Monitoring for Resistance to Organophosphorus and Pyrethroid Insecticides in Varroa Mite Populations

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INSECTICIDE RESISTANCE AND RESISTANCE MANAGEMENT Monitoring for Resistance to Organophosphorus and Pyrethroid Insecticides in Varroa Mite Populations

1,2 3 1 4 LAMBERT H. B. KANGA, JOHN ADAMCZYK, KEITH MARSHALL, AND ROBERT COX

J. Econ. Entomol. 103(5): 1797Ð1802 (2010); DOI: 10.1603/EC10064 ABSTRACT The occurrence of resistance in Varroa mite populations is a serious threat to the beekeeping industry and to crops that rely on the honey bee for pollination. Integrated pest management strategies for control of this pest include the judicious use of insecticides. To monitor Þeld populations of Varroa mite for insecticide resistance, a glass vial bioassay procedure was developed to use in the development of a resistance management strategy. Diagnostic concentrations needed to separate susceptible genotypes from resistant individuals were determined for cypermethrin (0.1 ␮g per vial), ßuvalinate (5.0 ␮g per vial), malathion (0.01 ␮g per vial), coumaphos (10.0 ␮g per vial), diazinon (5.0 ␮g per vial), methomyl (0.5 ␮g per vial), propoxur (0.1 ␮g per vial), and endosulfan (2.5 ␮g per vial). Resistance to organophosphorus insecticides (malathion, coumaphos) and pyrethroids (cypermetrhrin, ßuvalinate) was widespread in both La Media Ranch, TX, and Wewahitchka, FL, from 2007 to 2009. There was no resistance to endosulfan, diazinon, methomyl, and propoxur in Þeld populations of Varroa mite in the two locations where resistance was monitored. The seasonal patterns of resistance in Wewahitchka were different from those of La Media Ranch. In the former location, the frequency of resistance to all insecticides tested decreased signiÞcantly from 2007 to 2009, whereas it increased in the latter location. Resistance levels were unstable, suggesting that resistance could be successfully managed. The results validate use of the glass vial bioassay to monitor for resistance in Varroa mite and provide the basis for the development of a resistance management strategy designed to extend the efÞcacy of all classes of insecticides used for control of Varroa mite.

KEY WORDS Varroa destructor, Apis mellifera, monitoring insecticide resistance

The honey bee, Apis mellifera L. is essential for honey States (Sanford et al. 1999), but some resistance has production and crop pollination (Ͼ130 agricultural already developed to this compound (Elzen and West- plants in the United States are pollinated by honey ervelt 2002). bees). The ectoparasitic mite Varroa destructor Selection for mite resistant honey bees has shown Anderson & Trueman is currently a serious worldwide some success (Rinderer et al. 2000, Spivak and Reuter threat to beekeeping (De Jong et al. 1982, Rosenkranz 2001). However, chemical treatments are still neces- et al. 2010). Without adequate control of Varroa mite sary to ensure colony survival within the breeding infestations, bee mortality approaches 100% in un- programs for strains resistant to Varroa in North Amer- treated colonies, which can perish within a few weeks. ica and Europe (Bu¨ chler 1994, Rinderer et al. 1997). A major chemical control strategy is the use of Chemical controls, such as ßumethrin, amitraz, plastic strips impregnated with the pyrethroid ßuvali- cymiazole, and bromopropylate, leave toxic residues nate (Apistan, Wellmark International, Bensenville, (Wallner 1999), and are difÞcult to register in North IL). Although Apistan strips have been very effective America because of safety concerns for public health in controlling mites (Ferrer-Dufol et al. 1991), resis- and the environment. Although entomopathogenic tance has developed in several mite populations (El- fungi (Metarhizium anisopliae and Hirsutella thomp- zen et al. 1998, Milani 1999). In addition, these prod- sonii ucts leave residues in wax and honey (Cabras et al. ) offer promising avenues for biological control of 1997, Wallner 1999). More recently, plastic strips Varroa mite (Kanga et al. 2002, 2003), the develop- coated with the organophosphate coumaphos ment of a sustainable pest management strategy will (CheckMite, Bayer Shawnee Mission, KS) have been require the judicious integration of other control mea- used under emergency registration in the United sures. In this study, we report on the selection of diag- 1 Center for Biological Control, Florida A&M University, Tallahas- nostic concentrations of insecticides for use in resis- see, FL 32307-4100. tance monitoring and present results on monitoring 2 Corresponding author, e-mail: [email protected]. 3 USDAÐARS, BeneÞcial Insects Research Unit, Weslaco, TX 78596. Varroa mite populations for resistance to several in- 4 USDAÐARS, Honey Bee Research Unit, Weslaco, TX 78596. secticides.

0022-0493/10/1797Ð1802$04.00/0 ᭧ 2010 Entomological Society of America 1798 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 103, no. 5

Table 1. Responses of susceptible (S-CA) and ﬁeld-collected resistant (R-LMR) Varroa mite populations to different insecticides

Na Ϯ b c Insecticide Strain Slope SE LC50 (95% CL) RR (95% CL) Pyrethroid Cypermethrin SCA 125 1.17 Ϯ 0.42 0.020 (0.001Ð0.066) R-LMR 130 1.68 Ϯ 0.68 0.090 (0.016Ð0.190) 4.5 (3.05Ð5.98) Fluvalinate S-CA 120 1.69 Ϯ 0.45 0.100 (0.030Ð0.290) R-LMR 145 2.25 Ϯ 0.44 0.260 (0.110Ð0.420) 2.6 (1.88Ð4.98) Organophosphate Malathion S-CA 165 4.47 Ϯ 1.85 0.003 (0.002Ð0.004) R-LMR 160 3.21 Ϯ 0.95 0.011 (0.004Ð0.020) 3.7 (2.24Ð5.26) Coumaphos S-CA 147 1.36 Ϯ 1.81 0.150 (0.001Ð0.042) R-LMR 153 1.11 Ϯ 0.37 2.150 (0.278Ð9.343) 14.3 (11.76Ð19.59) Diazinon S-CA 124 1.16 Ϯ 0.44 0.040 (0.001Ð0.219) R-LMR 118 0.99 Ϯ 0.41 0.050 (0.002Ð0.337) 1.3 (0.78Ð2.14) Carbamate Methomyl S-CA 98 3.43 Ϯ 1.13 0.002 (0.001Ð0.005) R-LMR 122 3.49 Ϯ 0.87 0.002 (0.001Ð0.004) 1.0 (0.32Ð1.35) Propoxur S-CA 92 1.83 Ϯ 0.63 0.002 (0.001Ð0.007) R-LMR 129 1.97 Ϯ 0.62 0.003 (0.001Ð0.011) 1.5 (0.77Ð2.46) Cyclodiene Endosulfan S-CA 109 1.95 Ϯ 0.51 0.023 (0.008Ð0.740) R-LMR 146 1.92 Ϯ 0.53 0.034 (0.007Ð0.152) 1.5 (0.96Ð2.88)

a Number of mites tested. b Concentrations are expressed in micrograms per vial of the insecticide tested. c Resistance ratios (RR) calculated by dividing the LC50 for the R-LMR by the LC50 for the S-CA strain.

Materials and Methods mites was determined 18 h after exposure. Mites that were unable to walk for a short distance (Ͼ5 mm) after Insecticides. All insecticides tested were technical gentle probing with a Þne brush were considered dead. grade samples (Ϸ98% purity) as supplied by the man- Diagnostic Concentrations for Resistance Monitor- ufacturers. We used the organophosphorodithionate ing. Susceptible and resistant mite populations were malathion, the organophosphorothioate coumaphos, exposed to insecticides to establish diagnostic concen- and the pyrethroids cypermethrin and ßuvalinate trations that were estimated from the concentrationÐ (Chemical Service, West Chester, PA) for resistance mortality responses. At that concentration, all suscepti- monitoring. Other technical grade insecticides tested ble individuals were killed and only resistant phenotypes included the cylodiene endosulfan, the organophos- survived (Kanga and Plapp 1995). Concentration-re- phorothiolate diazinon, the oxime carbamate metho- sponse regressions for each strain and each insecticide myl, and the aryl carbamate propoxur. These chemi- were calculated with seven concentrations of insecti- cals were purchased either from Sigma (St. Louis, cides (plus an acetone control), with eight to 10 repli- MO) or Aldrich (Milwaukee, WI). cates of two to three mites per vial. Mites. Female mites were collected from infested Monitoring for Resistance. Assessments of the lev- frames of sealed brood taken from honey bee colonies els of resistance in Þeld populations were conducted maintained in or nearby Weslaco, TX, and in Wewa- using techniques as described by Kanga et al. (1997) hitchka, FL. Susceptible mites (S-CA) were collected and Elzen et al. (1998, 2002). Varroa mites collected from an apiary located in Cactus Alley (Old Marine from the apiaries were brought to the laboratory and base) in Weslaco. This apiary has feral honey bee exposed to residues of insecticide in treated vials to colonies that have never been treated with pesticides. determine the frequencies of resistant phenotypes. The resistant mites (R-LMR) were Þeld collected indi- Between 75 and 112 mites were tested at each di- viduals from La Media Ranch (near Weslaco) where agnostic concentration during an experimental run. high levels of resistance were reported. Drone and Statistical Analyses. Concentration-mortality data worker brood cells were opened and adult female mites were analyzed by Probit analysis (Russell et al. 1977). were collected from larvae and pupae, by using a camelÕs- Data from multiple experiments were pooled. Differ- hair brush. The mites were placed into glass scintillation ences among populations in response to insecticides vials (20 ml) containing honey bee larvae as a food were considered not signiÞcant if the 95% conÞdence source before insecticide bioassays. limit (CL) of the resistance ratio at the LC bracketed Insecticide Bioassays. The procedure used in this 50 1.0 (Robertson and Preisler 1992). study was described by Kanga and Plapp (1995). In brief, 20-ml glass scintillation vials were treated with 0.5-ml solutions of insecticide in acetone. The vials Results were rolled until the acetone evaporated and the insecticides were coated on the inner surfaces. Vials Laboratory Bioassays. Varroa mites displayed vary- treated with acetone only were used as controls. Two to ing levels of resistance within and between groups of three mites were placed in each vial and held at room insecticides tested (Table 1). The responses of S-CA temperature (27 Ϯ 1ЊC and 65% RH). Mortality of the and R-LMR strains to pyrethroids and two of the October 2010 KANGA ET AL.: RESISTANCE MONITORING OF VARROA MITE 1799

Table 2. Diagnostic concentrations for resistance monitoring in Varroa mite populations

Suggested concn Recommended concn Insecticide (␮g per vial) (␮g per vial) Cypermethrin 0.05Ð0.1 0.1 Fluvalinate 5.0Ð10.0 5.0 Malathion 0.005Ð0.01 0.01 Coumaphos 5.0Ð10.0 10.0 Diazinon 2.5Ð5.0 5.0 Methomyl 0.5Ð1.0 0.5 Propoxur 0.1Ð0.5 0.1 Endosulfan 2.5Ð5.0 2.5

organophosphorus insecticides tested were statisti- cally different based on failure of the 95% CL of the resistance ratio to bracket 1.0 (Robertson and Preisler 1992). The data indicated variable levels of responses to the different types of organophosphorus insecticides (i.e., 3.7-fold resistance to phosphorodithionate, 14.3-fold resistance to phosphorothioate, and 1.3-fold tolerance to phosphorothiolate). There was no resistance to oxime carbamate (1.0-fold) and aryl carbamate (1.5-fold). Resistance was 2.6Ð4.5-fold to the pyrethroid insecticides but was not signiÞcant to the cyclodiene endosulfan (1.5-fold). Diagnostic Concentrations for Resistance Monitor- ing. The responses of S-CA and R-LMR populations of Varroa mite to each insecticide were used to select diagnostic concentrations for resistance monitoring (Table 2). These concentrations were chosen to maximize differences in responses between susceptible and resistant mites (McCutchen et al. 1989, Kanga et al. 1999). For the organophosphorus insecticide mal- Fig. 1. (A) Responses of susceptible (S-CA) populations athion, data indicated that concentrations of 0.005 and of Varroa mites and Þeld-collected resistant (R-LMR) indi- ␮ viduals exposed to different concentrations of malathion. 0.01 g per vial (LC95Ð99) could be used to monitor for Arrows indicate proposed diagnostic concentrations for re- resistance in Þeld populations of Varroa mite (Fig. sistance monitoring. (B) Responses of S-CA populations of 1A); a single concentration, 0.01 ␮g of malathion per Varroa mites and Þeld-collected R-LMR individuals ex- vial (LC99), was chosen. Similarly, for coumaphos posed to different concentrations of coumaphos. Arrows (Fig. 1B), our data suggested that concentrations of 5.0 indicate proposed diagnostic concentrations for resistance ␮ and 10.0 g per vial (LC90Ð99) would separate sus- monitoring. ceptible from resistant individuals; we proposed 10.0 ␮g of coumaphos per vial for monitoring resistance in Varroa mite populations because at this concentration, tolerance to this insecticide; however, a single con- almost all susceptible individuals were killed and a few centration of 0.1 ␮g per vial was our best choice be- resistant phenotypes survived. For the pyrethroid in- cause at this concentration all susceptible individuals secticides, the concentrations of 0.05 and 0.1 ␮g per were killed. Thus, this concentration will maximize

vial (LC95Ð99) were found to be discriminating con- the responses to insecticides of the mixed popula- centrations for cypermethrin with 0.1 ␮g per vial tions of susceptible and resistant mites. Similarly, for ␮ (LC99) chosen for resistance monitoring in Þeld pop- methomyl, the best single concentration was 0.5 g ulations (Fig. 2A). For ßuvalinate, data indicated that per vial for the diagnostic concentrations of 0.5Ð1.0 ␮ ␮ concentrations of 2.5 and 5.0 g per vial (LC95Ð99) g per vial. For the organophosphorus insecticide could be used to identify resistance in Þeld popula- diazinon (2.5Ð5.0 ␮g per vial), 5.0 ␮g per vial was the tions of Varroa mite (Fig. 2B); we suggested a single best single concentration for tolerance monitoring. ␮ ␮ concentration of 5.0 g per vial (LC99) for resistance The concentrations of 2.5Ð5.0 g per vial of the monitoring in Varroa mite populations. cyclodiene endosulfan discriminated between sus- For the insecticides where resistance was not evi- ceptible and resistant individuals; however, a single dent, the concentration-regression lines for S-CA and concentration of 2.5 ␮g per vial could separate het- R-LMR mite populations overlapped. The patterns of erozygous and homozygous individuals (Kanga et al. responses of Varroa mite indicated that concentra- 1997).

tions at LC95Ð99 could be used to monitor for tolerance Monitoring for Miticide Resistance in Texas and to these insecticides. For the carbamate propoxur, Florida: 2007–2009. The percentage of survival of Var- 0.1Ð0.5 ␮g per vial provided a reliable indicator of roa mites to the diagnostic concentrations of cyper- 1800 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 103, no. 5

Fig. 3. Percentage of survival of Þeld-collected Varroa mites and exposed to diagnostic concentrations of cypermethrin (0.1 ␮g per vial), ßuvalinate (5.0 ␮g per vial), coumaphos (10.0 ␮g per vial), malathion (0.01 ␮g per vial), diazinon (5.0 ␮g per vial), methomyl (0.5 ␮g per vial), propoxur (0.1 ␮g per vial), and endosulfan (2.5 ␮g per vial) in La Media Ranch, TX. More than 1,375 mites were tested each year.

Fig. 2. (A) Responses of susceptible (S-CA) populations of Varroa mites and Þeld-collected resistant (R-LMR) indi- present in Þeld populations of Varroa mites in Wewa- viduals exposed to different concentrations of cypermethrin. hitchka. Arrows indicate proposed diagnostic concentrations for resistance monitoring. (B) Responses of S-CA populations Discussion of Varroa mites and Þeld-collected R-LMR individuals exposed to different concentrations of ßuvalinate. Arrows The overall data indicated that resistance to pyre- indicate proposed diagnostic concentrations for resistance throids and organophosphorus insecticides was monitoring. present in Þeld populations of Varroa mite in La Media Ranch and Wewahitchka. The similarity of the responses of Varroa mite to malathion and coumaphos in methrin (0.1 ␮g per vial), ßuvalinate (5.0 ␮g per vial), both sites tested from 2007 to 2009 suggested that the malathion (0.01 ␮g per vial), and coumaphos (10.0 ␮g same mechanisms may confer resistance to both in- per vial) indicated that resistance was wide spread in secticides. Several mechanisms of resistance have Þeld populations of Varroa mite in Texas from 2007 to been found in Varroa mite populations and included 2009 (Fig. 3). The frequencies of resistance varied esterase-mediated resistance (Sammataro et al. 2005), between classes of insecticides and increased for the monooxygenases by P450 (Hillesheim et al. 1996, organophosphorus insecticides malathion and couma- Mozes-Koch et al. 2000), and sodium channel gene phos over the past 3 yr. Resistance to both pyrethroid mutations (Wang et al. 2002, Liu et al. 2006). Knowl- insecticides (cypermethrin and ßuvalinate) declined edge of such resistance factors is critical in developing in 2008 and increased in 2009. There was no resistance a successful resistance management strategy. to endosulfan, diazinon, methomyl and propoxur in The patterns of frequency of resistance over the 3 Þeld populations of Varroa mite in La Media Ranch. yr in the two locations may indicate differences in Patterns of resistance to cypermethrin, ßuvalinate, chemical treatment regimes used by beekeepers. malathion and coumaphos in Wewahitchka were dif- However, beekeepers have not used malathion and ferent from those in La Media Ranch. Frequencies of cypermethrin for Varroa mite control; thus, resistance resistance to all insecticides tested decreased signiÞ- in mite populations between ßuvalinate and cyper- cantly from 2007 to 2009 in Florida (Fig. 4). The methrin and between coumaphos and malathion in- highest level of resistance was recorded in 2007 with dicated that cross-resistance has occurred in the ab- coumaphos. As in La Media Ranch, resistance to en- sence of selection pressure. Thus, there is merit of dosulfan, diazinon, methomyl and propoxur was not widening the search for effective groups of chemicals October 2010 KANGA ET AL.: RESISTANCE MONITORING OF VARROA MITE 1801

Acknowledgments We thank Carlos Gracia, Medrano Rodolfo and Henry Graham (USDAÐARS, Weslaco, TX) for technical assistance with this study. We are grateful to Katherine Aronstein (USDAÐ ARS, Weslaco), Stuart Reitz (USDAÐARS, Tallahassee, FL), and Janice Peters and Manuel Pescador (Florida A&M Univer- sity, Tallahassee, FL) for providing useful discussions and re- views of the manuscript.

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