Anopheles stephensi collection, rearing and identification
Anopheles stephensi larval and pupal collection sites
Larvae and pupae of An. stephensi were collected from Awash Subah Kilo Town (also spelled as Awash Sebat Kilo in other publications) and Haro Adi around Metehara from January 2021 to June 2021. Awash Subah Kilo Town is located in Administrative Zone 3 of the Afar Region, 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, lying at a longitude of 08°59′N 40°10′E at an elevation of 986 meters, with favourable climate and altitude for malaria vector breeding and optimal to parasite sporogonic cycle completion. Metehara is also a town in central Ethiopia; located in the East Shewa Zone of the Oromia Region, on a longitude of 08°54′N 39°55′E, 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 to the south of Metehara Town along Lake Beseka located about two kilometers away from Metehara Town (Table 1 and Fig. 1).
A total of 45 breeding sites/habitats, in and around the towns of Awash Subah Kilo and Metehara and Haro Adi village areas were visited for larval and pupal surveys. Of these, 31 breeding habitats were from Awash Subah Kilo Town, 7 from Metehara Town, and 7 from Haro Adi village. The survey of An. stephensi larvae and pupae was carried out in three sites, namely; Awash Subah Kilo Town, Metehara Town and Haro Adi around Metehara Town (Fig. 1). The survey for An. stephensi larvae and pupae was conducted in metal tanks near houses under construction, in jerry cans where water is reserved for daily household consumption, on cemented water banks for daily household consumption, on water reservoirs with geo-membrane plastic (near Metehara health center), overhead water tanks, and in cemented burrows of water reserved for production of cement blocks. These sites were selected based on the previous reports of the presence of An. stephensi (Ashine et al., 2020; Balkew et al., 2020). The sampling of breeding sites was managed based on the WHO guidelines for laboratory and field testing of mosquito larvicides (WHO, 2005). All natural and man-made breeding sites around the study areas were assessed for the presence or absence of An. stephensi larvae. All larval instars and pupae were collected and taken to a bio-contained facility in the insectary of Aklilu Lemma Institute of Pathobiology (ALIPB).
Larvae and pupae were collected using a WHO standard dipper and transferred into a plastic jar of five-litre capacity with a handle and a cover with plenty of holes to allow air circulation. The jar was used for handling and transporting the larvae and pupae. The scooped larvae and pupae were filtered using clean cheesecloth prepared for this purpose and transferred to a plastic jar. Then approximately 1-1.5 litres of water along with debris of plants, from their natural breeding sites was added for larvae to feed on until they reached the insectary.
Rearing Anopheles stephensi mosquitoes
The larvae and pupae collected from the field were transported and reared to adults in the insectary. During mosquito rearing all of the lab conditions, such as maintaining the temperature at 27 ± 2°C and 75 ± 10% relative humidity, were met and monitored. Upon arrival in the insectary, larvae were transferred into a white enamel plastic tray. Once larvae were removed from their natural water source in the plastic container using plastic micropipettes, a diet of baker’s yeast was added to the larval tray. After 5 minutes the tray was swirled to distribute the powder and prevent suffocation from undiluted/accumulated powder (EPHI, 2017). Larvae were provided food twice per day, and trays were checked to see if food remained unconsumed, and if larvae food remained unconsumed, no food was added.
Sorting of pupae from larvae was undertaken on a daily basis. Pupae were picked with plastic pipettes and transferred into a beaker with fresh deionized water and then transferred to adult holding cages. Adults in the cage were provided with sugar solution using soaked/wetted cotton ball placed on the top of the meshed cage. The cotton was maintained wet so that mosquitoes could feed on the sugar. The cotton balls were changed every 5 or 6 days, in order to avoid the growth of mold spores and/or fungus on the pad exposed to sugar (EPHI, 2017). Concurrent with sugar feeding, 3–7 days old female mosquitoes were fed on rabbit blood meals twice per week (ethical approval was obtained from Addis Ababa University-Aklilu Lemma Institute of Pathobiology (AAU-ALIPB) Ethical Review Board)). Water filled petri-dish and/or wet filter paper supported with cotton and placed on a petri-dish were provided for mosquitoes to lay eggs on. Breeding of wild-collected An. stephensi colonies continued until the end of the study. The tests were done on F1 and F2 generations of the field-collected larvae and pupae.
Anopheles stephensi species identification
Mosquito species identification was undertaken morphologically under a dissecting microscope. Before commencing any efficacy test of the selected larvicides against An. stephensi, 30 adult female mosquitoes were randomly aspirated from cages. Then these mosquitoes were transferred into a glass tube and exposed to chloroform by cotton ball wetted at tip. Each of these mosquitoes was laid under a stereomicroscope at 40X for morphological identification using the updated key to the females of Afro-tropical Anopheles mosquitoes, which includes An. stephensi (Coetzee, 2020). All were confirmed to be An. stephensi mosquito species. There were fewer Culex and Aedes species larvae as compared to An. stephensi, collected from the same habitats. Though there were a few Culex and Aedes species larvae collected with An. stephensi, all those aspirated from the cage were An. stephensi. The typical features with An. stephensi mosquito’s morphology are (i) the appearance with 3 pale bands in the palpus and the two apical pale bands are very broad with speckling on palpus segment 3 and (ii) in the 2nd main dark area on the vein 1of its wing, there are 2 pale interruptions (Coetzee, 2020). The specimens were not stored for further molecular confirmation because of financial limitations and the inability to preserve the specimens for a longer time. However, rearing of the colony in the insectary has continued.
Efficacy of Bacillus thuringiensis var. subspecies israelensis and temephos against An. stephensi larvae
Bacillus thuringiensis var. species israelensis (Bti); VectoBac WDG (FourStar®Briquets) of a solid form; produced by DBA FourStar Microbials LLC. 1501 East Woodfield Road, #200W (https://www.centralmosquetocontrol.com/all-products/fourstar/fourstar-briquet-180) in January 2019 and with expiry date of December 2023, were acquired from ICIPE/ILRI. The powder form of this bacterial larvicide was weighed on digital weighing scale and prepared in increasing doses of 0.05g, 0.1g and 0.2g, in such a way that it was to be applied in a container of 2000cm2 with one litre water volume until the dose mortality response was reached. Based on this design, the lowest prepared concentration of Bti (0.05g/l), was added to the water and remained for 48 hours by covering the container to prevent insects from landing or laying egg in it (Demissew et al., 2016). In order to assure no insects entered into the larvicide-treated water, the tray remained closed. The insectary had two secured doors, a double door at the entrance and each unit of the insectary had its own door and closed glass windows. The subsequent tests were done following the same procedure.
In preparation to expose the larvae to larvicides, late third to early fourth instar larvae were sorted in disposable cups with water using pipettes. Larvae were filtered first through cheesecloth on a separate container for this purpose. The filtered larvae were immediately transferred into plastic containers having an area of 2000cm2 and containing one litre of water treated with Bti of 0.05g, as per the application recommended for spot spray (BASF SE, n.d.). Batches of 25 larvae were exposed per testing container. Simultaneously an equal number of larvae (negative controls) were tested using untreated deionized water and with same number of larvae per container. The tests were done in four replicates. The experiment was repeated three times on different days. This test was repeated for larvae collected from each site. The two higher doses (0.1g and 0.2g) were not tested because larvae had already responded to the lowest dose of Bti (0.05g).
Temephos, an emulsifiable liquid concentrate containing 500g of active ingredient per liter, brand name BASF-Abate®500E, developed in Malaysia in 2018 (https://www.mkhardware.com.my/pages/pages_id/13613/) with no stated expiry date, was acquired from Ethiopian public health institute (EPHI), and tested against An. stephensi larvae. Following the same procedure used for Bti testing, temephos of 0.25ml/l, 0.5ml/l and 1ml/l was prepared to be tested in increasing concentrations, until the dose response was saturated. Temephos (0.25ml) was added to a container of 2000cm2 with one litre water volume using 1000ml capacity micropipette. Four replicates were set up for each concentration and each was run three times on different days. An equal number of negative controls were set up simultaneously with deionized water. The late third and early fourth stage larvae, collected from the field and from reared adults (F0, F1 and F2), were used for the larvicidal test. Larvae were first collected from the tray using pipettes into disposable plastic cups containing water. Then 25 larvae were filtered on and immediately transferred into the container of 1000ml water treated with 0.25ml of temephos. Larvae were confirmed susceptible to the lowest concentration of temephos (0.25ml/l) and the higher prepared concentrations were left not tested.
While conducting the efficacy tests of both larvicides, larval mortality was recorded after 24 hrs. Larvae that sank down to the bottom of water, in the case of temephos, and appeared floating on the water with swollen and blackened bodies, in the case of Bti, were considered dead. The WHO guidelines for laboratory and field testing of larvicides, states that the test should be rejected if the control mortality is > 20% or pupation is > 10% (WHO, 2005).
Data Analysis
The data were recorded using the WHO larvicide efficacy evaluation result recording form (WHO, 2005). The data from all replicates was pooled and entered into an excel spreadsheet for analysis using STATA version 14.0. Statistical analysis was not done because of the high susceptibility to the larvicides, with the exception of one larva exposed to Bti.
If the control mortality was between 5% and 20%, the mortalities of treated groups were corrected according to Abbott’s formula. Tests with control mortality greater than 20% or pupation greater than 10% were discarded.
The mortality of the test sample was calculated by summing the number of dead larvae across all exposure replicates and then expressing this as a percentage of the total number of exposed larvae.
Data Quality Assurance
The work to generate quality data started from strictly implementing the control of other factors, such as temperature, humidity, dose of larvicides, and conducting the test as per the laboratory procedures to gather all the important information from the study. 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, dated: Feb 10, 2021) from the IRB of Aklilu Lemma Institute of Pathobiology, Addis Ababa University.