Harmful Algae 2 (2003) 273281

Ichthyotoxicity of Chattonella marina (Raphidophyceae) to damselsh (Acanthochromis polycanthus): the synergistic role of reactive oxygen species and free fatty acids
Judith-Anne Marshall a, , Peter D. Nichols b , Brett Hamilton c , Richard J. Lewis c , Gustaaf M. Hallegraeff a
a c

School of Plant Science, University of Tasmania, G.P.O. Box 252-55, Hobart, Tasmania 7001, Australia b CSIRO Marine Research, G.P.O. Box 1538, Hobart, Tasmania 7001, Australia Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane, Qld 4072, Australia Received 18 December 2002; received in revised form 18 March 2003; accepted 28 March 2003

Abstract This investigation aimed to elucidate the relative roles of putative brevetoxins, reactive oxygen species and free fatty acids as the toxic principle of the raphidophyte Chattonella marina, using damselsh as the bioassay. Our investigations on Australian C. marina demonstrated an absence or only very low concentrations of brevetoxin-like compounds by radio-receptor binding assay and liquid chromatographymass spectroscopy techniques. Chattonella is unique in its ability to produce levels of reactive oxygen species 100 times higher than most other algal species. However, high levels of superoxide on their own were found not to cause sh mortalities. Lipid analysis revealed this raphidophyte to contain high concentrations of the polyunsaturated fatty acid eicosapentaenoic acid (EPA; 1823% of fatty acids), which has demonstrated toxic properties to marine organisms. Using damselsh as a model organism, we demonstrated that the free fatty acid (FFA) form of EPA produced a mortality and sh behavioural response similar to sh exposed to C. marina cells. This effect was not apparent when sh were exposed to other lipid fractions including a triglyceride containing sh oil, docosahexaenoate-enriched ethyl ester, or pure brevetoxin standards. The presence of superoxide together with low concentrations of EPA accelerated sh mortality rate threefold. We conclude that the enhancement of ichthyotoxicity of EPA in the presence of superoxide can account for the high C. marina sh killing potential. 2003 Elsevier B.V. All rights reserved.
Keywords: Ichthyotoxicity; Reactive oxygen species; Free fatty acids; Chattonella marina; Superoxide; Eicosapentaenoic acid

1. Introduction Several hypotheses have been proposed for the toxic mechanism of the sh killing raphidophyte Chattonella marina. The original theory put for Corresponding author. Tel.: +61-3-62261750; fax: +61-3-62262698. E-mail address: judi.marshall@utas.edu.au (J.-A. Marshall).

ward by Okaichi (1983) was the production of free fatty acids (FFAs) by Chattonella entering the blood stream via the gills, resulting in sh mortalities. Subsequent studies have investigated reactive oxygen species (ROS) production and its role in gill damage (Shimada et al., 1983; Oda et al., 1992a,b). Later investigations have centred on anoxia, mucus production, respiratory, ionoregulatory and cardiovascular physiology (Ishimatsu et al., 1990, 1991,

1568-9883/$ see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1568-9883(03)00046-5

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1997). C. marina has also been claimed to produce a fat-soluble toxin similar in structure to brevetoxin, rst characterised from the dinoagellate Karenia brevis (Onoue et al., 1990; Khan et al., 1995), but the raphidophyte toxin has yet to be fully characterised chemically. The ROS superoxide ( O2 ), which is produced by C. marina at levels 100-fold higher than that of most other alga (Marshall et al., 2002a), has also been implicated in sh mortalities through changes in gill pathology (Ishimatsu et al., 1997). Ecophenotypic differences in O2 production between Australian and Japanese strains have shown that higher O2 production in C. marina correlates with faster mortality in juvenile rainbow trout (Onchorrynchus mykiss) (Marshall et al., 2001). Furthermore, O is primarily controlled through photosynthesis 2 (Marshall et al., 2002a). However, when superoxide levels were suppressed in dark treatments, sh mortalities did not occur. This led to the hypothesis that a synergy between ROS and a toxic fraction could be responsible for sh mortalities (Marshall et al., 2001). C. marina has been found to produce high levels (10%) of FFAs, which have also been implicated in Chattonella sh toxicity (Okaichi et al., 1989). Both Australian and Japanese strains contain high levels of potentially toxic polyunsaturated fatty acids (PUFAs) including eicosapentaenoic acid (EPA, 20:53, 1823%; Marshall et al., 2002b). Arzul et al. (1995,1998) demonstrated that FFA including steraradonic acid (STA, 18:43) and EPA from the dinoagellate Karenia mikimotoi have an allelopathic effect on the diatom Chaetoceros gracile, and can result in haemolysis of horse red blood cells and mortality of Pecten maximus shellsh larvae. The present investigation aimed to elucidate the relative roles of putative brevetoxins, ROS and FFAs as the ichthyotoxic principle of C. marina. 2. Methods 2.1. Algal culturing A non-axenic culture of C. marina (UTCMPL03) isolated from Port Lincoln, South Australia, in 2002 was maintained in full strength seawater containing

GSe nutrients (Blackburn et al., 1989) at 25 C and 150 mol m2 s1 light. A culture of Dunaliella tertiolecta (CS-175) was used as an algal control in the sh exposure experiments. Experimental cultures were kept in exponential phase through the use of semi-continuous culture techniques to ensure nutrient limitation did not occur. 2.2. Fish bioassay Damselsh (Acanthochromis polycanthus Bleeker) weighing 6001200 mg were obtained from the University of Tasmania, School of Aquaculture, as three heterogeneous batches. Fish were exposed to early exponential stage cultures of C. marina at concentrations of 25035,000 cells ml1 , which were adjusted to cell concentrations to be tested 48 h prior to sh challenge to allow equilibrium of ROS levels. The experimental cultures were not aerated and oxygen levels were measured using a WTW Oxyguard probe. Pure FFA (EPA and STA) were obtained from Sigma Chemical Co. (St. Louis, MO). Other lipids used were an ethyl ester-enriched 22:63 (DHA) (66% of fatty acid fraction with 20% EPA) and tuna oil containing >99.5% tryglyceride (with 25% DHA and 8% EPA). Lipids were dissolved in 2 ml of methanol and introduced to 400 ml of seawater containing four damselsh at concentrations of 225 mg l1 . 2.3. Reactive oxygen species The oxygen radical superoxide ( O2 ) was measured using the luciferin analogue 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2- a]pyrazin-3-one (MCLA) as previously described by Oda et al. (1992a,b) at a concentration of 5 106 M and standardised against 5 106 M superoxide dismutase (SOD). All results were corrected for controls consisting of GSe media. Superoxide was generated though xanthine oxidase acting aerobically on a xanthine substrate resulting in the production of uric acid (Halliwell and Gutteridge, 1999) as shown in Fig. 1. Superoxide levels were maintained by periodically adding 5 106 M xanthine to 1030 units l1 of xanthine oxidase in ltered seawater, determined to be equivalent to the production of superoxide by C. marina and checked by MCLA luminescence.

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275

Xanthine
(substrate)

2H2O + 2O2 H2O2 + 2O2Xanthine Oxidase

(enzyme)

Toxicity of brevetoxins (PbTx-2 and PbTx-9) and FFA was calculated from the survival time of the sh and expressed as LT50 , the time it takes for 50% of sh to die at the prescribed concentration of toxin or algal cells.

3. Results 3.1. C. marina exposure and superoxide production

Uric Acid
(Keto form)

Fig. 1. Xanthine catalyses oxidation of xanthine to uric acid whilst reducing O2 to both superoxide (O2 ) and hydrogen peroxide (H2 O2 ).

2.4. Toxin analysis Chattonella cells were centrifuged, extracted in dichloromethane, evaporated and partitioned between methanol and hexane to remove fats and selected pigments, e.g. carotenoids. The residue for the methanol fraction was fractionated using silica ash chromatography as per Lewis et al. (1991) with chloroform/methanol (c:m) solutions of increasing polarity (1:0, 9:1, 7:3, 1:1, 2:8, 0:1). Fractions were assessed using a tritium labelled brevetoxin radio-labelled ligand binding (RLB) assay (Poli et al., 1986; Hamilton et al., 2002) at 0.5, 0.05, 0.005, 0.0005, 0.00005 and 0.000005%. Samples were further analysed using a gradient of 2% B per min (A: 0.1% formic acid (aq); B: 90% acetonitrile (aq) + 0.1% formic acid) established on an Agilent 1100 series high-performance liquid chromatography system coupled to a SCIEX Qstar (Quadrupole/time-of-ight) mass spectrometer (LC/MS). The Institute of Medical and Veterinary Science, South Australia, conducted mouse bioassays using IVMS method FH38. Duplicate mice were injected intraperitoneally with the equivalent of 3 106 cells of C. marina from Australia and Japan (strain NIES-118), extracted in methanol and suspended in 10% Tween solution. 2.5. Statistical analysis Results were analysed using a simple regression with a signicance level of 0.05.

Damselsh exposed to non-aerated cultures of C. marina less than 1000 cells ml1 rarely exhibited mortality. Fish exposed to algal concentrations of 10008000 cells ml1 suffered more rapid mortality times (89 6 min) than sh exposed to cell concentrations above 8000 cells ml1 (143 8 min; Fig. 2). A regression (ANOVA) model for Chattonella densities above 1000 cells ml1 suggests that sh mortality time is inversely correlated with cell density (P = 0.44) and closely correlated with superoxide levels (P = 0.67). Total superoxide levels were maximal in algal cultures with higher cell densities of C. marina, but the fastest sh mortality time correlated with low cell densities of 10008000 cells ml1 and was associated with levels of O2 production per cell greater than 40 chemiluminescence units (Fig. 3). No sh mortalities occurred during the 6 h experiments in aerated cultures. Dissolved oxygen (DO) levels in non-aerated experiments were maintained above that of the non-aerated control by oxygen released through photosynthesis of C. marina cells. The higher
200 LT50 (mins) 150 100 50 0 0 10000 20000 30000 Chattonella cell concentration (per ml) R = 0.67
2

Fig. 2. Time to death of 50% of experimental sh (LT50 ) as a function of cell density of C. marina, which was maintained under semi-continuous culture conditions in two separate experiments (n = 8 per treatment). Only one mortality was observed at cell densities <1000 cells ml1 (n = 20) over a 3 h period.

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200 180 160 140 120 100 80 60 40 20 0 50 100

Chemiluminesencse per cell

<1000 cells 1000-8000 cells >8000 cells

developed symptoms of hyperventilation, mucus excretion, vasodilation of the gills (as seen through the opercular cover), followed by an inability to maintain position in the water column. Pre-mortality response included gulping at the water surface before sinking and lying laterally prior to respiratory cessation. 3.2. Xanthine oxidase generated superoxide exposure

150 200 250 Mean mortality time (mins)

300

Fig. 3. Levels of superoxide per cell (determined by chemiluminescence) compared to mortality time of damselsh exposed to C. marina cultures. There is a clear distinction between low cell density (10008000 cells ml1 ) and rapid mortality time compared to high cell density (>8000 cells ml1 ) and longer mortality time. Only one mortality occurred at <1000 cells ml1 (n = 20).

C. marina density resulted in higher DO (r 2 = 0.83; data not shown). C. marina in the presence of sh produced signicantly higher O2 than C. marina not exposed to sh, regardless of whether the cultures were aerated (Fig. 4). Fish exposed to low concentrations of C. marina (10008000 cells ml1 ) rapidly

Superoxide was produced and maintained through periodic additions of the substrate xanthine to the enzyme xanthine oxidase for a period of 3 h. Levels of O produced, observed every 15 min did not deviate 2 signicantly from those assessed in the Chattonella control of 6000 cells ml1 (Fig. 5). Fish exposed to the xanthine oxidase generated O2 did not display stress behaviour for the rst 150 min when compared to the ltered seawater, the non-toxic Dunalliella and Chattonella controls. After this time, the sh appeared sluggish until termination of the experiment at 180 min. No sh mortalities resulted in the xanthine oxidase control trial, nor were any behavioural responses corresponding to Chattonella toxicity noted. 3.3. Brevetoxin analysis The RLB analysis did not indicate the presence of PbTx-like material in any of the fractions. The LC/MS experiments did not detect any of the major PbTxs (PbTx-1, -2, -3, -7, -9, -10). In the 7CHCl3 :1MeOH silica fraction, we observed: (1) [M + Na]+ at m/z 901.50, [M + NH4 ]+ at m/z 896.54, [M + H]+ at m/z 897.50, and [M + HH2 O]+ at m/z 861.50 at 29.1 min; (2) [M + Na]+ at m/z 843.47, [M + NH4 ]+ at m/z 838.47, [M + H]+ at m/z 821.45, and [M + HH2 O]+ at m/z 803.45 at 27.8 min; and (3) [M + Na]+ at m/z 899.48, [M + NH4 ]+ at m/z 894.55, and [M + H]+ at m/z 877.51 at 29.6 min. These chemical species were present at very low levels and the masses do not correspond to any known PbTxs. The residual masses (0.450.55 Da), a function of the relative number of mass decient elements such as oxygen present in each of the molecules, indicate that they share a very similar elemental composition to all known polyether marine toxins and perhaps are structurally similar. Mouse bioassays on lipophilic extracts of both Australian and Japanese strains of C. marina did not

Chemiluminescence Units

60 50 (x 10 )
4

40 30 20 0 50 100 Time (mins) 150 200

C. marina marina + Fish Aerated C. Non-aerated C. C.marina marina + Fish Aerated C. marina Non-aerated C. C. marina marina
Fig. 4. The effect of aeration on total superoxide production of C. marina cultures as determined by chemiluminescence, in the presence and absence of damselsh. Open symbols denote aerated cultures and closed symbols denote non-aerated cultures. Diamonds denote the presence of damselsh and triangles denote no sh present.

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277

90

Chemiluminesence Units

80 70 60

(x10 )

50 40 30 20 10 0 0 20 40 60 80

Time (mins)
Chattonella Chattonella Xanthine Oxidase + Xanthine Xanthine Oxidase + Xanthine every 15 mins
Fig. 5. Production of superoxide generated from the xanthine oxidase system compared to superoxide production by Chattonella marina at 6000 cells ml1 . A single application of xanthine shows a gradual reduction in superoxide levels, however regular additions of xanthine maintained high levels of superoxide.

result in mouse mortality (T = 24 h). There was no visible evidence of mouse liver abnormality or damage on post mortem examination. Fish exposed to pure PbTx-2 and -9 standards at 25 g l1 showed no signs of respiratory distress within the rst 6 h, but did display uncoordinated swimming behaviour (swimming laterally or up-side down) as described by Lewis (1992). 3.4. Free fatty acid exposure Exposure of damselsh to pure EPA resulted in a LC50 of 2.7 mg l1 (Fig. 6), with mortality of 50% of sh occurring at 155 min. Only 25% of sh died when exposed to 25 mg l1 of STA, but all sh showed symptoms of being adversely impacted by a toxic substance. Lethal and sub-lethal doses of EPA and STA resulted in the sh showing pronounced opercular movement (suggesting respiratory distress), production of mucus, inactivity, an inability to maintain position in the water column and a loss of righting reex. The gill region appeared red, suggestive of vasodilation. No symptomatic sh behaviour or mortalities occurred when the sh were challenged with the esteried fatty acids in TAG or the ethyl ester form, the latter containing 66% DHA (22:63).

Fish exposed to superoxide from xanthine oxidases system in conjunction with low levels of EPA (2 and 4 mg l1 ) displayed similar behavioural toxic symptoms to those sh exposed to Chattonella, but exhibited signicantly accelerated mortality rates when compared to sh exposed to EPA on its own (Table 1; Fig. 7a and b).

100 80 % Mortality 60 40 20 0 0 2 4 6 8 10 EPA concentration (mg per L)

Fig. 6. Toxicity of eicosapentaenoic acid (EPA) to damselsh calculated as percentage mortality. The tted polynomial curve of y = 1.77x2 + 28.90x 10.45 (r2 = 0.94) was used to calculate an LD50 of 2.7 mg l1 EPA.

y = -1.77x2 + 28.90x - 10.45 R2 = 0.94

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Table 1 The effect on damselsh exposed to sub-lethal (2 mg l1 ) and lethal (4 mg l1 ) concentrations of the free fatty acid form of eicosapentaenoic acid (EPA) in the presence or absence of superoxide, expressed as time to death (min) EPA treatment (mg l1 ) 2 4 Xanthine oxidase Time to death (min) (EPA only) 131 10 (n = 8) 82 13 (n = 8) 0 (n = 3) Time to death (min) (EPA + superoxide) 51 7 (n = 3) 44 5 (n = 3) EPA equivalent (mg l1 ) 4.9 5.8

The EPA equivalent is calculated from the equation tted to EPA exposure data shown in Fig. 7a.

4. Discussion 4.1. Brevetoxin-like compounds

200 180 160 140 120 100 80 60 40 20 0 0 2 4 6 EPA (mg L-1) EPA EPA + SO Time to death (mins) y = 213.64e
2 -0.18x

R = 0.87

10

(a)

200 180 160 140 120 100 80 60 40 20 0 0 2 4 6 EPA (mg l-1)

y = 58.79e
2

-0.07x

R = 1.00

Two independent investigations have failed to detect the presence of signicant quantities of fatsoluble brevetoxins in Australian strains of C. marina. Hallegraeff et al. (1998) detected only trace levels of brevetoxin-like compounds in an Australian strain (0.0060.03 fg per cell), using brevetoxin radioreceptor assays. In the present work, none of the major PbTxs were identied in an extract by either RLB or LC/MS. The RLB did not suggest the presence of any site 5 sodium channel toxin activator toxins, although it is possible that small amounts of lower potency toxins were present. Similarly, an intraperitoneal mouse bioassay produced negative results for neurotoxicity or liver damage for both the Australian or Japanese (NIES-118) strains. We therefore conclude that no PbTx-like compounds are detectable at signicant levels in the Australian strain of C. marina. This is supported by the observation that exposure of damselsh to pure PTx-2 resulted in sh behaviour comparable to that described by Lewis (1992). By contrast, sh behaviour when exposed to either C. marina cells or the FFAEPA and STAwas characterised by markedly different symptomology. 4.2. Superoxide If C. marina toxicity would be solely a function of a neurotoxin, one would expect a linear increase in sh toxicity with increasing Chattonella cell density, rather than the inverse relationship shown by this study (Fig. 2). These results have been previously replicated for the salmonid Onchorynchus mykiss (Marshall, unpublished data) and support the hypothesis of a synergistic effect of ROS with some other toxic

Time to death (mins)

10

(b)

Fig. 7. Toxicity of eicosapentaenoic acid (EPA) to damselsh. (a) The tted exponential curve produced the response equation y = 213.6e0.18x (r 2 = 0.87). (b) Combined data from exposure of sub-lethal (2 mg l1 ) and lethal (4 mg l1 ) concentrations of the free fatty acid form of EPA, in the presence of xanthine generated superoxide, was tted with a hypothetical exponential curve (y = 58.8e0.07x ) to predict the effect of low concentrations of EPA in the presence of superoxide.

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principle. Superoxide has been claimed to result in damage to the gill epithelium (Ishimatsu et al., 1997; Shimada et al., 1983), causing subsequent respiratory problems. Such toxic insult is thought to be localised at the gill membrane, where contact occurs between the gill epithelia and the Chattonella cells. If this were the case, the greater production of O2 per cell should produce more sh gill lamellae damage. In the present work, cell densities of less than 1000 cells ml1 did not appear to provide toxicity, possibly due to lower levels of algal cell to gill contact. Previous studies have also reported no lethality of Chattonella cultures at cell densities lower than 103 cells ml1 (Haque and Onoue, 2002, 2 103 cells ml1 ; Khan et al., 1996). Temperature also affects lethal cell density for C. ma-

rina, with higher temperatures apparently decreasing the toxic cell density threshold (Okaichi et al., 1989). Superoxide production is inversely related to cell density, possibly to maintain a minimum ambient environmental level of ROS as an allelopathic protection. 4.3. Dissolved oxygen DO self-evidently plays a role in the survivorship of the damselsh. The alleviation of respiratory distress may be instrumental in reducing the toxicity of C. marina. Hyperventilation during periods of low DO would increase ventilation volume and velocity of C. marina cells crossing the gill membranes, enhancing the incidence of gill contact with C. marina

Chattonella cells

Gill Impact

Hyperventilation

Chattonella cell rupture

ROS

Lipid peroxidation

FFA

Fish Death

Gill epithelium damage Reduction of PaO2 Osmoregulatory dysfunction

Fig. 8. Diagram summarising possible mechanisms of sh mortality when exposed to Chattonella derived FFA in combination with the ROS superoxide: Chattonella cells are brought into contact with the sh gill lamellae through ventilation. Cell contents released from broken Chattonella cells hydrolyse in high ROS environment to produce higher levels of FFA and ROS. Lipid peroxidation occurs on gill and other membranes resulting in reduced respiratory and osmoregulatory capacity and allowing the transfer of FFA and O2 into the blood stream. Damage to the chloride cells of the gills can also lead to reduced osmoregulatory capacity. Toxic mechanisms may occur in isolation or combination.

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cells. Aeration also prevented sh mortalities from the dinoagellate K. mikimotoi, but not for Gymnodinium cf. maguelonnense (Jenkinson and Arzul, 2001). Arzul et al. (1998) postulated that increased oxygen availability may be offset by greater free radical mediated cytotoxicity associated with haemolytic activity. In the present study, no signicant increase in O2 production resulted from aeration of algal cultures, but signicantly higher O2 was detected in the presence of sh, indicating that the presence of sh, not aeration, stimulates the greater O2 production by the cells, which may lead to more cytosolic activity. Lectins found in sh mucus have previously been found to stimulate O2 production in C. marina (Oda et al., 1998). 4.4. Free fatty acids We found that PUFAs in the FFA form play a predominant role in Chattonella ichthyotoxicity. The LC50 for EPA in the FFA form was calculated from the tted polynomial curve (Fig. 6) as 2.7 mg l1 for damselsh. Esteried PUFA, such as TAG or an ethyl ester enrichment of DHA (both also containing EPA), are not toxic to sh. Marshall et al. (2002b) found that levels of EPA in C. marina under optimal growth conditions are 1.52 mg l1 and that around 10% of the algal lipid is in the FFA form. By extrapolating the data for the toxicity of FFAs in the presence of O , using an exponential model (Fig. 7b), we pos2 tulate that small amounts of FFAs can become toxic to sh. Our model predicts that 0.2 mg l1 EPA in the presence of superoxide provides a LT50 of 83 min, which is equivalent to either 4 mg l1 of EPA or around 1000 cells ml1 of Chattonella culture. Both concentrations are toxic to sh. The rupture of fragile Chattonella cells may lead to a rapid increase in EPA concentration in the free form in the sh buccal cavity, exacerbated by sh hyperventilation. The liberation of the free form of EPA from cell lysis has been demonstrated with epilithic diatom biolms (Jttner, 2001). Free EPA from lysed diatom cells were shown to provide an allelopathic role against predator grazers, with an LC50 for zooplankton at 34 M (=10 mg l1 ). EPA has also been shown to provide an allelopathic protection of dinoagellates against diatoms at 1.5 mg l1 (Arzul et al., 1998). FFAs in combination with high levels of

ROS may lead to the peroxidation of the biological membranes (Halliwell and Gutteridge, 1999). The presence of FFAs with ROS damaged gill membranes may result in sh mortality through a number of causes: (a) breakdown of gill membranes resulting in reduced respiratory capacity; (b) absorption of FFA or superoxide into the blood stream resulting in reduced blood pH, leading to suppressed respiratory and/or osmoregulatory capacity; and/or (c) destruction of the chloride cells leading to reduced osmoregulatory capacity (Fig. 8). Fish mortality from C. marina cells is most likely due to a combination of all these factors. The above sh killing mechanism is believed to be a common factor in all C. marina strains, which produce high levels of ROS and contain abundant levels of PUFA such as EPA, regardless of brevetoxin concentration. Further research is in progress to more precisely determine the minimum toxic concentrations of FFA and ROS and elucidate the respiratory and cardiovascular sh pathology associated with fatty acid toxicity.

Acknowledgements We thank the following people from the University of Tasmania; Quinn Fitzgibbon and Prof. Ned Pankhurst from the School of Aquaculture for the culture of the damsel sh, Dr. Barry Munday of the School of Biomedical Science for advice on sh pathology, Dr. Tom Ross from the School of Agricultural Science for help with the xanthine oxidase system and models, Andrew Pankowski from IASOS for helpful discussion on oxygen radical chemistry, Helen Bond of the School of Plant Science for culturing assistance. We also thank the Institute of Medical and Veterinary Science, South Australia for the mouse bioassays. This work was supported by the Australian Research Council; Grant No. A00106134 and by the ARC Special Research Centre for Applied Genomics (University of Queensland). Experiments were conducted under the auspices of the University of Tasmania ethics approval No. A0006359. References
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