Dynamics of Falling Varroa Mites in Honeybee ( Apis mellifera ) Colonies Following Oxalic Acid Treatments

Gregorc A. , I . Planinc: Dynamics of Falling Varroa Mites in Honeybee (Apis mellifera) Colonies Following Oxalic Acid Treatments. Acta Vet. Brno 2004, 73: 385-391. The aims of the study were to establish the dynamics of the fallen mites after treating honeybee colonies and to establish the effectiveness of oxalic acid for the control of varroa in colonies during the bee-keeping season in order to reduce varroa populations to tolerable levels. This study presents data from the periodic checking of the number of mites that had fallen onto the bottom of hives in the pre-treatment periods and after treatments. Oxalic acid (2.9%) and sucrose-in-water solution (31.9%) (w/w) (OA) application in honeybee colonies triggered a significant increase (p < 0.01) in mite mortality. Two days after the August 8, 16 and 23 OA treatments, the mite mortality was estimated at 68.62 ± 12.29%, 65.31 ± 10.61%, and 33.35 % ± 13.99%, respectively. The mite mortality between the second and fourth day was estimated at 18.69 ± 7.43%, 22.98 ± 7.69%, and 14.06 ± 6.75%, respectively. Between the 2nd and 9th days after OA applications was a highly significant (p < 0.001) reduction and between the 9th and 11th days after the August 23 application the reduction in mite mortality was significant (p < 0.05). High correlation (R = 0.9896) was found between the natural mite-mortality and the number of mites that fell during three August OA treatments. Efficacy of treatments conducted in colonies with capped broods averaged 23.82 ± 1.52%. We can conclude that in colonies with less than 1 natural mite-death per day following treatments should reduce their mite population by approximately 40 %. Our results could be used to establish a mite treatment programme in honeybee colonies and to evaluate in further research. Varroa destructor, mite mortality, alternative control, biological control, bee parasites Varroa destructor, a parasite of Apis mellifera, can cause a collapse of untreated colonies within a few years. It is imperative to control the mite in order to maintain the populations of honeybee colonies in most bee-keeping regions around the world. The use of acaricides should be minimised in bee-keeping to avoid both the build-up of residues and their byproducts in honey and wax (Wallner 1999) and to reduce the potential of acaricideresistance (Ruij ter 1994). Currently, there is little pressure to resist selecting “natural acaricides” such as organic acids (Milani 1999), which do not accumulate in wax and their residue build-up in honey is limited and toxicologically insignificant (Imdorf et al. 1996). Counting the mites that drop from a colony onto a bottom board is a reliable diagnostic method to evaluate the efficacy of an acaricide treatment (Rit ter 1981; Fries et al. 1991; Gregorc and Jelenc 1996; Poklukar 1999). A relationship has been found between the number of mites in a hive’s debris and the mite population of a colony (Liebig et al. 1984). Experiments have been conducted to evaluate oxalic acid (OA) as a method for controlling the mites in colonies with and without brood (Imdorf et al. 1996; Brødsgaard et al. 1999; Nanet t i and Stradi 1997; Nanet t i 1999). During broodless periods, Radetzki (1994), Nanet t i et al. (1995) and Imdorf et al. (1997) found it to be highly effective in killing the mites and they estimated the elimination level at 97.3 %, 98.3 % and 99.5 %, respectively. When a capped brood was present, Mutinel l i et al. (1997) achieved 95 % efficacy after three ACTA VET. BRNO 2004, 73: 385–391 Address for correspondence: Dr. Ale‰ Gregorc Agricultural Institute of Slovenia Hacquetova 17 SI-1000 Ljubljana Slovenia Phone: + 386 1 2805150 Fax: + 386 1 2805255 E-mail: ales.gregorc@kis.si http://www.vfu.cz/acta-vet/actavet.htm treatments of 5 % OA and Brødsgaard et al. (1999) reported a 24 % efficacy of one spring treatment administered by trickling. Approximately 50 millilitres of OA solution was used to treat one normally developed colony. Three OA treatments had an efficacy of 39.2 % when a brood was present and 99.4 % when there was no brood (Gregorc and Planinc 2001). This paper presents data from the periodic checking of the number of mites that had fallen onto the bottom of hives to determine the natural mite-fall. The aim was to establish the effectiveness of OA as a single substance for controlling varroa in honeybee colonies by using a 2.9 %OA/31.95 % sucrose-in-water solution (Gregorc and Planinc 2002) throughout 2001. We also aimed to establish the dynamics of the fallen mites after treating colonies with capped broods and to establish a strategy for administering oxalic acid during the bee-keeping season in order to reduce varroa populations to tolerable levels. Materials and Methods Twenty-two Apis mellifera honeybee colonies, populated in national standard AÎ “back load” hives (Zde‰ar 1998) with nine combs (41 × 26 cm) in each brood and honey compartment, were located at one site near Vipava, Slovenia, which has a mild Mediterranean climate. In the spring of 2001, 38 cm × 29.8 cm metal sheets were placed on the floor of each of the hives in order to record the hives’ natural mite mortality. Wire screens above the sheets prevented the bees from coming into contact with the debris. Before the experiment, the colonies were equalised to occupy from 5 to 7 brood combs. On the sampling dates, the numbers of mites were recorded and the inserts emptied. The levels of natural mite “drop-down” in the colonies for periods before (April 21 to May 5 and June 23 to August 8) and after the OA treatments were recorded three, four and up to eleven times, respectively. The first treatment of the twenty-two colonies took place on August 8, after harvesting the honey. The colonies received 50 millilitres of a 2.9% OA and 31.9% sucrose-in-water solution (w/w), using oxalic acid dihydrate (Riedel-de Haën), sucrose (sugar) and de-mineralised water (Gregorc and Planinc 2001). OA solution was trickled over the combs, in situ, and bees in the brood compartment using a syringe. The same treatment was repeated on August 16 and 23, October 9 and November 11, with a final treatment of the broodless colonies on December 29. All colonies received the same OA/sugar-concentration solution. The outside temperatures during the experimental treatments were 30 °C on August 8, 34 °C on August 16, 32 °C on August 23, 25 °C on October 9, 13 °C on November 11 and 5 °C on December 29. The percentages of mites killed by the experimental treatments (FTB) were estimated using the formula: FTB = FOA1 / (FOA1 + FOA2) X 100 (Gregorc and Planinc 2001). FOA1 is the total number of mites that dropped during the treatments of colonies with capped broods and FOA2 is the number of mites that fell during the December 29 treatment of the broodless colonies. The efficacy of the treatments was also estimated by comparing the numbers of mites that fell before and after the treatments and the mite mortality between the consecutive OA treatments. The data analyses were performed by ANOVA (analysis of variance) with the aid of the Statgraphic (1991) programme. Results During the pre-treatment period of 55 days from April 21 to August 8, 2001, the average natural mite-death per day was estimated at 1.25 (± 1.37) and the mortality was estimated at 68.72 (± 75.20) mites. In this period 3.60 % (± 2.04%) of the total varroa mite population died naturally. The first OA treatment resulted in a significant increase (p < 0.01) in mite mortality. While the numbers of dead mites recorded between the consecutive brood-period treatments were not statistically different, the December 29 treatment of the broodless colonies resulted in a reduction in the mite mortality (p < 0.001) in comparison with the previous treatments (Fig. 1). Two days after the August 8, 16 and 23 OA treatments, the mite mortality was estimated at 68.62 ± 12.29%, 65.31 ± 10.61%, and 33.35 ± 13.99 %, respectively. The mite mortality between the second and fourth day after each of these treatments was estimated at 18.69 ± 7.43%, 22.98 ± 7.69 %, and 14.06 ± 6.75 %), respectively. Between the 2nd and 9th days after each OA application, a highly significant (p < 0.001) reduction in mite mortality was observed and between the 9th and 11th days after the August 23 application the reduction in mite mortality was also significant (p < 0.05). After September 3, the mite mortality in colonies with capped broods was constant at 6.25 ± 1.97% during the five weeks leading up to October 9 (Fig. 2). 386

The aims of the study were to establish the dynamics of the fallen mites after treating honeybee colonies and to establish the effectiveness of oxalic acid for the control of varroa in colonies during the bee-keeping season in order to reduce varroa populations to tolerable levels.This study presents data from the periodic checking of the number of mites that had fallen onto the bottom of hives in the pre-treatment periods and after treatments.Oxalic acid (2.9%) and sucrose-in-water solution (31.9%) (w/w) (OA) application in honeybee colonies triggered a significant increase (p < 0.01) in mite mortality.Two days after the August 8, 16 and 23 OA treatments, the mite mortality was estimated at 68.62 ± 12.29%, 65.31 ± 10.61%, and 33.35 % ± 13.99%, respectively.The mite mortality between the second and fourth day was estimated at 18.69 ± 7.43%, 22.98 ± 7.69%, and 14.06 ± 6.75%, respectively.Between the 2nd and 9th days after OA applications was a highly significant (p < 0.001) reduction and between the 9th and 11th days after the August 23 application the reduction in mite mortality was significant (p < 0.05).High correlation (R = 0.9896) was found between the natural mite-mortality and the number of mites that fell during three August OA treatments.Efficacy of treatments conducted in colonies with capped broods averaged 23.82 ± 1.52%.We can conclude that in colonies with less than 1 natural mite-death per day following treatments should reduce their mite population by approximately 40 %.Our results could be used to establish a mite treatment programme in honeybee colonies and to evaluate in further research.

Varroa destructor, mite mortality, alternative control, biological control, bee parasites
Varroa destructor, a parasite of Apis mellifera, can cause a collapse of untreated colonies within a few years.It is imperative to control the mite in order to maintain the populations of honeybee colonies in most bee-keeping regions around the world.The use of acaricides should be minimised in bee-keeping to avoid both the build-up of residues and their byproducts in honey and wax (Wallner 1999) and to reduce the potential of acaricideresistance (Ruijter 1994).Currently, there is little pressure to resist selecting "natural acaricides" such as organic acids (Milani 1999), which do not accumulate in wax and their residue build-up in honey is limited and toxicologically insignificant (Imdorf et al. 1996).
Counting the mites that drop from a colony onto a bottom board is a reliable diagnostic method to evaluate the efficacy of an acaricide treatment (Ritter 1981;Fries et al. 1991;Gregorc and Jelenc 1996;Poklukar 1999).A relationship has been found between the number of mites in a hive's debris and the mite population of a colony (Liebig et al. 1984).Experiments have been conducted to evaluate oxalic acid (OA) as a method for controlling the mites in colonies with and without brood (Imdorf et al. 1996;Brødsgaard et al. 1999;Nanetti and Stradi 1997;Nanetti 1999).During broodless periods, R adetzki (1994), Nanetti et al. (1995) and Imdorf et al. (1997) found it to be highly effective in killing the mites and they estimated the elimination level at 97.3 %, 98.3 % and 99.5 %, respectively.When a capped brood was present, M utinelli et al. (1997) achieved 95 % efficacy after three treatments of 5 % OA and Brødsgaard et al. (1999) reported a 24 % efficacy of one spring treatment administered by trickling.Approximately 50 millilitres of OA solution was used to treat one normally developed colony.Three OA treatments had an efficacy of 39.2 % when a brood was present and 99.4 % when there was no brood (Gregorc and Planinc 2001).
This paper presents data from the periodic checking of the number of mites that had fallen onto the bottom of hives to determine the natural mite-fall.The aim was to establish the effectiveness of OA as a single substance for controlling varroa in honeybee colonies by using a 2.9 %OA/31.95% sucrose-in-water solution (Gregorc and Planinc 2002) throughout 2001.We also aimed to establish the dynamics of the fallen mites after treating colonies with capped broods and to establish a strategy for administering oxalic acid during the bee-keeping season in order to reduce varroa populations to tolerable levels.

Materials and Methods
Twenty-two Apis mellifera honeybee colonies, populated in national standard AÎ "back load" hives (Zde‰ar 1998) with nine combs (41 × 26 cm) in each brood and honey compartment, were located at one site near Vipava, Slovenia, which has a mild Mediterranean climate.In the spring of 2001, 38 cm × 29.8 cm metal sheets were placed on the floor of each of the hives in order to record the hives' natural mite mortality.Wire screens above the sheets prevented the bees from coming into contact with the debris.Before the experiment, the colonies were equalised to occupy from 5 to 7 brood combs.On the sampling dates, the numbers of mites were recorded and the inserts emptied.The levels of natural mite "drop-down" in the colonies for periods before (April 21 to May 5 and June 23 to August 8) and after the OA treatments were recorded three, four and up to eleven times, respectively.
The first treatment of the twenty-two colonies took place on August 8, after harvesting the honey.The colonies received 50 millilitres of a 2.9% OA and 31.9%sucrose-in-water solution (w/w), using oxalic acid dihydrate (Riedel-de Haën), sucrose (sugar) and de-mineralised water (Gregorc and Planinc 2001).OA solution was trickled over the combs, in situ, and bees in the brood compartment using a syringe.The same treatment was repeated on August 16 and 23, October 9 and November 11, with a final treatment of the broodless colonies on December 29.All colonies received the same OA/sugar-concentration solution.
The outside temperatures during the experimental treatments were 30 °C on August 8, 34 °C on August 16, 32 °C on August 23, 25 °C on October 9, 13 °C on November 11 and 5 °C on December 29.
The percentages of mites killed by the experimental treatments (FTB) were estimated using the formula: FTB = FOA1 / (FOA1 + FOA2) X 100 (Gregorc and Planinc 2001).FOA1 is the total number of mites that dropped during the treatments of colonies with capped broods and FOA2 is the number of mites that fell during the December 29 treatment of the broodless colonies.
The efficacy of the treatments was also estimated by comparing the numbers of mites that fell before and after the treatments and the mite mortality between the consecutive OA treatments.The data analyses were performed by ANOVA (analysis of variance) with the aid of the Statgraphic (1991) programme.

Results
During the pre-treatment period of 55 days from April 21 to August 8, 2001, the average natural mite-death per day was estimated at 1.25 (± 1.37) and the mortality was estimated at 68.72 (± 75.20) mites.In this period 3.60 % (± 2.04%) of the total varroa mite population died naturally.The first OA treatment resulted in a significant increase (p < 0.01) in mite mortality.While the numbers of dead mites recorded between the consecutive brood-period treatments were not statistically different, the December 29 treatment of the broodless colonies resulted in a reduction in the mite mortality (p < 0.001) in comparison with the previous treatments (Fig. 1).
Two days after the August 8, 16 and 23 OA treatments, the mite mortality was estimated at 68.62 ± 12.29%, 65.31 ± 10.61%, and 33.35 ± 13.99 %, respectively.The mite mortality between the second and fourth day after each of these treatments was estimated at 18.69 ± 7.43%, 22.98 ± 7.69 %, and 14.06 ± 6.75 %), respectively.Between the 2 nd and 9 th days after each OA application, a highly significant (p < 0.001) reduction in mite mortality was observed and between the 9 th and 11 th days after the August 23 application the reduction in mite mortality was also significant (p < 0.05).After September 3, the mite mortality in colonies with capped broods was constant at 6.25 ± 1.97% during the five weeks leading up to October 9 (Fig. 2).
Each of the four treatments conducted in colonies with capped broods had an average efficacy of 23.82 ± 1.52%.The efficacy of the treatments increased during the experiment.The analyses of variance showed that the effectiveness of the October 9 OA treatment was significantly higher in comparison with the previous treatments (p < 0.001) and that the efficacy of December 29 treatment of the broodless colonies (89.21 ± 7.93%) was significantly higher (p < 0.01) than the previous OA treatments.The efficacy of the consecutive OA treatments is shown in Figure 6.

Discussion
The natural mite-mortality per day, which until the August OA treatments was estimated at an average of 1.25, increased up to a maximum of 9.57 mites per day.Within two days of the OA treatments in the capped-brood period approximately 70% of the mites had died and the remaining 30% died within next eight days.When there were longer periods between treatments the numbers of mites that fell also dropped to lower levels.Repeated OA treatments have similar mite-drop dynamics and can be used effectively in colonies after the harvesting season.It seems that an OA application in a colony with a brood effects the mites on the adult bees but not those under the brood cappings.When compared to a summer OA treatment an autumn treatment of a colony with a reduced capped-brood results in a higher efficacy due to the prolonged mite-drop.
The consecutive treatments of the colonies initially had an efficacy of 12% and 23% (August) which rose to 51% after the November treatment.The increase in efficacy indicates that the treatments have a higher impact in late autumn when the colonies are preparing for winter and have less brood present.Reducing a colony's mite population by approximately 60%, with three OA treatments in August, after the honey extraction, is essential to ensure its normal development.Gregorc andPlaninc (2001) andBr ødsgaard et al. (1999) previously established similar levels of OA efficacy against the mites and Gregorc and Poklukar (2003) established a level of approximately 21%.
The number of mites killed in the colonies, which were grouped into three categories after the three August OA treatments, correlated highly with the number of mites that died naturally in the same colonies before the treatments.The natural mortality rates of 0.65, 0.94 and 2.89 mites per day observed during the pre-treatment period correspond to 303, 748 and 1,966 dead mites post-treatment.The natural mite-mortality, which is indicative of the mite population (Liebig et al. 1984), could be effectively reduced during OA treatments.The high correlation between the natural mite-mortality and the mite mortality induced after the three OA applications during August, indicates that the continual monitoring of mites dropping in bee colonies is important.Summertime treatments  -16.8. 16.8.-23.8. 23.8.-9.10. 9.10.-5.11. 12.11.-29.12.
Mite mortality (%) Fig. 6.The relative efficacy of OA after each of the five consecutive treatments expressed as a percentage of dead mites.The OA treatments in August were conducted on colonies with capped broods and the October and November treatments on colonies with significantly reduced capped broods.Bars indicate standard deviations.
against the mites should be conducted in colonies when the natural mite-mortality and the threshold level of their mite infestation have been determined.In summertime it seems that colonies with less than 1 natural mite-death per day should have three OA treatments, which will reduce their mite population by approximately 40%.M utinelli et al. (1997) reduced the mite population in colonies with capped broods by 95% with three OA treatments and after trickling OA, when a brood was present, B r ø dsgaard et al. (1999) recorded a lower efficacy of 24%.Reducing the mite population ensures a colony's normal development and wintertime survival.Further experiments must be conducted to establish how a colony develops when more than one mite per day is found.Treating colonies without capped brood using OA is highly effective (Nanetti et al. 1995;Gregorc and Planinc 2001).

Fig. 1 .
Fig. 1.Mite mortality in the pre-treatment periods and following the oxalic acid applications.Between August 8 and October 8, three OA applications were performed.The sixth OA treatment was conducted on broodless colonies.Bars indicate standard deviations.

Fig. 2 .
Fig. 2. Mite-mortality dynamics after three consecutive OA treatments of colonies with capped broods.The treatments were conducted on August 8, 16 and 23.The majority of mites fell within two days of each treatment.Bars indicate standard deviations.

Fig. 3 .
Fig. 3.The average mite mortality of the colonies grouped after three oxalic acid treatments performed in August 2001.The number of mites that fell after the three OA treatments in August correlated with the mite mortality after the autumn OA treatments.Bars indicate standard deviations.