Orientation Of Culex Mosquitoes To Carbon Dioxide

Medical and Veterinary Entomology (2006) 20, 11–26

Orientation of Culex mosquitoes to carbon dioxidebaited traps: flight manoeuvres and trapping efficiency M . F . C O O P E R B A N D and R . T . C A R D E´ Department of Entomology, University of California, Riverside, U.S.A. Abstract. Females of Culex quinquefasciatus Say and Culex tarsalis Coquillet (Diptera: Culicidae) in the host-seeking stage were released and video recorded in three dimensions in a large field wind tunnel as they flew to four kinds of CO2-baited mosquito traps. The trapping efficiency (number of mosquitoes approaching compared to the number caught) was determined for each trap type. The Encephalitis Virus Surveillance (EVS), Mosquito Magnet Freedom (MMF) and Mosquito Magnet Liberty (MML) traps captured only 13–16% of approaching Cx. quinquefasciatus females, whereas the Mosquito Magnet-X (MMX) trap captured 58%. Similar results were obtained for Cx. tarsalis. Orientation behaviour and flight parameters of mosquitoes approaching the four traps were compared. Mosquitoes spent the most time orienting to the EVS trap. Flight speed decreased as mosquitoes entered the vicinity of each trap and a large portion of their time was spent within 30 cm downwind of the traps. Flights became highly tortuous downwind of the poorly performing traps and just upwind of the MMX trap. Differences between traps and possible explanations for the superior performance of the MMX trap are considered. Key words. Culex quinquefasciatus, Culex tarsalis, anemotaxis, carbon dioxide-

baited traps, host odours, trapping efficiency.

Introduction Female mosquitoes detect changes in CO2 concentration as minute as 50 p.p.m. via sensilla on their maxillary palps (Grant & O’Connell, 1996). When a host-seeking female encounters a plume of CO2, she orients upwind using optomotor anemotaxis (Kennedy, 1939; Daykin et al., 1965). The structure of the plumes of CO2 and other host-odour kairomones plays an important role in the attraction of Aedes aegypti (L.) mosquitoes. In a Y-tube olfactometer, orientation behaviour of Ae. aegypti varied with plume structure and odour, with a filamentous presentation of CO2 inducing improved upwind movement over a homogenous cloud of CO2 (Geier et al., 1999). In a dual choice flight chamber, fewer Ae. aegypti females entered ports emitting a homogenous CO2 plume than a turbulent plume, whereas more females entered ports emitting a homogenous plume of human skin odour than a turbulent

plume (Dekker et al., 2001). Dekker et al. (2005) also found that females were induced in a wind-tunnel assay to surge upwind by entering a concentration of CO2 as little as 0.5% above ambient, and that following a brief encounter with a single filament of CO2, mosquitoes were sensitized to fly upwind to lower concentrations of human skin odour than without such prior CO2 exposure. Public health and vector control programs designed to predict and control disease epidemics rely in part upon early detection of infected mosquitoes. This requires sampling of mosquito populations to detect population surges, delimit their distribution and establish the presence of the infected mosquitoes. Important tools in such monitoring programs are CO2-baited mosquito traps (Kline, 1999; Rueda et al., 2001; reviewed in Service, 1993). These traps have also been suggested as possible tools in mosquito control (Curtis, 1996). The release of CO2 by these traps attracts female mosquitoes in the host-seeking stage.

Correspondence: Miriam F. Cooperband, Entomology, University of California, Riverside, CA 92521, U.S.A. Tel.: þ 1 951 8274492; fax: þ 1 951 8273681; e-mail: [email protected] # 2006 The Authors Journal compilation # 2006 The Royal Entomological Society

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12 M. F. Cooperband and R. T. Carde´ Adding 1-octen-3-ol (octenol), an odour from ox breath to which tsetse flies are attracted (Vale & Hall, 1985; Takken & Kline, 1989; Kline et al., 1990; Kemme et al., 1993; Kline, 1994), or heat (Kline & Lemire, 1995; Makiya & Iwao, 2001), can improve trap capture for some mosquito species. CO2-baited traps have been compared under a wide variety of field conditions to evaluate the roles various configurations and odour release characteristics could play on trap capture (Hayes et al., 1958; Reisen & Pfuntner, 1987; Cummings & Meyer, 1999; Kline, 1999, 2002; Mboera et al., 2000; Reisen et al., 2000; Burkett et al., 2001). These studies generally have compared the numbers of different species of mosquitoes captured by different trap types using various combinations of visual or chemical cues such as light, CO2 and octenol. Because most of these studies were carried out in the field, there is no information on the trapping efficiency, that is, what proportion of mosquitoes lured to the vicinity of a given trap is actually captured. Vale & Hargrove (1979) addressed the question of field trapping efficiency for tsetse flies using electric nets (presumed invisible to the flies) strategically placed around a given trap configuration. This innovative method enabled comparison of the number of flies arriving in the vicinity of the trap with the number actually captured. Studies to determine trap efficiency with mosquitoes have released set numbers into a large, outdoor cage, in which a trap was operated from 90 min before dusk to 90 min after dawn (Kline, 1999, 2002). Because they deal with a constant number of mosquitoes, they provide useful comparisons of the relative success of traps. Such studies, however, cannot estimate trapping efficiency, because a mosquito enclosed within the cage for approximately 12 h will have multiple opportunities to approach a trap, whereas in the field a mosquito that approaches a trap but is not caught may subsequently leave its vicinity. In such instances, the reason approaching mosquitoes are not captured would be unknown. Trapping efficiency can be determined by examining the proportion of mosquitoes approaching traps that are actually caught. In our previous study (Cooperband & Carde´, 2006), we examined the differences between trap designs, CO2 plumes and other potential cues produced by the same four traps. In this study, Cx. quinquefasciatus and Cx. tarsalis are used to compare trapping efficiency and to examine the flight manoeuvres of female mosquitoes as they orientate upwind along the CO2 plumes emanating from these traps.

Materials and methods Wind tunnel Experiments were conducted in a field wind tunnel (Cooperband & Carde´, 2006), similar to the ‘pushing’ wind tunnel described by Carde´ et al. (1998). The steel frame of the wind tunnel was 2.5 m wide
1.85 m high
6.2 m long, and was covered with a translucent polyethylene plastic sheet which formed the ceiling and walls and

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had vertical zipper doorways on the sides near the middle and the upwind end. Air entering and exiting the wind tunnel passed through two screens made of HexcelÒ (aluminium sheet of hexagonal cells 5 cm thick with 1 cm I.D.) covered with black nylon insect screening to reduce turbulence. A fan (60 cm diameter blades) on a metal stand 0.8 m above the ground mixed and pushed outside air through the tunnel via a polyethylene pre-chamber which expanded over a distance of 2.5 m from the fan to the upwind laminizer. Except for the frame, which was fixed in the ground, the wind tunnel was assembled before each evening of experiments and at the end of experiments was dismantled and stored. Latex gloves were worn while setting up, running experiments and dismantling, and care was taken to avoid handling with bare skin anything to which mosquitoes would be exposed. Prior to set-up, the earth floor of the wind tunnel was cleared of any plants, twigs, rocks, leaves or other obvious visual cues to ensure similar conditions from day to day. Other large visual cues that would allow optomotor anemotaxis to take place remained, such as the wind tunnel frame, the video camera and tripod, the IR lighting, the laminizer screens, large stationary objects outside of the wind tunnel, and the traps themselves. A hot wire anemometer was used to measure the wind speed, which was adjusted to 50 cm/s.

Traps Four CO2-baited mosquito traps (see Cooperband & Carde´, 2006) were compared: encephalitis virus surveillance (EVS) trap, Mosquito MagnetÒ Freedom (MMF), Mosquito MagnetÒ Liberty (MML) and the Mosquito MagnetÒ X (MMX). The EVS trap consisted of an insulated plastic bucket (17 cm diameter by 20 cm high) to house dry ice with four 0.5 cm holes about 1 cm from the bottom to release sublimated CO2 (Rohe & Fall, 1979). Hanging below the bucket, three 1.5 V batteries powering the trap fan rested on a circular disc, attached 16 mm above the opening of a cylinder (9.5 cm high
11.5 cm diameter) which housed the fan. The battery-operated fan pulled mosquitoes through the 16mm opening into a net at the bottom of the cylinder. The CO2 outlet holes on the bucket were 20 cm above the trap entrance and the bucket contained 1.8 kg of dry ice. The MMF and MML were designed by American Biophysics Corp. (ABC) (North Kingstown, RI, U.S.A.). They utilize two fans, or ‘counter-flow technology’, in which one fan exhausts odours out the bottom outlet, while the other fan draws air from the bottom inlet and forces it out at the top, causing a suction at the bottom inlet that pulls in mosquitoes. Both traps rest on a stand on the ground and run on propane, which produces energy for the fans and releases CO2, heat and humidity as a by-product of combustion. The release of heat has been suggested as a way to improve trap efficacy (Kline & Lemire, 1995; Makiya & Iwao, 2001). The CO2 outlet is 10 cm below the trap entrance in all the Mosquito MagnetÒ traps. In

# 2006 The Authors 2006 The Royal Entomological Society, Medical and Veterinary Entomology, 20, 11–26

Orientation of Culex mosquitoes to traps the MMF and MML, heated, humidified CO2 is pushed out through that outlet. These traps differ slightly in size, shape, inlet and outlet airflow, CO2 concentration, heat, humidity and plume structure (see Cooperband & Carde´, 2006). The MMX counterflow geometry trap was designed by ABC for research purposes and is not available commercially. It was first described by Kline (1999) and was found to capture more mosquitoes in the field than other traps with similar designs (Kline, 2002). The fans were powered by a 12 V battery and 100% CO2 was provided by a pressurized cylinder releasing CO2 at 500 mL/min, which emulates the output of the above MMF trap (Alan Grant, pers. comm.). Air is pulled into the bottom of the trap, into a clear plastic jar (about 11.4 L), through a screen and pushed out through the top of the jar. Mosquitoes that are pulled in collect in the jar. Because this trap has a counterflow design similar to the MMF, except that it releases CO2 without heat or humidity, initially it was chosen to examine the difference between the presence and absence of these additional cues. Inserts containing octenol can be used with all three of the ABC traps and have been found to improve trap capture for some species of mosquitoes (Kemme et al., 1993). However, we wished to compare traps releasing only CO2. Moreover, addition of octenol to CO2 generally does not improve trap catch of ornithophagous species, including several Culex species (Gibson & Torre, 1999). Furthermore, octenol added to CO2-baited traps reduced capture of Culex pipiens pipiens L. (Burkett et al., 2001) and Cx. quinquefasciatus (Russell, 2004). To facilitate three-dimensional (3-D) video recording, pressurized tanks were placed outside of the wind tunnel for the MMF and MMX traps and against the inside wall of the wind tunnel for the MML trap. Analysis of the plume structures of CO2 and discussion of other physical differences between these four traps can be found in Cooperband & Carde´ (2006).

Insects A colony of anautogenous Cx. tarsalis was maintained in Boyden Laboratory at University of California Riverside at LD 16 : 8 h, 25 C and about 65% relative humidity. Eggs were collected and placed in plastic water pans, and alfalfa pellets for rabbits were added to the water as larval diet. Fourth instar larvae of Cx. quinquefasciatus were obtained weekly from the Walton Laboratory at University of California Riverside, where they were reared at LD 16 : 8 h, at about 26 C, with 50% relative humidity (see Georghiou & Wirth, 1997) and a larval diet consisting of one part brewer’s yeast to three parts ground mouse chow. Pupae of both species were collected daily and allowed to emerge in screened cages (30
30
30 cm) with access to 25% honey solution and water. Colonies were maintained by weekly blood feeding on live chicks, but mosquitoes used in experiments had no prior blood-feeding experience. In preliminary experiments it was determined that only 2%

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and 9% of 3-day-old Cx. tarsalis and Cx. quinquefasciatus females, respectively, flew upwind to EVS traps in the wind tunnel, whereas 75% and 93% of 7–10-day-old females flew upwind. Therefore, anautogenous female Cx. tarsalis or Cx. quinquefasciatus, about 10 days post-eclosion, were used. To allow mating, females were housed with males from eclosion until experiments.

Experimental set-up About 8–10 h prior to testing, mosquitoes were deprived of honey solution and water. About 1–2 h prior to experiments, females were aspirated into release cages made of PlexiglasÒ cylinders (5 cm
7 cm diameter) with mesh on one end and a slit near the open end into which a paper cover was inserted. Because most host-seeking behaviour by these species occurs soon after dusk (Meyer et al., 1985, 1986), they were allowed to acclimatize in their release cages outside in the shade at dusk, during which time the wind tunnel was constructed for that evening and recording equipment was set up. Mosquito releases began following sunset and experiments were conducted over the next 1–2 h. Only one species and one trap type were tested per night. A covered release cage containing four to six mosquitoes was placed in a 1 cm-wide rubber band stapled to the end of a 1.2 m-long wooden rod (1.5 cm diameter). It was then extended just inside the door of the wind tunnel with the mesh screen facing into the wind and held there for about 60 s to allow mosquitoes to rest on the mesh. The paper on the downwind end of the release cage then was removed gradually and the cage was slowly extended to the middle of the wind tunnel, keeping the same orientation. The cage was held for about 10 s in the centre of the CO2 plume and then slowly turned until the open end rotated to face upwind, at which point the mosquitoes flew out. The cage was held in place until the mosquitoes completed their flights upwind (to avoid making any movements that might interfere with their behaviour), after which it was removed and a new cage was placed in the rubber band.

Recording flights Each trap was set up in the centre of the upwind end of the wind tunnel, approximately 30 cm from the upwind screen, and mosquitoes were released 55 cm above the ground and 270 cm downwind of the traps. The mosquitoes were video-recorded using Sony Hi8 (EVO-550H) recorders and two Sanyo black and white video cameras (VCB3512T), with a shutter speed of 1/60 s and 6-mm lenses that were aimed from two different angles at mosquitoes as they flew upwind toward the traps. The two cameras were synchronized using an Event & Video Control Unit (Peak Performance Technologies Inc., Englewood, CO, U.S.A.). One video camera was always aimed from the side of the tunnel toward the trap, showing the XZ plane.

# 2006 The Authors Journal compilation # 2006 The Royal Entomological Society, Medical and Veterinary Entomology, 20, 11–26

14 M. F. Cooperband and R. T. Carde´ The other video camera was aimed about 90 from the first, and because the height of the trap being recorded varied, the second camera was positioned either on the ceiling of the wind tunnel (XY), in a hole in the ground below the trap (XY) or downwind near the release position (YZ), to obtain a clear view of mosquitoes approaching the traps. The two video cameras were aimed at the trap and the area directly downwind, capturing about the last 150 cm of the approach to the trap. Three Tracksys Ltd. (Nottingham, U.K.) Infrared LED Illuminator light sources (arrays of 90 LEDs with 880 nm peak output) were aimed upwind at the trap from behind the release point to illuminate the mosquitoes. A calibration object with 20 fixed 3-D coordinates was placed in the area just downwind of the trap entrance and video recorded prior to releasing the first mosquito. The coordinates provided by this object were later digitized and these allowed the software to calculate accurate 3-D coordinates of mosquitoes flying through the same space.

Environmental conditions Experiments were conducted at the edge of a wooded area of a University of California Riverside experiment station between May and November of 2000, 2001 and 2002. The average temperature at the start of data collection was 24.6 C, with a range between 20.6 and 28.1 C. The average relative humidity was 63.9%, ranging from 22 to 87%. The average barometric pressure was 760.2 mmHg, ranging between 757.4 and 763.5 mmHg.

Track discrimination Not all mosquito flights observed on the video monitor satisfied requirements for analysis. To be considered, the flight had to start downwind and end upwind, and be largely visible on both video records. Upwind flights were defined as those that started approximately 60 cm or more downwind of the trap and progressed to within 30 cm of the trap or closer. Tracks in which the mosquito flew from the upwind end of the tunnel to the downwind end were excluded. Similarly, tracks in which the mosquito reached the trap but the approach did not start far enough downwind were discarded. Of the tracks meeting the above requirements, not all were recorded with sufficient clarity for analysis. Because two cameras were used from different angles, on occasion a mosquito was visible in one camera, but behind the trap or out of view in the other camera. Because no.3-D coordinates could be calculated for those frames, this would appear as a gap when the final 3-D track was calculated by the computer program MOTUS (Peak Performance Technologies, Inc., Centennial, CO, U.S.A.). If more than 30% of all frames of a track were gaps, that track was excluded from further analysis. In the remaining tracks, the ‘interpolate gaps’ function of MOTUS was used to correct for missing coordinates. Following this procedure, the

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tracks were drawn by MOTUS and visually inspected and compared to the tracks prior to interpolation. If a large section of adjacent frames was missing in a given track, it sometimes resulted in an evident distortion of the flight path by the interpolation procedure, in which case such a track was also removed from the analysis.

Analysis of capture rates Each night of testing was considered a replicate for a given trap and species. Three sets of numbers for statistical comparisons for each trap and species were calculated: number of flights per number released, number caught (þ0.5) per number released and number caught (þ0.5) per number of flights. These ratios were square root arcsin transformed. A few instances occurred where the ratio of flights to number of mosquitoes released was greater than 1, in which case they were adjusted to 1 to permit the transformation. These means were compared using two-tailed t-tests with unequal variance (Microsoft Excel, 2002 SP3).

Three-dimensional analysis of flight tracks For each track, the resulting pairs of two-dimensonal (2-D) flight tracks from two views were entered as digital coordinates into MOTUS, which was then used to calculate the 3-D flight path taken by each mosquito. The 3-D flight coordinates were entered into an in-house computer program (TRACK 3-D, version 2.2.6, written by Josep Bau), which calculated a variety of flight parameters in one, two or three dimensions, such as: X speed (along wind velocity), Y speed (across wind velocity), Z speed (vertical velocity), track angle (XY), course angle (XY and 3-D), drift angle (XY and 3-D), inclination angle (XZ), front angle (YZ), ground speed (XY), airspeed (XY and 3-D), time elapsed, flight angle (3-D), flight speed (3-D), vertical position (Z) and tortuosity (XY, XZ, YZ and 3-D). Figures 1 and 2 provide diagrams of some 2-D and 3-D flight parameters. Track angle (XY), inclination angle (XZ) and front angle (YZ) represent the angle of movement of the mosquito in the three respective planes. The flight angle is the angle of movement of the mosquito in three dimensions (calculated

Fig. 1. The triangle of velocities as defined by Marsh et al. (1978) looking down from above a mosquito (in the XY plane).

# 2006 The Authors 2006 The Royal Entomological Society, Medical and Veterinary Entomology, 20, 11–26

Orientation of Culex mosquitoes to traps

Fig. 2. Definitions of angles and vectors used to calculate threedimensional flight parameters with the mosquito moving from the box at point 0 to point 1 (flight vector). Shadows indicate the mosquito’s location in the three planes.

using the flight vector). Drift angle is the angle of displacement of the mosquito between its heading and its actual trajectory as a result of the wind speed and direction. Tortuosity is a measure of path straightness and, when an organism is orienting toward a ‘goal’, indicates the level of efficiency of the orientation mechanism being used (Benhamou, 2004). In this study, tortuosity was calculated by dividing the distance travelled between the starting point and the end point by the straight distance between those two points. The tortuosity index, therefore, is always  1, and the higher the value, the more turning occurred.

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Flights tracks were divided into 10-cm increment bins along the X-axis, starting at the downwind edge of the trap, and flight parameters for each track were calculated for each bin. For each species, flight parameters were compared by trap. Flight parameters were also examined for each species and trap to look for differences between mosquitoes that were caught or not caught. Track durations for flight tracks of each trap and species were compared by date using the Games–Howell post hoc analysis of variance with P ¼ 0.05 (Games & Howell, 1976). This test was selected because it does not assume equal sample sizes or equal variances. No significant differences were found between dates for each trap and species, and so dates within each trap and species were combined and individual tracks were used as replicates. Means of flight parameters per bin were quantified for individual mosquitoes, grouped by species and by trap, and compared and tested for significance using the Games–Howell post hoc analysis of variance with a P-value of 0.05. Additionally, flight tracks of Cx. quinquefasciatus that were caught were compared to flight tracks of those that were not caught for each trap and bin using the Games– Howell post hoc analysis of variance with a P-value of 0.05. Complete track durations of Cx. quinquefasciatus mosquitoes that were caught were compared to those that were not caught using a Mann–Whitney test (P ¼ 0.05) for each trap.

Results Trap capture Table 1 provides numbers of mosquitoes released and number of release nights per trap and species (nine release nights were discarded due to technical problems).

Table 1. Proportions of female (a) Culex quinquefasciatus and (b) Culex tarsalis released, flying upwind and captured for four CO2-baited traps. Numbers in columns followed by the same letters are not significantly different from each other using two-tailed t-tests with unequal variance (P < 0.05). (a) Culex quinquefasciatus

EVS MMF MML MMX

Flights/releases

Captures/flights

Captures/releases

n mosquitoes released

N release nights

0.47a 0.44a 0.67a 0.54a

0.13a 0.16a 0.14a 0.58b

0.04a 0.05a 0.09a 0.26b

320 423 330 377

4 6 5 4

Flights/releases

Captures/flights

Captures/releases

n mosquitoes released

N release nights

0.75a 0.48a – 0.24

0.08a 0.37a – 0.71

0.06a 0.18a – 0.17

79 152 – 58

4 2 – 1

(b) Culex tarsalis

EVS MMF MML MMX

EVS, Encephalitis Virus Surveillance trap; MMF, Mosquito Magnet Freedom trap; MML, Mosquito Magnet Liberty trap; MMX, Mosquito Magnet-X trap.

# 2006 The Authors Journal compilation # 2006 The Royal Entomological Society, Medical and Veterinary Entomology, 20, 11–26

16 M. F. Cooperband and R. T. Carde´ Table 2. The number of tracks of each treatment used for the TRACK 3-D analysis of flight parameters.

with this species; the MMX trap was only tested on one evening, and so it was not included in the trap-capture comparison.

Trap Species

Caught?

EVS

MMF

MML

MMX

Culex quinquefasciatus

No Yes No Yes

72 3 42 –

42 3 19 4

61 7 – –

50 11 7 3

Culex tarsalis

EVS, Encephalitis Virus Surveillance trap; MMF, Mosquito Magnet Freedom trap; MML, Mosquito Magnet Liberty trap; MMX, Mosquito Magnet-X trap.

For each species and trap, the proportion of female mosquitoes that flew upwind and were caught are also indicated. Although approximately 50% of the mosquitoes released flew upwind toward the four trap types, only a small fraction of mosquitoes that were released, or of those that flew upwind, were captured. The MMX trap had the highest capture rate. For Cx. quinquefasciatus, 13–16% of upwind flights resulted in capture for the EVS, MMF and MML traps, whereas 58% of flights to the MMX trap resulted in capture (Table 1). For Cx. tarsalis, the percentage of upwind flights resulting in capture did not differ between the EVS and MMF traps. The MML trap was not tested

Number of tracks used Of 924 flights recorded (some mosquitoes returned to the release area and flew upwind again), 536 satisfied the digitizing criteria (where the mosquito started and finished and was visible in both camera views), and of those digitized, 324 were of sufficient clarity (< 30% gaps and tracks not distorted) to analyse for flight parameters. The number of tracks used in each treatment for 3-D flight track analysis is presented in Table 2. None of the flight tracks of Cx. tarsalis resulting in capture by the EVS trap satisfied the criteria for 3-D analysis. Because tracks differed in length and each started and ended at different distances from the trap, each bin contained a different number of tracks. The numbers of flight tracks per bin for each species and trap are given in Fig. 3. Track duration On average, both Cx. quinquefasciatus and Cx. tarsalis spent more time orienting to the EVS trap than to

Fig. 3. Number (N) of mosquito flight tracks per bin (distance downwind of trap over 10-cm increments) for (a) Culex quinquefasciatus and (b) Culex tarsalis used in analysis of flight parameters for different trap types. EVS, Encephalitis Virus Surveillance; MMF, Mosquito Magnet Freedom; MML, Mosquito Magnet Liberty; MMX, Mosquito Magnet-X.

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# 2006 The Authors 2006 The Royal Entomological Society, Medical and Veterinary Entomology, 20, 11–26

Orientation of Culex mosquitoes to traps Average Durations of Flights To Different Traps

30

a

a

20 Time (s)

Fig. 4. Average number of seconds (þ SE) mosquitoes spent in upwind flights to different traps for Culex quinquefasciatus and Culex tarsalis. Numbers under bars are the number of tracks analysed for each trap and species. Different letters above bars represent significant differences between traps for that species using the Games–Howell test (P < 0.05) (N.T. ¼ not tested). EVS, Encephalitis Virus Surveillance; MMF, Mosquito Magnet Freedom; MML, Mosquito Magnet Liberty; MMX, Mosquito Magnet-X.

b b

b

10

b c

0

N=

45 68 61 74 EVS MMF MML MMX Cx. quinquefasciatus

the MMF, MML and MMX traps (Fig. 4). For MMF and MML traps, tracks of Cx. quinquefasciatus females that were caught were significantly longer in duration than females that were not caught (Fig. 5). Similar trends were noted for EVS and MMX traps (Cooperband, 2005).

Significance of parameters Tracks of mosquitoes flying to different traps may or may not differ significantly, depending on the parameter considered. Additionally, significant differences detected between traps may be detected at one distance downwind of the trap but not at another. For each bin downwind of the traps, the parameters for which significant differences were found between traps are presented in Tables 3 and 4. Orientation behaviour did not appear to differ between traps at distances greater than about 100 cm downwind of the traps. For Cx. quinquefasciatus, the number of significantly different parameters was

42 EVS

23 N.T. 10 MMF MML MMX Cx. tarsalis

greatest between the trap and 30 cm downwind of the trap, and between 80 and 90 cm downwind of the traps. For Cx. tarsalis, the number of significantly different parameters was greatest between 20 and 30 cm downwind of the traps. As the mosquito approached, some parameters had more significantly different bins between traps than others. Likewise, the number of significantly different parameters between traps in each bin varied as well. For both species, parameters with the most bins containing significant trap differences were vertical position and speeds involving the X-axis (flight speed, ground speed, X speed, airspeed and 3-D airspeed). Fewer significant differences were seen in parameters such as time elapsed and angles associated with the XY plane (drift angle, 3-D drift angle, track angle, flight angle and 3-D course angle). The fewest differences were found in parameters involving the Z axis (Z speed, inclination angle and front angle) and tortuosity. Diagrams of parameter differences not shown here can be found in Cooperband (2005). Average Duration of Cx. quinquefasciatus Complete Flight Tracks to Traps

60

Caught

Track Duration (s)

Not caught

Fig. 5. Track durations for Culex quinquefasciatus mosquitoes that were caught compared to those that were not caught for individual traps. Significant differences are indicated with asterisks using Mann–Whitney test (P < 0.05). EVS, Encephalitis Virus Surveillance; MMF, Mosquito Magnet Freedom; MML, Mosquito Magnet Liberty; MMX, Mosquito Magnet-X.

17

*

40

*

20

0 EVS

MMF

MML

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MMX

18 M. F. Cooperband and R. T. Carde´ Table 3. Significant differences for various flight parameters as female Culex quinquefasciatus mosquitoes orientated to different traps. Traps are represented by the letters e (EVS), f (MMF), l (MML) and x (MMX). Data in columns are aligned between column headings, indicating where the bin measurements were taken. Dashes separate significantly different traps for the particular flight parameter and distance downwind of the trap using the Games–Howell test (P < 0.05), with significantly lower values on the left-hand side and higher values on the right-hand side of each dash. When no significant differences were detected between any of the traps, the space was left blank. Distances relate to the downwind edge of the trap entrance, and parameters for that column were calculated for each 10-cm increment downwind from that point. Culex quinquefasciatus Distance downwind of trap (cm) Parameter Flight speed (3-D) Ground speed (XY) X speed Airspeed (XY) 3-D airspeed 3-D drift angle Drift angle (XY) Time elapsed Track angle (XY) Flight angle (3-D) 3-D course angle Course angle (XY) Y speed Vertical position Z speed Inclination angle (XZ) Front angle (YZ)

150 140 130 120 110 x-l

100 90

x-ef

e-l e-lf e-l

80

70

f-l f-l f-l

f-lx ef-l f-lx f-lx; e-l ef-l ef-l f-lx ef-l f-l f-lx ef-l f-l f-lx ef-l f-l

l-x l-x

l-f l-f

60

50

l-ex; f-x l-ex l-x

30

20

ef-l; f-x ef-lx ef-lx e-flx ef-x ef-l ef-lx ef-lx e-flx e-fx; f-x ef-l ef-lx ef-lx e-flx e-flx; f-x ef-l ef-lx ef-lx e-flx e-flx; f-x ef-l ef-lx ef-lx e-flx e-flx; f-x x-e x-e f-e

x-f x-f

40

x-e

flx-e flx-e flx-e flx-e flx-e flx-e x-e fx-e fx-e x-l ex-l e-l x-l x-l

10

0

10 20

e-flx e-lx e-flx e-lx e-flx e-flx e-flx flx-e flx-e lx-e x-l x-l

x-f

el-x l-f f-e f-e f-el

3-D tortuosity XY tortuosity XZ tortuosity YZ tortuosity

l-efx e-lx x-e

e-lx e-x e-x

flx-e flx-e flx-e flx-e flx-e flx-e fl-e

EVS, Encephalitis Virus Surveillance trap; MMF, Mosquito Magnet Freedom trap; MML, Mosquito Magnet Liberty trap; MMX, Mosquito Magnet-X trap.

Velocity Mosquitoes reduced their speed as they approached traps. Several patterns were apparent when considering the number of parameters with significant differences at various distances downwind of the traps. At the farthest downwind distances, few parameters differed between traps for either species (Tables 3 and 4). Starting at 90 or 100 cm downwind of the traps, Cx. quinquefasciatus flew significantly faster to the MML trap than the MMF trap, and this continued until 20–30 cm downwind, where they both slowed to similar velocities. Flight speed, ground speed, 3-D airspeed and X speed were similar (Cooperband, 2005), and so only X speed is presented for Cx. quinquefasciatus and Cx. tarsalis (Fig. 6). As mosquitoes flew from 50 to 30 cm downwind of traps, they flew significantly slower along the X-axis to the EVS and MMF traps than to the MML or MMX traps, as indicated for all speed parameters involving the X-axis (Tables 3 and 4). Y speed and Z speed have few significant differences at different distances from traps (Tables 3 and 4). For all four

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traps, mosquitoes did not vary their speed along the Y-axis, but speed along the Z-axis increased from close to zero to about 10 cm/s, within 50 cm downwind of the traps, indicating that they flew upward more rapidly when close to the traps (Cooperband, 2005). As mosquitoes approached the traps, velocity parameters involving the X-axis show that both species flew more slowly to the EVS trap than to any of the others. This continued all the way to the leading edge of the traps (0 cm downwind of trap) for both species. There were no major patterns of differences between the traps for Cx. tarsalis upwind of the leading edge of the traps, but Cx. quinquefasciatus upwind of the EVS trap continued to have a significantly lower velocity than with the MML or MMX traps.

Angular headings Drift angle (XY) and 3-D drift angle seemed to reveal more differences between traps than track angle (XY), inclination angle (XZ) and front angle (YZ) for both

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Table 4. Significant differences for various flight parameters as female Culex tarsalis mosquitoes orientated to different traps. Traps are represented by the letters e (EVS), f (MMF), l (MML) and x (MMX). Data in columns are aligned between column headings, indicating where the bin measurements were taken. Dashes separate significantly different traps for the particular flight parameter and distance downwind of the trap using the Games–Howell test (P < 0.05), with significantly lower values on the left-hand side and higher values on the right-hand side of each dash. When no significant differences were detected between any of the traps, the space was left blank. Distances relate to the downwind edge of the trap entrance, and parameters for that column were calculated for each 10-cm increment downwind from that point Culex tarsalis. Distance downwind of trap (cm) Parameter

150

140

130

120

110

100

90

Flight speed (3-D) Ground speed (XY) X speed Airspeed (XY) 3-D airspeed 3-D drift angle Drift angle (XY) Time elapsed Track angle (XY) Flight angle (3-D) 3-D course angle Course angle (XY) Y speed Vertical position Z speed Inclination angle (XZ) Front angle (YZ)

f-e

f-e

f-e

80

70

60

50

40

30

20

10

e-fx e-fx e-fx e-fx e-fx

ef-x ef-x ef-x ef-x ef-x

e-x e-x e-x e-x e-x

ef-x ef-x ef-x ef-x ef-x

ef-x ef-x ef-x ef-x ef-x

ef-x ef-x ef-x ef-x ef-x

ef-x e-x ef-x ef-x ef-x

ef-x ef-x e-x e-x e-x

x-fe

x-fe x-fe

x-fe x-fe

x-fe x-fe x-fe e-f e-f e-f

x-fe x-e xf-e

x-e x-e xf-e

xf-e

fx-e

fx-e

fx-e

x-e

fx-e

fx-e

f-e

x-e

x-fe x-fe xf-e; x-f

f-e

f-e

fx-e

3-D tortuosity XY tortuosity XZ tortuosity YZ Tortuosity

0

10

20

x-e

xf-e

EVS, Encephalitis Virus Surveillance trap; MMF, Mosquito Magnet Freedom trap; MML, Mosquito Magnet Liberty trap; MMX, Mosquito Magnet-X trap.

species (Tables 3 and 4). For both species, the MMX drift angles are significantly smaller than the EVS drift angles, whereas the course angles are not. The difference in significance between drift angles and course angles can be explained by the fact that during upwind flight, without altering ground speed and wind speed, as the track angle approaches 90 , the drift angle will change much more than the course angle.

Vertical position For both species, although other flight parameters involving the Z-axis produced few significant differences between traps, the vertical position of mosquitoes as they flew upwind toward the traps produced a relatively large number of significant differences between traps (Tables 3 and 4; Fig. 7). Cx. quinquefasciatus females approached all four traps from below the trap entrances. Although Cx. tarsalis females followed the same approach pattern for the MMF and MMX traps, as they neared the EVS trap, they approached closer to the CO2 outlet.

Tortuosity Although tortuosity differed significantly in only a few bins, those bins are probably the most relevant in terms of insects being trapped, because the mosquitoes are then close to the trap entrance. Calculations of 3-D tortuosity can be seen for both species in Fig. 8, and significant differences between traps are noted in Tables 3 and 4. Both mosquito species have non-tortuous flights upwind until they arrive at the trap entrances. Cx. quinquefasciatus females have significantly more tortuous flights 10–30 cm downwind of the EVS trap than any other trap. Cx. tarsalis females follow a similar pattern, but the differences are not significant, probably due to the large variation and the small N.

Time elapsed From about 50 cm downwind to the leading edge of the traps, Cx. quinquefasciatus females spent significantly more time orienting toward the EVS trap than the MMF, MML

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20 M. F. Cooperband and R. T. Carde´

Fig. 6. Average velocity along the X-axis of (a) Culex quinquefasciatus and (b) Culex tarsalis females at distances of 10-cm increments downwind of four types of mosquito traps. EVS, Encephalitis Virus Surveillance; MMF, Mosquito Magnet Freedom; MML, Mosquito Magnet Liberty; MMX, Mosquito Magnet-X.

Fig. 7. Average vertical positions of (a) Culex quinquefasciatus and (b) Culex tarsalis females with respect to the trap entrance (0,0), at distances of 10-cm increments downwind of four types of mosquito traps. EVS, Encephalitis Virus Surveillance; MMF, Mosquito Magnet Freedom; MML, Mosquito Magnet Liberty; MMX, Mosquito Magnet-X.

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Fig. 8. Average three-dimensional (3-D) tortuosity of (a) Culex quinquefasciatus and (b) Culex tarsalis females at distances of 10-cm increments downwind of four types of mosquito traps. EVS, Encephalitis Virus Surveillance; MMF, Mosquito Magnet Freedom; MML, Mosquito Magnet Liberty; MMX, Mosquito Magnet-X.

or MMX traps (Fig. 9a, Table 3), and Cx. tarsalis females similarly spent significantly more time orienting toward the EVS or MMF traps than the MMX trap (Figs 4 and 9b, Table 4). For both species and all traps, more time was spent in bins near the trap entrances than further downwind.

Differences between caught and not caught For Cx. quinquefasciatus, flight tracks of mosquitoes that were caught were compared to those that were not caught for each trap. Table 5 shows which traps had differences between caught and not caught mosquitoes for each flight parameter and distance downwind of the trap. Again, parameters involving speed along the X-axis as well as vertical position often showed significant differences followed by angular headings involving the Y-axis, then other parameters involving the Z-axis, and finally tortuosity. Mosquitoes that were caught had significantly lower velocities and greater drift angles than those that were not caught. Diagrams comparing values for some of these flight parameters between caught and not caught mosquitoes can be found in Cooperband (2005). Table 5 shows that significant differences between flights for mosquitoes that were caught or not caught occurred in different bins for different traps. The EVS trap flights differed between 20 and 50 cm downwind of the trap, the MMF trap flights differed between 0 and 20 cm downwind of the trap, the MMX trap flights

differed from 10 cm downwind of the trap to 20 cm upwind of the trap, and the MML flights did not differ in as clear a pattern, but several differences occurred between 40 and 50 cm downwind of the trap. There were few differences between Cx. quinquefasciatus females that were caught and not caught in terms of the height relative to the trap entrance, except for those flying to the MML trap, in which the mosquitoes that were caught approached the trap from farther below the trap entrance than those that were not caught. For the MMF trap the XZ tortuosity 0–20 cm downwind of the trap was greater for mosquitoes that were caught than those that were not caught.

Discussion This is the first study to document the proportion of mosquitoes approaching CO2-baited traps that is captured and to describe their orientation behaviour during their approach. Some traps were surprisingly inefficient, capturing less than 10% of the Cx. quinquefasciatus and up to 18% of Cx. tarsalis mosquitoes released. The MMX trap was the most efficient for Cx. quinquefasciatus, with capture of 26% of the mosquitoes released. Additionally, 13–16% of Cx. quinquefasciatus females that flew upwind to EVS, MMF and MML traps, and 58% of those flying upwind to the MMX trap, were captured, and the results for Cx. tarsalis were similar. Similarly, in a field comparison, the MMF captured fewer mosquitoes than the counterflow geometry trap (MMX), although both

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Fig. 9. Average time elapsed for (a) Culex quinquefasciatus and (b) Culex tarsalis females at distances of 10-cm increments downwind of four types of mosquito traps. EVS, Encephalitis Virus Surveillance; MMF, Mosquito Magnet Freedom; MML, Mosquito Magnet Liberty; MMX, Mosquito Magnet-X.

captured more than a CDC-style trap similar to the EVS trap used in this study (Burkett et al., 2001). Flight tracks of both species to the EVS trap were significantly longer in duration than any of the other traps, suggesting that the EVS trap attracts mosquitoes, but not necessarily to the vicinity where they would be captured. Furthermore, Cx. quinquefasciatus mosquitoes that were caught spent more time orienting to traps than those that were not caught, suggesting that whether a mosquito is captured depends on how much time it is willing to invest flying around the trap before giving up (Figs 4 and 5). Although the MMX trap releases neither heat nor humidity, it still outperformed the EVS, MMF and MML traps with the two species tested. As shown in Cooperband & Carde´ (2006), the concentration of CO2 produced by the MMX was intermediate between that of the EVS trap and the other ABC traps, and its plume structure resembles that of the MMF trap. The greatest incidents of bursts (when CO2 concentration rises above background levels) for the three ABC traps generally occurred below the height of the trap entrances, whereas for the EVS trap burst numbers were greatest at the level of the trap entrance and above, especially at distances closer to the trap. The airspeed at the

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suction inlet and CO2 outlet was higher for the MMX trap than any of the other traps, which probably contributed to its higher trapping efficiency. The entrance of the EVS trap was 20 cm away from the CO2 source, as opposed to 10 cm for the ABC traps, but this did not seem to significantly reduce its efficiency compared to the MMF and MML traps, possibly because other factors such as visual cues may have reduced trap capture for those traps. For all trap types and both Culex species, flights were relatively straight and rapid at distances greater than 100 cm downwind of the trap. Within 100 cm of the trap, different mosquito orientation behaviour was observed with each trap type. Mosquitoes approaching the EVS and MMF traps tended to increase the tortuosity of their flight, and spent more time within 30 cm of reaching the trap. Mosquitoes approaching the MMX trap flew relatively straight until passing the leading edge of the trap, at which point they engaged in more tortuous flight. Figures in Cooperband (2005) show the angular headings for both species, how the 3-D drift angles of Cx. quinquefasciatus females are greater when flying to the EVS trap than the other traps from 40 cm downwind to the leading edge of the traps, and how for Cx. tarsalis females

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Table 5. Significant differences between caught and not caught Culex quinquefasciatus females for various flight parameters at different 10-cm increments downwind of four different traps. Traps are represented by the letters e (EVS), f (MMF), l (MML) and x (MMX). Data in columns are aligned between column headings, indicating where the bin measurements were taken. A letter indicates a significant difference between caught and not caught tracks for that trap, and for the particular flight parameter and distance downwind of the trap, using the Games–Howell test (P < 0.05). When no significant differences were detected, the space was left blank. Distances relate to the downwind edge of the trap entrance. Culex quinquefasciatus – differences between those caught and not caught Distance downwind of trap (cm) Parameter

100

90

80

Flight speed (3-D) Ground speed (XY) X speed Airspeed 3-D airspeed 3-D drift angle Drift angle (XY) Time elapsed Track angle Flight angle 3-D course angle Course angle Y speed Vertical position Z speed Inclination angle Front angle 3-D tortuosity XY tortuosity XZ tortuosity YZ tortuosity

l l

l

l

70

60

l l

x x

50

e e e e

40

e e e

30

20

10

e e e

fl f f f

fx fx fx fx fx fx f

e x

l

l l l l l

e e e e

e

l

l

l

0

10

20

x x

x x x

x x

e e

x

l

f ef l l l

f

x

EVS, Encephalitis Virus Surveillance trap; MMF, Mosquito Magnet Freedom trap; MML, Mosquito Magnet Liberty trap; MMX, Mosquito Magnet-X trap.

orienting to the MMX trap 3-D drift angle is lower than to the other traps from 70 cm downwind to the leading edge of the trap. Mosquitoes approaching the MML trap appear to have very little tortuosity. The MML trap presented the most logistical problems in terms of recording 3-D video near the trap entrance. This lowered the N near that entrance and could be partly responsible for the low tortuosity readings near the entrance of that trap. For all traps, few bins were significantly different for tortuosity, but this may be explained by the fact that generally the mosquitoes flew fairly straight tracks until they reached the trap, at which point they spent more time meandering around before being caught or leaving (see example track in Fig. 10). This appeared to be the case for all the traps except for the MMX. Mosquitoes seemed to fly directly to the entrance of the MMX and become captured, or fly past it and turn around. As some species of mosquitoes reach the source of CO2, they may shift orientation strategies and fly to prominent visual cues during their final approach (Gibson & Torr, 1999), and the fact that the MMX trap was mostly transparent may have played a role in its relative success. This is most

evident when looking at the time elapsed and tortuosities for Cx. quinquefasciatus. Mosquitoes spent more time slowly flying around and turning immediately downwind of the entrances of the EVS, MMF and MML traps, whereas this behaviour was rarely observed near the MMX trap. In fact, there is a marked increase in tortuosity after the mosquitoes passed the leading upwind edge of the MMX trap that is absent in approaches to the other three traps. This suggests that mosquitoes flying to the MMX trap may not perceive some cues that would cause them to reduce flight velocity, but after passing the trap, they exit the CO2 plume, and then switch to a ‘meandering’ behaviour that may improve their chance of regaining contact with the plume. Mosquitoes that continue to follow the plume, rather than perhaps flying to a prominent visual cue, would be ‘led’ along the plume to its source close to the trap entrance, increasing the likelihood of capture. We did not see a significantly different prominent peak in tortuosity downwind of traps for Cx. tarsalis. In fact, there is a peak just upwind of the EVS trap for this species. It is possible that this species relies more upon the odour plume than on visual cues, even during the final approach to the source.

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24 M. F. Cooperband and R. T. Carde´

XZ X

Z

Y

XY

YZ

Fig. 10. A schematic representation of an actual track of a Culex tarsalis female flying upwind to an Encephalitis Virus Surveillance (EVS) trap and not getting caught, showing the same track in a threedimensional view and in the three different planes.

Because Cx. tarsalis females approached all traps at the height of the CO2 outlet, it appears that they may be relying principally on the location of the CO2 plume, as opposed to Cx. quinquefasciatus females, which approached all traps from below the trap entrances (Fig. 7). Mosquitoes that approached the MMF trap were sometimes seen meandering slowly around its metal base, which may explain why upwind of this trap the average altitudes of both species were reduced considerably. Although Cx. pipiens (sensu lato) is reportedly highly ornithophagous (Bohart & Washino, 1978), the host preference of Cx. quinquefasciatus is more anthropophilic. Samuel et al. (2004) reported that 75% of Cx. quinquefasciatus females collected indoors had fed upon humans, and only about 2% had fed on birds. Of the combined Cx. quinquefasciatus mosquitoes captured both indoors and outdoors, Gomes et al. (2003) reported that about 52% and 22% had fed on humans and chickens, respectively. By contrast, many studies on the host feeding patterns of Cx. tarsalis show that this species feeds on a broad range of hosts but prefers birds (Bohart & Washino, 1978). In a field study, Wekesa et al. (1997) found that Cx. tarsalis fed upon birds and mammals about 71% and 27% of the time, respectively. If Cx. tarsalis females follow plumes to their source, relying more on olfactory and less on visual cues, this may aid in the location of birds as hosts, which provide less conspicuous visual cues in the dark at close range than humans. Similarly, if Cx. quinquefasciatus females rely more on visual cues when close to their hosts, this may aid in finding their preferred larger mammalian hosts and reduce their success at finding birds. Furthermore, the preference of biting sites around the human torso and legs by Cx. quinquefasciatus (de Jong & Knols, 1995) suggests that at some point during their approach to a human host, they would depart from the

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plume of CO2 originating from the head, which is the least preferred biting site for that species. The traps examined had multiple variables and therefore we can only consider which features correlate with particular orientation manoeuvres and we cannot verify cause and effect. Although there are several differences between the three ABC traps, a common feature was the CO2 outlet being positioned 10 cm below the trap entrance, whereas on the EVS trap it was located 20 cm above the trap entrance. This factor seemed to play a more important role in the orientation behaviour of Cx. tarsalis females, which appeared to follow the location of the CO2 plume with the highest burst numbers to the source which was 20 cm higher than the entrance in the case of the EVS trap, while Cx. quinquefasciatus females approached the trap on average from below the height of the trap entrance for all four traps. Our observations of approaching mosquitoes that were and were not captured by the EVS trap, suggest that capture might have been occurring randomly, in that a captured mosquito engaged in a meandering behaviour that could last several minutes just downwind of the trap, eventually happened to get too close to the entrance. Mosquitoes that ‘gave up’ sooner therefore tended not to be captured. Alternatively, some mosquitoes flying to the MMF, MML and especially the MMX trap sometimes flew directly into the trap entrance, without a long bout of first meandering back and forth. Difference in maximum concentration of CO2 between traps did not appear to affect orientation behaviour (Cooperband & Carde´, 2006). If the prediction by Gillies (1980) that the most important feature of the plume for orienting mosquitoes may be changes in CO2 concentration is correct, then burst number, which would be detected by the mosquito as a change in concentration, would be a more important feature of the plume than peak concentration. In

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Orientation of Culex mosquitoes to traps Cooperband & Carde´ (2006) we found that, at three CO2 thresholds, burst number was low 60 cm downwind of the EVS trap at the height below the trap entrance, whereas it remained high, at least at one of the three thresholds, for the ABC traps to 30 cm downwind of the traps at the height below the trap entrances. This comparatively low burst number frequency 60 cm downwind of the EVS trap below the trap entrance is likely caused by the CO2 outlet being farther away from the trap entrance on the EVS trap than on the ABC traps, and the fact that the CO2 is released passively rather than being blown downward. The low burst frequency may explain why mosquito flights downwind of the EVS trap became much more tortuous, resulting in more time elapsed prior to capture. Cooperband & Carde´ (2006) found that the plume of the MMX trap, unlike the MMF and MML plumes, dropped below the downward-pointing trap entrance, which may have contributed to the improved capture rate of that trap. Another possible explanation for the low capture efficiencies of these traps is that they may lack other important cues such as additional semiochemicals. Further behavioural studies are required in which some of the variables (such as relative plume location, suction strength, burst number, or visual appearance) can be varied systematically. Important caveats for this study are first that these findings apply only to the orientation of the two species studied, Cx. quinquefasciatus and Cx. tarsalis, in a light and unvarying wind. In relatively still air or in a turbulent flow, capture rates could be elevated or suppressed. Second, field trapping efficiencies could be higher than reported here, if mosquitoes that were not initially captured later reoriented to traps. Third, adding of other host odours to CO2 might change orientation behaviour, for example increasing the giving up time, which in turn might provide more opportunity for mosquitoes to meander close to the trap entrance and be captured. Acknowledgements Dr Alan Grant of American Biophysics Corporation and the Northwest Mosquito and Vector Control District generously provided the traps. We thank Dr Bill Walton for providing mosquitoes, Terence Fung, Ross Whittaker, Andrew Kuszynski, Brian Panama and Michelle Sanford for their assistance in experiments and data entry, Kris Tollerup and Dr Robert Beaver for statistical advice, Dr Josep Bau for his work on the TRACK 3-D computer program, Dr Kris Justus for training and advice, and Dr Marieta Braks for critical review. Drs Scott Ritchie and Alan Grant provided useful comments. We also acknowledge support from the University of California Systemwide Mosquito Research Program.

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Bohart, R.M. & Washino, R.K. (1978) Mosquitoes of California. Regents of the University of California, Berkeley, CA. Burkett, D.A., Lee, W.J. & Lee, K.W. et al. (2001) Light, carbon dioxide, and octenol-baited mosquito trap and host-seeking activity evaluations for mosquitoes in a malarious area of the Republic of Korea. Journal of the American Mosquito Control Association, 17, 196–205. Carde´, R.T., Staten, R.T. & Mafra-Neto, A. (1998) Behaviour of pink bollworm males near high-dose, point sources of pheromone in field wind tunnels: insights into mechanisms of mating disruption. Entomologia Experimentalis et Applicata, 89, 35–46. Cooperband, M.F. (2005) Carbon dioxide-baited traps and birdassociated odors: orientation behavior and chemical ecology of host-seeking Culex mosquitoes. PhD Thesis, University of California, Riverside, CA. Cooperband, M.F. & Carde´, R.T. (2006) Comparison of plume structures of carbon dioxide emitted from different mosquito traps. Medical and Veterinary Entomology, 20, 1–10. Cummings, R.F. & Meyer, R.P. (1999) Comparison of the physical parameters of four types of modified CDC-style traps in reference to their mosquito collecting efficiency. Proc. Calif. Mosq. Vector Control Assoc., 67, 38–44. Curtis, C.F. (1996) Introduction I: an overview of mosquito biology, behaviour and importance. Olfaction in Mosquito– Host Interactions, Vol. 200 (ed. by G. R. Bock and G. Cardew), p. 331. Ciba Foundation, Chichester. Daykin, P.N., Kellogg, F.E. & Wright, R.H. (1965) Host-finding and repulsion of Aedes aegypti. Canadian Entomologist, 97, 239–263. Dekker, T., Takken, W. & Carde´, R.T. (2001) Structure of hostodour plumes influences upwind flight and trap catch of Anopheles gambiae s.s. and Aedes aegypti in a dual-choice olfactometer. Physiological Entomology, 26, 124–134. Dekker, T., Geier, M. & Carde´, R.T. (2005) Carbon dioxide instantly sensitizes female yellow fever mosquitoes to human skin odours. Journal of Experimental Biology, 208, 2963–2972. Games, P.A. & Howell, J.F. (1976) Pairwise multiple comparison procedures with unequal n’s and/or variances: a Monte Carlo study. Journal of Educational Statistics, 1, 113–125. Geier, M., Bosch, O.J. & Boeckh, J. (1999) Influence of odour plume structure on upwind flight of mosquitoes towards hosts. Journal of Experimental Biology, 202, 1639–1648. Georghiou, G.P. & Wirth, M.C. (1997) Influence of exposure to single versus multiple toxins of Bacillus thuringiensis subsp. israelensis on development of resistance in the mosquito Culex quinquefasciatus (Diptera: Culicidae). Applied and Environmental Microbiology, 63, 1095–1101. Gibson, G. & Torr, S.J. (1999) Visual and olfactory responses of haematophagous Diptera to host stimuli. Medical and Veterinary Entomology, 13, 2–23. Gillies, M.T. (1980) The role of carbon dioxide in host-finding by mosquitoes (Diptera: Culicidae): a review. Bulletin of Entomological Research, 70, 525–532. Gomes, A.C., Silva, N.N., Marques, G. & Brito, M. (2003) Hostfeeding patterns of potential human disease vectors in the Paraiba Valley Region, State of Sao Paulo, Brazil. Journal of Vector Ecology, 28, 74–78. Grant, A.J. & O’Connell, R.J. (1996) Electrophysiological responses from receptor neurons in mosquito maxillary palp sensilla. Olfaction in Mosquito–Host Interactions, Vol. 200 (ed. by G. R. Bock and G. Cardew), pp. 233–253. Ciba Foundation, Chichester.

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26 M. F. Cooperband and R. T. Carde´ Hayes, R.O., Bellamy, R.E., Reeves, W.C. & Willis, M.J. (1958) Comparison of four sampling methods for measurement of Culex tarsalis adult populations. Mosquito News, 18, 218–228. de Jong, R. & Knols, B.G.J. (1995) Selection of biting sites on man by two malaria mosquito species. Experientia, 51, 80–84. Kemme, J.A., Van Essen, P.H.A., Ritchie, S.A. & Kay, B.H. (1993) Response of mosquitoes to carbon dioxide and 1-octen-3-ol in southeast Queensland, Australia. Journal of the American Mosquito Control Association, 9, 431–435. Kennedy, J.S. (1939) The visual responses of flying mosquitoes. Proceedings of the Zoological Society of London, Series A, 109, 221–242. Kline, D.L. (1994) Olfactory attractants for mosquito surveillance and control: 1-octen-3-ol. Journal of the American Mosquito Control Association, 10, 280–287. Kline, D.L. (1999) Comparison of two American Biophysics mosquito traps: the professional and a new counterflow geometry trap. Journal of the American Mosquito Control Association, 15, 276–282. Kline, D.L. (2002) Evaluation of various models of propane-powered mosquito traps. Journal of Vector Ecology, 27, 1–7. Kline, D.L. & Lemire, G.F. (1995) Field evaluation of heat as an added attractant to traps baited with carbon dioxide and octenol for Aedes taeniorhynchus. Journal of the American Mosquito Control Association, 11, 454–456. Kline, D.L., Wood, J.R. & Morris, C.D. (1990) Evaluation of 1-octen-3-ol as an attractant for Coquillettidia perturbans, Mansonia spp. and Culex spp. associated with phosphate mining operations. Journal of the American Mosquito Control Association, 6, 605–611. Makiya, K. & Iwao, K. (2001) New mosquito traps using carbon dioxide and heat. Medical Entomology and Zoology, 52, 241–247. Marsh, D., Kennedy, J.S. & Ludlow, A.R. (1978) An analysis of anemotactic zigzagging flight in male moths stimulated by pheromone. Physiological Entomology, 3, 221–240. Mboera, L.E.G., Knols, B.G.J., Braks, M.A.H. & Takken, W. (2000) Comparison of carbon dioxide-baited trapping systems for sampling outdoor mosquito populations in Tanzania. Medical and Veterinary Entomology, 14, 257–263. Meyer, R.P., Reisen, W.K., Eberle, M.W., Milby, M.M., Martinez, V.M. & Hill, B.R. (1985) A time segregated sampling device for determining nightly host-seeking patterns of female mosquitoes. Proceedings of the California Mosquito and Vector Control Association, 52, 162–166. Meyer, R.P., Reisen, W.K., Eberle, M.E., Milby, M.M. & Reeves, W.C. (1986) The nightly host-seeking rhythms of several culicine mosquitoes (Diptera: Culicidae) in the southern San Joaquin Valley of California. Proceedings of the California Mosquito and Vector Control Association, 54, 136.

Journal compilation

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Reisen, W.K. & Pfuntner, A.R. (1987) Effectiveness of five methods for sampling adult Culex mosquitoes in rural and urban habitats in San Bernardino County California USA. Journal of the American Mosquito Control Association, 3, 601–606. Reisen, W.K., Meyer, R.P., Cummings, R.F. & Delgado, O. (2000) Effects of trap design and CO2 presentation on the measurement of adult mosquito abundance using centers for disease controlstyle miniature light traps. Journal of the American Mosquito Control Association, 16, 13–18. Rohe, D.L. & Fall, R.P. (1979) A miniature battery powered CO2-baited trap for mosquito-borne encephalitis surveillance. Bulletin of the Society for Vector Ecology, 4, 24–27. Rueda, L.M., Harrison, B.A., Brown, J.S., Whitt, P.B., Harrison, R.L. & Gardner, R.C. (2001) Evaluation of 1-octen-3-ol, carbon dioxide, and light as attractants for mosquitoes associated with two distinct habitats in North Carolina. Journal of the American Mosquito Control Association, 17, 61–66. Russell, R.C. (2004) The relative attractiveness of carbon dioxide and octenol in CDC- and EVS-type light traps for sampling the mosquitoes Aedes aegypti (L.), Aedes polynesiensis Marks, and Culex quinquefasciatus Say in Moorea, French Polynesia. Journal of Vector Ecology, 29, 309–314. Samuel, P.P., Arunachalam, N., Hiriyan, J., Thenmozhi, V., Gajanana, A. & Satyanarayana, K. (2004) Host-feeding pattern of Culex quinquefasciatus Say and Mansonia annulifera (Theobald) (Diptera: Culicidae), the major vectors of filariasis in a rural area of south India. Journal of Medical Entomology, 41, 442–446. Service, M.W. (1993) Mosquito Ecology: Field Sampling Methods. 2nd edition. Wiley, New York. Takken, W. & Kline, D.L. (1989) Carbon dioxide and 1-octen-3-ol as mosquito attractants. Journal of the American Mosquito Control Association, 5, 311–316. Vale, G.A. & Hall, D.R. (1985) The role of 1-octen-3-ol, acetone and carbon-dioxide in the attraction of tsetse flies, Glossina spp. (Diptera: Glossinidae), to ox odor. Bulletin of Entomological Research, 75, 209–217. Vale, G.A. & Hargrove, J.W. (1979) Method of studying the efficiency of traps for tsetse flies (Diptera: Glossinidae) and other insects. Bulletin of Entomological Research, 69, 183–193. Wekesa, J.W., Yuval, B., Washino, R.K. & de Vasquez, A.M. (1997) Blood feeding patterns of Anopheles freeborni and Culex tarsalis (Diptera: Culicidae): effects of habitat and host abundance. Bulletin of Entomological Research, 87, 633–641. Accepted 18 December 2005

# 2006 The Authors 2006 The Royal Entomological Society, Medical and Veterinary Entomology, 20, 11–26