Trapping of field-collected S. oryzaeTo capture field-collected S. oryzae to evaluate how microbial vectoring over the course of a season in 2022, six commercial pitfall traps (Storgard, Trécé Inc., Adair, OK, USA) were baited with 5 g of wheat and the S. oryzae aggregation pheromone, (4S,5R)-5-hydroxy-4-methyl-3-heptanone (IL-703, Insects Limited, Westfield, IN, USA) and deployed 10 m apart. In 2023, 14 pitfall traps (Storgard, Trécé Inc.) were deployed, separated by 5–15 m and baited with the same stimuli. In addition, probe traps (WB Probe II, Storgard, Trécé Inc., Adair, OK, USA) were taken in identical locations in adjacent grain bins, but >90% of individuals came from the pitfall traps. Trapping took place at the Kansas State University Foundation Seed Farm (39°12'23"N, 96°35'42"W), and occurred on a weekly basis from 11 May 2022 to 28 Sep 2022 and 17 May 2023 to 1 Nov 2023. Live S. oryzae from each trap were placed in a sterile Ziplock bag and brought back to USDA-ARS Center for Grain and Animal Health in a cooler. Live weevils were stored at 23°C and 65% RH after brought back from the field. Within 24 h of being brought to the laboratory, S. oryzae were introduced to factitious, novel food patches as described below.Linking with weather dataWeather data was obtained from the Kansas Mesonet system (https://mesonet.k-state.edu/) at a weather station located in the same ___location as the trapping on the Kansas State University Agronomy Farm (39°12'26"N, 96°35'42"W). Air temperature was measured at 1.98 m above ground level (HMP155 probe, Vaisala, Vantaa, Finland) inside of a non-aspirated radiation shield within an error range of ± 0.1°C. Data was acquired every hour with a microprocessor (CR3000 series, Campbell Scientific Inc., Logan, UT, USA), which accurately measures to the microvolt level and controls peripheral devices. Mean maximum and minimum temperature was compiled for each 7-day period preceding each date of collection for S. oryzae in 2022 and 2023, and it was linked to microbial growth measures detailed below from the same date.Assessing microbial growth after dispersal to novel food patchesA total of n = 5 individual field-collected S. oryzae from each date were introduced individually onto petri dishes (100 ´ 20 mm) composed of potato dextrose agar as a factitious, novel food patch for 3 and 5 d to mimic dispersal (following the methods of Ponce et al. 2024). This was done within the confines of a permitted BSL2 (Permit# IBC-1693) space using a biosafety cabinet (75 × 73 × 95 cm L:H:W, #302381101, Labconco, Kansas City, MO, USA). After introduction of S. oryzae, the petri dishes were placed in an environmental chamber (Percival Scientific, Perry, IA, USA) set at constant conditions (30°C, 65% RH, and 14:10 L:D cycle). Petri dishes were photographed at 3 and 5 days of S. oryzae foraging in the novel food patch with a 3D-imaging system (Cognisys Inc., Traverse City, MI, USA) using a SLR camera (EOS 7D Mark II, Canon Inc., Tokyo, Japan) with a wide-angle lens (L series USM 17–40 mm, Canon Inc., Tokyo, Japan) and twin flash (MT-24X, Macro Twin Flash Lite, Canon Inc., Tokyo, Japan). Light was diffused using a partially cut frosted plastic jar (15.2 × 7.6 cm D:H). Images were processed using ImageJ v.1.53 (Wayne Rasband, National Institutes of Health, USA) individually by first subtracting the background, then finding edges, and converting the image to binary (white/black). As needed, erode and/or dilate was sparingly used to make sure the image reflected microbial growth in the original image. A circle encompassing only the Petri dish was created and the mean grayscale, standard deviation of the grayscale value, and count of pixels were measured as a surrogate for microbial growth on the dishes. This allowed a quantitative measure of microbial growth by creating an average in each image. The mean grayscale value could range from 0 (full white), indicating no microbial growth, to 255 (full black), indicating full microbial growth on the entire dish. Increased microbial growth was defined as higher mean grayscale values. Finally, visually, microbial morphospecies (alpha) richness was assigned to each image by two observers given the number of unique morphospecies on the plate as a proxy for community complexity. Where these numbers varied by observer (which was rare), both observers discussed together and came to a consensus on the number of morphospecies present in the petri dish. Microbial morphospecies have been prior used successfully to challenge the hypothesis “everything is everywhere, but, the environment selects” hypothesis (Telford et al. 2006).
