THE EFFICACY OF CERTAIN ESSENTIAL OILS AND THEIR MIXTURES ON THE PARASITIC MITE VARROA DESTRUCTOR (Anderson and Trueman)

Salah M. Hussein, Dahi M. Moustafa and A. R. Hassan

Plant Protection Dept., Faculty of Agriculture , Minia Univ.

Abstract

The ectoparasitic mite Varroa destructor (Anderson and Trueman) is considered one of the most serious

pests of beehives, causing tremendous damage to honey bees (Apis mellifera L) and great economic loses to the

beekeeping industry. Efficacy of six essential oils (Marjoram oil , Thymol,

Cinnamon, Clove, Eucalyptus, Citronella, and 15 of their mixtures were

evaluated against varroa mite infested the honeybee colonies for two

successive seasons. The tested oils were applied on the infested bee

colonies at 28 days intervals.

Results indicated that Cinnamon + clove and Marjoram oil gave the

reduction % against mites on adult (95.30% and 89.15% ) followed by

Thymol oil (87.5%), cinnamon oil (87.22%). From the mixtures Marjoram

+ clove oil gave the highest effect against Varroa on adult (84.47%).

Cinnamon oil and the mixture of Cinnamon and clove with concentration

of 1.5 ml / hive gave the highest reduction on mites parasite on adult bees

and brood mite infestation and highly knockdown thus they can be used as

a good elements in integrated mite control.

Introduction

Honey bee colonies are subject to infestation by insects, mites and diseases.

The ectoparasitic mite Varroa destructor (Anderson and Trueman) is considered one of the most serious

pests of beehives. Parasitism can result in a loss of up to 25% of adult weight, severe deformations of the wing and

reduced longevity of worker and drone honey bees. Colonies infested with Varroa destructor have significantly reduced

worker bee populations and eventually die if left without controlling.( Adams2001(

The widespread use of synthetic lipophilic acaricides has lead to the

accumulation of residues in beeswax, propolis and to a much lesser degree,

in honey (Hussein, 2009). The development of acaricide resistance in V.

destructor populations and the spectre of the contamination of hive products

provide considerable incentive to develop new treatment strategies that

minimize the potential for the rapid development of resistance and the

accumulation of residues.

Plant essential oils have potential as products for varroa mite control because

some of them are selective and have little or no harmful effects on non target

organisms. They may be applied to hiding places or host ( bees) in the same

way as other conventional acaricides. Many essential oils are known to

posses various bioefficacies such as ovicidal, various arthropod pests

repellent. Anti- feeding and biocidal activities against various arthropod pests

without any adverse effects on non target species. And they are found to be

highly effective against insecticide resistant pests .

Thus the present studies were carried out to Evaluate the efficacy of certain

essential oils and their mixtures on the parasitic mite Varroa

destructor( adult and brood infestation) to find out one of the natural

products or mixture which maximizing Varroa control and minimizing the

site effect on the honeybee colonies and environmental, with minimal costs.

MATERIALS AND METHOD

1-Experimental honeybee colonies:

Sixty six honeybee colonies of the first hybrid carnelian bees ( Apis

mellifera Carnica) were selected among the colonies of a private a piary

at Dahrout village. The selected colonies dequeened at August and three

days later, each received virgin sister queen. The successful natural mated

queens were marked on their thoraxes and were left in their colonies till

the end of the flow season. At the end of September, the colonies having

an equal strength and healthy marked queens were chosen for conducting

the experiments.

The experimental colonies were divided into groups, each having three

colonies as replicates. Average strength of the tested colonies was 8 combs

covered with bees housed in langstroth hive.

Types of the tested essential oils

Efficacy of six essential oils and 15 of their mixtures were evaluated

against varroa mite infested the honeybee colonies. Evaluation of the

tested oils were continued over two successive seasons the tested oils

(Table 1) were obtained from El- Gomhoria chemicals company, Cairo.

Preparation of the essential oils:

Emulsions of three concentrations (1.5, 0.75, and 0.38 ml/hive) of each

tested oil were prepared through mixing the specified concentration with

0.5 ml of an emulsifier ( Triton – X) and sufficient amount of water to

obtain 30 ml emulsion. Volume of the prepared emulsion was sufficient to

treat one infested bee colony (one hive). Preparation of the emulsions of

the tested oils have been done just before application.

Table (1): English and scientific name of the source of the essential oil:

English name Scientific name of plant source

1-Marjoram oil Majorana harensis

2-Citronella oil Cymbopogon nardul L

3- Cinnamon oil Cinnamomum zeylanicum Blume

4-Clove oil Eugenia caryophyllus L

5-Eucalyptus oil Eucalyptus globulis, labill

6-Thymol oil Thymus vulgaris L.

Application of the tested oils:

Hand sprayer was used for delivery of a particular concentration on the

combs covered with bees of the infested colony. Spraying had been done

on the end of the day hours to insure presence of most individuals of the

bee colony. Each concentration was replicated three times on three

infested colonies. Three colonies were used as control treatment, and

treated with emulsion of water and emulsifier. Treatments of the tested

oils were applied periodically at 28 days intervals

Evaluation the efficacy of the tested materials.

The efficacy of the tested materials were evaluated through estimation the

infestation percentage of Varroa mite on colony individuals and its brood.

Level of infestation on live bees was determined through randomly

collection of about 50 bees / colony in a gar partially filled with water and

few drops of detergent (Liquid soap). The collected bees were shaken for

30 seconds, then bee were filtered through muslin ( 8 to 12 mesh/ inch) to

remove the bees, and the passed adults of mite were then counted

(Komeill, 1988). This procedure was done before the application and after

24h, 48, 7; 14 and 28 post days application and the treatment was applied

new.

Also, infestation of brood was determined at the same time of examination

of the adult bees. One hundred of sealed brood worker per colony were

randomly chosen. The cell capping were removed by forceps and the

pupae were picked up to examine for mites presence ( Marcangeli et al.,

1992).

Besides above measurements, the hive debris were examined periodically

for determination the number of the natural mite fall per hive a day.

Amount of dead mites was used as indicator of mite population counting

the number of dead mites was done through providing the bottom brood of

each brood chamber with sticky white paper smeared with thin layer of

Vaseline.

For evaluation the efficacy of the tested essential oils in controlling Varroa

mite, two ways were considered, the first, estimation average effectiveness

period. While the second one was calculation reduction percentage of mite

infestation by applying Henderson and Telton 1955 equation.

The reduction rate of infestation =

( Ta X Cb)

= 100 x 1-

( Tb X Ca)

where:

Tb is % infestation of mite before treatment

Ta is % infestation of mite after treatment

Cb is % infestation of mite before treatment for the control.

Ca is % infestation of mite after treatment for the control.

Statistical analysis:

The collected data were subjected to one way analysis of variance

according to method of Mead et al. (1993). The statistical analyses were

conducted using the SPSS II and F- test. Differences among means were

determined by Duncan's multiple range test (P < 0.05 or < 0.01 ).

Results and Discussion

Efficacy of the tested essential oils with 1.5 ml./hive against Varroa

mite

The statistical analysis of the obtained data indicated that there were

significant differences among different treatments and concentrations.

Data shwed that the the concentration of 1.5 ml/hive gave the highest

effect as showen in Fig 1, 2 and 3 .

Data presented in Fig. 1 showed that the highest average reduction % of

mite infestation on adult bees (95.30%) was recorded with applying the oil

mixture of Cinnamon and Clove, followed by Marjoram oil ( 89.15%), and

Thymol oil (87.56%)

As regard to brood infestation the results showed much variation in

reduction infestation percentage, the highest reduction percentage ( 75%)

was obtained when the colonies treated with clove oil or the oil mixture of

Cinnamon and Eucalyptus.

On the other hand, recording the average of fallen mites for two seasons

showed that the highest number of dead varroa mite ( 17.25 mites/ colony)

was recorded with spraying Cinnamon oil, followed by Thymol oil ( 14.35

mites/ colony) and oil mixture of Cinnamon and Eucalyptus.

Fig.(1): Effect of spraying essential oils and their mixture on their

infestation level of the parasitic mite (Varroa destructor) with the highest

concentration (1.5 ml /hive

Fig.(2): Effect of spraying essential oils and their mixture on their

infestation level of the parasitic mite (Varroa destructor) with the

highest concentration (0.75 ml /hive)

Fig.(3): Effect of spraying essential oils and their mixture on their

infestation level of the parasitic mite (Varroa destructor) with the

highest concentration (0.36 ml /hive).

Table(2): Effect of spraying essential oils and their mixture on their infestation level of the parasitic mite (Varroa destructor) during two seasons with the highest concentration (1.5 ml /treatment).

Average effect on

TreatmentsReduction

% in varroa on adult

bees

Reduction % On brood

No. of Dead fallen Varroa mites

78.1125.006.88Marjoram oil

62.5837.507.63Citronella oil

73.7716.678.53Cinnamon oil

73.3850.0016.25Clove oil

76.8025.007.25Eucalyptus oil

75.5435.008.75Thymol oil

60.3225.003.75Marjoram oil + Citronella oil

69.7325.009.13Marjoram oil + Cinnamon oil

61.5035.008.75Marjoram oil + Clove oil

61.2050.004.35Marjoram oil + Eucalyptus oil

71.3437.507.50Marjoram oil + Thymol oil

64.0637.509.88Citronella oil+ Cinnamon oil

69.7332.504.25Citronella oil + Clove oil

67.9150.004.13Citronellal oil + Eucalyptus oil

67.4916.674.40Citronella oil + Thymol oil

79.0437.504.75Cinnamon oil + Clove oil

55.8242.509.13Cinnamon oil +Eucalyptus oil

69.3329.177.65Cinnamon oil + Thymol oil

68.5212.507.65Clove oil + Eucalyptus oil

66.8850.004.75Clove oil + Thymol oil

66.3737.505.15Eucalyptus oil + Thymol oil

0.000.001.50Control

From the data presented in fig1, 2 and 3 the maximized effect was

observed with spraying the high concentration 1.5 ml . The other

concentrations 0.75 and 0. 375 ml/ hive showed a little effect Fig 2 and 3

Thus we advise to use a high concentration which gave mortality more

than 90% reduction and gave highly mortality on mites. The low

concentration cannot used because it may develop the resistance of mite.

From our results, it is clear that Marjoram, Cinnamon, and Thymol

essential oils have biological activity against varroa mite and environment

friendly (non- polluting and lesser or no toxicological concerns ) (Isman,

2006).

The obtained results may permitted us to conclude the following points:

1-The tested essential oils in general were more effective in reduction of

Varroa mite population parasited on adult bees than those parasited on the

brood of the same colonies. These findings may be due to the direct

toxicity of these substances on the Varroa mite parasited on adult bees,

while this effect not reached rapidly, and directly to the brood and their

associated mite which lived together in the capped cells.

2-Application of the tested essential oils at 28 days with the concentration

1.5 ml/ hive, they showed various efficacy against Varroa mite which can

managed in the following descending order the oil mixture of cinnamon

and clove > Marjoram oil > Thymol oil > Cinnamon oil which

gave(95.30, 89.15, 87.56 and 87.22 reduction % on adult respectively.

These findings are in agreement with results of Richi et al., (1991) found

that the colonies treated by Apilife – Var ( Thymol represent the most

content) for a total 38 days resulted in 96.4% of the mites mortality and in

colonies treated for 79 days, 99.00%. Allam, (1999) found that the

essential oils treatments had higher effect on the Varroa mite, Less side

effect on brood rearing and Less hazards towards bees and queens, in spite

of some lost colonies during treatments. Imdorf et al., (1999) mentioned

that Thymol and Thymol blended with essential oils or essential oil

components provided the best results as mite mortality approached 100%.

Khattab (2001) found that the efficacy of the volatile oils were tested

against Varroa mite in Egypt i.e. Thymol and Clove oils as Varroacids in

controlling the Varroa mite were found to be highly effective in reducing

the Varroa mite infestation on the honeybee colonies.

Serag El- Dien and Eissa (2003) found that Clove oil ( 2 cm3/ hive) gave

the highest reduction ( 91.00%) of Varroa mite in comparing to menthol,

anise, citrus and camphor after four weeks of treatment.

Many essential oils are known to possess various bioefficacies such as

ovicidal, repllent, antifeeding and biocidial activities various arthropod

pests (Isman, 1999). Additionally, some plant extracts or phytochemicals

are found to be highly effective against insecticide – resistant mite and

insect pests. (Ahn et al., 1997) A carcidal activity has been reported from

some essential oils against V. destructor (Imdorf et al., 1999) and

Dermatophgoides fasinae and D. Pteronyssinus mites (Kim et al., 2003).

In this study, potencies varied according to oil type, concentration and

number of treatments. Additionally, differential susceptibility of the

essential oils from the same plant species to the adult mite was observed

in the two seasons. These differences may be due to the different in a

climatic conditions of the hive, and ecosystem. Thus the essential oils i.e.

( Majoroum oil, Thymol oil, and cinnamon oils gave highly average

reduction % on adult i.e 89.15, 87.56 and 87.22 % respectively.) and the

mixtures ( Cinnamon + clove, Majoroum + Cinnamon and Majoroum +

Clove)gave average reduction % 95.29, 84.72 and 84.47 % on adult)

might be good elements for naturally occurring varroa mite control agent

in honey bee colonies. Also from the general results Cinnamon oil gave

the highest aveg. Reductin % in mites in Adult and brood and gave the

highest aveg. Fallen no. of mite. And its mixture with Euchlyptous oil.

Investigation on mode of action of essential oils or chemical is important

for mite control because it may give useful information on the most

appropriate formulation and delivery means. Volatile compounds of many

plant extracts and essential oils are composed alkanes, alcohols, aldehydes

and terpenoids, particularly mono-terpenoids, and exhibit fumigant

activity Coats et al., 1991, Kown and Ahn, 2002 andf Kim et al., 2003).

Isman and Machial 2006 Bakkali et al., (2008) indicated that aromatic

plants and their essential oils have been used as antimicrobial, acaricidal

and insecticidal agents and to repel insect and mites or protect stored

products. These constitute effective alternatives to synthetic pesticides

without producing adverse effects or the environment (Isman 2000,

Isman and Machial, 2006). Moreover, the interest is essential oils has

regained momentum during the last decade primary due to their fumigant

and contact pesticidal activities and the less stringent regulatory approval

mechanisms for their exploration due to long history of use ( Isman,

2006).

the future potential of plant essential oils as an alternative to synthetic

acaricides for the control of this pest seems promising.

This variability could be overcome through careful sourcing of products,

identification of essential oils that are generally reliable in their toxicity or

controlled production practices. For example work suggests that the

techniques such as micro propagation and used it as dust application may

be use in reducing essential oil variability, through encouraging the

proliferation of selected, reliable plant genotypes (Garddulf et al. 2004).

In general New alternatives for the control of V. destractor are necessary

because of the rapid and widespread development of pythroid and organo-

phosphate resistant mite population and because of the potential for

contamination of hive products by these chemicals ( Hussein 2009)

Essentials, and especially components of essential oils, may serve as

alternatives or as adjuncts to traditional treatment measures. In our

screening tests, many oils and their mixtures show significant acaricide

activity against varroa mites. Some the them may be repellent to V

destructor, others may be alternative and majority of their caused mite

mortality and varied from season to another. This variation may be due to

variation in local environmental and colony conditions effect efficacy and

make it difficult to predict the outcome of many treatments. Only a

combination of Cinnamon and Clove has been successfully used for mite

control with minimum site effect on the bees. Also oils Clove, Thymol and

marjoram were highly effect against Varroa mite on adult and brood with

less effect on honey bee individuals and gave highly honey production with

highly annual profit colony.

Identifying compounds from these oils with acceptable acaricidal activity

with low toxicity to honey bees is essential for providing candidate

compounds for field trails.

Future research can assist in this effect by focusing on the characterization

of the dose response relationships between essential oils and mite /bee

toxicity and effects on mite behaviours. This procedure can serve as a

powerful screening technique because it guides subsequent field researches

into the most productive a venues.

Most essential oils are mixtures of more than 50 components (Imdorf,

1999). The depending on the individual partition coefficient of the

constituents, residues in honey and wax are to be expected. Residues in

honey can lead to adverse effects on taste, while residues in wax can render

it unsuitable for some applications.

REFERENCES

Adams R.,( 1989.): Identification of Essential Oils by Ion Trap Mass Spectroscopy, Academic Pres 1989.

Ahn, Y.J. Ahn, S.B. Lee, H.S. Lee and G.H. Kim,( 1998) Insecticidal and acaricidal activity of carvacrol and β-thujaplicine derived from Thujopsis dolabrata var. hondai sawdust, Journal of Chemical Ecology 24 (1998), pp. 81–90. Full Text via CrossRef

Allam, Sally, F.M. (1999): Studies on the honeybee parasite Varroa jacobsoni Oudemans ( Acari: Gamasida: Varroidae) in Egypt. Ph. D. Thesis, Acarology, Fac. Agric., Cairo University.

Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M., 2008. Biological effects of essential oils—a review. Food Chem. Toxicol. 46, 446–475.

Coats, J.R., Karr, L.L., Drewes, C.D., 1991. Toxicity and neurotoxic effects of monoterpenoids in insects and earthworms. in: Hedin, P.A. (Ed.), Naturally Occurring Pest Bioregulators. ACS Symposium Series No. 449, American Chemical Society, Washington, DC, pp. 305–316.

Garddulf, A., Wohlfart, I., Gustafson, R., 2004. A prospective cross-over field trial shows protection of lemon Eucalyptus extract against tick bites. J. Med. Entomol. 41, 1064–1067.

Imdorf, A.;S. Bogdanove;R. Ibjezocho and N.W.Calderone (1999): Use of essential oils for the control of varroa Jacobsoni Oud. In Honeybee colonies . Apidologie (30): 209-228.

Henderson, C. F. and Tilton (1955): Tests with acaricides against the brown mite J. Econ, Entomol. 48: 157- 161.

Hussein, S.M. (2009): Residues certain Varroacides in the honeybee products. The 6th International Arab Apicultural Conference, Abha Kingdom of Saudi Arabia. 17-19 Mar. 68p

Imdorf, A.;S. Bogdanove;R. Ibjezocho and N.W.Calderone (1999): Use of essential oils for the control of varroa Jacobsoni Oud. In Honeybee colonies . Apidologie (30): 209-228.

Isman, M.B., 2000. Plant essential oils for pest and disease management. Crop Prot. 19, 603–608.

Isman, M.B., 2006. Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world. Annu. Rev. Entomol. 51, 45–66.

Isman, M.B., Machial, C.M., 2006. Pesticides based on plant essential oils: from traditional practice to commercialization. In: Rai, M., Carpinella, M.C. (Eds.), Naturally Occurring Bioactive Compounds. Advances in Phytomedicine, vol. 3. Elsevier, pp. 29–44.

Khattab, M.M. (2001): The use of volatile oils as Varroacides for controlling Varroa jacobsoni infested honeybee colonies in Egypt ( Agric. Sc. Moshtohor. 39(4): 2491- 2498.

Kim,S., J. Yi, J. Tak and Y. Ahn, 2004: Acaricidal activity of plant essential oils against Dermanyssus gallinae (Acari: Dermanyssidae), Vet. Parasitol. 120 (2004), pp. 297–304.

Komeill, A.B. (1988): The impact of the Varroa mite on Iranian commercial beekeeping . Am. Bee. J., 126: 423- 424.

Marcangel; J.M. M.J. Eguaras, and N.A. Fernandez, (1992): Reproduciton of Varroa jacobsoni (Acardi: Mesostigmata: Varroidae in temperate dimates of Argentina. Apidologic, 23: 57- 60.

Mead, R. ; R.N. Curnow, and A.M. Harted, (1993): Statistical methods in agriculture and experimental Biology (2nd Ed.) chapman and Hall, London.

Richi, M.; A. Imdorf, and V. kilchenmann (1991): Treatment of Varroa disease using compounds of essential oils Apidologie 22(4): 417- 421

Serag El-Dien, F.Sh. and A.A. Eissa (2003): Efficiency of some natural oils and chemical substances against the mite, Varroa jacobsoni (Oud). Infesting honeybee in different localities in Egypt. J. Agric. Sci. Mansoura Univ. 28(11): 6921- 6925.

Sergio, R.; M. Maggi; C. Facerin and B. Susana (2007): Essential oils toxicity related to Varroa destructor and Apis millifera under laboratory conditions. Zootecnia Trop. Vol. 25 No.1 Maracay Jam.

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