Toxinological studies on the nematocystvenom of Chrysaora achlyos

Faisal F.Y. Radwana, Lisa-Ann Gershwinb, JosephW. Burnetta,*

aDepartment of Dermatology, University of Maryland, School of Medicine, 405 W. Redwood Street, 6th

Floor, Baltimore, Maryland 21201, USAbLaboratory of Integrative Biology, Museum of Paleontology, University of California, Berkeley, CA,

USA

Received 30 September 1999; accepted 11 December 1999

Abstract

Nematocyst venoms from both oral arms and lappets of Chrysaora achlyos were preparedand found to have factors producing mouse lethality, hemolysis and hepatocyte toxicity.These venoms had less potency than those of Chrysaora quinquecirrha a phylogenetic,

congeneric cousin. Envenomated bathers had signi®cant species-speci®c anti venom IgGand also cross-reacting antibody to Chrysaora quinquecirrha nematocyst venoms. Therewere similarities and contrasts in the capillary electropherograms and sodium dodecyl

sulfate (SDS) gels between C. achlyos nematocyst venoms and those of their C.quinquecirrha counterparts. 7 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Jelly®sh; Chrysaora; Venoms; Toxins

1. Introduction

Chrysaora achlyos (Ca ), a large new species of Scyphozoa has appeared on the

southwest coast of the United States and Northern Mexico at least three or four

0041-0101/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.

PII: S0041 -0101 (00)00092 -1

Toxicon 38 (2000) 1581±1591

www.elsevier.com/locate/toxicon

* Tel.: +410-328-5766; fax: +410-328-0098.

E-mail address: jburnett@umaryland.edu (Joseph W. Burnett).

times this century. It was ®rst described as a species in 1997 (Martin et al., 1997).At that time, only a few minor cutaneous stings had been experienced bycollectors and museum handlers (Martin and Kuck, 1991). Between mid July andlate August 1999, this animal reappeared between San Diego and Los Angeles,CA. Several hundred cases of mild cutaneous stinging occurred in bathers exposedto tentacles, which had been fragmented in the surf. The stings resulted inurticarial lesions of very short duration Ð less than an hour. There was noincidence of severe cutaneous or other systemic reactions. There was intermittentmild local pruritus and hyperpigmentation lasting about a week. A few incidencesof nausea were reported and one swimmer had brief shortness of breath.Lifeguards reported that immersion in mildly warm water would alleviate the painbut not shorten its duration (personal communication from California lifeguardsand marine biologists Ð Eric Johnson, Michael Shaadt, James D'Amato, B.Bass). Intact specimens of this jelly®sh were seen at sea o� the California coastand about 20 were collected to be shipped to various museums. Because of theintermittent appearance of this animal, and its noxious menace for beach users, atoxinological study was performed on the available specimens.

2. Materials and methods

2.1. Nematocyst preparation

Specimens of Ca were collected in California in 1999. Tentacles were manuallyseparated into two groups: (a) oral arms or mesenteric tentacles (MT) and (b)®shing tentacles plus lappets (FT), then removed from the bell. Tentaclenematocysts were then obtained by autolyzing the tissue for 1±3 days at 48C withrepeated changes of sea water (Burnett et al., 1992). After autolysis, thenematocysts were separated from the tissue by sedimentation and decanting, andthen larger tentacle parts were removed with forceps. This material was thenfrozen and shipped to Baltimore for analysis.

2.2. Toxin preparation

Each of the two nematocyst preparations were thawed, ®ltered and ruptured bysonication on ice using a Branson soni®er (Branson Instruments, CT, USA). Theresultant ¯uids were then clari®ed by centrifugation (13,500 g) for 1 h and used ascrude venom.

2.3. Protein determination

Protein concentrations were determined by the technique of Waddell (1956)comparing the absorbance of solutions at 225 and 215 nm on a Shimadzu UV-240recording spectrophotometer [protein (mg/ml)=(Ab 215ÿAb 225)� 144].

F.F.Y. Radwan et al. / Toxicon 38 (2000) 1581±15911582

2.4. Gel electrophoresis

Sodium dodecyl sulfate (SDS)±polyacrylamide gel electrophoresis was carriedout by the method of Laemmli (1970) in 7.5±10% discontinuous gels at 200 V for45 min in Tris±glycine bu�er at 48C and protein bands visualized with silver(Wray et al., 1981). Molecular masses were determined by comparison to Bio-RadBroad range standards: myosin 200 kDa; b-galactosidase 116.25 kDa;phosphorylase b 97.4 kDa; serum albumin 66.2 kDa; ovalbumin 45 kDa; carbonicanhydrase 31 kDa; trypsin inhibitor 21 kDa; lysozyme 14.4 kDa; aprotinin6.5 kDa.

2.5. Mice lethality

Mice i.v. lethality was determined by tail injections of 0.2±0.8 ml volume ofserial two-fold dilutions using at least two mice (20±25 male mixed bred, CharlesRiver) per dilution.

2.6. Enzyme-linked immunosorbent assay (ELISA)

An ELISA method, recently described for detecting antibody titers againstnematocyst venoms was used (Burnett et al., 1988). Brie¯y, 10 mg of the venom in50 ml 0.02 M phosphate-bu�ered saline (PBS), pH 7.2, was air-dried onto separatewells of a polystyrene microtiter plate. The plate was washed in PBS, 0.05%Tween 20, coated with 1% bovine serum albumin in distilled water (100 ml/well)for 30 min, again washed and 50 ml aliquots of di�erent dilutions of patient serumwere added to each. After 2 h incubation at 378C, the wells were washed and 50 mlof alkaline phosphatase labeled anti-human IgG was added to each well. After anadditional 1 h incubation at 378C, the plates were washed and each well reactedfor 15 min with 100 ml p-nitrophenyl phosphate substrate solution. The reactionwas terminated by the addition of 50 ml of 3N NaOH solution and the absorbanceat 405 nm was determined in a Bio Rad microplate reader (model 2550, Japan).Titers were then expressed as the reciprocal of the maximum dilution of the serawhich gave a positive colorimetric reaction.

2.7. Cytosensor analysis

The cells used were human liver cells (ATCC CCL-13), a non-malignant cellline established in 1954, as American Type Culture Collection.

The cytosensor microphysiometer (Molecular Devices, Sunnyvale, CA)measured the e�ects of various venom preparations on cellular metabolic rates.The instrument has four computer controlled independent channels which runsimultaneously, using one or two as control. The liver cells were plated in sterilecapsules (molecular devices) at a density of 2 � 105 cells/ml/capsule. Within 1 dayof plating, the capsules were placed in their microvolume chambers in thecytosensor and the cells perfused with minimum essential medium (MEM) (Gibco,

F.F.Y. Radwan et al. / Toxicon 38 (2000) 1581±1591 1583

BRL Products, Gaithersburg, MD), containing 5 mM glucose at a ¯ow rate of0.1 ml/min. Glucose metabolism produces lactic acid by glycolysis, or CO2 byoxidative respiration. The acidi®cation rates were measured automatically every2 min by halting the ¯ow of medium while recording the pH value every secondfor 30 s. The pH was recorded, and the acidi®cation rate (uV/s) was used as ameasure of metabolic activity as described previously (Cao et al., 1998).Acidi®cation rates of controls (i.e. untreated cells) were designated as 100% whenthe e�ects of treatments were compared as % of control.

Controlled metabolic activity levels of all cells in four channels were recordedfor 60 min prior to testing. Stock solutions of Ca FT and MT nematocyst venomswere prepared in test media and diluted to the desired concentrations immediatelybefore testing to guard against degradation of test agents by proteases or esterasespresent in the media or released by the cells. All tests were performed at leasttwice for a 30 s exposure time. Venoms were added at increasing proteinconcentrations of 1, 10, 35 mg/ml.

2.8. Hemolytic tests

The ability of crude nematocyst venoms to hemolyze red blood cells (RBCs)was monitored as described by Klug et al. (1989) as follows; RBCs from freshlydrawn human blood were washed several times in a bu�er containing 120 nMNaCl, 2 mM EDTA, 25 mM Na±phosphate (pH 7.4). Aliquots of 1 ml of a 0.5%RBC suspension were incubated overnight at 48C with di�erent proteinequivalents of nematocyst venoms at ®nal concentrations of 0.5±75 mg/ml. Theincubation was stopped by centrifuging the samples for 5 min (7000 g). Then thestill intact RBCs and the ghosts of the lysed specimens were separated from thereleased hemoglobin.

The concentration of the latter present in the supernatant was determinedspectrophotometrically at 545 nm. The lytic potency of the tested venoms isexpressed as % hemoglobin released compared to the total hemoglobin presentafter maximal lysis (measured after the addition of 2 ml of 0.5% SDS).

2.9. Capillary electrophoresis (CE)

CE experiments were performed on a P/ACE System 2100 (BeckmanInstruments, Fullerton, CA, USA) with temperature setting of 158C and UVdetection at 214 nm. The capillary used was 37 cm � 75 mm ID eCAPpolyethylene glycol internally coated fused silica. Samples were loaded by pressureinjections (5 psi/s) for 30 s and separations were performed under 5 kV in areverse polarity mode (anode at the outlet) for 30 min. The concentrations ofsamples were 50 mg/ml each in triple distilled water. The bu�er utilized was 0.5 Mphosphate adjusted to pH 7 and diluted to 0.05 M concentration. Before use, allbu�ers were ®ltered through a 0.45 mm pore size ®lter (Millipore, Bedford, MA,USA). Prior to each run, the capillary was rinsed with 1 M NaOH for 2 min,

F.F.Y. Radwan et al. / Toxicon 38 (2000) 1581±15911584

followed by a 5 min rinse with the running bu�er. Data were collected andanalyzed with System Gold Software (Beckman Instruments).

3. Results

Nematocysts of Ca had the same morphology (isorhizas and euryteles) and sizevariation as their relative, the sea nettle, C. quinquecirrha (Cq ) (Fig. 1). The CaMT and FT venoms had i.v. mouse lethality values of 8 and 6.5 mg protein/kgmouse respectively Ð values less toxic than those of Cq (Table 1). The 50%hemolysis value for the FT venom was slightly higher than that of the MT butcertainly below that of Cq FT (Table 1 and Fig. 2).

Sera from four patients were investigated. Two had been stung repeatedly byCq and two had been stung in 1999 by Ca. All four individuals had their highesttiter against the homologous venom but also cross reactivity to the heterogenousvenom (Table 2).

Major protein peaks appeared in the post-capillary electrophoresis eluatefollowing injection of Ca crude FT nematocyst venom after 4.8±10, 13 and 26 minof ¯ow time. This is in contrast with the results of the same procedure using CaMT nematocyst venom, where protein peaks appeared at 9 min then 14, 18 and

Fig. 1. Photomicrograph of nematocyst preparations from FT (a) and MT (b) of C. achlyos were

compared to those of C. quinquecirrha mixed tentacles (c). The specimens contain large isorhizas (large

arrows) and large euryteles (medium-sized arrows), plus numerous smaller type of euryteles (small

arrows). Magni®cation �400.

F.F.Y. Radwan et al. / Toxicon 38 (2000) 1581±1591 1585

Table

1

ComparisonofC.achlyos(C

a)andC.quinquecirrha(C

q)nem

atocyst

venom

toxinologicalactivities

Jelly®sh

Nem

atocyst

venom

preparationa

LD

50i.v.in

mice(mg/g)d

50%

Hem

olysis(mg/m

l)d

Minim

alcytosensorpotency

(mg/m

l)d

Ca

FT

6.5

150

10

MT

8220

35

Cq

FT

0.37

63

1.76

MT

1.75b

ND

cND

aFT

ÐFishingtentaclepluslappets;MT

Ðoralarm

s.bKelmanet

al.,1984.

cNotdonebutother

studiesrevealhem

olysisoflappetsmore

potentthen

thatoforalarm

s(BurnettandCalton,1976).ND

Ðnotdone.

dmg

designatesprotein.

F.F.Y. Radwan et al. / Toxicon 38 (2000) 1581±15911586

28 min. There was overlap in these two preparations (Fig. 3). The elution patternswere distinctly di�erent from those of the two tentacle nematocyst venoms of Cq(Bloom et al., submitted).

Cytosensor analysis of the FT nematocyst venom showed minimal cytologicdamage to cultured human liver cells after 30 s exposure to 10 mg protein/mlwhereas the MT nematocyst venom produced similar damage after 35 mg/mlexposure (Table 1).

SDS±gel electrophoresis showed that the FT or MT nematocyst venom from Cqhad a di�erent protein pattern than either of the two Ca preparations (Kelman etal., 1984; Cobbs et al., 1983). The most distinct bands seen on the Ca FT

Fig. 2. Comparison of hemolytic activities of both C. achlyos: Ca FT, Ca MT and sea nettle C.

quinquecirrha nematocyst venoms on human erythrocytes. Di�erent concentrations of each venom were

incubated with 0.5% RBC suspension for 18 h at 48C. Hemolysis percentages were calculated as

described in Section 2.8.

Table 2

Comparative titers of patients stung by Chrysaora jelly®sh

Titersa against venom from

Patients (age in years, sex) Stung by Interval since last sting Cq FTb Ca FTb Ca MTc

45 male Ca 1 month 450 3600 1800

32 male Ca 6 weeks 900 3600 3600

55 female Cq 1 year 900 450 0

58 male Cq 5 months 3600 1800 900

a Titers express the reciprocal of the maximum serum dilution still producing a positive colorimetric

reaction.b FT±®shing tentacles plus lappets.c MT±oral arms or mesentric tentacles.

F.F.Y. Radwan et al. / Toxicon 38 (2000) 1581±1591 1587

Fig. 3. Capillary electrophoresis chromatograms of C. achlyos FT nematocyst venom (a) and that from

MT (b). The elutions were performed on an eCAP silica-coated capillary (75 mm ID� 37 cm), at 158C,constant voltage of 5kV and UV detection at 214 nm. A sample of each venom (50 mg/ml) was injected

under pressure for 30 s in all runs. The running bu�er was 0.05 M phosphate, pH.7.

F.F.Y. Radwan et al. / Toxicon 38 (2000) 1581±15911588

nematocyst venom appeared near a molecular weight of 55 kDa whereas darkbands appeared in the MT nematocyst venom column after 35, 50, 55 and100 kDa (Fig. 4).

4. Discussion

C. achylos (Ca ) made its ®rst appearance in the Western Paci®c ocean severaldecades ago (Martin et al., 1997). It is the largest of the Chrysaora species andwas known to be venomous since a marine biologist who had previously handledpreserved specimens had experienced slight stings on his ®ngers. This year severalswimmers had noticed slight stings of short duration Ð a reaction thought to belimited because only fragmented tentacles survived in the surf. However, as Table 1clearly shows, the animals' nematocyst venom is not as toxic to mice, erythrocytesor hepatocytes as that of its smaller Eastern states cousin. This lesser potency

Fig. 4. SDS±PAGE analysis of C. achlyos venom constituents. A 7.5% polyacrylamide mini-slab gel

was prepared as described by Laemmli (1970). An aliquot of 10 mg protein for each sample was

injected into each well. Protein bands were stained by silver. Lanes: (1) Bio Rad protein standards; (2)

aliquot of C. achlyos MT nematocyst venom; (3) aliquot of C. achlyos FT nematocyst venom; (4)

aliquot of C. quinquecirrha ®shing tentacle nematocyst venom.

F.F.Y. Radwan et al. / Toxicon 38 (2000) 1581±1591 1589

appears to be fortunate for the unsuspecting Californian swimmers who mightcontact this large beast. It also shows that jelly®sh size and injurious potency arenot correlated. Additionally, it is another example that the venom's characteristicsnot the nematocyst's structure is the important factor determining toxicity sincethese two animals (Ca and Cq ) possess the same cnidom.

Ca FT nematocyst venom was more potent in our bioassays than its MTcounterpart (Table 1). This ®nding is in keeping with the present and pastobservations between the nematocyst venoms of Cq (Table 1; Burnett and Calton,1977, 1987).

Signi®cant amounts of IgG were present in the sera of two healthy adult malesstung 4±6 weeks previously by Ca. These individuals had cross reacting antibodiesto Cq, an animal they had never contacted. The reverse observations were true fortwo adult residents living 3000 miles away who were only exposed the ChesapeakeBay Cq. These investigations show that IgG can be stimulated within a few weeksthen persist and that the highest titers are directed toward epitopes found in thehomologous species.

The CE and SDS±gel electrophoresis experiments show both similarity andcontrasts in the location of venom protein contents. A major protein componentin Cq venom had a 19 kDa molecular weight (Burnett and Calton, 1976) whereasthat of Ca was 55 kDa.

The investigations reported above established a relationship between thenematocyst venoms of Ca and Cq and demonstrated the relatively increasedtoxinological potency of Cq. We were fortunate to obtain as much research dataas presented only because Ca came ashore this year in an area populated by alertmarine biologists such as Eric Johnson and Michael Shaadt (seeacknowledgment). This coincidence might not exist at future sightings Ðwhenever they occur.

Acknowledgements

The authors wish to acknowledge the aid of Eric Johnson of the BirchAquarium at Scripps Institute, La Jolla, CA; Michael Shaadt of the CabrilloAquarium, San Pedro, CA; the Solana Beach (CA) lifeguards, Holly A. Erdelyand Debra Modlin of Baltimore, MD. This research was supported in part fromfunds of the Egyptian Department of Missions.

References

Bloom, D.A., Eldefrawi, A.T., Radwan, F.F.Y., Burnett, J.W., Capillary electrophoresis fractionated

Chrysaora quinquecirrha (Desor) ®shing tentacle and Chironex ¯eckeri Southcott nematocyst

venoms. II Cytolytic e�ects, (submitted).

Burnett, J.W., Calton, G.J., 1976. A comparison of the toxicology of the nematocyst venom from sea

nettle ®shing and mesenteric tentacles. Toxicon 14, 109±115.

F.F.Y. Radwan et al. / Toxicon 38 (2000) 1581±15911590

Burnett, J.W., Calton, G.J., 1977. The chemistry and toxinology of some venomous pelagic

coelenterates. Toxicon 15, 177±196.

Burnett, J.W., Calton, G.J., 1987. Venomous pelagic coelenterates: chemistry, toxicology, immunology

and treatment of their stings. Toxicon 25, 581±602.

Burnett, J.W., Calton, G.J., Fenner, P.J., Williamson, J.A., 1988. Serological diagnosis of jelly®sh

envenomations. Comp. Biochem. Physiol. 91C, 79±83.

Burnett, J.W., Long, K.O., Rubinstein, H., 1992. Beachside preparation of jelly®sh nematocyst venom.

Toxicon 30, 794±796.

Cao, C.J., Eldefrawi, A.T., Burnett, J.W., Mioduszewski, R.J., Menking, D.E., Valdes, J.J., 1998.

Toxicity of sea nettle toxin to human hepatocytes and the protective e�ects of phosphorylating and

alkylating agents. Toxicon 36, 269±281.

Cobbs, C.S., Gaur, P.K., Russo, A.J., Warnick, J.E., Calton, G.J., Burnett, J.W., 1983.

Immunosorbent chromatography of sea nettle (Chrysaora quinquecirrha ) venom and

characterization of toxins. Toxicon 21, 385±391.

Kelman, S.N., Calton, G.J., Burnett, J.W., 1984. Isolation and partial characterization of a lethal sea

nettle (Chrysaora quinquecirrha ) mesenteric toxin. Toxicon 30, 139±144.

Klug, M., Weber, J., Tardent, P., 1989. Hemolytic and toxic properties of Hydra attenuata

nematocysts. Toxicon 27, 325±339.

Laemmli, U.K., 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4.

Nature 227, 680±685.

Martin, J.W., Gershwin, L., Burnett, J.W., Cargo, D.G., Bloom, D.A., 1997. Chrysaora achlyos, a

remarkable new species of scyphozoan from the Eastern Paci®c. Biol. Bull. 193, 8±13.

Martin, J.W., Kuck, H.G., 1991. Journal associates of an undescribed species of Chrysaora (Cnidaria,

Schyphozoa) in the Southern California Bight, with notes on unusual occurrences of other warm

water species in the area. Bull Southern California Acad. Sci. 90, 89±101.

Waddell, W.J., 1956. A simple ultraviolet spectrophotometric method for the determination of proteins.

J. Lab. Clin. Med. 48, 311±314.

Wray, W., Boulikas, T., Wray, V.P., Hancock, R., 1981. Silver staining of proteins in polyacrylamide

gels. Anal. Biochem. 118, 197±203.

F.F.Y. Radwan et al. / Toxicon 38 (2000) 1581±1591 1591