The Susceptibility of Varroa Destructor Against Oxalic Acid: a Study Case

Bulletin of Insectology 70 (1): 39-44, 2017 ISSN 1721-8861

The susceptibility of Varroa destructor against oxalic acid: a study case

1,2 1,2 3 1,2 1 Matías D. MAGGI , Natalia DAMIANI , Sergio R. RUFFINENGO , María C. BRASESCO , Nicolás SZAWARSKI , 1,2 4 5 1,2,6 1,2 Giulia MITTON , Fernando MARIANI , Diana SAMMATARO , Silvina QUINTANA , Martín J. EGUARAS 1Centro de Investigación en Abejas Sociales (Laboratorio de Artrópodos), Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Funes, Mar del Plata, Buenos Aires, Argentina 2CONICET, Consejo Nacional de Investigaciones Científicas y Técnicas, Rivadavia, Buenos Aires, Argentina 3Cátedra de Apicultura, Facultad de Ciencias Agrarias, Universidad Nacional de Mar del Plata, Balcarce, Buenos Aires, Argentina 4Beekeeping consultant, Victoria, Entre Ríos, Argentina 5USDA ARS, Carl Hayden Honey Bee Research Center, Tucson, Arizona, USA 6Laboratorio de Biología Molecular, Fares Taie Instituto de Análisis, Mar del Plata, Buenos Aires, Argentina

Abstract

Varroa destructor Anderson et Trueman is an ectoparasitic mite of the honey bee Apis mellifera L. and it must be controlled in managed bee colonies to maintain colony health. Taking into account that these mites are now resistant to synthetic acaricides worldwide, oxalic acid was suggested as an alternative for Varroa control. Oxalic acid is one of the most common natural miticides used against varroosis by spraying and sublimation administration techniques. It is a natural constituent of honey, very ac- tive against the Varroa mite, safe to use for beekeepers, and has no residue problems. Nevertheless, some authors have predicted that the risk of developing resistance to oxalic acid in mites is high. The objective of this research was to assess the susceptibility to oxalic acid of a V. destructor population belonging to a commercial apiary where 64 consecutive control treatments with this acid were performed. Bioassays to assess the oxalic acid susceptibility were performed on two mite populations: (1) a „focal‟ population consisting of mites previously exposed to oxalic acid treatments, and (2) a „naïve‟ population that was never exposed to this acid, which allows setting a reference in the absence of historical data on our „focal‟ mites. The results reported here sug- gest that the Varroa population exposed during 8 successive years to oxalic acid treatments remains susceptible to this acid.

Key words: Varroa destructor, susceptibility, oxalic acid, honey bee.

Introduction ganophosphate) and amitraz (formamidine) in strip formulations as an alternative treatment in areas in which Varroa destructor Anderson et Trueman is an obligate, the fluvalinate resistance prevailed (Elzen et al., 2000; ectoparasitic mite of the European honey bee Apis mel- Elzen and Westervelt, 2002). However, evidence of re- lifera L. (Anderson and Trueman, 2000). Varroa dam- sistance episodes to these pesticides has been also re- age is the main factor that increases the wintering losses ported in Europe and in the Americas (Rodriguez- of bee colonies (Akyol et al., 2006). High infestation Dehaibes et al., 2005; Maggi et al., 2009; 2011). levels may reduce the bee weight and lifespan; cause Natural pesticides based on plant extracts have been malformations; suppress the immune response; and de- suggested as an alternative for the control of this parasi- cline breeding production, adult bee population and tosis at the emergence of resistance (Milani, 1999; Ruf- honey storage in the colony (De Jong et al., 1982; finengo et al., 2014). Imdorf et al. (1999) has proposed Murilhas, 2002; Gregory et al., 2005). Because of that, that, the selection pressure for resistance against these mite population levels in beehives must be maintained “natural miticides”, should be low due to its rapid degra- below the economic injury (Delaplane et al., 2005) by dation inside the colony and its little use by the beekeep- means of acaricide treatments (Rosenkranz et al., 2010). ers which reduce the contact time with mites infesting Historically, synthetic acaricides such as pyrethroids bees. This is completely opposite to what has happened and organophosphates have been preferably chosen for with synthetic acaricides. Therefore, the residual effect Varroa control in the apiaries (Maggi et al., 2008). of a pesticide and their frequency of use in the hive are From the late 1980s to the early 1990s, the efficacy of important factors that modulate the emergence of resis- fluvalinate (pyrethroid) application was close to 100% tant mite populations (Medici et al., 2015). In addition, (Herbert et al., 1988). Its widespread use and, often its most vegetable extracts or even propolis are mixtures of misuse throughout those years have exerted a strong se- more than 150 components (Marcucci, 1995; Imdorf et lective pressure on mite populations. As a result, resis- al., 1999). In this way, it is unlikely that mites generate tant populations emerged in several countries worldwide resistance against all these components at the same time. (Milani, 1995; Elzen et al., 1998; Macedo et al., 2002) Despite their high bioactivity on the mites in laboratory and the treatments are no longer effective. Cross- conditions, very few of them have proven effective when resistance between pyrethroids was reported in V. de- were applied in hives during field trials. Considerable structor populations (Floris et al., 2001; Thompson et variation in local environmental and colony conditions al., 2002). Beekeepers began applying coumaphos (or- can affect the efficacy and make it difficult to predict the

outcome of many treatments. These reasons have dis- possible to detect resistance to these acaricides in ex- couraged beekeepers to use this kind of pesticides as posed mite populations (Maggi et al., 2009; 2010; 2011) over synthetic ones (Imdorf et al., 1999). The solution, before bee losses are reported. often, has been to use an effective single component iso- Theoretical and empirical works have been carried out lated from a natural extract as has occurred with thymol, to develop tactics to delay the emergence of resistance formic acid, oxalic acid (OA), among others (Calderone, in insects or mites (Denholm and Rowland, 1992). In 1999; Floris et al., 2004; Maggi et al., 2016). the case of V. destructor, tactics meant to preserve sus- Oxalic acid is an organic acid, natural constituent of ceptible mites (moderation tactics) avoiding an exces- the honey and very effective against the Varroa mite sive killing of mites, could be more suitable for slowing (Rademacher and Harz, 2006). It has been used by the the selection of resistant mites. The success of these beekeepers in the USA and Europe and the EU regula- strategies depends on the balance between the selection tions have permitted its utilization in biological bee- pressure resulting from the application of acaricides and keeping practices (EU Council Regulation, No. the disadvantage associated with resistance (Milani, 1804/1999). The OA is simple to use, cheap, safe for 1999), although this last is very variable and often small beekeepers, has no residue problems, is well tolerated (Roush and Daly, 1990). Both factors would cause that by bees and it is especially applied in broodless colonies the frequency of resistance genes is reduced in the in- (Mutinelli et al., 1997). These features make that this terval between treatments. However, for Varroa popula- acid is widely and frequently being used in some re- tions, where several generations of mites take place dur- gions. In this way, Milani (2001) has postulated that the ing a brief period of time, even a fitness decrease in the risk of emergence of resistance for OA is high if its use order of a few percent per generation would produce an is prolonged in time. In some European countries, it has appreciable disadvantage (Milani, 1999). On the other already been used intensively and with scarce rotation hand, the selection pressure increases dramatically when with other miticides (Charriére and Imdorf, 2002; the efficacy of treatments approaches 100% and the Nanetti et al., 2003). same acaricides are used repeatedly, or for prolonged Although the mode of action against Varroa mites is periods, reducing correspondingly the effect of any dis- not yet clearly understood, it seems that a direct contact advantage of the resistant strain. An example was the with the low pH of OA solutions has a deleterious effect high number of resistance phenomena to coumaphos on the mites and it would be a key factor (Nanetti and reported in Argentina and Uruguay, due the abusive use Stradi, 1997; Nanetti, 1999; Nanetti et al., 2003). The of this organophosphate (Maggi et al., 2009; 2011; OA has been conventionally applied in hives against the Medici et al., 2015). varroosis by spraying and sublimation administration For this study, we have selected a commercial apiary techniques. It is known that by mixing OA in sugar wa- where a high frequency of treatments with oxalic acid ter, the solution becomes attractive and is better distrib- was exerted during 8 consecutive years. For that, our uted by bees. Thus, it comes into contact easily with main objective was determinate the susceptibility to ox- bees and mites throughout the colony. In addition, Ali- alic acid in this „focal‟ mite population comparing it ano and Ellis (2008) performed a clever experiment: By with a Varroa population never exposed to this com- splitting single colonies with various dividers and ap- pound. We hypothesize that the „focal‟ population suf- plying OA only on one side, they found that it is appar- fered a high selection pressure for OA during the 8 ently transferred through the colony by contact among years. bees, rather than by trophallaxis, and it is well distrib- uted by this way (80% of killed mites on treated side, and 65% on untreated). Recently, in Argentina it has Materials and methods been approved a new commercial formulation based on OA which is applied in cellulose strips that remain 42 A commercial apiary composed by 54 colonies of a re- days within the colonies. Thus, by this application mode gional ecotype of A. mellifera located at Federal and action mechanism, it would be identical to the for- (30°57'4.42"S 58°47'55.78"W: Entre Rios province, Ar- mulations based on synthetic acaricides that many foci gentina) was selected as biological model for the assays. of resistant mites have generated (Maggi et al., 2016). In this apiary, the OA was topically applied as the sole High efficacy, ease of use, extremely low cost, lack of option for Varroa control during eight consecutive years temperature-dependence, and the shortage of alterna- (2000-2008). Thus, this V. destructor population was tives currently make that OA treatments become popu- considered as the „focal‟ population in our study. An lar (Nanetti et al., 2003; Maggi et al., 2016). average of eight treatment applications (± one) per year To date, there are no published data supporting resis- was made. The treatment solution was composed of 4% tance to any organic acid in V. destructor. There is also (w/v) oxalic acid (64 g oxalic acid dehydrate, 500 g little information on the susceptibility of mite popula- sugar, and distilled water to 1000 mL). The average tions that have ever been in contact with OA. This kind dosage was 50 mL per hive (5 mL per comb covered by of data would be useful for the design of studies esti- bees) applied on the top of each frame. According to the mating possible changes of susceptibility to this acid in beekeeper‟s personal observation, the colonies managed Varroa populations as it has occurred with the synthetic by applying only OA treatments must be maintained miticides. For example, the available data on the toxic- with a mite infestation level below than 2% to avoid ity to coumaphos and amitraz in mites from never colony weakness and honey production reduction. Thus, treated populations (Maggi et al., 2008; 2011) has made the monitoring of Varroa infestation levels was monthly

performed following the protocol of Marcangeli (2000). mites from the paper to a new clean Petri dish (Damiani The method involves collecting among 200 to 300 bees et al., 2010). Three adult bees were added to each of taken from both sides of three brood frames of each these new dishes as food for mites. Controls were made hive, in a container with water and detergent. After the by treating mites with a 55% (w/v) ethanol solution dur- sample shaking, mites are separated from bees. The per- ing the same contact time. Five replicates for each ex- centage of infestation is obtained by dividing the num- perimental unit were run. All treatments were carried ber of mites by the total number of sampled bees per out at room temperature (22-24 °C) and the treated one hundred. mites were incubated at 28 ± 1 °C and 60% RH. After A V. destructor population from Mar del Plata 24 h, mortality was evaluated by gently prodding each (38°10'06"S 57°38'10"W: Buenos Aires province, Ar- mite with a narrow paintbrush under a dissecting micro- gentina) which had never been treated with OA neither scope; lack of response to consecutive stimulus over other organic acid was used as control group. Thus, 1 min was considered an indication of death. Bioassays these mites constituted a „naïve‟ population. Varroa for the „naïve‟ mite population (Mar del Plata) and the control in this apiary was always performed using cou- „focal‟ mite population (Federal) were conducted simul- maphos, flumethrin, or amitraz in a rotation scheme. taneously. A χ2 test was carried out to determine statisti- The apiaries were at 900 km distance between them but cal differences on mite mortality between both popula- nevertheless both had similar geographic and climatic tions. This method has already been successfully ap- conditions. The queens, which were provided by a re- plied to evaluate the toxicity of propolis and ethanol bo- gional beekeeping house, do not stop egg laying during tanical extracts against populations of V. destructor winter in these regions. Both apiaries had an inter- (Damiani et al., 2010; 2014). localities distance higher than 5 km with regard to other apiaries, minimizing the bee drifting and the possibility of re-infestations. Results To perform the bioassays, five colonies from each apiary were selected during spring 2008. Capped brood Oxalic acid control on field combs were removed from these colonies and trans- The infestation level of V. destructor on adult bees be- ported to the laboratory. Mites were collected by opening fore and after applications of OA in the commercial api- and inspecting individual brood cells. Only mature fe- ary from Federal city during the year preceding the bio- male mites were sampled and transferred with a slender assay are reported in table 1. moistened paint brush to a plastic Petri dish with bee lar- vae as food for mites. Thus, they were kept at 28 ± 1 °C Bioassays and 70% RH for 1-3 h until used. A pool of mites from The percentage of dead mites recorded 24 h after ox- each population was exposed to increasing concentra- alic acid treatments for both mite populations is reported tions of OA (Cicarelli Laboratory, Argentina, Pro- in figure 1. For all the tested acaricide concentrations, analysis) solutions (0.05; 0.1; 0.2; and 0.4% w/v) pre- the mortality of mites from the „focal‟ population (Fed- pared in 55% (w/v) ethanol solution. These treatment eral city) was higher than from the „naïve‟ mite popula- concentrations were selected according to Toomemaa et tion (Mar del Plata city) (p < 0.01, figure 1). These mor- al. (2010). For treatment, 200 µl of each specified con- tality percentages were between 2.8-fold and 7.2-fold centration of OA was applied over six mites that remain higher for the „focal‟ mite population than for the con- on a piece of filter paper (3 × 3 cm). After 45 s, the control mite population. Mite mortality in controls was al- tact with the solution was stopped by removing the ways below than 10%.

Table 1. Infestation levels of V. destructor on adult bees before and after treatment applications of oxalic acid in the „focal‟ apiary studied.

Treatment V. destructor infestation on adult bees (%) Date of treatment application application event Before treatment a After treatment b 1 2 (0.98) 1.7 (1.1) 08/23/2006 2 1.7 (0.65) 0.9 (0.45) 08/30/2006 3 1.6 (1.1) 1.1 (0.58) 01/24/2007 4 1.1 (0.62) 0.4 (0.3) 01/31/2007 5 8.1 (3.57) 6 (2.6) 04/15/2007 6 6 (3.1) 4.8 (2.35) 04/22/2007 7 4.8 (2.35) 1.2 (0.73) 04/29/2007 8 3.9 (2.5) 0.5 (0.15) 06/15/2007

Varroa infestation rates were estimated in 10 of the 54 colonies each time. The mean values reported here represent the mite infestation in the apiary managed only with OA treatments during 8 successive years. Standard deviations are reported in brackets. a The same day of the treatment application. b One week after treatment.

yield different susceptibility levels to pesticides depend- ing on geographic location (Watkins, 1997). For example, although the LC50 values for amitraz on the mites from the study of Elzen et al. (2000) was 164 times higher than for mites from the Maggi et al. (2008) study, both Varroa populations were considered susceptible to the acaricide. Nevertheless, it is important to mention that we only were able to estimate the susceptibility to OA of the Varroa population from Federal apiary, after 8 years of continuous exposition to this acaricide (we could not sample in previous years). It would be interesting to have a study of susceptibility in this population, at time zero and year by year. Probably, with this kind of data, we might have been able to detect a possible change in the toxicity of OA on the mites through the

years. But even if this had happened, we hold that this = Mar del Plata apiary never treated with OA mite population would be even susceptible to the OA, = Federal apiary treated with OA for 8 successive years taking into account the bioassay results reported here and Figure 1. Mortality of V. destructor from both mite the efficacy in field conditions (table 1) that allowed populations subjected to increased concentrations of A. mellifera colonies to survive year after year. If the re- oxalic acid. Mite mortality is expressed as percentage sistance phenomenon to the OA has happened in the „fo- of dead individual ± standard deviation after 24 h of cal‟ apiary, we should find a significant change in the oxalic acid treatment. Five replicates for each experi- susceptibility compared to the „naïve‟ mites. Several mental unit were run (N = 125). P value from χ2 test studies have reported that susceptibility to an acaricide is compare mite mortality in both populations within the abruptly decreased when the Varroa mites develop resis- same treatment concentration. tance to it. A decreased susceptibility to amitraz around the order of 175 times was reported in Mexico (Rodri- guez-Dehaibes et al., 2005). For Europa, similar results Discussion and conclusions related to a reduction in the susceptibility to fluvalinate were published (Milani, 1995, Thompson et al., 2002). In this study, we found an ideal biological framework to In this context, our results indicate that there were no test the hypothesis of Milani (2001). This author postu- changes in susceptibility to OA in the „focal‟ mite popu- lated that the risk of emergence of resistance for the oxalic lation during the field treatments, and consequently, our acid is high if its use is prolonged in time. Here, we have main hypothesis could not be accepted: “the risk of compared the susceptibility of a Varroa population ex- emergence of resistance for the OA is high if its use is posed to 64 consecutive treatments with OA („focal‟ mite prolonged in time” (Milani, 2001). population) versus the susceptibility of a Varroa popula- Why a Varroa population does not modify their sus- tion never exposed to it („naïve‟ mite population) in order ceptibility against such selective pressure is an interest- to evaluate if resistance episodes to the OA happened. ing focus for future investigations. It is possible that the The methodology employed in the present study for mode of action of this acaricide and stability inside the conducting the bioassay allowed us to satisfactorily as- colony are the key to understanding the results presented sess the bioactivity of the OA on treated mites. Fur- here. A combination between the deleterious effects on thermore, it proved be quick and easy to implement mites by means of the low pH in aqueous solution when it was previously used for the evaluation of mor- (Nanetti and Stradi, 1997; Nanetti, 1999; Nanetti et al., tality by drugs against V. destructor populations. The 2003) and its fast degradation within the colony (Maggi mite mortality in control treatments did not exceed 10%, et al., 2016) generate that the selection pressure exerted thus complying with the standard norms for trials set in the „focal‟ Varroa population could be still lower forth by OECD (1998a; 1998b) and EPPO (2001). compared with the selection pressure generated by a syn- In our bioassay, the acaricide toxicity of the OA was thetic drug such as fluvalinate or coumaphos. Compared higher for mites from the „focal‟ apiary (Federal city) to OA, these acaricides possess a high stability and re- than for those mites belonging to the „naïve‟ population sidual effect inside honey bee colonies and a different never exposed to the OA (Mar del Plata city). These re- mode of action, killing mites by an over stimulation of sults indicate that the Varroa population from the Fed- its central nervous system (Wang et al., 2002; Johnson et eral apiary remained susceptible to the OA despite its al., 2009). A recent study has established a positive cor- prolonged use in time. It was also interesting that this relation between coumaphos residues in wax and the de- „focal‟ population resulted more susceptible to the acid velopment of Varroa resistance (Medici et al., 2015). than the „naïve‟ population with mites never exposed to However, residues of OA are not detected in wax after it. A possible explanation for these results is that mite acaricide treatments with this acid (Maggi et al., 2016). populations on which the toxic effects from a chemical Further investigations are needed to elucidate and under- are analyzed can show different degrees of natural sus- stand the mechanism of Varroa tolerance to the OA. ceptibility, a feature inherent of any population with re- In the case of V. destructor, tactics meant to preserve spect to a given variable: different mite populations may susceptible mites (“moderation tactics”), avoiding an

excessive death of mites, are suggested for slowing the References selection of resistant mites (Milani, 1999). The success of these tactics depends on the balance between the se- AKYOL E., KARATEPE B., KARATEPE M., KARAER Z., 2006.- lection pressure resulting from the application of acari- Development and control of the Varroa (Varroa destructor) cides and possible disadvantages associated with the re- in honey bee (Apis mellifera L.) colonies and effects on the colony productivity.- Uludag Bee Journal, 6 (4): 149-154. sistance, which would cause that the frequency of genes ALIANO N., ELLIS M., 2008.- Bee-to-bee contact drives oxalic acid for resistance decreases in the interval between treat- distribution in honey bee colonies.- Apidologie, 39 (5): 481-487. ments. The OA applied in sucrose solution would be a ANDERSON D., TRUEMAN J., 2000.- Varroa jacobsoni is more than reliable tool for a moderation tactic. First, if OA is ap- one species.- Experimental and Applied Acarology, 24: 165-189. plied only in one dose (as it was applied in the apiary BACANDRITSOS N., PAPANASTASIOU I., SAITANIS C., NANETTI studied here) its efficacy is rarely higher than 90%. Sev- A., ROINIOTI E., 2007.- Efficacy of repeated trickle applica- eral researchers have documented that OA should be tions of oxalic acid in syrup for varroosis control in Apis applied more than two times to obtain higher efficacy mellifera: influence of meteorological conditions and pres- (reviewed in Rademacher and Hartz, 2006). For the ence of brood.- Veterinary parasitology, 148 (2): 174-178. CALDERONE N., 1999.- Evaluation of formic acid and a thy- other hand, the efficacy could be even lower accord- mol-based blend of natural products for the fall control of ingly of the climate and the presence of brood, further Varroa jacobsoni (Acari: Varroidae) in colonies of Apis reducing the aggressiveness of treatment against mites mellifera (Hymenoptera: Apidae).- Journal of Economic En- (Bacandritsos et al., 2007). Second, the fast degradation tomology, 92 (2): 253-260. and the low residual of the OA make this product an in- CHARRIÉRE J., IMDORF A., 2002.- Oxalic acid treatment by teresting alternative acaricide. By this way, organic trickling against Varroa destructor: recommendations for miticides exert lower selection pressure against Varroa use in central Europe and under temperate climate condi- populations compared to synthetic compounds. In Ar- tions.- Bee world, 83 (2): 51-60. gentina, the low selection pressures exerted by organic DAMIANI N., FERNÁNDEZ N., MALDONADO L., ÁLVAREZ A., EGUARAS M., MARCANGELI J., 2010.- Bioactivity of propolis miticides is due to that this kinds of products is not used from different geographical origins on Varroa destructor very often by beekeepers, even when resistance phe- (Acari: Varroidae).- Parasitology Research, 107: 31-37. nomena to synthetic chemicals have been widely re- DAMIANI N., FERNÁNDEZ N., PORRINI M., GENDE L., ÁLVAREZ ported (Maggi et al., 2009; 2010). E., BUFFA F., BRASESCO C., MAGGI M., MARCÁNGELI J., Although no changes in susceptibility were detected in EGUARAS M., 2014.- Laurel leaf extracts for honey bee pest this research, it is strongly recommended to rotate this and disease management: antimicrobial, microsporicidal, and acaricide with other miticides and with nonchemical acaricidal activity.- Parasitology Research, 113: 701-709. control techniques applied in different seasons of the DE JONG D., DE JONG P., GONCALVEZ L., 1982.- Weight loss year, each one acting for a restricted period of time. In and other damage to developing worker honeybees (Apis mellifera) due to infestation with Varroa jacobsoni.- Journal Italy, since the spread of fluvalinate-resistant V. destruc- of Apicultural Research, 2: 165-167. tor strains, many beekeepers control this mite with a DELAPLANE K., BERRY J., SKINNER J., PARKMAN J., HOOD M., combination of essential oils at the end of summer and a 2005.- Integrated pest management against Varroa destruc- single treatment with an organophosphorus acaricide or tor reduces colony mite levels and delays treatment thresh- the OA in late autumn. A similar treatment schedule old.- Journal of Apicultural Research, 44: 157-162. based on acaricide rotation aimed to avoid resistance DENHOLM I., ROWLAND M., 1992.- Tactics for managing pes- episodes to the OA could be applied here. Regional in- ticide resistance in arthropods: theory and practice.- Annual tegrated pest management programs against V. destruc- Review of Entomology, 37: 91-112. tor should be performed in Argentina considering the ELLIS J., DELAPLANE K., HOOD W., 2001.- Efficacy of a bot- tom screen device, Apistan and Apilife VAR in controlling use or misuse of the available acaricides. Implicit in this Varroa destructor.- American Bee Journal, 141: 813-816. approach remain expectations that multiple tactics (1) ELZEN P., WESTERVELT D., 2002.- Detection of coumaphos reduce the likelihood that pests evolve resistance to any resistance in Varroa destructor in Florida.- American Bee one miticide, or (2) interact such that control is en- Journal, 142: 291-292. hanced or that a compensatory control can be provided ELZEN P., EISCHEN F., BAXTER J., PETTIS J., ELZEN G., WILSON W., if one component fails (Ellis et al., 2001; Rinderer et al., 1998.- Fluvalinate resistance in Varroa jacobsoni from several 2004; Sammataro et al., 2004; Delaplane et al., 2005). geographic locations.- American Bee Journal, 138: 674-676. ELZEN P., BAXTER J., SPIVAK M., WILSON W., 2000.- Control of Varroa jacobsoni Oud. resistant to fluvalinate and amitraz using coumaphos.- Apidologie, 31: 437-441. Acknowledgements EPPO, 2001.- PP 1/170 (3). Test methods for evaluating the side effects of plant protection products on honeybees.- Bul- The authors would like to thank the Universidad Na- letin OEPP/EPPO Bulletin, 31: 323-330. cional de Mar del Plata (UNMDP) and Consejo Na- FLORIS I., CABRAS P., GARAU V., MINELLI E., SATTA A., cional de Investigaciones Científicas y Técnicas TROULLIER J., 2001.- Persistence and effectiveness of pyre- (CONICET). This research was supported by a grant of throids in plastics strips against Varroa jacobsoni (Acari: ANPCyT, PICT 2011 to Matías D. Maggi. None of the Varroidae) and mite resistance in a Mediterranean area.- authors has any financial or personal relationships that Journal of Economic Entomology, 94: 806-810. could inappropriately influence or bias the content of FLORIS I., SATTA A., CABRAS P., GARAU V., ANGIONI A., 2004.- Comparison between two thymol formulations in the control the paper. Matías D. Maggi and Natalia Damiani con- of Varroa destructor: effectiveness, persistence, and residues.- tributed equally to this work. Journal of Economic Entomology, 97 (2): 187-191.

GREGORY P., EVANS J., RINDERER T., DE GUZMAN L., 2005.- ties. Concerted Action 3686. November 13-14, 1999. Agri- Conditional immune-gene suppression of honeybees parasi- cultural Research Centre-Ghent, Merelbeke, Belgium. tized by Varroa mites.- Journal of Insect Science, 5: 7-11. NANETTI A., STRADI G., 1997.- Varroasi: trattamento chimico HERBERT W., WITTHERELL P., SHIMANUKI H., 1988.- Control con acido ossalico in sciroppo zuccherino.- L’Ape Nostra of Varroa jacobsoni in queen cages and small laboratory Amica, 19 (5): 6-14. ® cages using amitraz, fluvalinate and Apitol .- American Bee NANETTI A., BÜCHLER R., CHARRIÈRE J., FRIES I., HELLAND S., Journal, 128 (4): 289-292. IMDORF A., KORPELA P., KRISTIANSEN P., 2003.- Oxalic acid IMDORF A., BOGNANOV S., IBAÑEZ OCHOA R., CALDERONE N., treatments for varroa control.- Apiacta, 38 (1): 81-87. 1999.- Use of essential oils for the control of Varroa jacob- OECD, 1998a.- Test No. 213: Honeybees, acute oral toxicity soni Oud. in honey bee colonies.- Apidologie, 30: 209-228. test.- OECD Publishing, Paris, France. JOHNSON R., POLLOCK H., BERENBAUM M., 2009.- Synergistic OECD, 1998b.- Test No. 214: Honeybees, acute contact toxic- interactions between in-hive miticides in Apis mellifera.- ity test.- OECD Publishing, Paris, France. Journal of Economic Entomology, 102 (2): 474-479. RADEMACHER E., HARZ M., 2006.- Oxalic acid for the control MACEDO P., ELLIS M., SIEGFRIED B., 2002.- Detection and of varroosis in honey bee colonies - a review.- Apidologie, quantification of fluvalinate resistance in Varroa mites in 37: 98-120. Nebraska.- American Bee Journal, 147: 523-526. RINDERER T., DE GUZMAN L., HARPER C., 2004.- The effects MAGGI M., RUFFINENGO S., GENDE L., EGUARAS N., SARDELLA of co-mingled Russian and Italian honey bee stocks and N., 2008.- LC50 baseline levels of amitraz, coumaphos, flu- sunny or shaded apiaries on varroa mite infestation level, valinate and flumethrin in populations of Varroa destructor worker bee population and honey production.- American from Buenos Aires Province, Argentina.- Journal of Apicul- Bee Journal, 144: 481-485. tural Research, 47 (4): 292-295. RODRIGUEZ-DEHAIBES S., OTERO-COLINA G., SEDAS V., MAGGI M., RUFFINENGO S., DAMIANI N., SARDELLA N., JIMENEZ J., 2005.- Resistance to amitraz and flumethrin in EGUARAS M., 2009.- A first detection of Varroa destructor Varroa destructor populations from Veracruz, Mexico.- resistance to coumaphos in Argentina.- Experimental and Journal of Apicultural Research, 44: 124-125. Applied Acarology, 47 (4): 317-320. ROSENKRANZ P., AUMEIER P., ZIEGELMANN B., 2010.-Biology MAGGI M., RUFFINENGO S., NEGRI P., EGUARAS M., 2010.- and control of Varroa destructor.- Journal of Invertebrate Resistance phenomena to amitraz from populations of the Pathology, 103: S96-S119. ectoparasitic mite Varroa destructor of Argentina.- Parasi- ROUSH R., DALY J., 1990.- The role of population genetics in tology Research, 107: 1189-1192. resistance research and management, pp. 97-152. In: Pesti- MAGGI M., RUFFINENGO S., YAMANDÚ M., OJEDA P., RAMALLO cide resistance in arthropods (ROUSH R., TABASHNIK B., G., FLORIS I., EGUARAS M., 2011.- Susceptibility of Varroa Eds).- Chapman and Hall, New York, USA. destructor (Acari: Varroidae) to synthetic acaricides in Uru- RUFFINENGO S. R., MAGGI M. D., FUSELLI S., DE PIANO F. G., guay: Varroa mites‟ potential to develop acaricide resis- NEGRI P., BRASESCO C., SATTA A., FLORIS I., EGUARAS M. J., tance.- Parasitology Research, 108: 815-821. 2014.- Bioactivity of microencapsulated essentials oils and MAGGI M., TOURN E., NEGRI P., SZAWARSKI N., MARCONI A., perspectives of their use in the control of Varroa destructor.- GALLEZ L., MEDICI S., RUFFINENGO S., BRASESCO C., DE Bulletin of Insectology, 67 (1); 81-86. FEUDIS L., QUINTANA S., SAMMATARO D., EGUARAS M., SAMMATARO D., DEGRANDI-HOFFMAN G., WARDELL G., 2016.- A new formulation of oxalic acid for Varroa destruc- FINLEY J., OSTIGUY N., 2004.- Testing a combination of con- tor control applied in Apis mellifera colonies in the presence trol tactics to manage Varroa destructor (Acari: Varroidae) of brood.- Apidologie, 47 (4): 596-605. population levels in honey bee (Hymenoptera: Apidae) MARCANGELI J., 2000.- Aplicación de una nueva técnica para colonies.- International Journal of Acarology, 30: 71-76. determinar los niveles de infección de Varroa jacobsoni en THOMPSON H., BROWN M., BALL R., BEW M., 2002.- First re- colmenas de Apis mellifera.- Natura Neotropicalis, 31 (1-2): port of Varroa destructor resistance to pyrethroids in the 81-85. UK.- Apidologie, 33: 357-366. MARCUCCI M., 1995.- Propolis: chemical composition, bio- TOOMEMAA K., MARTIN A., MÄND M., WILLIAMS I., 2010.- logical properties and therapeutic activity.- Apidologie, 26 Using oxalic acid in water solution in control of Varroa (2): 83-99. mites and its influence on honey bees.- Agronomy Research, MEDICI S., MAGGI M., SARLO E., RUFFINENGO S., MARIOLI J., 8 (Special Issue II): 345-350. EGUARAS M., 2015.- The presence of synthetic acaricides in WANG R., LIU Z., DONG K., ELZEN P., PETTIS J., HUANG Z., beeswax and its influence on the development of resistance 2002.- Association of novel mutations in a sodium channel in Varroa destructor.- Journal of Apicultural Research, 54 gene with fluvalinate resistance in the mite, Varroa destruc- (3): 267-274. tor.- Journal of Apicultural Research, 41: 17-25. MILANI N., 1995.- The resistance of Varroa jacobsoni Oud. to WATKINS M., 1997.- Resistance and its relevance to beekeep- pyrethroids: a laboratory assay.- Apidologie, 26: 415-429. ing.- BeeWorld, 78: 15-22. MILANI N., 1999.- The resistance of Varroa jacobsoni Oud. to

acaricides.- Apidologie, 30: 229-234. MILANI N., 2001.- Activity of oxalic and citric acids on the Authors’ addresses: Matías D. MAGGI (corresponding au- mite Varroa destructor in laboratory assays.- Apidologie, 32: 127-138. thor, e-mail: [email protected]), Natalia DAMIANI, María C. BRASESCO, Nicolás SZAWARSKI, Giulia MITTON, Silvina MURILHAS A., 2002.- Varroa destructor infestation impact on Apis mellifera carnica capped worker brood production, bee QUINTANA, Martín J. EGUARAS, Centro de Investigación en A- population and honey storage in a Mediterranean climate.- bejas Sociales, Facultad de Ciencias Exactas y Naturales, Uni- Apidologie, 33: 271-281. versidad Nacional de Mar del Plata, Funes 3350, 7600 Mar del MUTINELLI F., BAGGIO A., CAPOLONGO F., PIRO R., PRANDIN Plata, Buenos Aires, Argentina; Sergio R. RUFFINENGO, Cátedra L., BIASION L., 1997.- A scientific note on oxalic acid by de Apicultura, Facultad de Ciencias Agrarias, Universidad Na- topical application for the control of varroasis.- Apidologie, cional de Mar del Plata, 7620 Balcarce, Buenos Aires, Argenti- 28: 461-462. na; Fernando MARIANI, Beekeeping consultant, 3153 Victoria, NANETTI A., 1999.- Oxalic acid for mite control - results and Entre Ríos, Argentina; Diana SAMMATARO, USDA ARS, Carl review, pp. 6-11. In: Coordination in Europe of research on Hayden Honey Bee Research Center, Tucson, Arizona, USA. integrated control of Varroa mites in honey bee colonies. Proceedings of the Commission of the European Communi- Received April 18, 2015. Accepted November 17, 2016.