Previous Projects

The Australian Lions Stinger Research Foundation have funded a number of research projects. Some of our previous projects and their outcomes are listed below.

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Jodie is a PhD candidate at James Cook University's Reef and Ocean Ecology Laboratory in Townsville.

Check out Jodie's research profile for more information about her PhD research.

The box jellyfish, Chironex fleckeri, have potent, potentially life-threatening stings. They pose a significant risk to swimmers and other water users. It is notoriously hard to predict when and where box jellyfish will occur and this makes it difficult to manage the risks associated with them. Improving our understanding of their ecology, including their movements, could ultimately advance our understanding of the variability in their abundance [1]. This, in turn, could improve our ability to predict when and where they will occur, making it easier to manage the risks they pose to public health [1].

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Red Beach low tide

Red Beach, Mapoon, at low tide. Photo provided by Mark O’Callaghan.

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Before we can conduct this analysis, we need to understand how environmental conditions, namely temperature, effect the uptake of elements into box jellyfish statoliths. We conducted a controlled temperature holding experiment, following the work of Christopher Mooney and in conjunction with a similar experiment ran in December of 2015. Medusae were caught from Red Beach and transported back to Weipa. The Queensland Boating and Fisheries Patrol officers based out of Weipa kindly allowed us to use their shed to conduct the experiment. Thirty-Two box jellyfish medusae were held at constant temperatures for five days; eight were held at 20 oC, eight were held at 25 oC, eight were held at 27 oC and the final eight were held at 30 oC. The animals were preserved at the end of the experiment. The statoliths of the animals would have grown while they were held at the controlled temperatures. We will be able to evaluate the effect of temperature on the uptake of elements into statoliths by analysing the makeup of this new growth, at the very edge of the statoliths, and comparing it to the concentrations of elements in the water during the experiment. Water samples taken during the controlled temperature experiments conducted in 2015 and 2016 were sent to the Advanced Analytical Centre at James Cook University for analysis. Once the statoliths have been prepared for analysis, they will also be sent to the Advanced Analytical Centre.

Research boat
Retrieving our research boat, the Coryphaena. Photo provided by Mark O’Callaghan.

Catching bix jellyfish
Researcher Jodie Schlaefer in action, capturing a box jellyfish. Photo provided by Mark O’Callaghan.

We are confident that, once the statoliths are analysed, we will be able to reconstruct the movements of individual box jellyfish. As mentioned previously, this information could help to identify suitable polyp habitat, filling a critical gap in our current understanding of the box jellyfish lifecycle. It will improve our understanding of the ecology of box jellyfish by revealing their sources and movements. This could ultimately improve our ability to predict when and where they will occur, making it easier to manage the risk of stings [1]. My collaborators and I are would like to thank the Australian Lions Foundation for their substantial support. Without you, this work would not have been possible.

REFERENCES

1.  Kingsford MJ, Mooney CJ (2014) The ecology of box jellyfishes (Cubozoa). In: Pitt KA, Lucas CH (eds) Jellyfish Blooms. Springer, p 267-302
2.Hartwick RF (1991) Distributional ecology and behaviour of the early life stages of the box-jellyfish Chironex fleckeri. Hydrobiologia 216:181-188
3.Campana SE (1999) Chemistry and composition of fish otoliths: pathways, mechanisms and applications. Marine Ecology Progress Series 188:263-297
4.  Coates MM (2003) Visual ecology and functional morphology of Cubozoa (Cnidaria). Integrative and Comparative Biology 43:542-548
5.Gordon M, Hatcher C, Seymour J (2004) Growth and age determination of the tropical Australian cubozoan Chiropsalmus sp. Hydrobiologia 530:339-345
6.Mooney CJ, Kingsford MJ (2012) Sources and movements of Chironex fleckeri medusae using statolith elemental chemistry. Hydrobiologia 690:269-277


Medical research on marine species dangerous to humans

BSc (Hons), PhD AITHM, JCU Cairns

Dr Rob loves to wrestle deadly jellies, explore new research methods and appreciates a good meat pie. He is a fountain of knowledge and a go-to expert when it comes to jellyfish, lobsters, all sorts of weird animals and broken things.

Dr Rob's core expertise and the majority of his research focuses on the life cycle, ecology and physiology of box jellyfish with particular emphasis on the Irukandji jellyfish Carukia barnesi.

Check out Dr Rob's research profile on ResearchGate.

This study focuses on the Irukandji jellyfish Carukia barnesi Southcott, 1967. Little is known about the general ecology of C. barnesi; however, the medusa stage is considered oceanic, planktonic, has been found around coral reefs or islands, and under certain conditions, on beaches. Carukia barnesi is a relatively small box jellyfish species (bell size up to 35 mm), that are typically present during the summer monsoonal summer months between November and May in Queensland, Australia, which is commonly referred to as the 'stinger' season. Although not well defined, the distribution of this species is considered along the Great Barrier Reef and adjacent coastline, between Lizard Island and Fraser Island. There is evidence that the length of the Irukandji season in the Queensland region has progressively increased over the last 50 years, based on annual sting records, from 15 days long historically to over 150 days long currently, which has been speculated to be attributed to increased seawater temperatures. Similarly, there have been anecdotal reports that the southern distribution of C. barnesi has also increased over the last 50 years.

A sting from C. barnesi commonly results in Irukandji syndrome, which is often severely painful, potentially fatal, and frequently requires hospitalisation for treatment. The direct cost associated with treating envenomed victims, and the negative impact this species has on the Australian tourism industry through reduced revenue, are substantial (i.e., an estimated 65 million dollars in lost tourism revenue in 2002 alone). Further exacerbating the impact of this species is the simple fact that there are currently no methods in place for mitigating stings when this species is present other than through beach closures. Stinger exclusion nets are commonly used along the north-eastern coast of Queensland; however, these nets are designed to exclude large cubozoan species, primarily Chironex fleckeri, and do not exclude small species such as C. barnesi. Also, this species occurs with substantial spatial and temporal variability during the monsoonal summer months. Therefore, understanding the factors that contribute to this variability may facilitate the ability to model, and therefore predict, when and under what circumstances this species may be more prevalent. Currently, the ecological data required to produce a predictive model does not exist. Prior to the commencement of this research project, the early life history of C. barnesi had never been observed, or described, and nothing was known about the thermal and osmotic tolerance, or preference, of any of its life stages.

This study first describes the early life history C. barnesi, from egg fertilisation through to medusa production, and elucidates that this species develops an encapsulated planula stage that remains viable for six days to over six months. The polyps of C. barnesi asexually reproduce ciliated swimming polyps and produce medusae through monodisc strobilation. This study resulted in the first verified culture of C. barnesi polyps. With the polyp stage of the life cycle in culture, the opportunity to conduct manipulative temperature and salinity experiments were pursued, which provides new insights into potential polyp habitat suitability. Primary findings revealed 100% survivorship in osmotic treatments between 19‰ and 46‰, with the highest proliferation at 26‰. As salinity levels of 26‰ do not occur within the waters of the Great Barrier Reef or Coral Sea, it is concluded that the polyp stage of C. barnesi are probably found in estuarine environments, where these lower salinity conditions commonly occur.

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Further exploration of the diurnal behaviour pattern of C. barnesi medusae revealed that during light conditions, this species extends its tentacles and 'twitches' them frequently. This highlights the lure-like nematocyst clusters in the water column, which actively attract larval fish that are consequently stung and consumed. This fishing behaviour was not observed during dark conditions, presumably to reduce energy expenditure when they are not luring visually oriented prey. Larger medusae were found to have longer tentacles; however, the spacing between the nematocyst clusters was not dependent on size suggesting the spacing of the nematocyst clusters is important for prey capture. Additionally, larger specimens twitch their tentacles more frequently than small specimens, which correlate with their ontogenetic prey shift from plankton to larval fish. These results indicate that adult medusae of C. barnesi are not opportunistically grazing in the water column; instead, they utilise sophisticated prey capture techniques to specifically target larval fish.

This thesis also discusses the results of each of the experiments as a whole, and highlights areas where future research is required to predictively model the occurrence of this species. The overall focus of this thesis was to better understand the ecology and physiological limitations of C. barnesi to elucidate the factors that may contribute to the observed seasonal and distributional patterns. This research has also produced the baseline data for future research to build upon, with the expectation that the synthesis of these and future data will facilitate the ability to model, and therefore predict, the occurrence of this species in order to reduce the number of people stung.

LINK TO RESEARCH (published 2017)


How does venom from the Big Box Jellyfish (Chironex fleckeri) affect the human heart?

Anthea is a PhD candidate at JCU's Centre for Molecular Therapeutics (CMT). Anthea is based in the CMT's Biodiscovery Program which aims to isolate molecules of therapeutic potential produced by tropical flora and fauna, for the treatment of a range of infectious diseases and non-infectious human illnesses, including chronic disorders, allergies and autoimmune diseases as well as envenomations.

Check out Anthea's research portfolio on ResearchGate.

The project had two aims:

1) Purification (i.e. separation and isolation) of the key venom molecules involved in Box jellyfish envenomation.
2) Identification of the molecular mechanisms involved in fatal heart failure due to box jellyfish envenomation

Summary of findings:

Aim 1
Venoms are complex mixtures of proteins and other bioactive molecules. Each protein has a distinct set of physical and chemical properties. Those properties can be used as tools to purify a specific protein from a complex mixture while retaining the activity of that protein. Consequently each purification procedure has to be adjusted to each protein.

Previous studies had partially purified the crude toxic fractions CTF-α, -β and –γ (see Figure 1) from box jellyfish venom. However, partially purified proteins still have a high level of complexity, i.e. these fractions have far more than just a single component in them (rough estimate: ± 100 components). While the study of partially purified components can result in valuable data, resulting signals can be unreliable and inconclusive. However, to develop adequate treatment it is imperative to identify the specific components responsible for the box jellyfish induced heart failure.

Over the past year, with the help of Dr. Michael Smout, I have developed a purification procedure that substantially reduces the complexity of CTF-α and –β (see Figure 2 and 3). To develop this procedure several chromatographic columns, and the ideal order for the purification process, had to be tested. The procedure that attained the highest level of purity of the venom proteins, is as follows:

a) Size-exclusion chromatography: In this step, proteins are separated by size. This step uses the Superdex 200 Increase 10/300 GL that was funded by the Lions Foundation. This step has also been previously used to partially purify CTF-α, -β and –γ.

b) Ion-exchange chromatography: Here, proteins are separated by the electric charge state of the molecule. This step, achieves a better degree of separation than size-exclusion chromatography, because many proteins are of similar size but only very few have an equal charge state. For this step a Mono Q 5/50 GL was used (provided by the AITHM). The problem with this method is that the buffers used for ion-exchange chromatography are extremely high in salt, and this can have a damaging effect on the venom components. Therefore, it is important to add another chromatographic step immediately after the ion-exchange chromatography.

c) Size-exclusion chromatography: To remove the salt from the sample and increase the level of purity a final size-exclusion step was added to the procedure. The column used was a HiLoad Superdex 75 PG (provided by Prof. Norelle Daly).
This procedure results in a very low complexity sample of which the individual components can be identified via mass spectrometry (see new grant application).
A description of this procedure will be published shortly (within the next 6 months).


Aim 2
Following Aim 1, the purified samples were to be tested on a ProtoArray. Due to the extensive work associated with Aim 1 and the high level of sample purity required for Aim 2, this aim has not been completed yet. However, the ProtoArrays have been purchased and Aim 2 will be carried out within the next few months.

Additional work – Effect of box jellyfish venom on cancer cells
Because Aim 2 required completion of Aim1, a small additional side project was conducted while Aim 1 was carried out. This project used the size-exclusion column funded by the Lions Foundation.

This project tested the effects of whole venom as well as 3 partially purified fractions (CTF-α, -β and –γ) on a cancer cell line, to

1. test whether the fractions responsible for the heart failure in humans have an effect on other cells

2. identify fractions that have an effect on cancer cells but not on heart cells.

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The results from this project have been submitted for publication to the Journal of Venomous Animals including Tropical Diseases. The Lions Foundation was mentioned in the role of funding body for the study.

Read the article.


Resolving the human cardiotoxic components of the venom of the box jellyfish Chironex fleckeri.

Stephanie Chaousis was awarded a research grant as part of her Bachelor of Science (Zoology) Honours year at JCU.  Stephanie recently completed a PhD at Griffith University in molecular and environmental sciences. She has a passion for connecting people and ideas to create powerful collaborations and is interested in finding unique ways to apply academic research to industry outcomes.

The main goal for this research was to isolate the cardiotoxic component/s of Chiroex fleckeri (box jellyfish) venom and to elucidate the different effects the components have on human heart and skeletal muscle. These results will then aid in shaping new, novel and more effective first aid treatments for envenomation by this animal.

Abstract from research paper Rapid short term and gradual permanent cardiotoxic effects of vertebrate toxins from Chironex fleckeri (Australian box jellyfish) venom:

The vertebrate cardiotoxic components of the venom produced by the Australian box jellyfish, Chironex fleckeri, have not previously been isolated. We have uncovered for the first time, three distinct cytotoxic crude fractions from within the vertebrate cardiotoxic peak of C. fleckeri venom by monitoring viability of human muscle cells with an impedance based assay (ACEA xCELLigence system) measuring cell detachment as cytotoxicity which was correlated with a reduction in cell metabolism using a cell proliferation (MTS) assay. When the effects of the venom components on human cardiomyocytes and human skeletal muscle cells were compared, two fractions were found to specifically affect cardiomyocytes with distinct temporal profiles (labelled Crude Toxic Fractions (CTF), α and β). A third fraction (CTF-γ) was toxic to both muscle cell types and therefore not cardio specific. The vertebrate, cardio specific CTF-α and CTF-β, presented distinct activities; CTF-α caused rapid but short term cell detachment and reduction in cell metabolism with enhanced activity at lower concentrations than CTF-β. This activity was not permanent, with cell reattachment and subsequent increased metabolism of heart muscle cells observed when exposed to all but the highest concentrations of CTF-α tested. The cytotoxic effect of CTF-β took twice as long to act on the cells compared to CTF-α, however, the activity was permanent. Furthermore, we showed that the two fractions combined have a synergistic effect causing a much stronger and faster cell detachment (death) when combined than the sum of the individual effects of each toxin. These data presented here improves the current understanding of the toxic mechanisms of the Australian box jellyfish, C. fleckeri, and provides a basis for in vivo research of these newly isolated toxic fractions.

Read the full article on ScienceDirect.

Media release (Nov 2012): A heart-stopping sting - Stephanie Chaousis has discovered which part of box jellyfish venom will potentially kill humans.


Cloning and characterisation of the components of Irukandji jellyfish stings

Griselda Avila-Soria is a Molecular Biochemist with expertise in protein biochemistry, functional genomics, developmental and molecular biology. She has focused her studies in the phylum Cnidaria (Corals, anemones, hydras, true jellyfish and box jellyfish).

In 2006 when Dr Avila-Soria was awarded a Lions Foundation research grant she was a PhD candidate at JCU's Department of Biochemistry and Molecular Biology.

Since completing her PhD Griselda has worked as research fellow of the Mexican Council of Science and technology,  2014-2017 on a research program aimed to study the microbial assemblages associated to coral reefs in the central Pacific coast of Mexico. She is interested in gain information on how coral sense, co-exist and respond to microbes since a molecular and cellular point of view.

Currently Dr. Avil-Soria is working for Icaro Plant Science a private AgBiotechnology company developing multidisciplinary and  state of the art plant breeding techniques coupled with the development of organic agricultural approaches for plants displaying biological activity relevant for human health.

Find out more about Dr Avil-Soria on ORCID.

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Abstract from research paper Molecular characterisation of Carukia barnesi and Malo kingi, Cnidaria; Cubozoa; Carybdeidae:

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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-normalised cDNA library.

The cDNA libraries constructed were immunologically screened with species-specific antibodies, human sera and Chironex fleckeri anti-venom. 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-characterised and non-redundant EST resource from which were identified novel transcripts, several serine and zinc proteinases and their inhibitors, a pathogenic 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 characterised. Major findings not only included the identification of highly expressed defence genes but also, localisation of several of these using in situ hybridisation 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.

Read the full report.