Asha Poonia Book Final

7/31/2019 Asha Poonia Book Final

1/63

Dedicated to

My Parents


2/63


3/63

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


4/63

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


5/63

1

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


6/63

2

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


7/63

3

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


8/63

4

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


9/63

5

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


10/63

6

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


11/63

7

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


12/63

8

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).


13/63

9

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)


14/63

10

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


15/63

11

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,


16/63

12

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


17/63

13

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


18/63

14

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


19/63

15

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.


20/63

16

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


21/63

17

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.,


22/63

18

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).


23/63

19

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


24/63

20

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


25/63

21

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.


26/63

22

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.


27/63

23

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


28/63

24

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


29/63

25

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.


30/63

26

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%).


31/63

27

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


32/63

28

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.


33/63

29

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


34/63

30

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


35/63

31

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.


36/63

32

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


37/63

33

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.


38/63

34

Table 5: Efficacy of powdered sugar (2g/ frame) against Varroa


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


(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.



39/63

35

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


40/63

36

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.


41/63

37

Table 7: Efficacy of powdered sugar (3g/ frame) against Varroa


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


(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



42/63

38

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


43/63

39

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,


44/63

40

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


45/63

41

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,


46/63

42

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


47/63

43

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


48/63

44

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.


49/63

45

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).


50/63

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.

Alfredo F. 2004. Fungus provides Varroa mite relief for bees. October,www.ars.uspa.gov.

Aliano NP and Ellis MD. 2005. A strategy for using powdered sugar to reduceVarroa populations in honeybee colonies. Journal of Apicultural Research,44(2): 54-57.

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.

Alves SB, Flechtmann CH and Rosa AE. 1975. Varroa jacobsoni Oudemans,1904 (Acari: Mesostigmata, Varroidae) also in Brazil. Ecossistema, 3: 78-79.

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.


51/63

ii

Ball BV. 1989. Var

Date post:	05-Apr-2018
Category:	Documents
Upload:	asha-poonia
View:	218 times
Download:	0 times

Asha Poonia Book Final

Documents