This system of techniques measures pesticide efficacy against disease vector insects in the field and provides a unique way to visualize efficacy using maps. A key advantage of this system is that it is a uniform process that can be applied and compared across multiple environments against a range of target insects. At the core of this system is the use of an array of small cages with colony-reared sentinel mosquitoes strategically arranged in natural habitats and exposed to pesticide spray.
Spatial and temporal patterns of pesticide efficacy are derived from the percent mortality in sentinel cages and then mapped and visualized in a geographic information system. To construct the adult sentinel cages, first use a rolling disk cutter, straight edge, and cutting board to prepare two five-by-five-inch tulle mesh squares per sentinel cage from bulk tulle yardage and store the tulle mesh squares in batches of 50 in one-quart resealable plastic bags. Next, push the flat cardboard disks out of the bottoms of new half-pint cardboard ice cream cups to create open-ended two-by-three-inch cylinders and remove the cardboard disks from two ice cream cup lids to create one open-ended bezel for each end of the cylinder.
Then trap one tulle mesh square under one bezel to secure the mesh tightly to the cylinder. For each sentinel cage, fasten a hook-and-loop cable tie to a rubber band, and secure the rubber band around the bezel with the mesh square. Bag the sentinel cages in batches of 50 with a separate bag containing 50 bezels and 50 tulle mesh squares.
When the sentinel cages are ready, carefully shake 80 anesthetized mosquitoes out of a carbon dioxide anesthetization chamber evenly across a clean white 11 1/2 by 17 inch sheet of paper and set a timer for four minutes. Using a pistol aspirator, quickly select and transfer batches of 20 adult females into individual open sentinel cages. Immediately place a tulle mesh square with a bezel over the open upper end of the cage cylinder, and push down to complete the sentinel cage assembly.
Mosquitoes can die from stress. Therefore, for an accurate assessment of the pesticide toxicity, it is important to have sufficiently trained personnel for quickly producing and loading the sentinel cages into the storage coolers to minimize environmental stress. Once the tulle is in place, pivot the rubber band so that it lays across both mesh ends of the sentinel cage with the hook-and-loop cable tie positioned against the side of the cylinder.
Then squeeze the majority of a 10%sucrose solution out of a cotton ball so that it is still moist, just before the point of freely dripping, and gently tuck the cotton ball under the rubber band against the tulle mesh on one side of the sentinel cage so it is easily accessible to the mosquitoes. When all of the females have been removed, use a separate mechanical aspirator to collect all of the remaining male mosquitoes for freezing and load 10 to 12 cages in four to five layers in one 48-quart insulated picnic cooler for every 45 to 50 sentinel cages, slightly offsetting each layer so that the cages do not completely cover the cage below to promote air flow. Then cover the top layer with a folded 13-gallon trash bag and a wet towel, and use duct tape to fasten a folded piece of cardboard to the front corner of the upper lip of the cooler to promote air exchange and prevent overheating.
To establish the sentinel grid and control area, design a grid of locations for the sentinel cages that captures variations in the distance from the sprayer, distance along the spray line, vegetation and obstacles to spray penetration, and height above the ground. Use a 300-foot measuring tape reel to mark off convenient intervals from the sprayer and along the spray line. Install sentinel cage mounting poles into the ground according to the grid design with at least 48 inches of the pole above the ground and clearly label each pole with a code for its unique position with a permanent marker.
If the habitat does not naturally provide vegetation or obstacle challenges to the pesticide spray dispersion, consider placing a one-cubic-foot cardboard box on its side with the top open next to each sentinel cage pole to provide an additional sentinel location within the simulated refugia. The air flow through the boxes can be adjusted by drilling 1/2-inch holes in one or more of the sides, and each box should also be labeled with its unique position code and in a permanent marker. The basic length of the spray line should match the dimensions of the grid, although the start and stop points should be set well before and well after the grid.
Add approximately 10%of the basic length of the spray line to the beginning and end to increase the opportunity for the entire grid to be exposed given inevitable shifts and imperfections in wind patterns. Use the hook-and-loop cable tie to affix the sentinel cages to the poles, making that the sure cotton balls with 10%sucrose solution are still in place under the rubber bands, and label each cage with the location code of the pole and the pre-spray mortality of the cage. Places the cages for the boxes on their sides in the boxes so that both tulle mesh ends are perpendicular to the ground, and label the box cages in the same manner.
When all of the cages have been labeled and deployed in both the treatment and control areas and all of the key personnel have communicated readiness, have the spray operator conduct a final check on the wind speed and direction and initiate the aerial, ground, or portable sprayer pesticide application from the designated spray line or alternate spray line as needed. It is critical to have experienced applicators help design the field trials and to have a keen awareness of any potential meteorological changes to ensure the target area receives an appropriate dose of pesticide. Immediately after the post-spray holding time, record the post-spray mortality of the mosquitoes in each sentinel cage with a permanent marker as it is collected.
Six hours post spray, use disposable nitrile gloves to pick up and observe the cages one by one from the storage trays, calling out the unique location code, the pre-and post-spray mortality counts, and the six-hour mortality count from each cage for data recording. When all of the post-spray data have been recorded, use a geographic information system to map all of the unique sentinel cage locations from the code data for each cage, and create four columns for the Abbott-corrected mortality for the four mortality time periods in the attribute table for these locations. Create an interpolated coverage for mortality for each of the four mortality time periods across all of the unique sentinel locations in the treatment area, and add these interpolated coverages to the map using a stretched color legend on a blue-to-red spectrum and marking the sentinel cage locations with labels.
Enhance the map of the treatment area with an underlying satellite image, and add the appropriate map elements such as a north arrow, scale bar, legend, and title. Then add insets of a meteorological graph and a separate wind rose polar plot to show prevailing wind, wind speed, and variability throughout the spray period as well as a mortality color ramp so that the interpolated mortality coverage can be interpreted, and then export a finished map image for each mortality time period surface. In this representative series of sprays, the thermal fog pesticide sprayer, pesticide, and environment were kept constant, only varying the pesticide diluent such that water, diesel, and BVA13 mineral oil were used as the solvent in turn across the three trials.
Notably, almost no mortality was observed when the pesticide was diluted with BVA13, while a nearly 100%mortality was observed when the pesticide was diluted in water, with the effects of diesel as a diluent appearing to be affected by the geographic location of the sentinel cages. It should be noted that the inset wind rose diagram for this experiment revealed a wind angle that perfectly matched the angle of the 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 of the sentinel cages. If the mortality data are considered only in tabular form, it may be easy to miss this weakness in experimental design and to assign a lower overall efficacy to that pesticide application equipment biased by the zero mortality values from areas not contacted by the pesticide.
For the highest quality mortality data, take care when storing and handling the sentinel cages before and after the spray and to likewise ensure that the cages from both the treatment and control areas are equally exposed to the environment. For good-quality data tables, it is critical to carefully paste the data so that the correct information is aligned with the corresponding points in the correctly labeled columns, especially the data for each mortality time period. We've demonstrated this system using adult vector mosquitoes as sentinels, but immature mosquitoes can also be placed as sentinels in small 400-ml plastic cups filled with water to collect and evaluate sprayed larva sites in a variety of environments.
The sentinel and sex system is not an absolute measure of efficacy, but it proves the ability to compare the relative efficacy of a pesticide under different conditions or to compare different pesticides under the same conditions.
