Use of two-dimensional gel electrophoresis proteome reference maps of dinoflagellates for species recognition of causative agents of harmful algal blooms.

The sample preparation procedures established for Prorocentrum triestinum were adapted to cover both thecate and athecate dinoflagellates. Further, whether trichloroacetic acid (TCA) precipitation can be used to fix and preserve the harmful or nuisance species from local waters that they infest was tested. Optimized technical procedures developed were used to generate proteome reference maps for eight other local causative species of harmful algal blooms (HABs): Prorocentrum micans, Prorocentrum minimum, Prorocentrum sigmoides, Prorocentrum dentatum, Scrippsiella trochoidea, Karenia longicanalis, Karenia digitata and Karenia mikimotoi; together with one American species Karenia brevis (Florida, USA). These proteome maps were used to test their ability for species recognition in a mixed culture of dinoflagellates and whether such investigations will provide a comparative view at a global level. Comparisons of proteome profiles were made (i). between closely related species within the same family; (ii). between distantly related species belonging to different types, i.e., gymnodinioids, prorocentroids or peridinioids, or (iii). between different groups, i.e., thecate (armored) dinoflagellate cells against athecate (naked or unarmored) dinoflagellate cells. Species-specific two-dimensional electrophoresis (2-DE) protein profiles were observed in all ten species and it was possible to distinguish between even closely related species within the same family. To demonstrate the extent of reproducibility and usefulness of these 2-DE reference maps, 2-DE has been used to analyze three geographically distinct isolates of Prorocentrum dentatum, and to distinguish species composition in a mixed culture. Application of 2-D PAGE analysis to differentiate between taxonomically confused strains of a single species could be a powerful taxonomic tool.