Origin, evolution and functional diversification of a chemical arsenal: venom in bony and cartilaginous fish

Lead Research Organisation: Liverpool School of Tropical Medicine
Department Name: Molecular and Biochemical Parasitology

Abstract

Venoms represent a largely unstudied, yet valuable, natural resource. Venoms are also an important evolutionary innovation in the animal kingdom and have evolved separately in a number of different animals (e.g. spiders, scorpions, octopuses, reptiles, fish, mammals). Considering venoms induce extremely potent biological activities, a major limitation of venom research has been the very narrow range of venomous species investigated - surprisingly, entire groups of venomous animals remain virtually unstudied. One such group is fish, where extraordinarily little is known about the composition, evolutionary history or bioactivity of venoms found in different species, despite fish representing the largest group of venomous vertebrates after reptiles (>2,400 species).
In contrast to the predatory venoms of reptiles, fish venoms are thought to be defensive by acting to protect the individual from predation. Some recent morphological investigations into the evolutionary history of fish suggested that venom has evolved at least 15 times in different groups of bony and cartilaginous fish. However, tracing the evolution of venom with morphological characters can be complicated, because identifying the presence or absence of a venom apparatus is difficult and doesn't reveal whether multiple character changes (i.e. gain followed by loss) have occurred over time. In contrast to these studies, a number of independent observations combine to suggest that fish venoms may have evolved much earlier and less frequently than previously proposed, for example: (i) similar venom toxins are found in the venoms of very distinct fish lineages, (ii) the functional activities of different fish venoms appear to be largely conserved and (iii) antivenom raised against the venom of a single fish species is capable of neutralising the activity of venoms from completely different fish species. This evidence suggests the origin of fish venom may have occurred in an ancestral fish that existed prior to the evolutionary split of bony and cartilaginous fish.
The key aim of this study is to undertake a thorough investigation of venoms (including their toxin components and their functional activity) found in distinct fish lineages, including fascinating taxa such as the stingray, shark and lionfish and medically important species, including the stonefish and weeverfish (which are found in the waters surrounding the UK). By characterising the biodiversity of toxins found in the venoms of different fish, the evolutionary history of venom in this major vertebrate lineage can be revealed. Notably, the investigations proposed here will also determine the functional activities of different venoms and their components. Identifying the composition and bioactivity of different fish venoms will help us to: (i) understand the medical consequences that arise from the thousands of fish envenomings that occur annually and (ii) design effective new treatments that neutralise venom toxins from a wide range of fish species. Perhaps most importantly, venoms provide an exciting and largely unstudied resource for the discovery of new pharmacological diagnostics and therapeutics, particularly following the successes of drugs previously developed from snake and cone snail venoms. The research proposed here is highly likely to reveal novel targets for future pharmacological research following the identification and functional testing of toxins isolated from previously unstudied venoms.
The proposed research will predominantly be undertaken at Bangor University's School of Biological Sciences - this institute has extensive research experience in the fields of both fish biology and venom evolution. In addition, this application provides the opportunity to utilise international collaborations with experts based at the University of Queensland, Australia for the collection of venomous fish and Monash University, Australia for determining the functional activities of different fish venoms.

Planned Impact

The non-academic beneficiaries of the proposed research are three-fold: (i) commercial parties related to the pharmaceutical/biologicals industries, (ii) zoological institutions within the public sector and (iii) national health authorities and policy makers.

The development of novel pharmaceutical or diagnostic products from naturally-occurring toxins is of great current interest. The research proposed here will investigate the pharmacological activities of a taxonomically-diverse range of previously unstudied venom components - the results of this research is therefore highly likely to reveal novel compounds pertinent to future drug development programmes. In addition, antivenom manufacturing companies may also be interested in the outcomes of the research if the alternative hypothesis is supported. Commercial collaborations will be formed with UK-based pharmaceutical companies (where possible) willing to further the development of novel biological products.

Beneficiaries within the public sector include zoological institutions such as zoos, aquariums and museums. These institutions will be able to utilise the results of the scientific research proposed here to inform the public about the venoms different fish species possess, why fish are venomous and which fish are dangerous to humans. The advance in knowledge, communicated by lay film and print media, produced by this research project is likely to be noticeably beneficial to the public sector (particularly aquariums) and therefore also to the public themselves.

Thirdly, national health authorities and policy makers throughout the world are likely to be interested in the outputs of the proposed scientific research, particularly: (i) in regions of the world with a high incidence of fish envenoming and (ii) in the event that the alternative hypothesis is supported. The results of this study will provide a rigorous framework for determining which venomous fish species are potentially dangerous to humans and will therefore inform policies relating to the hospitalisation or delivery of antivenom therapy in the event of human envenomings. For example, at the local level, this project will greatly inform UK authorities on the function of the toxin components present in the venom of weeverfish - species capable of causing mortality that are responsible for the majority of the UK's medically-important fish envenomings.

Consequently, the scientific research proposed here has the potential to contribute to the UK's: (i) wealth, through the pharmaceutical development of novel toxins, (ii) culture, through public sector information and (iii) health, by informing national authorities on the potential dangers and requirements for hospitalisation following envenomings by certain fish species. In particular, the potential development of novel pharmaceutical or diagnostic tools from fish venoms may be valuable for the long-term economic competitiveness of the UK, especially when considering the precedents set by other pharmaceuticals developed from venom components (e.g. Captopril and Exenatide). Collaborations with UK-based pharmaceutical companies will be utilised in preference of non-UK companies where feasible. In line with NERC guidelines, in the short-term, immediate benefits are likely to be provided to beneficiaries in both the public and health sectors locally and internationally, resulting in the enhanced delivery of novel scientific information to the public and informing policy makers about the relative importance of fish envenomings occurring throughout the world.

Publications

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Casewell NR (2014) Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms. in Proceedings of the National Academy of Sciences of the United States of America

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Ujvari B (2015) Widespread convergence in toxin resistance by predictable molecular evolution. in Proceedings of the National Academy of Sciences of the United States of America

 
Description This research project has involved characterising the protein and/or gene composition of the venom of 25 species of fish. Fish venoms are almost completely unstudied, and our 2104 scientific paper on the venom of the blue-spotted stingray represented the first analysis of the gene and protein composition of fish venom. Since that time we have characterised the venom activity of a further six species (paper published in Toxins journal in 2017) and performed a thorough analysis of the venom composition and functional activity of fangblennies (paper published in Current Biology in 2017). Our broad conclusions from this project are that fish venoms have evolved independently on many occasions and therefore contain different bioactive venom constituents. However, the functional activity of these components are broadly similar, resulting in pain and cardiovascular and neurotoxic activity in envenomed victims and predators.
Exploitation Route We have shown that fish venoms are pharmacologically active and may be of interest for drug discovery programs.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description My current findings have been used to write scientific publications, engage in public engagement activities (e.g. public talks, scientific conference talks, media work) and deposit data to databases for the benefit of other scientists. In addition, my findings have been used to aid the creation of a "venomous animal" exhibit at the London Natural History Museum.
First Year Of Impact 2017
Sector Digital/Communication/Information Technologies (including Software),Culture, Heritage, Museums and Collections
Impact Types Societal

 
Title Development of VT Builder 
Description Development of a new computer programme (named VTBuilder) to help to better assemble venom gland transcriptomes 
Type Of Material Biological samples 
Year Produced 2015 
Provided To Others? Yes  
Impact Adoption by the venom community of this new tool fro assembling gene based DNA data from venomous animals 
URL http://www.lstmed.ac.uk/vtbuilder
 
Title New protein extraction method for fish proteins 
Description We devised a new sample preparation method to remove salt and mucous contamination from protein samples derived from fish. 
Type Of Material Biological samples 
Provided To Others? No  
Impact The research method was published in our recent scientific publication (see below) and we anticipate other academic end-users using it for their research purposes. Baumann K*, Casewell NR*, Ali SA, Jackson TNW, Vetter I, Dobson JS, Cutmore SC, Nouwens A, Lavergne V, Fry BG. 2014. A ray of venom: combined proteomic and transcriptomic investigation of fish venom composition using barb tissue from the blue-spotted stingray (Neotrygon kuhlii). Journal of Proteomics. 109C, 188-198. doi: 10.1016/j.prot.2014.06.004. *these authors contributed equally. 
 
Title Fish proteomes 
Description Databases containing details of the proteins identified in the venom and skin secretions of venomous fish 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact One of these databases were published in our recent scientific paper (see below), making them freely accessible for our researchers to use for their research purposes. The remaining databases will be made freely available following the publication of our results in other scientific papers over the coming 12 months. Baumann K*, Casewell NR*, Ali SA, Jackson TNW, Vetter I, Dobson JS, Cutmore SC, Nouwens A, Lavergne V, Fry BG. 2014. A ray of venom: combined proteomic and transcriptomic investigation of fish venom composition using barb tissue from the blue-spotted stingray (Neotrygon kuhlii). Journal of Proteomics. 109C, 188-198. doi: 10.1016/j.prot.2014.06.004. *these authors contributed equally. 
 
Title Fish transcriptomes 
Description Generation of gene expression data from the venom glands and skin of venomous fish. 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact One of these databases were published in our recent scientific paper (see below), making them freely accessible for our researchers to use for their research purposes. The remaining databases will be made freely available following the publication of our results in other scientific papers over the coming 12 months. Baumann K*, Casewell NR*, Ali SA, Jackson TNW, Vetter I, Dobson JS, Cutmore SC, Nouwens A, Lavergne V, Fry BG. 2014. A ray of venom: combined proteomic and transcriptomic investigation of fish venom composition using barb tissue from the blue-spotted stingray (Neotrygon kuhlii). Journal of Proteomics. 109C, 188-198. doi: 10.1016/j.prot.2014.06.004. *these authors contributed equally. 
 
Description Collaboration with A/Prof. Bryan Fry, University of Queensland, Australia 
Organisation University of Queensland
Country Australia 
Sector Academic/University 
PI Contribution We have contributed intellectually and in the data generation process to investigate the composition of fish venoms. This has included the generation of extensive molecular and proteomic data for >25 venomous fish species
Collaborator Contribution A/Prof. Fry has contributed to this collaboration by performing functional analyses of fish venom proteins supplied by myself. This has resulted in characterising novel bioactivities, including pain-inducing and neurotoxicity, for fish venom.
Impact The only outcome at this time is the following scientific publication. Baumann K*, Casewell NR*, Ali SA, Jackson TNW, Vetter I, Dobson JS, Cutmore SC, Nouwens A, Lavergne V, Fry BG. 2014. A ray of venom: combined proteomic and transcriptomic investigation of fish venom composition using barb tissue from the blue-spotted stingray (Neotrygon kuhlii). Journal of Proteomics. 109C, 188-198. doi: 10.1016/j.prot.2014.06.004. *these authors contributed equally. The collaboration is multi-disciplinary and the biological disciplines involved are: molecular biology, evolutionary biology, pharmacology and proteomics.
Start Year 2014
 
Description BBC filming for "Health Check" TV show 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Filmed an interview on venoms for BBC Health Check TV programme which aired on BBC World in September 2015
Year(s) Of Engagement Activity 2015
 
Description Participated in the content development for the London Natural History Museum exhibit entitled "Venoms: killer and cure" 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Participated in developing content for the NHM's venom exhibit which opened in November 2017 for six months. This included detailed discussions with Dr. Ronald Jenner (lead scientist on the exhibit), the loaning of biological specimens for the exhibit and the filming of video content (interviews) for the exhibit. The exhibit is anticipated to reach an audience of >30,000 people.
Year(s) Of Engagement Activity 2017,2018
URL http://www.nhm.ac.uk/visit/exhibitions/venom-killer-and-cure.html
 
Description Participation at the venoms to drugs conference 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Talk relating to the pharmacological development of therapeutic molecules from fish venoms stimulated discussions with academics and commercial pharma towards that end.

Impacts include forming new commercial contacts who will be valuable collaborators moving forward with my research.
Year(s) Of Engagement Activity 2014
 
Description Press release for fangblenny scientific paper 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Performed around 20 media interviews for a science paper on the venom of the fangblennies. This resulted in extensive coverage in international press including BBC, Le monde, wired, New Scientist, New York Times, etc.
Year(s) Of Engagement Activity 2017
URL https://www.nytimes.com/2017/03/30/science/fanged-blennies-fish-opioid-venom.html
 
Description University talks on fish venoms 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Talk generated many questions and discussion with interested students and lay persons.

I have had many expressions of interests from University students to visit my lab for intern purposes or for postgraduate study
Year(s) Of Engagement Activity 2012,2013,2014