TY - JOUR
AB - Abstract Efforts to develop mosquito attractants using vertebrate host volatiles have been well made under laboratory conditions but their attractiveness to mosquitoes in the wild still needs to be evaluated. In the present study, we evaluated the attraction of female Culex pipiens pallens Coquillett (Diptera: Culicidae) to 11 individual chemical compounds found in vertebrate host odors, and to synthetic blends, consisting of different combinations of the compounds. These tests were conducted under laboratory and field conditions using a Y-tube olfactometer and odor-baited traps, respectively. When delivered at concentrations ranging from 0.1 to 10.0 μg/kg, 9 of the 11 compounds were attractive to female mosquitoes under laboratory conditions. We developed 47 synthetic blends composed of the 6 most attractive compounds (propionic acid, hexanal, myristic acid, benzaldehyde, 1-octen-3-ol, and geranyl acetone) and 18 of them were significantly attractive to mosquitoes in the olfactometer. Most of the attractive blends contained two to four attractive compounds. In the field, 5 of the 18 blends captured significantly more mosquitoes than did control traps. The findings demonstrate that female mosquitoes can be attracted by single chemical compounds as well as some of their synthetic blends. The effectiveness of synthetic blends depended on specific combinations of several compounds, rather than simply increasing the number of attractive compounds in the blends. Synthetic blends may have potential for use in odor-baited traps for mosquito surveillance. host odors, Culex pipiens pallens, volatile chemical compounds, synthetic blends, odor-baited traps As a generalist feeder, Culex pipiens pallens Coquillett (Diptera: Culicidae) mainly feeds on the blood of avian hosts, humans, and other mammals (Tanaka et al. 1979, Molaei et al. 2006, Hamer et al. 2009). It is an important vector of mosquito-borne diseases, such as West Nile virus, filarial worms and avian malaria parasites (Perumalsamy et al. 2012; Ding et al. 2016; Fouad et al. 2016, 2017). In China, the most common mosquito control strategies rely heavily on chemical insecticides, which have consequently led to insecticide resistance, environmental contamination, and toxic hazards to human beings (Jones et al. 2012, Wassie et al. 2012). Mosquito host seeking depends mainly on an ability to identify specific compounds and cues released by the hosts including carbon dioxide, body heat, and odors (i.e., volatile compounds) (Bernier et al. 2000, Verhulst et al. 2016). More than 300 volatile compounds have been identified from human skin by gas chromatography/mass spectrometry, and most of them are carboxylic acids, aldehydes, and esters of carboxylic acids (Bernier et al. 2000). Although large numbers of host-produced volatile compounds have been identified, only a few are attractive to mosquitoes based on both laboratory and field tests (Hoel et al. 2007). The most attractive compounds include lactic acid and ammonia (Bernier et al. 2003, Smallegange et al. 2009). Culex quinquefasciatus showed behavioral preferences to some carboxylic acids, such as hexanoic acid (C6), tridecanoic acid (C13), and myristic acid (C14), in a Y-tube olfactometer assays (Puri et al. 2006). Ammonia was reported to be attractive to host-seeking Aedes and Anopheles mosquitoes (Geier et al. 1999, Smallegange et al. 2005). Mosquitoes have demonstrated enantiomeric selectivity, where in a behavioral assay (R) 1-octen-3-ol was significantly more attractive to Aedes aegypti than was (S)-1-octen-3-ol (Cook et al. 2011). Previous laboratory studies have provided scant evidence that odor blends were more attractive to mosquitoes than a single compound (Bosch et al. 2000, Bernier et al. 2007). In a Y-tube olfactometer test, Ae. aegypti was more attracted by the odors of a synthetic blend of ammonia, l-lactic acid, and carboxylic acid than to individual compound alone (Bosch et al. 2000). In a study using plant volatiles to attract sugar-feeding Cx. pipiens pallens, Yu et al. (2015) demonstrated that a reduced synthetic blend consisting of 6 attractive compounds was equally attractive to Cx. pipiens pallens as a blend consisting of 15 attractive compounds. These findings suggest that the most important factor in producing an effective attractant is the combinations of key synergistic compounds. Many studies have examined attractive host odors with the aim to improve mosquito control by luring mosquitoes to toxic bait traps (Bernier et al. 2007, Jawara et al. 2011, Owino et al. 2014). However, because of the complexity of odors in the outdoor environment and the complex physical properties of the active chemical compounds, the effectiveness of these compounds in attracting mosquitoes needs to be verified in the field. The objectives of the present study were to: 1) evaluate the attractiveness of selected chemical compounds of host odors and their blends to female Cx. pipiens pallens under laboratory conditions; 2) evaluate the effects of different combinations of active attractant compounds in synthetic blends to female Cx. pipiens pallens under laboratory and field conditions; and 3) determine the importance of the number and identity of active compounds in the formulation of synthetic blends that attract mosquitoes. Materials and Methods Mosquitoes Cx. pipiens pallens eggs were obtained from mosquito colonies reared in the laboratory of Urban Entomology, Institute of Insect Sciences, Zhejiang University where mosquitoes were maintained in an insectary at 26 ± 1°C, 75% RH, and a photoperiod of 14:10 (L:D) h for more than 50 generations. Adult mosquitoes were held in mesh-covered cages (30 × 30 × 30 cm) and fed a 5% sucrose solution ad libitum. Adult females were provided with a bloodmeal from a restrained mouse 5 d after emergence to sustain egg production (animal use approval protocol number: SYXK 2012-0178, Zhejiang University). Gravid females were allowed to oviposit in cups (5 cm in diameter by 7 cm depth) containing 100 ml of hay infusion water 1 wk after blood feeding. Egg rafts were transferred to plastic trays (25 cm in diameter by 12 cm depth) containing 3 liters of dechlorinated tap water. Larvae were reared in plastic trays (25 cm in diameter by 12 cm depth) with dechlorinated tap water and fed commercial rat food daily (0.3 g/100 larvae/d). Pupae were collected daily with a 60 mesh screen and transferred to holding cages. Chemicals Chemical compounds used in the experiments were selected based on the results from a study of human skin emanation by Bernier et al. (2000). Chemicals of high purity were used in the experiments: geranyl acetone (97% pure), propionic acid (≥99%), butyric acid (99.5%), heptanoic acid (98%), n-octanoic acid (98%), 1-octen-3-octenol (98%), p-cresol (98%), hexanal (97%), benzaldehyde (≥98%), and dimethyl sulfoxide (anhydrous solvent). Most were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Myristic acid (98% pure) and 6-methyl-5-hepten-2-one (≥98%) were purchased from Sigma-Aldrich Co. (Poole, Dorset, UK). Dual Choice Olfactometer Assays Tests of the attraction of female Cx. pipiens pallens to single chemical compounds and their synthetic blends were conducted in a Y-tube olfactometer (Fig. 1). The Y-tube olfactometer consists of three main parts: the release arm (diameter 3 cm, length 15 cm), the flight arm (diameter 3 cm, length 20 cm), and the selection arms (diameter 3 cm, length 20 cm). The airflow, produced by a compressed air pump, was purified by passing through charcoal. Two glass bottles (diameter 5 cm, height 15 cm) filled with distilled water were used to maintain humidity. Airflow was controlled by a flow meter and was 500 liters/h on each side. A glass dish inside the glass jar (diameter 8 cm, height 15 cm) contained 5 g of a selected solid odor source. Mosquitoes were used 5–7 d after emergence and had access to 5% sucrose before the tests. One 15 W red light bulb was placed above the Y-tube olfactometer for illumination (Yu et al. 2015). Tests were conducted from 1800 to 2100 hours at 26 ± 1°C and 70 ± 10% RH. Alternate tests were carried out by interchanging the control arm and treated arm before the treatments, and the results showed no difference between the two selection arms. Fig. 1. View largeDownload slide Schematic drawing (A) and lay-out (B) of the Y-tube olfactometer (not drawn to scale) used in the experiments. Fig. 1. View largeDownload slide Schematic drawing (A) and lay-out (B) of the Y-tube olfactometer (not drawn to scale) used in the experiments. In the tests, corn flour was used as dispenser. Both liquid and solid chemical compounds were dissolved with 100 μl dimethyl sulfoxide (DMSO), and then mixed with 5 g corn flour. Each compound was prepared as a concentration of 0.1, 1.0, 5.0, and 10.0 μg/kg. Corn flour treated with DMSO was used as control. Groups of 10 females were released into the release arm and allowed 5 min to adjust before exposure to the stimulus. Five grams of a selected mixture was placed on a glass dish at the end of one selection arm and a control was placed on the other. Thirty seconds later, the release arm was opened and the mosquitoes were allowed to enter the flight arm. The number of mosquitoes in the selection arms was counted 5 min later. Each dose for the selected mixture was replicated 10 times. The positions of the treatment and control arms were alternated between trials to eliminate positional biases. The tools were washed with ethanol and dried at 65°C for 6 h in an air-circulating oven. Attractiveness of Different Combinations of the Active Compounds We determined the attractiveness of different mixtures of six compounds (propionic acid, hexanal, myristic acid, benzaldehyde, 1-octen-3-ol, and geranyl acetone) that we found to be the most attractive to female Cx. pipiens pallens (Table 1). A total of 47 blends were formulated using the most attractive concentration for each compound (Supp Table 1 [online only]). The blends were divided into five groups (Groups A–E). Group A contained 15 blends that were composed of 2 active compounds (blend 1 to blend 15; Supp Table 1 [online only]). A third active compound was added to the Group A (2-compound) blends that were shown to elicit mosquito attraction (see preference index criteria below). Thus, this produced 3-compound blends (Group B; blend 16 to blend 28; Supp Table 1 [online only]). Groups C, D, and E were prepared by following the same protocol (Supp Table 1 [online only]). Group C consisted of 4-compound blends (blend 29 to blend 40), Group D consisted of 5-compound blends (blend 41 to blend 46; Supp Table 1 [online only]) and Group E was the full 6-compound blend (blend 47; Supp Table 1 [online only]). The attractant evaluation experiments were conducted using a Y-tube olfactometer with the same experimental protocol that is described above. Table 1. Optimal doses of single chemical compounds attracted to host-seeking female mosquitoes Compound Dose (μg/kg) df χ2 PI (%) P-value Propionic acid 5 1 18.77 43.67 ± 10.02 <0.001 Hexanal 1 1 14.44 38.00 ± 5.54 <0.001 Myristic acid 5 1 13.83 37.78 ± 8.01 <0.001 Benzaldehyde 10 1 13.46 37.48 ± 8.27 <0.001 1-Octen-3-ol 0.1 1 10.24 31.86 ± 10.81 0.001 Geranyl acetone 10 1 13.46 30.14 ± 10.78 <0.001 Butyric acid 10 1 7.67 29.11 ± 8.82 0.006 p-Cresol 10 1 7.36 27.78 ± 11.12 0.007 n-Octanoic acid 1 1 6.58 24.33 ± 14.18 0.01 Heptanoic acid 1 1 0.51 7.11 ± 12.45 0.477 6-Methyl-5-hepten-2-one 0.1 1 0.01 0.35 ± 15.47 0.919 Compound Dose (μg/kg) df χ2 PI (%) P-value Propionic acid 5 1 18.77 43.67 ± 10.02 <0.001 Hexanal 1 1 14.44 38.00 ± 5.54 <0.001 Myristic acid 5 1 13.83 37.78 ± 8.01 <0.001 Benzaldehyde 10 1 13.46 37.48 ± 8.27 <0.001 1-Octen-3-ol 0.1 1 10.24 31.86 ± 10.81 0.001 Geranyl acetone 10 1 13.46 30.14 ± 10.78 <0.001 Butyric acid 10 1 7.67 29.11 ± 8.82 0.006 p-Cresol 10 1 7.36 27.78 ± 11.12 0.007 n-Octanoic acid 1 1 6.58 24.33 ± 14.18 0.01 Heptanoic acid 1 1 0.51 7.11 ± 12.45 0.477 6-Methyl-5-hepten-2-one 0.1 1 0.01 0.35 ± 15.47 0.919 Ten female mosquitoes were used each replicate per concentration and each concentration was replicated 10 times for each compound. Total number of mosquitoes responded to single chemical compounds and control were analyzed by χ2 test. View Large Table 1. Optimal doses of single chemical compounds attracted to host-seeking female mosquitoes Compound Dose (μg/kg) df χ2 PI (%) P-value Propionic acid 5 1 18.77 43.67 ± 10.02 <0.001 Hexanal 1 1 14.44 38.00 ± 5.54 <0.001 Myristic acid 5 1 13.83 37.78 ± 8.01 <0.001 Benzaldehyde 10 1 13.46 37.48 ± 8.27 <0.001 1-Octen-3-ol 0.1 1 10.24 31.86 ± 10.81 0.001 Geranyl acetone 10 1 13.46 30.14 ± 10.78 <0.001 Butyric acid 10 1 7.67 29.11 ± 8.82 0.006 p-Cresol 10 1 7.36 27.78 ± 11.12 0.007 n-Octanoic acid 1 1 6.58 24.33 ± 14.18 0.01 Heptanoic acid 1 1 0.51 7.11 ± 12.45 0.477 6-Methyl-5-hepten-2-one 0.1 1 0.01 0.35 ± 15.47 0.919 Compound Dose (μg/kg) df χ2 PI (%) P-value Propionic acid 5 1 18.77 43.67 ± 10.02 <0.001 Hexanal 1 1 14.44 38.00 ± 5.54 <0.001 Myristic acid 5 1 13.83 37.78 ± 8.01 <0.001 Benzaldehyde 10 1 13.46 37.48 ± 8.27 <0.001 1-Octen-3-ol 0.1 1 10.24 31.86 ± 10.81 0.001 Geranyl acetone 10 1 13.46 30.14 ± 10.78 <0.001 Butyric acid 10 1 7.67 29.11 ± 8.82 0.006 p-Cresol 10 1 7.36 27.78 ± 11.12 0.007 n-Octanoic acid 1 1 6.58 24.33 ± 14.18 0.01 Heptanoic acid 1 1 0.51 7.11 ± 12.45 0.477 6-Methyl-5-hepten-2-one 0.1 1 0.01 0.35 ± 15.47 0.919 Ten female mosquitoes were used each replicate per concentration and each concentration was replicated 10 times for each compound. Total number of mosquitoes responded to single chemical compounds and control were analyzed by χ2 test. View Large Field Experiments The 18 synthetic blends that attracted to mosquitoes in the Y-tube olfactometer laboratory assays were field-tested in baited traps during June 2016 at Zhejiang University (30°16ʹN; 120°12ʹE), Hangzhou, China (Table 3). The blends tested in the field tests were dispensed following the same protocol used in the Y-tube olfactometer assays. Distilled water was added (40 ml) to each mixture of corn flour and compound blends (20 g for each trap). Table 3. Mean number ± SE of Culex pipiens pallens caught per night in traps baited with different synthetic blends under field conditions Blend number N n Trapped mosquitoes (mean ± SE) Composition 28 3 33 11.00 ± 2.08a 1-Octen-3-ol + hexanal + myristic acid 26 3 32 10.67 ± 2.33a Benzaldehyde + hexanal + myristic acid 22 3 29 9.76 ± 2.19ab Geranyl acetone + myristic acid + propionic acid 37 3 27 9.00 ± 1.00abc Geranyl acetone + benzaldehyde + hexanal + propionic acid 4 3 26 8.67 ± 1.33abc Geranyl acetone + propionic acid 23 3 17 5.67 ± 1.20bcd Benzaldehyde + 1-octen-3-ol + hexanal 5 3 17 5.67 ± 3.18bcd Geranyl acetone + 1-octen-3-ol 41 3 14 4.67 ± 2.82bcde Geranyl acetone + benzaldehyde + 1-octen-3-ol + hexanal + myristic acid 21 3 14 4.67 ± 1.20bcde Geranyl acetone + benzaldehyde + myristic acid 7 3 14 4.67 ± 1.20bcde Benzaldehyde + hexanal 19 3 13 4.33 ± 1.45cde Geranyl acetone + 1-octen-3-ol + propionic acid 31 3 12 4.00 ± 0.58cde Geranyl acetone + benzaldehyde + 1-octen-3-ol + hexanal 6 3 12 4.00 ± 1.53cde Benzaldehyde + 1-octen-3-ol 11 3 10 3.33 ± 0.33de 1-Octen-3-ol + myristic acid 29 3 8 2.67 ± 1.20de Geranyl acetone + benzaldehyde + 1-octen-3-ol + hexanal 38 3 7 2.33 ± 0.33de Benzaldehyde + 1-octen-3-ol + hexanal + propionic acid 32 3 7 2.33 ± 0.33de Geranyl acetone + benzaldehyde + hexanal + myristic acid 46 3 3 1.00 ± 1.00de Benzaldehyde + 1-octen-3-ol + hexanal + myristic acid + propionic acid Controls Water + corn flour 3 3 1.00 ± 1.00de Corn flour 3 1 0.33 ± 0.33f water 3 0 0f Blend number N n Trapped mosquitoes (mean ± SE) Composition 28 3 33 11.00 ± 2.08a 1-Octen-3-ol + hexanal + myristic acid 26 3 32 10.67 ± 2.33a Benzaldehyde + hexanal + myristic acid 22 3 29 9.76 ± 2.19ab Geranyl acetone + myristic acid + propionic acid 37 3 27 9.00 ± 1.00abc Geranyl acetone + benzaldehyde + hexanal + propionic acid 4 3 26 8.67 ± 1.33abc Geranyl acetone + propionic acid 23 3 17 5.67 ± 1.20bcd Benzaldehyde + 1-octen-3-ol + hexanal 5 3 17 5.67 ± 3.18bcd Geranyl acetone + 1-octen-3-ol 41 3 14 4.67 ± 2.82bcde Geranyl acetone + benzaldehyde + 1-octen-3-ol + hexanal + myristic acid 21 3 14 4.67 ± 1.20bcde Geranyl acetone + benzaldehyde + myristic acid 7 3 14 4.67 ± 1.20bcde Benzaldehyde + hexanal 19 3 13 4.33 ± 1.45cde Geranyl acetone + 1-octen-3-ol + propionic acid 31 3 12 4.00 ± 0.58cde Geranyl acetone + benzaldehyde + 1-octen-3-ol + hexanal 6 3 12 4.00 ± 1.53cde Benzaldehyde + 1-octen-3-ol 11 3 10 3.33 ± 0.33de 1-Octen-3-ol + myristic acid 29 3 8 2.67 ± 1.20de Geranyl acetone + benzaldehyde + 1-octen-3-ol + hexanal 38 3 7 2.33 ± 0.33de Benzaldehyde + 1-octen-3-ol + hexanal + propionic acid 32 3 7 2.33 ± 0.33de Geranyl acetone + benzaldehyde + hexanal + myristic acid 46 3 3 1.00 ± 1.00de Benzaldehyde + 1-octen-3-ol + hexanal + myristic acid + propionic acid Controls Water + corn flour 3 3 1.00 ± 1.00de Corn flour 3 1 0.33 ± 0.33f water 3 0 0f Numbers with different letter superscripts in the same column differ significantly (generalized linear models; P < 0.05). N is the number of replicates, and n is the total number of mosquitoes trapped. View Large Table 3. Mean number ± SE of Culex pipiens pallens caught per night in traps baited with different synthetic blends under field conditions Blend number N n Trapped mosquitoes (mean ± SE) Composition 28 3 33 11.00 ± 2.08a 1-Octen-3-ol + hexanal + myristic acid 26 3 32 10.67 ± 2.33a Benzaldehyde + hexanal + myristic acid 22 3 29 9.76 ± 2.19ab Geranyl acetone + myristic acid + propionic acid 37 3 27 9.00 ± 1.00abc Geranyl acetone + benzaldehyde + hexanal + propionic acid 4 3 26 8.67 ± 1.33abc Geranyl acetone + propionic acid 23 3 17 5.67 ± 1.20bcd Benzaldehyde + 1-octen-3-ol + hexanal 5 3 17 5.67 ± 3.18bcd Geranyl acetone + 1-octen-3-ol 41 3 14 4.67 ± 2.82bcde Geranyl acetone + benzaldehyde + 1-octen-3-ol + hexanal + myristic acid 21 3 14 4.67 ± 1.20bcde Geranyl acetone + benzaldehyde + myristic acid 7 3 14 4.67 ± 1.20bcde Benzaldehyde + hexanal 19 3 13 4.33 ± 1.45cde Geranyl acetone + 1-octen-3-ol + propionic acid 31 3 12 4.00 ± 0.58cde Geranyl acetone + benzaldehyde + 1-octen-3-ol + hexanal 6 3 12 4.00 ± 1.53cde Benzaldehyde + 1-octen-3-ol 11 3 10 3.33 ± 0.33de 1-Octen-3-ol + myristic acid 29 3 8 2.67 ± 1.20de Geranyl acetone + benzaldehyde + 1-octen-3-ol + hexanal 38 3 7 2.33 ± 0.33de Benzaldehyde + 1-octen-3-ol + hexanal + propionic acid 32 3 7 2.33 ± 0.33de Geranyl acetone + benzaldehyde + hexanal + myristic acid 46 3 3 1.00 ± 1.00de Benzaldehyde + 1-octen-3-ol + hexanal + myristic acid + propionic acid Controls Water + corn flour 3 3 1.00 ± 1.00de Corn flour 3 1 0.33 ± 0.33f water 3 0 0f Blend number N n Trapped mosquitoes (mean ± SE) Composition 28 3 33 11.00 ± 2.08a 1-Octen-3-ol + hexanal + myristic acid 26 3 32 10.67 ± 2.33a Benzaldehyde + hexanal + myristic acid 22 3 29 9.76 ± 2.19ab Geranyl acetone + myristic acid + propionic acid 37 3 27 9.00 ± 1.00abc Geranyl acetone + benzaldehyde + hexanal + propionic acid 4 3 26 8.67 ± 1.33abc Geranyl acetone + propionic acid 23 3 17 5.67 ± 1.20bcd Benzaldehyde + 1-octen-3-ol + hexanal 5 3 17 5.67 ± 3.18bcd Geranyl acetone + 1-octen-3-ol 41 3 14 4.67 ± 2.82bcde Geranyl acetone + benzaldehyde + 1-octen-3-ol + hexanal + myristic acid 21 3 14 4.67 ± 1.20bcde Geranyl acetone + benzaldehyde + myristic acid 7 3 14 4.67 ± 1.20bcde Benzaldehyde + hexanal 19 3 13 4.33 ± 1.45cde Geranyl acetone + 1-octen-3-ol + propionic acid 31 3 12 4.00 ± 0.58cde Geranyl acetone + benzaldehyde + 1-octen-3-ol + hexanal 6 3 12 4.00 ± 1.53cde Benzaldehyde + 1-octen-3-ol 11 3 10 3.33 ± 0.33de 1-Octen-3-ol + myristic acid 29 3 8 2.67 ± 1.20de Geranyl acetone + benzaldehyde + 1-octen-3-ol + hexanal 38 3 7 2.33 ± 0.33de Benzaldehyde + 1-octen-3-ol + hexanal + propionic acid 32 3 7 2.33 ± 0.33de Geranyl acetone + benzaldehyde + hexanal + myristic acid 46 3 3 1.00 ± 1.00de Benzaldehyde + 1-octen-3-ol + hexanal + myristic acid + propionic acid Controls Water + corn flour 3 3 1.00 ± 1.00de Corn flour 3 1 0.33 ± 0.33f water 3 0 0f Numbers with different letter superscripts in the same column differ significantly (generalized linear models; P < 0.05). N is the number of replicates, and n is the total number of mosquitoes trapped. View Large The experimental site was 300 × 300 m and surrounded with shrub plants and grasses. A few feral domestic animals, including chickens, dogs, and cats were present during the nighttime tests. The odor-baited traps used in the experiments are described in the study by Ding et al. (2016). Briefly, the traps consisted of a pedestal (diameter, upper 11.5 cm; lower, 9 cm; height, 3.6 cm) with three apertures (9.5 cm in length by 0.7 cm width) along the edge and a plastic container (10.5 cm in diameter by 10 cm height) with two sticky boards (9.2 cm in height by 8.9 cm width) inside (Fig. 2). A petri dish (8.6 cm in diameter) filled with a selected test mixture was placed in the pedestal. The odorants released by the mixture spread to the surrounding area through the three apertures. Two sticky boards were inserted into the plastic container to capture the attracted mosquitoes. Mosquitoes attracted by the odors released by the mixture entered the trap through one of the three apertures and were captured on one of the sticky boards. Three controls including distilled water alone, corn flour alone, and a mixture of corn flour and water (20 g/40 ml) were also tested. Trials were conducted simultaneously at three different sites. For each trial, a total of 21 traps (including 18 treatments and three controls) were placed in one site and traps were rotated between trial sites according to a random block design. The traps were placed 0.5–0.7 m above the ground and 10 m apart on the experimental site for 12 h (1800–0800 hours) (Fig. 2A). Each treatment was replicated three times. Fig. 2. View largeDownload slide The odor-baited traps used in field experiments. (A) The traps placed in the field. (B) Mosquitoes captured by the traps. Fig. 2. View largeDownload slide The odor-baited traps used in field experiments. (A) The traps placed in the field. (B) Mosquitoes captured by the traps. Ethical Statement All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The study protocol received approval from the local health and administrative authorities (SYXK 2012-0178). These studies did not involve endangered or protected species. Statistical Analysis The attractiveness of individual chemical compounds and the synthetic blends was evaluated by using a preference index (PI): PI= [(NT−NC)/(NT+NC)] ×100 where NT refers to the number of mosquitoes in the treatment arm and NC refers to the number of mosquitoes in the control arm of a Y-tube olfactometer (Carlsson et al. 1999, Yu et al. 2015). We considered a PI exceeding 30% as showing attraction to mosquitoes, and these compounds and blends were used to formulate more complex synthetic blends. Chi-square tests were used to compare the total number of mosquitoes that selected the treatment arm to those that selected the control arm. The number of mosquitoes captured by different traps in the field was subjected to negative binomial regression following the generalized linear models. The significance was taken at P < 0.05. All statistical analyses were conducted using SPSS v. 20 (SPSS Inc., Chicago, IL). Results Attraction of Mosquitoes to Individual Compounds in the Y-Tube Olfactometer Nine of the 11 tested chemical compounds of human odors were attractive to host-seeking female Cx. pipiens pallens in the Y-tube olfactometer tests (Table 1; Supp Fig. 1 [online only]). Six out of the 9 attractive compounds had a PI exceeding 30%. The most attractive concentration of each compound was 5 μg/kg of propionic acid, 10 μg/kg of benzaldehyde, 0.1 μg/kg of 1-octen-3-ol, 10 μg/kg of eranyl acetone, 1 μg/kg of hexanal, and 5 μg/kg of myristic acid. Mosquitoes were not significantly attracted by heptanoic acid and 6-methyl-5-hepten-2-one when compared with the control (Table 1; Supp Fig. 1 [online only]). Attraction of Mosquitoes to the Synthetic Blends in the Y-Tube Olfactometer Five of the 15 synthetic blends in the Group A were significantly attractive to mosquitoes with the PI exceeding 30%. Blend 6, consisting of benzaldehyde and 1-octen-3-ol, was the most attractive blend in this group and attracted 72% of the mosquitoes with a PI of 46.67% (Fig. 3; Table 2; Supp Table 1 [online only]). Other attractive blends included: blend 4 (χ2 = 9.707, df = 1, P = 0.002), blend 5 (χ2 = 11, df = 1, P = 0.001), blend 7 (χ2 = 19.755, df = 1, P < 0.001), and blend 11 (χ2 = 11.796, df = 1, P = 0.001). The other blends in this group were not significantly more attractive than the control (Table 2; Supp Table 1 [online only]). Table 2. Attractiveness of synthetic blends to the host-seeking female mosquitoes Group Blend number df χ2 PI (%) P-value A 1 1 6.90 26.67 ± 7.37 0.009 2 1 1.00 −10.00 ± 16.93 0.317 3 1 1.71 13.30 ± 8.78 0.19 4 1 9.71 30.44 ± 13.09 0.002 5 1 11.00 30.56 ± 4.97 0.001 6 1 21.59 46.67 ± 6.53 <0.001 7 1 19.76 45.11 ± 8.80 <0.001 8 1 4.00 20.00 ± 8.94 0.046 9 1 0.253 4.67 ± 6.74 0.615 10 1 8.34 29.79 ± 9.64 0.004 11 1 11.80 30.50 ± 7.24 0.001 12 1 8.50 28.89 ± 11.24 0.004 13 1 2.00 14.22 ± 5.97 0.157 14 1 0.01 1.43 ± 8.55 0.92 15 1 5.45 24.06 ± 9.62 0.02 B 16 1 0.52 7.56 ± 10.42 0.473 17 1 3.80 21.28 ± 11.71 0.051 18 1 0.04 1.78 ± 9.19 0.840 19 1 9.71 31.33 ± 7.95 0.002 20 1 1.96 14.00 ± 10.77 0.162 21 1 12.96 36.00 ± 7.18 <0.001 22 1 26.04 52.28 ± 11.03 <0.001 23 1 10.33 33.94 ± 11.29 0.001 24 1 0.01 0.44 ± 9.49 0.92 25 1 4.55 21.78 ± 13.94 0.033 26 1 9.91 31.33 ± 7.69 0.002 27 1 0.095 0.758 0.758 28 1 16.98 41.78 ± 7.45 <0.001 C 29 1 10.24 32.00 ± 8.00 0.001 30 1 0.26 5.56 ± 9.10 0.612 31 1 8.67 29.56 ± 8.91 0.003 32 1 15.04 34.07 ± 8.18 <0.001 33 1 8.50 29.56 ± 8.91 0.004 34 1 5.34 23.11 ± 4.19 0.021 35 1 4.55 23.33 ± 10.99 0.033 36 1 0.37 5.56 ± 9.01 0.544 37 1 13.22 36.67 ± 3.62 <0.001 38 1 15.68 40.00 ± 7.10 <0.001 39 1 0.04 1.78 ± 5.39 0.838 40 1 0.67 8.44 ± 7.96 0.414 D 41 1 14.11 38.56 ± 9.14 <0.001 42 1 2.98 18.33 ± 6.54 0.084 43 1 4.64 22.11 ± 8.21 0.031 44 1 4.08 20.67 ± 5.92 0.043 45 1 0.25 5.11 ± 5.92 0.615 46 1 14.74 38.67 ± 6.88 <0.001 E 47 1 0.38 5.72 ± 10.07 0.540 Group Blend number df χ2 PI (%) P-value A 1 1 6.90 26.67 ± 7.37 0.009 2 1 1.00 −10.00 ± 16.93 0.317 3 1 1.71 13.30 ± 8.78 0.19 4 1 9.71 30.44 ± 13.09 0.002 5 1 11.00 30.56 ± 4.97 0.001 6 1 21.59 46.67 ± 6.53 <0.001 7 1 19.76 45.11 ± 8.80 <0.001 8 1 4.00 20.00 ± 8.94 0.046 9 1 0.253 4.67 ± 6.74 0.615 10 1 8.34 29.79 ± 9.64 0.004 11 1 11.80 30.50 ± 7.24 0.001 12 1 8.50 28.89 ± 11.24 0.004 13 1 2.00 14.22 ± 5.97 0.157 14 1 0.01 1.43 ± 8.55 0.92 15 1 5.45 24.06 ± 9.62 0.02 B 16 1 0.52 7.56 ± 10.42 0.473 17 1 3.80 21.28 ± 11.71 0.051 18 1 0.04 1.78 ± 9.19 0.840 19 1 9.71 31.33 ± 7.95 0.002 20 1 1.96 14.00 ± 10.77 0.162 21 1 12.96 36.00 ± 7.18 <0.001 22 1 26.04 52.28 ± 11.03 <0.001 23 1 10.33 33.94 ± 11.29 0.001 24 1 0.01 0.44 ± 9.49 0.92 25 1 4.55 21.78 ± 13.94 0.033 26 1 9.91 31.33 ± 7.69 0.002 27 1 0.095 0.758 0.758 28 1 16.98 41.78 ± 7.45 <0.001 C 29 1 10.24 32.00 ± 8.00 0.001 30 1 0.26 5.56 ± 9.10 0.612 31 1 8.67 29.56 ± 8.91 0.003 32 1 15.04 34.07 ± 8.18 <0.001 33 1 8.50 29.56 ± 8.91 0.004 34 1 5.34 23.11 ± 4.19 0.021 35 1 4.55 23.33 ± 10.99 0.033 36 1 0.37 5.56 ± 9.01 0.544 37 1 13.22 36.67 ± 3.62 <0.001 38 1 15.68 40.00 ± 7.10 <0.001 39 1 0.04 1.78 ± 5.39 0.838 40 1 0.67 8.44 ± 7.96 0.414 D 41 1 14.11 38.56 ± 9.14 <0.001 42 1 2.98 18.33 ± 6.54 0.084 43 1 4.64 22.11 ± 8.21 0.031 44 1 4.08 20.67 ± 5.92 0.043 45 1 0.25 5.11 ± 5.92 0.615 46 1 14.74 38.67 ± 6.88 <0.001 E 47 1 0.38 5.72 ± 10.07 0.540 Ten female mosquitoes were used each replicate per concentration and each concentration was replicated 10 times for each blend. Total number of mosquitoes responded to each synthetic blend and control were analyzed by χ2 test. View Large Table 2. Attractiveness of synthetic blends to the host-seeking female mosquitoes Group Blend number df χ2 PI (%) P-value A 1 1 6.90 26.67 ± 7.37 0.009 2 1 1.00 −10.00 ± 16.93 0.317 3 1 1.71 13.30 ± 8.78 0.19 4 1 9.71 30.44 ± 13.09 0.002 5 1 11.00 30.56 ± 4.97 0.001 6 1 21.59 46.67 ± 6.53 <0.001 7 1 19.76 45.11 ± 8.80 <0.001 8 1 4.00 20.00 ± 8.94 0.046 9 1 0.253 4.67 ± 6.74 0.615 10 1 8.34 29.79 ± 9.64 0.004 11 1 11.80 30.50 ± 7.24 0.001 12 1 8.50 28.89 ± 11.24 0.004 13 1 2.00 14.22 ± 5.97 0.157 14 1 0.01 1.43 ± 8.55 0.92 15 1 5.45 24.06 ± 9.62 0.02 B 16 1 0.52 7.56 ± 10.42 0.473 17 1 3.80 21.28 ± 11.71 0.051 18 1 0.04 1.78 ± 9.19 0.840 19 1 9.71 31.33 ± 7.95 0.002 20 1 1.96 14.00 ± 10.77 0.162 21 1 12.96 36.00 ± 7.18 <0.001 22 1 26.04 52.28 ± 11.03 <0.001 23 1 10.33 33.94 ± 11.29 0.001 24 1 0.01 0.44 ± 9.49 0.92 25 1 4.55 21.78 ± 13.94 0.033 26 1 9.91 31.33 ± 7.69 0.002 27 1 0.095 0.758 0.758 28 1 16.98 41.78 ± 7.45 <0.001 C 29 1 10.24 32.00 ± 8.00 0.001 30 1 0.26 5.56 ± 9.10 0.612 31 1 8.67 29.56 ± 8.91 0.003 32 1 15.04 34.07 ± 8.18 <0.001 33 1 8.50 29.56 ± 8.91 0.004 34 1 5.34 23.11 ± 4.19 0.021 35 1 4.55 23.33 ± 10.99 0.033 36 1 0.37 5.56 ± 9.01 0.544 37 1 13.22 36.67 ± 3.62 <0.001 38 1 15.68 40.00 ± 7.10 <0.001 39 1 0.04 1.78 ± 5.39 0.838 40 1 0.67 8.44 ± 7.96 0.414 D 41 1 14.11 38.56 ± 9.14 <0.001 42 1 2.98 18.33 ± 6.54 0.084 43 1 4.64 22.11 ± 8.21 0.031 44 1 4.08 20.67 ± 5.92 0.043 45 1 0.25 5.11 ± 5.92 0.615 46 1 14.74 38.67 ± 6.88 <0.001 E 47 1 0.38 5.72 ± 10.07 0.540 Group Blend number df χ2 PI (%) P-value A 1 1 6.90 26.67 ± 7.37 0.009 2 1 1.00 −10.00 ± 16.93 0.317 3 1 1.71 13.30 ± 8.78 0.19 4 1 9.71 30.44 ± 13.09 0.002 5 1 11.00 30.56 ± 4.97 0.001 6 1 21.59 46.67 ± 6.53 <0.001 7 1 19.76 45.11 ± 8.80 <0.001 8 1 4.00 20.00 ± 8.94 0.046 9 1 0.253 4.67 ± 6.74 0.615 10 1 8.34 29.79 ± 9.64 0.004 11 1 11.80 30.50 ± 7.24 0.001 12 1 8.50 28.89 ± 11.24 0.004 13 1 2.00 14.22 ± 5.97 0.157 14 1 0.01 1.43 ± 8.55 0.92 15 1 5.45 24.06 ± 9.62 0.02 B 16 1 0.52 7.56 ± 10.42 0.473 17 1 3.80 21.28 ± 11.71 0.051 18 1 0.04 1.78 ± 9.19 0.840 19 1 9.71 31.33 ± 7.95 0.002 20 1 1.96 14.00 ± 10.77 0.162 21 1 12.96 36.00 ± 7.18 <0.001 22 1 26.04 52.28 ± 11.03 <0.001 23 1 10.33 33.94 ± 11.29 0.001 24 1 0.01 0.44 ± 9.49 0.92 25 1 4.55 21.78 ± 13.94 0.033 26 1 9.91 31.33 ± 7.69 0.002 27 1 0.095 0.758 0.758 28 1 16.98 41.78 ± 7.45 <0.001 C 29 1 10.24 32.00 ± 8.00 0.001 30 1 0.26 5.56 ± 9.10 0.612 31 1 8.67 29.56 ± 8.91 0.003 32 1 15.04 34.07 ± 8.18 <0.001 33 1 8.50 29.56 ± 8.91 0.004 34 1 5.34 23.11 ± 4.19 0.021 35 1 4.55 23.33 ± 10.99 0.033 36 1 0.37 5.56 ± 9.01 0.544 37 1 13.22 36.67 ± 3.62 <0.001 38 1 15.68 40.00 ± 7.10 <0.001 39 1 0.04 1.78 ± 5.39 0.838 40 1 0.67 8.44 ± 7.96 0.414 D 41 1 14.11 38.56 ± 9.14 <0.001 42 1 2.98 18.33 ± 6.54 0.084 43 1 4.64 22.11 ± 8.21 0.031 44 1 4.08 20.67 ± 5.92 0.043 45 1 0.25 5.11 ± 5.92 0.615 46 1 14.74 38.67 ± 6.88 <0.001 E 47 1 0.38 5.72 ± 10.07 0.540 Ten female mosquitoes were used each replicate per concentration and each concentration was replicated 10 times for each blend. Total number of mosquitoes responded to each synthetic blend and control were analyzed by χ2 test. View Large Figure 3. View largeDownload slide Attractiveness of the optimal blend of each group to female mosquitoes with their PIs. Group A: blends 1–15 (formulated by 2 preferred compounds), Group B: blends 16–28 (formulated by 3 compounds), Group C: blends 29–40 (formulated by four compounds) Group C: blends 41–46 (formulated by five compounds), and Group D: blend 47 (full blend). Asterisks denote significant differences in mean response to treatment versus control by chi-squared test (**P<0.01). Figure 3. View largeDownload slide Attractiveness of the optimal blend of each group to female mosquitoes with their PIs. Group A: blends 1–15 (formulated by 2 preferred compounds), Group B: blends 16–28 (formulated by 3 compounds), Group C: blends 29–40 (formulated by four compounds) Group C: blends 41–46 (formulated by five compounds), and Group D: blend 47 (full blend). Asterisks denote significant differences in mean response to treatment versus control by chi-squared test (**P<0.01). Six of the 13 synthetic blends in the Group B were significantly attractive to mosquitoes with a PI exceeding 30%. Blend 22, consisting of geranyl acetone, myristic acid and propionic acid, was the most attractive blend in this group with a PI of 52.28% (Fig. 3; Table 2; Supp Table 1 [online only]). Other significantly attractive blends in Group B included: blend 19 (χ2 = 9.707, df = 1, P = 0.002), blend 21 (χ2 = 12.960, df = 1, P < 0.001), blend 23 (χ2 = 10.333, df = 1, P = 0.001), blend 26 (χ2 = 9.907, df = 1, P = 0.002), and blend 28 (χ2 = 16.980, df = 1, P < 0.001) (Table 2; Supp Table 1 [online only]). Four of the 12 synthetic blends in the Group C were significantly attractive to mosquitoes with PIs exceeding 30%. Blend 38, a mixture of benzaldehyde, 1-octen-3-ol, hexanal, and propionic acid, showed the highest attraction in this group with a PI of 40.00% compared to the control (Fig. 3, Table 2; Supp Table 1 [online only]). Furthermore, blend 29 (χ2 = 10.240, df = 1, P = 0.001), blend 32 (χ2 = 15.042, df = 1, P < 0.001), and blend 37 (χ2 = 13.224, df = 1, P < 0.001) were also significantly attractive to mosquitoes (Table 2; Supp Table 1 [online only]). In Group D, blend 41, which consisted of geranyl acetone, benzaldehyde, 1-octen-3-ol, hexanal, and myristic acid (χ2 = 14.113, df = 1, P < 0.001) and blend 46, consisting of benzaldehyde, 1-octen-3-ol, hexanal, myristic acid, and propionic acid (χ2 = 14.735, df = 1, P < 0.001), were significantly attractive compared with the other synthetic blends in this group (Table 2; Supp Table 1 [online only]). The present data showed that the full blend, which consisted of all six attractive compounds of propionic acid, hexanal, myristic acid, benzaldehyde, 1-octen-3-ol, and geranyl acetone, was not significantly attractive to female mosquitoes compared with the control (χ2 = 0.375, df = 1, P = 0.540, Fig. 3; Table 2; Supp Table 1 [online only]). The Attractiveness of Odor-Baited Traps in Field Experiments In the field evaluation, 18 synthetic blends were tested, and a total of 363 mosquitoes were collected in 64 traps during a single night of trapping. The collections included 298 Cx. pipiens and 65 Aedes albopictus. The results indicate that traps containing blend 28 captured the highest number of female Cx. pipiens, with a mean of 11.00 ± 2.08 mosquitoes/trap. Traps containing blends 4, 22, 26, and 37 were moderately attractive to Cx. pipiens compared with the control mixture of corn and water. Traps containing the remaining blends did not capture significantly more mosquitoes than the control traps (Table 3). Discussion Host odors are important cues used by female mosquitoes to locate potential vertebrate hosts that may serve as a source of blood (Takken and Knols 1999, Takken and Verhulst 2013). The identification of these host odors has been a research focus for decades (Kline et al. 1990, Smallegange et al. 2005). The variety of preferred hosts and the blood feeding sites on those hosts to different mosquito species indicates that individual attractant compounds may play key roles in attracting mosquitoes (Mukabana et al. 2002, Torr et al. 2008, Verhulst et al. 2016). Multiple synthetic blends have been formulated as baits to attract different mosquito species (Bosch et al. 2000, Lyimo et al. 2013). Instead of using general chemical attractants isolated from individual hosts to formulate synthetic blends, previous studies indicated that specific chemical groups, such as ketones and aldehydes, play an important role in attracting mosquitoes (Okumu et al. 2010). Attractant-baited traps for mosquito surveillance and control are presently not widely used in developing countries. However, it is anticipated that this will change as highly efficacious attractants are developed, tested, and brought to market and that these attractants are expected to be more acceptable for use because of their safety and simplicity by communities in disease endemic regions (Silva et al. 2005, Mathew et al. 2013). Therefore, identification of compounds and blends that are attractive to mosquitoes will benefit vector control and public health programs around the globe. In this study, we examined the attractiveness of 11 individual compounds present in the human skin. Some of the compounds were previously reported to attract other mosquito species including Ae. aegypti, Anopheles gambiae, and Cx. quinquefasciatus (Bosch et al. 2000, Puri et al. 2006, Qualls and Mullen 2007). Six of the compounds (propionic acid, hexanal, myristic acid, benzaldehyde, 1-octen-3-ol, and geranyl acetone) were significantly attractive to female Cx. pipiens pallens to varying degrees with PIs exceeding 30% (Table 1). However, butyric acid, p-cresol, and n-octanoic acid were only slightly (PI < 30%) attractive to Cx. pipiens pallens when tested individually in the laboratory. In our study, we found that heptanoic acid was not attractive at low concentrations and showed repellent activity at high concentrations (Supp Fig. 1 [online only]). Heptanoic acid is a common compound found in human skin emanations and it elicited strong electrophysiological responses in female Cx. quinquefasciatus showing attraction at a concentration of 20 μg/ml (Puri et al. 2006) (Supp Fig. 1 [online only]). This discrepancy indicates that the overall attraction of specific compounds may vary between different mosquito species. In addition, our study showed that low concentrations of 6-methyl-5-hepten-2-one did not attract Cx. pipiens pallens, and was repellent at high concentrations (Supp Fig. 1 [online only]). In previous studies, 6-methyl-5-hepten-2-one was detected in both the fresh and incubated headspace of human sweat samples, and that 1 and 10% solutions elicited an electrophysiological response in female An. gambiae (Meijerink et al. 2000). However, our results are similar to the findings of Logan et al. (2008), which showed that 6-methyl-5-hepten-2-one significantly interfered in host-location by Ae. aegypti. However, the strong attraction of Cx. pipiens pallens to our other test compounds, such as propionic acid, 1-octen-3-ol, and geranyl acetone, confirms the findings of previous studies (Kline et al. 1990, Cook et al. 2011, Mathew et al. 2013). It has been proposed that blends of host odors are more attractive to mosquitoes than the individual compounds (Bosch et al. 2000). Thus, we hypothesized that all the blends formulated by using the six most attractive compounds should be attractive to mosquitoes and elicit greater attraction than the individual compounds by themselves. However, only 28 of the 47 blends were attractive to mosquitoes in laboratory tests, whereas only 18 of these blends had PI values exceeding 30% (Table 2). Blend 46, which consisted of five active compounds (benzaldehyde, 1-octen-3-ol, hexanal, myristic acid, and propionic acid), was strongly attractive to mosquitoes, supporting our hypothesis (Table 2). However, the full blend, formulated by adding geranyl acetone to blend 46, showed no significant attractiveness to mosquitoes (Table 2). A similar study by Mukabana et al. (2012), documented that the number of An. gambiae responding to a standard blend consisting of ammonia, (S)-lactic acid, tetradecanoic acid, and carbon dioxide was significantly reduced when three additional active compounds (isovaleric acid, 4,5-dimethylthiazole, and 3-methyl-1-butanol) were added. These data demonstrate that increasing the number of attractants in a blend does not necessarily enhance the effectiveness of that blend. Thus in some cases, there appears to be a competitive interplay between the individual chemicals that constitute attractant blends. The normal attractiveness of some chemicals in the blend is cancelled by the presence of other chemicals (Mukabana et al. 2012). Attractant-baited traps are important tools for mosquito surveillance programs. These include the Fay-Prince trap, the carbon dioxide-baited Centers for Disease Control trap, and the counter flow geometry trap (Silva et al. 2005, Williams et al. 2006). Attractant-baited traps are important for surveillance, but by themselves, these types of traps do not significantly impact mosquito populations and do provide an efficacious mosquito control strategy (Kline 2006). Human landing counts and landing collections are regarded as an efficient mosquito surveillance protocol, especially for species that are not attracted to traps (Williams et al. 2006, Owino et al. 2014). However, the use of human bait presents severe ethical issues. Some chemical baits and blends of chemicals can increase the attraction of mosquitoes to traps and are nearly as efficacious as human hosts (Mukabana et al. 2012). Thus, there is a continued need to find new and more efficacious chemical attractants for mosquito surveillance programs (Williams et al. 2006, Okumu et al. 2010). Our experiments reported here indicate that the effectiveness of synthetic blends differed between laboratory and field conditions. In addition to Cx. pipiens, the odor-baited traps in this study also captured Ae. albopictus. Both of these species are common in China (Peng et al. 2012, Jiang et al. 2014). The compounds and synthetic blends described in this study can be used as mosquito attractants under both laboratory and field conditions. Positive and negative chemical interactions may exist among some of these chemical compounds when they are combined. The most effective blends reported here consisted of mixtures of two to four compounds. Supplementary Data Supplementary data are available at Journal of Medical Entomology online. Acknowledgments We would like to thank Jinli Z for his help in making the figures and Lin C for his suggestions for the improvement the manuscript. 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TI - Laboratory and Field Evaluation of Multiple Compound Attractants to Culex pipiens pallens
JF - Journal of Medical Entomology
DO - 10.1093/jme/tjy015
DA - 2018-03-16
UR - https://www.deepdyve.com/lp/oxford-university-press/laboratory-and-field-evaluation-of-multiple-compound-attractants-to-zP9QhtSBzu
SP - 1
EP - 794
VL - Advance Article
IS - 4
DP - DeepDyve
ER -