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The annual toxicological profiles of two common puffer fish, Takifugu niphobles (Jordan and Snyder) and Takifugu alboplumbeus (Richardson), collected along Hong Kong coastal waters
Toxicon 40 (2002) 313±316
Short communication
www.elsevier.com/locate/toxicon
The annual toxicological pro®les of two common puffer ®sh, Takifugu niphobles (Jordan and Snyder) and Takifugu alboplumbeus (Richardson), collected along Hong Kong coastal waters Chun-Fai Yu*, Peter Hoi-Fu Yu Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, People's Republic of China Received 10 May 2001; accepted 3 August 2001
Abstract The toxicological pro®les of two local common puffer ®sh, Takifugu niphobles (Jordan and Snyder) and Takifugu alboplumbeus (Richardson), collected in Hong Kong waters were investigated continuously for 14 months (June 1997±August 1998). Their annual spawning seasons (as evident by the enlargement of gonads and presence of eggs in the ovary) were found to be from October to February (four consecutive months) and December to February (two consecutive months), respectively. The toxicities of their internal organs were determined by standard mouse bioassay and expressed in terms of mouse units (MU). The ovary (in both species) and liver (only in T. niphobles) were discovered to be moderately toxic (100±1000 MU/g) in their non-spawning seasons. Whilst all the intestine and skin were weakly toxic (between 10 and 100 MU/g), their ¯esh, however, was basically non-toxic (less than 10 MU/g) throughout the whole year. The testes, which were only developed in their spawning seasons, were also non-toxic. This study showed that the toxicological pro®les of both of the two species had prominent seasonal patterns which were highly related to their spawning seasons. Contrary to the most common belief, both species were discovered to be relatively less toxic during their spawning seasons. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Toxicological pro®le; Spawning season; Mouse bioassay; Mouse units
Hong Kong, like many other coastal cities in the world, faces the problems of marine pollution caused by both domestic and industrial wastewater discharge into the surrounding coastal waters and the rapid coastal developments along the shore line. The traditional ®sheries resources within Hong Kong waters have declined drastically in the past decades; puffer ®sh, however, can still be easily caught along the seashores. In 1968, a total of nine species of puffer ®sh was reported to be indigenous to local waters (Chan, 1968). The same number of species, belonging to two families and ®ve genera, has been collected and identi®ed by us (Yu and Yu, 1998). Among the nine local species, three were con®rmed to be toxic and one was nontoxic (Yu and Yu, 1997). Takifugu niphobles (Jordan and * Corresponding author. Fax : 1852-24793740. E-mail address: [email protected] (C.-F. Yu).
Snyder) and Takifugu alboplumbeus (Richardson) are the most common local puffer ®sh and can be collected in coastal waters throughout the whole year. However, some species can only be caught in the summer months as they migrate to the deeper seas in winter. Takifugu niphobles (Jordan and Snyder) and Takifugu alboplumbeus (Richardson) were, therefore, used in this study which aimed to ®nd out their spawning seasons and establish their annual toxicological pro®les. The puffer ®sh used in this study were collected by local ®shermen using ®sh traps placed along the coastal waters of the north-eastern outlying islands. Between June 1997 and August 1998, about 100 puffer ®sh specimens were collected in every two months for toxicological analysis. Ten female specimens (of similar sizes) of each species of Takifugu niphobles (Jordan and Snyder) and Takifugu alboplumbeus (Richardson) were randomly sorted out from the
0041-0101/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 0041- 010 1(01)00208- 2
314
C.-F. Yu, P.H.-F. Yu / Toxicon 40 (2002) 313±316
Fig. 1. The annual proportion of ovary to the total body weight and annual variation in toxicities in Takifugu niphobles (Jordan and Snyder) and Takifugu alboplumbeus (Richardson).
rest of the puffer ®sh. They were weighed and then dissected into different tissues (liver, intestine, ¯esh, skin and ovary), which would be pooled together for toxicological analysis. The total weight of the ovaries of the 10 specimens in each species were recorded so that the percentage of the ovary to the total body weight in each sampling month could be calculated. The mouse bioassays of the puffer ®sh toxicity basically followed two standard guidelines, (1) the assay method for tetrodotoxin (TTX, puffer ®sh principal toxin) in the Food Hygiene Examination Manual vol. 2 in Japan (Kawabata, 1978) and (2) the biological method of paralytic shell®sh poison in the Of®cial Methods of Analysis of the Association of Of®cial Analytical Chemists (AOAC) in the USA (Cunniff 1995). Lethal potency of TTX was expressed in mouse units (MU) and one MU was de®ned as the amount of toxin required to kill a 20 g white mouse in 30 min after intraperitoneal injection. The strain of the test mice used in this study was from the Institute of Cancer Research (ICR) and both males and females were used randomly. Each mouse was injected intraperitoneally with 1 ml of the
toxin extract from each puffer ®sh tissue, which has a concentration of 0.2 g/ml. The median death time of a group of three mice in each tissue sample in each puffer ®sh specimen was used to determine the tissue toxicity in MU/g. The annual toxicological pro®les of both puffer species were summarised in Fig. 1. Their respective spawning seasons (determined by the rapid enlargement of the ovaries and the presence of eggs) corresponded roughly to the width of the peaks of the lines showing the proportion of the ovary to the total body weight. The spawning season of Takifugu niphobles (Jordan and Snyder) was from October through February in the next year (about four consecutive months), whereas it was much shorter in Takifugu alboplumbeus (Richardson), starting from December to February of the next year (only about two consecutive months). The weight of the ovary of a Takifugu niphobles (Jordan and Snyder) specimen was only 1.13 ^ 0.45 (mean ^ standard deviation) g (or 1.56 ^ 0.70% of the body weight) in the non-spawning season but it would increase to 8.31 ^ 1.22 g (or 10.17 ^ 0.25% of the body weight) in
C.-F. Yu, P.H.-F. Yu / Toxicon 40 (2002) 313±316
the spawning season. The weight of ovary of a Takifugu alboplumbeus (Richardson) specimen increased from 0.51 ^ 0.13 g (or 1.02 ^ 0.32% of the body weight) to 3.50 ^ 0.52 g (or 7.4 ^ 0.3% of the body weight) in the spawning season. The variation in toxicities of ovary, liver and intestine from Takifugu niphobles (Jordan and Snyder) had a similar pattern. Their respective toxicities were observed to be lower from October to April, which was almost the same period as the spawning season. In the non-spawning season, the toxicities of ovary, liver and intestine were found to be 911.8 ^ 134.4, 136.0 ^ 37.4 and 55.8 ^ 24.4 MU/g, respectively. However, they would drop to 93.5 ^ 20.5, 17.0 ^ 13.5 and 13.3 ^ 7.3 MU/g in the period from October through April of the next year. No regular pattern in the annual toxicities of the skin and ¯esh could be determined and they were 47.8 ^ 27.7 and 7.0 ^ 1.0 MU/g, respectively for the whole study period. The testes developed only during the spawning season and its toxicity was 8.3 ^ 0.8 MU/g, which was considered to be non-toxic (Tani, 1945). The overall toxicity of Takifugu alboplumbeus (Richardson) was comparatively less than that of Takifugu niphobles (Jordan and Snyder). The toxicity of the ovary from Takifugu alboplumbeus (Richardson) was lower (105.8 ^ 45.7 MU/g) from December through June the next year, a period which contained its spawning season. In the non-spawning season, the toxicity of the ovary would increase to 385.0 ^ 86.6 MU/g. No regular pattern in the annual toxicities of the liver, intestine, skin and ¯esh could be determined and they were found to be 21.4 ^ 12.3, 24.9 ^ 24.2, 53.9 ^ 22.6 and 7.1 ^ 1.5 MU/ g, respectively for the whole study period. The testes developed only during the spawning season and its toxicity was 5.5 ^ 0.5 MU/g, which was also considered to be non-toxic (Tani, 1945). Among researchers, it has long been noticed that the toxicities of puffer ®sh vary with seasons and locations, even for the same species in the same region (Endo, 1984; Halstead, 1988; Khora et al., 1991; Nagashima et al., 2001; Tani, 1940, 1945; Yu and Yu, 1998). This study, to the best of our knowledge, is the ®rst ever to monitor continuously the toxicity of two puffer ®sh species together for over a year. The toxins in the puffer ®sh, mainly tetrodotoxin (TTX) and its derivatives, are said to protect themselves and their fertilised eggs from predators (Saito et al., 1984). TTX itself is a potent neurotoxin which blocks sodium channels with great speci®city and can produce rapid and violent death in humans and other animals even in small quantities. However, TTX can be accumulated inside puffer ®sh as they have a mutation in the protein sequence of their sodium channels, and, as a result, are not sensitive to TTX (Yoshida, 1994). Contrary to the most common belief that puffer ®sh should be more toxic in their spawning seasons in order to protect their fertilised eggs (Halstead, 1988; Hwang et al.,
315
1992; Jeong et al., 1994), the two species investigated in this study were actually less toxic, especially their ovaries, livers and intestines, the toxicity of which would decrease to about as much as 10% of the original during non-spawning season. The toxicities of the skin, ¯esh and testes did not have a prominent pattern related to the spawning seasons. The skin remained weakly toxic whilst the ¯esh was basically nontoxic throughout the whole year. The testes in both species developed only during the spawning season and their toxicities were also less than 10 MU/g. It is generally accepted that the toxins found in puffer ®sh are not actually produced by the ®sh themselves but rather by the bacteria living symbiotically inside their bodies (Do et al., 1990; Noguchi et al., 1987; Ritchie et al., 2000; Yotsu et al., 1987). The bacteria, also known as TTX-producing bacteria, can have a shelter to live and propagate or even obtain some nourishment from the ®sh in order to survive. The puffer ®sh themselves are not fast swimming and, therefore, accumulate the toxins produced by the bacteria inside their bodies so as to protect themselves from predators. The bacterial origin of the TTX found in the puffer ®sh bodies explains why their toxicities vary with seasons and locations. Further studies will be followed in this direction to elucidate the actual relationship between the toxicity of puffer ®sh and the symbiotic bacteria isolated from their bodies.
Acknowledgements The authors would like to express their special thanks to several local ®shermen for their effort in collecting all the puffer ®sh used in this investigation.
References Chan, W.L., 1968. Marine ®shes of Hong Kong. Hong Kong Government Press, Hong Kong. Cunniff, P. (Ed.), 1995. Of®cial methods of analysis 16th ed. The Association of Of®cial Analytical Chemists, Inc, Cunniff, pp. 46B±48. Do, H.K., Kogure, K., Simidu, U., 1990. Identi®cation of deep-seasediment bacteria which produce tetrodotoxin. Applied and Environmental Microbiology 56 (4), 1162±1163. Endo, R., 1984. Toxicological studies on puffer ®shes: comparison of toxicities on the various species. The Journal of Toxicological Sciences 9 (supplement I), 1±11. Halstead, B.W., 1988. Poisonous and Venomous Marine Animals of the World, 2nd revised ed. The Darwin Press, Inc., New Jersey. Hwang, D.F., Cao, C.Y., Yang, H.C., Jeng, S.S., Noguchi, T., 1992. Toxicity of puffer in Taiwan. Nippon Suisan Gakkaishi 58 (8), 1541±1547. Jeong, D.Y., Kim, D.S., Lee, M.J., Kim, S.R., Byun, D.S., Kim, H.D., Park, Y.H., 1994. Toxicity of several puffers collected at a ®sh market of Pusan, Korea. Bulletin of the Korean Fisheries Society 27 (6), 682±689.
316
C.-F. Yu, P.H.-F. Yu / Toxicon 40 (2002) 313±316
Kawabata, T., 1978. Food Hygiene Examination Manual, vol. 2. Japan Food Hygienic Association, Tokyo. Khora, S.S., Isa, J., Yasumoto, T., 1991. Toxicity of puffers from Okinawa, Japan. Nippon Suisan Gakkaishi 57 (1), 163±167. Nagashima, Y., Tanaka, N., Shimakura, K., Shiomi, K., Shida, Y., 2001. Occurrence of tetrodotoxin-related substances in the nontoxic puffer Takifugu xanthopterus. Toxicon 39, 415±418. Noguchi, T., Hwang, D.F., Arakawa, O., Sugita, H., Deguchi, Y., Shida, Y., Hashimoto, K., 1987. Vibrio alginolyticus, a tetrodotoxin-producing bacterium, in the intestine of the ®sh Fugu vermicularis vermicularis. Marine Biology 94, 625±630. Ritchie, K.B., Nagelkerken, I., James, S., Smith, G.W., 2000. Environmental microbiology: a tetrodotoxin-producing marine pathogen. Nature 404 (6776), 354. Saito, T., Noguchi, T., Harada, T., Murata, O., Hashimoto, K., 1984. Tetrodotoxin as a biological defense agent for puffers. Bulletin of the Japanese Society of Scienti®c Fisheries 51 (7), 1175±1180.
Tani, I., 1940. Seasonal changes and individual differences of puffer poison. Nippon Yakubutsugaku Zasshi 29 (1±2), 143±145. Tani, I., 1945. Toxicological studies in Japan. Teikoku-Tosho Co, Tokyo. Yoshida, S., 1994. Tetrodotoxin-resistant sodium channels. Cellular and Molecular Neurobiology 14 (3), 227±244. Yotsu, M., Yamazaki, T., Megura, Y., Endo, A., Murata, M., Naoki, H., Yasumoto, T., 1987. Production of tetrodotoxin and its derivatives by Pseudomonas sp. isolated from the skin of a puffer®sh. Toxicon 25 (2), 225±228. Yu, C.F., Yu, P.H.F., 1997. A preliminary study of puffer ®shes and their toxins found in Hong Kong waters. J. Food Hyg. Soc. Japan 38 (6), 460±463. Yu, P.H.F., Yu, C.F., 1998. The toxicological study of puffer ®shes in Hong Kong. Journal of Fisheries of China 22 (4), 382±384.
Short communication
www.elsevier.com/locate/toxicon
The annual toxicological pro®les of two common puffer ®sh, Takifugu niphobles (Jordan and Snyder) and Takifugu alboplumbeus (Richardson), collected along Hong Kong coastal waters Chun-Fai Yu*, Peter Hoi-Fu Yu Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, People's Republic of China Received 10 May 2001; accepted 3 August 2001
Abstract The toxicological pro®les of two local common puffer ®sh, Takifugu niphobles (Jordan and Snyder) and Takifugu alboplumbeus (Richardson), collected in Hong Kong waters were investigated continuously for 14 months (June 1997±August 1998). Their annual spawning seasons (as evident by the enlargement of gonads and presence of eggs in the ovary) were found to be from October to February (four consecutive months) and December to February (two consecutive months), respectively. The toxicities of their internal organs were determined by standard mouse bioassay and expressed in terms of mouse units (MU). The ovary (in both species) and liver (only in T. niphobles) were discovered to be moderately toxic (100±1000 MU/g) in their non-spawning seasons. Whilst all the intestine and skin were weakly toxic (between 10 and 100 MU/g), their ¯esh, however, was basically non-toxic (less than 10 MU/g) throughout the whole year. The testes, which were only developed in their spawning seasons, were also non-toxic. This study showed that the toxicological pro®les of both of the two species had prominent seasonal patterns which were highly related to their spawning seasons. Contrary to the most common belief, both species were discovered to be relatively less toxic during their spawning seasons. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Toxicological pro®le; Spawning season; Mouse bioassay; Mouse units
Hong Kong, like many other coastal cities in the world, faces the problems of marine pollution caused by both domestic and industrial wastewater discharge into the surrounding coastal waters and the rapid coastal developments along the shore line. The traditional ®sheries resources within Hong Kong waters have declined drastically in the past decades; puffer ®sh, however, can still be easily caught along the seashores. In 1968, a total of nine species of puffer ®sh was reported to be indigenous to local waters (Chan, 1968). The same number of species, belonging to two families and ®ve genera, has been collected and identi®ed by us (Yu and Yu, 1998). Among the nine local species, three were con®rmed to be toxic and one was nontoxic (Yu and Yu, 1997). Takifugu niphobles (Jordan and * Corresponding author. Fax : 1852-24793740. E-mail address: [email protected] (C.-F. Yu).
Snyder) and Takifugu alboplumbeus (Richardson) are the most common local puffer ®sh and can be collected in coastal waters throughout the whole year. However, some species can only be caught in the summer months as they migrate to the deeper seas in winter. Takifugu niphobles (Jordan and Snyder) and Takifugu alboplumbeus (Richardson) were, therefore, used in this study which aimed to ®nd out their spawning seasons and establish their annual toxicological pro®les. The puffer ®sh used in this study were collected by local ®shermen using ®sh traps placed along the coastal waters of the north-eastern outlying islands. Between June 1997 and August 1998, about 100 puffer ®sh specimens were collected in every two months for toxicological analysis. Ten female specimens (of similar sizes) of each species of Takifugu niphobles (Jordan and Snyder) and Takifugu alboplumbeus (Richardson) were randomly sorted out from the
0041-0101/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 0041- 010 1(01)00208- 2
314
C.-F. Yu, P.H.-F. Yu / Toxicon 40 (2002) 313±316
Fig. 1. The annual proportion of ovary to the total body weight and annual variation in toxicities in Takifugu niphobles (Jordan and Snyder) and Takifugu alboplumbeus (Richardson).
rest of the puffer ®sh. They were weighed and then dissected into different tissues (liver, intestine, ¯esh, skin and ovary), which would be pooled together for toxicological analysis. The total weight of the ovaries of the 10 specimens in each species were recorded so that the percentage of the ovary to the total body weight in each sampling month could be calculated. The mouse bioassays of the puffer ®sh toxicity basically followed two standard guidelines, (1) the assay method for tetrodotoxin (TTX, puffer ®sh principal toxin) in the Food Hygiene Examination Manual vol. 2 in Japan (Kawabata, 1978) and (2) the biological method of paralytic shell®sh poison in the Of®cial Methods of Analysis of the Association of Of®cial Analytical Chemists (AOAC) in the USA (Cunniff 1995). Lethal potency of TTX was expressed in mouse units (MU) and one MU was de®ned as the amount of toxin required to kill a 20 g white mouse in 30 min after intraperitoneal injection. The strain of the test mice used in this study was from the Institute of Cancer Research (ICR) and both males and females were used randomly. Each mouse was injected intraperitoneally with 1 ml of the
toxin extract from each puffer ®sh tissue, which has a concentration of 0.2 g/ml. The median death time of a group of three mice in each tissue sample in each puffer ®sh specimen was used to determine the tissue toxicity in MU/g. The annual toxicological pro®les of both puffer species were summarised in Fig. 1. Their respective spawning seasons (determined by the rapid enlargement of the ovaries and the presence of eggs) corresponded roughly to the width of the peaks of the lines showing the proportion of the ovary to the total body weight. The spawning season of Takifugu niphobles (Jordan and Snyder) was from October through February in the next year (about four consecutive months), whereas it was much shorter in Takifugu alboplumbeus (Richardson), starting from December to February of the next year (only about two consecutive months). The weight of the ovary of a Takifugu niphobles (Jordan and Snyder) specimen was only 1.13 ^ 0.45 (mean ^ standard deviation) g (or 1.56 ^ 0.70% of the body weight) in the non-spawning season but it would increase to 8.31 ^ 1.22 g (or 10.17 ^ 0.25% of the body weight) in
C.-F. Yu, P.H.-F. Yu / Toxicon 40 (2002) 313±316
the spawning season. The weight of ovary of a Takifugu alboplumbeus (Richardson) specimen increased from 0.51 ^ 0.13 g (or 1.02 ^ 0.32% of the body weight) to 3.50 ^ 0.52 g (or 7.4 ^ 0.3% of the body weight) in the spawning season. The variation in toxicities of ovary, liver and intestine from Takifugu niphobles (Jordan and Snyder) had a similar pattern. Their respective toxicities were observed to be lower from October to April, which was almost the same period as the spawning season. In the non-spawning season, the toxicities of ovary, liver and intestine were found to be 911.8 ^ 134.4, 136.0 ^ 37.4 and 55.8 ^ 24.4 MU/g, respectively. However, they would drop to 93.5 ^ 20.5, 17.0 ^ 13.5 and 13.3 ^ 7.3 MU/g in the period from October through April of the next year. No regular pattern in the annual toxicities of the skin and ¯esh could be determined and they were 47.8 ^ 27.7 and 7.0 ^ 1.0 MU/g, respectively for the whole study period. The testes developed only during the spawning season and its toxicity was 8.3 ^ 0.8 MU/g, which was considered to be non-toxic (Tani, 1945). The overall toxicity of Takifugu alboplumbeus (Richardson) was comparatively less than that of Takifugu niphobles (Jordan and Snyder). The toxicity of the ovary from Takifugu alboplumbeus (Richardson) was lower (105.8 ^ 45.7 MU/g) from December through June the next year, a period which contained its spawning season. In the non-spawning season, the toxicity of the ovary would increase to 385.0 ^ 86.6 MU/g. No regular pattern in the annual toxicities of the liver, intestine, skin and ¯esh could be determined and they were found to be 21.4 ^ 12.3, 24.9 ^ 24.2, 53.9 ^ 22.6 and 7.1 ^ 1.5 MU/ g, respectively for the whole study period. The testes developed only during the spawning season and its toxicity was 5.5 ^ 0.5 MU/g, which was also considered to be non-toxic (Tani, 1945). Among researchers, it has long been noticed that the toxicities of puffer ®sh vary with seasons and locations, even for the same species in the same region (Endo, 1984; Halstead, 1988; Khora et al., 1991; Nagashima et al., 2001; Tani, 1940, 1945; Yu and Yu, 1998). This study, to the best of our knowledge, is the ®rst ever to monitor continuously the toxicity of two puffer ®sh species together for over a year. The toxins in the puffer ®sh, mainly tetrodotoxin (TTX) and its derivatives, are said to protect themselves and their fertilised eggs from predators (Saito et al., 1984). TTX itself is a potent neurotoxin which blocks sodium channels with great speci®city and can produce rapid and violent death in humans and other animals even in small quantities. However, TTX can be accumulated inside puffer ®sh as they have a mutation in the protein sequence of their sodium channels, and, as a result, are not sensitive to TTX (Yoshida, 1994). Contrary to the most common belief that puffer ®sh should be more toxic in their spawning seasons in order to protect their fertilised eggs (Halstead, 1988; Hwang et al.,
315
1992; Jeong et al., 1994), the two species investigated in this study were actually less toxic, especially their ovaries, livers and intestines, the toxicity of which would decrease to about as much as 10% of the original during non-spawning season. The toxicities of the skin, ¯esh and testes did not have a prominent pattern related to the spawning seasons. The skin remained weakly toxic whilst the ¯esh was basically nontoxic throughout the whole year. The testes in both species developed only during the spawning season and their toxicities were also less than 10 MU/g. It is generally accepted that the toxins found in puffer ®sh are not actually produced by the ®sh themselves but rather by the bacteria living symbiotically inside their bodies (Do et al., 1990; Noguchi et al., 1987; Ritchie et al., 2000; Yotsu et al., 1987). The bacteria, also known as TTX-producing bacteria, can have a shelter to live and propagate or even obtain some nourishment from the ®sh in order to survive. The puffer ®sh themselves are not fast swimming and, therefore, accumulate the toxins produced by the bacteria inside their bodies so as to protect themselves from predators. The bacterial origin of the TTX found in the puffer ®sh bodies explains why their toxicities vary with seasons and locations. Further studies will be followed in this direction to elucidate the actual relationship between the toxicity of puffer ®sh and the symbiotic bacteria isolated from their bodies.
Acknowledgements The authors would like to express their special thanks to several local ®shermen for their effort in collecting all the puffer ®sh used in this investigation.
References Chan, W.L., 1968. Marine ®shes of Hong Kong. Hong Kong Government Press, Hong Kong. Cunniff, P. (Ed.), 1995. Of®cial methods of analysis 16th ed. The Association of Of®cial Analytical Chemists, Inc, Cunniff, pp. 46B±48. Do, H.K., Kogure, K., Simidu, U., 1990. Identi®cation of deep-seasediment bacteria which produce tetrodotoxin. Applied and Environmental Microbiology 56 (4), 1162±1163. Endo, R., 1984. Toxicological studies on puffer ®shes: comparison of toxicities on the various species. The Journal of Toxicological Sciences 9 (supplement I), 1±11. Halstead, B.W., 1988. Poisonous and Venomous Marine Animals of the World, 2nd revised ed. The Darwin Press, Inc., New Jersey. Hwang, D.F., Cao, C.Y., Yang, H.C., Jeng, S.S., Noguchi, T., 1992. Toxicity of puffer in Taiwan. Nippon Suisan Gakkaishi 58 (8), 1541±1547. Jeong, D.Y., Kim, D.S., Lee, M.J., Kim, S.R., Byun, D.S., Kim, H.D., Park, Y.H., 1994. Toxicity of several puffers collected at a ®sh market of Pusan, Korea. Bulletin of the Korean Fisheries Society 27 (6), 682±689.
316
C.-F. Yu, P.H.-F. Yu / Toxicon 40 (2002) 313±316
Kawabata, T., 1978. Food Hygiene Examination Manual, vol. 2. Japan Food Hygienic Association, Tokyo. Khora, S.S., Isa, J., Yasumoto, T., 1991. Toxicity of puffers from Okinawa, Japan. Nippon Suisan Gakkaishi 57 (1), 163±167. Nagashima, Y., Tanaka, N., Shimakura, K., Shiomi, K., Shida, Y., 2001. Occurrence of tetrodotoxin-related substances in the nontoxic puffer Takifugu xanthopterus. Toxicon 39, 415±418. Noguchi, T., Hwang, D.F., Arakawa, O., Sugita, H., Deguchi, Y., Shida, Y., Hashimoto, K., 1987. Vibrio alginolyticus, a tetrodotoxin-producing bacterium, in the intestine of the ®sh Fugu vermicularis vermicularis. Marine Biology 94, 625±630. Ritchie, K.B., Nagelkerken, I., James, S., Smith, G.W., 2000. Environmental microbiology: a tetrodotoxin-producing marine pathogen. Nature 404 (6776), 354. Saito, T., Noguchi, T., Harada, T., Murata, O., Hashimoto, K., 1984. Tetrodotoxin as a biological defense agent for puffers. Bulletin of the Japanese Society of Scienti®c Fisheries 51 (7), 1175±1180.
Tani, I., 1940. Seasonal changes and individual differences of puffer poison. Nippon Yakubutsugaku Zasshi 29 (1±2), 143±145. Tani, I., 1945. Toxicological studies in Japan. Teikoku-Tosho Co, Tokyo. Yoshida, S., 1994. Tetrodotoxin-resistant sodium channels. Cellular and Molecular Neurobiology 14 (3), 227±244. Yotsu, M., Yamazaki, T., Megura, Y., Endo, A., Murata, M., Naoki, H., Yasumoto, T., 1987. Production of tetrodotoxin and its derivatives by Pseudomonas sp. isolated from the skin of a puffer®sh. Toxicon 25 (2), 225±228. Yu, C.F., Yu, P.H.F., 1997. A preliminary study of puffer ®shes and their toxins found in Hong Kong waters. J. Food Hyg. Soc. Japan 38 (6), 460±463. Yu, P.H.F., Yu, C.F., 1998. The toxicological study of puffer ®shes in Hong Kong. Journal of Fisheries of China 22 (4), 382±384.