DOI: 10.15482/usda.adc/29645753.v1
title: Data from: Elucidating the behavioral ecology to conspecific emissions by the native red sunflower seed weevil, <i>Smicronyx fulvus</i>, a resurging pest of sunflower

description: <i>Insects</i>The<i> S. fulvus</i> used in this experiment were progeny of field-collected weevils. As described by Prasifka et al. (2015), adult weevils were collected from wild sunflowers in North Dakota, then used to artificially infest cultivated sunflower heads in Casselton, North Dakota. After <i>S. fulvus</i> oviposition and larval development, sunflower heads were cut and transported to the laboratory. Heads were suspended over plastic tubs to catch exiting weevils, and weevils were placed into moistened sand to overwinter in an environmental chambers set to a fluctuating thermal regime (see ‘FTR’ treatment description in Prasifka et al. 2015) for at least 90 d. Subsequently, larvae were allowed to pupate and emerge from the moistened sand at room temperature (23–25°C) in the laboratory. Emergence of new adults was checked on a daily basis, and the water balance of the sand was kept consistent by weighing the sand initially after placing on lab bench, and adding water on a weekly basis to ensure weight was constant. The experiments were conducted at the USDA Center for Grain and Animal Health Research in Manhattan, KS and weevils were checked daily for emergence and then used immediately for experiments.<i>Semiochemical-mediated movement assay</i>To elucidate the movement of <i>S. fulvus</i> in response to conspecific extracts, video-tracking was used to monitor individual male and female <i>S. fulvus</i> behavior. This was performed by tracking movement with a network camera (Basler AG, Ahrensburg, Germany) coupled with Ethovision XT (v. 14.0, Noldus Software, Leesburg, VA, US). Individuals of each sex were placed individually into arenas consisting of 90 mm (D) Petri dishes lined with 85 mm filter paper (Whatman #1 filter, GE Healthcare, Chicago, IL, USA), and covered with a lid to prevent flight. The camera was placed 80 cm above the arenas, and trials lasted 30 min. Each arena had four treatment zones: a stimulus half, stimulus zone (1.6 cm diameter zone in the center of the stimulus half of the dish with the treatment), control half, and control zone (1.6 cm diameter zone in the center of the control half of the dish without the treatment) (e.g., similar to the setup in Ponce et al. 2023). The control half/zone always contained a blank filter paper, while the stimulus half/zone contained a filter paper with a conspecific volatile extract (as above) or solvent only. To account for cursor bounce, an input filter was applied that discarded the accumulated distance if it was &gt;5 cm per s. Each trial was manually checked for irregularities, and any with unusual patterns in the accumulations of distance were re-run. At the end of the trial, the total distance moved (cm), the average instantaneous velocity (cm/s), frequency of entering each zone, duration in each zone, and latency to finding each zone were recorded. There were a total of n = 16 replicate females and n = 18 replicate males.<i>Volatile Collection</i>We used two volatile collection methods to sample cohorts of <i>S. fulvus</i>, and included the following treatments: mixed sex (8 males, and 8 females), female only (12 adult female weevils), male only (12 adult male weevils), empty control (unbaited), sunflower only (freshly cut sunflower head of dwarf sunflower inbred line HA 379), and mixed-sex <i>S. fulvus</i> on sunflowers (8 males and 8 females on a freshly cut dwarf sunflower head). The first method was solid phase micro-extraction (SPME), which utilized a 100 μm polydimethylsiloxane (PDMS) fiber to collect the headspace volatiles inside a container with a 250 mL capacity and removable PTFE septa for a 24 h period per replicate. Fibers were then inserted directly into the gas chromatograph-mass spectrometer (GC-MS) injection port to be desorbed. Fibers were always pre-conditioned at 230°C in a GC injection port for 5 min prior to sampling to eliminate any volatiles that were adsorbed prior to the experiment. There were n = 3 control, 3 female, 4 male, 5 mixed, and 3 sunflower only samples collected by SPME. Mixed treatments had 4 males and 4 females, while single-sex treatments had 8 male or female.For the second volatile collection method, in order to potentially quantify samples, we used a solvent extraction method with a volatile collection trap (VCT) attached to a volatile headspace chamber (500 mL capacity; 10.2 × 12.7 cm D:H). Central air was filtered and run through chemically inert PTFE tubing with airflow restricted to 1 L/min using a flow meter (Volatile Collection Systems, Gainesville, FL, USA) just prior to the headspace chamber. The headspace volatiles from the <i>S. fulvus</i> treatments (mixed sex, empty control, and sunflower only) were collected for 24 h on a VCT consisting of a drip tip borosilicate glass tube packed with 20 mg of absorbent Porapak-Q™ (Volatile Collection Systems, Gainesville, FL, USA) to adsorb volatiles with a stainless steel screen (No. 316) on one side, and held in place with a borosilicate glass wool plug followed by a PTFE Teflon compression seal. The volatiles on the traps were then eluted with 150 µL of dichloromethane (Millipore, Billerica, MA, USA) by pushing the solvent through with inert N<sub>2</sub> gas into 2-mL glass vials containing vial inserts with polymer feet and screwtop magnetic caps containing Teflon-lined septa. There were n = 7 Control, 6 Mixed male and female weevil, and 4 Sunflower only replicate samples. During processing, all samples for which no peaks were detected were removed, leaving 3 samples for each of the Control, Mixed, and Sunflower treatments. The VCTs were washed between runs with 700 μl of dichloromethane in triplicate. Samples were then stored at -20°C until batch analysis. Prior to analysis, 190.5 ng of tetradecane (99% purity, GC analytical grade, Millipore, Billerica, MA, USA) was added as an internal standard.<i>Gas chromatography coupled with mass spectrometry</i>All headspace collection sample extracts were run on an Agilent 7890B gas chromatograph (GC) (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with an Agilent Durabond HP-5 column (30 m length, 0.250 mm diameter and 0.25 μm film thickness) with helium as carrier gas at a constant 1.2 mL/min flow and 40 cm/s velocity, which was coupled with a single-quadrupole Agilent 5997B mass spectrometer (MS). Samples were injected with an autosampler using splitless mode. The compounds were separated by auto-injecting 1 μl of each sample into the inlet set at 250°C with flow rate of 18 ml/min. The oven temperature was programmed at 60°C and was immediately increased by 10°C/min to a final temperature of 280°C, which was held for 6 min. After a solvent delay of 3 min, mass ranges between 35 and 500 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. Compound peak areas relative to that of the internal standard were used to calculate the emission rates (ng/h of collection).