ASPECTS OF THE MORPHOLOGY AND ECOLOGY OF A DIPLOZOON SPECIES (MONOGENEA) FROM THE GILLS OF LABEO UMBRATUS IN THE VAAL DAM AND VAAL RIVER BARRAGE, GAUTENG, SOUTH AFRICA.

LAURETTE SEDDON

Supervisor: Prof. A. Avenant-Oldewage

Dissertation submitted in partial fulfilment of the requirements for the degree Master of Science in Zoology in the Faculty of Science of the Rand Afrikaans University

Johannesburg , May 2004

ABSTRACT

To date, 4 diplozoidae parasites have been described form Africa. Two belonging to the genus Diplozoon, namely D. aegyptensis and D. ghanense from sites in Northern Africa. One belonging to the genus Neodiplozoon, namely Neodiplozoon polycotyleus . The fourth monogenean is the concern of this study which aimed to determine the exact classification of the monogenean found on the gills of Labeo umbratus in the Vaal Dam and Vaal River Barrage respectively. The study was conducted over a 13-month period, with field data collections occurring every two to three months from January 1999 to February 2000. Host fishes were collected with the aid of gill nets with mesh sizes of 90, 110 and 130mm respectively. In-field measurements were taken regarding the total length, fork length, position of parasites on the gill arches and the host gender. All parasites collected were fixed in steaming AFA and stored in 70% ethanol. Laboratory measurements of whole mounts were completed with the aid of light microscope and drawing tube attachment. Staining methods employed included Boraxcarmine-iodine, Mayer’s Hematoxylin and Horen’s Trichrome. Scanning electron microscopy was used to gather information regarding the external morphology of the parasites. Both critical point drying and freeze drying preparation methods were used. For the investigation of the internal morphology serial sections of 5µm were made and stained with AZAN. Graphic reconstruction was completed using standard methods. All data collected was statistically analysed by the use of the SPSS (windows version 10.0) computer programme. Quantitative results showed greater prevalence in larger hosts, a preference for male hosts, no preference for attachment site, prevalence correlation to temperature only at the Vaal Dam site, prevalence correlation to pH values of the water, negative correlation between presence of parasite and host condition factor. The environmental factor proving to have the greatest impact is the turbidity levels. Measurement results are as follows: a total of 135 parasites were collected

i from 50 hosts. Measurements were taken from 62 parasites, equalling 46% of the total sample size. Data was obtained regarding the anterior length, posterior length, and total length of 57 parasites. The opisthaptor size (N=72) was length 190mm - 1000mm; width 50mm - 1000mm; the suckers (N=118) measured length 50mm - 150mm; width 50mm - 150mm. Between the left and right opisthaptor clamps, 1st clamps (N = 81) measured length 20mm - 110mm; width 70mm - 150mm, 2nd clamps (N = 83) with length 30mm - 100mm; width 50mm - 150mm, 3rd clamps (N = 82) with length 40mm - 90mm; width 40mm - 160mm and 4th clamps (N = 76) with length 10mm - 110mm; width 50mm - 150mm. No measurements could be made of clamp internal characteristics. Body of central hook 8 - 13µm in length, handle of central hook 2 - 5µm long. Only 7 eggs were measured with length 220 - 350µm and width 90 - 100µm. No diporpa larvae were collected. Graphic reconstruction confirmed the location of the reproductive organs in a ventral position in the body with the digestive tract occupying the dorsal region. Comparisons were made between the measurements obtained during this study, and measurements from other members of the genus Diplozoon and Paradiplozoon from both Africa and Europe. It is shown that although there is a certain measure of overlapping morphological sizes, physical structures distinguish it from other species and this is elucidated. The parasite dealt with in this study therefore is described here-in as a new species namely Diplozoon plicati.

KEYWORDS:

Monogenea, Diplozoon, Africa, Vaal River system, morphological measurements, condition factor, description, Labeo umbratus .

ii

OPSOMMING

Tot op hede is 4 verteenwoordigers behorende tot die Diplozoidae vanuit Afrika beskryf. Twee het tot die genus Diplozoon behoort, naamlik D. aegyptensis en D. ghanense vanaf Noord-Afrika. ‘n Derde een het tot die genus Neodiplozoon behoort, naamlik Neodiplozoon polycotyleus . Die vierde verteenwoordiger van die Monogenea is die fokus punt van hierdie studie wat poog om die taksonomie van die verteenwoordiger van die Monogenea op te klaar soos op die kieue van Labeo umbratus gevind is in die Vaaldam en Vaalrivierbarrage onderskeidelik. Die studie is uitgevoer oor ‘n tydperk van 13 maande met veldopnames wat elke twee tot drie maande vanaf Januarie 1999 tot Februarie 2000 plaasgevind het. Gasheervisse is met behulp van kieunette met maasgroottes van 90, 110 en 130mm onderskeidelik versamel. Afmetings met betrekking tot totale lengte, vurklengte, posisie van parasiet op kieue asook geslag van gasheer is geneem. Alle parasiete wat versamel is is in stomende AFA gefikseer en in 70% etanol gestoor. Afmetings van totaalpreparate is met behulp van ‘n ligmikroskoop en tekenbuisapparaat bestudeer. Boraks-Karmyn Jodium, Mayer se Hematoksalien en Horen se trichroom kleuring is gebruik vir die maak van preperate . Skandeerelektronmikroskopie is uitgevoer om inligting aangaande die uitwendige morfologie van die parasiete te versamel. Beide kritieke puntdroging en vriesdroging voorbereidingstegnieke is toegepas. Vir die ondersoek van die interne morfologie is seriesneë van 5µm gemaak en met AZAN gekleur. Grafiese rekonstruksie is voltooi deur die gebruik van standaard metodes. Alle data is statisties ontleed deur die SPSS (Windows weergawe 10.0) rekenaarprogram. Kwantitatiewe resultate dui op groter persentasie besmetting in groter gashere, ‘n voorkeur vir mannetjies, geen voorkeur vir posisie van aanhegting en ‘n persentasie besmetting korrelasie met temperatuur. Slegs by die Vaaldam studie area bestaan daar ‘n persentasie besmettings korrelasie met die pH waardes van die water. ‘n

iii Negatiewe korrelasie tussen die aanwesigheid van parasiete en die gasheer se kondisiefaktor is gevind. Die omgewings faktor wat blyk die grootste invloed te hê is turbiditeitsvlakke. Resultate is as volg: ‘n totaal van 135 parasiete is versamel van 50 gashere. Afmetings is geneem van 62 parasiete, gelykstaande aan 46% van die totale monstergrootte. Data met betrekking tot die anterior lengte, posterior lengte en totale lengte is verkry. Die opisthaptorgroottes (N=72) wys 190mm - 1000mm; wydte 50mm - 1000mm; die suiers (N=118) se lengte 50mm - 150mm; wydte 50mm - 150mm. Afmetings van die linker en regter opisthaptorklampe , 1ste klampe (N = 81) lengte 20mm - 110mm; wydte 70mm - 150mm, 2de klampe (N = 83) lengte 30mm - 100mm; wydte 50mm - 150mm, 3de klampe (N = 82) lengte 40mm - 90mm; wydte 40mm - 160mm en 4de klampe (N = 76) lengte 10mm - 110mm; wydte 50mm - 150mm. Geen afmetings kon gemaak word van interne klampstrukture nie. Liggaam van sentrale haak is 8-13µm lank en handvatsel meet 2-5µm lank (N = 8). Slegs 7 eiers is gemeet en die lengte was 220 - 350µm met wydte 90 - 100µm. Geen diporpa larwes is versamel nie. Grafiese rekonstruksies bevestig die posisie van die voortplantingsorgane wat ventraal in die liggaam geleë is met die spysverteringskanaal wat die dorsale area beslaan. Vergelykings is gemaak tussen die afmetings verkry gedurende hierdie studie en afmetings van ander lede van die genus Diplozoon en Paradiplozoon vanaf beide Afrika en Europa. Dit word gewys dat alhoewel daar ‘n sekere mate van oorvleuling van afmetings is, daar morfologiese strukture is wat dit onderskei van ander spesies. Die parasiet in hierdie studie bespreek, is beskryf as ‘n nuwe spesie naamlik Diplozoon plicati .

SLEUTELWOORDE:

Monogenea, Diplozoon, Vaalrivierstelsel, morfologiese afmetings, kondisiefaktor, beskrywing, Labeo umbratus .

iv

DECLARATION

I hereby declare that this report is my own original work. It is submitted in partial fulfilment of the requirements for the degree Master of Science in the Faculty of Science of the Rand Afrikaans University, Johannesburg, Gauteng, South Africa. It has never been submitted before for any other degree or examination at any other university or academic institution.

______

Laurette Seddon

v

ACKNOWLEDGEMENTS

With sincere thankfulness to:

ô My supervisor, Prof. Avenant-Oldewage for all her patience, guidance, ongoing encouragement and financial support.

ô Professor Swanepoel ex of Rand Afrikaans University Zoology Department for his translation of Khotenovsky’s keys to the Diplozoidea family and the description of Diplozoon paradoxum.

ô Mrs. Edie Lutsch of Zoology Department of the Rand Afrikaans Unive rsity for her guidance in producing microtome sections as well as with staining procedures.

ô The National Research Foundation, Pretoria, for their financial support of this project.

ô My immediate family, without whose support I could not have successfully completed this project.

vi

INDEX

ABSTRACT...... i KEYWORDS...... ii

OPSOMMING...... iii SLEUTELWOORDE...... iv

DECLARATION...... v

ACKNOWLEDGEMENTS...... vi

INDEX...... vii

LIST OF FIGURES...... xi

LIST OF TABLES...... xiii

ABBREVIATIONS...... xiv

CHAPTER 1: INTRODUCTION...... 1

CHAPTER 2: MATERIAL AND METHODS...... 5 2.1 MATERIAL COLLECTION...... 5 2.2 SCANNING ELECTRON MICROSCOPY...... 6 2.3 WHOLE MOUNTS……………………...... 7

vii 2.4 MORPHOLOGICAL MEASUREMENTS...... 8 2.5 SERIAL SECT IONING AND GRAPHIC RECONSTRUCTION..……………………...... 8 2.6 ECOLOGICAL STUDY...... 9

CHAPTER 3: CLASSIFICATION AND DISTRIBUTION OF THE DIPLOZOIDAE WITH TAXONOMY OF STUDIED SPECIES...... 14 3.1 INTRODUCTION...... 14 3.1.1 General Classification...... 14 3.1.2 . General classific ation of Monogenea…...... 16 3.1.3 Systematic position of the ‘family Diplozoidae’………………………………...…..…17 3.1.4 Classification of various members of the Diplozoidae family according to Khotenosvky (1985)…………………………………..……….…18 3.1.4.1 Neodiplozoinae…………….……...……18 3.1.4.2 Diplozoinae……………………..….…...19 3.1.5 Motivation for study…………………………...….28

3.2 CONCLUSION…………………………………………..28

CHAPTER 4: MORPHOLOGY AND DESCRIPTION...... 29 4.1 INTRODUCTION...... 29 4.1.1 General...... 29

4.2 MATERIAL AND METHODS...... 32

viii 4.3 RESULTS ……………………...... 33 4.3.1 Morphology of specimens studied...... 33 4.3.2 Taxonomy of specimens studied...... 36 4.3.3 Graphic reconstruction…...... 36 4.3.3 (a) Dorsal view of first individual……...….36 4.3.3 (b) Ventral view of second individual…….36

4.4 CONCLUSION...... 38 4.4.1 Species description………………………………39

4.5 ETYMOLOGY...... 40

CHAPTER 5: ECOLOGY...... 52 5.1 INTRODUCTION...... 52 5.1.1 Occurrence...... 54 5.1.2 Host specificity...... 55 5.1.3 Size and age of host...... 56 5.1.4 Host gender...... 57 5.1.5 Host behaviour and habitat...... 57 5.1.6 Seasonality and temperature...... 58 5.1.7 Water quality...... 59 5.1.8 Attachment sites...... 60 5.1.9 Condition factor...... 61

5.2 MATERIAL AND METHODS...... 62

5.3 RESULTS………………………...... 62 5.3.1 Occurrence...... 62 5.3.2 Host specificity...... 63

ix 5.3.3 Host size……………...... 63 5.3.4 Host gender...... 64 5.3.5 Seasonality and temperature...... 65 5.3.6 Water quality...... 66 5.3.7 Attachment sites...... 68 5.3.8 Condition factor...... 68

5.4 CONCLUSION...... 69

CHAPTER 6: DISCUSSION...... 74

REFERENCES...... 80

APPENDIX A: A.1STAINING TECHNIQUES…………………..93

A.2 SPECIES LIST OF MEMBERS OF THE FAMILY DIPLOZOIDAE PALOMBI, 1949, INCLUDING HOST, LOCALITY AND REFERENCE AS DISCUSSED IN CHAPTR 3………………………………….94

A.3 CLASSIFICATION DENDOGRAM PERTAINING TO KHOTENOVSKY’S PUBLISHED CLASSIFICATION FOR THE NEWLY DESCRIBED SPECIES OF THE GENUS DIPLOZOON ...... 98

x

LIST OF FIGURES

FIGURE 2.1 Map illustrating the location of sites, the Vaal Dam and Vaal River Barrage where bimonthly surveys were carried out to collect fishes infested with specimen……………….……...11 FIGURE 2.2 Illustrations of the separation of the gill arches, to enable the description of the distribution of parasites on the gills of the host……………………………………………………..…12 FIGURE 2.3 Illustrations to indicate method of measurements taken to obtain morphological data for use in identification……...………13

FIGURE 4.1 Scanning electron micrograph of Diplozoon plicati . ……………41 FIGURE 4.2 Micrographs of whole mount and serial sections of Diplozoon plicati. …………………………………………………………….....42 FIGURE 4.3 Parasite eggs with visible opercula lines which are not visible on SEM micrographs…………………………………………………..43 FIGURE 4.4 Various serial sections through major body structures of the Para site………………………………………………………...……44 FIGURE 4.5 Graphic reconstruction from serial sections has resulted in the Morphological image which shows the arrangement of organs from both a dorsal and ventral viewpoint……………………….45 FIGURE 4.6 Distribution map of Diplozoon in Africa, showing locality of the three recorded descriptions of Diplozoon on the continent...... 46 FIGURE 4.7 Comparative illustrations for type species of Diplozoon and Paradiplozoon, along with specimen from current study, to assist in classification……………………………………………………..47 FIGURE 4.8 Diagrammatic representation of transverse sections through (1) first part of ootype, (2) end part of ootype, (3) beginning of uterus and (4) uterine pore of Diplozoon plicati………………48

xi FIGURE 4.9 Diagrammatic representation of transverse sections through the (1) intestinum, (2) median ovovitelline duct, (3) mehlis gland of Diplozoon plicati……………………………………………………49 FIGURE 4.10 Diagrammatic representation of transverse sections through (1) Testis and vas deference, (2) spermatogonia………………….50 FIGURE 4.11 Diagrammatic representation of transverse sections through (1) Young oocyte, (2) median part of ovary, (3) anterior part of ovary with mature oocytes………………………………………..………51

FIGURE 5.1 Host gender preference of the specimen collected from Labeo umbratus in the Vaal Dam and Vaal River Barrage…………….70 FIGURE 5.2 Temperature fluctuations and the relationship to prevalence at the two sampling sites……………………………………………..71 FIGURE 5.3 Water quality data reflecting parasite prevalence at differing conditions at the Vaal Dam site…………………………..………72 FIGURE 5.4 Water quality data reflecting parasite prevalence at differing conditions at the Vaal River Barrage site……………………..…72 FIGURE 5.5 Condition factor analysis for A – Vaal Dam site, and B – Vaal River Barrage site………………………………………………….73

FIGURE 6.1 Characteristics as put forward by Mazourková, Matejusora, Koubková, & Gelnar (2003) for identification of Diplozoon species………………………………………..……………………..79

xii

LIST OF TABLES

TABLE 3.1 Table for determining genera (translated from Khotenovsky 1985: 106)……………………………………………………..…....19 TABLE 3.2 Descripti on of Diplozoon genus, with marks for applicability to current specimen……………………….…………………………..21 TABLE 3.3 Table for determining the species of Diplozoon (Khtenovsky, 1985: 227)…………………………………………………………..22 TABLE 3.4 Table for determining Paradiplozoon species, distributed through South-East Asia and Europe (from Khotenovsky, 1985:107) 23 TABLE 3.5 Table for determining Paradiplozoon species, distributed through Europe, West and Middle Asia and Far East (from Khotenovsky. 1985: 108)……………………………….…………25

TABLE 4.1 Morphological measurements as obtained from the specimen of Diplozoon plicati on Labeo umbratus ……………………...... 34

TABLE 5.1 Correlations existing between various host fish parameters and the total number of Diplozoon sp. found …………………………64 TABLE 5.2 Statistical data regarding gender preference of specimen Diplozoon plicati………………………………………………...….65

xiii

ABBREVIATIONS

Tl – total length Al – anterior length Pl – posterior length Ow – opisthaptor width Ol – opisthaptor length Sw – sucker width Sl – sucker length Pw – pharynx width Ew – egg width El – egg length Cw – clamp width Cl – clamp length CB – Central hook Body CH – Central hook Handle

xiv

CHAPTER 1: INTRODUCTION

Begon, Harper and Townsend (1996) defines a parasite as an organism that obtains its nutrients from one or a very few host individuals causing harm but not causing death immediately.

This is a very broad description of a relationship which exists naturally. As with all creatures, parasites are found in all morphological variations, both large and small, and throughout most of Earth’s natural environments. The parasite concentrated on in this study, has its own unique characteristics and way of life.

Members of the class Monogenea specifically the Diplozoidae have an expanded geographic range, including all continents except South America, Australia and Antarctica (no descriptions from these localities to date). Most species are host-specific and site-specific, requiring only one host to complete an entire life cycle (Reed, Francis-Floyd & Klinger, 2002).

Considering the large size of the African continent, as well as the mixture of climatic and geographic characteristics, it is surprising that it has only delivered four descriptions of monogeneans from the family Diplozoidae, namely: Paradiplozoon aegyptensis ( = Diplozoon aegyptensis Fischtal & Kuntz, 1963) in Egypt, Kenya and Uganda from Labeo forskalii, L. coubie, L. victorianus, L. cylindricus , Barilius loati and Barbus paludinosis (Fischtal & Kuntz 1963; Paperna 1979; Paperna 1996; Petr & Paperna 1979) and Paradiplozoon ghanense ( = Diplozoon ghanense Thomas, 1957) in Ghana from Alestes macrolepidotus and Alestes baremoze (Thomas 1957; Yamaguti 1963; Paperna 1969). Another mention was of an undescribed member of the

1 genus Diplozoon, on Alestes sp. in Uganda (Thurston 1970). The most recent description was made by Mashego (2000), concerning Neodiplozoon polycotyleus from Barbus marequensis, Barbus trimaculatus and Barbus neefi in the Limpopo province of South Africa.

The Diplozoidae are fish-gill ectoparasites comprising two individuals fused in permanent copula (Zurawski, Mair, Maule, Gelnar & Halton , 2003). When two unconnected diporpa larvae make contact on the host gill, their union triggers maturation, and the vas deferens from each individual grows through the body junction to the other. It is in this way that cross fertilization of the hermaphroditic organisms occurs.

This is the second record of a parasite with these familial characteristics found in the Vaal Dam and Vaal River Barrage. The first was of a parasite belonging to the genus Paradiplozoon. The ecology and morphology thereof was studied and recorded as part of an M.Sc dissertation completed under the supervision of the Zoology Departm ent of the Rand Afri kaans University in 2001.

The current study was initialised to clarify certain factors with regard to this second undescribed parasite as it was found to occur on Labeo umbratus . Questions arose as to the distribution of this parasite, the occurrence on the host and classification. Previous records of similar parasites show a localisation in Europe and Asia, if confirmed, this would be the first recording of a parasite on Labeo umbratus, and possible in the south of the African continent.

The objectives of the present study were to:

1. Present a complete species list in order to be able to compare the specimens found. 2. Study the external morphology of the parasite collected from L. umbratus and describe it. 3. Study the reproductive system and present a graphic reconstruction thereof.

2 4. Determine taxonomical status of this parasite. 5. Study occurrence of this parasite in a natural environment and determine the level of host specificity. 6. Determine whether the parasite exhibits preference for host gender, or size group. 7. Determine whether ecological factors, specifically season, temperature and water quality, influence the occurrence of this parasite on its host. 8. Ascertain whether this parasite has preferred sites for attachment. 9. Determine the effect on the host condition factor, if any, when infected with this parasite.

Different aspects of this investigation are discussed in separate chapters. Each chapter consists of an introduction and results section. The complete discussion and conclusions follow in the final chapter. The chapters are organised as follows:

Chapter 2: All the material and metho ds in order to avoid unnecessary repetition.

Chapter 3: The classification of this family, as well as their distribution is presented. The classificatio n system to be used was established. An English translation of original Russian keys to the species of this family composed by Khotenovsky (1985) is also included. A list was compiled which included the parasite species, synonyms used post 1985, fish hosts , the localities where parasites were collected, available morphological measurements, and a reference to the literature cited, this is reflected in Appendix A.2. The taxonomy of this specimen is included herein.

Chapter 4: Comprises an investigation into internal and external morphology by means of scanning electron microscopy, whole mounts and serially sectioned specimens used for graphic reconstruction. A species description is included.

3 Chapter 5: Concerns various aspects of the ecology of this parasite. This includes the ecological parameters such as prevalence, incidence, mean intensity, seasonality , size and gender of host and environmental variables.

Chapter 6: General discussion, conclusions and an overview of the study, including problems experienced and suggested solutions for any study initiated in the future.

4

CHAPTER 2: MATERIAL AND METHODS

2.1 MATERIAL COLLECTION

The Vaal Dam and Vaal River Barrage, located in the Vaal Triangle (Gauteng Province), was chosen as the collection site (FIGURE 2.1). Bimonthly surveys were carried out at one or both of the locations during a 13-month period starting in January 1999, and concluding in February 2000.

The host fish, Labeo umbratus (Smith, 1849), was collected with the aid of gill nets, which have a mesh size of 70, 90, 110 and 130mm respectively. Simultaneously other fish collected for related studies, included Labeobarbus aeneus (Burchell, 1822), Labeobarbus kimberleyensis (Gilchrist and Thompson, 1913), Labeo capensis (A. Smith, 1849), Cyprinus carpio (Linnaeus, 1758), Clarias gariepinus (Burchell, 1822) and Micropterus salmoides (Lacepéde, 1802).

The fish were placed in a temporary holding tank and supplied with circulating dam water. After collection, the fish were weighed in grams, using a Salter Model 235E scale. Both the fork length and the total length were recorded, in cm, using a calibrated measuring board. The fish were then killed by a single cut through the spinal cord and dissected to determine the gender.

The gills were removed by using dissection scissors. The left and right pairs of gills were placed in separate, distinctly marked Petri dishes and covered with dam water. The gills were examined with the aid of a dissection microscope. The number of parasites found in total per fish was determined, and the

5 position per gill filament was also noted according to a predetermined division of the gills (FIGURE 2.2).

The parasites were gently removed with the aid of a fine Camels hair brush (size:000) and placed in dam water in a Cyracause dish. Thereafter, they were brushed gently to remove fish slime and other debris. A drop of dam water was placed in the centre of a microscope slide and a small amount of petroleum jelly was placed on either side. The parasites were positioned centrally in the water and covered by a glass cover-slip which was carefully pressed down to avoid damage to the specimen. The parasites were fixed in this prostrate position in warmed aceto-formaldehyde alcohol for twenty minutes and p reserved in 70% ethanol.

2.2 SCANNING ELECTRON MICROSCOPY

The external morphology was studied with the aid of a JEOL JSM5600 scanning electron microscope, at acceleration voltages of 15 kV. Specimens prepared for scanning were either freeze dried or critical point dried.

The freeze dried specimens were prepared by gradual hydration from 70% ethanol to distilled water. A drop of 3.5% sodium hypochlorite was added and the parasite was gently brushed to remove fish slime. Specimens were placed into distilled water and into an Edwards Tissue dryer ETD4 for freeze drying. After drying, the specimens were sputter coated with gold in an Emscope Sc500.

During the critical point drying process, specimens were dehydrated with increasing ethanol concentrations, ra nging from 70% to 100%. The specimens were then cleaned with a fine artist’s brush and transferred to amyl acetate. Drying took place overnight in a Bio Rad Microscience Division CPD750 critical point drier. Thereafter the parasites were sputter coated with gold.

6 2.3 WHOLE MOUNTS

Whole mounts were prepared by using several staining techniques.

The first technique used, was the Boraxcarmine-iodine staining method. The parasites were removed from the 70% ethanol solution and placed in a saturated solution of iodine -ethanol (70%) for 6-7 hours. The parasitic worms were then transferred to 70% ethanol for a further 12 hours before counter- staining in 3% Boraxcarmine (Gaigher, 1984) in distilled water (Humason, 1979) for 2 hours. This was followed by dehydration with ethanol. Thereafter the parasites were cleared in xylene and mounted in Entellanä.

The second technique employed, was the Mayer’s Hematoxylin staining method (Humason, 1979), which was carried out by gradually hydrating the parasites from 70% etha nol and rinsing them with tap water. The parasites were then stained in Mayer’s Hematoxylin for approximately 20 minutes and differentiated in acid alcohol – 70% ethanol and hydrochloric acid – (Humason, 1979). After rinsing the parasites in tap water, the y were placed in Scott’s solution to blue (Humason, 1979). The specimens were rinsed a third time in water and gradually dehydrated to 100% ethanol. They were then cleared in xylene and mounted in Entellanä.

Horen’s Trichrome staining method, was the third technique used, as described in Manual of Veterinary Parasitological Techniques (1986). A stock solution was prepared and the parasites were transferred to a warm (60oC – 70oC) coloured lactophenol solution. The parasites were cleared and mounted in lactophenol and the cover slip was sealed with Glyceel (Hopkin & Williams ä).

See Appendix A.1 for summarized comparative staining technique table.

7 2.4 MORPHOLOGICAL MEASUREMENTS

Various measurements of the removed parasites were taken from the whole mounts as illustrated in FIGURE 2.3. Measurements of the whole mounts were made with the aid of a Zeiss standard 16 light microscope and drawing tube attachment. Measurements from preserved specimens were taken with the aid of a Wild M5 dissection microscope and drawing tube attachment. All measurements were compared to available data concerning other members of the Diplozoidae.

2.5 SERIAL SECTIONING AND GRAPHIC RECONSTRUCTION

Specimens were dehydrated in acetone prior to infiltration with fresh Transmit LM resin (TAABä ). Serial sections of the parasite were made with glass knives fixed to a rotary microtome. Transverse and sagittal sections were made at a thickness of 5mm. Sections were stained with AZAN stain, Mallory- Heidenhain (Koneff Modification, 1938; Humason, 1979). Time periods were modified as needed to ensure appropriate staining. Sections were studied and drawn with the aid of a Zeiss standard 16 light microscope and a drawing tube attachment in order to graphically reconstruct certain important structures according to the method of Pusey (1939).

Traditional transmission electron microscopy of ultra thin sections through various parts of specimens does produce high-resolution images of biological tissue, but most of the three-dimensional (3D) structural information is lost. This 3D structure can be recovered from serial sections.

8

2.6 ECOLOGICAL STUDY

A statistical analysis was carried out, using the data collected regarding the distribution and numbers of parasites found on the host gill filaments. The exact results shall be discussed in a later chapter.

The prevalence, intensity and mean intensity were calculated according to the method of Margolis, Esch, Holmes, Kuris and Schad (1982). The various results and comparisons shall also be presented in a later chapter.

The parameters are defined by Margolis et al. (1982) as:

Prevalence is the number of individuals of a host species infected with a particular parasite species divided by the number of hosts examined. (the prevalence can also be exp ressed as a percentage).

Intensity is the number of individuals of a particular parasite species in each infected host in a sample.

Mean intensity is the total number of individuals of a particular parasite species in a sample of a host species divided by the number of infected individuals of the host species in the sample.

Abundance is the total number of individuals of a particular parasite species in a sample of hosts divided by the total number of individuals of the host species in the sample.

9

The last parameter determined for each host, was the condition factor (CF). This was achieved through the use of the formula as stipulated in work completed by Klemm, Strober and Lazorchak (1992):

W x 105 Condition factor (CF) = L3

Where W is the weight (kg) and L is the total length (cm).

10

FIGURE 2.1 Map illustrating the location of sites, the Vaal Dam and Vaal River Barrage where bimonthly surveys were carried out to collect fishes infested with specimens.

11

LEFT ANTERIOR RIGHT ANTERIOR

Dorsal

Median Ventral 1

2

3

4

POSTERIOR

FIGURE 2.2 Illustrations of the separation of the gill arches, to enable the description of the distribution of parasites on the gills of the host. Gill arches numbered from most anterior (1), to second arch (2), third arch (3), and most posterior arch (4).

12

FIGURE 2.3 Illustrations to indicate positions where measurements were taken to obtain morphological data for use in identification. A – Adult coupled parasites with total length (Tl), posterior length (Pl), anterior length (Al), opisthaptor length (Ol), opisthaptor width (Ow), clamps numbered 1 to 4. B – Grouping of clamps with clamp length (Cl) and clamp width (Cw). C – Egg with part of filament, egg length (El) and egg width (Ew). D – Suckers at anterior end with pharynx width (Pw), sucker length (Sl) and sucker width (Sw). E – Central hook as found in distal area of opisthaptor with body (CB) and handle (CH). . F – Clamp stalk. G - Opisthaptor protrusion.

13

CHAPTER 3: CLASSIFICATION AND DISTRIBUTION OF THE FAMILY DIPLOZOIDAE WITH TAXONOMY OF STUDIED SPECIES

3.1 INTRODUCTION

This chapter’s purpose is to serve solely as background and summary of the existing taxonomy and distribution of monogeneans from the family Diplozoidae. This background will prove useful when a new species from this family will be examined and described.

3.1.1 General Classification

Monogenoidea van Beneden, 1858 (an independent class) was established by Bychowsky, 1937 with the subclasses Polyonchoinea and Oligonchoinea (Bychowsky, 1957). Latter subclass contains the order Mazocraeidea Bychowsky, 1937 and suborder Discocotylinea Bychowsky, 1957, with the family Discocotylinea which is divided into the subfamilies Discocotylinea Price, 1936 and Diplozooninae Palombi, 1 949 (Bychowsky, 1957).

Extensive revision of this family was done by Khotenovsky (1985) but unfortunately his manuscript of 262 pages is only available in Russian, and thus not widely known or acknowledged. The class Monogenea has a subclass Oligonchoidea with order Mazocraeidea and suborder Octomacrinae Khotenovsky, 1985 with two families. These families are Octomacridae Yamaguti, 1963 and Diplozoidae Palombi, 1949 (Khotenovsky, 1985).

14 A further division of this latter family into two subfamilies was completed by Khotenovsky (1985). The first being Diplozoinae Palombi, 1949 which encompass the genera Paradiplozoon Achmerov, 1974; Inustiatus Khotenovsky, 1978; Eudiplozoon Khotenovsky, 1985; Sindiplozoon Khotenovsky, 1985 and Diplozoon Nordmann, 1832. The second subfamily is Neodiplozoinae Khotenovsky, 1985 with only two genera, namely Neodiplozoon Tripathi, 1959 and Afrodiplozoon Khotenovsky, 1980 (cited in Khotenovsky, 1985).

For a number of years after the revision by Khotenovsky, Grabda-Kazubska & Pilecka-Rapacz (1987), Hirose, Akamatsu & Hibiya, (1987), Gusev (1985), Lebedev (1988), Lambert & Gharbi (1995) and Paperna (1996) all used the taxonomy of monogeneans as proposed by Bychowsky (1957). It is unclear whether the reason for this taxonomic decision was due to the fact that Khotenovsky’s work was rejected and ignored in publications, or was not known because knowledge of the Russian manuscript was lacking since no available English translation exists.

In the current study it was decided to adopt Khotenovsky’s (1985) classification system throughout the research to establish consistency.

Members of the family Diplozoidae have been recorded parasitizing numerous species of fishes worldwide. No records exist of the presence of Diplozoidae in Australia, Antarctica or South-America.

As new research is carried out in previously unexplored locations, the number of hosts, their distribution and new parasite species continue to increase. To demonstrate the distribution, a list of the parasite species, the host and the country where they were found is included in Appendix A.

In Appendix A, a change in genus name is indicated by the use of an asterisk (*) along with the original genus name. This change is indicated between the genus names of Diplozoon and Paradiplozoon only. No synonyms with Afrodiplozoon or Neodiplozoon were found to exist or be in used.

15

A total of 47 species belonging to the subfamily Diplozoidae were investigated in order to classify and describe the specimens collected in this study. Of these, 21 were transferred by Khotenovsky (1985) from the genus Diplozoon, to the genus Paradiplozoon. Of the original 47 species of Diplozoon, only two remain. The remaining 24 species which were originally classified as Diplozoon, have not been found in references as belonging to any other genus, and are thus regarded as being encompassed in the genus Diplozoon.

Mainly fish hosts of adult parasites are listed, but in the case of one example from Africa, the larval hosts were also included, as they correspond with hosts of the adult parasites. In cases where mention from literature is only made with regard to the parasite and host, with no location included, it is marked with a hash (# ).

3.1.2. General classification of Monogenea

According to Odhner (1912) in Price (1937), the division of the order Monogenea Carus, 1863 into two suborders was proposed based on the presence (Polyopisthocotylea) or absence (Monopisthocotylea) of a genito - intestinal canal.

In 1955 Hargis stated that the most widely accepted classification divides the subclass Monogenea into two orders, the Monopisthocotylea, in which the opisthaptor is without discrete multiple suckers or clamps, and the Polyopisthocotylea, with suckers or clamps on the opisthaptor.

According to Noble & Noble (1982) the flatworms are divided into four main classes namely Turbellaria, Trematoda, Cestoidea and Monogenea. They characterized monogenean subclasses Monopisthocotylea with an oral sucker either lacking or weakly developed, and Polyopisthocotylea as having a mouth surrounded by a prohaptor that consists of one or two suckers or pits. The polyopisthocotylean opisthaptor may of may not be armed, but always has

16 suckers or sucker-like bodies containing clamps and these parasites are almost exclusively blood feeders (Noble & Noble 1982).

The monophyly of each of the groups of Monogenea (Monopisthocotylea and Polyopisthocotylea) has been ascertained on morphological characters by Boeger & Kritsky, 1973; 1997 and 2000 in Mollaret, Jamieson & Justine, 2000. The monophyly has also been indicated by spermatological and molecular analyses (Justine, 1991 and Mollaret, Jamieson & Adler, 1997 in Mollaret et al., 2000.

3.1.3 Systematic position of the ‘family Diplozoidae’

Bychowsky (1957) proposed raising the taxon Monogenea to the status of a class: Monogenoidea containing two new subclasses, Polyonchoidea and Oligonchoidea. In the latter subclass, order Mazocraeidae, family Dacocotylidae (Price, 1937) and subfamily Diplozoinae (Palombi, 1949) contains the genus Diplozoon. Tripathi (1957) proposed the family Diplozoidae.

In 1963 Yamaguti split the order Monogenea into two suborders: Monopisthocotylea and Polyopisthocotylea and proposed placing Diplozoon and Neodiplozoon Tripathi, 1960 under the super family Diplozooidae and family Diplozoidae.

Members of the family Diplozoidae are the only monogeneans living in permanent pairs (Jovelin & Justine, 2001). Results obtained when investigating the phylogeny of this family, haves showed that Diplozoon nipponicum is a sister-taxon of the Microcotylinae (Jovelin & Justine, 2001). Further investigation showed that sperm structure is not able to throw light upon the phylogenetic position of the Diplozoidae (Jovelin & Justine, 2001).

The current status as proposed by Khotenovs ky (1985) split the family Diplozoidae into two subfamilies, namely Neodiplozoinae, with genera

17 Neodiplozoon and Afrodiplozoon; and Diplozoinae with genera Inustiatus , Sindiplozoon, Diplozoon, Eudiplozoon and Paradiplozoon.

This sub familial split is also shown by Spencer Jones & Gibson (1990) who compiled a list of monogenean genus-group names which were not included in Yamaguti’s Systema helminthum in 1963. They show the same Genus groupings as those used by Khotenovsky (1985), however mention is not made of the genus Diplozoon.

To ensure consistency during the present investigation it was decided to use Khotenovsky’s classification of this family.

A simplified dendogram is enclosed in Appendix A for ease of reference.

3.1.4 Classification of various members of the Diplozoidae family according to Khotenovsky (1985).

During 1985 Khotenovsky divided the family Diplozoidae into 2 subfamilies namely Neodiplozoinae and Diplozoinae.

3.1.4.1 Neodiplozoinae

This subfamily contains the genera Neodiplozoon and Afrodiplozoon, which are distinguished by the number of clamps, their arrangement and geographic areas of collection. Khotenovsky (1985) established that “Neodiplozoon has more than 1 pairs of clamps, which are arranged on the back parts of the body and is found in India”. In contrast, Khotenovsky (1985) noted that “Afrodiplozoon is found in Africa and has many (clamps), with fewer than 15 pairs of clamps situated laterally on the posterior body end which is entire”.

18 In consideration of the abovementioned subfamily characteristics, it is evident that specimens collected in the Vaal Dam and Vaal River Barrage from L. umbratus do not belong to this subfamily.

3.1.4.2 Diplozoinae

Keys for determining the genera of the subfamily Diplozoinae are provided in the manuscript of Khotenovsky (1985). Various morphological structures and distinguishing features, including their measurements are used.

Illustrations from Khotenovsky’s publication are redrawn and included here-in in order to become familiar with the re structures (FIGURE 3.1). TABLE 3.1 below contains an extract from Khotenovsky’s (1985) key for determining the genera of the Diplozoinae subfamily.

TABLE 3 .1 Table for determining genera (translated from Khotenovsky 1985: 106).

1. (2) The central region of the posterior part of the body does not show dilation. ______1. Paradiplozoon Achmerov 2. (1) The central region of the posterior part of the body shows a dilation of varying shapes. 3. (4) In anterior sucker of mouth / buccal cavity there are two large musculo-glandular organs. Dilation shows large lateral plicae. ______4. Eudiplozoon Khotenovsky 4. (3) Anterior sucker lacks musculo-glandular organ. Dilation deprived of plicae. 5. (6) Dilation disc-shaped. Intestinal branches form a dense network. External uterine opening lateral in the central region of the anterior part of the body. ______2. Inustiatus Khotenovsky 6. (6) Dilation cup-shaped. Intestinal branches do not form a dense network. External opening of uterus on the border between anterior and posterior body parts.

19 7. (8) Anterior region of posterior body part has plicae. ______5. Diplozoon Nordmann 8. (7) Anterior region of posterior body part does not have plicae. ______3. Sindiplozoon Khotenovsky

When evaluating the specimens collected from L. umbratus in the Vaal Dam and Vaal River Barrage against the above key, a degree of uncertainty exists.

The specimens collected fall into two genera classifications from the key, namely: 1. (2) The central region of the posterior part of the body does not show a dilation. ______1. Paradiplozoon Achmerov 7. (8) Anterior region of posterior body part has plicae. ______5. Diplozoon Nordmann

It is therefore necessary to investigate the descriptions of both genera in depth to determine the correct placement of specimens.

20 Genus Diplozoon Nordmann, 1832 (Nordmann, 1832:56; Khotenosvky, 1985:173) Type species Diplozoon paradoxum Nordmann, 1832, in Monogenea.

TABLE 3 .2 Description of Diplozoon genus, with marks for applicability to current specimen. v = applies. ? = does not apply

Posterior part of the body clearly / distinctly divided into three regions. ? The anterior region (of the posterior part of the body) has on its ventral v surface well developed folds / plicae. Folds on the dorsal surface are more shallow / smaller. v The central / middle region of the posterior part of the body forms a ? bowl-shaped enlargement / expansion, and the posterior region contains / carries the attachment clamps and v central hooks. v The intestines of the central region of the posterior part of the body v contain lateral branches (diverticulae) that are not interconnected (end freely). The gonads are located in the anterior region of the posterior part of v the body. The testes are uni ted / fused and fan-shaped. v The opening of the uterus, where the egg is expelled, is situated at the v level of the border between the anterior and posterior parts of the body. Egg with filament, attached to the lid end of the egg. v In the uterus the egg filament points forward (to external). v

The genus Diplozoon includes two species, widely spread through the Palaearctic region.

Observations: Nordmann (1832) established the genus Diplozoon describing them only as being paired worms.

21 Akhmerov (1974) divided the genus Diplozoon into two subgenera on the ground of the absence or presence of an enlargement in the middle region of the posterior part of the body.

Later Khotenovsky (1985) did not follow Akhmerov’s suggestion of dividing the genus into subgenera and proposed that the genus Diplozoon contains two species namely D. paradoxum and D. scardini.

TABLE 3.3 Table for determining the species of Diplozoon (Khtenovsky, 1985: 227).

1 (2) Folds / plicae of the anterior region of the posterior part of the body are large. Number of folds 4 – 8. Length of body of central hooks between 28 - 33µm ______D. paradoxum Nordmann 2 (1) Folds / plicae of the anterior region of the posterior part of the body are small / shallow. Number of folds 8 – 13. Length of body of central hooks between 22 - 26µm ______D. scardini Komarova

When considering the characteristics used for determining the species of Diplozoon in this study (folds large or shallow), the specimens collected could be grouped and named as D. paradoxum. But then the second characteristic is explored (number of folds / plicae) and the number of folds is determined as 5 – 16, which does not fit in either of the species.

The final characteristic, length of body of central hooks would then obviously prove to be the last characteristic to concentrate upon to determine this species and this is done in the chapter regarding the morphology.

The thoroughly completed identification keys compiled by Khotenovsky for the species belonging to the genus Paradiplozoon is enough of a guide to use as comparative information in this study. There are a number of initial shared characteristics which at first sight indicate the specimen belonging to this genus. Upon thorough comparison, it is however gleaned that the specimen

22 differs enough from the species of Paradiplozoon that it cannot be classified as a species of that genus.

The complete identification keys from Khotenovsky (1985) are included here in order to elucidate the differe nces.

TABLE 3.4 Table for determining Paradiplozoon species, distributed through South-East Asia and Europe (from Khotenovsky, 1985:107).

1 (2) Surface of the posterior part of the body is plicated. Posterior part of body approximately 1.5 times shorter than the anterior part of the body. Average / mean diameter of the 3rd clamp is 10 to 20 times smaller than the length of the poste rior part of the body. ______P. kashmirense (Kaw) 2 (1) Surface of the posterior part of the body is not plicated. 3 (6) Anterior and posterior parts of body are equal in length. 4 (5) Testes united into a single structure. _ _ _ _ P. barbi (Reichenback-Klinke) 5 (4) Testes divided into a number of lobes. _ P. tetragonopterini (Sterba) 6 (3) Posterior part of body is shorter than anterior part. 7 (16) Mean diameter of the 3rd clamp is from 3 to 8 times less than the 8 (11) length of the posterior part of the body. 9 (10) Median hooks are less than 20µm in length. 10 (9) Pharynx smaller than sucker. ______P. doi (Ha Ky) 11 (8) Pharynx longer than sucker.______P. malavense (Lim Lee Hon & Khotenovsky) 12 (15) Median hooks are more than 25µm in length. 13 (14) Width of 3rd clamp is more than 200µm in length. 14 (13) Intestinal diverticulae / branches of the anterior part of the body are arranged perpendicular to the longitudinal axis of the body. ______P. indicum (Dayal) 15 (12) Intestinal diverticulae / branches of the body are directed

23 backward. Testis is spirally winded. ______7. P. magnum (Lim LEE Hong & Khotenovsky) 16 (7) Width of attachment clamps is between 120 and 160 µm. ______9. P. ghanense (Thomas) 17 (24) Mean diameter of the 3rd clamp is from 10 to 38 times less than the length of the posterior part of the body. 18 (19) Mean diameter of the 3rd clamp is from 10 to 20 times less than the length of the posterior part of the body. Pharynx larger than suckers. ______1. P. soni (Tripathi) 19 (18) Pharynx is smaller or equal in size to the suckers. 20 (21) Width of clamps from 125 to 190µm. Eggs without filaments. ______4. P. cauveryi (Tripathi) 21 (20) Width of clamps from 80 to 117µm. Eggs with filaments. 22 (2 3) Length of suckers from 67 to 83µm. ______11. P. vietnamicum (Khotenovsky) 23 (22) Length of suckers from 95 to 125µm. ______8. P. aegyptense (Fischtal & Kuntz) 24 (17) Mean diameter of the 3rd clamp is from 25 to 38 times less than the length of the posterior part of the body. ______P. microclampi (Kulkarni)* * Paradiplozoon microclampi from Kulkarni’s 1971 description of Diplozoon microclampi, as reclassified by Khotenovsky in 1985. The Khotenovsky classification if followed to maintain clarity.

24

TABLE 3.5 Table for determini ng Paradiplozoon species, distributed through Europe, West and Middle Asia and Far East (from Khotenovsky. 1985: 108).

1. (12) Posterior part of body not plicated. 2. (7) Sclerites of clamps are robust. 3. (4) Posterior arch of clamp has well developed transverse striations . ______14. P. parabramisi (Ling) 4. (3) Posterior arch of clamp without transverse striations. 5. (6) Intestinal branches in anterior part of body clearly stand out against a yolky background. Marine parasites. ______21. P. schizothorazi (Iksanov). 6. (5) Intestinal branches in anterior part of body do not stand out against a yolky background. In posterior part of body intestine shows lateral branches. Parasites of Jazja, Chub (Leusiscus) and barbel. ______25. P. megan (Bychowsky and Nagibina). 7. (2) Sclerites of clamps are delicate. 8. (9) Suckers larger than pharynx or sometimes equal in size. Parasites of Black Amur Bream. _ _ _ _ 17. P. megalobramae Khotenovsky. 9. (8) Suckers smaller then pharynx. Parasites of inich fish. 10. (11) Anterior part of central plate shows a supplementary outgrowth; at the junction of the central plate and anterior arch of clamp two short sclerites are present. ______15. P. marinae (Achmerov). 11. (10) Anterior part of central plate does not show a supplementary outgrowth; at the junction of the central plate and anterior arch of clamp two long sclerites are present _ _ 18. P. hemiculteri (Ling). 12. (1) Posterior part of body pliated. 13. (16) Plicae small. Their width in the mid region of the posterior part of the body does not exceed 150µm. 14. (15) Anterior part of central plate connected to the arch of the clamp by a single short sclerite. Intestine in posterior part of body does not have lateral branches. Length of median hooks between 22 -

25 24µm. ______16. P. cyprini Khotenovsky. 15. (14) Anterior part of central plate connected to the arch of the clamp by two short sclerites. Intestine in posterior part of body does have branches. Length of median hooks between 27 - 30µm. ______26. P. rutili (Gläser). 16. (13) Plicae large. Their width in the mid region of the posterior part of the body exceeds 200µm. 17. (22) Intestine op posterior part of body is tubular and without lateral branches. 18. (21) Anterior part of median plate connected to arch of clamp by two sclerites. 19. (20) Number of plicae between 9 – 10. _ _ _ 24. P. minutum (Paperna) 20. (19) Number of plicae more than 40. ______30. P. pavlovskii (Bychowsky and Nagibina) 21. (18) Anterior part of median plate connected to arch of clamp by a single sclerite _ 23. P . tadzhikistanicum (Gavrilova and Dzhalilov) 22. (17) Intestine of posterior part of body has lateral branches. 23. (24) Clamp I is 2 to 2.5 times smaller than the other clamps. ______27. P. nagibinae (Gläser) 24. (23) Clamp I is less than 2 times smaller than the other clamps. 25. (26) Anterior part of median plate is continuous with the line formed by the arch of the clamp thus taking the appearance of an inverted letter “T”. ______29. P. bliccae (Reichenbach-Klinke) 26. (25) Anterior part of median plate is not continuous with the line formed by the arch of the clamp. 27. (28) Anterior part of median plate is connected to the arch of the clamp by one broad sclerite . ______22. P. capoetobrana (Gavrilova) 28. (27) Anterior part of median plate is connected to the arch of the clamp by two sclerites. 29. (36) Edge of anterior part of median plate is serrated. 30. (31) Pharynx is larger than the sucker. _ _ 36. P. alburni Khotenovsky 31. (30) Pharynx is smaller than sucker. 32. (35) Anterior part of median plate has a supplementary outgrowth.

26 33. (34) Testis lobulated. Parasite of Amur River Zerecha. ______19. P. amurense (Achmerov) 34. (33) Testis are a singular structure. Parasite os Amur River Jazja. ______20. P. skrjabini (Achmerov) 35. (32) Anterior part of median plate without supplementary outgrowth. ______33. P. tisae Khotenovsky 36. (29) Edge of anterior part of median plate is rounded / c urved. 37. (38) Length of median hooks exceed 25µm. Posterior part of median plate shows a rectangular outgrowth. _ _ 31. P. ergensi (Pejoch) 38. (37) Length of median hooks does not exceed 25µm. Posterior part of median plate does not have a rectangular outgrowth. 39. (40) Anterior part of median plate shows an additional arch-like piece that is connected to the arch of the clamps. _ _ _ 34. P. leucisci (Khotenovsky) 40. (39) Anterior part of median plate does not have an additional arch-like piece. 41. (42) Plicae on the ventral surface of the posterior part of the body do reach the level of the clamps…28. P. sapae (Reichenbach-Klinke) 42. (41) Plicae on the ventral surface of the posterior part of the body do not reach the level of the clamps. 43. (44) Plicae less than 15 in number……………...35. P. zeller (Gyntovt) 44. (43) Plicae more than 15 in number. 45. (46) Median plate robust. Its posterior part shows lateral extensions. The median sclerites of the anterior arch of the clamps are situated directly under the angle of the arch………..32. P. vojteki (Pejcoch) 46. (45) Median plate tender / delicate. Its posterior part does not show lateral extensions. The median sclerites of the anterior arch of the clamp are situated on the straight part of the arch only. 47. (48) Vitelline follicles lying in the anterior part of the body in a dense mass. Length of egg more than 260µm. …………….37a. P. homoion homoion (Bychowsky and Nagibina) 48. (47) Vitelline follicles lying dispersed in the anterior part of the body. ……………………..37b. P. homoion gracile (Reichenbach-Klinke)

27

3.1.5 Motivation for the study

Since these specimens does not fit any of the above species listed by Khotenovsky (1985) or any other description consulted, it can be concluded that it can be placed in the genus Diplozoon and that it is a new species. Although many authors have studied various members of the Diplozoidae family, no complete study has been undertaken using lightmicroscopy, scanning electron microscopy and serial sectioning of material for graphic reconstruction of the reproductive system. Most of the species descriptions are based on descriptions o f whole mounts.

3.2 CONCLUSION

As the specimen from the current study does not fully match any of the Paradiplozoon species listed in the keys above, it is concluded that it belongs to the genus Diplozoon as the most characteristics are shared with species from this genus.

28

CHAPTER 4: MORPHOLOGY AND DESCRIPTION

4.1 INTRODUCTION

4.1.1 General

The phylum Platyhelminthes can be divided into four main classes, namely: Turbellaria (mostly free living), Trematoda, Cestoidea and Monogenea (a ll parasitic) (Hickman et al 1993; www.encyclopedia.com).

All these members have characteristically dorsoventrally flattened bodies which show no segmentation, are acoelomate, bilaterally symmetrical, have protonefridial excretory systems, no definite anus, no respiratory or circulatory system (www.aber.ac.uk, 2002).

Most importantly, they are hermaphroditic and develop through either direct or indirect stages (Noble & Noble, 1982).

Members of the class Monogenea are characterised by sexual reproduction followed by direct development and a single host (Hargis, 1955). A simple metamorphosis is involved in this direct development and no interpolated sexual generations are found (Chandler & Read, 1961). Most monogeneans produce large eggs that hatch into larvae that immediately attack their definitive hosts (Chandler & Read, 1961). Monogenean egg capsules usually have terminal filaments (Hargis, 1971) that vary in length between the different genera.

29 Adult monogenea parasites attach to the host with an opisthaptor, a modification of the posterior end of the parasite (Noble & Noble, 1982) and are most commonly found on the skin and gills of various marine and fresh- water fish species (Price, 1937). Usually one or more suckers, cups or clamps are present on the opisthaptor (Noble & Noble, 1982). The adults range in length from less than 1mm, to 2 or even 3cm with the body outline varying from spindle -shaped to circular (Noble & Noble, 1982).

Monogeneans being primarily parasitic on fishes (Cone, 1995), occur on other aquatic animals (Price, 1937) such as eels. The majority are ecto -parasites and occur on specific sites such as the head, flank, fins, crypts of acoustolateralis system, surface of nasal epithelium, branchial arches (Cone, 1995), and by exception in the heart (Bychowsky, 1957). Paperna (1979) recorded Enterogyrus cichlidarum Paperna, 1963 from both Tilapia zillii and Tilapia aurea. Price (1937) found a few on crustaceans and cephalopods, whilst Oculotrema hippopotami lives beneath the eyelid of the hippopotamus (Whittington, Cribb, Hamwood & Halliday, 1998).

Tetrapods in aquatic or semi-aquatic environments, amphibians (anurans and urodeles) and chelonian reptiles (Whittington, 1998) have also been parasitized. Several species occur in the mouth and upper respiratory tract of turtles as well as the urinary bladder.

The class of monogeneans can either be sanguiform, polyopisthocotyleans or tissue grazing monopisthocotyleans (Cone, 1995).

Polyopisthocotyleans do not cause significant tissue damage due to their delicate manner of attachment to secondary gill lamellae and the subtle manner in which blood is drawn from the underlying blood vessels (Cone, 1995). Polyopisthocotyleans are sedentary parasites, since host blood is abundantly available (Noble & Noble, 1982) whilst monopisthocotyleans are browses on the skin, and need to move about on the host surface.

30 The monogean digestive tract consists of the mouth, pharynx, oesophagus and gut. The gut may be simple or branched, sometimes with innumerable small blind pouches or caeca (Noble & Noble, 1982). There is no anus present (Noble & Noble, 1982).

The Diplozoidae family of the monogenea is a unique one, where individuals actually occur in pairs consisting of two individuals in permanent copula with tissues fused into one functioning organic unit (Paperna, 1979) and have been found to parasitize the gills of freshwater fishes.

During the diporpa larval stages a small fleshy knob appears on the dorsal surface (Noble & Noble, 1982). Eventually this knob is inserted into a ventral sucker of another larval worm (Noble & Noble, 1982). The two worms become securely fused together and cannot be separated (Noble & Noble, 1982). Fusion of the reproductive organs becomes complete as the flukes mature, and cross-fertilization occurs as the occasion demands (Hargis, 1971). The gonads develop first, and finally the vagina of one individual opens in the region of the uterus, and the vas deferens of the other (Noble & Noble, 1982). This arrangement is mirrored, and cross-fertilization is thus enabled.

Gerasev (1994) found that the entry of the ‘seminal receptacle duct’ varied (entry either into vitelline reservoir or oviduct) not only within the genus Paradiplzoon, but also within one specific species, Diplozoon paradoxum.

During this study the reproductive system of the member of this family collected from Labeo umbratus in the Vaal Dam and Vaal River Barrage was investigated.

The genus Diplozoon was established with the description of D. paradoxum Nordmann, 1832 from the gills of bream and was subsequently recorded on a large number of fish species in Europe and Asia (Fotedar & Parveen, 1987). Thereafter various authors described an array of species from various fish hosts (see Appendix A).

31 Features found to be important for differentiating between the different species of Diplozoon are: (i) uniform range of measurements from a large number of specimens, (ii) the shape and size of the clamps on the opisthaptor, (iii) the average size ratio of the fore and hind -body and (iv) the way in which the distal part of the intestinal caecum terminates in the hind body (Fotedar & Parveen, 1987).

Paperna (1979) reported that Tripathi recorded 105 species of monogenea in 1957, from 86 fish species, and that majority of these appeared to be host- specific.

Thirteen years later, in 1982, Noble & Noble stated that around 435 species of monogenea were known. Monogeneans are considered to be amongst the most host-specific parasites (Whittington, 1998) and it is postulated that each species of fish may be parasitized by a single monogenean species. If accurate, this prediction leads to values of between 24 000 and 25 000 species of monogeneans in the world (Whittington, 1998). This statement is corroborated by Mashego (1983) who found three species of Dactylogyrus parasitizing Barbus paludinosis.

Descriptions of monogeneans from the African continent are few and far inbetween. This could be mainly due to the absence of monogenean taxonomists.

4.2 MATERIALS AND METHODS

As described in chapter 2.

Adult pairs (N – 62) were studied and measured with the aid of a dissection microscope. No diporpa larvae were found. 15 Adult pairs were prepared as stained whole mounts and studied with the aid of a light microscope. 5 Adult pairs were serially sectioned in resin, studied with the aid of a light microscope

32 and graphic reconstructions of the reproductive organs were made. Fifteen adult pairs were studied with the aid of a scanning electron microscope. The following results are based on the abovementioned material.

4.3 RESULTS

4.3.1 Morphology of specimens studied

Specimens of Diplozoon were found on the gills of Labeo umbratus (Smith, 1849) in both the Vaal Dam and Vaal River Barrage sites. Measurements of all known Diplozoon species, their hosts and localities together with available morphological measurements are presented in Appendix A: 2. Specific measurements obtained from the specimens studied during the period are given in TABLE 4.1.

33 TABLE 4.1 Morphological measurements as obtained from the specimen of Diplozoon plicati from Labeo umbratus.

Characteristic min max median std div n anterior length 1.75 4.41 3.08 1.90 41 posterior length 0.85 2.30 1.58 1.03 41 total length 2.60 6.71 4.66 2.91 41 sucker 1 width 0.05 0.15 0.1 0.10 67 sucker 1 length 0.05 0.13 0.09 0.10 67 sucker 2 width 0.05 0.13 0.09 0.10 51 sucker 2 length 0.05 0.15 0.1 0.10 51 opisthaptor width 0.05 1.00 0.53 0.70 72 opisthaptor length 0.19 1.00 0.60 0.6 0 72 clamp 1 width 0.07 0.15 0.11 0.02 72 clamp 1 length 0.02 0.11 0.07 0.01 81 clamp 2 width 0.05 0.15 0.12 0.02 74 clamp 2 length 0.03 0.10 0.06 0.01 83 clamp 3 width 0.04 0.16 0.12 0.02 73 clamp 3 length 0.04 0.09 0.06 0.01 82 clamp 4 width 0.05 0.15 0.12 0.02 66 clamp 4 length 0.01 0.11 0.07 0.02 76 Body of central hook 0.08 0.13 0.08 2.64 9 Handle f central hook 0.02 0.05 0.04 1.33 9 egg width 0.09 0.10 0.10 0.00 7 egg length 0.22 0.35 0.31 0.05 7

Morphological measurements obtained from 62 paired specimens collected in this study are reflected in TABLE 4.1. This shows that the ratio between posterior length and anterior length is 1:2.1 – 5.2 with a mean ratio of 1:1.9. The ratio between posterior length and total length is 1:3.1 – 7.9 (mean 1:2.9), and that between anterior length and total length is 1:1.5 – 3.8 (mean 1:1.5).

The posterior part of the body is divided into two parts, namely the opisthaptor with clamps and the remainder which shows 5 – 16 folds across both the dorsal and ventral surfaces (FIGURE. 4.1 D). Due to these folds, the body length shows great variation, but the ratio of all measurements remains constant.

34 The measurements of anterior suckers show that on average the suckers have equal measurements for length and width (50µm - 150µm, mean 100µm) and a minimum contraction diameter of 50mm. The diameter of the pharynx is less than that of the suckers, but it is longer. The suckers are well differentiated and clearly circular (FIGURE. 4.1 A).

The opisthaptor measurements show the width being equal to the length. The ratio of opisthaptor length to total posterior length from fusion point is 1:1.7 – 4.6 (mean 1:3) and the opisthaptor width to total posterior length is 1:4.5 – 12 (mean 1:2.6).

At the posterior terminal tip of the opisthaptor, a protrusion is present. This protrusion is present on both the dorsal and ventral surface of the opisthaptor and can be extended or c ontracted close to the body (FIGURE . 4.1 D and FIGURE. 4.2 B).

Mean values show that all clamps are equal in size. The maximum width of the clamps is 150mm (minimum = 40mm, mean = 120mm) (FIGURE. 4.1 B and FIGURE. 4.2 B). These sets of four clamps are carried on a stalk which can extend to the periphery of the opisthaptor (FIGURE . 4.1 B).

The reproductive opening is placed in a similar position to that of other Diplozoon species (FIGURE . 4.1 C).

The eggs bear an extremely long filament, which is 2000mm long or more (FIGURE. 4.1 E and FIGURE. 4.2 C). No more than three well developed eggs were present in each individual of the pair at one time (FIGURE . 4.2 A, C and D). The operculum of the egg is not always clearly visible. From the scanning electron microscope micrograph, no external slit, groove or other indication of an operculum is seen (FIGURE. 4.1 E). When searching for the operculum from light microscope micrographs, a possible opercula line is seen (FIGURE . 4.3 A).

35 The intestine clearly shows large diverticules, branching terminally and present to the point where the most anterior opisthaptor clamps occur (FIGURE. 4.2 B).

4.3.2 Taxonomy of specimens studied

Full taxonomy as after the detailed classification of the family with the new species added per Chapter 3. Dendogram included in Appendix A.

4.3.3 Graphic reconstruction

The serial sectioning and consequent reconstruction thereof was completed to describe the structures of the reproductive organs

Graphic reconstruction was completed of adult pairs in copula, in so doing gaining insight regarding the arrangement of internal structures from both a dorsal and ventral view respectively, as well as the connection between the two sets of reproductive organs

The following arrangement was clarified:

4.3.3 (a) Dorsal view of first individual: Below the intestinal tract, was the ovary where the youngest oocytes were found.

Ventral to the young ovary section, the section with more mature ova appeared and the organ located most ventrally proved to be the vas deferens.

4.3.3 (b) Ventral view of second individual: As found with the first individual, the ovary with young oocytes is located dorsally, and thus not very visible from this position.

36 Ventrally, but in some places placed parallel to the younger ovary is the male testis curving and being obscured greatly in whole mounts either by sections of young ovary, or the vas deferens itself. Serial sectioning made this reproductive section more clearly visible.

The uterine pore is located dorsally therefore It can only be viewed one at a time when seen from an external vantage point. Serial sectioning makes this structure and the accompanying uterus clearly visible (FIGURE 4.8)

The ovary with young oocytes follows numerous turns inside the posterior part of the body, all located anteriorly to the opisthaptor. Along these curves the oocytes mature and develop (FIGURE 4.11). The testis, containing spermatogonia with male reproductive cells have been formed and matured (FIGURE 4.10). The vas deference passes through to the second individual.

The vas deference and spermatogonia from the second individual opens into the oviduct of the first individual just anterior to the fusion point of the two individuals.

Upon maturation of the oocyte, shortly before fertilization and any contact with the vas deference contents, the vitelline glands open into the ovary through the common vitelline duct (FIGURE 4.9). The oocytes now traveling along the final lengths of the ovary are moved along with the help of elongated cilia.

The vitelline glands and the ir ducts enter the ovary in the anterior half of the body’s most posterior region.

The mehlis gland was located close to and emptied into the uterus shortly after the vas deferens united with the uterus (FIGURE 4.9). Prolific elongated

37 cilia were present in the oviduct as the egg cells matured and are presumed to aid in the movement of the egg towards the uterine pore.

The testes contain a large lumen (FIGURE 4.10). The testis and vas deference possess cuboidal epithelial walls, whilst most all other walls (ovary, uterus etcetera) have columnar epithelial cell structures with either brush borders or cilia for assistance in forward movement of larger oocytes and zygotes.

The zygotes that are formed travel along a uterus with the aid of ciliated epithelial cells. The uterus and formed egg with zygote are present in the most posterior part of the anterior section of the body. The uterus is visible in the reconstruction process from the most posterior starting position towards the anterior body where it curves distally. The egg is visible in the anterior body. The terminal lengths of the uterus curves around on itself to the posterior part of the body towards the reproductive opening where the egg will leave the parent body with filament pointing outward. The uterine pore is surrounded by a mesh network of pronounced striated muscles and is surrounded by a tegument fold (FIGURE 4.8).

4.4 CONCLUSION

Each individual which joins in permanent copula is proven to be a mirror image of its mate. The precise arrangement of the intestinal tract in the dorsal section of the body leaves a greater ventral space to accommodate the male and female ducts. These in turn are arranged in numerous curving patterns folding up on each other to facilitate development of both sperm and ova to full maturity before the fusion of reproductive parts from one individual to another and vice versa.

38

4.4.1 Species description

The following characteristics are used in the description and subsequent identification of the new species.

a) Mean ratio of posterior length to anterior length 1:1.9 b) Mean ratio of anterior length to total length 1:1.5 c) Posterior part of body only divided into 2 distinct regions., with the anterior region of the posterior part having5 – 16 folds both dorsally and ventrally. d) Mean ratio of opisthaptor length to total posterior length is 1:3. e) Body of the central hook 8 - 13µm in length. f) Terminating posterior region of opisthaptor elongated to dorsal and ventral positions to form protrusions extending outward from body. g) Clamps carried on stalks attached to distal part of posterior body section, stalk may extend laterally outward. h) Suckers positioned on most distal tip of anterior body section well- nigh circular. i) Folds on posterior body section more shallow on ventral surface. j) Reproductive opening situated mid-ventrally in fusion area. k) Eggs 10µm wide, 35µm long. l) Extremely elongated filament at tip of opercula cap of egg, extending outwards, exiting body first. m) Clamps one to four marginally equal in size. n) Intestinal tract diverticulate up to distal termination, marginal to origins of opisthaptor clamps.

39

4.5 ETYMOLOGY

This parasite has been classified as belonging to the genus Diplozoon, and as such has been described as a new specie belonging to this genus.

To complete this classification, the species name assigned to this specimen, is that of plicati, derived from the folds (plicae) that so characteristically occur on the posterior section of both individuals.

Full classification: Diplozoon plicati

40

FIGURE 4.1 : Scanning electron micrographs of Diplozoon plicati. A - suckers (a) on anterior end and pharynx protrusion (b). B – equal sized clamps (d) on prominent stalk (c). C – reproductive opening (e) of one mature Diplozoon in copula. D – opisthaptor protrusion (f) extending in dorsal and ventral direction as well as folds (g) covering both dorsal and ventral surfaces. E – Egg (h) containing zygote (i) extremely elongated filament (j).

41

FIGURE 4.2 : Micrograph of whole mount and serial sections of Diplozoon plicati. A – Posterior end of co-joined adults containing several eggs (a) as well as egg filaments (c). Ovaries are indicated by (b) and terminating intestinal diverticles by (d). B – Posterior end of co-joined adults clearly showing blood filled intestinal diverticles (d), equally sized clamps (e) and opisthaptor protrusion (f). C – Egg (a) with filament (c) and ovaries (b) as seen in stained whole mount. D - 5µm Section showing two eggs (a), one each in a fused individual.

42

FIGURE 4.3 : Parasite eggs with visible opercula lines which are not visible on SEM micrographs. A – Light micrograph of whole mount with filament (a), egg (b) and opercula line (c). B – Light micrograph of serial section with egg (b) and opercula line (c).

43

FIGURE 4.4 : Micrographs of body structures of Diplozoon plicati. A –section through the clamps and opisthaptor. B – fusion region showing reproductive organs. C – sagital section of complete body of single ind ividual. D – sagital section through opisthaptor showing protrusion of opisthaptor terminal border. E – cross section through anterior part of body in buccal area. 1 – opisthaptor protrusion. 2 – clamp position carried on stalk. 3 – intestinal tract. 4 – ovaries. 5 – vas deferens. 6 – anterior body region. 7 – testes. 8 – reproductive opening. 9 – anterior suckers. 10 – intestinal opening. 11 – pharynx.

44

FIGURE 4.5: Diagrammatic representation of reproductive structures from serial sections of Diplozoon plicati showing dorsal (individual one) and ventral views (individual two).(a) Relative position of vitelline gland and duct leading to mature oocytes; (b) Testis and vas deference; (c) Ovary with immature ova; (d) Ovary with mature ova; (e) Relative positioning of mehlis gland; (f) vas deference merging with posterior uterus; (g) uterine region leading to uterine pore containing developed and ripe egg.

45

FIGURE 4.6 : Distribution map of Diplozoon in Africa, showing locality of the three recorded descriptions of Diplozoon on the continent. # - Diplozoon g hanense; & - Diplozoon aegyptensis; @ - Diplozoon from current study.

46

FIGURE 4.7 : Comparative illustrations for type species of Diplozoon and Paradiplozoon, along with specimen from current study, to assist in taxonomic placement and classification. A – Paradiplozoon megan,. B – Diplozoon paradoxum ,. C – Diplozoon species from current study. i – Posterior part of body A, showing characteristic dilation immediately below clamps. ii & vii – Eggs with operculum visible, and origin of filament. iii & vi & xi – Central hook located parallel to clamps in opisthaptor. iv & viii & xii – Internal clamp structure. v – Posterior of body B with diverticulate intestinal tract. ix – Posterior of specimen C. x- Egg with filament origin and zygote .

47

FIGU RE 4.8 : Diagrammatic representation of transverse sections through (1) posterior part of uterus , (2) anterior part of uterus with egg; (3) uterine pore of Diplozoon plicati. (bb) brush border; (b.m.) basal membrane; (c.e.) columnar epithelial cell; (c.m.) circular muscle; (e.s.) egg shell; (l.s.) longitudinal section; (n.) nucleus; (s.) cilia; (t.) tegument.

48

FIGURE 4.9 : Diagrammatic representation of transverse sections through the (1) intestinum, (2) median ovovitelline duct, (3) mehlis gland of Diplozoon plicati. (b.m.) basal membrane; (c.e.) columnar epithelial cell; (g.c.n.) gland cells (unicellular) with nucleus; (l.c.n.) large cell with nucleus; (n.) n ucleus; (s.) cilia; (v.) vacuole.

49

FIGURE 4.10: Diagrammatic representation of transverse sections through (1) testis and vas deference, (2) spermatogonia. (b.m.) basal membrane; (t.) testis; (l.) lumen.

50

FIGURE 4.11: Diagrammatic representation of transverse sections through (1) young oocytes, (2 ) median part of ovary, (3) anterior part of ovary with mature oocytes. (b.b.) brush border; (b.m.) basal membrane; (c.e.) columnar epithelial; (c.m.) circular muscles; (l.m.) longitudinal muscles; (n.) nucleus .

51

CHAPTER 5: ECOLOGY

5.1 INTRODUCTION:

The intimate association which exi sts between all parasites and their respective hosts, has been the topic of numerous research papers in almost as many a field (ecology, zoology and physiology to name but a few). Due to certain parasite-host associations, the influence on the life, reprod uction (thus survival) and death of both the parasite and host individuals is a perfect focal point for any further investigation.

Hickman, Roberts & Larson (1993) define parasitism as being the condition of an organism living in or on another organism (host) at whose expense the parasite is maintained. It can be added that the obtaining of nutrients from the host normally causes harm, but does not cause death immediately (Begon, Harper & Townsend, 1996).

The perfect parasite -host association would of course ensure the continued survival of both host and parasite for the duration of the interaction. Both parties would in such a situation find at least the minimum of benefit (postponed mortality, fulfilled fecundity etcetera), but this is rarely if ever found in nature.

Begon et al. (1996) view the role of parasites in the shaping of community structure as least significant in communities where physical conditions are more severe, variable or unpredictable. For the present study, located in a pointedly stab le environment, it would thus seem to imply that small variations in conditions such as the geographic location, season, temperature, host

52 biology, size and habits of the host would be exposed in an obvious result, even if determined only statistically.

Because the host’s environment can be seen as the macro-environment of the parasite, it is quite obvious that the physical connection between parasite and host would constitute the micro -environment. All influences from this macro - environment would depend solely on the host, thus including factors such as gender, age, length and weight of the host (Barse, 1998). These are factors included in the current investigation on the occurrence of the Diplozoon sp. on Labeo umbratus .

Due to the exothermic nature of the fish, the parasite’ micro-environment can be highly affected by the temperature of the external environment. The cycles of seasons and temperatures have an obvious influence on the prevalence, mean intensity and abundance of parasites which choose to attach themselves to the exterior of a host in this changing macro-environment (Pilcher, Whitfield & Riley, 1989).

A decline in the physical water quality is shown to have a generally negative effect on ecto -parasite numbers (Koskivaara & Valtonen, 1991; Koskivaara, Valtonen & Prost, 1991; Bagge & Valtonen, 1996). Specifically the dissolved oxygen concentration, pH-values, salinity and temperature all have an influence on the parasites (Rohde, 1993; Barker & Cone, 2000; Chapman, Lanciani & Chapman, 2000).

Abovementioned factors are relevant also in affecting the preferred site attachment to the gill filaments. Due to the differing flow of water over the gills, different areas are exposed to differing degrees of factors such as pH, temperature etcetera. Numerous research projects have attempted to ascertain a clear connection between different factors and the preference for attachment site of Monogenean parasites (Buchmann, 1988; Buchmann, 1989; Gelnar, Hodová, Koubková, Simková & Zurawski, 2000).

53 Besides having preferred sites of attachment, clear preferences could exist with regard to the actual chosen host. Parasitism often leads to a fine scale of host specificity (Begon et al, 1996), and this would be influenced by the specific species of parasite and host involved in the interaction. For this reason the extent of host specificity of Diplozoon sp. is investigated.

According to Le Brun, Renaud & Lambert (1990), the host-specificity in the natural environment can be considered to express the realization of three independent but essential conditions: The first being the ecological condition, which implies that the host must be spatially accessible to the dispersal stages of the parasite. The second is an ethological condition where the behavioral characteristics of both host and parasite are essential to their encounter. A third mesological condition is necessary where the potential host must provide suitable physiological conditions for the parasite development.

With the majority of monogeneans being host specific (Noble & Noble, 1982; Poulin, 1992), and some also occurring on more that one host, it is an added incentive to establish the host range in order to place the Diplozoon sp. in the correct ‘category’ of monogeneans.

With the position and specificity cleared up, another factor to consider is the impact of the quantity of the parasite species on the physical condition of the host, i.e. the condition factor of the host. This study attempts to establish whether the impact of Diplozoon sp. on the condition factor of Labeo umbratus is negative, or negligent and then to supply further informative views on future in depth studies linked to the specific host parasite relationship and possibilities of aquaculture.

5.1.1 Occurrence

Investigation of the mono geneans parasite Pseudodactylogyrus anguillae occurring among wild eels (Anguilla rostrata) resulted in prevalence values of between 0.395 and 0.424, depending on the pH and temperature of the

54 location (Barker & Cone, 2000). Chapman et al (2000) determined the prevalence of Neodiplozoon polycotyleus in Barbus neumayeri. Of those hosts infected, 37.1% had one parasite individual, and 62.9% of the infected hosts had two parasite individuals.

In light of these previous results, the occurrence of the Diplozoon sp. is investigated and compared to that of other in the monogenean family.

5.1.2 Host specificity

The host specificity of a parasite takes into consideration the number of host species and the relative preference for each of these (Rohde, 1993). Many parasites and pathogens are host specific or at least have a limited range of hosts (Begon et al, 1996).

Begon et al (1996) also states that for any species of parasite the potential hosts are only a tiny subset of the available flora and fauna.

There may be detailed correspondence between specific genotypes of parasite and specific genotypes of the host (Begon et al, 1996). Parasites which infect a single host taxon or related taxa are said to exhibit phylogenetic host specificity (Rohde, 1993).

Ecological host specificity can also be found where a parasite with a wide host range exhibits certain host preferences which are usually determined by the ecological requirements of that host (Rohde, 1993). Host specificity varies widely among different taxa of parasites (Poulin, 1992).

Monogeneans tend to be more specific than Copepods, Digeneans, Acanthocephalans, Cestodes and Nematodes, with a larger proportion of species with only one known host (Poulin, 1992). Simková, Desdevises, Gelnar and Morand (2001) regard the monogenean species to be highly host specific when compared to other parasitic groups of fish. In recent years the question of parasite susceptibility transferal with hybrid fishes has been

55 raised, but greatly detailed genetic studies of both the involved hosts and parasites is a prerequisite for any continuing investigation into this phenomenon of the host specificity factor.

Other ectoparasites have been recorded from L. umbratus occurring in the Olifants River system. The parasite on record is a Crustacean skin parasite, Argulus japonicus (Avenant-Oldewage, 2001). This parasite causes severe localized skin damage (Avenant-Oldewage, 2001), whic h in turn would allow for an increase in susceptibility for parasitizing by other parasites, and could contribute to a decrease in condition factor.

5.1.3 Size and age of host

Age difference in the infection with parasites may often be due to a simple accumulation effect; where with increase in age comes an increase in the exposure to parasite infestation (Rohde, 1993).

Host body size was the factor most consistently correlated with parasite community richness (Poulin, 1995). Also stated by Poulin (1995) are possibilities that the larger host may consume greater quantities of food, that they may offer more space to parasites and may provide greater variety of niches for parasite occupation.

In a specific case where Kagel and Taraschewski (1993) inve stigated the host-parasite interface of Diplozoon paradoxum on Abramis brama, it was found that the greater prevalence of the parasite occurred on larger specimen of between 46 and 55cm. The prevalence of the adult D. paradoxum increased exponentially with an increase in host size.

The Diplozoon sp. found on Labeo umbratus was investigated with hopes of finding similar results.

56 5.1.4 Host gender

Chappell (1969) analyzed the parasitic incidence and intensity in male and female individuals of Gasterosteus asuleatus and found no significant differences, one of the parasite species he investigated in this case was Gyrodactylus rarus .

Arme and Halton (1972) observed the occurrence of Diclidophora merlangi, a monogenetic parasite of Gadus merlangus and found no differences in infection attributable to the sex of the host.

Gyrodactylus stephanus was studied as they occur on Fundulus heteroclitus and it was found that no significant difference in the densities of gyrodactylids existed between male and female fish (P>0.05) (Barse, 1998).

According to Rohde (1993), ecto parasites could infect the two genders differently because male and female fish often have different feeding habits.

Due to above -mentioned disparities the existence of any preference for host gender is investigated this case.

5.1.5 Host behaviour and habitat

According to Rohde (1993) plankton feeders have relatively few kinds and numbers of parasites and the frequency of infection is low, whereas carnivores have many kinds and numbers of parasites occurring at higher frequencies.

With L . umbratus being herbivorous and detrivorous (Gaigher, 1984; Dörgeloh, 1995), the present study aims to confirm or refute the above mentioned statement made by Rohde (1993). L . umbratus plays an important role in the energy cycle by utilizing detritus which is a stable food source in lentic systems (Gaigher, 1984).

57 Spawning of L. umbratus is polyandrous, where several males ‘drive’ a single female (Jackson and Coetzee, 1982; Mitchell, 1984; Tómasson, Cambray and Jackson, 1984; Skelton, 2002). L. umbratus is a confirmed flood plain spawner with a relatively high fecundity and long life cycle (Gaigher, 1984).

5.1.6 Seasonality and temperature

Most groups of animals and plants have more species in warm than in cold environments (Rohde, 1993). It is generally accepted that temperature is the most important single factor determining the distribution of marine organisms (Rohde, 1993).

Variations in temperature on and within the surface of the earth have a variety of causes: the effects of latitude and altitude, continental, seasonal and diurnal effects, microclimatic effects and, in soil and water, the effects of depth (Begon et al., 1996). Temperature will affect the behaviour of all the organisms that it interacts with, its competitors, parasites, symbionts and all other species that create of modify the physical environment in which it lives (Begon et al.. 1996).

Fluctuations in the temperature would thus not only affect the distributions of the host species, but also the infesting parasites and all relevant stages in the life -cycles of the parasites.

With the solubility of oxygen in water decreasing when temperatures rise (Begon et al., 1996) it could be increasingly important to ascertain the exact relationships between environmental temperatures and the completion of the life -cycles of the relevant parasite and the host.

For the monogenean Dicliphora merlangi occurring on the whiting, Merlangius merlangus, it was shown that there was a significant decrease in number of parasites during winter when compared to numbers in summer (Pilcher, Whitfield and Riley, 1989).

58 Due to L. umbratus having definite temperature preferences depending on season (Hattingh, 1975), this study investigated the influence of the season and temperature on the Diplozoon sp. of L. umbratus .

5.1.7 Water quality

Monogeneans are sensitive to envi ronmental changes (Bagge and Valtonen, 1996) and considering the fact that the examples of ecto parasites on the gills of the hosts are largely exposed to the changes in quality of the water that flows over the gills, this is an important matter to consider.

A decline in water quality will firstly influence the health status of the host, and this could in turn have two effects: it could negatively affect the occurrence of parasites on the host, or it could increase the numbers of parasites due to the negatively affected host which may have a lower immunological response to the parasites (Bagge and Valtonen, 1996).

Koskivaara and Valtonen (1991) found that the prevalence of Paradiplozoon homoion infection varied from 1.9 – 8.5% with the lowest value found in a polluted lake and the highest in an eutrophic lake.

Due to the significant correlation that was found by Burleson, Wilhelm and Smatresk (2001) to exist between fish mass and the level of dissolved oxygen during their study on the large mouth bass, it is important to keep in mind then the correlation that could exist between host size and prevalence of parasites. If a positive correlation could be found during this study between host size and prevalence, then surely also logically a correlation would exist between parasite prevalence and level of dissolved aquatic oxygen.

The water quality determination is then of great significance in the exact determination of the effect that pollutants would have on host individuals, their relevant parasites and the interactions that would follow.

59 Koskiva ara, Valtonen and Prost (1991) and Gelnar, Koubková, Plánková and Jurajda (1994), all found an increase in monogenean parasites in polluted localities studied, whilst Grabda-Kuzubzka and Pilecka – Rapacz (1987) found no indication that sewage pollution had a harmful or positive influence on monogenean parasites over a period of 50 years.

Due to the differing water qualities encountered at the two collection sites, a possible difference in prevalence, mean intensity and abundance of Diplozoon sp. on L. umbratus is investigated.

5.1.8 Attachment sites

The majority of monogeneans colonize the gill system (Lambert, 1990). Rohde (1977) showed that ecto-parasites on the gills of marine fish may partition their microhabita ts in the following ways: (i) transverse partitioning = preference for certain gill arches; (ii) longitudinal partitioning = preference for certain microhabitats along the longitudinal axis of the gills; (iii) vertical partitioning = preference for certain microhabitats along the axis extending from the tip pf the gill filaments to the bony parts of the gills; (iv) lateral partitioning = preference for external or internal gill filaments.

The gill micro environment is influenced by the macro environment, i.e. the biotic and abiotic conditions of the environment that surrounds the host- parasite system (Lambert, 1990). Most parasites select a site where they are not subjected to the full force of the macro environment (Wootten, 1974). When regarding the size of the gills alone, it could be speculated that in freshwater fishes the most posterior gills have less water flowing over then than those positioned more anteriorly (Paling, 1968).

Rohde (1991) studied the possible correlations between microhabitat width and number of parasite species on a host fish species, and found that none existed.

60 The majority (77%) of Neodiplozoon polycotyleus were located on filaments of the second gill arch, but the distribution did not differ between the right and left sides o f the branchial basket of Barbus neumayeri (Chapman et al, 2000).

Wootten (1974) found that Dactylogyrus amphibothrium showed a marked preference for the dorsal segment over the median and ventral segments.

In a study conducted by Rohde, Hayward, Heap and Gosper (1994) it was shown that the bony parts of the gills (gill arches) were never found to be infected, which indicates the availability of an empty niche.

The gills of L. umbratus were numbered and divided as indicated in the Materials and Methods section, and collection data was carefully studied to try and find any kind of possible preferred location on the microhabitat of the host.

5.1.9 Condition factor

Length-weight relationships are useful for fisheries research as they allow an estimate of the condition of the fish to be made (Stergiou and Moutopoulos, 2001). When this length-weight relationship is compared to the parasite load-length relationship, a clear picture can also be formed of the actual effect that the presence of a parasite can have on the condition of the host.

With the economic importance of L. umbratus rising in the last few years, it would be of great importance for freshwater-fish farmers to know exactly what effect the major fish parasites could have on their stocks. This it would save the cost of the implementation of possible unnecessary pest control plans if non-destructive parasites are found.

For this reason the condition factor is calculated in the present host-parasite interaction.

61 5.2 MATERIAL AND METHODS

All relevant methods applied to this section of the study have been discussed in Chapter 2, and will not be touched upon here again.

5.3 RESULTS

5.3.1 Occurrence

Prevalence, abundance and mean intensity of the Diplozoon sp. as collected from the L. umbratus hos t at both the Vaal Dam and Vaal River Barrage sites, was calculated as suggested by Margolis et al (1982). The subsequent values are compared and discussed.

From the Vaal Dam 12 of the 77 specimens of L. umbratus collected showed an infestation of Diplozoon sp. which yields a prevalence value of 0.156 or 15.6%. when compared to the collection from the Vaal River Barrage, 31 of 74 specimens collected were infested, thus with a prevalence value of 0.419 or 41.9%.

Due to the great difference in prevalence va lues between the two sites, the data from both localities were used separately, where statistically possible, to clarify the ecological specificities of the Diplozoon sp.

An average mean intensity for the Vaal Dam specimens showed a value of 1.56, with a maximum infestation of three parasites. A co-joined pair of adult Diplozoon sp. is viewed as one parasite. For the Barrage site, an average mean intensity of 4.07 was found, with a maximum infestation here of 10 parasites.

With regard to the abundance values, an average value of 0.207 for the Vaal Dam and 3.087 for the Barrage site are calculated.

62

Through light microscopy, adult parasites with eggs could be identified and it is shown that a total of 6 parasites from the Barrage site only, had obviously visible eggs. The highest egg percentage, 0.3%, was found in specimens collected during January, with specimen in October showing an egg containing percentage of 0.07%, and a value for June of 0.03%.

5.3.2 Host specificity

Of the 151 individual L. umbratus hosts collected, a total of 50 were found to be infested by the Diplozoon sp. Of the other fishes collected as discussed in Material & Methods, only Labeobarbus aeneus was found to have an unconfirmed infestation of Diplozoon sp. In-field identification from egg shape was done, and will be confirmed or refuted in further studies.

5.3.3 Host size

Due to the statistically small number of infected fish collected, the correlations for host size and parasitism are not split between catchment sites, and are thus regarded as a whole.

A particularly strong correlation at the 0.01 level was illustrated between weight, fork length and total length of the host fish. A weak correlation exists between the numbers of Diplozoon sp. specimen collected and the weight of the host fish. For the fork length and total lengths a strong correlation was indicated (at the 0.01 level) as can be seen in TABLE 5.1.

63 TABLE 5.1: Correlations existing between various host fish parameters and the total number of Diplozoon sp. found.

Parameter Correlation Weight (kg) Fork length Total length (cm) (cm) Weight Pearson 1.000 0.932 ** 0.918 ** Sig.(2-tailed) 0.000 0.000 Fork length Pearson 0.932 ** 1.000 0.988 ** Sig.(2-tailed) 0.000 0.000 Total length Pearson 0.918 ** 0.988 ** 1.000 Sig.(2-tailed) 0.000 0.000 Total number Pearson 0.182 0.377 ** 0.373 ** Diplozoon sp. Sig.(2-tailed) 0.205 0.007 0.008 ** Indicates that correlation is significant at the 0.01 level (strong) Absence of * indicates weaker correlation at 0.05 level.

5.3.4 Host gender

Of the total 151 host individuals collected, 87 were male, and 64 were female. Of these gender divisions, 36 male fish and 14 female fish were infected with Diplozoon sp. (Fig. 5.1). Once again, due to the statistically small values, no location division is made.

From the statistical data, it is indicated that preference is shown for male hosts, and not female. Greater clarity is provided in Table 5.2, with regard to the total number of parasites found.

64 TABLE 5.2: Statistical data regarding gender preference of Diplozoon sp. Gender Statistical parameter Statistical outcome ? Mean 3.18 Median 2.00 Variance 8.40 Std. deviation 2.90 Prevalence 0.39 Abundance 1.316 Maximum 12.00 ? Mean 1.43 Median 1.00 Variance 0.26 Std. deviation 0.51 Prevalence 0.22 Abundance 0.31 Maximum 2.00

5.3.5 Seasonality and temperature

Sampling sites were not separated for this part of the investigation, because the temperature between the sampling sites varied by only a few degrees. The results obtained are shown in FIGURE 5.2.

No definite seasonal occurrence of eggs could be clearly established, and the development of such could possibly thus rely on a number of other stimuli. The differing peaks in prevalence appear to be in no way connected to the fluctuations in temperature, and it is thus clear that other factors are the main influences of the reproductive cycles of this parasite.

65 5.3.6 Water quality

Forty-six percent of the total runoff from the Barrage catchment area is return flow, of which 75% is from sewage works, 11% from industries and 14% from mines. The main water quality problems then in this area are increased salinity and eutrophication (Braune and Rogers, 1987). Eutrophication in the enrichment of water with plant nutrients (mainly nitrates and phosphates) which stimulates the growth on undesirable aquatic plants such as algae and hyacinths (Department of Water Affairs and Forestry, 1995), this in turn detrimentally affects the water quality.

The Vaal River catchment area incorporates the rivers that drain the highly industrialized Witwatersrand. The effect can be seen in the abovementioned description of the Barrage site.

The best quality water is found in the catchment area of the Vaal Dam, which is the most important supplier of water to the Vaal River. Water quality is good with most mining and fuel processing plants having minimal return flown to the river (Braune and Rogers, 1987).

Agricultural activities such as cattle grazing on natural pastures and dry land cultivation are conducive to good water quality, but the use of unnatural fertilizers and pest control methods are a relative unknown factor which could easily circumvent any goodness coming from such agricultural activities. Another factor which should now be taken into account is the new influx of water from the Highlands water scheme in Lesotho. This water is not only algae rich, but is also bringing in different quantities of nutrients and possibly pollutants from distant and previously unconnected sources.

66 All data used is relevant for the bimonthly surveys and are the variables obtained as surface level. Temperature, pH, conductivity, oxygen content, salinity and turbidity were recorded.

The temperature between the two sites differed by a few degrees only.

At the Barrage site, the pH was generally higher, more alkaline. Levels for the conductivity, oxygen, salinity and turbidity were also generally higher at the Barrage than at the Vaal Dam.

Due to the differing nature of these two sites, the data sets regarding the water quality were split and investigated thoroughly. The results obtained can be seen from the graphs included in FIGURE 5.3 (for the Vaal Dam), and FIGURE 5.4 (for the Vaal River Barrage).

It can clearly be seen from the data sets that conductivity and salinity do not appear to have any influence on the values of parasite prevalence at either site. The conditions which do however appear to have an influence, are pH values and turbidity.

At the Vaal Dam site, the fluctuation in turbidity is minor, but the fluctuation for the pH levels is quite significant. The relationship between parasite prevalence and pH is seen clearly as the values change during the March and August sample periods. It would appear that with a rise in pH, a subsequent rise in prevalence can be expected.

The Vaal River Barrage site shows a similar increase in pH during the March sampling period, but not the August one. The change in pH also does not seem to be connected or influential to the prevalence values of the parasite.

67 At the Barrage site, the factor which appears to have the greatest influence is the turbidity levels. When the turbidity level is highest during the August sampling period, the prevalence value of the parasite is the lowest. There would thus appear to be an opposing connection between turbidity and parasite prevalence values.

5.3.7 Attachment sites

No oncomirasidia or diporpa were collected on any of the gills or gill arches.

On the left gill arch, the total number of parasites was the greatest on gill number one (15), the dorsal position showed to be the most preferred (total of 19)

When viewing data with regarding the right gill arch, the total number of parasites was the most on gill four (13), with the ventral position being preferred (total of 15).

Thus no significant preference for a specific gill arch or position is found.

5.3.8 Condition factor

A significant correlation at the 0.01 level (0.522) was found to exist between the weight of the host fish and the condition factor, but not between the condition factor and the fork length or total length.

When correlated with the number of parasites, a negative correlation to the condition factor is shown (-0.330).

When viewing the data sets separately for the condition factor at the two sampling sites, both sites show a negative correlation. This indicates that under

68 no influence from outside water qualities, the presence of this parasite is detrimental to the health of the host (FIGURE 5.5).

5.4 CONCLUSION

This parasite shows some rather restrictive ecological variations such as preference for gender, and also rather vague natural boundaries shown by turbidity being the only major environmental restrictor or agitator.

This apparent lack of rigid guides may be a reason for the previous unknown status of this parasite. If its ability to adapt to hosts and or environments is indicated by the results obtained, the distribution in the inland waters of South Africa could be greater than anticipated.

69

FIGURE 5.1: Histogramme showing prevalence values for host gender of Diplozoon plicati collected from Labeo umbratus in the Vaal Dam and Vaal River Barrage.

70

30

25

20

15

10 Temperature 5

0

1.2

1

0.8

0.6

0.4

Prevalence % 0.2

0 Jan'99 Mar'99 Jun'99 Aug'99 Oct'99 Feb'00 Sample dates

Vaaldam Barrage

FIGURE 5.2: Showing temperature fluctuations and the relationship to prevalence at the two sampling sites. December to February are the summer months. March to May are autumn months. June to August is winter, and September to November is spring.

71 Vaal Dam data Vaal River Barrage Data 50.0 150.0 40.0 30.0 100.0 20.0

Turbidity 10.0 50.0 Turbidity 0.0 9 0.0 8.8 9

8.6 8.4 8.5 8.2 8 8

pH values 7.8 7.5 7.6

pH Values 7.4 7 0.35 6.5 0.3 1.2

0.25 1

0.2 0.8

0.15 0.6

0.1 0.4 Relative values

0.05 Relative values 0.2

0 0 Jan'99 Mar'99 Jun'99 Aug'99 Oct'99 Feb'00 Jan'99 Mar'99 Jun'99 Aug'99 Oct'99 Feb'00 Sample dates Sample dates Cond. Sal. Prev. Cond. Sal. Prev.

FIGURE 5.3: Water quality data reflecting parasite FIGURE5.4: Water quality data reflecting parasite prevalence at differing conditions. prevalence at differing conditions.

72 A 1.40 1.20 1.00 0.80 0.60 0.40

Number of parasites 0.20 0.00 0 0.5 1 1.5 2 2.5

B 1.6

1.4

1.2

1

0.8

0.6 Number of parasites 0.4

0.2

0 0 2 4 6 8 10 12 14 Condition Factor value

C.F. Linear (C.F.)

FIGURE 5.5: Condition factor analysis for A – Vaal Dam site and B – Vaal River Barrage Site.

73

CHAPTER 6: DISCUSSION

After all information with regards to the specimens collected from the gills of Labeo umbratus has been gathered, the following can be ascertained:

This species is a new discovery from the Southern hemisphere, in particular South Africa.

It has been compared to all available descriptions of Diplozoon and Paradiplozoon. After completion of the comparison it is quite clear that although the specimen shares a number of characteristics, there is not one instance where all characteristics would place it within a particular known species.

It is determined that the species collected belongs to the genus Diplozoon due to the presence of the unique features expressed and emphasised in Chapter 4.

Mazourková, Matejusora, Koubková, & Gelnar (2003) emphasized the measurements of the marginal hooks found in the clamps in identification and distinguishing the species of the Diplozoon genus (FIGURE 6.1).

The important characteristics list is as follows: 1. median plate 2. anterior part of median plate 3. posterior part of median plate 4. trapeze spur 5. anterior joining sclerites of median plate

74 6. sclerites of the proximal tip of the median plate 7. sclerites of the distal tip of the median plate 8. spiky spur 9. anterior jaw 10. median sclerites of posterior jaw 11. lateral sclerites of posterior jaw

The morphological measurements taken are reflected in Chapter 4 and confirm the above placement within the existing genus Diplozoon and support the creation of the new species plicati

The shifting of almost all the once Diplozoon species to the genus Paradiplozoon, could be a case of taxonomic over-eagerness. Matìjusová, Koubková & Cunningham (2003) investigated the ITS2 rDNA by using RFLP Digestion. This resulted in the disclosure of a close relationship between P. sapae, P. nagibinae, P. bliccae and D. paradoxum. With a regard to this result, they question the validity of the genera Diplozoon and Paradiplozoon.

For completion of the host specificity study the difficulty in distinguishing between L. umbratus and L. capensis purely on the basis of haemoglobin electropherogram was found by Du Toit, Hattingh and Schabort in 1973. Van Vuuren, Mulder, Ferreira and Van der Bank (1990) completed a morphometric and electrophoretic analysis between L. capensis and L. umbratus from two localities in South Africa (one being The Vaal River Barrage), and found that the two species can be clearly separated on morphometric characters alone. Van Vuuren et al. did notice an insufficiency in data when the two species hybridise. The Diplozoon in question from this study was only ever found on the single host example, namely L. umbratus. No parasite specimens that remotely shared morphological traits with the current parasite were found occurring on a L. capensis.

75 If any hosts that were sampled were in fact the hybrid offspring from a L. umbratus and L. capensis cross the only way to investigate the specificity or host preference of this parasite would be to do a full study on the biochemical make- up (by use of gel electrophoresis amongst others), DNA sequencing, morphometric detail, etcetera of the hybridizing fishes.

Any indistinguishability found would perhaps provide grounds for a search of the parasite on the host L. capensis which will definitely put to rest the question of host specificity. If indeed these studies deliver results which show the presence of the current parasite on both the Labeo species mentioned, it would change the idea that most monogenean species are highly host-specific (Simková et al., 2001).

Upon completion of a host specificity study, further results supporting or refuting the male gender preference could be obtained. The male congregating behaviour during spawning, and subsequent disturbance of benthic constituents, could prove important in awakening perhaps dormant eggs from this or many other parasites to completion of their life cycles.

When studying specificity and host predictability, Sasal, Trouvé, Müller – Graf and Morand (1999) found a significant positive correlation between host body size and parasite richness for specialist species (P<0.02). Sasal et al. (1999) also found a significant correlation between host body size and percentage of infected hosts when working with specialist parasite species. These findings can serve to initiate multiple studies with regard to the current parasite species and their host, especially once the connection between the parasite and other hosts has been established.

The host size range is expected to remain as stated herein, but obvious confirmation will come from completion of further studies.

76 Site preference or attachment preference is also expected to remain as stated here. There would seem to be no explanation why the occurrence on either the left or right gill arches should be found in this parasite as infection would be entirely coincidental. The only fixed environmental factor to have any bearing on attachment positioning is the flow of current over the gills and their different parts. Different swimming techniques lend themselves to differing rates of flow as needed by the individual fish species. Therefore, if any preference for left or right side, or location between gill arches is found, the facilitator would be the rate of water flow.

If water flows faster over the gills, a greater amount of egg containing water could rush over the gills and in so doing possibly increase the number of attached parasites (if given enough time and opportunity to hatch to larval form, and fuse with another). No attachment preference is found to exist here.

To further investigate the seasonality of this parasite and in so doing determine the detail of its life cycle; further investigation would have to be completed. Also to be attempted if possible is laboratory completion of the life cycle with live captive host fish. The susceptibility to natural infestation could be investigated at the same time as well as the interrelation between infected and parasite-free individuals in order to determine means of distribution between individuals.

The influence of water quality on the prevalence of the parasite does seem to support the need for further investigation. More parasites were collected from the Vaal River Barrage site than the Vaal Dam site. Overall the factor which appeared to have the greatest influence upon the prevalence is the turbidity levels. With the rise in turbidity at the Vaal River Barrage site, two low points in prevalence are found, in opposition to the one high peak at the Vaal Dam where the turbidity is quite constant. The natural habits of the host L. umbratus as a bottom-feeder would indeed make it more likely to come across any parasite eggs lying dormant.

77

A possible trigger for egg laying in the parasites would appear to be a rise in turbidity levels. During agitation of the environment by the males these levels could be reached, and egg lying could commence. In so doing showing the reliance on levels of turbidity, the male spawning habit, and the connection with the prevalence levels from the localities. Further added information would help greatly to confirm such an influence.

When the condition factor analysis was completed it showed negative correlations between host health and parasite prevalence for both sites. This means that the presence of the parasite is negative to the health of the fish, but more so if the host is also found in a polluted environment where the combined effect of parasite and pollution increased the negative effect.

The importance of the host fish in food supply circles is not that wide spread, but with the interest in fish farming intensifying, the focus could very well shift in the near future. Members of the host genus like Labeo capensis and Labeo molybdinus are known to be taken as food (www.naturalhub.com, 2000). If then the fish farming community were to focus on this genus as a main source, the effect of a negative parasite presence through the ranks of the genus could prove costly. Özer & Erdem (1999) found that the overall prevalence of trichodinids and dactylogyrids was higher on farmed carp than on wild carp. This information alone should make anyone interested in fish farming more vigilant to the presence of ectoparasites.

78

FIGURE 6.1: Characteristics as put forward by Mazourková et al. (2003) for identification of Diplozoon species shown on clamp detail from Diplozoon plicati as in Chapter 4. a – median sclerit of posterior jaw. b – sclerites of the distal tip of the median plate. c – sclerites of the proximal tip of the median plate. d – posterior part of median plate.

79

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92

APPENDIX

A.1 For ease of reference a table has been made in summary of staining techniques used as discussed in Chapter 2.

Boraxcarmine-iodine Horen’s Trichrome Mayer’s Hematoxylin (Manual of Veterinary Parasitological (Humason, 1979) Techniques, 1986). Remove parasites from 70% Prepare stock solution of Gradually hydrate ethanol solution. lactophenol. parasites form 70% ethanol, rinse with tap water. Place in saturated iodine- Place parasites in warm, 60oC to Stain in Mayer’s ethanol (70%) for 6-7 hours. 70oC coloured lactophenol solution. Hematoxylin for 20 minutes. Transfer to 70% ethanol for Clear and mount parasites in Differentiate in acid further 12 hours. lactophenol. alcohol (70% ethanol & hydrochloric acid) Counter stain in 3% Seal cover slip with Glyceel Rinse in tap water. Boraxcarmine (Galigher, (Hopkin & Williamsä) 1934) in distilled water Place in Scott’s solution to (Humason, 1979) for 2 hours. blue. Rinse in water, gradually Dehydrate. dehydrate to 100% ethanol. Clear in xylene. Clear in xylene. Mount in Entellanä. Mount in Entellanä.

93 A.2 Species list of members of the Family Diplozoidae Palombi, 1949., including host, locality and reference as discussed in Chapter 3.

Table showing all previous discoveries of Diplozoon species throughout the world

Tot. Diplozoidae species Locality Host length(mm) Posterior(mm) Anterior(mm) Ratio Reference 1 P. aegyptensis Egypt, Labeo forskalii 3620-5760 Paperna, 1979 Lake Albert Labeo forskalii Paperna, 1996 Lake Albert Labeo forskalii Fischtal & Kuntz 1963 Ghana L. coubie Paperna, 1979 adult & larv. Egypt, W. Africa L. victorianus Petr & Paperna, 1979 Aswa, White adult & larv. Nile L. victorianus Petr & Paperna, 1979 adult & larv. Kenya L. victorianus Paperna, 1979 Uganda, Barilius loati Paperna, 1979 Aswa River Barilius loati Paperna, 1979 adult & larv. Tanzania L. cyclindricus Paperna, 1979 larv. Ruaha river L. cyclindricus Paperna, 1979 larv. Nzoia Rivert Barbus paludinosis Paperna, 1979 larv. Egypt Barbus paludinosis Petr & Paperna, 1979 larv. Aswa Barbus paludinosis Petr & Paperna, 1979 adult & larv. Lake Albert Alestes macrolepidotus Paperna, 1979 adult & larv. Butiaba Alestes macrolepidotus Paperna, 1979 adult & larv. Uganda Alestes macrolepidotus Paperna, 1979 2 P. agdamicum Azerbaijan Leuciscus cephalus orientalis Mikailov, 1973 3 P. balleri Ukraine Abramis ballerus 4000 1:1.2 - 1.9 Koval & Pashkevichute, 1973 4 P barbi U.S.A. Barbus semifasciolatus 1000 - 1300 Yamaguti, 1963 U.S.A. Puntius tetrazona Reichenbach-Klinke, 1954 Sumatra Barbus punctius 1000 - 1300 300 - 700 400 - 800 Tripathi, 1957 5 P. bergi Ukraine Abramis sapa 2600 - 5900 1:1 - 1.7 Koval & Pashkevichute, 1973 6 P. bliccae Ukraine Blicca bjoercna 3600 - 4500 Koval & Pashkevichute, 1973 7 P. bychowsky Soviet Union Koval & Pashkevichute, 1973 8 P. capeotobrama Soviet Union Koval & Pashkevichute, 1973 9 P. cauveryi India Cirrhinia cirrhosa 4780 - 5140 1500 - 1760 3110 - 3370 Tripathi, 1957 10 P. chazarichum Azerbaijan Rutilus friskii kutum Mikailov, 1973

94 11 P. diplodiscus Soviet Union Koval & Pashkevichute, 1975 12 P. ghanense Ghana Alestes macrolepidotus Thomas, 1957; Yamaguti, 1963 Ghana Alestes baremoze Paperna, 1969 Justine, Le Brun & Mattei, 1985; Lambert & Gharbi, 13 P. gracile France Gobio gobio 1995 France Barbus meridionalis Lambert & Gharbi, 1995 France Leuciscus soufia Lambert & Gharbi, 1995 France Phoxinux phoxinus Lambert & Gharbi, 1995 14 P. gracilis Germany Gobio gobio 2500 Koval & Pashkevichute, 1973; Bychowskii, 1962 15 P. halleri Soviet Union Koval & Pashkevichute, 1973 16 P. homoion Volga delta Rutilus rutilus 3000 - 5300 1:2 - 2.5 Bychowskii, 1962; Yamaguti, 1963 Ukraine Rutilus rutilus 4000 1:1.6 - 2 Koval & Pashkevichute, 1973 Leningrad Rutilus rutilus 3000 - 5300 1:2 - 2.5 Bychowskii, 1962; Yamaguti, 1963 Poland Phoxinus phoxinus 2340 - 3600 72.4 - 117 156 - 218 1:1.7 - 2.1 Prost, 1974 Poland Rutilis rutilius Prost, 1974 Russia Cyprinidae species Khotenovsky, 1977 17 P. homoion bliccae Reichenbach-Klinke, 1980 18 P. homoion gracile France Barbus meridionalis Macdonald & Jones, 1978 Poland Gobio gobio Prost, 1984 19 P. homoion homoion Reichenbach-Klinke, 1980 20 P. homoion sappae Reichenbach-Klinke, 1980 21 P indicum Lucknow Barbus sarana 6000 - 9600 2000 - 2900 4000 - 6700 Tripathi, 1957; Yamaguti, 1963 22 P. kashmirense Kashmere Schizothorax sp. 2300 - 4320 900 - 1720 1400 - 2640 Tripathi, 1957; Yamaguti, 1963 23 P. kurensis Azerbaijan Barbus lacerta cyri Mikailov, 1973 24 P. kuthkaschenichum Azerbaijan Alburnus filipii Mikailov, 1973 Azerbaijan Alburnus charusini hohenackeri Mikailov, 1973 25 P. markewitschi Ukraine Vimba vimba 5000 1:1.7 - 2 Koval & Pashkevichute, 1973 Ukraine Blicca bjoercna Koval & Pashkevichute, 1973 Poland Blicca bjoercna Wierzbicka, 1974 Poland Scardinius erythropthalmus Wierzbicka, 1974 Russia Cyprinidae species Khotenovsky, 1977 26 P. megan Volga delta Leuciscus idus 3500 - 7300 1:2.14 - 3.3 Bychowskii, 1962; Yamaguti, 1963 Ukraine Leuciscus idus 6000 1:2.2 - 3 Koval & Pashkevichute, 1973 Poland Leuciscus idus 6050 - 8480 1440 - 2450 4300 - 6050 Grabda-Kazubska & Pilecka-Rapacz, 1987 Leningrad Leuciscus idus 3500 - 7300 1:2.14 - 3.3 Bychowskii, 1962; Yamaguti, 1963 27 P. microclampi Kulkarni, 1971 28 P. mingetshauricum Azerbaijan Barbus capita Mikailov, 1973 29 P. minutum Israel Phoxinellus kervellei 770 - 1580 320 - 400 450 - 1180 1:1.41 - 2.96 Paperna, 1964; Paperna, 1996

95 Israel Tylognathus steinitziorum Paperna, 1964; Paperna, 1996 30 P. nagibinae Poland Abramis ballerus Wierzbicka, 1974 Ukraine Abramis ballerus 7000 1:1.6 - 2 Koval & Pashkevichute, 1973 31 D. nipponicum Japan, China Carassius vulgaris 6600 - 7300 Yamaguti, 1963 Japan, Cyprinus carpio 5000 - 7500 200 350 1:1.5 - 2 Bychowskii, 1962; Hirose, Akamatsu & Hibiya, 1987; Ukraine Cyprinus carpio Kamegai, 1968; Kamegai 1970 Russia Cyprinidae species Khotenosky, 1977; Koval & Pashkevichute, 1973 Tokyo city Carassius carassius 6600 - 7300 2750 - 2900 3700 - 4500 Tripathi, 1957; Kamegai, 1968 Japan Carassius auratus Hirose, Akamatsu & Hibiya, 1987 32 D. paradoxum Russia Cyprindae species Khotenovsky, 1977 Europe Cyprinus sp. 4000 - 11000 2220 2640 Tripathi, 1957 Europe Esox sp. Black sea Abramis brama 2200 - 10000 300 - 3000 1500 - 6400 Khotenovsky, 1985 Kagel & Taraschewski, 1993; Schmahl & Taraschewski, Germany Abramis brama 8000 - 10000 1987 Essex Abramis brama Anderson, 1974 Ukraine Abramis brama 3800 - 6700 1:1.5 Bychowskii, 1962; Koval & Pashkevichute, 1973 Poland Abramis brama Dzika, 1987; Wierzbicka, 1974 Morava River Abramis brama Gelnar, Koubkova, Plankova & Jurajda, 1994 Europe Rhodeus sp. Ukraine Rutilus sp. Koval & Pashkevichute, 1973 Ireland Rutilus rutilus Holland & Kennedy, 1997 Europe Chondrostoma sp. Ireland Gobio gobio Holland & Kennedy, 1997 Europe Cottas sp. Europe Lota sp. Europe Pelecus sp. Europe Perca sp. Europe Nemachilus sp. Poland Blicca bjoercna Wierzbicka, 1974 Europe Leucaspius sp. Ireland Scardinius erythrophthalmus Holland & Kennedy, 1997 Amur River Gnathopogon sp. Amur River Acanthorhodeus sp. Europe Megalobrana sp. Europe Parabramis sp. Europe Erythroculter sp. Europe Culter sp.

96 Europe Hemiculter sp. Europe Lefua sp. Europe Misgurnus sp. Volga Delta Acipenser güldenstädti Germany Squalius cephalus Schmahl & Taraschewski, 1987 33 P. pavlovskii Volga delta Aspius aspius 4000 - 5750 1:1.12 - 1.86 Bychowskii, 1962; Yamaguti, 1963 Poland Aspius aspius 5700 - 9300 1790 - 3720 3250 - 5580 Grabda-Kazubska & Pilecka-Rapacz, 1987 Leningrad Aspius aspius Yamaguti, 1963 Ukraine Aspius aspius 6750 1:1.2 - 3 Koval & Pashkevichute, 1973 34 P. persicum Azerbaijan Vimba vimba persicus Mikailov, 1973 35 D. plicati South Africa Labeo umbratus 2600-6710 850-2300 1750-4410 1:2.06-1.92 36 P. rutili France Rutilus rutilus Russia Cyprinidae species Khotenovsky, 1977 37 P. sapae Azerbaijan Abramis sapa bergi Mikailov, 1973 38 D. scardini Ukraine Scardinus eruth. rophthalmus 5000 Koval & Pashkevichute, 1973 39 P. schizothorazi Soviet Union Koval & Pashkevichute, 1973 40 P. schulmani Azerbaijan Alburnoides bipunctatus eichwaldi Mikailov, 1973 41 P. soni India Oxygaster bacaila 1350 - 1650 435- 536 580 - 1116 Tripathi, 1957; Yamaguti, 1963 42 P. strelkowi Soviet Union Koval & Pashkevichute, 1973 43 P. tadzhikistanicum Soviet Union Koval & Pashkevichute, 1973 Efrurter 44 P. tetragonopterini Aquarium Ctenobrycen spilurus 1350 - 1650 Yamaguti, 1963 Efrurter Aquarium Gymnocorymbus ternetzi Yamaguti, 1963 45 P. varicorhini Azerbaijan Varichorinus capoeta sevangi Mikailov, 1973 46 P. zeller Soviet Union Koval & Pashkevichute, 1973 Bulgaria Cyprinuc carpio 1070 - 1170 317 - 475 580-665 1:0.54 Nedeva & Babacheva, 1999 47 Diplozoon sp. Uganda Alestes sp. Thurston, 1970

no. 3 is identical to D. paradoxum ballerus Koval & Pashkevichute, 1973 no.5 is identical to D. paradoxum sapae Koval & Pashkevichute, 1973 no.25 is identical to P. gussevi, & D. paradoxum bliccae Koval & Pashkevichute, 1973 No.31, D. nipponicum, as used by Jovelin & Justine, 2001. synonym Eudiplozoon nipponicum as used by Matjusova, Koubkova & Cunningham, 2003.

97 A.3 Classification dendogram pertaining to Khotenovsky’s published classification for the newly described specie of the genus Diplozoon.

98