Extracts of fifty-seven newly isolated strains of dinoflagellates and raphidophytes were screened for protein phosphatase (PP2A) inhibition. Five strains, identified by rDNA sequence analysis as Prorocentrum rhathymum, tested positive and the presence of okadaic acid was confirmed in one strain by HPLC-MS/MS and by HPLC with fluorescence detection and HPLC-MS of the okadaic acid ADAM derivative. Quantitation of the ADAM derivative indicated that the concentration of okadaic acid in the culture medium is 0.153 μg/L.
KeywordsProrocentrum rhathymum; okadaic acid; diarrheic shellfish poisoning (DSP) toxins; LC-MS; ADAM derivative; HPLC-FLD The suite of marine toxins that includes okadaic (OA) acid and the dinophysistoxins (DTX) is collectively known as DSP (diarrheic shellfish poisoning) toxins. The acute symptoms of DSP include diarrhea, nausea, vomiting and abdominal pain. Outbreaks have been documented worldwide and are associated with the consumption of mussels, scallops, or clams tainted with OA, its analogs or derivatives. (Gestal-Otero, 2000). OA has been isolated from several dinoflagellates in the genera Prorocentrum and Dinophysis, including the species P. lima (Yasumoto et al., 1987), P. hoffmanianum (Murakami et al., 1982), P. concavum (Dickey et al., 1990), P. maculosum (Zhou and Fritz, 1993), P. belizeanum , P. faustiae (Morton 1998), P. arenarium (Ten-Hage et al., 2000), D. acuta (Draisci et al., 1998) and D. fortii (Murata et al., 1982). In addition to OA, several related polyethers were isolated from these dinoflagellates, including the dinophysis toxins, DTX-1 and DTX-2 which differ in the location and number of methyl substituents (Murata et al., 1982;Yasumoto, et al., 1985;Hu et al., 1993). OA, DTX-1 and DTX-2 are inhibitors of protein phosphatases PP1 and PP2A (Dounay and Forsyth, 2002 We recently reported the isolation of over fifty strains of dinoflagellates by viable high speed single-cell sorting ). Five of these strains (6-9 and 25) matched most closely with P. rhathymum in a BLAST (Altschul et al., 1990) analysis of their large-subunit ribosomal genes ) and tested positive for protein phosphatase (PP2A) inhibition in preliminary screening. The presence of OA was confirmed in one strain by HPLC with fluorescent detection of the ADAM (9-anthryldiazomethane) derivative (Quilliam et al., 1998) and by HPLC-MS and MS/MS experiments.Fifty-seven field strains were sequenced in the D1/D2 region of the LSU rDNA and compared with GenBank sequence data ). Dinoflagellate and raphidophyte genera identified included Akashiwo, Amphidinium, Heterocapsa, Cachonina, Chatonella, Coolia, Fragilidinium, Heterosigma, Karlodinium, Karenia, Protoceratium, Scripsciella and Prorocentrum. Field strains 6, 7, 8, 9, 23, 25 and identified strains CCMP687 (Prorocentrum mexicanum) and CCMP1591 (Prorocentrum micans) clustered within a clade of the genus Prorocentrum (Fig. 1). The sequences for strains 6, 7, 8 and 9 were identical to each other and matched with two sequences (AY259166 and AY259167) of P. rhathymu...