Itasca Microbial Observatory Educational Activities
One of the important aspects of any National Science Foundation funded research is the transfer of information and tools to a broad audience. This audience could include scientists who study different types or organisms or who may not even be biologists. On the other hand, the broader audience could include people who are not even scientists; that is, the general public.
Our research with the Itasca Microbial Observatory has resulted in the detection of many previously undescribed organisms; "new species." The discovery of new species is something that the general public can often get excited about. Although people may not recognize algal diversity as very interesting, the discovery of new species of any kind is almost always of interest. An important result from our work is that there are so many potential new species of algae that just about anybody with minimal training in biology can actually participate in the discovery of new species. We have developed this idea into a class project, suitable for upper-level undergraduate students or graduate students, and implemented the project in Botany 471/671, Phycology at NDSU. Karen Fawley taught this class during Fall semester, 2004, with 6 undergraduate students from a wide variety of backgrounds and interests including wildlife and fisheries management, biology education, and general biology.
The class activities included sampling the Red River of the North in Fargo, ND and performing several simple water chemical analyses on the samples. Each student performed algal isolations using our standard spread-plate isolation system and characterized several isolates by light microscopy. From these isolates, one isolate per student was selected for further characterization. The selected isolates were grown in liquid medium and analyzed for photosynthetic pigments by HPLC (high-performance liquid chromatography). DNA was extracted from each isolate using our standard procedure. Based on preliminary identifications from light microscopy, genes or gene regions appropriate for identification were selected. These loci were then amplified using PCR. The PCR products were then sequenced using appropriate primers, with sequencing performed by a commercial service (Northwoods DNA). Students performed all of the DNA isolations, PCR setup and analysis, and sequencing setup. DNA sequences were then evaluated using the Staden software package and compiled sequences were used for BLAST searches to identify the most similar sequence(s) in GenBank as a method of identification.
Detailed methods for each step of the exercise are available. If you have any questions or would like to try this exercise in your own class, send an e-mail to Karen Fawley.
All of the students produced usable results at every level of the exercise, except that one culture turned out to actually contain two different organisms. Here are some interesting results from the five successful characterizations.
1) Two students characterized isolates from the Desmodesmus/Scenedesmus lineage of the Chlorophyceae. The HPLC of photosynthetic pigments revealed one interesting result: some members of this lineage possess the xanthophyll loroxanthin and others do not. The only previous analysis of photosynthetic pigments from this lineage had indicated the presence of loroxanthin. From this result, it would be interesting to initiate a study of the distribution of loroxanthin in this group of organisms, which may prove to be an important biochemical character differentiating clades within this lineage. Both students produced ITS1, 5.8S rDNA, ITS2 sequences. One of the isolates' sequence matches perfectly with a Desmodesmus isolate from Itasca, which is of great interest to us because it indicates that this organism can be found in very different conditions (a river versus a lake). On the other hand, the sequence from the second isolate is so different from any other Scenedesmus or Desmodesmus sequence that it is difficult if not impossible to align. This organism may represent a new lineage within this group.
2) One student characterized an isolate identified by light microscopy as a Koliella sp. The rbcL sequence of this isolate was produced and analysis of this sequence clearly placed it in the Trebouxiophyceae, which is appropriate for Koliella. Unfortunately, no other rbcL sequences are available for Koliella, so this technique could not be used to determine if the isolate was a previously characterized species. However, very few isolates of Koliella are available in culture, so this isolate may be important in subsequent sequencing and taxonomic studies of this genus.
3) One student characterized a tiny coccoid green alga identified as Choricystis sp. Analysis of the rbcL sequence of this isolate showed that it was identical to the rbcL sequence of a Choricystis isolate from a lake in the Arrowwood National Wildlife Refuge, ND. This result is also interesting because our results on Choricystis indicate a great deal of diversity at the rbcL level, with little overlap among the communities found in Arrowwood and Itasca. The water chemistry and local geology of the Red River in Fargo and the James River in Arrowwood are very similar and it therefore is not surprising that the same "species" of Choricystis is present in both river systems. The rbcL sequence of this isolate is available from GenBank, with accession number AY822046.
4) The final student characterized a diatom identified as Nitzschia sp. The 18S rDNA sequence from this organism was generated and analyzed. This sequence turned out to be remarkably similar to the 18S rDNA sequence of the diatom "plastid" of the dinoflagellate Peridinium balticum. In fact, the two sequences differed by only 3 substitutions! This result is very interesting because it suggests that the tertiary endosymbiosis in P. balticum may have occurred relatively recently, and also raises the possibility that similar tertiary endosymbiotic events may have occurred multiple times in dinoflagellates.
Although only one sequence from this project has been submitted to GenBank so far, additional submissions will take place in the future. In addition, we expect to submit the unialgal isolates to a major culture collection once we have evaluated them further.
These results show that very interesting discoveries can be made with relative ease using these methods. As a result, this exercise is more than just a lab exercise or demonstration of techniques, the results are subject to interpretation and are potentially exciting and important.