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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 Columbia, 1  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE 1n  THE FACULTY OF GRADUATE STUDIES ( D e p a r t m e n t o f 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 a s c o n f o r m i n g to t h e r e q u i r e d s t a n d a r d  THE UNIVERSITY OF BRITISH COLUMBIA M a r c h , 1988  © K a t h l e e n G. B a r b e r , 1988  In  presenting  degree  this  at the  thesis  in  partial  fulfilment  University of  British  Columbia,  freely available for reference and study. copying  of  department publication  this or of  thesis by  for scholarly  his  this thesis  or  her  the  I agree  I further agree  purposes  may  representatives.  It  be is  requirements  for  an  Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  advanced  that the Library shall make it that permission granted  for extensive  by the head  understood  that  for financial gain shall not be allowed without  permission.  DE-6(3/81)  of  of  my  copying  or  my written  11 ABSTRACT  The following  research deals with the response of the small shore crabs,  Hemlgrapsus oreqonesls (PST).  These  shore  and Hemlgrapsus crabs  were  shown  resistance to administered saxitoxin was  found  after  administration  neurotoxin with s i m i l a r  nudus to  (STX). of  to  paralytic  develop  remarkable  toxins seasonal  No similar change 1n s e n s i t i v i t y  tetrodotoxin  actions to the PST.  a  shellfish  (TTX),  Resistance  another  marine  to STX in the small  shore crabs was linked to the presence of PST 1n the v i s c e r a , and t h i s 1n turn was  related  to  the  presence  of  toxic  dlnoflagellate  blooms  1n the  area.  Furthermore, t h i s research provides, for the f i r s t time, evidence of a protein component  (MW 145,000 daltons)  which appears  resistance to PST in the shore crab. shown to appear in sensitive  to be associated with acquired  In a d d i t i o n , this protein component was  crab extracts  doses of saxitoxin and tetrodotoxin in vivo.  after  the administration  of  low  111 TABLE OF CONTENTS Page ABSTRACT TABLE OF CONTENTS LIST OF TABLES  1i 11i v  LIST OF FIGURES  v1  ACKNOWLEDGEMENTS  vi 1  INTRODUCTION LITERATURE REIVEW 1. Human Intoxication 2. P a r a l y t i c S h e l l f i s h Toxins 3. Tetrodotoxin 4. Actions of Saxitoxin and Tetrodotoxin 5. Mechanism of Action 6. Organisms Elaborating Paralytic S h e l l f i s h Toxins 7. Bivalve Molluscs 8. Other Marine Animals 9. Tests for P a r a l y t i c S h e l l f i s h Toxins EXPERIMENTAL I. The Small Shore Crab (Hemigrapsus oregonesis) as a Bioassay for the Detection of P a r a l y t i c S h e l l f i s h Toxins in S h e l l f i s h II. Pattern of S e n s i t i v i t y and Resistance to Constant Doses STX and TTX in the Shore Crabs Hemigrapsus oregonesis and Hemigrapsus nudus III. Determination of Paralytic S h e l l f i s h Toxins in S h e l l f i s h and Shore Crabs IV. Gel Electrophoresis of Soluble Proteins in Visceral Extracts from the Shore Crabs Hemigrapsus oregonesis and Hemigrapsus nudus RESULTS AND DISCUSSION I. The Small Shore Crab (Hemigrapsus oregonesis) as a Bioassay for the Detection of P a r a l y t i c S h e l l f i s h Toxins in S h e l l f i s h 1. Determination of an Optimum Injectate Volume 1n Hemigrapsus oregonesis 2. Determination of Standard Curve for STX and TTX in the Small Shore Crab 3. The Crab Hemigrapsus oregonesis as a Test f o r the Presence of PST in S h e l l f i s h  1  4 5 8 9 10 11 16 24 27  33 36 38 41  45 45 50  1v II.  Pattern of S e n s i t i v i t y and Resistance to Constant Doses STX and TTX in the Shore Crabs Hemiqrapsus oreqonesis and Hemiqrapsus nudus 1. Long Term Fluctuation 1n S e n s i t i v i t y and Resistance to Constant Doses of STX and TTX 2. L e t h a l i t y Response of Hemiqrapsus nudus to Constant Doses STX and TTX III. Determination of P a r a l y t i c S h e l l f i s h Toxins in S h e l l f i s h and Shore Crabs 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 2. Sodium-Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)  52 52 53 59  63 64  CONCLUSIONS  75  GENERAL CONCLUSIONS  76  REFERENCES  85  APPENDICES I. II. III.  Sommer's Table - Death Time: Mouse Unit Relations for P a r a l y t i c S h e l l f i s h Toxins C a l i b r a t i o n Curve for Fluorescence vs. Saxitoxin Concentration C a l i b r a t i o n Curve for Absorbance vs. Protein Content  92 93 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 s h e l l f i s h Preparation of separation gels for various polyacrylamide strengths  Table 4. Table 5.  30 43  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 v a r i a b l e s : 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 Table 7. Table 8.  Table 9.  49  Determination of PST in s h e l l f i s h extracts using the crab (Hemigrapsus oregonesis) bioassay  51  A comparison of death times in two shore crabs (Hemigrapsus oregonesis) a f t e r administration of 0.05 ug STX and TTX  55  P a r a l y t i c s h e l l f i s h toxin content 1n samples of s h e l l f i s h and shore crabs collected from various B r i t i s h 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.  Figure 2.  Chemical structures of the p a r a l y t i c s h e l l f i s h toxins (PST)  6  Chemical structure of tetrodotoxin  8  Figure 3.  Injectate volume vs. death time In the crab Hemiqrapsus oreqonesis Figure 4a. L e t h a l i t y response to various doses of sax1toxin in the crab Hemiqrapsus oreqonesis 4b. L e t h a l i t y response to various doses of tetrodotoxin in the crab Hemiqrapsus oreqonesis Figure 5.  Figure 6.  46 47  47  Seasonal pattern of s e n s i t i v i t y and resistance to constant doses of saxitoxin and tetrodotoxin (0.05 yg) in the shore crab Hemiqrapsus oreqonesis  54  Sampling s i t e locations on the southern B.C. coast  58  Figure 7a. A comparison of the soluble v i s c e r a l proteins found in resistant and sensitive Hemiqrapsus oregonesis by SDS-PAGE ( 1 0 % )  65  7b. A comparison of the soluble v i s c e r a l 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  A comparison of the soluble v i s c e r a l proteins found in resistant Hemigrapsus oreqonesis from two B.C. locations by SDS-PAGE (7.5%)  69  Figure 1 0 . A comparison of the soluble v i s c e r a l proteins found in resistant and sensitive Hemigrapsus oregonesis and in s e n s i t i v e and resistant crabs and STX 1n vivo by SDS-PAGE (7.5%)  71  Figure 1 1 . A comparison of the soluble v i s c e r a l proteins found 1n resistant and sensitive Hemigrapsus oreqonesis and 1n s e n s i t i v e and resistant crabs and TTX in vivo by SDS-PAGE (6.0%)  72  Figure 12. A comparison of the soluble v i s c e r a l proteins found 1n sensitive Hemiqrapsus oregonesis, sensitive H. oreqonesis + varying doses of STX, and resistant H. oreqonesis by SDS-PAGE (6.0%)  77  Figure 1 3 . Phylogeny of invertebrates  79  Figure 9.  -  V11  -  ACKNOWLEDGEMENTS  The author would l i k e to thank her supervisors Dr. P.M. Townsley and Dr. D.D. K i t t s for t h e i r knowledgeable advice and creative input during the course of this research project and review of the t h e s i s . She also wishes to thank the members of the research committee, Dr. Powrie and Dr.  Vanderstoep both of  J.  the Department of  Food Science.  appreciation is also extended to Dr. A. Finlayson for his  W.D. Much  constant  assistance  expertise and  assistance  and constructive review of the t h e s i s . The  author  wishes  to  acknowledge  the valuable  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 t h e i r encouragement and support during  this  project. She is also grateful Karen  Chandler  for  their  to her parents Laurence and Edith Bamford, and s i s t e r support  and  understanding.  extended to John Gould for his patience and encouragement.  A  special  thanks  1s  1  INTRODUCTION  Paralytic  shellfish  poisoning  (PSP)  occurs  1n  humans  after  the  consumption of s h e l l f i s h which have been previously contaminated with certain marine  dinoflagellates  that  in  turn  contain  termed p a r a l y t i c s h e l l f i s h toxins (PST).  a potent  set  of  neurotoxins,  This type of poisoning can result in  death or a temporary incapacitating I l l n e s s .  There are no global  statistics  available on the true incidence of PSP, although reports of Illness and death after  consumption  of  contaminated  l i t e r a t u r e since early times.  shellfish  have  from  achieved  PSP  through  appears  relatively  PST t o x i c i t y values  tissue.  value  consumption.  small,  extensive monitoring  laboratories. Any  greater  Shellfish  reported  in  the  In North America, a total of 1600 PSP Incidents  and some 300 deaths were recorded by Prakash 1n 1974. deaths  been  procedures  are  than  control  reported  71  yg  is  Although the number of of  poisonings  carried in ug  out  PST/100  considered  poisonings are d i f f i c u l t  by  been  government g  unfit  to control  has  shellfish for  because  human of  the  unpredictable and sporadic occurrence of the d i n o f l a g e l l a t e s which produce the PST.  In  addition,  contaminated  shellfish  show no distinguishing  therefore cannot be separated from non-toxic and need for beds  and  trained personnel, monitoring  popular  approximately permanently  harvesting  14,000 miles  closed  to  the  of  areas.  is  of  and  Due to the expense  r e s t r i c t e d to the commercial  British  coastline  harvesting  shellfish.  signs and  an  Columbia  for  estimated  shellfish  for  example,  70% of  this  reason  this  has 1s  (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 s h e l l f i s h such as clams, mussels,  oysters  2 and scallops.  S h e l l f i s h become toxic within a few days of being exposed to a  'bloom' of poisonous accumulate  the  dinoflagellates  ingested  PST.  due to t h e i r unique a b i l i t y to bind and  With the exception of the Alaska  butter clam  which remains toxic for several years, contaminated s h e l l f i s h generally  lose  t h e i r t o x i c i t y in several weeks and again become safe for human consumption. In addition to the public health problem, the economic consequences of toxic dinof l a g e l l a t e blooms can be severe, especially in B.C. where s h e l l f i s h harvesting  is  leads  heavy  losses  for  and  retailers  as  to  wholesalers  a large  and growing  fishermen, well  as  The closure fish  related  shellfish  shellfish  industries  processors, as  tourism,  extensive.  After a survey of secondary effects caused by a red tide outbreak Canada,  Jensen  (1974)  e f f e c t of  such  beds  and  Eastern  The economic  and  of  recreation  1n  restaurants.  industry.  reported  that  red tide outbreaks  restaurants  noted  reduction in sales of seafood dishes, fishermen were unable to s e l l and  there was  25% to 50% decrease  in  sales  of  shellfish  a  is  50%  lobsters,  and f i n f i s h .  In  a d d i t i o n , the occurrence of a toxic bloom in one area can lead to a depression in demand for s h e l l f i s h  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 unclear  is  why  incomplete and  1n many  how certain  algae  basic are  areas. able  to  For  example,  produce the  it PST.  is  still Equally  unclear are the mechanisms of accumulation and excretion 1n resistant marine animals.  In a d d i t i o n , the mechanism of action causing poisoning  poorly understood.  Until  such fundamental questions  cannot hope to solve more p r a c t i c a l problems such as: humans,  an  shellfish,  a  acceptable  procedure  for  superior  alternative  to  the the  of PSP are answered, we an antidote for PSP in  detoxification official  in humans 1s  mouse  of  contaminated  bioassay  for  the  3 detection of PST 1n s h e l l f i s h  and a method for predicting when and where a  toxic bloom w i l l occur as well as how toxic i t w i l l be. The  original  objective  of  this  research  was  p o s s i b i l i t y of developing a more p r a c t i c a l bioassay the  prediction  of  PST  1n  shellfish.  The  to  Investigate  the  to the mouse bloassay  small  shore  oregonesis was chosen 1n this capacity because of Its  crab  for  Hemigrapsus  a v a i l a b i l i t y In  coastal  areas where s h e l l f i s h are commonly harvested, and because of i t s r e l a t i v e ease of handling  compared to other marine animals.  Preliminary  results  of  this  thesis indicated that t h i s small crab became seasonally resistant to saxitoxin (STX),  one  of  the  PST.  Although  considered a major disadvantage the mouse bioassay,  this  fluctuation  in  sensitivity  to the crab as a more practical alternative to  1t also provided the basis for another l i n e of  namely, the mechanism of  seasonal  Hemiqrapsus  Therefore,  oregonesis.  was  resistance the  to STX 1n the small  specific  objectives  of  inquiry,  shore crab this  study  were: 1. To  determine  saxitoxin  the  long-term  pattern  of  sensitivity  1n the green shore crab Hemiqrapsus  shore crab Hemigrapsus  and  oreqonesis,  resistance  to  and the purple  nudus.  2. To assay resistant and sensitive shore crabs for the presence of p a r a l y t i c shellfish 3. To  toxins.  compare  electrophoretic  profiles  resistant and sensitive shore crabs.  of  soluble  visceral  proteins  1n  4 LITERATURE REVIEW  1. Human Intoxication Paralytic  shellfish  poisoning may be diagnosed  easily  in humans by the  appearance of certain symptoms which can become apparent within a few minutes a f t e r consuming  toxic s h e l l f i s h .  Initially  there is  a tingling  or  burning  sensation of the l i p s , gums, tongue and face with a gradual progression to the neck, arms, f i n g e r t i p s , general  muscular  legs and toes.  incoordination.  In  Later, this  changes  to numbness  severe cases c o n s t r i c t i v e sensations  and of  the throat, ataxia, aphonia and incoherence of speech are prominent symptoms. Other associated symptoms Include:  weakness, d i z z i n e s s , malaise, prostration,  headache, s a l i v a t i o n ,  Intense t h i r s t ,  rapid pulse,  even temporary blindness.  impairment  Less common are gastrointestinal  of  vision  symptoms  and  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  become progressively  paralysis  more severe.  Death may  result  from respiratory paralysis within 2 to 24 hours depending on the magnitude of the dose of PST, and the i n d i v i d u a l .  If  one survives  24 hours the  prognosis  1s good and there appears to be no lasting e f f e c t s from the i l l n e s s . Because symptomatic. if  there Emesis  1s  no e f f e c t i v e antidote  for  PSP,  is  largely  should be induced immediately a f t e r symptoms appear and  respiratory d i f f i c u l t i e s appear, a r t i f i c i a l  and continued for several hours. anticurare drugs are useful  respiration  the PST (Murtha, 1960).  1n aiding a r t i f i c i a l  Digitalis  should be applied  Drug therapy has had variable success.  The  r e s p i r a t i o n , and oxlmes can  be used to counteract the acetylcholine esterase-11ke  Loubser, 1960).  treatment  Inhibitory  effects of  and alcohol are not recommended (Pepler and  5 2. P a r a l y t i c S h e l l f i s h Toxins  (PST)  a. Chemistry Knowledge  of  the  chemistry  of  paralytic  increased considerably in recent years. the  detection,  Until  isolation  and  shellfish  toxins  (PST)  has  Numerous advances have been made in  structural  determination of  the various  PST.  1975, i t was thought that saxitoxin (STX) was the sole toxin involved in  PSP.  STX was  determined i t s  isolated  in  a p u r i f i e d state  chemical properties.  by Schantz  et  al_.  (1957) who  STX is a c o l o r l e s s , hygroscopic  solid,  very soluble in water, partly soluble in methanol or ethanol but insoluble in most non-polar useful  solvents  absorption  such as  ethyl  in the u l t r a - v i o l e t  and petroleum ethers.  range and has an optical  130, and two 2 pKa's at pH's 8.2 and 11.5. the free  base  is  C H N 0 1 0  1 7  7  4  STX  shows no  rotation of  The molecular formula for STX as  with a molecular weight of 299.  often found in the form of the dihydrochloride s a l t  The toxin  is  ( C H N 0 « 2 H C 1 ) with a 1 0  1 7  7  4  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 c r y s t a l lization.  In  tetrahydropurine  1974,  Rapoport  derivative  and  and  associates  the  structure  determined was  that  finally  STX  was  elucidated  a by  Schantz et al_. (1975) using X-ray crystallography. It  became increasingly clear that many contaminated s h e l l f i s h and toxic  d i n o f l a g e l l a t e s contained other neutral or weakly basic toxins in addition to the highly basic STX.  A l l were shown to have actions similar to those of STX  although potencies varied considerably. date.  Twelve PST have been characterized to  The structures of the known PST can be found in Figure 1.  6  16  Figure 1. ref:  Rl  R2  R3  R4  1  H  H  H  H  Saxitoxin  2  H  H  H  SO3-  Bl  3  H  H  OSO3-  H  Gonyautoxln 2 (GTX2)  4  H  H  OSO3-  SO3-  CI  5  H  0S0 -  H  H  Gonyautoxln 3 (GTX3)  *6  H  OSO3-  H  SO3-  C2  7  OH  H  H  H  Neosaxltoxin (NEO)  8  OH  H  H  SO3-  B2  9  OH  H  OSO3-  H  Gonyautoxln 1 (GTX1)  10  OH  H  OSO3-  SO3-  C3  11  OH  OSO3-  H  H  Gonyautoxln 4 (GTX4)  12  OH  OSO3-  H  SO3-  C4  3  Structures of the p a r a l y t i c s h e l l f i s h toxins  (STX)  (PST)  Hall and Reichardt (1984)  The  twelve  derivatives  PST  formed  21-sulfogroups,  consist by  the  of  a  addition  respectively.  parent of  compound,  N-l-hydroxyl,  saxitoxin,  and  eleven  11-hydroxysulfate  STX contains two formal guanidinium groups  and (C's  1, 2, 3 and 7, 8, 9) which are strongly basic and behave as a d i - c a t i o n when the pH i s  less  than 8 (Hall  & Reichard, 1984).  It  has a purine base with a  3-carbon bridge l i n k i n g positions 3 and 9 and a methyl carbamate at position 6.  Compounds with 21-sulfo or 11-hydroxysulfate groups w i l l have a net charge  of +1 at pH's net charge.  less  than 8 and those with both groups w i l l  have l i t t l e or no  7 b. S p e c i f i c T o x i c i t i e s The s p e c i f i c t o x i c i t i e s of the PST are varied.  A t o x i c i t y value of 5500  mouse units (MU) per mL of the dihydrochloride s a l t was assigned to by Schantz et aj.. (1957).  It was also determined that 1 MU Is  approximately  dihyrodrochlorlde.  0.18  yg  STX  neosaxitoxin (NEO) and gonyautoxins by Geneuah and Shimizu (1981). a  toxicity  series.  relative  to  STX  (GTX) I,  GTX III of  The  II  and III  specific  saxitoxin  equivalent to toxicities  of  and IV were determined  has a s p e c i f i c t o x i c i t y of 5641 MU and  103, making  it  the most potent  of  the PST  GTX I has a s p e c i f i c t o x i c i t y of 3975 and a r e l a t i v e t o x i c i t y of 72,  while NEO and GTX's  II  and IV  have similar  t o x i c i t i e s 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  i t s corresponding carbamate (GTX's 1, 2, 3 & 4), and therefore the sulfamate toxins  when  present  in  contaminated  latent or c r y p t i c t o x i c i t y al_.  (1980)  found  that  (Hall  complete  shellfish,  & Reichardt hydrolysis  constitute  1984, Yasumoto of  the  a  reservoir  1985).  sulfamate  group  Hall  of et  can be  accomplished by heating s h e l l f i s h extracts to 100°C for 5 minutes in aqueous HC1 (0.1 M). it  1s  Due to the increase in t o x i c i t y associated with this conversion,  difficult  containing sulfo-group digestion.  to  sulfamate  accurately toxins.  determine  the  true  potencies  There 1s some concern that  of  hydrolysis  extracts of the  could take place under conditions of food storage, preparation or  8  3. Tetrodotoxin (TTX) Tetrodotoxin 1s a potent marine neurotoxin found c h i e f l y 1n the gonads and eggs of certain species of p u f f e r f i s h from the family Tetraodontidae. has also  It  been shown In the C a l i f o r n i a newt Taricha (Mosher et al.., 1984), a  goby Gobus e r i n i g e r  (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.  colorless prisms which are only sparingly acid solutions. solutions molecule  TTX  Its  soluble in water except in  At pH's below 3 or above 7 decomposition occurs. 1s  (Figure  degraded 2)  has  Into a  several  cyclic  quinazollne  hemllactal  grouping  and  ref:  Structure of tetrodotoxin  Kao 1983  are  slightly  In a l k a l i n e  compounds.  structure with a single guanidium cation and hemilactal anion.  Figure 2.  crystals  a  The TTX zwltterlon  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 Saxitoxin actions.  (STX) and t e t r o d o t o x i n  (TTX) e x h i b i t  similar  biological  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 o f e f f e c t o r c e l l s to autonomic agents.  These e f f e c t s a r e due t o an a b i l i t y o f the t o x i n s t o  prevent the i n c r e a s e o f 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 o f sodium 1ons r e q u i r e d f o r the generation o f an action potential.  The pharmacological a c t i o n o f STX and TTX 1s s i m i l a r l y  exerted on membranes that 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 o f STX and TTX t o b l o c k 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 o r on the a c t i v e sodium e x t r u s i o n process (Kao, 1967).  Other m o l e c u l a r components o f the nervous system which  are u n a f f e c t e d I n c l u d e : potassium channels, the sodium-potassium a c t i v a t e d ATPase o r 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 a r e 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 o f STX and TTX.  Whereas TTX lowers systemic a r t e r i a l p r e s s u r e (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 o f 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. r e s i s t a n t t o TTX.  These nerves a r e s u s c e p t i b l e t o STX but a r e h i g h l y  10 5. Mechanism of Action In  nerve and muscle c e l l s , an action potential  1s I n i t i a t e d  but transient Increase in membrane conductance to sodium. excitable membrane organization mediated by a s p e c i f i c dependent interior ions,  aqueous  consider  other  Current concepts of in  conductance to be  i n t r i n s i c membrane protein which provides  pathway  for  cations  (Weigele & Barchi, 1978).  and  the Increase  by a large  major  ionic  It  across is  the  membranes  thought that  channels  possess  a  hydrophobic  sodium and  some  voltage  potassium  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 parts of the outer membrane. who  provided  causing  the blockage  hypothetical group Bonding  evidence  for  the channel by bonding with adjacent  This theory has been challenged by Kao (1983) a  new model  to  explain  of the sodium channel.  In his  surface receptor s i t e for STX and TTX.  (planar)  projects  over  the  outside  orifice  the  physical  paper, Kao presented a The cationlc guanidinium of  the  sodium  forces would be e l e c t r o s t a t i c attraction between charged  groups and fixed anionic  charges  there would be weak bonding  around  forces  groups and the membrane receptor. C-12 hydroxy position  mechanics  the sodium channel.  channel.  guanidinium In  addition,  (probably H bonds) between twin hydroxy It  1s unlikely that covalent bonds at the  of STX and the membrane are involved, because reduced  STX which retains some i n t r i n s i c a c t i v i t y has an a l c o h o l i c configuration which cannot  form  covalent  bonds  (Koehn  et  al.,  1981;  Kao,  1983).  This  view  receives some support from the fact that both toxins are p o s i t i v e l y charged at pH 7 by virtue of t h e i r guanidinium groups and both contain many hydroxy and amino groups on the surface which are potential 1984).  H bond donors  (Strichartz,  11 During his  research on the mechanism responsible for the actions of STX  and TTX, Kao (1983) also Investigated the s t r u c t u r e / a c t i v i t y relationships the  toxins.  activity:  The  following  groups  of  STX were found to  7, 8, 9 guanidinium group 1n i t s  hydroxy group and the carbamyl groups  on  the  TTX molecule  groups  on the STX molecule.  was shown to be analogous  group.  were  be essential  of for  catlonlc charged form, the C-12  In addition, 1t was determined which  stereospedfically  similar  to  the  The 1, 2, 3 guanidinium group of TTX  to the 7, 8, 9 group of STX.  active  (charged)  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  comparable s t e r e o s p e d f i c  the  carbamyl  group  man are  largely  of  STX  were  located  1n  positions.  6. Organisms Elaborating PST a. Description of d i n o f l a g e l l a t e s Marine  protozoa  Mastigophorans, flagellates  poisonous  family  to  Peridiniidae  and  order  derived  Dinoflagellata.  proteins  marine food chain.  and fats  and are  Dinoflagellates  considered the  are microscopic,  cellulose  The  two  mobile  plates  flagella.  can be thrown off  In  thecate leaving  species  foundation  single  t h e t i c and often bioluminescent algae generally possessing and  class dino-  form an important part of ocean plankton as primary producers of  carbohydrates,  wall  from the  the  photosyn-  a heavy thecal c e l l  with  an "ecdaysal"  cell,  of  pellicles,  the  or temporary  cyst  which can synthesize a new theca 1n a short time.  Because the nutrition of  the  animals,  dinoflagellates  referred to as During  that  for  plants  and  they are  often  plant-animals.  their  c a l l e d 'blooms'  overlaps  periodic maxima,  can occur.  local  discolorations  of  coastal  waters  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. chlorophyll of  The red color is caused by a xanthophyll  (perldlnin 1n a  containing protein) which is part of the l i g h t harvesting antenna  photosystem  II  (Prezelen  and  Randall,  1978).  Red  tide  remarkably monospecific, with 90-95% of phytoplankton belonging species  (Sweeney,  1974).  There  are  three  general  blooms to a  hypotheses  are  single on  the  competitive advantage(s) which allows a single species to form a monospecific bloom.  These are,  phytoplankton  that the species may divide more rapidly than competing  because  of  the  dinoflagellates  may  competitors,  behavioral  affecting surface  or  blooms  population. stable  excrete  Include;  illumination  presence  a  substances  differences the  and  of  specific  which may  give  grazing  action  column are  an  known to  stimulate  the  factor,  the  toxic  growth  advantage.  of  Factors  turbulence, transparency,  of  An increase in temperature and l i g h t  water  Inhibit  temperature, water  the  growth  the  local  zooplankton  Intensity and a r e l a t i v e l y growth  of  blooms  (Prakash  1974). Dinoflagellates animals  are  of  responsible  the  genera  for  the  Gonyaulax  transfer of PST to certain marine (species  tamarensis-excavata and Pyrodinium (species  catenella,  acatenella  and  phoneus, bahamense var bahamense  and bahamense var compressa).  In 1979, Taylor proposed that a new genus be  formed  for  called  Gonyaulax. many  the  three  toxic  species  of  the  genus  The reason for this transfer is that these three species d i f f e r 1n  respects  epithelial  Protoqonyaulax,  from  plate  other  pattern,  displacement and cyst type.  species  1n the  hypothecal  genus  pattern,  Gonyaulax. apical  for  pore,  example  1n  degree  of  13 b. Location The world.  toxic  dinoflagellate  species  can  be  found  1n many  G. catenella 1s found along the P a c i f i c coast  parts  of  the  of North America from  southern C a l i f o r n i a 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 A f r i c a (Taylor, 1984).  G. tamarensls has been reported 1n Alaska, the North A t l a n t i c  from Long Island to the A r c t i c , and Portugal as well  as Venezuela (Reyes-Vasquez  north to Norway (Heimdal,  1983),  et aj.., 1979) and Japan (Fukuyu, 1979).  G^ acatenella appears to be s p e c i f i c to coasts  1n the north-western P a c i f i c .  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 d i n o f l a g e l l a t e s ,  also been shown to elaborate PST.  other aquatic organisms have  The occurrence of PST in the fresh water  cyanobacterium Aphanizomenon flos-aquae was reported by Mahmood and Carmlchael 1n 1986.  In  1983,  searching  gastropods  in Japan, Kotaki,  Janis as the toxin progenitor.  for  the  source  et aj..(1983)  of  PST  1n  coral  discovered a calcareous  crabs  and  red algae  This organism has also been shown to produce  tetrodotoxin (Yasumoto et a l . , 1986).  d. PST Levels and P r o f i l e s The p r o f i l e s and concentrations of PST in organisms responsible for PSP are  extremely  variable.  A  recent  report  by  White  (1986)  previous data on the toxin contents of various d i n o f l a g e l l a t e s laboratory conditions may be i n v a l i d .  suggests  that  cultured under  Concentrations of PST were determined  14 in 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  ug to 1.1 x 1 0  -6  -3  yg PST.  Another  finding  of t h i s study was t h a t 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. found 1n nearby blooms. and accumulate they may  Values were as high as those  Toxic c y s t s s i n k to the bottom of the water column  i n the f l o c c u l e n t l a y e r s at the sediment/water  overwinter.  i n t e r f a c e where  I t has been suggested t h a t 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 n a t u r a l blooms.  In t h i s study, a l l samples contained s e v e r a l  t o 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 p r e v i o u s 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 o f 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), t h a t the PST are  products o f non-toxic p r e c u r s o r s 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  s h e l l f i s h RNAase, a c t i n g on plankton RNA, Shimizu,  at  the  U n i v e r s i t y of  Rhode  algae.  Kodama proposed t h a t  r e s u l t e d 1n the formation of PST. I s l a n d , has  been  instrumental  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  1n  first  report (1984) d i s c u s s e d the o r i g i n of the unique perhydropuNne nucleus of the STX  derivatives.  Various feeding experiments  with  specifically  labelled  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 that 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) o f n e o s a x i t o x i n . T h i s was accomplished  by feeding [ l , 2 - C ] g l y c i n e , D,L-[3- *C] s e r i n e and l ,  L - [ S - m e t h y l - C ] methionine l,  1  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 o f the n e o s a x i t o x i n molecule. The  fundamental  possess PST remains d i s t i n c t algae.  question o f how these organisms to be s o l v e d .  come t o produce  PST a r e found i n s e v e r a l  or  taxonomically  A l l a r e known t o 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 extensive study on 40 s t r a i n s o f Gonyaulax from various 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  that t o x i g e n i c i t y i s inherent t o 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 o r some other minor g e n e t i c changes. Research  e f f o r t s by Shimizu  a r e 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 t o 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 regarding 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 o f 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.  that 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 .  II.  t h a t widely 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 a r e 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 Columbia.  in British  16 a  northern mainland coast  b  the s t r a i t between the northern half of Vancouver Island and the mainland  c iv.  the southwest coast of Vancouver Island.  that the 2 southern regions are most toxic in the summer/fall months, and the north region 1n winter months  v.  that  there i s  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 d i s t r i b u t i o n of toxic d i n o f l a g e l l a t e s Chiang  is very complex.  (1985) also examined the records of t o x i c i t y in B r i t i s h Columbia,  from 1963 to 1984 and developed a 'PSP A c t i v i t y Scale' figures.  The PSP a c t i v i t y level  index by an intensity index.  on the basis of these  is determined by multiplying an extensivity  The extensiveness of the area affected is  given  as the percentage of samples showing a positive response, and the intensity of a bloom  is  ug/lOOg and  the  ratio  those  between  showing  samples  levels  less  showing  PST  levels  than 80 ug/lOOg.  greater This  than 210  PSP A c t i v i t y  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 certain (cited  fact times  from  outbreak  of  research was phenomenon.  that of  mussels  can  the year  Sommer  et  poisonings started  has  al., in  contain  in earnest  potentially  been reported  1937).  July  a  of  However, 1927,  in  1n the  fatal  substance  literature  it  was  not  until  the  San  Francisco  since a  at 1793  massive  area,  that  to determine the nature and cause of  this  17 Until  1929, 1t had been thought  affected. this  In  time.  that mussels were the only marine organisms  f a c t , the toxin causing PSP was termed 'mussel  poison'  until  The fact that other bivalves, 1n addition to mussels, may become  contaminated was  demonstrated  consumption of the Washington  in August  clam Saxidomus  Further research on other clam species implicated:  horse  neck  clam -  showed that  (Sommer et al.., 1937). the following  nuttali,  little  patula. rock clam -  and the Pismo clam - T i v e l a stultorum. virtually all  three people died from  nuttali  Schizothaerus  Paphia staminea, razor clam - S i l i g u a  and his associates,  1929 a f t e r  were also  neck clam  -  Pholadidea penita  Since the pioneering work by Sommer  species of clam, mussel, oyster and scallop  have been shown to accumulate PST.  b. PST D i s t r i b u t i o n in Toxic S h e l l f i s h The toxin d i s t r i b u t i o n of the PST in s h e l l f i s h is v a r i a b l e , depending on the  type  of  bivalve  (mussel,  clam,  analyzed and the c o l l e c t i o n s i t e various role  clam species  oyster,  (see Table 1).  studied, saxitoxin  appears  in the t o x i c i t y of most s h e l l f i s h .  major toxins oyster  in  the mussel  regardless  California,  of  the  on the other  M.  edulis,  the  contained  second sample also containing saxitoxin.  tissue  to play a r e l a t i v e l y minor 1 and 2 appear as the  the two species The  specific  With the exception of the  Gonyautoxins  location. hand,  scallop),  mussel  of  M.  principally  scallop  and the  californianus  neosaxitoxin,  It was postulated  from  with a  (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  PST P r o f i l e  affected  (In order of  Reference  Importance) Mussel (Mytllus e d u l l s )  Oasle Bay,  G. catenella  not given  Japan V1go, Spain  GTX'S 1,2,5, STX  Shimizu, 1979  GTX'S 3,4 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,  C. catenella  GTX's 1,2,3,4,  Alaska 5,6, STX Mussel (Mytllus  Bodega Bay,  cal1forn1anus)  California  G. catenella  whole body  NEO, GTX's 1,2,3,  Whltefleet-  Site 1  4,  Smith et a l . , 1985  whole body Site 2  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  hepatopancreas  GTX's 1,2,3,4,5,  Maruyama et  (small amts.  STX, NEO  al_., 1983  also found 1n Mm, gonads and gill) Scallop (Patlnopecten  Ofunato Bay  yessoenls)  Japan  G. tamarensis  found 1n rectum, foot, gonads, gill  and mantle)  19  Table 1.  Summary of PST found 1n bivalve molluscs from various areas, (continued)  Bivalve Mollusc  Location  Dlnoflagellate  Body part  PST P r o f i l e  affected  ( i n order of  Reference  Importance) Oyster (Crassostrea  Senzak! Bay  glgas)  Japan  G. catenella  midgut gland  GTX's 1,2,3,5,  Onoue et a l .  STX, NEO  1980  neck tissues  STX, NEO  Whltefleet-  body  NEO, GTX'S 1,2,  Clam-butter (Saxldomus  Bodega Bay,  nuttal1)  California  G. catenel1 a  Smlth et al_., 1985  3,4 Clam-butter  —  —  siphon  STX  Schantz et al.., 1957  (Saxldomus glganteus)  Porpoise Island, Alaska  G. catenella  not given  STX, NEO 4  Puget Sound Washington  G. tamarensls  whole body  GTX'S 1,2, NEO  Essex,  G. tamarensls  not given  GTX'S 2,1, STX,  WhltefleetSmlth et al.., 1985  Clam-soft shell (Mya arenarla)  Clam-soft shell (Mya arenarla)  Massachusetts Hampton,  BI,  Jonas-Davles and U s t o n , 1985  Shimizu, 1979  GTX'S 3,4 G. tamarensls  GTX 2, STX,  Massachusetts  GTX'S 1,3  Clam-Man1la (Tapes joponlca)  Oase Bay Japan  G. catenella  not given  GTX's 1,2,5, STX, GTX'S 3,4  Shimizu, 1979  20 edulis sample from Spain. the  soft  shell  Whitefleet-Smith basic  toxin  clam  Gonyautoxlns  M.  et al..  arenaria  (1985)  1 and 2 were also the major toxins in  and  the  Manila  noted a striking  r a t i o among samples  of Saxidomus  clam  Tapes  v a r i a b i l i t y of  nuttal1.  Necks  japonlca.  basic/weakly  of S.  nuttali  contained the basic toxins NEO and STX exclusively while the bodies had equal proportions basic and weakly basic toxins. storage Since  sites the  for the various toxins  are  toxins  all  This suggests either d i f f e r e n t i a l  or degradative  closely  related  loss of certain toxins. structurally,  metabolic  interconversions of the toxins may be the reason for d i s s i m i l a r i t y  in those  toxin p r o f i l e s .  c. Physiology and Behavior The macroscopic appearance of toxic s h e l l f i s h does not d i f f e r from that of normal s h e l l f i s h . host  organisms and  For this reason, l i t t l e attention has been paid to these 1t  1s generally  e f f e c t on t h e i r well-being.  in  the mussel  Mytllus  cardioinhibitory  and  Kelloway  confirmed  (1935)  that  the PST have  However, the effects of  been reported by a number of that  assumed  researchers.  vascomotor  centers  these  findings  PST on s h e l l f i s h  Prinzmetal et a l .  californianus,  PST  depressed  and conduction and also  reported  extensive  mortality  and  morbundity  (1932)  showed the  the myocardium. a  rapid  to PST. in  have  respiration,  in  noted  systemic a r t e r i a l pressure a f t e r exposure of the mussel (1968)  l i t t l e or no  fall  in  Adams et al_.  shellfish  after  a  massive bloom of G. tamarensis off the coast of England, although no mortality was seen with the mussel Mytilus In  1985,  Shumway  et  al.  edulis. reported the oxygen  consumption,  shell-valve  a c t i v i t y , heart rates and f i l t r a t i o n rates of various bivalve molluscs.  The  results showed that there is no 'singular bivalve response' to the presence of  21 Gi  tamarensis,  individually  but  or  rather  a  complex  1n combination.  array  of  responses  Some displayed  no  that  change  can  1n  occur  shell-valve  a c t i v i t y while others either shut t h e i r valves and exhibited a swimming escape response (Placopecten magellanicus). or decreased t h e i r a c t i v i t y . shellfish  tested Increased  change 1n heart rate.  Its  None of the  shell a c t i v i t y , and none showed a  Oxygen consumption was more v a r i a b l e .  significant  Mytil us edul1s  and M. arenaria increased the amount of oxygen consumed, while others showed a s i g n i f i c a n t decrease.  Only M. arenaria s i g n i f i c a n t l y decreased i t s  rate a f t e r the addition of toxic d i n o f l a g e l l a t e s .  filtration  A l l others showed no change  in f i l t r a t i o n rate. One s t r i k i n g  feature of PSP testing  exposed to roughly accumulate  similar  is that d i f f e r e n t species of animals  amounts of toxic dinoflagellates  quite d i f f e r e n t amounts  of  toxin.  will  One explanation  frequently  is  selective  feeding; some species may p r e f e r e n t i a l l y feed on these organisms, while others may find them unpalatable. shown  that  the  mussel  Both situations  Mytilus  have been reported; 1t has been  californianus  dinof lagel l a t e s ,  even when they accounted for  community (Buley  1936 cited in G i l f i l l a n  will  feed  some 2% of  the  on  phytoplankton  the P a c i f i c  oyster  (Crassostrea gigas) does not readily accept G. washinqtonensis as food  (Norris  and Chen, 1974). that  feeding  dinoflagellates  1974), and that  selectively  Preliminary results published by Cued et al_. (1985) suggest rates are  unchanged or s l i g h t l y  of  bivalve  molluscs  species-specific. Increased  It  a  significant  tamarensis.  decrease  the  was  found  for the following  Placopectan magellanicus, and Mytilus edul1s. showed  in  in  feeding  presence that  bivalves:  of  rates Ostrea  toxic  remained edulis.  On the other hand, Mya arenaria  rate  after  exposure  to  toxic G_j_  22 These former studies Yamaguchi  (1974)  have pertinence, since  have suggested that  Investigations by Twarog &  the r e l a t i v e t o x i c i t i e s  attained by a  group of f i l t e r - f e e d i n g molluscs may be a result of d i f f e r i n g s e n s i t i v i t i e s 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.  individual fibres.  Resistance  nerve  fibres  The mussel  of  and was  Mytilus  were shown to be highly quantities  to PST and TTX was found to be the property of not due to a protective sheath around the  edulis and the scallop  resistant  the toxins.  Placopecten  magellanicus  to PST and were able to accumulate  The oyster  Crassostrea  virqinica  and  large  the  fresh  water clam El 1iptio complanata were highly sensitive to the PST and were able to bind only r e l a t i v e l y low amounts of PST.  Mya arenaria  (soft  shell  clam)  was of intermediate resistance.  d. Mechanism of Accumulation In most  shellfish  hepatopancreas  species,  the PST are accumulated p r i n c i p a l l y  and are then excreted or  released within  several  in the  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 d i s t r i b u t i o n  in the siphon of butter clams  corresponded to areas  that tip.  the PST  concentration decreased as  distance  of melanin pigment and  increased from the  siphon  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  d i s t r i b u t i o n of PST 1n the clam siphon corresponded with the d i s t r i b u t i o n of melanin ( i . e . 53% of siphon melanin also contained 46% of the PST). found  that  the  degree  of  binding  was  strongly  influenced by  They also  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  competition melanin.  desorption between  Increased  hydrogen  ions  with and  the the  Increasing PST  for  acidity,  binding  The exact mechanism by which the butter clams  Indicating  sites  on  the  retain PST is  not  known, however, Price and Lee have suggested that i t might be similar to the binding  of  findings  PST  on a weak  cation  exchanger.  This  theory  is  based on the  of White (1958), who showed that melanin can function as  a cation  exchanger by virtue of i t s free carboxyl and phenolic hydroxy groups.  At low  pH's, the weak a c i d i c groups in melanin are in the hydrogen form, which could explain the blocking of PST binding at a c i d i c pH's.  The e f f e c t 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 d i r e c t l y related to the valence cation.  of the  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 d e t o x i f i c a t i o n of poisonous s h e l l f i s h has not yet been developed. understanding excrete PST. degree of  This  concerning  is the  not surprising mechanism  by  in view of our current lack of which  shellfish  accumulate  and  Several methods have been put forward, but none has achieved any  success  on a commercial  level.  reported are ozonation and thermal shock.  The two most promising procedures Blogaslawski et a_l. (1979) provided  24 evidence that ozonation could remove low levels of PST from soft shell obtained bloom.  from Crow Harbour,  NB during  the i n i t i a l  stages of a G.  clams  excavata  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 d e t o x i f i c a t i o n .  These authors postulated that ozonation may be e f f e c t i v e  on s h e l l f i s h 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 i n e f f e c t i v e in d e t o x i f i c a t i o n . Heat treatment or thermal shock has been a popular idea for the removal of  PST  from contaminated  incidents  of  shellfish. effective clams. who  PSP  shellfish  illness  have  even  been  though  related  Metcoff et al.., 1947 showed that than  domestic  cooking  for  a to  large the  ingestion  commercial  reducing  the  percentage of  of  the  cooked  retorting was more  toxicity  in  soft  shell  The k i n e t i c s of PST destruction was determined by G i l l et al_., (1985),  established  a  mathematical  relationship  destruction and the time of heating  PST at various  The k i n e t i c s were found to be of the f i r s t most micro-organisms.  between  the  rate  processing  order as  of  thermal  temperatures.  t y p i c a l l y observed  for  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.  process  destruction  It of  PST  was  further  would  also  stated result  that in  a  a  significant  resulting  in  90%  reduction  in  the  n u t r i t i v e q u a l i t i e s of the processed s h e l l f i s h .  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. other marine animals  It was not u n t i l many years l a t e r that reports of  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 p r o f i l e ( i n order of significance)  Gonyaulax  San Francisco  New England  Part of Body  Reference  STX  digestive gland  Sommer et al_., 1937  STX  digestive  Foxall et  gland  al.., 1979  viscera  Kotakl et al.., 1981  Sand crab (Emerlta analoga)  Rock crab (Cancer i r r o r a t u s )  tamarensis  Gonyaulax tamarensis v i a contaminated shellfish Unknown  Japan  STX, GTX II, NEO  Coral crab (Zoslmus aeneus. Aterqatls f l o r l d u s , Platypodia granulosa)  Janla spp. (alga)  Japan  Z. Aeneus & whole body A. f l o r l d u s : appendages NEO, STX, GTX I & II P. granulosa: STX  Yasumoto et  Coral crab (Pllumnus vespertlUo,  Janla spp.  Japan  NEO, STX, whole body GTX I, II & III  Yasumoto et al.., 1983  Gonyaulax catenella via contaminated shellfish  Puget Sound  NEO, GTX II & III  viscera eggs  Jonas-Davles and Llston, 1985  Gonyaulax catenella v i a contaminated shellfish  Puget Sound  NEO, GTX II 8 III, STX  viscera eggs  Jonas-Davles and Llston, 1985  Snail (Turbo argyrostoma, Turbo marmorata, Tectus pyramls)  al.., 1981  Koyama et a].., 1981  Thalamlta spp. Erlphla scarbrlcula) Kelp crab (Pugettia  producta)  Rock crab (Cancer productus)  26  Table 2.  Occurrence of PST 1n various marine organisms (excluding bivalve molluses).  Marine Species  Causative Organism  Location  Shore crab  unknown  Puget Sound  (Hemigrapsus  PST p r o f i l e ( i n order of significance) GTX I & IV,  (continued)  Part of Body  Reference  whole body  Jonas-Davles and Llston, 1985  STX  oregonesis) Gonyaulax spp  Southern  STX  hepatopancreas Barber et a l . , 1988  Puget Sound  GTX II & II, NEO, GTX I & IV  whole body  Puget Sound  STX equivalents determined only  whole body  Jonas-Davles and Llston, 1985  B.C. Tubeworm (Eudlstrylla  Gonyaulax catenella  coast  spp.)  Starfish (Plaster ochraceus)  unknown  Jonas-Davles and Llston, 1985  Barnacle (Balanus spp.)  unknown  Puget Sound  STX equivalents determined only  whole body  Jonas-Davles and Llston, 1985  Crab larvae (cancer anthonyls)  Gonyaulax catenella  Los Angeles  STX equivalents only  larvae  Yazdandoust, 1985  27  began appearing 1n the l i t e r a t u r e . to accumulate PST.  Table 2 l i s t s the various organisms known  In addition to the many genera and species of crab  that  have been shown to contain PST, s n a i l s , tubeworms, s t a r f i s h and barnacles have also been shown to accumulate these toxins. The causative  organisms for  algae.  The red calcareous alga Jania is responsible for intoxication of coral  Infects coast,  are  either  such as the sand and coral  tubeworms  off  barnacles  feeders  crabs,  crabs  and  filter  toxic  dinoflagellates  the coasts of Japan while the d i n o f l a g e l l a t e  rock crabs is  from New England.  or  Gonyaulax  other  tamarensis  G. catenella, found along  the P a c i f i c  responsible for infecting tubeworms and barnacles d i r e c t l y and the  carnivorous  kelp  crabs,  rock  crabs  and  starfish  indirectly,  through  contaminated s h e l l f i s h . The source of PST in the shore crabs (from Washington & B.C.) snail  (from Japan)  is  unclear.  therefore not primary consumers carnivores,  ruling  possibility  is  dinoflagellate PST  (White  out  that cysts  1986).  These organisms are not of toxic d i n o f l a g e l l a t e s .  secondary  these  filter  intoxication  herbivores  become  via  feeders and  Neither are they  toxic  Infected  and marine  by  shellfish. consuming  which have been shown to contain s i g n i f i c a n t Another p o s s i b i l i t y  is  the existence of  One toxic  levels  of  another marine  species capable of producing PST, which is an item in the normal diet of sand crabs and s n a i l s . The  toxin  regardless  There are no l i t e r a t u r e reports of research in this area.  composition  of  the  various  species  is  quite  similar,  of l o c a t i o n , with the exception of the shore crab and one species  of coral crab.  The predominant toxin in most crabs  by STX in coral crabs and gonyautoxins rule the following STX, GTX I,  crab  II,  toxins  and III.  II  & III  are found in varying  is  neosaxitoxin  followed  1n kelp and rock crabs.  As a  amounts  NEO,  in a l l  crabs:  By comparison, the shore crab contains GTX's I and  28 IV and small appears  amounts of STX, and the coral crab P. granulosa only STX.  to be present  in larger quantities  in Japanese crabs  than  STX  in  crabs  from the P a c i f i c Northwest.  9. Tests f o r PST a. Mouse Bioassay The extracts,  method  currently  employed  the mouse bioassay,  is  for  the  routine  a modification of  described by Sommer and Meyer (1937).  assay  of  shellfish  a procedure  originally  B r i e f l y , an a c i d i c extract is prepared  from the s h e l l f i s h and a 1 mL aliquot is injected i n t r a p e r i t o n e a l l y 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 t o x i c i t y of the extracts is determined.  Values  are expressed in mouse units (MU) and then converted to ug STX/100 g s h e l l f i s h tissue.  A value  greater  consumption in Canada. that w i l l  kill  respiratory  g  is  considered u n f i t  One MU was further defined as  a 20 g mouse in  failure.  relationship:  than 71 y.g/100  Mouse  log dose (MU)=  15 minutes with  units  are  the amount of  symptoms  calculated  for  of  from  human poison  paralysis  the  or  following  145/t - 0.2, where t (time) is in seconds.  The  mouse unit as a measure of t o x i c i t y was developed because a reference toxin was unavailable at this time. In  1957,  Schantz  et  al_. prepared a  digestive glands of toxic mussels toxic  Alaska  toxicity  butter  clams  highly  purified  (Mytilus californianus)  (Saxidomas  giganteus).  This  toxin  the  and the siphons of toxin  of 5500 ± 500 MU/mg and was subsequently adopted as  standard for the o f f i c i a l mouse bioassay.  from  (STX)  had a  the reference  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 e f f e c t on the L D . I 0  mice were toxin.  shown to  be more  susceptible  than males  at  Female  higher doses of the  Acute L D ' s were determined using p u r i f i e d STX for several routes of 8 0  administration; 263 ug/kg for the o r a 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 t o x i c i t y .  These effects  were not additive and the sodium e f f e c t appeared to be the stronger, 1t did not a f f e c t the oral or intravenous t o x i c i t y .  although  It was concluded that the  median death time of mice as a c r i t e r i o n of t o x i c i t y  is  not r e l i a b l e at pH  levels above 4 or in the presence of sodium ions above 0.1 M. The mouse bioassay has sensitivity  1s  approximately  error of 20%.  Marginally  much as  Other  60%.  several  limitations.  30 ug/100 g  toxic  factors  For example, the l i m i t  shellfish  tissue  with  a  of  standard  s h e l l f i s h may also be underestimated by as  in  to  the  t o x i c i t y in mice, and the expense of maintaining a mouse colony is high.  In  a d d i t i o n , the mouse bioassay is toxicity  of  shellfish  extracts  shellfish  extracts  may  contribute  unable to accurately determine the potential containing  sulfamate  toxins.  Unfortunately,  the conditions used are not s u f f i c i e n t l y a c i d i c to ensure complete hydrolysis of the toxin complex to Its  corresponding  severely underestimate i t s potency.  carbamate state and therefore may  There have been many attempts to provide  an acceptable a l t e r n a t i v e to the mouse bioassay, remains the o f f i c i a l test for PSP.  but despite this e f f o r t  it  30 Table 3.  Summary of assays developed for the detection of PST in s h e l l f i s h .  Assay  Principle  Major limitations  Reference  Fluorometry  Alkaline oxidation of STX to fluorescent derivative  Only measures STX Complex and timeconsuming  Bates and Rapoport (1975, 1978)  Thin layer Chromatography  Alkaline oxidation of PST to fluorescent derivatives  Does not quantitate with s e n s i t i v i t y  Buckley et a l . (1976, 1978)  Electrophoresis  Alkaline oxidation of PST to fluorescent derivatives  Does not quantitate with s e n s i t i v i t y  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)  Radioimmunoassay  Measures STX via an anti STXOL antibody  Only measures STX Requires trained personnel Radioactivity  Carlson et a l . (1984)  Housefly Bioassay  Measures total t o x i c i t y by L D housef1ies  Detection l i m i t 20 ug/100 g Requires microtechniques  Siger et a l . (1984)  Chicken Embryo Bioassay  Measures total t o x i c i t y by % mortality in 96 hour chicken embryos  Time factor Expense in maintaining facility  Park et a l . (1986)  B0  1n  31 b. Other Assays Various assays (Table 3) have been developed for the purpose of detecting the toxins Rapoport later.  causing  (1975,  PSP.  1978)  The fluorometric technique developed by Bates and  provided the basis for several  other assays developed  It was found in these studies that STX can be oxidized with hydrogen  peroxide under a l k a l i n e  conditions  to the fluorescent derivative  hydroxymethyl-2-1minopurine-3(2H)-propion1c cleanup  of  the  shellfish  extract  by  add.  column  This procedure requires a  chromatography,  fluorescent measurement from which STX levels  8-amino-6-  followed  can be calculated.  by a  The major  l i m i t a t i o n 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 PST although Bates  and  at  Increased  Rapoport  levels  method  levels  of  less than 100 yg  t o x i c i t y was underestimated.  could  distinguish  between  However, the  acceptable  and  unacceptable s h e l l f i s h with some false positives and no f a l s e negatives  (Hall  1985). Subsequent form  studies  fluorescent  compounds.  extremely v a r i a b l e . fluorescing  Unfortunately  The N-l  compounds  creates a serious  have shown that other PST can also be derivatized to  hydroxy toxins  although  their  the  degree  (GTX's I,  specific  of  fluorescence  1s  IV and NEO) are poorly  toxicities  are  high.  This  l i m i t a t i o n for any assay based on fluorescence, because 1t  may underestimate the total t o x i c i t y of a sample by a considerable amount. Other  procedures  including  electrophoretic and high pressure based  on  Individual  the  measurement  compounds.  determine absolute  of  The  levels  of  the  thin  layer  chromatographic  l i q u i d chromatographic  fluorescence,  TLC and  but  1n  (HPLC) methods  addition  electrophoretic assays  the toxins  (TLC),  separate are  and are therefore useful  unable  are the to  only as a  32 scan of the toxin p r o f i l e .  The HPLC  technique is much more sophisticated.  This technique was o r i g i n a l l y reported by Sullivan et a l . (1983). Sullivan  et  aj..  (1985)  have  developed an  optimization  capable of detecting nanogram amounts of the N-l  Since then,  procedure which  hydroxy toxins.  This method  uses a binary gradient HPLC method with post column d e r i v a t i z a t i o n . chemistry i s based on the oxidation of a l l  HPLC represents  an extremely  Detection  toxins to fluorescent products but  the method requires two separate chromatographic conditions.  is  separations  sensitive  under d i f f e r e n t  and accurate  technique  which is suitable for handling large numbers of samples for routine monitoring and has the c a p a b i l i t y to determine very low levels of a l l the  equipment  is  expensive  and  not  commonly  found  the PST. in  However,  routine  testing  laboratories. Radioimmunoassay  procedures  are  sensitive,  inexpensive for the detection of STX in s h e l l f i s h .  simple  a first  step  in  the possible  relatively  Unfortunately, the lack of  antibody c r o s s - r e a c t i v i t y to the other PST severely l i m i t s a l t e r n a t i v e to the mouse bioassay at this time.  and  its  u t i l i t y as an  However, this test serves as  development of an enzyme immunoassay  for  the  entire PST s e r i e s . The housefly and chicken embryo assays do not have s u f f i c i e n t  advantage  over the mouse assay to be considered a l t e r n a t i v e s .  The housefly  inexpensive  the maintenance  but  relatively  insensitive  and  requires  assay of  is 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 s u f f i c i e n t advantage over the mouse bioassay to be considered as an a l t e r n a t i v e . Therefore, at present, the only accepted test PST in s h e l l f i s h is the mouse l e t h a l i t y  bioassay.  for the determination of  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,  (Division  of Microbiology,  100  ug/mL  1n  20%  ethanol,  >95%  purity  Food and Drug Administration, 1090 Tusculum  Ave., C i n c i n n a t i , OH, 45226)  b. Test Samples Toxic  shellfish  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 e d water. Injections  were made  Milne-Edwards  opening  with of  a  the  25  G 7/8  crab.  needle  A total  of  and  1 mL  syringe  at  10 male and female  the 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 t a c t i l e stimulation of the  eyes and walking legs e l i c i t e d no response.  34 b.  Determination of Dose/lethal 1ty Relationship  to STX and TTX 1n The Crab  H. oregonesis 1)  Toxin dose l e v e l s : STX - l x l O  - 3  TTX - 5 x l 0 -  ii)  Toxin standards  3  yg to l x l O  yg  - 2  yg to 5 x l 0 "  yg  2  were dried by means of a j e t of nitrogen gas  and then  d i l u t e d with deionized, d i s t i l l e d water just p r i o r to t e s t i n g .  111) Male and April  female  1985.  crabs  A total  were collected from Towers  of 20 crabs  (weighing  1.5  Beach,  to 3.5 g)  Vancouver  in  were used at  each toxin dose l e v e l .  iv)  Statistics:  One-way analyses  of variance (ANOVA) were performed on the  standard curve data followed by a regression  c. The  Crab  H.  oreqonesis  S h e l l f i s h Toxins (PST) Extracts Health  of  toxic  Protection  as  a  For  The  Determination  of  Paralytic  in S h e l l f i s h  shellfish  Branch  Test  analysis.  were obtained from Ms. May M i l l i n g  government  laboratory.  Aliquots  of  at the  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 t o x i c i t y of extracts was determined. from  the  The results of these tests were then compared to results obtained  government  laboratory  which  Tests were performed in July of 1985.  employs  the o f f i c i a l  mouse  bioassay.  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  procedure  for  worldwide.  AOAC method the  in  detection  Canada of  1n  1965.  paralytic  It  is  shellfish  The mouse bioassay measures total  the  toxins  only  accepted  1n  shellfish  t o x i c i t y when l e t h a l i t y time is  standardized against a saxitoxin standard solution. Body  tissues  homogenity.  A  100  hydrochloric acid gently  from g  (HC1)  frozen sample  shellfish  was  removed  (pH must be less  were and  removed added  than 4.0).  for 5 minutes and cooled to room temperature.  to  and  blended  100 mL of  to  0.1 N  The mixture was boiled Following t h i s , 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 destruction  of  d i s t i l l e d water. to s e t t l e u n t i l One mL of  this  addition  the  PST),  followed  by  dilution  a l k a l i n i z a t i o n and consequent to  200 mL with  deionized,  The mixture was returned to a beaker, s t i r r e d , and allowed the supernatant was translucent and free of s o l i d acid  (female, Swiss white). indicated  (to prevent local  by the last  extract was  administered  intraperitoneally  particles. to  3 mice  The time of Inoculation was noted and time of death gasping breath.  The extract was d i l u t e d so that  the  death time was in the range of 5 to 7 minutes. T o x i c i t y of the extracts was calculated a f t e r 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 (Division  dihydrochloride,  of Microbiology,  100  ug/mL  1n  20%  ethanol,  >95%  purity  Food and Drug Administration, 1090 Tusculum Ave.,  C i n c i n n a t i , OH, 45226)  Tetrodotoxin  in 0.1  N acetic a c i d , c i t r a t e 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 A p r i l 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. 24 hours of c o l l e c t i o n .  A l l tests were performed within  37 b. Additional Testing I)  Samples  of  Hemigrapsus  nudus  were  Beach during the same time period.  also  collected  from  Tsawwassen  A total of 10 male and female crabs  weighing 1.5 to 3.5 g were used for each l e t h a l i t y t e s t . II)  Samples (Powell  of  River)  Additional (Seshelt)  H. oregonesis 1n  samples  and H. nudus were collected from Okeover Arm  July of  H.  1986  during  oregonesis  a  toxic  were  dinoflagellate  gathered  in November 1986 during another toxic bloom.  at  bloom.  Porpoise  Bay  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  shellfish  8-amino-6-hydroxymethyl-2-iminopurine-3(2H)-propionic  acid,  the  extracts  to  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  reservoir, BioRad)  cm i . d . x 5 cm, medium f r i t t e d glass f i l t e r ,  45 mL  39 2. Methods a. Preparation of C a l i b r a t i o n Curve Several d i l u t i o n s of a stock solution of STX were used to establish a STX fluorescence solution  curve.  with  2 mL  Alkaline 1.0  oxidation was carried out by mixing 2 mL STX  N NaOH and  0.6  mL  1%  hydrogen  followed by an incubation at room temperature for 40 minutes. acid was then added to obtain the fluorescent product. was measured for the samples excited at 332 nm.  peroxide  (H2O2)  Glacial acetic  Fluorescence at 381 nm  A blank consisting of 0.6 mL  d i s t i l l e d water in place of hydrogen peroxide was subtracted from each sample value to obtain the standard curve (Appendix  II).  b. S h e l l f i s h Assay Bio-Rex by taking sulfuric  70 1on-exchange resin  200 mL (wet volume) acid  (H2SO4,  (50-100 mesh, H  form ) was equilibrated  and rinsing with water  3 X 600 mL), water  (NaOH, 3 X 600 mL) and water (3 X 600 mL). -  +  (600 mL),  (3 X 600 mL), 0.5 M 1 M sodium hydroxide  Rinsing was achieved by s t i r r i n g 5  10 minutes and decanting a f t e r the resin had s e t t l e d .  suspended  H2SO4, mL).  1n 0.2  M acetic acid  followed by rinsing  (600 mL)  The resin was then  and the pH adjusted  to  5.0  with  with 0.2 M pH 5 sodium acetate buffer (2 X 600  Resin was stored in this buffer at 4°C. Body  tissues  homogeneous.  from  frozen  A 2 gram aliquot  shellfish was  samples  were  removed and added  to  blended  until  2 mL of  0.5 M  t r i c h l o r o a c e t i c acid (TCA) which was freshly diluted from a solution of 2 M TCA.  A f t e r 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 i n g  u n t i l 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 glass  filter  and  45 mL reservoir)  cm 1.d.  containing  exchange resin and the effluent discarded.  X 5 cm with medium f r i t t e d equilibrated  Bio-Rex  70  1on  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, centrifuge tube. centrifuge to  1  portion.  Two mL of  while  water  1.2 was  M  NaOH and 0.05 mL of  substituted  for  the  H2O2 was  10%  H2O2  in  the  added other  Both portions were then centrlfuged at 1000 g for 1 minute and the  supernatants  H2O2  collected in a  This solution was mixed and divided in to 2 equal volumes in  tubes.  portion,  the effluent was  transferred  the solution  to cuvettes.  Forty minutes  after  the addition  was neutralized to pH 5 with 0.15 mL g l a c i a l  acetic  of  acid  and the fluorescence measured using excitation at 332 nm and emission at 381 nm.  The net absorbance was obtained by subtracting  unoxidized  blank  from  the  fluorescence  of  content of each sample was determined a f t e r (Appendix  II).  the  the fluorescence of the  oxidized portion.  The STX  reference to the standard  curve  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% g l y c e r o l , 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 U l t r a 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 c i t r a t e (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. and allowed to stand 5-10 min. allowed to stand 20-30 min.  This solution was mixed  Then, 0.25 mL Reagent D was added, mixed, and Color was red at 600 nm on a spectrophotometer  and amount protein determined a f t e r reference to a standard curve III).  (Appendix  43 b. Procedure for SDS  Polyacrylamide Slab Gels - Discontinuous  Buffer System  (based on U.K. Laemmli, 1970) 1)  Sample preparation Visceral  contents  from  10  homogenized and centrifuged at supernatant  (500  uL)  was  mercaptoethanol (10 uL). along  with  0.02%  (25  shore  crabs  5,000 g for  treated  with  were  5 min.  10% SDS  dissected  free,  An aliquot (25  uL)  and  of the 0.2 mM  Two drops of glycerol were added to the sample uL)  bromophenol  blue  solution  (tracking  dye).  Sample sizes of 25 uL were applied to the sample s l o t . ii)  Gel preparation (Bio-Rad) Separating gel - Three concentrations of polyacrylamide gels were used in these experiments: 6%, 7.5%, wide).  10% (0.2  See Table 4 for d e t a i l s .  Stacking gel  (3%) - The following  6.8 (3.75 mL); Acrylamide: Bis, d.d  cm thick x 11 cm long x 13.5 cm  reagents were used: 0.5 M TRIS-HC1, pH  30:1 (2 mL); SDS,  10% (0.30 mL); Water,  (8.8 mL); Ammonium persulfate, 10% (0.15 mL); TEMED, 0.00033%  mL).  This  solution  is  poured  over  the  separating  gel  once  it  (0.02 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 distilled).  solution - Acetic a d d , 7.5%; Methanol, 5%; water (deionized,  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 removed and stained for 1 hour. transferred vertical  to  a diffusion  position  with  a  hrs).  Gels were  The gels were then rinsed with water,  destainer  and destained  circulating  destaining  for 20 hours  1n a  solution  clarified  Preparation of separation gels for various polyacrylamide  strengths.  through a cartridge of activated carbon.  Table 4.  Amounts of Reagents to Use Reagent 1.5 M Tris-HCl pH 8.8 Acrylamide:Bis (30:1) 10% SDS H2O  (deionized distilled)  Concentration 0.375 M  — 0.1%  —  10% Ammonium Persulfate  0.05%  TEMED  0.00033%  6%  7.5%  10%  10 mL  10 mL  10 mL  8 mL  10 mL  13.4 mL  0.4 mL 21 mL 0.2 mL 14 uL  0.4 mL 19.4 mL 0.2 mL 14 uL  0.4 mL 16 mL 0.2 mL 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 l e t h a l i t y response in the crab, i t was necessary to determine an injectate volume which would not interfere with death times. For t h i s purpose, a variety of volumes were administered to the crab and the lethality  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 e d water were in excess of 60 minutes. after  As volumes  administration  uniform  injectate  increased past 100 uL, death times decreased to 5 min of  volume  400 uL water. of  On the  50 uL was  basis  subsequently  of used  these  results,  1n a l l  a  lethality  tests.  2. Determination of Dose-lethality Relationship  to STX and TTX in The Small  Shore Crab H. oreqonesis The standard death  times  in  curves depicting both STX and TTX dosages and  shore  crabs  obtained  presented In Figures 4a and 4b. the STX standard curve data.  from Towers  Beach  1n A p r i l  associated 1985 are  Analysis of variance (ANOVA) was performed on 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  Figure 4.  Lethality the  crab  (ugxIQ ) 2  response to various closes saxitoxin and tetrodotoxin in Hemigrapsus  tetrodotoxin (TTX)  oregonesis;  a  =  saxitoxin  (STX),  b  =  48  Table 5.  Results of analysis of variance on standard curve data for various doses STX in Hemiqrapsus oreqonesis.  Degrees of Freedom  Source  Sum of Squares  3 59  Between groups Within groups Total  Mean Squares 255.34  766.02 143.34 909.36  62  F ratio 105.10*  2.43  * (P<0.01)  The  results  of  one-way  analysis  of  variance  on  associated death times in the crab are shown in Table 5. (3,59)  is  4.10,  making  the  variance  Therefore, we must reject the null difference  among  the  treatment  ratio  dose  significant  that  there 1s no  and  conclude  STX  and  The c r i t i c a l F value  highly  hypothesis  means,  log  that  a  at  P<0.01.  statistical 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 e f f e c t on the r e s u l t s .  Results are presented in Table 6a and 6b.  Table 6a. Results of analysis of variance for the variable sex.  Degrees of Source Between groups Within groups Total * Not signficant  Freedom 1 61 62  Sum of Squares 1.55 11.72 13.27  Mean Squares 0.515 0.199  F ratio 1.59*  49 Table 6b. Results of analysis of variance for the variable weight.  Between groups Within groups  4 58  Total  62  * Not  Sum of Squares  D.F.  Source  Mean Squares  F ratio  0.342 0.531  0.644*  1.63 31.34 32.36  significant  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). crab was 1.59.  The F ratio for the variable sex versus death time in the  This value was not s i g n i f i c a n t at P<0.01 ( c r i t i c a l F value for  1 and 59 degrees of freedom = 7.12), indicating no e f f e c t on the death time of the crab due to sex.  S i m i l a r i l y , the variance ratio of 0.644 for the variable  weight versus death time, was not s i g n i f i c a n t at P<0.01 ( c r i t i c a l F value for 4 and 59 degrees  of freedom = 3.65).  Therefore, throughout  the  experiments, both male and female crabs were used, with weights  subsequent  ranging  from  1.5 to 3.5 g. Crab minutes  death  times  over a dose  after  range  administration  of  0.01  ug  STX  STX  ranged  to 0.001  ug  from  0.75  STX.  A  to  9.39  regression  analysis was performed on this data resulting in a correlation c o e f f i c i e n t (r) of  0.89  and a  c o e f f i c i e n t 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  dosages of 0.05 to 0.005 ug TTX.  TTX  from 2.86  to  8.71  minutes  for  The correlation c o e f f i c i e n t (r) of 0.83 gave  a c o e f f i c i e n t of determination ( r ) 2  regression equation of y = -4.55 -  ranged  of 0.69. 2.46x.  This response was described by  50 3. The Crab H. oregonesis as a Test for the Presence of PST 1n S h e l l f i s h The small bioassay  for  harvested, detecting  shore crab H, oregonesis PST.  and  was available  based  0.001  bioassay.  It  yg  Toxic  on  the  compared  shellfish  i n i t i a l l y appeared to be a promising  1n areas where s h e l l f i s h  standard to  0.3  curve yg  extracts  results,  PST  1t  detectable  prepared  are commonly  was  capable  using  the  by the government  of  mouse  fisheries  laboratory were used as the test material and aliquots of these extracts were administered  to  the  shore  crab.  Death  times  were  regression equation for a determination of PST content. then  compared to  official  government  performance of the mouse bioassay. a  bioassay  were  very  bioassay (Table 7). levels  of  results  PST  showed a  different  In a l l  (0.01  The  to  range  using  the  These results were  which were calculated after  PST values obtained using the crab as  from o f f i c i a l  results  based  on  the mouse  cases, the crab bioassay predicted extremely low  10.16 yg/100 of  results  analyzed  66 to  g)  240 yg  in  the  PST/100  extracts whereas g shellfish  official  tissue.  In  a d d i t i o n , the crab PST values showed no relationship to the mouse PST values; they did not increase as mouse values July  of  1985, and a f t e r  increased.  These tests were made in  reference to Figure 5 in the next section,  it  is  conceivable that at t h i s point, the shore crabs were resistant to administered PST. the  This assumption shore  crab was  cannot be confirmed because the l e t h a l i t y response of  not  tested during J u l y ,  although  in August  shore  crabs  showed a remarkable resistance to constant doses of STX with death times in excess of 20 min. consistently support time. of  the  to  The fact that the small shore crab, used as a test 1n July,  underestimated the assumption  the that  PST these  content crabs  of  shellfish  were resistant  extracts to  PST at  This change in s e n s i t i v i t y to STX 1s a severe disadvantage crab  as  a  bioassay  (obtained  detection of PST in s h e l l f i s h .  in  Its  natural  lends this  to the use  environment)  for  the  51 Table 7.  Sample #  Mean Death Time  Predicted PST Content*  in the shore crab (min)  (yg PST/100 g)  O f f i c i a l PST Content using mouse bioassay* (yg PST/100 g)  1  6.2  10.59  210  2 3 4  8.2 12.2 12.5 14.4  6.48 2.25 2.26  210  1.42  120  0.71  5  95 240  7  17.2 18.6  0.50  140 150  8  33.4  0.01  66  6  *  Determination of PST in s h e l l f i s h extracts using the crab bioassay.  Sample c a l c u l a t i o n - 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  lethality  tetrodotoxin  (TTX)  response  to  in the shore  constant  doses  of  crab H. oregonesis  shore  crab, H.  during the same time period.  its  lethality  and single  In addition, samples of  nudus, were collected from another  Vancouver area and tested for  (STX)  recovered from a  l o c a t i o n , was monitored over a period of 21 months. another  saxitoxin  response  at  location  selected  in  the  intervals  Because the Vancouver area is not tested for the  presence of toxic d i n o f l a g e l l a t e s , were taken from two additional  samples of both H. oreqonesis and H. nudus  areas along  the  southern  B.C.  coast  during  registered toxic blooms.  1. Long Term Fluctuation 1n S e n s i t i v i t y  and Resistance  to Constant  Doses of  Saxitoxin and Tetrodotoxin in the Shore Crab H. oregonesis The seasonal  pattern of crab s e n s i t i v i t y  to fixed dosages of  and tetrodotoxin are presented in Figure 5.  saxitoxin  Mean crab death times due to  tetrodotoxin injections remained r e l a t i v e l y constant (2 to 3 minutes) over the entire 21-month period, demonstrating no seasonal to this t o x i n .  Conversely, a wide seasonal  death times of crabs injected with saxitoxin.  change in crab  sensitivity  fluctuation was observed in mean Although the average death time  for a single dose of saxitoxin was approximately 1 minute 1n sensitive during  the spring  of  crabs  1985, a marked increase in death times approaching 30  minutes was observed in crabs saxitoxin  collected during August  resistance  to  injections  eventually  returned to sensitive  was  levels  found  to  (1 minute)  1985.  gradually  This  apparent  disappear  in November 1985.  and Crab  53 sensitivity until  to saxitoxin  the following  Increased times  injections persisted throughout the winter and f a l l  summer  (July  1986), when crabs  resistance to saxitoxin i n j e c t i o n s .  was  observed  in  August  1986  began to again  Peak resistance 1n crab death  and  reached  a  level  s u b s t a n t i a l l y smaller than the previous year (August 1985). same  temporal  pattern  of  relative  show an  resistance  to  which  was  Nevertheless, the  saxitoxin  Injections  was  observed and crabs were found to again lose their resistance to saxitoxin by October 1986.  2. L e t h a l i t y 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 lethality  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 l e t h a l i t y tests on H. nudus were  compared with those of H. oregonesis  (Table 8).  Throughout t h i s time period,  there was no change in s e n s i t i v i t y 1n H. nudus after administration of 0.05 yg TTX.  This  situation,  is  therefore similar  to that  of H.  oregonesis  where  death times remained r e l a t i v e l y constant (2-3 roin) during the entire 21 month period in response to TTX i n j e c t i o n s . resistance to STX Injections 1n H.  nudus  of  8.5  Conversely, both shore  crabs  showed  during August of 1985 with extended death times  min, and greater  than 20 min.  in  H.  oregonesis.  In  a d d i t i o n , both species of crab then returned to sensitive levels by the f a l l of 1985, although H. nudus  showed s l i g h t l y  1985 compared to H. oregonesis.  elevated death times  in October  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  D E A T H TIME tMIN)  10  Ap*  May  Jww  July  Aus  8«P« Oct  Nov  0*c  J*n  F«b  Apf  May  JIMM  July  At*  topi  Ocl  Mow 0«e  TIME OF YEAR  Figure 5. Seasonal  pattern of  sensitivity  and resistance to constant doses  tetrodotoxin in the shore crab Hemigrapsus  oregonesis.  (0.05 ug)  of saxitoxin and  55 subsequent  testings a f t e r 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 t h i s 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  aeneus (Noguchi et al_., 1985). paralytic  shellfish  (Koyama  et a_h,  These workers showed crab resistance to both  toxins and tetrodotoxin throughout the year, whereas the  present study showed a seasonal  resistance to STX only.  the  this  resistant  shore  crabs  in  study  remained  It  the  mechanism  1n  which  crabs  develop  is noteworthy that  relatively  tetrodotoxin despite reduced s e n s i t i v i t y to saxitoxin. that  1983) and Zosimus  sensitive  This finding  resistance  to  PSP  associated with the presence of the dominant toxin in the crabs'  waters  elaborating  all  year  round  (Kotakl  PST in the P a c i f i c Northwest  (Gaines and Taylor, 1985). southern B.C.  et  al_.,  1983),  show a seasonal  suggests could  be  environment.  While PSP and tetrodotoxin have been found in a marine macro-alga Japanese  to  inhabiting  dinof lagellates  pattern of t o x i c i t y  Moreover, tetrodotoxin does not occur naturally 1n  and therefore shore crabs are not l i k e l y to come into contact  with t h i s t o x i n .  Thus, the s e n s i t i v i t y of shore crabs to TTX year round may  result from t h e i r lack of exposure to this toxin. The  possibility  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.  c o a s t l i n e , namely Okeover Arm and Porpoise Bay  6) during  registered red tide blooms.  following  saxitoxin  (Figure  Similar extended death times of crabs  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  shellfish  respectively (Rudy Chaing, personal  for  Okeover  communication).  Arm  and  Porpoise  Bay,  57 Table 8. A comparison of death times 1n two shore crabs (Hemigrapsus nudus. Hemigrapsus oregonesis) a f t e r administration of 0.05 ug saxitoxin (STX) and tetrodotoxin (TTX).  Times of Year  Mean Death Time (min) H. nudus  August 1985 October January 1986 April July September October  Mean Death Time (min) H. oregonesis TTX STX  TTX  STX  1.7  8.5*  2.4  2.1 2.3 2.0 2.4 1.9  2.2 1.3 0.98  2.5 2.4 2.5  0.96  0.53 1.3 1.1  2.2 2.3 2.4  1.1 5.1* 1.6  2.1  * Resistance to STX, death times > 3 minutes.  > 20* 1.3 1.4  58  125°W  •* Death times #*  Figure 6.  30 minutes,Contamination level: 14,000 ugPST/100g  Death times 10 minutes, Contamination level: 1,700  ugPST/100g  Sampling s i t e locations on the southern B r i t i s h Columbia coast.  59 III.  DETERMINATION OF PARALYTIC SHELLFISH TOXINS IN SHELLFISH AND SHORE CRABS  During the time period A p r i l 1985 to December 1986, samples of sensitive and  resistant  toxins.  Two  crabs  were assayed  types  of  assay  for  were  the  used  presence to  of  determine  paralytic the  chemical fluorometric assay and the o f f i c i a l mouse bioassay. Table  9  for  the  fluorometric  assay  were  based  on  PST  shellfish content:  a  Results shown in  the  average  determinations, with each test consisting of 10 separate readings.  of  5  The mouse  bioassay was only done once using the average of results obtained from 3 mice for each sample. resistant  Samples of H. oregonesis and H. nudus were determined to be  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 s h e l l f i s h assayed for the presence of PST were clams, mussels and oysters. a  "bloom"  Mollusc response  of  Gonyaulax  off  the  B.C.  These molluscs were collected during coast  at  Okeover  Arm in  July  1986.  samples were assayed for the sake of comparing t h e i r response to the of  shore  dinoflagellates.  crabs  from the same location during  a toxic  outbreak  of  This comparison could not be made at Towers Beach because of  lack of PST testing in the greater Vancouver area. Fluorometric  readings  whereas mouse bioassay 100 g t i s s u e .  In a l l  fluorometric values. addition  to  STX  in  are  presented  in ug  saxitoxin  per  100 g  tissue  results are given In ug p a r a l y t i c s h e l l f i s h toxins per samples, bioassay values are greater than those of the These differences indicate the presence of other PST in  the  shellfish  extracts.  The heterogeneity  of  PST  in  s h e l l f i s h from B r i t i s h Columbia was reported by Bose et a_kt 1979.  Using the  same fluorometric technique and comparing the results  bioassay,  to the mouse  these researchers showed that a variety of PST were found 1n s h e l l f i s h , with STX contributing only p a r t i a l l y to the total t o x i c i t y .  60 Table 9. P a r a l y t i c s h e l l f i s h shore crabs. Sample  Hemigrapsus  toxin  content  of  various  Fluorometric Assay Mean ug STX/100 g ± S.D.*  B.C.  shellfish  and  Mouse Bioassay Mean yg PST/100 g  oreqonesis  (shore crab) (resistant to STX) Okeover Arm Towers Beach (sensitive to STX) Towers Beach 1  2  21.5 14.9  2.4 2.1  32 0**  0  0  Hemigrapsus nudus (shore crab) (resistant to STX) Okeover Arm (sensitive to STX) Tsawwassen 3  28.0  6.1  50  0  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  *  - Values were determined Appendix II).  after  reference  to  0** the  standard  curve  (See  * * - Mice showed signs of respiratory distress and muscular incoordination - Death times > 30 min. 1  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 t h e i r viscera a f t e r 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.  larger  nudus was  proportion of  slightly  STX.  more toxic while H. oregonesis  Resistant  crabs  from Towers  contained a  Beach in  Vancouver  (death times 5 min.) were shown to contain 14.9 yg STX a f t e r fluorometry but no PST a f t e r the mouse bioassay.  The mouse bioassay  is known to be Inaccurate  at low levels of PST because i t s lower l i m i t of s e n s i t i v i t y is in the range of 30-33 yg PST/100 g s h e l l f i s h  tissue and at these low l e v e l s ,  sodium ions  s h e l l f i s h extracts counteract the effect (Schantz et a_L, 1958). hand,  the  lower  STX/100 g.  of  sensitivity  for  the  On the other  fluorometric method  is  5 yg  Mice injected with extracts of resistant crabs from Towers Beach  exhibited motor  limit  in  distinct  neurological  Incoordination,  which  assay is more sensitive  symptoms  supports  the  such  as  respiratory  contention  than the mouse bioassay.  that  distress  the  Therefore,  and  fluorometric  it  Is  safe  to  conclude that low levels PST were present 1n these crabs. Extracts Beaches  at  therefore, crabs  taken  no  time  establish  from sensitive contained  shore  detectable  a relationship  (H. oregonesis,  HL nudus)  crabs  from Towers  amounts  of  between resistance  PST.  and  Tsawwassen  These  to STX in two  and the presence of PST in visceral  A relationship between s e n s i t i v i t y  results, shore  tissues.  to STX and the absence of accumulated PST  1s also apparent. Various  shellfish  (clams,  oysters,  mussels)  were also  collected  from  Okeover Arm during a toxic dinoflagel late bloom and assayed for the presence of PST.  Mussel  extracts  clams and then oysters.  contained the highest  levels of toxins followed by  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. the same argument  used for resistant  However,  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 H. nudus from Okeover Arm is molluscs  shore crabs Hemiqrapsus oreqonesis and  consistent with the presence of PST in  obtained from the same area.  bivalve  This presence was 1n turn, attributed  to the occurrence of a toxic d i n o f l a g e l l a t e bloom at t h i s same l o c a t i o n .  The  relationship between toxic dinoflagellates and toxic s h e l l f i s h has been known since 1928 a f t e r the pioneering work of Meyer and Sommer. of  intoxication  in  shore  crabs  is  far  from  clear.  However, the source These  crabs  are  not  f i l t e r - f e e d e r s , as are the bivalve molluscs, and neither are they carnivores, ruling out d i r e c t consumption of toxic s h e l l f i s h .  Shore crabs are herbivores  and perhaps  to  consume  toxic dinoflagellates  they may consume d i n o f l a g e l l a t e  washed  shore.  Alternatively,  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  resistance  to  the  Injected  possible  biochemical  mechanism(s)  STX 1n the shore crabs  responsible  H. oreqonesis  and H.  for  nudus.  extracts of v i s c e r a l tissues were subjected to electrophoretic separations and p r o f i l e s 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). 1n 25 uL extract  and milligrams  Values are given in milligrams  total protein  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. sensitive  H.  nudus.  This is not the case with samples of resistant and  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. extracts  contain  It  more protein  results are not conclusive.  would be tempting to conclude that than  sensitive  visceral  extracts  resistant but  these  64 Table 10. Protein content 1n visceral extracts of resistant and sensitive shore crabs (Hemlgrapsus oregonesis and Hemigrapsus nudus).  Sample  mg protein/25 UL.  H. oregonesis H. oregonesis  (res) (sen)  mg protein/ mg dry weight  0.884 0.984  0.883 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 p r o f i l e s  of  soluble visceral  and sensitive  shore crabs  (H. oregonesis)  most  feature of  this  striking  component  in  extracts  sensitive  crabs.  of  This  resistant  component  migrated only a very short conclusions in  this  gel  can be formed as because  10% gel  the  distance  is  that  it  is  of  Isolated because of Its  that  1s  absent  relatively  1n  located near the stacking Into the separating  proteins,  overloaded and therefore cannot be used as assumed  high  resistant  the presence of a large  to the molecular weight  standard  in  are presented in Figure 7a.  crabs  is  proteins  albumin  gel.  The  protein  extracts gel,  of  having  No s p e c i f i c  (MW) of t h i s component and BSA,  indicators.  MW compared to  were  heavily  However, i t the  other  can be proteins  slow migration through the g e l .  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 g e l , and the band 1s s l i g h t l y smaller than 1n Figure 7a due to a smaller sample s i z e .  Separation of the standard proteins 1s s t i l l  incomplete although we can now make a rough estimate of MW f o r 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 and sensitive Hemigrapsus oregonesis by SDS-PAGE (7.5%).  resistant  67  Figure 8.  A comparison of  the soluble visceral  proteins  found in  and sensitive Hemigrapsus nudus by SDS-PAGE (7.5%).  resistant  68 The electrophoretlc p r o f i l e s of resistant and sensitive extracts from the shore crab Hemigrapsus nudus are found 1n Figure 8. of  a  relatively  high  resistant H. nudus.  molecular  weight  protein  Again we see the presence compound  1n extracts  from  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. 3.5 hour time period.  Both gels are 7.5%, and both were run for the same  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 d i s s i m i l a r i t y  apparent  between p r o f i l e s  of H^.  oregonesis and H. nudus 1s the presence of a protein band in extracts from nudus sensitive to STX (Fig. 8). oregonesis  ( F i g . 7b).  Soluble v i s c e r a l  proteins in resistant H. oregonesis  were compared (Figure 9). 30 min)  This component 1s not found 1n sensitive H^  from two locations  The two locations were; Okeover Arm (death times >  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 d i f f e r e n t .  Crab extracts  from Okeover Arm contained a  larger amount of this protein compared to crab extracts from Towers Beach. could be speculated on the basis protein component produced is the  shore  crab.  Shore  of  these  results  that  the amount of  It this  related to the r e l a t i v e resistance acquired by  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 a d d i t i o n , showed a greater amount of the extra protein. other  hand,  resistant  crabs  from Towers Beach with  relatively  On the  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. in  Figure  extracts  10. of  Four samples  H.  oregonesis,  The results of this experiment can be found  were electrophoresed: as  well  previously dosed with STX in vivo.  as  It  resistant  resistant and  and  sensitive  sensitive  extracts  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.  other extracts  concentration  of  production  a protein band,  of  resistant  crabs,  this  All  after  component.  These  similar  administration  to of  showed the presence of a results  the STX  clearly  extra protein to  sensitive  After reference to the standard proteins run on this g e l ,  large  indicate  the  band found crabs  in  in  vivo.  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 s e l f ; w i l l sensitive The  the extra protein band appear 1n  crab extracts which have been Injected with tetrodotoxin in vivo?  answer  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.  is  obvious  to  that  component  1n  this  again  question  we  extracts  see of  can  the  be  found  production  sensitive  crabs  in  of  Figure  this  which  had  11 which  high  molecular  been dosed  It  weight  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 Hemiqrapsus oreqonesis and sensitive and resistant + STX in vivo by SDS-PAGE (7.5%).  H.  resistant oregonesis  72  •200,000  • • *-116,000 92,500 166,200 #5,000  R -Resistant to STX S  Figure 11.  Sensitive to STX  A comparison of the soluble visceral proteins found in Hemigrapsus oregonesis  and sensitive and resistant  + TTX in vivo by SDS-PAGE (6%).  H.  resistant oregonesis  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 e d water did not show the presence of the protein component. These  daltons)  results  show the  existence  of  a  protein  component  (MW 145,000  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  accumulated  related  in  the  to  viscera  the of  presence shore  of  PST,  crabs.  ingested  Neither  PST  naturally  and  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 a f t e r administration of STX  to  the  living  crab.  Therefore,  it  would  seem that  this  protein  1s  i n i t i m a t e l y Involved with STX, either accumulated naturally or administered In the laboratory by acute i n j e c t i o n . The appearance  of  the protein band  in  visceral  crabs given low doses of TTX jji vivo is i n t e r e s t i n g . earlier During  in t h i s this  thesis  sensitive  The shore crab was shown  to be sensitive to TTX over a 21 month time period.  same time period, shore crabs were shown to exhibit a  s e n s i t i v i t y to STX. TTX is  extracts of  While STX can be found in the crabs' habitat  varying  seasonally,  not found 1n these waters and the shore crab would therefore not be  l i k e l y to encounter t h i s marine toxin naturally. same protein sensitive  found  crabs  in  sensitive  injected with  crabs TTX.  It  1s curious then, that the  injected with A  logical  way  STX to  should  appear  investigate  in  this  74 phenomenon  would  resistance  to  be  to  TTX and  allow  the  crab  accumulations  of  to TTX  Ingest in  TTX  the  and  then  viscera,  test  followed  for by  electrophoretic separations of visceral proteins to determine the presence or absence of the 145 kd protein component. Intoxication pufferfish unclear. action  Is  unknown.  from  Japan,  In  any  case,  of TTX are very  It but  is  similar  macromolecule Isolated 1n this marine toxins.  found most commonly  where  since  Unfortunately,  the  they  encounter  the source of TTX  in the ovaries  the  TTX  is  structure, molecular weight  to those of  STX,  thesis has a general  it  1s  at  of the present  and mode of  plausible  that  the  s p e c i f i c i t y towards both  75 CONCLUSIONS  Several points of Interest concerning the nature of PST 1n marine animals were discovered during the course of these Investigations.  The shore  Hemiqrapsus oreqonesis and H. nudus were shown to become seasonally to  STX while  remaining  sensitive  to TTX throughout  occurred during mid to late summer with a gradual fall.  In  detectable  a d d i t i o n , visceral levels  of  PST  extracts  whereas  presence of PST in resistant  of  crabs  sensitive  the year.  crabs  resistant Resistance  return to s e n s i t i v i t y by the resistant  crab  to  extracts  STX did  contained not.  The  crabs was in turn associated with the presence  of toxic d i n o f l a g e l l a t e blooms in the area, although the route of intoxication remains visceral  unclear.  Furthermore, a novel protein component was  Isolated from  extracts of crabs resistant to STX and crabs injected with STX and  TTX in vivo.  During the course of this work, the following  Information  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 v i s c e r a l extracts of shore crabs given STX & TTX in vivo  has  76 GENERAL CONCLUSIONS  Before proceeding with a discussion  on the possible o r i g i n 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  p u r i f i c a t i o n of t h i s  novel protein component has since yielded the following  information;  approximate  (100°C,  1)  5 min)  the  resulted  MW is  145,000  daltons,  in a break down product  approximately 72,000 daltons,  or  2)  heat  subunit  with  treatment a MW of  3) this protein component is a c i d i c in nature.  In addition to these r e s u l t s , the 145 kd protein was shown to be induced in a dose dependent manner a f t e r acute studies with l i v i n g sensitive H. injected  with  varying  doses  of  STX  (Figure  12).  Visceral  oregonesis  extracts  of  sensitive control crabs given injections of water do not show the presence of the 145 kd protein (Lane 1). concentration) 3, 10 ng).  to  smallest  for the second lowest dose STX administered  (lane  The amount of protein then increased as the dose increased from 10  to 50 ng STX known  is  The size of the protein band (and therefore the  be  (lane  4).  resistant  Lane 5 shows the protein p r o f i l e of H. to  STX,  and again the  145 kd protein  is  oregonesis 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 i v i n g crab affects the amount of the 145 kd macromolecule produced. Consequently, present  in  shore  the crabs  results  to  date  suggest  that  exposed to STX may represent  the protein some  component  form of defence  Figure 12.  A comparison extracts  of  of the soluble visceral sensitive  Hemigrapsus  proteins found 1n visceral 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 w e l l . an  immune  response  1n  literature  (Cooper,  1974).  are a member, within The crustaceans annelids.  invertebrates  has  been  clearly  The existence of  established  The location of the crustaceans,  in  the  of which crabs  the broad taxonomic scheme can be found in Figure 13.  descend from the arthropods d i r e c t l y and p r i o r to that, the  Vertebrates  belong  to  the  chordats  which  branch  off  at  the  coelenterats. According phylogenetic  to  levels  cell-mediated immunity.  of  immunity  The  vertebrates,  Hildemann  first  while  and  Reddy  immuno-evolution: and  integrated  level  the  is  second  (1973)  there  are  quasi-immunorecognition, cell-mediated  characteristic (primordial)  of  1s  and all  vertebrates  possess  integrated  cell  antibody  Invertebrates  exemplified  mediated  major  primordial  humoral  coelomate invertebrates such as the annelids, and therefore the Only  three  and  by  and  advanced  crustaceans.  humoral  antibody  immunity. The immune response of  invertebrates such as  crustaceans, with a f l u i d  f i l l e d coelomic cavity has received some attention in the l i t e r a t u r e (Cooper, 1974).  The coelomic c a v i t i e s are f i l l e d and monitored by a complex group of  coelomocytes that sequester any Insulting foreign which  are  also  phagocytes,  become  substances.  immobilized and  analogous to opsonins, lyslns and agglutinins.  release  Coelomocytes, humoral  factors  This mixture of coelomic c e l l s  1s similar to vertebrate blood c e l l s , and coeloms are comparable to vertebrate bone marrow in that Many immunologists  they possess analogous leukocytic types  (Cooper,  1974).  believe that invertebrate coelomocytes are the evolutionary  precursors of a l l known vertebrate immunocytes (Acton and Weinhelmer, 1974). Invertebrate  humoral  active hemagglutinins  immunity  Involves  the  presence  of  biologically  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  such as bacteria and promote phagocytosis. the presence of  hemagglutinins  1n a l l  that coat p a r t i c u l a t e  There 1s substantial  coelomate  antigens  evidence for  invertebrates.  MacKay and  Jenkin (1970) believe that opsonins are present 1n the c r a y f i s h , an arthropod very  close  immunized activity  taxonomically with  than  weekly  the  doses  Crustacea.  of  in  an  (Acton  invertebrate  and  Weinhiemer,  Coelomycytes  endotoxin  in nonimmunized c r a y f i s h .  hemagglutinin literature  4  to  show  This  and  is  there  1974).  far  from  greater  crayfish phagocytic  one example of an induced  are  other  examples  Most hemagglutinins  in  form  the  strong  aggregates from 100 to 400,000 daltons.  The aggregation or d i s s o c i a t i o n  the  pH and  hemagglutinins  Calcium  ions  are  Sedimentation denaturing  is  dependent  important  is  for  dependent  solvents  for  on the stability  upon  is  the  concentration  and  dissociation  into  as  calcium  subunits  pH  ion  concentration.  range  they  of  of  7 to  require  (Marchalonls  8.  strong  & Edelman,  1968). Other enzymes.  components They  invertebrates  of  are  interest  important  and vertebrates  immune system.  Since  there  in  contained the  within  destruction  the of  and are therefore involved is  no evidence of  antibody  coelomycyte antigens in  in  are both  the defence or  production  in  the  invertebrate immune system, i t is l i k e l y that enzymes are of prime Importance in  the  destruction  of  foreign  matter  in  Invertebrates,  and  therefore,  in  crustaceans as w e l l .  Many s i m i l a r i t i e s exist between antibodies and inductive  enzymes (Table 11).  Both are r e l a t i v e l y large protein molecules  de  protein precursors.  novo  but  not  by  Both  have more or  synthesized  less  specific  a f f i n i t y for the substrate or antigen with which they react and by which they are  induced.  Anamnesis  enzymes and antibodies  or  memory  (Cooper, 1974).  capabilities  are  associated  with  both  Once primed to a certain antigen, an  enzyme or antibody is upon second challenge, f u l l y capable of responding in a  81 specific  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 f e r d r a s t i c a l l y  1s 1n the substances  capable of inducing t h e i r production.  Whereas enzymes  are Induced primarily by small molecules, antibodies are generally induced by macromolecules, e s p e c i a l l y proteins and conjugated proteins. there is  Unfortunately,  l i t t l e Information available on the presence of inductive enzymes in  invertebrates in general, and v i r t u a l l y no Information on Inductive enzymes in members of the crustaceans. Any attempt to make a v a l i d comparison between the novel protein isolated in this work and a component of the immune system in the crab would be tenuous at best. second,  F i r s t , we have very l i t t l e Information about this new p r o t e i n , and little  1s  known  about  the  response of coelomic invertebrates.  macromolecules  Involved  1n  the  Immune  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, about  this  protein component  after  PST  have  dinof l a g e l l a t e s neurotoxins  been  capable  introduced,  of  blocking  therefore  seem  it  either  the  pertinent  appears  in  naturally  information  crab visceral via  a  toxic  is  extracts bloom  of  We know that the PST are very potent Inward  movement  of  sodium  by being  and i t  able  reasonable,  to  1s generally assumed that  1ons  1n  they avoid the  accumulate and excrete the PST.  that  the  145 kd protein  Isolated  It  correct,  then  this  component may be  capable  of  would  here may be  Involved in protecting the crab from the lethal e f f e c t s of the PST. assumption  known  We also know that crabs and many s h e l l f i s h are r e l a t i v e l y  unaffected by these toxins effects  that  or in the laboratory.  excitable c e l l s .  toxic  1s  the only  If  binding  this or  82  Table 11.  A comparison of enzymes and antibodies.  Antibodies  Enzymes  Property Phylogenetlc d i s t r i b u t i o n  Unlqultous;  cells  A l a t e evolutionary acquisition; made only 1n vertebrates (and 1n c e r t a i n c e l l s of the lymphatic system)  Structure  Proteins with variable chemical and physical properties; an enzyme of a given s p e c i f i c i t y and from any p a r t i c u l a r organism 1s  A group of closely related proteins having a common multichain structure with the chains held together by -SSbonds. Molecules of a given s p e c i f i c i t y are heterogeneous 1n structure and function  homogeneous; crystal 1 zed  made  by  many  all  have  been  Constitutive  Yes  "Natural"  Inducible  Often  Yes  Function  S p e c i f i c reversible binding of Ugands* with breaking and forming covalant bonds  S p e c i f i c r e v e r s i b l e binding Ugands" without breaking forming covalent bonds  Reaction with Ugands*  Wide range of affinities; populations of enzyme molecules of a given specificity are uniform an a f f i n i t y f o r t h e i r Ugand  Wide range of a f f i n i t i e s ; but populations of antibody molecules of the same s p e c i f i c i t y are usually heterogenneous 1n a f f i n i t y f o r t h e i r Ugand  Affinity  Usually measured k l n e t l c a l l y  Usually at  antibodies?  measured with  equilibrium  of or  reactants  (because  the  reactions are so f a s t ) Number of s p e c i f i c Hgandblndlng s i t e s per molecule  Different 1n d i f f e r e n t enzymes, depending on number of polypeptide chains per molecules; usually one s i t e per chain  2 per molecule of the most prevalent type (NW 4r 150,000); each s i t e 1s formed by a pair of chains (a l i g h t plus a heavy chain)  Inducers  Primarily small molecules  Usually macromolecules, e s p e c i a l ly proteins and conjugated proteins  * Ugand • substrate or coenzyme, and antigen or hapten 1n case of antibodies, r e f : Cooper 1974  83  sequestering these very small molecules are small molecules component  The f a c t that the  PST  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 a t t h i s novel  1s a humoral  because they  (MW STX-372).  f a c t o r such as a b a c t e r i o c i d l n or  r e a c t with  f o r e i g n macromolecules.  p a r t i c u l a t e antigens  such as  hemagglutinin,  b a c t e r i a and  other  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 manner.  i n a dose dependent  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 o f phagocytes can occur w i t h i n minutes of antigen 1968,  Liebman, 1942).  challenge  1n i n v e r t e b r a t e s  (Evans et a j . ,  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 o n c l u s i o n s 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 PST.  immune response to  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.  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Mouse  Relations  for  Death Time*  Mouse Units  Death Time  1:00 10 15 20 25 30 35 40 45 50 55  100 66.2 38.3 264 20.7 16.5 13 9 11.9 10.4 9.33 8.42  5:00 05 10 15 20 30 40 45 50  1.92 1.89 1.86 1.83 1.80 1.74 1.69 1.67 1.64  2:00 05 10 15 20 25 30 35 40 45 50 55  7.67 7.04 6.52 606 5.66 5.32 5.00 4.73 4.48 4.26 4.06 3.88  6:00 15 30 45  1.60 1.54 1.48 1.43  7:00 15 30 45  1.39 1.35 1.31 1.28  8:00 15 30 45  1.25 1.22 1.20 1.18  3:00 05 10 15 20 2J  9:00 30 10:00 30  1.16 1.13 1.11 1.09  30 35 40 45 50 55  3.70 3.57 3.43 3.31 3.19 3.08 2 98 2.88 2 79 2.71 2 63 2.56  11:00 30  1.075 1.06  12:00  1.05  4:00 05 10 15 20 25 30 35 40 45 50 55  2.50 2.44 2.38 2.32 2.26 2.21 2.16 2.12 2.08 2.04 2.00 1.96  13 14 15 16 17 18 19 20 21 22 23 24 25 30 40 60  1.03 1.015 1.000 0.99 0.98 0.972 0.965 0.96 0.954 0.948 0.942 0.937 0.934 0.917 0.898 0.875  ' Minutes:Seconds.  ref: A0AC 1984  Unit  Mouse Units  Paralytic  Shellfish  93 APPENDIX  II  ug S T X  Calibration Curve for Fluorescence vs. Saxitoxin Concentration  94 APPENDIX  •10  III  T  CONCENTRATION  PROTEIN  C a l i b r a t i o n Curve for Absorbance vs. Protein Content  95 APPENDIX IV  Anatomy of Crab ref:  G.F. Warner 1977  

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