Honey bees in Asia: Problems and Challenges

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1 Honey bees in Asia: Problems and Challenges Dr. Panuwan Chantawannakul Department of Biology Faculty of Science, Chiang Mai University, Chiang Mai, Thailand

2 Apis mellifera A. dorsata A. laboriosa A. cerena A. florea A. andreniformis Data from Honey Asia

3 Domesticated bees Asian cavity nesting honey bee (native bee) European honey bee (the imported bee)

4 Migratory beekeeping

5 Bee loss

6 Apis mellifera bee (European honey bee) It was introduced in Thailand in 1980s Commercial bee

7 Modern

8 What is the Bee health status in Thailand?

9 Colony losses situation 10% loss (data collected from questionnaire: Thai beekeepers) Cause of losses (from the beekeepers) Mites (Varroa and Tropilaelaps) Wasp/ants Chalkbrood disease European foulbrood Flooding Unknown

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11 Bee diseases and parasites survey since 2002 in A. mellifera Chalkbrood and European foulbrood could be found in the rainy seasons No detection of American foulbrood Prevalence of Nosema cerenae Parasitic mites (both Varroa and Tropilaelaps) No Acarapis woodi, A. dorsalis

12 The most prevalent virus was Deformed wing virus (DWV), which occurred in seven provinces and in 33% of all samples, followed by Acute Bee Paralysis Virus (20%), Sacbrood Bee Virus (4%) and Kashmir Bee Virus (2%). Journal of Invertebrate Pathology ( 2008) 100(2)

13 Frequencies of simultaneous virus infections in honeybee sample No. of viruses Type of infection No. of sample Percentage Origin a LPU, 3LPA, 1PY, 2PH, 1CM 1 ABPV PY, 1CM DWV LPU, 2LPA, 2NAN, 3PH 2 ABPV,DWV CR, 1PY, 4NAN, 2CM 3 ABPV, DWV, KBV 1 2 1LPA ABPV, DWV, SBV LPA, 4CR 4 ABPV, DWV, KBV, SBV 2 4 2LPA a LPU, Lamphun; LPA, Lampang; CR, Chiang Rai; PY, Phayao; NAN, Nan; PH, Phrae; CM, Chiang Mai. Journal of Invertebrate Pathology ( 2008) 100(2)

14 Observations Twenty of 46 apiaries (43%) were infested with either Varroa destructor or Tropilaelaps sp. 15 of 46 (33%) were infested with both mites. Lampang apiaries were found positive for four viruses and also found with high infestations with Varroa mite Journal of Invertebrate Pathology ( 2008) 100(2)

15 Detection of bee viruses using Real-time PCR KBV KBV CBPV CBPV APV APV SBV SBV DWV DWV BQCV BQCV Journal of Invertebrate Pathology ( 2006) 69-73

16 Ct values (mites collected in athai apiary) Sample Number KBV CBPV AIV APV SBV DWV BQCV 16 S rrna IPC M1 M ± ± ± ± ± ± ± ± ± ± ±0.087 M12 M15 M ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.022 Journal of Invertebrate Pathology ( 2006) 69-73

17 Viruses in Native bees DWV Black Queen Cell Virus No AFB, EFB, and Chalkbrood diseases Varroa jacobsoni and Tropilaelaps spp.

18 Nosema disease Nosema ceranae and N. apis Microsporadia Nosema ceranae, tentatively linked to Colony Collapse disorder (CCD) Apis mellifera (worldwide) Apis dorsata Apis florea Apis cerana (first discovered, the original host)

19 Polymerase Chain Reaction (PCR) Detection Three pairs of primers were used in this study N. apis (16S rrna gene) N. ceranae (16S rrna gene) Apis COI gene (mitochondrial DNA as internal positive control)

20 Percent of N. ceranae infected colonies Percentage of N. ceranae infected colonies from four honeybee species collected from Northern Thailand % % 37.5% 45.4% 0 A. mellifera A. cerana A. dorsata A. florea Honeybee species Journal of Invertebrate Pathology ( 2010)

21 N.ceranae is also a common parasite in Asian honeybee species (A. cerana, A. dorsata and A. florea) The prevalence of N. ceranae infections in native feral honeybees seems to be lower than managed European honeybee

22 Phylogenetic analysis of Nosema ceranae isolated from European and Asian honey bees - The 16 S rrna gene sequence could not differentiate the nosema strains. We have designed a pair of primers based on polar tube protein gene (~780 bp product)

23 Single comb nesting honeybees Cavity nesting honeybees Phylogenetic analysis of N. ceranae isolates based on the PTP 1 gene. The maximum parsimony tree was generated by MEGA 4 software using the close-neighborinterchange algorithm. Encephalitozoon cuniculi was used as an outgroup for polar tube protein phylogenetic tree. The tree used 1,000 bootstrap replicates (Journal of Invertebrate Pathology, 2011).

24 Fewer bee pathogens in the native bees being observed What are defense mechanisms? - Native honey bees do not produce propolis (a resinous mixture that honey bee collect from tree buds, sap flows, or other botanical sources. It is used as a sealant for unwanted open spaces in the hive)

25 Defense mechanisms Behavioral response Immune response Some other factors

26 Grooming behavior: Resistance to mite Evaluation of the responses to Tropilaelaps mercedesae, the most widely spread species, using groups of caged bees A. cerana, A. dorsata (the indigenous host of Tropilaelaps), A. mellifera

27 After 6 hr, 2/3 of the inoculated mites on A. cerana were removed compared to only about 1/3 fora. dorsata and A. mellifera. The majority of the mites fell after 24 h from A. cerana, after 36 h from A. dorsata and after 48 h from A. mellifera Higher proportions of injured mites were also observed in cages with A. cerana and A. dorsata than in those with A. mellifera.

28 Immune responses Cellular immunity relates to the phagocytosis, encapsulation and melanization (Eater, a receptor for phagocytosis) Humoral immunity involves the synthesis of antimicrobial peptides (abaecin, apidaecin, defensin, and hymeoptaecin)

29 What are the other factors?

30 Symbiosis : Microflora of bees Metagemics of microflora in bee guts Conventional method: culture-dependent method

31 Differences between species Two species of honey bee located in the same area harbour different bacterial communities Principal component analysis (PCA) biplot showing the relationship among 270 midgut bacterial T-RFLP profiles from two species of Apis. FEMS Microbial Ecology (2011)

32 Differences between life stages Principal component analysis (PCA) biplot showing the relationship between variable and 135 T-RFLP profiles from three life stages of (a) Apis mellifera and (b) Apis cerana. FEMS Microbial Ecology (2011)

33 Traditional culture based technique

34 First group was the actinomycetes Actinomycetes are one of the most widely distributed groups of microorganisms in nature. Actinomycetes are one of the main microbial groups that produce bioactive compounds used as antibiotics Isolation from both honey bees and stingless bees Bee hives Bee Food source

35 Spore-bearing hyphae of some actinomycetes species Norcardiopsis alba Nonomuraea roseoviolacea Actinomadura sp. Streptomyces misawanensis Streptomyces badius Streptomyces fradiae World Journal of Microbiology (2009)

36 Physiological and biochemical characteristics -Melaninoid pigments production -Carbon utilization (D-Glucose, L-Arabinose, Sucrose, D-Xylose, myo-inositol, D- Mannitol, D-Fructose, Rhamnose and Raffinose) - NaCl tolerance (0-7%) - ph range of growth (4-12) - Temperature range (10-50 C) Chemotaxonomy - Determination of the isomers of DAP (Staneck & Roberts,1974 ) - Methyl ester of cellular fatty acids (MIDI, Sasser, 1990) - Whole cell sugar analysis (Mikami & Ishida, 1983) - Menaquinone analysis (Collins et al, 1977, 1984) - Polar lipid analysis (Minnikin et al.,1984 ) Molecular characteristics - 16S rrna gene (Nakajima et al. (1999) - G+C content of DNA (Tamaoka & Komagata, 1984) - DNA-DNA hybridization (Ezaki P. et Chantawannakul al., 1989)

37 Yeast communities a total of 186 yeast strains were isolated from 37 honey samples of 12 different bee species They belong to 20 species (19 ascomycetous and 1 basidiomycetous yeast species) Fungal diversity (2011)

38 New species in bee hive Actinomadura apis (IM17-1 T ) Streptomyces chiangmaiensis (TA4-1 T ) Streptomyces lannaensis (TA4-8 T ) Zygosaccharomyces siamensis Candida ludiana Candida suthepensis

39 Honey analysis

40 Antimicrobial activity Agar well diffusion method

41 Antibacterial and antioxidant properties of Thai honeys The MIC values ranged from 6% (v/v) - 22% (v/v) Longan honey showed the most effective antibacterial activity against S. aureus, MRSA, K. pneumoniae, S. marcescens, and P. acnes. Antioxidant properties, determined by average phenol content was in the range of ± , ± mg GAE/kg The DPPH radical scavenging assay was found to have an IC50 in the range of 5.8 ± ± 6.09 mg/ml

42 Physicochemical properties of Thai Moisture content: < 21% Diastase Activity: > 8 o Gothe Sucrose content: < 5% honeys Longan honey had the highest content of Glucose and fructose when compared with other types. Litchi honey had the lowest content of Glucose and fructose Average of HMF, ash, moisture, electrical conductivity, diastase activity, glucose fructose, sucrose and total acidity content were in the acceptable range of international standards. The results showed that all Thai honeys are of good quality, following the international honey standards.

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45 New window of opportunity

46 Thank you Waikato University (Bees n Trees project) Thailand Research Fund The Commission on Higher Education, Hitachi Scholarship Foundation Japan Collection of Microorganisms (JCM) RIKEN, Japan. York University, UK National Bee Unit, Central Science Laboratory, Ministry of Agriculture, Fisheries, and Food, UK USDA, Beltsville and Baton Rouge, USA All students in Bee protection center, Chiang Mai University Beekeepers and Northern Beekeeper Association of Thailand Department of Physics (Material Science Research Center) Department of Chemistry (Computer simulation group)