Study Area
The study was conducted in Lira and Kole Districts in Northern Uganda, (Fig. 1). Lira District has a projected population of 428,000 people and is bordered by Pader District to the north, Otuke district to the northeast, Alebtong District to the east, Dokolo District to the southeast, Apac District to the southwest, and Kole District to the west. The coordinates of Lira district are: 02 20N, 33 06E (Latitude: 02.3333; Longitude: 33.1000). Lira district is located 342 kilometers (212.81 mi), the capital city of Uganda. Kole district was formed from Apac District and has an estimated population of 231,900 as of July ‘2012’ and is composed of the following sub-counties; Aboke, Alito, Okwerodot, Akalo, Bala, Abeli, Ayer, and Kole town councils. The coordinates of Kole district are: 02 24N, 32 48E. It’s bordered by Lira District to the east, Apac District to the south, and Oyam District to the west. The district capital is located approximately 28 kilometers (17 mi) by road, northwest of Lira, the largest city in Lango sub-region. Both districts are in a malaria epidemic-prone area and experience a bimodal pattern of rainfall with a long rainy season (April—July), which triggers the peak malaria transmission period, short rainy season (October-November), and year-to-year variation. The hot and dry season is from December to March, which marks a low transmission period, and both districts are endemic for malaria. In both districts, economic activities include farming both animal and crop production. Concerning malaria transmission and malaria epidemic-prone areas, several malaria control interventions in both districts, such as case management, social behavior change communication (SBCC), use of insecticide-treated Nets, and Indoor residual spraying have been ongoing in Lira district since June 2014, expected to end in May 2023, and in Kole none IRS district, it started way back in 2009 and exited in 2013.
Study design
The study was cross-sectional in nature and involved quantitative data collection on species composition, indoor resting population density, and feeding patterns of malaria vectors over a period of six weeks from August to September 2022, in both the IRS and non-IRS districts of Lira and Kole respectively.
Sampling Procedure
Three sub-counties in each district and three parishes in each sub-county were randomly selected for the study, and finally, one village in each of these parishes was randomly selected. In each village, ‘20’small inadequately ventilated houses with thatched roofs and mud walls[9] were randomly selected for PSC. These types of houses provide a favorable, cool, resting place for endophilic and endophagic malaria vectors[10]. Two other houses were randomly selected from each village for human-baited catches, and two nights of mosquito collections were carried out simultaneously in the two districts. All female mosquitoes belonging to An. gambiae complex was identified based on morphological characteristics using standard identification keys adopted by Gillies and Coetzee[11]. Global Positioning System (GPS) coordinates were recorded on the data collection form for the sampled households using a handheld navigational system, to map the vector species composition, indoor resting population densities, and feeding pattern of malaria vectors in both districts.
Determination of indoor resting density of malaria vectors and species composition of mosquitoes
Pyrethrum Spray Catches (PSC)
The pyrethrum spray catch method[12] was conducted in 20 randomly selected houses throughout the study period, in each of the three selected villages per district. In each village, grass-thatched houses where people sleep and are not used for cooking were selected upon introduction of the team and explanation of the purpose and procedures of the activity to the household heads. Thatched roofs and mud walls provide better resting surfaces and more entry points for malaria mosquitoes[10], thus rendering a cool, favorable resting place for endophilic and endophagic malaria vectors.
The procedure (PSC) was conducted after preparing the household owners and houses by removing a few items from the house to allow the spreading of the white ground sheets to cover the ground surfaces where mosquitoes found resting indoors would fall dead or knocked down. Two vector control officers sprayed houses with aerosol insecticide (Kill it), with one spraying from inside a closed door, spraying the eaves and roof while moving in the opposite direction to the one spraying outside eaves to prevent any mosquitoes from escaping. The officer inside then came out and closed the door, timing for 7–10 minutes to ensure all the mosquitoes present indoors were either killed or knocked down. After 7–10 minutes, the doors were carefully opened, and the ground sheets that were laid on the ground were systematically folded and brought outside, starting with the one next to the door. All mosquitoes found either dead or knocked down on the ground sheets were carefully picked using forceps and put into a well-labeled petri dish with moist filter paper, starting from the first house to the twentieth house, and brought to the laboratory for further identification and analysis.
All mosquitoes collected were sorted and Identified in the field laboratory under a dissecting microscope to separate female Anopheles from male Anopheles and other insects, with the help of morphological keys developed by Gillies & Coetzee[11].
Determination of feeding/biting pattern
Mosquito trapping in both IRS and non-IRS districts was conducted using aspirators with trained personnel as bait in a double net weekly for two (2) consecutive nights for the six weeks at selected field sites. Both Indoor and outdoor biting mosquitoes were collected from 6.00 pm to 6.00 am by a two-person team of trained catchers using bed-net traps[13].
The bed net trap was made by making a 3 x 3-inch hole on each side of an untreated bed net, making a total of 4 to 6 holes on the net, and the bottom of the outer net was stretched tightly and tied to pegs in the ground leaving 15–20 cm between the ground and the lower edge of the net. People living in a room were each protected with an untreated net. The catchers sat under the bed net trap, which provided them with some protection, which was denied when the human-landing catch method was used. Outdoor human biting catches were carried out concurrently using the same method in the same household ten meters away[12]. Each of these collectors exchanged their position hourly and an interval of 5–10 minutes’ exchange period was given. The one outdoors moved indoors, while the one indoors moved outdoors, and each pair only worked for a period of four hours, making a total of six collectors per house and twelve collectors for the two houses in each Village and district. As hungry mosquitoes persisted in their attempts to look for a blood meal, they were approached, and as they got near the human-baited trap, were caught[14], by the human bait (collector) using an aspirator and a torch[12]. It was assumed that the mosquitoes that entered a trap during any hour were those actively seeking hosts and in most cases, would bite human hosts during the same hour and room/house if the bed net trap were absent[15]. The indoor and outdoor human-biting fractions of Anopheles mosquitoes were determined and recorded throughout the sampling period of six weeks for both the IRS and non-IRS districts. Each hourly catch was separately placed in a disposable polystyrene container pre-labeled with location, date, and time of capture and taken to the laboratory for identification. The mosquitoes collected were kept alive by providing them with a 10% sugar solution to feed on through a cotton wick[16].
Mosquito species identification
All mosquitoes collected were identified morphologically to species level in a field laboratory with the aid of a dissecting microscope and the use of morphological keys developed by Coetzee[11]. Female Anopheline mosquitoes were recorded in a form according to their abdominal status as unfed, freshly fed, half gravid, and gravid. Field specimens were individually placed in tubes containing silica gel desiccant and cotton immediately after collection and stored at room temperature until processing. Before homogenization, the dry field specimens were rehydrated at room temperature in 20 µL of double-distilled water for 20 minutes. Colony mosquitoes used as positive controls were killed by freezing at designated times post-feeding and were processed immediately. As these samples were fresh, no rehydration step was necessary.
Polymerase chain reactions for sibling species determination
DNA extraction
DNA was extracted from individual mosquitoes using the Chelex protocol by Musapa[17]. The legs and wing tissues were macerated by a sterile pipette tip and mixed with one hundred micro litres (100µl) of DNAase-free water, grinding was done until no large body parts were visible. Two hundred and fifty micro litres (250 µl) of grinding buffer containing 1xPBS (pH 7.4), 1% sarcosine, and 0.05% Tween 20 was added to the Eppendorf tube. The Eppendorf tubes wereclosed, pulsed in a micro-centrifuge, and stored at -20°C. The tubes with closed caps were placed in a PCR machine (Eppendorff Master Cycler gradient), and the lysate was boiled for 20 minutes at ninety-nine degrees centigrade (99°C) and then cooled for 1 minute. The tubes were placed with opened caps in the PCR machine at 90°C for 20 minutes, and briefly centrifuged and DNA was stored at -20°C. Female Anopheles mosquitoes were subsequently analyzed to identify sibling species within An. gambiae s.l. and An. funestus s.l. complex by standard PCR. After DNA extraction, Ribosomal DNA was amplified using the Martha protocol[18].
Amplified samples were run on a 1.5% agarose gel stained with ethidium bromide (EtBr). Gel electrophoresis was performed following the basic PCR protocol by Todd Lorenz to separate DNA fragments of species-specific base pairs using a Thermo EC105 Electron Corporation machine. A molecular weight ladder was loaded into the first lane of the gel, and samples were loaded into subsequent wells. The gel was run at a current of 10V/cm and a voltage of 80V for 1 hour. The DNA bands on the gel imager were visualized and the size of each band specific to each sibling species was determined using a DNA ladder[18].
Polymerase chain reaction amplification and species determination
To confirm the accuracy of morphological identification, PCR was used to separate An. gambiae complex samples. Samples caught indoors and outdoors were analyzed in both the intervention and non-intervention areas. All members of the An. gambiae complex was discriminated by the SNP-based PCR with primers that bind to the SNP sites utilizing Intentional Mismatch Primers (IMPs) within the intergenic spacer region, producing unique bands for each of the species and sub-forms[19]. One to two legs or wings of a single adult mosquito (as a DNA source) were placed in 22.0 µl of PCR reaction mixture. This mixture contained the following: /14.5 µl of double distilled water, 5.0 µl of 5X High Fusion reaction buffer (100 mM Tris-HCl pH 8.3, 500 mM MgCl2), 0.5 µl of 10 mM dNTPs,1.5 µl of UN, ME, AR, GA and QD primers (synthesized by Inqaba Biotechnical Industries, Pretoria, South Africa) and 0.5 µl of Phusion TM DNA polymerase. Finally, 3.0 µl of double distilled water was added to make a total volume of 25.0 µl, and the reaction mixture was agitated a few times [(MgCl2, Buffer, Phusion TM DNA polymerase and dNTPs. Negative controls containing PCR mixtures without DNA were added to each PCR experiment. The PCR reaction conditions were as follows: Initial denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, and final auto extension at 72°C for 5 min. A volume of 10.0 µl of the PCR product was mixed with 3.0 µl of ficoll dye (50% sucrose, 0.05 M EDTA pH 7, 0.1% bromophenol blue, 10% ficoll powder) and loaded on 2% agarose gel stained with 12.0 µl ethidium bromide (10 mg/100 ml), submerged in 1XTAE buffer and electrophoresed at 100 V for one hour. 5.0 µl of a molecular marker (Gene Ruler TM DNA ladder Mix, Cat. No. SM0331) was loaded on the first well of the agarose gel, followed by the wild samples in the next wells, the positive control in the second last well, and the negative control in the last well. The DNA fragments were visualized under ultraviolet (UV) light, and the size of the products was confirmed using a molecular ladder under the following cycling conditions: Initial denaturation at 98°C for 3 min, 39 cycles of denaturation at 98°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, and a final auto extension at 72°C for 7 min. Samples were electrophoresed on a 2% agarose gel for visualization using standard markers. Specificity of the An. gambiae sensu lato primers were checked by using three samples of An. gambiae s.s. positive control. The An. gambiae s.s. DNA-positive controls were obtained from the Vector Control Division, Ministry of Health.
Sub-species determination of the An. funestus complex by PCR
Identification of An. funestus complex was performed by PCR amplification following the modified protocol described by Koekemoer et al., (2002). PCR cycling conditions were as follows: 95°C/5min x 1 cycle (95°C/30sec, 50°C/30sec, 72°C/30sec) x 30 cycles, 72°C/5min x 1 cycle, 4°C hold run samples on a 2.5% agarose EtBr gel; load 7 µl sample. Universal and An. funestus primers create fragments of 587bp An. vaneedeni, 505bp An. funestus, 411bp An. rivulorum, 252bp An. parensis and 146bp An. leesoni[16]. The An. gambiae s.s. DNA positive control was obtained from the Vector Control Division Molecular Laboratory.
Data Analysis
The indoor resting density was calculated as the ratio of the number of Anopheles mosquitoes collected in the house to the number of structures sprayed in that study site. The difference in the indoor resting density of the vectors between the two districts was tested using an independent t-test. Endophagy, exophagy, and nocturnality (the different feeding behaviors exhibited by the proportion of mosquitoes collected between 6:00 pm to 6:00 am), were calculated for each collected mosquito species in both Lira -IRS and Kole- non-IRS districts. Statistical analyses were performed using SPSS software IBM Statistics 25.0 release (IBM Corporation). The species composition between IRS and Non-IRS was investigated using a non-parametric test (Mann-Whitney). A paired Mann-Whitney test was used to determine the difference between indoor and outdoor biting patterns of malaria vectors in both IRS and non-IRS districts. The level of significance was set at 5% (P < 0.05).