DOI: 10.15482/usda.adc/1529590
title: Data from: Microbial vectoring capacity by internal- and external-infesting stored product insects after varying dispersal periods between novel food patches: An underestimated risk

description: To determine whether colony populations of Lasioderma serricorne (cigarette beetle, CB) and Sitophilus oryzae (rice weevil, RW) vectored microbes, and to identify possible interactions with dispersal time, a vectoring assay was performed for each species. For the vectoring assay, the impact of dispersal (0, 24, or 72 h) and foraging time (3 or 5 d) on vectoring ability were tested.  Briefly, adult L. serricorne or S. oryzae were singly removed from colony containers with sterilized forceps and then placed immediately in the center of Petri dish containing agar for the 0 h dispersal period.  Alternatively, some insects were given a 24 or 72 h dispersal period in an autoclaved 4 L-capacity glass container and stored at constant conditions of 25°C, 60% RH, and 14:10 L:D photoperiod prior to being added to the PDA. Petri dishes were maintained at 30°C, 60% RH, and 14:10 L:D photoperiod for either 3 or 5 days, then photographed for microbial growth. Transfer of L. serricorne or S. oryzae adults from dispersal containers to agar at the conclusion of the dispersal period was performed inside the biosafety cabinet to prevent contamination of dishes.
Pictures of the agar dishes and corresponding microbial growth 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). Light was diffused using a partially cut frosted plastic jar (15.2 × 7.6 cm D:H) making a total of n = 60 replicates per treatment combination (of dispersal time, insect species, and foraging time in patch). The pictures taken were processed using ImageJ 1.53a (Wayne Rasband, National Institutes of Health, USA) to quantify the microbial growth in the agar dishes. The images had their backgrounds subtracted, then were processed using the "find edges" tool. Finally, they were converted to binary and either dilated or eroded to conform to the original image parameters. A circle encompassing the Petri dish was created and the mean grayscale, standard deviation of the grayscale value, and count of pixels was measured as a surrogate for microbial growth on the dishes. This allowed a quantitative measure of microbial growth by creating an average in a given 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. Finally, visually, microbial morphospecies (alpha) richness was assigned to each image given the number of unique morphospecies on the plate as a proxy for community complexity.
Treatments included those from microbially-enriched environments where Aspergillus flavus had been inoculated on wheat or flour (AF). To prepare the AF, 600 g of grain was added to a stainless-steel pot filled with water and placed on a hot plate at 500°C. Once boiling for 15 min, the water was drained and the grain was evenly spread out on sterile wipes (38.1 × 42.5 cm, 3 ply, Tech wipes, Skilcraft, NIB, Alexandria, VA) and allowed to dry inside a laminar fume hood (ca. 3 h). Afterwards, grain was evenly divided (~300 g) and placed in two separate autoclaved mason jars (950-mL capacity). A single hole was pierced through each lid and lined with a cotton ball. The jars were then sealed with aluminum foil and were autoclaved (533LS, Getinge, Rochester, NY, USA) for 30 min. To inoculate with A. flavus, a 3-inch strip of agar containing a pure culture of A. flavus grown on agar for 7 d at 30°C, 60% RH, and 14:10 L:D photoperiod was placed into each jar containing the grain. AF was then maintained at room temperature for roughly 10 d or until the A. flavus evenly covered as much the grain as possible. Batches of inoculated grain were used within 10–15 d of preparation. Grain was never used more than once for each replicate of every trial in each assay experiment to prevent cross contamination. A total of 75 insects were added to 300 g of AF in a 950-ml mason jar and allowed to forage for 2 weeks prior to use in the vectoring experiment. The same dispersal periods (0, 24, 72 h) and time in patch (3 and 5 d) described above were used for this experiment. The mean grayscale value and microbial morphospecies richness was recorded for each image. There were a total of n = 30 replicates per treatment combination.
Another treatment included field-collected individuals. To obtain sufficient numbers of adults, insects were caught at four different field sites around the area of greater Manhattan, KS including: 1) a site with a pre-harvest wheat field bordered by woodlands (39°14'26.2"N, 96°34'59.1"W), 2) local apartment complex consisting of end consumers (39°11'43.6"N, 96°36'07.4"W), 3) Kansas State University Agronomy Farm with storage silos (39°12'23.7"N, 96°35'43.2"W), and 4) a private residence adjacent to a working cattle farm (39°12'23.7"N, 96°35'43.2"W). In each location, a total of three 4-funnel Lindgren traps (Bioquip, Rancho Dominguez, CA, USA) were deployed at least 10 m apart at about 1 m height on rebar or hung from a tree along the perimeter of the location site, and were baited with a multi-species lure containing both L. serricorne sex pheromone and Sitophilus spp. pheromone (PTL bullet lure, #IL-108, and Sitophilus spp. bullet lure, #IL-703, Insects Limited, Westfield, IN, USA). In addition, three ground traps were deployed that consisted of commercially-available pitfall traps (Dome®, Storgard, Trécé, Adair, OK, USA) with two connectable pieces (Doud and Phillips 2020; Doud et al. 2021), containing a central well where a Sitophilus spp. lure was added along with a 5 g of whole maize as a kairomone bait. Pheromone lures were changed every 60 d. No kill mechanism was added because adults needed to be alive. Traps were checked on a daily basis for capture of new adults and brought immediately back into the laboratory in separate unused, sterilized containers for addition to agar dishes. Stored product insects were identified using taxonomic keys in USDA (1996). Dispersal period at 0 h and time in patch (3 and 5 d) as described above were used for this experiment.
Resources in this dataset:

Resource Title: Full CB & RW Vectoring Dataset
File Name: cb_rw_full_dataset_richness.csv