Comparison of freeze tolerance and phosphoglucose isomerase (PGI) within and among Habrotrocha rosa metapopulations

Background/Question/Methods

The bdelloid rotifer Habrotrocha rosa is a component of the inquiline community that thrives within the water-retaining pitcher-shaped leaves of Sarracenia purpurea.  This carnivorous plant ranges widely throughout the United States and Canada and is the only member of the genus that inhabits cold temperate climates.  We were interested in examining freeze tolerance in two rotifer life history types (fast vs. slow population growth rates) within a bog in southeastern Wisconsin.  Phosphoglucose isomerase (PGI), a dimeric enzyme that catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate, plays a key role in glucose metabolism and the resupply of ATP which could influence reproductive rates as well as freeze tolerance.  We hypothesized that variation in H. rosa life history is correlated with freeze tolerance and PGI isozymes.  Our sampling scheme was designed to provide samples across time as well as within habitats, between habitats and among plant locations on the bog.  Three rotifers were randomly selected from each pitcher sample, and each one became the foundress of a clone representing one H. rosa genotype that was present in the pitcher on the date of collection.  We examined PGI isozymes by electrophoresis and evaluated freeze tolerance at -20^oC and -80^oC.

Results/Conclusions

While no significant differences in recovery rates were found between fast and slow growers frozen at -20^oC (p > 0.05), there was a statistically significant difference at -80^oC for both time (p = 0.01) and life history (p = 0.006).  All of the H. rosa clones exhibited identical heterozygous genotypes for phosphoglucose isomerase.  There was slight variation in the intensity of the protein bands which we believe to be the result of differences in sample density.  While there is a statistically significant difference in freeze tolerance at -80^oC we do not feel that this is biologically relevant.  Our results for -20^oC contrast with those of Birkey et al. who were unable to achieve rotifer recovery after freezing at this temperature.  The difference most likely lies with our method of applying a gradual reduction in temperature prior to freezing which mimics the natural seasonal decrease in temperature and allows for the production of cryoprotectants.  Based on these results, we conclude that differences in population growth rates are not correlated with variations in freeze tolerance or PGI isozymes.  Further tests are scheduled to look for differences in other key metabolic enzymes which could account for life history differences.