Introduction

The colonies of the honey bee Apis mellifera are affected by a severe parasitosis evoked by the ectoparasite mite Varroa destructor (Anderson and Trueman 2000). To avoid its death, colonies must be treated against the disease. Synthetic acaricides have been the traditional way of control during the last years but resistant mites and residues in honey bee products have increased worldwide (Bogdanov et al. 1998; Milani 1999; Wallner 1999) even in Argentina (Maggi et al. 2009). For this reason, substances of botanical origin have emerged as natural alternative acaricides to diminish the population levels of V. destructor. For the control and prevention of honey bee pathologies, there is a recent inclination toward botanical substances. Several essential oils and botanical extract have been used with variable success against American foulbrood disease (Gende et al. 2009), chalkbrood (Dellacasa et al. 2003), Nosema infections (Pohorecka 2004) and varroosis (Damiani et al. 2009). Few studies have been made about propolis and honey bee diseases despite its botanical origin (Antúnez et al. 2008; Garedew et al. 2002; Gende et al. 2007).

Propolis consists of a mixture of resins and waxes that are collected by bees from various plant species, particularly of flowers and leaf buds. It is difficult to observe bees in their foraging task, therefore, the precise source from where the resins are obtained, is usually unknown. It has been observed bees scraping the protective resins from the flowers and leaf buds with their mandibles and then carrying them into the hive as pellets on their hind legs. In the process of gathering and elaborating of propolis, the resins are mixed with a little saliva and other secretions of bees and wax (Burdock 1998). These resins are used by bees to cover the cavities inside the nest and brood combs, to repair combs, to seal small cracks in the hive, to reduce the size of the hive entrance, to seal large dead animals within the hive and, perhaps most importantly, mixing small quantities of propolis with wax to close the brood cells (Bankova et al. 2000). Thus, propolis provides antibacterial and antifungal effects to the colony’s environment that strengthens protection against diseases.

Propolis should not only be free of pollutants, but also the percentage of inert substances in relation to its biological action, such as wax, insoluble particles and ash, must be recorded. However, the most important feature is its level of biological activity, which is essential to characterize the abundance of biologically active components present in a propolis sample. Because bees collect propolis from different plants depending on the geographic location and specificity of the local flora, a significant variation in the chemical composition of propolis complicate the quantification of their bioactive compounds. It is commonly believed that a sample of propolis is high quality if it contains a high percentage of flavonoids (Bonvehi and Coll 1994; Park et al. 1998). A literature review about the biological action of the components showed that this statement is, in some cases erroneous. Antibacterial substances with no-phenolic origin have been isolated from propolis collected in Brazil (Bankova et al. 1996). Thus, the biological activity of propolis is given by its high content of resins, primarily (but not exclusively) phenolic compounds, predominantly flavonoids (Bankova et al. 1983). The specific flora that is accessible for bees and geographical and climate features of the area where these resins have been collected by bees, determine a very variable composition of the propolis (Bankova 2005; Bedascarrasbure et al. 2006).

Numerous studies have proven the versatile pharmacological activity of propolis: bacteriostatic, bactericidal, antifungal, antiviral, cytotoxic, anti-inflammatory, antioxidant, antitumor, among others (Banskota et al. 2001; Marcucci 1995). In recent years, propolis has caught the attention of many researchers because of the multiple possibilities for use in human and veterinary medicine where its biological activity has been demonstrated against several parasites (Freitas et al. 2006; Higashi and de Castro 1994; Topalkara et al. 2007), herpes virus (Huleihel and Isanu 2002), HIV (Harish et al. 1997), and cancer (Oršolić et al. 2006). However, antecedents about acaricide and insecticide effects of propolis are very limited. Some researchers have demonstrated that the treatment with propolis decreases the larval growth and duration of the pupal metamorphosis, and are toxic to different developmental stages of the wax moth Galleria mellonella (Garedew et al. 2004; Johnson et al. 1994). Garedew et al. (2002) showed that V. destructor is sensitive to propolis solutions applied topically on it. The aims of the present work were to characterize propolis from different geographical locations of Pampean region from Argentina and to evaluate the bioactivity of the propolis extracts on V. destructor.

Experimental

Collection and analyses of propolis samples

The propolis samples were obtained making contact with beekeepers whose beehives are placed in different zones of Pampean region. In Table 1, the geographical location where each sample was collected is detailed.

Table 1 Geographical origin of propolis samples
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Upon receipt, each sample was inspected in order to find rests of bees, wood, plant, pupa of moth, among other. The major visible impurities were removed from the samples by hand. Each sample was weighed, frozen, ground with a mortar, and then stored at 4°C until use. The propolis were organoleptic and physicochemically characterized in the Agroindustries Laboratory, Famaillá Agricultural Experimental Station, National Institute of Agricultural Technology, Tucumán province. Appearance, consistency, visible impurities, aroma, flavor, and color were the organoleptic properties assessed. The physicochemical characterization was made in relation to the contents of water, ash and wax; mechanical impurities, total resins, total phenols, and total flavonoids (expressed as quercetine dihydrate) according to the protocol of IRAM-INTA norms (IRAM-INTA Norms 15935-1, 2008).

Extraction

For the bioassay, each single soft extract of propolis was obtained from a suspension elaborated from pulverized propolis and ethanol 70% at 1:9 (w/v) ratio according to Cunha et al. (2004). Thus, the suspension was extracted at 60°C for 2 h in constant shaking; then it was cooled at room temperature and filtered by suction. After filtrated, the solution free of wax and impurities was evaporated at 40°C up to obtained a soft extract. The humidity content of the soft extract was determined according to IRAM-INTA Norms 15935-1 (2008). The wet weight/dry weight ratio was used to prepare solutions with different concentrations of each single propolis.

Experimental animals

Adult female mites of V. destructor were obtained from A. mellifera colonies placed in an experimental apiary of the National University of Mar del Plata, Mar del Plata, Argentina (38º10′06″ S; 57º38′10″ W). All colonies had been left untreated for Varroa for the preceding 12-24 months. Parasitized brood combs were carried to the laboratory, and their healthy capped cells were opened and inspected in search of mites. To avoid starvation, the mites were kept in Petri dishes on bee larvae or pupae during the collection process. The mites that appeared recently molted, weak or abnormal were discarded because they may have a differential response during trials. All treatments were carried out at room temperature (22-24°C). The treated experimental animals were incubated at 28 ± 1°C and 60% R.H.

Topical application method

The soft extracts were dissolved in 55% ethanol. The treatment concentrations were 2.5%, 5%, 7.5%, and 10% (w/v). The topical applications on the mites were made using a methodology adapted from Garedew et al. (2002). For each trial, 200 μl of a specified concentration of propolis were applied on six mites placed on a piece of filter paper. Each treatment was stopped when mites were removing from the filter paper (3 × 3 cm), after they had remained in contact with the propolis during 30 s, minimum contact time required for that mites have a differential response (Damiani et al., unpublished data). Then, parasites were transferred to a clean Petri dish (90 × 15 mm). Five replicates for each experimental unit were done. Controls treating mites with 55% ethanol were used. All tests were carried out at room temperature (22°C) and treated mites were incubated at 28 ± 1°C and 60% RH. The activity of mites was observed under dissecting microscope at 10, 30, 60 min; and each 1 h for the next 7 h after the beginning of each treatment. Each individual mite was classified as mobile or inactive; it was considered inactive when did not show any movement in legs o the rest of the body when a stimulus was applied (Milani 1995). If a mite remained inactive after 8 h from the beginning of treatments, it was considered dead.

The proportion of inactive mites in each period of observation and for each concentration of treatment of every propolis sample was calculated. The treatments toxicity on mites was evaluated using SigmaStat 3.1 software. The effect on the mites of the different concentrations, over the total period of observation, of each individual propolis sample was analyzed by two-way RM analysis of variance (ANOVA) with observation time as a repeated measure. For multiple comparisons, the Tukey’s test was used. Comparisons between the effects of the propolis from different geographical origin were evaluated by two-way ANOVA (origin and concentration) followed by multiple comparisons of least squares means using the Tukey’s test.

Results

Analyses of propolis samples

The organoleptic features of each propolis sample are showed in Table 2. The physicochemical properties of each propolis extract are given in Table 3. The propolis extracts showed an average value of 66.86% of resins, 20.28% of total phenols, and 6.84% of flavonoids.

Table 2 Organoleptic characteristics of propolis samples
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Table 3 Physicochemical properties of propolis extracts
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UV spectrograms showed that the propolis extracts analyzed displayed a maximum absorbance range between 270 and 315 nm. The propolis from Manuel B. Gonnet, Tres Arroyos, Mar del Plata, Coronel Vidal, Campana, Islas del Ibicuy, Berisso, La Plata, Olmos and Escobar exhibited a main absorption peak at 292 nm; while in propolis from Balcarce, Lobos, Villa Paranacito and Capilla del Señor, it was at 294 nm.

Topical applications

The treatments with alcoholic extracts of propolis obtained from the Pampean region of Argentina showed mortality effects on the mite V. destructor. In Table 4, the results of the assessment of these effects of propolis of different geographic origin on mites are detailed. In general, a trend towards an increase in acaricide action as increased concentrations of the extracts was observed, except when the treatments were performed with the propolis extract of Villa Paranacito, where there was no difference between the concentrations tested (p > 0.05). The average percentage of mites killed by the treatments with 10% propolis solutions was 72.74%, varying between 56.7% (Propolis from Olmos) and 90% (Propolis from Campana), but these mortality rates were not significantly different between all propolis tested (p > 0.05).

Table 4 Percentage of dead mites ± SE after 8 h from treatment with different concentrations of propolis from different geographical origin
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In addition to mortality effects, treatments with propolis caused narcosis effects on V. destructor. This effect was evident when a high proportion of mites that remained in inactive state during the first hours after onset of treatments regained their activity. All propolis, regardless of geographical origin, narcotized to the mites in some degree during the treatments. In the treatments with higher concentrations (7.5% and 10%), a significant proportion of mites remained narcotized during the first 2 h after contact with propolis (all p < 0.05), except in the propolis from Paranacito Villa and Campana where these concentrations caused high mortality on the mites from the beginning of treatments. After treatment with low concentrations, the mites were recovered from the narcosis during the first hour (all p < 0.05). Not all mites were able to entirely recover from the narcosis. The mites that did not regain its activity during the first 8 h after treatment were considered dead. In the control group, the effects of narcosis and mortality were not observed (all p > 0.05).

Discussion

The biological activity of propolis on various microorganisms has been demonstrated (Burdock 1998). Moreover, its effects have been tested on certain parasites showing amoebic, antigiardial and tripanosomal activity (Freitas et al. 2006; Higashi and de Castro 1994; Topalkara et al. 2007). Recent researches have suggested the potential action of propolis extracts in the treatment of bee diseases, such as American foulbrood (Antúnez et al. 2008; Gende et al. 2007), the largest moth G. mellonella (Garedew et al. 2004) and the parasitic mite V. destructor (Garedew et al. 2002).

When propolis extracts are made in ethanol at 70%, the most biologically active components are obtained (Cunha et al. 2004). The main bioactive compounds found in the resinous fraction of propolis, are only soluble in alcohol solutions (Medana et al. 2008). For this reason, the parasites of honey bees are not affected by propolis applied on the walls of the hive by worker bees.

Organoleptic and physicochemical properties identified in the propolis samples used in these trials were consistent with data recorded in other propolis samples from the Pampean region (Bedascarrasbure et al. 2006). Due to the high content of biologically active compounds such as phenols and flavonoids, the propolis collected from colonies of A. mellifera from this geographical region have the highest quality in Argentina.

Concerning the effect of propolis extracts on V. destructor, a study showed that these mites are highly susceptible to propolis solutions (Garedew et al. 2002). In this previous research, propolis was applied topically on mites and treatment with a 10% solution resulted in 100% mortality. The toxicity rates were independent of time of exposure with the extract, indicating a high toxicity even with small contact times. However, in the present study, the percentage of mites killed by the treatment with propolis collected from different areas of the Argentinean pampas ranged between 60.5% and 90% after 30 s of exposure. The controversy between the results obtained by the German researchers and those presented here could be due to the difference in the botanical origin of propolis samples that determines a variable composition in the phenolic fraction of them. Thus, we can suppose that the acaricidal activity of propolis extracts is given by the bioactive components present in this fraction. In our study, the different propolis samples behaved statistically similar among them when their effects against V. destructor were tested. On the other hand, the propolis sample tested in the research of Garedew et al. (2002) was not characterized physicochemically therefore its composition is unknown; thus, ours results can not be fully comparative with yours. The samples used in the present research came from the Pampean region of Argentina and the results from their physicochemical analyses coincided with those obtained by Bedascarrasbure and his colleagues (2006) on 67 propolis samples from this same region (Table 5). On this basis, these issues suggest that all propolis from the Pampean region are homogeneous in composition and the bioactivity levels against mites are comparable among the different propolis samples.

Table 5 Mean values of physicochemical properties of propolis from this research and from bibliographical reference data
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Furthermore, a narcotizing effect was observed after the mites came in contact with propolis. Mites remained anesthetized during the first hours after topical treatment. The same effect was observed by Garedew et al. (2002). When planning a control treatment for varroosis in honey bee colonies, the narcosis generated by applying a substance, would result in the permanence of the mites in inactive state on the floor of the hive. In this situation, the parasites removed from their host, are unlikely to recover quickly and return back to parasitize a bee. It is possible that during this period of time, the bees will take it out of the hives during routine cleaning activities. Thus, the narcosis would be an added effect of propolis treatment that contributes positively to reducing the infestation levels of mites in the colonies. The power of narcosis plus the lethal effect caused when mites remain in contact with the propolis, demonstrate the potential of propolis extracts in controlling V. destructor by this method of administration.

The results suggest that propolis extracts from this area could be incorporated in honey bee colonies by spraying. Despite these promising results, the concentration, doses and the mechanism of action of propolis on mites need still to be adjusted so to optimize the acaricide potential observed in these bioassays. Garedew et al. (2002) suggested that contact with propolis solutions could lead to a weakening of the mites’ cuticle that could facilitate entry of the active compounds present in propolis. Moreover, the effect of propolis extracts on bees has been little investigated; only one report of Antúnez et al. (2008) showed that bees tolerated high concentrations of alcoholic extracts of propolis administered orally with syrup. In order to implement its use as an alternative control substance, further researches are required to obtain a full knowledge about the effects of propolis extracts on V. destructor and honey bees. The variable chemical composition according to phytogeographical origin, the effects on A. mellifera and the possible methods for extract administration in the hives, are the main factors to be taken into account in planning future work to incorporate propolis in a program of integrated management which reduces the amount of synthetic acaricides in the hives.