Comparative study on the cell toxicity and enzymatic activity of two northern scyphozoan species Cyanea capillata (L.) and Cyanea lamarckii (Péron & Léslieur)
Introduction
Species of the phylum cnidaria are major components of the pelagic system. The jellyfish Cyanea capillata and Cyanea lamarckii belong to the most abundant scyphocoan jellyfish in the northern coastal zone. Scyphomedusae are efficient predators. They express successful foraging behaviour and a wide dietary range. Studies on the trophodynamics have shown an enormous predation on fish eggs, fish larvae and small fish. Another important interaction is the potential competition on prey among cnidaria and zooplanktonivorous fish species especially when they occur as jellyfish blooms (Bamstedt et al., 1994; Purcell and Arai, 2001).
A further concern is the stinging capacity of jellyfish and the resulting public health hazard. At tropical and subtropical coast lines life-threatening and frequent contact between humans and jellyfish are an ongoing field of attention (Bailey et al., 2003).
Envenomations in the northern coastal territories are considered to be much less serious, although with the observed larger population numbers an increasing amount of stings is expected (Mills, 2001; Burnett, 2001). The cnidarian venom is preserved in specialized cell organelles (cnidocysts) which are harboured in cnidocytes situated as batteries at fishing and mesenteric tentacles. Adequate chemical and mechanical signals induce the fast discharge of cnidocysts resulting in immediate paralysis of its prey. Human can respond with cutaneous irritations and pain up to cardiovascular system failure after accidental contact with Cyanea spec. (Burnett, 2001). The reddish-brown pigmented C. capillata is more common and more information on its economic relevance and toxic potency is available (Burnett and Calton, 1987; Heeger et al., 1992). A few in vitro and in vivo studies with crude and partially purified C. capillata venom demonstrate cytolytic and cardiotoxic potencies (Walker, 1977a, Walker, 1977b; Walker et al., 1977; Long and Burnett, 1989). In these investigations mainly cnidocysts of tentacles were used and no special attention was paid on the effect of oral arms or of the surrounding tissue. The venom of C. capillata consists of a broad range of proteinaceous substances and the cardiotoxicity seems to be associated to a 70.000 Da protein whereas the hemolytic activity is supposed to be related to peptides (Walker, 1977a, Walker, 1977b; Long and Burnett, 1989), but causative substances could not finally be isolated and characterized. Comparable in vitro or in vivo studies for the toxicity of C. lamarckii are not performed, although the stinging capacity is specified as low.
The complex nature of the venom and the supposed multitude of pharmacological effects require the application of various in vitro assays to cover different modes of effects. The present paper is a comparative study comprising of two scyphozoan jellyfish species of different sizes, different types of toxigenic organs with a haemolysis assay detecting membrane activity and a cell line-based assay to measure cytotoxicity.
The haemolytic action of venoms is often accompanied by an enzymatic phospholipase A2 activity and a result of synergistic protein action (Hessinger and Lenhoff, 1976). Phospholipase A2 hydrolyzes phospholipids as components of cell membranes at the sn-2 position leading to the cleavage of lysophospholipids and free fatty acids. Metabolites of the fatty acid residue arachidonic acid are well known as secondary messengers in signal transduction and inflammation and control a wide variety of cell functions (Six and Dennis, 2000).
A number of marine invertebrates among them cnidaria have been screened for PLA2 activity. But there is inconsistent information on a PLA2 activity in C. capillata available and no information for C. lamarckii. Whereas Nevalainen et al. (2004a) described a relatively high activity in whole tissue extracts of C. capillata compared to other cnidaria Vaskovski and Suppes (1972) did not detect any activity with a less sensitive Thin Layer Chromatography method. Cnidarian PLA2 may be involved in physiological cell membrane lipid metabolism, in the irritation at the stinging site and in the enhancement of the haemolytic activity.
Section snippets
Material
Roswell Park Memorial Institute cell culture medium type 1640 (RPMI 1640), penicillin/streptomycin solution and phosphate buffered saline (PBS) (10×) were purchased from Invitrogen (Karlsruhe, Germany), Neutral Red, Bradford reagent, bovine serum albumin (BSA) faction V was obtained from Sigma (Munich, Germany) and foetal calf serum (FCS) from PAA Laboratories (Cölbe, Germany).
Organisms
Jellyfish of the species C. capillata (L.) and C. lamarckii (Péron & Léslieur) were collected at a research cruise to
Results
During the ships transect a sufficient amount of animals of both species could be obtained, whereas the C. lamarckii was more abundant and more than 200 organisms could be caught compared to 30 C. capillata (Table 1). Large animals of C. capillata (20–40 cm diameter) were collected in July around Helgoland.
Cnidocyst and extract preparation
The preparation and purification of cnidocysts from the toxigenic organs is a time consuming process. In order to maintain the venom activity the process has to be kept as fast as possible. Harsh physical or chemical conditions have to be avoided to prevent cnidocyst rupture. Basic principles for cnidocyst isolation are tissue extrusion in particular media, autolysis in sea or distilled water and mechanical techniques such as homogenization (McKay and Anderson, 1988). In the present study, a
Conclusion
Species of the genus Cyanea belong to the most abundant scyphozoa in the northern coastal territories. The stinging capacity of Cyanea spec. is described as low but in vitro studies to characterize the true toxic potency are not available for C. lamarckii and sporadic for C. capillata.
Comparing various cnidocyst isolation and tissue extraction procedures we found out that the isolation of intact cnidocysts by autolysis of untreated tissue in distilled water with gentle stirring prior to
Acknowledgement
The authors gratefully acknowledge Dr. I. Sötje (University of Hamburg, Biocentre Grindel and Zoological Museum, Section: Functional Morphology) and H. Döpke (AWI Bremerhaven, Station Helgoland) for the support in species taxonomy and tissue preparation.
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