insects Article Large-Scale Monitoring of Resistance to Coumaphos, Amitraz, and Pyrethroids in Varroa destructor Carmen Sara Hernández-Rodríguez 1, Óscar Marín 1, Fernando Calatayud 2, María José Mahiques 3, Ana Mompó 3, Inmaculada Segura 3, Enrique Simó 2 and Joel González-Cabrera 1,* 1 Instituto Universitario de Biotecnología y Biomedicina (BIOTECMED), Universitat de València, Dr. Moliner 50, 46100 Burjassot, Spain; [email protected] (C.S.H.-R.); [email protected] (Ó.M.) 2 Agrupación de Defensa Sanitaria Apícola APIADS, Calle Raval 75B, 46193 Montroi, Spain; [email protected] (F.C.); [email protected] (E.S.) 3 Agrupación de Defensa Sanitaria Apícola APICAL y APIVAL, C/Sants de la Pedra 75, 03830 Muro de Alcoy, Spain; [email protected] (M.J.M.); [email protected] (A.M.); [email protected] (I.S.) * Correspondence: [email protected]; Tel.: +34-963-543-122 Simple Summary: Varroa destructor, a parasitic mite of Apis mellifera, is causing severe damages to honey bee colonies worldwide. There are very few acaricides available to manage the parasite, and so the evolution of the mite’s resistance to acaricides poses a serious threat to controlling the mite. Using a combined approach that includes bioassays and genotyping, we estimated the expected efficacy of the treatments with acaricide products based on coumaphos, amitraz, and pyrethroids in apiaries from one of the most important beekeeping regions in Spain. This information was shared with the beekeeping community so that they can take informed and scientific-based decisions in the most convenient way to manage the parasite. Abstract: Varroa destructor is an ectoparasitic mite causing devastating damages to honey bee colonies Citation: Hernández-Rodríguez, C.S.; around the world. Its impact is considered a major factor contributing to the significant seasonal losses Marín, Ó.; Calatayud, F.; Mahiques, of colonies recorded every year. Beekeepers usually rely on a reduced set of acaricides to manage the M.J.; Mompó, A.; Segura, I.; Simó, E.; parasite, usually the pyrethroids tau-fluvalinate or flumethrin, the organophosphate coumaphos, and González-Cabrera, J. Large-Scale the formamidine amitraz. However, the evolution of resistance in the mite populations is leading Monitoring of Resistance to to an unsustainable scenario with almost no alternatives to reach an adequate control of the mite. Coumaphos, Amitraz, and Here, we present the results from the first large-scale and extensive monitoring of the susceptibility Pyrethroids in Varroa destructor. to acaricides in the Comunitat Valenciana, one of the most prominent apicultural regions in Spain. Insects 2021, 12, 27. https://doi.org/ Our ultimate goal is to provide beekeepers with timely information to help them decide what would 10.3390/insects12010027 be the best alternative for a long-term control of the mites in their apiaries. Our data show that there is a significant variation in the expected efficacy of coumaphos and pyrethroids across the Received: 23 November 2020 Accepted: 30 December 2020 region, indicating the presence of a different ratio of resistant individuals to these acaricides in each Published: 4 January 2021 population. On the other hand, the expected efficacy of amitraz was more consistent, though slightly below the expected efficacy according to the label. Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional clai- Keywords: acaricides; honey bees; bioassay; TaqMan; genotyping; acaricide resistance ms in published maps and institutio- nal affiliations. 1. Introduction Copyright: © 2021 by the authors. Li- The ectoparasitic mite Varroa destructor is considered a major pest of the Western honey censee MDPI, Basel, Switzerland. bee (Apis mellifera L.) [1]. This mite feeds mostly on the fat body of immature and adult bees This article is an open access article and vectors numerous lethal viruses, compromising the natural honey bee defenses [2,3]. distributed under the terms and con- These severe damages make V. destructor one of the main factors contributing to the many ditions of the Creative Commons At- seasonal losses of honey bee colonies around the world [4,5]. tribution (CC BY) license (https:// Varroa destructor shifted its host from the Eastern honey bee (Apis cerana F.) to the West- creativecommons.org/licenses/by/ ern honey bee in the late 1950s in Asia, but at present it is widely distributed throughout 4.0/). Insects 2021, 12, 27. https://doi.org/10.3390/insects12010027 https://www.mdpi.com/journal/insects Insects 2021, 12, 27 2 of 12 the world [6]. In Spain, V. destructor was first detected in 1985, and currently it can be found all over the country [7,8]. Varroa destructor reproduces throughout the spring and summer, and so the population is larger in autumn. Thus, treatments to control the mite are usually applied in that season to increase the possibility of overwintering success [9]. In this country, as in many others, it is mandatory to apply at least one acaricide treatment per year to manage the parasite (Royal Decree RD608/2006). However, beekeepers usually perform at least another treatment in the summer in case they detect mites in their colonies. The acaricides authorized to control V. destructor in Spain include “hard acaricides” (based on pyrethroids such as tau-fluvalinate and flumethrin, the formamidine amitraz, and the organophosphate coumaphos), together with “soft acaricides” (mostly based on formic or oxalic acid or the essential oil thymol) ([10]; www.aemps.gob.es/). Integrated pest management (IPM) strategies encourage the combined use of both types of acaricides and other beekeeping practices to reach better long-term control of the mite, but beekeepers have relied mainly on hard acaricides because they are faster and usually more effective [1]. The intensive use of pyrethroids to control Varroa for decades resulted in the evolution of the mite’s resistance to these acaricides in apiaries from several countries [11–15]. Since the emergence of resistance to pyrethroids, beekeepers switched to coumaphos as the best alternative to control the parasite, but the intensive treatment regime with this compound resulted in the evolution of resistance in many locations as well [16–18]. In this scenario, the alternatives to control V. destructor have been drastically reduced to a treatment with amitraz and soft acaricides. Currently, the extensive use of amitraz exerts an intense selec- tion pressure over populations, threatening them with the evolution of resistance to this compound. Indeed, a reduction in the efficacy of amitraz for Varroa control, which may be associated with the evolution of its resistance, has already been reported elsewhere [19–22]. The mechanism of resistance to pyrethroids in V. destructor is well known. It is as- sociated with mutations at the residue L925 of the major target site for pyrethroids—the voltage-gated sodium channel (VGSC) [12,23–25]. To detect these amino acid substitu- tions, TaqMan allelic discrimination assays have been developed [12,23,24]. This is a high throughput diagnostics technique capable of detecting mutations in individual mites. On the other hand, the molecular mechanisms causing the resistance to coumaphos and amitraz in V. destructor are still unknown, and so the reduction in the efficacy to control V. de- structor in apiaries using treatments based on these two acaricides can only be confirmed by bioassays with a direct exposition of mites to the acaricidal products [16–18]. The European Union (EU) is the world’s second largest honey producer after China. Spain is the country with the highest number of hives in Europe (more than three million hives) and the second largest producer of honey in the EU, with almost 30,000 tons per year (https://ec.europa.eu/info/food-farming-fisheries/animals-and-animal-products/ animal-products/honey_en). The Comunitat Valenciana is a Spanish extensive region of 23,255 km2 comprising three provinces with an important professionalized beekeeping sector. It has 358,327 hives in 2459 beekeeping operations, being the region with the second highest honey production in Spain. Almost all the hives (98%) are mobile, carrying out mi- gratory beekeeping throughout the year (https://www.mapa.gob.es/es/ganaderia/temas/ produccion-y-mercados-ganaderos/indicadoreseconomicossectordelamiel2018comentarios_ tcm30-419675.pdf). Despite the treatments to manage the mite, Varroa parasitism is far from being con- trolled, and it is a persistent problem in all honey-producing countries [1,26]. It seems plausible that the continuous presence of varroosis in beehives around the world is related to the resistance to acaricidal products. In Spain, this correlation has not been confirmed yet since there is no program to track the efficacy of the treatments or the evolution of resistance in Spanish apiaries. The lack of knowledge of the incidence and prevalence of varroosis in all the territories of this country led us to plan a systematic study in the Comunitat Valenciana region. The goal of this study is to determine the efficacy of the three groups of hard acaricides in V. destructor populations from apiaries located throughout the Insects 2021, 12, 27 3 of 12 three provinces of the region. This study is coordinated with the Department of Agricul- ture, Environment, Climate Change and Rural Development of the regional government (Generalitat Valenciana, www.gva.es) and Sanitary Defense Groups (abbreviated as ADS in Spanish) of the beekeeping sector. Our aim is to provide beekeepers with information about the impact of varroosis in their colonies and to estimate the expected efficacy for each apiary of acaricide treatments based on pyrethroids, coumaphos, and amitraz. 2. Materials and Methods 2.1. Mites Varroa destructor females were collected from apiaries located in the three provinces of the Comunitat Valenciana region (Spain): Castellón, Valencia, and Alicante. The collection of mites for this study was carried out in two consecutive annual beekeeping seasons: In the first period, 90 apiaries were sampled from April to July 2018, and in the second period, 89 different apiaries were sampled from November 2018 to July 2019 (Table1, Tables S1 and S2).