Source insects
Sitophilus zemais were from a field population collected in eastern Kansas in 2019, reared continuously on organic whole corn (Heartland Mills, Marienthal, KS, USA) at the Center for Grain and Animal Health Research in Manhattan, KS. In subculturing S. zeamais, 75 adults were allowed to mate and lay eggs on 200 g of maize in a pint mason jar (950 mL) for 7 d, then were removed and adults were used 4\u20135 weeks later after emergence. For experiments below, adults that were 4\u20136 weeks old were used. Prostephanus truncatus were originally collected from its endemic range in Mexico in 2011 and reared continuously on whole organic corn in an APHIS-approved quarantine facility (permit#526-23-58-76547) at the Center for Grain and Animal Health Research in Manhattan, KS. Individual P. truncatus aged 4\u20136 weeks old were used in the experiments below. Both species were reared at 14:10 L:D, and S. zeamais was reared at 27.5\u00b0C and 65% relative humidity (RH) in an environmental chamber (Percival Scientific, Perry, IA, USA), while P. truncatus was reared in a quarantine space at 23 \u00b1 0.1\u00b0C.
Lights and pheromones
The visual stimuli consisted of 3 mm opening fiber optic light emitting diodes (LEDs) that were labeled blue, green, and warm white (Patikil, Dragonmarts Co. Ltd., Hong Kong, China). Pheromones consisted of monitoring lures with the Sitophilus spp. pheromone, namely (4S,5R)-5-hydroxy-4-methyl-3-heptanone, and the P. truncatus 2-component pheromone, 1-methyiethyl (2E)-2-methyl-2-pentenoate and 1-methylethyl (E,E)-2,4-dimethyl-2,4-heptadienoate (bullet lures IL-703 and IL-953, Insects Limited, Westfield, IN, USA).
Wiring of lights for wavelength and intensity trials
To evaluate the individual wavelengths of lights, we used blue, green, and white LEDs at 100% brightness. These were wired in parallel along a solderless bread board (5.5 \u00d7 17 \u00d7 3.5 cm W:L:H; MB102-830, HandsOn Tech, Johor, Malaysia) and supplied with a 9 volt/1 amp switching AC/DC wall plug that was modified to remove the end connector to expose the internal wires for attaching to the bread board (Figure 1).
To determine the effect of intensity of a wavelength on insect behavior, we used a single wavelength of LED at a time at 1, 2, 10, and 100% of the total brightness. These were wired in parallel with resistors to control the flow of electricity (Figure 1). A 1.1 k\u2126 resistor was used for the 10%, three 1.1 k\u2126 resistors were used for the 2%, and single 10 k\u2126 resistor was used for the 1%. They were wired in the same bread board, and supplied with the same power plug as the single brightness.
Assessment of light spectra
A plug-and-play lux meter/spectrophotometer (AH-300 PAR, AquaHotri ApS, Denmark) was attached to a cell phone (e.g., Apple iPhone) and was controlled with the accompanying AquaHotri application for Apple. In total, n = 4 measurements were taken from each LED described above at a distance of 1 cm away. Spectra were captured from outputted graphs using the autotracer feature with the Plot Digitizer Pro app (Porbital).
Determining preference among light wavelengths using a 4-way choice assay
To evaluate the preference by S. zeamais and P. truncatus among light stimuli, a 4-way choice olfactometer (True Choice Olfactometer, Sigma Scientific LLC, Micanopy, FL, USA) was adapted to evaluate choice among light stimuli instead of olfactory stimuli (Figure 2). Briefly, LEDs were placed into the ends of 8.4 \u00d7 3 cm (L:D) cylindrical tubes attached to each cardinal direction of the inert PTFE body of the choice assay (29.7 \u00d7 29.7 \u00d7 4.5 cm L:W:H) that was elevated by four 15-cm long PTFE legs. For each round of testing, an adult P. truncatus or S. zeamais was added to a 29.6-mL capacity portion cup (3.1 \u00d7 4.4 cm H:D) with 5 mm diameter holes drilled every 2 cm around the circumference at floor level, and had a layer of white tape on the inside and outside to block the light except from the added holes. The release zone allowed insects to perceive all wavelengths of light equally and subsequently egress. A piece of 0.5 cm thick glass (29.7 \u00d7 29.7 cm L:W) was placed over the insect and arena. There was a distance of 12.3 cm from the release center zone to the termination of the internal arena, with a total internal arena area of 423.5 cm2. On each cardinal direction, there was one of four choices: no light (control), blue LED, white LED, or green LED. Adults were tested in an environmental chamber under constant conditions (25\u00b0C, 65% RH; Percival Scientific, Perry, IA, USA) under indirect red light. Sitophilus zeamais and P. truncatus were given 2 or 3 min, respectively, to make a decision, which was considered completed when an adult was less than 6.5 cm from the end of the arena (e.g., marked with tape) towards a treatment. Individuals that did not make a decision in that period were tracked but excluded from the statistical analysis. The chosen treatment was recorded, and the time to decision was noted with a stopwatch that began as soon as the adult left the release zone in the portion cup. The position of the assay was rotated by 90\u00b0 after every insect to prevent positional bias by adults. After every 5 replicates, the arena was rinsed with 70% ethanol and allowed to dry to prevent deposition of chemical cues by adults. In total, 400 4\u20136-wk old, mixed-sex adults were tested, with a total of n = 200 adults per species for the combination of treatments.
Determining preference among different light intensities using a 4-way choice assay
To evaluate preference by these species among different intensities of light, we used an identical 4-way choice as above with the following modifications. Blue, white, and green lights were tested separately. On each cardinal direction of the assay, an LED of the same wavelength at 1, 2, 10, or 100% brightness was used. This corresponded to 50, 100, 480, and 4600 lux, respectively. Sitophilus zeamais or P. truncatus were singly added following the procedure above. In each case, the chosen intensity was recorded, and time to decision was also noted with a stopwatch. In total, 1,200 adults were tested in these assays, with n = 200 adults per species and combination of intensities for a given wavelength of LED.
Assessing synergy of light with pheromones in a wind tunnel assay
To determine whether the combination of visual stimuli has a synergistic effect on attraction of conspecific pheromones by S. zeamais and P. truncatus, a wind tunnel assay was employed. The wind tunnel was identical to that in prior work (Van Winkle et al. 2022). Briefly, a mechanical wind turbine generated airflow at 0.38 m/s, which was forced through three successive sieves: an activated charcoal filter to scrub background volatiles, and two progressively smaller slatted-metal sieves (73 \u00d7 85 \u00d7 0.5 cm L:W:H) to create a laminar flow. A total of 13.5 cm upwind of the stimulus edge of the test arena, a single pheromone lure alone, one of the wavelengths of LEDs (at 100% brightness), or both kinds of stimuli were placed, which was 5 cm from the last sieve. A single S. zeamais or P. truncatus was placed in the center of the 21.6 \u00d7 27.9 cm test arena, and insects were given 2 or 3 min, respectively, to make a decision. The side on which the insects left the test arena was recorded as the stimulus edge (e.g., side closest to the stimulus), anti-stimulus edge (e.g., edge farthest away from the stimulus), or non-stimulus edge (other two sides). In addition, the time to decision was recorded with a stopwatch. For the analysis, the four edges were collapsed into the stimulus edge, and non-stimulus edge (other three sides). Insects that did not make a decision within the timeframe were tracked but excluded from the statistical analysis. In total, 640 adults were tested with n = 40 adults tested per species and stimulus.
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