Insect Strains and Rearing Two field-derived strains of T. castaneum from either Eastern Kansas, collected in 2012, or Riley County, KS, collected in 2019, were used to assess the effect of strain on the behavioral response to necromones. Except where noted, the 2012 field strain was used for each experiment. T. castaneum was reared on a mixture of 95% unbleached flour and 5% brewer’s yeast in an environmental chamber at 27.5ºC, 60% RH, and 14:10 L:D. Subculturing proceeded by adding 75 mixed-sex T. castaneum to a 947-ml mason jar filled two-thirds with mixed diet. Adults were removed after 72 h of oviposition. Mixed sex adults aged 4–8 weeks old were used in all assays. All experiments were performed between the years 2017–2020. Treatments Time of Death of Prior Captures on Behavioral Response For investigating the attraction to kairomone oil based on how long beetles were left in the oil, the following treatments were included: negative control (neg ctrl), 950μL of Trécé Storgard® Kairomone Oil (kairomone oil for the remainder of the manuscript; Adair, OK, USA) only, or 950 μL of kairomone oil plus 25 freshly killed, mixed sex T. castaneum adults aged in the oil for 1, 25, 48, 72, or 96 h. A second round of the beetles aged longer than 8 days was included with the following treatments: negative control (neg ctrl), 950μL of kairomone oil only, or 950 μL of kairomone oil plus 25 freshly killed, mixed sex T. castaneum adults aged in the oil for 8, 9, 10, or 11 d (Table 1). These experiments were performed in a combination of the wind tunnel, release-recapture assay, and two-choice olfactometer (Table 1). Treatments were added to 20 mL GC headspace vials (Gerstel, GmBH, Germany) for wind tunnel assays, while they were added to Trécé Storgard™ Dome® traps in the release-recapture assays. Influence of Density of Prior Captures on Behavioral Response In order to evaluate whether the behavioral response of T. castaneum modulates with different densities of conspecifics in traps, the following treatments for the density response study were used: the same negative control, 950 μl of kairomone oil only, or 950 μl of kairomone oil plus either 4, 10, 20, or 40 mixed sex T. castaneum adults that were allowed to incubate for 24 h or 96 h. These experiments were performed in a combination of the wind tunnel, release-recapture assay, and headspace collection/GC-MS (Table 1). Treatments were added to 20 mL GC headspace vials (Gerstel, GmBH, Germany) for wind tunnel assays, while they were added to Trécé Storgard™ Dome® traps in the release-recapture assays. Effect of Strain on Behavioral Response to Prior Captures To rule out losing the attraction behaviors from laboratory-rearing protocols, a more recent T. castaneum strain was used and tested against the strain from Eastern Kansas collected in 2012. Thus, both a 2012 and 2019 field-collected (from Riley Co., Kansas) population of T. castaneum were tested in these experiments. The treatments for the strain effect consisted of a negative control, kairomone oil only, and 950 μl of kairomone oil plus either 4, 10, 20, or 40 mixed sex T. castaneum adults, which were allowed to incubate for 24 h. Both strains were tested in the wind tunnel and a release-recapture assay (Table 1). Effect of Rancidity on Behavioral Response to Prior Captures We conducted an experiment to test if long-term storage of the kairomone oil may have caused it to become rancid, despite being stored at 4ºC as per the manufacturer’s instructions. Treatments included: 950 μl of the kairomone oil we have used for most of our other experiments (e.g., standard Storgard® kairomone oil, or SSO) only, Storgard® kairomone oil borrowed from a colleague at the Center for Grain and Animal Health Research (CGAHR) (e.g., BSO), corn oil purchased freshly from the market (e.g., CO), or one of each of these treatments + 25 dead T. castaneum (Table 1). Attraction behavior was assessed in the wind tunnel. Assay Methods Wind Tunnel Assay Wind tunnel assays were used to evaluate upwind attraction by T. castaneum to putative necromones (e.g., see Van Winkle et al. 2022 for a description). Briefly, air was generated with a fan (diameter: 36.5 cm) connected to an inlet to the wind tunnel, where the air passed through an activated carbon filter to eliminate impurities from the air, and two successively smaller slatted-metal sieves (73 × 85 cm) to create a laminar airflow, with an average airspeed of 0.38 m/s. A purified, constant, laminar flow of air was pushed over the treatments 13.5 cm upwind of a release arena (21.6 × 27.9 cm). The odor treatments (Table 1) were positioned level with the surface of the release arena in the wind tunnel and were housed in 20 mL glass headspace vials. Caps were removed from the vials when testing commenced. The adults were placed individually in the center of the release arena and were given 2 min to make a decision, including either leaving on the stimulus edge (upwind) or a non-stimulus edge (three other edges). Adults that did not respond within the timeframe were excluded from statistical analysis. Adults were never tested more than once. All treatments were represented equally in a bout of sampling. The trials were performed inside a walk-in environmental chamber at constant conditions (27.5ºC, 60% RH), with air on purge to vent build-up of odors. Behavior was evaluated using a behavioral response index (BRI) as follows: [(T-C)/N]*100, where T is the number of adults in the treatment leaving on the stimulus edge of the arena, C is the equivalent number for the control, and N is the total sample size for both groups. The BRI can vary from 100 (full attraction) to -100 (full repellency). A total of n = 60 replicate individuals were tested, depending on assay, experiment, and treatment. Release-Recapture Assay Prior to release, 100 mixed-sex T. castaneum were settled on an 8 × 8 cm slat of cardboard for 24 h. The cardboard containing the adults was then placed in the center of a walk-in environmental chamber (5 × 6 × 2 m) set at a constant 25°C, 65% RH, and 14:10 L:D. Paper was fully laid and carefully taped on the bottom of the chamber floor to allow for easy mobility by T. castaneum. A standard Trécé Dome Trap™ that held one of each treatment (Table 1) was positioned equidistantly along the chamber’s perimeter and randomized between replicates. After 24 h, trap capture totals were calculated equal to the additional number of T. castaneum found in the trap minus those seeded in the original treatment. Experimental treatments were run simultaneously. A total of n = 8 replicates per treatment and experiment combination were used. Two-Way Olfactometer Trapping To assess preference among stimuli, T. castaneum individuals were evaluated in a two-way olfactometer. The olfactometer arena consisted of a Petri dish (9 × 1.5 cm diameter:height) with two holes drilled through opposite sides of the base at equal distances from the edge and the center of the dish. A filter paper (85 mm diameter), bisected by a faint line, was placed on the surface of the olfactometer so that the holes were on opposite sides of the filter paper (as in Morrison et al. 2020). The putative necromones (Table 1) were placed in separate, smaller Petri dishes (3.5 cm diameter) below the release arena and centered under each hole. The position of the lure and necromones was randomized between each trial. A single adult was placed in the center of the arena and left for 24 h in an environmental chamber at constant conditions (30C, 65% R.H., 14:10 L:D). A total of n = 10 replicates per comparison were performed. The percent of adults choosing each stimulus and becoming trapped in the bottom petri dish was recorded. Headspace Collection Volatiles were collected from traps seeded with 0 (oil only), 4, 20, or 40 dead T. castaneum and aged 24 h or 96 h. Central airflow was first scrubbed with a charcoal filter, then restricted to 1 L/min with flow meters. Airflow was guided through PTFE tubing to 500 mL-capacity headspace glass containers with lids and an inlet for air. The containers also had an outlet with a Porapaq-Q trap that collected volatiles for 3 h. Volatiles were then eluted with 150 µL of dichloromethane. An internal standard of 1 µL of tetradecane was also added prior to being run on the GC-MS according to standard methodology. There were n = 8 replicates per treatment. Gas Chromatography Coupled with Mass Spectrometry All headspace collection sample extracts were run on an Agilent 7890B gas chromatograph (GC) equipped with an Agilent Durabond HP-5 column (30 m length, 0.250 mm diameter, and 0.25 μm film thickness) with He as the carrier gas at a constant 1.2 mL/min flow and 40 cm/s velocity. This was coupled with a single-quadrupole Agilent 5997B mass spectrometer (MS). The compounds were separated by auto-injecting 1 μl of each sample under splitless mode into the GC-MS at room temperature (approximately 23°C). The GC program consisted of 40°C for 1 min followed by 10°C/min increases to 300°C and then held for 26.5 min. After a solvent delay of 3 min, mass ranges between 50 and 550 atomic mass units were scanned. Compounds were tentatively identified by comparison of spectral data with those from the NIST 17 library and by GC retention index. Using the ratio of the peak area for the internal standard to the peak area for the other compounds in the headspace, the emission rates of samples were normalized in ng of volatile per 950 μl aliquot of oil, per μl of solvent, and per h of collection.