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Dedicated to
My Parents
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CONTENTS
Chapter Description Page(s)
I. INTRODUCTION 1-5
II. REVIEW OF LITERATURE 6-18
III. MATERIALS AND METHODS 19-25
IV. RESULTS 26-37
V. DISCUSSION 38-44
VI. SUMMARY AND
CONCLUSION
45
LITERATURE CITED I-XIV
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LIST OF TABLES
TABLE
NO.
PARTICULARS
1. Incidence ofVarroa destructoron Apis melliferabrood (May,2008 to April, 2009)
2. Effect of Varroa destructor incidence on perforated broodcells
3. Efficacy of Screen floor against Varroa destructor in Apismelliferacolonies
4. Effect of Screen floor on colony strength and stores in Apismelliferacolonies
5. Efficacy of Formic acid against Varroa destructor in Apis
melliferacolonies
6. Effect of Formic acid on colony strength and stores in Apismelliferacolonies
7. Efficacy of Powdered sugar (2g/frame) against Varroadestructorin Apis melliferacolonies
8. Effect of Powdered sugar (2g/frame) on colony strength andstores in Apis melliferacolonies
9. Efficacy of Powdered sugar (3g/frame) against Varroa
destructorin Apis melliferacolonies
10. Effect of Powdered sugar (3g/ frame) on colonystrength and stores in Apis melliferacolonies
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CHAPTER-I
INTRODUCTION
Apiculture forms an essential and vital component of sustainable
integrated rural development programme as it improves the economy of
farmers by enhancing the productivity of agricultural crops and honey
production. Despite its great potential, beekeeping industry is facing
several constraints, which needs immediate attention. Among these,
Varroa destructorAnderson and Trueman, an ectoparasitic mite of brood
and adult bees, is a serious pest of Apis melliferaL. It belongs to order
mesostigmata and family varroidae. Varroamite has been found on flower
feeding-insects Bombus pennsylvanicus (Hymenoptera: Apidae), Palpada
vinetorum (Diptera: Syrphidae), and Phanaeus vindex (Coleoptera:
Scarabaeidae) (Kevan et al., 1990). Although the Varroa mite cannot
reproduce on other insects, its presence on them may be a means by
which it spreads short distances. Among the bees that serve as hosts of
the Varroamite are Apis cerana, A. koshchevnikovi, A. mellifera mellifera,
A. m. capenis, A. m. carnica, A. m. iberica, A. m. intermissa, A. m. ligustica,
A. m. macedonica, A. m. meda, A. m. scutellata, and A. m. syriaca.
Varroa was first described on its native host, the Asian honey bee
(Apis ceranaFab.) in 1904 in Java (Oudemans, 1904). The mite, Varroa
jacobsonibegan its parasitic relationship with Asian honey bee by laying
eggs (up to six eggs) in drone brood cell. The patterns of speciation among
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the mites are mirrored in the patterns of speciation among the mites
native host A. cerana. This infers that mite and bees have been co-
evolving (Abrol, 2009). During this time the bees have developed several
behavioural traits to mitigate the harmful effect of mites. Some of these
traits such as tendency to swarm and willingness to abandon their hives
may have effectively countered the mite, but these traits also suggested
difficulty in domestication of this species.
To overcome these problems, the more reliable and productive bee
species Apis melliferawas introduced into Asia thirty five years ago. Not
long after this bees introduction Varroamite jumped hosts. Mite infested
A. mellifera was subsequently transported around the world via
quarantine incursions and normal practice of shipping live bees between
countries (Anonymous, 2006). Unfortunately, Varroa mite proved more
virulent to its new host and subsequent research revealed that genus
Varroaconsists of at least four but possibly seven distinct species (Munoz
et al., 2008; Abrol, 2009). Among the four recognized species, the most
destructive and largest among these is Varroa destructor (Anderson andTrueman, 2000) which is 1.1 mm long and 1.7 mm broad. The second is
V. jacobsoni (Oudemans, 1904)which is smalller than V. destructor (1.0
mm long and 1.5 mm wide) followed by V. rindereri (De Guzman and
Delfinado, 1996). V. underwoodi(Delfinado-Baker and Aggarwal, 1987) is
the smallest among these four species, female of which is 0.7 mm long
and 1.1 mm wide. These species are morphologically distinct and show
clear differences in their mitochondrial DNA sequences. They are
reproductively isolated and show differences in their host specificity and
geographical distribution (Anderson and Trueman, 2000). In this sense,
A. koshchevnikovi is parasitized by V. rindereri and A. cerana by V.
underwoodi, V. jacobsoniand V. destructorin Asia but A. melliferais only
parasitized by V. destructorworldwide.Several mitochondrial haplotypes
(17-18) of V. destructor have been described but only two of them are
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capable of reproducing on A. mellifera. These are the Korean (K) and
Japanese (J) haplotypes (Anderson and Trueman, 2000) which vary in
their virulence toward A. mellifera, with K type assumed to be more
virulent (De Guzman et al., 1997; 1999; Anderson and Trueman, 2000).
The K type infests A. mellifera worldwide but J type has only been
observed in Japan, America and Thailand (De Guzman et al., 1997;
Anderson and Trueman, 2000; Garrido et al., 2003).
V. destructor has spread all over the world except Australia and
central Africa causing severe losses of feral honeybee populations in USA
and worldwide (Kraus and Page, 1995; Llorente, 2003). In India, Varroa
was first reported on A. ceranafrom Delhi (Phadke et al., 1966) and later
from A. mellifera colonies from Himachal Pradesh (Kumar et al., 1988)
and Haryana (Sihag, 1988). It is reported to cause 30-40 per cent loss in
A. mellifera colonies (Anonymous, 2006). It has ravaged A. mellifera
colonies from Jammu and Kashmir, Himachal Pradesh, Punjab, Haryana,
Delhi, Rajasthan, Uttar Pradesh and Uttaranchal and is fast approaching
Bihar and West Bengal (Chhuneja, 2008). In the last three years,beekeeping in Haryana is adversely affected by this mite. In the present
scenario, 90 per cent apiaries and 50 per cent colonies are affected by
this mite (Gulati et al., 2009).
V. destructor feed upon haemolymph of adult and immature bees
during phoretic and reproductive life stages. It generally lives for seven days
to thirteen days on adult bees (Schulz, 1984). V. destructor has direct
impact on developing and adult bees, resulting in lowered body weights (De
Jong et al., 1982a; Bowen-Walker and Gunn, 2001) and reduced longevity
(De Jong and De Jong 1983; Kovac and Crailsheim, 1988). These impacts
translate into both lowered productivity and higher mortality at the colony
level (Murilhas, 2002). In addition to colony loss, reduced honey production
and a decrease in pollination efficiency as a consequence ofV. destructor
parasitism has also been observed (Calderon et al., 2007). V. destructor is
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also known to be associated with honeybee pathogens and are confirmed to
be vectors of diseases. Several experimental studies indicate that mites
transfer single stranded RNA viruses between bees such as to transmit
Hafnia alvei and Serratia marcescens, which cause Haffnosis and
Septicemia, respectively, in bees (Glinski and Jarosz, 1992). Varroa mites
are considered as most serious since they are threatening the survival of
Apisspecies in many regions of the world by feeding on larva, pupa or adult.
The potentiality of its damage can be imagined by its number in a bee hive.
Their number may reach up to 10,000 mites/ A. mellifera hive, 5 mites/
worker bee, 12 mites/ drone bee, 12 mites/ worker brood and 20 mites/
drone brood (Sharma, 2003). Grobov (1976) provided the figures on the
rapidity of the spread of the mite over short distance. The mites from
infested colony at a particular place were found in colonies 6-7 km away
from initial host colony after three months.
Several control measures are reported in literature which include use
of organic acids (formic acid, oxalic acid and lactic acid), chemicals
(Fluvalinate, Flumethrin, Amitraz, Cymiazole, Coumaphos,Bromoprophylate) and many vegetable oils. Formic acid (65%) at the rate of
300 ml is 95 per cent effective against mites (Calderone, 2000). Oxalic acid
(2-3%) when applied as spraying or trickling in the form of sugar solution
was 90 per cent effective (Charriere and Imdorf, 2002). Synthetic chemicals,
although most effective and reliable as they provide immediate relief but
cannot be used in organic honey production because of high residue levels
in honey (Kumari et al., 2003; Gulati and Kumari, 2005) and problem of
development of resistance in Varroa(Logelio and Plebani, 1992; Colin et al.,
1997). Therefore, attention is diverted for other alternatives (Gerson et al.,
1991) such as destruction of drone brood, caging of queen, use of
botanicals, biocontrol agents etc. In Haryana, as reported earlier, it has
come in devastated form causing severe losses to beekeepers. Systematic
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studies on its incidence and management are lacking from this area. In this
retrospective, present study was conducted with following objectives:
1. To study effect of mite incidence on brood2. To evaluate different control measures against Varroa destructor
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CHAPTER-II
REVIEW OF LITERATURE
Apiculture is a non-land based income generating activity and an
important component of sustainable integrated rural developmental
program. It has always been an important part of livelihood, cultural,
religious and natural heritage of rural communities of India and also
provides free ecosystem services in the form of cross pollination by
enhancing the productivity of agricultural crops and conservation of wild
flora. All beekeeping of India was based entirely on Apis ceranaup to 1983,
after that A. melliferawas introduced in India and it has almost completely
replaced A. ceranaand that brought sweet revolution in northern India.
But, A. mellifera is now facing its most dreaded enemy i.e. V. destructor,
which has caused severe losses to beekeeping industry of India. In this
chapter, an attempt has been made to review the information available onits host range, distribution, biology, symptoms of damage, pest potential
and management. All the information is complied in systematic form in this
chapter for ready reference.
2.1 Range and Species distribution ofVarroaIn 1904 in Java, Indonesia, Oudemans described the Varroa jacobsoni
mite in brood cells of drone larvae ofA. cerana(Oudemans, 1904). The mite
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was later found in European honey bee colonies in Japan (Tanabe and
Tamaki, 1986). Among the bees that serve as hosts of the varroa mite are
Apis cerana, A. koschevnikovi, A. mellifera mellifera, A. m. capenis, A. m.
carnica, A. m. iberica, A. m. intermissa, A. m. ligustica, A. m. macedonica, A.
m. meda, A. m. scutellata, and A. m. syriaca. (Anonymous, 2006). Varroa
mites have also been found on the wasps, flower-feeding insects Bombus
pennsylvanicus (Hymenoptera: Apidae), Palpada vinetorum (Diptera:
Syrphidae), and on Phanaeus vindex (Coleoptera: Scarabaeidae) (Kevan et
al., 1990).Although Varroacan only reproduce on honeybees, these insects
are a means of spreading the mite short distances. The Asian honey bee, A.
cerana, is less susceptible to this mite compared to A. mellifera. Mites can
attack only a few A. ceranabees as the adult workers kill and remove most
of them from the brood (Bailey and Ball, 1991). Secondly, A. mellifera is
preferred over A. cerana by Varroa because their hives temperature is
nearer to Varroaspreference than that of Asian bees (Le Conte and Arnold,
1988). Their haemolymph contains a greater quantity of juvenile hormone,
favourable to parasites development (Faucon and Fleche-Seben, 1988) andthey rear more drones than A. cerana(Noirot, 1988).
Studies have shown that Varroa consists of four species which are
morphologically distinct. These are Varroa destructor (most destructive and
largest species) (Anderson and Trueman, 2000), Varroa jacobsoni
(Oudemans, 1904), V. rindereri (De Guzman and Delfinado, 1996) and V.
underwoodi (Delfinado-Baker and Aggarwal, 1987). Studies made on the
genotypic, phenotypic and reproductive variation showed that Varroaforms
a complex of 18 different genetic variants that belong to different species.
Among these, V. jacobsoni infests A. cerana and V. destructor infests A.
mellifera.
Anderson and Trueman (2000) studied mt DNA Co-I gene sequences
of 18 haplotypes, out of whivh 9 haplotypes of V. jacobsoni has been
described that infest A. cerana in Malasia-Indonasia region including the
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java haplotype whereas 6 haplotypes of V. destructor infest A. mellifera
worldwide. Among these are Korean and Japan/Thailand haplotypes which
are more virulent than others. In India, Korean haplotype has been found to
infest A. mellifera(Chhuneja, 2008).
2.2 Worldwide Distribution ofVarroa
The Varroa mite has been found on honey bees in Asia, Europe,
America, New Zealand, North and South Africa. The mite has spread
worldwide in the last few decades due to the commercial transport of bees,
the migratory activities of beekeepers, drifting bees and swarms that may fly
long distances (Sumpter and Martin, 2004). The mite was initially observed
in Indonesia (Oudemans, 1904). Its occurrence has since been reported in
Singapore (Gunther, 1951), USSR (Breguetova, 1953), Hong Kong
(Delfinado, 1963), Philippines (Delfinado, 1963), The Peoples Republic of
China (Tzien, 1965), India (Phadke et al., 1966), North Korea (Tian, 1967),
Cambodia (Ehara, 1968), Japan (Ehara,1968), Vietnam (Stephen, 1968),
Thailand (Laigo and Morse, 1969), Czechoslovakia (Samsinak and Haragsim,
1972), Bulgaria (Velitchkov and Natchev, 1973), South Korea (Delfinado andBaker, 1974), Paraguay (Orosi, 1975), Romania (Orosi, 1975), Taiwan
(Akratanakul and Burgett, 1975), Argentina (Montiel and Piola, 1976),
Poland (Koivulehto, 1976), Uruguay (Grobov, 1976), Germany (Ruttner,
1977), Bangladesh (Marin, 1978), Myanmar (Marin, 1978), Brazil (Alves et
al., 1975), Hungary (Buza, 1978), Tunisia (Hicheri, 1978), Greece (Santas,
1979), Yugoslavia (Santas, 1979), Iran (Crane, 1979), Libya (Crane, 1979),
Turkey (Crane, 1979), Lebanon (Popa, 1980), USA (Wenner and Bushing,
1996), South Africa (Allsopp et al., 1997), New Zealand (Anonymous, 2002a)
and Hawaii (Anonymous, 2007). The only regions of the world where the
Varroamite has not yet spread to are Australia andthe central part of Africa
(Oldroyd, 1999).
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2.3 Causes of Rapid Spread ofVarroa
V. destructorhas now entered India and is now spreading in whole of
the country. It is very mobile and readily transfers between adult bees and
hence spread throughout colony and then between colonies and apiaries
when hive components (hive tool, gloves etc.), infested brood and adult bees
are interchanged during normal management apiary practices (Bessin,
2001). In India, the spread is fast over long distances because of the
migratory nature of the beekeeping industry. It has been known that that
mites are capable of transferring from one host to another during summer
robbing (Sakofski, 1980), during inter-colony drifting of workers and drones
(Sakofski and Koeniger, 1986), from drones to queens during mating (De
Jong et al., 1982b), from adult bees on to brood (Kovac and Crailsheim,
1988) and from newly emerged bees on to older bees (Kuenen and
Calderone, 1997).Moreover, importation of queen bees from infested areas
(Denmark et al., 2000) and transportation of infested bee colonies for
pollination led to the rapid spread of this mite in developed countries
(Anonymous, 1997) and is a major threat to the region where the mite is notyet present.
2.4 Losses and Symptoms Associated with Varroa
V. destructor can cause widespread losses in colonies in relatively
short periods of time. Due to weakening caused by the constant feeding of
the Varroamite on the haemolymph of developing honey bee larvae, pupae
and adults, a bee colony once attacked by the mite is at risk of being lost
within 3 to 4 years if left untreated(De Jong et al., 1982a).
Its symptoms include large numbers of unsealed brood cells, dead or
dying newly emerged bees with malformed wings, legs, abdomen, and thorax
at the entrance of affected colonies (De Jong, 1990; Duay et al., 2003).
Severe V. destructor in Apis melliferacolony causes 40 (Llorente, 2003) to
100 per cent loss within three years of initial infestation (Ritter et al., 1983),
resulting in decline feral bee population (Harbo and Hoopingarner, 1997)
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and managed colonies (Sammataro, 1997). Loss in pollination efficacy,
honey production due to V. destructor infestation is also reported
(Sammataro, 1997).
Physiological Effects caused by Varroa include loss in weight,
reduction in size, shortening of lifespan of bees (De Jong et al., 1982a;
Kovac and Crailsheim, 1988), changes in haemolymph, deformation of the
wings, damage in the brood, disorderly function of bee colonies degeneration
of glands, and death of bee colonies. Its pathological effects include fungal,
bacterial and viral disease some of which also contribute to the killing of the
bee populations (Bailey and Ball, 1991).
2.5 Varroa destructor: a Vector of Bee Viruses
Mites increase the incidence of honey bee diseases because they act
as vectors for honey bee pathogens (Ball, 1989). The V. destructorhas been
associated with the transmission of a number of bee viruses including APV
(Ball, 1989; Batuev, 1979), Slow paralysis virus(SPV) (Denholm, 1999) and
DWV (Bowen-Walker et al., 1999). Much of the pathology and mortality seen
in severely mite-infested bee colonies is linked to the mite-mediatedtransmission of viruses (Martin et al., 1998). Viruses are transmitted during
feeding when the mite injects a fluid, possibly salivary in origin into the
haemolymph (Gelbe et al., 1987). Alternatively, the open wound could also
offer a perfect setting for opportunistic infections by other pathogens to set
in. The term bee parasitic mite syndrome has been used to describe a
disease complex in which colonies are heavily infested with mites and
infected with viruses. Varroa mite, which may act as a vector to some of
these viruses (Sumpter and Martin, 2004) induces the accumulation of
viruses as a result of enhanced virus replication in the bee which often
leads to high mortality as seen in APV (Ball, 1989; Batuev, 1979), SPV, KBV
(Denholm, 1999) and DWV (Bowen-Walker et al., 1999). These four viruses
are also known to multiply rapidly when injected into bee pupae (Denholm,
1999). Recently, picorna-like virus particles (27 nm in diameter) were also
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isolated from a population of Varroa mites in beehives (Kleespies et al.,
2000).
2.6 Biology ofVarroamite
The ectoparasitic mite V. destructoris one of the most serious pests of
the honey bee A. mellifera. The adult female mite is reddish-brown in colour
and easily visible as flattened red/ brown elliptical dots. It shapes like a
scallop shell and has an oval and flat body measuring about 1.1 to 1.2 mm
long and 1.5 to 1.6 mm wide. Male mites are smaller, measuring about 0.7
mm by 0.7 mm, and are pale to light tan in colour (Bailey and Ball, 1991).
Female body is characterized by modified tarsus (lobed sucker), presence of
stiff hairs ventral side, modified chelicerae, lack of fixed digit and saw like
moveable digit (Delfinado and Baker, 1974). The mites reproduce in the
brood cell on pupae of worker bees and drones (De Jong, 1988) of which the
drone is distinctly preferred (Bailey and Ball, 1991). The reproduction cycle
starts when a female mite enters a brood cell just before it is sealed on 5th or
6th day (Infantidis, 1983). The female mite lays one unfertilized egg from
which a male hatches and a number of fertilized eggs from which femaleshatch (Rehm and Ritter, 1989). At 60 h after sealing, first unfertilized egg is
laid. Fertilized eggs are laid from 90 h onwards. Total development time is
6.5 daysin males and 5 - 5.5 days in females. In males, egg stage last for 30
h, protonymph for 52 h and deutonymph for 72 h but in female egg stage
last for 20-24 h, protonymph for 30 h and deutonymph for 75-80 h. Mating
occurs within the sealed cells. There are two theories on reproduction
behavior ofVarroa. One theory said that male doesnt feed and starts mating
with sister mites as soon as it matures and copulate once with each mite to
conserve its energy (Baker et al., 1984). However, other theory says male
feeds and mates several times with each sister mite to ensure fertilization
(Donze et al., 2007). Adult female mites are released when the developed bee
emerges from the cell or when workers remove the dead brood (Martin,
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2001). Males and immature daughters die inside brood cells (Oldroyd,
1999).
2.7 Detection ofVarroa
Proper and scientific detection ofVarroais as important as its control
because some of the symptoms of this disease are same as those of other
diseases. Various methods employed for detection ofVarroa are described
below.
Hive debris method is regularly employed which is a simplest, least
time consuming method, but it is shown 50-60 percent mites that fall from
colony into hive debris are live (Lobb and Martin, 1997; Webster et al., 2000)
which can climb back to brood frames and cause re-infestation. In modified
method, a white paper or plastic sheet covered with petroleum jelly or
another sticky agent is placed on the bottom board of a colony and the hive
is smoked with pipe tobacco in a smoker (Eischen, 1997). After closing the
hive for 10 to 20 minutes, the board is removed and the mites fallen are
counted.
Sticky paper method (Delaplane and Hood, 1999) and screen floors(PAM, 1993) are preferred by some researchers to monitor populations ofV.
destructor. Powdered sugar is also used by many workers to detect Varroa
population in A. melliferacolonies (Macedo et al. 2002; Fakhimzadeh, 2000;
Aliano and Ellis, 2005). The powder does not harm the bees but does
interfere with the mites ability to maintain its hold on the bee.
Mites can also be detected by pulling up capped brood cells using a
cappings scratcher (with fork-like tines); Varroa appears as brown or
whitish spots on the white pupae. Guanine, the fecal material ofVarroa, can
be seen as white spots on the walls of brood frames in highly infested
colonies (Anonymous, 2000b).
Alcohol, ether roll (Burgett et al., 1987; Calderone and Turcotte,
1996), hot water and soapy water (De Jong et al., 1982b) are also used by
researchers to count mites on adult bees but all the methods cause bee
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mortality. Hosmani et al. (2005) collected the bees in a jar and kept them in
a BOD for 2h to subdue their activity before examining mites on bee body.
Since no bee mortality occurs in this method, it seems to be better method
although it is comparatively more time consuming than other methods.
2.8 Management ofVarroa
Extensive research has been carried out to develop appropriate
technologies which include nonchemical and chemical methods. These are
reviewed below under different subheadings:
2.8.1 Use of traps
Mites trapped inside the brood cells can easily be removed from a
colony by heat treatment (Engles, 1994). Because Varroa prefer drones,
combs of drone brood are used to attract and trap the mites. The mites are
then removed by cutting out drone brood (Bailey and Fuchs, 1997; Bailey et
al., 1996). For 27days, if all the eggs laid by the queen are trapped then it
reduces the mite level up to 95 per cent in the hive (Calis et al., 1998). The
'freezing drone brood method' also offers good control but is labour intensive
and may weaken the colony. The method depends on the placement of aframe with drone brood comb in the central part of the brood nest. It is
removed when cells are capped and freezed for 24-48h. Another Varroamite
control method is the 'queen arrest method' where the queen is temporarily
confined to a single brood frame or portion. This method is labour intensive,
slows down colony development and may only be suitable for the dedicated,
small time beekeeper. Caging the queen ofA. ceranafor 35 to 40 days and
separating the brood frames helped interrupt the brood/mite cycle (Enayet
and Sharif, 1991) in A. cerana. Worker brood can also be removed to lower
the mite infestation level in the colony (Fries and Hansen, 1989).
2.8.2 Screen floor
Screen floors have also been employed by various workers to reduce
Varroapopulation (Pettis and Shimanuki, 1999; Ostiguy et al., 2000; Ellis et
al., 2001; Spear, 2002; Harris et al., 2003; Sammatro et al., 2004) in hive
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and brood. Ostiguy et al. (2000) reported that screen floor reduce the mite
population upto 44 percent. Likewise, Harbo and Harris (2004) also reported
that after nine weeks, colonies with screen floors had fewer mites in brood
cells. Delaplane et al. (2005) and Coffey (2007) also reported that use of
screen floors exert a modest restraint on mite population growth.
In screen used hives, increased brood production (Skubida and
Skowronel, 1995; Pettis and Shimanuki, 1999; Ellis et al., 2001) and adult
bee population (Ellis et al., 2003; Harbo and Harris, 2004) are reported as
compared in hives with wooden bottom board hives.
2.8.3 Smoke treatment
Partial control in lightly infested apiaries can be obtained with tobacco
smoke or smoke from other plant materials that cause mite knockdown
(Eischen, 1997). Smoke dislodges mites and can be used periodically to
remove emerging mites from brood cells. A sticky board used in conjunction
with smoke traps mites provide effective control. Pettis and Shimanuki
(1999) found Varroa that dropped to the bottom board of a hive were more
likely to remain there unless a bee passed within seven mm of it. Using ascreen to separate fallen Varroafrom bees may help keep mite levels lower.
2.8.4 Heat treatment
The mite succumbs around 46-48C, whereas sealed brood survives.
Using a combination of heat and lactic acid treatment, mite numbers were
reduced in Denmark (Brodsgaard and Hansen, 1994). In this method bees
are caged, placed in a chamber, and rapidly heated to 46-48C (RH 20%)
until mites stop falling from the bees (c. 2-15 min.). This technique is 23
(Hoppe and Ritter, 1986) to 95 per cent effective (Komissar, 1985; Akimov et
al., 1988). It is reported that heat treatment is much more effective when
combined with oil of wintergreen. Mites in brood cells failed to reproduce or
were killed when they were exposed to 40C for 24 h or to 42C for 18 h (Le
Conte et al., 1990). Engles (1994) recommended controlling mites by heating
combs of capped brood without adult bees. However, according to some
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workers, heat treatment is a risky procedure that can kill bees (Harbo,
2000). Bees do not survive for two days at 42C (Harbo, 1993). The Russian
technique (Komissar, 1985) recommended a low humidity of less than 20
per cent reduces the mortality of bees (Free and Booth, 1962).
2.8.5 Use of dusting material
Many chemicals are applied as dusts for managing Varroapopulations
in A. mellifera hives. Sulphur dusting is commonly applied in India to
control parasitic mites in A. melliferacolonies in India (Shivakoti and Bista,
2000). Powdered sugar dusting is also done on adult bees to remove mites.
Aliano and Ellis (2005) who reported 76.7 percent mites are fallen from
adult bees by application of powdered sugar whereas Macedo et al. (2002)
reported 92.9 percent mite fall. Fakhimzadeh (2000) hypothesized that the
dust adheres to the tarsal pads Varroa and prevents the mites from
attaching to bees. It is also thought that powdered sugar stimulates the
bees grooming behavior (Macedo et al., 2002). However, in a recent study,
powdered sugar (120g/application) dusted every two weeks for eleven
months did not provide significant V.destructorcontrol (Ellis et al., 2009).2.8.6 Use of Botanicals
Another approach is the use of volatile plant essential oils to control
bee mites (Colin, 1990; Gal et al., 1992; Imdorf et al., 1995). Some
botanicals found effective against V. destructorare neem oil (Melathopoulous
et al., 2000), vegetable oil (Le Conte et al., 1998), mineral oil (Imdorfet al.,
1999; Lindberg et al., 2000), thymol oil and canola oil (Sammataro et al.,
1998). Neem (5%), thymol (4.8 g thymol/l in 20% canola oil solution) and
canola (20% solution) demonstrated 60-90 per cent effectivity against Varroa
(Whittington et al., 1999). Neem also inhibit growth of bacterial honey bee
pathogens such as American Foul Brood (Rao et al., 1986; Williams et al.,
1998). Plant oils are complex compounds that may have unwanted side
effects on bees and beekeepers (Schaller and Korting, 1995) and could
contaminate hive products.
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2.8.7 Use of Resistant bee stocks
Suppressed mite reproduction (SMR) or Varroa Sensitive Hygiene
(Harris, 2007) is a trait of honey bees that provides resistance. Attributes
that enhance honey bee tolerance to Varroa are reviewed (Buchler, 1994).
Some beekeepers let all susceptible colonies die and then rear queens from
the survivors to head new colonies. Untreated Africanized colonies were
maintained in Arizona for several years with few tracheal and Varroamites
but resistant mechanisms were not discussed (Erickson et al., 1998).
Hygienic activity like removal of dead or dying bee (Boecking et al., 1999;
Spivak, 1996) reduces the mite levels in untreated colonies, which require
less chemical treatment to manage Varroa.
Defensive behaviors against Varroain races ofA. ceranawere studied
(Buchler et al., 1993; Rath, 1999; Sasagawa et al., 1999) and grooming is an
important component in mite reduction but it is highly variable in A.
mellifera(Buchler, 1994). Bees remove mites from each other and some even
kill them using their mandibles (Peng et al., 1987) but this trait may not be
heritable in some European bee stock (Harbo and Harris, 1999; Harbo andHoopingarner, 1997).
The pupal period influences the number of mites completing development.
Shortening this time results in fewer Varroareaching maturity; if the capped
cell stage is reduced by only six hours, fewer immature mites will become
adults. Two African bee races have a heritable (worker) post capping period
of only 10 days (Moritz, 1985), whereas European races require 11 to 12
days. Some researchers (De Jong, 1999; Moretto, 1999) suggest climate
plays a more important role in influencing the Varroapopulation but it is
difficult to maintain in A. melliferacolonies in northern regions.
2.8.8 Use of Organic acids
Environmentally safe chemicals, e.g. formic, oxalic and lactic acid can
be successfully applied to control Varroa (Ritter and Ruttner, 1980, Kraus
and Berg, 1994). Formic acid 65% (300 ml) of was found 95 per cent
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effective (Calis et al., 1998; Calderon, 2000) which kills mites on the adult
bees as well as in the sealed brood cells (Liebig et al., 1984). Formic acid
occurs naturally in honey (Crane, 1975) but application for mite control may
increase its concentration (Hansen and Guldborg, 1998). In addition, formic
acid treatment of colonies may damage uncapped brood, young bees and
may cause the losses of queens (Liebig et al., 1984; Fries, 1989; Bolli et al.,
1993).
Oxalic acid @ 2-3 g (2 treatment in 4 days) reduced mite level from 20
to 5 per cent and when applied as spraying or trickling of solution + sugar
solution was found 90 per cent effective (Charriere and Imdorf, 2002). Lactic
acid is weak acid than formic and oxalic acid and leaves less residues but
its efficacy is also less. It exists in small amounts naturally in honey
(Anonymous, 2002b). Its effectivity was evaluated by many workers but it
varies with concentration applied in the hive (Euteneuer, 1989; Kraus and
Berg, 1994).
2.8.9 Chemical treatment
While long-range, non-chemical controls are vigorously being sought,beekeepers need immediate relief from existing mite infestations. Fluvalinate
(99% effective), Flumethrin (95% effective), Cymiazole, Coumaphos,
Bromoprophylate and Amitraz (99% effective) (DEFRA, 2005) are used in
some parts of world for effective V. destructor control. The chemicals are
applied as pesticide-impregnated plastic strips, which are hung between
frames of bees in a hive. Applied in this manner, it is released slowly and
dispersed by adult bees (Burgett and Kitprasert, 1990). Among these,
Cymiazole, Coumaphos and Bromoprophylate are effective in brood less
condition. These chemical options for Varroa pose a serious problem
because repeated exposure to the same pesticides select for resistant mites
(Gerson et al., 1991).
Reports of fluvalinate-resistant mites have surfaced in Italy (Lodesani
et al., 1995; Loglio and Plebani, 1992; Milani, 1995), France (Colin et al.,
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1997), some U.S. states (Elzen et al., 1999; Pettis et al., 1998) and many
other parts of world. Coumaphos resistance is also reported in Italy (Vedova
et al., 1997) and the United States and in recent studies resistance to
Amritraz has also been found (Elzen et al., 1999). This resistance crisis is
being compounded by contamination of hive products including honey, wax
and propolis (Wallner, 1995). In addition, drone survival is found to be lower
in colonies treated with fluvalinate (Rinderer et al., 1999), which may also
affect their mating ability.
2.8.10 Biological control
Biological control agents of pests are naturally occurring predators or
parasites that will normally attack and kill a pest whilst sparing desirable
organisms. A strain of the fungus Metarhizium anisopliae was found as
effective as fluvalinate against Varroa (Jones, 2004). Coated plastic strips
with dry fungal spores exposed to all the bees within 5-10 minutes and
mites on adult bees die within 3-5 days. This fungus is safe to honeybees
and found no effect on queens production. It is effective even 42 days after
application (Jones, 2004). In another laboratory bioassay, the susceptibilityofVarroamites was measured to infection by of forty isolates of fungi from
six genera (Beauveria, Hirsutella, Paecilomyces, Metarhizium, Tolypocladium,
Lecanicillium) at 25OC and high humidity (> 95% RH) (Alfredo, 2004).
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CHAPTER-III
MATERIALS AND METHODS
The present investigations entitled Incidence of Varroa destructor
Anderson and Trueman (Acari: Varroidae) and its management in Apis
mellifera L. colonies were undertaken broadly in three phases. The firstphase covered the incidence and seasonal fluctuation in V. destructor
population in the University apiary, CCS Haryana Agricultural University,
Hisar (Haryana) emphasizing on biotic-biotic and biotic-abiotic interactions
during the year 2008-09. Random sampling from beekeepers outside the
University apiary was also done to get the idea of mite infestation in
Haryana apiaries. The second phase was concerned with the effect of mite
incidence on bee strength and colony stores. The third phase included
management of V. destructor in A. mellifera L. colonies by using different
control measures. Data on colony strength and stores were also recorded
before and after the termination of each experiment. These treatments were
applied in A. melliferacolonies with three replications each and compared
with control treatment. Materials used and methodology adopted for
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different experiments under laboratory and field conditions are outlined
below under relevant sub-headings.
Sampling of brood
The degree of brood infestation was ascertained by opening 50 each of
worker and drone sealed brood cells per hive on each sampling day. Drone
brood was available during the months of May to July and then from
November to March. Fortnightly sampling from six A. mellifera colonies
(three each of hive debris and sticky paper method) was done throughout
the study period. For sampling, brood cells were uncapped individually with
the help of needle; brood/pupae were removed with the help of forceps and
examined for the presence of mite. All perforated brood cells (a probable
symptom of mite infestation) in each colony were also counted visually and
examined for evidence of infestation by V. destructor. Counting of perforated
brood cells was based on number of exit holes present on brood cells.
Pattern of brood in relation to mite abundance was also recorded. Number
of worker and drone brood infested with V. destructorwere recorded in each
hive.
3.2.3 Per cent infestation ofVarroa destructor
Pest potential ofV. destructorin terms of per cent infestation was
calculated at each fortnight by using following formula:
No. of mites present on brood
% infestation= x100
Total no. of brood examined
Preference of host selection by V. destructorwithin worker (W) and drone (D)
brood was calculated by using simple ratio formula:
Per cent incidence on worker brood
W: D ratio =
Per cent incidence on drone brood
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Different control measures against Varroa destructor
Under this objective, various treatments were evaluated against V.
destructor in A. mellifera colonies and compared with control. The
treatments were screen floor, formic acid@ 5ml of 85 per cent/hive (cotton
swab method), dusting of powdered sugar (2 and 3g/ frame). Each
treatment had three replications (A. melliferacolonies). Untreated colonies
acted as control.
Colony Selection: All the experiments had three week duration and all test
colonies began with equalized colony strength, stores and mite population.
On the basis of pretreatment count, uniform pairing of treated and
untreated colonies was done having non significant mite, bee population
and brood, honey, pollen area between them. Prior to experimentation, the
worker populations were equalized for bees so that each hive contained
approximately 5 frames of bees. Brood, honey and pollen area were
quantified in square centimeters on all frames using wire grid having
squares of 2.5 cm on a side (Harbo and Harris, 2004). The data were
compared with V. destructorinfested colonies where no treatment was given.Pre treatment assessment: For pre treatment count, sticky paper was
inserted on to the bottom board of experimental colonies. Sticky papers
were removed three days later and mite drop was quantified (Ostiguy et al.,
2000).
Post treatment assessment: Fresh white sticky paper on the bottom board
was placed in each test colony. The number of mites in hive was estimated
on sticky paper at each observation period i.e. 7, 14 and 21 days after
treatment as per the method given under sub heading 3.1.2. At each
observation period, old sticky paper was replaced with new to avoid the
confusion in counting the number of earlier dropped mites over latest mite
drop per hive.
Final treatment assessment:To determine the efficacy of the experimental
treatments and to collect all the mites in the treated and untreated A.
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melliferacolonies, colonies were treated with formic acid (5 ml of 85%) by
cotton swab method after 21 days. Mites were collected from the bottom of
hives using sticky paper method in both treated and untreated groups. Per
cent efficacy and per cent reduction in mite population over control were
calculated by formulae following the method of Eguaras et al. (2005):
% Efficacy = 100 [It / (If+ It)]
Where It = Total number of mites at the sticky paper of the hive after
treatment Total number of mites at the sticky paper of the hive before
treatment
If= Total number of mites at the sticky paper of the hive final treatment
% reduction over untreated hives = [Ts - Cs]/ Ts 100
where Ts and Cs are the percentage of surviving mites in treated and
untreated hives, respectively
Details of each of these treatments are given under relevant
subheadings:
Screen floors
The effect of screen floors on bee colonies was evaluated in University
apiary during the year 2008. Pre treatment samples were collected from all
the treated and untreated hives to estimate the mean abundance of V.
destructor. After pre treatment, in three A. melliferacolonies having natural
infestation of V. destructor, commercially available screen floor/ bottom
board (10 mesh size) was placed below the hive. Sticky white paper was
placed on the sliding wooden tray of screen floor for the collection of mite.
These colonies had no air flow from the bottom. The air flow in the screen
floor treatment was only from the front entrance and was therefore similar
to control colonies in this respect. In three control colonies, wooden bottom
board was used. The two treatments were randomly arranged in the test
apiary.
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Observations on the number of mites per hive were recorded after 7,
14 and 21 days. After twenty one days, to record the residual mite
population, formic acid (5 ml of 85%) was applied by cotton swab method to
all the treated and untreated colonies. Final count of mites after this
treatment was recorded to calculate the per cent efficacy and per cent
reduction in mite population over control. The impact of treatment on bee
strength and area of brood, pollen and honey was also studied and
compared with the similar data on untreated hives.
Formic acid
The trial was carried out in University apiary by randomly distributing
the A. mellifera colonies of treated and untreated group. Before the trial,
mite infestation level, colony strength and stores were measured in both the
groups. Each group consisted of three hives. Formic acid (5 ml of 85%) was
applied through cotton swab method in three hives to keep the fumigation
evenly distributed (Plate V). Except for hive entrance, no other air flow
mechanism was there. After treatment, colonies were opened at 7th, 14th
and 21st
day. At each observation period, sticky paper was removed from thecolonies and number of mites were counted as per method described earlier
in this chapter. Fresh white sticky paper was placed on the bottom board
after removal of old sticky paper. Second application of 5 ml of formic acid
at 85 per cent was given after 21 days by cotton swab method to record the
remaining mite population in treated and untreated A. mellifera colonies.
The efficacy of the treatment was evaluated as per centage of mite mortality
and also as per centage of reduction in V. destructorpopulation over control.
At the end of study period (after 21 days), the state of the colony was
assessed by measuring the bee strength, brood, pollen and honey area in
treated and untreated colonies.
Sugar dusting (2g/ frame)
The effect of powdered sugar dusting on bee colonies was evaluated in
University apiary during the year 2008. Powdered sugar was prepared by
grinding the ordinary sugar in to fine powder form. Pre treatment samples
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were collected from all the treated and untreated hives to estimate the mean
abundance of V. destructor. After pre treatment, in three A. mellifera
colonies having natural infestation ofV. destructor, powdered sugar was
dusted on frames (2g/ frame) so that all the bees come directly in contact
with it (Plate VIII). In control colonies, no powdered sugar treatment was
given. Rest of the conditions was the same as that of treated colonies. V.
destructor population was quantified prior, 7, 14 and 21 days after
treatment by counting the number of mites on sticky paper placed on the
bottom board. After twenty one days, to record the remaining mite
population present in the colonies, formic acid (5 ml of 85%) was applied by
cotton swab method to all the treated and untreated colonies. Final count of
mites after this treatment was recorded to calculate the per cent efficacy
and per cent reduction in mite population over control. The impact of
treatment on bee strength and area of brood, pollen and honey was also
studied and compared with the similar data on untreated hives.
Sugar dusting (3g/ frame)
Efficacy of powdered sugar at higher dose (3g/ frame) was evaluatedin randomly distributed colonies by the same method as described under
3.5.9. Before the trial, mite infestation level, colony strength and stores were
measured in both the groups. Each group consisted of three hives.
Powdered sugar was dusted on the frames of treated hives (Plate VIII) and
observations on the number of mites per hive were recorded after 7, 14 and
21 days of treatment. After 21 days, formic acid (5 ml of 85%) application
was done in both the treated and untreated hives and residual mite
population collected after three days. The effect of the treatment on the
number of bees (frames), brood (cm2), honey (g) and pollen area (cm2) was
studied and compared with the control.
3.4 Statistical analysis
The seasonal incidence data was subjected to analysis of variance
(ANOVA) Critical difference (CD) was calculated to determine the difference
between seasons and sampling methods. Appropriate transformation of data
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was applied where ever was necessary. Correlation matrix was calculated
between V. destructor incidence and abiotic factors to see their effect on
population build up of mite.
Correlation analysis was run between mite infestation and perforated
brood/ deformed bees. The correlation coefficient r was also calculated to
see the effect of mite incidence on number of bees (frames), brood (cm2),
honey (g) and pollen area (cm2) during the study period. It is a measure of
the degree to which the regression equation of the dependent variable Y.
Correlation variables vary together and is defined by:
( )( )
( ) ( )
=22
YYXX
YYXXr
The significance of observed correlation coefficient was tested using
students t test. If tcal >ttab the observed correlation coefficient is significant
otherwise not, where t is estimated value of t for n-2 degree of freedom.
Experiments under objective III were conducted and analyzed using
Completely Randomized Block Design (CRBD). Critical difference (CD) was
calculated to know the efficacy of the different treatments in reducing the V.
destructorpopulation in hives. Effects of the treatments were also measured
on the colony strength and stores.
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CHAPTER-IV
RESULTS
The results of the present investigations entitled Incidence ofVarroa
destructorAnderson and Trueman (Acari: Varroidae) and its management in
Apis melliferaL. colonies have been presented under the relevant headings
of this chapter.
Incidence ofV. destructoron A. melliferabrood
Data pertaining to V. destructor incidence on A. mellifera brood is
presented in Table 1. Results showed that both worker and drone brood
were affected by V. destructoras almost similar number of worker and drone
broods were infested with mites. Out of 50 brood cells each of worker and
drone brood, maximum number of brood cells (worker 7.5, drone 8.0-8.5)
was infested with V. destructor in second fortnight of May. Per cent
infestation was 15, 16 and 15, 17 per cent in worker and drone brood ofA.
melliferain both the sampling methods, respectively (Table 1). It decreased
to zero in first fortnight of June for worker and drone brood in A. mellifera
colonies in which hive debris was collected. It again appeared in worker cellsin second fortnight of August (5 %) and then gradually increased to 7 per
cent in the second fortnight of September. However, in sticky paper method,
mites were present from first fortnight of May to second fortnight of
September in worker cells (4-15%) and from first fortnight of May to second
fortnight of July in drone cells (13-17%).
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Mean per cent mite incidence in brood ranged from 0 to 15.5 and 2 to
16 per cent in A. melliferacolonies in which natural mite fall was recorded
in hive debris and on sticky paper, respectively. From first fortnight of
October to second fortnight of April, no mites were recorded from brood
during the present investigation.
To know the preference of V. destructor, worker to drone brood
infestation ratio was calculated which ranged from 0.0 to 0.93 in hive debris
collected colonies (Table 1). Likewise, drone: worker brood ratio was 0.86 to
1.0 during the months of May to July in which sticky paper method was
used. The values indicated the preference ofV. destructortowards the drone
brood but the difference was non-significant as revealed by t-test.
Colonies infested with V. destructorshowed irregular pattern of brood
in comparison to healthy brood in uninfested colonies. In V. destructor
infested colonies, brood infested with mites, perforated brood cells and
abnormal bees were also noticed.
The data on the effect ofV. destructor incidence on brood perforation
showed heavy perforation of brood cells coinciding with the season of highmite infestation (Table 2). Perforation of brood was first detected in the first
fortnight of May (5 and 7 sealed brood cells) when V. destructorpopulation
was 36.5, 74.0 mites/ hive in hive debris and sticky paper method,
respectively. Brood perforation was highest in the second fortnight of May
(5.5, 7.5 sealed brood cells) in both methods of sampling. No perforated
brood cells were recorded from first fortnight of June to second fortnight of
July in hive debris method. Thereafter, it maintained a steady appearance
from first fortnight of August to second fortnight of September. In A.
melliferacolonies where sticky paper was used, brood perforation remained
static from first fortnight of June to first fortnight of August and ranged
from 4.5 to 5.5 sealed brood cells.
Per cent brood perforation ranged from 0 to 10 and 0 to 15 in hive
debris and sticky paper method, respectively (Table 2). Brood perforation
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and mite incidence showed significant positive correlation with two methods
of sampling, hive debris (r = 0.87) and sticky paper (r = 0.94). During the
period of study, increase/ decrease in V. destructor population led to
corresponding increase/ decrease in perforation of brood cells.
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Table 1: Incidence ofVarroa destructoron Apis melliferabrood (May,
2008 to April, 2009)
Brood cells with mites (Hivedebris) (N=50)
Brood cells with mites (Stickypaper) (N=50)
Period ofobservation
Broodcells
examined Workercells(W)
Dronecells(D)
Meanincidence(%)
W: Dratio
Workercells(W)
Dronecells(D)
Meanincidence(%)
W:Dratio
1st to 15th,May
50 7.00(14.00)
7.50(15.00)
7.25(14.50)
0.93 7.00(14.00)
7.00(14.00)
7.00(14.00)
1.00
16th to31st, May
50 7.50(15.00)
8.00(16.00)
7.75(15.50)
0.93 7.50(15.00)
8.50(17.00)
8.00(16.00)
0.88
1st to 15th,June
50 0.00(0.00)
0.00(0.00)
0.00(0.00)
0.00 6.50(13.00)
6.50(13.00)
6.50(13.00)
1.00
16th to30th, June
50 0.00(0.00)
0.00(0.00)
0.00(0.00)
0.00 6.50(13.00)
7.50(15.00)
7.00(14.00)
0.86
1st to 15th,July
50 0.00(0.00)
0.00(0.00)
0.00(0.00)
0.00 7.00(14.00)
7.50(15.00)
7.25(14.50)
0.93
16th to31st, July
50 0.00(0.00)
0.00(0.00)
0.00(0.00)
0.00 7.00(14.00)
7.50(15.00)
7.25(14.50)
0.93
1st to 15th,August
50 0.00(0.00)
- 0.00(0.00)
6.00(12.00)
- 3.00(6.00)
16th to31st,August
50 2.50(5.00)
- 2.25(2.50)
2.00(4.00)
- 1.00(2.00)
1st to 15th,September
50 3.00(6.00)
- 1.50(3.00)
3.00(6.00)
- 1.50(3.00)
16th to30th,September
50 3.50(7.00)
- 1.75(3.50)
2.00(4.00)
- 1.00(2.00)
Mean 50 2.35(4.70)
1.55(3.10)
1.95(3.90)
5.45(10.9)
4.45(8.90)
4.95(9.90)
tcal 1.31 (NS) 1.45 (NS)
No mites were noticed in brood cells from 1st fortnight of October to 2nd
fortnight of April
Figures in parentheses are per cent mite incidence in worker/drone brood
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Table 2: Effect ofVarroa destructor incidence on perforated brood
cells
No perforated brood cells from 1st fortnight of October to 2nd
fortnight of April were recorded
Figures in parentheses are per cent perforated brood cells
Hive debris Sticky paperPeriod of
observation
Brood
cellsexamined No.ofmites
No. ofperforatedbrood cells
No. ofmites
No. ofperforatedbrood cells
1st to 15th, May 50 36.50 5.00(10.0)
74.00 7.00(14.00)
16th to 31st, May 50 40.05 5.50(11.00)
86.50 7.50(15.00)
1st to 15th, June 50 16.00 0.00(0.00)
37.00 5.00(10.00)
16th to 30th, June 50 0.50 0.00(0.00)
24.50 5.00(10.00)
1st to 15th, July 50 22.50 0.00
(0.00)
47.00 5.50
(11.00)16th to 31st, July 50 24.00 0.00
(0.00)38.00 5.50
(11.00)1st to 15th, August 50 29.00 3.50
(7.00)30.00 4.50
(9.00)16th to 31st, August 50 25.00 5.00
(10.00)14.50 0.00
(0.00)1st to 15th,September
50 33.00 4.50(9.00)
23.5 2.50(5.00)
16th to 30th,September
50 17.00 5.00(10.00)
21.50 3.50(7.00)
Mean 50 24.35 2.80(5.70)
39.65 4.60(9.20)
r (Mite VsPerforation)
0.65 0.83
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Efficacy of formic acid
The total number of mites collected on sticky paper at the bottom board
before, during and after the formic acid treatment and its effectiveness
are presented in Table 3. The pretreatment count recorded was 26.0
mites per hive and 15.5 mites per hive in treated and untreated A.
melliferacolonies showing no significant difference with each other. More
number of mites were collected in first week of treatment (25 mites/hive)
which gradually declined to 21.6 and 20.0 mites/hive in the second and
third week, respectively. The natural mite fall in control colonies was
11.9, 9.0 and 11.0 in first second and third week, respectively (CD =
4.86;p = 0.05). In untreated A. mellifera colonies, significantly more
number of mites fall on sticky paper during the treatment (66.6
mites/hive) as compared to 31.9 mite fall/hive in untreated colonies.
Second application of formic acid after twenty one days for residual V.
destructorpopulation in both treated and untreated hives resulted in was
16.0 and 140.5 mite fall/hive, respectively which differed significantly
with each other (CD = 8.86;p = 0.05). The per cent efficacy and per cent
control over untreated hives was 71.73 and 85.36, respectively.
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Table 3: Efficacy of Formic acid against Varroa destructor in Apis
melliferacolonies
Number of mites/hive after treatment on sticky
paper
Treatment Pre
Treatment
7 DAT 14 DAT 21 DATTotalMean after
treatment
After final
Treatment
*
Formic acid (5 ml
of 85%)
26.00 25.00 21.60 20.00 66.60 22.20 16.0
Control 15.50 11.90 9.00 11.00 31.90 10.60 140.5
CD (p = 0.05) N.S. 4.86 8.86
% efficacy 71.73
% reduction over
control
85.36
DAT = Days after treatment*Formic acid (5 ml of 85%) was applied to record residual mite count
Table 4: Effect of Formic acid on colony strength and stores in Apis
melliferacolonies
TreatmentBee strength
(frames)
Brood Area
(cm2)Honey (g)
Pollen
Area(cm2)
Formic acid
(5 ml of 85%)
6.50 734.60 147.30 250.60
Control 6.00 760.00 158.30 248.30
Pre
treatment
CD (p = 0.05) N.S. N.S. N.S. N.S.
Formic acid
(5 ml of 85%)
6.50 939.00 105.30 263.30
Control 6.50 736.60 156.60 294.00After
treatment
CD (p = 0.05) N.S. 188.41 N.S. N.S.
NS = Non-significant
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Efficacy of powdered sugar (2g/frame)
In powdered sugar dusting (2g/frame) treatment, natural V.
destructor infestation in treated and untreated hives was 6.0 and 9.3
mites/hive, respectively before treatment which was comparable with
each other. Powdered sugar (2g/frame) application on frame led to
significant (CD = 8.04; p = 0.05) increase in natural fall ofV. destructor
(46.9 mites/hive) at the end three week period as compared to 21
mites/hive in untreated hives (Table 5). Week wise, post treatment count
recorded was 13.3, 16.6 and 17.0 mites/hive in first, second and third
week after treatment, respectively which was more than 8.5, 10.5 and 2
mites/hive in similar weeks in untreated A. mellifera colonies. The
residual treatment of formic acid (5 ml of 85%) resulted in significantly
higher mite fall (99.5 mites/hive) in untreated A. mellifera colonies as
compared to treated colonies (4.0 mites/hive) (CD = 4.65; p = 0.05) (Table
26). The per cent efficacy and per cent reduction in V. destructor
population over untreated hives was 87.21 and 81.75, respectively in
powdered sugar (2g/frame) treatment.
Over the course of this study, no significant difference was reported
colony strength and stores. Bee strength decreased from 4.1 to 4.0
frames and 5.0 to 4.5 frame in treated and untreated A. mellifera
colonies, although the difference between the treatments was non
significant (Table 6). Brood area although showed an increase from 244.0to 500.5 cm2 in treated hives but remained statistically comparable with
the brood area (750 cm2) in untreated hives. Similarly, comparable data
for honey was recorded in treated (303.1 g) and untreated (210.0 g) A.
mellifera colonies. Pollen area showed an increase from 162.3 to 222.0
cm2 in treated and 144.0 and 155.0 cm2 in untreated hives but difference
between the treatments was nonsignificant.
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Table 5: Efficacy of powdered sugar (2g/ frame) against Varroa
destructorin Apis melliferacolonies
Number of mites/hive after treatment on sticky
paper
Treatment Pre
Treatment
7
DAT
14 DAT21 DATTotal Mean after
treatment
After final
Treatment*
Powdered sugar
@2g/frame6.0 13.3 16.6 17.0 46.9 15.60 4.0
Control 9.3 8.5 10.5 2.0 21.0 7.00 99.5
CD (p = 0.05) N.S. 8.04 4.65
% efficacy 87.21
% reduction over
control81.75
DAT = Days after treatment
*Formic acid (5 ml of 85%) was applied to record residual mite count
Table 6: Effect of powdered sugar (2g/ frame) on colony strength and
stores in Apis melliferacolonies
TreatmentBee strength
(frames)
Brood Area
(cm2)Honey (g)
Pollen
Area(cm2)
Powderedsugar
@2g/frame
4.10 244.00 244.30 162.30
Control 5.00 750.00 200.30 144.00
Before
treatment
CD (p = 0.05) N.S. N.S. N.S. N.S.
Powderedsugar
@2g/frame
4.00 500.50 303.10 222.00
Control 4.50 750.00 210.00 155.00
After
treatment
CD (p = 0.05) N.S. N.S. N.S. N.S.
NS = Non-significant
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Efficacy of powdered sugar (3g/frame)
Powdered sugar dusting (3g/frame) in A. melliferahives and control hives
had an average pretreatment count of 16.5 and 13.3 mites/hive,
respectively (Table 7) which were at par with each other. The number of
mites dislodged from brood frames or bees during sugar dusting
(3g/frame), significantly differed from control treatment (CD = 42.90; p =
0.05). Significantly higher number ofVarroamites (59 mites/ hive) were
recorded from sticky paper of treated hives as compared to mites in
untreated A. melliferacolonies (15.60 mites/hive). The number of mites
fallen on sticky paper was more (78.5 mites/hive) in first week which
declined to 69, 29.5 mites/hive after second and third week, respectively.
In untreated hives, the natural fall during these weeks were 13.3, 16.6
and 17.0 mites/hive, respectively. Effectiveness of the treatment showed
significantly low residual V. destructor population (4 mites/hive) after
formic acid treatment in treated hives (CD = 8.86; p = 0.05). More
number of mites (135.5 mites/ hive) was recorded on sticky paper after
formic acid treatment at 21 days in untreated A. melliferacolonies. The
treatment showed an efficacy of 97.70 and 80.51 per cent reduction in V.
destructorpopulation over untreated hives.
During this study, equalization of colony strength and stores were done
in treated and untreated A. mellifera colonies prior to conduction of
experiment. With the result, in pre treatment, comparable data was
obtained for all the parameters in treated and untreated hives (Table 8).
Bee strength varied between 4.5 and 5.5 frames in both the treatment
showing no significant difference between them. A significant increase
(CD = 122.6; p = 0.05) in brood area (720.0 to 1005.5 cm2) was recorded
after sugar dusting (3g/frame) as compared to brood area in untreated A.
mellifera colonies (750.0 to 801 cm2) (Table 29). Pollen area decreased
from 128.0 to 118 cm2 in treated hives and from 173 to 8 cm2 in
untreated hives showing statistically significant differences between the
two parameters (CD = 62.6; p = 0.05). Honey increased from 161.2 to
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237.5 g in treated A. melliferacolonies as compared to 210.0 and 223.7 g
in control hives but the difference between them was non-significant.
Comparison of organic acids revealed that more number of mites
was recorded from trickling method of oxalic acid (3%) as compared toother methods of application of oxalic acid and formic acid (Fig. 7).
Formic acid and oxalic acid (cotton swab and top bar method) showed
similar mite fall after treatment. After final treatment, lesser number of
mites was observed in top bar method of oxalic acid (3%) which showed
its effectiveness over other treatments.
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Table 7: Efficacy of powdered sugar (3g/ frame) against Varroa
destructorin Apis melliferacolonies
Number of mites/hive after treatment on sticky paperTreatment Pre
Treatment7 DAT 14 DAT 21 DAT Total Mean after
treatment
After final
Treatment*
Powdered sugar
@3g/frame16.50 78.50 69.00 29.50 177.00 59.00 4.00
Control 13.30 13.30 16.60 17.00 46.90 15.60 135.50
CD (p = 0.05) NS 42.90 8.86
% efficacy 97.70
% reduction
over control80.51
DAT = Days after treatment
*Formic acid (5 ml of 85%) was applied to record residual mite count
Table 8: Effect of powdered sugar (3g/ frame) on colony strength and
stores in Apis melliferacolonies
TreatmentBee strength
(frames)
Brood Area
(cm2)Honey (g)
Pollen
Area(cm2)
Powdered
sugar
@3g/frame
5.50 720.00 161.20 128.00
Control 4.50 750.00 210.00 113.30
Before
treatment
CD (p = 0.05) N.S. N.S. N.S. N.S.
Powdered
sugar
@3g/frame
5.00 1005.50 237.50 118.00
Control 4.50 801.00 223.70 8.00
After
treatment
CD (p = 0.05) N.S. 122.60 N.S. 62.60
NS = Non-significant
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CHAPTER V
DISCUSSION
The purpose behind the present investigation was the occurrence
ofV. destructorin epidemic form in A. melliferacolonies of Haryana due
to which beekeepers have lost their colonies. In absence of specific
management strategies, beekeepers were using the unauthorized
pesticide impregnated strips resulting in more harm than good. Under
these circumstances, it seemed worthwhile to study the incidence of V.
destructorand its management with ecofriendly approaches. The studies
involved a schematic approach; weekly observations on the number of
mites per hive, efficacy of sampling methods, influence of environmental
conditions and evaluation of management practices against V. destructor
in A. melliferacolonies.
The results obtained on variable facets of this study have been
discussed and presented in this chapter under the light of available
literature on this subject.Incidence ofVarroa destructoron Apis melliferabrood
Present investigation showed maximum infestation ofV. destructor
in worker and drone brood (15 to 17%) during the month of August and
September, corresponding to the highest values of mite infestation in hive
debris and on sticky paper. During the lean period of V. destructor
population, no mites were recorded from worker/drone brood. Kokkinis
and Liakos (2004) also reported the occurrence of mites in brood duringseasonal occurrence ofV. destructorin A. melliferacolonies from April to
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September with peak in early November (25.2 5.4 mites/worker cell)
and mid August (36.0 4.9 mites/worker cell) in the first and second
year of study. The average number of mites in worker brood cell generally
fluctuated between values that were approximately twice as high as thoseobserved on adult bees at most observation dates. Similar trend was
noticed in the present study too, where 15-17 and 7.5-9.0 per cent
infestation in brood and on adult bees, respectively, was observed during
peak infestation period. In this study, comparatively lower counts of
mites were recorded from brood cells as compared to study conducted by
Kokkinis and Liakos (2004), which may be due to variation in
geographical location.It has been shown that in case of dead brood/ bees, mites are
capable of transferring from one host to another. Bowen-Walker and
Gunn (2001) reported that 26 per cent mites moved from one live host to
another within 7 days and when their host died, mites would remain on
the dead bees for an average of 48 26.5h before dismounting. Number
of mites in brood also depends on the size of brood cell. Message and
Goncalves (1995) compared the worker brood combs of Africanized bees
(4.5-4.6 mm) and Italian bees (A. mellifera ligustica, 4.9-5.1mm). They
observed that the smaller cells of Africanized bees contained fewer mites
resulting in lower reproduction. Piccirillo and De Jong (2003) also
reported significant positive correlation between cell width and mite
infestation. However, Taylor et al. (2007) reported no signigicant effect of
cell size on V. destructorinfestation and reproduction.
During the dearth period, when the brood area decreased due to
low colony stores, multiple infestations with V. destructorand T. clareae
were recorded. These observations are in conformity with earlier
observations which reported that in case of decreased brood area, an
increase in number of cells with multiple infestations is expected
(Marcongeli et al., 1992; Eguaras et al., 1994; Kokkinis and Liakos,
2004). In cells with multiple mite foundresses, it is observed that the
number of daughters per foundress decreases (Fuchs and Langenbach,
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1989; Donze et al., 1996) which ultimately leads to lower population of a
particular species in hive.
During the present study, brood perforation coincided with high V.
destructor infestation level, as measured with significant positivecorrelation (0.64 and 0.83 in hive debris and sticky paper method,
respectively). No reports came across for V. destructor infestation but
reports are available for T. clareaeinfestation. Hosmani et al. (2005a) and
Sihag (1990) recorded higher number of perforated brood cells during
peak infestation period (May) T. clareaepopulation in A. melliferacolonies
at Hisar (Haryana, India).
Worker and drone brood infestations were compared in the presentstudy. Although preference of drone brood by V. destructorwas observed
but it was not significant. Woyke (1987) reported that the mean
infestation rate of drone brood by V. jacobsoniwas 5.1 times than that of
worker brood whereas for T. clareae the rate was 1.5 times greater for
worker brood.
Boot et al. (1995) reported that in European honey bees, Varroa
mite invade drone cells up to 11.6 times more frequently than worker
brood. Le Conte and Arnold (1988) and Noirot (1988) reported the large
size of drone cell in which mite pheromone are diffused evenly, is the
possible reason for its preference over worker cells. The mite fertility in
drone brood varied from 92.2 (Fuchs and Langenbach, 1989), 93
(Ghamdi and Hoopingarner, 2003) to 95.1 per cent (Calderon et al., 2007)
in European bees. However, the average mite fertility in africanized honey
bees ranges from 50 to 77 per cent (Garrido et al., 2003), which is an
important factor related to bee tolerance.
5.3 Evaluation of Different Control Measures against Varroa
destructor
One of the explicit goals of investigators in the integrated pest
management of Varroa destructor is to reduce or eliminate beekeepers
reliance on synthetic acaricides. Several non-chemical strategies have
shown promise as control agents, either by (1) eliminating mites from
colony, or (2) slowing rate of mite population growth. With these facts in
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mind ten eco-friendly measures were evaluated against V. destructorin A.
mellifera colonies at Hisar and compared with control. The treatments
were use of screen floor, formic acid (5 ml of 85%), sugar (2g and
3g/frame). All treatments were applied to natural mite infestation. Beforeevaluation, A. mellifera colonies were equalized in terms of colony
strength and stores.
Efficacy of screen floor (Bottom board)
The effect of screen floor was determined by dividing the test
colonies into two test groups: normal wooden bottom board and screen
floors. The current study demonstrated a significantly (p = 0.05) higher
numerical count on sticky paper in screen floors used groups ascompared to normal wooden bottom board. Webster et al. (2000) reported
that in Varroainfested colonies, 39-50 per cent of the mite fall naturally
from honeybees are alive, mobile and capable of re-infesting the colony.
Screen floors act as a physical separator between fallen mites and bees,
thus reducing the risk ofV. destructorre-infestation. In the present case,
screen floors provided 98.81 per cent efficacy and 90.85 per cent
reduction in mite population over control. Ostiguy et al. (2000) also found
44 per cent lower mite population in colonies with bottom screen floors
as compared to untreated colonies. Harbo and Harris (2004) reported
that after nine weeks, colonies with screen floors had fewer mites, a low
percentage of mite population residing in brood cells and more cells of
capped brood, apparently by decreasing the rate at which founder mites
invade brood cells. Although present study was of three week duration
but effectiveness is depicted by significantly lower mite count after formic
acid treatment to collect remaining mite population in screen floor hives
as compared to higher residual population in wooden floor hives. Screen
floors have also been employed by various other workers to reduce Varroa
population in hive (Pettis and Shimanuki, 1999; Ostiguy et al., 2000;
Ellis et al., 2001; Sammataro et al., 2004) and brood (Harris et al., 2003).
Furthermore, its inclusion in any beekeeping management system is
further warranted as screen floors are also associated with increased
brood production (Skubida and Skowronel, 1995; Pettis and Shimanuki,
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1999; Ellis et al., 2001) and adult bee population (Ellis et al., 2003; Harbo
and Harris, 2004). However, in present study, no significant difference of
screen floors was witnessed on bee strength, brood, honey and pollen
area. Our studies are in agreement with earlier studies conducted byRinderer et al. (2003) who reported no effect on brood production and
adult bee population and Ellis et al. (2003) who reported no effect of
screen floors on pollen stores. Delaplane et al. (2005) reported that use of
screen floors reduced honey and pollen stores but favoured their use as
they exert a modest restraint on mite population growth. Moreover, their
cost benefit profile is considered good, based on an expected useful life of
10 years (Rice et al., 2004). Coffey (2007) concluded that screen floorsthough not sufficient as a stand alone treatment for Varrroa control,
could play an integral part in any integrated system.
Efficacy of formic acid
Formic acid has a strong acaricidal effect (Calderone and Nasr,
1999; Kochansky and Shimanuki, 1999; Calderone, 2000; Hood and
McCreadia, 2001; Underwood and Currie, 2004), low price, its occurrence
as a natural component of honey and has the advantage of killing mites
on adult bees as well as in sealed brood (Liebig et al., 1984; Fries, 1993).
During fumigation, the strong hydrogen bonds in formic acid cause the
vapours to act more like liquids than like gases (Laffitte, 2006).
Furthermore, it provides control for other honeybee parasites including
the honeybee tracheal mite, A. woodi (Engelsdorp and Otis, 2001), T.
clareae(Sharma et al., 2003) and possibly nosema disease (Sharma et al.,
2003; Underwood and Currie, 2004). The efficacy reported in literature
ranges from 29.6 per cent (Barbattini et al., 1994) to more than 90 per
cent (Calderone 2000; Eguaras et al., 2002) depending on doses,
modalities of application, and experimental or environmental
conditions.In the present study, formic acid 85% (5ml) applied by Cotton
swab method was found to be 71.7 per cent effective against V. destructor
and provided 85.3 per cent control over untreated hives. Three to four
applications of formic acid (65%) @ 300ml provided significant reduction
of Varroa infestation (Veen et al., 1998; Calderon et al., 2000). Formic
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acid (15ml) caused 55-60 percent mite mortality in brood cells (Calderon
et al., 2000) which was increased to 87-89 percent in trapped worker
brood (Calis et al., 1998). It is reported that generally as the
concentration of the fumigant increases, the amount of time necessaryfor effective pest control decreases and vice-versa (Harein and Krause,
1964).
Earlier studies indicated that treatment with formic acid can
increase queen mortality, damage to hatching bees (Fries, 1993) and have
detrimental effect on brood production (Westcott and Winsten, 1999;
Oserman and Currie, 2004), but this is likely to be due to a direct effect
of the acid on brood survival (Fries, 1991; Bolli et.al., 1993) and canaffect the physiology of the immature and young workers (Bolli et al.,
1993). However, in the present study of three weeks, no adverse effect on
colony strength (bees, brood) and colony stores (pollen, honey) were
observed. On the contrary, the brood area showed a significant increase
in formic acid treated A. mellifera colonies as compared to untreated
colonies. The results are in conformity with some studies which showed
that long term formic acid treatment did not damage brood and young
bees and did not limit colony development (Garg et al., 1984; Sharma et
al., 1994; Bernie and Winsten 1998; Westcott and Winsten, 1999).
Efficacy of formic acid is positively correlated with temperature and
relative humidity (Fries, 1993). It has been speculated that combination
of high temperatures and high concentrations of formic acid may
contribute to queen loss (VonPosern 1988, Underwood, 2005).
Efficacy of powdered sugar (2g/3g/frame)
Powdered sugar (Dowda Method), with a grain size between 5 and
15 micrometres does not harm the bees and becomes a small source of
feed, but does interfere with the mite's ability to maintain its hold on the
bee. It is believed to increase the bees' grooming behaviour, resulting in
greater percentage of mites to become dislodged (Macedo et al., 2002).
Combined effects of sugar and screen floor suggested that powdered
sugar works best as an amplifier of the effects of a screened bottom board
(Macedo et al., 2002). In the present study, the number of mites
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dislodged from brood frames/bees during sugar dusting (2 and
3g/frame), significantly differed from control treatment (CD = 8.0 and
42.9; p = 0.05). The number of mites fallen on sticky paper was more in
first week after application which declined in second and third week,respectively. The reason may be that in later weeks sugar may be
consumed by bees as food and thus less number of mites was dislodged
from bees. The treatment (3g/frame) showed an efficacy of 97.7 per cent
and reduced the V. destructorpopulation by 80.51 per cent as compared
to population in untreated hives. Aliano and Ellis (2005) reported that
mites continue to fall for several hours to days after dusting adult bees
with powdered sugar. Brood area was significantly increased after sugardusting (3g/frame) but no effect was observed on pollen area, honey and
bee strength.
Fakhimzadeh (2000) recorded a significantly greater mite fall per
hour in powdered sugar dusted colonies. The colonies dropped 0.17 and
5.8 mites per hour before and immediately after powdered sugar
application, respectively. Similar results were obtained by Aliano and
Ellis (2005) who reported 76.7 percent mites are fallen from adult bees by
application of powdered sugar (225g/hive). At a similar dose Macedo et
al. (2002) reported 92.9 percent mite fall in A. melliferacolonies and they
also used powdered sugar to detect and access Varroa populations in
honey bee colonies. Noirot (1988), however, cautioned that powdering
bees with dust (including talc, glucose) is not too safe for their respiratory
system.
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Chapter-VI
Summary and Conclusion
During present investigation, seasonal incidence of Varroa
destructor and its management through eco-friendly measures in Apis
mellifera colonies was undertaken. The results on above aspects are
summarized below.
Mite infestation in worker and drone brood was maximum (15 to 17%)during the month of August and September, corresponding to the highest
values of mite infestation in hive debris and on sticky paper.
Mite infestation showed a significant positive correlation withperforation of brood (r = 0.65; 0.83) (r = 0.97; 0.81) in both the sampling
methods.
Use of screen floor provided 90.85 percent reduction over traditionalwooden hives which was better in terms of efficacy over other methods.
Screen floors did not have significant effect on colony strength and stores
in A. melliferacolonies during the present study of 21 days.
Formic acid 85% (5 ml/hive) gave 71.73 and 85.36 per cent efficacy andcontrol over untreated hives, respectively with no adverse effect on colony
strength and stores.
Powdered sugar (3g/ frame) gave 97.70 and 80.51 per cent reduction inV. destructorpopulation over untreated hives whereas it was 87.21 and
81.75 at lower dose (2g/ frame).
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LITERATURE CITED
Abrol DP. 2009. Bees and Beekeeping in India. Kalyani Publishers, Ludhiana.205-360pp.
Akimov IA, Starovir IS, Yastrebtsov AV and Gorgol VT. 1988. The Varroamite the causative agent of varroatosis in bees. Morphological outline. NaukovaDumka Publishing House; Kiew, USSR: 120pp.
Akratanakul P and Burgett M. 1975. Varroa jacobsoni: A prospective pest ofhoney bees in many parts of the world. Bee World, 56: 119-121.
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Aliano NP, Ellis MD and Siegfried GD. 2006. Acute contact toxicity of Oxalicacid to Varroa destructor (Acari: Varroidae) and their Apis mellifera(Hymenoptera: Apidae) hosts in laboratory bioassays. Journal of economicEntomology, 99: 1579-1582.
Allsopp M, Govan V and Davison S. 1997. Bee health report Varroa in SouthAfrica. Bee World, 78: 171-174.
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Anderson DL and Trueman JWH. 2000. Varroa jacobsoni (Acari: Varroidae) ismore then one species. Experimental and Applied Acarology, 24: 165-189.
Anonymous 1994. An acaricidal composition and use thereof in disinfestingtreatments. www.patentsonline.com.
Anonymous 1996. How to make sticky boards. The speedy Bee.September: 5p.
Anonymous 2000b. Detecting Varroa. American Bee Journal, 5-6.Anonymous 2002a. http://www.newzeland.gov./apis/english/pdf.Anonymous 2002b. http://www.apis.admin.ch/ under Varroaand oxalic acid.Anonymous 2006. http://creatures.ifas.ufl.edu/misc/bees/varroa_mite. htm.Anonymous 2007. http://www.HawaiiBeekeepers.org/varroa.phpAntonious G, Bomford M and Vincelli P. 2009. Screening Brassica species for
glucosinolate content. Journal of Environmental Science and Health, 44(3):311-316.
Arculeo P. 1999. Trattamenti contro la Varroa con acido ossalico sperimentati in
Sicilia. LApe Nostra Amica, 4: 6-9.Bahreini R. Thamasebi GH, Nowzari J and Talebi M. 2004. A study of the
efficacy of formic acid in controlling Varroa destructor and its correlationwith temperature in Iran. Journal of Apicultural Research, 43(4): 158-161.
Bailey L and Ball BV. 1991. Honey Bee Pathology. Academic Press, San Diego 2:193pp.
Bailey SJ and Fuchs S. 1997. Experiments for the efficiency of Varroacontrolwith drone brood trapping-combs. Apidologie, 28: 184-186.
Bailey SJ, Fuchs S and Buechler R. 1996. Effectiveness of drone brood trappingcombs in broodless honeybee colonies. Apidologie, 27: 293-295.
Baker EW, Flechtmann CHW and Delfinado-Baker M. 1984. Acari domummeliponinarum Brasiliensium habitantes. 6. New species of BisternalisHunter (Laelapidae: Acari). International Journal of Acarology, 10(3): 181-189.
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Recommended