Anguillicoloides crassus: an invasive parasitic nematode infecting American eel (Anguilla rostratd) in Cape Breton, Nova Scotia by Cheryl Wall A Thesis Submitted to Saint Mary's University, Halifax, Nova Scotia in Partial Fulfillment of the Requirements for the Degree of Master of Science in Applied Science August 19, 2011, Halifax, Nova Scotia ©Cheryl Wall 2011 Approved: Dr. Katherine Jones Supervisor Approved: Dr. David Cone Supervisor Approved: Dr. Gary Conboy Examiner Approved: Mr. John MacMillan Supervisory Committee Approved: Dr. William Jones Supervisory Committee Approved: Dr. Kevin Vessey Dean of Graduate Studies Approved: Dr. Jeremy Lundholm Program Co-ordinator Date: August 19, 2011 Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington OttawaONK1A0N4 OttawaONK1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-81332-4 Our file Notre reference ISBN: 978-0-494-81332-4 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. 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While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. 1*1 Canada Certification Name: Cheryl Wall Degree: Master of Science in Applied Science Title of Thesis: Anguillicoloides crassus: an invasive parasitic nematode infecting American eel (Anguilla rostrata) in Cape Breton, Nova Scotia Examining Committee: Dr. Kevin Vessey, Dean of Graduate Studies Dr. Jeremy Lundholm, Program Co-ordinator Dr. Gary Conboy, External Examiner Dr. Katherine Jones, Supervisor Dr. David Cone, Supervisor Mr. John MacMillan, Supervisory Committee Dr. William Jones, Supervisory Committee TABLE OF CONTENTS ABSTRACT iv ACKNOWLEDGEMENTS v LIST OF FIGURES vi LIST OF TABLES viii CHAPTER 1 1 Ecology oi Anguillicoloides crassus infecting American eel (Anguilla rostrata) in Cape Breton, Nova Scotia CHAPTER 2 79 Inferring the origin of Anguillicoloides crassus infecting American eel (Anguilla rostrata) in Cape Breton, Nova Scotia using the cytochrome c oxidase I gene of the mitochondrial DNA SUMMARY 103 FUTURE RESEARCH 104 iii Anguillicoloides crassus: an invasive parasitic nematode infecting American eel (Anguilla rostrata) in Cape Breton, Nova Scotia Cheryl Wall August 19,2011 Abstract In 2007, Anguillicoloides crassus, an invasive, parasitic nematode found in the swimbladder of anguillid eels was documented for the first time in American eel in Canadian waters, including two sites in Cape Breton (CB) Island, Nova Scotia. That discovery necessitated this study on the distribution, ecology and origin of the parasite. It was found that sites throughout CB contain infected eels. Comparing the COI gene of the mitochondrial DNA among nematodes from CB, USA, Europe, and published sequences, it was determined that the CB infection likely originated from a direct transmission of the parasite from the USA or Japan via natural eel, intermediate or paratenic host movement and/or within the ballast water of boats. Infection occurred in both freshwater and estuarine sites, with intensity of infection increasing with length of eel. Pathological damage to the swimbladder was evident; however condition factor, liver somatic and spleen somatic indices were not affected by infection. iv Acknowledgements As with any research, this project would not be possible without the knowledge, support, advice and assistance of many individuals. I would like to sincerely thank all of those helping to make this thesis possible: Katherine Jones David Cone John MacMillan William Jones Timothy Frasier Jonathon Forest Dollie Campbell Stanley King Russell Easy Laurelle Saccary Christa Davis Alanna Khattar Carmen Cranley Heidi de Boer Mark Sullivan Dewayne Fox Lori Brown Francoise Daverat Florian Nagal Janek Simon Yu San Han Russell Poole Ciar O'Toole Jennie Dahlberg Hakan Wickstrom Susan Meek Susan Dore v LIST OF FIGURES CHAPTER 1 Figure 1 Taxonomy of Anguillicoloides crassus 29 Figure 2 ArcGIS map of sampling sites in Cape Breton, Nova Scotia: 31 eels caught Figure 3 ArcGIS map of Cape Breton, Nova Scotia: no eels caught 33 Figure 4 American eel 35 Figure 5 American eel length measurement 37 Figure 6 American eel weight measurement 39 Figure 7 American eel anal color 41 Figure 8 American eel swimbladder 43 Figure 9 Swimbladder yielding 50 Anguillicoloides crassus 45 Figure 10 Primary watersheds of Cape Breton, Nova Scotia 47 Figure 11 Scatter plot of intensity of infection vs. total length of 49 American eel in Cape Breton, Nova Scotia Figure 12 Scatter plot of intensity of infection vs. weight of 51 American eel in Cape Breton, Nova Scotia Figure 13 Scatter plot of condition factor vs. intensity for infected 53 American eels in Cape Breton, Nova Scotia Figure 14 Scatter plot of liver somatic index vs. intensity in infected 55 American eels in Cape Breton, Nova Scotia vi Figure 15 Scatter plot of spleen somatic index vs. intensity in infected 57 American eels in Cape Breton, Nova Scotia Figure 16 Histological section of American eel swimbladder 59 Figure 17 Histological section of American eel swimbladder 61 Figure 18 Bar graph of mean intensity vs. anal color in infected 63 American eel in Cape Breton, Nova Scotia CHAPTER 2 Figure 1 1.5% agarose gel showing serial dilutions of mtDNA used to 91 determine the optimal concentration for sequence analysis Figure 2 Maximum-Likelihood phylogenetic tree of Anguillicoloides 93 crassus cytochrome c oxidase I haplotypes vii LIST OF TABLES CHAPTER 1 Table 1 Sampling sites for American eels in Cape Breton, Nova 65 Scotia Table 2 Gross pathology of the swimbladder of American eels in 67 Cape Breton, Nova Scotia Table 3 Prevalence and mean intensity for the 10 primary 69 watersheds of Cape Breton, Nova Scotia Table 4 Prevalence and mean intensity for the 30 sites yielding 71 American eel in Cape Breton, Nova Scotia Table 5 Prevalence and mean intensity based on distance from 73 sea for American eel in Cape Breton, Nova Scotia Table 6 Prevalence and intensities of Anguillicoloides crassus 75 in European eels throughout Europe Table 7 Prevalence and mean intensities of Anguillicoloides 11 crassus in American eels from North American locations CHAPTER 2 Table 1 Sampling sites for American eels in Cape Breton, Nova 95 Scotia Table 2 Donated Anguillicoloides crassus samples by location 97 and sampler Table3 Location and number of samples of Anguillicoloides 99 crassus that underwent sequencing of the cytochrome c oxidase 1 gene Table 4 Identical sequences 101 Vlll Chapter 1: Ecology of Anguillicoloides crassus infecting American eel (Anguilla rostrata) in Cape Breton, Nova Scotia 1 Introduction American eel (Anguilla rostrata) are common in freshwater river systems and estuaries spanning the east coast of the western hemisphere. They occur as far north as Greenland, southward along the east coast of North America, and as far south as Guyana in South America (Scott & Crossman, 1973; Tesch, 1977, 2003). This anguillid species is catadromous, spawning in the Sargasso Sea, with its larvae drifting northward for approximately one year before entering estuaries and fresh water rivers of the east coast. In freshwater it pigments, grows, and develops sexually before returning to the sea to spawn (Scott & Crossman, 1973; Tesch, 1977; COSEWIC, 2006). American eel once supported valuable fisheries throughout much of their North American range, with high success occurring along the St. Lawrence estuary into the Maritime Provinces (Eales, 1968). Furthermore, these eels have high social and ceremonial significance for the First Nations of Eastern Canada, making American eel an important aspect of their culture (COSEWIC 2006; S. Denny, pers. comm.). Unfortunately, American eel has been in decline throughout North America during the last several decades, resulting in a recent (2006) listing of 'Special Concern' by the Committee on the Status of Endangered Wildlife in Canada (COSEWIC), and it is currently being considered for listing under the federal Species at Risk Act (COSEWIC 2006). It is believed that the North Atlantic Oscillation,