POSSIBLE CAUSES FOR THE COLONY COLLAPSE DISORDER (CCD) Zoran Stanimirović*, Nevenka Aleksić* and Jevrosima Stevanović*

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1 POSSIBLE CAUSES FOR THE COLONY COLLAPSE DISORDER (CCD) Zoran Stanimirović*, Nevenka Aleksić* and Jevrosima Stevanović* *The University of Belgrade, Faculty of Veterinary Medicine, Belgrade, Boulevard oslobodjenja 18, Serbia Abstract. The possible reasons for the diminishing of bees i.e. for the phenomenon known as the Colony Collapse Disorder (CCD) are given, as well as the biology, clinical findings, diagnosis and prevention/protection of honeybee colonies from American foulbrood, nosemosis, varoosis and certain viral infections. The importance of good condition of honeybee colonies, adequate diet supply, together with the reinforcing of hygienic and grooming behaviour which influence the higher resistance to aforementioned diseases is underlined. Further on, the biological means to combat the diseases of bees and bee colonies, which contribute to the development of ecological honeybee keeping and production of bee products devoid of residua of pharmaceuticals used in conventional bee keeping. Key words: CCD, American foulbrood, nosemosis, varroosis, honeybee viruses, biological means of combat, hygienic and grooming behaviour COLONY COLLAPSE DISORDER (CCD) Massive death of honeybees was described worldwide long ago; for example it happened in Ireland in 950, 992 and At the beginning of the 20th century, in spring of 1906, on the White Island (Great Britain) the majority of beekeepers lost their bee colonies. Furthermore, American beekeepers occasionally suffered from heavy losses. In the Cache Valley (Utah, USA) in 1903 two-thousand bee colonies died from mysterious phenomenon of disappearing throughout cold winter and spring. More recently, in 1995, beekeepers in Pennsylvania lost 53% of their bee colonies (Oldroyd, 2007). 1

2 Colony Collapse Disorder (CCD) is a phenomenon which was not known until recently but has become the most serious disease and cause of sudden death of honeybee colonies characterised by the disappearance of adult bees in and in front of beehives. Both honey and bee bread are usually present in abandoned beehives as well as the indications of recent brood rearing. Sometimes the queen and few bees can be found in the nest. Characteristically, in the hives without bees honey robbery appears late and the hives are invaded by usual pests (wax moth Galleria mellonella and small hive beetle Aethina tumida) more slowly than expected or are not at all. CCD is a multifactorial disease of honeybee colonies, i.e. it cannot be claimed to be caused by one agent only. The fact leads to the difficulty of recommendation of a single remedy which can prove the most efficacious (Stanimirović et al., 2008a,b,c). The most frequent causes of CCD are: - The deficiency in high-quality diet (bee bread and honey) - Bacterial infections (American foulbrood) - Fungal diseases (nosemosis and ascospherosis) - Parasitic infections of honeybee colonies, most often with Varroa destructor and Acarapis woodi - Mixed viral infections of honeybee colonies - Management in the apiary THE DEFICIENCY IN HIGH-QUALITY DIET (BEE BREAD AND HONEY) Global climatic changes, pollution and chemisation in all human activities, particularly in agricultural production, lead to disturbances of ecosystems, diminishing or problematic herbal production and, consequently, to reduced production of sufficient high-quality food for honeybee societies. Intensive (frequent and widespread) application of pesticides results in reduced production of high-quality pollen, which is the basis for high-quality bee bread preparation, an indispensable source of proteins. On 2

3 the other hand, the reduced livestock, especially the reduction in the number of sheep and goats, which means diminishing manure production, result in significant fall in high-quality nectar and pollen production since many herbal species have diminished or completely disappeared from the fields and pastures (Stanimirović et al., 2008a,b,c). For example, in the regions of Homolje and Peshter (Serbia) solely the number of domestic animals dramatically decreased being one tenth of the number in 1989, which also means similar decline in manure production. Having in mind that a sheep produces approximately 500 g of manure on average, the consequences are more easily understandable. On the other hand, the number of honeybee colonies rose meanwhile due to the idea that households can increase their budget by beekeeping. In attempt to earn as much as possible honeybee keepers do not mind the biological needs of their bees, i.e. they do not only deprive them of the surpluses in honey but also take away the one from the brood chambers. This honey is to belong to the bees only and it is not to be removed, since it is not nectar but represents a source of energy, essential amino acids, micro- and macroelements, vitamins and other active substances. It is a biologically active material manufactured from nectar with which it is mixed as well as with the secretion of bees glands. Besides, the honey from brood chambers comprises considerable amounts of pollen which under the influence of acids as well as of the enzymes of the bees exocrine glands bursts at some time and its content is mixed with the honey itself. That is why this honey is an extremely high-quality energetic proteinrich diet of the bees (Stanimirović et al., 2008a,b,c). It is the most important prerequisite for the wintering and rapid development of the colonies in spring. When depriving a colony of the honey from brood chambers beekeepers do severe damage to the bees as well as to themselves. Taking away this honey also means taking the residua of various preparations (amitraz, coumaphos, cymiazole hydrochloride, flumethrin, fluvalinate, dicyclohexylamine) used for the treatment of the bees in brood chambers (Stanimirović et al., 2003a,c, 2005a, 2006, 2007a,b; Pejin et al., 2006; Stevanović et al., 2006, 2008). Thus, the residua got into the honey either directly or from wax which had been contaminated previously. Such honey is not to be used by humans. In addition, apart from being deprived of honey bees are deprived of the highest-quality diet which strongly influences the ability of development and survival of the colonies and the immunological potential of each individual bee as well as the colony as a whole 3

4 (Stevanović, 2007; Stanimirović et al., 2008a,b,c). Due to the deprivation of honey the potential for development declines, the age polyethism is altered and by the addition of sugar as a substitute to the honey (by no means can sugar substitute honey) bees are exhausted additionally and the colony will not be ready for the main honey harvest. The destruction of the proportion of bees of different age in the colony leads to the decline in the number of cleaning bees (aged from 15 to 17 days, Arathi et al., 2000) which results in the diminished defensive potential to the infective agents always present in the hive (Paenibacillus larvae, Nosema apis, Nosema ceranae, Ascosphera apis, Varroa destructor and various viruses) (Stanimirović et al., 2005b, 2008a,b,c). Being capable of regulating humidity the honey from brood chambers influences the microclimate, temperature and the exchange of gases in hives. The excessive moisture, which can be detrimental to the thermoregulation in a hive, is absorbed by the honey, which helps the bees with the regulation of moisture and temperature in the brood. Honeybees efficaciously maintain the temperature in brood chambers at approximately 34.5 o C regardless of the environmental conditions. If the temperature is higher or lower, the bees will develop into seemingly normal adults, but with damaged reception of information and memory. Worker bees reared on temperatures lower than optimal tend to get lost in the field and are incapable of performing waggle dances efficaciously. If bee colonies fail to maintain the temperature and moisture in the brood continuously, symptoms similar to those of the CCD will develop (Oldroyd, 2007). If the humidity declines (in summer at high temperatures) the water from the honey in the brood chambers is released, which helps the maintenance of optimal moisture and temperature in the hives. Thus, the need for fanning bees and water carriers declines and worker bees can devote themselves to honey harvest and contribute to the honey yield (Stanimirović et al., 2008a,b,c). It is well-known that excessive moisture is a prerequisite for the appearance of fungi (the causative agents of chalk brood, Ascosphera apis, and nosemosis: Nosema apis and Nosema ceranae). In spring the temperature increases both outside and in the hives, the air becomes drier and if the bee colony is weak and there is no honey substantial decline in humidity is unavoidable. As a result, the brood dries, suffers from lack of moisture and dies thus providing conditions for bacterial infections. By all means Varroa destructor contributes to the 4

5 damage being not only an ectoparasite of the brood and adult bees but also a vector of mixed viral infections. In addition, apart from propolis, the honey from brood chambers has an antimicotic and bacteriostatic effect (Stanimirović et al., 2008a,b,c). Besides propolis and honey, pollen also contributes to the immunological properties and potential of honeybee colonies but in conditions described afore either there is a shortage of pollen or it is of weak quality. Pollen does not maintain its quality during the pasture season. The best is the one from early polliniferous plants (ephemeral flowering plants), the pollen of hazelnut, willow, from fruits, meadow grasses, corn, sophora etc. Global climatic changes have influenced the dynamics of flowering as well as the use of pollen of certain polliniferous plants. For example, these years there was little pollen of ephemeral flowering plants, hazelnut and plum and it was difficult to use due to bad weather conditions (Stanimirović et al., 2008a,b,c). Various pesticides can also influence the quality of pollen and nectar. Apart from numerous other pesticides used on vegetable, fruit and crop farming the use of imidacloprid- and fipronil-based pesticides has recently increased. These are poisonous for honeybees, acting by contact and after ingestion. Imidacloprid and fipronil are neonicotinoids. They are absorbed by plants through their roots and distributed into higher organs: flowers, fruits, leaves and seeds, where they remain for a long time and accumulate in the nectar and pollen (Šovljanski, 2008a,b). Neonicotinoids, such as imidacloprid, acts on acetylcholine receptors while fipronil influences the chlorine channels thus enhancing the permeability of neurons; in other words, fipronil is capable of blocking the passage of chloride ions through the GABA receptor and glutamategated chloride channels, components of the central nervous system of insects. While seeking for food and collecting nectar bees remember the scent of flowers and make some kind of maps which they use in the future. Unfortunately, the aforementioned insecticides do damage to the brain centres responsible for memory and orientation, which is why the bees become disoriented and incapable of returning to their hives; consequently they wander and eventually die of starvation (Oldroyd, 2007). In addition, the poisoned bees are agitated, their movements are uncoordinated; for example, they can hang from the sunflowers; at first they are very active but soon become apathetic, suffer cramps, droop and die in the end (Stanimirović et al. 2008a,b,c). 5

6 AMERICAN FOULBROOD Infective diseases of bees pose serious problems which influence the development of beekeeping in Serbia, as well as worldwide. Among them American foul brood (AFB), varroosis, nosemosis, viral and fungal diseases are of utmost importance. From a health protection and financial viewpoint American foulbrood is a hindrance claimed that had reached panzootic proportions a long time ago (Đuričić and Radojičić, 2000). It has been spread in various numbers of beehives in Serbia for several decades and is believed to have been present in all regions of the country (Đuričić et al., 2001; Laušević et al., 2001). American foulbrood is a highly contagious disease of brood, enzootic in the beginning, but can reach panzootic dimensions due to its assertiveness, capability of maintenance and slow spread in an apiary and the surroundings (Đuričić et al., 2001). Two forms of the bacterium which causes the disease can be distinguished: the mobile vegetative bacillary form and the spore incapable of any movements. Spores of the Paenibacillus larvae are extremely resistant to environmental factors and chemicals. The spores can survive in old hives as long as 35 years and still remain infective. At 110 o C (autoclave) they remain viable 30 minutes, in boiling wax at 125 o C 20 minutes and in dry soil they maintain their infectivity 228 days. The bacterium is in connection with the brood of honeybees (larvae) only. The infection occurs by spores of P. larvae that were brought into the brood by nursing bees. Vegetative forms develop from spores after the brood cells are closed. The infection of the diseased brood is extremely severe as the number of P. larvae per larva can exceed one billion, which is of utmost importance from the epizootic and healthcare viewpoint (OIE Manual of standards Diagnostic Tests and Vaccines, 2000). The diseased and dead bees, scales, honey, pollen and the interior of the hive of a diseases colony are the primary source of infection. Furthermore, the spores of P. larvae can be easily mechanically transmitted by Varroa destructor and adult wax moths. The honey from the honey chamber of infected hives is the secondary source of infection and is the cause of recrudescences. Young nursing bees disperse the spores inside the hives, and the infection is spread to other colonies by beekeepers when handling hives, moving weak and infected colonies to pastures, swarm trading, honey 6

7 robbery, lending/borrowing tools, preparing comb foundations from unsterile wax etc. The route of infection is oral, with spores only rather than with vegetative forms. Clinical signs can be seen on sealed brood as discoloration and changes in the configuration and integrity of cappings (Figure 1). The brood itself becomes incompact, scattered. After removing the cappings changes in the colour of the larvae become visible; the colour converts from as white as nacre to greyish yellow, creamy brown and dark brown in the end. Infected larvae lose their shape, are not sickle-like any more but grow into a soggy amorphous mass. If such a larva is stung with a match, 4-5 cm long sticky threads brown in colour stretch from its body; it is an indication for sending samples to the laboratory where the diagnosis is to be confirmed. Further on, as the disease progresses, scales which can hardly be removed from the bottom of the cells are formed. A B C Figure 1. Clinical diagnosis of American foulbrood A. A. Scattered sealed brood; B. Changes in the cappings of sealed worker bee brood (changes in colourlighter covers with perforations); C. Ropiness test : a larva is stung with a match, 4-5 cm long brown threads stretch from its body 7

8 The process of hydrolysis of diseased larvae and their disintegration into scales last approximately two months. The colony gets weaker and finally becomes the victim of numerous wax moths and honey robbery. The diagnosis is confirmed in the laboratory after the isolation of the pathogenic agent. The material for diagnosis is a frame of a diseased colony which is to be wrapped in paper. Sacbrood, European foulbrood and varroosis should be taken into consideration as a differential diagnosis. Unfortunately, recently beekeepers as well as some experts tend to take various prophilactic measures against American foulbrood such as administration of grease patties containing antibiotics which are sold over the counter (Đuričić et al., 2001). The preventive application of antibiotics is proposed not only by individuals, but also by pharmaceutical industry. In addition, private manufacturers of medicines for honeybees recommend the application of oxytetracycline in patties on the basis of the results of Wilson et al. and Kulinčević et al., which proved that sugary-oily patties with oxytetracycline can be used successfully to control American and European foulbrood when used at the beginning of the diseases (Mlađan and Živanov, 1996). Loss resulting from American foulbrood which has still been present proves that this is wrongly believed. In addition, the use of tetracyclines is prohibited. A team of Swedish and Serbian authors confirmed that there is no reason for the use of antibiotics to control American foulbrood. They found that there are four strains of Paenibacillus larvae (ERIC I, ERIC II, ERIC III and ERIC IV) which differ not only in resistance to high temperatures but also in their pathogenicity and clinical signs capable of inducing. It is to be pointed out that there are differences in germination (Figure 2), infectivity and the consequences of infections caused by various strains of P. larvae alone and in combination (Forsgren et al, 2008). The inspection of colonies for infection with American foulbrood takes place in September or October. There is no specific treatment of larvae which are diseased already, but the following measures of control according to law must be applied: closing the infected apiary; destruction of all infected and dilapidated hives together with the combs and bees (burning and burrowing); disinfection of tools and hives; prohibition of rearing colonies without queens and prevention of swarming in the infected apiary; disinfection of the apiary and tools used in the process (20 % formaldehyde or 6 % sodium hydroxide). Diagnostic testing for AFB must be done in all apiaries in the 8

9 vicinity of the infected (up to 3 km far). After two months the testing is repeated in the infected apiary and if the results are negative the infection is considered not to be present any more. Figure 2. Differences in germination of the four genotypes of Paenibacillus larvae at various temperatures and in the presence of antibiotics (Forsgren et al., 2008). The best means to control AFB is rearing strong colonies of autochthonous ecogenotypes with pronounced hygienic and grooming behaviour as well as taking care of biological needs of honeybees as live creatures (Stanimirović et al., 2002, 2003c). In addition, biological means of combat (replacement of old queens and old combs) and maintaining hygienic conditions of beekeeping contribute to the production of bee products free from residua but with all autochthonous biological features. Having in mind the aforementioned, it is clear that AFB is not connected directly with CCD (Oldroyd, 2007), but if present latently, which occurs frequently, leads to the disturbances in the immunity of bee colonies, altered age polyethism and thus the shortage in high-quality diet and weak hygienic conditions in the colony, which contributes to CCD (Stanimirović et al., 2008a,b,c). 9

10 NOSEMOSIS Nosemosis is a protozoal disease caused by the microsporidium Nosema apis (Figure 3) which is thought to be in cohabitation with the honeybee for over 60 million years. There are several successive stages in the lifecycle of Nosema apis: spora, planont, meront, sporont and sporoblast. The development of spores and sporulation depends on the temperature, the optimum being between 30 and 34 o C. Nosema apis develops in the cells of the adult bee s midgut (Mlađan et al., 2000). The infection is not only specific regarding the tissue, but also the type of the cells which are parasitised. Figure 3. Spores of Nosema apis Spores of Nosema apis remain infective in bees cadavers after being kept at 4 o C and % of relative humidity at least 81 days. Spores retain their infectivity in water or when dried even after 93 days. Collecting samples for laboratory analysis is of utmost importance since by clinical means putative diagnosis is obtained only. The best samples to collect are the bees from the flight entrance and from the top bars of the frames. The best time to identify the spores is after the winter, at the beginning of the main season. It is difficult to prove the presence of the spores in summer. There is a slight increase in the number of infected bees in autumn. Spreading of the disease occurs in the hive, among the colonies and among the apiaries. In each occasion, forage bees are the primary vectors of nosemosis. The infected bees lifespan is shorter when compared to the uninfected; it might even be 25 to 58 % shorter than expected (Mlađan et al., 1990). It leads to the replacement of 10

11 infected queens (clustering). In addition to this, orientation flights of young worker bees are delayed, the intake of nectar and pollen decreases or is even omitted, the bees are exhausted and the colony collapses. It is a common knowledge that faecal contamination of combs, frames, flight entrance and the walls of the hives is characteristic of nosemosis (Figure 4), as well as a huge number of dead bees on the bottom board (Figure 4). A B Figure 4. Clinical signs of nosemosis A. A frame with honey contaminated with faeces of diseased bees B. A contaminated flight entrance and the front side of a hive The discoloration of the midgut is a commonly seen pathological sign (Figure 5). In bees infected with N. apis the midgut is swollen and milky white in colour; as the disease progresses, the oedema diminishes and the colour changes into as white as lime. The discoloration is not observed at the beginning of the infection, but is present after some time; clinical signs are not present in bees younger than 21 days. The prerequisite for the prevention of nosemosis is the proper location of the apiary, strong colonies, availability of sufficient fresh drinking water and accurate diagnosis made in time, which enables the efficacy of control measures. Unfortunately, frequently it is not the case. For that reason large number of honeybees is infected in certain regions in Serbia (Mlađan et al., 1990). 11

12 Figure 5. Clinical changes on the midgut (colour changes: A, B) and midgut epithelium (C) of honey bee workers infected with Nosema apis. Comparative laboratory and field investigations on drugs for nosemosis (Furgala and Boch, 1970) revealed that bicyclohexylammonium fumagillin suppresses infection with N. apis without adverse effects, whilst the efficacy of paromomycin and sodium ethylmercuric thiosalicylate was negligible, the latter being highly toxic. Van Steenkiste and Jacobs (1980) after having completed researches on bicyclohexylammonium fumagillin, oxyquinoline sulphate and hexamethylenetetramine reported that only the first was efficacious against nosemosis in honeybees. It was proved that fumagillin strongly affected Nosema apis, which was not true for iodochlorhydroxyquin (Sugden and Furgala, 1979). Although being among many drugs possibly dangerous for humans (Stanimirović at al., 2007a,b, 2008c), if used properly fumagillin still remains the best medicine for suppressing nosemosis caused by Nosema apis. 12

13 A B Figure 6. Nosema spores (Fries et al, 2006) A. Nosema ceranae B. Nosema apis Nosemosis can be caused with another microsporidium, Nosema ceranae (Figure 6), which was proved worldwide, excepting in Australia, where it was not confirmed and in Africa, where it was not investigated. The findings of Klee et al. (2007) indicated its presence in certain regions of south Serbia. Extensive investigations on the topic are in progress. It is to be emphasized that Nosema ceranae induces unprecedented symptoms in honeybee colonies different from the ones that have ever been described in the infection caused by N. apis. The most affected in the hives are the worker bees especially in the period of intense activity. The diseased bees usually die far from the hives, which leads to progressive decline in their population without noticeable cadavers and can be detrimental for the colony due to reduced amounts of nectar and pollen. Spores of Nosema ceranae are capable of surviving for longer periods of time, similarly to those of N. apis, which contributes to quick spread of the disease. It is found that in the most affected regions re-infections are very frequent and occur after two to four months. Until recently, the diagnosis was difficult to obtain, but now it is possible due to molecular genetics methods. According to the proposals of European experts, the treatment of nosemosis must be completed with detailed disinfection of the equipment, tool and hives with flame and acetic acid. There are suggestions that N. ceranae is not a new pathogen but has always been present in the hives of the European honeybee and prevailed when its concurrent, N. apis, was defeated due to the inadequate use of fumagillin; thus N. ceranae occupied the empty ecological niche (Stanimirović et al., 2008c). 13

14 Nosema ceranae attack the guts of adult honeybees and when present in large bumbers, lead to their disorientation, which can be related to CCD (Oldroyd, 2007). In addition, this endoparasite provokes and maintains a high-level energetic stress, which results in high food consumption, continuous hunger and heavy agitation of bees (Mayack and Naug, 2009). They leave the hives even in bad weather conditions which contributes to a decrease in their number, alter the age polyethism and contributes to CCD (Stanimirović et al., 2008a,b,c). CHALKBROOD Chalkbrood is a disease of the honeybee which is caused by the fungus Ascosphaera apis. a) Symptoms on the larvae in the brood (Figure 7) At the beginning of the infection the larvae are pale yellowish in colour, soft, smooth, their shape varies and as the disease progresses they grow light yellow, become rough, their consistence is skinny and can be fragile. They are smothered in white mycelium wrappings which thicken rapidly and in a short time fill the whole space in the cells. The mycelium adheres to hind part of the larva whilst the head remains free, dry and resembles a button. Further on, the size of old mummified larvae decline due to dehydration and they seem to be transformed into chunks of chalk; hence, the name of the disease. The larvae in the advanced disease are dark dull green. b) Symptoms on the frames with the brood (Figure 8) Infected young larvae, which still seem healthy, are usually scattered among healthy brood, whilst the older ones, already mummified, are in sealed cells or sometimes in cells that the workers already opened. Generally, the cappings appear normal, but can be mottled or slightly concave. When the combs with sealed cells are cut lengthways, the mummified larvae (Figure 9) easily fall out. When the mycelium penetrates the cappings and covers them from outside, sealed broods seem as if it was sprinkled with flour, lime or greyish dust. 14

15 A Figure 7. The symptoms of chalkbrood on larvae in the brood A B The beginning of the infection: pale yellow larvae Advanced infection: dull dark green larvae B A B Figure 8. The symptoms of chalkbrood on the frame with brood A. Frame with scattered infected larvae among healthy brood B. Mummified larvae on the bottom board of a hive 15

16 The tentative diagnosis is based on the clinical signs, time of the appearance and the absence of the disease in adult worker bees. The accurate diagnosis can be obtained in the laboratory after microscopic assessment of mummified larvae (Figure 9) or by isolation of Ascosphaera apis in vitro. The samples for diagnosis are frames with mummified drone or worker brood or parts of brood sized 10 x cm. Each sample must be packed separately in a paper box. Figure 9. Mummified larvae suitable for the laboratory diagnosis of chalkbrood There is no registered chemical for the treatment of chalkbrood. If the disease is benign, it is sufficient to remove, destroy (burn) and replace frames with diseased brood, move the hives to dry sunny places, and maintain well ventilated colonies to keep the interior of hives dry and without fungus. In badly infected colonies re-queening is necessary and thorough cleaning and disinfection of the hives with flame. Combs with infected brood are to be burnt or remelted. In order to encourage hygienic behaviour supplemental feeding or sprinkling with sugar is recommended. Having in mind the aforementioned, it can be seen that chalkbrood is not connected directly with CCD (Oldroyd, 2007), but if present, contributes to disturbances in the immunity of the colonies, affects the age polyethism and results in the shortage in highquality diet and weak hygienic conditions in the colony, which lead to CCD (Stanimirović et al., 2008a,b,c). 16

17 VARROOSIS Varroa destructor (Figure 10), a tick first discovered in a drone brood of Apis cerana on Java, is the parasite that causes varroosis. It attacks Apis mellifera in both Europe and America, as well as Apis cerana and Apis mellifera in Asia, including the Far East. Figure 10. Varroa destructor - females When ovopositing, the female ticks prefer sealed drone cells (Figure 11). Only the first offspring of each female can reach maturity and mate before a new bee develops, approximately 12 days after the cells are sealed. The grooming behaviour of A. mellifera workerbees is less pronounced and they are more susceptible to varroosis in comparison with A. cerana. The selection in order to improve grooming behaviour or to shorten the development of sealed workerbee brood even for at least 24 h at honey harvest efficaciously prevents the ticks from completing their development. In addition, many of them die with adult bees on pasture (Stanimirović et al., 2002, 2003b, 2005b; Ćirković, 2002). If untreated, infested colonies of European subspecies of A. mellifera usually die in winter due to the large numbers of ticks. Sometimes, the infection in untreated colonies may last as long as three to four years. The high frequency of heavy infections with varroas in Europe in comparison with other parts of the world can be explained by high average density of colonies, cold winters and the presence of viruses which cause infections of the honeybee and are transmitted by varroas. Environmental factors have strong influence on the outcome of the infection. Significant seasonal differences in the effects of infestation and the lifespan of newly 17

18 developed adult bees were described. Climatic factors, the number of bees in a colony and food influence the severity and the course of infection. The populations of ticks preparing for wintering are much more resistant when compared to the ones in spring and summer. The reproduction of varroa is sustained in winter due to the lack of brood. Figure 11. The dynamics of bee treatment throughout the year Varroa destructor 18

19 Varroosis is the disease of both the brood and adult bees. Sources of infection are infected colonies, package bees, contact with the diseased bees, natural swarms, queens and brood. In summer, varoosis can spread as far as 11 km and more within three months. In heavy infections (more than 20 ticks per 100 bees in a hive) in autumn and summer death of the brood can be observed, as well as the discharge of dead larvae (drone and workerbee), young bees and drones. In autumn and winter, the bees in infected colonies are unsettled and frequently die at the beginning of winter. At first, the disease develops slowly, is unnoticed and does not influence the productivity of the colony. Clinical symptoms appear after 2-3 years. The ticks reduce the quantity of dry substance, total nitrogen, fatty acids and fat body in infected larvae, and increase the energy waste during respiration. There is a decrease in resistance to diseases and the strength of the colony. The symptoms appear if more than 20% of bees are infected. In winter unsettlement, buzzing, leaving the hives, diarrhoea and death occur. In spring and summer pupae die and the colonies strength lessens as a consequence of incapability of offspring for surviving. The fertility of queens diminishes significantly, they do not mate and the brood is scattered. Workerbees are inactive during honey harvest, which leads to reduced honey production and the incapability of bees to accommodate enough food for themselves. The spread of infection in the brood changes throughout the year. In spring and autumn, in the absence of drone brood, the workerbee brood is infected and vice versa. The majority of ticks is on the worker brood, which causes the emergence of high number of damaged bees incapable of flying. In summer female ticks reproduce in drone brood, where there is plenty of high-quality protein diet and the temperature is much lower than in the worker brood. The damage caused by varroas depends not only on the number of ticks in the attacked colony, but is also connected with secondary viral infections. The virus of acute bee paralysis is the most deleterious, at least in Europe. It leads to latent infections, not causing visible body damage. The ticks activate the viruses when infesting bees, transport them onto open and sealed broods, which show unspecific signs, especially in heavily infested colonies. Adult bees, in which the viruses are active, can infect young larvae when feeding them with the secretion of mandibular and thoracic glands. Having ingested sufficient quantities of viruses, larvae 19

20 die before their cells are sealed; those who survive continue development into latently infected adult bees. The virus of acute paralysis can sometimes be found even in the pollen collected by seemingly uninfected bees as well as in their thoracic salivary glands (Ponten and Ritter, 1992; Pohl and Ritter, 1997; Békési et al., 1999). Varroosis is nowadays the biggest problem in beekeeping in Serbia and, similarly, in the majority of the world. Each and every year it is necessary to treat bees in order to control the infection, in other words, to keep less than 3 % of the bees infected. It is of utmost importance that the number of ticks on bees is as little as possible at the beginning of winter. In Europe, several acaricides against varroa are approved, but all exert adverse effects on bees (Stanimirović et al., 2003a,c, 2005a). The problem is that acaricides cannot reach the ticks in the sealed brood. Systemic acaricides given to bees in food which can reach larvae in sealed brood via food are considered ideal (e.g. cymiazol hydrochloride). Short- or long-term application of the evaporated formic acid is deleterious to the majority of varroas including the ones in sealed cells. Geraniol, the component of Nasanov s glands of forager bees, repels the migrating mites, which was proved in the numerous experiments. Chemicals should be applied with great care due to their residua in bee products and possible toxic effects (Stanimirović et al., 2003a,c, 2005a, 2006, 2007a,b; Stevanović et al., 2008). In addition, the application of acaricides can result in resistance, as it is proven for fluvalinates. Manipulative treatment against varroosis includes diminishing drone broods in infested colonies (building frame, bait of drone and workerbee comb, TNT frames etc) in order to prevent the migration of female Varroa destructor into sealed cells, where they avoid chemical treatment. The drawbacks of this method are the destruction of monthly brood production, it is tedious and favours the effects of nosemosis and acarosis. Recently, a biophysical method, heating of the brood, is more frequently being applied in combat with varoosis. Huang (2001) applied temperature treatment of the hives themselves. The difficulty which arises from this treatment is the melting of wax, but it can be solved by replacing wax comb foundation with the one made of heatproof plastic. 20

21 Certain biological means of combat against varroas are recommended: the use of their natural enemies, pathogen fungi Hirsutella thompsonii and Metarrhizium anisopliae, which are quite efficacious against the ticks and are harmless to bees (Shaw et al., 2002; Kanga et al., 2002, 2003; Peng et al., 2002). It has been proved experimentally that treatment with precisely defined numbers of dry spores of these fungi do not influence the number of eggs laid by queens, and do no harm to the brood, larvae, pupae and adult bees. In laboratory conditions, ticks were infected with fungi while allowed to walk on the culture of H. thompsonii for five minutes. It was discovered by SEM that the membranous ambulacrae on the ticks legs were the places where the conidia of the fungi adhere and germinate (Figure 12). The infected ticks died from mycosis within 52.7 to 96.7 hours (LT50), which depended on the isolate of fungi. Figure 12. Distal parts of legs of Varroa destructor before and after the treatment with Hirsutella thompsonii spores VIRAL INFECTIONS Viruses are obligate intracellular parasites which can virtually be found in any living organism. They are incapable of any metabolic activity on their own and, thus, can live and reproduce only in live cells. Once in host cells viruses use their metabolism, machinery and components to produce their own offspring, the virions. This process 21

22 does harm to the host, leading to diseases or even death. Due to their powerful effects viruses are possibly the most serious challenge to the health of living organisms. In general, there are two means of transmission of viruses: horizontal and vertical. Horizontal transmission occurs among the organisms of the same generation and can be direct or indirect. Direct transmission is completed by contact, or via food, water and air. On the other hand, indirect transmission depends on vectors, among which varroas and nosemas are the most important. Vertical transmission occurs from mothers to the offspring via eggs (transovum). It is supposed that these various means of transmission influence the virulence of pathogens. Typically, horizontal transmission favours the onset of diseases and enhances the prevalence of infections under certain circumstances, for example in high-density populations (as are in beekeeping) and in cases where there is a high replication of pathogens. In contrast, vertical transmission is a mechanism that enables the long-lasting persistence and survival of viruses and favours the evolution of benign infections. The result of a viral infection can depend on the balance of these two means of its transmission. Similarly to other organisms, honeybees are exposed to various pathogens including viruses, which pose major threat to their health. Until now, at least eighteen viruses are described which attack bees worldwide and can dramatically influence their health under certain conditions (Martin, 2001). Due to dense populations and frequent contacts among the members of the society (feeding, chemical communication), honeybee colonies are especially prone to the transmission of diseases. Although there are gaps in the knowledge of the most important processes underlying in the dynamics of the transmission of viruses, casting light on the means of transmission of viruses among bees is being developed quickly, thus our knowledge of transmission and epidemiology of viral infection in bees has considerably improved during the last decade. There is no possible direct and efficacious treatment of the viral infection of honeybees. Some viral infections (acute paralysis) can be solved by re-queening with queens from different parts of the world, which, on the other hand, poses a high risk of importing exotic pathogens. Having considered that many viruses are connected with the varroa and that there is no known adequate medicine to combat them, it is plausible that only having defeated varroosis we can defeat viral infections. 22

23 Heavy infections with ticks result in the weakness of colonies and it can be claimed that varroas virtually lead to the decrease in the immunity of honeybee colonies. Untreated colonies usually die in 3-4 years. Very frequently viruses transmitted by varroas contribute to the collapse of a colony (Martin, 2001). However, it is asserted that in Europe there were many viruses detected in honeybee colonies even before the presence of the varroa, but clinical manifestations were observed only sporadically. Thus the presence of viruses did not influence the losses in beekeeping and was disregarded (Allen and Ball, 1996). The situation changed dramatically with the appearance of varroa ticks in Europe. Having in mind the direct correlation between the intensity of infection with varroas and the appearance of viral diseases, it is supposed that the presence of ticks plays the main role in the onset of clinical signs of viral diseases (Nordstrom et al., 1999). The ticks exhaust bees and, in addition, have negative consequences as biological and/or mechanical vectors and/or activators of other pathogens, especially viruses (Yue and Genersch, 2005; Shen et al., 2005a,b; Berényi et al., 2006) which lead to the collapse of bee colonies (CCD). The presence of viruses in ticks and their transmission by Varroa destructor have recently been proved by molecular methods, especially concerning the virus which causes the deformation of wings (DWV) (Genersch, 2005; Chen et al., 2005), the virus of acute bee paralysis (ABPV) (Bakonyi et al., 2002; Tencheva et al., 2004), the virus of sacbrood (SBV) (Chen et al., 2004; Shen et al., 2005a,b) and the virus of black queen cells (BQCV) (Chantawannakul et al., 2006). It was also proved that one single V. destructor can be infected with all aforementioned viruses. The co-egsistance of numeral viruses clearly proves their role in the transmission of viruses and development of viral diseases in bee colonies (Chantawannakul et al., 2006). Recently, the replication of Kashmir bee virus (KBV), SBV and DWV in varroas has experimentally been proved, as well as their presence in the ticks saliva, which undoubtedly confirms the role of these ectoparasites as biological vectors of the viruses that attack honeybees (Ongus et al., 2004; Shen et al., 2005a,b). The connection between viral infections and varroa-infestation in bee colonies is the most complicated aspect of parasitic relationship between bees and V. destructor. Nowadays, much attention is being given to this problem in order to cast light on the means of the transmission of viruses (Berényi et al., 2006; Chen et al., 2006). In Europe, 23

24 viruses the most often transmitted by varroas are DWV and acute paralysis virus (APV) (Tentcheva et al., 2006; Berényi et al., 2006). The most harmful is APV, which infects the bees latently not causing any visible damage to their bodies. The ticks activate the viruses when infesting bees and transmit them to both open and sealed brood which exerts non-specific signs. The lifespan of bees that were infected with APV in the pupal stage is shorter, thus they can only work as nursing bees for a short time (Békési et al., 1999). Adult bees with active viruses can infect young larvae most probably via mandibular and thoracic secretions when feeding the offspring. Larvae which ingested large quantities of viruses die before the brood is sealed; those who survive continue their development into latently infected adults. APV can sometimes be found even in pollen collected by seemingly healthy bees, as well as in their thoracic salivary glands. DWV replicates slowly and if present in large amounts leads to malformation of the bee s wings in praepupal stage (even before the pigmentation of eyes). However, the presence of this virus was also proved in bees with normal wings, but in numbers approximately ten times smaller than in the ones with deformities (Chen et al., 2005; Tentcheva et al., 2006). For unknown reasons, in some years, bee viruses appear scarcely ever. In the absence of viral infections connected with the ticks, honeybee colonies easily tolerate severalthousand populations of varroas. However, when the bees are attacked with both ticks and viruses, much lower number of varroas can result in CCD because the viruses enhance the effects of varroosis (Denholm, 1999). CCD and the Israeli virus of acute paralysis (IAPV). Recent findings of Cox-Foster et al. (2007) indicated the connection between CCD and a new virus, the Israeli virus of acute paralysis. This virus was found in all the colonies suffering from CCD, and, by contrast, was not identified in healthy ones. IAPV was first recognised in Israel and later in bees imported from Australia and royal jelly from China. The precise geographic origin of this virus is yet to be known. Is it proved that the IAPV is the reason for CCD? No, it is not for certain. It is claimed that IAPV is possibly connected to CCD, but further investigations into this problem are necessary to confirm or discard this assumption. It can be concluded that IAPV is a 24

25 marker for CCD but that, most possibly, other stressogens contribute to the onset of CCD, such as Varroa, other viruses, Nosema, fungi, pesticides, inadequate diet and management in the apiary. To conclude, multi-task care of honeybee colonies is necessary since it is the only way how to prevent the presence of complicated parasitic-viral infections. It should include: adequate hygienic-sanitary measures, application of drugs which varroas are not resistent to, the selection of honeybees in order to favour colonies with highly expressed hygienic and grooming behaviour, and selection of queens which posses the SMR gene responsible for the synthesis of proteins that influence the reproduction of adult female varroas (Harbo and Harris, 1999). MANAGEMENT IN THE APIARY Management in the apiary, from the choice of the place where to put it, the type of hives, the quality and timely replacement of the wax in the hives, to the choice and administration of medicines etc. is very important for the functioning of bee colonies. If any of these is omitted or is done wrongly, CCD is very likely to occur. Hereby, the importance of timely replacement of wax in the hives is emphasized. If the beekeepers had replaced only one third of old combs in a year, the losses caused by infestation with varroas would be reduced. In old combs (in cocoons of several generations hatched workerbees) there is a chemical originated from the cocoons of fifth-stage larvae, which stimulates the oviposition in female Varroa (Garrido and Rosenkranz, 2004). Clean wax from newly made combs is free of the substance since there were no brood in their cells. This was proved by low numbers of activated terminal oocytes in varroas tested in the presence of larvae in newly built combs. The only difference between new wax and the one in which several generations of bees were hatched is in the presence of exuvia (sheets discarded by moulting larvae) in the latter. The higher activation in the presence of larvae from combs that was used for several cycles is possibly the consequence of additive effects of cuticular substances of larvae and their exuvia in the cells (Stevanović, 2007). 25

26 What is to be done when CCD occurs? In the remaining hives: Control the infection with varroas Treatment against Nosema, if it is present Do not use anything from the abandoned hives What else can be done? Since not all the factors that contribute to CCD are known and there is no treatment against bee viruses, the best option is to maintain and support the health and strength of bee colonies, provide young and healthy queens and enough high-quality food, avoid import of reproductive material and swarms from regions where CCD was registered and regular control of reproductive and other bee material. REFERENCES: 1. Allen MF, Ball BV (1996) The incidence and world distribution of honey bee viruses. Bee World, 77, Arathi HS, Burns I, Spivak M (2000) Ethology of hygienic behaviour in the the honey bee, Apis mellifera (Hymenoptera: Apidae): Behavioural repertoire of hygienic bees. Ethology, 106 (4) Bakonyi T, Farkas R, Szendroi A, Dobos-Kovacs M, Rusvai M (2002) Detection of acute bee paralysis virus by RT-PCR in honey bee and Varroa destructor field samples: rapid screening of representative Hungarian apiaries. Apidologie, 33, Békési L, Brenda VD, Dobos-Kovàcs M, Bakonyi K, Rusvai M (1999) Occurence of acute paralysis virus of the honey bee (Apis mellifera) in a Hungarian apiary infested with the parasitic mite Varroa jacobsoni. Acta Veterinaria Hungarica, 47, Berényi O, Bakony T, Derakhshifar I, Köglberger H, Nowotny N (2006) Occurrence of Six Honeybee Viruses in Diseased Austrian Apiaries. Applied & Environmental Microbiology, 72, Chantawannakul P, Ward L, Boonham N, Brown M (2006) A scientific note on the detection of honeybee viruses using real-time PCR (TaqMan) in Varroa 26

27 mites collected from a Thai honeybee (Apis mellifera) apiary. Journal of Invertebrate Pathology, 91, Chen YP, Higgins JA, Feldlaufer MF (2005) Quantitative Real-Time Reverse Transcription-PCR Analysis of Deformed Wing Virus Infection in the Honeybee (Apis mellifera L.). Applied & Environmental Microbiology, 71, Chen Y, Evans J, Feldlaufer M (2006) Horizontal and vertical transmission of viruses in the honey bee, Apis mellifera. Journal of Invertebrate Pathology, 92, Cox-Foster DL, Conlan S, Holmes EC, Palacios G, Evans JD, Moran NA, Quan PL, Briese T, Hornig M, Geiser DM, Martinson V, vanengelsdorp D, Kalkstein AL, Drysdale A, Hui J, Zhai J, Cui L, Hutchison SK, Simons JF, Egholm M, Pettis JS, Lipkin WI. (2007) A metagenomic survey of microbes in honey bee colony collapse disorder. Science, 318 (5848) Ćirković D, 2002, Reproduktivno - produktivna i higijensko - negovateljska karakterizacija sjeničko-pešterskog ekotipa medonosne pčele. Magistarski rad, Fakultet veterinarske medicine, Univerzitet u Beogradu. 11. Đuričić Bosiljka, Radojičić Sonja, 2000, Uloga veterinarske struke u očuvanju zdravlja pčela i ljudi i razvoju pčelarstva. Zbornik radova 7. Savetovanja veterinara Republike Srpske sa mećunarodnim učešćem, jun, Teslić, Banja Vrućica. 12. Đuričić Bosiljka, Bošnjak Mirjana, Plavša Nada, 2001, Epizootiološka slika američke kuge pčelinjeg legla sa posebnim osvrtom na moguće greške u terapiji. Zbornik plenarnih radova. I Savetovanje o biologiji i zdravstvenoj zaštiti pčela, Dec 22, Beograd, Forsgren E, Stevanovic J, Fries I, Variability in germination and in temperature and storage resistance among Paenibacillus larvae genotypes, Veterinary Microbiology, 2008, 129 (3-4) Fries I, Martín R, Meana A, García-Palencia P, Higes M (2006) Natural infections of Nosema ceranae in European honey bees. Journal of Apicultural Research, 45 (4), Furgala B, Boch R (1970) The Effect of Fumidil-B, Nosemack and Humatin on Nosema apis. Journal of Apicultural Research, 9 (2) Garrido C, Rosenkranz P (2004) Volatiles of the honey bee larva initiate oogenesis in the parasitic mite Varroa destructor. Chemoecology, 14, Genersch E (2005) Development of a rapid and sensitive RT-PCR method for the detection of deformed wing virus, a pathogen of the honeybee (Apis mellifera). Veterinary Journal, 169, Harbo JR, Harris JW, 1999, Selecting honey bees for resistance to Varroa jacobsoni. Apidologie 30,