Indirect evidence that agricultural pesticides select for insecticide resistance in the malaria vector Anopheles gambiae

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1 34 Journal of Vector Ecology June 2016 Indirect evidence that agricultural pesticides select for insecticide resistance in the malaria vector Anopheles gambiae Djogbénou S. Luc *, Assogba Benoit, Djossou Laurette, and Makoutode Michel Institut Régional de Santé Publique, Université d Abomey Calavi, 01BP918 Cotonou, Bénin, ldjogbenou22002@yahoo.fr Received 23 July 2015; Accepted 25 September 2015 ABSTRACT: We investigated the possible relationship between the agricultural use of insecticides and the emergence of insecticide resistance. Bioassays were conducted using simulated mosquito larval habitats and well known Anopheles gambiae strains. Soil samples were collected from vegetable production areas in Benin, including one site with insecticide use, one site where insecticides had not been used for two months, and a third where insecticides had not been used. Pupation and emergence rates were very low in pyrethroidsusceptible strains when exposed to soil that had been recently exposed to insecticides. Pupation and emergence rates in strains with the kdr mutation alone or both the kdr and Ace-1 mutations were much higher. Overall, strains with the kdr mutation survived at higher rates compared to that without kdr mutation. Although this study is observational, we provide indirect evidence indicating that soils from agricultural areas contain insecticide residues that can play a role in the emergence of insecticide resistance in Anopheles. This aspect should be taken into account to better utilize the insecticide in the context of integrated pest management programs. Journal of Vector Ecology 41 (1): Keyword Index: Pesticide, selection pressure, vegetable crops, Anopheles gambiae, Benin. INTRODUCTION Urban agriculture is a common feature of Sub-Saharan Africa, contributing to the public policy goals of employment, income generation, poverty alleviation, and nutrition (De Haas and Gura 1996). Despite these benefits, the occurrence of health and environmental risks can also be associated with the use of insecticides in urban agriculture. Additionally, certain agricultural practices can also contribute to major health problems such as malaria, food-borne diseases, diet-related chronic diseases, and occupational health hazards (Aktar et al. 2009). In the case of malaria, one of the unforeseen consequences of the use of insecticides in agriculture is that it may select for resistance in mosquito vectors (Diabate et al. 2002, Djogbénou et al. 2011). Pesticide treatments used in agriculture may contaminate nearby mosquito larval habitats. If the amounts of insecticide in these sites reach sufficient levels as to be lethal to some mosquito larvae, this could result in the selection of resistance. Alternatively, exposure to sublethal doses of pyrethroids or other insecticides in the agricultural context can facilitate the response of mosquitoes to further exposure to a mosquitocidal treatment (David et al. 2014). Members of the Anopheles gambiae complex are the most important vectors of malaria in Sub-Saharan Africa. The complex consists of at least seven species that vary in their ability to transmit malaria (White 1974, Hunt et al. 1998). Malaria is the most important disease transmitted by Anopheles gambiae in West Africa. As no vaccine is yet available, vector control is an essential component of malaria control programs. The World Health Organization has reaffirmed the importance of vector control through the use of insecticide-treated bed-nets (ITNs) and indoor residual house spraying (IRS) (WHO 2012). While only pyrethroids have been recommended for use on bed nets, twelve other insecticides have been recommended for IRS, belonging to only four classes of chemicals (organochlorines, organophosphates, carbamates, and pyrethroids), and six of the twelve are pyrethroids (WHO 2006). Pyrethroids were initially developed for pest management in agriculture and are used in far larger quantities for crop protection and animal health than for public health purposes (Zaim and Guillet 2002). Resistance to one or more of these insecticides has been reported in several species of the Anopheles gambiae complex (Ranson et al. 2009). This resistance is a real threat to the use of insecticide-treated materials (WHO 2012, Toe et al. 2014). Recent findings have shown that pyrethroid resistance is widespread in West Africa with target-site mutations and metabolic resistance both implicated (Ranson et al. 2011). Mutations in the insecticide target site result in insensitivity termed knock-down resistance (kdr) (Chandre et al. 1999). Subsequent analyses have demonstrated that kdr is caused by two possible amino acid substitutions at the same positions (L1014F and L1014S) in the sodium channel of pyrethroid resistant insects (Martinez-Torres et al. 1998, Ranson et al. 2000). The kdr mutation has been observed in both An. gambiae and An. coluzzi in many West African countries (Santolamazza et al. 2008) and in Benin (Djogbénou et al. 2011). Pyrethroid resistance may also occur by metabolic detoxification through increased enzyme activities (monooxygenases, esterase, or glutathione S-transferase) (Hemingway and Karunaratne 1998). Organophosphates and carbamates are commonly used for IRS (Fanello et al. 2003). These two insecticide classes target the synaptic acetylcholinesterase (AChE) encoded by the Ace-1 gene. However, resistance to these insecticides has also been reported in wild An. gambiae s.s. from West Africa (N Guessan et al. 2003, Djogbénou et al. 2008), and insensitivity of the AChE enzyme to organophosphates and carbamates has been shown to result from a specific mutation of the Ace-1 gene in An. gambiae s.s. (Weill et

2 Vol. 41, no. 1 Journal of Vector Ecology 35 al. 2003, Weill et al. 2004). This mutation, named Ace-1 G119S or Ace-1 R, has been reported in An. gambiae s.s. from Ivory Coast (Djogbénou et al. 2009), Burkina Faso (Djogbénou et al. 2008, Dabire et al. 2009) and Benin (Djogbénou et al. 2011). In most cases, the challenges for urban agriculture in West Africa derive from a lack of official recognition and limited active support from national and city authorities. In several insecticide resistance management programs, it has also been stated that national and urban authorities, policy makers, and public health authorities need more evidence on the contribution of urban agriculture to the persistence of malaria through the selection of resistant mosquitoes. This information will further allow those involved in malaria control to better estimate the benefits and costs of urban agriculture. The present study aims to provide data from laboratory experiments to demonstrate the impact of residual insecticides present in vegetable growing areas on the response of mosquitoes to pyrethroids and to explain how such factors could affect vector control strategies in the future. Practically, this study investigates the possible relationship between the use of pyrethroids and the selection of insecticide resistance in An. gambiae. Bioassays were carried out in the laboratory to evaluate the effects of residual insecticides present in soil samples from vegetable farms of Houeyiho on insecticide-susceptible and resistant Anopheles gambiae s.s. MATERIALS AND METHODS Study areas This study was conducted in the Republic of Benin, West Africa. One vegetable growing area (Houeyiho) was used for the experiments in this study. The vegetable production area of Houeyiho was created in the 1970s with the aid of the Food and Agriculture Organization and the Dutch development agency Novib. The site is located at 6 45N and 2 31E in Cotonou near the airport. The site size increased from 5 ha in 1972 to 20 ha in 1997 and is, by far, the biggest vegetable growing area in Cotonou city. In Houeyiho, more than 300 farmers are involved in the cultivation of a large variety of vegetables such as cabbages, carrots, lettuce, amaranth, cucumbers, etc. Although most of the study site has been cultivated, small patches of water still remain in which the female mosquitoes can lay eggs. Several studies have been conducted using larvae of An. gambiae collected in these patches of water within the study area (Yadouleton et al. 2010a, Yadouleton et al. 2010b). Mosquito strains Four strains of An. gambiae s.s. (S-form) were used in this study: (1) An. gambiae Kisumu is a reference strain susceptible to pyrethroids, carbamates, and organophosphates. It was originally isolated in the Kisumu region of western Kenya in the early 1950s and has been maintained in the laboratory since that time (Shute 1956). (2) An. gambiae AcerKis is homozygous for the Ace-1 mutation and is resistant to both organophosphate and carbamate insecticides (Djogbénou et al. 2007). This strain has the same genetic background as the Kisumu strain and was created over the course of 19 generations of backcrossing and selection (with propoxur) between Kisumu and resistant An. gambiae caught in the Bobo-Dioulasso region of Burkina Faso in (3) An. gambiae KisKdr is homozygous for the kdr mutation (caused by amino acid substitutions at the same positions in the sodium channel of pyrethroid resistant insects: L1014F) and is resistant to both pyrethroids and DDT (Alout et al. 2013). This strain also shares the genetic background of Kisumu. It was obtained after several generations of backcrossing and selection (with permethrin 1 mg/liter) between Kisumu and a resistant An. gambiae (VKper) pyrethroid-resistant strain that was initially collected from the Valley du Kou in Burkina Faso and then selected repeatedly. (4) An. gambiae AcerKdrKis is homozygous for both Ace-1 and kdr (L1014F) mutations and is thus resistant to the main insecticide families used for the control of adult malaria vectors (pyrethroids, DDT, organophosphates, and carbamates). The strain AcerKdrKis was obtained by introgression of the two mutations (Ace-1 and kdr) and then purification. These resistant alleles were obtained from AcerKis and KisKdr strains after crossing and mass selection by exposing late 3 rd and early 4 th instars to appropriate insecticides. In the fourth generation, in which individuals with both resistant alleles were present, the strain was inbred and selected with chlorpyrifos methyl at 0.04 mg/liter to increase the Ace-1 R frequency. Finally, the purification of the Ace-1 RR homozygous strain was performed as follows. Pupae were held individually and adults that emerged were distributed in plastic cups (one male per three females) and allowed seven days to mate. After mating, the males of each cup were removed and their genotypes at the locus Ace-1 were determined (Weill et al. 2004). The females were removed and allowed to blood feed. After five days, females were placed individually in plastic cups to lay their eggs. Females were then killed and their genotypes for Ace-1 were determined. Only the eggs of homozygous Ace-1 RR females that were mated with homozygous Ace-1 RR males were kept. In short, all four strains are the S molecular form of An. gambiae and share the same genetic background as the Kisumususceptible reference strain but are homozygous for the resistance mechanisms they possess. Soil sample types We examined three soil sample types (soils were sampled once), which were selected based on the following criterion. Two of the soil samples (collected from Houeyiho, vegetable farms of specific farmers) had previous exposure to insecticides. The soil treated with insecticides within the previous week were labeled Freshly Treated Soil (FTS), while soil treated two months previously and not again were labeled Old Treated Soil (OTS). We also collected soil from two other areas where insecticides were not used, which were used as a control (CS, Control Soil). Each type of soil was collected and transported to the laboratory of Institut Régional de Santé Publique (IRSP) based in Ouidah. These soils were used to make artificial mosquito breeding sites in the laboratory. For each semi-natural habitat (a mixture of soil from each soil sample type and water), 25 g of dry soil was thoroughly mixed in 100 ml of deionized water in plastic containers. A total of 36 semi-natural habitats (three replicates, four mosquito strains, and three soil sample types) was created using plastic containers ( cm). The replicates were conducted simultaneously using mosquitoes from each strain. In each case, the parental populations of both strains had experienced the

3 36 Journal of Vector Ecology June 2016 A B Figure 1. Pupation rates (A) and adult emergence rates (B) for control soil (CS) for each strain of An. gambiae. Data are reported as the mean values and standard errors. A B Figure 2. Pupation rates (A) and adult emergence rates (B) for Old Test soil (OTS) for each strain of An. gambiae. Data are reported as the mean values and standard errors.

4 Vol. 41, no. 1 Journal of Vector Ecology 37 Figure 3. Pupation rates (A) and adult emergence rates (B) for Fresh Test Soil (FCS) for each strain of Anopheles gambiae. Data are reported as the mean values and standard errors. same laboratory conditions (27 C (± 1 C) and >80% relative humidity) and had been reared with the same protocol for several generations. Adult females blood-fed on restrained rabbits and the oviposition of the four strains was synchronized to generate matching cohorts of larvae. Fifty 1 st instar larvae were placed in randomly selected aquatic habitats in each of the four soil sample types. A total of 1,800 1 st instar larvae (50 larvae, three soil sample types, four mosquito strains, three replicates) was used. Larvae were fed daily with fish food at 2% (Tetramin BabyMin, Tetra Gmbh, Melle, Germany) (0.5 mg tetramin dissolved in 500 μl of water) until they either pupated or died. Pots of food were prepared each day. All experiments took place in a laboratory at the IRSP. We quantified the proportion of 1 st instar larvae reaching the pupal stage (pupation rate) and the proportion of 1 st instar larvae reaching the adult stage (emergence rate) for each strain on each of the three soil sample types tested. We counted the number of larvae surviving in each plastic container daily by carefully removing each larva using a pipette; all live larvae were placed back in the same plastic container. In the event of pupation, the date was recorded and the pupae were transferred to their own individual Drosophila tube (diam mm) containing 2 ml of deionized water. The tubes were sealed with foam plugs to prevent the emerging adults from escaping. In the event of adult emergence, the date was recorded. The number of individuals dying as either larvae or pupae was also recorded. The location of the plastic containers on the table were randomized on a daily basis to reduce positional effects. Statistical analyses Analyses were carried out using the R statistical package (version ). There were three replicates for each strain on each soil sample type. Each experiment involved 12 observations (three soil sample types, four mosquito strains) for a total of 36 artificial mosquito-breeding sites, which constituted the whole plot. The split-plots involved the effect of each soil sample on each mosquito strain. Data were normally distributed without transformation (Shapiro-Wilk W test: W=0.9550, P< ). To test if the experimental replicate affected the pupation and emergence rates, an analyses of variance (ANOVA) were used to test the fixed effects of soil sample type (Type), mosquito strain (Strain), and experimental effects (Exp) (model<-aov(y~type*strain+exp). Then, within each soil sample type, a one-way analysis of variance was carried out using the aov function to investigate possible significant differences between each strain (pupation and emergence rates). The model formula was: model<-aov (y ~ Strain, data) with y as the pupation or emergence rate.

5 38 Journal of Vector Ecology June 2016 RESULTS There was no significant variation between experiment replicates for pupation and emergence rates. The models show that only soil sample, strain, and their interaction influenced these two parameters (Table 1). When the control soil (CS) sample was used, no significant differences among strains were observed for pupation rate (F =1.05; p=0.42; df = 3) and these values were relatively high for all strains (from 77% for Acerkis to 85% for Kisumu) (Figure 1A). A similar observation was made when assessing the emergence rate (F=2.28; p=0.16; df = 3) with the control soil sample. (Figure 1B). When the Old Test soil (OTS) was used, a decrease was observed in the pupation and emergence rates recorded for the Acerkis strain (40%); these values were approximately 60% to 70% with AcerKdrKis, KisKdr, and Kisumu strains (Figure 2A). However, the differences among the data from the four strains were not significant (F=1.48; p= 0.29; for pupation rate and F=1.45; p=0.30 for emergence rate) (Figure 2A,B). In contrast to the other soil sample types (OTS, CS), the decrease in pupation and emergence rate for both Acerkis and Kisumu strains was considerable when the biological test was performed with the Fresh Test Soil (FTS). Both pupation and emergence rates were less than 1% for these strains but were still high for AcerKdrKis (60%) and KisKdr (70%) (F=23.59; p<0.001 for pupation rate and F=24.84; p<0.001 for emergence rate) (Figures 3A,B). DISCUSSION Mutations due to target site modification have been widely studied. Two distinct mutations in the S6 transmembrane segment of domain II of the VGSC at position 1014 have been identified, leading to amino acid residue changes from a leucine to a phenylalanine in West Africa (L1014F) and a leucine to a serine in East Africa (L1014S) (Martinez-Torres et al. 1998, Ranson et al. 2000, Donnelly et al. 2009). Recently, another mutation between domains IIIeIV of the VGSC (N1575Y) linked to the pyrethroid resistance phenotype in An. gambiae has been identified in west and central Africa (Jones et al. 2012). It was suggested that the selection of this mutation is associated with insecticide pressure in mosquitoes. Selective pressure is believed to be partly due to the use of insecticides in agriculture. Furthermore, this represents a threat to the efficacy of vector control programs (Diabate et al. 2002, Chouaibou et al. 2008, Yadouleton et al. 2009, Djogbénou et al. 2011, Yadouleton et al. 2011). The debate on the relative roles of agricultural and public health use of insecticides in the evolution of resistance in vectors remains ongoing. Some studies have attempted to confirm the hypothesis that agricultural practices result in the selection of insecticide resistance in malaria vectors. One such study was conducted in Benin at the same study site as the present study. In this study, only the Kisumu strain was used (Akogbeto et al. 2006) and it was clearly demonstrated that the soil samples tested contained inhibiting factors which contributed to the poor hatching rate of Anopheles eggs coupled with a retarded growth of larvae and a low yield of adult mosquitoes from hatched eggs. The limitations of these findings is that these inhibiting factors were not further characterized and there was no evidence that these factors represented insecticide residue or another toxic factors. Table 1. Summary results from analyses of variance on pupation and emergence rate. Pupation rate Emergence rate F value Pr (>F) F value Pr (>F) Type e e-07 Strain e e-05 Exp Type:Strain Type=Soil sample Type (CS, OTS & FTS) Strain= Mosquito colony (Kisumu, Acerkis, KisKdr & AcerKdrKis) Exp = Experiment F value= Fisher value The results from this study show a strong inhibitory effect of the Fresh Test Soil (FTS) on the pupation and emergence rates of Kisumu and AcerKis strains, which are without the kdr mutation (conferring a resistance phenotype to pyrethroids and DDT in the field). At the same time, the growth rate of KisKdr and AcerKdrKis strains (bearing kdr mutation) were less affected with the same type of soil. Knowing that the strains used in this experiment shared a majority of their genetic background, with the exception of the resistance alleles, we can conclude that the high survival of KisKdr and AcerKdrKis strains observed with FTS is due to the presence of the kdr allele. Given that the kdr allele confers cross-resistance to insects against pyrethroids and DDT, this result allowed us to speculate that the soil samples tested may contain relatively high levels of pyrethroids and organochlorine residues. Houeyiho site, 15 hectares in area, is located in the heart of the city of Cotonou, with an estimated farmer population of not less than 2,000. To protect their crops, vegetable-growing farmers used a large variety of synthetic pesticides for pest control (Assogba-Miguel 1999). Among the pesticides commonly used in vegetable farms in Bénin, pyrethroids appear to be most important. This reinforces our assumption that the greater association with the decrease of pupation and emergence in strains without the kdr mutation was with pyrethroid compounds. Residues from other sources acting on the same target as pyrethroids could be partially involved in this observation. Agricultural use of pesticides could potentially select metabolic mechanisms (detoxification enzymes) in mosquitoes. Further studies should investigate whether or not pesticides used for pest management in agriculture could select for mechanisms of metabolic resistance in An. gambiae. Synergists (e.g., piperonyl butoxide [PBO]) can be used in bioassays on insecticide-resistant strains with metabolic resistance mechanisms. This information could demonstrate how pesticides and other chemical products used for agricultural purposes might affect vector control programs. Some samples of soil and water from our study site were stored in the laboratory and will be analyzed by HPLC to verify the hypothesis and to identify the chemical compounds present in water and soil samples. We demonstrated that residual insecticide in soils from vegetable growing areas could be sufficient to select resistant individuals. Furthermore, the results provide more information on the quality of factors in the FTS that inhibited larval growth more than CS and OTS. We observed that there was no clear difference in the pupation and emergence rates of the four strains reared in Control Soil (CS) and Old Test Soil (OTS). As the OTS

6 Vol. 41, no. 1 Journal of Vector Ecology 39 was sampled two months after insecticide treatment, insecticide residues may have degraded and become less lethal for the Kisumu and Acerkis specimens. Permethrin (pyrethroid) residues have been shown to disappear rapidly in the soil even when large amounts are applied (Ismail and Kalithasan 2003). This could explain the high level of pupation and emergence rates observed with the OTS. Despite the limits of this study (absence of test using wild populations of An. gambiae from the same area where soils were collected and the analyses of soil composition) this study attempted to show, through biological tests, that agricultural activities can have a pronounced effect on the development of tolerance to insecticides in vectors such as mosquitoes. 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