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TANAWAT YEMOR
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
Figure Page
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
of adult life (Wang & Moeller, 1969). Ultrastructure may be seen in the glands of
workers 5 days after dosing with 5000 N. apis spores each and by 10 days which were
very noticeable (Wang & Moeller, 1971). There is some evidence that N. apis infected
bees showed atrophied hypopharyngeal glands that may have a foreshortened nursing
phase and begin foraging earlier than healthy bees (Hassanein, 1953). In order to the
infected bees are known to have lower levels of protein, resulting in a reduction size
of hypopharyngeal gland (Malone & Gatehouse, 1998; Wang & Moeller, 1970, 1971).
Many compounds have been tested for the control of Nosema, but the only effective
product is antibiotic fumagillin (Moffet, Lackett, & Hitchcock, 1969) which inhibits
the development of N. apis in the honey bee (Katznelson & Jamieson, 1952; Liu,
1973). Acetic acid was used as antibiotics for fumigation of combs for killing N. apis
spores even where treatments with fumagillin are possible, however there is the
problem of re-occurrence of the disease as according to only the vegetative forms of
the parasite are killed (Liu, 1973). Furthermore, the use of antibiotics including
sulpha drugs for the control of honeybee diseases represents a growing concern to the
honey market due to the increasing power of residue detection of laboratory
instruments. The development of new methods for the control of nosemosis is
therefore much hoped for by beekeepers and bee scientists. To approach this issue we
decided to evaluate the Nosema control potential of propolis substances of natural
origin with reported or possible inhibitory effect on the development of
microsporidians. In addition, there have been no published studies of using propolis to
control Nosema disease in any insect species in Thailand either.
As we known honeybee is the most important insect that play the pollination
role in the ecosystem, so the decreasing of honeybee population by nosemosis may
affect cross pollination of crops resulting the reduction of plants and animal diversity.
The effects of honeybee and stingless bee propolis propolis extract on nosemosis in
A. cerana might lead the valuable knowledge for honeybee pathologist and bee keeper
in term of the application for the control of nosemosis. The aims of this study are
(1) to investigate the effect of propolis from different regions and different species in
experiment infection N. ceranae spore on bee mortality, infectivity, infection ratio of
the ventricular cell, the protein production of hypopharyngeal glands of A. cerana and
(2) to investigate optimal dose of propolis extract to treat N. ceranae infected.
3
1.2 Objectives
The main objectives of this research are as follows:
1. To determine the differential infectivity and mortality of N. ceranae
spores infected A. cerana workers under doses of 80,000 spores/ bee, compare
between propolis from different region and species.
2. To examine differences in the infection ratio between infected cells and
non- infected cells of ventricular epithelial cells of A. cerana workers under doses of
80,000 spores/ bee, and treat with propolis from different region and species.
3. To determine the effect of propolis from different region and species on
the ventricular epithelia ultrastructural change in A. cerana workers infected
N. ceranae spores under doses of 80,000 spores/ bee.
4. To measure the protein contents and measuring acini diameters of
hypopharyngeal glands of A. cerana workers under doses 80,000 spores/ bee, and
treat with propolis from different region and species.
1.3 Hypotheses
To accomplish the objectives of this research the following hypotheses were
investigated.
Hypothesis I:
H0: Ethanolic extract propolis will decrease bee mortality, infection rate,
infectivity of N. ceranae, decrease deformality of ventricular epithelial cells and will
increase hypopharyngeal glands protein content when compare with control bees that
infected with Nosema bee but not given ethanolic extract propolis.
H1: Ethanolic extract propolis will not decrease bee mortality, infection rate,
infectivity of N. ceranae, decrease deformality of ventricular epithelial cells and will
not increase hypopharyngeal glands protein content when compare with control bees
that infected with N. ceranae bee but not given ethanolic extract propolis.
Hypothesis II:
H0: The ethanolic extract propolis from different species and different
regions will have different effects on nosemosis in A. cerana.
4
H1: The ethanolic extract propolis from different species of different regions
will not different effects on nosemosis in A. cerana.
The product that most people first associate with honey bee is honey,
although beekeeping generates much more than just honey. In 2005, worldwide
beekeeping can be produced honey more than 1.4 million tons mark with about 64.5
million beehives (Morandin & Winston, 2006). Honey is just one of several different
products that can be harvested. Others are beeswax, pollen, propolis, royal jelly and
venom, and the use of bees in apitherapy, which is medicine using honey bee products
(Riches & Harry, 2000). Beeswax is a valuable product that can provide a worthwhile
income in addition to honey. In 2009, the world production of beeswax is over 61.2
thousand tons. One kilogram of beeswax is worth more than one kilogram of honey,
usually around 4-10 US dollars per kilogram (Bradbear, 2009). Unlike honey,
beeswax is not a food product and is simpler to deal with, it does not require careful
packaging which this simplifies storage and transport. Beeswax has hundreds of uses,
around 40 percentage of the world trade in beeswax is used for the cosmetics industry,
which requires first class beeswax that has not been overheated, is pure and free from
propolis. While, around 30 percentage of world trade in beeswax is used by the
pharmaceutical industry (Riches & Harry, 2000). Beeswax also has many uses
worldwide, including the production of candles, electronics, lubricants, leather and
6
The widespread declines in honey bee populations have been reported across
the world. In 1947-2005, honey bee colonies and 25 percent colonies loss in central
Europe also have been reported (Potts et al., 2010). A huge massive decreased of
honey bee populations in last few decades have been partially assigned to a syndrome
named colony collapse disorder (CCD), which involves rapid loss of adult worker
bees (Runckel et al., 2011).The phenomenon of CCD is categorized by the rapid
vanishing of adult workers, leaving behind a queen along with few young workers and
lots of brood and food stores. Eventually the colony dies and oddly, the food stores
remain untouched by robbers and hive pests for unusually long periods of time
(Bromenshenk et al., 2010). Scientist proposed many factors are source of colony
8
of the microsporidia include the number of coils in the polar filament around the
periphery of the spore and the thickness of the polar filament (with isofilar and
anisofilar character states). A unique characteristic of all microsporidia is the long,
coiled polar filament present in the spore, which is used to inject the sporoplasm into
the host cell upon spore germination. The number of polar filament coils found inside
Nosema spores differs and provides a taxonomic basis for differentiating between
species (Burges, Canning, & Hulls, 1974). In the spore stage, the polar filament is
usually found as a series of tight coils, just below the plasma membrane. These are
observed in electron micrographs as a series of dots running just under the cell
membrane. The "dots" are cross-sections of the polar tube as it coils 5-15 times
around the entire circumference of the spore. The number of coils, their arrangement
relative to one another, and even the angle of helical tilt are conserved and diagnostic
for a particular species (Keeling & Fast, 2002b). The filament ranges from 0.1 to 0.2
m in diameter and 50 to 500m in length (Keeling & Fast, 2002b). They are
surrounded by massive arrays of ribosomes, particularly in immature cells (Bacchi et
al., 2002). Anteriorly, the polar filament passes through the polaroplast and attaches to
an anchoring disc at the apex of the cell. At infection, the polar filament will very
quickly elongate the polar tube (up to many times the length of the spore) (Fast &
Keeling, 2001). The polar tube or polar filament is composed of membrane and
glycoprotein layers (Keeling & Fast, 2002). It appears to have considerable internal
structure (Canning, Refardt, Vossbrinck, Okamura, & Curry, 2002). There is some
physical association between the end of the polar filament and the posterior vacuole,
but the precise nature and function of this contact are currently speculative (Keeling
& Fast, 2002).
Moreover, two rather different proteins have been isolated from the polar
tubes of Encephalitozoon. One is large (~50 kD) and proline-rich. The other is
smaller (~30Da), with a central region containing strings of lysine and an acidic
C-terminal region (Delbac, Peuvel, Metenier, Peyretaillade, & Vivares, 2001). The
ultrastructure of polar tube between N. apis and N. ceranae is also different, N. apis
spores have 30-44 polar filament coils (Fries, 1989) while N. ceranae spores
characteristically have 20-23 or 18-21 (Fries, Feng, da Silva, Slemenda, & Pieniazek,
1996; Chen et al., 2009). The morphology of microsporidian spores is highly varied
10
among species, overall spore sizes (between 1 and 10 m) and shape, and thickness of
exospore and endospore walls with structural variances even within species (Dunn &
Smith, 2001). Spore of N. apis and N. ceranae are related in size and shape, although
N. apis spores have been reported to be slightly bigger than those of N. ceranae, while
spore of N. ceranae is more curved (or rod-shaped) and inconsistent in shape (Fries
et al., 1996). The variation in size and form of N. ceranae isolated from different of
honey bee species have been reported; N. ceranae spores isolated from Apis mellifera
range between 2.0 - 2.5 m in width and 3.9 - 5.3 m in length (Chen et al., 2009).
In contrast, N. ceranae spores isolated from A. cerana can range between widths of
2.3 - 3.0 m with a lengths of 3.3 - 5.5 m (Fries et al., 1996). The microsporidian
spore wall functions to maintain the morphology of the spore and to protect against
the outer environment. The spore wall of N. ceranae is usually composed of three
layers, an electron-dense proteinaceous outer layer (exospore) with a thickness of
approximately 48-52 nm and an electron-transparent chitinous inner layer with 20-35
nm thick endospores. The endospore wall is connected to the plasma membrane
(Chen et al., 2009). The spore wall protein (SWP) is thought to interact directly with
the host cell and may play a key role in the microsporidia infection process. Currently,
two exosporal (SWP32 and SWP5) and three endosporal (SWP25, SWP26 and
SWP30) N. bombycis proteins have been identified (Cai, Lu, Qiu, Li, & Feng, 2011;
Li et al., 2009; Wu, li, Pan, Zhou, & Xiang, 2009). SWP26 has been shown to
facilitate spore adherence and host invasion processes (Li et al., 2009). SWP5
interacts with polar tube proteins and protects spores from phagocytic uptake by
cultured insect cells (Cai et al., 2011; Li et al., 2012). Chitin, a major component of
the endospore, was once thought to be fibrils and responsible for forming the bridges
across the endospore as part of the fibrillar system of exospores (Erickson &
Blanquet, 1969). Chitin is composed of b-1, 4-linked 2-acetamido-2-deoxy-b-D-
glucose and is the second most ubiquitous inartificial polymer of N acetylglucosamine
on earth (Dutta, Ravikumar, & Dutta, 2002). The exospore contains a protein which
cross-reacts with anti-keratin antibodies, but does not seem to have significant
homology with keratin (Bohne, Ferguson, Kohler, & Gross, 2000). Apparently, like
keratin, it has a C-terminal region with a repeated motif rich in glycine, serine, and a
number of conserved cysteine sites (Russell Hayman, Southern, & Nash, 2005). Some
11
groups also have an outer glycocalyx. In addition to the protein surrounding the
endospore is in the form of alpha chitin.
Motility organs of Nosema are similar to all fungi, but they lack flagella or
any other "9+2" Structure (Fast & Keeling, 2001). The polaroplast takes up most of
the anterior end of the cell. It is a transverse structure made up of tightly folded either
membranes or vesicles. It may also have a more loosely organized posterior region
variously referred to as the spongiform or tubular or vesicular polaroplast (Canning et
al., 2002; Keeling & Fast, 2002 ). Large vacuoles are usually found at the posterior
end of the cell. Their function is unknown. Like the polarosome, its principle purpose
may simply be to generate the directional pressures necessary to accomplish rapid
infection. In the spore stage, the polar filament is usually found as a series of tight
coils, just below the plasma membrane. These are observed in electron micrographs as
a series of dots running just under the cell membrane. The common features of all
microsporidia in the genus Nosema are being diplokaryotic (two nuclei) throughout
the entire life cycle (Sokolova et al., 2003).
Unfortunately, early identification of microsporidia species was mainly
based on spore morphology. This seemingly disproportionate number of Nosema
species may be due partly to incorrect identifications, resulted in the unnecessary
creation of new species (Malone & McIvor, 1995). Molecular phylogenic techniques,
based on rRNA and other genes, have been used to provide information for speciation
and classification of the microsporidia. Molecular phylogenetic data suggests that
microsporidia can be distinguished into three classes based on habitat: Aquasporidia,
Marinosporidia and Terresporidia. However, this division into three classes does not
characterize a final classification; it does make evolutionary relationships more
evident. According to the recent phylogenetic classification, the genus Nosema has
been allocated to the class Terresporidia, clade IV (Vossbrinck & Debrunner-
Vossbrinck, 2005).
can carry disease with them and further its distribution, or may become contaminated
by infectious spores from prior in habitants of the nesting site. Moreover, beekeeper
and human can distribute disease on a large scale, by use contaminated tools or hive
components and colonies translocation for pollination (Bigliardi & Sacchi, 2001;
Webster,1993).
Figure 2.1 Schematic representations of the early events in the life cycle of
N. ceranae. Spores extrude the polar tube which pierces the cell
membrane of the target cell followed by injection of the sporoplasm into
the host cell (a). The sporoplasm appears as small spherical body in the
host cell (b) and then develops into a spindle-shaped meront (c), which
begins to divide giving rise to paired meronts (d). These pairs of meronts
then undergo several rounds of cell division (e) until they separate and
develop into round to oval sporonts, which are condensed and
characterized by a thickened plasma membrane (f) (Gisder et al., 2010).
Microsporidia life cycle may be simple or complex and may involve sexual
or asexual reproduction, or both. Those with complex life cycles may have multiple
obligate hosts and as many as three different spore types. These forms tend to be
specialized to very specific host organisms and specific tissues. According to their
simple life cycles and often asexual reproduction, they have very broad tolerance and
are capable to infect many types of eukaryotic cells. There are two distinct phases in
the life cycle of microsporidia: a proliferative stage (merogony) and a sporogonic
phase (sporogony) (Bigliardi & Sacchi, 2001). The mature, infective microsporidian
14
spore is the only life stage that is sufficiantly environmentally resistant to survive
outside the living host cells. It was reported that spores of Encephalitozoan can
survive heating to 56 C for 60 min, a pH of 9 or 4 for 24 h, or storaged at 4 C for
2 years without losing infectivity (Hayman, Hayes, Amon, & Nash, 2001; Keeling &
Fast, 2002). The life cycle of Nosema begins after spore was ingested into honeybee
midgut, the free end of the tube inserts through the ventricular cell membrane of the
host serving as a pliable host through which the infectious sporoplasm is pumped into
the host cell in 15 to 500 msec (Keeling & Fast, 2002). This entire process is
completed in less than two seconds in the model systems in which it has been
measured (Delbac et al., 2001). Alternatively, the spore may be internalized by
phagocytosis (Hayman et al., 2001). The infectious dose for N. apis has been shown
to be approximately 100 spores per bee (Fries, 1988), and recent studies have
suggested the same dose for N. ceranae (Fries, 2010). Microsporidian ribosomes are a
large component of the sporoplasm which surround the cytoplasm. They promote a
very high rate of protein synthesis during the initial infective cycle when the
sporoplasm emerges from the tube, it has somehow already acquired a new cell
membrane (Bacchi et al., 2002). This is thought to derive from elements of the
polaroplast which precede the sporoplasm through the tube (Keeling & Fast, 2002).
Inside the host cell, the nuclear material in the sporoplasm replicates extensively,
either in direct contact with the host cytoplasm or inside a parasitophorous vacuole.
A typical microsporidian may replicate by merogony for some initial period inside the
host cell. During this period, in some cases, the nuclei may proliferate with or
without division into individual cells within the first 24-48 hours after the sporoplasm
has reached the host cell. It was reported that several rounds of division have
occurred, and then begin synthesizing of the coats and sporogony (Bacchi et al., 2002;
Canning et al., 2002). The transition to sporogony is marked by release of the
developing spore into the lumen of the vacuole and the accumulation of electron
dense material near the periphery of the cell of both merozonts and sporozonts which
showed little internal organization (Hayman et al., 2001). Electron dense extracellular
tubules have been observed surrounding developing spores during sporogony when
the spores completely fill the host cell cytoplasm after that the cell lyses and then
releases the spores to the surroundings (Bohne et al., 2000; Keeling et al., 2000).
15
Under normal conditions honey bee epithelial cells shed, burst, and release their
contents including digestive juices into the ventriculus (stomach). However, when the
cells are infected with N. apis, the parasite develops and multiplies in the cytoplasm
and form after about 5 days. Consequently, the spore-filled cells are shed into the
lumen, and some cells pass into the rectum. In order to they burst and release infective
spores rather than digestive juices (Morse & Shimanuki, 1990). Large numbers of
spores are produced in the host cell in 6 to 10 days. The parasite may also penetrate
and infect adjacent healthy cells. This spreads the infection further during the normal
digestive process of adult bees. Infected cells containing Nosema spores are shed into
gut lumen which can infect other healthy cells of the stomach lining cells. (Goodman,
2007; Malone et al., 1995; Matheson, 1993). In 2010, Gisder and his colleague had
studied Life cycle of N. ceranae in infected IPL-LD-65Y-cells to proof that the
heterologous, lepidopteran IPL-LD-65Y cells got truly infected by the honey bee-
specific pathogen N. ceranae, i.e. allowed the propagation of this pathogen, and to
analyses the time-course of the vegetative cycle them obtained a timed sequence of
micrographs demonstrating the entire infection process and the intracellular
vegetative cycle of N. ceranae. Germinating spores extruded the polar tube and hit the
target cells (Fig. 2.2 A and I) from a distance, followed by injection of the sporoplasm
into the host cell. The sporoplasm became visible as spherical body in the infected
cells shortly thereafter (Fig.2.2 B and J). Analysis of the time-course of the
intracellular life cycle revealed the following sequence of events: 16 h post
inoculation (p.i.) the injected sporoplasm developed into a hitherto undescribed
vegetative stage of N. ceranae, a spindle-shaped meront (Fig. 2.2 C and K).
Four hours later, merogonial replication of N. ceranae had started and the first pair of
spindle-shaped meronts could be seen (Fig. 2.2 D and L). These pairs of spindle-
shaped meronts then multiplied (Fig. 2.2 E, F, M and N) until the first condensed
structures (sporonts and sporoblasts) developing into primary spores could be detected
in infected cellsbetween 48 and 72 h p.i. (Fig. 2.2 F and O). At this stage, many pairs
of spindle-shaped meronts were still present. The primary spores (Fig.2.2F, G and O)
were characterized by a rather round shape as opposed to the oval form of
environmental spores (De Graaf et al., 1994a), which could be detected in infected
cells at about 96 h p.i. (Fig. 2.2 H and P). The morphological differences could be
16
Figure 2.2 Nosema ceranae life cycle observed in experimentally infected IPL-LD-
65Y (Lymantria dispar) cells. A-H. Vegetative stages of the bee pathogen
N. ceranae can be detected in IPL-LD-65Y cells after Giemsa staining. I-
P. Species-specific fluorescence in situ hybridization (FISH) analysis at
different time points post infection (p.i.). Cells were analysed applying a
Nosema-specific 16S rRNA-targeted (green fluorescence) and a universal
eukaryotic 18S rRNA-targeted (red fluorescence) oligonucleotide probe
and visualized by fluorescence microscopy. Eukaryotic nuclei were stained
with DAPI (blue fluorescence). The injected sporoplasm (marked with
arrows in B and J) develops into spindle-shaped meronts (marked with
17
arrows in C and K), which form pairs of meronts after the first cell
division (marked with arrows in D and L). These pairs continue
proliferation until infected cells contain high numbers of paired, spindle-
shaped meronts (F and N) and the first sporonts appear (F, G and O). The
vegetative cycle of N. ceranae is completed after approximately 96 h and
infected cells contain masses of primary and/or environmental spores
(H, P). Bars represent 10 mm (Gisder et al., 2010).
was the only known agent to cause nosemosis in A. mellifera until 2005, when
N. ceranae was first identified in samples from Taiwan (Huang et al., 2007). Other
A. mellifera samples were screened using SSUrRNA gene sequences, and N. ceranae
was also found in host samples from the Eastern USA collected in 2004 (Klee et al.,
2007), in 2005 from Europe (Higes et al., 2006), and in samples from Canada dating
as far back as 1994 (Currie, Pernal, & Novoa, 2010). N. ceranae has also been
identified in A. florea and A. dorsata from Thailand in 2010 (Chaimanee, Warrit,
& Chantawannakul, 2010) and in three species of bumblebees in Argentina (Plischuk
et al., 2009).
However, the exactly first time and place of N. ceranae cross infections in
A. mellifera is still mysterious (Fries, 2010). But they were likely unnoticed for a
while due to the fact that microsporidia are microscopic in size and infections rarely
cause death (Paxton, 2010). Paxton, Klee, Korpela, and Fries (2007) statement that
N. ceranae has been present in European honey bee, A. mellifera since at least 1998,
while Chen and his colleagues have discovered it in samples from the USA dating
back to 1995 (Chen, Evans, Smith, & Pettis, 2008). This indicates that N. ceranae was
transferred from A. cerana to A. mellifera earlier than previously thought; and
experimental descriptions of Nosema disease in A. mellifera since 1995 could have
involved N. ceranae without researchers being aware of its presence. Consequently,
these two microsporidia species have co-existed as parallel pathogens of A. mellifera
for nearly two decades (Fries et al., 2006). Experimental infection of the European
honey bee with the microsporidian, N. ceranae showed that the infectivity of this
parasite developed well in this host, and the intracellular life cycle was completed
within three days post infection. Therefore, this study also demonstrated the rapid
division of the parasite in A. mellifera, and suggests a high pathological potential in
early development of an infection (Fries, 1997). Additionally, this species is highly
pathogenic when experimentally inoculated into European honey bees and is
associated with reduced honey production and increased winter mortality (Higes et
al., 2007). The observations that N. ceranae causes a higher mortality than N. apis in
caged bees despite the same pathogen load (Higes et al., 2008; Paxton et al., 2007).
This suggests that the new species possibly has a higher virulence while this means
that N. ceranae could cause a particularly severe metabolic stress in its new host. Its
19
not only in honey bee the distribution of N. ceranae also have been report in other
insects. The presence of N. ceranae, an emerging honey bee pathogen, in three
species of Argentine native bumblebees was described using PCR technique by
Plischuk and his colleagues in 2009. PCR results showed that three of six bumble
bees species belonging to genus Bombus were positive to N. ceranae, and they
concluded the appearance of this pathogen in the context of the population decline of
this pollinators (Plischuk et al., 2009).
shortened the bee life span, with a resulting decrease in bee population (Buys, 1977;
Clark, 1980; Singh, 1975). Few data are available from N. ceranae infection in
A. cerana, although infected ventriculi that appear whitish and swollen have been
reported (Fries et al., 1996). N. ceranae infected bees do not fully develop their
hypopharyngeal glands resulting in up to 15 % of eggs in severely infected colonies
not producing mature larvae in early summer (Wang & Moeller, 1969). Queens are
generally superseded within 2 to 8 weeks after becoming infected (Moeller, 1962).
Infections of N. apis have a negative effect on the protein build-up of the fat body
(Bailey & Ball, 1991). Additionally, they start their foraging activity at a younger age
than healthy bees and collect significantly less pollen than uninfected colonies
(Anderson & Giacon, 1992).
activity remains high in honey kept at 4 C for several years and for at least 30 days at
30 C (Furgala & Sugden, 1985). Screening of natural compounds for the control of
Nosema disease in honey bee (A. mellifera) was evaluated using several substances of
natural origin; thymol, vetiver essential oil, lysozyme and resveratrol, with possible
inhibitory effect on the development of microsporidians. The results showed that
thymol and resveratrol undoubtedly have potential in the development of alternative
strategies for the control of Nosema disease (Maistrello et al., 2008). They have been
many reports that resveratrol can inhibit the development of the microsporidian
Encephalitazoon cunicoli in vitro experiments (Leiro, Cano, Ubeira, Orallo,
& Sanmartn, 2004) while thymol (3-hydroxy-p-cymene), a constituent of the
essential oil derived from thyme and many other plant species, has been shown to
suppress N. vespula disease in Helicoverpa armigera caterpillars and some evidence
suggested that thymol may suppress Nosema disease in honeybee colonies (Rice,
2001; Yucel & Dogaroglu, 2005). It is known to be effective in inhibiting the growth
of pathogenic bacteria and fungi, such as Salmonella typhimurium, Staphylococcus
aureus (Juven, Kanner, Schved, & Weisslowicz, 1994), Aspergillus flavus
(Mahmoud, 1999) and Cryptococcus neoformans (Viollon & Chaumont, 1994). In
apiculture, it is well known due its suppressive effects against the parasitic mite
V. destructor and honey bee are tolerant to its use through physical contact (Chiesa,
1991; Imdorf, Kilchenmann, Bogdanov, Bachofen, & Beretta, 1995). Also, recent
research has shown that thymol fed orally to adult bees is not toxic (Ebert, Kevan,
Bishop, Kevan, & Downer, 2007). Lysozyme was not at all able to reduce the
development of this microsporidians, but it can be used as antibacterial substance.
Protofil is a natural product obtains from different plants that prevents the
development cycle of N. apis. It also inhibits the intestinal pathogen flora and
stimulates the digest enzymatic secretion of honeybee larvae (Bogdan, Kilchenmann,
Imdorf, & Fluri, 1986; Chioveanu, Ionescu, & Mardare, 2004). An experiment stated
that acetic acid decreases the development of N. apis in the honeybee mid gut
(Mottoul, 1996), however, field studies performed in France demonstrated no impact
from acidified food on Nosema development (Vaillant, 1989). The chemical
composition of the food may have an impact on the spore germination of N. apis by
the way of when spore enters the mid gut of the honeybee, it germinates under the
23
2.6 Propolis
Propolis is a resinous substance that honeybee workers and stingless bee
collected from various types of plants containing 50-70 % resins and 10 % essential
oils, 30-50 % wax and 5-10 % pollen. It is used to seal any cracks and fissures in the
hive of honey bee and line their front door to prevent contamination as an antiseptic in
brood cells (Burdock, 1998). The chemical composition of propolis is highly variable
depending upon the insect species and plant species. There are more than 150
compounds have been identified from propolis (Greenaway, May, Scaysbrook, &
Whatley, 1990). The main chemical compositions are at least 38 flavanoids, phenolics
and various aromatic compounds (Schmitdt & Buchmann, 1999). Moreover, it
contains many of the B-complex vitamins, important minerals and trace elements. In
particularly, bioflavanoid content is now receiving attention according to its
antioxidant activity that play a major important role in the up taking of free radicals
24
which are produced in degenerative heart diseases, atherosclerosis, aging and effects
of toxic. It has been shown that propolis can stimulate various enzyme systems, cell
metabolism, circulation and collagen formation, as well as it improves the healing of
burn wounds resulting of the presence of arginine in its composition. It was reported
that propolis stimulated an immune response in mice by activating immune cells
producing cytokines (Young, 1987).
Bee propolis is one of the most promising extracts because it showed
properties of anti-viral, anti-bacterial, anti-inflammatory and immune stimulating
activities (Wang et al., 2005). One of the main chemicals is amitraz, [1, 5-di-(2, 4-
dimethylphenyl)-3-methyl-l, 3, 5-triaza-penta 1, 4-diene] which is a member of the
formamidine pesticides and used worldwide as insecticide and acaricide
(Hollingworth, 1976). Additionally, it is a veterinary medicinal product used by
beekeepers to control the ectoparasitic mite, Varroa destructor which is a wide spread
parasite feeds on hemolymph of mature and immature stages of honey bee that
damages beehives seriously. However, its toxicity has not been investigated at a
sufficient level (Grossman, 1993). Furthermore, propolis antimicrobial activity is one
of the most extensively investigated biological actions; some factors may influence its
inhibitory capacity. Several studies have been performed in various laboratories to
investigated its microbial activities (Fernandes Jr. Sugizaki, Fogo, Lopes, & Funari,
1997; Mirzoeva, Grishanin, & Calder, 1997).
Stingless bees are are highly eusocial bees. They are classified into the tribe
of Meliponini in the family Apidae, which are closely related to the common honey
bees and found in most tropical or subtropical regions of the world. Unlike other
eusocial bees, they do not sting but will defend by biting if their nest is disturbed.
They live usually in nests in hollow trunks, tree branches, underground cavities, or
rock crevices. There are 500 species of stingless bees are recorded and classified into
five genera: Melipona, Trigona, Meliponula, Dectylurina and Lestrimelitta. However,
only some species of two genera, Trigona and Melipona are the honey producing
bees. Trigona is the largest genus of stingless bees and have many subgenera which
is found extensively in tropical regions extending from Mexico to Argentina, India,
Sri Lanka to Taiwan, the Solomon Islands, South Indonesia and New Guinea, but no
members of the genus occur in Africa. There are more than 30 species of stingless
25
bees are reported in Thailand including two new species of Trigona binghami and
T. minor are newly added to the list of 30 species recorded earlier by Schwarz (1939),
and Michener and Boongird (2004) making a total of 32 stingless bees of Trigona
species currently recorded in Thailand. The newly recorded species were found in
HM Queen Sirikit Botanical Garden in Maerim Chiang Mai, Chanthaburi and Mae
Hong Son Provinces. The diversity of Trigona and their resin and gum collecting
behaviour mostly depended on environmental factors. The bees prefer to collect resin
and gum from 16 plant families including Anacardiceae, Dipterocarpaceae,
Euphobiaceae, Hypericaceae, Meliaceae and Moraceae. During the rainy season they
collected resin and gum all day, whilst during the dry season start from afternoon until
late in the day. T. apicalis collect resin and gum to make the largest number of
propolis compared with the other bee species. The honey broadly applied for different
purposes is mainly obtained from the western honeybee A. mellifera. In the tropical
and subtropical regions, however, honey is made not only by the aforementioned bee
species but also by several species of stingless bees that belong to the subfamily
Meliponinae and the genera Melipona and Trigona (Garedew, Schmolz, &
Lamprecht, 2003).
subsequently invading the gut epithelium (Bailey, 1981). Studies with Nosema apis
have shown that it imposes a high metabolic demand on the host (Wang and Moeller,
1970a; Liu, 1984; Malone and Gatehouse, 1998), and in 2009 Mayack and Naug have
been reported that foragers infected with N. ceranae incur an energetic stress, which
increases their hunger and compromises their survival that this could lead to infected
foragers being more likely to forage under adverse conditions, a higher hunger level
could also have a significant influence on the feeding behavior of bees within the
colony. Any such changes in feeding at the individual and the social level could result
in important changes in the transmission dynamics of an orally infecting pathogen
such as Nosema (Mayack and Naug, 2009). Honeybee foragers, which have some of
the highest recorded sugar levels of any insect show evidence of an energetic stress
when they are parasitized by the microsporidian Nosema ceranae (Mayack and Naug,
2009). Lysing the epithelial cells of the mid gut (Higes et al., 2007; Liu, 1984), these
microsporidians are in an ideal position to draw away the glucose and fructose that are
produced from the breakdown of dietary sucrose and as a result reduce the synthesis
of trehalose, the principal carbohydrate in insect hemolymph. Honey bees afflicted
with nosemosis start to forage earlier (Fries, 1995), while pathological changes of
their mid gut epithelial cells, as well as digestive and metabolic disorders (Hassanein,
1951). N. apis is a parasite of the bee digestive tract and is considered not to be highly
virulent and with a clear sesonality such that infections are least prevalent during the
summer months. In contrast, N. ceranae is highly pathogenic when experimentally
inoculated into European honey bees (Higes et al., 2007). However, there are usually
no symptoms of diarrhea or visible adult bee deaths, and there is a total lack of
seasonality in the diagnosis (Martin-Hernandez et al., 2007). The clinical and
epidemiological patterns of Nosema disease is a little known about the pathogenicity
in now a day.
processing (Simpson, Riedel, & Wilding, 1968). The development and activity of
these glands have been studied since early this century because they are intimately
involved in many important aspects of honeybee biology, including the division-of-
labour (Fluri, Lscher, Wille, & Gerig, 1982; Free, 1961; Halberstadt, 1970; Huang,
Otis, & Teal, 1989). Their acinar diameters were often used to describe the
physiological status of honeybee workers (Crailsheim & Stolberg, 1989; Free, 1961;
Maurizio, 1954; Moritz & Crailsheim, 1987).They are fully developed in nurse bees
and become atrophy or degenerate in elder foragers and guard bees (Wang & Moeller,
1969). The age at which the glands degenerate is quite flexible. It depends on the
colony conditions and the time of the year. In late summer workers still have well
developed glands compared to early summer, when bees of the same age have
degenerate ones (Maurizio, 1954; Moritz & Crailsheim, 1987). During winter, when
no or only small amounts of brood have to be nursed, these glands are well developed
in most of the workers (Brouwers,1983; Fluri et al., 1982; Lotmar, 1939; Moritz
& Crailsheim, 1987). Hypopharyngeal glands of Nosema infected bees showed a
reduction in their capacity to feed larvae in the colony with a royal jelly because they
fail to develop in size and eventually atrophy, whereas those of healthy bees increase
in volume over the first 2 weeks or so of adult life (Hassanein, 1951; Wang
& Moeller, 1969). The glandular degeneration have been seen in honeybee workers
5 and 10 days after dosing with 5000 N. apis spores/ bee (Wang & Moeller, 1971).
There is some evidence that N. apis infected bees, with their atrophied
hypopharyngeal glands, may have a fore shortened nursing phase and begin foraging
earlier than healthy bees (Hassanein, 1953). In order to complete the proper
development of both the glands and the fat body, newly emerged bees must digest
large amounts of protein over the first few days of adult life (Winston, 1987). It was
reported that newly emerged bees (kept in cages or hives) undergo a rapid rise in the
activity of gut proteolytic enzymes immediately after emergence and that this activity
peaks within 1 week of adults and then drops to a relatively steady state for the rest of
their lives (Burgess et al., 1996). There is evidence that bees may be exposed to
significant numbers of N. apis spores during this early part of adult life. In the hive
situation, most bees are thought to become infected by ingesting spores from the feces
of other bees while performing house-keeping duties in the early part of adulthood
28
(Bailey, 1981). Removing frames from hives and keeping in control room showed
that emerging adult bees are permitted to chew through the wax capping of their cells
as they emerge, they may become infected with N. apis, presumably by ingesting
spores contained within the wax. Thus it is likely that bees commonly become
infected at or around the time of emergence, when protein digestion is most
important, and that N. apis may exert its effect on the gland development by
disrupting proteolytic activity in the gut during this critical phase. In addition, the
gland abnormalities have been observed in bees only 5 days dosing of N. apis spores,
this suggests that the early proliferation of the vegetative stages of N. apis in
ventricular cells may cause this disruption before lyses (Wang & Moeller, 1971).
CHAPTER 3
RESEARCH METHODOLOGY
3.1 Instruments
- Camera (Nikon, Japan)
- Digital Orbital Shaker - Daihan (Daihan Scientific, Korea)
- Freezer (Krungthai, Thailand)
- Fume Hoods (Thermotek, Thailand)
- Incubator (Thermotek, Thailand)
- Incubator (WET binder, US)
- Light Microscope (Nikon, Japan)
- Micro centrifuge (WiseSpin, Scientific.co.ltd., Korea)
- Stereo Microscope (Nikon, Japan)
- Spectrophotometer (Shimadzu, Australia)
- Rotary microtome (Leica, Germany)
- Slides warmer (Fisher, USA)
- Magnetic stirrer and magnetic bars (Heidolph, Germany)
- Microtome blades (Feather, Japan)
- Warm plate (Fisher, USA)
3.3 Chemicals
3.3.1 Chemicals for Light Microscopy
- Absoluted Ethyl Alcohol (J.T Baker, Malaysia)
- Basic Fuchsin (Labchem, Australia)
- Distilled water
- Formaldehyde Solution (Univar, Australia)
- Glacial Acetic Acid (Analar, England)
- Hydrochloric Acid (J.K Baker, USA)
- Light Green (Fluka, Switzerland)
- Picric Acid (Labchem, Australia)
- Periodic Acid (Merck Chemical)
- Permount (Fisher Chemical, USA)
- Sodium Bisulfite (Merck Chemical)
- Xylene (Panreac, England)
- Ninhydrin (Merck, Germany)
- Distilled water
- Glycerine jelly (Ajax, Australia)
- Methanol (Sigma Chemical Company, USA)
- Phosphoric acid (Merck KGaA, Germany)
- Sodium Hydroxide (NSW 2147, Australia)
- Sodium chloride (Merck KGaA, Germany)
3.4 Specimens
A. cerana workers were obtained from the colonies of apiary in Samut
Songkhram Province, Thailand, from which representative samples of bees were
previously analyzed and the absence of Nosema spores was verified. While spores of
32
N. ceranae were prepared from heavily Noesma infected bees from infected colony
obtaining from local area of Samut Songkhram Province.
This study use propolis form Trigona apiscalis and Apis mellifera collected
from two different regions of Thailand, Chaing Mai and Chanthaburi Provinec
Figure 3.1 Map of Thailand represents propolis and honey bee specimens collected
sites (from http://www.dpt.go.th/provinces).
33
3.6 Methodology
3.6.1 Propolis extraction
Raw propolis from colonies of A. mellifera and T. apialis located in
Chanthaburi and Chiang Mai province, Thailand were collected for extraction. The
whole samples were dried in a hot air oven at 80 C for 72 hour and then frozen,
ground and homogenized prior to beginning extraction. For extraction, 30 g of fine
propolis was placed in an Erlenmeyer flask and extracted with 100 ml of 70 %
ethanol. The suspension was shaker for seven days in dark room at room temperature.
Then the suspensions were filtered, yielding a crude ethanol extract defined later as a
100 % propolis. Two concentrations of 50 % and 70 % propolis in distilled water
(v/v) were prepared for the experiment. After extraction, propolis extract from each
place and each species was evaluated the effects on Nosema spore in A. cerana, by
evaluate the effect of propolis extract on the development of N. ceranae, the
longevity, infectivity, infection ratio and on hypopharyngeal gland protein content of
honey beecompared between each group.
then the pellet were resuspended in 50 % sucrose solution (w/v) in water. The spore
concentration was determined by counting with a Haemocytometer, 40,000 spores per
l were prepared for inoculum dosage (80,000 spores/ bee).
3.6.3 Nosema free bees were divided into six groups as defined
follows;
Honeybee form 2 colonies were divided in 6 groups per colony, 50 bees
from each group were treated with different treatment, 3 replicates for each treatment
and defined each group as the following;
3.6.5 Infectivity
To obtain the Nosema spore counts, the mid gut of dead bee was removed
from each bee and placed into a sterile Eppendorf microcentrifuge tube in 200 l of
distilled water. Then the mid gut was grinding and the spore count for each bee was
calculated with a hemocytomete. In addition, after the end of each experiment, all
alive honey bee from each cage was freeze-killed, their entire gut were removed and
the present of Nosema spores in each bee also quantified on a hemacytometer under
light microscopic.
dead bees in each cage were recorded and bodies removed for storage at 0 C until
spore quantified. Bees that died within the first 24 h post inoculation were excluded
from analysis to eliminate handling effects. To evaluate mortality among the
treatments Kaplan-Meier survival curves were generated by plotting number of
surviving bees against days from initiation of the experiment (Le, 1997).
11) Stained with 1 % toluidine blue in sodium borate solution and studied
with light microscope.
12) After light microscopic selection of representative tissue areas, the Epon
block was trimmed for ultrathin (60 nm) sectioning.
13) Ultra-thin sections (90 nm) and floated in distilled water.
14) Tissues were collected on 200 mesh grids, stained by uranyl acetate and
contrast enhanced by lead citrate.
15) They were examined by Jeol 200SX (Tokyo, Japan)
CHAPTER 4
RESULTS
group of honey bee treated with 70 % propolis extract of A. mellifera from Chiang
Mai (70P4) with 28.674.67 %, respectively. The survival rate of honey bee groups
that treated with lower concentration of propolis extract or with 50 % (50P) were
showed slightly lower survival rate when compared to those of 70P groups; but
however, they were no significant differences (F11 = 19.49, P > 0.05). The survival
rate of 50P groups were as follow; group of honey bee treated with 50 % propolis
extract of A. mellifera from Chanthaburi (50P3) with 27.331.76 %, follow by group
of honey bee treated with 50 % propolis extract of T. apicalis from Chanthaburi
(50P1) with 26.02.31 %, group of honey bee treated with 50 % propolis extract of
A. mellifera from Chiang Mai (50P4) with 24.673.71 %, and group of honey bee
treated with 50 % propolis extract of T. apicalis from Chiang Mai (50P2) with
22.671.33 %, respectively (Fig. 4.2).
Figure 4.1 Kaplan-Meier curves show the cumulative mortality rate of honey bee
(A. cerana) after inoculated with N. ceranae for 30 days with 80,000
spores/ bee (80K) per bee, and treated with propolis from T. apicalis,
Chanthaburi (1); T. apicalis, Chiang Mai (2) A. mellifera, Chanthaburi (3)
and A. mellifera, Chiang Mai (4), with two different concentration 50 %
(50P) and 70 % (70P); infected bees without propolis 0 % (0P); infected
bees and treated with ethanol (CE); control propolis (CP) and control
without spores of N. ceranae (CO).
40
Figure 4.2 The survival rate (MeanSE) of honey bee (A. cerana) after inoculated
with N. ceranae for 30 days with 80,000 spores/ bee (80K), and treated
with propolis from T. apicalis, Chanthaburi (1); T. apicalis, Chiang Mai
(2) A. mellifera, Chanthaburi (3) and A. mellifera, Chiang Mai (4), with
two different concentration 50 % (50P) and 70 % (70P); infected bees
without propolis 0 % (0P); infected bees and treated with ethanol (CE);
control propolis (CP) and control without N. ceranae (CO). Vertical bars
with different letters represents significantly difference (ANOVA-
Duncans multiple range test, (F11 = 19.49, P < 0.0001)).
41
4.2 Infectivity
After 30 days post inoculation with 80,000 spores per bee of N. ceranae,
the alimentary tract of each bee from each treatment were taken out and macerated in
distilled water. A droplet of this suspension was placed onto a glass slide, and
examined at 400x magnification under the light microscope for the presence of
N. ceranae spores. The mean number of spores per individual bee from each group
was determine to evaluated the inhibited efficiency of propolis from each species and
regions on N. ceranae spore proliferation. As shown and presented graphically in
figure 4.3.
Figure 4.3 The infectivity (MeanSE) of A. cerana after inoculated with N. ceranae
spores for 30 days with 80,000 spores/ bee (80K), and treated with
propolis extract from T. apicalis, Chanthaburi (1); T. apicalis, Chiang Mai
(2) A. mellifera, Chanthaburi (3) and A. mellifera, Chiang Mai (4), with
two different concentration 50 % (50P) and 70 % (70P); infected bees
without propolis 0 % (0P);infected bees and treated with ethanol (CE);
control propolis (CP) and control without N. ceranae (CO). Vertical bars
with different letters represents significantly difference (ANOVA-
Duncans multiple range test, (F9= 19.46, P < 0.0001)).
42
50P and 70P were not significant different (F9 = 19.46, P > 0.05). The mean spore
per bee of honeybee treated with different species and regions also compared, the
resulted were showed no significant differences among the 2 species and 2 regions of
propolis extracted on mean spore per bee (Fig. 4.6). No mature spores were found in
both of control groups (CO and CP) (F9 = 19.46, P > 0.05).
ratio of 50P groups were found in group of honey bee that treated with 50 % propolis
extract of T. apicalis from Chanthaburi (50P1) with 63.20 % 4.79 of infected cells,
follow with group of honey bee treated with 50 % propolis extract of A. mellifera
from Chanthaburi (50P3) with 58.87 % 4.90, group of honey bee treated with
50 % propolis extract of T. apicalis from Chiang Mai (50P2) with 56.07 % 6.15,
and group of honey bee treated with 50 % propolis extract of A. mellifera from
Chiang Mai (50P4) with 55.001061.15 of infected cells, respectively. However,
when analyzed the infection ratio of honey bee that treated with propolis extract from
different species and region were not showed significant different on infection ratio.
All of propolis extract showed similar result of infection ratio, they were represent a
fewer infected cells than those of untreated groups (F9 = 18.79, P > 0.05) (Fig. 4.4).
Figure 4.4 The infection ratio 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 from T. apicalis, Chanthaburi (1); T. apicalis, Chiang Mai
(2) A. mellifera, Chanthaburi (3) and A. mellifera, Chiang Mai (4), with
two different concentration 50 % (50P) and 70 % (70P); infected bees
without propolis 0 % (0P); infected bees and treated with ethanol (CE);
control propolis (CP) and control without N. ceranae (CO). Vertical bars
with different letters represents significantly difference (ANOVA-
Duncans multiple range test, (F9 = 18.79, P < 0.0001)).
45
Figure 4.5 The light micrograph of ventricular epithelial cells of control bee (CO),
A. cerana worker on day 14 p.i. (PAS, 400x) showing well develop
microvilli at the apical part of the ventricular cells, a completed of an outer
muscular coat also present underneath of basement membrane.
(Abbreviations: br, brush border; bm, basement membrane; lu, lumen; n,
nucleus, mc, muscular; pm, perithrophic membrane).
46
Figure 4.6 The light micrograph of ventricular epithelial cells of control bee (CP),
A. cerana worker on day 14 p.i. (PAS, 400x) showing a simple columnar
together with pseudostratified columnar epithelium and well develop
microvilli at the apical part of the ventricular cells. (Abbreviations: br,
brush border; bm, basement membrane; lu, lumen; n, nucleus, mc,
muscular; pm, perithrophic membrane).
clearly observed at the bottom of the cells near the basement membrane. In heavily
infected bees from untreated propolis groups, bees had a much looser and not clearly
demarcated area of peritrophic membrane. In addition, it was observed that in the
presence of numerous N. ceranae spores the intestinal contents tended to be squeezed
into the center of the intestinal lumen probably impeding the germination of spores.
The spores were ovoid and rather uniform in shape and size and heterogeneously
stained. Mature spores were surrounded by a wall and immature spores were optically
empty. In heavily infected cells, different intracellular parasitic stages such as mature
and immature spores were found in the same cells (Fig. 4.7-4.10).
Figure 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) showing numerous of mature spore fully in ventricular
cell (pink granules). The mid gut epithelial layer is disrupted and most
cells have exploded. The nuclei have disappeared or diminished in three
cases, the cytoplasm inclusion is scattered and the cell membranes are
decayed completely. Some cells were separated from the basement
membrane. High number of mature spores distributed throughout the cell
cytoplasm (bm, basement membrane; lu, lumen; n, nucleus; sp, spore of
N. ceranae).
48
Figure 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) showing numerous of mature spore fully in ventricular
cell (pink granules). (bm, basement membrane; lu, lumen; n, nucleus; sp,
spore of N. ceranae).
Figure 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). (Abbreviations: bm, basement membrane; lu, lumen; n,
nucleus; sp, spore of N. ceranae).
49
Figure 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) showing Nosema spores distribute throughout the cell
cytoplasm, (Abbreviations: bm, basement membrane; lu, lumen; n,
nucleus; sp, spore of N. ceranae).
Figure 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 extract propolis of
T. apicalis collected from Chantaburi. sp, mature spore; bm, basement
membrane; lu, lumen; n, nucleus of ventricular cell ( PAS, 400x).
Figure 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) of ethanolic extract propolis of
T. apicalis collected from Chiang Mai. sp, mature spore; bm, basement
membrane; lu, lumen; n, nucleus of ventricular cell ( PAS, 400x).
51
Figure 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) of ethanolic extract propolis of
A. mellifera collected from Chantaburi. sp, mature spore; bm, basement
membrane; lu, lumen; n, nucleus of ventricular cell ( PAS, 400x).
Figure 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) of ethanolic extract propolis of
A. mellifera collected from Chiang Mai. sp, mature spore; bm, basement
membrane; lu, lumen; n, nucleus of ventricular cell ( PAS, 400x).
52
Figure 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) of ethanolic extract propolis from
T. apicalis, Chanthaburi. sp, mature spore; bm, basement membrane; lu,
lumen; n, nucleus of ventricular cell (PAS, 400x).
Figure 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) of ethanolic extract propolis from
T. apicalis, Chiang Mai. sp, mature spore; bm, basement membrane; lu,
lumen; n, nucleus of ventricular cell (PAS, 400x).
53
Figure 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) of ethanolic extract propolis of
A. mellifera collected from Chantaburi. sp, mature spore; bm, basement
membrane; lu, lumen; n, nucleus of ventricular cell ( PAS, 400x).
Figure 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) of ethanolic extract propolis of
A. mellifera collected from Chantaburi. sp, mature spore; bm, basement
membrane; lu, lumen; n, nucleus of ventricular cell ( PAS, 400x).
54
Figure 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), and treated
with propolis extract of T. apicalis collected from Chanthaburi (1);
T. apicalis collected Chiang Mai (2) A. mellifera collected Chanthaburi
(3) and A. mellifera collected Chiang Mai (4), with two different
concentrations 50 % (50P) and 70 % (70P); infected bees without
propolis 0 % (0P); infected bees and treated with ethanol (CE); control
propolis (CP) and control without N. ceranae (CO). Vertical bars with
different letters represents significantly difference (ANOVA-Duncans
multiple range test, (F9 = 18.79, P < 0.0001)).
higher protein content of HPGS than those of untreated groups (0P and CE). The
trends of protein content from all groups were slightly decreased when the time of
experiment increasing. The table 4.1 and figure 4.32 represented the mean protein
content of honey bee that treated with propolis extract of T. apicalis collected from
Chanthaburi. The highest protein concentration was found in un-infected control CO
group on day 6 post infection which was 532.93 24.31g per bee. In contrast, the
lowest protein concentration was found in CE group which were 309.7250.57g per
bee. However, bees that from propolis extract treated groups 50P1 and 70P1 were
showed higher protein content than those of untreated CE and 0P groups, and they
were significant difference in protein concentration (F2, 5 = 26.81, 8.86, P < 0.0001).
The highest protein content of honey bee that treated with propolis extract of
T. apicalis collected from Chiang Mai also found in un-infected CO group; follow
with CP group on day 6 post infections which were 530.7418.67 g per bee and
544.2154.18 g per bee, respectively (Table 4.2 and Fig. 4.33). The lowest protein
content still found in untreated with propolis extract group CE on day 14 p.i. which
was 316.3838.82 g per bee. Honey bee from group of 0P and CE are also
represented significantly lower protein content when compared to un-infected control
bee groups and propolis extract treated bees of 50P and 70P groups (F2, 5 = 16.11,
5.32, P < 0.0001). The highest mean total protein content of the hypopharyngeal
glands between honey bee treated with propolis extract of A. mellifera collected from
Chanthaburi was found in group of CO, un-infected honey bee control group which
532.9324.31 g per bee (Table 4.3 and Fig. 4.34). Protein concentration of all groups
of honey bee was decrease in concentration in later day post infection. The lowest
protein content was found in group of CE on day 14 p.i. which 309.7250.57 g per
bee. However, after treated with 50 and 70 percentage of propolis extract the protein
content were not significant difference from control CO and CP groups (F2, 5 = 11.88,
5.12, P > 0.05). There was significant difference in protein concentration of
hypopharyngeal glands among groups of honey bee treated with propolis extract of
A. mellifera collected from Chiang Mai (F2, 5 = 3.77, 0.23, P < 0.0327). The highest
protein content was found in un-infected CO group; follow with CP group on day 6
p.i. which was 514.4264.57 g per bee and 506.2737.27 g per bee, respectively
(Table 4.3 and Fig. 4.34).
64
Table 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.
Table 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.
Table 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.
Table 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.
electron dense external layer on the plasma membrane. Their shape gradually
transforms from spherical to ovoid, characteristic of the sporoblast. In the latter the
diplokaryotic nuclei are located along the long axis. Dividing sporonts were more
commonly observed than dividing meronts. The sporonts divided at once yielding two
sporoblasts that each developed into the final spore. The mature spores were
ovocylindrical, straight to slightly curved and formed free in the host cell cytoplasm
without any sporophorous vesicle. The spore wall consisted of a three layered
exospore, a lucent endospore and an internal plasma membrane that remained
unchanged from earlier developmental stages. The TEM observations revealed that
the number of polar filament coils was between 20 to 23, and most spores contained
22 polar filament coils and showed a two layer arrangements of polar filament coils in
the posterior part. The polar filament was isofilar with a width of 96-102 nm. The
transversely sectioned filament was stratified in four layers. The diplokarya of the
spore was located in the mid part of the spore and surrounded by the coiled polar
filament (Fig. 4.41). When present they were numerous and rarely associated with the
presence of sporogonic stages. Numerous sporonts were recognized with diplokarya
of similar size to those observed in daughter meronts. When sporonts were identified,
different intracellular parasite stages were found in the same cell, such as mature
spores, empty spores or merogonial stages. Infected cells were enlarged and the
cytoplasm contained a larger number of mitochondria and free ribosomes. Several
mitochondria were close to and surrounded the plasmalemma of meronts (Fig. 4.36)
while RER, usually ring-shaped, was commonly observed around sporonts. In cells
with sporogonialphases, the RER content was greater than when merogonial stages
were present.
Nosema spores were abnormal in appearance which could be seen in bees
treated with ethanolic extract propolis of T. apicalis collected from Chanthaburi,
T. apicalis, Chiang Mai, A. mellifera, Chanthaburi and A. mellifera collected from
Chiang Mai with all concentrations showed irregular in shaped in particularly at the
anchoring disk that became wrinkle (Fig. 4.44-4.51). Therefore, the different
developmental stages of spores were observed inside the cytoplasm of the ventricular
cells of bees treated with propolis. The disorganized cellular organelles also be found
in cytoplasm of ventricular cell of all treatments.
70
Figure 4.36 The transmission electron micrograph of infected ventricular cell filled
with different parasitic stages: brush border (bb), meronts (m), mature
spore (ms) and sporoblasst (sp).
Figure 4.38 The transmission electron micrograph showing the sporoblast divide by
binary fission, sometimes three or four cells form chains.
Figure 4.40 The transmission electron micrograph of ventricular cell with mature
spores distributed over the top of ventricular fold: brush border (bb),
mature spore (ms) and sporoblasst (sp).
Figure 4.50 The transmission electron micrograph longitudinal section of spore inside
the ventricular cell of bee treated with 70 percentages of ethanolic extract
propolis from A. mellifera, Chanthaburi on day 14 p.i showing abnormal
shape.
Honey bee are not only provided many kinds of valuable product but also
critical to agricultural production and maintenance of natural plant communities
(Gallai et al., 2009). However, widespread declines in honey bee populations have
become a cause of concern. Global honey bee colony loss occurs through interactions
between different stressors. Nosema ceranae are recognized as one of the potential
contributors to honey bee losses including the recent difficulty, colony collapse
disorder (CCD) (Cox-Foster et al., 2007). Nosema species (N. ceranae and N. apis)
are intracellular endoparasites of adult honey bees infecting the epithelial cells of the
mid gut (Forsgren & Fries, 2010). N. ceranae infection prevents immune system
function in honey bee which may decrease resistance of the host against other
pathogens. Additionally, infection with N. ceranae alters aspects of both honey bee
behavior and physiology, decreases bee longevity and consequently reduces colony
survival (Botias et al., 2013, Goblirsch et al., 2013). Nosemosis have been reported as
a serious disease of all three castes of adult honey bees from worldwide (Chen et al.,
2009).
The only known effective substance for the control of nosemosis is the
antibiotic fumagillin (Moffet et al., 1969). However, due to the risk of antibiotic
residues in honey bee products many countries in most of Europe fumagillin is
prohibited (Martin, 2003). Fumagillin residues have been proven to have adverse
genotoxic effects that may increase the risk of cancer and chromosomal anomalies in
mammals (Stanimirovic, Stevanovic, Bajic, & Radovic, 2007). The reoccurrence of
the disease after treated with fumagillin also report as a problem as according to some
authors only the vegetative forms of the parasite are killed. The development of new
methods for the control of nosemosis with ecologically-friendly products is therefore
much hoped for by beekeepers throughout the world. To approach this issue we
decided to evaluate the effect of propolis extract from Trigona apicalis and
Apis mellifera from two different regions of Thailand, Chiang Mai and Chanthaburi
province on the development of N. ceranae, the longevity infectivity, infection ratio
and on hypopharyngeal gland protein content of honey bee.
79
ones when they were fed with ad libitum sucrose (Mayack & Naug, 2009). However,
in our studied the mortality of infected bees was still higher than uninfected bees
although fed with ad libitum sucrose. From their studied Mayack and Naug concluded
that the lower survival of bees infected with N. ceranae is mainly due to the energetic
stress (Mayack & Naug, 2009). Many probable causes of the shortened life span
observed in infected bees were argued by scientists. For examples, the effects of
N. ceranae on metabolism of infected bees by degenerating epithelial ventricular
cells, thus influencing hemolymph levels of fatty acids and vitellogenin, affecting
enzymes secretion and protein content of hypopharyngeal glands ( Alaux et al., 2011;
Chaimanee et al., 2012; Goblirsch et al., 2013; Suwannapong et al., 2010).
Additionally, N. ceranae infection inhibits immune system function in honey bees
(Antunez et al., 2009) which may decrease resistance of the host against other
pathogens. Collectively, infection with N. ceranae decreases bee longevity and
consequently reduces colony survival (Botias et al., 2013; Goblirsch et al., 2013;
Higes et al., 2009).
Its was interestingly, the mortality of N. ceranae infected honey bee was
signicantly reduced after treated with propolis extract. Moreover, higher
concentration of propolis extracted showed slightly higher survival of infected honey
bee. However, propolis extract from T. apicalis and A. mellifera from Chantaburi and
Chiang Mai at the same concentration were not showed different results on honey bee
survival rate. Due to these result, we concluded different species and regions were not
significant different effect on honey bee survival rate. The results showed that
propolis extract at the used concentrations, caused no toxic effects on the adult bees,
nor anti-feedant properties, as pollen mix intake was similar throughout the
experimental groups. All the compounds were therefore considered suitable for
treating bees in experiments against nosema disease, at the highest of the tested
concentrations. In the case of propolis extract treated groups, higher survival might be
related to the lower spore load and specic life prolonging antioxidant properties of
this substance.
The mean number of spores per individual bee from each group was
determine to evaluated the inhibited efficiency of propolis from each species and
regions on N. ceranae spore proliferation. No spores were detected in control groups
81
(CO and CP) that inoculated with sucrose solution. Counts of N. ceranae spores in
mid gut tissues contents varied significantly among propolis extract treatments and
compared to untreated infected bees. We noted high variability of spore counts within
treatments, which may be due to variation in natural resistance or possibly patrilineal
genetic differences. Our results indicated that exposure to propolis extract had effects
on spore production. Indeed, in comparison to infected honey bee not exposed to
propolis extract, the spore production increased by double time whereas the spore
production decreased with propolis exposure. However, from my observation the
mean number of spore from dead bees were not corresponds with honey bee mortality
in some case. These results then, do not explain the mortality increase observed in the
presence of spore load and the spore overproduction did not seem sufficient to explain
the enhancement of honey bee' mortality. So, I propose propolis extract might not
only inhibited spore proliferations but it also another ways to increase honey bee
survival. Antunez and her colleague (2009) reported, infection by N. ceranae will
suppresses immune genes expression and enhances host susceptibility to other
pathogenic infections and decreases bee longevity. In our opinion one of the probably
mechanism that propolils increase the survival rate of honey bee after N. ceranae
infected is by stimulate immune genes expression.
Gisder and his co-worker (2011) reported N. ceranae can be completed life
cycle within four days while, Higes and his co-worker claim the rst intracellular
spore germination starts minimally 3 days post infection (Higes et al., 2007). This
reported related with our studied, the counts of spores from dead bees on days 3 and 4
after inoculation showed a higher mean spore than initiate dosage. According to
Bailey (1959) a quantitative relationship exists between number of spores fed to a bee
and number of spores that develop, the time between spore ingestion and formation of
new spores varies with temperature (Lotmar, 1943). They were 6 days, (Lotmar,
1943), 5 days (Bailey, 1981), 4-6 days (Jacobs et al., 1978) and 2 days (Kellner, 1980)
at 30 C for the parasite to complete its development within the host cell. The current
study showed the infectivity or mean number of spore per bee was propolis extract
dose dependence, the highest infectivity was founded in untreated bees 0P and CE
group with was 3.511060.13 and 3.461060.22 spore per bee respectively. In
contrast, it was decreased which increasing of propolis concentrations, the lowest
82
mean spores per bee show in group of bees treated with 70 % propolis with was
2.021060.04 spores per bee . However, its seem to at the same concentration of
propolis extract from different species and regions were not showed significant
different on the infectivity. At a lower concentration of propolis extract 50 % (50P)
represented a little higher of infectivity when compared to 70 % propolis treated
groups, but however the result is not significant different between these two
concentration. Thus I recommend using 50 % propolis extract instead of 70 %
propolis to save cost.
Almost dead bees contained average spore at 1.5x106 spores per bee, so we
can assume that this is the lowest dosage that can make bee die. However, Malone
and Giacon had studied the mean spore in Nosema infected bees, and reported that
spore-loads may vary greatly, with no clear relationship to survival time (Malone &
Giacon, 1996). Lotmar began quantitative determinations of N. apis spore levels in
the honey bee using hemacytometer counts found that N. apis infections are fully
developed after about 10 days with about 44x106 spores in the ventriculus at 30 C
(Lotmar, 1943). In the same experiment the rectum contained about 240x106 spores
42 days post infection (p.i.). Bailey (1981) states that 35x106-50x106 spores in the
ventriculus represents a fully developed infection, while Mussen (1978) mention that
quantitative measurements of Nosema spores may reach 180x106 spores per bee,
probably referring to collective samples of whole bees. Moreover, Weiss (1984)
reported that 250x106 spores have been found in the ventriculus of the honeybee.
Maistrello and his colleague (2008) suggested that defecation by monitored bees was
influenced on the differences in spore counts, these may cause of un-corrected and not
precious data to estimate the virulence level of nosimosis in honey bee.
The histological examination on the healthy controls honey bee (CO and CP)
revealed no mature spores of N. ceranae throughout the experiment. Our histological
examination was contrast from the polymerase chain reaction (PCR) studied by Chen
and his co-worker (2009), they reported the detection of N. ceranae in many tissues
such as hypopharyngeal glands, salivary glands, Malpighian tubules, and fat bodies of
infected bees. While, from our studied we did not find any of mature spores in others
tissues except in the ventriculus of infected bees. The ventricular epithelium can be
classified into different cell types such as columnar, regenerative, endocrine and
83
goblet cells. The most abundant type of epithelial cells is columnar cells that are
arranged in one layer and settled on the basement membrane, with well-developed
microvilli at the free end that stained red-pink with PAS, the green oval shaped
nucleus located at basal of cells. Another type of epithelium which could be observed
is regenerative cells; their terminals did not reach the lumen. While the mid gut
endocrine cells of insects, generally are not simply recognized by light microscope.
The columnar is function in digestion, while regenerative cells serve to regenerate the
damaged functional cells. All sections obtained from the sampled uninfected control
bees were characterized by an outer muscular coat (circular inwards, longitudinal
outwards) and median basement membrane coated with a stratum of soaring
cylindrical epithelium cells with small regenerator cells between them, and rabdorium
with perithrophic membrane. In addition, the cytoplasm was filled with fine and dense
homogeneous inclusions with intact and regular cell boundaries these were in
agreement with (da Cruz-Landim & Cavalcante, 2003).
The N. ceranae actions were evidently noticed on the mid gut epithelial cells
of the infected honey bee untreated with propolis extract. The abnormality of their
nuclei is clearly observed, their cytoplasm are granulated, vacuolated and cell
membrane disrupted. The mid gut epithelial layer is disrupted and most cells have
exploded. The nuclei have disappeared or diminished in three cases, the cytoplasm
inclusion is scattered and the cell membranes are decayed completely. Some cells
were separated from the basement membrane. There are no distinct cell boundaries
and the cell membranes are completely decayed. Each epithelial cell of bees in the CE
and 0P groups showed a high number of mature spores distributed throughout the cell
cytoplasm, but particularly at the apical part of the cell. Mature spores also were
clearly observed at the bottom of the cells near the basement membrane. In addition, it
was observed that in the presence of numerous N. ceranae spores the intestinal
contents tended to be squeezed into the center of the intestinal lumen probably
impeding the germination of spores. The spores were ovoid and rather uniform in
shape and size and heterogeneously stained. Mature spores were surrounded by a wall
and immature spores were optically empty. In heavily infected cells, different
intracellular parasitic stages such as mature and immature spores were found in the
same cells.
84
defense. Pathogens like N. ceranae entering the host through the gut first need to
overcome this barrier. If the peritrophic membrane is damaged or breached, the
pathogens may be more likely to infect the mid gut cells and reproduce. The
peritrophic matrix is secreted at the anterior end of the mid gut near the proventricular
valve. One possibility is that Nosema spores infecting the epithelial cells inhibit the
secretory cells from secreting peritrophic membrane. Once secretion is stopped,
spores could pass freely throughout the mid gut lumen via countercurrent flow
(Tlak Gajger, Kozaric, Berta, Nejedli, & Petrince, 2011). The results of the general
histological examination showed that the lumen of propolis extract treated bees was
coated with a firm layer peritrophic membrane, while heavily infected bees from
untreated propolis groups, bees had a much looser and not clearly demarcated area of
peritrophic membrane. From our observation, after treated with propolis extract the
peritrophic matrix were fully formed; while propolis untreated groups showed total
degradation of the peritrophic matrix enabling the N. ceranae to directly attack the
epithelial cells. We propose that propolis extract might induce the production and
secretion of mucous from the epithelial layer and additionally peritrophic membrane
to form a firm and resilient envelope. This mechanism might be one of the ways that
propolis extract can inhibited spores proliferation and results in honey bee mortality
and infectivity decrease. The similar results also reported by Tlak Gajger and her
colleagues (2011), they studied the effects of Nozevit extraxt on N. ceranae. Nozevit
is a natural extract of a specific species of oak bark, derived through a patented
process, and has been recognised as a rich source of tannin phenols. Based on their
result they conclude that the herbal preparation Nozevit plays a dual role of ensuring
protection from new infections with Nosema sp. spores and also against normal
physiological processes (Tlak Gajger, Kozaric, Berta, Nejedli, & Petrince, 2011).
Numerous findings have shown the production of reactive oxygen species (ROS) is
one of the most immediate immune response of the gut to fight bacterial infection
both in mammals and insects. ROS, which are efficient antimicrobial molecules, are
generally derived from oxidation-reduction process. A Gene Ontology analysis to
explore which functional components were affected by N. ceranae, was performed by
Dussaubat and her friends in 2012. They found that genes involved in oxidation
reduction were significantly overrepresented in the gene set upregulated upon spore
86
infection. This increase of oxidation reduction in the gut epithelia of bees parasitized
by N. ceranae would therefore indicate an enhanced generation of ROS in response to
the infection and suggests that ROS production is a general gut immune response to
microorganism infection, including microsporidia. Since residual ROS can cause
inflammatory disease, a balance between synthesis and elimination of ROS via
antioxidants is necessary to protect the host gut. Therefore, the antioxidant system
may play an essential role during gut infection (Dussaubat et al., 2012). Propolis is a
natural product produced by the honeybee, contains amino acids, phenolic acids,
phenolic acid esters, flavonoids, cinnamic acid, terpenes and caffeic acid. It possesses
several biological activities such as immunostimulatory, antiviral and antibacterial.
One of the most important biological activities is anti-inflammatory and antioxidant
activity. The investigated antioxidant activity of a propolis extract deprived of caffeic
acid phenethyl ester (CAPE) showed exhibited a strong antioxidant activity, higher
than galangi (Russo, Longo, & Vanella, 2002). Therefore, we noted propolis extract
plays an important role in the antioxidant activity by free radical scavenging and
prevented inflammatory disease in honey bee. These reasons could be explained why
propolis treated groups showed a fewer of damage cells.
Gut cells are usually renewed via the multiplication and differentiation of
stem cells in the basal cell layer that, once differentiated, move toward the lumen. In
insects, this renewal of intestinal stem cells is controlled by the canonical Wnt
signaling pathway. Dussaubat and her colleague (2012) reported the proliferation of
N. ceranae are inhibited and downregulated the expression of gene in tissue
homeostasis and morphogenesis. In addition, four main genes (frizzled2 (GB12765),
groucho (GB13456), basket (GB16401) and armadillo (GB12463) from canonical
Wnt signaling also downstream by the parasite (Dussaubat et al., 2012). This
suggesting that N. ceranae development inhibited the self-renewal of intestinal cells
of the host and caused a degeneration of the gut epithelium. Interestingly, the effect of
N. ceranae on tissue morphogenesis and integrity at the molecular level was
confirmed at the histological level as in our study. The epithelial cells of infected
honey bee showed major signs of degeneration, which are linked to the
downregulation of biological process. However, in all of propolis extract treated
groups were showed a minor signs of degeneration in gut epithelium. This supported
87
our believed, we believed propolis not only cured infected honey bee by antioxidant
activity but also, have immunostimulatory activity. The immunostimulatory activity
of propolis has been reported by many of researchers. Possamai, Honorio-Frana,
Reinaque, Frana, and Souto (2013) suggest that propolis adsorbed onto PEG
microspheres has immunostimulatory effects on phagocytes in human blood. The
effects of cinnamic acid from propolis extract were evaluated on human monocytes by
assessing the expression of Toll-like receptors (TLRs), HLA-DR, and CD80.
Cytokine production (TNF- and IL-10) and the fungicidal activity of monocytes
were evaluated as well. The results suggest that cinnamic acid modulated antigen
receptors, cytokine production, and the fungicidal activity of human monocytes
depending on concentration (Conti, Bfalo, Golim, Bankova, & Sforcin, 2013).
The hypopharyngeal glands in the honeybee are a paired organ, located in
the head of the worker bee, in front of the brain between the compound eyes. The
ducts open into the sub-oral part of the hypopharynx. Each of the glands is composed
of numerous small oval bodies or acini, short parts attached to axial or terminal
secretory duct. Each acinus is determined by 10 to 15 individual cellular bodies and
each cell is connected to the axial duct via a thin duct. . They are also the largest
glands in the head cavity. Hypopharyngeal glands (HPG) produce and secrete the
components of royal jelly which is fed to the larvae, queen, and drones. Thus, the
secretory activity and development of the hypopharyngeal glands directly affect the
workers behavior and indirectly may affect the strength of the colony (Al-Ghamdi,
Al-Khaibari, & Omar, 2011). Many factors such as age, dietary, insecticides and
pathogen have been studied and reported as a negative effect on hypopharyngeal
gland development. The effect of worker age on the hypopharyngeal glands
development was examined by Huang and Otis (1989). They reported the
hypopharyngeal glands are well developed when bees are nursed and degenerate
when bees become foragers at normal condition which depends on the age of workers,
the colony conditions and the time of the year (Huang & Otis 1989). Also, pollen
consumption is positively correlated with gland development (Darvishzadeh,
Hosseininaveh, Nehzati, & Nozari, 2015). Histological studies on hypopharyngeal
gland by Al-Ghamdi and his friends in 2011, showed that there were some differences
between the glands of the two races at the maximum developmental stage (9 days).
88
honey bee are increase. The same trend was find even in controls and in N. ceranae
infected groups. The highest protein content were represent on day 6 follow by day 10
and the lowest protein were find on day 14 post infection. However, all propolis
treated groups still showed higher protein content than untreated groups. From my
observation, we note propolis extract were directly or indirectly engaged in protein
synthesis (or royal jelly secretion), are well developed in the hypopharyngeal glands.
This propolis extract treated in infected bees showed marked higher of
hypopharyngeal glands protein content compared to untreated groups at the same age.
N. ceranae not only decrease protein content and reduce the size of honey
bee hypopharyngeal gland. Moreover, it has been shown that degeneration of these
glands. Our histological study of hypopharyngeal glands showed, Those of diseased
bees suffered from regressive changes; the volume of the lobules of the glands
decreased progressively; the nuclei disintegrated; the chromatin coalesced; and the
cytoplasm and the size of the secretory globules were extremely reduced, leading to
malformation of the hypopharyngeal gland. Our result was supported by the report of
Wang and Moeller (1969), they said the morphology and secretory cycle of the
hypopharyngeal glands of diseased (nosemosis) and healthy worker honey bees
(Apis mellifera) were dissimilar. The hypopharyngeal glands of infected worker bees
included fewer ribosomes on the nuclear envelope, a short cisternal endoplasmic
reticulum, small Golgi complex, myelinlike lysosome bodies, smaller mitochondria,
fewer and smaller microvilli in the secretory mass of the limited outer layer of
intracellular ductules, and the collapse of intracellular ductules (Wang & Moeller,
1969). The hypopharyngeal glands of 10 day old bees severely infected with Nosema
showed the disappearance of the rough endoplasmic reticulum, Golgi complex,
mitochondria, and pronounced myelinlike whorls of lysosome bodies. This caused the
cytoplasm of the glands to disintegrate and vacuolate and to become completely or
partially unable to secrete royal jelly to feed larvae and/or queens. The intracellular
tracheoles in the hypopharyngeal glands of 10 day old Nosema-infected bees showed
malformation of structure in both longitudinal and transverse section (Wang
& Moeller, 1971). The study of hypopharyngeal glands by electron microscope in
healthy honey bee show numerous electron-dense secretion granules, while honey bee
infected by N. apis, these granules appeared to have increased in size and lost their
91
electron density, possessing a core area that consisted of numerous smaller granules,
and a slightly electron dense fringe area, which in some cases possessed a crystalline
structure (Liu, 1990). Liu (1990), studied the effects of fumagillin treatment on the
hypopharyngeal glands of infected honey bee, she find the secretion granules were
also large and slightly electron dense, but the granular content was homogenous.
Some of these granules were also partially crystallized. She suggests these
ultrastructural changes in the secretion granules of diseased and fumagillin treated
bees, is probably associated with a change in secretory activity of the glands. From
this data we also suppose propolis extract might have the similar effect on N. ceranae
infected bees hypopharyngeal glands.
Thus, novel and sustainable treatment strategies against N. ceranae are
urgently needed to protect honey bee. The control of nosemosis in honey bee colonies
also requires treatments with an acceptable anti-microsporidial activity, without side
effect on honey bee and minimizing toxic residues in wax and honey. We here present
the screening assay of propolis extract for the identification of candidate agents active
against N. ceranae infections. Our results demonstrate that propolis extract of
T. apicalis and A. mellifera collectd from Chantaburi and Chiang Mai with two
concentrations are effective inhibiting development of N. ceranae in honey bee,
A. cerana. From above studied data, propolis extracts not only decreasing parasitosis
intensity but also enhances the survival or longevity and protein content of honey bee.
The significant antiparasitic activity of propolis extracts, coupled with its palatability
and no toxicity noticed during the assay, makes its inclusion feasible in a compound
for nosemosis control. Our 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. However, different hypotheses could be postulated multiple constituents
of plant extracts may be responsible of the therapeutic activity, being also possible
synergic, additive, and antagonistic effects among the different compounds. Due to,
the result of transmission electron microscope studied, we propose one of the
mechanism that propolis extracts inhibited N. ceranae proliferation by disturbed the
structure of N. ceranae spore wall. This result suggests that probably the chemical
92
composition in propolis might have direct toxic to N. ceranae that was supported by
the appearance of deformalities or abnormal in structure of N. ceranae spores
resulting to interfering or inhibiting spore growth and development that might reduce
effect of host cell destruction. The abnormal in appearance of N. ceranae mature
spore were finding distributed in cytoplasm of host cells. N. ceranae were abnormal
in appearance which could be seen in bees treated with all concentrations of propolis
extract showed irregular in shaped in particularly at the anchoring disk and spore wall
that became wrinkle. A lot of empty mature spores were finding in untreated propolis
extract groups while; in propolis extract treated groups were showed a younger stage
of development. These results means in propolis untreated groups were became
mature spore faster than treated groups, and pass to auto infection stage with quickly
than propolis treated groups. This is the reason why in propolis treated groups were
represented a lower infection ratio. The antimicrobial activity of propolis extract was
previously reported against another fungus species, such as Candida parapsilosis,
C. tropicalis, C. albicans, and other species. The propolis extract showed excellent
performance regarding its antifungal activity (Oliveira, Shinobu, Longhini, Franco, &
Svidzinski, 2006). 10 samples of propolis collected in different regions of Lithuania
were investigated the chemical composition and antimicrobial activity; all samples of
propolis ethanolic extract were active against gram-positive, gram-negative bacteria
and fungi. The antimicrobial activity was higher against gram-positive than against
gram-negative bacteria (Majiene, Trumbeckaite, Pavilonis, Savickas, & Martirosyan,
2007). Many studies have shown that propolis has a number of biological roles,
including antioxidant and antimicrobial activity, both conferred mainly by substances
belonging to the group of phenolic compounds, especially flavonoids, which make
propolis an important object of study for the most diverse pharmaceutical
applications, such as anti-aging and anti-acne cosmetics (Possamai et al., 2013). The
composition of highly variable according to the plant species visited by the bees, the
insect species and plant species, substances metabolized and secreted by the bees and
other components that are incorporated during its production. Despite this variability,
in general all samples have different antimicrobial activity ( Fernndez et al., 2008).
Although, in our experiments we used propolis extracts from different species of bees
and different regions, the results were showed in similar trends and on different sings
93
between propolis extract on N. ceranae. However, from our observation we noted the
higher concentration of propolis extract showed a betterr inhibited effectiveness on
N. ceranae proliferation. The correlated study have been reported by Kujumgiev and
his colleagues (1999), he collected propolis samples from different geographic
origins and investigated for their antibacterial (against Staphylococcus aureus and
Escherichia coli), antifungal (against Candida albicans) and antiviral (against Avian
influenza virus) activities. The activities of all samples were similar in spite of the
differences in their chemical composition. In samples from the temperate zone,
flavonoids and esters of phenolic acids are known to be responsible for the above
mentioned activities of bee glue; tropical samples did not contain such substances but
showed similar activities. Obviously, in different samples, different substance
combinations are essential for the biological activity of the bee glue (Kujumgiev
et al., 1999). The information from our studied could potentially help beekeepers
formulate appropriate treatment regimens for their colonies to mitigate N. ceranae
problems.
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APPENDIX
123
4. Karnowsky fixative:
Paraformaldehyde 2 g.
Distilled water 25 ml.
Glutaraldehyde (25 %) 10 ml
Fixing buffer 50 ml.
7. Toludine blue
Sodium borate 1 g.
Distilled H2O 100 ml.
Toludine blue 1 g.
1. Dissolve sodium borate in distilled water.
2. Stir gently and then add toluidine blue.
3. Mix until solution becomes homogenous.
4. Filter with Whatmann No. 1.
PROCEDURE
1. Xylene, 2 changes
2. Absolute alcohol
3. Alconol 95 90 70 %
4. Rinse in distilled water.
5. Periodic acid solution for 5 min (oxidizer).
6. Rinse in distilled water.
7. Place in Schiffs Leuco-fuchsin for 15 min.
8. Place in running tap water for 10 min for pink color to develop.
9. Stain in light green counter stain for 30 seconds.
10. Alconol 70 90 95 %
11. Absolute alcohol
12. xylene, 2 changes
13. Mount in permount
HARRIS HEMATOXYLIN
Hematoxylin crystals 0.5 g.
Alcohol, 95 % 50 ml.
Ammonium or potassium alum 100 g.
Distilled water 1000 ml.
Mercuric oxide 2.5 g.
Dissolve the hematoxylin in the alcohol, the alum in the water by the aid of
heat. Mix the two solutions. Bring the mixture to a boil as rapidly as possible and then
126
remove from the heat and add the mercuric oxide. Reheat the solution until it becomes
a dark purple, about 1 minute, and promptly remove the container from the flame and
plunge it into a basin of cold water. The solution is ready to use when cool. Add 2-4
ml. of glacial acetic acid to 100 ml. of solution if desired.
PROCEDURE
1. Xylene, 2 changes
2. Absolute alcohol
3. Treat with preheated 0.5 % ninhydrin in absolute ethyl alcohol at 37 C for 16-
20 hours.
4. Wash in running tap water for five minutes.
5. Place sections in Schiffs reagent for 30 minutes.
6. Wash running tap water.
7. Counterstained with haematoxylin. Differentiate and wash in tap water.
8. Dehydrate in alcohol, clear in xylene, and mount in a synthetic resin medium.
Results: Amino-group-pinkish-red or magenta
Table 1 Spores load per bee (Infectivity) of A. cerana after inoculated with spore of
N. ceranae 80,000 spores per bee and untreated with propolis extract (0P).
Table 2 Spores load per bee (Infectivity) of A. cerana after inoculated with spore of
N. ceranae 80,000 spores per bee and untreated with propolis extract (CE).
Table 3 Spores load per bee (Infectivity) of A. cerana after inoculated with spore of
N. ceranae 80,000 spores per bee and treated with 50 % propolis extract of
T. apicalis from Chanthaburi (50P1).
Table 4 Spores load per bee (Infectivity) of A. cerana after inoculated with spore of
N. ceranae 80,000 spores per bee and treated with 50 % propolis extract of
T. apicalis from Chiang Mai (50P2).
Table 5 Spores load per bee (Infectivity) of A. cerana after inoculated with spore of
N. ceranae 80,000 spores per bee and treated with 50 % propolis extract of
A. mellifera from Chanthaburi (50P3).
Table 6 Spores load per bee (Infectivity) of A. cerana after inoculated with spore of
N. ceranae 80,000 spores per bee and treated with 50 % propolis extract of
A. mellifera from Chiang Mai (50P4).
Table 7 Spores load per bee (Infectivity) of A. cerana after inoculated with spore of
N. ceranae 80,000 spores per bee and treated with 70 % propolis extract of
T. apicalis from Chanthaburi (70P1).
Table 8 Spores load per bee (Infectivity) of A. cerana after inoculated with spore of
N. ceranae 80,000 spores per bee and treated with 70 % propolis extract of
T. apicalis from Chiang Mai (70P2).
Table 9 Spores load per bee (Infectivity) of A. cerana after inoculated with spore of
N. ceranae 80,000 spores per bee and treated with 70 % propolis extract of
A. mellifera from Chanthaburi (70P3).
Table 10 Spores load per bee (Infectivity) of A. cerana after inoculated with spore of
N. ceranae 80,000 spores per bee and treated with 70 % propolis extract of
A. mellifera from Chiang Mai (70P4).
BIOGRAPHY
Education
2003 - 2006 Bachelor of Science (B.Sc.), Faculty of Science,
Burapha University, Chonburi, Thailand
2007 - 2009 Master of Science (M.Sc.), Faculty of Science,
Burapha University, Chonburi, Thailand
2010 -1015 Doctor of Philosophy (Biological science)
(Ph.D.), Faculty of Science, Burapha University,
Chon Buri, Thailand