Olfactory Attraction of Malaria Mosquitoes

Mosquitoes The colony of Anopheles gambiae sensu stricto (hereafter An. gambiae) was maintained at Wageningen University, The Netherlands, and originally obtained from Suakoko, Liberia (courtesy Prof. M. Coluzzi, Rome, Italy). The mosquitoes have been cultured in the laboratory since 1988 with blood meals from a human arm twice a week. The mosquito colony was kept in a climate room at 27 ± 1°C, 80 ± 5% RH and a photo-scotophase of 12/12 h L/D. Adult mosquitoes were maintained in 30 × 30 × 30 cm gauze cages with access to a 6% glucose solution on filter paper. Larvae were reared on tap water in plastic trays and fed daily with Tetramin® baby fish food (Melle, Germany). Pupae were collected daily and placed into adult cages for emergence.
Olfactometer A dual-port olfactometer, consisting of a flight chamber (1.60 x 0.66 × 0.43 m) with glass walls and a Luxan top, was used to study the behavioral responses of female mosquitoes to different odor stimuli. Pressurized air was charcoal filtered, humidified, and led through two Perspex mosquito trapping devices connected to two ports (4 cm diam and 28 cm apart) in the flight chamber (flow rate of 20.6 ± 1.4 cm/s). The light from one tungsten light bulb (75 Watt) was filtered and scattered through a screen of yellow cloth hanging ± 1 m above the flight chamber. This resulted in dim light of about 1 Lux in the olfactometer. The experimental room was maintained at 27 ± 1°C and a relative humidity of 61 ± 9% RH. The temperature inside the flight chamber was 27 ± 2.5°C and 64 ± 9% RH. The air flowing out of the ports was maintained above 80% RH and an air temperature of 27.4 ± 1°C.
Odor Stimuli Tested In The Olfactometer To insure a continuous and constant odor concentration for each compound within the odor plumes from start until the end of each experiment, we used air sample bags for ammonia, glass bottles for L-(+)-lactic acid, and 15 aliphatic carboxylic acids (Geier et al. 1999 (link); Bosch et al. 2000 (link)).Ammonia was supplied in a similar way as described in Smallegange et al. (2005 (link)). One day before the experiments, 250 μl of a 2.5% aqueous ammonia solution (25% in water; analytical grade, Merck) were injected into an 80 l Teflon air sample bag (SKC Gulf Coast Inc., Houston, TX, USA). Subsequently, the bag was filled with 60 l of warm, humidified, and charcoal filtered pressurized air at least 17 h prior to experiments to allow evaporation of the solution. This procedure resulted in an ammonia concentration of 136 ppm in the bag (Smallegange et al. 2005 (link)). During experiments, air pumps (Model 224-PCXR4, SKC Gulf Coast Inc., Houston, TX, USA) were used to lead air from the sample bag, through Teflon tubes (7 mm in diam; Rubber B.V., Hilversum, The Netherlands), and into the trapping devices at a flow of 230 ml/min. The flow of air was regulated by mechanical flow meters (Sho-Rate model GT1355; Brooks Instruments, Veenendaal, The Netherlands), and was mixed with the main air stream at a flow rate of approximately 23.5 l/min.L-(+)-lactic acid (90% aqueous solution, analytical grade, Purac Bioquimica or 88–92% aqueous solution, Riedel-de Haën) (henceforth termed lactic acid) was mixed with the main air stream by tapping lactic acid vapor (10 ml) from a 250 ml glass bottle (Fisher Scientific B.V., ‘s Hertogenbosch, The Netherlands) with Teflon tubing. The flow rate was regulated to 15 ml/min by flow meters (Gilmont, Fisher Scientific B.V., ‘s Hertogenbosch, The Netherlands), which caused a lactic acid release comparable to that from a human hand (Smith et al. 1970 (link); Geier et al. 1999 (link)). Since we applied lactic acid in a different way compared to previous experiments (Smallegange et al. 2005 (link)), we verified that it had no additional effect on the response of mosquitoes when added to ammonia.Fifteen saturated aliphatic carboxylic acids (C2–C16) of the highest purity grade available were used in the experiments: >99% acetic acid (Sigma), 99% propanoic acid (Sigma), 99% 2-methylpropanoic acid (Sigma), ≥99% 3-methylbutanoic acid (Sigma), >99% butanoic acid (Aldrich), >99% pentanoic acid (Sigma), ≥99% hexanoic acid (Sigma), 98% heptanoic acid (Sigma), ≥ 99% octanoic acid (Sigma), ≥ 97% nonanoic acid (Sigma), >99% decanoic acid (Sigma), ≥99% dodecanoic acid (Sigma), ≥ 98% tridecanoic acid (Sigma), >99% tetradecanoic acid (Sigma), ≥99% hexadecanoic acid (Sigma). Single, pure compounds [10 ml of a liquid compound (C2–C9) or 1 g of a solid compound (C10–C16)] were added to a 250 ml glass bottle (Fisher Scientific B.V., ‘s Hertogenbosch, The Netherlands). A charcoal-filtered, warm, humidified air stream was passed through the bottle and carried the vaporized compound at the desired flow rate through Teflon tubing and into the main stream through one of the trapping devices. Flow rates were regulated by Gilmont flow meters (Fisher Scientific B.V., ‘s Hertogenbosch, The Netherlands) at 0.5, 5, 50, and 100 ml/min. The calculated concentrations of the compounds in the air stream of the olfactometer are listed in Table S1 in the online supplement.When tripartite blends were tested, the three compounds were mixed just before entering the trapping device. When more than one aliphatic carboxylic acid was part of the blend, a Perspex ring with 10 holes was attached upstream of each trapping device to be able to release each odor into the main air stream individually (Fig. 1). Tripartite blends were tested against ammonia alone. During experiments with multi-component blends, the multi-component odor blends were tested against the ammonia + lactic acid blend. Initially, we wanted to examine whether we could increase the attractiveness of ammonia—our best kairomone at that time. For this reason, we used ammonia alone as a control. In addition, we had observed that ammonia + lactic acid + a mixture of 12 carboxylic acids did not attract more mosquitoes than ammonia + lactic acid, but was more attractive than ammonia alone (Smallegange et al. 2005 (link)). Once we determined which individual carboxylic acids augmented the attractiveness of ammonia + lactic acid (Table 1), we continued our experiments with ammonia + lactic acid as the control, as we aimed for a better result than was found in Smallegange et al. (2005 (link)).
Fig. 1

An olfactometer trapping device is composed of three parts: A: part with baffle where mosquitoes enter the device; B: middle part device; C: distal end sealed with metal gauze to prevent mosquito crossing. A Perspex ring (D) with 10 holes for separate odor delivery. The end of the tube running from the glass bottle with an odor was inserted through one of the holes. Charcoal filtered, warm, humidified, pressurized air is led into the trapping device through E (a: schematic representation of the couplings between the various parts, made to fit smoothly on each other to prevent air loss)

Table 1

Effect of adding an individual carboxylic acid, at four flow rates (ml/min), to ammonia + lactic acid tested against ammonia alone in the dual-choice olfactometer. The result of the χ²-test (P-value), trap entry response (%) and total number of mosquitoes released (n) are given for each two-choice test

Carboxylic acid	0.5 ml/min	5 ml/min	50 ml/min	100 ml/min
Acetic acid (C2)	P = 0.25	P = 0.24	P = 0.85	P = 0.56
	24.1%	22.5%	14.4%	15.3%
	n = 199	n = 262	n = 201	n = 177
Propanoic acid (C3)	P = 0.13	P < 0.001 A	P = 0.59	P = 0.001 A
	15.7%	21.2%	8.8%	20.8%
	n = 178	n = 156	n = 160	n = 154
2-Methylpropionic acid (2mC3)	P = 1.00	P = 0.38	P = 0.34	P = 0.22
	8.2%	18.9%	30.5%	39.9%
	n = 170	n = 175	n = 174	n = 198
Butanoic acid (C4)	P = 0.03 A	P = 0.69	P = 0.53	P = 0.47
	24.7%	11.6%	6.1%	11.0%
	n = 150	n = 225	n = 164	n = 155
3-Methylbutanoic acid (3mC4)	P = 0.008 A	P = 0.64	n.t.	n.t.
	16.6%	10.2%
	n = 169	n = 176
Pentanoic acid (C5)	P = 0.13	P = 0.32	P = 0.86	P = 0.01 A
	6.3%	20.6%	18.5%	39.4%
	n = 174	n = 175	n = 178	n = 175
Hexanoic acid (C6)	P = 0.003 R	P = 0.66	P = 0.88	P = 0.73
	19.7%	17.5%	26.3%	15.7%
	n = 234	n = 468	n = 179	n = 464
Heptanoic acid (C7)	P = 0.03 A	P = 0.005 R	P = 0.007 R	P = 0.85
	8.8%	11.0%	13.5%	15.4%
	n = 226	n = 164	n = 170	n = 175
Octanoic acid (C8)	P = 0.30	P = 0.47	P < 0.001 A	P = 0.23
	13.5%	16.0%	29.5%	29.2%
	n = 170	n = 187	n = 193	n = 195
Nonanoic acid (C9)	P = 0.78	P = 0.41	P = 0.16	P = 0.53
	7.6%	13.5%	4.5%	5.6%
	n = 170	n = 178	n = 177	n = 177
Decanoic acid (C10)	P = 0.32	P = 1.00	P = 1.00	P = 0.53
	4.1%	6.9%	3.7%	4.5%
	n = 219	n = 232	n = 215	n = 221
Dodecanoic acid (C12)	P = 0.47	P = 1.00	P = 1.00	P = 0.78
	9.4%	8.9%	4.4%	7.3%
	n = 180	n = 180	n = 180	n = 178
Tridecanoic acid (C13)	P = 0.26	P = 0.82	P = 0.09	P = 0.58
	23.4%	11.3%	25.4%	17.3%
	n = 167	n = 168	n = 169	n = 168
Tetradecanoic acid (C14)	P = 0.02 A	P = 0.04 A	P = 0.007 A	P = 0.01 A
	8.7%	11.2%	11.5%	12.7%
	n = 173	n = 170	n = 174	n = 173
Hexadecanoic acid (C16)	P = 0.06	P = 0.37	P = 0.74	P = 0.80
	5.7%	6.3%	5.2%	8.5%
	n = 175	n = 176	n = 172	n = 177

A: significantly more mosquitoes in the trapping device baited with the tripartite blend compared to the trapping device baited with ammonia (χ²-test, P < 0.05). R: significantly fewer mosquitoes in the trapping device baited with the tripartite blend compared to the trapping device baited with ammonia (χ²-test, P < 0.05). Calculated concentrations of the compounds in the odor plume are given in the online supplement (Table S1).

n.t. not tested

Olfactometer Tests Thirty female mosquitoes, 5–8 d-old, that had not received a blood meal, were randomly collected from their cage 14–18 h before the start of experiments. The mosquitoes were placed into a cylindrical release cage (8-cm diam, 10-cm high) with access to tap water from damp cotton wool placed on top of the cage. Experiments were performed during the last 4 h of the dark period, when An. gambiae is normally active (Haddow and Ssenkubuge 1973 ; Maxwell et al. 1998 (link); Killeen et al. 2006 (link)).In each trial, test compounds were released into the air stream and a group of mosquitoes was set free from a release cage placed at the downwind end of the flight chamber of the olfactometer, 1.60 m from the two ports. Mosquitoes were left in the flight chamber for 15 min. Female mosquitoes that had entered either of the trapping devices were counted at the end of the experiment, after anaesthetization with 100% CO₂. Mosquitoes remaining in the flight chamber were removed with a vacuum cleaner. After use, the trapping devices were washed with soapy water (CLY-MAX Heavy Duty Cleaner, Rogier Bosman Chemie B.V., Heijningen, The Netherlands), rinsed with tap water, and cleaned with cotton wool drenched in 70% ethanol (Merck).The operator wore surgical gloves (Romed®, powderfree vinyl) to avoid contamination of the equipment with human volatiles. Each trial started with new mosquitoes and clean trapping devices. An experiment testing a particular blend was repeated at least 6 times on different days. The sequence of test odors was randomized on the same day and between days. Test stimuli were alternated between right and left ports in different replicates to rule out any positional effects. Experiments in which only clean air was fed into the olfactometer through both ports were done to test the symmetry of the trapping system.
Trapping Experiments in Screen Cage To assess the performance of some of the blends as a lure, we conducted laboratory experiments with volatile baited MM-X traps in a textile screen cage (233 × 250 × 330 cm; Howitec Netting BV, Bolsward, The Netherlands) inside a climate-controlled room (22.7 ± 1.1°C and 52.4 ± 7.4% RH). Two Mosquito Magnet-X (MM-X) traps (American Biophysics Corp., U.S.A.) (Kline 1999 (link)) were placed inside the cage at 2 m distance from each other.Initially, a 9-compound blend (blend A) was tested. To compose this blend, 500 µl of a liquid pure compound or 500 mg of a solid compound (tetradecanoic acid) were put into individual low density polyethylene sachets (LDPE; 6 × 6 cm; Audion Elektro, The Netherlands; Torr et al. 1997 (link)) with a thickness of 0.1 mm (ammonia, lactic acid, heptanoic, octanoic, and tetradecanoic acid) or in a closed LDPE tube (32 × 14 mm, Kartell, 3.5 ml; Fisher Emergo, The Netherlands) within an LDPE sachet with a thickness of 0.2 mm (this delivery method was used for the 4 most volatile aliphatic carboxylic acids (i.e., propanoic, butanoic, 3-methylbutanoic, and pentanoic acids) to reduce their release to a higher extent than would have been possible with a sachet only). Only ammonia was diluted with distilled water (to 2.5%). The sachets were applied inside the central, black tube of the MM-X traps using odorless tape (3M™ Double Coated Tape 400). Air flow was created by a fan on top of this tube taking the headspace of the test blend downwards and outside the MM-X trap. Second, a tripartite blend was tested (blend B). Since we calculated that the release rates of the aliphatic carboxylic acids in blend A were at least two times higher than calculated for the multi-component blends tested in the optimization and subtraction olfactometer experiments, the 3 LDPE sachets (each with a thickness of 0.1 mm) containing the separate components of blend B were made as small as possible (2.5 × 2.5 cm) to reduce evaporation (Torr et al. 1997 (link)). One hundred µl of a liquid pure compound (ammonia, lactic acid) or 50 mg of a solid compound (tetradecanoic acid) were put into individual LDPE sachets. The amount of a compound within a sachet does not affect the evaporation rate of the compound through the LDPE material, whereas the surface of a LDPE sachet does (Torr et al. 1997 (link)). When blend B was applied, ammonia was not diluted (25.0%). Both blend A and B were tested against a blend of ammonia + lactic acid. Evaporation rates of the compounds were measured by weighing the LDPE sachets before and after experiments (see Table S1 in the online supplement).Fifty female mosquitoes, 5–8 d-old, which had not received a blood meal, were randomly collected 14–18 h before the start of experiments. They were placed into a cylindrical release cage (diam 8 cm, height 17.5 cm) with access to tap water from damp cotton wool placed on top of the cage. The mosquitoes were set free from the release cage in the center of the screen cage. After 4 h, the MM-X traps were closed and transferred into a freezer to kill the mosquitoes. These experiments were performed during the last 4 h of the dark period. Each two-choice test was repeated either 4 or 6 times, alternating the position of each treatment every experimental day. Surgical gloves were worn to avoid contamination of equipment with human volatiles. Experiments with unbaited traps in the MM-X setup were done 6 times to test the symmetry of the trapping system.
Statistical Analysis For each two-choice assay, a Chi-square test was used to analyze whether the total number (i.e., sum of all replicates; a comparison of data collected on different days revealed no heterogeneity) of mosquitoes trapped in the treatment trapping device (of either olfactometer or MM-X trap) and the total number that was trapped in the control trapping device (of olfactometer or MM-X trap) differed from a 1:1 distribution. Effects were considered to be significant at P < 0.05. The number of female mosquitoes caught in both trapping devices divided by the number of mosquitoes that flew out of the release cage is expressed as the trap entry response (TER).