dvm artur arszułowicz · 1 dvm artur arszułowicz field study documentation on apis plus part 2...

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DVMArturArszułowicz

FieldstudydocumentationonAPISPLUS

Part2

Comparativeassessmentofbeecolonies’hygienebehavior.

Łomża,6thSeptember2016

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I.INTRODUCTION

PreviousfieldresearchofthesubstancescontainedinAPISPLUSconfirmedthatthose substances allow bees to survive despite poisoning with the latestgeneration of plant protection products. During the tests Thiametoxam andImidaclopridbasedpesticideswereused.Concentrationofthepesticidewas375μgand125μgperoneliterofwater.Theexperimentshaveproventhatcoloniesprotectedwithpreparationweremuchsaferagainstthetoxicimpactofthemostdangerouspesticides–neonicotinoids,thantheoneswhichwerenotprotected.

Bees’ behavior was examined within: 2 hours, 24 hours and 13 days sincepesticide poisoning. Bees were divided into two groups: 1. control group; 2.protectedgroup.

In the control group, neonicotinoids caused death of the whole colonies orpartial death of colonies (depending on the pesticide dose). The bees whichsurvived, stopped taking care of bee brood and started to gather in groupssimilar to thoseas in thewinterseason.Furthermore,beesweresignificantlyweakeranddidnotpostanyguardsinfrontofthehivesanddidnotcollectpollenornectar.Beesrequiredfeeding.

Protectedbeesbehavedcompletelydifferent.Onlysmallpartofprotectedbeecolony died (approximately 5-15% of bees in the colony, depending on thepesticide dose). After 24 hours from the pesticide poisoning, the bees wereposting guards in front of the beehives. They also collected pollen, whichindicatesnomemoryloss.Generalappearanceofbeefamilygaveanindicationofahealthybeecolony.

Beehives,wherebeecoloniesdiedduetopesticidepoisoningweresettledwithnew,unprotectedcolonies.Thenewcoloniesalsodied.Itturnedoutthatthiskindofbeehivesarepoisonoustobeesevenafterafewweeks.Aftersettlingthesame beehives with protected colonies – the bees did not suffer from anynegativeeffectofpesticidepoisoning.

After applying APIS PLUS, increased movement inside beehive and defenseactivity of bees appeared. Increase of hygienic behavior of bee coloniesprotected with APIS PLUS as well as more flights were noted. Anotherexceptionallyimportantresultofstudywasobservedwhichwasincreaseoftheprocessof layingeggsby thequeenbeecomparedto thecontrolgroup.Thisimplies significant strength increase of bee colony just before winter, whichinfluencesitssurvivalrate.

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Hygienicbehaviorreferstotheabilitiesofbeestoidentify,detachandremovefromcells–sickordeadlarvae.Itisoneofthemostbasicdefensemechanismswhich grants bees resistance to parasites and pathogens. This research isfocusedonincreasedhygienicbehaviorofbeesafterapplyingAPISPLUS.

II.SUBJECTANDPLACEOFRESEARCH

Thecomparativeresearchonhygienicbehaviorofbeeswererunsincebeginningof May to the end of August 2016. Apiary was located in Lisno, Wiznamunicipality, Podlaskie Voivodeship. Apiary consisted of 10 bee families. Beefamilieswerebought inApril 2016 from randombeekeeper from theareaofSiedlce.Theywereplacedin10framed,Dadant-typebeehives.Afterplacement,beesweresystemicallyfeduntilfirstnectarwasshown.Whichwasdictatedbythelackoffeed.

Purchasedbeefamily,placedinDadant-typebeehive(6thofApril2016,16:34)

Examinedbeesbelonged toCarniolanhoneybee race (Apismellifera carnica).QueenbeeswereApismelliferacarnicacrossbreeds,notselected.

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During autumn 2015 all purchased bee families were combating againstvarroosiswithhomemademedicineinformoftwofibreboardpanelspercolony.FibreboardswereprobablyinfusedwithpreparationnamedKlartaninunknownquantity. They were stored in beehives since autumn 2015 until day ofpurchasing.Itisworthmentioningthathomemadeproductionofanymedicines,aswellas, itsdosingandstoring isunlawfuland incompatiblewithveterinarypractice.

FibreboardsinfusedwithpreparationnamedKlartan(6thApril2016,16:53)

Researchuncertainties:

1. DoesoverdosedKlartan,whichdeposedinbeeswaxduringlongstorageinbeehive,mayaltertheeffectsoftheAPISPLUS?

2. DomedicamentsusedagainstbeeandbeebrooddiseasesmayaltertheeffectsoftheAPISPLUS?

3. Domedicamentsusedagainstbeeandbeebrooddiseasesmayalterthehygienicinstinctofbees?

4. DoesAPISPLUSinteractswithneonicotinoidsinhoneyandbeebread?

III.RESEARCHMETHOD

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Themostcommonlyusedmethodsforexamininghygieneinstinctare:

1. Puncturingsealedbeebrood.Bee colonies, which will remove 95% punctured bee brood within 24hoursareconsideredtobehygienic.

2. Freezingsealedbeebrood.Beecolonies,whichwillremove95%frozebeebroodwithin48hoursaswellas in3-5days(thereareverydifferentviewsonthat issueamongscientists)areconsideredtobehygienic.

3. Placingmoistenedcardboardof50gweightbetweenframes.Beecolonies,whichwillremove(bybiting)cardboardwithin24hoursareconsideredtobehygienic.

4. Inseveralresearchcentersbeecoloniesare infectedwithAscosphaeraapis.

In field research on APIS PLUS, puncturing and freezingmethodswere used.Thosemethodsallowustocreatesimilarconditionsasactualbeebroodsdyingduetodisease. Inpuncturingmethod100sealedbeebroodswerepuncturedwith insulin needle ( with the smallest diameter). The smallest diameter ofneedleisusedbecauseoftheamountoflymphcomingoutofbeebroodbodyduring puncturing and because of the seal damage size. It is known that thesmallerdiameterof theneedle the lesshemolymphcomesoutofbeebrood.Thereby signal for locating dead bee brood is weaker. Secondly, the smallerdiameterthesmallerdamagedsurfaceoftheseal.Greatdamageofsealsurfaceeffectsinremovalofdeadbeebroodeveninunhygienicbeecolonies,whichdidnotremovefrozenbeebrood.

Within24,48and72hoursafterthetest,allremovedcellswerecounted

Threetestswererun.FirstinMay2016.SecondinJulyandthirdinAugust2016.Theresearchpurposewerecomparisonofdeadbeebroodsremovalpaceinbeecolonies protected byAPIS PLUS and unprotected ones during abundant andmoderatenectar inflowperiod.Thecomparisonwasalsomadeduringperiodwithoutnectarinflow.Nectarinflowabundancewastheresultoftheknowledgeofapiaryneighborhoodandamountofhoneycollectedbybeecoloniesduringtheresearch.

1. May2016–beebroodremovalpacewastestedduringabundantnectarinflow.

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2. July2016–beebroodremovalpacewastestedduringmoderatenectarinflow.

3. August 2016 – bee brood removal pace was tested during very weaknectarinflow.

Monthlyresearchoverviewhadtoanswerthequestionof:

1. Doesnaturalnectarinflowmaystimulatehygienicinstinctinbeecolony?

IV.TEST1

FourbeecolonieswereusedfortestinginMay2016(9-10thMay).Twocolonieswere protected with APIS PLUS in recommended dosage, 24 hours beforeinitiating the test.All frameswithbeeswere sprayedwithAPISPLUSusing agardensprayer.Puncturingmethodwaschoseninthefirstplaceusingtemplateof5x5cm(10x10cells).Weatherconditionswereverygood.Itwassunnyandtemperature was 20ºC. Dandelions and fruit trees were blossoming. Aroundtestedbeehiveswasexcessivemovementofbeeswithnectar.Threecastingwaxsheetswererebuilduntilthedayofresearch.

Bee colony participating in the research (9th Mat 2016, 15:14)

V.TEST1RESULTS

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Firstpair9-10THMay2016Unprotectedbeecolony(tab.no1)

BeecolonyprotectedwithAPISPLUS(blankanalysis)

(tab.no2)

Secondpair9-10THMay2016

Unprotectedbeecolony(tab.no3)

BeecolonyprotectedwithAPISPLUS(tab.no4)

Explanation

Beehiveno.

P T0 N0 N1 T1 N2 T2 N3 T3 NL HYG

1 100 16.10 6 100 16.10 100 16.10 100 16.10 12 12,0

Beehiveno.


2 100 16.30 16 100 16.30 100 16.30 100 16.30 0 12,0

Beehiveno.


1 100 17.00 13 100 17.00 100 17.00 100 17.00 0 12,0

Beehiveno.


2 100 17.30 9 100 17.30 100 17.30 100 17.30 0 12,0

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Symbol DescriptionBeginningofthetest0

P Numberofcellsontestedsurface(100)tO Dateofinitiatingthetest(date,hour)N0 Numberofcellswithoutlarvae

Firstcontrolwithin6hoursofinitiationT2 Within6hours(date,hour)N2 Numberofcellswithoutlarvae

Secondcontrolwithin12hoursofinitiationT2 Within12hours(date,hour)N2 Numberofcellswithoutlarvae

Thirdcontrolwithin24hoursofinitiationT2 Within24hours(date,hour)N2 Numberofcellswithoutlarvae

Ph.D. Kvetoslav Cermaks formula was used for calculating hygienic instinct(Pszczelarstwono5/2006).

HYG=(N1-N0)(T1/2) +(N2-N1)(T1 +T2)/2+(N3-N2)(T2 +T3)/2+(P-N3- P-NO-NL

NL)(T3+12)+NLxT3 (h) N3-N0

VI.TESTNO.1CONCLUSIONSIfbeecolonywillremoveduringitsfirstcontrol(within24hours)alldead

beebroods,andithastookplaceinbothprotectedandunprotectedcolonies,thenhygienicinstinctisconsideredtobe12hours.Thatistheamountoftimerequired for removalofeverydeadbeebrood.The less time is required forsuch removal thehigherhygienic instinct andvice-versa– themore time isrequired,thelowerhygienicinstinctis.

AllbeecoloniesdemonstratedhighlevelofhygienicInstinct.Puncturedbeebroodswereremovedin lessthan24hours.Thisconfirmsthathygienicbehaviorisnotregularbutvariable.Researchhasshownthatinbeecolonieswithunselectedbeequeenshygienicinstinctcanbeenhancedwithexternalfactor. External factor for unprotected bee colonies was abundant nectarinflow. Further research are required for answering the question whichexternalfactorhasenhancedhygienicinstinctmore–APISPLUSorabundantnectarinflow?

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12resealedcellsinunprotectedbeecolonywerecausedbyunsuccessfulkillingbeebroods.Iwasnotabletokill12beebroodsbypuncturingthemwithverythininsulinneedle.Chosendiameteroftheneedlewasverysmallandgiventhat Icouldnotpiercethroughhardcuticleofbeebroodsbutonlydefect itwithoutanydamagemadeforfuturebees.Giventhat,alivebeebroodsweresealedonceagain.Researchuncertainties:

1. WillAPISPLUSenhancehygienicinstinctofselectedbeequeens?2. DobeesprotectedwithAPISPLUSareabletodistinguishhealthysealed

beebroodsformthesickones?3. IsAPISPLUSabletoraisethelevelofmitepresencesensitivity(varroa)on

beebodyindifferentbeeraces?

VII.TEST2

Additionally freezingtestwasrun inMay.Twobeecolonieswereused in theresearch.FirstonewasprotectedwithAPISPLUS,theotherwasleftasablankmaterial.Beebroodwas cutoutand frozen for24hours in temperatureof -20°C.Thesametemplatewasusedasinpuncturingtests(10x100cells).24hoursbefore cutting out bee brood, bee colony was protected with APIS PLUS inrecommended dosage. Weather conditions were very good. Externaltemperaturewas20ºC.Dandelionsandfruit treeswereblossoming. Inflowofnectarwasabundant.Beecolonieshasrebuiltfourcastingwaxsheets.At20:00of 13th May 2016 frozen bee brood after obtaining room temperature wereplacedinexactsameplacesinframes.

VIII.TESTNO.2RESULTS

Readinghasnotbeentakenon14thofMayduetoweatherconditions–heavyrainsincemorning.

After48hours–on15thofMay2016at20o’clockreadinghasbeenmade.Bothprotectedandunprotectedbeecoloniesremovedallcellscontainingdamagedlarvaefrombothsidesofframe.

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Removedcellsinunprotectedbeecolony(15thMay2016,20.00)

RemovedcellsinAPISPLUSprotectedbeecolony(15thMay2016,20.00)

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IX.TESTNO.2CONCLUSIONSFreezing test confirmed conclusions from the puncture test. Both coloniesremoved all damaged cells within 48 hours. They presented high level ofhygienic instinct.However it isnecessarytotake intoconsiderationthefactthatdevelopmentofdiseasebeganwiththeendofnectarinflow.

X.TESTNO.3On21thofMay2016endofnectarinflowoccurred.Giventhat,anothertestwasrunontwobeecolonies–cellfreezing.On21thofMay2016beebroodhasbeecut out regarding template of 10x10. Bee brood has been sampled for theweakestcolonies.Freezingwasmadefor48hoursintemperatureof-20ºC.Beebroodwas freezing for48hoursbecauseofweather conditions–heavy rain.Warning (!) the freezing time does not affect hygienic instinct according toscientificliterature.Frozenbeebroodhasbeenplacedbackonitsplaceon23thofMay2016.

Frozenbeebroodhasbeenplacedbackonitsplace(23thofMay2016.12:09)

XI.TESTNO.3RESULTSForcalculatinglevelofinstinctusedthefollowingformula:Numberofremovedcells/sumofallcellsx100%

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Cellswhichhasbeenpartlyremoved(removedseal)duringcalculations,hasnotbeentakenintoaccountforthecalculation.Onlyfullyremovedcellshasbeentakenintoaccountforthecalculation.

APISPLUSprotectedcolony UnprotectedcolonyFirstside–75cellswithbeebroods.20emptycells.5cellswithbeebread.Secondside–76cellswithbeebroods.24emptycells.Bothsidestotal–151cellswithbeebroods.49emptycells(beebread).

Firstside–61cellswithbeebroods.38emptycells.1cellwithbeebread.Secondside–13cellswithbeebroods.87emptycells.Bothsidestotal–74cellswithbeebrood.126emptycells(beebread).

In unprotected colony, therewas no sign of cells removalwithin 12 hours.HoweverinAPISPLUSprotectedcolonytwocellshasbeenremovedduringthisperiod.Questions:

1. DoesAPISPLUSactuallyshortenstimetostimulatebeesforimmediatecellsremoving?Orstimulationiscausedbysugarsyrup(short-timeeffect)orgenetics?

Firstreadingwasmadeafter24hours.Protectedcolonyhasremoved12,5%ofcells.Unprotected–6,5%ofcells.Secondreadingwasmadeafter48hours.Protectedcolonyhasremoved25%ofcells.Unprotected–14%ofcells.Thirdreadingwasmadeafter96hours.Protectedcolonyhasremoved100%ofcells.Unprotected–65%ofcells.

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Unprotectedcolony,readingafter96hours(27thofMay2016,12:00)

Protectedfamily,readingafter96hours(27thofMay2016,12:15)

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BeesremovingcellsafterAPISPLUSapplication(6thJune2016,17:31)


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XII.TESTNO.3CONCLUSIONS

APISPLUSpreparationsignificantlystrengthenbeecolony’shygienic instinctduring period without nectar inflow. APIS PLUS protected bee colony hadapprox.100%moreremovedcells.Within96hoursprotectedbeecolonyhasremovedalmostallcells,onthecontraryunprotectedcolonycouldnothandleremovingevenhalfofthisresult.Questions:

1. Howbig isbee’senergyexpenditure,whichprotectedwithAPISPLUS,removescellssignificantlyfaster?

2. DoesAPISPLUSprotectedbee’senergyexpenditureaffectitslifespan?XIII.TESTNO.4Regarding the fact that abundant nectar inflow strengthens bee’s hygienicinstinct,adoubthasarisenwhethersugarsyrup(whereAPISPLUSisdissolved)strengthensbee’sinstinctinsteadofAPISPLUSorincooperationwithit.Forthispurpose further research has beenmade during 25th-31thof July 2016. 6 bee

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colonieswereexamined.Beecolonieshaspresentedsimilar“strength”toeachother.Beehivecontained10frames.5framesofbeebroodsand5framesofstocks–honeyandbeebread.Nectarinflowwasverylow.Theresearchwasrunafterlime tree had shed blossom. Hygienic instinct of 5 bee colonies has beenexaminedatfirstplaceasblankanalysis.

XIV.TESTNO.4RESULTS

Verificationofhygienicinstinctofall6beecolonieswasmadebypuncturingtest(tab.no.1)25-28thJuly2016.

Ph.D.KvetoslavCermaksformulawasusedforcalculatinghygienicinstinct.

Afterdeterminationofhygienicinstinctinallbeecolonies,colonieswerelinkedtogetherinpairs.Beecolonywithintensifiedinstinctwaspairedwiththeoneswithweakerinstinct.Purposeofthiswayoflinkingwastomaximizedistinctnessbetween results. Afterward paired colonies were examined with clear sugarsyrupin1:1ratio,sugarsyrupwithAPISPLUSandblanktest.

HygienicInstincttest–withoutAPISPLUSnorsugarsyrup(tab.no2)

28-31thJuly2016

Beehiveno


1 100 10.00 3 98 10.15 100 9.00 100 10.58 0 12,52 100 10.15 21 99 10.23 100 9.02 100 10.59 0 12,33 100 10.35 1 21 10.28 81 9.03 91 11.02 9 40,34 100 11.10 13 95 10.31 96 9.05 96 11.03 4 15.025 100 10.50 7 18 10.45 70 9.21 86 11.07 14 49,46 100 11.00 10 14 10.35 65 9.17 88 11.05 12 50,0

Beehiveno.


1 100 12.00 7 99 12.00 100 12.00 100 12.00 0 12,33 100 12.10 3 29 12.10 78 12.10 92 12.10 8 39,2

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HygienicInstincttest–withsugarsyrup

(tab.no3)28-31thJuly2016

HygienicInstincttest–withAPISPLUS

(tab.no4)28-31thJuly2016

All three testswere run in exact same time, exact sameweather conditions,exactsamebeehivesandexactsamenectarinflow.Ph.D.KvetoslavCermaksformulawasusedforcalculatinghygienicinstinct.

XV.TEST4CONCLUSIONS

1. Hygienicinstinctofcolonyno.1andno.3duringtests(25-28thJuly2016and 28-31th July 2016) was at the same level. It is because of theirgeneticpredispositions.Beecolonyno1–12,5hat25-28thJuly2016and12,3hat28-31thJuly2016Beecolonyno3–40,3hat25-28thJuly2016and39,2hat28-31thJuly2016

2. Aftersugarsyrupapplication,growthof instinctdidnotoccur.Duringtheverificationbeecolonyno.4hadinstinctonlevelof15,02h(tab.no1)whileaftersugarsyrupapplicationwasatthesamelevel15,03h(tab.no3).Thisprovesthatonlynaturalnectarinflowisabletostimulatethegrowthofhygienicinstinct.

3. TestswithAPISPLUSapplicationonbeecolonyno.2andno.6allowstodrawfollowingconclusions:

Beehiveno.


4 100 12.40 14 92 12.40 97 12.40 99 12.40 1 15.035 100 12.50 6 19 12.40 71 15.50 86 12.50 14 49,2

Nrula P T0 N0 N1 T1 N2 T2 N3 T3 NL HYG

2 100 12.20 17 99 12.20 100 12.20 100 12.20 0 12,36 100 12.30 11 39 12.30 74 12.30 91 12.30 9 43,8

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– when hygienic instinct is correct (due to genetic predispositions),applicationofAPISPLUSdoesnotaffectitsimprovementasitisalreadyverygood.APISPLUSdoesnotimprovegenetics.–incaseofabeecolonywithweakhygienicinstinct50,0h(tab.no1),applicationofthispreparationimprovesinstinctresultby6,2h(tab.no4)whichincasesofbeebrooddiseasesisveryimportant.

XVI.TESTNO.5

Further testsonAPISPLUS influenceonhygienic instinctwere run inAugust.Tests were run on 4 bee colonies using freezingmethod, which is similar tovarroosisattackonbeebroad.Weatherwassunnyduringthetest.Colonieswereinfulldevelopmentstage,7framesofbeebrood.Beequeenswereinthefullprocessoflayingeggs.Externaltemperature25°C.Honeywasnotcollectedfromthesebeehives.Totallackofnectarinflow.“Greendesert”inthefield.

Testwasrunonbeecoloniesno5andno6.withtheweakesthygienicinstinct.

XVII.TESTNO.5RESULTS

Bee brood for test purposeswas collected and frozen in the sameway as inprevioustests.Readingoffrozenbeebroodhastakenplaceafter24,48,72and96hours.

Beehiveno.


1 100 10.00 3 98 10.15 100 9.00 100 10.58 0 12,52 100 10.15 21 99 10.23 100 9.02 100 10.59 0 12,33 100 10.35 1 21 10.28 81 9.03 91 11.02 9 40,34 100 11.10 13 95 10.31 96 9.05 96 11.03 4 15.025 100 10.50 7 18 10.45 70 9.21 86 11.07 14 49,46 100 11.00 10 14 10.35 65 9.17 88 11.05 12 50,0

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APISPLUSprotectedbeecolony–beehiveno.5

Unprotectedbeecolony–beehiveno.6

Firstside–90cellswithbeebrood.10emptycells.Secondside–86cellswithbeebrood.14emptycells.Bothsidestotal–176cellswithbeebrood.24emptycells.

Firstside–88cellswithbeebrood.12emptycells.Secondside–82cellswithbeebrood.18emptycells.Bothsidestotal–170cellswithbeebrood.30emptycells.

Forcalculatinglevelofinstinctusedformula:Numberofremovedcells/sumofallcellsx100%

Results:1. After 24h protected colony removed 8% of cells. Unprotected

colonyremoved3%ofcells.2. After 48h protected colony removed 14% of cells. Unprotected





colonyremoved21%ofcells.

XVIII.TESTNO.5RESULTS

During the lackofnectar inflowperiod,APISPLUShas raisedhygienicinstinctincolonieswherewastheweakest.Thatrateofremovingcellsmaynotovercomediseasesbutmayminimizetheirprogression.Lotsof“unemployed”bees (nonectar inflow)were responsible for removingcellswithdeadbeebrood.HoneyproductionThree colonies (no. 1, 2, 4) characterized by high, natural hygienicInstinct,producedmorehoneythanotherbeecolonies.Approximately

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3 and half Dadant’s frame more, which is about 3 kilograms.MeasurementofhoneyproductionconcernedhoneyharvestinJune.Therefore, connection between high hygienic Instinct and honeyproductionisconfirmed.

XIX.FINALCONCLUSIONS

Themainaimoffieldstudieswastogiveananswertoquestionraisedatthebeginning: does APIS PLUS raises bees’ hygienic instinct? Conclusions of thisstudies show that preparation raises hygienic instinct. Bee colonies withincreasedhygienicinstinctarehealthycolonies,whichdevelopmentofdiseasesiseliminatedinnaturalway.Fromveterinarypointofview,beeswithincreasedhygienicinstinctarenecessarynowadays.Beecolonieswithhighinstinctallowtoreduceusageofmedications,simultaneouslydecreasingtheriskofcontainingbeeproducts(honey,honeybread,propolis).Regardingtheunknownmolecularmechanismofactionaswellasappearanceofmanydoubts,itisappropriatetorunfurtherlaboratoryandfieldstudiesbyspecializedresearchfacilities.Everyandeachpersonparticipatinginbees’lifeshallproudlysaytohimself:“Beewascreatedforme”.Itmeansthateveryoneofusisresponsibleforbees.Despitethere is no hierarchy of responsibility, there is hierarchy of realizing taskconnectedtoit.

TABLEOFCONTENT

I.INTRODUCTION

II.SUBJECTANDPLACEOFRESEARCH

III.RESEARCHMETHOD

IV.TESTNO.1

V.TESTNO.1RESULTS

VI.TESTNO.1CONCLUSIONS

VII.TESTNO.2

VIII.TESTNO.2RESULTS

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IX.TESTNO.2CONCLUSIONS

X.TESTNO.3

XI.TESTNO.3RESULTS

XII.TESTNO.3CONCLUSIONS

XIII.TESTNO.4

XIV.TESTNO.4RESULTS

XV.TESTNO.4CONCLUSIONS

XVI.TESTNO.5

XVII.TESTNO.5RESULTS

XVIII.TESTNO.5CONCLUSIONS

XIX.FINALCONCLUSIONS

DVMArturArszułowicz

dvm artur arszułowicz · 1 dvm artur arszułowicz field study documentation on apis plus part 2...

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