Pests and Pathogens. Varroa and Viruses. Orlando Yañez Peter Neumann. Institute of Bee Health University of Bern

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1 Pests and Pathogens Varroa and Viruses Orlando Yañez Peter Neumann Institute of Bee Health University of Bern

2 What is causing bees to decline? GM crops Pesticides Poor beekeeping management Pollution Electromagnetic waves UFOs Parasites and diseases

3 Varroa destructor (Acari: Varroidae) > Varroa destructor is a new parasite of European honey bees. > Since its introduction, it is the greatest threat for apiculture. > Without treatment, most colonies would collapse within a 2 3 year period. Photo by IBRA 29. Juni

4 Interspecific invasion from Apis cerana to Apis mellifera > Spread almost worldwide within a relatively short time period. As a new parasite, a balanced host-parasite relationship is lacking. 29. Juni

5 host parasite adaptation in the original host Apis cerana > Hygienic traits: Grooming behavior Uncapping and removal Entombing A. mellifera A. cerana > Population dynamic aspects (Reproduction only in drones) 29. Juni

6 Mite biology > Varroa is closely linked to its honey bee host and lacks a free living stage. Reproductive phase Phoretic phase Transportation and spread 6

7 Varroa as virus vector > It was though, that parasitism by Varroa was the solely cause for adult bee malformations. Bowen-Walker et al Juni

8 Varroa as virus vector > Adult bee malformations was associated to a honey bee virus. Non-deformed bees Mites and deformed bees Bowen-Walker et al Juni

9 Association between Varroa and viruses at colony level Survived colony Collapsed colony Varroa-virus associated with the collapse of honeybee colonies (Carreck et al 2010) 29. Juni

10 Honey Bee Viruses: Characterization Bailey & Ball - Rothamsted Research (UK) Symptoms sacbrood paralysis black queen cells deformed wings Serology injection assays - propagation inapparent picorna-like viruses ~18 viruses ~10 picorna-like viruses 29. Juni

Characterization Bailey & Ball - Rothamsted Research (UK) Symptoms sacbrood deformed wings paralysis black queen cells VIRUS Sacbrood virus Thai Sacbrood Chinese Sacbrood Deformed wing virus Kakugo

11 Characterization Bailey & Ball - Rothamsted Research (UK) Symptoms sacbrood deformed wings paralysis black queen cells VIRUS Sacbrood virus Thai Sacbrood Chinese Sacbrood Deformed wing virus Kakugo Varroa destructor-1 Slow bee paralysis virus Acute bee paralysis virus Kashmir bee Israeli bee paralysis Black queen cell virus Chronic bee paralysis virus Cloudy wing virus Bee virus bee virus X bee virus Y Arkansas bee virus Macula-like virus Apis iridescent virus Filamentous virus FAMILY Iflavirus Picorna-like Dicistrovirus (New virus family)??? Maculavirus Iridovirus Baculo/Ascovirus 29. Juni

12 Characterization Picornavirales > small > (+) Single-Strand RNA virus, > Nonenveloped > Icosahedral capsid, 30 nm 29. Juni

13 Characterization Picornavirales Iflavirus 9~10 kb IRES 5 LP VP3 VP1 VP2 helicase VPg 3C-pro RdRp 3 AAAAAAAA IRES Dicistrovirus 9~9.5 kb IRES helicase VPg 3C-pro RdRp VP2 VP3 VP1 SBV DWV/VDV-1 SBPV ABPV/KBV/IAPV BQCV CBPV 5 3 AAAAAAAA 29. Juni

Virus transmission routes Vertical transmission (next

low virulence useful for the host Pollen, and Larval for

beekeepers Horizontal Transmission (Same Generation) By

14 Virus transmission routes Vertical transmission (next Generation) Sperm X Egg No Symptoms (= latent) In general, low virulence useful for the host Pollen, and Larval for the viruses food, feces = No problems for bees and beekeepers Horizontal Transmission (Same Generation) By feeding, cleaning, robbering, drifting... from: Chen et al Juni

15 Varroa has changed the rules of the game because Varroa added a new transmission route

16 Viruses and Varroa destructor 1. The mite injects viruses directly into the bee haemolymph

Virus Black Queen Cell Virus Red: Transmitted by Varroa destructor ABPV SPV DWV

17 Viruses and Varroa destructor 100 nm Acute Bee Paralysis Virus Slow Paralysis Virus Deformed Wing Virus Sacbrood Virus Kashmir Bee Virus Israeli Acute Paralyis Virus Black Queen Cell Virus Red: Transmitted by Varroa destructor ABPV SPV DWV SBV KBV IAPV BQCV Elimination of the first defense mechanisms (e.g. intestinal lamina)

18 Viruses and Varroa destructor 1. The mite injects viruses directly into the bee haemolymph Chen et al % brood with KBV The more mites in a cell, the more likely a virus is transmitted (4 mites ~ 100% at KBV)

19 Viruses and Varroa destructor 1. The mite injects viruses directly into the bee haemolymph 2. Mite suppress the immune response of the bee i.e. Mosquito suppress blood clotting

20 Viruses and Varroa destructor 1. The mite injects viruses directly into the bee haemolymph 2. Mite suppress the immune response of the bee Suppressed immune response = increased susceptibility to viruses?

21 Viruses and Varroa destructor 1. The mite injects viruses directly into the bee haemolymph 2. Mite suppress the immune response of the bee 3. Virus replication in Varroa can promote virulence

22 Viruses and Varroa destructor DWV: Virus-function analysis in mites from bees with and without symptoms Yue & Genersch 2005 clear evidence for viral replication Virus + RNA Virus - RNA Bees showed only crippled wings, when the virus was able to successfully replicate in the mite

23 Viruses and Varroa destructor 1. The mite injects viruses directly into the bee haemolymph 2. Mite suppress the immune response of the bee 3. Virus replication in Varroa can promote virulence

24 Control For viruses of humans (influenza, HIV), there are medications (antivirals) Genome (+ RNA) Capsid Proteins Non-Structural Proteins Helicase Protease Replicase Prevent penetration into the host cell or stop replication in the host cell (i.e. blocking the protease enzymes) However, no specific drugs are available for the bee viruses. Currently, RNA interference technologies are being developed against honey bee viruses and Varroa (2020?)

Mello - Nobel Prize (2006) for their work on RNA interference.

25 Control Small Interfering RNA (sirna) > RNA interference is a vital part of the immune response to viruses. > Andrew Fire and Craig C. Mello - Nobel Prize (2006) for their work on RNA interference. Hammond (2005) Processing of dsrna Transfer to Effector Activated Effector 25

26 Control Small Interfering RNA (sirna) > RNA interference is a vital part of the immune response to viruses. > Andrew Fire and Craig C. Mello - Nobel Prize (2006) for their work on RNA interference. Foreign mrna Hammond (2005) sirna Effector Attach to target mrna Cleavage of Foreign mrna 26

27 Control Small Interfering RNA (sirna) > Protection of the honey bee against Deformed wing virus (DWV) Bees feed with DWV Bees feed with dsrna + DWV Desai et al Juni

28 Control Small Interfering RNA (sirna) > Protection of the honey bee against Israelí acute paralysis virus (IAPV) > Remebee TM by Beeologics > Laboratory assays. Maori et al Juni

29 Control Large-Scale Field Application of RNAi Technology against IAPV Honey production So, are we completely helpless against bee viruses until RNAi technology is commercially available? > Under FDA review for use in practice by feeding in syrup to hives. Hunter et al Juni

30 Prevention Minimize transmission between colonies (drifting, robbery)

31 Prevention Minimize transmission between colonies (drifting, robbery) A strict control of Varroa and other diseases; stress prevention

32 Prevention Minimize transmission between colonies (drifting, robbery) A strict control of Varroa and other diseases; stress prevention Support bees immune system? Bees use propolis, which has antiviral properties

33 Prevention Minimize transmission (drifting, robbery) More stringent criteria for the control of Varroa and other diseases; stress prevention Support bees immune system? Breeding Virus-tolerant bees?

34 Summary 1. Bee viruses are widespread, certainly underestimated because latent infections without symptoms (= healthy colonies) ABPV SPV DWV SBV Ellis & Munn KBV Ellis & Munn BQCV CPV CWV

35 Summary 1. Bee viruses are widespread, certainly underestimated because latent infections without symptoms (= healthy colonies) 2. Varroa destructor as a virus vector changes the rules of the game

36 Summary 1. Bee viruses are widespread, certainly underestimated because latent infections without symptoms (= healthy colonies) 2. Varroa destructor as a virus vector changes the rules of the game 3. Activation of viruses by Varroa destructor

37 Summary 1. Bee viruses are widespread, certainly underestimated because latent infections without symptoms (= healthy colonies) 2. Varroa destructor as a virus vector changes the rules of the game 3. Activation of viruses by Varroa destructor 4. No available remedies for viruses, certainly not at short term period. Antivirals

Summary 1. Bee viruses are widespread, certainly underestimated because latent infections without symptoms (= healthy colonies) 2. Varroa destructor as a virus vector changes the rules of the game 3.

38 Summary 1. Bee viruses are widespread, certainly underestimated because latent infections without symptoms (= healthy colonies) 2. Varroa destructor as a virus vector changes the rules of the game 3. Activation of viruses by Varroa destructor 4. No patent remedies for viruses, certainly not in the long term control 5. Prevention: Minimizing transmission, strict control Varroa + other diseases, breeding? Propolis?

39 Summary 1. Bee viruses are widespread, certainly underestimated because latent infections without symptoms (= healthy colonies) 2. Varroa destructor as a virus vector changes the rules of the game 3. Activation of viruses by Varroa destructor 4. No patent remedies for viruses, certainly not in the long term control 5. Prevention: Minimizing transmission, strict control Varroa + other diseases, breeding? Propolis?

40 Questions 40

41 Diagnostics MLPA Multiplex Ligation Dependent Probe Amplification Lina De Smet Detection of several viruses in one reaction LPO = Left Probe Oligo, RPO = Right Probe Oligo LHS = Left Hybridising Sequence, RHS = Right Hybridising Sequence 30. Juni

42 How works MLPA? 1. Denaturation: single stranded cdna 2. Hybridisation LPO RPO 5 3 Target DNA 1 5 LPO Target DNA 2 RPO 3 3. Ligation : Ligase-65 LPO RPO 5 Target DNA Target DNA PCR single primer pair LPO RPO Each target : different length 30. Juni

43 Diagnostics Housekeeping gene M S1 S2 S3 S4 S5 M S6 S7 S8 S9 S10 M 140 bp SBV 131 bp SBPV 122 bp BQCV 104 bp ABPV-family 95 bp DWV-family 88 bp CBPV M = Marker, S = Sample 30. Juni

- Determining the Causes - Dr. Diana Cox-Foster Pennsylvania State University

Colony Collapse Disorder - Determining the Causes - Dr. Diana Cox-Foster Pennsylvania State University Honey Bees in US Agriculture and the Colony Collapse Disorder Essential for pollination of over 100