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Home , Atergatis floridus, Decarbamoylsaxitoxin, Xanthidae, Zosimus aeneus

Fisheries Science 61(4), 659-662 (1995)

Paralytic Shellfish Profiles of Xanthid aeneus and floridus Collected on Reefs of Ishigaki Island

Osamu Arakawa,*1 Tamao Noguchi,*2 and Yoshio Onoue*1 *1Laboratory of Marine Botany and Environmental Science , Faculty of Fisheries, Kagoshima University, Shimoarata, Kagoshima 890, Japan *2Laboratory of Food Hygiene , Faculty of Fisheries, Nagasaki University, Bunkyo, Nagasaki 852, Japan (Received September 14, 1994)

Toxin compositions in xanthid crabs and were examined. Three specimens of each were collected on reefs of Ishigaki Island, Okinawa Prefecture, and were dissect ed into 6 parts; muscle of appendages, muscle of cephalothorax, skeleton of appendages, skeleton of cephalothorax, viscera, and gills. Toxicity of each tissue was determined by mouse bioassay and toxin compositions by high performance liquid chromatography (HPLC) with fluorometric detection. In both crabs, muscle of appendages gave the highest toxicities (1,100-5,900 MU/g) among the 6 tissues, and those of muscle of cephalothorax and gills were less than one tenth of it. In Z. aeneus, relative abun dances (mole%) of carbamoyl-N-hydroxyneosaxitoxin (hyneoSTX) and (neoSTX) were rather high, and 10~15% of (GTXs) were contained in addition to similar amounts of carbamoyl-N-hydroxysaxitoxin (hySTX), decarbamoylsaxitoxin (dcSTX), and (STX) ir respective of tissues except for gills. A. floridus possessed STX predominantly over the other STXs with no detectable amount of GTXs. In both crabs, gills had a somewhat different toxin profile from the other tissues with higher neoSTX and STX but lower hyneoSTX and hySTX. A good correlation was observed between the toxicities calculated from the results of HPLC analyses and those from mouse bioassay. Key words: paralytic shellfish poison, saxitoxin, , Zosimus aeneus, Atergatis floridus

Xanthid crabs Zosimus aeneus and Atergatis floridus land, Okinawa Prefecture. They were transported alive to have been known to possess potent paralytic . Com the laboratory of Kagoshima University, immediately positions of the toxic principle varied depending on the frozen, and kept below -30•Ž until use. habitat. The crabs living on reefs of Ishigaki Island, Okina wa Prefecture, mainly contained paralytic shellfish poison Preparation of Test Solutions (PSP) such as saxitoxin (STX), neosaxitoxin (neoSTX), Each specimen was partially thawed and dissected decarbamoylsaxitoxin (dcSTX), and gonyautoxins into 6 parts; muscle of appendages (MA), muscle of (GTXs),1-5)whereas in A. floridus inhabiting a small islet cephalothorax (MC), skeleton of appendages (SA), skele of Ishigaki Island and the Pacific coast of Kanto region, ton of cephalothorax (SC), 'viscera' including hepatopan puffer toxins were predominant.6-8) However, most of creas, reproductive organ and intestines (V), and gills (G). these studies were based on qualitative or semiquantitative To one gram each of these tissues was added 4 ml of 0.1 N analyses. HCl, and heated in a boiling water bath for 5 min. The Recently, we have separated two novel PSP compo mixture was centrifuged, and the supernatant was passed nents, carbamoyl-N-hydroxysaxitoxin (hySTX) and car through a YM1 ultrafiltration membrane (Amicon) to ob bamoyl-N-hydroxyneosaxitoxin (hyneoSTX), in addition tain a test solution. A portion of it was used for mouse to decarbamoylneosaxitoxin (dcneoSTX), from Ishigaki bioassay, and another portion for high performance liquid specimensof Z. aeneus.9) This finding and recent advances chromatographic analysis, as described below. in analytical technique of PSP using high performance liq uid chromatography10-12) prompted us to conduct a fully Mouse Bioassay quantitative analysis of the crab toxins, as a part of studies Mouse bioassay was performed according to the AOAC on the mechanism by which crabs accumulate or metabo method for PSP.13) Briefly, the test solutions were ap lize toxins. propriately diluted with 0.01 N HCl, and injected in traperitoneally into a group of three male mice of ddY Materials and Methods strain (19-20g). Lethal potency was calculated from the time required to kill the mice, and expressed in mouse Crab Specimens units (MU). One MU is defined as the amount of toxin In September 1993, three specimens of each of Zosimus which kills the mouse in 15 min after injection. aeneus (body weight, 52-96g) and Atergatis floridus (28 46g) were collected on reefs near Kabira Bay, Ishigaki - Is 660 Arakawa et al. High Performance Liquid Chromatographic Analysis (V), and gills (G)], MA gave highest toxicities Reverse phase high performance liquid chromatography (1, 100 5,900 MU / g as determined by mouse bioassay, (HPLC) was carried out on a Hitachi 655 HPLC system. A Fig. 6) irrespective of the specimens. Figs 2 shows the LiChroCART RP-18(e) column (4 x 250mm, Merck) was average and standard deviation of relative toxicity in tis used in combination with the two mobile phases (flow rate sues, which was calculated regarding the toxicity of MAas 0.8 ml/ min); (I) 2mM heptanesulfonic acid in 10mM am 1.0 for individual specimens. In both Z. aeneus and A. monium phosphate buffer (pH 7.3) for gonyautoxins floridus, the toxicity scores of SA, SC, and V were inter (GTXs), and (II) 2mM heptanesulfonic acid in 4% acetoni mediate (0.2~0.5), and those of MC and G were lessthan trile-30mM ammonium phosphate buffer (pH 7.3) for sax 0.1. itoxins (STXs).11,12)The eluate from the column was con HPLC analyses of standard toxins are shown in Fig. 3- tinuously mixed with 50 mm periodic acid (flow rate 0.4 A. Under the conditions described in "Materials and ml/ min) and with 0.2 N KOH containing 1 M ammonium Methods", 11 toxins including gonyautoxin 4 (GTX4,a), formate and 50% formamide (flow rate 0.4ml/ min), and gonyautoxin 1 (GTX1, b), dcGTX3 (c), dcGTX2 (d), GTX3 heated at 65°C for 1.5min.10) The fluorophors formed (e), GTX2 (f), hyneoSTX (g), neoSTX (h), hySTX (i), were monitored at 392nm with 336nm excitation. As ex dcSTX (j), and STX (k) were separable from each other, ternal standards, pure toxins which had been separated although dcneoSTX was indistinguishable from neoSTX from Ishigaki specimens of Z. aeneus (Fig. 1)9) were in this system (data not shown). Figures. 3-B and -C show employed. The following abbreviations were used for the representative chromatograms of the test solutions from toxins: saxitoxin, STX; neosaxitoxin, neoSTX; decar Z. aeneus and A. floridus. All of the Z. aeneus specimens bamoylsaxitoxin, dcSTX; carbamoyl-N-hydroxysaxitoxin, contained 4 components of GTXs (dcGTX3, dcGTX2, hySTX; carbamoyl-N-hydroxyneosaxitoxin, hyneoSTX; GTX3, and GTX2) and 5 components of STXs gonyautoxins 2 and 3, GTX2 and GTX3; decarbamoyl (hyneoSTX, neoSTX, hySTX, dcSTX, and STX) irrespec gonyautoxins 2 and 3, dcGTX2 and dcGTX3.- Toxin solu tive of the tissues, whereas only STXs were detectedin tions were calibrated separately by mouse bioassay and specimens of A. floridus. An unknown peak, as seenat the mixed at an appropriate ratio for the standard of HPLC retention time 12.0 min in C-(I), was observed in some analysis. The molar concentration of each toxin was esti chromatograms of both crab . mated from the reported specific toxicity listed in Fig. 1. Relative abundances (mole%) of individual toxins or toxin groups in tissues of Z. aeneus and A. floridus are Results shown in Figs. 4 and 5, respectively. Despite a largervaria tion in the relative abundance of toxin, some characteris Among the 6 crab tissues examined [muscle of append tics of toxin profiles in species or tissues could stillbe recog ages (MA), muscle of cephalothorax (MC), skeleton of ap nized. In Z. aeneus abundances of 1-N-hydroxy toxins pendages (SA), skeleton of cephalothorax (SC), viscera

Fig. 2. Relative toxicities in tissues of Z. aeneus and A. floridus. Fig. 1. Structures and specific toxicities of paralytic shellfish poisons. MA, muscle of appendages; MC, muscle of cephalothorax; SA, *From reference 12; **from reference 11; ***values remeasured skeleton of appendages; SC, skeleton of cephalothorax; V, viscera; on the toxins isolated in reference 9. G, gills. Toxin Profiles of Xanthid Crabs 661

Fig. 4. Relative abundances of individual toxins (mole%) in tissues of Z. aeneus. For abbreviations, MA, MC, SA, SC, V, and G, see legend of Fig. 2.

Fig. 5. Relative abundances of individual toxins (mole%) in tissues of A. floridus. For abbreviations, MA, MC, SA, SC, V, and G, see legend of Fig. 2.

amounts of hySTX and dcSTX as zero, resulting in appar ently dissimilar composition of MC to other tissues. Both in Z. aeneus and A. floridus, G had a somewhat different toxin profile from the other tissues with higher neoSTX and STX but lower hyneoSTX and hySTX. Toxicities in tissues of individual specimens calculated from the results of HPLC analyses were plotted against those of mouse bioassay (Fig. 6). A good correlation was observed between the two methods, where the correlation coefficient was calculated as 0.98, and the regression line, Y = 1.00x + 7.84, based on real numbers of MU/g.

Discussion Fig. 3. Chromatograms of standard toxins (A), and of the test solu tions prepared from muscles of appendages (MA) of a Z. aeneus As described above, little difference was observed be specimen (B) and of an As floridus specimen (C). tween Z. aeneus and A. floridus in distribution of toxicity (I) and (II) indicate the mobile phases used for analyses which are in the tissues, both showing an extremely high toxicity in referred to the text. a, GTX4; b, GTX1; c, dcGTX3; d, dcGTX2; e, appendages, but a very low toxicity in cephalothorax mus GTX3; f, GTX2; g, hyneoSTX; h, neoSTX; i, hySTX; j, dcSTX; k, STX. cles or gills. Similar results are seen in the literature.14) On the other hand, toxin compositions varied depend ing on the species. In Z. aeneus, abundances of 1-N (hyneoSTX and neoSTX) were relatively high, and hydroxy toxins (hyneoSTX and neoSTX) were relatively- 10~15% of GTXs (total of dcGTX3, dcGTX2, GTX3, and high, and GTXs were also present, whereas A. floridus pos GTX2) were contained in addition to similar amounts of sessed STX predominantly, with no detectable amount of hYSTX,dcSTX , and STX irrespective of tissues except for G GTXs. Kotaki et al.15) reported that separated . In A. floridus, STX was predominant in all tissues. As from the viscera of A. floridus transformed GTXs and toxicity of MC was extremely low, hySTX and dcSTX neoSTX into STX through a reductive elimination of were hardly detected. Consequently, the abundance of C-11 hydroxysulfate or N-1 hydroxyl moieties. This type each toxin in this tissue was calculated regarding the of transformation might be related to make the difference 662 Arakawa et al.

2) T. Yasumoto, Y. Oshima, M. Hosaka, and M. Miyakoshi: Analysis of paralytic shellfish toxins of xanthid crabs in Okinawa. Nippon Suisan Gakkaishi, 47, 957-959 (1981). 3) K. Koyama, T. Noguchi, Y. Ueda, and K. Hashimoto: Occurrence of neosaxitoxin and other paralytic shellfish poisons in toxic crabs belonging to the family . Nippon Suisan Gakkaishi, 47, 965 (1981). 4) T. Harada, Y. Oshima, and T. Yasumoto: Natural occurrence of decarbamoylsaxitoxin in tropical and bivalves. Agric. Biol. Chem., 47, 191-193 (1983). 5) K. Daigo, A. Uzu, O. Arakawa, T. Noguchi, H. Seto, and K. Hashimoto: Isolation and some properties of neosaxitoxin from a xanthid crab Zosimus aeneus. Nippon Suisan Gakkaishi, 51, 309 313 (1985). - 6) T. Noguchi, A. Uzu, K. Koyama, J. Maruyama, Y. Nagashima, and K. Hashimoto: Occurrence of as the major toxin in a xanthid crab Atergatis floridus. Nippon Suisan Gakkaishi, 49, 1887-1892 (1983). 7) T. Noguchi, O. Arakawa, K. Daigo, and K. Hashimoto: Local Fig. 6. Correlations between the toxicities determined by mouse bioas differences in toxin composition of a xanthid crab Atergatis floridus say and HPLC analyses. inhabiting Ishigaki Island, Okinawa. Toxicon, 24, 705-711 (1986). For abbreviations, MA, MC, SA, SC, V, and G, see legend of 8) O. Arakawa, T. Noguchi, Y. Shida, and Y. Onoue: Occurrenceof Fig. 2. 11-oxotetrodotoxin and 11-nortetrodotoxin-6(R)-ol in a xanthid crab Atergatis floridus collected at Kojima, Ishigaki Island. Fish eries Sci., 60, 769-771 (1994). in toxin compositions between the two species of xanthid 9) O. Arakawa, T. Noguchi, Y. Shida, and Y. Onoue: Occurrenceof carbamoyl-N-hydroxy derivatives of saxitoxin and neosaxitoxin in a crabs. xanthid crab Zosimus aeneus. Toxicon, 32, 175-183 (1994). Hydroxycarbamoyl toxins, which had been difficult to 10) Y. Nagashima, J. Maruyama, T. Noguchi, and K. Hashimoto: distinguish from carbamoyl toxins by the previous HPLC Analysis of paralytic shellfishpoison and tetrodotoxin by ion-pair method, were demonstrated here to be possessed in com ing high performance liquid chromatography. Nippon Suisan Gak mon by toxic reef crabs, although the mechanism of their kaishi, 53, 819-823 (1987). formation has not yet been clarified. 11) Y. Oshima, K. Sugino, and T. Yasumoto: Latest advances in A good correlation between the mouse bioassay and HPLC analysis of paralytic shellfish toxins, in "Mycotoxins and Phycotoxins '88" (ed. by S. Natori, K. Hashimoto, and Y. Ueno), HPLC analysis indicates little contribution of other tox Elsevier, Amsterdam, 1989, pp. 319-326. ins, such as puffer toxins to the toxicity of the crab speci 12) Y. Oshima, C. J. Bolch, and G. M. Hallegraeff: Toxin composition mens on reefs of Ishigaki Island. of resting systs of Alexandrium tamarense (Dinophyceae). Toxicon, 30, 153-1544 (1992). Acknowledgments The authors express sincere thanks to Mr. T. 13) S. Williams: Paralytic shellfish poison, in "Official Methods of Tohma, the director of Yaeyama Branch of Okinawa Fisheries Ex Analysis of the Association of Official Analytical Chemists", 14th perimental Station for his kind collaboration in collecting the crab speci Edn, Association of Official Analytical Chemists, Arlington, 1984, mens. pp. 344-345. 14) Y. Hashimoto: Toxic crabs, in "Marine Toxins and Other Bioactive Marine Metabolites", Japan Scientific Societies Press, Tokyo, References 1979, pp. 59-68. 15) Y. Kotaki, Y. Oshima, and T. Yasumoto: Bacterial transformation 1) T. Noguchi, S. Konosu, and Y. Hashimoto: Identity of the crab tox of paralytic shellfish toxins in crabs and a marine snail. in with saxitoxin. Toxicon, 7, 325-326 (1969). Nippon Suisan Gakkaishi, 51, 1009-1013 (1985).