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Molecular Techniques to Assess Fish Diet and Describe Prey Availability

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Molecular Techniques to Assess Fish Diet and Describe Prey Availability MOLECULAR TECHNIQUES TO ASSESS FISH DIET AND DESCRIBE PREY AVAILABILITY by Adam Daniel Clifford O’Dell B.Sc., University of Northern British Columbia, 2014 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN NATURAL RESOURCES AND ENVIRONMENTAL STUDIES (BIOLOGY) UNIVERSITY OF NORTHERN BRITISH COLUMBIA June 2018 © Adam O’Dell, 2018 ABSTRACT Determining ecological interactions between predators and prey is important for understanding population dynamics and nutrient and energy flow. Food webs, however, are challenging to describe, particularly for generalist predators such as juvenile fish whose diet consists largely of taxonomically diverse invertebrates. To gain a better understanding of predator-prey relationships for stream resident fish belonging to the families Salmonidae and Cottidae, I compared three methods of diet analysis – traditional taxonomy using morphological features, sequencing of individual specimens, and next generation sequencing of homogenized stomach contents. Morphological identification was often limited to the Order level but allowed for quantitative data, count and biomass. Genetic analysis of prey provided substantially greater taxonomic resolution, with next generation sequencing of stomach homogenates revealing a much more diverse diet in both fish species. I also used next generation sequencing to assess invertebrate biodiversity from environmental DNA samples from stream water, DNA from ethanol preservative from Surber samples, and from stomach homogenates of stream fish. Water samples detected terrestrial and aquatic invertebrate species. Surber ethanol revealed a similar invertebrate diversity as fish stomach homogenates and a subset of the taxa detected using eDNA. Fish, however, appeared to be generalist feeders and collectively accounted for the greatest number of unique taxa. Although substantial overlap was observed in the taxonomic composition of Salmonidae and Cottidae diets, detections of unique taxa in stomachs of species belonging to Salmonidae suggest a more opportunistic feeding behaviour and potentially exploitation of more available habitat for species within this family. i TABLE OF CONTENTS ABSTRACT .................................................................................................................................... i TABLE OF CONTENTS ............................................................................................................. ii LIST OF TABLES ....................................................................................................................... iv LIST OF FIGURES ................................................................................................................... viii ACKNOWLEDGEMENTS ........................................................................................................ xi PROLOGUE .................................................................................................................................. 1 CHAPTER 1 .................................................................................................................................. 5 ABSTRACT .................................................................................................................................................................................. 5 INTRODUCTION ....................................................................................................................................................................... 6 METHODS AND MATERIALS ............................................................................................................................................... 8 Morphological Identification of Prey Items ................................................................................................................... 10 Sanger Sequencing of Individual Prey Items ................................................................................................................. 11 Next generation Sequencing of Homogenized Stomach Samples ........................................................................ 15 RESULTS .................................................................................................................................................................................... 21 Morphological identification ................................................................................................................................................ 21 Individual sequencing of prey items .................................................................................................................................. 24 Next generation Sequences from stomach samples ................................................................................................... 27 Comparison of Approaches .................................................................................................................................................... 29 DISCUSSION ............................................................................................................................................................................. 39 Morphological analysis ............................................................................................................................................................ 40 Individual sequencing .............................................................................................................................................................. 42 Ion Torrent Next Generation Sequencing ....................................................................................................................... 44 Conclusion ...................................................................................................................................................................................... 45 CHAPTER 2 ................................................................................................................................ 48 ABSTRACT ................................................................................................................................................................................ 48 INTRODUCTION ..................................................................................................................................................................... 48 METHODS AND MATERIALS ............................................................................................................................................. 52 Sample Collection ....................................................................................................................................................................... 54 DNA Extraction ........................................................................................................................................................................... 56 ii Selection of universal invertebrate primers .................................................................................................................. 59 Preparation for Ion Torrent Sequencing ......................................................................................................................... 59 Sequence analysis ....................................................................................................................................................................... 62 Taxonomic Assignment of OTUs .......................................................................................................................................... 66 Analysis ........................................................................................................................................................................................... 66 RESULTS .................................................................................................................................................................................... 69 Taxon Composition – Lowest possible level ................................................................................................................... 70 Taxon Composition – genera and species ....................................................................................................................... 76 Comparison of Taxonomic Composition Between Sample Types......................................................................... 87 DISCUSSION ............................................................................................................................................................................. 95 Taxonomic resolution ............................................................................................................................................................... 98 Limitations to the approach............................................................................................................................................... 100 Consistency in the approach .............................................................................................................................................. 103 Ecological significance.......................................................................................................................................................... 105 EPILOGUE ............................................................................................................................... 108 LITERATURE
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