Data from: Flight capacity and behavior of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) in response to kairomonal and pheromonal stimuli

Kairomonal and pheromonal stimuli

The pheromone stimuli consisted of a proprietary blend (IL-103, Insects Limited, Westfield, IN, USA) of the sex pheromone for E. kuehniella, including less than 5% concentration by weight of (Z, E)-9,12-tetradecadienyl acetate (e.g., ZETA). The kairomonal stimuli consisted of 20 g of a food cue mixture comprised of cracked wheat, wheat shorts, of wheat germ, and of brewer’s yeast in a 23:7:2:1 ratio. These were the dry components of the E. kuehniella diet.

Flight Mill Apparatus

Flight mills were assembled after Smith et al. (2012) and Ruiz et al. (2022) (Figure 1). The base assembly consisted of a Delrin rod (95.25 mm length × 38.1 mm diameter) mounted to a 6.35-mm thick equilateral triangular Plexiglass base (each side 13.6 cm long), with screws used as levels. At the top of the rod, a 1.5 cm length (0.72 mm, 22 ga) hypodermic needle was mounted into a hole, and a 5 mm circular magnet was glued to each end of dual 35 mm long metal #1 insect pins at the top of the rod around the axle pin. A unipolar digital Hall effect sensor (OHN3120U, Optek, Inc., Carrollton, TX) was wired to a coupler with one wire for the 5V DC, one ground, and one output signal. This sensor assembly was attached to the top of the Delrin rod with a Plexiglass assembly and thumbscrews. The rotation flight arm assembly consisted of a Teflon bar (2.0 cm length × 1.1 cm diameter) with circular magnets (polarity conserved) glued to the bottom and a hole bored into the bottom to accept the other end of the axle pin that was mounted in the Delrin rod. A hypodermic needle (28 cm length × 0.72 mm diameter) was threaded through a hole in the flight arm assembly, with the ends bent downward at a 95° angle. As a result, the rotation flight arm assembly fit onto the base assembly axle pin, and the circular magnets on the base and bottom of the flight arm assemblies configured to the same polarity kept the arm suspended over the axle, allowing it to spin on the Teflon bearing with very little friction. When the small magnet facing down from the flight arm assembly rotated over the Hall effect sensor, a signal was sent through the output signal wire.

The six flight mills were situated on a 71.1 cm × 91.4 cm piece of plexiglass to minimize vibrations. Each mill was spaced 39 cm apart in tandem (e.g., three in a row) with 25.4 cm between rows. Each mill contained three wires connected to the main connector board (#777101-01, National Instruments, Austin, TX, USA). A DC Power Supply (QW-MS3010D, QW, Tampa, FL) was set to the site and connected to the main wiring block to provide power. A 50-pin ribbon cable (180524-10, National Instruments) ran from the main wiring connector block into a specially made PC port (#77690-01, National Instruments), where a computer with the software Labview 2017 (version 17.0.1f3, National Instruments) automatically recorded the data from the mills. Data intervals for Hall effect sensors were set to 1 ms. Hall sensors were triggered when a magnet from the flight arm passed over, and this was recorded by the software as a voltage change. Schematics, photographs, wiring diagrams, ordering information, and data acquisition programs for this flight mill setup are all available online (Jones et al. 2010)(Flight Mill Studies n.d.).

Flight Capacity Experiments

Six adults blocked by sex were run simultaneously on the six flight mills described above (15-FMASM SDP Unit, Crist Instrument Co., Hagerstown, MD) to test flight capacity. Freshly eclosed moths were used for the experiments to standardize age, and were likely unmated. A 14-gauge copper wire was stripped into individual threads and cut into 4 cm segments. The copper wire thread was wrapped around an insect pin (#6 insect pin, BioQuip Products, Rancho Dominguez, CA) embedded in modelling clay to form a loop. The loop was pinched with standard metal forceps, the excess wire was ablated on the shorter end with scissors, and the loop was flattened relative to the plane of gravity. The moths were placed singly on a metal mason jar lid suspended on ice and restrained with a 2 oz plastic lid until sessile. Subsequently, the pronotum of E. kuehniella was descaled lightly with an artist’s paintbrush. The copper loop was dipped in instant adhesive (#347908, Evo Stik Multi-Purpose Impact Adhesive, Bostik, Ltd., Leicester, United Kingdom) and affixed onto the pronotum of the adult, ensuring not to impair proper wing functioning in addition to avoiding the eyes and antennae of the adults. The individual was tethered by inserting the point of the copper wire into the end of the hypodermic needle attached to the rotation arm assembly of each flight mill.

Each trial was started between 15:00-18:00 by gently blowing by mouth over the back of the insects to initiate flight to provide the opportunity for flight (per the established guidance for tethered flight mill systems: Naranjo 2019) and flight was recorded over initiate the 24 h observation period. Any and all flight bouts were recorded during this time. A flight bout was defined as consecutive rotations of the flight arm of the flight mill by E. kuehniella separated by a pause of 3 s in rotation. Flight mills were housed in a dedicated space in the laboratory under constant conditions (at 21.6 ± 0.01°C, 43 ± 0.2% RH, 14:10 L:D photoperiod). Air flow was gently vented at a mean velocity of 4.57 m per min. Treatments included an unbaited control, kairomone (e.g., food cue), and pheromone (e.g., all as above). Treatment stimuli sources were placed on a platform located directly in the center of the set of flight mills. There was a total of n = 18 reps per treatment combination of stimulus. At the end of a trial, insects were detached from the apparatus and weighed on a balance. Data were streamed in real-time to a computer containing the software, which was used to automatically record the flight parameters, namely distance flown, as well as the number of tandem flight bouts over the sampling interval (flights lasting more than 1 s), and their average length, and average distance flown per bout. To conservatively estimate flight distance, all flight distances consisting of a singleton flight bout were eliminated from the datasets. In addition, to support data quality and avoid the false accumulation of data, revolutions were eliminated if the sum of revolutions on the flight mill divided by the total time spent flying was larger than 3.6 s (the conservatively slowest time E. kuehniella could make a revolution and still be actively flying) and the number of revolutions was greater than 30. Data were also parsed by time of day to determine the time of maximum dispersal. Data were analyzed with R software (v. 2022.02.1 Build 461) (R Core Team 2022). Ggplot2 was used for some of the figures (Wickham et al. 2016).

Cage Assay

To test flight initiation patterns of E. kuehniella, a cage assay was utilized. Cohorts of 20 adult moths blocked by sex were collected in a plastic bag from colony jars, then briefly anesthetized for no longer than 30 s with N2 gas. Moths were gently placed in a petri dish (100 × 15 mm) lined with polytetrafluoroethylene (PTFE) (MilliporeSigma, Burlington, MA, USA), which was then situated in a randomly chosen corner of a mesh cage (28 × 28 × 28 cm, Bug Dorm, MegaView Science Co. Ltd., Taiwan) and covered with a funnel (height: 12.5 cm; large opening: 15 cm D; small opening: 2.6 cm D). The semiochemical treatments listed above were placed randomly in one of the three remaining corners. Cages were placed in a walk-in environmental chamber under constant conditions (at 25 ℃, 65% RH, 14:10 L:D photoperiod) and given 24 h to respond to the stimuli. The proportion of adults leaving the funnel was measured at the end of the trial period. In total, n = 8 replications were performed with each treatment, translating to 480 total E. kuehniella individuals tested.