TY - JOUR
AU - Koppenhöfer, Albrecht, M
AB - Abstract The annual bluegrass weevil, Listronotus maculicollis (Kirby) (Coleoptera: Curculionidae), is the most difficult to control insect pest on golf courses in eastern North America. Insecticide resistance, particularly to pyrethroids, is a serious and expanding issue in its management. Optimal diagnostic tools for resistance detection are crucial for efficient resistance monitoring and mitigation. Developed vial and Petri dish assays clearly separated different resistance levels among weevil populations. With the pyrethroid bifenthrin, susceptible, moderately resistant (resistance ratios, RR50s 12.2–95.7), and highly resistant (RR50s 258.2–1760.9) populations were distinguished. With the organophosphate chlorpyrifos, susceptible, tolerant (RR50s 2.4–6.7), and resistant (RR50s 8.8–120.7) populations were distinguished. In validation assays, several bifenthrin and chlorpyrifos concentrations were needed to separate resistance levels in Petri dish (bifenthrin: 112.2 and 336.3 or 3,362.5 mg AI/m2; chlorpyrifos: 3.4 and 33.6 mg AI/m2) and vial (bifenthrin: 112.1 or 1,120.8 mg AI/m2; chlorpyrifos: 2.2 and 11.2 mg AI/m2) assays. The Petri dish assay with formulated bifenthrin and chlorpyrifos was the best option for L. maculicollis resistance detection and monitoring. It demonstrated sufficient discriminating power, accurately reflected resistance levels, and was easier to conduct. A single diagnostic concentration sufficed to separate susceptible and resistant populations. To determine different resistance or tolerance levels, two to three concentrations were necessary. Listronotus maculicollis, resistance, annual bluegrass weevil, diagnostic concentration, diagnostic assay Developing tools for insecticide resistance detection and monitoring is a key component of resistance management (Roush and Miller 1986, ffrench-Constant and Roush 1990). Resistance monitoring programs usually serve several important purposes: documenting or confirming resistance at locations with reported control failures, determining whether or not other factors (application error and wrong timing) caused the reduced product efficacy, monitoring changes in the resistance level of populations, which were previously reported to be resistant, and resistance detection at new locations (Roush and Miller 1986). The precision of a detection and monitoring tool is crucial for the subsequent steps in resistance management and for the choice of control strategies. Traditionally, dose- or concentration-response curves are developed, and LD50s are calculated and compared to separate susceptible and resistant populations (Halliday and Burnham 1990). However, this approach is labor intensive, requiring large sample sizes from the populations tested, and often is impractical in the field or in diagnostic laboratories. Therefore, for many insect species, an assay based on one or two diagnostic or discriminating doses is developed to make resistance monitoring feasible (Halliday and Burnham 1990). The annual bluegrass weevil, Listronotus maculicollis (Coleoptera: Curculionidae), is the most difficult to control insect pests of short-mown golf course turf in the Northeast and Mid-Atlantic regions of the United States and southeastern Canada. Annual bluegrass weevil larvae can cause severe damage to tees, fairways, collars, and greens on golf courses. High turf quality standards on golf courses often lead to heavy reliance on conventional insecticides for annual bluegrass weevil management. Annual bluegrass weevil populations were first controlled with chlorinated hydrocarbons starting in the late 1960s, which was later followed by the use of organophosphates. Starting in the 1980s, pyrethroids became the primary insecticides because of their high levels of efficacy against annual bluegrass weevil adults (Schread 1970). Application of adulticides (mostly pyrethroids) in early spring can prevent annual bluegrass weevil oviposition and subsequent damage caused by larval feeding. Due to excessive pyrethroid use, by the 2000s, many golf courses started to observe a decrease in the efficacy of this strategy. Resistance to pyrethroids was first documented by Ramoutar et al. (2009a,b) in adults from several annual bluegrass weevil populations from southern New England. According to a recent survey (McGraw and Koppenhöfer 2017), conducted throughout the pest’s range (all states of Mid-Atlantic and Northeast regions of the United States and southeastern Canada), on average 19% of responding golf courses (10% participation) suspected (or had confirmed) to have insecticide-resistant annual bluegrass weevil populations, with higher rates around the epicenter of the weevil’s distribution (e.g., 55% Long Island, NY; 48% Connecticut). Kostromytska et al. (2018) confirmed pyrethroid resistance from many golf courses around the New York Metropolitan area, throughout New Jersey, and in eastern Pennsylvania with resistance ratios (RR50s) as high as 343 compared with the most susceptible populations. Furthermore, an extensive summary of field efficacy tests suggested that the efficacy of most insecticides currently available for annual bluegrass weevil management was reduced (15–57%) against pyrethroid-resistant populations (Koppenhöfer et al. 2012); only spinosad (Koppenhöfer et al. 2012, 2018) and cyantraniliprole (Koppenhöfer et al. 2018) applied as larvicides seem to be unaffected thus far. Pyrethroid resistance in annual bluegrass weevil seems to be at least in part due to enhanced enzymatic detoxification (Ramoutar et al. 2009b), a rather nonspecific mechanism that breaks down active ingredients before they reach their target sites in the organism. The specter of the expanding threat of insecticide resistance in annual bluegrass weevil warrants continued efforts on the development of more sustainable management practices such as plant resistance (Kostromytska and Koppenhöfer 2014, 2016), biological control (McGraw et al. 2010, Koppenhöfer and Wu 2017, Wu et al. 2017), and improving annual bluegrass weevil monitoring. However, with the continued high demands on turf quality, the use of insecticides will remain an important part of annual bluegrass weevil management. Hence, effective detection and monitoring of resistance levels in field populations may serve as a cornerstone not only for development of effective resistance management programs but also for developing more site-specific management recommendation at any given golf course. Optimal tools for resistance detection are necessary to determine whether insecticide resistance or other factors affect insecticide performance and to help monitor the development and expansion of resistance. The main goal of this study was to develop effective, practical, and reliable resistance diagnostic bioassays and several diagnostic concentrations, which will allow fast and accurate determination of the resistance levels in samples of annual bluegrass weevil populations. Materials and Methods Insects Adult annual bluegrass weevils were collected from eight populations at different golf courses in New Jersey, eastern Pennsylvania, southeastern New York, and southwestern Connecticut during 2014–2015. The resistance levels of the populations had been determined in previous topical and greenhouse assays (Kostromytska et al. 2018). Two populations were relatively pyrethroid-susceptible (PB, Manalapan, NJ; HP, Farmingdale, NJ) and five populations demonstrated various degrees of tolerance or resistance to pyrethroids (CN, Easton, CT; JC, Cheltenham, PA; LI, Glen Cove, NY; GB, Somers Point, NJ; EW, River Vale, NJ). Overwintering generation annual bluegrass weevil adults were collected from hibernation sites in late October or early November. They were kept in groups of 200 in 840-ml plastic containers filled with pasteurized moist sand (5% w/w) for 2–4 mo in an incubator (10 h light at 6°C: 14 h dark at 4°C) to break their reproductive diapause (Wu et al. 2017). Prior to bioassays, adults were extracted and kept in groups of 100 in ventilated clear plastic containers on moist sand in an incubator (14 h light at 21°C: 10 h dark at 14°C) for 14 d to allow them to become sexually mature after diapause termination (Wu et al. 2017). They were provided with black cutworm artificial diet (Bio-Serv, Frenchtown, NJ) and annual bluegrass, Poa annua L., clippings as food. Spring generation adults were vacuumed from fairways on golf courses and Rutgers Horticulture Farm No. 2 (HF, North Brunswick, NJ) in late June to early July using a modified leaf blower vacuum. After collection, the adults were placed into 840-ml containers with black cutworm artificial diet and P. annua sprigs as food sources and kept in an incubator (14 h light at 24°C: 10 h dark at 16°C) for at least 1 wk prior to use in experiments. Toxicity of Bifenthrin and Chlorpyrifos in Petri Dish Assays The toxicity of the formulated products Talstar Pro (AI: bifenthrin, FMC, Philadelphia, PA) and Dursban 50W (AI: chlorpyrifos, Dow AgroSciences, Indianapolis, IN) was determined in 9-cm-diameter Petri dishes. The bottom of the dishes was lined with a single layer of filter paper and treated with aqueous solution (1 ml) of one of the insecticide concentrations using a pipette. Untreated controls received water only (1 ml). Then 10 adults (not sexed) were introduced into the dishes. Mortality was evaluated at 24, 48, and 72 h after treatment. Weevils were considered dead if no movement was observed even after probing and moribund if twitching or uncoordinated movements were observed but the weevils were not able to walk. The bioassays were conducted in 2014 and 2015. In both years, the experiment was replicated three times (three runs with one replicate per run). In 2014, spring generation adults of four annual bluegrass weevil populations (LI, EW, PB, and HP) were exposed to four (EW) or five (LI, PB, and HP) concentrations of bifenthrin (0.34 mg to 6.72 g AI/m2) and chlorpyrifos (3.36 mg to 0.34 g AI/m2). In 2015, the Petri dish assay was repeated with overwintering generation adults using two susceptible (PB and HP) and five resistant (CN, EW, GB, JC, and LI) populations with five concentrations of bifenthrin (0.34 mg to 6.72 g AI/m2) and chlorpyrifos (3.36 mg to 0.34 g AI/m2) for each population. Toxicity of Bifenthrin and Chlorpyrifos in Vial Assays Vial bioassays were conducted in 16-ml glass vials with an internal surface area of 35.5 cm2. Technical-grade bifenthrin and chlorpyrifos (>95% purity) were dissolved in acetone and applied to the vials’ inner surface assuming that weevils would be exposed to insecticides through direct contact with the treated glass surface and by ingestion through preening. Technical-grade AI dissolved in 1-ml acetone (bifenthrin: 0.34 mg to 6.72 g AI/m2; chlorpyrifos: 0.11 mg to 0.34 g AI/m2) was added to the vials that were then manually rolled at a 10° angle until most of the acetone had evaporated (~60 s). Vials were then rolled horizontally on a hot dog roller until completely dry (~1 h). Control vials were treated with acetone only. Ten adults (not sexed) were introduced into each vial. Then the vials were plugged with a cotton ball and held horizontally in the incubator (14 h light at 25°C: 10 h dark at 20°C). Mortality was evaluated at 24, 48 and 72 h after adult introduction. The assays were conducted with overwintering generation adults using two susceptible (PB and HP) and four resistant (LI, EW, CN, and GB) populations and were replicated three times (three runs with one replicate per run). Diagnostic Concentration Validation in Assays With Susceptible and Resistant Populations Using mortality data obtained in the Petri dish and vial bioassays, concentration-response curves were developed and concentrations required to kill 50% (LC50), 95% (LC95), and 99% (LC99) of the tested populations were estimated using Probit analysis (SAS Institute 2011). The selection of the candidate diagnostic concentrations for both assay types aimed to differentiate not only susceptible from resistant populations but also different resistance levels. The resistance level of the tested populations had been determined in a previous study using a topical assay (Kostromytska et al. 2018). For the Petri dish assay, three concentrations each of bifenthrin (Talstar Pro) and chlorpyrifos (Dursban 50W) were chosen and tested. For the vial assay, three and two concentrations of technical-grade bifenthrin and chlorpyrifos, respectively, were used. Assays were conducted using the same methodology for treatment application and insect introduction as reported in the sections describing vial and Petri dish assays. The experiment was conducted three times with two Petri dishes or vials (with 10 tested weevils each) per experimental run. Statistical Analysis Lethal concentrations (LCs), or concentrations causing 50% mortality of the tested annual bluegrass weevils, were determined by conducting Probit analyses PROC PROBIT (SAS Institute 2011), which automatically adjusted all variates and covariates by a homogeneity factor if the data did not fit the Probit model. In instances when control mortality was observed, data were corrected using Abbot’s formula (Abbott 1925). Replicates with control mortality >10% were excluded from the analysis. Resistance ratios were calculated (RR50 = LD or LC50 of resistant/LD or LC50 of susceptible population) and their significances determined according to Robertson et al. (2007). Differences of percent mortality among the populations in diagnostic dose assays were determined by analyses of variance (GLM procedure, SAS Institute [2011]) with subsequent LS means multiple comparisons with Tukey-Kramer α-level adjustments. Results Toxicity of Bifenthrin and Chlorpyrifos in Petri Dish Assays Results of the 2014–2015 Petri dish experiments demonstrated different resistance levels among the tested populations (Table 1). Populations PB and HP were relatively susceptible, with similar LC50 values (RR50 did not significantly differ based on Robertson et al. [2007] method). The populations EW and JC in 2014 (RR50s relative to the most susceptible population [HP] ranged 367–659) and GB, CN, and EW in 2015 (RR50s ranged 12.2–51.5) had a moderate resistance level to bifenthrin. The population LI was the most resistant to bifenthrin in the Petri dish assays (RR50s ranging 2,294–2,504 in 2014 and 352.8–372.5 in 2015). Notably, the RR50 values for resistant population were significantly higher in 2014 (in some cases ~10-fold) than in 2015 (Table 1). However, the LC50s for susceptible populations were similar in both years. Table 1. Toxicity of bifenthrin to adult Listronotus maculicollis from different populations determined with formulated product (Talstar Pro, LC50 expressed in mg AI/m2) in Petri dish assays evaluated at 24, 48, and 72 h after treatment in 2014 and 2015 Evaluation Population LC50 95% CI RR50a Slope ± SE Pb 2014  24 h PB 26.7 10.0–328.0 3.1 0.88 ± 0.22 0.45 HP 8.6 4.9–22.6 NCc 1.15 ± 0.22 0.59 JC 3,857.4 1,643.6–4.8E4 450.0* 0.78 ± 0.23 0.19 EW 5,647.0 2,004.3–4.1E5 659.0* 0.69 ± 0.24 0.22 LI 19,655.9 6,824.5–4.3E5 2,294.0*,d 0.77 ± 0.21 0.39  48 h PB 5.2 3.9–7.5 1.1 1.12 ± 0.11 0.33 HP 4.5 2.8–9.4 NC 1.12 ± 0.20 0.33 JC 2,042.2 NC 448.0* 0.78 ± 0.23 0.03 EW 2,691.7 1,370.8–1.2E5 591.0* 0.93 ± 0.24 0.15 LI 11,404.9 4,999.5–7.9E4 2,504.0* 0.87 ± 0.21 0.49  72 h PB 2.8 2.1–3.9 0.8 1.04 ± 0.10 0.31 HP 3.6 2.2–6.7 NC 1.15 ± 0.19 0.23 JC 1,885.2 NC 520.9* 0.71 ± 0.37 <0.01 EW 1,307.3 804.8–2,636.0 367.1*` 1.17 ± 0.24 0.35 LI 8,849.5 4,195.4–44,620.4 2,484.8* 0.89 ± 0.21 0.31 2015  24 h PB 32.3 17.0–109.7 3.5* 1.04 ± 0.22 0.52 HP 9.2 6.2–14.5 NC 1.46 ± 0.22 0.55 GB 272.1 157.1–452.9 39.7* 1.02 ± 0.17 0.75 CN 262.1 166.5–403.7 28.7* 1.25 ± 0.19 0.83 EW 471.2 270.6–869.4 51.5* 0.92 ± 0.17 0.50 LI 3,406.5 2,291.2–6,255.4 372.5* 1.34 ± 0.25 0.84  48 h PB 6.4 4.2–10.4 1.0 1.25 ± 0.19 0.75 HP 6.6 4.4–10.1 NC 1.43 ± 0.21 0.58 GB 154.1 91.8–237.9 21.7* 1.23 ± 0.19 0.84 CN 186.7 117.8–281.9 28.5* 1.32 ± 0.19 0.77 EW 177.6 106.6–275.3 27.1* 1.21 ± 0.19 0.95 LI 2,406.8 1,685.2–3,819.2 367.7* 1.42 ± 0.25 0.93  72 h PB 4.1 2.8–6.2 1.5 1.39 ± 0.20 0.67 HP 3.3 2.2–5.0 NC 1.36 ± 0.19 0.76 GB 58.9 29.3–93.6 17.7* 1.34 ± 0.24 0.64 CN 109.6 65.9–165.1 33.0* 1.37 ± 0.22 0.13 EW 40.7 11.1–80.9 12.2* 0.93 ± 0.19 0.99 LI 1,171.7 816.7–1,635.0 352.8* 1.56 ± 0.26 0.25 Evaluation Population LC50 95% CI RR50a Slope ± SE Pb 2014  24 h PB 26.7 10.0–328.0 3.1 0.88 ± 0.22 0.45 HP 8.6 4.9–22.6 NCc 1.15 ± 0.22 0.59 JC 3,857.4 1,643.6–4.8E4 450.0* 0.78 ± 0.23 0.19 EW 5,647.0 2,004.3–4.1E5 659.0* 0.69 ± 0.24 0.22 LI 19,655.9 6,824.5–4.3E5 2,294.0*,d 0.77 ± 0.21 0.39  48 h PB 5.2 3.9–7.5 1.1 1.12 ± 0.11 0.33 HP 4.5 2.8–9.4 NC 1.12 ± 0.20 0.33 JC 2,042.2 NC 448.0* 0.78 ± 0.23 0.03 EW 2,691.7 1,370.8–1.2E5 591.0* 0.93 ± 0.24 0.15 LI 11,404.9 4,999.5–7.9E4 2,504.0* 0.87 ± 0.21 0.49  72 h PB 2.8 2.1–3.9 0.8 1.04 ± 0.10 0.31 HP 3.6 2.2–6.7 NC 1.15 ± 0.19 0.23 JC 1,885.2 NC 520.9* 0.71 ± 0.37 <0.01 EW 1,307.3 804.8–2,636.0 367.1*` 1.17 ± 0.24 0.35 LI 8,849.5 4,195.4–44,620.4 2,484.8* 0.89 ± 0.21 0.31 2015  24 h PB 32.3 17.0–109.7 3.5* 1.04 ± 0.22 0.52 HP 9.2 6.2–14.5 NC 1.46 ± 0.22 0.55 GB 272.1 157.1–452.9 39.7* 1.02 ± 0.17 0.75 CN 262.1 166.5–403.7 28.7* 1.25 ± 0.19 0.83 EW 471.2 270.6–869.4 51.5* 0.92 ± 0.17 0.50 LI 3,406.5 2,291.2–6,255.4 372.5* 1.34 ± 0.25 0.84  48 h PB 6.4 4.2–10.4 1.0 1.25 ± 0.19 0.75 HP 6.6 4.4–10.1 NC 1.43 ± 0.21 0.58 GB 154.1 91.8–237.9 21.7* 1.23 ± 0.19 0.84 CN 186.7 117.8–281.9 28.5* 1.32 ± 0.19 0.77 EW 177.6 106.6–275.3 27.1* 1.21 ± 0.19 0.95 LI 2,406.8 1,685.2–3,819.2 367.7* 1.42 ± 0.25 0.93  72 h PB 4.1 2.8–6.2 1.5 1.39 ± 0.20 0.67 HP 3.3 2.2–5.0 NC 1.36 ± 0.19 0.76 GB 58.9 29.3–93.6 17.7* 1.34 ± 0.24 0.64 CN 109.6 65.9–165.1 33.0* 1.37 ± 0.22 0.13 EW 40.7 11.1–80.9 12.2* 0.93 ± 0.19 0.99 LI 1,171.7 816.7–1,635.0 352.8* 1.56 ± 0.26 0.25 a RR50s calculated with HP population as susceptible. bP values indicate goodness of fit of mortality data to an expected Probit model (α = 0.05). c Not calculated. d RR50s marked with an asterisk differ significantly from the susceptible population. Open in new tab Table 1. Toxicity of bifenthrin to adult Listronotus maculicollis from different populations determined with formulated product (Talstar Pro, LC50 expressed in mg AI/m2) in Petri dish assays evaluated at 24, 48, and 72 h after treatment in 2014 and 2015 Evaluation Population LC50 95% CI RR50a Slope ± SE Pb 2014  24 h PB 26.7 10.0–328.0 3.1 0.88 ± 0.22 0.45 HP 8.6 4.9–22.6 NCc 1.15 ± 0.22 0.59 JC 3,857.4 1,643.6–4.8E4 450.0* 0.78 ± 0.23 0.19 EW 5,647.0 2,004.3–4.1E5 659.0* 0.69 ± 0.24 0.22 LI 19,655.9 6,824.5–4.3E5 2,294.0*,d 0.77 ± 0.21 0.39  48 h PB 5.2 3.9–7.5 1.1 1.12 ± 0.11 0.33 HP 4.5 2.8–9.4 NC 1.12 ± 0.20 0.33 JC 2,042.2 NC 448.0* 0.78 ± 0.23 0.03 EW 2,691.7 1,370.8–1.2E5 591.0* 0.93 ± 0.24 0.15 LI 11,404.9 4,999.5–7.9E4 2,504.0* 0.87 ± 0.21 0.49  72 h PB 2.8 2.1–3.9 0.8 1.04 ± 0.10 0.31 HP 3.6 2.2–6.7 NC 1.15 ± 0.19 0.23 JC 1,885.2 NC 520.9* 0.71 ± 0.37 <0.01 EW 1,307.3 804.8–2,636.0 367.1*` 1.17 ± 0.24 0.35 LI 8,849.5 4,195.4–44,620.4 2,484.8* 0.89 ± 0.21 0.31 2015  24 h PB 32.3 17.0–109.7 3.5* 1.04 ± 0.22 0.52 HP 9.2 6.2–14.5 NC 1.46 ± 0.22 0.55 GB 272.1 157.1–452.9 39.7* 1.02 ± 0.17 0.75 CN 262.1 166.5–403.7 28.7* 1.25 ± 0.19 0.83 EW 471.2 270.6–869.4 51.5* 0.92 ± 0.17 0.50 LI 3,406.5 2,291.2–6,255.4 372.5* 1.34 ± 0.25 0.84  48 h PB 6.4 4.2–10.4 1.0 1.25 ± 0.19 0.75 HP 6.6 4.4–10.1 NC 1.43 ± 0.21 0.58 GB 154.1 91.8–237.9 21.7* 1.23 ± 0.19 0.84 CN 186.7 117.8–281.9 28.5* 1.32 ± 0.19 0.77 EW 177.6 106.6–275.3 27.1* 1.21 ± 0.19 0.95 LI 2,406.8 1,685.2–3,819.2 367.7* 1.42 ± 0.25 0.93  72 h PB 4.1 2.8–6.2 1.5 1.39 ± 0.20 0.67 HP 3.3 2.2–5.0 NC 1.36 ± 0.19 0.76 GB 58.9 29.3–93.6 17.7* 1.34 ± 0.24 0.64 CN 109.6 65.9–165.1 33.0* 1.37 ± 0.22 0.13 EW 40.7 11.1–80.9 12.2* 0.93 ± 0.19 0.99 LI 1,171.7 816.7–1,635.0 352.8* 1.56 ± 0.26 0.25 Evaluation Population LC50 95% CI RR50a Slope ± SE Pb 2014  24 h PB 26.7 10.0–328.0 3.1 0.88 ± 0.22 0.45 HP 8.6 4.9–22.6 NCc 1.15 ± 0.22 0.59 JC 3,857.4 1,643.6–4.8E4 450.0* 0.78 ± 0.23 0.19 EW 5,647.0 2,004.3–4.1E5 659.0* 0.69 ± 0.24 0.22 LI 19,655.9 6,824.5–4.3E5 2,294.0*,d 0.77 ± 0.21 0.39  48 h PB 5.2 3.9–7.5 1.1 1.12 ± 0.11 0.33 HP 4.5 2.8–9.4 NC 1.12 ± 0.20 0.33 JC 2,042.2 NC 448.0* 0.78 ± 0.23 0.03 EW 2,691.7 1,370.8–1.2E5 591.0* 0.93 ± 0.24 0.15 LI 11,404.9 4,999.5–7.9E4 2,504.0* 0.87 ± 0.21 0.49  72 h PB 2.8 2.1–3.9 0.8 1.04 ± 0.10 0.31 HP 3.6 2.2–6.7 NC 1.15 ± 0.19 0.23 JC 1,885.2 NC 520.9* 0.71 ± 0.37 <0.01 EW 1,307.3 804.8–2,636.0 367.1*` 1.17 ± 0.24 0.35 LI 8,849.5 4,195.4–44,620.4 2,484.8* 0.89 ± 0.21 0.31 2015  24 h PB 32.3 17.0–109.7 3.5* 1.04 ± 0.22 0.52 HP 9.2 6.2–14.5 NC 1.46 ± 0.22 0.55 GB 272.1 157.1–452.9 39.7* 1.02 ± 0.17 0.75 CN 262.1 166.5–403.7 28.7* 1.25 ± 0.19 0.83 EW 471.2 270.6–869.4 51.5* 0.92 ± 0.17 0.50 LI 3,406.5 2,291.2–6,255.4 372.5* 1.34 ± 0.25 0.84  48 h PB 6.4 4.2–10.4 1.0 1.25 ± 0.19 0.75 HP 6.6 4.4–10.1 NC 1.43 ± 0.21 0.58 GB 154.1 91.8–237.9 21.7* 1.23 ± 0.19 0.84 CN 186.7 117.8–281.9 28.5* 1.32 ± 0.19 0.77 EW 177.6 106.6–275.3 27.1* 1.21 ± 0.19 0.95 LI 2,406.8 1,685.2–3,819.2 367.7* 1.42 ± 0.25 0.93  72 h PB 4.1 2.8–6.2 1.5 1.39 ± 0.20 0.67 HP 3.3 2.2–5.0 NC 1.36 ± 0.19 0.76 GB 58.9 29.3–93.6 17.7* 1.34 ± 0.24 0.64 CN 109.6 65.9–165.1 33.0* 1.37 ± 0.22 0.13 EW 40.7 11.1–80.9 12.2* 0.93 ± 0.19 0.99 LI 1,171.7 816.7–1,635.0 352.8* 1.56 ± 0.26 0.25 a RR50s calculated with HP population as susceptible. bP values indicate goodness of fit of mortality data to an expected Probit model (α = 0.05). c Not calculated. d RR50s marked with an asterisk differ significantly from the susceptible population. Open in new tab LC50s were significantly higher after 24 h than after 72 h for PB (7.1-fold) and EW (4.3-fold) populations, but not statistically different for HP (2.1-fold) and LI (2.6-fold) in 2014. In 2015, LC50s were significantly lower after 72 h than after 24 h for all tested population, with difference ranging 2.9- to 11.7-fold. In both years, fewer moribund weevils were observed and the fiducial limits (FL) of the LC50 values were narrower after 72 h than after 24 h (Table 1). Results of the Petri dish assays with chlorpyrifos in 2015 demonstrated that toxicity of chlorpyrifos was reduced for pyrethroid-resistant populations (Table 2). The population with the highest pyrethroid resistance level (LI) was also the most tolerant to formulated chlorpyrifos with RR50s of 120.7 (after 24 h), 8.8 (after 48 h), and 12.5 (after 72 h) compared with the most sensitive population (PB). The moderately pyrethroid-resistant populations (GB, CN, and EW) had significantly higher LC50s than the susceptible PB population (RR50s ranging 2.4–6.7). The greatest difference between the most pyrethroid-resistant and most susceptible population was observed after 24 h. At this time, the most resistant population (LI) was clearly discriminated from the moderately resistant populations, which had LC50 values 17.9- to 36.9-fold lower than for the LI population. Between the 24- and 72 h evaluation, LC50s significantly decreased for the susceptible PB population (3.3-fold), the moderately resistant populations (EW, 2.0-fold; CN, 2.5-fold; GB, 4.1-fold), and, most notably, the most resistant population (LI, 32.2-fold). As a result, the LI population could no longer be differentiated from the moderately resistant populations (EW, CN, and GB) after 72 h with LC50s only 2.3- to 3.9-fold lower than for LI. Table 2. Toxicity of chlorpyrifos to adult Listronotus maculicollis from different populations determined with formulated product (Dursban 50W, LC50 expressed in mg AI/m2) in Petri dish assays evaluated at 24, 48, and 72 h after treatment (n = 180) in 2015 Evaluation Population LC50 95% CI RR50a Slope ± SE Pb 24 h PB 0.75 0.6–0.9 NCc 2.72 ± 0.41 0.88 HP 0.89 0.3–1.9 1.2 2.27 ± 0.50 0.08 GB 2.96 2.5–3.5 3.9*,d 3.69 ± 0.53 0.79 CN 3.02 2.4–3.8 5.9* 2.29 ± 0.34 0.18 EW 5.08 3.7–7.5 6.7* 1.60 ± 0.28 0.12 LI 91.0 37.7–1,054 120.7* 0.54 ± 0.20 0.64 48 h PB 0.52 0.4–0.6 NC 2.99 ± 0.43 0.99 HP 0.61 0.5–0.8 1.2 2.93 ± 0.44 0.72 GB 1.24 0.9–1.5 2.4* 3.77 ± 0.79 0.95 CN 2.47 1.9–3.1 3.3* 2.28 ± 0.36 0.38 EW 1.51 1.2–1.8 2.9* 3.33 ± 0.59 0.85 LI 4.55 0.6–10.3 8.8* 0.86 ± 0.22 0.35 72 h PB 0.23 0.2–0.3 NC 3.54 ± 0.64 0.94 HP 0.45 0.3–0.6 2.0 3.23 ± 0.46 0.67 GB 0.73 0.2–1.0 3.2* 3.11 ± 0.99 0.88 CN 1.01 0.6–1.3 4.5* 2.84 ± 0.67 0.80 EW 1.22 0.9–1.4 5.4* 4.35 ± 0.96 0.99 LI 2.82 0.1–4.8 12.5* 2.42 ± 0.95 0.99 Evaluation Population LC50 95% CI RR50a Slope ± SE Pb 24 h PB 0.75 0.6–0.9 NCc 2.72 ± 0.41 0.88 HP 0.89 0.3–1.9 1.2 2.27 ± 0.50 0.08 GB 2.96 2.5–3.5 3.9*,d 3.69 ± 0.53 0.79 CN 3.02 2.4–3.8 5.9* 2.29 ± 0.34 0.18 EW 5.08 3.7–7.5 6.7* 1.60 ± 0.28 0.12 LI 91.0 37.7–1,054 120.7* 0.54 ± 0.20 0.64 48 h PB 0.52 0.4–0.6 NC 2.99 ± 0.43 0.99 HP 0.61 0.5–0.8 1.2 2.93 ± 0.44 0.72 GB 1.24 0.9–1.5 2.4* 3.77 ± 0.79 0.95 CN 2.47 1.9–3.1 3.3* 2.28 ± 0.36 0.38 EW 1.51 1.2–1.8 2.9* 3.33 ± 0.59 0.85 LI 4.55 0.6–10.3 8.8* 0.86 ± 0.22 0.35 72 h PB 0.23 0.2–0.3 NC 3.54 ± 0.64 0.94 HP 0.45 0.3–0.6 2.0 3.23 ± 0.46 0.67 GB 0.73 0.2–1.0 3.2* 3.11 ± 0.99 0.88 CN 1.01 0.6–1.3 4.5* 2.84 ± 0.67 0.80 EW 1.22 0.9–1.4 5.4* 4.35 ± 0.96 0.99 LI 2.82 0.1–4.8 12.5* 2.42 ± 0.95 0.99 a RR50s calculated with PB population as most susceptible. bP values indicate goodness of fit of mortality data to an expected Probit model (α = 0.05). c Not calculated. d RR50s marked with an asterisk differ significantly from the most susceptible population. Open in new tab Table 2. Toxicity of chlorpyrifos to adult Listronotus maculicollis from different populations determined with formulated product (Dursban 50W, LC50 expressed in mg AI/m2) in Petri dish assays evaluated at 24, 48, and 72 h after treatment (n = 180) in 2015 Evaluation Population LC50 95% CI RR50a Slope ± SE Pb 24 h PB 0.75 0.6–0.9 NCc 2.72 ± 0.41 0.88 HP 0.89 0.3–1.9 1.2 2.27 ± 0.50 0.08 GB 2.96 2.5–3.5 3.9*,d 3.69 ± 0.53 0.79 CN 3.02 2.4–3.8 5.9* 2.29 ± 0.34 0.18 EW 5.08 3.7–7.5 6.7* 1.60 ± 0.28 0.12 LI 91.0 37.7–1,054 120.7* 0.54 ± 0.20 0.64 48 h PB 0.52 0.4–0.6 NC 2.99 ± 0.43 0.99 HP 0.61 0.5–0.8 1.2 2.93 ± 0.44 0.72 GB 1.24 0.9–1.5 2.4* 3.77 ± 0.79 0.95 CN 2.47 1.9–3.1 3.3* 2.28 ± 0.36 0.38 EW 1.51 1.2–1.8 2.9* 3.33 ± 0.59 0.85 LI 4.55 0.6–10.3 8.8* 0.86 ± 0.22 0.35 72 h PB 0.23 0.2–0.3 NC 3.54 ± 0.64 0.94 HP 0.45 0.3–0.6 2.0 3.23 ± 0.46 0.67 GB 0.73 0.2–1.0 3.2* 3.11 ± 0.99 0.88 CN 1.01 0.6–1.3 4.5* 2.84 ± 0.67 0.80 EW 1.22 0.9–1.4 5.4* 4.35 ± 0.96 0.99 LI 2.82 0.1–4.8 12.5* 2.42 ± 0.95 0.99 Evaluation Population LC50 95% CI RR50a Slope ± SE Pb 24 h PB 0.75 0.6–0.9 NCc 2.72 ± 0.41 0.88 HP 0.89 0.3–1.9 1.2 2.27 ± 0.50 0.08 GB 2.96 2.5–3.5 3.9*,d 3.69 ± 0.53 0.79 CN 3.02 2.4–3.8 5.9* 2.29 ± 0.34 0.18 EW 5.08 3.7–7.5 6.7* 1.60 ± 0.28 0.12 LI 91.0 37.7–1,054 120.7* 0.54 ± 0.20 0.64 48 h PB 0.52 0.4–0.6 NC 2.99 ± 0.43 0.99 HP 0.61 0.5–0.8 1.2 2.93 ± 0.44 0.72 GB 1.24 0.9–1.5 2.4* 3.77 ± 0.79 0.95 CN 2.47 1.9–3.1 3.3* 2.28 ± 0.36 0.38 EW 1.51 1.2–1.8 2.9* 3.33 ± 0.59 0.85 LI 4.55 0.6–10.3 8.8* 0.86 ± 0.22 0.35 72 h PB 0.23 0.2–0.3 NC 3.54 ± 0.64 0.94 HP 0.45 0.3–0.6 2.0 3.23 ± 0.46 0.67 GB 0.73 0.2–1.0 3.2* 3.11 ± 0.99 0.88 CN 1.01 0.6–1.3 4.5* 2.84 ± 0.67 0.80 EW 1.22 0.9–1.4 5.4* 4.35 ± 0.96 0.99 LI 2.82 0.1–4.8 12.5* 2.42 ± 0.95 0.99 a RR50s calculated with PB population as most susceptible. bP values indicate goodness of fit of mortality data to an expected Probit model (α = 0.05). c Not calculated. d RR50s marked with an asterisk differ significantly from the most susceptible population. Open in new tab Toxicity of Bifenthrin and Chlorpyrifos in Vial Assays Mortality data obtained in the vial assays were consistent with the results obtained in the Petri dish assays and previous studies (topical and greenhouse bioassays, Kostromytska et al. [2018]) and effectively separated resistant and susceptible populations (Table 3). For bifenthrin, it was possible to discriminate resistant and susceptible populations based on mortality data obtained after 24, 48, or 72 h (Table 3). After 24 h, populations PB and HP were relatively susceptible to bifenthrin. Their LC50 values did not differ statistically from each other but were lower than those of all other tested populations. The remaining populations could be separated into moderately resistant populations (EW, CN, GB; RR50s ranged 24.7–62.2) and a highly resistant population (LI; RR50 = 258.3). The LC50 calculated for the LI population was significantly higher than the LC50s for populations GB (10.4-fold), CN (5.7-fold), and EW (4.2-fold). The same trend was observed after 48 and 72 h except that the difference between the most susceptible (PB) and the most resistant population (LI) was greater with RR50s of 935.8 and 1,760.9, respectively. Table 3. Toxicity of bifenthrin and chlorpyrifos to adult Listronotus maculicollis from different populations determined with technical-grade AI (LC50 expressed in mg AI/m2) in vial assays evaluated at 24, 48, and 72 h after treatment (n = 180) in 2015 Evaluation Population LC50 95% CI RR50a Slope ± SEM Pb Bifenthrin  24 h PB 36.3 11.4–1,579 2.8 0.50 ± 0.16 0.59 HP 12.8 4.9–126.3 NCc 0.50 ± 0.15 0.69 GB 316.2 88.1–1,034 24.7*,d 0.48 ± 0.15 0.94 CN 580.1 198–3,144 45.4* 0.47 ± 0.15 0.92 EW 795.3 262–9,173 62.2* 0.43 ± 0.15 0.88 LI 3,302 1,832–8,625 258.2* 0.88 ± 0.18 0.72  48 h PB 1.35 0.5–2.7 NC 0.71 ± 0.16 0.91 HP 1.83 0.4–4.6 1.2 0.53 ± 0.15 0.58 GB 33.9 8.1–69.5 25.1* 0.94 ± 0.20 0.51 CN 129.1 46.6–236.5 90.1* 0.79 ± 0.16 0.96 EW 51.3 3.5–138.9 37.9* 0.54 ± 0.16 0.79 LI 1,266 719–2,383 935.8* 0.90 ± 0.17 0.14  72 h PB 0.81 0.3–1.5 1.5 0.85 ± 0.17 0.94 HP 0.53 0.1–1.2 NC 0.67 ± 0.16 0.89 GB 16.3 1.5–41.9 30.8* 0.87 ± 0.22 0.58 CN 50.7 10.9–109.5 95.7* 0.75 ± 0.17 0.95 EW 15.4 0.3–53.3 29.2* 0.59 ± 0.17 0.89 LI 933.3 426–1,984 1,760.9* 0.71 ± 0.17 0.68 Chlorpyrifos  24 h PB 0.51 0.38–0.67 NC 2.33 ± 0.30 0.78 HP 0.60 0.46–0.77 1.2 2.68 ± 0.34 0.72 GB 1.77 1.52–2.05 3.5* 4.59 ± 0.62 0.59 CN 2.60 2.15–3.21 5.1* 2.93 ± 0.41 0.21 EW 2.68 2.17–3.42 5.2* 2.54 ± 0.38 0.11 LI 10.47 8.55–12.69 20.5* 2.93 ± 0.40 0.84  48 h PB 0.31 0.25–0.39 NC 3.11 ± 0.46 0.87 HP 0.37 0.26–0.47 1.2 3.40 ± 0.51 0.95 GB 1.34 1.13–1.57 4.2* 4.08 ± 0.56 0.40 CN 1.60 1.34–1.89 5.0* 3.55 ± 0.48 0.89 EW 1.81 1.48–2.20 5.7* 2.89 ± 0.40 0.50 LI 6.12 5.08–7.22 19.2* 3.97 ± 0.61 0.53  72 h PB 0.23 0.17–0.31 NC 2.41 ± 0.37 0.90 HP 0.32 0.25–0.40 1.4 3.72 ± 0.61 0.93 GB 1.13 0.95–1.31 4.8* 4.46 ± 0.66 0.46 CN 1.37 1.14–1.63 5.9* 3.50 ± 0.50 0.59 EW 1.39 1.16–1.64 5.9* 3.66 ± 0.51 0.44 LI 4.34 3.23–5.32 18.5* 3.38 ± 0.62 0.93 Evaluation Population LC50 95% CI RR50a Slope ± SEM Pb Bifenthrin  24 h PB 36.3 11.4–1,579 2.8 0.50 ± 0.16 0.59 HP 12.8 4.9–126.3 NCc 0.50 ± 0.15 0.69 GB 316.2 88.1–1,034 24.7*,d 0.48 ± 0.15 0.94 CN 580.1 198–3,144 45.4* 0.47 ± 0.15 0.92 EW 795.3 262–9,173 62.2* 0.43 ± 0.15 0.88 LI 3,302 1,832–8,625 258.2* 0.88 ± 0.18 0.72  48 h PB 1.35 0.5–2.7 NC 0.71 ± 0.16 0.91 HP 1.83 0.4–4.6 1.2 0.53 ± 0.15 0.58 GB 33.9 8.1–69.5 25.1* 0.94 ± 0.20 0.51 CN 129.1 46.6–236.5 90.1* 0.79 ± 0.16 0.96 EW 51.3 3.5–138.9 37.9* 0.54 ± 0.16 0.79 LI 1,266 719–2,383 935.8* 0.90 ± 0.17 0.14  72 h PB 0.81 0.3–1.5 1.5 0.85 ± 0.17 0.94 HP 0.53 0.1–1.2 NC 0.67 ± 0.16 0.89 GB 16.3 1.5–41.9 30.8* 0.87 ± 0.22 0.58 CN 50.7 10.9–109.5 95.7* 0.75 ± 0.17 0.95 EW 15.4 0.3–53.3 29.2* 0.59 ± 0.17 0.89 LI 933.3 426–1,984 1,760.9* 0.71 ± 0.17 0.68 Chlorpyrifos  24 h PB 0.51 0.38–0.67 NC 2.33 ± 0.30 0.78 HP 0.60 0.46–0.77 1.2 2.68 ± 0.34 0.72 GB 1.77 1.52–2.05 3.5* 4.59 ± 0.62 0.59 CN 2.60 2.15–3.21 5.1* 2.93 ± 0.41 0.21 EW 2.68 2.17–3.42 5.2* 2.54 ± 0.38 0.11 LI 10.47 8.55–12.69 20.5* 2.93 ± 0.40 0.84  48 h PB 0.31 0.25–0.39 NC 3.11 ± 0.46 0.87 HP 0.37 0.26–0.47 1.2 3.40 ± 0.51 0.95 GB 1.34 1.13–1.57 4.2* 4.08 ± 0.56 0.40 CN 1.60 1.34–1.89 5.0* 3.55 ± 0.48 0.89 EW 1.81 1.48–2.20 5.7* 2.89 ± 0.40 0.50 LI 6.12 5.08–7.22 19.2* 3.97 ± 0.61 0.53  72 h PB 0.23 0.17–0.31 NC 2.41 ± 0.37 0.90 HP 0.32 0.25–0.40 1.4 3.72 ± 0.61 0.93 GB 1.13 0.95–1.31 4.8* 4.46 ± 0.66 0.46 CN 1.37 1.14–1.63 5.9* 3.50 ± 0.50 0.59 EW 1.39 1.16–1.64 5.9* 3.66 ± 0.51 0.44 LI 4.34 3.23–5.32 18.5* 3.38 ± 0.62 0.93 a RR50 calculated using population with the lowest LC50 as most susceptible. bP values indicate goodness of fit of mortality data to an expected Probit model (α = 0.05). c Not calculated. d RR50 marked with an asterisk differ significantly from the susceptible population. Open in new tab Table 3. Toxicity of bifenthrin and chlorpyrifos to adult Listronotus maculicollis from different populations determined with technical-grade AI (LC50 expressed in mg AI/m2) in vial assays evaluated at 24, 48, and 72 h after treatment (n = 180) in 2015 Evaluation Population LC50 95% CI RR50a Slope ± SEM Pb Bifenthrin  24 h PB 36.3 11.4–1,579 2.8 0.50 ± 0.16 0.59 HP 12.8 4.9–126.3 NCc 0.50 ± 0.15 0.69 GB 316.2 88.1–1,034 24.7*,d 0.48 ± 0.15 0.94 CN 580.1 198–3,144 45.4* 0.47 ± 0.15 0.92 EW 795.3 262–9,173 62.2* 0.43 ± 0.15 0.88 LI 3,302 1,832–8,625 258.2* 0.88 ± 0.18 0.72  48 h PB 1.35 0.5–2.7 NC 0.71 ± 0.16 0.91 HP 1.83 0.4–4.6 1.2 0.53 ± 0.15 0.58 GB 33.9 8.1–69.5 25.1* 0.94 ± 0.20 0.51 CN 129.1 46.6–236.5 90.1* 0.79 ± 0.16 0.96 EW 51.3 3.5–138.9 37.9* 0.54 ± 0.16 0.79 LI 1,266 719–2,383 935.8* 0.90 ± 0.17 0.14  72 h PB 0.81 0.3–1.5 1.5 0.85 ± 0.17 0.94 HP 0.53 0.1–1.2 NC 0.67 ± 0.16 0.89 GB 16.3 1.5–41.9 30.8* 0.87 ± 0.22 0.58 CN 50.7 10.9–109.5 95.7* 0.75 ± 0.17 0.95 EW 15.4 0.3–53.3 29.2* 0.59 ± 0.17 0.89 LI 933.3 426–1,984 1,760.9* 0.71 ± 0.17 0.68 Chlorpyrifos  24 h PB 0.51 0.38–0.67 NC 2.33 ± 0.30 0.78 HP 0.60 0.46–0.77 1.2 2.68 ± 0.34 0.72 GB 1.77 1.52–2.05 3.5* 4.59 ± 0.62 0.59 CN 2.60 2.15–3.21 5.1* 2.93 ± 0.41 0.21 EW 2.68 2.17–3.42 5.2* 2.54 ± 0.38 0.11 LI 10.47 8.55–12.69 20.5* 2.93 ± 0.40 0.84  48 h PB 0.31 0.25–0.39 NC 3.11 ± 0.46 0.87 HP 0.37 0.26–0.47 1.2 3.40 ± 0.51 0.95 GB 1.34 1.13–1.57 4.2* 4.08 ± 0.56 0.40 CN 1.60 1.34–1.89 5.0* 3.55 ± 0.48 0.89 EW 1.81 1.48–2.20 5.7* 2.89 ± 0.40 0.50 LI 6.12 5.08–7.22 19.2* 3.97 ± 0.61 0.53  72 h PB 0.23 0.17–0.31 NC 2.41 ± 0.37 0.90 HP 0.32 0.25–0.40 1.4 3.72 ± 0.61 0.93 GB 1.13 0.95–1.31 4.8* 4.46 ± 0.66 0.46 CN 1.37 1.14–1.63 5.9* 3.50 ± 0.50 0.59 EW 1.39 1.16–1.64 5.9* 3.66 ± 0.51 0.44 LI 4.34 3.23–5.32 18.5* 3.38 ± 0.62 0.93 Evaluation Population LC50 95% CI RR50a Slope ± SEM Pb Bifenthrin  24 h PB 36.3 11.4–1,579 2.8 0.50 ± 0.16 0.59 HP 12.8 4.9–126.3 NCc 0.50 ± 0.15 0.69 GB 316.2 88.1–1,034 24.7*,d 0.48 ± 0.15 0.94 CN 580.1 198–3,144 45.4* 0.47 ± 0.15 0.92 EW 795.3 262–9,173 62.2* 0.43 ± 0.15 0.88 LI 3,302 1,832–8,625 258.2* 0.88 ± 0.18 0.72  48 h PB 1.35 0.5–2.7 NC 0.71 ± 0.16 0.91 HP 1.83 0.4–4.6 1.2 0.53 ± 0.15 0.58 GB 33.9 8.1–69.5 25.1* 0.94 ± 0.20 0.51 CN 129.1 46.6–236.5 90.1* 0.79 ± 0.16 0.96 EW 51.3 3.5–138.9 37.9* 0.54 ± 0.16 0.79 LI 1,266 719–2,383 935.8* 0.90 ± 0.17 0.14  72 h PB 0.81 0.3–1.5 1.5 0.85 ± 0.17 0.94 HP 0.53 0.1–1.2 NC 0.67 ± 0.16 0.89 GB 16.3 1.5–41.9 30.8* 0.87 ± 0.22 0.58 CN 50.7 10.9–109.5 95.7* 0.75 ± 0.17 0.95 EW 15.4 0.3–53.3 29.2* 0.59 ± 0.17 0.89 LI 933.3 426–1,984 1,760.9* 0.71 ± 0.17 0.68 Chlorpyrifos  24 h PB 0.51 0.38–0.67 NC 2.33 ± 0.30 0.78 HP 0.60 0.46–0.77 1.2 2.68 ± 0.34 0.72 GB 1.77 1.52–2.05 3.5* 4.59 ± 0.62 0.59 CN 2.60 2.15–3.21 5.1* 2.93 ± 0.41 0.21 EW 2.68 2.17–3.42 5.2* 2.54 ± 0.38 0.11 LI 10.47 8.55–12.69 20.5* 2.93 ± 0.40 0.84  48 h PB 0.31 0.25–0.39 NC 3.11 ± 0.46 0.87 HP 0.37 0.26–0.47 1.2 3.40 ± 0.51 0.95 GB 1.34 1.13–1.57 4.2* 4.08 ± 0.56 0.40 CN 1.60 1.34–1.89 5.0* 3.55 ± 0.48 0.89 EW 1.81 1.48–2.20 5.7* 2.89 ± 0.40 0.50 LI 6.12 5.08–7.22 19.2* 3.97 ± 0.61 0.53  72 h PB 0.23 0.17–0.31 NC 2.41 ± 0.37 0.90 HP 0.32 0.25–0.40 1.4 3.72 ± 0.61 0.93 GB 1.13 0.95–1.31 4.8* 4.46 ± 0.66 0.46 CN 1.37 1.14–1.63 5.9* 3.50 ± 0.50 0.59 EW 1.39 1.16–1.64 5.9* 3.66 ± 0.51 0.44 LI 4.34 3.23–5.32 18.5* 3.38 ± 0.62 0.93 a RR50 calculated using population with the lowest LC50 as most susceptible. bP values indicate goodness of fit of mortality data to an expected Probit model (α = 0.05). c Not calculated. d RR50 marked with an asterisk differ significantly from the susceptible population. Open in new tab Overall, bifenthrin LC50s decreased significantly for all tested populations between 11.4- and 51.4-fold from the 24-h to the 72-h evaluation. LC50 values decreased significantly from 24 to 48 h after treatment, which was followed by an additional less pronounced decrease from 48 to 72 h after treatment. Additionally, after 72 h fewer moribund weevils were observed and the data fit the model better, and consequently the FLs of the obtained LCs were narrower than after 24 h. For chlorpyrifos, most of the mortality occurred 24 h after exposure. LC50s did not change significantly over time even though they decreased numerically for every population from 24 to 72 h between 1.9- and 2.4-fold (Table 3). Pyrethroid-susceptible populations did not differ statistically in their susceptibility to chlorpyrifos (LC50 values 0.23 and 0.32 mg AI/m2 for PB and HP, respectively, with RR50 = 1.4). Populations with moderate pyrethroid resistance demonstrated increased tolerance to chlorpyrifos if compared with pyrethroid-susceptible populations (RR50s ranging 3.5–5.9). The most pyrethroid-resistant population had the highest level of resistance or tolerance to chlorpyrifos (RR50s relative to the most susceptible populations PB ranged 18.5–20.5 depending on the evaluation time). Diagnostic Concentration (LC) Validation in Assays With Susceptible and Resistant Populations Diagnostic LC Validation in Petri Dish Assay Based on the developed bifenthrin and chlorpyrifos concentration–response curves for susceptible and resistant populations, candidate concentrations were developed. Results of the Petri dish assay yielded concentration–response curve with LC50 values significantly separated (Fig. 1). According to the results of the Probit analysis, three concentrations, which theoretically could separate susceptible and populations with different resistance levels, were chosen: 112.1 (~2-fold of LC95 of susceptible population), 336.3 (~2-fold of LC99 of susceptible populations), and 3,362.5 (the highest LC95 value among moderately resistant populations) mg AI/m2 (Fig. 1). In the experiments with chlorpyrifos, only data obtained 24 and 48 h after treatment application could be considered; after 72 h, high mortality made it difficult to discriminate between susceptible and resistant populations and especially between different resistance levels (Fig. 2). Because tolerance levels to chlorpyrifos were relatively low, concentrations of 3.4 (~LC95 of susceptible populations), 11.2 (~ LC99 of susceptible populations), and 33.6 mg AI/m2 (~LC95 of moderately resistant population) were used (Fig. 2). Fig. 1. Open in new tabDownload slide Bifenthrin concentration–mortality curves obtained in the Petri dish bioassay for adults from susceptible (PB and HP) and pyrethroid-resistant Listronotus maculicollis populations (CN, EW, GB, and LI) evaluated after 24 (A), 48 (B), and 72 h (C). Vertical lines indicate candidate diagnostic concentrations. 1 Diagnostic concentrations, mg AI/m2. Fig. 1. Open in new tabDownload slide Bifenthrin concentration–mortality curves obtained in the Petri dish bioassay for adults from susceptible (PB and HP) and pyrethroid-resistant Listronotus maculicollis populations (CN, EW, GB, and LI) evaluated after 24 (A), 48 (B), and 72 h (C). Vertical lines indicate candidate diagnostic concentrations. 1 Diagnostic concentrations, mg AI/m2. Fig. 2. Open in new tabDownload slide Chlorpyrifos concentration–mortality curves obtained in the Petri dish bioassay for Listronotus maculicollis adults from susceptible (PB and HP) and pyrethroid-resistant populations (CN, EW, GB, and LI) evaluated at 24 (A), 48 (B), and 72 h (C) after treatment. Vertical lines indicate candidate diagnostic concentrations. 1 Diagnostic concentrations, mg AI/m2. Fig. 2. Open in new tabDownload slide Chlorpyrifos concentration–mortality curves obtained in the Petri dish bioassay for Listronotus maculicollis adults from susceptible (PB and HP) and pyrethroid-resistant populations (CN, EW, GB, and LI) evaluated at 24 (A), 48 (B), and 72 h (C) after treatment. Vertical lines indicate candidate diagnostic concentrations. 1 Diagnostic concentrations, mg AI/m2. In the experiment with bifenthrin (Talstar Pro), separation of populations with different resistance levels was observed at 24 h (F = 21.8; df = 14, 89; P < 0.01), 48 h (F = 16.8; df = 14, 89; P < 0.01), and 72 h after treatment (F = 24.3; df = 14, 89; P < 0.01). At 24 h after treatment, the lowest bifenthrin concentration (112.1 mg AI/m2) caused 75% mortality of susceptible weevils (PB population), which was significantly higher than the mortality observed for all moderately and highly resistant populations (EW, CN, and LI) except for the GB population (Fig. 3A). At the concentrations of 336.3 and 3,362.5 mg AI/m2, no differences were observed among susceptible and moderately resistant populations. The most resistant population (LI) had lower mortality than all other populations. Fig. 3. Open in new tabDownload slide Mortality (%mean ± SEM) of adult Listronotus maculicollis from susceptible (PB) and moderately (EW, CN, and GB) and highly pyrethroid-resistant (LI) populations caused by three bifenthrin concentrations (FP) in a diagnostic Petri dish assay evaluated at 24 (A), 48 (B), and 72 h (C) after treatment. Fig. 3. Open in new tabDownload slide Mortality (%mean ± SEM) of adult Listronotus maculicollis from susceptible (PB) and moderately (EW, CN, and GB) and highly pyrethroid-resistant (LI) populations caused by three bifenthrin concentrations (FP) in a diagnostic Petri dish assay evaluated at 24 (A), 48 (B), and 72 h (C) after treatment. At 48 h after treatment with bifenthrin, differences among populations were less clear (Fig. 3B). The susceptible population (PB) and only the GB population among the moderately resistant populations had significantly higher mortality than the most resistant population (LI) at all three bifenthrin concentrations but did not differ significantly from each other. The other moderately resistant populations (CN and EW) did not differ significantly from the most susceptible population (PB) and only at the 336.3 mg AI/m2 concentration from the most resistant population (LI). At 72 h after treatment with bifenthrin (Fig. 3C), the lowest bifenthrin concentration (112.1 mg AI/m2) caused the highest mortality for the susceptible population (90%), significantly lower mortality for the moderately resistant populations (42–53%), and the lowest mortality for the highly resistant population (3%). A similar pattern was observed at the 336.3 mg AI/m2 concentration, but mortality did not differ statistically between the susceptible population and the moderately resistant GB and EW populations. At the highest tested concentration (3,362.5 mg AI/m2), mortality did not differ statistically between the susceptible and all moderately resistant populations (88–100%) but was significantly lower for the LI population (43%) than all other populations. In the Petri dish diagnostic assays with formulated chlorpyrifos (Dursban), differences among populations with different resistance levels were detected at 24 h (Fig. 4A: F = 23.9; df = 14,74; P < 0.01) and 48 h (Fig. 4B: F = 17.1; df = 14,74; P < 0.01). At 72 h after treatment, mortality was too high to allow for clear separation among populations and thus was not shown. The lowest tested chlorpyrifos concentration (3.4 mg AI/m2) provided clear separation between the susceptible and resistant populations at 24 h (80% vs 0–14%) and 48 h (90% vs 0–32%) but gave no significant separation between moderately and highly resistant populations. Fig. 4. Open in new tabDownload slide Mortality (% mean ± SEM) of adult Listronotus maculicollis from susceptible (PB) and moderately (EW, CN, and GB) and highly pyrethroid-resistant (LI) populations caused by three chlorpyrifos (FP) concentrations in a diagnostic Petri dish assay evaluated at 24 (A), 48 (B), and 72 h (C) after treatment. Fig. 4. Open in new tabDownload slide Mortality (% mean ± SEM) of adult Listronotus maculicollis from susceptible (PB) and moderately (EW, CN, and GB) and highly pyrethroid-resistant (LI) populations caused by three chlorpyrifos (FP) concentrations in a diagnostic Petri dish assay evaluated at 24 (A), 48 (B), and 72 h (C) after treatment. The 11.2 mg AI/m2 chlorpyrifos concentration clearly separated the susceptible from the highly resistant population at 24 h after treatment (96% vs 2%) and 48 h (100% vs 4%), but separation of the moderately resistant populations from either the susceptible or highly resistant populations was less clear. At 24 h, all resistant populations had lower mortality than the susceptible population but only the GB population had significantly higher mortality than the highly resistant population. At 48 h, the GB population did not differ significantly from the susceptible population and the EW population did not differ significantly from the highly resistant population. The highest chlorpyrifos concentration (33.6 mg AI/m2) effectively separated the moderately resistant from the highly resistant population after 24 h (54–72% vs 2%) and 48 h (62–84% vs 14%). But the susceptible population (100% at 24 and 48 h) separated from the moderately resistant populations only for EW after 48 h. Diagnostic LC Validation in Vial Assay Data obtained in the vial bioassays with bifenthrin (see Section Toxicity of Bifenthrin and Chlorpyrifos in Vial Assays) yielded concentration–response curves with wide FLs especially for the LC95 and LC99. Thus, three concentrations of technical-grade bifenthrin were chosen based on the concentration–response curves developed in the assays described above in 2.3 and 3.2: 33.6, 112.1, and 1,120.8 mg AI/m2 (Fig. 5). In the vial bioassays with chlorpyrifos, two concentrations were sufficient to separate populations with different tolerance levels: 2.2 (~LC99 of susceptible populations) and 11.2 mg AI/m2 (~LC99 of moderately resistant populations; Fig. 6). Fig. 5. Open in new tabDownload slide Bifenthrin concentration–mortality curves obtained in the vial bioassay for adults from susceptible (PB and HP) and resistant Listronotus maculicollis populations (CN, EW, GB, and LI) evaluated at 24 (A), 48 (B), and 72 h (C) after treatment. Vertical lines indicate candidate diagnostic concentrations. 1 Diagnostic concentrations, mg AI/m2. Fig. 5. Open in new tabDownload slide Bifenthrin concentration–mortality curves obtained in the vial bioassay for adults from susceptible (PB and HP) and resistant Listronotus maculicollis populations (CN, EW, GB, and LI) evaluated at 24 (A), 48 (B), and 72 h (C) after treatment. Vertical lines indicate candidate diagnostic concentrations. 1 Diagnostic concentrations, mg AI/m2. Fig. 6. Open in new tabDownload slide Chlorpyrifos concentration–mortality curves obtained in the vials for adults from susceptible (PB and HP) and resistant Listronotus maculicollis populations (CN, EW, GB, and LI) evaluated at 24 (A), 48 (B), and 72 h (C) after treatment. Vertical lines indicate candidate diagnostic concentrations. 1 Diagnostic concentrations, mg AI/m2. Fig. 6. Open in new tabDownload slide Chlorpyrifos concentration–mortality curves obtained in the vials for adults from susceptible (PB and HP) and resistant Listronotus maculicollis populations (CN, EW, GB, and LI) evaluated at 24 (A), 48 (B), and 72 h (C) after treatment. Vertical lines indicate candidate diagnostic concentrations. 1 Diagnostic concentrations, mg AI/m2. In the vial bioassays with bifenthrin, only data collected at 24 h after treatment provided a clear separation of different resistance levels (Fig. 7). Two bifenthrin concentrations (112.1 mg AI/m2 and 1,120.8 mg/m2) effectively separated susceptible population (95 and 98% mortality, respectively), moderately resistant populations (43–51% and 63–71%, respectively), and highly resistant population (12 and 30%, respectively; Fig. 7A: F = 27.9; df = 6, 71; P < 0.01). The lowest concentration (33.6 mg AI/m2) effectively separated the susceptible population (87%) from all resistant populations but did not separate the moderately resistant populations (22–27%) from the highly resistant population (0%). At 48 h, similarly to to the evaluation at 24 h, the mortality of the susceptible population (90%) was higher than the mortality in the moderately resistant populations (50–55%) and the highly resistant population (12%). However, the statistical effect was not significant (F = 1.4; df = 6, 71; P = 0.23). At the 72-h evaluation, mortality in the susceptible population was only different from that in the highly resistant population (F = 2.1; df = 6, 71; P = 0.06). Fig. 7. Open in new tabDownload slide Mortality (%mean ± SEM) of adults from susceptible (PB) and resistant Listronotus maculicollis populations (CN, EW, and LI) caused by diagnostic concentrations of technical-grade bifenthrin (A) and chlorpyrifos (B) in vial assays evaluated at 24 h after treatment. Fig. 7. Open in new tabDownload slide Mortality (%mean ± SEM) of adults from susceptible (PB) and resistant Listronotus maculicollis populations (CN, EW, and LI) caused by diagnostic concentrations of technical-grade bifenthrin (A) and chlorpyrifos (B) in vial assays evaluated at 24 h after treatment. At 24 h after treatment with chlorpyrifos, the lower concentration (2.24 mg AI/m2) clearly separated the susceptible (93%) from the resistant populations, but did not separate the moderately pyrethroid-resistant populations (15–20%) from the population with the highest resistance level of the pyrethroid resistance (2%; Fig. 7B: F = 34.6; df = 3, 47; P < 0.01). At the higher concentration (11.2 mg AI/m2), susceptible and moderately resistant populations were not separated (90–100%) but all differed significantly from the highly resistant population (25%). At 48 h after treatment with chlorpyrifos, mortality was too high to effectively separate the susceptible and moderately and highly resistant populations (F = 2.7; df = 3, 47; P = 0.06). The lower concentration (2.24 mg AI/m2) caused on average 98.3% mortality of the susceptible population, which significantly differed from the mortality in only one of moderately (EW, mortality 66.7%) and highly resistant (45%) populations. No difference was observed among resistant populations. At the higher concentration (11.2 mg AI/m2), mortality did not differ statistically among populations (range 80–100%). A similar trend was observed 72 h after treatment when mortality at the lower concentration was higher in the susceptible population than in populations EW (75%) and LI (53.3%), but not in population CN (76.7%); no differences among populations were observed at the higher concentration (F = 4.4; df = 3, 47; P < 0.01). Discussion The availability of diagnostic methods that are both accurate and practical is crucial in the development of pesticide resistance monitoring and management programs. Our study is the first to develop such diagnostic tools for a turfgrass insect pest, the annual bluegrass weevil. The two methods developed, both using baseline diagnostic doses, were a Petri dish assay with the formulated products and a vial assay with the technical-grade active ingredients. Our methods targeted the two most commonly used adulticides in annual bluegrass weevil management, bifenthrin and chlorpyrifos, both contact insecticides. Adults are a major target in annual bluegrass weevil management, and high resistance levels to bifenthrin and other pyrethroids (Ramoutar et al. 2009a, Koppenhöfer et al. 2018, Kostromytska et al. 2018) and elevated tolerance to chlorpyrifos (Clavet et al. 2010, Koppenhöfer et al. 2018, Kostromytska et al. 2018) have been observed. An acceptable diagnostic method for resistance monitoring has to satisfy several important criteria. First, the assay has to exaggerate or at least reflect the differences between susceptible and resistant populations. Second, the assay has to reflect field conditions (ffrench-Constant and Roush 1990). To become widely used, the procedure also needs to be feasible for practitioners and diagnostic laboratories for fast, cost-effective and reliable resistance screening. The topical LD assay method through the direct application of exact doses onto the insect is the most precise and discriminative for evaluating bifenthrin and chlorpyrifos toxicity against annual bluegrass weevil adults (Kostromytska et al. 2018). It is a standard procedure for many insect species (Halliday and Burnham 1990). However, it is labor intensive and requires special skills, large insect samples, and thus, it is not feasible for monitoring of multiple field populations. The results of the previous topical test with annual bluegrass weevil adults (Kostromytska et al. 2018) were used as a reference to evaluate the discriminative power of the here developed bioassays. Compared with the results of the topical bioassay with bifenthrin, LC50 values and RR50s calculated based on the data obtained in the Petri dish assays (2015) tended to underestimate the resistance level of moderately resistant populations at 24, 48, or 72 h after treatment application (RR50s ranging 12.2–51.5 in Petri dish assay vs 30.5–148.4 in topical assay). Differences between susceptible and moderately resistant populations were the smallest after 72 h (RR50s ranging 12.2–33.0). The RR50 values of the most resistant population were similar in the topical and Petri dish assays (depending on the evaluation time, 343.1–448.3 in topical assay and 352.8–372.5 in Petri dish assay). It is possible that other ingredients included in the formulation affect toxicity and diminish differences between susceptible and moderately resistant populations. However, a similar trend was observed for the vial bioassay with bifenthrin, in which obtained LC values and RR50s tended to underestimate the resistance levels at 24 h after treatment application (RR50s ranging 24.7–258.2). After 48 and 72 h, the RR50 of the most resistant population (343.1, topical assay) was overestimated (935.8 and 1,760.9 at 48 and 72 h, respectively, vial assay). However, the RR50s for the moderately resistant populations were lower than the RR50s obtained in the topical assay. Data obtained in the Petri dish assay with bifenthrin in 2014 yielded RR50s for moderately (367–591) and highly resistant (2,294–2,504) populations significantly higher than RR50s obtained in topical assay (Kostromytska et al. 2018) and the Petri dish assay in 2015. This could be at least in part due to the use of phenologically different annual bluegrass weevil populations, spring generation adults in 2014 versus overwintering generation adults in 2015, which might differ physiologically with respect to resistance or tolerance. A similar trend was observed in a previous toxicity study with annual bluegrass weevil (O. S. Kostromytska, unpublished data). More studies are needed to better understand the effect of annual bluegrass weevil phenological generation on the insecticides’ toxicity and its potential role in the development of accurate monitoring techniques. Overall, the Petri dish and vial assays were sensitive enough to discriminate susceptible, moderately resistant, and highly resistant annual bluegrass weevil populations and can be employed for resistance detection and monitoring. The subsequent step was the selection and dose validation for both bioassay types. In Petri dish and vial validation assays, susceptibility to bifenthrin was more clearly separated at 72 h after treatment than at 24 and 48 h. At 24 and 48 h, many weevils were moribund or knocked down, and it was challenging to distinguish them from dead weevils. In addition, differences between susceptible and moderately resistant populations were not always clear. In Petri dish assays, the lowest bifenthrin concentration (112.2 g AI/m2) allowed to distinguish between susceptible and resistant populations (including the population with the lowest resistance level, GB), whereas the two higher concentrations separated moderately and highly resistant populations at 72 h after treatment. For chlorpyrifos, two doses were sufficient for detecting the level of resistance or tolerance. Practically, Petri dish assay could be used as a two-step monitoring strategy for both bifenthrin and chlorpyrifos. In the first step, annual bluegrass weevils are exposed to the lowest concentration (112. 2 mg AI/m2 of bifenthrin; 3.4 mg AI/m2 of chlorpyrifos) to separate susceptible from resistant populations. In the second step, moderately and highly resistant populations could be further separated by exposure to one of the higher concentrations (336.3 or 3,362.5 mg AI/m2 of bifenthrin; 33.6 mg/m2 of chlorpyrifos). Mortality assessment can be made at 24–72 h after exposure. However, for bifenthrin, 72-h evaluation is recommended to avoid difficulties in separating moribund from dead weevils. The vial assay has acceptable discriminative power for bifenthrin and chlorpyrifos. Separation of different levels of bifenthrin resistance could be achieved with one diagnostic concentration (either 112.1 or 1,120.8 mg AI/m2) using the vial assay. Two concentrations are necessary for chlorpyrifos tolerance detection: 1) a lower concentration to separate susceptible and resistant populations (2.2 mg AI/m2); and 2) a higher concentration to separate populations with moderate tolerance from populations with high tolerance (11.2 mg AI/m2). However, the vial assay is more time and labor consuming and requires the use of technical-grade active ingredients in organic solvent, which results in accumulation of hazardous waste. Vial drying takes additional time and extra equipment and labor to ensure uniform distribution. In addition, evaluation inside the vials is harder to perform especially if moribund weevils are present. Conclusions Based on our findings and considering the main criteria for resistance diagnostic assays, the Petri dish assay that uses formulated bifenthrin and chlorpyrifos is a better candidate than the vial assay as a diagnostic assay for annual bluegrass weevil resistance detection and monitoring. The Petri dish assay demonstrated sufficient discriminating power, accurately separated populations with moderate and high resistance levels, and at the same time is relatively easy to use by practitioners like diagnostic laboratories and consultants. In addition, a single diagnostic concentration is sufficient to separate susceptible and resistant populations. However, for more accurate and precise determination of the resistance or tolerance levels, a multistep procedure with two concentrations is necessary. This diagnostic assay will help gather broader and more precise information about the extent of annual bluegrass weevil pyrethroid resistance and organophosphate tolerance. In addition, the obtained diagnostic information will help optimize annual bluegrass weevil management and resistance mitigation programs. Acknowledgments We appreciate the technical assistance of John T. Sanders and Richard Keller. We are grateful for expertise and assistance provided by M. E. Scharf. We thank Nassau CC, Glen Cove, NY; JC Melrose CC, Cheltenham, PA; Greate Bay GC, Somers Point, NJ; Ridgewood CC, Paramus, NJ; Edgewood CC, River Vale, NJ; The Connecticut GC, Easton, CT; Plainfield CC, Edison, NJ; Pine Brook GC, Manalapan Township, NJ; Howell Park GC, Howell, NJ; and Rutgers University Horticultural Farm No. 2 for providing collection sites. This research was supported by grants from the Golf Course Superintendents Assn. of America and supporting Chapters (Connecticut AGCS, GCSA of New Jersey, Greater Pittsburgh GCSA, Hudson Valley GCSA, Long Island GCSA, Metropolitan GCSA, Mountain & Valley GCSA, New Jersey Turfgrass Assn., Pocono Turfgrass Assn.), the U.S. Golf Assn., the New York State Turfgrass Assn., the O.J. 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TI - Diagnostic Dose Assays for the Detection and Monitoring of Resistance in Adults From Listronotus maculicollis (Coleoptera: Curculionidae) Populations
JF - Journal of Economic Entomology
DO - 10.1093/jee/toy167
DA - 2018-09-26
UR - https://www.deepdyve.com/lp/oxford-university-press/diagnostic-dose-assays-for-the-detection-and-monitoring-of-resistance-aXjXEO2Oym
SP - 2329
VL - 111
IS - 5
DP - DeepDyve
ER -