Research Article Received: 26 February 2015
Revised: 6 July 2015
Accepted article published: 14 July 2015
Published online in Wiley Online Library: 7 August 2015
(wileyonlinelibrary.com) DOI 10.1002/ps.4076
Fungicides affect Japanese beetle Popillia japonica (Coleoptera: Scarabaeidae) egg hatch, larval survival and detoxification enzymes Glen R Obear,a,b* Adekunle W Adesanya,c Patrick J Liesch,a R Chris Williamsona and David W Heldc Abstract BACKGROUND: Larvae of the Japanese beetle, Popillia japonica (Coleoptera: Scarabaeidae), have a patchy distribution in soils, which complicates detection and management of this insect pest. Managed turf systems are frequently under pest pressure from fungal pathogens, necessitating frequent fungicide applications. It is possible that certain turfgrass fungicides may have lethal or sublethal adverse effects on eggs and larvae of P. japonica that inhabit managed turf systems. In this study, eggs and first-, second- and third-instar larvae were treated with the fungicides chlorothalonil and propiconazole, and survival was compared with that of untreated controls as well as positive controls treated with the insecticide trichlorfon. RESULTS: Chlorothalonil reduced survival of first-instar larvae treated directly and hatched from treated eggs. Propiconazole delayed egg hatch, reduced the proportion of eggs that successfully hatched and reduced survival of first-instar larvae treated directly and hatched from treated eggs. Sublethal doses of the fungicides lowered the activities of certain detoxification enzymes in third-instar grubs. CONCLUSIONS: Fungicide applications to turfgrass that coincide with oviposition and egg hatch of white grubs may have sublethal effects. This work is applicable both to high-maintenance turfgrass such as golf courses, where applications of pesticides are more frequent, and to home lawn services, where mixtures of multiple pesticides are commonly used. © 2015 Society of Chemical Industry Keywords: white grubs; pesticides; P450; carboxylesterase; glutathione S-transferase
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INTRODUCTION
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Japanese beetle (Popillia japonica Newman) larvae, like other soil-dwelling larvae, have a patchy distribution in soils, which complicates detection and management. Female Japanese beetles are active during summer and have numerous periods of oviposition during their 4–6 week activity period.1,2 In the soil, females lay eggs which must absorb water to hatch successfully. Larvae spend 9–10 months in soil, and movement and survival are influenced by environmental conditions and turfgrass culture. When used, insecticides are commonly applied at or just before egg hatch (preventive) or when larger third-instar grubs are present (curative).2 Soil moisture from rainfall or supplemental irrigation of amenity grasses positively influences larval weight, abundance and development of Japanese beetle larvae.3 The presence and performance of white grubs are also affected by turfgrass culture.3 Japanese beetle larvae are rarely found in golf course putting greens, but infestations can occur in areas directly adjacent to putting greens with identical soils and plant characteristics. The impacts of mowing height vary, depending on species, but there is limited evidence that grass height is a significant factor in survival of Japanese beetle larvae.3,4 Golf course putting greens often receive the most agrochemical inputs among areas on golf courses Pest Manag Sci 2016; 72: 966–973
owing to high golfer standards and pest pressure, where numerous fungal diseases can damage turf under a variety of environmental conditions throughout the growing season.5 It is possible that commonly applied turfgrass fungicides may adversely affect larvae of P. japonica. Fungicides, although not intended to control insects, may cause physiological responses in insects. The chloronitriles, specifically the active ingredient chlorothalonil, and the demethylation inhibitor (DMI) fungicides are commonly and frequently applied to amenity turfgrass for disease management. The results of studies on insects with chloronitrile fungicides are varied, with some reporting toxicity of chlorothalonil6 and other studies reporting
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Correspondence to: GR Obear, Department of Agronomy and Horticulture, University of Nebraska – Lincoln, Lincoln, NE 68583, USA E-mail: [email protected]
a Department of Entomology, University of Wisconsin – Madison, Madison, WI, USA b Department of Agronomy and Horticulture, University of Nebraska – Lincoln, Lincoln, NE, USA c Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, USA
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Fungicides affect Japanese beetle egg hatch and larval survival no toxicity.7,8 The exact mechanism of toxicity is unknown, but chlorothalonil has been reported to bind to sulfhydryl enzymes, which are important for cellular respiration, bind and deplete cellular glutathione (GSH) and inhibit glycolysis by binding with glyceraldehyde 3-phosphate dehydrogenase (GAPDH).9 Demethylation inhibitor fungicides can decrease the activity of P450 enzymes in insects,10,11 which are important for growth, reproduction, hormone synthesis and detoxification of foreign chemical compounds.12 Annual bluegrass weevils, Listronotus maculicollis (Coleoptera: Curculionidae), a pest of cool-season turfgrass, that have prior exposure to DMI fungicides have increased susceptibility to synthetic pyrethroids. Resistance to pyrethroid insecticides is common among populations of annual bluegrass weevil in the northeastern United States, and fungicides that inhibit detoxification enzymes may increase the efficacy of insecticide applications.13 It is possible that fungicides applied to turfgrass may inhibit detoxification enzymes in other turfgrass insects. Furthermore, suppression of these enzymes may leave individuals more susceptible to insecticide applications that are otherwise not directly toxic.14 Japanese beetle grubs are facultative monophages2 and likely rely on multiple detoxification enzymes for the metabolism of xenobiotics such as plant toxins and pesticides.15 Ahmad16 reported that there is significant P450 activity in the third-instar grubs of Japanese beetle. It is unknown whether fungicides encountered by Japanese beetle grubs affect the activities of widely studied detoxification enzymes such as cytochrome P450, glutathione S-transferase (GST) and carboxylesterase (CoE). The overall objective of this study was to gain a better understanding of how two commonly applied turf fungicides affect eggs and larvae of the Japanese beetle. This study specifically examined the influence of propiconazole and chlorothalonil on immature Japanese beetle survival and their effect on the activities of detoxification enzymes in third-instar larvae.
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MATERIALS AND METHODS
2.1 Insect rearing and collection A series of laboratory assays were conducted to investigate the potential lethal effects of commonly applied fungicides labeled for use in managed turfgrass. Adult male and female Japanese beetles were collected in traps (Pherocon® JB trap; Trécé, Adair, OK) baited with a floral lure (2-phenyl-ethyl propionate, eugenol
www.soci.org and geraniol; 3:7:3 ratio) from field sites with no insecticide use for the preceding 12 months. Traps were hung on metal stands at a height of 1.5 m at 9:00 and collected at 15:00 on the same day. Eggs and first-instar larvae were reared in the laboratory. Adults from traps were emptied into six different 26 L plastic tubs containing moistened silt loam potting soil (River Run Products Corp., Custer, WI) with a depth of approximately 10 cm, and each tub contained approximately 1000 mixed-sex beetles. To collect eggs, adults were allowed 48 h to mate and oviposit into the soil, and then the soil in each tub was searched for eggs. First-instar larvae were collected using the same method, except the tubs with eggs were left undisturbed for 2 weeks so the eggs could hatch. Second- and third-instar larvae were collected from infested Poa pratensis turfgrass on golf courses in Wisconsin. The areas where larvae were collected had not been treated with pesticides for at least 12 months. 2.2 Experimental conditions and treatments Experimental treatments were delivered topically to eggs and larvae. The chemical treatments evaluated were two fungicides (propiconazole as Banner MAXX II, and chlorothalonil as Daconil Weather Stik; Syngenta Crop Protection, Greensboro, NC), a formulation blank (FB) or formulation less the active ingredient, an insecticide (trichlorfon, Dylox; Bayer Environmental Sciences, Research Triangle Park, NC) and an untreated control (Table 1). To simulate exposure in a field setting, application rates were calculated on the basis of the surface area of the bottom of the container (petri dish or plastic cup) that held the egg or larva. Both fungicides were prepared in solution using deionized water and mixed at the maximum labeled rate for controlling Sclerotinia homoeocarpa, a common fungal disease of turfgrass in the midwestern United States.5 Trichlorfon was prepared in solution using deionized water and mixed at the maximum labeled rate for white grubs. Control larvae were treated with deionized water. All chemical treatments and controls were replicated 20 times in two independent assays using different cohorts of eggs or larvae. All experiments were conducted using a completely randomized design in a controlled growth chamber set at 23 ∘ C and 50% relative humidity with no supplemental lighting. 2.3 Topical application to eggs and first-instar larvae Eggs or larvae were collected and placed individually in 47 mm diameter petri dishes containing moistened filter paper (Fisher
Table 1. Chemical treatments applied to eggs and first-, second- and third-instar larvae of the Japanese beetle Ratea, b Treatment Untreated control Banner MAXX IId Banner MAXX II FBe Daconil Weather Stikd Daconil Weather Stik FBe Dylox 420 SLf
Active ingredient – 14.3% propiconazole – 54% chlorothalonil – 37.3% trichlorfon
kg product ha−1
Solution concentrationc kg AI ha−1
– 6.9 6.9 13.8 13.8 24.8
– 1.0 – 7.5 – 9.3
mg product mL−1 – 2.0 2.0 3.9 3.9 6.9
mg AI mL−1 – 0.3 – 2.1 – 2.6
a All products prepared at maximum labeled rate for use on managed turfgrass. b Rate calculated on the basis of the area of the container holding individual eggs or larvae. c Applied at a volume of 0.7 mL per individual egg or larva. d Syngenta Crop Protection, Greensboro, NC.
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e Formulation blank (FB) contains the same inert ingredients without active ingredients. f Bayer Environmental Sciences, Research Triangle Park, NC.
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www.soci.org Scientific, Waltham, MA). Treatments were applied directly with a micropipette to the surface of eggs or the abdominal pleuron of the larvae at a volume of 0.7 mL. This application volume was selected because it was sufficient to cover eggs and larvae without leaving standing water in dishes. An additional 0.7 mL of deionized water was added to petri dishes to wet the filter paper and prevent desiccation. Eggs were monitored daily for hatch, and the date of egg hatch was recorded. Any eggs that had not hatched after 26 days were assumed to be dead. After hatching, 5 g of autoclaved silt loam potting soil was added to the petri dishes and moistened with 0.7 mL of deionized water. A slice of organic carrot was added to each dish at that time. Larvae or eggs were monitored daily and characterized as alive or dead. The assay was concluded when mortality in the control larvae exceeded 50% in the egg assay and 70% in the first-instar assay. 2.4 Topical application to second- or third-instar larvae Field-collected larvae were placed into individual 0.26 L cups with holes poked in the bottom for drainage. Larvae were acclimated for a 24 h period and then subjected to the same treatments as eggs and first-instar larvae (Table 1). Treatments were applied topically to the abdominal pleuron of each larva using the same methods and volume as previously described. Following treatment application, 30 g of autoclaved silt loam potting soil was added to each cup. An additional 1.2 mL of deionized water was added to each cup, followed by a slice of carrot for food. Holes were poked in plastic lids which were then placed on top of cups. Larvae were monitored daily and characterized as alive or dead, and the assay was concluded when mortality in the control larvae exceeded 40% in the second-instar assay and 50% in the third-instar assay. 2.5 Effects of chemical treatment on activities of detoxification enzymes in third-instar larvae 2.5.1 Topical treatment of grubs Third-instar larvae were placed into individual 47 cm diameter petri dishes with moistened filter paper and a slice of carrot as previously described. Larvae were treated with Banner MAXX II, Daconil Weather Stik and Dylox at the maximum labeled rates listed in Table 1. A control group was treated with deionized water. Treatments were applied to the abdominal pleuron via pipette with a water volume of 0.7 mL. An additional 0.7 mL of deionized water was added to petri dishes to moisten the filter paper and prevent desiccation. Larvae were removed from the growth chamber 24 h after treatment, flash frozen with dry ice and stored at −80 ∘ C until enzyme extractions were performed.
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2.5.2 Enzyme extraction and determination of protein content Following treatment, the bodies of four whole larvae for each of the chemical and control treatments (Table 1) were homogenized in 10 mL of ice-cold homogenization buffer [0.1 M phosphate buffer, pH 7.5, 10% glycerol, 1 mM of ethylenediaminetetraacetic acid (EDTA), 0.1 mM of dithiothreitol (DTT), 1 mM of phenylmethylsulfonyl fluoride (PMSF) and 1 mM of phenylthiourea (PTU)]. The homogenate was filtered through a funnel with a cheesecloth into a 15 mL centrifuge tube. The filtrate (homogenate) was centrifuged (AllegraTM 25R; Beckman Coulter, Inc., Brea, CA) at a speed of 10 000 × g for 30 min at 4 ∘ C using a type TA-14-50 rotor. The pellet was discarded, and the supernatant was carefully transferred into another centrifuge tube and spun in an ultracentrifuge equipped with a TI 865 rotor (Sorvall Discovery 90SE; Thermo Scientific, Waltham, MA) at a speed of 100 000 × g for 1 h at 4 ∘ C. The
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supernatant was used as the source of enzyme for GST and CoE activity assay, while the pellets (microsomes) were dissolved in a resuspension buffer (0.1 M phosphate buffer, pH 7.5, 20% glycerol, 1 mM of EDTA, 0.1 mM of DTT and 1 mM of PMSF). All the steps described were performed on ice. The microsomes and cytosol were temporarily stored in a −80 ∘ C freezer. The amount of protein present in each sample was determined using bovine serum albumin as the standard.17 A total of 3–4 determinations were done for each sample using a UV–vis spectrophotometer (Du 640; Beckman Coulter, Inc., Pasadena, CA).
2.5.3 Determination of P450 activity The activity of P450 was assayed using two chemical reactions, i.e. ethoxyresorufin O-demethylation (EROD) and ethoxycoumarin O-deethylation (ECOD) in a NADPH-dependent reaction using modified methods of Lee and Scott.18 A sample (30–50 μL) of microsomes and 40 μL of ECOD buffer (50 mM of Tris-base, 150 mM of KCl and 1 mM of EDTA, pH 7.8) containing 4 nM of 7-ethoxycoumarin (7-EC) were added to each well of a 96-well plate (black flat-bottom, BD Falcon; BD Biosciences, Franklin Lakes, NJ). For P450 EROD activity, another sample of 30–50 μL of microsomal fraction was added to a well, followed by the addition of 0.3 pmol of ethoxyresorufin. EROD buffer (0.1 M phosphate buffer, 0.1 mM of EDTA, 5 mM of MgCl2 , pH = 7.8) was added to each well for a final volume of 100 μL. Both P450 reactions were started with the addition of 10 μL of 0.01 M NADPH (freshly prepared) to each well, then incubated for 30 min at 30 ∘ C. For P450 ECOD, the fluorescence of NADPH was removed by adding 0.3 μM of oxidized glutathione and 0.5 units of glutathione reductase (Sigma-Aldrich, St Louis, MO) to each well. The reaction was stopped after 10 min at 25 ∘ C by adding 120 μL of stop solution (50% acetonitrile and 50% TRIZMA-base buffer). P450 ECOD activity is defined as the measured amount of 7-hydroxycourmarin produced at a wavelength of 390 nm and 465 nm for excitation and emission respectively. The fluorescence intensity for P450 EROD was measured at 530 and 580 nm for excitation and emission respectively. Control reactions used NADPH and substrate without the microsomal fraction. Product standard curves were also generated for resorufin and 7-hydroxycoumarin using the respective wavelengths for each product. P450 activity is expressed in pmol product formed min−1 mg−1 protein.
2.5.4 Determination of GST activity GST activity was assayed using the cytosolic fractions of the extract to catalyze the conjugation of glutathione (GSH) and chloro-2,4-dintrobenzene (CDNB) using a modification of Habig et al.19 For each reaction, freshly prepared GSH was dissolved in a phosphate buffer (0.1 M, pH 6.5) containing EDTA (1 mM), PMSF (1 mM) and DTT (1 mM). A quantity of 5 μL of the enzyme source was added to the GSH–buffer solution in a final volume of 200 μL (final GSH concentration 2.5 mM) in a 96-well plate. Reactions were incubated for 3 min at 25 ∘ C, after which 5 μL of 60 mM CDNB (dissolved in alcohol, freshly prepared) was added. The control reaction is the non-enzymatic reaction between GSH and CDNB under the same conditions as those used for the enzymatic reaction. The mixture was shaken, and the change in absorbance was measured for 5 min at a wavelength of 340 nm. GST activity was expressed as millimol CDNB conjugated min−1 mg−1 protein using an extinction coefficient of 5.3 mM−1 cm−1 for S-(2,4-dinitrophenyl) glutathione.
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Fungicides affect Japanese beetle egg hatch and larval survival 2.5.5 Determination of CoE activity CoE was determined using the hydrolysis of 𝛼- and 𝛽-naphthylacetate (𝛼- and 𝛽-NA) to 𝛼- and 𝛽-naphthol using the modified protocol of Van Asperen.20 The working concentration of the substrates (𝛼- and 𝛽-naphthylacetate) was 0.3 mM (containing 0.3 mM of eserine), diluted from their respective 0.03 M stock. Each assay reaction contained 60 μL of phosphate buffer (0.04 M, pH 7.0), 50 μL of the supernatant (enzyme source) and 80 μL of 0.3 mM substrate solutions. The reaction was incubated at 37 ∘ C for 30 min and stopped by the addition of 20 μL of stop solution (two parts 1% Fast Blue BB and five parts 5% sodium dodecyl sulfate). The colour developed at room temperature for 15 min, and then absorbance was measured at 600 nm for the hydrolysis of 𝛼-NA and at 550 nm for 𝛽-NA using a microplate reader (Cytation 3; Bio-Tek® , Winooski, VT). The product (𝛼- and 𝛽-naphthol) standard curve was generated by measuring optical absorbance at 600 and 550 nm at different concentrations. 2.6 Statistical analysis Analysis of variance (ANOVA) was used to determine whether chemical treatments significantly affected the length of time required for eggs to hatch, and a Student’s t-test was used to separate treatment means. A nominal logistic model was used to determine whether chemical treatments affected the total proportion of eggs that successfully hatched, and odds ratios were used to make pairwise comparisons between treatments. Data from each assay were subjected to survival analysis, and the Kaplan–Meier method was used to calculate survival functions for each treatment group. The log-rank test was used to test whether the survival functions were different among all treatment groups in each assay. The Holm–Sidak method was used to make multiple pairwise comparisons between survival functions. ANOVA was also used to test whether chemical treatment significantly affected the titers (activities) of P450, GST and CoE. Student’s t-test was employed to separate the means of each activity (P450, GST and CoE) using JMP software (v.11).
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RESULTS
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Table 2. Japanese beetle egg hatch timing and proportion affected by treatment with trichlorfon, propiconazole and chlorothalonil (data pooled from two cohorts; n = 240)a
Treatment Dyloxb Banner MAXX IIc Daconil Weather Stikd Untreated control Daconil FBe Banner FBe
Days to hatch 21.3 a 19.1 b 17.9 c 17.5 cd 17.3 d 17.1 d F 5,194 = 37.899, P < 0.001
Proportion of eggs hatched 0.60 b 0.625 b 0.85 a 0.925 a 0.90 a 0.95 a 𝜒 2 = 30.761, P < 0.001
a Numbers in columns followed by different letters are significantly different. b 37.3% trichlorfon. c 14.3% propiconazole. d 54% chlorothalonil. e Formulation blank (FB) contains the same inert ingredients without active ingredients.
P < 0.001), Daconil Weather Stik (𝜒 2 = 46.114, P < 0.001) and the Daconil Weather Stik FB (𝜒 2 = 70.574, P < 0.001). Larvae from eggs treated with Daconil Weather Stik began to perish 3 DAH, and survival was significantly reduced relative to the untreated control (𝜒 2 = 26.437, P < 0.001). Survival was also significantly lower from Daconil Weather Stik treatment compared with the Daconil Weather Stik formulation blank (𝜒 2 = 21.241, P < 0.001). Larvae from eggs treated with Banner MAXX II began to perish 5 DAH, and survival was significantly reduced relative to the untreated control (𝜒 2 = 13.153, P = 0.002). However, survival was not significantly different between Banner MAXX II treatment and the Banner MAXX II formulation blank (𝜒 2 = 1.707, P = 0.346). The Banner MAXX formulation blank reduced survival relative to the untreated control (𝜒 2 = 7.905, P = 0.024).
3.2 Survival of first-instar larvae following topical treatment Survival of first-instar larvae (n = 240) was significantly affected by chemical treatments (𝜒 2 = 243.962, P < 0.001). Untreated controls began to perish 7–8 days after treatment (DAT) (Fig. 1B). First-instar larvae treated with Dylox perished within 1–3 DAT, and survival was significantly reduced relative to the untreated control (𝜒 2 = 79.375, P < 0.001), to Banner MAXX II (𝜒 2 = 62.003, P < 0.001), to the Banner MAXX II FB (𝜒 2 = 79.255, P < 0.001), to Daconil Weather Stik (𝜒 2 = 79.560, P < 0.001) and to the Daconil Weather Stik FB (𝜒 2 = 83.638, P < 0.001). Larvae treated with Daconil Weather Stik began to perish 4 DAT, and survival was significantly reduced relative to the untreated control (𝜒 2 = 18.670, P < 0.001). Survival was also reduced with Daconil Weather Stik treatment relative to the Daconil Weather Stik formulation blank (𝜒 2 = 6.973, P = 0.072). Treatment with Banner MAXX II significantly reduced survival relative to the untreated control (𝜒 2 = 6.278, P = 0.083), but survival was not different from the Banner MAXX II FB (𝜒 2 = 0.0632, P = 0.801). The Banner MAXX formulation blank reduced survival relative to the untreated control (𝜒 2 = 6.582, P = 0.0795).
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3.1 Hatch and neonate survival following topical treatment of eggs Eggs treated with Dylox and Banner MAXX II took 2–4 days longer to hatch on average (F 5,194 = 37.899, P < 0.001) relative to untreated controls and the formulation blank (Table 2). The time required to hatch for eggs treated with Daconil Weather Stik was not significantly different from that of untreated controls or the formulation blank. Similarly, the total proportion of eggs treated with Dylox or Banner MAXX II that hatched was about 30% less (𝜒 2 = 30.761, P < 0.001) relative to untreated controls and the Banner MAXX II formulation blank. Daconil Weather Stik did not significantly reduce the proportion of eggs hatched relative to untreated controls or the Daconil Weather Stik formulation blank. Survival of first-instar larvae hatched from treated eggs (n = 195) was significantly affected by chemical treatments (𝜒 2 = 208.281, P < 0.001). Untreated controls began to perish 7 days after hatch (DAH) and began to decline rapidly around 9 DAH (Fig. 1A). First-instar larvae from eggs treated with Dylox perished within 1–2 DAH, and Dylox significantly reduced survival relative to the untreated control (𝜒 2 = 71.902, P < 0.001), Banner MAXX II (𝜒 2 = 39.041, P < 0.001), the Banner MAXX II FB (𝜒 2 = 72.524,
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Figure 1. Survival of Japanese beetle eggs and larvae treated topically with Dylox, Banner MAXX II or Daconil Weather Stik. Data from each life stage assay were pooled together from two cohorts. FB, formulation blank. (A) First-instar larvae hatched from treated eggs (n = 195; 𝜒 2 = 208.281, P < 0.001). Eggs that did not hatch were not included in this analysis. (B) First-instar larvae (n = 240; 𝜒 2 = 243.962, P < 0.001). (C) Second-instar larvae (n = 240; 𝜒 2 = 245.792, P < 0.001). (D) Third-instar larvae (n = 240; 𝜒 2 = 168.543, P < 0.001).
3.3 Survival of second-instar larvae following topical treatment The survival of second-instar larvae (n = 240) was significantly affected by chemical treatments (𝜒 2 = 245.752, P < 0.001). Untreated larvae began to perish 8 DAT and steadily declined until the assay was concluded at 23 DAT (Fig. 1C). Larvae treated with Dylox began to perish 1 DAT, and survival was significantly reduced relative to the untreated control (𝜒 2 = 87.373, P < 0.001), to Banner MAXX II (𝜒 2 = 87.373, P < 0.001), to the Banner MAXX II FB (𝜒 2 = 87.373, P < 0.001), to Daconil Weather Stik (𝜒 2 = 89.033, P < 0.001) and to the Daconil Weather Stik FB (𝜒 2 = 87.373, P < 0.001). Survival was not significantly reduced from treatment with Daconil Weather Stik (𝜒 2 = 0.674, P = 0.958), Banner MAXX II (𝜒 2 = 3.045, P = 0.570) or either formulation blank relative to the untreated control.
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3.4 Survival of third-instar larvae following topical treatment Survival of third-instar larvae (n = 240) was significantly affected by chemical treatments (𝜒 2 = 168.543, P < 0.001). Untreated controls began to perish 3 DAT and steadily declined until the assay was concluded 13 DAT (Fig. 1D). Larvae treated with Dylox began to perish 1 DAT, and survival was significantly reduced relative to
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the untreated control (𝜒 2 = 65.792, P < 0.001), to Banner MAXX II (𝜒 2 = 62.435, P < 0.001), to the Banner MAXX II FB (𝜒 2 = 53.019, P < 0.001), to Daconil Weather Stik (𝜒 2 = 72.786, P < 0.001) and to the Daconil Weather Stik FB (𝜒 2 = 61.269, P < 0.001). Survival was not significantly reduced from treatment with Daconil Weather Stik (𝜒 2 = 4.595, P = 0.278), Banner MAXX II (𝜒 2 = 1.413, P = 0.882) or either formulation blank relative to the untreated control. 3.5 Effects of chemical treatment on activities of detoxification enzymes in third-instar larvae Untreated third-instar grubs had the highest mean P450 ECOD and EROD activities compared with other treated grubs (Fig. 2). Banner MAXX and Dylox (F 3,32 = 5.07, P = 0.005) significantly reduced P450 ECOD activity but not Daconil Weather Stik relative to the untreated grubs. The chemical treatments also lowered P450 EROD activity, but only the reductions by Daconil Weather Stik and Dylox were significant (F 3,32 = 3.44, P = 0.028). Grubs treated with Banner MAXX had highest activity of GST (F 3,12 = 5.07, P = 0.0114), while there was no significant difference in mean GST activity among control grubs and grubs treated with Daconil Weather Stik and Dylox. There was a 2.38-fold increase in GST activity by Banner MAXX relative to untreated grubs (Fig. 3). Although the chemically treated grubs had lower CoE activity (Fig. 4),
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Fungicides affect Japanese beetle egg hatch and larval survival
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Figure 2. Mean (± SE) P450 ethoxycoumarin O-deethylation (ECOD) (upper) and ethoxyresorufin O-demethylation (EROD) (lower) activity in Japanese beetle third-instar grubs topically dosed with fungicides (Banner MAXX and Daconil) or insecticide (Dylox). ANOVA (F 3,32 = 5.07, P < 0.01). Bars with the same letter were not significantly different (Student’s t-test, 𝛼 = 0.05).
Figure 3. Mean (± SE) GST CDNB activity in Japanese beetle third-instar grubs topically dosed with fungicides (Banner MAXX and Daconil) or insecticide (Dylox). ANOVA (F 3,6 = 5.73, P < 0.01). Bars with the same letter above were not significantly different (Student’s t-test, 𝛼 = 0.05).
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DISCUSSION
The findings of our study provide evidence that both propiconazole and chlorothalonil may have lethal or sublethal adverse effects on eggs and larvae of P. japonica. Previous studies14,21 have also reported sublethal effects of fungicides on larval and adult Coleoptera and Hymenoptera respectively. In our study, Daconil Weather Stik reduced survival of first-instar larvae treated directly, and fewer first-instar larvae hatched from treated eggs compared
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only grubs that were exposed to Dylox had significantly lower CoE activity compared with untreated control grubs (F 3,6 = 8.4, P = 0.0144). Treatment with Banner MAXX and Daconil Weather Stik fungicides decreased CoE activity by 27 and 34% compared with untreated control grubs, while Dylox treatment reduced CoE activity by 72%. This evidence suggests that the formulated fungicide products both reduce activity of detoxification enzymes.
Figure 4. Mean (± SE) 𝛼-napthylacetate CoE activity of Japanese beetle third-instar grubs topically dosed with fungicides (Banner MAXX and Daconil) or insecticide (Dylox). ANOVA (F 3,6 = 8.4, P < 0.01). Bars with the same letter above were not significantly different (Student’s t-test, 𝛼 = 0.05).
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with the control. Survival was also reduced compared with the Daconil Weather Stik formulation blank, suggesting that the active ingredient chlorothalonil was responsible for the adverse effects. Banner MAXX II significantly increased the length of time required for egg hatch and reduced the proportion of successfully hatched eggs compared with the control and the Banner MAXX II formulation blank. This suggests that the active ingredient propiconazole was responsible for the adverse effects. Banner MAXX II also reduced survival of first-instar larvae treated directly or hatched from treated eggs, but survival in these assays was not significantly different from the Banner MAXX II formulation blank, suggesting that the formulation carrier had some adverse effect in addition to the active ingredient. In spite of evidence of adverse effects from both chlorothalonil and propiconazole, these chemicals do not appear to have utility as insecticides, considering that neither chemical reduced survival to the same degree as trichlorfon. However, these fungicides may result in mortality of P. japonica if applications coincide with periods of oviposition and egg hatch. In the present study, neither fungicide significantly reduced survival in older larvae (i.e. second- or third-instar larvae). Survival in response to trichlorfon was lower for all life stages, but mortality was delayed for third-instar larvae. Later instars of P. japonica are known to be more resilient to certain chemicals than early instars. For example, application of imidacloprid, which is lethal to first instars almost immediately after egg hatch, only causes behavioral impairment in second- and third-instar larvae.22 This may be due simply to an exposure response or an increasing biosensitivity to the chemicals. For example, a third-instar Japanese beetle grub has approximately 80 times the mass of a first-instar grub,1 which changes the physiological dose to the same application rate. However, fall armyworm larvae have decreased susceptibility to insecticides, as they age independently of dose.23 In the case of fall armyworm, detoxification enzyme activities increase with larval age. The present study used only third-instar Japanese beetle grubs; however, experiments are under way to characterize the life stage variability in enzyme activity in Japanese beetles. Untreated third-instar grubs had higher activities of P450 ECOD, EROD and CoE compared with treated grubs. This suggests that the tested dose of propiconazole, chlorothalonil or trichlorfon inhibited the activities of P450 and CoE, similarly to the findings of Darvas et al.10 and Brattsten et al.11 Banner MAXX II induced the activity of GST compared with other treatment groups, similarly to its reported effect on the caterpillar Mamestra brassicae (Lepidoptera: Noctuidae).24 The elevated GST activity in the presence of propiconazole may be metabolically expensive to Japanese beetle larvae and cause reductions in their fitness. Future studies could evaluate the consequences (e.g. weight gain or successful pupation) when fungicides are present during larval development. Propiconazole and chlorothalonil may have lethal or sublethal adverse effects on eggs and first-instar larvae of P. japonica. Enzymatic assays revealed that these fungicides inhibit the activity of detoxification enzymes of third-instar P. japonica larvae. However, neither fungicide affected oviposition choice by female Japanese beetles.25 This work is relevant to high-maintenance turfgrass sites that either receive frequent application of fungicides, such as golf courses, or where multiple pesticides are mixed together for convenience (lawns). Fungicide applications, especially when coinciding with oviposition and egg hatch, may cause significant local mortality of P. japonica. However, the findings from this study need to be validated in a field setting. In this study, application rates
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were representative of those applied in the field setting, but eggs and larvae likely received a greater exposure than when exposed in context. Soil processes, including binding of chemicals by soil as well as microbial degradation of chemicals over time, could serve to reduce exposure in a field setting.
ACKNOWLEDGEMENTS We thank Jerry Kershasky of University Ridge Golf Course, Bill Kohoss of Coldwater Canyon Golf Course and Jon Hegge of Evansville Golf Club for allowing us to collect grubs from their facilities. We also thank Rebecca Le Beau, Andrew Le Beau, Joshua Horman and John Gillis for providing technical assistance. Drs Douglas J Soldat, Daniel K Young and Zachary J Reicher reviewed an earlier version of this manuscript. This paper is based upon work supported by the Alabama Agricultural Experiment Station through Hatch funding from the National Institute of Food and Agriculture, United States Department of Agriculture. The Wisconsin Turfgrass Association provided additional financial support.
REFERENCES 1 Fleming WE, Biology of the Japanese beetle. US Department of Agriculture Technical Bulletin 1449 (1972). 2 Potter DA and Held DW, Biology and management of the Japanese beetle. Annu Rev Entomol 47:175–205 (2002). 3 Potter DA, Powell AJ, Spicer PG and Williams DW, Cultural practices affect root-feeding white grubs (Coleoptera: Scarabaeidae) in turfgrass. J Econ Entomol 89:156–164 (1996). 4 Dobbs EK and Potter DA, Conservation biological control and pest performance in lawn turf: does mowing height matter? Environ Pest Manag 53:648–659 (2014). 5 Smiley RW, Dernoeden PH and Clarke BB, Compendium of Turfgrass Diseases, 3rd edition. APS Press, St Paul, MN, 167 pp. (2005). 6 Idris AB and Grafius E, Differential toxicity of pesticides to Diadegma insulare (Hymenoptera: Ichneumonidae) and its host, the diamondback moth (Lepidoptera: Plutellidae). J Econ Entomol 86:529–536 (1993). 7 Livingston JM, Yearian WC and Young SY, Insecticidal activity of selected fungicides: effects on three lepidopterous pests of soybean. J Econ Entomol 71:111–112 (1977). 8 Takahashi Y, Kojimoto T, Nagaoka H, Takagi Y and Oikawa M, Tests for evaluating the side effects of chlorothalonil and spinosad on the parasitic wasp (Aphidius colemani). J Pestic Sci 30:11–16 (2005). 9 Caux PY, Kent RA, Fan GT and Stephenson GL, Environmental fate and effects of chlorothalonil: a Canadian perspective. Crit Rev Environ Sci Technol 26:45–93 (1996). 10 Darvas B, Rees HH, Hoggard N, Eldin MHT, Kuwano E, Bélai I et al., Cytochrome P-450 inducers and inhibitors interfering with ecdysone 20-monooxygenases and their activities during postembryonic development of Neobellieria bullata Parker. Pestic Sci 36:135–142 (1992). 11 Brattsten LB, Berger DA and Dungan LB, In vitro inhibition of midgut microsomal P450s from Spodoptera eridania caterpillars by demethylation inhibitor fungicides and plant growth regulators. Pest Biochem Physiol 49:234–243 (1994). 12 Feyereisen R, Insect P450 enzymes. Annu Rev Entomol 44:507–533 (1999). 13 Ramoutar D, Cowles RS, Requinta E, Jr, and Alm SR, Synergism between demethylation inhibitor fungicides or gibberellin inhibitor plant growth regulators and bifenthrin in a pythrethroid-resistant population of Listronotus maculicollis (Coleoptera: Curculionidae). J Econ Entomol 103:1810–1814 (2010). 14 Johnson RM and Percel EG, Effect of a fungicide and spray adjuvant on queen-rearing success in honey bees (Hymenoptera: Apidae). J Econ Entomol 106:1952–1957 (2013).
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Fungicides affect Japanese beetle egg hatch and larval survival 15 Xie W, Wang S, Wu Q, Feng Y, Pan P, Jiao X et al., Induction effects of host plants on insecticide susceptibility and detoxification enzymes of Bemisia tabaci (Hemiptera: Aleyrodidae). Pest Manag Sci 67:87–93 (2010). 16 Ahmad S, Mixed-function oxidase activity in a generalist herbivore in relation to its biology, food plants, and feeding history. Ecology 64:235–243 (1983). 17 Bradford MM, A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Analyt Biochem 72:248–254 (1976). 18 Lee SST and Scott JG, An improved method for preparation, stabilization, and storage of house fly (Diptera: Muscidae). J Econ Entomol 82:1559–1563 (1989). 19 Habig WH, Michael JP and Jakoby WB, Glutathione S-transferase: the first enzymatic step in mecapturic acid formation. J Biol Chem 249:7130–7139 (1974). 20 Van Asperen K, A study of housefly esterase by means of a sensitive colorimetric method. J Insect Physiol 8:401–406 (1962).
www.soci.org 21 Patterson M and Alyokhin A, Survival and development of Colorado potato beetles on potato treated with phosphite. Crop Prot 61:38–42 (2014). 22 George J, Redmond CT, Royalty RN and Potter DA, Residual effects of imidacloprid on Japanese beetle (Coleoptera: Scarabaeidae) oviposition, egg hatch, and larval viability in turfgrass. J Econ Entomol 100:431–439 (1997). 23 Yu SJ, Induction of detoxifying enzymes by allelochemicals and host plants in the fall armyworm. Pestic Biochem Physiol 19:330–336 (1983). 24 Johansen NS, Moen LH and Egaas E, Sterol demethylation inhibitor fungicides as disruptors of insect development and inducers of glutathione S-transferase activities in Mamestra brassicae. Comp Biochem Physiol C 145:473–483 (2007). 25 Obear GR, Williamson RC and Liesch PJ, Oviposition preference of the Japanese beetle (Coleoptera: Scarabaeidae) in golf putting greens under different soil moisture and fungicide regimes. Appl Turfgrass Sci 11. DOI:10.2134/ATS-2014-0034-RS (2014).
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Revised: 6 July 2015
Accepted article published: 14 July 2015
Published online in Wiley Online Library: 7 August 2015
(wileyonlinelibrary.com) DOI 10.1002/ps.4076
Fungicides affect Japanese beetle Popillia japonica (Coleoptera: Scarabaeidae) egg hatch, larval survival and detoxification enzymes Glen R Obear,a,b* Adekunle W Adesanya,c Patrick J Liesch,a R Chris Williamsona and David W Heldc Abstract BACKGROUND: Larvae of the Japanese beetle, Popillia japonica (Coleoptera: Scarabaeidae), have a patchy distribution in soils, which complicates detection and management of this insect pest. Managed turf systems are frequently under pest pressure from fungal pathogens, necessitating frequent fungicide applications. It is possible that certain turfgrass fungicides may have lethal or sublethal adverse effects on eggs and larvae of P. japonica that inhabit managed turf systems. In this study, eggs and first-, second- and third-instar larvae were treated with the fungicides chlorothalonil and propiconazole, and survival was compared with that of untreated controls as well as positive controls treated with the insecticide trichlorfon. RESULTS: Chlorothalonil reduced survival of first-instar larvae treated directly and hatched from treated eggs. Propiconazole delayed egg hatch, reduced the proportion of eggs that successfully hatched and reduced survival of first-instar larvae treated directly and hatched from treated eggs. Sublethal doses of the fungicides lowered the activities of certain detoxification enzymes in third-instar grubs. CONCLUSIONS: Fungicide applications to turfgrass that coincide with oviposition and egg hatch of white grubs may have sublethal effects. This work is applicable both to high-maintenance turfgrass such as golf courses, where applications of pesticides are more frequent, and to home lawn services, where mixtures of multiple pesticides are commonly used. © 2015 Society of Chemical Industry Keywords: white grubs; pesticides; P450; carboxylesterase; glutathione S-transferase
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Japanese beetle (Popillia japonica Newman) larvae, like other soil-dwelling larvae, have a patchy distribution in soils, which complicates detection and management. Female Japanese beetles are active during summer and have numerous periods of oviposition during their 4–6 week activity period.1,2 In the soil, females lay eggs which must absorb water to hatch successfully. Larvae spend 9–10 months in soil, and movement and survival are influenced by environmental conditions and turfgrass culture. When used, insecticides are commonly applied at or just before egg hatch (preventive) or when larger third-instar grubs are present (curative).2 Soil moisture from rainfall or supplemental irrigation of amenity grasses positively influences larval weight, abundance and development of Japanese beetle larvae.3 The presence and performance of white grubs are also affected by turfgrass culture.3 Japanese beetle larvae are rarely found in golf course putting greens, but infestations can occur in areas directly adjacent to putting greens with identical soils and plant characteristics. The impacts of mowing height vary, depending on species, but there is limited evidence that grass height is a significant factor in survival of Japanese beetle larvae.3,4 Golf course putting greens often receive the most agrochemical inputs among areas on golf courses Pest Manag Sci 2016; 72: 966–973
owing to high golfer standards and pest pressure, where numerous fungal diseases can damage turf under a variety of environmental conditions throughout the growing season.5 It is possible that commonly applied turfgrass fungicides may adversely affect larvae of P. japonica. Fungicides, although not intended to control insects, may cause physiological responses in insects. The chloronitriles, specifically the active ingredient chlorothalonil, and the demethylation inhibitor (DMI) fungicides are commonly and frequently applied to amenity turfgrass for disease management. The results of studies on insects with chloronitrile fungicides are varied, with some reporting toxicity of chlorothalonil6 and other studies reporting
∗
Correspondence to: GR Obear, Department of Agronomy and Horticulture, University of Nebraska – Lincoln, Lincoln, NE 68583, USA E-mail: [email protected]
a Department of Entomology, University of Wisconsin – Madison, Madison, WI, USA b Department of Agronomy and Horticulture, University of Nebraska – Lincoln, Lincoln, NE, USA c Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, USA
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Fungicides affect Japanese beetle egg hatch and larval survival no toxicity.7,8 The exact mechanism of toxicity is unknown, but chlorothalonil has been reported to bind to sulfhydryl enzymes, which are important for cellular respiration, bind and deplete cellular glutathione (GSH) and inhibit glycolysis by binding with glyceraldehyde 3-phosphate dehydrogenase (GAPDH).9 Demethylation inhibitor fungicides can decrease the activity of P450 enzymes in insects,10,11 which are important for growth, reproduction, hormone synthesis and detoxification of foreign chemical compounds.12 Annual bluegrass weevils, Listronotus maculicollis (Coleoptera: Curculionidae), a pest of cool-season turfgrass, that have prior exposure to DMI fungicides have increased susceptibility to synthetic pyrethroids. Resistance to pyrethroid insecticides is common among populations of annual bluegrass weevil in the northeastern United States, and fungicides that inhibit detoxification enzymes may increase the efficacy of insecticide applications.13 It is possible that fungicides applied to turfgrass may inhibit detoxification enzymes in other turfgrass insects. Furthermore, suppression of these enzymes may leave individuals more susceptible to insecticide applications that are otherwise not directly toxic.14 Japanese beetle grubs are facultative monophages2 and likely rely on multiple detoxification enzymes for the metabolism of xenobiotics such as plant toxins and pesticides.15 Ahmad16 reported that there is significant P450 activity in the third-instar grubs of Japanese beetle. It is unknown whether fungicides encountered by Japanese beetle grubs affect the activities of widely studied detoxification enzymes such as cytochrome P450, glutathione S-transferase (GST) and carboxylesterase (CoE). The overall objective of this study was to gain a better understanding of how two commonly applied turf fungicides affect eggs and larvae of the Japanese beetle. This study specifically examined the influence of propiconazole and chlorothalonil on immature Japanese beetle survival and their effect on the activities of detoxification enzymes in third-instar larvae.
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MATERIALS AND METHODS
2.1 Insect rearing and collection A series of laboratory assays were conducted to investigate the potential lethal effects of commonly applied fungicides labeled for use in managed turfgrass. Adult male and female Japanese beetles were collected in traps (Pherocon® JB trap; Trécé, Adair, OK) baited with a floral lure (2-phenyl-ethyl propionate, eugenol
www.soci.org and geraniol; 3:7:3 ratio) from field sites with no insecticide use for the preceding 12 months. Traps were hung on metal stands at a height of 1.5 m at 9:00 and collected at 15:00 on the same day. Eggs and first-instar larvae were reared in the laboratory. Adults from traps were emptied into six different 26 L plastic tubs containing moistened silt loam potting soil (River Run Products Corp., Custer, WI) with a depth of approximately 10 cm, and each tub contained approximately 1000 mixed-sex beetles. To collect eggs, adults were allowed 48 h to mate and oviposit into the soil, and then the soil in each tub was searched for eggs. First-instar larvae were collected using the same method, except the tubs with eggs were left undisturbed for 2 weeks so the eggs could hatch. Second- and third-instar larvae were collected from infested Poa pratensis turfgrass on golf courses in Wisconsin. The areas where larvae were collected had not been treated with pesticides for at least 12 months. 2.2 Experimental conditions and treatments Experimental treatments were delivered topically to eggs and larvae. The chemical treatments evaluated were two fungicides (propiconazole as Banner MAXX II, and chlorothalonil as Daconil Weather Stik; Syngenta Crop Protection, Greensboro, NC), a formulation blank (FB) or formulation less the active ingredient, an insecticide (trichlorfon, Dylox; Bayer Environmental Sciences, Research Triangle Park, NC) and an untreated control (Table 1). To simulate exposure in a field setting, application rates were calculated on the basis of the surface area of the bottom of the container (petri dish or plastic cup) that held the egg or larva. Both fungicides were prepared in solution using deionized water and mixed at the maximum labeled rate for controlling Sclerotinia homoeocarpa, a common fungal disease of turfgrass in the midwestern United States.5 Trichlorfon was prepared in solution using deionized water and mixed at the maximum labeled rate for white grubs. Control larvae were treated with deionized water. All chemical treatments and controls were replicated 20 times in two independent assays using different cohorts of eggs or larvae. All experiments were conducted using a completely randomized design in a controlled growth chamber set at 23 ∘ C and 50% relative humidity with no supplemental lighting. 2.3 Topical application to eggs and first-instar larvae Eggs or larvae were collected and placed individually in 47 mm diameter petri dishes containing moistened filter paper (Fisher
Table 1. Chemical treatments applied to eggs and first-, second- and third-instar larvae of the Japanese beetle Ratea, b Treatment Untreated control Banner MAXX IId Banner MAXX II FBe Daconil Weather Stikd Daconil Weather Stik FBe Dylox 420 SLf
Active ingredient – 14.3% propiconazole – 54% chlorothalonil – 37.3% trichlorfon
kg product ha−1
Solution concentrationc kg AI ha−1
– 6.9 6.9 13.8 13.8 24.8
– 1.0 – 7.5 – 9.3
mg product mL−1 – 2.0 2.0 3.9 3.9 6.9
mg AI mL−1 – 0.3 – 2.1 – 2.6
a All products prepared at maximum labeled rate for use on managed turfgrass. b Rate calculated on the basis of the area of the container holding individual eggs or larvae. c Applied at a volume of 0.7 mL per individual egg or larva. d Syngenta Crop Protection, Greensboro, NC.
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e Formulation blank (FB) contains the same inert ingredients without active ingredients. f Bayer Environmental Sciences, Research Triangle Park, NC.
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www.soci.org Scientific, Waltham, MA). Treatments were applied directly with a micropipette to the surface of eggs or the abdominal pleuron of the larvae at a volume of 0.7 mL. This application volume was selected because it was sufficient to cover eggs and larvae without leaving standing water in dishes. An additional 0.7 mL of deionized water was added to petri dishes to wet the filter paper and prevent desiccation. Eggs were monitored daily for hatch, and the date of egg hatch was recorded. Any eggs that had not hatched after 26 days were assumed to be dead. After hatching, 5 g of autoclaved silt loam potting soil was added to the petri dishes and moistened with 0.7 mL of deionized water. A slice of organic carrot was added to each dish at that time. Larvae or eggs were monitored daily and characterized as alive or dead. The assay was concluded when mortality in the control larvae exceeded 50% in the egg assay and 70% in the first-instar assay. 2.4 Topical application to second- or third-instar larvae Field-collected larvae were placed into individual 0.26 L cups with holes poked in the bottom for drainage. Larvae were acclimated for a 24 h period and then subjected to the same treatments as eggs and first-instar larvae (Table 1). Treatments were applied topically to the abdominal pleuron of each larva using the same methods and volume as previously described. Following treatment application, 30 g of autoclaved silt loam potting soil was added to each cup. An additional 1.2 mL of deionized water was added to each cup, followed by a slice of carrot for food. Holes were poked in plastic lids which were then placed on top of cups. Larvae were monitored daily and characterized as alive or dead, and the assay was concluded when mortality in the control larvae exceeded 40% in the second-instar assay and 50% in the third-instar assay. 2.5 Effects of chemical treatment on activities of detoxification enzymes in third-instar larvae 2.5.1 Topical treatment of grubs Third-instar larvae were placed into individual 47 cm diameter petri dishes with moistened filter paper and a slice of carrot as previously described. Larvae were treated with Banner MAXX II, Daconil Weather Stik and Dylox at the maximum labeled rates listed in Table 1. A control group was treated with deionized water. Treatments were applied to the abdominal pleuron via pipette with a water volume of 0.7 mL. An additional 0.7 mL of deionized water was added to petri dishes to moisten the filter paper and prevent desiccation. Larvae were removed from the growth chamber 24 h after treatment, flash frozen with dry ice and stored at −80 ∘ C until enzyme extractions were performed.
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2.5.2 Enzyme extraction and determination of protein content Following treatment, the bodies of four whole larvae for each of the chemical and control treatments (Table 1) were homogenized in 10 mL of ice-cold homogenization buffer [0.1 M phosphate buffer, pH 7.5, 10% glycerol, 1 mM of ethylenediaminetetraacetic acid (EDTA), 0.1 mM of dithiothreitol (DTT), 1 mM of phenylmethylsulfonyl fluoride (PMSF) and 1 mM of phenylthiourea (PTU)]. The homogenate was filtered through a funnel with a cheesecloth into a 15 mL centrifuge tube. The filtrate (homogenate) was centrifuged (AllegraTM 25R; Beckman Coulter, Inc., Brea, CA) at a speed of 10 000 × g for 30 min at 4 ∘ C using a type TA-14-50 rotor. The pellet was discarded, and the supernatant was carefully transferred into another centrifuge tube and spun in an ultracentrifuge equipped with a TI 865 rotor (Sorvall Discovery 90SE; Thermo Scientific, Waltham, MA) at a speed of 100 000 × g for 1 h at 4 ∘ C. The
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supernatant was used as the source of enzyme for GST and CoE activity assay, while the pellets (microsomes) were dissolved in a resuspension buffer (0.1 M phosphate buffer, pH 7.5, 20% glycerol, 1 mM of EDTA, 0.1 mM of DTT and 1 mM of PMSF). All the steps described were performed on ice. The microsomes and cytosol were temporarily stored in a −80 ∘ C freezer. The amount of protein present in each sample was determined using bovine serum albumin as the standard.17 A total of 3–4 determinations were done for each sample using a UV–vis spectrophotometer (Du 640; Beckman Coulter, Inc., Pasadena, CA).
2.5.3 Determination of P450 activity The activity of P450 was assayed using two chemical reactions, i.e. ethoxyresorufin O-demethylation (EROD) and ethoxycoumarin O-deethylation (ECOD) in a NADPH-dependent reaction using modified methods of Lee and Scott.18 A sample (30–50 μL) of microsomes and 40 μL of ECOD buffer (50 mM of Tris-base, 150 mM of KCl and 1 mM of EDTA, pH 7.8) containing 4 nM of 7-ethoxycoumarin (7-EC) were added to each well of a 96-well plate (black flat-bottom, BD Falcon; BD Biosciences, Franklin Lakes, NJ). For P450 EROD activity, another sample of 30–50 μL of microsomal fraction was added to a well, followed by the addition of 0.3 pmol of ethoxyresorufin. EROD buffer (0.1 M phosphate buffer, 0.1 mM of EDTA, 5 mM of MgCl2 , pH = 7.8) was added to each well for a final volume of 100 μL. Both P450 reactions were started with the addition of 10 μL of 0.01 M NADPH (freshly prepared) to each well, then incubated for 30 min at 30 ∘ C. For P450 ECOD, the fluorescence of NADPH was removed by adding 0.3 μM of oxidized glutathione and 0.5 units of glutathione reductase (Sigma-Aldrich, St Louis, MO) to each well. The reaction was stopped after 10 min at 25 ∘ C by adding 120 μL of stop solution (50% acetonitrile and 50% TRIZMA-base buffer). P450 ECOD activity is defined as the measured amount of 7-hydroxycourmarin produced at a wavelength of 390 nm and 465 nm for excitation and emission respectively. The fluorescence intensity for P450 EROD was measured at 530 and 580 nm for excitation and emission respectively. Control reactions used NADPH and substrate without the microsomal fraction. Product standard curves were also generated for resorufin and 7-hydroxycoumarin using the respective wavelengths for each product. P450 activity is expressed in pmol product formed min−1 mg−1 protein.
2.5.4 Determination of GST activity GST activity was assayed using the cytosolic fractions of the extract to catalyze the conjugation of glutathione (GSH) and chloro-2,4-dintrobenzene (CDNB) using a modification of Habig et al.19 For each reaction, freshly prepared GSH was dissolved in a phosphate buffer (0.1 M, pH 6.5) containing EDTA (1 mM), PMSF (1 mM) and DTT (1 mM). A quantity of 5 μL of the enzyme source was added to the GSH–buffer solution in a final volume of 200 μL (final GSH concentration 2.5 mM) in a 96-well plate. Reactions were incubated for 3 min at 25 ∘ C, after which 5 μL of 60 mM CDNB (dissolved in alcohol, freshly prepared) was added. The control reaction is the non-enzymatic reaction between GSH and CDNB under the same conditions as those used for the enzymatic reaction. The mixture was shaken, and the change in absorbance was measured for 5 min at a wavelength of 340 nm. GST activity was expressed as millimol CDNB conjugated min−1 mg−1 protein using an extinction coefficient of 5.3 mM−1 cm−1 for S-(2,4-dinitrophenyl) glutathione.
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Fungicides affect Japanese beetle egg hatch and larval survival 2.5.5 Determination of CoE activity CoE was determined using the hydrolysis of 𝛼- and 𝛽-naphthylacetate (𝛼- and 𝛽-NA) to 𝛼- and 𝛽-naphthol using the modified protocol of Van Asperen.20 The working concentration of the substrates (𝛼- and 𝛽-naphthylacetate) was 0.3 mM (containing 0.3 mM of eserine), diluted from their respective 0.03 M stock. Each assay reaction contained 60 μL of phosphate buffer (0.04 M, pH 7.0), 50 μL of the supernatant (enzyme source) and 80 μL of 0.3 mM substrate solutions. The reaction was incubated at 37 ∘ C for 30 min and stopped by the addition of 20 μL of stop solution (two parts 1% Fast Blue BB and five parts 5% sodium dodecyl sulfate). The colour developed at room temperature for 15 min, and then absorbance was measured at 600 nm for the hydrolysis of 𝛼-NA and at 550 nm for 𝛽-NA using a microplate reader (Cytation 3; Bio-Tek® , Winooski, VT). The product (𝛼- and 𝛽-naphthol) standard curve was generated by measuring optical absorbance at 600 and 550 nm at different concentrations. 2.6 Statistical analysis Analysis of variance (ANOVA) was used to determine whether chemical treatments significantly affected the length of time required for eggs to hatch, and a Student’s t-test was used to separate treatment means. A nominal logistic model was used to determine whether chemical treatments affected the total proportion of eggs that successfully hatched, and odds ratios were used to make pairwise comparisons between treatments. Data from each assay were subjected to survival analysis, and the Kaplan–Meier method was used to calculate survival functions for each treatment group. The log-rank test was used to test whether the survival functions were different among all treatment groups in each assay. The Holm–Sidak method was used to make multiple pairwise comparisons between survival functions. ANOVA was also used to test whether chemical treatment significantly affected the titers (activities) of P450, GST and CoE. Student’s t-test was employed to separate the means of each activity (P450, GST and CoE) using JMP software (v.11).
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RESULTS
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Table 2. Japanese beetle egg hatch timing and proportion affected by treatment with trichlorfon, propiconazole and chlorothalonil (data pooled from two cohorts; n = 240)a
Treatment Dyloxb Banner MAXX IIc Daconil Weather Stikd Untreated control Daconil FBe Banner FBe
Days to hatch 21.3 a 19.1 b 17.9 c 17.5 cd 17.3 d 17.1 d F 5,194 = 37.899, P < 0.001
Proportion of eggs hatched 0.60 b 0.625 b 0.85 a 0.925 a 0.90 a 0.95 a 𝜒 2 = 30.761, P < 0.001
a Numbers in columns followed by different letters are significantly different. b 37.3% trichlorfon. c 14.3% propiconazole. d 54% chlorothalonil. e Formulation blank (FB) contains the same inert ingredients without active ingredients.
P < 0.001), Daconil Weather Stik (𝜒 2 = 46.114, P < 0.001) and the Daconil Weather Stik FB (𝜒 2 = 70.574, P < 0.001). Larvae from eggs treated with Daconil Weather Stik began to perish 3 DAH, and survival was significantly reduced relative to the untreated control (𝜒 2 = 26.437, P < 0.001). Survival was also significantly lower from Daconil Weather Stik treatment compared with the Daconil Weather Stik formulation blank (𝜒 2 = 21.241, P < 0.001). Larvae from eggs treated with Banner MAXX II began to perish 5 DAH, and survival was significantly reduced relative to the untreated control (𝜒 2 = 13.153, P = 0.002). However, survival was not significantly different between Banner MAXX II treatment and the Banner MAXX II formulation blank (𝜒 2 = 1.707, P = 0.346). The Banner MAXX formulation blank reduced survival relative to the untreated control (𝜒 2 = 7.905, P = 0.024).
3.2 Survival of first-instar larvae following topical treatment Survival of first-instar larvae (n = 240) was significantly affected by chemical treatments (𝜒 2 = 243.962, P < 0.001). Untreated controls began to perish 7–8 days after treatment (DAT) (Fig. 1B). First-instar larvae treated with Dylox perished within 1–3 DAT, and survival was significantly reduced relative to the untreated control (𝜒 2 = 79.375, P < 0.001), to Banner MAXX II (𝜒 2 = 62.003, P < 0.001), to the Banner MAXX II FB (𝜒 2 = 79.255, P < 0.001), to Daconil Weather Stik (𝜒 2 = 79.560, P < 0.001) and to the Daconil Weather Stik FB (𝜒 2 = 83.638, P < 0.001). Larvae treated with Daconil Weather Stik began to perish 4 DAT, and survival was significantly reduced relative to the untreated control (𝜒 2 = 18.670, P < 0.001). Survival was also reduced with Daconil Weather Stik treatment relative to the Daconil Weather Stik formulation blank (𝜒 2 = 6.973, P = 0.072). Treatment with Banner MAXX II significantly reduced survival relative to the untreated control (𝜒 2 = 6.278, P = 0.083), but survival was not different from the Banner MAXX II FB (𝜒 2 = 0.0632, P = 0.801). The Banner MAXX formulation blank reduced survival relative to the untreated control (𝜒 2 = 6.582, P = 0.0795).
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3.1 Hatch and neonate survival following topical treatment of eggs Eggs treated with Dylox and Banner MAXX II took 2–4 days longer to hatch on average (F 5,194 = 37.899, P < 0.001) relative to untreated controls and the formulation blank (Table 2). The time required to hatch for eggs treated with Daconil Weather Stik was not significantly different from that of untreated controls or the formulation blank. Similarly, the total proportion of eggs treated with Dylox or Banner MAXX II that hatched was about 30% less (𝜒 2 = 30.761, P < 0.001) relative to untreated controls and the Banner MAXX II formulation blank. Daconil Weather Stik did not significantly reduce the proportion of eggs hatched relative to untreated controls or the Daconil Weather Stik formulation blank. Survival of first-instar larvae hatched from treated eggs (n = 195) was significantly affected by chemical treatments (𝜒 2 = 208.281, P < 0.001). Untreated controls began to perish 7 days after hatch (DAH) and began to decline rapidly around 9 DAH (Fig. 1A). First-instar larvae from eggs treated with Dylox perished within 1–2 DAH, and Dylox significantly reduced survival relative to the untreated control (𝜒 2 = 71.902, P < 0.001), Banner MAXX II (𝜒 2 = 39.041, P < 0.001), the Banner MAXX II FB (𝜒 2 = 72.524,
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Figure 1. Survival of Japanese beetle eggs and larvae treated topically with Dylox, Banner MAXX II or Daconil Weather Stik. Data from each life stage assay were pooled together from two cohorts. FB, formulation blank. (A) First-instar larvae hatched from treated eggs (n = 195; 𝜒 2 = 208.281, P < 0.001). Eggs that did not hatch were not included in this analysis. (B) First-instar larvae (n = 240; 𝜒 2 = 243.962, P < 0.001). (C) Second-instar larvae (n = 240; 𝜒 2 = 245.792, P < 0.001). (D) Third-instar larvae (n = 240; 𝜒 2 = 168.543, P < 0.001).
3.3 Survival of second-instar larvae following topical treatment The survival of second-instar larvae (n = 240) was significantly affected by chemical treatments (𝜒 2 = 245.752, P < 0.001). Untreated larvae began to perish 8 DAT and steadily declined until the assay was concluded at 23 DAT (Fig. 1C). Larvae treated with Dylox began to perish 1 DAT, and survival was significantly reduced relative to the untreated control (𝜒 2 = 87.373, P < 0.001), to Banner MAXX II (𝜒 2 = 87.373, P < 0.001), to the Banner MAXX II FB (𝜒 2 = 87.373, P < 0.001), to Daconil Weather Stik (𝜒 2 = 89.033, P < 0.001) and to the Daconil Weather Stik FB (𝜒 2 = 87.373, P < 0.001). Survival was not significantly reduced from treatment with Daconil Weather Stik (𝜒 2 = 0.674, P = 0.958), Banner MAXX II (𝜒 2 = 3.045, P = 0.570) or either formulation blank relative to the untreated control.
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3.4 Survival of third-instar larvae following topical treatment Survival of third-instar larvae (n = 240) was significantly affected by chemical treatments (𝜒 2 = 168.543, P < 0.001). Untreated controls began to perish 3 DAT and steadily declined until the assay was concluded 13 DAT (Fig. 1D). Larvae treated with Dylox began to perish 1 DAT, and survival was significantly reduced relative to
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the untreated control (𝜒 2 = 65.792, P < 0.001), to Banner MAXX II (𝜒 2 = 62.435, P < 0.001), to the Banner MAXX II FB (𝜒 2 = 53.019, P < 0.001), to Daconil Weather Stik (𝜒 2 = 72.786, P < 0.001) and to the Daconil Weather Stik FB (𝜒 2 = 61.269, P < 0.001). Survival was not significantly reduced from treatment with Daconil Weather Stik (𝜒 2 = 4.595, P = 0.278), Banner MAXX II (𝜒 2 = 1.413, P = 0.882) or either formulation blank relative to the untreated control. 3.5 Effects of chemical treatment on activities of detoxification enzymes in third-instar larvae Untreated third-instar grubs had the highest mean P450 ECOD and EROD activities compared with other treated grubs (Fig. 2). Banner MAXX and Dylox (F 3,32 = 5.07, P = 0.005) significantly reduced P450 ECOD activity but not Daconil Weather Stik relative to the untreated grubs. The chemical treatments also lowered P450 EROD activity, but only the reductions by Daconil Weather Stik and Dylox were significant (F 3,32 = 3.44, P = 0.028). Grubs treated with Banner MAXX had highest activity of GST (F 3,12 = 5.07, P = 0.0114), while there was no significant difference in mean GST activity among control grubs and grubs treated with Daconil Weather Stik and Dylox. There was a 2.38-fold increase in GST activity by Banner MAXX relative to untreated grubs (Fig. 3). Although the chemically treated grubs had lower CoE activity (Fig. 4),
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Figure 2. Mean (± SE) P450 ethoxycoumarin O-deethylation (ECOD) (upper) and ethoxyresorufin O-demethylation (EROD) (lower) activity in Japanese beetle third-instar grubs topically dosed with fungicides (Banner MAXX and Daconil) or insecticide (Dylox). ANOVA (F 3,32 = 5.07, P < 0.01). Bars with the same letter were not significantly different (Student’s t-test, 𝛼 = 0.05).
Figure 3. Mean (± SE) GST CDNB activity in Japanese beetle third-instar grubs topically dosed with fungicides (Banner MAXX and Daconil) or insecticide (Dylox). ANOVA (F 3,6 = 5.73, P < 0.01). Bars with the same letter above were not significantly different (Student’s t-test, 𝛼 = 0.05).
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DISCUSSION
The findings of our study provide evidence that both propiconazole and chlorothalonil may have lethal or sublethal adverse effects on eggs and larvae of P. japonica. Previous studies14,21 have also reported sublethal effects of fungicides on larval and adult Coleoptera and Hymenoptera respectively. In our study, Daconil Weather Stik reduced survival of first-instar larvae treated directly, and fewer first-instar larvae hatched from treated eggs compared
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only grubs that were exposed to Dylox had significantly lower CoE activity compared with untreated control grubs (F 3,6 = 8.4, P = 0.0144). Treatment with Banner MAXX and Daconil Weather Stik fungicides decreased CoE activity by 27 and 34% compared with untreated control grubs, while Dylox treatment reduced CoE activity by 72%. This evidence suggests that the formulated fungicide products both reduce activity of detoxification enzymes.
Figure 4. Mean (± SE) 𝛼-napthylacetate CoE activity of Japanese beetle third-instar grubs topically dosed with fungicides (Banner MAXX and Daconil) or insecticide (Dylox). ANOVA (F 3,6 = 8.4, P < 0.01). Bars with the same letter above were not significantly different (Student’s t-test, 𝛼 = 0.05).
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with the control. Survival was also reduced compared with the Daconil Weather Stik formulation blank, suggesting that the active ingredient chlorothalonil was responsible for the adverse effects. Banner MAXX II significantly increased the length of time required for egg hatch and reduced the proportion of successfully hatched eggs compared with the control and the Banner MAXX II formulation blank. This suggests that the active ingredient propiconazole was responsible for the adverse effects. Banner MAXX II also reduced survival of first-instar larvae treated directly or hatched from treated eggs, but survival in these assays was not significantly different from the Banner MAXX II formulation blank, suggesting that the formulation carrier had some adverse effect in addition to the active ingredient. In spite of evidence of adverse effects from both chlorothalonil and propiconazole, these chemicals do not appear to have utility as insecticides, considering that neither chemical reduced survival to the same degree as trichlorfon. However, these fungicides may result in mortality of P. japonica if applications coincide with periods of oviposition and egg hatch. In the present study, neither fungicide significantly reduced survival in older larvae (i.e. second- or third-instar larvae). Survival in response to trichlorfon was lower for all life stages, but mortality was delayed for third-instar larvae. Later instars of P. japonica are known to be more resilient to certain chemicals than early instars. For example, application of imidacloprid, which is lethal to first instars almost immediately after egg hatch, only causes behavioral impairment in second- and third-instar larvae.22 This may be due simply to an exposure response or an increasing biosensitivity to the chemicals. For example, a third-instar Japanese beetle grub has approximately 80 times the mass of a first-instar grub,1 which changes the physiological dose to the same application rate. However, fall armyworm larvae have decreased susceptibility to insecticides, as they age independently of dose.23 In the case of fall armyworm, detoxification enzyme activities increase with larval age. The present study used only third-instar Japanese beetle grubs; however, experiments are under way to characterize the life stage variability in enzyme activity in Japanese beetles. Untreated third-instar grubs had higher activities of P450 ECOD, EROD and CoE compared with treated grubs. This suggests that the tested dose of propiconazole, chlorothalonil or trichlorfon inhibited the activities of P450 and CoE, similarly to the findings of Darvas et al.10 and Brattsten et al.11 Banner MAXX II induced the activity of GST compared with other treatment groups, similarly to its reported effect on the caterpillar Mamestra brassicae (Lepidoptera: Noctuidae).24 The elevated GST activity in the presence of propiconazole may be metabolically expensive to Japanese beetle larvae and cause reductions in their fitness. Future studies could evaluate the consequences (e.g. weight gain or successful pupation) when fungicides are present during larval development. Propiconazole and chlorothalonil may have lethal or sublethal adverse effects on eggs and first-instar larvae of P. japonica. Enzymatic assays revealed that these fungicides inhibit the activity of detoxification enzymes of third-instar P. japonica larvae. However, neither fungicide affected oviposition choice by female Japanese beetles.25 This work is relevant to high-maintenance turfgrass sites that either receive frequent application of fungicides, such as golf courses, or where multiple pesticides are mixed together for convenience (lawns). Fungicide applications, especially when coinciding with oviposition and egg hatch, may cause significant local mortality of P. japonica. However, the findings from this study need to be validated in a field setting. In this study, application rates
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were representative of those applied in the field setting, but eggs and larvae likely received a greater exposure than when exposed in context. Soil processes, including binding of chemicals by soil as well as microbial degradation of chemicals over time, could serve to reduce exposure in a field setting.
ACKNOWLEDGEMENTS We thank Jerry Kershasky of University Ridge Golf Course, Bill Kohoss of Coldwater Canyon Golf Course and Jon Hegge of Evansville Golf Club for allowing us to collect grubs from their facilities. We also thank Rebecca Le Beau, Andrew Le Beau, Joshua Horman and John Gillis for providing technical assistance. Drs Douglas J Soldat, Daniel K Young and Zachary J Reicher reviewed an earlier version of this manuscript. This paper is based upon work supported by the Alabama Agricultural Experiment Station through Hatch funding from the National Institute of Food and Agriculture, United States Department of Agriculture. The Wisconsin Turfgrass Association provided additional financial support.
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