Phosphorus 30 CH to control Varroa population in Apis mellifera colonies

(1)

Research

paper

Phosphorus

30 CH

to

control

Varroa

population

in

Apis

mellifera

colonies

Francesca

Moscatelli

a

,

Marco

Pietropaoli

a

,

Giuseppina

Brocherel

a

,

Andrea

Martini

b

,

Giovanni

Formato

a,

_*

a

IstitutoZooproﬁlatticoSperimentaledelleRegioniLazioeToscana,ViaAppiaNuova1411,00178Rome,Italy

b

DipartimentodiScienzedelleProduzioniAgroalimentariedell'Ambiente(DISPAA),UniversitàdiFirenze,PiazzaledelleCascine18,Florence,Italy

ARTICLE INFO

Articlehistory:

Received27November2015 Receivedinrevisedform12June2016 Accepted13June2016 Availableonlinexxx Keywords: Phosphorus30CH Varroadestructor Homeopathy Honeybee ABSTRACT

Introduction:VarroadestructorisconsideredtobethemaincauseofEuropeanhoneybee(Apismellifera

ssp. Linnaeus 1758) colony losses. The use of homeopathic products in veterinary practices has

consistently increased in the last 50 years, but limited data are available on the application of

homeopathictreatmentstohoneybees.

Theaimsofthisstudyweretoinvestigatetheacaricideefﬁcacyandtolerabilityforhoneybeestreatedfor

35dayswiththehomeopathicproductPhosphorus30CH.

Methods:Theclinicaltrialwascarriedoutduringthesummerof2012inCentralItaly.Twenty-four

honeybeecolonieswereevenlydividedintotwodifferentgroups:onetreatedwithPhosphorus30CH(12

colonies)andoneleftuntreated(12colonies).Themitemortalityrateoftheremedywasevaluatedby

countingthenumberoffallenmitesonthestickyboardsplacedonthebottomtrayofthehivesevery

3days.Oxalicacidadministrationinanabsenceofbroodwasusedtoestimatethenumberofsurviving

mites.Toassessthehoneybeetolerabilitytothetreatment,immediatelybeforeandafterthePhosphorus

30CHadministration,anevaluationoftheadulthoneybeepopulationwasperformed.

Results: The resultsrevealedthat no efﬁcacydifferences (p-value=0.079,U=23; EV=45; Variance

(U)=150),nordifferencesinhivestrengths(p-value=0.118;U=25.5;EV=45;Variance(U)=147.76)were

observedinthetreatedgroupcomparedtotheuntreatedgroup.

Conclusion:Ourresultsareconsistentwithotherstudiesconductedusinghomeopathicremediesto

controlvarroamitesinApismelliferacolonies.Furtherstudiesareneededtocompareourdatawith

differenttreatmentdurations,differentadministrationmethodsandpotencyoftheremedy.

ã2016ElsevierGmbH.Allrightsreserved.

1.Introduction

Homeopathywasdevelopedin1796bySamuelHahnemann.It isasystemofalternativemedicinebasedontheprinciplethatlike cureslike,accordingtowhich,asubstancecausingthesymptomsof adiseaseisabletocurethedisease[9].Theuseofhomeopathic productsinveterinarypracticeshasconsistentlyincreasedinthe last50years[10,17].

Today, Varroa destructor(V. destructor), considered themain parasiteoftheEuropeanhoneybee(ApismelliferaLinnaeus1758), hasspreadtobeehivesworldwide,withtheexceptionofAustralia, andisamajorthreatforapiculture[21,8].

V.destructoris considereda crucial factorin thedecreasing numbers of honeybee colonies and beekeepers [3]. Increased tolerancetodifferentsyntheticactivemoleculeshasbeenreported

in strains of V. destructor that are resistant to ﬂuvalinate, ﬂumethrin and amitraz [1,5]. The use of natural treatments instead of hard chemicals and synthetic molecules has the advantageof limitingthedrug resistancewithinthepathogens offarmanimals[25,18,8],avoidingtheenvironmental contamina-tion and persistence of active compounds residues or their metabolites in the honeybee products. Moreover,homeopathic products can also decrease the cost of procedures and reduce health hazardstohumans duringtreatmentof animals[13,24]. Finally, the application of the homeopathic remedies should encourage beekeepers to adopt good farming methods [14], simultaneouslyenhancing thehoneybees’ welfareand immune systems[16].

Few data are available regarding the application of homeo-pathic treatments for honeybee diseases. In their preliminary studies,Sassolietal.[23]testedtheefﬁcacyofCalcareaSulphurica inimprovingtheresistancetoVarroamitesinbeehives.Lottiand Martini[15]testedtheefﬁciencyofCalcareasulphurica200CHand ApiBioxal1 toimprove the resistance tovarroosis of honeybee

* Correspondingauthor.

E-mailaddress:giovanni.formato@izslt.it(G.Formato).

http://dx.doi.org/10.1016/j.eujim.2016.06.007

1876-3820/ã2016ElsevierGmbH.Allrightsreserved.

EuropeanJournalofIntegrativeMedicinexxx(2016)xxx–xxx

GModel

EUJIM557No.ofPages4

Pleasecitethisarticleinpressas:F.Moscatelli,etal.,Phosphorus30CHtocontrolVarroapopulationinApismelliferacolonies,Eur.J.Integr.Med. (2016),http://dx.doi.org/10.1016/j.eujim.2016.06.007

ContentslistsavailableatScienceDirect

European

Journal

of

Integrative

Medicine

(2)

familiestreatedinanabsenceofbrood.Withthesamepurpose, Persanoand Marinelli [19] testedthree homeopathic products: Eureka,Apeas plus, and Apedin Vapor. Other studies have been conductedbyRuizEspinozaandGuerreroSalinas[22] thathave attemptedtobuildupanexperimentalprotocolforthecontrolofV. destructor,includingthefrequencyofapplicationandthepotency (202CH)ofSulphurandVarroaNosode.TocontrolVarroamitesin 2005,Flores [7] appliedEquisetum spp. 60CH withorwithout succussiondirectlytothefeedadministeredtothehoneybees.

Duetothelackofinformationontheapplicationof homeopa-thy to honeybee diseases, this study aimed to investigate the tolerabilityofthePhosphorus30CHtreatmentsonhoneybeesand itsefﬁcacyinimprovingresistanceagainstV.destructor.

2.Methods

TheﬁeldtrialaimedtoverifyefﬁcacyofPhosphorus30CHin improvingresistanceofhoneybeestoV.destructorafter35daysof treatment in presence of brood and to verify the honeybees’ tolerabilityofthistreatment.Theclinicaltrialwascarriedoutfrom June2012totheendofJuly2012inRomeprovince(CentralItaly) on24honeybeecolonies,locatedinthesameapiary(41₄₃0_28.100_N

1242057.100E),housedin10-frameDadant-Blattbeehivesandfree ofanyothersymptomaticdisease.

The24hivesweredividedintotwohomogeneousgroups:(1)12 coloniestreatedwithPhosphorus30CH(“Phosphorus”group)and (2) 12 colonies left untreated (“Control” group). To evenly distributethecoloniesinthetwogroupsaccordingtotheVarroa populationineachcolonyandthehivestrength,theinitialVarroa infestationlevelofthehiveswasevaluatedbythenaturalmitefall countsrecordedfortwoweeksbeforestartingthetrials[2],while theadult honeybeepopulationwas estimated by theLiebefeld estimationmethod[11].

TheprotocolisshowninFig.1forthe“Phosphorous”groupand

Fig.2forthe“Control”group.

ThePhosphorus30CHadoptedremedywasselectedaccording toscientiﬁcliteratureonsymptomstransferredfromhumansto Apismelliferaethologicalcharacteristics[4,12].Weconsideredas conceivablecharacteristicsforhoneybees:irritability;sensitivity (asreportedbyKent[12]:“thepatientphosphorusisverysensitive to all external sensations”); busy; perseverance; apprehensive during the “storm”. One gram of Phosphorus 30 CH (Boiron laboratoryLtd,20124Milan,Italy)wasdilutedin10mlofwater andthemixturewasdissolvedina1:1ratioofsucrose,avoiding theuseofmetaltoolstomixtheproduct.Onceaweek,1mlofthe Phosphorus30CHsugarsolutionwaspouredonalumpofsucrose positionedontheframesinthebroodbox(Fig.3),foratotalof5 dosestocover35daysoftreatment.

To assess the honeybees’ tolerability to the treatment, immediately before the ﬁrst Phosphorus 30 CH administration (day0)andoneweekafterthe5thtreatment(day35),theadult

honeybeepopulationwasevaluatedusingtheLiebefeldestimation method[11].Finally,thehoneybeequeens’vitalitywasrecorded weeklyduringthe35daysofthetreatment.

The experimental protocol (Figs. 1 and 2) in the apiary for testingtheefﬁcacyofPhosphorus30CHagainstV.destructorwas conductedaccordingtotheEUguidelines[6].For theﬁeldtrial period,themitefallwasrecordedevery3daysthroughtheuseof stickyboardsputonthebottomtrayofthehives.

To estimatethenumberof surviving mitesaftera periodof 35days of treatment administration, we counted Varroa fall producedbytheadministrationofasingledoseoftrickledoxalic acidsolution(Apibioxal,ChemicalsLaifs.r.l.)after24daysofqueen caging with VAR-CONTROL1 cages (Api-Mo.Bru, Campodoro, Padova, Italy – http://www.apimobru.com/en/ppe/ppe.htm)and consideringatreatmentefﬁcacyof90%[20].

The percentage of acaricide efﬁcacy (AE) in each hive was evaluatedusingtheformula:

AE¼ VPhosph30CH

VTotðPhosph30CHþOxalicacidÞ90

where VPhosphis thetotalnumber of miteseliminatedwiththe treatment with Phosphorus 30 CH, and VTot(Phosph30CH+Oxalicacid) representsthetotalnumberofmitesremovedbythePhosphorus 30CHtreatment,plustheoxalicacidtreatments.

The mean acaricide efﬁcacy in the two groups (AE) was evaluatedusingtheformula:

AE¼

S

VPhosph30CH

S

VTotðPhosph30CHþOxalicacidÞ

90

Statisticalanalysiswasperformedtocomparetheefﬁcacyof the treatment and the strength of the hives betweenthe two experimentalgroups.Theanalysisonlyincludedcoloniesthathad alevelofinfestationbetween300and3,000mitespercolony,as indicated in the guideline on veterinary medicinal products controllingV.destructorparasitosisinbees[6].Consideringthat thedatadonotcomefromanormallydistributedpopulation,the comparisonsbetweengroupswerecarried outwitha nonpara-metricMann-Whitneytestforindependentsamples.Thesigni ﬁ-cance level was set to 0.05. All the statistical analyses were performedusingXLSTATsoftware.

3.Results

Thetwogroups,placedinthesamesitebeforestartingthetrial, presentedhomogeneousresults,havingasimilarinfestationlevel (p-value=0.540; U=37.0; EV=45; Variance (U)=150.00) and strength (p-value=0.901; U=47.0; EV=45; Variance (U)=145.395).

Theacaricideefﬁcacyobservedinthe“Phosphorus”groupwas 46.1%,whiletheacaricideefﬁcacyobservedinthe“control”group

Fig.1.Schemeoftheprotocoladoptedforthe“Phosphorus”group. 2 F.Moscatellietal./EuropeanJournalofIntegrativeMedicinexxx(2016)xxx–xxx

GModel

EUJIM557No.ofPages4

(3)

was 54.9%(Fig.4).The standarddeviation(SD)was 4.0%inthe Phosphorus groupand12.1%in theControlgroup.No statistical differenceswereobserved(p-value=0.079,U=23;EV=45; Vari-ance(U)=150)betweenthetwogroups.Consideringthetoxicityof thetreatmentwithPhosphorus30CHontheadulthoneybees,in the “Phosphorus” group, after the treatmentit was possible to observeanincreaseintheadulthoneybeepopulationof19.74%, withan SD of31.2%,comparingtoan increasein the“control” groupof34.23%withanSDof42.6%(Table1).Eveninthiscase,no statisticaldifferenceswereobservedbetweenthetwogroups (p-value=0.118; U=25.5; EV=45; Variance (U)=147.76). After the treatment (day35),thedeaths of3 queensinthe_{“Phosphorus”} groupand1inthecontrolgroupwereobserved.

4.Discussion

Inourevaluationofthetolerabilityofthetreatment,wedidnot observe any toxic effect on the adult honeybee populations, conﬁrmingtheharmlessnessofthehomeopathicremedies.

However,weobservedunsatisfactoryresultswithPhosphorus 30CHinthecontroloftheVarroapopulation,perhapsduetothe methodsofuseweadopted:durationofthetreatment(35days), methodofadministration(thelump ofsugaronceaweek) and potencyoftheremedy(30CH).Manyfactorsthatmayinterfere with the evaluation of the ef_ﬁcacy of homeopathic treatments must beconsidered, including the remedy, the potency of the remedy,themethodofadministration,theclimaticconditions,the durationofthetreatmentandthehoneybeesubspecies[19,22,23]. Furtherstudiesmodifyingoneormoreofthefactorsmentioned aboveareneededtoverifyconceivableimprovementinthecontrol oftheparasite.

Itshouldalsobetakenintoconsiderationthatthehomeopathic remedymustnothaveanyacaricideeffects,butaimstocreatea balancebetweenhostandparasitewithinfestationdecrease.

Sassoli et al. [23] and Lotti and Martini [15] treated the honeybee families with Calcarea sulphurica, associating the treatment with Api-Bioxal1, and observed that the acaricide efﬁcacydidnotdifferbetweenthetreatedanduntreatedgroup. PersanoandMarinelli[19]showedthatafterthetreatmentswith the homeopathic remedies Eureka and Apedin Vapor (water, ethanol 19%, lactose and plant extracts: Echinacea angustifolia, Thuya occidentalis, Spiraea ulmaria and Oxalis acetosella)

Fig.2.Schemeoftheprotocoladoptedforthe“Control”group.

Fig.3.Remedyadministrationtothehoneybeesbyalumpofsugar.

Fig.4. Acaricideefﬁcacy.

Table1

EvaluationofthestrengthofthehoneybeehivesinthetwoexperimentalgroupsbeforeandafterthePhosphorus30CHtreatment.

Pre-treatment(honeybees) Post-treatment(honeybees) Increase

“Phosphorus”group 51082.5 59190.59 54008.09(+19.74%) “Control”group 55136.44 70542.21 15405.77(+34.23%) F.Moscatellietal./EuropeanJournalofIntegrativeMedicinexxx(2016)xxx–xxx 3

GModel

EUJIM557No.ofPages4

(4)

administered by dripping and Apeas plus (administered by spraying),thenumberofmitesinthehoneybeecoloniespersisted athighlevels.

Ruiz Espinoza and Guerrero Salinas [22] tried to control V. destructorbytheapplicationofSulphur202CHtothehoneybees throughimpregnatedsugarglobulesandnotednoeffectivecontrol ofthemites.Finally,in2005,Flores[7]foundthatEquisetumspp. 60CHshowedanefﬁcacyof 57%incontrollingvarroamitesby directadministrationtothefeedofthehoneybees.Ourresultsare consistentwiththosepresentedintheabove-mentionedstudies. References

[1]J.Baxter,F.Eischen,J.Pettis,W.T.Wilson,H.Shimanuki,Detectionof ﬂuvalinateresistantV.mitesinU.S.honeybees,Am.BeeJ.138(4)(1998)291. [2]M.R.Branco,N.A.C.Kidd,R.S.Pickerd,Acomparativeevaluationofsampling

methodsforVarroadestructor(Acari:Varroidae)populationestimation, Apidologie37(2006)452–461.

[3]P.delaRua,R.Jaffe,R.Dall’Olio,I.Munoz,J.Serrano,Biodiversity,conservation andcurrentthreatstoEuropeanhoneybees,Apidologie40(2009)263–684. [4]H.Duprat,MateriaMedicaOmeopatica,FratelliPalombiEditori,Roma,1983. [5]P.J.Elzen,J.R.Baxter,M.Spivak,W.T.Wilson,ControlofVarroajacobsoniOud. resistanttoﬂuvalinateandamitrazusingcoumaphos,Apidologie31(2000) 437–441.

[6]EuropeanMedicinesAgency,CommitteeforMedicinalProductsforVeterinary UseGuidelineonVeterinaryMedicinalProductsControllingVarroadestructor ParasitosisinBees,(2008) EMA/CVMP/EWP/459883/2008.Availableat:

http://www.ema.europa.eu/docs/en_GB/document_library/ Scientiﬁc_guideline/2010/11/WC500099137.pdf.(accessed23.06.14). [7]S.Flores,EvaluaciónDeLaDinamizaciónHomeopáticaDeColaDeCaballo

(Equisetumarvense)ParaElControlDeácaro(Varroajacobsoni)EnAbejas(Apis mellifera),DepartamentodeAgroecología,UACh,Chapingo,México,2005,pp. 56.

[8]V.Goswami,M.S.Khan,Managementofvarroamite,Varroadestructorby essentialoilandformicacidinApismelliferaLinn.colonies,J.Nat.Prod.6 (2013)206–210.

[9]S.Hahnemann,J.Kunzli,A.Naude,P.Pendleton,OrganonofMedicine, Gollancz,London,1983.

[10]K.E.Hammarberg,Animalwelfareinrelationtostandardsinorganicfarming, ActaVet.Scand.Suppl.95(2001)17–26.

[11]A.Imdorf,L.Gerig,Courseindeterminationofcolonystrength,SwissFederal DairyResearchInstitute,LiebefeldCH-3003BernSwitzerland(afterL.Gerig,

1983.LehrgangzurErfassungderVolksstärke).SchweizBienen-Zeitung106: (2001)199–204.

[12]J.T.Kent,Materiamedicaomeopatica–LelezioniclassichediJ.TKentsui179 rimediessenzialidell'omeopatia(2003).

[13]A.Kijlstra,I.A.J.Eijck,Animalhealthinorganiclivestockproductionsystems:a review,NJAS–WageningenJ.LifeSci.54(1)(2006)77–94.

[14]K.Johnoson,Bees?:morequestionsthananswers,Homeopathyinpractice, (2009)p.60–63.

[15]C.Lotti,A.Martini,Calcareasulphuricaprotectsbeesagainstvarroasis, Homeopathyinpractice,Summer(2013).

[16]A.Martini,V.Ferrante,S.Barbieri,EdizioneItalianadeltestoacuradi,Salutee benessereanimaleinagricolturabiologica,Edagricole,Bologna,IT,in:S. Vaarst,V.Lund,W.Lockeretz(Eds.),AnimalHealthandWelfareinOrganic Agriculture,CABIPublishing,2006,pp.266.

[17]R.Mathie,L.Hansen,M.Elliott,J.Hoare,Outcomesfromhomeopathic prescribinginveterinarypractice:aprospective,research-targeted,pilot study,Homeopathy96(1)(2007)27–34.

[18]M.B.Molento,Parasitecontrolintheageofdrugresistanceandchanging agriculturalpractices,Vet.Parasitol.163(3)(2009)229–234.

[19]L.PersanoOddo,E.Marinelli,Impiegodiprodottiomeopaticinellalottacontro VarroadestructorAnderson&Trueman.InAttiXIXCongressoNazionaledi Entomologia,Catania,Italia,(2002)10–15Giugno,2002.Ministerodelle PoliticheAgricoleeForestaliIstitutoSperimentaleperlaZoologiaAgraria, SezionediApicoltura,Roma.

[20]M.Perugini,A.Giacomelli,M.Milito,M.Pietropaoli,M.Pizzariello,F.Scholl,G. Formato,L'encagementdesreines:unpremierbilan,Proc.JourneeSceintiﬁque Apicole(2013)68–73.

[21]P.Rosenkranz,P.Aumeier,B.Ziegelmann,BiologyandcontrolofVarroa destructor,J.Invertebr.Pathol103(Suppl.1)(2010)S96–S119. [22]F.J.RuizEspinoza,J.I.GuerreroSalinas,Resultadodeinvestigaciónde

Homeopatíaenabejas(Apismelliferam.L.).FaseIControlhomeopáticode ácaros(VarroadestructorOud.)enabejas,LahomeopatiadeMexico74(637) (2005)112–120.

[23]C.Sassoli,C.Lotti,A.Martini,D.Pradella,G.Ragona,G.Brajon,Valutazionedi trattamentiomeopaticiperilrafforzamentodellaresistenzaallepiu’comuni patologiedifamigliediapiallevateconmetodobiologico,Quaderno SOZOOALP7(2012)143–152.http://www.sozooalp.it/ﬁleadmin/superuser/ quaderni/quaderno_7/Quaderno_SZA7_Completo.pdf.

[24]S.M.Tabassam,Z.Iqbal,A.Jabbar,Z.Sindhu,A.I.Chattha,Efﬁcacyofcrude neemseedkernelextractsagainstnaturalinfestationofSarcoptesscabieivar. ovis,J.Ethnopharmacol.115(2)(2008)284–287.

[25]H.M.Thompson,M.A.Brown,R.F.Ball,M.H.Bew,FirstreportofVarroa destructorresistancetopyrethroidsintheUK,Apidologie33(4)(2002)357– 366.

4 F.Moscatellietal./EuropeanJournalofIntegrativeMedicinexxx(2016)xxx–xxx

GModel

EUJIM557No.ofPages4

View publication stats View publication stats