1. Bioassay Components

1. Terrestrial arena construction
   1. 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).
   2. 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.
   3. 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.
   4. 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.
2. 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.
   1. 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.
   2. For the aerial arena's collection containers, use screw top polymethylpentene containers (125 mL, 64 mm outer diameter, 74 mm height) and drill the bottoms (15 mm diameter).
   3. Into each of the containers' bottoms, affix threaded pipes (15 mm diameter, 60 mm length).
   4. 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.
   5. 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.
3. 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).
   1. For all experiments, use standard 5 mm LEDs with positive and negative leads. The LEDs can be narrow in their range of wavelength ± 5 nm or can be large in their wavelength range ± 50 nm.
   2. 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.
   3. 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.

2. Arena Preparation

1. 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.
   1. 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.
   2. Whenever the arena must be handled, wear nitrile gloves to avoid introducing human odors onto the surfaces of the arena.
2. 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.
3. Types of experiments
   NOTE: This setup is capable of single and competitive light testing.
   1. For single light testing, use one light emitting on a single cloverleaf while the rest of the clover leaves have nothing emitting from them.
   2. 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.
   3. Rotate LED positions after every replicate to control the potential effect of light interference between opposing light sources and any environmental preferences.
   4. 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.
4. Arena setup
   1. 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.
   2. Place the base plate of the arena on top of this pedestal. Assemble each piece of the arena on top of this base plate.
   3. 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.
   4. Install the light emitting diodes (LEDs) into the four collection container's LED holders.
   5. Set up the electrical equipment to control the lights.

3. Starting Bioassays

1. 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).
   1. 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 (< -20 °C for midges to -4.0 °C for mosquitoes).
   2. 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.
   3. 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.

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.

1. Record environmental conditions.
2. Stop recordings such as the infrared camera, if used.
3. 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.
4. 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.