Brocherel, Giuseppina

Formato, Giovanni

Martini, Andrea

Moscatelli, Francesca

Pietropaoli, Marco

English

Florence Research

European Journal of Integrative Medicine xxx (2016) xxx–xxxG ModelEUJIM 557 No. of Pages 4Research paperPhosphorus 30 CH to control Varroa population in Apis mellifera coloniesFrancesca Moscatellia, Marco Pietropaolia, Giuseppina Brocherela, Andrea Martinib,Giovanni Formatoa,*a Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Via Appia Nuova 1411, 00178 Rome, ItalybDipartimento di Scienze delle Produzioni Agroalimentari e dell'Ambiente (DISPAA), Università di Firenze, Piazzale delle Cascine 18, Florence, ItalyA R T I C L E I N F OArticle history:Received 27 November 2015Received in revised form 12 June 2016Accepted 13 June 2016Available online xxxKeywords:Phosphorus 30 CHVarroa destructorHomeopathyHoneybeeA B S T R A C TIntroduction: Varroa destructor is considered to be the main cause of European honeybee (Apis melliferassp. Linnaeus 1758) colony losses. The use of homeopathic products in veterinary practices hasconsistently increased in the last 50 years, but limited data are available on the application ofhomeopathic treatments to honeybees.The aims of this study were to investigate the acaricide efficacy and tolerability for honeybees treated for35 days with the homeopathic product Phosphorus 30 CH.Methods: The clinical trial was carried out during the summer of 2012 in Central Italy. Twenty-fourhoneybee colonies were evenly divided into two different groups: one treated with Phosphorus 30 CH (12colonies) and one left untreated (12 colonies). The mite mortality rate of the remedy was evaluated bycounting the number of fallen mites on the sticky boards placed on the bottom tray of the hives every3 days. Oxalic acid administration in an absence of brood was used to estimate the number of survivingmites. To assess the honeybee tolerability to the treatment, immediately before and after the Phosphorus30 CH administration, an evaluation of the adult honeybee population was performed.Results: The results revealed that no efficacy differences (p-value = 0.079, U = 23; EV = 45; Variance(U) = 150), nor differences in hive strengths (p-value = 0.118; U = 25.5; EV = 45; Variance (U) = 147.76) wereobserved in the treated group compared to the untreated group.Conclusion: Our results are consistent with other studies conducted using homeopathic remedies tocontrol varroa mites in Apis mellifera colonies. Further studies are needed to compare our data withdifferent treatment durations, different administration methods and potency of the remedy.ã 2016 Elsevier GmbH. All rights reserved.Contents lists available at ScienceDirectEuropean Journal of Integrative Medicinejourna l home page : www.e l sev ier .com/euj im1. IntroductionHomeopathy was developed in 1796 by Samuel Hahnemann. Itis a system of alternative medicine based on the principle that likecures like, according to which, a substance causing the symptoms ofa disease is able to cure the disease [9]. The use of homeopathicproducts in veterinary practices has consistently increased in thelast 50 years [10,17].Today, Varroa destructor (V. destructor), considered the mainparasite of the European honeybee (Apis mellifera Linnaeus 1758),has spread to beehives worldwide, with the exception of Australia,and is a major threat for apiculture [21,8].V. destructor is considered a crucial factor in the decreasingnumbers of honeybee colonies and beekeepers [3]. Increasedtolerance to different synthetic active molecules has been reported* Corresponding author.E-mail address: giovanni.formato@izslt.it (G. Formato).http://dx.doi.org/10.1016/j.eujim.2016.06.0071876-3820/ã 2016 Elsevier GmbH. All rights reserved.Please cite this article in press as: F. Moscatelli, et al., Phosphorus 30 CH to c(2016), http://dx.doi.org/10.1016/j.eujim.2016.06.007in strains of V. destructor that are resistant to fluvalinate,flumethrin and amitraz [1,5]. The use of natural treatmentsinstead of hard chemicals and synthetic molecules has theadvantage of limiting the drug resistance within the pathogensof farm animals [25,18,8], avoiding the environmental contamina-tion and persistence of active compounds residues or theirmetabolites in the honeybee products. Moreover, homeopathicproducts can also decrease the cost of procedures and reducehealth hazards to humans during treatment of animals [13,24].Finally, the application of the homeopathic remedies shouldencourage beekeepers to adopt good farming methods [14],simultaneously enhancing the honeybees’ welfare and immunesystems [16].Few data are available regarding the application of homeo-pathic treatments for honeybee diseases. In their preliminarystudies, Sassoli et al. [23] tested the efficacy of Calcarea Sulphuricain improving the resistance to Varroa mites in beehives. Lotti andMartini [15] tested the efficiency of Calcarea sulphurica 200 CH andApiBioxal1 to improve the resistance to varroosis of honeybeeontrol Varroa population in Apis mellifera colonies, Eur. J. Integr. Med.2 F. Moscatelli et al. / European Journal of Integrative Medicine xxx (2016) xxx–xxxG ModelEUJIM 557 No. of Pages 4families treated in an absence of brood. With the same purpose,Persano and Marinelli [19] tested three homeopathic products:Eureka, Apeas plus, and Apedin Vapor. Other studies have beenconducted by Ruiz Espinoza and Guerrero Salinas [22] that haveattempted to build up an experimental protocol for the control of V.destructor, including the frequency of application and the potency(202 CH) of Sulphur and Varroa Nosode. To control Varroa mites in2005, Flores [7] applied Equisetum spp. 60 CH with or withoutsuccussion directly to the feed administered to the honeybees.Due to the lack of information on the application of homeopa-thy to honeybee diseases, this study aimed to investigate thetolerability of the Phosphorus 30 CH treatments on honeybees andits efficacy in improving resistance against V. destructor.2. MethodsThe field trial aimed to verify efficacy of Phosphorus 30 CH inimproving resistance of honeybees to V. destructor after 35 days oftreatment in presence of brood and to verify the honeybees’tolerability of this treatment. The clinical trial was carried out fromJune 2012 to the end of July 2012 in Rome province (Central Italy)on 24 honeybee colonies, located in the same apiary (4143028.100N1242057.100E), housed in 10-frame Dadant-Blatt bee hives and freeof any other symptomatic disease.The 24 hives were divided into two homogeneous groups: (1) 12colonies treated with Phosphorus 30 CH (“Phosphorus” group) and(2) 12 colonies left untreated (“Control” group). To evenlydistribute the colonies in the two groups according to the Varroapopulation in each colony and the hive strength, the initial Varroainfestation level of the hives was evaluated by the natural mite fallcounts recorded for two weeks before starting the trials [2], whilethe adult honeybee population was estimated by the Liebefeldestimation method [11].The protocol is shown in Fig. 1 for the “Phosphorous” group andFig. 2 for the “Control” group.The Phosphorus 30 CH adopted remedy was selected accordingto scientific literature on symptoms transferred from humans toApis mellifera ethological characteristics [4,12]. We considered asconceivable characteristics for honeybees: irritability; sensitivity(as reported by Kent [12]: “the patient phosphorus is very sensitiveto all external sensations”); busy; perseverance; apprehensiveduring the “storm”. One gram of Phosphorus 30 CH (Boironlaboratory Ltd, 20124 Milan, Italy) was diluted in 10 ml of waterand the mixture was dissolved in a 1:1 ratio of sucrose, avoidingthe use of metal tools to mix the product. Once a week, 1 ml of thePhosphorus 30 CH sugar solution was poured on a lump of sucrosepositioned on the frames in the brood box (Fig. 3), for a total of 5doses to cover 35 days of treatment.To assess the honeybees’ tolerability to the treatment,immediately before the first Phosphorus 30 CH administration(day 0) and one week after the 5th treatment (day 35), the adultFig. 1. Scheme of the protocol adoptPlease cite this article in press as: F. Moscatelli, et al., Phosphorus 30 CH to (2016), http://dx.doi.org/10.1016/j.eujim.2016.06.007honeybee population was evaluated using the Liebefeld estimationmethod [11]. Finally, the honeybee queens’ vitality was recordedweekly during the 35 days of the treatment.The experimental protocol (Figs. 1 and 2) in the apiary fortesting the efficacy of Phosphorus 30 CH against V. destructor wasconducted according to the EU guidelines [6]. For the field trialperiod, the mite fall was recorded every 3 days through the use ofsticky boards put on the bottom tray of the hives.To estimate the number of surviving mites after a period of35 days of treatment administration, we counted Varroa fallproduced by the administration of a single dose of trickled oxalicacid solution (Apibioxal, Chemicals Laif s.r.l.) after 24 days of queencaging with VAR-CONTROL1 cages (Api-Mo.Bru, Campodoro,Padova, Italy – http://www.apimobru.com/en/ppe/ppe.htm) andconsidering a treatment efficacy of 90% [20].The percentage of acaricide efficacy (AE) in each hive wasevaluated using the formula:AE ¼ VPhosph 30 CHVTot ðPhosph 30 CH þOxalic acidÞ 90where VPhosph is the total number of mites eliminated with thetreatment with Phosphorus 30 CH, and VTot(Phosph30CH+Oxalicacid)represents the total number of mites removed by the Phosphorus30 CH treatment, plus the oxalic acid treatments.The mean acaricide efficacy in the two groups (AE) wasevaluated using the formula:AE ¼ SV Phosph 30 CHSVTot ðPhosph 30 CHþOxalic acidÞ 90Statistical analysis was performed to compare the efficacy ofthe treatment and the strength of the hives between the twoexperimental groups. The analysis only included colonies that hada level of infestation between 300 and 3,000 mites per colony, asindicated in the guideline on veterinary medicinal productscontrolling V. destructor parasitosis in bees [6]. Considering thatthe data do not come from a normally distributed population, thecomparisons between groups were carried out with a nonpara-metric Mann-Whitney test for independent samples. The signifi-cance level was set to 0.05. All the statistical analyses wereperformed using XLSTAT software.3. ResultsThe two groups, placed in the same site before starting the trial,presented homogeneous results, having a similar infestation level(p-value = 0.540; U = 37.0; EV = 45; Variance (U) = 150.00) andstrength (p-value = 0.901; U = 47.0; EV = 45; Variance(U) = 145.395).The acaricide efficacy observed in the “Phosphorus” group was46.1%, while the acaricide efficacy observed in the “control” grouped for the “Phosphorus” group.control Varroa population in Apis mellifera colonies, Eur. J. Integr. Med.Fig. 2. Scheme of the protocol adopted for the “Control” group.Fig. 3. Remedy administration to the honey bees by a lump of sugar.Fig. 4. Acaricide efficacy.Table 1Evaluation of the strength of the honeybee hives in the two experimental groups befoPre-treatment (honeybees) “Phosphorus” group 51082.5 “Control” group 55136.44 F. Moscatelli et al. / European Journal of Integrative Medicine xxx (2016) xxx–xxx 3G ModelEUJIM 557 No. of Pages 4Please cite this article in press as: F. Moscatelli, et al., Phosphorus 30 CH to c(2016), http://dx.doi.org/10.1016/j.eujim.2016.06.007was 54.9% (Fig. 4). The standard deviation (SD) was 4.0% in thePhosphorus group and 12.1% in the Control group. No statisticaldifferences were observed (p-value = 0.079, U = 23; EV = 45; Vari-ance (U) = 150) between the two groups. Considering the toxicity ofthe treatment with Phosphorus 30 CH on the adult honeybees, inthe “Phosphorus” group, after the treatment it was possible toobserve an increase in the adult honeybee population of 19.74%,with an SD of 31.2%, comparing to an increase in the “control”group of 34.23% with an SD of 42.6% (Table 1). Even in this case, nostatistical differences were observed between the two groups (p-value = 0.118; U = 25.5; EV = 45; Variance (U) = 147.76). After thetreatment (day 35), the deaths of 3 queens in the “Phosphorus”group and 1 in the control group were observed.4. DiscussionIn our evaluation of the tolerability of the treatment, we did notobserve any toxic effect on the adult honeybee populations,confirming the harmlessness of the homeopathic remedies.However, we observed unsatisfactory results with Phosphorus30 CH in the control of the Varroa population, perhaps due to themethods of use we adopted: duration of the treatment (35 days),method of administration (the lump of sugar once a week) andpotency of the remedy (30 CH). Many factors that may interferewith the evaluation of the efficacy of homeopathic treatmentsmust be considered, including the remedy, the potency of theremedy, the method of administration, the climatic conditions, theduration of the treatment and the honeybee subspecies [19,22,23].Further studies modifying one or more of the factors mentionedabove are needed to verify conceivable improvement in the controlof the parasite.It should also be taken into consideration that the homeopathicremedy must not have any acaricide effects, but aims to create abalance between host and parasite with infestation decrease.Sassoli et al. [23] and Lotti and Martini [15] treated thehoneybee families with Calcarea sulphurica, associating thetreatment with Api-Bioxal1, and observed that the acaricideefficacy did not differ between the treated and untreated group.Persano and Marinelli [19] showed that after the treatments withthe homeopathic remedies Eureka and Apedin Vapor (water,ethanol 19%, lactose and plant extracts: Echinacea angustifolia,Thuya occidentalis, Spiraea ulmaria and Oxalis acetosella)re and after the Phosphorus 30 CH treatment.Post-treatment (honeybees) Increase59190.59 54008.09 (+19.74%)70542.21 15405.77 (+34.23%)ontrol Varroa population in Apis mellifera colonies, Eur. J. Integr. Med.4 F. Moscatelli et al. / European Journal of Integrative Medicine xxx (2016) xxx–xxxG ModelEUJIM 557 No. of Pages 4administered by dripping and Apeas plus (administered byspraying), the number of mites in the honeybee colonies persistedat high levels.Ruiz Espinoza and Guerrero Salinas [22] tried to control V.destructor by the application of Sulphur 202 CH to the honeybeesthrough impregnated sugar globules and noted no effective controlof the mites. Finally, in 2005, Flores [7] found that Equisetum spp.60 CH showed an efficacy of 57% in controlling varroa mites bydirect administration to the feed of the honeybees. Our results areconsistent with those presented in the above-mentioned studies.References[1] J. Baxter, F. Eischen, J. Pettis, W.T. Wilson, H. Shimanuki, Detection offluvalinate resistant V. mites in U.S. honeybees, Am. Bee J. 138 (4) (1998) 291.[2] M.R. Branco, N.A.C. Kidd, R.S. 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Phosphorus 30 CH to control Varroa population in Apis mellifera colonies

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Phosphorus 30 CH to control Varroa population in Apis mellifera colonies

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