DOI: 10.15482/usda.adc/28339499.v1
title: Data from: Developing a growing degree day model to guide integrated pest management of <i>Eucosma giganteana, </i>a pest of a novel perennial oilseed crop

description: Lower activity threshold studyTo evaluate the lower activity threshold, <i>E. giganteana</i> larvae were collected starting in the first week of April to May 21st, 2023, at the Land Institute, Salina, KS (38.768402, -97.567081). The larvae were collected from outdoor potted <i>S. integrifolium </i>plants. The top six to eight centimeters of soil within each pot was removed and sifted through a 0.635 cm mesh screen to remove all loose soil. Any lepidopteran hibernacula in the remaining debris were removed and placed into a plastic screw-top container with a mesh bottom to allow airflow. All collected hibernacula (and the larvae within) were transported to the USDA-ARS Center for Grain and Animal Health Research (Manhattan, KS, USA) in an insulated ice chest. If they were unable to be transported the same day they were collected, they were instead kept in a refrigerator at 4.4℃. A total of 97 <i>E. giganteana </i>larvae were collected across 10 dates (2 April, 7 April, 8 April, 12 April, 14 April, 3 May, 4 May, 17 May, 19 May, 21 May).Once in the lab all hibernacula were opened and the larvae were counted, and any non-<i>E. giganteana </i>larvae were excluded from the study. Larvae were then sorted into Petri dishes (100 × 15 mm, diameter: height) and labeled according to when they had been collected, the date they were placed in the environmental chambers (Percival Scientific Inc., Perry, IA, USA), and the temperature (7, 12.5, 18, 27.5, and 30 ℃). The Petri dishes were buried in a larger container (300 × 150 × 100 mm length:width:height) containing potting soil to mimic their natural environment, insulation, and humidity. The soil-filled container was watered whenever the soil had dried to mimic the natural moisture cycle. Beginning May 11th, <i>E. giganteana </i>larvae in the chambers were checked every one to three days, newly constructed hibernacula were counted, and dead larvae were documented and removed. No further larvae were placed in the chamber after May 16th as they had all rapidly died.To determine their lower activity threshold (LAT), two <i>E. giganteana </i>larvae from the same chamber were removed from their initial environmental chamber (e.g., 7, 12.5, 18, or 27.5 ℃) and placed in a separate environmental chamber with a different temperature (e.g., 5, 6, 8, 9, 10, 11, 14, 17, and 20℃) as a common garden experiment. This new temperature was intended to mimic a change in temperature that the larvae would experience during the spring in their natural environment. In the chamber, each larva’s s movements were recorded for 30 min in a smaller Petri dish (35 × 15 mm diameter: height) using a Dino-Lite camera (AF4135ZTE, Dino-Lite, VA, USA) attached to a Dino-Lite stand (RK-06A Dino-Lite, VA, USA) using a fully rotating clip before returned to its chamber of origin. Video was streamed live to a nearby laptop and captured with DinoCapture 2.0 (v.1.5.48.A, AnMo Electronics, New Taipei City, Taiwan). Each larva was only used once in each temperature. The selection of larvae for a given temperature was randomized. There was a total of n = 6–24 replicates per common chamber temperature. The range of replicate numbers was due to larval death during the duration of this experiment. Video files were uploaded manually into Ethovision software (v.16.0, Noldus Inc., Leesburg, VA, USA), which was then used to track and quantify the movement of each larva in the recordings (n= 233 total, 6,990 minutes). The total distance moved (cm) and velocity (cm/s) was recorded.Weather data and GDD ModelWeather data was provided by The Land Institute through a weather station positioned on their property (38.80000, -97.60000). Shielded air temperature was measured using a Vantage Pro2 Plus weather station (Davis Instruments, Hayward, CA, USA) that fed its data to WeatherLink. The station has been in continuous operation for more than 10 years. This weather station provided readings of the high and low temperature every 30 minutes. GDD were calculated based on the Baskerville-Emin method (Baskerville and Emin, 1969). Briefly, the temperature diurnal time course in a 24-h period is approximated by a sine wave using the high and low temperature readings from the weather station. The area above the lower activity threshold (from the study above), but below the daily maximum, approximated by the sine wave, was integrated for the resulting GDD. The biofix date for the GDD was set to the 60<sup>th</sup> day of the year (e.g., March 1<sup> </sup>in most years).Phenological DataThere were two sources of phenological data for the GDD model. One was from historical trap capture data from 2019 and 2020 at The Land Institute (Ruiz et al. 2022). Another was from this study, which was conducted in six fields, three each in 2023 and 2024. All fields were located on The Land Institute's property in Salina, Kansas (Table 1 and Table 2). This trapping data was used to pair key milestones of adult <i>E. giganteana</i> development to GDDs. The key milestones we examined included beginning of flight, peak flight, middle of flight, and end of flight. Data from 2019 was used to develop predictions, while data in the three other years (e.g., 2020, 2023, 2024) was used to validate the model.Phenological Data from 2023Field trapping was done according to the methodology in Ruiz et al. (2022). The fields were located in North-Central Kansas at the Land Institute (Table 1). No pesticides were applied to these fields during this experiment. Starting the first week of June, six transects were set out with two in each <i>S. integrifolium </i>field. Each transect contained seven 30.4 cm × 30.4 cm clear sticky card traps (Alpha Scents, Canby, OR, USA) folded in half and affixed to the top of a 1.27 cm diameter, 91.4 cm PVC pole that was hammered into the ground making the top about 80 cm above ground. 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 then placed inside the PVC pipe. Two large binder clips were also used to anchor the sticky card to its card holder (Figure 1).The sticky traps in each transect were spaced 10 m apart around the perimeter of the field. For each transect, all traps were baited with one of three treatments. Two to three of the seven traps were baited with a control of 50 µl of acetone inside a LDPE 3-mL dropping bottle (Wheaton, DWK Life Sciences, Millville, NJ, USA). The remaining traps were baited with 50 µl of diluted (<i>E</i>)-8-dodecenyl acetate (Alfa Chemistry, Ronkonkoma, NY, USA). The low concentration of (<i>E</i>)-8-dodecenyl acetate was made by diluting 5.75 µl of (<i>E</i>)-8-dodecenyl acetate in 5 ml of acetone. A doubled concentration of (<i>E</i>)-8-dodecenyl acetate was made by diluting 11.5 µl in 5 ml of acetone. In all cases, the baited dropping bottle was placed in the top of the PVC pipe by the base of the sticky card (Figure 1). The sticky cards were collected and replaced biweekly until the first <i>E. giganteana </i>individual was caught, at which time it was changed to weekly. The lures and control bottles were replaced biweekly, and the treatment positions were rotated at that time.When collected, the sticky cards were held in a 7.6 L (=2 gal) labeled Ziploc<sup>®</sup> bag for transport back to USDA-ARS. All collected sticky traps were placed in a freezer for approximately twenty-four hours prior to counting. The total number of <i>E. giganteana</i> and nontarget Lepidoptera per trap was recorded. 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.Phenological Data from 2024Field 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 2). The pesticides (methoxyfenozide and chlorantraniliprole) were applied once during the season directly to one of the fields and adjacent to one of the other fields. Three transects were set out in each of the three fields used. Each transect contained four traps for a total of 36 traps. The traps were assembled similarly as in 2023, however hand-made sticky cards were used instead of manufactured ones, because of a noticeable decrease in efficacy in capturing <i>E. giganteana</i>. 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 (Trécé Adhesives Division, Adair, OK, USA). The sticky sides were covered with wax paper for transport. In the field, each sticky card was enclosed in a chicken wire cage (2in mesh) to reduce capture of birds and other large nontarget species. In each transect, one of the traps was baited with a control of 50 µl of acetone inside a 3-mL dropping bottle, the rest were baited with 50 µl diluted (<i>E</i>)-8-dodecenyl acetate at three concentrations. A low concentration (5.75 µl of (<i>E</i>)-8-dodecenyl acetate in 5 ml of acetone), a medium concentration (78.5 µl of (<i>E</i>)-8-dodecenyl acetate in 5 ml of acetone), and at a high concentration (580.4 µl of (<i>E</i>)-8-dodecenyl acetate in 5 ml of acetone). Traps were replaced weekly, and the baited dropping bottles were replaced every two weeks at which point the position of the treatment was rotated. The total number of <i>E. giganteana</i> and the number of nontarget lepidoptera was recorded for each trap. For both years, captures are averaged across baited treatments to generalize phenological events for GDD model.Egg to larvae GDDDuring the summer of 2024, adult <i>E. giganteana </i>moths were collected between the hours of 22:00-24:00 in one of the <i>S. integrifolium </i>fields at The Land Institute (38.769622, -97.598576). All captured <i>E. giganteana </i>were sexed and placed into individual small deli cups. Cups with female <i>E. giganteana </i>moths were checked daily for the presence of eggs. Cups with eggs laid in them were dated and then checked daily for larval emergence. A data logger (HOBO Temperature/Relative Humidity Data Logger, Onset, Bourne, MA, USA) recording the temperature and relative humidity in the surrounding area was placed beside all the cups. The temperature data collected from the data loggers was used along with the GDD model created above using the date eggs were laid as the phenological event triggering the beginning of GDD accumulation. A total of n = 25 egg clutches yielded larvae and were used for this.Damage threshold<i>Silphium integrifolium </i>root crowns were dug up at the beginning of April from a site managed by The Land Institute in Colby, KS. All <i>S. integrifolium </i>plants from this site were half siblings and had no prior record of <i>E. giganteana </i>damage. More than one hundred root crowns were retrieved and transported back to The Land Institute in Salina, KS. The root crowns were placed in pots (15.8 cm in diameter × 20.5 cm height) and allowed time to establish themselves and sprout approximately three weeks later. This was done outdoors where they were regularly checked on and watered. Once resprouted, <i>S. integrifolium </i>were re-potted into 11.4-L cloth pots that were used for the experiment. The plants were subsequently brought outside to continue maturing in self-contained cages (68.58 cm × 68.58 cm × 182.88 cm length:width:height) for the experiment starting in June to prevent other pests from interfering with the plants. There were a total of 20 replicate cages, each containing four plants, for a total of 80 plants. The cages were arranged in a grid over watering mats (WaterPulse<sup>TM</sup>, Los Gatos, CA) at a field site at The Land Institute. Water was supplied to watering mats with 1.59 cm drip tape and watered for 30 min per day.The <i>E. giganteana</i> larvae were collected in the middle of August by pulling them off damaged <i>S. integrifolium </i>flower heads at The Land Institute. Each flower head was dissected and any <i>E. giganteana</i> larvae found within were removed and placed on cups with artificial diet. They remained on the diet cups for two weeks before being re-located in the <i>S. integrifolium </i>pots above. <i>Eucosma giganteana</i> diet was made according to a pre-existing recipe (Murrell et al. 2023). The diet consisted of 76.6 g <i>S. integrifolium </i>flower heads collected fresh from the field, 20 g agar, 30 g wheat germ, 11.7 g sucrose, 10 g Wesson’s salt mixture (Wesson 1932), 15 g vitamin diet fortification mix, 2 g sorbic acid, 2 g methyl paraben, 11.7 g casein, 0.37 g aureomycin, and 850 L water (600 L boiling water to dissolve agar and 250 L room temperature to cool the mixture). The diet mixture was allotted into individual cups and allowed to dry for about a week before larvae were added to the cups. A total of 4–7 caterpillars were added to each cup.Cages were randomly assigned to one of five treatments, with varying densities of <i>E. giganteana</i> larvae: control (e.g., zero larvae), 10, 20, 30, and 40 larvae per cage. The number of larvae were distributed among the four plants within the cage (0, 2–3, 5, 7–8, 10 larvae per pot) and labeled accordingly. A metal mesh top was constructed to cover the pots, and to prevent the larvae from escaping once they were placed inside. There were 16 replicates of each larval density. Cages were left out until the first week of December 2023 to allow the larvae sufficient time to first descend into the root crowns (presumably to feed) and then exit the root crowns to prepare for overwintering. The pots containing the root crowns, and the larvae were held in a cold storage room at 4°C until spring to sift through the samples. During April and May of 2024, each potted sample was taken and the dirt surrounding the root crown was sifted using a 27-gauge hardware cloth to locate any remaining larvae and empty hibernacula. The root crown was washed of all remaining debris soil, allowed to dry, and placed in a labeled bag.Prior to being X-rayed, each root crown was weighed and then cut into slices smaller than 10 × 10 cm L: W and 2 cm thick. Each slice was placed in the bottom half of a 10 × 10 cm square petri dish and X-rayed using a Faxitron radiography system (Faxitron X-ray Corp., Marlborough, MA, USA) on two of its sides to determine if there was tunneling by caterpillars. After X-raying was complete, the best image from each slice was imported into ImageJ v.1.53 (Wayne Rasband, National Institutes of Health, USA; Abramoff et al. 2004), and the two-dimensional area of the root crown slice was taken as well as the number and two-dimensional area of the tunnels. Using this information, we were able to determine the proportion of the area of each root crown that was tunneled.Baskerville GL, Emin P. 1969. Rapid estimation of heat accumulation from maximum and minimum temperatures. Ecology. 50(3):514–517. https://doi.org/10.2307/1933912.Ruiz K, Bruce A, Chérémond N, et al. 2022. Field trapping and flight capacity of <i>Eucosma </i><i>giganteana</i> (Riley) (Lepidoptera: Tortricidae) in response to behaviorally active congeneric semiochemicals in novel silflower agroecosystems. Insects 13(4):350. https://doi.org/10.3390/insects13040350.Wesson LG. 1932. A modification of the Osborne-Mendel salt mixture containing only inorganic constituents. Science. 75(1943):339–340. https://doi.org/10.1126/science.75.1943.339.