DOMOIC‐ACID‐PRODUCING DIATOMS: ANOTHER GENUS ADDED!

A turning point in the history of harmful algal bloom (HAB) events occurred in November, 1987, when over 100 illnesses and at least three deaths were traced to the consumption of mussels from eastern Prince Edward Island (PEI), Canada. This was a novel syndrome, amnesic shellfish poisoning (ASP). The culprit toxin was rapidly identified as the neurotoxin domoic acid (DA), a low molecular weight amino acid; the source organism took several more months to confirm (Bates et al. 1989). To everyone's surprise, it turned out to be a pennate diatom. This was unprecedented, because up to that time most toxicity from HAB events had been caused by dinoflagellates or cyanobacteria. Indeed, this was the first time that a diatom was shown to be a toxin producer. That DA-producing diatom appeared in the literature as Nitzschia pungens forma multiseries, but is now called Pseudo-nitzschia multiseries(Fig. 1). Scanning electron micrographs of the domoic-acid-producing diatom Pseudo-nitzschia multiseries, sampled from Cardigan Bay, Prince Edward Island, Canada, in September, 1998. (A) Inner view of a valve; (B) outer view of another valve. Scale bars = 20 μm. The images are montages, created by using 11 (panel A) or 9 (panel B) separate digital images at a magnification of 10,000x (images courtesy of J. Ehrman, Digital Microscopy Facility, Mount Allison University, Sackville, NB, Canada; image A and montage technique used available at http://www.mta.ca/~jehrman/pnmulti.htm). This issue of the Journal of Phycology includes two papers that build on a growing body of research on the source organisms of DA (Lundholm and Moestrup 2000, Kotaki et al. 2000). However, these papers take us on a slightly different path because a different genus—Nitzschia—is now implicated, widening the breadth of DA-producing organisms; for this reason the papers are especially significant. We must first understand some of the taxonomic and nomenclatural changes that have occurred within the genus Nitzschia (see Hasle 1965, 1993, 1994, 1995, Taylor 1993. The changed names will again appear in my discussion of DA-producing species and Table 1. The genus Nitzschia was described by Hassall in 1845. As Lundholm and Moestrup (2000) discuss (from Mann 1986, this is the second largest diatom genus, comprising about 900 species. The genus Pesudo-nitzschia (as Pseudo-Nitzschia) was established, for Nitzschia seriata, N. fraudulenta and N. sicula, because the cells of the former two, at least, were distinguished by more sharply pointed tips that also overlapped to form a chain (Peragallo and Peragallo 1897–1908). However, because of other morphological arguments (e.g. its raphe was only partially reduced compared to Fragilariopsis, and its capability of movement was at least partially retained), Hustedt (1958) reduced this genus to a section of Nitzschia in 1958. With the discovery that DA was produced by certain of these diatoms, it became even more important to review some of the taxonomic questions. This is a case in which ongoing work on diatom morphology by Prof. Grethe Hasle (University of Oslo, Norway) has paid enormous dividends. Reexamining the available information on the complex genus Nitzschia and based in part on its “stepped colonies” (chains formed by overlapping cell tips) observed by the Peragallo brothers, Hasle (1994) recognized Pseudo-nitzschia as a genus distinct from Nitzschia. She then compared Pseudo-nitzschia pungens forma pungens (then considered to be nontoxic, see below) and the DA-producer Pseudo-nitzschia pungens forma multiseries and raised the latter in rank from forma to species, based on her own morphological observations and considering differences in immunofluorescence, molecular biology, and physiological ability to produce DA (Hasle 1995). All the DA-producing pennate diatoms (except one, see below) then belonged to the genus Pseudo-nitzschia. Thanks to the work of Prof. Hasle, the taxonomy of the genus Pseudo-nitzschia has been clarified. To further place the papers by Lundholm and Moestrup (2000) and Kotaki et al. (2000) into context, we must examine the flurry of activity that resulted from the original ASP incident in 1987 and the discovery of P. multiseries as the first DA-producing diatom. During the search for the DA source in eastern PEI, a pennate diatom identified as Amphora coffeaeformis was isolated from the contaminated mussels. It was shown to produce low amounts of DA (Shimizu et al. 1989, Maranda et al. 1990). This was the second diatom genus reported to be capable of producing DA; however, two other isolates of A. coffeaeformis from the Center for Culture of Marine Phytoplankton (CCMP) were shown to be nontoxic (Bates et al. 1989), as well as an isolate from sediments in Nivå Bay, Denmark (N. Lundholm and J. Skov, personal communication). A review of the morphology of this diatom group revealed that A. coffeaeformis has often been misidentified and that the identity of the toxigenic species from PEI is uncertain (Sala et al. 1998). In light of this, an increased effort should be made to test other A. coffeaeformis isolates for toxicity and to verify their taxonomic identity. After the 1987 episode, countries around the world became concerned about ASP outbreaks along their own coasts. Canada's expanded monitoring program discovered high levels of DA in molluscan shellfish from the Bay of Fundy in 1988. The source of the DA in that episode was another representative of the genus Pseudo-nitzschia, P. pseudodelicatissima (Martin et al. 1990). Curiously, other isolates of this species from Galveston Bay (TX), Massachusetts Bay (MA), Monterey Bay (CA), Denmark, and Australia failed to produce DA (see Bates et al. 1998. An exception was one isolate of P. pseudodelicatissima from Danish waters, but it was toxic in only three out of six combinations of light and temperature (Lundholm et al. 1997). Recently, isolates of what is reported to be this species from the Gulf of Mexico (Pan et al. 2001) and coastal waters of Washington (Adams et al. 2000) were shown to be toxin producers. A third species from the genus Pseudo-nitzschia, P. australis, was identified as the producer of DA that killed Brandt's cormorants and brown pelicans in Monterey Bay in 1991 (Garrison et al. 1992). Once again, the nomenclature of a species in this genus was questioned. The nomenclatural history is as follows (Hasle 1965, 1993, 1994, Taylor 1993. It was first described in Argentinean waters by Frenguelli (1939) as Pseudonitzschia australis (no hyphen). When transferred to the genus Nitzschia, it could not be named Nitzschia australis, because that name already existed for another diatom, so Nitzschia pseudoseriata was proposed because of its resemblance to N. seriata (Hasle 1965). When transferred back to Pseudo-nitzschia, it was once again correctly called Pseudo-nitzschia australis (Hasle 1993). Because this species was thought to be restricted to the southern hemisphere (hence its name), and because of its resemblance to Pseudo-nitzschia seriata, there were some uncertainties when it was first identified as a DA producer in Monterey Bay. Indeed, P. australis from Californian waters was likely misidentified in the past as P. seriata (see Villac et al. 1993, Fryxell et al. 1997. Sar et al. (1998) have authenticated type material of P. australis. Pseudo-nitzschia australis is now surpassing P. multiseries as the DA producer causing the most problems around the world. It was the DA source responsible for the deaths, in 1999, of over 400 California sea lions (Scholin et al. 2000), which had consumed DA-contaminated northern anchovies in Monterey Bay (Lefebvre et al. 1999). Given that this species was once thought to be restricted to the southern hemisphere, it is interesting that P. australis is now appearing as a DA producer in European waters. It was first suspected as the source of DA in cultured mussels from Galicia, northwest Spain, in 1994 (Míguez et al. 1996), and Galician isolates were subsequently shown to produce the toxin (Fraga et al. 1998). It is also suspected of being the source of the DA found in king scallops (Pecten maximus) and queen scallops (Chlamys opercularis) in Scottish waters in 1999 and 2000 (Gallacher et al. 2001, C. Bolch, Dunstaffnage Marine Laboratory, personal communication), the occurrence of which resulted in the world's largest area closed to shellfishing. The remaining Pseudo-nitzschia species reported to produce DA, although not all isolates are toxic, are given in Table 1. The latest described species, P. multistriata, was originally found in Japanese waters (Takano 1993, 1995); isolates from the Gulf of Naples were recently shown in a preliminary report to produce DA (Sarno and Dahlmann 2000). This brings the total to nine toxigenic Pseudo-nitzschia species. Each of these is known from most parts of the world and is, therefore, cause for concern. Isolates of P. multistriata from New Zealand, however, are reported to be nontoxic (Rhodes et al. 2000). Likewise, P. pungens has consistently been shown to be nontoxic in waters of the Atlantic, Gulf of Mexico, and Europe. However, several isolates (but not all) have been observed to produce low concentrations of DA from New Zealand (Rhodes et al. 1996), Washington (Trainer et al. 1998), and Monterey Bay (see Bates et al. 1998. Until the identity of DA can be unambiguously confirmed by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) or by proton NMR spectroscopy, reports of toxin production by P. pungens should still be considered with caution. Up to now, therefore, diatoms from two genera (Pseudo-nitzschia and Amphora) were known sources of DA. This is in addition to the original DA producer discovered in the 1950s, the rhodophycean macroalga Chondria armata and later Chondria baileyana, Alsidium corallinum, Amansia glomerata, Digenea simplex, and Vidalia obtusiloba (see Bates et al. 1998. Now, there is a third diatom genus that is conclusively shown, in this issue, to be a DA producer: Nitzschia(Fig. 2). Interestingly, the newly noted producer in this genus is also a newly described species, Nitzschia navis-varingica, isolated from a shrimp-culture pond in Vietnam. Girdle view of the marine diatom Nitzschia navis-varingica from Vietnam, discovered to be a producer of the neurotoxin domoic acid. (Courtesy of N. Lundholm and Ø. Moestrup.) The papers by Lundholm and Moestrup (2000) and Kotaki et al. (2000) should become a case history in how to tackle the problem of characterizing a new source of DA. These two research groups from different continents coordinated their efforts, the former documenting the morphology and, therefore, the identity of the new species, and the latter the identity of the toxin and the dynamics of its production. Kotaki et al. (2000) first screened the ability of five isolates of N. navis-varingica to produce DA, using a now-standard analytical technique applied to phytoplankton, that is, precolumn derivatization of the amino acid with fluorenylmethoxycarbonyl (FMOC), followed by HPLC and detection of the fluorescent compound (Pocklington et al. 1990). To identify DA, many studies have simply relied on the coincidence of retention times for the unknown and authentic DA peaks on the HPLC chromatogram. However, it is always possible that other compounds may appear with the same retention time as DA. Further verification steps could be to determine the absorbance spectrum of DA by diode array detection (DAD) or to apply a DA-specific antibody (see Table 1). Nevertheless, ambiguities remain with each of these approaches. Kotaki et al., therefore, repeatedly purified the compound by liquid chromatography (LC) and then looked for the characteristic parent ion peak of DA at m/z 312, using electrospray ionization mass spectrometry (ESI MS) (Quilliam et al. 1989). Even this may not be sufficient, if other compounds have the same mass/charge ratio (i.e. m/z 312) as DA. They, therefore, carried out additional tests, which together with LC purification and ESI MS data, provided selectivity and supporting evidence for the identity of DA. These tests included UV absorbance detection at 242 nm, UV absorbance spectra (200–350 nm), and high performance silica gel TLC in comparison with authentic DA. A further step, following LC and after obtaining a mass spectrum for DA, would be to fragment the parent ion and to generate a second or tandem mass spectrum (LC-MS/MS). The presence of diagnostic daughter or fragment ions at m/z 161 and 266 in the tandem mass spectrum then provides unambiguous confirmation that the substance is DA. An alternative approach is proton NMR spectroscopy, which was recently used by Kotaki et al. (1999) to confirm DA production by P. multiseries in Japanese waters. That approach requires more than the 38 μg of purified DA they obtained from Nitzschia navis-varingica (Y. Kotaki personal communication). Kotaki et al. (2000) provide further information about the dynamics of DA production in a time-course experiment, whereby successive samples are taken during the exponential and stationary phases in batch culture (cf. Bates et al. 1991. This enabled a comparison with similar experiments carried out with at least 3 of the 11 diatom species reported to produce DA. They found essentially the same toxin dynamics as for P. multiseries and P. seriata, that is, DA production beginning during late exponential phase and continuing more rapidly into stationary phase (see Bates 1998. However, this differs from P. australis (Garrison et al. 1992) and P. pseudodelicatissima (Pan et al. 2001), which show production during most of the exponential phase and not during stationary phase. The information to date for P. pseudodelicatissima is not entirely consistent, because an isolate from Washington waters produced DA during the late exponential as well as stationary phase (Adams et al. 2000). Clearly, more such studies are required with this and the remaining toxigenic species. Kotaki et al. (2000) also compared cellular DA levels with other DA-producing Pseudo-nitzschia species. Levels were comparable to those in P. multiseries and P. seriata, less than in P. australis, but more than in P. turgidula and P. pungens. Another significance of their paper is that they looked for additional commonalities with other studies by investigating the influence of bacteria on toxin production. Although they confirmed that axenic cultures produced less DA than bacteria-containing cultures, the effect was not as pronounced as was found for P. multiseries (see Bates 1998. Such comparative studies are an important first step in understanding any differences in mechanisms of DA production among species and, now, even genera. The significance of the accompanying paper by Lundholm and Moestrup (2000) is that the newly discovered producer of DA, Nitzschia navis-varingica, is now well described by light microscopy and by transmission and scanning electron microscopy and placed into a taxonomic context. It will, therefore, be possible for others to identify this and possibly other similar species and, therefore, be alerted to the possibility of DA production. The species clearly belongs to the genus Nitzschia, as the solitary cells are characterized by two chloroplasts (one in each half of the cell) and by a more or less eccentric raphe raised on a keel and supported by fibulae. It is distinguished from the genus Pseudo-nitzschia by not forming stepped colonies, by the presence of areolae in the raphe canal, and by possessing a raphe raised on a keel, not in the valvar plane. It is worrisome that this new species is found in ponds used for shrimp aquaculture. As Lundholm and Moestrup (2000) point out, diatoms represent the preferred food organism in shrimp-pond aquaculture, and species of Nitzschia are one of the most common diatom groups in those ponds. There are still, however, many unresolved questions. How many other shrimp ponds in tropical coastal areas harbor N. navis-varingica? What proportion of the shrimp-pond phytoplankton flora does this species compose, and what is its seasonal cycle? Does it actually produce DA in shrimp ponds and, if so, under what conditions? In what other habitats is this species found? The genus Nitzschia is found in fresh water, in soil, and in brackish and marine waters. It is, therefore, possible that additional DA producers are present in other than marine or brackish environments. In summary, the two papers highlighted in this issue point out several important lessons and directions for future research. First, they illustrate how international cooperation and sharing of expertise permit solid scientific achievement in an area of toxic algal research that potentially has world-wide implications. Second, they show the importance of basic taxonomic studies that lay the foundation for further ecophysiological work. The monographs on Pseudo-nitzschia taxonomy produced by Prof. Hasle 35 years ago have stood the test of time and can now be augmented by molecular studies. A further illustration is the morphological work of Sala et al. (1998), which points out how frequently Amphora coffeaeformis has been misidentified in the past. Similarly, the misidentification of P. australis as P. seriata has been clarified by the morphological work of Hasle (1965) and Fryxell et al. (1997). Could we have missed other morphological subtleties that distinguish toxic from nontoxic isolates of presumably the same species? Interestingly, isolates of P. pseudodelicatissima from the Black Sea and from the CCMP culture collection failed to reproduce sexually when male and female clones were mixed together (Davidovich and Bates 1998). We must encourage the training of taxonomists and the publication of their work if we are to know with what species we are dealing and to compare results from different laboratories. Finally, these papers make us look for commonalities in those algae that produce DA. Although the ecological or physiological role of DA is still not certain (see Bates 1998, it is interesting that N. navis-varingica and the other toxigenic Pseudo-nitzschia species spend part of their life history on the benthos and that the rhodophycean macroalgae and Amphora coffeaeformis are benthic. Is there a relationship between toxin production and mode of life, irrespective of genus? Kotaki et al. provide solid evidence for DA production, and Lundholm and Moestrup give convincing arguments for placing N. navis-varingica in the genus Nitzschia. We shall await molecular work to see where this new species fits into the phylogeny of other Nitzschia as well as Pseudo-nitzschia species, DA producing or otherwise. I thank R. Horner, V. Trainer, I. Kaczmarska, N. Lundholm, C. Cusack, G. Hasle, G. Doucette, and M. Quilliam for their comments. I especially thank G. Fryxell, who engaged me in many thought-provoking discussions; her work has shown the importance of integrating studies on diatom morphology and ecology.

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