Dedicated to My Parents

CONTENTS

Chapter I. II. III. IV. V. VI.

Description INTRODUCTION REVIEW OF LITERATURE MATERIALS AND METHODS RESULTS DISCUSSION SUMMARY AND CONCLUSION LITERATURE CITED

Page(s)

1-5 6-18 19-25 26-37 38-44 45

I-XIV

LIST OF TABLES

TABLE NO. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

PARTICULARS

Incidence of Varroa destructor on Apis mellifera brood (May, 2008 to April, 2009) Effect of Varroa destructor incidence on perforated brood cells Efficacy of Screen floor against Varroa destructor in Apis mellifera colonies Effect of Screen floor on colony strength and stores in Apis mellifera colonies Efficacy of Formic acid against Varroa destructor in Apis mellifera colonies Effect of Formic acid on colony strength and stores in Apis mellifera colonies Efficacy of Powdered sugar (2g/frame) against Varroa destructor in Apis mellifera colonies Effect of Powdered sugar (2g/frame) on colony strength and stores in Apis mellifera colonies Efficacy of Powdered sugar (3g/frame) against Varroa destructor in Apis mellifera colonies Effect of Powdered sugar (3g/ frame) strength and stores in Apis mellifera colonies on colony

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 destructor Anderson and Trueman, an ectoparasitic mite of brood and adult bees, is a serious pest of Apis mellifera L. It belongs to order mesostigmata and family varroidae. Varroa mite 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 Varroa mite 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 cerana Fab.) in 1904 in Java (Oudemans, 1904). The mite, Varroa jacobsoni began its parasitic relationship with Asian honey bee by laying eggs (up to six eggs) in drone brood cell. The patterns of speciation among

the mites are mirrored in the patterns of speciation among the mites native host A. cerana. This infers that mite and bees have been coevolving (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 mellifera was introduced into Asia thirty five years ago. Not long after this bees introduction Varroa mite 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 Varroa consists 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 and Trueman, 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. jacobsoni and V. destructor in Asia but A. mellifera is only parasitized by V. destructor worldwide. Several mitochondrial haplotypes (17-18) of V. destructor have been described but only two of them are

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. cerana from 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 of V. destructor parasitism has also been observed (Calderon et al., 2007). V. destructor is

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 Apis species 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

studies on its incidence and management are lacking from this area. In this retrospective, present study was conducted with following objectives:

1. 2.

To study effect of mite incidence on brood To evaluate different control measures against Varroa destructor

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 cerana up to 1983, after that A. mellifera was introduced in India and it has almost completely replaced A. cerana and 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 on its 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 of Varroa In 1904 in Java, Indonesia, Oudemans described the Varroa jacobsoni mite in brood cells of drone larvae of A. cerana (Oudemans, 1904). The mite

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 Varroa can 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. cerana bees 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 Varroas preference 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) and they 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 Varroa forms 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

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 of Varroa 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 and Baker, 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 Varroa mite has not yet spread to are Australia and the central part of Africa (Oldroyd, 1999).

2.3 Causes of Rapid Spread of Varroa V. destructor has 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 not yet 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 Varroa mite 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 mellifera colony 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)

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. destructor has 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-mediated transmission 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 of Varroa mite The ectoparasitic mite V. destructor is 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 females hatch (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 days in 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 of Varroa. 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 of Varroa Proper and scientific detection of Varroa is 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 of Varroa 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 of V. destructor. Powdered sugar is also used by many workers to detect Varroa population in A. mellifera colonies (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 of Varroa, 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 of Varroa 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 27 days, 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 a frame 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 Varroa mite 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 of A. cerana for 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 Varroa population (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 a screen to separate fallen Varroa from 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 Varroa populations in A. mellifera hives. Sulphur dusting is commonly applied in India to control parasitic mites in A. mellifera colonies 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. destructor control (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. destructor are neem oil (Melathopoulous et al., 2000), vegetable oil (Le Conte et al., 1998), mineral oil (Imdorf et 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 Varroa mites 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 Varroa in races of A. cerana were 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 and Hoopingarner, 1997). The pupal period influences the number of mites completing development. Shortening this time results in fewer Varroa reaching 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 Varroa population but it is difficult to maintain in A. mellifera colonies 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 susceptibility of Varroa mites 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).

18

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 first phase 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. mellifera colonies with three replications each and compared with control treatment. Materials used and methodology adopted for

19

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. destructor were recorded in each hive. 3.2.3 Per cent infestation of Varroa destructor Pest potential of V. destructor in terms of per cent infestation was calculated at each fortnight by using following formula: No. of mites present on brood % infestation= Total no. of brood examined Preference of host selection by V. destructor within 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 x100

20

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. mellifera colonies). 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. destructor infested 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.

21

mellifera colonies, 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. mellifera colonies 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.

22

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 the colonies 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. destructor population 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
23

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 of V. 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 evaluated in 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
24

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:
r=

(X X )(Y Y ) (X X ) (Y Y )
2

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. destructor population in hives. Effects of the treatments were also measured on the colony strength and stores.

25

CHAPTER-IV

RESULTS
The results of the present investigations entitled Incidence of Varroa destructor Anderson and Trueman (Acari: Varroidae) and its management in Apis mellifera L. colonies have been presented under the relevant headings of this chapter. Incidence of V. destructor on A. mellifera brood 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. destructor as 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 of A. mellifera in 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 cells in 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%).

26

Mean per cent mite incidence in brood ranged from 0 to 15.5 and 2 to 16 per cent in A. mellifera colonies 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 of V. destructor towards the drone brood but the difference was non-significant as revealed by t-test. Colonies infested with V. destructor showed 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 of V. destructor incidence on brood perforation showed heavy perforation of brood cells coinciding with the season of high mite infestation (Table 2). Perforation of brood was first detected in the first fortnight of May (5 and 7 sealed brood cells) when V. destructor population 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. mellifera colonies 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

27

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.

28

Table 1: Incidence of Varroa destructor on Apis mellifera brood (May, 2008 to April, 2009)
Period of observation
Brood cells examined Brood cells with mites (Hive debris) (N=50) W: D Worker Drone Mean cells cells incidence ratio (%) (D) (W) 7.00 7.50 7.25 0.93 (14.00) (15.00) (14.50) 7.50 8.00 7.75 0.93 (15.00) (16.00) (15.50) 0.00 0.00 0.00 0.00 (0.00) (0.00) (0.00) 0.00 0.00 0.00 0.00 (0.00) (0.00) (0.00) 0.00 0.00 0.00 0.00 (0.00) (0.00) (0.00) 0.00 0.00 0.00 0.00 (0.00) (0.00) (0.00) 0.00 0.00 (0.00) (0.00) 2.50 2.25 (5.00) (2.50) 3.00 (6.00) 3.50 (7.00) 1.50 (3.00) 1.75 (3.50) 1.95 (3.90) Brood cells with mites (Sticky paper) (N=50) W:D Worker Drone Mean cells cells incidence ratio (%) (D) (W) 7.00 7.00 7.00 1.00 (14.00) (14.00) (14.00) 7.50 8.50 8.00 0.88 (15.00) (17.00) (16.00) 6.50 6.50 6.50 1.00 (13.00) (13.00) (13.00) 6.50 7.50 7.00 0.86 (13.00) (15.00) (14.00) 7.25 0.93 7.00 7.50 (14.50) (14.00) (15.00) 7.00 7.50 7.25 0.93 (14.00) (15.00) (14.50) 6.00 3.00 (12.00) (6.00) 2.00 1.00 (4.00) (2.00) 3.00 (6.00) 2.00 (4.00) 1.50 (3.00) 1.00 (2.00) 4.95 (9.90)

1st to 15th, May 16th to 31st, May 1st to 15th, June 16th to 30th, June 1st to 15th, July 16th to 31st, July 1st to 15th, August 16th to 31st, August 1st to 15th, September 16th to 30th, September Mean tcal

50 50
50 50 50 50 50 50

50 50

50

2.35 1.55 (4.70) (3.10) 1.31 (NS)

5.45 4.45 (10.9) (8.90) 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

29

Table 2: Effect of Varroa destructor incidence on perforated brood cells

Period of observation Brood cells examined 50 50 50 50 50 50 50 50 50 50 50 Hive debris No. No. of of perforated mites brood cells 36.50 5.00 (10.0) 40.05 5.50 (11.00) 16.00 0.00 (0.00) 0.50 0.00 (0.00) 22.50 0.00 (0.00) 24.00 0.00 (0.00) 29.00 3.50 (7.00) 25.00 5.00 (10.00) 33.00 4.50 (9.00) 17.00 5.00 (10.00) 24.35 2.80 (5.70) 0.65 Sticky paper No. of No. of mites perforated brood cells 74.00 7.00 (14.00) 86.50 7.50 (15.00) 37.00 5.00 (10.00) 24.50 5.00 (10.00) 47.00 5.50 (11.00) 38.00 5.50 (11.00) 30.00 4.50 (9.00) 14.50 0.00 (0.00) 23.5 2.50 (5.00) 21.50 3.50 (7.00) 39.65 4.60 (9.20) 0.83

1st to 15th, May 16th to 31st, May 1st to 15th, June 16th to 30th, June 1st to 15th, July 16th to 31st, July 1st to 15th, August 16th to 31st, August 1st to 15th, September 16th to 30th, September Mean r (Mite Vs Perforation)

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

30

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. mellifera colonies 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. destructor population 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.

31

Table 3: Efficacy of Formic acid against Varroa destructor in Apis mellifera colonies
Treatment Pre Treatment Number of mites/hive after treatment on sticky paper 7 DAT 14 DAT 21 DAT Total Mean after After final treatment Treatment * Formic acid (5 ml 26.00 of 85%) Control CD (p = 0.05) % efficacy % reduction over control

25.00 11.90

21.60 9.00

20.00 66.60 11.00 31.90 4.86 71.73 85.36

22.20 10.60

16.0 140.5 8.86

15.50 N.S.

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 mellifera colonies
Treatment Formic Pre acid Bee strength Brood Area (frames) (cm2) Honey (g) Pollen Area(cm2)

6.50 6.00 N.S. 6.50 6.50 N.S.

734.60 760.00 N.S. 939.00 736.60 188.41

147.30 158.30 N.S. 105.30 156.60 N.S.

250.60 248.30 N.S. 263.30 294.00 N.S.

(5 ml of 85%)

treatment Control CD (p = 0.05) Formic After treatment acid

(5 ml of 85%) Control CD (p = 0.05)

NS = Non-significant

32

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 of V. 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 244.0 to 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.

33

Table 5: Efficacy of powdered sugar (2g/ frame) against Varroa destructor in Apis mellifera colonies
Treatment Pre Treatment 7 DAT Powdered sugar @2g/frame Control CD (p = 0.05) % efficacy % reduction over control Number of mites/hive after treatment on sticky paper 14 DAT 21 DAT Total Mean after treatment After final Treatment*

6.0 9.3 N.S.

13.3 8.5

16.6 10.5

17.0 2.0

46.9 21.0 8.04

15.60 7.00

4.0 99.5 4.65

87.21 81.75

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 mellifera colonies
Treatment Powdered sugar @2g/frame Control CD (p = 0.05) Powdered sugar @2g/frame Control CD (p = 0.05) Bee strength Brood Area (frames) (cm2) Honey (g) Pollen Area(cm2)

4.10 5.00 N.S. 4.00 4.50 N.S.

244.00 750.00 N.S. 500.50 750.00 N.S.

244.30 200.30 N.S. 303.10 210.00 N.S.

162.30 144.00 N.S. 222.00 155.00 N.S.

Before treatment

After treatment

NS = Non-significant

34

Efficacy of powdered sugar (3g/frame)

Powdered sugar dusting (3g/frame) in A. mellifera hives 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 of Varroa mites (59 mites/ hive) were recorded from sticky paper of treated hives as compared to mites in untreated A. mellifera colonies (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. mellifera colonies. The treatment showed an efficacy of 97.70 and 80.51 per cent reduction in V. destructor population 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

35

237.5 g in treated A. mellifera colonies 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 to other 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.

36

Table 7: Efficacy of powdered sugar (3g/ frame) against Varroa destructor in Apis mellifera colonies
Treatment Pre Number of mites/hive after treatment on sticky paper After final

Treatment 7 DAT 14 DAT 21 DAT Total Mean after

treatment Treatment* Powdered sugar @3g/frame Control CD (p = 0.05) % efficacy % reduction over control

16.50 13.30 NS

78.50 13.30

69.00 16.60

29.50 177.00 17.00 46.90 42.90

59.00 15.60

4.00 135.50 8.86

97.70 80.51

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 mellifera colonies
Treatment Powdered sugar Before treatment @3g/frame Control CD (p = 0.05) Powdered sugar After treatment @3g/frame Control CD (p = 0.05) Bee strength Brood Area (frames) (cm2) Honey (g) Pollen Area(cm2)

5.50 4.50 N.S. 5.00 4.50 N.S.

720.00 750.00 N.S. 1005.50 801.00 122.60

161.20 210.00 N.S. 237.50 223.70 N.S.

128.00 113.30 N.S. 118.00 8.00 62.60

NS = Non-significant

37

CHAPTER V

DISCUSSION
The purpose behind the present investigation was the occurrence of V. destructor in epidemic form in A. mellifera colonies 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. destructor and 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. mellifera colonies. 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 of Varroa destructor on Apis mellifera brood Present investigation showed maximum infestation of V. 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 during seasonal occurrence of V. destructor in A. mellifera colonies from April to
38

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 those observed 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. destructor infestation and reproduction. During the dearth period, when the brood area decreased due to low colony stores, multiple infestations with V. destructor and 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,

39

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 positive correlation (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. clareae infestation. Hosmani et al. (2005a) and Sihag (1990) recorded higher number of perforated brood cells during peak infestation period (May) T. clareae population in A. mellifera colonies at Hisar (Haryana, India). Worker and drone brood infestations were compared in the present study. Although preference of drone brood by V. destructor was observed but it was not significant. Woyke (1987) reported that the mean infestation rate of drone brood by V. jacobsoni was 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

40

mind ten eco-friendly measures were evaluated against V. destructor in 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. Before evaluation, 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 as compared to normal wooden bottom board. Webster et al. (2000) reported that in Varroa infested 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 of V. destructor re-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,

41

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 by Rinderer 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 floors though 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

42

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 necessary for 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 can affect 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

43

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. destructor population 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 sugar dusting (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. mellifera colonies 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 with

perforation 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 traditional wooden 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. mellifera colonies during the present study of 21 days. Formic acid 85% (5 ml/hive) gave 71.73 and 85.36 per cent efficacy and control 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 in V. destructor population over untreated hives whereas it was 87.21 and 81.75 at lower dose (2g/ frame).

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