Nippon Suisan Gakkaishi 53(5),881-884 (1987) Resistibilityof Two Xanthid Crabs Zosimus aeneus and Daira perlata Against Paralytic Shellfish Poison and Tetrodotoxin Kinue Daigo,* Osamu Arakawa,* Tamao Noguchi,* AtsushiUzu,* and Kanehisa Hashimoto* (AcceptedSeptember 12, 1986) Two xanthidcrabs, toxic species Zosimus aeneus and nontoxicspecies Daira perlata were examinedfor resistibility against paralytic shellfish poison (PSP) and tetrodotoxin(TTX). In theformer crab, the minimum lethaldose (MLD) ofscallop PSP consistingmainly of gonyautoxins was estimatedtobe 10,000MU/20g body weight,and MLD ofTTX 1,000MU/20g, bothMLD valuesbeing comparable to thecorresponding values in anothertoxic xanthid crab Atergatis floridusreported previously. On theother hand, D. perlatawas not resistibleagainst scallop PSP and TTX, both MLDs beingaround 1 MU/20g. However,some specimensof thiscrab werefound to survivewhen crabPSP consistingmainly of saxitoxinswas administeredat levels of 100-500MU/20g. In the sea,there inhabitrather many animals 1984. Live specimens of another xanthid crab havingtetrodotoxin (TTX) and paralyticshellfish D. perlata("kanoko-ogigani") were collectedin poison (PSP). They have been causing human IshigakiIsland, Okinawa, in August 1985. poisoningswith a high lethalitysporadically. These specimenswere transportedto our labora- However, littleinformation is availableon the tory and acclimatizedin an aquarium for at least mechanismof theirtoxification. one week. Among those animals,some xanthidcrabs such On the other hand, Z. aeneus specimenswere. asAtergatis floridus and Zosimus aeueusspecifical- also collectedfrom IshigakiIsland, Okinawa, in lyare endowed with both toxins.1-3)In thiscon- 1982, and specimens of the scallopPatinopecten nection,the tropicalspecimens of A.floriduscon- yessoensisfrom Ofunato Bay, Iwate, in 1979. tainedPSP as the major toxin and TTX as the Both of them were transportedto our laboratory minor,4)whereas the specimens inhabitingthe and kept frozen untilbeing used to preparePSP. Pacificcoast of Japan Proper had TTX as.the Pufferovaries used for preparationof TTX were major,2) obtainedfrom frozenFugu vermicularisporphyreus In a previouspaper,5) we reportedthat Japan ("mafugu") specimens. Proper specimens of A. floridusshowed high resistibilitiesto both toxins,which remind us of PartialPurification of PSP and TTX the observations with some TTX-containing Two types of PSP sample were prepared,one animalssuch as puffers6)and a tropicalgoby.7) being rich in gonyautoxins(GTXs) and the other In thissituation, we examined thistime PSP and rich in saxitoxins(STXs) as follows. GTX-rich TTX resistibilityof a tropicalxanthid crab Z. PSP sample was partiallypurified from the scallop aeneus,along with Daira perlatawhich also in- digestiveglands by the previously reported habittropical reefs and is a "nontoxic" speciesof method9)except thatBio-Rex 70 chromatography Xanthidae.8) was omitted. STX-rich PSP sample was prepared from the Ishigakispecimens of Z. aeneus by the Materialsand Methods procedure of purificationof PSP using Bio-Rex 70 chromatography.9) Both samples thus pre- Materials pared are referredto as "scallopPSP" and "crab Live specimens of a xanthid crab Z. aeueus PSP" below. TTX was partiallypurified from the ("umore-ogigani")were collectedin Cebu lsland, pufferovaries by the method previouslyreport- Philippines,in October 1983 through February ed.10) The purityof both PSP preparationswas * Laboratory of Marine Biochemistry , Faculty of Agriculture, University of Tokyo, Bunkyo, Tokyo 113, Japan (醍 醐 絹 江,荒川 修,野 口 玉 雄,宇 津 敦,橋 本 周 久:東 京 大 学 農 学 部). 882 Daigo et al. calculated to be 10-20%, and that of TTX pre- Table 1. Resistibilityof Z. aeneus against PSP and parations about 50%, from specifictoxicity, TTX Each toxin sample was dissolved in3% Na Dose Body Death CIto affordvarious concentrations, and used in the Toxin Sex*1 weight time followingresistibility experiments. (MU/20g) (g) (h)*2 M 39.0 1 Measurement of PSP and TTX resistibility 20,000 M 26.1 0.6 F 33.9 0.4 A calculatedvolume (0.08-0.98ml)of thetoxin solutionsthus prepared was injectedat the carpus M 36.9 (一) of the rightchela of each crab taken from the Scallop PSP 10,000 F 17.8 (一) F 39.9 <24 aquarium. Administered doses of scallopPSP and TTX were 5,000-20,000MU/20g and 500- M 42.5 (一) 5,000MU/20g body weight, respectively,in Z. 5,000 F 35.8 (一) aeneus,and were 1-100 MU/20g and 1-10 MU/ F 39.2 (一) 20g body weight,respectively, in D.perlata. In D 5,000 F 45.8 0.2 . perlata,crab PSP was also administeredat F 49.2 0.3 dosesof 1-500MU/20g body weight. This toxi M 53.1 <24 n was not administeredto Z. aeneus due to the 2,000 F 46.1 <24 scarcityof the sample available. F 59.8 <24 TTX Immediately after injection,the Crabs were M 55.3 (一) returnedinto the aquarium and observedfor any 1,000 F 48.3 (一) signsinduced, or responsesto physicalstimuli F 69.0 <24 with a glassrod or tweezers,usually for 24h (Z. M 75.8 (一) aeneus)or for one hour (D. perlata).The crab 500 M 75.6 (一) was judgedto have been killedwhen itdid not F 63.0 (一) respirenor respondat all,and the timerequired *1 M=male , F=female, was recorded. *2 (一): Survival . Results andDiscussion specimensused were estimatedto be 10,000MU/ 20g and 1,000MU/20g body weight,respectively. The resultsobtained for Z, aeneus are sum These valuesare rathercomparable to the MLDs - marized in Table 1. As to scallopPSP, all the of respectivetoxins in another xanthid crab A threecrab specimens died within one hour after. floridus,5,000-10,000 MU/20g and 1,000-2,000 injection,at 20,000MU/20g body weight.One MU/20g body weight.5) This seems to suggest out of the three specimens died within 24h at that PSP or TTX resistibilityis not significantly 10,000MU/20g, whereas all the three survived species-specificnor depends upon the toxincom- at 5,000Mu/20g. The common signs evoke position,as faras thesetwo crabsare concerned. d were floppinglegs, rapid respiration,paralysis at The results ofresistibility experimentswith D. the rightchela and legsirrespective of the dose of perlatawhere two types of PSP were used are administration,and followedby death in severe summarized in Table 2. When scallopPSP was cases. administered,all the threespecimens died within When administeredwith TTX, on the other 23min at 100 MU/20g body weight. At 10 hand, both specimens died within 0.3h at 5,000 MU/20g, two out of the three specimens died MU/20g body weight. All the three injecte within21min, but one survived. A similarresult d died within 24h at 2,000MU/20g, and one out was obtainedat 1 MU/20g. As to crab PSP, only of the threespecimens died with in 24h at 1,000 one out of the threeadministered died in 2 min at MU/20g. All specimens were found to survive a dose of 500 MU/20g. Two out of the three at 500 MU/20g. The common signsobserved were died within 14min at 100 MU/20g and one out lossof body balance,difficulty in walking, paralysis of the three in 55min at 10 MU/20g. At 1 at the rightchela and legs,and followedby death MU/20g, no specimenswere killed.The common insevere cases. signs ofthe survivedD. perlataevoked by a high From those data, the minimum lethal doeses levelof crab PSP (100, 500 MU/20g) were es- (MLDs) of scallop PSP and TTX in the Z. aeneus sentiallythe same as those in Z. aeneus. It was PSP-and TTX-resistibilityof Two Xanthid Crabs 883 Table2. Resistibilityof D. perlataagainst PSP and resistibleto both toxins than those nontoxic crabs, TTX since the former crab generally required a longer death time than the lattercrabs at the same dose Dose Body Death Toxin Sex*1 weight time level. (MU/20g) (g) (min)*2 On the other hand, the MLD of crab PSP in M 13.5 2 D. perlata was estimated to be 10-100 MU/20g. 100 F 34.7 23 It was noted that two of the three specimens F 28.4 2 survived even when administered at 500 MU/20g M 42.5 21 (Table 2). As a matter of fact, a toxicityassay Scallop PSP 10 M 34.6 10 demonstrated that some specimens of D. perlata F 23.6 (一) were as toxic as 2-12 MU/g, which may allow us M 45.2 (一) to regard this crab as a weakly toxic species(Daigo 1 F 36.2 15 et al., unpublished). All those results suggest F 19.2 38 that there are two strainsin D. perlata,one which, M 13.8 2 to some extent,is resistibleto eithertoxin and can 500 F 23.7 (一) be toxified,and the other which is not at all. F 20.3 (一) In passing, PSP and TTX resistibilityof each crab seemed not to differbetween both sexes. M 19.5 6 100 F 40.5 (一) The mechanism which makes xanthid crabs such F 32.0 14 as Z. aeneus and A. floridusso resistibleto both Crab PSP PSP and TTX, and the mechanism which makes M 16.6 55 D. perlata more resistibleto crab PSP than to 10 F 36.3 (一) scallop PSP, remain to be solved. F 29.4 (一) M 16.1 Acknowledgements (一)1 F 34.0 (一) F 22.8 (一) The expenses of the present study were defrayed M 21.6 4 in part by a Grant-in-Aid from the Ministry of 10 F 36.2 2 Education, Science and Culture. F 34.0 2 TTX M 20.7 (一) References 1 F 36.9 26 F 17.6 5 1) K. Koyama, T. Noguchi, Y. Ueda, and K. *1 M=male Hashimoto: Nippon Suisan Gakkaishi,47, 965 , F=female. *2 (一): survival . (1981). 2) T. Noguchi, A. Uzu, K. Koyama, J. Maruyama, noteworthy that some specimens of D.