,Experimental Insects,Four to eight-week mixed-sex adults of a field strain from Eastern Kansas (collected in 2022, hereafter FS-22) and pyrethroid-resistant strain collected from Juiz de Fora County in the state of Minas Gerais, southeastern Brazil in 2006 (hereafter, Brazil-resistant) S. zeamais were used in this study. The Brazil-resistant strain exhibits high pyrethroid resistance and low fenitrothion resistance and has been used in prior studies (Guedes et al., 2006a). These strains were reared and maintained on tempered organic maize at 25–27.5 °C, 65% RH, and 16:8 (L:D) h photoperiod.,Synergist-coated glass vials,In this study, we used one of the most effective synergists, piperonyl butoxide (PBO, Tokyo Chemical Industry Co. Ltd., Tokyo, Japan). Briefly, each of 20-ml glass scintillation vials was coated with 0.5 ml of PBO solution in acetone (solvent) at 0.1 mg/ml by using a Roto-Torque Heavy Duty Rotator (Model 7637, Cole-Parmer Instrument Company, Vernon Hills, IL, USA). For the control, vials were treated with 0.5 ml of acetone (solvent) only.,LLIN treatment,We used 0.34% alpha-cypermethrin based LLIN (63.2 mg/m2 active ingredient (a.i.), 40 deniers, 100 holes/cm2; Carifend®, BASF, Ludwigshafen, Germany) and a netting physically identical but without insecticide (Casa Collection, Mesh White, 1721-9668; Jo-Ann's, Hudson, OH, USA) as a control netting in our study.,Effects of synergists on LLIN against S. zeamais,A cohort of 20 mixed-sex S. zeamais adults was first pre-exposed to each scintillation vial coated with PBO or acetone (control) for 60 min (1 h) or 180 min (3 h). The pre-exposed adults were then transferred to each plastic Petri dish (9 × 9 cm square) containing either LLIN or control netting and were exposed for 60 min or 180 min. The inside walls of the dishes were coated with a polytetrafluoroethylene (PTFE) preparation (e.g., fluon, 60 wt% dispersion in water, MilliporeSigma GmbH, Steinheim, Germany) to prevent insects from escaping. After exposure, insects were placed in an environmental chamber under constant conditions (30°C, 65% RH, and 16:8 L:D). A total of n = 5 replicates were performed per treatment combination of strain, exposure time, netting type, and synergist. Immediate mortality was recorded directly after exposure, as well as delayed mortality at 24, 48, 72, and 168 h later. Insect conditions were recorded as alive, affected, or dead as described by Ranabhat et al. (2022). Specifically, insects moving normally were considered alive, whereas they were considered affected if they moved in an uneven pattern and/or exhibited twitching of tarsi or antennae or showed lethargic or drunken movements. On the other hand, insects were considered dead if no visible movement was observed after disturbance with a fine brush.,Lethal exposure assay to determine the susceptibility of S. zeamais,For this assay, a cohort of 20 mixed-sex adults of laboratory (pyrethroid-susceptible, FS-22) or Brazil pyrethroid-resistant strain of S. zeamais was exposed each 20-mL glass scintillation vial coated with a 2 mg/ml deltamethrin solution in acetone (solvent) or acetone only (control) at constant conditions (27.5° ± 0.1 C, 65% RH, 16:8 L:D) in an environmental chamber. Each of the three insect conditions including alive, affected, or dead as described above was recorded at each of 12 time points (i.e., 1, 2, 4, 6, 24, 48, 72, 96, 144, 168, 192, and 216 h) after the exposure. To examine the insect’s conditions, the exposed adults from each vial were transferred to each plastic Petri dish (90 mm in diameter; 59.4 cm2 bottom surface area) with a lining of a filter paper (85 mm D, Grade 1, GE Healthcare, Buckinghamshire, United Kingdom) that was adhered to the bottom using double-sided tape. The inside walls of the dishes were covered with a polytetrafluoroethylene preparation (Fluon, 60 wt% dispersion in water, MilliporeSigma GmbH, Steinheim, Germany) to prevent insects from escaping. The insect conditions were

,Insects,Adult P. truncatus were obtained from insect colonies kept in the Laboratory of Entomology and Agricultural Zoology (LEAZ), at the Department of Agriculture, Crop Production and Rural Environment, University of Thessaly, Greece, on whole maize kernels, at 26°C and 55% relative humidity (RH) and continuous darkness.,Insecticide-Incorporated Netting,The experiments were carried out in plastic petri dishes 90 mm in diameter (50.4 cm2 bottom surface). The inside of each petri dish was covered with two types of LLIN (0.4% deltamethrin, D-Terrence, Vestergaard INC., Lausanne Switzerland; and 0.34% alpha-cypermethrin, Carifend, BASF Ag, Ludwigshafen, Germany) and a polytetraflurorethylene preparation (Fluon, 60 wt% dispersion in water, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) to prevent insects from escaping. An additional series of dishes with physically identical control netting were prepared without an insecticide treatment to serve as the control. Twenty P. truncatus were then exposed on the insecticide-treated netting in petri dishes for 60, 90, 120, 240 min, 1, 3, and 5 days.,Mortality and Recovery,After exposure, insects were evaluated for mortality and individual P. truncatus that remained alive or knocked down (not dead) were then place into clean Petri dish arenas with a small amount of clean cracked maize kernels and evaluated for delayed mortality after 7 days. Using a stereomicroscope (SMZ-18, Nikon Inc., Tokyo, Japan) under 60× magnification, P. truncatus were classified as alive (moving normally, is able to right itself when flipped over, no twitching), affected (moving sluggishly or erratically, unable to right itself, twitching of antennae or legs may be present), or dead (completely immobile even after prodding) according to prior published definitions. There were three 3 replicates for each time treatment and netting type and 3 subreplicates for a total of 9 replicates for each treatment combination.,Explanation of files,The file "Greece Net Data 2023_All" includes the raw mortality ratings, whereas "Greece Net Data 2023_Recovery" includes calculated recovery values by P. truncatus, where recovery was calculated to "alive" from the initial reading (0 d) in "Greece Net Data 2023_All".,

,Aim of Dataset,In this work, we performed a two-year latitudinal biosurveillance program for Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae), related bostrichids, and Sitophilus spp. (Coleoptera: Curculionidae) in and around grain production and some natural areas to evaluate how landscape elements, latitude, and season affected their spatiotemporal dynamics.,Sampling locations & traps,The biosurveillance program was conducted by use of a trapping network in central North America in 2021 and 2022 and also in Greece in 2022. Trapping locations were selected along a latitudinal series across major grain-producing states in central North America from 19.6 to 46.8° N, including Estado de México in México, Texas, Oklahoma, Kansas, Nebraska, South Dakota, and North Dakota (Figure 1; Supplementary Table 1). The number of sites was expanded in 2022 compared to 2021 to provide a more comprehensive picture. At each location, we set up three-trap transects in each of two to three habitats: (1) near row crops (e.g., wheat, maize or soybean), (2) near a food storage facility (e.g. bins, elevator, or processor), and (3) in a natural habitat with no grain source nearby. Pitfall traps (Storgard Dome™ traps, Trécé, Inc., Adair, OK, USA) and 4-funnel Lindgren traps (Bioquip, Rancho Dominguez, CA, USA) spaced 5–10 m apart with a vented collection cup (9.5 × 15.2 cm D:H) at the base were used. The Lindgren traps included a 9 cm (D) piece of 0.4% w/w deltamethrin-incorporated netting or a 1-inch piece of No-Pest Strip (Hot Shot, Reynold’s Consumer Products, Lake Forest, IL) as the kill mechanism, as these have successfully been used in traps in the past (Wilkins et al. 2021). There were either three or four Lindgren or four pitfall traps in a given transect. The Lindgren traps were baited with a commercial formulation of male-produced P. truncatus aggregation pheromone (IL-953, Insects Limited, Westfield, IN, USA), Sitophilus spp. aggregation pheromone separately (IL-703, Insects Limited), multi-species pheromone lures for the cigarette beetle, Lasioderma serricorne (F.) (Coleoptera: Anobiidae), the Indian meal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae), and Trogoderma spp. (Coleoptera: Dermestidae) (IL-708, Insects Limited), and a R. dominica pheromone septa (Item#3158, Trece, Inc., Adair, OK, USA). A batch of lures was purchased in May 2021, and another batch was purchased in April 2022. The pitfall trap only contained the Sitophilus spp. and/or P. truncatus lure. We also added a small amount of maize or wheat to keep insects in the pitfall trap based on synergized response with food cues + pheromones for Sitophilus spp. (Trematerra and Girgenti 1989). The traps were deployed for 7-d periods either on a weekly or monthly basis depending on location from 14 June to as late as 7 Dec 2021 and 4 May to 6 Dec 2022. In Greece, the same protocol as above was utilized in a compressed timeframe consisting of 4 weeks during the key maize harvest in September 2022 at 4 sites between Volos and Thessaloniki (Central and Northern Greece).,Insect identification and specimen deposition,Insects were identified to species or genus where possible for all specimens using the USDA and Canadian taxonomic keys for stored product insects (Bousquet 1990; USDA 1991). Each trap capture was noted separately along with identifying information, and the abundance of P. truncatus, P. punctatus, other Bostrichidae, and Sitophilus spp. (including S. zeamais and S. oryzae) were recorded. Insects were identified using a dissecting microscope (SMZ18, Nikon Inc., Tokyo, Japan) at 30 x magnification. All specimens for project were deposited at the Kansas State University Museum of Entomological and Prairie Arthropod Research in the Department of Entomology.,,

,The aim of the current study was to track the movement of phosphine-resistant and -susceptible adults of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), which is a major pest of stored products, after brief exposures to phosphine. Exposures were followed for extended intervals to assess the recovery patterns, and how those patterns are related to known resistance to phosphine. A video-tracking procedure coupled with Ethovision software was used to assess movement after exposure.,Two strains of T. castaneum were used, one susceptible and one resistant to phosphine. The susceptible T. castaneum strain had been maintained in continuous culture without any known exposure to phosphine for >30 years at the USDA-ARS Center for Grain and Animal Health Research (CGAHR), in Manhattan, KS, USA. The phosphine-resistant strain of T. castaneum was collected from wheat in Palmital, Brazil during 1988 (BRZ-5). The rearing media consisted of 95% organic, unbleached, wheat flour plus 5% brewer's yeast. Tribolium castaneum were reared under laboratory conditions of 27.5°C, and 65% relative humidity (R.H.), 14:10 L:D. Adults, of mixed sex and <1 month old, were used in the exposure bioassays.,The protocol that was used in our bioassays to generate phosphine was the Phosphine Tolerance Test (Detia Degesch GmbH, Laudenbach, Germany) with some modifications, as performed by Agrafioti et al. 2021. In particular, the phosphine was generated within a plastic canister (5 L capacity) by adding 50 mL of water to two kit magnesium phosphide pellets. The concentration of phosphine gas inside the plastic canister was determined by using several dosimeter Draeger glass tubes (Draeger 25A, 0–10 000 ppm, Draeger Safety AG & Co., USA). Ten adults of each strain were placed in a plastic syringe of 100 mL with separate syringes used for each species and strain. Then, a specific gas quantity was removed from the canister with the syringe and blended with fresh air to produce a 100-mL volume with a concentration of either 1000 or 3000 ppm and compared to phosphine-free controls (0 ppm). The insects inside the syringe were held at the concentrations above for a 5 min exposure, while additional syringes containing only fresh air and insects were used as negative controls.,To understand the propensity for movement after a 5 min phosphine exposure, a video-tracking procedure was used. After exposure of phosphine-resistant or phosphine-susceptible T. castaneum for 5 min, adult movement was evaluated immediately after exposure or 24 h later under the same environmental chamber conditions as the colonies (see Source Insects), but held without supplemental food. Movement was recorded for 3 h immediately after phosphine exposure but binned into 30 min intervals (e.g., 0–30, 30–60, 60–120, 120–150, and 150–180 min) in order to evaluate how movement varied over the measured time period. Movement was also recorded 24 h after exposure for periods of 1 h (binned by 30 min intervals). Movement measures of adults was tracked in six replicate Petri dishes (90 × 15 mm D:H) with a piece of filter paper (85 mm D, Grade 1, GE Healthcare, Buckinghamshire, United Kingdom) lining the bottom using a network camera (GigE, Basler AG, Ahrenburg, Germany) affixed 80 cm above the dishes. The Petri dishes were backlit using a LED light box (42 × 30 cm W:L, LPB3, Litup, Shenzhen, China) to increase contrast and affixed in place with white foam board with holes specifically cut to size for the petri dishes. Video was streamed to a nearby computer and processed in Ethovision (v. 14.0.1322, Noldus Inc., Leesburg, VA). The software was used to calculate the total distance moved (cm) and the mean instantaneous velocity (cm/s) for each adult. Each adult was considered a replicate and was never used more than once. Only adults classified as alive (normal movement speed and activity), or affected (sluggish movements or on back with legs twitching) were used in this assay.

,Experimental Insects,Adult Tribolium castaneum and Rhyzopertha castaneum were obtained from insect colonies kept at the United States Department of Agriculture (USDA) Center for Grain and Animal Health Research in Manhattan, Kansas. Tribolium castaneum was continuously reared on 95% unbleached, organic wheat flour with 5% brewer’s yeast added, while R. dominica was reared on organic wheat, and both were held in an environmental chamber (Percival Scientific, Model CTH-811, Perry, IA, USA) set at a temperature of 30 °C, relative humidity (RH) of 65%, and a 16/8 h light/dark photoperiod. For the bioassays below, 3- to 4-week-old T. castaneum and R. dominica adults were used.,Synergist-coated Vials,Insecticide synergists tested in the bioassays were: piperonyl butoxide (PBO, Tokyo Chemical Industry Co. Ltd., Tokyo, Japan), diethyl maleate (DEM, Thermo Scientific Chemicals, Waltham, MA, USA), and triphenyl phosphate (TPP, Sigma-Aldrich, St. Louis, MO, USA). PBO, DEM, and TPP were dissolved in acetone, separately, and the concentrations used for them were all 0.1 mg/ml. Glass scintillation vials (27 × 61 mm, D × H, Wheaton Science Products, Millville, NJ, USA) were coated with 0.5 ml of synergist solution by rolling the vials on a Roto-Torque Heavy Duty Rotator (Model 7637, Cole-Parmer Instrument Company, Vernon Hills, IL, USA) to spread the synergist across the inner surface until all visible signs of liquid had disappeared. The vials were then left open to evaporate the acetone residues. In parallel, vials were coated with 0.5 ml of acetone as a control.,Long-lasting Insecticide-incorporated Netting (LLIN),Carifend® LLIN incorporating 0.34% (w/w) alpha-cypermethrin (BASF, Ludwigshafen, Germany) was used in this study. This netting was cut into squares and secured to the bottoms of square Petri dishes (100 × 100 × 15 mm, L × W × H, VWR, Radnor, PA, USA) as LLIN exposure arenas.,Effects of Synergists on LLIN against T. castaneum and R. dominica,Synergists and LLIN exposure bioassays for T. castaneum and R. dominica adults were conducted as below. Twenty adults were pre-exposed in a glass scintillation vial coated with one of the three synergists (PBO, DEM, TPP, or acetone as a control) for 1 h, and then transferred to a LLIN exposure arena. For T. castaneum, twenty adults were exposed to LLIN for 6, 12, 24, 48, 72, 96, 120, 144, and 168 h, respectively, and then examined for adult condition (unaffected, affected, and dead). For R. dominica, preliminary experiments showed that adults were very susceptible to LLIN, so twenty adults were exposed to LLIN for 0.5, 1, 2, 6, 12, 24, and 48 h, respectively. The adults were then transferred to a 35 × 10 mm Petri dish (Falcon, Franklin Lakes, NJ, USA) and adult condition was examined after 24 h. T. castaneum and R. dominica adults were observed and recorded as unaffected if they were active and behaving normally with coordinated walking and species-specific movements; affected if they had uncoordinated waliking and sluggish movements or were on their backs with legs twitching, and/or could not right themselves after being prodded (i.e., knocked down); finally, recorded as dead if they were completely motionless even after prodding. There were 4 replicates for each treatment combination of synergist type × LLIN exposure time.,Effects of Food on Efficacy of Synergists and LLIN Combined Exposures in T. castaneum and R. dominica,Twenty adults were pre-exposed in a vial coated with PBO, DEM, TPP, or acetone as a control for 1 h, and then exposed to LLIN for 72 h (T. castaneum) and 1 h (R. dominica). After exposure, the adults were transferred from LLIN exposure arena to a recovery arena, consisting of Petri dish (35 × 10 mm, D × H, Falcon, Franklin Lakes, NJ, USA) with 0.5 g of wheat flour or without flour as a control. Adult condition was assessed as number of unaffected, affected and dead at 1, 3, 5, and 7 d post-exposure. The delayed mortality and recovery rates were expressed as percentages,

,Insecticide Netting In this study, we focused on two types of long-lasting insecticide netting (LLIN) that have been found to be effective for managing various stored product insect pests. One is an LLIN consisting of a polyethylene netting (2 × 2 mm mesh, D-Terrence, Vestergaard, Inc., Lausanne, Switzerland) with 0.4% deltamethrin active ingredient (a.i.), while the second one is Carifend® net (40 deniers with mesh size 97 knots/cm2; BASF AG, Ludwigshafen, Germany) containing 0.34% α-cypermethrin (a.i.).,Foundational Model We used a standard Lefkovitch matrix model to project population growth for Tribolium castaneum, with four life stages (e.g., egg, larva, pupa, and adult;(Lefkovitch,1965). In equation (1), the Leftkovitch matrix L matrix (4 × 4) represents the life-stage structure of T. castaneum which has an egg, larvae, pupae, and an adult, where only the adults contribute to the fecundity, F. By multiplying L with the population vector ni(t), where t is time step (e.g., generation) and i is a life stage, we obtain the resultant vector ni(t + 1), which reveals the distribution of individuals across different life stages in the subsequent time period. In equation (1), P1 represents the probability of staying in the egg stage and G1 is the probability of moving from the egg to the larval stage, P2 is the probability of staying in the larval stage, G2 is probability of moving from the larval stage to pupal stage, P3 is the probability of staying in the pupal stage, G3 is probability of moving from the pupal stage to adult, while P4 is the probability of staying in the adult stage (Figure 1).,Model Parameterization and Scenarios We simulated population outcomes for up to 15 generations by using the life table data for T. castaneum using the R package popbio. Survivorship, fecundity, and transition information for each stage were derived from the literature (summarized in Table 1). The developmental duration of eggs, larvae, and pupae were 3.82 ± 0.005, 22.81 ± 0.67, and 6.24 ± 0.071 days (Kollros,1944). The average life duration of the adult used in this study was 221.16 days (Park et al., 1961). We used 94 offspring for fertility from the study Park et al.,(1965) and 99% rate of eclosion from pupae to adult. In order to explore the sensitivity of the base model to changes in mortality and fecundity, both of these parameters were systematically varied from near zero to their maximum value given in the base model (e.g., F = 94, P4 = 0.871). The parameters were varied alone or in combination and the resulting population growth was plotted. All plots were created using ggplot2 (Wickham, 2016) in R software (R Core Team, 2022). Three empirical scenarios from the literature were modeled containing estimates of fecundity reduction only, survivorship reduction only, or both fecundity and survivorship reduction when using LLIN (R.V. Wilkins et al., 2021; Gerken et al., 2021;Scheff et al., 2021, Scheff et al., 2023; Table 2). An individual projection matrix was constructed for each of the three scenarios and combinations of the reductions in fecundity, survivorship, or both. Population growth and proportion in each life stage was projected for 15 generations for each case, including the base model. Overall variation and oscillation were calculated to compare trends among proportion of life stages in each case. In order to compare differences in population sizes between cases for all generations and for generation 15 only, population sizes for each generation were bootstrapped 1000 times to provide iterative replication. The bootstrapped data were then compared one case to another using proc ttest in SAS (Version 9.4) for all generations and for generation 15 only. In addition, a sensitivity analysis was performed to determine which stage should be targeted to most greatly affect the population growth after exposure to the netting. Moreover, a mortality function based on empirical data with LLIN exposure collected in the laboratory

,Four to six-week-old larvae of Trogoderma variabile and Trogoderma inclusum were used for the experiment. Both strains were originally obtained from the field in north-central Kansas in 2016 and 2012, respectively. Colonies of these species were reared under controlled conditions in an environmental chamber set to a temperature of 27.5 °C, 65% RH, and 14:10 (L:D) h photoperiod. Both species were fed 300 g of ground dog food (SmartBlend, Lamb flavor, PurinaOne, St. Louis, MO, USA) with oats sprinkled on top and a moistened, crumpled paper towel placed on the surface in a 950-ml mason jar.,Treatments The long-lasting insecticide-incorporated polyethylene netting (2 × 2 mm mesh, D-Terrance, Vestergaard Inc., Lausanne, Switzerland) included 0.4% deltamethrin, or control netting that was identical in physical properties but without insecticide. These were used with the movement assay. We assessed the movement in the vicinity of important pheromonal and food kairomones after exposure to LLIN or control netting. Food consisted of 0.01 g of organic, unbleached flour (Heartland Mills, Marienthal, KS, USA), and pheromonal stimuli included a broad spectrum, multi-species lure (PTL lure, IL-108-10, Batch#1288200321, Insects Limited, Westfield, IN, USA), including Trogoderma spp pheromone (Ranabhat et al. 2023a). In each replicate, we used a single pellet (white color), and affixed it in place so it did not move in a Petri dish using a 1 × 1 mm square of parafilm. For each replication of testing, we used a fresh lure.,Movement Assay The movement of larvae after exposure to the 0.4 % deltamethrin LLIN or a control netting in response to food cues (using 0.01 g of flour) or with conspecific sex pheromones (using a single bead from a disaggregated PTL lure held in place with a small square of parafilm), was tracked in six individual arenas (100 × 15 mm D: H) with a piece of filter paper (85 mm D, Ahlstrom-Munksjö, Helsinki, Finland) lining the bottom for 30 min using a network camera (GigE, Basler AG, Ahrenburg, Germany) affixed 76 cm above and centered over the dishes. The Petri dishes were backlit using a LED light box (42 × 30 cm W:L LPB3, Litup, Shenzhen, China) to increase contrast and affixed in place with white foam board. The video was streamed to a computer and processed in Ethovision (v.14.5 Noldus Inc., Leesburg, VA, USA). Prior to use in the movement assay, larvae of T. variabile or T. inclusum were exposed to the 0.4% deltamethrin LLIN or a control netting for 1 min in a 21 × 21 cm square Petri dish, then their movement was tracked individually after a post-exposure holding duration of 1 min or 24 h. A small 1.1 cm hidden stimulus zone encircled each stimulus, midway and centered on each half of the arena wherein movement was tracked separately from each half of the arena (control vs. treatment). The total distance moved (cm), instantaneous velocity (cm/s), frequency of entering each half of the petri dish and stimulus zone, cumulative duration spent in each zone (s), and latency of entering each zone (s) over a 30 min trial period was logged after exposure to a given treatment. The control side of the arena remained empty. A total of n = 16 replicates were run per treatment combination for both species,No-Choice Release-Recapture Assay A release- recapture experiment was conducted for the larvae of both T. variabile and T. inclusum where larvae were exposed to the 0.4% deltamethrin LLIN and control netting for 1 min. After exposure, treated insects were released at one corner of the sanded plastic bin (60 × 41.6 × 16.5 cm L:W:H ). A commercial pitfall trap (Dome Trap™, Trécé, Inc., Adair, OK, USA) that contained a PTL lure (used only white beads as above), or 0.01 g flour, or no stimuli (unbaited for control), was deployed in the opposite corner, diagonally across from the release point in the bin. The bins were located in a large (4.8 × 2.1 × 6 m, L:W:H) walk-in environmental chamber (Percival Instruments, Dallas County, IA,

Wild bee and butterfly samples were collected from the margins of agricultural fields located on five Conservation Areas in Missouri. In 2016 and 2017, samples were collected and composited by genera for a total of 90 samples. Samples were extracted via pressurized liquid extraction and solid phase extraction cleanup. Samples were analyzed for 168 pesticides and degradates using both gas and liquid chromatography-tandem mass spectrometry. Overall, 16 pesticides were detected. Pesticides detected in greater than 2% of the composite samples included: metolachlor (24%), tebuconazole (22%), atrazine (18%), imidacloprid desnitro (13%), bifenthrin (9%), flumetralin (9%), p,p’-DDD (6%), tebupirimfos (4%), fludioxonil (4%), flutriafol (3%), cyproconazole (2%), and oxadiazon (2%). Concentrations for individual pesticides ranged from 2 to 174 ng/g. Results indicate that wild pollinators are exposed to a wide variety of pesticides.

Wild bee and butterfly samples were collected from the margins of agricultural fields located on five Conservation Areas in Missouri. In 2016 and 2017, samples were collected and composited by genera for a total of 90 samples. Samples were extracted via pressurized liquid extraction and solid phase extraction cleanup. Samples were analyzed for 168 pesticides and degradates using both gas and liquid chromatography-tandem mass spectrometry. Overall, 16 pesticides were detected. Pesticides detected in greater than 2% of the composite samples included: metolachlor (24%), tebuconazole (22%), atrazine (18%), imidacloprid desnitro (13%), bifenthrin (9%), flumetralin (9%), p,p’-DDD (6%), tebupirimfos (4%), fludioxonil (4%), flutriafol (3%), cyproconazole (2%), and oxadiazon (2%). Concentrations for individual pesticides ranged from 2 to 174 ng/g. Results indicate that wild pollinators are exposed to a wide variety of pesticides.

,Experimental Insects,The field strains of T. castaneum and R. dominica (F.) were used in this study. The former originates from Eastern Kansas in 2012, and the latter is also from Eastern Kansas but from 2019. For all species, four to eight-week-old adults were used. Rearings were kept at the USDA Center for Grain Animal Health Research in Manhattan, KS. Tribolium castaneum was reared on a mixture of 95% unbleached, organic flour and 5% brewer’s yeast, while R. dominica was reared on tempered organic whole wheat. Colonies were maintained at 27.5°C, 65% RH, and 14:10 for maintenance or 16:8 (L:D) h photoperiod for the experiment.,Treatments,Treatments included exposure to three different types of long-lasting insecticide-incorporated netting (LLIN). These consisted of 1) Carifend®, LLIN with 0.34% alpha-cypermethrin (40 deniers, BASF, Ludwigshafen, Germany), 2) D-Terrence, LLIN with 0.4% deltamethrin (2 × 2 mm mesh, Vestergaard SA., Lausanne, Switzerland), and 3) 8% etofenprox LLIN (AgBio, Inc, CO, USA), and for control, we used netting identical to the Carifend or Vestergaard netting but lacking insecticide.,Direct Lethality Assessments,Cohort of 20 mixed-sex adult beetles were exposed for 5, 60, or 120-min intervals on netting affixed to a 9 × 9 cm2 petri dish in the laboratory. After exposure, we took the evaluated condition after 0, 24, 72, or 168 h as alive, affected, or dead condition (Figure 1), according to the definitions described in Ranabhat et al. (2022) in Petri dishes without netting containing 8.5 cm D filter paper. Briefly, living adults were defined as moving with normal speed and activity and able to right themselves if flipped. By contrast, affected adults exhibited sluggish or drunken movements, could not right themselves if flipped, and some or all of their limbs exhibited twitching. Dead adults were completely immobile. For post-exposure treatment, adults were held under the same environmental chamber conditions as the colonies but without supplemental food after exposure. We performed a total of n = 4 replications per treatment combination for each species.,Baseline Mobility Assay after Exposure to LLINs,Based on the observation of the lethality assay, we focused our baseline mobility assay on Carifend® and D-Terrence LLIN. Using only alive adults, we assessed their movement in six individual Petri dishes (100 × 15 mm D: H) that consisted of a filter paper (85 mm D, Grade 1, GE Healthcare, Buckinghamshire, United Kingdom) lining. Treatments included a negative control (e.g., filter paper only), one of the two LLINS, or an identical netting to the Carifend or Vestergaard netting but without insecticide (e.g., as a positive control). Their movement was tracked for 60-min using a network camera (GigE, Basler AG, Ehrenburg, Germany) affixed 80 cm above the dishes. The Petri dishes were backlit using a LED light box (42 × 30 cm W: L, LPB3, Litup, Shenzhen, China) to increase contrast and affixed in place with white foam board. The video was streamed to a computer and processed in Ethovision (v.14.0, Noldus Inc., Leesburg, VA). The program automatically calculated the total distance moved (cm) and the instantaneous velocity (cm/s) over the 60-min period for each adult. Each adult was considered a replicate and was never used more than once. In total, n = 18 replicates were performed per treatment combination.,Comparison of Sublethal Effects among LLINs,For the sublethal movement assay, mixed-sex adult beetles were exposed to the Carifend®, D-Terrence LLIN, or control net as mentioned above. Cohorts of 5–10 adults were exposed for 5- or 60-min intervals on LLINs affixed to a 9 × 9 cm2 Petri dish in the laboratory. After exposure, the effects of the LLINs on adult movement were assessed either immediately or after 72 h in Petri dishes under the same environmental chamber conditions as the colonies but without supplemental food and then assayed using the video-tracking system described above by using Ethovision