Dr. Panuwan Chantawannakul

Department of Biology

Faculty of Science,

Chiang Mai University,

Chiang Mai, Thailand

Email: panuwan@gmail.com

Honey bees in Asia:

Problems and Challenges

A. dorsata

A. laboriosa

A. cerena

A. florea

A. andreniformis

Apis mellifera

Data from Honey Asia

Domesticated bees

Asian cavity nesting honey bee

(native bee)

European honey bee (the imported bee)

•

Migratory beekeeping

Bee loss

Apis mellifera bee (European honey bee)

• It was introduced in Thailand in 1980s

• Commercial bee

P. Chantawannakul

Modern beekeeping @BEEP

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What is the Bee health status in Thailand?

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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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P. Chantawannakul

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

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• 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) 116-119

P. Chantawannakul

Frequencies of simultaneous virus infections in honeybee sample

No. of viruses

Type of infection No. of sample

Percentage Origina

0 8 17 1LPU, 3LPA, 1PY,

2PH, 1CM

1 ABPV 7 15 6PY, 1CM

DWV 15 33 8LPU, 2LPA, 2NAN, 3PH

2 ABPV,DWV 8 17 1CR, 1PY, 4NAN, 2CM

3 ABPV, DWV, KBV 1 2 1LPA

ABPV, DWV, SBV 5 11 1LPA, 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) 116-119 P. Chantawannakul

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) 116-119 P. Chantawannakul

Detection of bee viruses using Real-time PCR

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39

KBV

KBV

CBPV

CBPV

APV

APV

SBV

SBV

DWV

DWV

BQCV

BQCV

Journal of Invertebrate Pathology ( 2006) 69-73 P. Chantawannakul

Ct values (mites collected in aThai

apiary) Sample

Number

KBV CBPV AIV APV SBV DWV BQCV 16 S rRNA

IPC

M1

M9

M12

M15

M16

34.608

±0.077

37.575

±1.174

37.395

±0.154

38.874

±0.138

36.932

±0.899

40

40

40

40

40

40

40

40

40

40

35.082

±0.079

36.278

±0.327

36.302

±0.109

37.407

±0.629

35.262

±0.799

35.670

±0.146

31.917

±0.223

27.945

±0.119

29.624

±0.049

32.121

±0.068

29.164

±0.015

27.607

±0.628

29.199

±0.522

33.920

±0.227

24.348

±0.481

37.499

±0.151

40

40

38.257

±0.840

28.375

±0.269

19.240

±0.107

19.585

±0.087

19.795

±0.027

22.409

±0.033

22.372

±0.022

Journal of Invertebrate Pathology ( 2006) 69-73

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Viruses in Native bees

• DWV

• Black Queen Cell Virus

• No AFB, EFB, and Chalkbrood diseases

• Varroa jacobsoni and Tropilaelaps spp.

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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) P. Chantawannakul

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)

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Percentage of N. ceranae infected colonies from four honeybee species collected from Northern Thailand

0

20

40

60

80

100

A. mellifera A. cerana A. dorsata A. florea

Perc

en

t o

f N

. cera

nae

infe

cte

d

co

lon

ies

Honeybee species

77.5%

22.2%

37.5% 45.4%

Journal of Invertebrate Pathology ( 2010)

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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

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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)

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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-neighbor-

interchange 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).

Cavity nesting honeybees

Single comb

nesting honeybees

• 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)

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Defense mechanisms

• Behavioral response

• Immune response

• Some other factors

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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

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• 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.

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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)

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What are the other factors?

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Symbiosis : Microflora of bees

• Metagemics of microflora in bee guts

• Conventional method: culture-dependent method

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Differences between species

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Principal component analysis (PCA) biplot showing the

relationship among 270 midgut bacterial T-RFLP profiles from two species of Apis.

Two species of honey bee

located in the same area

harbour different bacterial

communities

FEMS Microbial Ecology (2011)

Differences between life stages

P. Chantawannakul

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)

Traditional culture based technique

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• 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

P. Chantawannakul

Streptomyces misawanensis Streptomyces fradiae

Norcardiopsis alba Nonomuraea roseoviolacea Actinomadura sp.

Spore-bearing hyphae of some actinomycetes species

Streptomyces badius

World Journal of Microbiology (2009)

P. Chantawannakul

• Molecular characteristics

- 16S rRNA gene (Nakajima et al. (1999) - G+C content of DNA (Tamaoka & Komagata, 1984)

- DNA-DNA hybridization (Ezaki et al., 1989)

• 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 )

P. Chantawannakul

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)

P. Chantawannakul Fungal diversity (2011)

New species in bee hive

Actinomadura apis (IM17-1T)

Streptomyces chiangmaiensis (TA4-1T)

Streptomyces lannaensis (TA4-8T)

Zygosaccharomyces siamensis

Candida ludiana

Candida suthepensis

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Honey analysis

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Antimicrobial activity

Agar well diffusion method

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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 493.79 ± 34.78-1,160.39 ± 348.66 mg GAE/kg

• The DPPH radical scavenging assay was found to have an IC50 in the range of 5.8 ± 1.55-19.76 ± 6.09 mg/ml

P. Chantawannakul

Physicochemical properties of Thai honeys

• Moisture content: < 21% • Diastase Activity: > 8 oGothe • Sucrose content: < 5% • 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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New window of opportunity

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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)