policy to provide the residents with new LLINs every three years of use while maintaining a high level of LLINs coverage and usage. There is also need for urgent development and deployment of non-pyrethroid based vector control tools.


Despite global efforts to malaria control, malaria remains a major public health problem particularly in Africa where more than 80 % of the cases are reported [1]. Long lasting insecticide nets (LLINs), indoor residual spraying (IRS) and case management using artemisinin-combination therapy are the key tools currently used for malaria control [1]. There are indications of variations in compliance and insecticide resistance in Kenya [2, 3], however the role of insecticide decay in LLINs and IRS is not well understood in the context of high insecticide resistance. The efficacy of LLIN and IRS for malaria control depends on the adherence to the specified insecticide application procedure, insecticide resistance in mosquito population and the persistence of the insecticides on the sprayed surface. Mosquito resistance to insecticide, which reduces the efficacy of LLINs, has been intensively studied across Africa, and resistance has been detected in all major African malaria vectors and resistance to multiple classes of insecticides is not uncommon [415]. However, insecticides may lose their effectiveness due to chemical degradation over time and bioavailability of the insecticide on the sprayed surface which may be affected by the porosity of the sprayed surface [16]. Reduced effectiveness of ITN/LLIN and IRS control programs can contribute to an upsurge of malaria incidence [17, 18].

The decay of insecticidal efficacy with time in IRS program has been previously studied. For example, re-spray was not needed after 6 to 12 months of DDT spray, but houses sprayed with lambda-cyhalothrin would need to be re-sprayed every 3–4 months to maintain acceptable efficacies [19]. Insecticidal efficacy decay is less clear in LLINs, particularly when mosquitoes are modestly resistant to pyrethroids. Three classes of LLINs are recommended by WHO: 1) permethrin-incorporated net - a LLIN made of high density polyethylene monofilament yarn blended with 2 % permethrin, 2) deltamethrin-coated net – a LLIN made of multifilament polyester netting treated with deltamethrin, and 3) alphacypermethrin-coated net – a LLIN made of multifilament polyester netting treated with alphacypermethrin [20]. Currently 11 brands of LLINs are recommended by WHO for public use: 1) DawaPlus 2.0 - Deltamethrin coated on polyester; 2) Duranet - Alpha-cypermethrin incorporated into polyethylene; 3) Interceptor - Alpha-cypermethrin coated on polyester; 4) LifeNet - Deltamethrin incorporated into polypropylene; 5) MAGNet - Alpha-cypermethrin incorporated into polyethylene; 6) Olyset Net - Permethrin incorporated into polyethylene; 7) Olyset Plus - Permethrin and PBO incorporated into polyethylene; 8)PermaNet 2.0 - Deltamethrin coated on polyester; 9) PermaNet 3.0 - Combination of deltamethrin coated on polyester with strengthened border (side panels), deltamethrin and PBO incorporated into polyethylene (roof); 10) Royal Sentry- Alpha-cypermethrin incorporated into polyethylene; and 11) Yorkool LN - Deltamethrin coated on polyester [20]. Duration of protective efficacy should be at least 3 years under recommended conditions of use. Indeed, a number of studies reported reduced efficacy of LLINs after several years of use [18, 21, 22]. Some nets have been shown to be less effective in inhibiting mosquito feeding [23], and wear and washing of nets may cause further insecticidal efficacy decay [24].

Intensive pyrethroid-based malaria vector control program in the past decade in Africa has led to rapid rise of pyrethroid resistance in malaria vectors across Africa. For example, pyrethroid resistance was reported in An. gambiae in 27 sub-Saharan African countries, in An. arabiensis in 14 sub-Saharan countries, and An. funestus in at least 4 countries [14]. In addition to being geographically widespread, knockdown resistance (kdr) mutations have reached to extremely high frequency levels for An. gambiae throughout Africa [13, 2531]. The scale up of ITN and IRS programs will select for higher insecticide resistance [32]. Important questions related to the first-line malaria vector control tools include: how effective are old LLINs when mosquito vectors are modestly or highly resistant to the insecticide used in the intervention, and how often should a house be re-sprayed after one round of IRS?

The objectives of this study were to assess the impact of insecticidal decay effect in the context of modest to high insecticide resistance. Comprehensive information on bioefficacy and persistence of the insecticide on the LLINs and indoor residual sprayed walls in the context of vector insecticide resistance is important for making operational decisions in malaria control programmes, such as the frequency of net replacement and re-spray in IRS program under the current situation of modest to high pyrethroid resistance in the malaria vector populations.


Study sites

The study was conducted in seven villages in western Kenya: Iguhu, Emakakha, Chulaimbo, Emutete, Bungoma, Ahero and Kisian (Fig. 1). Bungoma, Emutete, Iguhu and Emakakha are in the highland-fringe malaria epidemic area whereas Chulaimbo, Ahero and Kisian are in the basin region of Lake Victoria (low land) where malaria is endemic. An. gambiae is the predominant species in Bungoma, Emutete, Iguhu, Emakakha, Kisian and Chulaimbo, whereas An. arabiensis is the predominant species in Ahero [3335], however there was a significant malaria vector species composition change recently [36]. Bungoma and Emutete were previously used for other insecticide resistance studies [33, 37]. Maize is the primary agricultural crop for all sites except that Ahero has irrigation and rice is a main crop.

Fig. 1

A map of the study sites in western Kenya

Scientific and ethical clearance

This study was approved by the Ethical Review Board of the Kenya Medical Research Institute (KEMRI) and Institutional Review Board of the University of California, Irvine. The area chief, sub-chief and village elders were sensitized on the study activities planned, household heads provided written consent authorizing the spraying of their houses in the indoor residual spraying programmes. For mosquito collection, oral consent was obtained from the field owners in each location. These locations were not protected land, and the field studies did not involve endangered or protected species.

WHO tube resistance bioassay

Mosquito larvae were collected from the seven study sites, and brought to the insectary of KEMRI and reared to adults. Upon pupation, they were transferred to cages and allowed to emerge as adults, and then provided with 10 % sucrose in cotton swabs. Three to five days old female adults were used for resistance bioassays. Female mosquitoes aged 2–5 days old were exposed to diagnostic doses of lambdacyhalothrin (0.05 %), deltamethrin (0.05 %), and permethrin (0.75 %). The bioassays were carried out using standard WHO testing protocol [38]. The number of surviving mosquitoes was recorded after the 24 h recovery period. A total of 200 mosquitoes per site, for each insecticide was tested.

Species identification

Susceptible and resistant mosquitoes preserved after the bioassays were identified to species level using species-specific PCR assay following procedure described by Scott et al. [39]. At least 50 mosquitoes from every site preserved from the bioassays were used. A total of 1002 specimens within An. gambiae species complex were molecularly identified by PCR. DNA was extracted from the combined legs and wings of each specimen using ethanol precipitation [40]. The rDNA-PCR method was used to distinguish between the two sibling species of the An. gambiae s.l. species complex native to western Kenya, An. gambiae s.s. and An. arabiensis [40].

Characterization of kdr mutation

PCR-based assay was carried out to check for kdr gene mutations in randomly selected individuals. A total of 570 mosquitoes were genotyped for the 7 populations. Extraction of DNA followed the procedure by Collins et al. [40]. Real-time TaqMan assay was used to quantify the genotype at amino acid position L1014S of the voltage gated sodium channel, following the methods of Bass et al. [41] as modified by Mathias et al. [31]. Samples were genotyped for the wild-type (susceptible) allele using probe 5´-CTTACGACTAAATTTC-3´, and for the 1014S kdr allele using probe 5´-ACGACTGAATTTC-3´.

Evaluation of insecticidal decay effect of LLINs

Olyset® LLINs, the most commonly used LLINs in western Kenya, were collected from the field and used for insecticidal decay test. Olyset® is made of single filament polyethylene, blended with permethrin 2 % as active ingredient at a concentration of 1000 mg permethrin per m2 [42]. Twenty LLINs were collected randomly from two villages, and the household heads were surveyed for the age of the nets, and then provided with new LLINs. The net age was further checked by questionnaire survey to the spouse of the household heads on the months that started to use the LLIN, and by check with village chiefs and health center administrators on LLIN mass distribution years and months. The nets were packed individually in polythene bags, and then wrapped in aluminum foil and stored at 4 °C. Six pieces of netting materials, 30 × 30 cm2, were cut with two pieces each from the roof panel, upper side panel and lower side panel from each net [43]. Three pieces from each position were used for mosquito bioassays to determine their insecticidal effects, and the remaining three pieces for insecticide concentration analysis.

The insecticidal decay effect of LLINs was measured using the standard WHO cone bioassay [43]. Mosquitoes reared from field-collected larvae were used, and the susceptible Kisumu strain was used as a control. Ten female mosquitoes were released in each cone and exposed to the netting material for 3 min. They were then transferred to paper cups and held for 24 h. The number of mosquitoes knocked down was recorded 60 min after exposure, and the mortality rates scored after the 24 h recovery period. New unused LLINs were used as positive control. Each Net was tested with twelve replicates and 120 mosquitoes from each site.

Net insecticide concentration analysis

To determine the insecticide concentration in the LLINs, 3 pieces of netting materials, 25 cm2 each, were cut from the roof panel, upper side panel and lower side panel, and placed in a glass test tube. The netting materials were immersed in the extraction solvent (4:1 hexane: chloroform solution) and then vortexed for 1 min. The netting materials were incubated at room temperature for 10 min, and then filtered through 0.45 μm PTFE (Polytetrafluoroethylene) membrane. The contents were diluted to a final volume of 10.0 ml. An aliquot of the 10.0 ml solution was analyzed by gas chromatography [44]. Molecular grade of permethrin and deltamethrin with known concentration (1 mg/ml in 4 hexane: 1 chloroform, stabilized with 0.1 % acetic acid) was used as internal standard to quantify the concentrating of permethrin and deltamethrin. Three independent insecticide extractions and gas chromatography analyses were conducted for each net.

Insecticidal decay of insecticides in the IRS program

The IRS program in Kenya commonly uses lambda-cyhalothrin (ICON 10CS). 100 g of lambda-cyhalothrin (Trade name ICON 10cs 100 g/liter) was diluted in 1 L water, then 62.5 ml of the diluted insecticide was further diluted in 10 L water which was sprayed on the wall as recommended by the manufacturer. The insecticides were sprayed using Hudson X-pert compression sprayer (10 L capacity). ICON 10CS is designed by the manufacturer to be effective on the wall for up to six months [20]. To determine the insecticidal effects of insecticides used in IRS, five houses were randomly selected in the area where the IRS with ICON was on going. Mud slabs of 5 cm diameter were prepared with the same mud used for house walls by the local residents. Eight mud slabs in lid tops were attached to the wall in each selected house. Study houses were then sprayed using ICON 10CS capsule suspension. For each house, one mud slab was removed immediately after spraying, then each month for insecticidal activity bioassay in the subsequent seven months after spray.

The bioassays were conducted using the standard WHO cone bioassay with adult mosquitoes raised from field-collected larvae from the seven study sites. Twenty female An. gambiae mosquitoes were released into each cone at the vertical position. The mosquitoes were exposed to the treated surfaces of mud slab for 30 min. Mosquitoes were transferred to a recovery cup, and the number of surviving mosquitoes was recorded after the 24 h recovery period. Laboratory reared susceptible Kisumu strain, a WHO designated susceptible reference strain, was used as a control. For every month 5 replicates were used.

Data analysis

The mortality rates of mosquitoes in the standard WHO tube resistance bioassay was calculated and adjusted using the Abbot’s formula [45]. Susceptibility status of a mosquito population was classified according to the WHO criteria (98–100 % mortality indicates susceptibility, 90–97 % mortality suggests possibility of resistance that needs to be confirmed, and <90 % mortality suggests resistance) [38]. LLINs were grouped into four classes: new net, <1 year, 1 ~ 2 years and >2 years. The mortality rates in the cone bioassays were calculated for each net age class for each of the two study sites. One-way analysis of variance (ANOVA) and Tukey-Kramer HSD test were used to determine the statistical difference between LLINs in different age groups. Finally, insecticidal decay effects of insecticides in the IRS were determined by comparing mortality rate in the cone bioassay among mud slabs at different months after spray. As in the LLIN insecticidal decay effect studies, one-way ANOVA and Tukey-Kramer HSD test were used to determine the statistical difference between mud slabs in different months after spray. Heterozygous and homozygous mutation rates of kdr gene were calculated. To determine if these genotypes were under selection, Hardy-Weinberg equilibrium test for kdr genotypes was performed, and χ 2 test was used to determine the significance of the departure from Hardy-Weinberg equilibrium.


Insecticide resistance status of the study populations

WHO tube resistance bioassay found the Kisumu susceptible strain was highly susceptible to pyrethroids, but all 7 study populations were resistant as mortality rates were all below 90 % by WHO standards (Fig. 2) [38]. In particularly, 4 out 7 populations (Chulaimbo, Emakakha, Emutete and Bungoma) were highly resistant to deltamethrin as evidenced by <80 % mortality in the bioassay. Similarly, 6 out of 7 populations were highly resistant to permethrin, and 5 out of 7 populations highly resistant to lambda-cyhalothrin. Ahero population in rice-irrigation area was least resistant to the three insecticides tested.

Fig. 2

Mortality rates of Anophlese gambiae and An. arabiensis in the standard WHO tube insecticide susceptibility test. Standard diagnostic dosage was used for each insecticide: deltamethrin (0.05 %), permethrin (0.75 %) and lambda-cyhalothrin (0.05 %). The 95 % confidence interval is shown

Species identification

PCR analysis showed that An. gambiae was the predominant species in Bungoma comprising 90.0 % specimens within An. gambiae species complex. An. gambiae remained the dominant species in the highland: Iguhu (88.0 %), Emutete (94.0 %) and Emakakha (93.4 %; Table 1). In the lowland An. arabiensis was dominant two sites: Kisian (64.4 %) and Ahero (89.3 %). Overall, 3.6 % of the samples tested were not successfully amplified.

Table 1 Species composition (%) at the seven study sites in western Kenya

Kdr genotyping

Table 2 summarizes kdr genotyping results. Overall, kdr genotyping failed in 2.8 % of the samples. Homozyzygous kdr mutations of L1014S ranged from 73 to 88 % in An. gambiae s.s. dominant populations, whereas L1014S mutations were relatively low (7–31 %) in An. arabiensis dominant populations. In addition, in An. gambiae most of the mutations were homozygous (Table 2). Hardy-Weinberg analysis found that, for the four An. arabiensis populations tested for Hardy-Weinberg equilibrium, only one population (Chulaimbo) showed significant deviation and the deviation resulted from heterozygosity deficiency (Table 2). On the other hand, for An. gambiae, five out of the six populations tested showed significant departure from Hardy-Weinberg equilibrium, and all were caused by heterozygosity deficiency.

Table 2 Genotype and allele frequencies of kdr at the seven study sites in western Kenya

Insecticidal decay of LLINs with age

A total of 21 LLINs were tested using the WHO cone bioassay for their insecticidal activities. No surviving mosquitoes were observed in the cone bioassay, and there was no decrease in insecticidal activities in the LLINs for up to 3 years against the laboratory susceptible Kisumu strain (100 % mortality) (Fig. 3). However, mortality rate was decreased to 77.5 – 85.0 % when the new LLINs were tested against field collected mosquitoes, suggesting that the LLINs had limited killing efficacy on mosquitoes predisposed with insecticide resistance genes. The insecticidal activity in LLINs of 1 ~ 2 years and >2 years old was similar for both populations (66.7 % for Emutete and 59.6 % for Bungoma), but it was significantly lower than the new LLINs (85 % for Emutete and 77.5 % for Bungoma, P < 0.05) (Fig. 3).

Fig. 3

Mortality rates of field mosquito populations (Emutete and Bungoma) and laboratory susceptible Kisumu strain exposed to LLINs of various ages in the standard WHO cone bioassay. The 95 % confidence interval is shown

The chemical concentration analysis found that new LLINs had the highest concentration at 0.14 μg/ml (Fig. 4). Significantly lower concentration (0.07 μg/ml) was found in LLINs of 1 ~ 2 years and >2 years old, in comparison to LLINs of <1 year old (0.09 μg/ml, P < 0.05), suggesting reduced availability of the active insecticide ingredient in the nets over time (Fig. 4).

Fig. 4

The net chemical content of the LLINs. The 95 % confidence interval is shown. The target concentration of permethrin for new LLINs was 0.14 % of total weight

Insecticidal decay effect in mud walls sprayed with insecticides

The mud slabs sprayed with lambda-cyhalothrin in the IRS program killed all susceptible An. gambiae for at least 7 months after the spray in the cone bioassay (Fig. 5). The mortality rate of freshly sprayed mud slabs ranged from 93.5 to 84.6 % for the 7 study populations, and such a reduction from 100 % mortality rate observed in the susceptible mosquito population likely resulted from insecticide resistance in the mosquito populations. Within the first four months after the spray, small reduction in the mortality rate was observed (71.1 – 88.2 %). Significant decay in insecticidal activities was observed 6 months after spray for most populations (72.5 -80.7 %, P < 0.05). Bungoma population, the most resistant population, consistently exhibited the lowest mortality rate during the test period (72.5 – 86.6 %), whereas the least resistant Ahero population showed the highest mortality rates during the testing periods (80.7 – 93.5 %).

Fig. 5

Bioassay mortality rates of seven mosquito populations and the susceptible Kisumu strain exposed to mud slabs treated with ICON (lambda-cyhalothrin). The 95 % confidence interval is shown


Malaria transmission reduction by indoor residual spray and insecticide-impregnated nets is primarily through decreasing vector abundance and human-vector contact rate. Insecticidal efficacy of the nets and residual insecticides in IRS is critical to the efficacy of the malaria vector control program. Insecticidal efficacy may be affected by insecticide resistance in the mosquito vectors, gradual loss of chemical bioavailability and chemical degradation. This study was carried out to assess insecticidal efficacy of LLINs that have been used by residents for several years and to determine the efficacy of residual insecticides in the IRS program in the context of various insecticide resistance levels. Our data supports three conclusions: 1) Prior to the indication of decay, there was evidence that insecticide resistant mosquitoes were less likely to be killed after exposure to LLINS and IRS; 2) the LLINs we tested (Olyset®) showed time-dependent decay of insecticide concentration, and this was collaborated by reduced mortality in the older nets compared to new nets and by the insecticide chemical analysis in the nets; and 3) residual insecticide of IRS with lambda-cyhalothrin (ICON 10CS) showed decay in insecticidal efficacy 5 months after the spray in the field. Decay of pyrethroid insecticidal efficacy was indicated by bioassay mortality rates, instead of chemical assays as there were challenges in extracting pyrethroids from the sprayed surfaces.

An. gambiae from all study sites showed resistance to pyrethroids except An. arabiensis mosquito population in Ahero which was moderately susceptible to pyrethroids. The observed pyrethroid resistance in these regions could be linked to the public health use of insecticides for LLINs and IRS [31]. An. arabiensis in Ahero are largely exophilic and zoophilic [46], indicating its low contact with LLINs and sprayed walls and thus less exposure to the insecticide. Previous studies in these sites have reported high abundance of An. gambiae or complete lack of An. arabiensis in Bungoma, Emutete and Iguhu [33, 36, 47], the present study found 3–8 % of An. arabiensis, suggesting An. arabiensis was spreading and establishing the populations in western Kenya highlands. We found high frequencies of kdr mutations in An. gambiae and low frequencies in An. arabiensis, consistent with the finding of Ochomo et al. [33] which reported fixation of L1014S in Bungoma An. gambiae population and absence in An. arabiensis population. However, Kawada et al. [48] reported a high frequency and wide distribution of L1014S in An. arabiensis in Suba district, western Kenya, suggesting a patchy distribution pattern of kdr mutation in An. arabiensis in western Kenya.

The mortality rates of field mosquito populations against residual insecticide in IRS were significantly lower than the susceptible Kisumu strain, and decreased significantly with time. Lambda-cyhalothrin (ICON CS10) is known to persist on sprayed surfaces for six months [49]. Thus, reduced mortality against field mosquito populations in the freshly sprayed surface was resulted from insecticide resistance. On the other hand, insecticidal efficacy decay was evident five months after the spray. The magnitude of the effects of insecticide resistance and insecticidal decay were comparable, about 20 % less mortality (compare to control) in resistance and 15-20 % less mortality in insecticidal decay (compare to newly sprayed walls with after 6 months of spray). The lower mortality rates observed on treated mud wall could also be due to less bioavailability of the insecticides on the sprayed surfaces. Studies from different countries such as Tanzania and Vietnam also found that the material of walls affected the durability of insecticides sprayed on the wall [16, 5053], a factor should be considered when choosing the insecticides for indoor residual spraying as many rural areas in tropical Africa are shifting from building mud walled houses to modern cemented houses. For example, studies in Tanzania with ICON 10 CS recorded 100 % mortality of An. gambiae up to seven months on sprayed mud wall surfaces [52], whereas in Vietnam, the residual effect of ICON lasted for up to four months on wood, five months on bamboo, and three months on bricks in bioassays against An. dirus [53]. A WHOPES supervised trial in Benin reported the residual effect of CS and WP ICON formulations with 30 mg/m2 to be up to two months only, and in India trials reported persistence up to four to six months [53]. The bioefficacy and persistency of insecticides, as revealed by mosquito mortality, depends on the type of surface, the dosage, and the age of spray deposits [5055]. We speculate that microclimate conditions may also play a role.

Net cone bioassays and net chemical analysis confirmed the degradation of insecticides and its impact on mosquito mortality. Earlier studies have indicated the reduced efficacy of the nets with the number of washes, physical condition of the nets and insecticide resistance [24, 56]. Our finding is consistent with Toe et al. [57] who reported reduced susceptibility of field collected mosquitoes to LLINs, compared to laboratory reared Kisumu strain. Whether the observed insecticidal decay and insecticide resistance may lead to operational failure of LLINs is not clear. For example, whether this resistance could result in operational ITN or IRS malaria control failure in the field is unknown. For example, a report from Malawi found that the use of ITNs reduced the incidence of clinic malaria incidence by 30 % in children in an area with moderate levels of pyrethroid resistance and considerable malaria transmission [58]. A thorough assessment of the impact of this resistance on the efficacy of LLINs and IRS on malaria disease and transmission will give a clear indication to the decision makers on the need to shift or not from pyrethroids to alternative carbamate or organophosphate insecticides [59].


In conclusion, this study used seven sites in malaria endemic and epidemic areas of western Kenya and confirmed that insecticide decay and developing levels of resistance have an important negative contribution to insecticidal efficacy of LLINs and IRS. This finding is congruent with several studies from this region over the past several years [3, 33, 48, 6063]. Pyrethroid resistance poses a major threat to the current malaria control strategies in Africa. Due to increased residual malaria transmission and rebound of malaria transmission in many sites in Africa, transmission control is required despite of moderate and high insecticide pyrethroid resistance. In order to mitigate against the role of insecticide resistance and decay, new nets should be redistributed every three years as recommended by WHO while maintaining a high level of LLIN coverage and usage. There is also need for urgent development and deployment of non-pyrethroid based vector control tools and non-insecticide based ecological tools.


  1. 1.

    WHO. World Malaria Report. Geneva: WHO; 2012.

    Google Scholar 

  2. 2.

    Atieli HE, Zhou G, Afrane Y, Lee M-C, Mwanzo I, Githeko AK, et al. Insecticide-treated net (ITN) ownership, usage, and malaria transmission in the highlands of western Kenya. Parasites & Vectors. 2011;4(1):113.

    Article  Google Scholar 

  3. 3.

    Ochomo EO, Bayoh NM, Walker ED, Abongo BO, Ombok MO, Ouma C, et al. The efficacy of long-lasting nets with declining physical integrity may be compromised in areas with high levels of pyrethroid resistance. Malar J. 2014;12:368.

    Article  Google Scholar 

  4. 4.

    Coetzee M, Koekemoer LL. Molecular systematics and insecticide resistance in the major African malaria vector Anopheles funestus. Annu Rev Entomol. 2013;58:393–412.

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Protopopoff N, Matowo J, Malima R, Kavishe R, Kaaya R, Wright A, et al. High level of resistance in the mosquito Anopheles gambiae to pyrethroid insecticides and reduced susceptibility to bendiocarb in north-western Tanzania. Malar J. 2013;12:149.

    PubMed Central  Article  PubMed  Google Scholar 

  6. 6.

    Abdalla H, Wilding CS, Nardini L, Pignatelli P, Koekemoer LL, Ranson H, et al. Insecticide resistance in Anopheles arabiensis in Sudan: temporal trends and underlying mechanisms. Parasites & Vectors. 2014;7:213.

    Article  Google Scholar 

  7. 7.

    Riveron JM, Yunta C, Ibrahim SS, Djouaka R, Irving H, Menze BD, et al. A single mutation in the GSTe2 gene allows tracking of metabolically based insecticide resistance in a major malaria vector. Genome Biol. 2014;15(2):27.

    Article  Google Scholar 

  8. 8.

    Randriamaherijaona S, Briët OJ, Boyer S, Bouraima A, N'Guessan R, Rogier C, et al. Do holes in long-lasting insecticidal nets compromise their efficacy against pyrethroid resistant Anopheles gambiae and Culex quinquefasciatus? Results from a release-recapture study in experimental huts. Malar J. 2015;14(1):332.

    PubMed Central  Article  PubMed  Google Scholar 

  9. 9.

    Cisse MB, Keita C, Dicko A, Dengela D, Coleman J, Lucas B, et al. Characterizing the insecticide resistance of Anopheles gambiae in Mali. Malar J. 2015;14(1):327.

    PubMed Central  Article  PubMed  Google Scholar 

  10. 10.

    Wanjala CL, Mbugi JP, Ototo E, Gesuge M, Afrane YA, Githeko AK, et al. Pyrethroid and DDT-resistance and organophosphate susceptibility among Anopheles mosquito Species Western Kenya. Emerging Infectious Diseases. (in press).

  11. 11.

    Mutunga JM, Anderson TD, Craft DT, Gross AD, Swale DR, Tong F, et al. Carbamate and pyrethroid resistance in the akron strain of Anopheles gambiae. Pesticide Biochemistry Physiology. 2015;121:116–21.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Glunt KD, Abílio AP, Bassat Q, Bulo H, Gilbert AE, Huijben S, et al. Long-lasting insecticidal nets no longer effectively kill the highly resistant Anopheles funestus of southern Mozambique. Malar J. 2015;14:298.

    PubMed Central  Article  PubMed  Google Scholar 

  13. 13.

    Ranson H, Jensen B, Vulue JM, Wang X, Hemingway J, Jollins. Identification od a point mutation in the voltage-gated sodium channel gene of Kenyan Anopheles gambiae associated with resistance to DDT and pyrethroids. Insect Mol Biol. 2000;9:491–97.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Diabate A, Baldet T, Chandre E, Dabire KR, Simard F, Quedraogo JB, et al. First report of a kdr in Anopheles arabiensis from Burkina Faso, West Africa. J Am Mosq Control Assoc. 2004;20:195–96.

    CAS  PubMed  Google Scholar 

  15. 15.

    Edi CV, Koudou BG, Jones CM, Weetman D, Ranson H. Multiple-insecticide resistance in Anopheles gambiae mosquitoes Southern Côte d'Ivoire. Emerg Infect Dis. 2012;18(9):1508–11.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  16. 16.

    Mulambalah CS, Siamba DN, Ngeiywa MM, Vulule JM. Evaluation of Lambda-Cyhalothrin Persistence on Different Indoor Surfaces in a Malaria Epidemic-Prone Area in Kenya. Res J Biological Sci. 2010;5(3):258–63.

    Article  Google Scholar 

  17. 17.

    Zhou G, Yaw AA, Amruta D, Harrysone EA, Ming-Chieh L, Christine LW, et al. Modest additive effects of integrated vector control measures on malaria prevalence and transmission in western Kenya. Malar J. 2013;12:256.

    PubMed Central  Article  PubMed  Google Scholar 

  18. 18.

    N’Guessan R, Corbel V, Akogbeto M, Rowland M. Reduced efficacy of insecticide treated nets and indoor residual spraying for malaria control in pyrethroid resistance area, Benin. Emerging Infecious Diseases. 2007;13:199–206.

    Article  Google Scholar 

  19. 19.

    WHOPES. WHO recommended insecticides for indoor residual spraying against malaria vectors. 2015.

  20. 20.

    WHO. Report of the fifth WHOPES Working Group meeting. Review of Olyset Net, bifenthrin 10% WP. Geneva, 30–31 October 2001. Geneva. 2001.

  21. 21.

    Lindblade KA, Dotson E, Hawley WA, Bayoh N, Williamson J, Mount D, et al. Evaluation of long-lasting insecticidal nets after 2 years of household use. Trop Med Int Health. 2005;10(11):1141–50.

    Article  PubMed  Google Scholar 

  22. 22.

    Graham K, Kayedi MH, Maxwell C, Kaur H, Rehman H, Malima R, et al. Multi country field trials comparing wash resistance of PermaNet and conventional insecticide treated nets against anopheline and culicine mosquitoes. Med Vet Entomol. 2005;19(1):72–83.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Okia M, Ndyomugyenyi R, Kirunda J, Byaruhanga A, Adibaku S, Lwamafa DK, et al. Bioefficacy of long-lasting insecticidal nets against pyrethroid-resistant populations of Anopheles gambiae ss from different malaria transmission zones in Uganda. Parasites & Vectors. 2013;6:130.

    Article  Google Scholar 

  24. 24.

    Atieli FK, Munga SO, Ofulla AV, Vulule JM. The effect of repeated washing of long-lasting insecticide-treated nets (LLINs) on the feeding success and survival rates of Anopheles gambiae. Malar J. 2010;9(304):29.

    Google Scholar 

  25. 25.

    Ranson H, Abdallah H, Badolo A, Guelbeogo WM, Kerah-Hinzoumba C, Yangalba-Kalnona E, et al. Insecticide resistance in Anopheles gambiae: data from the first year of a multi-country study highlight the extent of the problem. Malar J. 2009;8(1):299.

    PubMed Central  Article  PubMed  Google Scholar 

  26. 26.

    Santolamazza F, Calzetta M, Etang J, Barrese E, Dia I, Caccone A, et al. Distribution of knock-down resistance mutations in Anopheles gambiae molecular forms in west and west-central Africa. Malar J. 2008;7:74.

    PubMed Central  Article  PubMed  Google Scholar 

  27. 27.

    Yadouleton A, Martin T, Padonou G, Chandre F, Asidi A, Djogbenou L, et al. Cotton pest management practices and the selection of pyrethroid resistance in Anopheles gambiae population in northern Benin. Parasites & vectors. 2011;4:60.

    Article  Google Scholar 

  28. 28.

    Dabire RK, Namountougou M, Sawadogo SP, Yaro LB, Toe HK, Ouari A, et al. Population dynamics of Anopheles gambiae s.l. in Bobo-Dioulasso city: bionomics, infection rate and susceptibility to insecticides. Parasites & vectors. 2012;5:127.

    Article  Google Scholar 

  29. 29.

    Koffi AA, Alou LP, Adja MA, Kone M, Chandre F, N'Guessan R. Update on resistance status of Anopheles gambiae s.s. to conventional insecticides at a previous WHOPES field site, "Yaokoffikro", 6 years after the political crisis in Cote d'Ivoire. Parasites & vectors. 2012;5:68.

    CAS  Article  Google Scholar 

  30. 30.

    Adasi K, Hemingway J. Susceptibility to three pyrethroids and detection of knockdown resistance mutation in Ghanaian Anopheles gambiae sensu stricto. J Soc Vector Ecology. 2008;33(2):255–62.

    Article  Google Scholar 

  31. 31.

    Mathias DK, Ochomo E, Atieli F, Ombok M, Bayoh MN, Olang G, et al. Spatial and temporal variation in the kdr allele L1014S in Anopheles gambiae s.s. and phenotypic variability in susceptibility to insecticides in Western Kenya. Malar J. 2011;10:10.

    PubMed Central  Article  PubMed  Google Scholar 

  32. 32.

    Stump AD, Atieli FK, Vulule JM, Besansky NJ. Dynamics of the pyrethroid knockdown resistance allele in western Kenyan populations of Anopheles gambiae in response to insecticide-treated bed net trials. Am J Trop Med Hyg. 2004;70(6):591.

    CAS  PubMed  Google Scholar 

  33. 33.

    Ochomo E, Bayoh MN, Brogdon WG, Gimnig JE, Ouma C, Vulule JM, et al. Pyrethroid resistance in Anopheles gambiae s.s. and Anopheles arabiensis in western Kenya: phenotypic, metabolic and target site characterizations of three populations. Med Vet Entomol. 2012;10(1111):1365–2915.

    Google Scholar 

  34. 34.

    Obala AA, Kutima HL, Nyamogoba HDN, Mwangi AW, Simiyu CJ, Magak GN, et al. Anopheles gambiae and Anopheles arabiensis population densities and infectivity in Kopere village, Western Kenya. J Infection in Developing Countries. 2012;6(08):637–43.

    Article  Google Scholar 

  35. 35.

    Ototo EN, Githeko AK, Wanjala CL, Scott TW. Surveillance of vector populations and malaria transmission during the 2009/10 El Nino event in the western Kenya highlands: opportunities for early detection of malaria hyper-transmission. Parasites & Vectors. 2011;4:144.

    Article  Google Scholar 

  36. 36.

    Bayoh MN, Mathias DK, Odiere MR, Mutuku FM, Kamau L, Gimnig JE, et al. Anopheles gambiae: historical population decline associated with regional distribution of insecticide-treated bed nets in western Nyanza Province Kenya. Malar J. 2010;9(1):62.

    PubMed Central  Article  PubMed  Google Scholar 

  37. 37.

    Bonizzoni M, Afrane Y, Dunn WA, Atieli FK, Zhou G, Zhong D, et al. Comparative transcriptome analyses of deltamethrin-resistant and-susceptible Anopheles gambiae mosquitoes from Kenya by RNA-Seq. PLoS ONE. 2012;7(9), e44607.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  38. 38.

    WHO. Test procedures for insecticide resistance monitoring in malaria vector mosquitoes. Geneva: World Health Organization; 2013. p. 6–17.

    Google Scholar 

  39. 39.

    Scott JA, Brogdon WG, Collins FH. Identification of single specimens of the Anopheles gambiae complex by the polymerase chain reaction. Am J Trop Med Hyg. 1993;49(4):520.

    CAS  PubMed  Google Scholar 

  40. 40.

    Collins FH, Mendez MA, Rasmussen MO, Mehaffey PC, Besansky NJ, Finnerty V. A ribosomal RNA gene probe differentiates member species of the Anopheles gambiae complex. Am J Trop Med Hyg. 1987;37(1):37.

    CAS  PubMed  Google Scholar 

  41. 41.

    Bass C, Nikou D, Donnelly M, Williamson M, Ranson H, Ball A, et al. Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods. Malar J. 2007;6(1):111.

    PubMed Central  Article  PubMed  Google Scholar 

  42. 42.

    WHOPES. Report of the thirteenth WHOPES working group meeting. Geneva: WHO/HQ; 2009. Review of Olyset LN, Dawaplus 20 LN, Tianjin Yorkool LN, 2009.

    Google Scholar 

  43. 43.

    WHO. Guidelines for laboratory and field testing of long-lasting insecticidal mosquito nets. Geneva: World Health Organization; 2005.

    Google Scholar 

  44. 44.

    You J, Lydy MJ. A solution for isomerization of pyrethroid insecticides in gas chromatography. J Chromatogr. 2007;1166:181–90.

    CAS  Article  Google Scholar 

  45. 45.

    Abbott WS. A method of computing the effectiveness of an insecticide. J Econ Entomol. 1925;18(2):265–7.

    CAS  Article  Google Scholar 

  46. 46.

    Githeko AK, Adungo NI, Karanja DM, Hawley WA, Vulule JM, Seroney IK, et al. Some observations on the biting behavior of Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus and their implications for malaria control. Exp Parasitol. 1996;82(3):306–15.

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    Mushinzimana E, Munga S, Minakawa N, Li L, Feng C, Bian L, et al. Landscape determinants and remote sensing of anopheline mosquito larval habitats in the western Kenya highlands. Malar J. 2006;5(1):13.

    PubMed Central  Article  PubMed  Google Scholar 

  48. 48.

    Kawada H, Dida GO, Ohashi K, Komagata O, Kasai S, Tomita T, et al. Multimodal pyrethroid resistance in malaria vectors, Anopheles gambiae ss, Anopheles arabiensis, and Anopheles funestus ss in western Kenya. PLoS ONE. 2011;6(8):e22574.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  49. 49.

    WHO. Cyhalothrin and Lambda-cyhalothrin Health and Safety Guide’ Health and Safety Guide No. 38, International Programme on Chemical Safety. Geneva: World Health Organization; 1990.

    Google Scholar 

  50. 50.

    Raghavendra K GS, Eapen A, Tiwari SN, Satyanarayan TS, Ravindran J, Sreehari U et al. Field evaluation of lambda-cyhalothrin (ICON 10 CS) indoor residual spraying against Anopheles culicifacies in India. J Vector Borne Diseases. 2011

  51. 51.

    Villarreal C, Rodriguez MH, Bown DN. Low-volume application by mist-blower compared with conventional compression sprayer treatment of houses with residual pyrethroid to control the malaria vector Anopheles albimanus in Mexico. Med Vet Entomol. 1995;9:187–94.

    CAS  Article  PubMed  Google Scholar 

  52. 52.

    Curtis C, Maxwell CA, Finch RJ. A comparison of use of a pyrethroid either for house spraying or for a bednet treatment against malaria vectors. Trop Med Int Health. 1998;3:619–31.

    CAS  Article  PubMed  Google Scholar 

  53. 53.

    WHOPES. Review of Spinosad 0.5 % GR and 12 SC, Lambda-cyhalothrin 10 % CS, K-O Tab 1-2-3 and Interceptor. webcite Geneva: World Health Organization. 2007: 22–36.

  54. 54.

    Rozilawati H, Lee HL, Mohd Masri S, Mohd Noor IS. Filed bioefficacy of deltamethrin residual spraying against dengue vectors. Trop Biomed. 2005;22:143–8.

    CAS  PubMed  Google Scholar 

  55. 55.

    Giga DP, Canhao SJ. Relative toxicity and persistence of pyrethroid deposits on different surfaces for the control of Prostephanus truncates and Sitophilus zeamais. J Stored Prod Res. 1991;27(3):153–60.

    CAS  Article  Google Scholar 

  56. 56.

    Dabire RK, Diabate A, Baldet T, Pare-Toe L, Guiguemde RT, Ouedraogo JB, et al. Personal protection of long lasting insecticide-treated nets in areas of Anopheles gambiae ss resistance to pyrethroids. Malar J. 2006;5(6):12.

    PubMed Central  Article  PubMed  Google Scholar 

  57. 57.

    Toe KH, Jones CM, N'Fale S, Ismail HM, Dabiré RK, Ranson H. Increased pyrethroid resistance in malaria vectors and decreased bed net effectiveness Burkina Faso. Emerg Infect Dis. 2014;20(10):1691–6.

    PubMed Central  Article  PubMed  Google Scholar 

  58. 58.

    Multambalah CS, Siaba DN, Ngeiywa MM, Vulule JM. Evaluation of lamda-mcyhalothrin persistence on differenct indoor surface in the malaria epidemic-prone area of Kenya. Res J Biol Sci. 2010;(5)3:258–63.

  59. 59.

    Kleinschmidt I, Mnzava AP, Kafy HT, Mbogo C, Bashir AI, Bigoga J, et al. Design of a study to determine the impact of insecticide resistance on malaria vector control: a multi-country investigation. Malar J. 2015;14:282.

    PubMed Central  Article  PubMed  Google Scholar 

  60. 60.

    Kawada H, Ohashi K, Dida GO, Sonye G, Njenga SM, Mwandawiro C, et al. Preventive effect of permethrin-impregnated long-lasting insecticidal nets on the blood feeding of three major pyrethroid-resistant malaria vectors in western Kenya. Parasites & Vectors. 2014;7:383.

    Article  Google Scholar 

  61. 61.

    Vulule JM, Beach RF, Atieli FK, McAllister JC, Brogdon WG, Roberts JM, et al. Elevated oxidase and esterase levels associated with permethrin tolerance in Anopheles gambiae from Kenyan villages using permethrin-impregnated nets. Med Vet Entomol. 1999;13(3):239–44.

    CAS  Article  PubMed  Google Scholar 

  62. 62.

    Chen H, Githeko AK, Githure JI, Mutunga J, Zhou G, Yan G. Monooxygenase Levels and Knockdown Resistance (kdr) Allele Frequencies in Anopheles gambiae and Anopheles arabiensis in Kenya. J Med Entomol. 2008;45(2):242–50.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  63. 63.

    Ochomo E, Bayoh NM, Kamau L, Atieli F, Vulule J, Ouma C, et al. Pyrethroid susceptibility of malaria vectors in four Districts of western Kenya. Parasites & Vectors. 2014;7:310.

    Article  Google Scholar 

Download references


The authors wish to thank the staff in the Climate and Human Health Research Unit, Centre for Global Health Research, Kenya Medical Research Institute, Kisumu, Kenya, for assistance with field mosquito collection. This study was supported by grants from the National Institutes of Health (R01 AI050243, D43 TW001505 and RO1 AI094580). This paper is published with permission from the director of Kenya Medical Research Institute.

Author information



Corresponding author

Correspondence to Guiyun Yan.

Additional information

Competing interest

The authors declare that they have no competing interest.

Authors’ contributions

CLW participated in the study design, performed laboratory work, data collection, data analysis and drafted the manuscript. JM Participated in study design and contributed manuscript writing, JS Participated in study design and contributed manuscript writing, YAA participated in the study design and writing of the manuscript. EO participated in data collection and study design, MG participated in data collection and study design, HA participated in the study design and writing of the manuscript. GZ participated in study design, data analysis, and manuscript writing, AKG and GY designed the study and supervised data collection and contributed to writing of the manuscript. All authors have read and approved the final manuscript.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wanjala, C.L., Zhou, G., Mbugi, J. et al. Insecticidal decay effects of long-lasting insecticide nets and indoor residual spraying on Anopheles gambiae and Anopheles arabiensis in Western Kenya. Parasites Vectors 8, 588 (2015).

Download citation


  • Malaria
  • Insecticidal decay
  • Insecticide resistance
  • Indoor residual spraying
  • Long-lasting insecticide nets