Proceedmgs of the 7th Internauona! Worktng Conference on Stored-p'I'oouct Proteciu»i - Volume 1

Sensitivity of the Greater Wax moth Galleria mellonella tocarbon dioxide enriched modified atmospheres

E J. Donahaye, S Navarro, Mmam Rmdner and A. Aznelll

Abstract

Galleria mellonella (L ), is a cosmopolitan pest, causingsenous econorruc damage to honey-bee combs dunngstorage. Efforts are bemg made in Israel to replace existmgcontrol methods against this pest based on fumigation, withan environmentally friendly treatment that affords long-termprotection For this purpose, application of modifiedatmospheres (MAs) by smgle shot flushmg with carbondioxide (CO2) has been developed using flexible treatmentchambers, and modified transport contamers Thislaboratory study was made to provide information on theeffect of exposure penod on wax moth mortality over therange of MA concentrations and temperatures encountered mpractice.Three MAs, namely 80 %, 70 % and 60 % CO2 m air,

were supphed at 75 % r. h to exposure chambers held at26°C and 30°C Eggs (24 - 48 h), larvae (12 d), pupae (0- 48 h) and adults (0 - 24 h) were exposed in the chambersfor penods ranging from eight hours to twelve days.Mortalities were recorded and subjected to probit analysisexcept for eggs where exposure time till no-survival wasrecorded. For all treatments, adults were the mostsensitrve, and eggs the least sensitive. With sensitrvrtybeing consistently higher at 30°C. It was concluded that 10days exposure to a range of 90 - 60 % CO2Will give completecontrol of all stages

Introduction

The greater wax moth, GalIena mellonella (L ) is acosmopolitan insect that causes considerable economic lossesto beekeepers by damagmg wax combs Infestation can begmwithm the hive but IS particularly evident over the penodbetween the close of the bee-keeping (honey flow) seasonwhen supers are removed from the hive, and dunngstorage, until the next season (Eckert, 1951, Smith, 1990,and Singh, 1962) In warm chma tes m particular, all combs

1 Department of Stored Products, Agncultural Research Orgamzanon ,The Volcani Center, P 0 B 6, Bet Dagan, Israel, Fax: 972 - 3- 9604428, E -Mail: Jondon@netVlslon net II

should be treated and then protected dunng storage againstwax moth damage Failure to do so can result m rapid andcomplete destruction of the unprotected combs. Since theempty combs can only be built-up by the honeybeesthemselves, and a ready supply of empty combs dunng thebrood-rearing and honey-flow season is essential for thewell-bemg of the hive , wax moth mfestations can turn bee-keepmg busmesses mto losing ventures The most usualpractices by commercial beekeepers to control the wax mothare based either on fumigation - formerly With EDB and nowWith methyl bromide or phosphme , or by wrappmg theempty combs With cellophane to prevent the entrance of themoth, as well as treatmg the empty combs With sulfur and/or naphthalene or paradichlorobenzme crystals (Burgess,1978) Both methods are relatively meffective while theformer method mvolves a potential health hazardConsequently the adverse effect of the wax moth on growthof the colony and honey production poses a senous threat tothe economic viability of the bee-keeping mdustryThere is therefore an urgent need to fmd alternative

technologies for controlling the wax moth, With preferencetowards the use of non- tOXiCmethods that pose no threat toheal th of opera tor or consumer, and which areenvironmentally fnendly The use of modified atmospheres(MAs) is one of the more prormsmg methods. It has beendeveloped as an alternative to fumigation of storedagncultural commodities m that it retains the specialcapacity of in-situ treatment to control storage pests(Navarro and Donahaye , 1990) It has been successfullyapphed m Israel, to wax moth control, using speciallydesigned flexible treatment chambers and also modifiedtransport contamers (Yakobson et al , 1997) However,there is only scanty mformation on the effect of exposurepenod and MA concentrations on wax moth mortahty(Cantwell et al , 1972, Greatn and D'Agaro , 1992).Therefore this study was undertaken to provide the datanecessary to enable the preparation of dosage schedules forroutme treatments of honey combs and supers agamst theravages of the wax moth

Materials and Methods

Modified atmospheres

In commercial scale trials (Yakobson et al , 1997) the

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Proceedmqs of the 7th International Worhng Conference on Stored-product Protectwn - Volume 1

treatment procedure we chose was by single-shot flushmg ofthe storage chambers with carbon dioxide (COz) to initialconcentrations rangmg from 60% to 95%, after whichconcentrations dropped progressively over the storageperiod. Therefore, m the laboratory, we chose three MAs,namely 80% , 70% and 60% COzin air, m order to providedata on wax moth mortality that would span the range ofhigh COzconcentrations used m practice.These gas compositions were obtamed from supply

cylmders of oxygen (Oz), mtrogen (Nz), and COzusmg agas-mixmg apparatus descnbed by Donahaye (1990). Thisconsisted of the three component gases supplied m tubmg atrates regulated by a series of valves and gas-flow meters,that enabled them to be mixed m the desired combmations.After the gas supplies converged, the gas mixtures m thecommon supply-lines were led to temperature controlledtreatment chambers, and passed through wash-bottlescontammg sulphuric acid to obtam a constant relativehumidity of 75%. Then the gas mixtures were delivered Viadistribution chambers to a senes of 100ml Erlenmeyer flasksthat served as exposure chambers arranged in-parallel. Gassupply was designed to provide a flow rate through theexposure chambers of 7.5 to 10 ml/rmn. To periodicallycheck the MAs, gas samples were withdrawn from theexposure chambers and analysed usmg a gas chromatographequipped With twm thermal conductrvity cells and dualcolumns packed With 'Poropak Q' and 'Molecular SIeve5a' .

Temperatures

To cover the ambient temperatures prevalent under fieldconditions, the experiments were earned out in CT roomsmamtamed at 26°C and 30°C.

Insects

Rearmg techmque: All stages of G. mellonella wereobtamed from cultures reared at 30°C and 70% relativehumidity (r. h ) m the dark on a diet of maize flour (22%),wheat flour (11 %), ground wheat (11 %), dned milk(11%), dried yeast (5.5 %), beeswax (17.5 % ), honey(11 %) and glycenn (11 %) (Sehnal, 1965). The stockmatenal was collected from a field population mfestmg bees-wax m the apiary of the ARO, Israel. Cultures were startedfrom eggs obtamed by placmg 6 to 10 freshly emerged adultsm a petri dish together With pieces of folded and dampenedpaper. About 500 eggs layed on the paper were thentransferred to 500ml culture Jars contammg 150 ml of foodmedium. Larvae were retamed m these culture Jars until the4th mstar Then they were transferred m groups of 50, to500 ml Jars contammg 300 ml of culture medium which waschanged every three to five days Before pupation, a bagmade from nylon mesh and contammg sections of PE tubmg,

each 4mm i. d. , and 25 mm long, was placed in the cultureJars. Larvae that pupated m these tubes were then removedand placed m petn dishes, pnor to use m expenments, or morder to obtain freshly emerged adults.Preparation of stages before exposure to treatments: Eggs

were obtamed as descnbed above. They were held until 24- 48h old, and then rafts contammg about 50 eggs were cutfrom the paper and placed m an exposure chamber fortreatment Larvae for expenments were removed fromculture Jars 12 days after the cultures had been set-up.Pupae were removed from culture Jars every 2 days andexposed to treatments aged 0 - 48 h. Newly emerged adultswere exposed to treatment aged 0 - 24 h

Experimental procedure

For larvae, pupae and adults, groups of 25 - 30 insectswere placed m sets of exposure chambers, and linked to thegas mixture apparatus. For eggs, pieces of paper contammga raft of eggs were placed m each exposure chamber. Anadditional flask contammg msects held under atmosphencconditions served as control. Periodic removal of flasks wasbased on prelmunary trials to cover the time ranges overwhich msect mortality was found to occur for each stage,gas-mixture and temperature. Due to heterogeneity ofresponse, each set of exposures was repeated several times.For larvae, pupae and adults, mortality results weresubjected to probrt analysis (Daum, 1979).At the end of each exposure time, one flask was removed

from the apparatus and mcubated at 30 ± 1°C and 70 ± 5% rh. until mortality was recorded. For larvae, food mediumwas added, and mortality of larvae was considered ascompnsmg those msects that failed to pupate, while pupalmortah ty consisted of those that failed to emerge as adultsAdults that failed to recover after 24 h were considered asdead. For eggs, due to the great difficulty m countmg egghatch, and a delayed hatchmg effect caused by thetreatment, the rafts of eggs were mcubated With culturemedium, and for exposures at which no larvae wererecorded after 20 days, total kill of eggs was considered tohave been obtamed.

Results and Discussion

The exposure times required to produce 99% mortality forlarvae pupae and adults, and 100% mortality for eggs ateach of the three MAs and at the two temperatures are givenm Figs 1 and 2. For companson of sensrtivity between theinsect stages to the different treatments, results at theLT95 and LT99 levels, including 95% confidence limits,are given m Table 1

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Proceedings of the 7th Iniernatumol Working Conference on Stored-product Protectum. - Volu-me 1

90-r------------------------------~y = 434.196xCO 368) r = 0.934y= 224 517x(0 245) r = 0 862

y= 212.981xCO 257) r= 0.973

y= 104.048x(0 110) r= 0 71080-+---------........-------=:~-------....:..-:.---:-~------i

EggLarvaPupa

Adult

o 70U~

60-+---------------------=:::::.......:::::------------1

50~--,.-..,..__.,..-.,____r_-.,__.....,..-...____T""-r____r_-r___r_---,~..,...___,-...,___,__l10 20 30 40 50 60 70 80 90 100110120130140150160170180190200

Time in hours

Fig.l. Concentration/time mortality curves for the sensrtrvrties of larvae, pupae and adults of Galleria mellon ella to carbondioxide at 26'C at the L1'99 level, and eggs at LTlOO

90,--------------------------------,y = 1521 873x(0 645) r x: 0 999y = 125 173x(0 155) r= 0 978

y= 145 475x(0 248) r = 0 979

y= 125 033x(0 172) r = 0929801-----------------~--------------1

EggLarva

PupaAdult

o 70-t------''c--~~-~;:---------------~~---------1U~

60+------.,;~-------:::::._.."..-------==---"""'=='------"'---__1

50+--...-- ......------------ ......---..,...- .......-...,....-..,.----110 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160

Time in hours

Fig.2. Concentratron/time mortality curves for the sensitrvrties of larvae, pupae and adults of Gallerza mellonella to carbondioxide at 30'C at the L1'99 level, and eggs at LTlOO

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Proceedmqs of the 7th Internatwnal Working Conference on Stored-product Protectwn - Volume 1

Table 1. Summary of sensitrvrtes at the LT95 and LT99 levels of three stages of Galiena mellonella , and time to total kill ofthe egg stage, at three modified atmopheres and two temperatures.

ModifiedAtmosphere

StageTemp Se SlopeSEslope

LT95(h)

95% Conf.lmuts

LT99(h)

95% Conf.lmuts

60% CO2

EggLarvaPupaAdultEggLarvaPupaAdultEggLarvaPupaAdultEggLarvaPupaAdultEggLarvaPupaAdultEggLarvaPupaAdult

70% CO2

80% CO2

1.001.001.00

1 001.001.00

1. 701.001.00

1.001.001 00

1.671.001.00

1.001.001.00

4.383.052.37

3.655.33.71

3.657.762.02

3.594.876.57

4.6210.213.4

5.17.738.03

0.480.790.63

0.421.30.89

0.981.810.47

0.521.020.9

1.071.952.73

0.881.101. 73

192*11485.527.6

150*80.455.624.3

166*10353 536.7

120*22.315.313.2

96*47.54312.1

96*15.615.210.1

96 -11462-17116 -114

16314353.5

130-22692-46426-445

92 -19353 -18023 -117

96 - 95053 -11136 -628

24 -61.116 - 40.614-23

43 - 33143-6812-18

16-3816-2410-19

* time to total kill

From the figures It can be seen that for the three MAs andboth temperatures, the egg was the most resistant stage.For all stages, both CO2concentration.and temperature hada marked mfluence on lethal times- the higher the CO2concentration and the higher the temperature, the shorterthe lethal time. The 99% lethal times ranged from 15 h foradults at 80% CO2and 30°C, to 184 h (app. 8 days) foreggs at 60% CO2and 26°CBoth figures also provide 99% mortahties at intermediate

MAs, as well as more tentative extrapolation to lower CO2concentrations than those used m these expenmentsThese fmdmgs may be compared With those of Cantwell et

al (1972) who studied sensitrvrties of all stages of thegreater wax moth to high CO2 concentrations attemperatures rangmg from 21 to 37 .8°C. They concludedthat the larval stage was the most resistant to high CO2,followed by eggs, whereas our results showed that eggswere the most resistant They found that at 37 .8°C (100°F)exposures to obtam complete kill were greatly shortened,requmng a maximum of 28 hours for larvae exposed to

64 -11243 -10417-53

12474.537.1

73 4% CO2m air. However, tlus would require artificialheatmg of the treatment chamber, and because this IS notavasilable to most apiculturahsts the highest temperatureused in our expenments was 30°C Although their work wascarried out under different temperatures and CO2regimesand most of the study was undertaken on larvae, which theyconsidered as the most resistant stage, where therrconditions were similar to ours, the results were alsocomparable though their larvae seemed less susceptible. Forexample at 27.8°C and 60% CO2 they obtained 42%mortality after 4 days exposure, while our results at 26°Cand 60% CO2gave 90% larval mortality after 4 days. Ourresults do not seem to conform WIth those of Greatti andD'Agaro (1992) who flushed CO2 at a rate of 8L/mmthrough a 0.49 rrr' chamber contammg mfested supers andobtamed complete kill after 3 mm.Our fmdmgs support those of the field tnals (Yakobson et

al. 1997) m which complete kill was recorded dunng smgle-shot treatments m flexible lmers and modified transportcontainers that were momtored for the first 10 days, dunng

695

71- 33945-7821-154

15865.579.6

17-3512-2411-17

34.521.116.8

34-14438-5411-15

66.750.113.6

13-2413-189-14

21.218 612.3

Proceedings of the 7th Iniernatumal Worktng Conference on Stored-product Protection - Yoiume 1

wluch time mitral high COz concentrations (96 - 65%) ,dropped to near the 40% level. Jay et al. (1972) carriedout COz fumigations of comb honey in serru-trailer vans byflushing to a concentration of 98. 6% COz WhICh was thenheld for 10 to 12 hours, They obtamed 92.7 -100% kill oftest larvae. However, this technology requires the use of aCOz supply vessel and other logistical back-up and IS not

Widely apphcable to the small scale bee keeper.Furthermore, the advantage of treatmg the supers mpermanently mstalled modified con tamers or flexibleenclosures, IS that after the treatment, the wax combscontmue to be protected from remfestation within theenclosed space until required for use m the field.

References

Burges, M. D 1978. Control of wax moth: Physical,chemical and biologicalmethods. Bee World 59 (4), 129-139Cantwell, G E., Jay, E. G , Pearman, G. Jr andThompson, J. V. 1972. Control of the greater wax mothGalIena mellonella (L.) in comb honey WIth carbondioxide. Parts I and II, Amencan Bee Journal 112(9) , 302- 303; 342 - 344.

Daum R J 1979. A reVISIOnof two computer programs forprobit analysis Bulletm of the Entomological Society ofAmenca 16, 10 -15.Donahaye, E. 1990. Laboratory selection of resistance bythe red flour beetle, Tnbohum castaneum (Herbst), to anatmosphere of low oxygen concentration. Phytoparasitica,18, 189- 202.

Eckert, J. E. 1951. Beekeepmg in Hawau. Gleanmgs in beeculture 79, 393 - 400, 468 - 472, 509Greatti, M. and D'Agaro, M. 1992. Control of Galleriamellonella and Achroia gnsella with carbon dIOXIde (inItahan). Apicoltore Moderno 83, 123- 128Jay, E G, Cantwell, G E Pearman, G. C. andThompson, J. V. 1972. Control of the greater wax mothGalIena mellonella (L.) in comb honey WIth carbondIOXIde. Amencan Bee Journal, 112: 342, 344.Navarro, S. and Donahaye , E. 1990. Generation andapplication of modified atmospheres and fumigants for thecontrol of storage insects. In: Champ, B R., HIghley,E., and Banks, H. J. ed., Fumigation and controlledatmosphere storage of grain: proceedings of anmternational conference, Smgapore, 14 - 18 February1989. ACIAR Proceedmgs No. 25, 152-165.Sehnal, F. 1965. Kntisches studium der bionorrue undbiometrik der in verschiedenen lebensbedmgungengezuchteten wachsmotte GalIena mellonella L. Z. WISS.Zool 174, 53 - 82Singh, S. 1962. Beekeping m India Indian Council ofAgncultural Research, New Dehli.Smith, F. G. 1960. Beekeping in the TroPICS Longmans,Green, London.Yakobson, B. A. , Navarro, S. , Donahaye, E. , Aznelr, A. ,Slavezky, Y. and Ephrat , H. 1997 Control of beewaxmoths usmg carbon dIOXIde in flexible plastic and metalstructures. In: Donahaye, E J., Navarro, SandVarnava, A. ed., proceedmgs mternational conferencecontrolled atmosphere and fumigation in stored products,21- 26 Apnl 1996. Pnntco Ltd. NiCOSIa, Cyprus, 169 -174.

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