Biotechnology Advances 25 (2007) 176–194 www.elsevier.com/locate/biotechadv Research review paper Biotechnological significance of toxic marine dinoflagellates ⁎ F. Garcia Camacho a, , J. Gallardo Rodríguez a, A. Sánchez Mirón a, M.C. Cerón García a, E.H. Belarbi a, Y. Chisti b, E. Molina Grima a aDepartment of Chemical Engineering, University of Almería, 04120 Almería, Spain b Institute of Technology and Engineering, Massey University, Private Bag 11 222, Palmerston North, New Zealand Received 2 February 2006; accepted 28 November 2006 Available online 30 November 2006 Abstract Dinoflagellates are microalgae that are associated with the production of many marine toxins. These toxins poison fish, other wildlife and humans. Dinoflagellate-associated human poisonings include paralytic shellfish poisoning, diarrhetic shellfish poisoning, neurotoxic shellfish poisoning, and ciguatera fish poisoning. Dinoflagellate toxins and bioactives are of increasing interest because of their commercial impact, influence on safety of seafood, and potential medical and other applications. This review discusses biotechnological methods of identifying toxic dinoflagellates and detecting their toxins. Potential applications of the toxins are discussed. A lack of sufficient quantities of toxins for investigational purposes remains a significant limitation. Producing quantities of dinoflagellate bioactives requires an ability to mass culture them. Considerations relating to bioreactor culture of generally fragile and slow-growing dinoflagellates are discussed. Production and processing of dinoflagellates to extract bioactives, require attention to biosafety considerations as outlined in this review. © 2006 Elsevier Inc. All rights reserved. Keywords: Algal toxins; Ciguatera fish poisoning; Diarrhetic shellfish poisoning; Dinoflagellates; Marine toxins; Neurotoxic shellfish poisoning; Paralytic shellfish poisoning Contents 1. Introduction ...................................................... 177 2. Harmful algal blooms (HABs) ............................................ 177 3. Health impact of dinoflagellates ........................................... 179 Abbreviations: AOAC, Association of Official Analytical Chemists; ASP, Amnesic shellfish poisoning; AZAs, Azaspiracids; AZP, Azaspiracid shellfish poisoning; bFGF, Basic fibroblast growth factor; CFP, Ciguatera fish poisoning; CTXs, Ciguatoxins; DGGE, Denaturing gradient gel electrophoresis; DNA, Deoxyribonucleic acid; DSP, Diarrhetic shellfish poisoning; DTX, Dinophysis toxins; DTXn, DTXs (n=1–3); ELISA, Enzyme-linked immunosorbent assay; EU, European Union; FISH, Fluorescent in situ hybridization; GNTX2/3, Gonyautoxin 2/3; HABs, Harmful algal blooms; HPLC, High-performance liquid chromatography; IC50, Concentration required to produce half the maximal inhibition; IOC, Intergovernmental Oceanographic Commission; LC-MS, Liquid chromatography coupled mass spectroscopy; MAbs, Monoclonal antibodies; MBA, Mouse bioassay; MTXs, Maitotoxins; NSP, Neurotoxic shellfish poisoning; PbTx, Brevetoxins; PbTx-n, Brevetoxin n (n=1–10); PSP, Paralytic shellfish poisoning; PTXs, Pectenotoxins; PTX2, Pectenotoxin-2; RIA, Radioimmunoassay; RNA, Ribonucleic acid; STXs, Saxitoxins; TT, Tetanus toxoid; TTH, Tachypleus tridentatus hemocyanin; TTX, Tetrodotoxin; YTXs, Yessotoxins; ZTs, Zooxanthellatoxins. ⁎ Corresponding author. Fax: +34 950015484. E-mail address: [email protected] (F.G. Camacho). 0734-9750/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2006.11.008 F.G. Camacho et al. / Biotechnology Advances 25 (2007) 176–194 177 4. Dinoflagellate biotoxins ................................................ 180 4.1. Potential applications .............................................. 180 4.2. Detection of toxins ............................................... 182 4.2.1. Paralytic shellfish poisoning (PSP) .................................. 183 4.2.2. Diarrhetic shellfish poisoning (DSP).................................. 184 4.2.3. Neurotoxic shellfish poisoning (NSP) ................................. 184 4.2.4. Ciguatera fish poisoning (CFP) .................................... 185 4.3. Reference standards ............................................... 185 5. Detection and identification of HAB species...................................... 186 6. Bioreactor culture of dinoflagellates .......................................... 186 7. Biosafety considerations ................................................ 188 8. Concluding remarks .................................................. 188 Acknowledgements ..................................................... 189 References .......................................................... 189 1. Introduction toxins. The latter are of potential medical and commercial significance. Bioactive compounds from Phytoplanktons are responsible for a large proportion nondinoflagellate phytoplanktons are reviewed else- of the photosynthetically produced biomass that sup- where (Metting and Pyne, 1986; Schwartz et al., 1990; ports zooplankton and other higher lifeforms. Filter- Borowitzka, 1995, 1999; Codd, 1995; Shimizu, 1996, feeding organisms such as bivalve shellfish and larvae 2003; Moore, 1996, 2005; Tringali, 1997; Tyagi et al., of commercially important crustaceans rely almost 1999; Lebeau and Robert, 2003a,b; Singh et al., 2005). completely on phytoplankton for food. Mass prolifera- tions of phytoplankton are known as algal blooms. Such 2. Harmful algal blooms (HABs) blooms contain high concentrations of algal biomass. Blooms occur typically in localized regions that are HABs are a natural phenomena, but their frequency, temporarily rich in inorganic nutrients such as nitrogen, geographic range and intensity appear to have increased phosphorus and iron. Algal blooms are more frequent in since the 1970s and their economic impact is greater summers when the sunlight levels are high. Most algal now than in the past. HABs appear to be stimulated by blooms are harmless and can actually benefit aqua- nutrient discharges in domestic, industrial and agricul- culture and wild fisheries; however, under exceptional tural wastes (Lam and Ho, 1989). Such discharges alter circumstances, high-density blooms of normally bene- ratios of key nutrients in coastal waters to favor ficial algae can cause indiscriminate kill of fish and development of HABs (Smayda, 1990; Riegman et al., invertebrates through oxygen deprivation. In addition, 1992). Nitrogenous runoffs have been clearly linked nontoxic algal blooms can make finfish more vulnerable with the formation of large phytoplankton blooms in the to disease (Rensel and Whyte, 2003). Gulf of California (Beman et al., 2005). The increased Certain microalgae are toxic. Blooms of toxic algae economic impact of HABs is probably linked with the are known as harmful algal blooms, or HABs. Effects of increased consumption of seafood and growth in coastal HABs on aquatic organisms have been reviewed by populations (Hallegraef, 2003). Landsberg (2002). HABs causing red discoloration of HABs can develop quite suddenly. Dormant cysts of water are commonly known as “red tides”. At least 90 most of the red tide dinoflagellate species survive in ocean species of marine microalgae are known to produce sediments for years (Pfiester and Anderson, 1987). Under toxins. Of these species, 70 are dinoflagellates (Taxo- appropriate environmental conditions, the cysts germinate nomic Reference List of Toxic Plankton Algae of to produce HABs. Climatic changes that appear to be Intergovernmental Oceanographic Commission (IOC); taking place may be a contributory factor to increased http://ioc.unesco.org/hab/data.htm). Dinoflagellates frequency of HABs. In past warmer geological periods, occur widely in aquatic environments. Only about half some of the HAB-associated dinoflagellates occurred of the dinoflagellate species are photosynthetic. World- over larger ranges than they do today, as reflected in the wide, marine algal toxins cause more than 60,000 fossil records of the resting cysts. A warmer climate may poisoning events annually with an associated mortality favor their return to former ranges (Edwards and rate of 1.5% (Gill et al., 2003). This review is focused Richardson, 2004). MacLean (1989) presented strong on biotechnology aspects of dinoflagellates and their circumstantial evidence for a coincidence between 178 F.G. Camacho et al. / Biotechnology Advances 25 (2007) 176–194 blooms of the dinoflagellate Pyrodinium bahamense and dinoflagellate cysts has been discussed by Marret and El Niño–Southern oscillation climatological events. Zonneveld (2003). Examples of temporarily altered ranges of dinoflagellates Transport of toxic dinoflagellates and their cysts in include human shellfish poisonings in New Zealand that ship ballast waters appears to contribute to their spread were associated with blooms of algal species similar to to nonnative regions. Moving contaminated shellfish Karenia brevis (Jasperse, 1993) that is considered stocks from one region to another, is a further likely endemic to the Gulf of Mexico and the east coast of contributor. Bivalves can harbor viable dinoflagellate Florida. These blooms coincided with the unusual cells and sometimes can also contain resistant resting climatic conditions associated with an El Niño event. cysts (Scarratt et al., 1993; Schwinghamer et al., 1994). Other similar cases have been documented (Dale and Barely any effort is made currently to control marine Nordberg, 1993). Geographic