Project 1
Biochemical Analysis of Kairomones and
Developmental Biology of Inducible Defenses in Daphnia
(PIs: B.
De Stasio, J. Lokensgard, N. Wall)
Work by
others has suggested that the freshwater waterflea Daphnia is affected by kairomones from several sources. In
particular, it has been shown that a substance released by larvae of the
phantom midge fly Chaoborus affects
the development of Daphnia, and that
this material probably consists primarily of a water-soluble compound whose
molecular mass is under 500 D. This project has three main goals:
1)
Establish
a laboratory population of Daphnia
minnehaha and develop a system for producing induced animals so that
bioassay tests of unknown substances can be performed
2)
Identification
of kairomone compounds by concentration, derivatization, and identification
using GC-MS (gas chromatography combined with mass spectrometry)
3)
Determination
of the cellular and molecular basis of this morphological induction using a
detailed comparison of the histology of kairomone treated embryos to that of
control embryos.
Summary of Research Results
The initial goal of the summer project was to find local
populations of Daphnia minnehaha that
could provide a source of animals for our laboratory cultures. Laura Guili took
the lead on this aspect of the project, sampling eight potential
locations. Daphnia minnehaha were found in two of these locations. Individuals from one of these locations (a
pond off of Highway M in Outagamie County, WI) were used to establish multiple
cultures in the laboratory as the experimental stocks. All three students (Michael Bolt, Laura Guili
and Farzin Haque) collected samples from Bullhead Lake, Manitowoc County, WI to
obtain larvae of the phantom midge Chaoborus. These animals were fed small prey in the
laboratory to cause them to produce the kairomone that induces changes in Daphnia morphology. In the laboratory, Laura Guili perfected
culturing protocols and experimental methods for the developmental bioassay analysis
of kairomones. Her laboratory cultures
provided animals with a range of morphologies from uninduced to four neckteeth
per individual (see Lawrence University Merck/AAAS website at
www.lawrence.edu/dept/biology/). This
part of the project required all of the Merck Scholars to collaborate and
interact on a nearly daily basis with each other and the faculty mentors to
discuss needs and discoveries.
Farzin Haque first prepared a medium based on nanopure
water in which the Chaoborus could be
maintained and fed. He ran eight samples, with a wide range of larval
population densities, filtering and extracting (SPE) the water in each case.
Gradient elution (methanol/water) of the SPE columns provided nine fractions,
which were concentrated and silylated using trimethylsilyl cyanide under
conditions that should convert any hydroxylic functional groups or amines to
trimethylsilyl esters, ethers or amines. The silylated extracts were subjected
to gc-ms analysis, while corresponding un-silylated samples were used for
developmental bioassays. The last
fraction eluted from the SPE columns was highly active, which may mean that
additional active material was actually left on those columns. That will be
explored as the project continues into the summer of 2003. The gas chromatogram of the silylated sample
most closely corresponding to the bioactive fraction contains a large number of
peaks, of which the most promising have been selected by selective ion
monitoring. The mass spectra of those peaks are being analyzed, and will
provide the starting point for the next stage of the project.
We performed a histological analysis of the tissues of
uninduced Daphnia over the summer,
and are currently finishing the histological analysis of the tissues of induced
Daphnia. Michael Bolt worked for much of the summer
determining the best combination of tissue fixation, processing, and staining
techniques to get good quality histological samples. He has archived these sections as digital images
and labeled the tissues. He is currently
documenting the developmental timing of neckteeth expression in both uninduced
and induced daphnids. This is a
necessary piece of information for molecular analysis of altered gene
expression. Our next step will be to
produce a large stock of kairomone for future induction of Daphnia and for the
generation of cDNA pools from induced organisms so that we can begin our
molecular analysis of the induction process.