Mode of application of acaricides against the ectoparasitic mite (Varroa destructor) infesting honeybee colonies, determines their efficiencies and residues in honey and beeswax

1 Introduction

Honeybees provides highly valued pollination services for a wide variety of agricultural crops (Al Naggar et al., 2018). However, over the last few years, a lot of studies have been published about the losses of honeybee colonies and the declining population of native and wild bees (Goulson et al., 2015; Jacques et al., 2017). Several factors are implicated behind these losses including pathogens and parasites, poor nutrition, beekeeping management, climate change, and pesticides (Kerr et al., 2015; Al Naggar and Baer, 2019). However, a combination of Varroa and viruses are now frequently implicated in collapsing colonies (Martin et al., 2012).

The parasitic mite, Varroa destructor, became the single greatest threat to honey bee health, the apiculture and pollination industries, since it spread from its native host, the Asian honey bee (Apis cerana) to the naive European honey bee (A. mellifera). (reviewed in, Traynor et al., 2020). Varroa infestation occur mainly during winter and causes significant impacts on honeybee colony health as a consequence of its transmission of a cocktail of viruses while feeding on honeybee haemolymph and fat bodies (Martin et al., 2012; Wilfert et al., 2016; Ramsey et al., 2019). Highly infested weak colonies facilitate mite dispersal and disease transmission to stronger and healthier colonies. In untreated honeybee colonies, heavy Varroa infestation causes 100% mortality in a few weeks (Kanga et al., 2010; Rosenkranz et al., 2010).

Chemical treatments against V. destructor are almost inevitable for commercial beekeeping. Synthetic acaricides such as fluvalinate, flumethrin, amitraz, coumaphos, and cymiazole have been successfully used to control V. destructor (Al Naggar et al., 2016; Gracia et al., 2017). However, the use of acaricides inside beehives implies a risk of contamination of honey and other hive products (beeswax for example) (Wallner, 1999; Martel et al., 2007; Calatayud-Vernich et al., 2018). Acaricides mostly contaminate beeswax due to its non-polar nature, while honey remains relatively free of contaminants (Calatayud-Vernich et al., 2018).

Honeybees colonies require regular Varroa control during the whole year which means repeated application of varroacides that led to contamination of many hive products (Abou-Shaara, 2014; Pohorecka et al., 2018) Moreover, the effect of using these acaricides to control Varroa mites has long been a concern to the beekeeping industry due to unintended negative impacts on honeybee health (reviewed in Tihelka, 2018). Thus, selecting the best acaricide application or delivery method became a necessity not only for controlling Varroa mite but also to safe bees from unintended negative impacts and minimizing the residues accumulated in honey and other bee matrices. Moreover, beekeepers all over the world pay a great attention to novel treatments and application methods of acaricides due to the developed resistance of honeybees against these inhive chemicals (Tihelka, 2018).

Here we assessed the effect of the different mode of application on efficiency of two acaricides; Mavrik 2F (fluvalinate) and Vapcozin-20 (amitraz) against Varroa mite in brood and adult honeybees. To do so, we applied these varroacides using strips or direct spraying or carton paper methods. We also quantified acaricides residues in honey and beeswax at different time points to investigate the effect of the delivery method on acaricides levels.

2 Materials and methods

2.1

2.1 Acaricides

We used two commercial acaricides: Mavrik 2F (22% w/w of fluvalinate as active ingredient), produced by AAKO B.V.- Holland and Vapcozin-20 (20% amitraz as active ingredient), produced by Veterinary and Agricultural products Mfg. Co. Ltd – Jordan. The two chemical acaricides were used in concentration 2% (adding 20 mL/litter of water) for controlling Varroa mites. Treatments were applied in three forms: 1) as stripes of cotton (2 × 20 cm) which impregnated for 24 h in each tested acaricide solution and held in one comb between the brood nests of each tested colonies; 2) with direct spraying (5 mL for each colony) on the honey bee combs including mature and immature stages of bees and 3) using carton paper (20 × 20 cm) which impregnated for 5 min in each tested acaricide solution and put directly on the top of brood combs (Fig. 1). All treatments were applied twice only at month interval.

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Fig. 1

Application methods of acaricides against Varroa mite. A) Cotton strips; (2 × 20 cm) which impregnated for 24 h in each tested acaricide solution and held in one comb between the brood nests. B) Spraying (5 mL of each acaricide solution for each colony) on the honey bee combs including mature and immature stages of bees and C) Cartoon paper: (20 × 20 cm) which impregnated for 5 min in each tested acaricide solution and put directly on the top of brood combs.

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3 Results

3.1

3.1 Efficiency of tested acaricides against Varroa infestation

At the beginning of the experiment, mean rates of infestation with phoretic Varroa destructor of adult and pupae (brood) of honeybee workers of all experimental colonies were 29.52 MPHB and 29.47 MPFP, respectively. There were no significant differences (p > 0.05) in rates of infestation with V. destructor among replicates assigned to experimental groups prior to treatment. The infestation rates with Varroa mite decreased significantly (P < 0.05) in brood and adult honeybee workers of colonies treated with Mavrik 2F as compared to only non-treated (control) colonies (Fig. 2a, c). The different application methods of Mavrik 2F against V. destructor showed however, relatively similar efficiencies in both brood and adult honeybees that range from 93.8 to 100 (Table 1).

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Fig. 2

Rate of infestation with V. destructor (mean ± SD; n = 3 colonies) of honey bee brood and adult before and after treatment with Mavrik 2F and Vapcozin-20 acaricides. Means with different letters are significantly different (one-way ANOVA with tukey post hoc test, p < 0.05).

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Table 1 Efficiencies (%) of different methods of application of Mavrik 2F (fluvalinate) and Vapcozin-20 (amitraz) acaricides against varroa mite infesting brood and adult honeybee workers.

Acaricide/M. of Application	Mavrik 2F		Vapcozin-20
	Brood	Adult	Brood	Adult
C. Strips	100	97.51	83.75	90.94
Spraying	100	100	100	90.66
C. Paper	100	93.81	88.7	96.37

Similarly, the mean rates of infestation with Varroa mite decreased significantly (P < 0.05) in both brood and adult honey bee workers of treated colonies with Vapcozin-20 using the three different application methods compared to non- treated control colonies, with efficiencies ranged from 83.75 to 96.37 (Table 1; Fig. 2b, d). In the other hand, the direct spraying of Vapcozin-20 on honeybee colonies was the only method that significantly suppressed the level of Varroa infestation in honeybee brood with 100% efficiency (Table 1; Fig. 2b). There were no significant differences in terms of efficiency against Varroa mites between the different application methods of either Mavrik 2F or Vapcozin-20 acaricides (Table 1).

4 Discussion

Varroa mite (Varroa destructor) represents the major challenge for beekeeping worldwide. Various methods and materials have been therefore suggested and tested for its control that included plant extracts, essential oils, biological agents, mechanical methods, and some chemicals (Abou-Shaara, 2014; Abou-Shaara et al., 2016; Gajger et al., 2020; Masry et al., 2020). Here, we tested the efficiency of two acaricides Mavrik 2f (fluvalinate) and Vapcozin-20 (amitraz) against Varroa mite using three different application methods (direct spraying, cotton strips and carton paper) and quantified their residues in honey and beeswax. Both acaricides showed great efficacy against Varroa mites regardless of the application method. However, the use of carton papers was the most appropriate application method for these compounds because it showed very minimal acaricides residues in honey and beeswax.

Our results showed clearly that Mavrik 2F and Vapcozin-20 are highly effective against Varroa mites over the winter in Egypt. Although the different application methods of both acaricides showed relatively similar efficiencies against Varroa mite, the direct spraying method demonstrated the best efficiency. Our results are in agreement with Sajid et al. (2020) who evaluates the effectiveness of five miticides (fluvalinate, flumethrin, amitraz, formic acid, and oxalic acid) on Apis mellifera colonies and reported similar findings. The use of thymol as dust achieved the best reduction % of Varroa infestation followed by vermiculite blocks and dilution in sugar syrup (Emsen and Kelly, 2007). In the same context, three oxalic acid application methods (trickling, spraying, and sublimation) at three or four (sublimation) doses, using 110 broodless colonies in early January 2013 have been compared against Varroa mite (Al Toufailia et al., 2015). The authors found that applying oxalic acid via sublimation in broodless honeybee colonies in winter is a highly successful way to manage Varroa destructor and does not cause any damage to the colonies. Both the dose of acaricide and application method have been showed to affect colony performance and Varroa mortality and these effects were also correlated with different climate conditions (Al Toufailia et al., 2015).

Recently, Gajger et al. (2020) also evaluated seven products for controlling Varroa mite, six were treated during Summer and one was treated during Autumn. The authors found that, treating colonies with CheckMite+ (coumaphos) as strips achieved the best Varroa reduction % while the least reduction % was in colonies treated with Apiguard (thymol) that was applied in an aluminum tray and coated sheet placed on the top bar of frames. Therefore, it could be concluded that, the overall efficacy of an acaricide could be affected by numerous factors, including the concentration of the compound involved, treatment period, and the colony and apiary environment (Gracia et al., 2017). Moreover, the efficacy of some compounds depends on the evaporation pressure within the colony; therefore, the time of year or the ambient temperature during treatment application can influence the effectiveness of the treatment (Calderone and Spivak, 1995). Consequently, the use of appropriate application method or/and the chemical state or form of an acaricide (solid, liquid or gas) is very important for better Varroa management and to avoid any hazard on honeybee health.

Veterinary drugs of varying chemical nature are used worldwide (except Australia) to regulate V. destructor. If these varroacids are fat-soluble and non-volatile, the risk of contamination of bee products increases with repeated applications over the years (Wallner, 1999). In the current study, residues of fluvalinate detected in honey and beeswax were higher than amitraz residues in the three different application methods. This might be related to the instability of amitraz in in honey and beeswax (Bogdanov, 1989; Korta et al., 2001; Pohorecka et al., 2018). Amitraz residues were completely degraded after 10 days of treatment in honey and after one day in beeswax (Korta et al., 2001). However, the different application methods could affect the acaricides residues levels in honey and other hive matrices. For example, fumigation of beehives with amitraz results in contamination of honey stored in combs (Pohorecka et al., 2018). In the present study, samples of honey and beeswax collected from colonies treated with Vapcozin-20 (amitraz) using the direct spraying method, contained the highest residues compared to the levels found in colonies treated with other application methods. This might be due to the direct exposure of honey and beeswax with the chemical as reported earlier (Martel et al., 2007). On the other hand, previous studies showed that the solubility of fluvalinate in wax is very high, and it was detected in wax at concentrations reaching 8000 times greater than honey and these residues were stable for more than a year in beeswax (Bogdanov et al., 1998; Mullin et al., 2010).

The use of acaricides is likely to increase due to resistance developed by mites which requires increased treatments in the future. This undoubtedly will increase the levels of acaricides residues in honey and other bee products. Therefore, beekeepers should apply miticides by away that achieve good efficiency against the Varroa mites and of minimal residues. In the current study, residues of fluvalinate and amitraz have been detected in only wax samples collected from colonies treated with the strip method after three months of acaricides treatment. Moreover, fluvalinate residues detected in both honey and wax collected from colonies treated by this method, exceeded the maximum residue limits (0.05 ppm for both honey and wax) according to EU Pesticides database. This acaricide is applied by beekeepers through pesticide-impregnated plastic strips and is subsequently distributed throughout a colony by nestmate interaction and trophallaxis. Therefore, it could adversely impact honeybee health because fluvalinate has already been reported as impacting queen and drone performance and competitiveness (Sokol, 1996; Rinderer et al., 1999) and increasing susceptibility to viral pathogens(Locke et al., 2012).

On the other hand, treating colonies with either fluvalinate or amitraz using cartoon paper as a delivery method led to very minimal or non-detectable residues in both honey and wax in the current study. This new delivery method, looks promising, efficient, very simple and cheap. Further studies are however still required to test this method with other acaricides and under different apiary and environmental conditions.

5 Conclusion

The efficiency of two acaricides Mavrik 2f (fluvalinate) and Vapcozin-20 (amitraz) against Varroa mites have been tested using three different application or delivery methods and their residues in honey and beeswax have been quantified. Both acaricides showed great efficacy against Varroa mites regardless of the application method. However, the use of carton papers was the most appropriate delivery method for these compounds because it showed very minimal acaricides residues in honey and beeswax. Beekeepers should avoid treating their colonies using fluvalinate strips, because it can easily accumulate in colonies to reach unsafe levels.