Are Pfiesteria species toxicogenic? Evidence against production of ichthyotoxins by Pfiesteria shumwayae

Abstract

The estuarine genus Pfiesteria has received considerable attention since it was first identified and proposed to be the causative agent of fish kills along the mid-Atlantic coast in 1992. The presumption has been that the mechanism of fish death is by release of one or more toxins by the dinoflagellate. In this report, we challenge the notion that Pfiesteria species produce ichthyotoxins. Specifically, we show that (i) simple centrifugation, with and without ultrasonication, is sufficient to "detoxify" water of actively fish-killing cultures of Pfiesteria shumwayae, (ii) organic extracts of lyophilized cultures are not toxic to fish, (iii) degenerate primers that amplify PKS genes from several polyketide-producing dinoflagellates failed to yield a product with P. shumwayae DNA or cDNA, and (iv) degenerate primers for NRPS genes failed to amplify any NRPS genes but (unexpectedly) yielded a band (among several) that corresponded to known or putative PKSs and fatty acid synthases. We conclude that P. shumwayae is able to kill fish by means other than releasing a toxin into bulk water. Alternative explanations of the effects attributed to Pfiesteria are suggested.

Fig 1.

Fish-killing activity of P. shumwayae grown on algae and tilapia, plus supernatant and lysate, were assayed with C. variegatus (n = three per well × two wells per treatment). Cell-free lysates and supernatants were from raw tank water cultured on tilapia prey and centrifuged with or without ultrasonication. For lysate, no data were collected after 4 days, because the water in the wells evaporated. MeOH and dichloromethane (DCM) extracts of lyophilized raw tank water were tested against G. holbrooki (n = two fish per beaker × two beakers per treatment); no mortality was observed in this case up to 14 days (last 7 days not shown). “Untreated” and “ASW only” controls refer to fish tested in ground water (for G. holbrooki) or ASW (for C. variegatus), respectively.

Fig 2.

Gel (2% agarose) showing a lack of PCR amplification with genomic DNA from P. shumwayae with primers specific to ketosynthase domain of type I and II PKSs (PKSI and PKSII, lanes 1 and 2, respectively). Shown is positive amplification of 1.8-kb (lane 3) and 100-bp (lane 4) products, respectively, with “universal” 18S primer (35) and P. shumwayae-specific primers (PfBSSU/16s-B) confirm the presence of P. shumwayae DNA. Lane 5 is a 100-bp ladder.

Fig 3.

Gel (1% agarose) of RT-PCR products showing lack of expression of type I or II PKS genes by P. shumwayae. No amplification was observed with cDNA from P. shumwayae using type I PKSI (lane 2) or type II PKSII (lane 3) primer. Expression, likewise, is not seen with primers PKSI and PKSII (lanes 5 and 6, respectively) for the same culture exposed to larval fish (C. variegatus) 18 h before RNA extraction or with negative control of the algal food, R. salina, only (lanes 8 and 9, respectively). Positive amplification was observed with universal eukaryotic 18S primers (35) for CCMP 2089 (lane 4), CCMP 2089 exposed to C. variegatus (lane 7), and R. salina (lane 10). Amplification of the type I PKS sequences was observed for positive control plasmids PL29 (lane 11) and AK112 (lane 12). Negative controls of no template (lanes 13, 14, and 15, respectively) showed no amplification with PKSI, PKSII, or 18S primers. Lane 1 is a 1-kb DNA ladder.

Fig 4.

Gel (2% agarose) of RT-PCR products showing amplification with P. shumwayae-specific primer. Amplification of the expected 400-bp sequence was observed with cDNA from P. shumwayae (CCMP 2089) as well as the same culture exposed to larval C. variegatus 18 h before RNA extraction using the primers PfBSU/PfBITSR201L (lanes 2 and 5), confirming the presence of P. shumwayae cDNA. No amplification was seen (lane 8) with the algal food, R. salina. Positive amplification was also observed for all positive controls of P. shumwayae genomic DNA (lanes 3, 6, and 9). No amplification was observed for any of the negative controls of no template (lanes 4, 7, and 10). Lanes 1 and 11 are 100-bp DNA ladders.