**4. Insecticide resistance studies in Botswana**

#### **4.1. Mosquitoes**

#### *4.1.1. Esterases in Culex mosquitoes*

The global spread of resistant genes acts as an example of evolution in action showing how selective forces, genetic variability, gene flow, migration, and life history can interact to produce changes in gene frequency. Two types of esterases are known and coded for loci est-A and est-B, corresponding to the production of esterases A and B, respectively [19]. These two elevated esterases have been shown to be overproduced as a result of amplification. This being said, several copies of one gene found on the same genome of the structural genes coding for them may happen in isolation or together [20, 21]. However, this is the major mechanism associated with organophosphate resistance in Culicine mosquitoes [22]. The association of these esterases has been found to be globally widespread in *Culex pipiens* complex mosquitoes, with highly active esterases A2 and B2 being shown to be strongly associated with organo‐ phosphate resistance in strains of *Culex quinquefasciatus* from California [23], West Africa [24], Kenya [25], Thailand and South Africa [26], and Vietnam [27]. Esterase B1 has been linked to North America and the Far East in *Culex* mosquitoes collected in Foshan [28]. Resistance by this massive overproduction of these esterases is conferred in a way that enzymes are able to detoxify the ester-based organophosphorus insecticides by hydrolysis to produce a nontoxic ionic metabolite. At the same time, the increased production of esterases can effectively sequester the insecticide and prevent it from reaching its target site [29].

A study was conducted in order to establish the concept of migration and the widespread of these esterases and whether they are present in mosquitoes sampled in Botswana. Their presence will be a clear indication of the possibility of resistance demonstrated by the mos‐ quitoes due to the selection pressure from the use of insecticides.

The mosquito larvae were collected from two areas in the southern part of Botswana: Gaborone and Molepolole. The areas are at least 50 km apart and have different economic activities. Gaborone is a city whilst Molepolole is a village. Single larvae at third and fourth instar and the adults were used for the experiment. The adults were identified as that of *Culex* specie from both collection sites. The presence of esterases was determined by subjecting each homoge‐ nised larvae and adult to a nondenaturing 7.5% PAGE gel to reveal the esterase bands.

Esterases identified as A2–B2 were revealed in the Molepolole strain, in both the adults and the larvae (Figure 1)

Esterase activity was determined by carrying out esterase and protein assays on the same homogenates of the single larvae and adults. The method used was described by Callaghan [30]. The results showed that esterase activity also varied in the larval and the adult stages for these mosquitoes from the two areas. In the Gaborone strain, the esterase activity for the *Culex* larvae was up to 50 μmol of β-naphthol per microgram of protein, whilst that of the adults ranged from 101 to 150 μmol of β-naphthol per microgram of protein. The results revealed that esterase activity increased in almost 35% of the population tested upon reaching the adult

**4. Insecticide resistance studies in Botswana**

The global spread of resistant genes acts as an example of evolution in action showing how selective forces, genetic variability, gene flow, migration, and life history can interact to produce changes in gene frequency. Two types of esterases are known and coded for loci est-A and est-B, corresponding to the production of esterases A and B, respectively [19]. These two elevated esterases have been shown to be overproduced as a result of amplification. This being said, several copies of one gene found on the same genome of the structural genes coding for them may happen in isolation or together [20, 21]. However, this is the major mechanism associated with organophosphate resistance in Culicine mosquitoes [22]. The association of these esterases has been found to be globally widespread in *Culex pipiens* complex mosquitoes, with highly active esterases A2 and B2 being shown to be strongly associated with organo‐ phosphate resistance in strains of *Culex quinquefasciatus* from California [23], West Africa [24], Kenya [25], Thailand and South Africa [26], and Vietnam [27]. Esterase B1 has been linked to North America and the Far East in *Culex* mosquitoes collected in Foshan [28]. Resistance by this massive overproduction of these esterases is conferred in a way that enzymes are able to detoxify the ester-based organophosphorus insecticides by hydrolysis to produce a nontoxic ionic metabolite. At the same time, the increased production of esterases can effectively

A study was conducted in order to establish the concept of migration and the widespread of these esterases and whether they are present in mosquitoes sampled in Botswana. Their presence will be a clear indication of the possibility of resistance demonstrated by the mos‐

The mosquito larvae were collected from two areas in the southern part of Botswana: Gaborone and Molepolole. The areas are at least 50 km apart and have different economic activities. Gaborone is a city whilst Molepolole is a village. Single larvae at third and fourth instar and the adults were used for the experiment. The adults were identified as that of *Culex* specie from both collection sites. The presence of esterases was determined by subjecting each homoge‐ nised larvae and adult to a nondenaturing 7.5% PAGE gel to reveal the esterase bands.

Esterases identified as A2–B2 were revealed in the Molepolole strain, in both the adults and

Esterase activity was determined by carrying out esterase and protein assays on the same homogenates of the single larvae and adults. The method used was described by Callaghan [30]. The results showed that esterase activity also varied in the larval and the adult stages for these mosquitoes from the two areas. In the Gaborone strain, the esterase activity for the *Culex* larvae was up to 50 μmol of β-naphthol per microgram of protein, whilst that of the adults ranged from 101 to 150 μmol of β-naphthol per microgram of protein. The results revealed that esterase activity increased in almost 35% of the population tested upon reaching the adult

sequester the insecticide and prevent it from reaching its target site [29].

quitoes due to the selection pressure from the use of insecticides.

**4.1. Mosquitoes**

266 Insecticides Resistance

the larvae (Figure 1)

*4.1.1. Esterases in Culex mosquitoes*

**Figure 1.** Gel showing the presence of A2B2 esterases from the adult Molepolole strain of *Culex* mosquitoes. The A and B esterases preferentially hydrolyse α- and β-naphthyl acetate, respectively. In the Gaborone strain, both the adults and the larvae revealed the presence of one esterase band B1, which preferentially hydrolysed β-naphthyl acetate (Fig‐ ure 2a and b).

**Figure 2.** (a). Gel showing the presence of B1 esterases from the adult Gaborone strain of *Culex* mosquitoes. (b). Gel showing the presence of B1 esterases from the larvae of the Gaborone strain of *Culex* mosquitoes.

stage. However, contrary to the results of the Gaborone strain, the esterase activity was found to be high at the larval stage.

The origin of the amplified A2–B2 esterases is yet to be identified. The appearance of resistance to organophosphates among populations of *C. pipiens* complex mosquitoes in Africa was reported in 1967 [31] and in Asia around 1968 [32]. On the basis of earlier reports of organo‐ phosphate resistance, Africa is thought to be the origin of amplification, but the first use of organophosphates in the continent is not well documented. According to Raymond et al. [33], the occurrence of overproduced esterase B1 is relegated to Asia and North America, but not in Africa. The distribution of B1 unlike the A2 and B2, which have been shown to be closely linked, has not yet been studied to elucidate the cause of its geographical spread and impor‐ tance in organophosphate insecticide resistance [20, 34]. However, the presence of B1 esterases as shown in the *Culex* mosquitoes collected from Molepolole indicated that esterase B1 has slowly found its way into Southern Africa.

The distribution of A2–B2 esterases in Africa, Asia, and North America may have been attributed to migration events [33]. This spread was also inevitable in the city of Gaborone. Botswana shares borders with South Africa where the presence of A2–B2 has been reported in organophosphate resistant strain of *Culex pipiens* [26]. This spread could have also been due to frequent migration of people between the two countries through visits, tourism, and trade. It is also not surprising that the spread of esterase B1 found itself into every town and village in the country through the Asian and Chinese traders, where only esterase A2–B2 was known to be dominant, such as in Africa. Thus, the presence of esterase B1 in Gaborone is as a result of migration of the esterase genes from areas of known prevalence such as North America, Asia, and China. Gaborone is an urban area where domestic and industrial pesticide spraying is randomly carried out. The high esterase activity displayed by the adults in the Gaborone strain may have been acquired initially at the larval stage and increased in the adults due to increase in the resistance from the kill of these chemicals.

The study has also shown that the possibility of resistance in mosquitoes is not restricted to one developmental stage. However, the esterase patterns in the developmental stages were the same, indicating that the same esterase genes are responsible for resistance throughout development from the larvae to adult stages, in both strains. Reasons for the increase in esterase activity in the larvae of Molepolole strain could be attributed to the fact that the area is a village within which there are farms lands and rearing of livestock. There is an extensive amount of agricultural practices whereby the application of insecticides on crops or acaricides on livestock is bound from time to time. These may have found their way into the nearby streams and gutters, which make good breeding sites for the mosquitoes.

#### *4.1.2. Susceptibility tests on malaria vector, Anopheles arabiensis*

Malaria is distributed in the northern part of the country, and this is a disease that is of public health priority to the government of Botswana, as it accounts for over 95% of malaria cases in Botswana [36]. *A. arabiensis* is the main malaria vector, and studies conducted in 2006 revealed that the species is distributed in all malaria areas of Botswana.

In order to reduce malaria transmission, the government of Botswana has engaged in what is called integrated vector management (IVM), which involves the utilisation of different interventions, including environmental management, safe, careful, and thoughtful use of insecticides. One such intervention is the indoor residual spraying (IRS) of insecticides, which goes back to the 1940s when spraying of human dwellings was initiated [37]. In the 1950s, the use of diethyl-dichloro-trichloroethane (DDT) started in Botswana for the malaria vector control using IRS [35]. In 1997, Botswana then introduced insecticide-treated nets (ITNs) to complement IRS as part of the IVN initiative [38]. Between 1971 and 1973, fenitrothion, which is an organophosphate, briefly replaced DDT. However, due to the poor efficacy of fenitro‐ thion, DDT was reinstated as the main insecticide to serve together with IRS as Botswana's principal vector control intervention against malaria.

The WHO global strategy for the Malaria control is to break the malaria parasite transmission by using indoor residual spraying or pyrethroid impregnated materials such as bed nets. It is during such programmes that the annual vector susceptibility studies are carried out in Botswana.

Similar studies were conducted [39] to confirm the presence of pyrethroid resistance among *Anopheles gambiae* from West Africa (Benin and Burkina Faso), Central Africa (Cameroon), and *A. arabiensis* from Southern Africa (Botswana). WHO test kits for resistance tests were used with the adult mosquitoes being subjected to exposure to permethrin, deltamethrin, and DDT. From the results, permethrin resistance were detected in Benin, Burkina Faso, and deltameth‐ rin resistance was detected also in Cote d' Ivoire. Botswana, on the other hand, showed susceptibility of *A. arabiensis* towards permethrin and DDT. The results obtained by Coetzee [40] on the susceptibility status of *A. Arabiensis* in Botswana using the same three insecticides were found to be in agreement with the susceptibility tests conducted annually for vector susceptibility by the Ministry of Health in Botswana. Table 2 presents the summary of the results.


**Table 2.** Percentage susceptibility levels of *Anopheles arabiensis* towards insecticides.

as shown in the *Culex* mosquitoes collected from Molepolole indicated that esterase B1 has

The distribution of A2–B2 esterases in Africa, Asia, and North America may have been attributed to migration events [33]. This spread was also inevitable in the city of Gaborone. Botswana shares borders with South Africa where the presence of A2–B2 has been reported in organophosphate resistant strain of *Culex pipiens* [26]. This spread could have also been due to frequent migration of people between the two countries through visits, tourism, and trade. It is also not surprising that the spread of esterase B1 found itself into every town and village in the country through the Asian and Chinese traders, where only esterase A2–B2 was known to be dominant, such as in Africa. Thus, the presence of esterase B1 in Gaborone is as a result of migration of the esterase genes from areas of known prevalence such as North America, Asia, and China. Gaborone is an urban area where domestic and industrial pesticide spraying is randomly carried out. The high esterase activity displayed by the adults in the Gaborone strain may have been acquired initially at the larval stage and increased in the adults due to

The study has also shown that the possibility of resistance in mosquitoes is not restricted to one developmental stage. However, the esterase patterns in the developmental stages were the same, indicating that the same esterase genes are responsible for resistance throughout development from the larvae to adult stages, in both strains. Reasons for the increase in esterase activity in the larvae of Molepolole strain could be attributed to the fact that the area is a village within which there are farms lands and rearing of livestock. There is an extensive amount of agricultural practices whereby the application of insecticides on crops or acaricides on livestock is bound from time to time. These may have found their way into the nearby streams

Malaria is distributed in the northern part of the country, and this is a disease that is of public health priority to the government of Botswana, as it accounts for over 95% of malaria cases in Botswana [36]. *A. arabiensis* is the main malaria vector, and studies conducted in 2006 revealed

In order to reduce malaria transmission, the government of Botswana has engaged in what is called integrated vector management (IVM), which involves the utilisation of different interventions, including environmental management, safe, careful, and thoughtful use of insecticides. One such intervention is the indoor residual spraying (IRS) of insecticides, which goes back to the 1940s when spraying of human dwellings was initiated [37]. In the 1950s, the use of diethyl-dichloro-trichloroethane (DDT) started in Botswana for the malaria vector control using IRS [35]. In 1997, Botswana then introduced insecticide-treated nets (ITNs) to complement IRS as part of the IVN initiative [38]. Between 1971 and 1973, fenitrothion, which is an organophosphate, briefly replaced DDT. However, due to the poor efficacy of fenitro‐ thion, DDT was reinstated as the main insecticide to serve together with IRS as Botswana's

slowly found its way into Southern Africa.

268 Insecticides Resistance

increase in the resistance from the kill of these chemicals.

and gutters, which make good breeding sites for the mosquitoes.

*4.1.2. Susceptibility tests on malaria vector, Anopheles arabiensis*

that the species is distributed in all malaria areas of Botswana.

principal vector control intervention against malaria.

Both studies have been able to show that the malaria vector *A. arabiensis* does not have an indication of resistance towards the insecticides used in Botswana as it is fully susceptible to DDT and pyrethroids. DDT still remains the most sensitive insecticide, when tested against pyrethroids. Baseline studies were carried out on insecticide resistance in five Southern African countries including Botswana. The results showed that there was also complete susceptibility of *A. arabiensis* to DDT and pyrethroids [40].
