Journal of Economic Entomology Advance Access published December 26, 2015 Journal of Economic Entomology, 2015, 1–5 doi: 10.1093/jee/tov348 Insecticide Resistance and Resistance Management Research article Monitoring for Insecticide Resistance in Asian Citrus Psyllid (Hemiptera: Psyllidae) Populations in Florida Lambert H. B. Kanga,1,2 Julius Eason,1 Muhammad Haseeb,1 Jawwad Qureshi,3 and Philip Stansly3 1Center for Biological Control, CAFS, Florida A&M University, Tallahassee, FL 32307 ([email protected]; [email protected] com; [email protected]), 2Corresponding author, e-mail: [email protected], and 3Department of Entomology and Nematology, Southwest Florida Research and Education Center, University of Florida, 2685 State Road 29 North, Immokalee, FL 34142 (jawwadq@ufl.edu; pstansly@ufl.edu) Received 30 July 2015; Accepted 15 November 2015 Downloaded from Abstract The development of insecticide resistance in Asian citrus psyllid, Diaphorina citri Kuwayama, populations is a serious threat to the citrus industry. As a contribution to a resistance management strategy, we developed a glass vial technique to monitor field populations of Asian citrus psyllid for insecticide resistance. Diagnostic concentrations needed to separate susceptible genotypes from resistant individuals were determined for http://jee.oxfordjournals.org/ cypermethrin (0.5 lg per vial), malathion (1.0 lg per vial), diazinon (1.0 lg per vial), carbaryl (1.0 lg per vial), carbofuran (0.1 lg per vial), methomyl (1.0 lg per vial), propoxur (1.0 lg per vial), endosulfan (1.0 lg per vial), imidacloprid (0.5 lg per vial), acetamiprid (5.0 lg per vial), chlorfenapyr (2.5 lg per vial), and fenpyroximate (2.5 lg per vial). In 2014, resistance to two carbamate insecticides (carbaryl and carbofuran), one organophos- phate (malathion), one pyrethroid (cypermethrin), and one pyrazole (fenpyroximate) was detected in field populations of Asian citrus psyllid in Immokalee, FL. There was no resistance detected to diazinon, methomyl, propoxur, endosulfan, imidacloprid, and chlorfenapyr. The levels of insecticide resistance were variable and unstable, suggesting that resistance could be successfully managed. The results validate the use of the glass vial bioassay to monitor for resistance in Asian citrus psyllid populations and provide the basis for the develop- by guest on May 11, 2016 ment of a resistance management strategy designed to extend the efficacy of all classes of insecticides used for control of the Asian citrus psyllid. Key words: Asian citrus psyllid, Diaphorina citri, monitoring, insecticide resistance The presence of the Asian citrus psyllid, Diaphorina citri the most serious problem facing the entire citrus industry (Bove´ Kuwayama (Hemiptera: Psyllidae), has seriously affected citrus pro- 2006). Currently, chemical control of the psyllid vector is considered ductivity and profitability in Florida. The Asian citrus psyllid is a one of the most effective methods to manage this pest in citrus. vector of the lethal bacterial disease, huanglongbing (HLB), also Several classes of insecticides (pyrethroid, organophosphate, neoni- known as “citrus greening.” Infected plants exhibit nonsymptomatic cotinoid, or carbamate) have been used extensively to control the and symptomatic characteristics (Halbert and Manjunath 2004). vector. Frequent applications of conventional and microbial pesti- The disease infiltrates vascular tissue of the plants, which results in cides have led to the development of insecticide resistance in Asian blockages that disrupt the flow of nutrients from the leaves to roots. citrus psyllid populations (Tiwari et al. 2011, Wells and Stelinski The leaves on infected citrus express chlorosis, and the fruits are sig- 2011). nificantly reduced in size, misshapen, or lopsided, and retain a sour Therefore, there is an urgent need to assess the incidence and or bitter taste. Infected citrus trees display stunted growth, offseason severity of insecticide resistance in field populations of Asian citrus bloom, die-back, and death (Bove´2006). These HBL-infected trees psyllid and develop a resistance management strategy for Asian cit- typically die within 5–6 yr of infection. The average infection rate in rus psyllid that is designed to extend the efficacy of all classes of Florida is estimated to be 1.6% but can reach up to 100% in the insecticides used for control. southern and eastern parts of the state, and currently, there is no In this study, we report on the selection of diagnostic concentra- effective treatment of the disease (Tiwari et al. 2010). tions of insecticides for use in resistance monitoring and present Although relatively new to North America, citrus greening is results on monitoring Asian citrus psyllid populations for resistance considered the oldest and the most destructive disease of citrus and to several insecticides. VC The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: [email protected] 1 2 Journal of Economic Entomology, 2015, Vol. 0, No. 0 Materials and Methods resistant phenotypes and reflected more accurately the resistance gene frequencies (Kanga et al. 1999). Concentration–response Insecticides regressions for each strain and each insecticide were calculated with All insecticides were technical-grade samples (>98% purity). These five or six doses of insecticides (plus an acetone control) with 10–12 include three pyrethroids (cypermethrin, b-cyfluthrin, and fenpropa- replicates of five psyllids per vial. In total, 11,545 psyllids were thrin), four carbamates (carbaryl, carbofuran, propoxur, and tested during these experimental runs. methomyl), one cyclodiene (endosulfan), and three organophos- phates (diazinon, malathion, and dimethoate). The carbamates were purchased from Chemical Service (West Chester, PA), and the Monitoring for Resistance pyrethroid, cyclodiene, and organophosphate insecticides from Assessments of the levels of resistance in field populations of Asian Sigma-Aldrich (St. Louis, MO). The other insecticides tested citrus psyllid were conducted using techniques as described by included four neonicotinoids (imidacloprid, acetamiprid, clothiani- Kanga et al. (1999, 2010). Asian citrus psyllids were collected from din, and thiamethoxam), one pyrazole (fenpyroximate), and one citrus groves in summer of 2014 and brought to the laboratory. pyrrole (chlorfenapyr) that were purchased from Sigma-Aldrich. The psyllids were exposed to a diagnostic dose of an insecticide in treated vials to determine the frequencies of resistance. During an experimental run, between 60 and 85 psyllids were tested at each Insects diagnostic concentration of insecticides in glass vials along with The susceptible culture (S-LAB) of adult Asian citrus psyllids came control vials treated with acetone only. Mortality was recorded after from a population continuously reared at the Southwest Florida 24 h exposure as described above. Research and Education Center (SWFREC), University of Florida. The culture was established in 2006 using field populations which Statistical Analyses Downloaded from had not been subjected to insecticides. The culture was maintained The concentration–mortality data were subjected to Probit analysis on orange jasmine, Murraya paniculata (L.) Jack (Sapindales: (POLO-PC, Le Ora Software, Berkeley, CA) (Russell et al. 1977). Rutaceae), without exposure to insecticides in an air-conditioned Data were corrected for control mortality using Abbott’s (1925) for- glasshouse maintained at 27 6 4C and 70 6 10% relative humidity mula. Differences among populations in response to insecticides (RH) under natural light. The field populations (R-IMOK) were a were considered not significant if the 95% confidence limit (CL) of mixture of susceptible and resistant adult Asian citrus psyllids. They http://jee.oxfordjournals.org/ the resistance ratio at the LC bracketed 1.0 (Robertson and were collected using an aspirator from commercial citrus groves in 50 Priesler 1992). Resistance ratios were obtained by dividing the LC Immokalee, FL (26 4100400 N, 81 2602000 W). They were trans- 50 of the resistant population by that of the susceptible population. ported to the laboratory at SWFREC, placed in plastic mesh cages (BioQuip, Rancho Dominguez, CA), and fed on young seedlings of Citrus macrophylla Wester until used for bioassays. Results Laboratory Bioassays Insecticide Bioassay Levels of insecticide resistance in Asian citrus psyllid populations by guest on May 11, 2016 The procedure used in this study was described by Kanga and Plapp varied within and between classes of insecticides tested (Table 1). (1995). Briefly, glass scintillation vials (20 ml) were treated with The responses of susceptible (S-LAB) and field-collected (R-IMOK) 0.5-ml solutions of insecticides in acetone, and the vials were hand Asian citrus psyllid populations to one pyrethroid (cypermethrin), rolled until acetone evaporated and the insecticides coated evenly on an organophosphate (malathion), two carbamates (carbaryl and the inner surfaces. Control vials were treated with acetone only. carbofuran), and one pyrazole (fenpyroximate) were statistically dif- 1 8 Insecticide dilution ratios from 1:10 to 1:10 were tested, and vials ferent based on the failure of the 95% CL of the resistance ratio to treated with a concentration of insecticides were replicated a mini- bracket 1.0 (Robertson and Preisler 1992). Data indicated variable mum of