Discovery of a scorpaenitoxin gene in Cephalopholis argus
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A brief portion of my dissertation research on scorpaenitoxins
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Discovery of a scorpaenitoxin gene in Cephalopholis argus
- 1. Discovery of a Scorpaeniform toxin gene in Cephalopholis argus: implications for ciguatoxin detection Christie Wilcox Hawaii Institute of Marine Biology
- 7. Ciguatera in Roi Dierking & Campora 2008. Pacific Science 63(2):193-204
- 8. Ciguatera in Roi Dierking & Campora 2008. Pacific Science 63(2):193-204
- 9. Ciguatera in Roi Dierking & Campora 2008. Pacific Science 63(2):193-204 39% false + 50% false - Wong et al. 2005. Toxicon 46: 563-571
- 10. Ciguatera in Roi Dierking & Campora 2008. Pacific Science 63(2):193-204
- 11. Ciguatera in Roi Dierking & Campora 2008. Pacific Science 63(2):193-204 26% false + 15% false - Bienfang et al. 2011. Food and Nutrition Sciences 2: 594-598
- 12. Grouper Taxonomy Smith & Wheeler (2006). Journal of Heredity 2006:97(3):206–217
- 13. Grouper Taxonomy Groupers Smith & Wheeler (2006). Journal of Heredity 2006:97(3):206–217
- 14. Grouper Taxonomy Scorpionfishes & Lionfishes Groupers Stonefishes & Waspfishes Smith & Wheeler (2006). Journal of Heredity 2006:97(3):206–217
- 15. Grouper Taxonomy Scorpionfishes & Lionfishes Groupers Stonefishes & Waspfishes Smith & Wheeler (2006). Journal of Heredity 2006:97(3):206–217
- 16. Scorpaenitoxins
- 17. Ciguatoxin Scorpaenitoxin Mouse Bioassay: Weakness, paralysis, death Neuroblastoma Assay: Cytotoxicity Rapid Hemolysis Assay: Hemolysis Guinea Pig Atrial Assay: positive inotropy
- 18. Ciguatoxin Scorpaenitoxin Mouse Bioassay: Weakness, paralysis, death Neuroblastoma Assay: Cytotoxicity Rapid Hemolysis Assay: Hemolysis Guinea Pig Atrial Assay: positive inotropy
- 19. Ciguatoxin Scorpaenitoxin Mouse Bioassay: Weakness, paralysis, death Neuroblastoma Assay: Cytotoxicity Rapid Hemolysis Assay: Hemolysis Guinea Pig Atrial Assay: positive inotropy
- 20. Ciguatoxin Scorpaenitoxin Mouse Bioassay: Weakness, paralysis, death Neuroblastoma Assay: Cytotoxicity Rapid Hemolysis Assay: Hemolysis Guinea Pig Atrial Assay: positive inotropy
- 21. Ciguatoxin Scorpaenitoxin Mouse Bioassay: Weakness, paralysis, death Neuroblastoma Assay: Cytotoxicity Rapid Hemolysis Assay: Hemolysis Guinea Pig Atrial Assay: positive inotropy
- 22. Research Question Do roi possess the ability to produce scorpaenitoxins?
- 23. Methods
- 24. Sequence Comparison 1 10 20 30 40 50 Roi Tx CCAAA TA TCTGGA TGA TTCTAA GAAA TTCAA GAA TCA GA GTCGA GTGA CA CTTC PaTx-b .................A GG.................................. Lionfish PvTx-b .................A GG.................................. neoVTX-b ....G......A ..A ..CA G.............................U.... Stonefish ....G......A ..A ..CA G.............................U.... SnTX-b 120 130 140 150 160 Roi Toxin (554 bp) Roi Tx A GAA TA TTCTGAA TA TTTTCA GAA CCTCGA GGCAA CTCA TGTA GTCA TA GGGA T PaTx-b 3% sequence divergence ...................................................... PvTx-b Lionfish toxins ...................................................... 4% amino acid divergence neoVTX-b ..............UA ...G.....A ..C................G........ SnTX-b ..............UA ...G.....A ..C................G........ 230 240 14% sequence divergence 250 260 270 Stonefish toxins 18% amino acid divergence Roi Tx GTTCA GGA CA TCCA GGGCA GCA TGGAA GCTGCGA TAAA GAA GA TTCCCTCCGTT PaTx-b ...............................U......................
- 25. Research Question Do roi possess the ability to produce scorpaenitoxins?
- 26. Research Question Do roi possess the ability to produce scorpaenitoxins? YES.
- 27. Future Directions • Expression Profile • Affect on Assays
- 28. Implications
- 29. Acknowledgements • Bryan Fry (University of Queensland) • Paul Bienfang (SOEST) • Brian Bowen & the ToBo Lab • Michelle Gaither, Kira Krend
- 30. Questions ?
Editor's Notes
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- Many of you are probably familiar with the Peacock Grouper, Cephalopholis argus, locally known as roi. \nThey were introduced in introduced in the 1950s with the hopes of creating a fishery\nThey established well, and have spread throughout the main hawaiian islands and far north as French Frigate Shoals\nLocal populations have flourished, and roi are now the most abundant nearshore reef predators in the main Hawaiian islands. As such, roi are impacting the biodiversity of coral ecosystems through top-down control of reef fish recruitment\nDreams of a fishery have yet to be realized because although there are plenty of roi, no one will eat them because they fear ciguatera poisoning\n\n\n\n
- Many of you are probably familiar with the Peacock Grouper, Cephalopholis argus, locally known as roi. \nThey were introduced in introduced in the 1950s with the hopes of creating a fishery\nThey established well, and have spread throughout the main hawaiian islands and far north as French Frigate Shoals\nLocal populations have flourished, and roi are now the most abundant nearshore reef predators in the main Hawaiian islands. As such, roi are impacting the biodiversity of coral ecosystems through top-down control of reef fish recruitment\nDreams of a fishery have yet to be realized because although there are plenty of roi, no one will eat them because they fear ciguatera poisoning\n\n\n\n
- Many of you are probably familiar with the Peacock Grouper, Cephalopholis argus, locally known as roi. \nThey were introduced in introduced in the 1950s with the hopes of creating a fishery\nThey established well, and have spread throughout the main hawaiian islands and far north as French Frigate Shoals\nLocal populations have flourished, and roi are now the most abundant nearshore reef predators in the main Hawaiian islands. As such, roi are impacting the biodiversity of coral ecosystems through top-down control of reef fish recruitment\nDreams of a fishery have yet to be realized because although there are plenty of roi, no one will eat them because they fear ciguatera poisoning\n\n\n\n
- Many of you are probably familiar with the Peacock Grouper, Cephalopholis argus, locally known as roi. \nThey were introduced in introduced in the 1950s with the hopes of creating a fishery\nThey established well, and have spread throughout the main hawaiian islands and far north as French Frigate Shoals\nLocal populations have flourished, and roi are now the most abundant nearshore reef predators in the main Hawaiian islands. As such, roi are impacting the biodiversity of coral ecosystems through top-down control of reef fish recruitment\nDreams of a fishery have yet to be realized because although there are plenty of roi, no one will eat them because they fear ciguatera poisoning\n\n\n\n
- Many of you are probably familiar with the Peacock Grouper, Cephalopholis argus, locally known as roi. \nThey were introduced in introduced in the 1950s with the hopes of creating a fishery\nThey established well, and have spread throughout the main hawaiian islands and far north as French Frigate Shoals\nLocal populations have flourished, and roi are now the most abundant nearshore reef predators in the main Hawaiian islands. As such, roi are impacting the biodiversity of coral ecosystems through top-down control of reef fish recruitment\nDreams of a fishery have yet to be realized because although there are plenty of roi, no one will eat them because they fear ciguatera poisoning\n\n\n\n
- Many of you are probably familiar with the Peacock Grouper, Cephalopholis argus, locally known as roi. \nThey were introduced in introduced in the 1950s with the hopes of creating a fishery\nThey established well, and have spread throughout the main hawaiian islands and far north as French Frigate Shoals\nLocal populations have flourished, and roi are now the most abundant nearshore reef predators in the main Hawaiian islands. As such, roi are impacting the biodiversity of coral ecosystems through top-down control of reef fish recruitment\nDreams of a fishery have yet to be realized because although there are plenty of roi, no one will eat them because they fear ciguatera poisoning\n\n\n\n
- Many of you are probably familiar with the Peacock Grouper, Cephalopholis argus, locally known as roi. \nThey were introduced in introduced in the 1950s with the hopes of creating a fishery\nThey established well, and have spread throughout the main hawaiian islands and far north as French Frigate Shoals\nLocal populations have flourished, and roi are now the most abundant nearshore reef predators in the main Hawaiian islands. As such, roi are impacting the biodiversity of coral ecosystems through top-down control of reef fish recruitment\nDreams of a fishery have yet to be realized because although there are plenty of roi, no one will eat them because they fear ciguatera poisoning\n\n\n\n
- Many of you are probably familiar with the Peacock Grouper, Cephalopholis argus, locally known as roi. \nThey were introduced in introduced in the 1950s with the hopes of creating a fishery\nThey established well, and have spread throughout the main hawaiian islands and far north as French Frigate Shoals\nLocal populations have flourished, and roi are now the most abundant nearshore reef predators in the main Hawaiian islands. As such, roi are impacting the biodiversity of coral ecosystems through top-down control of reef fish recruitment\nDreams of a fishery have yet to be realized because although there are plenty of roi, no one will eat them because they fear ciguatera poisoning\n\n\n\n
- Many of you are probably familiar with the Peacock Grouper, Cephalopholis argus, locally known as roi. \nThey were introduced in introduced in the 1950s with the hopes of creating a fishery\nThey established well, and have spread throughout the main hawaiian islands and far north as French Frigate Shoals\nLocal populations have flourished, and roi are now the most abundant nearshore reef predators in the main Hawaiian islands. As such, roi are impacting the biodiversity of coral ecosystems through top-down control of reef fish recruitment\nDreams of a fishery have yet to be realized because although there are plenty of roi, no one will eat them because they fear ciguatera poisoning\n\n\n\n
- Ciguatera is a paralytic lipid toxin produced by certain dinoflaggelate species in tropical and subtropical waters. Ingestion of the toxin leads to neurological and muscular symptoms including paralysis and death. Currently, there is no cure, and a high enough dose of the toxin is fatal. CTX estimated to affect as many as 500,000 ppl per year\n\nThe toxins are heat-stable, colorless, and odorless, and are thus impossible to detect without biochemical investigation. Because the toxins are not inactivated through any normal means of fish preparation, detection of fish with high toxin concentration is the only way to prevent poisoning. In communities where ciguatera is endemic, large, predatory reef fish are assumed to pose a health risk, and thus are avoided, reducing the number of fish available for sustenance and increasing the fishing pressure on other fish species.\n\n
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- Studies examining roi, however, have produced confusing results. Dierking & Campora [8] found that between 8% and 24% of the roi had unsafe CTX levels, but found no correlation to length or weight, which doesn’t make sense for a bioaccumulating toxin. However, these results were obtained using a potentially unreliable test for ciguatoxicity, the membrane immunoassay, marketed commerically as ciguacheck. Studies which have compared the results of this method to conventional ciguatera assays have found extraordinarily high false negative and false positive rates. However, there might be another confounding variable affecting the detection of ciguatera in roi in the bioassays used to evaluate the ciguacheck test - one that is based on a unique characteristic of roi taxonomy\n
- Studies examining roi, however, have produced confusing results. Dierking & Campora [8] found that between 8% and 24% of the roi had unsafe CTX levels, but found no correlation to length or weight, which doesn’t make sense for a bioaccumulating toxin. However, these results were obtained using a potentially unreliable test for ciguatoxicity, the membrane immunoassay, marketed commerically as ciguacheck. Studies which have compared the results of this method to conventional ciguatera assays have found extraordinarily high false negative and false positive rates. However, there might be another confounding variable affecting the detection of ciguatera in roi in the bioassays used to evaluate the ciguacheck test - one that is based on a unique characteristic of roi taxonomy\n
- Studies examining roi, however, have produced confusing results. Dierking & Campora [8] found that between 8% and 24% of the roi had unsafe CTX levels, but found no correlation to length or weight, which doesn’t make sense for a bioaccumulating toxin. However, these results were obtained using a potentially unreliable test for ciguatoxicity, the membrane immunoassay, marketed commerically as ciguacheck. Studies which have compared the results of this method to conventional ciguatera assays have found extraordinarily high false negative and false positive rates. However, there might be another confounding variable affecting the detection of ciguatera in roi in the bioassays used to evaluate the ciguacheck test - one that is based on a unique characteristic of roi taxonomy\n
- Studies examining roi, however, have produced confusing results. Dierking & Campora [8] found that between 8% and 24% of the roi had unsafe CTX levels, but found no correlation to length or weight, which doesn’t make sense for a bioaccumulating toxin. However, these results were obtained using a potentially unreliable test for ciguatoxicity, the membrane immunoassay, marketed commerically as ciguacheck. Studies which have compared the results of this method to conventional ciguatera assays have found extraordinarily high false negative and false positive rates. However, there might be another confounding variable affecting the detection of ciguatera in roi in the bioassays used to evaluate the ciguacheck test - one that is based on a unique characteristic of roi taxonomy\n
- Studies examining roi, however, have produced confusing results. Dierking & Campora [8] found that between 8% and 24% of the roi had unsafe CTX levels, but found no correlation to length or weight, which doesn’t make sense for a bioaccumulating toxin. However, these results were obtained using a potentially unreliable test for ciguatoxicity, the membrane immunoassay, marketed commerically as ciguacheck. Studies which have compared the results of this method to conventional ciguatera assays have found extraordinarily high false negative and false positive rates. However, there might be another confounding variable affecting the detection of ciguatera in roi in the bioassays used to evaluate the ciguacheck test - one that is based on a unique characteristic of roi taxonomy\n
- Studies examining roi, however, have produced confusing results. Dierking & Campora [8] found that between 8% and 24% of the roi had unsafe CTX levels, but found no correlation to length or weight, which doesn’t make sense for a bioaccumulating toxin. However, these results were obtained using a potentially unreliable test for ciguatoxicity, the membrane immunoassay, marketed commerically as ciguacheck. Studies which have compared the results of this method to conventional ciguatera assays have found extraordinarily high false negative and false positive rates. However, there might be another confounding variable affecting the detection of ciguatera in roi in the bioassays used to evaluate the ciguacheck test - one that is based on a unique characteristic of roi taxonomy\n
- Studies examining roi, however, have produced confusing results. Dierking & Campora [8] found that between 8% and 24% of the roi had unsafe CTX levels, but found no correlation to length or weight, which doesn’t make sense for a bioaccumulating toxin. However, these results were obtained using a potentially unreliable test for ciguatoxicity, the membrane immunoassay, marketed commerically as ciguacheck. Studies which have compared the results of this method to conventional ciguatera assays have found extraordinarily high false negative and false positive rates. However, there might be another confounding variable affecting the detection of ciguatera in roi in the bioassays used to evaluate the ciguacheck test - one that is based on a unique characteristic of roi taxonomy\n
- Groupers originally perciformes, but a landslide of molecular evidence has suggested that instead they belong among the venomous scorpaeniformes.\n\nThis is a small tree of the scorpaeniformes. Don’t worry about the species names - what I want to draw your attention to is that this one, right here, is a grouper. There have now been several molecular studies which have all agreed that the grouper family seranidae belong right here, in the middle of the scorpaeniform tree.\n\nThis is important because this places them as close relatives of lionfishes and scorpionfishes, one of the two most venomous groups in this lineage, known for their a highly potent scorpaenitoxin. further data has shown that the scorpaenitoxins from lionfishes are highly similar to those their distant relatives, the stonefishes, suggesting that this toxin originated with the earliest scorpaeniforms and is synapomorhic to the group as a whole. This means that groupers, too, may possess scorpaenitoxins. \n
- Groupers originally perciformes, but a landslide of molecular evidence has suggested that instead they belong among the venomous scorpaeniformes.\n\nThis is a small tree of the scorpaeniformes. Don’t worry about the species names - what I want to draw your attention to is that this one, right here, is a grouper. There have now been several molecular studies which have all agreed that the grouper family seranidae belong right here, in the middle of the scorpaeniform tree.\n\nThis is important because this places them as close relatives of lionfishes and scorpionfishes, one of the two most venomous groups in this lineage, known for their a highly potent scorpaenitoxin. further data has shown that the scorpaenitoxins from lionfishes are highly similar to those their distant relatives, the stonefishes, suggesting that this toxin originated with the earliest scorpaeniforms and is synapomorhic to the group as a whole. This means that groupers, too, may possess scorpaenitoxins. \n
- Groupers originally perciformes, but a landslide of molecular evidence has suggested that instead they belong among the venomous scorpaeniformes.\n\nThis is a small tree of the scorpaeniformes. Don’t worry about the species names - what I want to draw your attention to is that this one, right here, is a grouper. There have now been several molecular studies which have all agreed that the grouper family seranidae belong right here, in the middle of the scorpaeniform tree.\n\nThis is important because this places them as close relatives of lionfishes and scorpionfishes, one of the two most venomous groups in this lineage, known for their a highly potent scorpaenitoxin. further data has shown that the scorpaenitoxins from lionfishes are highly similar to those their distant relatives, the stonefishes, suggesting that this toxin originated with the earliest scorpaeniforms and is synapomorhic to the group as a whole. This means that groupers, too, may possess scorpaenitoxins. \n
- Groupers originally perciformes, but a landslide of molecular evidence has suggested that instead they belong among the venomous scorpaeniformes.\n\nThis is a small tree of the scorpaeniformes. Don’t worry about the species names - what I want to draw your attention to is that this one, right here, is a grouper. There have now been several molecular studies which have all agreed that the grouper family seranidae belong right here, in the middle of the scorpaeniform tree.\n\nThis is important because this places them as close relatives of lionfishes and scorpionfishes, one of the two most venomous groups in this lineage, known for their a highly potent scorpaenitoxin. further data has shown that the scorpaenitoxins from lionfishes are highly similar to those their distant relatives, the stonefishes, suggesting that this toxin originated with the earliest scorpaeniforms and is synapomorhic to the group as a whole. This means that groupers, too, may possess scorpaenitoxins. \n
- Groupers originally perciformes, but a landslide of molecular evidence has suggested that instead they belong among the venomous scorpaeniformes.\n\nThis is a small tree of the scorpaeniformes. Don’t worry about the species names - what I want to draw your attention to is that this one, right here, is a grouper. There have now been several molecular studies which have all agreed that the grouper family seranidae belong right here, in the middle of the scorpaeniform tree.\n\nThis is important because this places them as close relatives of lionfishes and scorpionfishes, one of the two most venomous groups in this lineage, known for their a highly potent scorpaenitoxin. further data has shown that the scorpaenitoxins from lionfishes are highly similar to those their distant relatives, the stonefishes, suggesting that this toxin originated with the earliest scorpaeniforms and is synapomorhic to the group as a whole. This means that groupers, too, may possess scorpaenitoxins. \n
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- the presence or absence of scorpaenitoxins in roi is important because it is possible that these toxins are throwing off ciguatera bioassays. There are four major bioassays used to evaluate the presence of ciguatoxin in fish - mouse, neuro, hemo, guinea. \n\ninotropy = increase in contractility\nWhat all this means is that if roi do possess a scorpaenitoxin, our tests for ciguatoxin may be thrown off. \n
- the presence or absence of scorpaenitoxins in roi is important because it is possible that these toxins are throwing off ciguatera bioassays. There are four major bioassays used to evaluate the presence of ciguatoxin in fish - mouse, neuro, hemo, guinea. \n\ninotropy = increase in contractility\nWhat all this means is that if roi do possess a scorpaenitoxin, our tests for ciguatoxin may be thrown off. \n
- the presence or absence of scorpaenitoxins in roi is important because it is possible that these toxins are throwing off ciguatera bioassays. There are four major bioassays used to evaluate the presence of ciguatoxin in fish - mouse, neuro, hemo, guinea. \n\ninotropy = increase in contractility\nWhat all this means is that if roi do possess a scorpaenitoxin, our tests for ciguatoxin may be thrown off. \n
- the presence or absence of scorpaenitoxins in roi is important because it is possible that these toxins are throwing off ciguatera bioassays. There are four major bioassays used to evaluate the presence of ciguatoxin in fish - mouse, neuro, hemo, guinea. \n\ninotropy = increase in contractility\nWhat all this means is that if roi do possess a scorpaenitoxin, our tests for ciguatoxin may be thrown off. \n
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- i used pcr to amplify scorpaenitoxin gene fragments from eight different roi specimens using primers designed against the published scorpaenitoxin from Synanceia horrida. Four of the specimens produced clean enough amplification for sequencing, and I aligned and compared those sequences to published toxin sequences using Geneious.\n
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- 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.\n\nSecondly, I think it is important to quantify the affect of scorpaenitoxins on the four major bioassays used to examine ciguatoxin levels, like the guina atrial assay and the hemolysis assay.\n
- Understanding the expression of roi scorpaenitoxins and how they affect ciguatera assays will allow us to develop a more accurate test for ciguatera in this potentially lucrative species, allowing us to reopen the fishery which would mitigate the negative ecological affects of the introduction while protecting the public from ciguatera poisoning.\n
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