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. These differ among species in the number of hooks and spines they are adorned with as well as in the shape of the various spines (Rushton-Mellor, 1994a). The third pair of appendages is the maxillules or suckers which consist of rods made up of sclerites.
The number of rods varies in the different species (Rushton-Mellor, 1994a). The fourth pair of appendages is the mandibles which are contained in the proboscis or mouth tube, a structure situated posterior to the pre-oral spine between the suckers.
Posterior to the suckers, is the next pair of appendages occurs, i.e. the maxillae.
These are characterized by the shape of the basal plate, the number of setae and the number of scales present (Piasecki and Avenant-Oldewage, 2008). On the thorax, four pairs of biramous swimming legs occur (Figure 1.2). The legs of males bear the accessory copulatory structures which vary morphologically between the different species(Rushton-Mellor 1994a)
The posterior tagma is the abdomen. The abdomen has a distinctive shape in each species (Rushton-Mellor 1994a).
CHAPTER 1 4 General Introduction
Figure 1.2. Schematic drawing of the ventral view of a female Argulus japonicus redrawn and adapted from Fryer (1982). an antenna, ar anterior respiratory area, as antennule, bp basal plate, ms maxillules, mt mouth tube, mx maxillae, pr posterior respiratory areas, ps preoral spine, sl biramous swimming legs, sp spermatheca.
1.2 Introduction to the reproductive features of Argulus Müller, 1785
In general, the main difference observed in males and females is the presence of eggs in the thoracic region of the female and their absence in males. Specific differences between males and females include that the abdomen of the male that contains the elongated testes (Figure 1.3. A), which are milky white with sperm in live specimens, and dark in preserved specimens, while the abdomen of the female houses the round spermathecae which are always dark in colour. Furthermore, in females, the spermathecal spines occur anterior to the natatory lobes (Wilson, 1902).
In males, the second, third, and fourth pair of swimming legs (Figure 1.3. B) are adorned with accessory copulatory structures which are species specific in shape and position (Wilson, 1902). The structures on the second pair of legs are protrusions situated on the ventral side of the legs. The indentations on the ventral side of the third pair of legs are known as sockets. The protrusions situated on the dorsal side of the fourth pair of legs are intricately adorned with scales, setae, and
CHAPTER 1 5 General Introduction spines and are known as the pegs. They are also situated in such a way as to be able to fit into the socket on the third pair of legs (Wilson, 1902).
Figure 1.3. Schematic drawing of Argulus japonicus redrawn from Fryer (1982); A. Abdomen of the male; B. Second, third and fourth swimming legs of the male to show the accessory copulatory structures. ap accessory protrusion, pg peg, st socket, ts testis.
The morphological features discussed in brief in this introductory chapter, provides the background information needed for this dissertation. The morphological characteristics of the reproductive structures of both male and female A. japonicus specimens were considered in order to understand copulation in this species for the first part of the study. This resulted in the description of the mechanism of sperm transfer involving these structures in A. japonicus. Furthermore, the general morphology of the genus was considered for the identification of specimens collected from Malaysia. Although an identification key was not used, comparisons to other species formerly identified and described from the Orient were used to confirm the identification of Argulus coregoni Thorell, 1866.
CHAPTER 1 6 General Introduction 1.3 Objectives of this study
Although Argulus is a well studied group as far as morphology and development is concerned, certain gaps exist in the general knowledge. Detailed descriptions are available on the accessory copulatory structures of A. japonicus; however, the actual function of these structures is unknown. In addition, the process of sperm transfer is somewhat enigmatic even though copulation has been witnessed (Shafir and
Oldewage, 1992) and there are three hypothesis or explanations as to how sperm is transferred in Argulus japonicus. These are discussed in a future chapter.
Therefore the first objective of this study is to conduct a detailed study of copulating pairs by:
a. Keep a breeding colony of Argulus japonicus.
b. Observe specimens in copula and freeze them in this state.
c. Study the specimens frozen in copula using SEM and light microscopy.
d. Identify the mechanism of sperm transfer.
The hypothesis for this part of the study is that: sperm is transferred with the aid of the swimming legs of the male.
The second part of this study began during 2007, when specimens of an unidentified fish louse were collected in a restaurant in Malaysia and were donated to Prof.
Avenant-Oldewage at the University of Johannesburg by a former student Mrs
Elmine Knight. As it was collected off the fish that was being served to eat, a study was proposed to identify the fish louse.
CHAPTER 1 7 General Introduction Therefore the second objective of this study is to:
a. Study the specimens donated from Malaysia.
b. Compare the various morphological features to that of other species described
from the Orient.
c. Identify the species of Argulus.
d. Provide an updated description of the species by the use of scanning electron
microscopy and light microscopy where need.
The hypothesis for this part of the study is that: this is a new record for this species in
Malaysia.
The specimens were identified as A. coregoni because of the morphological features such as the shape of the abdomen and the accessory copulatory structures of the male. Scanning electron microscopy was used to improve the description of the mouthparts and additional morphological structures previously only described from light microscopy.
1.4.1. Outline of the dissertation
Based on these objectives and hypotheses a study was planned and it is presented in the chapters that follow. Different aspects of this study are discussed in separate chapters. The document is organised as follows:
Chapter 1 is a general introduction to the morphology of Argulus Müller, 1785.
Each chapter succeeding chapter 1 consists of an introduction, materials and methods, discussion, and conclusion. To minimise the repetition of references, all the references are included at the end of the document.
CHAPTER 1 8 General Introduction Chapter 2 describes aspects of copulation and sperm transfer in Argulus japonicus
Thiele, 1900.
Chapter 3 describes a first record of Argulus coregoni Thorell, 1866 a fish ectoparasitic crustacean from Malaysia and additional notes on the morphology.
This chapter has been accepted for publication in Malaysian Applied
Biology, 38(2): 61-71.
Chapter 4 contains a brief general discussion of the study and gives suggestions for future research on Argulus sp.
Chapter 5 is a combined reference list of all the literature cited in the different chapters of this dissertation.
In addition to the publication mentioned above, the following outputs were delivered.
1.4.2. Oral presentations
1. Everts, L. Aspects of the biology of Argulus spp. Postgraduate symposium,
Department of Zoology, University of Johannesburg. 26 March 2009.
2. Everts, L. and Oldewage, A. 2009. Aspects of copulation in Argulus japonicus.
The 38th Annual Conference of the Parasitological Society of Southern Africa.
Magaliesberg. 20 - 23 September 2009.
3. Everts, L. and Oldewage, A. 2009. Aspekte van paring by Argulus japonicus.
Die Suid-Afrikaanse Akademie vir Wetenskap en Kuns, Jaarkongres: Afdeling
Biologiese Wetenskappe, Eeufeesjaar. Die skool vir omgewingswetenskap en
ontwikkeling, North-West University, Potchefstroom. 2 October 2009.
4. Everts, L. Aspects of copulation in Argulus japonicus. Postgraduate
symposium, Department of Zoology, University of Johannesburg. 30 October
2009.
CHAPTER 1 9 General Introduction 1.4.3. Published abstracts:
1. Everts, L. and Oldewage, A. 2008. Study of an ectoparasitic crustacean from
Malaysia with light and electron microscopy. Journal of the South African
Veterinary Association, 80(2): 126-140.
2. Everts, L. and Oldewage, A. In press. Aspects of copulation in Argulus
japonicus. Journal of the South African Veterinary Association.
3. Everts, L. and Oldewage, A. In press. Aspekte van paring by Argulus
japonicus. Tydskrif vir Natuurwetenskap en Tegnologie.
1.4.4. Special awards received during this study
1. Next Generation Scholarship. A bursary initiated by the University of
Johannesburg.
2. University of Johannesburg merit bursary received on the basis of BSc.
Honours marks.
3. Recipient of the Senior Oral Presentation Award presented by the
Parasitological Society of Southern Africa. For the presentation: Aspects of
copulation in Argulus japonicus, at the 38th Annual Conference of the
Parasitological Society of Southern Africa. Magaliesberg. 20 - 23 September
2009.
4. Recipient of the best Masters Student Presentation Award presented by Die
Suid-Afrikaanse Akademie vir Wetenskap en Kuns, Jaarkongres: Afdeling
Biologiese Wetenskappe, Eeufeesjaar. For the presentation: Aspekte van
paring by Argulus japonicus, at Die skool vir omgewingswetenskap en
ontwikkeling, North-West University, Potchefstroom. 2 October 2009.
Date of first registration February 2009.
CHAPTER 1 10 Sperm transfer in A. japonicus
CHAPTER 2
Sperm transfer by the means of a spermatophore in Argulus japonicus Thiele, 1900
CHAPTER 2 11 Sperm transfer in A. japonicus
2.1. Introduction:
Argulus Müller, 1785 is a serious pathogen in natural and intensive fish culture (Allum and Hugghins, 1959; Kruger et al., 1983; Menezes et al., 1990; Shafir and Oldewage,
1992; Buchmann and Bresciani, 1997; Northcott et al., 1997; Avenant-Oldewage,
2001; Cengizler et al., 2001; Taylor et al., 2006; Piasecki and Avenant-Oldewage,
2008). Argulus japonicus Thiele, 1900 is an opportunist (Shafir and Oldewage, 1992;
Avenant-Oldewage, 2001) and infections reach severe proportions in a very short time leading to catastrophic fish kills (Kruger et al., 1983; Menezes et al., 1990;
Northcott et al., 1997; Avenant-Oldewage, 2001; Taylor et al., 2006). Egg-laying has been studied in various species (Wilson, 1902; Shimura and Egusa, 1980; Shafir and van As, 1986; Shafir and Oldewage, 1992; Ikuta et al., 1997; Ikuta and Makioka,
1997; Gault et al., 2002; Hakalahti et al., 2004a; Harrison et al., 2006; Taylor et al.,
2009a), along with the egg hatching dynamics (Shimura, 1983a; Sundara Bai et al.,
1988; Pasternak et al., 2000; Mikheev et al., 2001; Fenton and Hudson, 2002;
Hakalahti and Valtonen, 2003; Hakalahti et al., 2004b; Fenton et al., 2006; Taylor et al., 2009b) and this has shown that organisms in this genus are prolific reproducers.
The subsequent development of larvae has also been documented in various species (Tokioka, 1936a; Stammer, 1959; Thomas, 1961; Shimura, 1981; Shafir and van As, 1986; Rushton-Mellor and Boxshall, 1994; Lutsch and Avenant-Oldewage,
1995; Pasternak et al., 2004a; Pasternak et al., 2004b; Lester and Hayward 2006;
Møller et al., 2007). However, the process of sperm transfer is still somewhat enigmatic even though copulation has been witnessed (Shafir and Oldewage, 1992).
An understanding of sperm transfer requires that the reproductive systems of both the male and female be understood first. The female reproductive organs consist of the ovary which is situated in the thorax and the paired seminal receptacles
CHAPTER 2 12 Sperm transfer in A. japonicus
(spermathecae) in the anterior portion of the abdomen (Wilson, 1902; Debaisieux,
1953). The male reproductive system is more complex. It consists of two testes in the abdomen connected to two vasa efferentia leading to a common seminal vesicle in the thorax, and two vasa defferentia leading posterior to the two ejaculatory ducts which is linked to a common genital aperture. There are also two glands of the prostate complex which run into the prostate reservoirs and then into the ducts of the prostate reservoir, these run parallel to the vas efferens and join to the ejaculatory duct (Avenant-Oldewage and Swanepoel, 1993). Wingstrand (1972) described the sperm of Argulus foliaceus as being filiform in appearance. Based on his descriptions and figures, the appearance of the sperm would differ depending on the angle and section of the sperm being viewed. Accessory copulatory structures are borne on the swimming legs of the male, and consist of an indentation known as a socket on the postero-ventral surface of the third pair of legs, and a peg on the antero-dorsal surface of the fourth pair of legs (Jurine, 1806; Leydig, 1850; Grobben, 1908; Martin,
1932; Debaisieux, 1953; Avenant-Oldewage and Swanepoel, 1993). The accessory structures are not connected to the reproductive ducts (Avenant-Oldewage and
Swanepoel, 1993).
The method of sperm transfer has received sporadic attention and three explanations have been provided. The first is that the accessory copulatory structures of the male, borne on the swimming legs, serve to transfer sperm during copulation (Jurine, 1806;
Leydig, 1850; Claus, 1875; Grobben, 1908). Jurine (1806) was the first to identify the accessory copulatory structures of the male and considered the peg of the fourth leg as the penis of the male (“je considère comme le pénis du mâle”) and the socket as a vesicle containing a transparent liquid aimed at fertilization (“une vésicule située sur le bord postérieur du premier anneau de la troisième paire de pates, laquelle est
CHAPTER 2 13 Sperm transfer in A. japonicus remplie d’un liquide transparent, qui paroît être destiné à la fécondation”). Leydig
(1850) added that the accessory copulatory structures acted as a sperm hook (peg) and that the socket was employed as a sperm capsule. Claus (1875) agreed with
Leydig (1850) and added that the accessory copulatory protrusion, that is the peg, also serves to open the semen capsule, the socket, (“dem Haken des letzten Beines das Werkzeug sehen, welches zur oeffnung der kapsel während der Anfűlling mit
Sperma dient”). However, for the second explanantion Martin (1932) provided a morphological review of the genus and concluded that the accessory copulatory structures had no function in sperm transfer but, instead grasp the female’s swimming legs and hold her in place, while sperm is transferred directly from aperture to aperture. Debaisieux (1953) agreed with Martin (1932) about the fact that the accessory copulatory structures were used to grasp the female but instead,
Debaisieux was of the opinion that the spermathecal spines of the female play a role in the transfer of sperm from male to female (“la femelle introduit les épines alternativement, dans l’orifice génital mâle et que, faisant agir les muscles rétracteurs elle aspire ou pompe les spermatozoids”). Following their study of the male reproductive system where a possible connection between the accessory structures and seminal ducts was sought, and included a reconstruction, Avenant-Oldewage and Swanepoel (1993) observed a membrane covering the entrance of the genital aperture of the male which corroborated this argument and they speculated that the spermathecal spines of the female probably serve to puncture the membrane to allow the transfer of sperm directly into the spermathecae. They showed that there is no connection between the seminal ducts and the accessory copulatory structures and came to the conclusion that accessory structures would then only serve to hold the female during copulation. This formed the third explanation.
CHAPTER 2 14 Sperm transfer in A. japonicus
Therefore, the mechanical process of sperm transfer and the function of the accessory copulatory structures of the male is still mostly unknown. The purpose of this study is to provide information about the copulation process in A. japonicus through histological and scanning electron microscopy studies of copulating pairs.
2.2. Materials and methods:
Specimens of Argulus japonicus were collected from ornamental fish farms in the
Nelspruit and Modjadjiskloof regions of South Africa. These were transported to the laboratory and placed in a tank with goldfish, Carassius auratus (L). The specimens were left to lay eggs. These eggs were then monitored for development while a constant supply of gold fish was provided to replace succumbed fish specimens and to establish a breeding colony of parasites. As soon as the Argulus reached a size visible with the naked eye (an average of 3mm), they were transferred with the fish to a smaller tank for easier observation. Once eggs were observed on the side of this tank, the Argulus were monitored more closely to observe them in copula. Whole mounts of a male (Figure 2.1) and a female (Figure 2.2) were made for comparison to whole mounts of pairs. When copulation was spotted on a fish, the fish was removed from the tank and copulating pairs were immediately frozen by the application of a Cryo freeze aerosol (AGAR Scientific) (Krenn et al., 2008).
Thereafter, some pairs were fixed in acetic acid-formalin-alcohol (AFA), that is a mixture of 12,5ml glacial acetic acid, 20ml 40% formalin, 125ml 95% ethanol, and
112,5ml distilled water, at room temperature. These were then transferred to 70% ethanol prior to dehydration in an ascending series of acetone, and then embedded in resin and sectioned at 5μm. Sections were stained with azocarmine-aniline blue
(AZAN), periodic acid Schiff (PAS), or Hematoxylin and Eosin (Humason, 1979). The
CHAPTER 2 15 Sperm transfer in A. japonicus slides were studied with a compound microscope either with a drawing tube or a camera. Alternatively, frozen pairs were transferred to 70% ethanol and then to lactophenol and stained with a few crystals of lignin pink. They were mounted in lactic acid and studied with a dissection microscope.
The remainder of the specimens were transferred to 70% ethanol, rehydrated in a descending series of ethanol, then freeze dried and sputter coated with gold for scanning electron microscopy. Observations were done at 4 to 15kV on a JEOL
5600 SEM. None of the pairs used for SEM were cleaned during preparation to prevent the loss of sperm.
The holding of, and experimentation with animals was in accordance to the specifications of and approved by the Faculty Ethics committee of the University of
Johannesburg.
2.3. Results:
2.3.1. Observation results
Observations of 30 pairs in copula, showed that copulation in A. japonicus could last between 30 to 180 minutes and commences once the male attaches to the female, and terminates at the pair’s separation. Adherence is followed by flapping of the male’s abdomen in a dorsoventral plane and appears to assist positioning. When the abdominal flapping ceases, the male’s abdomen is positioned on either the left-hand or the right-hand side, parallel to the female’s abdomen. The male’s third and fourth legs, on the side where the female is positioned, are then placed ventral to the female’s abdomen, while the remainder of his body is on her dorsal side (Figure 2.3 a and b).
CHAPTER 2 16 Sperm transfer in A. japonicus
2.3.2. Whole mount results
Whole mounts were made of 5 pairs. These whole mounts showed that the male’s peg on the fourth leg is inserted into the socket of the third leg but, they are not used to hold the female’s legs in place. Additionally, the peg and socket mechanisms are in close proximity to the spermathecal spines which are situated on the female’s abdomen (Figure 2.3 a and b).
2.3.3. Histological results
Histological sections were made of 6 pairs. These sections of copulating pairs show that both the female’s spermathecae contain sperm (Figure 2.4) but that sperm is only present in the socket situated on the side where the female’s body is situated
(Figure 2.5). This sperm is surrounded by secretions similar in substance to that from the gland of the prostate complex. During copulation, the peg of the fourth leg is inserted in to the socket of the third leg (Figure 2.6 LM and Figure 2.7 SEM).
2.3.4. Scanning electron microscopy results
Scanning electron microscopy was conducted on 17 pairs. These micrographs of the accessory copulatory structures of the male show the peg on the fourth leg while the socket’s opening flap is visible (Figure 2.8). Scanning electron micrographs of the spermathecal spines of the female illustrate that the terminal tip is sharp (Figure 2.9) with a sub-terminal opening (Figure 2.10).
From the live observations, wholemounts, histological sections and the SEM micrographs, the following information is obtained. Three main phases of copulation were identified namely pre-ejaculation, ejaculation, and post-ejaculation, with multiple sub-stages marking each stage (Table 2. 1).
CHAPTER 2 17 Sperm transfer in A. japonicus
Pre-ejaculation:
The first stage of this phase is marked by the adherence of the male with his suckers dorsally to the female’s carapace; the male is seen to be obscured by the female from a ventral view. This is followed by alignment, during which the male’s abdomen moves anterior to that of the female. During the third stage, attachment, the male establishes his position on the female and cannot be removed without force. The male’s third and fourth legs and abdomen are positioned ventral to the female’s abdomen, while the remainder of his body remains dorsal to her body. The male’s peg is observed as inserted in the socket confirming observations in the sections.
Ejaculation:
The fourth stage is marked by the contraction of the muscles in the abdomen surrounding the testes which has the effect that the male’s testes are compressed rhythmically presumably to force sperm into the vas efferens and eventually into the seminal vesicle. The testes area contracts or sinks into the abdomen (Figure 2.11).
The fifth stage is marked by engorgement, during which the muscles surrounding the male’s genital aperture contract to cause compression of the sperm and glandular secretions which is observed as bulging of the genital aperture area (Figure 2.12).
Thereafter a spermatophore or sperm packet is released from the genital aperture in a globular form marking stage 6, ejaculation. This globular emission consists of secretions from the gland of the prostate complex which surrounds the sperm, forming a malleable spermatophore or sperm packet (Figure 2.13, 2.14, 2.15) containing sperm (Figure 2.16).
Post ejaculation:
During stage seven, the male remains attached to the female. During stage eight the male remains adhered to the female but is no longer attached. While during stage
CHAPTER 2 18 Sperm transfer in A. japonicus nine, the male remains adhered but may change position on the female, often staying for up to 20 minutes still adhered but in a different position, that is realigned. During stage ten the male completely abandons the female.
Table 2.1: A breakdown of the stages of copulation in Argulus japonicus.
Phase 1: Pre-ejaculation Stage 1 Adherence Stage 2 Alignment Stage 3 Attachment Phase 2: Ejaculation Stage 4 Contraction Stage 5 Engorgement Stage 6 Ejaculation Phase 3: Post-ejaculation Stage 7 Attached Stage 8 Adhered Stage 9 Realignment Stage 10 Abandonment
CHAPTER 2 19 Sperm transfer in A. japonicus
Figure 2. Light Micrographs of Argulus japonicus. 1. Light micrograph of a male specimen of Argulus japonicus cleared in lactic acid. Scale bar 1100μm. sv seminal vesicle, ts testis. 2. Light micrograph of a female specimen of Argulus japonicus cleared in lactic acid. Scale bar 1750μm. arrow natatory lobes, block location of spermathecal spines, ov ovary, sp spermatheca. 3a. Light micrograph of a whole-mount of a copulating pair in stage 3. Scale bar 1100μm. Stained with lignin pink in lactophenol. 3b. Diagrammatic representation of the whole- mount of a copulating pair in stage 3. Scale bar 1100μm. f female, m male, pg peg, sp spermatheca, st socket, ts testis. 4. Light micrograph of a section through a female’s spermatheca. Scale bar 20μm. Stained with Azan. gs secretion of the prostate gland, se sperm, sl cuticle lining of the spermatheca. 5. Light micrograph of a section through the male’s socket. Scale bar 20μm. Stained with Azan. gs secretion of the prostate gland, se sperm, sw cuticle lining of the socket wall. 6. Light micrograph of a histological section of the male’s socket with the peg in it. Scale bar 20μm. gs secretion of the prostate gland, pg peg, se sperm, sw cuticle lining of the socket wall.
CHAPTER 2 20 Sperm transfer in A. japonicus
Figure 2 Scanning electron micrographs of Argulus japonicus: 7. Scanning electron micrograph showing the peg on the right fourth leg inserted into the socket of the right third leg of the male. Scale bar 100μm. pg peg, st socket, 3 leg 3, 4 leg 4. 8. Scanning electron micrograph showing the left peg and socket of the male on legs 3 and 4. Scale bar 100μm. pg peg, st socket, 3 leg 3, 4 leg 4. 9. Scanning electron micrograph showing the spermathecal spines of the female situated on the abdomen dorsal to the natatory lobes. Scale bar 10μm. Block indicates figure 10, ss spermathecal spine. 10. Scanning electron micrograph of a spermathecal spine to show the spine opening. Scale bar 5 μm. so spine opening. 11. Scanning electron micrograph of the male’s contracted abdomen. Scale bar 200μm. ca contracted abdomen, 4 right 4th leg.12. Scanning electron micrograph of the male’s genital perimeter to demonstrate the contraction of muscles prior to the spermatophore being released. Scale bar 100μm. cm contracted muscle, ga genital aperture, 4 right 4th leg.
CHAPTER 2 21 Sperm transfer in A. japonicus
Figure 2. Scanning electron micrographs of Argulus japonicus: 13. Scanning electron micrograph of the male’s genital aperture to show the spermatophore emerging. Scale bar 100μm. cm contracted muscle, ga genital aperture, sm spermatophore, 4 right 4th leg. 14. Scanning electron micrograph of the male’s genital aperture to show the second stage of spermatophore secretion. Scale bar 100μm. cm contracted muscle, ga genital aperture, sm spermatophore, 4 right 4th leg. 15. Scanning electron micrograph of the male’s genital aperture to show the whole spermatophore. Scale bar 100μm. block indicates figure 16, cm contracted muscle, ga genital aperture, sm spermatophore, 4 right 4th leg. 16. Scanning electron micrograph of the sperm contained by the complete spermatophore. Scale bar 55μm. gs glandular secretions, se sperm.
CHAPTER 2 22 Sperm transfer in A. japonicus
2.4. Discussion:
Different methods of sperm transfer occur in branchiurans. Spermatophores are employed for sperm transfer in Dolops ranarum (Fryer, 1958; 1960; 1969). These spermatophores are malleable when first secreted to allow the male to manoever it onto the spermathecal spines of the female. Once in contact with water however, it hardens and this requires that the female sheds her exoskeleton to be free of the spermatophore (Fryer, 1960). The spermatophores secreted by Argulus japonicus and Dolops ranarum therefore differ, in A. japonicus the prostate secretions are mixed with the sperm allowing the sperm to occur on the outside as well as within the spermatophore, whereas the spermatophore secretions in D. ranarum are secreted to form an outer shell that surrounds the sperm entirely (Fryer, 1960).
Literature on Dipteropeltis Calman, 1912 is depauperate and in fact only females have been documented (Calman, 1912; Ringuelet, 1943; 1948; Weibezahn and
Cobo, 1964; Møller and Olesen, 2009). The method of sperm transfer is thus unknown.
In Chonopeltis Thiele, 1901 copulation was documented in 1984 by van Niekerk who wrote his unpublished doctoral thesis in afrikaans on the biology of Chonopeltis australis Boxshall, 1976 and described copulation and sperm transfer. He observed that sperm is transferred directly from the male’s genital aperture to the female’s spermathecal apertures and that contraction of the males muscles surrounding the genital aperture is required for this to occur.
The mechanism of sperm transfer in Argulus japonicus is comparable to that of
Dolops ranarum in the extrusion of spermatophores. Jurine (1806) explained that the socket of the third leg changed from transparent to milky white, supposedly carrying a liquid aimed at fertilization. This observation was corroborated in the present
CHAPTER 2 23 Sperm transfer in A. japonicus study, in which sections of copulating pairs revealed sperm in the sockets. It is now known that the male’s legs are used to attach to the female by placing them ventral to the female’s abdomen; mostly close to the natatory lobes. The role of the spermathecal spines in sperm transfer or copulation has not been clarified, however it is evident that sperm has to pass through the spines in to the spermathecae. The presence of sperm on the accessory copulatory structures as well as Jurine’s (1806) hypothesis of sperm transfer suggests that the spermatophore is manoevred by the fourth leg into the socket of the third leg using the peg, however it is achieved via a spermatophore or sperm packet. Thereafter, the placement of the peg and socket mechanism close to the spermathecal spines, aligns the spermatophore with the spermathecal spines of the female. This alignment would allow the female to receive the sperm from the male, into the spermathecae. The observation of a spermatophore offers additional information to the original observations made by
Jurine (1806) who noticed the sperm in the socket. It therefore corroborates the first explanation for sperm transfer.
CHAPTER 2 24 Argulus coregoni in Malaysia
CHAPTER 3
First record of Argulus coregoni a fish ectoparasitic crustacean from
Malaysia and additional notes on the morphology
CHAPTER 3 25 Argulus coregoni in Malaysia
3.1. Introduction
Since 1909, there have been numerous reports on Argulus species in the oriental region. A literature survey has shown 28 species (Table 3.1). Three of these were first reported or described from Europe i.e. Argulus coregoni Thorell, 1866, Argulus foliaceus Linnaeus, 1758, and Argulus japonicus Thiele, 1899.
During November 2007, seven specimens of A. coregoni were collected in a restaurant in Langat, Selangor in Malaysia and subsequently studied and compared to previous literature. Previous studies of A. coregoni were mainly done using light microscopy. The first description was done by Thorell (1866) thereafter, Tokioka’s
(1936b) re-description followed. However, he did not describe the scales on the periphery of the carapace, the thorax and maxillae but provided a rib count for the sucker. Gurney (1948) provided drawings of the ventral view of a male specimen, including the antennules, antennae, mandibles, toothed labrum, a section of sucker ribs and the basopodites of the second, third and fourth swimming legs of the male, as part of his paper reporting the presence of Argulus species found in Britain.
Hoshina (1950) provided drawings of the antennae, antennules, and the basopodites of the swimming legs of the male, a dorsal view of the female and the ventral view of a male. Romanovsky (1955) provided similar drawings with the addition of the respiratory areas of this species as well as the endopodite of the first thoracic leg of the female. Thereafter, Roland (1963) provided drawings, including the scales on various structures and Penczak (1972) concentrated on the accessory copulatory structures of the males. Okland (1985) provided drawings of the dorsal and ventral aspects and a side view of a male and female.
CHAPTER 3 26 Argulus coregoni in Malaysia
Table 3.1: A list indicating the species, location, host and the reference of freshwater Argulus species reported in Asia and the surrounding islands. The fish species is recorded as it appears in the original reference and the valid species name according to Fishbase (Froese and Pauly, 2009) is also given Species Location Host name (from reference) Valid host name References A. belones Sumatra Belone schismatorhynchus Ablennes hians Van Kampen, 1909 van Kampen, 1909 (Valenciennes, 1846) A. bengalensis West Bengal Not known Not Known Ramakrishna, 1951 Ramakrishna, 1951 A. caceus Misaki, Japan Spheroides sp Spheroides sp Yamaguti, 1963 Wilson, 1922 A. cheni Canton, China Ctenopharyngodon idellus Ctenopharyngodon idella (Valenciennes, Shen, 1948 Shen, 1948 1844) A. chenensis Soochow, China Ophiocephalus argus Channa argus argus Yamaguti, 1963 Ku et Yang, 1955 (Cantor, 1842) Mylopharyngodon aethiops Mylopharyngodon piceus (Richardson, 1846) Leiocassis sp Leiocassis sp A. coregoni Otsu, Japan Acheilognathus moriokae Acheilognathus melanogaster (Bleeker, Tokioka, 1936b Thorell, 1865 1860) Tokyo, Japan Salvelinus fontinalis Salvelinus fontinalis Hoshina, 1950 (Mitchell. 1814) Salmo irideus Oncorhynchus mykiss (Walbaum, 1792) Plecoglossus altivelis Plecoglossus altivelis altivelis (Temminck & Schlegel, 1846) Soochow, China Mylopharyngodon aethiops Mylopharyngodon piceus Wang, 1958 (Richardson, 1846) Tokyo, Japan Salmo gairdneri Oncorhynchus mykiss Shimura and Inoue, (Walbaum, 1792) 1984 Onchorynchus masou Oncorhynchus masou masou (Brevoort, 1856) Honshu, Japan Oncorhynchus masou ishikawai Oncorhynchus masou masou (Brevoort, Nagasawa & Ohya, 1856) 1996a Honshu, Japan Plecoglossus altivelis Plecoglossus altivelis altivelis (Temminck & Nagasawa & Ohya, Schlegel, 1846) 1996b Yoshiga, Japan Salvelinus leucomaenis imbrius Salvelinus leucomaenis imbrius Nagasawa & Kawai, Jordan & McGregor, 1925 2008 A. foliaceus Nuwara Eliya, Sri Mirror carp Cyprinus carpio carpio Kirtisinghe, 1964 Linnaeus, 1758 Lanka Linnaeus, 1758
CHAPTER 3 27 Argulus coregoni in Malaysia
Table 3.1 continued Species Location Host name (from reference) Valid host name References A. giganteus Not known Not known Not known Ramakrishna, 1951 Ramakrishna, 1951 Mahim Creek, Tetrodon oblongus Takifugu oblongus Rangnekar, 1957 Bombay (Bloch, 1786) A. indicus Bangkok, Thailand Fighting fish Betta splendens Wilson, 1927 Weber, 1892 Regan, 1910 West Bengal Ophiocephalus punctatus Channa punctata Ramakrishna, 1951 (Bloch, 1793) Tasik Temengor, Ophiocephalus micropeltes Channa micropeltes Seng, 1986 Perak (Cuvier, 1831) Hyderabad Major carps Major carps Jafri & Ahmed, 1991 A. japonicus Japan Cyprinus carpio Cyprinus carpio carpio Tokioka, 1936b Thiele, 1900 Linnaeus, 1758 Carassius carassius Carassius carassius (Linnaeus, 1758) Yaizu Goldfish Carassius auratus auratus Yamaguti, 1937 (Linnaeus, 1758) Malacca Ctenopharyngodon idellus Ctenopharyngodon idella (Valenciennes, Seng, 1986 1844) Penang Leptobarbus hoeveni Leptobarbus hoevenii Seng, 1986 (Bleeker, 1851) Hyderabad Labeo rohit Labeo rohita Jafri &Ahmed, 1991 (Hamilton, 1822) Catla catla Gibelion catla (Hamilton, 1822) Cirrhina mrigala Cirrhinus cirrhosus (Bloch, 1795) A. kumingensis Kunming, China Free Swimming Free Swimming Shen, 1948 Shen, 1948 A. kusafugu Aichi, Japan Spheroides niphobles Takifugu niphobles Yamaguti, 1963 Yamaguti et Yamasu, (Jordan & Snyder, 1901) 1959 A. mangalorensis Nethravarthy Free Swimming Free Swimming Natarajan, 1982 Natarajan, 1982 estuary, Mangalore A. matuii Chiba, Japan Parapristipoma trilineatum Parapristipoma trilineatum (Thunberg, Yamaguti, 1963 Sikama, 1938 1793)
CHAPTER 3 28 Argulus coregoni in Malaysia
Table 3.1 continued Species Location Host name (from reference) Valid host name References A. mongolianus Manchuria Not known Not known Yamaguti, 1963 Tokioka, 1939 A. nativus Sri Lanka Promicrops lanceolatus Epinephelus lanceolatus Kirtisinghe, 1964 Kirtisinghe, 1964 (Bloch, 1790) A. onodai Japan Spheroides alboplumbus Takifugu alboplumbeus Tokioka, 1936b Tokioka, 1936 (Richardson, 1845) A. papuensis Papua New Bunaka herwerdeni Ophieleotris aporos Rushton-Mellor, 1991 Rushton-Mellor, 1991 Guinea (Bleeker, 1854) A. plecoglossi Japan Plecoglossus altivelis Plecoglossus altivelis altivelis (Temminck & Yamaguti, 1937 Yamaguti, 1937 Schlegel, 1846) A. puthenveliensis India Esomus danrica Esomus danricus Thomas, 1961 Ramakrishna (Hamilton, 1822) Puntius vittatus Puntius vittatus Day, 1865 Macropodus cupanus Pseudosphromenus cupanus (Cuvier, 1831) Panchax panchax blochii Aplocheilus blockii (Arnold, 1911) A. rothschildi Tring Reservoir Abramis brama Abramis brama Leigh-Sharpe, 1933 Leigh-Sharpe, 1933 (Linnaeus, 1758) A. scutiformis Japan Not known Not known Thiele, 1899 Thiele,1900 Misaki, Japan Mola mola Mola mola Tokioka, 1936b (Linnaeus, 1758) Hokkaido Not known Not known Siripur, Bihar Labeo rohita Labeo rohita (Hamilton, 1822) Himalayas Not known Not known Saurashtra Murrel Channa striata (Bloch, 1793) Calcutta Ophiocephalus punctatus Channa punctata Yamaguti, 1963 (Bloch, 1793) Bihar Labeo rohita Labeo rohita (Hamilton, 1822)
CHAPTER 3 29 A. coregoni in Malaysia
Table 3.1 continued Species Location Host name (from reference) Valid host name References A. siamensis- Rajahmundry Not known Not known Ramakrishna, 1951 peninsularis Ramakrishna, 1951 Not known Ambassis ranga Parambassis ranga Yamaguti, 1963 (Hamilton, 1822) A. taliensis Lake Tali, China Free Swimming Free Swimming Shen, 1948 Shen, 1948 A. tientsinensis Tientsin, China Pseudobargrus fulvidraco Pelteobagrus fulvidraco Ku and Wang, 1956 Ku and Wang, 1956 (Richardson, 1846) A. yui Soochow, China Cyprinus carpio Cyprinus carpio carpio Yamaguti, 1963 Wang, 1958 Linnaeus, 1758 Mylopharyngodon aethiops Mylopharyngodon piceus (Richardson, 1846) A. yunnanensis Kunming Lake, Free Swimming Free Swimming Shen, 1948 Shen, 1948 China
CHAPTER 3 30 A. coregoni in Malaysia
Shimura (1983b) used scanning electron microscopy to describe the mouth tube and preoral sting of this species in comparison with that of Argulus japonicus. The aim of this study was to provide micrographs of the various morphological structures and include previously unknown information on this species. This study also documents a new locality of A. coregoni in the Orient.
3.2. Materials and Methods
Specimens of Argulus coregoni first seen on the 9 November 2007, were collected on the 19 November 2007 off the body surfaces of red tilapia at the “Langat Fishing,
Seafood and Beer Garden” Restaurant on the Langat River in Selangor, Malaysia.
Seven specimens, five male and two female, were collected by Mrs. E. Knight, a fish parasitologist (see acknowledgements). The specimens were preserved in 70% ethanol and donated to the second author (Prof. Oldewage).
Light microscopy
Lactophenol and lignin-pink were used as the clearing and staining agent respectively on two males and one female. A compound and dissection microscope each with drawing tubes attached was used to examine and draw specimens.
Micrographs were taken.
Scanning Electron Microscopy
Specimens were rehydrated in a descending ethanol series and eventually transferred to distilled water. A drop of sodium hypochlorite was added to the distilled water and the parasites were then cleaned with a Camel’s hair paintbrush to remove mucous that remained from the fish host. Three males and one female were freeze dried, the water was sublimated and then the parasites were mounted onto carbon-tape, ventral side facing up. The specimens were sputter coated with gold
CHAPTER 3 31 A. coregoni in Malaysia and normal scanning electron microscopy (SEM) procedures were then followed using a JEOL5600 microscope. Micrographs were taken at 4 to 15kV.
3.3. Results
Argulus coregoni is recognisable by the abdomen lobes which have sharply pointed terminal ends and are not covered by the carapace (Figure 3. 1).
The carapace of this species has a band of sharp triangular scales along the outer edges (Figure 3. 2) and it tapers towards the abdomen on either side. The scales face towards the posterior
The antennules are devoid of ornamentation in the form of scales but the basal podomere bears a large posterior spine, the second bears an anterior spine, the third bears a post median spine, and the last podomere terminates in a hook (Figure 3. 3).
The three spines on the antennules are sharp and recurved but vary in size. A small projection occurs laterally and bears a terminal group of setae. These seem to concur with the depictions by previous authors in their illustrations (Gurney, 1948;
Hoshina, 1950; Romanovsky, 1955). The antennae bear a single short but sharp spine on the basal podomere, while the rest of the podomeres bear setae. The second podomere bears three, the third one, the fourth four, the fifth ten and the sixth has three simple but long setae
The suckers contain approximately 62 rods (Figure 3. 4); this number is not exact because some areas of the sucker sustain damage during the parasite’s life causing damage to the rods which thus alters the count. The sclerites which make up the rods, are generally rounded with a proximal dent (Figure 3. 5).
The maxillae are composed of five podomeres (Figure 3. 6), with the basal podomere bearing three blunt, rectangular shaped spines that terminate dorsoventrally flattened, of which one is shorter than the others. The basal plate
CHAPTER 3 32 A. coregoni in Malaysia bears blunt triangular scales (Figure 3. 7) and two simple setae pointing towards the posterior. The last podomere of the maxillae terminates in a blunt fused extension, below which are two recurved sharp claws (Figure 3. 8). The dorsal surface of the second, third, fourth and fifth segments are adorned with pectinate scales arranged on delineated areas. Some scales have finely bristled ends, while others exhibit coarsely bristled ends (Figure 3. 9).
Accessory copulatory structures are present on the second, third and fourth swimming legs of the male. The second leg bears three terminally rounded projections, the two ventral projections differ in size from each other while a smaller one occurs dorsally (Figure 3. 10), and these are covered in blunt ovular scales and sensory setae (Figure 3. 11). The third legs contain an indentation (socket) on the ventral side, in which the peg of the fourth leg may fit (Figure 3. 12I&II). This socket has ridges which resemble fingerprints lining the inside of it and scales surrounding the entrance of the socket. The peg on the fourth leg consists of only one projection with its terminal end having four clear sharp spines (Figure 3. 13). The ventral side of this peg is devoid of ornamentation, but at the connection to the leg, there is a bulbous swelling (Figure 3. 13), while on the dorsal side and the spines of this peg there are ovular scales (Figure 3. 14). These scales would come into contact with the ridges on the inside of the indentation. Sensory setae occur on the dorsal side of the peg. All the swimming legs are made up of an endo and an exopodite both of which have plumose setae, on which setules are present and the legs bear ovular scales.
The preoral spine (Figure 3. 15) is situated anterior to the proboscis; it is retractable and terminates in a single opening. The proboscis terminates in the mouth which is surrounded by an upper and a lower lip (Figure 3. 16). The upper lip terminates in
CHAPTER 3 33 A. coregoni in Malaysia two protrusions laterally and the base of the indentation bears what appear to be two sensory pits. The sensory pits are deep and have a furrow that rotates towards the inverted centre. The lower lip is crescent shaped and the terminal ends extend anteriorly past the origin of the upper lip. It is covered by rows of small protrusions organized in short lines. In the centre of the lower lip a protrusion appears which bears sensory protrusions in the middle. Enclosed within the mouth are two biramous mandibles (Figure 3.16 and 3.17). Each mandible consists of a crescent- shaped structure that protrudes medially and a second structure that extends anteriorly. The latter is only visible after micro-dissection to remove the upper lip.
The crescent-shaped parts each bear a single row of denticles proximally. The denticles face towards the posterior end of the body (Figure 3. 16). Two labial spines are present between the mandibles, each terminating in an opening.
Two respiratory areas occur on the ventral surface of the carapace, the anterior is smaller and roughly ovular in shape which is flat posteriorly. The second is a much larger kidney bean shaped posterior region very similar to that depicted in Penczak’s
(1972) report. What became apparent during the SEM is that these are clearly defined by a ridge delineating the margins and the surface structure differs in appearance when compared to the rest of the carapace (Figure 3. 18) in that, the surface in the respiratory areas are somewhat smoother than the carapace, and the creases that are evident in both are not as pointy in the respiratory areas as they are in the remainder of the carapace.
CHAPTER 3 34 A. coregoni in Malaysia
Figure 3.1.Light micrograph of a wholemount specimen of a male Argulus coregoni stained with lignin pink. Specimen is 4mm long. Scanning electron micrographs of Argulus coregoni: 2. Dentate scales that face towards the posterior and occur along the edge of the carapace. 3. Antennule and antennae -a antenna, as anterior spine, lp lateral projection, ps posterior spine, th terminal hook. 4. Section of the sucker- d rods. 5. Rod with sclerites. 6. Maxillae- bp basal podomere. 7. Basal plate of the maxilla. 8. Maxilla terminates in a blunt fused extension below which are two recurved sharp claws -arrows show claws, e extension. 9. Maxilla scales- cb coarsely bristled scales, fb finely bristled scales.
CHAPTER 3 35 A. coregoni in Malaysia
Figure 3. Scanning electron micrographs of Argulus coregoni. 10. Second swimming leg of male with accessory copulatory projections, block indicates projections. 11. Male second leg projection with scales and sensory setae, arrow shows a seta. 12. Male third swimming leg socket. 12I. Scales on the socket. 12II. Scales magnified. 13. Peg on the fourth leg of the male with the four sharp spines, ventral side. 14. Peg on the fourth leg of the male showing scales on the dorsal side. 15. Preoral spine. 16. Mouth- arrow sensory pits, lp lower lip, ls labial spines, sp sensory protrusions, up upper lip. 17. Mouth with the upper and lower lips removed to show mandibles- white arrows indicate crescent shaped mandibles, black arrow indicate second section of mandible. 18. Anterior respiratory area and a part of the posterior respiratory area - ar anterior respiratory area, pr posterior respiratory area.
CHAPTER 3 36 A. coregoni in Malaysia
3.4. Discussion
The SEM study revealed scales on the carapace that were not previously described.
These are presumably used to aid attachment to the host. When the argulids attempt to move around on the host, the entire anterior end of the carapace is lifted, the suckers are replaced and then the carapace is flattened against the surface.
While the setae of the antennae have previously been shown in drawings, counts were not specified. In this study, it was found that there are 21 setae visible from a ventral view on the antennae.
Roland (1963) paid close attention to the shape of the scales on the maxillae. In a light microscopy study, she depicted that the majority of the scales were coarsely pectinate however, in this study, it was shown that some of the pectinate scales have finely bristled ends. In addition, the scales on the maxillae that were depicted as tear drop shaped scales were found to be finely bristled pectinate scales instead.
The accessory copulatory structures have garnered many opinions about the functions thereof. Leydig (1850) postulated that with Argulus foliaceus the socket of the third leg was a semen capsule into which the peg of the fourth leg would transfer sperm before depositing the sperm into the spermathecae of the female. The ridges which are present, however, seem to suggest a mechanism for the peg of the fourth leg to slide in more easily, assisted by the scales present on the dorsal side of this peg. The fact that the scales are facing towards the posterior suggest that their function is to aid the peg from slipping from the socket. Furthermore, in A. japonicus, it was shown that there is no connection between the peg and the reproductive organs (Avenant-Oldewage and Swanepoel, 1993).
The term respiratory areas have been used to refer to the areas on the ventral surface of the carapace. This function was ascribed to the structures by Grobben
CHAPTER 3 37 A. coregoni in Malaysia
(1908), but Haase (1975a, b) has suggested that the function is rather ion exchange or osmotic regulation based on ultra structural investigation of the structure. These publications have unfortunately not resulted in a change in terminology in the prominent literature in this group.
Concerning the distribution of A. coregoni, it was first described by Thorell (1866) from the lakes of Scandinavia. Subsequently, Tokioka (1936b) found specimens in
Otsu in Japan on Acheilognathus moriokae Jordan and Thompson. These were the first to be reported in Asia. Gurney’s (1948) specimens of A. coregoni in 1948 were restricted to water systems in the United Kingdom on trout and pike. Hoshina (1950) reported a second sighting of the parasite in Japan. Next, Romanovsky (1950) worked with Argulus species from 120 different locations in Czechoslovakia, one of the species was A. coregoni. Following that report, Roland (1963) provided a comprehensive redescription of A. coregoni on Salmo fario L., from a river in France.
Thereafter, Penczak (1972) reported it in Poland on Leuciscus cephalus (L.). Finally,
Okland (1985) published a record of A. coregoni in central and southern Norway with a list of the lake types as well as the fish hosts on which this parasite was found.
Thereafter, the places where the parasite was seen were repititions of these first sightings.
Red tilapia results from a cross between Oreochromis species (Popma and Masser,
1999). Crosses have been reported in Taiwan between a mutant red Oreochromis mossambicus and either a mutant Oreochromis niloticus or wild Oreochromis species. Other crosses have been reported in Israel, and in Florida in North America
(Popma and Masser, 1999).
Fish infected with Argulus will exhibit extreme flicking of the fins (Yildiz and
Kumantas, 2002), shoaling behaviour (Northcott et al., 1997), reduction in feeding
CHAPTER 3 38 A. coregoni in Malaysia
(Taylor et al., 2006) and jumping out of the water (Taylor et al., 2006). Other signs of infection, if the parasite has detached when the fish is caught are scale loss, fin damage, (Northcott et al., 1997) and haemorrhaging. Adults are visible on fish as motile black or green spots on the body, but on closer inspection the abdomen and eyes are easily visible.
Argulus coregoni cause a hemorrhagic response through the excretion of toxic substances into the skin of its host while feeding; in order to facilitate the haemorrhaging. In severe infections this could lead to hemorrhagic anaemia
(Shimura and Inoue 1984). Argulus coregoni has also been linked to secondary infections. It is the intermediate host for skrjabillanid nematodes in Russia
(Tikhomirova 1970, 1980) and has been linked to secondary bacterial infections, namely furunculosis caused by Aeromonas salmonicida (Shimura et al., 1983a), and
Columnaris disease caused by Flavobacterium columnare (Bandilla et al., 2006). In other argulid species it was shown that the immune system of fish becomes unable to fight a secondary infection due to the suppression of the immune response from a first stressor (Ruane et al., 1999; van der Salm et al., 2000). In the case of the aforementioned bacterial infections, the immune response of the fish might have been weakened by the first infection with A. coregoni and it therefore becomes more susceptible to the bacterial infections. Other problems associated with argulid infections is a reduced rate of growth and an impact on the aesthetic value of the fish host which results in economic losses in fisheries (Northcott et. al., 1997; Taylor et al., 2006).
This species has not previously been reported in Malaysia, nor has it been reported on red tilapia in any other locality. It is suggested that this A. coregoni was imported
CHAPTER 3 39 A. coregoni in Malaysia into Malaysia, either with another fish species already known to be a host and then transferred to red tilapia or, through the importation of red tilapia.
3.5. Conclusion
Ten days passed between the time of first sighting and collection, which suggests that the occurrence of A. coregoni in the Langat River is probably not incidental and that the Langat River has been contaminated with this parasite. Fish farmers utilising water from this river should take note of the risks associated with infection by this parasite and the potential diseases it transmits.
CHAPTER 3 40 General Discussion
CHAPTER 4
Summative discussion and future
CHAPTER 4 41 General Discussion
4.1. Sperm transfer in Argulus japonicus Thiele, 1900
The objectives set for this part of the study were achieved. The hypothesis that sperm is transferred with the aid of the swimming legs of the male was proven true.
It was shown that a spermatophore which is formed and extruded during copulation is manoeuvred into the socket structure before the sperm is transferred to the spermathecal spines of the female. This is the first description of a spermatophore in
Argulus and offers an alternative explanation for sperm transfer. This explanation also provides the first successful explanation for the presence of the various structures on legs 2 to 4 of the male. This was achieved by combining serial sections and scanning electron microscopy with observations of live specimens.
4.2. Argulus coregoni Thorell, 1866 in Malaysia.
The objectives set for the second part of the study were also achieved. The hypothesis for this part of the study was that this was a new record for this species in
Malaysia and it was also proven true. The specimens collected in Malaysia were identified as A. coregoni based on morphological features such as the shape of the abdomen and the accessory copulatory structures of the male. Scanning electron microscopy was used to improve the description of the mouthparts and additional morphological structures previously only described from light microscopy. The specimens were most likely introduced into Malaysia.
4.3. Future research.
As far as future research in this group is concerned, the morphology and distribution of Argulus has been documented well but, there are other aspects that need urgent attention, such as the pathology of the genus. In this respect, Bower-Shore (1940)
CHAPTER 4 42 General Discussion suggested that Argulus foliaceus caused destruction to the colour pigments of a stickleback fish; and that the infection by A. foliaceus damaged the mucous layer thus making the fish susceptible to infection with fungi. Thereafter, Pfeil-Putzien
(1977) and Pfeil-Putzien and Baath (1978) showed that Spring Viraemia of Carp
(SVC) could be transmitted by A. foliaceus, while at the same time Moravec (1978) showed that A. foliaceus is the intermediate host to Molnaria erythrophthalmi, a skrjabillanid nematode. Shimura et al., (1983b) studied the haematological changes caused by Argulus coregoni on trout (Oncorhynchus masou) and noted significant changes in infected fish. Ahne (1985) then confirmed that A. foliaceus is the vector for SVC. Watson and Avenant-Oldewage (1996) conducted a brief, scanning electron microscopy study on the damage cause by A. japonicus, the results of this study provided evidence that epidermal cells were removed and proboscis damage was caused. Molnár and Moravec (1997) followed up on Moravec’s (1978) earlier study to show even second and third stage larvae nematodes present in A. foliaceus.
Thereafter, Molnár and Székely (1998) drew the conclusion that the high prevalence of skrjabillanid nematodes in Hungary can be correlated to the high infection of A
.foliaceus, and Moravec et al., (1999) showed that Argulus mexicanus is the intermediate host to Daniconematid nematodes in Mexico. A study on the stress caused by infection with Argulus revealed that feeding cortisol to fish infected with
Argulus japonicus will reduce the stress induced by the parasite on the host (van der
Salm, et al., 2000; Haond et al., 2003). It is now generally known and accepted that infections by Argulus cause ulcerations and haemorrhagic changes to the host (Yildiz and Kumantas, 2002; Akter et al., 2007) and make the host susceptible to bacterial disease (Bandilla et al., 2006). Most recently, Forlenza et al., (2008) conducted a transcriptional analysis of the common carp infected by A. japonicus larvae. Within
CHAPTER 4 43 General Discussion this study changes in gene transcription in peripheral blood leucocytes and skin of carp infected by larval A. japonicus was studied. They showed that the response to infection is at first restricted to the site of attachment, and thus feeding, but is then extended throughout the skin, as a whole organ, as the infection continues. A description to cellular level of the infection was provided, but the lack of such a description for adult A. japonicus seems to be a possible avenue to follow in the future. This question could be studied through the use of wound cultures studied via light and scanning electron microscopy. Furthermore, while chemical analysis has been conducted on the mouth extract of Argulus coregoni (Shimura and Inoue,
1984), and the haematological effects caused by Argulus infection (Shimura et al.,
1983b), the direct effects of this parasite have yet to be described. In a future study, a breeding colony of parasites on a fish colony can provide material for scanning electron microscopy and light microscopy sections to study the cellular response to feeding of Argulus. The study could be comparable to that of Dezfuli et al, (2008) for
Dentitruncus truttae, a monogenean or that of Avenant-Oldewage (1994) for Dolops ranarum.
Another aspect worthy of a study in Argulus is the effect of pheromones secreted by the females to attract males and to what extent these chemical cues play a role in mate recognition. Bandilla (2007) studied mate and host recognition cues but acknowledged that the extent to which the chemical cue of the female works is unknown because the eye-sight of Argulus sp. is well-developed (Mikheev et al.,
1998; Meyer-Rochow et al., 2001). This study may require the use of a maze tank to judge the reactions that occur in the males. Knowledge about how effective pheromones are, may provide another possible control mechanism whereby the
CHAPTER 4 44 General Discussion chemical cue can be disrupted or used as a lure to destroy parasites on commercial fish farms.
Furthermore, studies on different aspects of species of the other three genera,namely Dolops, Chonopeltis and the lesser knownDipteropeltis (Piasecki and
Oldewage, 2008) would be worthwhile. This would involve complete morphological comparisons similar to that conducted by Møller et al, (2008). This comparison would require obtaining specimens of each genus namely, Argulus, Dolops,
Dipteropeltis and Chonopeltis. The comparison would have to include morphological, reproductive, and behavioural aspects, resulting in a comprehensive comparison for use by stakeholders affected (fish farmers, fishermen, and researchers). The significance of such a study would be that the mechanisms used by various genera could be compared to get to a better understanding of this group and its phylogenetic position. A thorough understanding of the biology will also contribute to the development of effective treatments.
CHAPTER 4 45 General References
CHAPTER 5
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