DOI: 10.15482/usda.adc/24851613.v1
title: Data from: The capacity of the larger grain borer and the maize weevil to vector microbes after foraging at novel food patches

description: Adult <i>Prostephanus truncatus</i> and <i>Sitophilus zeamais</i> were obtained from insect colonies kept at the United States Department of Agriculture (USDA) Center for Grain and Animal Health Research facility in Manhattan, KS. Both insects were reared on whole kernels of maize and held in incubators under constant conditions at 27 ± 0.1°C (mean ± SE), 64 ± 1% RH, and 14:10 (L:D) h photoperiod for <i>S. zeamais</i> and at 27.2 ± 0.1°C, 37 ± 1% RH, and continuous darkness for <i>P. truncatus</i> as mandated by the applicable USDA-APHIS PPQ-approved permit (P526-191021-020). Colony individuals were used for all tests described below. Insect Dispersal and Agar Agar was prepared using 1000ml autoclave-safe glass jars. We mixed 900ml of deionized H2O with 32 grams of potato dextrose agar (PDA, NEOGEN, Lansing, Michigan, USA) in the 1000 ml glass jar with a magnetic stirring rod and autoclaved (533LS, Getinge, Rochester, NY, USA) the media for 30 min and then stirred on a stir plate until the agar media had cooled to 45 – 50 degrees celsius. A biosafety cabinet ( 75 x 73 x 95 cm L:H:W, #302381101, Labconco, Kansas City, MO, USA) was prepared by sanitizing with 70% ethanol and exposure to UV light for 10 min. Then petri dishes (100 x 15mm) were poured with agar media and allowed to solidify overnight in the biosafety cabinet. New petri dishes were prepared for each experimental replicate. Colony <i>P. truncatus</i> and <i>S. zeamais</i> were placed into autoclaved 4-L glass containers that were stored at constant conditions (25°C, 60% RH, and 14:10 L:D) for 0, 24, or 72 hours. After the dispersal (0, 24, or 72 h) period in the sterilized jar, the insects were transferred individually onto Petri dishes that had been prepared with potato dextrose agar. For the 0 h dispersal period, adults were immediately placed into the agar dish instead of holding in the autoclaved jars. After the insect was placed into the petri dish, the dishes were sealed with parafilm and transferred to an insect growth chamber under constant conditions at 30± 0.1°C (mean ± SE), 64 ± 1% RH, and 14:10 (L:D) h. The insects were given 3 and 5 days to disperse on the agar “novel food patch.” At 3 and 5 days, petri dishes were briefly opened to photograph fungal growth and then promptly resealed and placed back into the incubator. There were a total of n = 30 replicate individuals per treatment combination (insect species by dispersal period by time in patch) (methodology adapted from Ponce et al. 2024). It is important to note that Ponce et al. 2024 demonstrated that the brief opening of petri dishes for photography did not significantly contaminate petri dishes with microbes. Photography and Image processing Prior to imaging, light was diffused using a partially cut occluded plastic jar (14.5 × 6 cm D:H). Pictures of the Petri dish were taken using a DSLR camera (EOS 7D Mark II, Canon, Tokyo, Japan) mounted to 3D imaging StackShot (CogniSys, Inc., Traverse City, MI, USA) equipped with a dual flash (MT-26EX-RT, Canon, Tokyo, Japan). A zoom lens (EF 17-40 mm 1:4 L USM, Canon, Tokyo, Japan) was used to focus on the dish so that it filled the area of view. A single photo was taken of the Petri dish, and then subsequent dishes were imaged in quick succession, and labelled appropriately on the computer with treatments. The images were processed using ImageJ v.1.53k (Schneider et al. 2012) to quantify the microbial growth in the new food patches. The grey background of the images was first subtracted from the rest of the image, then the images were processed using find edges. Afterwards, images were converted to binary, and adjusted with erode or dilate as appropriate based on the original parameters of the microbial growth on the agar medium. By selecting a circle encompassing only the petri dish with microbial growth, the mean greyscale value, standard deviation grayscale value, and count of pixels were recorded in a separate spreadsheet with explanatory variables. This allowed a quantitative measure of microbial growth by creating an average of pixels in a given picture. The mean greyscale value could range from 0 (full white), indicating no microbial growth, to 255 (full black), indicating full microbial growth on the entire dish.