Caiqing Mo ® J. Douek ® B. Rinkevich Development of a PCR strategy for thraustochytrid identification based on 18S rDNA sequence Received: 4 April 2001 / Accepted: 15 December 2001 / Published online: 21 March 2002 Ó Springer-Verlag 2002 Abstract The identification and characterization of the thraustochytrids, an emerging economically and bio- technologically important group of marine heterotrophic protists, is usually based on morphological characters. In this research we used molecular markers to identify thraustochytrids. We designed three sets of primers based on the 18S rDNA sequence alignment of known strains and employed a PCR (polymerase chain reaction) strategy to identify unknown thraustochytrid isolates. DNA from 26 thraustochytrids (three isolated from primary cell cultures of the tunicate Botryllus schlosseri and 23 from a coral holding aquarium) were amplified by these primers, revealing 21 isolates with three bands each, which were assigned to two groups according to PCR fragment sizes. Taxonomic characterizations were de- duced by comparing with GenBank data. Four isolates were further studied by sequencing their 18S rDNA. Sequence alignments and phylogenetic analysis revealed that isolates from the coral aquarium (7-5 and 8-7) were highly similar to each other and 95.0–97.0% similar to Thraustochytrium multirudimentale and Schizochytrium minutum. Isolates from the tunicate primary cell cultures (BS1 and BS2) were also closely related to each other and 84.3–86.0% similar to labyrinthulid quahog parasite and Thraustochytrium pachydermum. AFLP (amplified frag- ment-length polymorphism) analysis revealed 2.5–3.6% differences within the genomic DNA of each group, showing that each isolate is different, although isolates within each group may belong to the same species, in spite of differences found in the general morphology. Introduction The thraustochytrids, a common group of marine he- terotrophic protists, are known as pathogens of edible invertebrates (Bower 1987; Mass et al. 1999), as pre- dominant contaminants of marine invertebrate cell cul- tures (Rinkevich 1999, and literature therein), and as potential sources for valuable bioactive compounds (Lewis et al. 1998; Bowels et al. 1999). This last charac- teristic has been further illuminated by our results on primary cell cultures of sponges and tunicates (Rinkevich and Rabinowitz 1993; Ilan et al. 1996), revealing that thraustochytrid-infected cultures resist bacterial con- tamination better than freshly prepared cultures. An area of research we are currently pursuing is the isolation of thraustochytrids that produce novel bioactive com- pounds. A major obstacle, however, is the identification and proper systematic classification of different isolates. Thraustochytrids belong to the phylum Labyrint- hulomycota (Porter 1990) and were studied in the past mainly by mycologists (Barr 1992). Cells form naked globose or colonial structures, associated with thin or more-developed, scaled cell walls. Many of them develop ectoplasmic networks generated by a unique organelle termed the sagenogen (or sagenogenetosome). Some growing cells exhibit a gliding mobility associated with the ectoplasmic networks. Their reproduction activities involve the formation of heterokont, biflagellate zoosp- ores (Porter 1990). The phylum Labyrinthulomycota contains a single class, Labyrinthulea, and a single order, the Labyrint- hulida, with two families, the Labyrinthulidae and the Thraustochytriidae (Olive 1975). Eight genera have been described in the family Thraustochytriidae through the use of morphological characterizations: Althornia, Apla- nochytrium, Corallochytrium, Japonochytrium, Laby- rinthuloides, Schizochytrium, Thraustochytrium and Ulkenia, comprising more than 30 species (Raghukumar 1987; Porter 1990). However, Corallochytrium was later characterized by its 18S rRNA gene sequence and Marine Biology (2002) 140: 883–889 DOI 10.1007/s00227-002-0778-9 Communicated by R. Cattaneo-Vietti, Genova Caiqing Mo ® J. Douek ® B. Rinkevich (&) Minerva Center for Marine Invertebrate Immunology and Developmental Biology, Israel Oceanographic and Limnological Research, National Institute of Oceanography, P.O. Box 8030, Tel Shikmona, Haifa 31080, Israel E-mail: buki@ocean.org.il Fax: +972-4-8511911