Discovery of a Scorpaeniform Toxin Gene in Cephalopholis argus

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Slides for my talk at the International Society for Toxinology World Conference and Venom Week 2012.

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  • Venoms represent unique evolutionary adaptations that possess potent biological activities which can lead to novel pharmeceuticals. So far, most venom research has focused on terrestrial species like snakes and spiders, largely ignoring the over 1200 estimated venomous fishes. Research that has focused on fish venoms has gone straight for the throat, taking a largely biochemical approach and attempting to isolate toxins from highly venomous species. In fish in particular, such methods are fraught with difficulties, as toxins have proven extremely labile and difficult to isolate. But it was Halstead in 1988 who suggested that before these approaches can be truly fruitful, we need to take a step back. He said:
  • But Halstead in 1988, suggested that before these approaches can be truly fruitful, we need to take a step back. He wrote: . Thus halstead advocated an evolutionary approach to studying fish venoms
  • Taking halstead up on his call for a evolutionary approach, smith and wheeler produced a phylogenetic roadmap to understanding venoms in fish. When it comes to toxic fish, we know that highly venomous species are interspersed phylogenetically among less toxic relatives. To date, little research has looked at these non-venomous fish to try and understand the evolution – and, at the lack of a better word, de-evolution – of venoms. I call special attention to these non-venomous fishes because they, too, represent may evolutionary novelty. Some have lost all venom characteristics, while others may possess genes and thus the ability to be venomous, but these genes aren’t currently expressed. But equally likely is the idea that some of these do possess and express toxin genes that, through evolutionary changes, have simply become less potent. These weak varieties allow us a unique way to study toxicity. By comparing highly potent toxins to less potent ones within a family of toxins, we can garner information about what parts of a toxin are most pharmacologically relevant, particularly in species where isolation and characterization of toxins has proven difficult.One such group is the Scorpaeniformes. This order, in particular, may give us unique insight into the evolution of toxicity. Within this group, there are 3 venomous lineages – the gunard perches, the scorpionfishes and lionfishes, and the stonefishes and waspfishes.
  • Though there is considerable diversity of delivery apparatus within this order, there is strong evidence that all three toxic possess highly similar venoms. First of all, they all have similar clinical manifestations. Envenomations from each of these three groups present with intense, radiating pain, local edema, and neuromuscular side effects. But more importantly, crude extracts from all three lineages cross-react with Stonefish Antivenom, suggesting that the similar protein toxins exist in all venomous fish in this order. To simplify nomenclature, I refer to this family of related protein toxins as Scorpaenitoxins. In vivo and in vitro, scorpaenitoxins have potent cytolytic and hemolytic activities, as well as neuromuscular effects which appear to stem from either their ability to activate sodium channels or to form ion pores. The first scorpaenitoxin to be isolated was Stonustoxin from synanceiahorrida, and since then, several similar proteins have been isolated from three species of stonefish, one scorpionfish and two species of lionfish. But while we know a lot about scorpaenitoxins and their activities, we do not have a crystal structure for the protein. Interestingly, we so have mRNA sequences for several of these proteins, allowing us to examine the protein from a purely genetic perspective.
  • From an evolutionary perspective, the fact that scorpaenitoxins are present in three distinct clades suggests that the presence of this toxin is an ancestral trait and synapomorphic to the group as a whole, which begs the question: what happened to this toxin in these other, “harmless” lineages? Do they possess the genes for scorpaenitoxins, too? And if they do, why aren’t they considered venomous? Do they not express the toxin, or are their versions less potent than their relatives?
  • As a pilot study, I chose to investigate the presence of scorpaenitoxins in groupers. While groupers were once placed in the order Perciformes, a wealth of molecular and anatomical evidence supports their proper placement in the Scorpaeniformes as a sister taxon of the venomous scorpionfishes and lionfishes. Though close relatives of one of the most venomous groups of fish in the world and well-nested in the venomous order, there is no evidence that groupers are venomous themselves. For my research, I chose to investigate whether they posses a toxin similar to the scorpaenitoxins present in othterscorpaeniform fishesMy particular study organism is the peacock grouper, cephalopholis argus, locally known as Roi. These voracious introduced piscivores are the most abundant reef predators here in Hawaii, making them an readily accesible member of this family to study.
  • To look for scorpaenitoxins in roi, I isolated genomic dna from several roi specimens as well as three lionfish species as positive controls. Using primers designed against the known mRNA sequenes for stonefish, I used polymerase chain reaction to amplify a portion of a scorpaenitoxin gene from my grouper and lionfish specimens. PCR products were cleaned and sequenced at the UH sequencing core. Sequences were assembled and examined by eye using the program geneious. I blasted each sequence against the NCBI database to see if it could possibly belong to any other protein. I aligned and compared the produced sequences to the known mRNAs for stonefish and lionfish, and calculated crude divergence rates. Last but not least, I used the neighbor-joining tree plug in in geneious to construct a crude phylogenetic tree of the scorpaenitoxin sequences.
  • I was able to amplify a 578 base pair portion of genomic DNA that appears homologous to a chunk of the stonustoxin beta subunit from both my lionfish positive controls and my grouper specimens. Here you can see a small portion of this sequence. The top four sequences represent the previously known scorapenitoxin sequences. On top are scorpaenitoxin mRNAs from two stonefish species, followed by the mRNA sequences for the two pterois scorpaenitoxins. Below are my three new scorpaenitoxin genes, from two other genera of lionfishes and the grouper. Differences between sequences are highlighted. As you can see, the sequences I obtained are highly similar to the previously known mRNA sequences. When I conducted a blast query, my amplified chunks only matched scorpaenitoxins, and thus are unlikely to represent accidental amplification of any other proteins. The sequence for my grouper, roi, was less than 6% divergent from any lionfish species, and appeared to be more similar to Pterois toxins than the two other genera. As expected from the scorpaeniform phylogeny, stonefish sequences were much more divergent from both the grouper and lionfish sequences. The crude phylogenetic tree supports the predicted relationship between these species, with the two stonefish species divergent from the other lionfishes and the grouper. It’s interesting that the grouper sequence fell within the lionfish clade, however, I hestitate to draw any strong phylogenetic conclusions from such a small section of DNA. I would like to isolate and amplify the entire gene sequence to get a better idea of the similarities and differences within this group of related fishes.
  • Of course, just because they have the gene doesn’t mean they use it. For the next steps in my research, I intend to determine the expression profile of scorpaenitoxin in roi using two methods. Since all other known scorpaenitoxins cross react with the antibody to stonefish antivenom, i will first use a western blotting protocol to detect the protein in roi tissues. Second, I will extract and isolate the messenger RNA from different tissues to look for expression of the scorpaenitoxin gene genetically. Very specifically, I want to look for expression throughout the body, not just in the spines. We have never asked for whether these toxins are produced solely in spine tissues. But there is a secondary reason I want to study toxin expression in the body tissues.
  • Though perhaps only a coincidence, both roi and the invasive lionfish in the caribbean have been investigated recently for the presence of ciguatera, which is limiting our ability to establish a fishery for these highly invasive and potentially lucrative species. As many of you know, ciguatera is a small lipid poison present in many tropical fishes, particularly large, reef-associated predators. While ciguatera and scorpaenitoxins may look nothing alike, there is evidence that in a lab they could appear quite similar.
  • So in addition to understanding the expression of scorpaenitoxins , I also intend to investigate whether scorpaeniform venoms affect ciguatera assays. Understanding the expression of scorpaenitoxins and how these venoms affect ciguatera assays will allow us to develop a more accurate test for ciguatera in this lineage of fishes, allowing us to re-evaluate commercially and ecologically important fisheries.
  • Non-assay had 15-50% false negative and false positive rates
  • Discovery of a Scorpaeniform Toxin Gene in Cephalopholis argus

    1. 1. Christie WilcoxDiscovery of a Scorpaeniform Toxin Gene University of Hawaii at Manoa in Cephalopholis argus Cell and Molecular Biology Hawaii Institute of Marine Biology
    2. 2. Venom Evolution
    3. 3. Venom EvolutionUntil such fundamentals as theanatomical distribution of fish venomshave been determined, thepharmacological and chemicalcharacterization of these compounds willcontinue to be unstudied.– Halstead (1988) in the introduction to his treatise onvenomous marine organisms
    4. 4. Scorpaeniformes Smith & Wheeler (2006). Journal of Heredity 2006:97(3):206–217
    5. 5. Scorpaenitoxins
    6. 6. Scorpaeniformes Smith & Wheeler (2006). Journal of Heredity 2006:97(3):206–217
    7. 7. Serranidae Family Serranidae (Groupers) Cephalopholis argus The Peacock Grouper
    8. 8. Methods
    9. 9. Sequence Comparison Roi Other LionfishPterois spp 1-2 % 5-7 %Other Lionfish 5-6 % 5.5 %Stonefish 12-14 % 14-16 %
    10. 10. Future DirectionsExpression Profile
    11. 11. Invasive Species
    12. 12. Ciguatoxin Scorpaeniform VenomsMouse Bioassay:weakness, paralysis, deathNeuroblastoma Assay:cytotoxicityRapid Hemolysis Assay:hemolysisGuinea Pig Atrial Assay:positive inotropy
    13. 13. Future DirectionsExpression ProfileAffect on Assays
    14. 14. AcknowledgementsBryan Fry (University of Queensland)Paul Bienfang (SOEST)Brian Bowen & the ToBo LabMichelle Gaither, Kira KrendFunding: Sigma Xi GIAR, EECB Maybelle Roth
    15. 15. Questions ?

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