MATERIALS AND METHODS

Survey Studies and Assessment of Infestation Levels of Varroa destructor in Different Sites during 12 Months Period in Qassim Region
Seven apiaries in 4 districts (3 bee hives each, hybrid craniolian and identical in strength) were randomly chosen to survey the infestation levels of Varroa mite in bee hives during the period from January 2006 to January 2007. Apiaries included in this study were two in Buraydah, Melida and Onayzah and one apiary in Bakeriah. The apiary of Agricultural Technology College (Buraydah-1) and Al-Mezini apiary (Buraydah-2); the apiary of Agricultural Experimental Station of the Faculty of Agriculture and Veterinary Medicine, Qassim University (Melida-1) and Al-Shwehi apiary (Melida-2); Al-Qounasi apiary (Onayzah-1) and Al-Garbouh apiary (Onayzah-2); Al-Bazehi apiary (Bakeriah). Survey studies dealt with mite individuals living on worker bees and those found on the bottom of bee hives.

Assessment of Mite Infestation Levels on the Bottom of Bee Hives
A strong white sheet, coated with vaseline, was monthly inserted on hive's floor. After 1 week, the sheet was removed and the fallen or dead mites were visually inspected and counted. The earlier technique was repeated for 12 months.

Assessment of Infestation Levels of Mites Living on Worker Bees
Three samples of 30 worker bees per sample were monthly collected (for 12 months) from each tested apiary by removing a frame from the center of each colony. Worker bees were swept with a brush into a beaker containing water and soap as a washing liquid. Each beaker was gently shacked for 30 sec then the bees were collected using a wire net. Varroa mites, which fell from bees during washing, were collected and counted for further data analysis (Ritter, 1981).

Control of Varroa Mites by Essential Oil
Plant Materials and Isolation of Essential Oil

Preparing Solutions of Essential Oil
Serial dilutions ranging from 0.0 to 750 g L^-1 from all extracted oil was prepared using sugar solution (1:1) in the presence of Tween 40 as an emulsifier. After that, the prepared oil were carefully mixed to obtain homogenous solutions. In all cases, the volume of each concentration was completed to 5 m L^-1 with sugar solution (1:1).

Effect of Essential Oil on Worker Bees
A. mellifera worker bees were collected from the apiary of the College of Agriculture and Veterinary Medicine, Qassim University. Three replicates of 10 worker bees for each concentration, was kept separately in plastic cups covered with cloth netting and secured with a rubber band to prevent bees from escaping. Groups of untreated bees were subsequently treated with sugar solution plus Tween 40 and used as a check. Different concentrations of each oil were immersed in small piece of cotton and placed on cloth netting for bee feeding. Dead and survived bees were recorded 24 h after treatments to exclude any concentrations that been recorded to have fatal effects on bees. From the earlier test, the following concentrations of tested extracts shown in Table 1 were examined against Varroa mites under laboratory conditions.

Table 1:	Names and concentrations of the tested essential oil, safe to worker bees, used against Varroa mites

Effect of Essential Oil on Varroa Mites
Ten worker bees were collected and subjected to be infested with 10 adult females of Varroa mite and kept in a plastic cup as previously mentioned. Bees in this treatment were fed on sugar solution containing 6.25% of camphor extract and replicated for 5 times. Five other groups of infested worker bees were fed on 6.25, 3.125, 12.5 and 3.125% of aloe, cloves and black seed while the fifth group was fed on sugar solution only and used as a check. In all cases, numbers of dead mites were counted 1 and 7 days after treatments.

Data Analysis
Analysis of Variance test (ANOVA) one way for the completely randomized design was used to compare means of Varroa mite individuals collected from beehive's bottom and those living on worker bees during 12 months in seven apiaries of four sites in Qassim Region. In all cases, means were compared using Student-Newman-Keuls Test (LSD) (p = 0.005) (Costat Statistical Software, 1990).

With regard to the effect of essential oil extracts on worker bees and Varroa mites, numbers of living and dead worker bees and mites were counted and the mortality percentages were corrected according to Abbott's (1925) formula.

RESULTS

Survey Study
Survey generally showed that infestation levels of the ectoparasitic mite V. destructor significantly fluctuated during the 12 month experiment and significantly affected by sites.

Infestation Levels of V. destructor Found on the Bottom of Bee Hives
Data in Table 2 showed that mite population found on the bottom of beehives started from low numbers in February (48.7 mite hive^-1) and March (63.86 mite hive^-1), which presented 5 and 7% of the total annual mite population (Fig. 1). Mite population significantly increased by an average of 121.14 and 147.71 mite hive^-1 in June and July, respectively, which presented 14%, while mite populations during August and September were sub-equal (146.80 and 120.85 mite hive^-1, respectively) and presented 12% of the annual mite population during 12 months. The infestation level was significantly declined in October, November and December which presented only 7, 5 and 3% of the total annual mite population, respectively (Table 2, Fig. 1).

Table 2:	Average numbers of Varroa mite individuals found on the bottom of bee hives and those living on worker bees during 12 months (in different sites)
Mean values within each column followed by the same letter(s) aren't significantly different

Fig. 1:	Infestation percentage of Varroa mites found on the bottom of bee hives during 12 months (in different sites)

Table 3:	Average numbers of Varroa mite individuals found on the bottom of bee hives and those living on worker bees in different sites (during 12 months)
Mean values within each column followed by the same letter(s) aren't significantly different

On the other hand, collecting mite individuals during the whole period of experiment showed that apiaries in Melida-1 significantly recorded the highest level of infestation by an average of 167.4 mite hive^-1 (Table 3). The infestation levels significantly decreased in Buraydah-1 followed by Onayzah-1, Bakeriah, Onayzah-2, Melida-2, Buraydah-2 by an average of 140.8, 91.16, 64.6, 63.0, 47.5 and 27.0 mite hive^-1, respectively. From the earlier results, it was noticed that mite infestation level in Melida-1 apiary presented about 28%, while the infestation levels declined in apiaries of Buraydah-1, Onayzah-1, Bakeriah, Onayzah-2, Melida-2, Buraydah-2 by 23, 15, 10, 11, 8 and 8% of the total annual infestation (LSD = 0.466) (Table 3, Fig. 2).

Infestation Level of V. destructor Living on Worker Bees
Data presented in Table 2 and Fig. 3 showed that population of V. jacobsoni living on worker bees was low in winter time and gradually increased from April and May with an average of 15.28 and 16.0 mite bee^-1 (presented 8 and 9% of the total annual number of mites), respectively. In July, the average number of mites parasitizing worker bees reached its peak of an average of 24.57 mite bee^-1 which is almost 14% of the total infestation (Fig. 3). Reduction in infestation level was significant in later months (from August till October) where each bee was infested by an average of 19.57, 18.06 and 13.85 mites, respectively (Table 2). These values presented 11, 10 and 8% of the total mite infestation, respectively (Fig. 3).

Fig. 2:	Infestation percentage of Varroa mites found on the bottom of bee hives different sites (during 12 months)

Fig. 3:	Infestation percentage of Varroa mites found/30 worker bees during 12 months (in different sites)

On the other hand, data in Table 3 obviously showed that there were no significant differences between infestation levels in Buraydah-1; Buraydah-2 and Onayzah-1 where each worker bee was infested by an average of 12.16, 12.83 and 12.41 mite individuals presented about 12% of the total mite infestation on worker bees during 12 months, respectively (LSD = 0.465). The infestation level significantly increased in Melida-2, Bakeriah, Onayzah-2 and reached its highest level in Melida-1. In the earlier sites, the infestation percentage averaged 13, 16, 17 and 18% where each worker bee was noticed to be infested by an average of 13.58, 17.0, 17.5 and 18.91 mite individuals, respectively (LSD = 0.465) (Fig. 4).

Effect of Essential Oil Extracts on Varroa Mite Infestation Affecting Honey Bees
The effectiveness of local essential oil (safe to worker bees), aloa, camphor, garlic, black seed and cloves were tested against Varroa mite individuals living and feeding on worker bees hymolymph in laboratory. Data in Table 4 and Fig. 5 showed that cloves (3.125%) was the most effective essential oil causing 62 to 77.4% Varroa mite mortality at 1 and 7 days after treatment. Extracts of garlic (3.125%), camphor (6.25%) and black seed (12.5%) came second, third and fourth in mite infestation reduction percentages with 51, 47 and 43% at 1 day after treatment, respectively.

Fig. 4:	Infestation percentage of Varroa mites/30 worker bees in different sites (during 12 months)

Fig. 5:	Reduction percentages of Varroa infestation on worker bees 1 and 7 days after treatment with essential oil

Table 4:	Corrected mortality (%) of V. destructor parasitizing worker bees 1 and 7 days after treatment in laboratory
Mean values within each column followed by the same letter(s) aren't significantly different

Seven days after treatment, black seed was significantly more effective than garlic and camphor where they reduced mite infestation to 72, 66 and 56%, respectively. Aloe extract was the weakest extract with reduction in mite infestation by only 34 and 45% at 1 and 7 days after treatment, respectively. In general, it was noticed that mortality percentage was positively correlated with time after treatment (r = 0.93). Therefore, it can be recommended to repeat the application for two or three times as needed according to the infestation level.

DISCUSSION

For the first time in Qassim Region, survey studies showed that the infestation levels, which were represented by numbers of Varroa mites collected from the bottom of beehives or parasitizing worker bees, affected either by season or locality were differed significantly. The highest level of mites was obtained during the period extending from June until September. Apiaries in Melida-1 recorded the highest level of mite infestation and significantly decreased in Buraydah-1 followed by Onayzah-1, Bakeriah, Onayzah-2, Melida-2, Buraydah-2. Concerning Varroa mites parasitizing worker bees, results showed that the infestation level of V. destructor infesting worker bees gradually increased from April and reached its peak in July. These findings may be due to adult female mites that find bee brood to start laying eggs and build up their population. Similar results were obtained by Ritter (1981), Cervanica and Aspiras (1987) and Contzen et al. (2004), who stated that Varroa mites began to lay eggs in bee brood of A. mellifera during spring and summer time. Also, Fathy and Fouly (1997) found that V. jacobsoni was found in great numbers in June and July. Moreover, it was noticed that Varroa mite reached its highest rate of infestation to honeybees in New Zealand during April-June (Stevenson et al., 2005).

The use of essential oil plants of the tested substances as natural acaricides is of immense significance in view of the environmental and toxicological implications of the indiscriminate use of synthetic acaricides and overcoming or reducing the problem of increasing Varroa resistance. Accordingly, the essential oil tested against Varroa mite showed that cloves was the most effective essential oil followed by garlic, camphor and black seed. Black seed was significantly more effective than garlic and camphor especially seven days after treatment, while aloe extract was the weakest substance. Fathy and Fouly (1997) noticed that the aqueous sugar solution of rosemary, fennel and marjoram essential oil were more effective than extracts of lemongrass, sag and thymus, that means there are certain essential oil plants which are considered to be a promising source of natural acaricides against Varroasis. Therefore, the efficiency of an extract may mainly due to its contents. Batisha et al. (2008) found that Eucalyptus oil rich in cineole was very effective against Varroa mites in India.

From the present results, it can be also concluded that mortality percentage of V. destructor was positively correlated with time after treatment (r = 0.93). Therefore, it is highly recommended to repeat the application for two or three times as needed according to the infestation level especially in winter time when no brood in bee colonies. Similarly, Fathy and Fouly (1997) found that mite mortality positively increased by repeating the treatments in broodless bee hives. Contradictory, Gregorc and Poklukar (2003) found that treatments with rotenone and oxalic acid in aqueous sugar solution can be administrated during summer in July-August. However, they achieved 98.65% mite mortality by using three treatments of oxalic acid in broodless colonies and significantly decreased in colonies with capped brood. While, Wachendorfer et al. (1985) and Bacandritos et al. (2007) repeated the application of formic and oxalic acid against V. destructor for four successive times (approximately one every 16 days) in Greece. Bacandritsos et al. (2007) obtained 65.33% cumulative mite mortality in November after the first three treatments. In December, when no brood in hives, the mite mortality obtained by the fourth treatment increased to 77.3%.

In general, the effect of natural substances such as volatile or essential oil plants, even if the treatments were repeated, can't completely eliminate Varroa invasion from bee colonies especially in countries like Saudi Arabia where sealed brood cells are present almost all around the year. Further studies are highly needed with regard to the effect of natural substances on Varroasis.

ACKNOWLEDGMENT

This study was financially supported by the Deanship of Scientific Research at Qassim University, Saudi Arabia. The authors wish also to express their appreciation to Dr. K.A. Osman, Department of Plant Production and Protection, Faculty of Agriculture and Veterinary Medicine, Qassim University, Saudi Arabia for his effort in preparing oil solutions and concentrations during the course of this study.