Identification of euglenoid algae that produce ichthyotoxin(s).

Reported incidences of harmful algal blooms (HABs) have increased during the past 20 years, suggesting either increased occurrence or more rigorous monitoring efforts (Landsberg 2002). Several toxic compounds are produced, including neurotoxins, hepatotoxins, dermonecrotics and/or gastroenterics. Direct HAB effects include animal mortalities from toxin ingestion and/or oxygen depletion following algal senescence. Indirect impacts from HABs include alterations of normal trophic transfer resulting from food web alterations, fish mortalities from secondary stressors (disease/ indirect oxygen effects) and lost revenue from tourist and recreational industry cancellations (Anderson, Kaoru & White 2000). Diatoms, dinoflagellates, pelagiophytes and prymnesiophytes are the only divisions of microalgae known to produce toxins (Wehr & Sheath 2003); cyanobacteria additionally are known to produce at least three classes of toxins (Chorus 2001). Most toxin-producing taxa occur in marine/ estuarine systems; but herein we describe the production of an unknown toxin by freshwater Euglena species. Unexplained fish mortalities were reported in July–August 2002 at a commercial aquaculture facility in North Carolina. Fish mortalities were reported from three 1.2 ha striped bass, Morone saxatilis (Walbaum), ponds; two of these ponds had total mortality. Over 21 000 striped bass died and revenue lost from this single event exceeded $100 000. No clear pathology was evident in dead fish, with the exception of reddening of the gill tissue. No water quality factors (nitrates, ammonia, pH, temperature) were abnormal and dissolved oxygen was above 4 mg L during fish mortalities. Water samples from the second mortality event examined microscopically contained >99% Euglena by numeric counts. When exposed to the pond water, laboratory-reared channel catfish, Ictalurus punctatus (Rafinesque), were killed within 6.5 min. Pond water was serially fractionated to separate dissolved compounds, bacteria, and algal fractions using 0.22, 2.0 and 10 lm nitrocellulose filters (Costar, Cambridge, MA, USA). Both dissolved and algal fractions were toxic to fish. The dissolved fraction maintained toxicity following treatment with a proteinase, suggesting the compound was non-protein. The toxin was stable when heated to 30 C for 10 min and retained activity when frozen at )80 C for 60 days. Euglena cells were isolated from the water samples and brought into unialgal culture in AF-6 media (http://www.nies.go.jp/biology/mcc/medium. htm#AF-6). Light microscopic analyses verified the species identity as E. sanguinea, a cosmopolitan species found in many shallow, relatively calm, eutrophic freshwater systems (Fig. 1a). Journal of Fish Diseases 2004, 27, 115–117