1. Healthcare

Explorations in impacts of fumagillin and tylosin treatments on the
honey bee (Apis mellifera L.) midgut microbiome in vivo
Ann Bernert and Ramesh Sagili, PhD. Oregon State University, Corvallis, OR
Materials and Methods
Purpose
The purpose of this research project if to identify if tylosin
and/or fumagillin decrease levels of symbiotic
Lactobacillus species in the honey bee midgut.
Image 1: Recollecting marked bees from
the mother colony. The economic value of
pollination by honey bees in the United
States is a $15 billion1
Image 2: Newly emerged bees were
painted and released into mother colony
to be inoculated with natural hive
microbes, painted bees were then
recaptured for in vivo experiment
Background Information
Honey bees are vital to modern agriculture for their economically
significant pollination services.1,2 The health of honey bee colonies is
essential for maintaining these important services. The honey bee
microbiome is a newly recognized and important aspect of colony
health.3,4,5
The honey bee microbiome develops appropriate immune
responses in the honey bee and helps outcompete pathogenic
microbes.3 Symbiotic honey bee microbes inhibit the growth of
pathogenic microbes and play an important part in making honey
and digesting pollen.6,7 It has been shown that Lactobacillus species
are some of these very important symbiotic microbes.2
Tylosin, a treatment for American Foulbrood disease, a bacterial
pathogen, and fumagillin for the control of Nosema, a
microsporidian pathogen are two commonly used chemicals to
combat honey bee diseases.8,9 This research investigates how these
chemicals affect the number of Lactobacillus species in the honey bee
midgut. Our hypothesis is that tylosin and fumagillin may
deplete symbiotic microorganisms in the honey bee midgut. By
understanding how these chemicals affect the symbiotic microbes in
the honey bee gut, it will be possible to create pathogen control
programs with minimal detrimental side effects.
Treatments: Concentration was per manufacturers recommended dosage
1) Control, 2) T,ylosin, 3) Fumagillin, 4) Tylosin and Fumagillin
In vitro experimentation
A well diffusion test using MRS agar, a Lactobacillus specific media, and a
solution of honey bee midgut microbes that was spread across the surface of
the media in a petri plate. 50 µl of prepared treatment solutions was placed
in the well. After 72 hours of incubation, the distance between the bacterial
field of growth and the chemical filled well was measured.
In vivo experimentation
Bees were inoculated with normal hive microflora and then randomly
assigned to one of the four treatment groups. Each treatment group was
replicated four times. Each sample day, 10 bees were randomly selected,
midguts were dissected and plated according to 100x dilution series
replicated twice. Bacterial Colony Formation Units were counted after 72
hours of incubation.
Ashrafun Nessa – For work coordination and all the guidance with caged bees
Ken Johnson & Todd Temple – For help with plate dilution series experiment
Ellen Toptizhofer – For the background on experimental design
Cameron Jack – For all the help working in the field with the colonies, collecting
frames, painting and recollecting bees
Many thanks to the undergraduate student worker bees- For the help with
sampling and data collection
Tylosin reduced Lactobacillus sp. growth both in vitro and in vivo
Fumagillin did not affect Lactobacillus sp. in vitro but did decrease
Lactobacillus Colony Formation Units in vivo suggesting additional
microbial interactions occurring in vivo
While these chemicals do decrease the quantity of symbiotic
bacteria in honey bee midguts in laboratory settings, they may not
deplete Lactobacillus sp. as greatly in a field setting
This data suggests treating hives with tylosin and fumagillin
should be done in consideration with the effect they have on
symbiotic microbes
Further research is needed to fully understand the interactions
between honey bee treatments and the hive microbiome
Results
In vitro Experiment
Tylosin significantly reduced bacteria from honey bee midguts and
fumagillin did not reduce growth. There was no synergistic affect when
tylosin and fumagillin were used together
Inhibition zone
Image 5: Symptom of Nosema infection- the
pathogen causes severe energetic distress8
Inhibition zone
Image 3: Tylosin, labelled “Tylan”, on left
and control plates, labelled “C”, on right
Image 4: Fumagillin plates, labelled
“Fum”, and fumagillin & tylosin plates,
labelled “F+T”, on the right
In vivo Experiment
 The average Colony Formation Units of all replications per treatment (n=
80 per treatment, 160 counts total) was used for graphs
100%
100
90%
90
80%
80
10
6J.
Evans, T. Armstrong (2006) Antagonistic interactions between honey bee symbionts and
implications for disease. BMC Ecology 6 (4)
0
Control
Tylosin
Fumagillin
Tylosin &
Fumagillin
Before treatment
0%
Control
Tylosin
5 days after treatment
Fumagillin Tylosin &
Fumagillin
12 days after treatment
Graph 1: Percent Reduction in Colony
Formation Units on Lactobacillus selective
media after treatment
Lautenbach, R. Seppelt, J. Liebscher, C. Dormann (2012) Spatial and Temporal Trends of
Global Pollination Benefit. PLoS ONE 7 (4) e35954. doi:10.1371/journal.pone.0035954
Anderson, T Sheehan, B. Eckholm, B, Mott, G. DeGrandi-Hoffman (2011) An emerging
paradigm of colony health: microbial balance of the honey bee and hive (Apis mellifera).
Insectes Sociaux
20
10%
2S.
5K.
30
20%
Sagili, D. Burgett (2011) Evaluating Honey Bee Colonies for Pollination: A Guide for
Commercial Growers and Beekeepers. A Pacific Northwest Extension Publication, PNW 623
Marttila, D. Rios, V. Walker-Sperling, G. Roeselers, I. Newton (2012) Characterization of
the Active Microbiotas Associated with Honey Bees Reveals Healthier and Broader
communities when Colonies are Genetically Diverse. PLoS ONE 7 (3) e32962
40
30%
1R.
4H.
50
50%
References
DeGrandi-Hoffman, B. Eckholm, K. Anderson (2012) Honey Bee Health: The potential
Role of Microbes. In: Sammataro, D. and Yoder, J., editors. Honey Bee Colony Health:
Challenges and Sustainable Solutions. Boca Raton, FL. CRC Press. p. 1-12.
60
60%
Image 6: Symptom of
American Foulbrood- a
spore-forming bacteria9
3G.
70
70%
40%
Acknowledgements
Conclusion
5 days after treatment
12 days after treatment
Graph 2: Comparison of the average
Colony Formation Units between
treatments at specified sampling times
7F.
Reynald, M. DeGiusti, A. M. Alippi (2004) Inhibition of the growth of Ascosphaera apis by
Bacillus and Paenibacillus strains isolated from honey. Revista Argentintina de Microbiologia (36)
52-55
8Z.
Huang (2012) Effects of Nosema on Honey Bee Behavior and Physiology. eXtension
http://www.extension.org/pages/60674/effects-of-nosema-on-honey-bee-behavior-andphysiology#.UzuZP_ldWSo
9A.
Flores (2007) Helping Beekeepers Beat American Foulbrood. USDA ARS
http://www.ars.usda.gov/is/ar/archive/jul07/bee0707.htm