Toxicity and paralytic shellfish toxin profiles of the xanthid crabs, Lophozozymus pictor and Zosimus aeneus, collected from some Australian coral reefs

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0101!89 T3 .00+ .00 .y1 19R9 Pergamon Press pk

Toriron Vol. 27 . Tio. 5, pp . 59(r000, l9ä9 . Printed in Great Britain.

TOXICITY AND PARALYTIC SHELLFISH TOXIN PROFILES OF THE XANTHID CRABS, LOPHOZOZYMUS PICTOR AND ZOSIMUS AENEUS, COLLECTED FROM SOME AUSTRALIAN CORAL REEFS L. E. LLEWELLYNI and R. ENDEAN Z 'School of Biochemistry, University of New South Wales, PO Box L, Kensington, New South Wales, Australia, and 2 Department of Zoology, University of Queensland, St Lucia, Brisbane, Queensland, Australia (Acceptedfor publication 6 December 1988)

L. E. LLewt:LLnv and R. ENDEAN . Toxicity and paralytic shellfish toxin profiles of the xanthid crabs, Lophozozymus pictor and Zosimus aeneus, collected from some Australian coral reefs. Toxicon 27, 59600, 1989 .-Purification of toxic aqueous extracts from the xanthid crabs Zosimus aeneus and Lophozozymus pictor, collected from Australian waters, yielded paralytic shellfish toxins, including saxitoxin (STX), neosaxitoxin (neoSTX) and gonyautoxins 1, 2 and 4 (GTXI,z,4) . No more than two paralytic shellfish toxins were found in any of the purified extracts from any specimen . Four specimens of Z. aeneus and one specimen of L. pictor each contained more toxic material than the suggested human oral lethal dose . The moult of a specimen of L. pictor was toxic, which may indicate a route in crabs for toxin removal . Tr-tE cxAas Zosimus aeneus and Lophozozymus pictor (Xanthidae) have been implicated in human poisonings (Host-nMOTO et al., 1967 ; Mom et al., 1970; GONZnLFS and ALCALA, 1977 ; ALCALA, 1983). Specimens of Z. aeneus contain in their flesh, paralytic shellfish toxins (PSTs) (KOYAMA et al ., 1981), as well as tetrodotoxin (TTX) and some of its derivatives (YASUMURA et al., 1986), whilst L. pictor has been shown to contain palytoxin (YASUMOTO et al., 1986). L. pictor and Z. aeneus were first recorded as toxic in Australia by LLEWELLYN and ENDEAN (1987) . Some of the toxins present in the Australian crabs belonging to these species have now been characterized and this paper describes their isolation and characterization. Specimens of Z. aeneus were collected from exposed surfaces and amongst rubble at Heron Island Reef (23°26'8, 151 °5TE), Capricorn Group, Great Barrier Reef. Specimens of L. pictor were also collected from Heron Island and from nearby Wistari Reef (23°29'8, 151 °53'E) as well as from Bird and Goat Island Isthmus (27°30'S, 153°23'S), Moreton Bay, south-east Queensland . Crabs were frozen for storage until required . One specimen of L. pictor from Moreton Bay was maintained in an aquarium until the crab moulted. Both crab and moult were then extracted. Crabs were extracted as described by LLEWELLYN and ENDEAN (1988) . This method involves the use of acidified 70% ethanol (pH 2.0 with concentrated HCl) to extract toxins from homogenized crabs on a vibrating sand bath at 120°C. Solvent volume during extraction was maintained at 2 ml solvent/g of crab material . 596

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After decanting the solvent, the extract was centrifuged at 16,500 x g for 1 hr followed by filtering through Whatman Grade 1 filter paper. The extract was then evaporated in vacuo with a Buchi rotary evaporator prior to ultra-filtration of the aqueous extract through an Amicon PIv~ 10 membrane ( > 10,000 mol.wt) to produce the final extract. Purification of toxins extracted from the crabs was achieved by utilizing a modification of the method mf VII-nT~LI~r-S1~uTx et al. (1985) as previously described by LLSWELLYN and ENDEAN (1988) . Separation of the acetic acid eluents of Amberlite CG-50 (NH4+ or Na+ form, 6 x 20 cm, Rohm and Hass) and Bio-Gel P-2 columns (6 x 15 cm, Bio-Rad Laboratories, 20000 mesh) was involved initially. Further separation was then achieved with a 1 .5 x 100 ctn Bio-Rex 70 (H+ form) column (Bio-Rad Laboratories) followed by purification of toxic peaks on a 1 .0 x 55 cm Bio-Gel P-2 column in 2 mM HCI. Toxins were characterized by thin layer chromatography (TLC) (YASUMOTO et al., 1981 ; NOGUCHI et al., 1982) and cellulose acetate membrane electrophoresis (FnLLON and SHIMiZU, 1977). Saxitoxin (U .S . Food and Drug Administration) was used as a reference for calculating R, values after electrophoresis and as a standard for TLC. Toxic extracts from specimens of the one species collected at the same time from the same collection zone were combined for purification. Crude extracts and column fractions from their subsequent purification were assayed by i.p . injection into three to six male or female Quackenbush mice weighing 18-23 g. Lethality was determined according to the A.O .A .C. procedur e of HORWITZ (1980), which defines one mouse unit (MU) as the amount of toxin required to kill a 20 g mouse in 15 min. Tesr.E 1 . LECFtAL117F3 a+ eQuDOUS nrra~crs oP sracu~nts oF Zosimus aeneus eNn LophozozymL.s pictor

Collection date

Sex

Crab weight (p~

Total toxicity (MU)

Toxicity (MU/g)

Heron Island

May 1983

Heron Island

Nov. 1983

Heron Island

Feb. 1985

Heron Island

Dec. 1985

M M M F F F M F F M M F M M M F F

144.3 115.5 114.3 91 .7 86 .6 116.4 190.3 52 .0 109.2 33 .2 73 .8 (09.5 59 .1 75 .6 99 .1 78 .9 53 .3

303.0 716.1 5223.5 2613.5 5975 .4 6087 .7 589.9 2605.2 5547.4 57 .9 155.2 511 .8 27 .2 50 .2 288.7 30 .5 60 .0

2.1 6.2 45 .7 28 .5 69.0 52.3 3.1 50.1 50.8 1.7 2.1 4.7 0.5 0.7 2.9 0.4 1.1

F M F M F M M

150.4 146.5 215.7 28 .8 106.9 129.8 108.3

1082 .9 2768 .9 156.5 28 .0 26 .8 23 .2 101 .5

7.2 18.9 0.7 1 .0 0.2' 0.2 0.9

Collection area Zosimus aeneus

Lophozozymrv pictor

Heron Island

Nov. 1983

Heron Island Wisten Reef Morston Bay

Feb. 1985 Feb. 1984 May 1984 Oct.1985

'This crab was maintained in an aquarium until it moulted and then the crab and moult were extracted.

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Toxicities for crude extracts were expressed as total number of MU in an extract (total extract toxicity) and as MU/g of crab material (see Table 1). Apart from these toxic extracts, three specimens of L. pictor and nine specimens of Z. aeneus contained no detectable toxicity . The moult of the specimen of L. pictor maintained in an aquarium contained 28.9 MU (=0.3 MU/g). The toxins characterized in extracts from the crabs are listed in Table 2. Six combined groups of toxic extracts including five groups of extracts from Z. aeneus and one group of extracts from L. pictor were purified. Toxins chromatographically and electrophoretically similar to the paralytic shellfish toxins, saxitozin (STX), neosaxitoxin (neoSTX), gonyautoxin-1 (GTX,), gonyautoxin-2 (GTX~ and gonyautoxin-4 (GTX4) were variously found in the extracts . None of the toxins possessed R t or Rm values that corresponded with TTX or TTX derivatives. Neither were any toxins detected with methanolic 10°1° KOH, indicating the absence of TTX or any of its derivatives (MosllElt et al., 1964). Two of the combined toxic extracts from Z. aeneus and the extract from L. pictor contained one paralytic shellfish toxin and the remaining two extracts contained two paralytic shellfish toxins . ENnEnN et al. (1983) found only one toxic peak in extracts of Australian specimens of A .1loridus which was chromatographically similar to TTX. LLEWELLYN and ENDEAN (1988) reported only one paralytic shellfish toxin in extracts of Australian specimens of the portunid crab, Thalamita stimpsoni. Previously, up to six paralytic shellfish toxins have been found in some crabs (Rn.l et al., 1983), and paralytic shellfish toxins and TTX co-occur in some crabs (YnsuMUxn et al., 1986). The reduced number of toxins found in Australian crabs may indicate that a simpler toxin profile is present in an organism from which the toxins may be accumulated or that metabolic toxin conversion may be influenced by environmental factors producing a simple toxin profile. ILv et al. (1983) showed that Z. aeneus specimens from Okinawa and Fiji exhibited similar paralytic shellfish toxin profiles and suggested that the toxin profile was species specific and unrelated to region . This suggestion is not supported by the present study because of the toxin profiles of Z. aeneus found in this study and those found by RnJ et al. (1983) are dissimilar . Only palytoxin has been previously found in extracts of L. pictor (YASUMOTO et al., 1986) and this report is the first record of paralytic shellfish toxins in this species of crab . A specimen of Z. aeneus had the highest total toxicity of about 6088 MU, which is higher than the suggested lethal oral dose of paralytic shellfish toxins for humans of 2500 MU T~t.t: 2. P~t.rnc cctar.i mosr ~roxnvs rx~mvr nv ncriucts oc Z. aeneus exn L.pictor cor t~ct~ Pxoe~ I-IOeoty lsuxn, Gxe~r HNenrnx lti~ Species L. pictor

Collection date

Z. asneus

Nov. Nov. Nov . Dec.

Z. asneus

Feb. '85

Z. asneus Z. asneus

'83 '83 '83 '85

R,'

R,z

Rm

PSTs present

Proportion of toxicity

0.77 0.78 0.58 0.77 0.77 0.69 0.86

0.60 0.62 0.33 0.44 0.44 0.34 0.7I

0.55 0.57 1 .00 0.66 0.05 0.68 0.32

GTX2 GTX= STX GTX, GTX, neoSTX GTX,

100% 100% 100% 2T% 73% 63% 37%

STX, saxitoxin; neoSTX, neosaxitoxin; GTX, gonyautoxin; Rs', toxin's Rr after TLC in pyridine~thyl acetate-acetic acid-water (75 : 35 :15 : 30); Rs~, toxin's Rs after TLC in t-butanol-acetic acidJuvater (2 :1 : 1); Rm, toxin's relative mobility compared to pure saxitoxin standard after cellulose acetate electrophoresis; PST, paralytic shellfish toxin.

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(HALSTEAD, 1978) and 3000 MU (HAS1uMOTO, 1979). Three other extracts from Z. aeneus contained more than 3000 MU and another two had more than 2500 MU . One extract of L. pictor contained more than 2500 MU but less than 3000 MU . Several other crabs had relatively high levels of toxin (1000-2500 MU) and could be considered to be dangerous if eaten by small or weak persons or if more than one crab were eaten. The toxicity of Z. aeneus is lower than reported toxicities from outside Australian waters . KOYAMA et al. (1983) reported a toxicity of 16,500 MU/g from a 45 g specimen of Z. aeneus collected from Ishigaki Island, Okinawa. The toxic material in the moult of a specimen of L. pictor may indicate the existence of an indirect detoxification mechanism involving removal of toxins via the carapace of crabs. The exoskeletons of some species of crabs are known to contain high levels of toxins (Koxosu et al., 1969; GARTH and ALCALA, 1977 ; YASUh(OTO et al., 1986) and the finding by this study of toxic material in the moult of a specimen of L. pictor suggests that removal of a detectable amount of toxins occurs at the time of moult. However, the actual importance of this detoxification mechanism remains to be determined .

Acknowledgements-The authors thank the Australian Research Council, Australian Coral Reef Society and University of Queensland for financial support .

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