Monitoring for Insecticide Resistance in Asian Citrus Psyllid (Hemiptera: Psyllidae) Populations in Florida

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

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 organophosphate (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.

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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 organophosphates (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 10 times during an experimental run. Five psyllids were levels of responses to different types of organophosphorus insecti- placed in each control and treated vial and held at room temperature cides with 5.7-fold resistance to a phosphorodithioate (malathion), (27 6 1 C) and 65% RH; the vial served as a replicate for each con- but only 1.0-fold tolerance to a phosphorothioate (diazinon) and centration tested. Mortality was determined 24 h after exposure. 0.44-fold tolerance to another phosphorodithioate (dimethoate). Psyllids unable to walk (>5 mm) after probing with a fine brush Similarly, there were variable levels of responses to carbamate insec- were recorded as dead. ticides [i.e., 1.5-fold tolerance to oxime carbarmate (methomyl); 2.14-fold and 2.08-fold resistance to the aryl carbamates, carbaryl Diagnostic Concentrations for Resistance Monitoring and carbofuran, respectively; and 1.1-fold tolerance to propoxur]. Susceptible (S-LAB) and resistant (R-IMOK) psyllid populations There was no resistance to the neonicotinoids imidacloprid were exposed to insecticides to establish diagnostic concentrations, (0.34-fold), acetamiprid (0.43-fold), clothianidin (0.94-fold), and which were estimated from the concentration–mortality responses. thiamethoxam (1.1-fold). Field populations of Asian citrus psyllid Where the concentration–response regression lines for the suscepti- were resistant to the pyrazole fenpyroximate (2.5-fold), and susceptible to the pyrrole chlorfenapyr (0.86-fold). ble and resistant populations did not overlap, we used the LC99 of the susceptible populations as the diagnostic concentration. At that concentration, almost all susceptible individuals were killed and Diagnostic Concentrations for Resistance Monitoring only resistant phenotypes survived. Where the concentration– for Conventional Insecticides response regression lines for susceptible and resistant strains The responses of S-LAB and R-IMOK populations of Asian citrus overlapped, we used an approximate LC85–95 for the susceptible psyllid to each insecticide were used to select diagnostic concentra- populations as the diagnostic concentration. In this range, most tions for resistance monitoring (Table 2). These concentrations were resistant insects survived and most susceptible insects were killed. chosen to maximize differences in responses between susceptible However, this lower concentration allowed more survival of and resistant psyllids (McCutchen et al. 1989). The concentrations Journal of Economic Entomology, 2015, Vol. 0, No. 0 3

Table 1. Responses of susceptible (S-LAB) and ﬁeld-collected (R-IMOK) Asian citrus psyllid populations to different insecticides

a b c Insecticide Strain N Slope 6 SEM LC50 (95%CL) RR

Pyrethroid Cypermethrin S-LAB 264 1.19 6 0.18 0.008 (0.001–0.02) – R-IMOK 257 1.43 6 0.22 0.020 (0.004–0.038) 2.5 (1.21–5.26) b-Cyﬂuthrin S-LAB 338 1.96 6 0.27 0.001 (0.0009–0.002) – R-IMOK 330 1.69 6 0.25 0.002 (0.001–0.003) 2.0 (0.96–4.21) Fenpropathrin S-LAB 296 1.30 6 0.138 0.028 (0.016–0.053) – R-IMOK 287 0.903 6 0.139 0.056 (0.005–0.037) 2.0 (0.87–3.98) Organophosphate Diazinon S-LAB 367 1.50 6 0.29 0.074 (0.008–0.241) – R-IMOK 306 1.27 6 0.22 0.075 (0.004–0.279) 1.0 (0.56–2.04) Dimethoate S-LAB 309 3.34 6 0.61 0.197 (0.086–0.308) R-IMOK 300 3.420 6 0.66 0.087 (0.066–0.118) 0.44 (0.14–0.88) Malathion S-LAB 419 3.91 6 0.96 0.448 (0.31–0.58) – R-IMOK 250 4.07 6 0.75 2.56 (1.90–3.33) 5.7 (4.22–7.33) Carbamate Carbaryl S-LAB 356 3.6 6 1.45 0.15 (0.056–0.269) – R-IMOK 319 2.82 6 0.69 0.336 (0.154–0.471) 2.14 (1.20–4.28) Carbofuran S-LAB 360 1.64 6 0.22 0.012 (0.003–0.474) –

R-IMOK 263 2.10 6 0.50 0.025 (0.003–0.121) 2.08 (1.18–4.24) Downloaded from Methomyl S-LAB 373 1.29 6 0.19 0.077 (0.026–0.132) – R-IMOK 287 1.60 6 0.45 0.119 (0.0312–0.423) 1.5 (0.08–0.31) Propoxur S-LAB 369 1.72 6 0.22 0.16 (0.018–0.425 – R-IMOK 303 2.21 6 0.33 0.18 (0.02–0.417) 1.1 (0.62–2.20) Cyclodiene

Endosulfan S-LAB 364 1.02 6 0.12 0.068 (0.039–0.122) – http://jee.oxfordjournals.org/ R-IMOK 350 1.77 6 0.22 0.025 (0.015–0.041) 0.37 (0.21–0.85) Neonicotinoid Imidacloprid S-LAB 310 1.35 6 0.20 0.035 (0.01–0.124) – R-IMOK 289 1.71 6 0.13 0.012 (0.004–0.04) 0.34 (0.16–0.72) Acetamiprid S-LAB 415 6.10 6 2.51 1.923 (1.13–3.966) – R-IMOK 358 1.46 6 0.28 0.818 (0.404–1.246) 0.43 (0.24–0.88) Clothianidin S-LAB 257 2.29 6 0.43 0.018 (0.0009–0.002) – R-IMOK 248 2.09 6 0.39 0.017 (0.005–0.015) 0.94 (0.60–1.93)

Thiamethoxam S-LAB 352 1.05 6 0.27 0.021 (0.012–0.030) – by guest on May 11, 2016 R-IMOK 349 1.24 6 0.25 0.023 (0.013–0.037) 1.1 (0.54–2.45) Pyrazole Fenpyroximate S-LAB 482 1.25 6 0.20 0.120 (0.078–0.173) – R-IMOK 574 1.04 6 0.25 0.301 (0.208–0.409) 2.5 (1.41–4.76) Pyrrole Chlorfenapyr S-LAB 417 3.038 6 0.063 0.556 (0.212–0.991) R-IMOK 427 1.28 6 0.247 0.477 (0.10–0.92) 0.86 (0.46–1.64)

a Number of Asian citrus psyllids tested. b Concentrations are expressed in micrograms of the insecticides per vial. c Resistance ratio (RR) calculated by dividing the LC50 for R-IMOK by the LC50 for the S-LAB strain.

of 0.5 and 1.0 lg per vial (LC95–99) were found to be discriminating in the field. This concentration should maximize the response to insec- concentrations for the pyrethroid cypermethrin; however, a single ticides of mixed populations of susceptible and resistant psyllids. concentration of 1.0 lg of cypermethrin per vial was our best choice Similarly, for the cyclodiene endosulfan, 1.0 lg per vial (LC99)was for resistance monitoring because all susceptible individuals were the best single concentration for tolerance monitoring (Fig. 4). killed and resistance phenotypes survived at this concentration For the insecticides where resistance was not evident, the concen- (Fig. 1). For the organophosphorus insecticide malathion, concentra- tration-regression lines for S-LAB and R-IMOK psyllid populations tions of 1.0 and 2.5 lg per vial (LC95–99) could be used to monitor for overlapped. The patterns of responses of Asian citrus psyllid indi- resistance in field populations of Asian citrus psyllid (Fig. 2)andthe cated that concentrations at LC95–99 could be used to monitor for single concentration, 1.0 lg of malathion per vial (LC95), was chosen. tolerance to these insecticides. For the carbamate carbaryl (Fig. 3), data suggested that concentra- For the neonicotinoid insecticide imidacloprid, concentrations of tions of 0.5–1.0 lg per vial (LC95–99) provided an indication of resist- 0.5 and 1.0 lg per vial (LC95–99) could be used as diagnostic concen- ance; however, a single concentration of 1.0 lg of carbaryl per vial trations, with 0.5 lg per vial chosen for resistance monitoring was the best choice, as it could separate heterozygous and homozy- (Fig. 5). Similarly, data suggest that concentrations of 5.0 and gous individuals (Kanga et al. 1999). For the oxime carbamates 10.0 lg per vial (LC95–99) will separate susceptible from resistant methomyl and propoxur, a concentration of 1.0 lg per vial (LC99) individuals for acetamiprid. For the pyrrole chlorfenapyr and the could be used as a diagnostic concentration to monitor for resistance pyrazole fenpyroximate, we suggest the diagnostic concentrations of 4 Journal of Economic Entomology, 2015, Vol. 0, No. 0

Table 2. Diagnostic concentrations for resistance monitoring in 100 Asian citrus psyllid populations

Insecticides Suggested Recommended 80 concentrations concentrationa (mg per vial) (mg per vial) 60 Cypermethrin 0.5–1.0 1.0 R-IMOK Malathion 1.0–2.5 1.0 40

Diazinon 1.0–2.5 1.0 Mortality % Carbaryl 0.5–1.0 1.0 Carbofuran 0.5–1.0 1.0 20 Methomyl 0.5–1.0 1.0 S-LAB Propoxur 0.5–1.0 1.0 Endosulfan 0.5–1.0 1.0 0 Imidacloprid 0.5–1.0 0.5 0.001 0.01 0.1 1 10 100 Acetamiprid 5.0–10.0 5.0 Malathion (µg/vial) Fenpyroximate 2.5–5.0 2.5 Chlorfenapyr 2.5–5.0 2.5 Fig. 2. Responses of susceptible populations (S-LAB) of Asian citrus psyllid Clothianidin 0.5–1.0 0.5 and ﬁeld-collected resistant (R-IMOK) individuals exposed to different con- b-Cyﬂuthrin 0.05–0.10 0.1 centrations of Malathion. Arrow indicates proposed diagnostic concentra- Dimethoate 1.0–2.5 1.0 tions for resistance monitoring. Fenpropathrin 1.0–2.5 1.0 Downloaded from Thiamethoxam 1.0–2.5 1.0 100 a Recommended concentration for monitoring resistance if only a single diagnostic concentration will be used. 80 R-IMOK http://jee.oxfordjournals.org/ 100 60

80 R-IMOK 40 S-LAB % Mortality

60 20

40 by guest on May 11, 2016

% Mortality 0 0.001 0.01 0.1 1 10 20 S-LAB Carbaryl (µg/vial)

Fig. 3. Responses of susceptible populations (S-LAB) of Asian citrus psyllid 0 and ﬁeld-collected resistant (R-IMOK) individuals exposed to different con- 0.001 0.01 0.1 1 10 centrations of Carbaryl. Arrow indicates proposed diagnostic concentrations Cypermetrhin (µg/vial) for resistance monitoring.

Fig. 1. Responses of susceptible populations (S-LAB) of Asian citrus psyllid 100 and ﬁeld-collected resistant (R-IMOK) individuals exposed to different concentrations of Cypermethrin. Arrow indicates proposed diagnostic concentrations for resistance monitoring. 80 R-IMOK

2.5 and 5.0 lg per vial (LC ) for resistance monitoring in the 95–99 60 field populations of Asian citrus psyllid.

Monitoring for Resistance in Field Populations of Asian % Mortality Citrus Psyllid Using the established diagnostic concentrations, we monitored for 20 S-LAB insecticide resistance in field populations (R-IMOK) of Asian citrus psyllid in the summer of 2014 (Fig. 6). The percent survival of Asian 0 citrus psyllid to the diagnostic concentrations of carbaryl (1.0 lg per 0.001 0.01 0.1 1 10 vial), carbofuran (1.0 lg per vial), methomyl (1.0 lg per vial), pro- µ poxur (1.0 lg per vial), malathion (1.0 lg per vial), diazinon (1.0 lg Endosulfan ( g/vial) per vial), cypermethrin (1.0 lg per vial), and endosulfan (1.0 lg per Fig. 4. Responses of susceptible populations (S-LAB) of Asian citrus psyllid vial) indicated that resistance was present in field populations of and ﬁeld-collected resistant (R-IMOK) individuals exposed to different con- Asian citrus psyllid in Immokalee, FL. The frequency of resistance centrations of Endosulfan. Arrow indicates proposed diagnostic concentra- varied between classes of insecticides and was the highest for the tions for resistance monitoring. Journal of Economic Entomology, 2015, Vol. 0, No. 0 5

100 gluthione-s-transferase (Tiwari et al. 2011). Knowledge of such resistance factors is critical in developing a successful resistance management strategy. The patterns of resistance in Asian citrus psyllid 80 R-IMOK populations varied between classes of insecticides, which might be due to the mode of action of these insecticides. Asian citrus psyllid 60 populations were susceptible to diazinon, methomyl, dimethoate, propoxur, and endosulfan. In addition, Asian citrus psyllid populations were indistinguishable from the susceptible colony in their responses 40 % Mortality to neonicotinoids (imidacloprid, acetamiprid, clothianidin, and thiamethoxam) and pyrrole (chlorfenapyr). Imidacloprid and to a lesser S-LAB 20 extent thiamethoxam and clothianidin are used for Asian citrus psyllid control in Florida citrus, whereas chlorfenapyr has never been labeled for citrus or any other food crop in the United States. 0 This study led to the development of a fast, simple, and user- 0.001 0.01 0.1 1 10 friendly resistance monitoring technique needed to maximize the Imidacloprid (µg/vial) likelihood of success in resistance management programs for Asian citrus psyllid populations. In the future, citrus growers who wish to Fig. 5. Responses of susceptible populations (S-LAB) of Asian citrus psyllid monitor for resistance could be provided with glass vials containing and ﬁeld-collected resistant (R-IMOK) individuals exposed to different concentrations of Imidacloprid. Arrow indicates proposed diagnostic concentra- diagnostic concentrations by extension personnel or consultants. This technique will provide data needed to make informed decisions

tions for resistance monitoring. Downloaded from on an overall integrated pest management strategy.

Propoxur 80 Endosulfan Diazinon Acknowledgments Methomyl Carbofuran We thank Rodrigue Pierre (Florida A&M University, Tallahassee,

Cypermethrin http://jee.oxfordjournals.org/ 60 Carbaryl FL) for his technical assistance with this study. We are also grateful Malathion to Susie Legaspi, Stuart Reitz (USDA-ARS, Tallahassee, FL), Janice Peters and Manuel Pescador (Florida A&M University) for provid- 40 ing useful discussions and reviews of the manuscript. % Survival

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