Viral Diseases in Honey Bees

BLANK REVERSE PAGE “The happiness of the bee and the dolphin is to exist. For man, it is to know that and to wonder at it.” - Jacques Yves Cousteau

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Cover Photo: Colony inspection during early autumn, at the experimental apiary in Flakkebjerg, Denmark. Viral Diseases in Honey Bees

Ph. D. Thesis Roy Mathew Francis

Principal Supervisor: Per Kryger, Senior Scientist Department of Agroecology Science and Technology Aarhus University, Denmark

Co-supervisor: Steen Lykke Nielsen, Head of Section Department of Agroecology Science and Technology Aarhus University, Denmark

Assessment Committee: Bernd Wollenweber, Associate Professor Department of Agroecology Science and Technology Aarhus University, Denmark

Yves Le Conte, Directeur de la recherché Institut National de la Recherche Agronomique (INRA) Avignon, France

Annette Bruun Jensen, Associate Professor Centre for Social Evolution University of Copenhagen, Denmark BLANK REVERSE PAGE Preface • 07

Preface

his Ph.D. dissertation is hereby submitted in partial fulfilment of the requirements Tfor obtaining the Ph.D. degree from the Graduate School of Science and Technology (GSST), Science and Technology, Aarhus University. The work presented in this report was carried out during the time period between October 2009 and October 2012, at the Depart- ment of Agroecology, Aarhus University, Research centre in Flakkebjerg, Slagelse, Denmark. This Ph.D. project focuses on fundamental research in the field of honey bee virology. Honey bees are one of the most important insects, beneficial to human beings. They provide us with several commercial products such as honey and wax, but more importantly carries out the invaluable laborious work of pollination. Honey bees are on the decline as a result of pests, diseases, stress and other environmental factors. Of the numerous pathogens of honey bees that are being studied, viruses are probably the least understood. Their role in bee health is now found to be intricate and extensive. Remarkable advancements in molecular methods have enabled us to peer into the tiny and elusive world of bee viruses. This work expands our knowledge in understanding the influence and impact of viral diseases on bee health. The core of this dissertation is made up of three main parts: The background information on the research topic, papers/manuscripts resulting from the project, followed by general discussion, and perspectives. This project has resulted in three manuscripts. Manuscript I describes the development and testing of novel primers to detect three popular honey bee viruses ABPV, KBV and IAPV in a single assay. These primers allow fast and cost-effective screening of large number of samples qualitatively or quantitatively. These primers were used in the two experiments that followed. Manuscript II explores the distribution and quantifica- tion of two well-studied viruses in five different tissues of honey bee queens. Manuscript III describes a large year-long study exploring viral infection in honey bee workers and varroa mites from 23 hives under three treatment conditions across eight months. I would like to express my sincere gratitude to several people who has been invaluable in the successful completion of this project. Firstly, I wish to thank my principal supervisor, Per Kryger for providing me the opportunity to work on this project, and showering me with his insightful wisdom. He was an incredible person, with creative ideas, inspiring thoughts, extremely patient, supportive and a great friend. I wish to thank my co-supervisor, Steen Lykke Nielsen for his support, motivation and encouragement. I wish to thank colleagues from the COLOSS network for their support and cooperation. I would like to thank lab technicians, Henriette Nyskjold, Tina Tønnerson, Lene Hasmark Andersen, Trine Søberg Nielsen and Hanne Birgitte Christiansen for all their hard work, dedication and professionalism. I wish to thank Sonja Graugaard, Jette Broeng Hansen and Kirsten Brahe Larsen for their tremen- dous help in tackling administrative issues and making my life in Denmark easier. Special thanks to ‘eagle-eye Hans’ Hans A Pedersen for extensive proof-reading of the thesis. A big thanks to the canteen staff Elnor, Jette, and Mette for the great food and endless supply of coffee. I would like to thank Per Kristiansen and Flemming Vejsnæs for their help and support with field work and public relations respectively. I thank all the wonderful Danish beekeepers who have taken the effort of providing me with samples on time. I wish to thank all my friends and colleagues who have made my stay here a pleasure. I should mention the calm and productive environment at the institute was remarkable and the facilities and equipment were more than adequate. Finally, my heartfelt thanks to the funding bodies AU, EU Honey Program and Promilleafgiftsfonden. BLANK REVERSE PAGE Summary • 09

Summary

oney bees (Apis mellifera L.) play a vital role in terrestrial ecosystems by maintaining Hfloral biodiversity and provide sustainable agriculture through pollination thereby ensuring food security. Honey bees are under constant threat by numerous pathogens and pests including bacteria, fungus, viruses, microsporideans, mites, insects, pesticides and environmental changes. The interactions among these along with possibly other unknown factors are causing widespread decline in honey bee populations across the planet. Colony Collapse Disorder (CCD) is a recent phenomenon characterised by sudden disappearance of bees from the colony, which has perplexed beekeepers and researchers worldwide. The field of honey bee pathology is an active area of research. Numerous scientists and researchers across the globe, work together to understand a complex and constantly evolving plethora of pathogens and diseases. Many parasites and pathogens have been controlled through better management strategies, breeding programs, chemical control or simply through host-parasite co-evolution. The single greatest threat to beekeeping is the recently introduced invasive mite Varroa destructor. In addition to the damage caused by feeding on the bees, the mites are also effective transmitters of viruses. Viral prevalence and virus related mortality has significantly risen following the rapid spread of varroa mites. Viruses are the most recent and still poorly understood pathogens of the honey bees. Varroa mites along with specific viruses have been suspected to play a major role in CCD. This dissertation aims to unravel some of the mysteries surrounding the infection and transmission of some of these viruses. This project involves three different studies examining different aspects of viral infection in honey bees. One of the most important groups of viruses often associated with dwindling colonies is the ABPV-KBV-IAPV complex. These are closely related viruses showing high genome similarity. While these three viruses are considered damaging to bee health, their prevalence in the Danish bee population is relatively low. Viral diagnosis of honey bee samples typi- cally requires separate assays for each virus tested. A short stretch of sequence was identified in the genome which was conserved across the three viruses. Specially designed consensus primers (AKI primers) enabled the detection of these three different viruses in a single assay thus economising time and expense. Eighty-six different samples from several countries were tested using AKI primers and virus-specific primers to confirm the reliability of the primers. The health of the queen is critical for the well-being of the colony. Queen failure is often reported as a cause for colony losses by beekeepers. Although the queen is seldom reported to be sick, her role in intra-colony viral transmission is unknown. Therefore, the second study quantitatively investigated the distribution of viral infection in five tissues of 86 honey bee queens. Deformed Wing Virus (DWV) was highly prevalent in the queen tissues while AKI viruses were comparatively less prevalent. The thorax was found to be the most frequently infected tissue and the highest viral titre was found in the spermatheca. The ovaries showed the lowest viral titre and was the least frequently infected. In this study, we also tried to address the question of sexual transmission of these viruses which in turn influences their virulence. The third study aimed to create a comprehensive overview on the progression of viruses and mites over the course of a year. This study examined eight collections of 200 bees from 23 hives under three treatment conditions to reveal trends and patterns in viral infection of two 10 • Summary bee viruses (AKI and DWV) including mite infestation. Growth of varroa mites were partly suppressed by treatment, but viral titres still increased. Untreated colonies showed higher mite and virus levels than treated colonies. Most colonies that collapsed over the winter had significantly higher AKI and DWV titres in October compared to survivors. A novel sampling method was introduced to estimate prevalence of virus-infected workers in the colony. The results are put into context with previously developed varroa-virus models. This project has fulfilled its objectives in providing us with fresh insight and a better understanding of the complex nature of honey bee health. This work has also incited novel questions and opened doors for new and exciting areas of research. Sammendrag • 11

Sammendrag

onningbier (Apis mellifera L.) spiller en afgørende rolle i terrestriske økosystemer. HBestøvning af blomstrende planter er med til at sikre biodiversiteten og give et bære- dygtigt landbrug. Honningbier sikrer dermed fødevaresikkerheden. Honningbier er under konstant pres af mange skadegørere, herunder bakterier, svampe, virus, mikrosporidier, mider, insekter, pesticider og miljømæssige forandringer. Vekselvirkninger mellem disse skadegørere, eventuelt sammen med endnu ukendte faktorer, har ført til omfattende tab af honningbier over hele kloden. Colony Collapse Disorder (CCD) er et nyt fænomen karakter- iseret ved pludselig forsvinden af bier fra bifamilien, som har udfordret biavlere og forskere fra hele verden. Honningbiens patologi er et aktivt forskningsområde. Talrige videnskabsfolk over hele verden arbejder sammen for at forstå de komplekse og dynamiske sammenhænge mellem honningbiens patogener og sygdomme. Mange parasitter og patogener er blevet kontrolleret ved hjælp af bedre biavlsteknik, ved avlsprogrammer, kemisk bekæmpelse eller blot vært-parasit co-evolution. Den mest betydningsfulde trussel mod biavl er den introducerede invasive mide Varroa destructor. Ud over de skader, som forårsages af fodring på bierne og deres yngel, er mider også effektive spredere af virus. Prævalens af virus og deraf følgende dødelighed er steget efter den hurtige udbredelse af varroamiderne. Virus er de senest beskrevne patogener, og er den gruppe patogener vi har mindst viden om. Varroamider er sammen med specifikke virus mistænkte for at spille en vigtig rolle i CCD. Denne afhandling søger at opklare nogle af de uafklarede problemstillinger, der er knyttet til virusinfektion og overførsel. Projektet omfatter tre forskellige studier, der undersøger flere aspekter af virusinfektion i honningbier. En af de mest vigtige grupper af virus, som ofte er forbundet med bitab i bifamilier, er ABPV- KBV-IAPV komplekset. Disse tre virus er nært beslægtede og udviser høj grad af genom lighed. Selvom disse tre virus betragtes som skadelige for biers sundhed, synes deres udbredelse i den danske bibestand forholdsvis lav. Diagnosticering af prøver af honningbier for virus kræver typisk separate analyser for hvert virus man ønsker at teste. Korte sekven- sstykker i genomet er bevaret imellem de tre virus. Specielt designede konsensus primere (AKI primere) muliggør detektion af disse tre forskellige virus i et enkelt assay, hvilket sparer både tid og penge. 86 forskellige prøver fra flere lande blev testet ved anvendelse af AKI primer og andre specifikke primere for hvert virus for at bekræfte pålideligheden af de nye primere. Dronningens sundhed er essentiel for bifamiliens velfærd. Dronningetab bliver ofte angivet som årsag til tab af bifamilier hos biavlere. Dronningens rolle i overførelse af virus i bifamilien er dårligt kendt. I det andet studie undersøgte jeg kvantitativt fordelingen af virusinfektion i fem væv hos 86 honningbidronninger. Deform Vinge Virus (DWV) var hyppigt fore- kommende i dronningevæv, mens AKI virus var mindre udbredt. Dronningens thorax viste sig at være det hyppigst inficerede væv, mens det højeste virusantal blev fundet i sædgemmet. Æggestokkene havde laveste virusantal og var sjældent inficeret. I denne undersøgelse har jeg forsøgt at behandle spørgsmålet om seksuel overførsel af disse to virus, som igen hænger sammen deres virulens. Den tredje undersøgelse havde til formål at skabe et samlet overblik over sammenhæng i udviklingen af virus og mider i løbet af et år. I alt otte indsamlinger á 200 bier fra 23 bistader 12 • Sammendrag med tre forskellige behandlingsformer blev undersøgt. Jeg forsøgte at kortlægge tendenser og mønstre vedrørende virusinfektion af to bivirus (AKI og DWV) sammenholdt med mide- angreb. Varroamidernes vækst blev noget hæmmet af behandlingen, men virusantal øgedes indtil oktober. Ubehandlede bifamilier havde højere mide- og virusniveauer end behand- lede bifamilier. De fleste bifamilier, der brød sammen i løbet af vinteren havde signifikant højere AKI og DWV antal i oktober i forhold til overlevende. Jeg har afprøvet en ny prøveud- tagningsmetode for at vurdere forekomsten af virusinficerede arbejdsbier i bifamilien. Re- sultaterne diskuteres med udgangspunkt i ældre varroavirus modeller. Contents • 13

Contents

A. Preface 07 B. Summary (English) 09 C. Sammendrag (Dansk) 11 D. Background 1. The Honey Bee 17 1.1 The Honey Bee Colony 1.2 Life Cycle 1.3 Sex Determination 1.4 The Beekeeping Industry 1.5 Bee Pollination 2. Honey Bee Pathology 22 3. Viruses in Honeybees 24 3.1 Dicistroviridae 3.2 Iflaviridae 3.3 Transmission, Pathology and Virulence 3.4 Diagnostics 4. The Varroa Mite 33 4.1 Life Cycle 5. Host Defence 37 6. Colony Collapse Disorder 38 7. Sampling Strategies 40 8. Aims and Objectives 41

E. Papers 1. Paper I – Single assay detection of ABPV, KBV and IAPV 45 2. Paper II – Patterns of viral infection in honeybee queens 57 3. Paper III – Varroa-virus interaction in collapsing honey bee colonies 79

F. General Discussion and Perspectives 105 G. Appendix 109 H. References 115 BLANK REVERSE PAGE Section D BACKGROUND BLANK REVERSE PAGE The Honey Bee • 17

1 The Honey Bee sata, Apis cerana and Apis florae bees are oney bees along with other bees, natively found in south and south-east Hwasps and ants belong to the order Asia. Apis koshenikovi and Apis nigroc- Hymenoptera within class Insecta and incta are found highly localised on the family Apidae. Their closest relatives in- Indonesian archipelago (HEPBURN and clude the orchid bees (Euglossini), the RADLOFF 2011). Apis mellifera in Europe bumble bees (Bombini) and stingless and Apis cerana in Asia have been exten- bees (Meliponinae). Most members of sively used in commercial beekeeping. Apidae societies are workers, identifi- The European honey bees are much pre- able by the presence of pollen basket on ferred in beekeeping due to their boun- the hind legs to carry pollen (WINSTON tiful honey yield and gentle nature. This 1987). Honey bees belong to the genus work will focus only on Apis mellifera Apis and are characterised by their abil- Linnaeus bees natively found on main- ity to collect and store nectar from flow- land Europe. ers and produce honey to meet their energy requirements. Honey bees are be- 1.1 The Honey Bee Colony lieved to have evolved about 30 million Honey bees are highly social insects that years ago and make up only a small frac- live in large congregated clusters, typi- tion of the 17,000 species of known bees (MICHENER 2000). There are currently 10-11 families of bees, of which honey bees belong to the advanced long-tongue lineage. Nine species of honey bees are currently recognised, including Apis adreniformis, Apis cerana, Apis dorsata, Apis florea, Apis koshevnikovi, Apis laboriosa, Apis Fig. 1: Apis mellifera queen, drone and worker (left to right). mellifera, Apis nigrocincta, and Note the comparative difference in size and morphology. Apis nuluensis. Of these, Apis mellifera (European honey bee) is natively cally in natural caverns such as trees re- distributed across Europe, the Middle ferred to as a bee colony or a bee hive. A East, Asia and Africa. Apis mellifera in typical summer beehive is inhabited by Europe is composed of up to 10 sub- 50,000 to 60,000 worker bees, few hun- species based on morphometric and dred drones and a single queen (TAUTZ genetic data. The European honey bees 2008). The queens and workers are fe- are thought to have originated in Africa male while the drones are males. The or the Middle East and evolved through queen is usually the only reproductive four lineages, M (west and north Eu- female and the only one with highly de- rope), C (east Europe), O (east and cen- veloped ovaries. The worker bees on the tral Asia) and A (Africa) (RUTTNER other hand have underdeveloped ova- 1988; WHITFIELD et al. 2006). Apis dor- ries, causing them to be reproductively 18 • The Honey Bee

Fig. 2: Development of a typical honey bee worker. Honey bees show complete metamorphosis from an egg through larvae and pupae into adult. Based on drawings by (TOFILSKI 2012). sterile females. The queen’s role involves hive will also begin to start producing egg-laying, which is constant renewal drones that mate with virgin queens at of the hive population, and secretion nearby drone congregation areas (DCA). of queen pheromones, which is vital to By mid-summer, the hive is booming maintain the social order. Male drones with bees and they quickly run out of ex- are seen in the hive only during the mat- pandable space. At this point, queen cells ing season. The only role of the drones are created by workers in preparation for is to mate with the queen and propagate a swarm. About half of the worker bees their genetic material. The various castes form a group with the newly emerged within the hive have different work roles queen and prepare to swarm (LINDAUER which change over the time of their life 1971). span. Apart from reproduction, all ac- Aided by scout bees, the new swarm tivities are carried out by the workers, heads out to find a suitable nest in a including nurturing and feeding the hollow tree trunk or a fissure in a rock young, maintaining hygiene, building (BRITTON et al. 2002). Comb building combs, defence and foraging. and foraging starts almost immediately The population of a honey bee colony after the bees have moved in to their varies drastically from summer to win- new home. To construct the character- ter in northern latitudes. A spring hive istic symmetric hexagonal shaped cells, which has successfully overcome winter the worker bees produce thin wax scales may contain anywhere between 5000 from wax glands on the ventral side their and 15000 workers, one queen and usu- abdomen A single comb may contain up ally no drones. The rising temperature to 200,000 cells and these serve a vari- and budding blossoms of spring time are ety of functions such as shelter, stor- welcome signs for the members of a hive age (honey, pollen) and brood nursery and lead to a frenzy of activities within (WINSTON 1987). and outside the hive. Nectar and pollen stores rapidly accumulate as foragers ex- 1.2 Life Cycle plore fresh resources. When the queen The honey bee undergoes a complete begins egg-laying, the hive is quickly metamorphosis (holometamorphosis) repopulated with worker bees. A typical with four distinct life stages: egg, larvae, hive can rise from 6000 bees to 40,000 pupae and the adult. The life of a honey bees in just 10 weeks. At this stage, the bee starts as a white elongate-oval egg The Honey Bee • 19 produced by the queen. A fertilised egg pendents on temperature and nutrition. is a result of a fusion of the male drone The lifespans of worker bees greatly dif- sperm and the queen egg while an unfer- fer from summer to winter. The summer tilised egg is solely from the queen and bees live between three to four weeks does not involve the drone. A fertilised (LINDAUER 1971) while the winter bees egg gives rise to a diploid female worker can survive for up to four months. whereas the unfertilised egg gives rise to The active duties of the worker bee a haploid male drone. The size and type through its lifespan can be divided into of the brood cell created by the work- brood activities, nest activities and field ers determine the type of egg laid by the activities. Brood activities include lar- queen and subsequently developing bee val feeding and capping brood cells in caste. the first few days. Three-day old work- A single egg is positioned erectly at the ers start to develop their hypopharyn- bottom of the cell. The egg develops into geal and mandibular glands to feed the a larva which is fed royal jelly by the larvae. This period of nursing occurs nurse worker bees. Almost the entire between the age of 6 and 13 days. It has body cavity of the larvae is taken up by been observed that an average larva is the digestive system. Queen larvae are inspected nearly 1300 times a day. Nest fed higher quantities of royal jelly than activities happen between the age of 10 worker larvae, and this induces queen and 20 days involving cleaning, under- development. The larva undergoes five taking, pollen packing, and guarding. moults during the larval stage. During Other activities include food handling, the larval stage, the brood gains 900- ventilation, queen-tending and orien- 2300 times the weight of the egg. Before tation flights. At age 18 to 20 days, the the final moult, the brood cell is capped bees are ready for field activities such as by the workers. The larva then spins a foraging and scouting (JOHANNSMEIER cocoon and stretches out to fill the cell 2001). The drones can usually be seen in with the head towards the cap. During the hive during summer months. They the pupal stage, internal organs undergo are fed by the workers and do not ac- extensive development including col- tively contribute to the workings of the oration of the cuticle. The capped pupa colony. Their only role is to fly out and metamorphoses through various stages mate with queens from other colonies. of development named as the white-eye Drones either die after they mate, die stage, purple-eye stage, red-eye stage naturally, or are killed by the workers. and finally emerges out as an adult bee. The queen bee has the longest lifespan A developing worker bee spends three of more than 5 years. A virgin queen is days as an egg, six days as a larva and 12 mated by 7 to 45 drones. The sperm is days as a pupa. The total developmental stored in a small spherical organ called time of a worker bee is 21 days, a drone the spermatheca. Except during swarm- is 24 days and a queen bee is 16 days. A ing, the mated queen remains in the hive newly emerged bee is hairy and relative- for the rest of her life and lays eggs using ly smaller. The time of development de- the stored sperm for fertilisation. 20 • The Honey Bee

1.3 Haplodiploidy Sex Determination male and her daughter is 1:2. The full- The honey bees have a haplodiploidy sys- sister workers are more closely related tem of sex determination which is com- to one another than a worker is related mon to many hymenopterans (CHAR- to her own daughter. This is thought to LESWORTH 2003; HEIMPEL and DE BOER be a reason why workers do not produce 2008). In this system, the males are hap- their own offspring, but rather help their loid while the females are diploid. The mother (queen) produce more sisters honey bees, more specifically arrhenoto- (workers). Evolution of eusociality is fa- kous where the fertilised eggs develop as voured when sisters are more closely re- females and the unfertilised eggs devel- lated than worker-offspring. op into males. In honey bees, the diploid The sex determining gene csd (BEYE worker and queen have 16 pairs of chro- et al. 2003) is located on LG3. A single mosomes while the haploid drone has 16 copy (haploid) of this gene forms a drone chromosomes. Depend- ing on the size of the cell, the queen will lay a fertilised egg or an unfertilised egg. The unfertilised drone is haploid and entirely derived from a selection of the queen’s own genes. Therefore the drones have no fa- ther. The fertilised genome is half derived from the queen’s genes and the rest is derived from one of the multiple drones. All workers arising from a particu- lar drone’s sperms are full-sisters while workers Fig. 3: Arrhenotokous haplodiploidy system of reproduction in honey bees. arising from one of the The queen is polyandrous thus mating with several drones. Unfertilized queen eggs produce haploid drones. Eggs fertilized by sperm produce queen’s egg and a dif- diploid workers. Feeding excess of royal jelly to diploid offspring produces ferent drone’s sperm are queens. half-sisters to the first group of workers. Therefore, workers while two different copies (heterozygotic produced by a queen are half-sisters or diploid) create a worker. The two copies full-sisters due to multiple drone mating of the sex locus which creates a female by the queen. must be composed of two different al- The relatedness ratio between full-sisters leles. This is critical, as two identical is 3:4 while relatedness between a fe- alleles, although present in two copies The Honey Bee • 21

(homozygotic diploid) will give rise to Honey bees obtain the majority of their non-fertile diploid drones. Hence vari- energy from plant sugars in the floral ability on the sex locus is highly impor- nectar. Nectar is mostly composed of su- tant in honey bees. More than 100 differ- crose, glucose and fructose. With their ent sex alleles have been identified and specialised mouth parts, worker bees col- this number continues to rise (MUNK lect nectar which is stored in a reservoir et al. unpublished). The multiple drone called the crop. This is then transferred mating ensures higher genetic diversity to the nest workers where enzymes from and better fitness within the bees (MAT- the hypopharyngeal glands are added TILA and SEELEY 2007). Inbreeding is a (WINSTON 1987). The sugars are broken serious issue in honey bees and leads to down, water is evaporated and the con- production of diploid drones and gener- tents are placed into cells. When water ally reduced fitness in the colony. This is content is reduced below 18%, the honey often a dilemma with honey bee conser- is capped for storage using wax. Honey vation programs, as very strict conserva- bees are the only insects that produce tion of small bee populations results in food consumed by man. Honey is an im- inbreeding depression. portant international commodity with global production of 1.5 million metric 1.4 The Beekeeping Industry tonnes in 2010 (FAO 2012). In Denmark, The honey bee is man’s smallest domestic 3000 tonnes of honey is harvested an- animal. Human beings have artificially nually estimated at a value of 60 million nurtured and maintained honey bees in DKK (BRADBEAR 2009). The world’s logs, skeps, mud pots and wooden boxes largest producers of honey (2010) are for thousands of years. Development of China, Turkey, USA, Ukraine and Ar- movable and reusable comb hives started gentina (FAO 2012). with Lorenzo Langstroth in the 1850s. Honey bees collect pollen as a nutrition- The growth of large-scale commercial al source as it contains up to 28% pro- honey bee management started hereaf- tein. Pollen is rich in essential fatty ac- ter, although the ancient Egyptians were ids, amino acids, vitamins, minerals and known to maintain hundreds of hives. enzymes, and low in cholesterol. It is a The economic might of the beekeeping popular supplement among athletes and industry revolves around bee products sportsmen because of its ability to reju- such as honey and beeswax, pollina- venate the body and accelerate the rate tion services, queen rearing, breeding of recovery. Workers 8-17 days old have and beekeeping merchandise. The total highly developed wax glands on the ven- number of managed bee colonies in the tral side of the abdomen which produce world was estimated to be 72.6 million beeswax used in comb building and cell in 2007 (VAN ENGELSDORP and MEIXN- capping. Beeswax is used in a huge vari- ER 2010). Honey bees produce a diverse ety of products including candles, soaps, variety of products suitable for human lubricants, water-proofing, polishes, pre- use including honey, pollen, propolis, servatives and skin care products. Royal wax and royal jelly. jelly is produced from the hypopharyn- 22 • Honey Bee Pathology geal gland by the worker bees, which bee can visit thousands of flowers in a is fed to the queen larvae. Royal jelly is single day. The abundance and diversity used in skin care, medical products and of pollinators such as bees are declining research. in various parts of the world. Agrono- mists and farmers often lack informa- 1.5 Bee Pollination tion on the critical role of pollinators in The evolution and divergence of bees food production, which may lead to poor is closely linked to angiosperms, with management practices and indiscrimi- plants evolving flowers with odour, nate use of pesticides. This concern has shapes, colours, nectar and pollen re- been widely reported as it may affect the wards to attract the bees. And the bees world food supply. (AIZEN et al. 2009; in turn provide a mechanism to transfer GARIBALDI et al. 2011). Decline in polli- pollen between plants (WINSTON 1987). nating insects is found to lead to dimin- One-third of all plants or plant prod- ishing plant species that rely on them ucts consumed by human beings are (BIESMEIJER et al. 2006). Several factors thought to be insect pollinated. 35 out of have been suggested as causes including 107 globally leading crops directly used rising pesticide use, combination of in- for human consumption are pollinated secticides, neonicotinoids, monoculture by insects - mostly honey bees (KLEIN etc. et al. 2007). Almost 90% of fruit trees are dependent on honey bees for pollination (MORSE and CALDERONE 2000). 2 Honey Bee Pathology About 84% of the 264 crops grown in Just like any other living organism, hon- the EU depend largely on insect pol- ey bees are susceptible to a whole range lination (WILLIAMS 1994). By the value of pathogens and diseases. Pathogens af- of their pollination, honey bees are the fecting honey bees can be grouped into third most valuable domestic animal in endoparasites, ectoparasites and preda- Europe. The value of pollination is esti- tors. Endoparasites live within the host mated to be 50 times the value of honey body and these include bacteria, fun- in Western Europe. In Denmark, the gus, microsporidiae, viruses and mites value of pollination services by bees is such as tracheal mites. Ectoparasites are estimated to be 600-1000 million Dan- parasites that live or feed on the host ex- ish Kroner (AXELSEN et al. 2011). ternally, such as varroa mites and tropi- Honeybees are extremely effective pol- laelaps mites. Predators such as Aethina linators due to their great numbers. A tumida (small hive beetle) and Vespa ve- single colony can visit up to a million lutina prey on honey bees. flowers in a single day. Honey bees are The most common bacterial diseases are aware of the different types of flowers American foulbrood (AFB) and Euro- and their time and duration of bloom- pean foulbrood (EFB)(GENERSCH 2007). ing. They are also very consistent for- AFB is a highly contagious quarantine agers such that they rarely switch plant disease caused by the bacterium Paeni- during a single foraging trip. A single bacillus larvae. The bacterium exists in a Honey Bee Pathology • 23 vegetative and an infectious spore phase. Other low-profile parasites of the honey The spores are extremely robust and bee include the trypanosomatid Crithid- practically indestructible in nature. The ia mellificae, the bacterium Spiroplasma spores are easily spread through infected spp. and the phorid fly Apocephalus( bo- honey, pollen or by trophallaxis. AFB is a realis) (RUNCKEL et al. 2011). Crithidia brood disease and the spores germinate mellificae is a trypanosomatid parasite in the gut of young larvae, proliferating of the honey bee identified in 1967, but rapidly and consuming the tissue from is not known to show any pathological within, inducing mortality in young lar- effect (LANGRIDGE and MCGHEE 1967). vae. AFB can be identified by its char- Phorid flies are parasitic on honey bees, acteristic ropy texture and foul odour. bumble bees and wasps. Adult female European Foulbrood is a similar brood phorid flies oviposit eggs in the abdo- disease caused by the bacterium Meliso- men of worker bees and the larvae of coccus plutoneus (FORSGREN 2010). The the phorid fly emerge out through the EFB leaves creamy white brood which is junction between the head and thorax. less ropy than AFB. They are reported to manipulate the be- Fungal diseases include nosemosis, haviour of the host to abandon the bee chalkbrood and stonebrood. Nosemo- hive and are now being suspected as new sis is caused by two fungal microspo- threat inducing colony collapse (CORE et ridea, either Nosema apis or Nosema al. 2012). ceranae (FRIES 2010). Nosema spores Ectoparasites include several insects germinate in the gut of adult bees, and and mites (SAMMATARO et al. 2000) causes diahorrea, malnutrition and re- such as the varroa mite, wax moth, bee duced lifespan. Chalkbrood is a brood lice and the tropilaelaps mite. Tropilae- disease induced by Ascosphaera apis laps (Tropilaelaps spp.) mites are serious which causes larvae to appear white and parasites affecting brood and adult bees chalky in appearance (ARONSTEIN and (TOMASZEWSKA 1987). They are native MURRAY 2010). Stonebrood is caused by parasites on Asian dorsata bees. They fungus Aspergillus spp. causing larvae have not yet been found in Europe. Bee to appear mummified and eventually lice (Braula coeca) are a minor pest of turning black, green or yellow. Tracheal the European honey bee found world- mites (Acarapis woodi) live in the trache- wide. Braula is a wingless fly that lives al system of the honeybee resulting in on the bodies of honeybees and feeds ill health and reduced lifespan (BAILEY on their food. Braula is not known to 1958). Extensive infestation can render cause any major damage to bees and the bee unable to fly. Malpighamoeba has been wiped out due to varroa treat- mellificae is a protozoan amoeba-like ment. Other minor pests include bee ta- parasite that invades the gut of adult chinids (Rondanioestrus apivorus) and bees, causing the formation of cysts in bee conopids (Physocephala fascipennis) the malpighian tubules. The symptoms which have been identified on bees from are similar to nosemosis including diar- South Africa (JOHANNSMEIER 2001). The rhoea and inability to fly. varroa mite is the most serious pest of 24 • Viruses In Honey Bees

South Africa. The bee scorpion Elling ( - senius fulleri) is an interesting creature often found clinging on to honey bees in Africa but their impact on bees and bee colonies is not known. The Asian hornet Vespa ( velutina) was accidently introduced into France from China before 2004 (ROME et al. 2012). It is a serious predator preying on honey bees and other social insects. It has rapidly spread across France and into sev- Fig. 4: Stereomicroscopic image of Small hive eral neighbouring countries. beetle (Aethina tumida), a predatory pest of honeybees. the honey bee and will be discussed in 3 Viruses In Honey Bees more detail later. Viruses are non-living, opportunistic, Predators actively prey on honey bees. obligate cellular parasites. They are com- The small hive beetle (SHB) Aethina ( posed of a nucleic acid genome (RNA or tumida) (Fig. 4) is brown-black in col- DNA) and a protein coat called the cap- our and measures 7 mm long by 3 mm sid. Some enveloped viruses may have a wide. It is a pest of the honey bee and second coat providing extra protection. causes considerable damage to beekeep- Viruses need the host cellular machin- ing across the United States, Canada, ery for transcription, translation and Australia and Africa. SHBs can cause replication. Rapid advances in molecular extensive harm to bee hives by eating diagnostic methods have led to the dis- brood, honey and wax, especially when covery and characterisation of approxi- in large numbers (HUANG and LIN 2001). mately 18 honey bee viruses. Most of Adult SHBs reproduce in the bee hive by these viruses have been detected only in laying up to 1000 eggs in combs, crev- honey bees while a few have been identi- ices, pollen or brood. SHBs have not yet fied in other bees and ants too. Viruses been found in Europe. The larger Afri- can attack bees at various stages of their can counterpart of SHB is the large hive life and this in turn may alter the symp- beetle (Hoplostoma spp.) found in sandy toms and outcome of the infection. and savanna areas of southern Africa. The most commonly studied honey They feed on wasp and bee brood, pol- bee RNA viruses include the Acute Bee len and honey. Ants and termites are a Paralysis Virus (ABPV), Kashmir Bee major honey bee pest in several parts Virus (KBV), Israeli Acute Paralysis of the world, but are not considered an Virus (IAPV), Black Queen Cell Vi- issue in Europe. Bee pirate wasps such rus (BQCV), Chronic Bee Paralysis as the banded bee pirate (Palarus lati- Virus (CBPV), Deformed Wing Virus frons), and yellow bee pirate (Philanthus (DWV) and Sacbrood Virus (SBV). diadema) are extremely destructive in Other reported viruses include Kakugo Viruses In Honey Bees • 25

Virus (KV), Varroa Destructor Virus-1 Almost all honey bee viruses have single (VDV-1) Bee Virus X (BVX), Bee Virus stranded RNA genomes except the Fila- Y (BVY), Cloudy Wing Virus (CWV), mentous Virus (FV), which has a DNA Slow Bee Paralysis Virus (SBPV), Arkan- genome. All honey bee RNA viruses sas Bee Virus (ABV), Macula-like Virus except CBPV are members or show fea- (MaLV), Berkeley Bee Virus (BBV), Thai tures of the order Picornavirales. Within Sacbrood Virus (TSBV), Aphid Lethal this order, based on the structure and ge- Paralysis Virus (ALPV), Big Sioux Riv- nome organisation, most honey bee vi- er Virus (BSRV) and Lake Sinai Virus ruses are grouped into the two families (LSV-1/LSV-2). For the complete list of Dicistroviridae and Iflaviridae. viruses with characteristics, see Appen- dix. 3.1 Dicistroviridae The most common bee viruses have Dicistroviruses are ~30 nm icosahedral RNA based genomes and these viruses non-enveloped viruses with a bicistronic use their own RNA-dependent RNA monopartite genome. The viral capsid is polymerases to replicate their genomes. composed of 60 copies of three different These enzymes lack the proof-reading viral proteins: VP1, VP2 and VP3. Viral capability seen in their DNA counter- entry into the host cell occurs via clath- rin-mediated endocytosis (BONNING and MILLER 2010). This family consists of two genera Aparavirus and Cripavirus. Members of genus Aparavirus include ABPV, IAPV, KBV and closely related viruses SINV-1 (Solenopsis Invicta Vi- rus-1) and TSV (Taura Syndrome Virus). ABPV is the type species for Aparavirus. Fig. 5: Electron micrograph of (a) Acute Bee Cripavirus includes BQCV and the type Paralysis Virus (BAILEY 1963) and (b) Kashmir et al. species is CrPV (Cricket Paralysis Virus). Bee Virus (BAILEY et al. 1979). Dicistroviridae as the name suggests, parts, and this leads to high error rates. have a characteristic bicistronic genome As a result, a supposedly single strain of having separate open reading frames viral population is composed of several (ORF) for structural and non-structur- different genotypic variants. This gives al proteins (Fig. 6A). The two ORFs are the virus a remarkable evolutionary rate, seperated by about 200 bases, including often enabling it to overcome extreme se- an intergenic internal ribosome entry lective pressure. This mixed population site (IRES). The 5’ end of ORF1 includes of mutant viral genotypes is referred to the first IRES, a 5’ untranslated region as a quasi-species (AUBERT et al. 2005). (UTR) and the 5’ CAP covalently linked Despite the quasi-species nature of RNA to a small viral protein (VPg). The 5’ viruses, molecular characterisation, leader is followed by the non-structural taxonomy and phylogenetic analysis is ORF coding for the enzymes helicase, mostly based on sequence data. protease and RNA-dependent RNA 26 • Viruses In Honey Bees p oly me r- ase (RdRp). The structural ORF located on the 3’ end codes for the four viral proteins VP1 Fig. 6: Genome composition of two common honey bee virus families (A) Dicistroviridae to VP4, and (B) Iflaviridae. of which three proteins form the sub-units of the non-AUG codons. capsid. The 3’ end after ORF2 is com- A 428bp IAPV fragment was found to posed of 150-500 bases 3’ UTR linked be integrated into the honey bee genome to a polyA tail. The genome sizes of thus rendering the bee resistant to IAPV ABPV, KBV and IAPV are 9491 bases infection (MAORI et al. 2007b). Con- (NC_002548), 9524 bases (AY275710) and versely, 41 bases of bee sequence were 9499 bases (NC_009025) respectively. found integrated in a defective IAPV The IRES is an important mechanism RNA. Thus reciprocal exchange of ge- employed by the virus to regulate vi- netic material between host and virus ral translation (BONNING and MILLER has been arguably demonstrated in a 2010). The virus may disrupt normal non-retroviral honey bee system. Two host cell cap-dependent translation studies, (FIRTH et al. 2009; SABATH et and instead initiate translation at the al. 2009) based on prediction tools, have viral IRES requiring fewer translation suggested the existence of a functional initiation factors. Unlike other Picor- overlapping gene named pog (predicted naviruses, Dicistroviridae has two IRES overlapping gene) in IAPV and its rela- (5’ UTR IRES and IGR-IRES) allowing tives ABPV, KBV and SINV-1. The pog differential regulation of its two ORFs gene translates in a shifted reading frame independently. The 5’ UTR IRES of di- of the structural polyprotein gene. Over- cistroviruses has an RNA structure that lapping genes allow more information to recruits ribosomes directly and transla- be compressed into a genome of limited tional translocation can proceed with- length. This gene was shown to be under out the two translation initiation factors positive selection which also meant that eIF1 and eIF4E. The 5’ UTR IRES and this region is highly conserved across picornaviral IRES requires all transla- these viruses. Lab experiments failed to tion initiation factors except two while detect POG protein in SINV-1 infected the IGR-IRES requires no initiation fac- ants (VALLES and SABATH 2012). tors. The IGR-IRES structure is highly The presence of thepog gene is supported conserved among dicistroviruses and is by the fact that this region is highly con- also reported to initiate translation at served and positively selected to main- Viruses In Honey Bees • 27

tain the exact sequence. This aided us in mites (BALL 1983; BALL 1985) and impli- designing primers in this short stretch, cated in varroa-associated colony losses which was conserved across three vi- (BEKESI et al. 1999; BERENYI et al. 2006; ruses hence, enabling their detection in FAUCON et al. 1992).The ABPV genome a single assay. was sequenced in 2000 (GOVAN et al. 2000). 3.1.1 The ABPV-KBV-IAPV Complex KBV was first identified in Apis cerana ABPV, KBV and IAPV viruses are the bees from northern India (BAILEY and most important honey bee viruses in WOODS, 1977). KBV is serologically and the family Dicistroviridae. The ABPV- genetically similar to ABPV, but the cap- KBV-IAPV complex (DE MIRANDA et sid protein profiles were reported to be al. 2010a) is a group of closely related different (ALLEN and BALL, 1995). KBV single stranded viruses commonly exist- is generally less prevalent (NIELSEN et al. ing as covert low-titre infections. They 2008; SIEDE et al. 2005; TENTCHEVA et are picorna-like viruses that belong to al. 2004), but has been detected in work- the same family and thus share a simi- ers, queens, honey, pollen, royal jelly and lar genetic makeup and similar biologi- brood food (SHEN et al. 2005a). KBV cal features, such as high virulence and has been found in the faeces of workers horizontal transmission routes. ABPV and queens (HUNG 2000). KBV was de- was discovered in 1963 (BAILEY et al. tected in eggs but not in queens (CHEN 1963). Observed symptoms include pa- et al. 2006b). KBV seems to be the most ralysis, trembling and rapid death in 1-2 virulent of all known honey bee viruses days post infection. ABPV can be readily (BAILEY et al. 1979). KBV was detected detected in immuno-based infectivity in varroa mites (HUNG and SHIMANUKI tests and also detected in bumble bees 1999; SHEN et al. 2005b). Varroa mites (BAILEY and GIBBS 1964). ABPV is viru- have been shown to vector KBV by ex- lent, a fast killer and is correlated with posing KBV-free pupae to varying num- mite infestation. Based on a modelling bers of KBV-positive mites (CHEN et al. approach (MARTIN 2001b), ABPV can 2004a). KBV-positive mites were found destroy a colony only in the presence of to transmit KBV to pupae 70% of the a large existing mite population. ABPV time. The mite to mite transmission rate has been detected in bees while absent was 51%. KBV has been found in the in mites from the same colony, and this faeces of workers and queens (HUNG finding supports transmission between 2000), queen ovaries and eggs (CHEN bees (TENTCHEVA et al. 2004). ABPV et al. 2005b). Few studies have reported has been detected in the brain and hy- KBV in mite-associated colony losses popharyngeal glands (BAILEY and (HUNG et al. 1995; HUNG et al. 1996a; MILNE 1969), and faeces implying oral TODD et al. 2007). The KBV genome was transmission (CHEN et al. 2006a). ABPV sequenced in 2004 (DE MIRANDA et al. has been detected in semen implying 2004). vertically transmission (YUE et al. 2006). Israeli Acute Paralysis Virus (IAPV) ABPV has been shown to be vectored by was extracted and identified from dead 28 • Viruses In Honey Bees

70% sequence similarity) but can be distinguished by PCR and serology. IAPV has been strongly implicated in CCD syndrome (COX-FOSTER et al. 2007) and studies have also associated IAPV with collapsing colonies (ANTUNEZ et al. 2006; BLANCHARD et al. 2008). Var- roa mites have recently been shown to be effective transmitters of IAPV (DI PRISCO et al. 2011a). IAPV has been shown to be silenced by ingestion of IAPV-dsRNA through the RNAi pathway (MAORI et al. 2009). ABPV, KBV and IAPV show a worldwide distribution (AL- LEN and BALL 1996b; ELLIS and MUNN 2005). ABPV is one of most common honey bee viruses in Europe (BAKONYI et al. 2002; GAUTHIER et al. 2007; NIELSEN et al. 2008; TENTCH- EVA et al. 2004) and has been implicated in winter losses (SIEDE et al. 2008). The prevalence of ABPV and KBV fluctu- ates over the season with peak titres in late summer or autumn DE IRANDA Fig. 7: Whole genome unrooted dendogram relating the ABPV and ( M et al. 2010a). In DWV complexes to their families. The tree was created in MEGA Denmark during 2006-2007, 5 based on a neighbour-joining algorithm using the p-distance ABPV was detected in 11 out method. Node values correspond to percentage bootstrap support of 96 apiaries, and KBV in 1 after 10000 replications. S, G and F denote species, genus and apiary (NIELSEN et al. 2008). family respectively. Honey bee viruses are underlined. Asterix (*) corresponds to the type species of the respective genus. Refer to Although IAPV has been asso- the appendix for the full list of viruses in this figure. ciated with collapsing colonies in the US (COX-FOSTER et al. bees in collapsing colonies from Israel 2007), the prevalence of IAPV in Eu- (MAORI et al. 2007a). IAPV is closely rope has been relatively low: 13-25% in related to ABPV and KBV (showing 60- Spain (ANTUNEZ et al. 2012) and 14% in Viruses In Honey Bees • 29

France (BLANCHARD et al. 2008). IAPV tal stages of the bee including egg (CHEN was first reported in Denmark in 2010 et al. 2005a), larvae, pupae (GAUTHIER et from a single bee sample (FRANCIS and al. 2007), adults and all castes including KRYGER 2012). queens (CHEN et al. 2005b; GAUTHIER et al. 2011), workers and drones (CHEN 3.2 Iflaviridae et al. 2004b; YANEZ et al. 2012). Trans- Iflaviridae consists of a single genus, mission of DWV takes place through Iflavirus, which includes DWV, SBV, trophallaxis between adult bees as well KV and VDV-1. The genome sizes of as between nurse bees and larvae (CHEN DWV, SBV, KV and VDV-1 are 10,140 et al. 2005a; CHEN et al. 2006b; FIEVET bases (NC_004830), 8838 bases (AF092924), et al. 2006; MÖCKEL et al. 2011; YUE and 10,152 bases (NC_005876) and 10,112 bas- GENERSCH 2005; YUE et al. 2006). Infec- es (NC_006494) respectively. tion of queen offspring from infected se- Iflaviruses have a single ORF, encoding men by artificial insemination has been capsid proteins (VP1-VP4) towards the demonstrated (DE MIRANDA and FRIES 5’ end and the non-structural proteins 2008). DWV is known to be transmit- towards the 3’ end (Fig. 6B). The 5’ end is ted by varroa mites (BALL 1983) and is covalently linked to viral protein (VPg), strongly correlated with mite infestation followed by the IRES, leader protein level in bee colonies (BOWEN-WALKER (L-protein), capsid proteins (VP1-VP4) et al. 1999). DWV has been reported in and non-structural proteins including drone tissues (FIEVET et al. 2006), and helicase, protease and RdRP. The VPg semen (YUE et al. 2006) suggesting that is a small protein seen in several posi- queens may be infected during mating. tive ssRNA viruses that binds to the 5’ DWV has been found to infect the queen end of the genome and aids in stability, head, adipose tissues, gut and ovaries replication, translation and movement. (FIEVET et al. 2006), as well as worker The Lp protein is thought to have multi- brains (SHAH et al. 2009). Both posi- ple functions including protease activity. tive- and negative-strand DWV has been The translated polyprotein is cleaved by found in the head, thorax and abdomen viral 3C-protease to produce functional of crippled workers, but only in the proteins. thorax and abdomen of asymptomatic bees (YUE and GENERSCH 2005). DWV 3.2.1 DWV and other Iflaviruses has been reported to replicate in varroa DWV, KV and VDV-1 are closely related mites based on the detection of negative viruses that belong to the genus Iflavirus strands in the mite (GISDER et al. 2009; within the family Iflaviridae. ONGUS et al. 2004b), while other studies Deformed Wing Virus was first isolated have reported the absence of DWV rep- from Japanese bees (BALL 1983). DWV is lication in mites (SANTILLAN-GALICIA et associated with very characteristic symp- al. 2010; ZHANG et al. 2007). The DWV toms of wing deformity and reduced genome was sequenced in 2006 (LANZI body size (YUE and GENERSCH 2005). et al. 2006). DWV has also been report- DWV has been found in all developmen- ed to be silenced by ingestion of dsRNA 30 • Viruses In Honey Bees

(DESAI et al. 2012). Sacbrood Virus (SBV) is an extensively Kakugo Virus (KV) was isolated from studied honey bee virus that belongs the brains of aggressive worker guard to the family Iflaviridae. Since it is not bees (FUJIYUKI et al. 2004). KV is closely closely related to the viruses in this related to other picornaviruses and ifla- project, it will not be discussed here. viruses. The KV genome shows 98% homology to the DWV genome, but is 3.3 Transmission, Pathology & Virulence thought to have different pathogenicity since wing deformity has not been as- Viruses are known to be diverse in their sociated with KV. KV prevalence differs infection and transmission strategies. from hive to hive for the same season Based on the type of infection, viruses and also depends on the health state of can be classified into overt and covert the colony. KV was claimed to be found infections (DE MIRANDA and GENERSCH only in attackers and not in foragers (FU- 2010). Overt infections show obvious dis- JIYUKI et al. 2006). It was detected most- ease symptoms which may be diagnostic ly in the head, thorax and abdomen. KV and show causal relationship. Overt in- was also detected in varroa mites but it is fections can be further divided to acute not known if KV is mite-mediated or if it and chronic infections. Acute infection causes disease in bees. involves the production of huge numbers Varroa Destructor Virus-1 (VDV-1/ of viral particles in a short time, which VaDV) was identified in Varroa destruc- quickly kills the host with intense ob- tor tissues during microscopy. VDV-1 is servable symptoms. Chronic infections 84% similar to DWV in nucleotide simi- mean slow, but sustained production of larity and both are reported to replicate viral particles over the life time of the in varroa mites (ONGUS et al. 2004b). host, resulting in observable symptoms. DWV and VDV-1 recombinants have While both acute and chronic infections been reported in mites implying inde- have significant negative impact on host pendent evolution of modular genomes fitness causing obvious symptoms, the (MOORE et al. 2011). difference lies in the duration and inten- DWV is widespread in Europe, highly sity of infection. correlated with mite infestation (BO- Covert infections are characterised WEN-WALKER et al. 1999; HIGHFIELD by the absence of obvious observable et al. 2009; MARTIN et al. 2010; NORD- symptoms, but may still affect the host STROM 2003), and studies continue to adversely. The infected host cell is not show its role in colony collapse (DAINAT destroyed by the attacking virus. Covert et al. 2012b; MARTIN 1998; MARTIN et infections can be classified into latent al. 2012). DWV has also been detected and persistent infections. Latent infec- in bumble bees (GENERSCH et al. 2006). tion requires the integration of the viral In Danish bees, DWV is the most com- genome into the host DNA or its exist- monly found virus (in 78 of 96 apiaries ence as an extrachromosomal episome, (NIELSEN et al. 2008)) while KV and thereby releasing viral particles through- VDV-1 have not yet been surveyed. out the lifetime of the host. Latent infec- Viruses In Honey Bees • 31

Fig. 9: DWV-infected bee. Dorsal and lateral view of a DWV infected worker bee showing extreme wing deformity. tions are usually seen in retroviruses and no true latency has been demonstrated in honeybees. During a persistent infection, viral particles are generated in the infected cell at a low constant rate without destroying the host. This is possible only if the virus successfully evades the host immune response and regulates gene expression for stable persistence. Certain studies have shown the re-emergence of covert infections as overt outbreaks (RIBIERE et al. 2002). These outbreaks could be induced by various factors such as environment (temperature, humidity), colony conditions (crowd- ing, dead bees) or stress. Based on the mode of transmission, viruses may be horizontally or vertically transmitted. Horizontal transmission involves movement of viral particles between individuals of the same generation. This occurs through various modes such as oral feeding Fig. 8: Viral transmission pathway. Colony level (trophallaxis), contaminated food transmission of viruses occurs through drifting, robbing sources, contaminated wax, honey, or swarming. Within a hive, transmission between the nectar, comb parts, open wounds or queen, drones are workers is far more complex. Tissues are abbreviated as Ep-Endophallus, Tt-Testis and Sp- by vector-mediated transmission. Spermatheca. Viruses in bees are not known to be 32 • Viruses In Honey Bees air-borne. Certain horizontal routes ob- wiped out of the colony. The hosts are served in honey bees include transmis- killed off quickly, thus preventing verti- sion by cannibalism of dead infected cal transmission (FRIES and CAMAZINE individuals, and the faecal-oral route by 2001). At the colony level, most diseases ingestion of contaminated faeces. are therefore low-virulence. Widespread Vertical transmission involves the trans- colony collapse due to disease usually fer of viral particles from one genera- happens only if the colonies are heav- tion to the next. Sometimes the verti- ily infested by mites and the disease is cal transmission is referred to as sexual mite-vectored. Several honey bee viruses mode of transmission (venereal), as this ABPV, KBV, IAPV, DWV and SBPV are usually occurs during mating when in- varroa-associated and have been shown fected sperm from drones is transferred to be horizontally transmitted by mites to queen, who then produces infected while a few viruses have been speculated eggs. The difference between horizontal to be vertically transmitted. A complete and vertical transmission is not very well list of viruses and transmission routes is defined and is often a topic of discus- shown in Appendix. sion. For example, if the infected sperm Some viruses have been reported to be infects the queen, then the transmission associated with other microorganisms, is horizontal and sexual. Overtly infec- mostly parasites. BQCV, BVY and FV tious viruses are usually spread horizon- are reported to be associated with Nose- tally along with other possible routes. It ma spp. while BVX is reported to be as- is hypothesised for a host-parasite rela- sociated with protozoan Malpighamoeba tionship that horizontal transmission se- mellificae. lects for lethal high-virulence parasites, while vertical transmission selects for 3.4 Diagnostics benign low-virulence parasites (FRIES Viral diagnostics in bees have pro- and CAMAZINE 2001). gressed from classical observation of Viral transmission and dynamics is a symptoms to molecular methods. Diag- different picture at the colony level. Hor- nostic methods have advanced remark- izontal inter-colony transmission occurs ably fast with the development of novel through robbing, drifting or by foraging. molecular techniques. The diverse exist- Drifting occurs when bees accidently ing technologies have their own merits enter neighbouring colonies especially and demerits. Comparisons of diagnos- with drones, as they are readily ac- tic methods must consider all or many of cepted. When the honey flow is limited, the following aspects: sensitivity, specifi- worker bees may try to rob honey from city, accuracy, precision, reproducibility, neighbouring colonies. Vertical inter- simplicity, time and expense. colony transmission occurs through Symptomatic observation is still widely swarming (colony level reproduction). used by beekeepers and researchers to Viruses which are highly virulent at identify infective agents. Certain viruses the individual level are not virulent at have clearly defined and demonstrated the colony level as the virus is quickly symptoms that can be reliably and accu- The Varroa Mite • 33 rately used for diagnostics. SBV shows a handling, storage and downstream data definite fluid-filled yellow sac symptom processing are all critical in establishing in the larval stage while the twisted, de- a baseline to compare results. Biologi- formed or missing wings on adults are cal samples used to diagnose viral in- characteristic of DWV. High levels of fection could include egg, larvae, pupae infection by paralysing viruses usually and adult bees and hive materials such results in bees showing trembling, paral- as comb parts, wax, pollen or honey. ysis, shiny bloated abdomen and inabil- Isolation of live virus for infectivity ity to fly. The disadvantage is that viral studies must ensure the viral material infections are often inapparent: Disease is obtained from fresh live or frozen bee may not be observed in all castes and life material. Nucleic acid based detection stages, and multiple infections may yield can work on materials in which a vi- confusing observations. rus is killed or rendered non-infective. Serology-based bioassays such as ELISA All samples in this study were live bees utilise the antigen-antibody interaction which were frozen and/or lyophilised of the virus to detect and quantify viral immediately on collection. titre. Bioassays are currently outdated, as they are laborious, time-consuming and less sensitive. Electron microscopy 4 The Varroa Mite in combination with serology (immuno- Varroa mites (Varroa spp.) are arachnids histochemistry) or nucleic acid tech- that belong to subclass Acari and subor- niques (in situ hybridisation/in situ PCR) der Mesostigmata. Varroa mites are par- can be useful in detection and localisa- asites on their tropical host Apis cerana. tion of viral particles. Global honey bee trade and transport of The most sensitive, advanced and pop- bees has introduced the mite to Europe, ular diagnostic tools are nucleic acid Africa, America, and other places, thus detection methods. These involve de- successfully transferring them to the tection of nucleic acid such as DNA by new host, the Apis mellifera bee. Apis binding with fluorescent dyes such as cerana bees have a long history of co- SYBR Green or Taqman probes. The tar- evolution with varroa mites leading to a get molecules are PCR amplified using balanced host-parasite relationship. The specific primers and detected or quanti- Apis mellifera bees are highly suscepti- fied by FRET (Fluorescence Resonance ble, and their hygienic traits are insuf- Energy Transfer). The advantage of ficient to control the mites. Up to four these methods includes high sensitivity, species of varroa mites have being iden- specificity and reproducibility. On the tified, namely Varroa destructor (AN- downside, these are relatively expensive DERSON and TRUEMAN 2000), Varroa and require specialised equipment. Most jacobsoni Oudemans (OUDEMANS 1904), recent high-throughput workflows rely Varroa rindereri, and Varroa underwoo- on microarrays and sequencing. di. Varroa destructor Korean haplotype Apart from the standardisation of di- is the most common species found on agnostic methodologies, the sample Apis mellifera bees. 34 • The Varroa Mite

Fig. 10: The varroa mite. The most serious pest of honeybees worldwide is the ectoparasitic mite Varroa destructor. (A) Lateral view of an adult female mite resting on the head of a pupae. (B) Dorsal view of an adult female mite. (C) An immature female mite. (D) An immature male. (E) An adult female on a pupae. Photographs used with permission © Gilles San Martin. The Varroa Mite • 35

Fig. 11: (Previous page) Development of the ing widespread colony destruction and varroa mite. The life cycle of the varroa mite is in economically damaging the beekeeping synchrony with the development of the honey bee. Most mite cycles only produce a single viable adult industry. Several remedies have been female. The probability of a fertile female emerging recommended to control the varroa successfully is shown as percentage values. The population in beehives including chemi- male mites do not exit the developmental cell. cal and physical treatments. Chemical Numbers on the left denote developmental stage of the bee in days. methods include the use of acaricides or natural products to control mites. Or- Varroa mites are 8-legged ectoparasites ganic compounds such as oxalic acid, that infest honey bees and bee colonies. lactic acid and formic acid are popular, as They have flat oval reddish-brown bod- they do not contaminate the honey yield, ies and measure 1 – 1.8 mm in length. leave minimal residues and show low tol- They attach themselves to the bodies of erance development. Formic acid is the adult bees using their tiny suction feet only agent demonstrated to kill mites and feed on the bee haemolymph using inside sealed brood (FRIES 1991). Other their highly modified piercing mouth chemical aciricides include organophos- parts. The mites snuggle underneath the phates (coumaphos) and pyrethroids abdominal sternites to pierce the soft (tau-fluvalinate, flumethrin). Several inter-segmental tissues and also remain commercially available acaricides use hidden from the hygienic surveillance these compounds or derivatives. Some of the bees. At the individual level, this of the popular brand names used in parasitisation results in weight loss lead- Denmark include Bayticol Pour-on Vet, ing to reduced lifespan, immune sup- and Apiguard. Physical approaches to pression (YANG and COX-FOSTER 2007), mite control include wire-netted sticky decreased learning capabilities and boards, powdered sugar, heating, and other disorders depending on the level biological control such as brood removal and time of infestation. Varroa mites and the fungus Beauveria spp. (MEIKLE have been shown to be an effective vec- et al. 2008; STEENBERG et al. 2010). tor transmitting several RNA viruses, Even the most meticulous of hive man- causing more frequent outbreaks of viral agement strategies do not effectively infection. At the colony level, the overall eliminate the mite population. The nat- fitness is reduced due to lower numbers ural selection strategy can be slow, eco- of swarms (FRIES and CAMAZINE 2001), nomically non-viable and can lead to less decreased flight performance in infect- desirable traits such as increased aggres- ed drones (DUAY et al. 2002), scattered sive behaviour (FRIES and BOMMARCO brood, crippled bees and reduced bee 2007). Untreated colonies have been re- population. ported to survive for more than 11 years Extensive infestation of varroa mites without any mite suppression measures, and the associated impact on the bee is suggesting an integrated varroa man- referred to as varoosis (BOECKING and agement strategy (LE CONTE et al. 2007). GENERSCH 2008). Varoosis is the most More long-term varroa control strate- devastating disease of honey bees caus- gies include selective breeding of bees 36 • The Varroa Mite for improved hygienic behaviour such as life stage, the female mite clings on to the better grooming and quicker removal of body of the adult bee and feeds on the parasitised brood (MORITZ et al. 2010). haemolymph of the bee by piercing the This idea has been actively pursued for soft inter-segmental tissue between the the last few years by the USDA which sternites. During this phase, the mite is successfully developed VSH (varroa sen- able to shift to new host individuals in sitive hygiene) bees which are extremely close proximity within the hive or dur- sensitive to brood parasitised by varroa ing foraging etc. (ROSENKRANZ et al. mites. VSH bees are claimed to prefer- 2010). entially remove mite infested pupae less As the adult female mite approaches re- than five days post capping (DANKA et productive maturity, she transfers herself al. 2011). to nurse bees and is quickly transported Varroa mites were initially suspected of to the brood cells. The mites are attract- being a potential viral vector on the ob- ed to the larvae by means of chemical servation that they transmitted ABPV signals (LE CONTE et al. 1989). The mite and DWV several hours after removal of enters a brood cell before the larvae is the infected host, negating the possibil- capped and moves to the bottom of the ity of contaminated mouthparts (BALL cell, consuming the larval food and feed- 1983). Varroa mites have now been dem- ing on the larval haemolymph. The first onstrated to transmit several viruses in- egg is usually unfertilised and develops cluding DWV (BOWEN-WALKER et al. into a haploid male. All further eggs 1999), ABPV (BALL 1983), KBV (CHEN et are fertilised and develop into females. al. 2004a; SHEN et al. 2005b) and IAPV The males are smaller, pear-shaped and (DI PRISCO et al. 2011b). Some studies have yellowish colouration. An adult show that apart from the translocation female mite produces 4-6 offspring in of the virus, the mite feeding may in- a single worker cell. The mite offspring duce viral activation due to immunosup- pass through well-defined developmen- pression (YANG and COX-FOSTER 2007). tal stages, namely larvae, protonymph, Certain studies have also reported DWV deutonymph and finally the adult stage. replication in mites (ONGUS et al. 2004a; The immobile stages are referred to as YUE and GENERSCH 2005) while others protocrysalis and deutocrysalis. The claim to find no evidence for viral repli- females gradually change from an ob- cation in mites (SANTILLAN-GALICIA et long shape to an elliptical shape in the al. 2008). final phase of change, which includes a changing colouration to reddish-brown. 4.1 Life Cycle The developmental time of the offspring The life cycle of the varroa mite is very from egg to adult is between 5-6 days. closely associated with its host, the The now matured offspring females mate honey bee. The mite has two distinct with the single matured male to produce stages – the phoretic phase on the adult further offspring. The life of the male bee and the reproductive phase in the mite is short and does not have a phoret- capped brood cells. During the phoretic ic stage. The female mites (usually only Host Defence • 37 one) crawl out from the uncapped cells munity as the primary line of defence. and find new host individuals (IFANTI- The physical barrier includes the outer DIS 1983; IFANTIDIS and ROSENKRANZ exoskeleton, cuticular linings, external 1988). anti-microbial secretions and chitinous membranes. Classification into humoral and cellular responses is only for con- 5 Host Defence venience, as in reality they are highly in- Honey bees have evolved diverse defence tertwined and overlap in function. mechanisms to counter-attack assault by Cellular immunity consists of haemo- pathogens. Social insects have individ- cyte-mediated responses that come into ual level and group-level (colony-level) play with the onset of microbial infec- defences. tion. Special immune receptor proteins The honey bee defence starts at the col- called pattern recognition receptors ony level. Honey bee colonies are always (PRRs) recognise pathogen-associated confined cavities with narrow entrances molecular patterns (PAMPs) on the sur- patrolled by guard bees helping to keep face of microorganisms and trigger a intruders away. The hygienic behaviour proteolytic cascade. This activates anti- (ROTHENBUHLER 1964) of worker bees microbial peptide expression pathways is important in the rapid detection and and several other mechanisms such as removal of dead or diseased brood. The phagocytosis, encapsulation, and nodu- grooming behaviour is useful in keeping lation. During phagocytosis, circulat- individuals clean and healthy. The anti- ing plasmatocytes, activated by surface microbial properties of propolis, certain receptors, engulf pathogens and de- pollen and royal jelly aid in excluding stroy them intracellularly. Bacteria are bacteria, viruses and other microbes. known to be destroyed by phagocytosis, Female worker bees have a modified ovi- but different bacteria may evoke differ- positor which is used as sting to inject ent immune responses as phagocytosis toxic venom into large predators such as is a complex process involving several bears or humans. Apis cerana japonica is sequential signal transduction events known to kill attacking hornets by sur- (TSAKAS and MARMARAS 2010). Nodu- rounding and heating them to death. lation is a method of entrapping bacte- Most reported internal defence path- rial clusters by aggregates of multicel- ways in honey bees are plausible predic- lular haemocytes. Encapsulation works tions based on Drosophila or Anopheles against larger targets such as protozoa or studies. Insects are not known to possess nematodes to which haemocytes attach, antibodies or immune memory (CHEN forming a capsule layer and killing it. and SIEDE 2007). Individual host de- The humoral immune response involves fence mechanisms can be classified to melanisation, haemolymph clotting and three categories: physical barriers, hu- secretion of anti-microbial peptides moral response and cellular response. (VILMOS and KURUCZ 1998). The black The physical and chemical barrier of the pigment melanin is transported to the individual bee confers non-specific im- cuticle, regions of injury or encapsulated 38 • Colony Collapse Disorder parasites. The enzyme phenoloxidase is duction of AMPs was observed, suggest- responsible for the catalysis of melanin, ing suppression of the humoral immune which is triggered by the serine pro- response by the virus. tease cascade. Anti-microbial peptides RNA interference (RNAi) is an impor- (AMPs) are synthesised in several sites tant mechanism by which eukaryo- such as fat bodies, haemocytes, epithe- tic cells regulate gene expression at the lial cells, etc. Haemolymph clotting in- transcriptomic level (FIRE et al. 1998; volving the clottable proteins lipophorin HANNON 2002). RNAi is a critical anti- and vitellogenin has been identified in viral defence pathway especially useful cockroaches and locusts. Rapid pro- against positive ssRNA viruses (LI et al. duction of anti-microbial factors from 2002; VOINNET 2005). Replication of the fat bodies, epithelial cells, and salivary viral genome forms dsRNA intermedi- glands kill target cells by attacking dif- ates, which are recognised by the endo- ferent components of the bacterial cell nuclease enzyme Dicer. Dicer cleaves wall causing lysis. These are activated in the dsRNA into short interfering RNAs the event of tissue injury or microbial in- (siRNA) of 20-25bp. These siRNAs form fection. Several candidate genes involved the RNA-induced silencing complex in immune response have been studied (RISC), which can degrade specific tar- in honey bees following other insect or get mRNA or viral RNA. Three differ- vertebrate studies (BONCRISTIANI et al. ent RNAi pathways have been defined 2012; NAZZI et al. 2012). in Drosophila (TSAKAS and MARMARAS The immune response in honey bees is 2010). Natural resistance to IAPV was thought to be mediated through four reported in honey bees harbouring a non-autonomous pathways, namely the ~1000nt fragment of IAPV sequence in Toll pathway, the Immune deficiency their genome (MAORI et al. 2007b). Arti- pathway (Imd), the JAK/STAT path- ficial ingestion of short dsRNA to honey way and the JNK pathway (EVANS et al. bee workers was reported to suppress 2006). The Toll pathway is activated by infection of IAPV (MAORI et al. 2009), fungal and gram-positive bacterial in- Chinese Sacbrood (LIU et al. 2010) and fections. Specific peptidoglycan recogni- DWV (DESAI et al. 2012). This method tion proteins (PGRPs) recognise bacte- was improvised to be used therapeuti- rial peptidoglycans and activate serine cally against viral infection, and large protease cascades. The Imd pathway is scale field studies in the US have shown activated by gram-negative bacteria. An- significant reduction of IAPV in treated ti-viral responses are thought to be con- bees (HUNTER et al. 2010). trolled via the Toll, Imd and JAK/STAT pathways. Bacterial and viral infections do not induce the same pathways. Hon- 6 Colony Collapse Disorder ey bee larvae injected with ABPV do not Colony Collapse Disorder (CCD) was produce a humoral immune response in first described by VAN ( ENGELSDORP et contrast to Escherichia coli infected con- al. 2009) to explain large scale colony trol bees (AZZAMI et al. 2012). No pro- losses (50-90%) in the US during the Colony Collapse Disorder • 39 winter of 2006-2007 and again in 2007- imported bees. No CCD events have 2008 (VAN ENGELSDORP et al. 2008). been reported from Australia, which This sudden disappearance of bees was is, interestingly, free of varroa mites. A distinct from regular winter losses in study by (CHEN and EVANS 2006) uncov- several respects. The notable features ered the historic presence of IAPV in the were rapid loss of adult workers from the US although IAPV was first identified hives, absence of dead bees in or around and reported in Israel in 2007 (MAORI the hive, presence of capped brood, pres- et al. 2007a). IAPV was soon reported ence of pollen, sufficient food stores in several studies (ANTUNEZ et al. 2012; and a queen remaining with few work- BLANCHARD et al. 2008) but was not ers. Losses from CCD syndrome were implicated in CCD. Furthermore CDD quickly reported from Europe (DAINAT was reported from IAPV-free colonies. et al. 2012c), Middle East and Asia (CAR- Varroa-virus models suggest IAPV kills RECK and NEUMANN 2010). The world- infected individuals quickly such that wide monetary losses in apiculture and the prevalence at colony level remains agriculture due to CCD is estimated to low (MARTIN 2001a). Nosema ceranae be 75 billion USD (SWINTON et al. 2007). was implicated in CCD (HIGES et al. The COLOSS (Prevention of honey bee 2009; PAXTON 2010), in stark contrast to COlony LOSSes) COST Action network (STEVANOVIC et al. 2011) who reported was set up with more than 150 mem- no CCD symptoms in colonies with high bers from 40 countries to investigate the titres of Nosema ceranae. A study (BRO- phenomenon (CARRECK and NEUMANN MENSHENK et al. 2010) which claimed 2010). Nosema and iridoviruses to drive colony The causes of CCD are debated intensely collapse was refuted in several follow- by the scientific community. Postulated up studies (FOSTER 2011; TOKARZ et al. reasons range from pathogens, pesticides 2011). A combination of varroa mites and nutritional deficiency to immune and bee health has been suggested in a stresses, migratory beekeeping (EVANS few studies (AMDAM et al. 2004; LE CON- and SCHWARZ 2011; OLDROYD 2007) TE et al. 2010; VEJSNÆS et al. 2010). and even climate change (LE CONTE and Although no single causal factor could NAVAJAS 2008). Several pathogens have be identified and Koch’s postulate re- been short-listed in several studies that mains to be fulfilled, it is becoming clear could be potential cause of CCD. One that CCD may be multi-factoral and of the earliest studies that investigated a complex (NAZZI et al. 2012). It is possi- wide range of pathogens including bacte- ble that CCD deaths are overemphasised ria, fungi, protozoans and viruses found by combining bee deaths from common Israeli Acute Paralysis Virus (IAPV) pathogens into a single unknown cause to be highly associated with collapsing (WILLIAMS et al. 2010). Altenatively, colonies (COX-FOSTER et al. 2007). This CCD may be just an innate response to consequently led to country-wide sam- highly unfavourable colony conditions, pling and surveys studying the intro- leading to so called adaptive suicide duction of IAPV from Australia though (SEAVER 2011). Nevertheless, many re- 40 • Sampling Strategies cent studies seem to point to the Varroa- sampled as a scoop of individuals and DWV interaction as a lethal combina- then diagnosed as pooled samples or in- tion leading to the collapse of honey bee dividuals. Bees are sampled for various colonies (CARRECK et al. 2010a; DAINAT reasons, such as to estimate mite load in et al. 2012a; DAINAT et al. 2012b; HIGH- the colony, to estimate the presence of FIELD et al. 2009; MARTIN et al. 2012). nosema, viruses, etc. This is important High DWV variant diversity was found in order to enable beekeepers to estimate in colonies where varroa mites were still the health of their bees to make deci- absent. These are natural DWV vari- sions on treatment and apiary manage- ants seldom causing wing deformity and ment in general. never resulting in colony death. Intro- The often unregulated use of acaricide duction of varroa mites reduced this treatments had lead to drastic rise in tol- diversity to a single DWV variant in 2-3 erance to these chemicals. This means years. This single selected DWV strain the only way to control mites would to was thereafter adapted for mite-mediat- be further increase the dosage or fre- ed transmission and may have different quency of these treatments. This cannot virulence thus contributing to colony be a long-term strategy since it is envi- collapse (MARTIN et al. 2012). ronmentally harmful and unsustain- There is sufficient recent literature to able. Hence, a proper sampling strategy suggest that the varroa-virus interaction can allow beekeepers to only treat their plays a major role in honey bee disease. hives if the pathogen of interest is above It is therefore reasonable that we have a specified threshold. One methods of mostly focussed our efforts on studying sampling involves estimating female the role of varroa mites and viruses (AKI mites in a colony from mite densities on and DWV) and their interaction with adult bees and pupae (MARTIN 1998). A honey bees. second method involves regression on mites caught on sticky boards (BRANCO et al. 2006). Although, these methods 7 Sampling Strategies are good at estimating colony level mite To study the effect of pathogens on a host densities, they are not practical for apiar- organism, it is important to sample the ies or large-scale studies. host organism to isolate the pathogen of A recent large practical sampling study interest. The sampling must ensure that (LEE et al. 2010) decided to deal with sampled material contains the patho- sampling in a different way, which gen of interest, is sampled at favourable would suit small and large-scale opera- times, sampling quantity is practical and tions with different levels of precision. financially feasible, and that sampling The sampling plan for beekeepers with leads to statistically robust results. a precision of C ≤ 0.25 would require 5 Since honey bees are social insects, the x 35 bees or 1 x 175 bees from separate survival of the colony is more important frames. The sampling plan for research- than the survival of every single indi- ers with a precision of C ≤ 0.1 would re- vidual. Therefore honey bees are usually quire 21 x 35 bees or 3 x 300 bees from Aims And Objectives • 41 separate frames. To account for mites in number of bees sampled was too low to the capped brood, the mite count is mul- determine mite levels accurately but ap- tiplied by a factor 2. propriate for estimating viral load. The method used to sample the bees and mites also has profound repercussions on the state of the colony. Some of the 8 Aims and Objectives methods include bottling followed by As explained in the introduction, it is washing, immersion in alcohol or anaes- clear that honey bees are facing a diffi- thetising using CO2, powdered sugar, cult period and therefore, any improve- etc. Sampling large numbers of worker ment in diagnostics, understanding and bees using lethal methods can have det- control of the factors involved, can help rimental effect on the colony. Conse- us to better fight honey bee pathogens. quent removal of mites also affects the As a diagnostic lab performing viral dynamics of the mite population, pos- analysis on honey bee samples from sibly influencing the experiment. The beekeepers on a regular basis, it was im- powdered sugar method is a popular portant to improve the existing screen- non-lethal technique but is not thought ing methods. Hence the objective for the to be as effective. A few devices based on first experiment was to design primers to pumps and vacuums have been devised, detect ABPV, KBV and IAPV viruses in but they are cumbersome or impractical. a single assay. Since these are low-preva- An improved technique or device would lence viruses in Denmark, separate PCR greatly enhance the current workflow in runs are time-consuming and wasteful. bee sampling. Viral infection in honeybee queens have Manuscript III deals with varroa and vi- not been studied extensively and its role rus levels in 23 hives over 8 months. A in venereal transmission is therefore, single bottle of ~200 bees was sampled still uncertain. The aim of the second from every hive once a month. The bees experiment was thus, to investigate the were anaesthetised using CO2 and mites distribution of viral infection in various were shaken off. The total mites were tissues of honey bee queens. We were in- counted as an approximate estimation terested in the general trend of AKI and of colony-level mite infestation. To esti- DWV virus infection, including differ- mate viral prevalence in the colony, the ences in frequency, titre and preference 200 bees were split into 10 sub-samples between tissues. We also investigated and viral analysis was carried out sepa- whether the viruses replicated in all tis- rately. The method of splitting into sub- sues. samples allows estimation of the preva- The third study monitored varroa and lence of virus infected workers in the viruses in 23 colonies under three treat- colony. Therefore, a system of fixed sam- ment conditions over the span of a year. pling quantity was maintained through- The aim of this study was to capture the out the experiment although the mite seasonal trend and fluctuations in varroa and viral prevalence was later found to and virus titres in response to different vary drastically through the season. The treatments. The varroa-virus interaction 42 • Aims And Objectives with the honey bee host was the para- mount concern. A new strategy of splitting the bee samples into sub-samples was used to get a clearer picture of colony-level viral prevalence. Section E PAPERS BLANK REVERSE PAGE Paper 1 • 45

PAPER 1 Single assay detection of ABPV, KBV & IAPV BLANK REVERSE PAGE

BLANK REVERSE PAGE Paper 2 • 57

PAPER 2 Patterns of viral infection in honey bee queens (Manuscript resubmitted after Review) BLANK REVERSE PAGE 1

BLANK REVERSE PAGE

Paper 3 • 79

PAPER 3 Varroa-virus interaction in collapsing honey bee colonies (Manuscript) BLANK REVERSE PAGE 1

Table S2 A K I A01 A02 A03 A04 A05 A06 A07 A08 A09 A10 A11 B01 B02 B03 B04 B05 B06 B07 B08 B09 C01 C02 C03 100000000000 3 00000000000 2 00000000000000000000000 3 00000000000000000000000 4 00000000000000000000000 5 000000000000000000000 2 0 6 00000000000000000000000 7 00000000000000000000000 April 2011April 8 00000000000000000000000 9 00000000000000000000000 10 000000000 3 0 0 0 0 0 0 0 0 0 0 3 0 0 VAR 0 0 0 0 0 0 0 0 0 0 3 0 1 00000000000000000000000 2 00000000000000000000000 3 00000000000000000000000 4 00000000000000000000000 5 00000000000000000000000 6 00000000000000000000000

May 2011May 7 00000000000000000000000 8 00000000000000000000000 9 00000000000000000000000 10 0 0 0 0 0 000000000000000 VAR 0 0 0 0 0 0 0 0 0 0 3 0 1 00000000000000000 2 0 3 0 0 2 2 00000000000000000 3 0 4 0 0 0 3 0 0 0 0 0 3 00000000000 3 0 4 0 0 0 4 00000000000000000 3 0 6 0 0 0 5 0 0 0 0 0 0 7 0 0 0 0 0 0 0 0 0 0 3 0 5 0 0 0 6 00000000000000000 4 0 4 0 0 0

June 2011 7 00000000000000000 3 0 7 0 0 0 8 00000000000000000 4 0 4 0 0 0 9 00000000000000000 4 0 3 0 0 0 100000000000000 2 0 0 0 3 0 3 0 0 0 VAR 0 0 0 0 0 0 0 5 1 0 0 0 0 0 0 3 0 0 0 0 3 3 0 3 0 0 4 3 4 0 0 0 2 0 0 0 0 0 0 0 0 2 0 0 3 3 0 0 0 3 4 4 4 0 0 0 3 0 3 0 0 0 0 0 0 0 0 0 5 2 0 2 3 0 7 4 4 0 0 0 4 0 0 0 0 0 0 0 0 5 0 0 3 3 0 2 3 0 4 4 5 0 0 0 5 0 0 0 0 0 0 3 0 3 0 0 0 0 0 0 3 0 4 4 4 5 3 0 6 0 2 3 0 3 0 0 0 3 0 0 6 0 0 0 3 0 4 4 4 0 0 0 7 0 3 0 0 0 0 3 0 3 0 0 4 3 0 0 0 0 5 4 4 0 0 0 July 2011July 8 0 0 0 0 0 0 3 0 3 0 0 3 0 0 0 0 0 4 3 4 0 0 0 9 0 0 0 3 3 0 0 0 3 0 0 0 3 0 3 0 0 4 4 8 0 0 0 10 0 0 0 0 0 0 3 0 3 0 0 3 3 0 0 0 2 4 5 3 2 0 0 VAR 0 0 3 3 0 0 3 3 0 3 3 3 0 0 0 1 0 3 0 6 0 0 0 0 3 0 0 0 3 0 3 0 4 3 3 0 0 0 3 2 0 0 4 0 0 0 0 0 3 0 0 3 0 0 4 0 3 3 3 0 0 3 4 3 0 0 3 0 0 0 0 0 4 0 0 3 3 0 0 0 3 0 3 0 0 0 6 4 0 0 0 0 0 0 0 3 3 0 0 0 0 0 0 0 3 3 3 0 0 0 3 5 0 0 0 0 0 0 0 0 3 0 0 3 0 0 0 0 4 3 4 0 0 3 5 6 0 0 0 3 0 0 0 0 3 0 0 0 0 0 0 0 3 4 3 3 0 0 6 7 0 0 0 0 0 0 0 0 3 0 0 3 3 3 5 0 4 3 3 4 0 3 5 August 2011August 8 0 0 0 0 0 0 0 0 3 0 0 0 0 3 3 0 4 4 3 3 0 0 6 9 0 0 3 0 0 0 0 3 4 0 0 0 0 0 4 0 5 4 4 4 0 3 4 10 0 0 3 0 0 0 0 2 3 0 0 0 0 0 3 0 4 4 3 3 0 0 4 VAR 0 0 0 0 0 3 3 0 5 0 0 3 5 2 4 0 1 0 0 0 7 0 0 0 0 0 0 0 0 0 0 2 3 0 4 0 0 9 4 8 2 0 0 0 8 0 3 0 0 0 0 0 0 0 2 0 3 0 3 0 0 9 5 5 3 4 0 0 5 0 0 2 0 0 0 0 0 0 3 0 0 0 0 2 0 10 4 6 4 0 0 0 7 0 0 0 0 0 0 3 0 0 0 0 0 0 3 0 3 9 5 6 5 0 0 0 8 0 0 0 0 2 0 3 0 0 3 0 0 0 3 3 3 10 4 6 6 0 0 3 5 00000000000 3 0 3 0 3 9 5 6 7 0 0 3 5 0 0 0 0 0 0 0 0 0 3 2 4 3 0 0 0 10 4 7 8 0 0 3 6 0000000000000000 9 6 6 September 2011 September 9 0 3 3 9 0 0 0 0 0 0 3 0 0 3 2 4 0 0 3 3 9 4 6 10 0 0 0 5 0 0 0 0 0 0 3 0 3 0 0 0 4 0 3 3 10 4 6 VAR 0 0 3 7 4 3 3 0 4 3 3 3 0 3 0 0 9 7 5 1 0 0 4 9 8 4 7 4 6 3 0 6 0 0 4 0 2 3 4 8 9 8 2 0 0 3 9 8 3 6 3 5 0 0 6 0 0 0 0 0 0 3 0 9 7 3 0 0 0 9 8 3 6 4 5 0 0 5 0 0 0 3 3 0 3 0 9 7 4 0 0 0 9 7 3 6 4 5 0 0 5 0 3 0 0 0 0 0 3 9 7 5 0 0 3 8 8 0 6 3 4 0 0 5 0 3 3 0 3 0 0 3 10 7 6 0 0 0 8 5 0 6 4 5 0 0 5 0 0 0 0 0 3 3 0 9 6 7 0 0 3 8 9 0 6 4 5 0 0 5 0 2 0 0 0 0 0 3 9 8 8 0 0 3 8 9 3 7 5 5 0 4 5 0 0 0 3 3 3 0 0 9 6 October 2011October 9 3 0 3 9 8 4 7 4 5 0 0 5 0 0 0 3 3 3 3 2 8 7 10 0 2 4 9 7 3 6 4 5 0 3 5 0 0 0 0 3 3 3 7 9 6 VAR 3 4 3 7 5 4 5 2 0 4 4 0 0 3 3 6 4 3 4 8 7 1 4 4 3 3 0 0 0 0 0 0 0 3 0 0 0 0 2 4 3 3 3 0 0 0 0 0 0 0 0 3 0 5 3 3 4 3 3 000 000000000 3 4 4 4 3 2 0 0 0 0 0 0 0 0 3 0 0 3 5 4 4 3 0 3 0 0 0 0 0 0 2 0 0 0 0 6 4 4 3 0 0 0 0 0 0 0 0 0 0 3 0 0

April 2012April 7 4 4 0 2 0 0 0 0 0 3 0 0 0 4 0 0 8 4 4 0 0 0 0 0 0 0 0 3 0 0 4 0 0 9 4 4 3 0 0 0 3 0 0 0 3 0 0 4 0 0 10 3 4 3 2 0 0 0 0 0 3 0 0 0 3 0 0 ** * **** ORGANIC FLUMETHRIN UNTREATED VARROA-FREE 0 Null 2 Low 5 Medium 9 High Table S2 DWV A01 A02 A03 A04 A05 A06 A07 A08 A09 A10 A11 B01 B02 B03 B04 B05 B06 B07 B08 B09 C01 C02 C03 10000000000 5 0 6 6 0 0 6 0 0 4 9 4 0 2 0 4 0 5 0 0 0 0 0 0 0 0 4 4 0 0 7 0 0 0 5 4 0 3 0 0 0 4 0 0 0 0 0 0 0 0 4 4 0 0 7 0 0 4 5 5 0 4 0 0 0 4 0 0 0 0 0 0 0 0 0 4 0 0 5 0 0 4 5 5 0 5 0 0 0 5 0 0 0 0 0 0 0 0 5 4 0 0 5 0 0 4 7 5 0 6 0 0 0 4 0 0 0 0 0 0 0 0 4 4 0 0 6 0 0 5 0 7 0 7 4 5 0 5 0 0 0 0 0 0 0 0 6 4 0 6 7 0 0 5 5 4 0 April 2011April 8000000000000 9 5 0 0 9 0 4 4 4 5 4 9 0 4 5 5 0 0 0 5 0 0 4 0 0 5 0 4 9 0 4 5 5 5 0 10 0 0 0 6 0 0 0 5 0 0 5 0 0 4 0 0 7 0 0 4 5 7 0 VAR 0 4 0 0 4 4 0 0 0 0 6 0 1 0 0 0 0 0 0 0 4 0 0 0 0 5 4 0 0 6 0 0 5 0 4 0 2 0 0 0 0 0 0 4 5 0 0 0 0 6 4 0 0 4 0 0 4 4 0 0 3 0 0 0 4 0 0 0 4 0 0 0 0 7 4 0 0 0 0 0 8 4 0 0 4 0 0 0 0 0 0 0 4 0 0 0 0 5 4 0 0 4 0 0 6 5 6 0 5 0 0 0 4 0 0 0 4 0 0 0 0 4 5 0 0 4 0 0 5 4 4 0 6 0 0 0 5 0 0 0 4 0 0 0 0 5 4 0 0 4 0 0 5 4 4 0

May 2011May 7 0 0 0 0 0 0 0 5 0 0 0 0 5 5 0 0 6 4 0 6 5 5 4 8 0 0 0 5 0 0 0 4 0 0 0 0 4 4 0 0 5 0 0 8 5 4 0 9 0 0 0 0 0 4 0 0 0 0 0 0 5 5 0 0 8 0 0 9 5 4 0 10 0 0 0 0 0 0 0 0 0 4 4 0 0 4 4 0 5 4 0 4 VAR 0 0 0 4 0 0 0 4 0 0 5 0 1 0 0 0 4 0 0 0 0 0 4 0 0 4 0 0 0 5 0 0 5 4 10 0 2 0 0 5 0 0 0 0 0 0 0 0 0 5 0 0 0 5 0 0 5 5 5 0 3000000000000 4 0 0 0 5 0 0 6 5 5 0 4 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 5 0 0 6 5 4 0 5 000000000000000 6 6 0 0 5 7 5 0 6000000000000 4 0 0 0 5 0 0 6 7 6 4

June 2011 7 0 0 0 0 4 0 0 0 0 0 0 0 0 0 4 0 6 0 0 7 5 6 4 800000000000 4 0 4 0 4 6 0 0 5 6 5 0 9 0 0 0 0 0 0 0 0 0 5 0 0 4 0 5 4 5 0 0 8 6 6 4 10 0 0 0 0 0 4 0 0 0 0 0 0 0 4 4 0 5 0 0 5 5 4 4 VAR 0 0 0 0 0 4 6 0 1 0 0 0 0 5 7 4 4 0 6 0 5 6 4 5 5 6 9 5 7 8 7 4 2 0 0 0 4 5 6 0 4 0 8 0 5 6 4 4 5 5 7 0 7 7 9 5 3 0 0 0 0 5 7 5 8 0 6 0 5 6 5 5 6 6 8 0 9 7 6 0 4 0 0 0 0 5 6 4 4 0 5 4 5 6 6 5 5 7 7 5 7 9 9 4 5 0 0 0 0 5 7 0 6 0 6 0 5 5 5 5 6 6 7 0 6 8 6 0 6 0 0 0 0 5 6 0 5 0 5 0 5 6 5 4 5 6 7 0 8 9 8 0 7 0 0 0 0 5 6 4 4 0 6 0 5 6 5 5 6 5 8 4 8 9 6 0 July 2011July 8 0 0 0 0 5 6 5 8 0 6 0 5 6 5 4 5 5 7 4 6 8 7 0 9 0 5 0 0 5 6 6 5 0 9 4 5 6 5 5 6 6 7 0 8 9 9 4 10 0 0 0 0 7 6 0 7 0 6 0 5 7 5 5 5 5 7 4 6 8 7 4 VAR 4 4 5 6 0 4 5 5 4 5 7 9 9 5 0 1 0 0 5 6 8 4 0 6 0 6 0 4 9 5 5 4 6 8 5 7 9 6 9 2 0 0 0 7 6 5 0 6 0 6 0 5 7 4 4 4 9 9 6 8 9 5 9 3 0 0 0 6 6 4 0 6 0 6 0 4 8 6 5 4 9 8 5 8 5 4 10 4 0 0 0 5 7 4 0 6 0 6 0 5 8 5 6 4 6 8 5 8 5 4 9 5 0 0 0 5 6 5 0 9 5 6 5 5 7 5 5 4 6 10 6 8 6 5 9 6 4 0 0 6 6 4 0 8 0 6 0 0 6 5 4 5 6 9 5 9 5 5 10 7 0 0 0 4 6 4 6 6 0 5 5 9 6 6 11 6 6 9 8 8 7 7 8 August 2011August 8 0 4 0 4 7 5 0 9 4 6 4 4 8 5 6 0 8 7 7 10 9 6 9 9 0 0 0 0 8 4 0 6 4 8 5 4 7 6 5 4 4 9 6 8 6 5 10 10 0 0 0 5 8 4 0 7 0 5 4 5 9 5 6 6 7 9 0 10 8 5 10 VAR 0 4 0 6 7 7 5 9 6 8 7 6 9 5 9 0 1 4 6 6 8 9 5 0 10 0 4 0 4 7 6 4 4 4 10 9 9 10 6 8 2 0 7 6 8 9 5 4 7 4 0 0 4 9 5 0 4 9 10 9 9 10 7 9 3 0 5 6 7 9 5 0 7 0 0 5 4 9 4 4 5 4 10 6 10 11 6 9 4 0 7 6 9 9 5 0 7 4 0 4 4 10 5 5 0 4 9 5 9 11 9 9 5 4 6 8 8 9 8 6 7 4 4 6 4 9 5 7 5 5 9 10 10 11 7 9 6 4 5 6 8 9 5 0 7 0 4 4 4 8 4 4 5 5 9 9 9 11 9 8 7 4 6 6 7 9 6 4 8 0 4 4 4 9 5 4 0 5 9 10 10 11 9 9 8 5 5 6 7 9 4 4 7 0 4 4 4 8 6 4 0 4 10 9 8 11 7 8 September 2011 September 9 4 6 6 9 9 6 4 9 4 5 5 6 7 5 5 6 4 9 9 10 11 9 9 10 0 6 5 9 10 6 4 7 4 4 5 5 9 6 4 5 5 9 10 9 11 9 8 VAR 6 8 6 9 10 7 9 8 5 4 5 7 6 9 9 10 12 8 11 1 4 7 9 10 10 7 8 6 6 5 5 8 8 6 5 5 5 9 7 11 11 9 2 6 7 9 9 10 8 7 6 6 5 5 7 8 5 0 5 5 7 10 10 11 9 3 6 7 9 10 10 7 8 10 6 10 0 7 8 5 0 4 6 6 9 10 11 9 4 5 9 9 8 10 7 8 6 6 7 5 7 8 6 4 4 5 7 9 10 11 10 5 5 8 9 9 10 9 8 9 6 5 5 7 8 6 5 6 5 7 10 11 11 9 6 4 6 7 8 9 6 8 8 5 5 7 7 8 6 4 5 6 7 7 11 11 9 7 7 9 9 10 11 7 8 7 6 5 4 7 9 5 0 4 4 6 7 10 10 10 8 6 9 9 9 10 10 8 8 6 5 6 7 8 5 4 4 5 8 10 10 10 9 October 2011October 9 5 9 8 8 10 7 8 7 6 5 5 7 9 5 4 5 7 7 10 10 10 9 10 5 9 9 10 10 6 7 6 5 5 6 6 6 5 5 4 5 8 6 11 11 9 VAR 5 8 10 10 10 6 9 8 4 6 7 11 7 5 5 8 10 9 10 12 11 1 4 5 7 4 4 0 0 4 5 4 0 0 0 6 4 5 2 4 5 7 9 0 0 5 0 4 0 4 0 0 5 4 5 3 5 4 6 0 0 4 4 0 0 0 0 0 0 7 4 4 4 4 5 6 0 0 5 5 4 0 4 0 0 0 6 4 5 5 5 5 5 0 0 4 0 0 4 5 0 5 0 4 5 4 6 4 5 5 4 0 5 6 0 4 5 0 6 0 5 0 5

April 2012April 7 4 5 6 4 0 4 5 0 0 5 4 0 4 5 4 4 8 5 5 6 4 0 4 5 0 4 5 0 4 0 6 4 4 9 5 5 5 4 0 0 4 0 0 10 4 0 4 4 5 5 10 5 5 6 4 4 0 4 0 0 4 0 0 0 4 5 5 ** * **** ORGANIC FLUMETHRIN UNTREATED VARROA-FREE 0 Null 2 Low 5 Medium 9 High

BLANK REVERSE PAGE General Discussion • 105

General Discussion and Perspectives

lthough this project has provided us with great insight into the world of honey Aviruses, this has also raised several unanswered questions along with the need for improved methodologies. Conventional methods of disease diagnostics such as immuno-assays and ELISA have paved way to more specific and sensitive nucleic acid based methods for instance, realtime-PCR and microarrays. Better, faster and high-throughput quantitative methods like digital PCR, nanochips and integrated microfluidic circuits are being investigated to diagnose bee materials more efficiently and effectively. Some of the most recent high throughput systems in honey bee immunity and disease include Beepath qPCR array (EVANS 2006), biochip viral microarray (GLOVER et al. 2011), EST & cDNA microarray for brain and behaviour study (WHITFIELD et al. 2002) and MLPA-based BeeDoctor (SMET et al. 2012; in press) developed at Ghent University, Belgium. The AKI primers developed in this study now allows faster screening of samples and have proven instrumental to further our understanding of varroa-virus interactions as demonstrated in our study. We strive to improve the use of these AKI primers to differentiate variants using high resolu- tion melting (HRM). This would be extremely useful as it would eliminate the need for confirmatory virus-specific PCRs. This brings us to the topic of defining viral species. How is one virus different from another? The classical methods involve electron microscopy of the structure, immune-assays, host preference and phenotypic manifestation of the disease and symptoms. Viral species are now increasingly classified based on genome divergence. If so, what dissimilarity in the genetic composition defines a threshold for declaring new viral species? We know that viruses exist as quasi-species and therefore it is difficult to say if the ABPV-KBV-IAPV complex is three different viruses or a mix of different strains. The same argument also applies to DWV-KV-VDV1 complex. But ultimately, their effect on the health of honey bees is our concern at a biological scale. The social structure of honey bees dictates that no individual bee is isolated from the numerous transmission routes exhibited by viruses. This impacts on our capacity to study bees and viruses in vitro. The study of viral infection in honey bees is further limited by the lack of established honey bee tissue cultures. Therefore, our understanding of the development and the spread of viral infection in honey bee tissues is restricted to PCR-based methods. Given the small size of honey bees, determining tissue-specific infections is demanding. Some of the advanced methods to localise viral particles in the bee tissues include in situ PCR, fluorescent in situ hybridisation (FISH) or using GFP markers. While we know that infected drones successfully reach DCAs, we still do not know whether they mate with potential queens. The sexual transmission of any virus from the drone (sperm) to queen has not yet been demonstrated in a natural setting. The polyandrous behaviour of the queens means that her chances of acquiring viruses from an infected drone increases with every 106 • General Discussion mating. Does the benefit of increased diversity for colony fitness outweigh the risk of viral infection for the queen? The study on queens have shed light on the prevalence and titres of AKI and DWV viruses in various queen tissues. Most of the 86 queens tested were healthy and free of any symptomatic infection. This would suggest that the risk of infection for the queen during mating is negligible. Those few queens that exhibited high viral titres came from colonies with high varroa and virus load, point- ing to a worker-queen transmission of virus. It still remains to be shown why or how the queen is generally able to stay healthy despite being the longest living member of the bee colony. Sick worker bees are known to abandon their colony to restrict the spread of disease. This behaviour remains unreported for queens. CCD colonies often have the queen remaining behind with just a few workers. We intend to continue these studies along with field experiments to answer some of these questions. The large-scale field study has delivered a considerable amount of information, helping us to better understand varroa-virus interaction within honey bee colonies. While our findings are congruent with previously developed varroa-virus models, certain details such as the continued prevalence of AKI late in the season remains unexplained. This study helped us in defining an optimal sampling strategy for varroa and viruses in future experiments. It is now clear that a fixed sampling strategy for the whole year is inadequate, due to high differences in prevalence of viruses through the season. An better sampling methodology would mean low-density sampling in spring time, when the virus is low-prevalence, and increased density in late summer-autumn when the viral prevalence is much higher. This is also one of the first studies to analyse viruses by splitting samples into subsamples allowing us to estimate prevalence of virus-infected workers in the colony. An improved sampling strategy is being put to test currently in a follow-up study relating to varroa-virus interaction in Danish apiaries. The varroa-virus study clearly demonstrates that the ABPV-KBV-IAPV complex still plays a significant role in colony losses in Denmark. AKI and DWV viruses were found to be highly prevalent in the Danish bee population, especially during autumn. The beekeepers who fail to observe this problem at colony level would risk severe winter losses. It is imperative that a varroa-virus threshold be defined for targeted treatment to ensure successful overwintering. Specific knowledge of the varroa load and viral prevalence can help the beekeepers to formulate a safe treatment strategy to ensure the health of their colonies. Simple and rapid diagnostic tools for varroa and viruses are important for beekeepers to predict colony vitality. The prevalence of viruses seen in Denmark, extends to the queen. Given the complex transmission routes between queens and workers, and vice versa, a prerequisite to future establishment of virus-free colonies, would be the production of virus-free queens by breeders. Im- plementing such programs require close mutual co-operation between beekeepers and researchers. New and innovative ways to counter pests and diseases are constantly transforming the landscape of honey bee pathology. Varroa mites and consequently viruses are General Discussion • 107 predominantly controlled by chemical application. Organic and biological methods of control seem to have gained popularity over chemical methods in most of Europe and especially in Denmark. While this is a positive trend, it cannot be a long-term strategy due to build-up of resistance from recurring treatment. In contrast, ingestion of dsRNA has been demonstrated to control IAPV and DWV in bees. Although, feeding dsRNA on a regular basis can also not be a sustainable practice, it is possible to integrate the sequence into the honey bee genome to obtain stable expression. The integration of viral genome into the host has been demonstrated in several studies to happen naturally, yet the idea of genetically modified bees though targeted inser- tion of a transgene is petrifying to most people. In 1998, the use of GM crops (corn, cotton and soya beans) reduced the use of pesticides by 3.5% (WOLFENBARGER and PHIFER 2000). The traditional method of producing high quality bees were through breeding programs. This is a highly laborious, time-consuming and not guaranteed to yield satisfactory results. While certain desirable traits such as hygienic behaviour can be selected, this may give rise to undesirable features such as aggressiveness. This random shuffling of genetic material aiming to achieve the right combination is widely accepted as the conventional method, although it is a gamble. Gene-specific or targeted mutation has been studied for over a decade in various organisms from plants to insects and higher animals. Although gene expression studies in honey bees are on the rise, no studies have been carried out to regulate the expression of suitable genes to achieve desirable traits. GM organisms are often a hot topic of debate and only better understanding and safer technologies can instil public confidence in biotechnology. This project has fulfiled its objectives in providing us with greater knowledge and deeper understanding of the subject. This has also incited novel questions and opened doors for new and exciting areas of research. It is vital that honey bees are in good health to ensure that they continue to play their key role in ecology and food production for the benefit of human beings, animals and plants that rely on them. It is important for honey bee biologists that we strive to maintain and preserve the honey bees for the future generations as their contribution to the ecosystem is invaluable. 108 • General Discussion Appendix • 109

Appendix

Table. 1: Complete list of honey bee viruses with references.

Virus Abbr. Reference 1 Acute Bee Paralysis Virus ABPV (BAILEY et al. 1963; BAKONYI et al. 2002; DE MIRANDA et al. 2010a) 2 Kashmir Bee Virus KBV (BAILEY et al. 1979; CHEN et al. 2004a; HUNG et al. 1996b) 3 Israeli Acute Paralysis Virus IAPV (MAORI et al. 2007a) 4 Black Queen Cell Virus BQCV (BAILEY and WOODS 1977) 5 Deformed Wing Virus DWV (BALL 1983; DE MIRANDA and GENERSCH 2010) 6 Kakugo Virus KV (FUJIYUKI et al. 2004; FUJIYUKI et al. 2005) 7 Varroa Destructor Virus-1 VDV-1 (ONGUS et al. 2004a) 8 Sacbrood Virus SBV (BAILEY and FERNANDO 1972; BAILEY et al. 1964) 9 Slow Bee Paralysis Virus SBPV (BAILEY and WOODS 1974; DE MIRANDA et al. 2010b) 10 Chronic Bee Paralysis Virus CBPV (BAILEY et al. 1963; BURNSIDE 1945; RIBIERE et al. 2010) 11 Cloudy Wing Virus CWV (BAILEY et al. 1980a; CARRECK et al. 2010b) 12 Bee Virus X BVX (BAILEY and WOODS 1974) 13 Bee Virus Y BVY (BAILEY et al. 1980b) 14 Arkansas Bee Virus ABV (BAILEY and WOODS 1974; LOMMEL et al. 1985) 15 Berkeley Bee Virus BBPV (LOMMEL et al. 1985) 16 Macula-like Virus MaLV 17 Filamentous Virus FV (BAILEY et al. 1981; CLARK 1978) 18 Apis Iridiscent Virus AIV (BAILEY and BALL 1978) 19 Aphid Lethal Paralysis Virus ALPV (VAN MUNSTER et al. 2002; WILLIAMSON et al. 1988) 20 Big Sioux River Virus BSRV (RUNCKEL et al. 2011) 21 Lake Sinai Virus LSV-1 (RUNCKEL et al. 2011) Honey Bee Virus Reviews (ALLEN and BALL 1996a; AUBERT et al. 2005; BAILEY 1967; CHEN and SIEDE 2007; DE MIRANDA et al. 2010a) 110 • Appendix

Table. 2: List of viruses in Fig. 7. Type species of the genus is marked by asterix (*).

Abbrv. Name Accession Genus Family ABPV Acute Bee Paralysis Virus* NC_002548 Aparavirus Dicistroviridae IAPV Israeli Acute Paralysis Virus NC_009025 KBV Kashmir Bee Virus NC_004807 SINV-1 Solenopsis Invicta Virus - 1 NC_006559 TSV Taura Syndrome Virus NC_003005 ALPV Aphid Lethal Paralysis Virus NC_004365 Cripavirus BQCV Black Queen Cell Virus NC_003784 CrPV Cricket Paralysis Virus* NC_003924 DCV Drosophila C Virus NC_001834 HiPV Himetobi P Virus NC_003782 HoCV Homalodisca coagulate Virus - 1 NC_008029 PSIV Plautia Stali Intestine Virus NC_003779 RhPV Rhopalosiphum Padi Virus NC_001874 TrV Triatoma Virus NC_003783 DWV Deformed Wing Virus NC_004830 Iflavirus Iflaviridae EoPV Ectropis Obliqua Picorna-like Virus NC_005092 IFV Infectious Flacherie Virus* NC_003781 KV Kakugo Virus NC_005876 PNV Perina Nuda Virus NC_003113 SBPV Slow Bee Paralysis Virus NC_014137 SBV Sacbrood Virus NC_002066 VDV-1 Varroa Destructor Virus - 1 NC_006494 EV-109 Human Enterovirus 109 NC_014336 Enterovirus Picornaviridae FMDV Food and Mouth Disease Type A NC_011450 Aphthovirus PV Poliovirus NC_001489 Enterovirus Appendix • 111

List of Figures Fig 01 - Queen, Worker and Drone – Comparative figure Pg. 17 Fig 02 - Development of a honey bee worker Pg. 18 Fig 03 - Haplodiploidy system of reproduction in honey bees Pg. 20 Fig 04 - Small Hive Beetle Pg. 24 Fig 05 - Electronmicrograph of ABPV & KBV Pg. 25 Fig 06 - Genome composition Pg. 26 Fig 07 - Virus whole genome dendogram Pg. 28 Fig 08 - Viral transmission pathway Pg. 31 Fig 09 - DWV infected bee with deformed wings Pg. 31 Fig 10 - Varroa Mite Pg. 34 Fig 11 - Development of the varroa mite Pg. 34 Fig 12 - Micrographs of three honey bee pests Pg. 114

List of Abbreviations

ABPV Acute Bee Paralysis Virus ABV Arkansas Bee Virus AFB American Foulbrood ALPV Aphid Lethal Paralysis Virus AMP Antimicrobial Peptide AIV Apis Iridiscent Virus BBV Berkeley Bee Virus BRSV Big Sioux River Virus BQCV Black Queen Cell Virus BVX Bee Virus X BVY Bee Virus Y CBPV Chronic Bee Paralysis Virus CCD Colony Collapse Disorder COST European Cooperation in Science and Technology COLOSS Prevention of honey bee Colony Losses CWV Cloudy Wing Virus DCA Drone Congregation Area DNA Deoxyribonucleic Acid dsRNA double stranded RNA DWV Deformed Wing Virus EFB European Foulbrood FRET Fluorescence Resonance Energy Transfer FV Filamentous Virus IAPV Israeli Acute Paralysis Virus IRES Internal Ribosome Entry Site KBV Kashmir Bee Virus 112 • Appendix

Lp Leader Protein LSV Lake Sinai Virus MaLV Macula-like Virus MLPA Multiplex Ligation Probe-dependent Amplification ORF Open Reading Frame PAMP Pathogen Associated Molecular Patterns PGRP Peptidoglycan Recognition Proteins pog predicted overlapping gene PRR Pattern Recognition Receptors RdRp RNA Dependent RNA Polymerase RNA Ribonucleic Acid RNAi RNA Interference RT-PCR Real-Time Polymerase Chain Reaction SBPV Slow Bee Paralysis Virus SBV Sacbrood Virus SINV Solenopsis Invicta Virus TSBV Thai Sacbrood Virus TSV Taura Syndrome Virus USDA United States Department of Agriculture UTR Upstream Untranslated Region VDV Varroa Destructor Virus VP/Vp Viral Protein

List of Scientific Names

Acarapis woodi (Tracheal mite) Aethina tumida (Small hive beetle) Anopheles spp. (Anopheles mosquito) Apis cerana (Asian honey bee) Apis dorsata (Giant honey bee) Apis florae (Dwarf honey bee) Apis koshevnikovi Apis laboriosa (Giant honey bee) Apis mellifera (European honey bee) Apis nigrocincta Apocephalus borealis Ascosphaera apis (Chalkbrood) Aspergillus spp. (Stonebrood) Beauveria spp. Braula coeca (Bee lice) Crithidia mellificae Drosophila melanogaster (Fruit fly) Appendix • 113

Ellingsenius fuller (Bee scorpion) Escherichia coli Hyplostoma spp. (Large hive beetle) Malpighamoeba mellificae Melisococcus plutoneus (European foulbrood) Nosema apis Nosema ceranae Paenibacillus larvae (American foulbrood) Palarus latifrons (Banded bee pirate) Philanthus diadema (Yellow bee pirate) Physocephala fascipennis (Bee conopid) Rondanioestrus apivorus (Bee tachinid) Spiroplasma spp. Tropilaelaps spp. (Tropilaelaps mite) Varroa destructor (Common varroa mite) Varroa jacobsoni (Varroa mite) Varroa rindereri (Varroa mite) Varroa underwoodi (Varroa mite) Vespa velutina (Asian hornet)

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Table. 3: Complete list of viruses showing various characteristics. Fig. 12: Micrographs of tracheal mites, tropilaelaps mite and varroa mite taken during the photomicrography course, held at Umeå University, Umeå, Sweden. 114 • Appendix References • 115

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