Copyright By Ivette Yañez 2019 2 Investigation of genes coding for potential tetrodotoxin binding proteins in Canthigaster valentini and Taricha granulosa. By Ivette Yañez, B.A. A Thesis Submitted to the Department of Biology California State University Bakersfield In Partial Fulfillment for the Degree of Master of Science, Biology Summer 2019 3 4 Table of Contents ACKNOWLEDGEMENTS ........................................................................................................................................ 6 ABSTRACT ................................................................................................................................................................. 7 KEYWORDS ............................................................................................................................................................... 8 INTRODUCTION ....................................................................................................................................................... 9 INTRODUCTION TO TETRODOTOXIN................................................................................................................................. 9 HYPOTHESIS OF TTX ORIGIN IN TOXIC ORGANISMS .................................................................................................. 12 USES OF TTX........................................................................................................................................................................... 14 RESISTANCE OF TTX ............................................................................................................................................................ 15 TTX AS A PHEROMONE ....................................................................................................................................................... 16 PUFFERFISH SAXITOXIN AND TETRODOTOXIN BINDING PROTEINS (PSTBP) ...................................................... 17 RESEARCH PURPOSE …............................................................................................................................ ........................... 20 MATERIALS AND METHODS.............................................................................................................................. 27 DISSECTION ............................................................................................................................................................................ 27 COMPETITIVE INHIBITION ENZYMATIC IMMUNOASSAY (CIEIA) ........................................................................... 27 DNA EXTRACTION................................................................................................................................................................. 27 PCR ANALYSIS ....................................................................................................................................................................... 28 CLONING OF CANTHIGASTER VALENTINI AMPLICONS ................................................................................................ 29 COLONY PCR OF CLONED CANTHIGASTER VALENTINI AMPLICONS ........................................................................ 29 SEQUENCING OF CLONED CANTHIGASTER VALENTINI AMPLICONS ........................................................................ 29 CONTIG ASSEMBLY OF LARGE PRODUCT (5.1) ............................................................................................................. 30 TARICHA GRANULOSA ........................................................................................................................................................... 30 RESULTS .................................................................................................................................................................. 33 IDENTIFICATION OF TTX IN CANTHIGASTER VALENTINI ............................................................................................. 33 IDENTIFICATION OF TTX-BP GENES IN CANTHIGASTER VALENTINI ........................................................................ 33 INVESTIGATION OF TTX-BP GENES IN TARICHA GRANULOSA ................................................................................... 34 INVESTIGATION OF BACTERIA IN TARICHA GRANULOSA .......................................................................................... 35 DISCUSSION ............................................................................................................................................................ 55 INVESTIGATION OF TTX IN CANTHIGASTER VALENTINI .............................................................................................. 55 INVESTIGATION OF TTX-BP GENES IN CANTHIGASTER VALENTINI .......................................................................... 55 FUTURE WORK IN RESEARCH OF TTX BINDING PROTEINS IN CANTHIGASTER VALENTINI ............................... 56 INVESTIGATION OF TTX BINDING PROTEIN GENES IN TARICHA GRANULOSA ..................................................... 57 FUTURE WORK IN RESEARCH OF TTX BINDING PROTEINS IN TARICHA GRANULOSA ........................................ 58 INVESTIGATION OF BACTERIAL PRESSENCE IN TARICHA GRANULOSA ................................................................. 58 CONCLUSION ......................................................................................................................................................................... 60 LITERATURE CITED ............................................................................................................................................. 61 5 Acknowledgements I would like to express my gratitude to my thesis advisor and committee chair, Dr. Szick for her guidance and encouragement during these past three years. Support from Dr. Szick has allowed to pursue multiple research projects that have sprouted form my original research objective, thank you. I would also like to acknowledge and thank the members of my thesis committee Dr. Stokes and Dr. Francis for their support and guidance during my thesis research. Their patience and guidance have helped me become a better scientist. I would like to extend my gratitude to the Student Research Scholars Grant, the Grants, Research and Sponsored Programs (GRaSP), the Graduate Student-Faculty Collaborative Initiative, and the Graduate Student Center at California State University, Bakersfield for its support of my research through grants and scholarships. Their support also provided me with opportunities to present my work at competitions and prepare for my defense. I would like to thank Xochilt Ramirez for her contributions to this work as a research assistant. I would also like to thank Linsi Gutierrez and Aaron Baumgardner. This project would not have been possible without their help, support, and friendship. Quisiera darle agradecimiento a mi familia. El apoyo de parte de mi madre Gloria Valencia y hermanos, Rubi Contreras, Perla Cantu, y Salvador Yañez Valencia ha hecho toda le diferencia en mi carrera académica. Quisiere también reconocer a mi padre Salvador Yañez Morales, aunque no pudo estar conmigo durante mis estudios, espero honorar su memoria con lo que he logrado durante estos tres años. 6 Abstract Tetrodotoxin (TTX) is a powerful neurotoxin that selectively binds to voltage gated sodium channels in excitable tissues causing paralysis, asphyxiation, and death. TTX is found in a variety of unrelated taxa where multiple species have been reported to produce TTX binding proteins. Pufferfish in the Tetraodontidae family (mainly Takifugu spp.) have been demonstrated to accumulate high TTX concentrations however, the presence of TTX is still unknown for many pufferfish species. Canthigaster valentini is a species of pufferfish thought to be toxic but has yet to be assayed for the presence of TTX. Other species such as Taricha granulosa are also capable of accumulating high concentrations of TTX, however, the origin of TTX in this species is still debated. This study has multiple objectives i) determine if TTX is present in C. valentini liver tissues using CIEIA analysis, ii) identify possible TTX binding protein genes in C. valentini using PCR analysis with TTX binding protein gene specific primers, iii) identify possible TTX binding protein genes in T. granulosa using PCR analysis with TTX binding protein gene specific primers, and iv) to identify bacterial flora present on skin, intestine, liver, and ovary tissues using PCR analysis with 16S rDNA specific primers. In this study, the first instance of TTX identification and quantification in C. valentini is reported. Also, BLAST analysis of amplified products identified gene fragments similar to PSTBPs found in Takifugu spp. and their ancestral TBT-bp2 genes in pufferfish. Here, it is suggested that C. valentini may possess TTX binding proteins similar to those found in Takifugu spp. pufferfish. PCR amplification of T. granulosa DNA using TTX binding protein gene specific primers produced an amplicon of 615 bp however, BLAST analysis did not produce a significant alignment. Although bacterial DNA was amplified from total DNA extracts from T. granulosa tissues, metagenomic analysis did not produce data. 7 Keywords Tetrodotoxin, Tetrodotoxin binding protein, Taricha granulosa, Canthigaster valentini 8 Introduction Introduction to Tetrodotoxin Tetrodotoxin (TTX) is a potent neurotoxin found to be over 1000 times more toxic to humans than