Visualizing Efficacy of Pesticides Against Disease Vector Mosquitoes in the Field

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

1. Constructing adult sentinel cages
   1. Determine the total number of sentinel cages needed for the field trial by including treatment and control areas and the number of replicates planned, then add 5% to offset losses due to damage or unusual mortality (see step 2.1). Prepare 5 x 5 in tulle mesh squares (2 per sentinel cage) using a rolling disk cutter, straight edge, and cutting board with bulk tulle yardage. Store the tulle mesh squares in batches of 50 in 1 qt resealable plastic bags.
   2. Prepare the sentinel cages by carefully pushing the flat cardboard disks out of the bottoms of new ½ pt cardboard ice cream cups to create open-ended 2 x 3 in cylinders. For each cup, remove the cardboard disks out of 2 lids to create open-ended bezels (1 for each end of the cylinder). Secure one tulle mesh square to one end of the cylinder by trapping it under the bezel as the bezel becomes tightly fitted to the cylinder.
   3. For each sentinel cage, fasten a hook-and-loop cable tie to a rubber band, and secure the rubber band around the bezel that is in place. Bag the sentinel cages in batches of 50, with a separate bag containing 50 bezels and a bag of 50 tulle mesh squares.
      Note: Tulle mesh size should be the minimum gap size to contain the particular sentinel species to be placed in the cage. For example, Aedes aegypti mosquitoes will require a smaller mesh size than Culex quinquefasciatus mosquitoes.

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. Scheduling and rearing colony insects
   1. For sentinel insects, select a species that is most appropriate for the environment, season, and medical threat in the location where the trial will take place. Consider the colony capabilities and development time to adult for that species; for instance, consider if the colony will be able to provide the number of specimens needed for the number of sentinel cages required in the time frame.
   2. For all rearing, ensure population rearing cages are kept in a controlled environment and are large enough for the number of adults planned, and that a feeding and cleaning regime is established and carefully followed to reduce later stress-related mortality that will confound field efficacy trials. For extensive field trials, plan to rear sequential batches in separate well-labeled population cages.
   3. Use only female mosquitoes in sentinel cages, preferably 3-5 d post-eclosion. Mosquito pupae can be sexed under a dissecting scope or physically separated using a glass pupal separator to reduce sorting effort with adults at the sentinel cage loading step.
      Note: A colony must be able to supply double the number of mosquitoes planned for use in sentinel cages, because only females are used for sentinels. Use 10 adult females for each sentinel cage, as this number is quick to load, quick to count for mortality, and easy to determine whether any have escaped. It is essential to back-plan from the proposed spray date so that 3- to 5-day-old adult mosquitoes are available. However, the 3-5 d spread does provide some margin in case a weather hold prevents spraying.
3. Loading sentinel cages
   1. Prepare a CO₂ chamber by lining the bottom of a new ½ gal plastic food storage container with cotton balls. Direct one end of a 6-ft length of polyurethane tubing (¼ inch diameter) into the cotton balls through a tight-fitting hole punched low in one side of the container. Attach a 20 lb CO₂ bottle with a regulator to the other end of the tube and place the lid loosely on the container to allow gas to escape after diffusing through the cotton balls and circulating through the container. Tape the tube to the table top to prevent the lightweight chamber from being pulled or knocked to the floor. Set a flow rate appropriate to the species being used (start at ~7 L/min and make adjustments as needed).
   2. Prepare 10% sucrose solution in a new ½ gal plastic food container and submerge 100 cotton balls so they become fully saturated. Lay out the sentinel cages from step 1.1 in batches of 25 or 50 as space permits, with the tulle mesh squares and loose bezels on hand nearby.
   3. Remove mosquitoes from the population cage with a mechanical aspirator capable of channeling mosquitoes directly into removable ventilated collection tubes. No more than ~10 mosquitoes should be aspirated into each tube, and no more tubes than can be processed within 10 min should be filled to mitigate stress. Place 2-4 tubes at a time into the CO₂ chamber and set a timer for a 4-min exposure to anaesthetize the mosquitoes for subsequent sexing and transfer to sentinel cages.
   4. Once the timer chimes, remove all tubes from CO₂ chamber and carefully shake out anaesthetized mosquitoes from the tubes to scatter them evenly across a clean white 11.5 x 17-in sheet of paper. Set the timer for 4 min, which is the approximate time before mosquitoes will become active again. Use a pistol aspirator^15,16 or soft-touch insect forceps to pick out batches of 20 adult females and immediately place them in the waiting open sentinel cages. Once all females have been picked out of the anaesthetized batch, use a separate mechanical aspirator to collect all remaining male mosquitoes which will be destroyed by freezing.
   5. As each waiting open sentinel cage receives a batch of 20 adult female mosquitoes, trap 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. 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. Squeeze the majority of 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, easily accessible to the mosquitoes.
   6. Repeat steps 1.3.3-1.3.5 until all the sentinel cages are loaded.
      Note: The cage loading procedure should include a minimum of two personnel: one to conduct all mosquito handling steps and another to conduct all cage handling steps.
4. Loading sentinel cage coolers
   1. Prepare a 48-qt insulated picnic cooler for every 45-50 sentinel cages (depending on the make/size of cooler) by placing a wet 18 x 24-in cotton towel at the bottom covered by a 13-gal tall kitchen trash bag. The wet towel should be wrung out to the point of saturation but not dripping. Fold and arrange the 13-gal bag to completely cover the bottom of the cooler and at least 3 in from the bottom on all sides.
   2. Load the cooler by carefully placing sentinel cages in stacked layers with the cotton balls facing up. Depending on the make of the cooler, there should be about 10-12 cages per layer and 4-5 layers. If possible, slightly offset each layer so that a cage does not completely cover the cage below to promote air flow. Cover the top layer with a second 13-gal trash bag (folded like the bottom trash bag) and spread a second wet towel on top of this bag.
   3. Use duct tape to fasten a folded piece of cardboard in a front corner of the upper lip of the cooler, such that the lid is propped open about 3/8 in to promote air exchange and prevent overheating. In each cooler include a 1-qt resealable plastic bag with 10 spare cotton balls soaked in 10% sucrose solution, and a separate bag with 5 spare rubber bands and 5 hook-and-loop cable ties (these components are occasionally lost from sentinel cages during deployment in the field). Throughout the time in the coolers in the lab and field, top off the sucrose solution in all cotton balls on cages as needed, using a dropper and 10% sucrose solution stored in a rugged container.
      Note: With this method, typical colony mosquitoes may be loaded into sentinel cages and stored in coolers 24-28 h before use in field trials.
5. Transporting sentinel cage coolers to the field
   1. During transport to field site keep sentinel cage coolers in a vehicle with air conditioning but protected from direct sunlight. At field sites in mild to warm temperate environments, the coolers can be stacked outdoors to acclimatize or kept in vehicles with air conditioning running; but in both cases, keep coolers out of direct sunlight. Watch for any ambient conditions outdoors that could affect sentinels in the coolers (e.g., vehicle exhaust, trash fires, etc.)
   2. In extreme hot environments, keep coolers in vehicles with air conditioning running out of direct sunlight, but acclimatize the sentinels gradually to outdoor conditions before deployment to the treatment area to avoid death from shock. In all environments, consider testing one or two coolers of sentinel cages to determine how long they can be left out or how quickly they can be acclimatized to local outdoor conditions.

2. Preparation of the Field Site

1. Establishing the sentinel grid and control area
   1. Select a study area that provides sufficient space for dispersal of the planned mode of pesticide application (aerial, truck, or portable) with a clear path for movement of the spray device, and clear of human settlement or activity. If possible, include a variety of vegetation density and general heterogeneity of the habitat to challenge spray penetration.
   2. Design a grid of locations for sentinel cages that captures variations in distance from the sprayer, distance along the spray line, vegetation/obstacles to spray penetration, and height above the ground. Use a 300 ft measuring tape reel to mark convenient intervals from the sprayer and along the spray line; for example, use multiples of 25 ft intervals.
   3. Produce a map of the grid by marking up a printout of a satellite image with a clear numbering code for sentinel cage locations and distribute it to the field team. Whenever possible, obtain GPS coordinates for each cage location or at least coordinates of prominent features that can later be used for registration points to build a spatially coherent map in the GIS.
   4. Mark sentinel cage grid locations on the ground with flagging tape, glow sticks, and/or pin flags. Pesticide sprays are often conducted at dusk or night: attempt to clear distinct walking paths to all sentinel locations, including extra flagging tape and/or glow sticks to mark trip hazards or other significant obstacles.
   5. Install sentinel cage mounting poles into the ground according to the grid design, and clearly label each pole with a code for its unique position with a permanent marker. At least 48 inches of the pole should be above ground. If the habitat does not naturally provide vegetation/obstacle challenges to pesticide spray dispersion, consider placing a 1 ft³ cardboard box on its side with the top open next to each sentinel cage pole, to provide an additional sentinel location within simulated refugia. Air flow through the box can be adjusted by drilling ½-in holes in one or more sides, and boxes should be labeled with a unique position code with a permanent marker.
   6. If there is a risk of ants attacking sentinel mosquitoes, install sentinel cage mounting poles through a tight-fitting hole drilled in the center of an 18 x 18-in adhesive floor tile, with the sticky side facing upwards to trap ants before they can reach sentinels. The 1 ft³ boxes from step 2.1.5 can also be placed on the sticky side of such tiles to impede ants, and also to keep boxes in place in windy conditions.
   7. Establish an upwind control area (consisting of a similar habitat and meteorology as the treatment area) for the deployment of untreated control sentinel cages exposed to the environment on poles and/or in 1 ft³ boxes for the same amount of time as sentinels in the treatment area. A specific team of personnel with separate coolers of sentinel cages should be dedicated to handling all control sentinels.
   8. Resources permitting, design a distribution of appropriate traps throughout treatment area, areas adjacent to treatment area, and control areas to conduct surveillance of local natural populations of mosquitoes that are closest to the species selected in 1.2.1. Begin daytime or overnight surveillance, depending on the species, during one or more of the 12-24 h periods before the spray trials are to take place. Continue surveillance throughout the spray trials, followed by surveillance for one or more periods after the spray trials.

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.

2. Practicing safety
   1. Establish a communication system among the spray team, treatment area sentinel handling team, control area sentinel handling team, etc., via mobile phones (voice/text) or handheld radios.
   2. Supply personal protective equipment (PPE) to all personnel that are appropriate for their roles and appropriate for the pesticide formulation and application equipment.
   3. Establish a pesticide storage and mixing/loading area protected with secondary containment and stocked with emergency equipment, such as shovels and/or fire extinguishers for thermal fog applications, towels to contain spills or drips, waste containers, clean water, first aid supplies, and portable eyewash stations. Importantly, establish protocols in the cases of spills or exposure of personnel to chemicals.
3. Marking the spray line and recording meteorology
   1. For driving or hand-carry applications, make sure the spray line is clear and that obstacles are identified and clearly marked with flagging tape, pin flags, and/or glow sticks. For aerial applications, make all appropriate arrangements with air traffic control.
   2. The basic length of the spray line should match the dimensions of the grid; however, set the start and stop points 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 (see representative results).
   3. Whenever possible, anticipate major wind shifts, set up alternate spray line(s), and design the sentinel grid to be informative from both spray directions. The spray operator should be prepared to stop spraying briefly if the wind starts to gust badly or suddenly shifts, which can create a safety hazard. The operator can resume spraying when appropriate, and the trial can still produce usable efficacy data as long as controls are kept in the environment for a similar extended period.
   4. Place at least one weather station with a data recorder capable of measuring temperature, relative humidity, wind speed, and wind direction in the spray area and another in the control area. Also, set up a simple wind indicator ribbon (about 2-3 ft of bright colored flagging tape) on a spare sentinel cage pole in view of the spray operator and field team to increase overall awareness of conditions. For aerial applications, the aircraft should ideally be capable of reporting meteorology at release height.
      Note: Collaborate with a university, public health organization, mosquito and vector control district, or other agency with knowledge of the area proposed to conduct field trials, such that existing partnerships and understandings with landowners or land stewards may be built upon and help to develop the research program. Existing partnerships translate to site security, long-term use, and in some cases, critical buy-in and acceptance by the public. With structured grids, it is straightforward for local collaborators to develop their own numbering/labeling schemes for rapid and repeatable deployment, collection, and mortality checks of sentinel cages. Once the grid, spray line, and meteorology are set up, the materials can be left in place (for example, overnight until a convenient spray time or if there is a weather hold).
      CAUTION: Cardboard boxes can deform when wet if it rains, and strong winds can move the heavy adhesive floor tiles.

3. Deploying the Sentinel Cages

1. Determine in advance the positioning of sentinel cages at each sentinel point in the grid, such as the height of cage on the mounting pole (including multi-height, where one might be placed at the bottom of the pole and another at the top), or whether cages will be placed in the 1 ft³ boxes.
2. Assign 2-person sentinel cage deployment teams: one to hold the cooler and another to pull out sentinel cages as needed. Alternatively, assign individuals to carry and place multiple cages depending on the environment and size of the grid. Always include extra sentinel cages in case one is dropped or stepped on, tulle is torn, or one is lost while moving through the grid.
3. Per the positioning regime as described in step 3.1, use the hook-and-loop cable tie to affix sentinel cages to poles and make sure cotton balls with 10% sucrose solution are still in place under the rubber band. Place cages on their sides in the 1 ft³ boxes so that both tulle mesh ends are perpendicular to the ground. Orientation of cages on poles may be specified in advance; for instance, such that mesh tulle sides are in line with the planned spray direction and anticipated wind direction. In hot desert environments, angle the cages on the poles such that the insects are not in direct sunlight and may move into the shade of the side of the cage cylinder.
4. In both treatment and control areas, clearly label each sentinel cage with a permanent marker as it is put in place with two pieces of information: (1) the pole and/or box unique position code for later mapping in the GIS, and (2) pre-spray mortality for later correction for spray-induced mortality or mortality from exposure to the environment regardless of spray. For example, if pre-spray mortality is zero, write “pre-0”; if 2 specimens are dead, write “pre-2”, etc.

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

1. Assign a timekeeper to track application spray duration (start/stop times) and hold times using a stopwatch, clock, or digital video with time/date stamps. The timekeeper can also be assigned a scribe to capture multiple key variables including the time at which sentinel cage deployment is completed, flying, driving, walking speed, application device flow rate, pesticide concentration and diluent, spray time, hold time, time at which cage collection is completed, and general habitat observations.
2. Prepare the pesticide application equipment per manufacturer guidelines and load the pesticide (and diluent if necessary), using the minimum amount to cover the target area plus a pre-determined amount to account for spray line volumes and pesticide reservoir sump to prevent cavitation during spray. Do not exceed application rates specified on the pesticide label and do not use pesticides not approved by the U.S. Environmental Protection Agency, unless an experimental use permit specific to the location of the field trial has been secured.
3. Establish a sufficient post-spray hold time that will allow pesticide spray plume to disperse throughout the sentinel cage grid before the sentinel cages are collected. Hold time is based on a balanced consideration of habitat/vegetation density, meteorological conditions (in particular extreme heat or wind), application device, application mode (aerial, ground vehicle, portable), target insect, and pesticide formulation, and may require preliminary trials to develop effectively.
4. Once sentinel cages are labeled and deployed in both treatment and control areas and all key personnel have communicated readiness, have the spray operator conduct a final check on 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. Set the flying, driving, or walking speeds depending on application device flow rate and pesticide label rate.
5. Once the spray application is complete, have the timekeeper mark the beginning of the hold time and notify all personnel 1-2 min before the hold time will elapse, so that all personnel can move into position and prepare for collecting the sentinel cages.
   CAUTION: Observe all safety precautions specified on the pesticide label in addition to standard pesticide applicator best practices. Pesticide applications should be conducted by a certified pesticide applicator with, if possible, additional certification in the public health pesticide category. Before the spray application ensure all personnel are upwind of application area, or in vehicles with air conditioning on recirculate if they must be in the target area, and/or are wearing appropriate PPE. Ensure all sentinel cage coolers are closed and that inside closed vehicles parked upwind of the spray application have the air conditioning on recirculate.

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

1. Use the same sentinel cage teams to collect cages throughout the grid once the hold time has elapsed. All personnel handling post-spray cages should wear disposable nitrile gloves to minimize human contact with pesticides. Return post-spray cages to the protective environment of the sentinel cage coolers as soon as possible, stored in a cool vehicle out of direct sunlight, and returned to the lab for processing as soon as possible.
2. In both treatment and control areas, it is critical to record post-spray mortality on each sentinel cage with a permanent marker as it is collected. Post-spray mortality count should include all dead mosquitoes in the cage, and this number should be written directly adjacent to the “pre-“ count, but circled clearly enough to distinguish it from the pre-exposure mortality count. Later, the pre-count will be subtracted from the post-count to obtain the actual mortality from exposure to the pesticide (spray area) or environment (control area). Do not attempt to conduct this math in the field while hurrying to collect sentinel cages.
3. Attempt to return sentinel cages to the lab workspace within 6 h post-spray so that the 6 h mortality count can be recorded. In the lab, transfer all cages into plastic trays, discarding all rubber bands, hook-and-loop cables ties, and cotton balls. Each tray should hold approximately 20 sentinel cages stored with the mesh tulle parallel to the ground. Place a new 10% sucrose solution cotton ball on all cages and cover each tray with a new moist cotton towel, similar to those used in the sentinel cage coolers. Stack the trays on a lab bench or in an incubator using ½-in PVC pipe spacers. Store the trays with cages from treatment areas well-separated or in different incubators that control cage trays.
4. At this point, transfer pre- and post-spray mortality data from each cage to data forms as the 6 h mortality count is conducted: one person wearing disposable nitrile gloves should take sentinel cages out of trays one-by-one for each cage, calling out the 1) unique location code, then the 2) pre-count and the immediate post-count read from the cage itself, followed by the 3) current 6 h count observed directly. A second person should record these data on pre-prepared forms having a row for each unique position code from the trial (Table 1). Repeat this process at 12 h and 24 h post-spray, storing the trays in stable conditions in between mortality counts.
5. Once the 24 h mortality count is complete, freeze all trays of the sentinel cages to kill all specimens, and then conduct final counts for each cage to obtain an accurate denominator for calculating percent mortality. If a specimen is missing from a cage, look directly below it in the tray, where it may have dried out and fallen through. Dispose of all sentinel cages, cotton towels, and plastic trash bag liners in sealed garbage bags in normal trash, and thoroughly clean all coolers and trays with soap and water and air-dry them in direct sunlight.
   Note: Larval tray spacers are easily made from PVC stock pipe, with a notch cut in each end so they cannot roll off the trays. Spacers allow stacking of larval trays to save space in vehicles, incubators, or lab benches. For some field sites, it may not be possible to return to the lab within 6 h; in which case, transfer cages from coolers to trays in the field using the above protocol and materials and stack them carefully in the air-conditioned vehicle. Recording mortality in each cage immediately after the post-spray hold time, and at approximately 6 h, 12 h, and 24 h post-spray will provide information on knockdown (short-term) vs. long-term effects of the pesticide.

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

1. Transfer all hand-recorded mortality data from the forms into an electronic spreadsheet and calculate 4 new columns for post-spray, 6 h, 12 h, and 24 h mortality (all corrected for pre-spray mortality).
2. Correct all treatment area mortality data for control mortality in 4 additional new columns using the Abbott correction¹⁷: calculate a mean control mortality across all control sentinels for each time period that mortality was recorded (i.e., post-spray, 6 h, 12 h, and 24 h; all corrected for pre-spray mortality), and use this mean to Abbott-correct the mortality in each treatment area sentinel cage for each corresponding time period. The threshold for acceptable control mortality may be arbitrarily determined based on the species of sentinel mosquito used but should not exceed 25%¹⁸.
3. In a geographic information system map all unique sentinel cage locations using the GPS data gathered in step 2.1.3 and create 4 columns for Abbott-corrected mortality for the 4 mortality time periods in the attribute table for these locations. Create an interpolated coverage for mortality for each of the 4 mortality time periods across all unique sentinel locations in the treatment area. Add these interpolated coverages to the map and use a stretched color ramp on a blue to red spectrum oriented low (blue) to high (red).
4. Enhance the map of the treatment area with an underlying satellite image and add sentinel cage positions (distinguishing treatment and control sentinels), spray line, driving direction, scale bar, north arrow, interpolated mortality color ramp key, key to symbols, and a detailed title with date, habitat type, pesticide and diluent used, application equipment used, and target species. Export a map image for each mortality time-period surface.
5. Add meteorological information as an inset graph and include a separate wind rose (polar plot) to show prevailing wind, wind speed, and variability throughout the spray period. Also, provide separate meteorological data for release height in the case of aerial applications.
6. Graph local natural population surveillance data as an index of the number of females of each species collected per trap per night. Output a series of graphs partitioned by location (adjacent to treatment area, inside treatment area, and inside control area) and period (sampled prior to, during, or following the spray trials) and place them separately as insets in the main GIS map to show effects on natural populations over time within, adjacent to, and away from the pesticide application.