Note: This protocol is written specifically for field trials targeting adult mosquitoes. Information on modifications necessary for immature mosquitoes, other adult sentinel insect species, and unique scenarios is included in the discussion.
1. Sentinel Insect Rearing and Sentinel Cage Preparations
Figure 1: Sentinel cage preparation. (A) Shown here are two personnel loading sentinel cages with anaesthetized mosquitoes spread on a large white sheet of paper. The stack of tulle mesh squares in the foreground ready for placement on the waiting open sentinel cages should be noted. (B) Shown are several loaded sentinel cages, awaiting placement of 10% sucrose cotton balls and realignment of the rubber band. (C) Shown are sentinel cages in a cooler, ready for deployment in the field. Please click here to view a larger version of this figure.
2. Preparation of the Field Site
Figure 2: Prepare field site with sentinel cage poles. Two scenarios of sentinel cage poles distributed in (A) irrigated tropical microhabitat in a hot-arid region and (B) hot-arid desert. In (A), three kinds of sentinel apparatus are shown: the "ladder" on the left is a series of cotton ribbons suspended between PVC pipe to capture pesticide at various heights above ground; the pole in the middle is for sentinel mosquito cages; and the pole on the right supports a slide spinner to capture pesticide droplets. In (B) there is a similar cotton ribbon to capture pesticide, but it should be noted that the right-hand support of the apparatus in the foreground in the open doubles as a sentinel cage pole (the sentinel cage attached about one-third of the way down); in contrast, the apparatus in the background is placed so that the ribbon and the sentinel cage (indicated with yellow arrow) are sheltered within vegetation. The aircraft in the background is conducting a ULV pesticide spray. Please click here to view a larger version of this figure.
3. Deploying the Sentinel Cages
Figure 3: Placement of sentinel cages indoors and outdoors. Examples of pole placements and 1 ft3 boxes (A) indoors in a simulated rural residence, (B) outdoors in a simulated neighborhood, and (C) in a date palm grove in a hot-arid zone. Also shown are the sticky tiles under the box and under the sentinel cage placed in the open in (A), and the sentinel cage pole pin pushed through the sticky tile next to the box in (B) to reduce incursion of ants into sentinel cages. The plywood square with upright PVC mount to the left of the box in (A) is a support for a slide spinner. A similar apparatus may be used to support a sentinel cage pole to allow placement of sentinels at varying heights. Also shown is the slide spinner with slides on a mount to the left of the sentinel cage pole and box (B). In (C) is shown a high-low placement of two sentinel cages to investigate movement of the pesticide at different levels. The top of the opened sentinel cage cooler is just visible in the foreground. Please click here to view a larger version of this figure.
Note: Depending on size of the grid, ensure there are sufficient personnel to deploy all cages in an appropriate time frame to minimize environmental effects on sentinel mortality. It may be necessary to conduct practice runs with empty cages to determine whether enough personnel are present. It is understood that there will unavoidably be some spread in the exposure time of sentinel cages to the environment, owing to set-up time with the given number of cages per person. In very large sentinel grids (e.g., for some aerial applications), plan on coordinating multiple teams in several vehicles to place the sentinel cages within a reasonable time frame. It is inevitable that some mortality will occur across the sentinel cage mosquito population, so it must be accounted for with careful cage labeling prior to environmental exposure outside the coolers and prior to pesticide application.
4. Conducting the Pesticide Application
Figure 4: Vehicle mounted thermal fog spraying a grid in warm-arid equatorial location. A truck mounted ULV sprayer driving along a spray line directing pesticide spray through a grid of sentinel cages in an open field (A). Similar to Figure 2, each sentinel position is fitted with a second pole to support a cotton ribbon to collect pesticide droplets for later analysis with gas chromatograph/mass spectrometry. Close-ups of sentinel cages (B) in a warm temperate environment before spray and (C) in a hot-arid environment after spray. Please click here to view a larger version of this figure.
5. Collecting the Sentinel Cages and Recording Mortality Data
Figure 5: Processing sentinel cages post-spray. Two scenarios of transferring post-spray cages into stacked trays during the 6 h mortality check: (A) in a hotel room near a remote field site and (B) in a convenient parking lot returning to the lab from a distant field site. Note the PVC spacers in the inset photo in (A), moist towels covering cages laid out in trays in the main photo of (A), and an example of a unique location code and pre- and circled post-spray mortality annotated directly on a sentinel cage in the inset photo in (B). Please click here to view a larger version of this figure.
6. Processing, Analyzing, and Mapping Mortality Data
Here are representative results presented from two unpublished field studies that included the core of the methods described above. In these studies, two aspects of adulticide efficacy against sentinel disease vector insects were investigated.
The first study (unpublished data 2010-2012) investigated whether diluent might influence efficacy of pesticide against mosquitoes in a hot-arid desert environment applied with a thermal fog device. We conducted three separate applications with a synergized permethrin adulticide capable of being diluted in either oil or water. Each of the applications was conducted using a truck-mounted thermal fog generator with a different diluent: either water, BVA13 mineral oil, or diesel. Then, a grid of at least 20 sentinel mosquito cages placed on poles in an open arid-land area (Figure 6) was used and meteorology was recorded using a portable weather recorder.
The second study (unpublished data 2011) investigated the relative efficacy of a single pesticide formulation applied simultaneously with two kinds of sprayers (ULV and thermal fog) in a hot equatorial environment against sand flies. We used two adjacent grids of 25 sentinel sand fly cages placed on poles in low grass-forb habitat of varying density in a large field in a hot equatorial valley basin (Figure 7). Meteorology was recorded using a portable weather recorder positioned between the two grids. One truck carried the ULV device and another carried the thermal fog generator, and both initiated sprays along the spray lines at the same time, each moving from east to west at a speed appropriate to the label-specified application rate and given flow rate of the sprayer.
In both field studies, after a 10-min hold time post-spray, we collected all sentinel cages from treatment and control areas, simultaneously initiating the process of recording post-spray mortality. Percent mortality data was then coded, corrected for observed background control sentinel mortality, and placed into a GIS coverage consisting of georeferenced points corresponding to the locations of the sentinel cages in the treatment area grid (Figure 6 and Figure 7). Despite the ideal situation in which a pesticide spray produces a 100% kill throughout the target area, the realistic threshold for acceptable efficacy of a pesticide is arbitrary. Expectations of efficacy could vary with distance from the sprayer (e.g., setting a threshold of 95% mortality 50 ft from the spray line, with 80% mortality at 250 ft).
The results from both representative studies naturally show the spectrum of positive to negative outcomes, because the color ramp represents areas of 0 to 100% mortality (see color ramps in Figure 6 and Figure 7). All mortality data in the treatment area are normalized by the background mortality in the untreated control area to a threshold of 25% control mortality18, above which would have discarded due to excessive environmental or colony effects on mortality. The utility of the electronic mapping approach for visualizing mortality data is evident here: the researcher and (later) the reader can instantly understand the relative efficacy of the focal pesticides and diluents (Figure 6) or the focal pesticide sprayers (Figure 7) against mosquitoes or sand flies, respectively. It is valuable to compare Figure 6 to the underlying mortality data that would traditionally be presented in tabular form (Table 2) and require much more internal conceptualization, with commensurate increased potential for error, by researcher and reader alike. The electronic map, if supplemented with meteorological data and a satellite photo base coverage, also facilitates rapid assessment of potential effects of the habitat on the pesticide applications. It should be noted that the inset wind rose diagram in Figure 7 has a wind angle that perfectly matches the angle of spatial mortality in the west grid, indicating that the spray truck should have started further to the east so that pesticide would have an opportunity to reach all sentinel cages. If mortality data is only been considered in tabular form, it may be easy to miss this weakness in the experimental design and assign a lower overall efficacy to that pesticide application equipment, biased by zero mortality values from areas not contacted by the pesticide.
In our experience, the majority of pesticide applications in the field produce a gradient of mortality through the target area sentinel cages, which automatically demonstrates that the application was valid. However, if zero mortality is observed throughout the treatment area and the spray event is valid (i.e., the spray cloud moving through the target sentinel area is observed), it can be inferred that the pesticide is not effective with that species at the rate applied, in that environment, and with that application equipment. Of course, this is given that the pesticide batch is not expired nor has been stored inappropriately. On the other hand, some aerial pesticide applications in particular may produce no visible or detectable spray cloud impacting the target area, and zero mortality throughout sentinels may mean that the spray missed the target area. It is advised to anticipate this scenario by setting up a series of extra sentinel cages that brackets the target impact area to some distance upwind and downwind (e.g., 50 ft intervals for at least one swath width in each direction) so that some indication of touchdown of the pesticide may be gleaned if the target area is missed.
Figure 6: Representative interpolated sentinel cage mortality data from a field trial in hot-arid desert conditions targeting mosquitoes. In this series of sprays, the thermal fog pesticide sprayer, pesticide, and environment were kept constant, varying among pesticide diluent to (A) water, (B) diesel, and (C) BVA 13 mineral oil across the three trials. Please click here to view a larger version of this figure.
Figure 7: Representative interpolated sentinel cage mortality data from a field trial in hot-equatorial conditions targeting sand flies. In this set of two simultaneous spray applications the pesticide and environment were kept constant, but the pesticide sprayer was different between the two grids: thermal fog (west grid) and ultra-low volume (ULV, east grid). Note that the wind direction indicated in the wind rose diagram perfectly matches the angle of spatial mortality in the west grid, indicating that the spray truck should have started further to the east so that pesticide would have an opportunity to reach all sentinel cages. Please click here to view a larger version of this figure.
PROJECT: | ||||||
ECOLOGICAL ZONE: | ||||||
LOCATION: | ||||||
FIELD SITE: | ||||||
DATE OF APPLICATION: | ||||||
SPRAY EQUIPMENT: | ||||||
PESTICIDE FORMULATION: | ||||||
DILUENT: | ||||||
SENTINEL INSECT SPECIES: | ||||||
LIFE STAGE OF SENTINELS: | ||||||
MORTALITY CHECKS | ||||||
[copied directly from sentinel cages; data observed in field] | [observed from sentinel cages stored in trays] | |||||
Sentinel Cage Code | No. PRE-DEAD | No. DEAD POST-SPRAY/HOLD TIME | No. DEAD 6 hr POST-SPRAY | No. DEAD 12 hr POST-SPRAY | No. DEAD 24 hr POST-SPRAY | |
Treatment Area | A1 | |||||
A2 | ||||||
A3 | ||||||
A4 | ||||||
A5 | ||||||
B1 | ||||||
B2 | ||||||
B3 | ||||||
B4 | ||||||
B5 | ||||||
C1 | ||||||
C2 | ||||||
C3 | ||||||
C4 | ||||||
C5 | ||||||
D1 | ||||||
D2 | ||||||
D3 | ||||||
D4 | ||||||
D5 | ||||||
E1 | ||||||
E2 | ||||||
E3 | ||||||
E4 | ||||||
E5 | ||||||
Control Area | Control 1 | |||||
Control 2 | ||||||
Control 3 | ||||||
Control 4 | ||||||
Control 5 | ||||||
Control 6 | ||||||
Control 7 | ||||||
Control 8 | ||||||
Control 9 | ||||||
Control 10 |
Table 1: Sample mortality data form. The form has spaces for general information about the field trial in the top left, which is critical to manage data from multiple field projects. The main section of the form has spaces for pre- and post-spray mortality (both copied directly from the sentinel cages), through the later 6 h, 12 h, and 24 h checks. Handwritten data on this form are entered into a similar electronic spreadsheet, with added columns to correct mortality data for pre-spray dead and to correct spray area mortality for any environmentally-induced mortality in the control area.
PERCENT MORTALITY (ABBOTT-CORRECTED) | |||
Sentinel Cage Code | (A) Aqualuer + Water | (B) Aqualuer + Diesel | (C) Aqualuer + BVA13 |
A1 | 1 | 0.78 | 0.54 |
A2 | 1 | 1 | 0.05 |
A3 | 1 | 0.29 | 0 |
A4 | 1 | 0.04 | 0.03 |
A5 | 1 | 0.04 | 0 |
B1 | 1 | 0.79 | 0 |
B2 | 1 | 0.82 | 0 |
B3 | 1 | 0.15 | 0 |
B4 | 1 | 0.21 | 0.15 |
B5 | 1 | 0 | 0 |
C1 | 1 | 0.93 | 0 |
C2 | 1 | 0.71 | 0.01 |
C3 | 1 | 0.32 | 0 |
C4 | 1 | 0.26 | 0 |
C5 | 1 | 0.46 | 0 |
D1 | 1 | 0.67 | 0 |
D2 | 0.78 | 0.24 | 0.01 |
D3 | 0.97 | 0.5 | 0 |
D4 | 1 | 0.27 | 0.03 |
D5 | 1 | 0.33 | 0 |
E1 | 1 | 0.82 | – |
E2 | 1 | 0.88 | – |
E3 | 1 | 1 | – |
E4 | 1 | 0.79 | – |
E5 | 1 | 0.78 | – |
Table 2: Underlying mortality data used to create the interpolated efficacy map in Figure 6. It should be noted that some spatial information can be captured in this table; for instance, this can be done by separating rows and columns by distance from sprayer and distance along spray line, respectively. However, temporal changes in mortality corresponding to immediate post-spray, 4 h post-spray, and 12 h post-spray, for example, would require a more complex table or additional tables. Similarly, separate tables for each diluent used in the trials are necessary for clarity if the tables are partitioned by space and time.
Bioassay racks: | |||
plastic tube lid | Visipak | 192224 | |
1.25-in PVC coupler SCH-40 | Lowes | PVC 00100 0800 | |
1/4-in OD brass rod | K&S Engineering | 1165 | |
Name | Company | Catalog Number | Comments |
Bioassays: | |||
FDA silicone o-rings S-500-70 | Alltek seal and packing | PA-2127-12 | |
fine screening | Walmart | 40310-0000-063 white | T-310 |
cotton balls | Fisher Brand | Large cotton balls (non-sterile) | |
plastic tubes | Visipak | 775674 | |
regulator | Norgren | R83-200-RNEA | |
reguilator gauge | Wika Instrument Corp | 4315031 | |
CO2 canister | 20 lb capacity | ||
CO2 chamber | Mainstays | Modified tupperware container (16 cup) | |
1/4-in tygon tubing | |||
maglite aspirator and tubes | Bioquip | 2809D | D-cell maglite aspirator |
modified PVC pipe for o-rings | Lowes | PVC 07112 0600 | SCH-40 pipe modified by cutting tool on inner surface to accommodate bioassay tube |
Pupal separator | John W. Hock Co. | 5412 | |
Name | Company | Catalog Number | Comments |
Field sentinel cages: | |||
1/2 pt cardboard can body | Neptune | 295 | |
1/2 pt cardboard cup lid | Neptune | 295A | |
coarse screening | Walmart | 41721-0540-063 white | T-1721 |
Velcro cable ties 8-in x 1/2-in | Velcro Brand | VEL91140 | |
rubber band | National Institutes for the Blind | 7510-01-058-9974 | |
cotton balls | Fisher Brand | Large cotton balls (non-sterile) | |
PVC spacers | Lowes | PVC 04010 0600 | Modified by cutting into 18-in length pieces and cutting half off of the end (lengthwise) |
Tray totes | Blue Ridge Thermalforming | 400-3N-WHT-ABS | |
Name | Company | Catalog Number | Comments |
Field bioassay set-up equipment: | |||
60-in tread-in post | Jeffers.com | T8T4 | |
1 ft3 cardboard boxes | USP | S-18344 | |
Deli cups | WNA Inc. | APCOMBO16 | |
18-in x 18-in linolium tiles | Lowes | LSS4307BPS | |
Name | Company | Catalog Number | Comments |
Sentinel cage transport: | |||
48 qt Island Breeze cooler | Igloo | ||
16-in x 19-in. terry towels | Ability One | 7920-01-454-1150 | |
garbage bags | 13 gal (kitchen size) |
Efficacy of public health pesticides targeting nuisance and disease-vector insects such as mosquitoes, sand flies, and filth-breeding flies is not uniform across ecological zones. To best protect public and veterinary health from these insects, the environmental limitations of pesticides need to be investigated to inform effective use of the most appropriate pesticide formulations and techniques. We have developed a research program to evaluate combinations of pesticides, pesticide application equipment, and application techniques in hot-arid desert, hot-humid tropical, warm and cool temperate, and urban locations to derive pesticide use guidelines specific to target insect and environment. To these ends we designed a system of protocols to support efficient, cost-effective, portable, and standardized evaluation of a diverse range of pesticides and equipment across multiple environments. At the core of these protocols is the use of an array of small cages with colony-reared sentinel mosquitoes (adults and immatures) and sand flies (adults), strategically arranged in natural habitats and exposed to pesticide spray. Spatial and temporal patterns of pesticide efficacy are derived from percent mortality in sentinel cages, then mapped and visualized in a geographic information system. Maps of sentinel mortality data may be statistically compared to evaluate relative efficacy of a pesticide across multiple environments, or to study multiple pesticides in a single environment. Protocols may be modified to accommodate a variety of scenarios, including, for example, the vertical orientation of sentinels in canopy habitats or simultaneous testing of ground and aerial application methods.
Efficacy of public health pesticides targeting nuisance and disease-vector insects such as mosquitoes, sand flies, and filth-breeding flies is not uniform across ecological zones. To best protect public and veterinary health from these insects, the environmental limitations of pesticides need to be investigated to inform effective use of the most appropriate pesticide formulations and techniques. We have developed a research program to evaluate combinations of pesticides, pesticide application equipment, and application techniques in hot-arid desert, hot-humid tropical, warm and cool temperate, and urban locations to derive pesticide use guidelines specific to target insect and environment. To these ends we designed a system of protocols to support efficient, cost-effective, portable, and standardized evaluation of a diverse range of pesticides and equipment across multiple environments. At the core of these protocols is the use of an array of small cages with colony-reared sentinel mosquitoes (adults and immatures) and sand flies (adults), strategically arranged in natural habitats and exposed to pesticide spray. Spatial and temporal patterns of pesticide efficacy are derived from percent mortality in sentinel cages, then mapped and visualized in a geographic information system. Maps of sentinel mortality data may be statistically compared to evaluate relative efficacy of a pesticide across multiple environments, or to study multiple pesticides in a single environment. Protocols may be modified to accommodate a variety of scenarios, including, for example, the vertical orientation of sentinels in canopy habitats or simultaneous testing of ground and aerial application methods.
Efficacy of public health pesticides targeting nuisance and disease-vector insects such as mosquitoes, sand flies, and filth-breeding flies is not uniform across ecological zones. To best protect public and veterinary health from these insects, the environmental limitations of pesticides need to be investigated to inform effective use of the most appropriate pesticide formulations and techniques. We have developed a research program to evaluate combinations of pesticides, pesticide application equipment, and application techniques in hot-arid desert, hot-humid tropical, warm and cool temperate, and urban locations to derive pesticide use guidelines specific to target insect and environment. To these ends we designed a system of protocols to support efficient, cost-effective, portable, and standardized evaluation of a diverse range of pesticides and equipment across multiple environments. At the core of these protocols is the use of an array of small cages with colony-reared sentinel mosquitoes (adults and immatures) and sand flies (adults), strategically arranged in natural habitats and exposed to pesticide spray. Spatial and temporal patterns of pesticide efficacy are derived from percent mortality in sentinel cages, then mapped and visualized in a geographic information system. Maps of sentinel mortality data may be statistically compared to evaluate relative efficacy of a pesticide across multiple environments, or to study multiple pesticides in a single environment. Protocols may be modified to accommodate a variety of scenarios, including, for example, the vertical orientation of sentinels in canopy habitats or simultaneous testing of ground and aerial application methods.