Comments on Microalgae

Compiled by Robert Andersen

1. What is the best way to "census" marine phytoplankton?

The consensus is that there is no "one" or "best" method because the different microalgae require different methods. By analogy, one does not have one method for censusing invertebrates because of their diversity. Plankton nets are applicable only for large, tough species. Whole water samples collect all species, but post-sampling treatment varies. Chemical fixation can preserve some but not all species for later identification. Small and morphologically indistinct species require an alternative to preservation. Flow cytometry will be inadequate for species identification because of its low discrimination power, however, flow cytometry is perfect for counting the numbers of cells belonging to broad taxonomic groups (e.g., cyanobacteria, picoeukaryotes and cryptophytes). Flow cytometry is excellent for counting individuals in larger taxonomic groups (e.g., pico-and coccoid phytoplankton). This provides good estimates of cell numbers but species identifications are very limited or impossible.

Molecular approaches such as environmental PCR and DGGE will provide extensive information about diversity, but the sequences may be "orphans" (i.e., if the species has not been sequenced for the gene, the sequence will not be tied to a known species). There are more SSU rRNA sequences than for any other gene. DNA amplification from single cells of Lugol's preserved samples has been used with success. Fluorescent in situ hybridization with oligonucleotide probes can now be applied routinely for specific groups (e.g. Not, F., Simon, N., Biegala, I. C. & Vaulot, D. 2002. Application of fluorescent in situ hybridization coupled with tyramide signal amplification (FISH-TSA) to assess eukaryotic picoplankton composition. Aquat. Microb. Ecol. 28:157-166.). This technique can be targeted to orders, families, genera and species, but requires sequence data for the taxon (i.e., if the species has not been sequenced, then no probe can be constructed). Another technique very suitable to automation is quantitative PCR (see Suzuki, M. T., Preston, C. M., Chavez, F. P. & DeLong, E. F. 2001. Quantitative mapping of bacterioplankton populations in seawater: field tests across an upwelling plume in Monterey Bay. Aquat. Microb. Ecol. 24:117-127.).

Electron microscopy is useful for scaly organisms (e.g., coccolithophores) and diatoms. Properly preserved samples can be examined later in the laboratory.

Cultures of microalgae are perhaps the most useful means for identifying organisms, but this approach is hampered in several ways. It requires substantial work to isolate single cells from samples, although flow cytometry sorting procedures would likely result in more isolates than those established by traditional manual isolation using a micropipette or agar plating. Some species may prove difficult or impossible to culture. Culturing, as a basis for the census, would require extensive work and culture collections may not be able to maintain all the isolates.

Cryopreservation in the field should be a simple and easy process, but the survivability of cells upon thawing in the laboratory will vary. Prokaryotes, picoplankters and small diatoms are likely to cryopreserve rather well, but larger cells (e.g., large diatoms and dinoflagellates) and certain taxonomic groups (e.g., cryptophytes) will probably not survive. It should be possible to obtain DNA sequences from cells whether or not they live and grow after thawing.

Census of benthic microalgae generally follows the methods outlined above for phytoplankton.

If limited to one approach, a molecular sequencing approach is probably the best choice. It will result in numerous "orphan" sequences, but it will provide the most data on diversity per dollar expended.

2. Can these methods be carried out by parataxonomists (= technicians)?

Technicians can carry out sampling after a short training period. DNA sequencing and other molecular techniques can be carried out by technicians - even robots. Species identifications requiring microscopy are more specialized, and these may require a knowledgeable taxonomist for accurate identifications. Flow cytometry sorting or analysis requires a skilled technician.

3. What are the traditional methods for preserving and archiving phytoplankter specimens?

There is no "one best" traditional method for preserving and archiving specimens. Liquid preservation with glutaraldehyde and Lugol's iodine fixed cells can be used when the cells have morphologically distinct features. However, the liquid samples are not likely to be useful as long term preserved samples. Glutaraldehyde fixed cells can also be thin-embedded on microscope slides and examined later. This provides a permanent record but involves some toxic chemicals. Filtered samples can be used to examine scaly species with SEM, but the long term preservation of filters in a museum is not possible. Larger diatoms can cleaned and mounted with a hyrax-type mounting medium, and these are routinely maintained for long periods in museums.

Digital photography provides an alternative means for "preserving" the microalgal image in cases where the actual cell is not amiable to long term preservation.

Cryopreserved samples could be maintained indefinitely, but they can be analyzed only after thawing.

4. What culture methods should be used?

There is no one best method for isolating algae into culture, and some species are much easier to culture than others. A portion of the sample should be enriched with dilute culture medium (media) in the field, and a portion of the sample should be returned to the laboratory for isolation. A high speed sorting flow cytometer (e.g., FACSAria) can be used at sea or in the laboratory. Cryopreserved samples can be returned to the laboratory and upon thawing, live cells can be isolated into culture.

5. What types of forward-looking protocols can be applied (e.g., automated technology)?

DNA microarrays have potential, but they are limited to taxa whose sequences are known. In situ flow cytometry may be a forthcoming technology (Olson, R. J., Shalapyonok, A. & Sosik, H. M. 2003. An automated submersible flow cytometer for analyzing pico- and nanophytoplankton: FlowCytobot. Deep-Sea Res. I 50:301-315.). Similarly, in situ FlowCam can provide continuous monitoring. FlowCam provides a digital image of the cell as it passes through the chamber, and these images can be sent to anywhere in the world via satellites. The most promising techniques, however, are probably molecular techniques that can take advantage of high-throughput sequencing. Initially, these will generate a large number of orphan sequences, but over time these can be tied to specific species.

6. What are the major databasing and website projects? Are they linked to OBIS, the website used by the Census of Marine Life?

The culture collections often provide some of the best data on microalgae (including links to GenBank), but the data are not usually entered in a manner that is beneficial for community ecology. A few collections have global distribution maps that probably can be linked to IBIS, but the collection information (temperature, depth, etc.) may prove difficult to extract. Also, algal culture collections usually include information about abiotic factors affecting growth, but they rarely if ever have information about top-down ecology (e.g., predation).

AlgaeBase and the Index Nominum Algarum, together with the more broadly-based Index Nominum Genericorum (Plantarum) () provide valuable nomenclatural information - but the two algal sites are incomplete and under resourced. Phylogenetic navigators, such as the Tree of Life Web Project have few algae. Micro*scope provides a generic taxonomic scheme, photographs, and links to GenBank. culture collections, etc. TreeBase, the checklist of phytoplankton in the Skagerrat-Kattegat and the census of freshwater algae in Australia are other web sites containing algal information.

A major effort is needed to provide the database structure that Census of Marine Life (OBIS) require. Phycologically-oriented databasers could get together with persons working on database issues in other biodiversity-related activities (e.g. research funded by NSF Biodiversity Surveys and Inventories, Tree of Life, Knowledge and Distributed Intelligence, PEET, Microbial Observatories) and hammer out a common strategy.