,Data collected by Sabita Ranabhat from early Sep 2020 - Jun 2021. It consists of two experiments: one evaluating mobility of Tribolium castaneum and Rhyzopertha dominica after exposure to differently formulated controlled release materials for 0-60 min, and a second experiment looking at median lethal time after exposure to cheesecloth treated with permethrin, deltamethrin, indoxacarb, and dinotefuran between 0-168 h. Tested strains include phosphine-resistant and phosphine-susceptible populations.,,

,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,

,Insecticide: Two insecticides were used in this study: an existing formulation (tradename: Diacon IGR+ R ; Central Life Sciences, Schaumberg, IL, USA), and a new formulation with synergist (tradename: Gravista ). Diacon IGR+ contains 11.4% methoprene and 4.75% deltamethrin, with a label rate of 0.12 kg AI/L and 0.05 kg AI/L. The label rate as a residual surface treatment gives a range of 28.5 mL AI/L−171 mL AI/L H2O to cover 94 m2 for both compounds. We used the maximum labeled rate of 24 mg AI/m2 for deltamethrin and 57 mg AI/m2 for methoprene. This corresponded to 0.3 ml of the formulation in 25 ml H2O, sprayed at the rate of 0.3 ml per 50.3 cm2 arena, using an artist’s air brush (Badger 100 series, Badger Corporation, Franklin Park, IL, US) for each treatment. Each replicate was evenly applied to the concrete dish using a compressor pump. The new Gravista formulation has one labeled rate of 684 ml formulation/L H2O to cover 92.9 m2. To achieve this, we mixed 0.5 ml of the new formulation in 10 ml H2O. This was sprayed at the same rate as the other compound. Distilled water was used for the control arenas at 0.3 mL per arena. The arenas were given 8 h to dry prior to use in experiments. Insects (20 of each species per replicate) were exposed on the insecticide-treated petri dishes for either 4 or 24 h. After exposure, individual Prostephanus truncatus and Sitophilus zeamais were removed and placed into clean Petri dish arenas and evaluated for condition. Using a stereomicroscope (SMZ-18, Nikon Inc., Tokyo, Japan) under 60× magnification, P. truncatus and S. zeamais 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 (Ranabhat et al., 2022).,,Dispersal and Mortality: To test dispersal capacity to new food patches, a dispersal apparatus was employed. Species-specific cohorts of 20 adults (P. truncatus or S. zeamais) were exposed to Gravista, IGR+, or an untreated control as above for 4 or 24 h, then given 48 h to disperse across 30 or 70 cm standardized sections of PVC pipe (3.175 cm ID). After exposure to insecticide formulations, insects were evaluated for condition after exposure before placing them in the dispersal apparatus. The ends of both sides of the PVC pipe were sealed with mesh (425 μm) to prevent escape. At the far end of the pipe, a hole (2 cm D) was drilled and centered over a glass jar (5 × 6.5 cm D:H) to create a pitfall trap design. The glass jar contained 20 g of whole maize kernels, representing a novel food patch, to induce insects to disperse with food kairomones. Untreated, clean, and uninfested yellow maize was used in the experiments. Grain was sourced from Heartland Mills (Marienthal, KS, USA), and frozen for 72 h prior to use to ensure no prior insect infestation was present. At the end of the sampling period, the number of insects in the jar and their mortality was scored as alive, affected or dead. In addition, the position of each individual was recorded as residing in zone 1 (at the release point), zone 2 (in first half of tube), zone 3 (in second half of tube), or zone 4 (collection jar with maize). In total, there were n = 12 replicate cohorts for each species and combination of distance and treatment. In total, 1,440 P. truncatus and 1,440 S. zeamais were tested in this experiment.,

,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,

,2.1 Experimental insects,A laboratory colony of T. castaneum maintained at the USDA Center for Grain and Animal Health Research for over 30 years free of exposure to insecticide was used for these studies. The colony was kept in 0.95-L glass jars with filter paper lids for air flow with a rearing diet consisting of 95% unbleached, organic flour with 5% brewer's yeast added. Jars were held in an environmental chamber set at a temperature of 30 °C, relative humidity (RH) of 65%, and a 16:8 h light/dark photoperiod.,2.2 Manipulating diet nutritional quality,Unbleached, organic wheat flour was mixed with microcrystalline cellulose (Thermo Fisher Scientific, Waltham, MA, USA), a nonnutritive and nontoxic filler that has a similar particle size to wheat flour to produce three diet treatments that differed in nutritional quality: 0% flour (0 flour:100 cellulose), 25% flour (25:75), and 100% flour (100:0). A treatment with no flour or cellulose was also included, as a no-food control.,2.3 Long-lasting insecticide-incorporated polyethylene netting,Two types of Long-lasting insecticide-incorporated nettings (LLINs), both commercially labeled in the USA, were used: CarifendTM (BASF, Ludwigshafen, Germany) and D-TerrenceTM (Vestergaard, Lausanne, Switzerland),. Carifend netting incorporated 0.34% (w/w) α-cypermethrin while D-Terrence netting incorporated 0.4% (w/w) deltamethrin. Control netting was physically identical to the Carifend netting or D-Terrence netting but lacked any insecticide.,2.4 Effects of diet on recovery of T. castaneum adults after exposure to LLIN,The netting was cut into squares and secured to the bottoms of square Petri dishes (100 × 100 × 15 mm, L × W × H, VWRTM, Radnor, PA, USA) with double-sided tape and label tape. 20 mixed-sex adults (2- to 3-week-old) were exposed to one of the three netting types (Carifend, D-Terrence, and control) for 2, 24, 48, 72, 96, 120, 144, and 168 h, respectively, with a total of 24 combinations of netting and exposure time treatment. Immediately following exposure, 20 adults were examined for health condition (unaffected, affected and dead) and transferred from the netting arena to recovery arenas. Recovery arenas were Petri dishes (35 × 10 mm, D × H, FalconTM, Franklin Lakes, NJ, USA) with 0.5 g of one of the three flour:cellulose diets (0:100, 25:75, and 100:0)). In addition, arenas without any food were also included as controls. There were 4 replicates for each combination of netting × exposure time × diet.,The condition of T. castaneum were assessed as number of unaffected, affected and dead at 0, 1, 2, 3, 4, 5, 6, and 7 d post-exposure to netting. Adults were recorded as unaffected if they were active and behaving normally with coordinated walking and species-specific movements; affected if they had uncoordinated walking 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, dead if they were completely motionless even after prodding. During exposure and recovery, insects were kept in an environmental chamber under the same conditions as described above. Because no T. castaneum adults were observed to be affected and the number of dead was very low after exposure to control netting (Fig. 1), recovery experiments on control netting were not included in rest of the study.,2.5 Effects of diet and netting exposure on mobility of T. castaneum adults,Eighteen newly emerged (≤ 2-day-old), mixed-sex adults that had been reared on standard lab diet were transferred to 0.5 g of one of the assigned diets (0:100, 25:75, and 100:0 flour:cellulose) in Petri dishes (35 × 10 mm, D × H, FalconTM, Franklin Lakes, NJ, USA) for one week. As above, there was also a no food treatment as a control. After the one-week time period, 18 adults from each of the diet treatments were exposed to netting (Carifend, D-Terrence, or control) for 10, 30, 60, or 90 min. Only adults rated as unaffected or affected

,Reproductive output and behavioral data from stored-product pest insects exposed to long-lasting insecticide treated netting. Includes offspring output and survival for Trogoderma variabile (treated as larvae) and Tribolium castaneum (treated as adults).,,

,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 or 16:8 (L:D) h photoperiod.,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, 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 software. Only adults classified as alive or

,Our goals were to 1) isolate, and culture two fungal morphotypes, 2) characterize the volatile emissions from grain inoculated by each fungal morphotype (Aspergillus flavus or Fusarium spp.) compared to uninoculated and sanitized grain, and 3) understand how MVOCs from each morphotype affects mobility, attraction, and preference by L. serricorne. Headspace collection revealed that the Fusarium- and A. flavus-inoculated grain produced significantly different volatiles compared to sanitized grain or the positive control. Changes in MVOC emissions affected close-range foraging during an Ethovision assay, with a greater frequency of entering and spending time in a small zone with kernels inoculated with A. flavus compared to other treatments. In the release-recapture assay, MVOCs were found to be attractive to L. serricorne at a longer distances in commercial pitfall traps. While there was no preference shown among semiochemical stimuli in a still-air, four-way olfactometer, it is possible that methodological limitations prevented robust interpretation from this assay. Overall, our study suggests that MVOCs are important for close- and long-range orientation of L.serricorne during foraging, and that MVOCs may have the potential for inclusion in behaviorally-based tactics for this species.,

,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