Paralytic Shellfish Poisoning Toxins: Biochemistry and Origin

Aqua-BioScience Monographs Vol. 3, No. 1, pp. 1–38 (2010) www.terrapub.co.jp/onlinemonographs/absm/

Masaaki Kodama

Laboratory of Marine Biochemistry, Department of Aquatic Biosciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan e-mail: [email protected]

Abstract Plankton feeders such as bivalves often become toxic. Human consumption of the toxic bivalve Received on September 8, 2009 causes severe food poisoning, including paralytic shellfish poisoning (PSP) which is the most Accepted on October 13, 2009 dangerous because of the acuteness of the symptoms, high fatality and wide distribution throughout Published online on the world. Accumulation of PSP toxins in shellfish has posed serious problems to public health April 9, 2010 and fisheries industry. The causative organisms of PSP toxins are known to be species of dinoflagellates including those belonging to the genus Alexandrium, Gymnodinium catenatum Keywords and Pyrodinium bahamense var. compressum. Bivalves accumulate PSP toxins during a bloom • paralytic shellfish poisoning toxins of these dinoflagellates. Thus, the dinoflagellate toxins have been considered as being concen- • saxitoxin trated in bivalves through food web transfer. However, field studies on the toxin level of bivalves • gonyautoxin in association with the abundance of toxic dinoflagellates could not support the idea. A kinetics • tetrodotoxin study on toxins by feeding experiments of cultured dinoflagellate cells to bivalves also showed • dinoflagellate similar results, indicating that toxin accumulation of bivalves is not caused by simple accumula- • Alexandrium tamarense tion of toxins due to food-web transfer. Based on the discovery of toxin-producing bacterium in • bivalve the cells of toxic dinoflagellates, this study suggests that PSP toxins in toxic dinoflagellates are • intracellular bacteria catabolites of bacterial substance in dinoflagellate cells. On the other hand, tetrodotoxin (TTX), • bacterial infection a puffer toxin, is reported to be produced by some species of bacteria. Thus, the origin of TTX of puffer is considered to be bacteria. However, the mechanism for puffer to possess TTX through bacteria is unknown. The present study revealed that organisms bearing either TTX or PSP toxins possess both toxins, although the proportion of both is different among the specimens. In fact, a significant level of TTX is detected in A. tamarense. In addition, TTX-like toxin-producing bacteria are found to be infected in the liver of toxic specimens of puffer. These findings strongly suggest that a similar mechanism between bacteria and toxic organisms is involved in the production of TTX and/or PSP toxins in toxic organisms.

1. Introduction as bivalves are carried out and often result in the prohibi- tion of harvesting these products. Thus, the contamina- Saxitoxin (STX) and its analogs (STXs) are potent tion of bivalves with STXs has posed severe problems neurotoxins that block voltage-gated sodium channels on for the bivalve culture industries as well as for public excitable cells (Kao 1966). These toxins are produced by health. Many studies have been carried out on the toxin- several species of dinoflagellates such as Alexandrium producing dinoflagellates and the toxins they produce and tamarense and A. catenella (Schantz 1986). During a there is much known about their taxonomy and ecology bloom of these species, filter-feeding bivalves become (Steidinger 1993). The chemical structures and pharma- toxic by ingesting them. Human consumption of toxic cological properties of the toxins have been well defined shellfish results in paralytic shellfish poisoning (PSP) (Schantz 1986; Kao 1966). On the biosynthesis of the owing to its main symptom, paralysis. Bivalve farming toxin, Shimizu et al. (1984) proved that the skeleton of areas infested by these species run costly monitoring pro- STXs is synthesized from arginine and acetate based on grams to check for the shellfish toxicity and the causa- the tracer experiments using PSP-producing cyanobac- tive dinoflagellates in the water. When these are present, terium Aphanizomenon flosaque, and proposed the unique regular tests for toxins in associated seafood products such biosynthetic pathway. Very recently, this pathway has been

Fig. 1. Structures of PSP toxins

proved to be true with slight modification through chemi- tricyclic ring system with two guanidium moieties and a cal and biochemical studies (Kellmann et al. 2008a, b). hydrated ketone. Subsequently, various derivatives of However, these data are also obtained from toxin- STX, such as neoSTX and gonyautoxin (GTX) 1–4, and producing cyanobacteria. Currently there is no informa- B and C toxins, have been isolated from contaminated tion on the biosynthesis of PSP toxins in toxin-producing shellfish as well as the causative dinoflagellates, and their dinoflagellates, although PSP toxins have been detected chemical structures have been identified (Oshima et al. in bacteria isolated from toxin-producing dinoflagellates 1977; Shimizu et al. 1978; Hall et al. 1980; Kobayashi (Kodama et al. 1988; Doucette and Trick 1995; Gallacher and Shimizu 1981; Shimizu and Hsu 1981; Wichmann et et al. 1997). Studies on the role of toxin-producing bac- al. 1981; Harada et al. 1982; Koehn et al. 1982). Cur- teria in the marine ecosystem are considered to be be- rently, more than 20 derivatives are known (Oshima coming more important. 1995a). The toxin in shellfish and toxic dinoflagellates is usually a mixture of two or more toxin components. 2. PSP toxins found in bivalves and dinoflagellates Among them, STX is rather a minor component. Figure 1 shows the structures of the components commonly found STX and its derivatives belong to a group of in toxin-contaminated shellfish and the causative neurotoxins which block voltage-gated sodium channels dinoflagellates. All of these are derivatives of STX. As on excitable cells (Kao 1966). These toxins are known to shown in Fig. 1, the toxins can be classified into two cat- be produced by several species of dinoflagellates such as egories: N1-H type and N1-OH type groups. The sim- Alexandrium tamarense and A. catenella (Schantz 1986). plest component of the former group is STX, and that of During a bloom of these species, bivalves, filter-feeding the latter is neoSTX. Another structural variation in both mollusks, accumulate toxins by ingesting them. The PSP groups is the presence or absence of an O-sulfate at C11 toxins consist of a group of toxin components, the struc- position and an N-sulfate on the carbamoyl groups. These tures of which are similar to each other. These compounds variations greatly influence the physical and chemical are water soluble alkaloids. A typical toxin is STX which characters as well as the pharmacological characters. was first isolated from the toxic Alaskan butter clam Generally, the toxins are considered to be water soluble Saxidomus giganteus (Schantz et al. 1957). As STX is and heat stable. The stability varies greatly, depending highly hygroscopic and hard to crystallize, extended ef- on the pH and the structures (Shimizu 2000). At an alka- forts were required to elucidate its chemical structure. line pH, all the toxin components degrade quickly, even Finally, Schantz et al. (1975) succeeded in determining at room temperature. Generally the toxins are stable units chemical structure, which possesses a characteristic der acidic conditions and it is noteworthy that the po-

Table 1. Specific toxicity of each toxin component (MU·µmol–1)*.

Component Specific toxicity Component Specific toxicity STX 2483 dcGTX2 1617 neoSTX 2295 dcGTX3 1872 dcSTX 1274 GTX5 160 GTX1 2468 C1 15 GTX2 892 C2 239 GTX3 1584 C3 33 GTX4 1803 C4 143

*MU: mouse unit; one MU is a dose of toxin which kills one 20-g mouse (ddY strain) in 15 min. According to Oshima, Y. Post-column derivatization HPLC methods for paralytic shellfish poisons. In: Hallegraeff GM, Anderson DM, Cembella AD (eds). Manual on Harmful Marine Microalgae. IOC Manuals and Guides No. 33. Intergovern- mental Oceanographic Commission, UNESCO, Paris. 1995; pp. 81–111.

tency of a toxin is different among toxin components. (neoSTX). Fukuyo (1979) observed that toxic Table 1 shows the specific toxicity of each toxin compo- dinoflagellates in Ofunato Bay presented three peaks in nent expressed as mouse units (MU) per µm mole of toxin, abundance in 1979. They found that the first and second in which 1 MU is a dose of toxin sufficient to kill a male peaks appearing in spring and summer, respectively, con- mouse (ddY strain) in 15 min (Oshima 1995a). sisted of Alexandrium tamarense (former Protogonyaulax tamarensis) and the third peak in autumn consisted of 3. Accumulation of paralytic shellfish poisoning- A. catenella (former P. catenella). toxins in bivalve during a bloom of toxic In order to make clear any possible relationship dinoflagellates in the field between the appearance of toxic dinoflagellates and the toxification of the scallop feeding on those plankton, the PSP is a dangerous food poison because of its high occurrence of Alexandrium spp. in Ofunato Bay was fatality, acuteness in developing symptoms, and world- monitored in association with the toxin level of scallop wide occurrence. It is confirmed by many authors that Patinopecten yessoensis in Ofunato Bay (Ogata et al. bivalves become toxic during blooms of toxic 1982). Seawater samples for monitoring the cell density dinoflagellates. PSP has long been known along the Pa- of A. tamarense were collected, usually twice a week, cific and Atlantic coasts of North America and Canada, from different depths of water (2 m-intervals) at Shizu and many fatal cases have been recorded (Prakash et al. (St. S. 24 m depth) and Yamaguchi (St. Y. 14 m depth) in 1967). Poisoning occurred in September of 1972 and in Ofunato Bay (Fig. 2). Some 500 mL of seawater was con- June and September of 1974 after large-scale red tides of centrated by gravity filtration using a membrane filter. A. tamarense occurred along the East Coast, and 26 per- The concentrate was made up to 20 to 40 mL and each sons were intoxicated during the 1972 incidence. It was 1 mL portion was used for microscopic counting of prohibited to take shellfish along a 3200 km coastline, Alexandrium cells. When the population of Alexandrium which resulted in great economic loss (LoCicero 1975). tended to increase, seawater samples were collected al- In Japan, PSP had not been recognized before the inci- most every day. Nontoxic scallop Patinopecten yessoensis dent at Toyohashi in 1948 (Hashimoto et al. 1950). Since were transplanted from Mutsu Bay, Aomori Prefecture, then, a few cases of PSP have been observed and so de- to the two stations at a 10 m depth. Five scallop speci- tailed surveys on the causative dinoflagellates and bivalve mens were collected for testing for toxicity whenever the toxicity have not been conducted. Ofunato Bay is one of seawater sampling was done. Digestive glands were ex- the places where PSP due to consumption of scallop cised from them, combined and measured for PSP toxic- Chlamys nipponensis akazara occurred in 20 people in- ity by the AOAC method (Horowitz 1984). cluding one fatal case in 1961 (Kawabata et al. 1962). Alexandrium appeared twice in spring and again The toxin in toxic shellfishes, as well as possibly causa- in autumn in this bay. Similar results were obtained from tive organisms in the bay, has been examined by some both stations. The results from St. S in Fig. 3 show that investigators (Murano 1975), though not satisfactorily. relatively small number of Alexandrium began to appear After the 1977 incident, Fukuyo (1979) isolated an in February and March, and disappeared in April at both Alexandrium species from the bay, and identified it as stations. The plankton appeared again in May and sharply Gonyaulax excavate (Alexandrium tamarense). Oshima increased its density, showing large fluctuations. It di- and Yasumoto (1979) showed that the toxin from cultured minished by the end of June at both stations. Alexandrium A. tamarense consisted of GTX1, 2, 3, 4 and neosaxitoxin appeared again in September and reached maximum

Fig. 3. Seasonal changes in cell number of Alexandrium (—) and in toxicity level of scallop (---) at St. Shizu of Ofunato Fig. 2. Map of Ofunato Bay showing two sampling station. Bay. Reprinted with permission from Bulletin of the Japanese Reprinted with permission from Bulletin of the Japanese Soci- Society of Scientific Fisheries, 48, Ogata et al., The occurrence ety of Scientific Fisheries, 48, Ogata et al., The occurrence of of Protogonyaulax spp. in Ofunato Bay, in association with the Protogonyaulax spp. in Ofunato Bay, in association with the toxification of the scallop Patinopecten yessoensis. 563–566, toxification of the scallop Patinopecten yessoensis. 563–566, Fig. 3, © 1982, the Japanese Society of Fisheries Science. Fig. 1, © 1982, the Japanese Society of Fisheries Science.

abundance in mid-October at both stations. It disappeared the scallop. As shown in Fig. 4, it has been confirmed by by the end of October, showing that it appeared three times the toxicity change of highly toxic scallop which was in 1980. The Alexandrium which occurred in spring and reared in an aquarium (Oshima et al. 1982a). When the summer was identified as A. tamarense while that oc- highly toxic scallop was reared in Alexandrium-free wa- curred in autumn was identified as A. catenella. In a con- ter, around two thirds of toxin was lost within 1 week. tinuous survey in the bay over 10 years, similar patterns However, a considerable amount of toxin was maintained were observed. Toxicity of scallop increased in associa- for a long period. As described above, the species ap- tion with the occurrence of Alexandrium. However, the pearing in spring was A. tamarense while that in autumn ratio of the toxicity level to the cell number significantly was A. catenella. The scallop toxicity was correlated with varied, even in the same species. The ratio for the spring the abundance of A. tamarense (Fig. 4), whereas the as- A. tamarense was generally larger than for that of sum- sociation of A. catenella with shellfish toxicity was not mer, suggesting that both organsms differed substantially clear in this bay. from each other in the amount of toxin per cell. It has Interestingly, some curious phenomena were ob- been reported that the amount of toxin contained in a cul- served during the monitoring. One of them is that there tured A. tamarense cell varied depending upon growth was no marked toxicity increase in the shellfish when conditions (Ogata et al. 1987a). The toxin contents of A. tamarense reached the maximum abundance. The natural cells in the field seemed to reflect their physi- toxicity of scallop increased to the maximum, then the ological conditions. Once intoxicated, scallop did not population of A. tamarense was decreasing. The time- become nontoxic in July and August, even during sev- lag between the two maxima was about one week. An- eral months after the responsible plankton disappeared. other is that the scallop showed a repeated rise and fall in They still maintained considerable amounts of toxin, in- toxicity during the period, when the supply of the toxin dicating that the toxin can not be eliminated easily from seemed to be apparently suppressed. These apparently

ins due to exposure to toxic bloom of dinoflagellate, become toxic because of ingestion of toxic feces from toxic scallop (Kikuchi et al. 1996). These facts show that scallop possibly accumulate toxins from toxic feces of the toxic scallop as well as from toxic dinoflagellates. Thus, it seems to be difficult to understand the kinetics of toxins accumulated in scallop from dinoflagellates from the data on field survey.

4. Toxin production of Alexandrium species

Toxin production of the toxic dinoflagellate has been indicated to be affected by a variety of physiological or environmental factors: the toxicity of A. tamarense is different in its various growth stages (Prakash 1967; White and Maranda 1978; Oshima and Yasumoto 1979; Singh et al. 1982; Boyer et al. 1985); salinity affects the toxin production of A. tamarense (White 1986); toxin production of A. tamarense changes depending on the different growth stage of the cells (Kodama et al. 1982a). Here we describe the effect of water temperature and light intensity on growth rate and toxicity change in A. tamarense. A clonal culture was obtained from a single vegeta- tive cell of A. tamarense isolated from Ofunato Bay, Iwate Fig. 4. Decline of the toxicities of scallop Patinopecten Prefecture, Japan in March, 1984, when the species was yessoensis (a) and mussel Mytilus galloprovincialis (b) in labo- blooming. The clone obtained was mass-cultured and ratory tanks. Reprinted with permission from Bulletin of the maintained in enriched seawater T1 medium (Ogata et Japanese Society of Scientific Fisheries, 48, Oshima et al., Fea- tures of paralytic shellfish poison occurring in Tohoku district. al. 1987b) at 15°C under an illumination of 3000 lux with 525–530, Fig. 4, © 1982, the Japanese Society of Fisheries an LD cycle of 16 h:8 h. This clone was cultured under Science. different temperatures and light intensities after acclimation of the clonal population during a period of 20 days. An aliquot of the cells from each culture was taken every day to count the cell density under a micro-

scope. The growth rate (µ2 was estimated from cell counts curious phenomena may be due to the difference of the in the exponential phase according to Fukazawa et al. toxin content of the cells under different environmental (1980). and/or physiological conditions. Accurate analysis of the The growth rate decreased with the decrease of parameters such as toxin contents of the moving plank- water temperature and light intensity. The toxicity of the ton cells is actually difficult. Thus the one week time-lag cell increased with a decrease of growth rate (Fig. 5). In- observed between the peaks of shellfish toxicity and abun- terestingly, this growth rate was decreased by lowering dance of toxic dinoflagellate may be within the limit al- the temperature rather than by lowering the light inten- lowed usually in the fixed point observation on the sea. sity. This fact coincided well with our observation that However, similar phenomena have been observed almost the toxicity of A. tamarense at a lower temperature was every year in the monitoring in Ofunato Bay (Sekiguchi higher than that at a higher temperature (Ogata et al. et al. 1989) and in Okirai Bay in the same prefecture us- 1987a). This fact suggests that photosynthesis is neces- ing mussel Mytilus galloprovincialis as a monitoring bi- sary for the production of toxin in A. tamarense. In order valve (Oshima et al. 1982a). to confirm the effect of light on the toxin production of This phenomenon is difficult to explain if the tox- A. tamarense, we inhibited growth by lowering either ins in the dinoflagellates transfer to the shellfish via the water temperature or light intensity during mid-exponen- food chain. It is hard to evaluate the exchange of the toxin tial phase, and then examined the toxicity of the cells amount between dinoflagellates and shellfish in a field before and after treatment. As shown in Fig. 6, growth survey in which samples of shellfish and plankton are stopped when either temperature or light intensity was collected periodically at a station set in the field, even lowered. After this treatment, toxicity of the cells (the though the frequency of the sampling is increased. In growth of which was slowed by lowering temperature) addition, it is pointed out that nontoxic scallop introduced increased, while that by lowering light intensity did not to the scallop culture area where scallop accumulated tox- (see Table 2). These facts indicate that photosynthesis is

Glover et al. (1975) showed that amino acids were produced during short periods of photo-assimilation. We

added NO3 as a nitrogen source in the culture experiments. Therefore, de novo synthesis of amino acids should be

carried out utilizing incorporated NO3. The NO3 assimilation of microalgae is reported to be light-dependent (Morriss 1974; Syrett 1981), and this was also confirmed in A. tamarense (MacIsaac et al. 1979). Therefore, a decrease of light intensity at mid-exponential phase of our experiments seems to significantly retard de novo synthesis of amino acids by suppressed nitrogen assimila- Fig. 5. Alexandrium tamarense. Relation between toxicity and tion. The interruption of toxin production observed dur- growth rates measured in different water temperature (a) and ing the light retardation at the mid-exponential phase light intensity (b). Different toxicity formulas are used in A seems to depend on suppressed de novo synthesis of amino and B. Reprinted with kind permission from Springer Science acids because of the decrease of NO3 assimilation. From + Business Media: Marine Biology, Effect of water tempera- these, de novo synthesis of amino acids by photo-assimi- ture and light intensity on growth rate and toxicity change in lation is thought to play an important role in the toxin Protogonyaulax tamarensis, 95, 1987, 217–220, Ogata, production of A. tamarense. Ishimaru and Kodama, Fig. 3.

5. Transformation of toxins in dinoflagellates and bivalves

PSP toxins have been detected in several species of Alexandrium, Gymnodinium catenatum and Pyrodinium bahamense var. compressum. These species are photosynthetic dinoflagellates. Thus, PSP toxins are considered to be produced by these dinoflagellates. This view is supported by the fact that toxin production has been confirmed in axenic cultures of these species (Burke et al. 1960; Singh et al. 1982). That metabolism of toxins, including biosynthesis, occurs in the cells of these dinoflagellates was indicated by these experiments. Vari- ous toxin components found in the toxic species of dinoflagellates could be metabolites of toxin metabolism that occurred in the dinoflagellates. Interestingly, the toxin profile of cultured cells does not change significantly under different culture conditions such as different temperatures, light intensities, and levels of nutrients, Fig. 6. Alexandrium tamarense. Growth curves obtained by if the culture is monoclonal (Boyer et al. 1986, 1987; lowering the temperature (a) and light intensity (b) in the mid- Cembella et al. 1987; Ogata et al. 1987b). Therefore, the exponential phase. Reprinted with kind permission from toxin profiles of dinoflagellates isolated from various ar- Springer Science + Business Media: Marine Biology, Effect of eas have been elucidated by many research groups. These water temperature and light intensity on growth rate and toxic- studies indicate that the toxin profile is often specific to a ity change in Protogonyaulax tamarensis, 95, 1987, 217–220, species of dinoflagellates. For example, G. catenatum Ogata, Ishimaru and Kodama, Fig. 5. shows a characteristic toxin profile which consists of N-sulfocarbamoyl toxins such as B and C toxins as major components (Oshima et al. 1990). GTXs are rarely detected in the species. On the other hand, P. bahamense necessary for the toxin production of A. tamarense. They var. compressum contains B toxins and their carbamate also explain well the observation of the field survey that counterparts, STX and neoSTX, as dominant toxins. A the toxin content of A. tamarense occurred in March and significant amount of decarbamoyl saxitoxin (dcSTX) is higher than that occurred in May to June (Ogata et al. (see the structure in Fig. 1) is also found in this species 1982). (Harada et al. 1982, 1983). A difference in toxin profile Shimizu et al. (1984) showed that STX analogs were has also been observed among species belonging to the synthesized from arginine and acetic acid in A. tamarense. genus Alexandrium. C2 and GTX4 are the dominant

Table 2. Alexandrium tamarense (OF84423D3). Effect of slowdown of the growth rate on toxicity.

Treatment Harvesting Toxicity Total toxin concentrationa MU/104 cell MU/mm3 (nmol/104 cells) Ab Before treatment 0.68 2.30 0.491 After treatment 1.63 4.84 1.007

Bc Before treatment 0.64 2.71 0.398 After treatment 0.68 3.13 0.494

aCalculated from the toxin composition bTreatment A: Growth rate was slowed by lowering temperature at the mid-exponential phase cTreatment B: Growth rate was slowed by lowering light intensity at the mid-exponential phase Reproduced with kind permission from Springer Science + Business Media: Marine Biology, Effect of water temperature and light intensity on growth rate and toxicity change in Protogonyaulax tamarensis, 95, 1987, 217–220, Ogata et al., Table 3.

components in Alexandrium (Protogonyaulax) tamarense in some clones of A. tamarense that catalyzes the oxida- and A. catenella (Oshima et al. 1990), while GTX1–4 tion of N1-H of GTX2,3 to form GTX1,4. However, are dominantly found in A. tamiyavanichii (Kodama et this enzyme is not always present in all the isolates of al. 1988; Ogata et al. 1990). This species was isolated A. tamarense. On the other hand, N-sulfotransferase that from the Gulf of Thailand and identified as A. cohorticula, converts carbamate toxins to the corresponding C toxins but it was reclassified as a new species A. tamiyavanichii has been detected in G. catenatum (Oshima 1995b; by Balech: A. ostenfeldii showed a toxin profile consist- Yoshida et al. 1996). The occurrence of these enzymes ing of B toxins and their carbamate counterparts as domi- may explain the characteristic toxin profile of some nant toxins (Hansen et al. 1992). dinoflagellate species. The hereditary character of the Strains of the same species isolated from different toxin profile in dinoflagellates supports the role of en- geographical areas often show the different toxin profile. zymes in the characteristic toxin profile of dinoflagellates. Oshima et al. (1993a) showed that a Tasmanian iso- Various toxin components found in the dinoflagellates late of G. catenatum contains C4, which was absent in may be metabolites derived from a de novo product in the same species isolated from other areas (Ikeda et the biosynthesis of the STX skeleton, though so far this al. 1989). Furthermore, they detected novel compo- is not known with certainty. It is plausible that enzymes nents, 13-deoxydecarbamoyl derivatives, in some strains are involved in the transformation of the de novo toxin to of G. catenatum (Oshima et al. 1993b). A similar phe- various toxin components. nomenon was also observed among different strains of The profile of the toxins accumulated in shellfish A. minutum. Most of the isolate of A. minutum from vari- during a bloom of toxic dinoflagellates reflects that of ous areas show a characteristic toxin profile consisting the causative dinoflagellate. However, the toxin compo- of GTX1–4 (Oshima et al. 1989; Franco et al. 1994). In nents accumulated in the shellfish change after the causa- contrast, neoSTX, STX, and C toxins were detected in an tive dinoflagellates disappear from the environment isolate from New Zealand (Chang et al. 1997). Another, (Oshima 1995a). The shellfish themselves are not in- similar phenomenon was observed among clones of volved in biosynthesis of the toxins, and thus the toxin A. tamarense (=Protogonyaulax tamarensis) isolated components transferred from the dinoflagellates undergo from different areas of Japan (Oshima et al. 1982b). metabolic transformation in the shellfish. In an earlier It is noteworthy that nontoxic strains are often study, Shimizu and Yoshioka (1981) showed that trans- found in toxic species. Some strains of A. tamarense formation of PSP toxins occurs in the shellfish. They in- are reported to be nontoxic (Loeblich and Loeblich III cubated an homogenate of the scallop Patinopecten 1975). Oshima et al. (1993b) also reported that a clonal magellanicus contaminated with neoSTX and GTX2,3, culture of G. catenatum established from a cyst isolated and found that the proportion of STX increased after in- from Tasmania is nontoxic. These facts may indicate that cubation. These facts showed that neoSTX and GTXs are PSP toxins are not essential for the survival of these converted to STX in the shellfish. From these data, they dinoflagellates. Although little is known about the me- suggested the presence of enzymes in the shellfish which tabolism of toxins in dinoflagellates, some enzymes in- are involved in the transformation of toxin components, volved in toxin transformation have been reported. though little is known about such enzymes. The rate of Oshima (1995b) showed the presence of oxidase activity the transformation is very low indicating that enzyme

doi:10.5047/absm.2010.00301.0001 © 2010 TERRAPUB, Tokyo. All rights reserved. 8 M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010 activity in the scallop is low, if the reaction is enzymatic. Oshima (1995b) screened the enzyme activities which transform C toxins in the shellfish, and detected the activity to hydrolyze N-sulfocarbamoyl of C toxins to form dcGTXs in two species of clams, Mactra chinensis and Peronidia venulosa. Enzyme activity to hydrolyze carbamate toxins was also detected in some species of shellfish (Sullivan et al. 1983). However, the enzyme involved in the transformation of GTXs to STX has not been found in shellfish, though this transformation is observed generally in the shellfish.

6. Non-enzymatic transformation of PSP toxins: formation of unique intermediate conjugates of PSP toxins and thiols

As described above, GTXs accumulated in shellfish are gradually transformed to STXs. The rate of transformation is very slow (Oshima 1995a), suggesting that the reaction is not enzymatic. On the other hand, Kotaki Fig. 7. Change of toxin components and glutathione (GSH) in et al. (1985) reported that a part of GTXs incubated with the mixture of gonyautoxins (GTX) 2,3 and bacterial bacterial cells are were transformed to STXs, showing homogenates during incubation. GTX2,3 (10 µmol·L–1) was that these bacteria possess an ability to transform GTXs incubated with the homogenates at 30°C. Change of toxin to STXs. They also indicated that the factors involved in components in the homogenate with 0.1 M phosphate buffer, toxin transformation occur widely in the ecosystem. Thus, pH 7.4. (b) Change of toxin components in the homogenate we incubated GTXs in the water-soluble extract of a bac- with 0.1 M borate buffer, pH 7.4 containing 100 mM L-ser- ᭺ ᭹ terium Moraxella sp. (PTB-1) (Sakamoto et al. 2000) and ine. , GTX2,3; , saxitoxin; white bar, GSH level. PSP, paralytic shellfish toxins. Reprinted from Sakamoto et al., Forma- found that the extract prepared with 0.1 M phosphate tion of intermediate conjugates in the reductive transformation buffer (pH 7.4) often converted GTXs to STXs. How- of gonyautoxins to saxitoxins by thiol compounds. Reprinted ever, this transformation activity was not always observed, with permission from Fisheries Science, 66, Oshima et al., indicating that the activity of the extract is not stable. Features of paralytic shellfish poison occurring in Tohoku dis- However, once the activity was obtained from the bacte- trict. 136–141, Fig. 2, © 2000, the Japanese Society of Fisher- rium, it is very stable, even after heat treatment. These ies Science. facts indicate that the activity is not due to the enzyme. On the other hand, Asakawa et al. (1987) reported that glutathinone (GSH), a biological reductant widely occurring in organisms, transform GTX1, 2, 3 to STX. Bacte- the cells were homogenized (Fig. 7, lower). This level ria are known to possess a membrane-bound enzyme that also decreased slightly, but 0.21 mM of GSH remained degradates GSH (Nakayama et al. 1984). If the activity in the extract, even when the homogenate was incubated is due to GSH, a part of GSH seems to be degradated by for 24 h. These results show that GSH in bacterial cells is the enzyme during preparation of water-soluble extract easily decomposed by the GSH degradating enzyme dur- of the bacterium. Thus, the GSH levels in the extracts ing extraction and incubation, unless the extract was pre- with and without L-serine, the inhibitor of GSH pared in the presence of the inhibitor against the enzyme degradating enzyme, were analyzed. At the same time, (Sakamoto et al. 2000). transformation of GTXs to STXs was also analyzed Figure 7 also shows the change of GTX2,3 added (Sakamoto et al. 2000). to these homogenates during incubation. When GTX2,3 Figure 7 shows the change of GSH level in the ex- was incubated with the cell homogenate in the presence tracts prepared from the homogenates with and without of the inhibitor, more than 80% of GTX2,3 disappeared the inhibitor. The GSH level in the extract in the absence within 0.5 h, although no STX was detected (Fig. 7, of the inhibitor was 0.21 mM when the extract was pre- lower). After 1 h incubation, most of GTX2,3 disappeared pared just after the cells were homogenated (Fig. 7, up- and a trace amount of STX appeared. The level of STX per). However, this level decreased gradually when the increased gradually during further incubation and reached homogenate was incubated at 30°C. After 24 h incuba- 2.2 mM after 24 h incubation, showing that about 20% of tion, the level was decreased to 0.06 mM. In contrast, GTX2,3 added to the homogenate was transformed to GSH levels in the extract in the presence of the inhibitor STX. This phenomenon also suggests the formation of were 0.31 mM when the extract was prepared just after an intermediate compound which cannot be detected by

HPLC in the transformation reaction of GTX2,3 to STX (Sakamoto et al. 2000). When GTX2,3 was added to the homogenate without the inhibitor, about 20% of toxins also disappeared within 0.5 h (Fig. 7, upper). However, the level of GTX2,3 did not change during further incubation. No STX nor other known PSP toxin components were detected during incubation. These results show that the bacterial activity to transform GTX2,3 to STX is mainly due to GSH. Unstable activity of bacterial extract prepared in the absence of the inhibitor seems to be caused by degradation of GSH by the GSH degradating enzyme in the bacterial homogenate during extraction. Membrane-bound protein fraction prepared by 2-mercaptoethanol (ME) contain- Fig. 8. Change of toxin components in the mixture of ing a buffer also showed a significant activity to trans- gonyautoxins (GTX) 1,4 and glutathione (GSH) during incu- form GTX2,3 to STX. This activity was stable under heat- bation. GTX1,4 (4 µM) was incubated with 1 mL of 8 mM ᮀ ing and protease treatment, showing that it is not an GSH in 0.1 M phosphate buffer, pH 7.4, at 70°C. , GTX1,4 in the reaction mixture; ᭿, neoSTX in the reaction mixture; enzymatic reaction. The activity was also observed in the ᭺, GTX1,4 in the phosphate buffer as a control. Reprinted with extraction buffer, suggesting that it is the effect of 2-ME permission from Fisheries Science, 66, Sakamoto et al., For- contained in the buffer. mation of intermediate conjugates in the reductive transforma- Figure 8 shows the change of toxin components in tion of gonyautoxins to saxitoxins by thiol compounds. 136– the mixture of GTX1,4 and GSH during incubation at 141, Fig. 3, © 2000, the Japanese Society of Fisheries Science. 70°C in 0.1 M phosphate buffer (pH 7.4). GTX1,4 decreased markedly and disappeared from the mixture within 2 h. In contrast, neoSTX appeared at 30 min and gradually increased during incubation for 2 h to 35% of the amount of GTX1,4 added to the reaction mixture. GTX1,4 in the phosphate buffer decreased slightly after 12 h, but no neoSTX was detected by HPLC. Similar results were obtained when GTX2,3 was incubated with GSH. In this case, however, STX was formed instead of neoSTX. Figure 9 shows the change of toxin components in the reaction mixture of GTX1,4 and 2-ME during incubation at 70°C in the phosphate buffer (Sakamoto et al. 2000). In this case, most of GTX1,4 disappeared within 2 h while a small amount of neoSTX appeared. The amount of neoSTX increased gradually by 15% of the original amount of GTX1,4. Similar results were obtained Fig. 9. Change of toxin components in the mixture of from the reaction mixture of GTX2,3 and 2-ME except gonyautoxins (GTX) 1,4 and 2-mercaptoethanol (2-ME) dur- that STX was formed instead of neoSTX. ing incubation. GTX1,4 (4 µM) was incubated with 1 mL of 8 mM 2-ME in 0.1 M phosphate buffer, pH 7.4, at 70°C. These results show that thiol compounds transform ᮀ ᭿ GTXs to STXs. However, the sum of GTXs and STXs in , GTX1,4 in the reaction mixture; , neoSTX in the reaction mixture; ᭺, GTX1,4 in the phosphate buffer as a control. Re- the reaction mixtures was less than half of the original printed with permission from Fisheries Science, 66, Sakamoto amount of GTXs, showing that more than half of GTXs et al., Formation of intermediate conjugates in the reductive added to the reaction mixtures disappeared. This suggests transformation of gonyautoxins to saxitoxins by thiol com- that the conjugates of GTXs and thiol compounds are pounds. 136–141, Fig. 4, © 2000, the Japanese Society of Fish- formed in the course of the transformation. Thus, the 2 h eries Science. incubation mixtures of GTXs and GSH in which GTXs disappeared, were heated in 1 M 2-ME at 100°C for 10 min. HPLC analysis showed that almost 100% of the added GTXs was recovered as STXs, indicating that Table 2 summarizes the results of TLC of the GTXs and thiol compounds form conjugates and that ad- reaction mixture of GTXs and GSH before and after ditive molecules of thiol compounds are necessary to re- incubation. After incubation, spots of GTXs disappeared lease STX from the conjugates. and those of STXs appeared instead. In addition to these,

doi:10.5047/absm.2010.00301.0001 © 2010 TERRAPUB, Tokyo. All rights reserved. 10 M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010 spots different from those of toxin components and GSH STXs consists of a two-step reaction (Fig. 11). In the first appeared at Rf 0.10 in the mixture of GTX1,4 and GSH step, the sulfur atom of the thiols attacks the electrophilic and 0.04 in the mixture of GTX2,3 and GSH, respectively, C12 of GTXs to form a thiohemiketal, followed by the indicating the formation of the conjugates of GTXs and formation of stable thioether conjugates by a 1,2 shift. GSH. A spot corresponding to GSH became very faint This is an intramolecular reaction in which GTXs easily after incubation, suggesting that excess amounts of GSH react with a low concentration of thiols at a low tempera- were oxidized to GSSG during incubation which could ture. The second step is an intermolecular reaction be- not be detected by this system (Sakamoto et al. 2000). tween the conjugates and thiols. Therefore, it requires In a trial to purify the conjugate by chromatogra- more energy than the first-step reaction. In order to com- phy on Bio-Gel P-2, it was isolated in a pure form (Sato plete the second-step reaction within a short period, heat- et al. 2000). The high resolution FABMS and ing with an excess amount of thiols is necessary. Actu- NMR(HMBC) spectrum of the purified substances ally, the conjugates are formed, even in a solution of GTXs showed that the compounds are conjugates of neoSTX in a low concentration of thiols, such as 1 mM or lower at and thiols in which the C11 atom of neoSTX is covalently room temperature. However, only a trace amount of STXs bound with a sulfur atom of thiols (Fig. 10). When is obtained under this condition. In contrast, almost 100% GTX2,3 was incubated with thiols, the corresponding of STXs in the conjugates can be recovered when conju- conjugates of STX and thiols were obtained (Sato et al. gates dissolved in 1 M or higher concentrations of thiols 2000a). are heated for several minutes. There has been no report on the reductive elimina- Reduction of N1-OH type toxins to N1-H type tox- tion of sulfate ester by thiols. Thus, the transformation of ins was not observed in the reaction of GTXs and thiols. GTXs to STXs via the intermediate conjugates is consid- This fact does not coincide with the results of Asakawa ered to be a quite unique reaction. Formation of the con- et al. (1987) in which GSH transformed GTX1 to STX. jugates does not occur under acidic conditions. Further- In their experiments, they used partially purified toxins more, it was confirmed that GTXs-12-ol reported in toxin- containing GTX2,3 with a trace amount of GTX1. The producing cyanobacterium (Onodera et al. 1997) and a reaction mixtures were analyzed by electrophoresis. Prob- dinoflagellate species from tropical area (Lim et al. 2007), ably, most of GTXs in the reaction mixture formed con- analogues in which the gem diol at C12 is reduced, did jugates with GSH and disappeared from the mixture. The not react with thiols at all. These facts indicate that the amount of neoSTX derived from the conjugate was too characterstic keto-gem diol structure at C12 of the toxins low to be detected by electrophoresis. is essential for the reaction. When the equilibrium mix- Discovery of stable 11-thiol derivatives of STX is ture of α- and β-O-sulfate toxins is incubated with particularly important in terms of the metabolism of PSP thiols, the α-O-sulfate toxin is consumed faster than toxins. These findings show that STX derivatives are more the β-O-sulfate toxin in the formation of the conjugates, reactive than previously have been considered. The re- showing that α-O-sulfate toxin is a “reactive isomer” action between GTXs and thiols is useful for the de- (Fig. 11). velopment of biochemical probes such as antibody against Treatment of the conjugates with an excess amount PSP toxins which would facilitate the study of more de- of thiols results in the formation of disulfides and STX or tailed mechanisms of the reactions in which the toxins neoSTX, indicating that the transformation of GTXs to are involved.

Fig. 10. Structure of the intermediates in the transformation of GTX1,4 into neoSTX. Left: gluthathione-neoSTX adduct; right: 2-mercaptoethanol-neoXTX adduct. Reprinted from Bioorganic & Medical Chemistry Letters, 10, Sato, Sakai and Kodama, Identification of thioether intermediates in the reductive transformation of gonyautoxins into saxitoxins by thiols, 1787–1789, © 2000, with permission from Elsevier.

Fig. 11. Mechanism of reductive transformation of GTX1,4 mixture to neoSTX by thiol. Reprinted from Bioorganic & Medical Chemistry Letters, 10, Sato, Sakai and Kodama, Identification of thioether intermediates in the reductive transformation of gonyautoxins into saxitoxins by thiols, 1787–1789, © 2000, with permission from Elsevier.

7. Accumulation and depuration kinetics of PSP tox- ingested cells. At the same time, specimens of each ani- ins in the scallop fed A. tamarense mal were transferred to an aquarium with freshly prepared filtered seawater, to which was added the culture As described above, observation of the field survey of A. tamarense. The shellfish were then reared in the on shellfish toxicity in association with the abundance of same way. Specimens of each animal were thus fed on causative dinoflagellates suggests that the mechanism by A. tamarense once a day for eight days. After feeding which bivalves accumulate PSP toxins is more complex was completed, the specimens were further reared in the than those which have been considered based on the food same way without feeding for 12 days. During rearing, web transfer theory. However, it is almost impossible to three specimens of each animal were sampled every two understand how the shellfish accumulate and depurate days for the analysis of the toxins by HPLC. PSP toxins during a bloom of dinoflagellates from the All the animals ingested most of the cells fed within field data. Therefore, we examined this problem by the 24 h. More than 90% of toxin components of the cells kinetics of toxin accumulation in shellfish based on the consisted of C1,2 and GTX1,4, and this profile was al- feeding experiments of dinoflagellate to the shellfish. most constant throughout the feeding period. Figure 12 In a trial, interspecific difference in the ability to shows the change of toxin contents accumulated in each accumulate PSP toxins were examined by rearing five shellfish specimen in a molar base. The amount of each species of bivalves, scallop Patinopecten yessoensis, oys- toxin component is also shown in each bar. Average val- ter Crassostrea gigas, mussel Mytilus galloprovincialis, ues of accumulated toxins and those of incorporated toxin short-necked clam Ruditapes philippinarum, and ascidian amounts in a specimen calculated from the toxin con- Halocynthia roretzi (Sekiguchi et al. 2001a). The speci- tents and ingested cell number of A. tamarense, are also mens of each species were reared in separate tanks with shown in the figure. 500 L of seawater and fed a known number of cultured All the specimens became toxic, but the toxin level A. tamarense cells. After 24 h, the number of A. tamarense of the specimens showed remarkable individual variation, cells in the tank was counted to determine the number of even among the specimens sampled on the same day. To

Fig. 12. Toxin accumulation in four species of bivalves and an ascidian fed on cultured cells of Alexandrium tamarense. (a) Scallop, (b) mussel, (c) oyster, (d) short-necked clam and (e) ascidian. Reprinted with permission from Fisheries Science, 67, Sekiguchi et al., Accumulation of paralytic shellfish poisoning toxins in bivalves and an ascidian fed on Alexandrium tamarense cells, 301–305, Fig. 1, © 2001, the Japanese Society of Fisheries Science.

the contrary, no significant difference was observed in specimens sampled at the same stage of the experiment, toxin composition of the specimens sampled on the same the average toxin levels showed a trend toward increas- day. Toxin composition of each animal closely reflected ing during the feeding experiments. No significant dif- that of A. tamarense, that is, more than 90% of accumu- ference was observed in the maximum toxin levels among lated toxins consisted of C1,2 and GTX1,4. The propor- the mussel, the short-necked clam, and the oyster which tion of C1,2 to GTX1,4 was almost constant during the were fed on almost equal amounts of A. tamarense feeding experiment. These results indicate that there is (Fig. 12). However, the ascidian accumulated about no difference in the deterioration rate among the toxin twice more toxins than other bivalves, although they components, although C1,2 are reported to be rapidly ingested equal amounts of A. tamarense. Although eliminated over five days in the scallop Patinopecten scallop were fed on three time more than dinoflagellate, yessoensis exposed to a natural bloom of A. catenella in they accumulated nearly the same level of toxins as the which C2 was dominant (Oshima et al. 1990). The dif- ascidian. ference in the results between feeding experiments and After the feeding had been stopped, toxin levels of the field study may be due to the difference between natu- each species decreased. However, a considerable high ral and laboratory conditions. level was often observed in the scallop and the ascidian, After the feeding had been stopped, amounts of even after the cessation of feeding. These results show STXs (STX, neoSTX and decarbamoyl-STX) increased that accumulated toxins in these animals are hardly elimi- slightly. This trend was observed when the specimens nated. This observation coincided well with our previous were reared for a longer period. These changes in toxin results that scallops maintained a high toxin level for a profile were similar to those observed by Oshima et al. long time after the disappearance of the causative (1990). Although a marked individual difference was dinoflagellate in nature. observed in the accumulated toxin levels of the three

Many studies have been carried out to discover the kinetics of toxin accumulation of bivalves (Bricelj et al. 1990, 1991, 1996; Wisessang et al. 1991). In these studies, many shellfish specimens are kept in the same tank and fed on cultured cells of toxic dinoflagellates, and the shellfish toxicity was analyzed by using a homogenate of several specimens to minimize the error of individual differences. The current study unexpectedly revealed that bivalves showed considerable individual variations in toxicity. These differences are considered to be caused by different feeding behaviors among the specimens, probably due to the difference in feeding behavior owing to the sensitivity of handling or a sudden change of environment among the specimens. Thus, the feeding experiments should be designed to take feeding behavior of Fig. 13. Change in amounts of PSP toxins accumulated in scal- shellfish into consideration. lop specimens reared separately in individual tanks. The inte- grated amounts of toxins released from a single specimen and As handling of the shellfish during the experiments those supplied to each specimen by feeding Alexandrium was found to largely affect the feeding behavior of the tamarense cells are also shown. The sample was accidentally shellfish (Sekiguchi et al. 2001a), a single specimen of lost during analysis. Reprinted with permission from Marine shellfish was reared in a single tank, and the known Ecology Progress Series, 220, Sekiguchi et al., Accumulation amount of cultured cells of dinoflagellate was fed to the and depuration kinetics of paralytic shellfish toxins in the scal- shellfish specimen (Sekiguchi et al. 2001b). Shellfish lop Patinopecten yessoensis fed Alexandrium tamarense, 213– accumulate toxins by ingesting the toxic dinoflagellate. 218, Fig. 3, © 2001, Inter-Research. Occurrence of toxins in rearing water showed that a part of the toxins was released to the environmental water, even while ingesting the dinoflagellate. When the feeding stopped, the shellfish continuously excreted the tox- such as those with a cysteine residue are possibly involved ins. The profile of excreted toxins was similar to that ac- in the formation of the conjugates with the toxins. These cumulated in the shellfish, indicating that shellfish re- thiol-bound toxins possibly react with other thiols in the lease the toxin components non-selectively. shellfish and dinoflagellates to release toxins. Chemical Interestingly, the amount of toxins accumulated in transformation of the toxins observed in in vitro experi- the shellfish was not parallel to that of the dinoflagellate ments could be involved in the transformation in cells fed to the shellfish (Fig. 13). At the earlier period of dinoflagellates as well as in shellfish. These findings sug- the experiment when shellfish were ingesting the cells, gest that PSP toxins undergo a complex metabolism in the amount of toxins accumulated in the shellfish was shellfish as well as in dinoflagellates. more than the supplied toxins from dinoflagellates fed to the shellfish. In the later period when the feeding was 8. Change of toxin productivity in Alexandrium cells stopped, the sum of the toxins in the shellfish and rearing water had decreased to a level which was less than that There are various data on the toxicity of different introduced from the dinoflagellates cells, showing that strains of A. tamarense isolated from various regions of some of the toxins disappeared from the experimental the world (White and Maranda 1978; Oshima and system. However, this level recovered to almost the same Yasumoto 1979; Kodama et al. 1982a; Singh et al. 1982; level as that derived from the fed cells of the Boyer et al. 1985; Ogata et al. 1987a). As the toxicity of dinoflagellate, when they were further reared. These facts A. tamarense is affected by physiological or environmen- indicate that a part of the toxins was transformed to an tal factors, these data cannot be directly compared. How- unknown form in the shellfish which could not be de- ever, the toxicities of these strains seem to differ from tected by chemical analysis such as HPLC applied in our each other. White (1986) reported the high toxin content study. The unknown form of the toxins is gradually trans- of A. tamarense in nature. He also demonstrated that the formed to toxins again in the shellfish which can be de- toxicity of the cell in nature fluctuated during the bloom, tected by chemical analysis. The unexpected high level suggesting that the toxicity of the cells is different in dif- of toxins accumulated in the shellfish, more than the fed ferent water masses, even if they bloom in the same area amount of toxins, may show that an unknown form also during the same period. Alternatively, these data suggest occurs in the dinoflagellate fed to the shellfish. Thiol com- that the toxicity differs in different clones from one an- pounds of biological origin, such as GSH, are thought to other. Therefore, we examined the toxicity of various be involved in the appearance and disappearance of the clones isolated from the same area during the same sea- toxins in shellfish and dinoflagellate. Proteinous thiols son, by culturing them under the same conditions (Ogata

Fig. 14. Toxicity of various clones of Alexandrium tamarense Fig. 15. Toxicity of subclones from a single clone of as a function of growth rate. These clones were grown at 15°C Alexandrium tamarense (OF84423D-3) as a function of growth under 3000 lux in medium T1 (Ogata et al. 1987b). Toxicity is rate. These subclones were grown at 15°C under 3000 lux in expressed in mouse unit (MU) according to Horwitz (1984) medium T1. The toxicity (MU) and the growth rate (µ2) are as where 1 MU represents the dose to kill a 20 g male mouse within defined as in Fig. 14. Reprinted from Toxicon, 25, Ogata,

15 min. The growth rate (µ2) is estimated from cell counts dur- Kodama and Ishimaru, Toxin production in the dinoflagellate ing the exponential phase according to the method of Fukazawa Protogonyaulax tamarensis, 923–928, © 1987, Pergamon Jour- et al. (1980). Reprinted from Toxicon, 25, Ogata, Kodama and nal Ltd. with permission from Elsevier. Ishimaru, Toxin production in the dinoflagellate Protogonyaulax tamarensis, 923–928, © 1987, Pergamon Journal Ltd. with permission from Elsevier. tary character but an acquired one, unless mutation occurred during the culture experiments. In other words, the information on toxin production is not coded in the et al. 1987a). Figure 14 shows the toxicity plotted genes of A. tamarense. against their growth rate (µ2) for various clones of These experiments were carried out within one year A. tamarense which were grown under the same condi- after the isolation of the clone from which subclones were tions. The data points are randomly scattered, showing prepared. Formation of the zygote cannot occur during that the growth characteristics and toxin production were culture because of the heterothallism of A. tamarense different, depending on the clone. The maximum differ- (Turpin et al. 1978; Anderson 1980; Yoshimatsu 1992), ences in toxicity and growth rate were about 100- and 4- indicating that the genetic type of each subclone was iden- fold, respectively. HPLC-analysis of the toxin components tical during our experiments. If mutation occurred, there revealed that these clones had toxin profiles similar to should have been some groups of clones with different each other. These results indicate the occurrence of vari- characteristics. However, the toxicities of subclones were ous clones of A. tamarense which possess different growth different from each other, suggesting that the variation in characteristic and toxin productivity. This can be ex- the toxicity among the subclones is unlikely because of plained by the heterothallism of A. tamarense (Turpin mutation. et al. 1978; Anderson 1980; Yoshimatsu 1992). Accord- In conclusion, these results suggest that toxin pro- ing to this theory, toxin productivity as well as the growth duction of A. tamarense is not a hereditary characteris- rate of A. tamarense could be inheritance. tic, i.e. the information on toxin production is not In order to know the heredity of the toxin pro- coded in the genes of A. tamarense. This idea means ductivity of A. tamarense, we established 20 subclones that toxin production in A. tamarense is an acquired char- from a single clone isolated from Ofunato Bay. These acteristic. subclones were grown under the same conditions, as Recently, two groups reported separately the same described above, and the toxicity and growth rate were results on nontoxic subclones obtained from a toxic sin- analyzed. In Fig. 15, the toxicities of the subclones are gle clone of Alexandrium. Martins et al. (2004) described plotted against their growth rate. Most of the subclones that possible explanations for the change include genetic showed a similar growth rate. In contrast, their toxicity mutations or the effects of prolonged treatment of the toxic varied significantly. The maximum difference in toxicity culture with antibiotics. Omura et al. (2003) describe that of the subclones was about 20 fold. These results suggest they prepared many subclones from non-axenic parent that the toxin production of A. tamarense is not a heredi- culture of A. tamarense obtained from a single cell

doi:10.5047/absm.2010.00301.0001 © 2010 TERRAPUB, Tokyo. All rights reserved. M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010 15 collected from Ofunato Bay in the same way as described above. Among these subclones, about half were non-toxic. They proposed that this ratio is too large to explain that loss of toxin production in subclones is caused by mutation. Some external factors may be involved in the toxin productivity of toxic dinoflagellates. No significant difference was observed in growth and other physiological characteristics between toxic and non-toxic clones. These facts suggest that PSP toxins are not essential for the survival of these dinoflagellates, but rather are foreign substances for them.

9. Bacterial production of saxitoxin Fig. 16. Identification of the bacterial toxin as saxitoxin by HPLC-fluorometric analysis. A, saxitoxin standard (0.2 MU); It has been described above that a large difference B, bacterial toxin (0.3 MU). Reprinted from Agricultural and is observed in the toxicity among various subclones ob- Biological Chemistry, 52, Kodama et al., Bacterial production tained from a single clone. This finding strongly suggests of saxitoxin, 1075–1077, © 1988, the Agricultural Chemical that toxin production of A. tamarense is not a hereditary Society. characteristic. Bacterial symbiosis or infection is commonly observed in higher plants. Thus, it is plausible that the bacteria are involved in toxin production of A. tamarense. including the A. lusitanicum strain noted above (Gallacher A strongly toxic clone of A. tamarense was treated et al. 1997), using the capillary electrophoresis-mass with a mixture of antibiotics to obtain the axenic culture. spectrometry (CE-MS) method. The culture was tested for the presence of bacteria and On the other hand, Silva (1979) presented the hy- confirmed that the culture medium was bacterium-free. pothesis that dinoflagellate toxicity is due to intracellular A. tamarense cells were harvested from thus-obtained bac- bacteria. We also suggested that toxin production by teria-free culture, washed several times with sterilized A. tamarense is not of hereditary characteristics (Ogata seawater, lightly homogenized, and then inoculated onto et al. 1987b). The finding of toxin-producing bacterium agar plates of a nutrient medium. After incubation for in the cells of A. tamarense support both Silva’s hypoth- several days, bacterial colonies were observed on the esis and our previous results. The toxin production by plates. The obtained bacterial strain was mass-cultured our bacterium was considerably lower than that by and bacterial cells were extracted for toxin analysis. As A. tamarense, suggesting that symbiosis or parasitism the toxicity was detected by mouse bioassay, the extract with A. tamarense enhances the toxin production by this containing crude toxin was prepared from more than bacterium. In addition, only STX could be detected in 70 L of culture. The toxic substance was isolated by us- Moraxella sp., whereas the main toxin components of ing several types of chromatographies. The purified sub- A. tamarense are GTXs (Ogata et al. 1987a, b). Thus, the stance was indistinguishable from authentic STX in the toxin production of Moraxella sp. under various condi- analysis by different types of chromatographies such as tion was surveyed. As shown in Fig. 17, Moraxella sp. in TLC, electrophoresis, and HPLC for PSP toxins (Kodama shaking culture grew faster than those in standing or ro- et al. 1988). Typical results from HPLC analysis are tatory culture at 25 and 37°C. Figure 18 shows time course shown in Fig. 16. The peak height in HPLC to the mouse of toxicity in the cell extract of Moraxella sp. grown un- toxicity ratio also coincided well with that of standard der various culture conditions. No significant toxicity STX, showing that the obtained bacterium produces STX. was observed in any culture during 64 h. At 25°C, it This toxigenic bacterium (PTB-1) was tentatively identi- increased during 24 h, whereas it decreased after 24 h. fied as “Moraxella sp.” [later classified as a Pseudomonas/ The cells shaken in Tryptic-Soy Broth (TSB; BBL Co. Alteromonas species (Doucette and Trick 1995)] and most Ltd. Tokyo) at 25°C also showed weak toxicity at 64 h recently as a new genus within the α-proteobacteria which disappeared after 168 h incubation. On the other (Kopp et al. 1997). Doucette and Trick (1995) and hand, Moraxella sp. showed considerable toxicity in all Franca et al. (1995) employed HPLC methods to exam- the culture conditions when cultured in Marine Broth ine toxin production characteristics of Moraxella sp. and (MB; Difco) at 25°C for 168 h. another bacterium obtained from the toxic dinoflagellate Inoculates of Moraxella sp. in 1% MB and seawater A. lusitanicum. Both groups reported PSP toxins in bac- became slightly turbid after 10 days incubation, showing terial cell extracts. Gallacher and co-workers have made that the bacterium grew only slightly in these media. an important step in confirming the synthesis of PSP tox- Figure 19 illustrates HPLC chromatograms of the extract ins by bacteria isolated from five Alexandrium cultures, of Moraxella sp. grown in the seawater for 10 days. Peaks

Fig. 17. Growth of Moraxella sp. Under various culture conditions. Moraxella sp. Was cultured in Heart Infusion (᭺), Brain Heart Infusion (᭹), Tryptic-Soy Broth (᭝), CAYE medium containing 1% Casamic acid and 1% yeast extracts (᭡), and Marine Broth (᭿) at 25 and 37°C by standing, shaking and rotatory culture methods. Upper, 25°C, Lower 37°C; A, standing culture; B, shaking culture; C, rotatory culture. Reprinted from Toxicon 28, Kodama et al., Production of paralytic shellfish toxins by a bacterium Moraxella sp. isolated from Protogonyaulax tamarensis. 707–714, © 1990, Pergamon Press plc. with permission from Elsevier.

Fig. 18. Time course of toxin production by Moraxella sp. under various conditions. The toxicity of cells grown under various conditions (refer to Fig. 17) was assayed by mouse neuroblast cell culture method (Kogure et al. 1988; Sato et al. 1988). Upper, 25°C; Lower, 37°C; A, standing culture; B, shaking culture; C, rotatory culture. Reprinted from Toxicon 28, Kodama et al., Production of paralytic shellfish toxins by a bacterium Moraxella sp. isolated from Protogonyaulax tamarensis. 707–714, © 1990, Pergamon Press plc. with permission from Elsevier.

Fig. 20. Changes of total toxin amount during culture. ᭹: cul- Fig. 19. HPLC-fluorometric analysis of the extract from ture in 1% Marine Broth; ᭺: culture in natural seawater. Re- Moraxella sp. cells cultured in natural seawater for 10 days at printed from Toxicon 28, Kodama et al., Production of para- 20°C, using a develosil C8 column according to the method of lytic shellfish toxins by a bacterium Moraxella sp. isolated from Oshima et al. (1987). Mobile phase in (A) and (C) is 2 mM Protogonyaulax tamarensis. 707–714, © 1990, Pergamon Press 1-heptanesulfonic acid in 10 mM phosphoric acid (pH 7.2). plc. with permission from Elsevier. Mobile phase in (B) and (D) is 2 mM 1-heptanesulfonic acid in 10 mM phosphoric acid containing 10% acetonitrile (pH 7.2). (A), GTX standards: GTX1 (5 pmoles); GTX2 (1.3 pmoles); GTX3, (0.5 pmoles); GTX4, (12 pmoles). (B): neoSTX and These results show that Moraxella sp. required so- STX standards; neoSTX, (9 pmoles); STX, (5 pmoles). (C) and dium chloride for its growth and grew best in shaken cul- (D): extract of Moraxella sp. (10 µL of extract corresponding to cells in 5 mL of culture). Reprinted from Toxicon 28, Kodama tures, indicating that it is an aerobic marine heterotrophic et al., Production of paralytic shellfish toxins by a bacterium bacterium. This fact is also supported by stable growth Moraxella sp. isolated from Protogonyaulax tamarensis. 707– under various conditions in MB which is prepared from 714, © 1990, Pergamon Press plc. with permission from artificial seawater. However, during 64 h, it did not pro- Elsevier. duce significant amount of toxin under these culture conditions. Only cells of the standing culture in Heart Infu- sion (HI) at 25°C showed significant toxicity although the culture grew poorly under this condition. Interestingly, corresponding to GTX1,4 followed by those of GTX2,3 once produced, toxin often disappeared during incuba- appeared. A faint peak corresponding to neoSTX was also tion. Although it is poorly understood about the metabo- observed. These results show that main toxin components lism of PSP toxins, these findings suggest that the toxin of Moraxella sp. grown under these conditions are simi- is an intermediate which is metabolized to non-toxic lar to the GTXs. Figure 20 and Table 3 show the changes substance(s). Chemical reactions between GTXs and bio- of total toxin amounts per 1 L of culture and proportion logical thiols such as GSH are considered to be involved of toxin components which were calculated from the re- in these phenomena. On the other hand, the toxin appeared sults of HPLC-fluorometric analysis. In the seawater, in MB cultures which were grown for 168 h at 25°C. The Moraxella sp. produced PSP toxins within 24 h, how- cells at 168 h incubation should be under a starved ever, the amount was low for one week, but increased condition, because they were left under a nutrition de- considerably by the 10th day. The pattern of toxicity ficient environment for more than 100 h. In order to ob- change in 1% MB was similar to that in the seawater, tain energy for survival under starved conditions, although the toxicity did not increase as in the seawater. Moraxella sp. possibly catabolizes its biological Throughout the incubation period the toxin profile substance(s). PSP toxins might be a kind of catabolites changed (Table 3). However, the main components were of the metabolism (Kodama et al. 1990b). always similar to GTXs, especially GTX1,4. In the ex- treme cases, most of the toxin was GTX1,4. STX and 10. Bacteria in the cells of toxic dinoflagellates neoSTX were scarcely observed. These findings suggest that transformation of toxin components occurs in Several studies indicate that axenic, or bacteria-free, Moraxella sp. dinoflagellate cultures retain the ability to produce these

Table 3. Toxin composition of Moraxella sp. cultured seawater and 1% marine broth.

Toxin composition* Duration GTX1 GTX2 GTX3 GTX4 neoSTX STX Culture medium (days) mole % Natural seawater 1 41.0 11.8 2.7 44.5 — — 2 25.6 8.0 2.2 64.2 — — 3 100.0 — — — — — 4 50.5 8.7 1.8 28.7 — — 5 100.0 — — — — — 6 70.5 23.7 5.8 — — — 7 56.7 — — 43.3 — — 10 10.0 0.5 1.4 87.3 0.7 —

1% Marine Broth 1 28.1 — 2.5 69.4 — — 2 20.3 6.2 1.8 71.7 — — 3 37.0 — — 63.0 — — 4 1.0 0.1 3.4 95.5 — — 5 — — — 100.0 — — 6 34.8 4.4 — 60.8 — — 7 90.7 9.3 — — — — 10 40.7 10.4 1.6 47.3 — —

*Toxin composition was calculated by comparing peak heights of samples against standard PSP toxins and expressed as mole %. This article was published in Toxicon, 28, Kodama, Ogata, Sakamoto, Sato, Honda and Miwatani, Production of paralytic shellfish toxins by a bacterium Moraxella sp. isolated from Protogonyaulax tamarensis, 707–714, © Elsevier (1990).

toxins at a level similar to those found in nonaxenic cultures (e.g. Kim et al. 1993). If bacteria are involved in toxin production in dinoflagellates, they should be present within the dinoflagellates. Several investigators including us have reported the direct observation of intracellular bacteria in toxic species of dinoflagellates by electron microscopy (Silva 1979; Kodama and Ogata 1988; Franca et al. 1996), although the bacterial numbers were generally few and such bacteria were not always discernible Fig. 21. Western blot analysis of the extract of Alexandrium even with cells of the same algal culture. Although toxi- tamarense prepared by a Teflon homogenizer using antigenic bacteria could be isolated from toxic dinoflagellates, Moraxella sp. antibody. A: extract of Moraxella sp., B: extract it was not clearly proven whether the isolated bacterial of A. tamarense. Reprinted from Kodama et al., Symbiosis of strains and the corresponding intracellular bacteria were bacteria in Alexandrium tamarense. In: Yasumoto, Oshima and Fukuyo (eds). Harmful and Toxic Algal Blooms. Intergovern- the same. mental Oceanographic Commission of UNESCO, Paris. 1996, In order to know the presence of intracellular bac- pp. 351–354, Fig. 1. teria in the toxic A. tamarense, we used an antibody against a toxin-producing bacterium to perform Western blots of an extract from the “host,” an A. tamarense isolate (axenic culture) (Kodama et al. 1996a). Increased some of which corresponded with those of Moraxella sp. cross reactivity was observed after the apparent disrup- extract, suggesting the presence of protein components tion of residual (=accumulation) bodies present within of Moraxella sp. in A. tamarense cells. However, the the algal cells. Figure 21 shows the results from western number of the bands was too small to show clearly the blot analysis using antibody against Moraxella sp. and presence of bacterial protein components in A. tamarense the extract of A. tamarense cells which was extracted cells. As the protein components were considered to be mildly with phosphate buffered saline (PBS) using a not sufficiently extracted, the homogenate to prepare the Teflon homogenizer. Several faint bands were observed, extract was observed under light microscope. As shown

in Fig. 22, most of the cells were disrupted, but many intact organelle were observed. These organelle showed fluorescence when activated by ultraviolet light, showing that the organelle were hard to be homogenized by the method applied. Therefore, homogenate of A. tamarense was sonicated for 1, 5 and 10 min in phosphate buffered saline (PBS) in the same buffer containing 2% SDS. Figure 23 shows the observation of the homogenates under fluorescent microscope. Although most of the cells were disrupted by 1 min sonication, many fluorescent organelle were not disrupted by the procedure in both buffers. Significant number of these organelle were still observed even after 10 min sonication. However, the number decreased considerably, showing that they were disrupted as the sonication time became longer (Fig. 23). SDS was effective for the disruption of the organelle. Western blot analysis of the extracts prepared from these homogenates showed that the stain- ing of the bands with antibody against Moraxella sp. became stronger as the sonication time of the cells in SDS- containing buffer for longer time was effective to extract the fluorescent organelle. Therefore, a large number of the cells (5 × 107 cells) were extracted with 0.5 mL of 1.25 M tris containing 10% SDS to prepare the concen- Fig. 22. Microscopic observation of the homogenate prepared trated extract. The extract thus obtained showed clear by a Teflon homogenizer. Upper: light microscopic observa- bands corresponding to almost all the protein components tion; lower: fluorescent microscopic observation. Reprinted of Moraxella sp. in western blot analysis (Fig. 24). These from Kodama et al., Symbiosis of bacteria in Alexandrium tamarense. In: Yasumoto, Oshima and Fukuyo (eds). Harmful results show that protein components which reacted with and Toxic Algal Blooms. Intergovernmental Oceanographic antibody against protein extracts of Moraxella sp. are Commission of UNESCO, Paris. 1996, pp. 351–354, Fig. 2. contained in these fluorescent organelle. A. tamarense

Fig. 23. Fluorescent microscopic observation of homogenates prepared under different conditions. Upper: Cells were sonicated for 1, 5, 10 min in SDS-containing buffer; lower: Cells were sonicated for 1, 5, 10 min in PBS. Bar: 100 µm. Reprinted from Kodama et al., Symbiosis of bacteria in Alexandrium tamarense. In: Yasumoto, Oshima and Fukuyo (eds). Harmful and Toxic Algal Blooms. Intergovernmental Oceanographic Commission of UNESCO, Paris. 1996, pp. 351–354, Fig. 3.

Fig. 24. Western blot analysis of the extract of Alexandrium tamarense prepared by sonication in SDS-containing buffer. A: extract of Moraxella sp.; B: extract of A. tamarense. Re- printed from Kodama et al., Symbiosis of bacteria in Alexandrium tamarense. In: Yasumoto, Oshima and Fukuyo (eds). Harmful and Toxic Algal Blooms. Intergovernmental Oceanographic Commission of UNESCO, Paris. 1996, pp. 351– 354, Fig. 4. Fig. 25. Microscopic observation of Alexandrium tamarense cell. Left: light microscopic observation; right: fluorescent microscopic observation. Reprinted from Kodama et al., Sym- biosis of bacteria in Alexandrium tamarense. In: Yasumoto, clone used in the present study was grown and main- Oshima and Fukuyo (eds). Harmful and Toxic Algal Blooms. Intergovernmental Oceanographic Commission of UNESCO, tained for more than a year under axenic condition. No Paris. 1996, pp. 351–354, Fig. 5. bacteria were observed in the cell-free medium of the clone in monthly test for detection of bacteria. There- fore, the presence of bacterial substances in the extract of A. tamarense clone implies that bacteria are living in the cells, probably in the fluorescent organelle. axenic condition in which no bacteria are supplied from Figure 25 shows the light and fluorescent the environment, a part of bacteria growing in the lyso- microscopy of A. tamarense cell harvested at late station- some-like organelle are considered to be digested in the ary phase. Autofluorescent organelle is seen under the organelle to provide an organic substance to A. tamarense nucleus. The organelle showed blue-white fluorescence for its growth. The relation between A. tamarense and under UV light excitation, green fluorescence under blue endocellular bacteria may be mutualism. light excitation with a red cut-off filter and yellow with- These results and the facts that bacteria could be out the red cut-off. These characteristics are similar to isolated from an axenic culture of this dinoflagellate only those of autofluorescent organelle called PAS/accumula- after disruption of the residual bodies, led us to contend tion bodies observed in other dinoflagellates which are that intracellular bacteria exist within the residual bodies considered to be lysosome. Franca et al. (1995) and and these retain the ability to reproduce within the algal Mascarenhas et al. (1995) have suggested that residual cells. This idea is consistent with electron microscopic bodies of A. lusitanicum which seem to be the same as observations showing partially digested bacteria in the PAS/accumulation bodies, contain remnant of bacteria. residual bodies of several toxic dinoflagellates (Franca They observed living bacteria in the space between the et al. 1995). The fact that apparently stable association of outer cellular membrane and the thecal complex, which certain bacteria with an algal isolate is observed over many seem to originate from environment. From these facts, years also support the idea. Generally, bacteria responsi- they suggested that incorporated bacteria are trapped by ble for infection are killed by defense mechanisms of the food vacuole and transferred to the residual body to be host organism. However, some specific infectious bac- digested. The digested bacteria might be a nutritive source teria are known to possess escape mechanisms allow- for Alexandrium. On the contrary, the results of the present ing them to survive within cells of the host (Groisman study show that bacteria are living in the Alexandrium 1992). It is hypothesized that the growth of such bacteria residual body. Therefore, a part of the bacteria incorpo- inside residual bodies may be controlled by digestive proc- rated into the inside the cells seem to survive and grow in esses, thereby harmonizing bacterial growth with that of the organelle while others are digested in the organelle. the dinoflagellate and allowing extended maintenance of It is well known that organic substances such as soil ex- the intracellular-bacterial flora. tracts and vitamins are essential for the growth of Alexandrium. We suggest that A. tamarense can grow by 11. Utilization of organic substances for growth and utilizing bacteria as well as organic substances under limi- toxin production by Alexandrium tamarense tation of nutrient or light intensity (Ogata et al. 1996). These findings suggest that Alexandrium spp. require or- There have been many dinoflagellate species re- ganic substances which originate from bacteria. Under ported to possess a heterotrophic behavior such as

T1 and SWII medium (nitrate; 0.71 mM) (Iwasaki 1961) were used for unialgal and axenic cultures, respectively. Semi-continuous culture was established by repeating dilution of culture with an equal volume of fresh medium when the cell density increased to the double. An aliquot of the cells was taken to measure cell density under a

microscope. The growth rate (µ2) was estimated by the method of Fukazawa et al. (1980). Toxin contents of the cultured cells were analyzed by HPLC according to Oshima (1995b). As shown in Fig. 26, the semi-con- Fig. 26. Growth curve of Alexandrium tamarense cultured tinuous culture acclimated to N-limited condition was es- semicontinuously in N-limited T1 medium in which nitrate level tablished by cultivating A. tamarense in N-limited T1 was reduced to 2% of the normal medium. A half volume of medium in which nitrate was reduced to 2% of the nor- culture was diluted with equal volume of fresh medium suc- mal medium. During the first 30 days, the growth of cessively when the cell density increased to double. Reprinted A. tamarense was unstable, but it showed stable growth from Ogata et al., Utilization of organic substances for growth with growth rate of 0.25 division/day after 30 days. The and toxin production by Alexandrium tamarense. In: Yasumoto, Oshima and Fukuyo (eds). Harmful and Toxic Algal Blooms. culture maintained exponential phase, showing that cells Intergovernmental Oceanographic Commission of UNESCO, grew continuously under N-limited condition. As shown Paris. 1996, pp. 343–346, Fig. 1. in Fig. 27, toxin content of the cell dropped to about 1/10 that of those in the initial culture cultivated in normal T1 medium as indicated previously (Boyer et al. 1987; Anderson et al. 1980). When nitrate (1.0 mM) or yeast auxotrophy or phagotrophy (Gaines and Elbrachter extract (DIFCO; 20 mg·L–1) was added to the culture (in- 1987). However, dinoflagellates which produce PSP dicated by arrows in Fig. 27), the cells grew well. The toxins such as A. tamarense have until now been regarded maximum yield of the culture increased remarkably in as photosynthetic organisms. Therefore, their growth both cases. At the same time, toxin production of the cells physiology and toxin production have been studied was enhanced to almost 5 times by these treatments. Ni- from the standpoint that they are autotrophic organisms. trate and ammonia in yeast extract used here were meas- As described above, we gave a clear evidence that ured to be 0.055 and 1.095 µmol·g–1, respectively, be- photosynthesis is necessary for the toxin production of ing too low to explain the growth after the addition of A. tamarense (Ogata et al. 1987b). The fact also supports yeast extract. These results show that A. tamarense can the autotrophic character of A. tamarense. On the other use organic N-substance in yeast extract as the nitrogen hand, Alexandrium spp. require some organic substances source instead of nitrate. such as vitamins (Iwasaki 1979), indicating that they also Figure 28 also shows the effect of bacterium, possess heterotrophic characters. However, little is Moraxella sp., on the growth and toxin production of known about this process on these species. We found that A. tamarense cultured under N-limited condition semi- A. tamarense possesses bacteria in the cells and that the continuously. At the dilution of the culture in the maxi- isolated bacteria produce toxins, though the productivity mum density, 107 cells of live or autoclaved Moraxella is low (Kodama et al. 1988). Based on these results, we sp. cells were added with 100 mL of the fresh medium. propose a hypothesis that these bacteria are necessary for The maximum cell number of A. tamarense increased A. tamarense to produce toxins. Thus it is based on a significantly by addition of autoclaved bacterial cells, premise that A. tamarense utilizes organic substance. Such as in the case of yeast extract. This increase in growth heterotrophic character is considered to be also impor- was repeatedly observed. Toxin production of cells tant to understand the growth of these species in the natu- was also induced by this treatment. In contrast, growth ral environment. Despite numerous reports on the rela- of A. tamarense became lower by the addition of live tionship between the occurrence of Alexandrium spp. and Moraxella sp. cells. However, when the addition of bac- environmental factors such as inorganic nutrient concen- terial cells was repeated, A. tamarense started to grow trations, it seems difficult to correlate the growth of this well as in the case of autocraved cells (0.39 division/day). dinoflagellate solely to these factors. The utilization of Toxicity of cells also increased gradually by the treat- organic substance(s) of A. tamarense was studied in rela- ment. These results indicate that A. tamarense also pos- tion with growth and toxin production of this species sess phagotrophic characters and utilize environmental (Ogata et al. 1996). bacteria for their growth and toxin production. As men- The unialgal and axenic cultures were main- tioned above, Franca et al. (1995) observed bacteria in tained under irradiation of 60 µmol photons·m–2·s–1 the space between the outer cellular membrane and the (cool-white fluorescent lamps) with 16 h:8 h LD cycle. thecal complex, and suggested that these bacteria might

Fig. 27. Change of growth (lower) and toxin production (upper) of Alexandrium tamarense after addition of nitrate (1.0 mM) or yeast extract (20 mg·L–1) under N-limited condition. Nitrate and yeast extract were added at a time indicated by arrow. N: Culture supplemented with nitrate, Y: Culture supplemented with yeast extract, B: Control culture. Reprinted from Ogata et al., Utilization of organic substances for growth and toxin production by Alexandrium tamarense. In: Yasumoto, Oshima and Fukuyo (eds). Harmful and Toxic Algal Blooms. Intergovernmental Oceanographic Commission of UNESCO, Paris. 1996, pp. 343–346, Fig. 2.

Fig. 28. Change of growth (lower) and toxin production (upper) of Alexandrium tamarense after addition of autoclaved (AM) or live (LM) Moraxella sp. cells under N-limited condition. Bacterial cells were added at times indicated by arrows. Reprinted from Ogata et al., Utilization of organic substances for growth and toxin production by Alexandrium tamarense. In: Yasumoto, Oshima and Fukuyo (eds). Harmful and Toxic Algal Blooms. Intergovernmental Oceanographic Commission of UNESCO, Paris. 1996, pp. 343–346, Fig. 3.

be a nutritive source of the dinoflagellate. Our results grow by utilizing organic substances such as yeast ex- support their observations. tract, if they are available, even when it can not grow Previously, we have reported that de novo synthe- autotrophically under low light condition. sis of toxins in A. tamarense is repressed under low Cultures used in these experiments were not axenic. light condition (Ogata et al. 1987b). Therefore, the ef- It could be possible that bacteria contaminating the cul- fect of organic substance on the toxin production un- ture decomposed the added organic substances to inor- der low light condition was examined. As shown in ganic nutrients which are available for A. tamarense. Fig. 29, cell division of A. tamarense stopped at mid- Therefore, the effect of organic substance on the growth exponential phase by lowering light intensity from 60 was examined by using an axenic culture. As shown in to 5 µmol photons·m–2·s–1. No significant toxin produc- Fig. 30, the axenic strain grew well in SWII medium with tion was observed during 4 days under this condition. the maximum yield of 8 × 104 cells·mL–1. Cells grown When the yeast extract (20 mg·L–1) was added to the cul- in N-depleted SWII medium (SWII-N) showed a simi- ture at the time of lowering light intensity, cells were ob- lar growth, but the maximum yield was much depressed served to keep growing though the growth rate became to 2 × 103 cells·mL–1. When the culture was spiked with lower. During the period, the toxin contents of cell in- nitrate (1.0 mM) at the stationary phase, N-limited cul- creased. These results suggest that A. tamarense can ture induced remarkable cell division, resulting in the re- covery of cell yield of 8 × 104 cells·mL–1. Growth of the cells in SWII-N was also induced by the addition of yeast extract (20 mg·L–1), though the growth rate and cell yield were lower than those of nitrate addition. These results clearly show that A. tamarense is mixotrophic organism. It can grow autotrophically under the conditions suitable for photosynthesis. When the environment is not suitable for photosynthesis, it can grow using energy

Fig. 29. Changes of growth (lower) and toxin production (up- Fig. 30. Effect of supplemented yeast extract on the growth of per) of Alexandrium tamarense by addition of yeast extract axenic Alexandrium tamarense cells. Cells were grown in SWII (20 mg·L–1) under light limited condition. Culture was divided medium (ᮀ), N-depleted SWII (᭡), SWII supplemented with to two parts after lowering light intensity at mid-exponential nitrate (1.0 mM) (᭜), and SWII supplemented with yeast ex- phase. A: Control culture, B: culture supplemented with yeast tract (20 mg·L–1) (᭺). Nitrate and yeast extract were added at extract at the time indicated by arrow. Reprinted from Ogata et the time indicated by arrow. Reprinted from Ogata et al., Utili- al., Utilization of organic substances for growth and toxin pro- zation of organic substances for growth and toxin production duction by Alexandrium tamarense. In: Yasumoto, Oshima and by Alexandrium tamarense. In: Yasumoto, Oshima and Fukuyo Fukuyo (eds). Harmful and Toxic Algal Blooms. Intergovern- (eds). Harmful and Toxic Algal Blooms. Intergovernmental mental Oceanographic Commission of UNESCO, Paris. 1996, Oceanographic Commission of UNESCO, Paris. 1996, pp. 343– pp. 343–346, Fig. 4. 346, Fig. 5.

doi:10.5047/absm.2010.00301.0001 © 2010 TERRAPUB, Tokyo. All rights reserved. 24 M. Kodama / Aqua-BioSci. Monogr. 3: 1–38, 2010 heterotrophically supplied. It is striking that A. tamarense the shellfish toxicity during the period seemed to be due produces toxins even when it grows heterotrophically. to the toxins associated with these particles. However, This fact is very important to understand the blooming shellfish toxicity did not increase remarkably, although mechanism of Alexandrium species. high levels of STXs were detected in the fractions con- Although a number of survey have been conducted taining particles with a size smaller than dinoflagellates. on the environmental factors for the growth of natural It seems quite strange that shellfish toxicity increased sig- population of A. tamarense, little is known about their nificantly as A. catenella grew to high density; yet no blooming mechanisms. In the field survey in Ofunato Bay, significant amount of toxin was detected in A. catenella the bloom of A. tamarense was often observed under the (Sakamoto et al. 1993). environment in which the level of inorganic N such as Although definitive proof of a bacterial source of nitrate or ammonia was low (unpublished data). During the toxin is lacking, STXs detected in particles smaller in the bloom, this species was found in higher densities at size than dinoflagellates are thought to originate from around 10 m depth where light intensity was as low as bacteria grown in seawater, and thus the particles may be 30 µmol photons·m–2·s–1 even during sunny day. These living on the surface of detritus which is favorable for facts indicate that natural population of A. tamarense their growth (Kodama et al. 1990a). Probably, as the large grows in an environment in which its autotrophic activ- number of bacterial cells are attaching on the detritus, ity is limited. Jacobsen and Anderson (1996) presented the amount of toxin in the detritus is often greater than electron micrographs clearly showing that A. ostenfeldii the amount associated with free-living bacteria. However, ingests the plankton as food. These findings suggest that even though the detritus fraction (5–20 µm fraction) heterotrophic characters as well as autotrophic ones play showed high toxicity, shellfish toxicity did not increase an important role for the growth and toxin production of remarkable. These findings may indicate that bacterial the natural population of Alexandrium species. toxins are not directly associated with shellfish toxicity. It is also noteworthy that, although shellfish became toxic 12. Toxin-producing bacteria in marine ecosystem during a bloom of A. catenella, no significant toxicity was detected in the A. catenella cells from this location Since the first finding that a bacterium (PTB-1) iso- (See Fig. 31). These findings support the idea that the lated from A. tamarense produces STX (Kodama et al. toxicity of shellfish cannot be attributed solely to trans- 1988; Doucette and Trick 1995), several strains of STX- port of dinoflagellates toxins via the food chain. Toxin- producing bacteria have been isolated from dinoflagellates producing bacteria may possess “biological component” (Franca et al. 1996; Gallacher et al. 1997). Also we have in which toxin is incorporated as a part; the “biological detected STXs in fractions of particles smaller in size than component” which releases toxin by digestion. dinoflagellates (Sakamoto et al. 1993). The distribution of STX-producing bacteria in the In a monitoring survey on shellfish toxicity, we marine environment appears to be quite widespread. In- analyzed STXs in particles suspended in seawater after vestigators in increasing numbers are now reporting the fractionation of the particles according to size by passing isolation of such bacteria from locations that are spatially them through sieves of different pore sizes. Figure 31 and temporally distinct from the locations of the shows data obtained for samples from Tanabe Bay where dinoflagellates. Levasseur et al. (1996) examined the shellfish became toxic during a bloom of A. catenella potential for bacterial production of STXs in the St. Law- (Sakamoto et al. 1992, 1993). Surprisingly, no signifi- rence estuary, Canada. They screened bacterial strains cant amount of shellfish toxicity increased considerably isolated from the St. Lawrence estuary by HPLC and (indicated by arrows in Fig. 31). In contrast, significant found four strains positive for STXs, indicating that pu- toxicity was detected in association with particles smaller tatively toxigenic bacteria are present in that region. than A. catenella such as those in the size ranges of 5–20 Interestingly, these four bacterial strains isolated from and 0.45–5 µm by HPLC analysis. The 5–20 µm fraction the St. Lawrence estuary by Levasseur et al. (1996) origi- showed the highest level of toxicity when the cell den- nated from sites exhibiting no concurrent PSP activity, sity of A. catenella was very low. The toxins in this and three of these locations had no prior history of bloom fraction were highly purified and identified as STX by formation. Although free-living and particle associated mass spectrometry. These findings clearly indicate that bacteria capable of producing STXs appear to exist in an unknown STX-producing organism(s), smaller than the St. Lawrence estuary, only the toxins associated with A. catenella, occurs together with A. catenella in the larger size fractions accounted for shellfish toxicity dur- seawater. Similar results were obtained for samples from ing this Alexandrium bloom. These results indicate that Ofunato Bay during a bloom of A. tamarense (Kodama STX-producing bacteria ingested by shellfish could not et al. 1990a). A slight increase in shellfish toxicity was be directly responsible for the STXs in the shellfish, al- observed when fractions containing particles smaller than though a contribution of toxin-producing bacteria to dinoflagellates showed considerable toxicity. As no sig- shellfish toxicity could not be ruled out. nificant toxicity was observed in other particle fractions,

Fig. 31. Seasonal variation of Alexandrium catenella abundance, scallop toxicity and the toxicity of particle fractions from January to June 1990 in Tanabe Bay. (A) Vertical distribution of A. catenella. Cell density (cells L–1): (ᮀ) 0 to 99; (ᮀ) 100 to 999; (ᮀ) 1000 to 4999; (ᮀ) 5000 to 9999; (ᮀ) >10000. (B) Abundance of A. catenella (᭹) (no of cells) and scallop toxicity (᭺, MU g–1). A. catenella were collected from 1 L samples taken every 2 m from the surface to 8 m depth. Arrows: peaks of A. catenella abundance where shellfish toxicity is decreasing. (C) Toxicity of particle fractions: (ᮀ) > 20 µm; (᭡) 5–20 µm; () 0.45–5 µm. Reprinted with permission from Marine Ecology Progress Series, 89, Sakamoto et al., Causative organism of paralytic shellfish toxins other than toxic dinoflagellates, 229–235, Fig. 1, © 1992, Inter-Research.

Information on the distribution of STX-producing dinoflagellates and their associated shellfish but also in bacteria in marine ecosystems is important to solve the phylogenically diverse species of animals. Noguchi et al. problem of the association between these bacteria and (1969) showed for the first time that the xanthid crab the STXs of shellfish and other organisms. In order to Atergatis floridus, a non-plankton feeder animal accumu- prove the toxigenicity of bacteria, more detailed chemi- lates a high level of STX which could not be associated cal evidence such as analytical data obtained by mass with dinoflagellates. After this discovery, high levels of spectrometry or nuclear magnetic resonance (NMR) are STXs were detected in various animals, such as the horse- required. However, because of the low toxin productiv- shoe crab, Carcinoscorpius rotundicauda (Fusetani et al. ity of bacteria, various investigators have applied indi- 1982) and several species of marine snails (Kotaki et al. rect methods for toxin identification such as HPLC and 1981). We also found that a low level of STX is detected bioassay of crude extracts. When crude extracts are in puffer Takifugu pardalis in addition to a large amount analyzed by fluorometric-HPLC, ghost peaks with reten- of tetrodotoxin (TTX) (Kodama et al. 1983a). In 1988, a tion times identical to those of standard toxins often ap- food poisoning including fatal cases by ingestion of fresh- pear (Onodera et al. 1997). However, these peaks can be water puffer Tetraodon fangi occurred (Laobhripart et al. distinguished from toxin peaks by changing the reaction 1990). We analyzed the puffer by mouse assay and found reagent for post-column derivatization to water; the ghost that the puffer possesses a potent neurotoxin, though no peaks do not disappear as a result of this treatment, but TTX was detected by chemical analysis (Sato et al. 1997). the toxin peaks disappear. In addition, these impurities In the analysis of PSP toxins by HPLC, however, a large can be mostly removed by pretreatment of the samples, amount of a toxin was detected instead of TTX which such as by partial purification of the toxins by Bio-Gel P- was identified as STX (Sato et al. 1997) (Fig. 32). Al- 2 (Bio-Rad Laboratories, Richmond, CA) or activated though no intensive survey on the PSP toxins-bearing charcoal treatment. HPLC analysis of bacterial toxins after organisms was conducted, toxins were not detected in such treatment will provide more detailed information freshwater bivalves collected from the area where the concerning the toxigenicity of the bacteria. puffer were observed. Thus, the source of STX found in freshwater puffer is unknown. 13. STXs in organisms other than dinoflagellates and In order to know the occurrence of STXs in puffer, their associated organisms we surveyed toxins in tropical species of puffer on which there is few knowledge (Sato et al. 2000a). Seven spe- It is well known that STXs are found not only in cies of puffer including 27 specimens of Arothron

Fig. 32. Fast bombardment mass spectrum of freshwater puffer toxin. A fast atom bombardment mass spectrum (FAB-MS) of the purified toxin was measured in glycerol as a matrix through a magnetic scan from 25 to 500 mass units. The toxin exhibited an ion peak at m/z = 300, 282 and 374, which correspond with a pseudomolecular ion (M + H)+ of authentic STX, + + (M + H – H2O) and (M + H + glycerol – H2O) , respectively. Reprinted from Toxicon, 35, Sato, Kodama, Ogata, Saitanu, Furuya, Hirayama and Kakinuma, Saxitoxin as a toxic principle of a freshwater puffer Tetraodon fangi, in Thailand, 137–140 © 1997, Pergamon Press plc. with permission from Elsevier.

Fig. 33. Toxicity of various organs of puffers collected from Masinloc Bay, Philippines. The toxicity was measured by mouse bioassay and expressed as mouse unit (MU) of TTX. L: liver, I: intestine, M: mussel, S: skin. (a) Number of specimens which did not kill mice (ND): 2, (b) 2, (c) 1, (d) 3, (e) 1, (f) 2, (g) 1, (h) 5, (i) 1. Reprinted from Toxicon, 38, Sato, Ogata, Borja, Gonzales, Fukuyo and Kodama. Frequent occurrence of paralytic shellfish poisoning toxins as dominant toxins in marine puffer from tropical water, 1101–1109, © 2000, Pergamon Press plc, with permission from Elsevier.

mappa, 25 of A. manillensis, 20 of Chelonodon patoca, 6 of A. nigropunctatus, 5 of A. hispidus, one A. stellatus and one A. reticularis were collected at Masinloc Bay, Luzon, Philippines over the year of 1992. The tissues excised from partially thawed specimens were subjected to extraction and analyzed by HPLC for PSP toxins as well as TTX. Figure 33 shows the toxicity of the extracts analyzed by mouse bioassay. Most of the extracts killed mice with typical signs of TTX and STXs. In the chemical analysis, a considerable level of STXs was detected in addition to TTX. Although the number of the specimens was not enough, the major toxin of these puffer seemed to be STXs. In Fig. 34 is shown one of the results obtained from A. mappa specimen by HPLC analysis. In Fig. 34. HPLC-chromatograms of a skin extract of A. mappa. addition to TTX, significant level of neoSTX, (A) Analysis by HPLC specially designed for TTX. For de- decarbamoyl STX, GTX5 and STX were detected. Most tail conditions see Yotsu et al. (1989). (B) and (C) analysis by of the puffer specimens showed similar profiles, sug- HPLC specially designed for PSP toxins. For detail conditions gesting that the major toxin in puffer Masinloc Bay is see Oshima (1995a). Reprinted from Toxicon, 38, Sato, Ogata, STX (Fig. 35). Borja, Gonzales, Fukuyo and Kodama. Frequent occurrence of The Masinloc Bay where these puffer specimens paralytic shellfish poisoning toxins as dominant toxins in ma- were collected is well known for the bloom of Pyrodinium rine puffer from tropical water, 1101–1109, © 2000, Pergamon bahamense var. compressum, a tropical PSP toxins- Press plc, with permission from Elsevier.

a long period. Meanwhile, the xanthid crab is known to release STXs (Noguchi et al. 1985). Arakawa et al. (1998) showed that a large proportion of the STXs injected into the xanthid crab disappeared within a short period. This finding implies that the rate of release of STXs from the xanthid crab is significantly high. Therefore, it is unlikely that the xanthid crab accumulates a high level of STXs by continuous ingestion of Jania sp. with a low concentration of STX, even over a long period. Interestingly, these STX-bearing animals often possess tetrodotoxin (TTX) as well, a potent neurotoxin which binds to the same biological receptor as STXs (Kao 1966). This suggests that organisms known to possess either STXs or TTX may possess both of these toxins. This speculation is supported by the fact that a significant amount of TTX has been detected in A. tamarense, a representative dinoflagellate capable of producing STXs. During a bloom of A. tamarense, considerable amounts of TTX accumulate in shellfish together with STXs (Kodama et al. 1993). As shown in Fig. 35 the levels of TTX in the shellfish was shown to be parallel with the levels of STXs (Kodama et al. 1993). These findings indicate that TTX found in the shellfish origi- Fig. 35. Mol% of PSP toxins to total toxin (TTX + PSP tox- nates from A. tamarense. Actually, a significant level of ins) in each organ of puffer. L: liver, I: intestine, M: muscle, TTX was identified in cultured cells of A. tamarense S: skin. Reprinted from Toxicon, 38, Sato, Ogata, Borja, Gonzales, Fukuyo and Kodama. Frequent occurrence of para- (Kodama et al. 1996b). This is the first case to be shown lytic shellfish poisoning toxins as dominant toxins in marine that A. tamarense could be the causative organism for puffer from tropical water, 1101–1109, © 2000, Pergamon Press TTX-bearing animals. plc, with permission from Elsevier. It is well known that TTX is produced by bacteria, although the productivity is very low (Noguchi et al. 1986; Yasumoto et al. 1986). It is noteworthy that TTX- producing bacteria and STX-producing bacteria display producing dinoflagellate (Montojo et al. 2006). Bivalve similar characteristics in terms of toxin productivity. The in the bay become toxic almost every year. Interest- toxin productivity of TTX-producing bacteria is very low ingly, the toxin profile of puffer was close to that of and the TTX levels in the TTX-producing bacteria in- P. bahamense var. compressum and its associated crease significantly under starvation conditions (Noguchi bivalves. On the other hand, Zaman et al. (1997) reported et al. 1986; Yasumoto et al. 1986). Although the evidence that toxin profile of freshwater and/or brackishwater is circumstantial, various phenomena suggest that these puffer Tetraodon cutctia and C. patoca collected from bacteria are widely distributed in natural ecosystems. The the rivers and tributary in Dhaka, Bangladesh, was also origin of the STXs in toxic animals not associated with close to that of P. bahamense var. compressum, though dinoflagellates seems to be bacteria, as in the case of TTX, P. bahamense var. compressum can not grow in fresh- and the mechanism by which the organisms become toxic water. Thus the PSP toxins found in puffers from owing to the presence of TTX and STXs derived from Masinloc Bay was not derived from P. bahamense bacteria seems to be similar in each instance. var. compressum. The source of STXs found in the Phil- ippine puffers is unknown. 14. Possible mechanism by which pufferfish become Yasumoto et al. (1981) concluded that the STXs in toxic: a model based on the mechanism by which the xanthid crab are not derived from dinoflagellates, as accumulation of STXs occurs in dinoflagellates no STXs were detected in bivalves living in the same area where the toxic xanthid crab is inhabiting. Instead, It is well known that wild puffer possess a potent they detected a low level of STXs in Jania sp., a neurotoxin named as tetrodotoxin (TTX). Generally, TTX carcacious alga which is often found in the stomach of is contained in various toxic species of puffer. However, the toxic xanthid crab, and suggested that the origin of TTX is not always contained in all the specimens of toxic STXs in the xanthid crab is this alga (Kotaki et al. 1983). species. Non-toxic specimens are often observed among It is possible that the xanthid crab accumulates a high the toxic species of puffer. It has been a mystery why level of STXs by continuous ingestion of Jania sp. over puffer possess the potent toxin, TTX. Although the

reason is still not clear, it is known by fish culturists that nal bacterial flora of nontoxic cultured puffer T. rubripes. puffer which are artificially hatched and fed an artificial We analyzed TTX levels in the liver, the main toxic or- diet are nontoxic. Matsui et al. (1982) demonstrated that gan of the puffer, and in the intestine of cultured puffer nontoxic cultured puffer Takifugu rubripes become toxic specimens of 60 days and 6 months old, applying a sensi- when fed a diet containing TTX, and indicated that TTX tive method of analysis of TTX (Sato et al. 1990). No in the puffer is an exogenous substance which originates TTX was detected in the liver of either juvenile or adult from the diet. On the other hand, TTX has been shown to puffer specimens, whereas a low level of TTX, 0.2 mouse be produced by bacteria (Noguchi et al. 1986; Yasumoto unit (MU)·g–1 (where one MU is a dose required to kill a et al. 1986) and thus the source of TTX in TTX-bearing 20-g male mouse within 30 min), apparently due to in- animals such as puffer is considered to be bacteria. Hence, testinal bacteria, was detected in the intestine of both sam- TTX of bacterial origin is considered to accumulate in ples. These observations show that the level of TTX in the puffer via food webs (Yasumoto et al. 1992). The the intestine is too low for accumulation by the puffer. screening of various marine organisms for TTX revealed TTX- and STX-bearing animals have been re- that this toxin is widely distributed in marine organisms, ported to be resistant to these toxins (Koyama et al. some species of which could be components of the diet 1983). However, these animals die when a substantial of puffer (Noguchi et al. 1983; Miyazawa et al. 1986). amount of the toxin (about 1000 times the lethal dose for These studies also showed that the trumpet shell Charonia common animals) is injected (Koyama et al. 1983). These sauliae accumulates TTX by ingesting the TTX bearing observations show that the toxins are also toxic to these starfish Astropecten polyacanthus (Narita et al. 1984). animals. Although the mechanism is not known, puffer However, such a case clearly supporting the food chain sometimes accumulate TTX at levels much higher than theory of TTX accumulation in toxic animals is rare. The their lethal dose (Kodama et al. 1984). In this regard, distribution of toxic components of the diet of the puffer species of puffer are secreting this dangerous toxin into rarely overlaps with that of toxic puffer, indicating that environmental water continuously (Kodama et al. 1985). the puffer toxin cannot be accumulated via a simple food As shown in Fig. 36, puffer possess developed TTX-se- web mechanism. On the contrary, Noguchi et al. (1987) creting glands in the skin which consist of specific TTX- proposed that puffer become toxic by absorbing a low secreting cells (TTX cells) (Kodama et al. 1986). When level of TTX produced by intestinal bacteria over a long stimulated, the puffer secretes a considerably high amount period. This idea seems plausible, because TTX- of TTX into the environment, that is, 4 mg of TTX is producing Vibrio alginolyticus is a dominant species in often secreted on a single stimulation (Kodama et al. the intestinal bacterial flora of toxic puffer (Noguchi et 1985), showing that the rate of TTX secretion is signifi- al. 1987). However, Sugita et al. (1988) reported that cantly high. A similar phenomenon is also observed in V. alginolyticus is also a dominant member of the intesti- the case of the TTX bearing newt (Shimizu and Kobayashi

Fig. 36. Light micrographs of the skin of T. pardalis. (A) Longitudial section of a large alveolar secretory gland, with an opening (arrow) to the skin surface. The cytoplasm of the scretory cells (TTX cells) are almost empty. The bar equals 100 µm. (B) Epidermis, with mucous cells, sacciform cells and free TTX cells. The bar equals 10 µm. Helly’s fluid, PAS—hematoxylin stain. SG, secretory gland; E, epidermis; D, dermis; L, lumen of the gland; MC, mucous cell; FC, free TTX cell. Reprinted from Toxicon, 24, Kodama, Sato, Ogata, Suzuki, Kaneko and Aida, Tetrodotoxin secreting glands in the skin of puffer fishes. 819–829, © 1986, Pergamon Journal Ltd. with permission from Elsevier.

1983). These findings indicate that TTX-bearing animals (Sato et al. 1997). Therefore, the approach taken in in- actively release TTX from a specifically developed or- vestigation of toxins from dinoflagellates was applied to gan and the rate of release of the toxin is significantly puffer toxin, that is, attempts were made to isolate bacte- high. Given that the TTX present in the puffer is accu- ria from the liver tissue of toxic and nontoxic specimens mulated via food webs, the rate of uptake of TTX should of puffer. Interestingly, bacteria were isolated only from be greater than the high rate of release, that is, a consid- toxic specimens (Kodama et al. 1995). Polyclonal anti- erably large amount of TTX must be supplied to the body against the bacterium isolated from the liver reacted puffer continuously. This seems consistent with our re- with an extract of the liver from which the bacterium was sults indicating that the cultured puffer T. rubripes did isolated. Electron microscopic observations also sup- not accumulate TTX, although a low level was detected ported the view that bacteria were present in the liver in the intestine. The supply of TTX from the intestinal cells (Fig. 37). An extract of the isolated bacteria was bacteria was too low for accumulation by the puffer. found to display sodium channel blocking activity and Watabe et al. (1987) reported that tritium-labeled gave a TTX-like peak on HPLC analysis. These findings TTX injected intraperitoneally into toxic puffer is accu- suggest that bacterial symbiosis in the liver cells is in- mulated in the liver for a short period and then trans- volved in the mechanism of production of puffer toxin as ported to the skin and gallbladder to be released. About in the case of toxic dinoflagellates. Most of the bacteria 70% of the injected TTX disappeared within 6 days. We isolated from the liver of the puffer were gram-positive, also observed that TTX injected into the nontoxic cul- which are usually not found in seawater but rather in the tured puffer T. rubripes, was accumulated in the liver first, sediments (Kodama et al. 1995). Polyclonal antibody moved to the skin, and then was released into the envi- against the bacterium isolated from the liver reacted with ronment (unpublished data). About 50% of the injected an extract of the liver from which the bacterium was iso- TTX disappeared within a few weeks. These data show lated (Fig. 38). It is well known that puffer tend to hide that TTX taken up by the puffer is not retained for a long themselves in the bottom sediment (Katayama and Fujita period because of the high rate of secretion of TTX. Al- 1965). It seems plausible that bacterial infection may though no significant amount of TTX has been detected occur in this habitat of the puffer. This may explain why in the diet of the puffer, some specimens of wild puffer puffer cultured in a cage, having no contact with bottom accumulate substantial amounts of TTX (Kodama et al. sediments, are nontoxic. 1984). These observations lead us to contend that puffer As described above, bacteria which produce STXs accumulate TTX not via food webs but through some or STX-like substances are widely distributed in natural other mechanism which remains unknown. ecosystems. This is also true for bacteria which produce Interestingly, a considerable amount of TTX was TTX or TTX-related compounds (Noguchi et al. 1987; detected in the muscle of cultured puffer T. rubripes speci- Simidu et al. 1990). Shewanella alga, the species found mens 2 weeks after injection of TTX (unpublished data). to produce TTX, was shown to produce STXs as well Watabe et al. (1987) also observed that injected tritium (Doucette et al. 1988). This species shows wide labeled TTX is detected in significant concentrations phylogenetic and spatial distribution in natural ecosys- in the muscle of the puffer. In contrast, no TTX is de- tems. These observations imply that most aquatic ani- tected in the muscle of wild specimens of T. rubripes. mals are exposed to these bacteria. Therefore, we screened Tani (1945) reported that the muscle of T. rubripes is the viscera of various common aquatic animals for the nontoxic, although considerable toxicity is often detected presence of STXs and TTX (Sato et al. 1993). HPLC in the liver and other organs. Based on analyses of the analyses and bioassays of the toxins partially purified toxicity of various specimens, the muscle of this species from the viscera showed that all species examined has been designated as a safe food after careful removal showed the presence of both TTX and STXs, although of the organs prone to contamination with the toxin by the level were very low (Table 4). For further confirma- licensed chefs in Japan. The results of these toxicity analy- tion, an extract of the chum salmon, Onchorhyncs keta, ses indicate that natural specimens of T. rubripes never in which low levels of TTX, STX, and neoSTX were de- take in such an amount of TTX at once as that adminis- tected by HPLC analysis, was prepared from a large tered in the experiments. The results also tend to support amount of the viscera (62 kg) of specimens collected at a the view that TTX in the puffer is not accumulated via river mouth during the homing period. A toxin with a re- food webs. tention time identical to that of TTX was detected in the As described above, we have suggested that sym- HPLC analysis, but was lost during the purification biotic bacteria in dinoflagellates are involved in the pro- process; however, toxins corresponding to STX and duction of substantial amounts of STX. Most of the toxic neoSTX were obtained in a highly pure state. Mass species of puffer possess STXs as well as TTX (Sato et spectrometry and NMR analyses clearly showed that al. 1993). In specimens of the freshwater puffer, Tetraodon these toxins were STX and neoSTX (Sato et al. 1998). fangi, which caused food poisoning including lethal cases These findings show that TTX and STXs are not sub- in Thailand, only STX was detected as the toxic agent stances found only in specific organisms but are widely

Fig. 37. Electron micrograph of hepatocytes from Takifugu poecilonotus. (a) There are three hepatocytes in the electron micrographs. Each cell contain many microorganism-like components (arrows). No abnormal bioreactions are seen in every hepatocyte. F, fat droplet. Bar = 2 µm. (b) Enlarged part of (a). Three round microorganisms are seen. These are surrounded by double membranes from the cytoplasms of the hepatocytes. Numerous ribosomes (R) and the thick septum (S) are demon- strable. Bar = 0.5 µm. Reprinted with permission from Kodama, Shimizu, Sato, Ogata and Terao. The infection of bacteria in the liver cells of toxic puffer—A possible cause for organisms to be made toxic by tetrodotoxin in association with bacteria. Harmful Marine Algal Blooms—Proceed. 6th Intern. Conference Toxic Marine Phytoplankton, Nantes, October 1993, P. Lassus et al., © Technnique et Documentation—Lavoisier, 1995.

Fig. 38. Double immunodiffusion of the extracts of the liver and isolated bacteria against antibody P and puffer liver. 1 and 4: the liver extract of a toxic specimen of the pufferfish Takifugu porphyreus; 2 and 6: the extract of bacteria isolated from the liver, the concentration of the extracts is different; 3 and 5: the extract of antigenic bacteria to prepare antibody P. (a) Center: antibody P, (b): Center the serum of puffer specimen T. porphyreus from which the liver (1 and 4) and bacteria (2 and 6) were prepared. Antibody P was prepared by immunizing rabbit with homogenate of bacteria isolated from the liver of another toxic specimen of T. porphyreus. Reprinted with permission from Kodama, Shimizu, Sato, Ogata and Terao. The infection of bacteria in the liver cells of toxic puffer—A possible cause for organisms to be made toxic by tetrodotoxin in association with bacteria. Harmful Marine Algal Blooms—Proceed. 6th Intern. Conference Toxic Marine Phytoplankton, Nantes, Octo- ber 1993, P. Lassus et al., © Technnique et Documentation—Lavoisier, 1995.

Table 4. Occurrence of TTX and STXs in common aquatic animals.

Species Part TTX Toxin composition (mg mL–1 solution*)

GTX1,4 GTX2,3 neoSTX STX Shark Lemna ditropis Int 2.3 2.3 2.3 0.1 Liv 1.0 0.3 0.5 0.1 Salmon Oncorhynchus keta Int 1.1 4.2 1.9 Liv 0.7 0.3 0.1 Conger eel Conger myriaster Vis 1.3 Saury Cololabis saira Vis 1.0 Cod Gadus macrocephalus Int 0.6 0.6 0.2 3.1 Liv 0.2 2.1 Parrotfish Ypsiscarus ovifrons Int 1.9 0.9 0.7 0.2 0.1 Liv 1.6 0.1 0.4 Skipjack Katuwonus pelamis Liv 1.7 0.9 Filefish Nevodom modestus Int 0.8 0.3 0.3 0.1 Liv 2.5 1.8 0.1 Crayfish Procambarus clarkii Hep 0.4 1.0 0.1 Rock crab Hemigrapsus sanguineus Hep 0.1 Rock crab Pachygrapsus crassipes Hep 0.1 Marine snail Neptunea arthritica Dig 0.2 1.0 0.9 0.3 0.1

Int, intestine; Liv, liver; Vis, viscera; Hep, hepatopancreas; Dig, digestive gland. *1 mL solution is equivalent to 100 g of organ. This article was published in Toxic Phytoplankton Blooms in the Sea (Smayda and Shimizu, eds.), Sato, Ogata and Kodama, Wide distribution of toxins with sodium channel blocking activity similar to tetrodotoxin and paralytic shellfish toxins in marine animals, pp. 429–434, © Elsevier (1993).

distributed in common aquatic animals at low levels. As cera did not originate from the diet. These findings sup- shown in Table 4, no significant difference was observed port the view that bacteria which produce TTX and STXs in the levels of these toxins in the viscera of various com- are widely distributed in natural ecosystems. mon animals, indicating that these toxins are not concen- trated via the food web but rather they originate from 15. Hypothesis on toxin accumulation in toxic or- intestinal bacteria. More than 90% of the homing chum ganisms salmon returning close to their home river do not con- sume any diet, and thus their stomachs are empty (Ueno As described above, bacteria which produce low 1993). All of the stomachs in the viscera used in the above levels of toxins live in cells of the puffer and in experiments were empty, thus the STXs found in the vis- dinoflagellates which display high levels of TTX and/or

production by the bacteria increases significantly under P-limiting conditions (Doucette and Trick 1995) as well as under starved conditions (Kodama et al. 1990b). Toxic animals and dinoflagellates are hosts which possess the enzyme(s) required to digest the “unknown substance” and release the toxins. If the “unknown substance” in question is RNA, the enzyme required to digest this substance would be an RNase, an enzyme which occurs widely in living organisms. Toxic organisms might possess a specific type of RNase capable of digesting foreign RNA which contains unusual components, STXs, and/or TTX. Some strains of toxic dinoflagellates are known to be nontoxic. The RNase in these strains might lack the ability to digest foreign RNA containing unusual Fig. 39. Hypothesis on the mechanism by which toxic organ- components which are not essential for their growth. isms become toxic in association with infectious bacteria. References Anderson DM. Effect of temperature conditioning on develop- ment and germination of Gonyaulax tamarensis STXs. Toxic shellfish also possess these bacteria in their (Dinophyceae) hypnozygotes. J Phycol 1980; 16: 166–172. cells, carried by the vector dinoflagellates. These bacte- Arakawa O, Noguchi T, Onoue Y. Transformation of ria grow in cells of the host organisms. The host organ- gonyautoxins in the xanthid crab Atergatis floridus. Fish isms kill and digest some of the bacteria that have prolif- Sci 1998; 64: 334–337. erated within the host cells and thereby maintain a suit- Asakawa M, Takagi M, Iida A, Oishi M. Studies on the conver- able number of bacterial cells for their survival. That is, sion of paralytic shellfish poison (PSP) components by biochemical reducing reagents. Eiseikagaku 1987; 33: 50–55 direct biochemical interactions between the bacteria and (in Japanese). the host occur within the cells in the host organisms. The Boyer GL, Sullivan JJ, Andersen RJ, Harrison PJ, Taylor FJR. finding that the number of bacteria found in the host is Toxin production in three isolates of Protogonyaulax sp. few tends to support this idea. Based on these findings, In: Anderson DM, White AW, Baden DG (eds). Toxic the hypothesis shown in Fig. 39 is proposed, suggesting Dinoflagellates. Elsevier, New York. 1985; pp. 281–286. that TTX and STXs are not the final products of their Boyer GL, Sullivan JJ, Andersen RJ, Taylor FJR, Harrison PJ, metabolisms but are intermediate products synthesized Cembella AD. Use of high-performance liquid chroma- by some bacterial substance which seems to be impor- tography to investigate the production of paralytic shell- tant for their survival. Recently, the biosynthetic path- fish toxins by Protogonyaulax spp. in culture. Mar Biol way of STX proposed by Shimizu et al. 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