The Honey Parasite, ceranae: Biology, Management and Future Directions

Stephen F. Pernal Abdullah Ibrahim, Johan van den Heever and Andony Melathopoulos AAFC Beaverlodge Research Farm Beaverlodge, Alberta Nosema – : Highly specialized parasitic fungi Nosema Reduces:

Nursing

Lifespan

Pollen collection

Queen fertility

Queen acceptance

Leads to wintering mortality, poor spring build-up and loss of honey production. What is Nosema ceranae? • Isolated from in China in 1996.

• Reported from Apis mellifera in Taiwan 2005 and later confirmed in Spain. Later confirmations from Germany and France.

• In Spain, N. ceranae characterized by N. ceranae N. apis gradual depopulation, higher fall/winter mortality, low honey production but not (Fries et al. 2006) dysentery or crawling seen with N. apis.

• Above symtoms similar to “dry nosemosis” seen in France.

• Widepsread in the U.S., Canada and other countries. Managing Nosema Unknowns

• Susceptibility to fumagillin.

• Disinfection.

• Seasonal Phenology.

• Alternative Therapies. Fumagillin Fumagillin dicyclohexylammonium

Medivet Pharmaceuticals DIN 02231180 (21 mg/g) Fumagillin Efficacy Experiment N. ceranae-infected overwintered singles: 22 April 2009

Pre-experimental infection level: 4.3 ± 0.5 million spores per bee Fumagillin Efficacy Experiment Treatment Formulations – Spring 2009

DUSTING PATTY Icing Sugar Dust Pollen, Soy Flour, Syrup

SYRUP 1:1 Sucrose Drench vs. Full Colonies assessed Volume 30 June, 26 Aug 09. Spring Fumagillin Efficacy Experiment 2009 Treatments applied 22 April, 6 May; Total a.i. = 100 mg

133,000,000 12 Drench Dust 10 8 Patty

)

6 Syrup Untreated a Untreated ab Patty, Syrup, Drench 6 b Dust * a 4 a a

a Untreated ab Patty, Syrup

Spores / Bee (x 10 (x / Bee Spores 2 b Dust, Drench b b b * 0

5-Oct 15-Jul 29-Jul 9-Sept 22-Apr29-Apr06-May13-May20-May27-May03-Jun 11-Aug 23-Sept 19-Nov Comb Disinfection Experiment Inoculating Comb

4.51 × 108 N. ceranae spores per colony Comb Disinfection Experiment

Electron Beam Irradiation Acetic Acid Disinfection Heat Treatment

10 kGy 120 mL 80% acetic acid 49 ± 1°C for 24 h per brood chamber

Comb Disinfection Experiment Sampling Bees for Spores

Weekly samples of 100 foragers (2 May - 4 June), then bi-weekly (monthly during winter).

Spores enumerated microscopically at 400x.

Comb Disinfection Experiment Hived with New Zealand Packages

* a Inoculated 65 b Fumigated Inoculated bc Heated 6 60 bc Irradiated 55 x 10 Heated c Uninoculated (Heated) 55 Fumigated 13.3x 106 Irradiated 50 (Heated) SE) Uninoculated

+

6 12 a 10

a Inoculated a Heated a Inoculated 8 ab Heated ab Uninoculated ab Heated

a ab Irradiated b Inoculated ab Fumigated 6 b Fumigated b Fumigated b Uninoculated a b Uninoculated b Irradiated b Irradiated

Spores / Bee ( x10 ( / Bee Spores 4 *

b * 2 * b 0 2 8 4 2 7 15 21 16 16 30 12 28 10 24 22 13 17 17 17 17 15 29 15 28 16 29 16 29 18 May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug 2009 2010 Comb Disinfection Experiment

N. apis N. ceranae N. apis + N. ceranae 100

80

60

40

% Colonies 20

0

Heated Heated Heated Heated Heated Irradiated Inoculated Irradiated Irradiated Irradiated Irradiated Fumigated InoculatedFumigated InoculatedFumigated InoculatedFumigated InoculatedFumigated Uninoculated Uninoculated Uninoculated Uninoculated Uninoculated May 2 Jun 4 Oct 7 Apr 29 Aug 18 2009 2010 Comb Disinfection Experiment Cumulative Mortality 18 Aug 2010

50 χ2=10.088; df=4.58; P=0.039 40

30

20

% Dead Colonies 10

0

Heat

Irradiated Fumigated Inoculated Uninoculated Alternatives to Fumagillin

Silkmoth Cultivation

Cage Experiments Screening for alternative compounds

Screen for Anti-Nosema Antibiotics (Highest concentrations: liquids 10 mg/kg, powders 1 mg/kg; n = 2-4)

No Inoculum Rifampicin Fumagillin Genistein Naturally-derived Thiabendazole Sulfaquinoxaline Synthetic Carbendazim Nozevit Phenyl salicylate Quinine hemisulfate Itaconazole Apigenin Vita Gold Ofloxacin Chloroquine Benomyl No Treatment Resveratrol Lomefloxacin Vita Green Acetic Acid Honey B Hlthy Cinnamon Oil Thymol Quercetin Aniseed Oil Quinidine sulphate Metronidazole Albendazole Eucalyptus Oil Eugenol

0 5 10 15 20 25 30 35 Spores (x 106) + SE Synthetic Compounds Against N. ceranae Compounds with significant concentration response Antibiotics against to N. ceranae Compounds with significant concentration response

5 ns 4 ns Carbendazim 3 2 * 1 0 0.004 0.04 0.4

SE) 5

+ 4 JP-P1-7a 3 2 1

(millions (millions 0

Spores per beeSpores 5 0.004 0.04 0.4 4 Fumagillin 3 2 1 Not tested 0 0.004 0.04 0.4 Dose (mMol) Summary • Irradiation is the most effective method of disinfecting comb contaminated with N. ceranae.

• Fumagillin is effective at suppressing active infections of N. ceranae. Sampling is critical during spring and fall.

• In northern temperate climates, N. ceranae spore levels appear to naturally decline during mid-summer, similar to patterns seen for N. apis.

• Predictive relationships between N. ceranae infection levels and measures of productivity remain elusive.

• Synthetic analogues of fumagillin may hold promise as an alternative to conventional therapies.

Future Directions

• Transmission?

• Spore survival?

• Treatment thresholds?

• Differential Virulence?

• Competition with N. apis?

• Mechanisms for Resistance?

Acknowledgements

• CANPOLIN • Medivet Pharmaceuticals • Alberta Beekeepers Commission • Canadian Bee Research Fund • Bee Maid Honey • Matching Investment Initiative (AAFC) • Iotron Industries Canada ltd.