Osmotic stress does trigger brevetoxin production in the dinoflagellate Karenia brevis

Although a number of factors may influence its production, the physiological role of brevetoxin in the dinoflagellate Karenia brevis is still open to debate. Not to be left out of the discussion, Sunda et al. (1) challenge our suggestion that salinity stress may be a possible trigger for brevetoxin production. Their “repeat” of our experiment is not an actual replication (extraction protocols, analytical method, K. brevis isolates, and culturing conditions differed among their three laboratories). Moreover, a number of experimental details, including use of internal standards and preparation of toxin standards, are omitted. Internal standards are important because extraction efficiency varies among samples and could affect results. Nevertheless, both reports show that there is no long-term increase in brevetoxin production after salinity stress (1, 2) and that low-toxin cultures [SP1 (2) and “nontoxic” Wilson (1)] do not increase brevetoxin production in response to hypoosmotic stress.

Our report does show that there is a short-term response in brevetoxin production: after a salinity shift from 35 to 27, brevetoxin concentration per cell increased from 10% to 53% within 5 d (2). Only laboratory B (1) performed a similar experiment, and their results confirmed our observation: after salinity stress, brevetoxin per cell increased ∼25% (figure 3 in ref. 1; Wilson isolate). Laboratory A also demonstrated increased brevetoxin cell quota in SP3 by ∼15% after 12 d. The remaining isolates were not used in our experiments, and, as previously suggested (3), different isolates may have a different environmental responses.

The error in our original calculation (2) was a concern for us, so we repeated the experiment, using controls that received seawater with nutrient additions at the same time as hypoosmotic stress treatments (similar to laboratory A in ref. 1). We confirmed all previous results: controls remained constant between replicates, and hypoosmotic stress (salinity of 35 to 27) produced an increase in brevetoxin cell concentration; Wilson isolate increased 28%, TXB4 isolate increased 59%, and SP3 isolate increased 21% by 2 d after the treatment.

In our paper, we demonstrate that cell size shows a synchronous diel change (supporting information in ref. 2). Therefore, brevetoxin on a per volume basis will vary over the day. Sunda et al. (1) do not address this diel periodicity, nor indicate exact time that cell size was determined. In addition, we note that these measurements were determined for glutaraldehyde-preserved cells by laboratory C, not live cells as we reported. Preservation is well-known to distort cell shape and volume. Although our measurements were based on images of single cells only, the Coulter Multisizer particle counter cannot distinguish between K. brevis cells and other particles in the sample, nor can it distinguish between single and dividing cells.

Sunda et al. (1) imply that defensive mechanisms are a well-known function of brevetoxins; however, two of the three citations concern species other than K. brevis and the third was published in 2012, after our publication (2). We have suggested a functional role in osmoregulation while not ruling out other possible uses for brevetoxins.

Finally, we agree that there is no known mechanism for brevetoxin to facilitate osmoregulation. However, it is well established that ion movement is in the direction of the current flow as directed by the electrical potential (4). In the case of hypoosmotic stress, Na⁺ would flow out of the cell, as determined by the membrane potential.