effects of propolis of apis mellifera linneause, 1787 and trigona apicalis smith, 1857 from different

148 Pages·2016·4.9 MB·English

EFFECTS OF PROPOLIS OF APIS MELLIFERA LINNEAUSE, 1787 AND
TRIGONA APICALIS SMITH, 1857 FROM DIFFERENT REGIONS OF
THAILAND ON EXPERIMENTAL INFECTION OF NOSEMA CERANAE IN THE
ASIATIC HONEYBEE APIS CERANA FABRICIUS, 1793

TANAWAT YEMOR

A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DOCTOR DEGREE OF PHILOSOPHY
IN BIOLOGICAL SCIENCE
FACULTY OF SCIENCE
BURAPHA UNIVERSITY
JUNE 2016
COPYRIGHT OF BURAPHA UNIVERSITY

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude and deep appreciation to my
major advisor, associate Professor Dr. Guntima Suwannapong, and my Co. advisor,
Professor Dr. Robert John Paxton, for their excellent instruction, patience and
encouragements through the years. Without this kindness and understanding this work
could not be accomplished.
Great appreciation is given to Dr. Nittaya Chaiyanate and Associate
Professor Dr. Mark Eric Benbow as examining committee, for all of their guidance,
and valuable advice throughout this study.
I also would like to sincerely thank the Department of Biology, Faculty of
Science which provided facility to complete this work and thank to Agricultural
Occupation Promotion and Development Center Chanthaburi and Chiang Mai
Provincial (Bee) for providing propolis of both of A. mellifera and T. apicalis for this
study. Kindly thanks are extended to all members of Bees and Blowflies Biology
Laboratory for their help and friendship. I also would like to thank Nation Research
Program Management: NRPM 34502, NRPM 222691 and Science Achievement
Scholarship of Thailand (SAST) for financial support for this study.
Finally, greatest respect also goes to my parents for their loving, patience,
prayer, caretaking, attention, and support throughout my study.

Tanawat Yemor
53810073: MAJOR: BIOLOGICAL SCIENCE; Ph.D.
KEYWORDS: APIS CERANA/ MID GUT/ NOSEMA/ PROPOLIS/ INFECTIVITY
TANAWAT YEMOR: EFFECTS OF PROPOLIS OF APIS MELLIFERA
LINNEAUSE, 1787 AND TRIGONA APICALIS SMITH, 1857 FROM DIFFERENT
REGIONS OF THAILAND ON EXPERIMENTAL INFECTION OF NOSEMA
CERANAE IN THE ASIATIC HONEYBEE APIS CERANA FABRICIUS, 1793.
ADVISORY COMMITTEE: GUNTIMA SUWANNAPONG, Ph.D., ROBERT
JOHN PAXTON, Ph.D. 137 P. 2016.

Honey bees are not only provided many valuable products but also serve as
pollinator for varieties crop plants. Recently, there are reports of sudden disappeared
of colony population raising cause of concern. One of the serious problems causes of
colony population losses is Nosema ceranae infection. The effective substance for the
control of Nosema is fumagillin, but it prohibited due to the risk of antibiotic residues
in honey and it is difficult to degraded that cause human health issue. Thus, novel and
sustainable treatment strategies against N. ceranae are urgently needed. To approach
this issue I decided to evaluate the effect of propolis extract from Trigona apicalis and
Apis mellifera collected from two different regions of Thailand, Chiang Mai and
Chanthaburi provinces on the ultrastructure of Nosema spores, the survival rate,
infectivity, infection ratio and on hypopharyngeal gland protein content of
Apis cerana after individually inoculated with 80,000 N. ceranae spores per bee, and
then each group were treated with 50 % and 70 % propolis extract compared to
control bees. The results showed that propolis extract not only decreasing parasitosis
intensity, but also enhances the survival and protein content of hypopharyngeal gland.
The significant anti-parasitic activity of propolis extracts, coupled with its palatability
and no toxicity noticed in this study, makes its inclusion feasible in a compound for
nosemosis control. My investigations were not sufficient to explain which
mechanisms are involved on N. ceranae reduction when propolis extracts
supplementation on infected honey bee is applied. Therefore, the ways of action
remain unclear and further research is necessary to determine the identity of the active
compounds.
CONTENTS

Page
ABSTRACT.………………………………………………….………………......... iv
CONTENTS…………….…………………………………………..……………... v
LIST OF TABLES……………………………..…………………………...…....... vi
LIST OF FIGURES………………………………..……………………….……… vii
CHAPTER
1 INTRODUCTION……………………………………………………..……. 1
1.1 Statements and significance of the problem………………………… 1
1.2 Objectives…………………………………………………………… 3
1.3 Hypotheses…………………………………………………………. 3
1.4 Contribution to knowledge…………………………………………. 4
1.5 Scope of study………………………………………………………. 4
2 LITERATURE REVIEWS………………………………………………….. 5
3 MATERIALS AND METHODS……………………………………………. 29
4 RESULTS……………………………………………………………………. 38
4.1 Survival rate....................................................................................... 38
4.2 Infectivity........................................................................................... 41
4.3 Infection ratio...................................................................................... 43
4.4 Light Microscopy for examination of honey bee mid gut.................. 45
4.5 Histological study the structure of honeybees hypopharyngeal
glands………………………………………………………………. 54
4.6 Measuring acini diameters of hypopharyngeal glands....................... 61
4.7 Protein content of the hypopharyngeal glands of bees...................... 62
4.8 Transmission microscopic study of honeybees ventricular................ 68
5 CONCLUSION AND DISSCUSSION……………………………………… 78
REFERENCES……………………………………………………………………. 94
APPENDIX………………………………………………………………………… 122
BIOGRAPHY……………………………………………………………………… 137
LIST OF TABLES

Table Page
4.1 The protein content of hypopharyngeal gland of A. cerana dissected
from bee infected 80,000 spores per bee on day 6, 10 and 14 p.i. and
treated with propolis extract of T. apicalis collected from
Chanthaburi………………………………………………………………. 64
4.2 The protein content of hypopharyngeal gland of A. cerana dissected
from bee infected 80,000 spores per bee on day 6, 10 and 14 p.i. and
treated with propolis extract of T. apicalis collected from Chiang
Mai……………………………………………………………………….. 65
4.3 The protein content of hypopharyngeal gland of A. cerana dissected
from bee infected 80,000 spores per bee on day 6, 10 and 14 p.i. and
treated with propolis extract of A. mellifera collected from
Chantaburi…………………………………………………….................. 66
4.4 The protein content of hypopharyngeal gland of A. cerana dissected
from bee infected 80,000 spores per bee on day 6, 10 and 14 p.i. and
treated with with propolis extract of A. mellifera collected Chiang
Mai……………………………………………………………………….. 67
LIST OF FIGURES

Figure Page

2.1 Schematic representations of the early events in the life cycle of
N. ceranae. …………………………………………………………….. 13
2.2 Nosema ceranae life cycle observed in experimentally infected IPL-
LD-65Y (Lymantria dispar) cells. ……………………………………... 16
3.1 Map of Thailand represents propolis and honey bee specimens
collected sites…………………………………………………………… 32
4.1 Kaplan-Meier curves show the cumulative mortality rate of honeybees
(A. cerana) after inoculated with N. ceranae for 30 days with 80,000
spores/ bee (80K) per bee, and treated with propolis of T. apicalis…... 39
4.2 The survival rate (MeanSE) of honeybees (A. cerana) survival rate
after inoculated with N. ceranae for 30 days with 80,000 spores/ bee
(80K), and treated with propolis.............................................................. 40
4.3 The infectivity (MeanSE) of A. cerana after inoculated with
N. ceranae spores for 30 days with 80,000 spores/ bee (80K), and
treated with propolis extract ………………………………………….. 41
4.4 The infection ratio cells of A. cerana mid gut epithelial cell after
inoculated with N. ceranae for 14 days with 80,000 spores/ bee
(80K), and treated with propolis extract………………………………. 44
4.5 The light micrograph of ventricular epithelial cells of control bee
(CO), A. cerana worker on day 14 p.i. (PAS, 400x)…………………... 45
4.6 The light micrograph of ventricular epithelial cells of control bee
(CP), A. cerana worker on day 14 p.i. (PAS, 400x)….……………….. 46
4.7 The light micrograph of ventricular epithelial cells of N. ceranae
infected bees and untreated with propolis extract (CE), A. cerana
worker on day 14 p.i. (PAS, 400x) …………………………………… 47
4.8 The light micrograph of ventricular epithelial cells of N. ceranae
infected bees and untreated with propolis extract (CE), A. cerana
worker on day 14 p.i. (PAS, 400x)……………………………………. 48
viii

LIST OF FIGURES (CONTINUED)

Figure Page

4.9 The light micrograph of ventricular epithelial cells of N. ceranae
infected bees and untreated with propolis extrac (0P), A. cerana
worker on day 14 p.i. (PAS, 400x)……………………………………. 48
4.10 The light micrograph of ventricular epithelial cells of N. ceranae
infected bees and untreated with propolis extrac (0P), A. cerana
worker on day 14 p.i. (PAS, 400x)……………………………………. 49
4.11 The light micrograph of ventricular epithelial cells of A. cerana
worker on day 14 after inoculated with N. ceranae dosages 80,000
spores per bee and treated with 50 percentages (50P1) of ethanolic 50
extract propolis of T. apicalis collected from Chantaburi. …...............
4.12 The light micrograph of ventricular epithelial cells of A. cerana
worker on day 14 after inoculated with N. ceranae dosages 80,000
spores per bee and treated with 50 percentages (50P2)……………….. 50
4.13 The light micrograph of ventricular epithelial cells of A. cerana
worker on day 14 after inoculated with N. ceranae dosages 80,000
spores per bee and treated with 50 percentages (50P3)……………….. 51
4.14 The light micrograph of ventricular epithelial cells of A. cerana
worker on day 14 after inoculated with N. ceranae dosages 80,000
spores per bee and treated with 50 percentages (50P4)………………. 51
4.15 The light micrograph of ventricular epithelial cells of A. cerana
worker on day 14 after inoculated with N. ceranae dosages 80,000
spores per bee and treated with 70 percentages (70P1)……………….. 52
4.16 The light micrograph of ventricular epithelial cells of A. cerana
worker on day 14 after inoculated with N. ceranae dosages 80,000
spores per bee and treated with 70 percentages (70P2)……………….. 52
4.17 The light micrograph of ventricular epithelial cells of A. cerana
worker on day 14 after inoculated with N. ceranae dosages 80,000
spores per bee and treated with 70 percentages (70P3)………………. 53
ix

LIST OF FIGURES (CONTINUED)

Figure Page
4.18 The light micrograph of ventricular epithelial cells of A. cerana
worker on day 14 after inoculated with N. ceranae dosages 80,000
spores per bee and treated with 70 percentages (70P3)……………….. 53
4.19 The light micrograph of hypopharyngeal gland of an A. cerana worker
on day 14 p.i., control (CO))…………………………………………... 55
4.20 The light micrograph of hypopharyngeal gland of an A. cerana worker
on day 14 p.i, control (CP)…………………………………………….. 55
4.21 The light micrograph of hypopharyngeal gland of an of an A. cerana
worker on day 14 p.i, (0P)…………………………………………….. 56
4.22 The light micrograph of hypopharyngeal gland of an A. cerana worker
on day 14 p.i, (CE)…………………………………………………….. 56
4.23 The light micrograph of hypopharyngeal gland of an A. cerana worker
on day 14 p.i, and treated with 50 percentages (50P1)………………... 57
4.24 The light micrograph of hypopharyngeal gland of an A. cerana worker
on day 14 p.i, and treated with 50 percentages (50P2)………………... 57
4.25 The light micrograph of hypopharyngeal gland of an A. cerana worker
on day 14 p.i, and treated with 50 percentages (50P3)………………... 58
4.26 The light micrograph of hypopharyngeal gland of an A. cerana worker
on day 14 p.i, and treated with 50 percentages (50P4)………………... 58
4.27 The light micrograph of hypopharyngeal gland of an A. cerana worker
on day 14 p.i, and treated with 70 percentages (70P1)………………... 59
4.28 The light micrograph of hypopharyngeal gland of an A. cerana worker
on day 14 p.i, and treated with 70 percentages (70P2)………………... 59
4.29 The light micrograph of hypopharyngeal gland of an A. cerana worker
on day 14 p.i, and treated with 70 percentages (70P3)………………... 60
4.30 The light micrograph of hypopharyngeal gland of an A. cerana worker
on day 14 p.i, and treated with 70 percentages (70P4)………………... 60
4.31 The average size of hypopharyngeal glands of A. cerana after
inoculated with N. ceranae for 14 days with 80,000 spores/ bee (80K). 62
x

LIST OF FIGURES (CONTINUED)
Figure Page
4.32 The protein content (meanSE) of hypopharyngeal gland of A. cerana
dissected from bee infected 80,000 spores per bee on day 6, 10 and 14
p.i. and treated with propolis of T. apicalis collected from
Chanthaburi…………………………………………………………… 64
4.33 The protein content (meanSE) of hypopharyngeal gland of A. cerana
dissected from bee infected 80,000 spores per bee on day 6, 10 and 14
p.i. and treated with propolis of T. apicalis collected from Chiang
Mai…………………………………………………………………… 65
4.34 The protein content (meanSE) of hypopharyngeal gland of A. cerana
dissected from bee infected 80,000 spores per bee on day 6, 10 and 14
p.i. and treated with propolis of A. mellifera collected from
Chantaburi…………………………………………………………… 66
4.35 The protein content (meanSE) of hypopharyngeal gland of A. cerana
dissected from bee infected 80,000 spores per bee on day 6, 10 and 14
p.i. and treated with propolis of A. mellifera collected from Chiang
Mai……………………………………………………………………... 67
4.36 The transmission electron micrograph of infected ventricular cell
filled with different parasitic stages…………………………………… 70
4.37 The transmission electron micrograph of ventricular cells of A. cerana
on day 14 after infection with N. ceranae……………………………... 70
4.38 The transmission electron micrograph showing the sporoblast divide
by binary fission………………………………………………………. 71
4.39 The transmission electron micrograph showing sporoblast with
developing polar filament……………………………………………… 71
4.40 The transmission electron micrograph of ventricular cell with mature
spores distributed over the top of ventricular fold…………………….. 72
4.41 The transmission electron micrograph of longitudinal section of
N. ceranae spore……………………………………………………….. 72
4.42 The transmission electron micrograph of ventricular cells of A. cerana 73