Unraveling the Fish Kill Mechanism(S) of the Harmful Alga Chattonella Marina, from the Perspective of Osmotic Disturbance

CITY UNIVERSITY OF HONG KONG 香港城市大學

Unraveling the fish kill mechanism(s) of the harmful alga Chattonella marina, from the perspective of osmotic disturbance

有害海洋褐胞藻的魚毒性 — 對魚類滲透壓調節的干擾

Submitted to Department of Biology and Chemistry 生物及化學系 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 哲學博士學位

Xu Jingliang 徐景亮

February 2010 二零一零年二月 i

ABSTRACT

The harmful algal bloom (HAB) species, Chattonella marina, has caused severe economic loss to marine fisheries worldwide. In the past three decades, suffocation or respiratory disorder has been seen as the major mechanism responsible for fish kills from C. marina. However, recent studies have shown that osmotic distress is another probable cause of death of fish exposed to the toxic C. marina. Changes in gill chloride cells (both in density and number) and blood osmolality have been reported in goldlined seabream (Rhabdosargus sarba) exposed to C. marina.

However, the precise toxic mechanism(s), i.e. precisely how C. marina induces such physiological impairments in marine fish is virtually unknown. Consequently the generally accepted reason(s) for the fish deaths must be seen as highly controversial.

The present study attempted to identify the fish kill mechanisms linked to this toxic

HAB species.

We hypothesized that the effects of C. marina on fish are due to a disruption of osmotic homeostasis. More specifically, we proposed that plasma osmolality would increase following exposure to C. marina via one or more of the following pathways: i) inhibition of NaCl secretion in fish gill; ii) disruption to paracellular tight junction of fish gill and gastrointestinal (GI) tract epithelium, and/or iii) inhibition of the water uptake process at the GI tract. By comparing fish susceptibility to C. marina using fish species with different osmoregulatory and respiratory capacities, we would be able to identify the major cause of fish kills by C. marina.

Three local marine fish species, goldlined seabream (Rhabdosargus sarba),

Russell’s snapper (Lutjanus russellii), and green grouper (Epinephelus coioides) ABSTRACT ii were chosen for the fish susceptibility study. The osmoregulatory and respiratory capacities of these species were compared by subjecting the fish to acute hypertonic seawater and hypoxia exposure. Based on the mortality results (LT50), the order of hypoxia tolerance was identified as: green grouper > Russell’s snapper > goldlined seabream; while the hypertonic seawater tolerance was comparable in all three species with similar LT50 around 3 h.

When the three fish species were exposed to bloom concentration of C. marina (10,000 cells/ml), the goldlined seabream was found to be most susceptible to C. marina (LT50 = 5.1 h), followed by the Russell’s snapper (LT50 = 7.5 h). Fish that died as a result of C. marina exposure all exhibited a significant elevation of plasma osmolality. However, the relationship between blood partial pressure of oxygen (pO2) and fish mortality was not consistent. For example, significant decline of pO2 was found in C. marina stressed goldlined seabream, but not in C. marina stressed Russell’s snapper. The green grouper was most tolerant to C. marina. No fish mortality or osmotic disruption was observed in this species throughout the exposure period (48 h), despite the fact that the blood pO2 decreased by up to 68%.

These findings suggest that disturbance of osmotic balance is an important mechanism involved in fish kills by C. marina, and that this disruption is independent of decreases in pO2.

No significant change was observed in gill Na+-K+-ATPase (NKA) activity in goldlined seabream following exposure to C. marina. Therefore, inhibition of NaCl secretion from gills is unlikely to be the cause of abnormal elevation of Na+, Cl- levels in the plasma of fish exposed to C. marina. In the green grouper, a C. marina tolerant species, the levels of gill cyclic adenosine monophosphate (cAMP, a major component of osmoregulatory signal transduction) and plasma cortisol (a crucial ABSTRACT iii osmoregulatory hormone) increased 5.4 and 1.3-fold respectively within 1 h of exposure to C. marina, and returned to the control levels after 6 h. Activation of these energetic regulatory processes could stimulate rapid NaCl secretion from the gills, which may be a response by the green grouper to the osmotic stress induced by

C. marina. The green groupers also secreted copious amount of mucus which covered their gills and which may prevent direct contact with the toxic C. marina.

To further investigate whether C. marina could directly affect the integrity of paracellular tight junction (TJ) in fish epithelia, the esophageal epithelium of goldlined seabream was isolated and mounted onto an Ussing system. The tissue was subsequently exposed to C. marina and changes in transepithelial resistance (TER) were recorded. The TER data confirm that the paracellular tight junction on the esophageal epithelium could be loosened by direct exposure to C. marina. Two modes of action for C. marina on TJ integrity were discovered – one is fast-acting and reversible (C. marina cells at the exponential growth phase), while the other is accumulative and irreversible (C. marina cells at the stationary growth phase).

Loosening of paracellular TJs between fish blood and hypertonic external seawater will undoubtedly accelerate the entry of seawater ions into fish blood and at the same time promote loss of water content from blood into seawater. As a result, the plasma osmolality in fish will increase rapidly and significantly.

In addition, we found a concomitant increase of gut fluid osmolality in goldlined seabream during C. marina exposure, suggesting the desalination process could be inhibited, and insufficient H2O absorption from the gut might be an additional cause of elevation of plasma osmolality.

Overall, findings of the present study provide a major breakthrough on understanding of the fish kill mechanisms linked to the harmful algal species, C. ABSTRACT iv marina. Strong links between fish death and increased osmolality have been observed. Metabolic processes involved in NaCl secretion from the gills may be a mechanism for dealing with osmotic stress following exposure to C. marina. NaCl secretion from the gills is unlikely to be the cause of this increased plasma osmolality.

Importantly, this study provides new evidence that elevation of plasma ions and osmolality in fish exposed to C. marina is mediated directly via a disruption of epithelial paracellular tight junction. Further studies on the direct effects of isolated

C. marina toxins on major TJ core components are therefore suggested. vii

TABLE OF CONTENTS

ABSTRACT i ACKNOWLEDGEMENTS v TABLE OF CONTENTS vii LIST OF FIGURES x LIST OF TABLES xiii ACRONYMS AND ABBREVIATIONS xiv CHAPTER 1 GENERAL INTRODUCTION 1 1.1 Harmful algal blooms and their toxic effects on fish 1 1.2 Severe global economic losses caused by raphidophytes 4 1.3 The toxicity of raphidophytes 6 1.3.1 Are raphidophytes toxic to all kinds of fish? And only to fish? 6 1.4 Chattonella marina – the typical raphidophyte 7 1.4.1 Proposed C. marina toxins and the fish kill mechanisms 9 1.4.1.1 Brevetoxins 9 1.4.1.2 Hemolysin and hemagglutinin 10 1.4.1.3 Reactive oxygen species 12 1.4.1.4 Nitric oxide 14 1.4.1.5 Polyunsaturated fatty acid 14 1.4.2 Two proposed fish kill pathways by C. marina – respiratory disorder vs. osmotic distress 16 1.4.2.1 Recent findings in our research group 17 1.4.2.2 Discrepancy in blood osmolality data 17 1.5 Research hypothesis 20 CHAPTER 2 SUSCEPTIBILITY OF MARINE TELEOSTS TO CHATTONELLA MARINA 21 2.1 Relationship between physiological properties of fish and their susceptibility to C. marina 21 2.2 Research hypothesis 24 2.3 Materials and methods 25 2.3.1 Fish maintenance 25 2.3.2 Mass culture of algae 25 2.3.3 Exposure experiment setup 26 2.3.3.1 Hyperosmotic stress – double strength seawater (DSW) treatment 26 2.3.3.2 Hypoxia (HY) Treatment 26 2.3.3.3 C. marina (CM) treatment 27 2.3.3.4 Seawater (SW) control group 27 2.3.4 Blood collection 27 2.3.5 Blood analysis 28 TABLE OF CONTENTS viii

2.3.6 Statistical analysis 28 2.4 Results 29

2.4.1 Mortality and LT50 in different treatments 29 2.4.2 Fish tolerance to abrupt hyperosmotic shock 31 2.4.3 Fish tolerance to hypoxia 31 2.4.4 Fish tolerance to C. marina 32 2.4.5 Changes of blood pH 32 2.5 Discussion 37 2.5.1 Hypertonic and hypoxic tolerances of selected fish species 37 2.5.2 Fish susceptibility to C. marina is related to their tolerance to hypoxia 37 2.5.3 Osmotic balance is the key for fish survival in C. marina exposure 38 2.5.4 Deduction of fish killing mechanism of C. marina 39 CHAPTER 3 PLASMA OSMOLALITY IN CHATTONELLA MARINA STRESSED FISH 42 3.1 Osmoregulation in marine teleost 42 3.2 Gill chloride cell and sodium/chloride secretion in marine teleosts 42 3.3 The gastrointestinal tract of marine teleosts is important for water absorption 45 3.4 Esophageal desalination 47 3.5 Water absorption in the intestine 48 3.6 Research hypotheses 49 3.7 Methodology 51 3.7.1 Fish maintenance 51 3.7.2 Mass culture of algae 52 3.7.3 Exposure setup 52 3.7.4 Tissue sampling and storage method 53 3.7.5 Detecting osmolality and ion (Na+, K+, Ca2+, Mg2+ and Cl-) levels 54 3.7.6 NKA activity 54

3.7.7 Blood pO2, pH and hematocrit 55 3.7.8 Gill cAMP level and plasma cortisol concentration 55 3.7.9 Statistical analysis 55 3.8 Results 56 3.8.1 Change of plasma osmolality and ion levels 56

3.8.2 Hematocrit, blood pO2 and pH 63 3.8.3 Gill NKA activity, gill cAMP level and plasma cortisol concentration 63 3.8.4 Intestine NKA activity, gut fluid osmolality and ion levels in goldlined seabream 64 3.9 Discussion 64 3.9.1 Increased plasma osmolality in goldlined seabream is not due to the inhibition of gill NKA activity 64 3.9.2 Green grouper shows higher osmoregulatory ability than goldlined seabream in C. marina exposure 66 TABLE OF CONTENTS ix

3.9.3 Insufficient water uptake from the intestine in goldlined seabream might be a cause of the increased plasma osmolality 67 - - 3.9.4 Cl /HCO3 exchange activity along the gastrointestinal tract might be inhibited in goldlined seabream 68 CHAPTER 4 DISRUPTION OF PARACELLULAR TIGHT JUNCTIONS BY CHATTONELLA MARINA CAN BE A DIRECT CAUSE OF OSMOTIC DISTRESS 70 4.1 Relationship between paracellular tight junction and fish osmohomeostasis 70 4.1.1 Tight junction and paracellular transport 70 4.1.2 The structure and function of paracellular tight junctions on teleost branchial epithelium 73 4.1.3 The structure and function of paracellular tight junctions on teleost esophageal epithelium 75 4.2 Disruption of TJ structure and function by microbial toxins 76 4.3 Research hypothesis 80 4.4 Materials and methods 81 4.4.1 C. marina growth curve in culture flasks 81 4.4.2 Preparation of C. marina culture (cm) and cell free C. marina medium (cf) 82 4.4.3 C. marina toxicity test using marine medaka 82 4.4.4 Ussing chamber set up and TER measurement 83 4.4.5 Procedures of esophagus exposure 84 4.4.6 QA/QC for esophagus integrity 87 4.4.7 Statistical analysis 87 4.5 Results 88 4.5.1 Growth curve of C. marina and its toxicity to medaka 88 4.5.2 Transepithelial electrical resistance of goldlined seabream esophagus exposed to C. marina 89 4.6 Discussion 92 4.6.1 C. marina toxicity in different growth stages 92 4.6.2 C. marina toxin(s) and the decrease of epithelial TER 93 4.6.3 Pillar cells, tight junctions and damage of gill lamellae 95 CHAPTER 5 GENERAL DISCUSSION AND CONCLUSION 98 5.1 Osmotic disturbance and fish kills by C. marina 98 5.2 Abnormal elevation of plasma osmolality was not due to an inhibition of NKA activity on fish gill 99 5.3 Impairment of paracellular tight junction on gill epithelium might be the major cause of abnormal plasma osmolality elevation in C. marina stressed goldlined seabream 99 5.4 Insufficient water uptake from intestine may lead to an abnormal elevation of plasma osmolality in goldlined seabream 102 5.5 Conclusion 103 REFERENCE 105

LIST OF FIGURES

Fig. 1.1 Biocompounds produced by C. marina and the speculated fish kill mechanisms. HL = hemolysin, HA = hemagglutinin, ROS = reactive oxygen species, NO = nitric oxide, EPA = eicosapentaenoic acid. 8 Figs. 1.2A&B Blood collection methods used in Tang’s studies (A) and the present study (B). Fig 1.2A Fish blood was collected directly from tail using a 1.7 ml eppendorf containing 0.1 ml heparin solution (170u/ml). Fig 1.2B Fish blood was collected by a 1 ml syringe rinsed with heparin (5000 u/ml) and remaining heparin solution was pushed out from the syringe before use. CM: Chattonella marina treatment; SW: seawater control; DT: non-toxic alga Dunaliella tertiolecta control (see text for details). 19 Fig. 2.1 Osmotic distress and respiratory disorder are the two probable causes of fish death upon exposure to C. marina 22

Fig. 2.2 Median lethal time (LT50) of goldlined seabream, Russell’s snapper and green grouper exposed to double strength seawater (DSW), hypoxia (HY) and the toxic Chattonella marina (CM). NM: no mortality. 30

Figs. 2.3A-D Comparison of plasma osmolality (A-C) and blood partial oxygen level (pO2) (D-F) between double strength seawater (DSW) treatment and seawater (SW) control in goldlined seabream (A & D), Russell’s snapper (B & E) and green grouper (C & F). ali = alive fish, sym = fish sampled when showing stressed symptom. Data are expressed as mean ± standard deviation (n = 15 in SW control, n = 6-11 in DSW treatments) (*, p < 0.05; **, p < 0.01). 33

Figs. 2.4A-E Comparison of blood partial oxygen level (pO2) (A-C) and plasma osmolality (D-F) between hypoxia (HY) treatment and seawater (SW) control for goldlined seabream (A & D), Russell’s snapper (B & E) and green grouper (C & F). ali = alive fish, sym = fish sampled when showing stressed symptom. Data are expressed as mean ± standard deviation (n = 15 in SW control, n = 6-11 in HY treatments) (*, p < 0.05; **, p < 0.01). 33

Figs. 2.5A-E Comparison of plasma osmolality (A-C) and blood partial oxygen level (pO2) (D-F) between C. marina (CM) treatment and seawater (SW) control for goldlined seabream (A, D), Russell’s snapper (B, E) and green grouper (C, F). ali = alive fish, sym = fish sampled when showing stressed symptom. Data are expressed as mean ± standard deviation (n = 15 in SW control, n = 6-11 in CM treatments) (*, p < 0.05; **, p < 0.01). 34 Figs. 2.6A-I Comparison of blood pH between double strength seawater (DSW), aquatic hypoxia (HY), C. marina (CM) treatments and seawater (SW) control for goldlined seabream (A-C), Russell’s snapper (D-F) and green grouper (G-I). ali = alive fish, sym = fish sampled when showing stressed symptom. Data are expressed as mean ± standard deviation (n = 15 in SW control, n = 6-11 in DSW/HY/CM treatments) (*, p < 0.05; **, p < 0.01). 35 Fig. 2.7 Proposed fish kill mechanism of C. marina. 41 Fig. 3.1 Current model of NaCl secretion in the gill of marine teleost (Modified from Marshall, 2002). CC = chloride cell, AC = accessory cell, PVC = pavement cell. 45 Fig. 3.2 The progressive change in the composition of imbibed seawater along the gastrointestinal tract in unfed typical marine teleost fish. Data are derived from a total of 24 different marine or seawater acclimated euryhaline teleosts (Marshall and Grosell, 2006). 46 Fig. 3.3 Current model of NaCl and water absorption in the intestine of marine teleost (Modified from Grosell, 2006). 49 LIST OF FIGURES xi

Fig. 3.4 Proposed causes of elevation of plasma osmolality in C. marina stressed fishes. 51 Figs. 3.5A-L Temporal changes of plasma osmolality and Na+, Cl-, Ca2+, Mg2+, K+ concentrations in goldlined seabream (A-F) and green grouper (G-L) upon exposure to the toxic Chattonella marina (CM), the seawater (SW) and the non-toxic alga Dunaliella tertiolecta (DT) control groups. CM sym = goldlined seabream sampled when stress symptom (loss of body balance) developed. Data are expressed as mean ± standard deviation (n = 15 in SW and DT controls, n = 10 for each time point in CM treatment). A different letter of the alphabet indicates significant difference (p < 0.05) from other treatments within each set of experiment. 58 Figure 3.5A-L (continued) 59

Figs. 3.6A-F Temporal changes of hematocrit, blood pO2 and pH in goldlined seabream (A-C) and green grouper (D-F) upon exposure to the toxic Chattonella marina (CM), the seawater (SW) and the non-toxic alga Dunaliella tertiolecta (DT) control groups. CM sym = goldlined seabream sampled when stress symptom (loss of body balance) developed. Data are expressed as mean ± standard deviation (n = 15 in SW and DT controls, n = 10 for each time point in CM treatment). A different letter of the alphabet indicates significant difference (p < 0.05) from other treatments within each set of experiment. 60 Figs. 3.7A-F Temporal changes of gill NKA activity, gill cAMP level and blood cortisol in goldlined seabream (A-C) and green grouper (D-F) upon exposure to the toxic Chattonella marina (CM), the seawater (SW) and the non-toxic alga Dunaliella tertiolecta (DT) control groups. CM sym = goldlined seabream sampled when stress symptom (loss of body balance) developed. Data are expressed as mean ± standard deviation (n = 15 in SW and DT controls, n = 10 for each time point in CM treatment). A different letter of the alphabet indicates significant difference (p < 0.05) from other treatments within each set of experiment. 61 Figs. 3.8A-F Temporal changes of gut fluid osmolality, Na+, Cl-, Ca2+, Mg2+ concentrations and intestine NKA activity (A-F) in goldlined seabream upon exposure to the toxic Chattonella marina (CM), the seawater (SW) and the non-toxic alga Dunaliella tertiolecta (DT) control groups. CM sym = goldlined seabream sampled when stress symptom (loss of body balance) developed. Data are expressed as mean ± standard deviation (n = 15 in SW and DT controls, n = 10 for each time point in CM treatment). A different letter of the alphabet indicates significant difference (p < 0.05) from other treatments within each set of experiment. 62 Fig. 4.1 Epithelial tight junction (TJ) complex of vertebrates. ZO-1= zonula occluden-1, JAM = junction adhesion molecule, CAR = coxsackievirus and adenovirus receptor. (modified from O’Hara and Buret, 2008). 71 Fig. 4.2 Change of gill tight junction structure in teleost in acclimation between seawater (SW) and freshwater (FW). PVC = pavement cell, AC = accessory cell, CC = chloride cell (or mitochondria rich cell, more precise in fresh water) (modified from Karnaky, 2001). 75 Fig. 4.3 Mechanism utilized by pathogens to act on epithelial tight junctions. ZO-1= zonula occluden-1, JAM = junction adhesion molecule, CAR = coxsackievirus and adenovirus receptor, PKA = protein kinase A, ZO-1 = zonula-occluden-1, PKC = protein kinase C, MLC = myosin light chain, MLCK = myosin light chain kinase. (modified from O’Hara & Buret, 2008). 78 Fig. 4.4 Ussing chamber setup for esophageal epithelium exposure to C. marina. TER = transepithelial electrical resistance, cm = C. marina culture, cf = cell free C. marina culture. 84 Fig. 4.5 Growth curve of C. marina in 25cm2 culture flask. (n=3 for each day). 88 LIST OF FIGURES xii

Fig.4.6 Mortality of marine medaka to day-5 (CM-D5) and day-10 (CM-D10) C. marina culture (n=3 in each treatment). (**, p<0.01). 88 Fig. 4.7 Patterns of typical transepithelial electrical resistance (TER) change in day-5 cell free C. marina culture control (CF-D5) and day-5 C. marina culture treatment (CM-D5). AD = adaptation, S1-5 = step 1-5. 89 Figs. 4.8A-F Comparison of the change of transepithelial electrical resistance (ΔTER) of esophageal epithelium in the CM treatments and CF controls. CF-D5 = day-5 cell free C. marina culture control, CM-D5 = day-5 C. marina culture treatment, CF-D10 = day-10 cell free C. marina culture control, CM-D10 = day-10 C. marina culture treatment, cf1-5 = 1st-5th addition of cell free C. marina culture medium, cm1-2 = 1st – 2nd addition of C. marina culture. In A-D, * indicates significant different (p<0.05) between two adjacent steps in the same treatment; In E & F, * indicates significant difference (P < 0.05) between the same step from different treatments; ns = no significant difference. 91 Fig. 4.9 Speculated toxic mechanisms of C. marina on marine fishes. 95 Figs. 4.10A&B Gill filaments of goldlined seabream, TEM micrographs. Fig. 4.10A Non- toxic D. tertiolecta algal control for 6 h. Lamellae stand upright on the filament with intact pillar cells supporting the respiratory epithelia. Fig. 4.10B 2000 cells/ml C. marina for 6 h. Breakdown of pillar cells (arrows) and collapse of lamellae (arrowheads) are commonly found. Scale bars in A and B = 10 μm. (From Tang, unpublished data). 96 Fig. 4.11 Deduction of fish kill mechanism of C. marina. 97

xiii

LIST OF TABLES

Table 1.1 Typical harmful algal species and their adverse effects on fish. 3 Table 1.2 Documented Chattonella blooms from 1970 to 2004. 5 Table 2.1 A comparison among marine teleosts of their susceptibility to Chattonella marina (LT50) with reference to their physiology and natural habitat. 23 Table 2.2 Mortality of goldlined seabream, Russell’s snapper and green grouper upon 24 h exposure to double strength seawater (DSW), hypoxia (HY) and C. marina (CM) 30 Table 2.3 A summary of fish tolerance to abrupt hyperosmotic shock (DSW). 35 Table 2.4 A summary of fish tolerance to abrupt hypoxia. 36 Table 2.5 A summary of fish tolerance to C. marina. 36 Table 2.6 A summary of fish blood pH alteration in different treatments. 36 Table 4.1 Microbial impairment on tight junction structure and function. 77 Table 4.2 Stepwise changes of culture added to apical side of esophageal epithelium mounting on Ussing chamber. CF-D5 = day-5 cell free C. marina culture control, CM-D5 = day-5 C. marina culture treatment, CF-D10 = day-10 cell free C. marina culture control, CM-D10 = day-10 C. marina culture treatment. (n = 5-7 epithelia in each treatment). 86

xiv

ACRONYMS AND ABBREVIATIONS

AC Accessory cells

AD Adaptation

ASP Amnesic shellfish poisoning

B Brackish water

C. marina Chattonella marina ca. circa, in approximately

Ca2+ Calcium ion cAMP Cyclic adenosine triphosphate

CAR Coxsackievirus and adenovirus receptor

CC Chloride cell

CCFA Chloride cell fractional area cells/ml Cells per millilitre cf Cell free Chattonella marina culture

CFTR Cystic fibrosis transmembrane regulator

Cl- Chloride ion cm Centimetre cm Chattonella marina culture

CM Chattonella marina

CPE Clostridium perfringens enterotoxin

CTX Ciguatoxins

CY Cylindrospermopsin

DA Domoic acid

DNA Deoxyribonucleic acid

DO Dissolved oxygen

DSP Diarrhetic shellfish poisoning ACRONYMS AND ABBREVIATIONS xv

DST Dinophysistoxins

DSW Double strength seawater

DT Non-toxic

e.g. exempli gratia, for example

EPA Eicosapentaenoic acid

EPEC Enteropathogenic E. coli

et al. et alii, and others

F Fresh water

FFA Free fatty acid

g Gram

GI Gastrointestinal

g/L Milligram per litre of volume

h Hour

H2O Water

H2O2 Hydrogen peroxide

HA Hemagglutinin

HABs Harmful algal blooms

HL Hemolysin

HY Hypoxia

JAM Junction adhesion molecule

K+ Potassium ions

L Litre

LT50 Median lethal time

lux The International System unit of illumination, equal to one lumen per square meter

M Marine

MC Microcystins

MDCK Madin-Darby Canine Kidney ACRONYMS AND ABBREVIATIONS xvi

mg/L Milligram per litre of volume

mg/ml Milligram per millilitre of volume

Mg2+ Magnesium ions

Mg2+-ATPase Magnesium ATPase

min Minute

MLC Myosin light chain,

MLCK Myosin light chain kinase

mm Millimetre

mM Millimolar

mmHg Millimetre of Mercury

mOsmol/kg Milliosmole per kilogram

mRNA Messenger ribonucleic acid

MRC Mitochondria rich cells

n Nucleus

n.a. Information not available

NA no LT50

n.r. Data are not reported in the literature

n.t. nontoxic to fish

Na+ Sodium ion Na+,K+-ATPase Sodium potassium ATPase

NKA Na+,K+-ATPase

NKCC Na+-K+-2Cl- co-transporter

nm One billionth (10-9) of a metre

NO Nitric oxide

NSP Neurotoxic shellfish poisoning

OA Okadaic acid

O· Singlet oxygen

O2 Oxygen ACRONYMS AND ABBREVIATIONS xvii

- O2· Superoxide anion radical

OH· Hydroxyl radical

PbTx Brevetoxins

PD Transepithelial potential difference

pO2 Partial pressure of oxygen

PSP Paralytic shellfish poisoning

PKA Protein kinase A

PKC Protein kinase C

PUFA Polyunsaturated fatty acid

PVC Pavement cell

ROS Reactive oxygen species

spp. Species

STX Saxitoxin

TER Transepithelial resistance

TJ Tight junction

USA United States of America

ZO-1 Zonula-occluden-1

µM One thousandth (10-6) of a metre

ΔTER the change of TER