DOI: 10.15482/usda.adc/28264451.v1
title: Data from: Multiple infestation of a grain mass by <i>Sitophilus oryzae</i> L. (Coleoptera: Curculionidae) and the fungus, <i>Aspergillus flavus,</i> optimizes abiotic conditions for improved insect fitness

description: <i>Source Insects and Microbes</i><i>Sitophilus oryzae</i> that were 4–8-week-old and collected from a food facility in eastern Kansas in 2012 were used. Adults were reared continuously in the laboratory at CGAHR and subcultured on tempered whole organic wheat, with 75 mixed-sex adults added to 300 g of wheat. Adults were sieved off after a 1-week oviposition period. Colonies were maintained in a chamber set at 27.5˚C, 65% RH, and 14:10 (L:D) h photoperiod. The fungus, <i>Aspergillus flavus</i>, was isolated from field-collected <i>S. oryzae</i> after allowing adults to forage on potato dextrose agar dishes (100 × 15 mm) for 5 d. From the initial dish, <i>A. flavus</i> was re-plated on a new dish in multiple successive rounds until it was a pure isolate. This was used to create <i>A. flavus</i>-inoculated grain, which was then used for the treatments below.<i>Treatments and Preparation of Grain Masses</i>At the outset of the experiment, the grain moisture of the wheat for the experiments was determined to be 10.8% using a moisture meter (DICKEY-john, GAC2100, Auburn, IL, USA). A total of 300 g of organic, whole untampered organic wheat (Heartland Mills, Marienthal, KS, USA) was added to each pint mason jar (950 mL; 8.5 D × 17 cm H). In order to assess the effect of singly or multiply infested grain by insects and microbes on the microclimate and fitness outcomes, each grain mass was assigned to one of our treatments: control (no insects or added microbes; Ctrl), the addition of 75 mixed-sex <i>S. oryzae</i> adults only (SO), inoculation with 11.6 g of <i>A. flavus</i> inoculated grain only (AF, details below), and finally the addition of both <i>S. oryzae</i> (75 adults) and <i>A. flavus</i> (11.6 g of inoculated grain; SO + AF). In each grain mass, a datalogger (Hobo® U10-003 Temp/RH Data Logger, Onset, Bourne, MA, USA) was attached below the lid and recorded temperature and relative humidity every 5 min. The experiment was allowed to run for 60 d in an environmental chamber set to 30°C, 60% RH, and 14:10 L:D photoperiod. At the end of the period, all jars were immediately frozen to stop halt reproduction and microbial activity. There were n = 5 replicate grain masses per treatment. For the purposes of looking at changes in abiotic variables, the 60 d period was divided into equal 20 d increments, and labeled early (first 20 d), mid (second 20 d), and late (final 20 d).<i>Preparation of A. flavus-inoculated grain</i>In order to inoculate treatments with <i>A. flavus</i>, an inoculum was prepared from wheat that had already undergone a complete colonization process. Briefly, 600 g of grain was added to a stainless-steel pot filled with water and placed on a hot plate at 500°C. It was allowed to boil for 15 min, then 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). The grain was allowed to dry inside a laminar fume hood (for ca. 3 h). Subsequently, the grain was divided (in 300 g lots) and placed in two separate sterile mason jars (950-mL capacity). A single hole was drilled 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 <i>A. flavus</i>, a 3-inch strip of agar containing a pure culture of <i>A. flavus</i> grown on PDA for 7 days at 30°C, 60% RH, and 14:10 L:D photoperiod as above (Ponce et al., 2023; Ponce et al., 2024) was placed into each jar containing the grain. The jars were maintained at room temperature for roughly 10 days or until the <i>A. flavus</i> evenly covered as much of the grain as possible. Inoculated grain was used within 10–15 days of preparation. A total of 11.6 g of this inoculated grain was then added to 300 g for the <i>A. flavus</i> treatments above.<i>Grain Moisture & Progeny Production</i>At the end of 60 d, grain moisture readings were taken from 20 g of every replicate and each treatment after allowing grain masses to reach room temperature by using a moisture meter (DICKEY-john, GAC2100, Auburn, IL, USA). In addition, the number of F<sub>1</sub> progeny produced was measured by subtracting the 75 initial adults from the final total adults counted in each grain mass. Progeny production was used as the measure of fitness for <i>S. oryzae</i>. Frass was sieved (No. 30 sieve, 594 × 594 um mesh, W.S. Tyler Co., Cleveland, OH, USA) from grain by excluding adults and grain, then weighing using a tared balance (Quintix2102-1S, Sartorius AG, Göttingen, Germany) and weigh boats (Thomas Scientific, Chadds Ford Township, PA, USA).