DOI: 10.15482/usda.adc/28055111.v1
title: Data from: Behavioral and physiological response of <i>Eucosma giganteana </i>to semiochemicals from conspecifics and <i>Silphium integrifolium</i>

description: <i>Trapping in 2023 with a linear set of dosages of </i>(E)<i>-8-dodecenyl acetate</i>Field trapping was done according to the methodology in Ruiz et al. 2022. The fields were located in North-Central Kansas at the Land Institute near Salina, KS. No pesticides were applied to these fields during the experiment in 2023. Starting the first week of June, six transects were set out, two in each <i>Silphium integrifolium </i>field. Each transect contained seven 30.4 cm x 30.4 cm sticky card traps (Alpha Scents, Canby, OR, USA) affixed to the top of a 1.27 cm diameter, three foot in length PVC pole that was hammered into the ground until sturdy. The cards were affixed using a 271 cm long sticky card ring holder (Olson Products Inc., Medina, OH, USA) that was bent to a 90° angle and placed inside the PVC pipe. Two large binder clips were also used to anchor the sticky card to its card holder.The sticky traps in each transect were spaced 10 meters apart around the perimeter of the field. Within each transect, traps were baited with a linear increase in concentrations in 2023, including either a control (50 µl of acetone), a low concentration (50 µl of a solution made by mixing 5.75 µl of (<i>E</i>)-8-dodecenyl acetate in 5 ml of acetone), or a doubled concentration (11.5 µl of (<i>E</i>)-8-dodecenyl acetate diluted in 5 ml of acetone) of (<i>E</i>)-8-dodecenyl acetate (Alfa Chemistry, Ronkonkoma, NY, USA). All lures were added to a 3-ml LDPE dropping bottle (Wheaton, DWK Life Sciences, Millville, NJ, USA). The clear sticky card traps were collected and replaced biweekly until the first <i>E. giganteana </i>adult was caught, then traps were changed weekly. The lures and control bottles were replaced once every two weeks (with lure emissions confirmed out to 14 d in Ruiz et al. 2022) and their position in the field rotated at each change. Each lure was in each position twice over the course of the season.When collected, the sticky cards were held in a 7.6 L (=2 gal) labeled Ziploc<sup>©</sup> bag transported back to USDA-ARS. All collected sticky traps were placed in a freezer for approximately 24 h. The total number of <i>E. giganteana</i> per trap and their distance from the lure in millimeters was recorded. In addition, the number of nontarget lepidoptera was recorded on each trap. Individual <i>E. giganteana </i>and non-target lepidoptera were only counted if more than half of the specimen was remaining on the sticky trap at the time of counting to ensure positive identification.<i>Trapping in 2024 with an exponential set of concentrations of </i>(E)<i>-8-dodecenyl acetate</i>Field trapping in 2024 was conducted similarly to that in 2023 with the following modifications. Three different fields located at the Land Institute were used (Table 1). [HS1] Pesticides were applied once to one of the fields and adjacent to one of the others. Three transects were deployed in each of the three fields. Each transect contained four traps for a total of 36 traps. The traps were assembled similarly to those used in 2023, but a hand-made sticky card was used instead of a manufactured one to improve captures. These sticky cards were made of a laminated 21.6 × 27.9 cm (=8.5 by 11 in) piece of white cardstock paper (Astrobright, Neenah, WI, USA) coated on both sides with TAD<sup>Ⓡ</sup> all-weather adhesive (Trécé Adhesives Division, Adair, OK, USA). The sticky sides were covered with wax paper for ease of travel. Additionally, the sticky cards had a chicken wire cage placed over them in the field to try to prevent the capture of birds and other nontargets on the traps. Traps in 2024 were baited with an exponential set of concentrations of (<i>E</i>)-8-dodecenyl acetate. In each transect, there was a solvent only control (50 µl of acetone), a low concentration equivalent to the 2023 treatment (50 µl of a solution made of 5.75 µl of (<i>E</i>)-8-dodecenyl acetate diluted in 5 ml of acetone), a medium concentration (50 µl of a solution made of 78.5 µl of (<i>E</i>)-8-dodecenyl acetate diluted in 5 ml of acetone), and a high concentration (50 µl of a solution made of 580.4 µl of (<i>E</i>)-8-dodecenyl acetate diluted in 5 ml of acetone). The traps were replaced weekly, and the lures were replaced biweekly, as well as rotated positions in the transect. Each lure was in each position twice over the course of the season.<i>Eucosma giganteana and Silphium integrifolium collections from the field</i><i>Eucosma giganteana</i> cannot yet be reared successfully in the laboratory, thus we sourced all specimens from the field. Adult <i>E. giganteana </i>individuals were carefully captured by hand in one of the fields planted to <i>Silphium integrifolium</i> at the Land Institute (38.769622, -97.598576) between 22:00 and 24:00 five times a week from June to August 2024. Moths were immediately sexed and individually placed in small deli cups with appropriate labels. They were brought back to the USDA-ARS Center for Grain and Animal Health (39.1955486, -96.5987334) for the experiments described below. Once in the lab but prior to use in experiments, moths were kept in a quiet environment at approximately 23 ± 0.1℃ and 16:8 L:D photoperiod. Importantly, no lures were used to capture insects to avoid biasing the results of the experiments below. <i>Silphium integrifolium</i> flower heads were cut 1 cm below the flower and brought back on a weekly basis during the same timeframe and stored at 4°C until needed for experiments. Flower heads were never more than 4 days old prior to use.<br><i>Headspace Characterization</i>Headspace was collected from the following treatments: 10 <i>E. giganteana</i> male moths only, 10 <i>E. giganteana</i> female moths only, an even mix of male and female moths (5:5), flower cuttings of <i>S. integrifolium</i>, and a blank control. For the <i>E. giganteana</i> treatments, only alive, healthy adult moths that were collected within five days were used. For the <i>S. integrifolium </i>collections, approximately 25 grams of flower heads cut the same week as collections were used.For each treatment, <i>E. giganteana</i> or <i>S. integrifolium</i> were placed in a clean 100-mL beaker. To prevent moth escapees, a metal mesh top was constructed and affixed to the opening of the beaker. The beaker was then placed in one of eight 500-mL glass headspace collection containers with a PTFE septum and lid. A Pora-Pak Q volatile collection trap (VCT) was inserted in the output end. The VCT consisted of an angled drip-tip collection point borosilicate glass tube with a mesh (Stainless Steel #316 screen), packed with 20 mg of PoraPak-Q™ chemical absorbent held in place with a borosilicate glass wool plug, and followed by a PTFE Teflon™ compression seal. A PTFE tube spanned from the flow meter (CADS-4CPP, Clean Air Delivery System, Sigma Scientific, LLC, Micanopy, FL, USA) to the input end of the headspace container at a flow rate of 1 L/min. Prior to that, the air was scrubbed with an activated carbon filter and was pumped in using the central air pump for the center. Samples ran for 24 h. Each volatile collection trap was collected and eluted with 150 µl of dichloromethane in a fume hood into a 2-mL GC vial containing a 250 µl glass insert with polymer feet. The solvent was gently pushed through the volatile collection trap with N<sub>2</sub> gas. At the end of collecting all the samples, 1 µl of an internal standard, tetradecane (190.5 ng), was added to each of the samples. The samples were then all capped with a magnetic screw top lid and secured with PTFE tape before being placed in a freezer at -20 ℃ until they could be run. All headspace samples were collected within 5 weeks. After each replication, the headspace collection containers were all washed with methanol and then hexane. VCTs were rinsed in triplicate with dichloromethane. A total of at least n = 5 replicates were tested for each treatment.<br><i>Gas Chromatography Coupled with Mass Spectrometry</i>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. The GC 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 into the GC–MS at room temperature (approximately 23 °C). The flow rate was 18 ml/min. 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 14 library and by GC retention index. The samples were normalized according to the following formula: (Pk<sub>sam</sub> – Pk<sub>min</sub>)/(Pk<sub>max</sub> – Pk<sub>min</sub>), where Pk<sub>sam</sub> is the peak area from the sample, Pk<sub>min </sub>is the global minimum peak area, and Pk<sub>max</sub> is the global max peak area.<br><i>Electroantennography of E. giganteana</i>All<i> </i>electroantennogram (EAG) recordings of <i>E. giganteana</i> were taken from 19:00 to 23:00 which corresponded to the peak activity period of <i>E. giganteana</i> based on prior literature (Ruiz et al. 2022). Prior to recordings, the machine and software were powered on and given 30 min to warm up. Only field-captured moths within three days were used for the recordings. The moths were sexed prior to recordings and knocked down in a freezer for 1–2 min. The moth’s antenna was then ablated, alternating between left and right antennae for each recording, at the pedicle as close to the scape as possible. Then, using electroactive gel (Spectra 360 Electrode gel, Parker Laboratories INC., Fairfield, NJ, USA), the ablated antennae was placed on an antenna holder (Syntech GmbH, Buchenbach, Germany) and covered with gel to ensure connection and prevent desiccation. The antennal holder was then slotted into the EAG probe (EAG Combi Probe, Syntech GmbH, Buchenbach, Germany). The software was opened to evaluate the EAG trace and ensure the antenna was connected correctly. The recording was started once the line showing the antennal baseline had stabilized. Subsequently, the antenna was given an additional minute or two to ensure the baseline remained stable. A flow meter (CS-55, Syntech GmbH, Buchenbach, Germany) was triggered to puff air at a rate of 0.3 μl s<sup>-1</sup> through a Pasteur pipette and across the antennae. Once the flow meter was triggered, there was a brief delay before the first puff, then a 1 min delay before the second puff.The treatments included a control of blank air, neat concentration of (<i>E</i>)-8-dodecenyl acetate (Alfa Chemistry, Ronkonkoma, NY, USA), neat dichloromethane solvent control, diluted concentration of (<i>E</i>)-8-dodecenyl acetate (e.g., consisting of 11.5 µl of neat (<i>E</i>)-8-dodecenyl acetate diluted with 5 ml of dichloromethane), 150 μl dichloromethane + 1 μl tetradecane (190.5 ng μl<sup>-1</sup>) control, female <i>E. giganteana </i>headspace (see above), <i>S. integrifolium </i>headspace (see above), and neat acetone solvent control. For each treatment, 6 µl of the treatment was pipetted into the center of a small filter paper (2.5 cm diameter), which was then folded and placed into the top of a pasteur pipette. The treatments were presented in the order given above to each antenna for standardization. Apart from the blank control which was puffed four times at the beginning of each recording and twice at the conclusion of each recording, all other treatments were only puffed twice. The Pasteur pipettes containing the treatments were replaced after an hour to ensure the antennae was supplied with a fresh odor source. There was a total of n = 15 replicate individuals for female moths and n = 17 replicate individuals for male moths for each treatment.<i>Flight Mill Apparatus</i>[RM2]The flight mill was based on the design found in Attisano et al. (2015) and Stowe et al. (2022). In short, there were 25.5 × 24.5 × 12 cm (length × width × height) plexiglass shelves stacked 4 by 4 vertically adjacent to each other. A hypodermic needle (19-gauge non-magnetic hypodermic steel tubing) 22 cm long comprised the flight arm assembly and was affixed to a 10 uL micropipette tube in a center pivot with hot glue, and held weightless by two strong neodymium magnets ventrally and dorsally. The flight rod is horizontal with one end for a flag and the other end to hold an insect. The flag is used to interrupt the LED/PT sensor (OPB800W, Optek Technology Inc., Texas USA) which changes the analog signal from low voltage to higher voltage. In each shelf, there was a sensor that measured when the signal was broken by the revolution of the flight arm to record flight using a datalogger (DATAQ Model DI-2108-P data acquisition interface and software, DATAQ Instruments, Ohio, USA) and streamed to an attached computer using customized software. The interface has +5 VDC and ground terminals for supplying power to the sensors. The hardware includes an array of screw-terminals for connecting the wires from the 8-LED/PT sensors. This electronics is interfaced to a PC via a USB cable. The analog signals are converted to digital signals and transferred to the computer for data collection and processing.<br><i>Flight Capacity of E. giganteana in Response to Semiochemicals</i>Only moths captured within 1–3 days of the start of flight mill recording were used. Ten to 12 moths were prepared at a time for the eight flight mills. The 89-mL (=3 oz) solo cups containing these moths were placed in an ice bath to partially knock them down. Individual moths were then removed, and their scales were gently brushed off their pronotum using a small silicone brush to allow better adherence of the glue. A small dollop of T-7000 glue was applied to the blunt end of a size 2 entomology pin (Bioquip, Rancho Dominguez, CA, USA) and this was pressed against the pronotum for 1 min to allow time for the glue to dry. The pins with moths were inserted into an elevated piece of polystyrene until the moths were ready to be placed on the flight mill. Eight moths were run simultaneously on separate, individual flight mills, choosing moths to run that were more active (after Attisano et al. 2015). Moths that could not properly beat their wings due to the glue or wing placement were not used on the flight mills. The tip of the pin of each moth was inserted into the arms of the flight mill (19-gauge center pivot) which were weightlessly supported between two neodymium magnets and positioned to fly counterclockwise. Each moth was encouraged to fly by initially gently blowing on its abdomen to make sure the arm with the moth on it could pass through the sensor. The rotations of the moth were recorded through the breaking of an infrared beam produced by an IR sensor (OPB800Q, Optek Technology, Texas USA) mounted on the side of each flight mill cell. Once all moths were set up and vetted, the automated software (WINDAQ, DI- 149, Ohio, USA) was started, and flight was logged continuously. Moths were given the opportunity to fly in a 24-h period. After the end, the moths were removed from the flight mill arms, their condition was noted, they were weighed on a microbalance (QUINTIX2102-1S, Sartorius AG, Göttingen, Germany), and then they were frozen. The moths were weighed to evaluate whether size affects flight capacity. For each round of the flight mill, each sex was run independently (blocked) to determine if flight capacity was altered in the presence of the other sex. An additional block included both sexes being run, which consisted of four male and four female moths running simultaneously. The semiochemical treatments included a low concentration of (<i>E</i>)-8-dodecenyl acetate (made with 11.5 µl of (<i>E</i>)-8-dodecenyl acetate diluted with 5 ml of acetone) and a high concentration of (<i>E</i>)-8-dodecenyl acetate (made with 580.4 µl of (<i>E</i>)-8-dodecenyl acetate diluted with 5 ml of acetone). For all semiochemical treatments, a total of 25 µl was pipetted onto a piece of filter paper (10 cm diameter) and placed in a Petri dish (100 × 15 mm diameter: height). The Petri dish was left uncovered in front of and centered adjacent to the flight mill. In total, there were two blocks of eight moths used for each of the treatments, so n = 16 behavioral replicates for each treatment combination of sex and semiochemical.