integrated management for ectoparasitic mites varroa destructor

INTEGRATED MANAGEMENT FOR ECTOPARASITIC MITES VARROA
DESTRUCTOR (ANDERSON AND TRUEMAN) AND TROPILAELAPS
CLAREAE (DELFINADO AND BAKER) OF HONEY BEE APIS
MELLIFERA L. IN RELATION TO HONEY YIELD
RASHID MAHMOOD
07-arid-01
Department of Entomology
Faculty of Crop and Food Sciences
Pir Mehr Ali Shah
Arid Agriculture University, Rawalpindi
Pakistan
2012

INTEGRATED MANAGEMENT FOR ECTOPARASITIC MITES VARROA
DESTRUCTOR (ANDERSON AND TRUEMAN) AND TROPILAELAPS
CLAREAE (DELFINADO AND BAKER) OF HONEY BEE APIS
MELLIFERA L. IN RELATION TO HONEY YIELD
by
RASHID MAHMOOD
(07-arid-01)
A thesis submitted in the partial fulfillment of
the requirements for the degree of
Doctor of Philosophy
in
Entomology
Department of Entomology
Faculty of Crop and Food Sciences
Pir Mehr Ali Shah
Arid Agriculture University, Rawalpindi
Pakistan
2012

CERTIFICATION
I hereby undertake that this research is an original one and no part of this thesis
falls under plagiarism. If found otherwise, at any stage, I will be responsible for the
consequences.
Name: Rashid Mahmood Signature:__________________
Registration No: 07-arid-01 Date: _____________________
Certified that the contents and form of thesis entitled “Integrated
Management for ectoparasitic mites Varroa destructor (Anderson and Trueman)
and Tropilaelaps clareae (Delfinado and Baker) of Honey Bee Apis mellifera L. in
Relation to Honey yield” submitted by “Mr. Rashid Mahmood” has been found
satisfactory for the requirement of the degree.
Supervisor: _______________________
(Dr. Ata-ul-Mohsin)
Co-Supervisor: ______________________
(Dr. Elizabeth Stephen)
Member: _______________________
(Dr. Muhammad Naeem)
Member: _______________________
(Dr. Ghazala Kaukab)
Chairman: _________________________
Dean: _________________________
Director, Advanced Studies: ___________________________
ii

iii

Dedicated
To
Holy Prophet MUHAMMAD
(Peace be upon Him)
and
to my
beloved MOTHER
iv

CONTENTS
v
PAGE
LIST OF TABLES viii
LIST OF FIGURES ix
PUBLICATIONS xi
ACKNOWLEDGMENTS xii
ABSTRACT xiv
1. INTRODUCTION 01
1.1. OBJECTIVES 07
2. RIVIEW OF LITERATURE 08
2.1. MITES, APISTAN AND RESISTANCE 08
2.2. THYMOL AND FORMIC ACID FOR MITE CONTROL 12
2.3. PLANT OILS/ EXTRACTS FOR MITE CONTROL 16
2.4. OXALIC ACID FOR MITES CONTROL 22
2.5. INTEGRATED PEST MANAGEMENT FOR MITES CONTROL 26
3. MATERIALS AND METHODS 31
3.1. ADULT BEES INFESTATION ASSESSMENT 31
3.2. BROOD INFESTATION ASSESSMENT 32
3.3. MITE INFESTATION ASSESSMENT 32
3.4. CONTROL OF TROPILAELAPS CLAREAE USING THYMOL
AND FORMIC ACID 33
3.5. THE EFFECTIVENESS OF DIFFERENT CONCENTRATIONS

OF OXALIC ACID SOLUTION FOR VARROA DESTRUCTOR
CONTROL 34
3.6. FIELD TRIAL FOR ECTOPARASITIC MITES CONTROL WITH
THYMOL AND OXALIC ACID SOLUTION 36
3.7. CONTROL OF VARROA DESTRUCTOR WITH PLANT
OILS/ EXTRACT S 38
3.7.1. Oil extraction by soxhlet apparatus 38
3.7.2. Preparation of tobacco water extracts 39
3.7.3. Laboratory bioassay 39
3.7.4. Field tests in bee hives 40
3.8. THE EFFECTIVENESS OF INTEGRATED CONTROL OF
TROPILAELAPS CLAREAE AND VARROA DESTRUCTOR
WITH DIFFERENT TREATMENTS 41
3.9. HONEY YIELD 43
3.10. STATISTICAL ANALYSIS 44
4. RESULTS AND DISCUSSION 45
4.1. CONTROL OF MITES USING THYMOL AND FORMIC ACID 45
4.2. THE EFFECTIVENESS OF DIFFERENT CONCENTRATIONS OF
OXALIC ACID SOLUTION FOR CONTROLLING OF
VARROA DESTRUCTOR. 52
4.3. FIELD TRIAL OF TROPILAELAPS AND VARROA MITES
CONTROL WITH THYMOL AND OXALIC ACID SOLUTIONS 60
4.4. CONTROL OF VARROA MITES WITH PLANT OILS/EXTRACTS 68
vi

4.5. THE EFFECTIVENESS OF INTEGRATED CONTROL OF
TROPILAELAPS CLAREAE AND VARROA DESTRUCTOR
WITH DIFFERENT TREATMENTS 82
4.5.1. Integrated pest management for control of bee mites 90
SUMMARY 93
CONCLUSION 97
RECOMMENDATIONS 97
LITERATURE CITED 98
APPENDICES 127
COPY OF THE PUBLICATION 138
vii

LIST OF TABLES
Table No. Page
4.1 Efficacy of thymol in Apis mellifera colonies 48
4.2 Efficacy of formic acid in Apis mellifera colonies 48
4.3 Simple Linear Regression Model Results 50
4.4 Mean Comparisons, F-test and Coefficient of variance 51
4.5 Simple Linear Regression Model Results 57
4.6 Multiple Comparisons of different concentrations of oxalic acid
with control against Varroa mites 59
4.7 Simple Linear Regression Model Results 65
4.8 Multiple Comparisons of different treatments of thymol and oxalic acid
with Control 66
4.9 Mean mortality of mites by different combinations of oils/extracts 72
4.10 Acaricides’ efficacy of different essential oils/extract in Apis mellifera
colonies. 73
4.11 Mean honey yield produced from Apis mellifera colonies treated with
different plant oils/extract. 75
4.12 Simple Linear Regression Model Results 78
4.13 Multiple Comparisons of plant oils/ extracts with control
against Varroa mortality 79
4.14 Multiple Comparisons of different treatments for the control of mites 88
viii

LIST OF FIGURES
Figure No. Page
4.1 Mortality of T. clareae in bee colonies treated with thymol and formic acid. 49
4.2 Mean efficacy of acaricides at the end of experiment. 49
4.3 Mean amount of honey produced from colonies treated with thymol and
formic acid against T. clareae 50
4.4 Mite mortality (%) after treating bee colonies with thymol and formic acid 51
4.5 Mean number of mites found dead in colonies after using different
concentrations of oxalic acid 55
4.6 Mean efficacy of oxalic acid with different concentrations observed
at the end of experiment 56
4.7 Mean amount of honey produced from colonies various treatment
of oxalic acid 57
4.8 Mites mortality (%) after treating bee colonies with different concen-
trations of oxalic acid 59
4.9 The mean number of mites fallen for T. clareae and V. destructor
bars for different treatments of thymol and oxalic acid 64
4.10 The mean % efficacy for T. clareae (plain) and V. destructor bars
for different treatments of thymol and oxalic acid. 64
4.11 The mean amount of honey produced from colonies treated with different
treatments of thymol and oxalic acid 65
4.12 T. clareae mite mortality (%) after treating bee colonies with
ix

different treatments. 67
4.13 V. destructor mite mortality (%) after treating bee colonies with
different treatments. 67
4.14 Mean mite mortality as affected by plant oils/ tobacco extract. 71
4.15 Mean mite mortality as affected by different concentrations of plant
oils/tobacco extract concentrations 71
4.16 Mean mite mortality as affected by different concentrations of plant
oils/extracts 78
4.17 Acaricides mite mortality by different essential oils/extract in Apis
mellifera colonies. 80
4.18 Efficacy of different essential oils/extract in Apis mellifera colonies. 80
4.19 Honey yield by application of different essential oils/extract in
Apis mellifera colonies. 81
4.20 The mean number of mites fallen for T. clareae and V. destructor
bars for different treatments. 86
4.21 The mean % efficacy for T. clareae and V. destructor bars
for different treatments. 86
4.22 The mean amount of honey produced from colonies treated with different
treatments 87
4.23 T. clareae mite mortality (%) after treating bee colonies
with different treatments. 89
4.24 V. destructor mite mortality (%) after treating bee colonies
with different treatments. 89
x

PUBLICATIONS
1. Mahmood, R., E.S. Wagchoure, S. Raja, G. Sarwar and M. Aslam. 2011. Effect
of thymol and formic acid against ectoparasitic brood mite Tropilaelaps
clareae in Apis mellifera colonies. Pak. J. Zool., 43(1):91-95.
2. Mahmood, R., E.S. Wagchoure, A.U. Mohsin, S. Raja and G. Sarwar. 2012.
Control of ectoparasitic mite Varroa destructor in honeybee (Apis mellifera L.)
colonies by using different concentrations of oxalic acid. J. Anim. Plant Sci.
22(1):72-76.
xi

ACKNOWLEDGEMENTS
If oceans turn into ink and all of the wood becomes pens, even then the praises
of “Allah Almighty” cannot be expressed. He, Who created the universe and knows
whatever, is there in it, hidden or evident and Who bestowed upon me the intellectual
ability and wisdom to search for the secrets. I must bow my head before Allah
Almighty Who is Compassionate and Merciful and Whose help enabled me to
complete this job, which marks an important turning point in my life.
Countless salutations be upon the “Holy Prophet Muhammad (Peace Be
Upon Him), the city of knowledge who has guided his “Umma” to seek knowledge
from cradle to grave.
I am extremely grateful and indebted to express my deepest sense of
appreciation and devotion to my ever-affectionate and worthy supervisor Associate
Professor, Dr. Ata-ul-Mohsin, Department of Entomology, Pir Mehr Ali Shah, Arid
Agriculture University, Rawalpindi. I am particularly indebted to him for his scholastic
and sympathetic attitude, inspiring guidance, generous assistance, constructive
criticism, and timely advice during the course of this degree program.
I wish to extend my thanks to my co-supervisor Dr. Elizabeth Stephen,
Director/ Principal Scientific Officer, Honeybee Research Institute, NARC, Islamabad,
for her obligation, well wishes and encouragement during the course of my research
studies and presentation of this manuscript.
I am also very grateful to the committee members, Dr. Muhammad Naeem,
Professor/ Chairman Department of Entomology and Dr. Ghazala Kaukab, Associate
xii

Professor, Department of Biochemistry, Pir Mehr Ali Shah, Arid Agriculture
University, Rawalpindi for their encouragement, help, guidance, and cooperation
during the entire study.
I would like to record my sincerest thanks to all my fellows Mr. Muhammad
Siddique Munawar, Dr. Shazia Raja, Dr. Farida Iftikhar, Mr. Ghulam Sarwar and Mr.
Asif Ghuman for their friendly cooperation.
The support provided in the field and laboratory work by all the staff members
of PMAS-Arid Agriculture University, Rawalpindi and Honey Bee Research Institute
particularly M/S Qurban Ali, Muhammad Riaz, Zafar Iqbal and Umar Draz is also
appreciated.
Last but not the least I pay my cordial thanks to my brothers, sisters, wife, kids,
particularly my respected Parents and my well wishers for their love, prayers, moral
encouragement and continuous support throughout my life.
xiii
RASHID MAHMOOD

ABSTRACT
The efficacy of different organic acids, plant oils and extract was evaluated a
series of experiments for the control of ectoparasitic mites Varroa destructor Anderson
& Trueman (Acrina: Varroidae) and Tropilaelaps clareae Delfinado and Baker
(Acrina: Laelapidae), a big threat to honeybee, Apis mellifera ligustica (Hymenoptera:
Apidae) populations world-wide. All the experiments were maintained using modified
bottom board trays (mechanical control) and maintaining test colonies with regular re-
queening with hygienic queens (genetic control).
Effectiveness of 4gm thymol and 20 ml formic acid (65%) against T. clareae
mite on honeybee colonies was calculated and it was found that formic acid killed
significantly higher number of T. clareae mite as compared to thymol and control
group. The total honey production harvested from colonies treated with formic acid
was higher (14.33 kg) as compared to other groups.
Different concentrations of oxalic acid (OA) were tested for their effectiveness
against V. destructor mite populations. Average efficacy of OA recorded with 3.2, 4.2
and 2.1 % was 95, 81 and 46 % respectively. The honey produced was also found
maximum (23 kg) in 3.2% OA treatment.
Different amounts of thymol with 3.2% oxalic acid (OA) on both mite
populations in honeybee colonies were also determined. It was found out that average
efficacy of 2, 4 and 6 gm thymol with 3.2 % OA for controlling T. clareae was 26, 40,
35 % and for V. destructor it was 93, 99 and 94 %, respectively. The results clearly
showed that the 3.2 % OA with 4gm thymol was the best treatment for controlling
xiv

these mites. The honey produced was also found maximum in (21 kg) 3.2% OA+ 4 gm
thymol treatment.
The fourth study was conducted in laboratory as well as in bee hives to
evaluate the acaricidal effects of some plant oils on Varroa mites. In the laboratory
experiments with different oils/extracts, clove oil in combination with tobacco extract
proved very effective against under study mites. The treatments were significantly
effective when applied in 5 % as compared to 10 and 15 % concentrations. In the
second experiment using only 5 % concentration for 24 hrs, the most effective
combination was clove oil and tobacco extract. The field experiment with all the
oils/extracts individually and in all the previously tested combinations confirmed the
lab results as clove oil + tobacco extract the best combination with 96.48 % efficacy.
The honey produced was also found maximum (20.5 kg) in clove oil + tobacco extract
treatment.
In view of the findings of previous studies, the fifth and final experiment
regarding integrated management was carried out to determine the effects of three
different treatments. i.e. 4gm thymol + 3.2% OA and 65% formic acid (T1), 5% clove
oil + Tobacco extract and 4gm thymol+3.2% OA (T2) and 5% clove oil + Tobacco
extract and 65 % formic acid (T3) to manage ectoparasitic mites i.e. T. clareae and V.
destructor populations in honeybee A. mellifera colonies round the year. Average
efficacy was calculated and it was found that T1 had the highest efficacy 86 and 97.75
% for both the mites, respectively. The total honey production harvested from colonies
treated with different acaricides was also determined and significantly more amount of
honey was produced (30 kg) from the hives treated with 4gm thymol + 3.2% OA and
xv

65% formic acid. It was observed that during all experiments treatment cause no effect
upon queen and adult honey bee activities.
xvi

INTRODUCTION
1
Chapter 1
Honeybees are beneficial insects which produce products like royal jelly, honey
and other value added products. Honey is a sort of nutritive food to human being.
Additionally, its also contain greatest value in preparing of pharmaceuticals, health
food products and some famous industrial products (Wakhal, et al., 1999). The Holy
Quran in sub-section 16, The Bee (Al-Nahl) also mentions stress upon the beneficial
role of honey bee in human life. In the modern world of science honey has been proved
as a remedy for human health disorders. In Pakistan, beekeeping is a profitable
business. It is reported that there are more than 4,000 beekeepers rearing Apis mellifera
in the beehives, about 400,000 colonies of A. mellifera has been producing 10,000 MT
honey annually and 27000 families are being benefited from beekeeping (Annual
Report, PARC 2010-11).
Besides their medicinal and nutritional benefits honeybees can be a source of
balance in the environment by pollinating and proliferating many plant species. They
are also an important source of bio-diversity. They can play an important role in
increasing the yield of crops up to 20 times more than the cost of honey they can
produced. Honeybee pollination do have some significant effects and can help in
improving the shape, color, size, taste and shelf life of the fruits (Atwal and Goyal,
1971).
In addition to a source of income and food, honeybees are also improving the
environment through their valuable pollination of medicinal plants, forest plant species,
landscapes and wastelands, however, in Pakistan, very few farmers have awareness

2
about importance of honeybees and their role in pollinating the crops. It is also
estimated that honeybees are able to do about 80% of all the pollination activity, along
with that they are responsible for ensuring about one third of the food supply. They also
proved to be an important economical pollinator of crop monocultures world over
(Watanabe, 1994). Crops that depend upon honeybee pollination and its benefits
include alfalfa, cherries, apple, oranges, plums, pears, almonds, melons, berries, and
pumpkins (Hoff, 1995; Ahmad, 1987). It is also proved that in absence of honeybee
activities, yield of some crops, fruits, nut and seed crops would decrease up to 90%
(Southwick and Southwick, 1992).
In the western end of Pakistan three species of honey bee are naturally found in
abundance (Ruttner, 1988). Pakistan Agricultural Research Council (PARC) in late
1980s introduced the western honeybee A. mellifera from Australia among the
commercial beekeepers because least out put of A. cerana (Waghchour and Martin,
2008). From 1992 to onwards, beekeepers started reporting heavy colony losses and
many of these colonies were found to be deceased by the ectoparasitic mite T. clareae.
The natural host of T. clareae is A. dorsata (Laigo and Morse, 1968; Delfinado and
Baker, 1985), which is found in the mountain regions throughout Pakistan and migrate
to the plain areas during the Acacia flow in spring. This honey flow is also exploited by
migratory beekeepers that shift large numbers of A. mellifera colonies on Acacia
modesta flora. That causes the shift of T. clareae from A. dorsata to A. mellifera
(Stephen, 1968).
Ectoparasitic mites’ infestations results in low yield of honey, swarming and
absconding of bee colonies. The two mite species V. destructor and T. clareae are
considered as the cause of continued increasing infestation among the Apis mellifera

3
colonies in Asia (De Jong et al. 1982). Each year a considerable damage among in bee
colonies were caused by mite infestation. As a result, capital flight was observed among
the beekeeping industry (Khan et al., 1987). Besides the economical loss of both honey
bees and honey yield, it was expected; infested colony may migrate or die (Needham,
1988).
Tropilaelaps clareae have been found for the first time as an ectoparasitic mite
of honey bee A. mellifera in the Peoples Republic of Philippines (Delfinado and Baker,
1961). The primary host of T. clareae was A. dorsata. This mite had the ability to shift
on A. mellifera (Laigo and Morse, 1968; Anderson and Morgan, 2007). Tropilaelaps
mite found in Asia and can infect all Apis species (Bailey & Ball, 1991; Schmid, 1998).
At the same time it was also found that they can be a source of disease transfer in honey
bee (Laigo and Morse, 1969; De Jong et al., 1982; Burgett et al., 1983). Ectoparasitic
mites can suck haemolymoph, causing brood loss (De Jong et al., 1982; Burgett and
Akratanakul, 1985).
T. clareae mite is known to have a wide distribution all over in Asia extending
from eastwards of Iran to Papua New Guinea (Matheson, 1995).The infestation caused
by T. clareae can be observed in abundance in between February, March to April with a
decease in infestation from May to August (Camphor et al., 2005). T. clareae is
parasitic on bee brood, adult and causes brood malformation, absconding or death of
the bees with gradual colony decline. Mites’ development requires almost one week to
disperse on honey bees. They are small in size and could not be easily seen by naked
eye. Poor management of bee colonies along with hive microclimate can increase the
chances of infestation of T. clareae mites in bee colonies (Mahavir and Gupta, 1999).
Their attack can cause 30-70% colony loss of A. mellifera along with decrease in honey

4
yield (Woo and Lee, 1997). T. clareae mite was also found responsible for the physical
loss of 50% colonies in Philippines and India (Laigo and Morse, 1968; Atwal and
Goyal, 1971).
Another ectoparasitic mite called Varroa destructor which can cause great
losses to honey bees (Apis mellifera L.) along with great economic loss to the
beekeeping industry (Abbadi and Nazar, 2003). Oudemans (1904) described presence
of Varroa mite on A. cerana. Varroa mite was found on A. mellifera in Philippines and
Hong Kong during 1962-63 (Delfinado, 1963). Just after the introduction of A.
mellifera in Pakistan during 1977-78, V. destructor mite became a serious pest of this
newly introduced A. mellifera and attacked over a large number of honeybee colonies
(Ahmad, 1988). Varroa mites’ growths depend on all honeybee stages from larva to
adult bees. A large number of adult bees were found in front of bee hive attacked by
mites. Colonies heavily infected by Varroa mite become unproductive (Ritter, 1981).
The V. destructor as serious ectoparasitic mites are subject of concern to beekeepers
worldwide. This mite which was feed on haemolymph of brood and adult bees causes
colony disorder, decreasing brood and deforming immature and mature bees. It can also
reduce the ability of bees to pollinate plants (De Jong, 1984).
Varroa mites, which infest bee colonies, are a threat to the beekeeping industry.
Without having adequate control measures, they can destroy almost an entire colony
within a few months. This destructive mite is now present in colonies across the world
except in Australia (Abrol and Sharma, 2009).
Presently, synthetic acaricides i.e. chlorobenzilate, sulphur, phenothiazine,
amitraz or different pyrethroids can bee used to control these mites. Different kinds of
acaricides were used effectively to control mites’ infestation but with the passage of

5
time mite population started getting resistant against many successful acaricides
(Loglio and Plebani, 1992). No dought, the certain sub lethal acaricides doses can be a
reason for these mites these problems, like their application within the colony tends to
contaminate the wax and honey. The excessive use of chemical caused pollution in
environment. Therefore, the utmost need of the time is to promote the suitable
pesticides which can kill the target organisms and at the time no effect on other living
organisms. Organic assets could be used to have healthy and safe environment.
Keeping in mind importance of safe and effective methods to suppress mite
populations in beehives, the present study aimed at determining the efficacy of formic
acid, thymol, oxalic acid and plant oil/extract against T. clareae and V. destructor
mites.
Thymol extracted from thyme plants is an essential oil that can be used to
intoxicate bee mites when ever evaporated in apiary. It is the quality of thymol that it
can affect on un-sealed brood. We can use thymol up to eight week for the control of
bee mites. However no reports of resistance among bee mites against the use of thymol.
Formic acid, being an organic chemical can damage the respiratory system of
mites and thus kills them. It can kill phonetic mites with chances to kills mites inside
brood cells. The chance of mites developing resistance against formic acid is very low
as it takes part in the metabolism of all organisms and the important thing is that
“Formic acid occurs naturally in honey”.
Oxalic acid (OA) is also an organic chemical and is found to be precipitated as
crystals on bees and mites, when its solution evaporates. OA can affect the mites in
brood less condition. It is found to be a natural constituent of honey and very effective

6
against the Varroa mite and its use has been increasing in last years (Charriere and
Imdorf, 2002).
Oxalic acid is found to be safe in use, has no residual problems, cheap and no
case of honeybee toxicity have been reported (Mutinelli et al., 1997; Rademacher and
Harz, 2006). High potency applied in winter is extremely effective with strong
influence over development in spring; however with the lower concentrations, have no
bad effect at the same time. Yet to be cleared that whether the potency of the solutions
matter in achieving high rate of efficacy or it is the low concentrations that can reduce
the mite infestation.
In the present scenario of friendly pesticides, some successful attempts have
been made to include substances such as plant extracts, essential oils, secondary
metabolites from microorganisms, hormones, plant derived pesticides, pheromones and
genes used to help crops to transform resistance to pests. It is reported that some of the
essential plant oils can be used as repellent and at same time as fumigant insecticide
against specific pests, and fungicidal actions against some important plant pathogens
(Kordali et al., 2005).
Volatile oil constituents of Mentha species showed highly effective results
against Tribolium castanum and Callosobruchus maculates (Tripathi et al., 2000).
Essential oils derived from eucalyptus and lemongrass has been found effective as
animal repellents. It is also found that essential oils of Ocimum sanctum caused 20%
mortality among 3rd instar S. litura larvae (Sharma et al., 2001). Essential oil of Lippia
alba induces growth inhibition, where both relative growth and feeding consumption
rates of S. litura were conspicuously reduced (Tripathi et al., 2003). Many studies
conducted upon the usage of some extracts of natural essential oil of various plants like

7
rosemary, mint, lemongrass, thyme, camphor, marjoram, santonica seeds, clove, ginger
and eucalyptus (Fathy and Fouly, 1997; Gregorc and Poklukar, 2003; Batish et al.,
2008).
In short, we can say that control of honeybee ectoparasitic mites by using
conventional pesticides resulted in the form of pesticidal residues in honey which is not
acceptable all over the world. World Trade Organization and World Health
Organization have set some quality standards which are to be met by bee keepers to
avoid huge economic losses.
While designing the present study an integrated management approach was
taken into consideration for the control of mites. In this regard various concentrations
of organic chemicals and plant oil/ extract will be tested individually and in various
combinations to evaluate their efficacy against mite populations of honeybees. As a
result a set of new recommendations would be approached to be practiced by the
beekeepers to manage the mite population and will improve the honey yield and
indirectly will impact the livelihood and economic condition of the beekeepers.
1.1 Objectives
Keeping in view the importance of safe and non-contaminated methods in order
to suppress mite populations efficacy in beehives to increase honey yield, the present
study was aimed with following objectives:
To minimize the use of pesticides or synthetic acaricides inside bee colonies to
control the parasitic bee mites.
To monitor mite population growth in the colonies by regular sampling and
applying IPM to control mites.
Management of Apis mellifera for high honey yield.

8
REVIEW OF LITERATURE
Chapter 2
As all sorts of scientific studies needs to take a deep look into the previous
work done in the relevant field; likewise an extensive exercise has been carried out
on the management of honeybee mites in relation to honey production. In this
chapter some of the relevant literature is reviewed.
2.1 MITES, APISTAN AND RESISTANCE
In Apis mellifera L. colonies, mortality from V. destructor infestation can
reach 100% in two years if left untreated. (De Jong, 1990). Tropilaelaps clareae
has been proved to be a more serious pest of A. mellifera in Southeast Asia.
Infestation of Tropilaelaps can be recognized either visually or by examining bee
debris. Dead or malformed immature and bees with malformed wings that crawl at
the hive's entrance and especially the presence of fast-running, red-brown,
elongated mites on the combs are diagnostic for the presence of T. clareae. An
early diagnosis can be made after opening brood cells and finding immature and
adult mites therein. The hive (colony) may be treated with various chemicals that
cause the mites to drop off combs and bees. The debris can then be examined
visually or by using a flotation procedure (Burgett et al., 1983).
The mite, T. clareae was responsible for the loss of 50% of the brood in A.
mellifera colonies in Philippines and India. In A. mellifera colonies this mite is
considered as a serious pest, making control treatments necessary (Laigo and
Morse, 1968; Atwal and Goyal, 1971).
8

9
Colonies infested with V. destructor have significantly reduced worker bee
populations which eventually die if left without controlling. The development of
infested brood is also affected because emerged bees have a low weight and shorter
life span (De Jong et al., 1982).
Like other animals honeybees are also affected by various pests and
diseases. One of the most common pests is V. destructor that provokes big losses in
apiculture. It feeds on haemolymph of larva, pupa and adult bees during the whole
life. Contamination of colony with Varroa leads to decreasing of body weight,
deformation and even death (Ritter, 1981; Mosaddeg and Komeyli-Birjond 1988).
With the introduction of A. mellifera in Pakistan in 1977-78, Varroa mite became a
serious pest of this newly introduced honeybee and destroyed a large number of
colonies (Ahmad, 1988).
T. clareae is a more serious pest of A. mellifera than Varroa mites
(Wongsiri et al., 1989). Chemical, cultural and combinations of chemical and
cultural methods provide control of parasitic mites in bee colonies (Tangkanasing
et al., 1988), In addition, these methods are either labor intensive, costly, reduce
bee populations or contaminate bee products. Thus, finding honey bees with natural
defenses against parasitic mites generally and especially against T. clareae is
highly desirable (Wongsiri et al., 1989).
The control of Varroa is especially difficult as the majority of mites stay in
the sealed brood for reproduction and are therefore well protected from different
forms of treatments (Hoppe et al., 1989). The use of acaricides smoke such as
Fluvalinate and amitraz is more rapid, but contaminates honey and may accelerate

10
the development of resistance to these chemicals by the mites (Ellis et al., 1988;
Herbert et al., 1989; Witherell and Bruce, 1990).
Beekeepers have been forced to combat the parasitic mites with acaricides,
coumaphos, synthetic Pyrethroid and Fluvalinate (Apistan strip), the most common
remedies used for this purpose. During the last 10 years resistances against the
synthetic acaricides have increased in medication (Elzen, et al., 1998).
The parasitic mite V. destructor is the most disturbing pest in honey bee
colonies worldwide. Its impact has been compounded because these mites quickly
became defiant to the chemicals viz., Fluvalinate (Apistan) and coumaphos
(Check-Mite), the two most common and effective controls available (Elzen et al.,
1998; Milani, 1999; Elzen et al., 2000). In many countries the mites have
developed resistance to coumaphos, amitraz and Pyrethroids which are employed
in most of the commonly used treatments (Lodesani et al., 1995; Milani, 1999;
Miozes-Koch et al., 2000; Floris et al., 2001).
The efforts towards controlling the Varroa mite have been mainly focused
on the use of synthetic miticides, which provide a good degree of control.
However, these hard chemicals have the disadvantages of leaving residues in honey
and wax (Wallner, 1999) and of allowing the mites to rapidly develop resistance to
their active ingredient (Milani, 1999). Furthermore, acaricides residues have been
detected in honey and beeswax products (Milani, 1995; Wallner, 1999).
Unfortunately, Varroa have developed resistance to numerous classes of
synthetic acaricides in several geographic areas (Milani, 1999; Elzen et al., 2000;
Spreafico et al., 2001). V. destructor is a mite parasite which causes tremendous
damage to honey bees. Varroa mites can kill honey bee colonies within 1-2 years if

11
left untreated. Various chemicals have been used to control the mite, but
unfortunately chemicals can potentially harm the bees and also contaminate honey
if not used carefully. The mite pest is also developing resistance to chemicals.
Apistan strip losing its potency in treating mites because of mite-resistance
(Zachary, 2001).
V. destructor is the most destructive parasite of honey bees. Although the
susceptibility of honey bees to Varroa infestation is influenced by heritable
characters (Harbo and Harris, 1999). Most of the commercial colonies die
following 1-2 years of consecutive invasion of V. destructor without treatment
(Martin et al., 1998; Downey and Winston, 2001). Beekeepers rely heavily on
synthetic acaricides to decrease Varroa populations to non-damaging levels (Finley
et al., 1996; Caron, 1999; Melathopoulos and Farney, 2002).
Repeated use of chemicals has resulted in resistance to fluvalinate
(Apistan). Apistan-resistant Varroa have been detected in Europe (Lodesani et al.,
1995) and the U.S. (Baxter et al., 1998; Elzen et al., 1998; Pettis et al., 1998).
More recently, coumaphos resistance has been detected in the US (Elzen and
Westervelt, 2002; Pettis, 2004). Honey bee mites are considered as major factors in
beekeeping. Among honey bee mites T. clareae Delfinado and Baker is a
predominant ectoparasitic mite associated with five Apis species in Asia and
causing 50 to 100 percent loss of bee colonies (Hosamani et al., 2006).
The population changes of T. clareae in A. mellifera colonies were
investigated for a period of 14 month in Islamabad, Pakistan. The environmental
conditions resulted in honey bee brood being present throughout the year, which
allowed T. clareae to breed continuously. The phoretic period of T. clareae was

12
very short as the infestation of the brood (8.1 %) was 20 times greater than that of
the adult workers (0.4 %). There were rapid increases in the T. clareae population
during March and April (Elizabeth and Martin, 2009).
Stimulation effects on the sensory and defensive behaviors of Egyptian
honey bees towards Varroa invasion were studied through remedied honeybee
colonies with the essential oils. Astonishing results to the grooming and hygienic
behaviors consequence of the sensory responses enhanced the defense behavior of
honey bee colonies against Varroa mite (Allam and Zakaria, 2009).
The studies revealed that mite infestation had a pronounced influence on the
body weight of developing worker and drone brood and emerging adults. Similar
reductions were found in pupae of drones and workers infested with Varroa mite.
Evidently, infested colonies had weak workers and drones and exhibited reduced
honey gathering and pollination activities (Kotwal and Abrol, 2009).
2.2. THYMOL AND FORMIC ACID FOR MITE CONTROL
Thymol was harmless to honeybees but effective against Varroa mite
(Bollhalder, 1998; Calderone, 1999). Thymol, a secondary plant metabolite
composed of terpinoids (Karpouhtsis et al., 1998) was chosen because of its
regulatory acceptability as a food-grade botanical compound (Imdorf et al., 1996;
Calderone et al., 1997).
In a study it was recommended not to use the powdered thymol on weak
colonies at high temperature (higher than 27-30 C). In this condition, bees can
abandon the hive (Mikityuk and Grobov, 1979).

13
Thymol is a volatile monoterpenoid found among many species of plants,
and is lethal to Varroa at doses safe to their honey bee hosts (Imdorf et al., 1994;
Lindberg et al., 2000). Applied as a fumigant, thymol can set off synthetic
acaricides because of its proven high to medium effectiveness at killing Varroa,
(Imdorf et al., 1999).
The use of organic substances in controlling the Varroa mite is increasing
and is a highly desirable alternative to chemical controls as it avoids mite resistance
and leaves far less residue in the hive products (Wallner, 1999).
Many natural products have been tested to control Varroa infestations in
honey bee colonies, but few of them have shown promise as potential ideal
miticides; Thymol and oxalic acid are among them (Imdrorf et al., 1999).Thymol is
contained in several commercially available medicinal products and numbers of
studies have demonstrated its efficacy at controlling mite infestations in honey bee
colonies, but with variable results (Imdorf et al., 1996; Calderone et al., 1997;
Imdorf et al., 1999).
The use of thymol and formic acid, both are equally effective and left no
residues in honey. Formic acid and thymol have shown some promising results for
mites control (Hoppe et.al., 1989; Imdorf et al., 1996; Calderone et al., 1997;
Feldlaufer et al., 1997; Andermatt, 1999; Kochansky and Shimannki, 1999; Mattila
and Otis, 1999; Mattila et al., 2000; Whittington et al., 2000).
Two formic acid autumn treatments, gel packets and impregnated paper
wick were tested in apiary to evaluate their effectiveness against V. destructor and
their residues in honey in a Mediterranean region (Sardinia, Italy). Both treatments
were efficient in the apiary control of the varroasis, with values of percentage of

14
mite mortality ranging between 93.6 and 100%, without statistical differences
between them (Satta, et al., 2005).
To evaluate the effectiveness of Apiguard® treatments against V. destructor
twenty one colonies of A. mellifera L. in Dadant-Blatt hives were used. Two
groups of seven colonies each were treated and one group was left as untreated
controls. Two aluminum trays of Apiguard® were installed in the hives with a two
week interval between treatments. The trays of one of the treatments (group 1)
were covered with a plastic mesh which only allowed the bees’ legs and
mouthparts to contact the product. The plastic mesh allowed evaporation but
reduced bee contact and product removal. The other Apiguard® treated group
(group 2) received uncovered trays as recommended by the manufacturer.
Apiguard® trays remained there in the hives for 30 days. The percent effectiveness
(E %) was significantly higher in the uncovered trays (93.34 ± 1.18%) than in the
covered trays (87.23 ± 1.80%) (Palmeri, et al., 2007).
The percentage efficacy of Apiguard® and Exomite Apis under Irish
weather conditions was examined from August –September 2005. Total mite drop
was counted and the percentage efficacy was estimated by treating all colonies with
Bayvarol®. Variation in floor type reduced mite population growth early in the
foraging season, but the effect was not significant. In contrast, a significant benefit
was realized by the inclusion of drone brood trapping as a colony management
strategy for reducing Varroa mite populations. Although colony development was
not affected by drone brood trapping. Apiguard® was more effective than
Exomite Apis as an autumn treatment under Irish weather conditions. The
recorded percentage efficacy was 85% (Mary, 2007).

15
A study was carried out to determine thymol and formic acid residues in
honey in case a honey super is placed on a hive immediately after termination of a
Varroa control with formic acid or thymol in early spring. The thymol and formic
acid residues in the honey exceeded significantly the thymol and formic acid
residues in the honey from the control group. However, the thymol concentration
was always below the taste threshold and the formic acid concentration was most
of the time below the taste threshold (Donders et al., 2007).
Effectiveness of two natural miticides, formic acid and thymol, for
controlling infestations of V. destructor in honey bee colonies was studied. The
highest effectiveness was obtained with two applications of 12.5 g of thymol
(92.1%), whereas with the formic acid the effectiveness was 66.4%. Both miticides
killed a significant number of mites but their effectiveness decreased after the first
application (Espinosa-Montano and Guzman-Novoa 2007).
It was suggested that the miticides like thymol and oxalic acid might be
able to solve this problem in beekeeping industry, if they are applied regularly and
according to the recommendations. Introduction of these scientifically approved
miticides would be beneficial to beekeepers and could enhance the production and
export of high quality honey (Pichai et al., 2008).
The results suggest that formic acid is an effective alternative to Apistan as
a fall treatment for Varroa mites in temperate climates. (Calderone, 2000). Two
organic compounds (thymol and oxalic acid) with three delivery methods (dust,
trickled and vermiculite) were applied to 30 infested honey bee colonies to
investigate the effects of treatments on colony development and to determine
residues in honey. Bee population, number of mites in brood cells and brood area

16
of groups were determined in autumn, before and after the application. It was
observed that treatments did not cause damage to amount of brood and bee
population (Emsen and Dodologlu, 2009).
Effectiveness of two synthetic (Bayvarol and Apivar) and two natural
acaricides (Apiguard and ApiLife Var) against V. destructor were evaluated with
use of infested colonies of A. mellifera, kept in Langstroth standard hives. All
acaricides significantly reduced the levels of Varroa mite infestation on adult
honeybees and worker brood, but the efficacy was higher for Apiguard (93–97 %)
and ApiLife Var (94–98 %) compared to Bayvarol (85–90 %) and Apivar (82–88
%). Overall, the data indicated that essential oils like Apiguard and ApiLife Var
can be recommended in the control of V. destructor, while synthetic varroacides
like Bayvarol and Apivar should be minimized due to increased mite resistance for
these products. (Loucif-ayad, et al., 2010).
2.3. PLANT OILS/ EXTRACTS FOR MITE CONTROL
The efficiency of Azadirachta indica (Neem), Cucuma longa (Turmeric),
Acorus calamus (Sweet flag) and citrus species (Grape fruit) was tested against
mites in the laboratory with bioassays and in the bee colonies/ hives and found
effective against bee mites (Eischen and Wilson, 1997; Calderone and spivak,
1995; Elzen et al., 1999).
The botanical oils neem, thymol, and canola were examined for control of
parasitic mites in A. mellifera colonies. Neem oil spray (5% solution) killed 90.6%
of Varroa mites, three times more than died in the untreated group. Thymol-oil

17
spray, thymol in vermiculite and canola oil spray (20% solution) killed 79.8%,
68.6%, and 65.6% of the Varroa mites, respectively. Neem and thymol-oil spray
treatments were detrimental to bees, as they both had 50% queen loss, and colonies
treated with neem oil had one-third as many adult bees and one-sixth as much
brood as untreated colonies at the end of the experiment (Whittington, et al., 2000).
Tetradifon strips, naphthalene and leaves of plant species such as Premna
odorata Blanco (Ialagaw), Blumea balsamifera (L.) DC, (sambong), Gliriciddia
sepium (Jacq) Steud, (madre de cacao) and Hyptis suaveolens Poir, (suob kabayo)
were tested for the control of bee mites. High mortalities were observed in
naphthalene and Tetradifon treated colonies and among the plant materials
evaluated. H. suaveolens and G. sepium (Cervancia and Aspiras, 1987).
Dung et al. (1992) suggested that the control of Varroa mite in the future
should be based on biotechnical control methods such as trapping of mites in the
drone brood and using heat treatment (42-44 °C for 20-30 min) to reduce the mite
population inside the colony. On the other hand, several people used essential oils
to control the pest (Fathy and Fouly, 1993, 1995). Nowadays in some countries,
these are so attempts to replace medicinal plant with chemical treatments. Pepper
and mint etc are some of the involved medicinal plants (Rajiter, 1983; Ariana, et
al., 2000, Hagigation, 2000). Beekeepers are increasingly turning to alternative
treatments incorporating essential oils and organic acids (Mutinelli, et al., 1997;
Melathopoulos and Gates, 2003; Gregorc and Poklukar, 2003).
Worm wood flowers, clove oil and peppermint oil can be considered
promising agents for the control of V. destructor. Good results can be obtained
when the above materials are used two to three times each month during the

18
infestation period. Effective time of application depends on the mite level of
infestation and the life cycle of the mite. The natural control agents used in this
work seemed to have no adverse effects on bees. (Al-Abbadi and Nazer, 2003)
Numerous plant derived substances have demonstrated physiological and
behavioral activity against insect pests, and they can provide new sources for the
development of natural pesticides (Geroge et. al., 2008; Isman 2006). Products
with botanical origin have shown a wide range of biological activities including
toxicity properties (Aivazi and Vijayan 2009; Banchio et al., 2003, 2005; Ciccia et
al., 2000; Ferrero et al., 2006; Jbilou et al., 2006).
Spearmint, thyme, eucalyptus, marjoram, cumin, garlic, basil, orange,
geranium, menthol and eugenol were tested against V. destructor. Basil, geranium
and eugenol oil exhibited noticeable varroacidal activity and were selected for
different tests during winter, spring, and autumn seasons. Results showed that all
treatments were significantly effective against Varroa in treated colonies compared
to untreated ones. Counts of dropped mites were also significantly high. Some
adverse effects including reduced brood area, colony disturbance and bee mortality
occurred in some treatments, especially those with eugenol oil, which seemed to be
harmful to bees. Therefore, the IPM approach is recommended to combat Varroa
mites (Abd El-Halim et al., 2006).
Cineole, clove oil, formic acid, marjoram oil, menthol, oregano oil, oxalic
acid, sage oil, thymol, and wintergreen were tested at several concentrations in
sugar syrup fed to bees over several days, and dead bees were counted daily.
Oxalic acid was the most toxic of the products tested. Menthol and cineole had
mortality levels not different from controls fed plain syrup after 8 days of

19
treatment. At 14 days of treatment, wintergreen was found the least toxic. Results
indicated that all the tested products could be used safely for treating bees orally if
dose is carefully managed in the hive (Ebert, et al.2007).
Water extracts of three botanicals, garlic leaves, liquorices leaves, turmeric
rhizomes with three concentrations (2, 1, and 0.5%) and sulphur powder were
evaluated against T. clareae in A. mellifera L. colonies. A single spray of garlic
extract at 2% provided significantly more dead mites in hive debris with brood
frames with bees (72.39%) and without bees (63.04%) than in the untreated group.
Extracts of liquorices (2%) and turmeric (2%) killed 29.03, 14.61 per cent and
28.07, 14.26 per cent in colonies where the solution was sprayed on brood frames
with bees and without bees, respectively. Among the treatments, significantly more
dead mites (18.17) were observed in garlic treated colonies than sulphur treated
ones (15.4 mites) after seven days of the treatment (Hosamani, et al., 2007).
Neem (Azadirachta indica) aqueous extract of seed at different
concentrations, viz., 0.5, 1, 2, 3 and 4%, were tested against Greater wax moth,
(Galleria mellonella L.). Post spray mean mortality (83.33%) of the pest insect was
with 4% aqueous neem, followed by 73.33%, 56.67%, 50% and 50 % with 3, 2, 1
and 0.5 per cent, respectively as compared to control (3.33%) (Izhar-ul-Haq, et al.,
2008).
The efficacy rate of geranium oil, lemon oil, mixture of (geranium, lemon,
chamomile and mint oils) and mixture of (lemon, black cumin and mint oils), in
comparison with Mitac was 37.27%, 32.56%, 37.59%, 36.27% and 53.64%
respectively. This study indicates that using mixture of essential oils was more
suitable with cotton strips in control Varroa mite. Cultural control by adding lemon

20
oil to sugar solution, used in feeding the colonies, especially in winter, putting the
colonies in citrus garden and cultivation of geranium or chamomile around apiaries
(these new cultural control) were added to IPM programmes for control of Varroa
mite (Hassan, et al.2008).
By applying a topical dose of 100 μg/larvae, greater then 90% larval
mortality has been reported when essential oil of Satoreja hortensis, Thymus
serpyllum and Origanum creticum (LD50= 48.4–53.4) were applied to 3rd instars S.
litura (Isman, et al., 2001). Sharda and Rao (2000) reported essential oil of
Ageratum conyzoides caused 43.0–68.75% mortality and Tripathi, et al. (2003) also
reported toxicity of essential oil of Aegle marmelos by topical application to S.
litura larvae. Essential oil of Lippia alba induced growth inhibition, where both
relative growth and feeding consumption rates of S. litura were conspicuously
reduced (Tripathi, et al., 2003). Many studies had been carried out using some
extracts of natural essential oil of various plants such as rosemary, lemongrass,
camphor, thyme, majoram, mint, clove, ginger, roots, santonica, seeds, fennel and
eucalyptus (Fathy and Fouly, 1997; Gregorc and Poklukar, 2003; Batish et al.,
2008).
Insecticidal effect of volatile oils from peppermint (Mentha piperita), basil
(Ocimum basilicum), lemon (Citrus limon) and orange (Citrus sinensis) against two
museum insect pests black carpet beetle and cigarette beetle was evaluated by
fumigation test among tested/ evaluated oils. Peppermint oil offered the highest
toxicity to adults and larvae of the black carpet beetle and cigarette beetle at LD50
level and orange oil was less toxic to adult and larvae of both species (Bakrl, et al.,
2010).

21
The fumigant toxicity of a large number of essential oils extracted from
various spices and herb plants was assessed against several major stored-product
insects. Tribolium castaneum (Herbst) was found to be the most resistant,
compared with Sitophilus oryzae (L.), Rhyzopertha dominica (F.) and Oryzaephilus
surinamensis (L.), to most essential oils tested. Edible oils are potential control
agents against Callosobruchus maculatus (F.) and to a lesser extent against
Sitophilus zeamais Motsch., S. oryzae and Sitotroga cerealella (Oliv.). These
materials could be very useful on the farm level in developing countries. Plantoils
can play an important role in stored-grain protection and reduce the need for, and
risks associated with, the use of insecticides (Shaaya, et al., 1997).
Eggs and young larvae of C. maculatus can be affected by extracts of Piper
guineense whereas older larvae are usually less susceptible. Piper guineense also
caused egg mortality of C. maculatus (Mbata, et al., 1992). Schoonhoven (1978)
indicated that 100 ml of selected vegetable oils effectively protected cowpea
against the pulse beetle Zabrotes subfasciatus.
Under control/ Laboratory condition extracts of four plants, Mucuna
pruriens (Fabaceae), Adenium obesum (Apocynaceae), Azadirachta indica
(Meliaceae) and Calotropis procera (Asclepiadaceae) testd against the migratory
locust (Locusta migratoria) evalutated through contact and stomach poisioning for
their locusticidal properties. Mucuna extracts act both as contact and stomach
poisons cause up to 99% mortality by direct suprayin as well as feed on wheat
seedling (Abdalla et al., 2009).
Foraging activity, gathering of pollen & nectar, food stock inside the bees

22
colony and count of bee individuals is more when low numbers of Varroa inside
honeybee colony. It is therefore recommended that the use of volatile plant oils,
which are safer to bees and bee products of pesticide pollution in the case of the
use of pesticides in the fight against Varroa mite. Using of natural materials such as
volatile oils give good results when used for control and the results were not good
as of pesticides, but the pesticides cause serious damage to human health ( Nageh,
et al., 2011).
2.4. OXALIC ACID (OA) FOR MITE CONTROL
Spraying honey bee colonies with 2 or 3% oxalic acid water solution has
been used to control the Varroa mite. 2% oxalic acid, at a dose of 100–150 ml per
colony, was found to be very effective in mite control and harmless to bees
(Takeuchi and Harada, 1983).
Varroa mites can be effectively controlled by using OA dripping method
(Nanetti and Stradi, 1997).
Oxalic acid with different dosage, ways and times of application has been
tested on bee colonies against Varroa mite and reported highly satisfactory results
for the control of mites (range of mean effectiveness: 66.0-99.5%) (Imdorf et.al.,
1999).
A single treatment in autumn by applying 3% oxalic acid dihydrate solution
(30-50 ml per hive) reached efficacies between 56 and 98%. The 3.5% solution
generally achieved 95% efficacy (Charriere and Imdorf, 2002; Nanetti et al., 2003).
Applying concentrations of oxalic acid above 3.5% there observed no evident
increase in the efficacy, although the bees tolerated concentrations up to 4.5%

23
(Charriere and Imdorf, 2002; Nanetti et al., 2003). Higher concentration of oxalic
acid can cause harm to the colonies (Liebig, 1998; Nanetti et al., 2003). Multiple
treatments during summer or autumn, as well as summer treatments in artificial
swarms, showed reduced efficacy and caused high bee mortality (Liebig, 1999).
Oxalic acid (OA) has been found to be highly effective in treating colonies
without brood (Imdorf et al., 1997; Brodsgaard et al., 1999). Efficacy of 95%was
achieved after three treatments using a 5% OA solution (Mutinelli et al.,1997) and
efficacy of 24% after one springtime treatment administered by trickling when a
capped brood was present (Brodsgaard et al.,1999).
Trickling method using a sugar solution of 4.2% O.A applied distributed in
the colony by a syringe with a dosage of 5 ml, maintains an 80% efficacy against
Varroa in combs partially or fully occupied by the bees under broodless conditions.
(Marinelli, et al., 2000). However, it was found that 2.8% and 3.5% oxalic acid
dihydrate solution (40-50 ml/hive) killed the mites about 55% and 90%
respectively. 3.5% oxalic acid dihydrate solution is the recommended concentration
for practicing beekeepers (Nasr, et al., 2001).
The two oxalic acid applications removed 60 ± 12% of Varroa mites
adhering to adult honey bees, while the natural fall of mites measured in control
colonies (for a period of 40 days) was 32 ± 4% (Hatjina and Haristos ,2005).
Oxalic acid has been used extensively to control Varroa mite infestations,
but its use has resulted in variable levels of mite mortality (Nanetti, et al., 2003).
Oxalic acid has been applied with spraying, evaporating and trickling methods for
mite control (Charriere and Imdorf, 2002).

24
In an experiment 2.9% Oxalic acid (OA) treatment in September resulted in
25% mite mortality. OA treatments in October and November resulted in
approximately 97% mite mortality. These results suggested that OA is effective
during the broodless period and less effective when applied to colonies with capped
broods (Gregorc and Planinc, 2002).
A single autumn application of 4.5 or 6% oxalic acid dihydrate (30 ml per
hive or 5ml per bee way) reached 92.2 and 92.6% mite mortality. The
investigations showed higher tolerance by the bees of the 4.5% OA concentration
(Fries, 2007; Nanetti, et al., 2003). According to the one s study three OA
treatments of colonies with brood had average efficacy of 39.2%, and 99.4%when
there was no brood present. So OA was found highly effective in broodless periods
(Gregore and Planinc, 2001; 2004).
Aqueous oxalic acid solutions in concentrations 1.7 and 1.4% have been
reported to kill Varroa mites whereas a 0.7% aqueous solution had no effect
(Takeuchi and Sakai, 1983). Oxalic acid is reported as a safer agent, has no residue
problems, cheap and has no case of honeybee toxicity (Mutinelli, et al., 1997;
Rademacher and Harz, 2006).
Oxalic acid field trials for the control of V. destructor were carried out in an
apiary. The colonies received four successive applications with 4.2% oxalic acid
(OA) and 60% sugar solution by trickling method with two alternative types of
syringes from the broodright to broodless period. The results indicated that the
first three applications (from 6th October to 25th November—broodright period)
resulted in 65.3% cumulative mite mortality, while only the last application (after

25
26th November—broodless period) resulted in 77.3% mite mortality. (Nicolaos, et
al., 2007)
Autumn treatments with 5 or 6% oxalic acid dihydrate (5 ml per bee way)
reached efficacies from 89.7-96.7% and multiple treatments using 6 and 7% oxalic
acid dihydrate were still tolerated (Baggio and Mutinellio, 2003a, 2003b; Ferrero et
al., 2004; Mutinelli and Baggio, 2002; Mutinelli et al., 1997; Nanetti and Stradi,
1997; Nanetti et al., 2003). The efficacy of the oxalic acid (OA) sugar solution was
evaluated against varroa mites in brood-right honeybee colonies. In laboratory
trials, each comb was sprayed with 4ml solution containing 0-4%OA and 30%
sugar. 3% OA treatment gave the best results and caused 81.8±6.3% mite mortality
at 48 hours post-treatment. In field trials honeybee colonies received five
successive applications (at intervals of 3-4 days) with 3% OA syrup by spraying
method with two alternative doses (2 ml or 4 ml OA per comb). These treatments
resulted in 72.6 ± 11.3% and 82.4 ± 3.8% mite mortality, respectively. Results
suggested that repeated spray applications of OA syrup was effective for the
control of varroa mites in brood-right honeybee colonies ( Yue-Wen Chen and Pao-
Liang Chen, 2008).
The toxicity of various concentrations of oxalic acid dihydrate on bees and
Varroa mites was determined by spraying honey bee colonies with no brood or
little brood in beehive conditions. A water solution of 0.5% OA gave effective
control of the mite and was not toxic to bees whereas higher concentrations of OA
(1.0 and 1.5%) were highly toxic to bees. In autumn, spraying bee colonies had
little capped brood once or twice with a 0.5% OA solution gave effective mite
control (92.94 ± 0.01% and 91.84 ± 0.02%, respectively) with no noticeable

26
toxicity to bees (Toomemaa, et al., 2010). Applying an optimum volume of 3 .0 ml
of 2.8% OA solution per 1000 bees to package for effective mite control with
minimum adult bee mortality. (Nicholas and Ellis, 2009). The three oxalic acid
treatments resulted in an efficacy of mite mortality in between 47.68% and 98.83%
with a mean of 70.12% (Skerl, et al., 2011)
2.5. INTEGRATED PEST MANAGEMENT FOR MITE CONTROL
Hygienic bees are selected for their ability to detect and remove diseased or
mite-infested brood more quickly than non-hygienic bee stock. The speed with
which hygienic bees removed infested brood prevents the mite from completing
their reproductive cycle, and this technique can provide between 40 and 60%
control (Spivak 1996; Spivak & Reuter, 2001). If the bees in the colony removed
more than 75% of the dead brood in two consecutive 24 hrs periods, the colony
was considered hygienic. In earlier tests for hygienic behavior, it was found that a
75% removal in a 24 hrs period two times in a row was equivalent to a 95%
removal over a 48 hour period (Spivak, 1996).
The use of dishwashing detergent solutions and ethanol are the most
common solutions being used. The use of detergent solution with mechanical
agitation as a single wash was the most effective (97%) way of detecting Varroa
mites (De Jong et al., 1982; Rinderer et al., 2004). T. clareae infestation varied
from an average of 56.4 mites per comb to 90.3 mites per comb. All hives were
dusted daily with sulphur (15g/colony) for 15 days and mite mortality was
assessed. Daily mite fall before dusting was 4.0-5.0, whereas after treatment the
average number of dead mites per hive was 90.1-121.2. However, all colonies

27
remained infested after sulphur dusting (Jyothi, 1996).The hygienic behavior of
honey bees (Apis spp.) is a natural defense against diseases and parasitic mites
(Park, 1937; Gilliam, et al., 1983; Boecking and Drescher, 1991). Hygienic honey
bees detect, uncap and remove diseased or mite-infested brood from the colonies
and limit the population growth of both Varroa destructors (Boecking et al., 1992;
Spivak and Reuter, 1998) and Tropilaelaps clareae mites (Ritter and Schneider-
Ritter, 1988; Boecking and Drescher, 1990; Boecking, et al., 1992).
Various biotechnical methods are used by professional beekeepers to
control V. jacobsoni and T. clareae. For control of Tropilaelaps, a broodless period
has to be created. Varroa is controlled by trapping methods. The methods suffice to
successfully control both mite species without the use of chemicals (Dung, et al.,
1997).
Efforts to control Varroasis have been focused on the use of control
synthetic miticides; however these miticides have some disadvantages: they may
promote the mites to develop resistance against their active ingredient; they are
toxic to bees and humans and may leave chemical residues in honey which is a
product for human consumption (Miozes-Koch et al., 2000). Mites can be
controlled by using natural miticides, which have low toxicity and low
environmental impact, because no residues are left in honey or because these
breakdown or volatilized rapidly. Few natural products have shown effectiveness
against Varroa; formic acid, oxalic acids and thymol essential oil are among them
(Imdorf et al., 1999).
A laboratory bioassay was developed to evaluate miticides to control
Varroa jacobsoni (Oudemans), an important parasite of the honey bee, Apis

28
mellifera L. Bees and mites were exposed to applications of essential oil
constituents in Petri dishes (60 by 20 mm). The six most selective of the 22
treatments tested (clove oil, benzyl acetate, thymol, carvacrol, methyl salicylate,
and Magic3) were further evaluated to estimate LD50 values and selectivity ratios
(A. mellifera LD50/ V. jacobsoni LD50) at 24, 43, and 67 h after exposure. These
results indicated that essential oil constituents alone may not be selective enough to
control Varroa under all conditions, but could be a useful component of an
integrated pest management approach to parasitic mite management in honey bee
colonies (Lindberg, et al.,2000).
The Varroa Treatment Device (VTD) filled with 85% formic acid (FA) was
field tested for honey bee parasitic mite control. Three apiaries with 28 honey bee
colonies were used in this test. Two VTD/FA treatments, one Apistan treatment
and one control were replicated seven times. Although the results of this test
indicated that the VTD/FA is less effective than Apistan in controlling varroa
mites, the VTD/FA provides a viable alternative varroa mite control in combination
with other mite control measures, especially as an early season treatment (Hood, et
al., 2001).
Four acaricides viz., 85% formic acid (5ml/colony), sulphur (500mg/comb),
fluvalinate (3ml quantity of 5 ppm solution) and amitraz (2 strips/ colony) were
tested against ectoparasitic mite T. clareae infesting A. mellifera colonies. All the
treated colonies became mite free within 22-25.5 days and these chemicals had no
adverse effect on the brood and bees or queens (Sharma, et al., 2003). V. destructor
is the most serious pest attacking honey bees. Without control measures applied, an
entire apiary can collapse in two years. Both Fluvalinate and coumaphos have

29
given excellent control in the past, but now there is clear evidence of Varroa
resistance to these compounds (Eischen, 1998; Elzen et al., 1999; Elzen and
Westervelt, 2002; Pettis, 2004).
The 50% formic acid fumigator (FAF) for varroa mite control was
developed. The fumigator was evaluated for five years on 123 colonies in five bee
yards in Connecticut, Maryland and West Virginia (USA). Treatments eliminated
all mites on adult bees and 90-95% of mites in sealed brood cells. Very few brood
or new young adult bees were injured by the treatment. The 50% FAF used with
other essential oil treatments including salt-grease patties with wintergreen, feeding
1:1 syrup with Honey-B-Healthy7 (spearmint and lemongrass essential oils), and
use of screened bottom boards, together provide a synergistic effect to keep mite
numbers at a relatively low level, as part of an integrated pest management (IPM)
system (Amrine and Noel, 2007).
Fluvalinate (one strip colony-1), Formic acid 80% (10 ml colony-1),
Menthol (10 gm colony-1), and sulfur powder (10 gm colony-1) were studied
against varroa mites in bee colonies. Fluvalinate proved the best with 89%
reduction of mite population after one week of the treatment during 2004-05 and
95% during 2005-06. It was followed by formic acid, menthol and sulfur with 75,
69, 55 % mite reduction during 2004-05 and 73, 65, 50 % during 2005-06
respectively (Saleem, et al., 2008).
Non-chemical control such as modified bottom boards that can catch mites
when they drop from bees (Pettis and Shimanuki, 1999). Hygienic queen bee stock
whose workers often remove mite-infested brood from pupal cells (Spivak 1996;

30
Spivak and Reuter, 2001) use of drone-brood to trap mites (Calis, et al., 1999) and
heat treatment (Huang, 2000) provide some mite population suppression, but not
sufficiently to maintain bee colonies at low mite levels for multiple season. Some
research has been conducted to develop a multi-component integrated pest
management (IPM) approach to V. destructor control (Tangkanaasing, et al., 1988;
Manino et al., 1996; Ellis, 2001; Sammataro, et al., 2004). Main attribute of any
IPM system is the use of a combination of control methods involving hygienic bees
(genetic control), modified bottom boards (a cultural control) and thymol
application (a chemical control) as well as the use of the commonly applied
miticides, Apistan (Rice, et al., 2004).
Varroa destructor mites collected from sealed drone brood (Apis mellifera)
placed in Petri dishes were prepared in advance with filter paper impregnated with
4.2% solution of oxalic acid, formic acid 60%, 15% lactic acid, Bee Vital and
menthol and thymol crystals. Study showed that 15% lactic acid ranks first with
thymol followed in descending order of 60% formic acid, oxalic acid 4.2% and
Vital Bee Hive Clean. All substances tested acaricides have good activity but Vital
Bee Hive Clean product has all the qualities in the safety application and obtaining
organic bee Products (Balint, et al., 2010).

31
MATERIALS AND METHODS
Chapter 3
The research work was carried out at Honeybee Research Institute (HBRI) of
National Agricultural Research Centre, Islamabad, Pakistan on Apis mellifera colonies
naturally infected with ectoparasitic mites. Treatments were given randomly to all
experimental colonies. Modified bottom boards and hygienic new queens were used in
all colonies during the experiments. The mite collection trays (mite excluders) were
kept under bottom boards for assessing the population of mites. The rate of
ectoparasitic mites’ infestation and treatment efficacy was estimated by counting falling
mites on mite collection tray and by counting the dead mites in the sealed worker and
drone brood before and after treatment. Treatments in all replications were applied with
Complete Randomize Design (CRD).
3.1. ADULT BEES INFESTATION ASSESSMENT
The adults and sealed brood populations of test colonies were assessed for
infestation week before the treatments’ application. To collect the sample (150-250
bees/colony) of mites’ infestation the alcohol wash technique was used (De Jong et al.,
1982). To assess the infestation level on adult honeybees, a sample of 250 bees was
taken with the help of iron funnel from each experimental honeybee colony. The bees
were kept in plastic jars and these jars were placed in the refrigerator for 2-3 hours in
order to calm the bees. To wash the bees a detergent solution was prepared by
dissolving two-table spoon of powder detergent in one litre hot water. Then bees were
washed in semi hot detergent water solution in order to detach the mites sticked with
the bees’ body. Each bee jar was kept for 5-10 minutes and then shaked well before
31

32
pouring all the material on a muslin cloth in a steel container. After washing the muslin
cloth it was examined under the electronic magnifying glass in order to count the
detached mites. The washed bees were also examined critically by magnifying glass to
count if any mite remained sticked to the body of bees. (De Jong, et al., 1982).
3.2. BROOD INFESTATION ASSESSMENT
The sealed worker brood population of test colonies was assessed for infestation
a week earlier to the application of treatments. The mite infestation was assessed by
opening 100 cells of sealed brood from each of the test colony before and after
treatment application. For brood infestation examination, the central areas of the brood
frames were selected and with the help of a fine forceps each larva was removed. The
larvae were kept in the Petri dishes (90 mm dia) containing 4-5 ml alcohol in
refrigerator at10 ˚C for one hour. After 1-2 hours, the larvae were examined under the
electric magnifying glass to count the mites detached from larvae (De Jong et al.,
1982).
3.3. MITE INFESTATION ASSESSMENT
Census of mite population were required the use of mite excluders kept below
the bee frame in the bottom of the bee hive. Mite population count was done after 24
hrs (Fries, et al., 1991; Devlin, 2001). At the end, all the experimental colonies were
applied with Apistan (Fluvalinate) strips. The mite population was measured using mite
collection tray (mite excluders) placed on the bottom boards of each test bees colony
after 24 hrs period. Colony survival was monitored throughout the experiment. Honey
production was measured by weighing of each hive body used for honey collection
before and after the honey extraction process. The weight difference was considered as

33
the amount of harvestable honey. Thus, five different experiments were designed for
control the ectoparasitic mites’ population; the detail is given as below:
3.4. EXPERIMENT 1: CONTROL OF TROPILAELAPS CLAREAE USING
THYMOL AND FORMIC ACID
The experiment was conducted at HBRI of NARC, Islamabad on honey bee
colonies infested with the T. clareae. Treatments were given randomly to all
experimental colonies which were re-queened with hygienic queens prior to the start of
the experiment.
Twelve queen right honeybee colonies in Langstroth hives were used which
consist of ten bee frame, five brood frame and equal mite infestation levels. The hives
were placed at a distance of 5 meters from each other. Colonies were divided into 3
groups of 4 colonies each. One group was treated with finely grinded thymol (T) and
the second group received formic acid (F. A). Group one received four treatments (4
gm) each with a weekly interval, testing a total amount of 16 gm thymol crystals
placed in Petri dishes (80 mm dia) on top of the brood frame under the top cover of
hives. Second group received 4 treatments of 65 % formic acid (20 ml each) applied on
card board placed in the mite collection trays placed in the deep bottom board of the
hive. Total 80 ml formic acid was applied at weekly interval for 28 days and third
group served as control with no treatment. At the end, all the experimental colonies
were applied with Apistan (Fluvalinate) strips. Each honeybee colony was equipped
with a modified bottom board for placing mite collection trays (mite excluders) through
the back side of the hive without disturbing the bees. Thymol and formic acid efficacy
and rate of mite damage was calculated on count of collected mites in debris.

34
The mite fall was counted on the mite collection trays at weekly interval for one
month. Mite mortality was examined weekly in debris collected in mite collection trays
placed under the screen. In order to evaluate total mite population an Apistan
(Fluvalinate) strip was applied to the colonies. Apistan strip was removed from the
colonies after 30 days and dropped dead mites were counted (Marcangeli and Garcia,
2004). Treatment efficacy was calculated for each colony by using following formula
(Higes, et al., 1997):
Where,
VD+7+VD+14+VD+21+VD+28
E (%) = __________________________________ × 100
VT
E = Efficacy of thymol and formic acid
V D+n = Mites collected per week
VT= Total number of mites collected
3.5. EXPERIMENT 2: THE EFFECTIVENESS OF DIFFERENT
CONCENTRATIONS OF OXALIC ACID SOLUTION FOR VARROA
DESTRUCTOR CONTROL
About 50 adult and sealed brood populations of Honeybee Research Institute
apiaries were assessed for infestation before selecting the experimental colonies. To
collect the sample (250 bees/ colony) of mite infestations the alcohol wash technique
was used (De Jong et al., 1982). The mite infestation was evaluated by opening 100

35
cells of sealed brood before treatment (Burgett and Burikam, 1985) while for the
assessment of mite population in debris, mite collection trays were below the bee frame
in the bottom of the bee colony and removed after 24 hrs to count the mites (Devlin,
2001). Finally, twenty queen right honeybee colonies in Langstroth hives were used on
mite infestation levels. The colonies were placed in HBRI premises in December 2008
with mean outside temperature of 3 0 C.
Each honeybee colony was equipped with a modified bottom board and a mite
collection tray (mite excluder) which was placed through the back side of the hive,
without disturbing the bee colony. OA treatment efficacy and the rate of ectoparasitic
mite infestation was calculated on count of falling mites in debris. The honeybee
colonies of each group were placed at an appropriate distance of 5 meters. Colony
strength (number of combs covered with bees, brood areas, and amount of food) was
almost equal. Colonies were divided into 4 groups of 5 colonies each and were applied
with different concentrations of oxalic acid (OA). First group (T1) was treated with
4.2% OA solution. Second group (T2) received 3.2 % OA solution, the third group (T3)
was treated with 2.1% OA solution and the fourth group served as control (T4) with no
treatment.
Oxalic acid was applied in sugar syrup. To obtain 4.2%, 3.2% and 2.1 % OA
solution, 100, 75 and 50 gm oxalic acid dehydrate was mixed with 1 liter of sugar water
(1:1) (Prandin, et al., 2001). Treatments were only delivered to frame spaces that
contained bees; any empty frame was not treated. The 5 ml mixture was trickled
directly on the adult bees in between two frames using a syringe as recommended by
Imdorf et al., (1997) and Brodsgaard et al., (1999).

36
All groups received oxalic acid solution with three doses at five days interval.
At the end, all the experimental colonies were applied with Fluvalinate (Apistan) strip
for knockdown. Apistan strips were removed from the colonies after four weeks and
dropped dead mites were counted (Marcangeli and Garcia, 2004). All the colonies were
checked for dead worker bees and queens at the end of treatment application. The
efficacy of the OA treatments was calculated by using following formula (Marinelli, et
al., 2004):
No. of mites fallen for each treatment
Efficacy of oxalic acid (%) =
______________________________
× 100
Total number of fallen mites
3.6. EXPERIMENT 3: FIELD TRIAL FOR ECTOPARASITIC MITES
CONTROL WITH THYMOL AND OXALIC ACID SOLUTION
About 50 honeybee colony populations (adult and sealed brood) of apiary were
assessed for infestation prior to selecting the experimental colonies. To collect the
sample (250 bees/ colony) of mite infestations the alcohol wash technique was used (De
Jong, et al., 1982). To get an accurate adult bee count the colonies were inspected at
sunrise before the bees started foraging. The mite infestation was evaluated by opening
100 cells of sealed brood before treatment (Burgett and Burikam, 1985) while for the
assessment of mite population in debris mite collection trays were kept for 24 hrs in the
bottom of the bee colony. Mites fell in debris were counted (Devlin, 2001).
Finally, twenty queen right honeybee colonies in Langstroth hives were used on
mite infestation levels. The hives were placed at a distance of 5 meters from each other.
The experiment was started in the month of December 2009 (i.e. the peak time of mite

37
population) when mean outer temperature was 3 0 C. Colonies were divided into 4
groups of 5 colonies each. One group was treated with 2 gm finely grinded thymol plus
3.2 % OA (T1), the second group received 4 gm finely grinded thymol plus 3.2% OA
(T2), the third group was treated with 6gm finely grinded thymol plus 3.2% OA (T3)
and the fourth group served as control group (C). All groups received three treatments
with a weekly interval. Thymol crystals (finely grinded) were placed in Petri dishes (80
mm dia) on top of the brood frame under the top cover of hives. Oxalic acid was
applied in sugar syrup. To obtain 3.2 % OA solution, 75 gm oxalic acid dehydrate was
mixed with 1 liter of sugar water (1:1) (Prandin, et al., 2001). Treatments were only
delivered to frame spaces that contained bees; any empty frame was not treated. All
three groups received 3.2% oxalic acid solution with three doses at seven days interval.
The 5 ml mixture was trickled directly on to the adult bees in between two frames using
a syringe as recommended by Imdorf, et al., (1997) and Brodsgaard, et al., (1999).
Each honeybee colony was equipped with a modified bottom board. Mite
collection trays (mite excluders) were placed through the back side of the hive covered
by a wire screen to prevent the bees from coming into contact with the debris. The rate
of both ectoparasitic mites damage and thymol & formic acid treatment efficacy was
calculated by count of falling mites in debris through magnifying lamp/glass in the
laboratory. At the end, all the experimental colonies were given Fluvalinate (Apistan)
strip for knockdown. Apistan strips were removed from the colonies after four weeks
and dropped dead mites were counted (Marcangeli and Garcia, 2004). All the colonies
were checked for dead worker bees and queens at the end of treatment application. The
efficacy of all the treatments was calculated by using following formula (Marinelli, et
al., 2004):

38
No. of mites fallen for each treatment
Efficacy of thymol and oxalic acid (%) =
______________________________
× 100
Total number of fallen mites
3.7. EXPERIMENT 4: CONTROL OF V. DESTRUCTOR WITH PLANT
OILS/ EXTRACT
The experiment was conducted at HBRI of NARC, Islamabad. About 100 adult
and sealed brood populations of apiary were assessed for infestation prior to selecting
the experimental colonies. To collect the sample (250 bees/ colony) of mite infestations
the alcohol wash technique was used (De Jong et al., 1982). Procedure to get an
accurate adult bee count, evaluation of mite infestation and assessment of mite
population in debris etc was followed as mentioned in experiment # 3. The plants oils
tested for their efficacies were Neem oil (Azadirachta indica), Garlic oil (Allium
sativum), Clove oil (Syzygium aromaticum), Olive oil (Olea europaea) and tobacco
(Nicotiana tabacum).
3.7.1. Oil Extraction by Soxhlet Apparatus
Four gm of moisture free seed sample of the test plants was taken, with washed
plugged into with absorbent cotton. After that the thimble was placed in an extractor
fixed in condenser for extraction purpose. 150 ml of the seed sample oil placed in a
receiving flask attached with the apparatus. Solvent evaporating, over the time and
collected in the extractor. When the flask reached to maximum level, then was poured
into a receiving flask and the solvent again evaporated and passed through the sample
for second extraction. This was a continuous cyclic process until all the ether and

39
hexane soluble materials have been utilized. The flask was heated for 10 hours at 3-4
drops/sec condensation rate. The extraction time was 12-14 hrs.
3.7.2. Preparation of Tobacco water extracts
Two kilograms of dried tobacco leaves samples were collected from the local
market and were converted into powder form. The sample was tied in a cotton cloth in
the form of a bag and dipped in five liter of water at 80 0
C for 16 hours. In this way
20% concentrated solution attained which was diluted as 5%, 10 % and 15% for further
use in the trials.
3.7.3. Laboratory Bioassay
The compounds were assessed for their efficacy as miticides by exposing adult
mites to volatile fumes of the oils. Adult mites were removed from sealed cells of
worker and drones and placed in glass petri dish with five mites/ petri dish. A damp
tissue paper was placed on the bottom of Petri dishes and covered with a sheet of Para
film ‘M’ laboratory film. Small holes were punched in the film to allow evaporation of
the water from the tissue paper below. A piece of filter paper equal in size to the
diameter of the dish (9.0 cm) was placed on top of the Para film sheet. Forty micro
liters of 5, 10 and15 % solution of each plant essential oil diluted in methanol was
placed on the second piece of filter paper (4.25 cm diameter) attached to the lid of the
Petri dish. In control dishes only distilled water was poured on the filter paper. Each of
five treatments i.e. Neem oil (Azadirachta indica), Garlic oil (Allium sativum), Clove
oil (Syzygium aromaticum), Olive oil (Olea europaea) and tobacco (Nicotiana
tabacum) extract were replicated 4 times. Where as methanol was used as base in
formulations.

40
Dead and alive mites were counted after one and two days interval treatment.
Bee mortality count was done by separating each mite sophisticatedly irresponsive mite
towards stimulus considered an indication of increase in death tool. All bees were
indepently checked for the existence of mites.
3.7.4. Field Tests in bee hives
Best combination of promising oils/extract was tested on A. mellifera colonies.
Treatments were given randomly to all experimental colonies which were re-queened
with hygienic queens prior to the start of the experiment.
Forty eight queen right honeybee colonies in Langstroth hives were used on
mite infestation levels. The hives were placed at a distance of 5 meters from each other.
Colonies were divided into 16 groups of 3 colonies each. Each group of three colonies
received the most effective concentration as determined previously alone (garlic, neem,
clove, olive oil and tobacco extract) and in combination (garlic + neem, garlic + clove,
garlic + olive, garlic + tobacco, neem + clove, neem + olive, neem + tobacco, clove +
olive, clove + tobacco, olive + tobacco). Treatments were applied every evening when
the honeybees were present in the hives. 15 ml of each test material was sprayed by
using a simple plastic sprayer (500 ml). Solutions of all extract concentration were
sprinkled over the bees inside the colonies (Zaitoon, 2001). Mite and worker bee
mortalities were recorded every day at 10 am as if they were moved to the new hives
and the mite collection trays were removed and transmitted to the lab for counting the
mortalities.
Total 60 ml plant oil/ extract was applied at 5 days interval for 20 days. At the
end, all the experimental colonies were given Apistan (Fluvalinate) strips. Each
honeybee colony was equipped with a modified bottom board for placing mite

41
collection trays (mite excluders), through the back side of the hive. Efficacy of plant
oil/extract and the rate of V. destructor infestation was calculated by count of falling
mites in debris.
The mite fall were counted on the mite collection trays. Mite mortality was
examined after five day in debris collected in mite collection trays placed under the
screen. In order to evaluate total mite population an Apistan strip was applied to the
colonies. Apistan strip was removed from the colonies after 30 days and dropped dead
mites were counted (Marcangeli and Garcia, 2004).
Treatment efficacy was calculated for each colony by using following formula
(Higes et al., 1997):
Where,
VD+5+VD+10+VD+15+VD+20
E (%) = __________________________________ × 100
VT
E = Efficacy of plant oil / extract
V D+n = Mites collected on different days
VT= Total number of mites collected
3.8. EXPERIMENT 5: THE EFFECTIVENESS OF INTEGRATED
CONTROL OF TROPILAELAPS CLAREAE AND VARROA
DESTRUCTOR WITH DIFFERENT TREATMENTS
About 150 adult and sealed brood populations of Honeybee Research Institute
apiaries were assessed for infestation before selecting the experimental colonies. To
collect the sample (250 bees/ colony) of mite infestations the alcohol wash technique
was used (De Jong et al., 1982). The mite infestation was evaluated by opening 100

42
cells of sealed brood before treatment (Burgett and Burikam, 1985) while for the
assessment of mite population in debris mite collection trays kept for 24hrs in the
bottom board of bee hive and mites was counted (Devlin, 2001). Finally, thirty queen
right honeybee colonies in Langstroth hives were used on mite infestation levels.
The colonies were placed in HBRI premises and at different locality of
beekeeping areas. Each honeybee colony was equipped with a modified bottom board
and a mite collection tray (mite excluder) which was placed through the back side of
the hive, without disturbing colony. Efficacy and rate of infestation was calculated by
count of falling mites in debris. The honeybee colonies of each group were placed at
appropriate distance of 5 meters. Colony strength (number of combs covered with
bees, brood areas, and amount of food) were equal.
Colonies were divided into three groups of ten colonies each. Treatments were
only delivered to frame One group (T1) was tested with three applications of 4 gm
thymol + 3.2 % oxalic acid solution in the November-December, 2010 and two
application of 65% formic acid in July,2011. Thymol crystals (finely grinded) were
placed in Petri dishes (80mm) on top of the brood frame under the top cover of hives
spaces that contained bees. Oxalic acid was applied in sugar syrup. To obtain 3.2 % OA
solution, 75 g oxalic acid dehydrate was mixed with 1 liter of sugar water (1:1)
(Prandin et al., 2001). Treatments were only delivered to frame spaces that contained
bees; any empty frame was not treated. The 5 ml mixture was trickled directly on the
adult bees in between two frames using a syringe as recommended (Imdorf et al., 1997;
Brodsgaard et al., 1999). 65% formic acid (20 ml) applied on each card board placed in
the mite collection trays placed in the deep bottom board of the hive.

43
The second group (T2) received three application of 5% clove oil + Tobacco
extract in March, 2010 and two applications of 4gm thymol+3.2% OA solution in
December, 2010 and January,2011. Thymol crystals (finely grinded) were placed in
Petri dishes (80 mm via) on top of the brood frame under the top cover of hives spaces
that contained bees. 3.2% Oxalic acid was applied in sugar syrup.
While the third group (T3) was treated with three applications of 5% clove oil +
Tobacco extract in July, 2010 and two applications of 65 % formic acid in March,
2011. Treatments were applied every evening when the honeybees were present in the
hives. 15 milliliters of clove oil and tobacco extract was sprayed by using plastic
sprayer (500 ml). Solutions of all clove oil and tobacco extract concentrations were
sprinkled over the bees inside the colonies (Zaitoon, 2001).
At the end; all the experimental colonies were given Fluvalinate (Apistan) strip
for knockdown. Apistan strips were removed from the colonies after four weeks and
dropped dead mites were counted (Marcangeli and Garcia, 2004). All the colonies were
checked for dead worker bees and queens at the end of treatment application. The
efficacy of the treatments was calculated by using following formula (Marinelli et al.,
2004):
No. of mites fallen for each treatment
Efficacy (%) = ______________________________ × 100
Total number of fallen mites
3.9. HONEY YIELD
Honey was harvested after all experiments with the help of manual honey
harvester and compared honey yield of treated and control honeybee colonies. Honey

44
production was measured by taking the weight of each hive body used for honey
collection before and after the honey extraction process. The weight difference was
considered as the amount of harvestable honey.
3.10. STATISTICAL ANALYSIS
All data collected by performing experiments were statistically being analyzed
through MSTAT C computer based software (Freed and Eisensmith, 1986). Analysis of
Variance was used at 5% probability level (Montgomery, 2011).

45
RESULTS AND DISCUSSION
Chapter 4
4.1. EXPERIMENT 1: CONTROL OF MITES USING THYMOL AND
FORMIC ACID
A range of organic compounds that occur naturally and are present in honey can
be used to control parasitic mites. Few of them including formic acid and thymol have
shown potential effectiveness against these mites, which have no negative effect on the
development of colonies (Melathopoulos and Gates, 2003; Floris et al., 2004).
The results obtained from the experiment are shown in Table 4.1 and Table 4.2.
Significant number of mites mortality was found in both groups treated with thymol
and formic acid showing colonies with different levels of infestation at 5% level of
significance. Thymol is the main constituent of several commercially available
medicinal products and a number of studies have demonstrated its efficacy at
controlling mite infestations in honey bee colonies, but with variable results
( Calderone et al., 1997; Imdorf, et al., 1999).
In the results, number of mites fallen for the thymol ranged between 244-317
with a mean value of 277.75+16.19 (Mean+ SE), while the range and mean number
values for formic acid treatment and control were between 313-450, 47-52 and
376.50+ 34.11, 49.25+ 1.03 (Mean+SE) respectively (Fig 4.1) which is not in
agreement with Imdorf et al. (1995) who demonstrated that thymol had the highest
varroacidal activity at concentrations well tolerated by the bees but is in confirmation
with Harold et al. (1989) who found that mites were best controlled by placing formic
acid plates at the bottom board of the colonies and after four treatments at four days
45

46
intervals 94% of the mites were killed and the most effective treatment (62% of mites
killed) was with 40 ml of 65% formic acid (Greatti, et al., 1993). Bollhalder (1998) and
Calderone (1999) reported that thymol was very effective for the control of bee mites
and no side effects on honeybees.
Also, many researchers recorded some adverse effects on bees after treating
essential oils or their components; Lensky, et al. (1996) found that the use of pure
origanum oil during summer was harmful to the bees, and 30% thymol was also
harmful depending on dose and ambient temperature. This was found also by Chiesa
(1991) and Gal, et al. (1992). Mattila and Otis (1999) reported that honey production
was reduced by 30% during the Apiguard® treatment. On contrast, Mutinelli, et al.
(1996) reported low or absent bee mortality in all tests of formic acid, lactic acid or
Apilife-VAR®, and also El-Shaarawy (1999) claimed that honey yield increased after
colonies treated with Apiguard® or formic acid.
The range of efficacy in colonies treated with thymol was 60.50% to 62.15%
while for formic acid the range was 77.59% to 82.87%. The mean value of efficacy for
thymol, formic acid and control were 61.49%, 79.52% and 16.95%, respectively. When
compared among thymol, formic acid and control. They were found significantly
different at 5% level of significance (Fig 4.2). Three application of formic acid per hive
showed high percentage of mite mortality (Mutenelli et a1., 1994; Van Veen, et al.,
1998) which is also confirmed by our experiment where we used four doses of formic
acid.
The range of honey yield in colonies treated with thymol was 11.24-12.55 while
13.12-15.11 and 4.5-6.15, for formic acid and control, respectively. The honey
produced from different hives when treated with acaricides was also weighed at the end

47
of experiment. The mean amount of honey produced in kg from thymol, formic acid
treated colonies and control was 11.81 + 0.28, 14.33 + 0.47 and 5.39 + 0.36 (Mean+
SE) respectively (Fig 4.3). The honey produced was also compared between thymol,
formic acid and control and the results were found significant at 5% level of
significance.
It can be concluded from the experiment that since formic acid is also effective
against Acarapis woodi (Sharma et al., 1983), it can be used safely without any side
effects in controlling both endo and ectoparasitic mites infesting honey bee colonies.
In regression analysis dependent variable is Honey yield and independent
variable is Mites mortality, regressed Mites mortality on Honey yield. The coefficient
5.554 which is intercept indicating that in natural environment without applying any
treatment we can get on an average a yield of 5.554537 kg of honey per hive. The
second coefficient in the regression line is slope which shows that due to mortality of
one Tropilaelaps mite there is on the average an increase of 0.021132 kg in the yield by
applying treatments. (Table 4.3).
Less value of CV indicates high precision of the experiment in efficacy and
honey yield as compared to mite mortality on thymol and formic acid (Table 4.4).
As described in pie chart (fig. 4.4 ) it is clear that on an average 54%, 39% and
7% mites were killed by applying formic acid, thymol and by natural environment,
respectively in mite collection trays (control group).

Colony
number
48
Table 4.1 Efficacy of thymol in Apis mellifera colonies
Mites killed
by thymol
Mites killed
by apistan
Total mortality
of mites/colony
1 290 180 470 61.70
2 260 162 422 61.61
3 317 193 510 62.15
4 244 156 398 60.50
Mean 61.49
Table 4.2 Efficacy of formic acid in Apis mellifera colonies
Colony number Mites killed by
formic acid
Mites killed
by apistan
Total mortality
of mites/colony
Efficacy (%)
1 322 93 415 77.59
2 315 85 400 78.59
3 419 111 530 79.05
4 450 93 543 82.87
Mean 79.52
Efficacy (%)

Mite Mortality
600
500
400
300
200
100
0
49
Thymol Formic Acid Control
Treatments
Mite Mortality
Fig.4.1 Mortality of T. clareae in bee colonies treated with thymol and formic acid.
Efficacy (% )
100
90
80
70
60
50
40
30
20
10
0
Thymol Formic Acid Control
Treatments
Fig.4.2 Mean efficacy of acaricides at the end of experiment
Efficacy (% )

Honey Y ield (k g .)
25
20
15
10
5
0
50
Thymol Formic Acid Control
Treatments
Honey Yield
(k )
Fig.4.3 Mean amount of honey produced from colonies treated with thymol and formic
acid against T. clareae
Table 4.3 Simple Linear Regression Model Results
Mite mortality = MM
Coefficients
Standard
Error t Stat P-value
Intercept 5.554537 1.442565 3.85046 0.00321
MM Variable 0.021132 0.00526 4.017145 0.00245

Table 4.4 Mean Comparisons, F-test and Coefficient of variance
51
Treatment Mite Mortality Efficacy Honey Yield
Thymol 277.75b 61.490b 11.81b
Formic Acid 376.50a 79.525a 14.33a
Control 49.250c 16.953c 5.39c
LSD (0.05) 69.76 2.31 1.22
F- test 59.3** 1987** 146**
C.V 18.60 2.74 7.27
**Highly significant at 0.1%.
Fig. 4.4 Mite mortality (%) after treating bee colonies with thymol and formic acid

52
4.2. EXPERIMENT 2: THE EFFECTIVENESS OF DIFFERENT
CONCENTRATIONS OF OXALIC ACID SOLUTION FOR
CONTROLLING OF VARROA DESTRUCTOR.
The results of experiment showed that the efficacy of different concentrations of
oxalic acid in all colonies with 3.2% concentration yielding the highest mites’
mortality. Significant numbers of mites fall in all the groups showing colonies with
different levels of infestation at 5% level of significance. In results the number of mites
fallen for the T1 (4.2 % OA) T2 (3.2 % OA), T3 (2.1 % OA) and T4 (Control) ranged
between 931-1123, 1188-1348, 200-225 and 49-80, respectively as shown in Fig 4.5
and mean number 1023.4+ 32.14, 1242+28.45, 215.80+4.36 and 66.60+ 5.99
(Mean+SE), respectively.
Generally, OA showed high efficacy against mites which is in accordance with
the results (Radetzki, 1994; Nanetti et al., 1995; Nanetti and Stradi 1997; Gregorc and
Planinc, 2002; Gregorc and Poklular 2003; Marinelli et al., 2004; Gregorc and Planinc
2004; Rademacher and Harz 2006) showing that OA is very effective against V.
destructor.
Nanetti, et al. (2003) considered OA a good method for controlling the mites
but stated that it may cause reduction in the brood, conversely Imdorf et al. (1997)
reported that OA did not show any significant decrease on brood area which is in
confirmation with experiment’s results as the brood also showed no lasting loss and the
slight damage to eggs and larvae can be tolerated as it hardly influences the total
population of the bee colonies. No raised bee mortality was observed during the
application of treatments. Neither loss of queens after any of the treatments as other

53
authors have indicated nor supersedure of the queens was found (Wachendorfer et al.,
1985).
After several trials conducted by various scientists in different countries
regarding testing of different combinations between OA and sucrose concentrations, it
was observed that 4.2 % OA is the most effective; nonetheless 3.2 % option gave
similar results while 2.1 % OA did not yield sufficient mite mortality (Nanetti et al.,
2003). Findings obtained at the end of experiment were in accordance with these
findings up to the extent as 3.2% OA was found the best concentration not the 4.2 %
OA . The least effective concentration found in the experiment’s results as well other
findings was the same i.e. 2.1%.
Results are also in accordance with (Nicolaos et al., 2007) as the 4.2% OA by
trickling method in broodless period resulted in 77.3% mite mortality. The range of
efficacy in colonies treated with T1, T2, T3 and T4 was 77.91-82.74%, 93.67-96.29%,
42.11-47.91% and 17.82-23.77% respectively. The mean value of efficacy for the said
treatments also varied between different treatments and found significantly different at
5% level of significance (Fig 4.6).
The honey produced from different hives when treated with different
concentrations of OA was also weighed at the end of experiment. The mean amount of
honey produced in kg from T1, T2, T3 and T4 was 14.6 + 0.40, 23.0 + 0.44, 9.0 + 0.32
and 3.4 + 0.24 (Mean+ SE), respectively (Fig 4.7). The honey produced was also
compared but the results were significant as well at 5% level of significance.
Presently, synthetic acaricides are regularly used for the control of Varroa
destructor, however, due to the persistent nature they accumulate in honey and wax
(Bogdanov et al., 2002). Mites resistance to acaricide reported in many countries (Elzen

54
et al., 1999). These problems have initiated the development of non toxic substances
i.e. organic acids and essential oils.
As an alternative control strategy winter treatment is very important because
most of Varroa which are likely to appear in the next year population are destroyed in
this way. These are the mites that survived the autumn season treatment. For the winter
treatment Oxalic acid offers a promising opportunity.
Oxalic acid is a very promising candidate chemical for the control of Varroa
mites. It has many advantages like simple in use, cheap, non-toxic toward beekeepers.
It also causes low or no honeybee toxicity and there is no record of queen loss or brood
/adult bee mortality. It is a natural constituent of honey and many vegetables, and no
significant residues have been found in hive products in Europe (Del Nozal et al., 2000;
Bernardini and Gardi, 2001).
3.2% OA killed high number of mite. These results were also confirmed by
(Fries et al., 2000) that the six frame bee colonies required 30 ml of 3.2% OA for mite
control.
3.2% OA was the required dose as proved by applying both quantities (4.2 % &
3.2% OA) simultaneously on the same size of bee colonies. 3.2% OA dose will develop
the grooming behavior in honey bees and increase in bee to bee contact which will
increase the grooming behavior among honeybees resulting in fall of more number of
both the mite species and an increase in honey yield. Application of highly
concentrated OA i.e., more than 3.2% OA did not give increased efficacy.

M ite m o rta lity
1600
1400
1200
1000
800
600
400
200
0
55
Mite mortality
4.2 % OA 3.2% OA 2.1 % OA Control
Treatments
Fig.4.5. Mean number of mites found dead in colonies after using different
concentrations of oxalic acid

E ffic a c y (% )
120
100
80
60
40
20
0
56
Efficacy (% )
4.2 % OA 3.2% OA 2.1 % OA Control
Treatments
Fig. 4.6. Mean efficacy of oxalic acid with different concentrations observed at the
end of experiment

Honey y ield (k g .)
30
25
20
15
10
5
0
57
4.2 % OA 3.2% OA 2.1 % OA Control
Treatments
Honey yield (kg.)
Fig. 4.7. Mean amount of honey produced from colonies various treatment oxalic acid
Table 4.5 Simple Linear Regression Model Results
Coefficients Standard Error t Stat P-value
Intercept 0.459239 1.562506 0.293912 0.772187
MM Variable 0.014349 0.001636 8.772711 6.43E-08
MM= Mite mortality

58
In regression analysis dependent variable is Honey yield and independent
variable is Mites mortality, regressed Mites mortality on Honey yield.
The coefficient 0.459239 which is intercept indicating that in natural
environment without applying any treatment we can get on the average a yield of
0.459239kg of honey per hive. The second coefficient in the regression line is slope
which shows that due to mortality of one Varroa mite there is on an average increase of
0.014349 kg in the honey yield by applying treatment (Table 4.5).
Less value of C.V. indicates high precision of the experiment in mite mortality,
efficacy and honey yield as compared to control on different concentrations of oxalic
acid (Table 4.6).
As shown in the pie chart (Fig. 4.8) it is clear that on an average 40%, 49% , 8%
and 3% mites were killed by applying 4.2% OA, 3.2% OA, 2.1% OA and by natural
environment in mite collection trays (control group).

59
Table 4.6. Multiple Comparisons of different concentrations of oxalic acid with
control against Varroa mites
Treatment Mite Difference Efficacy Difference Honey
Mortality
Yield
Difference
2.1% OA 215.8 149.2* 45.950 24.376* 9.000 5.600*
3.2% OA 1242.0 1175.4* 94.508 72.934* 23.000 19.600*
4.2% OA 1023.4 956.8* 80.940 59.366* 14.60 11.200*
Control 66.6 21.574 3.400
LSD (0.05 79.851 3.16 1.32
F-test 715** 1480** 538**
C.V. 7.65 3.17 6.45
**Highly significant at 0.1%.
*Pairs that are significantly different are flagged with an asterisk.
Fig 4.8 Mites mortality (%) after treating bee colonies with different
concentrations of oxalic acid

60
4.3. EXPERIMENT 3: FIELD TRIAL OF TROPILAELAPS AND VARROA
MITES CONTROL WITH THYMOL AND OXALIC ACID SOLUTION
The number of Tropilaelaps mites fallen for the T1 (2 gm thymol + 3.2% OA),
T2 (4 gm thymol + 3.2% OA), T3 (6 gm thymol + 3.2% OA) and control ranged
between 39-50, 55-80, 51-54 and 26-31, respectively. In case of Varroa the ranges for
T1, T2, T3 and control was 1068-1122, 1487-1613, 1124-1311 and 43-69. The mean
number of Tropilaelaps and Varroa fallen for each treatment were shown in Fig 4.9.
In the results number of mites fallen for T1, T2, T3, And T4 with a mean value
for Tropilaelaps mites was 44.6+ 2.25, 62.60+ 4.50, 53.00+ 2.21 and 28.4+ 0.81 and for
Varroa mite was 1112.8+ 28.67, 1560.4+ 21.96, 1230.4+ 31.08 and 47.80+ 5.45,
respectively. When different treatments were compared for Tropilaelaps mite a highly
significant difference was found for the number of fallen mites at 5% level of
significance. A significant difference was found between all the treatments. The
number of Varroa mite fell for each treatment was also compared and the results
obtained were in accordance with the Tropilaelaps mites. The highest number of mites
fell in T2 and when different treatments were compared it was found that only T2 was
significantly different from all the other treatments, which clearly showed T2 to be the
most effective miticide against Varroa mites at 5% level of significance.
In case of Tropilaelaps mite the range of efficacy in colonies treated with T1,
T2, T3 and control was 26-28, 34-48, 31-38 and 7-9 %, respectively. The mean value of
efficacy for the said treatments also varied between different treatments. The
percentages were arcsine square root transformed and when compared were found to be

61
significantly different (at 5% level of significance) where T2 was found to have the
highest efficacy (Fig 4.10).
For the Varroa mites the efficacy range for T1, T2, T3 and control was 92-94,
98-99, 92-95 and 15-23%. The results showed a highly significant difference between
efficacies (at 5% level of significance). The T2 again showed the highest efficacy of
99% (Fig 4.10).
The honey produced from different hives when treated with different treatments
was also weighed at the end of experiment. The mean amount of honey produced in kg
from T1, T2, T3 and T4 was 10+ 0.32, 21.00+ 0.32, 12.00+ 0.32 and 5.60+ 0.25 (Fig
4.11) and results showed a significant high amount of honey from the colonies treated
with T2 (at 5% level of significance).
From the range of available organic compounds occurring naturally we selected
a combination of thymol and oxalic acid. Thymol is the main constituent of several
commercially available medicinal products and numbers of studies have demonstrated
its efficacy in controlling mite infestations in honey bee colonies, but with variable
results (Imdorf et al., 1995; Calderon et al., 1997).
recently evaluated several essential oils and related compounds including
Thymol, methyl silicate and benzyl acetates were less effective for mites control rather
best option if integrated against bee mites (Lindberg et al.,2000; Ali et al., 2002)
The effectiveness of OA against mite was famous by the end of 1980. OA was
effective, simple to apply, helpfulness in result and least cost. Lactic acid and oxalic
acid were approved as an alternative mite treatment (Popov et al., 1989; Charriere and
Imdorf, 2002). It has also been observed from pervious experimental studies that 3.2 %
oxalic acid could be effectively used for controlling the honeybee mites, therefore for

62
the present trial combination of 3.2 % oxalic acid with different quantities of thymol
was tried against honeybee mite’s i.e. T. clareae and V. destructor which is in
accordance with, Fries (2007) who answered an important question regarding
application of OA or in used quantity of OA. The use of 3.2% OA solution generate 92-
2% rate of efficacy by trickling method while OA solution of 1.6% can attain only
68.3% rate of efficacy (Fries, 2007).
It can be concluded from the experiment that since thymol and OA both are
effective against mites both can be safely used together without any side effects in
controlling both species of mites.
In regression analysis dependent variable is Honey yield and independent
variable is Mites mortality, regressed Mites mortality on Honey yield. The coefficient -
0.23838 which is meaningless as p-value is 0.937. The second coefficient in the
regression line is slope of Tropilaelaps mite which shows that due to mortality of one
mite there is on an average an increase of 0.153872 kg in the yield by applying
treatment T. clareae by supposing other factor constant but it is a non-significant. The
3 rd coefficient in the regression line is the slope of Varroa mites which shows that due
to mortality of one mite there is on the average an increase of 0.005196 kg in the yield
by applying V. destructor by supposing other factor constant and it is significant (Table
4.7).
Value of C.V. indicates high precision of the experiment in mite mortality,
efficacy and honey yield as compared to control on different treatments (Table 4.8). As
shown in the pie chart (Fig. 4.12, 4.13) it is clear that on an average 24%, 33%, 28%
15% T. clareae and 28%, 40%, 31% and 1% V. destructor mites were killed by

63
applying 2 gm thymol + 3.2%OA, 4 gm thymol + 3.2%OA, 6 gm thymol + 3.2%OA,
and by natural environment in mite collection trays (control group).
Generally insect mortality is dose and exposure time dependent. In present
study a higher dose of organic acids (6gm thymol/3.2% OA) caused comparatively low
mortality of mite than 4gm thymol/3.2% OA treatment. One possible reason for this
contraditiction may be due to leaving of bees from beehive due to fumigant effect of the
high dose organic acid and as bees along with mites left the hive it also reduced the
exposure time of mites to the treated higher dose of thymol which resulted in low
mortality.

2000
1800
1600
1400
1200
1000
800
600
400
200
0
2 g Thymol +
3.2 % OA
64
4 g Thymol +
3.2 % OA
Treatments
6 g Thymol +
3.2 % OA
Control
T. clareae
V. destructor
Fig 4.9. The mean number of mites fallen for T. clareae and V. destructor bars for
E ffic ac y (% )
120
100
80
60
40
20
0
2 g Thymol +
3.2 % OA
different treatments of thymol and oxalic acid
4 g Thymol +
3.2 % OA
Treatments
6 g Thymol +
3.2 % OA
Efficacy (% ) against T.
clareae
Efficacy (% ) against V.
des tructor
Control
Fig 4.10. The mean % efficacy for T. clareae and V. destructor bars for
different treatments of thymol and oxalic acid

Honey y ield (k g .)
25
20
15
10
5
0
2 g Thymol +
3.2 % OA
65
4 g Thymol +
3.2 % OA
T reatments
6 g Thymol +
3.2 % OA
Honey yield (kg.)
Control
Fig 4.11 The mean amount of honey produced from colonies treated with different
treatments of thymol and oxalic acid
Table 4.7. Simple Linear Regression Model Result
Coefficients Standard Error t Stat P-value
Intercept -0.23838 2.976307 -0.08009 0.937099
T. clareae
Variable 0.153872 0.10088 1.525299 0.145573
V. destructor
Variable 0.005196 0.002435 2.134164 0.04769

66
Table 4.8. Multiple Comparisons of different treatments of thymol and oxalic acid
with Control
Treatment Mite Mortality Efficacy Honey Difference
T. claerae Difference Varroa Difference T. claerae Difference Varroa Difference
Yield
2gm Thymol + 44.60
3.2% OA
16.200* 1112.8 1065.0* 26.398 17.924* 93.250 75.450* 10.00 4.400*
4gm Thymol + 62.60
3.2% OA
34.200* 1560.4 1512.6* 40.040 31.566* 98.834 81.034* 21.00 15.400*
6gm Thymol + 53.00
3.2% OA
24.600* 1230.4 1882.6* 34.852 26.378* 93.970 76.170* 12.00 6.400*
Control 28.40 - 47.8 - 8.47 - 17.80 - 5.60 -
LSD (0.05) - 10.187 - 87.91 - 5.1522 - 2.751 - 1.09
F-test 27.2** - 746** - 98.1** - 2681** - 466** -
C.V 13.18 - 5.43 - 11.38 - 2.21 - 5.52 -
**Highly significant at 0.1%.
*Pairs that are significantly different are flagged with an asterisk.

28%
15%
T. clareae
1
24%
33%
2gm thymol+3.2% OA
4gm thymol+3.2% OA
6gm thymol+3.2% OA
Control
Fig 4.12 T. clareae mite mortality (%) after treating bee colonies with
31%
1%
different treatments.
Varroa destructor
40%
28%
2gm thymol+3.2% OA
4gm thymol+3.2% OA
6gm thymol+3.2% OA
Control
Fig 4.13 Varroa destructor mite mortality (%) after treating bee colonies with
different treatments.

68
4.4. EXPERIMENT 4:- CONTROL OF VARROA MITES WITH PLANT
OILS/ EXTRACT
The results obtained from lab experiment showed that the essential oils/extract
had a significant effect on the mite mortality (at 5% level of significance). The clove
oil and tobacco extract both proved to be most effective against mites (Fig 4.14),
followed by garlic, olive and neem oil, respectively. The most effective combination
was clove oil and tobacco extract and the least effective treatment after control was
garlic and tobacco extracts. The results are in agreement with Allam- Sally (1999) and
El-Zemity (2006) who stated that the clove oil gave good results in controlling mites
and Fouly and Al-Dehhairi (2009) who found clove killed 62% Varroa mites. Hussein,
et al. (2001) also used six plant oils to control Varroa mites in honeybee colonies and
rated clove oil best among the other essential oils. Similar results were also observed by
Rajiter (1983), who found 50-79 % mite mortality when applied different amounts of
tobacco as fumigant. Abdol-Ahad Shaddel-Telli et al. (2008) who concluded that
tobacco extract without harmful effect against honeybees and decreased Varroa mite
population. There are different researchers that reported a positive effect of tobacco on
varroa mite mortalities (Rijiter 1982, Rijiter 1983, Rijiter and Eijnd 1984).The
percentage concentrations and timings of treatment were also found significantly
different (at 5% level of significance). In the past the essential oil (neem) was applied
for the control of mites by avoiding their direct contact with them but the results of
those trials showed no positive effect on Varroa population (Bunsen, 1991). The
conclusion was the effective substances in neem are not volatile. Keeping in view the
work of previous researchers the aim was to bring the essential oils in different
concentrations into contact with mites. The results of experiment were also found in
confirmation with Melathopoulos et al. (2000) who observed that when bees and mites

69
were brought into contact with neem oil that it was spread on a surface it resulted in a
95% success. Hassan et al. (2008) indicated that neem oil efficacy rate is only 4.95%
against Varroa mite and not suitable for Varroa control in Egyptian bee race.
The results/ findings of the experiment shows that the overall mean mortality
for the number of mites for different concentrations i.e. 5%, 10% and 15% of essential
oils/ extract were 4.15+0.23, 2.95+0.16 and 2.35+0.18 (Mean+ SE) (Fig 4.15)
respectively, which clearly showed that 5 % is the most effective as compared to 10 and
15% concentrations, which is against the findings of Abdel Rahman and Rateb (2008)
who found that the highest concentrations of lemon juice (10, 25, 50, 75 and 100 %)
caused high number of fallen dead mites and Zaitoon (2001) who found that the highest
concentrations (500 ppm) of Rhazya stricta caused 100% mite mortality. Results are
confirmed by Abd El-Wahab and Ebada (2006) who recorded significant differences
between the sour orange, lemon grass and citronella oils in different concentrations.
In the second part of laboratory experiments the 5 % concentrations of different
oils/extracts were applied in various combinations following the same procedure and it
was found that even the combinations also killed mites in a significantly different
manner (at 5% level of significance). The most effective combination was clove oil and
tobacco extract with the mean mortality of 5.00 + 1.01 (Mean + SE) and the least
effective treatment after control was garlic and tobacco extract (0.25 + 0.21 (Mean+
SE). The detailed results are presented in Table 4.9.
In our study that higher concentration of plant oil treatment was less effective in
honeybee colonies for mites control as compared to use of lower concentrations of plant
oil. One possible reason for this may be due to absconding of honey bees from bee
hives (Imdorf, et al.,2003), as a result treated higher concentration of plant oil treatment

70
mite present in bee hive exposed for less time compared to lower concentration of
plant oil treatment.
Generally insect mortality is dose and exposure time dependent. In present
study a higher concentration of plant oil (15%) caused comparatively low mortality of
mite than lower concentration of plant oil treatment. One possible reason for this
contraditiction may be due to leaving of bees from beehive due to fumigant effect of the
high dose plant oil and as bees along with mites left the hive it also reduced the
exposure time of mites to the treated higher dose of thymol which resulted in low
mortality.

71
Fig 4.14. Mean mite mortality as affected by plant oils/ tobacco extract.
Fig 4.15. Mean mite mortality as affected by different concentrations of plant
oils/tobacco extract concentrations

72
Table 4.9 Mean mortality of mites by different combinations of oils/extract
Treatments (5 %) Mean mite mortality SE
Neem +Garlic 3.25 0.25
Neem +Clove 3.75 0.47
Neem +Tobacco 3.50 0.86
Neem +Olive 3.50 0.95
Garlic + Clove 3.75 0.75
Garlic + Tobacco 0.25 0.21
Garlic + Olive 2.5 0.28
Clove +Tobacco 5.00 1.01
Clove + Olive 2.75 0.31
Tobacco + Olive 2.5 0.50
Control 0.00 0.00

Table 4.10. Acaricides efficacy of different essential oils/extract in Apis
mellifera colonies.
73
Plant oils/extrac Range of efficacy (%) Mean efficac
%
Minimum Maximum
Neem 79.80 89.79 85.36 2.94
Garlic 78.90 93.98 87.83 4.54
Clove 80.85 92.68 88.01 3.63
Tobacco 82.47 88.61 85.68 1.78
Olive 84.32 89.86 87.11 1.60
Neem+Garlic 80.63 91.82 86.86 3.29
Neem+Clove 71.58 89.73 81.38 5.29
Neem+Tobacco 84.96 89.62 87.90 1.47
Neem+Olive 83.08 88.87 85.78 1.67
Garlic+Clove 79.15 87.41 84.07 2.51
Garlic+Tobacco 76.21 82.82 80.05 1.98
Garlic+Olive 80.35 87.17 83.94 1.98
Clove+Tobacco 95.48 97.24 96.48 0.52
Clove+Olive 84.27 89.44 87.19 1.53
Tobacco+Olive 83.40 89.50 86.70 1.78
Control 22.91 25.40 23.98 0.74
S.E

74
The range of efficacy in colonies treated with different oils/extract and their
combinations were 71.58% to 97.24%. In field experiment using only 5% concentration
alone & in combination all oils/ extract individually and in all the previously tested
combinations confirmed the lab results as clove oil + tobacco extract the best
combination with mean value of 96.48% efficacy. The percentages were compared
between different treatments the results were significantly different (at 5% level of
significance) (Table 4.10, Fig. 4.18).
From the results obtained from this study, it can be concluded that the clove oil
alone or in combination with tobacco extract at 5 % concentrations can be considered a
promising agent for the control of V. destructor (Fig. 4.17)
The honey produced from different hives when treated with essential
oils/extracts was also weighed at the end of experiment. The mean amount of honey
produced in kg from different treatments (Table 4.11) was also compared and the
results were significantly different as shown in Fig. 4.19. (At 5% level of significance).

75
Table 4.11. Mean honey yield produced from Apis mellifera colonies treated with
different plant oils/extract.
Plant oils/extract Mean honey yield
(kg)
S.E.
Neem oil 12.30 0.35
Garlic oil 14.83 0.17
Clove oil 16.53 0.26
Tobacco extract 14.57 0.29
Olive oil 13.23 0.39
Neem +Garlic oil 15.23 0.39
Neem +Clove oil 15.13 0.47
Neem oil +Tobacco extract 17.80 0.20
Neem +Olive oil 13.13 0.47
Garlic + Clove oil 12.53 0.29
Garlic oil+ Tobacco extract 12.33 0.33
Garlic + Olive oil 13.00 0.50
Clove oil +Tobacco extract 20.50 0.29
Clove + Olive oil 16.13 0.41
Tobacco extract + Olive oil 14.50 0.29
control 6.23 0.39

76
The researchers tend to return to investigations involving plant extracts for a
natural control of parasites (Semmler et al., 2009) and pests (Islam 2006; George et al.,
2008) with importance in agricultural and veterinary industries. Botanical extracts
obtained from different plant species have also shown a broad spectrum of biological
activity in relation with mite population management (Ciccia et al., 2000; Banchio et
al., 2003, 2005; Ferrero et al., 2006; Jbilou et al., 2006; Aivazi and Vijayan 2009).
As far as the timings of application are concerned (Fig.4.16) the number
of dead fallen mites were higher after 24hrs, than 48 hrs. These results are supported by
the findings of Shoreit and Hussein (1994), who found that the maximum mean number
of dead mites was observed after the first treatment with coriander extract after that it
was gradually decreased. Abdel Rahman and Rateb (2008) resulted that the numbers of
dead fallen Varroa mites were comparatively higher after 24 hrs, than after 48 hrs and
after 72 hrs which exhibited the lowest number. This is contrary to the findings of
Calderone and Spivak (1995) and El-Zemity (2006) who found that essential oils
showed good result against varroa mite after 48 hrs of exposure.
In Argentina, use of plant substance (Propolis) extract as natural substitute for
control of Varroa mite was found common (Damiani et al., 2009, 2010 a, b). But
criticis were off the view that all such treatments would have very narrow range of
doses used against Varroa mites (Kraus et al., 1994). It was observed that the use of
extract from tobacco along with plant oils could caused an increase in mite mortality
ratio and safer to be use in bee hives.
Acaricides can caused environmental degradation then comparative to plant
extracts who are more beneficial to species and to the nature at the same time
(Mansaray 2000; Ottaway 2001; Isman and Machial 2006).

77
In regression analysis dependent variable is Honey yield and independent
variable is Mites mortality, we regressed Mites mortality on Honey yield. The
coefficient 8.403449 which is intercept indicating that in natural environment without
applying any treatment we can get on the average a yield of 8.403449 kg of honey per
hive. The second coefficient in the regression line is slope which shows that due to
mortality of one varroa mite there is on the average an increase of 0.028413 kg in the
yield by applying treatment, and both coefficients are significant (Table 4.12).
Value of C.V. indicates high precision of the experiment in mite mortality;
efficacy and honey yield as compared to control on different plant oils/ extract
applications (Table 4.13).

78
Fig 4.16. Mean mite mortality as affected by different concentrations of plant
oils/extracts.
Table 4.12. Simple Linear Regression Model Results
Coefficients Standard Error t Stat P-value
Intercept 8.403449 1.127462 7.453422 1.91E-09
MM Variable 0.028413 0.005214 5.449031 1.92E-06
MM= Mites Mortality

79
Table 4.13 Multiple Comparisons of plant oils/ extracts with control against
Varroa mite mortality
Treatment Varroa Difference Efficacy
(%)
Difference Honey
yield
(Kg)
Difference
Neem Oil 278.33 204.00* 85.367 61.387* 12.300 6.067*
Garlic Oil 201.33 127.00* 87.830 63.850* 14.833 8.600*
Clove Oil 199.67 125.33* 88.010 64.030* 16.533 10.300*
Tobacco extract 180.33 106.00* 85.680 61.700* 14.567 8.333*
Olive Oil 220.33 146.00* 87.113 63.133* 13.233 7.000*
Neem+Garlic Oil 167.67 93.33* 86.857 62.877* 15.233 9.000*
Neem+Clove Oil 167.00 92.67* 81.380 57.400* 15.133 8.900*
Neem Oil
+Tobacco extract
187.33 113.00* 87.897 63.917* 17.800 11.567*
Neem+Olive Oil 222.67 148.33* 85.783 61.803* 13.133 6.900*
Garlic+Clove Oil 202.00 127.67* 84.070 60.090* 12.533 6.300*
Garlic Oil
+Tobacco extract
169.00 94.67* 80.047 56.067* 12.333 6.100*
Garlic+Olive Oil 197.33 123.00* 83.937 59.957* 13.300 6.767*
Clove Oil
+Tobacco extract
381.67 307.33* 96.483 72.503* 20.500 14.267*
Clove+Olive Oil 227.00 152.67* 87.190 63.210* 16.133 9.900*
Tobacco+Olive Oil 216.33 142.00* 86.707 62.727* 14.500 8.267*
Control 74.33 - 23.980 - 6.233 -
LSD (0.05) - 77.408 11.271 1.512
F-test 12.1** - 36.3** - 74.4** -
C.V. 15.32 - 5.57 - 4.32 -
**Highly significant at 0.1%.
*Pairs that are significantly different are flagged with an asterisk.

Mite m ortality of V arroa
450
400
350
300
250
200
150
100
50
0
Neem oil
80
G arlic oil
Clove oil
Tobacco
extract
Olive oil
Neem+Garlic
oil
Neem
+Clove oil
Neem+
Tobacco
Neem+ Olive
oil
Garlic +
Clove oil
G a rlic
+T obbaco
G a rlic +Olive
Oil
Clove oil +
Tobacco
Clove + O liv e
oil
Tobacco
extract+
Control
Treatments
Mite mortality of V arroa
Fig 4.17. Acaricides mite mortality by different essential oils/extract in Apis
Efficacy (% )
120
100
80
60
40
20
0
mellifera colonies.
Neem oil
G arlic oil
Clove oil
Tobacco extra ct
Olive oil
Neem+Garlic oil
Neem +Clove oil
Neem+ Tobacco extract
Neem+ Olive oil
Garlic + Clove oil
G arlic +T obbaco extract
Garlic +Olive Oil
Clove oil + Tobacco extract
Clove + Olive oil
T oba cco extract+ Olive oil
Control
Efficacy(%)
Treatments
Fig 4.18. Efficacy of different essential oils/extract in Apis mellifera colonies.

Honey yield (kg .)
25
20
15
10
5
0
81
Treatments
Neem oil
Garlic oil
Clove oil
T oba cco extra ct
Olive oil
Neem+G a rlic oil
Neem +Clove oil
Neem+ Tobacco extract
Neem+ Olive oil
Garlic + Clove oil
Garlic +Tobbaco extract
Garlic +Olive Oil
Clove oil + Tobacco extract
Clove + Olive oil
T obacco extra ct+ Olive oil
Control
Fig 4.19. Honey yield by application of different essential oils/extract
in Apis mellifera colonies.
Honey yield (kg.)

82
4.5. EXPERIMENT 5: THE EFFECTIVENESS OF INTEGRATED
CONTROL OF TROPILAELAPS CLAREAE AND VARROA
DESTRUCTOR WITH DIFFERENT TREATMENTS
The experiment shows that the number of Tropilaelaps mites fallen for the T1,
T2 and T3 ranged between 113-127, 61-79 and 79-100, respectively. In the case of
Varroa the ranges for T1, T2 and T3 were 705-756, 165-185 and 177-208. The mean
number of Tropilaelaps and Varroa fallen for each treatment were shown in Fig 4.20.
When different treatments were compared for Tropilaelaps mite a highly significant
difference was found for the number of fallen mites (at 5% level of significance).
Moreover, a significant difference was found between all the treatments. The
number of Varroa mite fell for each treatment was also compared and the results
obtained were in accordance with the Tropilaelaps mites. The highest number of mites
fell in T1 and when different treatments were compared it was found that only T1 was
highly significantly different from all the other treatments, which clearly showed T1 to
be the most effective against Varroa mites (at 5% level of significance).
Results are confirmed by Amrine and Noel, 2007 concluded that 50% formic
acid, spearmint, lemongrass, essential oils with use of screen bottom boards trays
collectively effective for the control of Varroa mites. Harold et al. (1989) reported that
94% mites were killed by application of 4 treatments of formic acid and the most
effective treatment (62% of mites killed) was with 40 ml of 65% formic acid (Greatti et
al., 1993). Thymol effectual against mites but safe to honeybees (Bollhalder 1998;
Calderone 1999).

83
Chiesa (1991) and Gal et al. (1992) Lensky et al. (1996) reported that 30%
thymol was harmful to bee colonies during summer. Mattila and Otis (1999) showed
that honey yield was reduced by 30% during the formic acid treatment. On contrast,
Mutinelli et al. (1996) reported low bee mortality in all tests of formic acid and also El-
Shaarawy (1999) found that honey yield increased when apiaries treated with formic
acid.
Oxalic acid was found very effective for control of mites is in confirmation with
the results showing that OA is very effective against V. destructor (Gregorc and Planinc
2001, 2002; Gregorc and Poklular 2003; Marinelli et al., 2004).
Imdorf et al., (1997) claimed that OA did not effect on brood area and
Wachendorfer et al., (1985) observed no bee mortality, no loss of queen and no
supersedure by the application of OA.
Further in case of Tropilaelaps the range of efficacy in colonies treated with T1,
T2 and T3 were 84-88, 78-85 and 68-83%, respectively. The mean value of efficacy for
the said treatments also varied between different treatments and when compared was
found to be significantly different (at 5% level of significance) where T1 was found to
have the highest efficacy. For the Varroa mites the efficacy range for T1, T2 and T3
was 97-98, 91-93 and 81-86 %. The results showed a highly significant difference
between efficacies (at 5% level of significance). The T1 again showed the highest
average efficacy of 81 % respectively (Fig 4.21).
Three application of formic acid in bee hive shown good efficacy against mite
control (Mutenelli et a1., 1994; Van Veen et al., 1998) which is also confirmed by the
experiment where 2-3 doses of formic acid were used. The honey produced from
different hives when treated with different treatments was also weighed at the end of

84
experiment. The range of honey in colonies treated with T1, T2, T3 was 28-33, 21-24
and 20-24 kg, respectively. The mean amount of honey produced in kg from T1, T2 and
T3 is shown in Fig 4.22 and results showed a significantly more amount of honey
(29.80 kg) from the colonies treated with T1 (at 5% level of significance). Less value of
C.V. indicates high precision of the experiment in mite mortality, efficacy and honey
yield in first group (4gm thymol + 3.2% OA and 65% formic acid) as compared to
other groups (Table 4.14).
As shown in the pie chart (Fig. 4.23, 4.24) it is clear that on an average 43%,
24%, 32% T. clareae and 66%, 16% and 18% V. destructor mites were killed by
applying different treatments. Formic acid, oxalic acid and thymol are very effective for
the control of Varroa mite but nontoxic to honeybees (Imdorf, et al.,1999).
Thymol treatment before honey flow in spring does not affect the taste of the
honey (Donders et al., 2006). Stoya et al. (1986) showed that long-term formic acid
treatment in autumn according to the prescriptions will not increase honey acidity
above the required limit. The oxalic acid content remained unchanged, even after two
successive treatments during the same autumn. No rise of free acidity was encountered
after a combined treatment with formic and oxalic acid during the three trial years
(Bogdanov, et al., 2002).
The main objective of the study was to improve the honey yield with the control
over mite effect. Treatments were conducted in off season just to strengthen the bee
colonies and improve in honey yield during the production season. Secondly there was
no difference of taste in honey was found before and after applying the organic acid.
The residue level of OA in honey was 76.3 were found in honey after autumn treatment
but were still within the natural content levels of honey form various botainical origions

85
likewise using high concentrations of OA (7%, 20-30ml/ hive) did not even raise OA
content of the honey after the treatment (Nanetti, et al., 2003). By using thymol, OA
and mixture of two products residues were below the taste threshold (Berna and
Dodologlu, 2009). The replacement of synthetic acaricide treament by OA and thymol
minimize the risk of residues in bee products such as honey, wax and Propolis
(Bogdanov et al., 1999; Moosbeckhofer et al., 2003).

E ffic a c y (% )
900
800
700
600
500
400
300
200
100
0
4 g Thymol + 3.2 % OA
& 65% Formic acid
86
5% Clove oil + Tobacco
extract & 4 g Thymol +
3.2 % OA
Treatments
T. clareae
V. destructor
5% Clove oil + Tobacco
extract & 65% Formic
acid
Fig 4.20. Mean number of mites fallen by various treatments for T. clareae and
140
120
100
80
60
40
20
0
4 g Thymol + 3.2 % OA
& 65% Formic acid
V.destructor bars.
5% Clove oil + Tobacco
extract & 4 g Thymol +
3.2 % OA
Treatments
5% Clove oil + Tobacco
extract & 65% Formic
acid
T. clareae
V. destructor
Fig 4.21. Mean efficacy (%) by various treatments for T. clareae and V.destructor
bars.

H o n e y Y i e l d (k g )
40
35
30
25
20
15
10
5
0
4 g Thymol + 3.2 %
OA & 65% Formic
acid
87
5% Clove oil +
Tobacco extract & 4
g Thymol + 3.2 % OA
Treatments
5% Clove oil +
Tobacco extract &
65% Formic acid
Honey yield (kg)
Fig 4.22. Mean amount of honey produced from colonies treated with different
treatments

88
Table 4.14. Multiple Comparisons of different treatments for the control of
mites
Treatment Mite Mortality (#) Efficacy (%) Honey
4gm thymol + 3.2% OA
and 65% formic acid
5% clove oil+ tobacco
extract and 4gm thymol +
3.2% OA
T. clareae V. destructor T. clareae V. destructor
Yield
(Kg)
120.80a 724.30a 86.000a 97.748a 29.800a
66.90c 175.00c 81.037b 92.044b 22.700b
5% clove oil+ tobacco
extract and 65 % formic acid
88.90b 195.00b 75.404c 82.875c 21.900b
LSD (0.05) 5.377 9.829 2.802 0.979 1.256
F-test 214** 8458** 30.1** 494** 101**
C.V. 6.36 2.94 3.78 1.17 5.52
**Highly significant at 0.1%.
*Pairs that are significantly different are flagged with an asterisk.

32%
24%
89
T. clareae
44%
3 applications of 4gm
thymol +3.2%OA & 2
appliacations of 65%
formic acid
3 applications of 5% clove
oil+ Tobacco and 2
applications of 4gm thymol
+3.2%OA
3 applications of 5% clove
oil+ Tobacco and 2
applications of 65% formic
acid
Fig. 4. 23. T. clareae mite mortality (%) after treating bee colonies
with different treatments.
16%
Varroa destructor
18% 3 applications of 4gm
thymol +3.2%OA & 2
appliacations of 65%
formic acid
3 applications of 5%
clove oil+ Tobacco and 2
applications of 4gm
thymol +3.2%OA
3 applications of 5%
66%
clove oil+ Tobacco and 2
applications of 65%
formic acid
Fig. 4. 24. Varroa destructor mite mortality (%) after treating bee colonies
with different treatments.

90
4.5.1 Integrated Pest management for control of bee mites
Objective of the present study was to establish an efficient strategy against
ectoparasitic bee mites Tropilaelaps clareae and Varroa destructor in honey bee, Apis
mellifera colonies. Synthetic acaricides such as fluvalinate, coumaphos, and amitraz
are very efficient but due to their repeated use mites developed resistance against them,
therefore researchers decided to develop an integrated pest management strategy (IPM)
that would reduce dependence on synthetic pesticides.
IPM approach was based on a target with the use of commonly existing
practices that could be more effective and environment friendly.
In the recent studies, IPM strategy was also based on the use of treatment
thresholds and the singly and combined use of organic acids i.e. oxalic and formic acid,
thymol and plant oils/extract at different time periods of the year. Different mite
population estimators including natural mite fall using mite collection trays, alcohol
wash, and mites counts in adult honey bee were used.
Mid summer treatments with plant oils/extract reduced mite populations while
spring treatment with formic acid and thymol were found to be the most efficient in
reducing mite populations. A late fall oxalic acid treatment had a high efficacy when
queen laying and brood populations were at their lowest, which also helped in avoiding
the use of Apistan strip treatments.
Since an IPM approach does not have to be non-chemical, but it includes a
combination of different control strategies, also tested approaches involving hygienic
bees (a genetic control), modified bottom boards (a cultural control), and thymol,
formic, and oxalic acid application (a chemical control), plant oils/extract as well as the
use of the commonly applied miticide Apistan.

91
IPM based beekeeping was safe practice that could increase the specialty of
beekeepers. In all experiments Apistan strip did not used repeatedly and recommending
its use only at suitable time before honey flow and at the end of every treatment in this
way all mites in the hive will be killed easily without harmful effect on bee and
beekeepers. It is advised that the use of thymol and formic acid at the start of spring
before honey flow. In case of heavy infestation, a mid spring/summer treatment is also
recommended.
Integrated approach of plants oils can reduce suffocation in mites as well as
among honey bees. As because oils effectiveness over mites relies on suitable
temperature, season and colony status.
A screen bottom board with a sticky board below was also proved to be quite a
good tool for mite’s control and same is true for using hygienic lines of bees as the
mites will have less chance to overwhelm a strong, healthy colony of bees than they do
in a similarly strong, healthy colony on non-hygienic line of bees.
It is therefore recommended to use all treatments, which are safer to bees and
bee products. Hygienic bee, screen bottom board, chemical control, apistan, organic
acid (oxalic acid, formic acid,), essential oil (thymol), plant oil/extract can be used for
controlling ectoparasitic mites of honey bee, while synthetic Fluvalinate (apistan)
should be minimized due to possible development of resistance in mites. Use of
modified bottom boards and hygienic bee stock as it facilitates the colony management
and only require a small amount of extra maintenance.
The treatments mentioned above can also be used to maintain mite’s
populations below the economic injury level in large apiaries, however, some of the
application methods are laborious and may require investment in time for

92
implementation, and some might need multiple visits to the apiary for application. This
causes an increased production costs for beekeepers, of especially for professional
beekeepers that manage hundreds or even thousands of hives, but still the output they
receive will be far greater than the input.

93
SUMMARY
The proposed research work was conducted at HBRI, NARC on A. mellifera
honeybee colonies infested with the V. destructor and T. clareae. Keeping in view the
importance of safe and non-contaminated methods to suppress mite populations in
beehives and to escape from resistance problem, different organic compounds and
essential oils in various concentrations were applied in laboratory and field conditions.
The results are summarized as under:
All the experiments were maintained using modified bottom board trays
(mechanical control) and maintaining test colonies with regular re-queening with
hygienic queens (genetic control). For the first experiment 3 groups of 4 honeybee
colonies were used. Group 1 was tested with finely grinded thymol and the second
group received formic acid. Group 1 received four treatments (4 gm) with a weekly
interval, testing a total amount of 16gm thymol crystals placed in Petri dishes (80 mm
dia) on top of the brood frame under the top cover of hives. Second group received 4
treatments of 65% formic acid (20ml each) applied on card board placed in the mite
collection trays put in the deep bottom board of the hive. Total 80 ml formic acid was
applied at weekly interval for 28 days. The third group was served as control and the
rate of T. clareae infestation, treatment efficacy was estimated
Honey yield of treated colonies was compared. The results showed that formic
acid killed significantly higher number of mites (315-450) and average honey yield was
higher (14.33 kg) as compared to thymol and control.
In the second experiment the effects of oxalic acid (OA) in different
concentrations in reducing V. destructor populations in honeybee colonies were
determined. Twenty honeybee colonies were used in this experiment. Colonies were
93

94
divided into 4 groups of 5 colonies each. The OA with different concentrations i.e. 4.2,
3.2 and 2.1 % was applied thrice on different dates. Oxalic acid was applied in sugar
syrup. The 5 ml solution was applied by tricking over adults honeybee in two frames at
a time using a syringe.
As shown in the results high efficacy of OA is critical to its concentrations.
Average efficacy of OA with 3.2, 4.2 and 2.1 % was 95, 81 and 46 % respectively.
Experiments created that success by retaining the Queen, with no loss among brood and
honey bees. The honey produced was also found maximum (23 kg) for 3.2 % OA.
In the third experiment the effectiveness of 3.2% oxalic acid with 2, 4 and 6 gm
of thymol was used to control mite infestation among brood less condition. Colonies
were divided into 4 groups of 5 colonies each. One group was treated with 2gm finely
grinded thymol plus 3.2% OA, the second group received 4gm finely grinded thymol
plus 3.2% OA, the third group was treated with 6gm finely grinded thymol plus 3.2%
OA and the fourth group served as control group (C). All groups received three
treatments with a weekly interval.
Average efficacy of 2, 4 and 6 gm thymol with 3.2% OA for controlling T.
clareae was 26, 40 and 35 and for V. destructor was 93, 99 and 94% respectively. The
results clearly showed that the 3.2% OA with 4gm thymol was the best treatment for
controlling these mites. The honey yield was maximum (21 kg) in colonies treated with
3.2 % OA + 4gm thymol.
In the fourth experiment the acaricidal effects of some plant oils i.e. clove,
neem, garlic and olive along with tobacco extract used alone and in combinations for
controlling the Varroa mite in lab. and field were evaluated. In lab. 40 micro liters of 5-
15 % solution of plant oil thinned in methanol was applied to mites placed in Petri

95
dishes. In the control dishes only distilled water was poured on the filter paper. The
results in the lab showed that clove oil and tobacco extract both proved to be equally
effective against mites. We did not observe any bee mortality because of these oils. The
treatments were significantly effective when applied in 5% as compared to 10 and 15 %
concentrations. It was also determined that oils/extracts mostly killed the mites during
first 24 hrs.
In the second part of this experiment using only 5% concentration for 24 hrs the
most effective combination was clove oil and tobacco extract. The field experiment
with all the oils/extracts individually and in all the previously tested combinations
confirmed the lab results as clove + tobacco extract the best combination with 96.48%
efficacy.
In view of the findings of above studied, an integrated management approach
based upon regular requeening, using of modified bottom board trays and periodic
applications of various miticides was carried out to determine the effects of three
different treatments i.e. 4gm thymol + 3.2% OA and 65% formic acid (T1), 5% clove
oil + Tobacco extract and 4gm thymol+3.2% OA (T2) and 5% clove oil + Tobacco
extract and 65 % formic acid (T3) on both ectoparasitic mites in honeybee colonies
round the year. Average efficacy was calculated and it was found that T1 had the
highest efficacy for both the mites. The total honey production harvested from colonies
treated with different acaricides was also determined and significantly more amount of
honey (30 kg) was produced from the hives treated with 4gm thymol + 3.2% OA and
65% formic acid.
Conclusively, it is said that all the treatments used i.e. thymol, formic acid,
oxalic acid and plant oils/ extract are effective in controlling the honeybee mites on

96
adult bees and brood of honeybees. So it was concluded that tested miticides and
essential oils/extracts can provide beekeepers an effective and safer solution to control
mites in bee colonies, which ultimately help to boost the production of high quality
honey.
In fact, organic acids and plant essential oils do have several important benefits.
Due to their volatile nature, there is a much lower level of risk to the environment than
with current synthetic pesticides. Predator, parasitoid and pollinator insect populations
will be less impacted because of the minimal residual activity, making these acid
compatible with integrated pest management programs. It is also obvious that resistance
will develop more slowly to organic /essential-oil based pesticides owing to the
complex mixtures of constituents. Ultimately, it is in developing countries where the
source plants are endemic that these pesticides may ultimately have their greatest
impact in integrated pest management strategy.

97
CONCLUSION
In first experiment average honey yield was increased by 10 kg/colony treated
with 65% formic acid as compared to control. In second experiment average honey
yield was increased by 19 kg/colony treated with 3.2% oxalic acid as compared to
control. In experiment 3 average honey yield was increased by 15 kg/colony treated
with 4gm thymol + 3.2% oxalic acid as compared to control. In fourth experiment
average honey yield was increased by 14 kg/colony treated with 5% Clove oil +
tobacco extracts as compared to control. In the fifth experiment average honey
harvested was 30 kg but honey yield increased by 7 kg /colony treated with 4gm
thymol+3.2% oxalic acid in winter and 65% formic acid in summer as compared to
other two groups.
It is concluded from integrated mite management trial that 4gm thymol + 3.2%
oxalic acid and 65% formic acid applied through out the year along with maintaining
regular re-queening and use of modified bottom board trays have shown 2 times in T.
clareae and 4 times in V. destructor best results on mite mortality. Average honey yield
increased up to 30 kg in these treated colonies.
RECOMMENDATIONS
Based on research findings, it is recommended that regular re-queening with
hygienic queen, use of modified bottom boards along with three applications of 4 gm
thymol+3.2% Oxalic acid in mid winter at weekly intervals, two application of 65%
formic acid in mid summer fortnightly and apistan strips should be hanged in late
summer for a period of one month for successfully controlling both ectoparasitic mites
(Tropilaelaps clareae and Varroa destructor) of Apis mellifera and for the production
of quality honey yield.
97

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APPENDICES
Experiment 1 Effect of Thymol and Formic acid on mite mortality and honey yield
Treatment
Thymol
65%
Control
Bee Mortality of Tropilaelaps clareae Mites Total No. Efficacy Honey
hive
killed by of mites (%) Yield
#. Week Week Week Week Total Apistan mortality ( Kg)
1 2 3 4
1 50 75 90 75 290 180 470 61.7 11.56
2 53 72 98 37 260 162 422 61.61 11.89
3 61 82 105 69 317 193 510 62.15 12.55
4 47 69 80 46 244 156 398 60.5 11.24
2 71 81 110 53 315 85 400 78.59 15.09
3 97 111 129 82 419 111 530 79.05 14.01
Formic acid 1 75 89 125 33 322 93 415 77.59 13.12
4 100 121 142 87 450 93 543 82.87 15..11
1 12 14 14 12 52 259 311 16.72 5.15
2 14 12 12 11 49 275 324 17.82 4.5
3 12 12 11 12 47 289 336 16.26 5.75
4 12 12 13 12 49 288 337 17.01 6.15

Experiment 1
128
Completely Randomized ANOVA for Tropilaelaps clareae mortality
Source DF SS MS F P
Treatment 2 225409 112704 59.3 0.0000
Error 9 17119 1902
Total 11 242527
CV 18.60
Completely Randomized ANOVA for Efficacy of thymol
and formic acid
Source DF SS MS F P
Treatment 2 8298.89 4149.45 1987 0.0000
Error 9 18.80 2.09
Total 11 8317.69
CV 2.74
Completely Randomized ANOVA for Honey yield
Source DF SS MS F P
Treatment 2 170.166 85.0830 146 0.0000
Error 9 5.251 0.5834
Total 11 175.417
CV 7.27

129
Experiment 2. Effect of different concentrations of Oxalic acid on mite mortality
and honey yield
Treatment Bee hive
#
4.2% OA
3.2% OA
2.1% OA
Control
Mortality of Varroa destructor Mites killed Total No.
Apistan of
Week
1
Week
2
Week
3
Total
mite
mites killed
Efficacy
(%)
1 490 372 130 992 207 1199 82.74 14
2 501 399 114 1014 231 1245 81.45 14
3 519 401 137 1057 235 1292 81.81 15
4 485 351 95 931 264 1195 77.91 16
5 544 427 152 1123 267 1390 80.79 14
1 666 475 207 1348 52 1400 96.29 22
2 601 402 196 1199 81 1280 93.67 23
3 619 411 216 1246 84 1330 93.68 24
4 612 401 216 1229 71 1300 94.54 24
5 599 397 192 1188 71 1259 94.36 22
1 114 70 41 225 255 480 46.86 9
2 101 65 34 200 275 475 42.11 10
3 109 67 42 218 237 455 47.91 8
4 111 69 42 222 257 479 46.35 9
5 117 62 35 214 246 460 46.52 9
1 35 28 17 80 300 380 21.05 3
2 20 18 11 49 226 275 17.82 4
3 27 22 18 67 233 300 22.33 4
4 32 24 23 79 266 345 22.9 3
5 23 18 17 58 186 244 23.77 3
Honey
yield (Kg)

Experiment 2
130
Completely Randomized ANOVA for varroa mortality
Source DF SS MS F P
Treatment 3 5090478 1696826 715 0.0000
Error 16 37945 2372
Total 19 5128423
CV 7.65
Completely Randomized ANOVA for Efficacy of oxalic acid
Source DF SS MS F P
Treatment 3 16505.2 5501.73 1480 0.0000
Error 16 59.5 3.72
Total 19 16564.7
CV 3.17
Completely Randomized ANOVA for Honey yield
Source DF SS MS F P
Treatment 3 1048.60 349.533 538 0.0000
Error 16 10.40 0.650
Total 19 1059.00
CV 6.45

Treat. Bee
hive
#
1 15 520 12 410 12 192 39 1122 100 84 139 1206 28.06 93.03 10
2gm Thymol+
3.2% OA
1 19 770 20 534 23 235 62 1539 100 15 162 1554 38.27 99.03 21
4gm Thymol+
3.2% OA
1 16 612 19 499 19 200 54 1311 90 89 144 1400 37.50 93.64 12
6gm Thymol+
3.2% OA
Control
Experiment 3.
131
Effect of Thymol and Oxalic acid on mite mortality and honey yield
Mortality of mites Total No. of
mite mortality
Week 1 Week 2 Week3
Mites killed byTotal
number of mEfficacy
Apistan killed (%)
Tc Vd Tc Vd Tc Vd Tc Vd Tc Vd Tc Vd Tc Vd
2 20 488 18 387 12 193 50 1068 139 90 189 1158 26.46 92.23 9
3 21 480 14 410 10 188 45 1078 130 86 175 1164 25.71 92.61 11
4 22 650 16 370 11 201 49 1221 141 78 190 1299 25.79 94.00 10
5 17 500 12 389 11 186 40 1075 114 64 154 1139 25.97 94.38 10
2 18 789 18 546 19 238 55 1573 95 14 150 1587 36.67 99.12 22
3 38 712 22 541 20 234 80 1487 87 21 167 1508 47.90 98.61 20
4 18 760 18 550 21 280 57 1590 76 21 133 1611 42.86 98.70 21
5 21 711 20 635 18 267 59 1613 112 21 171 1634 34.50 98.71 21
2 15 600 16 455 17 180 48 1235 98 82 146 1317 32.88 93.77 13
3 18 590 18 499 15 177 51 1266 112 73 163 1339 31.29 94.55 11
4 20 600 15 356 16 168 51 1124 97 90 148 1214 34.46 92.59 12
5 28 701 16 345 17 170 61 1216 99 60 160 1276 38.13 95.30 12
1 13 35 9 19 9 15 31 69 312 234 343 303 9.04 22.77 6
2 10 14 9 12 9 12 28 38 300 176 328 214 8.54 17.76 5
3 9 18 10 14 10 14 29 46 368 247 397 293 7.30 15.70 6
4 8 16 8 15 10 12 26 43 299 198 325 241 8.00 17.84 5
5 8 17 9 13 11 13 28 43 267 245 295 288 9.49 14.93 6
Honey
Yield
(kg)

Experiment 3
132
Completely Randomized ANOVA for Tropilaelaps clareae mortality
Source DF SS MS F P
Treatment 3 3154.95 1051.65 27.2 0.0000
Error 16 617.60 38.60
Total 19 3772.55
CV 13.18
Completely Randomized ANOVA for varroa mortality
Source DF SS MS F P
Treatment 3 6429753 2143251 746 0.0000
Error 16 45994 2875
Total 19 6475747
CV 5.43
Completely Randomized ANOVA for efficacy of Tropilaelaps
Source DF SS MS F P
Treatment 3 2872.46 957.488 98.1 0.0000
Error 16 156.15 9.760
Total 19 3028.62
CV 11.38
Completely Randomized ANOVA for efficacy of varroa
Source DF SS MS F P
Treatment 3 22645.5 7548.52 2681 0.0000
Error 16 45.1 2.82
Total 19 22690.6
CV 2.21
Completely Randomized ANOVA for Honey Yield
Source DF SS MS F P
Treatment 3 629.350 209.783 466 0.0000
Error 16 7.200 0.450
Total 19 636.550
CV 5.52

133
Experiment 4. Effect of plant oils and extract on Varroa destructor mortality
Neem Oil
Garlic Oil
Clove Oil
Treatment Bee
5
days
Hive #
Tobacco extract
Olive
Neem+garl. Oil
Neem+clove Oil
Neem Oil +
tobacco extract
Neem+olive oil
Garl+clove oil
Garlic oil +
tobacco extract
Garlic + olive oil
Clove oil
+tobacco extracts
Clov+ olive oil
Tobacco extract
+olive oil
Control
10
days
15
days
20
days
Varroa
Mite
Mites killedTotal
Apistan No. of
mite
Efficacy
(%)
Honey
yield (kg)
1 89 78 76 56 299 34 333 89.79 12
2 80 78 70 67 295 46 341 86.51 13
3 65 66 60 50 241 61 302 79.80 11.9
1 56 57 23 33 169 45 214 78.97 14.5
2 78 43 46 34 201 21 222 90.54 15
3 89 69 43 33 234 15 249 93.98 15
1 60 69 56 34 219 23 242 90.50 16.5
2 70 45 34 41 190 15 205 92.68 17
3 78 45 34 33 190 45 235 80.85 16.1
1 60 45 34 21 160 34 194 82.47 14
2 56 45 44 34 179 23 202 88.61 15
3 69 56 44 33 202 33 235 85.96 14.7
1 65 75 33 22 195 22 217 89.86 14
2 69 56 55 44 224 33 257 87.16 13
3 89 76 44 33 242 45 287 84.32 12.7
1 68 34 44 33 179 43 222 80.63 14.7
2 78 23 22 23 146 13 159 91.82 16
3 56 55 34 33 178 24 202 88.12 15
1 67 34 23 12 136 54 190 71.58 14.4
2 77 45 45 34 201 23 224 89.73 16
3 77 32 33 22 164 34 198 82.83 15
1 80 45 43 22 190 22 212 89.62 18
2 67 45 45 23 180 22 202 89.11 18
3 68 65 45 14 192 34 226 84.96 17.4
1 58 46 45 34 183 23 206 88.83 14
2 78 65 55 23 221 45 266 83.08 12.4
3 76 45 67 76 264 45 309 85.44 13
1 69 56 56 55 236 34 270 87.41 12.6
2 78 34 47 44 203 34 237 85.65 13
3 76 23 23 45 167 44 211 79.15 12
1 66 23 27 57 173 54 227 76.21 12
2 68 23 33 22 146 34 180 81.11 12
3 87 23 45 33 188 39 227 82.82 13
1 65 56 55 55 231 34 265 87.17 14
2 66 34 33 44 177 33 210 84.29 12.5
3 69 45 47 23 184 45 229 80.35 12.5
1 99 98 102 123 422 12 434 97.24 21
2 98 99 90 98 385 13 398 96.73 20.5
3 89 80 80 89 338 16 354 95.48 20
1 52 54 23 23 152 21 173 87.86 16
2 67 66 67 88 288 34 322 89.44 16.9
3 66 65 55 55 241 45 286 84.27 15.5
1 67 54 44 56 221 44 265 83.40 14
2 86 43 34 33 196 23 219 89.50 15
3 88 43 56 45 232 34 266 87.22 14.5
1 34 12 11 23 80 235 315 25.40 7
2 23 12 23 11 69 223 292 23.63 6
3 11 19 32 12 74 249 323 22.91 5.7

Experiment 4
134
Completely Randomized ANOVA for varroa mortality
Source DF SS MS F P
Treatment 15 179930 11995.4 12.1 0.0000
Error 32 31820 994.4
Total 47 211750
CV 15.32
Completely Randomized ANOVA for efficacy of plant oil/extracts
Source DF SS MS F P
Treatment 15 11486.1 795.742 36.3 0.0000
Error 32 674.6 21.080
Total 47 121.60.7
CV 5.57
Completely Randomized ANOVA for honey yield
Source DF SS MS F P
Treatment 15 423.627 28.2418 74.4 0.0000
Error 32 12.153 0.3798
Total 47 435.780
CV 4.32

135
Experiment 5. Effect for Integrated Management on mite mortality and honey yield
Treatment 1
4g thymol+3.2% 65% formic
Bee Oxalic acid
Acid
Total
mite
hive
mortality
#
Mites Total
killed by number
Efficacy(%)
Apistan of Mite
mortality
Honey
24-11-10 1-12-10 8-12-10 6-7-11 22-7- 11
yield
29-7-11
(Kg)
Tc Vd Tc Vd Tc Vd Tc Vd Tc Vd Tc Vd Tc Vd Tc Vd Tc Vd
1 20 310 15 214 14 120 42 31 31 30 122 705 17 15 139 720 87.77 97.92 29
2 18 315 17 217 12 125 44 30 29 28 120 715 20 17 140 732 85.71 97.68 28
3 24 319 20 219 10 119 45 29 28 29 127 715 22 16 149 731 85.23 97.81 30
4 24 324 16 222 12 117 40 31 29 30 121 724 21 17 142 741 85.21 97.71 29
5 22 321 14 229 11 121 41 32 30 31 118 734 21 19 139 753 84.89 97.48 29
6 21 334 12 213 14 121 41 32 31 30 119 730 19 13 138 743 86.23 98.25 31
7 23 321 11 222 12 111 42 33 33 29 121 716 21 18 142 734 85.21 97.55 32
8 24 312 12 234 15 123 43 31 29 29 123 729 22 19 145 748 84.83 97.46 28
9 22 322 14 212 12 124 39 30 26 31 113 719 16 16 129 735 87.60 97.82 29
10 21 333 16 234 11 125 44 33 32 31 124 756 18 17 142 773 87.32 97.80 33
Treatment 2
Bee 5%clove oil+
4g Thymol+ Total mite Mites Total Efficacy Honey
hive Tobaaco extract 3.2% OA mortality killed by number (%) Yield
#
Apistan of mite
(Kg.)
2-3-10 9-3-10 16-3-10 28-12-10 14-1-11 21-1-11 mortality
1
Tc Vd Tc Vd Tc Vd Tc Vd Tc Vd Tc Vd Tc Vd Tc Vd Tc Vd
16 25 11 25 21 67 11 47 11 21 70 185 15 14 85 199 82.35 92.96 23
2 21 20 11 22 13 56 10 49 12 22 67 169 16 17 83 186 80.72 90.86 23
3 15 22 10 20 12 68 24 50 13 19 74 179 17 16 91 195 81.32 91.79 23
4 13 22 16 21 11 69 12 49 12 19 64 180 17 15 81 195 79.01 92.31 24
5 14 24 13 23 12 55 11 48 12 20 62 170 17 16 79 186 78.48 91.40 23
6 11 26 16 24 12 54 11 45 11 16 61 165 14 14 75 179 81.33 92.18 22
7 19 26 13 26 21 56 15 47 11 15 79 170 16 15 95 185 83.16 91.89 21
8 16 23 15 25 14 66 12 51 5 17 62 182 17 13 79 195 78.48 93.33 23
9 17 26 12 28 13 59 21 47 5 18 68 178 12 15 80 193 85.00 92.23 24
10 11 25 16 22 11 62 16 43 8 20 62 172 15 16 77 188 80.52 91.49 21

Treatment 3
Bee 5% clove oil+
65% Formic Total mite Mites Total Efficacy
HiveTobacco
extract acid
mortality killed by number (%)
#
Apistan of mite
6-7-10 13-7-10 20-7-10 01-03-11 17-03-11 24-03-11 mortality
Tc Vd Tc Vd Tc Vd Tc Vd Tc Vd Tc Vd Tc Vd Tc Vd Tc Vd
136
1 24 45 24 75 10 30 15 30 12 16 85 196 30 40 115 236 73.91 83.05 23
2 23 47 27 75 15 32 16 31 10 17 91 202 29 42 120 244 75.83 82.79 22
3 27 46 26 74 14 35 16 32 11 16 94 203 30 42 124 245 75.81 82.86 20
4 23 45 22 70 12 30 15 33 10 15 82 193 27 46 109 239 75.23 80.75 20
5 29 50 21 73 12 37 16 32 10 16 88 208 29 45 117 253 75.21 82.21 22
6 22 45 23 56 11 34 11 29 13 13 80 177 24 42 104 219 76.92 80.82 22
7 26 46 23 65 16 33 17 24 11 15 93 183 44 29 137 212 67.88 86.32 23
8 31 51 28 66 17 39 13 34 11 16 100 206 23 43 123 249 81.30 82.73 24
9 28 44 30 71 13 31 12 31 14 14 97 191 44 41 141 232 68.79 82.33 22
10 24 44 21 69 12 32 12 33 10 13 79 191 16 34 95 225 83.16 84.89 21
Honey
Yield
(Kg.)

Experiment 5
137
Completely Randomized ANOVA for Tropilaelaps mortality
Source DF SS MS F P
Treatment 2 14689.4 7344.70 214 0.0000
Error 27 927.4 34.35
Total 29 15616.8
CV 6.36
Completely Randomized ANOVA for varroa mortality
Source DF SS MS F P
Treatment 2 1940963 970482 8458 0.0000
Error 27 3098 115
Total 29 1944061
CV 2.94
Completely Randomized ANOVA for efficacy on Tropilaelaps
Source DF SS MS F P
Treatment 2 562.124 281.062 30.1 0.0000
Error 27 251.888 9.329
Total 29 814.012
CV 3.78
Completely Randomized ANOVA for efficacy on varroa
Source DF SS MS F P
Treatment 2 1126.04 563.021 494 0.0000
Error 27 30.79 1.140
Total 29 1156.83
CV 1.17
Completely Randomized ANOVA for Honey yield
Source DF SS MS F P
Treatment 2 378.200 189.100 101 0.0000
Error 27 50.600 1.874
Total 29 428.800
CV 5.52

138
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