Anopheles stephensi larval and pupal collection sites
Larvae and pupae of An. stephensi were collected from Awash Subah Kilo Town (also called Awash Sebat Kilo Town) and Haro Adi village around Metehara from January 2021 to June 2021, which is mostly a dry season, except the minor rains in March-April, in these areas. Awash Subah Kilo Town (08◦59'24.50"N, 40◦9'54.46"E) is located in Administrative Zone 3 of the Afar Regional State, just above a gorge of the Awash River, after which it is named. The town lies on the Addis Ababa–Djibouti Railway line at about 217 km from Addis Ababa. This town is the largest settlement in Awash Fentale district, situated at an elevation of 986 meters. Haro Adi village is located near Metehara Town. Metehara Town (08◦52'35.29"N, 39◦55'8.58"E) is located in central Ethiopia in the East Shewa Zone of the Oromia Regional State, situated at an elevation of 947 meters above sea level. Haro Adi village, from where the larvae and pupae of An. stephensi were collected, is a village located approximately two kilometers to the south of Metehara Town along Lake Beseka (Fig.1). The odds of mortality from insecticides were calculated by taking alpha-cypermethrin 0.05% insecticide and Awash Subah Kilo Town as constant, for comparing with their counterparts.
Rearing Anopheles stephensi mosquitoes
The larvae and pupae collected from the field were transported in plastic containers and reared to adults in the insectary at Aklilu Lemma Institute of Pathobiology, Addis Ababa University (AAU-ALIPB). The insectary has two secured doors, with a double door at the entrance and each separate unit of the insectary has its own door and sealed glass windows, which prevent mosquitoes from escaping. During mosquito rearing temperature and relative humidity of the lab were monitored. In the insectary, larvae were transferred into white plastic trays which were covered with mesh to prevent rapidly emerging adults. Larvae were fed by adding baker’s dry yeast (Saf-instant®), approximately half of teaspoon at a time, to the tray, and 5 minutes later, the tray was swirled to distribute the yeast powder and prevent suffocation from undiluted/accumulated powder [14].
Pupae were removed with plastic pipettes and transferred into a beaker with fresh deionized water and then transferred to adult holding cages daily. Adults in the cage were provided with 10% sugar solution using wetted cotton placed on the top of the mesh cage. The cotton was maintained wet so that mosquitoes could feed on the sugar ad libitum. The cotton balls were changed every 5 to 6 days, in order to avoid growth of mold spores and/or fungus [14]. Concurrent with sugar feeding, 3-7 days old female mosquitoes were fed on live rabbit blood meals twice per week (ethical approval was obtained from AAU-ALIPB Ethical Review Board). Water filled petri dishes and/or wet filter papers supported with cotton and placed on petri dishes were provided for mosquitoes to lay eggs on. The bioassay tests were conducted up to the seventh generation, with intention to have susceptible colony to be tested against LLINs products.
Anopheles stephensi species identification
Morphological identification of mosquitoes was done using dissecting microscope. Before commencing any bioassay tests 30 adult female mosquitoes were randomly aspirated from cages and transferred into paper cup. Then these mosquitoes exposed to and killed by chloroform (LABORT®). Dead mosquitoes were transferred to petri-dish and placed under a stereomicroscope at 40X and identified using identification key [15]. The identification was repeated on all exposed mosquitoes after reading the result. The specimens were neither stored nor sequenced for further molecular confirmation because resource of limitations.
Efficacy of long-lasting insecticide treated net products against adult stage of Anopheles stephensi
The LLINs for test were obtained both from Aklilu Lemma Institute of Pathobiology (ALIPB) and Amibara district health office in Afar Regional State. Four net products, MAGNet™, PermaNet® 2.0 DuraNet© and SafeNet®, were used for the test. The first three were obtained from those imported for distribution through the national program in 2020. These LLINs were collected from the distribution point prior to distribution to the community. SafeNet®, was obtained from ALIPB from the LLIN products collected for efficacy testing purposes. The manufacturing locations of the nets were Indian, Vietnam and China, respectively. DuraNet© product release and expiry dates were August 2020 and August 2023, respectively. MAGNet™ product release and expiry dates were March 2020 and February 2023, respectively.
MAGNet™ was impregnated with alpha-cypermethrin of 5.8 g/kg (261 mg/m2) and DuraNet© was impregnated with alpha-cypermethrin of 5.8 g/kg. SafeNet® is also impregnated with alpha-cypermethrin of 5.0 g/kg (200 mg/m2). PermaNet® 2.0 was impregnated with deltamethrin of 1.4 g/kg (56 mg/m2). Due to the SafeNet® and PermaNet® 2.0 product tags not including insecticide information, details on insecticide impregnation were obtained via WHO PQ/manufacturer websites. The tags on all net products did not include information of denier.
Five samples, one from each net panel (upper, head, feet, right and left), were taken with the tag referenced as the head position. The size of each sample taken from each net panel was 25 cm by 25 cm. Two WHO bioassay cones were used for each of the net samples taken from the aforementioned positions. Five sugar-fed 2-5 day old female adult An. stephensi mosquitoes were placed in each cone, resulting in ten mosquitoes tested per net sample, and were exposed for three minutes [16]. Simultaneously, as a negative control, two groups of ten sugar-fed 2-5 day old female adult An. stephensi mosquitoes were exposed via WHO cone bioassay to an untreated net sample (a net not treated with any insecticide). After the three minutes exposure time, mosquitoes then were transferred to holding cups and were immediately provided with 10% sugar solution. Knockdown and mortality was recorded at 60 minutes and 24 hours post exposure. The test was repeated, simultaneously with control groups, on two other nets of the same product on different days, resulting in a total of three nets tested for each net product. The investigation was done on unused and unwashed nets and conducted under controlled laboratory conditions.
Susceptibility test of Anopheles stephensi
Six insecticide impregnated papers (propoxur 0.1%, bendiocarb 0.1%, pirimiphos-methyl 0.25%, deltamethrin 0.05%, alphacyper-methrin 0.05% and permethrin 0.75%), all with impregnation date of February 2020 and date of expiry in February 2023, were obtained from Ethiopian Public Health Institute (EPHI).
The study was conducted as per the WHO standard procedures in conducting susceptibility test [17]. Six tubes for holding (labeled green), four tubes for treatment (labeled red) and two control tubes (labeled yellow) were set up prior to testing. Batches of 25 (3-5 day old non-blood fed) female mosquitoes were taken from rearing cages using mouth aspirators and transferred into the tubes. At the end of 1 hour resting period, they were transferred and exposed for 1 hour [17]. For the pyrethroids insecticides, mosquitoes unable to stand or move in a coordinated manner, or unable to fly were recorded as knocked down. After 1 hour, mosquitoes were transferred back into holding tubes with untreated papers and 10% sugar water. During the 24 hour post-exposure holding period, provided with 10% sugar water, tubes were kept in a cardboard shelter in the lab with maintained temperature and relative humidity. Mortality was recorded at 24 hours post-exposure and the result was interpreted as per the WHO guideline [17].
Data Analysis
Data were recorded using the WHO susceptibility test result recording form. Data from all replicates was pooled and entered into an Excel spreadsheet for analysis using STATA version 14.0. Logistic regression for mortality or survival of mosquitoes as an effect of exposure to insecticides and odds ratio was calculated for insecticides and by sites of mosquito origin.
Data Quality Assurance
Data quality was maintained by strictly implementing the control of other factors, such as temperature, humidity and conducting the test as per the laboratory procedures. In addition, data were rechecked for proper capturing at recording, organizing, cleaning and analysis steps.
Ethical Consideration
This study involved no human subjects and it was implemented after obtaining ethical clearance (Ref. No.: ALIPB IRB/40/2013/21) from the IRB of Aklilu Lemma Institute of Pathobiology, Addis Ababa University.