Molecular characterization of Carukia barnesi and Malo kingi, Cnidaria; Cubozoa; Carybdeidae

This thesis reports the molecular characterization of C. barnesi Southcott, 1967 and Malo kingi Gershwin, 2007; two Australian box jellyfish responsible to trigger, a complex and variable illness in humans, termed -Irukandji syndrome-. This is characterised by a 30 min delay before severe systemic symptoms occur including severe pain, catecholamine effects and in some cases cardiopulmonary decompensation. Between 100 to 200 cases of Irukandji syndrome are reported each year and in 2002, Irukandji jellyfish caused the death of two people in North Queensland, Australia. M. kingi is presumed to be responsible for one of these fatalities. Currently there is no cure or antivenom for Irukandji box jellyfish stings. and studies have been hampered by the Irukandji small size and seasonality. In addition, sparse Cubozoa taxonomy has contributed to a general lack of knowledge of these medusae. Samples of Irukandji box jellyfish were collected during the 2003 and 2005 summers at North Queensland beaches and on the Great Barrier Reef. The mRNA of one specimen of C. barnesi was in vitro-amplified and used for the construction of an expression cDNA library, which was then amplified once. The mRNA of two adult M. kingi specimens was combined to generate a non-normalized cDNA library. The cDNA libraries constructed were immunologically screened with species-specific antibodies, human sera and Chironex fleckeri antivenom. The antibodies were antigenic against native cubozoan venom proteins, but failed to specifically bind venom proteins expressed in bacteria. However, the antibodies were reactive against several proteins exhibiting structural, catalytic or chaperone activity. Although these results were unexpected, they forced us to reconsider our early perceptions of some of the toxic protein properties. The M. kingi cDNA library proved to be a good quality molecular tool that allowed the establishment of a well-characterized and non-redundant EST resource from which were identified novel transcripts, several serine and zinc proteinases and their inhibitors, a phatogenic like gene, allergens, two neurotoxin-like genes (CbX and Mk- 332), two cytolysins: MkTX-A and B homologous to those previously reported. In addition, several of the encoded proteins were expressed in a bacterial system and further characterized. Major findings not only included the identification of highly expressed defence genes but also, localization of several of these using in situ hybridization techniques indicated their putative involvement in the box jellyfish defence system. During the analysis of the Cubozoan sequence data, a significant number of genes displaying high similarities with genes from plants, prokaryotes, viruses and even unicellular eukaryotes were also identified. Surprisingly, some genes were phylogenetically more closely related to higher animals such as primates than to invertebrate animals similar to jellyfish such as ctenophores or sponges. It was interesting to find that Cubozoans, simple, brainless, and venomous animals, display such an elevated level of gene sophistication. Jellyfish research has been previously hampered due to limited availability of biological specimens, and this was overcome during this work. The molecular information presented in this thesis represents a basis for future investigations