2.1 Experimental Insects
A field strain of the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) and the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) obtained from Pottawatomie Co, KS, and eastern KS, respectively, were used in this study. We used four to eight-week-old adults of both species. Cultures of R. dominica and T. castaneum have been maintained in the laboratory since 2019 and 2012, respectively at the USDA Center for Grain Animal Health Research in Manhattan, KS. Ryzopertha dominica were reared on tempered organic whole wheat, while T. castaneum were reared on a mixture of 95% unbleached, organic flour and 5% brewer’s yeast. The colonies were subcultured on a monthly basis. The colonies were maintained at 27.5°C, 65% RH, and 14:10 (L:D) h photoperiod in environmental chambers (Percival Scientific, Perry, IA, USA).
2.2 Experimental Arena and its Production
The experimental arena consisted of a 63 cm × 15.5 cm × 9.5 cm L:W: H metal frame. This arena also contained four square blocks of concrete (e.g., Rockite, Hartline Products Co., Cleveland, OH, USA) measuring 15.24 cm × 15.24 cm × 1.5 cm L:W: H to create a testing platform that mimics the surface of a food facility (Figure 1). The concrete was prepared by first mixing tap water and Rockite cement mix in a large water pitcher. The tap water was added to the dry Rockite mixture and combined until a thick paste consistency was achieved. The slurry was poured into a 1.1 L volume silicone square mold (15.24 cm × 15.24 cm). The slurry was poured 1.5 cm thick. The cement concrete squares were left to dry and solidify at room temperature for 2–3 d. Cement squares were modular and new ones were used with each replicate performed for the assay.
In each experimental arena, there were four cement squares (Figure 1). We applied tape (VWR International, LLC Radnor, PA, USA) on the wall of the metal frame before placing the concrete to make it easier to remove the concrete after each replicate was conducted, and so the metal forms could be re-used. Adhesive caulk (DAP Kwik Seal, DAP Products Inc., Baltimore, MD, USA) was applied to fill any gaps between the concrete squares or between the concrete and the metal frame. The concrete at the distal end of the experimental arena was filled with 20 g of whole, organic, unbleached flour (Heartland Mills, Marienthal, KS, USA) for T. castaneum or organic whole wheat (Heartland Mills, Marienthal, KS, USA) for R. dominica. Insects were released on the concrete square at the opposite end. The two middle concrete slabs were reserved for one or two treatments as below. The inner wall of the metal frame was coated with fluon (polytetrafluoroethylene, Sigma-Aldrich Co., St. Louis, MO, USA) to prevent insect escape.
2.3 Insecticide formulation for concrete
We used CentanylTM EC (formulation consisting of 49.13 mg deltamethrin/mL), and Evergreen® (formulation consisting of 8.39 mg pyrethrins/mL) which were obtained from Central Garden & Pet Company (Schaumburg, IL, USA) and MGK® Company (Aspen Lane North, MN, USA), respectively. In this study, we used a high labeled rate of CentanylTM EC (45 ml in one gallon of diluent to cover 930,000 cm2), and a high labeled rate of Evergreen® (650.618ml of product in 3785.412 ml of water to cover 700,000 cm2) (Table 1.). A water-only treated control was used for each replication.
For CentanylTM EC, each lot was formulated by adding 0.23 ml of product in 18.9 ml of water in a 25-ml volumetric flask. For Evergreen®, each lot was formulated by adding 4.3 ml of product and 25.1 ml of water in a 50-ml volumetric flask. We evenly sprayed 1 ml of the final formulation of CentanylTM EC per concrete square (area: 232.3 cm2) and 1.5 ml of Evergreen® formulation per concrete square by using an artist’s airbrush (Badger 100 series, Badger Corporation, Franklin Park, IL). For the negative control, we sprayed 1.5 ml of tap water with the artist’s airbrush. We allowed the sprayed concrete squares to dry at room temperature for 24 h prior to using them for the experiment.
2.4 Treatment
We used the following six treatments: 1) untreated controls with netting identical to long-lasting insecticide-incorporated netting (LLIN) but lacking insecticide (Casa Collection, Mesh White, 1721-9668; Jo-Ann’s, Hudson, OH, USA), and/or concrete sprayed only with distilled water, 2) LLIN only (Carifend® net, 0.34% alpha-cypermethrin, BASF Corp., Ludwigshafen, Germany) applied near the insect release point (15.24 cm × 15.24 cm; same size strip of the net were used for whole experiment), 3) CentanylTM EC only applied near the insect release position, 4) Evergreen® only applied near the insect release position, 5) a strip of the LLIN and CentanylTM EC (LLIN laid sequentially directly before the insecticide to intercept immigrating insects: Morrison et al. 2018, Wilkins et al. 2021), and 6) a strip of the LLIN material and Evergreen® (LLIN laid sequentially directly before the insecticide to intercept immigrating insects).
We assembled the experimental arenas for each treatment replicated on separate days. In each experimental arena, we released 30 mixed-sex adults of either R. dominica or T. castaneum (just a single species per arena) at the far end of the testing platform opposite the food source. Each dispersal apparatus was placed into a sterilite bin (86.3 × 30.5 × 39.4 cm L:H:W) to prevent insect escape and effects from neighboring apparatuses, and placed on the shelf of a walk-in environmental chamber at the same conditions as the rearing colonies. After a 48 h dispersal period, we counted the number of insects on each concrete square and we also checked their conditions in clean petri dishes lined with filter paper. The condition of the adults was checked under the dissecting microscope and rated as alive, affected, or dead by following the definitions in Morrison et al. 2018. Briefly, the alive were moving around normally without impairment, while those that were affected showed uncertain movements, twitching of extremities, and/or were not able to right themselves after being gently prodded with a paintbrush. Those that were classified as dead were completely immobile. We collected the food source by removing the flour or wheat. Subsequently, the commodity was gently sieved using two sieves (#10 sieve, 2.0 × 2.0 mm mesh, W.S. Tyler, Mentor, OH; and #20 sieve, 0.841 × 0.841 mm mesh; W.S. Tyler, Cleveland, OH), making sure no eggs were harmed during the process and all food dust and detritus was transferred to progeny production containers. All the adults were removed from the commodity and the commodity was retained for 6 weeks to evaluate progeny production. There were 5–6 replications for each treatment and species combination.
