J Arthropod-Borne Dis, December 2016, 10(4): 501–509 N Adjlane et al.: Evaluation of Oxalic … 501 http://jad.tums.ac.ir Published Online: October 04, 2016 Original Article Evaluation of Oxalic Acid Treatments against the Mite Varroa destructor and Secondary Effects on Honey Bees Apis mellifera *Noureddine Adjlane 1, 2, El-Ounass Tarek 3, Nizar Haddad 4 1Department of Biology, University M’Hamed Bougara of Boumerdes, Boumerdès, Algeria 2Laboratory of Biology, and Physiology Animal, ENS Kouba, Algiers, Algeria 3Institut Technique des Elevages, Baba Ali, Alger, Algérie 4Department of Bee Research, National Center for Agriculture Research and Extension, Baqa’a, Jordan (Received 22 Sep 2013; accepted 13 July 2015) Abstract Background: The Varroa destructor varroasis is a very serious parasite of honeybee Apis mellifera. The objective of this study was to evaluate the effectiveness of Varroa treatment using organic acid (oxalic acid) in Algeria identify- ing its side effects on bee colonies. Methods: Treatment was conducted in one apiary consisting 30 colonies kept in Langstroth hives kind. Oxalic acid dripped directly on bees 5ml of this solution of oxalic acid per lane occupied by a syringe. Three doses were tested: 4.2, 3.2 and 2.1% oxalic acid is 100, 75 and 50 g of oxalic acid dehydrate in one litter of sugar syrup (1water to1 surge) concentration. Results: The percentage of average efficiency obtained for the first dose was 81%, 72.19% for the second dose, and 65% for third one, while the dose of 100 g oxalic acid causes a weakening of honey bee colonies. Conclusion: The experiments revealed that clear variation in the treatment efficiency among colonies that this might be related to brood presence therefore in order to assure the treatment efficiency oxalic acid should be part of a big- ger strategy of Varroa treatment. Keywords: Varroa destructor, Apis mellifera, Oxalic acid, Effectiveness, Algeria Introduction Varroa Disease is a parasitic mite of adult and brood stages of honeybees due to an ex- ternal parasitic mite, Varroa destructor An- derson and Trueman, 2000. Varroa is re- sponsible for an outbreak in Apis mellifera since its transfer from the Asian honeybee, A. cerana, the original host (Colin 1999). It is considered as the major factor responsible for colony losses worldwide (Martin et al. 2012, Nazzi et al. 2012). Parasitism of the bee by this mite causes deformation and weakness of the young work- ers. Heavy infestation causes death before the emergence of nymphs and the birth of mutilated bees (Boecking and Genersch 2008). Varroa heavily decrease the general weight up to 30% of individuals (Bowen-Walker and Gunn 2001), the total hemolymph volume and its protein content (Bowen-Walker et al. 1999). It was estimated to be between 10% and 50% reduction in total protein of par- asitized nymphs (Dandeu et al. 1991). The work of Yang and Cox-Foster (2005) clearly show that the Varroa weakens the immune system of bees and makes it more susceptible to viral and bacterial infections. Varroa by its role as a vector injects the vi- rus it carries directly into the hemolymph of the honeybee. On the other hand, as an acti- vator through the bite of Varroa allows the activation of certain viruses present in a la- tent state in the hemolymph of the honeybee (Tentcheva et al. 2004). In Algeria, the Varroa mite has become a *Corresponding authors: Dr Noureddine Adjlane, E-mail: adjlanenoureddine@hotmail. com J Arthropod-Borne Dis, December 2016, 10(4): 501–509 N Adjlane et al.: Evaluation of Oxalic … 502 http://jad.tums.ac.ir Published Online: October 04, 2016 major concern of a beekeeper since the dis- covery of the first cases of infestation with Varroa in the country. Many acaricide were used to fight against this disease (Adjlane et al. 2012). Today, chemical control with strips containing pyréthriniode (Apistan ®, Bayva- rol ®) can be used to a limited extent be- cause these products are no longer optimal efficiency due to mites resistant against these products (Adjlane et al. 2013). It is becoming crucial to provide beekeep- ers with new treatments for control Varroa mite, oxalic acid is considered as one the natural products that became widely used as alterative treatment. In addition, it was used for this purpose since the early '80s. In early tests, a reduction of 20 to 30% was observed in Varroa infestation after spraying it on each side of populated frame with honeybees (Charrière and Imdorf 1995). However, this method of treatment was criticized due to the bees disrupting after the frames moving, the danger oxalic acid vapours on the beekeep- ers Respiratory system in addition to being a very laborious work and time-consuming Charriére et al. (1998). However, the sugar role in the oxalic acid treatment is not clear, it is proposed that the sugar addition increase the solution oral absorption by honeybees and improves its adhesion to their bodies. Many experiments worldwide were con- ducted to test the efficiency of this acid in Varroa treatment (Nanetti et al. 1995, Nan- etti and Stradi 1997, Imdorf and Charriere 1998, Higes 1999, Gregorc and Planinc 2001, 2002, Moosbeckhofer et al. 2003, Nozal et al. 2003, Hatjina and Haristos 2005, Bacan- dritsos et al. 2007, Girişgin and Aydin 2010, Giovenazzo and Dubreuil 2011, Mert and Yucel 2011, Gregorc and Planinc 2012). These studies showed that the population density, amount of brood, climatic condi- tions, honey availability, migratory beekeep- ing, hive management and the development of Varroa population do directly influence the treatment efficiency. Thus, there are no scientific studies under Algerian conditions. The aim of this work was to study the ef- fectiveness of oxalic acid against Varroa mites, and to determine the side effects of the application of this treatment on bee colonies Materials and Methods The experiment was conducted in the north- central Algeria in the region of Boumerdes, this province is one of the wettest regions in the country. Annual rainfall varies between 500 mm and 1300 mm per year (36° 46 '00 "N, 3° 29' 00" E) (Fig.1). The relief of Bou- merdes is divided into several physical units: the plains and valleys to the north, the hills and plateaus in the middle part and the mountains south. Winter temperatures vary between 8 °C and 15 °C. They climb to 25 °C in May to an average of 28 °C to 32 °C in July and August. The average relative humidity is high enough, it varies between 75–90%. Thirty Apis mellifera intermissa colonies kept in standard Langstroth hives. In each colony, a mobile bottom board was installed with wire screen to count dead mites and to avoid mite removal by honeybees. The natu- ral mite mortality was equal for the thirty honeybee colonies. The application of oxalic acid is produced by the drip method (Imdorf et al. 1997), dripped directly on bees 5 ml of this solution of oxalic acid per lane occupied by a syringe (Imdorf et al. 1997, Brødsgaard et al. 1999). Oxalic acid is an organic compound with the formula H2C2O4. It shows in the form of colourless crystals of oxalic acid dehydrate (71.4% active ingredient). Oxalic acid dehy- drate is partially soluble in water (12.5% by weight at 25 °C) and some organic solvents (ethanol, diethyl ether). It has a molecular weight of 126,07g / mol, a density of 1.653, a melting point at 101.5 °C and sublimed at 150 °C (INRS 2005). The colonies were treated with three dif- ferent concentrations: 4.2, 3.2 and 2.1% ox- J Arthropod-Borne Dis, December 2016, 10(4): 501–509 N Adjlane et al.: Evaluation of Oxalic … 503 http://jad.tums.ac.ir Published Online: October 04, 2016 alic acid is 100, 75 and 50 g of oxalic acid dehydrate in one litter of sugar water (1.1). For each colony, two applications were made at weekly intervals. Dead Varroa were collected and counted every 2 days throughout the trial period us- ing diapers greased covering the bottom of the hive. To determine the number of mites remain- ing after treatment with oxalic acid, a control treatment with Apivar was made two weeks later. The effectiveness of oxalic acid is cal- culated as follows: The strength of each treated colony was estimated to analyze the possible impact of oxalic acid on the development of the colony. The data was analysed using SPSS ver- sion 5.0 (Chicago, IL, USA) and analysis of variance (ANOVA). Results Efficacy results and mortality of Varroa during treatment with oxalic acid and control (Apivar) for the three experimental groups are shown in Tables 1, 2, 3 and Fig. 1. Treat- ment with oxalic acid applied by the drop- by-drop method gives an efficiency of the dose used. The solution of 100 g of oxalic acid was a higher compared to the other two doses (70 and 50 g) efficiency. The most efficient dose was 100 g of ox- alic acid is 81% (Table 1), while 70 g of oxalic acid efficiency rate varies between 57 and 87% (Table 2), the dose of 50 g of oxalic acid was 65% (Table 3). Statistical analysis revealed a significant difference between the three treatments (F= 7.87, df= 10, P= 0.002). Fig. 1. The test area on the map of Algeria Table 1. Determination of the effectiveness of oxalic acid applied by drip for colonies of group I (100g of oxalic acid in 1 liter of syrup) (August-September 2012-Boumerdes area) Number of colony Number of dead Varroa (oxalic acid) (A) Number of dead Var- roa (Apivar®) (B) Total number of dead Varroa (A+B) (A) Effectiveness of oxalic acid (B) / (A+B)*100 1 345 271 616 78,50 2 245 251 496 86,34 3 167 15 182 91,65 4 390 114 504 77,31 5 401 175 576 69,54 6 290 65 355 81,65 7 189 61 250 75,45 8 365 178 543 67,20 9 423 54 477 88,65 10 289 19 308 93,76 J Arthropod-Borne Dis, December 2016, 10(4): 501–509 N Adjlane et al.: Evaluation of Oxalic … 504 http://jad.tums.ac.ir Published Online: October 04, 2016 Table 2. Determination of the effectiveness of oxalic acid applied by drip for colonies of group II (70g of oxalic acid in 1 liter of syrup) (August-September 2012-Boumerdes area) Number of colony Number of dead Varroa (oxalic acid) (A) Number of dead Var- roa (Apivar®) ( B ) Total number of dead Varroa ( A+B ) Effectiveness of oxalic acid (C) / (A+B ) *100 11 411 194 605 67,87 12 309 124 433 71,23 13 233 115 348 66,89 14 245 54 299 81,87 15 523 387 910 57,44 16 298 166 464 64,12 17 203 57 260 77,98 18 453 93 545 83,09 19 314 179 493 63,65 20 234 33 266 87,65 Table 3. Determination of the effectiveness of oxalic acid applied by drip for colonies of group III (50g of oxalic acid in 1 liter of syrup) (August-September 2012-Boumerdes area) Number of colony Number of dead Varroa (oxalic acid) (A) Number of dead Var- roa (Apivar®) ( B ) Total number of dead Varroa ( A+B ) (D) Effectiveness of oxalic acid (E) / (A+B ) * 100 21 198 102 300 65,90 22 311 247 558 55,65 23 382 150 532 71,78 24 254 160 414 61,34 25 412 275 687 59,89 26 256 101 357 71,65 27 201 113 314 63,87 28 399 290 689 57,88 29 421 93 514 81,90 30 345 229 574 60,1 0 10 20 30 40 50 60 70 80 90 100 100g OA 70 g OA 50 g OA Dose of oxalic acid E ff ec ti ve n es s o f o xa lic a ci d ( % ) Fig. 2. The average efficiency obtained for each treatment. Different letters indicate a significant difference between the three groups (<0.05, Newman-Keuls test at the 5% level) (August-September 2012-Boumerdes area) J Arthropod-Borne Dis, December 2016, 10(4): 501–509 N Adjlane et al.: Evaluation of Oxalic … 505 http://jad.tums.ac.ir Published Online: October 04, 2016 Discussion Charriere and Imdorf (2001) reported in trials conducted by dripping on colonies with- out brood in Switzerland showed an average 97% efficiency for the solution of 45 g of oxalic acid, against 96% efficiency for the treatment of 30 g of oxalic acid. In slovenia, the efficacy of oxalic acid treatments be- tween 8 August and 16 September was 41% (Gregorc and Planinc 2012). A lower dose of 10–15 mL/hive (3.5%) showed a good acari- cidal effect (98%) when smaller colonies were treated (Moosbeckhofer 2001). Gregorc and Planinc (2001) treated colo- nies with brood three times in the summer by applying 4.1–5.2% oxalic acid dehydrate in varying sugar solutions w/w corresponding to approximately 4.5–5.9% oxalic acid dehydrate w/v. An efficacy of 39–52% was achieved. In this experiment, the efficiency is relat- ed to the presence of a quantity of brood in the colonies. In northern Algeria, the brood is present almost throughout the year but with a very small amount in July–August. In- deed, several authors recommend applying this treatment period of absence of brood (Nanetti et al. 1995, Nanetti and Stradi 1997, Imdorf and Charriere 1998, Higes 1999, Gregorc and Planinc 2001, 2002) as oxalic has no effect on Varroa staying in sealed brood cells acid. Investigations from Canada in a climatic region comparing with Central Europe con- firmed the results of autumn treatments: 2.8 % oxalic acid dihydrate solution (40–50 mL/ hive) killed about 55% of the mites, 3.5% about 90% in colonies with remaining brood (Nasr et al. 2001). Efficiency of up to 100% was reached only in broodless artificial swarms (Büchler 1998). Experts recommend treatment with oxalic acid using spray application compared to treat- ment with dripping mode. This treatment guar- antees a very good efficacy against Varroa coupled with good tolerance to bees. Indeed, several trials reported efficiency greater than 95% for the spray application of 30 g of oxalic acid in one litter of water (Colin 1997, Imdorf et al. 1997, Charriére et al. 1998). The mode of action of treatment with ox- alic between passes and the amount of frames allows the bees to absorb the acid solution, and therefore the treatment penetrates and flows in their hemolymph. Treatment meth- od using (oxalic acid+sugar) is reported to be efferent due to adding sugar, and it re- duces the variation among colonies (Charri- ere and Imdorf 1999). Regarding the influence of these treat- ments on the development of colonies, no abnormalities were detected immediately af- ter treatment with oxalic acid. Studies with a higher concentration of 5% described dou- bled bee mortality in autumn, bad overwin- tering (reduced colony strength) of treated colonies and impaired spring development (Charrière 2001). Colonies treated twice with 3% oxalic acid dihydrate solution (7.3 and 6.4 g per comb side, one gram corresponds here approximately to one millilitre) resulted in high bee mortality averaging 170 dead bees after each administration and queen loss in one colony (Nanetti et al. 1995). The ef- fect of oxalic acid on bee larvae was inves- tigated by Gregorc et al. (2004). A solution of 6.5 g oxalic acid dihydrate/ 50 g sugar/ 100 mL water sprayed on honeybee larvae (0.121 mg/ larvae) affected the columnar cells of the midgut, leading to necrosis. In order to determine the tolerance of bees to these treatments, and avoid a possible weak- ening of colonies post treatment with oxalic acid, monitoring of treated colonies is per- formed by repeated checks the strength of the colony during autumn and winter. In gen- eral, we observed a significant drop in bee populations during the winter. The colonies treated with oxalic acid solution of 100 g group are weakened by 25%. However, the J Arthropod-Borne Dis, December 2016, 10(4): 501–509 N Adjlane et al.: Evaluation of Oxalic … 506 http://jad.tums.ac.ir Published Online: October 04, 2016 colonies of the second group treated with a solution of 70 g of oxalic acid have lost 15% of their bee populations. With the third pro- cessing, distribution, there has been a weak- ening of 10%. It is therefore apparent that the higher the oxalic acid content increases, winter losses more significant. Although different experimental condi- tions make direct comparisons difficult, as- sessments made in Switzerland and Germany tends to a higher loss of bees during winter of 45 g oxalic acid solutions. Lower concen- trations have provided better wintering colo- nies (Nanetti 1999). Higes et al. (1999) showed that oxalic acid has a negative effect on the development of the brood; these authors re- ported that three queens treated colonies died after applying a solution of 45 g of oxalic acid by spraying. The bad weather and low temperatures prevent persistent bees collect pollen and nectar pollen insufficient reserves generate a slow evolution of colonies (Im- dorf et al. 1996). The number of individuals populating the colony is a direct function of floristic conditions, they even dependent on climatic conditions (Vandame 1996). From January, all colonies showed a slight recov- ery in parallel with the improved weather conditions, and thus flora. However, there were much colonies in group 2 (70 g of ox- alic acid) that had resulted in better recovery from the first group. Conclusion The experiments revealed clear variation in the treatment efficiency among colonies that this might be related to brood presence there- fore in order to assure the treatment efficien- cy oxalic acid should be part of a bigger strat- egy of Varroa treatment. The solution of 100 g of oxalic acid ap- pears to be the most efficient comparing to the other two doses but it was poorly toler- ated by the honey bees while the (70 and 50 g of oxalic acid) solutions where less effi- cient in Varroa treatment. Therefore, under the similar conditions we recommend the treatment with 70 g of oxalic acid. Acknowledgements The authors thank the Association of Beekeepers Boumerdes for its material and technical support during this study. 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