Aspects of the Biology of Selected Monogenean Parasites from Fish in the Vaal Dam, South Africa

ASPECTS OF THE BIOLOGY OF SELECTED MONOGENEAN PARASITES FROM FISH IN THE VAAL DAM, SOUTH AFRICA

DIONNE CRAFFORD

THESIS

SUBMITTED IN FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE

PHILOSOPHIAE DOCTOR

ZOOLOGY

IN THE

FACULTY OF SCIENCE

AT THE

UNIVERSITY OF JOHANNESBURG

PROMOTOR: PROF. A. AVENANT-OLDEWAGE (UNIVERSITY OF JOHANNESBURG)

CO-PROMOTOR: PROF. W. LUUS-POWELL (UNIVERSITY OF LIMPOPO)

FEBRUARY 2013

Dedication

To Nicolette, Iwan and Larindi.

If I were to attempt composition of an ode describing my love and appreciation, any amount of words would only miserably fail at conveying its true extent…

Thank you!!

ii Acknowledgements

 Christ through whom all things are possible;  Nicolette Crafford, my wife, whom unselfishly gave so much of her time, effort and dreams to help realize mine;  The University of Johannesburg (UJ) for financial and logistical support;  The National Research Foundation (NRF) for financial support;  The University of Limpopo (UL) for logistical support;  Prof. Annemarie Avenant-Oldewage (University of Johannesburg) and Prof. Wilmien Luus-Powell (University of Limpopo), my study supervisors for this project, for practical assistance, scientific insight and support on so many levels throughout the entire process;  Dr. Richard Greenfield (University of Johannesburg) for his technical and scientific expertise in the field during planning and execution of field trips for fish collection;  Students from the University of Johannesburg (specifically students from the Parasitology laboratory) whom, under the leadership of Prof. Avenant- Oldewage, assisted with field work preparation and actual fish collection. I do not have the words to explain how much I appreciate their assistance;  Staff of the University of Johannesburg Island (Vaal Dam), where the field work was conducted, for excellent service;  Mr. Frederik van der Walt from the University of Johannesburg (Department of Statistics) whose kind assistance went far beyond what was originally expected;  Prof. WJ Henderson from the University of Johannesburg, for his help with formulating correct Latin names where new species were described;  Staff at the University of Limpopo, in particular Dr. Moses Matla, for practical guidance with reference to identification of parasites;  Prof. Dawie Kok (ClinVet International (Pty) Ltd) for encouragement and logistical arrangements with matters pertaining to ClinVet involvement (i.e. use of laboratory facilities and equipment where required);  My parents, whom never stopped believing in me.

iii Table of Contents

1 SYNOPSIS: FROM AIM TO ABSCISSION ...... 1 1.1. Introduction ...... 1 1.2. Problem statement ...... 1 1.3. Project aims / objectives / hypotheses ...... 1 1.3.1. Project aims / objectives ...... 1 1.3.2. Hypotheses formulated from objectives ...... 2

1.4. Thesis outlay ...... 3 1.4.1. Notes on chapter delineation and section cross-referencing ...... 3 1.4.2. Notes on referencing methodology ...... 3 1.4.3. Organization of chapters...... 4 1.4.4. Study outputs ...... 7 1.4.4.1. Conferences ...... 7 1.4.4.2. Published papers ...... 7

2 GENERAL INTRODUCTION...... 8 2.1. A note concerning nomenclature ...... 9 2.2. Why parasites? ...... 10 2.2.1. Aquaculture and ornamental fish trade ...... 11 2.2.2. Parasitological studies in “wild” versus cultured fish populations: Finding a common link . 12 2.2.3. Fisheries management ...... 13 2.2.4. Parasites as biological models and indicators ...... 13 2.2.5. Environmental management, ecosystem integrity and biodiversity ...... 14 2.2.6. Host taxonomy, translocation history and phylogenetic relationships ...... 16 2.2.7. Relevance of aspects mentioned to the current study as reflected in this thesis ...... 17

2.3. Terms and terminology: Constructing an ecological framework for discussion ...... 19 2.3.1. Defining “ecology” as a distinct field of science ...... 19 2.3.2. Application of the term “ecology” in the current study ...... 20 2.3.3. Infection statistics ...... 21

2.4. Meet the Monogenea: A fleeting introduction ...... 21 2.4.1. The bigger picture: a phylogenetic view ...... 21 2.4.2. A “typical” monogenean: morphology and biology ...... 24 2.4.3. Genera included in the current study ...... 26 2.4.4. Gyrodactylus von Nordmann, 1832 ...... 26 2.4.5. Dactylogyrus Diesing, 1850 ...... 27 2.4.6. Dogielius Bychowsky, 1936 ...... 27 2.4.7. Diplozoon von Nordmann, 1832 and Paradiplozoon Akhmerov, 1974 ...... 28 2.4.8. Quadriacanthus Paperna, 1961 ...... 28 2.4.9. Structures used for species descriptions / identification ...... 28

2.5. Morphological variability and phylogeny – a challenging interpretation ...... 29 2.6. Species identification, description and phylogeny: Morphometric vs. molecular approaches 30

iv 2.7. Host specificity ...... 31 2.8. Site specificity ...... 33 2.9. Host effects on infection parameters ...... 34 2.10. Effect of abiotic and environmental variables on infection parameters ...... 37 2.10.1. Temperature ...... 38 2.10.2. Chemical characteristics ...... 38 2.10.3. Other factors ...... 39

2.11. Notes on the study of parasite communities...... 40 2.12. Review of monogenean research in southern Africa ...... 42 2.13. Problem statement ...... 43 3 MATERIALS AND METHODS ...... 48 3.1. Study site description ...... 48 3.1.1. River system and catchment description ...... 48 3.1.2. Vaal Dam ...... 49

3.2. Fish collection ...... 51 3.2.1. Method of collection ...... 51 3.2.2. Species collected ...... 52

3.3. Host necropsy and parasite recovery ...... 52 3.3.1. Identification and taxonomic nomenclature of hosts examined ...... 52 3.3.2. Host weight measurement ...... 53 3.3.3. Preparation of mucous smears ...... 53 3.3.4. Euthanasia ...... 53 3.3.5. Host length measurements and condition factor calculations ...... 53 3.3.6. Host necropsy procedure...... 54 3.3.7. Division of gills for subsequent examination...... 54 3.3.8. Examination of gills ...... 55 3.3.9. Parasite removal from gill scrapings and mucous smears ...... 56

3.4. Parasite identification and description ...... 56 3.4.1. Illustrations of sclerotized structures ...... 56 3.4.2. Standard methodology for measuring sclerotized structures ...... 56 3.4.3. A note on terminology used with reference to sclerotized structures of different genera ... 57 3.4.3.1. Dactylogyrus Diesing, 1850 and Dogielius Bychowsky, 1936 ...... 57 3.4.3.2. Quadriacanthus Paperna, 1961 ...... 58 3.4.3.3. Gyrodactylus von Nordmann, 1832 ...... 58

3.5. Calculation of infection statistics ...... 61 3.6. Statistical analysis ...... 62 3.6.1. Descriptive statistics ...... 62 3.6.2. Grouping of measured variables / morphometric analyses to evaluate the separate species versus forms of the same species concept (Chapter 4) ...... 62 3.6.3. Description of ecological aspects of monogeneans infecting Labeo spp. hosts as examined during the winter (June / July 2009) survey (Chapter 6) ...... 63

v 3.6.4. Description and seasonal comparison of ecological aspects of monogeneans infecting Labeo spp. as examined during a winter (June / July 2009) and summer (January 2010) survey (Chapter 7) ...... 64 3.6.5. Description of ecological aspects of monogeneans infecting other host species (Chapters 8 to 12) ...... 64

4 EVALUATION OF VARIANCE OF FORMS WITHIN A SINGLE PARASITE SPECIES, VERSUS SEPARATE SPECIES OF DACTYLOGYRUS Diesing, 1850 AND DOGIELIUS Bychowsky, 1936 ON LABEO Cuvier, 1817 HOSTS ...... 66 4.1. Introduction ...... 66 4.2. Materials and methods ...... 68 4.3. Results and discussion ...... 69 4.3.1. Dactylogyrus Diesing, 1850 analyses ...... 69 4.3.2. Dogielius Bychowsky, 1936 analyses ...... 77 4.3.3. General discussion and conclusion ...... 84

5 MONOGENEAN PARASITE SPECIES DESCRIPTIONS FROM LABEO Cuvier, 1817 HOSTS IN THE VAAL DAM, SOUTH AFRICA, WITH A REVIEW OF RELATED PARASITE SPECIES ...... 86 5.1. Introduction ...... 86 5.2. Materials and methods ...... 87 5.3. Results and discussion ...... 88 5.3.1. Host species ...... 88 5.3.2. Parasite species ...... 88 5.3.3. Dactylogyrus Diesing, 1850 ...... 88 5.3.3.1. Species A (Dactylogyrus iwani n. sp., Figure 5-1) ...... 88 5.3.3.2. Species B (Dactylogyrus larindae n. sp., Figure 5-2) ...... 89 5.3.3.3. Species C (Dactylogyrus nicolettae n. sp., Figure 5-3) ...... 103 5.3.3.4. Species D (n. sp., Figure 5-4) ...... 104 5.3.4. Dogielius Bychowsky, 1936 ...... 107 5.3.4.1. Species E (Dogielius intorquens n. sp., Figures 5-7 and 5-8) ...... 107 5.3.5. General discussion and conclusion ...... 113

6 ASPECTS OF THE ECOLOGY OF MONOGENEAN PARASITES OF LABEO CAPENSIS (Smith, 1841) AND LABEO UMBRATUS (Smith, 1841) IN THE VAAL DAM, GAUTENG PROVINCE, SOUTH AFRICA COLLECTED DURING A WINTER SURVEY ...... 115 6.1. INTRODUCTION ...... 115 6.2. MATERIALS AND METHODS ...... 116 6.3. RESULTS AND DISCUSSION ...... 116 6.3.1. Host species ...... 116 6.3.2. Parasite species ...... 117 6.3.3. Biological and ecological aspects ...... 118 6.3.3.1. Comments on statistical tests employed ...... 118 6.3.3.2. Infection statistics and host specificity ...... 118 6.3.3.3. Effect of host variables ...... 122

vi 6.3.3.4. Environmental variables ...... 122 6.3.4. General discussion ...... 123 6.3.4.1. Host species: condition factor values and macroscopic pathology ...... 123 6.3.4.2. Ecological aspects: numbers and distribution ...... 124 6.3.4.3. Infection statistics: host preference ...... 125 6.3.4.4. Infection statistics: site preference ...... 127 6.3.4.5. Effect of host variables ...... 131 6.3.4.6. Effect of environmental variables ...... 135 6.3.5. Summary and conclusion ...... 137

7 ASPECTS OF THE ECOLOGY OF MONOGENEAN PARASITES OF LABEO CAPENSIS (Smith, 1841) AND LABEO UMBRATUS (Smith, 1841) IN THE VAAL DAM, GAUTENG PROVINCE, SOUTH AFRICA COLLECTED DURING A SUMMER SURVEY AS COMPARED WITH THE PRECEDING WINTER SURVEY ...... 139 7.1. INTRODUCTION ...... 139 7.2. MATERIALS AND METHODS ...... 139 7.3. RESULTS AND DISCUSSION ...... 140 7.3.1. Host species ...... 140 7.3.2. Parasite species ...... 140 7.3.3. Biological and ecological aspects ...... 141 7.3.3.1. Infection statistics and host specificity ...... 141 7.3.3.2. Effect of host variables ...... 142 7.3.3.3. Environmental variables ...... 145 7.3.4. Seasonal comparison ...... 145 7.3.4.1. Infection statistics ...... 145 7.3.4.2. Site preference on gill ...... 149

7.4. CONCLUSION ...... 151 8 ASPECTS OF THE ECOLOGY OF QUADRIACANTHUS AEGYPTICUS El-Naggar and Serag, 1986 FROM CLARIAS GARIEPINUS (Burchell, 1822) IN THE VAAL DAM, GAUTENG PROVINCE, SOUTH AFRICA WITH DESCRIPTION OF VARIATION OBSERVED IN HAPTORAL SCLERITES AND MALE COPULATORY ORGAN ...... 152 8.1. INTRODUCTION ...... 152 8.2. MATERIALS AND METHODS ...... 153 8.3. RESULTS AND DISCUSSION ...... 153 8.3.1. Host species ...... 153 8.3.2. Parasite species ...... 154 8.3.3. Measurement of haptoral sclerites and male copulatory organ (MCO) ...... 154 8.3.4. Comparing Quadriacanthus aegypticus morphology as described in previous studies .... 155 8.3.5. Morphological variation observed in the current study ...... 162 8.3.6. Morphological variability observed – summary and conclusion ...... 165 8.3.7. Biological and ecological aspects ...... 166 8.3.7.1. Infection statistics and host specificity ...... 166 8.3.7.2. Effect of host variables and evaluation of site specificity on hosts ...... 166

vii 8.3.7.3. Environmental variables ...... 168 8.3.7.4. Host species: condition factor values and macroscopic pathology ...... 168

8.4. SUMMARY AND CONCLUSION ...... 169 9 ASPECTS OF THE ECOLOGY OF A SPECIES OF GYRODACTYLUS von Nordmann, 1832 FROM CLARIAS GARIEPINUS (Burchell, 1822) IN THE VAAL DAM, GAUTENG PROVINCE, SOUTH AFRICA ...... 170 9.1. INTRODUCTION ...... 170 9.2. MATERIALS AND METHODS ...... 171 9.3. RESULTS AND DISCUSSION ...... 171 9.3.1. Host species ...... 171 9.3.2. Parasite species ...... 172 9.3.3. Biological and ecological aspects ...... 176 9.3.3.1. Infection statistics and host specificity ...... 176 9.3.3.2. Effect of host variables and evaluation of site specificity on hosts ...... 178 9.3.3.3. Environmental variables ...... 178

9.4. SUMMARY AND CONCLUSION ...... 179 10 ASPECTS OF THE ECOLOGY OF MONOGENEAN PARASITES OF CYPRINUS CARPIO Linnaeus, 1758 IN THE VAAL DAM, GAUTENG PROVINCE, SOUTH AFRICA ...... 180 10.1. INTRODUCTION ...... 180 10.2. MATERIALS AND METHODS ...... 181 10.3. RESULTS AND DISCUSSION ...... 181 10.3.1. Host species ...... 181 10.3.2. Parasite species ...... 183 10.3.3. Biological and ecological aspects ...... 187 10.3.3.1. Infection statistics and host specificity ...... 187 10.3.3.2. Effect of host variables and evaluation of site specificity on hosts ...... 189 10.3.3.3. Environmental variables ...... 192 10.3.3.4. Host species: condition factor values and macroscopic pathology ...... 193

10.4. SUMMARY AND CONCLUSION ...... 194 11 ASPECTS OF THE ECOLOGY OF A MONOGENEAN PARASITE OF CTENOPHARYNGODON IDELLA (Valenciennes, 1844) IN THE VAAL DAM, GAUTENG PROVINCE, SOUTH AFRICA ...... 195 11.1. INTRODUCTION ...... 195 11.2. MATERIALS AND METHODS ...... 196 11.3. RESULTS AND DISCUSSION ...... 196 11.3.1. Host species ...... 196 11.3.2. Parasite species ...... 197 11.3.3. Measurement of haptoral sclerites and male copulatory organ (MCO) ...... 198 11.3.4. Comparing morphology and measurements as described in previous studies ...... 199 11.3.5. Morphological variation observed in the current study ...... 199 11.3.6. Biological and ecological aspects ...... 201 11.3.6.1. Infection statistics and host specificity ...... 201

viii 11.3.6.2. Effect of host variables and evaluation of site specificity on hosts ...... 203 11.3.6.3. Environmental variables ...... 205 11.3.6.4. Host species: condition factor values and macroscopic pathology ...... 206

11.4. SUMMARY AND CONCLUSION ...... 207 12 ASPECTS OF THE ECOLOGY OF MONOGENEAN PARASITES OF LABEOBARBUS AENEUS (Burchell, 1822) AND LABEOBARBUS KIMBERLEYENSIS (Gilchrist and Thompson, 1913) IN THE VAAL DAM, GAUTENG PROVINCE, SOUTH AFRICA ...... 208 12.1. INTRODUCTION ...... 208 12.2. MATERIALS AND METHODS ...... 209 12.3. RESULTS AND DISCUSSION ...... 210 12.3.1. Host species ...... 210 12.3.2. Parasite species ...... 210 12.3.2.5. Monogeneans from Labeobarbus aeneus (Burchell, 1822): Dactylogyrus sp. G...... 213 12.3.3. Morphological variation observed in the current study ...... 215 12.3.4. Infection statistics ...... 215 12.3.5. Infection statistics related to host variables ...... 217 12.3.6. Environmental variables and seasonal comparison ...... 224 12.3.7. Host species: condition factor values and macroscopic pathology ...... 225

12.4. SUMMARY AND CONCLUSION ...... 226 13 GENERAL DISCUSSION ...... 227 14 REFERENCES ...... 289

ix List of Appendices

Appendix A: Notes on how apparent morphometric variation and slight rotation of Dogielius Bychowsky, 1936 (originally denoted “Species E” but later described as Dogielius intorquens Crafford, Luus-Powell and Avenant-Oldewage (2012) anchor can affect point to point measurements ...... 345

Appendix B: Notes on the application of additional point to point measurements, adapted from Gyrodactylus von Nordmann, 1832, to Dactylogyrus Diesing, 1850 and Dogielius Bychowsky, 1936...... 353

Appendix C: Preliminary measurements showing the gills of Labeo capensis (Smith, 1841) to be more robust than that of Labeo umbratus (Smith, 1841) (Chapter 4 – Form versus species evaluation) ...... 371

List of Tables

Table 2-1: Comparison of ecological terms. Throughout this thesis the partial revision by Bush, Lafferty, Lotz and Shostak (1997) were followed...... 23

Table 2-2: Summary of published papers dealing with monogeneans in southern Africa...... 44

Table 4-1: Univariate descriptive statistics of Dactylogyrus Diesing, 1850 structure component measurements (parasites collected from Labeo capensis (Smith, 1841) and Labeo umbratus (Smith, 1841))...... 70

Table 4-2: Bivariate statistics results of Dactylogyrus Diesing, 1850. Structure component measurements (parasites collected from Labeo capensis (Smith, 1841) and Labeo umbratus (Smith, 1841)): Pearson’s correlation coefficient, blocked by component with significant level indicators...... 71

Table 4-3: Principal component analysis results of Dactylogyrus Diesing, 1850 structure component measurements (parasites collected from Labeo capensis (Smith, 1841) and Labeo umbratus (Smith, 1841)) showing the first three principal components. ... 74

Table 4-4: Cluster centres of all the measures from Dactylogyrus Diesing, 1850 in the original scale after performing a k-means cluster analysis with k=2...... 75

Table 4-5: Non-parametric Mann-Whitney U test results – Dactylogyrus Diesing, 1850...... 75

Table 4-6: Univariate descriptive statistics of Dogielius Bychowsky, 1936 structure component measurements (parasites collected from Labeo capensis (Smith, 1841) and Labeo umbratus (Smith, 1841))...... 78

Table 4-7: Bivariate statistics results of Dogielius Bychowsky, 1936 structure component measurements (parasites collected from Labeo capensis (Smith, 1841) and Labeo umbratus (Smith, 1841)): Pearson’s correlation coefficient, blocked by component with significant level indicators...... 79

x Table 4-8: Principal component analysis results of Dogielius Bychowsky, 1936, structure component measurements (parasites collected from Labeo capensis (Smith, 1841) and Labeo umbratus (Smith, 1841)) showing the first two principal components...... 81

Table 4-9: Cluster centres of all the measures in the original scale after performing a k-means cluster analysis with k=2...... 81

Table 4-10: Non-parametric Mann-Whitney U test results – Dogielius Bychowsky, 1936...... 82

Table 5-1: Summary description: Dactylogyrus pseudanchoratus Price and Géry, 1968 / Dactylogyrus cf. pseudanchoratus Paperna, 1979 / Dactylogyrus pseudanchoratus micronchus Paperna, 1979 / Dactylogyrus helicophallus Paperna, 1973 “species complex” (from Paperna 1979)...... 92

Table 5-2: Dactylogyrus pseudanchoratus Price and Géry, 1968 / Dactylogyrus cf. pseudanchoratus Paperna, 1979 / Dactylogyrus pseudanchoratus micronchus Paperna, 1979 / Dactylogyrus helicophallus Paperna, 1973 “species complex” measurement (all in micrometers) summary table...... 93

Table 5-3: Summary of hard part structure measurements (all in micrometers) of selected species previously compared to the Dactylogyrus pseudanchoratus Price and Géry, 1968 / Dactylogyrus helicophallus Paperna, 1973 species complex...... 95

Table 5-4: Comparison matrix of most obvious differences between selected species within / comparable to the Dactylogyrus pseudanchoratus Price and Géry, 1968 species group, as noted by various authors...... 97

Table 5-5: Summary of hard part structure measurements (all in micrometers) of other selected species showing resemblances (in terms of hard part structures) with Dactylogyrus species listed in Table 5-2...... 99

Table 5-6: Summary of morphometric variation in hard part structures (anchor, bar(s), marginal hook, copulatory organ) of selected species within / comparable to the Dactylogyrus pseudanchoratus Price and Géry, 1968 species group...... 101

Table 5-7: Summary of hard part structure measurements (all in micrometers) of other selected species of Dogielius Bychowsky, 1936 showing resemblances Dogielius intorquens n. sp...... 111

Table 5-8: Summary of morphometric variation in hard part structures of selected species of Dogielius Bychowsky, 1936 comparable to Dogielius intorquens n. sp...... 112

Table 6-1: Summary description of sampled Labeo Cuvier, 1817 population from the Vaal Dam...... 117

Table 6-2: Length category and gender distribution of sampled Labeo Cuvier, 1817 population from the Vaal Dam...... 117

xi Table 6-3: Monogenean parasite infection statistics for species of Labeo Cuvier, 1817 from the Vaal Dam: Labeo umbratus (Smith, 1841)...... 120

Table 6-4: Monogenean parasite infection statistics for species of Labeo Cuvier, 1817 from the Vaal Dam: Labeo capensis (Smith, 1841)...... 121

Table 6-5: Environmental (physical) and water quality variables from the Vaal Dam (source: Rand Water, sampling point identification number C-VD1l)...... 123

Table 7-1: Summary description of sampled Labeo Cuvier, 1817 host population from the Vaal Dam (Summer survey, January 2010)...... 140

Table 7-2: Weight category gender distribution of sampled Labeo Cuvier, 1817 host population from the Vaal Dam (Summer survey, January 2010)...... 141

Table 7-3: Monogenean parasite infection statistics for species of Labeo Cuvier, 1817 from the Vaal Dam collected during a summer (January 2010) survey: Labeo umbratus (Smith, 1841)...... 143

Table 7-4: Monogenean parasite infection statistics for species of Labeo Cuvier, 1817 from the Vaal Dam collected during a summer (January 2010) survey: Labeo capensis (Smith, 1841)...... 144

Table 7-5: Environmental (physical) and water quality variables from the Vaal Dam (source: Rand Water, sampling point identification number C-VD1l)...... 145

Table 7-6: Summary of key infection statistics for selected monogenean species from species of Labeo Cuvier, 1817 calculated for the winter and summer surveys respectively. .... 146

Table 7-7: Summary results (p-values) following statistical comparison of infection statistics between seasons: monogenean species collected from species of Labeo Cuvier, 1817 during a winter (June / July 2009) and summer (January 2010) survey respectively. Statistically significant values (i.e. p < 0.05) are indicated in shaded blocks...... 147

Table 7-8: Summary results for gill site preference (including Chi-square p-values) as recorded from species of Labeo Cuvier, 1817 during a winter (June / July 2009) and summer (January 2010) survey respectively. Statistically significant values (i.e. p < 0.05) are indicated in shaded blocks...... 150

Table 8-1: Summary description of sampled Clarias gariepinus (Burchell, 1822) host population from the Vaal Dam during summer (January 2010) survey...... 154

Table 8-2: Length category gender distribution of sampled Clarias gariepinus (Burchell, 1822) host population from the Vaal Dam during summer (January 2010 survey)...... 154

Table 8-3: Selected standard measurements (all in micrometers) for Quadriacanthus Paperna, 1961 from literature: summary table (1 of 4) ...... 156

xii Table 8-4: Selected standard measurements (all in micrometers) for Quadriacanthus aegypticus El-Naggar and Serag, 1986 (from literature and from the current study) and Quadriacanthus agnebiensis N'Douba, Lambert and Euzet, 1999 only...... 159

Table 8-5: Complete set of standard measurements (all in micrometers) as recorded for Quadriacanthus aegypticus El-Naggar and Serag, 1986 in the current study...... 160

Table 8-6: Quadriacanthus aegypticus El-Naggar and Serag, 1986 and unknown species infection statistics for Clarias gariepinus (Burchell, 1822) from the Vaal Dam ...... 167

Table 9-1: Summary of species of Gyrodactylus von Nordmann, 1832 described from Clarias gariepinus (Burchell, 1822)...... 171

Table 9-2: Summary description of sampled Clarias gariepinus (Burchell, 1822) host population from the Vaal Dam during summer (January 2010) survey...... 172

Table 9-3: Length category gender distribution of sampled Clarias gariepinus (Burchell, 1822) host population from the Vaal Dam during summer (January 2010 survey)...... 172

Table 9-4: Selected standard measurements (all in micrometers) for species of Gyrodactylus von Nordmann, 1832 reported from Clarias gariepinus (Burchell, 1822): summary table (1 of 2)...... 174

Table 9-5: Gyrodactylus von Nordmann, 1832 infection statistics for Clarias gariepinus (Burchell, 1822) from the Vaal Dam ...... 177

Table 10-1: Summary description of monogenean parasites occurring on Cyprinus carpio Linnaeus, 1758 as recorded in published papers...... 182

Table 10-2: Summary description of sampled Cyprinus carpio Linnaeus, 1758 host population from the Vaal Dam in January 2010...... 185

Table 10-3: Length category gender distribution of sampled Cyprinus carpio Linnaeus, 1758 host population from the Vaal Dam during January 2010...... 185

Table 10-4: Monogenean parasite infection statistics for Cyprinus carpio Linnaeus, 1758 from the Vaal Dam during January 2010...... 190

Table 11-1: Summary description of sampled Ctenopharyngodon idella (Valenciennes, 1844) host population from the Vaal Dam during summer (January 2010 survey)...... 197

Table 11-2: Length category gender distribution of sampled Ctenopharyngodon idella (Valenciennes, 1844) host population from the Vaal Dam during summer (January 2010 survey)...... 197

Table 11-3: Dactylogyrus lamellatus Achmerow, 1952 (from Ctenopharyngodon idella (Valenciennes, 1844)) measurement (all in micrometers) summary table...... 200

Table 11-4: Monogenean parasite infection statistics for Ctenopharyngodon idella (Valenciennes, 1844) from the Vaal Dam ...... 202

xiii Table 11-5: Environmental (physical) and water quality variables from the Vaal Dam (source: Rand Water, sampling point identification number C-VD1l)...... 205

Table 12-1: Summary descriptions of sampled Labeobarbus kimberleyensis (Gilchrist and Thompson, 1913), Labeobarbus aeneus (Burchell, 1822) and a suspected hybrid of these species host populations from the Vaal Dam during winter (June / July 2009) and summer (January 2012) surveys...... 211

Table 12-2: Length category gender distributions of sampled species of Labeobarbus Rüppell, 1836 host populations from the Vaal Dam during winter (June / July 2009) and summer (January 2010) surveys...... 212

Table 12-3: Sclerite dimensions (measured in micrometers) of Dactylogyrus varicorhini Bychowsky, 1958 as recorded by Paperna (1961)*, as well as measurements (all in micrometers) of the apparent “D. varicorhini species group / type” specimens collected during the current study...... 214

Table 12-4: Monogenean parasite infection statistics for Labeobarbus Rüppell, 1836 from the Vaal Dam: Labeobarbus kimberleyensis (Gilchrist and Thompson, 1913) – Winter (June 2009) survey...... 219

Table 12-5: Monogenean parasite infection statistics for Labeobarbus Rüppell, 1836 from the Vaal Dam: Labeobarbus kimberleyensis (Gilchrist and Thompson, 1913) – Summer (January 2012) survey...... 220

Table 12-6: Monogenean parasite infection statistics for Labeobarbus Rüppell, 1836 from the Vaal Dam: Labeobarbus aeneus (Burchell, 1822) – Winter (June 2009) survey...... 221

Table 12-7: Monogenean parasite infection statistics for Labeobarbus Rüppell, 1836 from the Vaal Dam: Labeobarbus aeneus (Burchell, 1822) – Summer (January 2010) survey. .... 222

Table 12-8: Monogenean parasite infection statistics for Labeobarbus Rüppell, 1836 from the Vaal Dam: Labeobarbus suspected hybrid – Summer (January 2010) survey #...... 223

Table 12-9: Environmental (physical) and water quality variables from the Vaal Dam (source: Rand Water, sampling point identification number C-VD1l)...... 225

Table 13-1: List of all host and parasite species collected during the current study in the Vaal Dam...... 228

Table 13-2: Summary of illustrations for collected parasite species and new parasite species described for each of the host fish species examined in the Vaal Dam...... 229

Table 13-3: Summary of infection statistics of monogenean parasites collected, as calculated from host species examined from the Vaal Dam, Gauteng Province, South Africa...... 242

xiv Table 13-4: Summary of general trends in host effects (gender) and site specificity / site preference (position on gills) for monogenean composite communities (dactylogyrid parasites except for Clarias gariepinus (Burchell, 1822) as indicated in the footnote) on various fish hosts from the Vaal Dam, Gauteng Province, South Africa. Statistically significant (p<0.05) p-values are indicated with bold text in shaded blocks...... 271

Table 13-5: Summary of existing parasite species collected and new parasite species described for each of the host fish species examined...... 279

xv List of Figures

Figure 3-1: A diagram depicting the location of the study site. Map 1 shows the position of South Africa on the continent of Africa (A). Map 2 shows the position of the Vaal Dam in Gauteng Province (C bottom and shaded province top) within South Africa (B). Map 3 shows the position of the Vaal Dam (D), with the University of Johannesburg Island study site indicated as “UJ Eiland” (coordinates S 26 52.249, E 28 10.249), within Gauteng Province, South Africa...... 50

Figure 3-2: The Vaal River catchment showing the division into four zones on the basis of water quality. The Vaal Dam is located in water quality zone 1 in Gauteng Province. Please refer to Figure 3-1 to see the position of Gauteng Province relative to the rest of South Africa. “RWB Barrage” refers to the “Vaal River Barrage” also indicated on Map 3; Figure 3-1.This illustration was redrawn from the following original source: Braune, E. and Rogers, K.H. (1987)...... 51

Figure 3-3: An illustration showing the division of gill sets (L = Left hemisphere, R = Right hemisphere, numbers 1 to 4 denotes gill arch numbers) and individual gill arches (ah = anterior hemibranch, ph = posterior hemibranch, A = dorsal segment position, B = median segment position, C = ventral segment position)...... 55

Figure 3-4: An illustration depicting measurements (for Dactylogyrus Diesing, 1850 and Dogielius Bychowsky, 1936) employed as adopted from Guégan and Lambert (1991), Musilová, Řehulková and Gelnar (2009), A = Dactylogyrus spp. anchor; B = Dogielius spp. anchor; C = Dactylogyrus spp. dorsal bar; D = Marginal hook (applicable to both Dactylogyrus spp. and Dogielius spp.); E = Dogielius spp. dorsal bar; F = Dactylogyrus spp. male copulatory organ (MCO); G = Dogielius spp. MCO; a = anchor total length; b = anchor shaft length; c = length of outer root; d = length of inner root; e = length of tip / point; f = anchor aperture; g = length of dorsal bar; h = width of dorsal bar; i = marginal hook total length; j = accessory piece length; k = penis tube trace length...... 59

xvi Figure 3-5: An illustration depicting (for Quadriacanthus Paperna, 1961) measurement methodology employed as adopted from N’Douba, Lambert and Euzet (1999), El- Naggar and Serag (1986): A = Ventral bar component; B = Dorsal bar component; C = Dorsal accessory sclerite; D = Dorsal anchor; E = Marginal hook; F = Ventral accessory sclerite; G = Ventral anchor; H = Male copulatory organ (MCO); a = anchor total length; c = anchor base width; e = length of tip / point; f = length of ventral bar component; g = width of ventral bar component; h = marginal hook total length; i = penis tube trace length; j = accessory piece length; l = dorsal accessory sclerites width; m = dorsal accessory sclerites length; n = ventral accessory sclerites length; o = measurements of dorsal bar component length (median length); p = width of dorsal bar component; q = dorsal bar component base width; r = dorsal bar component median process length...... 60

Figure 3-6: An illustration depicting (for Gyrodactylus von Nordmann, 1832) measurement methodology employed as adopted from Christison, Shinn and van As (2005), Přikrylová, Matĕjusová, Jarkovský and Gelnar (2008), Přikrylová, Blažek and Vanhove (2012): A = Anchor; B = Dorsal bar; C = Ventral bar; D = Marginal hook; a = Anchor total length; b = Anchor point length; c = Anchor shaft length; d = Anchor root length; e = Ventral bar median length; f = Ventral bar membrane length; g = Ventral bar width; h = Dorsal bar length; i = Dorsal bar width; j = Marginal hook total length; k = Marginal hook sickle length; l = Marginal hook handle length; m = Marginal hook sickle distal width; n = Marginal hook sickle proximal width; o = Marginal hook sickle aperture distance; OLMH = Overall length of hook section (of the marginal hook) itself...... 61

Figure 4-1: Visual representation of bivariate statistics results of Dactylogyrus Diesing, 1850 structure component measurements (parasites collected from Labeo capensis (Smith, 1841) and Labeo umbratus (Smith, 1841)): Scatter matrix of all measurements...... 72

Figure 4-2: Scree plot of the principal component analysis (PCA) – Dactylogyrus Diesing, 1850...... 73

Figure 4-3: Kernel density estimation of principal components (PC) 1 and 2 demonstrates bimodal grouping of specimens – Dactylogyrus Diesing, 1850...... 74

Figure 4-4: Histogrammes of the first principal component grouped by cluster – Dactylogyrus Diesing, 1850...... 76

Figure 4-5: Three-dimensional scatter plot with axis given by principal components and the grouping variable is the cluster number – Dactylogyrus Diesing, 1850...... 76

Figure 4-6: Three-dimensional scatterplot with axis given by principal components and the grouping variable is the two different host species – Dactylogyrus Diesing, 1850. .... 77

xvii Figure 4-7: Visual representation of bivariate statistics results of Dogielius Bychowsky, 1936 structure component measurements (parasites collected from Labeo capensis (Smith, 1841) and Labeo umbratus (Smith, 1841)): Scatter matrix of all measurements...... 80

Figure 4-8: Scree plot of the principal component analysis (PCA) – Dogielius Bychowsky, 1936...... 80

Figure 4-9: Histogram of the first principal component – Dogielius Bychowsky, 1936...... 82

Figure 4-10: Histograms of the first principal component grouped by cluster – Dogielius Bychowsky, 1936...... 83

Figure 4-11: Three-dimensional scatterplot with axis given by principal components and the grouping variable is the cluster number – Dogielius Bychowsky, 1936...... 83

Figure 5-1: Dactylogyrus iwani n. sp. (species A): A = Anchors; B = Dorsal bar; C = Marginal hooks; D = Male copulatory organ (MCO) showing variation in the position of the accessory piece; E = Variation in accessory piece structure observed depending on orientation; i to iv = Marginal hook position (following Guegan and Lambert, 1991). . 91

Figure 5-2: Dactylogyrus larindae n. sp. (species B): A = Anchors; B = Dorsal bar; C = Marginal hooks; D = Male copulatory organ (MCO); i to iv = Marginal hook position (following Guegan and Lambert, 1991)...... 91

Figure 5-3: Dactylogyrus nicolettae n. sp. (species C): A = Anchors; B = Dorsal bar; C = Marginal hooks; D = Male copulatory organ (MCO) showing variation in position of accessory piece; i to iv = Marginal hook position (following Guegan and Lambert 1991)...... 104

Figure 5-4: Dactylogyrus sp. D n. sp.: A = Anchors; B = Dorsal bar; C = Marginal hooks; D = Male copulatory organ (MCO); i to iv = Marginal hook position (following Guegan and Lambert 1991)...... 106

Figure 5-5: Dactylogyrus sp. D n. sp.: Illustration depicting variations observed in terms of male copulatory organ (MCO) sclerite positions...... 106

Figure 5-6: Dactylogyrus sp. D n. sp.: Illustration depicting variations observed in terms of transverse bar orientation...... 106

Figure 5-7: Dogielius intorquens n. sp.: From Labeo umbratus (Smith, 1841): A = Anchors; B = Dorsal bar; C = Marginal hooks; D = Male copulatory organ (MCO); E = vagina; I to iv = Marginal hook position (following Guegan and Lambert 1991)...... 110

Figure 5-8: Dogielius intorquens n. sp.: From Labeo capensis (Smith, 1841): A = Anchors; B = Dorsal bar; C = Marginal hooks; D = Male copulatory organ (MCO); I to iv = Marginal hook position (following Guegan and Lambert 1991)...... 110

xviii Figure 8-1: An illustration of the haptoral sclerites and male copulatory organ Quadriacanthus aegypticus El-Naggar and Serag, 1986 (redrawn from El-Naggar and Serag 1986). A – Ventral bar; B – Dorsal bar; C – Dorsal anchor; D – Marginal hooks; E – Ventral anchor; F – Male copulatory organ...... 155

Figure 8-2: An illustration of the haptoral sclerites and male copulatory organ of Quadriacanthus aegypticus El-Naggar and Serag, 1986 as drawn during the current study with the aid of a drawing tube. A – Ventral bar; B – Dorsal bar; C – Dorsal anchor; D – Marginal hooks; E – Ventral anchor; F – Male copulatory organ...... 161

Figure 8-3: An illustration of the haptoral sclerites and male copulatory organ Quadriacanthus aegypticus El-Naggar and Serag, 1986, as redrawn from Kritsky and Kulo (1988). A – Ventral bar; B – Dorsal bar; C – Dorsal anchor; D – Marginal hooks; E – Ventral anchor; F – Male copulatory organ...... 163

Figure 8-4: An illustration to depict the extend of variation in shape and size of haptoral sclerites and male copulatory organ (MCO) of Quadriacanthus aegypticus El-Naggar and Serag, 1986 observed during the current study: A – MCO; B – Ventral bar; C – Dorsal bar; D – Ventral anchor with associated sclerite...... 164

Figure 9-1: An illustration of the haptoral sclerites of Gyrodactylus von Nordmann, 1832 as drawn during the current study with the aid of a drawing tube. A – Hamuli; B – Bars (Ventral bar with membrane bottom; Dorsal bar top); C – Marginal hooklet...... 176

Figure 10-1: Dactylogyrus extensus Mueller and Van Cleave, 1932 from Cyprinus carpio Linnaeus, 1758 in the Vaal Dam, South Africa (current study): A = Anchors; B = Dorsal bar; C = Marginal hooks; D = Male copulatory organ (MCO)...... 186

Figure 10-2: Dactylogyrus minutus Kulwiec, 1927 from Cyprinus carpio Linnaeus, 1758 in the Vaal Dam, South Africa (current study): A = Anchors; B = Dorsal bar; C = Marginal hooks; D = Male copulatory organ (MCO)...... 186

Figure 10-3: Gyrodactylus kherulensis Ergens, 1974 from Cyprinus carpio Linnaeus, 1758 in the Vaal Dam, South Africa (current study): A = Anchors; B = Dorsal bar; C = Marginal hooks...... 187

Figure 11-1: Dactylogyrus lamellatus Achmerow, 1952 from Ctenopharyngodon idella (Valenciennes, 1844) in the Vaal Dam, South Africa (current study): A = Anchors; B = Dorsal bar; C = Marginal hooks; D = Male copulatory organ (MCO)...... 198

Figure 11-2: Dactylogyrus lamellatus Achmerow, 1952 from Ctenopharyngodon idella (Valenciennes, 1844) redrawn from Gussev (1962): A = Anchors; B = Dorsal bar; C = Marginal hooks; D = Male copulatory organ (MCO)...... 199

Figure 11-3: Variation observed in the orientation of the MCO of Dactylogyrus lamellatus Achmerow, 1952 from Ctenopharyngodon idella (Valenciennes, 1844) in the Vaal Dam, South Africa...... 201

xix Figure 12-1: An illustration depicting variation observed in the sclerite morphology of Dactylogyrus sp. G collected from Labeobarbus aeneus (Burchell, 1822). The shape of the anchors (A) and bars (B) and large size of the marginal hooks (C) are reminiscent of the “Dactylogyrus varicorhini Bychowsky, 1958 species group / type”. Unfortunately the male copulatory organ (D) was not clearly visible in any of the specimens collected from L. aeneus...... 216

Figure 12-2: An illustration depicting variation observed in the sclerite morphology of Dactylogyrus sp. G collected from Labeobarbus kimberleyensis (Gilchrist and Thompson, 1913). Once again the shape of the anchors (A) and bars (B) and large size of the marginal hooks (C) are reminiscent of the “Dactylogyrus varicorhini Bychowsky, 1958 species group / type”...... 217

xx Summary Fish parasites may cause disease and lead to commercial losses. In order to construct practical management systems to mitigate or manage potential adverse effects, information on basic biological variables is required. Compared to the number of internationally published papers on the subject, little is known about the monogenean parasite fauna of South African fishes. Monogeneans are largely ectoparasitic, thin, flattened, host- and site specific parasites with a simple life cycle involving a single host (often a fish). They range in size between 0.3 mm and 20 mm and are mostly bilaterally symmetrical with the body subdivided into a number of regions. Attachment organs are a necessary feature both anteriorly and posteriorly, with the morphology of the prominent posterior attachment organ (opisthaptor) highly variable between genera. It may contain suckers, clamps or large hooks (anchors) as well as marginal hooks. In some genera anchors (one to two pairs) are associated with spikes or accessory sclerites and are supported by a connecting bar. All these structures, together with the sclerotized male copulatory organ, are of taxonomic significance.

The aim of the current study was generation of baseline data on monogenean parasite infections of several fish species in the Vaal Dam (S 26 52.249, E 28 10.249), Gauteng Province, South Africa, during two surveys performed during winter (June / July 2009) and summer (January 2010) respectively.

Fish species (Cyprinus carpio Linnaeus, 1758; Micropterus salmoides (Lacepède, 1802); Clarias gariepinus (Burchell, 1822); Labeobarbus aeneus (Burchell, 1822); Labeo capensis (Smith, 1841); Labeo umbratus (Smith, 1841); Ctenopharyngodon idella (Valenciennes, 1844) and Labeobarbus kimberleyensis (Gilchrist and Thompson, 1913)) were collected using gill nets of varying mesh sizes. Fish were randomly removed from the holding tank and processed. Each fish was allocated a numerical field number, weighed and measured. Approximately 3 to 6 mucous smears were prepared from random areas on the skin and fins using microscope slides and subsequently examined with the aid of a stereo microscope.

Fish were killed humanely by severing the spinal cord and the gills were removed, separated into left and right hemispheres and placed in water in separate petri dishes labelled with the fish number and side (i.e. left or right).

xxi If the fish was not further processed in the field, each hemisphere together with a pencil-written label was placed in a glass bottle containing 70% ethanol. Gills were divided into areas according to standard published procedure. The mucus and epithelial lining of each respective area was scraped onto a microscope slide (with the aid of a second microscope slide) and examined with the aid of a stereo microscope. Parasites were removed with the aid of a stout eye lash hair (glued to a wooden handle) and subsequently mounted in either Malmberg’s solution (GAP) or glycerine jelly. Parasites were examined, sclerotized hard parts drawn with the aid of a drawing tube and compound microscope and measurements made directly from drawings according to standard published methodology (i.e. to allow parasite identification and description).

Gyrodactylus von Nordmann, 1832 and Dactylogyrus Diesing, 1850 are considered to be two of the most common monogenean genera on fishes throughout the world, a supposition supported through the results of the current study (especially with regard to the latter for which several species were identified and described). Other genera encountered included species of Dogielius Bychowsky, 1936; Diplozoon von Nordmann, 1832; Paradiplozoon Akhmerov, 1974 and Quadriacanthus Paperna, 1961.

Other than expected, monogenean parasites were not recorded from all host species examined. No parasites were collected from M. salmoides as only two host specimens were examined. All the other fish species harboured at least one monogenean parasite species as predicted. Furthermore it was postulated that all host species would harbour more than one species of each monogenean genus encountered. However, more than one species of the same genus (Dactylogyrus spp. in this case) was only recorded from Labeo spp. and C. carpio (i.e. the other host species only harboured one species per monogenean genus).

Morphological plasticity in size and even shape of sclerites was evident in Quadriacanthus aegypticus El-Naggar and Serag, 1986 (from C. gariepinus) as well as Dactylogyrus spp. and Dogielius intorquens Crafford, Luus-Powell and Avenant- Oldewage, 2012 (from the host species Labeo Cuvier, 1817).

xxii External variables such as geographical variation or temperature effects are thought to be unlikely causative agents, while the effect of host and parasite size may require additional experimental study.

It is postulated that parasite phylogeny (i.e. genotype) is responsible for observed variation possibly reflecting the existence of species complexes. Experimental infection studies combined with molecular biology research may aid in making definitive taxonomic determinations when dealing with suspected species complexes. Such an approach should be considered in future studies to evaluate the above statement regarding phylogeny / genotype.

While prevalence did exceed 50% in a number of cases during summer, this was not the case during winter. Furthermore mean intensity of infection rarely exceeded 10 in winter with only a few cases exceeding 20 during summer. There was a significant difference in parasite numbers when comparing summer and winter surveys for L. umbratus (i.e. prevalence was higher in summer for Dactylogyrus spp. and Dogielius sp. but higher in winter for Diplozoon sp.). In terms of mean intensity and mean abundance, significant seasonal differences (much higher in summer) were recorded only for Dactylogyrus larindae Crafford, Luus-Powell and Avenant-Oldewage, 2012. Gyrodactylus sp. was recorded from C. gariepinus (summer survey only) and C. carpio (summer survey only) in low numbers. The small data set confounds any seasonal comparisons and hence no generalizations can be proposed as to the effects of season on gyrodactylid infection statistics. Temperature most likely did contribute to the higher dactylogyrid numbers / infection statistics recorded during summer due to an increased rate of development.

A statistically significant preference (monogenean component community) for male fish from the host species Labeo Cuvier, 1817 during summer was recorded in only two instances. The inconclusive pattern observed may be the result of a sampling artefact (i.e. low numbers of hosts collected for one or both host genders).

Additional sampling is required to investigate possible seasonal trends, but a significant host gender preference is deemed unlikely.

xxiii All monogenean species exhibited specificity with regard to host genus. Most parasites also demonstrated apparent host species specificity. In instances where monogenean parasites did occur on more than one closely related species a clear host preference was still evident.

A number of new parasite species have been described from endemic Labeo spp. Additional sampling (i.e. examination of a larger number of samples) is required to clarify species identification of monogeneans occurring on endemic Labeobarbus spp., but at least one new monogenean species description from this host genus is anticipated.

Both C. carpio and C. idella harboured monogenean parasites that are known to occur globally on the same host species (i.e. the parasites were translocated together with the original fish stock when introduced into South Africa).

Site specificity was found not to be distinct where more than one species co-existed on the gills. Neutral interactions (i.e. random distribution due to water current / flow over the gills) thus apparently had a greater effect on site preference than negative interactions (e.g. competition). This was especially clear from parasite distribution on the gills of Labeo spp., where no clear differentiation of preferred selection sites between the various monogenean species collected was observed.

This study contributes to a baseline understanding of the monogenean component community occurring on fish hosts in the Vaal Dam, paving the way for further taxonomic (additional species descriptions and refinement of descriptions provided herein) and applied (in terms of ecological, physiological and experimental aspects) research.

xxiv Opsomming

Visparasiete kan siektetoestande veroorsaak en lei tot ekonomiese verliese. Basiese grondvlakdata is nodig om praktiese bestuurstelsels te ontwerp wat die effek van negatiewe impak kan verminder of bestuur. Vergeleke met die aantal internasionale publikasies oor hierdie onderwerp, is daar betreklik min bekend oor die voorkoms van die Monogenea groep in Suid-Afrika.

Die organismes is meestal dun, afgeplatte, gasheerspesifieke ektoparasiete wat dikwels ook ‘n voorkeur toon vir bepaalde aanhegtingsposisies. Die lewenssiklus is eenvoudig en daar is net een gasheer (meestal ‘n vis) betrokke. Die parasiete wissel in lengte van 0.3 mm to 20.0 mm en is bilateraal simmetries. Beide kante (anterior en posterior) van die liggaam bevat aanhegtingsstrukture. Die posterior struktuur is mees prominent en word ‘n haptor genoem. Die morfologie van die struktuur wissel na gelang van parasietgenus en bevat groot en klein hake, suiers of klampe. In sommige genera word die hake (waarvan daar gewoonlik een of twee pare is en wat deur ‘n staaf verbind en ondersteun word) met penne of addisionele ondersteunende strukture geassosieer. Hierdie strukture, tesame met die harde strukture van die manlike kopuleringsorgaan, is van taksonomiese belang.

Die doel van die huidige studie was om basislyndata in te win oor die stand van Monogenea-infeksies op verskeie visspesies in die Vaaldam (S 26 52.249, E 28 10.249), Gauteng Provinsie, Suid-Afrika, soos bepaal tydens twee opnames wat onderskeidelik in die winter (Junie / Julie 2009) en somer (Januarie 2010) uitgevoer is.

Visspesies (Cyprinus carpio Linnaeus, 1758; Micropterus salmoides (Lacepède, 1802); Clarias gariepinus (Burchell, 1822); Labeobarbus aeneus (Burchell, 1822); Labeo capensis (Smith, 1841); Labeo umbratus (Smith, 1841); Ctenopharyngodon idella (Valenciennes, 1844) en Labeobarbus kimberleyensis (Gilchrist en Thompson, 1913)) is versamel deur gebruik te maak van kiefnette van verskeie groottes. Visse is ewekansig uit die houtenk verwyder vir ondersoek (m.a.w. ewekansig ondersoek en nie na gelang van gasheerspesie-identiteit nie). ‘n Veldnommer is aan elke vis toegeken waarna dit geweeg en gemeet is.

xxv Ongeveer drie tot ses slymsmere is vanaf verskeie areas op die vel en vinne gemaak met behulp van glasmikroskoopplaatjies, waarna die smere ondersoek is met behulp van ‘n disseksiemikroskoop.

Alle visse is menslik gedood deur die rugstring deur te knip agter die kop. Die kieue is verwyder, verdeel in linker en regter helftes en in water binne aparte petri-bakkies (wat gemerk is met die visnommer en kant van die kop) geplaas.

Indien die vis nie verder bewerk kon word in die veld nie, is elke kieuhelfte saam met ‘n potlood-geskrewe etiket in ‘n glasbottel wat 70% etanol bevat, geplaas. Elke kieu is opgedeel in areas volgens standaard gepubliseerde prosedures. Die slym- en epiteellaag van elke area is afgeskraap op ‘n mikroskoopplaatjie (deur van ‘n tweede plaatjie gebruik te maak) en ondersoek met behulp van ‘n disseksiemikroskoop. Parasiete is vanuit die smere verwyder met behulp van ‘n stewige wimperhaar (wat aan ‘n dun houthandvatsel vasgeplak is) en is daarna monteer in Malmberg se oplossing (Gliserien-Ammonium-Pikraat of te wel GAP) of gliserienjellie. Parasiete is ondersoek en harde dele van taksonomiese belang geteken met behulp van ‘n tekenbuis en ligmikroskoop. Mates (om sodoende parasiet-identifikasie en beskrywings waar nodig moontlik te maak) is direk vanaf sketse gemeet volgens standaard, gepubliseerde metodes.

Die genera Gyrodactylus von Nordmann, 1832 en Dactylogyrus Diesing, 1850 word gereken as twee van die algemeenste groepe Monogenea op visse dwarsoor die wêreld. Die aanname is bevestig in die huidige studie, veral met verwysing na die laaste genus waarvan ‘n hele aantal spesies beskryf en identifiseer is. Ander genera wat versamel is sluit Dogielius Bychowsky, 1936; Diplozoon von Nordmann, 1832; Paradiplozoon Akhmerov, 1974 en Quadriacanthus Paperna, 1961 in.

Anders as wat verwag is, het Monogenea nie voorgekom op al die visspesies wat ondersoek is nie. Geen parasiete (Monogenea) is byvoorbeeld gevind op swartbaars (M. salmoides) nie. Ander visspesies wat ondersoek is, het egter minstens een Monogenea spesie op hulle gehad soos wat voorspel is. Verder was die verwagting dat alle gasheerspesies meer as een spesie Monogenea sou huisves.

Daar is egter net meer as een spesie van dieselfde genus (Dactylogyrus in hierdie geval) gevind op Labeo spp. en C. carpio.

xxvi Morfologiese variasie in grootte en selfs vorm van harde strukture is opgemerk in Quadriacanthus aegypticus El-Naggar en Serag, 1986 (versamel vanaf C. gariepinus) sowel as Dactylogyrus spp. en Dogielius intorquens Crafford, Luus- Powell en Avenant-Oldewage, in druk (vanaf spesies van Labeo Cuvier, 1817).

Dit is onwaarskynlik dat eksterne omgewingsinvloede soos geografiese variasie of temperatuureffekte die variasie veroorsaak, maar die effek van beide gasheer- en parasietgroottes (en enige korrelasies wat mag bestaan) vereis verdere ondersoek. Daar word voorgestel dat parasietgenotipe verantwoordelik is vir die variasie en dat moontlike kriptiese spesiekomplekse bestaan. Vir toekomstige projekte moet eksperimentele infekteringstudies, gekombineer met ‘n molekulêre aanslag, oorweeg word aangesien dit uitsluitsel oor spesie-identifikasies moontlik sal maak.

Alhoewel persentasie voorkoms 50% oorskry het in ‘n aantal gevalle tydens die someropname, was dit nie die geval vir die winteropname nie. Gemiddelde intensiteit van besmetting het in die winter slegs in ‘n paar gevalle oor 10 gestrek, terwyl ‘n paar gevalle tydens die someropname ‘n waarde van 20 oorskry het. Daar was statisties betekenisvolle verskille in parasietgetalle tussen die opnames (en dus ook seisoene) vir L. umbratus, waar meer Dactylogyrus spp. en Dogielius sp. in die somer gekry is, terwyl Diplozoon sp. getalle weer hoër was in die winter. Wat gemiddelde intensiteit van besmetting betref is statisties betekenisvolle seisoenale verskille slegs gekry vir Dactylogyrus larindae Crafford, Luus-Powell en Avenant- Oldewage, in druk. Gyrodactylus sp. is slegs tydens die someropname versamel vanaf C. gariepinus en C. carpio in betreklike lae getalle. Die klein datastel verhoed egter dat enige veralgemenings of slotsom rakende die effek van seisoene op die voorkoms van Gyrodactylus sp. geformuleer kan word. Dit wil wel voorkom of hoër temperature tydens die somer wel bygedra het tot hoër Dactylogyrus sp. getalle deurdat dit die ontwikkelingstempo van die parasiete verhoog.

‘n Statisties betekenisvolle voorkeur vir manlike gashere is in twee gevalle tydens die someropname aangeteken. Dit was egter nie deur die bank die geval vir alle gashere nie en word moontlik toegeskryf aan te klein steekproefgroottes vir die onderskeie geslagte. ‘n Gasheergeslagvoorkeur word as hoogs onwaarskynlik geag maar toekomstige studies moet die moontlike voorkoms daarvan, veral in terme van moontlike seisoenale patrone, verder ondersoek.

xxvii Alle parasietspesies was spesifiek in voorkoms tot ‘n bepaalde gasheergenus. Meeste parasiete was spesifiek tot ‘n gasheerspesie, of het ‘n defnitiewe voorkeur getoon vir ‘n spesifieke gasheerspesie (waar ‘n parasiet van dieselfde spesie op nabyverwante gashere voorgekom het).

‘n Aantal nuwe parasietspesies is beskryf vanaf inheemse Labeo gashere. Verdere ondersoek (groter steekproefgetalle) is nodig om die spesies te identifiseer wat op inheemse Labeobarbus spp. voorkom. Na verwagting sal minstens een nuwe spesie van die gasheer af beskryf kan word.

Daar kom op beide C. carpio en C. idella in die Vaaldam Monogenea-parasiete voor wat al wêreldwyd op dieselfde gashere gevind is. Die logiese afleiding is dus dat die parasiete saam met die visse ingevoer is.

Waar daar meer as een Monogenea-spesie op die kieue voorgekom het, kon geen voorkeur vir ‘n spesifieke plek van aanhegting bespeur word nie. Neutrale / ewekansige effekte soos watervloeipatrone oor die kieue het oënskynlik ‘n groter effek op vashegtingvoorkeure as negatiewe interaksies soos kompetisie. Dit kon veral duidelik gesien word op die kieue van Labeo spp., waar daar geen aanhegtingsplekvoorkeure onderskei kon word vir / tussen die talle parasietspesies wat saam op die kieue voorgekom het nie.

Basislyninligting oor die samestelling van die Monogenea-gemeenskap in die Vaaldam wat tydens die huidige studie opgeteken is, sal positief bydra tot die beplanning van verdere taksonomiese (nuwe spesiebeskrywings en verfyning van bestaande beskrywings) en toegepaste (in terme van ekologiese, fisiologiese en eksperimentele aspekte) studies.

xxviii

CHAPTER 1

1 - SYNOPSIS: FROM AIM TO ABSCISSION

1.1. Introduction

The aim of this first chapter is to provide an overview of how this thesis is structuredIn the sections that follow the general problem statement shall be defined, followed by more specific project aims or objects and a synopsis of how this thesis is organized,i.e. chapter outlay.

1.2. Problem statement

Advancement of research on the parasitic monogeneans or Monogenea of freshwater fishes in South Africa is, at least to some extent, hampered by the lack of published accounts of species composition and infection level baseline data.

1.3. Project aims / objectives / hypotheses

The project aims / objectives set in section 1.3.1 were envisioned to aid in guiding the scope of data collection, analyses and finally thesis outlay.

From these (i.e. objectives) a number of hypotheses were compiled (section 1.3.2) to aid in setting the scope for investigation / discussion in the thesis. It was thus not intended as an exhaustive list of all topics to be investigated, but rather guidelines as to what was considered central issues to be investigated in the current project.

1.3.1. Project aims / objectives

1) Parasite collection, identification and description from various fish host species;

2) Calculation of infection parameters / variables (prevalence, mean intensity and mean abundance);

3) Comparison of infection parameter statistics between surveys / seasons where applicable / feasible;

4) Discuss / evaluate the potential effects of environmental / abiotic variables on infection parameter statistics;

5) Comment on the effect of host related factors on infection parameter statistics;

6) Comment on host- and site specificity;

7) Comment on monogenean parasite species richness and species composition.

1.3.2. Hypotheses formulated from objectives

Hypothesis 1-1: Each fish species examined shall harbour at least one monogenean parasite species.

Hypothesis 2-1: Both prevalence and intensity range of infection shall be high (>50% and 20-100 parasites respectively) in the majority of cases.

Hypothesis 3-1: There shall be a significant difference in infection values between seasons.

Hypothesis 3-2: Higher infection values shall be recorded for dactylogyrids in summer, while the opposite shall be true for gyrodactylids.

Hypothesis 4-1: Water temperature effects shall surpass potential host immunological effects (also see Hypothesis 3-1 and 3-2).

Hypothesis 5-1: Larger / older fish shall harbour a greater number of parasites.

Hypothesis 5-2: Larger / older fish shall harbour a greater number of parasite species.

Hypothesis 5-3: Host gender shall have no effect on parasite infection statistics.

Hypothesis 6-1: Monogenean parasites encountered shall exhibit a high degree of host specificity.

Hypothesis 6-2 Examination of previously unexamined (for monogenean parasites) fish species shall reveal novel monogenean species.

Hypothesis 6-3: Exotic (non-endemic to South Africa) fish species with a global translocation history shall harbour monogenean parasites with the same global distribution pattern.

Hypothesis 6-4: Monogenean parasites from such alien fish species shall not occur on endemic fish species (i.e. host switching shall not have occurred).

Hypothesis 6-5: Site specificity shall not be distinct where more than one species co-exist on the gills, i.e. neutral interactions (random distribution) shall have a greater effect than negative interactions (e.g. competition).

Hypothesis 7-1: More than one species from at least one genus shall occur on the same fish host.

Hypothesis 7-2: More than one monogenean genus shall be recovered from every host species examined.

1.4. Thesis outlay

1.4.1. Notes on chapter delineation and section cross-referencing

The thesis consists of a number of chapters (section 1.4.3) dealing with particular aspects. It was decided to largely use host genus identification to delineate chapters. Monogenean parasites are notoriously host specific which would make such delineation feasible. The sections within each chapter have been allocated a clear section number that starts with the chapter number. This allows easy cross- referencing between chapters. Methodology of host and parasite collection and data analyses were obviously very similar for all species. For this reason a single, detailed materials and methods chapter was compiled.

In the chapters that follow, reference was made to specific sections in the methods and materials chapter to avoid unnecessary repetition / duplication of information. The majority of aspects investigated (section 1.3) are applicable to all monogenean parasites. The same argument (i.e. the need to avoid unnecessary duplication of information) was thus relevant to certain discussion sections in various chapters (i.e. sections that have bearing on discussions recorded in other chapters). Once again such cross-references (i.e. to sections) were inserted where deemed applicable / necessary. Within the chapters recommendations are made for future research efforts / directions. Such recommendations are consolidated in a discussion on future research needs in Chapter 13.

1.4.2. Notes on referencing methodology

A complete reference list was not provided at the end of each chapter. To avoid unnecessary duplication of information a single, complete reference list of all references used in the thesis was compiled and constitutes a chapter by itself (Chapter 14), as is described in the next section. This reference list contains complete references for all literature cited in discussions (i.e. references that were actually read / consulted by the author).

1.4.3. Organization of chapters

The section shall provide details on the way chapters were organized within this thesis, as well as a short overview of what the chapters entail.

Chapter 1: Synopsis - Current chapter that provides an overview of thesis structure / organization and hence the broad scope of investigation.

Chapter 2: General introduction – This chapter provides additional information on the aspects to be investigated, thus further elucidating the scope of investigation.

Chapter 3: Materials and methods – This chapter provides detailed information on materials used and methods / procedures employed. This is not repeated in subsequent chapters, but cross-references (i.e. section numbers) are provided to the relevant sections in the materials and methods chapter.

Chapter 4: Evaluation of variance of forms within a single parasite species, versus separate species of Dactylogyrus Diesing, 1850 and Dogielius Bychowsky, 1936 on Labeo Cuvier, 1817 hosts – This chapter provides an overview of procedures followed to reach a decision regarding the species status of closely related parasite species / “forms” collected from two closely related host species.

During these examinations some practical challenges were encountered. Discussions on these are included as appendices (Appendix A and Appendix B) at the end of the thesis.

Chapter 5: Monogenean parasite species descriptions from Labeo spp. in the Vaal Dam, South Africa, with a review of related parasite species – This chapter provides species descriptions for a number of new monogenean species encountered, following the species status decision reached in Chapter 4.

Chapter 6: Aspects of the ecology of monogenean parasites of Labeo capensis (Smith, 1841) and Labeo umbratus (Smith, 1841) in the Vaal Dam, Gauteng Province, South Africa collected during a winter survey – This is the first chapter dealing with parasite infection statistics of a particular host genus (i.e. Labeo spp.). In this chapter results (infection statistics as well as host and site specificity data) for a winter survey is discussed.

Chapter 7: Aspects of the ecology of monogenean parasites of Labeo capensis (Smith, 1841) and Labeo umbratus (Smith, 1841) in the Vaal Dam, Gauteng Province, South Africa collected during a summer survey as compared with the preceding winter survey – This chapter deals with the same host genus but reports on a subsequent summer survey. Some of the analyses performed for the winter survey data was found not to provide any meaningful contributions (e.g. attempts at analyzing data within length classes) and was thus not repeated for the summer survey. Applicable results are also statistically compared between seasons in order to comment on any seasonal effects in infection statistics and / or distribution (i.e. site preference on gills).

Chapter 8: Aspects of the ecology of Quadriacanthus aegypticus El-Naggar and Serag, 1986 from Clarias gariepinus (Burchell, 1822) in the Vaal Dam, Gauteng Province, South Africa with description of variation observed in haptoral sclerites and male copulatory organ – This chapter deals with Q. aegypticus collected from sharptooth catfish (C. gariepinus). This host was only encountered during the summer survey sampling and infection statistics are reported on. Variation in the size and shape of sclerites (haptoral as well as the male copulatory organ) are also reported on and discussed in terms of previous descriptions from literature.

Chapter 9: Aspects of the ecology of a species of Gyrodactylus von Nordmann, 1832 from Clarias gariepinus (Burchell, 1822) in the Vaal Dam, Gauteng Province, South Africa – This chapter also deals with the parasite fauna of sharptooth catfish (C. gariepinus). Unequivocal species identification of Gyrodactylus sp. parasites is often dependent on additional molecular analyses as well as studies on the finer structure (i.e. shape) of very small sclerites such as marginal hooklet sickles.

As such analyses did not fall within the scope (i.e. surveys were conducted to collect baseline data on the occurrence of monogenean parasites) of the current project, identification to species level was not performed. However, infection statistics are reported on and preliminary haptoral sclerite measurements are compared to previously published accounts from other Gyrodactylus spp. collected from the same host in Africa. Unequivocal species identification shall, however, form part of another project.

Chapter 10: Aspects of the ecology of monogenean parasites from Cyprinus carpio Linnaeus, 1758 in the Vaal Dam, Gauteng Province, South Africa – This chapter deals with the occurrence of previously described monogenean parasites collected from common carp (C. carpio). This host was only encountered during the summer survey sampling and infection statistics are reported on.

Chapter 11: Aspects of the ecology of a monogenean parasite from Ctenopharyngodon idella (Valenciennes, 1844) in the Vaal Dam, Gauteng Province, South Africa – This chapter deals with the occurrence of a previously described monogenean parasite collected from grass carp (C. idella). This host was only encountered during the summer survey sampling and infection statistics are reported on.

Chapter 12: Aspects of the ecology of a monogenean parasite from Labeobarbus aeneus (Burchell, 1822) and Labeobarbus kimberleyensis (Gilchrist and Thompson, 1913) in the Vaal Dam, Gauteng Province, South Africa – This chapter deals with the occurrence of monogenean parasites collected from smallmouth (L. aeneus) and largemouth (L. kimberleyensis) yellowfishes. Mention is made of possible hybridization but data was found to be inadequate to make any meaningful deductions. The hosts were encountered during both winter and summer survey sampling and infection statistics are reported on. Due to low prevalence additional seasonal statistical comparisons were not performed.

Chapter 13: General discussion – This chapter aims to consolidate results and conclusions from the previous chapters into a single, coherent discussion. This shall include additional comparisons with trends recorded in the literature and ultimately result in a condensed summary of findings, from which future research needs shall be inferred.

Chapter 14: References – This chapter includes the complete list of references cited in this thesis.

Appendix A - This appendix contains discussions on the effect of anchor orientation on standard measurements applied to Dogielius spp., as well as recommendations on how anchor orientation could be standardized.

Appendix B - This appendix contains discussions on additional examinations relating to evaluation of measurement methodologies employed for Dactylogyrus and Dogielius spp.

Appendix C - This appendix provides preliminary results on gill morphology differences between L. umbratus and L. capensis.

1.4.4. Study outputs

1.4.4.1. Conferences

Posters were presented at the following conferences:

1) Crafford, Luus-Powell and Avenant-Oldewage: “Ecological aspects of some monogenean parasites from fishes in the Vaal River system South Africa – Preliminary surveys”. Presented at the 6th International Symposium on Monogenea, 3 to 7 August 2009, Marine and Coastal Management National Research Aquarium, Cape Town, South Africa.

2) Crafford, Luus-Powell and Avenant-Oldewage: “Does size matter? The case of the lesser-endowed monogenean….”. Presented at the 8th International Symposium of Fish Parasites, 26 to 30 September 2011, Vina del Mar, Chile, South America.

1.4.4.2. Published papers

The following paper has been accepted for publication but no further information (e.g. issue and page numbers) was available at the time of submission of this thesis:

Crafford, D., Luus-Powell, W.J. and Avenant-Oldewage, A. (2012) Monogenean parasite species descriptions from Labeo spp. hosts in the Vaal Dam, South Africa. African Zoology 47(2): 216-228.

The content of this paper is reflected in Chapter 5.

CHAPTER 2

2 - GENERAL INTRODUCTION

“Few would disagree with the proposition that nature is immensely complex. However, if we wish to understand this complexity, we will be well advised to abstract relatively simple facets from nature and examine these first”.

Begon and Mortimer (1994)

In the following chapters there is considerable overlap in some of the basic variables (or underlying principles applicable to the better understanding of such variables) under discussion. In an attempt to avoid unnecessary duplication of information, applicable background information relevant to many of the chapters following, is briefly consolidated within this introduction chapter. The various sections are clearly numbered for easy cross referencing in subsequent chapters. The subjects discussed within this chapter are varied and information may appear “fragmented” to some extent. Choice of variables was, however, dictated by the scope of the current project, as delineated in Chapter 1. Every attempt was made to present information on chosen variables within this chapter in a logical manner as described below.

Elaborate discussion on historical debate with regard to nomenclature fall outside the scope of this project. Awareness of these developments is, however, essential. Furthermore terminology used throughout this thesis shall be defined.

The need for “a bigger picture” applies and as a result the importance (and possible practical applications) of parasitological studies, with emphasis on aquatic systems and monogeneans in particular, will be briefly reviewed.

Following this general review, ecology as a science shall be defined and the use of the term delineated within the framework of the current study (i.e. ecological aspects to be examined in subsequent chapters). Finally terminology to be used shall be clarified.

With a larger framework for discussion in place, the subject of this thesis (i.e. Monogenea) shall be introduced with reference to general taxonomy, basic morphology and genera encountered.

Subsequently host and site specificity, a trait often exhibited by monogeneans with obvious ecological and phylogenetic relevance, shall be discussed. With the former in mind one may expect seasonal variation in terms of parasite numbers and distribution, which shall then link to the next section.

There exists an intricate interplay between hosts, parasites and habitat in any system, with such interactions proving to be complex even within species-poor systems (Choudhury, Hoffnagle and Cole 2004). As a result the possible effects that selected host and abiotic / environmental variables may have on monogenean parasite numbers and distribution shall be examined.

With this framework firmly in place, the current situation with regard to monogenean research in South Africa will be discussed. This shall elucidate the problem statement and subsequent objectives to be met through the research reflected in this thesis, as already summarized in Chapter 1. In view of these objectives a number of hypotheses (where relevant / applicable) can be formulated that shall ultimately be applied to results obtained and thoroughly discussed in the general discussion chapter.

2.1. A note concerning nomenclature

Since 1859 the phylum Platyhelminthes has played a central role in discussions of metazoan phylogeny (Baguñà and Riutort 2004). With reference to the class comprising monogenean flatworms, the early part of the 20th century saw the initiation of a sporadic debate on whether this class of Platyhelminthes should be referred to as Monogenea or Monogenoidea.

This debate was rekindled by a publication by Boeger and Kritsky (1993) revising the classification of the group. This, in turn, led to a publication by Wheeler and Chisholm (1995) where they argue that (and justify why) priority and stability of nomenclature, as well as consensus among a number of consulted specialists, favour the use of Monogenea instead of Monogenoidea. A detailed description / historical review of this debate and research surrounding it, falls outside the scope of the current thesis. The thorough review and excellent discussions by Wheeler and Chisholm (1995) on this subject, however, reveals the following highlights in the progression of this debate.

Van Beneden was the first to recognize monogeneans as distinct taxon in 1858 using French terminology, though Carus first used the name in Latinised form (i.e. Monogenea) in 1863. Bychowsky proposed that the name of this group be changed to Monogenoidea in 1937 after he changed the taxonomic rank from order to that of class. This led to use of both terms and sparked the initial debate. In 1978 a round table discussion was held at the Fourth International Congress of Parasitology (ICOPA IV) where Monogenea was decided on as the preferred name of this parasite group. Though this decision was rejected by some authors (e.g. Boeger and Kritsky 1993), the term Monogenea is still preferred and used by the majority of parasitologists.

Throughout this thesis the term “Monogenea” shall be used to refer to the taxonomic class, but reference to “monogenea” or “monogeneans” as a general term (i.e. “common name”) shall also be employed when referring to this group of parasites.

2.2. Why parasites?

Parasites may cause disease and lead to commercial losses. However, if the purpose and significance of parasitological studies on fish parasites relied solely on therapeutic and clinical efficacy studies (e.g. Willomitzer 1980b; Schmahl and Taraschewski 1987; Schmahl and Mehlhorn 1988; Schmahl 1991, 1993; Abo-Esa 2008; Kayis, Ozcelep, Capkin and Altinok 2009), research presented in this thesis would indeed have little practical significance. Fact remains that in order to find practical applications for specific variables (or generate practical management systems to mitigate or manage potential adverse effects for that matter), one needs to identify and define such variables. Parasites are no different.

In the current study the aim is to generate such “baseline data” to hopefully stimulate further interest in this parasite group in South Africa. In the sections that follow a brief overview shall be provided of the relevance and broader implications / applications of general parasitological research within the greater realm of aquatic biology and also biodiversity studies. Throughout this general overview emphasis shall be on the use of data on monogeneans within this larger framework. Finally the relevance of the current research within the reviewed framework shall be commented upon.

2.2.1. Aquaculture and ornamental fish trade

A review by Hecht and Endemann (1998) revealed a low-level intensity of aquaculture in sub-Saharan Africa. Does this then not make all potential data on fish parasites irrelevant? Hecht and Endemann (1998) tend to disagree as they point out that a priori information may be used to draft and implement proactive measurements as opposed to reactive research aimed at crisis control.

Indeed, parasites (including monogeneans) cause huge economic losses in the agricultural industry globally, with aquaculture being no exception. Furthermore parasites may be translocated through the ornamental fish trade (e.g. Tamaru, Cole, Bailey and Brown 1997; Thilakaratne, Rajapaksha, Hewakopara, Rajapakse and Faizal 2003). Under culture conditions parasitism may lead to lower growth and other adverse physiological alterations, the latter often translating in increased host susceptibility to parasites (Ghiraldelli, Martins, Yamashita and Jerônimo 2006). Cloutman (1976) confirms that differences in host susceptibility to monogenean infection may be physiological rather than ecological. Increased mucous and hyperplasia may not only interfere with further infection and make transfer through mucous energetically too costly for parasites (or remove them through sloughing) (Jones 2001), but also affect site selection with reference to mucous cell density (Buchmann and Bresciani 1998). Some monogenean ectoparasites apparently reduce mucous cell density in host skin which may be an attempt to minimize inhibitory effects (Jones 2001). With reference to the immune mechanism in fish skin, Buchmann (1999) proposed an excellent model.

Pathology is, however, not restricted to direct physiological effects (e.g. enhanced mucous secretion associated with epithelial cell hyperplasia) or mortality, but may also translate into reduced fitness through indirect effects. As a result large amounts of time and resources are spent finding ways to control or manage such infections. These research avenues are diverse and range from conventional chemical treatment (e.g. Székely and Molnár 1987) to neuromuscular (e.g. Halton 2004) and genetic studies. The latter is applicable to studies as diverse as parasite resistance of potentially suitable fish species for aquaculture (e.g. Flajshans, Kocour, Gela and Piackova 2004), as well as studies on parasites as a tool for monitoring drug resistance development and improving management practises (Collins, Miller and Cunningham 2004).

On a larger scale population genetic studies have also been suggested to document the evolutionary origin of drug resistance in pathogens (Huyse, Poulin and Théron 2005). Furthermore, the helminth neuromuscular system is increasingly considered to be an exploitable target in novel drug development (Halton 2004). However, before one can endeavour to pursue such specialized research fields, one obviously first need to identify suitable subjects and collect baseline data on biology, numbers and distribution.

2.2.2. Parasitological studies in “wild” versus cultured fish populations: Finding a common link

Ayotunde, Ochang and Okey (2007) state that parasite and / or disease conditions under natural conditions may serve as a basis for information on the potential risk of diseases expected under intensive fish culture. Such an approach is exemplified by Al-Samman, Molnár and Székely (2006) whom surveyed both cultured and freshwater fish with regards to monogenean infection. Parasites (including monogeneans such as Gyrodactylus von Nordmann, 1832) can and do have an impact on important African aquaculture species such as Clarias gariepinus (Burchell, 1822) (Abo-Esa 2008). Apart from direct and / or indirect pathology (e.g. Appleby, Mo and Aase 1997; Arafa, El-Naggar and El-Abbassy 2009) leading to economic losses, losses and energy expenditure related to host response must also be considered.

Furthermore such studies (i.e. examining defence mechanisms in fish against parasites) may play a crucial role in the future development of vaccines against piscine parasitoses (Alvarez-Pellitero 2008).

Parasites may also be introduced to wild populations through introduction of cultured fish, an example being Dactylogyrus minutus Kulwiec, 1927 and Dactylogyrus anchoratus (Dujardin, 1845) which was introduced to Africa with Cyprinus carpio Linnaeus, 1758 (Hecht and Endemann 1998).

In a review by Klein (2003) it becomes clear that social behaviours in vertebrates change following parasite infection (i.e. some parasites exhibit the ability to exploit the proximate mechanisms that mediate the expression of social behaviours to increase transmission).

One example the author mentions is a reduction in courtship behaviour of guppies following infection with Gyrodactylus turnbulli Harris, 1986. While the proximate mechanisms involved were as yet unknown, this example once again serve to illustrate the “indirect” negative effect (i.e. as opposed to the “direct” effect posed by mortality) monogeneans have on the aquaculture and ornamental fish trade.

2.2.3. Fisheries management

Management of commercial fish species (including freshwater fisheries management) relies on data on age, growth and mortality of stock (e.g. Adeyemi, Bankole, Adikwu and Akombu 2009). These variables are all potentially influenced by parasite infections. Barber, Hoare and Krause (2000) state that, as parasites may have both a socio-economic and human health impact, a full understanding of the widely varying effects of fish parasites on their hosts are central to the development and management of global fisheries. Securing a firm baseline database on all parasites present in a particular system may aid greatly in the interpretation of fisheries data. In this regard the ecological role and impact of parasites have long been underestimated and models incorporating them underutilized. In fact, Williams, MacKenzie and McCarthy (1992) state that opportunities for further work on parasites (and the application thereof) extend beyond immediate value to fisheries research. It is thus not only in the realm of ecology (e.g. Morand and Krasnov 2008) and fisheries management where the study of parasites has proven to be an exceptional model.

2.2.4. Parasites as biological models and indicators

According to Peeler, Murray, Thebault, Brun, Giovaninni and Thrush (2007), risk analyses (i.e. reducing complex biological processes to a simplified series of events to investigate the probability and potential consequences of undesirable events) has only recently been regularly employed in the management of aquatic animal health. Using studies on Gyrodactylus salaris Malmberg, 1957 as a model, the authors demonstrated that risk analyses can support animal health policy development over a broad spectrum (e.g. fisheries management and aquaculture). They, however, warn that a long term constraint for quantitative analysis is the lack of information about the disease-hazard of a very large number of fish species traded.

The fact that a lack of baseline data is currently considered the main constraint to the application of risk analysis in aquatic health, once again highlights the need to gather baseline data on poorly (at least in some hosts and localities) studied parasites such as monogeneans. An exception (i.e. a well-studied monogenean) is G. salaris, a major problem in salmon (both wild and farmed) in Norway since its presumed introduction from Sweden in the 1970’s (Scholtz 1999). To aid in risk analysis (e.g. Peeler, Thrush, Paisley and Rodgers 2006) and drafting of practical environmental management practises, research on this parasite was conducted in very diverse fields. This included development and evaluation of morphological means of identification (e.g. Shinn, Hansen, Olstad, Bachmann and Bakke 2004; Shinn, Collins, García-Vásquez, Snow, Matĕjusová, Paladini, Longshaw, Lindenstrøm, Stone, Turnbull, Picon-Camacho, Vázquez Rivera, Duguid, Mo, Hansen, Olstad, Cable, Harris, Kerr, Graham, Monaghan, Yoon, Buchmann, Taylor, Bakke, Raynard, Irving and Bron 2010), examination of molecular variation amongst different populations (Cunningham and Mo 1997) and examination of immune mechanisms (e.g. Kania, Larsen, Ingerslev and Buchmann 2007). It is, however, not only within the arena of environmental management where parasites are employed in models.

Huyse et al. (2005) state that, because parasites have a high potential for specialization and diversification, they represent ideal models for studying speciation processes.

Furthermore some groups live in conditions that are ripe for sympatric speciation (Huyse et al. 2005). Applications for the use of parasites to study phylogenetic relationships are further discussed in section 2.2.6.

The use of parasites as indicators for environmental degradation or biological diversity is further investigated in the next section.

2.2.5. Environmental management, ecosystem integrity and biodiversity

There is a worldwide concern over the management and assessment of impacted ecosystems (Laurenson, Hocutt and Hecht 1989; Morley 2009). This includes, but is not limited to, the role of invasive fish species (e.g. Laurenson et al. 1989). Invasive fish species often harbour invasive parasite species (e.g. Dove and Ernst 1998), a fact that is often neglected.

Monogeneans are generally considered to be very host-specific (e.g. Bakke, Harris and Cable 2002), yet the severe pathology and mortality caused by G. salaris clearly demonstrate the dangers exotic translocated monogeneans may pose (e.g. Appleby et al. 1997). It thus comes as no surprise that Kunz (2002) states that biodiversity research plays a key role in parasitology.

Fish and other aquatic organisms (e.g. Casazza, Silvestri and Spada 2002), including monogenean fish parasites, have been proven to be valuable biological indicators to evaluate the impact of both organic (e.g. Escher, Wahli, Bűttner, Meier and Burkhardt-Holm 1999; Khan and Billiard 2007; Madanire-Moyo and Barson 2010) and chemical or metal pollution (e.g. Jooste, Luus-Powell and Polling 2005; Pettersen, Vøllestad, Flodmark and Poléo 2006; Gheorghiu, Cable, Marcogliese and Scott 2007; Khanna, Sarkar, Gautam and Bhutiani 2007; Bayoumy, Osman, El-Bana and Hassanain 2008; Blanar, Munkittrick, Houlahan, MacLatchy and Marcogliese 2009; Retief, Avenant-Oldewage and du Preez 2009; Watson, Crafford and Avenant- Oldewage 2012) aquatic biota. Complex patterns of parasite infection are influenced by variables such as: 1) Differences in sensitivity of parasites to different types of wastewater (i.e. direct detrimental effect on the parasite); 2) Wastewater induced host immune response inhibition resulting in higher parasite susceptibility (i.e. indirect positive effect on the parasite) (Escher et al. 1999). In aquatic ecosystems fish parasite communities reflect interactions with the aquatic environment as well as fish and invertebrate communities, all three being involved in the parasite life cycle (Kadlec, Šimková, Jarkovský and Gelnar 2003b).

Despite the myriad of ecological variables involved, parasites are still considered to be effective indicators of environmental changes in terms of population size and community structure (Hassan 2008), including aspects such as the ratio of endoparasites to ectoparasites (e.g. Lafferty 1997; Crafford and Avenant-Oldewage 2009).

In terms of ecosystem integrity (and related interactions and processes), changes in parasite population sizes and community organization are also drivers of further changes in ecosystem structure and function (Hassan 2008), with obvious implications with regard to biodiversity.

Choudhury et al. (2004) also drive this realization home when stating that continuous studies are critical in assessing future impacts of parasites and is an important issue in maintaining biotic integrity, as parasites can have a direct effect on ecosystem functions (Ondračkova, Dávidová, Gelnar and Jurajda 2006). As such, studies on parasites form an integral part of research and monitoring programs in areas where conservation of biodiversity is given high priority (e.g. Barson, Bray, Ollevier and Huyse 2008).

Once again baseline data on various systems may be useful when combined in larger datasets. Allan and Flecker (1993), for example, concluded that rivers and streams (i.e. running water) are in dire need of restoration and preservation. They also conclude that current ability to address these issues is to a degree limited by an inadequate knowledge base.

2.2.6. Host taxonomy, translocation history and phylogenetic relationships

Williams et al. (1992) provide an excellent review on the use of parasites as biological indicators of fish population biology, migrations, diet and phylogenetics. As a point of interest they state that one of the first attempts to use parasite indicators (i.e. parasites as “tags” or “markers”) to infer aspects of fish biology, was a study on sturgeon stock discrimination using monogenean parasites. Although the suitability of this group (i.e. Monogenea) as biological tags are well recognized (Lambert and El Gharbi 1995), monogeneans are admittedly not useful markers under all conditions as 1) most ectoparasites are easily transmitted horizontally from host to host (irrespective of original area of infection) and 2) are more susceptible to changes in various abiotic parameters (Bush, Fernández, Esch and Seed 2001).

Despite these possible shortfalls, parasites have since those initial attempts been successfully employed as biological tags to verify or elucidate the taxonomic relationship between sympatric sibling fish host species (Cloutman 1976). Huyse et al. (2005), for example, state that isolated and small demes (adult reproducing parasites that usually inhabit an individual host organism) of parasites with a direct life cycle (such as monogeneans), are likely to exhibit population genetics patterns similar to that of the host. They conclude that this may result in host-parasite cospeciation at the macro-evolutionary scale.

Monogenean parasite fauna can also shed light on fish host life history and aspects such as probable translocations (or the lack thereof, e.g. Ernst, Fletcher and Hayward (2000). Studies on host specificity with reference to taxonomically related hosts (e.g. family level) may also reveal much of interest concerning the processes involved in parasite speciation (Harris and Lyles 1992; Malmberg 1998).

Comparison of host and parasite phylogenies can furthermore shed light on probable evolutionary patterns and events with regard to aspects like host switching and co- evolution (also known as “Fahrenholz’s rule” stating that “parasite phylogeny mirrors host phylogeny”) (Guégan and Agnése 1991; Nieberding and Olivieri 2006). Guégan and Agnése (1991), for example, also demonstrated that both evolution by descent (i.e. phylogenetic evolution) and sequential colonizations (non-phylogenetic evolution) were displayed following an analysis of African Labeo host species and their dactylogyrid parasites.

2.2.7. Relevance of aspects mentioned to the current study as reflected in this thesis

Aquaculture: Commercial overfishing, particularly with regard to marine fish stocks, results in a reduction in fish density, selective removal of large fish and an adverse impact on ecological system functions (e.g. food web complexity) (Wood, Lafferty and Micheli 2010). All of this may also be driving a global reduction in fish parasite diversity (Wood et al. 2010). It is, however, the continuing need for a constant supply of protein rich food, in conjunction with dwindling natural fish stocks that drives expansion and further development of the global aquaculture industry. During the past few years there has also been continued interest in South Africa in evaluating endemic fish species for aquaculture purposes.

A very pertinent example is Labeo umbratus (Smith, 1841), commonly known as moggel. This species shows promise as a suitable aquaculture species in small impoundments (e.g. Potts, Booth, Hecht and Andrew 2006). No monogenean parasites have previously been described from this species. Should the aquaculture status of moggel in future evolve from proverbial obscurity to established industry, it would obviously be beneficial to have baseline data available on possible threats that may be encountered under intensive culture conditions.

Fisheries management: There are several examples of studies dealing with evaluation of water systems (predominantly still waters) in South Africa with regard to suitability for the establishment of commercial fisheries (e.g. Potts and Khumalo 2005). These studies deal mostly with host (i.e. fish) biology on a community ecological scale. Such data has not been linked with intensive ecological studies on impacts of pathogens (i.e. parasites). Contributing baseline data to poorly studied parasites groups (i.e. monogeneans in this case) may help pave the way for additional ecological studies, where such organisms may well be found to play a greater ecological role in fisheries management than expected.

Biological indicators, environmental management and phylogenetic relationships: South Africa’s aquatic history is marred with introductions of non- endemic species, the majority (e.g. Oncorhynchus mykiss (Walbaum, 1792); Salmo trutta Linnaeus, 1758; Micropterus salmoides (Lacepède, 1802); Micropterus dolomieu (Lacepède, 1802) and C. carpio) being lawfully imported for angling purposes (Skelton 2001). Apart from these planned introductions, there have been many others that resulted from the ornamental fish trade (Skelton 2001), aquaculture (e.g. D’Amato, Esterhuyse, van der Waal, Brink and Volckaert 2007) and unlawful translocations by anglers (personal observations by author). The results include a very real threat of extinction through hybridization (e.g. Oreochromis mossambicus (Peters, 1852)) threatened by introductions of Oreochromis niloticus (Linnaeus, 1758) (D’Amato et al. 2007), cases of possible loss of endemic species through predation and uncertainty as to the possible effects and distribution of pathogens associated with these introductions (e.g. Mouton, Basson and Impson 2001).

While monogeneans are renowned for their high degree of host-specificity (e.g. Bakke et al. 2002), some species have been known to spread from an introduced to an endemic host species (e.g. Al-Samman et al. 2006).

Baseline data on this group will obviously help shed more light on possible effects introduced pathogens may have on biological diversity (amongst other things).

With regard to hybridization studies, monogeneans are known “biological tags” (e.g. Flajshans et al. 2004).

As such they may possibly be employed, together with traditional molecular techniques in hybridization quantification studies within endemic (to South Africa) Labeo Cuvier, 1817 spp. and species of Labeobarbus Rüppell, 1836 respectively (see Dupont and Crivelli 1988 for an example where monogeneans were employed in a host hybridization study). Once again plausible species for use in such studies would first have to be identified through basic research.

Several studies on fish health have been employed in southern Africa as a means to monitor water quality and hence biological integrity (e.g. Crafford and Avenant- Oldewage 2009; Watson et al. 2012). Very few of these incorporated monogeneans (for a recent exception see Madanire-Moyo and Barson 2010; Madanire-Moyo, Luus- Powell and Olivier 2012), yet the use of monogeneans as indicators of pollution have been thoroughly demonstrated (e.g. Pečinková, Matĕjusová, Koubková and Gelnar 2005). Baseline data on previously unknown monogeneans in various river systems may help raise awareness so this group can be more widely included in biological monitoring studies in South Africa.

2.3. Terms and terminology: Constructing an ecological framework for discussion

2.3.1. Defining “ecology” as a distinct field of science

The term “ecology” is widely abused. It is not only used with reference to office parks or other developments containing at least one tree (e.g. so called “eco-office parks”), but also by scientists discussing basic biology or the life-history of organisms. In the introduction to their book, Begon, Harper and Townsend (1990) state that the ultimate subject matter of ecology relates to where organisms occur, how many there are and what they do. Ecology (particularly with reference to community level) as a field of science in its own right may thus be defined as:

The study of interactions and processes that determine the number (i.e. abundance) and distribution of organisms.

From this fairly broad definition a number of key words can be identified as will subsequently be discussed.

Interactions: Two pertinent examples are predation and competition, most often deemed the two most important interactions determining organism numbers and distribution.

Processes: This category includes aspects such as births, deaths, immigration and emigration, disturbances (i.e. disruptive “interaction” between the biotic and abiotic environment, such as pollution) also sort under this category. This is because the environment (i.e. consisting of all those factors and phenomena outside the organisms that influence it) retains a central position in the definition of ecology as a science (Begon et al. 1990).

Numbers and distribution: Simply stated (with reference to any particular organism):

How many are there and can any distinct distribution patterns be identified? From what we have reviewed thus far, this is in effect the proverbial niche that basic research (of which the current study is to a large extent an example) wishes to fill, i.e. to collect baseline data.

2.3.2. Application of the term “ecology” in the current study

Animals do not truly have an “ecology” (as it relates to the study of interactions and processes in a community setting, i.e. emphasis on interactions and processes) but rather a “biology” (life-history traits, behaviour, habitat preference etc., i.e. emphasis on the species or organism under investigation). This is also reflected in the title of this thesis. Where the term “ecology” is used in the current study, it does not refer to the study of interactions or processes, but rather the effect thereof in terms of observed patterns in numbers (i.e. infection statistics) and distribution (i.e. host and site specificity). As a result no “cause and effect” relationships were experimentally examined and thus cannot be commented upon. In some instances it may, however, be apt to comment on possible interactions or processes that may potentially have played a role in creating observed patterns (with reference to parasite numbers and distribution).

2.3.3. Infection statistics

Throughout this thesis terminology and calculations relating to infection statistics follow that suggested by Bush, Lafferty, Lotz and Shostak (1997), as adapted from Margolis, Esch, Holmes, Kuris and Schad (1982). For ease of comparison definitions from these two publications are tabulated (Table 2-1) on the next page. Throughout this study the term “infection” (as opposed to “infestation”) shall be used.

2.4. Meet the Monogenea: A fleeting introduction

2.4.1. The bigger picture: a phylogenetic view

The major phyla comprised by the term helminths (a miscellaneous group of free living and parasitic worms) are the flatworms (Platyhelminthes) and round-worms (Nematoda) (Halton 2004). The former is relevant to this introduction.

Flatworms are usually dorso-ventrally flattened, hermaphroditic, acoelomate and bilaterally symmetrical, generally lack an anus and are thought to form the root stock of Bilateria in the phylogenetic scheme (Halton 2004). They represent a strategic evolutionary milestone being the most primitive animals in existence to exhibit bilateral symmetry with attendant cephalisation and condensation of neurons into a central nervous system (Halton 2004). Research indicates that this nervous system serves in motor and behavioural activities essential to the life cycle, but also plays an integrative role in regulating and coordinating reproduction and development events (Halton 2004).

The Neodermata (parasitic Platyhelminthes) consists of three classes, namely the Cestoda, Trematoda and Monogenea. There are some morphological similarities between these groups with regard to structures such as secretory glands (Poddubnaya, Scholz, Kuchta, Levron and Gibson 2008). The evolutionary relationship between (and even within) these classes is much debated (e.g. Mollaret, Jamieson, Adlard, Hugall, Lecointre, Chombard and Justine 1997; Justine 1998; Olson and Littlewood 2002; Baguñà and Riutort 2004; Perkins, Donnellan, Bertozzi and Whittington 2010) and is thought to restrict understanding of the evolution of parasitism and contingent adaptations (Perkins et al. 2010). Such debate is not surprising as Bush et al. (2001) state that continuous testing, modification and refinement of phylogenetic hypotheses forms the basis of phylogenetic studies.

Monogenea differ from the other two classes in that they have a direct life cycle (i.e. no intermediate host), predominantly parasitize the external surfaces of fish and are traditionally divided into two subclasses (resulting from two distinctive feeding strategies (Perkins et al. 2010). These are the Polyopisthocotylea (blood feeding) and the Monopisthocotylea (epithelium feeding) (Perkins et al. 2010). In an attempt to test monogenean monophyly and infer the evolution of diet in the Neodermata, Perkins et al. (2010) phylogenetically analysed available platyhelminth genomes from all three classes. This very interesting study produced three major findings: (1) the Cercomeromorphae (i.e. Cestoda plus Monogenea) was rejected and a Digenea plus Cestode clade was supported; (2) the Monogenea is paraphyletic (i.e. the Monopisthocotylea and Polyopisthocotylea have long and independent evolutionary histories); (3) the Monopisthocotylea may not be monophyletic. The common ancestor (of Cestoda and Digenia) is considered to have been monogenean-like and most likely sanguinivorous (Perkins et al. 2010). From there dietary specializations most probably evolved to suit diverse microhabitats in the final vertebrate hosts.

Apart from molecular approaches, other scientific fields such as ultrastructure of spermiogenesis and spermatozoa (e.g. Watson and Rohde 1995), have also been employed in the past to investigate and comment on possible phylogenetic relations. A detailed review of all other variables examined and different results obtained, however, falls beyond the scope of the current study.

Table 2-1: Comparison of ecological terms. Throughout this thesis the partial revision by Bush, Lafferty, Lotz and Shostak (1997) were followed.

Terms defined by Margolis, Esch, Holmes, Kuris and Schad (1982) Partial revision as suggested by Bush, Lafferty, Lotz and Shostak (1997) Term Expressed Definition / Concept Definition / Concept as:

Prevalence Percentage Number of individuals of a host species Number of hosts infected with 1 or more individuals of a particular parasite infected with a particular parasite species species (or taxonomic group) divided by the number of hosts examined for

divided by Number of hosts examined. that parasite species (% used descriptively; proportion used mathematically)

Incidence Cases per unit Number of new cases of a disease or

of time infection appearing in a population within Number of new hosts that become infected with a particular parasite

a given time period divided by Number during a specified time interval divided by the number of uninfected hosts

of uninfected individuals in the population present at the start of the time interval.

at the beginning of the time period.

Intensity Numerical Number of individuals of a particular Number of individuals of a particular parasite species in a single infected range parasite species in each infected host host, i.e. the number of individuals in an infrapopulation.

(i.e. in an infrapopulation) in a sample.

Mean intensity Numerical Total number of individuals of a particular Average intensity of a particular species of parasite among the infected range parasite species in a sample of a host members of a particular host species (i.e. total number of parasites of a

species divided by Number of infected particular species found in a sample divided by the number of hosts

individuals of the host species in the infected with that parasite). Should always be reported in conjunction

sample. with prevalence.

Density Unit must be Number of individuals of a particular Number of individuals of a particular parasite species in a measured specified parasite species per unit area, volume, sampling unit taken from a host or habitat, e.g. units of area, volume or weight.

or weight of infected host tissue or organ.

Relative density/ Numerical Total number of individuals of a particular Abundance: Number of individuals of a particular parasite in/on a single Abundance range parasite species in a sample of hosts host regardless of whether or not the host is infected (i.e. can be "0") divided by Total number of individuals Mean abundance: Total number of individuals of a particular parasite species

of the host species (infected and in a sample of a particular host species divided by the total number of hosts of

uninfected) in the sample. that species examined (infected and uninfected hosts).

Infrapopulation Not applicable All individuals of a species of parasite Includes all individuals of a species in an individual host at a particular occurring in an individual host. time.

Suprapopulation Not applicable All individuals of a species of parasite in Includes all developmental phases of a parasite at a particular place and time. all stages of development within all hosts (Emended definition to include free-living phases by dropping the "within"

in an ecosystem. all hosts" phrase).

Site / Location Not applicable The tissue, organ, or part of the host in The topological or spatial location in a host where a particular sample of which a parasite was found (site is parasites are collected (i.e. anatomical parallels to geographic locality). Suggests

preferable because of the possibility of "habitat" can refer to organs and tissues (i.e. typical local environment). "Niche"

confounding "location" and "locality"). refers to the role of the parasite and how it fits within a community.

2.4.2. A “typical” monogenean: morphology and biology

Monogenea are largely ectoparasitic with a simple life cycle involving a single host, the latter often being a fish (Barnes, Calow, Olive and Golding 1993). They are renowned for the high degree of host and site specificity they exhibit (Bush et al. 2001). Most monogeneans range in size between 0.3 mm to 20 mm and are thin and flattened (Bush et al. 2001). They are mostly bilaterally symmetrical with partial asymmetry superimposed on a few species (particularly involving the opisthaptor) (Schmidt and Roberts 1977). The body can be subdivided into a number of regions: cephalic region (anterior to pharynx), trunk (body proper), peduncle (portion of body tapered posteriorly) and finally the haptor (Schmidt and Roberts 1977).

As they attach to mobile hosts, attachment organs (e.g. suckers, hooks and clamps) are a necessary feature both anteriorly and posteriorly (e.g. Arafa et al. 2009). The morphology of the prominent posterior attachment organ (called an opisthaptor or simply haptor) (Barnes et al. 1993) is highly variable between genera. It may contain suckers (often in various stages of development), clamps or large hooks (also called anchors or hamuli) and small hooks (remnants of the larval stage that are called marginal hooks or hooklets) (Barnes et al. 1993). In some genera anchors are associated with additional structures like spikes (e.g. Agrawal, Tripathi and Shukla 2005) or sclerites (e.g. El-Naggar and Serag 1986; Galli and Kritsky 2008). There are most often one (e.g. Chinabut and Lim 1992) to two (e.g. Agrawal, Tripathi and Devak 2006) pairs of anchors which are more often than not supported by accessory sclerites or a connecting bar that may be of taxonomic significance (e.g. Amine and Euzet 2005). On the anterior surface of the body there may be up to four pigmented “eyespots” (photoreceptors) (e.g. Jain 1959). Monogeneans are monoecious but cross-fertilization appear to be the rule in most cases as the reproductive systems are not connected, necessitating cross-fertilization in the majority of cases (Bush et al. 2001). In many species the ejaculatory duct is thickened to form a penis-like structure, often with an associated sclerotized copulatory organ (simple or elaborate and complex) that joins with the ejaculatory duct (e.g. Gussev, Jalali and Molnár 1993). Further discussions in this thesis shall concentrate on the morphology of the sclerotized structures of the opisthaptor and copulatory organ as these are characteristic of each species.

This is discussed in more detail in section 2.4.9. Further details on structures such as adhesive apparatus (e.g. El-Naggar and Kearn 1980) and neuromasculature (e.g. El-Naggar, Arafa, El-Abbassy, Stewart and Halton 2004) fall outside the scope of the current study.

Monogenean life cycles are generally relatively straightforward. The shelled eggs release free-swimming ciliated larvae (oncomiracidia) upon hatching (Barnes et al. 1993) which disperse and allow exploitation of new hosts. The life span of these larvae is generally 12 to 48 hours at 20 to 28°C, apparently losing the ability to reach hosts after 4 to 6 hours (Paperna 1996). This mode of reproduction differs from that of the Gyrodactylidae. Gyrodactylus spp. give birth to fully developed adults with intra-uterine embryos that in turn also already contain second and often third generation embryos (Paperna 1996).

As mentioned previously, the Monogenea is also traditionally divided into two subclasses according to feeding strategies: the Polyopisthocotylea (blood feeding) and the Monopisthocotylea (epithelium feeding) (Perkins et al. 2010). In the latter case the epidermis of the host is often eroded by a proteolytic secretion produced in the feeding organ by gland cells (Kearn 1963). Apart from these generalized differences in feeding behaviour, a number of other differences between Monopisthocotylea and Polyopisthocotylea have been recorded. The former has a single opisthaptor but paired attachment organs anteriorly, while the latter has a divided opisthaptor with a pair of eversible buccal suckers or a single oral sucker (Barnes et al. 1993). Monopisthocotylea furthermore exhibit great variation with regard to detailed events of spermiogenesis and sperm structure, while Polyopisthocotylea are generally more uniform in these characteristics (Watson and Rohde 1995). Kingston, Dillon and Hargis (1969) also describe a number of generalized differences between monopisthocotylid (MP) and polyopisthocotylid (PP) eggs and larvae (oncomiracidia). Monopisthocotylea eggs are generally angular or pyramidal with a single (typically short) abopercular filament. In comparison PP eggs are generally bipolar and fusiform but may also be unipolar and globular. The larvae of both MP and PP bear cilia generally situated in three zones and are pyriform to fusiform in body shape. Their digestive tract structure is also similar (pharynx located in the middle third of the body leading to a rhabdocoel-like intestine).

Two pairs of eye-spots are generally present in MP oncomiracidia while PP specimens examined varied in this character.

2.4.3. Genera included in the current study

Gyrodactylus and Dactylogyrus Diesing, 1850 (both monopisthocototyleans) are considered to be two of the most common monogenean genera on fishes throughout the world (Hassan 2008). This supposition was also supported through the results of the current study, with a single Dogielius Bychowsky, 1936 species also encountered. Furthermore species of Diplozoon von Nordmann, 1832 as well as Paradiplozoon Akhmerov, 1974 were also collected, but as they are the focus of a separate study further identification to species level was not performed during the current study. Finally a representative from the genus Quadriacanthus Paperna, 1961 was also collected.

2.4.4. Gyrodactylus von Nordmann, 1832

The fusiform body of gyrodactylids anteriorly possess two conspicuous cephalic processed that bear spike sensilla as well as adhesive glands, thought to both have a sensory function and be involved in attachment (Bakke, Cable and Harris 2007). Posteriorly the opisthaptor is armed with anchors, bars and marginal hooks (Bakke et al. 2007), the latter being 16 in number (e.g. Wellborn and Rogers 1967). The pair of anchors is supported by a dorsal and ventral bar (Price 1967) and consists of a keratin-like protein (Shinn, Gibson and Sommerville 1993). Eye spots are lacking (Price 1967). Gyrodactylids occur widely on fish where they are economically significant but, however, also infect aquatic tetrapods and cephalopods (Harris, Shinn, Cable, Bakke and Bron 2008). The group is unique amongst the monogeneans because they are viviparous (Harris 1993). Evidence presented by Harris (1998a) suggests strongly that the first two births of Gyrodactylus gasterostei Gläser, 1974 occur by non-sexual means (either asexual or parthenogenetic). Polyembryony is likely to occur when the parent is not well equipped to sense the ecological circumstances of the offspring and / or it is constrained to produce fewer eggs than a favourable environment can bear (Craig, Slobodkin, Wray and Biermann 1997).

Because a swimming oncomiracidium is absent transmission is linked to development of a hostile environment (host immune response or host death), occasional physical contact between hosts by chance or resulting from periodically reoccurring behaviour, density-dependent mechanisms within the parasite population itself or accidental dislodgement (Bakke et al. 2007).

2.4.5. Dactylogyrus Diesing, 1850

The genus Dactylogyrus with more than 900 nominal species, is considered the largest helminth genus (Gibson, Timofeeva and Gerasev 1996). The life cycle follows a typical monogenean pattern: an egg followed by a free-swimming oncomiracidium (Schmidt and Roberts 1977). It is common to find more than one species on a single host (e.g. Mizelle and McDougal 1970) and in such cases the species may exhibit marked site specificity (Bush et al. 2001). This is however not always the case, as was shown by Dzika and Szymanski (1989). This genus shares some similarities with the genus Dogielius, most obvious being a single pair of anchors and the presence of 14 marginal hooks (Price and Yurkiewicz 1968).

Some sources indicate marginal hooks as numbering 16 (e.g. Mizelle and Price 1963; Price 1967), but this eighth pair of marginal hooks is very small and, more often than not, not considered to be of taxonomic value in species descriptions. In Dactylogyrus a simple transverse bar supports the bases of the anchors, with a vestigial ventral bar either present or absent (Price 1967). Four eyespots are usually present and the copulatory complex (as is the case with Dogielius) consists of a tubular penis and associated accessory piece.

2.4.6. Dogielius Bychowsky, 1936

This genus is very closely related to the cosmopolitan genus Dactylogyrus (Price and Yurkiewicz 1968) as was described above. In Dogielius the anchors and bars are located ventrally while the same structures are located dorsally in Dactylogyrus. As in Dactylogyrus the bases of the anchors are supported by a simple haptoral bar. Furthermore, there is a characteristic re-curving near the distal aspect of the anchors of Dogielius (Price and Yurkiewicz 1968).

2.4.7. Diplozoon von Nordmann, 1832 and Paradiplozoon Akhmerov, 1974

Members of the Diplozoidea are unique amongst monogenean parasites with regard to reproduction physiology (Schmidt and Roberts 1977). Juveniles (called diporpa) grow independently and shall die if it does not meet another juvenile. When they do make contact, however, they attach to each other and fuse permanently (in the shape of an “X”) to stimulate sexual maturation (Bush et al. 2001). Taxonomy of these two genera falls outside the scope of this project (dealt with in a separate project) and only infection statistics for the respective genera shall be reported on.

2.4.8. Quadriacanthus Paperna, 1961

Members of this genus possess 14 haptoral hooks and two pairs of anchors, the bases of each pair joined by a dissimilar transverse bar (Price 1967). The latter consists of a solid base to which narrower appendages are attached (Price 1967). The ventral bar is V-shaped while the dorsal bar may be either T or Y-shaped (Tripathi, Agrawal and Pandey 2007). Two accessory plates are attached to the solid, single root of each anchor (Price 1967). “Eyes” may be present or absent, with granules usually scattered in the cephalic area (anterior trunk) (Tripathi et al. 2007).

2.4.9. Structures used for species descriptions / identification

As parasites, adult helminths require secure attachment (Shinn, Bron, Sommerville and Gibson 2003). As ectoparasites of fish face the danger of being swept off the body of their host by water currents, this is particularly important (Halton 2004). The proverbial “tools of the attachment trade” are usually hooks or clamps, often exquisitely “designed” to fit the appropriate attachment site (e.g. gills or skin) on a sucker-like structure (opisthaptor or haptor) (Halton 2004). These sclerotized attachment structures, as well as other sclerotized structures such as the male copulatory organ and vagina, have traditionally been used for species identification (e.g. Hanek, Molnar and Fernando 1975; Mo and Appleby 1990; Shinn, Gibson and Sommerville 1993).

Pouyaud, Desmarais, Deveney and Pariselle (2006) state that the use of morphology of the haptoral sclerites is more suitable to infer phylogenetic relationships than genitalia morphology.

They continue by saying that the latter seems to be more useful to resolve species- level identifications, presumably because of its faster rate of change. Jarkovský, Morand, Šimková and Gelnar (2004) concluded that specialist parasites exhibit more similarity in attachment apparatus because of specialisation to the same host. Furthermore they found that species similarity in copulatory organs within infra- communities tend to exhibit a random pattern, with the attachment apparatus being less variable than the copulatory organs (which may be due to reproductive isolation).

As many monogeneans have elongated bodies, capable of great extension and contraction, total length and shape of the body are of limited taxonomic significance (Khalil and Mashego 1998).

Chaetotaxy (mapping of argentophilic surface sensory structures) also shows potential as a reliable and simple method to distinguish between species of Gyrodactylus (e.g. Shinn, Gibson and Sommerville 1997, 1998).

2.5. Morphological variability and phylogeny – a challenging interpretation

The challenge of quantifying morphometric variability within a described species effectively, yet allow differentiation between closely related and morphologically very similar species, remain a tremendous challenge (also see discussions on morphometric versus molecular techniques, section 2.6).

Dmitrieva, Gerasev and Pron’kina (2007), for example, studied the morphometric variability of Ligophorus llewellyni Dmitrieva, Gerasev and Pron’kina 2007 (a monogenean) and its most similar congeners from the Black Sea. Using a principal component analysis approach the authors examined 30 morphometric characters. They subsequently identified 22 that were most useful as diagnostic criteria permitting differentiation between morphologically similar species of Ligophorus Euzet and Suriano, 1977.

The question “but what factors give rise to morphometric variation?” inevitably arises. Size differences of sclerotized parts in specimens from the same species, collected from different localities, indicate that geographical variation does exist (Huyse, Malmberg and Volckaert 2004). To determine whether this is a result of genotype / historical phylogeny or due to shorter term abiotic or host effects, however, is more difficult to determine unambiguously.

For example, temperature may also affect the size of parasites and sclerotized attachment structures (particularly that of Gyrodactylus spp., e.g. Appleby 1996).

2.6. Species identification, description and phylogeny: Morphometric vs. molecular approaches

The useful phylogenetic application of morphological data have been reduced by the fact that different parasites face similar selection pressures, which in turn have led to abundant convergent evolution (Perkins et al. 2010). Huyse and Volckaert (2002) also state that morphological parameters may have a different mode of evolution in different species groups. As a result morphometric differences (e.g. of haptoral hard parts) of two morphologically similar species could be interpreted as intraspecific variation (Huyse and Malmberg 2004).

Progress in sequencing technology is thought to make molecular data a more rapid, efficient and cost-effective way to produce robust phylogenetic hypotheses (Perkins et al. 2010) or simply facilitate parasite identification (e.g. Santos, Sasal, Vernau and Lenfant 2006). Furthermore, traditional morphology-based phylogeny could only rely on calibration by fossil records, while the concept of a “molecular clock” in molecular phylogenetics provides a novel opportunity of dating evolutionary events (Hypša 2006).

This, however, does not imply that morphometric approaches are redundant. The studies by McHugh, Shinn and Kay (2000) and Shinn, des Clers, Gibson and Sommerville (1996) (both on Gyrodactylus spp.) demonstrate exactly how relevant morphometric approaches still are. Perkins et al. (2010) state that while morphology may have limited applications in phylogenetics, it is vital to (1) distinguish the plethora of monogenean species already described and (2) to provide detailed descriptions of undescribed taxa.

These two approaches should thus complement each other, as is indeed exemplified by studies on Gyrodactylus spp. (e.g. Huyse et al. 2004). Gyrodactylus salaris, for example, are morphologically similar to Gyrodactylus thymalli Žitňan 1960, yet the latter pose no threat to salmonid fisheries (Collins et al. 2004). Furthermore all DNA sequences obtained from the ribosomal array are virtually identical (Collins et al. 2004).

In such cases the application of molecular tests (including examination of more useful molecular regions / markers, e.g. Collins et al. (2004) used in conjunction with morphometric analysis is invaluable.

The study by Collins et al. (2004) demonstrates the importance of choosing the correct molecular regions or genes for molecular analysis. While some DNA regions may be almost identical between species, the presence of different isotypes of a gene within the same species also translates into more extensive analysis before full characterization of a species can be achieved. Hypša (2006) also state that morphological changes connected to the parasitic life cycle tend to obscure phylogenetic affiliations in some instances, making utilization of molecular characters the only solution in such cases. The author, however, warns that factors such as ambiguity or scarcity of phylogenetic information in sequences employed, intricacy of gene relationships at low phylogenetic levels and a lack of criteria when deciding among several competing co-evolutionary scenarios, may make molecular phylogenetics based on tree inference from primary nucleotide sequences insufficient (or even improper).

Both morphometric and molecular approaches thus appear to be fraud with potential errors and pitfalls, making a combined approach more suitable. However, comparative results obtained using morphological and molecular traits respectively often do not concur (e.g. Justine 1998; Huyse, Audenaert and Volckaert 2003), clouding the issue even further. Luckily the purpose of this section was not to diffuse this debate or solve the issue, but merely to make the reader aware of the various arguments!

Blažek, Bagge and Valtonen (2008a) state that thousands of worms are often handled in ecological studies, making molecular identification of each parasite individual impractical in such cases. The current study is a point in case. For the purposes of this study all monogeneans were described and identified according to standard morphological procedures based only on sclerotized structures.

2.7. Host specificity

Poulin and Keeney (2007) consider host specificity to be the most fundamental property of parasites.

They argue that it is also of general interest to parasitologists, as it not only appears to be a key determinant of the probability of extinction, but also reflects the ability of a parasite to colonize new host species as and when the opportunity arises. Barger and Esch (2002) also argue that host specificity does not act simply through the accumulation of sites at which a suitable piscine host is present. It rather mediates the probability, given the presence of one or more suitable fish hosts, that a population of parasites persist at that particular site.

Despite the apparent importance of host specificity, the mechanisms involved in host specificity are poorly understood but may include mechanical, behavioural and chemical factors that affect parasite attraction, attachment, feeding, reproduction and ultimately host response (Buchmann, Madsen and Dalgaard 2004). Of these, chemical factors appear to be more important than abiotic physical factors such as water current (Buchmann et al. 2004). More specifically, specific attachment and establishment of gyrodactylids to the appropriate fish seems to be mediated by a host factor possibly related to mucous and epithelial cells (Buchmann et al. 2004).

Traditionally host specificity has simply been quantified by the list of host species actually used by a parasite in nature (Poulin and Keeney 2007). Bakke et al. (2002), however, state that gyrodactylids should not simply be classified as specialists or generalists purely in terms of host identity. They believe that other features (e.g. stability of the host resource) also influence specialism in parasites. As a result recent indices attempt to take into account evolutionary and ecological differences among the host species used by the parasite (Poulin and Keeney 2007).

Pouyaud et al. (2006), for example, inferred that the physiological traits of hosts are insufficient to entirely explain parasite host-specificity. Bush et al. (2001) state that phylogeny must be considered first when evaluating what factors may limit host range. These authors speculate (as inferred from evolutionary ecology theory) that two reasons why parasites do not specialize are: 1) it possibly and quite simply does not come into contact with other hosts and / or 2) colonization of new hosts would be maladaptive to the parasite. The scope and nature of the current study shall, however, restrict discussions to the traditional definition (i.e. list of host species actually used by a parasite in nature) in chapters and discussions that are to follow.

Blažek et al. (2008a) warn that monogeneans may exhibit more generalized host preferences under conditions suitable to infrapopulation growth. Kearn (1994) also believes that host switching may be more common in monogeneans that spread by direct transfer. King and Cable (2007) for example, experimentally demonstrated that G. turnbulli is not a strict host specialist. Poulin and Keeney (2007) warn that genetic studies revealed complexes of cryptic species in many taxa of parasites. As a result many “generalist” species may in fact be sets of highly host-specific species that could not be distinguished on morphological grounds. This statement was confirmed by Freeman and Ogawa (2010) with specific reference to udonellids (a group of marine monogeneans).

The situation remains complex: the usefulness of molecular studies is sometimes overstated whilst experimental studies (as was demanded by King and Cable 2007) are often performed outside an ecological context (i.e. in an unnatural context) (Poulin and Keeney 2007). While neither of these two approaches (i.e. molecular or experimental) is reflected in this thesis, deductions made from observations do allow for some speculation and identification of future research needs.

2.8. Site specificity

Once a parasite encounters a host it has two options. Firstly, it could select where it wants to attach (or settle and develop where it is released / make first contact with the host) (e.g. Blažek and Gelnar 2006). Secondly, it could migrate to specific sites often far removed from where it attached initially (e.g. Cone and Cusack 1989; Buchmann and Uldal 1997).

The extend of site specificity may also be influenced by the presence of other parasite species (i.e. bispecific infection conditions, e.g. Baker, Pante and de Buron 2005).

Site specificity often transcends host specificity (i.e. a given parasite may be found in a wide spectrum of hosts but always in the same organ or even site within that organ) (Bush et al. 2001).

But why would parasites be site specific? Bush et al. (2001) speculate that being adapted to a specific site may possibly increase the fitness of the parasite in that particular site (i.e. site specificity should be adaptive).

Site specificity may thus well be related to aspects such as reproduction, feeding and withstanding differential ventilating currents (Buchmann and Bresciani 1998). Spatial distribution studies on other parasites (e.g. larval stages of mussels) have shown host behaviour and habitat use, as well as respiratory current, to be more important than an active choice of attachment site (Blažek and Gelnar 2006). In some dactylogyrids motility of infective stages (oncomiracidia) may thus potentially be overridden by other (abiotic) factors such as water current or water flow dynamics over the gills (e.g. Paling 1968), also precluding an active choice for a preferred site. Baker et al. (2005) have indeed shown that both neutral (i.e. random distribution through, for e.g. water flow dynamics) and negative (i.e. competition) interactions could play a role in determining parasite distribution on the gills.

Gyrodactylid parasite occurrence has, however, been correlated to mucous cell density, indicating that mucous cell contents play a decisive role in active gyrodactylid site selection (Buchmann and Bresciani 1998). Increased mucous production may interfere with further infection and remove parasites through sloughing (Jones 2001). As a result some monogenean ectoparasites apparently reduce mucous cell density in host skin which may be an attempt to minimize these inhibitory effects (Jones 2001).

2.9. Host effects on infection parameters

Host-related factors may include host species and host biology / ecology (e.g. Guégan, Lambert, Lévêque, Combes and Euzet 1992), host age and / or host size (e.g. Cable and van Oosterhout 2007), host diet / feeding habits (Rohde and Rohde 2005), site in the host and the host’s immune response (Bush et al. 2001).

Host species biology, behaviour and habitat: Given the discussion in section 2.7 (i.e. a characteristically high degree of host specificity within the Monogenea), the importance of host species is self-explanatory. Bush et al. (2001), however, also warns that even within a single host population not all hosts are “created equal”. Different host species do exhibit more pronounced differences in behavioural, reproductive and feeding biology. Demersal fish hosts that form large shoals and are in continuous contact with bottom sediment will for example allow gyrodactylids to benefit from a limited, 2-dimensional spatial distribution (Boeger, Kritsky, Pie and Engers 2005).

Shoaling behaviour will obviously also facilitate direct transmission between hosts for gyrodactylids (Boeger et al. 2005), yet the oncomiracidia of dactylogyrids will most certainly also benefit from host shoaling behaviour. In fact, larvae of particular parasite species often react only to specific hatching factors released by particular host species. In other words the parasite hatching rhythms are adapted to the host’s behaviour (Rohde and Rohde 2005).

In streams dispersal of parasites by stream fish depends on factors such as stream structure and resultant home ranges of the piscine host (Barger and Esch 2002). The authors argue that dispersal might be more important to parasite community structure in streams with a low degree of heterogeneity. Furthermore, as distance between sampling sites relates to the probability that fish at different sampling sites recruit parasites from different species pools, the relationship between proximity of sites and community similarity is largely predictive (Barger and Esch 2001).

Host diet: As monogeneans have a direct life cycle and do not make use of an intermediate host, the role of predation in parasite transmission should be negligible. Gyrodactylus salaris parasites drifting in the water column were shown to have been transferred to salmon, with potential transfer through predation being suggested (Bakke et al. 2002).

Host age: Longevity of pelagic fish hosts are thought to be one of the factors that determine parasite richness (Bush et al. 2001). This makes inherent sense: the longer a fish lives, the more opportunity for infection with parasites shall present itself. In a study by Cable and van Oosterhout (2007), larger fish indeed carried the highest parasite loads and experienced the highest mortality rates.

On the other hand, physiological changes over time as the host matures (which may include development of an immunological response against certain parasites) may confound any general trends.

Apart from host immunological effects, abiotic variables may also mask effects. Hodneland and Nilsen (1994) state that it is unclear whether water temperature or host age / size was most important in determining infrapopulation size of Gyrodactylus pterygialis Bychowsky and Poljansky, 1953.

Host size: The “species-area relationship” dictates that the number of species encountered increases systematically in samples from larger areas, but that the rate of increase in the number of species encountered decreases with progressively larger areas (i.e. after reaching a “plateau” in species richness the number of species remains constant despite an increase in area sampled) (Bush et al. 2001). Bush et al. (2001) continues by saying that the gills of fish represent an ideal system for examining such relationships. Fish length can also be used as an indicator of gill size. Some studies do indeed demonstrate a positive correlation between host body size and parasite species richness (e.g. Guégan and Hugueny 1994; Dávidová, Ondračková, Jurajda and Gelnar 2008). However, negative correlations between intensity of infection and fish size have also been reported for monogeneans (e.g. Cusack 1986). Neutral correlations have also been recorded with regard to fish parasites (e.g. Ward, Hoare, Couzin, Broom and Krause 2002 found no relationship between trematode abundance / prevalence and body length). The larger a fish is, the older it should be. This observation then obviously clashes with the “host age hypothesis” mentioned earlier. The same applies to monogeneans found on the body surface. Cusack (1986), for example, found a negative correlation between fish size and intensity of infection with Gyrodactylus colemanensis Mizelle and Kritsky, 1967. Bush et al. (2001) conclude that area (in terms of host and gill size but also geographical range of the host) is not predominant in determining species richness. The effect of area is probably overridden by factors such as evolutionary time, richness of the group, phylogeny and historical processes (Bush et al. 2001).

Site in the host: The majority of monogeneans reside either on the gills or on the skin of fish (though exceptions do exist). Exposure of naïve sticklebacks to detached Gyrodactylus sp. demonstrated that, in addition to direct attachment to the fins, parasites may attach to the head region and even inside the mouth (i.e. drawn into the buccal cavity of the fish as a result of respiration) (Grano-Maldonado 2009). The latter parasites, if not completely ingested, can temporarily attach to the lining of the mouth or pharynx. From there they may migrate to preferred sites on the skin (Grano-Maldonado 2009). Oncomiracidia of dactylogyrids either actively attach to the skin of the host fish and then migrate to the gills, or become attached when washed with swallowed water through the gills (Paperna 1996).

Immune response: Due to increasing economic losses in aquaculture and fisheries, an interest in host defence mechanisms has developed (Alvarez-Pellitero 2008). A typical host response against a monogenean gill parasite may include the appearance of lymphocytes, mucoid secretions and hyperplasia of the tissue at the attachment site (Arafa et al. 2009). The increased mucous production may result in sloughing off parasites together with shed mucous (Lester 1972). Buchmann (1999), proposing a model for skin immune mechanisms against monogeneans in fish, also identified mucous cells, epithelial cells and leucocytes as important cellular components of the model. In short cytokines are released (following injury to epithelial cells) that both attracts macrophages and neutrophils and affect mucous cell secretions. Furthermore leukotrienes are thought to be involved in inflammatory reactions with production of humoral substances involved in subsequent anti- parasitic response (Buchmann 1999). Buchmann and Uldal (1997) actually demonstrated that fish with a high density of mucous cells had the lowest susceptibility to parasites. Lindenstrøm, Secombes and Buchmann (2004) also demonstrated constitutive expression of immune relevant genes in the skin of rainbow trout. Some monogeneans may migrate to areas where this skin immune response is less severe (e.g. Gyrodactylus derjavini Mikailov, 1975 avoid skin areas by migrating to the cornea) (Sitjá-Bobadilla 2008). Others (e.g. Pseudactylogyrus bini (Kikuchi, 1929)) actually exploit the host immune reaction, more specifically the occurrence of hyperplasia, to improve attachment by imbedding in host tissue (Sitjá- Bobadilla 2008). Some data on the acquisition of acquired protection against monogeneans is available (Alvarez-Pellitero 2008), suggesting that physiological changes related to host age and / or associated temporal differences in parasite exposure may indeed affect host infection statistics.

2.10. Effect of abiotic and environmental variables on infection parameters

A number of abiotic and environmental variables continuously restrict or encourage the growth, development and transmission of parasites (Hassan 2008). These may include temperature, water quality, mechanical barriers, food supply (Hassan 2008), latitude, altitude, salinity, depth, light intensity, frequency and intensity of physical disturbances (Bush et al. 2001) and currents (Paperna 1996).

2.10.1. Temperature

Water temperature has been identified by several authors as a critical environmental parameter for parasites of fishes as it affects parasite survival, growth and timing of transmission, distribution and host hormone cycles (Hassan 2008). It can affect the size of gyrodactylid parasites and their sclerotized attachment structures (e.g. Appleby 1996) as well as their reproduction rates (e.g. Blažek et al. 2008a). Seasonal trends in monogenean numbers correlate well with temperature, probably due to the fact that incubation time is temperature dependent (Paperna 1996). Blažek, Jarkovský, Koubková and Gelnar (2008b) found that gyrodactylids peaked in spring (water temperature above 6°C) and Dactylogyrus cryptomeres Bychowsky, 1943 in summer (water temperature above 14°C). Dactylogyrids thus appear to be more thermophilous, but this may obviously differ between genera and species. Bhuiyan, Akther and Musa (2007), examining the composite parasite population (i.e. all parasites encountered including monogeneans) of Labeo rohita (Hamilton, 1822), also found the maximum infection during the pre-winter period. Lamková, Šimková, Palíková, Jurajda and Lojek (2007), however, warn that diverse genera of the monogenean parasite group respond differently to water temperature changes. Dactylogyrus spp. eggs, for example, hatch within two to six days at temperatures of 20 to 28°C (Paperna 1996). Temperature may, however, not only influence abundance of individual species, but also community structure (i.e. species composition) as a whole (e.g. Šimková, Sasal, Kadlec and Gelnar 2001).

Temperature also affects fish physiology and immunology (Lamková et al. 2007), so changes in host immune response may also play a role. Le Roux, Avenant- Oldewage and van der Walt (2011), for example, expected that most parasites would occur in winter as it is generally accepted that fish’s immunity is compromised during this period. The authors confirmed this during the sampling period (14 monthly surveys), but also demonstrated that sample size may greatly influence mean intensity of infection across a sampled host population. They warn that this could mask seasonal effects.

2.10.2. Chemical characteristics

Salinity tolerance has been shown to influence the occurrence of monogenean gill parasites (e.g. Baker et al. 2005).

Gyrodactylus spp. on salmonids are for example stenohaline, resulting in unsuitable hosts being able to support parasite population growth in moderately saline water bodies whereas the same parasite would be killed in freshwater (Bakke et al. 2002). Toxicants and other matter that can affect monogenean distribution include eutrophication, humic substances content, hypoxia, acidification (low pH), aqueous aluminium (Bakke et al. 2002) and waterborne zinc (Gheorghiu et al. 2007). Paperna (1996) states that oxygen levels may also affect reproductive rates and species richness (e.g. adverse living conditions may accelerate oviposition of undeveloped eggs). Madanire-Moyo and Barson (2010) also found that fluctuation in parasite prevalence and diversity could be attributed to changes in levels of nutrients, dissolved oxygen and conductivity. Furthermore they speculated that the monogenean Macrogyrodactylus clarii Gussev, 1961 (collected from C. gariepinus) may in fact be sensitive to high levels of conductivity, nutrients and hypoxia.

2.10.3. Other factors

The number of species of monogeneans and digeneans tend to increase towards the tropics, a trend that appears to be correlated (at least to some extend) with the increase in the number of available host species (Bush et al. 2001). This is not surprising, as most monogeneans are restricted to a single fish genus or species (Rohde 1979). Apart from this “geographical gradient”, richness of free-living organisms generally decreases with increasing depth (an example of a macrohabitat variable where a clear distinction between macrohabitat and geographical range is not possible, as explained by Rohde (1979). Bush et al. (2001) explained that this is presumably because of a decreasing temperature and light, increasing pressure and lack of seasonal change. They continue by saying that, as a result, the overall parasite fauna of benthic fishes in deep water is less diverse than in shallow water. Apart from depth, water levels may also play a role. Kadlec et al. (2003b), for example, demonstrated that flood conditions can affect parasite community species composition on some fish hosts.

Another factor that influences parasite distribution and richness is schooling behaviour of especially mid-water fishes (Bush et al. 2001). In mesopelagic fishes larval parasites common to pelagic prey are often encountered.

Adult helminths (such as monogeneans) are rare and larval cestodes and especially larval nematodes are dominant (probably due to low host specificity) (Bush et al. 2001).

2.11. Notes on the study of parasite communities

A number of criteria, aimed at identifying parasites sharing common attributes, often used to try and create subsets of parasite species within the community milieu are discussed by Bush et al. (2001).

The most frequently used application is that of a host generalist as opposed to a host specialist. This obviously relates to host specificity discussed earlier. Though not applicable to monogeneans, Bush et al. (2001) warned that one must consider the life cycle and particularly each host species (i.e. intermediate or final) separately. Their second warning, however, is applicable to monogeneans: to unambiguously identify a parasite as being a host generalist or host specialist, information is required about the occurrence of the parasite in all potential hosts. This statement is illustrated by the fact that parasites may also exhibit “ecological host specificity”, i.e. they may occur on hosts sharing ecological requirements irrespective of taxonomic relationship (Rohde 1979). This statement once again underlines the importance of gathering baseline data on parasite distribution and infection statistics.

Other criteria used to describe parasite communities, relate to the degree of parasite species host and site specificity as the latter is considered to be a fundamental property of parasitic organisms (Poulin and Keeney 2007). These aspects have been described in sections 2.7 and 2.8. Within a community context this can also be viewed in terms of niche restriction (i.e. use of different niches by different parasite species).

Bush et al. (2001) discussed a number of categories: niche restriction by descent (i.e. phylogeny), niche restriction by adaptation (e.g. morphological adaptations), niche restriction due to predation (e.g. “cleaning symbioses”) and niche restriction due to competition. The most important parameters or niche dimensions, as defined by Rohde (1979), are host species, geographical range, macrohabitat, microhabitat, host gender, season and hyperparasitism.

Bush et al. (2001), however, concluded by saying that many mechanisms (some possibly yet unidentified) may influence the determination of infracommunity patterns.

The notion of core and satellite species (so called “core / satellite species dichotomy”) is often used in community descriptions (e.g. Hanski 1982; Ulrich and Zalewski 2006). According to Bush et al. (2001) the use thereof is somewhat controversial as other mechanisms may produce similar patterns. Mehranvar and Jackson (2001) for example also warned that such models should be tested with various taxonomic groups and at different, carefully defined spatial scales before formally testing the predictions of the models. Bush et al. (2001) explain the implication of this dichotomy when applied to parasite communities: If an inverse relationship exists between the abundance of a parasite species and the probability of extinction, or there exists a random variation in transmission or extinction (or both), then each parasite in a community will tend towards one of two states. Core species colonize most host individuals in high numbers (i.e. regionally common and locally abundant). In contrast satellite species colonize few host individuals and in low numbers (i.e. regionally uncommon and locally rare). Species considered a satellite species with respect to abundance may be a core species with respect to another variable, such as biomass. For this reason one cannot simply classify core and satellite species based simply on high or low prevalence respectively.

Another criterion that may be used to classify constituents of communities is the notion of “guilds”, i.e. partitioning data into what may be more meaningful subsets based on functional similarity and not taxonomic classifications (Pedersen and Fenton 2007).

Yet another distinction that could be made relates to how parasites colonize and maintain themselves in a host: allogenic species mature in birds or mammals (or both) while autogenic parasites mature in fishes (Bush et al. 2001). When only considering the monogenean parasite community, such a distinction is obviously not applicable.

Species richness or abundance is another obvious criterion that should be investigated (e.g. Poulin and Justine 2008).

Screens determine which (and hence also the numbers of) species can be found in a particular component community. Screens may reflect biotic features such as phylogeny, interactions between species, host immunology and host food habits, or abiotic features such as acid rain, episodic droughts or extinctions (Bush et al. 2001). A variety of measures (e.g. Rózsa, Reiczigel and Majoros 2000) and diversity indices (e.g. Williams, Witkowski and Balkwill 2005) can be applied in an attempt to quantify observed species diversity. These include Simpson’s Index, Shannon-Wiener Index and Brillouin’s Index (Peet 1974; Williams et al. 2005). Species richness has also been used as indicator of water quality (e.g. Galli, Crosa, Mariniello, Ortis and D’Amelio 2001). Dušek, Gelnar and Šebelová (1998) reported (at the component community level) a decrease in the number of parasite species with a more equal distribution of their abundances in a polluted site compared with a control site.

2.12. Review of monogenean research in southern Africa

Paperna (e.g. Paperna 1973; Paperna 1996) made invaluable contributions to descriptions of Monogenea from inland water fish in Africa (e.g. Uganda, Ghana, Tanzania and Kenya). Monogenean parasites of fish have also been collected from African countries such as Botswana (Christison, Shinn and van As 2005; Modise, King, Baker and Van As 2006), Ivory Coast (N’Douba, Lambert and Euzet 1999), Burkina-Faso, Cameroon, Guinea, Niger, Senegal (Pariselle, Bilong Bilong and Euzet 2003; Přikrylová and Gelnar 2008), Egypt, Gabon, Ethiopia, Zaïre, Zimbabwe, Sudan, Gambia, Mali, Togo, Morocco, Sierra Leone, Tunisia, Benin, Malawi, Congo, Zambia and Chad (as summarized in Khalil and Polling 1997).

In comparison, published papers dealing with other parasite fauna from fish in South Africa often refers to the presence of some monogenean parasites (e.g. Mouton et al. 2001), but rarely are dedicated to representatives of this group (e.g. Luus-Powell, Mashego and Khalil 2003). Ecological applications for representatives from the group as possible taxonomic indicators (Lambert and El Gharbi 1995) have, however, also been examined in South Africa (Milne, Walsh, Avenant-Oldewage and van der Bank 2006). Furthermore some representatives of the group also negatively impacts fisheries and aquaculture on a global scale (e.g. Johnsen 2006).

The importance of the group in the South African aquaculture industry has also been recognized (e.g. Luus-Powell, Theron and Hattingh 2006; García-Vásquez, Hansen, Christison, Rubio-Godoy, Bron and Shinn 2010; García-Vásquez, Hansen, Christison, Bron and Shinn 2011; Vaughan and Chisholm 2010a; Vaughan and Chisholm 2010b).

From a South African fisheries point of view it is clear that this parasitic group already has an economic impact, yet the importance of the group is presently not reflected in the amount of research dedicated to them.

But why do an apparent lack of baseline data exist in South Africa? Monogeneans do not pose a zoonotic problem for humans and under natural conditions most of them are not pathogenic (Bush et al. 2001). As a result very little is known about the biology of most species. Relative sampling intensity also remains a huge problem when evaluating aspects such as host specificity (Bakke et al. 2002). As mentioned previously many routine parasite investigations have been performed in South African water systems. Monogenean parasites are mentioned in very few of them. The parasites are very small and often more elaborate and time-consuming methods are required for effective monogenean parasite discovery, recovery, fixing and identification compared to standard / routine methodology.

A chronological summary of published articles (i.e. published presentation and poster abstracts excluded) dealing with monogenean research in southern Africa is summarized in Table 2-2.

2.13. Problem statement

Fletcher and Whittington (1998) made an attempt to conservatively predict the biodiversity of the monogenean fauna of Australia’s 180 species of freshwater fishes. Their estimates indicate that only 5% of an estimated 500 species of Monogenea in Australia have been described at that point. Given the lack of basic research on monogeneans in South Africa with regard to previously unexamined (for monogenean parasites) freshwater host species and localities, the situation in South Africa may be very similar. Estimates for marine fishes in China reflect that between 3000 and 9000 monogenean parasites still need to be described (Jianying, Tingbao, Lin and Xuejuan 2003). Once again the situation in South Africa may be similar.

Table 2-2: Summary of published papers dealing with monogeneans in southern Africa.

Author Locality Content and comments (Date of publication)

Price and Yurkiewicz Bubi River, Zimbabwe Recorded Dogielius junorstrema Price and Yurkiewicz, 1968 (1968) (formerly Rhodesia) from Labeo ruddi Boulenger, 1907

Described two species of Dactylogyrus Diesing, 1850: Dactylogyrus jubbstrema Price, Korach and McPott, 1969 Price, Korach and Kwa-Zulu Natal, South from the gills of Glossogobius giuris (Hamilton, 1822) and McPott (1969) Africa Dactylogyrus pienaari Price, Korach and McPott, 1969 from the gills of Labeo rosae Steindachner, 1894.

Recorded two species of Dactylogyrus: Dactylogyrus myersi Price, McClellan, Price and Géry, 1968 from the gills of Barbus trimaculatus Lydenburg, South Druckenmiller and Peters, 1952 and Dactylogyrus varicorhini Bychowsky, 1958 Africa Jacobs (1969) from the gills of Labeobarbus kimberleyensis (Gilchrist and Thompson, 1913).

Prudhoe and Hussey Central Transvaal, Described Gyrodactylus transvaalensis Prudhoe and (1977) South Africa Hussey, 1977 from Clarias gariepinus (Burchell, 1822).

Described three new species of Dactylogyrus (Dactylogyrus teresae Mashego, 1983, Dactylogyrus enidae Mashego, 1983 and Dactylogyrus dominici Mashego, 1983) from Mashego (1983) Limpopo, South Africa Barbus paludinosus Peters, 1852 and Barbus neefi Greenwood, 1962; Recorded new host (Barbus trimaculatus Peters, 1952 and Labeobarbus marequensis (Smith, 1841)) records for a number of other Dactylogyrus species.

Reported on monogeneans of three Siluriform fishes (C. gariepinus, Synodontis zambezensis Peters, 1852 and Schilbe intermedius Rüppell, 1832); Parasites recovered included Quadriacanthus bagrae Paperna, 1979, Quadriacanthus aegypticus El-Naggar and Serag, 1986, Douëllou and Chishawa Lake Kariba, Quadriacanthus numidus Kritsky and Kulo, 1988, (1995) Zimbabwe Macrogyrodactylus congolensis karibae Douellou and Chishawa, 1995, Synodontella zambezensis Douellou and Chishawa, 1995 , Schilbetrema undinula Kritsky and Kulo, 1992 and Schilbetrema quadricornis Paperna and Thurston, 1968.

Compiled a check list of the helminth parasites recorded Khalil and Polling (1997) Southern Africa from African freshwater fishes. This list also includes monogenean representatives from southern Africa.

Reviewed the African monogenean genus Macrogyrodactylus Malmberg, 1957, which also constituted Middle Letaba and Khalil and Mashego the first record of species of this genus (Macrogyrodactylus Mokgoma Matlala (1998) congolensis Prudhoe, 1957, Macrogyrodactylus clarii Dams, South Africa Gussev, 1961 and Macrogyrodactylus karibae Douellou and Chishawa, 1995).

Described a new genus (Mormyrogyrodactylus) and new species (Macrogyrodactylus gemini Luus-Powell, Mashego Luus-Powell, Mashego Northern Province, and Khalil, 2003) from Marcusenius macrolepidotus (Peters, and Khalil (2003) South Africa 1852). This was the fifth genus of the Gyrodactylidae to be described from Africa.

Table 2-2 (continued): Summary of published papers dealing with monogeneans in southern Africa.

Author Locality Content and comments (Date of publication)

Described Gyrodactylus thlapi Christison, Shinn and van As, 2005 from Pseudocrenilabrus philander philander (Weber, Christison, Shinn and Botswana (Okavango 1897). This brought the number of gyrodactylids described van As (2005) Delta) from the African continent to 18, as was reviewed by the authors in the same manuscript.

Reported the first confirmed incidence of Pseudodactylogyrus anguillae (Yin and Sproston, 1948) from Anguilla mossambica Peters, 1852. Their very thorough study included both morphological and molecular (DNA Christison and Baker Eastern Cape, South extraction, PCR and sequence analysis) analyses of (2007) Africa collected parasites. The authors mention the general lack of parasite data for this host species, supporting the notion that a lack of baseline data may exists for many other South African fish species.

Recorded three species of Macrogyrodactylus (M. clarii, M. Barson, Bray, Ollevier Save-Runde River karibae and M. congolensis) from C. gariepinus. The authors and Huyse (2008) floodplain, Zimbabwe provided morphological measurements and compared it to the original descriptions.

Examined monogenean parasites on freshwater fish hosts in. While representatives from a wide range of genera have been collected (e.g. Dactylogyrus, Dogielius Bychowsky, 1936, Quadriacanthus Paperna, 1961, Macrogyrodactylus, Olivier, Luus-Powell and Cichlidogyrus Paperna, 1960, Enterogyrus Paperna, 1963 Limpopo, South Africa Saayman (2009) and Scutogyrus Pariselle and Euzet, 1995), many could not be identified to species level. This demonstrates a lack of baseline data with regard to the identity of monogenean parasites on freshwater fish species endemic to South Africa.

Examined the diversity of metazoan parasites of C. gariepinus. They recovered M. clarii and demonstrated that Madanire-Moyo and Upper Manyame this parasite may be a useful indicator of water quality as it Barson (2010) catchment, Zimbabwe might be sensitive to high levels of conductivity, nutrients and hypoxia.

Performed a phylogenetic study on Macrogyrodactylus spp. One specimen collected from Zimbabwe displayed Zimbabwe Barson, Přikrylová, morphological features intermediate between (other samples also Vanhove and Huyse Macrogyrodactylus heterobranchii N'Douba and Lambert, collected from Senegal (2010) 1999 and M. clarii. Molecular analyses suggested a hybrid and Kenya) origin which hints at historical sympatry of the two host species.

Provided a checklist of the 85 species in the genus Cichlidogyrus, including hosts, localities and authors. They also commented on host specificity and distribution. They Le Roux and Avenant- South Africa (amongst conclude that additional studies on aspects such as Oldewage (2010b) other countries) morphology, ecology and pathological effects of members of this genus are required. This would include examination of other possibly infected, previously unexamined cichlid hosts in South Africa.

Table 2-2 (continued): Summary of published papers dealing with monogeneans in southern Africa.

Author Locality Content and comments (Date of publication)

Gyrodactylus spp. parasitising Oreochromis Günther, 1889 Garcìa-Vásquez, Stellenbosch, South were sampled from fourteen countries and compared with Hansen, Christison, Africa each other and type material of known Gyrodactylus Rubio-Godoy, Bron and (amongst other species (morphological and molecular analyses). Parasite Shinn (2010) countries) collected included a morphologically cryptic group from South Africa.

Vaughan and Chisholm Southern Cape coast, Described Neoheterocotyle robii Vaughan and Chisholm, 2010a South Africa 2010 from the gills of Rhinobatos annulatus (Müller and Henle, 1841).

Cape Town (Two Vaughan and Chisholm Described Heterocotyle tokoloshei Vaughan and Chisholm, Oceans Aquarium), 2010b 2010 from the gills of Dasyatis brevicaudata (Hutton, 1875) South Africa kept in captivity.

Described Gyrodactylus ulinganisus Garcìa-Vásquez, Hansen, Christison, Bron and Shinn, 2011 from Oreochromis mossambicus (Peters, 1852). Molecular Stellenbosch, South Garcìa-Vásquez, methods were employed to distinguish this monogenean Africa Hansen, Christison, Bron species from other closely related species. The authors (amongst other and Shinn (2011) warned that very little is known regarding host-specificity, countries) ecology and population dynamics of Gyrodactylus spp. on cichlid hosts and that further research is required. This would include additional baseline data on the monogenean fauna of other cichlids in South Africa.

Discussed aspects of the ecology of Cichlidogyrus philander Douellou, 1993 from P. philander philander. The authors’ Le Roux, Avenant- state: baseline data on parasite infections within natural Oldewage and van der Gauteng, South Africa water systems may serve as a reference point for Walt (2011) development of management strategies. This statement confirms the need for similar baseline studies on monogenean parasites from other endemic piscine hosts.

Reported on the population dynamics and spatial distribution of monogenean parasites on the gills of O. mossambicus. The aim of the study was to address the shortfalls in information concerning monogenean ecology on this host Madanire-Moyo, Matla, species. They continue by saying that such baseline data Olivier and Luus-Powell Limpopo, South Africa may be essential in avoiding catastrophic losses in intensive (2011) aquaculture. Once again this statement confirms the need for similar baseline studies on monogenean parasites from other endemic piscine hosts that show promise for future use in aquaculture (e.g. Labeo umbratus (Smith, 1841)).

Milne and Avenant- Vaal Dam, South Reported on a method to examine the internal sclerites of Oldewage (2006) Africa the attachment clamps through fluorescent detection.

Reported on seasonal growth of the attachment clamps of Milne and Avenant- Vaal Dam, South this parasite species using geometric morphometric Oldewage (2012) Africa methods.

From the previous section (i.e. based on publication output) it is obvious that research interest in South African monogeneans has already increased starting from the 1990’s. Many of these publications, however, still stress the fact that further research is required and that the current knowledge base is lacking. It is from this viewpoint that the following problem statement has been formulated:

“Advancement of research on the parasitic monogenea of freshwater fishes in South Africa is hampered by the lack of species composition and infection level baseline data”.

Objectives were already listed in Chapter 1 and will not be repeated here.

CHAPTER 3

3 - MATERIALS AND METHODS

3.1. Study site description

The study site was an area of the Vaal Dam (S 26 52.249, E 28 10.249) surrounding the University of Johannesburg Island (previously RAU Island), Vaal River system, Gauteng Province, South Africa.

3.1.1. River system and catchment description

The Vaal River (Figure 3-1) rises in the vicinity of Lake Chrissie near Breyton (Department of Water Affairs and Forestry 1993) on the western slopes of the Drakensberg escarpment (Braune and Rogers 1987). It flows in a west-south-west direction across the interior plateau (Braune and Rogers 1987) and joins the Orange River near Douglas after some 1200 km (Department of Water Affairs and Forestry 1993). The Orange River in turn spills into the Atlantic Ocean at Alexander Bay (Department of Water Affairs and Forestry 1993). The Vaal River has a catchment area of 192 000 km² which can be divided into four zones (Figure 3-2) on the basis of water quality problems (Braune and Rogers 1987). These are, in order of decreasing water quality, the Vaal Dam, the Vaal River Barrage, the Bloemhof Dam and the Douglas weir subcatchments (Figure 3-2) (Braune and Rogers 1987). Rainfall also decreases in this order, as, proceeding down the Vaal River from its headwater region, the climate becomes progressively more arid and warmer (Helgren 1979).

The Drakensberg area (subcatchment one) exhibits the highest rainfall (800 to 1 000 mm per annum) as well as the lowest evaporation (Braune and Rogers 1987). This is also the major catchment area. Rainfall decreases and evaporation increases steadily westward. The lower reaches of the river receive approximately 300 mm rain per annum and thus largely depend on the eastern catchments for water supply (Braune and Rogers 1987). As a result of the change in climate a change in vegetation also occurs.

The river is thus characterized by a diversity of landscapes that results in a diversity of scenic characteristics, fauna and flora life (Department of Water Affairs and Forestry 1993).

3.1.2. Vaal Dam

The Vaal Dam is the fourth largest storage reservoir in South Africa (Department of Water Affairs and Forestry 1993) with a surface area of 32,107 hectares and capacity of 2,535.5  106m3 (Braune and Rogers 1987). The dam supplies high quality water for power station requirements in the Vaal Dam subcatchment (Wepener, van Dyk, Bervoets, O’Brien, Covaci and Cloete 2011). The Vaal Dam catchment is important for future water supply in South Africa. It is estimated that annual runoff is approximately proportional to rainfall over the catchment raised to a power between four and five (Kriel 1992). Thus an increase of 10% in the annual rainfall would lead on average to an increase of about 50 % in annual runoff. This prompted suggestions concerning continuing feasibility studies on artificial rainfall stimulation (Kriel 1992). The area between the Vaal Dam and the Vaal River Barrage is subject to mining activities and municipal run-off, resulting in a sharp decrease in water quality and associated sustained pollution exposure and stress conditions (Wepener et al. 2011). Potential long-term threats to this important Vaal Dam catchment are pollution from diffuse agricultural sources, further industrial development and atmospheric pollution (Braune and Rogers 1987).

Map 1 Map 2 Map 3

3.2. Fish collection

Fish were only collected after obtaining the required permit and ethical clearance from the Ethics Committee of the University of Johannesburg.

3.2.1. Method of collection

Fish were collected using gill nets of varying mesh sizes (90, 110 and 130 mm stretched mess sizes respectively).

Once removed from the nets, fish were placed in a container containing circulating dam water and transported back to shore. Fish were subsequently placed in a canvas holding tank through which water was circulated using a water pump.

3.2.2. Species collected

The following fish species were collected and subsequently examined:

Common carp Cyprinus carpio Linnaeus, 1758 Grass carp Ctenopharyngodon idella (Valenciennes, 1844) Orange River Mudfish Labeo capensis (Smith, 1841) Moggel Labeo umbratus (Smith, 1841) Smallmouth yellowfish Labeobarbus aeneus (Burchell, 1822) Largemouth yellowfish Labeobarbus kimberleyensis (Gilchrist and Thompson, 1913) Sharptooth catfish Clarias gariepinus (Burchell, 1822) Largemouth bass Micropterus salmoides (Lacepède, 1802)

3.3. Host necropsy and parasite recovery

Fish showing distress from gill net injury were removed from the holding tank first and processed. Following that fish were randomly caught with a hand-held net and processed in no particular order (i.e. specimens belonging to the same species were not necessarily processed sequentially). As each fish was removed from the holding tank, it was allocated a numerical number (starting with the number one and then adding one for each subsequent fish).

3.3.1. Identification and taxonomic nomenclature of hosts examined

Fish were identified by research personnel during handling and necropsy as the particular site has been well sampled in the past. No specialized identification techniques (e.g. scale counts etc.) were performed.

It would, however, appear that some cross-breeding between L. aeneus and L. kimberleyensis are taking place, as a few specimens exhibited morphological characteristics that are intermediate between the species (most notably with reference to the position of the eye relative to the mouth). Such specimens were simply referred to as “L. kimberleyensis hybrid”. Please note that this has not been confirmed by molecular analyses and the taxonomic status (i.e. hybrid) of such fish should thus be viewed as preliminary.

Nomenclature (and characters used for identification) were according to Skelton (2001) and Fishbase (Froese and Pauly 2011).

The species name and / or common name were subsequently recorded next to the field identification number allocated (see section 3.3).

3.3.2. Host weight measurement

Immediately after being removed from the holding tank fish were weighed with a suitable scale. The weight for each individual fish was recorded in grams.

3.3.3. Preparation of mucous smears

Once weighed, approximately 3 to 6 mucous smears from the skin and fins were immediately prepared. A microscope slide was used to scrape slime from a few random areas on the sides of the body, adjacent to fins and from fins (particularly back, tail, pectoral and anal) themselves. This was then transferred to other slides as required (i.e. frequency of transfer depending on amount of mucous collected on the first slide). After spreading the mucous evenly over the slide, it was examined with the aid of a stereo microscope. Both weighing and mucous smear preparation was performed as speedily as possible to avoid unnecessary suffering. Mucous smear preparation prior to euthanasia was however critical to avoid blood-mixed smears that complicate parasite recovery.

3.3.4. Euthanasia

Fish were killed humanely by severing the spinal cord behind the head with a suitable instrument (i.e. kitchen scissors or sharp knife). Care was taken not to allow blood flow over or into the gill chambers to avoid clogging of gills with clotting blood.

3.3.5. Host length measurements and condition factor calculations

Once euthanized, fish were placed on an enclosed (on one end so the fish’s snout rested against the zero position) measuring board. This allowed accurate measurements to be taken (in centimetres) of the standard length, fork length (where applicable) and total length.

Fultons condition factor (K) was calculated as W/L3 (with W = weight in grams and L = total length in centimetres) with a scaling factor of 100 applied to bring the factor close to unity (Froese 2006; Nash, Valencia and Geffen 2006). The formula employed was thus: K = 100 x (W/L3)

3.3.6. Host necropsy procedure

After being measured, the fish was transferred to a stainless steel dissection trough. Using kitchen scissors, knives and standard dissection equipment the gills were carefully removed. Gills were separated into left and right hemispheres. Each hemisphere was placed in a separate petri dish containing water. Each dish contained a pencil-written label indicating the field identification number of the fish, as well as the hemisphere it held (i.e. “L” for left or “R” for right). As an example a typical label would read “56L”, indicating fish number 56 (to which the species identification, weight and length data has already been linked) and the left hemisphere of the gill set belonging to that fish. If the fish was not further processed in the field, the hemisphere (i.e. four gill arches from either the left or right side of the fish) together with the pencil-written label was placed in a glass bottle containing 70% ethanol. In species other than C. gariepinus (which possess an external genital papilla in males) the abdomen was also cut open to determine the gender of the fish (i.e. presence of the characteristically white testes indicated male gender).

3.3.7. Division of gills for subsequent examination

Gills placed in petri dishes could immediately be further processed in the field. However, as monogenean parasite recovery and subsequent mounting is cumbersome and time consuming; only a minority of samples collected could immediately be processed in the field.

Gills placed in 70% ethanol (together with the pencil-written label indicating fish field number and gill set hemisphere) in the field were transferred to a petri dish containing water in the laboratory. Gill arches were subsequently separated in sequence (i.e. one to four) (Figure 3-3). The sequence of the four gill arches on each hemisphere (i.e. left or right) could be easily deduced (i.e. gill arch 1 being proximally located in the fish, i.e. nearest to the operculum).

Subsequent division of each gill arch (Figure 3-3) was adapted from Blažek and Gelnar (2006). For each gill arch the following were identified and examined: (1) Three segments – dorsal, median and ventral; (2) both the anterior (“front” of gill facing water current) hemibranch and posterior (i.e. “back” of gill) hemibranch.

To a large extent, only the outer areas were examined due to the parasite removal method (see section 3.3.8) employed. For the same reason the gill was also not divided into distal, central and proximal areas.

3.3.8. Examination of gills

After placing the gill to be examined on a microscope slide in a second petri dish, the mucus and epithelial lining of the dorsal segment on the anterior hemibranch was scraped onto the slide with the aid of a second microscope slide.

The gill arch was again placed in the petri dish containing water, the scraping spread evenly over the slide and the slide examined using a stereo microscope. This process was repeated for each of the segment areas examined on both the anterior and posterior hemibranch's. In total six gill scrapings were thus examined per gill arch processed.

3.3.9. Parasite removal from gill scrapings and mucous smears

Parasites were removed with the aid of a stout eye lash hair (glued to a wooden match stick) and subsequently mounted (cover slip sealed with clear nail varnish) in either Malmberg’s solution (glycerine ammonium picrate or GAP) or glycerine jelly. Both glycerine jelly (e.g. Hargis 1953) and GAP (e.g. Wong, Brennan, Halton, Maule and Lim 2008) are routinely used as both fixing and mounting media in monogenean research.

3.4. Parasite identification and description

Recommendations made by Gussev (1979) were considered during examination and illustration of relevant structures reported on in this thesis.

3.4.1. Illustrations of sclerotized structures

Sclerotized hard parts (anchors, bars, marginal hooks and male copulatory organ consisting of a penis and accessory piece) were drawn with the aid of a Zeiss drawing tube (model number 47 46 20-9900) and Zeiss compound microscope (model number 4709506). Measurements were made directly from drawings.

3.4.2. Standard methodology for measuring sclerotized structures

Types of measurements required for species identification and description differ between monogenean genera.

In all cases measurement of haptoral sclerites and male copulatory organ (MCO) employed during this study was adopted (and at times adapted) from relevant published accounts. In the chapters that are to follow the methodology applicable to each individual genus or species are dealt with (though only with reference to other published papers without any illustrations in some cases).

To provide a clear visual overview, the various measurement methodologies employed for the respective genera examined are discussed below. Measurements were subsequently tabulated (using micrometer as unit in all cases) and used for species description / identification and comparison.

3.4.3. A note on terminology used with reference to sclerotized structures of different genera

For different genera different terms are often used to refer to similar structures. Even within the same genus different terms are often used interchangeably. Large hooks are often referred to as hook, anchor, hamulus or gripus. Small hooks are often referred to as marginal hooks, marginal hooklets or simply hooklets. Bars are often referred to as dorsal bar or ventral bar depending on orientation / position, or simply transverse bar if only one bar is present. An additional sclerite associated with these bars is often called accessory sclerite or cuneus. The male copulatory organ (MCO) consists of a copulatory tube (called a cirrus or penis) and an accessory piece.

To cause the least amount of confusion, terminology used in this thesis shall be standardized as follows: Anchors, marginal hooks, bar (dorsal or ventral depending on position), accessory sclerite, penis, accessory piece and male copulatory organ (with reference to the latter two structures combined) for species other than Gyrodactylus von Nordmann, 1832. For the latter (Chapter 9) the term hamulus shall be used with reference to the anchor.

3.4.3.1. Dactylogyrus Diesing, 1850 and Dogielius Bychowsky, 1936

Measurement of haptoral sclerites (Figure 3-4) was adopted from that suggested by Guégan and Lambert (1991) (i.e. overall anchor length, length of shaft, length of outer root, length of inner root, length of tip, width and length of dorsal bar) and Musilová, Řehulková and Gelnar (2009) (i.e. accessory piece total length and penis tube trace-length) (Figure 3-4). An additional measurement (anchor aperture) was adopted from Shinn, Harris, Cable, Bakke, Paladini and Bron (2010).

These standard measurements were used in subsequent chapters, in order for results obtained to be comparable to that obtained in other published studies.

Additional sclerites are associated with the roots of the anchors of Dactylogyrus lamellatus Achmerow, 1952 from grass carp (C. idella).

As the orientation of these sclerites vary greatly they were not measured and only standard anchor measurements were employed. Furthermore the male copulatory organ (MCO) bears a simple curved penis and an elaborate accessory piece structure.

The latter proved to be prone to manipulation during mounting and as a result orientation varied greatly. Following Paperna (1961), for ease of comparison, the dimension of the entire MCO was measured (i.e. measurement “j” in Figure 3-4 but representing the greatest length of the entire MCO for this particular parasite species).

During measurement of especially Dogielius sp. it was found that the degree of rotation in terms of anchor view can greatly affect measurements and will therefore also have an effect on repeatability and comparability. This aspect (and the methodology employed to standardise measurements during this study) is elucidated in Appendix A.

Measurements from another group (see section 3.4.2.3 – Gyrodactylus spp.) were also adapted and applied to Dactylogyrus spp. and Dogielius sp., to evaluate if these additional measurements would add any value to descriptions of species or morphological forms (also see Chapters 4 and 5). As this is, however, not pertinent to the remainder of this section, it is discussed in further detail in Appendix B.

3.4.3.2. Quadriacanthus Paperna, 1961

Measurement methodology (Figure 3-5) was adopted from N’Douba, Lambert and Euzet (1999) and El-Naggar and Serag (1986)

3.4.3.3. Gyrodactylus von Nordmann, 1832

Measurements for this group (Figure 3-6) were adopted from Přikrylová, Matějusová, Jarkovský and Gelnar (2008) for anchors and bars, as well as Christison, Shinn and van As (2005) for marginal hooks (as was suggested by Přikrylová, Blažek and Vanhove 2012).

Figure 3-5: An illustration depicting (for Quadriacanthus Paperna, 1961) measurement methodology employed as adopted from N’Douba, Lambert and Euzet (1999), El-Naggar and Serag (1986): A = Ventral bar component; B = Dorsal bar component; C = Dorsal accessory sclerite; D = Dorsal anchor; E = Marginal hook; F = Ventral accessory sclerite; G = Ventral anchor; H = Male copulatory organ (MCO); a = anchor total length; c = anchor base width; e = length of tip / point; f = length of ventral bar component; g = width of ventral bar component; h = marginal hook total length; i = penis tube trace length; j = accessory piece length; l = dorsal accessory sclerites width; m = dorsal accessory sclerites length; n = ventral accessory sclerites length; o = measurements of dorsal bar component length (median length); p = width of dorsal bar component; q = dorsal bar component base width; r = dorsal bar component median process length.

3.5. Calculation of infection statistics

See section 2.3.3 (Chapter 2) for a tabulated summary. Infection statistics were either calculated using the Microsoft Excel formula function or Quantitative Parasitology 3.0 software (Rozsa, Reiczigel and Majoros 2000).

3.6. Statistical analysis

3.6.1. Descriptive statistics

Arithmetic mean, standard deviation and range (minimum and maximum values) were calculated for (a) all measurement data from all species described to facilitate species descriptions; (b) other data (e.g. fish host body weight and length) where applicable (i.e. where such information needed to be summarized / described).

3.6.2. Grouping of measured variables / morphometric analyses to evaluate the separate species versus forms of the same species concept (Chapter 4)

For both Dactylogyrus spp. and Dogielius spp. the grouping of measured variables on species of Labeo Cuvier, 1817 were analyzed (University of Johannesburg, Department of Statistics) as described below (see Chapter 4).

Univariate statistics: Univariate descriptive statistics were performed to summarize the number of observations, range (minimum and maximum) and mean for the various structures measured.

Bivariate statistics: The Pearson’s correlation coefficient was calculated for each pair of variables. To ensure that variables are always greater than zero the measures were transformed with a log-transform (ln(x+1) where x is the measurement and ln is the natural logarithm). The resulting correlation matrix was also visually represented as a scatter matrix.

Principal component analysis: A principal component analysis (PCA) was performed on the data. As PCA is a variable reduction technique the aim was to summarise the ten measurements in only the top two or three components. PCA is also useful in identifying outliers and the components are always orthogonal (i.e. have zero correlation).

PCA was performed on the log-transformed variables after they had been standardized and replaced missing values by the mean.

A scree plot depicting the eigenvalues of the PCA was compiled to determine if the principal components have eigenvalues greater than one (i.e. if they would suffice to describe the data). Where applicable kernel density was estimated on selected principal components to investigate grouping.

Cluster analysis: A cluster analysis was performed on the standardized log- transformed measures to see whether there is an underlying grouping of specimens. Specifically, a k-means cluster analysis with k=2 was performed. Where principal components were known to be orthogonal and specimens were assumed to be independent, a non-parametric Mann-Whitney U test was performed to determine whether the principal components have different distributions for different clusters. This was graphically displayed as (1) histograms of the applicable principal component grouped by cluster; (2) three dimensional scatter plots where the axis were given by the principal components and the grouping variable was the cluster number.

3.6.3. Description of ecological aspects of monogeneans infecting Labeo spp. hosts as examined during the winter (June / July 2009) survey (Chapter 6)

Statistical analyses were performed by Statcon (University of Johannesburg statistical analysis support division) using SPSS version 18.0 software. Descriptive statistics were calculated for host data. Cramer’s V value was calculated to test for an association between fish gender and fish species. Infection statistics were calculated for each described monogenean species. The Shapiro-Wilk test was used to test for normality. The test was applied to both original and log-transformed data with no difference in result. The Levene statistic test was used to test for homogeneity of variance between host species for the length and weight data as well as parasite infection data. Spearman’s test was used for measuring correlation between host length and weight data, as well as correlations between the different parasite species in the component community. The Omnibus test was used to evaluate if the model can successfully predict variation in the respective dependent variables. The Wald Chi-square test was used to evaluate which variable(s) most successfully predict variation in each respective dependent variable (i.e. a test of model effect).

While this procedure assumes a normal distribution, the test was considered to be robust enough to also apply to non-normal data. The procedure was applied to both original and log-transformed data with no difference in result. Furthermore both the T-test (parametric independent samples test) and Mann-Whitney test (non- parametric test) were applied to weight, length and parasite data per host species. This was done to compare results (with regards to differences between species) in order to evaluate the use of parametric tests. As there was no difference in outcome the statistician suggested that parametric test employed were robust enough for use in the study. The Pearson Chi-Square statistic was used to test (at parasite species level) for (a) equal proportion of parasite on left and right gill; (b) on gill arch 1, 2, 3 or 4, (c) on front or back of the gill and (d) on the dorsal, median or ventral positions on the gill.

3.6.4. Description and seasonal comparison of ecological aspects of monogeneans infecting Labeo spp. as examined during a winter (June / July 2009) and summer (January 2010) survey (Chapter 7)

SOFA (Statistics Open For All) software, version 0.9.22 (www.sofastatistics.com) was used to apply the Mann-Whitney test (non-parametric test) applied to parasite numbers (i.e. for comparison between seasons).

QP3.0 (Quantitative Parasitology Version 3.0, Reiczigel and Rózsa 2005, http://www.zoologia.hu/qp/qp.html) was used to compare infection statistics between seasons. Only L. aeneus, L. kimberleyensis, suspected Labeobarbus spp. hybrids, L. capensis and L. umbratus were collected during both winter and summer surveys. Of these sufficient numbers of only the Labeo spp. were collected to warrant statistical analysis. Seasonal differences were investigated for all parasites (i.e. “component community” that also includes unidentified species) occurring on these host fish. Seasonal differences were statistically investigated only for parasite species of which adequate numbers were collected during both surveys.

3.6.5. Description of ecological aspects of monogeneans infecting other host species (Chapters 8 to 12)

Statistical analyses were performed using SOFA (Statistics Open For All) software, version 0.9.22 (www.sofastatistics.com). The Mann-Whitney test (non-parametric test) was applied to weight, length and parasite data per host species.

The Chi-Square statistic was used to test (at parasite species level) for (a) equal proportion of parasite on left and right gill; (b) on gill arch 1, 2, 3 or 4, (c) on front or back of the gill and (d) on the dorsal, median or ventral positions on the gill. Negative cases (i.e. fish on which no parasites were found) were excluded from the data sets prior to performing the analyses. Unknown scores were either indicated as “missing values” in the data sets (for Mann-Whitney U test analyses), or deleted from the dataset (for Pearson Chi-square analyses).

CHAPTER 4

4 - EVALUATION OF VARIANCE OF FORMS WITHIN A SINGLE PARASITE SPECIES, VERSUS SEPARATE SPECIES OF DACTYLOGYRUS Diesing, 1850 AND DOGIELIUS Bychowsky, 1936 ON LABEO Cuvier, 1817 HOSTS

4.1. Introduction

Quantifying morphometric variability within a described species effectively, yet allowing differentiation between closely related and morphologically very similar species remain a tremendous challenge (also see section 2.5).

Freeman and Ogawa (2010), with reference to udonellids being a more species-rich group than previously recognized, for example concluded that earlier descriptions of species that have been synonymised with Udonella caligorum Johnston, 1835, may in fact have represented true new species. The result is a wide range of morphological descriptions for U. caligorum, casting a proverbial shadow of doubt over the usefulness of morphological data for the entire group. Pseudothoracocotyla ovalis (Tripathi, 1956) was also recognized as a possible species complex by Hayward and Rohde (1999). The authors are of the opinion that the genetically isolated worms (from different host species) have not had sufficient time for evolutionary development of changes in phenotype (also refer to section 2.2.6 for examples of how parasites have been used to infer phylogenetic relationships). In both these cases apparent morphological variation / plasticity resulted in an underestimation of the number of species.

With regard to species of Gyrodactylus von Nordmann, 1832 the opposite appear to have occurred: a very large number of species have been described, with many of the species appearing to be synonyms due to a large amount of morphological plasticity resulting from variables such as temperature (see section 2.10.1). In this case apparent morphological variation / plasticity thus resulted in an over-estimation of the number of species.

Barson, Přikrylová, Vanhove and Huyse (2010) also warns that, with increased application of advanced molecular geno-typing procedures, previously described species-complexes may in fact shown to be complexes of hybrid species. As a result morphometric approaches are often combined with molecular approaches when examining species of Gyrodactylus (e.g. Huyse, Audenaert and Volckaert 2003; Huyse, Malmberg and Volckaert 2004; Huyse and Malmberg 2004) (also see section 2.6).

To aid in morphological discrimination between very similar Gyrodactylus spp. a number of additional measurements have been proposed (e.g. Harris, Shinn, Cable, Bakke and Bron 2008; Shinn, Harris, Cable, Bakke, Paladini and Bron 2010). With the aid of statistical analyses using tools such as principal component analysis (PCA); such measurements have been proven to effectively discriminate between closely related species (e.g. Shinn, des Clers, Gibson and Sommerville 1996).

The challenge of morphometric variation and use of statistical analysis techniques to quantify such variation is, however, not only restricted to the genus Gyrodactylus. Dmitrieva, Gerasev and Pron’kina (2007), for example, studied the morphometric variability of Ligophorus llewellyni Dmitrieva, Gerasev and Pron'kina, 2007 and its most similar congeners from the Black Sea using a similar approach.

During this project a number of additional measurement variables, originally developed for Gyrodactylus spp. (Shinn et al. 2010), were evaluated when applied to apparently closely related forms / species of Dogielius Bychowsky, 1936 and Dactylogyrus Diesing, 1850 (both collected from Labeo umbratus (Smith, 1841) and Labeo capensis (Smith, 1841), see Appendix B). Standard measurements used to characterise the respective genera (see section 3.4.2.1) were, however, found to adequately distinguish between the forms / species encountered (see Appendix B). To evaluate if the specimens collected represented different forms or separate species, the specimens were measured and subsequent measurements later statistically analyzed according to parasite genus. For the purposes of this chapter the species status of the genera shall thus be referred to as “sp.” (i.e. assuming a single species).

The Dogielius sp. specimens collected resemble Dogielius kabaensis Guegan and Lambert, 1991, while the Dactylogyrus sp. specimens share some similarities with the Dactylogyrus pseudanchoratus Price and Géry, 1968 species group. With regard to both Dogielius sp. and Dactylogyrus sp. two apparent “morphotypes”, differing in size of sclerotised structures and each respective type apparently associated with a particular species of Labeo Cuvier, 1817 could be visually differentiated during microscopic examination. Detailed descriptions (including measurement values and illustrations of sclerotised structures), comparison of morphology and information on fish hosts are discussed in Chapter 5.

Before such formal descriptions could be compiled, these apparent “morphotypes” had to be statistically evaluated to (1) confirm that different forms / species, based on visual inspection and subsequent morphological descriptors (i.e. measurements), can indeed also be statistically differentiated; (2) assist in making a decision as to taxonomic status (i.e. are the “morphotypes” in fact forms of the same species or separate species).

4.2. Materials and methods

For study site description refer to section 3.1.

For fish collection, necropsy and parasite recovery procedures refer to sections 3.2 and 3.3.

Data used in this chapter originated from the first survey (winter, June / July 2009). Twenty-six specimens (of which 21 were male) of L. umbratus were collected, compared to 13 (of which four were male) L. capensis.

For details on parasite description and measurement procedures refer to section 3.4 (more specifically section 3.4.2.1 and Figure 3-4). Representatives from the monogenean genera Dactylogyrus and Dogielius were collected from these host species.

For details on statistics employed for purposes of morphometric analyses (i.e. grouping of measured variables) refer to section 3.6.

4.3. Results and discussion

Note that species descriptions of the new species referred to in this chapter are provided in Chapter 5. This chapter (Chapter 4) only serves to illustrate the statistical analyses used to confirm the existence of two forms / species for each of the respective genera.

4.3.1. Dactylogyrus Diesing, 1850 analyses

Grouping of measured variables: Preliminary morphometric analysis was performed as is discussed below. Further detailed morphometric (and possibly also molecular studies) need to be performed on larger data sets to further elucidate this aspect.

Univariate statistics: The number of measurements made on each of the 10 variables measured on the 42 specimens, together with the minimum, maximum, mean and standard deviation values are shown in Table 4-1.

The male copulatory organ (MCO) exhibited the most missing (i.e. unobtainable) values. As measurements obtained from the projection of curved structures are likely to be more variable than those of linear features of a similar size (Shinn et al. 1996), penis tube trace length was measured in an attempt to mitigate such projection errors. However, with reference to standard deviation values, the penis tube trace length appeared most variable. As indicated by the large number of missing values, the MCO is not always clearly visible and it is often challenging to discern the exact length of the delicate, curved penis. This most likely contributed to variation observed. The marginal hook length was calculated as the average length of all marginal hooks I to VII for both sets.

Table 4-1 also lists the variables measured for each of the structure components (i.e. hamulus, transverse bar, marginal hook and MCO). Second to penis tube trace length, the total hamulus length also appeared variable (apparently to a large extend resulting from variation in inner root length and shaft length).

Table 4-1: Univariate descriptive statistics of Dactylogyrus Diesing, 1850 structure component measurements (parasites collected from Labeo capensis (Smith, 1841) and Labeo umbratus (Smith, 1841)).

Structures / Variable N Range Mean Std. deviation Components Statistic Min Max (μm) (μm) (μm) (μm)

Hamulus Total hamulus length 42 29.80 46.80 36.76 4.62

Hamulus shaft length 41 18.20 30.90 24.71 2.68

Hamulus outer root length 40 0.70 1.80 1.29 0.36

Hamulus inner root length 41 10.90 20.20 15.19 3.08

Hamulus tip length 41 8.00 14.50 10.29 1.37

Transverse Transverse bar length 40 12.30 22.70 16.53 2.28 bar Transverse bar width 40 1.80 5.00 2.76 0.77

Marginal Marginal hook total 30 14.20 20.40 16.70 1.35 Hook length(average)

MCO Accessory piece length 23 10.90 16.40 13.34 1.44

Penis/Penis tube trace length 28 16.40 36.40 25.29 5.48

Shinn et al. (1996) found root length to be most variable and also the softest part of the hamulus in Gyrodactylus sp. They continue by saying that lesser sclerified (i.e. “soft”) features are prone to exhibit relatively higher size plasticity. This may partly explain the degree of variation observed. Preliminary measurements of host gill structure (filament and lamellae length) indicated the gills of L. capensis to be more robust (see Appendix C). It may thus be that the variation (size plasticity) is related to host gill structure resulting in the two forms described in the preceding section.

Bivariate statistics: Table 4-2 contains the correlations coefficients and significance levels. The grouping of the measures by different components (hamulus, transverse bars, marginal hook and MCO) has been blocked for ease of interpretation.

The highly significant correlation coefficient values calculated when comparing total hamulus length with inner root length and shaft length, confirms that variation in both the latter results in differences in total hamulus length. A visual representation of the correlation matrix (i.e. a scatter matrix) is depicted in Figure 4-1.

The histograms of the total hamulus length and hamulus inner root length clearly indicate that bi-modal distribution might be applicable. Furthermore, it would seem that the different components have a high correlation within the component and lower correlations between different components.

Correlations of log- transformed measurements

bar length bar

Hamulus tip length tip Hamulus Total hamulus length Total hamulus Transverse bar width bar Transverse Hamulus shaft length shaft Hamulus Transverse Accessory piece length piece Accessory Hamulus inner root length root inner Hamulus length total hook Marginal Hamulus outer root length root outer Hamulus Penis/Cirrus tube trace length trace tube Penis/Cirrus

Total hamulus length 1 .937** .530* .905** .153 .740** .624** .244 .303 -.263

Hamulus shaft length .937** 1 .487* .722** .326 .616** .553* .458 .209 -.015

Hamulus outer root length .530* .487* 1 .532* .198 .249 .078 .192 .407 -.211

Hamulus inner root length .905** .722** .532* 1 -.058 .790** .625** -.009 .284 -.499*

Hamulus tip length .153 .326 .198 -.058 1 .099 -.009 .522* .255 .185

Transverse bar length .740** .616** .249 .790** .099 1 .636** .078 .375 -.291

Transverse bar width .624** .553* .078 .625** -.009 .636** 1 .001 .242 -.014

Marginal hook total length .244 .458 .192 -.009 .522* .078 .001 1 -.022 .239

Accessory piece length .303 .209 .407 .284 .255 .375 .242 -.022 1 -.248

Penis/Cirrus tube trace -.263 -.015 -.211 -.499* .185 -.291 -.014 .239 -.248 1 length

**. Correlation is significant at the 0.01 level (2-tailed). *. Correlation is significant at the 0.05 level (2-tailed).

4 Total hamulus length 3.5 3 3.5 Hamulus shaft length 3 2.5 1.5 Hamulus outer root length 1 0.5 4 Hamulus inner root length 3 2 3 Hamulus tip length 2.5 2 3.5 Transverse bar length 3 2.5 2 Transverse bar width 1.5 1 3.5 Marginal hook total length 3 2.5 3 Accessory piece length 2.5 2 4 Penis/Penis tube trace length 3 2 3 3.5 4 2.5 3 3.50.5 1 1.5 2 3 4 2 2.5 3 2.5 3 3.5 1 1.5 2 2.5 3 3.5 2 2.5 3 2 3 4

Principal component analysis (PCA): The scree plot in Figure 4-2 depicts the eigenvalues of the PCA. It would seem as if the first three principal components have eigenvalues greater than one and would suffice to describe the data.

In fact, the first component describes 42.1% of the data variability, the first and second described 59.42% of the data and the first three components described 70.4% of the data variability. Hence, the number of measurements has been reduced from ten to three.

Table 4-3 contains the first three principal components. It is very clear that the first component is a function of the hamulus measurements and the transverse bar. The second component is a function of the marginal hook and the third component is proportional to the penis tube trace length, but inversely related to the accessory piece length.

Eigenvalue

Figure 4-2: Scree plot of the principal component analysis (PCA) – Dactylogyrus Diesing, 1850.

This is to be expected since the correlation between these two measurements was - 0.248. Hence, the third component is a function of the MCO. Given apparent variability and difficulty in measuring exact penis tube trace length discussed earlier, the use of this component as a diagnostic feature to distinguish between two possible forms should be viewed with caution. Interestingly the first principal component seemed to be bi-modal. In Figure 4-3 a kernel density was estimated on principal component one (PC 1) and principal component two (PC 2). Clearly there seems to be some grouping of the specimens.

Cluster analysis: Table 4-4 contains the cluster centres of all the measures in the original scale. Since the principal components are known to be orthogonal and specimens are assumed to be independent, we may perform a non-parametric Mann-Whitney U test to see whether the principal components have different distributions for different clusters. The results are in Table 4-5. Clearly the first principal component shows that the distribution for cluster one’s first principal component is different from cluster two’s.

Structure Principal Component Measurement component 1 2 3 Total hamulus length .961 .044 -.138 Hamulus shaft length .891 -.083 -.092 Hamulus Hamulus outer root length .818 -.063 -.026 Hamulus inner root length .805 .227 -.203 Hamulus tip length .688 -.047 -.237 Transverse bar Transverse bar length .676 -.128 .170 Transverse bar width .121 .841 .081 Marginal hook Marginal hook total length .132 .816 .301 Accessory piece length -.344 .523 -.440 MCO Penis/Penis tube trace length .305 -.038 .808

Note: MCO = Male copulatory organ.

Figure 4-3: Kernel density estimation of principal components (PC) 1 and 2 demonstrates bimodal grouping of specimens – Dactylogyrus Diesing, 1850.

Table 4-4: Cluster centres of all the measures from Dactylogyrus Diesing, 1850 in the original scale after performing a k-means cluster analysis with k=2.

Cluster Measurement 1 2 Total hamulus length 34 42 Hamulus shaft length 23 27 Hamulus outer root length 1 2 Hamulus inner root length 13 19 Hamulus tip length 10 10 Transverse bar length 15 19 Transverse bar width 2 3 Marginal hook total length(average) 17 17 Accessory piece length 13 13 Penis/Penis tube trace length 28 22

Table 4-5: Non-parametric Mann-Whitney U test results – Dactylogyrus Diesing, 1850.

This is graphically displayed in two ways. Figure 4-4 represents the histograms of the first principal component grouped by cluster. For the remaining principal components the differences between the distributions were negligible.

A three-dimensional scatter plot (Figure 4-5) was also employed where the grouping is made apparent by the colour coding of each specimen (the axis are given by the principal components and the grouping variable is the cluster number). One can clearly see the grouping within the data.

Figure 4-4: Histogrammes of the first principal component grouped by cluster – Dactylogyrus Diesing, 1850.

Figure 4-5: Three-dimensional scatter plot with axis given by principal components and the grouping variable is the cluster number – Dactylogyrus Diesing, 1850.

Figure 4-6 shows the same plot, but this time with the colour grouping made by fish species. It would seem as if the clusters correspond to different fish species, although there are four observations that were different.

Figure 4-6: Three-dimensional scatterplot with axis given by principal components and the grouping variable is the two different host species – Dactylogyrus Diesing, 1850.

The morphometric analyses thus confirmed clear groupings. This supports the description of two forms of a single parasite species / two separate parasite species that occur on both closely related Labeo host species, yet shows a distinct preference for a particular host species. Furthermore the distinction of the two forms / species based primarily on hamulus length has been confirmed by the morphometric analyses presented above.

4.3.2. Dogielius Bychowsky, 1936 analyses

Grouping of measured variables: Preliminary morphometric analysis was performed as is discussed below. Due to the small sample size statistical accuracy is admittedly seriously hampered. For this reason further detailed morphometric (and possibly also molecular studies) need to be performed on larger data sets to further elucidate this aspect.

Univariate statistics: The number of measurements made on each of the seven variables measured on the 22 specimens, together with the minimum, maximum, mean and standard deviation values are shown in Table 4-6. Once again the MCO exhibited the most missing (i.e. unobtainable) values and a high standard deviation.

Table 4-6: Univariate descriptive statistics of Dogielius Bychowsky, 1936 structure component measurements (parasites collected from Labeo capensis (Smith, 1841) and Labeo umbratus (Smith, 1841)).

Structures Variable N Range Mean Std. Deviation

Statistic Min Max (μm) (μm) (μm) (μm)

Hamulus Total hamulus length 22 22.70 34.10 29.17 3.21

Hamulus shaft length 22 30.00 44.30 36.97 4.44

Hamulus tip length 22 16.80 28.20 23.05 3.11

Transverse bar Transverse bar length 22 33.20 52.70 44.95 5.37

Transverse bar width 22 3.20 7.30 5.54 1.05

Marginal hook total Marginal Hook 20 15.90 20.60 18.07 1.52 length(average)

MCO Accessory piece length 17 14.10 31.40 25.22 4.50

Note: MCO = Male copulatory organ.

The length of the transverse bar, however, exhibited the highest standard deviation. The marginal hook length was calculated as the average length of all marginal hooks I to VII for both sets. Table 4-6 also lists the variables measured for each of the structure components (i.e. hamulus, transverse bar, marginal hook and MCO).

Bivariate statistics: Table 4-7 contains the correlations coefficients and significance levels. The grouping of the measures by different components (hamulus, transverse bars, marginal hook and MCO) has been blocked for ease of interpretation. There appears to be no significant correlations between the copulatory apparatus length and hamulus tip length respectively and any other covariate.

A visual representation of the correlation matrix (i.e. a scatter matrix) is depicted in Figure 4-7. The histograms of the total hamulus length and hamulus inner root length once again indicate that bi-modal distribution might be applicable.

Principal component analysis (PCA): The scree plot in Figure 4-8 depicts the eigenvalues of the PCA. It would seem as if the first two principal components have eigenvalues greater than one and would suffice to describe the data.

In fact, the first component describes 51.1% of the data variability and the first two components described 71.4% of the data variability. Hence, the number of measurements has been reduced from seven to two.

Correlations of log- transformed variables

length

Hamulus total Transverse width bar Hamulusshaft length Transverse length bar Marginal hook total lengthMarginal hook total Copulatoryapparatus length Hamulus Length of tip /Hamulus tip point of Length

Hamulus total length 1.000 .940** -.110 .810** .557** .577** .403

Hamulus shaft length .940** 1.000 -.061 .790** .600** .602** .313

Hamulus Length of tip / -.110 -.061 1.000 .097 .257 -.387 .109 point

Transverse bar length .810** .790** .097 1.000 .495* .527* .278

Transverse bar width .557** .600** .257 .495* 1.000 .244 .155

Marginal hook total length .577** .602** -.387 .527* .244 1.000 -.106

Copulatory apparatus length .403 .313 .109 .278 .155 -.106 1.000

*. Correlation is significant at the 0.05 level (2-tailed). **. Correlation is significant at the 0.01 level (2-tailed).

Table 4-8 contains the first two principal components. It is very clear that the first component is a function of the hamulus total length and shaft length. The coefficients of the transverse bar measurements are also quite high. Interestingly, the length of the hamulus tip length is not a covariant driving the first component, but rather the second. This was seen previously: the correlations of the hamulus tip length with the other covariates are not significantly different from zero.

The second component is thus a function of the hamulus tip length, negatively related to the marginal hook length and positively related to the MCO length. The first principal component seemed to be bi-modal.

4 Hamulus total length 3. 5 3 4 Hamulus shaft length 3. 5 3 3. Hamulus Length of tip / point 5 3

2. 5 4 Transverse bar length 3. 8 3. 6 3 Transverse bar width 2. 5 2 1. 5 1 Marginal hook total length (average)3. 23. 1 3 2. 92. Copulatory apparatus length 3.8 5 3

2. 5 3 3. 4 3 3. 4 2. 3 3. 3. 3. 4 1 1. 2 2. 3 2. 2. 3 3. 3. 2. 3 3. 5 5 5 5 6 8 5 5 8 9 1 2 5 5

Figure 4-7: Visual representation of bivariate statistics results of Dogielius Bychowsky, 1936 structure component measurements (parasites collected from Labeo capensis (Smith, 1841) and Labeo umbratus (Smith, 1841)): Scatter matrix of all measurements.

Figure 4-8: Scree plot of the principal component analysis (PCA) – Dogielius Bychowsky, 1936.

In Figure 4-9 the histogram of the first principal component is depicted. It does seem as if there is some grouping amongst variables present.

Table 4-8: Principal component analysis results of Dogielius Bychowsky, 1936, structure component measurements (parasites collected from Labeo capensis (Smith, 1841) and Labeo umbratus (Smith, 1841)) showing the first two principal components.

Structure Principal Component Measurement component 1 2 Total hamulus length .957 -.032 Hamulus Hamulus shaft length .960 -.007 Hamulus tip length -.006 .863 Transverse bar length .874 .074 Transverse bar Transverse bar width .653 .339 Marginal Hook Marginal hook total length .674 -.575 MCO Accessory piece length .309 .466

Cluster analysis: Table 4-9 contains the cluster centres of all the measures in the original scale.

Table 4-9: Cluster centres of all the measures in the original scale after performing a k-means cluster analysis with k=2.

Measurement Cluster

1 2

Hamulus total length 26 31

Hamulus shaft length 32 40

Hamulus Length of tip / point 23 23

Transverse bar length 40 49

Transverse bar width 5 6

Marginal hook total length 17 19

Copulatory apparatus length 23 27

As the principal components are known to be orthogonal and specimens are assumed to be independent, a non-parametric Mann-Whitney U test was performed to see whether the principal components have different distributions for different clusters. The results are in Table 4-10. Clearly the distribution of the first principal component is different for each cluster.

This is graphically displayed in two ways. Figure 4-10 represents the histograms of the first principal component grouped by cluster. For the second principal components the differences between the distributions were not significant.

Table 4-10: Non-parametric Mann-Whitney U test results – Dogielius Bychowsky, 1936.

Figure 4-9: Histogram of the first principal component – Dogielius Bychowsky, 1936.

A three-dimensional scatterplot (Figure 4-11) was also employed where the grouping is made apparent by the colour coding of each specimen (the axis are given by the principal components and the grouping variable is the cluster number).

Figure 4-12 shows the same plot, but this time with the colour grouping made by fish species. It would seem as if the clusters to a large extend correspond to different fish species.

Figure 4-10: Histograms of the first principal component grouped by cluster – Dogielius Bychowsky, 1936.

Figure 4-11: Three-dimensional scatterplot with axis given by principal components and the grouping variable is the cluster number – Dogielius Bychowsky, 1936.

The morphometric analyses thus once again support the description of two forms of a single parasite species that occurs on both closely related Labeo host species, yet each form shows a distinct preference for a particular host species.

Figure 5

Figure 14-12: Three-dimensional scatterplot with axis given by principal components and the grouping variable is the two different host species – Dogielius Bychowsky, 1936.

4.3.3. General discussion and conclusion

Lambert and El Gharbi (1995) state that the complex structure of the sclerotized MCO is a good specific diagnostic criterion, as it is less subject to convergence phenomena and adaptive pressures compared to the haptoral sclerites. Pouyaud, Desmarais, Deveney and Pariselle (2006) state that the use of morphology of the haptoral sclerites is more suitable to infer phylogenetic relationships when compared to the morphology of the MCO. They continue by saying that the latter is more useful to resolve species-level identifications, resulting from its faster rate of change. This statement then implies, in contrast to the statement by Lambert and El Gharbi (1995) that the MCO is more subject to adaptive selection pressures compared to haptoral sclerites. Both, however, agree that MCO structure is an important determinant when deciding on new species status. Given the fact that the MCO morphology of both the penis and accessory piece for the Dactylogyrus specimens collected in this study are very similar (see Chapter 5), the statement by both Lambert and El Gharbi (1995) and Pouyaud et al. (2006) implies that description of separate forms of the same species may be more appropriate. The same applies to the Dogielius specimens collected. Jarkovský, Morand, Šimková and Gelnar (2004) concluded that greater similarity in attachment apparatus amongst specialist parasites should be evident due to specialisation to their host.

They continue by saying that a random pattern is exhibited with regard to species similarity in MCO morphology within infra-communities, but that MCOs are generally more variable than the haptoral sclerites. They attribute reproductive isolation as a probable cause of this observed pattern. The high degree of similarity in MCO structure observed between the Dactylogyrus spp. collected in this study is indeed rare, once again hinting at two forms of the same species.

Yet there are marked differences in haptoral sclerite (particularly anchor shape and size) morphology which, coupled with distinct host preference of the two Dactylogyrus sp. “forms”, pointing to two separate Dactylogyrus species. Referring to Gyrodactylus spp., Appleby (1996) states that morphological variation / plasticity will result in differences in anchor size, but rarely in anchor shape. As there is a difference in anchor shape, resulting from inner root length and shaft length, it was decided to describe two separate Dactylogyrus spp. (a suggestion also made by an anonymous reviewer following submission of a manuscript for publication). As the two Dogielius forms / species exhibited the same anchor and MCO morphology in terms of shape, but only differed with regard to size depending on the host species from which they were collected, two forms of the same species were described (see Chapter 5).

Conclusion: Statistical analysis indicated that two forms / species of Dactylogyrus and Dogielius respectively could be distinguished depending on fish host identity (i.e. a clear host preference is evident). It is concluded that the morphological variation observed warrants the description of two species of Dactylogyrus and one species of Dogielius exhibiting two distinct forms.

CHAPTER 5

5 - MONOGENEAN PARASITE SPECIES DESCRIPTIONS FROM LABEO Cuvier, 1817 HOSTS IN THE VAAL DAM, SOUTH AFRICA, WITH A REVIEW OF RELATED PARASITE SPECIES

5.1. Introduction

The importance of monogenean infections of freshwater fishes is widely recognized (e.g. Bakke, Harris, Jansen and Hansen 1992; Thilakaratne, Rajapaksha, Hewakopara, Rajapakse and Faizal 2003; Galli, Strona, Benzoni, Crosa and Stefani 2007; Abo-Esa 2008). Compared to the rest of Africa (e.g. Paperna 1973; El Gharbi, Birgi and Lambert 1994; Paperna 1996; N’Douba, Lambert and Euzet 1999; Pariselle, Bilong Bilong and Euzet 2003; Christison, Shinn and van As 2005; Přikrylová and Gelnar 2008; Přikrylová, Matĕjusová, Musilová and Gelnar 2009a; Přikrylová, Matĕjusová, Musilová, Gelnar and Harris 2009b to name but a few), fairly few publications deal with especially freshwater monogeneans from South Africa (e.g. Luus-Powell, Mashego and Khalil 2003; Christison and Baker 2007; Le Roux and Avenant-Oldewage 2010a, 2010b; Le Roux, Avenant-Oldewage and van der Walt 2011; Madanire-Moyo, Matla, Olivier and Luus-Powell 2011). Limited availability of South African baseline data appears to be an important current constraint with regards to general advancement in parasitic monogenean research on freshwater fish in this country. Due to the relatively strict host specificity of monogeneans, study of endemic host species (not previously specifically examined for monogenean parasites) is expected to yield a number of new monogenean species (Gussev, Jalali and Molnár 1993).

The current chapter forms part of a project that aims to contribute to such “baseline data” on monogenean parasite infections on fish hosts occurring in the Vaal Dam (Vaal River system, Gauteng province, South Africa). While the parasite fauna of fish from this study site (and the river system within which it lies) has been investigated previously (e.g. Bertasso and Avenant-Oldewage 2005; Tsotetsi, Avenant-Oldewage and Mashego 2005), none focussed on monogenean parasites.

The monogenean fauna of the moggel (Labeo umbratus (Smith, 1841)) and the Orange River mudfish (Labeo capensis (Smith, 1841)) is reported on in this chapter, the first published account (Crafford, Luus-Powell and Avenant-Oldewage, 2012) on the monogenean fauna on species of Labeo Cuvier, 1817 from this locality. Both species are endemic to South Africa but not restricted to the Vaal River system (Skelton 2001).

Thus far a single dactylogyrid (Dactylogyrus pienaari Price, Korach and McPott, 1969) has been described from a Labeo species (Labeo rosae Steindachner, 1894) in South Africa (Makambosi Pan, Kwazulu-Natal Province) (Price, Korach and Mc Pott 1969). More recently Olivier, Luus-Powell and Saayman (2009) reported the presence of Dactylogyrus Diesing, 1850 and Dogielius Bychowsky, 1936 on L. rosae and Labeo ruddi Boulenger, 1907 and Dactylogyrus spp. on Labeo cylindricus Peters, 1868 and Labeo molybdinus Peters, 1868 from Middle Letaba Dam, Limpopo Province, South Africa. None of these parasites were, however, identified to species level. A number of papers deal with the dactylogyrid monogenean fauna of Labeo species from other countries in Africa, including: Paperna (1969, 1973 and 1979) in Ghana, Kenya, Uganda and Tanzania; Guégan, Lambert and Euzet (1988, 1989) in Mali and Senegal; Guégan and Lambert (1990, 1991) in Mali, Guinea, Senegal, Sierra Leone, Ivory coast and Ghana; Musilová, Řehulková and Gelnar (2009) in Senegal. Khalil and Polling (1997) listed a total of 24 species of Dactylogyrus and 15 species of Dogielius reported from the following species of Labeo in Africa: Labeo alluaudi Pellegrin, 1933; Labeo coubie Rüppell, 1832; L. cylindricus, Labeo forskalii Rüppell, 1835; Labeo parvus Boulenger, 1902; Labeo rosae Steindachner, 1894; Labeo rouaneti Daget 1962; Labeo ruddi Boulenger, 1907; Labeo senegalensis Valenciennes, 1842 and Labeo victorianus Boulenger, 1901. This changed to 27 species of Dactylogyrus following description of three new species from L. coubie in Senegal by Musilová et al. (2009).

The objective of the current study was to identify, describe and compare the monogenean parasite fauna of two sympatric cyprinids from the genus Labeo (L. capensis and L. umbratus) occurring in the Vaal Dam, South Africa.

5.2. Materials and methods

For study site description refer to section 3.1.

For fish collection, necropsy and parasite recovery procedures refer to sections 3.2 and 3.3.

For details on parasite description and measurement procedures refer to section 3.4 (more specifically section 3.4.2.1 and Figure 3-4).

For details on descriptive statistics employed refer to section 3.6.1.

5.3. Results and discussion

This chapter was not intended and should not be viewed as a comprehensive and complete overview of Dactylogyrus and Dogielius species recorded from African Labeo spp. Comparisons with previously described species are restricted to those showing resemblances in morphology of the hard parts (most notably the copulatory organ) with the new species described here. Within this wider selection, emphasis is placed on those species that have been compared to each other by previous authors working on monogeneans of Labeo spp. in Africa.

5.3.1. Host species

Twenty-six specimens (of which 21 were male) of L. umbratus were collected, compared to 13 (of which four were male) L. capensis.

5.3.2. Parasite species

Representatives from the monogenean genera Dactylogyrus and Dogielius were collected and are described. All measurement values are given in micrometers (μm) unless otherwise indicated.

5.3.3. Dactylogyrus Diesing, 1850

Four new species of Dactylogyrus were encountered. Two species occur on both L. umbratus and L. capensis but exhibit a clear host preference. A third species was collected only from L. capensis and a fourth from L. umbratus.

5.3.3.1. Species A (Dactylogyrus iwani n. sp., Figure 5-1)

Type hosts and locality: Labeo capensis, Vaal Dam (Vaal River System) close to University of Johannesburg Island, S 26 52.249, E 28 10.249, Gauteng Province, South Africa.

Other hosts: Labeo umbratus, Vaal Dam (Vaal River System) close to University of Johannesburg Island, S 26 52.249, E 28 10.249, Gauteng Province, South Africa.

Site: Gill lamellae

Type specimens: Holotype: SAM A61783 - one whole mounted specimen in glycerine jelly.

Paratypes: SAM A61784 and SAM A61785 - two specimens in GAP and glycerine jelly

Material examined: Fifteen specimens from L. capensis in glycerine ammonium picrate and glycerine jelly (all flattened to some degree).

Description: Body length 222.9 (152.4-373.8); greatest width 45.8 (31.0-69.0). Single pair of anchors with rounded inner root (slightly truncate in some specimens). Total anchor length 42.0 (38.0-46.8); anchor shaft length 27.2 (23.6-30.9); anchor outer root length 1.5 (1.1-1.8); anchor inner root length 19.0 (17.0-20.2); anchor point length 10.2 (8.2-12.3); anchor aperture 22.4 (19.8-24.5). One dorsal bar (ranging in shape from “bow-shaped” to “dumbbell-shaped). Dorsal bar length 18.4 (15.0-21.4); dorsal bar width 3.4 (2.7-5.0). Seven pairs of marginal hooks. Total marginal hook length (average for all seven marginal hook pairs) 16.4 (14.6-17.9). MCO consists of curved penis and simple accessory piece (shaft with widening / flaring terminal section). Accessory piece shaft extends into the flaring distal portion to form a “ridge” on the edge of the widening terminal section of the accessory piece. Depending on orientation this “ridge” may appear on varying positions on the flaring / widening terminal section (e.g. central). Accessory piece length 13.3 (11.8 - 14.0); penis length (tube trace length) 21.7 (16.4 to 28.2).

Remarks: Refer to comments following description of Species B (Dactylogyrus larindae n. sp.). The haptoral and MCO sclerites are illustrated in Figure 5-1.

5.3.3.2. Species B (Dactylogyrus larindae n. sp., Figure 5-2)

Type hosts and locality: Labeo umbratus, Vaal Dam (Vaal River System) close to University of Johannesburg Island, S 26 52.249, E 28 10.249, Gauteng Province, South Africa.

Other hosts: Labeo capensis, Vaal Dam (Vaal River System) close to University of Johannesburg Island, S 26 52.249, E 28 10.249, Gauteng Province, South Africa.

Site: Gill lamellae

Type specimens: Holotype: SAM A61786 - one whole mounted specimen in glycerine jelly.

Paratypes: SAM A61787 and SAM A61788 - two specimens in

GAP and glycerine jelly

Material examined: 23 specimens from L. umbratus and 3 specimens from L. capensis in glycerine ammonium picrate and glycerine jelly (all flattened to some degree).

Description: Body length 201.7 (135.7-250.0); greatest width 39.3 (23.8-61.9). Single pair of anchors with truncate inner root. Total anchor length 32.3 (29.8-37.3); anchor shaft length 22.3 (18.2-25.7); anchor outer root length 1.2 (0.7-1.8); anchor inner root length 12.7 (10.9-15.5); anchor point length 10.0 (8.0-14.5); anchor aperture 17.2 (15.0-21.4). One dorsal bar (ranging in shape from “bow-shaped” to “dumbbell-shaped). Dorsal bar length 15.3 (12.3-17.3); dorsal bar width 2.3 (1.8-3.2). Seven pairs of marginal hooks. Total marginal hook length (average for all seven marginal hook pairs) 16.4 (14.2-19.8). MCO consists of curved penis and simple accessory piece (shaft with widening / flaring terminal section). Accessory piece shaft extends into the flaring distal portion to form a “ridge” on the edge of the widening terminal section of the accessory piece in flattened specimens. Accessory piece length 12.8 (10.0 - 15.5); penis length (tube trace length) 25.4 (17.3 to 33.6).

Remarks: The two new species (Figures 5-1 and 5-2) show some similarities (anchor morphology and MCO structure, more specifically the presence of a curved penis) with the Dactylogyrus pseudanchoratus Price and Géry, 1968 species group described by Paperna (1979).

A brief, comparative description of applicable species within this group is provided in Table 5-1 with measurement values summarized in Table 5-2.

Reference cited Taxon Summary description Taxon author

Anchors with long inner roots, short or even vestigial outer roots, characteristic small knob on inner rim of shaft/tip junction; cirrus tubiform and slightly curved (or also Dactylogyrus pseudanchoratus Paperna 1973, Paperna 1979 coiled if longer), accessory piece is simple, bifurcating distally (i.e. Y-shaped or of species group Type species: Price and Géry, 1968 similar shape evolved from Y-shape). Species in this group differ from each other mainly in the relative size of the anchors and copulatory organ, length of the cirrus and details of the accessory piece.

Paperna 1979 Large anchors; anchors and bars conform to the species group characteristics. Dactylogyrus pseudanchoratus Funnel of the cirrus triangular in shape, cirrus tube tapering distally to a delicate Price and Géry, 1968 narrow tube. Accessory piece short and bifid distally.

Paperna 1979 Differs from typical specimen (D. pseudanchoratus) in the larger size of the cirrus, in Dactylogyrus cf. pseudanchoratus Price and Géry, 1968 these specimens (unlike the type) the vagina is opened through a sclerotinoid tube.

Closely related to D. helicophallus. Cirrus longer than that of typical specimen (D. pseudanchoratus) but not as long and coiled as in D. helicophallus – this apparently Dactylogyrus pseudanchoratus place these forms in an intermediate position between the two species (D. Paperna, 1979 micronchus pseudanchoratus and D. helicophallus – see comment by Paperna (1979) below). The bar and accessory piece of the copulatory organ remain within the size range of the typical specimens.

Paperna 1979 Anchors, bars and hooklets conform to that of the species group description. Cirrus Dactylogyrus helicophallus whip shaped, tapering distally and strongly curved or even coiled. Accessory piece Paperna, 1973 stout, the distal bifurcation is barely distinct (“T-shaped”).

Remark (Paperna 1979): “Among the specimens of D. pseudanchoratus typ. were also observed specimens with smaller anchors which could be intermediate between D. pseudanchoratus typ. and D.p. micronchus. This may evident the existence of a whole range of intermediate forms between D. pseudanchoratus to D. helicophallus”.

Type Anchor Literature Marg. Acc. Taxon Type / main host locality L W Inner Outer Bar Cirrus referenced L Shaft Tip hooks piece (Dactylogyrus) (Other hosts) (Other (Mean) (Mean) root root (Mean) (Mean) Taxon author (Mean) (Mean) (Mean) (Mean) (Mean) localities) (Mean) (Mean) Barbus Cuvier and Price and Géry, 210- Gabon 56 52-61 - - - - 23-28 18-20 20-25 15-20 Cloquet, 1816 1968 240 Barbus macrolepis 130- Paperna 1979 70-100 50-56 24-25 1-3 29-32 14-20 26-27 16-20 24-25 17-18 Pfeffer, 1889 Tanzania Price and Géry, 310 Barbus occidentalis 1968 330 100 40-42 19 - - - 26 19 - - (Boulenger, 1920) Dactylogyrus Barbus petitjeani pseudanchoratus Daget, 1962

Barbus sacratus (# = Forma 48-49 24-33 2-5 30-36 15-20 22-28 Daget, 1963 (54#) (31#) (3#) (33#) (18#) (25#) “petitjeanii”; Guegan and Barbus waldroni 420- ** = Forma Lambert 1990 70-140 25-30 14-22 Norman, 1935 Gabon 770 15-20 “complexa”) Price and Géry, (100) (27) (19) Barbus parawaldroni (550) Lévêque, Thys van den 1968 Audenaerde and Traoré, 48-62 21-31 2-5 30-38 15-20 29-35 1987 (56**) (25**) (3**) (34**) (17**) (32**) Barbus wurtzi Pellegrin, 1908 Dactylogyrus cf. Barbus altianalis 250- Uganda Paperna, 1979 70-110 49-60 20-28 3-5 32-38 11-18 27-30 17-18 25-37 18-23 pseudanchoratus (Boulenger, 1900) 440 Dactylogyrus Labeo 220- pseudanchoratus Tanzania Paperna, 1979 60-100 31-34 12-13 2-4 22-25 9-12 25-26 9-17 28-38 14-17 Cuvier, 1817 240 micronchus Labeo forskalii 180- 15-17; Uganda 40 33-35 15 2 20-22 14-19 16 15-17 72-82 Dactylogyrus Rüppell, 1835 (Labeo Paperna 1979 200 20-25 (Kenya, helicophallus victorianus Boulenger, Paperna, 1973 200- 6-10; 7- Tanzania) 50-70 30-35 13-17 1-2 19-21 10-12 18-20 - 32-39 1901, Labeo sp.) 290 16 a Labeo capensis Crafford, Luus- 152- 14-21 (Smith, 1841) South Powell and 31-69 38-45 17-20 1-2 24-29 8-11 15-23 16-32 12-16 Species A and B 374 (16- (Labeo umbratus Africa Avenant-Oldewage, (46) (42) (19) (1.5) (27) (10) (19) (22) (13) n.sp. (228) 18*) (Smith, 1841)) 2012 (A = Dactylogyrus Crafford, Luus- iwani; b 135- 14-21 L. umbratus South Powell and 24-62 30-37 11-15 1 18-26 8-15 12-17 17-33 11-16 B = D. larindae) 250 (15- (L. capensis) Africa Avenant-Oldewage, (39) (34) (13) (1) (23) (10) (15) (28) (13) (212) 18*) 2012 L = Length; W = Width, Marg hooks = Marginal hooks; Acc piece = Accessory piece; * = range of average values for all seven marginal hook pairs

Both differ from D. pseudanchoratus in terms of bar and anchor size (larger in D. pseudanchoratus) and accessory piece morphology (bifid according to Paperna 1979 or trifid according to Guegan and Lambert 1990 in D. pseudanchoratus – this discrepancy in description illustrates the need for the re-description of the type material of D. pseudanchoratus in future studies).

Measurements of the new species (especially D. larindae n. sp.) conform well to that of D. pseudanchoratus micronchus and D. helicophallus. As with these species the penis is whip shaped, tapers distally and is strongly curved. The penis of both new species are, however, generally shorter than that recorded for D. helicophallus, although the accessory piece structure is very similar. A distal bifurcation is barely distinct in D. helicophallus but absent in the new species. The accessory piece shaft extends into the flaring distal portion to form a “ridge” on the edge of the widening accessory piece. Depending on the orientation of the shaft relative to the flaring portion, the position at which this “ridge” is observed may vary (e.g. more central or apparently extending over the distal edge). Average penis length of the new species is more comparable to that of D. pseudanchoratus micronchus, yet the accessory piece structure differs (more distinctly bifid in D. pseudanchoratus micronchus). The two new species also differ from both above-mentioned species in terms of transverse bar shape. In the majority of specimens the transverse bar appeared slightly “bow-shaped”, yet often also appeared more slender and “dumbbell shaped”. Morphologically the two species described here appears to be intermediate between D. helicophallus and D. pseudanchoratus micronchus, supporting Paperna’s (1979) notion that they may form part of a species complex consisting of a range of intermediate forms.

Morphology of the two new species was also compared to that of other species described from Labeo spp. hosts in Africa (Paperna 1973; Paperna 1979; Guegan et al. 1988; Guegan and Lambert 1990; Guegan and Lambert 1991). Table 5-3 shows measurement values for species that have also been compared to the D. pseudanchoratus species group by other authors. A comparison matrix highlighting differences in morphology between those species and the new species (D. larindae and D. iwani) encountered in this study is provided in Table 5-4.

Bamako, Mali Guegan and 320- 60- 15-20 34-39 14-18 22-24 23-27L (Missira, Lambert 1991 520 100 1-3 12-14 14-17 18-23 (20- (36) (16) (23) 2-4w Dlaba) Paperna 1973 (400) (80) 24*)

17-23 280- (penis) Labeo coubie Guegan Lambert 40-80 30-40 12-19 23-27 11-16 24-28L Niger, Baoulé 470 1-2 12-16 4-5 17-25* Rüppell, 1832 and Euzet, 1988 (60) (36) (16) (25) (14) 3-5w (370) (basal bulb) Dactylogyrus [Labeo parvus falcilocus Boulenger, 1902 Barbus wurtzi 260- Pellegrin, 1908] Guegan and 50-80 Mali # 480 30-43 12-21 1-3 22-29 11-16 23-29 14-21 17-23 10-20* Lambert 1990 (60) (380)

324- Musilová et al. 69-90 36-39 15-18 1-2 26-28 13-16 22- Senegal 471 26-29L 14-18 20-25* 2009 (77) (38) (16) (2) (27) (14) 24** (417)

Labeo rouaneti 250- 40- Dactylogyrus The Republic Guegan and 47-55 22-29 26-33 22-26 Daget, 1962 630 120 3-6 17-18 18-22 40-47 26-30 jucundus of Guinea Lambert, 1991 (50) (26) (30) [8-10] (490) (90)

20-27 230- 17 (penis) Dactylogyrus Labeo senegalensis Guegan, Lambert 40-80 42-48 15-19 31-35 Mali 470 2-4 (16- 17-21L 16-30 10-12 - rastellus Valenciennes, 1842 and Euzet, 1988 (60) (45) (17) (33) (340) 18) (basal bulb) * = Total length of copulatory apparatus ; L = Length; w = width; Acc piece = Accessory piece; transv. = transverse; vest = vestigial bar; ** = tube trace-length (length of bulb plus penis) # = Summary of range observed in parasites collected from three different host species (Guegan and Lambert 1990)

Table 5-3 (continued): Summary of hard part structure measurements (all in micrometers) of selected species previously compared to the Dactylogyrus pseudanchoratus / Dactylogyrus helicophallus species complex.

Dorsal Type locality Literature Anchor Taxon Type / main host transv Marg. Cirrus Acc. (Other referenced L W (Dactylogyrus) (Other hosts) Inner Outer bar hooks / Penis piece localities) Taxon author L Shaft Tip root root [vest.] 30-35 230- (penis) 40-80 40-48 18-22 26-31 17-19 21-26L Guegan, Lambert 400 2-3 14-16 8-10 29-34* Dactylogyrus Mali and Euzet, 1988 (60) (43) (20) (28) (18) 2-4W L. coubie (320) (basal retroversus (Senegal) bulb)

Musilová et al. 23-24L 34- - - 42 18-19 2-3 29 17-18 15-16 30-34* 2009 3W 35** 180- 22-25L Dactylogyrus 40-80 39-44 17-22 26-30 14-19 18- L. senegalensis Mali Guegan, Lambert 380 3-4 3-4W [6- 15-23 23-26* tubarius and Euzet, 1988 (60) (42) (20) (28) (16) 25** (310) 7] * = Total length of copulatory apparatus ; L = Length; w = width; Acc piece = Accessory piece; transv. = transverse; vest = vestigial bar; ** = tube trace-length (length of bulb plus penis)

Dactylogyrus Dactylogyrus Dactylogyrus Dactylogyrus Dactylogyrus retroversus Dactylogyrus falcilocus jucundus rastellus tubarius Dactylogyrus Guegan, iwani n. sp. and Guegan, Guegan and Guegan, Guegan, Diesing, 1850 Lambert and Dactylogyrus Lambert and Lambert, 1991 Lambert and Lambert and Euzet, 1988 larindae n. sp. Euzet, 1988 (Df) (Dj) Euzet, 1988 (Dr) Euzet, 1988 (Dt) (Dre) 1Dre: Larger 1 3 Dl: Bar shorter, Dactylogyrus Dr: Size genitalia, Dt: Accessory copulatory organ pseudanchoratus (smaller) and morphology of piece has simple - - morphology Price and Géry, morphology of accessory piece bifid form (trifid differs (not bifid / 1968 (Dp) anchors differ. differ, absence in Dp) trifid) of vagina. Dactylogyrus Dl: Bar not pseudanchoratus 1,2 Dpm: curved, micronchus sclerotized - - - - accessory piece Paperna, 1979 vagina absent morphology (Dpm) differs (not bifid) Dl: Bar has no 2 median knob, Dj: Male cirrus shorter, Dactylogyrus copulatory accessory piece helicophallus structures and - - - - morphology Paperna, 1973 hamuli larger; differs (Dh) shorter non- (bifurcation spiral penis. completely absent) Dl: Bar, anchor Dl: Bar, anchor Dl: Bar, anchor D. iwani n. sp. and copulatory and copulatory and copulatory and D. larindae n. organ Dl: Only 1 bar organ organ Dl: Only 1 bar sp. morphology morphology morphology differs differs differs 1 = Guegan Lambert, A. and Euzet 1988; 2 = Guegan and Lambert 1991; 3 = Guegan and Lambert 1990

Measurement values for other described Dactylogyrus spp. that also show some resemblance to D. iwani n. sp. and D. larindae n. sp. are summarized in Table 5-5.

The new species differ from the other species described in terms of size and morphology of the copulatory organ and anchors, as well as the size, morphology and number of transverse bars observed (illustrated in Table 5-6).

The two new species differ from each other based on anchor inner root structure (often more rounded in D. iwani n.sp. and more truncate in D. larindae n. sp.). Furthermore both anchor shaft length and inner root length is greater in D. iwani n. sp. compared to D. larindae n. sp., resulting in a larger total anchor length in the former. Because of the shorter inner root and shaft length, the “gape” (i.e. distance between anchor tip and anchor shaft) may appear larger in D. larindae n. sp. However, anchor aperture length was greater for D. iwani n. sp. compared to D. larindae n. sp., indicating that this is not the case. Though MCO structure is similar, the penis of D. larindae n. sp. is slightly longer than that of D. iwani n. sp. For details of statistical analyses of measured variables refer to Chapter 4.

Anchor Dorsal Type locality Literature Taxon Type / main host transv Marg. Cirr. / Acc. (Other referenced L W (Dactylogyrus) (Other hosts) Inner Outer bar hooks Penis piece localities) Taxon author L Shaft Tip root root [vest.]

250- 17-20L Dactylogyrus Labeo alluaudi Guegan and 40-80 40-45 19-21 24-28 40-46 48-54 Sierra Leone 460 1-3 12-14 3-5w 15-19 longiphalloides Pellegrin, 1933 Lambert, 1991 (60) (43) (20) (25) (370) [8-10]

220- 40- 19-23L Dactylogyrus Labeo rouaneti Guegan and 39-43 18-21 23-27 13-15 Rep Guinea 670 110 3-5 3-5w 16-23 35-42 - sematus Daget, 1962 Lambert, 1991 (41) (20) (25) (14) (370) (70) [8-10] 15-20 Guegan 240- (penis Niger, 40-70 29-35 12-16 21-24 18-12 14-17L 24- Lambert and 350 1-2 14-18 ) 6-7 Baoulé (50) (32) (14) (22) (11) 3-4w 28* Euzet, 1988 (280) (basal Dactylogyrus Labeo coubie bulb) jaculus Rüppell, 1832 Musilová, 16-19L 284- Řehulková, 79-98 33-36 14-17 2 22-24 10-12 3w 25- 26- Mali 415 15-21 and Gelnar (86) (35) (15) (1-2) (23) (11) [7-8L 27** 29* (345) 2009 1w]

Guegan et al. 350- 16-20L 18-21 Dactylogyrus Labeo parvus 40-80 32-43 15-22 23-28 10-15 Niger 1988 500 1-4 3-4w 12-16 25-32 (29- brevicirrus Boulenger, 1902 (60) (39) (18) (26) (12) Paperna, 1973 (420) [6-7] 37)

* = Total length of copulatory apparatus; L = Length; w = width; Acc piece = Accessory piece; transv. = transverse; vest = vestigial bar; ** = tube trace-length (length of bulb plus penis)

Table 5-5 (continued): Summary of hard part structure measurements (all in micrometers) of other selected species showing resemblances (in terms of hard part structures) with Dactylogyrus species listed in Table 5-2

Type Anchor Dorsal Literature Taxon Type / main host locality Out transv Marg. Cirr. / Acc. referenced L W Inner (Dactylogyrus) (Other hosts) (Other L er Shaft Tip bar hooks Penis piece Taxon author root localities) root [vest.] Labeo victorianus Boulenger, 1901 [Labeo forskalii Rüppell, 1835 Uganda Guegan and 280- Barbus cf. kersteni 40-80 35-41 16 20-26 10-13 16-19L (Kenya, Lambert 1991 480 2-3 14-20 - 31-40 Peters, 1868 (70) (38) (14-18) (23) (11) [5-8] Tanzania) Paperna, 1973 (390) Barbus altianalis (Boulenger, 1900) Labeo Cuvier, 1817 Dactylogyrus L. parvus] longiphallus Uganda 190- L. victorianus Paperna 1979 50-80 34-41 16-22 1-4 20-24 11-17 15-24 10-15 45-63 32-40 and Kenya 240 120- L. forskalii L. Albert Paperna 1979 35-40 32-35 13-15 1-4 20-23 10-12 16-18 10-15 38-40 30 250 180- 50- B. cf. kersteni Ruwenzori Paperna 1979 31-33 11-15 1-4 21-22 11-15 17-20 12-17 36-40 34-35 310 100 B. altianalis Uganda Paperna 1979 210 100 40 17 4 25 7 20 20-24 40 36 Labeo sp.1 and 220- 80- Tanzania Paperna 1979 43-44 20-25 2-5 24-25 10-14 20-22 13-22 35-38 22-28 sp.2 330 100 Dactylogyrus Barbus ablabes 210- longiphallus Ghana Paperna, 1979 70-80 37-39 18-19 1-2 20-23 8-11 22-26 11-15 27-36 12-15 (Bleeker, 1863) 240 gracilis 20-23L 218- Dactylogyrus Musilová et al. 54-66 36-40 16 26-28 16-18 2-4w 27- 26- L. coubie Senegal 309 2-3 17-24 dembae 2009 (61) (38) (15-17) (27) (17) [5-7L 34** 30* (271) 1w] 20-23L Musilová, 326- Dactylogyrus 48-75 36-42 17 26-29 12-14 3-4w 28- 28- L. coubie Senegal Řehulková, and 456 3-4 15-23 leonis (60) (39) (16-19) (28) (12) [6-8L 34** 32* Gelnar 2009 (365) 1-2w] * = Total length of copulatory apparatus; L = Length; w = width; Acc piece = Accessory piece; transv. = transverse; vest = vestigial bar; ** = tube trace-length (length of bulb plus penis)

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5.6a - Dactylogyrus pseudanchoratus 5.6b – Dactylogyrus pseudanchoratus 5.6c –Dactylogyrus helicophallus 5.6d – D. iwani n. sp. (redrawn from Paperna 1979) michronchus (redrawn from Paperna 1979) (current study) (redrawn from Paperna 1979)

5.6e – D. larindae n. sp. 5.6f – Dactylogyrus falcilocus 5.6g – Dactylogyrus jucundus 5.6h – Dactylogyrus rastellus (current study) (redrawn from Guégan Lambert and Euzet 1988) (redrawn from Guégan and Lambert 1991) (redrawn from Guégan et al. 1988)

5.6i – Dactylogyrus retroversus 5.6j – Dactylogyrus tubarius 5.6k – Dactylogyrus longiphalloides 5.6l – Dactylogyrus sematus (redrawn from Guégan et al. 1988) (redrawn from Guégan et al. 1988) (redrawn from Guégan and Lambert 1991) (redrawn from Guégan and Lambert 1991)

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Table 5-6 (continued): Summary of morphometric variation in hard part structures of selected species within / comparable to the Dactylogyrus pseudanchoratus species group.

5.6m - Dactylogyrus jaculus 5.6n – Dactylogyrus brevicirrus 5.6o – Dactylogyrus longiphallus 5.6p – Dactylogyrus dembae (redrawn from Guégan et al. 1988) (redrawn from Guégan et al. 1988) (redrawn from Guégan and Lambert 1991) (redrawn from Musilova et al. 2009)

5.6q – Dactylogyrus leonis (redrawn from Musilová, Řehulková and Gelnar, 2009)

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5.3.3.3. Species C (Dactylogyrus nicolettae n. sp., Figure 5-3)

Type hosts and locality: Labeo capensis, Vaal Dam (Vaal River System) close to University of Johannesburg Island, S 26 52.249, E 28 10.249, Gauteng Province, South Africa.

Site: Gill lamellae

Type specimens: Holotype: SAM A61789 - one whole mounted specimen in glycerine jelly.

Paratypes: SAM A61790 and SAM A61791 - two specimens in GAP and glycerine jelly

Material examined: 10 specimens in Malmberg’s solution and glycerine jelly (all flattened to some degree).

Description: Body length 232.1 (169.0-285.7); greatest width 65.5 (42.9-90.5). Single pair of stout (i.e. appear short and thick) anchors with rounded inner root. Inner root with a characteristic bend (bending towards the hook tip) and outer root absent or greatly reduced. Total anchor length 31.7 (28.6-34.5); anchor shaft length 18.8 (117.3-21.4); anchor outer root length 0.3 (0-1.4); anchor inner root length 19.4 (15.5-20.9); anchor point length 11.1 (8.2-12.7); anchor aperture 14.2 (11.8-15.5). One dorsal bar with median knob present. Dorsal bar length 17.5 (14.1-19.1); dorsal bar width 3.2 (1.8-4.5). Seven pairs of marginal hooks. Total marginal hook length 14.9 (13.6-16.9). MCO consists of curved penis and simple accessory piece (shaft with widening / flaring terminal section). Accessory piece length 14.9 (13.6 - 16.8); penis length (tube trace length) 21.8 (19.1 - 22.7).

Remarks: It is the distinct shape (i.e. robust appearance with short, thick shaft and thick inner root, the latter with a distinct bend, outer root reduced or absent) of the anchors (Figure 5-3) that most clearly differentiate this parasite from other Dactylogyrus spp. described from Labeo hosts. The transverse bar also has a distinct median knob. The copulatory organ is very similar to that described for D. iwani n. sp. and D. larindae n. sp. (both in terms of general morphology and measurements). This is very uncharacteristic, as closely related monogenean species are often differentiated based on differences in copulatory organ morphology (e.g. Paraguassú, Luque and Alves 2002; Tingbao, Gibson and Bijian 2005).

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The ridge originating from the accessory shaft is however less distinct, apparently curving around the edge of the flaring distal end of the accessory piece.

5.3.3.4. Species D (n. sp., Figure 5-4)

Type hosts and locality: Labeo umbratus, Vaal Dam close to University of Johannesburg Island, S 26 52.249, E 28 10.249, Gauteng Province, South Africa.

Other records: None (new description)

Site: Gill lamellae

Material examined: 11 specimens in GAP and glycerine jelly (all flattened to some degree).

Description: Body length 238.1 (159.5-288.1); greatest width 80.4 (35.7-129.8). Single pair of anchors with distinct shape (see discussion): total length 40.8 (36.4- 43.6); inner root 18.8 (13.2-22.5); outer root 2.2 (1.6-2.7); shaft 24.8 (23.0-25.9); tip 13.0 (11.6-14.8). One transverse bar: 19.1 (14.5-21.8) long; 4.3 (3.6-5.5) wide. Marginal hooks: seven pairs, similar in shape and size; hook lengths (based on average values for each of the seven pairs measured) 18.6 (10.9-23.6). Sclerotized vagina not observed.

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The copulatory organ consists of a tube (cirrus), a predominantly “v-shaped”, seemingly articulated accessory piece consisting of two sections, an associated “flange” and third section varying in position. Accessory piece lengths: Length of two respective sections forming the “v” shape) 10.2 (7.3-11.8) and 9.6 (6.4-11.8) respectively; length of associated section 8.4 (7.3-10.0); greatest width of “flange” 4.1 (3.6-5.5); cirrus tube-trace length 30.5 (22.7-37.3).

Remarks: In terms of general anchor shape, this parasite (species D n. sp.) more closely resembles D. iwani n. sp. (and to a lesser extend D. larindae n. sp.) than D. nicolettae n. sp., all from the same locality. There is however a number of distinct characteristics that distinguish species D n. sp. from other Dactylogyrus spp. described from Labeo hosts thus far. The first relates to anchor morphology which is reminiscent of the anchor morphology of D. pseudanchoratus micronchus (Paperna 1979). In the majority of other species the anchor shaft bends evenly as it turns upwards to form the anchor tip. (i.e. “smoothly” rounded gape bend). In species D there are often two slight “kinks” or sharper bends visible within the gape bend: the first where the shaft starts curving at the base of the anchor bend and the second where the bend turns upwards towards the tip (Figure 5-4). This is more distinct in some specimens. These “kinks” are a result of a thickening of the shaft at the base of the root, followed by a second thickening of the shaft where it turns into the hook bend. The second more significant difference relates to copulatory organ morphology. Once again (as was the case with other Dactylogyrus spp. described from the same locality and host species) a curved / slightly coiled penis with basal bulb is evident. The structure of the accessory piece however differs completely in that it has a distinct v- shape (Figure 5-4). This “v” shape was clearly visible in most specimens. However, two other structures that appear less distinct were also observed. The first would best be described as a “flange” covering the “v-shaped” area described previously. The other structure is similar in length and appearance to the sections forming the “v” shape. It appears to be associated with one of these sections but the position in which it was observed, varied greatly (Figure 5-5). In similar fashion the position of the cirrus relative to the copulatory organ also varied. No sclerotized vagina observed. It must also be noted that the general shape of the bar differs in appearance depending from what angle it is observed. It often appears slightly bent with a knob-like projection protruding from the middle in a lateral view.

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From a dorsal view the knob-like projection may appear to be a central, ridge-like structure running over the width of the bar, while it may appear less distinct from yet another angle (Figure 5-6).

Figure 5-4: Dactylogyrus sp. D n. sp.: A = Anchors; B = Dorsal bar; C = Marginal hooks; D = Male copulatory organ (MCO); i to iv = Marginal hook position (following Guegan and Lambert 1991).

Figure 5-5: Dactylogyrus sp. D n. sp.: Illustration depicting variations observed in terms of male copulatory organ (MCO) sclerite positions.

Figure 5-6: Dactylogyrus sp. D n. sp.: Illustration depicting variations observed in terms of transverse bar orientation.

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5.3.4. Dogielius Bychowsky, 1936

Only one species of Dogielius, collected from both L. umbratus and L. capensis, was recorded from the Vaal Dam. Two apparent forms (differing in the size but not shape of haptoral sclerites and MCO) of the same species exhibiting host preference were encountered (see Chapter 4).

5.3.4.1. Species E (Dogielius intorquens n. sp., Figures 5-7 and 5-8)

Type hosts and locality: Labeo umbratus, Vaal Dam (Vaal River System) close to University of Johannesburg Island, S 26 52.249, E 28 10.249, Gauteng Province, South Africa.

Other hosts: Labeo capensis, Vaal Dam (Vaal River System) close to University of Johannesburg Island, S 26 52.249, E 28 10.249, Gauteng Province, South Africa.

Site: Gill lamellae

Material examined: 11 specimens from L. capensis and 11 specimens from L. umbratus in Malmberg’s solution and glycerine jelly (all flattened to some degree).

Description: For measurements of specimens recorded from the two host species respectively, refer to Table 5-7. Species description combined for both host species as follows: Body length 257.0 + 44.3 (169.0-333.3); greatest width 65.9 + 14.2 (38.1- 92.9). Single pair of anchors. Inner root of anchor subdivided to form a median root in addition to the inner and outer roots. Total anchor length 29.2 + 3.2 (22.7-34.1); anchor shaft length 37.0 + 4.4 (30.0-44.3); anchor point length 23.1 + 3.1 (16.8- 28.2); anchor aperture 35.7 + 4.2 (27.7-41.35). One smooth, evenly curved dorsal bar with rounded ends containing distinctly shape “ridges” or “sockets” in which the anchors presumably articulates. Dorsal bar length 44.9 + 5.4 (33.2-52.7); dorsal bar width 5.5 + 1.0 (3.2-7.3). Seven pairs of marginal hooks (range based on arithmetic averages for all marginal hook pairs) 17.8 + 2.9 (13.4-19.7). MCO has a distinctly “twisted” appearance, particularly at the base of the structure. Consists of slightly curved penis and simple accessory piece (shaft with widening / flaring terminal section). Accessory piece shaft bifurcates. Curved, slightly hook-like structure lies adjacent to the termination of the whip-shaped cirrus and terminates against bifurcated accessory piece (often flaring slightly at the base).

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Second similar structure (without terminal hook shape of the first structure) originates from the distal termination of the same bifurcated accessory piece shaft. Accessory piece length 25.5 + 5.1 (16.0-30.9); penis length (tube trace length) 23.0 + 5.5 (13.6 - 30.9).

Remarks: The size and morphology of the bar and anchors are very similar to that of Dogielius kabaensis Guégan and Lambert, 1991, while the structure of the copulatory organ and sclerotized vagina also shares some resemblances. The new species, however, exhibits a single accessory piece “shaft”, while the accessory piece in D. kabaensis divides proximally (closer to the penis bulb) to form two “shafts” (i.e. double accessory piece). The distal morphology of the MCO shares a number of similarities. The one accessory shaft in D. kabaensis bifurcates to form two distinct structures, while the other widens to form a flaring structure. Guégan and Lambert (1991) compared D. kabaensis to Dogielius junorstrema victorianus Paperna, 1979 and Dogielius parvus Guégan, Lambert and Euzet, 1989. The new species differ from D. junorstrema victorianus and D. parvus in the morphology of the dorsal bar and the fact that D. intorquens does not have a double accessory piece. The total length range of the marginal hooks is, however, similar to that of D. junorstrema victorianus. The bar and anchor morphology of the new species is also similar to that of Dogielius dublicornis Paperna, 1973, but the structure of the copulatory organ (particularly the distal structure of the accessory piece) and bar length (much longer in D. dublicornis) differ. In some specimens the anchor shaft exhibited a more distinct bend directly below the roots, resulting in variation in general anchor shape. However, as such variation was even observed between the two anchors of a single worm, it is interpreted as an artefact of the mounting and fixing process (i.e. pressure exerted on the cover slip). Furthermore, while size differences between the apparent forms were noted, general shape appeared similar (Figures 5-7 and 5-8). The measurements for the Dogielius sp. specimens from L. umbratus all appear slightly larger compared to that recorded for L. capensis. The differences hint at two parasite forms of the same species exhibiting a clear host preference. As onchomiracidia that hatch from eggs have an equal chance to attach to either host species, an active host selection / preference mechanism is considered unlikely.

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Rather it is postulated that differences in gill filament and gill lamellae morphology result in different rates of sclerite development (and hence size) on the respective host species (preliminary measurements indicate that the finer gill structure of L. capensis may indeed be more robust, Appendix C). However, additional morphometric analyses of both gills and parasite sclerite measurements, preferably combined with an experimental infection study, needs to be performed to further investigate this hypothesis. It is also conceivable that chemical cues may attract or repulse potential parasites and such reactions may form the basis of host selection (e.g. Buchmann, Madsen and Dalgaard 2004).

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Table 5-7: Summary of hard part structure measurements (all in micrometers) of other selected species of Dogielius Bychowsky, 1936 showing resemblances Dogielius intorquens n. sp.

Anchor Dorsal bar Acc. piece Type locality Length Cirrus / Taxon Type / main host Literature referenced Marg. (Copu- (Other Width Penis (Dogielius) (Other hosts) Taxon author Length Shaft hooks latory localities) Tip (e) Length Width (Mean) (a) (b) apparatus) Paperna 1979 Dogielius Labeo Rhodesia Price and Yurkiewicz, 178-243 46-72 - - - 47-55 - 17-29 19-24 17-22 junorstrema Cuvier, 1817 (Zimbabwe) 1968 Labeo victorianus Dogielius Boulenger, 1901 junorstrema (Barbus altianalis Kenya Paperna, 1979 140-230 50-80 34-37 38-40 4-6 44-49 - 13-21 36-42 33-43 victorianus (Boulenger, 1900))

Dogielius Labeo cylindricus junorstrema Peters, 1868 Tanzania Paperna, 1979 150-210 70-100 32-37 35-36 - 42-48 - 17-22 22-30 16-25 ruahae Labeo sp. 1

Dogielius Paperna 1979 L. cylindricus Tanzania 150-200 80 38-40 38-40 4-5 60-70 - 12-15 45-55 15-18 dublicornis Paperna, 1973

Labeo parvus The Republic 330-420 60-80 33 (30- 43(41- 16(14- 19-22 50-56 5-7 16-22 22-30 Boulenger, 1902 of Guinea (370) (70) 36) 45) 17) (28-31) Dogielius Guegan and Lambert, kabaensis 1991 Labeo alluaudi Bagbwe, Loffa 280-380 50-80 20-24 28-30 34-37 17-20 40-42 4-5 16-20 25-30 Pellegrin, 1933 Basin (320) (60) (30-32)

Dogielius Labeo parvus Guegan, Lambert and 250-350 60-90 29-33 33-38 18-22 Mali 38-47 5-9 16-21 25-30 (31-34) parvus Boulenger, 1902 Euzet, 1989 (300) (70) (31) (35) (20)

Crafford, Luus-Powell Labeo capensis 169-279 48-83 23-31 31-39 17-28 33-47 3-6 11-23 South Africa and Avenant- - (22-31) (Smith, 1841) (231) (63) (27) (33) (23) (41) (5) (13-20*) Dogielius Oldewage, 2012 intorquens n. Crafford, Luus-Powell sp. Labeo umbratus 250-369 38-93 29-34 37-44 18-28 46-53 5-7 12-23 South Africa and Avenant- - (21-30) (Smith, 1841) (285) (69) (32) (41) (24) (49) (6)* (13-21*) Oldewage, 2012

L = Length; W = Width, Marg hooks = Marginal hooks; Acc piece = Accessory piece; * = range of average values for each respective marginal hook pair

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Table 5-8: Summary of morphometric variation in hard part structures of selected species of Dogielius Bychowsky, 1936 comparable to Dogielius intorquens n. sp.

10a – Dogielius junorstrema victorianus 10b – Dogielius junorstrema ruahae 10c – Dogielius dublicornis 10d – Dogielius kabaensis (redrawn from Paperna 1979) (redrawn from Paperna 1979) (redrawn from Paperna 1979) (redrawn from Guégan and Lambert 1991)

10e – Dogielius parvus 10f – Dogielius intorquens (redrawn from Guégan et al. 1989) (Current study)

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5.3.5. General discussion and conclusion

Morphologically D. iwani n. sp. and D. larindae n. sp. appears to be intermediate between D. helicophallus and D. pseudanchoratus micronchus, supporting Paperna’s (1979) notion of a species complex consisting of a range of intermediate forms. While descriptions of new species within apparent species complexes have been published (e.g. Hinsinger and Justine 2006) and is considered valid in this case, the need for further studies to quantify ranges of variability / existence of further intermediate forms is acknowledged. Barson, Přikrylová, Vanhove and Huyse (2010), for example, warn that previously described species-complexes may in fact be complexes of hybrid species, a possibility that could only be investigated by means of geno-typing. In ecological studies where thousands of worms are handled, molecular identification of each respective individual parasite is in practice impossible (e.g. Blažek, Jarkovský, Koubková and Gelnar 2008b). For monogeneans in general (e.g. Pariselle et al. 2003; Přikrylová and Gelnar 2008) and dactylogyrids in particular (e.g. Musilová et al. 2009), identification and description are based on measurements and morphology of sclerotized hard parts. This is reflected by the fact that most higher-level monogenean diversity relates principally to morphological specialization for attachment by the haptor (Cribb, Chisholm and Bray 2002). Yet a large degree of morphometric variability is also observed within species of Dactylogyrus (e.g. Šimková, Pečínková, Řehulková, Vyskočilová and Ondračková 2007). Paperna (1979) implied the existence of D. pseudanchoratus / D. helicophallus as well as D. junorstrema complexes. Guégan and Lambert (1991) compare D. kabaensis to D. junorstrema victorianus and mention similarities of morphology and size. They then continue to make distinction between two forms of D. kabaensis from two Labeo spp., stating that they probably represent a complex of species which is difficult to characterize with usual anatomical criteria. This may well be the case for D. intorquens n. sp. found on both L. capensis and L. umbratus in the current study.

Future studies could focus on larger, more frequent sample sizes possibly combined with measurements of additional morphological variables (e.g. Mizelle and Kritsky 1967a) and molecular work (e.g. Šimková, Morand, Jobet, Gelnar and Verneau 2004; Šimková, Ottavá and Morand 2006; Přikrylová, Matĕjusová, Jarkovský and Gelnar 2008; Přikrylová et al. 2009a; Přikrylová et al. 2009b).

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Molecular phylogeny of monogenean parasites is receiving increasing levels of attention as morphological based taxonomy of highly derived parasite groups is likely to poorly reflect their evolutionary relationships (Perkins, Donnellan, Bertozzi, Chisholm and Whittington 2009). Once the relationship of these parasites found on both L. capensis and L. umbratus in the Vaal river system has been clarified and possible seasonal trends in measurement variations evaluated, the same can be done for material from the apparently related species complexes from elsewhere in Africa for comparison.

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CHAPTER 6

6 - ASPECTS OF THE ECOLOGY OF MONOGENEAN PARASITES OF LABEO CAPENSIS (Smith, 1841) AND LABEO UMBRATUS (Smith, 1841) IN THE VAAL DAM, GAUTENG PROVINCE, SOUTH AFRICA COLLECTED DURING A WINTER SURVEY

6.1. INTRODUCTION

Ecological aspects of monogenean fauna of the moggel (Labeo umbratus (Smith, 1841)) and the Orange River mudfish (Labeo capensis (Smith, 1841)) in the Vaal Dam, as recorded during a winter sampling survey, is reported on in this chapter.

Both species are endemic to South Africa with L. umbratus having a slightly wider distribution compared to L. capensis (the latter being restricted to the Orange-Vaal River systems) (Skelton 2001). Labeo umbratus prefers standing or gently flowing water and thrives in shallow impoundments (e.g. farm irrigation dams where they reproduce successfully (Skelton 2001). In similar fashion L. capensis prefers running waters (i.e. rivers) but also does well in large impoundments where it may enjoy a greater power of dispersal due to spawning habits (does not require longitudinal spawning migration) (Tómasson, Cambray and Jackson 1984; Skelton 2001). Both are bottom feeders, with L. umbratus feeding on detritus and soft sediment while L. capensis prefers to graze from the firm surfaces of rocks and plants (Froese and Pauly 2011).

Bush, Fernández, Esch and Seed (2001) state that host species that share common ancestors are likely to share morphological attributes, occupy similar niches, be subject to similar evolutionary constraints and therefore might also exhibit similarities with regard to parasite communities harboured. As outlined in Chapters 1 and 2 the aim of the project is to identify and describe the monogenean fauna of fish in the Vaal dam (Vaal River system), collect baseline data in order to identify future research needs and in doing so to stimulate further research on freshwater monogenean parasites in South Africa.

Within this larger framework the objectives of the current chapter are defined as follows:

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(1) Examine and compare aspects of monogenean biology, in terms of infection statistics and site preference as determined during a winter survey, within an ecological framework (i.e. numbers and distribution of parasites); (2) Discuss the possible effects of selected host and environmental variables on patterns of numbers and distribution identified above.

6.2. MATERIALS AND METHODS

For study site description refer to section 3.1.

For fish collection, necropsy and parasite recovery procedures refer to sections 3.2 and 3.3. The current chapter only reports on data collected during the winter (June / July 2009) survey.

For details on parasite identification and description refer to Chapter 4 and Chapter 5.

In this chapter species code names (i.e. A to E), as used in Chapter 4, are replaced with the scientific names where applicable (i.e. A, B, C and E) as allocated in Chapter 5. For a complete tabulated summary of species codes (and taxonomic status / scientific names where applicable) used in this manuscript, refer to Chapter 13.

For details on calculation of infection statistics refer to sections 3.5 and 2.3.3.

For details on statistical tests employed refer to section 3.6.3.

6.3. RESULTS AND DISCUSSION

6.3.1. Host species

Twenty-six specimens (of which 21 were male) of L. umbratus were collected, compared to 13 (of which four were male) L. capensis. Table 6-1 summarizes average weight, total length and condition factor values for fish hosts collected. Table 6-2 summarizes the gender distribution within length class categories. The majority of L. umbratus (n=16) resided within the > 40 < 45 cm length class, making this the length class within which the majority of fish (n = 22) resides when combining data for the two host species.

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6.3.2. Parasite species

Representatives from the monogenean genera Dactylogyrus Diesing, 1850, Dogielius Bychowsky, 1936 and Diplozoon von Nordmann, 1832 were collected. Specimens from the former two genera were previously discussed in Chapters 4 and 5. Please note that for Dactylogyrus only Dactylogyrus iwani Crafford, Luus-Powell and Avenant-Oldewage, 2012, Dactylogyrus larindae Crafford, Luus-Powell and Avenant-Oldewage, 2012 and Dactylogyrus nicolettae Crafford, Luus-Powell and Avenant-Oldewage, 2012 (i.e. species A to C) were used for analysis as very few (n=2) specimens of species D were encountered during the winter survey. Throughout this chapter we shall only refer to Diplozoon sp. as description thereof forms part of another project.

Table 6-1: Summary description of sampled Labeo Cuvier, 1817 population from the Vaal Dam.

Host Labeo umbratus (Smith, 1841) Labeo capensis (Smith, 1841) Average Average Average Average Average Average length condition length condition Variable Number weight (g) Number weight (g) (cm) factor (cm) factor * [SD] [SD] [SD] [SD]* [SD] [SD] 43.17 0.94 38.30 0.85 Male 21 781 [293] 4 475 [96] [3.53] [0.13] [1.19] [0.17] 1030 45.22 0.95 40.07 1.02 Female 5 9 672 [179] [741] [6.59] [0.33] [3.11] [0.09] 43.56 0.94 39.52 0.97 Combined 26 829 [408] 13 612 [180] [4.19] [0.17] [2.74] [0.14] * = Fulton’s condition factor: K = 100 x W / L3 (calculated for all individual fish with average value determined using calculated condition factor values) Key SD = Standard deviation Average length = calculated using individual total fish length

Table 6-2: Length category and gender distribution of sampled Labeo Cuvier, 1817 population from the Vaal Dam.

Number of fish collected

Length category Labeo umbratus (Smith, 1841) Labeo capensis (Smith, 1841)

Male Female Male Female

> 35 to < 40 cm 1 1 4 5

> 40 to < 45 cm 16 2 0 4

> 45 to < 50 cm 3 0 0 0

> 50 to < 55 cm 1 2 0 0

Total 21 5 4 9

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Skelton (2001) states (referring to standard lengths) that L. umbratus reach 10 cm after a year, with males reaching 15 cm and females 25 cm after 2 to 3 years when they reach sexual maturity. The fish attains a length of 50 cm (total length) and reach ages of five to six years (Skelton 2001). From this we can deduce that all L. umbratus examined in this study were between three and six years of age. Potts, Booth, Hecht and Andrew (2006), examining L. umbratus in Eastern Cape (South Africa) reservoirs, however, found fish of 350 mm in length to be between six and ten years old depending on locality.

The age range of fish in this study may thus be anything between three to greater than 10 years.

6.3.3. Biological and ecological aspects

6.3.3.1. Comments on statistical tests employed

Data analysis methodology is outlined in section 3.6.3. In the majority of cases results of the T-test (parametric independent samples test) and Mann-Whitney test (non-parametric test), applied to weight, length and parasite data per host species, correlate with regard to different host species. It was thus concluded that use of parametric tests is justified in this case.

6.3.3.2. Infection statistics and host specificity

This paper investigates four monogenean parasite species recorded from Labeo spp. in the Vaal Dam. Three species (D. iwani, D. larindae and D. nicolettae as described in Chapter 5) belonged to the genus Dactylogyrus, considered to be the largest helminth genus (Gibson, Timofeeva and Gerasev 1996). The other two species belongs to the genera Diplozoon and Dogielius (Dogielius intorquens Crafford, Luus-Powell and Avenant-Oldewage, 2012 as described in Chapter 5) respectively.

The species D. iwani and D. larindae occurred on both L. capensis and L. umbratus but exhibited a clear host preference. Diplozoon sp. were found predominantly on L. umbratus but also occurred on L. capensis (single specimen) while D. intorquens was also found on both host species. In comparison D. nicolettae was only recovered from L. capensis.

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Infection statistics and inter-specific associations / correlations are summarized in Tables 6.3 (L. umbratus) and 6.4 (L. capensis). Parasite infection data were not normally distributed for both L. capensis and L. umbratus (Shapiro-Wilk test). Unequal variance between species was found in all instances (Levene statistic). Differentiation between D. iwani and D. larindae from the two fish host species is clear when comparing prevalence, mean intensity (most pronounced) and mean abundance values for these parasites. Whilst D. nicolettae was only found on L. capensis, it exhibits an intermediate prevalence and mean intensity compared to that recorded for D. iwani and D. larindae. In turn D. intorquens was found on both hosts and was the most prevalent parasite on L. capensis. An opposite trend was observed for the Diplozoon sp. (most prevalent on L. umbratus). On L. umbratus there was a strong positive correlation between D. larindae and D. intorquens, but a slight negative correlation between the former species and Diplozoon sp. The latter trend was also observed in L. capensis. There were, however, apparently stronger positive inter-monogenean species correlations on this host. Dactylogyrus iwani was positively correlated with D. nicolettae, while D. intorquens was positively correlated with both aforementioned species. These correlations are obviously based solely on statistical analyses, but biological relevance (or the lack thereof) is further discussed in section 6.3.4.3.

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Table 6-3: Monogenean parasite infection statistics for species of Labeo Cuvier, 1817 from the Vaal Dam: Labeo umbratus (Smith, 1841).

Host species Labeo umbratus (Smith, 1841) Monogenean species Dl a Dl b Dl c Dl spp. D e Dip. sp. ?* CC Distribution Total 8 332 0 16 51 53 28 488 1 4 81 0 1 10 26 7 129 2 2 99 0 7 12 4 5 129 Gill arch number 3 1 89 0 4 20 6 10 130 4 1 63 0 4 9 17 6 100 Left 0 142 0 3 27 25 17 214 Gill set Right 8 190 0 11 24 28 11 272 (side of head) Side unknown 0 0 0 2 0 0 0 2 Dorsal (D) 5 85 0 1 16 3 8 118 Medial (M) 3 129 0 2 21 4 8 167 Gill arch region Ventral (V) 0 106 0 4 11 4 4 129 Unknown 0 12 0 9 3 42 8 74 Anterior 2 166 0 4 29 - 9 210 Gill orientation Posterior 3 145 0 3 16 - 11 178 Unknown 3 21 0 9 6 53 8 100 Infection levels Prevalence (%) 19.2 38.5 0.0 34.6 38.5 57.7 26.9 76.9 Mean intensity 1.6 33.2 0.0 1.8 5.1 3.5 4.0 24.4 Intensity range: Minimum 1 4 0 1 1 1 1 1 Intensity range: Maximum 3 110 0 3 16 7 6 141 Mean abundance 0.3 12.8 0.0 0.5 2.0 2.0 1.1 18.8 P (%) 19.1 33.3 0.0 28.6 33.3 71.4 28.6 76.2 Host gender: Male MI 1.8 38.7 0.0 1.3 5.9 3.6 3.3 25.2 MA 0.3 12.9 0.0 0.4 2.0 2.6 1.0 19.2 P (%) 20.0 60.0 0.0 60.0 60.0 0.0 20.0 80.0 Host gender: MI 1.0 20.3 0.0 1.7 3.3 0.0 2.0 20.5 Female MA 0.2 12.2 0.0 1.0 2.0 0.0 0.4 16.4 Inter-specific associations (Spearman’s rho test correlation) Dactylogyrus iwani Crafford, Luus- 1.000 0.678 0.678 - 0.540 0.021 - - Powell and Avenant-Oldewage, 2012 Dactylogyrus larindae Crafford, Luus- - 1.000 1.000 - 0.818 -0.200 - - Powell and Avenant-Oldewage, 2012 Dactylogyrus nicolettae Crafford, Luus-Powell and Avenant-Oldewage, ------2012 Dactylogyrus sp. D - - - - 0.468 -0.201 - - Dogielius intorquens Crafford, Luus- - - - - 1.000 0.035 - - Powell and Avenant-Oldewage, 2012 Diplozoon von Nordmann, 1832 - - - - - 1.000 - - Key (species codes used) Dl a – Dactylogyrus iwani Dl b – Dactylogyrus larindae Dl c – Dactylogyrus nicolettae D e – Dogielius intorquens Dip sp. – Diplozoon. Dl spp. - Dactylogyrus spp. not identified *? – Specimens that could not be to species level (damaged / lost during identified to genus level (damaged / lost CC = Component community mounting) during mounting)

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Table 6-4: Monogenean parasite infection statistics for species of Labeo Cuvier, 1817 from the Vaal Dam: Labeo capensis (Smith, 1841).

Host species Labeo capensis (Smith, 1841) Monogenean species Dl a Dl b Dl c Dl spp. D e Dip. sp. ?* CC Distribution Total 194 17 30 33 36 1 14 325 1 46 6 4 4 7 0 2 69 2 51 3 8 15 13 1 6 97 Gill arch number 3 68 4 12 9 14 0 6 113 4 29 4 6 5 2 0 0 46 Left 86 6 12 13 22 1 9 149 Gill set Right 108 11 18 20 14 0 5 176 (side of head) Unknown 0 0 0 0 0 0 0 0 Dorsal (D) 59 6 9 3 13 0 3 93 Medial (M) 76 8 12 11 13 0 3 123 Gill arch region Ventral (V) 43 2 8 6 6 0 0 65 Unknown 16 1 1 13 4 1 8 44 Anterior 94 9 21 3 18 0 5 150 Gill orientation Posterior 84 7 7 6 14 0 1 119 Unknown 16 1 2 24 4 1 8 56 Infection levels Prevalence (%) 76.9 61.5 38.5 46.2 84.6 7.7 38.5 100 Mean intensity 19.4 2.1 6.0 6.0 3.3 1.0 2.8 25.2 Intensity range: Minimum 5 1 2 1 1 1 1 3 Intensity range: Maximum 50 5 10 18 8 1 5 75 Mean abundance 14.9 1.3 2.3 2.8 2.8 0.1 1.1 25.2 P (%) 75.0 75.0 50.0 25.0 75.0 0.0 25.0 100 Host gender: Male MI 23.3 2.3 7.5 1.0 4.0 0.0 3.0 27.0 MA 17.5 1.75 3.8 0.3 3.0 0.0 0.8 27.0 P (%) 77.8 55.6 33.3 55.6 88.9 11.1 44.4 100 Host gender: MI 17.7 2.0 5.0 7.0 3.0 1.0 2.8 24.4 Female MA 13.8 1.1 1.7 3.9 0.1 2.7 1.2 24.4 Inter-specific associations Dactylogyrus iwani Crafford, Luus- 1.000 0.765 0.785 - 0.790 -0.389 - - Powell and Avenant-Oldewage, 2012 Dactylogyrus larindae Crafford, Luus- - 1.000 0.482 - 0.664 -0.328 - - Powell and Avenant-Oldewage, 2012 Dactylogyrus nicolettae Crafford, Luus-Powell and Avenant-Oldewage, - - 1.000 - 0.721 -0.220 - - 2012. Dactylogyrus Diesing, 1850 ------Dogielius intorquens Crafford, Luus- - - - - 1.000 -0.234 - - Powell and Avenant-Oldewage, 2012. Diplozoon von Nordmann, 1832 - - - - - 1.000 - - Key (species codes used) Dl a – Dactylogyrus iwani Dl b – Dactylogyrus larindae Dl c – Dactylogyrus nicolettae D e – Dogielius intorquens Dip sp. – Diplozoon sp. Dl spp. - Dactylogyrus spp. not identified *? – Specimens that could not be to species level (damaged / lost during identified to genus level (damaged / lost CC = Component community mounting) during mounting)

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6.3.3.3. Effect of host variables

Tables 6-1 and 6-2 summarize host length, weight and gender data. A Cramer’s V value of 0.491 (p=0.002) was calculated, indicating a strong association between host gender and host species. As gender ratio and sample sizes differ between host species collected, the observed association is most probably an artefact resulting from the sampling method employed. Length and weight data was found to be normally distributed for L. capensis but not for L. umbratus (Shapiro-Wilk test). Equal variance between species was found for length data but not weight data (Levene statistic). There was a strong correlation between weight and length (standard, fork and total) data for both species (Spearman’s rho test), indicating that use of only total length in subsequent discussions is warranted.

Infection levels (number of parasites collected) in terms of site selection / distribution on gills, host gender and host size classes are summarized in Tables 6-3 and 6-4. For all parasite species, more parasites were found on the second and third gill arches. This trend (i.e. unequal proportion of parasites) was statistically significant (Pearson Chi Square, p=0.002) for L. umbratus where most parasites were generally found on the third gill arch. Slightly more parasites were found on the right set of gill arches (except for D. intorquens). This trend was however not statistically significant (Pearson Chi Square, p > 0.2). Furthermore, for all parasite species, most parasites were found on the medial position of the gill arch (Pearson Chi Square, p = 0.000 for both host species) while more parasites were recovered from the anterior hemibranch (Pearson Chi Square, p = 0.000 for both host species). With the exception of Diplozoon sp., infection statistics were generally higher for female hosts compared to male hosts. This trend was however not statistically significant (Mann- Whitney U test, p = 0.200). While it would appear that the first two size classes were most heavily parasitized, this is probably an artefact resulting from the sampling effort as the last two size classes contained few fish.

6.3.3.4. Environmental variables

Selected physical and other water quality parameter values are summarized in Table 6-5. Water analysis data was obtained from Rand Water (sampling point reference C-VD1l, monthly water sampling).

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As samples were not taken on the precise dates of fish host sampling, results are provided for analyses conducted within approximately two months prior to the sampling effort as well as two months thereafter.

Table 6-5: Environmental (physical) and water quality variables from the Vaal Dam (source: Rand Water, sampling point identification number C-VD1l).

DO Temp Cond TDS CaCO3 Cr Cu Pb Date pH (%) (°C) (mS/m) (mg/L) (mg/L) (µg/L) (µg/L) (µg/L) 26 May 09 57.2 15.8 20.0 140.0 8.2 59.0 < 10.0 < 10.0 13.0 23 June 09 76.6 17.3 17.0 150.0 7.0 65.0 < 10.0 10.0 < 8.0 28 July 09 60.2 16.5 <1.0 ** 145.0 8.1 68.0 < 10.0 10.0 < 8.0 25 Aug 09 63.8 19.6 21.0 145.0 7.7 66.0 < 10.0 10.0 < 8.0

DO = Dissolved oxygen; Temp = Temperature; Cond = Conductivity; CaCO3 = Calcium carbonate; Cr = Chrome; Cu = Copper; Pb = Lead.

6.3.4. General discussion

6.3.4.1. Host species: condition factor values and macroscopic pathology

For comprehensive reviews on Fulton’s condition factor used in this study, refer to Froese (2006) and Nash, Valencia and Geffen (2006). While more female L. umbratus were collected compared to males, there were no significant difference (p=0.856, t-test) in mean condition factor values between genders (Table 6-1). L. capensis on the other hand, exhibited a slightly lower mean condition factor for males compared to females. This difference was statistically significant (p=0.026, t- test). Differences between genders with regards to infection statistics were slight (Tables 6-3 and 6-4). It is thus unlikely that parasite infections observed influenced condition factor values. Some studies do show a negative correlation between condition factor values and monogenean infection (e.g. Stojanovski, Hristovski, Cakić, Cvetkovic, Atanassov and Smiljkov 2009). However, monogenean infections in wild (e.g. Cusack 1986; Le Roux, Avenant-Oldewage and van der Walt 2011) and even some cultured (e.g. Cone and Cusack 1988) populations often have little negative impact on host variables such as growth and survival and often produce no clinical signs of disease. This generalization obviously does not ring true for parasites like Gyrodactylus salaris Malmberg, 1957 in geographical areas outside its native range (e.g. Scholz 1999).

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The interaction between parasitism, water contamination and condition factor is complex. It is influenced not only by variables such as environmental and other ecological factors (Thomas, Guégan, Michalakis and Renaud 2000; Arafa, El- Naggar and El-Abbassy 2009), physical and chemical factors, health status and susceptibility of hosts (Escher, Wahli, Büttner, Meier and Burkhardt-Holm 1999), host gender (Pervin and Mortuza 2008), but in all probability also host genetic and physiological factors (Lamková, Šimková, Palíková, Jurajda and Lojek 2007; Tavares-Dias, Moraes and Martins 2008). The results of Galli, Crosa, Mariniello, Ortis and D’ Amelio (2001) for example suggest that host condition factor was affected by neither infection indices nor pollution level. Given the variability in parasite / condition factor correlations observed in the literature, identification of a correlation does thus not necessarily translate into a “cause-and-effect” relationship. Variation in the condition factor also reflects the state of sexual maturity and the degree of nourishment (Williams 2000; Froese 2006). The fact that Ali, Iqbal, Rana, Athar and Iqbal (2006) found condition factor not to be influenced by feeding regime in terms of feed cycling, may be an indication that it may be a better measure of the state of sexual maturity. The current study survey was conducted in winter while both species reproduce in spring and early summer. Females were thus not burdened with the physiological stress of egg production and spawning which may explain the condition factor results (i.e. very little difference between genders) obtained.

Macroscopic pathology / abnormal conditions resulting from monogenean infection may include sporadic haemorrhages (Martins, Moraes, Fujimoto, Onaka, Nomura, Silva and Schalch 2000) and pale gills with excessive mucous secretion (Buchmann 1999; Arafa et al. 2009). Mucous secretion appears to play a role in host specificity / parasite attraction and subsequent host response against the parasites it attracted (Buchmann and Bresciani 1998). None of the afore-mentioned conditions were however observed macroscopically during handling and processing of fish.

6.3.4.2. Ecological aspects: numbers and distribution

Rohde and Rohde (2005) state that host range, micro and macro habitat preference and selection, geographical range, sex and age of host, season, food and hyperparasites are sufficient variables to characterise the ecological niche (also referred to as a “multi-dimensional hyper volume”) within which a species can exist.

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Pariselle, Bilong Bilong and Euzet, (2003) also mentioned the effects of historical events on the host population (to explain variability observed) and the role of inheritance from ancestral host species (to explain sharing of parasite species). Only some of these biotic and abiotic variables that fall within the scope of the current chapter will subsequently be discussed.

6.3.4.3. Infection statistics: host preference

Of the monogenean parasite species discussed here, one appears to be a strict “specialist” (D. nicolettae on L. capensis) while the remaining four (D. iwani, D. larindae, D. intorquens and Diplozoon spp.) infected both host species examined (Table 6-3 and 6-4). The use of the terms “specialist” and “generalist” should in this case be used tentatively. Bush et al. (2001) state that, in order to unambiguously classify a parasite as being a host generalist or specialist, one need to know much about the parasites on all potential hosts. With regards to the monogenean fauna of the Vaal River system this is obviously not yet the case. Diplozoon sp. is a good example. Further studies are most definitely required to unequivocally illustrate and further evaluate the degree of host specificity of monogeneans on fish in the Vaal River system.

The two species D. iwani and D. larindae share many morphological characteristics and were initially considered to possibly be two forms of a single species (see Chapter 4). Šimková, Ottavá and Morand (2006) state that generalist Dactylogyrus spp. tend to prefer one host species and infect other host species in low abundance or only occasionally. While both parasite species occur on both host species, there was a clear preference for a particular host (Table 6-3 and 6-4). While variation in morphometric measurements for the same parasite species from different hosts and / or geographical areas have been reported previously for monogeneans (e.g. Guégan and Lambert 1991; Pariselle, Lim and Lambert 2002; Rubtsova, Balbuena, Sarabeev, Blasco-Costa and Euzet 2006) and digeneans (e.g. Chibwana and Nkwengulila 2010), it was decided to describe species A (D. iwani) and B (D. larindae) as separate species (Chapters 4 and 5). As there was no clear difference in site preference on the gills where these two species occur together on the same host species, ecological factors such as competitive exclusion is unlikely to be responsible for the apparent host segregation / preference observed.

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The latter may possibly also be related to co-evolutionary development of parasite and host, as Bush et al. (2001) warns that phylogenetic factors should not be ignored whenever host-parasite interactions are studied. In fact, aspects of vertebrate host evolution have been inferred from the phylogeny of monogenean parasites (Paugy, Guégan and Agnèse 1990; Lambert and El Gharbi 1995; Verneau, Bentz, Sinnappah, du Preez, Whittington and Combes 2002), as host specificity is thought to have acted as a guiding force of co-evolution (Kearn 1994). Compared to parasite- host co-evolution, host switching and ecological transfer however appears to be the more common mechanism of speciation in gyrodactylid monogeneans (Bakke, Harris and Cable 2002) and may also be considered in this case. Šimková et al. (2006) has, however, shown that host specificity is not necessarily a major factor with regard to the phylogenetic congruence between hosts and Dactylogyrus spp. parasites. Šimková and Morand (2008) consider being a strict specialist an ancestral trait for Dactylogyrus spp. and attribute speciation to association by descent and host-switching events. Guégan and Agnèse (1991) conclude that both non- phylogenetic evolution (host switching and sequential colonization) and phylogenetic evolution were apparent, but that it is difficult to evaluate contributing proportions in a complex of parasite species. Indeed, mechanisms involved in host specificity are often poorly understood as it may include behavioural, mechanical and chemical factors affecting parasite attraction, attachment, feeding, reproduction and host responses (Buchmann, Madsen and Dalgaard 2004).

Blažek, Bagge and Valtonen (2008a) warn that monogenean species apparently exhibiting host specificity, may exhibit more generalized host preferences under conditions (temperature and host immunity status) more suitable for infrapopulation growth. This implies that “host specificity” may in fact theoretically differ between different localities within the same river system, depending on sampling site conditions (e.g. pollution levels and geographical influences such as temperature) on both parasites and hosts. However, continuous transmission in association with other putative apomorphic and plesiomorphic features are thought to enhance colonization of new hosts by viviparous gyrodactylids (Boeger, Kritsky, Pie and Engers 2005).

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As dactylogyrids do not share this “reproductive advantage” (in terms of the potential for new host colonization), the occurrence of host-switching phenomena differing between localities within the Vaal River system is thought to be unlikely. Apart from this, species from the genus Dactylogyrus are renowned for their level of host specificity (e.g. Dupont and Crivelli 1988; Molnár 2009), though exceptions have been recorded (e.g. Shamsi, Jalali and Aghazadeh Meshgi 2009). As relative sampling intensity remains a problem in discussions on host specificity (Bakke et al. 2002), the apparent host specificity observed in the Vaal Dam thus still needs to be confirmed by further sampling. Future studies should compare the “symbiota” (parasite species associated to a certain host species in a given area as a whole) (Galli, Stefani, Benzoni and Zullini 2005) of these two host species (L. capensis and L. umbratus) from various areas and habitat types within the same system (Vaal River). Such studies will also allow evaluation of possible relationships between levels of aggregation and host specificity (e.g. Krasnov, Stanko, Miklisova and Morand 2006). While dactylogyrids are renowned for their host specificity, King and Cable (2007) warn that host specificity can never be assumed or even inferred from natural parasite distributions unless experimentally tested. Gyrodactylids lend themselves to such an experimental approach, but the latter may prove more difficult when working with dactylogyrids. Gyrodactylids are larger and being viviparous they can be experimentally transferred to new hosts and number of daughters given birth to (and their physical position) visually monitored under host anaesthesia without killing the host. Dactylogyrids lay eggs and it is therefore much more difficult to experimentally control infection rate by the swimming oncomiracidia. Furthermore dactylogyrids are much smaller and accurately examining the number of parasites and their position on the gills without killing the host is very difficult. Nonetheless, further development of suitable models to experimentally examine the true extent of the apparent host specificity observed may be worthwhile.

6.3.4.4. Infection statistics: site preference

Bush et al. (2001) state that site specificity should be adaptive in that it should increase the fitness of the parasite in that particular site (i.e. when compared to fitness obtained in some other site).

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Site preference by monogeneans is thought to be related to feeding (food source availability), reproduction (transmission probability), population dynamics (e.g. parasite density), taxonomy (i.e. ecological and morphological differences between monogenean species) and differences in water current over different parts of the gill surface (e.g. Cone and Cusack 1989; Turgut, Shinn and Wootten 2006). Furthermore, Kadlec, Šimková and Gelnar (2003a) concluded that species abundance could be a factor influencing microhabitat preference in the case of congeneric species. Rohde and Rohde (2005) however, state that such niche preferences are to a large extend also determined genetically. They believe interspecific competition (as argued by e.g. Paperna 1964b) to probably be of less importance than often believed and state that niche restriction to facilitate mating and segregation of niches to avoid interspecific hybridization, are likely to be more important. This view is accommodated in the following list of categories, listed by Bush et al. (2001), in answer of the question “What mechanism might we invoke to explain niche restriction (site selection) in parasites?”: descent; mating; adaptation; predation and competition. The authors conclude that many mechanisms (some as yet unidentified) may determine infracommunity patterns. The strong positive correlations observed between the Dogielius sp. and several Dactylogyrus spp. in the current study (Tables 6-3 and 6-4) also indicate a lack of interspecific competition. Such conclusions should, however, be interpreted with caution. As smears / scrapings were made to facilitate parasite collection, no comment can be made on parasite distribution on distal, central and proximal areas of the gill as divided by Blažek and Gelnar (2006).

The question as to what mechanisms may then give rise to such positive correlations arises. Current experimental design provided no means to provide answers to this question. One hypothesis may be that one species may alter the gill habitat (physical alteration or systemic effects resulting from a host immune response) to facilitate more successful establishment by another species. Tombi and Bilong-Bilong (2004) states that perforations caused by monogenean hooks may result in the rupture of myxosporidian cysts (i.e. example of physical alteration). Baker, Pante and de Buron (2005) showed that both neutral and negative interactions may affect site preference correlations with reference to monogeneans and copepods. Such effects have as yet not been conclusively demonstrated between monogenean species.

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In most instances all species are thought to have identical opportunities for settling on the host (Dzika and Snymański 1989). It would appear that site preference / separation between monogenean species often decrease with an increase in epidemiological parameters of infection (i.e. niche breadths increase with an increase in parasite infection, e.g. Yang, Liu, Gibson and Dong 2006 and Matejusová, Simková, Sasal and Gelnar 2003). With a decrease in site specificity one would most probably also encounter changes in species correlations with regard to specific sites. Such statistical correlations may well be mere chance “by-products” of factors such as water flow, parasite density, ecological and morphological differences between monogenean species (Turgut et al. 2006). With regard to the latter variable, it may simply be that the physical dimensions of gills is suitable for both species so that they inadvertently occur together. Gill asymmetry may also result in a preference for either side of the head. This could be further investigated in future studies (i.e. gill morphometrics and experimental investigations). Some monogeneans may also react to specific “hatching factors” (i.e. hatching rhythms have adapted to host behaviours such as schooling during spawning season) (Rohde and Rohde 2005). This may create a “swarm effect” where parasites triggered by the same factors may well infect the host together and as such demonstrate positive statistical correlation. Such effects would, however, be best studied using an experimental infection approach.

Only slightly more parasites were found on the right set of gill arches (except for D. intorquens). Studies on Dactylogyrus spp. by Özer and Öztürk (2005), Turgut et al. (2006) and Le Roux et al. (2011) also demonstrated no significant differences in distribution between the right and left sets of gills.

In the present study the majority of parasite specimens (all species) were found on the median position of the gill arch. Turgut et al. (2006) found statistically significant differences in gill arch position preference of Dactylogyrus extensus Mueller and Van Cleave, 1932 (median and ventral), Dactylogyrus difformis Wagener, 1857/ Dactylogyrus difformoides Gläser and Gussev, 1971 (dorsal) and Dactylogyrus auriculatus Nordmann, 1832 (ventral). Le Roux et al. (2011) found that Cichlidogyrus philander Douellou, 1993 occurred less often on the ventral region of the gill, with prevalence on the dorsal and median areas being similar.

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More parasites were recovered from the anterior (i.e. “outer” or closer to the operculum) hemibranch. Anderson, Blažek, Percival and Janovy (1993) also found that statistically significantly more Dactylogyrus banghami Mizelle and Donahue, 1944 were recorded from the front (i.e. anterior) compared to the rear (posterior) arch faces. Turgut et al. (2006) however found a greater number of D. extensus and D. auriculatus on the inner (i.e. posterior) side of the hemibranch.

The fact that, for all parasite species examined, more parasites were found on the second and third gill arches probably relate to current (water flow over the gills), as was also concluded by Le Roux et al. (2011). Anderson et al. (1993) (with reference to D. banghami) and Blažek and Gelnar (2006) (with reference to glochidial larval stages) also recorded more parasites on the second and third arches. Paling (1968) demonstrated that most of the respiratory current appears to flow through the third pair of gill slits, with smaller yet appreciable volumes flowing through the second and fourth pairs of slits. The first and last gill slits were shown to carry comparatively little of the water flow. Turgut et al. (2006) confirmed this trend by demonstrating a preference for the second and third gill arches in three of the parasite species examined and state that differences in water volume also influence available attachment surface area as well as aerobic conditions. Blažek and Gelnar (2006) conclude that spatial distribution (in glochidial larval stages) is not due to active choice of attachment site, but rather a result of respiratory current and host fish behaviour and habitat use. The same reasoning may well apply to the spatial distribution patterns of Dactylogyrus spp. observed in the current study. Baker et al. (2005) postulate that some monogeneans (e.g. Metamicrocotyla macracantha (Alexander, 1954)) may prefer gill arch number 1 as the current flow is minimal and parasites may be the least precariously attached. Matejusová et al. (2003), however, warn that gill preference is the consequence of a complex process that cannot be adequately explained by any individual model or single hypothesis. Fish respiration, for example, involves two stages. During the first stage the mouth is opened and water stream into the mouth cavity. Following this the mouth and opercula is closed and the pressure inside the gills chamber is increased to force water flow across the filaments (also in reverse to allow O2 / CO2 exchange). Slight modifications of this pattern (e.g. amount of pressure exerted by different fish species may influence flow over the gills) will have an influence on parasite larvae distribution.

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As was also recorded by Dzika and Szymański (1989) there was no clearly defined separation among the present study species. This indicates a lack of competition for space and food. Apparent site preferences once again most probably relate to water flow over the gills. Šimková, Gelnar and Morand (2001) also found that negative interspecific interactions are not important within dactylogyrid communities. Microhabitat preference is however also dependent on / influenced by species abundances (Šimková et al. 2001; Kadlec et al. 2003a; Matejusová et al. 2003) and intensity of infection (Yang et al. 2006). This implies that such preferences may vary seasonally, stressing the need for further surveys within the Vaal River system.

6.3.4.5. Effect of host variables

Bush et al. (2001) state that host-related factors (such as species, age diet, attachment site, immune response and parasite component community composition) are known to affect fecundity of parasites. For the purposes of this chapter we shall only discuss two factors, namely host size (related to host age) and host gender. Furthermore one can distinguish between possible host size effects on the community level (species richness) and population level (infection statistics).

Effect of host size: Larger fish are generally also older and were thus exposed to possible parasitic infection for a longer period than younger fish. With advancing host age and size an increase in parasite species richness and / or intensity is often observed (e.g. Willomitzer 1980a, 1980b; González et al. 2001; Le Roux et al. 2011). Intensity of parasite infection may, however, also decrease with host age (e.g. Cone and Cusack 1988), most probably due to a better developed immune response in older fish. Larger fish will theoretically have larger surface areas available for infection by parasites. This is however not always the case as negative correlations between intensity of infection and fish size have been reported for monogeneans (e.g. Cusack 1986). Bush et al. (2001) also concluded that factors related to phylogeny and historical processes that affect the host can override the effects of area, as larger fish with larger gills do not always have more monogenean parasites.

Evaluating host size – length, age and weight: In this paper total length was preferred above weight as indicator of host size, as there may be poor agreement in length-weight correlations in Labeo spp. above a certain length (e.g. Jhingran 1952).

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This probably results from the fact that some Labeo spp. exhibits a tendency to increase more in mass than in size (e.g. Tómasson, Bruton and Hamman 1985; Montchowui Laleye, Moreau, Philippart and Poncin 2009). Host length is furthermore widely considered to be a potentially important biological variable influencing ectoparasite infection statistics (e.g. Guégan and Hugueny 1994; Cable and van Oosterhout 2007; Dávidová, Ondračková, Jurajda and Gelnar 2008). Large differences in terms of length-age relationships exist between and within Labeo spp. (e.g. Legendre and Albaret 1991, Weyl and Booth 1999, Potts et al. 2006), resulting from a number of confounding factors (Narejo, Mastoi, Lashari, Abid, Laghari and Mahesar 2009). From available length-age data (Laurenson, Hocutt and Hecht 1989; Skelton 2001; Potts et al. 2006) we can deduce that all L. umbratus and L. capensis specimens examined were older than three years. Various methods may be employed to determine fish age (e.g. Phelps, Edwards and Willis 2007; Plug 2008), with growth zones in otoliths most often employed in L. capensis (e.g. Laurenson et al. 1989) and L. umbratus (e.g. Potts et al. 2006). The scope of the current study, however, did not warrant such procedures. During future studies it may be interesting to perform age determination on a sub-set of fish for each species from the Vaal Dam during sampling for parasite recovery. This may provide the opportunity to further assess and compare the use of length and age data in parasite infection comparisons.

Population level host size effects: Differences in parasite infection levels between the size classes examined in this study were not expected. Apart from surface area available and increased probability of infection as host age increases, host immunity to parasite infection must also be considered. Animals within the same age / length class from the same locality most probably share the same history with regards to pathogen exposure and immunological development. Bhuiyan, Akther and Musa (2007) successfully examined the effect of host length on parasite occurrence in three size classes of Labeo rohita (Hamilton, 1822): < 18 cm, 18 to 28 cm and > 28 cm. In the current study no small fish (< 35 cm) were collected and more than 50% of fish fell within a single size category (> 40 to < 45 cm).

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Community level host size effects: When examining wide host size ranges representing developing parasite communities, it is likely that a greater proportion of nested communities will be encountered (Rohde, Worthen, Heap, Hugueny and Guégan 1998). Rohde et al. (1998) ascribe this to “differential colonization probabilities”: i.e. it is likely that parasite species are acquired in a certain order depending on changing habitat and food preferences as fish grow. Furthermore, older fish may have had more time to accumulate the rarer parasite species. Matějusová, Morand and Gelnar (2000) also found a positive relationship between parasite species richness and total fish length. All fish in the current study were estimated to be older than three years with very narrow size ranges. Thus unpredictable, unstructured, depauperate and clumped parasite assemblages could be expected (Rohde et al. 1998). The fact that the last two size classes contained very few fish may, however, give rise to sampling artefacts and false trends (e.g. that the largest fish exhibited poorest parasite species richness). While the current data (in terms of weight class range adequacy) is admittedly largely insufficient to evaluate the latter trend, acquired host resistance may well contribute to a trend for higher infection levels in smaller fish (e.g. Hodneland and Nilsen 1994; Al-Zubaidy 2007; Onyedineke, Obi, Ofoegbu and Ukogo 2010). In other cases larger fish were found to carry the highest parasite loads (e.g. Tombi and Bilong Bilong 2004; Cable and van Oosterhout 2007; Nachev and Sures 2009), while Bhuiyan et al. (2007) recorded highest infection in medium sized fish. Öztürk and Altunel (2006) demonstrated that the relationship between abundance and host length can differ between species of the same genus (Dactylogyrus spp.), while Özer and Erdem (1998) found no correlation in the level of trichodinid infections and the length of the host. In similar fashion, Tekin Özan, Kir and Barlas (2008) found that distribution of infrapopulations of Dactylogyrus minitus Kulwiec, 1927 did not vary significantly with size class of fish (though it peaked in larger size classes). There thus exists an apparent discrepancy from literature in observed correlation between parasite load and fish size. This may be partially explained by the fact that parasite-mediated selection against large body size may be counteracted by other evolutionary and ecological factors favouring larger body size in naturally-occurring fish populations (Cable and van Oosterhout 2007). In the current study only D. nicolettae on L. capensis was found to be a good predictor of host total length (Omnibus and Wald chi-square tests).

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In a nested community structure, the species comprising depauperate assemblages represent non-random subsets of progressively richer communities (Rohde et al. 1998). Poulin and Guégan (2000) however warns that even if species assemblages are considered as not nested, it does not imply that these assemblages are not structured in some other way. They propose a nestedness/anti-nestedness continuum and advocate a possible link between the latter and a positive mean intensity-prevalence relationship. As statistical investigation of aspects such as parasite richness, sampling effort and host range requires analysis of large databases (e.g. Guégan and Kennedy 1996); the data gathered in the current study is considered not suitable for commenting on such a relationship.

There has also been some speculation regarding the relative contributions of environmental factors, host age/size and other parasite traits (e.g. degree of host specificity) to observed infection statistics. Potentially confounding factors discussed include environmental conditions such as temperature (Hodneland and Nilsen 1994; Dávidová et al. 2008) and the degree of host specificity (Matějusová et al. 2000). This is in partial agreement with a study by Guégan, Lambert, Lévêque, Combes and Euzet (1992), whom state that host ecology together with host species size are deciding factors for explaining monogenean species richness in West African cyprinid fishes. We thus suggest that further ecological studies (e.g. Barger and Esch 2001, 2002) should be conducted comparing various host sizes from various habitats, collected during different seasons within the Vaal River system.

Effect of host gender: Infection statistics calculated in the present study did not differ with regards to host gender for the majority of parasite species. González, Acuña and Oliva (2001) also found that prevalence and mean intensity of the monogenean Neoheterobothrium Price, 1943 were not affected by host sex. In the current study the prevalence on female fish was slightly higher compared to male fish. For L. umbratus, Diplozoon sp. was, however, a good predictor of host gender (Omnibus and Wald chi-square tests) emphasizing a possible gender effect. Higher prevalence of parasite infection in female fish was also recorded in studies performed by Tombi and Bilong Bilong (2004) and Özer and Öztürk (2005). Body length of infested females was generally larger than that of males.

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While there appeared to be no significant preference for any particular length class, the correlation observed between length and gender makes it difficult to conclude if observed preference was for gender, length or possibly a combination of both variables. Lamková et al. (2007) stated that fish physiology and immunology can alter parasite infection parameters. They found no differences in gender for immunological parameters in fish outside periods of reproduction. Infection statistics may thus well differ with regards to host gender during early spring and summer compared to the winter survey in the current study. This aspect should be investigated in future studies.

Parasite data was also statistically evaluated (Omnibus and Wald chi-square tests) in terms of prediction value. The species D. iwani, D. nicolettae and Diplozoon sp. was found to be good predictors of host species. For L. umbratus, Diplozoon sp. was a good predictor of host gender. For L. capensis, D. nicolettae was a good predictor of host total length.

6.3.4.6. Effect of environmental variables

Hassan (2008) states that habitat alteration; climate change and pollution have complex effects on parasites making any meaningful generalizations difficult. Some of the environmental factors that affect distribution and abundance of species are latitude, altitude, salinity, depth, light intensity and frequency and intensity of physical disturbances (Bush et al. 2001). Temperature, however, appears to be the single most important variable affecting monogenean (especially gyrodactylid) parasite biology (e.g. Lester and Adams 1974; Gelnar 1991; Harris 1993; Özer and Erdem 1999; Šimková, Sasal, Kadlec and Gelnar 2001; Tinsley 2001; Bakke et al. 2002; Öztürk and Altunel 2006; Bakke, Cable and Harris 2007; Blažek, Jarkovský, Koubková and Gelnar 2008b) and morphology. The latter aspect is particularly true for the genus Gyrodactylus von Nordmann, 1832, where large variation in sclerotized hard parts measurements (i.e. size but not shape) are often recorded (e.g. Hanek and Furtado 1973; Harris 1998b) and have been shown to be strongly correlated with water temperature (e.g. Mo 1991a, 1991b, 1993; Hodneland and Nilsen 1994; Appleby 1996) and host type (i.e. primary or secondary host, e.g. Dmitrieva and Dimitrov 2002).

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Variability in attachment apparatus shape in turn appears to be related to the degree of host specificity (i.e. specialists versus generalists) (Jarkovský, Morand, Šimková and Gelnar 2004). Lamková et al. (2007) state that while temperature variation does influence fish parasite life cycles directly, it also affect fish physiology and immunology that indirectly alters parasite infection. They found parasite diversity and infection parameters in parasite communities to be highest in spring and early summer. The current paper reports on a single winter survey. Hossain, Hossain, Rahman, Akter and Khanom (2008) found winter to be the season when with the highest ectoparasite (including monogeneans) prevalence was recorded. This was also the result expected by Le Roux et al. (2011). The authors state that it is generally accepted that fish’s immunity is compromised during winter. This was indeed the case for C. philander during one winter survey but the trend was not consistent. They state that sample size may have had an effect on the observed mean intensity of the parasite, concluding that seasonal water temperature variations at the sampling site may have been too small to have an effect on parasite intensity.

The importance of other physical variables, such as salinity, has also been discussed by several authors (e.g. Bakke et al. 2002; Hassan 2008). This makes intuitive sense as other invertebrates also show varying tolerances to salinity concentration (e.g. Browne, Palmer, Muller and Davies-Coleman 2004). Other studies (e.g. Le Roux et al. 2011), however, found no correlation between prevalence or abundance and salinity levels. Total dissolved salts (TDS), which is directly proportional to electrical conductivity (EC), are both indicative of the salt content of water (Anderson and Cummings 1999). As natural waters contain varying quantities of TDS (influenced by characteristics of geological formations etc.), South African guidelines for aquatic ecosystems state that (1) TDS concentrations should not be changed by >15% from the normal cycles of the water body under unimpacted conditions at any time of the year and (2) the amplitude and frequency of natural cycles in TDS concentrations should not be changed (Department of Water Affairs and Forestry 1996). Salinity (based on TDS) remained fairly constant over the four month period during autumn / winter (Table 6-5).

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Comparing these concentrations to values recorded during a number of surveys in 1998 to 2000 (Crafford and Avenant-Oldewage 2009), the current TDS values is slightly lower yet in the same order (186 to 217 mg/L compared to the current 140 to 150 mg/L). TDS values in the Vaal River Barrage (end 1998 to beginning 2000) ranged between 478 and 584 mg/L (Crafford and Avenant-Oldewage 2009), indicating poorer water quality within the same system. Water quality, with particular reference to pollutants such as heavy metals, may however affect monogenean parasite numbers and distribution (e.g. Dušek, Gelnar and Šebelová 1998; Galli et al. 2001; Marcogliese 2004; Blanar, Munkittrick, Houlahan, MacLatchy and Marcogliese 2009). Heavy metal concentrations at the study site, however, appear low, as was previously confirmed by Crafford and Avenant-Oldewage (2010). A fish health assessment index and associated parasite index have previously been applied to the Vaal River system (Crafford and Avenant-Oldewage 2009). It would be interesting to compare trends in biological environmental monitoring results using monogenean data from various sites, to results obtained using the less cumbersome existing parasite index in future studies. The “pollution ecology” of the monogenean parasites encountered, as well as the suitability of the two Labeo spp. hosts as models, will have to be determined / evaluated in the process. Galli et al. (2001) for example found Dactylogyrus vistulae Prost, 1957 to show good resistance to pollution stresses. Such an evaluation may well be combined with a detailed ecological study investigating both seasonal, habitat type and water quality effects on monogenean infections in the greater Vaal River system.

6.3.5. Summary and conclusion

The apparent host specificity observed for the Dactylogyrus and Dogielius species needs to be confirmed. Differences in parasite infection levels between the size classes examined in this study were not expected nor observed. No small fish were collected and more than 50% of fish fell within a single size category. In terms of gill arch specificity on the gill apparatus, an unequal proportion of parasites on the various areas examined were found to be statistically significant (Pearson Chi Square) in some cases. More specifically there appeared to be a preference for the median position on the anterior side of the third gill for the majority of parasite species examined.

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While the parasites encountered thus did not appear to find a fairly homogenous environment on the entire branchial apparatus of both Labeo spp. examined, a lack of competitive interactions can be inferred as the apparent preference was the same for most parasite species. It is believed that the apparent preference may rather relate to water flow over the gills. The parasite prevalence on female fish was slightly higher compared to male fish but this trend was not statistically significant. Future ecological studies should compare parasite community composition (including site preferences of the various species), infection statistics between wider host length classes, seasons and various habitats (e.g. rapids, glide sections, deep pools, shallow backwaters etc.) within the Vaal River system, to that obtained in the Vaal Dam.

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CHAPTER 7

7 - ASPECTS OF THE ECOLOGY OF MONOGENEAN PARASITES OF LABEO CAPENSIS (Smith, 1841) AND LABEO UMBRATUS (Smith, 1841) IN THE VAAL DAM, GAUTENG PROVINCE, SOUTH AFRICA COLLECTED DURING A SUMMER SURVEY AS COMPARED WITH THE PRECEDING WINTER SURVEY

7.1. INTRODUCTION

The preceding chapter (Chapter 6) reported on a winter (June / July 2009) survey conducted at the Vaal Dam, Gauteng Province, South Africa. The current chapter reports on the results of a survey conducted during summer (January 2010). Seasonal effects (notably temperature) may have a significant impact on the numbers of monogeneans, as discussed by Kir and Tekin Özan (2007) in which Dactylogyrus minitus Kulwiec, 1927 development and hence reproduction rate was found to increase during summer.

Refer to section 6.1 for a short introduction to the host species and how the study of their monogenean parasites contributes to the greater aim of the current project.

The objectives of the current chapter are defined as follows: (1) Examine and compare aspects of monogenean biology, in terms of infection statistics and site preference as determined during a summer survey, within an ecological framework (i.e. numbers and distribution of parasites on gills); (2) Discuss the possible effects of selected host and environmental variables on patterns of numbers and distribution identified above; (3) Compare the results obtained in the first two points with that obtained for the winter survey (reported on in Chapter 6).

7.2. MATERIALS AND METHODS

For study site description refer to section 3.1.

For fish collection, necropsy and parasite recovery procedures refer to sections 3.2 and 3.3.

For details on parasite identification and description refer to Chapter 4 and Chapter 5.

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For details on calculation of infection statistics refer to sections 3.5 and 2.3.3.

For details on statistical tests employed refer to section 3.6.3.

7.3. RESULTS AND DISCUSSION

7.3.1. Host species

Twenty-one specimens (of which 15 were male) of Labeo umbratus (Smith, 1841) were collected, compared to 20 (of which nine were male) Labeo capensis (Smith, 1841). Table 7-1 summarizes average weight, average total length and average condition factor values for fish hosts collected. Table 7-2 summarizes the gender distribution within length class categories. As was the case for the first (winter) survey, the majority of L. umbratus (n=18) resided within the > 40 < 45 cm length class. The majority of the L. capensis specimens collected (n=18), could be found in the > 30 < 35 cm length class. For a general discussion on host species condition factor values and macroscopic pathology, refer to section 6.3.4.1.

7.3.2. Parasite species

Representatives from the monogenean genera Dactylogyrus Diesing, 1850, Dogielius Bychowsky, 1936 and Diplozoon von Nordmann, 1832 collected during the winter (June / July 2009) survey (see Chapters 4 to 6), were encountered during the summer (January 2010) survey.

Table 7-1: Summary description of sampled Labeo Cuvier, 1817 host population from the Vaal Dam (Summer survey, January 2010).

Host Labeo umbratus (Smith, 1841) Labeo capensis (Smith, 1841) Average Average Average Average Average Average length condition length condition Variable Number weight (g) Number weight (g) (cm) factor * (cm) factor * [SD] [SD] [SD] [SD] [SD] [SD] 44.31 0.77 208 32.90 0.54 Male 15 680 [193] 9 [2.54] [0.10] [123] [2.53] [0.17] 542 41.60 0.74 1901 32.03 0.58 Female 6 11 [120] [1.26] [0.10] [44] [1.53] [0.09] 640 43.54 0.76 199 32.42 0.56 Combined 21 20 [183] [2.55] [0.10] [86] [2.03] [0.13] * = Fulton’s condition factor: K = 100 x W / L3 (calculated for each individual fish with the average value presented here) Key SD = Standard deviation Average length = Total length

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From available length-age data (Laurenson, Hocutt and Hecht 1989; Skelton 2001; Potts, Booth, Hecht and Andrew 2006) we can deduce that all L. umbratus and L. capensis specimens examined were older than three years. Given the narrow size ranges and the fact that many of the length classes contain very few or no fish, additional infection statistics calculations based on length class or age would provide no meaningful results (as was demonstrated in Chapter 6) and were thus not performed.

Table 7-2: Weight category gender distribution of sampled Labeo Cuvier, 1817 host population from the Vaal Dam (Summer survey, January 2010).

Number of fish collected Length category Labeo umbratus (Smith, 1841) Labeo capensis (Smith, 1841) Male Female Male Female < 30 cm 0 0 0 1

> 30 to < 35 cm 0 0 8 10

> 35 to < 40 cm 0 0 1 0

> 40 to < 45 cm 12 6 0 0

> 45 to < 50 cm 2 0 0 0

> 50 to < 55 cm 1 0 0 0

Total 15 6 9 11

7.3.3. Biological and ecological aspects

7.3.3.1. Infection statistics and host specificity

As was the case during the winter survey, Dactylogyrus iwani Crafford, Luus-Powell and Avenant-Oldewage, 2012 and Dactylogyrus larindae Crafford, Luus-Powell and Avenant-Oldewage, 2012 occurred on both L. capensis and L. umbratus, but again exhibited the same host preference as before. Dactylogyrus nicolettae Crafford, Luus-Powell and Avenant-Oldewage, 2012 was once again only recovered from L. capensis. Diplozoon sp. was found predominantly on L. umbratus but once again also occurred on L. capensis (very low prevalence as recorded during the first survey). Dogielius intorquens Crafford, Luus-Powell and Avenant-Oldewage, 2012 was again found on both host species.

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What is most evident from Table 7-3 is that a very large number of worms (especially with regards to D. larindae) were collected during the summer survey. From Table 7- 6 it is clear that greater numbers of parasites were generally collected during summer when compared with numbers collected during winter.

For a general discussion on numbers and distribution of organisms refer to section 6.3.4.2.

7.3.3.2. Effect of host variables

Infection levels (number of parasites collected) in terms of site selection / distribution on gills, host gender and host size classes are summarized in Tables 7-3 and 7-4.

For all parasite species (i.e. with reference to the component community column in the tables referred to above), more parasites were found on the second and third gill arches.

This trend (i.e. unequal proportion of parasites) was statistically significant (Pearson Chi Square, p=0.005), the result thus being in agreement with that obtained during the first survey.

Slightly more parasites were found on the left set of gill arches (compared to more parasites on right gill arches during the first survey). This trend was not statistically significant during the first survey, but was found to be significant (Pearson Chi Square, p = 0.000) for the second survey.

Furthermore, as for the first survey, most parasites were found on the medial position of the gill arch (Pearson Chi Square, p = 0.228). During the first survey this difference was, however, found to be statistically significant (which was not the case in the second survey).

More parasites were recovered from the anterior hemibranch (Pearson Chi Square, p = 0.114). This echoes the trend observed during the first survey. However, during the first survey the trend was found not to be statistically significant.

For a general discussion on host and site preference effects, refer to sections 6.3.4.3 and 6.3.4.4. For a general discussion on the possible effects of host variables on monogenean infections, refer to section 6.3.4.5.

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Table 7-3: Monogenean parasite infection statistics for species of Labeo Cuvier, 1817 from the Vaal Dam collected during a summer (January 2010) survey: Labeo umbratus (Smith, 1841).

Host species Labeo umbratus (Smith, 1841) Dl Dl Dip. Monogenean species Dl a Dl b Dl d D e ? CC c spp. sp. Distribution Total 65 2380 0 23 225 70 15 79 2857 1 13 450 0 6 59 17 7 21 573 2 19 647 0 4 49 13 0 15 747 Gill arch 3 23 612 0 3 56 20 2 22 738 number 4 7 481 0 3 57 16 6 20 590 Unknown 3 190 0 7 4 4 0 1 209 Left 37 1292 0 9 116 30 9 34 1522 Gill set (side of Right 33 1088 0 14 109 40 6 45 1335 head) Unknown 0 0 0 0 0 0 0 0 0 Dorsal (D) 24 676 0 8 61 20 6 18 813 Gill arch Medial (M) 27 926 0 4 91 26 0 33 1107 region Ventral (V) 11 574 0 3 67 20 9 27 711 Unknown 3 204 0 8 6 4 0 1 226 Anterior 32 1196 0 8 108 19 0 37 1400 Gill Posterior 30 961 0 7 109 47 0 41 1195 orientation Unknown 3 223 0 8 8 4 0 1 247 Not applicable 0 0 0 0 0 0 15 0 15 Infection levels Prevalence (%) 95.24 95.24 0 47.62 85.71 80.95 23.81 57.14 100.00 Mean intensity 3.25 119.00 0 2.30 12.50 4.12 3.00 6.58 136.05 Intensity range: Minimum 1 17 0 1 1 1 1 1 2 Intensity range: Maximum 8 335 0 8 40 10 6 13 364 Mean abundance 3.10 113.33 0 1.10 10.71 3.33 0.71 3.76 136.05 P (%) 93.33 100.00 0 46.67 93.33 73.33 26.67 66.67 100.00 Host gender: MI 2.93 122.07 0 2.29 12.50 4.45 2.5 6.30 146.27 Male MA 2.73 122.67 0 1.07 11.67 3.27 .067 4.20 146.27 P (%) 100.00 83.33 0 50.00 66.67 100.00 16.67 33.33 100.00 Host gender: MI 4.00 108.00 0 2.33 12.50 3.50 5.00 8.00 110.50 Female MA 4.00 90.00 0 1.17 8.33 3.50 0.83 2.67 110.50 Key (species codes used) Dl a – Dactylogyrus iwani Dl b – Dactylogyrus larindae Crafford, Dl c – Dactylogyrus nicolettae Crafford, Luus-Powell and Luus-Powell and Avenant-Oldewage, Crafford, Luus-Powell and Avenant- Avenant-Oldewage, 2012 2012 Oldewage, 2012

D e – Dogielius intorquens Crafford, Dip sp. – Diplozoon Dl d – Dactylogyrus sp. D Luus-Powell and Avenant-Oldewage, 2012 von Nordmann, 1832 Dl spp. - Dactylogyrus spp. not *? – Specimens that could not be identified to species level identified to genus level (damaged / CC = Component community (damaged / lost during lost during mounting) mounting)

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Table 7-4: Monogenean parasite infection statistics for species of Labeo Cuvier, 1817 from the Vaal Dam collected during a summer (January 2010) survey: Labeo capensis (Smith, 1841).

Host species Labeo capensis (Smith, 1841) Dl Dip. Monogenean species Dl a Dl b Dl c Dl d D e ? CC spp. sp. Distribution Total 204 18 53 0 45 31 11 18 380 1 49 3 11 0 13 6 6 3 91 2 59 8 16 0 15 11 1 7 117 Gill arch 3 53 3 17 0 8 8 4 1 94 number 4 39 4 8 0 9 5 0 7 72 Unknown 4 0 1 0 0 1 0 0 6 Left 102 12 29 0 21 17 9 15 205 Gill set Right 98 6 23 0 24 13 2 3 169 (side of head) Unknown 4 0 1 0 0 1 0 0 6 Dorsal (D) 51 5 13 0 9 8 2 4 92 Medial (M) 113 6 20 0 23 12 6 8 188 Gill arch region Ventral (V) 36 7 19 0 13 10 2 6 93 Unknown 4 0 1 0 0 1 1 0 7 Anterior 109 12 26 0 17 10 0 11 185 Posterior 74 5 22 0 27 16 0 7 151 Gill orientation Unknown 21 1 5 0 1 5 0 0 33 Not applicable 0 0 0 0 0 0 11 0 11 Infection levels Prevalence (%) 85.00 45.00 70.00 0 55.00 65.00 5.00 35.00 85.00 Mean intensity 12.00 2.00 3.79 0 4.09 2.38 11.00 2.57 22.35 Intensity range: Minimum 2 1 1 0 1 1 11 1 4 Intensity range: Maximum 35 4 11 0 10 5 11 5 69 Mean abundance 10.20 0.90 2.65 0 2.25 1.55 0.55 0.90 19.00 P (%) 100.00 66.67 77.78 0 44.44 77.78 11.11 22.22 66.67 Host gender: MI 14.78 2.33 4.29 0 8.25 2.86 1.00 6.5 42.33 Male MA 14.78 1.56 3.33 0 3.67 2.22 1.22 1.44 28.22 P (%) 72.73 27.27 63.64 0 45.45 54.55 0 18.18 72.73 Host gender: MI 8.88 1.33 3.29 0 2.4 1.83 0 2.5 15.75 Female MA 6.45 0.36 2.09 0 1.09 1.00 0 0.45 11.45 Key (species codes used) Dl a – Dactylogyrus iwani Crafford, Dl b – Dactylogyrus larindae Crafford, Dl c – Dactylogyrus nicolettae Luus-Powell and Avenant- Luus-Powell and Avenant-Oldewage, Crafford, Luus-Powell and Avenant- Oldewage, 2012 2012 Oldewage, 2012

D e – Dogielius intorquens Crafford, Dip sp. – Diplozoon Dl d – Dactylogyrus sp. D Luus-Powell and Avenant-Oldewage, 2012 von Nordmann, 1832

Dl spp. - Dactylogyrus spp. not *? – Specimens that could not be identified to species level (damaged identified to genus level (damaged / CC = Component community / lost during mounting) lost during mounting)

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7.3.3.3. Environmental variables

Selected physical and other water quality parameter values are summarized in Table 7-5. Water analysis data was obtained from Rand Water (sampling point reference C-VD1l, monthly water sampling). As samples were not taken on the precise dates of fish host sampling, results are provided for analyses conducted within approximately two months prior to the sampling effort as well as two months thereafter.

Table 7-5: Environmental (physical) and water quality variables from the Vaal Dam (source: Rand Water, sampling point identification number C-VD1l).

DO = Dissolved oxygen; Temp = Temperature; Cond = Conductivity; CaCO3 = Calcium carbonate; Cr = Chrome; Cu = Copper; Pb = Lead.

For a general discussion on the possible effects of environmental variables on monogenean infections, refer to section 6.3.4.5.

7.3.4. Seasonal comparison

Significantly more parasites were collected during the summer (i.e. second) survey compared to the winter (i.e. first) survey. (Mann-Whitney U test, p = 0.001).

7.3.4.1. Infection statistics

Infection statistics, as calculated for both the winter and summer surveys, are summarized in Table 7-6. For D. iwani infection statistics increased during summer on both hosts (i.e. L. umbratus and L. capensis). For D. larindae, infection statistics increased during summer on what appears to be the “primary host” (i.e. L. umbratus). The higher prevalence of Dactylogyrus spp. during summer (also see D. nicolettae) is in agreement with trends reported for other Dactylogyrus spp. (e.g. also see sections 10.3.6.3 and 11.3.6.3 for discussions on especially temperature effects on Dactylogyrus spp. reproduction rate). Increased parasite reproduction combined with host schooling during spawning in spring and early summer (i.e. facilitating parasite transfer / increased probability of infection) may well result in higher infection statistics during surveys performed later in summer.

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Interestingly enough Diplozoon sp. was less prevalent on both host species during the summer survey.

There was, however, a marked increase in mean intensity of infection on L. capensis but not on L. umbratus. One must, however, keep in mind that only a single L. capensis specimen was infected with this parasite during each of the surveys. The apparent “seasonal trend” observed is most likely an artefact of this very small sample size. It is more likely that no clear seasonal trend exists as reflected by data for L. umbratus.

Table 7-6: Summary of key infection statistics for selected monogenean species from species of Labeo Cuvier, 1817 calculated for the winter and summer surveys respectively.

Survey 1 (Winter) Survey 2 (Summer) Monogenean Labeo capensis Labeo umbratus L. capensis L. umbratus species (Smith, 1841) (Smith, 1841) P MI MA P MI MA P MI MA P MI MA D. iwani 76.9 19.4 14.9 19.2 1.6 0.3 85.00 12.00 10.20 95.24 3.25 3.10 D. larindae 61.5 2.1 1.3 38.5 33.2 12.8 45.00 2.00 0.90 95.24 119.00 113.33 D. nicolettae 38.5 6.0 2.3 0.0 0.0 0.0 70.00 3.79 2.65 0.0 0.0 0.0 D. intorquens 84.6 3.3 2.8 38.5 5.1 2.0 65.00 2.38 1.55 80.95 4.12 3.33 Diplozoon sp. 7.7 1.0 0.1 57.7 3.5 2.0 5.00 11.00 0.55 23.81 3.00 0.71 All specimens 100 25.2 25.2 76.9 24.4 18.8 85.00 22.35 19.00 100.00 136.05 136.05

Key: D. iwani = Dactylogyrus iwani Crafford, Luus-Powell and Avenant-Oldewage, 2012; D. larindae = Dactylogyrus larindae Crafford, Luus-Powell and Avenant-Oldewage, 2012; D. nicolettae = Dactylogyrus nicolettae Crafford, Luus-Powell and Avenant-Oldewage, 2012; D. intorquens = Dogielius intorquens Crafford, Luus-Powell and Avenant-Oldewage, 2012; All specimens = Component community, i.e. all parasites collected including damaged and unidentified specimens (only identified to genus level).

Mean intensity of D. intorquens infection decreased slightly during the summer survey for both host species. Prevalence of infection, however, decreased considerably for L. capensis as opposed to the considerable increase in the same infection statistic for L. umbratus. This parasite species exhibit two forms (based on sclerite size but not shape differences) exhibiting an apparent host preference (see Chapter 5). The question arises if differences in seasonal trends may be form specific. Additional experimental studies are suggested to investigate this aspect further.

Quantitative Parasitology 3.0 software (Rozsa, Reiczigel and Majoros 2000) was used to compare infection statistics between surveys. Analyses were only performed for parasite species of which adequate numbers were collected during both surveys.

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The results (p-values) of various statistical tests performed are summarized in Table 7-7. For L. umbratus there was a statistically significant difference in prevalence between seasons (i.e. higher in summer for Dactylogyrus spp. and Dogielius sp. but higher in winter for Diplozoon sp.). In terms of mean intensity and mean abundance, significant seasonal differences (much higher in summer) were recorded only for D. larindae.

For L. capensis no statistically significant seasonal differences in calculated prevalence, mean intensity or mean abundance values were recorded for any of the parasites for which analyses were performed.

Labeo umbratus (Smith, 1841) Survey 1 (Winter) versus Survey 2 (Summer): p - values Monogenean Prevalence Survey 2 (Summer)Mean intensity Mean abundance species Bootstrap 2-sample Bootstrap 2-sample Chi-square Fisher’s Exact t-test t-test D. iwani N/A N/A N/A N/A D. larindae 0.000 0.000 0.0050 0.0010 D. nicolettae N/A N/A N/A N/A D. intorquens 0.003 0.007 0.5430 0.1765 Diplozoon sp. 0.020 0.037 0.5430 0.1765 All specimens 0.018 0.026 0.0010 0.0010 Labeo capensis (Smith, 1841) Survey 1 (Winter) versus Survey 2 (Summer): p - values Monogenean Prevalence Survey 2 (Summer)Mean intensity Mean abundance species Bootstrap 2-sample Bootstrap 2-sample Chi-square Fisher’s Exact t-test t-test D. iwani 0.557 0.659 0.1940 0.3535 D. larindae N/A N/A N/A N/A D. nicolettae 0.073 0.148 0.2565 0.7925 D. intorquens 0.216 0.263 0.2960 0.1320 Diplozoon sp. N/A N/A N/A N/A All specimens 0.143 0.261 0.7355 0.3500

Key: D. iwani = Dactylogyrus iwani Crafford, Luus-Powell and Avenant-Oldewage 2012; D. larindae = Dactylogyrus larindae Crafford, Luus-Powell and Avenant-Oldewage, 2012; D. nicolettae = Dactylogyrus nicolettae Crafford, Luus-Powell and Avenant-Oldewage, 2012; D. intorquens = Dogielius intorquens Crafford, Luus-Powell and Avenant-Oldewage, 2012; All specimens = Component community, i.e. all parasites collected including damaged and unidentified specimens (only identified to genus level). ys).

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A number of interesting points arise from the results summarized in Table 7-7. Highly significant seasonal differences (increase during summer) were recorded for D. larindae on L. umbratus but not on L. capensis. From this it can be deduced that temperature (i.e. leading to an increase in parasite reproductive capabilities) could not solely be responsible for higher parasite numbers in summer, as this would also have been reflected in statistics for L. capensis.

The fact that infection statistics increased drastically in one host species (L. umbratus) but remained the same (mean intensity) or actually decreased (prevalence) in another (L. capensis), suggests that factors such as parasite host specificity or host population dynamics most probably also play an important role. The dactylogyrid D. larindae did show a preference for L. umbratus during the first (winter) survey, a preference that was again confirmed in the second (summer) survey. Commenting on the possible effect of host population dynamics may be more difficult. From the sampling efforts it was clear that these two species were the most prevalent fish species at the locality sampled. The sampling effort was, however, not conducted in a manner that would allow calculation of any population related variables (e.g. population size or age cohorts) that would allow detailed comparison of host species population differences.

On the other hand D. iwani demonstrated a preference for L. capensis during both seasons. The occurrence of this parasite (i.e. in terms of prevalence) also increased on this host from winter to summer, but numbers (i.e. mean intensity) did not increase (these trends were not statistically significant). Infections statistics for D. iwani increased significantly from winter to summer as recorded from L. umbratus (i.e. the “non-preferred” host). This is in stark contrast to the situation encountered in the preceding paragraph (i.e. where D. larindae infection statistics increased significantly in the preferred host only). From this it may be deduced that host preference may also not play a decisive role in the seasonal trends observed.

As discussed in sections 10.3.6.3 and 11.3.6.3, increased temperature generally result in higher reproductive rates for dactylogyrids. This is indeed evident from current results (Table 7-7). Yet, from the discussions above, it is clear that such seasonal trends cannot be attributed to simple temperature differences alone.

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Host preference effects may also affect seasonal trends, but the discrepancy discussed in the previous paragraph suggests that it may also not be paramount in determining such trends. It is postulated that the interaction between parasite and host population dynamics may be largely responsible for the results (i.e. no clear trend on parasite genus level with regard to seasonal infection statistics) obtained. Labeo capensis prefers to spawn in shallow rocky rapids while ideal spawning sites for L. umbratus is defined as flooded grassy banks of rivers and shallow rocky stages (Skelton 2001; Froese and Pauly 2011). One can thus assume that, while some overlap in spawning sites (i.e. rocky areas) most probably does occur, the two fish species are to a degree separated with regard to spawning behaviour. It may be that some of the parasite species (e.g. D. larindae) have developed more effective strategies (i.e. compared to that of closely related species such as D. iwani) to synchronize reproduction with such schooling behaviour of the preferred host. With host populations separated to an extend during spawning events, the preferred host population would be subjected to higher numbers of infective oncomiracidia compared to the challenge experienced by other possible hosts during the same time period.

Future ecological studies examining seasonal effects should ideally include specifically designed sampling efforts that would allow calculation of host population variables and descriptions of population dynamic differences between the host species examined. Such field studies could possibly also be combined with laboratory studies examining parasite reproductive rates at different temperatures as recorded from different hosts. Such an approach may shed light on parasite population dynamics that could be insightful when discussed in light of field study data collected as described above.

7.3.4.2. Site preference on gill

Gill site preference (pooled parasite component community), together with the Chi- square p-value to indicate if it was statistically significant, is summarized in Table 7-8 for both surveys.The only gill position for which the same statistically significant preference was recorded during both surveys, relates to gill arch preference (i.e. second and third arches). This was expected, as it relates to water flow over the gills as has been discussed in detail in section 6.3.4.3.

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For both surveys parasites exhibited a preference for the medial position of the gill as well as the anterior face of the gill. This is once again most probably the result of water flow and water pressure on the dispersion of parasites during the respiration process. This preference was found to be statistically significant during the winter survey only.

Winter Summer (June / July 2009) (January 2010) Gill position variable Preference p-value Preference p-value

Gill arch Second and third 0.002 Second and third 0.005 (1 to 4) Side of head Right > 0.2 Left 0.000 (left / right) Position on gill arch Medial 0.000 Medial > 0.2 (dorsal, median or ventral) Side of gill Anterior 0.000 Anterior 0.114 (anterior / posterior)

During winter a statistically insignificant preference was recorded for the right side of the head, while parasites appeared to prefer the left gill set during summer (statistically significant). Previous studies (e.g. Turgut, Shinn and Wootten 2006; Le Roux, Avenant-Oldewage and van der Walt (2011) also indicated no significant differences in gill set preference. It is thus believed that the significant preference observed in summer may have been the result of a random effect. Blažek and Gelnar (2006) (with reference to Rutilus rutilus (Linnaeus, 1758), Perca fluviatilis Linnaeus, 1758 and Rhodeus sericeus (Pallas, 1776) fish host species) state that gill site preference may not be due to active choice for a particular attachment site, but rather a result of fish respiration (as mentioned above) as well as fish behaviour and habitat use. Another option may thus be that fish would change their behaviour in summer months that may facilitate a preference for the left gill set. One explanation may be that fish may have an inclination to twist to one side (i.e. left) during spawning when releasing eggs or fertilizing them. This then would bring the left side in closer contact with the sediment where oncomiracidia may rest. From a biological viewpoint this is highly unlikely.

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Firstly no reference to such a behavioural adaptation could be found in the literature. Secondly, both fish species have sedentary habits so they would be in close contact with the sediment for long period in any event. If such a “twisting preference” does exist, it would only happen for brief periods that are unlike to significantly affect probability of infection in any way.

7.4. CONCLUSION

Significantly higher numbers of parasites were collected during the summer survey compared to the winter survey. This also generally translated into higher prevalence values being recorded during the summer survey. This was expected as dactylogyrids are known to have higher reproduction rates at higher temperatures (e.g. sections 10.3.6.3 and 11.3.6.3). However, apparent discrepancies (i.e. not in agreement with the general trend of increased infection statistics during summer) were recorded for individual species and are possibly related to host preference.

Furthermore site preference remained similar (with the exception of gill set, i.e. side of head) in both seasons. This was once again expected as dactylogyrid distribution on the gills is most likely determined by current during the respiration process (see section 6.3.4.3).

In both instances (i.e. infection statistics affected largely by temperature and site preference affected largely by respiration current), it is postulated that the apparent discrepancies observed relate to aspects of host biology and behaviour. The experimental design employed did, however, not allow investigation of aspects of host population dynamics or site specific (i.e. Vaal Dam) differences in behaviour between host species.

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CHAPTER 8

8 - ASPECTS OF THE ECOLOGY OF QUADRIACANTHUS AEGYPTICUS El-Naggar and Serag, 1986 FROM CLARIAS GARIEPINUS (Burchell, 1822) IN THE VAAL DAM, GAUTENG PROVINCE, SOUTH AFRICA WITH DESCRIPTION OF VARIATION OBSERVED IN HAPTORAL SCLERITES AND MALE COPULATORY ORGAN

8.1. INTRODUCTION

The genus Quadriacanthus Paperna, 1961 is characterized by the fact that species have an accessory sclerite on both ventral and dorsal anchors, with a dorsal bar lacking an anterior shield (Kritsky and Kulo 1988). The two pairs of anchors each have a short single root with the dorsal anchors larger than ventral anchors. Furthermore, the dorsal bar has two posterolateral arms and a funnel-like process projecting from its posterior extremity. The ventral bar is V-shaped and consists of two arms that articulate at the proximal ends. Seven pairs of marginal hooks are present (El-Naggar and Serag 1986).

El-Naggar and Serag (1986) described Quadriacanthus aegypticus from the gills of Clarias lazera Valenciennes, 1840 (a synonym of Clarias gariepinus (Burchell, 1822)) from the Nile Delta waters in Egypt. Kritsky and Kulo (1988) subsequently reviewed eight species of Quadriacanthus from the same host species in the Nile River. Amongst these are Q. aegypticus, which is fairly easily distinguished from the other species by the unique structure of the male copulatory organ (MCO). The accessory piece is characterized by a distinct hooked termination and two distinctive lateral outgrowths. Similar structures were described for Quadriacanthus agnebiensis (from Heterobranchus isopterus Bleeker, 1863) by N’Douba, Lambert and Euzet (1999). These authors continue by saying that this species differ from Q. aegypticus by having a smaller penis and accessory piece. Based on their illustration it would also appear that the lateral outgrowths are less distinctive, as is the curve in the accessory piece shaft where these outgrowths originate.

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El-Naggar and Serag (1986) described two positions in which the MCO can be most often observed. In the first the accessory piece is roughly parallel to the penis (copulatory tube) so that the two hooks lie adjacent to the distal end of the penis.

In the second the accessory apparatus is pulled posteriorly so that the two hooks lie adjacent to the middle region of the copulatory tube.

During a survey conducted in January 2010, Q. aegypticus specimens were recovered from the gills of C. gariepinus from the Vaal Dam, Gauteng Province, South Africa, constituting a new locality record for this parasite. The aim of this study was to investigate aspects of the ecology of Q. aegypticus from C. gariepinus in the Vaal Dam and comment on the degree of apparent morphological plasticity observed with regard to shape and size of sclerotized structures.

8.2. MATERIALS AND METHODS

For study site description refer to section 3.1.

For fish collection, necropsy and parasite recovery procedures refer to sections 3.2 and 3.3.

For details on parasite description and measurement procedures refer to section 3.4 (more specifically section 3.4.2.2 and Figure 3-5).

For details on calculation of infection statistics refer to sections 3.5 and 2.3.3.

8.3. RESULTS AND DISCUSSION

8.3.1. Host species

Eleven specimens (of which six were male) of C. gariepinus were collected collected during January 2010. Table 8-1 summarizes average weight, total length and condition factor values for fish hosts collected. Table 8-2 summarizes the gender distribution within length class categories (correlated with approximate age class based on published length / age correlations). The 11 fish collected had an unequal distribution with regard to numbers of fish in the three length classes (between two and six fish per length class). The first length class contained only males, with both the second and third length classes containing more males than females.

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8.3.2. Parasite species

A single representative from the monogenean genus Quadriacanthus was collected (see next section). This parasite was identified as Q. aegypticus, a parasite known to occur on C. gariepinus. Twenty-four parasite specimens could not be identified to genus or species level as they were damaged during collection / mounting. The identity of these parasites is indicated as “unknown” in Table 8-6.

Table 8-1: Summary description of sampled Clarias gariepinus (Burchell, 1822) host population from the Vaal Dam during summer (January 2010) survey.

Host Clarias gariepinus (Burchell, 1822) Average condition Average weight (g) Average length (cm) Variable Number factor* [SD] [SD] [SD]

Male 6 1267 [475] 56.67 [5.80] 0.67 [0.08]

Female 5 1100 [779] 53.18 [9.15] 0.64 [0.11]

Combined 11 11901 [602] 55.08 [7.32] 0.65 [0.09]

* = Fulton’s condition factor: K = 100 x W / L3 (calculated individually for each fish with calculated arithmetic average reflected here) Key SD = Standard deviation Average length = Total length

Table 8-2: Length category gender distribution of sampled Clarias gariepinus (Burchell, 1822) host population from the Vaal Dam during summer (January 2010 survey).

Corresponding age Clarias gariepinus (Burchell, 1822) Length category category * Male Female

> 40 to < 50 cm ~ 2 to 3 years 0 2

> 50 to < 60 cm ~ 4 to 5 years 4 2

> 60 to < 70 cm ~ > 5 years 2 1

Total Not applicable 6 5

* = Based on mean length / year class table in Yalçin, Solak and AkYurt (2002)

8.3.3. Measurement of haptoral sclerites and male copulatory organ (MCO)

Results from selected standard measurements taken and recorded in literature for Quadriacanthus spp. are summarized in Table 8-3.

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Table 8-4 summarizes measurements obtained for Q. aegypticus in the current study, as well as measurements for Q. aegypticus and Q. agnebiensis recorded by other authors for comparison.

Table 8-5 summarizes the complete set of measurement values (i.e. measurements as depicted in Figure 3-5) obtained for Q. aegypticus in the current study.

8.3.4. Comparing Quadriacanthus aegypticus morphology as described in previous studies

The original description of Q. aegypticus, especially the unique structure of the MCO and ventral accessory sclerite (see Figure 8-1 as redrawn from El-Naggar and Serag 1986), corresponds with that observed in the current study (Figure 8-2). The majority of average measurement values also clearly correspond well with that recorded for this species in the literature (Table 8-3), despite the fact that measurement ranges (i.e. minimum and maximum values) appear much wider for selected variables (e.g. MCO) measured in this study.

Figure 8-1: An illustration of the haptoral sclerites and male copulatory organ Quadriacanthus aegypticus El-Naggar and Serag, 1986 (redrawn from El-Naggar and Serag 1986). A – Ventral bar; B – Dorsal bar; C – Dorsal anchor; D – Marginal hooks; E – Ventral anchor; F – Male copulatory organ.

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Table 8-3: Selected standard measurements (all in micrometers) for Quadriacanthus Paperna, 1961 from literature: summary table (1 of 4)

Taxon Type locality Ventral anchor Dorsal anchor Ven- No Type / main host Literature referenced Dorsal Acc. (Quadriacanthus Synonyms (Other Length Width Base Base tral. Cirrus . (Other hosts) Taxon author Length Length bar. piece Paperna, 1961) localities) width width bar. Kritsky and Kulo 1988 398 102 ** Clarias lazera Lim, Timofeeva and 32 10 49 14 53 63 25 21 (343- (71- Valenciennes, 1840 Quadriacanthus. c. Israel Gibson 2001 (29-34) (9-11) (47-51) (12-15) (42-65) (52-72) (22-31) (17-26) Quadriacanthus (Uganda, 444) 134) 1 (Heterobranchus clariadis Paperna, 1961 clariadis Ghana, Egypt, isopterus Paperna, 1979 180 India) Tripathi, Agrawal and 82 (70- 24 10 40 10 44 50 20 22 Bleeker, 1863) (160- Pandey 2007 95) (22-26) (9-11) (36-42) (9-12) (40-46) (42-54) (18-22) (21-24) 230) Quadriacanthus Kritsky and Kulo 1988 clariadis Paperna, Lim, Timofeeva and 394 93 Quadriacanthus Egypt 38 10 47 14 47 57 43 39 ** Clarias lazera 1961; Anacornuatus Gibson 2001 (313- (76- aegypticus (Zimbabwe) (36-44) (9-12) (43-51) (13-18) (39-53) (45-74) (40-52) (33-49) aegypticus (El-Naggar El-Naggar and Serag, 502) 105) 2 and Serag, 1986) 1986 497 134 Quadriacanthus El-Naggar and Serag 41 46 44 41 ** Clarias lazera - Egypt (378- (120- - - - - aegypticus 1986 (35-43) (42-49) (40-47) (35-47) 630) 157) Quadriacanthus clariadis Kritsky and Kulo 1988 438 104 Quadriacanthus allobychowskiella Uganda Lim, Timofeeva and 29 11 48 16 50 63 45 36 3 ** Clarias lazera (380- (88- allobychowskiella Quadriacanthus kearni (Egypt) Gibson 2001 (26-31) (8-13) (44-51) (15-18) (44-56) (51-74) (41-48) (30-43) 499) 115) (El-Naggar and Serag, Paperna, 1979 1985) ** Clarias lazera Kritsky and Kulo 1988 Quadriacanthus (Clarias Lim, Timofeeva and 7 34 9 40 51 22 24 4 - Ghana - - 27-28 voltaensis camerunensis Gibson 2001 (6-8) (33-36) (8-10) (38-42) (48-53) (20-23) (20-31) Lönnberg, 1895) Paperna, 1965 Kritsky and Kulo 1988 # Bagrus docmac 410 99 Quadriacanthus Uganda Lim, Timofeeva and 32 10 39 13 52 63 25 19 (* Bagrus bayad) (287- (80- clariadis bagrae (Tanzania) Gibson 2001 (30-34) (9-12) (33-42) (12-16) (42-64) (51-78) (23-27) (17-21) Quadriacanthus (Forsskål, 1775) 481) 126) 5 Paperna, 1979 bagrae 330 (Clarias gariepinus Tripathi, Agrawal and 75 27 9 32 12 52 55 - India (260- - - (Burchell, 1822)) Pandey 2007 (65-95) (24-29) (7-10) (29-35) (10-13) (48-54) (40-58) 365) Quadriacanthus ## Tilapia esculenta Uganda Kritsky and Kulo 1988 *** May be a senior synonym of Q. bagrae with holotype representing a specimen (accidental infestation) with 6 See *** tilapiae Graham 1928 (Kenya) Paperna, 1973 deformed anchor bases as a result of coverslip pressure. Lim, Timofeeva and 399 90 Quadriacanthus 37 10 36 11 29 45 39 33 7 ** Clarias lazera - Egypt Gibson 2001 (329- (78- papernai (33-41) (8-13) (34-37) (9-13) (24-33) (40-49) (35-43) (29-37) Kritsky and Kulo, 1988 461) 108) ** Clarias lazera = Clarias gariepinus; # Bagrus docmac = Bagrus docmak; * Bagrus bayad = Bagrus bajad; ## Tilapia esculenta = Oreochromis esculenta

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Table 8-3 (continued): Selected standard measurements (all in micrometers) for Quadriacanthus spp. from literature: summary table (2 of 4)

Ventral anchor Dorsal anchor Taxon Type / main host Type locality Literature referenced Ven- Dor- Acc. No. Synonyms Length Width Base Base Cirrus (Quadriacanthus) (Other hosts) (Other localities) Taxon author Length Length tral bar sal bar piece width width 383 132 37 14 40 13 38 64 41 34 Quadriacanthus 8 ** Clarias lazera - Egypt Kritsky and Kulo, 1988 (340- (101- (35- (12- (37- (12- (33- (56- (38- (28- ashuri 431) 165) 38) 15) 42) 14) 45) 75) 43) 39) Lim, Timofeeva and Gibson 392 99 42 11 45 14 46 46 30 22 Quadriacanthus Egypt 9 ** Clarias lazera - 2001 (346 - (80- (40- (10- (42- (12- (44- (44- (28- (20- numidus (Zimbabwe) Kritsky and Kulo, 1988 432) 121) 44) 13) 47) 15) 48) 47) 32) 24) Quadriacanthus 10 - - 24-25 8-9 43-44 12-13 30-31 45-46 38-39 - sp 1 Heterobranchus - Ghana Kritsky and Kulo 1988 28 37 38 46 Quadriacanthus isopterus 9 11 - - (26- (36- 9-10 (35- (42- 23-24 - sp 2 (8-10) 30) 38) 39) 51) Lim, Timofeeva and Gibson Heterobranchus 580 138 38 48 15 54 39 39 40 Quadriacanthus 2001 9 12 longifilis - Ivory coast (470- (100- (33- (42- (13- (50- (33- (35- (38- longifilisi N’Douba, Lambert and (8-11) Valenciennes, 1840 795) 185) 41) 55) 17) 60) 43) 42) 43) Euzet, 1999 Lim, Timofeeva and Gibson 748 148 29 68 18 56 51 52 60 Quadriacanthus Heterobranchus 2001 8 13 - Ivory coast (540- (130- (27- (63- (17- (52- (48- (49- (54- thysi longifilis (7-9) N’Douba, Lambert and 820) 165) 31) 72) 19) 61) 54) 56) 65) Euzet, 1999 Lim, Timofeeva and Gibson 424 96 26 29 36 28 38 32 Quadriacanthus Heterobranchus 2001 6 9 14 - Ivory coast (380- (80- (25- (27- (34- (27- (35- (30- ayameensis isopterus N’Douba, Lambert and (5-7) (8-10) 480) 125) 27) 30) 39) 32) 43) 35) Euzet, 1999 Lim, Timofeeva and Gibson 410 99 31 42 12 46 31 29 31 Quadriacanthus Heterobranchus 2001 8 15 - Ivory coast (300- (70- (28- (37- (10- (43- (28- (28- (28- agnebiensis isopterus N’Douba, Lambert and (7-9) 670) 130) 33) 44) 14) 49) 34) 31) 34) Euzet, 1999 Lim, Timofeeva and Gibson 553 125 27 35 12 46 33 27 25 Quadriacanthus Heterobranchus 2001 8 16 - Ivory coast (485- (70- (24- (33- (11- (41- (29- (26- (24- simplex isopterus N’Douba, Lambert and (6-9) 660) 170) 29) 37) 13) 49) 36) 28) 28) Euzet, 1999 Lim, Timofeeva and Gibson 464 114 30 31 28 22 36 42 Quadriacanthus Heterobranchus 2001 9 9 17 - Ivory coast (356- (82- (27- (28- (24- (20- (34- (40- gourenei isopterus N’Douba, Lambert and (8-10) (8-10) 554) 164) 32) 34) 32) 23) 39) 45) Euzet, 1999 Lim, Timofeeva and Gibson 754 199 32 41 15 52 34 59 63 Quadriacanthus Heterobranchus 2001 18 - Ivory coast (605- (170- (31- - (40- (12- (48- (31- (58- (62- macrocirrus isopterus N’Douba, Lambert and 875) 220) 33) 42) 16) 54) 36) 60) 64) Euzet, 1999

** Clarias lazera = Clarias gariepinus

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Table 8-3 (continued): Selected standard measurements (all in micrometers) for Quadriacanthus spp. from literature: summary table (3 of 4)

Lim, Timofeeva and Gibson 2001; Tripathi, Agrawal and 28 Anacornuatus indus Clarias batrachus Quadriacanthus indus India Pandey 2007; ------Dubey, Gupta and Agarwal, 1992

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Table 8-3 (continued): Selected standard measurements (all in micrometers) for Quadriacanthus spp. from literature: summary table (4 of 4)

Table 8-4: Selected standard measurements (all in micrometers) for Quadriacanthus aegypticus El-Naggar and Serag, 1986 (from literature and from the current study) and Quadriacanthus agnebiensis N'Douba, Lambert and Euzet, 1999 only.

Ventral anchor Dorsal anchor Taxon Type / main host Locality Literature referenced Ven- Dor- Acc. No. Synonyms Length Width Base Base Cirrus (Quadriacanthus) (Other hosts) (Country) Taxon author Length Length tral bar sal bar piece width width Heterobranchus Lim, Timofeeva and Gibson 410 99 31 42 12 46 31 29 31 Quadriacanthus 8 15 # isopterus - Ivory coast 2001; N’Douba, Lambert (300- (70- (28- (37- (10- (43- (28- (28- (28- agnebiensis (7-9) Bleeker, 1863 and Euzet, 1999 670) 130) 33) 44) 14) 49) 34) 31) 34) Kritsky and Kulo 1988 ** Clarias lazera Quadriacanthus clariadis Lim, Timofeeva and Gibson 394 93 38 47 14 47 57 43 39 Quadriacanthus Egypt 10 Valenciennes, 2001 (313 - (76- (36- (43- (13- (39- (45- (40- (33- aegypticus Paperna, 1961 (Zimbabwe) (9-12) 1840 Anacornuatus aegypticus (El-Naggar and Serag, 502) 105) 44) 51) 18) 53) 74) 52) 49) 1986)

497 134 41 46 44 41 Quadriacanthus 2 ** Clarias lazera - Egypt El-Naggar and Serag, 1986 (378- (120- (35- - (42- - (40- (35- - - aegypticus 630) 157) 43) 49) 47) 47)

431 122 37 48 15 45 41 46 43 Quadriacanthus Clarias gariepinus Current study 9 - South Africa (562- (151- (33- (54- (12- (36- (34- (36- (32- aegypticus (Burchell, 1822) El-Naggar and Serag, 1986 (7-13) 323) 104) 41) 41) 16) 54) 50) 64) 63)

** Clarias lazera = Clarias gariepinus; # The male copulatory organ of Q. agnebiensis morphologically most closely resemble that of Q. aegypticus of all the species mentioned thus far – for ease of comparison measurements are thus repeated here.

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Table 8-5: Complete set of standard measurements (all in micrometers) as recorded for Quadriacanthus aegypticus El-Naggar and Serag, 1986 in the current study.

Ventral anchor Dorsal anchor Marginal hooks Ventral bar Dorsal bar

Tip / Tip / Acc. Species Length Width Total Base Total Base Base Com- Cirrus point point I to III IV V to VII Length Width Length Width piece length width length width width po-nent length length

431 122 18 31 (21- 14 43 Quadriacanthus 37 9 12 48 15 5 45 7 41 16 29 17 46 (562- (151- (11-23) 36) (8-20) (32- aegypticus (33-41) (7-13) (5-15) (54-41) (12-16) (3-6) (36-54) (5-9) (34-50) (10-23) (23-38) (14-21) (36-64) 323) 104) 63) 19 (8-36)

Table 8-5 (continued): Complete set of standard measurements (all in micrometers) as recorded for Quadriacanthus aegypticus in the current study.

Ventral Dorsal accessory accessory sclerite sclerite Species

Length Width Length

Quadriacanthus 15 8 9 aegypticus (6-21) (5-16) (8-12)

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From these figures (8-1 and 8-2) it, however, also becomes clear that the shape of the dorsal ventral sclerite as well as anchor shape appear to differ considerably. The shape illustrated in Figure 8-2 rather resembles illustrations provided by Kritsky and Kulo (1988) (Figure 8-3). A number of shape and size differences become evident when comparing Figures 8-2 and 8-3: in the illustration from Kritsky and Kulo (1988) the dorsal bar is larger while the posterior edges of the ventral bar sections (where they meet) appear to be more “rough” in that it forms a distinct upper “ridge”.

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Furthermore Kritsky and Kulo (1988) illustrated additional “membrane-like” structures associated with both the dorsal and ventral bars that were also not illustrated in the original description. The authors also do not mention these structures in subsequent descriptions or comparisons with other species. Despite these apparently gross morphological differences in size and shape, Kritsky and Kulo (1988) state that this species was adequately described by El-Naggar and Serag (1986). One can infer that these authors found the morphological variation / plasticity observed with regard to bar and anchor size and shape to be acceptable / satisfactory to retain same species status, leading to the conclusion that the shape of the MCO (distinct hooked termination and two distinctive lateral outgrowths) is considered the most characteristic / important feature when identifying this species.

8.3.5. Morphological variation observed in the current study

Such variation is, however, not only evident when viewing results obtained in different studies / publications, but also within the current study. Figure 8-4 serves to illustrate the degree of variation in shape and size observed for some of the sclerotized structures.

Male copulatory organ (MCO): Variation in the accessory piece position (i.e. either roughly parallel to the penis so that the two hooks lie adjacent to the distal end of the penis or pulled posteriorly so that the two hooks lie adjacent to the middle region of the copulatory tube), as also described by El-Naggar and Serag (1986), were observed. However, apart from this already documented variation considerable variation was observed during the current study in the size of the MCO. As is evident from Table 8-4 the penis and accessory piece length ranges recorded were greater than those reported in previous studies. However, the characteristic features (distinct hooked termination and two distinctive lateral outgrowths) corresponds with that reported by El-Naggar and Serag (1986) and Kritsky and Kulo (1988), which led to the conclusion that this species was indeed Q. aegypticus. Two other species of Quadriacanthus show some resemblance to Q. aegypticus with regard to MCO structure. The first is Q. gourenei described from Heterobranchus isopterus (N’Douba et al. 1999).

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However, the ventral anchors of Q. gourenei exhibit a very distinct and characteristic “kink” in the anchor tip, a shape described as “sigmoid” by N’Douba et al. (1999). This sigmoid tip is absent in specimens examined in the current study.

In comparison the haptoral sclerites of Q. agnebiensis, also described from Heterobranchus isopterus, are similar in shape to that of Q. aegypticus. It also shows similarities with regard to MCO structure, the accessory piece also having a terminal hook and two (though apparently less distinct) lateral outgrowths. As can be seen from Table 8-4 the MCO for this species (Q. agnebiensis) is much smaller than that of Q. aegypticus.

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Ventral bar: The ventral bar differed between individual parasites in length of the two ventral bar components, orientation of these components and the shape of the proximal ends where the two ventral bar components articulate.

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Differences in ventral bar component orientation were probably a result of the mounting process (i.e. pressure exerted on the cover slip). Ventral bar component proximal end shape ranged from rounded to more truncate (as described by El- Naggar and Serag 1986), often with a “ridge” or uneven edge (as illustrated by Kritsky and Kulo 1988). In the current study this “ridge” and the degree of “roughness” thereof also varies from absent, to less distinct to very distinct. It would thus appear that the ventral bar may be of little taxonomic significance, as a very large degree of variation occurs.

Dorsal bar: The dorsal bar also slightly differed between individual parasites with regard to shape but more so in size. Once again slight differences in shape were most probably a result of manipulation during fixing and mounting of the parasites.

Anchors: The anchors (to a larger degree the ventral anchors) also exhibited differences with regard to shape and size. Shapes reminiscent of those illustrated by both El-Naggar and Serag (1986) and Kritsky and Kulo (1988) were observed, with the latter shape predominating (i.e. most representative of the species in the current study).

8.3.6. Morphological variability observed – summary and conclusion

During the current study apparent variation in the size of the MCO, as well as size and shape of haptoral sclerites of Q. aegypticus have been observed. Comparing descriptions by El-Naggar and Serag (1986) and Kritsky and Kulo (1988), as well as the comment by the latter authors that the species was adequately described by the former, it would appear that such variation in size (MCO and haptoral sclerites) and shape (haptoral sclerites) is deemed acceptable to retain species status. Based on the MCO shape and characteristic features (distinct hooked termination and two distinctive lateral outgrowths) the species in the current study was identified as Q. aegypticus. It can be distinguished from Q. agnebiensis based on sclerite size (much smaller in Q. agnebiensis) (N’Douba et al. 1999). Apparent morphological plasticity was also encountered in Dactylogyrus spp. from Labeo spp. hosts during this study (see Chapters 4 and 5). In the latter case sclerite (specifically anchors as well as MCO) shape remained constant with variation in predominantly anchor length.

For Q. aegypticus variation in both sclerite size and shape appear evident.

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Given this large degree of morphological plasticity observed, future studies should consider statistical morphometric analyses using large databases with consideration of season. As a large degree of variation was observed in parasites collected from a single season in the current study, a seasonal or temperature effect or correlation with sclerite shape or size is deemed unlikely. The main aim of such analyses would, however, be to identify and quantify “morphological groupings” within the Vaal Dam Q. aegypticus parasite community. Molecular techniques may then be employed to evaluate if the “morphological variants” do in fact belong to a single species. Such a study may be extended to include comparison with Q. agnebiensis.

8.3.7. Biological and ecological aspects

8.3.7.1. Infection statistics and host specificity

Infection statistics are summarized in Table 8-6. Quadricathus aegypticus prevalence exceeded 90% with a mean intensity of 18.20.

8.3.7.2. Effect of host variables and evaluation of site specificity on hosts

Tables 8-1 and 8-2 summarize host length, weight and gender data. Infection levels (number of parasites collected) in terms of site selection / distribution on gills, host gender and host size classes are summarized in Table 8-6.

Both prevalence and mean intensity were higher for male hosts compared to female hosts. There was, however, no statistically significant difference (Mann-Whitney U test, p = 0.448) between male and female host with regard to the number of parasites collected. The small sample size (n=11) confounds any generalized conclusions.

The middle size class (i.e. 50 to 60 cm) exhibited the lowest parasite prevalence but highest mean infection. There was no difference in prevalence of monogenean infection between the other two size classes (i.e. greater than 40 to 50 cm and greater than 60 to less than 70 cm respectively) with a calculated value of 100% in both cases. Intensity of infection appears to be highest in the larger size class and lowest in the small size class. The small sample size (only 11 host specimens collected) again confounds any unequivocal generalizations. Future studies should include more size classes with larger sample sizes.

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Table 8-6: Quadriacanthus aegypticus El-Naggar and Serag, 1986 and unknown species infection statistics for Clarias gariepinus (Burchell, 1822) from the Vaal Dam

Host species Clarias gariepinus (Burchell, 1822) Quadriacanthus aegypticus Monogenean species ?* CC El-Naggar and Serag, 1986 Distribution Total 182 24 206 1 53 7 60 2 46 10 56 Gill arch number 3 43 5 48 4 30 2 32 Unknown 10 0 10 Left 92 9 101 Gill set Right 80 15 95 (side of head) Unknown 10 0 10 Dorsal (D) 45 6 51 Medial (M) 65 5 70 Gill arch region Ventral (V) 62 12 74 Unknown 10 1 11 Anterior 94 13 107 Gill orientation Posterior 77 11 88 Unknown 11 0 11 Infection levels Prevalence (%) 90.91 54.55 90.91 Mean intensity 18.20 4.00 20.60 Intensity range: Minimum 1 1 2 Intensity range: Maximum 37 6 43 Mean abundance 16.55 2.18 18.73 P (%) 100.00 50.00 100.00 Host gender: Male MI 22.33 4.33 24.50 MA 22.33 2.17 24.50 P (%) 80.00 60.00 80.00 Host gender: MI 12.00 3.67 14.75 Female MA 9.60 2.20 11.80 P (%) 100.00 100.00 100.00 > 40 to < 50 cm MI 4.50 3.00 7.50 (n = 2 fish) MA 4.50 3.00 7.50 P (%) 83.33 50.00 83.33 > 50 to < 60 cm MI 24.60 4.33 27.20 (n = 6 fish) MA 20.50 2.17 22.67 P (%) 100.00 33.33 100.00 > 60 to < 70 cm MI 16.67 5.00 18.33 (n = 3 fish) MA 16.67 1.67 18.33 Key (species codes used) * = ? – Specimens that could not be identified to genus CC = Component community level (damaged / lost during mounting)

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As monogenean eggs and oncomiracidia often descent to the bottom (Molnár 1971a), it may be assumed that catfish (with its sedentary habits) may be subjected to greater concentrations of parasites, resulting in the high prevalence values encountered.

More parasites were found on the first and second gill arches. This trend (i.e. unequal proportion of parasites) was statistically significant (Pearson Chi Square, p = 0.001). This is in agreement with the fact that distribution of dactylogyrids on the gills is thought to be related to water flow over the gills (largest volume passes of the second gill arch - see section 6.3.4.5 for a detailed discussion).

More parasites were found on the left gill set but the trend was not statistically significant (Pearson Chi Square, p = 0.133). Furthermore, most parasites (though not statistically significant, Pearson Chi Square, p = 0.227) were found on the ventral position of the gill arch. More parasites were recovered from the anterior hemibranch (statistically insignificant, Pearson Chi Square, p = 0.239).

8.3.7.3. Environmental variables

For a brief discussion on the potential effects of environmental variables as recorded in literature refer to section 6.3.4.5. Selected physical and other water quality parameter values were summarized in Table 7-5 (refer to section 7.3.3.3 for details).

Monogenean (other than Gyrodactylus von Nordmann, 1832) infection is often optimal during summer (e.g. Kir and Tekin Özan 2007). This may once again help explain the high prevalence of infection during this survey. During this study C. gariepinus was only encountered during the summer survey.

Collections were attempted during winter, but due to the lowered metabolism and thus also reduced vertical movement of this sedentary warm water fish species, catching them in floating gill nets during this season proved to be challenging. Future studies should attempt more intensive winter collections for seasonal comparisons.

8.3.7.4. Host species: condition factor values and macroscopic pathology

Very similar condition factor values (not statistically significant different, p=0.211 Mann Whitney U test) were calculated for both males and females (Table 8-1).

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8.4. SUMMARY AND CONCLUSION

The apparent variation in sclerite structure observed for Q. aegypticus in the current study, as well as apparent observation evident from descriptions provided in the literature, needs to be confirmed and further described. More comprehensive morphometric analyses should be performed in future studies to quantify variation and possibly describe different forms. These could then possibly be examined by molecular means to determine if these forms represent a single species, more than one distinct species or a complex of various closely related species.

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CHAPTER 9

9 - ASPECTS OF THE ECOLOGY OF A SPECIES OF GYRODACTYLUS von Nordmann, 1832 FROM CLARIAS GARIEPINUS (Burchell, 1822) IN THE VAAL DAM, GAUTENG PROVINCE, SOUTH AFRICA

9.1. INTRODUCTION

Gyrodactylus von Nordmann, 1832 contains more than 400 species of which approximately 59% were recorded from single host species (Harris, Shinn, Cable and Bakke 2004). For a brief introduction to the genus Gyrodactylus refer to section 2.4.4.

Up till present only a single species from the genus Gyrodactylus (Gyrodactylus transvaalensis Prudhoe and Hussey, 1977) have been described from Clarias gariepinus (Burchell, 1822) from South Africa (confluence of the Elands and Olifants Rivers (Prudhoe and Hussey 1977) (Table 9-1)). A number of representatives of this genus have, however, also been described from C. gariepinus from other African countries, as is summarized in Table 9-1. Apart from these published accounts of site localities and taxonomic descriptions, the muscle and nervous systems of Gyrodactylus rysavyi Ergens, 1973 have also been described by Arafa, El-Naggar, El-Abbassy, Stewart and Halton (2007).

This chapter reports on preliminary examinations of gyrodactylid specimens from the same host species collected from the Vaal Dam in South Africa. As molecular analysis falls outside the scope of the current project, the preliminary sclerite measurements are only compared to that reported for other gyrodactylid species from the literature but unequivocal species identification was not possible. Further molecular analysis and detailed study of marginal hooklet sickle morphology is suggested for species identification (or new descriptions).

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Table 9-1: Summary of species of Gyrodactylus von Nordmann, 1832 described from Clarias gariepinus (Burchell, 1822).

Locality from where originally Taxon author reference Parasite species described (Re-descriptions) (Other records) Gyrodactylus alberti Uganda Paperna, 1973 Gyrodactylus clarii Ergens, 1973 Egypt Gyrodactylus rysavyi (Přikrylová, Blažek and Vanhove (Mozambique) 2012) Gyrodactylus groschafti Egypt Ergens, 1973 Gyrodactylus transvaalensis South Africa Prudhoe and Hussey, 1977 Přikrylová, Blažek and Vanhove, Gyrodactylus turkanaensis Kenya 2012 Přikrylová, Blažek and Vanhove, Gyrodactylus alekosi Mozambique 2012

9.2. MATERIALS AND METHODS

For study site description refer to section 3.1. For fish collection, necropsy and parasite recovery procedures refer to sections 3.2 and 3.3.

For details on parasite description and measurement procedures refer to section 3.4 (more specifically section 3.4.2.3 and Figure 3-6). For details on calculation of infection statistics refer to section 3.5 and 2.3.3.

This chapter only aims to provide a preliminary examination of Gyrodactylus sp. specimens collected during this study. It is not intended as a detailed species description but only serves to compare preliminary measurements with those of other described species from the same host. The need for further detailed morphometric studies (especially with regard to the finer structure of the marginal hooklets, more specifically sickle morphology) combined with molecular analyses is acknowledged and suggested to facilitate unequivocal species description / identification.

9.3. RESULTS AND DISCUSSION

9.3.1. Host species

Eleven specimens (of which six were male) of C. gariepinus were collected collected during January 2010. Table 9-2 summarizes average weight, total length and condition factor values for fish hosts collected.

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Table 9-3 summarizes the gender distribution within length class categories (correlated with approximate age class based on published length / age correlations). The 11 fish collected had an unequal distribution with regard to numbers of fish in the three length classes (between two and six fish per length class). The first length class contained only males, with both the second and third length classes containing more males than females.

Table 9-2: Summary description of sampled Clarias gariepinus (Burchell, 1822) host population from the Vaal Dam during summer (January 2010) survey.

Host Clarias gariepinus (Burchell, 1822) Average condition Average weight (g) Average length (cm) Variable Number factor* [SD] [SD] [SD]

Male 6 1267 [475] 56.67 [5.80] 0.67 [0.08]

Female 5 1100 [779] 53.18 [9.15] 0.64 [0.11]

Combined 11 1191 [602] 55.08 [7.32] 0.65 [0.09]

* = Fulton’s condition factor: K = 100 x W / L3 (calculated individually for each fish with calculated arithmetic average reflected here) Key SD = Standard deviation Average length = Total length

Table 9-3: Length category gender distribution of sampled Clarias gariepinus (Burchell, 1822) host population from the Vaal Dam during summer (January 2010 survey).

Corresponding age Clarias gariepinus (Burchell, 1822) Length category category * Male Female

> 40 to < 50 cm ~ 2 to 3 years 0 2

> 50 to < 60 cm ~ 4 to 5 years 4 2

> 60 to < 70 cm ~ > 5 years 2 1

Total Not applicable 6 5

* = Based on mean length / year class table in Yalçin, Solak and AkYurt (2002)

9.3.2. Parasite species

A representative from the monogenean genus Gyrodactylus was collected from C. gariepinus (Figure 9-1). Results from selected standard measurements taken from this and other species collected from C. gariepinus are summarized in Table 9-4.

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Prudhoe and Hussey (1977) described G. transvaalensis from South Africa (confluence of the Elands and Olifants Rivers). It would thus be the most logical starting point when attempting species identification for the specimens collected during the current study. The excellent re-descriptions provided by Přikrylová, Blažek and Vanhove (2012) (Table 9-4) provide a solid base for preliminary comparison between the current species collected and other species (including G. transvaalensis) described from Africa.

The current specimen resembles G. turkanaensis Přikrylová, Blažek and Vanhove, 2012 with regard to measurements of hamulus total length, hamulus point length, hamulus shaft length, ventral bar median length, dorsal bar length and marginal hook sickle length. The current specimen also resembles some of the previously described G. rysavyi specimens with regard to hamulus total length, hamulus root length and ventral bar median length. The ventral bar membrane length and ventral bar width falls within the range of that recorded for G. turkanaensis, but exhibits a smaller average value. The marginal hook total length appears to fall in the range of that described for G. groschafti Ergens, 1973. Both dorsal bar width and marginal hook handle length fall in the range of that described for G. transvaalensis. Future studies should further elucidate the finer structure of the marginal hook sickle.

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Table 9-4: Selected standard measurements (all in micrometers) for species of Gyrodactylus von Nordmann, 1832 reported from Clarias gariepinus (Burchell, 1822): summary table (1 of 2).

1 Gyrodactylus 3 4 5 Gyrodactylus Variable Gyrodactylus alekosi 2 Gyrodactylus rysavyi Gyrodactylus rysavyi Gyrodactylus rysavyi 6 turkanaensis groschafti HTL 51.1 (50.5-53.0) 82.3 (77.0-87.5) 81.0 (81.0-91.0) 91.8 (90-93) 98.6 (89.0-106.5) 35.0 (35.0-37.0)

HPL 26.4 (25.0-27.5) 36.4 (34.0-38.5) 42.0 (41.0-48.0) 46.4 (45.0-47.5) 48.9 (45.5-53.0) 17.0 (17.0-19.0)

HSL 29.0 (28.5-30.0) 49.3 (47.5-51.0) 61.0 (56.0-61.0) 57.8 (57.5-58.5) 59.7 (54.5-64.0) 22.0 (22.0-23.0)

HRL 26.3 (25.0-29.0) 42.2 (34.0-44.5) 36.0 (36.0-43.0) 42.5 (40.0-44.0) 49.0 (38.0-57.0) 17.0 (17.0-18.0)

VBL 5.3 (4.5-5.5) 7.4 (5.5-8.5) 6.0-8.0 6.0 (5.5-6.5) 8.3 (6.0-10.5) 4.0-5.0

VBML 8.9 (8.0-10.0) 19.3 (17.0-21.0) 19.0 (18.0-19.0) 19.7 (19.0-21.5) 21.8 (16.0-27.0) 6.0

VBW 15.5 (13.5-17.5) 20.1 (18.0-22.5) 28.0-32.0 29.9 (28.0-31.5) 33.4 (28.5-38.5) 10.0-11.0

DBL 1.9 2.2 (2.0-2.5) 2.0-3.0 2.3 (2.0-2.5) 2.8 (1.8-3.5) 1.0

DBW 14.6 18.5 (16.5-21.0) 16-19 21.5 (19.5-22.5) 23.7 (19.0-28.0) 9.0

MHTL - 30.8 (29.0-32.0) 28.0-30.0 30.7 32.6 (30.5-36.5) 20.0 (19.0-20.0)

MHSL 4.7 (4.5-5.0) 4.0 (3.5-5.0) 5.0-6.0 4.3 (4.0-4.5) 4.2 (4.0-5.0) 5.0

MHHL 18.5 (18.0-19.0) 27.4 (26.0-28.5) - 26.8 (26.5-27.0) 28.4 (26.2-32.0) -

MHSDW 3.6 (3.5-4.0 3.9 (3.0-4.5) - - 3.5 (3.0-4.0) -

MHSPW 4.0 (4.0-4.5) 3.4 (3.0-3.5) - 3.4 (3.0-4.0) 3.6 (3.0-4.0) -

MHSAD 5.1 (5.0-5.5) 5.4 (4.5-6.0) - 4.3 (4.0-5.0) 4.5 (4.0-5.0) - HTL = Hamulus total length; HPL = Hamulus point length; HSL = Hamulus shaft length; HRL = Hamulus root length; VBL = Ventral bar median length; VBML = Ventral bar membrane length; VBW = Ventral bar width; DBL = Dorsal bar length; DBW = Dorsal bar width; MHTL = Marginal hook total length; MHSL = Marginal hook sickle length; MHHL = Marginal hook handle length; MHSDW = Marginal hook sickle distal width; MHSPW = Marginal hook sickle proximal width; MHSAD = Marginal hook sickle aperture distance. 1 = Original description by Přikrylová et al. (2012); 2 = Original description by Přikrylová et al. (2012); 3 = Original description by Ergens (1973); 4 = Re-description of original paratypes by Přikrylová et al. (2012); 5 = Re-description of new specimens by Přikrylová et al. (2012); 6 = Original description by Ergens (1973).

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Table 9-4 (continued): Selected standard measurements (all in micrometers) for species of Gyrodactylus reported from C. gariepinus: summary table (2 of 2).

Gyrodactylus Gyrodactylus Gyrodactylus 9 10 11 Variable 6 7 8 Gyrodactylus alberti Gyrodactylus clarii Gyrodactylus sp. transvaalensis transvaalensis transvaalensis

HTL 45.0-55.0 (without root) 41.5-45.0 43.5 (41.5-44.5) 113-120 53.0-54.0 78.1 (74.5-82.7)

HPL 20.0-25.0 20.5-23.0 22.3 (20.0-23.0) - - 34.7 (30.5-38.2)

HSL 25.0-30.0 27.5-28.5 27.8 (25.5-30.0) - - 47.9 (42.3-50.5)

HRL 15.0-20.0 14.5-17.0 20.0 (20.0-22.0) - - 36.8 (32.3-43.2)

VBL 15.0 4.5 3.9 (3.5-4.5) 6.0-9.0 - 6.0 (6.0-8.0)

VBML - - 8.1 (7.5-9.1) - 16.8 (15.5-18.2)

VBW - 10.5 11.4 (9.5-13.5) 21.0-23.0 21.0 19.5 (19.1-20.0)

DBL 12.0 - 1.1 (1.0-1.5) - - 2.2 (1.8-2.7)

DBW - - 13.2 (12.0-14.5) - - 14.3 (11.8-16.8)

MHTL 25.0 23.0 21.5 (20.5-22.0) 20.0-24.0 22.0-28.0 17.5 (16.4-20.0)

MHSL 5.0 5.0-5.5 5.0 (4.5-5.5) - - 3.4 (2.7-3.6)

MHHL - - 16.0 (15.5-17.0) - - 14.1 (12.7-16.4)

MHSDW - - 4.0 (4.0-4.5) - - -

MHSPW - 4.0 3.7 (3.5-4.0) - - -

MHSAD - 5.5-5.7 5.7 (5.5-6.0) - - -

RAR - - - 0.39-0.41 0.22 - HTL = Hamulus total length; HPL = Hamulus point length; HSL = Hamulus shaft length; HRL = Hamulus root length; VBL = Ventral bar median length; VBML = Ventral bar membrane length; VBW = Ventral bar width; DBL = Dorsal bar length; DBW = Dorsal bar width; MHTL = Marginal hook total length; MHSL = Marginal hook sickle length; MHHL = Marginal hook handle length; MHSDW = Marginal hook sickle distal width; MHSPW = Marginal hook sickle proximal width; MHSAD = Marginal hook sickle aperture distance; RAR = Root / anchor ratio. 6 = Original description by Prudhoe and Hussey (1977); 7 = Re-description of original paratypes by Přikrylová et al. (2012); 8 = Re-description of new specimens by Přikrylová et al. (2012); 9 = Original description by Paperna (1973); 10 = Original description by Paperna (1973); 11 = Preliminary measurements from current study.

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9.3.3. Biological and ecological aspects

9.3.3.1. Infection statistics and host specificity

Infection statistics are summarized in Table 9-5. Gyrodactylus sp. prevalence exceeded 50% with a mean intensity of 10.50.

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Table 9-5: Gyrodactylus von Nordmann, 1832 infection statistics for Clarias gariepinus (Burchell, 1822) from the Vaal Dam

Host species Clarias gariepinus (Burchell, 1822) Monogenean species Gyrodactylus von Nordmann, 1832 Distribution Total 63 1 14 2 6 3 22 Gill arch number 4 6 Unknown 0 N/A (Skin) 15 Left 21 Gill set Right 27 (side of head) Unknown 0 N/A (Skin) 15 Dorsal (D) 8 Medial (M) 18 Gill arch region Ventral (V) 22 Unknown 0 N/A (Skin) 15 Anterior 36 Posterior 12 Gill orientation Unknown 0 N/A (Skin) 15 Infection levels Prevalence (%) (P) 54.55 Mean intensity (MI) 10.50 Intensity range: Minimum 2 Intensity range: Maximum 40 Mean abundance (MA) 5.73 P (%) 83.33 Host gender: MI 11.40 Male n=6 MA 9.50 P (%) 20.00 Host gender: MI 4.00 Female n=5 MA 0.80 P (%) 0.00 > 40 to < 50 cm MI 0.00 (n = 2 fish) MA 0.00 P (%) 66.67 > 50 to < 60 cm MI 13.00 (n = 6 fish) MA 8.67 P (%) 66.67 > 60 to < 70 cm MI 4.50 (n = 3 fish) MA 3.00

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9.3.3.2. Effect of host variables and evaluation of site specificity on hosts

Tables 9-2 and 9-3 summarize host length, weight and gender data. Infection levels (number of parasites collected) in terms of site selection / distribution on gills, host gender and host size classes are summarized in Table 9-5. In total 63 parasites were collected, 48 from the gills and 15 from the skin.

Both prevalence and mean intensity were higher for male hosts compared to female hosts. Gyrodactylus sp. was, however, collected from five males and only a single female. Any gender effects observed are thus likely to be artefacts of inadequate sampling.

Parasites were only collected from the middle and larger size classes (i.e. > 50 to < 60 cm and > 60 to< 70 cm respectively), with a prevalence of 66.67% calculated for both groups. Intensity of infection was slightly higher in the middle size class. The small sample size (only 11 host specimens collected) confounds any general conclusions. Future studies should include more size classes with larger sample sizes.

More parasites were found on the third gill arch. This trend (i.e. unequal proportion of parasites) was not statistically significant (Pearson Chi Square, p = 0.624). More parasites were found on the right gill set but the trend was not statistically significant (Pearson Chi Square, p = 0.154). Furthermore, most parasites (though not statistically significant, Pearson Chi Square, p = 0.660) were found on the ventral position of the gill arch. More parasites were recovered from the anterior hemibranch (statistically insignificant, Pearson Chi Square, p = 0.933).

9.3.3.3. Environmental variables

For a brief discussion on the potential effects of environmental variables on monogeneans as recorded in literature refer to section 6.3.4.5. Selected physical and other water quality parameter values were summarized in Table 7-5 (refer to section 7.3.3.3 for details).

Blažek, Jarkovský, Koubková and Gelnar (2008b) found that gyrodactylids peaked in spring (water temperature above 6°C) and Dactylogyrus cryptomeres Bychowsky, 1943 in summer (water temperature above 14°C).

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One would thus expect higher number of Gyrodactylus sp. parasites during early spring or even late winter with water temperatures ranging between 6 and 14°C. During the current study no C. gariepinus specimens were collected during the winter survey. Future studies should thus include both winter and spring sampling efforts to further comment on possible seasonal effects.

Temperature can also affect the size of gyrodactylid parasites and their sclerotized attachment structures (e.g. Appleby 1996). Seasonal sampling, possibly combined with controlled laboratory experiments, would thus also enable investigation of temperature effects on sclerite morphology.

9.4. SUMMARY AND CONCLUSION

Prudhoe and Hussey (1977) described G. transvaalensis from South Africa. The specimens collected in the current survey shares very few characteristics with this previously described species. The current specimens appear to be more closely related to G. turkanaensis and to a lesser extend G. rysavyi (Table 9-4). Further sampling to collect additional specimens for molecular analysis, combined with detailed morphomteric analyses (particularly the finer structure of the marginal hooklet sickle) is required for unequivocal species description.

The specimens collected occurred both on the gills and skin, but the small sample size confounds any significant conclusions with regard to site preference. Results of the preliminary examination results reported here suggest that it is indeed the same gyrodactylid species occurring on both skin and gills.

This supposition should be viewed as preliminary pending on further investigation (i.e. marginal hook sickle morphometrics and molecular analyses) as described above.

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CHAPTER 10

10 - ASPECTS OF THE ECOLOGY OF MONOGENEAN PARASITES OF CYPRINUS CARPIO Linnaeus, 1758 IN THE VAAL DAM, GAUTENG PROVINCE, SOUTH AFRICA

10.1. INTRODUCTION

The natural distribution range of carp (Cyprinus carpio Linnaeus, 1758) includes Central Asia to the Black Sea, as well as the Danube in Europe (Skelton 2001). While the Chinese have cultured carp for the past 2400 years, the Romans were the first to culture this species in Europe (Skelton 2001). Since those early days this species has been widely transported around the world (Skelton 2001). It is thought that carp were already introduced in South Africa in the 1700’s, with many introductions recorded in the 1800’s (Skelton 2001). Importation of the ornamental koi variety is still ongoing (Mouton, Basson and Impson 2001). Consequently this fish species (and with it many parasites associated with it) are now found in countries as diverse as the United States of America (USA) (Cone and Dechtiar 1986), Turkey (Özer and Erdem 1998), Mozambique (Boane, Cruz and Saraiva 2008), Iraq (Al- Zubaidy 2007), Syria (Al-Samman, Molnár and Székely 2006), Bangladesh (Akter, Hossain and Rahman 2007), Uganda (Paperna 1979), Macedonia (Stojanovski, Hristovski, Cakic, Cvetkovic, Atanassov and Smiljkov 2008), Iran (Shamsi, Jalali and Aghazadeh Meshgi 2009), Sri Lanka (Thilakaratne, Rajapaksha, Hewakopara, Rajapakse and Faizal 2003), Mexico (Pérez-Ponce de Leόn, Rosas-Valdez, Aguilar- Aguilar, Mendoza-Garfias, Mendoza-Palmero, García-Prieto, Rojas-Sánchez, Briosio-Aguilar, Pérez-Rodriguez and Dominguez-Dominguez 2010), Iberian Peninsula (García-Berthou, Boix and Clavero 2007), South America (Brazil) (Ghiraldelli, Martins, Yamashita and Jerônimo 2006), Australia (Dove and Ernst 1998) and Indonesia (Buchmann, Slotved and Dana 1995). In many of these countries it is considered an invasive species (e.g. Zambrano, Martínez-Meyer, Menezes and Peterson 2006) due to the effects (e.g. increases in turbidity, nitrogen and ammonia in the water column) of its benthic foraging behaviour (Winker 2010) as is described below.

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Cyprinus carpio is described as a hardy species with a wide environmental condition tolerance range (Skelton 2001). It favours large slow-flowing or large standing water bodies with soft bottom sediment. This habitat preference relates to diet, as this omnivorous species feeds on organic matter mainly by grubbing in the sediment (Skelton 2001).

They are known to play host to a large number of monogenean parasites as summarized in Table 10-1. The aim of this chapter is to report on monogenean parasite infections found on C. carpio during a summer (January 2010) survey in the Vaal Dam, Gauteng Province, South Africa. Collections were attempted during winter, but due to the lowered metabolism and thus also reduced vertical movement of this sedentary fish species, catching them in floating gill nets during this season proved to be challenging.

10.2. MATERIALS AND METHODS

For study site description refer to section 3.1.

For fish collection, necropsy and parasite recovery procedures refer to sections 3.2 and 3.3.

For details on calculation of infection statistics refer to sections 3.5 and 2.3.3.

For details on statistical tests employed refer to section 3.6.5.

10.3. RESULTS AND DISCUSSION

10.3.1. Host species

Thirteen specimens (of which eleven were male) of C. carpio were collected. Table 10-2 summarizes the average weight, total length and condition factor values for fish hosts collected. Table 10-3 summarizes the gender distribution within length class categories. The thirteen fish collected had a similar distribution with regard to numbers of fish in the three length classes (i.e. four or three fish per length class). All length classes contained more male fish with the third length class containing no females at all.

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Table 10-1: Summary description of monogenean parasites occurring on Cyprinus carpio Linnaeus, 1758 as recorded in published papers.

Parasite species Locality References

USA Cone and Dechtiar 1986;

Gyrodactylus katharineri Malmberg, 1964 Not stated Harris, Shinn, Cable and Bakke 2004 Czech Svobodová, Máchová, Kroupová, Smutná and Groch Republic 2007 Van Cleave 1921; Putz and Hoffman 1963; Gyrodactylus fairporti Van Cleave, 1921 USA Mizelle and Kritsky 1967b Gyrodactylus carpio Kritsky and Mizelle, 1967 USA Mizelle and Kritsky 1967b

Bulgaria Nedeva and Babacheva 1999 Gyrodactylus derjavini Mikailov, 1975 Not listed Harris, Shinn, Cable and Bakke 2004 Gyrodactylus sprostonae Ling, 1962 Japan Ogawa and Egusa 1978 Gyrodactylus menschikowi Gvosdev, 1950 Iraq Al-Zubaidy 2007 Cengizler, Aytac, Sahan, Ozak and Genç 2001; Öztürk Gyrodactylus elegans von Nordmann, 1832 Turkey 2005

Gyrodactylus bimicroforatus Jin, 1993, Gyrodactylusgurleyi Price, 1937 Gyrodactylus longoacuminatus, Zitfian, 1964; Gyrodactylus medius Kathariner, 1894; Gyrodactylus nagibinae Gussev, 1962; Not listed Harris, Shinn, Cable and Bakke 2004 Gyrodactylus ophiocephali Gussev, 1955; Gyrodactylus procerus Lux, 1990; Gyrodactylus shulmani Ling, 1962; Gyrodactylus vimbi Shulman, 1954

Gyrodactylus stankowici Ergens, 1970 Germany Kappe 2004 Gyrodactylus shulmani, Ling, 1962 Germany Kappe 2004 Germany Kappe 2004 Gyrodactylus cyprini Diarova, 1964 Bavaria Dzika, Dzikowiec and Hoffmann 2009 Mongolia and Ergens 1974 Russia Czech Gelnar and Lux 1991; Šefrová and Laštůvka 2005 Gyrodactylus kherulensis Ergens, 1974 Republic Japan Ogawa and Egusa 1978; Ogawa 1994 Germany Kappe 2004; Harris, Shinn, Cable and Bakke 2004 Jarkovský, Morand, Šimková and Gelnar 2004; Jalali and Dactylogyrus achmerovi Gussev, 1955 Iran Barzegar 2005; Shamsi, Jalali and Aghazadeh Meshgi 2009 Monaco and Mizelle 1955 Jarkovský; Morand, Šimková Dactylogyrus crassus Kulviec, 1927 Not listed and Gelnar 2004 Dactylogyrus auriculatus (Nordmann, 1832) Dactylogyrus cryptomeres Bychowsky, 1943 Dactylogyrus difformes Wagener, 1857 Dactylogyrus dujardinianus (Siebold, 1849) Dactylogyrus falculatus Guegan, Lambert and Not listed Monaco and Mizelle 1955 Euzet, 1988 Dactylogyrus fallax Wagener, 1857 Dactylogyrus formosus Kulwiec, 1927 Dactylogyrus mollis (Wedl, 1858) Dactylogyrus wegeneri Kulwiec, 1927

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Table 10-1 (continued): Summary description of monogenean parasites occurring on Cyprinus carpio Linnaeus, 1758 as recorded in published papers.

References Parasite species Locality (listed by) Shamsi, Jalali and Aghazadeh Meshgi 2009; Jalali and Dactylogyrus sahuensis Ling, 1965 Iran Barzegar 2005 Not listed Monaco and Mizelle 1955; Dactylogyrus vastator Nybelin, 1924 Turkey Cengizler, Aytac, Sahan, Ozak and Genç 2001 Iran Shamsi et al. 2009; Jalali and Barzegar 2005 Not listed Monaco and Mizelle 1955; Mizelle and McDougal 1970 Dactylogyrus anchoratus (Dujardin, 1845) Syria Al-Samman, Molnár and Székely 2006 Iran Shamsi et al. 2009; Jalali and Barzegar 2005 Not listed Monaco and Mizelle 1955; Mizelle and McDougal 1970 Japan Ogawa and Egusa 1977 Uganda Paperna 1979 Indonesia Buchmann, Slotved and Dana 1995 Dactylogyrus minutus Kulwiec, 1927 South Africa Mashego 2003 Kir and Tekin Özan 2007; Aksoy, Saglam and Dorucu Turkey 2006 Stojanovski, Hristovski, Cakic, Cvetkovic, Atanassov and Macedonia Smiljkov 2008 Not listed Mizelle and McDougal 1970 Japan Ogawa 1994 Indonesia Buchmann et al. 1995 Australia Dove and Ernst 1998 South Africa Mashego 2003 Thilakaratne, Rajapaksha, Hewakopara, Rajapakse and Sri Lanka Faizal 2003 USA Choudhury, Hoffnagle and Cole 2004 Dactylogyrus extensus Turkey Öztürk 2005; Aksoy et al. 2006 Mueller and Van Cleave, 1932 Syria Al-Samman et al. 2006 Macedonia Stojanovski et al. 2008 Bangladesh Hossain, Hossain, Rahman, Akter and Khanom 2008 Iran Shamsi et al. 2009; Jalali and Barzegar 2005 Pérez-Ponce de Leόn, Rosas-Valdez, Aguilar-Aguilar, Mendoza, Mendoza-Palmero, García-Prieto, Rojas- Mexico Sánchez, Briosio-Aguilar, Pérez-Rodríguez and Domínguez-Domínguez 2010; Kohn, Cohen, Salgado- Maldonado 2006 Dactylogyrus ersinensis Iraq Al-Zubaidy 2007 Spasskij and Rojtmann, 1960 Diplostomum phoxini (Faust, 1918) Turkey Kayis, Ozcelep, Capkin and Altinok 2009 Diplozoon nipponicum Goto, 1891 Japan Ogawa 1994 Eudiplozoon nipponicum (Goto, 1891) Macedonia Stojanovski et al. 2008 Paradiplozoon zeller (Gyntovt, 1967) Bulgaria Nedeva and Babacheva 1999 Paradiplozoon homoion Turkey Kayis et al. 2009 (Bychowsky and Nagibina, 1959)

10.3.2. Parasite species

Two representatives from the monogenean genus Dactylogyrus were collected.

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These parasites were identified as Dactylogyrus extensus Mueller and Van Cleave, 1932 (Figure 10-1) and Dactylogyrus minutus Kulwiec, 1927 (Figure 10-2). These two species have previously been reported from the same host and locality (Vaal Dam) by Mashego (2003). Both species appear to be very prevalent globally (or at least most often reported - see Table 10-1) and as a result D. extensus has also been used for development of imaging protocols (Galli, Strona, Villa, Benzoni, Fabrizio, Maria and Kritsky 2006).

Molnár (2009) states that D. extensus was most probably the first (of several) monogeneans introduced to Europe together with carp native to the Amur region. Using parasite data he demonstrated that carp stocks currently residing in the USA has a mixed origin (i.e. imported from both Europe and Asia). Thilakaratne et al. (2003) listed D. extensus as one of the most common parasites in ornamental fish species prepared for export from Sri Lanka. The apparently high prevalence of the parasite within the ornamental fish trade shall obviously help facilitate global spread of the parasite.

One representative from the monogenean genus Gyrodactylus was collected (Table 10-4). A characteristic feature of collected specimens is a deeply notched dorsal bar. This is evident in a number of Gyrodactylus spp., such as Gyrodactylus trematoclithrus Rogers, 1967 from Lucania goodei Jordan, 1880 (Rogers, 1967), Gyrodactylus stableri Hathaway and Herlevich, 1973 from Fundulus kansae Garman, 1895 (Hathaway and Herlevich 1973) and Gyrodactylus aurorae Mizelle, Kritsky and McDougal, 1969 from Rana a. aurora Baird and Girard, 1852 (Mizelle, Kritsky and McDougal 1969). None of these species have, however, been found on carp and the remaining sclerites (particularly the ventral bar) differ in morphology to that of specimens collected in the current study. Species of Gyrodactylus recorded from carp have been listed in the introduction section (Table 10-1). Of these species two have notched dorsal bars (Gyrodactylus kherulensis Ergens, 1974 and Gyrodactylus cyprini Diarova, 1964). Dzika, Dzikowiec and Hoffman (2009) also recovered G. cyprini while investigating D. extensus. These authors state that G. cyprini is the only gyrodactylid described from carp that has an oval plate associated with the anchors. No such plate was observed during examination of the specimens collected during the present study.

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The specimens are, however, characterized by a dorsal bar that is very deeply notched (appears to be divided yet with the two sections in close contact in some specimens) and a very distinct heel on the terminal shaft section of the anchor. The illustrations accompanying the original description by Ergens (1974) confirm considerable variation in the degree of notching of the dorsal bar as well as shaft root structure (though the latter was not as distinct as was recorded for the current specimens). This parasite collected during the current study was thus identified as G. kherulensis (Figure 10-3).

Six parasite specimens could not be identified to genus or species level as they were damaged during collection / mounting. The identity of these parasites is indicated as “unknown” in Table 10-4.

Table 10-2: Summary description of sampled Cyprinus carpio Linnaeus, 1758 host population from the Vaal Dam in January 2010.

Host Cyprinus carpio Linnaeus, 1758 Average condition Average weight (g) Average length (cm) Variable Number factor* [SD] [SD] [SD]

Male 11 450 [457] 30.31 [8.35] 1.22 [0.49]

Female 2 100 [0] 25.50 [0.99] 0.61 [0.07]

Combined 13 396 [437] 29.57 [7.84] 1.13 [0.50]

* = Fulton’s condition factor: K = 100 x W / L3 (Calculated for each individual and average as represented here determined from all calculated values) Key SD = Standard deviation Average length = Average total length

Table 10-3: Length category gender distribution of sampled Cyprinus carpio Linnaeus, 1758 host population from the Vaal Dam during January 2010.

Cyprinus carpio Linnaeus, 1758 Length category Approximate age * Male Female

> 20 to < 25 cm ~ 1 to 3 years 4 1

> 25 to < 30 cm ~ 3 to 4 years 3 1

> 30 to < 50 cm > 4 years 4 0

Total 11 2

* Derived from age at length correlation table compiled by Karataş, Çiçek, Başusta and Başusta (2007)

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Figure 10-3: Gyrodactylus kherulensis Ergens, 1974 from Cyprinus carpio Linnaeus, 1758 in the Vaal Dam, South Africa (current study): A = Anchors; B = Dorsal bar; C = Marginal hooks.

10.3.3. Biological and ecological aspects

10.3.3.1. Infection statistics and host specificity

For a brief discussion of host specificity in Dactylogyrus spp. parasites as recorded in previous studies refer to section 6.3.4.2. During the current study all three monogenean species (i.e. D. minutus, D. extensus and G. kherulensis) was only found on C. carpio.

Jarkovský, Morand, Šimková and Gelnar (2004) classify D. extensus as a “specialist” restricted to C. carpio. This sentiment is echoed by Kappe (2004) and the results of the current study support this notion. However, Shamsi et al. (2009) stated that both D. anchoratus as well as D. extensus exhibit low host-specificity and tolerate a wide range of temperature and salinity, making them successful invading species. This exact same opinion is shared by Dove and Ernst (1998). Adaptation of D. extensus to both oxygen and salinity levels has been demonstrated by Paperna (1964a).

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Apart from C. carpio the parasite has indeed also been recorded from species such as Micropterus dolomieu (Lacepède, 1802) (smallmouth bass) (Mizelle and McDougal 1970) as well as Carassius auratus (Linnaeus, 1758) (goldfish) (Shamsi et al. 2009). In this study the parasite has not been collected from other fish species (including Micropterus salmoides (Lacepède, 1802), largemouth bass), but the possibility of host switching should be monitored in further studies and may even be examined by experimental infection studies.

Infection statistics are summarized in Table 10-4. Ghiraldelli et al. (2006) reported that Dactylogyrus spp. is often the most prevalent monogeneans on C. carpio. The statement is confirmed in the current study for one of the Dactylogyrus sp recorded. Dactylogyrus extensus was most prevalent (76.92%). Mashego (2003) recorded 100.00% prevalence at the same locality. The lower prevalence recorded during the current study corresponds well to the 73.60% prevalence Öztürk (2005) recorded for the same parasite species in Turkey. Stojanovski et al. (2008) reported a lower prevalence of 32.00% for D. extensus from carp in Macedonia.

Dactylogyrus minutus and G. kherulensis were found to be equally prevalent (7.69%) during the current study. Mashego (2003) recorded a prevalence of 40.00% for D. minutus at the same locality, similar to the 38.09% prevalence recorded by Kir and Tekin Özan (2007) for the same parasite in Turkey. Stojanovski et al. (2008) reported a prevalence of 24.00% for D. minutus from carp in Macedonia.

Öztürk (2005) recorded a prevalence of 67.10% for G. elegans on carp in Turkey, while Al-Zubaidy (2007) reported prevalence of 32.00% and 12.60% (for G. menschikowi and G. derjavini respectively) from carp in Iraq. Furthermore Hossain,Hossain, Rahman, Akter and Khanom (2008) calculated Dactylogyrus spp. prevalence to be 19.00%. The prevalence values recorded for these two parasite species (D. minutus and G. kherulensis) during the current study can thus be considered low compared to the norm as reflected in published literature. Such low prevalence is, however, not completely unheard of. Akter et al. (2007), for example, calculated a prevalence of 7.19% for Gyrodactylus sp. and 14.39% for Dactylogyrus sp. on C. carpio in Bangladesh.

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Mean intensity (current study) for individual species ranged between 1.00 and 3.90 with a mean intensity of 5.10 calculated for the component community (i.e. for all parasite species combined, including unidentified / damaged specimens).

This is much less than the mean intensities of 27.00 and 53.21 recorded by Mashego (2003) and Öztürk (2005) for D. extensus, but in line with the mean intensity value of 2.00 Mashego (2003) calculated for D. minutus. It is, although somewhat higher, also still comparable to the low mean intensity values (0.66 and 0.13) Al-Zubaidy (2007) recorded for G. menschikowi and G. derjavini, respectively, from carp in Iraq. It is, however, much lower than the mean intensity value of 67.75 Öztürk (2005) recorded for G. elegans on carp in Turkey.

10.3.3.2. Effect of host variables and evaluation of site specificity on hosts

For a brief discussion of host specificity in Dactylogyrus spp. parasites as recorded in previous studies refer to section 6.3.4.4. Tables 10-2 and 10-3 summarize host length, weight and gender data. Infection levels (number of parasites collected) in terms of site selection / distribution on gills, host gender and host size classes are summarized in Table 10-4.

Both prevalence and mean intensity were higher for male hosts compared to female hosts. There was, however, no statistically significant difference (Mann-Whitney U test, p = 0.103) between male and female hosts with regard to the number of parasites collected. Given the small sample size for female hosts (n=2) no generalized conclusion is possible.

Stojanovski et al. (2008) found that the condition factor for infected fish were lower (1.06) when compared to uninfected fish (1.12). They conclude that this was due to the monogenean parasite infections encountered. They, however, also vaguely mention that physiological changes during spawning (which should transpire into gender differences) would also affect the condition factor value. The latter is more likely as they recorded relatively low intensity of infection (the highest intensity of infection recorded was only 18.0). Unfortunately no information is given about the size of fishes collected (i.e. smaller fish may be affected more than larger fish). The correlation observed may thus not necessarily be attributed to a cause (monogenean parasite burdens) and effect (decreased condition factor value in infected fish) correlation.

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Furthermore, if decreased condition factor is attributed to parasite burdens one would have to consider all parasites and not only monogeneans. The small sample size of the current study confounds any conclusions to be drawn.

Table 10-4: Monogenean parasite infection statistics for Cyprinus carpio Linnaeus, 1758 from the Vaal Dam during January 2010.

Host species Cyprinus carpio Linnaeus, 1758 D. G. D. minutus Dact. spp. ?* CC Monogenean species extensus kherulensis Distribution Total 39 3 4 3 2 51 1 13 0 3 0 16 2 5 0 1 1 7

Gill arch number 3 6 0 0 Found applicable: Not on skin 1 7 4 5 0 0 0 5 Unknown 10 3 0 0 13 Left 14 0 2 1 17 Gill set Right 15 0 2 1 18 (side of head) Unknown 10 3 0 0 13 Dorsal (D) 5 0 0 1 6 Medial (M) 19 0 4 0 23 Gill arch region Ventral (V) 5 0 0 1 6 Unknown 10 3 0 0 13

Anterior 15 0 2 1 18 Gill orientation Posterior 14 0 2 1 17 Unknown 10 3 0 0 13 Infection levels Prevalence (%) 76.92 7.69 15.38 7.69 15.38 76.92 Mean intensity 3.90 3.00 2.00 3.00 1.00 5.10 Intensity range: Minimum 1 3 1 3 1 1 Intensity range: Maximum 10 3 3 3 1 14 Mean abundance 3.00 0.23 0.31 0.23 0.15 3.92 P (%) 81.82 9.09 18.18 9.09 18.18 81.82 Host gender: MI 4.11 3.00 2.00 3.00 1.00 5.44 Male MA 3.36 0.27 0.36 0.27 0.18 4.45 P (%) 50.00 0.00 0.00 0.00 0.00 50.00 Host gender: MI 2.00 0.00 0.00 0.00 0.00 2.00 Female MA 1.00 0.00 0.00 0.00 0.00 1.00 P (%) 40.00 0.00 0.00 0.00 20.00 40.00 > 20 to < 25 cm MI 3.50 0.00 0.00 0.00 1.00 4.00 (n = 5 fish) MA 1.40 0.00 0.00 0.00 0.20 1.60 P (%) 100.00 0.00 0.00 0.00 0.00 100.00 > 25 to < 30 cm MI 1.50 0.00 0.00 0.00 0.00 1.50 (n = 4 fish) MA 1.50 0.00 0.00 0.00 0.00 1.50 P (%) 100.00 25.00 50.00 25.00 25.00 100.00 > 30 to < 50 cm MI 6.50 3.00 2.00 3.00 1.00 9.25 (n = 4 fish) MA 6.50 0.75 1.00 0.75 0.25 9.25 Key (species codes used) D. extensus = Dactylogyrus extensus D. minutus = Dactylogyrus minutus Dact. spp. = Dactylogyrus spp. that could not be G. kherulensis = Gyrodactylus kherulensis identified to species level (damaged during mounting) * = ? – Specimens that could not be identified to genus CC = Component community level (damaged / lost during mounting)

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An attempt at statistical analysis of differences between size classes and gender was deemed inadequate given the small sample size and shall not be discussed. Jalali and Barzegur (2005) found that D. extensus was more prevalent on what they termed adult fish (18 to 29 cm) while D. vastator and D. anchoratus were more prevalent on fingerlings (3 to cm). Tekin Özan, Kir and Barlas (2008) found very little variation in host size classes with regard to D. minutus infections, but found intermediate sized fish (i.e. approximately 30 to 40 cm) to be slightly less infected. Future studies should include more size classes (providing possibilities for experimental infection studies) to examine such possible host size effects.

Al-Zubaidy (2007) (with reference to D. ersinensis, G. menschikowi and G. derjavini infecting carp) concluded that prevalence was negatively correlated with fish size (i.e. smaller fish were more susceptible to infection). The age of carp can be most accurately determined from asteriscus otoliths (Phelps, Edwards and Willis 2007). In future studies otoliths may be retained for accurate age differentiation in order to compare infection statistics between length classes to that obtained using age classes (i.e. to determine if an accurate correlation exists between age and length class).

Host biology and behaviour may also play a role to ensure successful infection. Molnár (1971a) showed that Dactylogyrus lamellatus Achmerow, 1952 oncomiracidia preferred to localize near the sediment / substratum, as motile miracidia would often descend to rest. This is in concurrence with Kir and Tekin Özan (2007) whom states the dactylogyrid eggs fall to the bottom of the water body in question.

One can thus assume that fish with sedentary habits (in this case carp that exhibit sedentary feeding behaviour) will be subjected to greater concentrations of hatching D. extensus and D. minutus oncomiracidia.

For a brief discussion of site specificity in Dactylogyrus spp. parasites as recorded in previous studies refer to section 6.3.4.5. While certain trends do emerge from Table 10-4 (e.g. more parasites were found on the first gill arch), statistical analysis and further discussion of these results was not deemed justifiable given the small sample sizes of both host and parasite specimens. Furthermore the large number of unknown cases may confound general trends.

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Turgut, Shinn and Wootten (2006) examined dactylogyrid site specificity on carp and found fewer D. extensus on the first gill arch compared to the other arches (this was not statistically significant though). Furthermore they found no statistical difference in the number of D. extensus parasites on the left or right set of gill arches. They did, however, find more parasites on the median position of the gill arch and the posterior hemibranch.

Paperna (1964b) demonstrated competitive exclusion of D. extensus by D. vastator. The mechanism by which this happens appears to be histological changes to the gills that make the habitat unsuitable for D. extensus. Such negative interactions are very rare and this appears to be the only published account. Given the high prevalence of D. extensus encountered during the current study it is obvious that it is not being excluded by D. minutus.

While the opposite may be true the limited sample size does not allow any analyses to test this hypothesis. Both these monogenean species are reported in the literature as occurring together on gills, but no mention has been made of suspected negative interactions. This possibility is thus considered to be highly unlikely.

10.3.3.3. Environmental variables

Kir and Tekin Özan (2007), with reference to D. minutus, state that parasite infection is optimal during summer. They explain this by saying that temperature affects dactylogyrid egg development (positive correlation). This trend was confirmed by Tekin Özan et al. (2008). Paperna (1964a) has shown that egg hatching for D. extensus is still unimpaired at temperatures ranging between 24 and 28 °C. Özer and Erdem (1998) examined the occurrence of monogenean parasites of carp in relation to temperature. They divided their study period into three respective periods based on temperatures encountered: Period 1 (approximately 25.5 to 26.2 °C, i.e. high); Period 2 (approximately 12.7 to 13.6°C, i.e. low); Period 3 (approximately 14.8 to 16.1°C, i.e. intermediate). Temperatures for the current study falls between periods 1 and 3.

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The authors indeed found the highest prevalence of infection for D. extensus during these two periods. Özer and Erdem (1998) furthermore state that physiological stress caused by spawning may further contribute to increased parasite burdens during spring and summer. During spawning fish also congregate that would result in increased infection opportunities for the parasite.

Future studies should include more regular seasonal sampling to elucidate the effect temperature may have on prevalence and intensity of monogenean parasites in the Vaal Dam. From results discussed in Chapter 7 (i.e. Dactylogyrus Diesing, 1850 from Labeo Cuvier, 1817 hosts), it is postulated that C. carpio shall also exhibit increased levels of parasite infection during summer. This would be the result of increased reproduction (resulting from shorter development periods) for parasites such as D. extensus, which could be compounded by a reduced host immunological state resulting from physiological spawning related stress. An opposite trend is expected for G. kherulensis. Gyrodactylid numbers often decrease at higher temperatures and as a result higher numbers of this parasite may be expected to occur in winter.

Paperna (1964a) has also shown that D. extensus is resistant to prolonged (up to six days) lowered oxygen levels (down to 0.4 mg/L) and salinities up to 1.485% NaCl. It is clear that this parasite would not have been inhibited by dissolved oxygen and salinity conditions encountered during the current study. Paperna (1964a) concludes that this parasite may develop strains that are well adjusted to a wide range of environmental conditions it may encounter, once emphasizing the invasive abilities of this parasite.

10.3.3.4. Host species: condition factor values and macroscopic pathology

The very small sample size (n=2 females) preclude any statistical analysis, discussion and hence conclusive generalization. Further studies (including larger sample sizes and greater seasonal sampling variation), to investigate the potential contribution of spawning associated changes to fish health as opposed to parasite induced changes, are required.

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No macroscopic pathology / abnormal conditions resulting from monogenean infection (e.g. sporadic haemorrhages and pale gills with excessive mucous secretion) (Buchmann 1999; Martins, Moraes, Fujimoto, Onaka, Nomura, Silva and Schalch 2000; Arafa, El-Naggar and El-Abbassy 2009) were observed. This is in accordance with results obtained by Jalali and Barzegar (2005). They found no abnormalities when examining naturally (i.e. in wild fish) occurring monogenean infections, but did record disease symptoms (and associated gill changes as described above) in infected fry of cultured carp.

10.4. SUMMARY AND CONCLUSION

The apparent host specificity observed for especially D. extensus on C. carpio in the Vaal Dam needs to be confirmed / monitored, as it has been shown to be a potential invasive species (i.e. with regard to host switching to indigenous fish). Differences in parasite infection levels between gender and size classes examined could not be statistically evaluated due to sample size constraints. Differences between size classes have, however, been reported in other studies and need to be further investigated. Future ecological studies should thus compare infection statistics between wider host length classes as well as seasons.

In terms of gill arch specificity on the gill apparatus, the small sample size and large number of unknown cases (with regard to position on the gills) precludes any meaningful discussion.

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CHAPTER 11

11 - ASPECTS OF THE ECOLOGY OF A MONOGENEAN PARASITE OF CTENOPHARYNGODON IDELLA (Valenciennes, 1844) IN THE VAAL DAM, GAUTENG PROVINCE, SOUTH AFRICA

11.1. INTRODUCTION

While grass carp (Ctenopharyngodon idella (Valenciennes, 1844)) is endemic to the Amur River system (i.e. China extending through to eastern Siberia), the species has been widely transported around the world (Froese and Pauly 2011). The species now almost has a cosmopolitan distribution and has been reported from countries as diverse as Cuba (Mendoza-Franco, Vidal-Martinez, Cruz-Quintana and Leόn 2006), Mexico (Kohn, Cohen and Salgado-Maldonado 2006), Syria (Al-Samman, Molnár and Székely 2006), Bangladesh (Hossain, Hossain, Rahman, Akter and Khanom 2008), Iran (Jalali and Barzegar 2006), Turkey (Uzbìlek and Yildiz 2002), Hungary (Molnár 1971b) and Canada (Cudmore and Mandrak 2004). As it prefers large slow- flowing or standing water bodies with vegetation, it often can be found in lakes, ponds, pools and backwaters of large rivers (Froese and Pauly 2011) where they are tolerant of a wide range of temperatures (approximately 0° to 38°C) (Froese and Pauly 2011) and oxygen levels (as low as 0.2 mg/l) (Cudmore and Mandrak 2004). Older fish are also very tolerant of salinities up to 17.5 ppt (Cudmore and Mandrak 2004). These fish are often employed for weed control purposes (e.g. Uzbilek and Yildiz 2002) as it feeds on higher aquatic plants and submerged grasses (approximately 95% of the diet are made up of macrophytes) (Cudmore and Mandrak 2004). It, however, shall also feed on detritus, insects and other invertebrates (Froese and Pauly 2011).

They are known to play host to a number of monogenean parasites, including Gyrodactylus ctenopharyngodontis Ling, 1962; Gyrodactylus elegans von Nordmann, 1832; Gyrodactylus katharineri Malmberg, 1964; Gyrodactylus medius Kathariner, 1894; Gyrodactylus wageneri von Nordmann, 1832; Dactylogyrus aristichthys Long and Yu, 1958; Dactylogyrus ctenopharyngodontis Achmerow, 1952; Dactylogyrus hypophthalmichthys Akhmerov, 1952; Dactylogyrus inexpectatus Isjumova, 1955; Dactylogyrus lamellatus Achmerow, 1952; Dactylogyrus

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magnihamatus Achmerow, 1952; Dactylogyrus nobilis Long and Yu, 1958; Dactylogyrus scrjabini Achmerow, 1952 (Cudmore and Mandrak 2004); Dactylogyrus vistulae Prost, 1957 (Galli, Strona, Benzoni, Crosa and Stefani 2007) and Diplozoon paradoxum Nordmann, 1832 (Willomitzer 1980a). Of these D. lamellatus appear to be most prevalent / widely distributed (e.g. Šefrová and Laštůvka 2005; Al-Samman et al. 2006; Jalali and Barzegar 2006) and often result in mortalities during grass carp fry and fingerling production (e.g. Shamsi, Jalali and Aghazadeh Meshgi 2009). Because of the important impact this parasite has on grass carp production, research has also been conducted on development of effective control measures (e.g. Molnár 1971c, Willomitzer 1980b).

The aim of this chapter is to report on D. lamellatus infection found on C. idella during a summer (January 2010) survey in the Vaal Dam, Gauteng Province, South Africa.

11.2. MATERIALS AND METHODS

For study site description refer to section 3.1. For fish collection, necropsy and parasite recovery procedures refer to sections 3.2 and 3.3. For details on parasite description and measurement procedures refer to section 3.4 (more specifically section 3.4.2.1 and Figure 3-4 with reference to Dactylogyrus Diesing, 1850). For details on calculation of infection statistics refer to sections 3.5 and 2.3.3 and for details on statistical tests employed refer to section 3.6.5.

11.3. RESULTS AND DISCUSSION

11.3.1. Host species

Twelve specimens (of which seven were male) of C. idella were collected. Table 11- 1 summarizes average weight, total length and condition factor values for fish hosts collected. Table 11-2 summarizes the gender distribution within length class categories. The twelve fish collected had an equal distribution with regard to numbers of fish in the three length classes (four fish per length class). The first length class contained more male fish with equal gender distribution in the other two length classes.

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11.3.2. Parasite species

A single representative from the monogenean genus Dactylogyrus was collected (Table 11-3). This parasite was identified as D. lamellatus, a parasite known to be specific to C. idella (Pugachev, Gerasev, Gussev, Ergens and Khotenowsky 2010). Ten parasite specimens could not be identified to genus or species level as they were damaged during collection / mounting. The identity of these parasites is indicated as “unknown” in Table 11-3.

Table 11-1: Summary description of sampled Ctenopharyngodon idella (Valenciennes, 1844) host population from the Vaal Dam during summer (January 2010 survey).

Host Ctenopharyngodon idella (Valenciennes, 1844) Average condition Average weight (g) Average length (cm) Variable Number factor* [SD] [SD] [SD]

Male 7 450 [240] 37.27 [4.57] 0.79 [0.22]

Female 5 780 [822] 42.18 [11.55] 0.78 [0.20]

Combined 12 588 [553] 39.32 [8.14] 0.79 [0.20]

* = Fulton’s condition factor: K = 100 x W / L3 Key SD = Standard deviation Average length = Total length

Table 11-2: Length category gender distribution of sampled Ctenopharyngodon idella (Valenciennes, 1844) host population from the Vaal Dam during summer (January 2010 survey).

Ctenopharyngodon idella Corresponding age Length category (Valenciennes, 1844) category * Male Female

> 30 to < 35 cm ~ 4 years 3 1

> 35 to < 40 cm ~ 4 years 2 2

> 40 to < 61 cm ~ 5 to 6 years 2 2

Total Not applicable 7 5

* = Based on mean length / year class figure in Cudmore and Mandrak (2004)

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11.3.3. Measurement of haptoral sclerites and male copulatory organ (MCO)

The haptoral sclerites as well as the MCO are depicted in Figure 11-1. Measurement values obtained for sclerites (n = 11 parasites measured) are summarized in Table 11-3.

Interestingly enough Molnár (1971a) found that the final body size (i.e. length and width measurement) of D. lamellatus depends on the dimensions of its host. Fingerlings (less than 10 cm long) were found to play host to parasites up to a maximum size of 400 μm in length and 120 μm in width. Larger fish (more than 30 cm long) was found to carry parasites as large as 580 μm in length and 160 μm in width. Tabulated sclerite measurements, however, indicated that the size of chitinous organs was not affected by differences in body size. It would thus appear that the parasites size correlates with the host size but the parasite’s sclerotized attachment structures / organs does not. This implies that total body length and width measurements of the parasite may be of little taxonomic importance.

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11.3.4. Comparing morphology and measurements as described in previous studies

Basic morphology corresponds with that recorded by Gussev (1962) as is exemplified by Figure 11-2 when compared with Figure 11-1. The MCO was, however, found to be prone to manipulation and subsequent distortion during the preservation and mounting process (see section 11.3.5 and Figure 11-3).

Measurements of sclerites also compare well to that previously recorded by Pugachev et al. (2010) (Table 11-3).

11.3.5. Morphological variation observed in the current study

As mentioned the MCO (particularly the position and orientation of the structures comprising the fairly elaborate accessory piece) appears to be prone to manipulation. The characteristic shape of the MCO illustrated in Figures 11-1 and 11-2 is thus not always clearly visible, as illustrated in Figure 11-3:

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Table 11-3: Dactylogyrus lamellatus Achmerow, 1952 (from Ctenopharyngodon idella (Valenciennes, 1844)) measurement (all in micrometers) summary table

Anchor Marg. Literature L W Inner Outer Bar Penis MCO* Form description L Shaft Tip hooks referenced (Mean) (Mean) root root (Mean) (Mean) (Mean) (Mean) (Mean) (Mean) (Mean) (Mean) (Mean) 226.9- 63.5- 32.7- 10.9- 2.5- 23.6- 15.5- 20.9- 18.2- 20.9- 28.2- Current study # Not applicable 384.6 138.5 36.8 13.6 4.5 26.8 19.1 29.1 33.2 30.0 50.9 (Vaal Dam, South Africa) (304.3) (100.3) (34.8) (12.3) (3.4) (25.8) (17.0) (26.2) (25.0) (26.5) (37.5) Pugachev et al. Up to Up to Palaearctic and Amur regions 31-44 10-14 3-5 25-30 14-18 27-36 19-35 20-28 35-50 (2010) 530 130 L = Length; W = Width, Marg hooks = Marginal hooks; Acc piece = Accessory piece; * = Total length of male copulatory organ (MCO), i.e. length of penis and accessory piece combined; # = MCO’s that appeared intact and in correct orientation ranged between 45.5 to 50.9 micrometers.

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Figure 11-3: Variation observed in the orientation of the MCO of Dactylogyrus lamellatus Achmerow, 1952 from Ctenopharyngodon idella (Valenciennes, 1844) in the Vaal Dam, South Africa.

11.3.6. Biological and ecological aspects

11.3.6.1. Infection statistics and host specificity

For a brief discussion of host specificity in Dactylogyrus spp. parasites that were recorded in previous studies are presented in section 6.3.4.3. During the current study D. lamellatus was only found on C. idella. Strict host specificity of this parasite was demonstrated by Molnár (1971a) whom attempted to infect (by subjecting disease-free fry to larvae in water) grass carp, common carp, silver carp, bighead carp and a common carp x grass carp hybrid with D. lamellatus larvae. Only the grass carp fry were infected.

Infection statistics are summarized in Table 11-4. The high prevalence (83%) obtained is in agreement with result obtained by Al-Samman et al. (2006) (100%) in Syria. Molnár (1971a) demonstrated that oviposition rate increases with a rise in temperature, which may help explain the high prevalence recorded during this summer survey (January 2010). As no C. idella were collected during the winter survey (June/July 2009) no seasonal trends can be discussed here. Molnár (1971b) found intensity of infection to be very high (80 to 250 D. lamellatus parasites per fish) on grass carp fry (3.5-4.5 cm) under cultured conditions. Fish showing signs of clinical disease (behavioural changes or physical lesions) always had more than 150 parasites on them.

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Table 11-4: Monogenean parasite infection statistics for Ctenopharyngodon idella (Valenciennes, 1844) from the Vaal Dam

Ctenopharyngodon idella Host species (Valenciennes, 1844) Dactylogyrus Monogenean species lamellatus ?* CC Achmerow, 1952 Distribution Total 73 10 83 1 16 7 23 2 29 3 32 Gill arch number 3 21 0 21 4 7 0 7 Gill set Left 36 9 45 (side of head) Right 37 1 38 Dorsal (D) 28 3 31 Medial (M) 31 2 33 Gill arch region Ventral (V) 13 5 18 Unknown 1 0 1 Anterior 51 2 53 Gill orientation Posterior 22 8 30 Infection levels Prevalence (%) 83.33 8.33 83.33 Mean intensity 7.30 10.00 8.30 Intensity range: Minimum 1 10 1 Intensity range: Maximum 31 10 31 Mean abundance 6.08 0.83 6.92 P (%) 71.43 14.29 71.43 Host gender: MI 13.00 10.00 15.00 Male MA 9.29 1.43 10.71 P (%) 100.00 0 100.00 Host gender: MI 1.60 0 1.60 Female MA 1.60 0 1.60 P (%) 50.00 0 50.00 > 30 to < 35 cm MI 2.50 0 2.50 (n = 4 fish) MA 1.25 0 1.25 P (%) 50.00 0 50.00 > 35 to < 40 cm MI 23.50 0 23.50 (n = 4 fish) MA 11.75 0 11.75 P (%) 50.00 25.00 50.00 > 40 to < 61 cm MI 10.50 10.00 15.50 (n = 4 fish) MA 5.25 2.50 7.75 Key (species codes used) ?* – Specimens that could not be identified to genus CC = Component community level (damaged / lost during mounting)

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He also found that intensity of infection decreased during winter. Such high intensities of infection are typical of crowded aquaculture conditions and were thus not expected in the current study. Then again such small fish were not examined and in future studies infection intensities relating to size differences could be experimentally examined or by additional sampling within natural populations.

11.3.6.2. Effect of host variables and evaluation of site specificity on hosts

For a brief discussion of host specificity in Dactylogyrus spp. parasites as recorded in previous studies refer to section 6.3.4.6. Tables 11-1 and 11-2 summarize host length, weight and gender data. Infection levels (number of parasites collected) in terms of site selection / distribution on gills, host gender and host size classes are summarized in Table 11-4.

Prevalence was higher for female hosts compared to male hosts with mean intensity of infection showing an opposite trend (i.e. higher for male hosts). There was, however, no statistically significant difference (Mann-Whitney U test, p = 0.335) between male and female host with regard to the number of parasites collected. There was also no difference in prevalence of monogenean infection between size classes with a calculated value of 50% in all three cases. Mean intensity of infection appears to be highest in the middle size class. The small sample size (only twelve host specimens collected) and little variation between size classes (e.g. no fish smaller than 30 cm were collected and examined with two of the size classes evaluated in 5 cm increments), however, confound any unequivocal generalizations.

Molnár (1971a) discusses the correlation between grass carp age and establishment and further development of D. lamellatus infection. Frequent failure of the parasites to infect very young fish has been noted and may be related to either the physical dimensions (i.e. size of gills) or state of development of the gill apparatus (i.e. a function of fish age). Both fish size and age may thus play a role and in very young fish the larvae tend to settle on surrounding tissues (e.g. arches or pharyngeal epithelium) and not on the gill lamellae. During this study this phenomenon could not be studied as such very small fish have not been collected. Willomitzer (1980a) noted that parasite species richness (with specific reference to dactylogyrosis and diplostomosis) tend to increase with advancing age in grass carp fry.

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In the current study only monogenean parasites were examined and only a single species were encountered.

Host size also appears to affect mortality rate of infected fish. Molnár (1971b) state that surface gill area affects the point at which D. lamellatus intensity of infection becomes lethal. He continues by saying that temperature and water oxygen content also contributes (e.g. small fish with massively infected gills shall die from oxygen depletion at increased temperatures at a faster rate than less infected fish).

Host biology and behaviour may also play a role to ensure successful infection. Molnár (1971a) showed that oncomiracidia preferred to localize near the bottom as they would often descent to rest. Grass carp are herbivorous and shall feed at various depths depending on the availability of food. As very little aquatic vegetation was visible in the shallows and none on the water surface during the survey when fish was collected (personal observation), one can only but assume that the majority of fish at this site fed on the bottom. The fact that coarse anglers often catch fish at this venue (Vaal Dam) on soft dough baits fished on the bottom (personal observation) further supports this statement. Habitat preference in the Vaal Dam may thus also contribute to increased infection probability. Future studies could investigate habitat and feeding preferences in different bodies of water (e.g. floating vegetation versus sediment/bottom vegetation). This could be combined with an experimental approach during which fishes could be maintained at different depths (e.g. separated by mesh of appropriate size to allow eggs and oncomiracidia to pass through) to evaluate to what degree depth stratification may affect infection statistics.

For a brief discussion of site specificity in Dactylogyrus spp. parasites as recorded in previous studies refer to section 6.3.4.5. More parasites were found on the second gill arch during the present study. This trend (i.e. unequal proportion of parasites) was not statistically significant (Pearson Chi Square, p=0.587) and is thought to be related to water flow over the gills (see section 6.3.4.5 for a detailed discussion). This is confirmed by Molnár (1971a) whom states that infection of C. idella with D. lamellatus most frequently takes place by passive infection (i.e. larvae swept passively through the gills with water). The other two routes he mentions are eggs that become fixed to the gills as well as active migration of larvae to the gills via the skin. Slightly more parasites were found on the left set of gill arches for grass carp from the Vaal Dam.

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This trend was also not statistically significant (Pearson Chi Square, p = 0.139). Furthermore, most parasites (though not statistically significant, Pearson Chi Square, p = 0.353) were found on the medial position of the gill arch, while significantly (Pearson Chi Square, p = 0.042) more parasites were recovered from the anterior hemibranch. Molnár (1971a) found that the parasites occur on any position in older fish but seem to prefer a terminal position (i.e. closer to gill filament tips) in younger fish. The method used for parasite recovery (gill scrapes) in the current study, however, did not allow differentiation of such positions.

11.3.6.3. Environmental variables

For a brief discussion on the potential effects of environmental variables as recorded in literature refer to section 6.3.4.6. Selected physical and other water quality parameter values are summarized in Table 11-5. Water analysis data was obtained from Rand Water (sampling point reference C-VD1l, monthly water sampling). As samples were not taken on the precise dates of fish host sampling, results are provided for analyses conducted within approximately one month prior to the sampling effort as well as one month thereafter.

Table 11-5: Environmental (physical) and water quality variables from the Vaal Dam (source: Rand Water, sampling point identification number C-VD1l).

DO Temp Cond TDS CaCO3 Cr Cu Pb Date pH (%) (°C) (mS/m) (mg/L) (mg/L) (µg/L) (µg/L) (µg/L) 22 Dec 2009 80.4 20.3 18 145 8.23 57 <0.010 0.01 <8.00 26 Jan 2010 71.9 23.4 18 170 8.22 56 <0.010 <0.010 <8.00 23 Feb 2010 77.3 19.5 17 175 7.348 55 <0.010 0.02 <8.00 DO = Dissolved oxygen; Temp = Temperature; Cond = Conductivity; TDS = Total dissolved solids; CaCO3 = Calcium-carbonate; Cr = Chrome; Cu = Copper; Pb = Lead. As indicated previously, Molnár (1971a) demonstrated an increased oviposition rate in D. lamellatus with an increase in temperature. He states that the optimum temperature in Hungary (with reference to oviposition rate) is 17 to 28°C. Water temperatures recorded over the study period all fall within that optimal range. Increase in temperature also results in a decrease in time required to reach sexual maturation in developing parasites (Molnár 1971a). Furthermore he states that failure of larval development was often not due to temperature influences, but rather other factors like fungal or bacterial growth and oxygen depletion.

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Dissolved oxygen levels remained high during the study period which would most probably have resulted in a good larval development rate.

11.3.6.4. Host species: condition factor values and macroscopic pathology

For comprehensive reviews on Fulton’s condition factor used in this study, refer to Froese (2006) and Nash, Valencia and Geffen (2006) (also see section 3.3.5). While more male C. idella were collected compared to females, there were no significant difference (p=0.468 Mann Whitney U test) in mean condition factor values between genders (Table 11-1). Despite the statistically insignificant gender differences (in terms of prevalence and intensity of infection) discussed in section 11.3.6.2, it is thus unlikely that parasite infections observed influenced condition factor values.

For a brief overview of how condition factors relate to monogenean infections from previous studies, refer to section 6.3.4.1. In this section it was also postulated that, as the first study survey was conducted in winter and fish generally reproduce in spring and early summer, females were not burdened with the physiological stress of egg production and spawning which may explain the condition factor results (i.e. very little difference between genders) obtained. Results from the current chapter are a reflection of a survey performed later in summer (January). The same argument may thus ring true (i.e. the period post-spawning was long enough to mitigate any effects spawning and associated physiological changes may have had on the condition factor). Future studies could also include a spring survey, to further investigate the potential contribution of spawning associated changes to fish health as opposed to parasite induced changes.

No macroscopic pathology / abnormal conditions resulting from monogenean infection (e.g. sporadic haemorrhages and pale gills with excessive mucous secretion) (Buchmann 1999; Martins, Moraes, Fujimoto, Onaka, Nomura, Silva and Schalch 2000; Arafa, El-Naggar and El-Abbassy 2009) were observed. Signs observed by Molnár (1971b), with specific reference to D. lamellatus infection in C. idella, include fish gathering at the surface or pond edges showing lethargic behaviour or compulsive circular movements, as well as pale anaemic gills with macroscopically visible lesions covered by copious amounts of mucous and surrounded by haemorrhage.

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In future studies, disease status of sampled fish (with particular reference to protozoan gill parasites in especially fry) could be established and correlated with prevalence and intensity of infection statistics as these parasites apparently have an antagonistic effect (Molnár 1971a).

Such an approach should also include studies on histopathological gill changes, as has been done by Molnár (1972).

11.4. SUMMARY AND CONCLUSION

Differences in parasite infection levels between gender and size classes examined were not expected nor observed. Future ecological studies should compare parasite community composition (including site preferences of the various species), infection statistics between wider host length classes, seasons and various habitats (e.g. rapids, glide sections, deep pools, shallow backwaters etc.) within the Vaal River system, to that obtained in the Vaal Dam.

In terms of gill arch specificity on the gill apparatus, an unequal proportion of parasites on the various areas examined were found to be statistically significant (Pearson Chi Square) in one case (more parasites present on the anterior hemibranch). Though not statistically significant, there also appeared to be a preference for the medial position on the second gill arch. It is believed that the apparent preference may rather relate to water flow over the gills.

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CHAPTER 12

12 - ASPECTS OF THE ECOLOGY OF MONOGENEAN PARASITES OF LABEOBARBUS AENEUS (Burchell, 1822) AND LABEOBARBUS KIMBERLEYENSIS (Gilchrist and Thompson, 1913) IN THE VAAL DAM, GAUTENG PROVINCE, SOUTH AFRICA

12.1. INTRODUCTION

The genus Barbus Cuvier and Cloquet, 1816 is one of the largest fish genera in the world and initially contained both South African barbs (minnows) and yellowfishes. Some of the South African yellowfishes now have been allocated their own genus, namely Labeobarbus Rüppell, 1836 (Skelton 2001). From a parasitological view this distinction may well have merit. El Gharbi, Birgi and Lambert (1994) examined fish from two subgenera (Barbus (Barbus) spp. as well as Barbus (Labeobarbus) spp.) in North Africa. They concluded that, apart from one Dactylogyrus species occurring on both, the two groups each have their own distinct monogenean fauna. Such data may thus be applied to infer phylogenetic relationships amongst these fish host species. Lévéque and Guégan (1990) followed a similar approach and used morphological characteristics of the hosts combined with monogenean species data, to elucidate the phylogenetic relationship between African species of the genus Barbus.

Labeobarbus aeneus (Burchell, 1822) (smallmouth yellowfish) were originally endemic to the Orange-Vaal River system but has also been introduced into the Gouritz (South Cape) and Olifants (Limpopo) River systems (Froese and Pauly 2011). This omnivorous bottom feeder occurs in both rivers and impoundments (with rocky or sandy substrates) but prefers clear-flowing waters. Here they migrate upstream to spawn over suitable gravel beds from spring to midsummer after the first good rains (Froese and Pauly 2011).

Labeobarbus kimberleyensis (Gilchrist and Thompson, 1913), the largest scale- bearing indigenous fish species in southern Africa, occurs in the Orange-Vaal River system (Froese and Pauly 2011). This predator prefers flowing water below rapids or in deep channels but also does well in impoundments.

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It is known to breed over gravel beds in running water from mid-summer to late summer (Froese and Pauly 2011). According to Froese and Pauly (2011) the IUCN Red List Status of this species is indicated as “Near Threatened (NT)”.

As is the case with some other endemic species, little is known about the monogenean fauna of representatives of the genus Labeobarbus in South Africa. Mashego (1983) described a number of Dactylogyrus spp. from barbs (Barbus paludinosus Peters, 1852, Barbus neefi Greenwood, 1962 and Barbus trimaculatus Peters, 1952) and identified another (Dactylogyrus spinicirrus (Paperna and Thurston, 1967)) from largescale yellowfish (Labeobarbus marequensis (Smith, 1841)). Price, McClellan, Druckenmiller and Jacobs (1969) reported D. varicorhini Bychowsky, 1958 from L. kimberleyensis (largemouth yellowfish). These are also the only two records of monogenean parasites from South African yellowfishes as listed by Khalil and Polling (1997). Hempel, Avenant-Oldewage and Mashego (2000), however, also reported Paradiplozoon Akhmerov, 1974 from L. aeneus (smallmouth yellowfish) in the Vaal Dam. Furthermore Matla, Mokgalong and Mashego (2010) reported dactylogyrids on Labeobarbus, Labeo and Barbus spp. from Lake Tzaneen, while Mbokane, Luus-Powell, Matla and Theron (2010) reported dactylogyrids from Labeobarbus and Barbus spp. from Nwanedi-Luphephe Dams.

The current chapter reports on monogenean parasites recovered from L. aeneus, L. kimberleyensis and suspected hybrids of these two species collected from the Vaal Dam, South Africa.

12.2. MATERIALS AND METHODS

For study site description refer to section 3.1.

For fish collection, necropsy and parasite recovery procedures refer to sections 3.2 and 3.3.

For details on parasite description and measurement procedures refer to section 3.4 (more specifically sections 3.4.2.1 and 3.4.2.3 as well as Figures 3-4 and 3-6).

For details on calculation of infection statistics refer to sections 3.5 and 2.3.3.

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12.3. RESULTS AND DISCUSSION

12.3.1. Host species

Winter survey: Three specimens (of which all were male) of L. kimberleyensis were collected, while 16 specimens (of which 14 were male) of L. aeneus were collected. Only one suspected hybrid (male) was collected during this survey.

Summer survey: Four specimens (of which one was male) of L. kimberleyensis were collected, while 15 specimens (of which five were male) of L. aeneus were collected. Two suspected hybrids were collected during this second survey (one male and one female).

Table 12-1 summarizes average weight, total length and condition factor values for fish hosts collected. Table 12-2 summarizes the gender distribution within length class categories. The fish collected exhibited unequal distribution with regard to numbers of fish in the different length classes, as fish of similar size were collected in most instances. In similar fashion gender distribution was also unequal as more males than females were collected.

12.3.2. Parasite species

Two genera were recovered from yellowfish during the current study: Paradiplozoon and Dactylogyrus (Table 11-3). A previous study dealt with Paradiplozoon sp. from yellowfish (Milne and Avenant-Oldewage 2006 and 2012) and for the purposes of this study reference is only made to genus level. The next section shall deal with the possible identity of the Dactylogyrus sp. Some parasite specimens could not be identified to genus or species level as they were damaged during collection / mounting. The identity of these parasites is indicated as “unknown” in the tables that follow.

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Labeobarbus kimberleyensis (Gilchrist and Thompson, 1913) - Winter survey (June / Host July 2009)

Average weight (g) Average length (cm) Average condition Variable Number factor* [SD] [SD] [SD] Male 3 650 [229] 39.47 [6.42] 0.92 [0.15]

Female 0 - - -

Combined 3 650 [229] 39.47 [6.42] 0.92 [0.15]

Host L. kimberleyensis - Summer survey (January 2010)

Male 1 950 [N/A] 47.60 [N/A] 0.88 [N/A]

Female 3 842 [391] 45.67 [7.48] 0.83 [0.05]

Combined 4 869 [324] 46.15 [6.19] 0.85 [0.04]

Host Labeobarbus aeneus (Burchell, 1822) - Winter survey (June / July 2009)

Male 14 511 [265] 37.31 [5.12] 0.90 [0.14]

Female 2 975 [530] 44.25 [7.42] 1.06 [0.07]

Combined 16 569 [324] 38.18 [5.66] 0.92 [0.15]

Host L. aeneus - Summer survey (January 2010)

Male 5 470 [120] 36.66 [3.30] 0.94 [0.03]

Female 10 493 [233] 37.35 [4.63] 0.88 [0.18]

Combined 15 485 [198] 37.12 [4.12] 0.90 [0.14]

Host Labeobarbus sp. suspected hybrid - Winter survey (June / July 2009)

Male 1 600 [N/A] 41.20 [N/A] 0.86 [N/A]

Female 0 - - -

Combined 1 600 [N/A] 41.20 [N/A] 0.86 [N/A]

Host Labeobarbus sp. suspected hybrid - Summer survey (January 2010)

Male 1 650 [N/A] 43.90 [N/A] 0.77 [N/A]

Female 1 550 [N/A] 39.60 [N/A] 0.89 [N/A]

Combined 2 600 [701] 41.75 [3.04] 0.83 [0.08]

* = Fulton’s condition factor: K = 100 x W / L3 (calculated for each individual fish with the calculated average provided in the table) Key SD = Standard deviation Average length = Total length N/A = Not applicable

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Labeobarbus kimberleyensis Corresponding age Length category (Gilchrist and Thompson, 1913) – June / July 2009 category * Male Female

> 30 to < 46 cm ~ 6 to 7 years 3 0

Corresponding age L. kimberleyensis – January 2010 Length category category * Male Female

> 30 to < 40 cm ~ 6 to 7 years 0 1

> 45 to < 55 cm ~ 9 to 11 years 1 2

Total ~6 to 11 years 1 3

Labeobarbus aeneus Corresponding age Length category (Burchell, 1822) – June / July 2009 category * Male Female

> 20 to < 30 cm ~ 3 to 4 years 1 0

> 30 to < 40 cm ~ 5 to 7 years 12 1

> 40 to < 50 cm ~ 7 to 8 years 1 1

Total ~ 3 to 8 years 14 2

Corresponding age L. aeneus – January 2010 Length category category * Male Female

> 29 to < 35 cm ~ 5 to 6 years 2 3

> 35 to < 40 cm ~ 5 to 7 years 2 4

> 40 to < 45 cm ~ 7 to 8 years 1 3

Total ~ 5 to 8 years 5 10

Corresponding age Labeobarbus sp.** – June / July 2009 Length category category * Male Female

41.2 ~ 7 years *** 1 0

Corresponding age Labeobarbus sp.** – January 2010 Length category category * Male Female

> 39 to < 45 cm ~ 7 to 8 years 1 1

* = Based on mean length / year class tables in Mulder (1973b) ** = Suspected hybrid (L. kimberleyensis x L. aeneus as determined by macroscopic observation only) – hybrid status should be considered preliminary until it can be confirmed by molecular and more detailed morphometric means in future studies. *** = Based on length vs. age data for L. aeneus.

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12.3.2.5. Monogeneans from Labeobarbus aeneus (Burchell, 1822): Dactylogyrus sp. G

As mentioned previously D. varicorhini was reported to occur on L. kimberleyensis (Price et al. 1969). In the current study specimens of the “D. varicorhini species group / type” was recovered from both L. kimberleyensis (Figure 12-2) and L. aeneus (Figure 12-1). This is in accordance with El Gharbi et al. (1994) whom stated that the “type varicorhini” specimens are restricted to Labeobarbus spp. This was confirmed thorough sampling by Mashego (1983) who did not recover any specimens of this type from South African barbs (Barbus spp.).

In discussions that follow the “D. varicorhini species group / type” specimens shall be referred as “Dactylogyrus sp. G”. While it shares several characteristics with the “D. varicorhini species group / type”, too few adequate (in terms of visibility of sclerites) specimens were collected to allow unequivocal species identification.

Morphological features that are characteristic of the “D. varicorhini species group / type” include the characteristic shape of the anchors (Price et al. 1969), the characteristic shape of the bar (El Gharbi et al. 1994) and the large size of the marginal hooks (El Gharbi et al. 1994). Unfortunately the MCO (male copulatory organ) was not clearly visible in the majority of the specimens collected during the current study.

However, the long, curved penis observed in some specimens is typical of several species belonging to the varicorhini type (e.g. El Gharbi et al. 1994; Jalali, Papp and Molnár 1995; Guégan and Lambert 1990).

Preliminary measurements from four specimens indicate that sclerite (anchors, marginal hooks and bars) size ranges also fall within that previously recorded for D. varicorhini by Paperna (1961) (Table 12-3). Although the current specimens could not be identified to species level, it is concluded that it most definitely belongs to the “D. varicorhini species group / type” (i.e. based solely on anchor and bar shape and size). Furthermore, as D. varicorhini has been collected from L. kimberleyensis in South Africa previously, additional sampling of both yellowfish species is required to unequivocally determine if 1) it is indeed a single parasite species infecting both host species and 2) identify / describe the parasite(s) to species level.

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Current and pilot Current study “Minor” variety from study “Major” variety from adult fish (200 to Tadjikistan (Vaal Dam) juvenile fish (70 to 80 mm (Vaal Dam) 400 mm in length)in open water variety (n=3 specimens in length) in shallow water (n=4 specimens * * measured) Variables measured * measured) # (unit = μm) # Varicorhinus Labeobarbus Varicorhinus Barbus canis Labeobarbus capoeta kimberleyensis damascinus Valenciennes, V. damascinus B. canis aeneus Güldenstädt, (Gilchrist and Valenciennes, 1842 1842 (Burchell, 1822) 1773 Thompson, 1913) Total length (entire body) 450 - 720 870 200-320 260-300 310-560 245-464 (334) 431-469 (447) Total width (entire body) 80-110 100 50-80 50-70 70-150 52-91 (76) 119-142 (135) Anchor length 60-83 60-61 35-50 45 43-46 48-55 (51) 52-58 (56) Inner root length 17-20 25-26 12-20 17-18 14-17 12-19 (15) 18-23 (20) Outer root length 6-8 9-11 3-7 5 4-7 3-7 (5) 4-9 97) Shaft length 39-45 40-50 30-35 32-35 31-33 37-39 (38) 41-44 (43) Tip length 15-20 17 5-20 11 13-15 9-16 (12) 14-16 (15) Bar one length 39-44 44 25-31 30 23-26 28-31 (29) 26-35 (30) Bar two length 25-30 24-31 20-28 29 23-25 17-25 (20) 16x15x9 *** Marginal hook length 30-47 35-50 17-37 22-35 26-37 16-38 (29) 27-40 (33) 50-59 (55)**** Copulatory organ 20-40 x 30-34 39 - 20-22 28-35 ** 15-18 x 4-9 (16x7) ***** * = Measurements obtained from Paperna (1961); # = Measured in current study (ranges with average value given in brackets); ** = Male copulatory organ not clearly visible / measurable in any of the specimens; L. = Labeobarbus; *** = A second “y-shaped” bar was visible in only one specimen. Measurements indicate the length of the three “legs” of the structure. If this structure was seen from the side it may possibly be perceived as a single bar; **** = Penis tube trace length; ***** = Accessory piece structure greatest length and width dimensions.

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12.3.3. Morphological variation observed in the current study

Figure 12-1 depicts some of the variation observed in the “D. varicorhini species group / type” specimens collected from L. aeneus. It would appear that, particularly with regard to anchors, the apparent “variation” relates more to orientation of sclerites rather than true morphological differences.

A similar scenario is apparent when viewing Figure 12-2 (illustrating variation observed in the “D. varicorhini species group / type” specimens collected from L. kimberleyensis). This further emphasizes the need for additional sampling to elucidate both specie(s) identification and subsequently to quantify and describe variation where applicable. This is especially relevant were the same parasite appears to be found on both L. kimberleyensis and suspected Labeobarbus spp. hybrids. Future studies may, for example, determine if potential differences in host gill morphology may result in morphological variation in parasite attachment structures. Apart from possible differences between host species, differences between size classes of the same species also need to be examined (see data from Paperna 1961 in Table 12-1). This will be further discussed in section 12.3.5.

12.3.4. Infection statistics

Infection statistics for the parasites collected are summarized in Tables 12-4 to 12-8. As Paradiplozoon sp. is found in-between the two sets (i.e. anterior and posterior sides of the gill) of gill lamellae, gill orientation is indicated as “unknown” for this parasite species.

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Figure 12-1: An illustration depicting variation observed in the sclerite morphology of Dactylogyrus sp. G collected from Labeobarbus aeneus (Burchell, 1822). The shape of the anchors (A) and bars (B) and large size of the marginal hooks (C) are reminiscent of the “Dactylogyrus varicorhini Bychowsky, 1958 species group / type”. Unfortunately the male copulatory organ (D) was not clearly visible in any of the specimens collected from L. aeneus.

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12.3.5. Infection statistics related to host variables

For a brief discussion of host specificity in Dactylogyrus spp. parasites as recorded in previous studies refer to section 6.3.4.6. Tables 12-1 and 12-2 summarize host length, weight and gender data. Infection levels (number of parasites collected) in terms of site selection / distribution on gills, host gender and host size classes are summarized in Tables 12-4 to 12-8.

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There was no statistically significant difference (Mann-Whitney U test, p = 0.251, pooled for both Labeobarbus spp. and both surveys) between male and female host with regard to the number of parasites collected.

Small sample sizes within different size classes, as well as small increment differences between size classes (i.e. 5 or 10 cm increments), confound meaningful generalized deductions. In some instances there was only one size class (e.g. Tables 12-4 and 12-8). In others there were no differences in prevalence between smallest and largest size classes (e.g. Table 12-6), while a higher prevalence was recorded in the largest size class in two instances (Tables 12-5 and 12-7).

Based on sclerite dimensions, Paperna (1961) identified two forms of D. varicorhini that occurred on adult and juvenile fish respectively. Based on length-age estimates in Table12-2, all fish in the current study may well be considered adult as they were older than three years. From Table 12-3 it appears that a difference in sclerite dimensions may well exist between specimens collected from L. aeneus and L. kimberleyensis respectively, with monogeneans from the latter presenting with slightly larger dimensions. Whether this constitutes separate species or forms of the same species need to be determined in future studies through more intensive sampling.

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Table 12-4: Monogenean parasite infection statistics for Labeobarbus Rüppell, 1836 from the Vaal Dam: Labeobarbus kimberleyensis (Gilchrist and Thompson, 1913) – Winter (June 2009) survey.

Host species Labeobarbus kimberleyensis (Gilchrist and Thompson, 1913) (Winter survey June 2009) Monogenean species Paradip. sp Dact. G Dact. spp. ? * CC Distribution Total 0 0 2 0 2 1 0 0 1 0 1 2 0 0 0 0 0 Gill arch number 3 0 0 1 0 1 4 0 0 0 0 0 Unknown 0 0 0 0 0 Left 0 0 1 0 1 Gill set Right 0 0 1 0 1 (side of head) Unknown 0 0 0 0 0 Dorsal (D) 0 0 2 0 2 Medial (M) 0 0 0 0 0 Gill arch region Ventral (V) 0 0 0 0 0 Unknown 0 0 0 0 0 Anterior 0 0 0 0 0 Gill orientation Posterior 0 0 0 0 0 Unknown 0 0 2 0 2 Infection levels Prevalence (P) (%) 0 0 25.0 0 25.0 Mean intensity (MI) 0 0 2.0 0 2.0 Intensity range: Minimum 0 0 2.0 0 2.0 Intensity range: Maximum 0 0 2.0 0 2.0 Mean abundance (MA) 0 0 0.5 0 0.5 P (%) 0 0 33.3 0 33.3 Host gender: MI 0 0 2.0 0 2.0 Male MA 0 0 0.67 0 0.67 P (%) Host gender: MI No female fish collected. Female MA P (%) 0 0 33.3 0 33.3 > 30 to < 46 cm MI 0 0 2.0 0 2.0 (n = 4 fish) MA 0 0 0.67 0 0.67 Key (species codes used) Paradip. – Paradiplozoon Akhmerov, 1974 Dact. G – Dactylogyrus sp. G Dact spp. = Dactylogyrus Diesing, * ? = Specimens that could not be 1850 not identified to species level identified to genus level (damaged / CC = Component community (damaged / lost during mounting) lost during mounting)

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Table 12-5: Monogenean parasite infection statistics for Labeobarbus Rüppell, 1836 from the Vaal Dam: Labeobarbus kimberleyensis (Gilchrist and Thompson, 1913) – Summer (January 2012) survey.

Labeobarbus kimberleyensis (Gilchrist and Thompson, 1913) Host species (Summer survey January 2012) Monogenean species Paradip. sp Dact. G Dact. spp. ? * CC Distribution Total 6 3 0 0 9 1 3 2 0 0 5 2 1 0 0 0 1 Gill arch number 3 1 0 0 0 1 4 1 1 0 0 2 Unknown 0 0 0 0 0 Left 1 2 0 0 3 Gill set Right 5 1 0 0 6 (side of head) Unknown 0 0 0 0 0 Dorsal (D) 6 2 0 0 8 Medial (M) 0 0 0 0 0 Gill arch region Ventral (V) 0 1 0 0 1 Unknown 0 0 0 0 0 Anterior 0 3 0 0 5 Gill orientation Posterior 0 0 0 0 0 Unknown 6 0 0 0 4 Infection levels Prevalence (P) (%) 50.0 25.0 0 0 50.0 Mean intensity (MI) 3.0 3.0 0 0 4.0 Intensity range: Minimum 3 3 0 0 3 Intensity range: Maximum 3 3 0 0 6 Mean abundance (MA) 1.5 0 0 0 2.3 P (%) 0 0 0 0 0 Host gender: MI 0 0 0 0 0 Male MA 0 0 0 0 0 P (%) 66.7 33.3 0 0 66.7 Host gender: MI 3 3 0 0 4.5 Female MA 2.0 1.0 0 0 3.0 P (%) 0 0 0 0 0 > 30 to < 40 cm MI 0 0 0 0 0 (n = 1 fish) MA 0 0 0 0 0 P (%) 66.7 33.3 0 0 66.7 > 45 to < 55 cm MI 3.0 3.0 0 0 4.5 (n = 3 fish) MA 2.0 1.0 0 0 3.0 Key (species codes used) Paradip. – Paradiplozoon Akhmerov, 1974 Dact. G – Dactylogyrus sp. G Dact. spp. = Dactylogyrus Diesing, * ? = Specimens that could not be 1850 not identified to species level identified to genus level (damaged / CC = Component community (damaged / lost during mounting) lost during mounting)

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Table 12-6: Monogenean parasite infection statistics for Labeobarbus Rüppell, 1836 from the Vaal Dam: Labeobarbus aeneus (Burchell, 1822) – Winter (June 2009) survey.

Host species Labeobarbus aeneus (Burchell, 1822) (Winter survey June 2009) Monogenean species Paradip. sp Dact. G Dact. spp. ? * CC Distribution Total 35 3 9 3 50 1 8 1 6 3 18 2 4 2 3 0 9 Gill arch number 3 6 0 0 0 6 4 5 0 0 0 5 Unknown 12 0 0 0 12 Left 12 1 9 2 25 Gill set (side of head) Right 14 2 0 1 18 Unknown 9 0 0 0 9 Dorsal (D) 2 1 4 0 9 Medial (M) 2 1 2 2 7 Gill arch region Ventral (V) 2 1 3 1 7 Unknown 29 0 0 0 29 Anterior 0 2 5 2 9 Gill orientation Posterior 0 1 3 1 5 Unknown 35 0 1 0 3 Infection levels Prevalence (P) (%) 68.8 12.5 12.5 6.3 75.0 Mean intensity (MI) 3.2 1.5 4.5 3.0 4.2 Intensity range: Minimum 1 1 1 3 1 Intensity range: Maximum 5 2 8 3 14 Mean abundance (MA) 2.2 0.2 0.6 0.2 3.1 P (%) 64.3 7.1 7.1 0 85.7 Host gender: Male MI 3.2 1.0 1.0 0 2.6 MA 2.1 0.1 0.1 0 2.2 P (%) 100.0 50.0 50.0 50.0 100.0 Host gender: Female MI 3.0 2.0 8.0 3.0 9.5 MA 3.0 1.0 4.0 1.5 9.5 P (%) 100.0 0 0 0 100.0 > 20 to < 30 cm (n = 1 fish) MI 3.0 0 0 0 3.0 MA 3.0 0 0 0 3.0 P (%) 61.5 7.7 0 0 69.2 > 30 to < 40 cm (n = 13 fish) MI 3.4 1.0 0 0 3.1 MA 2.1 0.1 0 0 2.2 P (%) 100.0 50.0 100.0 50.0 100.0 > 40 to < 50 cm (n = 2 fish) MI 2.5 2.0 4.5 3.0 9.5 MA 2.5 1.0 4.5 1.5 9.5 Key (species codes used) Paradip. – Paradiplozoon Akhmerov, 1974 Dact. G – Dactylogyrus sp. G Dact spp. = Dactylogyrus Diesing, *? – Specimens that could not be 1850 not identified to species level identified to genus level (damaged / CC = Component community (damaged / lost during mounting) lost during mounting)

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Table 12-7: Monogenean parasite infection statistics for Labeobarbus Rüppell, 1836 from the Vaal Dam: Labeobarbus aeneus (Burchell, 1822) – Summer (January 2010) survey.

Host species Labeobarbus aeneus (Burchell, 1822) (Summer survey January 2010) Monogenean species Paradip. sp Dact G Dact. spp. ? * CC Distribution Total 60 0 0 2 62 1 22 0 0 2 24 2 12 0 0 0 12 Gill arch number 3 9 0 0 0 9 4 17 0 0 0 17 Unknown 0 0 0 0 0 Left 34 0 0 2 36 Gill set Right 26 0 0 0 26 (side of head) Unknown 0 0 0 0 0 Dorsal (D) 21 0 0 0 21 Medial (M) 33 0 0 2 35 Gill arch region Ventral (V) 6 0 0 0 6 Unknown 0 0 0 0 0 Anterior 0 0 0 2 2 Gill orientation Posterior 0 0 0 0 0 Unknown 60 0 0 0 60 Infection levels Prevalence (P) (%) 73.3 0 0 13.3 80.0 Mean intensity (MI) 5.5 0 0 1.0 5.2 Intensity range: Minimum 1 0 0 1 1 Intensity range: Maximum 14 0 0 1 14 Mean abundance (MA) 4.0 0 0 0.1 4.1 P (%) 80.0 0 20.0 20.0 100.0 Host gender: MI 8.8 0 1.0 1.0 7.4 Male MA 7.0 0 0.2 0.2 7.4 P (%) 70.0 0 0 0 70.0 Host gender: MI 3.6 0 0 0 3.6 Female MA 2.5 0 0 0 2.5 P (%) 40.0 0 0 20.0 60.0 > 29 to < 35 cm MI 4.5 0 0 1.0 3.3 (n = 5 fish) MA 1.8 0 0 0.2 2.0 P (%) 83.3 0 16.7 0 83.3 > 35 to < 40 cm MI 5.8 0 1.0 0 6.0 (n = 6 fish) MA 4.8 0 0.2 0 5.0 P (%) 100.0 0 0 0 100.0 > 40 to < 45 cm MI 5.5 0 0 0 5.5 (n = 4 fish) MA 5.5 0 0 0 5.5 Key (species codes used) Paradip. – Paradiplozoon Akhmerov, 1974 Dact. G – Dactylogyrus sp. G Dact. spp. = Dactylogyrus Diesing, *? – Specimens that could not be 1850 not identified to species level identified to genus level (damaged / CC = Component community (damaged / lost during mounting) lost during mounting)

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Table 12-8: Monogenean parasite infection statistics for Labeobarbus Rüppell, 1836 from the Vaal Dam: Labeobarbus suspected hybrid – Summer (January 2010) survey #.

Host species Labeobarbus aeneus (Burchell, 1822) (Summer survey January 2010) Monogenean species Paradip. sp Dact. G Dact. spp. ? * CC Distribution Total 2 0 0 0 2 1 0 0 0 0 0 2 1 0 0 0 1 Gill arch number 3 0 0 0 0 0 4 1 0 0 0 1 Unknown 0 0 0 0 0 Left 1 0 0 0 1 Gill set Right 1 0 0 0 1 (side of head) Unknown 0 0 0 0 0 Dorsal (D) 0 0 0 0 0 Medial (M) 2 0 0 0 2 Gill arch region Ventral (V) 0 0 0 0 0 Unknown 0 0 0 0 0 Anterior 0 0 0 0 0 Gill orientation Posterior 0 0 0 0 0 Unknown 2 0 0 0 2 Infection levels Prevalence (P) (%) 100.0 0 0 0 100.0 Mean intensity (MI) 1.0 0 0 0 1.0 Intensity range: Minimum 1 0 0 0 1 Intensity range: Maximum 1 0 0 0 1 Mean abundance (MA) 1.0 0 0 0 1.0 P (%) 100.0 0 0 0 100.0 Host gender: MI 1.0 0 0 0 1.0 Male MA 1.0 0 0 0 1.0 P (%) 100.0 0 0 0 100.0 Host gender: MI 1.0 0 0 0 1.0 Female MA 1.0 0 0 0 1.0 P (%) 100.0 0 0 0 100.0 > 39 to < 45 cm MI 1.0 0 0 0 1.0 (n = 2 fish) MA 1.0 0 0 0 1.0 Key (species codes used) Paradip. – Paradiplozoon Akhmerov, 1974 Dact. G – Dactylogyrus sp. G Dact. spp. = Dactylogyrus Diesing, *? – Specimens that could not be 1850 not identified to species level identified to genus level (damaged / CC = Component community (damaged / lost during mounting) lost during mounting) # Note: No parasites were collected from the single suspected hybrid specimen examined during the winter (June 2009) survey and as such only a table for the summer survey is included.

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For a brief discussion of site specificity in Dactylogyrus spp. parasites as recorded in previous studies refer to section 6.3.4.5. Generally more parasites were found on the first gill arch followed by the second gill arch. This trend (i.e. unequal proportion of parasites on different gill arches) was not statistically significant (Pearson Chi Square, p=0.630, pooled for all Labeobarbus spp. and both seasons) and is thought to be at least partially related to water flow over the gills (see section 6.3.4.5 for a detailed discussion). With the latter in mind one would expect more parasites on the second and third gill arches. Lower prevalence of parasites (compared to that recorded from Labeo spp.) combined with a large number of unknown positions (e.g. Table 12-6) possibly mask general trends.

In a few instances slightly more parasites were found on the left set of gill arches (Tables 12-6 and 12-7) while there was no difference in distribution in others (Tables 12-4 and 12-8). In one instance slightly more parasites were found on the right side of the head (Table 12-5). This trend (unequal distribution of parasites between left and right gill sets) was not statistically significant (Pearson Chi Square, p=0.176, pooled for all Labeobarbus spp. and both seasons).

Most parasites were found on the dorsal (Tables 12-4, 12-5 and 12-6) or medial (Tables 12-7 and 12-8) positions of the gill arch. The unequal distribution observed was statistically significant (Pearson Chi Square, p = 0.032, pooled for all Labeobarbus spp. and for both surveys). Once again the large proportion of unknown positions (Table 12-6) confounds any generalized conclusions.

More parasites (yet not statistically significantly, Pearson Chi Square, p = 0.083, pooled for all Labeobarbus spp. and for both surveys) were recovered from the anterior hemibranch.

12.3.6. Environmental variables and seasonal comparison

For a brief discussion on the potential effects of environmental variables as recorded in literature refer to section 6.3.4.6. Selected physical and other water quality parameter values are summarized in Table 12-9. Water analysis data was obtained from Rand Water (sampling point reference C-VD1l, monthly water sampling). As samples were not taken on the precise dates of fish host sampling, results are provided for analyses conducted within approximately one month prior to the sampling effort as well as one month thereafter.

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Table 12-9: Environmental (physical) and water quality variables from the Vaal Dam (source: Rand Water, sampling point identification number C-VD1l).

DO Temp Cond TDS CaCO3 Cr Cu Pb Date pH (%) (°C) (mS/m) (mg/L) (mg/L) (µg/L) (µg/L) (µg/L) Winter survey (June/July 2009) 26 May 09 57.2 15.8 20.0 140.0 8.2 59.0 < 10.0 < 10.0 13.0 23 June 09 76.6 17.3 17.0 150.0 7.0 65.0 < 10.0 10.0 < 8.0 28 July 09 60.2 16.5 <1.0 ** 145.0 8.1 68.0 < 10.0 10.0 < 8.0 25 Aug 09 63.8 19.6 21.0 145.0 7.7 66.0 < 10.0 10.0 < 8.0 Summer survey (January 2010) 22 Dec 2009 80.4 20.3 18 145 8.23 57 <0.010 0.01 <8.00 26 Jan 2010 71.9 23.4 18 170 8.22 56 <0.010 <0.010 <8.00 23 Feb 2010 77.3 19.5 17 175 7.35 55 <0.010 0.02 <8.00

For discussions on how environmental variables (especially temperature) can affect Dactylogyrus spp. infection statistics, refer to sections 10.3.6.3 and 11.3.6.3.

Based on these discussions (sections mentioned above) one would expect higher parasite numbers during the summer survey. This was indeed the case, with 52 parasites being collected during winter (June/July 2009) from 20 fish compared to 73 parasites collected during summer (January 2010) from 29 fish (Pearson Chi Square, p=0.001, pooled for all Labeobarbus spp.).

12.3.7. Host species: condition factor values and macroscopic pathology

For comprehensive reviews on Fulton’s condition factor used in this study, refer to Froese (2006) and Nash, Valencia and Geffen (2006) (also see section 3.3.5).

While more male fish (25) were collected compared to females (16), there were no significant difference (p=0.072 Mann Whitney U test, pooled for all Labeobarbus spp. and both surveys) in mean condition factor values between genders (Table 12-1). As there was also no statistically significant difference (Mann-Whitney U test, p = 0.251, pooled for all Labeobarbus spp. and both surveys) between male and female host with regard to the number of parasites collected, it is thus unlikely that parasite infections observed influenced condition factor values.

For a brief overview of how condition factors relate to monogenean infections from previous studies, refer to section 6.3.4.1.

For a discussion on the possible effect reproductive cycles may have on condition factor values, refer to sections 10.3.6.4 and 11.3.6.4.

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As also suggested for Cyprinus carpio carpio Linnaeus, 1758 and Ctenopharyngodon idella (Valenciennes, 1844) additional seasonal sampling (e.g. during the spring period) may help elucidate the role played by reproductive status in affecting condition factor when also compared with parasite burdens.

12.4. SUMMARY AND CONCLUSION

The taxonomy (species status) of the “D. varicorhini species group / type” specimens collected from both L. kimberleyensis and L. aeneus needs to be confirmed. Of particular interest is the observation of a “y” shaped bar in one specimen from L. kimberleyensis. As sclerites (anchors and bars and particularly the MCO) of some specimens from especially L. aeneus were not clearly visible, further study is needed to clarify the structure of especially bars and the MCO. As was the case with two “varieties” of D. varicorhini being described by Paperna (1961) (Table 12-3), it would appear that two varieties (if subsequent studies confirm that these are indeed the same species) may exist on the two Labeobarbus spp. (with slightly larger sclerite dimensions recorded for monogeneans from L. kimberleyensis). This may prove to be an interesting tool in future studies examining possible hybridization between these two species, as monogeneans have been used to clarify taxonomy of large Barbus spp. from West Africa (Lévèque and Guégan 1990).

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CHAPTER 13

13 - GENERAL DISCUSSION

In the general introduction chapter (Chapter 2) it was ascertained that an apparent lack of published baseline data (i.e. on host species parasite fauna and locality records) exists in South Africa with regard to monogenean research.

In the context of this thesis, “baseline data” could also be translated into a simple question: “Which monogenean parasite taxa occur in the Vaal Dam in South Africa and at which infection levels?” This implies parasite identification (and description where necessary) with consideration of parasite and host biology (i.e. aspects of ecology) and calculation of infection statistics.

This has been done as reported on in preceding chapters. In the pages that follow the various aspects shall be consolidated and influencing variables identified, as guided by the objectives previously defined. The chapter is concluded with recommendations for future studies.

The monogenean parasite species collected from the Vaal Dam is tabulated in Table 13-1.

A visual summary of these monogenean species encountered (together with information on taxonomic status and host species) is provided in Table 13-2. It is intended as a simple “quick reference guide” to aid in discussions that follow.

As was expected, examination of endemic fish species previously not intensively examined for monogenean parasites (e.g. Labeo capensis (Smith, 1841) and Labeo umbratus (Smith, 1841)), resulted in the description of new parasite species (as was also predicted by Gussev, Jalali and Molnár 1993). The lists in Tables 13-1 and 13-2 demonstrate the high degree of host specificity / host preference monogeneans are renowned for. Closely related hosts (e.g. species of Labeo Cuvier, 1817) harbour apparently closely related parasites, a phenomenon emphasising the potential use of such parasites in evolutionary phylogenetic studies.

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Table 13-1: List of all host and parasite species collected during the current study in the Vaal Dam.

Survey 1: Winter (June / July 2009) Survey 2: Summer (January 2010) List of all host List of all host List of all parasite List of all parasite species species species species (n = 6 in total) (n = 4 + one hybrid) (n = 8 + one hybrid) (n = 14 in total)

Dactylogyrus iwani D. iwani Crafford, Luus-Powell and D. larindae Avenant-Oldewage, 2012 L. capensis D. nicolettae Dactylogyrus larindae Crafford, Luus-Powell and Avenant-Oldewage, 2012 Labeo capensis Dactylogyrus nicolettae (Smith, 1841) Crafford, Luus-Powell and Dactylogyrus sp. D Avenant-Oldewage, 2012 (Undescribed) L. umbratus Dogielius intorquens D. intorquens Crafford, Luus-Powell and Diplozoon sp. Avenant-Oldewage, 2012 Diplozoon Quadriacanthus aegypticus von Nordmann, 1832 Clarias gariepinus El-Naggar and Serag, 1986 (Burchell, 1822) Gyrodactylus sp. G

(Undescribed) Dactylogyrus iwani Dactylogyrus extensus Crafford, Luus-Powell and Mueller and Van Cleave, 1932 Avenant-Oldewage, 2012 Cyprinus carpio Dactylogyrus minutus Dactylogyrus larindae Linnaeus, 1758 Kulwiec, 1927 Crafford, Luus-Powell and Gyrodactylus kherulensis Labeo umbratus Avenant-Oldewage, 2012 (Smith, 1841) Ergens, 1974 Dactylogyrus sp. D (Undescribed)

Dogielius intorquens Crafford, Luus-Powell and Ctenopharyngodon Avenant-Oldewage, 2012 Dactylogyrus lamellatus idella Diplozoon Achmerow, 1952 (Valenciennes, 1844) von Nordmann, 1832

Dactylogyrus sp. L

Labeobarbus aeneus L. aeneus (belonging to the Dactylogyrus (Burchell, 1822) L. kimberleyensis varicorhini Bychowsky, 1958 L. aeneus x L species group / type Labeobarbus kimberleyensis kimberleyensis Paradiplozoon (Gilchrist and Thompson, suspected 1913) No parasites collected Akhmerov, 1974

L. aeneus x L. Micropterus No parasites collected kimberleyensis salmoides

suspected hybrid (Lacépède, 1802)

With only two surveys performed in one locality within a single river system, this list (Table 13-1) is by no means considered to be exhaustive with regard to the monogenean fauna of the South African fish species examined.

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Table 13-2: Summary of illustrations for collected parasite species and new parasite species described for each of the host fish species examined in the Vaal Dam.

Species code * / Taxonomic status # / Illustration Scientific name ** Host ##

# New species: Crafford, Luus-Powell * Species A and Avenant-Oldewage, in-press ** Dactylogyrus iwani n. sp. ## Labeo capensis (Smith, 1841) and Labeo umbratus (Smith, 1841)

# New species: Crafford, Luus-Powell * Species B and Avenant-Oldewage, in-press ** Dactylogyrus larindae n. sp. ## L. capensis and L. umbratus

# New species: * Species C Crafford, Luus-Powell and Avenant-Oldewage, ** Dactylogyrus nicolettae in-press n. sp. ## L. capensis

# New species: Description not yet published (manuscript to * Species D be prepared for (Dactylogyrus sp.) publication).

## L. umbratus

* = Species code allocated used in this study (used throughout this manuscript if the parasite has not been described previously, or used where taxonomic status, i.e. species or form of same species, were evaluated, e.g. Chapter 4); ** = Species name used in this manuscript if the species has been described previously / description proposed in this manuscript has been accepted and published in a peer-reviewed journal; # = Description of taxonomic status (e.g. new species / existing species / uncertain); ## = Scientific name of host(s) from which parasite was collected.

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Table 13-2 (continued): Summary of illustrations for collected parasite species and new parasite species described for each of the host fish species examined in the Vaal Dam.

Species code * / Taxonomic status # / Illustration Scientific name ** Host ##

# New species: Crafford, Luus-Powell * Species E and Avenant-Oldewage, in-press ** Dogielius intorquens n. sp.) ## L. capensis and L. umbratus

# * Species F Known species (i.e. species already ** Quadriacanthus described) aegypticus ## El-Naggar and Serag, Clarias gariepinus 1986 (Burchell, 1822)

# Taxonomic status uncertain: To be further investigated by * Species G molecular and more (Gyrodactylus sp.) detailed morphometric means.

## C. gariepinus

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Table 13-2 (continued): Summary of illustrations for collected parasite species and new parasite species described for each of the host fish species examined in the Vaal Dam.

Species code * / Scientific Taxonomic status # / Illustration ## name ** Host

# Known species * Species H (i.e. species already described) ** Dactylogyrus extensus Mueller and Van Cleave, ## Cyprinus carpio 1932 Linnaeus, 1758

# Known species * Species I (i.e. species already

described) ** Dactylogyrus minutus

Kulwiec, 1927 ## C. carpio

* Species J # Known species (i.e. species already ** Gyrodactylus described) kherulensis Ergens, 1974 ## C. carpio

# Known species (i.e. species already * Species K described)

** Dactylogyrus ## Ctenopharyngodon lamellatus idella Achmerow, 1952 (Valenciennes, 1844)

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Table 13-2 (continued): Summary of illustrations for collected parasite species and new parasite species described for each of the host fish species examined in the Vaal Dam.

Species code * / Scientific Taxonomic status # / Illustration name ** Host ##

# Taxonomic status uncertain: To be further * Species L investigated by more

detailed morphometric Dactylogyrus sp. L means (especially of male copulatory organ) in (belonging to the future studies. Dactylogyrus varicorhini Bychowsky, 1958 species ## Labeobarbus aeneus group / type) (Burchell, 1822)

With further sampling many additional parasite species may be uncovered, similar to that suggested biodiversity estimates calculated for both Australia (Fletcher and Whittington 1998) and China (Jianying, Tingbao, Lin and Xuejuan 2003). Based on the large number of monogenean parasites already identified from these two surveys, it is hypothesized that there must still be a large number of undescribed monogenean species on South African fishes. Based on the results in Table 13-1, an estimate of 2 to 6 monogenean parasite species per unexamined (for monogenean parasites) fish species remaining in South Africa is proposed.

Al-Samman, Molnár and Székely (2006) found that only eight out of 15 fish species were infected with dactylogrid monogeneans. No monogeneans were recovered from largemouth bass, Micropterus salmoides (Lacépède, 1802) in the current study. Factors that may result in false negative cases when searching for monogenean parasites include the small size of parasites, combined with low prevalence and intensity and inappropriate preservation techniques (Ernst, Fletcher and Hayward 2000).

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With only two host specimens examined during the current study, this (i.e. absence of monogenean parasites) may come as no surprise, especially if parasite prevalence was low. Furthermore recovery, fixation and mounting of monogeneans are labour intensive and time consuming. As a result routine sampling effort often does not accommodate their recovery (i.e. the parasites may be inadvertently overlooked) (Pérez-Ponce de León, Rosas-Valdez, Aguilar-Aguilar, Mendoza- Garfias, Mendoza-Palmero, García-Prieto, Rojas-Sánchez, Briosio-Aguilar, Pérez- Rodríguez and Domínguez-Domínguez 2010), especially if collection takes place under challenging environmental conditions (e.g. Rokicka 2009). Representatives of the monogenean genus Urocleidus Mueller, 1934 are known to occur in the urinary bladder of M. salmoides. In both cases the urinary bladder was not examined. It may thus be that this parasite was in fact overlooked. Other monogenean species that are known to occur on largemouth bass are Actinocleidus Mueller, 1937; Acolpenteron Fischthal and Allison, 1940; Haplocleidus Mueller, 1937 and Onchocleidus Mueller, 1936 (Kohn, Cohen and Salgado-Maldonado 2006). Future studies should confirm the monogenean parasite status of this fish species in the Vaal Dam and / or Vaal River system.

Hypothesis 1-1: Each fish species examined shall harbour at least one monogenean parasite species.

Evaluation outcome: Inconclusive. No parasites were collected from the gills or skin of M. salmoides (only two specimens were examined and the urinary bladders were not processed / examined). All of the other fish species harboured at least one monogenean parasite species as predicted.

Identification (and description in some cases) of the parasite species listed in Tables 4-1 and 4-2 involved point to point measurements. Such measurements often reflected a surprising degree of morphological variation / morphometric plasticity within species. For the majority of specimens for which measurements were recorded, there was variation in body length and width measurements. This was expected as many monogeneans have elongated bodies that are capable of great contraction and extension (Khalil and Mashego 1998). Poulin (2005) argued that there is an evolutionary trend of decreasing size in monogeneans, but the limited dataset generated during the current study does not allow any investigation of such a relationship.

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Monogenean parasite species are traditionally distinguished by the shape and size of sclerotized parts of both the opisthaptoral and copulatory organ complexes (e.g. Mizelle 1962; Kritsky and Kulo 1992; Paraguassu, Luque and Alves 2002; Pariselle, Bilong Bilong and Euzet 2003). Yet morphological plasticity appears to be fairly common and thus potentially makes studies on taxonomy challenging (e.g. Prost 1984). Chapter 4, for example, dealt with morphology of closely related Dactylogyrus Diesing, 1850 and Dogielius Bychowsky, 1936 species from Labeo hosts (i.e. examining whether they in fact represent different forms of the same species or different species all together). Quantifying morphometric variability within a described species effectively, yet allowing differentiation between closely related and morphologically very similar species, proved to be challenging. This would best be done combining morphological and molecular approaches (as has become the norm for gyrodactylids, e.g. Cunningham 1997; Cunningham, Aliesky and Collins 2000; Huyse, Audenaert and Volckaert 2003; Huyse, Malmberg and Volckaert 2004; Huyse and Malmberg 2004; Přikrylová, Matějusová, Jarkovský and Gelnar 2008; Přikrylová, Matějusová, Musilová and Gelnar 2009a; Přikrylová, Matějusová, Musilová, Gelnar and Harris 2009b). Employing both allow hypotheses from one type of data to be tested on the other (Blair, Campos, Cummings and Laclette 1996) as exemplified by Přikrylová et al. (2008). However, at present morphological differentiation is still the norm for dactylogyrid taxonomy and molecular techniques fell beyond the scope of the current project. Initially the use of additional morphometric measurements was evaluated to determine if they may help distinguish between specimens (Appendix B). The variables that did show promise was subjected to a principal component analysis approach (e.g. Dmitrieva, Gerasev and Pron’kina 2007) and the most diagnostic criteria were identified (total anchor length and anchor shaft length). Different forms belonging to the same species was initially suspected for Dactylogyrus Diesing, 1850 due to the similarities in male copulatory organ (MCO) morphology, as Pouyaud, Desmarais, Deveney and Pariselle (2006) state that morphology of the genitalia seems to be more useful to resolve species-level identifications. This is in agreement with Jarkovský, Morand, Šimková and Gelnar (2004) who concluded that specialist parasites possess more similarity in attachment apparatus, with the MCO being more variable (which may be due to reproductive isolation).

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The other sclerites (especially anchors) were, however, found to differ in both morphology (i.e. shape) and size and as a result two species were described (Dactylogyrus iwani Crafford, Luus-Powell and Avenant-Oldewage, 2012 and Dactylogyrus larindae Crafford, Luus-Powell and Avenant-Oldewage, 2012). This criteria (similar male copulatory organ but differences in attachment sclerites) was also employed by Price and Mizelle (1964) with regard to Dactylogyrus macrolepidotus Price and Mizelle, 1964 and Dactylogyrus banghami Mizelle and Donahue, 1944. The sclerites for the Dogielius Bychowsky, 1936 specimens, on the other hand, were similar in morphology but only differed in size. As a result a single species (Dogielius intorquens Crafford, Luus-Powell and Avenant-Oldewage, 2012), exhibiting two forms, was described. Chapter 8 described wide variation in size of the MCO as well as apparent variation in both size and shape of other sclerites (especially anchors) of Quadriacanthus aegypticus El-Naggar and Serag, 1986. Based on previous work performed within this group, MCO structure (which is unique to Q. aegypticus) was given priority and it was decided that all examined specimens belong to the same species. In future studies it could be interesting to compare the degree of variation observed in the current study, with possible variation from another locality such as Lake Kariba (e.g. Douёllou and Chishawa 1995).

But what variables could possibly be responsible for such variation observed in the current study?

Temperature is known to affect gyrodactylid anchor size which (e.g. Mo 1991a, 1991b, 1993; Hodneland and Nilsen 1994; Appleby 1996), has been shown to surpass heritability of sclerite dimensions (Harris 1998b) and must thus be considered in investigations of apparent morphological plasticity (Huyse and Volckaert 2002). However, given that such variation was observed in specimens collected during the same season, this is unlikely to be the case for both D. intorquens and Q. aegypticus. All fish were collected from the same locality, so phylogenetic geographical variation (e.g. Huyse et al. 2004) is also not applicable. Shorter term abiotic (e.g. environmental apart from temperature) or host effects may play a roll, but no evidence indicating such influences was uncovered in the present study.

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Barson, Bray, Ollevier and Huyse (2008), studying species of Macrogyrodactylus Malmberg, 1957 on Clarias gariepinus (Burchell, 1822), reported broad measurement ranges (e.g. marginal hook length ranges were in some cases completely different from the original description). They postulated that such ranges may be temperature dependent, yet they do not rule out that variations may be indicative of new species that would require further morphometric and molecular analysis. The same reasoning applies to Q. aegypticus in the current study, even more so given the fact that temperature appears not to be a causative (of observed sclerite morphology variation) variable.

Host (Sebastes melanops Girard, 1856) body size differences could be correlated to body size differences (total length) of Microcotyle sebastis Goto, 1894 by Thoney (1986a). Total reproductive capacity of the worm is in turn determined by the size of the worm (Trouvé, Sasal, Jourdane, Renaud and Morand 1998), the latter also an important determinant of abundance (Poulin and Justine 2008). The size of haptoral sclerites may thus also be related to host size. Huyse et al. (2004) reported that marginal hook, body, anchor and ventral bar measurements were smaller in Gyrodactylus arcuatus Bychowsky, 1933 from small fish hosts when compared to specimens from larger fish hosts. The presence of small haptoral hard parts may in fact favour the secondary adaptation of Gyrodactylus von Nordmann, 1832 to small fish hosts (Huyse and Malmberg 2004). Huyse and Volckaert (2002) also reported apparent host-specific variation (i.e. populations were morphologically adapted to their respective hosts) in Gyrodactylus rugiensoides Huyse and Volckaert, 2002. Thus, apart from host body size per se, other host species-specific attributes (e.g. gill morphology or site on host as discussed in subsequent paragraphs) also appear to play a role. In the current study little variation in terms of host size classes were observed, confounding any attempt that correlating host size with parasite morphological variation. This is mainly due to the sampling method employed (i.e. large gill mesh sizes).

Apart from host size, host status from the parasite’s perspective (i.e. primary versus secondary host) may also play a role.

Dmitrieva and Dimitrov (2002) stated that gyrodactylid sclerites were generally larger in parasites infecting primary hosts compared to specimens of the same species infecting secondary (i.e. “non-preferred”) hosts.

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Šimková, Pečinkova, Řehulková, Vyskočilová and Ondračková (2007) found significant differences in the attachment and copulatory sclerite morphology of Dactylogyrus dyki Ergens and Lucky, 1959 collected from different Barbus Cuvier and Cloquet, 1816 host species. Furthermore Šimkova and Morand (2008) stated that specialists generally have larger anchors and live on larger or longer-lived hosts (this may also relate to physical dimensions and morphology of attachment sites such as gills, as will be discussed later).

Huyse and Malmberg (2004) identified gill and fin genotypes for the parasite Gyrodactylus harengi Malmberg, 1957. They stated that further investigations are needed to assess whether or not morphological differences between these genotypes can be detected. This implies that intra-specific morphological variation may be encountered on the same host depending on parasite attachment site on the host. Apart from some Gyrodactylus sp. from C. gariepinus all parasites were found on the gills. Preliminary examination suggests that the Gyrodactylus sp. specimens found on both the skin and gills of C. gariepinus belong to the same species. In future studies this needs to be verified by both molecular analysis (i.e. to evaluate genotype) as well as more detailed morphometric analysis (i.e. to compare morphotype / phenotype results with genotype results).

Wong, Brennan, Halton, Maule and Lim (2008) found that the anchors and associated grooves of two species of Bravohollisia Bychowsky and Nagibina, 1970, co-existing on the same fish species, were morphologically different in size and shape. This is reminiscent of differences in the anchors of D. iwani and D. larindae reported on in this thesis. While these parasites do occur on both Labeo sp. hosts examined, there was a clear host preference in terms of especially mean intensity of infection. Differences in fine host gill structure are thought to play a role in the apparent host preference (and hence also morphological variation) observed in the current study. Future studies should also combine parasite sclerite morphometric studies (in particular that of L. capensis and L. umbratus) with host gill structure morphometric studies.

An experimental approach would be most suitable for such an investigation, as one would be able to accurately evaluate different host size classes with known infection periods.

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Such an approach (i.e. experimental approach in which subsamples of the same parasite population can be sampled at various time points) would also allow evaluation of sclerite morphology in parasites of different ages, as seasonal growth of attachment clamps have been demonstrated for Paradiplozoon Akhmerov, 1974 (Milne and Avenant-Oldewage 2012). Hayward and Rohde (1999), with reference to the wide range of clamp measurements they recorded for Thoracotyle crocea MacCallum, 1913 and Pseudothoracocotyla ovalis (Tripathi, 1956), conclude that these measurements appear to vary considerably depending on worm size (i.e. variation in characters and measurements can be expected when examining parasite specimens of different sizes). Such a size related effect was, however, not evident during the current study. Appleby (1996), with reference to Gyrodactylus callariatis Malmberg, 1957, also stated that there was no statistically significant difference when comparing the length of the hard parts between young and older worms.

Fixation methods (e.g. the use of ambient versus hot formalin, Viozzi and Brugni 2004) may thus also affect the morphology of soft tissues. The same methods (i.e. GAP or glycerine jelly) used for all parasite species fixing / mounting procedure in the current study excluded a process to “relax” the worms (due to their small size and fragility). Worms were thus mounted in whatever position they were fixed in, with the mounting process often distorting soft tissues (i.e. due to cover slip pressure and addition of heat where glycerine jelly was used). For this reason total body length and width measurements are considered to be of very little taxonomic importance. As a result it is also difficult to accurately assess the true size of the worm in relation to sclerite measurements. The variation in sclerite (i.e. attachment sclerites on the haptor as well as the male copulatory organ sclerites) measurements occurred in both specimens fixed in both GAP and glycerine jelly. It is thus concluded that the use of two different fixing agents could not affect sclerotized structures and hence could not be responsible for the variation observed.

Tripathi, Agrawal and Pandey (2007) postulated that premature fixation (i.e. fixation while worms were alive compared to worms that were first killed in 4% formalin) may also result in measurement differences. In the current the study the majority of worms were killed in 70% ethanol and only recovered from the gills and mounted later.

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Future studies could statistically compare the amounts of variation observed between worms fixed and mounted (i.e. in GAP or glycerol-jelly) while still alive and those killed in 70% ethanol and later fixed in GAP or glycerol-jelly.

Another variable that may result in morphological variation is water pollution. Gheorghiu, Cable, Marcogliese and Scott (2007) for example demonstrated that the longer the exposure of Gyrodactylus turnbulli Harris, 1986 to zinc, the smaller the parasite. The concentrations of zinc used during the experiment also affected size (i.e. higher concentrations resulted in even smaller parasites). However, as mentioned in section 3.1 the Vaal Dam is situated in the upper catchment of the Vaal River system and is characterized by good water quality. This is also reflected by the water analysis data (Rand Water) presented in this thesis. It is thus unlikely that pollution levels are responsible for any morphological variation observed.

Yet another possible reason for morphological variation relate to parasite phylogeny (i.e. genotype). Morphological plasticity may be a reflection of the initial steps of speciation (often associated with geographical isolation that shall be discussed shortly) (Meinilä, Kuusela, Ziętara and Lumme 2004). Huyse and Volckaert (2002), with reference to classical morphological analysis, state that the occurrence of cryptic speciation may lead to underestimation of the number of species. An example is provided by Freeman and Ogawa (2010) with reference to Udonella caligorum Johnston, 1835. Mendoza-Franco, Vidal-Martínez, Cruz- Quintana and León (2006) also found the species of Salsuginus Beverley-Burton, 1984 to be morphologically and metrically extremely similar.

They concluded that experimental infections and/or molecular analyses were necessary for definitive species determination. The opposite may obviously also be true (i.e. species “overestimation” compared to the “underestimation” scenario described above). Large numbers of species belonging to the same “species group” may be described (e.g. Chien 1971, 1974a, 1974b), with differences possibly resulting from morphological plasticity and not necessarily reflecting true species differences.

With regard to the current study it may be that D. iwani and D. larindae, D. intorquens and Q. aegypticus respectively may in fact constitute cryptic species complexes.

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Differentiation between closely related parasite species in field studies where mixed infections are encountered remains problematic (e.g. Hayward and Rohde 1999; Matejusová, Koubková, Gelnar and Cunningham 2002; Ziętara and Lumme 2003; Tingbao, Gibson and Bijian 2005; Dmitrieva et al. 2007; Freeman and Ogawa 2010). This could at least partly be attributed to the fact that selection pressures may result in very similar phenotypes (Kunz 2002). In the preceding chapters additional morphometric analyses (e.g. McHugh, Shinn and Kay 2000; Rubtsova, Balbuena, Sarabeev, Blasco-Costa and Euzet 2006), combined with a molecular approach (e.g. Ziętara, Arndt, Geets, Hellemans and Volckaert 2000; Matejusová, Gelnar, McBeath, Collins and Cunningham 2001; Šimková, Plaisance, Matějusová, Morand and Verneau 2003; Huyse and Volckaert 2002; Baguñá and Riutort 2004; Huyse and Malmberg 2004; Huyse et al. 2004; Šimková, Morand, Jobet, Gelnar and Verneau 2004; Šimkova, Matějusová and Cunningham 2006; Malmberg, Collins, Cunningham and Jalali 2007; Wu, Zhu, Xie, Wang and Li 2008) have also been suggested previously in this thesis to investigate this possibility (i.e. existence of species complexes).

Another confounding factor may be mode of reproduction. Harris (1993), with reference to Gyrodactylus spp., stated that sexually reproducing species exhibit less heterogeneity between populations and may be more variable compared to possible asexually reproducing species. With reference to Gyrodactylus gasterostei Gläser, 1974, Harris (1998a) states that clonal reproduction is suggested. He continues by saying that such asexual reproduction makes normal criteria for the definition of species difficult to apply, as numerous morphological variants of gyrodactylid species actually represent asexually reproducing morphotypes. Though nothing was known about the possible offspring of such pairings at the time of publication, Huyse et al. (2003) stated that hybridization between Gyrodactylus spp. (e.g. G. arcuatus and G. gasterostei) is possible. This could potentially lead to morphometric variability. None of the species in the current project for which morphological variation have been observed (i.e. D. iwani; D. larindae; D. intorquens and Q. aegypticus) belong to the genus Gyrodactylus and hence do not reproduce asexually.

Such a possible effect should, however, be investigated with regard to the Gyrodactylus spp. collected from Clarias gariepinus (Burchell, 1822) and Cyprinus carpio Linnaeus, 1758 in future studies with greater parasite sample sizes.

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Geographic variation (e.g. Huyse et al. 2004) in sclerite morphology has also been reported. Williams, MacKenzie and McCarthy (1992), in a review of studies using parasites as indicators in marine fish populations, state that anchor length of the marine monogenean Kuhnia scombri (Kuhn, 1829) differed significantly on host fish (Scomber australasicus Cuvier, 1832) collected from two different localities (New Zealand and New South Wales). The authors concluded that different populations of S. australasicus may exist at the two localities. Pariselle, Lim and Lambert (2002) link such effects to co-evolutionary relationships between parasites and hosts (i.e. geographic isolation of a host would automatically isolate the parasite), thus leading to the development of sub-species. Nonetheless Williams et al. (1992) also caution that any deductions regarding morphometric differences between populations should be viewed with caution, as intraspecific variation with respect to season and temperature also need to be considered. In similar fashion Chibwana and Nkwengulila (2010) found clearly distinguished “between-population” morphometric differences in freshwater diplostomid metacercariae. It is also possible that geographical differences in parasite fauna may reflect genetic differences within the host, as Khalali and Amirkolaie (2010) demonstrated that the genetic make-up of C. carpio sampled from three localities differed. In the current study all host fish were recorded from the same locality (i.e. same component community population) so variation cannot be attributed to population differences. Future studies could sample host populations from different localties / geographical regions within the same larger river system (i.e. Vaal River or even Vaal-Orange Rivers system), but also from other systems (e.g. Olifant River system) to determine if differences in morphological plasticity differ between the parasite populations. Such monogenean parasite data has also been employed to examine host systematic and evolutionary trends (e.g. Pouyaud et al. 2006). Experimental studies employing dactylogyrid infections, could possibly also examine if egg morphology (e.g. Pamplona-Basilio, Kohn and Feitosa 2001) exhibit morphological plasticity, or if this structure may in fact be of added morphological species differentiation value when combined with traditionally employed sclerite measurements.

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Table 13-3: Summary of infection statistics of monogenean parasites collected, as calculated from host species examined from the Vaal Dam, Gauteng Province, South Africa.

Monogenean Host Winter (June / July 2009) Summer (January 2010)

Number Number Species Species P MI MA P MI MA collected * collected *

Dactylogyrus iwani 194+204 76.90 50.00 14.90 85.00 35.00 10.20 Crafford, Luus-Powell and Avenant-Oldewage, 2012 Dactylogyrus larindae 17+18 61.50 5.00 1.30 45.00 4.00 0.90 Crafford, Luus-Powell and Avenant-Oldewage, 2012 Labeo capensis 12 + 20 Dactylogyrus nicolettae 30+53 (Smith, 1841) 38.50 10.00 2.30 70.00 11.00 2.65 Crafford, Luus-Powell and Avenant-Oldewage, 2012 = 32

Dogielius intorquens 36+31 84.60 8.00 2.80 65.00 5.00 1.55 Crafford, Luus-Powell and Avenant-Oldewage, 2012 Diplozoon von Nordmann, 1832 1+11 7.70 1.00 0.10 5.00 11.00 0.55

D. iwani 8+65 19.20 3.00 0.30 95.24 8.00 3.10

D. larindae 332+2380 38.50 110.00 12.80 95.24 335.00 113.33 Labeo umbratus 27 + 21 Dactylogyrus sp. D 0+23 (Smith, 1841) - - - 47.62 8 1.10 = 48

D. intorquens 51+70 38.50 16.00 2.00 80.95 10.00 3.33

Diplozoon sp. 53+15 57.70 7.00 2.00 23.81 6.00 0.71

0+182 - - - 90.91 37.00 16.55 Quadriacanthus aegypticus El-Naggar and Serag, 1986 Clarias gariepinus 0 + 11 (Burchell, 1822) = 11 Gyrodactylus sp. G 0+63 - - - 54.55 40.00 5.73

* = Winter (June/July 2009, survey 1) + Summer (January 2010, survey 2) = Total number of specimens collected P = Prevalence; MI = Mean intensity; MA = Mean abundance.

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Table 13-3 (continued): Summary of infection statistics of monogenean parasites collected, as calculated from host species examined from the Vaal Dam, Gauteng Province, South Africa.

Monogenean Host Winter (June / July 2009) Summer (January 2010)

Number Number Species Species P MI MA P MI MA collected * collected *

Dactylogyrus extensus 0+39 - - - 76.92 10.00 3.00 Mueller and Van Cleave, 1932 0 + 13 Dactylogyrus minutus 0+3 Cyprinus carpio - - - 7.69 3.00 0.23 Kulwiec, 1927 Linnaeus, 1758 = 13

Gyrodactylus kherulensis 0+3 - - - 7.69 3.00 0.23 Ergens, 1974 Ctenopharyngodon 0 + 12 Dactylogyrus lamellatus 0+73 idella - - - 83.33 31.00 6.08 Achmerow, 1952 (Valenciennes, = 12 1844)

3+0 12.50 2.00 0.20 - - - Dactylogyrus sp. L Labeobarbus 16 + 20 aeneus = 36 Paradiplozoon Akhmerov, 1974 35 +60 (Burchell, 1822) 68.80 5.00 2.20 73.30 14.00 4.00

Labeobarbus 3 + 7 kimberleyensis Paradiplozoon sp. 0+6 - - - 50.00 3.00 1.50 (Gilchrist and = 10 Thompson, 1913)

L. aeneus x L. 1 + 2 Paradiplozoon sp. 1+2 0 0 0 100.00 1.00 1.00 kimberleyensis = 3 Micropterus 0 + 2 None Not applicable salmoides = 2 (Lacepède, 1802) * = Winter (June/July 2009, survey 1) + Summer (January 2010, survey 2) = Total number of specimens collected P = Prevalence; MI = Mean intensity; MA = Mean abundance.

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Once parasite identification (or description where applicable) was completed. infection statistics were calculated for all parasites and reported on in preceding chapters. A summary is provided in Table 13-3.

Paperna (1996) stated that, in most fish species associated with monogenean parasites, prevalence is high (approaching 100% as was also recorded by Soylu and Emre 2005 as well as Galli and Kritsky 2008), with intensities of 20 to 100 parasites (or more) per fish being common. This was not always the case in the current study. Prevalence often exceeded 50% (especially in summer), but mean intensity were below 20 in the majority of cases. D. larindae from L. umbratus was the only monogenean species for which truly large numbers of parasites were collected (i.e. mean intensity exceeding 100 during both seasons). Al-Samman et al. (2006) also reported low prevalence and intensity of infection values for the majority of the 15 fish species they examined in Syria. Several other authors also reported monogenean infections ranging between 16 and 50% prevalence (Hossain, Hossain, Rahman, Akter and Khanom 2008; Stojanovski, Hristovsk, Cakic, Nedeva, Karaivanova and Atanasov 2009).

Hassan (2008) also found that parasitic infection was more common in native fish when compared to exotic fish (with regard to mean prevalence of infection with any species of parasite). This general trend was observed to some extend (more specifically with regard to the native Labeo spp.), but was not evident in other native fish species (more specifically Labeobarbus spp.).

Hypothesis 2-1: Both prevalence and average intensity of infection shall be high (>50% and 20-100 parasites respectively) in the majority of cases.

Evaluation outcome: Rejected. While prevalence did exceed 50% in a large number of cases during summer, this was not the case during winter. Furthermore mean intensity of infection rarely exceeded 10 in winter with only a few cases exceeding 20 during summer.

From the evaluation of hypothesis 2-1 presented above, it is clear that seasonal differences (i.e. between the two surveys) were noted. Huyse, Poulin and Théron (2005) stated that many parasite populations may experience strong seasonal fluctuations in size. As a result significant seasonal fluctuations with regard to monogenean infection statistic parameters were indeed expected.

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The only host species for which adequate numbers of both fish and parasites were collected to allow meaningful seasonal comparison were L. capensis and L. umbratus (Chapter 7).

Dzika and Szymanski (1989) found that species richness (i.e. expressed as number of species in “species groups”) generally increase with mean intensity of infection. This was not observed in the current study with regard to Labeo spp., most probably indicating that the populations were adequately sampled during both surveys.

Greater numbers of parasites were collected during the summer survey compared to the winter survey which also generally represented higher prevalence values being recorded during summer.

This is in agreement with results reported by Stojanovski, Kulišic, Baker, Hristovski, Cakič and Hristovski (2004) and Koyun (2011). A number of authors (e.g. Öztürk and Altunel 2006; Bhuiyan, Akther and Musa 2007; Hossain, Hossain, Rahman, Akter and Khanom 2008), however, reported highest prevalence of infection to generally occur in winter. Hossain et al. (2008) stated that this effect (i.e. infection peaking in winter) may have been the result of host immune effects (i.e. reduced host immune response in winter) or poor water quality. However, gonad development during spring and early summer may result in elevated steroid hormone levels that could also suppress the host’s immune function (Lamková, Šimková, Paliková, Jurajda and Lojek 2007). In this study higher infection statistics in summer was expected as dactylogyrids are known to have higher reproduction rates at higher temperatures (e.g. sections 10.3.6.3 and 11.3.6.3). The possible effects of temperature will be discussed in detail at a later stage. The remainder of this section, however, shall focus on other reasons (i.e. apart from temperature) for seasonal variation observed in the current study.

Apparent discrepancies (i.e. not in agreement with the general trend of increased infection statistics during summer as one would expect from temperature effects) were recorded for individual species and were possibly related to host preference. The latter, in turn, is most probably a combination of evolutionary phylogeny (i.e. genotype) as well as shorter term interaction between parasite and host population dynamics. This view is confirmed by the results reported by Kadlec, Šimková, Jarkovský and Gelnar (2003b).

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They examined the parasite fauna of three different host fish species (perch, bitterling and roach). In two of these (perch and bitterling) seasonal and flood conditions had no effect on parasite (both endo- and ecto- combined) species diversity. Flood conditions did, however, result in an increase in parasite species diversity in roach. When analyzing ectoparasites separately, a decrease in monogenean parasite species abundance was recorded in roach and perch. An opposite trend (i.e. increase in ectoparasite abundance following the flood) was recorded in bitterling due to a high abundance of Gyrodactylus spp. From this we can deduce that the same host species and parasite populations (within the same component parasite communities), subjected to the same environmental and seasonal conditions may indeed react differently (as was also found in the current study). Kadlec et al. (2003b) concluded that these differences observed may be the result of changes in host life history strategies or immune response under conditions of stress.

From the above it can be concluded that temperature, intuitively considered a key ecological variable when pondering seasonal differences, is not solely responsible for seasonal patterns in monogenean infection statistics. Madanire-Moyo, Matla, Olivier and Luus-Powell (2011), for example, recorded peaks in parasite infection statistics in both winter and summer.

Gelnar (1991) warned that field studies dealing with seasonal changes in monogenean parasite numbers often fail to provide definite answers as to the effect of this single ecological variable (i.e. temperature). He demonstrated that, even within the same genus (Gyrodactylus), there exist different temperature preferences. He concluded that experimental studies that allow controlled conditions is extremely important in providing information on the effects of temperature on monogenean life cycles. Although his study dealt with gyrodactylids, the same principle applies here. Future studies could include an experimental aspect in an attempt to mitigate some of the confounding effects host population dynamics and behaviour (e.g. spawning habitat and interaction with closely related species etc.) may have on true temperature effects.

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Hypothesis 3-1: There shall be a significant difference in infection statistic values between seasons.

Evaluation outcome: Hypothesis accepted. There was a significant difference in parasite numbers when comparing summer and winter surveys for L. umbratus (i.e. prevalence was higher in summer for Dactylogyrus spp. and Dogielius sp. but higher in winter for Diplozoon von Nordmann, 1832). In terms of mean intensity and mean abundance, significant seasonal differences (much higher in summer) were recorded only for D. larindae.

Seasonal environmental effects (most notably temperature) are intuitively considered to be the main driving force behind seasonal variation in infection statistic parameters. Abiotic and environmental variables that can either restrict or encourage the growth, development and transmission of parasites include temperature, water quality, mechanical barriers, food supply (Hassan 2008), latitude, altitude, salinity, depth, light intensity, frequency and intensity of physical disturbances (Bush, Fernández, Esch and Seed 2001) and currents (Paperna 1996). Selected variables shall subsequently be discussed and their possible involvement (or the lack thereof) in the current project evaluated.

Water temperature is considered a critical environmental parameter as it affects parasite growth / size (e.g. Appleby 1996), reproduction rates (e.g. Blažek, Jarkovský, Koubková and Gelnar 2008b), survival, timing of transmission, distribution, host hormone cycles (Hassan 2008) and longevity (Coutant and Talmage 1976; Lester and Adams 1974).

Seasonal trends in monogenean numbers can most like be ascribed to the fact that both dactylogyrid egg incubation time (increase with increasing temperature, e.g. Paperna 1996) and gyrodactylid survival (decrease with increasing temperature, e.g. Coutant and Talmage 1976; Gelnar 1991; Winger et al. 2008) largely depend on temperature.

Reed, Francis-Floyd and Klinger (2002), for example, stated that time required for maturation of Dactylogyrus spp. eggs may vary from a few days to five or six months depending on temperature.

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Such a drastic temperature effect on reproductive / developmental rate is not applicable to all monogenean genera, as Thoney (1986b) for example found that temperature did not correlate with the development period of Microcotyle sebastis Goto, 1894.

Dávidová et al. (2005) reported a negative relationship between intensity and prevalence of Gyrodactylus rhodei Zitnan, 1964 and temperature. Cone and Cusack (1988) found increased intensity of gyrodactylid infection during winter with a peak in spring. Blažek et al. (2008b) also found that gyrodactylids numbers peaked in spring (water temperature above 6 °C) and Dactylogyrus cryptomeres Bychowsky, 1943 in summer (water temperature above 14 °C). Paperna (1964a) reported that Dactylogyrus extensus Mueller and Van Cleave, 1932 eggs hatch within four to six days at temperatures of 20 to 28 °C (yet failed to hatch at 37 °C). Molnár (1971a) also reported an increased oviposition rate with an increase in temperature for Dactylogyrus lamellatus Achmerow, 1952 and also indicate a temperature range of 17 to 28 °C as optimal for this parasite. Dactylogyrids thus appear to be more thermophilous (as reflected in this study with particular reference to D. larindae), but this may obviously differ between genera and species (as was discussed in section 14.5). Whilst Bhuiyan et al. (2007) (examining all parasites encountered including monogeneans) from Labeo rohita (Hamilton, 1822), found maximum infection to occur pre-winter, Lamková et al. (2007) warned that diverse genera of the monogenean parasite group respond differently to water temperature changes. Possible reasons for this have been discussed in the preceding section.

Temperature may also affect community structure (i.e. species composition) (e.g. Šimková, Sasal, Kadlec and Gelnar 2001). This was not the case in the current study where the same parasite species were recorded from Labeo spp. hosts during both the winter and summer surveys.

Temperature also affects fish physiology and immunology (Lamková et al. 2007). Fishes have been shown to respond to parasite infections by activating both acquired and innate immune mechanisms (Alvarez-Pellitero 2008). Activation of such responses is thought to be compromised during winter (Le Roux, Avenant- Oldewage and van der Walt 2011). This could then theoretically result in higher parasite infection number during this period.

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In the current study this was not the case and the combination of temperature, genotype (evolutionary phylogeny) and interaction between parasite biology / population dynamics and host species biology / population dynamic (as was also recorded for other parasites such as Argulus japonicus Thiele, 1900 by Shafir and Oldewage 1992) appear to have overridden any immunological effects.

Hypothesis 3-2: For dactylogyrids higher infection statistics shall be recorded in summer, while the opposite shall be true for gyrodactylids

Evaluation outcome: Inconclusive. Gyrodactylus spp. were recorded from C. gariepinus (summer survey only) and C. carpio (summer survey only) in low numbers. The small data set confounds any seasonal comparisons and hence no generalizations can be proposed. As discussed in section 14.5, temperature most likely did contribute to higher dactylogyrid numbers / infection statistics recorded during summer (as did other factors as discussed in the same section).

Second to temperature, water quality (i.e. chemical characteristics) is another variable that may affect infection statistic parameters between seasons (or localities). As indicated previously water quality in the Vaal Dam is considered to be fairly good compared to other catchment areas in the same system (e.g. Vaal River Barrage). Water quality in the Vaal Dam remained good during both winter and summer survey and it is unlikely that water quality / chemical characteristics affected infection statistics.

Future studies should compare monogenean fauna of the same fish species, within the same system yet at different localities exhibiting differences in water quality (e.g. Vaal Dam versus Vaal River Barrage, Crafford and Avenant-Oldewage 2011). Water quality variable / chemical characteristics that have been shown to affect the occurrence of monogeneans include salinity (e.g. Bakke, Harris and Cable 2002; Baker, Pante and de Buron 2005; Johnsen 2006; Hassan 2008), eutrophication, dissolved humic substances, hypoxia (oxygen levels), conductivity (Paperna 1996; Madanire-Moyo and Barson 2010), acidification (low pH), aqueous aluminium (Bakke et al. 2002) and waterborne zinc (Gheorghiu et al. 2007). Many of these water quality challenges (most notably salinity, high conductivity and eutrophication) are known to occur in the Vaal River Barrage.

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Labeo umbratus have been shown to grow faster in eutrophic systems (Potts and Khumalo 2005). As host age / length relationships can also affect monogenean infection probabilities, such an effect may contribute to differences in the monogenean parasite communities of the Vaal Dam compared to that of the Barrage.

Bakke et al. (2002) stated that salinity (amongst other factors) could not only potentially negatively affect monogenean parasites directly, but could also influence the spectrum of available hosts. Some monogeneans such as D. extensus have been shown to exhibit a very high tolerance to salinity (Paperna 1964a; Jalali and Barzegar 2005).

Future studies could determine if these variables have a direct negative effect on monogeneans (and to what extend these effects differ amongst species encountered), or possibly a positive effect through reduced host immunity.

Apart from temperature and chemical characteristics (i.e. water quality), a number of other factors can affect infection statistics parameters. The existence of a “geographical gradient” (Bush et al. 2001) in terms of increase in parasite numbers towards the tropics cannot be commented upon due to the small geographical scale of the current project. Barger and Esch (2001), however, found that the relationship that exists between the proximity of sampling sites and parasite community similarity is predictive (i.e. the distance between these sites related to the probability of recruiting parasites from different geographically separated species pools). As this can be observed on a much smaller geographical scale (the study in question was performed in an Appalachian stream), a similar approach (e.g. comparing monogenean parasite fauna at several sites both above and below the Vaal River Barrage) could be applied to the Vaal River system in future studies.

The possible effects of depth can also not be commented upon as the host sampling procedure (floating gill nets) did not allow quantification of this variable (i.e. sampling at different depths). Bush et al. (2001) states that bottom-dwelling fishes at any depth exhibit greater parasite richness than pelagic fishes at similar depths. This effect is expected to be more pronounced in the ocean (i.e. where greater depths and greater variation in depth may be encountered), yet may also be applicable to freshwater systems.

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Oncomiracidia sink and rest on the bottom / sediment (e.g. Molnár 1971a). It follows that fish with sedentary habits should be more exposed to infection. This most definitely appear to be the case for the Labeo spp. examined.

Apart from depth, water levels may also play a role. Kadlec et al. (2003b), for example, demonstrated that flood conditions can affect parasite community species composition on some fish hosts. This is because turbulence and the strong current may prevent large numbers of parasites from attaching to a host (Cloutman 1976; Choudhury, Hoffnagle and Cole 2004) or flush parasite infective stages away all together Hassan (2008). Seasonal studies on the same river system (i.e. Vaal River) should investigate the effect of flood events on monogenean parasites further. Sampling in different habitats within the river (e.g. mild riffles, very fast rapids and deep yet fast glides) may also help to investigate the effect of water current on monogenean infection statistics.

Anthropogenic effects on aquatic systems in general may include introduction of exotic species, biodiversity loss due to climate change, habitat alteration and pollution (Hassan 2008). Freshwater parasites in general (e.g. Sures 2001) and monogeneans and digeneans in particular, (e.g. Soleng, Poléo and Bakke 2005; Pettersen, Vollestad, Flodmark and Poléo 2006; Gheorghiu et al. 2007; Blanar, Munkittrick, Houlahan, MacLatchy and Marcogliese 2009) may be good indicators of metal pollution. During previous environmental monitoring studies, fish health of fish in the Vaal Dam (unpolluted site) was compared to that in the Vaal River Barrage (polluted site) (Crafford and Avenant-Oldewage 2010).

The authors recorded the highest concentrations of heavy metals in C. gariepinus gill (filaments and arches) tissue followed by muscle, liver and lastly skin. As Q. aegypticus was recorded from gills and Gyrodactylus sp. from skin of C. gariepinus in the Vaal Dam, it is possible that higher pollution levels in the Vaal River Barrage may negatively affect parasite numbers at these sites. Metal pollution may, however, also increase opercular movement (Rani, Milton, Uthiralingam and Azhaguraj 2011) which could indirectly increase chances of exposure to gill parasites such as Quadriacanthus spp. This, in combination with the possible effects of pollution on fish health and immunity in general (e.g. Escher, Wahli, Büttner, Meier and Burkhardt-Holm 1999; Golovanova 2008), could be further investigated in future studies.

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Hypothesis 4-1: Temperature effects shall surpass potential host immunological effects (also see Hypothesis 3-1 and 3-2).

Evaluation outcome: Inconclusive. Much higher infection statistics (for Dactylogyrus spp.) were calculated during summer from Labeo spp. This observation supports the hypothesis. Given low numbers of Gyrodactylus spp. collected (also see Hypothesis 3-2), for which an opposite trend was expected, this hypothesis could, however, not be rigorously tested. The hypothesis statement itself also has a potential flaw in that the two variables (temperature and immunological response) are not completely independent. For gyrodactylids, for example, increased temperature may lead to reduced numbers through a direct effect (parasite mortality) or indirect effect (more effective host immunological response) (Blažek et al. 2008b).

In the preceding paragraphs the influence of abiotic variables on infection statistic parameters were discussed. In the paragraphs that follow the effect of biotic variables shall be discussed. Host-related factors may include host species, host age, host body size, host diet and feeding habits, host biology (e.g. social behaviour) and habitat preference (e.g. benthic or pelagic), site in the host (e.g. skin versus gill) and the host’s immune response (Bush et al. 2001; Luque and Poulin 2004). Only selected factors, deemed to be applicable to data collected during the current project, shall be discussed hereafter. Different host species exhibit pronounced differences in behavioural, reproductive and feeding biology. Dávidová Ondračková, Jurajda and Gelnar (2008) demonstrated that the character of the host habitat (e.g. gravel pits versus rivers) can significantly affect parasite assemblages. In the current study many of the fish species exhibit bottom-feeding behaviour (e.g. C. gariepinus, Labeo spp. and C. carpio but also to a lesser extend species of Labeobarbus Rüppell, 1836 and Ctenopharyngodon idella (Valenciennes, 1844)). Apart from these inter-species differences, Garcίa-Berthou (2001) also demonstrated size- and depth variation in the habitat and diet within the same species (C. carpio).

Host size or age thus can also indirectly affect parasite fauna through changes in the habitat preference and behaviour of the host. Boeger, Kritsky, Pie and Engers (2005) stated that demersal fish hosts forming large shoals while in continuous contact with the bottom sediment, will allow monogeneans to benefit from the limited, 2- dimensional spatial distribution (i.e. shoaling behaviour would facilitate more effective parasite transmission).

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This is evident in the study where especially both Labeo spp. were prone to shoaling (personal observation of numbers encountered in the same net(s) during sampling). The largest numbers of monogenean parasites were also recovered from these species (especially L. umbratus). During spawning season shoaling generally escalates dramatically in the Vaal River (personal observation). Larvae of particular parasite species have been shown to react to “hatching factors” released by particular host species (i.e. endogenous parasite hatching rhythms appears to be adapted to the host’s behaviour with host specific factors attracting larvae, Rohde and Rohde 2005). It is postulated that such a situation may be in operation during fish spawning where dactylogyrids would benefit from increased shoaling. This may also help explain the higher infection statistics encountered for Labeo spp. during the current study. If increased infection is indeed related to host spawning behaviour, Labeobarbus spp. infection statistics should be higher in rivers compared to dams situated in the same river systems. This is because these fish species prefer to spawn over gravel beds in running water and as a result are more prolific spawners in a river habitat compared to a still water habitat (Skelton 2001).

Another host related factor that may be considered, is host status in terms of introductions. The Labeo spp., Labeobarbus spp. and C. gariepinus are all endemic to South Africa. C. carpio, C. idella and M. salmoides are introduced species. New host introductions imply introduction of parasites associated with them (Galli, Stefani, Benzoni and Zullini 2005; Galli, Strona, Benzoni, Crosa and Stefani 2007). Hassan (2008) states that exotic species typically start with small founder populations which (by chance) often do not have the full parasite complement of the source area. This was also observed in the current study. While C. carpio is known to be infected by a very large number of monogenean parasites (see Chapter 10 for a summary), only three monogenean parasite species were found on this host during the current study. Though deemed unlikely due to a high degree of host specificity, especially when a phylogenetic dissimilarity exists between endemic and introduced species (Dove and Ernst 1998), introduced parasites may transfer from exotic to native hosts (e.g. Mizelle and McDougal 1970; Jalali and Barzegar 2006; Galli et al. 2007; Shamsi, Jalali and Aghazadeh Meshgi 2009). In such cases they may be more damaging to the new (endemic) host (i.e. the new host may lack appropriate evolutionary defences).

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Dactylogyrus anchoratus (Dujardin, 1845), for example, was shown by Shamsi et al. (2009) to transfer from carp to Barbus sharpeyi Günther, 1874.

Host gender, age and size effects may also affect infection parameter statistics. There appeared to be a preference for male hosts (statistically significant in only two cases, Table 13-4). Hassan, Akinsanya and Adegbaju (2010) also recorded more parasites as well as higher prevalence of infection from male hosts (C. gariepinus) compared to female hosts. It might be postulated that, since both statistically significant male host preferences (Labeo spp. and C. gariepinus) in the current study was recorded for summer, male hosts may exhibit some physiological or behavioural difference compared to females (e.g. different spawning behaviour) during this season. This trend (i.e. a male host gender preference during summer) was, however, not evident for all host species (e.g. C. idella) examined. If such a seasonal host gender preference does exist, it might thus be host species specific as it would most probably be the result of host behaviour and / or host habitat use. Johnson, Lafferty, van Oosterhout and Cable (2011) for example found higher gyrodactylid infections in female guppies and attributed it to the fact that females have a higher propensity for shoaling. Tombi and Bilong-Bilong (2004) also argued that higher infection statistics (Dactylogyrus spp., Dogielius spp. and Gyrodactylus sp.) observed in females relate to the shoaling behaviour of gravid Barbus martorelli Roman, 1971. A similar effect may be applicable to the males of other fish species. Additional, multiple seasonal sampling occasions would have to be performed for each host species in question, to determine if trends observed in this study were the result of chance or if a seasonal host gender preference does exist. Le Roux, Avenant-Oldewage and van der Walt (2011) for example found no seasonal or host gender effects when they examined Cichlidogyrus philander Douëllou, 1993.

Hinsinger and Justine (2006) found that Pseudorhabdosynochus cyathus Hinsinger and Justine, 2006 occurs only on young (i.e. smaller) hosts, whilst Pseudorhabdosynochus venus Hinsinger and Justine, 2006 occurred on older hosts. Guégan, Lambert, Lévêque, Combes and Euzet (1992) stated a combination of host size and host ecology largely explains monogenean species richness in West African cyprinid fishes.

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Age-specific costs of resistance to parasites to the host, as well as age-specific parasite preferences have indeed been suggested as subjects for further study by Michalakis and Hochberg (1994).

In previous chapters attempts were made to quantify host age and size effects (i.e. division into size classes and allocation of appropriate host age based on published age-length data results). However, in all instances no meaningful deductions were possible due to sampling constraints.

Gill net mesh sizes used resulted in hosts of a similar size being collected. The result was size classes exhibiting very small increment changes (e.g. 5 cm) with one or two classes containing many host specimens and others very few.

Hassan (2008) found, with regard to both parasite prevalence of infection as well as parasite community diversity, that differences among fish species were not related to fish size, host size and age effects. Madanire-Moyo et al. (2011) also found that monogenean parasite infection statistics were not influenced by fish (Oreochromis mossambicus (Peters, 1852)) size. Yet such affects have been reported in other studies as briefly discussed below. The longer a fish lives the more opportunity for infection with parasites shall present itself, i.e. larger fish may be expected to carry the highest parasite loads (e.g. Cloutman 1976; González, Acuña and Oliva 2001; Choudhury et al. 2004; Seddon 2004; Özer and Öztürk 2005; Cable and van Oosterhout 2007; Tekin Özan, Kir and Barlas 2008; Nachev and Sures 2009). Furthermore, larger fish may then also harbour a greater number of parasite species (Guégan and Hugueny 1994; Matějusová, Morand and Gelnar 2000; Dávidová, Ondračková, Jurajda and Gelnar 2008; Šimková, Lafond, Ondračkova, Jurajda, Ottova and Morand 2008). Larger fish would also possess larger gills that would constitute a larger surface area for parasites to attach to (Bush et al. 2001). Tekin Özan et al. (2008) found no statistically significant host size effects, but stated that infection did peak in larger size classes. Yet development of immunological response against certain parasites as fish grow older may confound any general trends. Cone and Cusack (1988) as well as Winger et al. (2008) recorded a general decrease in intensity of infection with host age (and hence also host size). Ekanem, Eyo and Sampson (2011) as well as Hassan et al. (2010), found more parasites on smaller (20 to 29 cm and 19.0 to 21.9 cm respectively) C. gariepinus.

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Gyrodactylus anguillae Ergens, 1960, also appeared to be more prevalent on smaller eels (Ernst et al. 2000) and Al-Zubaidy (2007) found smaller carp to be more susceptible to both dactylogyrids and gyrodactylids. These authors also recommended that more accurate surveys for this parasite should encompass broader host size ranges.

To add to this apparent confusion, Bhuiyan et al. (2007) reported highest levels of infection from medium sized fish and Hodneland and Nilsen (1994) reported that no host size differences in parasite infection was evident. Future studies in the Vaal Dam should thus also employ smaller mesh sizes to more accurately reflect predetermined (i.e. after consulting literature for size ranges commonly examined to allow for meaningful comparison) target size classes for investigation of potential fish host size effects (if any) on monogenean parasite infection statistics. In this regard the “gear selectivity” mathematical function (Booth and Potts 2006) would have to be employed to ensure that a representative proportion of fish in each size class is collected. Apart from numbers of parasites, the number of parasite species present may also be affected by host age. Willomitzer (1980a, 1980b) found that Dactylogyrus sp. infect C. idella fry from three weeks of age, yet Diplozoon paradoxum Nordmann, 1832 was found to occur almost exclusively on fingerlings 35 weeks of age (Willomitzer 1980a).

It would be interesting to investigate, in future studies, if such age effects would also occur in host species in the Vaal Dam where both Dactylogyrus spp. as well as Diplozoon spp. (i.e. on Labeo spp. hosts) or Paradiplozoon spp. (i.e. on Labeobarbus spp. hosts) are present. Age may be determined using otoliths following necropsy or by non-lethal methods such as the use of pectoral fin rays (Phelps, Edwards and Willis 2007; Gόmez-Márquez, Peña-Mendoza, Salgado- Ugarte and Arredondo-Figueroa 2008; Winker, Ellender, Weyl and Booth 2010).

Host size may even affect parasite morphology. Hayward and Rohde (1999), for example, postulated that anomalies in posterior clamp size of Thoracocotyle crocea MacCallum, 1913 may be a response of the worm to the rapid growth of gill lamellae in young hosts. Huyse et al. (2004) stated that body and haptor sclerite dimensions were distinctly smaller in G. arcuatus from small fish hosts compared to specimens from larger fish hosts. Morphological variation observed during the current study has been discussed in section 13.3.3.

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As all fish were of a very similar size, morphological plasticity in terms of sclerite measurements in the current study cannot be attributed to host size effects. As mentioned previously it is rather postulated that finer gill structure may be responsible for the morphological plasticity observed.

The previous discussion on fish length links to the notion of condition factor values. The latter involves fish weight. This in turn warrants a discussion on weight / length relationships and how it relates to fish age.

Macroscopic pathology / abnormal conditions resulting from monogenean infection may include proliferative epithelial reaction (Molnár 1971b; Molnár 1972; Appleby, Mo and Aase 1997), sporadic haemorrhages (Martins, Moraes, Fujimoto, Onaka, Nomura, Silva and Schalch 2000), pale gills with excessive mucous secretion (Buchmann and Bresciani 1998; Buchmann 1999; Arafa, El-Naggar and El-Abbassy 2009) and scale loss with associated surface lesions (Tokşen, Gamsiz and Nemli 2007; Kassaye and Tadesse 2009). This result in abnormal clinical signs such as displacement in gill lamellae due to base fusion as well as oedema and swelling of secondary lamellae (Rahanandeh, Sharifpour, Jalali, Kazemi, Fatideh and Sabet 2010). Obviously such severe reactions could potentially decrease host fitness and result in lower condition factor values for infected fish. However, none of the aforementioned conditions were observed during handling and processing of fish during the current study. In the preceding chapters it was thus concluded that any negative effects of parasite infections on observed condition factor values was highly unlikely, as monogenean infections in wild populations often have very little negative impact on host growth and survival (e.g. Cusack 1986; Reed et al. 2002). Furthermore aspects such as feeding frequency, efficacy of food conversion (Ali, Iqbal, Rana, Athar and Iqbal 2006; Abid and Ahmed 2009), feeding intensity and maturation of gonads (Khan and Siddiqui 1973) shall also affect condition factor values.

Seddon (2004) found a negative correlation between Diplozoon sp. infection and condition factor of L. umbratus at two sampling sites in the Vaal River system. As one exhibited poor water quality (Vaal River Barrage) and the other good water quality (Vaal Dam), the author concluded that: “under no influence from outside water qualities, the presence of this parasite is detrimental to the health of the host”.

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Molnár (1971c) stated that infection with D. lamellatus often causes considerable loss of condition in C. idella (even in sub-lethal infections), indicating that the statement made by Seddon (2004) does have merit. Yet Le Roux, Avenant- Oldewage and van der Walt (2011) actually found more parasites on fish with a higher condition factor studying C. philander from Pseudocrenilabrus philander philander (Weber, 1897).

The apparent discrepancy between these studies (i.e. Seddon 2004 and the current study) can be explained by the fact that the interaction between parasitism, water contamination and condition factor is often complex, being influenced by physiological, physical and chemical factors as well as parasite and host variables including genotype (and obviously the myriad of interactions between all the aforementioned factors) (e.g. Escher et al. 1999; Barber, Hoare and Krause 2000; Šimkova, Jarkovský, Koubková, Baruš and Prokeš 2005; Froese 2006; Lamková et al. 2007; Pervin and Mortuza 2008; Tavares-Dias, Moraes and Martins 2008).

Legendre and Albaret (1991) confirmed that the growth process is a complex activity influenced by a multitude of interactive factors. This implies that length / weight relationships or ratios may also vary considerably within single species or populations. Furthermore length / weight relationship differences between species may also be a confounding factor. Jhingran (1952) for example reported that there was poor agreement between recorded weights and predicted weights (based on length) once L. rohita exceeded 490 mm in length. Furthermore Montchowui, Laleye, Moreau, Philippart and Poncin (2009) stated that Labeo parvus Boulenger, 1902 exhibited a tendency to increase more in mass than in size (i.e. length). Similar trends recorded for Labeo calbasu (Hamilton, 1822) led Narejo, Mastoi, Lashari, Abid, Laghari and Mahesar (2009) to conclude that the length frequency age determination method should be verified / employed in conjunction with other techniques of age determination. Such effects may also apply to Labeo spp. in the current study as a change in body shape between length groups was recorded by Mulder (1973a). This could be further investigated in future studies.

As fish condition (in terms of body weight) would vary with the degree of gonad development, the condition factor is considered to be a good indicator of physiological stress relating to reproduction (Neff and Cargnelli 2004; Froese 2006).

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Variation in the condition factor may thus reflect both the degree of nourishment (e.g. Chaiyapechara, Casten, Hardy and Dong 2003) as well as the state of sexual maturity (Williams 2000).

The results reported by Ali et al. (2006) (i.e. condition factor was not influenced by feeding regime), suggests that the condition factor may be a better measure of the state of sexual maturity. The current study surveys were conducted in winter and summer respectively.

The fish species examined reproduce in spring and early summer. Females were thus not burdened with the physiological stress of egg production and spawning in winter. The summer study survey was performed in January 2010, with the commencement of spawning behaviour expected to have already commenced in September or October 2009 (i.e. spring and early summer). January could already be considered mid-summer and the majority of spawning behaviour was expected to have been completed by then (also see discussion in the next paragraph). It is thus postulated that females may have largely recovered from the physiological stress related to spawning. This line of reasoning is confirmed by Froese (2006) whom provides the following general pattern in seasonal variation of condition of adult fishes: decrease during times of low temperature and / or low availability of food followed by an increase towards the spawning season. Following that there is again a sharp decline after spawning (especially in females) and then a second increase after spawning. It is postulated that both sampling occasions (i.e. mid-winter and mid-summer) reflects the last situation in the seasonal patterns described (i.e. a reflection of the second increase following spawning). In the current study this then resulted in very little difference in condition factor values between genders during both winter and summer surveys.

Spawning is often related to cues such as temperature and rain (i.e. increase in the amount of running water / current strength) and as a result may also vary somewhat according to locality. For example, fish were observed to start spawning from as early as the end of August in the Lowveld region, while spawning in the Free State and Gauteng typically occurs from end September or October (personal observation).

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Relative time points of commencement of spawning for the different fish species in the Vaal Dam could be examined in future studies and possibly combined with ecological studies on the impact of spawning behaviour (e.g. increased shoaling) on monogenean parasite infection statistics.

It was previously recommended that smaller mesh sizes should also be employed to sample a greater variety of size classes. When combining such an approach with studies on the effects of parasitism and / or spawning on condition factor values, changes in body shape and size as fish grow should be considered. For example, Labeobarbus kimberleyensis (Gilchrist and Thompson, 1913) and Labeobarbus aeneus (Burchell, 1822) exhibit an increase in body depth above 20 cm in length which led Mulder (1973a) to employ an arbitrary length group division at 25 cm. Mulder (1973b) also found a change in body shape for L. capensis and L. umbratus with increasing length intervals. It would thus make more sense to calculate and compare condition factor values within each length class for each species examined. Such data can then be complemented with age determination techniques to more accurately correlate length to age data for the specific locality.

In this regard Winker et al. (2010) confirmed that growth zones in astericus otoliths of both smallmouth yellowfish (L. aeneus) and Orange River mudfish (L. capensis) can be interpreted as annuli (i.e. growth zones were deposited annually). Plug (2008) demonstrated that fish bones may also be used to estimate length (from which ages can then be inferred using length / age relationship data).

As monogeneans have a direct life cycle and do not make use of an intermediate host, the direct role of predation (i.e. host diet) in parasite transmission in the current study is considered to be negligible.

Gyrodactylus salaris Malmberg, 1957 parasites drifting in the water column were shown to have been transferred to salmon, with potential transfer through predation being suggested (Bakke et al. 2002). In the current Gyrodactylus spp. were recorded from C. carpio and C. gariepinus. Cyprinus carpio is an omnivore feeding on organic matter. Clarias gariepinus is a predator with a largely piscine diet (though they shall also take large invertebrates, Skelton 2001). It is thus deemed unlikely that they shall feed on dislodged Gyrodactylus spp. in the water column.

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As discussed previously host feeding behaviour and associated host biology / habitat preference may play a more important role. No seasonal variation in the food composition for Labeo coubie Rüppell, 1832 was found by Ayotunde, Ochang and Okey (2007). In the current study the more sedentary Labeo spp. harboured more monogenean parasite species, as well as greater numbers of parasites, compared to Labeobarbus spp. which are less frequently associated with sediment. It is thus postulated that habitat selection plays a larger role than host diet (with regard to potential effects on monogenean infection).

Another biotic variable that needs to be considered relates to immunology. A typical host immune response to parasites (the mechanisms of which are discussed by Buchmann 1999 and Sitjá-Bobadilla 2008) against a monogenean gill parasite, may include mucoid secretions, hyperplasia of the tissue at the attachment site and the appearance of lymphocytes (Arafa et al. 2009) as well as intact complement factors (Buchmann 1998). Fish skin is not only an important source of pro-inflammatory molecules, but contains immune response genes (Lindenstrøm, Secombes and Buchmann 2004) and actively modulates local inflammation during ectoparasite infections (González, Buchmann and Nielsen 2007).

This early inflammatory response (resulting from parasite induced injury) involves the influx of cells (most notably neutrophilic granulocytes) (González, Huising, Stakauskas, Forlenza, Verburg-van Kemenade, Buchmann, Nielsen and Wiegertjes 2007). Some data suggests acquisition of acquired protection against monogeneans (e.g. Alvarez-Pellitero 2008), indicating that physiological changes in the host and / or associated differences in temporal parasite exposure may affect host infection statistics.

Pollution may also suppress the immune response and lead to susceptibility of fish to infectious agents (Bernet, Schmidt-Posthaus, Wahli and Burkhardt-Holm 2000). Furthermore monogenean infections itself may result in cortisol production in trout, leading to depressed immune response mechanisms that may in turn enhance bacterial invasion (Busch, Dalsgaard and Buchmann 2003; Bandilla, Valtonen, Suomalainen, Aphalo and Hakalathi 2006). While neither of these scenarios were observed (or are suspected to have occurred), it serves to demonstrate the complexity associated with the effects of immune response in fishes.

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Furthermore concurrent endoparasite infections (e.g. anisakids) could activate the host immune system and result in negative effects on monogenean parasites (Larsen, Bresciani and Buchmann 2002). Future studies could include recovery of the entire parasite fauna of each host examined to investigate if any such correlations exist.

The current study does not allow any comments to be made on possible immune response effects. As stated previously, narrow size classes with small increment differences confounded any speculation on possible physiological changes associated with host age.

Furthermore data on temporal parasite exposure is also required as stated above, with two sampling occasions (during which no physiological host responses were examined) considered to be inadequate for any temporal analysis of host immune response.

An experimental approach would be better suited to this kind of study where, for example, blood and tissue chemistry (e.g. De Azevedo, Martins, Bozzo and Moraes 2006; Ghiraldelli, Martins, Yamashita and Jerônimo 2006; Tavarez-Dias et al. 2008; Marijić and Raspor 2010) of infected host fish of different sizes and reproductive status can be monitored over time with sub-sampling performed to monitor parasite burdens. Such blood analyses could also help evaluate if immune response reaction holds promise for development of vaccines, as has been done for microbial diseases in fish (e.g. Osman, Mohamed, Rahman and Soliman 2009; Toranzo, Romalde, Magariños and Barja 2009; Noor El Deen, Nagwa Sad and Abd E Aziz 2010).

Hypothesis 5-1: Larger / older fish harbour a greater number of parasites.

Evaluation outcome: Inconclusive. The current experimental design and collection method employed (large gill net mesh sizes) resulted in narrow size classes (some of which contained very few fish) differing with very small increments, thus confounding any meaningful analysis.

Hypothesis 5-2: Larger / older fish harbour a greater number of parasite species.

Evaluation outcome: As for hypothesis 5-1.

Hypothesis 5-3: Host gender shall have no effect on parasite infection statistics.

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Evaluation outcome: Inconclusive. A statistically significant preference for male fish during summer was recorded in two instances. The inconclusive pattern observed may be the result of a sampling artefact (i.e. low numbers of hosts collected for one or both host genders). Additional sampling is required to investigate possible seasonal trends.

Once parasites were identified and described, infection statistics calculated and the factors that may have influenced the results observed considered, additional biological aspects relating to parasite occurrence on the various hosts could be examined further.

Central to many studies on monogeneans are the aspects of site and host specificity. Host specificity can be defined as a measure of the capacity of a parasite to survive in or on species other than the definitive host (Jones 2001) and is considered to be of central importance to parasites and parasitologists alike (Poulin and Keeney 2007). According to Jones (2001) host specificity must not be viewed as an indirect indicator of innate resistance where data are based on field observations (natural infections), as was indeed the case in the current study. Specificity of parasitism thus not only depend on host immune response (e.g. Secombes and Chappell 1996), but rather on history (or phylogeny), morphology, physiology and ecology (i.e. interactions) (Stunkard 1970; Schmidt and Roberts 1977; Williams et al. 1992; Jovelin and Justine 2001), a statement mirrored in previous discussions on host- parasite effects in this thesis. The current study focuses mainly on ecological aspects, yet there are many physiological / morphological aspects (relating to especially host recognition) that would require investigation in future studies.

Halton (2004), with reference to ultrastructure studies, proposes four functions for the helminth tegument: a) absorption of exogenous material; b) synthesis and secretion of endogenous materials; c) osmoregulation and excretion and d) provision of sensory input. It is the latter that may be related to host recognition and hence a mechanism to explain host specificity in monogeneans (e.g. Buchmann and Lindenstrøm 2002). Innervations of head lobes (also called anterior adhesive sacs or apparatus, e.g. El-Naggar and Kearn 1980) with nerves in some monogeneans are associated with the presence of numerous sensilla, the latter possibly involved with contact stimulation and chemoreception (e.g. Watson and Rohde 1994; El-Naggar, Arafa, El-Abbassy, Stewart and Halton 2004).

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During attachment these sacs are everted in order to expose the enclosed duct openings and microvilli so secretions (e.g. Whittington, Armstrong, Chisholm and Cribb 2004; Wong et al. 2008) are extruded. El-Naggar and Kearn (1980) postulate that such ciliary structures (inside the adhesive sacs) may serve as contact receptors which may initiate the flow of these adhesive secretions. Wong et al. (2008) also found unciliated structures and state that they are probably sensory receptors also related to secretion.

It is postulated that such sensory structures may also be involved in host recognition / selection.

Experimental evidence of a sensory function (i.e. tangoreception, photoreception, rheoreception or chemoreception) is however restricted by the small size of the presumed receptor (Halton 2004). This makes ablation and electrophysiological studies technically difficult and as a result experimental evidence for such sensory functions is lacking (Halton 2004). Apart from examination of the possible functions of surface sensillary structures, the structures themselves may be of taxonomic value to determine between species (Shinn, Gibson and Sommerville 1997; Shinn, Gibson and Sommerville 1998).

Future studies should include staining of parasites with different types of stains that would also allow detailed study of different types of soft tissues (e.g. Lyons 1970). Such examinations would allow determination of the presence of adhesive sacs (with associated ciliated structures) or possibly other sensory structures on the monogenean species encountered during this study. Additionally, such an approach (i.e. “whole mount” illustration also detailing soft tissues) may also positively contribute to the quality of taxonomic descriptions in general as argued by Price, McClellan, Druckenmiller and Jacobs (1969) and Gussev (1979). This approach (i.e. whole mount drawings in addition to traditional sclerite drawings) was indeed employed by a number of authors (e.g. Thapar 1948; Tripathi 1957; Jain 1959; Price and Pike 1969; Ogawa and Egusa 1977; Timmons and Rogers 1977; Kritsky, Kulo and Boeger 1987; Agrawal, Tripathi and Shukla 2005; Amine and Euzet 2005; Agrawal, Tripathi and Devak 2006; Bilong Bilong, Nack and Euzet 2007; Tripathi, Agrawal and Pandey 2009).

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This could also be combined with SEM or TEM study of the tegument to further investigate the presence of microvillous-like projections or transversal ridges (Cohen, Kohn and Baptista-Farias 2001). Similar approaches are often employed for other parasite groups such as copepods (Lutsch and Avenant-Oldewage 1995; Marx and Avenant-Oldewage 1996; Robinson and Avenant-Oldewage 1996; Kruger, Avenant- Oldewage, Wepener and Oldewage 1998).

Rohde, Worthen, Heap, Hugueny and Guégan (1998) concluded that unstructured, unpredictable, clumped and depauperate parasite assemblages are generally the rule (with reference to deductions made from large ecological databases). With the amount of data available following only two surveys, the current study does not allow any comments on this subject.

Additional sampling of fish in wider size ranges would have to be performed at the same locality, different localities within the system and even different systems to compile a species composition database from which such possible relationships could be inferred. Parasites were found to be host genera specific (e.g. on Labeo spp. and Labeobarbus spp.), but some were host species specific (e.g. on C. carpio, C. gariepinus and C. idella). Galli and Kritsky (2008) stated that it is not uncommon for a single fish host species to host multiple species of a monogenean genus. This was also observed in the current study with regard to Dactylogyrus spp. on the two Labeo spp.

This also in agreement with a statement by Huyse et al. (2004) that closely related hosts are often parasitized by closely related monogenean species. Rohde and Rohde (2005) stated that the phylogenetic position of the hosts should thus also be considered when discussing observed parasite host specificity.

Poulin and Keeney (2007) state that host specificity may hint at the probability of local extinction (as was recorded for Dactylogyrus amphibothrium Wagener, 1857 by Zhokov, Pugatcheva, Molodozhnikova and Mironovskii 2006) but also reflects the ability of a parasite to colonize new host species. In the current study preference for more than one closely related host species within the same genus will increase probability of successful host infection and hence reduce the probability of extinction. It would also increase the probability of infection of other hosts within the same genus should the parasite be accidentally translocated with a host.

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For strict host specialists the inverse probabilities are expected (i.e. increased chance of extinction and decreased chance of infecting new host species).

According to Barger and Esch (2002), host specificity mediates the probability (given the presence of one or more suitable fish hosts) that a population of parasites persist at any particular site. This statement imparts predictive value to host specificity baseline data, as the parasite fauna of new areas may be predicted in advance. This would allow development of models on the possible effect of monogenean parasites on for example aquaculture activities planned in a particular area. Future studies should evaluate the accuracy of such species composition predictions (based on baseline data collected during the current study) by sampling the same host species in other geographical areas / river systems.

Despite the apparent importance of host specificity, the mechanisms involved in host specificity are poorly understood but may include behavioural, mechanical and chemical factors (Buchmann, Madsen and Dalgaard 2004) of which chemical cues appear most important (Buchmann et al. 2004). This obviously closely links with the study of sensory structures discussed previously.

In the context of the current study, future experimental studies could examine host selection by, for example, impregnating membranes with the slime of different fish species and observing if motile oncomiracidia demonstrate a preference for any particular membrane. Oncomiracidia separated in this manner could then be exposed to fish of that species (and fish of other species) to evaluate of the preference observed actually transpires into successful colonization of that particular host. This could help confirm the notion of “host preference” versus “host specialization” (the former for example being described for D. larindae and D. iwani and the latter for D. nicolettae in the current study) as is also discussed by Šimkova, Verneau, Gelnar and Morand (2006). This may also confirm if D. intorquens represent a single form exhibiting differences in sclerite size because of host gill differences, or if it actually constitute two separate forms (with inherent differences in sclerite size) that exhibits a host preference. King and Cable (2007) also warned that host specificity cannot be assumed until it was experimentally proven.

More generalized host preferences may be observed for monogeneans under conditions suitable to infrapopulation growth (Blažek, Bagge and Valtonen 2008a).

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It may then be postulated that the host preference for Labeo spp. may differ along a gradient of conditions that would either promote or restrict infrapopulation growth. Future studies should thus also attempt to sample localities (where these host species occur separately and others where they occur together but with different abundances) exhibiting varying degrees of suitability with regard to host needs and evaluate whether any differences in host preference can be observed.

The usefulness of molecular studies is sometimes overstated when viewed from an community ecological perspective, whilst experimental studies (as was demanded by King and Cable 2007) are also often performed outside an ecological context (i.e. in an unnatural context) (Poulin and Keeney 2007). Neither of these two approaches (i.e. molecular or experimental) was applied in the current project.

Future studies could employ both and also combine it with field observations, in order to comment on the effect of possible influencing factors (determined by experimental and molecular means) on what was actually observed in the field.

Hypothesis 6-1: Monogenean parasites encountered shall exhibit a high degree of host specificity.

Evaluation outcome: Accepted. All parasite species exhibited specificity with regard to host genus. Most parasites also demonstrated apparent host species specificity. In instances where monogenean parasites did occur on more than one closely related species (e.g. D. larindae and D. iwani) a clear host preference was evident.

Hypothesis 6-2 Examination of previously unexamined (for monogenean parasites) fish species shall reveal novel monogenean species.

Evaluation outcome: Accepted. A number of new parasite species have been described from endemic Labeo spp. hosts. Additional sampling (i.e. examination of a larger number of samples) is required to clarify species identification of monogeneans occurring on endemic Labeobarbus spp., but at least one new monogenean species description from this host genus is anticipated.

Hypothesis 6-3: Alien (non-endemic to South Africa) fish species with a global translocation history shall harbour monogenean parasites with the same global distribution pattern.

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Evaluation outcome: Accepted. Both C. carpio and C. idella harboured monogenean parasites that are known to occur globally on the same host species.

Hypothesis 6-4: Monogenean parasites from such alien fish species shall not occur on endemic fish species (i.e. host switching shall not have occurred).

Evaluation outcome: Accepted. No host-switching appear to have occurred.

With regard to parasite distribution on / in a host (i.e. site specificity), Kassai (1999) stated that the term “site” is indeed preferable to “location” as the latter may also be used to refer to a geographic locality. In free-living environments it is difficult to assess correlations between organismal success (i.e. in terms of any particular site imparting increased advantage to the organism choosing to reside there based on critical resources) and environmental variations (Sukhedo and Bansemir 1996).

Examining such effects in parasites, however, is a lot more productive as the habitat is replicated (at least to a large extend) in the individuals belonging to a particular host species (Sukhedo and Bansemir 1996). Furthermore fishes are available in large numbers, fairly few parasite species are involved (making it easier to detect community structuring processes through the study of site specificity) and gills are considered a quantifiable, natural habitat (Janovy 2002).

Gyrodactylus spp. were found on both skin and gills of Clarias gariepinus. Harris and Lyles (1992) studied Gyrodactylus spp. infections on the skin of guppies (Poecilia reticulata Peters, 1859). They found that G. turnbulli were predominantly posteriorly distributed, whereas Gyrodactylus bullatarudis Turnbull, 1956 exhibited a more anterior distribution. Interestingly enough it would appear that site preference may potentially vary according to the age of the hosts (i.e. mature versus immature) (Richards and Chubb 1996).

Cone and Cusack (1989) also identified site selection preference (fin margins) for Gyrodactylus colemanensis Mizelle and Kritsky, 1967 on rainbow trout. In the current study random (in terms of position) mucous smears were made from the skin of each fish. Skin scrape methodology followed in the current study thus did not allow any comment to be made on site specificity on skin. In future studies certain standard sites for mucous smears should be identified (e.g. base of tail, back area adjacent to / along back fin, stomach area from base of cloaca to opercular slits, area on side of fish directly behind operculum etc.).

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Parasite (Gyrodactylus spp.) distribution on different fish hosts (C. gariepinus and C. carpio) could then be examined and compared. As gyrodactylid parasite occurrence has been correlated with mucous cell density, mucous cell contents may play a decisive role in active gyrodactylid site selection (Buchmann and Bresciani 1998). This is potentially related to a strategy by parasites attempting to evade areas on the fish where a strong immune response is likely (González et al. 2007; Sitja-Bobadilla 2008), as parasites are often removed by sloughing of mucous (e.g. Lester 1972). Site preferences observed in future studies could be combined with histopathological examination of skin tissue from the respective sites, to determine if such a correlation (i.e. between mucous cell density and site preference) can also be identified.

Other sites from where Gyrodactylus spp. have been recovered in other studies include the mouth and nasal cavities (Xiao-Qin, Wei-Jun and Cheng-Ping 2000). In future studies these sites should also be specifically examined.

Apparent gill preferences observed during the current study, are summarized in Table 13-4. During preceding chapters it was shown that different parasite species on the same host did not exhibit spatial separation based on site preference. As a result general trends in site preference as recorded for the parasite component communities on the different hosts are summarized in Table 13-4.

Gills were divided into particular areas and examined by scraping as explained in Chapter 3. Baker et al. (2005) found that Metamicrocotyla macracantha (Alexander, 1954) preferred the first gill arch. As this arch experiences minimal current flow they postulated that the parasite attaches here in order to secure itself on the gill. Halton (2004) found Diclidophora merlangi (Kuhn in Nordmann, 1832) to most frequently occur on the first gill of the whiting, whilst Diclidophora luscae (van Beneden and Hesse, 1864) in turn seemed to prefer the second and third gills of the pout (Trisopterus luscus (Linnaeus, 1758)). Le Roux et al. (2011) reported that C. philander from P. philander philander are also more likely to infect the second and third gill arches.

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Matejusová, Šimková, Sasal and Gelnar (2003) reported the same trend for Pseudodactylogyrus anguillae (Yin and Sproston, 1948) and Pseudodactylogyrus bini Kikuchi, 1929 from Anguilla anguilla (Linnaeus, 1758), as did Yang, Liu, Gibson and Dong (2006) for Polyabris mamaevi Ogawa and Egusa, 1980 and Tetrancistrum nebulosi Young, 1967 from Siganus fuscescens (Houttugn, 1782). Özer and Öztürk (2005) found that Dactylogyrus cornu Linstow, 1878 prefer the second gill arch of Vimba vimba tenella (Nordmann, 1840). The same preference (i.e. second and third gills) was also observed most often (and was also found to be statistically significant most often, Table 13-3) in the current study and is believed to be related to water flow over the gills during respiration as the largest volume passes over the second gill arch (Paling 1968; Blažek and Gelnar 2006).

There appeared to be no clear preference for either the left or right side of the head (i.e. gill set), with only a single statistically significant result (for the left side) being recorded for Labeo spp. during summer (Table 13-3). Baker et al. (2005) also recorded no preference for any particular side of the head for Metamicrocotyla macracantha (Alexander, 1954). Le Roux et al. (2011) confirmed this trend when they examined C. philander from P. philander philander.

One possible reason for parasites preferring a particular gill set may be gill asymmetry (Rohde 1979). Ayoade, Sowunmi and Nwachukwu (2004) examined Labeo ogunensis Boulenger, 1910 and found that gill filaments and rakers from both sides were not significantly different. Furthermore fish gender and size were shown to exert minimal influence on fluctuating asymmetry. It is thus unlikely that gill asymmetry would have had an effect on site preference in the current study, but this could be examined further / confirmed in future studies.

It might once again be that changes in seasonal behaviour (e.g. body orientation during feeding or spawning that would favour increased exposure of one side of the head to sediment where resting oncomiracidia may gather) could be responsible for this observation. This is considered to be an unlikely explanation as mention of such behaviour could not be found in literature and was not observed.

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Table 13-4: Summary of general trends in host effects (gender) and site specificity / site preference (position on gills) for monogenean composite communities (dactylogyrid parasites except for Clarias gariepinus (Burchell, 1822) as indicated in the footnote) on various fish hosts from the Vaal Dam, Gauteng Province, South Africa. Statistically significant (p<0.05) p-values are indicated with bold text in shaded blocks.

Variable

1 Host gender Gill arch Gill set Gill arch region Gill orientation Host Season p- p- p- p- p- Preference Preference Preference Preference Preference value 2 value 3 value3 value3 value 3

nd rd Labeo Winter Female 0.200 2 and 3 0.002 Right > 0.200 Medial 0.000 Anterior 0.000 Cuvier, 1817 Summer Male 0.001 2nd and 3rd 0.005 Left 0.000 Medial 0.228 Anterior 0.114 Summer 4 Male 0.448 1st and 2nd Left 0.133 Ventral 0.227 Anterior 0.239 Clarias gariepinus 0.001 (Burchell, 1822) Summer 5 Male 0.048 3rd 0.624 Right 0.154 Ventral 0.660 Anterior 0.933

Cyprinus carpio Summer Male 0.103 1st 0.186 Right 0.282 Medial 0.755 Anterior 0.282 Linnaeus, 1758 Ctenopharyngodon nd idella Summer Female 0.335 2 0.587 Left 0.139 Medial 0.353 Anterior 0.042 (Valenciennes, 1844)

Labeobarbus aeneus 6 Pooled (Burchell, 1822)

Dorsal/ Labeobarbus (i.e. winter st kimberleyensis Male 0.251 1 0.630 Left 0.176 0.032 Anterior 0.083 Medial (Gilchrist and and Thompson, 1913) summer combined) Labeobarbus sp. suspected hybrid

Micropterus salmoides Not applicable (Lacepède, 1802)

1 = Season: Winter = Survey 1 (June / July 2009); Summer = Survey 2 (January 2010); 2 = Mann-Whitney U test; 3 = Pearson Chi square; 4 = As recorded for the dactylogyrid parasite Quadriacanthus aegypticus; 5 = As recorded for Gyrodactylus spp.; 6 = Pooled for both seasons and all species due to small sample size / low parasite prevalence.

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There was a very clear preference for the medial gill arch region in all host species examined for all species with the exception of C. gariepinus (though only statistically significant in one case, Table 13-3). Apparent preference for the medial position is also documented for D. banghami (Anderson, Blažek, Percival and Janovy 1993). For C. philander a preference for the dorsal region was observed followed by the medial region (Le Roux et al. 2011). In the current study a similar distribution pattern is also thought to be related to water flow dynamics over the gills (Paling 1968).

The gills of C. gariepinus are morphologically completely different from that of the other fish species examined. The arch is much longer and hence the length of the filaments relative to the length of the gill arch much shorter. Furthermore the gill basket is much wider due to the large, broad head resulting in a “less dense” appearance of the gills within the gill basket.

It is postulated that water flow dynamics over the gills of this fish species differ from that observed in other fish species and that this resulted in the different (compared to other host species) gill arch region preference observed. Future studies could further examine this aspect experimentally in the laboratory.

In terms of gill orientation, there was also an apparent preference for the anterior face of the gill in all host species examined (though only statistically significant in two cases, 95% confidence interval, Table 13-3). This could also possibly be related to water flow dynamics. The gills are slightly convex (i.e. rounded on the surface that faces the operculum) as they overlap each other. As parasites pass over the gills, water pressure is likely to be highest against the convex, anterior face of the gill and as a result they shall be more likely to attach to this side (i.e. “front” or “outside” surface facing towards the opercula) of the gill.

With the discussions in the preceding paragraphs in mind, a number of conclusions may be drawn with regard to site specificity. Kadlec, Šimková and Gelnar (2003a) postulated that microhabitat preference might be related to species abundance (parasites tend to be segregated in case of low abundance to increase mating opportunities). Buchmann and Bresciani (1998) also stated that site specificity may be related to aspects such as reproduction, withstanding differential ventilating currents and feeding.

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This statement is in agreement with Blažek and Gelnar (2006), whom argued that host behaviour and habitat use (in conjunction with water flow dynamics over the gills) may be more important in determining observed trends in parasite site specificity (i.e. not necessarily an active choice for a preferred site by the parasite).

Furthermore Baker et al. (2005) reported that neutral (i.e. random distribution resulting from water flow dynamics as reported by Paling 1968) as well as negative (i.e. competition as was reported by Paperna (1964b) as well as Chung, Lin and Kou 1984) interactions could play a role in determining parasite distribution on the gills. However, as negative interspecific interactions are very rarely encountered between monogeneans (e.g. Šimková, Desdevises, Gelnar and Morand 2000; Šimková, Gelnar and Morand 2001; Šimková, Sasal, Kadlec and Gelnar 2001), it is unlikely to influence parasite distribution (Mouillot, Šimková, Morand and Poulin 2005).

Paperna (1996) stated that oncomiracidia of dactylogyrids could either actively attach to the skin of the host fish before migrating to the gills, or become attached when washed through the gills. One could see from the above that one recurring variable are deemed paramount: water flow dynamics.

Results obtained in the current study indeed suggest that neither negative interactions, nor active selection of sites resulting from migratory behaviour following attachment played a role and that the site preferences were a result of neutral interactions (predominantly water flow over the gills). Furthermore, as similar trends were observed in a number of different fish species, it can also be deduced that the effect of water current may transcend that of biological (e.g. behaviour and habitat preference) differences between fish species. Considering that all the species either have sedentary habits (e.g. Labeo spp., C. carpio and C. gariepinus) or at least feed on the bottom from time to time (e.g. Labeobarbus spp. and C. idella) suggest that they do share some biological attributes which may explain the similarity in monogenean site preference observed. A seasonal preference with regard to host gender possibly exists, but this needs to be confirmed in future studies.

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Hypothesis 6-5: Site specificity shall not be distinct where more than one species co-exist on the gills, i.e. neutral interactions (i.e. random distribution) shall have a greater effect than negative interactions (e.g. competition).

Evaluation outcome: Accepted. This is especially clear from parasite distribution on the gills of Labeo spp., where there appears to be no clear differentiation of preferred selection sites between the various monogenean species collected.

In the previous paragraphs first host and then site specificity were discussed. In the discussion that follows the link between these two aspects shall be further investigated. Reproductive isolation may occur with or without geographical isolation or physical barriers (Coyne 2007). Huyse et al. (2005) stated that small, isolated demes (random mating populations) of parasites with a direct life cycle are likely to show population genetics patterns similar to that of their host. This may result in host-parasite cospeciation at a macroevolutionary scale, explaining why most monogenean parasites exhibit a high degree of host specificity.

Huyse et al. (2005) further stated that habitat selection (resulting in apparent site specificity) also relates to parasite population fragmentation along three scales: space, host species and host individual (Huyse et al. 2005). The second scale (host species) shall in turn result in observed host specificity, with fragmentation along these spatial scales complicating the ecological genetics of parasites (as well as the study thereof).

During the current study a single sampling point within a very large river system was sampled. Within such a large system is difficult to imagine the existence of “small, isolated demes”. Future studies should consider also sampling truly isolated host populations. Examples may include Labeo spp. introduced to small irrigation impoundments receiving no water from a river system, or points on a small spruit where large weirs permanently separated host populations. However, terminology like “host-parasite cospeciation” and “macroevolutionary scale” suggest very long time periods, so very little difference in host and site specificity is anticipated between isolated demes that have not been separated for a suitable (in terms of an evolutionary time scale) length of time.

A logical conclusion is that host and site specificity, possibly contributing to a process of speciation, may result in apparent morphological plasticity.

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It is once again Huyse et al. (2005) whom warned that it is also very important to quantify and compare the phylogeographic variation in both the host and parasite throughout their geographical range (microevolutionary scale). Sampling of various populations / “isolated demes” throughout the host’s geographical range as suggested in the previous section may thus well be more applicable to a microevolutionary scale. Speciation in parasites could be triggered or accelerated by coevolutionary arms races, adaptive radiations (e.g. after host switching) or non- adaptive processes (e.g. genetic drift and Wahlund effects) (Huyse et al. 2005). It is thus possible that, through the process of speciation, morphological plasticity may become evident. This may be particularly applicable to Gyrodactylus spp. where the tempo of parasite speciation is much faster than speciation amongst hosts (Ziętara and Lumme 2002). Additional sampling over a wider geographical range is encouraged to quantify the degree of morphological plasticity observed in different populations with varying degrees of phylogeographic isolation.

With regard to monogenean sampling over large geographic areas, poor quality of monogenean descriptions may lead to erroneous identifications and hence confound the determination of true monogenean distribution (e.g. Harris, Shinn, Cable, Bakke and Bron 2008; Galli and Kritsky 2008). Such additional sampling should thus also include morphometric (and molecular in the case of gyrodactylids, e.g. García- Vásquez, Hansen, Christison, Bron and Shinn 2011) examinations to amend existing descriptions where necessary, or to compile thorough descriptions for new species that may be encountered.

Following examination of biological aspects of individual species, discussion on the species composition of the Vaal Dam monogenean parasite community as a whole is a next logical step. Both historical and ecological constraints govern the structure of monogenean parasite communities (Morand, Šimkova, Matejusová, Plaisance, Verneau and Desdevises 2002). One would obviously require large databases to make any meaningful comments on patterns of evolutionary ecology that may have shaped such communities.

Furthermore Barrantes and Sandoval (2009) warn that diversity indices, though extensively used, have statistical and conceptual problems that make accurate comparisons of species abundance or richness across communities virtually impossible.

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They continue by saying that a very simple method, such as reporting the number of species per taxonomic category, may in fact be a lot more meaningful (i.e. provide more information) than a diversity index value. Given the small sample size (two surveys) and geographical restrictions (one locality), it is agreed that diversity index calculations may be of limited value in this case. For the purposes of the current study the community was thus simply described in terms of species composition as suggested by Barrantes and Sandoval (2009). A summary of the number of parasite species encountered, as related to host species from which they were collected, are provided in Table 13-5.

Species richness (i.e. number of species encountered) are probably the most simple variable used to describe communities. Guégan and Kennedy (1996), however, warns that it is greatly influenced by both sampling effort and area sampled (i.e. geographical considerations).

Monogenean species richness is often reduced at polluted localities (Dušek, Gelnar and Šebelova 1998), hence an ecosystem rich in parasite species are generally deemed to be a healthy system (Hudson, Dobson and Lafferty 2006). Fourteen parasite species were collected from eight host species in the current study. In the Amazon five monogenean species per species of host is considered a reasonable estimate (Kritsky cited in Fletcher and Whittington 1998 as “personal communication”). Fletcher and Whittington (1998) as well Jianying, Tingbao, Lin and Xuejuan (2003) proposed a conservative estimate of three monogenean species per fish species in Australia and China respectively. These estimates also seem to be in agreement with results obtained in the present study (i.e. between one and five parasite species per host fish examined, with the exception of M. salmoides where no parasites were recovered). One can but assume that, with examination of other endemic species in South Africa not yet rigorously studied for monogenean fauna, many more monogenean species await discovery. Based on the results in Table 13- 1, an estimate of two to six monogenean parasite species per unexamined (for monogenean parasites) fish species remaining in South Africa is proposed.

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Fletcher and Whittington (1998) speculated that reasons for the paucity of described monogenean species from Australian freshwater fish is the result of a combination of several factors: the relatively impoverished freshwater fish fauna, huge geographical areas to be sampled, the relatively short history of scientific research in the nation and the fact that field excursions to collect specimens in remote regions are logistically difficult (i.e. restrictions regarding suitable laboratories to work from). At least some of these (e.g. relatively short history of intermittent scientific research on specifically monogenean parasites in conjunction with fairly little published data on work performed in South Africa) may also be applicable to the South African and even African context.

The notion of host specificity is also reflected in the classification of host generalists as opposed to host specialists: Šimková, Kadlec, Gelnar and Morand (2002) found that generalists had a higher abundance and were more widely distributed among hosts compared to specialists.

Of 14 species collected, seven were found to occur on only one fish host species, three were found to occur on two closely related species and the status of four remain contentious (two are thought to occur on two host species, one is thought to occur on a single host species and the host specificity of one remains inconclusive – these have to be confirmed by additional sampling and investigation). At least 50% of the species were thus host specific to a single host species. All parasites species, however, were specific to a single host genus even when they infected two species. Bush et al. (2001) warns that, to identify a parasite as being a host generalist or host specialist, a researcher would need to know much about the parasites in all potential hosts (i.e. sufficient data should be available, a sentiment shared by Abowei and Ezekiel 2011). As indicated above additional sampling is most definitely required to confirm the trends described above. Species-poor communities are thought to be composed mostly of host generalists (Bush et al. 2001). With fourteen species encountered during the current study, the component community could be considered species rich and as a result the high degree of host specificity observed was expected.

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Hypothesis 7-1: More than one species from at least one genus shall occur on the same fish host.

Evaluation outcome: Rejected. More than one species of the same genus (Dactylogyrus spp. in this case) was only recorded from Labeo spp. and C. carpio (i.e. the other host species only harboured one species per monogenean genus).

Hypothesis 7-2: More than one monogenean genus shall be recovered from every host species examined.

Evaluation outcome: Rejected. For some host species (e.g. C. idella) representatives from only one monogenean genus were recorded.

Before defining specific future research needs, the practical relevance of this project in a South African context shall first be briefly evaluated.

Compared to international output, very little research has been published on parasites and diseases hampering development of African aquaculture (Hecht and Endemann 1998). Labeo umbratus, commonly known as moggel, shows promise as a suitable aquaculture species in small impoundments (e.g. Potts, Booth, Hecht and Andrew 2006). During this study it was shown that this fish species carries the heaviest monogenean parasite burdens when compared to other fish species in the Vaal Dam.

Under intensive aquaculture conditions often associated with environmental stress (e.g. Jeney and Jeney 1995; Svobodová, Máchová, Kroupová, Smutná and Groch 2007; Öktener, Eğribaş and Başusta 2008), monogenean infections often escalate and subsequently result in mortalities (Martins et al. 2000; Uzbilek and Yildiz 2002; Eiras, Segner, Wahli and Kapoor 2008). Future studies may possibly also investigate effective treatment (e.g. Székely and Molnár 1987; Schmahl and Taraschewski 1987; Schmahl and Mehlhorn 1988; Schmahl 1991; Schmahl 1993; Athanassopoulou, Pappas and Bitchava 2009) against parasites encountered during this project with the possibility of future aquaculture developments in mind. In this regard Huyse et al. (2005) suggested that studies on drug resistance be combined with population genetic studies.

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Table 13-5: Summary of existing parasite species collected and new parasite species described for each of the host fish species examined.

Monogenean species: Number of Common name Scientific name host fish Number of examined * Name species

1) Dactylogyrus iwani Crafford, Luus-Powell and Avenant-Oldewage, 2012 2) Dactylogyrus larindae Crafford, Luus-Powell and Avenant-Oldewage, 2012 Labeo capensis Orange River Mudfish 12 + 20 = 32 4 3) Dactylogyrus nicolettae (Smith, 1841) Crafford, Luus-Powell and Avenant-Oldewage, 2012 4) Dogielius intorquens Crafford, Luus-Powell and Avenant-Oldewage, 2012 (*** Diplozoon von Nordmann, 1832)

1) D. iwani 2) D. larindae Labeo umbratus 27 + 21 Moggel 4 5) Dactylogyrus sp. D = 48 (Smith, 1841) 4) D. intorquens 6) Diplozoon sp.

7) Quadriacanthus aegypticus Clarias gariepinus 0 + 11 Sharptooth catfish 2 El-Naggar and Serag, 1986 (Burchell, 1822) = 11 8) Gyrodactylus sp. G

9) Dactylogyrus extensus Mueller and Van Cleave, 1932 Cyprinus carpio 0 + 13 10) Dactylogyrus minutus Common carp 3 Linnaeus, 1758 = 13 Kulwiec, 1927 11) Gyrodactylus kherulensis Ergens, 1974 Ctenopharyngodon 0 + 12 12) Dactylogyrus lamellatus Grass carp idella 1 = 12 Achmerow, 1952 (Valenciennes, 1844)

13) Dactylogyrus sp. L (belonging to Labeobarbus aeneus the Dactylogyrus varicorhini 16 + 20 Smallmouth yellowfish (Burchell, 1822) 2 Bychowsky, 1958 species group / = 36 type) 14) Paradiplozoon sp.

Labeobarbus 3 + 7 At least Largemouth yellowfish kimberleyensis (Gilchrist See ** (table footnote) = 10 one and Thompson, 1913)

Suspected yellowfish L. aeneus x L. 1 + 2 1 14) Paradiplozoon Akhmerov, 1974 hybrid kimberleyensis = 3

Note: Urinary bladder (which also Micropterus salmoides 0 + 2 Largemouth bass 0 harbours monogeneans in this host (Lacepède, 1802) = 2 species) was not examined.

* Pooled for both the 2009 winter (June / July) and 2010 summer (January) surveys in following format: Survey 1 + Survey 2 = Total ** Inconclusive: A) All monogeneans found on the gills of this species during the current study was damaged and could only be identified to genus level (Dactylogyrus sp.) because of presence of eye-spots; *** Found on only two specimens of Labeo capensis - to be confirmed in future studies.

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Several still waters in South Africa have been evaluated for the establishment of commercial fisheries (e.g. Tomasson, Bruton and Hamman 1985; Potts and Khumalo 2005; Richardson, Booth and Weyl 2009). Management of fish stocks not only requires understanding of ecology, biology and establishment success of fish species (Weyl, Stadtlander and Booth 2009), but also parasite fauna and potential impact on fish health (e.g. Loot, Poulet, Reyjol, Blanchet and Lek 2004). In this regard monogeneans are considered to constitute a major group of parasites (e.g. Lerssutthichawal and Supamattaya 2005). The current study provides baseline data on the monogenean fauna of one such still water (i.e. Vaal Dam). A geographical parasite database may have wider applications. Aguilar-Aguilar, Contreras-Medina and Salgado-Maldonado (2003), for example, examined the distributional patterns of fish helminths (including monogeneans) to generate hypotheses on the relationships between hydrological systems in Mexico.

The fact that fish helminthology in southern Africa is considered by Barson and Avenant-Oldewage (2006a and 2006b) to be much less widely-studied when compared to other aspects of fish biology and aquatic parasitology, demonstrate the need for additional research. The study by Bertasso (2004), though not including monogenean helminths, is yet another example of such an approach.

South Africa’s aquatic history is marred with introductions of non-endemic species (e.g. M. salmoides, C. idella and C. carpio in the case of the current study). Russell (2011), for example, stated that 13 of the national parks in South Africa contain aquatic systems which support 63 indigenous and 11 alien fish species. Inbreeding (i.e. hybridization) has been shown to contribute to declines in wild fish populations (Neff and Avise 2004). In the current study these introduced species do not appear to pose a very real threat of extinction of endemic species through hybridization. Ironically enough it is possible hybridization within endemic genera (i.e. within Labeobarbus spp. and Labeo spp. respectively) that may threaten genetic biodiversity (e.g. van Vuuren, Mulder, Ferreira and van der Bank 1990).

On the level of macroscopic identification, occurrence of suspected hybridization L. aeneus and L. kimberleyensis has been observed in the current study. Given the conservation status of largemouth yellowfish (Impson, Bills and Wolhuter 2008) this is an obvious cause for concern.

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In future studies this could be further investigated on a molecular level combined with the use of monogeneans as “biological tags” (e.g. Dupont and Crivelli 1988; Williams et al. 1992; Lambert and El Gharbi 1995), as hybridization of fish species in the natural environment is poorly documented (D’Amato, Esterhuyse, van der Waal, Brink and Volckaert 2007). Though originally collected from L. aeneus (Hempel, Avenant-Oldewage and Mashego 2001), it would appear that Paradiplozoon sp. occurs on both host species as well as the suspected hybrid. Future studies should confirm that this is in fact the same species. Dupont and Crivelli (1988) stated that, as a general rule, it is not feasible to attempt prediction of the parasitism of a hybrid by knowing the parasitism of its parents. They continue by saying that it is at least partially possible to qualitatively do so for Dactylogyrus spp., as they found the complete set of Dactylogyrus species on the hybrid that was also found on the “parental” species of fish they examined.

Furthermore Birgi and Lambert (1987) found that Barbus jae Boulenger, 1903 can be distinguished from other Barbus spp. based solely on fauna, whilst El Gharbi, Birgi and Lambert (1994) found that Labeobarbus spp. and Barbus spp. each have unique Dactylogyrus spp. fauna that makes this genus suitable for biogeographical phylogenetic and taxonomic indicators. This has already previously been applied in studies by Guégan and Lambert (1990, 1991) as well as Guégan and Agnése (1991). Future parasitic investigations examining hybridization with the genus Labeobarbus should thus rather focus on the genus Dactylogyrus, in combination with both host morphometric and genetic / molecular approaches as exemplified by Paugy, Guégan and Agnèse (1990).

Parasites in fishes are considered to be excellent indicators of environmental impact (e.g. Sures 2004; Khan and Billiard 2007; Lafferty 2008; Madanire-Moyo and Barson 2010). Parasites have, for example, been used to illustrate the effects of habitat degradation / alteration (weir construction) on fish health and the parasite component community itself (Loot, Reyjol, Poulet, Šimková, Blanchet and Lek 2007). Such an approach could be applied to the Vaal River system (e.g. comparing the monogenean parasite component community composition above and below the Vaal River Barrage).

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Furthermore the potential use of monogenean parasites as indicators of water quality is well documented (e.g. Zharikova 1993; Pečinková, Matějusová, Koubková and Gelnar 2005; Pech, Vidal-Martinez, Aguirre-Macedo, Gold-Bouchot, Herrera-Silveira, Zapata-Pérez and Marcogliese 2009; Madanire-Moyo and Barson 2010). Galli, Crosa, Mariniello, Ortis and D’Amelio (2001) demonstrated that species within the same monogenean genus may exhibit variable sensitivities to pollution levels. Such possible effects should be investigated and elucidated in future studies with regard to species encountered during this study.

Monogenean parasites can also be used to test behavioural ecology models. An example is the study by Wedekind (1992) where infection with Diplozoon spp. were successfully employed to test a sexual ornamentation (as indicator of overall male condition) theory. Similar studies may be considered for Labeo spp. in future.

When compared between localities, monogenean parasites may also be used as markers to reveal and compare aspects of fish behaviour and ecology in different systems (or isolated parts within the same system) (e.g. Williams et al. 1992; Wilson, Muzzall and Ehlinger 1996; Weichman and Janovy 2000; Smale, Langlois, Kendrick, Meeuwig and Harvey 2010).

The question “Which monogenean parasite taxa occur in the Vaal Dam in South Africa and at which infection levels?” was formulated to capture the core objective of this project. This was indeed achieved through parasite identification (and description where necessary), calculation of infection statistic parameters and discussion on the possible effects of both abiotic and biotic (i.e. parasite and host biology) factors on the results obtained. The major findings as discussed in this final chapter are provided in the summary section (following the Table of Contents in the beginning of this thesis).

Despite the main objective (i.e. collection of baseline data with regard to the monogenean fauna of the Vaal Dam) being met, pulling at the loose threads of biological complexity did indeed result in the discovery of many other loose ends. These unanswered questions need to be formulated and further investigated in future studies. Through this journey from aim to abscission a number of future research needs were thus identified, as is discussed on the following page.

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The first relates to methodology. More specifically, the method used to examine gills and recover parasites (i.e. preservation in ethanol and subsequently scraping). Gills may also be frozen and then thawed and scraped (Wong et al. 2008). In ecological studies where the aim is to obtain accurate infection statistic data, all parasites present needs to be recovered. To evaluate suitability of the method employed the following approach may be considered. Firstly, examine a gill arch using a traditional / standard “paging” method (i.e. using pinsette and / or needles to “page” through filaments) and remove all parasites found. Following this, scrape the same gill unto microscope slides, examine the scraping and remove any parasites found. Finally place the same gill in a suitable closed container (e.g. sample bottle with lid) in water, shake vigorously and examine the resulting content in the container (once again removing parasites observed, if any). This may be repeated for several gill arches in different combinations.

Quantifying the sequential ratio of worms recovered from each respective method should provide adequate data to comment on the most suitable / preferential methodology.

The second suggestion pertaining to methodology is that future studies could focus on the evaluation of different stains and staining methods that would possibly allow for more comprehensive descriptions (i.e. examination structures apart from the obvious, diagnostic sclerotized structures used in this thesis).

For many of the parasites recovered (e.g. Q. aegypticus), apparent morphological variation in haptoral sclerite shape and size proved to be a practical challenge in terms of identification and description. A paramount need to be addressed in future studies is more comprehensive quantification of the variation observed. This could be done by examining and measuring more specimens (i.e. increase the examined population size) and then apply comprehensive qualitative morphometric analyses (e.g. PCA and Cluster Analysis) using the traditional point to point measurements. This could possibly also be combined with the use of qualitative descriptors of anchor shape and / or area. Such data may potentially be used to identify recurring trends in morphological variation (if at all present) and to identify and quantify “morphological groupings” within the Vaal Dam parasite community.

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This may allow the researcher to comment on questions such as: Are morphological plasticity “random” and probably caused by mechanical damage during fixing /mounting, or might other factors contribute to more predictable patterns of morphological plasticity? This could be combined with seasonal examinations to investigate the possible effects of temperature. In the case of the apparently closely related Dactylogyrus spp. from Labeo spp., examination of other host species in the same genus would also allow more accurate quantification of the degree of variation observed within a phylogenetic framework. Quantification of morphological variation may in future also prove to be an interesting tool for examining possible hybridization (e.g. the occurrence of the “Dactylogyrus varicorhini Bychowsky, 1958 type” specimens on Labeobarbus hosts). In this regard additional measuring methodology (e.g. Appendix B) should be further investigated for the different parasite species and the use thereof evaluated using more sophisticated statistical analyses (e.g. principal component analysis and cluster analysis).

Challenges faced when employing traditional morphometric analyses inevitably results in consideration of molecular alternatives. Molecular analyses fell outside the scope of the current study (i.e. collection of baseline data) but should most definitely be considered in future studies. This is particularly relevant to the Gyrodactylus sp. collected from C. gariepinus, but should also be investigated for the dactylogyrids collected from both Labeo spp. and Labeobarbus spp. Molecular techniques may also be employed to evaluate if the “morphological variants” or “morphological groupings” for parasites such as Q. aegypticus do in fact belong to a single species. This can then, for example, be compared to molecular results for Quadriacanthus agnebiensis N'Douba, Lambert and Euzet, 1999, a species showing striking resemblances with Q. aegypticus with regard to MCO structure.

Experimental approaches could be incorporated in future studies to validate baseline data results in terms of degree of host specificity observed (and hence also the possibility of host switching). Due to mixed species infections encountered this may prove to be a challenge. However, exposing uninfected (e.g. by anthelmintic treatment) fish of different species (e.g. two different Labeo spp.) to infected fish of a single species, may well allow evaluation of the degree of host specificity and/or host preference.

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Such an experimental approach would also allow for studies on egg and larvae morphology to be conducted, as information on differences between species or forms appear to be lacking.

Experimental studies may also be designed to investigate if seasonal trends may be species specific. For example, mixed populations of naturally infected L. capensis and L. umbratus hosts could be maintained. One option would be to alter the temperature in the same tank over time (to simulate seasons) and to perform a necropsy (parasite recovery) on sub-samples of the population at different time points. A number of replicate experiments may be run in a number of tanks (i.e. simulating a series of “seasons” within each tank). Aggregation is an almost universal phenomenon in parasites (Krasnov, Stanko, Miklisova and Morand 2006) and may be a confounding factor with such an experimental approach, even when attempting to standardize abiotic conditions for all groups. The reason for this in the scenario under discussion would be differences in genotypes of individual hosts. The researcher should thus also attempt to mitigate such effects by, for example, maintaining and using a single laboratory fish strain of similar genotype.

Furthermore a biostatistician should also be consulted to evaluate the degree of aggregation in order to increase the number of replicates if necessary, so that a statistical model can be employed on the data with consideration of / to correct for the possible effects of observed aggregation. Another option would be to keep several replicate groups in tanks at different temperatures (i.e. simulate only a single “season” in each tank). Once again a number of replicates would be required for each “season” to allow statistical evaluation. Experimental studies as suggested above may also be used as an opportunity to examine and describe development of the parasite (including sclerite development) at different time points on different hosts. It would also allow experiments evaluating how the disease status of fish affects subsequent monogenean infections and vice versa (e.g. protozoan gill parasites in C. idella fry, Molnár 1971a). Histopathological studies to describe microscopic changes (e.g. Molnár 1972) should form part of such an approach. Furthermore the effects of host behaviour on probability of infection (and subsequent behavioural changes following infection) could be experimentally investigated. Molnár (1971a) showed that oncomiracidia preferred to localize near the bottom as they would often descent to rest.

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Fish could thus be maintained at different depths (e.g. separated by mesh of appropriate size to allow eggs and oncomiracidia to pass through) to evaluate to what degree depth stratification may affect infection statistics.

Such an approach may further contribute to the experimental work on epizootiology (with focus on temperature, host age and previous exposure to parasite effects) performed by Molnár (1971b). Molnár (1971b), however, do mention behavioural changes as well. Fry were normally found to gather at the bottom of the pond, but infected fry were found more often in the water surface layer (especially near the sides of the pond). Further experimental studies could thus focus on depth stratification effects both in terms of infection rates but also host behavioural changes related to infection.

More intensive sampling at the same locality is needed to clarify a number of findings. During the first (winter) survey a single Diplozoon sp. specimen was collected from L. capensis. During the second (summer) survey more parasites (n=11) was collected, but once again only from a single host specimen. Seddon (2004) reported Diplozoon sp. only from L. capensis from the same locality (Vaal River system).

Additional sampling is required to confirm Diplozoon sp. infection on L. capensis and to determine if this is in fact a different species to the parasite of the same genus infecting L. umbratus (i.e. same species exhibiting host preference or two separate species all together).

Future studies should also sample additional localities, both within the same river system, but also in other river systems. It would be beneficial to have additional (i.e. for localities other than the Vaal Dam) baseline data available on the monogenean parasite fauna of endemic species for which intensive aquaculture systems may still be developed. This would allow risk analysis and pro-active planning to avoid or manage possible threats that may be encountered under intensive culture conditions. To truly and unambiguously identify a parasite as being a host generalist or host specialist, the researcher would need to know much about the parasites in all potential hosts. Relative sampling intensity, however, remains a problem in discussions on host specificity (Bakke et al. 2002).

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Future studies should thus compare monogenean parasite species associated to a certain host species in a given area (Vaal River system) as a whole (i.e. with consideration of habitat types within the same system). Such studies will also allow evaluation of relationships between levels of aggregation and host specificity (e.g. Krasnov et al. 2006).

This study employed two sampling occasions representative of two different seasons. For all species examined additional seasonal sampling and comparison is required. Temperature may affect sclerite morphology, yet, as a large degree of variation was observed in parasites collected during a single season in the current study, a seasonal or temperature effect or correlation with sclerite shape or size is deemed unlikely. This needs to be verified in future studies employing more comprehensive morphometric analyses as discussed previously.

This could be investigated in conjunction with some of the experimental approaches already suggested. Furthermore no (e.g. C. gariepinus, C. idella and C. carpio) or fewer (Labeobarbus spp.) specimens were collected from some host species during the winter survey. Future studies should attempt more intensive winter and spring collections to allow comment on possible seasonal effects in terms of infection statistics, site preference and host biology / physiology (e.g. effect of spawning).

If performed at different localities, such data will also have biological environmental monitoring applications. This could be compared to results obtained using the less cumbersome existing parasite index (associated with the fish health assessment index, e,g, Crafford and Avenant-Oldewage 2009) in future studies.

Host effects on monogenean parasite infections also require further study. In the current study small sample size and little variation between size classes confound unequivocal generalizations with regard to host size effects. Future studies should include more size classes – this could possibly be combined with experimental infection studies as proposed above. During future studies it may also be interesting to perform age determination on a sub-set of fish for each species from the Vaal Dam during sampling for parasite recovery. This may provide the opportunity to further assess and compare the use of length and age data in parasite infection comparisons.

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Furthermore infection statistics may possibly differ with regards to host gender during early spring and summer (due to physiological changes in especially females relating to spawning activity) compared to the winter and mid-summer surveys in the current study. This aspect should be investigated in future studies and links strongly to the proposed seasonal studies already proposed.

The aim of this project was to collect baseline data on monogenean parasite fauna from a locality previously not intensively investigated with regard to this specific parasite group.

It is believed that, through this journey from aim to abscission, this objective was reached. However, reaching the top of the hill allows surveillance of the many hills and mountains that still lie ahead.

It is my wish that others shall tread these paths, as yet less travelled, to construct their own journeys from aim to abscission...

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CHAPTER 14

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APPENDIX A

Appendix A: Notes on how apparent morphometric variation and slight rotation of Dogielius Bychowsky, 1936 (originally denoted “Species E” but later described as Dogielius intorquens Crafford, Luus-Powell and Avenant- Oldewage, 2012 anchor can affect point to point measurements

A1. Introduction

Identification of species (and the methodology followed to derive comparable characteristics) is dependent on review of previous descriptions. For this reason not only results but also methods should be reflected accurately and in suitable detail. While reviewing literature on standard measurements employed for Dogielius Bychowsky, 1936 specimens, a dilemma presented itself. The anchor shape, more specifically the hook bend, of the Dogielius sp. encountered was not as distinct as that illustrated in the published examples. As a result the exact point of measurement was difficult to determine. While an illustration was indeed available depicting standard methodology, the actual point to point measurements employed were to a large extend still subjective for two reasons: 1) Slight rotation of the anchor could influence points used and hence resulting measurements; 2) Slight variations in anchor shape between species may also confound standard points of measurements as illustrated in the literature to some extend. This appendix aim to elucidate the difficulties encountered and describe the procedure followed to standardize measurements used in this study.

When this parasite was first encountered it was simply identified as “Dogielius sp. E”. Later it was described (Crafford, Luus-Powell and Avenant-Oldewage 2012) as Dogielius intorquens n. sp. (see Chapter 5). However, as this discussion is also applicable to other Dogielius spp., subsequent discussions shall use D. intorquens n. sp. as example yet refer to Dogielius spp. in general discussions.

A2. Materials and methods

Guegan and Lambert (1991) was consulted with regard to standard point to point measurements when examining Dogielius spp. The publication consulted provides the illustration shown in Figure A1 (C) and Figure A3 (C).

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With Chapter 3 in mind, where only the standardized measurements as used in subsequent publications are depicted, Figure A3 shows all measurements discussed here for comparison.

It was soon found that fairly slight changes in anchor orientation can effect measurement (especially e, the tip length) as depicted in Figure A2. The reason for this is the fact that Dogielius spp. anchors exhibit a much more gradual bend compared to the much more pronounced bend of the anchor in Dactylogyrus spp. Diesing, 1850 (see Figure A3). This pronounced bend, together with the elongated inner root, makes it much easier to gauge the “highest” and “lowest” (i.e. most distal and proximal) parts of the anchor (i.e. inner root tip and anchor bend edge) in Dactylogyrus spp.

This orientation-induced variation led to the method depicted in Figure A1 (A), in which the “tip length” is restricted to only the very tiny terminal hook-like structure. As this structure has a definite bend (more comparable to that of Dactylogyrus spp.) it is easier to standardize a measuring point.

Figure A1 (B) shows the measurements employed based on the standard proposed measurement illustrated in Figure A1 (C). In order to standardize orientation (and hence also subsequent measurements), the anchor was orientated so that, if the top edges of the anchor roots were joined, the resulting line would lie at an angle of 180 degrees. A second line, parallel to the first, was then moved down until it touched the bottom line of the anchor. The centre of this area was the considered to be the “anchor bend measuring” point.

In order to comment on the usefulness of the optional / additional hook terminal approach versus the standard approach, results were compared with reference to the ability to distinguish between the two forms found on the respective hosts (two separate forms were described exhibiting a clear host preference – see Chapter 5). For each set of measurements a Mann Whitney U test was performed (Statistics Open For All open-source statistics, analysis and reporting package software, version 1.1.1) to see if the measurements differed significantly between parasites found on the different host species. Only average measurements and Mann Whitney U test p-values were summarized in tables (Tables A1 and A2).

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Figure A1 An illustration depicting possible point to point measurements for identification and description of species from the genera Dogielius Bychowsky, 1936: A = Terminal hook tip (i.e. optional / additional) method examined in this project in an attempt to more clearly define a standard point from where to measure length e (tip length); B = Standard method employed (as adapted from C) to allow comparison with previous publications; C) suggested standard method from literature as illustrated by Guegan and Lambert (1991).

Figure A2 An illustration depicting how slight changes in the orientation (A, B and C) of the same Dogielius Bychowsky, 1936 anchor can affect measurements: a = anchor total length; b = anchor shaft length; e = length of tip / point

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Figure A3 A comparative illustration depicting standard (A,C and D) and experimental (B) measurements employed for identification and description of species from the genera Dactylogyrus Diesing, 1850 and Dogielius Bychowsky, 1936 (from Labeo hosts - adopted from Guégan and Lambert 1991; Musilová, Řehulková and Gelnar 2009): A = Dactylogyrus spp. anchor; B, C and D = Dogielius spp. anchor; E = Dactylogyrus spp. transverse bar; F = Marginal hook (applicable to both Dactylogyrus spp. and Dogielius spp.); G = Dogielius spp. transverse bar; H = Dactylogyrus spp. copulatory organ; I = Dogielius spp. copulatory organ; a = anchor total length; b = anchor shaft length; c = Length of outer root; d = Length of inner root; e = length of tip / point; f = length of transverse bar; g = width of transverse bar; h = Marginal hook total length; i accessory piece length; j = tube trace length.

A3. Results

A3.1. Terminal hook tip method

A summary of measurements obtained (arithmetic average values) using this method is summarized in Table A1.

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Table A1 “Terminal hook tip method” measurements (arithmetic average values): a = anchor total length; b = anchor shaft length; e = length of tip / point. Mann Whitney U test p- values indicate whether measurements differed significantly between parasite forms collected from different host fish species (p-values < 0.05 are indicated in bold text and represents a statistically significant difference).

p-value Number of Average in Standard (Mann Host Variable measurements micrometers deviation Whitney U test) Lc 11 32.8 3.1 a < 0.001 Lu 11 39.9 1.9 Lc 11 45.5 3.8 b < 0.001 Lu 11 55.0 2.3 Lc 11 1.8 0.3 e 0.037 Lu 11 2.0 0.2

A3.2 Adapted standard method:

A summary of measurements obtained (arithmetic average values) using this method is summarized in Table A2.

A4. Discussion:

A4.1. Terminal hook tip method (Fig. A1 (A))

This method was found not to be preferable for two reasons.

Firstly the terminal hook tip is very short and as such it is very difficult to accurately measure the length. Despite the difficulty experienced, the terminal hook tip length was still found to differ significantly between the two parasite forms from the two different host fish species. No general deduction can be made from the small pilot study sample size. It may, however, be worthwhile to perform a more elaborate morphometric analysis (e.g. PCA combined with cluster analysis) on a larger dataset to determine if this variable could actually help distinguish between the two parasite forms. As differences between forms in both anchor total and shaft length are highly significant, the contribution of this third variable in terms of form discrimination would most probably be negligible.

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Table A2 Standard method measurements (arithmetic average values): a = anchor total length; b = anchor shaft length; e = length of tip / point; Lc = Labeo capensis (Smith, 1841); Lu = Labeo umbratus (Smith, 1841). Mann Whitney U test p-values indicate whether measurements differed significantly between parasite forms collected from different host fish species (p-values < 0.05 are indicated in bold text and represents a statistically significant difference).

p-value Number of Average in Standard (Mann Host Variable measurements micrometers deviation Whitney U test) Lc 11 26.6 2.1 a < 0.001 Lu 11 31.8 1.6 Lc 11 33.3 2.5 b < 0.001 Lu 11 40.6 2.4 Lc 11 22.6 3.7 e 0.669 Lu 11 23.6 2.5

Secondly, the measurements obtained are not comparable to previous measurements recorded in the literature. This confounds any scientific attempt at comparison for the purpose of parasite identification / evaluation of morphometric plasticity.

A4.2. Adapted standard method (Fig. A1 (B))

This method, based on the standard method (Fig. A1 (A)) gleaned from Guegan and Lambert (1991), was found to be adequate as it at least allowed comparison with previously published results. One can, however, only speculate as to how rotation of Dogielius spp. anchors have influenced measurements in the past, once again stressing the importance of adequate drawings.

A4.3. Comments on variation in anchor shape / morphometric plasticity

In the preceding section the importance of accurate drawings was mentioned. During the course of this project and submission of papers resulting from it, reviewers instructed that drawings of anchors should show a mirror image in terms of size and shape. In reality this is not always the case (e.g. Figures A4 and A5). Admittedly such variation may also be the result of various confounding factors, such as damage to anchors during the fixing or mounting process.

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It thus makes sense to examine several specimens and reflect the most common situation in descriptive drawings. While variation in anchor shape will also be reflected in measurement ranges, the author is of the opinion that such variation should also be better illustrated and discussed in publications. The reviewers, however, argued that such illustrated variation could cause confusion, as shape differences may indicate different species. As mentioned such variation was, however, often observed in a single specimen.

Figure A4 Initial drawing showing variation in anchors observed – such variation was even observed in Dogielius Bychowsky, 1936 anchors from the same specimen (also see amended drawing submitted for publication in Figure 3A2.5).

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Figure A5 Amended drawing with anchors showing mirror images as requested by reviewers (also see initial drawing showing observed variation in Figure 3A2.4).

A5. Conclusions

In order to retain repeatability for research that are to follow (and hence also comparability with previous publications), well-standardized method descriptions are required. While a picture may well replace a thousand words, simple instructions on standardizing measurement methodology (e.g. in terms of anchor orientation where a distinct point of measurement is lacking) may well still be required. When measuring structures it is thus of paramount importance to standardize such methods across all measuring points if and where standardized methodology is lacking.

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APPENDIX B

Appendix B: Notes on the application of additional point to point measurements, adapted from Gyrodactylus von Nordmann, 1832, to Dactylogyrus Diesing, 1850 and Dogielius Bychowsky, 1936

B1. Introduction:

Monogenean species are traditionally described based on structure of sclerotized structures (e.g. haptor and copulatory organ). In recent years molecular approaches have also become an invaluable additional tool, especially with regard to the larger monogeneans (e.g. Gyrodactylus von Nordmann, 1832). Yet even with this approach there appears to be a degree of overlap between closely related Gyrodactylus spp., necessitating even more involved and comprehensive measuring techniques and additional variables to be measured.

As haptor structures often differ widely between different genera, a set of standard measurements have evolved for each respective genus over time. For both Dactylogyrus Diesing, 1850 and Dogielius Bychowsky, 1936 two apparently closely related forms (in both instances described as a single species) exhibiting distinct host preference was found in the current study (see Chapters 4 and 5).

The Dogielius sp. was initially designated “Dogielius sp. E” but later described (Crafford, Luus-Powell and Avenant-Oldewage, 2012) as Dogielius intorquens n. sp.

The Dactylogyrus spp. were initially considered to possibly be a single species representing two forms, the latter designated as “forma capensis” (FC,) (i.e. predominantly found on Labeo capensis (Smith, 1841)) and “forma umbratus” (FU) (i.e. predominantly found on Labeo umbratus (Smith, 1841)) respectively. Following further examination (current appendix as well as Chapter 4) it was, however, decided that the forms represent two species (designated “Dactylogyrus sp. A” and “Dactylogyrus sp. B” respectively). Both were subsequently describe (Crafford et al. 2012) as two new species namely Dactylogyrus iwani n. sp. (i.e. previously FC / “Dactylogyrus sp. A”) and Dactylogyrus larindae n. sp. (i.e. previously FC / “Dactylogyrus sp. B”).

The current appendix report on initial investigations on attempting to differentiate between the apparently closely related forms (see previous

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paragraph). At the time this appendix was compiled, species / form status was uncertain and as a result subsequent discussions shall refer to the terminology employed at that stage of the project (e.g. “Dactylogyrus spp. and Dogielius spp. forms”, “FC”, “FU” etc.).

During these initial investigations it was decided to, apart from standard measurement methodology for these parasite genera (Dactylogyrus spp. and Dogielius spp.), also apply additional measurements (as developed for Gyrodactylus spp.) to the standard measurement “repertoire” to see if these additional measurements would result in better discrimination between the forms.

B2. Materials and methods:

Standard measuring methodology is described in the main body of Chapter 3. Adapted methodology for use when measuring Dogielius spp. has been discussed in Appendix 3-2.

Standard measurements as well as additional measurements for the two parasite forms (from each respective monogenean genus, i.e. Dactylogyrus and Dogielius) are depicted in Figure 3A3.1 and summarized in Table 3A3.1.

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Figure B1 An illustration depicting measurements (standard as adopted from Guégan and Lambert 1991; Musilová Řehulková and Gelnar 2009, as well as additional measurements for Gyrodactylus von Nordmann, 1832 adopted from Shinn Harris, Cable, Bakke, Paladini and Bron 2010). For description of measurement codes and explanation as to which measurements are considered standard and which are considered additional, please refer to Table 3A3.1.

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Table B1 Summary of standard (shaded) and additional measurements employed to measure sclerotized structures of Dactylogyrus Diesing, 1850 and Dogielius Bychowsky, 1936 forms.

Measurement description Measurement code Dactylogyrus Diesing, 1850 Dogielius Bychowsky, 1936

a Anchor total length (i.e. overall hook length)

b Anchor shaft length Length of shaft and inner root

c Length of outer root Width of root

d Length of inner root Not applicable

e Length of tip / point

f Anchor aperture Outer root to anchor shaft aperture

g Distal shaft width

h Proximal shaft width Shaft width perpendicular to outer root end

Ø Anchor aperture angle

∞ Anchor point curve angle Not applicable

B Inner anchor aperture angle Not applicable

i Length of transverse bar

j Width of transverse bar

k Marginal hook total length

l Marginal hook root length

m Marginal hook shaft length

n Marginal hook tip length

o Penis bulb width Not applicable

p Accessory piece length Copulatory apparatus length

q Not applicable Accessory piece width

ct Penis tube trace length Not applicable

L Body length

W Body width

For each set of measurements (within each respective genus) a Mann Whitney U test was performed (Statistics Open For All [SOFA] open-source statistics, analysis and reporting package software, version 1.1.1) to see if the measurements differed significantly between parasites found on the different host species.

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This was interpreted as a measure of the variable’s ability to distinguish / discriminate between forms. Only average measurements and Mann Whitney U test p-values were summarized in tables (Tables 3A3.2 to 3A3.4). Summary results of analysis (i.e. a summary of which variables differed significantly between forms) is provided in Table 3A3.5.

B3. Results:

For both the Dactylogyrus sp. and Dogielius sp. standard anchor (with the exception of tip length) and transverse bar measurements differed significantly between forms (Table 3A3.5). Other variables that shows promise to be of discriminatory use are anchor aperture / outer root to anchor shaft aperture (“f”), proximal shaft width / shaft width perpendicular to outer root end (“h”) and anchor aperture angle (Ø) (Table 3A3.5).

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Table B2 Summary of standard (shaded) and additional measurements of Dactylogyrus Diesing, 1850 forms (arithmetic average values). Mann Whitney U test p-values indicate whether measurements differed significantly between the two parasite forms (p-values < 0.05 are indicated in bold text and represents a statistically significant difference).

Number of Average in Standard p-value (Mann Parasite form Variable measurements micrometers deviation Whitney U test) Fc 15 42.0 2.4 a < 0.001 Fu 27 33.8 2.4

Fc 14 27.2 1.7 b < 0.001 Fu 27 23.4 2.1

Fc 14 1.5 0.2 c < 0.001 Fu 26 1.2 0.4

Fc 14 19.0 1.0 d < 0.001 Fu 27 13.2 1.6

Fc 14 10.2 1.0 e 0.606 Fu 27 10.3 1.5

Fc 14 22.4 1.4 f < 0.001 Fu 27 18.2 2.1

Fc 14 1.7 0.3 g 0.868 Fu 27 1.7 0.3

Fc 14 7.1 0.6 h 0.036 Fu 27 6.5 0.8

Fc 14 63.3 3.3 Ø 0.012 Fu 27 58.7 5.9

Fc 14 52.3 4.3 B 0.255 Fu 27 49.5 6.1

Fc 14 11.0 3.7 Ø - B 0.007 Fu 27 9.2 2.3 Fc = “Forma capensis” (later described as Dactylogyrus iwani Crafford, Luus-Powell and Avenant-Oldewage, 2012); Fu = “Forma umbratus” (later described as Dactylogyrus larindae Crafford, Luus-Powell and Avenant- Oldewage, 2012); a = anchor total length; b = anchor shaft length; c = length of outer root; d = length of inner root; e = length of tip / point; f = anchor aperture; g = distal shaft width; h = proximal shaft width; Ø = anchor aperture angle; B = inner anchor aperture angle. ө = anchor aperture angle; B = inner anchor aperture angle.

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Table B2 (continued): Summary of standard (shaded) and additional measurements of Dactylogyrus Diesing, 1850 forms (arithmetic average values). Mann Whitney U test p-values indicate whether measurements differed significantly between the two parasite forms (p-values < 0.05 are indicated in bold text and represents a statistically significant difference).

Number of Average in Standard p-value (Mann Parasite form Variable measurements micrometers deviation Whitney U test) Fc 14 5.7 2.2 ∞ 0.860 Fu 27 6.2 3.0

Fc 14 18.4 1.6 i < 0.001 Fu 26 15.5 1.9

Fc 14 3.4 0.8 j < 0.001 Fu 26 2.4 0.5

Fc 12 15.8 1.8 ki 0.564 Fu 16 17.1 2.5

Fc 11 15.2 2.2 kii 0.979 Fu 15 15.8 1.4

Fc 11 17.2 1.6 kiii 0.279 Fu 15 16.9 1.5

Fc 12 16.6 2.0 kiv 0.808 Fu 15 16.7 2.1

Fc 14 16.0 1.4 kv 0.803 Fu 16 16.9 3.0

Fc 14 17.0 2.1 kvi 0.756 Fu 16 17.4 2.1

Fc 13 16.6 1.1 kvii 0.073 Fu 16 18.0 1.9

Fc = “Forma capensis” (later described as D. iwani; Fu = “Forma umbratus” (later described as D. larindae; ∞ = anchor point curve angle; i = length of transverse bar; j = width of tranverse bar; ki to kvii = marginal hook total length (respective averages for pairs 1 to 7).

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Table B2 (continued): Summary of additional measurements of Dactylogyrus Diesing, 1850 forms (arithmetic average values). Mann Whitney U test p-values indicate whether measurements differed significantly between the two parasite forms (p-values < 0.05 are indicated in bold text and represents a statistically significant difference).

Number of Average in Standard p-value (Mann Parasite form Variable measurements micrometers deviation Whitney U test) Fc 12 5.6 0.9 li 0.578 Fu 16 5.9 1.6

Fc 11 5.5 1.2 lii 0.067 Fu 15 5.0 1.1

Fc 11 6.1 1.5 liii 0.643 Fu 15 6.1 1.1

Fc 13 5.8 0.9 liv 0.913 Fu 16 6.0 1.3

Fc 14 5.8 1.4 lv 0.308 Fu 16 5.7 1.9

Fc 14 5.7 1.1 lvi 0.519 Fu 16 6.3 1.7

Fc 13 6.1 1.0 lvii 0.827 Fu 16 6.6 1.6

Fc 12 6.1 0.8 mi 0.188 Fu 16 6.8 0.7

Fc 11 5.5 1.5 mii 0.025 Fu 15 6.5 0.7

Fc 11 6.3 0.5 miii 0.421 Fu 15 6.5 0.8 Fc = “Forma capensis” (later described as D. iwani; Fu = “Forma umbratus” (later described as D. larindae; li to lvii = marginal hook root length (respective averages for pairs 1 to 7); mi to miii = marginal hook shaft length (respective averages for pairs 1 to 3);

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Number of Average in Standard p-value (Mann Parasite form Variable measurements micrometers deviation Whitney U test) Fc 13 6.1 0.6 miv 0.948 Fu 16 6.2 0.8

Fc 14 6.0 0.6 mv 0.269 Fu 16 6.6 0.9

Fc 14 6.7 1.3 mvi 0.467 Fu 16 6.6 1.0

Fc 13 6.1 0.4 mvii 0.013 Fu 16 6.8 0.6

Fc 12 4.2 0.7 ni 0.799 Fu 16 4.4 0.7

Fc 11 4.3 0.4 nii 0.622 Fu 15 4.3 0.6

Fc 11 4.5 0.6 niii 0.570 Fu 15 4.3 0.6

Fc 12 4.7 0.9 niv 0.715 Fu 15 4.4 0.6

Fc 14 4.2 0.6 nv 0.379 Fu 16 4.4 0.7

Fc 14 4.6 0.6 nvi 0.546 Fu 16 4.2 0.9

Fc = “Forma capensis” (later described as D. iwani); Fu = “Forma umbratus” (later described as D. larindae); miv to mvii = marginal hook shaft length (respective averages for pairs 4 to 7); ni to nvi = marginal hook tip length (respective averages for pairs 1 to 6);

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Number of Average in Standard p-value (Mann Parasite form Variable measurements micrometers deviation Whitney U test) Fc 13 4.4 0.4 nvii 0.156 Fu 16 4.6 0.6

Fc 10 5.3 0.6 o 0.106 Fu 19 5.0 0.6

Fc 10 13.4 1.4 p 0.780 Fu 13 13.3 1.5

Fc 10 4.2 0.8 q 0.215 Fu 13 4.4 0.7

Fc 12 22.3 4.4 ct 0.013 Fu 16 27.6 5.2

Fc 13 222.9 64.0 L 0.977 Fu 11 218.8 38.4

Fc 13 45.8 10.7 W 0.083 Fu 10 39.2 9.7

Fc = “Forma capensis” (later described as D. iwani); Fu = “Forma umbratus” (later described as D. larindae; nvii = marginal hook tip length (average for pair 7); o = penis bulb width; p = accessory piece length; q = accessory piece width; ct = penis tube trace length; L = body length; W = body width

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Table B3 Summary of adapted (shaded variables) and additional measurements of Dogielius Bychowsky, 1936 forms* (arithmetic average values). Mann Whitney U test p-values indicate whether measurements differed significantly between parasite forms collected from different host fish species (p-values < 0.05 are indicated in bold text and represents a statistically significant difference).

Number of Average in Standard p-value (Mann Fish species Variable measurements micrometers deviation Whitney U test) Lc 11 32.8 3.1 a < 0.001 Lu 11 39.9 1.9

Lc 11 45.5 3.8 b < 0.001 Lu 11 55 2.3

Lc 11 14.4 1.6 c 0.003 Lu 11 16.8 1.3

Lc 11 1.8 0.3 e 0.037 Lu 11 2.0 0.2

Lc 11 23.7 1.4 f 0.002 Lu 11 26.8 2.1

Lc 11 0.8 0.2 g 0.162 Lu 11 1.0 0.2

Lc 11 2.4 0.4 h 0.006 Lu 11 3.1 0.6

Lc 11 73.2 6.8 ө 0.033 Lu 11 78 3.4

Lc 11 44.6 7.9 B 0.974 Lu 11 44.7 10.5

Lc 11 28.6 9.0 ө - B 0.324 Lu 11 33.3 13.0

* = Later described as a single species (Dogielius intorquens Crafford, Luus-Powell and Avenant-Oldewage, 2012) exhibiting two forms; Lc = Labeo capensis; Lu = Labeo umbratus; a = anchor total length; b = anchor shaft length; e = length of tip / point; f = anchor aperture; g = distal shaft width; h = proximal shaft width; ө = anchor aperture angle; B = inner anchor aperture angle.

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Table B3 (continued): Summary of standard (shaded) and additional measurements of Dogielius Bychowsky, 1936 forms* (arithmetic average values). Mann Whitney U test p-values indicate whether measurements differed significantly between parasite forms collected from different host fish species (p-values < 0.05 are indicated in bold text and represents a statistically significant difference).

p-value Number of Average in Standard (Mann Parasite form Variable measurements micrometers deviation Whitney U test) Lc 11 41.0 4.4 i < 0.001 Lu 11 48.9 2.5 Lc 11 4.8 0.8 j < 0.001 Lu 11 6.2 0.7 Lc 9 15.1 3.0 ki 0.230 Lu 5 16.8 1.7 Lc 2 16.5 3.7 kii 1.000 Lu 2 16.3 5.3 Lc 5 16.8 3.3 kiii 0.041 Lu 7 19.9 1.0 Lc 7 16.8 2.8 kiv 0.431 Lu 6 18.3 1.5 Lc 5 17.8 3.5 kv 0.927 Lu 6 18.0 3.3 Lc 9 19.8 2.3 kvi 0.052 Lu 9 18.1 1.2 Lc 10 18.4 1.4 kvii 0.120 Lu 10 19.5 1.6 * = Later described as a single species (D. intorquens) exhibiting two forms; Lc = Labeo capensis; Lu = Labeo umbratus; i = length of transverse bar; j = width of tranverse bar; ki to kvii = marginal hook total length (respective averages for pairs 1 to 7).

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Table B3 (continued): Summary of additional measurements of Dogielius Bychowsky, 1936 forms* (arithmetic average values). Mann Whitney U test p-values indicate whether measurements differed significantly between parasite forms collected from different host fish species (p-values < 0.05 are indicated in bold text and represents a statistically significant difference).

Number of Average in Standard p-value (Mann Parasite form Variable measurements micrometers deviation Whitney U test) Lc 9 3.8 1.5 li 0.693 Lu 6 3.7 1.7

Lc 2 4.1 0.6 lii 1.000 Lu 2 3.0 2.6

Lc 5 4.6 0.5 liii 0.463 Lu 7 4.3 1.7

Lc 7 3.8 1.7 liv 0.315 Lu 7 4.6 0.6

Lc 6 3.9 1.8 lv 0.489 Lu 9 4.6 0.6

Lc 9 4.6 1.7 lvi 0.616 Lu 10 4.7 0.7

Lc 10 4.2 1.5 lvii 0.127 Lu 10 5.0 0.7

Lc 7 7.5 2.4 mi 0.283 Lu 6 8.6 1.8

Lc 2 8.4 3.5 mii 0.691 Lu 2 8.1 4.0

Lc 5 7.8 2.6 miii 0.116 Lu 7 10.2 1.9 * = Later described as a single species (D. intorquens) exhibiting two forms; Lc = Labeo capensis; Lu = Labeo umbratus; li to lvii = marginal hook root length (respective averages for pairs 1 to 7); mi to miii = marginal hook shaft length (respective averages for pairs 1 to 3);

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Number of Average in Standard p-value (Mann Parasite form Variable measurements micrometers deviation Whitney U test) Lc 6 7.7 2.6 miv 0.388 Lu 8 9.0 1.0

Lc 5 8.3 2.7 mv 0.558 Lu 8 9.1 2.1

Lc 7 9.2 1.6 mvi 0.590 Lu 10 8.7 1.2

Lc 9 8.9 1.1 mvii 0.232 Lu 10 9.5 1.6

Lc 7 3.9 0.9 ni 0.351 Lu 6 4.1 0.5

Lc 2 4.0 0.8 nii 0.691 Lu 2 3.6 1.3

Lc 5 4.5 0.8 niii 0.221 Lu 7 5.1 0.9

Lc 6 4.4 0.8 niv 0.280 Lu 7 5.0 0.9

Lc 5 4.8 0.6 nv 0.883 Lu 8 4.9 1.3

Lc 7 5.2 0.6 nvi 0.141 Lu 10 4.8 0.8

* = Later described as a single species (D. intorquens) exhibiting two forms; Lc = Labeo capensis; Lu = Labeo umbratus; miv to mvii = marginal hook shaft length (respective averages for pairs 4 to 7); ni to nvi = marginal hook tip length (respective averages for pairs 1 to 6).

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Number of Average in Standard p-value (Mann Parasite form Variable measurements micrometers deviation Whitney U test) Lc 9 5.0 0.4 nvii 0.620 Lu 10 5.1 0.7

Lc 7 4.2 0.4 o 0.869 Lu 5 4.2 1.0

Lc 7 24.7 4.1 p 0.223 Lu 9 26.9 2.9

Lc 8 5.2 2.0 q 0.767 Lu 5 4.9 1.0

Lc 11 231.4 33.3 L 0.002 Lu 10 285.2 38.0

Lc 10 63.1 13.1 W 0.405 Lu 10 68.7 15.4

* = Later described as a single species (D. intorquens) exhibiting two forms; Lc = Labeo capensis; Lu = Labeo umbratus; nvii = marginal hook tip length (average for pair 7); o = penis bulb width; p = copulatory organ length; q = accessory piece width; L = body length; W = body width.

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Table B4 Summary of standard measurements (for selected anchor variables) of Dogielius Bychowsky, 1936 forms* (arithmetic average values). Mann Whitney U test p-values indicate whether measurements differed significantly between parasite forms collected from different host fish species (p-values < 0.05 are indicated in bold text and represents a statistically significant difference).

Number of Average in Standard p-value (Mann Fish species Variable measurements micrometers deviation Whitney U test) Lc 11 26.6 2.1 a <0.001 Lu 11 31.8 1.6

Lc 11 33.3 2.5 b <0.001 Lu 11 40.6 2.4

Lc 11 22.6 3.7 e 0.669 Lu 11 23.6 2.5

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Table B5 Summary of Mann-Whitney U-test comparisons of standard (shaded) and additional measurements employed to measure sclerotized structures of Dactylogyrus Diesing, 1850 and Dogielius Bychowsky, 1936 forms.

Significant (i.e. p < 0.05) Measurement description difference between forms? Code (Dactylogyrus Diesing, 1850 / Dogielius Bychowsky, 1936) Dact. spp. Dog. spp.

a Anchor total length (i.e. overall hook length) Yes Yes *

b Anchor shaft length / Length of shaft and inner root Yes Yes *

c Length of outer root / Width of root Yes Yes

d Length of inner root / Not applicable Yes N/A

e Length of tip No Yes **

f Anchor aperture / Outer root to anchor shaft aperture Yes Yes

g Distal shaft width No No

Proximal shaft width / Shaft width perpendicular to outer root h Yes Yes end

Ø Anchor aperture angle Yes Yes

∞ Anchor point curve angle / Not applicable No No

B Inner anchor aperture angle / Not applicable No No

i Length of transverse bar Yes Yes

j Width of transverse bar Yes Yes

Yes k Marginal hook total length No (1 out of 7 sets only)

l Marginal hook root length No No

Yes m Marginal hook shaft length (2 out of 7 sets No only)

n Marginal hook tip length No No

o Penis bulb width / Not applicable No N/A

p Accessory piece length / Copulatory apparatus length No No

ct Penis tube trace length / Not applicable Yes N/A

L Body length No Yes

W Body width No No

Dog. = Dogielius; Dact. = Dactylogyrus; N/A = Not applicable; * = For both standard and adapted measurements (see Appendix 3-2); ** = For adapted measurement only.

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B4. Discussion

The only three additional variables measured that do appear to distinguish between the two forms in both genera, are related to anchor structure. As the standard measurements employed already clearly distinguish between these forms, the application of additional measurements may be counterproductive in this case.

B5. Conclusion

For the two forms of the same species of Dactylogyrus and Dogielius respectively, as recorded from two closely related host species, this pilot study indicate that standard measurements are adequate in distinguishing between these forms. It would appear that additional measuring methodology, as adapted from that used for representatives of the genus Gyrodactylus, provides very little additional advantage with regard to discrimination between closely related forms of Dactylogyrus and Dogielius. As a result only standard methods were employed for description and identification purposes throughout the remainder of this thesis.

Future studies could use larger sample sizes and employ more sophisticated statistical analyses (e.g. principal component analysis and cluster analysis) to confirm this preliminary finding.

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APPENDIX C

Appendix C: Preliminary measurements showing the gills of Labeo capensis (Smith, 1841) to be more robust than that of Labeo umbratus (Smith, 1841) (Chapter 4 – Form versus species evaluation)

C1. Introduction

The occurrence of different forms of the same species / apparently closely related species exhibiting a host preference, raised the question as to whether differences in the gill morphology / size of the closely related host species may in fact give rise or contribute to this apparent preference. As a result a number of preliminary measurements were made and compared as described below. The aim was thus not to prove gill morphology to be a driving force for host selection in this particular case, but rather to investigate size differences and comment on the need for further more elaborate research on the topic.

C2. Materials and methods

The gills from one fish of each species were used for measurements. The same (third) gill arch was used from each fish. The height of each gill arch was measured. The length of filaments in the centre of the dorsal, medial and ventral areas of each gill arch was measured and the average calculated.

Figure C1: Figure showing preliminary gill measurement methodology: a = total gill arch height; (b+c+d)/3 = average gill filament length.

Several measurements (four from each area i.e. 12 in total) were made of lamellae length on these filaments and an average value calculated.

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To correct for fish size differences, average filament length was expressed as a ratio: Average filament length divided by total gill arch height.

In similar fashion average lamellae length were expressed as: Average lamellae length divided by total gill arch height.

C3. Results and discussion

Results are summarized in Table C1.

Table C1 Measurements from the third gill arch of two closely related host fish species.

Labeo capensis Labeo umbratus Variable (Smith, 1841) (Smith, 1841) Fish total length (cm) 37.5 41.9

Fish total weight (g) 500 650

Gill arch height (GAH) (μm) 24000.00 45000.00

Filament length (FL) dorsal (μm) 11000.00 6500.00

Filament length (FL) medial (μm) 14500.00 9500.00

Filament length (FL) ventral (μm) 12000.00 7000.00

Average filament length (AFL) (μm) 15375.00 17000.00

AFL / GAH 0.64 0.38

462.50 525.00

412.50 287.50

275.00 418.75

337.50 225.00

456.25 306.25

487.50 800.00 Lamellae length (LL) (μm) 450.00 662.50

475.00 750.00

362.50 637.50

450.00 362.50

362.50 387.50

287.50 481.75

Average lamellae length (ALL) 401.56 487.02

ALL/GAH 0.017 0.011

As can be seen from the calculated filament and gill lamellae ratios, the gills of L. capensis appear to be more robust.

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It is concluded that differences in gill morphology and size needs to be further investigated as it may play a role in monogenean host preference (i.e. may in fact be dictated by the physical dimensions of the gills and not host species identity per se). However, such morphological adaptation combined with strict host specificity (i.e. “isolation” on any particular host) may potentially drive speciation.

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