Study sites
Field studies were carried out in Kilifi County at the Kenyan coast and Busia County in Western Kenya (Figure 1). An outbreak of dengue was reported in Malindi, Kenya in 1982 [14] and previous seroprevalence studies have shown that dengue infection was prevalent in Malindi area of Kilifi, with chikungunya infection occurring in Busia County [15].
Kilifi County experiences a bimodal kind of rainfall- the long monsoon rains (April-July) and the short rains (October-December) that averages annual rainfall of 950 mm. The temperatures range from a minimum of 21°C and a maximum of 32°C. Busia County on the other hand has an average annual rainfall of 1500 mm. The rainfall pattern is also bimodal; long rains (March-June) and short rains (October-December). Temperatures range from minimum of 14°C and maximum of 30°C.
In Busia County, traps were set up in villages in the rural area namely Obekai (0 30.875 N, 34 12.293 E), Kamosin (0 31.530 N, 34 13.125 E) and Kalwa (0 30.190 N, 34 14.020E). These locations occur at approximately 1189 m above sea level (asl). The main vegetation in these areas consists of large, tall eucalyptus trees that form thick canopies. The local inhabitants are mainly small-scale farmers growing maize, millet and cassava as food crops while a few grow sugarcane and coffee as cash crops. They also keep a few animals mainly cattle, sheep, goats, pigs, chicken and guinea fowls.
In Kilifi county, traps were set up at three sites located in the urban area namely Kenya Medical Research Institute (KEMRI) campus, Kilifi hospital (3 37.800 S, 39 51.483 E) and Mnarani estate (3 38.368 S, 39 50.824 E). These locations occur at approximately 30.5 m asl. The inhabitants in the urban area mainly engage in small businesses or work in offices. They also grow maize, cassava and sweet potatoes and keep a few animals mainly goats.
The traps were set up during the wet seasons at both sites. In Busia, traps were set up in November 2013 and in Kilifi in December 2013 and April 2014.
Odor collection from trunk and feet of volunteers
Four adult volunteers, 2 males and 2 females, between the ages of 25 and 40 years were identified and enrolled to participate in the study after obtaining informed consent. The two males had participated in our previous study and one of the males was more attractive than the other [12]. The volunteers were each requested to put on clean new cotton T shirts and clean new cotton socks (Lux Industries Ltd 39 K.K Tagarest, Kolkata-700-007) issued to them by the researchers for 18 hrs. The worn socks and T shirts from the volunteers were individually wrapped in at least 4 layers of aluminium foil and stored in cool boxes (10°C) for immediate transportation to the laboratory for odor trapping using the volatile entrainment system as described below.
Headspace trapping of odors trapped in worn socks and T shirts
The socks and T shirts obtained from the volunteers were held in tightly sealed volatile collection jars (ARS, Gainesville, FL, USA) and odors collected on Super Q adsorbent (30 mg, Alltech, Nicholasville, KY) traps for 24 hr. The Super Q filters were eluted with 150 μl dichloromethane Sigma-Aldrich Corporation (3050 Spruce Street, St. Louis, Missouri 63103 USA) and stored at −80°C until use.
Mosquitoes
Mosquitoes used in this study were obtained from two different populations; (i) An inbred generation reared at the International Centre of Insect Physiology and Ecology (icipe), Duduville campus, Nairobi, established in 2001 from blood-fed and gravid Ae. aegypti caught at Rabai, Kilifi County, and (ii) A first filial (F1) generation of Ae. aegypti established from eggs collected from Rabai, Kilifi in 2013 and reared in a separate insectary at icipe’s Duduville campus. In both cases, Ae. aegypti were reared at a mean temperature and relative humidity of day, 28°C, 70% RH and night, 26°C, 80% RH; and a reversed circadian rhythm of light (15:01–3:00) and darkness (3:01–15:00). The newly emerged adult females were maintained on glucose (6% solution ad libitum) (Sigma®) continuously available on filter paper and no blood meal. On the experimental days the mosquitoes were deprived of glucose for 6 hrs before the experiments.
Gas chromatography/electroantennographic detection (GC/EAD)
Volatiles collected from the feet and trunk of volunteers were analyzed by coupled GC/EAD analysis using a Hewlett-Packard (HP) 5890 Series II gas chromatograph equipped with an HP-5 column (30 m × 0.25 mm ID × 0.25 μm film thickness, Agilent, Palo Alto, California, USA). Nitrogen was used as the carrier gas at 1.2 ml/min. Volatiles were analyzed in the splitless mode at an injector temperature of 280°C venting at 0.8 min. The oven temperature was held at 35°C for 5 min, then programmed at 10°C/min to 280°C and maintained at this temperature for 10 min. The column effluent was split 1:1 after addition of make-up nitrogen gas for simultaneous detection by flame ionization detector (FID) and EAD. For EAD detection, silver-coated wires in drawn-out glass capillaries (1.5 mm I.D.) filled with Ringer saline solution [16] served as reference and recording electrodes.
Antennal preparations were made by decapitating 4–7 days old females of Ae. aegypti at the base of the head and slicing off the tip of the last antennal segment with a scalpel under a dissecting microscope. The antenna was then mounted on to the micromanipulator such that the base of the head was connected to the reference electrode, and the cut tip of the antenna was connected to the recording electrode. The analog signal was detected through a probe (INR-II, Syntech, Hilversum, the Netherlands), captured and processed with a data acquisition controller (IDAC-2, Syntech, the Netherlands), and later analyzed with soft- ware (EAG 2000, Syntech) on a personal computer. An aliquot (5 μl) of the Super Q-trapped volatile extract from each volunteer’s feet and trunk was analyzed using fresh female antennae in at least three replicate runs.
Coupled gas chromatography/mass spectrometry (GC/MS)
GC/MS analysis of volatiles was carried out on an Agilent system (Agilent Technologies, Inc., Santa Clara, CA, USA) consisting of a 7890A gas chromatograph, a 5975C Mass spectrometer with a triple Axis detector and an Agilent ChemStation data system. The GC column was an HP-5 MS fused silica capillary (30 m × 0.25 mm × 0.25 μm film thickness) (J&W, Folsom, CA, USA). The carrier gas was helium with a column head pressure of 8.827 psi and flow rate of 1.2 mL/min. Inlet temperature was 270°C and MSD detector temperature was 280°C. The oven temperature was held at 35°C for 5 min and then increased at 10°C/ min to a final temperature of 280°C, which was held for 10.5 min. The identity of each component in the extracts of the volatiles was determined by comparison with references from mass spectral libraries (NIST05, Agilent Technologies [NIST05, Agilent Technologies NIST database, G1033A, revision D.05.01, ChemStation data system (G1701EA, version E.02.00). An aliquot (1 μl) of the volatile extract from each volunteers’ feet or trunk and of synthetic authentic compounds was injected into the GC-MS for analysis.
GC/EAD-active components were identified both by comparing their mass spectral data with those recorded in the Mass Spectral Library NIST 2005 and by co-injection with authentic standards.
Chemicals
Hexanal, heptanal, hexanoic acid, octanal, nonanal, decanal and undecanal were obtained from (Sigma-Aldrich Chemie (GmbH, Germany) while propionic acid, 3-methylbutyric acid, and 6,10-dimethyl-5,9-undecadien-2-one (geranyl acetone) were sourced from Sigma-Aldrich Corporation (3050 Spruce Street, St. Louis, Missouri 63103 USA). Purities of the compounds ranged between 95% and 99%. The BG lure used in this study was purchased from Biogent, with an expiry date of December 2015. It mainly contains lactic acid, hexanoic acid and ammonia [13].
Field testing of EAG-active compounds
Experiment 1
Study design
For field testing in both Kilifi and Busia, mosquitoes were collected using six BG sentinel traps baited with carbon dioxide plus (i) Blend 1; 3-methylbutyric acid and propionic acid each at 0.05 mg/μl at a ratio of 1:1 (ii) Blend 2; nonanal and octanal each at 0.05 mg//μl at a ratio of 1:1(iii) Blend 3; nonanal, octanal, 3-methylbutyric acid and propionic acid each at 0.05 mg/μl dispensed separately at a ratio of 1:1:1:1 (iv) BG-lure (v) worn socks and (vi) worn T shirts.
Traps baited with human odors
Odors were obtained from the feet and trunk of a male volunteer aged 32 years old in Busia and a male volunteer aged 30 years old in Kilifi. Both of them had donated odors for the GC/EAD tests. The volunteers were requested to put on new, clean, 100% cotton socks and T shirts (Lux Industries Ltd 39 K.K Tagarest, Kolkata-700-007) to trap odors from their feet and trunk for 18 hrs daily for a period of 12 days. New socks and T shirts were provided daily. The volunteers were also provided with odorless soap to bathe with daily and requested to avoid the use of deodorants and perfumes. The socks and T shirts once removed by the volunteers were wrapped in at least 4 layers of aluminium foil and stored in cool boxes at 10°C and transferred into the laboratory and then into −80°C freezer until use. The worn socks and T shirts were used daily to bait BG sentinel traps by hanging them on the rails of the BG sentinel trap inner structure as described in Owino et al. [12].
Traps baited with synthetic chemicals
Preliminary trials to determine the possible range of attractive doses of, nonanal, octanal, propionic acid, 3-methylbutyric acid and hexanoic acid, were conducted in the field at icipe’s Nairobi campus. These chemicals were identified as the consistent EAD-active components that were most commonly shared amongst the different volunteers. Concentrations of individual compounds, including 0.005, 0.01, 0.02 and 0.05 mg/μl were evaluated in three replicate trials. Trap captures showed that the optimal attractive dose of nonanal and octanal to Ae. aegypti was 0.05 mg/μl while hexanoic acid, 3-methylbutyric acid and propionic acid were effective between 0.01 and 0.05 mg/μl/ [data not shown]. Hexanal and decanal did not show strong attraction to Ae. aegypti at the tested concentrations.
To obtain stock concentrations, 100 mg of each EAD-active compound was diluted in 1 ml of hexane. Ten milligrams (10% of the concentration of individual component) of the antioxidant, 2, 6-di-tert-butyl-4-methylphenol (butylated hydroxytoluene, BHT, Aldrich) was then added to the aldehyde stocks to prevent oxidization to their respective corresponding fatty acids. To bait the traps, 50 μl of each compound was transferred from the stock and diluted in hexane to make 100 μl. The solution was adsorbed on cotton wicks measuring 5 mm × 30 mm wrapped in a nylon stocking material measuring 12 mm × 30 mm. The cotton wicks and BG-lure were then inserted into the odor pockets of the BG sentinel traps. Each compound was dispensed from its own cotton wick.
Mosquito sampling
At each of the study sites, Kilifi and Busia, six different locations were randomly chosen around homesteads. Traps were set up at approximately 100 m away from the nearby house (occupied or unoccupied). The six BG sentinel traps baited as described above were randomly set up at each of the six locations with a distance of at least 100 m between traps. The traps were hung at 0.2 m above the ground and attached to each was a Bioquip igloo that dispensed carbon dioxide in the form of dry ice [12]. To offset any positional bias, traps were rotated every experimental day. The traps were set up at 9.00 am and left to run until 5.00 pm. Trapped mosquitoes were collected and transported to the laboratory where they were freeze-killed and identified under a dissecting microscope to species level using morphological keys [17-19].
Experiment 2
Study design
Comparison of the efficacies of various EAD-active carboxylic acids in attracting Ae. aegypti at the two field sites, Kilifi and Busia (Experiment 1), showed that only hexanoic acid strongly attracted this mosquito species. Previously, we had detected it in the odors released from the BG lure (12). It was therefore selected for further evaluation in Kilifi which compared to Busia had a higher density of Ae. aegypti (see Results section). This experiment was carried out in five locations in Kilifi. Mosquitoes were collected in the field using five BG sentinel traps baited with carbon dioxide plus either (i) hexanoic acid at 0.05 mg/μl (ii) Blend 2; octanal and nonanal each at 0.05 mg/μl at a ratio of 1:1 (iii) Blend 4; hexanoic acid, nonanal and octanal each at 0.05 mg/μl at a ratio of 1:1:1 (iv) BG-Lure (v) carbon dioxide only. The compounds were dispensed from rubber septa which were inserted into the odor pockets of the BG sentinel traps instead of the cotton wicks wrapped in Nylon materials like in experiment 1. In traps baited with more than one compound, each compound was prepared individually as already described and dispensed separately from rubber septa. The average release rate of the hexanoic acid was 0.7 mg/hr over the 7 hr trapping period [Additional file 1] while the average release rate of hexanoic acid from the BG lure was calculated as 1.9 mg/hr over the same period. [Additional file 2]. The release rates were calculated based on GC/MS peak area comparison with those of authentic standards.
Mosquito sampling
The five BG sentinel traps were randomly set up at each of the five locations just as described in experiment 1 above after which captured mosquitoes were freeze- killed and identified to species using appropriate keys [17-20].
Data analysis
The daily mosquito counts in the different traps were subjected to negative binomial regression following the generalized linear models (GLM) procedures in R 3.1.0 [21]. The trap baited with the BG commercial lure was used as the control and the reference category in both field experiments 1 and 2. The incidence rate ratios (IRR), a likelihood measure that mosquito species chose other treatments instead of the reference category, and corresponding P-values were estimated. The Pearson’s chi-square test was applied to evaluate differences between proportions of fed and gravid mosquitoes per treatment trap against the reference category. The tests were performed at 5% significance level.
Ethics statement
The study was approved by the national ethics review committee based at the Kenya Medical Research Institute (KEMRI) and informed consent was obtained from each of the participants. Different sampling locations were randomly chosen around homesteads after obtaining oral consents from the heads of the homes.