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Response of the shore crabs Hemigrapsus oregonesis and Hemigrapsus nudus to paralytic shellfish toxins Barber, Kathleen Gladys 1988

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RESPONSE OF THE SHORE CRABS HEMIGRAPSUS OREGONESIS AND HEMIGRAPSUS NUDUS TO PARALYTIC SHELLFISH TOXINS by KATHLEEN G. BARBER Sc. (Ag.Sc.) Honours, U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE 1n THE FACULTY OF GRADUATE STUDIES (Department of Food S c i e n c e ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA March, 1988 © K a t h l e e n G. B a r b e r , 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) 11 ABSTRACT The following research deals with the response of the small shore crabs, Hemlgrapsus oreqonesls and Hemlgrapsus nudus to paralytic shel l f i sh toxins (PST). These shore crabs were shown to develop a remarkable seasonal resistance to administered saxitoxin (STX). No similar change 1n sensit iv ity was found after administration of tetrodotoxin (TTX), another marine neurotoxin with similar actions to the PST. Resistance to STX in the small shore crabs was linked to the presence of PST 1n the viscera, and this 1n turn was related to the presence of toxic dlnoflagellate blooms 1n the area. Furthermore, this research provides, for the f i r s t time, evidence of a protein component (MW 145,000 daltons) which appears to be associated with acquired resistance to PST in the shore crab. In addition, this protein component was shown to appear in sensitive crab extracts after the administration of low doses of saxitoxin and tetrodotoxin in vivo. 111 TABLE OF CONTENTS Page ABSTRACT 1i TABLE OF CONTENTS 11i LIST OF TABLES v LIST OF FIGURES v1 ACKNOWLEDGEMENTS vi 1 INTRODUCTION 1 LITERATURE REIVEW 1. Human Intoxication 4 2. Paralytic Shel l f ish Toxins 5 3. Tetrodotoxin 8 4. Actions of Saxitoxin and Tetrodotoxin 9 5. Mechanism of Action 10 6. Organisms Elaborating Paralytic Shel l f ish Toxins 11 7. Bivalve Molluscs 16 8. Other Marine Animals 24 9. Tests for Paralytic Shel lf ish Toxins 27 EXPERIMENTAL I. The Small Shore Crab (Hemigrapsus oregonesis) as a Bioassay for the Detection of Paralytic Shel l f ish Toxins in Shel l f ish 33 II. Pattern of Sensit iv ity and Resistance to Constant Doses STX and TTX in the Shore Crabs Hemigrapsus oregonesis and Hemigrapsus nudus 36 III. Determination of Paralytic Shellf ish Toxins in Shel l f ish and Shore Crabs 38 IV. Gel Electrophoresis of Soluble Proteins in Visceral Extracts from the Shore Crabs Hemigrapsus oregonesis and Hemigrapsus nudus 41 RESULTS AND DISCUSSION I. The Small Shore Crab (Hemigrapsus oregonesis) as a Bioassay for the Detection of Paralytic Shel l f i sh Toxins in Shel lf ish 1. Determination of an Optimum Injectate Volume 1n Hemigrapsus oregonesis 45 2. Determination of Standard Curve for STX and TTX in the Small Shore Crab 45 3. The Crab Hemigrapsus oregonesis as a Test for the Presence of PST in Shel l f ish 50 1v II. Pattern of Sensit ivity and Resistance to Constant Doses STX and TTX in the Shore Crabs Hemiqrapsus oreqonesis and Hemiqrapsus nudus 52 1. Long Term Fluctuation 1n Sensit ivity and Resistance to Constant Doses of STX and TTX 52 2. Lethality Response of Hemiqrapsus nudus to Constant Doses STX and TTX 53 III. Determination of Paralytic Shel l f ish Toxins in Shel l f ish and Shore Crabs 59 IV. Comparison of Soluble Visceral Proteins in Sensitive and Resistant Shore Crabs (Hemiqrapsus oreqonesis, and Hemiqrapsus nudus) using Gel Electrophoresis 1. Protein Content of Visceral Extracts from Resistant and Sensitive Shore Crabs 63 2. Sodium-Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) 64 CONCLUSIONS 75 GENERAL CONCLUSIONS 76 REFERENCES 85 APPENDICES I. Sommer's Table - Death Time: Mouse Unit Relations for Paralytic Shel l f ish Toxins 92 II. Calibration Curve for Fluorescence vs. Saxitoxin Concentration 93 III. Calibration Curve for Absorbance vs. Protein Content 94 V LIST OF TABLES Page Table 1. Summary of PST found 1n bivalve molluscs taken from various areas 19 Table 2. Occurrence of PST in various marine organisms 26 Table 3. Summary of assays developed for the detection of PST in shel l f i sh 30 Table 4. Preparation of separation gels for various polyacrylamide strengths 43 Table 5. Results of analysis of variance on saxitoxin standard curve data from the crab Hemigrapsus oregonesis 48 Table 6a. Results of analysis of variance for the variables: sex in the crab (Hemigrapsus oregonesis) and dose STX 48 6b. Results of analysis of variance for the variables: weight of the crab (Hemigrapsus oregonesis) and dose STX 49 Table 7. Determination of PST in shel l f i sh extracts using the crab (Hemigrapsus oregonesis) bioassay 51 Table 8. A comparison of death times in two shore crabs (Hemigrapsus oregonesis) after administration of 0.05 ug STX and TTX 55 Table 9. Paralytic shel l f i sh toxin content 1n samples of shel l f i sh and shore crabs collected from various Br it ish Columbia locations 60 Table 10. Protein content in visceral extracts of resistant and sensitive shore crabs (Hemigrapsus oregonesis and Hemigrapsus nudus) 64 Table 11. A comparison of antibodies and Inductive enzymes 82 v1 LIST OF FIGURES Page Figure 1. Chemical structures of the paralytic shel l f i sh toxins (PST) 6 Figure 2. Chemical structure of tetrodotoxin 8 Figure 3. Injectate volume vs. death time In the crab Hemiqrapsus oreqonesis 46 Figure 4a. Lethality response to various doses of sax1toxin in the crab Hemiqrapsus oreqonesis 47 4b. Lethality response to various doses of tetrodotoxin in the crab Hemiqrapsus oreqonesis 47 Figure 5. Seasonal pattern of sensit iv i ty and resistance to constant doses of saxitoxin and tetrodotoxin (0.05 yg) in the shore crab Hemiqrapsus oreqonesis 54 Figure 6. Sampling site locations on the southern B.C. coast 58 Figure 7a. A comparison of the soluble visceral proteins found in resistant and sensitive Hemiqrapsus oregonesis by SDS-PAGE (10%) 65 7b. A comparison of the soluble visceral proteins found 1n resistant and sensitive Hemiqrapsus oreqonesis by SDS-PAGE (7.5%) 66 Figure 8. A comparison of the soluble visceral proteins found in resistant and sensitive Hemiqrapsus nudus by SDS-PAGE (7.5%) 67 Figure 9. A comparison of the soluble visceral proteins found in resistant Hemigrapsus oreqonesis from two B.C. locations by SDS-PAGE (7.5%) 69 Figure 10. A comparison of the soluble visceral proteins found in resistant and sensitive Hemigrapsus oregonesis and in sensitive and resistant crabs and STX 1n vivo by SDS-PAGE (7.5%) 71 Figure 11. A comparison of the soluble visceral proteins found 1n resistant and sensitive Hemigrapsus oreqonesis and 1n sensitive and resistant crabs and TTX in vivo by SDS-PAGE (6.0%) 72 Figure 12. A comparison of the soluble visceral proteins found 1n sensitive Hemiqrapsus oregonesis, sensitive H. oreqonesis + varying doses of STX, and resistant H. oreqonesis by SDS-PAGE (6.0%) 77 Figure 13. Phylogeny of invertebrates 79 - V 1 1 -ACKNOWLEDGEMENTS The author would l ike to thank her supervisors Dr. P.M. Townsley and Dr. D.D. Kitts for their knowledgeable advice and creative input during the course of this research project and review of the thesis. She also wishes to thank the members of the research committee, Dr. W.D. Powrie and Dr. J . Vanderstoep both of the Department of Food Science. Much appreciation is also extended to Dr. A. Finlayson for his constant assistance and constructive review of the thesis. The author wishes to acknowledge the valuable expertise and assistance provided by Sherman Yee, senior technician 1n the Department of Food Science. Many thanks are extended to Sam Broese van Groenou, Chris Hansen, Sara Weintraub and Eleanore Wellwood for their encouragement and support during this project. She is also grateful to her parents Laurence and Edith Bamford, and sister Karen Chandler for their support and understanding. A special thanks 1s extended to John Gould for his patience and encouragement. 1 INTRODUCTION Paralytic shel l f ish poisoning (PSP) occurs 1n humans after the consumption of shel l f ish which have been previously contaminated with certain marine dinoflagellates that in turn contain a potent set of neurotoxins, termed paralytic shel lf ish toxins (PST). This type of poisoning can result in death or a temporary incapacitating I l lness. There are no global stat ist ics available on the true incidence of PSP, although reports of Illness and death after consumption of contaminated shel l f i sh have been reported in the l i terature since early times. In North America, a total of 1600 PSP Incidents and some 300 deaths were recorded by Prakash 1n 1974. Although the number of deaths from PSP appears relat ively small, control of poisonings has been achieved through extensive monitoring procedures carried out by government laboratories. PST toxicity values are reported in ug PST/100 g shel l f ish tissue. Any value greater than 71 yg is considered unfit for human consumption. Shellf ish poisonings are d i f f i c u l t to control because of the unpredictable and sporadic occurrence of the dinoflagellates which produce the PST. In addition, contaminated shel l f ish show no distinguishing signs and therefore cannot be separated from non-toxic she l l f i sh. Due to the expense and need for trained personnel, monitoring is restricted to the commercial beds and popular harvesting areas. Br it ish Columbia for example, has approximately 14,000 miles of coastline and an estimated 70% of this 1s permanently closed to the harvesting of shel l f i sh for this reason (Lutz, 1984). The relationship between PST and certain plankton organisms of the genus Gonyaulax was i n i t i a l l y discovered by Sommer et a l . (1937). Dinoflagellates are a rich source of food for many shel l f ish such as clams, mussels, oysters 2 and scallops. Shel l f ish become toxic within a few days of being exposed to a 'bloom' of poisonous dinoflagellates due to their unique ab i l i t y to bind and accumulate the ingested PST. With the exception of the Alaska butter clam which remains toxic for several years, contaminated shel l f i sh generally lose their toxic i ty in several weeks and again become safe for human consumption. In addition to the public health problem, the economic consequences of toxic dinof lagellate blooms can be severe, especially in B.C. where shellf ish harvesting is a large and growing industry. The closure of shel l f ish beds leads to heavy losses for fishermen, f ish and shel l f i sh processors, wholesalers and retai lers as well as related industries such as tourism, recreation and restaurants. The economic effect of red tide outbreaks is extensive. After a survey of secondary effects caused by a red tide outbreak 1n Eastern Canada, Jensen (1974) reported that restaurants noted a 50% reduction in sales of seafood dishes, fishermen were unable to sel l lobsters, and there was 25% to 50% decrease in sales of shel l f i sh and f in f i sh . In addition, the occurrence of a toxic bloom in one area can lead to a depression in demand for shel l f i sh from other unaffected areas. A decrease of 25% was reported for clam sales in areas not stricken with the toxic bloom. Despite the public health and economic implications associated with PSP, knowledge is incomplete 1n many basic areas. For example, i t is s t i l l unclear why and how certain algae are able to produce the PST. Equally unclear are the mechanisms of accumulation and excretion 1n resistant marine animals. In addition, the mechanism of action causing poisoning in humans 1s poorly understood. Until such fundamental questions of PSP are answered, we cannot hope to solve more practical problems such as: an antidote for PSP in humans, an acceptable procedure for the detoxification of contaminated she l l f i sh , a superior alternative to the o f f i c i a l mouse bioassay for the 3 detection of PST 1n shel l f i sh and a method for predicting when and where a toxic bloom wi l l occur as well as how toxic i t wi l l be. The original objective of this research was to Investigate the poss ib i l i ty of developing a more practical bioassay to the mouse bloassay for the prediction of PST 1n she l l f i sh. The small shore crab Hemigrapsus  oregonesis was chosen 1n this capacity because of Its ava i lab i l i ty In coastal areas where shel l f ish are commonly harvested, and because of i ts relative ease of handling compared to other marine animals. Preliminary results of this thesis indicated that this small crab became seasonally resistant to saxitoxin (STX), one of the PST. Although this fluctuation in sensit iv ity was considered a major disadvantage to the crab as a more practical alternative to the mouse bioassay, 1t also provided the basis for another l ine of inquiry, namely, the mechanism of seasonal resistance to STX 1n the small shore crab Hemiqrapsus oregonesis. Therefore, the specif ic objectives of this study were: 1. To determine the long-term pattern of sensit iv i ty and resistance to saxitoxin 1n the green shore crab Hemiqrapsus oreqonesis, and the purple shore crab Hemigrapsus nudus. 2. To assay resistant and sensitive shore crabs for the presence of paralytic shel l f i sh toxins. 3. To compare electrophoretic profi les of soluble visceral proteins 1n resistant and sensitive shore crabs. 4 LITERATURE REVIEW 1. Human Intoxication Paralytic shel l f i sh poisoning may be diagnosed easily in humans by the appearance of certain symptoms which can become apparent within a few minutes after consuming toxic she l l f i sh. In i t ia l ly there is a tingling or burning sensation of the l ip s , gums, tongue and face with a gradual progression to the neck, arms, f ingertips, legs and toes. Later, this changes to numbness and general muscular incoordination. In severe cases constrictive sensations of the throat, ataxia, aphonia and incoherence of speech are prominent symptoms. Other associated symptoms Include: weakness, dizziness, malaise, prostration, headache, sal ivation, rapid pulse, Intense th i r s t , impairment of vision and even temporary blindness. Less common are gastrointestinal symptoms such as nausea, vomitting, diarrhea and abdominal pain, while muscular twitchings and convulsions are rare. In the terminal stages of the disease, motor weakness and muscular paralysis become progressively more severe. Death may result from respiratory paralysis within 2 to 24 hours depending on the magnitude of the dose of PST, and the individual. If one survives 24 hours the prognosis 1s good and there appears to be no lasting effects from the i l lness . Because there 1s no effective antidote for PSP, treatment is largely symptomatic. Emesis should be induced immediately after symptoms appear and i f respiratory d i f f i cu l t i e s appear, a r t i f i c i a l respiration should be applied and continued for several hours. Drug therapy has had variable success. The anticurare drugs are useful 1n aiding a r t i f i c i a l respiration, and oxlmes can be used to counteract the acetylcholine esterase-11ke Inhibitory effects of the PST (Murtha, 1960). Dig i ta l i s and alcohol are not recommended (Pepler and Loubser, 1960). 5 2. Paralytic Shel l f ish Toxins (PST) a. Chemistry Knowledge of the chemistry of paralytic shel l f i sh toxins (PST) has increased considerably in recent years. Numerous advances have been made in the detection, isolation and structural determination of the various PST. Until 1975, i t was thought that saxitoxin (STX) was the sole toxin involved in PSP. STX was isolated in a purified state by Schantz et al_. (1957) who determined i ts chemical properties. STX is a colorless, hygroscopic sol id, very soluble in water, partly soluble in methanol or ethanol but insoluble in most non-polar solvents such as ethyl and petroleum ethers. STX shows no useful absorption in the ultra-v io let range and has an optical rotation of 130, and two 2 pKa's at pH's 8.2 and 11.5. The molecular formula for STX as the free base is C 1 0 H 1 7 N 7 0 4 with a molecular weight of 299. The toxin is often found in the form of the dihydrochloride salt (C 1 0 H 1 7 N 7 0 4 «2HC1) with a molecular weight of 372. Early studies on the chemical structure of STX were hampered by the highly polar nature of the molecule which prevented attempts at STX crysta l -l i zat ion. In 1974, Rapoport and associates determined that STX was a tetrahydropurine derivative and the structure was f ina l l y elucidated by Schantz et al_. (1975) using X-ray crystallography. It became increasingly clear that many contaminated shel l f i sh and toxic dinoflagellates contained other neutral or weakly basic toxins in addition to the highly basic STX. Al l were shown to have actions similar to those of STX although potencies varied considerably. Twelve PST have been characterized to date. The structures of the known PST can be found in Figure 1. 6 Rl R2 R3 R4 1 H H H H 2 H H H SO3-3 H H OSO3- H 4 H H OSO3- SO3-5 H 0S03- H H 16 * 6 H OSO3- H SO3-7 OH H H H 8 OH H H SO3-9 OH H OSO3- H 10 OH H OSO3- SO3-11 OH OSO3- H H 12 OH OSO3- H SO3-Saxitoxin (STX) Bl Gonyautoxln 2 (GTX2) CI Gonyautoxln 3 (GTX3) C2 Neosaxltoxin (NEO) B2 Gonyautoxln 1 (GTX1) C3 Gonyautoxln 4 (GTX4) C4 Figure 1. Structures of the paralytic shel l f i sh toxins (PST) ref: Hall and Reichardt (1984) The twelve PST consist of a parent compound, saxitoxin, and eleven derivatives formed by the addition of N-l-hydroxyl, 11-hydroxysulfate and 21-sulfogroups, respectively. STX contains two formal guanidinium groups (C's 1, 2, 3 and 7, 8, 9) which are strongly basic and behave as a di-cation when the pH is less than 8 (Hall & Reichard, 1984). It has a purine base with a 3-carbon bridge linking positions 3 and 9 and a methyl carbamate at position 6. Compounds with 21-sulfo or 11-hydroxysulfate groups wi l l have a net charge of +1 at pH's less than 8 and those with both groups wi l l have l i t t l e or no net charge. 7 b. Specific Toxicit ies The specif ic tox ic i t ies of the PST are varied. A toxic i ty value of 5500 mouse units (MU) per mL of the dihydrochloride salt was assigned to saxitoxin by Schantz et aj.. (1957). It was also determined that 1 MU Is equivalent to approximately 0.18 yg STX dihyrodrochlorlde. The specif ic tox ic i t ies of neosaxitoxin (NEO) and gonyautoxins (GTX) I, II and III and IV were determined by Geneuah and Shimizu (1981). GTX III has a specif ic toxic ity of 5641 MU and a toxic i ty relative to STX of 103, making i t the most potent of the PST series. GTX I has a specif ic toxicity of 3975 and a relative toxic ity of 72, while NEO and GTX's II and IV have similar tox ic i t ies of 30 to 40% that of STX. c. Cryptic Toxins Each of the sulfamate toxins (CI, C2, C3 and C4) is far less potent than its corresponding carbamate (GTX's 1, 2, 3 & 4), and therefore the sulfamate toxins when present in contaminated she l l f i sh, constitute a reservoir of latent or cryptic toxic i ty (Hall & Reichardt 1984, Yasumoto 1985). Hall et al_. (1980) found that complete hydrolysis of the sulfamate group can be accomplished by heating shel l f i sh extracts to 100°C for 5 minutes in aqueous HC1 (0.1 M). Due to the increase in toxicity associated with this conversion, i t 1s d i f f i c u l t to accurately determine the true potencies of extracts containing sulfamate toxins. There 1s some concern that hydrolysis of the sulfo-group could take place under conditions of food storage, preparation or digestion. 8 3. Tetrodotoxin (TTX) Tetrodotoxin 1s a potent marine neurotoxin found chief ly 1n the gonads and eggs of certain species of pufferfish from the family Tetraodontidae. It has also been shown In the Cal i fornia newt Taricha (Mosher et al.., 1984), a goby Gobus eriniger (Noguchi and Hashimoto, 1973), two species of parottflsh Ypsiscarus ovifrons and Scarus pjbbus, an angelfish Pmacanthus semicirculatus, and two species of xanthid crabs Aterqatis floridus and Zosimus aeneus (Yasumoto et aj.., 1986). Tetrodotoxin 1s an amino perhydroqulnazal1ne compound with an empirical formula of C11H17N3O8 and a molecular weight of 319.3. Its crystals are colorless prisms which are only sparingly soluble in water except in s l ight ly acid solutions. At pH's below 3 or above 7 decomposition occurs. In alkaline solutions TTX 1s degraded Into several quinazollne compounds. The TTX molecule (Figure 2) has a cycl ic hemllactal grouping and a zwltterlon structure with a single guanidium cation and hemilactal anion. Figure 2. Structure of tetrodotoxin ref: Kao 1983 9 4. A c t i o n s o f S a x i t o x i n and T e t r o d o t o x i n S a x i t o x i n (STX) and t e t r o d o t o x i n (TTX) e x h i b i t s i m i l a r b i o l o g i c a l a c t i o n s . S y s t e m a t i c a l l y they produce a neuro-muscular b l o c k , hypotension and r e s p i r a t o r y d e p r e s s i o n but do not a l t e r the responses of e f f e c t o r c e l l s to autonomic agents. These e f f e c t s are due to an a b i l i t y o f the t o x i n s to prevent the i n c r e a s e of the e a r l y t r a n s i e n t 1on1c p e r m e a b i l i t y which allows the downhill Inward movement of sodium 1ons r e q u i r e d f o r the generation of an a c t i o n p o t e n t i a l . The pharmacological a c t i o n o f STX and TTX 1s s i m i l a r l y e x e r t e d on membranes t h a t respond with r e g e n e r a t i v e s p i k e processes. These Include motor axons and muscle membranes or common e x c i t a b l e c e l l s . The a b i l i t y of STX and TTX to block a c t i o n p o t e n t i a l s occurs without a l t e r a t i o n o f the r e s t i n g p o t e n t i a l . B l o c k i n g can be p a r t i a l o r complete, and i t i s r e v e r s i b l e a t low doses STX or TTX (Kao, 1964). N e i t h e r STX nor TTX have an e f f e c t on the c h o l i n e s t e r a s e system or on the a c t i v e sodium e x t r u s i o n process (Kao, 1967). Other molecular components of the nervous system which are u n a f f e c t e d Include: potassium channels, the sodium-potassium a c t i v a t e d ATPase or post s y a p t i c r e c e p t o r s (Benzer and R a f e r t y , 1973). There are some i n t e r e s t i n g d i f f e r e n c e s between the a c t i o n s of STX and TTX. Whereas TTX lowers systemic a r t e r i a l p ressure (hypotension) a t a l l doses which cause neuromuscular weakening, STX 1n low doses does not a f f e c t blood p r e s s u r e (Kao, 1967). The f u l l s i g n i f i c a n c e o f t h i s d i f f e r e n c e i s not known because the mechanism causing hypotension 1s not c l e a r . Another d i f f e r e n c e i n the a c t i o n s of STX and TTX Involves t h e i r e f f e c t s on the c e n t r a l nervous system. TTX 1s a powerful emetic and hypothermic agent, which has not been r e p o r t e d f o r STX. The emetic a c t i o n 1s b e l i e v e d t o be exerted on the hypothalmus (Kao, 1967). TTX and STX a l s o d i f f e r 1n t h e i r e f f e c t s on tetrodon and t a r l c h a nerves. These nerves are s u s c e p t i b l e to STX but are h i g h l y r e s i s t a n t t o TTX. 10 5. Mechanism of Action In nerve and muscle ce l l s , an action potential 1s Initiated by a large but transient Increase in membrane conductance to sodium. Current concepts of excitable membrane organization consider the Increase in conductance to be mediated by a specif ic intr ins ic membrane protein which provides a voltage dependent aqueous pathway for cations across the membranes hydrophobic interior (Weigele & Barchi, 1978). It is thought that sodium and potassium ions, and other major ionic channels possess some definite spatial relationship to each other (Kao, 1983). Until recently the most common assumption has been that the positively charged guanidinium group of STX and TTX enters Into the sodium channel and that the rest of the molecule obstructs the channel by bonding with adjacent parts of the outer membrane. This theory has been challenged by Kao (1983) who provided evidence for a new model to explain the physical mechanics causing the blockage of the sodium channel. In his paper, Kao presented a hypothetical surface receptor site for STX and TTX. The cationlc guanidinium group (planar) projects over the outside or i f ice of the sodium channel. Bonding forces would be electrostatic attraction between charged guanidinium groups and fixed anionic charges around the sodium channel. In addition, there would be weak bonding forces (probably H bonds) between twin hydroxy groups and the membrane receptor. It 1s unlikely that covalent bonds at the C-12 hydroxy position of STX and the membrane are involved, because reduced STX which retains some intr ins ic act iv i ty has an alcoholic configuration which cannot form covalent bonds (Koehn et a l . , 1981; Kao, 1983). This view receives some support from the fact that both toxins are posit ively charged at pH 7 by virtue of their guanidinium groups and both contain many hydroxy and amino groups on the surface which are potential H bond donors (Strichartz, 1984). 11 During his research on the mechanism responsible for the actions of STX and TTX, Kao (1983) also Investigated the structure/activity relationships of the toxins. The following groups of STX were found to be essential for act iv i ty : 7, 8, 9 guanidinium group 1n i ts catlonlc charged form, the C-12 hydroxy group and the carbamyl group. In addition, 1t was determined which groups on the TTX molecule were stereospedf ica l ly similar to the active groups on the STX molecule. The 1, 2, 3 guanidinium group of TTX (charged) was shown to be analogous to the 7, 8, 9 group of STX. Likewise the C-9 and C-10 hydroxys of TTX were shown to be similar to the C-12 hydroxy of STX, and the C-8 hydroxy of TTX and the carbamyl group of STX were located 1n comparable stereospedfic positions. 6. Organisms Elaborating PST a. Description of dinoflagellates Marine protozoa poisonous to man are largely derived from the class Mastigophorans, family Peridiniidae and order Dinoflagellata. The dino-f lagel lates form an important part of ocean plankton as primary producers of carbohydrates, proteins and fats and are considered the foundation of the marine food chain. Dinoflagellates are microscopic, single c e l l , photosyn-thetic and often bioluminescent algae generally possessing a heavy thecal cel l wall and two mobile f lage l la . In thecate species with pe l l i c les , the cellulose plates can be thrown off leaving an "ecdaysal" or temporary cyst which can synthesize a new theca 1n a short time. Because the nutrition of the dinoflagellates overlaps that for plants and animals, they are often referred to as plant-animals. During their periodic maxima, local discolorations of coastal waters called 'blooms' can occur. Blooms may be yellow, brown, green, black, milky 12 or red. Blooms of Gonyaulax species are referred to as a 'red t i d e ' . The number of organisms needed to produce a red tide 1s numerous, approaching 20 to 30,000 per mL. The red color is caused by a xanthophyll (perldlnin 1n a chlorophyll containing protein) which is part of the l ight harvesting antenna of photosystem II (Prezelen and Randall, 1978). Red tide blooms are remarkably monospecific, with 90-95% of phytoplankton belonging to a single species (Sweeney, 1974). There are three general hypotheses on the competitive advantage(s) which allows a single species to form a monospecific bloom. These are, that the species may divide more rapidly than competing phytoplankton because of the presence of a specif ic growth factor, toxic dinoflagellates may excrete substances which Inhibit the growth of competitors, or behavioral differences may give an advantage. Factors affecting blooms Include; the temperature, water turbulence, transparency, surface illumination and the grazing action of the local zooplankton population. An increase in temperature and l ight Intensity and a relat ively stable water column are known to stimulate the growth of blooms (Prakash 1974). Dinoflagellates responsible for the transfer of PST to certain marine animals are of the genera Gonyaulax (species catenella, acatenella and tamarensis-excavata and Pyrodinium (species phoneus, bahamense var bahamense and bahamense var compressa). In 1979, Taylor proposed that a new genus be formed called Protoqonyaulax, for the three toxic species of the genus Gonyaulax. The reason for this transfer is that these three species d i f fer 1n many respects from other species 1n the genus Gonyaulax. for example 1n epithel ia l plate pattern, hypothecal pattern, apical pore, degree of displacement and cyst type. 13 b. Location The toxic dinoflagellate species can be found 1n many parts of the world. G. catenella 1s found along the Pacif ic coast of North America from southern Cal i fornia to south eastern Alaska (Neal, 1982), on the south-eastern coast of Japan (Hashimoto et a l . , 1976) and on the west coast of South Africa (Taylor, 1984). G. tamarensls has been reported 1n Alaska, the North Atlantic from Long Island to the Arct ic , and Portugal north to Norway (Heimdal, 1983), as well as Venezuela (Reyes-Vasquez et aj.., 1979) and Japan (Fukuyu, 1979). G^ acatenella appears to be specif ic to coasts 1n the north-western Paci f ic. P_^  bahamense var compressa has been reported in Japan, Brunei, Palau and Papau New Guinea as well as in Indonesia and Malaysia (Taylor, 1984). c. Other Aquatic Organisms In addition to the toxic dinoflagellates, other aquatic organisms have also been shown to elaborate PST. The occurrence of PST in the fresh water cyanobacterium Aphanizomenon flos-aquae was reported by Mahmood and Carmlchael 1n 1986. In 1983, searching for the source of PST 1n coral crabs and gastropods in Japan, Kotaki, et aj..(1983) discovered a calcareous red algae Janis as the toxin progenitor. This organism has also been shown to produce tetrodotoxin (Yasumoto et a l . , 1986). d. PST Levels and Profi les The prof i les and concentrations of PST in organisms responsible for PSP are extremely variable. A recent report by White (1986) suggests that previous data on the toxin contents of various dinoflagellates cultured under laboratory conditions may be inval id. Concentrations of PST were determined 14 i n Gonyaulax tamarensis var excavata o c c u r r i n g n a t u r a l l y and values were found to be 4 to 20 times higher than any values p r e v i o u s l y obtained i n l a b o r a t o r y c u l t u r e d c e l l s , from 2.7 x 1 0 - 6 ug to 1.1 x 1 0 - 3 yg PST. Another f i n d i n g of t h i s study was that the PST amounted to approximately 4 per cent of the dry weight of the c e l l s . White (1986) a l s o found very high t o x i n l e v e l s i n the r e s t i n g c y s t s of G tamarensis var excavata. Values were as high as those found 1n nearby blooms. Toxic c y s t s sink to the bottom of the water column and accumulate i n the f l o c c u l e n t l a y e r s at the sediment/water i n t e r f a c e where they may overwinter. I t has been suggested that they may be a s i g n i f i c a n t f a c t o r i n the spread of PSP. Shimizu (1979) analyzed the t o x i n contents and p r o f i l e s of G. tamarensis and G. c a t e n e l l a c u l t u r e d i n the l a b o r a t o r y and c o l l e c t e d from natural blooms. In t h i s study, a l l samples contained several to x i n s with STX p l a y i n g a r e l a t i v e l y minor r o l e i n the t o t a l t o x i c i t y , which confirmed previous f i n d i n g s that t o x i n p r o f i l e s and l e v e l s vary c o n s i d e r a b l y among and between d i f f e r e n t s p e c i e s . e. B i o s y n t h e s i s In s p i t e of advances i n the chemistry of the PST, u n t i l very r e c e n t l y l i t t l e was known about the b i o s y n t h e t l c o r i g i n of these b i o l o g i c a l l y important molecules. I t has been suggested by Kodama et a l . , (1982), that the PST are products of non-toxic precursors which are hydrolyzed i n the d i g e s t i v e t r a c t of s h e l l f i s h a f t e r consumption of contaminated algae. Kodama proposed that s h e l l f i s h RNAase, a c t i n g on plankton RNA, r e s u l t e d 1n the formation of PST. Shimizu, at the U n i v e r s i t y of Rhode I s l a n d , has been instrumental 1n e l u c i d a t i n g the b i o s y n t h e t l c pathway of the s a x i t o x i n analogs. His f i r s t r eport (1984) discussed the o r i g i n of the unique perhydropuNne nucleus of the STX d e r i v a t i v e s . Various feeding experiments with s p e c i f i c a l l y l a b e l l e d 15 p r e c u r s o r s on Aphanizomenon flos-aquae (a f r e s h water blue green algae) and Gonyaulax tamarensis, showed t h a t e i t h e r o r t h i n i n e or a r g i n i n e serve as the b i o g e n e t i c p r o g e n i t o r s f o r the t o x i n nucleus. In a l a t e r study (1985) t h i s group determined the o r i g i n o f the s i d e chain carbon (C-13) of n e o s a x i t o x i n . T h i s was accomplished by f e e d i n g [ l , 2 - l , C ] g l y c i n e , D,L-[3- 1*C] s e r i n e and L - [ S - m e t h y l - l , C ] methionine to A. flos-aquae which r e s u l t e d i n a d i s t i n c t enrichment a t the C-13 p o s i t i o n of the n e o s a x i t o x i n molecule. The fundamental q u e s t i o n o f how these organisms come to produce or possess PST remains to be s o l v e d . PST are found i n s e v e r a l t a x o n o m i c a l l y d i s t i n c t a l g a e . A l l are known to have non t o x i c s t r a i n s as w e l l . Shimizu (1986) c a r r i e d out an ex t e n s i v e study on 40 s t r a i n s o f Gonyaulax from v a r i o u s l o c a t i o n s i n an attempt to determine the o r i g i n o f the PST. R e s u l t s showed th a t t o x i g e n i c i t y i s inhe r e n t to s p e c i f i c s t r a i n s and i t was p o s t u l a t e d that t h i s phenomenon was due to plasmids or some other minor g e n e t i c changes. Research e f f o r t s by Shimizu are now focused on the i d e n t i f i c a t i o n o f the p a r t i c u l a r g e n e t i c f a c t o r common to a l l organisms known to e l a b o r a t e the PST. f . P r e d i c t i o n o f Blooms There i s very l i t t l e i n f o r m a t i o n i n the l i t e r a t u r e r egarding p r e d i c t i o n s of when and where t o x i c d i n o f l a g e l l a t e blooms w i l l occur. Gaines and T a y l o r (1985) analyzed the records of t o x i c i t y i n B r i t i s h Columbia from 1955 to 1982 and found the f o l l o w i n g p a t t e r n s : I. t h a t there was no p r o g r e s s i v e i n c r e a s e i n t o x i c i t y over the y e a r s . I I . t h a t w i d e l y separated areas became t o x i c i n s i m i l a r y e a r s . H i . t h a t there are 3 regions o f c o n s i s t e n t l y high t o x i c i t y i n B r i t i s h Columbia. 16 a northern mainland coast b the s t ra i t between the northern half of Vancouver Island and the mainland c the southwest coast of Vancouver Island. iv. that the 2 southern regions are most toxic in the summer/fall months, and the north region 1n winter months v. that there is a 7 year pattern of PSP (the massive blooms of 1986 in B.C. were predicted by these researchers on the basis of these patterns). These researchers concluded that the mechanism of incubation and distribution of toxic dinoflagellates is very complex. Chiang (1985) also examined the records of toxicity in Br it ish Columbia, from 1963 to 1984 and developed a 'PSP Act iv ity Scale' on the basis of these figures. The PSP act iv i ty level is determined by multiplying an extensivity index by an intensity index. The extensiveness of the area affected is given as the percentage of samples showing a positive response, and the intensity of a bloom is the ratio between samples showing PST levels greater than 210 ug/lOOg and those showing levels less than 80 ug/lOOg. This PSP Act iv ity Scale is a very general index and as yet has not been used with any degree of success except to indicate when a very bad year may occur. 7. Bivalve Molluscs a. Description The fact that mussels can contain a potentially fatal substance at certain times of the year has been reported 1n the l i terature since 1793 (cited from Sommer et a l . , 1937). However, i t was not until a massive outbreak of poisonings in July of 1927, in the San Francisco area, that research was started in earnest to determine the nature and cause of this phenomenon. 17 Until 1929, 1t had been thought that mussels were the only marine organisms affected. In fact, the toxin causing PSP was termed 'mussel poison' until this time. The fact that other bivalves, 1n addition to mussels, may become contaminated was demonstrated in August 1929 after three people died from consumption of the Washington clam Saxidomus nuttali (Sommer et al.., 1937). Further research on other clam species showed that the following were also implicated: horse neck clam - Schizothaerus nutta l i , l i t t l e neck clam -Paphia staminea, razor clam - Sil igua patula. rock clam - Pholadidea penita and the Pismo clam - Tivela stultorum. Since the pioneering work by Sommer and his associates, v i r tua l ly a l l species of clam, mussel, oyster and scallop have been shown to accumulate PST. b. PST Distribution in Toxic Shel l f ish The toxin distribution of the PST in shel l f i sh is variable, depending on the type of bivalve (mussel, clam, oyster, scallop), the specif ic tissue analyzed and the col lection site (see Table 1). With the exception of the various clam species studied, saxitoxin appears to play a relat ively minor role in the toxic i ty of most shel l f i sh. Gonyautoxins 1 and 2 appear as the major toxins in the mussel M. edulis, the two species of scallop and the oyster regardless of the location. The mussel M. californianus from Cal i forn ia, on the other hand, contained principal ly neosaxitoxin, with a second sample also containing saxitoxin. It was postulated (Whitefleet-Smith et aj.., 1985) that the STX 1n the one mussel sample could be a result of the reductive conversion of NEO to STX which has been previously shown in the soft shell clam Mya arenaria (Shimizu, 1977) and the scallop Placopecten  magellanicus (Shimizu and Yoshloka, 1981) which was also shown to reduce GTX's 1-4 to STX. This conversion may also explain the presence of STX in the M^ . 18 Table 1. Summary of PST found In bivalve molluscs from various areas. Bivalve Mollusc Location Dlnoflagellate Body part affected PST Prof i le (In order of Importance) Reference Mussel (Mytllus edulls) Oasle Bay, G. catenella not given Japan GTX'S 1,2,5, STX GTX'S 3,4 Shimizu, 1979 V1go, Spain Gonyaulax spp STX GTX's 2,5,1, 4,3, Haines, Alaska G. catenella GTX's 1,2,3,4, 5, NEO, GTX 6 E l f i n Cove, Alaska C. catenella GTX's 1,2,3,4, 5,6, STX Mussel (Mytllus Bodega Bay, cal1forn1anus) Cal i fornia G. catenella whole body Site 1 whole body Site 2 NEO, GTX's 1,2,3, Whltefleet-4, Smith et a l . , 1985 NEO Seal lop (Placopecten Bay of Fundy G. tamarensis hepatopancreas GTX 1,2, NEO, Hsu et a l . . magellanlcus) Canada Cyst form (small amts. STX, GTX 7 1979 also found 1n Mm, gonads and g i l l ) Scallop (Patlnopecten Ofunato Bay G. tamarensis  yessoenls) Japan hepatopancreas GTX's 1,2,3,4,5, Maruyama et (small amts. STX, NEO al_., 1983 found 1n rectum, foot, gonads, g i l l and mantle) 19 Table 1. Summary of PST found 1n bivalve molluscs from various areas, (continued) Bivalve Mollusc Location Dlnoflagellate Body part affected PST Prof i le ( in order of Importance) Reference Oyster (Crassostrea glgas) Senzak! Bay Japan G. catenella midgut gland GTX's 1,2,3,5, STX, NEO Onoue et a l . 1980 Clam-butter (Saxldomus nuttal1) Bodega Bay, Cal i fornia G. catenel1 a neck tissues body STX, NEO NEO, GTX'S 1,2, 3,4 Whltefleet-Smlth et al_., 1985 Clam-butter — — siphon STX Schantz et al.., 1957 (Saxldomus glganteus) Porpoise Island, Alaska G. catenella not given STX, NEO 4 Whltefleet-Smlth et al.., 1985 Clam-soft shell (Mya arenarla) Puget Sound Washington G. tamarensls whole body GTX'S 1,2, BI, NEO Jonas-Davles and Uston, 1985 Clam-soft shell (Mya arenarla) Essex, Massachusetts G. tamarensls not given GTX'S 2,1, STX, GTX'S 3,4 Shimizu, 1979 Hampton, Massachusetts G. tamarensls GTX 2, STX, GTX'S 1,3 Clam-Man1la (Tapes joponlca) Oase Bay Japan G. catenella not given GTX's 1,2,5, STX, Shimizu, 1979 GTX'S 3,4 20 edulis sample from Spain. Gonyautoxlns 1 and 2 were also the major toxins in the soft shell clam M. arenaria and the Manila clam Tapes japonlca. Whitefleet-Smith et al.. (1985) noted a striking var iab i l i ty of basic/weakly basic toxin ratio among samples of Saxidomus nuttal1. Necks of S. nuttali contained the basic toxins NEO and STX exclusively while the bodies had equal proportions basic and weakly basic toxins. This suggests either di f ferentia l storage sites for the various toxins or degradative loss of certain toxins. Since the toxins are a l l closely related structural ly, metabolic interconversions of the toxins may be the reason for d iss imi lar i ty in those toxin prof i les . c. Physiology and Behavior The macroscopic appearance of toxic shellf ish does not d i f fer from that of normal she l l f i sh . For this reason, l i t t l e attention has been paid to these host organisms and 1t 1s generally assumed that the PST have l i t t l e or no effect on their well-being. However, the effects of PST on shel l f i sh have been reported by a number of researchers. Prinzmetal et a l . (1932) showed that in the mussel Mytllus californianus, PST depressed respiration, the cardioinhibitory and vascomotor centers and conduction in the myocardium. Kelloway (1935) confirmed these findings and also noted a rapid f a l l in systemic arter ia l pressure after exposure of the mussel to PST. Adams et al_. (1968) reported extensive mortality and morbundity in shel l f i sh after a massive bloom of G. tamarensis off the coast of England, although no mortality was seen with the mussel Mytilus edulis. In 1985, Shumway et a l . reported the oxygen consumption, shell-valve act iv i ty , heart rates and f i l t r a t i on rates of various bivalve molluscs. The results showed that there is no 'singular bivalve response' to the presence of 21 Gi tamarensis, but rather a complex array of responses that can occur individually or 1n combination. Some displayed no change 1n shell-valve act iv i ty while others either shut their valves and exhibited a swimming escape response (Placopecten magellanicus). or decreased their act iv i ty . None of the shel l f i sh tested Increased Its shell act iv i ty , and none showed a significant change 1n heart rate. Oxygen consumption was more variable. Mytil us edul1s and M. arenaria increased the amount of oxygen consumed, while others showed a signif icant decrease. Only M. arenaria s ignif icantly decreased its f i l t r a t i on rate after the addition of toxic dinoflagellates. Al l others showed no change in f i l t r a t i on rate. One striking feature of PSP testing is that different species of animals exposed to roughly similar amounts of toxic dinoflagellates wi l l frequently accumulate quite different amounts of toxin. One explanation is selective feeding; some species may preferential ly feed on these organisms, while others may find them unpalatable. Both situations have been reported; 1t has been shown that the mussel Mytilus californianus wi l l feed selectively on dinof lagel lates, even when they accounted for some 2% of the phytoplankton community (Buley 1936 cited in G i l f i l l a n 1974), and that the Pacif ic oyster (Crassostrea gigas) does not readily accept G. washinqtonensis as food (Norris and Chen, 1974). Preliminary results published by Cued et al_. (1985) suggest that feeding rates of bivalve molluscs in the presence of toxic dinoflagellates are species-specif ic. It was found that rates remained unchanged or s l ight ly Increased for the following bivalves: Ostrea edulis. Placopectan magellanicus, and Mytilus edul1s. On the other hand, Mya arenaria showed a signif icant decrease in feeding rate after exposure to toxic G_j_ tamarensis. 22 These former studies have pertinence, since Investigations by Twarog & Yamaguchi (1974) have suggested that the relative tox ic i t ies attained by a group of f i l ter - feeding molluscs may be a result of d i f fer ing sensit iv i t ies of each of the species nerves to PST. Without exception, 1t was found that those species more resistant to PST accumulate toxins to a greater extent than those less resistant. Resistance to PST and TTX was found to be the property of individual nerve fibres and was not due to a protective sheath around the f ibres. The mussel Mytilus edulis and the scallop Placopecten magellanicus were shown to be highly resistant to PST and were able to accumulate large quantities of the toxins. The oyster Crassostrea virqinica and the fresh water clam El 1iptio complanata were highly sensitive to the PST and were able to bind only relat ively low amounts of PST. Mya arenaria (soft shell clam) was of intermediate resistance. d. Mechanism of Accumulation In most shel l f i sh species, the PST are accumulated principal ly in the hepatopancreas and are then excreted or released within several weeks. An exception to this rule is the Alaska butter clam Saxidomus giganteus 1n which the PST move from the hepatopancreas to the siphon where they can remain for several years (Schantz, 1969). Quayle (1969) showed that the PST distribution in the siphon of butter clams corresponded to areas of melanin pigment and that the PST concentration decreased as distance increased from the siphon t i p . Price and Lee (1971, 1972) in a series of studies on the nature of PST accumulation 1n the siphons of Alaska butter clams, determined that the distr ibution of PST 1n the clam siphon corresponded with the distribution of melanin ( i .e . 53% of siphon melanin also contained 46% of the PST). They also found that the degree of binding was strongly influenced by pH, with an 23 Increase 1n binding to melanin as the pH Increases from 2 to 5. The reversible nature of the binding was also demonstrated by these researchers. The rate of desorption Increased with the Increasing ac id i ty, Indicating competition between hydrogen ions and the PST for binding sites on the melanin. The exact mechanism by which the butter clams retain PST is not known, however, Price and Lee have suggested that i t might be similar to the binding of PST on a weak cation exchanger. This theory is based on the findings of White (1958), who showed that melanin can function as a cation exchanger by virtue of i ts free carboxyl and phenolic hydroxy groups. At low pH's, the weak acidic groups in melanin are in the hydrogen form, which could explain the blocking of PST binding at acidic pH's. The effect of cations on the binding of PST to clam melanin was also determined by Price and Lee. It was found that cations interfered with the binding of PST to clam melanin and that the degree of interference was direct ly related to the valence of the cation. As the valence increased, so did the desorption of the bound PST from melanin, suggesting competition for binding sites on melanin. This increased competition with increasing valence is consistent with the preference of weak acid cation exchangers for cations of increasing valence (Bruenger and Atherton, 1967). e. Detoxification A viable procedure for the detoxification of poisonous shel l f ish has not yet been developed. This is not surprising in view of our current lack of understanding concerning the mechanism by which shel l f i sh accumulate and excrete PST. Several methods have been put forward, but none has achieved any degree of success on a commercial leve l . The two most promising procedures reported are ozonation and thermal shock. Blogaslawski et a_l. (1979) provided 24 evidence that ozonation could remove low levels of PST from soft shell clams obtained from Crow Harbour, NB during the i n i t i a l stages of a G. excavata bloom. However, White et al.. (1985) found that ozonation of soft shell clams, which had retained PST for long periods of time, did not result in any degree of detoxif ication. These authors postulated that ozonation may be effective on shel l f i sh which have freshly acquired the toxins, whereas toxins stored for long periods of time may be bound, bioconverted or shunted into certain organs or tissues so that ozone is ineffective in detoxif ication. Heat treatment or thermal shock has been a popular idea for the removal of PST from contaminated shel l f i sh even though a large percentage of the incidents of PSP i l lness have been related to the ingestion of cooked she l l f i sh . Metcoff et al.., 1947 showed that commercial retorting was more effective than domestic cooking for reducing the toxic ity in soft shell clams. The kinetics of PST destruction was determined by G i l l et al_., (1985), who established a mathematical relationship between the rate of thermal destruction and the time of heating PST at various processing temperatures. The kinetics were found to be of the f i r s t order as typical ly observed for most micro-organisms. The PST were found to be much more stable to heat than any of the common bacteria or spores of spoilage organisms of public health significance. It was further stated that a process resulting in 90% destruction of PST would also result in a significant reduction in the nutrit ive qualit ies of the processed she l l f i sh. 8. Other Marine Animals Sommer et al.. (1937) were the f i r s t to report the presence of PST in the sand crab Emerita analoga. It was not until many years later that reports of other marine animals which contained PST, in addition to bivalve molluscs, 25 Table 2. Occurrence of PST In various marine organisms (excluding bivalve noil uses) Marine Species Causative Organism Location PST pro f i le Part of Reference (in order of Body significance) Sand crab (Emerlta analoga) Gonyaulax tamarensis San Francisco STX digestive Sommer et gland al_., 1937 Rock crab Gonyaulax tamarensis New England STX digestive Foxall et (Cancer irroratus) via contaminated gland al.., 1979 shel l f i sh Snail Unknown Japan STX, GTX II, viscera Kotakl et (Turbo argyrostoma, NEO al.., 1981 Turbo marmorata, Tectus pyramls) Coral crab Janla spp. Japan Z. Aeneus & whole body Yasumoto et (Zoslmus aeneus. (alga) A. f lor ldus: appendages al.., 1981 Aterqatls f lor ldus, NEO, STX, GTX Platypodia I & II granulosa) P. granulosa: Koyama STX et a].., 1981 Coral crab Janla spp. Japan NEO, STX, whole body Yasumoto et (Pllumnus GTX I, II & III al.., 1983 vespert lUo, Thalamlta spp. Erlphla scarbrlcula) Kelp crab Gonyaulax catenella Puget Sound NEO, GTX II & viscera Jonas-Davles (Pugettia producta) via contaminated III eggs and Llston, shel l f i sh 1985 Rock crab Gonyaulax catenella (Cancer productus) via contaminated shel l f i sh Puget Sound NEO, GTX II 8 viscera III, STX eggs Jonas-Davles and Llston, 1985 26 Table 2. Occurrence of PST 1n various marine organisms (excluding bivalve molluses). (continued) Marine Species Causative Organism Location PST prof i le Part of (in order of Body significance) Reference Shore crab (Hemigrapsus oregonesis) unknown Puget Sound GTX I & IV, whole body STX Jonas-Davles and Llston, 1985 Gonyaulax spp Southern B.C. coast STX hepatopancreas Barber et a l . , 1988 Tubeworm (Eudlstryl la spp.) Gonyaulax catenella Puget Sound GTX II & II, whole body NEO, GTX I & IV Jonas-Davles and Llston, 1985 Starfish (Plaster ochraceus) unknown Puget Sound STX equivalents whole body determined only Jonas-Davles and Llston, 1985 Barnacle (Balanus spp.) unknown Puget Sound STX equivalents whole body determined only Jonas-Davles and Llston, 1985 Crab larvae (cancer anthonyls) Gonyaulax catenella Los Angeles STX equivalents larvae only Yazdandoust, 1985 27 began appearing 1n the l i terature. Table 2 l i s t s the various organisms known to accumulate PST. In addition to the many genera and species of crab that have been shown to contain PST, snai ls, tubeworms, starf ish and barnacles have also been shown to accumulate these toxins. The causative organisms for f i l t e r feeders such as the sand and coral crabs, tubeworms and barnacles are either toxic dinoflagellates or other algae. The red calcareous alga Jania is responsible for intoxication of coral crabs off the coasts of Japan while the dinoflagellate Gonyaulax tamarensis Infects rock crabs from New England. G. catenella, found along the Pacific coast, is responsible for infecting tubeworms and barnacles d irect ly and the carnivorous kelp crabs, rock crabs and starfish indirect ly, through contaminated she l l f i sh . The source of PST in the shore crabs (from Washington & B.C.) and marine snail (from Japan) is unclear. These organisms are not f i l t e r feeders and therefore not primary consumers of toxic dinoflagellates. Neither are they carnivores, ruling out secondary intoxication via toxic she l l f i sh . One poss ib i l i ty is that these herbivores become Infected by consuming toxic dinoflagellate cysts which have been shown to contain signif icant levels of PST (White 1986). Another poss ib i l i ty is the existence of another marine species capable of producing PST, which is an item in the normal diet of sand crabs and snai ls. There are no l iterature reports of research in this area. The toxin composition of the various crab species is quite similar, regardless of location, with the exception of the shore crab and one species of coral crab. The predominant toxin in most crabs is neosaxitoxin followed by STX in coral crabs and gonyautoxins II & III 1n kelp and rock crabs. As a rule the following toxins are found in varying amounts in a l l crabs: NEO, STX, GTX I, II, and III. By comparison, the shore crab contains GTX's I and 28 IV and small amounts of STX, and the coral crab P. granulosa only STX. STX appears to be present in larger quantities in Japanese crabs than in crabs from the Pacif ic Northwest. 9. Tests for PST a. Mouse Bioassay The method currently employed for the routine assay of shel l f ish extracts, the mouse bioassay, is a modification of a procedure original ly described by Sommer and Meyer (1937). Br ief ly , an acidic extract is prepared from the shel l f i sh and a 1 mL aliquot is injected intraperitoneally into white Swiss mice weighing 18-22 g. The death time is measured to the nearest 5 seconds and the mean death time of the mice is then referred to Sommers table (Appendix 1) from which the toxicity of the extracts is determined. Values are expressed in mouse units (MU) and then converted to ug STX/100 g shel l f ish tissue. A value greater than 71 y.g/100 g is considered unfit for human consumption in Canada. One MU was further defined as the amount of poison that wi l l k i l l a 20 g mouse in 15 minutes with symptoms of paralysis or respiratory fa i lure. Mouse units are calculated from the following relationship: log dose (MU)= 145/t - 0.2, where t (time) is in seconds. The mouse unit as a measure of toxicity was developed because a reference toxin was unavailable at this time. In 1957, Schantz et al_. prepared a highly purif ied toxin from the digestive glands of toxic mussels (Mytilus californianus) and the siphons of toxic Alaska butter clams (Saxidomas giganteus). This toxin (STX) had a toxic ity of 5500 ± 500 MU/mg and was subsequently adopted as the reference standard for the o f f i c i a l mouse bioassay. One mouse unit (MU) was established as being equivalent to 182 ug STX dihydrochloride. 29 Wiberg and Stephenson (1960) conducted a series of experiments to determine 1f such factors such as sex, route of administration, pH or presence of sodium ions Influenced the acute median lethal dose (LD,„) of the toxin in mice. The sex of the mice was found to have an effect on the L D I 0 . Female mice were shown to be more susceptible than males at higher doses of the toxin. Acute LD 8 0 ' s were determined using purif ied STX for several routes of administration; 263 ug/kg for the ora l , 10.0 ug/kg for intraperitoneal and 3.4 ug/kg for the intravenous routes. Increases in pH above 4.0 or the addition of sodium ions were found to reduce intraperitoneal toxic i ty. These effects were not additive and the sodium effect appeared to be the stronger, although 1t did not affect the oral or intravenous toxic i ty. It was concluded that the median death time of mice as a cr iterion of toxic ity is not reliable at pH levels above 4 or in the presence of sodium ions above 0.1 M. The mouse bioassay has several l imitations. For example, the l imit of sensit iv i ty 1s approximately 30 ug/100 g shel l f ish tissue with a standard error of 20%. Marginally toxic shel l f i sh may also be underestimated by as much as 60%. Other factors in shel l f i sh extracts may contribute to the toxic i ty in mice, and the expense of maintaining a mouse colony is high. In addition, the mouse bioassay is unable to accurately determine the potential toxic i ty of shel l f i sh extracts containing sulfamate toxins. Unfortunately, the conditions used are not suf f ic ient ly acidic to ensure complete hydrolysis of the toxin complex to Its corresponding carbamate state and therefore may severely underestimate i t s potency. There have been many attempts to provide an acceptable alternative to the mouse bioassay, but despite this effort i t remains the o f f i c i a l test for PSP. 30 Table 3. Summary of assays developed for the detection of PST in shel l f i sh. Assay Principle Major limitations Reference Fluorometry Alkaline oxidation of STX to fluorescent derivative Only measures STX Complex and time-consuming Bates and Rapoport (1975, 1978) Thin layer Chromatography Alkaline oxidation of PST to fluorescent derivatives Does not quantitate with sensit iv ity Buckley et a l . (1976, 1978) Electrophoresis Alkaline oxidation of PST to fluorescent derivatives Does not quantitate with sensit iv ity Fallon and Shimizu (1977) High Pressure Liquid Chromatography Alkaline oxidation of PST to fluorescent derivatives Expensive Requires expert personnel Sullivan and Iwaoka (1983) Jonas Davles et a l . (1984) Sul1 Ivan et a l . (1985) Radioimmuno-assay Measures STX via an anti STXOL antibody Only measures STX Requires trained personnel Radioactivity Carlson et a l . (1984) Housefly Bioassay Measures total toxic i ty by LD B 0 1n housef1ies Detection l imit 20 ug/100 g Requires micro-techniques Siger et a l . (1984) Chicken Embryo Bioassay Measures total toxicity by % mortality in 96 hour chicken embryos Time factor Expense in maintaining fac i l i t y Park et a l . (1986) 31 b. Other Assays Various assays (Table 3) have been developed for the purpose of detecting the toxins causing PSP. The fluorometric technique developed by Bates and Rapoport (1975, 1978) provided the basis for several other assays developed later. It was found in these studies that STX can be oxidized with hydrogen peroxide under alkaline conditions to the fluorescent derivative 8-amino-6-hydroxymethyl-2-1minopurine-3(2H)-propion1c a d d . This procedure requires a cleanup of the shel l f i sh extract by column chromatography, followed by a fluorescent measurement from which STX levels can be calculated. The major l imitation of this technique 1s that only the STX content 1s determined. In Norway this method was used and compared to the mouse bioassay for 2 years and a good correlation was found between the two at levels of less than 100 yg PST although at Increased levels toxicity was underestimated. However, the Bates and Rapoport method could distinguish between acceptable and unacceptable shel l f i sh with some false positives and no false negatives (Hall 1985). Subsequent studies have shown that other PST can also be derivatized to form fluorescent compounds. Unfortunately the degree of fluorescence 1s extremely variable. The N-l hydroxy toxins (GTX's I, IV and NEO) are poorly fluorescing compounds although their specif ic toxic i t ies are high. This creates a serious l imitation for any assay based on fluorescence, because 1t may underestimate the total toxic i ty of a sample by a considerable amount. Other procedures including the thin layer chromatographic (TLC), electrophoretic and high pressure l iquid chromatographic (HPLC) methods are based on the measurement of fluorescence, but 1n addition separate the Individual compounds. The TLC and electrophoretic assays are unable to determine absolute levels of the toxins and are therefore useful only as a 32 scan of the toxin prof i le . The HPLC technique is much more sophisticated. This technique was orig inal ly reported by Sullivan et a l . (1983). Since then, Sullivan et aj.. (1985) have developed an optimization procedure which is capable of detecting nanogram amounts of the N-l hydroxy toxins. This method uses a binary gradient HPLC method with post column derivatization. Detection chemistry is based on the oxidation of a l l toxins to fluorescent products but the method requires two separate chromatographic separations under different conditions. HPLC represents an extremely sensitive and accurate technique which is suitable for handling large numbers of samples for routine monitoring and has the capabil ity to determine very low levels of a l l the PST. However, the equipment is expensive and not commonly found in routine testing laboratories. Radioimmunoassay procedures are sensitive, simple and relat ively inexpensive for the detection of STX in she l l f i sh. Unfortunately, the lack of antibody cross-reactivity to the other PST severely l imits i ts u t i l i t y as an alternative to the mouse bioassay at this time. However, this test serves as a f i r s t step in the possible development of an enzyme immunoassay for the entire PST series. The housefly and chicken embryo assays do not have suff ic ient advantage over the mouse assay to be considered alternatives. The housefly assay is inexpensive but relat ively insensitive and requires the maintenance of a housefly colony and specialized techniques for injecting very small amounts of toxin. The embryo test requires incubation periods of 96 hours, and therefore does not have suff ic ient advantage over the mouse bioassay to be considered as an alternative. Therefore, at present, the only accepted test for the determination of PST in shel l f i sh is the mouse lethal i ty bioassay. 33 EXPERIMENTAL I. THE SMALL SHORE CRAB (HEMIGRAPSUS OREGONESIS) AS A BIOASSAY FOR THE DETECTION OF PARALYTIC SHELLFISH TOXINS IN SHELLFISH 1. Materials a. Chemical Saxitoxin dihydrochloride, 100 ug/mL 1n 20% ethanol, >95% purity (Division of Microbiology, Food and Drug Administration, 1090 Tusculum Ave., Cincinnati, OH, 45226) b. Test Samples Toxic shel l f i sh extracts (Health Protection Branch, Microbiology Laboratory, Vancouver, B.C.) c. Test Animals Hemigrapsus oregonesis (shore crab) Swiss white mice (Baulb-c) 2. Methods a. Determination of an Optimum Injectate Volume in The Small Shore crab \L oregonesis Volumes tested ranged from 50 uL to 400 uL of deionized d i s t i l l ed water. Injections were made with a 25 G 7/8 needle and 1 mL syringe at the Milne-Edwards opening of the crab. A total of 10 male and female crabs weighing 1.5 to 3.5 g were used at each of the eight volumes tested and death times recorded. Death times were determined when tact i le stimulation of the eyes and walking legs e l i c i ted no response. 34 b. Determination of Dose/lethal 1ty Relationship to STX and TTX 1n The Crab H. oregonesis 1) Toxin dose levels: STX - l x l O - 3 yg to l x l O - 2 yg TTX - 5x l0- 3 yg to 5xl0" 2 yg i i ) Toxin standards were dried by means of a jet of nitrogen gas and then diluted with deionized, d i s t i l l ed water just prior to testing. 111) Male and female crabs were collected from Towers Beach, Vancouver in April 1985. A total of 20 crabs (weighing 1.5 to 3.5 g) were used at each toxin dose level . iv) S tat i s t ics : One-way analyses of variance (ANOVA) were performed on the standard curve data followed by a regression analysis. c. The Crab H. oreqonesis as a Test For The Determination of Paralytic Shel l f i sh Toxins (PST) in Shel l f ish Extracts of toxic shel l f i sh were obtained from Ms. May Mil l ing at the Health Protection Branch government laboratory. Aliquots of 50 yL were administered to a total of 10 crabs for each sample extract tested, and death times noted. Using the regression equation obtained from a regression analysis performed on the standard curve data, the toxic i ty of extracts was determined. The results of these tests were then compared to results obtained from the government laboratory which employs the o f f i c i a l mouse bioassay. Tests were performed in July of 1985. 35 Mouse bioassay The mouse bioassay was developed by Sommer and Meyer (1937) and adopted as an o f f i c i a l AOAC method in Canada 1n 1965. It is the only accepted procedure for the detection of paralytic shel l f ish toxins 1n shel lf ish worldwide. The mouse bioassay measures total toxicity when lethal i ty time is standardized against a saxitoxin standard solution. Body tissues from frozen shel l f i sh were removed and blended to homogenity. A 100 g sample was removed and added to 100 mL of 0.1 N hydrochloric acid (HC1) (pH must be less than 4.0). The mixture was boiled gently for 5 minutes and cooled to room temperature. Following th is , the pH was adjusted to pH 2.0 to 4.0 with either 5 N HC1 or 0.1 N sodium hydroxide (NaOH) by dropwise addition (to prevent local alkal inization and consequent destruction of the PST), followed by di lution to 200 mL with deionized, d i s t i l l ed water. The mixture was returned to a beaker, s t i r red, and allowed to settle until the supernatant was translucent and free of solid part ic les. One mL of this acid extract was administered intraperitoneally to 3 mice (female, Swiss white). The time of Inoculation was noted and time of death indicated by the last gasping breath. The extract was diluted so that the death time was in the range of 5 to 7 minutes. Toxicity of the extracts was calculated after reference to Sommer's Table (Appendix I) to determine mouse units (MU). 36 II. PATTERN OF SENSITIVITY AND RESISTANCE TO CONSTANT DOSES OF SAXITOXIN AND TETRODOTOXIN IN THE SHORE CRABS HEMIGRAPSUS OREGONESIS AND HEMIGRAPSUS  NUDUS 1. Materials a. Chemical Saxitoxin dihydrochloride, 100 ug/mL 1n 20% ethanol, >95% purity (Division of Microbiology, Food and Drug Administration, 1090 Tusculum Ave., Cincinnati, OH, 45226) Tetrodotoxin in 0.1 N acetic acid, citrate free (Sigma Chemicals, St. Louis, MO) b. Test Samples Hemiqrapsus oregonesis (shore crab) Hemiqrapsus nudus (shore crab) 2. Methods a. Monitoring Experiment The small shore crab H. oreqonesis was collected at low tide from Towers Beach, Vancouver at monthly intervals from April 1985 to December 1986. Each sampling consisted of 10 male and female crabs weighing from 1.5 to 3.5 g. A constant dose of 0.05 ug STX and TTX was Injected into each crab at the Milne Edwards opening and the death times recorded. Al l tests were performed within 24 hours of col lect ion. 37 b. Additional Testing I) Samples of Hemigrapsus nudus were also collected from Tsawwassen Beach during the same time period. A total of 10 male and female crabs weighing 1.5 to 3.5 g were used for each lethal i ty test. II) Samples of H. oregonesis and H. nudus were collected from Okeover Arm (Powell River) 1n July 1986 during a toxic dinoflagellate bloom. Additional samples of H. oregonesis were gathered at Porpoise Bay (Seshelt) in November 1986 during another toxic bloom. 38 III. DETERMINATION OF PARALYTIC SHELLFISH TOXINS IN SHELLFISH AND SHORE CRABS Chemical Assay This assay, developed by Bates et a l . (1975, 1978), Involves the alkaline hydrogen peroxide oxidation of saxitoxin in shel l f i sh extracts to 8-amino-6-hydroxymethyl-2-iminopurine-3(2H)-propionic acid, the fluorescence of which was measured at pH 5. 1. Materials a. Chemical Saxitoxin dihydrochloride, 100 ug/mL in 20% ethanol, >95% purity. BioRex 70 ion exchange resin, 50-100 mesh, H + form (Sigma Chemicals, St. Louis, MO). b. Test samples Hemigrapsus oregonesis (green shore crab) Hemigrapsus nudus (purple shore crab) Mytilus edulis (mussel) Crassostrea gigas (oyster) Tapes japonica (clam) c. Equipment Shimadzu RF0450 Spectrofluorophotometer (Kyoko, Japan) Janke and Kunkel Ultra Turrax Homogenizer RC2-B Sorvall Centrifuge Glass columns (0.8 cm i .d . x 5 cm, medium f r i t ted glass f i l t e r , 45 mL reservoir, BioRad) 39 2. Methods a. Preparation of Calibration Curve Several dilutions of a stock solution of STX were used to establish a STX fluorescence curve. Alkaline oxidation was carried out by mixing 2 mL STX solution with 2 mL 1.0 N NaOH and 0.6 mL 1% hydrogen peroxide (H2O2) followed by an incubation at room temperature for 40 minutes. Glacial acetic acid was then added to obtain the fluorescent product. Fluorescence at 381 nm was measured for the samples excited at 332 nm. A blank consisting of 0.6 mL d i s t i l l ed water in place of hydrogen peroxide was subtracted from each sample value to obtain the standard curve (Appendix II). b. Shel l f ish Assay Bio-Rex 70 1on-exchange resin (50-100 mesh, H + form ) was equilibrated by taking 200 mL (wet volume) and rinsing with water (3 X 600 mL), 0.5 M sulfuric acid (H2SO4, 3 X 600 mL), water (600 mL), 1 M sodium hydroxide (NaOH, 3 X 600 mL) and water (3 X 600 mL). Rinsing was achieved by st irr ing 5 - 10 minutes and decanting after the resin had settled. The resin was then suspended 1n 0.2 M acetic acid (600 mL) and the pH adjusted to 5.0 with H2SO4, followed by rinsing with 0.2 M pH 5 sodium acetate buffer (2 X 600 mL). Resin was stored in this buffer at 4°C. Body tissues from frozen shellf ish samples were blended until homogeneous. A 2 gram aliquot was removed and added to 2 mL of 0.5 M tr ichloroacetic acid (TCA) which was freshly diluted from a solution of 2 M TCA. After mixing, this extract was heated to an internal temperature of 85-90°C for 10 minutes, followed by cooling 1n an ice bath to 20°C, NaOH (10%) was then added, with s t i r r ing until a constant pH of 5 to 5.5 was reached. The solution was then centrifuged at 12,000 g for 10 minutes. The supernatant 40 was then applied to a glass column (0.8 cm 1.d. X 5 cm with medium fr i t ted glass f i l t e r and 45 mL reservoir) containing equilibrated Bio-Rex 70 1on exchange resin and the effluent discarded. The column was subsequently eluted with 30 mL of 0.2 M pH 5.0 sodium acetate buffer, 25 mL of water and 1.0 mL of 0.05 M hydrochloric acid (HC1) and the effluent again discarded. After eluting with 4 mL of 0.5 M HC1, the effluent was collected in a centrifuge tube. This solution was mixed and divided in to 2 equal volumes in centrifuge tubes. Two mL of 1.2 M NaOH and 0.05 mL of 10% H2O2 was added to 1 portion, while water was substituted for the H2O2 in the other portion. Both portions were then centrlfuged at 1000 g for 1 minute and the supernatants transferred to cuvettes. Forty minutes after the addition of H2O2 the solution was neutralized to pH 5 with 0.15 mL glacial acetic acid and the fluorescence measured using excitation at 332 nm and emission at 381 nm. The net absorbance was obtained by subtracting the fluorescence of the unoxidized blank from the fluorescence of the oxidized portion. The STX content of each sample was determined after reference to the standard curve (Appendix II). 41 IV. GEL ELECTROPHORESIS (SDS-PAGE) OF SOLUBLE PROTEINS IN VISCERAL EXTRACTS FROM THE SHORE CRABS HEMIGRAPSUS OREGONESIS AND HEMIGRAPSUS NUDUS 1. Materials a. Chemical* i) Bio-Rad High Molecular Weight Standards, 50% glycerol, 0.5 M NaCl and 1% SDS in 200 uL total volume. Contents - Myosin MW 200,000 Daltons B-galactosidase MW 116,250 Phosphorylase b MW 92,500 Albumin (BSA) MW 66,200 Ovalbumin MW 45,000 11) Acrylamide Methylene-bis-acrylamide Ammonium persulfate Tetramethylethylenediamine (TEMED) Sodium dodecyl sulfate (SDS) 2-mercaptoethanol Bromophenol blue Coomassie blue (Hydroxymethyl) amino methane (TRIS) * A l l chemicals purchased from Bio-Rad Laboratories 42 b. Test Samples Hemigrapsus oregonesis (shore crab) Hemigrapsus nudus (shore crab) c. Equipment Atto SJ 1060 SDH Electrophoresis Unit Janke and Kunkel Ultra Turrax Homogenizer RC2-B Sorvall Centrifuge 2. Methods a. Determination of Protein Content in Visceral Extracts (Lowry Method) Reagent A - Copper sulfate (0.5 g) and sodium citrate (1.0 g) were dissolved in 100 mL water Reagent B - Sodium carbonate (20 g) and sodium hydroxide (4.0 g) were dissolved in 1 L water Reagent C - To 50 mL Reagent B, 1 mL Reagent A was added Reagent D - To 10 mL Folin - Ciocalteau reagent, 10 mL water was added To 0.5 mL sample, 2.5 mL Reagent C was added. This solution was mixed and allowed to stand 5-10 min. Then, 0.25 mL Reagent D was added, mixed, and allowed to stand 20-30 min. Color was red at 600 nm on a spectrophotometer and amount protein determined after reference to a standard curve (Appendix III). 43 b. Procedure for SDS Polyacrylamide Slab Gels - Discontinuous Buffer System (based on U.K. Laemmli, 1970) 1) Sample preparation Visceral contents from 10 shore crabs were dissected free, homogenized and centrifuged at 5,000 g for 5 min. An aliquot of the supernatant (500 uL) was treated with 10% SDS (25 uL) and 0.2 mM mercaptoethanol (10 uL). Two drops of glycerol were added to the sample along with 0.02% (25 uL) bromophenol blue solution (tracking dye). Sample sizes of 25 uL were applied to the sample s lot. i i ) Gel preparation (Bio-Rad) Separating gel - Three concentrations of polyacrylamide gels were used in these experiments: 6%, 7.5%, 10% (0.2 cm thick x 11 cm long x 13.5 cm wide). See Table 4 for detai ls . Stacking gel (3%) - The following reagents were used: 0.5 M TRIS-HC1, pH 6.8 (3.75 mL); Acrylamide: Bis, 30:1 (2 mL); SDS, 10% (0.30 mL); Water, d.d (8.8 mL); Ammonium persulfate, 10% (0.15 mL); TEMED, 0.00033% (0.02 mL). This solution is poured over the separating gel once i t has polymerized. Electrode buffer (pH 8.3) - T r i s , 0.025 M; glycine, 0.192 M; SDS, 0.1%; water (deionized, d i s t i l l e d ) . Stain - Trichloroacetic acid, 50%; Coomassie blue R-250, 0.1%; water (deionized, d i s t i l l e d ) . Destaining solution - Acetic a d d , 7.5%; Methanol, 5%; water (deionized, d i s t i l l e d ) . 44 Gel electrophoresis was performed at room temperature with a constant voltage of 90 volts for a time period that required the tracker dye marker to migrate one cm from the gel bottom (=3.5 hrs). Gels were removed and stained for 1 hour. The gels were then rinsed with water, transferred to a diffusion destainer and destained for 20 hours 1n a vertical position with a circulating destaining solution c lar i f ied through a cartridge of activated carbon. Table 4. Preparation of separation gels for various polyacrylamide strengths. Amounts of Reagents to Use Reagent Concentration 6% 7.5% 10% 1.5 M Tris-HCl pH 8.8 0.375 M 10 mL 10 mL 10 mL Acrylamide:Bis (30:1) — 8 mL 10 mL 13.4 mL 10% SDS 0.1% 0.4 mL 0.4 mL 0.4 mL H2O (deionized d i s t i l l ed ) — 21 mL 19.4 mL 16 mL 10% Ammonium Persulfate 0.05% 0.2 mL 0.2 mL 0.2 mL TEMED 0.00033% 14 uL 14 uL 14 uL 45 RESULTS AND DISCUSSION I. THE SMALL CRAB (HEMIGRAPSUS OREGONESIS) AS A BIOASSAY FOR THE DETECTION OF PARALYTIC SHELLFISH TOXINS IN SHELLFISH 1. Determination of an Optimum Injectate Volume 1n H. oreqonesis Prior to testing of the lethal i ty response in the crab, i t was necessary to determine an injectate volume which would not interfere with death times. For this purpose, a variety of volumes were administered to the crab and the lethal i ty responses recorded (see Figure 3). The mean death times for crabs injected with 50 and 100 uL deionized d i s t i l l ed water were in excess of 60 minutes. As volumes increased past 100 uL, death times decreased to 5 min after administration of 400 uL water. On the basis of these results, a uniform injectate volume of 50 uL was subsequently used 1n a l l lethal i ty tests. 2. Determination of Dose-lethality Relationship to STX and TTX in The Small  Shore Crab H. oreqonesis The standard curves depicting both STX and TTX dosages and associated death times in shore crabs obtained from Towers Beach 1n April 1985 are presented In Figures 4a and 4b. Analysis of variance (ANOVA) was performed on the STX standard curve data. To create a linear response between death time 1n the crab and dose STX and TTX administered, 1t was necessary to apply a logarithmic transformation. 46 Figure 3 . Injectate volume vs death time in the crab Hemigrapsus oreqonesis 47 15 i c b. Dose TTX (ugxIQ2) Figure 4. Lethality response to various closes saxitoxin and tetrodotoxin in the crab Hemigrapsus oregonesis; a = saxitoxin (STX), b = tetrodotoxin (TTX) 48 Table 5. Results of analysis of variance on standard curve data for various doses STX in Hemiqrapsus oreqonesis. Source Degrees of Freedom Sum of Squares Mean Squares F ratio Between groups 3 766.02 255.34 105.10* Within groups 59 143.34 2.43 Total 62 909.36 * (P<0.01) The results of one-way analysis of variance on log dose STX and associated death times in the crab are shown in Table 5. The c r i t i c a l F value (3,59) is 4.10, making the variance ratio highly s ignif icant at P<0.01. Therefore, we must reject the null hypothesis that there 1s no s tat i s t ica l difference among the treatment means, and conclude that a significant difference exists between death time 1n the crab and dose STX administered. Additional one-way analyses of variance were performed in order to establish i f factors such as sex or weight in the shore crab had an effect on the results. Results are presented in Table 6a and 6b. Table 6a. Results of analysis of variance for the variable sex. Source Degrees of Freedom Sum of Squares Mean Squares F ratio Between groups 1 1.55 0.515 1.59* Within groups 61 11.72 0.199 Total 62 13.27 * Not signficant 49 Table 6b. Results of analysis of variance for the variable weight. Source D.F. Sum of Squares Mean Squares F ratio Between groups 4 1.63 0.342 0.644* Within groups 58 31.34 0.531 Total 62 32.36 * Not s ignif icant The c r i t i c a l F values for 1 and 59 degrees of freedom are 4.00 (P=0.05) and 7.12 (P=0.01). The F ratio for the variable sex versus death time in the crab was 1.59. This value was not signif icant at P<0.01 (c r i t i ca l F value for 1 and 59 degrees of freedom = 7.12), indicating no effect on the death time of the crab due to sex. S imi lar i ly , the variance ratio of 0.644 for the variable weight versus death time, was not significant at P<0.01 (c r i t i ca l F value for 4 and 59 degrees of freedom = 3.65). Therefore, throughout the subsequent experiments, both male and female crabs were used, with weights ranging from 1.5 to 3.5 g. Crab death times after administration STX ranged from 0.75 to 9.39 minutes over a dose range of 0.01 ug STX to 0.001 ug STX. A regression analysis was performed on this data resulting in a correlation coeff icient (r) of 0.89 and a coeff icient of determination (r 2 ) of 0.79. The regression equation (y = a + bx) was found to be y = -17.97 + 9.38x where y = crab death time and x = log dose of STX. Similarly, death times for TTX ranged from 2.86 to 8.71 minutes for dosages of 0.05 to 0.005 ug TTX. The correlation coeff ic ient (r) of 0.83 gave a coeff ic ient of determination (r 2 ) of 0.69. This response was described by regression equation of y = -4.55 - 2.46x. 50 3. The Crab H. oregonesis as a Test for the Presence of PST 1n Shel l f ish The small shore crab H, oregonesis i n i t i a l l y appeared to be a promising bioassay for PST. It was available 1n areas where shel l f i sh are commonly harvested, and based on the standard curve results, 1t was capable of detecting 0.001 yg compared to 0.3 yg PST detectable using the mouse bioassay. Toxic shel l f i sh extracts prepared by the government fisheries laboratory were used as the test material and aliquots of these extracts were administered to the shore crab. Death times were analyzed using the regression equation for a determination of PST content. These results were then compared to o f f i c i a l government results which were calculated after performance of the mouse bioassay. The PST values obtained using the crab as a bioassay were very different from o f f i c i a l results based on the mouse bioassay (Table 7). In a l l cases, the crab bioassay predicted extremely low levels of PST (0.01 to 10.16 yg/100 g) in the extracts whereas o f f i c i a l results showed a range of 66 to 240 yg PST/100 g shel l f ish tissue. In addition, the crab PST values showed no relationship to the mouse PST values; they did not increase as mouse values increased. These tests were made in July of 1985, and after reference to Figure 5 in the next section, i t is conceivable that at this point, the shore crabs were resistant to administered PST. This assumption cannot be confirmed because the lethal i ty response of the shore crab was not tested during July, although in August shore crabs showed a remarkable resistance to constant doses of STX with death times in excess of 20 min. The fact that the small shore crab, used as a test 1n July, consistently underestimated the PST content of shel lf ish extracts lends support to the assumption that these crabs were resistant to PST at this time. This change in sensit iv i ty to STX 1s a severe disadvantage to the use of the crab as a bioassay (obtained in Its natural environment) for the detection of PST in she l l f i sh . 51 Table 7. Determination of PST in shel l f i sh extracts using the crab bioassay. Sample # Mean Death Time Predicted PST Content* Of f i c ia l PST Content in the shore crab using mouse bioassay* (min) (yg PST/100 g) (yg PST/100 g) 1 6.2 10.59 210 2 8.2 6.48 210 3 12.2 2.25 95 4 12.5 2.26 240 5 14.4 1.42 120 6 17.2 0.71 140 7 18.6 0.50 150 8 33.4 0.01 66 * Sample calculation - y = -17.97 - 9.38x y = death time in crab x = log dose PST 52 II. PATTERN OF SENSITIVITY AND RESISTANCE TO CONSTANT DOSES OF SAXITOXIN AND TETRODOTOXIN IN THE SHORE CRABS HEMIGRAPSUS OREGONESIS AND HEMIGRAPSUS  NUDUS The lethal i ty response to constant doses of saxitoxin (STX) and tetrodotoxin (TTX) in the shore crab H. oregonesis recovered from a single location, was monitored over a period of 21 months. In addition, samples of another shore crab, H. nudus, were collected from another location in the Vancouver area and tested for its lethality response at selected intervals during the same time period. Because the Vancouver area is not tested for the presence of toxic dinoflagellates, samples of both H. oreqonesis and H. nudus were taken from two additional areas along the southern B.C. coast during registered toxic blooms. 1. Long Term Fluctuation 1n Sensitivity and Resistance to Constant Doses of  Saxitoxin and Tetrodotoxin in the Shore Crab H. oregonesis The seasonal pattern of crab sensitivity to fixed dosages of saxitoxin and tetrodotoxin are presented in Figure 5. Mean crab death times due to tetrodotoxin injections remained relatively constant (2 to 3 minutes) over the entire 21-month period, demonstrating no seasonal change in crab sensit iv ity to this toxin. Conversely, a wide seasonal fluctuation was observed in mean death times of crabs injected with saxitoxin. Although the average death time for a single dose of saxitoxin was approximately 1 minute 1n sensitive crabs during the spring of 1985, a marked increase in death times approaching 30 minutes was observed in crabs collected during August 1985. This apparent resistance to saxitoxin injections was found to gradually disappear and eventually returned to sensitive levels (1 minute) in November 1985. Crab 53 sens i t iv i ty to saxitoxin injections persisted throughout the winter and fa l l until the following summer (July 1986), when crabs began to again show an Increased resistance to saxitoxin injections. Peak resistance 1n crab death times was observed in August 1986 and reached a level which was substantially smaller than the previous year (August 1985). Nevertheless, the same temporal pattern of relative resistance to saxitoxin Injections was observed and crabs were found to again lose their resistance to saxitoxin by October 1986. 2. Lethality Response of Hemigrapsus nudus to Constant Doses of Saxitoxin and  Tetrodotoxin At selected intervals during the time period August 1985 to October 1986, samples of another small shore crab (H. nudus) were collected and subjected to letha l i ty tests in the same manner as H. oregonesis. Hemigrapsus nudus did not inhabit the Towers Beach area and was therefore collected from a nearby area - Tsawwassan Beach. The results of the lethality tests on H. nudus were compared with those of H. oregonesis (Table 8). Throughout this time period, there was no change in sensit iv ity 1n H. nudus after administration of 0.05 yg TTX. This s ituation, is therefore similar to that of H. oregonesis where death times remained relat ively constant (2-3 roin) during the entire 21 month period in response to TTX injections. Conversely, both shore crabs showed resistance to STX Injections during August of 1985 with extended death times 1n H. nudus of 8.5 min, and greater than 20 min. in H. oregonesis. In addition, both species of crab then returned to sensitive levels by the fa l l of 1985, although H. nudus showed s l ight ly elevated death times in October 1985 compared to H. oregonesis. In September 1986, H. oregonesis again showed resistance to STX but H. nudus did not. It remained sensitive to STX at a l l STX TTX DEATH TIME tMIN) 10 Ap* May Jww July Aus 8«P« Oct Nov 0*c J*n F«b TIME OF YEAR Apf May JIMM July At* topi Ocl Mow 0«e Figure 5. Seasonal pattern of sensitivity and resistance to constant doses (0.05 ug) of saxitoxin and tetrodotoxin in the shore crab Hemigrapsus oregonesis. 55 subsequent testings after August of 1985. As the source of intoxication of shore crabs is unclear at this time, 1t 1s Impossible to speculate as to the reason for this discrepancy. These observation that the small shore crabs, Hemiqrapsus oreqonesis and Hemiqrapsus nudus, exhibit resistance to PST is supported by previous research on the xanthid crabs, Atergates floridus (Koyama et a_h, 1983) and Zosimus  aeneus (Noguchi et al_., 1985). These workers showed crab resistance to both paralytic shel l f i sh toxins and tetrodotoxin throughout the year, whereas the present study showed a seasonal resistance to STX only. It is noteworthy that the resistant shore crabs in this study remained relat ively sensitive to tetrodotoxin despite reduced sensit iv ity to saxitoxin. This finding suggests that the mechanism 1n which crabs develop resistance to PSP could be associated with the presence of the dominant toxin in the crabs' environment. While PSP and tetrodotoxin have been found in a marine macro-alga inhabiting Japanese waters a l l year round (Kotakl et al_., 1983), dinof lagellates elaborating PST in the Pacif ic Northwest show a seasonal pattern of toxicity (Gaines and Taylor, 1985). Moreover, tetrodotoxin does not occur naturally 1n southern B.C. and therefore shore crabs are not l ike ly to come into contact with this toxin. Thus, the sensit iv ity of shore crabs to TTX year round may result from their lack of exposure to this toxin. The poss ib i l i ty that the resistance of shore crabs to saxitoxin administration in late summer was attributed to the presence of dinoflagellate blooms at Towers Beach could not be confirmed because of the lack of PST testing in that area. Therefore, small shore crabs were also collected at two locations on the B.C. coastline, namely Okeover Arm and Porpoise Bay (Figure 6) during registered red tide blooms. Similar extended death times of crabs following saxitoxin injections were observed in H. oreqonesis and H. nudus 56 collected from Okeover Arm (30 minutes) and Porpoise Bay (10 minutes). These crab responses corresponded to PSP contamination levels of 14,000 yg PSP and 1,700 yg PSP per 100 g shel l f i sh for Okeover Arm and Porpoise Bay, respectively (Rudy Chaing, personal communication). 57 Table 8. A comparison of death times 1n two shore crabs (Hemigrapsus nudus. Hemigrapsus oregonesis) after administration of 0.05 ug saxitoxin (STX) and tetrodotoxin (TTX). Times of Year Mean Death Time (min) Mean Death Time (min) H. nudus H. oregonesis TTX STX TTX STX August 1985 1.7 8.5* 2.4 > 20* October 2.1 2.2 2.5 1.3 January 1986 2.3 1.3 2.4 1.4 Apri l 2.0 0.98 2.5 0.96 July 2.4 0.53 2.2 1.1 September 1.9 1.3 2.3 5.1* October 2.1 1.1 2.4 1.6 * Resistance to STX, death times > 3 minutes. 58 125°W •* Death times 30 minutes,Contamination level: 14,000 ugPST/100g #* Death times 10 minutes, Contamination level: 1,700 ugPST/100g Figure 6. Sampling site locations on the southern Br i t i sh Columbia coast. 59 III. DETERMINATION OF PARALYTIC SHELLFISH TOXINS IN SHELLFISH AND SHORE CRABS During the time period April 1985 to December 1986, samples of sensitive and resistant crabs were assayed for the presence of paralytic shel l f ish toxins. Two types of assay were used to determine the PST content: a chemical fluorometric assay and the o f f i c i a l mouse bioassay. Results shown in Table 9 for the fluorometric assay were based on the average of 5 determinations, with each test consisting of 10 separate readings. The mouse bioassay was only done once using the average of results obtained from 3 mice for each sample. Samples of H. oregonesis and H. nudus were determined to be resistant i f mean death times were in excess of 3 minutes. This value was reached on the basis of the monitoring study just presented. In addition to resistant and sensitive shore crabs, other shel l f i sh assayed for the presence of PST were clams, mussels and oysters. These molluscs were collected during a "bloom" of Gonyaulax off the B.C. coast at Okeover Arm in July 1986. Mollusc samples were assayed for the sake of comparing their response to the response of shore crabs from the same location during a toxic outbreak of dinoflagel lates. This comparison could not be made at Towers Beach because of lack of PST testing in the greater Vancouver area. Fluorometric readings are presented in ug saxitoxin per 100 g tissue whereas mouse bioassay results are given In ug paralytic shel l f i sh toxins per 100 g tissue. In a l l samples, bioassay values are greater than those of the fluorometric values. These differences indicate the presence of other PST in addition to STX in the shel l f i sh extracts. The heterogeneity of PST in shel l f i sh from Br it i sh Columbia was reported by Bose et a_kt 1979. Using the same fluorometric technique and comparing the results to the mouse bioassay, these researchers showed that a variety of PST were found 1n shel l f i sh, with STX contributing only part ia l ly to the total tox ic i ty. 60 Table 9. Paralytic shel l f i sh toxin content of various B.C. shel l f i sh and shore crabs. Sample Fluorometric Assay Mean ug STX/100 g ± S.D.* Mouse Bioassay Mean yg PST/100 g Hemigrapsus oreqonesis (shore crab) (resistant to STX) Okeover Arm1 Towers Beach2 (sensitive to STX) Towers Beach 21.5 14.9 0 2.4 2.1 32 0** 0 Hemigrapsus nudus (shore crab) (resistant to STX) Okeover Arm3 (sensitive to STX) Tsawwassen 28.0 0 6.1 50 0 Mvtllus edulls (mussel) Okeover Arm 80.99 4.8 196 Tapes .iaponica (clam) Okeover Arm 65.39 11.4 87 Crassostrea qiqas (oyster) Okeover Arm 33.09 6.3 0** * - Values were determined after reference to the standard curve (See Appendix II). * * - Mice showed signs of respiratory distress and muscular incoordination 1 - Death times > 30 min. 2 - Death times 5 min. • - Death times > 30 min. 61 Shore crabs known to be highly resistant to STX contained detectable levels of PST 1n their viscera after fluorometry and the mouse bioassay were performed (Table 9). Samples of H. oregonesis and H. nudus from Okeover Arm (with death times of > 30 min) contained similar low levels of toxins by both assays. H. nudus was s l ight ly more toxic while H. oregonesis contained a larger proportion of STX. Resistant crabs from Towers Beach in Vancouver (death times 5 min.) were shown to contain 14.9 yg STX after fluorometry but no PST after the mouse bioassay. The mouse bioassay is known to be Inaccurate at low levels of PST because its lower l imit of sensit iv ity is in the range of 30-33 yg PST/100 g shel l f i sh tissue and at these low levels, sodium ions in shel l f i sh extracts counteract the effect (Schantz et a_L, 1958). On the other hand, the lower l imit of sensit iv ity for the fluorometric method is 5 yg STX/100 g. Mice injected with extracts of resistant crabs from Towers Beach exhibited d ist inct neurological symptoms such as respiratory distress and motor Incoordination, which supports the contention that the fluorometric assay is more sensitive than the mouse bioassay. Therefore, i t Is safe to conclude that low levels PST were present 1n these crabs. Extracts taken from sensitive shore crabs from Towers and Tsawwassen Beaches at no time contained detectable amounts of PST. These results, therefore, establish a relationship between resistance to STX in two shore crabs (H. oregonesis, HL nudus) and the presence of PST in visceral tissues. A relationship between sensit iv i ty to STX and the absence of accumulated PST 1s also apparent. Various shel l f i sh (clams, oysters, mussels) were also collected from Okeover Arm during a toxic dinoflagel late bloom and assayed for the presence of PST. Mussel extracts contained the highest levels of toxins followed by clams and then oysters. Clam extracts contained a very high percentage of STX 62 (75%) compared to mussels (41%). Oysters showed lower levels of PST after fluorometric determinations although the mouse bioassay showed none. However, the same argument used for resistant crabs from Towers Beach can be applied here, and consequently i t can be assumed that oysters did contain low levels of PST. The presence of PST in the small shore crabs Hemiqrapsus oreqonesis and H. nudus from Okeover Arm is consistent with the presence of PST in bivalve molluscs obtained from the same area. This presence was 1n turn, attributed to the occurrence of a toxic dinoflagellate bloom at this same location. The relationship between toxic dinoflagellates and toxic shel l f i sh has been known since 1928 after the pioneering work of Meyer and Sommer. However, the source of intoxication in shore crabs is far from clear. These crabs are not f i l ter - feeders , as are the bivalve molluscs, and neither are they carnivores, ruling out direct consumption of toxic she l l f i sh. Shore crabs are herbivores and perhaps consume toxic dinoflagellates washed to shore. Alternatively, they may consume dinoflagellate cysts which accumulate at the sediment/water Interface and have been shown to contain high PST levels (White, 1986). 63 IV. COMPARISON OF SOLUBLE VISCERAL PROTEINS IN SENSITIVE AND RESISTANT SHORE CRABS USING GEL ELECTROPHORESIS To elucidate the possible biochemical mechanism(s) responsible for resistance to Injected STX 1n the shore crabs H. oreqonesis and H. nudus. extracts of visceral tissues were subjected to electrophoretic separations and prof i les of resistant and sensitive crabs were compared. 1. Protein Content of Visceral Extracts from Resistant and Sensitive Shore  Crabs The protein content in visceral extracts of resistant and sensitive shore crabs was determined (Table 9). Values are given in milligrams total protein 1n 25 uL extract and milligrams protein per mg dry weight. The visceral extracts used for these determinations were prepared in the same way as those used for electrophoretic separations. The protein content of sensitive H.  oreqonesis was the highest of the extracts on a wet basis but lowest on a dry basis. Whereas extracts of resistant H. oreqonesis showed more protein on a dry basis than sensitive extracts, on a wet basis sensitive crabs were shown to contain more protein. This is not the case with samples of resistant and sensitive H. nudus. Both on a wet and a dry basis, resistant extracts contained approximately 100 mg more protein than sensitive extracts. Perhaps additional proteins may have been picked up in the sediment when aliquots were drawn from the supernatant. It would be tempting to conclude that resistant extracts contain more protein than sensitive visceral extracts but these results are not conclusive. 64 Table 10. Protein content 1n visceral extracts of resistant and sensitive shore crabs (Hemlgrapsus oregonesis and Hemigrapsus nudus). Sample mg protein/25 UL. mg protein/ mg dry weight H. oregonesis (res) 0.884 0.883 H. oregonesis (sen) 0.984 0.667 H. nudus (res) 0.643 0.857 H. nudus (sen) 0.538 0.742 2. Sodium-dodecvl-sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) The electrophoretic profi les of soluble visceral proteins in resistant and sensitive shore crabs (H. oregonesis) are presented in Figure 7a. The most str iking feature of this 10% gel is the presence of a large protein component in extracts of resistant crabs that 1s absent 1n extracts of sensitive crabs. This component is located near the stacking gel, having migrated only a very short distance Into the separating gel. No specif ic conclusions can be formed as to the molecular weight (MW) of this component in this gel because the standard proteins, albumin and BSA, were heavily overloaded and therefore cannot be used as indicators. However, i t can be assumed that i t is of relat ively high MW compared to the other proteins Isolated because of Its slow migration through the gel. In order to get a clearer view of this extra protein band in resistant crab extracts, duplicate samples were run through a 7.5% polyacrylamide gel (Figure 7b). The large MW protein band from resistant crabs has migrated further Into the stacking gel, and the band 1s s l ight ly smaller than 1n Figure 7a due to a smaller sample s ize. Separation of the standard proteins 1s s t i l l incomplete although we can now make a rough estimate of MW for this protein component as greater than 100,000 daltons. 65 R S R S R S R-Resistant to STX S-Sensitive to STX Figure 7a. A comparison of the soluble visceral proteins found 1n resistant and sensitive Hemiqrapsus oregonesis by SDS-PAGE (10%). R - Resistant to STX S- Sensitive to STX Figure 7b. A comparison of the soluble visceral proteins found in resistant and sensitive Hemigrapsus oregonesis by SDS-PAGE (7.5%). 67 Figure 8. A comparison of the soluble visceral proteins found in resistant and sensitive Hemigrapsus nudus by SDS-PAGE (7.5%). 68 The electrophoretlc prof i les of resistant and sensitive extracts from the shore crab Hemigrapsus nudus are found 1n Figure 8. Again we see the presence of a re lat ively high molecular weight protein compound 1n extracts from resistant H. nudus. Although the standard proteins are again overloaded, this compound 1s located 1n a very similar position to the component found 1n H^ . oregonesis from Figure 7b. Both gels are 7.5%, and both were run for the same 3.5 hour time period. The distance between the extra protein compound and the band at the end of the electrophoretic run in Figures 7b and 8 1s exactly the same - 7.3 cm. These results lend support to the assumption that the same protein component Is present 1n extracts of two species of shore crab known to be resistant to STX. The diss imi lar i ty apparent between prof i les of H^ . oregonesis and H. nudus 1s the presence of a protein band in extracts from nudus sensitive to STX (Fig. 8). This component 1s not found 1n sensitive H^ oregonesis (Fig. 7b). Soluble visceral proteins in resistant H. oregonesis from two locations were compared (Figure 9). The two locations were; Okeover Arm (death times > 30 min) and Towers Beach (death times 5 min). Resistant crab extracts from both locations showed the presence of a high MW protein component but the size of the bands were di f ferent. Crab extracts from Okeover Arm contained a larger amount of this protein compared to crab extracts from Towers Beach. It could be speculated on the basis of these results that the amount of this protein component produced is related to the relative resistance acquired by the shore crab. Shore crabs from Okeover Arm exhibited a much higher tolerance to saxitoxin as shown by the death times which were in excess of 30 min, and in addition, showed a greater amount of the extra protein. On the other hand, resistant crabs from Towers Beach with relat ively lower death times of 5 minutes contained lower levels of the protein component. 69 Figure 9. A comparison of the soluble visceral proteins found in resistant Hemiqrapsus oreqonesis from two B.C. locations by SDS-PAGE (7.5%). 70 The next step in this research was to see 1f the extra protein component found in resistant crab extracts would appear 1n sensitive crabs administered with a low dose of STX in vivo. The results of this experiment can be found in Figure 10. Four samples were electrophoresed: resistant and sensitive extracts of H. oregonesis, as well as resistant and sensitive extracts previously dosed with STX in vivo. It is clear from this gel that the only lane showing an absence of the high molecular weight protein 1s the extract of sensitive H. oregonesis. Al l other extracts showed the presence of a large concentration of this component. These results clearly indicate the production of a protein band, similar to the extra protein band found in resistant crabs, after administration of STX to sensitive crabs in vivo. After reference to the standard proteins run on this gel , 1t 1s now possible to determine the approximate molecular weight of this protein component, which appears to be about 140-150,000 daltons. Another question presented i t se l f ; wi l l the extra protein band appear 1n sensitive crab extracts which have been Injected with tetrodotoxin in vivo? The answer to this question can be found in Figure 11 which shows the electrophoretic patterns of resistant and sensitive H. oregonesis as well as sensitive H. oregonesis which had been given a low dose of TTX in vivo. It is obvious that again we see the production of this high molecular weight component 1n extracts of sensitive crabs which had been dosed with TTX. Therefore, the administration of either STX or TTX to sensitive shore crabs In vivo caused the production of a protein component similar to the one found 1n shore crabs known to be resistant to STX but sensitive to TTX. The results of the electrophoretic experiments are summarized below: 1. A protein component (MW « 145,000 daltons) appeared in visceral extracts of Hemigrapsus oregonesis and H. nudus known to be resistant to STX, and known to contain PST accumulated naturally. 71 Figure 10. A comparison of the soluble visceral proteins found in resistant Hemiqrapsus oreqonesis and sensitive and resistant H. oregonesis + STX in vivo by SDS-PAGE (7.5%). 72 R -Resistant to STX S Sensitive to STX •200,000 • • *-116,000 92,500 166,200 #5,000 Figure 11. A comparison of the soluble visceral proteins found in resistant Hemigrapsus oregonesis and sensitive and resistant H. oregonesis + TTX in vivo by SDS-PAGE (6%). 73 2. This component 1s absent from extracts of H. oreqonesis and H. nudus known to be sensitive to STX. 3. This protein component appeared in visceral extracts of sensitive H^ . oreqonesis administered with low doses STX and TTX in vivo. 4. Control crabs Injected with d i s t i l l ed water did not show the presence of the protein component. These results show the existence of a protein component (MW 145,000 daltons) which appears to be associated with acquired resistance to STX in two shore crabs, Hemiqrapsus oreqonesis and H. nudus. Resistance to STX has in turn been related to the presence of PST, ingested naturally and accumulated in the viscera of shore crabs. Neither PST nor the high MW protein component were found 1n shore crabs sensitive to STX. The appearance of the protein band in electrophoresed extracts of shore crabs given low doses of STX in vivo of considerable importance. This component appeared in extracts within several minutes after administration of STX to the l iv ing crab. Therefore, i t would seem that this protein 1s initimately Involved with STX, either accumulated naturally or administered In the laboratory by acute injection. The appearance of the protein band in visceral extracts of sensitive crabs given low doses of TTX jji vivo is interesting. The shore crab was shown ear l ier in this thesis to be sensitive to TTX over a 21 month time period. During this same time period, shore crabs were shown to exhibit a varying sensit iv i ty to STX. While STX can be found in the crabs' habitat seasonally, TTX is not found 1n these waters and the shore crab would therefore not be l ike ly to encounter this marine toxin naturally. It 1s curious then, that the same protein found in sensitive crabs injected with STX should appear in sensitive crabs injected with TTX. A logical way to investigate this 74 phenomenon would be to allow the crab to Ingest TTX and then test for resistance to TTX and accumulations of TTX in the viscera, followed by electrophoretic separations of visceral proteins to determine the presence or absence of the 145 kd protein component. Unfortunately, the source of TTX Intoxication Is unknown. It is found most commonly in the ovaries of the pufferfish from Japan, but where they encounter the TTX is at present unclear. In any case, since the structure, molecular weight and mode of action of TTX are very similar to those of STX, i t 1s plausible that the macromolecule Isolated 1n this thesis has a general spec i f i c i ty towards both marine toxins. 75 CONCLUSIONS Several points of Interest concerning the nature of PST 1n marine animals were discovered during the course of these Investigations. The shore crabs Hemiqrapsus oreqonesis and H. nudus were shown to become seasonally resistant to STX while remaining sensitive to TTX throughout the year. Resistance occurred during mid to late summer with a gradual return to sens i t iv i ty by the f a l l . In addition, visceral extracts of crabs resistant to STX contained detectable levels of PST whereas sensitive crab extracts did not. The presence of PST in resistant crabs was in turn associated with the presence of toxic dinoflagellate blooms in the area, although the route of intoxication remains unclear. Furthermore, a novel protein component was Isolated from visceral extracts of crabs resistant to STX and crabs injected with STX and TTX in vivo. During the course of this work, the following Information has been gained on this novel protein component: 1. MW « 145,000 daltons 2. Found in visceral extracts of shore crabs resistant to STX 3. Found in visceral extracts of shore crabs given STX & TTX in vivo 76 GENERAL CONCLUSIONS Before proceeding with a discussion on the possible origin and function of this novel protein component, a summary of subsequent research carried out by Donna Smith 1n this laboratory will be presented. The partial puri f icat ion of this novel protein component has since yielded the following information; 1) the approximate MW is 145,000 daltons, 2) heat treatment (100°C, 5 min) resulted in a break down product or subunit with a MW of approximately 72,000 daltons, 3) this protein component is acidic in nature. In addition to these results, the 145 kd protein was shown to be induced in a dose dependent manner after acute studies with l iv ing sensitive H. oregonesis injected with varying doses of STX (Figure 12). Visceral extracts of sensitive control crabs given injections of water do not show the presence of the 145 kd protein (Lane 1). The size of the protein band (and therefore the concentration) is smallest for the second lowest dose STX administered (lane 3, 10 ng). The amount of protein then increased as the dose increased from 10 to 50 ng STX (lane 4). Lane 5 shows the protein prof i le of H. oregonesis known to be resistant to STX, and again the 145 kd protein is present, although the size of the band was smaller than extracts containing 50 ng STX. After administration of 5 ng STX, there was no change in the electrophoretic pattern compared with extracts of sensitive control crabs (Lane 2). The protein component however, did appear when higher doses STX were administered, in a dose dependent manner. Therefore, 1t appears that the amount of STX administered to the l iv ing crab affects the amount of the 145 kd macromolecule produced. Consequently, the results to date suggest that the protein component present in shore crabs exposed to STX may represent some form of defence Figure 12. A comparison of the soluble visceral proteins found 1n visceral extracts of sensitive Hemigrapsus oregonesis. sensitive H^ oregonesis + varying doses of STX, and resistant H. oregonesis by SDS-PAGE (6%). ref: D. Smith, 1987 78 mechanism or immune response to the PST, and to TTX as wel l . The existence of an immune response 1n invertebrates has been clearly established in the l i terature (Cooper, 1974). The location of the crustaceans, of which crabs are a member, within the broad taxonomic scheme can be found in Figure 13. The crustaceans descend from the arthropods d irect ly and prior to that, the annelids. Vertebrates belong to the chordats which branch off at the coelenterats. According to Hildemann and Reddy (1973) there are three major phylogenetic levels of immuno-evolution: quasi-immunorecognition, primordial cell-mediated immunity and integrated cell-mediated and humoral antibody immunity. The f i r s t level is characteristic of a l l Invertebrates and vertebrates, while the second (primordial) 1s exemplified by advanced coelomate invertebrates such as the annelids, and therefore the crustaceans. Only vertebrates possess integrated cel l mediated and humoral antibody immunity. The immune response of invertebrates such as crustaceans, with a f lu id f i l l e d coelomic cavity has received some attention in the l iterature (Cooper, 1974). The coelomic cavities are f i l l e d and monitored by a complex group of coelomocytes that sequester any Insulting foreign substances. Coelomocytes, which are also phagocytes, become immobilized and release humoral factors analogous to opsonins, lyslns and agglutinins. This mixture of coelomic cells 1s similar to vertebrate blood ce l l s , and coeloms are comparable to vertebrate bone marrow in that they possess analogous leukocytic types (Cooper, 1974). Many immunologists believe that invertebrate coelomocytes are the evolutionary precursors of a l l known vertebrate immunocytes (Acton and Weinhelmer, 1974). Invertebrate humoral immunity Involves the presence of biologically active hemagglutinins that occur naturally or whose synthesis may be induced. 79 Figure 13. Phylogeny of invertebrates ref: Cooper, 1974 80 Opsonins are substances such as hemagglutinins that coat particulate antigens such as bacteria and promote phagocytosis. There 1s substantial evidence for the presence of hemagglutinins 1n a l l coelomate invertebrates. MacKay and Jenkin (1970) believe that opsonins are present 1n the crayf ish, an arthropod very close taxonomically to the Crustacea. Coelomycytes from crayfish immunized with 4 weekly doses of endotoxin show far greater phagocytic act iv i ty than in nonimmunized crayfish. This is one example of an induced hemagglutinin in an invertebrate and there are other examples in the l i terature (Acton and Weinhiemer, 1974). Most hemagglutinins form strong aggregates from 100 to 400,000 daltons. The aggregation or dissociation of the hemagglutinins is dependent on the pH and calcium ion concentration. Calcium ions are important for s tabi l i ty as is the pH range of 7 to 8. Sedimentation is dependent upon concentration and they require strong denaturing solvents for dissociation into subunits (Marchalonls & Edelman, 1968). Other components of interest contained within the coelomycyte are enzymes. They are important in the destruction of antigens in both invertebrates and vertebrates and are therefore involved in the defence or immune system. Since there is no evidence of antibody production in the invertebrate immune system, i t is l ikely that enzymes are of prime Importance in the destruction of foreign matter in Invertebrates, and therefore, in crustaceans as wel l . Many s imi lar i t ies exist between antibodies and inductive enzymes (Table 11). Both are relatively large protein molecules synthesized de novo but not by protein precursors. Both have more or less specif ic a f f i n i t y for the substrate or antigen with which they react and by which they are induced. Anamnesis or memory capabilities are associated with both enzymes and antibodies (Cooper, 1974). Once primed to a certain antigen, an enzyme or antibody is upon second challenge, fu l ly capable of responding in a 81 specif ic heightened manner. There 1s a rapid rise in response, or the increased production of antibodies or enzymes, upon a second exposure to the same antigen. One property 1n which enzymes and antibodies d i f fer drast ical ly 1s 1n the substances capable of inducing their production. Whereas enzymes are Induced primarily by small molecules, antibodies are generally induced by macromolecules, especially proteins and conjugated proteins. Unfortunately, there is l i t t l e Information available on the presence of inductive enzymes in invertebrates in general, and v irtual ly no Information on Inductive enzymes in members of the crustaceans. Any attempt to make a valid comparison between the novel protein isolated in this work and a component of the immune system in the crab would be tenuous at best. F i r s t , we have very l i t t l e Information about this new protein, and second, l i t t l e 1s known about the macromolecules Involved 1n the Immune response of coelomic invertebrates. However, 1t is possible to speculate 1n a general sense on the function of this protein component, and how 1t may relate to a defence mechanism involving PST 1n the shore crab. Other than the molecular weight, the only pertinent information known about this protein component 1s that i t appears in crab visceral extracts after PST have been introduced, either naturally via a toxic bloom of dinof lagellates or in the laboratory. We know that the PST are very potent neurotoxins capable of blocking the Inward movement of sodium 1ons 1n excitable ce l l s . We also know that crabs and many shel l f i sh are relatively unaffected by these toxins and i t 1s generally assumed that they avoid the toxic effects by being able to accumulate and excrete the PST. It would therefore seem reasonable, that the 145 kd protein Isolated here may be Involved in protecting the crab from the lethal effects of the PST. If this assumption is correct, then this component may be capable of binding or 82 Table 11. A comparison of enzymes and antibodies. Property Enzymes Antibodies Phylogenetlc distr ibution Unlqultous; made by a l l ce l l s Structure Proteins with variable chemical and physical properties; an enzyme of a given spec i f ic i ty and from any particular organism 1s homogeneous; many have been crystal 1 zed A late evolutionary acquisition; made only 1n vertebrates (and 1n certain ce l l s of the lymphatic system) A group of closely related proteins having a common multichain structure with the chains held together by -SS-bonds. Molecules of a given spec i f i c i ty are heterogeneous 1n structure and function Constitutive Inducible Function Reaction with Ugands* Yes Often Af f in i ty Number of specif ic Hgand-blndlng sites per molecule Inducers Specif ic reversible binding of Ugands* with breaking and forming covalant bonds Wide range of a f f i n i t i e s ; populations of enzyme molecules of a given speci f ic i ty are uniform an a f f in i ty for their Ugand Usually measured k lnet lca l ly Different 1n different enzymes, depending on number of polypeptide chains per molecules; usually one s i te per chain Primarily small molecules "Natural" antibodies? Yes Specif ic reversible binding of Ugands" without breaking or forming covalent bonds Wide range of a f f i n i t i e s ; but populations of antibody molecules of the same spec i f i c i ty are usually heterogenneous 1n a f f i n i t y for their Ugand Usually measured with reactants at equilibrium (because the reactions are so fast) 2 per molecule of the most prevalent type (NW 4r 150,000); each s i te 1s formed by a pair of chains (a l ight plus a heavy chain) Usually macromolecules, especial-ly proteins and conjugated proteins * Ugand • substrate or coenzyme, and antigen or hapten 1n case of antibodies, ref: Cooper 1974 8 3 sequestering these very small molecules (MW STX-372). The f a c t that the PST are small molecules would tend to e l i m i n a t e the p o s s i b i l i t y t h at t h i s novel component 1s a humoral f a c t o r such as a b a c t e r i o c i d l n or hemagglutinin, because they react with p a r t i c u l a t e antigens such as b a c t e r i a and other f o r e i g n macromolecules. On the other hand, enzymes are known to react with small molecules by r e v e r s i b l e binding 1n a very s p e c i f i c manner (Table 11). An i n t e r e s t i n g property of the novel macromolecule I s o l a t e d 1s that i t appears w i t h i n minutes of acute a d m i n i s t r a t i o n of STX i n a dose dependent manner. One would not expect an Induced enzyme to be produced 1n such large q u a n t i t i e s i n such a short time by de novo s y n t h e s i s . There are, however, reports i n the l i t e r a t u r e showing that defence r e a c t i o n s of phagocytes can occur w i t h i n minutes of antigen challenge 1n i n v e r t e b r a t e s (Evans et a j . , 1968, Liebman, 1942). Whether an analagous s i t u a t i o n e x i s t s 1n the crab Is unknown, but such a phenomena could e x p l a i n the i n d u c t i o n of r e s i s t a n c e to STX a f t e r exposure to the PST. Therefore, on the b a s i s of the preceding d i s c u s s i o n , i t would seem more l i k e l y that the p r o t e i n component I s o l a t e d 1n t h i s study was an enzyme rather than a humoral f a c t o r but there i s l i t t l e information with which to back up t h i s assumption. I t 1s c l e a r that we do not have adequate knowledge of the p r o t e i n component i s o l a t e d i n t h i s study to form v a l i d c onclusions as to I t s o r i g i n or f u n c t i o n as a defence component of the crabs immune response to PST. Before such comparisons can be made, 1t 1s necessary to complete the p u r i f i c a t i o n and c h a r a c t e r i z a t i o n of t h i s macromolecule and e s t a b l i s h without question that i t does indeed bind 1n some manner with the PST. In summary, the i s o l a t i o n of the macromolecule 1n t h i s t h e s i s may provide a promising lead f o r f u r t h e r research on the mechanism of acquired r e s i s t a n c e to PST i n marine animals. An understanding of how c e r t a i n marine animals are 84 a b l e to withstand the p o t e n t i a l l y f a t a l e f f e c t s of PST may subsequently o f f e r a c l u e 1n the search f o r an e f f e c t i v e a n tidote to p a r a l y t i c s h e l l f i s h p o i s o n i n g i n humans. 85 REFERENCES Acton R.T. and Weinheimer P.F. 1974. Hemagglutinins: primitive receptor molecules operative in invertebrate defence mechanisms. In: E.L. Cooper (ed) Contemporary Topics in Immunobiology. Plenum Press, New York p. 271-287. 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Death Mouse Death Mouse Time* Units Time Units 1:00 100 5:00 1.92 10 66.2 05 1.89 15 38.3 10 1.86 20 264 15 1.83 25 20.7 20 1.80 30 16.5 30 1.74 35 13 9 40 1.69 40 11.9 45 1.67 45 10.4 50 1.64 50 9.33 55 8.42 6:00 1.60 15 1.54 2:00 7.67 30 1.48 05 7.04 45 1.43 10 6.52 15 606 7:00 1.39 20 5.66 15 1.35 25 5.32 30 1.31 30 5.00 45 1.28 35 4.73 40 4.48 8:00 1.25 45 4.26 15 1.22 50 4.06 30 1.20 55 3.88 45 1.18 3:00 3.70 9:00 1.16 05 3.57 30 1.13 10 3.43 10:00 1.11 15 3.31 30 1.09 20 3.19 2J 3.08 11:00 1.075 30 2 98 30 1.06 35 2.88 40 2 79 12:00 1.05 45 2.71 50 2 63 13 1.03 55 2.56 14 1.015 15 1.000 4:00 2.50 16 0.99 05 2.44 17 0.98 10 2.38 18 0.972 15 2.32 19 0.965 20 2.26 20 0.96 25 2.21 21 0.954 30 2.16 22 0.948 35 2.12 23 0.942 40 2.08 24 0.937 45 2.04 25 0.934 50 2.00 30 0.917 55 1.96 40 0.898 60 0.875 ' Minutes:Seconds. ref: A0AC 1984 93 APPENDIX II ug STX Calibration Curve for Fluorescence vs. Saxitoxin Concentration 94 APPENDIX III •10T CONCENTRAT ION PROTE IN Calibration Curve for Absorbance vs. Protein Content 95 APPENDIX IV Anatomy of Crab ref: G.F. Warner 1977 

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