Discrimination between domoic acid producing and non toxic forms of the diatom Pseudo-nitzschia pungens using immunofluorescence

Separate polyclonal antibodies were developed against cell surface antigens of the 2 forms of the pennate diatom Pseudonitzschia (previously known as Nitzschia) pungens, i.e. forma multiseries (the domoic-acid-producing form) and forma pungens (the nontoxic form). Positive antigenic reactions were visualized with epifluorescence microscopy, using a fluorescein isothiocyanate (FITC) indirect immunofluorescence assay. The assay successfully distinguished 31 clones of f. multiseries from the 17 clones of f. pungens tested, with no cross reactions of the antisera between the 2 forms. The antisera were active against P. pungens cells isolated from Prince Edward Island and Nova Scotia, Canada, and Massachusetts, Rhode Island, Texas and Washington, USA. Of the 27 other clones tested from the genera Pseudonitzschia and Nitzschia, 2 other domoic-acid-producing species (P. australis and P. pseudodelicatissima) and 2 nontoxic species (P. subcurvata and P. fraudulenta) showed a slight positive response to the antisera, as did the domoic-acid-producing pennate diatom Amphora coffaeiformis. These reactions are not great enough to cause concern about misidentification, but pose questions about phylogenic relationships. Other representatives from the class Bacillariophyceae and from 8 other major classes of phytoplankton did not cross react with the antisera. Excellent labelling was obtained with live cells and those frozen at -60-degrees-C, or preserved in 2% glutaraldehyde-paraformaldehyde, 2% borate-buffered formalin or 2% paraformaldehyde. Immunofluorescence shows great promise as a technique to distinguish between the 2 forms of P. pungens for research and monitoring purposes.