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Development and analysis of primary cultures from the midgut of the honey bee, Apis mellifera

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Laura Evins Willard (Creator)
Institution
The University of North Carolina at Greensboro (UNCG )
Web Site: http://library.uncg.edu/
Advisor
Olav Rueppell

Abstract: The European honey bee, Apis mellifera, is an important research species of tremendous agricultural and economic importance. However, relatively few in vitro techniques are described for investigating honey bee physiology at the cellular or molecular level. This project describes a new technique for developing primary cell lines from the midgut of adult honey bees and characterizes the impact of donor age on the longevity, differentiation, and proliferation of intestinal stem cells within these cultures. Additionally, this study characterizes the effects of 20-hydroxyecdysone on intestinal stem cell proliferation and differentiation in vitro. Suspension cultures of midgut cells obtained through non-enzymatic dissociation were cultured in WH2 media with 1 µg/mL penicillin/streptomycin and 0.25 µg/mL Amphotericin B survived for up to 15 days. Age of donor had no significant effect on the longevity of cultures or rate of cell differentiation. However, the number of viable intestinal stem cells was significantly different between cultures from pre-eclosion pharate adults and cultures from workers. A 5-bromo-2'-deoxyuridine (BrdU) assay found very low levels of proliferation in cultures from all ages with the exception of foragers. Addition of 20-hydroxyecdysone did not affect the either longevity of the culture or the rate of cell differentiation or proliferation. These results suggest that the behavior of stem cells in culture is impacted by the age of the donor while differentiation is independent of age. Further, this work supports the idea that intestinal stem cells lose their proliferative capacity with age. The lack of effect of 20-hydroxyecdysone on measured parameters may indicate a physiological difference in the midguts of Apis mellifera and lepidopteran species and warrants further investigation. These cultures provide a starting point for future use as a model for honey bee midgut molecular and cellular physiology as well as in vitro studies of honey bee intestinal health.

Additional Information

Publication
Thesis
Language: English
Date: 2012
Keywords
Honey Bee, Apis mellifera, Physiology
Subjects
Honeybee $x Physiology
Honeybee $x Genetics
Honeybee $x Molecular aspects