Paralytic shellfish toxins in the xanthid crab Atergatis floridus collected from Australian coral

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1 Journal of Wilderness Medicine 2, (1991) ORIGINAL ARTICLE Paralytic shellfish toxins in the xanthid crab Atergatis floridus collected from Australian coral reefs L.E. LLEWELLYN* and R. ENDEAN Department ofzoology, University ofqueensland, St. Lucia, Australia, 4067 One hundred and nine specimens of Atergatis floridus (Xanthidae) were collected from coral reefs of the Capricorn Group in the southern region of the Great Barrier Reef, Queensland, Australia. Sixty-five acidified 70% ethanol extracts of whole crabs contained detectable toxicity, quantified with an assay which defines 1 mouse unit (MU) as the intraperitoneal dose which kills a 20 g mouse in 15 min. The most toxic extract contained 3838 MU (108 MUg-I of crab), which exceeds the suggested human lethal dose. Chromatography and electrophoresis detected toxins similar to the paralytic shellfish toxins (PSTs), namely saxitoxin, neosaxitoxinand gonyautoxins 1 and 2. All of the purified toxins exhibited pharmacological activity consistent with that exhibited by the PSTs. No more than two of these toxins were present in any extract. Statistical analysis demonstrated there were geographic and temporal patterns in the variation of crude toxicity levels. Combined foregut contents from 42 of the crabs comprised fish, crustacean and algal remains, from which 279 MU were extracted from 1.4 g of material (= 199 MUg-I) Key words: poisonous crabs, paralytic shellfish toxins, Atergatis floridus Introduction Human deaths after eating poisonous crabs have been reported from the Philippines [1-4], Palau Islands [5], Japan [6], Mauritius [7] and Singapore [8]. The species implicated in these poisonings were Zosimus aeneus, Lophozozymus pictor, Carpilius masculatus, Demania toxica, D. reynaudii and Eriphia sebana, which all belong to the family Xanthidae. Xanthid crabs are generally bulky, slow-moving walking crabs found in or near coral reef areas. The symptoms exhibited by victims include vomiting, diarrhea, respiratory difficulty, trembling, dizziness, numbness and/or spasms of the mouth, face and limbs. Limb paralysis, convulsions, and abdominal and general pain may follow. Finally, shock may precede death.. To date, saxitoxin (STX) and its derivatives, collectively called the paralytic shellfish toxins (PSTs) [9], tetrodotoxin (TTX) and TTX analogues [10] and palytoxin [11], have been characterized from poisonous crabs. One of these species of poisonous crabs, Atergatis floridus, was first reported as toxic when water-soluble extracts of specimens from the Ryukyu and Amami Islands, Japan killed mice when administered by intraperitoneal *Present address: DepartmentofPharmacology, Yale University School ofmedicine, Sterling Hall of Medicine, POB 3333, New Haven CT , USA /91 $ Chapman and Hall Ltd

2 Toxic crabs from Australian coral reefs 119 injection [12]. Since that time, many of the PSTs and TTX have been found in representatives of this species collected from Fiji, Okinawa, Taiwan and Japan [10, 13-15]. Specimens of Atergatis floridus from Moreton Bay and Heron Island, Australia, have been reported as toxic [16,17] with a toxin similar to TTX occurring in the crabs from Moreton Bay [16]. In contrast, PSTs and not TIX occurred in the xanthid crabs Lophozozymus pictor, Zosimus aeneus and Eriphia sebana, as well as in the portunid crab Thalamita stimpsoni, collected from the Great Barrier Reef [18-20]. It was therefore decided to investigate which toxins may occur in specimens of A. floridus from Heron Island and neighbouring reefs within the Great Barrier Reef. Methods Collection andpreparation ofaqueous extracts Specimens were collected at low tide from the reef crests of Heron Island Reef (23 26'S, 'E), Wistari Reef (23 29'S, 'E) and ErskineIsland Reef (23 18'S, 'E), Capricorn Group, Great Barrier Reef. Heated, acidified 70% ethanol (ph 2.0 with concentrated HCI) was used to extract homogenized crabs. Solvent volume during extraction was maintained at 2 ml g-l of crab. After decanting the solvent, the extract was centrifuged at g for 1 h, followed by filtering through Whatman Grade 1 filter paper. The final crude extract was produced by evaporation in vacuo with a Buchi Type R rotary evaporator, followed by ultrafiltration through an Amicon PMlO membrane ( > mol.wt) [18]. Statistical analysis ofcrude extract assay results The Statistical Analysis Systems package [21] for personal computers was used to analyse for any temporal or geographic variation of crude extract toxicity levels (both total extract toxicity and MUg-l values) or whether any relationship existed between sex, weight or carapace width and length with toxicity. Relationships were tested between collection zone, date or sex with toxicity with the unbalanced two-way ANOVA method in the SAS General Linear Models procedure [22]. Relationships between carapace length, carapace width, and weight and toxicity levels were tested with the SAS correlation procedure [22]. Similar analyses were performed with suitable data previously published for toxicity of individual A. floridus extracts [12,14,23]. Examination and extraction offoregut contents Foreguts of 42 crabs were dissected prior to extraction of the crabs. Presence of food items was recorded prior to storage in 70% alcohol. Gut contents were pooled and filtered through a Whatman Grade 1 filter paper, dried and weighed. The ethanolic filtrate was retained. Foregut contents were homogenized in acidified 70% ethanol (ph 2.0 with HCI) with an IKA-WERK Ultra-Turrax tissue homogenizer. This homogenate was filtered through a Whatman Grade 1 filter paper and the filtrate was combined with the ethanol used for storing the gut contents. The gut contents were re-extracted and the filtrate was added to the extract. Ethanol was removed and the remaining water extract concentrated in vacuo with a Buchi rotary evaporator. Toxin purification and characterization Toxic extracts of crabs collected at the same time from the same area were combined for

3 120 Llewellyn and Endean purification. Separation of the acetic acid eluents of Amberlite CG-50 (NH 4 + or Na+ form, 6 cm X 20 cm, Rohm and Hass) and Bio-Gel P-2 columns (6 cm x 15 cm, Bio-Rad Laboratories, mesh) was involved initially. Further separation was achieved with a step-wise HCI gradient (168 ml HzO, 728 mil mm HCI, 490 ml20 mm HCI) on a 1.5cm x 100cm Bio-Rex 70 column (H+ form; Bio-Rad Laboratories) [18]. Two peaks of toxicity can be eluted from the Bio-Rex 70 column, which correspond with basic and weakly basic PSTs [24]. Toxins occurring in these peaks were separated on a 1.0 cm X 55 cm Bio-Gel P-2 column in 2 mm HCI [18]. Toxins were characterized by thin layer chromatography (TLC) [25,26] and cellulose acetate membrane electrophoresis [27]. TLC was performed on 5 cm X 20 cm, 25 mm thick silica gel 60 glass-backed plates (Sigma Chemical Company, MO, USA). Separate plates were run in the two solvent systems of pyridine-ethyl acetate-acetic acid-water (75:35:15:30) and t-butanol-acetic acid-water (2:1:1). Electrophoresis was performed on 5 cm X 30 cm cellulose acetate strips (Chemetron) with 0.08 MTris-HCI buffer (ph 8.7) at 0.87 ma cm- I for 30 min. Toxins were visualized by spraying with 1% HzOz and 10% KOH in methanol and heating the plates at 120 C for 15 min and viewing under UV light. STX (US Food and Drug Administration) was the standard for TLC and electrophoresis. Non-STX toxins were identified by comparison with ~ and R m values reported for PSTs, TTX and TTX derivatives [25,26,28,29]. Pharmacological characterization ofpurified toxins The pharmacological actions of the purified PSTs were examined by testing the ability of the toxins to reduce the compound action potential (CAP) of the amphibian sciatic nerve [30-32] and their ability to affect isolated mammalian ileum [33]. Specimens of the toad Bufo marinus were pithed and the sciatic nerve dissected. The nerve was placed in a Ringer's solution of the following composition (mm): NaCl; 1.88 KCI; 1.1 CaCl z ' 2HzO; 2.38 NaHC0 3 ; 0.08 NaH z P0 4 ' 2HzO; 11.1 D (+)-glucose. The preparation was laid across four platinum electrodes in the cavity of a flat perspex holder. The CAPs were created by square-wave stimulation of the nerves provided by a Grass S88 stimulator (Grass Instruments, Massachusetts, USA) at a rate of 120 pulses sec-i, with a pulse width of lo- z msec and not allowed to exceed 2V in strength. The CAP was recorded with a Tektronix Model No oscilloscope. Toxin (0.01 MU) was added to the bath. Male or female rats ( g) were killed by a blow to the head and exsanguination. Segments of ilea were removed, cleaned and placed in Ringer's solution of the following composition (mm): NaCl; 2.7 KCI; 2.0 CaCl z 2HzO; 1.03 MgCl z 6HzO; 11.9 NaHC0 3 ; 1.8 NaH z P0 4 ' 2HzO; 5.55 D (+)-glucose, bubbled with carbogen (95% oxygen, 5% carbon dioxide) and maintained at 37 C. A resting tension of 1 g was applied to each preparation. Contractions of the ileal segments were recorded via a Narco Bio-Systems isotonic myograph transducer connected to a Riken Denshi Model SP-H3C chart recorder. Toxin (0.5 MU ml- I ) was added to the organ bath after the preparation had equilibrated and consistently responded to the same dose of acetylcholine (10-6 M). Assay ofextract toxicity to mice Extracts of the foregut contents, the crab extracts and column fractions from their subsequent purification were assayed by intraperitoneal (Lp.) injection of between three and

4 Toxic crabs from Australian coral reefs 121 six male or female Quackenbush mice weighing g. Toxicity was estimated with a PST assay described by the Association of Official Analytical Chemists [34], which defines one mouse unit (MU) as the amount of toxin required to kill a 20 g mouse in 15 min. One of these mouse units corresponds with the approximate LD so dose of saxitoxin in mice of this size [35]. Toxicity values were expressed as the total number of MU in an extract (total extract toxicity) and as MU g-l of crab material. Results Sixty-five of the 109 crab extracts contained detectable toxicity. The combined foregut contents weighed 1.4 g and contained 279 MU (= 199 MU g-l). Intoxicated mice displayed shaking and uneasiness, wobbling gait, cyanosis, strong spasms and jumping prior to death stemming from respiratory paralysis. These signs were consistent with PST intoxication [35,36]. The highest toxicity was 3838 MU (108 MU g-l) in a crab collected from Heron Island in May, No extracts of Erskine Island crabs contained detectable toxicity (see Table 1). Only hard remains of the crab's diet remained identifiable and of the 42 guts examined, 6 contained fish remnants, 22 contained crustacean remains and 2 contained obvious algal remains, which were only identifiable as a green and a brown alga. All the guts contained sand. No significant correlations were found between carapace length, width or crab weight with crude toxicity values from this study or in data analysed from other studies. Significant relationships were detected between collection zone and collection date with both Table 1. Toxicities of crude extracts of Atergatisfloridus Collection area Heron Island Wistari Reef Erskine Island Collection date No. andsex Min. and max. Min. and max. (No. toxic) extract total toxicity(mu g-l) toxicity May F (4) M (5) Nov IF (1) M (2) Feb F (8) M (11) Aug F (0) 7M (3) Feb F (6) M (6) July F (1) M (0) Dec IF (1) M (0) Feb F (4) M (7) Dec F (1) M (5) Feb F (0) 4M (0)

5 122 Llewellyn and Endean total extract toxicity and MU g-l values in this study. Analyses of previously published data revealed a similar significant relationship between collection zone and date with toxicity values for crabs collected from the Ryuku and Amami Islands [12] but not for crabs from Fiji [14] and the Philippines [23]. Eight groups of extracts from crabs collected at the same time from the same area were combined for purification. A ninth group of extracts contained the most toxic extract detected. This extract was purified separately and found to contain a single toxin similar to STX. The remaining extracts of this ninth group of extracts were then combined for purification and contained two toxins which behaved like STX and gonyautoxin 2 (GTX z ). In all, 10 purifications were performed. Although toxins resembling STX, neostx, gonyautoxin 1 (GTX 1 ) and GTX z were detected, no more than two toxins ever co-occurred in any of the groups of extracts examined (see Table 2). Like PSTs, toxins fluoresced under UV light after spraying with 1% HzOz and heating. No toxins were detected with methanolic 10% KOH, indicating the absence of TTX or TTX derivatives [37]. All the toxins behaved similarly to the PSTs by reducing nerve CAPs and the resting tension and magnitude of spontaneous contractions of the guinea-pig ileum. Table 2. Toxins purified from extracts of Atergatisfloridus Collection area Collection date PSTs present],2 Proportion oftotal PSTs (%) present! Heron Island Wistari Reef May STX May GTX STX Nov STX neostx Feb STX GTX 1 Feb STX neostx Feb neostx Aug GTX 1 Feb STX Feb GTX 1 Dec STX neostx Rl, toxin's R f after TLC in pyridine-ethyl acetate-acetic acid-water (75:35: 15:30) Rl, toxin's R f after TLC in t-butanol-acetic acid-water (2:1:1) R m, toxin's relative mobility to STX after cellulose acetate membrane electrophoresis. ISTX, saxitoxin; neostx, neosaxitoxin; GTX, gonyautoxin. 2Toxin identification of non-stx toxins based upon previous reported Rr and ~ values [15,16,18,19]. 3A single highly toxic extract purified separately from the remaining extracts of crabs collected at the same time from the same area which were combined for purification.

6 Toxic crabs from Australian coral reefs Discussion Ingestion of large xanthid crabs (e.g. Zosimus aeneus and Lophozozymus pietor) has caused fatal human poisonings [1-6]. One of the 109 extracts prepared in this study contained 3838 MU, which is greater than the suggested oral lethal PST doses of 2500 and 3000 MU [38,39]. Several other crabs contained high levels of toxin ( MU) and could be considered dangerous. In the main, toxicity of individual crabs in this study is lower than that of specimens of A. floridus from outside Australia. Toxicities of up to 400 MU g-l were reported for specimens of A. floridus from Ishigaki Island, Okinawa [25]. In another study, 8 of 15 specimens of A. floridus individuals also collected from Ishigaki Island contained more than 1000 MU g-t, including one crab with 9000 MU g-t, and no crab had less than 100 MU g-l [11]. Fijian specimens of A. floridus had toxicities ranging from MU g-t, with all 35 crabs collected being toxic [14]. Not all crabs in this study were toxic and the highest toxicity detected was far lower than previously reported maximum toxicities of non-australian crabs. Reef crabs from Australia have a variable toxin content, which statistical analysis indicates has significant temporal and geographic relationships. If the toxin source is dietary, as is likely, this relationship could be explained by the geographic distribution of the source organism and seasonal variation of the toxicity level in the source organism. Such seasonal variation has been demonstrated for the red alga, Jania sp., which is a reputed PST source organism [40]. Regionality of toxicity has also been observed in trumpet shells, which had reputedly accumulated TTX from their diet [41]. As was initially suggested to account for variation in crab toxicities, the toxin source for coral reef crabs is probably dietary [42]. This is supported by the detection in this study of toxicity in the crab gut contents. The toxicity of the gut contents was far greater than the maximum value of 1.5 MU g-l obtained for the red alga, Jania sp., which was suggested as a primary source of PSTs in reef crabs [40,43]. If there is only one exogenous source, the toxicity of the pooled gut contents would be lower than the actual toxicity of the toxic component of the crab's diet, since the toxic component of the crab's diet would be a fraction of the total gut content's mass. PSTs are probably accumulated along a food chain, with Jania sp. being one possible starting point of the toxification food chain. Toxic bacteria in detritus have been implicated as the source of TTX in A. floridus [15]. The presence of sand in all of the gut contents of crabs examined in this study and in a high proportion of crabs in an earlier study [44] may support this suggestion. Intake of coral reef sediment however, may also allow intake of toxic dinoflagellates or their cysts which occur in benthic environments and reefal sediments [45,46]. Toxins similar to paralytic shellfish toxins, as judged by their ~ and R m values, positive responses to 1% HzOz and heating, and their pharmacological actions, were present in extracts of Australian specimens of A. floridus. PST toxin composition of crabs has been suggested to be specific to species, but not to the region of their collection [14]. This suggestion is not supported by this study, as no pattern in the crabs' toxin profiles was apparent. Variation of species' toxin profiles was demonstrated by this study, as one crab extract contained a toxin like STX, while extracts of other specimens collected from the same zone at the same time contained a GTXz-like toxin along with a toxin similar to STX. This finding suggests variation within the same group of crabs collected at the same time from the same area. Similarly, markedly different toxin profiles were present in A. 123

7 124 Llewellyn and Endean floridus from two areas separated by a strait only 100 m wide [15]. Also, A. floridus toxin profiles obtained from crabs collected outside Australian waters include STX, neostx, GTX 2, dcstx [14]; STX, neostx, GTX 2 [25]; and TTX co-occurring with PSTs [13,47]. Five of the ten groups of extracts possessed only one toxin and the remaining groups contained only two toxins. Similarly, only one toxin was detected in extracts of A. floridusfrom Moreton Bay, Australia [16]. Simpler toxin profiles in Australian specimens of A. floridus may indicate a simple toxin profile in a toxin source organism or that metabolic toxin conversion may produce the toxin profile. Conversely, the more complex profiles previously reported for non-australian specimens of A. floridus may result from combining a large number of crabs for extraction, each of which may have had simple, yet different, toxin profiles. Acknowledgements The authors are grateful to the Australian Research Council, Australian Coral Reef Society and University of Queensland for providing financial support. References 1. Gonzales, RB. and Alcala, AC. Fatalities from crab poisoning on Negros Island, Philippines. Toxicon 1977; 15, Alcala, AC., Alcala, L.c., Garth, J.S., Yasumura, D. and Yasumoto, T. Human fatality due to ingestion of the crab Demania reynaudii that contained a palytoxin-like toxin. Toxicon 1988; 26, Alcala, AC. and Halstead, B.W. Human fatality due to ingestion of the crab Demania sp. in the Philippines. Clin Toxicol1970; 3, Alcala, AC. Recent cases of crab, cone shell and fish intoxication on Southern Negros Island, Philippines. Toxicon 1983; Suppl. 3, Mote, G.E., Halstead, B.W. and Hashimoto, Y. Occurrence of toxic crabs in the Palau Islands. Clin Toxicol1970; 3, Hashimoto, Y., Konosu, S., Yasumoto, T., Inoue, A. and Noguchi, T. Occurrence of toxic crabs in Ryukyu and Amami Islands. Toxicon 1967; 5, Halstead, B.W. and Cox, K.W. Case of poisoning by the red spotted crab Carpilius maculatus (Linnaeus) in Mauritius. Proc R Soc Arts Sci Maurit 1973; 4, Tan, C.T.T. and Lee, E.J.D. A fatal case of crab toxin (Lophozozymus pictor) poisoning. Asia Pacific J Pharmacol1988; 3, Noguchi, T., Konosu, S. and Hashimoto, Y. Identity of the crab toxin with saxitoxin. Toxicon 1969; 7, Noguchi, T., Koyama, K., Uzu, A and Hashimoto, K. Local variation of toxicity and toxin composition in a xanthid crab Atergatis floridus. Bull Jpn Soc Scient Fish 1983; 49, Yasumoto, T., Yasumura, D., Ohizumi, Y., Takahashi, M., Alcala, AC. and Alcala, L.C. Palytoxin in two species of xanthid crab from the Philippines. Agric Bioi Chem 1986; 50, Inoue, A, Noguchi, T., Konosu, S. and Hashimoto, Y. A new toxic crab, Atergatis floridus. Toxicon 1968; 6, Noguchi, T., Usu, A., Koyama, K., Maruyama, J., Nagashima, Y. and Hashimoto, K. Occurrence of tetrodotoxin as the major toxin in a xanthid crab Atergatis floridus. Bull Jpn Soc Scient Fish 1983; 49, Raj, u., Haq, H., Oshima, Y. and Yasumoto, T. The occurrence of paralytic shellfish toxins in

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9 126 Llewellyn and Endean 37. Mosher, H.S., Fuhrman, F.A Buchwald, H.D. and Fischer, H.G. Tarichatoxin-tetrodotoxin: a potent neurotoxin. Science 1964; 144, Halstead, B.W. Poisonous and Venomous Marine Animals of the World Princeton, NJ: Darwin Press, Hashimoto, Y. Marine Toxins and Other Bioactive Marine Metabolites. Tokyo: Japan Scientific Societies Press, Kotaki, Y., Tajiri, M., Oshima, Y. and Yasumoto, T. Identification of a calcareous red alga as the primary source of paralytic shellfish toxins in coral reef crabs and gastropods. Bull Jpn Soc Scient Fish 1983; 49, Noguchi, T., Jeon, J.K., Maruyama, J., Sato, T., Saisho, T. and Hashimoto, K. Toxicity of trumpet shells inhabiting the coastal waters of Kagoshima Prefecture, along with identification of the responsible toxin. Bull Jpn Soc Scient Fish 1985; 51, Koyama, K., Noguchi, T., Uzu, A and Hashimoto, K. Individual, local and size-dependent variations in toxicity of the xanthid crab, Zosimus aeneus. Bull Jpn Soc Scient Fish 1983; 49, Yasumoto, T., Oshima, Y., Tajiri, M. and Kotaki, Y. Paralytic shellfish toxins in previously unrecorded species of coral reef crabs. Bull Jpn Soc Scient Fish 1983; 49, Saisho, T., Noguchi, T., Koyama, K., Uzu, A., Kikuta, T. and Hashimoto, K. Examination of stomach contents in xanthid crabs. Bull Jpn Soc Scient Fish 1983; 49, Oshima, Y., Singh, H.T., Fukuyo, T. and Yasumoto, T. Identification and toxicity ofthe resting cysts of Protogonyaulax found in Ofunato Bay. Bull Jpn Soc Scient Fish 1982; 48, Yasumoto, T., Oshima, Y., Marukami, Y., Nakajima, I., Bagnis, R. and Fukuyo, Y. Toxicity of benthic dinoflagellates found in coral reef. BullJpn Soc Scient Fish 1980; 46, Noguchi, T., Uzu, A, Daigo (Koyama), K., Shida, Y. and Hashimoto, K. A tetrodotoxin-like substance as a minor toxin in the xanthid crab Atergatisfloridus. Toxicon 1984; 22,