Aquatic Parasite Information – a Database on Parasites of Freshwater and Brackish Fish in the United Kingdom A thesis submitted to Kingston University London for the Degree of Doctor of Philosophy by Bernice BREWSTER BSc (Hons.) London June 2016 Declaration This thesis is being submitted in partial fulfilment of the requirements of Kingston University, London for the award of Doctor of Philosophy. I confirm that all work included in this thesis was undertaken by myself and is the result of my own research and all sources of information have been acknowledged. Bernice Brewster 2nd June 2016 I Abstract A checklist of parasites of freshwater fish in the UK is an important source of information concerning hosts and their distribution for all aspects of scientific research. An interactive, electronic, web-based database, Aquatic Parasite Information has been designed, incorporating all freshwater and brackish species of fish, parasites, taxonomy, synonyms, authors and associated hosts, together with records for their distribution. One of the key features of Aquatic Parasite Information is this checklist can be updated. Interrogation of Aquatic Parasite Information has revealed that some parasites of freshwater and brackish species of fish, such as the unicellular groups or those metazoans that are difficult to identify using morphological characters, are under reported. Aquatic Parasite Information identified the monogenean family Dactylogyridae and the cestodes infecting UK freshwater fish as under-represented groups, owing to the difficulties identifying them morphologically. Both the Dactylogyridae and cestodes have implications for pathology, outbreaks of disease and morbidity in freshwater fish in the UK, therefore accurate identification is critical. Studies were undertaken using both standard morphological techniques of histology and molecular techniques to identify dactylogyrid species and tapeworms commonly found parasitizing fish in the UK. Morphological studies demonstrated that histological processes could lead to distortion of the specimens and permanent mounting may affect the orientation which may obscure vital characters. Molecular techniques were successfully employed using ITS1 for the Dactylogyridae and cox1 and r28s for the cestodes, to demonstrate genetic variability for the interspecific identification of species. Histology, scanning electron microscopy and molecular techniques have also ii identified an Atractolytocestus sp. tapeworm, parasitizing carp in the UK, as a potentially new species. Analysis of parasite records extracted from Aquatic Parasite Information has implicated freshwater fishery management policies as impacting on the dissemination and distribution of parasites, resulting in the spread of some species and decline of others. iii Acknowledgments I gratefully acknowledge the support, guidance and dedication of my Supervisors Dr Ruth Kirk, Dr Scott Lawton, Dr James Denholm-Price, the late Professor Angela Russell-Davies and Dr Chris Williams. I am indebted to Mr Sivasankara Desikan for designing the front-ends for Aquatic Parasite Information, enabling the content of the database to be accessible on the internet and for his patience throughout the process. I am also indebted to Amy Reading and Nigel Hewlett, Environment Agency, National Fisheries Laboratory for providing me with cestode specimens for molecular work and data relating to fish movements. This project could never have taken place without all the fish traders and fish farmers who have unknowingly contributed by delivering samples of fish for examination for movement consent. Finally, a special thanks to my family and friends for putting up with the fish stuff, my constant mess and chaos and also to Finzi who has had to wait ‘forever’, every day for a walk. iv Table of Contents Declaration i Abstract ii Acknowledgements iv Table of contents v List of figures x List of tables xv Chapter 1. General Introduction and rationale for the project 1.1 Current legislation and regulations 1 1.2 Importation of freshwater fish 4 1.2.1 Aquaculture 4 1.2.2 Ornamental market 5 1.2.3 Recreational angling 8 1.3 Introduction of novel parasites 10 1.4 Aquaculture in the UK 12 1.5 Changing populations of fish predators 16 1.6 Aquatic Parasite Information 19 1.6.1 Database design 20 1.6.2 Interrogating the database 21 1.7 Morphological and molecular study of species of Dactylogyrus (Monogenea; Dactylogyroidea) associated with coarse fish 21 1.8 Cestodes of freshwater fish in the UK 22 1.9 Identification of Atractolytocestus species (Cestoda: 23 Caryophyllidea; Lytocestidae) infecting common carp (Cyprinus carpio) in the UK 1.10 Concluding remarks 24 v 1.11 Aims and Objectives 26 Chapter 2. Aquatic Parasite Information database 2.1 Introduction 27 2.2 Database software design 29 2.3 Data sources 31 2.4 Data overview 32 2.5 Concluding remarks 49 Chapter 3. Aquatic Parasite Information Database, Category 2 parasite distribution 3.1 Introduction 51 3.2 Methods 57 3.3 Results and discussion 58 3.4 Concluding remarks 93 Chapter 4. Morphological and molecular study of species of Dactylogyrus (Monogenea: Dactylogyroidea) associated with coarse fish 4.1 Introduction 94 4.2 Materials and methods 98 4.2.1 Collection of dactylogyrids from UK freshwater fish 98 4.2.2 Preparation of Dactylogyrus specimens for 98 morphological study 4.2.3 DNA extraction and PCR amplification 101 4.2.4 Sequence assembly, initial identification and 102 phylogenetics vi 4.2.5 Markers for species identification and inter- and 104 intraspecies molecular diversity 4.3 Results 4.3.1 Morphometric analysis of Dactylogyrus species from 105 carp, bream, roach and tench 4.3.2 Morphological description of Dactylogyrus species 105 4.3.3 Initial identification of sequences using BLASTn 114 4.3.4 Phylogenetic reconstruction 115 4.3.5 Diversity and phylogenetic power of ITS1 in resolving 120 Dactylogyrus taxonomy 4.4 Discussion of results 124 4.5 Concluding remarks 130 Chapter 5. Cestodes of freshwater fish in the UK 5.1 Introduction 132 5.2 Materials and methods 133 5.2.1 Collection of cestodes from UK freshwater fish 133 5.2.2 Preparation of cestodes for morphological study 134 5.2.3 DNA extraction and PCR amplification 135 5.2.4 Assembly of Caryophyllidea and Bothriocephalidea 136 cox1 and r28s rDNA fragments, molecular identification of species and phylogenetic analysis 5.2.5 Markers for species identification, inter- and 141 intra-species molecular diversity 5.3 Results 141 5.3.1 Morphology of cestodes identified from UK species 141 of freshwater fish vii 5.3.2 Caryophyllidea morphology 142 5.3.3 Bothriocephalidea morphology 148 5.3.4 Sequence assembly and initial comparison of 149 species and phylogenetics based on Caryophyllidea cox1, r28s and Bothriocephalidea r28s 5.3.5 Caryophyllidea phylogeny 152 5.3.6 Bothriocephalidea phylogeny 153 5.3.7 Diversity and phylogenetic power of cox1 and 164 r28s in resolving Caryophyllidea and Bothriocephalidea taxonomy 5.4 Discussion 171 5.4.1 Caryophyllidea 171 5.4.2 Freshwater Bothriocephalidea 175 5.5. Concluding Remarks 177 Chapter 6. Identification of Atractolytocestus (Cestoda: Caryophyllidea: Lytocestidae) species parasitizing common carp (Cyprinus carpio) in the United Kingdom 6.1 Introduction 179 6.2 Materials and methods 182 6.2.1 Histological preparation of Atractolytocestus species 183 6.2.2 Preparation of Atractolytocestus for scanning electron 183 microscopy viii 6.2.3 DNA extraction and amplification 184 6.2.4 Assembly of Atractolytocestus cox1, molecular 185 identification of species and phylogenetic analysis 6.2.5 Intra-species molecular diversity 186 6.3 Results 186 6.3.1 Morphology of Atractolytocestus huronensis and 186 Atractolytocestus sp. from Pease Pottage, West Sussex 6.3.2 Initial comparison of Atractolytocestus sequences 190 and phylogenetics based on cox1 6.3.3 Intraspecific molecular variance of Atractolytocestus 191 cox1 sequences 6.4 Discussion 195 6.5 Concluding remarks 199 Chapter 7. Summary 200 Appendix 1. Database software design 207 Appendix 2. Fish hosts and associated parasites 212 Appendix 3. Freshwater fish parasite distribution in the UK 221 References 240 ix List of Figures Fig. 1.1 Coldwater and tropical freshwater fish tonnage imported into 6 the UK 2004 – 2014 (Source: Ornamental Aquatic Trade Association) Fig. 1.2 Life cycle of Philometroides sanguinea 11 Fig. 1.3. World carp and cyprinid production 2005 – 2012 (Source FAO) 13 Fig. 1.4 Scottish farmed rainbow trout escapes 1998 – 2014 14 (Source Scotland Aquaculture) Fig. 1.5 Otter predation of carp 18 Fig. 2.1 Entities forming the conceptual design of the relational 30 database for the parasites of freshwater fish Fig.2.2 Annual records of carp infected with Sanguinicola inermis, 48 extracted from Aquatic Parasite Information Fig. 2.3 Example of emaciated carp removed from an over-stocked fishery 49 Fig. 3.1 Aquatic Parasite Information parasite search engine offering 57 multiple choices for mining information Fig. 3.2 Aquatic Parasite Information advanced search engine, facilitating 58 search for records in specific fields within the database Fig. 3.3 Aquatic Parasite Information annual records 1967 – 2016, for 61 Ergasilus sieboldi and E. briani Fig. 3.4 Ergasilus sieboldi and Ergasilus briani host records, data 62 from Aquatic Parasite Information Fig. 3.5.Prevalence of infection with Ergasilus sieboldi sites A to E, 63 mixed coarse fisheries and Site E a reservoir (data from Aquatic Parasite Information records ) Fig. 3.6 Mean intensity of Ergasilus sieboldi sites A to D, mixed coarse 63 fisheries and Site E a reservoir, data from Aquatic Parasite Information Fig.3.7 Prevalence of Ergasilus briani infection from sites B, D, J, K, L, M, 64 N, P, R, and S mixed coarse fisheries, data from Aquatic Parasite Information x Fig. 3.8 Mean intensity of Ergasilus briani on sites B, D, J, K, L, M, 66 N, P, R, and S mixed coarse fisheries (Data from Aquatic Parasite Information) Fig. 3.9 Prevalence of Ergasilus sieboldi and Ergasilus briani on sites 67 B & D (Data from Aquatic Parasite Information) Fig.