DOI: 10.15482/usda.adc/29538218.v1
title: Data from: Odor Preference, Feeding, Survival, and Reproductive Fitness of the Invasive Larger Grain Borer Prostephanus truncatus (Horn) on Acorns of Three Native North America Oak Species

description: Insects Prostephanus truncatus adults were obtained from an insect colony maintained at the United States Department of Agriculture (USDA), Agricultural Research Service, Center for Grain and Animal Health Research facility in Manhattan, Kansas, USA. The individuals were originally collected from Mexico, in native range of P. truncatus. Insects were reared on whole maize kernels in jars with screened lids, kept in a room at 23°C and 45% relative humidity (RH) under continuous darkness.  Acorns and grains Acorns from black oak (Quercus velutina), red oak (Quercus rubra), and bur oak (Quercus macrocarpa) were collected from the ground beneath trees in Manhattan, Kansas, USA at two locations (39.18°N, -96.59°W and 39.20°N, -96.62°W) during the fall of 2022–2024. We removed the caps and discarded any acorns that had cracks or holes in the shells since these may already be infested with other insects. Intact acorns (without cracks or holes) from healthy oak trees were used in the experiments. Organic yellow maize (Zea mays) and hard red winter wheat (Triticum aestivum) (Heartland Mills, Marienthal, KS, USA) were also used in the experiments. Odor preference assays with acorns and grains₋ P. truncatus odor preference assays were conducted in a Petri dish (100 × 15 mm, D × H, VWR, Radnor, PA, USA), with one individual per dish. Two holes, spaced 5 cm apart and centered over each half of the dish, were carefully drilled on the bottom using a 7/64-inch (2.8-mm) drill bit. Holes were also drilled through a piece of filter paper (85 mm, Ahlstrom-Munksjö, Mt. Holly Springs, PA, USA) affixed with double-sided tape to the bottom of the arena to facilitate movement of P. truncatus. The holes allowed the diffusion of host volatiles into the arena (e.g., confirmed with a smoke test) so that there were point sources emissions of volatiles but no visual cues. Two plastic cups (30 × 50 mm, D × H) were placed directly below each of the holes containing the food stimulus with pairwise comparisons: a single intact acorn of black oak, red oak or bur oak, 1.5 g of maize or wheat kernels, and empty (i.e., blank control). Prostephanus adults were placed individually in the center of the arena and they were given 16 h to respond to an odor source. At the end of the trial, the number of insects choosing each treatment was recorded. There were n = 74 replicates per treatment combination.  Headspace solid phase microextraction and gas chromatography-mass spectrometry (HS-SPME/GC-MS) analysis of acorns and grains Volatile organic compounds (VOCs) from acorns and grains were analyzed by HS-SPME/GC-MS according to previously developed methodologies (Buśko et al. 2016; De Flaviis et al. 2021) with some modifications, as described below. Maize, wheat, and acorns of bur oak, black oak and red oak were ground separately with a dedicated coffee grinder (Hamilton Beach, Model 80335G, Glen Allen, VA, USA) into a ﬁne whole ﬂour. Before use, the coffee grinder was thoroughly rinsed with water, followed by repeated rinsing with hexane to avoid cross contamination of samples. A total of 2 g of the flour from each sample (n = 5 replicates per treatment) was transferred into a 20-mL headspace vial (Gerstel, Linthicum Heights, MD, USA) and a vial with no flour served as a negative control. The vials were then completely sealed with magnetic screw caps with a Teflon septum. Solid phase microextraction (SPME) was performed using an automated program on an MPS autosampler (Gerstel, Linthicum Heights, MD, USA). The flour samples were incubated at 60°C on a heating block for 15 min in the pre-equilibration stage, and then a 50/30 µm DVB/Carboxen/PDMS fiber (Supelco 57298-U, St Louis, MO, USA) was exposed for 30 min at 60°C to extract the VOCs. After the extraction step, the SPME fiber was desorbed in the injection port of the GC-MS for 3 min at 260°C in a pulse splitless mode of 40 psi followed by a purge flow of 15 mL/min. VOCs were separated using an Agilent 7890B gas chromatograph (Agilent, Santa-Clara, CA, USA) coupled to a single quadrupole mass spectrometer (Agilent 5997B), using a Durabond HP-5 capillary column (30 m × 0.25 mm × 0.25 μm) (Agilent). The GC oven ran initially 1 min at 40°C followed by ramping from 40°C to 80°C at a rate of 7 °C/min, then from 80°C to 130°C at a rate of 1 °C/min, and finally from 130°C to 260°C at a rate of 10 °C/min. The He flow rate was held constant at 1.5 mL per min. Spectra were acquired at 50 spectra/s within a range of 40–380 amu. The transfer line temperature was 280°C and the ion source temperature was 200°C. Compounds were tentatively identified by comparison of spectral data with those from the NIST 17 library and by GC retention index.  Mobility in close-range food preference assays Following the odor preference for bur oak acorns observed in the choice assays, insect mobility in close-range food preference assays was recorded over a 60-min period using a video-tracking apparatus combined with Ethovision software (v. 16.0, Noldus Information Technology Inc., Leesburg, VA, USA). The equipment was set up to track six arenas (100 × 15 mm, D × H, VWR, Radnor, PA, USA) simultaneously, and within each arena, a single adult was observed. The arenas were placed in cutouts on white foamboard to affix position that was 80 cm below a network camera (GigE, Basler AG, Ahrenburg, Germany) and backlit with a LED light box (42 × 30 cm, W × L, LPB3, Litup, Shenzhen, China). A piece of filter paper (85 mm, Ahlstrom-Munksjö, Mt. Holly Springs, PA, USA) was taped to the bottom of the arena to facilitate mobility. Two tiny notches exactly halfway on each side of the arena marked where food was to be placed (i.e., 2.1 cm from the edge of the arena). On either side of the arena, we left unbaited (i.e., blank control), or placed a single kernel of maize, a single kernel of wheat, or a piece of bur oak acorn (approximately the same mass as a maize kernel). Stimuli were affixed in place with a 3 × 3 mm square of parafilm (Parafilm M, Bemis Co., Davisburg, MI, USA). In total, there were 6 food comparisons, including: maize vs wheat, maize vs bur oak, wheat vs bur oak, maize vs blank, wheat vs blank, and bur oak vs blank. For each individual trial, the arena was lined with a new filter paper. After tracking, the program calculated the frequency of visits and time spent in each zone adjacent to the food source (e.g., within a 1.6-cm diameter circle) for each adult. There were n = 18 replicates per treatment. Prostephanus truncatus feeding and survival on acorns Acorns from black oak, red oak and bur oak were tested for their ability to support adult P. truncatus feeding and survival. We determined P. truncatus feeding, survival and reproductive fitness on both damaged and undamaged acorns. To simulate damage, three holes that were equally spaced apart were drilled on the adaxial surface of each acorn shell with a Dremel multi-tool (Model 300, Racine, WI, USA) equipped with a 5/64-inch (2-mm) drill bit. A piece of filter paper (60 mm, Whatman, GE Healthcare Life Sciences, Buckinghamshire, UK) was taped to the bottom of a glass jar (118 mL, Ball Quilted Crystal, Atlanta, GA, USA). A total of 20 adults were added to a single damaged or undamaged acorn (red oak, black oak, or bur oak) in the jar with a screened lid in a no-choice assay. A jar with no food served as a negative control and a jar with 2.5 g of maize kernels served as a positive control. The jars were left undisturbed for 2 and 4 weeks to allow the adults to feed on the acorns at 23°C and 45% RH in continuous darkness. Afterwards, each acorn was dissected to check for the presence of alive or dead adults, including those found inside or outside the acorn. The rates of adult survival and presence inside acorns were expressed as percentages, dividing the number of individuals that were alive or present inside acorns by the total number of adults for the sample, respectively. At the end of each period, an X-ray imaging system (MX-20, Faxitron, Wheeling, IL, USA) was also used to examine the acorns for evidence of insect tunneling and feeding activity. Feeding damage was quantified based on the amount of fines produced, and the fines were weighed using a precision balance (B120S, Sartorius Basic, Göttingen, Germany). In total, there were 8 treatments, including: acorns (damaged or undamaged) from bur oak, black oak and red oak, maize as a positive control, and no food as a negative control. There were n = 5 replicates per treatment. Prostephanus truncatus reproductive fitness on acorns Damaged and undamaged acorns of black oak, red oak and bur oak were tested for their ability to support production of P. truncatus F<sub>1</sub> progeny. A piece of filter paper (60 mm, Whatman, GE Healthcare Life Sciences, Buckinghamshire, UK) was taped to the bottom of a glass jar (118 mL, Ball Quilted Crystal, Atlanta, GA, USA) and 20 adults were added to the jar with a single damaged or undamaged acorn in a no-choice test. The resulting jars were left undisturbed under the same conditions as above for four weeks, after which the adults were removed. Subsequently, the acorns were maintained for six weeks. At the end of this time period, the X-ray imaging system was used to examine the acorns for evidence of insect reproductive success. Then, the acorn was dissected to count the total number of P. truncatus F<sub>1</sub> progeny, including larvae, pupae and adults produced. There were 6 treatments that were damaged or undamaged acorns: black oak, red oak, and bur oak, each with n = 25 replicates per treatment.