Description of the study area
The study was conducted in Arba Minch district in Southern Nation, Nationalities and Peoples’ Regional state (SNNPR) of Ethiopia. The study village is found north of Arba Minch at about 16 km and south of Addis Ababa at about 470 km. The village is located at 6°6.666´ N and 37°35.775´E and at altitude of 1,206masl. It is one of the malarious villages in Arba Minch district. The climate is hot and humid which is suitable for malaria vectors. The inhabitants are mixed farmers as they are ranching cattle and growing crops. They grow cash crops such as mangoes and bananas as the main source of income. LLINs and IRS are the principal malaria vector control tools. An. arabiensis is the principal malaria vector in the area (Massebo et al., 2013). (Fig. 1).
An experimental study was conducted to investigate the host-seeking behavior of malaria vectors. The first experiment was conducted in the shore of Lake Abaya with a high potential of mosquito breeding from October to December 2016. Four tents were constructed and placed close to the shore of the Lake Abaya (Fig. 2). The tents have mosquito entry points which can be closed and opened by zips. The zip of each tent was closed at day time to prevent damages by wind. During the mosquito collection time (18:00–24:00) the zip was remaining open to allow mosquito entering. Mosquito collection was done until the mid-night due to the high wind wave after mid-night. The shore of the Lake was selected to avoid the interference of other domestic/wild animals by attracting mosquitoes as the experiment was conducted in open area where other wild animals are usually not stay. (Fig. 2.)
The tents were designed to sample mosquitoes by human land catches (HLC) from human alone and cattle baited tents to see the mosquito biting tendency. The distance between tents was 20 m. The tents for human alone and cattle bait were selected randomly on the first night, and then rotated to reduce the bias driven by variation in attractiveness to mosquitoes, collecting abilities, and spatial variation due to location of the tents. Four human volunteers (above 18 years) were recruited from the local people to collect mosquitoes entering the tents. The volunteers who had collection experience, and gave written consent were included. Two persons sat inside tents with calves (one person and calve in each tent). The other two persons sat inside the other two tents (one person in each tent) without calves. There was two collection shifts in one cycle which was completed in eight nights. In the first four consecutive nights, collectors were rotated daily between tents, and after four consecutive night collection, there was two days break for collectors. Then, in the next four consecutive nights, we rotated only calves.
The second trial was done by keeping calves outside the tents at 1 m distance from the tents (Fig. 3). In this case two human collectors sat inside the two tents (one in each tent) separately without calves outside the tent at one meter distance. And inside the other two tents two human collectors sat separately in each tent and two calves (one outside each tent) were kept at one meter distance. In the first night, the tents were randomly selected for calves and humans. In the first four days, the calves were rotated and in the second four days the collectors were rotated to minimize the bias due to location and collection skills.
The third activity was done within the village to assess the biting rhythm and identify the peak biting hours of the malaria vectors. Indoor and outdoor mosquito collection was done in two houses with human occupants using HLCs. In each house, a pair of collectors were interchanged their positions between indoor and outdoor every hour. A pair of collectors at one house in the first night was rotated to the second house in the next collection night and the cycle continues until the end to minimize the bias due to collection skills and attractiveness of the collectors. The distance between the two houses was about 200 m. The collection was done from 18:00–06:00 for seven nights during the study period.
Each collector sat on a chair with the legs exposed from foot to knee and caught mosquitoes as soon as they land on the exposed legs before they start biting by aspirator. Each hour collection was kept separately in labeled paper cups for species identification and further analysis. A strong supervision was made to avoid the bite of mosquito on collectors. (Fig. 3.)
Identification of Anopheles mosquitoes
The collected Anopheles mosquitoes were brought to Medical Entomology Laboratory at Arba Minch University. They were killed via refrigerator and the species were identified through microscope based on morphological characteristics using an identification key(Gillies and Coetzee, 1987). Then, the identified female Anopheles mosquitoes were placed in vials over silica gel for circum-sporozoite proteins (CSPs) test.
Mosquitoes processing for CSPs detection
The CSP detection was performed by Enzyme-Link Immuno Sorbent Assay (ELISA) technique (Beier et al., 1987) at Arba Minch University Medical Entomology laboratory. The head and thorax of female An. pharoensis was used for P. falciparum and P. vivax-210CSP detection. Two separate 96-well micro-titer plates were coated with 50 µl solution of P. falciparum and P. vivax-210 monoclonal antibodies (MAB). The plates were covered and incubated for overnight at room temperature. Then the contents of plates were sucked, emptied and filled with 200 µl blocking buffer (BB) and further incubated for one hour at room temperature. During the incubation time, mosquitoes were grounded individually in 50 µl grinding solution and the final volume brought to 250 µl by rising with 100 µl BB twice. BB was removed from plate after one hour and 50 µl of each homogenized mosquito triturate was added to each of the two test wells. P. falciparum and P. vivax-210 positive samples and a wild-colony of An. pharoensis were used as both positive and negative controls. Plates were incubated for two hours and washed with phosphate-buffered saline (PBS)-Tween 20 twice. Horseradish peroxidase-conjugated monoclonal antibody was then added to each well and incubated for one hour, and the wells were washed three times with PBS-Tween 20. Finally, 80 µl of preoxidase substrate was added per well and incubated for 30 minutes. The wells were observed visually for green color and also their optical density determined at 414 nm in microplate ELISA reader. Sample with green with color and with optical values of greater than two times the average optical density of the negative controls were considered positive.
The first primary outcome variable was the host-seeking tendency and hourly biting rhythm of malaria vectors to hosts. The secondary outcome variable was the species composition of Anopheles mosquitoes.
The host-seeking tendency of malaria vectors was calculated by A Generalized Estimating Equations (GEE) with a negative binomial distribution. The mean ratio of the number of Anopheles species collected from cattle baited tents and human alone was used to determine the host preference of mosquitoes and the impact of the proximity of the cattle on human exposure. The Anopheles mosquito peak biting time was also determined by GEE with negative binomial distribution. The p-value < 0.05 was used to determine the peak biting hours and the feeding tendency of malaria mosquitoes. The data were entered into excel and analyzed using SPSS version 20.
The study was reviewed and approved by the ethical review committee of Arba Minch University (Ref.No.1480/11).The objective of the study was explained to human volunteers. Participation in mosquito collection was based on their willing and only those adequately trained on the collection process was included. Verbal and written consent was obtained from human volunteers. The volunteers for field trial were informed to be alert and catch the coming mosquitoes as soon as landing on their exposed legs. Malaria prophylaxis was given to prevent infection.