Previous Projects

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].

The Australian Lions Foundation generously awarded me $7,000 last year to research the movements of box jellyfish medusae (the free-swimming stage of the jellyfish lifecycle). These funds helped to pay for a field trip to Weipa, on the west coast of Cape York Peninsula, far north Queensland. We packed a LandCruiser with our equipment and drove it the 1200 km from Townsville to Weipa, towing a boat with us all the way. We made multiple trips to Red Beach, Mapoon, Port Musgrave, which is approximately 90 km north of Weipa, to observe box jellyfish in their natural habitat. We observed medusae swimming back and forth parallel to the shore. The medusae could swim at speeds that greatly exceeded the current speeds in the shallow waters where they are typically found. We also observed how they reacted to obstacles placed in their field of view (they have complex eyes similar in structure to the eyes of vertebrates). These observations were used to generate a model of medusae behaviour. This behavioural model was paired with a model of the currents in Port Musgrave in a technique known as biophysical modelling. The modelling suggested that there was little potential for box jellyfish to move out of the Port Musgrave system. Consequently, the population inhabiting Port Musgrave, and other populations inhabiting similar estuarine bays, are probably very isolated from each other. The findings also suggested that the considerable swimming ability of the medusae allowed them to stay in shallow waters close to shore, near people. The isolation of populations could contribute to the high variability in box jellyfish abundance, which makes their occurrence so hard to predict. This research will be published in a forthcoming themed section of the scientific journal Marine Ecology Progress Series titled “Jellyfish Bloom Research: Advances and Challenges”. The section will include cutting edge research on jellyfish from international contributors and it will have an international readership.

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

Furthermore, there is uncertainty surrounding the lifecycle of box jellyfish. Box jellyfish medusae metamorphose from polyps that are sessile and live attached to hard substrates (e.g. rocks) [2]. The polyps are thought to inhabit tidal estuaries, but only one wild population of polyps has been documented [2]. Metamorphosis from polyps to medusae probably first occurs at the start of the wet season [2]. Medusae are then thought to move into coastal waters but there is little evidence to support this theory [2]. One way to investigate the sources and movements of medusae would be to track the movements of individuals from metamorphosis to capture. This could potentially be done with a technique called elemental chemistry analysis. The elemental composition of estuarine and marine water is different. As animals move between these waters, they will take different concentrations of elements into their bodies [3]. Box jellyfish have hard structures called statoliths that help with orientation [4]. These structures are laid down continuously throughout the life time of the animals [5]. By analysing the elemental make up of box jellyfish statoliths along a transect from the core to edge, we can track the movements of individuals from when they were juvenile medusae (core) to when they were captured as adults (edge) [6].

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.

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

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

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.

With the relationship of temperature and salinity on the polyp stage known, focus was shifted to exploration of these factors on the medusa stage. The thermal and osmotic tolerance of C. barnesi medusae were investigated to determine if environmental parameters drive the marked seasonality of this species. By exploring oxygen consumption over a range of temperatures, the minimum thermal requirement for C. barnesi was estimated at 21.5ºC, which does not explain the seasonal occurrence of this species. The optimum temperature for swimming pulse rate was determined to occur between 27.5ºC and 30.9ºC and the optimum temperature was estimated at 29.2ºC, which encompasses the typical summer thermal regime in situ. This research concludes that reduced fitness associated with environmental temperatures that departure from optimum may better explain the seasonal pattern of this species. Conversely, departure from optimum temperature did not explain the southern distribution limits of this species, suggesting that C. barnesi could theoretically persist further south than their loosely defined southern distribution limits. The optimum salinity of C. barnesi medusae was estimated at 35.8‰ and fitness was reduced as salinity levels reduced below 29‰, adding further support that C. barnesi medusae are oceanic and cannot persist in estuarine environments, where low salinity conditions commonly occur. The respiration rate of C. barnesi was significantly suppressed at night, providing evidence that this species is less active during night conditions, presumably to conserve energy.

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)

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.

This project showed that the heart specific fractions CTF-α and –β had no or very little effect on cancer cells. CTF–γ on the other hand had a substantial effect on cancer cells, but little effect on heart cells (See Figure 4). These results suggest the presence of several distinct components in the box jellyfish venom that could have a potential as drug targets and should thus be further investigated (Manuscript attached). (Because synthetic drug production is expensive and laborious, several animal venoms are currently under investigation as natural drug source for a vast variety of diseases.)

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.

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.