Amitraz Resistance –Rumors and Realities

Dr. rer. nat. Ulrike Marsky – September 10th 2019

CONTENTS

➢ Introduction to acaricide resistance

➢ Amitraz as an active compound against V. destructor

➢ Research on low efficacy / suspected amitraz resistance

➢ Amitraz efficacy and winter mortality analyses in countries with a long history of amitraz use in beekeeping

➢ Experiences with low efficacy and suspected amitraz resistance from the point of view of a pharmaceutical producer

➢ Research projects on amitraz resistance

➢ Conclusion: Current status and outlook on amitraz resistance

Dr. rer. nat. Ulrike Marsky – September 10th 2019

WHAT IS RESISTANCE?

Coles & Dryden, 2014. Insecticide/acaricide resistance in fleasand ticks infesting dogs and cats. Parasites and Vectors: 7:8.

“… the selection of a specific heritable trait (or traits) in apopulation of arthropods, due to that population’s contact with achemical, that results in a significant increase in the percentageof the population that will survive a standard dose of thatchemical (or a closely related chemical in the case of crossresistance).”

Dr. rer. nat. Ulrike Marsky – September 10th 2019

WHAT IS RESISTANCE?

Martin, S. J. "Acaricide (pyrethroid) resistance in Varroadestructor." Bee world (2004).

“Pesticide resistance is the ability of a pest to survive normallylethal rates of a pesticide. …”

Dr. rer. nat. Ulrike Marsky – September 10th 2019

WHAT IS RESISTANCE?

➢ Contact with the active substance (“a chemical” or “pesticide”)

➢ Significant increase of surviving individuals within a population of the target species over time / change in sensitivity of a pest population

➢ Resulting in a failure of a correct application / standard dose of the pesticide

➢ Due to the selection of heritable traits

➢ Mechanisms of resistance1:➢ Behavioral changes (avoidance)➢ Reduced penetration (cuticle thickening)➢ Increased detoxification of the pesticide by enzymes➢ Target site desensitivity (modifications of action site)

1Sammataro, Diana, et al. "The resistance of varroa mites (Acari: Varroidae) to acaricides and the presence of esterase." International Journal of Acarology 31.1 (2005): 67-74.

Dr. rer. nat. Ulrike Marsky – September 10th 2019

➢ First indicator: Treatment efficacy of a correct application➢ Field assay, rough estimation: Mite kills below 50% indicate

approaching resistance, kills below 25% indicate full resistance.1

➢ Statistically significant differences in enzyme levels of susceptible and resistant mites, between resistance ratios of mite populations

➢ Frequency of resistance mutations in populations

QUANTIFYING RESISTANCE

Treatment efficacy

Field assay Pettis test

LC50/LC90LD50/LD90

Enzyme levels

Genome analysis

1Suszkiw, Jan. "Easy-to-use bioassay spots Varroa resistance." Agricultural Research53.4 (2005): 19.

Dr. rer. nat. Ulrike Marsky – September 10th 2019

CASES OF ACARICIDE RESISTANCE IN V. DESTRUCTOR

tau-fluvalinate© Gilles San Martin

➢ Pyrethroid

➢ Registered in Europe in 1980s, Italy: 1989

➢ First reports of resistance in Italy: 1992/1993 in Lombardy:

variation in efficacy between 4% and 89.5%; mean = 44%1

➢ LC50 of resistant mite populations was 25-50 x higher than in

susceptible populations2

➢ Mechanisms of resistance suggested: Mutations in voltage-gated

sodium channels3, 4, increase in levels of detoxification enzymes

1 Lodesani, M., M. Colombo, and M. Spreafico. "Ineffectiveness of Apistan® treatment against the mite Varroa jacobsoni Oud in several districts of Lombardy (Italy)." Apidologie 26.1 (1995): 67-72.

2 Milani, Norberto. "The resistance of Varroa jacobsoni Oud to pyrethroids: a laboratory assay." Apidologie 26.5 (1995): 415-429.

3 Hubert, Jan, et al. "Point mutations in the sodium channel gene conferring tau‐fluvalinate resistance in Varroa destructor." Pest management science 70.6 (2014): 889-894.

4 González-Cabrera, Joel, et al. "Novel mutations in the voltage-gated sodium channel of pyrethroid-resistant Varroa destructor populations from the Southeastern USA." PloS one

11.5 (2016): e0155332.

Dr. rer. nat. Ulrike Marsky – September 10th 2019

CASES OF ACARICIDE RESISTANCE IN V. DESTRUCTOR

Coumaphos

➢ Organophosphate

➢ Perizin® registered in Germany in 1985, Checkmite+® used in in

the US since 2000

➢ Field assays in Florida and Maine demonstrated mite mortalities

of 6.3% and 13%, respectively (Year: 2001)1,2

➢ Mode of action: irreversibly inhibits acetylcholinesterase (AChE,

enzyme involved in neurotransmission)3,4

1 Pettis, J. S., and Tony Jadczak. "Detecting coumaphos resistance in Varroa mites." American Bee Journal 145.12 (2005): 967-970.

2 Elzen, P. J., and D. Westervelt. "Detection of coumaphos resistance in Varroa destructor in Florida." American Bee Journal 142.4 (2002): 291-292.

3 Lodesani, Marco, and Cecilia Costa. "Limits of chemotherapy in beekeeping: development of resistance and the problem of residues." Bee World 86.4 (2005): 102-109.

4 Williamson, Sally M., et al. "Exposure to acetylcholinesterase inhibitors alters the physiology and motor function of honeybees." Frontiers in physiology 4 (2013): 13.

Dr. rer. nat. Ulrike Marsky – September 10th 2019

CASES OF RESISTANCE REVERSION IN V. DESTRUCTOR

➢ Pesticide resistance can be associated with fitness costs -> decline in

the frequency of alleles for resistance1,2

➢ Italian mite strain resistant to fluvalinate – resistant mites decreased

by factor 10 in three years without contact to fluvalinate1,2

➢ Example: In English mite populations, the frequency of a mutation

responsible for fluvalinate resistance was low in populations not

exposed to recent treatments

➢ indicating the mutation may not survive well in the absence of

pyrethroid selection3

1 Milani, Norberto. "The resistance of Varroa jacobsoni Oud. to acaricides." Apidologie 30.2-3 (1999): 229-234.

2 Milani, Norberto, and Giorgio Della Vedova. "Decline in the proportion of mites resistant to fluvalinate in a population of Varroa destructor not treated with pyrethroids." Apidologie 33.4 (2002): 417-422.

3 Gonzalez-Cabrera, Joel, et al. "An amino acid substitution (L925V) associated with resistance to pyrethroids in Varroa destructor." PLoS One 8.12 (2013): e82941.

Dr. rer. nat. Ulrike Marsky – September 10th 2019

AMITRAZ AND IT’S USE IN BEEKEEPING

Amitraz

➢ Formamidine

➢ White to yellowish crystalline substance

➢ Active compound of several veterinary medicines and pesticides,

targets mainly insects, mites, ticks

➢ Application with strips, fumigation: Apivar®, Apitraz®, Biowar®,

Varidol®, etc.

© Gilles San Martin

Dr. rer. nat. Ulrike Marsky – September 10th 2019

AMITRAZ – MODE OF ACTION

AMITRAZ

PENETRATION through the cuticle

or by ingestion

VARROA

blocking the Octopamine receptors involved in

neuronal transmission

EXCESSIVE STIMULATION

OF THE CENTRAL NERVOUS

SYSTEM

(PARALYSIS)

Varroa mites fall off bees

VARROA Death• DUE TO STARVATION

• INABILITY TO PENETRATE A NEW CELL AND REPRODUCE

Dr. rer. nat. Ulrike Marsky – September 10th 2019

AMITRAZ AND IT’S USE IN BEEKEEPING

Amitraz

➢ Mechanisms of resistance suggested: Mutations in octopamine

receptors1, detoxification2, but mechanism has not been described

in V. destructor yet3

➢ Although amitraz resistance in V. destructor has been reported in

several countries, amitraz remains largely effective3

➢ Amitraz resistance has been confirmed genetically only in other

species (example: Boophilus microplus) : Cattle ticks have been

controlled with amitraz for >40 years, but prevalence of resistant

populations remains low4

© Gilles San Martin

1 Kamler, Martin, et al. "Comparison of tau-fluvalinate, acrinathrin, and amitraz effects on susceptible and resistant populations of Varroa destructor in a vial test." Experimental and applied acarology 69.1 (2016): 1-9.

2 Johnson, Reed M., et al. "Pesticides and honey bee toxicity–USA." Apidologie 41.3 (2010): 312-331.

3 Evans, Jay D., and Steven C. Cook. "Genetics and physiology of Varroa mites." Current opinion in insect science 26 (2018): 130-135.

4 Jonson & Hope (2007) Progress in the epidemiology and diagnosis of amitraz resistance in the cattle tick Boophilusmicroplus. Veterinary Parasitology, Volume 146, Issues 3–4, Pages 193-198.

Dr. rer. nat. Ulrike Marsky – September 10th 2019

AMITRAZ –RESISTANCE DEVELOPMENT IN V. DESTRUCTOR

➢ Amitraz resistance reported in several countries: US, Czech Republic,

France, Argentina, Portugal and others

➢ Mechanism of resistance in V. destructor not described yet1

➢ Increase of LC50 lower compared to other acaricides2 and reduced

efficacy in the field less pronounced than in other acaricides3

➢ Meta-Analysis (Poland): No statistical differences in efficacy of

amitraz treatments in the field between the years of 1981-1985,

1986-2000 and 2001-2009 (93.6 ± 3.73%, 95.4 ± 3.43% and 93.2 ±

6.59%, respectively)4

➢ Use of amitraz-based varroacides associated with the lower winter

losses in the US than any other varroacide products5

1Evans, Jay D., and Steven C. Cook. "Genetics and physiology of Varroa mites." Current opinion in insect science 26 (2018): 130-135.

2Rodríguez-Dehaibes, Sóstenes R., et al. "Resistance to amitraz and flumethrin in Varroa destructor populations from Veracruz, Mexico." Journal of Apicultural Research 44.3 (2005): 124-125.

3Elzen, Patti J., et al. "Control of Varroa jacobsoni Oud. resistant to fluvalinate and amitraz using coumaphos." Apidologie 31.3 (2000): 437-441.

4Dzierżawski, A., and W. Cybulski. "Evaluation of the efficacy of Apiwarol AS." Medycyna Weterynaryjna66.7 (2010): 475-479.

5Ariela I Haber, Nathalie A Steinhauer, Dennis vanEngelsdorp, Use of Chemical and Nonchemical Methods for the Control of Varroa destructor (Acari: Varroidae) and Associated Winter Colony Losses in U.S. Beekeeping Operations, Journal of Economic Entomology, Volume 112, Issue 4, August 2019, Pages 1509–1525

Dr. rer. nat. Ulrike Marsky – September 10th 2019

AMITRAZ – efficacy and winter mortality analyses in countries with a long history of amitraz use in beekeeping

Product Country Year (of data collection)

Type Authors / Study directed by

Efficacy (%) /winter mortality (%)

Apivar Bulgaria 2011 Efficacy Gurgulova et al. 2015National Diagnostic and Research Veterinary Medical Institute, 1000 Sofia, Bulgaria

>99

Apivar France 2007-2018 Efficacy FNOSAD La Fédération

Nationale des Organisations Sanitaires Apicoles Départementales

95.75

Apivar France 2010 - 2018 Winter mortality

ADAGE Association for the

Development of Beekeeping Grand Est

Mean (Apivar): 15.1 Mean (all): 24.7

Apivar Greece 2018 Efficacy Prof. Paschalis Harizanis Agricultural University, Athens

97.33

Apivar Spain 2018 Efficacy Euromiel S. Coop. de Segundo Grado

99.43

Amitraz USA 2013-2017 Winter mortality

Haber et al. University of Maryland, BIP

Amitraz: 18.8Average: 23

Dr. rer. nat. Ulrike Marsky – September 10th 2019

EXPERIENCES WITH LOW EFFICACY AND SUSPECTED AMITRAZ RESISTANCE

Common factors associated with low efficacy reports:➢ Under-dosing➢ Incorrect application➢ Unfinished treatments (early removal, or report during treatment period)➢ Monitoring-related problems:

➢ No pre-treatment monitoring➢ Misinterpretation of monitoring results➢ Treatment applied although monitoring results indicate very low

infestation➢ Fixed routines (annual treatment schedule is not adapted to the season)➢ Re-infestation (sometimes related to short treatment period)

Other possible factors associated with low efficacy or resistance development:➢ Repeated / continuous use of the same active ingredient➢ Not acknowledging climate / temperature / seasonal changes

Dr. rer. nat. Ulrike Marsky – September 10th 2019

RECOMMENDED IMP (INTEGRATED PEST MANAGEMENT) ACTIONS

➢ Regular monitoring of mite infestation starting in spring (once a month)

➢ Treat only if necessary

➢ Monitoring before and after a varroa treatment

➢ Stay informed about local infestation thresholds

➢ Renew brood comb every 2-(max.!)3 years

➢ Treat all colonies in an apiary at the same time

➢ Rotate treatments (active ingredients!)

➢ Respect the product labels and instructions

Dr. rer. nat. Ulrike Marsky – September 10th 2019

RESEARCH PROJECTS CONCERNED WITH AMITRAZ RESISTANCE

➢ Dr. Jeffrey S. Pettis:

Refinement and validation of a field assay to detect amitraz resistance in

Varroa destructor

➢ Ramesh Sagili, Ph.D. (Oregon State University) & Shelley Hoover (Plant and

Bee Health Surveillance Section, Government of Alberta):

Evaluating and comparing amitraz resistance in Varroa mite populations

in the United States and Canada

Dr. rer. nat. Ulrike Marsky – September 10th 2019

RESEARCH PROJECTS CONCERNED WITH AMITRAZ RESISTANCE

Dr. Joel Gonzales-Cabrera

ERI BIOTECMED. Department of Genetics

University of Valencia

• Pesticide resistance• Tau-fluvalinate & coumaphos in Spain• Collecting & analyzing mite samples suspected of amitraz resistance• New project on potential fitness costs associated with resistance

Dr. rer. nat. Ulrike Marsky – September 10th 2019

CONCLUSION: CURRENT STATUS AN AND OUTLOOK ON AMITRAZ RESISTANCE

➢ Although amitraz resistance in V. destructor has been reported in several countries, amitraz remains largely effective1; this includes countries with long-term use of amitraz in beekeeping

➢ IPM and respecting the product label & instructions optimizes treatment efficacy (not only of amitraz-based varroa treatments!)

➢ Resistance is a only question of time, but it is also a question of monitoring and treatment practices in the field

➢ We as pharmaceutical producers welcome discussions and exchange about resistance development with the honey bee research community

1Evans, Jay D., and Steven C. Cook. "Genetics and physiology of Varroa mites." Current opinion in insect science 26 (2018): 130-135.

Dr. rer. nat. Ulrike Marsky – September 10th 2019

Thank you

Dr. rer. nat. Ulrike Marsky –September 10th 2019 APV-90-EN-N01-09/19