Insecticide resistance in Anopheles stephensi in Somali Region, eastern Ethiopia

Our samples were collected in Kebri Dehar, a small town in the Somali Regional State, as previously detailed [4]. This town has a tropical semiarid climate with typically bimodal rainfall patterns. The area is also known to experience recurrent droughts. The population size is over 100,000 individuals, many of whom are pastoralists.

Larvae samples were collected from Kebri Dehar small town in Somali Region in eastern Ethiopia as previously detailed (Fig. 1) [4]. This region is predominantly lowland plain, with sparse vegetation including trees and shrubby. Larvae and pupae of Anopheles were collected from likely larval breeding habitats including man-made water containers, fresh water pools, stream margins, discarded tires, plastic containers, cisterns, barrels. In the study area water is stored in a container locally called “Birka” and it is constructed from cement and stone, the local people used for drinking and construction purposes. Briefly, immature stages of An. stephensi were collected from breeding sites in Kebri Dehar town using the dipping method according to WHO guidelines and were reared to adulthood in the field laboratory (Fig. 2). Collections took place from November to December 2016. In the field laboratory, the larvae and pupae were maintained at 28 ± 2 °C and 70 ± 10% relative humidity. The pupae were sorted and transferred with pipettes from the enamel trays to beakers with small amounts of water. Each beaker was placed inside a cage and was provided with 10% sugar solution for rearing them in the cage (Fig. 2). After two to three days, the pupae emerged to adults and the cages were put in safe place protected from contamination, ants, and other insects. The laboratory reared females of An. stephensi were used for different insecticide susceptibility test using WHO bioassay.

Species identification of the mosquitoes was conducted using standard morphological keys [24, 25] and molecular analysis of ITS2 and COI loci as reported previously for An. stephensi [4].

Insecticide susceptibility tests were carried out following WHO insecticide susceptibility test procedure [26]. A total of 700 non-blood fed adult female An. stephensi (2–3 day-old) were exposed to insecticide impregnated papers with discriminating concentrations of DDT (4%), Pirimiphos-methyl 0.25%, malathion (5%), deltamethrin (0.05%), permethrin 0.75%, bendiocarb (0.1%), and propoxur (0.1%). Batches of 25 mosquitoes in four replicates were exposed to insecticide impregnated papers for 1 h in WHO test tubes. The knockdown effects for all tested insecticides were recorded at 10, 15, 20, 30, 40, 50, and 60 min. A control in two replicates (50 female An. stephensi were used for each insecticide), each with equal number of mosquitoes, exposed to papers impregnated with oil was run concurrently. Mosquitoes were then transferred into holding tubes with untreated papers, where they were supplied with 10% sucrose solution. And, mortality was recorded 24-h post exposure. When mosquito mortality rate in the control is between 5 and 20%, mortality was corrected using Abbott’s formula [27]. All survived and dead specimens following bioassay were kept individually in Eppendorf tubes in silica gel for further molecular identification and kdr PCR assays. Determination of resistance was based on WHO criteria as follows: 98–100% mortality indicates susceptibility, 90– 97% mortality indicates resistance candidate (more investigation is needed) and less than 90% mortality suggests resistance [26].

In order to evaluate the presence of insecticide resistance mutations in the An. stephensi collected, a portion of An. stephensi from each experimental arm were selected for genotyping. For kdr mutation analysis, PCR was used to amplify the region of the vgsc gene that housed the L1014 alleles. Mosquito legs were used as templates for DNA. Mosquitoes were PCR amplified and sequenced individually. Analysis of the vgsc was completed according to Singh et al. [20]. PCR amplification of a portion the acetylcholinesterase gene (ace-1), associated with resistance to organophosphates and carbamates [28] was also performed for An. stephensi according to the protocol detailed in [29]. Vgsc and ace-1 PCR reactions were performed at 25 μl total with 2X Promega Hot Start Master Mix (Promega, Madison, W), with 1 μl template DNA, and the primer conditions listed in Table 1. Temperature protocols for vsgc amplification were as follows 95 for 5 min, 35 cycles of 95 for 30 s, 50 for 30 s, 72 for 45 s, 72 for 7 min. Temperature protocols for ace-1 were as follows: 94 for 5 min, 35 cycles of 94 for 30 s, 54 for 30 s, and 72 for 30 s, 72 for 5 min. PCR products were run on 2% agarose gel for 1 h at 100 V to confirm successful PCR amplification.

Sequences were submitted as queries to the National Center for Biotechnology Information’s (NCBI) Basic Local Alignment Search Tool (BLAST) to confirm correct loci were amplified. Sequences were then aligned to identify kdr L1014 and ace-1R G1109 mutations. The allele and genotype frequencies of these mutations were then calculated.

A total of 1200 An. stephensi larvae and pupae were collected from the breeding sites. Anopheles stephensi larvae occurred more frequently in cemented water reservoir and plastic water reservoir for construction. Anopheles stephensi positive habitats were mainly located close to human dwelling. Other Larvae and pupae of Aedes and Culex mosquitoes were visually detected and coexisted with An. stephensi, but not recorded.

A total of 700 An. stephensi were tested with different insecticides based on WHO protocol. The results of the susceptibility status of populations of An. stephensi are presented in Table 2. Overall, the percent mortality after exposure to insecticides ranged from 14% (pirimiphos-methyl) to 67% (deltamethrin). Using the WHO mortality threshold of above 98%, An. stephensi demonstrated resistance to bendiocarb, propoxur, DDT, malathion and permethrin.

A total of 51 mosquitoes were selected randomly from each research arm to represent the natural population of An. stephensi, including 19 that were tested for resistance to deltamethrin, permethrin, or DDT. Of these, eight were resistant to one of these insecticides. Of the 51 An. stephensi examined for kdr mutations, none carried the L1014 mutation. In addition, 30 An. stephensi were analysed for ace1 mutations. Of these, 20 had been tested for resistance to bendiocarb, propoxur, or malathion and eight were found to be resistant. Overall, none of the An. stephensi genotyped carried the ace1R G119S mutation.