Aspects of the reproductive biology of Argulus japonicus and the morphology of Argulus coregoni from Malaysia. by LOURELLE ALICIA MARTINS EVERTS DISSERTATION Submitted in partial fulfilment of the requirements for the degree MASTER OF SCIENCE in ZOOLOGY in the FACULTY OF SCIENCE at the UNIVERSITY OF JOHANNESBURG SUPERVISOR: PROF. A. AVENANT-OLDEWAGE JANUARY 2010 ACKNOWLEDGEMENTS I would like to acknowledge the following people: - Professor Annemarié Avenant-Oldewage for your never-ending encouragement, guidance, and inspiration throughout this study as well as the opportunity to work with you. - My family and friends for all your support and encouragement. - Edie Lutsch, for your patience, help and support with the preparation of the microscope slides. - Dr. Willie Oldewage for your patience and help with the scanning electron microscope. - The National Research Foundation and the University of Johannesburg for funding this study. - I would also like to acknowledge the support and assistance of: Ebrahim Karim (for help on the photo-plates), Johan Therone (for help on the field excursion) and Mrs Elmine Knight (for collecting the specimens in Malaysia). ABSTRACT A general introduction provides the foremost morphological characteristics of the genus. A breeding colony of Argulus japonicus was kept under laboratory conditions in order to study sperm transfer. Pairs in copula were studied with histology and scanning electron microscopy. Sections of copulating pairs revealed sperm on the accessory copulatory structures of the male; and scanning electron microscopy showed that sperm transfer occurs in three phases which can be differentiated to ten different stages. Sperm transfer occurs via a spermatophore in A. japonicus. This is the first observation of a spermatophore in Argulus. For the second part of this study, seven specimens of an unknown freshwater ectoparasitic crustacean were collected from red tilapia fish, kept for consumption at the “Langat Fishing, Seafood and Beer Garden” Restaurant just off the Langat River in Selangor, Malaysia. Initial investigation showed that the specimens were of the genus Argulus. Light and scanning electron microscopical studies were subsequently used to identify the species. A comparison with all Argulus species formerly described from Asia and the surrounding islands was conducted. The species was identified as Argulus coregoni, due to the presence of the roughly triangular shaped anterior respiratory areas and the kidney bean shaped posterior respiratory areas. Additionally, the abdomen with sharply pointed terminal ends as well as the presence of characteristic accessory protrusions on the second i swimming leg of the male specimens confirmed this identification. This species has not previously been described from Malaysia. The final chapter of this dissertation contains an overall summative discussion of the different parts of this study and highlights future possible research avenues. ii OPSOMMING ʼn Algemene inleiding verskaf die vernaamste morfologiese eienskappe van die genus. ʼn Teel-kolonie van Argulus japonicus is aangehou onder laboratoriumtoestande om pare in copula te bestudeer, ten einde spermoordrag te beskryf. Kopulerende pare is deur middel van histologie en skandeerelektronmikroskopie bestudeer. Sneë van kopulerende pare het getoon dat sperme wel teenwoordig is op die aksessoriese strukture op die swempote van die mannetjies; en die SEM-studie bewys dat spermoordrag in drie fases plaasvind wat in tien verskillende stadiums onderskei kan word. Spermoordraging vind plaas deur ʼn spermatofoor in A. japonicus. Dit is die eerste keer wat ʼn spermatofoor in Argulus opgemerk is. Vir die tweede deel van hierdie studie, is sewe organismes van ʼn onbekende varswater ektoparasitiese Krustaseër versamel vanaf rooi tilapia vis, by die “Langat Fishing, Seafood and Beer Garden” Restaurant in die Langatrivier in Selangor, Maleisië. Die aanvanklike ondersoek het getoon dat die organismes van die genus Argulus is. Lig- en SEM-studies is gebruik om die spesies te identifiseer. Vergelyking met al die Argulus spesies wat voorheen in Asië en die omringende eilande beskryf is, is gedoen. Die spesie is as Argulus coregoni gëidentifiseer omdat dit ʼn driehoekige voorste asemhalingsarea en ʼn boontjie-vormige agterste asemhalingsarea het. Voorts is die identifikasie bevestig deur die abdomen wat terminaal skerp eindig en kenmerkende aksessoriese uitsteeksels op die tweede swempoot van iii die mannetjies. Hierdie spesies was nog nooit vanuit Maleisië beskryf nie. Die laaste hoofstuk van hierdie verhandeling bevat ʼn samevatende bespreking van die verskillende dele van hierdie studie en dit lig moontlike toekomstige navorsing vrae uit. iv TABLE OF CONTENTS ABSTRACT i OPSOMMING iii TABLE OF CONTENTS v LIST OF FIGURES vii LIST OF TABLES vii CHAPTER 1: General Introduction 1 1. General Introduction 2 1.1 Introduction to the morphology of Argulus Müller, 1785 2 1.2 Introduction to the reproductive features of Argulus Müller, 1785 5 1.3 Objectives of this study 7 1.4.1 Outline of the dissertation 8 1.4.2 Oral presentations 9 1.4.3 Published abstracts: 10 1.4.4 Special awards received during this study 10 CHAPTER 2: Sperm transfer by the means of a spermatophore in Argulus japonicus Thiele, 1900 11 2.1. Introduction 12 2.2. Materials and methods: 15 2.3. Results: 16 2.3.1 Observation results 16 2.3.2 Whole mount results 17 2.3.3 Histological results 17 v 2.3.4 Scanning electron microscopy results 17 2.4. Discussion 23 CHAPTER 3: First record of Argulus coregoni a fish ectoparasitic crustacean from Malaysia and additional notes on the morphology 25 3.1. Introduction 26 3.2. Materials and Methods 31 3.3. Results 32 3.4. Discussion 37 3.5. Conclusion 40 CHAPTER 4: Summative discussion and future research 41 4.1. Sperm transfer in Argulus japonicus Thiele, 1900 42 4.2. Argulus coregoni Thorell, 1866 in Malaysia 42 4.3. Future research 42 CHAPTER 5: General References 46 vi LIST OF FIGURES Figure 1.1. A-C: Schematic diagram of argulids to illustrate the shape of the carapace 3 Figure 1.2: Schematic drawing of the ventral view of a female Argulus japonicus 5 Figure 1.3. A and B: Schematic drawing of Argulus japonicus 6 Figure 2.1-6: Light micrographs of Argulus japonicus 20 Figure 2. 7-12: Scanning electron micrographs of Argulus japonicus 21 Figure 2. 13-16: Scanning electron micrographs of Argulus japonicus 22 Figure 3. 1-9: Scanning electron micrographs of Argulus coregoni 35 Figure 3. 10-18: Scanning electron micrographs of Argulus coregoni 36 LIST OF TABLES Table 2.1: A breakdown of the stages of copulation in Argulus japonicus 19 Table 3.1: A list indicating the species, location, host and the reference of freshwater Argulus species reported in Asia and the surrounding islands 27 vii General Introduction CHAPTER 1 General Introduction CHAPTER 1 1 General Introduction 1. General Introduction Argulus Müller, 1785 is one of only four genera belonging to the class Branchiura, most commonly known as the fish lice, a class of crustacean arthropods. Branchiura are characterized by their dorsoventrally flattened bodies and the possession of carapace lobes which may or may not cover their biramous thoracic legs (Piasecki and Avenant-Oldewage, 2008). They have a pair of compound eyes, four pairs of swimming legs and an unsegmented abdomen (Poly, 2008). Argulus is a serious pathogen of fish in natural (Allum and Hugghins, 1959; Kruger et al., 1983; Menezes et al., 1990; Shafir and Oldewage, 1992; Northcott et al., 1997; Avenant-Oldewage, 2001; Cengizler et al., 2001; Taylor et al., 2006 ) and intensive fish cultures (Menezes et al., 1990; Northcott et al., 1997; Buchmann and Bresciani, 1997; Cengizler et al., 2001; Taylor et al., 2006 ). Argulus is the best known genus in this group and Tam (2005) summarised the extent of research done on this genus in Africa. He indicated that A. japonicus Thiele, 1899, A.coregoni Thorell, 1866, and A. foliaceus Linnaeus, 1758 are the most studied species and that a lot of work is lacking in african species. For the purposes of this study, only the morphology of the genus Argulus will be considered. 1.1. Introduction to the morphology of Argulus Müller, 1785 While attached to their hosts, specimens of Argulus appear as dark coloured spots on lighter coloured fish hosts and milky white to grey coloured spots on darker coloured fish hosts, either way, the eyes are apparent. In order to identify a species, a number of features need to be considered, as identified by Rushton-Mellor (1994a) for the African species. CHAPTER 1 2 General Introduction Firstly, the carapace and its shape are typical. Argulids have horseshoe-shaped cephalic shields which extend into the carapace lobes (Piasecki and Avenant- Oldewage, 2008). The carapace shape varies, for instance, in A. coregoni, the carapace lobes cover only the first three pairs of legs (Figure 1.1.A), whereas in other species such as A. personatus Cunnington, 1913 only the first two pairs of legs are covered (Figure 1.1.B), while in A. japonicus; the carapace lobes cover all four pairs of swimming legs (Figure 1.1.C). Where the cephalic shield meets the carapace lobes a slight indentation occurs. Figure 1.1. Schematic diagram of argulids to illustrate the shape of the carapace; A. Argulus coregoni female redrawn and adapted from Fryer (1982); B. Argulus personatus male redrawn from Rushton-Mellor (1994b); C. Argulus japonicus female redrawn and adapted from Fryer (1982). ab abdomen, cs cephalic shield, cl carapace lobes, es eye spots, sp spermatheca. Ventrally, on the carapace, two pairs of respiratory areas occur (Figure 1.2) which are species specific and may be used for identification based on their shape CHAPTER 1 3 General Introduction (Rushton-Mellor 1994a). They are clearly delineated and are covered by a thinner cuticle than the rest of the carapace (Walker et al., 2004). Five pairs of appendages occur on the cephalothorax (Piasecki and Avenant-Oldewage, 2008). The first and second appendages are the antennules and the antennae respectively.