Tracking toxins in venomous Vermes: comparative venomics of polychaete annelids

Although most lay people have little problem in identifying creatures like jellyfish, wasps, spiders, and scorpions as potentially dangerous venomous organisms, far fewer know that iconic animals such as the Komodo dragon, or the inconspicuous centipedes possess potent venoms. Similarly, although digging up bloodworms-a group of marine polychaete annelids-for use as fish bait is a multi-million dollar cottage industry along the coasts of the Northeastern United States and Canada, it is doubtful whether unsuspecting fishermen realize that they are hooking worms that possess potent neurotoxic venom. Bloodworms and several groups of the related scale-worm have two pairs of hardened jaw, associated with venom gland. Although these venoms presumable play a crucial role in the life of the animals in subduing prey, we know almost nothing about the make-up of their venoms. This leaves not only a large hole in our understanding of an important group of organisms-representing possibly as many as 1,000 species of venomous worms-but it also compromises our general understanding of venom biology and evolution in the animal kingdom. This study aims to remedy this ignorance by performing the first extensive and intensive analysis of the composition of polychaete venoms.

This project uses a combination of cutting edge techniques to determine venom toxin composition on both the genetic and protein levels. Eight species of worms have been selected-4 species of bloodworms and 4 species of scale-worms-to represent a cross section of the major groups they represent. For each species, up to half a million mRNA sequences will be characterized. These precursor molecules are the templates for the production of toxin proteins. In addition, the toxin proteins themselves will also be determined directly from extracted venom. Together these data will reveal what toxins are expressed in the venom glands of the worms, providing an important contribution towards elucidating the precise identity of one toxin that is known to exist in bloodworm venom. This is important because this toxin has a very specific mode of action-paralyzing and killing crustacean prey in the wild-that is already of use to researchers who study neuronal processes in the laboratory.

By comparing venom toxins between different groups of worms and other venomous animals we can also start to address important questions relating to the evolution of venoms and venomous organisms. For example, venoms are a prime example of convergent evolution, the repeated independent evolution of similar adaptations in different taxa. Since venoms have evolved many times independently, a comparison of their composition can reveal shed light on the rules under which convergent evolution operates.

Lastly, the outstanding feature of venoms, and the source of our fascination with them, is of course the physiological effects they have. This project will test specific types of pharmacological activities of the worm venoms, such as neurotoxity. This will lead to a better understanding of the biology of the organisms, but perhaps even more importantly, understanding the bioactivities of toxins can have valuable downstream applications. Already seven prescription drugs have been developed from components of animal venoms, from animals as different as cone snails and the Gila monster. Since these are used to treat serious conditions in humans, including diabetes and hypertension, potentially many people could benefit from mining the hidden biodiversity of venoms.

Identify the toxins expressed in the venom glands of 8 species of polychaete annelids
- Use 454 GS FLX Titanium technology to generate the first comprehensive polychaete venom gland transcriptomes
- Use nanoLC-MS/MS to identify toxin proteins to generate the first comprehensive polychaete proteome data
- Compare the transcriptome and proteome data to shed light on the poorly understood relationship between venom gland transcriptomes and proteomes
- Use the new data as an important step towards elucidation of the complete sequence of alpha-glycerotoxin, an unusual and useful neurotoxin from bloodworms

Understand whether more close related species convergently recruit venom toxins from more similar toxin families than distantly related species
- Use standard phylogenetic methods (principally model-based methods such as Bayesian analysis and ML) to estimate toxin gene trees including the polychaete toxins and those of other studied venomous animals to produce the first explicit assessment of the role of phylogeny in shaping venom composition across phylogenetic levels ranging from homologous venoms in closely related species to convergent venoms in species of more or less closely related families to convergent venoms between phyla and major bilaterians clades
- Use the new data and resulting trees to re-assess current hypotheses about the evolution of toxin gene families across Metazoa, for example the importance the importance of gene duplications in contributing to venom complexity.

Reveal the pharmacological activities of polychaete venoms
- Use enzymatic and non-enzymatic in vitro and in vivo essays to obtain a better understanding of the toxic activities of bloodworm venoms, and generate the first data on venom activity in scale-worms

Because venomics is richly interdisciplinary in terms of its concepts, methods, data and the nature of the questions it addresses, this project should be of interest to the wider community of biologists. Also, because venoms have proven to be rich natural resources for bioactive compounds that have been used for developing commercially successful drugs, venom research is very interesting to the pharmaceutical industry. Moreover, venomous animals have long played important roles in cultures the world over, and they are widely used in traditional medicine. As reservoirs of a great diversity of useful bioactive compounds venomous animals are therefore medically and economically valuable natural resources. The first step in safeguarding the medical and economic potential of native venomous species is to understand what bioactive compounds are present in their venoms, and what pharmacological effects these may have, which is the project's explicit focus. Therefore, the conservation and protection of native venomous species in the wild deserves special attention. As an illustration of the need for this, the harvesting of the bloodworm Glycera dibranchiata is a multi-million dollar business along the coasts of the United States and Canada. The worms are sold as bait, and are exported also to Europe, often as live individuals. This introduces the risk that such species establish themselves as invasive species, which may have detrimental effects on native populations of close related European species. Finally, venomous animals have a unique 'cool' factor for general audiences, and venom biology has a great potential to engage them with some of the most fundamental questions in biology in an informal and attractive way.

The impact of the research for the broader research community will be secured by disseminating the results through the customary channels of publishing scientific papers, presenting talks at international meetings, and through deposition of the data in the appropriate public databases. The potential impact of the research for the economic and medical competitiveness of the UK will be realized by consulting with two research groups specializing in the development of new drugs from bioactive compounds found in nature, to explore ways to exploit specific pharmacological activities of venom components commercially. The results of this study will also be disseminated to the appropriate people at Defra (Department for Environment, Food and Rural Affairs) to alert them to the value of venoms as a unique natural resource in the UK, and the need to think seriously about their conservation. Finally, the results of the research and the importance of venoms for fundamental and applied science will be communicated to general audiences in several ways. The NHM is an ideal place to do this, with about 5 million visitors per year. First, we will develop and present six presentations on venoms for the popular series Nature Live. These are interactive events in which researchers present their work to Museum visitors, using multimedia and real specimens from the collections. Second, a website on the research will be created and hosted by the Museum's servers for people to look up details. Third, I have made contact with the Head of Biology at St. Paul's secondary school in London (Dr Jon Bennett), and agreed to give several presentations on the research and venoms in general to the pupils. This will expose them to the excitement of science, and how fundamental science and societal benefits can go hand in hand. Fourth, we will arrange a meeting in the Museum with the appropriate people to explore the possibilities of setting up an exhibition on venomous animals in one of the Museum's galleries reserved for temporary (typically several months to a year) exhibits. The Museum's collections are rich in material for this purpose, and should allow a professional and attractive exhibit to be put together.