この博士論文は内容の要約のみの公開（または一部非公開）になっています筑波大学 (University of Tsukuba)201

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Sympatric and Longitudinal Comparison of
Midgut/Pyloric  Bacterial Population in
European Honey Bee (Apis mellifera) and
Japanese Honey Bee (Apis cerana japonica)
著者 STOIC Marko
内容記述 この博士論文は内容の要約のみの公開（または一部
非公開）になっています
year 2017
その他のタイトル セイヨウミツバチとニホンミツバチの中腸腸内細菌
叢の同所的縦断的比較
学位授与大学 筑波大学 (University of Tsukuba)
学位授与年度 2017
報告番号 12102甲第8323号
URL http://hdl.handle.net/2241/00150074
 
 
 
Sympatric and Longitudinal Comparison of Midgut/Pyloric 
Bacterial Population in European Honey Bee (Apis mellifera) 
and Japanese Honey Bee (Apis cerana japonica) 
 
 
 
 
 
 
 
 
 
 
 
 
April 2017 
 
 
 
 
Marko STOIC 
 
 
  
Sympatric and Longitudinal Comparison of Midgut/Pyloric 
Bacterial Population in European Honey Bee (Apis mellifera) 
and Japanese Honey Bee (Apis cerana japonica) 
 
 
 
 
 
 
 
A Dissertation Submitted to  
the Graduate School of Life and Environmental Sciences,  
the University of Tsukuba  
in Partial Fulfilment of the Requirements  
for the Degree of Doctor of Philosophy in Agricultural Science  
(Doctoral Program in Biosphere Resource Science and Technology)  
 
 
 
 
Marko STOIC 
 
1 
 
Sympatric and Longitudinal Comparison of Midgut/Pyloric  
Bacterial Population in European Honey Bee (Apis mellifera)  
and Japanese Honey Bee (Apis cerana japonica) 
(セイヨウミツバチとニホンミツバチの中腸腸内細菌叢の同所的縦断的比較) 
STOIC Marko 
Apis mellifera honey bees are the most valuable pollinators of fruits and crops, giving a 
significant contribution to world’s agriculture. Nonetheless, major increases in food production, 
alongside bacterial, viral, parasitic, and fungal diseases, have started to inflict weakening and 
losses in honey bee populations. To resist environmental stressors, honey bees developed 
mechanical, physiological, and immune defences. Alongside those barriers, antibiotics are still 
used against bacterial pathogens. Scarce number of studies was performed on honey bee’s 
ability to use food sources as a mean of protection against various pathogens. Further, 
particular bacterial isolates, which are a part of honey bee’s gut microbial community, have 
exhibited antibacterial activity through certain experiments. Due to those properties, gut bacteria 
recently gained large interest in honey bee research. Nowadays, gut bacteria are mainly 
surveyed in European honey bee. There is still no research of a possible sympatric effect of two 
bee species, kept in the same apiary, on their microbial population. Therefore, amongst 
previously mentioned disease threats and assorted defence mechanisms, the objective of this 
research was explained by focusing on antibacterial potency of chestnut honey as a food 
source, and defining the status of microbial population in two bee species, in the beginning and 
in the end of foraging season. 
Four chestnut honey samples from Japan and four from Croatia, along with two non-
chestnut honey samples, were tested for their antibacterial property against Paenibacillus larvae 
2 
 
bacteria, the causative agent of a highly contagious American foulborood disease (AFB). 
Samples which possessed the highest amounts of chestnut pollen were characterised as a high 
purity chestnut honey, and those samples expressed strong, significant inhibitory properties. 
Within the chestnut group, certain honey samples showed noticeably better activity than the 
others. After comparing analysed measurements of inhibition zones caused by Japanese and 
Croatian honey samples, I concluded that the Croatian group inhibited the growth of P. larvae 
more effectively than the Japanese group. The results showed that antimicrobial potency of raw 
chestnut honey depended on its purity. Various levels of antibacterial activity were indentified 
and varied from samples obtained from different locations. By using artificial honey, a substance 
made with the same ratio of sugars as in real honey, it was shown that sugar content did not 
influence the sizes of inhibition zones. Additionally, non-chestnut honey samples were also less 
effective against bacterial growth. Although pH level did influence honey’s activity, the purity, 
based on the amount of chestnut pollen, was a significantly stronger factor in suppressing 
bacterial growth. Results provided in this experiment could benefit as a first step in finding an 
alternative approach in fighting highly pathogenic diseases like AFB. 
To establish the status of symbiotic bacteria between two sympatric honey bee species, 
samples of worker bees from April and September were analyzed by using metagenomic 
analysis of 16S-rRNA gene. Only apiaries which contained European honey bee (A. mellifera) 
and Japanese honey bee (A. cerana japonica) were used for sampling. The part of digestive 
system used for this analysis was midgut and pyloric section, due to their highly dynamic 
environment. Metagenomic analysis of V1 and V3 variable regions of 16S rRNA gene analysis 
identified that in the group of non-dominant bacterial species the most abundant were 
Pasteurella pneumotropica with 31.4%, followed by Actinobacillus pleuropneumoniae (25.2%), 
Lactobacillus apis (8.4%) and Chromobacterium subtsugae (6%)  With the usage of BLAST 
(Basic Local Alignment Search Tool) to obtain more thorough results of particular bacterial 
3 
 
families showed that the most dominant bacterial family was Pasteurelaceae (34%), with its 
most abundant species Gilliamella apicola (49%), which remained dominant in both bee species 
and depended on location. Snodgrasella alvi was the third most abundant bacteria (4%), a 
member of third most abundant family (Neisseriaceae). Its abundance as a core gut member 
was low in both samplings. Flavobacteriaceae were the second most abundant family (12%) 
with its member Apibacter adventoris (31%), mostly present in A. cerana japonica. Similar, but 
opposite situation was noticed with bacteria Frischella perrara (12%), a member of 
Pasteurellaceae family, which occurred mostly in A. mellifera. Lactobacillus family was present 
in less than 3.5% and depended only on the factor of location. All bacteria were equally 
abundant in the beginning and in the end of foraging season. Results of this bacterial research 
show that even though midgut and pyloric sections of honey bee digestive system present 
somewhat dynamic medium, particular specificities may be detected. Presence of P. 
pneumotropica, A. pleuropneumoniae and C. subtsuage was documented as a result of their 
frequent occurrence. They were not the most dominant species, yet the most detectible, non-
core microbiota. Fluctuations of gut microbiota are to be expected regarding location since bees 
are subjected to various living conditions. Establishing certain patterns in their change may 
prove to be beneficial in developing certain approaches in disease prevention and immunity 
enhancement. 
The importance of environment regarding honey bee food provision and its effect on 
microbial population has already been discussed. It remains to be established if foraging 
location could provide bees with food sources, like chestnut flowers in spring, which could help 
them inhibit the growth of P. larvae. The importance of pollen source and diversity still remains 
an important factor. Although the precise reason for the insignificance of season with gut 
microbiota still remains unclear, a possibility of constant temperature between two sample 
periods remains as a feasible explanation. The results of midgut and pyloric analysis showed 
4 
 
that G. apicola, bacteria connected with pollen digestion, remained the most abundant species 
from two sampling in both bee species. Same apiaries have expressed different trends in 
microbial population. Results from this survey were closer to previous results of other 
researchers, who found no significant shifts in observed bacteria. In this survey, only A. 
adventoris and F. perrara depended on their sympatric bee hosts as influential factors. 
It was concluded that honey properties and bacterial abundance of different members 
may diverge in various samples due to location and traits in species. Chestnut honey was 
established effective in inhibiting the growth of a pathogen closely connected to honey bee 
development. Nevertheless, further actions should be continued in elucidating the exact factors, 
which could contribute in establishing chestnut honey in bee diet as a valid antibacterial product. 
This dissertation presented location as a factor that can condition changes. Various locations 
may contain various food sources, which can consequently influence symbiotic bacteria. Further 
studies should definitely confirm what particular aspects of a location contribute to the 
abundance of symbionts and what steps can be taken in improving the honey bee’s food 
sources. 
 


セイヨウミツバチとニホンミツバチの中腸腸内細菌叢の同所的縦断的比較

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セイヨウミツバチとニホンミツバチの中腸腸内細菌叢の同所的縦断的比較

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