University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Dissertations & Theses in Natural Resources Natural Resources, School of 5-2015 Acute Toxicity of β-N-Methylamino-L-Alanine (BMAA) to Fathead Minnow (Pimephales promelas) and Zebrafish (Danio rerio) Jiayi Wang University of Nebraska-Lincoln, [email protected] Follow this and additional works at: http://digitalcommons.unl.edu/natresdiss Part of the Marine Biology Commons, Natural Resources Management and Policy Commons, Pharmacology, Toxicology and Environmental Health Commons, Terrestrial and Aquatic Ecology Commons, and the Water Resource Management Commons Wang, Jiayi, "Acute Toxicity of β-N-Methylamino-L-Alanine (BMAA) to Fathead Minnow (Pimephales promelas) and Zebrafish (Danio rerio)" (2015). Dissertations & Theses in Natural Resources. 116. http://digitalcommons.unl.edu/natresdiss/116 This Article is brought to you for free and open access by the Natural Resources, School of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Dissertations & Theses in Natural Resources by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. ACUTE TOXICITY OF β-N-METHYLAMINO-L-ALANINE (BMAA) TO FATHEAD MINNOW (PIMEPHALES PROMELAS) AND ZEBRAFISH (DANIO RERIO) by Jiayi Wang A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science Major: Natural Resource Sciences Under the Supervision of Professors Kyle Hoagland and Daniel Snow Lincoln, Nebraska May, 2015 ACUTE TOXICITY OF β-N-METHYLAMINO-L-ALANINE (BMAA) TO FATHEAD MINNOW (PIMEPHALES PROMELAS) AND ZEBRAFISH (DANIO RERIO) Jiayi Wang, M.S. University of Nebraska, 2014 Advisors: Kyle Hoagland and Daniel Snow β-N-methylamino-L-alanine (BMAA) is a neurotoxic amino acid produced by most species of cyanobacteria. Exposure to BMAA has been hypothesized as a cause of ALS and possibly Parkinson’s and Alzheimer’s diseases for several decades. Both in vitro and in vivo experiments revealed that exposure to elevated concentrations of BMAA can damage motor neurons and cause motor dysfunctions. However, the exact mechanism of BMAA-induced neurotoxicity has not been well understood. Based on the available literature and in spite of its water-soluble and non-protein nature, BMAA appears to be able to bioaccumulate in organisms. The ubiquity of cyanobacteria and the potential for bioaccumulation of BMAA, arouse wide human health concern. Previous investigations into toxicity of BMAA mainly focused on various mammalian test models, including rats, mice and primates. However, the toxic effect of BMAA on aquatic organisms has attracted limited attention. Due to the potential for widespread BMAA production in aquatic ecosystems, it is important to understand the toxicity of BMAA in aquatic organisms, such as fish, from an ecotoxicological perspective Because limited information exists about how exposure to BMAA influences fish, we investigated the acute toxic effect of β-N-methylamino-L-alanine (BMAA) on two widely adopted model fish: fathead minnow (Pimephales promelas) and zebrafish (Danio rerio). The 96 h toxicity tests revealed that BMAA is not acutely lethal to fathead minnow (juveniles: 96 h LC50 > 10 mg/L; larvae: 96 h LC50 > 10 mg/L) or zebrafish (larvae: 96 h LC50 > 10 mg/L; embryos: 10-day LC50 > 100 mg/L) at concentrations well above those reported in the literature. However, exposure to BMAA at 100 mg/L significantly affected the heart rate of zebrafish embryos. Additionally, in the zebrafish embryo-larvae behavioral assay, a range of locomotor function anomalies in zebrafish embryo-larvae exposed to BMAA were observed. On one hand, BMAA was found to accelerate the onset of spontaneous contractions and enhanced touch-evoked contractions. On the other hand, BMAA reduced performance in touch-evoked escape, free swimming and startle response. To our knowledge, we are the first to investigate the acute toxicity of BMAA in fathead minnow and its behavioral effects on fish. The finding that BMAA influenced locomotor behaviors of fish is of both neurotoxicological and ecological importance. iii ACKNOWLEDGEMENTS First, I would like to thank my advisors Dr. Kyle Hoagland and Dr. Daniel Snow. I am so grateful that I could be a student of Dr. Hoagland, a nice and responsible advisor. Without him, I could not have been able to work on this project and pursue my M.S. degree in this country. Thank you to Dr. Daniel Snow for his valuable advice on my research design and academic writing. I would like to thank Dr. Blair Siegfried and Dr. Ayse Kilic, for being willing to be my committee members. Thank you both for your support and suggestions on my thesis. I would also like to thank all staff in the Water Science Lab, especially Dr. David Cassada. His patient and professional guidance helped me to have a better understanding of HPLC analysis. Besides, I want to thank Vicky Samek and Sara Findrick in the Life Sciences Annex, for their assistance in establishment and maintenance of the lab. I want to thank Drs. Mark Pegg, Steve Thomas and Amy Burgin, for their help in providing equipment needed for my experiment. I also want to thank Kelly Heath and Kathy Pinkerton for their useful suggestions on animal care. Especially, please allow me to thank Dr. John Carroll, Dr. Ayse Kilic, and Dr. John Holz and Mr. Tadd Barrow (HABAquaticSolutions) for their generous financial help when I had trouble to continue my study. Without your support, I could never have had the chance to finish my research. Finally, I want to say thank you to my family and my girlfriend Mary Wang. It was they who gave me the strength to finish my degree. I could not imagine how I could iv be here without their care and encouragement. v TABLE OF CONTENTS Abstract ..........................................................................................................................i ACKNOWLEDGEMENTS .........................................................................................iii TABLE OF CONTENTS ..............................................................................................v LIST OF FIGURES ......................................................................................................ix ABBREVIATIONS .......................................................................................................x CHAPTER ONE. LITERATURE REVIEW 1.1 β-N-methylamino-L-alanine (BMAA)..................................................................... 1 1.1.1 ALS/PDC and the discovery of BMAA ................................................................ 1 1.1.2 Structure and biochemistry of BMAA .................................................................. 2 1.2 Neurotoxicity of BMAA .......................................................................................... 3 1.2.1 BMAA ALS/PDC hypothesis ............................................................................... 3 1.2.2 Bioaccumulation of BMAA .................................................................................. 4 1.2.3 Experimental evidences of BMAA-induced neurotoxicity ................................... 5 1.2.4 Mechanisms of BMAA-induced neurotoxicity ..................................................... 7 1.3 BMAA in ecosystems .............................................................................................. 9 1.3.1 Source of BMAA .................................................................................................. 9 1.3.2 Occurrence of BMAA in diverse environments.................................................. 11 1.3.3 Bioaccumulation of BMAA in wild organisms .................................................. 11 vi 1.4 Ecotoxicity of BMAA ............................................................................................ 13 1.4.1 Toxic effects of BMAA on terrestrial organisms ................................................ 13 1.4.2 Toxic effects of BMAA on aquatic organisms .................................................... 15 CHAPTER TWO: MATERIALS AND METHODS 2.1 Introduction…........................................................................................................ 18 2.2 Chemicals…………………………………………………………………………....................22 2.3 Test organisms and maintenance ............................................................................ 23 2.4 Acute fathead minnow juvenile test ....................................................................... 24 2.4.1 Lab conditions ..................................................................................................... 24 2.4.2 Preparation before exposure ............................................................................... 24 2.4.2.1 Handling of fathead minnow juveniles ............................................................ 24 2.4.2.2 Acclimation of fathead minnow juveniles ....................................................... 25 2.4.3 Range-finding test ............................................................................................... 25 2.5 Acute fathead minnow larvae test .......................................................................... 26 2.5.1 Lab conditions ..................................................................................................... 26 2.5.2 Preparation before exposure ............................................................................... 26 2.5.2.1 Handling of fathead minnow larvae