PHYSIOLOGICAL ECOLOGY
Mechanism of Resistance Acquisition and Potential Associated Fitness Costs in Amyelois transitella (Lepidoptera: Pyralidae) Exposed to Pyrethroid Insecticides MARK DEMKOVICH,1,2 JOEL P. SIEGEL,3 BRADLEY S. HIGBEE,4 AND MAY R. BERENBAUM1
Environ. Entomol. 44(3): 855–863 (2015); DOI: 10.1093/ee/nvv047
ABSTRACT The polyphagous navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae), is the most destructive pest of nut crops, including almonds and pistachios, in California orchards. Management of this insect has typically been a combination of cultural controls and insecticide use, with the latter increasing substantially along with the value of these commodities. Possibly associated with increased insecticide use, resistance has been observed recently in navel orangeworm populations in Kern County, California. In studies characterizing a putatively pyrethroid-resistant strain (R347) of navel orangeworm, susceptibility to bifenthrin and b-cyfluthrin was compared with that of an established colony of susceptible navel orangeworm. Administration of piperonyl butoxide and S,S,S-tributyl phosphorotrithioate in first-instar feeding bioassays with the pyrethroids bifenthrin and b-cyfluthrin produced synergistic effects and demonstrated that cytochrome P450 monooxygenases and carboxylesterases contribute to resistance in this population. Resistance is therefore primarily metabolic and likely the result of overexpression of specific cytochrome P450 monooxygenases and carboxylesterase genes. Resistance was assessed by median lethal concentration (LC50) assays and maintained across nine generations in the laboratory. Life history trait comparisons between the resistant strain and susceptible strain revealed significantly lower pupal weights in resistant individuals reared on the same wheat bran-based artificial diet across six generations. Time to second instar was greater in the resistant strain than the susceptible strain, although overall development time was not significantly different between strains. Resistance was heritable and may have an associated fitness cost, which could influence the dispersal and expansion of resistant populations in nut-growing areas in California. KEY WORDS Amyelois transitella, detoxification, insecticide, resistance, pyrethroid The navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae), is considered the most destructive pest of introduced nut crops in California orchards (Connell 2001, Bentley et al. 2008, Zalom et al. 2012). The geographic range of navel orangeworm extends from the southern tier of the United States, through Mexico and Central America, and into South America (Heinrich 1956). Although this insect pest was initially described as feeding on fallen fruits of Citrus sinensis (L.) Osbeck (Rutaceae), the introduction of additional nonindigenous fruit crops allowed the navel orangeworm to expand its range and establish itself as a pest in California orchards by the 1940s (Wade 1961). Navel orangeworm causes economic damage to almonds (Prunus dulcis Batsch), pistachios (Pistacia vera L.), figs (Ficus carica L.), pomegranates (Punica granatum L.), and walnuts (Juglans spp.) in the Central Valley of California, demonstrating its capacity to
1 Department of Entomology, University of Illinois at UrbanaChampaign, Urbana, IL, 61801. 2 Corresponding author, e-mail:
[email protected]. 3 USDA-ARS, San Joaquin Valley Agricultural Sciences Center, Parlier, CA, 93648. 4 Paramount Farming Company, 6801 E. Lerdo Highway, Shafter, CA, 93263.
consume host plants with distinctly different chemistries (Bentley et al. 2008). Navel orangeworm is an internal feeder with a preference for fallen and mummy (unharvested) fruit or injured and diseased fruits (Heinrich 1956). Neonates tunnel into nuts, resulting in consumption of the nutmeat and the production of large quantities of frass and webbing. Infestation by navel orangeworm results in an increased susceptibility to infection by Aspergillus species, many of which produce aflatoxins, a carcinogenic crop contaminant that causes millions of dollars in losses each year (Campbell et al. 2003, Molyneux et al. 2007, van Egmond et al. 2007, Palumbo et al. 2014). Management of this insect pest has typically been a combination of cultural control (removal of unharvested fruits) and insecticides, but insecticide use has increased substantially along with the value of these commodities. Usage of bifenthrin, one of the top five most frequently applied insecticides in almond orchards, increased 14.5-fold in almonds from 2007 to 2010 and 9.3-fold in pistachios from 2008 to 2010 (California Department of Pesticide Regulation [CDPR] 2010). Insecticides sprayed to control navel orangeworm in heavily infested orchards are usually applied in rotation when the hulls split and the kernel is exposed to both larval feeding and infection by
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Aspergillus species (Niu et al. 2012). Insecticide rotational practices may enhance the risk of resistance acquisition if rotated insecticides share a common route of detoxification. Navel orangeworm resistance to standard applications of the pyrethroid insecticide bifenthrin has recently been reported in almond orchards in Kern Country, California (B. Higbee, Paramount Farming Company, personal observation). The primary mechanisms involved in insecticide resistance to pyrethroids involve target site insensitivity, increased metabolism, or a combination of both (Khambay and Jewess 2005, Li et al. 2007, Feyereisen 2011). Target site resistance to pyrethroids occurs through the inheritance of point mutations in the para-type sodium channels, which are the binding sites for this insecticide class (Casida et al. 1983, Davies et al. 2007). Metabolic resistance often occurs through contributions of cytochrome P450 monooxygenases (P450s), glutathione-S-transferase, and carboxylesterase (COE) enzymes (Feyereisen 2005, Oakeshott et al. 2005, Ranson and Hemingway 2005, Liu 2011) and results in a decrease in the effective dose of insecticide available at the target site. The acquisition of resistance to synthetic insecticides often carries an associated fitness cost when resources generally directed toward fitness-enhancing traits are redirected toward production and maintenance of resistance (Carrie`re et al. 1994). Costs associated with insecticide resistance such as increased development time of larval and pupal stages have been observed in many lepidopteran pests, including the tobacco budworm (Heliothis virescens) (F.) (Lepidoptera: Noctuidae) (Sayyed et al. 2008), the Old World cotton bollworm (Helicoverpa armigera) (Hu¨bner) (Lepidoptera: Noctuidae) (Wang et al. 2010), and the obliquebanded leafroller (Choristoneura rosaceana) (Harris) (Lepidoptera: Tortricidae) (Carrie`re et al. 1994). A compelling form of evidence for the contribution of an enzyme system in resistance is enhancement of toxicity in the presence of an insecticide synergist that compromises the enzyme in question (Feyereisen 2011). In this experiment, the synergists piperonyl butoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF) were assessed in bioassays with the pyrethroids bifenthrin and b-cyfluthrin to determine if P450s or COEs, respectively, contribute to pyrethroid resistance in a field-derived population of navel orangeworm (R347) from Kern County, California. A susceptible laboratory colony (CPQ) of navel orangeworm was used to compare the effects of the synergists and to calculate resistance level differences. Life history traits were compared in these two strains to identify possible fitness costs associated with the initial development of resistance to pyrethroids and to determine if resistance was maintained in the absence of bifenthrin selection pressure. Materials and Methods Chemicals. PBO was purchased from Tokyo Kasei Kogyo (Tokyo, Japan). b-Cyfluthrin, acetone, and methanol were purchased from Sigma (St. Louis, MO).
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Bifenthrin and S,S,S-tributyl phosphorotrithioate were purchased from Chem Service Inc. (West Chester, PA). Methanol was used as a solvent for bifenthrin, and acetone as a solvent for b-cyfluthrin. All stock solutions were stored at 20 C. Insects. A susceptible colony of A. transitella designated as CPQ (J. Siegel, U.S. Department of AgricultureAgricultural Research Service [USDAARS], Parlier, CA) and a resistant colony designated as R347 (B. Higbee, Paramount Farming Company, Bakersfield, CA) were kept in an incubator and maintained at conditions of 27 6 4 C without light. Adults from the R347 population were collected in the field, and the colony was reared for two generations before experiments were initiated. Larvae were mass-reared until pupation in 500-ml Mason jars containing 200 g of a wheat bran-based artificial diet modified from Finney and Brinkman (1967). For every 1,000 g of wheat bran (Great River Organic Milling, Fountain City, WI), 360 ml of honey, 360 ml of glycerin, 320 ml of water, 40 g of brewer’s yeast, and 4 ml of Vanderzant vitamins (1%) solution were added. Fifty larvae were reared in each jar. Adults were transferred to additional 500-ml Mason jars with tissue paper on the inside and covering the top to serve as an oviposition substrate. Eggs were collected every 48 h from these jars. First-instar larvae were selected within 24 h of egg hatch. Four larvae were gently transferred into each 28-ml (1-oz) plastic cups containing 5 g of unaugmented (control) diet or diet mixed with different concentrations of insecticide in the bioassays. In total, 20 larvae (five cups) were prepared for each treatment with or without insecticide application. Insecticide Preparation. Insecticides were mixed in with a standard semisynthetic artificial diet and then poured into separate 28-ml (1-oz) cups to harden (Niu et al. 2012). The concentrations tested in the susceptible strain were 50, 200, 300, and 500 ng/g for bifenthrin and 50, 100, 150, 500 ng/g, and 1 m/g for b-cyfluthrin. The concentrations tested in the resistant strain were 500 ng/g, 1, 1.5, 2, and 4 mg/g for bifenthrin and 50, 100, 500 ng/g, 1, and 2 m/g for b-cyfluthrin. In each bioassay, 20 neonates were exposed to each concentration of insecticide. Mortality levels were assessed and recorded at each concentration after 48 h. Bioassays were replicated three times. Larvae that did not move when touched with a soft brush were scored as dead. Bioassays with Synergists. Median lethal concentrations determined for b-cyfluthrin and bifenthrin within each strain of navel orangeworm were mixed into the artificial diet in the presence or absence of PBO or DEF. A concentration of 200 mg/g for PBO was previously established by Niu et al. (2012) as the maximum concentration determined to be nonlethal for first-instar larvae. A concentration of 100 mg/g was used for DEF because it produced Fig. 1). For bifenthrin, the LC50 was on average 8.7-fold greater (ranging from 6.2- to 11.7-fold) in the resistant strain than in the susceptible strain, and for b-cyfluthrin, it was 11-fold greater (ranging from 4.7- to 17.8-fold) in the resistant strain than in the susceptible strain. The relationships between mortality and time in assays with pyrethroids are described in Table 1. In the presence of insecticide, the relationship between mortality and time was logarithmic, while the relationship between mortality and time in the controls was either linear or flat. In the CPQ bifenthrin assays, mortality levels differed (P < 0.0001) between insecticide treatments and the control across all time points examined (cmagenta>Fig. 2A). Both PBO and DEF synergized the toxicity of bifenthrin at 72 h through 144 h (F 84.198; df ¼ 3, 310; P 0.0001). The treatments including either PBO or DEF with bifenthrin did not
Fig. 1. Median lethal concentration (LC50) values in a susceptible (CPQ) and a resistant (R347) (F4) strain of navel orangeworm after 48 h for the pyrethroid insecticides bifenthrin and b-cyfluthrin. Error bars represent the 95% confidence limits in the LC50 for each insecticide.
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Table 1. Regression data for bioassays monitoring mortality over time with the pyrethroids bifenthrin and b-cyfluthrin and the synergists PBO and DEF in the susceptible strain (CPQ) and the resistant strain (R347) Treatment CPQ Bifenthrin Control CPQ Bifenthrin CPQ Bifenthrin þ PBO CPQ Bifenthrin þ DEF R347 Bifenthrin Control R347 Bifenthrin R347 Bifenthrin þ PBO R347 Bifenthrin þ DEF CPQ b-Cyfluthrin Control CPQ b-Cyfluthrin CPQ b-Cyfluthrin þ PBO CPQ b-Cyfluthrin þ DEF R347 b-Cyfluthrin Control R347 b-Cyfluthrin R347 b-Cyfluthrin þ PBO R347 b-Cyfluthrin þ DEF
Equation
R2
F
P
Mortality ¼ 0.027 þ 0.001 time Mortality ¼ 0.039 þ 0.342 log (time þ 1) Mortality ¼ 0.008 þ 0.475 log (time þ 1) Mortality ¼ 0.004 þ 0.434 log (time þ 1) Mortality ¼ 0.005 þ 0.001 time Mortality ¼ 0.014 þ 0.252 log (time þ 1) Mortality ¼ 0.035 þ 0.462 log (time þ 1) Mortality ¼ 0.047 þ 0.401 log (time þ 1) Mortality ¼ 0.024 þ 0.001 time Mortality ¼ 0.046 þ 0.270 log (time þ 1) Mortality ¼ 0.027 þ 0.463 log (time þ 1) Mortality ¼ 0.042 þ 0.385 log (time þ 1) Mortality ¼ 0.018 þ 0.0001 time Mortality ¼ 0.037 þ 0.244 log (time þ 1) Mortality ¼ 0.073 þ 0.367 log (time þ 1) Mortality ¼ 0.073 þ 0.391 log (time þ 1)
0.268 0.736 0.936 0.910 0.263 0.745 0.871 0.877 0.219 0.846 0.890 0.858 0.047 0.773 0.821 0.831
(1, 54) ¼ 19.805 (1, 26) ¼ 72.585 (1, 26) ¼ 379.073 (1, 26) ¼ 261.362 (1, 26) ¼ 9.259 (1, 26) ¼ 75.953 (1, 26) ¼ 175.793 (1, 26) ¼ 184.961 (1, 54) ¼ 15.165 (1, 26) ¼ 142.970 (1, 26) ¼ 211.057 (1, 26) ¼ 156.973 (1, 26) ¼ 1.279 (1, 26) ¼ 88.681 (1, 26) ¼ 118.980 (1, 26) ¼ 127.379
