The Gembe East area in the Mbita District of Nyanza Province in western Kenya was used as the study area. The area is drier in the eastern part of the district, and becomes progressively wetter towards the higher altitudes in the western parts of Gwassi Hills and Mfangano Island. In the highlands, the rainfall ranges between 800–1900 mm per annum, while the lowlands receive slightly less at 800–1200 mm each year. The rainfall pattern in the area is bimodal, with the long rainy season occurring from March through May, and the short rainy season occurring in November and December. Malaria infection rates rise steadily between September and February and peak briefly in June, following the long rains. The Mbita and Suba districts are 2 focal points identified as high vector transmission areas in Kenya, and more than 50% of the population is exposed to malaria at a rate of ≥40% Pf PR2-10 (Pf parasite rate corrected to a standard age-range of 2 to less than 10 years old). Efforts have recently been renewed in this area to increase the use of effective preventative measures such as ITNs and combined vector control approaches. The Akado Medical Centre Project Mosquito Net, the Power of Love Foundation in partnership with World Swim Against Malaria, and the Ministry of Health distributed 6000 ITNs to children under 5 years of age and to pregnant women in the Gembe area. This increased the ITN coverage rate by at least 35%, from 17% to 52%.
Adult mosquito collection and rearing of F1 progenies
Indoor adult collections took place in Nyandago, Nyaroya, Akuot, Alala, Alero, Kirindo, Ngou, and Uwi village in the Gembe East area on the shores of Lake Victoria. The house residents were informed about the study and their written consent was obtained before mosquito collection. Collections were performed during February 5–11, 2010; April 19–June 10, 2010; September 13–October 28, 2010; and January 27–February 4, 2012. Collections were done with a battery-powered aspirator (C-cell Aspirator, BioQuip Products, Rancho Dominguez, CA, USA) between 07:00–09:00 by 3 persons. After collection, gravid females were individually confined into a 20 ml glass vial containing 2 ml of water. A strip of filter paper (ca 3 × 4 cm) was placed inside each vial to collect eggs. Hatched larvae were reared with dechlorinated tap water until adult emergence. Larvae were fed a mixture of powdered animal food (CE-2; Clea Inc., Tokyo, Japan.) and dried yeast (Ebios®; Mitsubishi Tanabe Pharma, Tokyo, Japan). Grass leaves were added to rearing water containing An. funestus s.s. and An. rivulorum larvae to provide a resting place. The containers were exposed to sunlight to facilitate phytoplankton growth as a larval food resource. Unfed F1 female adults were used for insecticide susceptibility tests.
Adult mosquito collection by miniature light traps equipped with collection bottle rotators
Indoor mosquito trapping was done with 6 sets of the Center for Disease Control (CDC) miniature light trap (Model 512) equipped with a collection bottle rotator (Model 1512) (John W. Hock Co., Gainesville, FL, USA) in 3 houses in Nyaroya village and 4 houses in Nyandago village from September 16–October 5, 2010 and July 11–18, 2012. The house residents were informed about the study and their written consent was obtained before mosquito collection. The collection bottle rotator, which has 8 separate plastic collection bottles, was programmed to collect active mosquitoes at 2-hour intervals between 16:00–08:00. The traps were placed in the corner of the living room as far apart from the places where people sleep as possible. Female mosquitoes were identified and classified as unfed, blood-fed, and gravid. The abdominal contents of fed females were used for DNA extractions in order to identify the blood source.
Identification of meal sources in blood-fed mosquitoes
Mosquitoes collected in the field were individually placed in 1.5 ml plastic tubes containing silica gel desiccant and stored at −10°C until processed. Engorged abdomens were separated from the rest of the body for blood source identification.
DNA was extracted from the blood in each abdomen using the REDExtract-N-Amp™ Tissue PCR Kit (SIGMA, St. Louis, MO, USA) per the manufacturer’s instructions. Species identification of the host blood source was performed by 2 different methods; multiplex PCR as described by Kent and Norris, or direct sequencing as described by Sawabe et al..
Multiplex PCR was carried out using published cytochrome-B primers for human (Human741F, ggcttacttctcttcattctctcct) and cow (Cow121F, catcggcacaaatttagtcg) and a universal reverse primer (UNREV1025, ggttgtcctccaattcatgtta). A 20 μl cocktail consisting of 0.5 μM of each primer and previously diluted (300 times) 1 μl of the DNA template was placed in a 0.2 mL cell containing lyophilized AccuPower™ PCR Premix (BIONEER, Daejeon, Korea). The PCR was performed under the following conditions: a hot start at 95°C for 5 min; 35 cycles of template denaturing at 95°C for 1 min, primer annealing at 54°C for 1 min, and amplicon extension at 72°C for 1 min; and a final extension at 72°C for 7 min. Human and cow blood was detected by agarose gel electrophoresis (2% TAE) as 334 bp and 561 bp bands, respectively.
PCR amplification to generate amplicons for sequencing was done using published primers VerU-1 (aagacgagaagacccyatgga) and VerU-2 (cctgatccaacatmgaggtcgta) designed to detect the cytochrome-B gene of vertebrates; and Mammalian-1 (tgayatgaaaaaycatcg) and Mammalian-2 (tgtagttrtcwgggtckccta) cytochrome-B designed to detect the cytochrome-B gene of mammals. The PCR mixture contained 4 μl of REDExtract-N-Amp™ ReadyMix cytochrome-B (SIGMA, St. Louis, MO, USA), 0.5 μM of each primer, and 1 μl of the DNA template in a total volume of 10 μl. PCR was conducted under the following conditions: a hot start of 94°C for 2 min; 35 cycles of template denaturing at 94°C for 30 sec, primer annealing at 55°C for 30 sec, and amplicon extension at 72°C for 1 min; and a final extension at 72°C for 4 min. Direct sequencing was performed with a 3730 DNA Analyzer (Applied Biosystems, Carlsbad, CA, USA). The results were analyzed by MEGA 4.0 public domain software (http://www.megasoftware.net/). Sequences were used to perform basic local alignment searches against Genbank (National Center for Biotechnology Information websitehttp://ww.ncbi.nlm.nih.gov/BLAST/) to determine the identities of the host species.
Detection of P. falciparum in mosquitoes
Evidence of the presence of P. falciparum was first determined by enzyme-linked immunosorbent assay (ELISA). The head and thorax of each mosquito was homogenized in phosphate buffered saline (pH 7.4) and tested for the circumsporozoite antigen using a monoclonal antibody ELISA. ELISA positive samples were confirmed by a Plasmodium specific PCR. DNA extraction was performed on the QIAcube (kit AIAamp DNA Micro Kit 56304, Qiagen, Tkyo, Japan) following the manufacturer’s instructions and the methods by Durnez et al.. A 20 μl aliquot of the ELISA-lysate was added to 80 μl of ATL-buffer and extracted in the QIAcube machine, which eluted the DNA in 50 μl AE-buffer. The pestles used in the extraction had been sterilized in HCl solution to prevent contamination. The nested PCR was carried out according to the methods by Snounou et al.. The first amplification was done using published primers rPLU-5 (cctgttgttgccttaaacttc) and rPLU-6 (ttaaaattgttgcagttaaaacg). The PCR mixture contained 0.4 μl of Mighty Amp DNA Polymerase (Takara Bio, Inc., Tokyo, Japan), 0.6 μM of each primer, and 1 μl of the DNA template in a total volume of 20 μl. PCR was conducted under the following conditions: a hot start of 98°C for 2 min; 35 cycles of template denaturing at 98°C for 10 sec, primer annealing at 58°C for 15 sec, and amplicon extension at 68°C for 1 min; and a final annealing at 57°C for 2 min and extension at 72°C for 4 min. The 2nd amplification was done using Plasmodium falciparum specific primers rVIV1 (cgcttctagcttaatccacataactgatac) and rVIV2 (acttccaagccgaagcaaagaaagtcctta). The PCR mixture contained 0.2 μl of Mighty Amp DNA Polymerase (Takara Bio, Inc., Tokyo, Japan), 0.3 μM of each primer, and 1 μl of the DNA template in a total volume of 10 μl. PCR was conducted under the following conditions: a hot start of 98°C for 2 min; 35 cycles of template denaturing at 98°C for 10 sec, primer annealing at 58°C for 15 sec, and amplicon extension at 68°C for 30 sec; and a final annealing at 57°C for 2 min and extension at 72°C for 4 min.
Insecticide susceptibility tests using World Health Organization test tubes for F1 progenies
Adult susceptibility tests to insecticides was done using World Health Organization (WHO) test tube kits for F1 progenies and performed according to WHO instructions (WHO/CDS/CPC/MAL/98.12). Papers impregnated with either 0.75% permethrin, 0.05% deltamethrin, 4% DDT, 1% fenitrothion, or 0.1% propoxur were used for the tests. F1 larvae from the separate egg batches oviposited by field collected females were pooled in one batch to get adult females. Ten F1 1–3-day-old female mosquitoes were released into WHO test tubes for exposure to insecticide-impregnated paper for one hour, and the time to knockdown was recorded. One hr exposure to fenitrothion was performed in our bioassay, although 2 hr is stipulated in WHO instructions. Insects were then transferred to a clean tube, fed via cotton soaked with a 5% glucose solution, and mortality was recorded after one day. KT50 (time required for 50% knockdown) was obtained and average mortality was calculated. Two to 4 replications were performed for each insecticide. Control test was performed in each replication.
Mosquito adults were examined microscopically to distinguish An. funestus s.l. from other anophelines based on identification keys developed by Gillies and Coetzee. Individual species within An. funestus s.l. were identified using the multiplex polymerase chain reaction (PCR) method described by Koekemoer et al..
KT50 was calculated by Bliss' probit method. Mosquito densities were calculated as the mean number of female mosquitoes collected per collection time (2 hours) per house. The data for each replication were excluded from analysis when the total number of mosquitoes collected during a night was less than 5.