Morphotaxonomy, genetic affinities and ecology of Australian and Antarctic populations of the potentially fish killing, heterotrophic dinoflagellates Cryptoperidiniopsis brodyi and Pfiesteria piscicida

The heterotrophic dinoflagellate Cryptoperidiniopsis brodyi and closely related species were investigated from Australia-wide marine environments with regard to their morphology, phylogenetic relationships, interactions with shellfish larvae, and natural abundance. Nine isolates of C. brodyi and two isolates of Pfiesteria piscicida were collected and cultured from Australia and ballast water originating from Indonesia. Scanning electron microscopy (SEM) and molecular sequence analyses of SSU, LSU, ITS, and 5.8S rDNA regions revealed that Australian C. brodyi strains have identical morphological features but include two different genetic variants. Isolates of C. brodyi from Australia, comprised the two ITS genotypes A and B which diverged 16.2% and 6.6%, respectively, of the ITS genotype from the U.S. type locality. Genotype A was widespread whereas genotype B thus far has only been found in Tasmania. Pfiesteria piscicida was cultured from ballast water indicating a potential inflow of foreign harmful algae into Australian waters. Previous studies using PCR-based assays claimed a wide distribution of Pseudopfiesteria shumwayae in Australia including in Brunswick River in Western Australia. However, isolates from the Brunswick River samples were identified in the present work as C. brodyi. Nonspecific reactions from P. shumwayae SSU rDNA-based primers were confirmed with Australian C. brodyi. This suggests that P. shumwayae presence in Australia has been overestimated by previous molecular detection methods. A species-specific real-time PCR assay using the TaqMan\\(^¬¨vÜ\\) probe system was developed for rapid detection and quantification of C. brodyi in environmental samples. Specific PCR primer-probes for C. brodyi were designed against the ITS2 rDNA regions and tested for selectivity, specificity and sensitivity of detection. The assay was able to detect the presence of less than 1 cell per PCR reaction, did not respond to non-target species, and accurately quantified C. brodyi in natural water samples. This assay was used together with previously reported P. piscicida- and P. shumwayae-specific real-time PCR assays to investigate the temporal variation in C. brodyi, P. piscicida, and P. shumwayae in the Derwent estuary, Tasmania from November 2004 until April 2006. Cryptoperidiniopsis brodyi occurred throughout the years at very low abundances (below 112 cells L\\(^{-1}\\)), while P. piscicida was detected only once and P. shumwayae was never detected during the 18 month survey (36 sampling dates). A further 8 cultures established from the Chapman River, Western Australia, during September 2005 were also all confirmed as C. brodyi. The species specific real-time assays were also used to examine Ace Lake water samples collected from Antarctica. Pfiesteria piscicida cells were detected and confirmed by partial DNA sequences of the PCR amplicons. Feeding behaviour and interactions of C. brodyi and P. piscicida with Pacific oyster (Crassostrea gigas) larvae and brine shrimp (Artemia salina) nauplii were examined in a micro-scale bioassay format. The zoospores became active and showed aggressive feeding response toward oyster larvae. Micropredation resulted in deaths of planktonic oyster larvae (below Imm size), but zoospores were less attracted to brine shrimp and no deaths of brine shrimp were observed. Given the very low abundances of C. brodyi and P. piscicida in the Derwent estuary, adverse effects from micropredatory feeding by these dinoflagellates on planktonic larvae may rarely occur in this environment. This study demonstrates the geographic and temporal variations in C. brodyi, P. piscicida and P. shumwayae by using species-specific real-time PCR, microscopy and sequence analyses. Continued studies of additional isolates and field samples over a range of temporal and spatial scales would allow a better understanding of phylogeographic and ecological aspects of C. brodyi, Pfiesteria and Pseudopfiesteria species.