Sugar-fermenting yeast as an organic source of carbon dioxide to attract the malaria mosquito Anopheles gambiae

Female mosquitoes used for the laboratory experiments were collected from a culture of Anopheles gambiae s.s. (hereafter referred to as An. gambiae) (Suakoko strain) kept at Wageningen University, The Netherlands. The culture has been reared by blood-feeding on human arms since 1988. Larvae were kept in tap water and fed on Tetramin^® baby fish food. Pupae were collected daily and transferred to 30 cm cubic gauze cages for emergence. Adult mosquitoes were kept at 27°C, 80% RH and a photo:scotophase of 12:12 h, respectively. A 6% glucose solution was provided ad libitum on filter paper.

The semi-field experiments were conducted using the Mbita strain of An. gambiae. The mosquitoes have been reared under ambient climatic conditions at insectaries belonging to the Thomas Odhiambo campus of the International Centre of Insect Physiology and Ecology (ICIPE) located at Mbita Point, western Kenya, since 2001. Adult insects were kept in 30 cm cubic gauze cages and provided with a 6% glucose solution ad libitum. Blood feeding took place on human arms. Larvae were kept in filtered water from Lake Victoria and fed on Tetramin^® baby fish food. Upon pupation, insects were transferred to adult cages for emergence.

Mosquito Magnet-X counter flow geometry traps (MM-X; American Biophysics Corp., USA, [21], see also [22, 23]), were suspended from metal or wooden stands, with the odour outlet 15 cm above ground level [12, 13]. The bullet-shaped cartridges within the lower end of the odour outlet tube of the traps were removed. The electric ventilators in the MM-X traps operated on 12 V batteries. During the experiments performed in the MalariaSphere [24] also CDC miniature light traps (Model 512; John W. Hock Company, USA, [25]) were used. These traps were run on 6 V batteries (Gaston Battery Industrial Ltd, China). After removing the caught mosquitoes, each trap was cleaned with 10% ethanol.

Yeast-produced carbon dioxide was produced by mixing dry yeast (Dr. Oetker, The Netherlands, used in the laboratory experiments carried out in Wageningen or Angel Yeast Co. Ltd., China, used in the semi-field and field experiments in Kenya), sugar (Van Gilse Kristalsuiker, Suiker Unie, The Netherlands, in the laboratory experiments or Sony Sugar, South Nyanza sugar Co. Ltd., Kenya, in the (semi-)field experiments) and tap water [16] in two plastic bottles of 1.5 L or 5 L, connected with each other by silicon tubing, or one plastic container of 25 L. Mixing took place 1-1½ h before mosquitoes were released, at ambient temperature, until the dry yeast was dissolved. No additional stirring or mixing took place during the experiments. A 0.5 L respectively 1 L bottle was put in between the 1.5 L respectively 5 L bottles with the mixtures and the MM-X trap to prevent foam produced by the mixtures entering the trap (Figure 1A-C). Holes were drilled into the original screw caps of the bottles and into the side of the small bottles; silicon tubing (Ø 7 mm; Rubber B.V., The Netherlands) fitted through these holes to connect the bottles. The smaller bottle was connected to the MM-X trap using the original MM-X tubing (micron filter and orifice removed) and the Luer connection at the underside of the trap's top lid. The connections were sealed by Teflon tape and held under water to check for leakage. Several combinations of bottle size and amount of yeast, sugar and water were used. The carbon dioxide output was estimated by measuring the volume of water displaced from a submerged measuring cylinder (Table 1). For this purpose, the tubing that was attached to the MM-X traps during the mosquito trapping experiments was now led into a measuring cylinder which was held in a bucket of water (Figure 1D).

Industrial carbon dioxide (≥ 99.9%) was released from pressurized gas cylinders (Linde Gas Benelux B.V., The Netherlands in laboratory experiments or Carbacid Investments Ltd., Kenya, in (semi-)field experiments) and supplied to the MM-X traps through silicon tubing (Ø 7 mm; Rubber B.V., The Netherlands). The Luer connection at the underside of the trap's top lid was used to release the gas directly into the odour outlet tube of the trap. A flow meter (Sho-Rate model GT1350 or GT1355, used in laboratory and semi-field experiments; Brooks Instruments, The Netherlands) or an orifice (American Biophysics Corp., USA; used in field experiments) regulated the flow rate of CO₂. During the laboratory experiments, CO₂ was led through a 0.5 L bottle before it was released into a MM-X trap. This bottle was filled for 50% with a 10% sugar solution.

Human foot odour was released from nylon socks (40 Den, 100% polyamide, HEMA, The Netherlands) worn by WHS (laboratory experiments) or KJvR (semi-field and field experiments) for 12 h prior to the experiments [6, 12, 13, 26‐29]. A clean nylon sock served as a control. Socks were placed along the odour outlet tube of the MM-X trap without blocking the airflow and held in position by odourless tape (3M™ Double Coated Tape 400; used in laboratory experiments) or by a small metal wire (in (semi-)field experiments).

Two MM-X traps were placed in a textile screen cage (330 × 250 × 233 cm; Howitec Netting BV, The Netherlands, [30]) at approximately 2.5 m distance from each other inside a climate-controlled room (22.2 ± 1.6°C and 52.6 ± 7.8% RH). The CO₂ cylinder and the yeast-produced CO₂ bottles were positioned within the sluice of the cage. Either two 1.5 L bottles or one 25 L container contained the yeast-sugar solution. In each 1.5 L bottle, 7 g of dry yeast and 100 g of refined household sugar were dissolved in 1 L of tap water. In the 25 L container, a mixture of 70 g of dry yeast, 1 kg of sugar and 10 L of tap water was prepared. In contrast to the 1.5 L bottles, which were used during a single experiment only, the 25 L container was used during two consecutive days without adding additional yeast, sugar or water. During the time it was not used, the container was closed and stored at room temperature. Worn socks were used to test the effect of human emanations on the attractiveness of yeast-produced CO₂. The flow rate of industrial CO₂ was set at 15 ml/min, a flow rate within the range (up to 20 ml/min) that was previously measured to be produced by two 1.5 L bottles each containing a mixture of 7 g of dry yeast, 100 g sugar and 1 L of tap water.

Experiments were conducted in the last 4 h of the dark phase when An. gambiae is normally searching for a blood host [31‐33]. For each replicate, 50 mosquitoes were released from the centre of the screen cage and left in it for 4 h. After this period, the release cage and the traps were closed, the mosquitoes killed by freezing, and counted. The dual-choice experiments conducted are listed in Table 3. Treatments were alternated between the two positions to rule out any positional effect. In addition, experiments with two unbaited MM-X traps were conducted to test for positional effects. Each dual-choice experiment was replicated 6-8 times. Surgical gloves were worn by the operator to avoid contamination of equipment with human volatiles.

The field experiments were conducted in Lwanda, a rural village at an altitude of 1169 m above sea level in the basin region of Lake Victoria, Nyanza Province, western Kenya. The area has a main rainy season from March to May and a short rainy season from October to December. Experiments were conducted at the end of the short rainy season, in December 2008. Lwanda has a variety of mosquito breeding habitats [37, 38].

Based on several criteria (household, location of cooking site, roof construction, vegetation around the house and all houses at walking distance from each other) four approximately similar houses in Lwanda were selected. The occupants of the houses were sleeping under bed nets. Each house was provided with a MM-X trap, a car battery and a CO₂ cylinder. The MM-X traps were hung outdoors, 15 cm above ground level, under the overhanging thatched roof, at the window side of the house [13]. Vaseline petroleum jelly was used around the tubing, suspension cable and electrical cables to prevent ants from reaching the mosquitoes caught in the MM-X trap.

Two series of each eight nights (i.e. two blocks of a 4 × 4 Latin square) were run. In the first series the following four treatments were tested: unbaited, industrial CO₂ at a flow rate of 250 ml/min (the amount of CO₂ released by a human, [2]), and yeast-produced CO₂ at two different flow rates, using either a mixture of 17.5 g of dry yeast (Angel), 250 g sugar (Sony) and 2½ L of tap water or 35 g of dry yeast (Angel), 500 g sugar (Sony) and 2½ L of tap water in each 5 L bottle. In the second series the effect of the combination of CO₂ and human emanations on the trap catches was examined by testing industrial CO₂ at a flow rate of 250 ml/min with or without the addition of a worn sock, and yeast-generated CO₂ produced by 35 g of dry yeast (Angel), 250 g sugar (Sony) and 2½ L of tap water in each 5 L bottle with or without the addition of a worn sock. Each experiment ran from 8:30 pm until 6:30 am, after which the mosquitoes in the traps were killed by placing the traps in a freezer and counted. Surgical gloves were worn to avoid contamination of equipment with human volatiles.

The mosquitoes caught in each trap during one night were morphologically identified and counted. Culicines were identified to genus, anophelines to species. Female An. gambiae sensu lato mosquitoes were placed in a 2 ml Eppendorf tube with dry silica gel and a piece of cotton wool. These mosquitoes were transported to the Laboratory of Entomology of Wageningen University for species identification. The Bender buffer method [39] was used to extract DNA from a mosquito leg and part of the abdomen of each mosquito, followed by polymerase chain reaction (PCR) analysis [40].

The concentration of yeast-generated CO₂ produced by 17.5 g of dry yeast, 250 g sugar and 2½ L of tap water in each 5 L bottle and flowing from a MM-X trap was measured in the laboratory using a Xentra 4100 CO₂ analyser (Servomex, The Netherlands). The data were transferred to a PC using Das Wizard 2.0 software (Measuring Computing Corporation, USA). The analyser measured at 1 Hz and was programmed to shift to the next measuring point after 60 successive readings. The average of these 60 readings was plotted in a graph. The analyzer measured over a range of 0-1030 ppm with an accuracy of 0.1 ppm.

Three series of readings, each for a duration of 20 min, were taken at different times after mixing the yeast-sugar solution (1½, 25½ and 49½ h). For each series of readings, three measuring points were positioned at different distances from the MM-X trap (Table 2). To make a comparison with concentrations flowing from a MM-X trap baited with industrial CO₂ another three series of readings, at different distances from the MM-X trap, were made (Table 2). For this comparison the human equivalent of CO₂ percentage present in breath (5%) and the amount released (250 ml/min) were chosen [1, 2].

For each dual-choice test (laboratory and semi-field experiments) a χ²-test was used to test whether the distribution of the total number of mosquitoes caught in the treatment or control trap over all replicates differed from a 1:1 distribution. A χ²-test was also used to compare the total number of mosquitoes found inside (total number caught by the two CDC light traps + found resting inside the house) and caught outside (by the MM-X trap) the house in the MalariaSphere. Effects were considered to be significant when P < 0.05.

Of the mosquitoes caught during the field experiments, the rarely caught male mosquitoes were discarded from the data. Due to many zeros, the numbers of anopheline and Aedes females were transformed (natural logarithm of (x+1)) before subjection to a Generalized Linear Model (GLM; Genstat^® release 12.1; Normal distribution, fitted terms: night, house, treatment, and when significant the interaction between house and treatment). Mansonia, Culex and total counts were not transformed before subjection to a GLM (Poisson distribution, linked in log, dispersion estimated to account for heterogeneity, fitted terms: night, house, treatment, and when significant the interaction between house and treatment). Two-sided t-probabilities were calculated to test pairwise differences of means. Effects were considered to be significant when P < 0.05.

Experiments with two unbaited MM-X traps revealed no positional effect within the cage (P = 0.24, n = 297; Table 3). In total, 15.5% of the mosquitoes were caught by the two traps. A trap baited with a worn sock caught significantly more mosquitoes than a trap baited with a clean sock (P < 0.001, n = 277). The two traps together caught on average 41.6% of the mosquitoes that flew out of the release cage.

A trap baited with yeast-produced CO₂, produced by a mixture of 7 g of dry yeast, 100 g sugar and 1 L of tap water in each 1.5 L bottle, caught significantly more mosquitoes than an unbaited trap (P < 0.001, n = 279). During these experiments, on average 77.0% of the mosquitoes released were caught. Also when the other trap was baited with industrial CO₂ (15 ml/min) led through two 1.5 L bottles each filled with 1 L of sugar water, the trap baited with yeast-produced CO₂ (two 1.5 L bottles with each 7 g dry yeast + 100 g sugar + 1 L water) caught significantly more mosquitoes (P < 0.001, n = 298, in total 51.6% caught).

Significantly more mosquitoes were caught by traps baited with yeast-produced CO₂ combined with a worn sock than traps baited with a worn sock only. This was observed when two 1.5 L bottles each containing 7 g dry yeast+100 g sugar+1 L water were used for the production of yeast-produced CO₂ and when one 25 L container with 70 g of dry yeast, 1 kg of sugar and 10 L of tap water was used (P = 0.007, n = 278 respectively P < 0.001, n = 371). In total a mean of 55.5% and 78.8%, respectively, of the mosquitoes that had left the release cage were caught during these experiments.

In the first series of field experiments 392 and in the second series 486 female mosquitoes were caught over eight nights in traps hanging next to the four selected houses. The majority consisted of Mansonia spp. mosquitoes: 48.7% and 66.0% in series 1 and 2, respectively. Also Culex spp. females were caught in high proportions: 34.7% respectively 23.3% of the total number of female mosquitoes found in the traps. Of the anophelines (12.2% and 9.7%, respectively) 3.8% respectively 5.1% were An. gambiae s.l. females. PCR tests revealed that all (except five specimens that could not be identified) of the An. gambiae s.l. specimens were Anopheles arabiensis. The majority of the anophelines were Anopheles coustani females; only a few Anopheles funestus (1%) were found in the traps. In addition, 4.3% respectively 1.0% of the mosquitoes caught were Aedes spp.

GLM analysis showed that both in series 1 and 2 the average number of mosquitoes caught by the four traps hardly varied during the eight nights, whereas the location of the trap (i.e. house) often significantly affected the number of mosquitoes trapped during a night (Table 7). In the first series a significant effect of treatment was found for Culex and Mansiona spp., as well as for Culex spp. in the second series.

In the case of An. gambiae s.l., the effect of the different baits (treatment) on the number of mosquitoes caught depended on the location of the trap (P_interaction = 0.04 in series 1 and P_interaction = 0.03 in series 2). In the first series, traps baited with industrial CO₂ caught significantly more An. gambiae s.l. than unbaited traps (P = 0.02), but similar numbers as traps baited with yeast-produced CO₂ (P = 0.14 and 0.33, respectively; Table 7). The second series of experiments showed that, overall, adding a worn sock to either yeast-produced or industrial CO₂ significantly increased the number of mosquitoes caught (P = 0.003 and 0.002, respectively). Traps baited with yeast-produced CO₂ plus a worn sock also caught more mosquitoes than industrial CO₂ alone (P = 0.02). The majority of the An. gambiae s.l. females were trapped next to house #1 (P < 0.05).

Taking all mosquito species caught during the first series together, unbaited traps caught significantly fewer mosquitoes than odour-baited traps (P < 0.05). Traps baited with yeast-produced CO₂ at the lowest flow rate caught significantly fewer mosquitoes than traps baited with yeast-produced CO₂ at the highest flow rate and traps baited with industrial CO₂ (P = 0.009 and 0.003, respectively). Traps baited with the latter two baits caught similar numbers of mosquitoes (P = 0.74). In the second series the location of the traps determined the total numbers of mosquitoes caught (P = 0.003), independent of treatment.

The carbon dioxide concentrations measured at different distances from a MM-X trap are summarized in Figure 3. It shows clearly the distance effect on the concentration of CO₂, the further away from the MM-X trap the lower the CO₂ concentration, independent of its source (CO₂ cylinder or yeast-sugar solution 1½, 25½ or 49½ h post mixing). Concentrations measured at a distance of 200 cm or at a height of 100 cm were between 400 and 500 ppm. Measurements taken 1½ hours after mixing the yeast-sugar solution, within or close to the trap (0 and 30 cm from the trap, 5 cm above ground level) also showed CO₂ levels between 400 and 500 ppm.

Carbon dioxide concentrations produced by yeast-sugar solutions 25½ or 49½ h post mixing, measured inside or 10 cm below the trap outlet, was higher (600-850 ppm) than when industrial CO₂ (5%, 250 ml/min) was used to bait the trap (500-600 ppm). At 30 cm from the trap and 5 cm above ground level, CO₂ levels were similar for industrial and yeast-sugar solutions 25½ and 49½ h after mixing (450-550 ppm).