We would like to thank the scientists and technicians at the Coachella Valley Mosquito and Vector Control District and the U.S. Army Medical Research Directorate-Kenya for expert production of colony insect specimens and collaboration on the field studies producing the unpublished data presented in the representative results. This research was supported by the U.S. Department of Agriculture (USDA)-Agricultural Research Service and the U.S. Department of Defense (DoD) Deployed War-Fighter Protection Program (DWFP). Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA, DoD, or DWFP. The USDA is an equal opportunity provider and employer.

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<li>Abbott, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+method+of+computing+the+effectiveness+of+an+insecticide%5BTitle%5D)+AND+%22Journal+of+Economic+Entomology%22%5BJournal%5D)">A method of computing the effectiveness of an insecticide.</a> Journal of Economic Entomology. 18, 265-267 (1925).</li>
<li>Healy, M. J. R. <a target="_blank" href="https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1744-7348.1952.tb00899.x">A table of Abbott's correction for natural mortality.</a> The Annals of Applied Biology. 39 (2), 211-212 (1952).</li>
<li>Britch, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+ULV+and+thermal+fog+mosquito+control+applications+in+temperate+and+desert+environments%5BTitle%5D)+AND+%22Journal+of+the+American+Mosquito+Control+Association%22%5BJournal%5D)">Evaluation of ULV and thermal fog mosquito control applications in temperate and desert environments.</a> Journal of the American Mosquito Control Association. 26 (2), 183-197 (2010).</li>
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</ol>

The authors have no conflicts of interest to disclose.

Combining the classic sentinel cage approach with electronic mapping of interpolated mortality data is a unique and powerful method to evaluate pesticides in the field, and it supports comparative pesticide efficacy studies across multiple environments and diverse configurations of pesticides and application techniques. Although the basic sentinel cage method is not new, the visualization of sentinel cage mortality patterns in a GIS is an advancement conducive to deeper analysis of patterns of flow of aerosol pesticide sprays. The interpolation of point measurements of pesticide efficacy into a color-coded map coverage is similar to adding smoke effusions to a wind tunnel to visualize air flow around an automobile, and it is a major improvement for reporting spatial and temporal mortality data in a series of tables.
Some parts of pesticide sprays can be seen with the naked eye, and pesticide droplets from invisible portions of sprays can be captured on glass slides or other media which, like sentinel cages, are long-established protocols. Evaporative products from droplets and droplets themselves can be captured with cotton ribbons and analyzed with a gas chromatograph/mass spectrometer, which provides even more information about the fate of a pesticide spray. However, the realized efficacy of the spray and spatial patterns of actual pesticide-induced mortality in the target area (which may include both droplet and evaporative product components) can only be definitively measured by sentinel insects.
Furthermore, the temporal component of efficacy can only be definitively measured by a series of mortality observations in sentinel insects that capture quantified indices of rapid knockdown versus long-term morbidity and mortality on the target species. Again, a series of interpolated color-coded maps can be used to clearly visualize the evolution of post-spray mortality over time, with an explicit spatial component, in a way that a series of tables is not able to communicate to a reader. The series of maps can be animated in a loop to reproduce the progress of the spray and its effects on sentinel insects, further enhancing the understanding of efficacy not only for a single trial, but in comparisons among pesticides, techniques, application equipment, target insects, and ecological zones.
For the highest quality mortality data in this method, great care should be taken throughout the start-to-finish handling and observations of the sentinel insects. Exposure time and conditions of sentinel insects to the environment across both the treatment and control areas before the pesticide spray should be as equal as practical. This exposure should include a period of acclimation to the ambient conditions and a uniform hold time post-spray for both treatment and control insects. Observations of mortality before the spray, after the hold time (i.e., during retrieval of cages), and for the designated periods post-spray should be carefully tracked on data forms. Care should also be taken to retrieve all sentinels from the field before departing for the lab. Control and treatment sentinel cages should be physically separated throughout the protocol. Reliable observations of background baseline mortality from the control cages at all designated time periods are critical to appropriate correction of observed mortality in the treatment zone. Accurate, precise, comparable, and meaningful efficacy maps can only be gained from high-quality mortality data input to the geographic information system.
The sentinel insect method is naturally flexible to be relevant in a variety of scenarios - anywhere a small cage can be placed, mortality data can be collected. For example, we have conducted pesticide trials with sentinel cages placed in and around simulated urban and rural buildings10 and U.S. military tents (unpublished data 2017-2018), in addition to multiple scenarios of desert, temperate, and tropical vegetation19,20,21,22 [including hoisting cages up to 60 ft into pine canopy to measure vertical mortality following a large aerial application (unpublished data 2011-2017)]. If the ground is too hard to place sentinel cage poles or it is preferred to place them on concrete or asphalt areas, simple stands or concrete blocks can be constructed to support the poles. For scenarios to investigate sprayed liquid larvicides, the protocol can be modified to place empty plastic disposable 1 qt cups to capture larvicide across sentinel locations. These cups can be later filled with water and mosquito larvae to measure efficacy of the application7,23,24,25. Use floor tiles with the sticky side up to keep cups in place in wind and keep lids nearby to rapidly cap and collect following the post-spray hold time. Alternatively, cups can be left in place to weather naturally or left open in a controlled environment to investigate the longevity of a residual larvicide treatment.
To investigate droplet density and droplet spectra throughout the application area, slide spinners can be placed near sentinel insect positions - though exercise caution that the vortex from the spinning slides does not affect the flow of the pesticide spray to the sentinel insects. Similar to mapping mortality, additional columns in the attribute table for the sentinel locations can be added for droplet and dye parameters to derive interpolated coverages. Note that adding droplet collection aspects will demand an increase in the number of personnel in the field, with dedicated teams for example to carefully collect slides and assist the spray operator with dye additives. With additional materials and teams of personnel, these methods may be merged to conduct simultaneous trials using larval and adult sentinels, multiple application modes (aerial, ground, portable), or pesticides side-by-side (see representative results).
Although the main protocol was written for mosquitoes, we have successfully conducted field trials with sand flies and filth-breeding flies as sentinels with only minor modifications to the sentinel cages and overall protocol. For example, it is not practical to sex adult sand flies or filth breeding flies so that mixed-sex batches are used in sentinel cages, as this will reduce load on the colony because fewer specimens are needed than when working with mosquitoes. For sand flies, a very fine mesh must be used for the sentinel cages; furthermore, sand flies are not anaesthetized but instead added with aspirators directly into fully assembled cages through a rubber slit glued over a hole cut in the side of the cylinder.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58440/58440fig8.jpg" /> 
Figure 8: Additional scenarios demonstrating the flexibility of the sentinel system. The sentinel protocol for investigating pesticide efficacy in the field is very flexible, as shown in sentinel cages hoisted at intervals up to 60 ft through pine canopy (A) and a nearby open area (B) to investigate capability of aerial ULV pesticide spray to penetrate canopy. The sentinel system can easily be adjusted to examine larvicide sprays targeting immature mosquito stages using disposable plastic cups to capture droplets (C) indoors and (D) outdoors in a simulated urban area. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58440/58440fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The efficacy mapping method is naturally flexible because it is based on interpolation which is a standard process in most GIS programs26. Generally, interpolation uses known data at set points to estimate data at nearby unsampled points. There exist several types of interpolation techniques27 which may be selected based on the spatial spread and density of the point mortality data. We have used inverse distance weighting (IDW), which assigns higher statistical weight to known data from points closer to the unknown points being estimated. Troubleshooting for the field portion of the method is centered on control sentinel mortality (i.e., if mortality is &#62; 25% in controls, and there is certainty that the application did not impact the control area, something in the environment other than the pesticide is causing mortality, which will confound analysis; then, the trial will need to be repeated or moved to another location). The most common vulnerabilities&#160;in the mapping portion of the method are production of&#160;quality data tables in the GIS, so it is critical to carefully paste data so that the right data are aligned with the right points (reading frame) and correctly label columns for each mortality time period, pesticide, application equipment, etc.
The sentinel insect method is not designed to be an absolute measure of efficacy. Rather, the method provides the ability to compare relative efficacy of a pesticide (under a given environment, application equipment, diluent, target insect, and technique) to the same pesticide under different conditions, or to compare different pesticides under the same conditions. The method does not include pesticide droplet or active ingredient capture, though apparatuses for investigating these aspects can easily be placed in the grid adjacent to sentinel cages. The sentinel cage method does not measure efficacy of pesticide sprays against vector insects in flight, which is possible but not practical28. A lively controversy exists whether the kind of mesh on sentinel cages effects measurements of pesticide spray efficacy14,29,30,31. However, this is not hugely relevant to our objective of investigating relative efficacy of a formulation across environments or several formulations within an environment (which can be conducted using a standard cage with a standard mesh type).
For example, a recent study compared sentinel cage mortality results across three similar aerial pesticide application experiments separated by decades, each using different sentinel cage methods, all with comparable results10. Similarly, controversy exists over whether to transfer sentinel insects to &#34;clean&#34; cages (i.e., cages that have not been sprayed). Again, data from sentinel insect mortality should be considered relative and not absolute and are not concerned with additional mortality resulting from sentinel insects contacting pesticide adhering to the cage or mesh. In fact, in natural environments, pesticide sprays will also adhere to natural surfaces that target insects may contact. We have previously found that mortality induced from handling mosquitoes, to include CO2- or cold-based anaesthetization, may exceed mortality from contacting a pesticide that could have been present on cages (unpublished data 2008). Another limitation is that the relevance and applicability of sentinel cage mortality results in the local natural populations of the target insect still not being fully known; however, the closer genealogically the colony-reared sentinel insects are to the local species, the stronger the applicability of the efficacy maps is to local populations.
In future variations of this method, it would be beneficial to include modifications to accommodate pesticide applications with unmanned aerial systems (UAS). Current developments with UAS use in operational vector control include pesticide applications (in particular, larvicide formulations targeted at immature mosquito habitat) over and through highly inaccessible locations. To gain relevant information from sentinel trials, sentinel insect stations would need to be placed throughout inaccessible locations to the greatest extent possible. An example of relevant information is testing the capability of UAS effectively reaching a certain area with larvicide, with the only line-of-sight piloting by an operator who cannot directly observe the target area. This scenario may require development of other UAS to deploy and retrieve sentinel cages or larvicide collection cups and others to record meteorology in these inaccessible target areas. Mortality data from such a scenario can be analyzed in the GIS as with established scenarios, with added map features such as effects of natural obstacles, microhabitat, and micrometeorology that may differ from such effects during more standard applications with truck, aircraft, or portable sprayers. Advanced capabilities of GIS such as visualization of 3-dimensional interpolations of mortality from enhanced sentinel placement in vertical and horizontal grids are also possibilities for both standard and emerging application technology.

Efficacy of public health pesticides targeting nuisance and disease-vector insects such as mosquitoes, sand flies, and filth-breeding flies is not uniform across desert, tropical, temperate, or urban ecological zones1. Certain key species in these three groups of insects are important vectors of parasites, viruses, filarial worms, and bacteria that cause significant diseases in humans, pets, and livestock worldwide. To best protect public and veterinary health, the environmental limitations of pesticides must be investigated to inform effective use of the most appropriate pesticide formulations and techniques. Public health pesticide manufacturers are not required by the U.S. Environmental Protection Agency to specify the expected efficacy of a formulation across a range of environments or target insects, yet these pesticides are used for mosquito and vector control across multiple ecological zones in the U.S. and around the world.
We have developed a research program to evaluate numerous combinations of pesticides and pesticide application equipment and techniques in hot-arid desert, hot-humid tropical, warm and cool temperate, and urban locations, in order to derive pesticide use guidelines specific to the target insect and environment1. In this program, we evaluate pesticides that target adult stages of mosquitoes and sand flies (adulticides) and immature stages of mosquitoes (larvicides) using pesticide application equipment that is hand carried, truck- or aircraft-mounted, and installed in fixed locations. Then, four major outdoor pesticide application techniques are evaluated: (1) ultra-low volume (ULV) or thermal fog aerosol space sprays of adulticides designed for rapid knockdown of target insects, (2) a variant of the first technique in which liquid larvicides are applied with ULV or thermal fog for short- or long-term suppression of immature stages of target insects, (3) timed misting sprays from fixed locations designed to repel or kill, and (4) low volume (LV) cold mist sprays of residual pesticides designed to apply long-lasting toxic or repellent coatings on a variety of natural or artificial substrates. Presented here are the detailed methods for conducting techniques (1) and (2) mentioned above. Methods for (3) will be presented in separate studies, and techniques in (4) are described in brief in earlier publications2,3,4.
To accomplish this complex research program, we designed a system of protocols to support efficient, cost-effective, portable, and repeatable/standardized evaluation of aerosol adulticide and larvicide techniques with diverse pesticides/equipment combinations across multiple environments. At the core of these protocols is the use of colony-reared sentinel mosquitoes (adults and immatures) and sand flies (adults) to indicate spatial and temporal patterns of pesticide efficacy. In the case of adulticide applications, sentinel adult mosquitoes or sand flies are contained in small single-use disposable cages distributed in structured arrays through the target area and an untreated control area. For larvicide applications, small single-use disposable cups are similarly distributed to collect sprayed larvicide droplets for later introduction of water and sentinel colony-reared immature mosquitoes. Next, we record percent mortality in sentinel cages, or percent adult development in sentinel cups, at set intervals post-spray and use these data to produce electronic maps of spatial and temporal efficacy in a geographic information system (GIS) that may be quantitatively compared between and among environments.
Using sentinel cages of colony reared insects to evaluate pesticide efficacy in the field is a long-established practice5,6, and using empty plastic sentinel cups for collecting sprayed larvicide is emerging in the literature7. However, our electronic mapping of efficacy to visualize spatial and temporal patterns of mortality is an innovation that greatly improves investigation of mortality otherwise presented in flat table formats. Also, the high throughput cage loading system and the modular cage deployment system adaptable to diverse scenarios described here are unique to our program. Other research programs approach evaluation of pesticide applications in the field differently. Current popular methods include capturing and analyzing dye-labeled pesticide droplets from sprays in the field on spinning glass slides8 or acrylic rods9, which is a long-established process producing data that can be electronically mapped and visualized.
One drawback is that droplet size and density measurements from collection media are only estimated from a small proportion of the total collection surface, with software-assisted microscopy of fields of view that are unfortunately highly subjective. Also, maps of droplet distribution and density do not fully illustrate pesticide efficacy, because the assumption is that the presence of a threshold number of droplets of a certain size automatically indicates target insect mortality. This assumption does not account for mortality from evaporative products from droplets through the target area that may also induce mortality10, or that a lower number of droplets or other droplet sizes may kill some proportion of target individuals. The original rationale10,11,12 is that an aerosol pesticide is designed to impinge small droplets on actively flying target insects. However, our observations in the field, including reductions in natural populations after spraying when target insects are not actively flying, suggest that droplets or evaporative products from droplets are reaching targets that are not flying but rather hidden in resting refugia (unpublished data 2011). Also, we have observed in analysis of a field spray application (via simultaneous droplet capture, pesticide active ingredient capture, and sentinel cages) that maps of droplet distribution, active ingredient distribution, and mortality are not concordant (unpublished data 2010).
Another popular approach to evaluating pesticide efficacy is deploying sentinel cages in a grid delineated in a flat mowed homogeneous field with no obstructions to the pesticide plume, and under near ideal meteorological conditions (e.g., consistent winds &#60;10 mph and wind direction perpendicular to the spray line). Still, others approach this by measuring efficacy with sentinels placed in wind tunnels14. These approaches provide one perspective on pesticide efficacy, but are less likely to realize operational efficacy under non-ideal field conditions (heterogeneous habitats that include obstructions to pesticide flow and variable if not under sub-optimal meteorological conditions). Seeking evidence in support of absolute efficacy is not realistic. Operational conditions are rarely ideal, and choosing formulations based on tests in wind tunnels or direct application in engineered parks may be misleading.
In our research, we use natural field sites and the given meteorological conditions (but not in rain or extreme winds that are outside the limits of any operational program). This is likely more informative for operational vector control when observing reasonable efficacy in a pesticide formulation despite poor environmental conditions, heterogeneous habitat, and obstructions to pesticide flow. Whenever possible, it is recommended to supplement sentinel insect mortality data with before- and after-surveillance of natural target insect populations in the treatment and control areas, as a bridge between controlled exposure and realized exposure to the focal pesticide. However, surveillance of natural populations is not sufficient to determine whether pesticide application produces mortality in a target population or whether target insects actually move from the target area after detecting oncoming pesticide aerosols.
Regardless of the caveats to any evaluation of pesticide sprays in the field, electronic mapping of mortality data in a GIS (as opposed to flat presentation of mortality data in tables) retains the quantitative attributes capable of rigorous comparison across trials and also provides a means for quick, visual assessment. With data captured in the GIS, researchers can set thresholds for pesticide efficacy and visualize the relative capability of a focal pesticide across multiple environments, or they can compare capabilities of multiple pesticides within a single environment across a variety of application equipment and techniques.

<table><tbody><tr><td>&lt;strong&gt;Bioassay racks: &lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>plastic tube lid</td><td>Visipak</td><td>192224</td><td></td></tr><tr><td>1.25-in PVC coupler SCH-40</td><td>Lowes</td><td>PVC 00100 0800</td><td></td></tr><tr><td>1/4-in OD brass rod</td><td>K&amp;amp;S Engineering</td><td>1165</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Bioassays: &lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>FDA silicone o-rings S-500-70</td><td>Alltek seal and packing</td><td>PA-2127-12</td><td></td></tr><tr><td>fine screening</td><td>Walmart</td><td>40310-0000-063 white</td><td>T-310</td></tr><tr><td>cotton balls</td><td>Fisher Brand</td><td></td><td>Large cotton balls (non-sterile)</td></tr><tr><td>plastic tubes</td><td>Visipak</td><td>775674</td><td></td></tr><tr><td>regulator</td><td>Norgren</td><td>R83-200-RNEA</td><td></td></tr><tr><td>reguilator gauge</td><td>Wika Instrument Corp</td><td>4315031</td><td></td></tr><tr><td>CO2 canister</td><td></td><td></td><td>20 lb capacity</td></tr><tr><td>CO2 chamber</td><td>Mainstays</td><td></td><td>Modified tupperware container (16 cup)</td></tr><tr><td>1/4-in tygon tubing</td><td></td><td></td><td></td></tr><tr><td>maglite aspirator and tubes</td><td>Bioquip</td><td>2809D</td><td>D-cell maglite aspirator</td></tr><tr><td>modified PVC pipe for o-rings</td><td>Lowes</td><td>PVC 07112 0600</td><td>SCH-40 pipe modified by cutting tool on inner surface to accommodate bioassay tube</td></tr><tr><td>Pupal separator</td><td>John W. Hock Co.</td><td>5412</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Field sentinel cages: &lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1/2 pt cardboard can body</td><td>Neptune</td><td>295</td><td></td></tr><tr><td>1/2 pt cardboard cup lid</td><td>Neptune</td><td>295A</td><td></td></tr><tr><td>coarse screening</td><td>Walmart</td><td>41721-0540-063 white</td><td>T-1721</td></tr><tr><td>Velcro cable ties 8-in x 1/2-in</td><td>Velcro Brand</td><td>VEL91140</td><td></td></tr><tr><td>rubber band</td><td>National Institutes for the Blind</td><td>7510-01-058-9974</td><td></td></tr><tr><td>cotton balls</td><td>Fisher Brand</td><td></td><td>Large cotton balls (non-sterile)</td></tr><tr><td>PVC spacers</td><td>Lowes</td><td>PVC 04010 0600</td><td>Modified by cutting into 18-in length pieces and cutting half off of the end (lengthwise)</td></tr><tr><td>Tray totes</td><td>Blue Ridge Thermalforming</td><td>400-3N-WHT-ABS</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Field bioassay set-up equipment:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>60-in tread-in post</td><td>Jeffers.com</td><td>T8T4</td><td></td></tr><tr><td>1 ft&lt;sup&gt;3&lt;/sup&gt; cardboard boxes</td><td>USP</td><td>S-18344</td><td></td></tr><tr><td>Deli cups</td><td>WNA Inc.</td><td>APCOMBO16</td><td></td></tr><tr><td>18-in x 18-in linolium tiles</td><td>Lowes</td><td>LSS4307BPS</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Sentinel cage transport: &lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>48 qt Island Breeze cooler</td><td>Igloo</td><td></td><td></td></tr><tr><td>16-in x 19-in. terry towels</td><td>Ability One</td><td>7920-01-454-1150</td><td></td></tr><tr><td>garbage bags</td><td></td><td></td><td>13 gal (kitchen size)</td></tr></tbody></table>

visualizing efficacy of pesticides against disease vector mosquitoes in the field

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.

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.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58440/58440fig6.jpg" /> 
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. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58440/58440fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58440/58440fig7.jpg" /> 
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. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58440/58440fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>PROJECT:</td>
			<td colspan="5"></td>
		</tr>
		<tr>
			<td></td>
			<td>ECOLOGICAL ZONE:</td>
			<td colspan="5"></td>
		</tr>
		<tr>
			<td></td>
			<td>LOCATION:</td>
			<td colspan="5"></td>
		</tr>
		<tr>
			<td></td>
			<td>FIELD SITE:</td>
			<td colspan="5"></td>
		</tr>
		<tr>
			<td></td>
			<td>DATE OF APPLICATION:</td>
			<td colspan="5"></td>
		</tr>
		<tr>
			<td></td>
			<td>SPRAY EQUIPMENT:</td>
			<td colspan="5"></td>
		</tr>
		<tr>
			<td></td>
			<td>PESTICIDE FORMULATION:</td>
			<td colspan="5"></td>
		</tr>
		<tr>
			<td></td>
			<td>DILUENT:</td>
			<td colspan="5"></td>
		</tr>
		<tr>
			<td></td>
			<td>SENTINEL INSECT SPECIES:</td>
			<td colspan="5"></td>
		</tr>
		<tr>
			<td></td>
			<td>LIFE STAGE OF SENTINELS:</td>
			<td colspan="5"></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="5">MORTALITY CHECKS</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">[copied directly from sentinel cages; data observed in field]</td>
			<td colspan="3">[observed from sentinel cages stored in trays]</td>
		</tr>
		<tr>
			<td></td>
			<td>Sentinel Cage Code</td>
			<td>No. PRE-DEAD</td>
			<td>No. DEAD POST-SPRAY/HOLD TIME</td>
			<td>No. DEAD 6 hr POST-SPRAY</td>
			<td>No. DEAD 12 hr POST-SPRAY</td>
			<td>No. DEAD 24 hr POST-SPRAY</td>
		</tr>
		<tr>
			<td rowspan="25">Treatment Area</td>
			<td>A1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A2</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A3</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A4</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B2</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B3</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B4</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C2</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C3</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C4</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D2</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D3</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D4</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E2</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E3</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E4</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="10">Control Area</td>
			<td>Control 1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Control 2</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Control 3</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Control 4</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Control 5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Control 6</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Control 7</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Control 8</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Control 9</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Control 10</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
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.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">PERCENT MORTALITY (ABBOTT-CORRECTED)</td>
		</tr>
		<tr>
			<td>Sentinel Cage Code</td>
			<td>(A) Aqualuer + Water</td>
			<td>(B) Aqualuer + Diesel</td>
			<td>(C) Aqualuer + BVA13</td>
		</tr>
		<tr>
			<td>A1</td>
			<td>1</td>
			<td>0.78</td>
			<td>0.54</td>
		</tr>
		<tr>
			<td>A2</td>
			<td>1</td>
			<td>1</td>
			<td>0.05</td>
		</tr>
		<tr>
			<td>A3</td>
			<td>1</td>
			<td>0.29</td>
			<td>0</td>
		</tr>
		<tr>
			<td>A4</td>
			<td>1</td>
			<td>0.04</td>
			<td>0.03</td>
		</tr>
		<tr>
			<td>A5</td>
			<td>1</td>
			<td>0.04</td>
			<td>0</td>
		</tr>
		<tr>
			<td>B1</td>
			<td>1</td>
			<td>0.79</td>
			<td>0</td>
		</tr>
		<tr>
			<td>B2</td>
			<td>1</td>
			<td>0.82</td>
			<td>0</td>
		</tr>
		<tr>
			<td>B3</td>
			<td>1</td>
			<td>0.15</td>
			<td>0</td>
		</tr>
		<tr>
			<td>B4</td>
			<td>1</td>
			<td>0.21</td>
			<td>0.15</td>
		</tr>
		<tr>
			<td>B5</td>
			<td>1</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>C1</td>
			<td>1</td>
			<td>0.93</td>
			<td>0</td>
		</tr>
		<tr>
			<td>C2</td>
			<td>1</td>
			<td>0.71</td>
			<td>0.01</td>
		</tr>
		<tr>
			<td>C3</td>
			<td>1</td>
			<td>0.32</td>
			<td>0</td>
		</tr>
		<tr>
			<td>C4</td>
			<td>1</td>
			<td>0.26</td>
			<td>0</td>
		</tr>
		<tr>
			<td>C5</td>
			<td>1</td>
			<td>0.46</td>
			<td>0</td>
		</tr>
		<tr>
			<td>D1</td>
			<td>1</td>
			<td>0.67</td>
			<td>0</td>
		</tr>
		<tr>
			<td>D2</td>
			<td>0.78</td>
			<td>0.24</td>
			<td>0.01</td>
		</tr>
		<tr>
			<td>D3</td>
			<td>0.97</td>
			<td>0.5</td>
			<td>0</td>
		</tr>
		<tr>
			<td>D4</td>
			<td>1</td>
			<td>0.27</td>
			<td>0.03</td>
		</tr>
		<tr>
			<td>D5</td>
			<td>1</td>
			<td>0.33</td>
			<td>0</td>
		</tr>
		<tr>
			<td>E1</td>
			<td>1</td>
			<td>0.82</td>
			<td>-</td>
		</tr>
		<tr>
			<td>E2</td>
			<td>1</td>
			<td>0.88</td>
			<td>-</td>
		</tr>
		<tr>
			<td>E3</td>
			<td>1</td>
			<td>1</td>
			<td>-</td>
		</tr>
		<tr>
			<td>E4</td>
			<td>1</td>
			<td>0.79</td>
			<td>-</td>
		</tr>
		<tr>
			<td>E5</td>
			<td>1</td>
			<td>0.78</td>
			<td>-</td>
		</tr>
	</tbody>
</table>
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.

Watch this Scientific Journal Video about Visualizing Efficacy of Pesticides Against Disease Vector Mosquitoes in the Field at JoVE.com

Visualizing Efficacy of Pesticides Against Disease Vector Mosquitoes in the Field

The efficacy of public health pesticides targeting nuisance and disease-vector insects is not uniform across different ecological zones. Here we present a system of techniques using captive vector insects as sentinels for pesticide efficacy to derive electronic maps supporting the standard evaluation of pesticides across multiple environments.

Efficacy of public health pesticides targeting nuisance and disease-vector insects such as mosquitoes, sand flies, and filth-breeding flies is not uniform ...

visualizing-efficacy-pesticides-against-disease-vector-mosquitoes

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8.5K Views.  United States Department of Agriculture, Agricultural Research Service Center for Medical, Agricultural, & Veterinary Entomology. The efficacy of public health pesticides targeting nuisance and disease-vector insects is not uniform across different ecological zones. Here we present a system of techniques using captive vector insects as sentinels for pesticide efficacy to derive electronic maps supporting the standard evaluation of pesticides across multiple environments.

Video: Visualizing Efficacy of Pesticides Against Disease Vector Mosquitoes in the Field

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the development of new and innovative methods of mosquito control based around the Sterile Insect Technique (SIT)1-3. A control method known as RIDL&#160;(Release of Insects carrying a Dominant Lethal)4, is based around SIT, but uses genetic methods to remove the need for radiation-sterilization5-8. A RIDL&#160;strain of Ae. aegypti was successfully tested in the field in Grand Cayman9,10; further field use is planned or in progress in other countries around the world.
Mass rearing of insects has been established in several insect species and to levels of billions a week. However, in mosquitoes, rearing has generally been performed on a much smaller scale, with most large scale rearing being performed in the 1970s and 80s. For a RIDL&#160;program it is desirable to release as few females as possible as they bite and transmit disease. In a mass rearing program&#160;there are several stages to produce the males to be released: egg production, rearing eggs until pupation, and then sorting males from females before release. These males are then used for a RIDL&#160;control program,&#160;released as either pupae or adults11,12.
To suppress a mosquito population using RIDL&#160;a large number of high quality male adults need to be reared13,14. The following describes the methods for the mass rearing of OX513A, a RIDL&#160;strain of Ae. aegypti 8, for release and covers the techniques required for the production of eggs and mass rearing RIDL&#160;males for a control program.

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

To achieve population suppression of Aedes aegypti using the RIDL&#174; (Release of Insects carrying a Dominant Lethal) system, large numbers of male mosquitoes need to be released. This requires the use of mass rearing techniques and technology to provide reliable systems to obtain the maximum number of high quality male mosquitoes.

New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the ...

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling ...

Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect.
ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP2,9.
Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission7. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.

Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection.  ATSs are derived from cDNA clones of ...

Immunology and Infection

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

Biology

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. Insecticide resistance surveillance of mosquito populations is typically done through Centers for Disease Control and Prevention (CDC) bottle bioassays or World Health Organization (WHO) tube tests. However, these methods can result in a high degree of variability in mortality data due to variable insecticide contact with&#160;the insect, the relatively small numbers of organisms tested, extensive variation in mass between populations, and constantly changing environmental conditions, leading to variable outcomes. This paper presents the topical application bioassay, adapted as a high-throughput phenotypic bioassay for both mosquitoes and fruit flies, to test large numbers of insects along&#160;a range of insecticide concentrations.
This assay 1) ensures consistent treatment and insecticide contact with every organism, 2) produces highly specific dose-response curves that account for differences in average mass between strains and sexes (which is particularly important for field-collected organisms), and 3) allows for the calculation of statistically rigorous median lethal doses (LD50), which are necessary for resistance ratio comparisons-an alternative surveillance approach from diagnostic dose mortality, which is also used for larvicide resistance surveillance. This assay will be a complementary tool for accurately phenotyping mosquito populations and, as illustrated using fruit flies, is easily adaptable for use with other insects. We argue that this assay will help fill the gap between genotypic and phenotypic insecticide resistance in multiple insect species.

We describe the methodology and importance of the topical application bioassay to measure insecticide susceptibility in mosquitoes and fruit flies. The presented assay is high-throughput, utilizes insect mass-thus allowing for calculating a mass-relativized lethal dose instead of concentration-and likely has lower variability than other similar methods.

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. ...

Non-invasive Electropenetrography to Study Mosquito Probing Behaviors

AC-DC Electropenetrography for the Study of Probing and Ingestion Behaviors of Culex tarsalis Mosquitoes on Human Hands

Mosquitoes transmit pathogens that negatively affect human and animal health. A greater understanding of their blood-feeding biology and interactions with hosts and pathogens could be exploited to develop new targets for controlling mosquito-borne diseases. Unfortunately, probing (i.e., biting) behaviors of mosquitoes are poorly understood because they occur inside host tissues. Here, a non-invasive procedure is described for using AC-DC electropenetrography (EPG) to indirectly visualize and quantify mosquito feeding behaviors by recording changes in electrical signals generated when probing and ingesting on human hands. Thin gold wires are attached to the mosquitoes using conductive silver glue and connected to the EPG instrument. The human host holds a substrate voltage probe in their hand. Probing of the host by the wired mosquito completes the electrical circuit, and electrical signals are recorded on a computer as &#34;waveforms&#34; that can then be measured and enumerated for analysis. This methodology has been used to characterize the probing and ingestion behaviors of Aedes aegypti and Culex tarsalis mosquitoes and can be applied to other mosquito species. EPG can be used to study the effects of pathogens, insecticides, and other factors on mosquito feeding behaviors.

Procedures are described for studying the probing and ingestion behaviors of Culex tarsalis&#160;mosquitoes on human hands using AC-DC electropenetrography.

Mosquitoes transmit pathogens that negatively affect human and animal health. A greater understanding of their blood-feeding biology and interactions with ...

Topical Testing Assay: A Technique to Evaluate the Insecticidal Potential of Dopamine Receptor Antagonists on Adult Female Mosquitoes by Topical Application

Source: Brito-Sierra, C.A. et al., <a href="https://jove.unicartagenaproxy.elogim.com/v/57768">Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes.</a> J. Vis. Exp. (2019).
In this video, we demonstrate the topical testing assay to determine the toxicity of a potential insecticide on adult female mosquitoes. Using a dopamine receptor antagonist that causes lethal inhibition of neurotransmission in mosquitoes, this method describes a way to evaluate the toxic effects of insecticides upon topical application.

Source: Brito-Sierra, C.A. et al., Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes. J. Vis. Exp. (2019).
In this video, ...

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Biochemical assays

Larval Contact Assay: A Procedure to Evaluate Mosquito Larval Mortality following Direct Contact with Test Larvicidal Compound

To begin, label the wells of a clear 24-well tissue culture plate. Use an analytical balance to weigh the test compound. Then, dissolve the compound in sterile, double-distilled water in a 1.5-milliliter tube, resulting in an 80-millimolar stock solution. Next, serially dilute the stock solution using double-distilled water to prepare working stock solutions of the desired concentrations.
Note that care must be taken when the larvae are added to the wells, during the removal of excess water, and when the test chemistry is added, to avoid physical damage to the larvae. Injury may increase mortality, and thus, produce false positive results or invalidate the assay.
Use a wide-bore plastic transfer pipette to transfer five third-instar larvae to each well of the plate. Then, use a 1-milliliter pipette to gently remove the water, and replace it with the desired volume of double-distilled water.
Take care not to touch the larvae when removing the water. Work quickly to ensure the larvae do not desiccate, and gently add the test chemistry by pipetting against the opposite side of the plastic well.
Add an appropriate volume of testing solution to each well, and gently rotate the plate to ensure uniform mixing. Place the plate in a growth chamber, maintaining 12-hour light-dark cycle at 25 degrees Celsius with 75% to 85% relative humidity.
To check larval movement, gently tap the plate. If no movement is observed, gently touch the larva with a sterile toothpick. Score the larva as dead if no response is noticed, and use a score sheet to record the total number of dead larvae in each well at the time points described in the text protocol.

EoE Sub Articles

Mosquito Feeding Assay: A Method to Evaluate Test Compound Effects on Mortality and Fecundity of Adult Female Mosquitoes

Collect approximately 150 four- to five-day-old adult female mosquitoes with an aspirator, and transfer them to a separate cage. Remove the source of sugar 1 to 24 hours prior to the feeding assay.
Prepare an 80-millimolar stock solution of the testing compound in water. Then, serially dilute it with water to obtain working stock solutions of the desired concentrations.
To obtain desired testing concentrations, add 40 microliters of each dilution to 960 microliters of defibrinated rabbit blood in a 1.5-milliliter tube, and mix by pipetting. Place a membrane filter on a feeding unit, and seal it with a rubber ring.
Next, use a pipette to transfer 1 milliliter of the blood with testing solution through the delivery port located on the reverse side of the feeding unit. Attach the feeding unit to a heating unit. Then, swab the membrane surface gently with freshly prepared 10% lactic acid solution.
Place the feeding unit in the cage. Cover the cage with a dark cloth, and allow the mosquitoes to feed for one hour. After the mosquitoes feed, place the cage in a refrigerator at 4 degrees Celsius for five minutes to anesthetize the mosquitoes. Then, count and record the total number of mosquitoes in the cage.
By examining the abdomen, count and record the total number of fully- and partially-fed female mosquitoes. A minimum of 50 blood-fed mosquitoes should be obtained per dose. Remove any mosquitoes that have not fed. Next, transfer the cage to a growth chamber and use a score sheet to record the number of dead mosquitoes at the appropriate time points.
On day 3 post-blood feeding, place an egg cup in the cage for 72 hours. Use a dissecting microscope to count the total number of eggs produced per treatment.

Visualizing Efficacy of Pesticides Against Disease Vector Mosquitoes in the Field

Summary

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Alphavirus Transducing System: Tools for Visualizing Infection in Mosquito Vectors

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

AC-DC Electropenetrography for the Study of Probing and Ingestion Behaviors of Culex tarsalis Mosquitoes on Human Hands

Topical Testing Assay: A Technique to Evaluate the Insecticidal Potential of Dopamine Receptor Antagonists on Adult Female Mosquitoes by Topical Application

Larval Contact Assay: A Procedure to Evaluate Mosquito Larval Mortality following Direct Contact with Test Larvicidal Compound

Mosquito Feeding Assay: A Method to Evaluate Test Compound Effects on Mortality and Fecundity of Adult Female Mosquitoes