1. Bioassay Components
<ol>
	<li>Terrestrial arena construction
	<ol>
		<li>Use metal flashing material strips of 2.54 cm length. Bend each strip into a half circle having a diameter of 15.24 cm (Figure 1).</li>
		<li>Connect the ends of each half circle and form a cloverleaf shape from the four pieces. Connect a locking nut and a #10 machine screw to the ends of each half circle to hold them together.</li>
		<li>Drill a hole at the midpoint of each semicircle 0.79 cm from the bottom of the arena. Affix 5 mm LED holders to the holes drilled in the middle of each semicircle.</li>
		<li>With the purpose of reducing reflection, spray matte black spray paint throughout the arena14 to cover the metal flashing. Use a liquid texture (e.g., Fluon) to prevent insects from climbing out of the arena over the spray paint.</li>
	</ol>
	</li>
	<li>Aerial arena construction 
	NOTE: The aerial arena has a similar size and dimension to the terrestrial arena; however, polyacrylic material was used (Figure 2). The clear plastic allows all light to pass through. The clear plastic prevents reflection that interferes with the experiment. The clear plastic also enables the experiment to be filmed.
	<ol>
		<li>Thread the furthest points of each semicircle to allow collection containers to screw onto the main arena. The cloverleaf shape sends insects back to the middle. No external corners encourage insect congregation; however, the collection cups for aerial insects have all internal corners and no external corners.</li>
		<li>For the aerial arena&#39;s collection containers, use screw top polymethylpentene containers (125 mL, 64 mm outer diameter, 74 mm height) and drill the bottoms (15 mm diameter).</li>
		<li>Into each of the containers&#39; bottoms, affix threaded pipes (15 mm diameter, 60 mm length).</li>
		<li>Attach a 5 mm LED holder to the lids of each container. Thread each collection cage lid onto the large orifice of the collection cages.</li>
		<li>Seat the threaded pipe from the arena in the small orifice on the opposite side of the collection cage. Ensure that the entire threaded pipe end is flush with the walls inside the arena and fits tight to the collection cage. 
		NOTE: The threaded pipe was made of Teflon. The Teflon glows the color of the LED that its collection cage holds. The threaded pipe was the only element that glows with respect to the insects in the arena due to a grey plastic substance at the base of each collection cage.</li>
	</ol>
	</li>
	<li>Electronics preparation 
	NOTE: There are various colors (wavelengths) of LEDs are dependent on chemicals used to construct the LED and therefore a broad variety of colors are possible (Table 1).
	<ol>
		<li>For all experiments, use standard 5 mm LEDs with positive and negative leads. The LEDs can be narrow in their range of wavelength &#177; 5 nm or can be large in their wavelength range &#177; 50 nm.</li>
		<li>Define the viewing angle as the maximum conical angle at which a display can be viewed. These are otherwise known as through hole LEDs. Through hole LEDs require either through hole slots on a PCB, a wiring harness, or wires soldered to the negative and positive terminals. Surface mount LEDs require proper PCB design and solder to incorporate them.</li>
		<li>Incorporate variable resistors into the electronics to control the LED power intake (LED intensity) (Figure 3). Use a light spectrometer to verify the intensity (W/m2) and wavelength (nm) of the LEDs for each experiment.</li>
	</ol>
	</li>
</ol>
2. Arena Preparation
<ol>
	<li>Before and between each replicate, carefully disassemble and clean the arena using an odorless, nonabrasive soap in warm water to remove any odors or unwanted attractants. Use a sponge with a low abrasive level to avoid scratching the arena.
	<ol>
		<li>Thoroughly dry the arena and set it aside to finish air drying in preparation for the next trial. This will prevent water spots from developing. Scratches and water spots can cause refraction on those points on the arena. Distortions create error in results.</li>
		<li>Whenever the arena must be handled, wear nitrile gloves to avoid introducing human odors onto the surfaces of the arena.</li>
	</ol>
	</li>
	<li>Record the following environmental conditions: humidity, temperature, barometric pressure, date, start/end time, external light sources, and LED positions in the arena. Record these values and monitor their trends from experiment to experiment. This ensures proper uniform experimental replicates, record the environmental conditions before and after the replicates.</li>
	<li>Types of experiments 
	NOTE: This setup is capable of single and competitive light testing.
	<ol>
		<li>For single light testing, use one light emitting on a single cloverleaf while the rest of the clover leaves have nothing emitting from them.</li>
		<li>For competitive experiment, emit light from all four cloverleafs with different characteristics in competition with each other. 
		NOTE: Other experiments can assess the importance of insect state (fed, starved, teneral, mated, blood fed, etc.) and life history stage. Behavioral recording/analysis software can be used to record and quantify behavior. For nocturnal experiments, infrared cameras can be used to view the insects, which will glow white in the IR recording in contrast to the dark arena.</li>
		<li>Rotate LED positions after every replicate to control the potential effect of light interference between opposing light sources and any environmental preferences.</li>
		<li>To count the number of collections for insects who do not go into holes, use infrared LEDs, an infrared camera, and software14. The video recording will show the number of beetle visits to each LED. A collection is not counted unless the insect moves from the center of the arena toward an LED as opposed to following an edge past an LED.</li>
	</ol>
	</li>
	<li>Arena setup
	<ol>
		<li>Set up a pedestal with four identical mason jars and place a black linen cloth on top of them. The linen cloth is black to keep light from reflecting off the bottom of the arena.</li>
		<li>Place the base plate of the arena on top of this pedestal. Assemble each piece of the arena on top of this base plate.</li>
		<li>Place the cloverleaf arena centrally around the release point in the base plate. Keeping this central allows the insects to emerge from the center of the experiment, giving them no initial preference.</li>
		<li>Install the light emitting diodes (LEDs) into the four collection container&#39;s LED holders.</li>
		<li>Set up the electrical equipment to control the lights.</li>
	</ol>
	</li>
</ol>
3. Starting Bioassays
<ol>
	<li>Place the clear lid of the arena over the arena parallel to the baseplate. If insects are released through the baseplate, the arena lid should already be on the arena. This contains insects and allows visual assessment or video recording (terrestrial insects).
	<ol>
		<li>If necessary by species (aerial insects), temporarily immobilize the insects to allow extraction from their (emergence) cages and allow arena introduction. Knocking the insects down can be accomplished with temporarily with carbon dioxide or a cold temperature (&#60; -20 &#176;C for midges to -4.0 &#176;C for mosquitoes).</li>
		<li>Using an aspirator, extract the desired sex and count of insects from the knocked down insects. Then, introduce the insects into the arena through the base plate. Use a pipe or other aspiration tool for insect extraction. Too much handling or long exposures will reduce survival.</li>
		<li>Start bioassay recordings/assessment before acclamation to confirm the insects are responding only to the light and not exhibiting an escape response. To avoid escape response, an provide an acclimation time of 1 h to the insects before powering on the electronics. Insects orient towards specific wavelengths of light during their escape response when placed into a new environment.</li>
	</ol>
	</li>
</ol>
4. Ending and Quantifying Bioassays
NOTE: The duration of each experimental replicate will depend on insect behavior and response timing, in general use a longer exposure, more responses tend to be more informative.
<ol>
	<li>Record environmental conditions.</li>
	<li>Stop recordings such as the infrared camera, if used.</li>
	<li>In the case of using collection chambers: after each replicate, place the cloverleaf arena into a freezer to kill the insects for quantification. The arena should not be left in the freezer for too long because the freezing environment may cause the plastic to crack.</li>
	<li>Quantify insect behavior by counting insect responders in collection cages or analyzing video. Insects that remained in the cloverleaf arena were counted as having made no choice. For example, Culicoides were found to be most attracted to UV light compared with making no choice7.</li>
</ol>

<ol>
	<li>Briscoe, A. D., Chittka, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+color+vision+in+insects.">The evolution of color vision in insects.</a> Annu. Rev. Entomol. 46, 471-510 (2001).</li><li>Srinivasan, M. V., Chahl, J. S., Nagle, M. G., Zhang, S. W., Srinivasan, M. V., Venkatesh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embodying+natural+vision+into+machines.">Embodying natural vision into machines.</a> From living eyes to seeing machines. , 249-265 (1997).</li><li>Srinivasan, M., Moore, R. J. D., Thurrowgood, S., Soccol, D., Bland, D., Barth, F. G., Humphrey, J. A. C., Srinivasan, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Biology+to+engineering:+insect+vision+and+applications+to+robotics.">From Biology to engineering: insect vision and applications to robotics.</a> Frontiers in sensing. , 19-39 (2012).</li><li>Allan, S. A., Day, J. F., Edman, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+ecology+of+biting+flies.">Visual ecology of biting flies.</a> Annu.Rev. Entomol. 32, 297-316 (1987).</li><li>Brown, A. W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+the+responses+of+the+female+Aedes+mosquito+Part+V.+The+role+of+visual+factors.">Studies of the responses of the female Aedes mosquito Part V. The role of visual factors.</a> Bull. Entomol. Res. 44, 567-574 (1953).</li><li>Brown, A. W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+responses+of+the+female+Aedes+mosquito+Part+VI.+The+attractiveness+of+coloured+cloths+and+Canadian+species.">Studies on the responses of the female Aedes mosquito Part VI. The attractiveness of coloured cloths and Canadian species.</a> Bull. Entomol. Res. 45, 67-78 (1954).</li><li>Snyder, D., Cernicchiaro, N., Cohnstaedt, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sugar-feeding+status+alters+biting+midge+photoattraction.">Sugar-feeding status alters biting midge photoattraction.</a> Med. Vet. Entomol. 30, 31-38 (2016).</li><li>Menzel, R., Shmida, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ecology+of+flower+colours+and+the+natural+colour+vision+of+insect+pollinators:+The+Israeli+flora+as+a+study+case.">The ecology of flower colours and the natural colour vision of insect pollinators: The Israeli flora as a study case.</a> Biological Reviews. 68, 81-120 (1993).</li><li>Walker, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+selected+thysanoptera+to+colored+surfaces.">Responses of selected thysanoptera to colored surfaces.</a> Environ. Entomol. 3, 295-304 (1974).</li><li>Lelito, J. P., Fraser, I., Mastro, V. C., Tumlinson, J. H., Baker, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+visual-cue-based+stickytraps+for+monitoring+of+emerald+ash+borers,+Agrilus+planipennis+(Col.,+Buprestidae).">Novel visual-cue-based stickytraps for monitoring of emerald ash borers, Agrilus planipennis (Col., Buprestidae).</a> J. Appl. Entomol. 132, 668-674 (2008).</li><li>Diclaro, J. W., Cohnstaedt, L. W., Pereira, R. M., Allan, S. A., Koehler, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+Physiological+Response+of+Musca+domestica+to+Colored+Visual+Targets.">Behavioral and Physiological Response of Musca domestica to Colored Visual Targets.</a> J. Med. Entomol. 49 (1), 94D100 (2012).</li><li>Steverding, D., Troscianko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+role+of+blue+shadows+in+the+visual+behaviour+of+tsetse+flies.">On the role of blue shadows in the visual behaviour of tsetse flies.</a> Proc. R. Soc. Lond. B. 271, 16-17 (2004).</li><li>Cohnstaedt, L. W., Gillen, J. I., Munstermann, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-emitting+diode+technology+improves+insect+trapping.">Light-emitting diode technology improves insect trapping.</a> J. Am. Mosq. Control Assoc. 24, 331-334 (2008).</li><li>Duehl, A. J., Cohnstaedt, L. W., Arbogast, R. T., Teal, P. E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+light+attraction+to+increase+trap+efficiency+for+Tribolium+castaneum+(Coleoptera:+Tenebrionidae).">Evaluating light attraction to increase trap efficiency for Tribolium castaneum (Coleoptera: Tenebrionidae).</a> J. Economic Entomol. 104, 1430-1435 (2011).</li><li>Schmid, R. B., Snyder, D., Cohnstaedt, L. W., McCornack, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hessian+Fly+(Diptera:+Cecidomyiidae)+Attraction+to+Different+Wavelengths+and+Intensities+of+Light-EmittingDiodes+in+the+Laboratory.">Hessian Fly (Diptera: Cecidomyiidae) Attraction to Different Wavelengths and Intensities of Light-EmittingDiodes in the Laboratory.</a> Environ. Entomol. 46 (4), 895-900 (2017).</li><li>Cohnstaedt, L. W., Snyder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+features+of+a+proposed+insecticidal+sugar+trap+for+biting+midges.">Design features of a proposed insecticidal sugar trap for biting midges.</a> Vet. Ital. 52 (3-4), 265-269 (2016).</li></ol>

Photo-attraction bioassays are an important tool to determine the optimal attractive color(s) and minimize the options for field trials of these colors. However, several factors must be considered when optimizing the bioassay for a specific insect including: Single Light vs. competitive light experiments, brightness, optimal spectral range, ambient light interference, state of the insects, and natural behaviors that may limit the possible responses.
Most insects have some phototaxis, which may be an innate escape mechanism causing the insect to move towards the light. This can be tested by providing a single light source in the arena and leaving the other three sides dark. However, a competitive test will have four colored lights and demonstrates color preference based on the insect response to each light. Bioassay users must determine if they are testing for light attraction or light preference. The competitive arena can be set up to look for repulsion as well. Remember that the insects may still not make a choice of light color, if they stay in the arena and do not orient towards a light. These no choice insects must be accounted for in the results.
Light emitting diode brightness must always be considered, and arena lights must be increased or decreased to the same intensity; therefore, it is important to test the brightness of the LEDs before each trial with a photo spectrometer. The potentiometers are important for controlling the voltage to each LED, which in turn adjusts the brightness. Commercially produced LEDs vary in voltage response and so even within a group of LEDs with the same spectra, each different LED must be evaluated, and the potentiometer adjusted before use. Even with this technique neutral density filters are sometimes needed to reduce the intensity of very bright bulbs. Snyder et al. (2016)8 and Schmidt et al. (2017)10 found brightness to be a significant factor in biting midge and Hessian fly collections with the brighter lights collecting proportionally more insects, although wavelength was the most important factor followed by brightness.
Bioassay users will benefit by testing narrow wavelength spectra LEDs. Snyder et al. (2016)8 found C. sonorensis able to differentiate between wavelengths (10 nm apart) and these elicited very different behavioral responses. Narrow wavelength LEDs will therefore be necessary to determine the optimal narrow wavelength of light for a given behavior.
External light can interfere with light attraction. Schmidt et al. (2017)10 found Hessian flies much more attracted to colors when given a dark arena than during a lighted one. However, in a crepuscular arena (partially lit), the lights worked the best. A dark arena blocks 100% of external light and is used to test nocturnal insects in their more natural visual environment. The arenas can also be used in natural light to simulate the visual environment of a diurnal insect, an important factor for ensuring attraction under real world trapping conditions.
Although visual attraction is important, olfactory attractants (pheromones, kairomones) can be added as in Duehl et al.(2010)16. This synergistic attraction increased trap collection. A long-distance attractant can aid in bringing individuals closer to the attractive light source and will greatly increase trap attraction14. For example, the pheromone used to attract the red flour beetles was a female sex pheromone. However, testing various stages such as fed, unmated, newly emerged, ovipositing, food/host seeking or other states may be important because they may have unique attractions as the may the various life history stages such as larvae, pupae, or adults. The trapping environment should also be considered, in food rich environments like flour mills food odor based attractants will be less efficacious.
Arenas may alter or influence insect behaviors even under controlled conditions such as fixed light levels, humidity, and temperature. The small areas or openings may be restrictive to natural insect movements. For example, in one trial Culex tarsalis mosquitoes did not enter the narrow openings in the collection cages (LW Cohnstaedt, personal observation) and house flies would not enter dark areas11. In some cases, these can be overcome by using sticky paper and catching the insects that go near the lights but will not enter cages or videotaping the insect behaviors. Therefore, all laboratory bioassay results need to be confirmed with field tests.
The light bioassay arena and protocol described are unique because they can be adapted to any terrestrial or aerial insect species. The arenas design accounts for high activity and low activity insects (the cloverleaf shape) and the lights are flexible for various competitive and non-competitive assays. Lastly this method can also accommodate most any life history trait (such as starved, sugar/host seeking, life history stage, etc.). These reasons help make this light bioassay a universal and flexible protocol for minimal time or money invested.

Almost all entomological surveillance depends on olfaction or visual attractants and often both. Volatile olfactory attractants may disperse throughout the environment resulting in a large attractive area. However, visual attractants may have a more limited range because of the invertebrate compound eye resolving images1,2,3. Therefore, visual attractants must be optimized to the insect of interest to maximize attraction and the trap designed to take advantage of the target insect&#39;s natural behaviors.
Visual attraction is based on wavelengths from the sun or other sources of light that are absorbed or reflected by an object&#39;s surface; organisms view this absorption/refraction of wavelengths as color. Insect vision has been found to include blue, green, and ultraviolet (UV) wavelengths1. Insects use their vision to aid in finding mates, food, and shelter4. Insects can visually define object sizes, colors, shapes, movements and contrasts5,6. Nocturnally active insects are generally attracted to light of differing contrast and intensity4, whereas diurnal insects can resolve colors and images, in addition to contrast because of greater photon availability during the day. Monitoring traps use the insect&#39;s visual cues to their advantage to optimize attraction and capture7.
The most common method of evaluating photo-attraction was observation of insect movement towards various colored shapes such as flowers8&#160;or objects (such as sticky cards9,10). Visual bioassays using colonized insects can help identify the optimal range of wavelengths and/or intensities, which reduces the number of field trials. Visual bioassays such as the &#34;Two-Sided Light Tunnel&#34; were designed for testing flies11. The problem with two sided light tunnels are that they do not account for insects that are not collected. Most insects will get stuck on internal corners and along edges in arenas. Also only two colors can be tested at one time. Other assays include the methods of Steverding &#38; Troscianko (2004)12, which narrowed tsetse fly attraction to broad bands (&#177;50 nm) of light colors. Light emitting diodes (LEDs) have been incorporated into traps to improve insect attraction by optimizing the wavelengths of emitted light1,13,14. Optimizing the visual attraction of these traps or monitoring devices will improve insect collection efficiency by using the insect&#39;s innate behaviors to lure insects. In this way, bioassay results are used to optimize existing trapping technology. The &#34;Terrestrial Arthropod Trap&#34; that improved the industry standard dome-type trap for red flour beetle surveillance (US patent# US8276314B2)) and the &#34;Method and Compositions for Improved Light Traps&#34; that incorporated of light emitting diodes into aerial insect traps (US patent# US2009/0025275A1). The two patents use LED technology that was optimized using the bioassay results to significantly improve insect traps.
This study describes a photo attraction bioassay arena and methods that allow investigators to evaluate the insect response to narrow wavelengths as a competitive or single attractive color. Equipment and experimental modifications are presented for nocturnal, diurnal, terrestrial, and aerial insects.

<table><tbody><tr><td>metal flashing material</td><td></td><td></td><td></td></tr><tr><td>#10 stainless steel machine screw</td><td></td><td>Stock</td><td></td></tr><tr><td>#10 stainless steel locking nut</td><td></td><td>Stock</td><td></td></tr><tr><td>5 mm LED holder</td><td>Radio Shack Corp</td><td>276-080</td><td></td></tr><tr><td>matte black spray paint</td><td></td><td>Stock</td><td></td></tr><tr><td>Fluon</td><td></td><td>Stock</td><td></td></tr><tr><td>molded polyacrylic</td><td></td><td></td><td></td></tr><tr><td>screw top Nalgene</td><td>Thermo Fisher Scientific</td><td>Nunc polymethylpentene</td><td>125 mL, 64 mm outer diameter, 74 mm height</td></tr><tr><td>Threaded Teflon pipes</td><td></td><td>Stock</td><td>15 mm diameter, 60 mm length</td></tr><tr><td>StellarNet light spectrometer</td><td>Stellar Net, Inc</td><td>BLACK Comet C-SR-25</td><td></td></tr><tr><td>LED infrared light source</td><td>Tracksys LTD</td><td></td><td></td></tr><tr><td>infrared video camera</td><td>Panasonic Corp</td><td>WV-BP330 Panasonic CCTV camera</td><td></td></tr><tr><td>MEDIACRUISE software</td><td>Canopus Corp</td><td></td><td></td></tr></tbody></table>

key elements of photo attraction bioassay for insect studies or monitoring programs

Optimized visual attractants will increase insect trapping efficiency by using the target insect&#39;s innate behaviors (positive photo-taxis) as a means to lure the insect into a population control or monitoring trap. Light emitting diodes (LEDs) have created customizable lighting options with specific wavelengths (colors), intensities, and bandwidths, all of which can be customized to the target insects. Photo-attraction behavioral bioassays can use LEDs to optimize the attractive color(s) for an insect species down to specific life history stages or behaviors (mating, feeding, or seeking shelter). Researchers must then confirm the bioassay results in the field and understand the limited attractive distance of the visual attractants.
The cloverleaf bioassay arena is a flexible method to assess photo attraction while also assessing a range of natural insect behaviors such as escape and feeding responses. The arena can be used for terrestrial or aerial insect experiments, as well as diurnal, and nocturnal insects. Data collection techniques with the arena are videotaping, counting contact with the lights, or physically collecting the insects as they are attracted towards the lights. The assay accounts for insects that make no-choice and the arenas can be single (noncompetitive) color or multiple (competitive) colors. The cloverleaf design causes insects with strong thigmotaxis to return to the center of the arena where they can view all the options in a competitive LED tests. The cloverleaf arena presented here has been used with mosquitoes, bed bugs, Hessian fly, house flies, biting midges, red flour beetles, and psocids. Bioassays are used to develop accurate and effective insect traps to guide the development and optimization of insect traps used to monitor pest population fluctuations for disease vector risk assessments, the introduction of invasive species, and/or be used for population suppression.

The terrestrial arena has been used to improve pest monitoring traps for red flour beetles14 and the aerial arenas for hessian flies15 and biting midges7. Although the cloverleaf arenas were similar, the conditions for each insect species were different and accommodated the evaluation of nocturnal or diurnal insects that can crawl or fly. More importantly these lab studies translated into field applications for monitoring insect pest population changes, introduction of invasive species, population suppression, and/or disease vector risk assessments.
The red flour beetles, a stored product pest, were evaluated in the terrestrial arena and filmed using an infrared camera14. Responses were considered positive for a color, if a beetle moved towards and contacted the LED. The arena setup was a competitive style with four lights or three lights and a dark blank for control. The trial data indicates the beetles were most attracted to near UV LED (390 nm) (Figure 4). This information was used to make a better red flour beetle trap using an octagonal UV LED array, which resulted in a 20% increase in collection compared to a 1% capture rate with the original pheromone attractant alone.
Hessian flies, wheat field crop pests were evaluated for photo attraction using the aerial arena with a diurnal setting15. Hessian flies were most attracted to green wavelengths with high intensities (Figure 5). Females preferred the green spectra of 502 and 525 nm. However, both sexes preferred high intensity light (16 W/m2). This is the first report of Hessian fly attraction to select emitted wavelengths and intensities from LEDs under controlled conditions. These results are being used to develop a better Hessian fly detection trap for uninfested wheat fields.
The disease vector biting midge, Culicoides sonorensis can transmit viruses, which in cervids, ovids, and bovids may result in epizootic hemorrhagic disease or blue tongue disease. C. sonorensis were tested using the aerial arena under nocturnal conditions to determine the optimal colors that attracted sugar seeking biting midges7. The highest proportions of biting midges were attracted to ultraviolet (UV) light and light intensity was important with the brightest lights being most attractive (Figure 6). Sugar-seeking and escape behaviors were triggered by 355 nm and 365 nm in wavelength respectively and the biting midges distinguished between the two-colored lights. Using these wavelengths, the attraction of C. sonorensis to light traps can be improved and the lights have been incorporated into insecticidal sugar traps16.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57445/57445fig1.jpg" /> 
Figure 1: This drawing reflects the dimension of the Terrestrial arena. The release point at the middle of the arena as well as points of LED attachments at the apex of each half circle are labeled. Also presented is an example of a conical light projection from an LED. The optimal viewing angle of the LEDs is 45&#176; although the arena design allows for more narrow or broad viewing angles as the half circles will limit light crossover except at the middle of the arena. The terrestrial arena has a lower profile compared to the aerial arena because the insects do not need space to fly, which helps video recordings stay focused on the insects. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57445/57445fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57445/57445fig2.jpg" /> 
Figure 2: The aerial light assay arena constructed from clear acrylic although it has all the same design benefits of the terrestrial arena but allows for more vertical space for flying insect evaluation. Four collection containers have LEDs of various wavelengths illuminating their respective apex of the cloverleaf. This figure shows the arena set up competition style with red, green, blue, and UV lights. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57445/57445fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57445/57445fig3.jpg" /> 
Figure 3: The electrical schematic of a 6 V DC power source attached to variable resistors (potentiometers) that control the power to each LED (light emitting diode) so the intensity of each LED can be adjusted independently. Neutral density paper can also be used to reduce the intensity without altering the emitted wavelengths. Wavelength and wavelength range are adjusted by selecting different LED chemistries. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57445/57445fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57445/57445fig4.jpg" /> 
Figure 4: (Top) The movement of ten red flour beetles for 5 min was assessed in the cloverleaf arena. A visit was defined movement towards a color resulting in touching the LED. Tested colors were blue (410 nm) and UV (390, 380, and 360 nm). Standard error bars are indicated and significant differences are denoted by letters (p&#60;0.0001), different letters indicate significantly different means. (Bottom) Further evaluation of movement with lower intensity colors was similar to above but with the colors UV (390 nm), green (555 nm), red (655 nm), and yellow (587 nm). (Figure 4 was reprinted from Duehl et al. 2011 with permission.) <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57445/57445fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57445/57445fig5.jpg" /> 
Figure 5: Males and female Hessian flies were evaluated for photo attraction separately to prevent cofounding factors. (A-C) are female fly responses and (D-F) are male. Significant differences are indicated by different letters (P &#60;0.05), different letters indicate significantly different means. (A and D) Both males and females were significantly attracted to green (527 nm) compared to red (624 nm), amber (590 nm), and blue (472 nm). (B and E) Within the green spectra 502-525 nm was most attractive and (C and F) intensity of light was important. (Figure was reprinted from Schmid et al. 2017 with permission.) <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57445/57445fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57445/57445fig6.jpg" /> 
Figure 6: (Top) Culicoides sonorensis were attracted significantly more to UV light than blue, green, or red. Different letters indicate significantly different means (P &#60;0.05), different letters indicate significantly different means. Sugar meals were provided prior to each replicate. (Bottom) Attraction to light intensity was assessed using Culicoides sonorensis movement towards the same UV light, but at different intensities (4, 8, and 12 watts) and a blue light (24 watts). (Figure was reprinted from Snyder et al. 2016 with permission.) <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57445/57445fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Table 1: General LED table for wavelengths. More narrow LED wavelengths do exist; this list just shows broad ranges of LEDs that exist in the insect&#39;s vision spectra. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57445/Table_1.docx">Please click here to download this file.</a>

Watch this Scientific Journal Video about Key Elements of Photo Attraction Bioassay for Insect Studies or Monitoring Programs at JoVE.com

Key Elements of Photo Attraction Bioassay for Insect Studies or Monitoring Programs

Photo-attraction bioassay arenas are used to determine the optimal light color(s) to maximize insect attraction; however bioassays and methods are specific to target insect behaviors and habitats. Customizable equipment and modifications are explained for nocturnal or diurnal and terrestrial or aerial insects.

Optimized visual attractants will increase insect trapping efficiency by using the target insect&#39;s innate behaviors (positive photo-taxis) as a means ...

key-elements-photo-attraction-bioassay-for-insect-studies-or

Nanyang Technological University

Research

JoVE Journal

Behavior

7.7K Views.  Agricultural Research Service, United States Department of Agriculture. Foto-attractie bioassay arena's zijn gebruikt om te bepalen van de optimale lichte kleur(en) te maximaliseren insect attractie; bioassays en methoden zijn echter specifiek te richten op insecten gedrag en habitats. Aanpasbare apparatuur en wijzigingen worden uiteengezet voor nachtelijke of dagverloop en terrestrische of luchtfoto insecten.

Video: Key Elements of Photo Attraction Bioassay for Insect Studies or Monitoring Programs

A Wind Tunnel for Odor Mediated Insect Behavioural Assays

Olfaction is the most important sensory mechanism by which many insects interact with their environment and a wind tunnel is an excellent tool to study insect chemical ecology. Insects can locate point sources in a three-dimensional environment through the sensory interaction and sophisticated behavior. The quantification of this behavior is a key element in the development of new tools for pest control and decision support. A wind tunnel with a suitable flight section with laminar air flow, visual cues for in-flight feedback and a variety of options for the application of odors can be used to measure complex behaviour which subsequently may allow the identification of attractive or repellent odors, insect flight characteristics, visual-odor interactions and interactions between attractants and odors lingering as background odors in the environment. A wind tunnel holds the advantage of studying the odor mediated behavioural repertoire of an insect in a laboratory setting. Behavioural measures in a controlled setting provide the link between the insect physiology and field application. A wind tunnel must be a flexible tool and should easily support the changes to setup and hardware to fit different research questions. The major disadvantage to the wind tunnel setup described here, is the clean odor background which necessitates special attention when developing a synthetic volatile blend for field application.

Here, we describe the construction and use of a wind tunnel for odor mediated behavioural assays with insects. The wind tunnel design facilitates the release of odor sources by several methods, with and without visual stimuli. Wind tunnel experiments are important methods to identify behaviorally active volatile chemicals.

Olfaction is the most important sensory mechanism by which many insects interact with their environment and a wind tunnel is an excellent tool to study ...

Low-Cost Automated Flight Intercept Trap for the Temporal Sub-Sampling of Flying Insects Attracted to Artificial Light at Night

Sampling methods are selected depending on the targeted species or the spatial and temporal requirements of the study. However, most methods for passive sampling of flying insects have a poor temporal resolution because it is time-consuming, costly and/or logistically difficult to perform. Effective sampling of flying insects attracted to artificial light at night (ALAN) requires sampling at user-defined time points (nighttime only) across well-replicated sites resulting in major time and labor-intensive survey effort or expensive automated technologies. Described here is a low-cost automated intercept trap that requires no specialist equipment or skills to construct and operate, making it a viable option for studies that require temporal sub-sampling across multiple sites. The trap can be used to address a wide range of other ecological questions that require a greater temporal and spatial scale than is feasible with previous trap technology.

To study the impacts of artificial light at night (ALAN) on nocturnal flying insects, sampling needs to be confined to nighttime. The protocol describes a low-cost automated flight intercept trap that allows researchers to sample at user-defined periods with increased replication.

Sampling methods are selected depending on the targeted species or the spatial and temporal requirements of the study. However, most methods for passive ...

Environment

Vertical T-maze Choice Assay for Arthropod Response to Odorants

Given the economic importance of insects and arachnids as pests of agricultural crops, urban environments or as vectors of plant and human diseases, various technologies are being developed as control tools. A subset of these tools focuses on modifying the behavior of arthropods by attraction or repulsion. Therefore, arthropods are often the focus of behavioral investigations. Various tools have been developed to measure arthropod behavior, including wind tunnels, flight mills, servospheres, and various types of olfactometers. The purpose of these tools is to measure insect or arachnid response to visual or more often olfactory cues. The vertical T-maze oflactometer described here measures choices performed by insects in response to attractants or repellents. It is a high throughput assay device that takes advantage of the positive phototaxis (attraction to light) and negative geotaxis (tendency to walk or fly upward) exhibited by many arthropods. The olfactometer consists of a 30 cm glass tube that is divided in half with a Teflon strip forming a T-maze. Each half serves as an arm of the olfactometer enabling the test subjects to make a choice between two potential odor fields in assays involving attractants. In assays involving repellents, lack of normal response to known attractants can also be measured as a third variable.

A vertical, T-maze olfactometer is described for assaying the behavioral response of arthropods. The olfactometer allows the experimenter to measure choices performed by test subjects when subjected to two potential odor fields. Both attraction to and repulsion from odorants can be measured with this device.

Given the economic importance of insects and arachnids as pests of agricultural crops, urban environments or as vectors of plant and human diseases, ...

Neuroscience

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

Biology

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

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 ...

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model to study animal avoidance behavior. This protocol introduces a light-spot assay to investigate larval phototactic behavior. The experimental set-up includes two main parts: a visual stimulation system that generates the light spot, and an infrared light-based imaging system that records the process of larval light avoidance. This assay allows tracking of the behavior of larva before entering, during encountering, and after leaving the light spot. Details of larval movement including deceleration, pause, head casting, and turning can be captured and analyzed using this method.

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

This protocol introduces a light-spot assay to investigate Drosophila larval phototactic behavior. In this assay, a light spot is generated as light stimulation, and the process of larval light avoidance is recorded by an infrared light-based imaging system.

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model ...

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. ...

Assessment of Phytochemical Effects on Helicoverpa armigera Through Feeding Assay

Developing a Feeding Assay System for Evaluating the Insecticidal Effect of Phytochemicals on Helicoverpa armigera

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and agricultural productivity. In response, plants produce several phytochemicals that negatively impact the insect's growth and survival. This protocol demonstrates an obligate feeding assay method to evaluate the effect of a phytochemical (quercetin) on insect growth, development, and survival. Under controlled conditions, the neonates were maintained until the second instar on a pre-defined artificial diet. These second-instar larvae were allowed to feed on a control and quercetin-containing artificial diet for 10 days. The insects' body weight, developmental stage, frass weight, and mortality were recorded on alternate days. The change in body weight, the difference in feeding pattern, and developmental phenotypes were evaluated throughout the assay time. The described obligatory feeding assay simulates a natural mode of ingestion and can be scaled up to a large number of insects. It permits one to analyze phytochemicals' effect on the growth dynamics, developmental transition, and overall fitness of H. armigera. Furthermore, this setup can also be utilized to evaluate alterations in nutritional parameters and digestive physiology processes. This article provides a detailed methodology for feeding assay systems, which may have applications in toxicological studies, insecticidal molecule screening, and understanding chemical effects in plant-insect interactions.

Author Spotlight: Development and Challenges of Feeding Assays for Insect Pest Management 

This protocol describes the obligate feeding assay to evaluate the potentially toxic effect of a phytochemical on the lepidopteran insect larvae. This is a highly scalable insect bioassay, easy to optimize the sublethal and lethal dose, deterrent activity, and physiological effect. This could be used for screening eco-friendly insecticides.

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and ...

Light Spot Assay: A Method to Investigate Drosophila Phototactic Behavior

Source: Sun, Y., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/60235/" target="_blank">Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis</a>. J. Vis. Exp. (2019).
Drosophila larvae are light-sensitive and respond to changes in surrounding light. This video describes an assay that tests their ability to avoid light, called the light spot assay. The featured protocol demonstrates how to set up the assay with a blue light LED and shows how to record the animal&#39;s behavior in response to the light encounter.

Source: Sun, Y., et al. Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis. J. Vis. Exp. (2019).
Drosophila larvae are light-sensitive ...

Key Elements of Photo Attraction Bioassay for Insect Studies or Monitoring Programs

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