Effectiveness of combining certain biotechnical methods with thymol treatment against Varroa destructor infestation

In this study, the effectiveness of combining various biotechnical methods with thymol was investigated against the mite, Varroa destructor during late summer. Experimental colonies were randomly selected and six study groups were formed with nine colonies in each group. Experimental colonies were created as follows: colonies of renewed queen bees (RQ); colonies in which the queen is trapped on one comb, but worker bees can come and go to carry out their duties (CT); colonies in which ten grams of powdered thymol was added to 90 g of the bee cake, and 100 g of the bee cake with thymol was applied to the colonies (TY); colonies in which the requeen method plus the thymol method were used (RQ+TY); colonies in which the comb trapping method plus the thymol method were used (CT+TY); and untreated control colonies (CC). During the late summer period, the mite infestation level, sealed brood areas, bee population, and effectiveness of applications were determined in the groups. There was no significant difference in the infestation rate, sealed brood areas, and bee populations in the treatment groups before brood interruption. The efficacy of the requeen method, the comb trapping method, the thymol method, the requeen plus thymol method, and comb trapping plus other groups against V. destructor infestation were 40.23, 39.76, 80.45, 98.28 and 97.93%, respectively. These results showed that combining biotechnical methods with thymol is a safe, easy and effective alternative to late summer therapy against V. destructor.


INTRODUCTION
The mite Varroa destructor has many deleterious impacts on honey bee colonies (Gunes et al., 2017).Therefore, mite infestation in honey bee colonies needs to be constantly controlled by acaricides.To date, synthetic compounds such as coumaphos, amitraz, fluvalanite and flumethrin have been used against mite invasion.However, these synthetic compounds leave residues in honey and honey wax, and mites gradually develop resistance to their active ingredient over time (Wallner, 1999;Medici et al., 2015).In recent years, soft acaricides E-mail: mcengiz@atauni.edu.tr.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License such as thymol which do not lead to mite resistance and do not show any toxic effect on honey bees (Gregorc et al., 2018), have started to be used worldwide (Imdorf et al., 1999;Cengiz, 2012;Giacomelli et al., 2016).
Thymol, found naturally in honey, has high acaricidal efficacy against mites when used both alone or in combination with other biotechnical control methods (Coffey, 2007;Giacomelli et al., 2016).Investigations show that the acaricidal effect of thymol is low in the presence of brood, but high in the absence of brood (Akyol and Yeninar, 2008;Rosenkranz et al., 2010).Thymol has been used by a number of researchers to reduce mite infestations (Calderone, 1999;Cengiz, 2012;Brasesco et al., 2016;Dar et al., 2017).According to Giacomelli et al. (2016), biotechnical methods are sustainable approaches for mite treatment.Biotechnological applications, such as brood removal and caging the queen are cause of artificially broodless period in honey bee colonies and were already successfully used to V. destructor infestation control in combination with products based on thymol (Calderone, 2005;Rosenkranz et al., 2010;Gregorc et al., 2017a).The techniques of requeening and comb trapping also allow one to create a broodless period in the colony to be created.
The first objective of this research was to determine effects of requeening and comb trapping on the efficacy of V. destructor control using thymol.The second aim was to determine the positive and negative effects of these procedures on the pre-winter performance of the colonies.The results of this research will enhance alternative application among beekeepers in their fight against V. destructor in honey bee colonies.

MATERIALS AND METHODS
This study was carried out on a total of 54 colonies including 45 experimental colonies and 9 controls in Narman, Erzurum (40°21'3.70"E longitude, 41°52'39.09"N latitude, and 1650 m above sea level).The experiment was conducted from August 11 to September 19, 2017.Before the experiment, the colonies were equalized for brood area (3879.45±70.94cm 2 ), bee population (41883.18±453.15pcs) and for the quantity of food (two frames full of honey and pollen).At the beginning of the research, all the colonies were queenright and had brood present.
The natural mite mortality was monitored three times, three weeks prior to the beginning of the experiment.Two weeks prior to the start of thymol treatment, the level of infestation of the experimental colonies was determined using sticky bottom boards.The colonies were randomly divided into six groups according to applied treatment; colonies with renewed queen bees (RQ); colonies where the queen is trapped on one comb, but worker bees can come and go to carry out their duties (CT); colonies where thymol in sugar bee candy was applied (TY); colonies in which the requeen plus thymol methods were used (RQ+TY); colonies in which the comb trapping plus thymol methods were used (CT+TY); and untreated control colonies (CC).
For the treatment with thymol, bee cake was prepared (18 kg Ice sugar + 3 kg honey + 400 g pollen + 2 L water).Ten grams of powdered thymol was added to 90 g of the bee cake.One hundred grams of the sugar bee candy with thymol was applied to TY, RQ plus TY and CT+TY groups.The thymol treatments were applied weekly for three weeks.The requeen, comb trapping, and control colonies received thymol free sugar bee candy.
In the requeening group, queen bees were caged at the beginning of the experiment.Old caged queens were removed from the hive after 20 days, and young mated queens were introduced to the colonies by using the cages.The queens, who were kept in the cage for one day, were released.In the comb trapping groups, the queens were imprisoned in a single frame and released after 21 days in the frame.Combs of caged queen bees were taken from the hive, and combs were destroyed.There were no drone brood in this period in the colonies, and, after 21 days, the sealed brood remained.These two treatments led to the emergence of sealed brood in the colonies prior to thymol treatment.The exposure of many mites to thymol therapy increased the efficacy of thymol.Approximately 300 bees were collected from each colony to determine the adult bee infestation level in the experimental colonies.Worker honey bees from the brood combs were brushed into glass jars from which the circular center portion of the lids had been removed and replaced with a 3.1 mm mesh screen (Gregorc et al., 2017b).Then, about two tablespoons (15 g) of powdered sugar was added to the bees, and the glass jar was turned for one min to ensure proper mixing of the powder sugar and honey bees.The honey bees were vigorously shaken over a white paper plate for about four min, and the dislodged V. destructor were then counted.
The capped brood area was measured by the PUCHTA method (S = 3.14 × A/2 × a/2) in cm² (Akyol et al., 2014;Cengiz and Erdoğan, 2017).Before and after treatments, the bee population was estimated as the number of combs covered with bees and multiplied by 2972 method by Gris Valle et al. (2004).
Oxalic acid treatment was used as a finisher treatment to test the efficacy of the treatments.100 g of oxalic acid dihydrate were diluted in a 1:1 sugar solution (1000 g sucrose + 1000 mL water), and 4.2% oxalic acid solution was prepared.Oxalic acid solution was trickled using a large-scale syringe so that a 5 mL solution would be placed on each comb covered with bees (Gregorc et al., 2016).
Climatic temperatures were recorded during the trial period for the evaporation of thymol in parallel with the increase in temperature (Imdorf et al., 1999).Thymol is most efficacious at a temperature range between 15 and 35°C.Air humidity was not recorded, as thymol evaporation is not influenced by this parameter (Giacomelli et al., 2016).Sucrose syrup was prepared at ratio of 2:1 autumn feeding and each trial colony was given 0.5 L syrup for the 15-day autumn feeding period.

Statistical analysis
The SPSS 20.0 for Windows package program was used for statistical evaluation, and multiple comparison tests were performed for significant components.Prior to the analysis of variance, regarding the arcsiney/100 transformation to the percent efficiency of the organic compounds used in the investigation; a logarithmic transformation (log10) was applied to the amount of the sealed brood area and a bee population that did not fit the normal distribution.

RESULTS AND DISCUSSION
The external temperatures in the tested apiaries mostly remained within the optimum temperature range described for thymol during the 20-days of treatment  (Figure 1).In fact, the mean temperature was 22.4°C and the maximum was never over 35°C.However, the mean minimum temperature during the trial was 13.8°C.This mean temperature was slightly below ideal temperature (15°C).
There was no significant difference in the infestation rate and number of mites on bees among the treatment groups before brood interruption (F 5.48 = 0.36; P < 0.01).The treatment groups ranged from a high of 10.22±1.53mites on bees to a low of 8.22±1.78mites on bees.However, there was an increase in the number of Varroa on adult bees in the break in the brood cycle groups after three weeks (F 5.48 = 11.06;P < 0.01, Table 1).The difference in the increase in number of mites observed between groups was also found to be statistically significant (F 5.48 = 11.06;P < 0.01).Thymol treatment samples showed that the mite infestation rate decreased significantly in the requeen plus thymol and comb trapping plus thymol treatment groups.The infestation rate decreased from 16.37±2.47%to 1.44±0.66%and 15.07±2.16% to 1.51±0.62%for requeen plus thymol and comb trapping plus thymol group, respectively.
The total number of mites fall during treatment was different from the other groups in the requeen plus thymol and comb trapping plus thymol groups.The differences in the weekly mite fall observed between groups were also found to be statistically significant (F 5.48 = 163,98; P<0.01).The lowest mite fall after the application of oxalic acid was determined in the requeen plus thymol (10.33±0.52)and comb trapping plus thymol (12.33±0.89)groups (Table 2).
There was no significant difference between the brood area (F 5.48 = 0.31; P < 0.01) and bee populations (F 5.48 = 0.66; P < 0.01) of the pre-treatment colonies, while the difference in brood areas of the requeen and the requeen plus thymol groups after treatment was statistically   significant (F 5.48 = 27.85;P < 0.01).On the other hand, there was a decrease in the bee population in all groups except in the thymol and control groups (Table 3).The differences in the bee population between the observed groups were also found statistically significant (F 5.48 = 21.24;P < 0.01).The differences in treatment efficacy between the observed groups were also found to be statistically significant (F 5.48 = 874.25;P < 0.01).
The reduction rates of mite infestation for the requeen and comb trapping groups alone were determined as 40.23 and 39.76%, respectively.The treatment efficacy of requeen plus thymol (98.28%) and comb trapping plus thymol (97.93%) was different from other methods (Figure 2).
The application of the requeening and comb trapping methods in late summer reduced the mite destruction level in honey bee colonies.These two methods were not as effective as co-administration with thymol, although they caused a significant reduction in V. destructor infestation.The efficacy rate was 80.45% for the colonies treated with thymol.The average efficacy of thymol in the experimental colonies were found to agree with previously reported results by Emsen et al. (2007), (informed as 83.15%), but were lower than the result of another study Montano and Guzman-Novoa (2007), (informed as 92.1%).The average efficacy of thymol in the present study was higher than in previously reported finding by Cornelissen and Gerritsen (2006), (informed as Means with no common superscript in a column within a parameter differ (P<0.05).

70.7%
).This is thought to be related to the colonies' sealed brood areas.This research shows that artificial brood interruption with requeening and comb trapping applications is effective in increasing the efficacy of thymol treatment.However, the number of varroa on the bees increased in the groups in which the brood was artificially interrupted.Actually, the report that a large majority of the Varroa mites were in the sealed brood was consistent with the results of this study (Mondet et al., 2018).
The average efficacy of the requeen plus thymol and the comb trapping plus thymol groups was calculated as 98.28 and 97.93%, respectively.The efficacy values of these groups in the study are consistent with previously reported result by Giacomelli et al. (2016) (informed as 96.8%).At the end of the study, the decrease in the population of bees was considerably higher in the break in the brood cycle groups than in the other groups.This is due to the artificial brood interruption of brood production.In the requeen and requeen plus thymol groups, the sealed brood area after treatment was significantly higher than in the other groups.In other words, the sealed brood areas in the requeening have increased.Many researchers have reported that young queen bees produce more brood (Woyke, 1984;Akyol et al., 2007;Koç and Karacaoğlu, 2011;Öztürk, 2013;Büchler et al., 2013).

Conclusions
Colony management techniques used in this study have many advantages in the fight against V. destructor.The results show that an artificial brood interruption in the brood cycle can be used successfully to enhance the efficacy of different treatments.Particularly with the requeening combining thymol application, beekeepers will not only be successful in the fight against the V. destructor but also have young queen bee for the next season.When an adequate autumn feed is made in the requeen colonies, the production of brood in the colonies can be increased, and the colonies can be successfully overwintered.As a result, the application of the requeen plus thymol and comb trapping plus thymol methods efficacy against V. destructor was found similar.However, it can be said that the requeen plus thymol method is more advantageous than the comb trapping plus thymol method in terms of increasing brood production.

Figure 1 .
Figure 1.Temperatures recorded in Erzurum city during the trial (°C).
common superscript in a column within a parameter differ (P<0.05).

Figure 2 .
Figure 2. Efficacy of treatment groups on control of Varroa destructor.

Table 1 .
Number of mites on bees and infestation rate on bees.

n Number of mites on bees at various stages of the experiment (
X ± X S ) Infestation Rate on Bees (%) ( X ± X S )

Before brood interruption After three weeks of brood interruption Before brood interruption After three weeks of brood interruption
a,b,c Means with no common superscript in a column within a parameter differ (P<0.05).

Table 2 .
Number of fallen mites during treatment with thymol and oxalic acid.

Table 3 .
Effects of applications on sealed brood areas and bee populations.