Study sites, sample collection and preparation
Adult female mosquitoes used in this study had previously been collected from three areas: Kerio Valley (Baringo county), Rabai (Kilifi county) and Nguruman (Kajiado county) (Fig. 1), as part of vector-borne disease surveillance project and stored at – 80 °C at the International Centre of Insect Physiology and Ecology (icipe). The mosquitoes were surveyed between August 2019 and May 2020. Nguruman is an agropastoral area located in Kajiado county at the southern end of the Kenyan Rift Valley bordering Tanzania. The area has a semi-arid climate characterized by erratic rains, extreme temperatures, and cyclic and prolonged droughts30. The vegetation is dominated by bushland, grassland and open woodlands along seasonal river valleys. Specific indicator data for malaria is not available for Nguruman except for estimates pertaining to the larger Kajiado county which as of 2019 indicates a malaria incidence rate of 5 per 1000 population31. Collections in Kerio Valley (Baringo county within the Rift Valley) were conducted in Kapluk and Barwesa, both agro-pastoral areas with arid and semi-arid ecology. Malaria is a major vector-borne disease in the areas with report of perennially occurrence in neighboring riverine areas32. Rabai is one of the seven administrative sub-counties of Kilifi county in the coastal region of Kenya where malaria is endemic. The main economic activities in the area include subsistence agriculture, casual labor, crafts and petty trading. The weather patterns at the sites during the sampling period were as follows: Kerio Valley (mean daily temperature: 21.2 °C, mean daily rainfall: 4.1 mm, mean relative humidity: 73.4%); Rabai (mean daily temperature: 26.4 °C, mean daily rainfall: 2.1 mm; mean relative humidity: 78.1%) and Nguruman (mean daily temperature: 22.5 °C, mean daily rainfall: 0.9 mm, mean relative humidity: 61.2%).
Mosquito survey and processing
Host seeking mosquitoes were trapped using CDC light traps baited with dry ice (carbon dioxide) attractive to several mosquitoes. Traps were set outdoors about 10–15 m away from randomly selected homesteads from 18:00 h to 06:00 h. After collection, the mosquitoes were anesthetized with trimethylamine and temporarily stored in liquid nitrogen before transportation to the Emerging Infectious Disease (EID) laboratory at icipe and later stored at − 80 °C. Anopheline mosquitoes were morphologically identified to species level using published taxonomic keys15,33.
DNA extraction and Anopheles species discrimination
DNA was extracted from the head/thorax of individual mosquitoes using ISOLATE II Genomic DNA Extraction kit (Bioline, UK) following the manufacturer’s instructions and used for species discrimination and screening for P. falciparum infection and Gste2 mutations (described below).
Cryptic sibling species of the Anopheles funestus and Anopheles gambiae complexes were identified using conventional PCR34,35 and/or sequencing. PCR for An. funestus complex in a 15 µl reaction volume comprised 0.5 µM of each primer targeting: Anopheles funestus s.s, Anopheles vaneedeni, Anopheles rivulorum, Anopheles parensis, Anopheles leesoni, Anopheles longipalpis A and Anopheles longipalpis C, 3 µl of 5X HOT FIREPol Blend Master Mix Ready to Load (Solis BioDyne, Estonia) and 2 µl of DNA template. The cycling conditions were initial denaturation at 95 °C for 15 min, and then 30 cycles of denaturation at 95 °C for 30 s, annealing at 46 °C for 30 s and extension at 72 °C for 40 s and final extension at 72 °C for 10 min. Size fragments of each species were scored after separation in 1.5% agarose gel electrophoresis stained with ethidium bromide against a 1 Kb DNA ladder (HyperLadder, Bioline, London, UK).
For An. gambiae s.l., PCR in a 10 µl volume consisted of 2 µl of 5X Evagreen HRM Master Mix (Solis BioDyne, Estonia), 1 µl of DNA template and 10 µM concentration of each primer targeting An. gambiae s.s and An. arabiensis. The thermal cycling conditions included initial denaturation for 15 min at 95 °C followed by 40 cycles of denaturation at 95 °C for 20 s, annealing at 61 °C for 15 s and extension at 72 °C for 20 s followed by final extension at 72 °C for 7 min.
A subset of An. funestus s.l. samples that failed to amplify using the established protocol, was further amplified and sequenced targeting the internal transcribed spacer 2 (ITS2) region of the ribosomal DNA (rDNA)36. This target has shown utility in discriminating closely related mosquito species including anophelines12 and sequences from diverse species for this marker are well represented in reference databases (e.g. GenBank). PCR volumes for rDNA ITS2 were 15 µl containing 0.5 µM of the forward and reverse primers, 3 µl of 5X HOT FIREPol Blend Master Mix Ready to Load (Solis BioDyne, Estonia) and 2 µl of DNA template. The cycling conditions were initial denaturation at 95 °C for 15 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s and extension at 72 °C for 45 s and final extension at 72 °C for 7 min. ExoSAP IT rapid cleanup kit (Affymetrix Inc., Santa Clara, CA, USA) was used to clean the PCR product as per the manufacturer’s guideline, and then outsourced for bidirectional Sanger sequencing to Macrogen, South Korea.
Detection of malaria parasites
Plasmodium falciparum sporozoites in individual mosquitoes (head/thorax) were detected by analyzing high resolution melting (HRM) profiles generated from real time PCR products of non-coding mitochondrial sequence (ncMS)37. A P. falciparum DNA from National Institute for Biological Standards and Control (NIBSC; London, UK) was used as a reference positive control. PCR was carried out in a 10 µl volume consisting of 2 µl of 5X Evagreen HRM Master Mix (Solis BioDyne, Estonia), 1 µl of DNA template and 10 µM of each primer. PCR cycling conditions were initial denaturation for 15 min at 95 °C followed by 40 cycles of denaturation at 95 °C for 20 s, annealing at 61 °C for 15 s and extension at 72 °C for 20 s followed by final extension at 72 °C for 7 min. A fraction of RT-PCR-HRM positive samples were further analyzed using conventional PCR in a 10 µl volume consisting of 2 µl of 5X HOT FIREPol Blend Master Mix Ready to Load (Solis BioDyne, Estonia), 1 µl of DNA template and 10 µM of each primer. The cycling conditions comprised initial denaturation for 15 min at 95 °C followed by 40 cycles of denaturation at 95 °C for 20 s, annealing at 61 °C for 15 s and extension at 72 °C for 20 s followed by final extension at 72 °C for 7 min. PCR product of samples positive by RT-PCR were purified using ExoSAP- IT (USB Corporation, Cleveland, OH, USA) and outsourced for sequencing to Macrogen, South Korea. All sporozoite-positive mosquitoes were molecularly identified to species by PCR of the ITS2 region as described above.
Genotyping for L119F-GSTe2 mutation and sequencing
Two outer and two inner primers in a PCR assay were used to genotype the L119F-GSTe2 mutations that confer resistance of An. funestus mosquitoes to pyrethroids/DDT19 as described previously28. Thus, only An. funestus s.l. was screened using this assay. Briefly, PCR in a 15 µl reaction volume consisted of 10 µM of each primer, 3 µl of 5X HOT FIREPol Blend Master Mix Ready to Load (Solis BioDyne, Estonia), and 2 µl of DNA template. The cycling conditions were initial denaturation at 95 °C for 15 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 59 °C for 30 s and extension at 72 °C for 40 s and final extension at 72 °C for 7 min. Amplicons were resolved in a 1.5% agarose gel stained with ethidium bromide (Sigma-Aldrich, GmbH, Germany) against a 1 Kb DNA ladder (HyperLadder, Bioline, London, UK). The amplicons were scored as either homozygous susceptible (SS) at 312 bp, homozygous resistant (RR) at 523 bp or heterozygous (RS) when both bands were visualized.
Representative GSTe2 allele positive samples were sequenced for the GSTe2 gene using the Gste2F and Gste2R primers as described previously38. PCR comprised a reaction volume of 15 µl in MyTaq DNA Polymerase Kit (Bioline, London, UK) containing 10 µM of each primer, 5X My Taq reaction buffer, 2 µl of My taq DNA polymerase and 1 µl of DNA template. PCR conditions were: initial denaturation of 5 min at 95 °C, followed by 30 cycles of 94 °C for 30 s, 58 °C for 30 s and 72 °C for 1 min, with a final extension at 72 °C for 10 min. Cleaning and sequencing of amplicons were performed as described above.
Sequence and polymorphism analysis
Sequences (mosquito, P. falciparum, GSTe2) were viewed and cleaned in Geneious Prime39 and queried in GenBank using Basic Local Alignment Search Tool (BLastn). Parasite sequences were assigned as P. falciparum after > 98% percentage identity. MAFFT in Geneious Prime39 was used to perform multiple sequence alignments with default parameters. Maximum likelihood (ML) trees were inferred for mosquito ITS2 sequences using the best fit model of sequence evolution with nodal support for different groupings evaluated through 1000 bootstrap replications. GSTe2 gene polymorphism analysis was performed in Geneious Prime and ML tree reconstructed from MAFFT alignment using PhyML v. 2.2.4. Haplotype distribution network was constructed using Templeton-Crandall Sing (TCS) program v. 1.2140.
Statistical analysis
Relative abundance was used to estimate the outdoor composition of the anopheline mosquitoes. Daily counts of female mosquito/trap/night for An. funestus s.l. and An. gambiae s.l. were compared for each area using generalized linear models (GLM) with negative binomial error structure based on best-fit model residuals. The mean catches/trap/night was computed for each of the species complexes. The P. falciparum sporozoite infection rates (Pfsp) were expressed as the number of positive specimens of the total number of specimens examined. The distribution of L119F-GSTe2 mutations was assessed by determining allelic frequencies in different species. Infection status among the resistant mosquitoes was compared using the Fisher’s Exact Test. Data were analyzed using R v 4.1.0 software at 95% confidence limit.
Ethical considerations
Ethical review and approval of the study was granted by the Scientific and Ethical Review Unit (SERU) of the Kenya Medical Research Institute (KEMRI) (Protocol No. SSC 2787). Prior to data collection, the purpose of the study, procedures and associated benefits/risks were provided to the local leadership at county and community levels. Additionally, informed verbal consent to trap mosquitoes around homesteads was obtained from household heads.
Source: Ecology - nature.com
