Introduction

Varroa destructor (Anderson and Trueman 2000) (Acari: Varroidae) is the most serious pest of honeybee (Apis mellifera), causing considerable economic losses such as malformation, weight loss, and a shortened life span in the bee throughout the world. It also serves as a vector of diseases that may lead to 100 % bee mortality (Kanga and James 2002). There are several synthetic acaricides against V. destructor. The most effective agents are the organophosphate coumaphos (Checkmite®, Asuntol®, Perizin®), the pyrethroids tau-fluvalinate (Apistan®, Klartan®, Mavrik®) and flumethrin (Bayvarol®), and the formamidine amitraz (Milani and Barbattini 1988; Milani and Della Vedova 1996; Ritter 1988). Although these compounds are very effective in most situations, significant drawbacks including contamination of honey, wax, and pollen have been reported (Chauzat et al. 2009; Johnson et al. 2009; Lodesani et al. 2008; Martel et al. 2007; Wallner 1999). Furthermore, much evidence of drug resistance was recorded in many cases (Milani and Della Vedova 1996; Milani and Vedova 2002; Sammataro et al. 2005). Therefore, investigations on appropriate alternative compounds to develop new treatment strategies are necessary (Lodesani 2004). Recently, natural compounds such as plant-derived products (PDPs) were introduced as good candidates to safer control agents that may provide acaricidal activity and few drawbacks (Aydin et al. 2007; Batish et al. 2008; Cakmak et al. 2006; De Ruijter 1982; Eischen and Vergara 2004; Gregorc and Poklukar 2003; Satta et al. 2008; Shaddel-Telli et al. 2008). The acaricidal activities of some PDPs or essential oil-based products against honeybee mite were investigated (Calderone et al. 1997; Semmler et al. 2009). Lepidium latifolium is known as perennial pepper weed and belongs to family Brassicaceae. This plant is native to southern Europe and Asia (Francis and Warwick 2007; Hultén and Fries 1986). The members of genus Zataria are widely distributed in Iran, Afghanistan, and Pakistan (Mahmoudabadi et al. 2007; Shaiq Ali et al. 2000), and Z. multiflora is one the most common herbal medicines in Iranian traditional medicine (Khalili and Vahidi 2006). Several studies have been performed on different medicinal properties of Z. multiflora, and its antimicrobial, antitoxigenic, insecticidal, and scolicidal activities has been confirmed (Lindberg et al. 2000; Moazeni and Roozitalab 2012; Najari et al. 2014). Several studies have been done on acaricidal potential of PDPs (Calderone et al. 1997; Flamini 2003 ; Floris et al. 2004; Mattila et al. 2000), such as compounds from L. Latifolium and Z. multiflora (Mohagheghzadeh et al. 2000; Navaei and Mirza 2007). The objective of the present study was the field assessment of L. latifolium and Z. multiflora methanolic extracts against V. destructor in A. mellifera colonies.

Materials and methods

The study area

The present cross-sectional study was carried out on A. mellifera colonies in Shahrekord (32° 19′ 32″ N, 50° 51′ 52″ E), capital city of Chaharmahal and Bakhtiari Province, Iran, in summer 2014. Its weather is dry, cold in winter, and mild in summer. The annual average temperature in Shahrekord is about 5.11 °C, but the minimum and maximum absolute temperatures during the last 30 years have been −32 °C and 42 °C, respectively (http://www.weatherzone.com.au/world/middle-east/iran/shahrekord).

Plant materials and extraction

Methanolic extracts of L. latifolium and Z. multiflora (Fig. 1) were prepared as follows:

Fig. 1
figure 1

L. latifolium (a) Z. multiflora (b). L. latifolium (perennial pepper weed) is an herbaceous perennial that can grow up to 1.5 m (5 ft.) tall. Plants emerge from thick, minimally branched roots or semi-woody crowns. In the late fall to early spring, a rosette of leaves develops with 4–12 in. (10–30 cm) long and 1–2 in. (2.5–5 cm) wide, toothed leaves. Lepidium latifolium is native to southeast Europe, North Africa, and southwest Asia. Z. multiflora is a genus of flowering plant in the Lamiaceae family. It is a shrub to a height of 40–80 cm, with twisted branches, native to southwestern Asia (Iran, Afghanistan, Pakistan, and Kashmir). Small leaves are almost rounded and in the surface are covered with a large number of follicles. Plant powder is silvery green with aromatic smell and astringent and spicy taste. Its harvest time is in June when it is blossoming

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After removing and collecting the leaves of both plants, they were dried in the shade. After that, the dried leaves were ground into powder by electric blender, and then 100 g of each was dissolved in 400 ml of pure methanol separately. The solution was kept for 24 h at room temperature after mixing with a magnetic stirrer. The solvent was removed by evaporator system after filtering. Finally, the dry powder was obtained using lyophilization of the semisolid material. The obtained residue was placed in a sterile glass container and stored at 4 °C till use (Moazeni and Nazer 2010).

Insects

Thirty hybrid honeybee colonies, A. mellifera infested with V. destructor, were used in the present study. Each colony consisted of ten full-depth combs. The hives were placed in a farm apiary near Shahrekord, capital city of Chaharmahal and Bakhtiari Province, Iran

Mean infestation rate

The percentage of Varroa infestation in the tested colonies before and after treatments was determined in approximately 100 living worker bees taking directly from the combs using icing sugar (sugar shake method) (Mark and Cliff 2001). We calculated the infestation rate by Alloui et al. (2002) formula (Alloui et al. 2002).

Field application and experimental design

In this study, the acaricidal effect of L. latifolium and Z. multiflora methanolic extracts were tested and compared with Apistan chemical strip (ACS) against V. destructor. Four concentrations (100, 200, 400, and 500 ppm) of the methanolic extracts were used, and each concentration was evaluated in three colonies (El Zalabani et al. 2012). Treatments were performed by means of 500-ml plastic sprayer. To treat each colony, different dilutions of plant extracts (100 ml each) were sprinkled over the bees inside the colonies (three colonies for each concentration). Every comb in the colony were exposed to test sample by rising up separately and sprinkled over the bees which covered the comb. ACS by slow release polymer strip formulation was applied in form of polymer beehive strips. Each 8 g (±0.8 g) strip containing 824 mg tau-fluvalinate, which was hanged between the middle combs as positive control. Three colonies were used as a control. Technical floors were used in the hives to record natural mite mortality. The mites which fallen from the adult bees and showed no movements were considered dead. The mean of mortality of mites and bees following treatment by different concentrations were recorded 4, 8, and 12 days after treatment. Three colonies were treated by distilled water using the same method and were regarded as negative control (El Zalabani et al. 2012).

Statistical analysis

Statistical analysis was performed using SPSS software version 21. One-way ANOVA and Duncan tests were used for comparison of measured parameters among control and treatment groups. Kruskal-Wallis, a nonparametric test, was used to compare the mortality rates of bees and mites among different groups. All values in tables were presented as mean and standard error of mean (SEM), and P < 0.05 was considered as statistically significant.

Results

To evaluate the acaricidal effect of L. latifolium and Z. multiflora methanolic extracts, the different dilutions of two plant extracts were applied to all 30 colonies and the effect of each dilution was evaluated in three colonies. Treatments were carried out at 12-day intervals and repeated three times for each extract. The mortality rate of Varroa and honeybees was calculated 4, 8, and 12 days after treatment. The colonies treated by ACS and distilled water were regarded as positive and negative controls, respectively.

The preliminary phytochemical screening of both plants showed a similarity in response. Our results showed that both extracts were effective against Varroa in a dose-dependent manner and had a negligible effect on bees. The extract of L. latifolium was the most effective, since its 500 ppm concentration caused 100 % mortality in mites after 12 days compared to Z. multiflora and ACS-treated colonies during the same period. The Varroa mortality rates after treatment with different extracts are presented in Table 1. The data in Table 1 showed that the high concentration of L. latifolium and Z. multiflora extracts had significant acaricidal effects on Varroa with the lowest mortality rate on bee colonies. The results of field application of two extracts are shown in Table 2 and their respective effects on honeybees in Table 3.

Table 1 Mean mortality of mites following treatment by different concentrations of L. latifolium and Z. multiflora methanolic extracts compared to controls
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Table 2 Mean infestation rate of bees before and after treatment by different concentrations of L. latifolium and Z. multiflora extracts compared to controls
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Table 3 Mean mortality of bees following treatment by different concentrations of L. latifolium and Z. multiflora methanolic extracts compared to controls
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The data in Table 2 showed that the mean infestation rate of colonies before treatment with 500 ppm of L. latifolium (18.66 ± 0.72) was decreased to zero after 12 days with minimal effect on bees compared to the other groups (see Table 3). This finding means that compared to ACS and Z. multiflora, L. latifolium extract had more and safer therapeutic effects.

The number of mite drop is depicted in Fig. 2. The highest mite drops occurred 12 days after treatment by 500 ppm of L. latifolium which was significantly higher than the other groups.

Fig. 2
figure 2

The number of mite drop after treatment with different concentrations of L. latifolium and Z. multiflora extracts compared to controls. L. latifolium: 100, 200, 400, and 500 ppm were demonstrated by A1, A2, A3, and A4, respectively. Z. multiflora: 100, 200, 400, and 500 ppm were demonstrated by B1, B2, B3, and B4, respectively

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The effects of two plant extracts and ACS on honeybees are shown in Fig. 3. L. latifolium had the least mortality effect on bees compared with Z. multiflora and ACS. Compared to control, the mortality effect of L. latifolium was significantly decreased by increasing concentration and time.

Fig. 3
figure 3

The number of dead bees after treatment with different concentrations of L. latifolium and Z. multiflora extracts compared to controls. L. latifolium: 100, 200, 400, and 500 ppm were demonstrated by A1, A2, A3, and A4, respectively. Z. multiflora: 100, 200, 400, and 500 ppm were demonstrated by B1, B2, B3, and B4, respectively

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Discussion

The application of PDPs is a new alternative method for the control of arthropod infestation, especially against Varroa mite in bee colonies (Calderone et al. 1997; El Zalabani et al. 2012). Natural products such as essential oils and methanolic extracts offer a highly desirable alternative to synthetic products. These substances are commonly used because they are generally inexpensive and safe to both man and honeybees (Eischen and Vergara 2004; El Zalabani et al. 2012; Gregorc and Poklukar 2003; Shaddel-Telli et al. 2008). Recently, some extracts have been tested for the control of different honeybee pathogens (Damiani et al. 2009; El Zalabani et al. 2012). Thyme, mint, lemon juice, camphor, eucalyptus essential oils, and tobacco smoke were demonstrated to be effective against Varroa with no or low harmful effects on bees (Abdel-Rahman 2008; Calderone 1999; Calderone et al. 1997; Damiani et al. 2009; De Ruijter 1982).

The control of parasites and pathogens in A. mellifera colonies can be performed more effectively by integrated pest management (IPM). According to the IPM strategies, pest control drugs should be environmentally friendly. In all cases, controls of Varroa mite by natural PDPs are recommended more than chemical acaricides (Dimetry et al. 2005; Flamini 2003; Ruffinengo et al. 2015), not only to keep the social life of honeybee away from any harmful effect but also for food safety and economic issues.

Nowadays, many researchers tend to return to investigations involving plant extracts for safe control of parasites and pest (Damiani et al. 2009; Semmler et al. 2009). Numerous investigations about the biological activity of PDPs against V. destructor and other pathogens in bee colonies have previously been performed (Damiani et al. 2011; George et al. 2008; Porrini et al. 2011; Umpiérrez et al. 2013).

In the present study, the acaricidal activities of botanical extracts obtained from L. latifolium and Z. multiflora were evaluated on V. destructor. Furthermore, the lethal effects of such extracts on bees were also examined.

Based on our results, compared to ACS which is used frequently as commercially available products, L. latifolium and also Z. multiflora with fewer side effects were more effective to treat infested colonies and in effective levels had no significant mortality on treated bees. Compared to ACS, L. latifolium extract was more effective, since its 500 ppm concentration caused 100 % acaricidal effect after 12 days. In the present study, Z. multiflora showed less acaricidal activity than L. latifolium, but more than ACS.

Several studies have been performed on different medicinal properties of Z. multiflora and its antimicrobial, antitoxigenic, insecticidal, and scolicidal activities have been confirmed (Lindberg et al. 2000; Moazeni and Roozitalab 2012; Najari et al. 2014). Our findings showed that the infestation rate was decreased by increasing concentration of both extracts, and this was in agreement with other studies (Abdel-Rahman 2008; El Zalabani et al. 2012). In addition, the results (Table 3) showed that both extracts appeared to be nontoxic for honeybees.

Conclusion

Our study showed that both L. latifolium and Z. multiflora at high concentrations are nontoxic for bee colonies and can decrease the overall population of Varroa mites, significantly. However, L. latifolium was more effective and its application can be considered as a simple, effective, safe, and cheap treatment for controlling Varroa in bee colonies.