Gustatory Responses to Tetrodotoxin and Saxitoxin in

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Gustatory Responses to Tetrodotoxin and Saxitoxin in Fish: e Mechanism for Avoiding Marine Toxins Kunio Yamamori and Moritaka Nakamura School sf Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-01, japan

Department of Fisheries, Faeubty of Agriculture, University of Tokyo, Pskyda 1 13, japan

and Toshiaki 1. Haral Department of Fisheries and Oceans, Central and Arctic Region, Freshwater Institute, 50 1 University Crescent Winnipeg, Man. R3P 2N6

Yamamori, K., M. Nakamura, T. Matsui, and 8.1. Hara. 1988. Gustatory responses to tetrdotoxin and saxitoxin in fish: a possible mechanism for avoiding marine toxins. Can. 1. Fish. Aquat. Sci. 45: 2182-21 86. The gustatory responses ts tetrsdotoxin (PTX) and saxitoxin (STX) recorded from the palatine nerve (Vllth cranial nerve) were studied electsophysiologicaIly in rainbow trout (Sagma gairdneri) and Arctic char (Salvdhus alpinus). Both toxins were highly effective gustatory stimuli in both species. In rainbow trout, TTX had a threshold concentration 2 x 18-7 mollb and at ? 8 - 5 moll!- evoked a response four times that of 10-3 mol L-prolinelb, the most potent amino acid for this species. The threshold for STX was lower moll!-), but unlike PTX the response magnitude reached a maximum at 1 0 - 6 msl/L. The reverse occurred in Arctic char; lower threshold for PBX (18 - m d l L ) than STX (10-7 rnolIL) and the response magnitude never exceeded that of 1 mol L-proline18. Cross-adaptation experiments indicated that the receptsrfs) for TTX are distinct from those which detect amino acids and bile salts and that TTX and STX do not share the same receptor populations, Furthermore, the integrated response to TTX or SPX was a fast-adapting, phasic response and rapidly returned to baseline even with continued stimulation. Perfusion of the gustatsry organs with these toxins had little toxic effect. The sensitive, specific gustatory receptor system for the toxins suggests the existence of a mechanism for avoiding poisonous prey organisms that has adaptive advantage to the receiver (predator). bes r6pnses gustatives 5 la tcfitrodotoxine fTTX) et 5 la saxitoxine (STX) enregistrees i3 partir du nerf palatin (Vile ned cranien) ont 6t6 6tudiees 6lectrophysiologiquementchez la truite arc-en-ciel fSa!rns gairdneri) et chez I'smble de l'arctique (Salve%inus a!pinus). Ces deux toxines sont des stimulis gustatifs tr&s efficaces chez les deux esp6ces. Chez la t r ~ i t e arc-en-ciel, la TTX avait erne concentration seuil de 2 x 10-' moll8 et 5 1 0 4 moll!-, e l k provoquait une rkponse quatre fois suMrieure 2 celle de la L-proline A 10-3 mslll, l'acide amin4 dont l'effet est le plus puissant pour cette esp6ce. Le seuil pour [a STX 6tait inf6rieur f18-8 molIL), mais contrairement A ce qu'on a observe pour la TTX, la rkponse a atteint un maximum 2 1 8-6 mslIL. O n a observe le ph6nom&ne inverse chez l'omble de l'arctique, soit un seuil inferieur pour la TTX (I mol/L) et avec la STX (10-7 rnolIL), et Ifimportance des reponses ne d6passait jarnais ceIle de la L-proline i3 10-3 mol/L. Les experiences d'adaptation crois6e ont indique que le ou les recepteurs de la BTX sont differents de ceux qui d4tectent les acides amin6s et sels biliaires, et que la BBX et la STX ne partagent pas les m$mes populations de recepteurs. En outre, la r6ponse int6gr6e 3 la TBX ou 2 la STX ktait une r6pnse en phase A adaptation rapide, avec un setsur rapide 5 la ligne de base, meme avec une stimulation continue. La pedusion des organes gustatifs avec ces toxines avait peu d'effets toxiques. Le syst&me de r6cepteur gustatif particulier et sensible pour ces toxines sugggre ['existence d'un mkcanisrne permettant d'eviter des proies toxiques, qui constitue un avantage d'adaptation pour le recepteur (predateur).

Received November 17, 1987 Accepted duly 24, 1988

(J9487)

uffer toxin, tetrdotoxin (TTX), is one s f the most potent neurotoxins known, with its Eehd toxicity to humans 308 times that of cyanide. TTX exerts its toxic action by specifically blocking the voltage-sensitive sodium channels in nene and muscle membranes (Nxahashi 1994; Kao 1981). TTX was once believed to be found exclusively in tetraodontid fishes, 16 species altogether including 12 species of the genus Fugu (Tani 1945), but its distribution seems much wider than 'Author to whom esmespondence should be addressed.

originally thought. It has been shown to be present in a variety of aquatic organisms: California newt (Tarica torosa) (Buchwald et d. 1964), goby (Gobius criniger) ((Nognchi and Hashimoto 1973), Costa Ricm atelopid frogs (Atekopus sp.) (Kim et al. 1995), blue-ringed octopus (Octopus ma~ukosus)(Scheurnack et al. 1998), Japanese ivory shell (Babjrlonia japone'ca) (Nogeaehi et A. 198 1; Yasurnoto et al. 198 I), trumpet shell (Chsmnia smliae) (Narita et A. 1981), Japanese sta15sh (Astropecten polyacanthus) (Noguchi et al. 1982), xanthid crab (Atergatis floridus) (Noguchi et al. 19831, frog shell (Tutufa Can. J. Fish. &reat, Sci., VoE. 45, 1988


&issostom)(Noguchi et d. 19841, and flatwoms (Jeon et al. 1986). Wether TTX in puffers is synthesized endogenously or accumulated exogenously through the food chain is not clearly understood, but the finding that puffers cultured with artificial foods become nontoxic suggests m exogenous origin (Matsui et d. 1981, 1982). Isolation of TTX-producing bacteria Ebaio sp. from the intestinal flora sf the xmthid crab further supports this view (Noguchi et al. 1986). Puffers, when stimulated by hadling or electrical shwk, secrete TTX into the surrounding water from a unique glandlike structure in the skin containing a high concentration of TTX (Kodama et al. 1986). This response is usually accompanied by swelling of the abdomen (Kodama et d. 1985; Saito et al. 1985). TTX has been implicated in repelling predators. In addition, preliminary khavioural studies demonstrated that some fish species (rainbow trout, striped beak perch, greenlings, etc .) reject toxic puffer livers and artificial food pellets containing TTX ( Y a m m o ~et al. 1980). These findings suggest that fish may detect TTX via the gustatory system and thereby protect themselves from poisonous prey, Although earlier studies have shown that TTX did not induce electrical responses in the gustatory systems of frog and rat (Ozeki a d Noma 19721, no investigation has been reported in fish. The present study was designed to investigate the gustatory sensitivity of rainbow tmut (Salmo gaiadneri) and Arctic char (Salvelinus abinus) to TTX by measuring the integrated electrical responses from the palatine nerve innervating the palate and inside the upper lip. "These species were chosen primarily because of laboratory convenience; however, they are both anadromous and could encounter marine toxins in nature. Furthermore, the gustatory sensitivity and specificity for amino acids md bile salts have been well investigated in both species ( M m i et al. 1983; Sveinsson 1985). Saxitoxin (STX), a paralytic shellfish toxin having similar pharmacological actions to TTX, was also tested. STX is believed to be only one of 1012 closely related naturally occurring toxins produced by dinoflagellates of the genus GonyauBm; a massive bloom causes the phenomenon known as 'red tide9 in comtd waters.

Materials and Methods Rainbow trout and Arctic char, 20-27 cm in body length, were obtained from the Rockwood Hatchery, Freshwater Institute. Fish were held in fiberglass tanks supplied with dechlorinated Winnipeg city water (water temperature 11.5- 12.5"C). Gustatory responses were recorded from palatine nerves (VHth cranial nerve) innervating palatal taste buds and inside the upper lip, using the method of M m i et d.(1983). Briefly, the fish were paralyzed with an intramuscular injection of Flaxedil (gallmine triethiodide, 5 mg/kg body weight) and positioned in a plexiglass trough with dechlorinated water perfusing the gills throughout the experiments. The eyeball was removed to expose a nerve branch running though the bottom of the eye socket to the oral roof, The freed nerve bundle was severed centrally and its peripheral end was placed on bipolar platinumiridium electrodes. Mineral oil was added to the orbit to prevent w i n g of the nerve preparation during recording. The electricd activity of the whole nerve bundle was mplified (Grass 7P5 1I), integrated (Grass 7P3; time constant 8.5 s), and recorded on a polygraph (Grass 7B). The response magnitude was measured as the height of integrated responses over the stimulus duration of 5 s and expressed as a percentage of a response to a standard msl L-proline/L.Each stimulus was tested twice stimulus, Can.J. Fish. Aqua. Sci., &lo/.45, 1988

STX

lo"

If 5xlf 5x10~ 10' FIG. 1, Integrated gustatmy responses to increasing concentrations of tetrodstoxin (TTX) md smitswin (STX) in (A) rainbow trout and (B) Arctic char. Tracings at the far left in each column represent the response to the standard stimulus, lorn3 mol E-prolindk. Concentrations are shown in m V L . Time signal, each division = 5 s. consecutively, with a standard time interval of 2 min, md the mean responses were obtained. For delivering chemical stimuli, an apparatus described previously was used (Mami et al. 1983; Evans and H x a 1985). In cross-adaptation experiments, the gustatory organs were pegfused with % 8- "01 L-proline/%, 1W 3 mol L-a-amino-pgumidinoprspionic acid (AGPA)/L, 18-"01 ktaine/L, or 10- mol taurolithocholic acid (TLCA)/L, and a concentration series of TTX, dissolved in respective adapting stimulant solutions, were tested. Response magnitudes were compared with those prior to adaptation (control). In the second series of crossadaptation experiments, the receptors were adapted to or 5 >< TTWL and responses to varied concentrations of STX were tested. The reverse were also ex Stock solutions (lo-' moUL) of TTX and STX were made with distilled water and stored in a refrigerator. TTX (3.19 mg) was initidly dissolved in 0.1 d of 6% acetic acid solution to which distilled water was added to obtain the final stock concentration. All final test solutions were freshly prepared, just before each application, with the same water used to perfuse the gills a d palate. The pH of these test solutions ranged from 7.3 to 7.7, and therefore no adjustment was made. TTX was donated by S d y o Co., Ltd. (Tokyo). STX (diacetate form) and AGPA were purchased from CaIbiochem-Behriang Gorp.

e Uwadqted


-

-8

-7

-6 -5 Log Concentration (rnolbL)

x Adapted to L-Proline o Adapted to Betaine A Adapted to T LCA

-4

-

-7

-6 -5 Log Csneentratisn Qmol BL)

-4

FIG. 3. Semi-logarithmic plots of the concentration-respnse relationships to tetrodotoxin (TTX) before (@) md during adaptation to lo-" mol L-proline/L ( x ), I0 - 3 mo1 betainell and mol taurolithochslic acid (TLCA)/L (A). Average (N = 3) response magnitude is represented as a percentageof that induced by the standard stimulus, 10- mol L-pro'C3lineIL.

(n),

trout (Fig. 2B). Because the response magnitude to 10-"oI L-poBine/L was greater in Arctic char than in rinbow trout, the relative response magnitudes to both TTX m d STX appeared smaller, never exceeding that of the standard (Fig. 2B). Log Concentration (rnol/b)

FIG. 2. Semi-logarithmicplots sf the concentration-response relationships to tctrodotoxin (TTX)and saxitoxin (ST%)in (A) rainbow trout and (B)k c t i c char. Average response magnitude is represented as a percentage of that induced by the standard sthu1mt, ma% L-pra%ine/L. Points represent mean SE of 7-14 fish.

(La Solla, CA), and TLCA and other m i n o acids were from Sigma Chemical Co. (St. Louis, MO). All chemicals were of the highest purity available from co

Results Response Characteristics to TTX and STX Typical gustatory responses of rainbow trout and Arctic char to increasing concentrations of TTX m d STX are presented in Fig. I. The integrated response to TTX and STX, a fastadapting a d phasic response, rapidly returned to the baseline even with continued stimulation. The concentratio+response (C-R) relationships were generdly sigmoidal, when plotted sedsgarithmicaIly (Fig. 2). In rainbow trout, a clear response to TTX appeared at 2 x 18- moUL and the magnibde reached six times that of the standard 10- mol t-pmline1L response at 5 x moIIL. The threshold for STX was lower, 10-* mol1L. Unlike TTX the response magnitude reached a maximum at 5 x moUL m d no further increase occurred with higher concentrations (saturation; Fig. 2A). In Arctic char, the threshold concent~ationfor TTX was lower than that for STX (10- * versus 10- mol1L). However, the general shapes of C-R relationships were similar to those observed in rainbow

To evaluate whether there are different types of gustatory receptors, or transduction processes for TTX (and STX) and other h o w n gustatory stimuli (amino acids and bile salts), cross-adaptation experiments were performed. If two stimuli share the s m e receptors, gustatory responses will be reduced when the receptors are adapted to one of the stimuli. Adaptation to lo-' msl L-prslineIL, 10-%ol betainell, or mol TLCNL had little effect on responsiveness to TTX in rainbow bout (Fig. 3). The responses elicited by TTX during L-proline or TLCA adaptation were slightly reduced, but the reduction was not significant. Similarly, in Arctic char, adaptation to Lpmline, which elicited greater responses than TTX at d l concentrations (cf. Fig. 2B), had no significant effect on the TTX respsnse (data not presented). Conversely, adaptation to 5 x 10-"ol TTWL patidly inhibited L-proline responses (60 9 35% SD), but had little effect on responses to I O - b o l AGPNL (98 iz 16% SD)and 10- 9 mol/L TLCAIL (82 k 22% SD). ma%-L-"TX had little In rainbow trout, adaptation to effect on TTX responses (Fig. 4A). Adaptation to 10-"ol TTWL had no effect on STX responses at concentrations between 10-"nd 5 X mo%L(Fig. 4B).Perfusion with 5 x 10m6mol TTWL had no effect on STX response at 5 x 163-%oVL (99 9 30% SD), at which both chemicals elicited approximately equal response magnitudes.

Discussion The extreme gustatory sensitivity of the trout and char to the marine toxins TTX and STX corroborates our earlier behavCan. J- Fish. Aquwt. Sci., Vok. 45, 6988

I !A


e Unadapted o Adapted to ST%

-6

-7

-5

Log Concentration (rndlL)

-7

-8

-6

Log Concentrat ion (rnollL) FIG. 4. (A) Semi-logarithmic plots s f the concentration-response relationships to tetrsdotoxin (TTX) before ( 8 ) and during (0) adapmol saxitoxin (STX)/L. (B) Semi-logarithmic plots of tation to the concentration-response relationships to saxitoxin (STX) before ( 8 ) and during (0) adaptation to 90-"01 tetrodotoxin (TTX)/L. In both graphs, average (N = 3) response magnitude is repkesented as a permol centage of that induced by the standard stimulus, L-proline/L.

ioural findings that fishes detect these toxins through the garstatory system arid the suggestion that they might protect themselves from poisonous prey (Yarnamofi et al. 1980). The response obtained is fast adapting and phasic, similar to the gustatory amino acid responses ( M m i et al. 1983; Sveinsssn 1985). The estimated threshold concentrations of the toxins in Can. J. Fish. Aqu~b.Sci., h1.45, 1988

the present study are comparable with that of L-proline, the most stimulatory amino acid for rainbow trout ( M m i et al. 1983). A somewhat lower threshold value (10-'I mol/E) has been h o w n for TLCA (Ham et al. 1984). This is the first study to demonstrate that T'FX and STX stimulate the gustatory organ of fish. In contrast, TTX even at 3.08 x 10-%ol/L did not stimulate the gustatory systems (receptor cells and ehorda tympani nerve fibers) of fmg and rat, nor did it inhibit the generation of receptor potential in response to gustatory stimuli (Ozeki and Noma 1972). The following two observations clearly indicate that there exists a separate class s f specific receptor sites for TTX and xposaare of the gustatory organ to h o w n stimuli such as L-pro ( I ?ine, = betaine, AGPA, and TECA had minimal effects ow the responsiveness to TTX and (2) when TTX was used as an adapting stimulus, it failed to inhibit responses induced by all of the above-hown gustatory stimulants except L-proline. However, the inability of L-proline and betaine to interfere with TTX receptors could partly be due to lesser potency of adapting stimuli relative to testing stimuli (e.g. 100% for 1W3 mol Lprolinell versus 200% and over for TTX at casncentrations 2 x mol/L and above). The fact that exposure to TTX at 5 x 10-"OVL significant%ydepresses L-proline responses, but not vice versa, suggests some phamalogicd effect on the gustatory transduction processes. Furthermore, evidence that TTX and bile salt stimulations are mutually independent and that amino acids m d bile sdts stimulate separate gustatory fiber types (S. Ymashita a d T. J. H a a , unpubl. data) suggests different trmsduction mechanisms for bile salt stimulation. The sensitivity to TTX was unaffected by adaptation to STX, m d vice versa. These results, combined with the fact that they have distinct C-R relationships, provide strong evidence that the receptm(s) for TTX are different from those for STX in rainbow trout. The apparent differences in the C-R relationships between TTX a d STX in Arctic char d s o support this contention. In this species, TTX receptors appear to have higher affinities (C-R curve shifted one order of magnitude to the left) than those for STX (shifted to the right). Further support to this is the recent experimental result that the gustatory sytem of a strain of Japanese rainbow trout lacks sensitivity to STX at all, while responding normally to TTX at concentrations eomparable with those found in the present study (a specific anosrnia; K. Ymamori, unpubl. data). Despite differences in the chemicd structure, STX exerts essentially the same effect on nerve membranes as TTX, selectively blocking transient sodium channels (Naahashi 1974). One of the differences at the cellular level is the faster rate of action and recovery after STX than after TTX (Hille 1975). However, the finding that, unlike TTX, STX does not interact with the effects of chikquitoxin, another TTX analog, has been interpreted as that the receptor for STX is different, but partially overlapping with that for TTX (Kao 1981). Further research is necessary to establish whether separate types of receptors for TTX and STX exist. How can the TTX-containing animals such as puffers tolerate a concentration of TTX in their bodies, fatal enough for d l otherwise n o m d animals'? Studies have shown that puffers are more resistant to TTX than other fishes, e.g. %$bCb-300times that of goldfish (Saito et al. 1984). The nerve of the Atlantic puffer S p h e ~ ~ i rnaculabkt~s de~ is less sensitive to TTX than the frog nerve by a factor of 1000, but is highly sensitive to STX (Naahashi 1974). There appear to be at least two Binds of sodium channels, the common ones which are blocked by TTX a d others which are not affected by TTX (Jacques et al. 1978). 2185


T.No~ucrai,W.NAR~TA, S. MATSUBARA, M. NARA, JEON,J. K., K. MIYAZAWA, K. I m , AND K. H a s H r ~ o ~ o1986. . &mererace of paralytic toxicity in marine flatworms. Bull. 3pn. S w . Sci. Fish. 52: 1065-1069. U Q , e. Y . 1981. Tetrodotoxin, saxitoxin, chiriquitoxin: mew perspectives on ionic channels. Fed. km.40: 30-35. KIM,Y.H.,G. B. BROWN, H. S. M o s m ~AND , F. A. FUHRMAN. 1975. Tetradotoxin: occurrence in atelopid frogs of Costa Rica. Science (Wash., DC) 189: 151-152. KODAMA,M.. T. ~ G A T A , 3. SATO.1985. External secretion of tetrdotoxin AND from puffer fishes stimulated by electric shock. Mar. Biol. 87: 199-202. KBDAMA, M., 'k. OGATA,S. SATO, Y.SUZUKI, T. KANBKO, AND K . A D A .1986. Tetrodotoxin secreting glands in the skin of puffer fishes. Toxicon 24: 819-829. MARUI,T., R. E. EVANS,B. ZELINSKI,AND T. J. HARA. 1983. Gustatory responses of the rainbow trout (%almsgcsirdnsra') palate to amino acids and derivatives. J. Comp. Physiol. A 153: 423433. MATSUI,T., S. HAMADA,ANH) S. KONOSU.1981. Difference in accumulation of puffer fish toxin and crystalline tetmdotoxin in the puffer fish, Fuged mbripes rubripes. Bull. Jpn. Soc. Sci. Fish. 44: 535-537. MATSUI,T., H. SATQ,S. HAMADA, m~ C. SHIMEU.1982. C o m p ~ s o nof toxicity of the cultured m d wild puffer fish Fugu niphobles. Bull. Jpn. SK. Sci. Fish. 48: 253. NAWAHASHI, T. 1974. Chemicals as tools in the study s f excitable membrane. Physiol. Rev. 54: 813-889. NARITA,M., T. NOGUCHI, J. MARUYAMA, Y . UEDA,K. H A S H I M QY~.,WATANm ~ AND , K. MDA. 1981. Occurrence of tetrodstsxin in a trumpet shell, "Bsshubora" Charonia sauliae. Bull. Jpn. Soc. Sci. Fish. 47: 935-941. trout and Arctic char detect extremely N ~ U C KT., I , m~ Y. H A S H K ~ Q 1943. ~ . Isolation of tetrodotsxin from a goby toxins TTX and STX via the specialGobbus crtnbger. Toxicon 11: 305-304. e results of cross-adaptation Y. SHIDA, Nmucpra, T., I - K . JEON,0.AMKAWA,H. SUGITA,Y. DEGUCMI, nct C-R relationships, are relexperiments, together w AND K. HASHMOTO. 1986. Occurrence of tetr-odotoxin and anhydrotetrodotoxim in Vls'brio sp. isolated from the intestines of a xanthid crab, Abert to the interpretation that these receptors are distinct from gatis floridus, J. Biwhern. 99: 311-3 14. hose which detect other h o w n gustatory stimuli such as amino 1984. OccurNWUCHI,T., J. MARUYAMA, H. NAMTA,AND K. HASHHMOTO. evidence is obtained to indirence of tetrodotoxin in the gastropod mollusk Tufifa lissostoma (frog s for TTX are distinct from shell). Toxicon 22: 2 19-226. those for STX. Apparent lack sf toxic effect of TTX on N m u c ~ rT., , J. MARUYAMA, Y. UEDA,K.H A S K ~ O TAND Q , T. HARADA. 1981. Occumnce of tetrodotoxin in the Japanese ivory shell BaBPybnicajaponic~. s d t stimulation sugg Bull. Jpn. Soc. Sci. Fish. 47: 909-913. between amino acids m~ K. HASHMOTO.1982. TetroN m u c ~ r T., , H. N ~ T AJ. , MARUYAMA, tern for the toxins tion sf the sensitive dotoxin in the s ~ i s Asfropecten h po&ysrcanthus,in association with toxprovides adaptative significance for predators. This is the first ification of a trumpet shell, '%oshubora" & z r o n i a sauliae. n possible dud functions of marine toxins as semiSoc. Sci. Fish. 48: 1173-1 1'9'9. N m u c a ~ 'F., , A. UZU, K. KOYAMA, 3. M ~ U Y A MYA. ,NAGASHIMA, AND K. als providing adaptive si ificmce both to p HASHIMOTO. 1983. Occurrence of tetrodotoxin as the major toxin in a and receiving organisms. xmthid crab Atergatisflm-ihs. Bull. Jpn. Soc. Sci. Fish. 49: B 887-1 892. Omm, M., AND A. NOMA.1972. The actions of tetrodotoxin, procaine and acetylcholine on gustatory receptions in frog and rat. Spn. 3. Physiol. 22: 467475. obert E. Evans, Freshwater Institute, for technical assisS. KANOH,J.-K. JEON,T. NWUCHH, T. HARADA, 0. SMTO,T., 3. MARUYAMA, tance and P. V. Cassidy for typing. K. Y. was supported by a travel M ~ T AAND , K. HASHIMOTO. 1984. Toxicity of the cultured pufferfish grant from the School sf Fisheries Sciences, Kitasato University. Fugu rubripex rubripes along with their resistibility against tetrdotoxin. Bull. Jpn. SOC.Sci. Fish. 50: 1573-15'75. 0. M~TRATA, AND K. HASHIMOTO. 1985. SAITO,T.,T. NQGUCH,T.HARADA, Tetrodotoxin as a biological defense agent for puffers. Bull. Jpn. Soc. BUCHWALD, H. D., L. D u m m , H. G. FISHER,R. HAMA, H. S. M O S ~ R , Sci. Fish. 51: 1175-1180. C. Y.U O , AND E A. WHRMAN. 1964. Identity of tarichatoxin and tetroS C ~ U M A CD. K ,D.,M. E. H. HOWEN, I. SPNCE, ~m R. J. QUINN.1978. dstoxin. Science (Wash., BC) 143: 474475. Maculotoxin: a neurotoxin from the venurn glands of the octopus PHapaEEVANS,R. E., AND T. J. HM. 1985. The characteristics sf the electroolfacochiaencs m u l o s a . Science (Wash., DC) 199: 188-189. togram (EOG): its loss and recovery following olfactory nerve section in SWSSON, T. 1985. Elmbrsphysiological and behavioral studies on chemsrainbow trout (Salmteo gairdneri). Brain Wes. 330: 65-75. reception in Arctic char (Sdvebims alpinus). M.Sc. thesis, University of H m , 2'. JayS. MACDQNA~.D~ R. E. EVANS,T. M ~ u HMD , S. WH. 1984. a, Winnipeg, Man. 177 p. Mopholine, bile acids and skin mucus as possible chemical cues in sal5. Tsxicologicd studies on Japanese puffers. Teikoku TFosho, m n i d homing: electrophysiologicd re-evaluation, p. 363-378. BIZ J. D. Tokyo. 103 p. (In Japanese) McCleave, G . P. h o l d , J. D. W s o n , and W. H. Neill [ed.] Mechanisms YAMAMQRI, K., M. NAKAMURA, AND H. KAMIYA. 1980. Behavioral responses of migration in fishes. Plenum Publishing, New York, NY. of fishes to puffafk& toxin, tetrodotoxin. Pmsenkd at Awnu. Meet. Jpn. HUE, B. 1975. The receptor for tetrodotoxira and saxitoxin. A structural Ssc. Sci. Fish., Tokyo, 3-5 April, 1980. hypthesis. Biophys. Ja 15: 615419. AND S. M I Y A K O S1981. ~ . Occumence YASUMOTO, T., Y. 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It seems reasonable to assume that puffers might have evolved e of sodium channel as the f TTX. The inability of 'ITX to inhibit the other stimulants may d s o sugm channels are not primarily ction process in rainbow trout. It is generally accepted that puffers release TTX into the environment to prevent predators $om attacking. Certain species secrete mounts as high as mol TTX into the surding water, while being sti d by repeated handling electric shock (Saito et al. ; K o d m a et al. 1985). Cultured, nontoxic puffers do not release any toxin; however, they become toxic and can release up to 2 X lW7mol TTX when fed with toxic puffer livers @&to et al. 1985). Histological studies have shown that a unique exocrine gland exists in the skin sf some puffers (Rodma et d. 1986). The gland is a alveolar secretory and bran d structure surrounded by ning to the skin searface. ement membrane, with an cal stimulation causes c s tion of microfilaments in the epithelial cells, resulting in secretion of th which a high concentration of TTX has been

Can. J. Fish. Aquaa. Sci., Vil. 45, 1988