ASPECTS OF THE MORPHOLOGY OF CHONOPELTIS THIELE,1900 (CRUSTACEA: BRANCHIURA) WITH SPECIAL REFERENCE TO THE REPRODUCTIVE SYSTEMS
by
Madeleina Jeanetta Grundlingh
THESIS
Submitted in partial fulfilment of the requirements for the degree
DOCTOR OF PHILOSOPHY
in
ZOOLOGY
in the
FACULTY OF NATURAL SCIENCES
at the
ND AFRIKAANS UNIVERSITY
Promotor : Prof. J.H. Swanepoel Co-promotor : Dr. A. Oldewage
March 1996 This thesis is dedicated with love and appreciation to my parents, Hennie and Rita Blignaut. " The earth is the Lord's, and the fulness thereof; the world, and they that dwell therein. For he bath founded it upon the seas, and established it upon the floods. "
- Psalm 24 :1-2 LED
II WOULD LIKE TO EX SS MY G TITUDE TO
My Maker, for granting me the mercy, ability and strength to complete this study. Prof. J.H. Swanepoel who gave me the opportunity to undertake this study, and to complete it under his competent guidance. Dr. A. Oldewage for her greatly appreciated assistance and constructive criticism during this study. The personnel at the Zoology Department of the RAU, especially Edie Lutsch for her much appreciated friendship, support and assistance in histochemical preparation procedures. Dr. Andrew Deacon, Gerhard Strydom and other Kruger National Park staff members for their assistance in the field work. Hester Roets, Karien Brink and Vernie Naidoo for their time and aid in preparing the bromides and micrographs. Dr. Louis Swanepoel and Rika Nel for their much appreciated assistence during the final preparation of the thesis. My Mother, Rita Blignaut, and Tienie van der Berg for the linguistic attention to the thesis. Ester Le Grange for the typing of the thesis and Hendre van der Berg for the typographic finishes of the thesis. My family, especially my husband, Martin, and daughter, Maritza, for their continuous patience, encouragement and support. The RAU, FRD and Sanlam for financial support during this study.
ll Branchiuran crustaceans are fairly common parasites of freshwater fishes in Africa and are presently represented by not more than 200 described species belonging to four genera, namely: Argulus Miiller,1785; Dolops Audouin,1837; Chonopeltis Thiele,1900; and Dipteropeltis Calman,1912. Chonopeltis is, however, the only one of these genera which is endemic to Africa and species of this genus have been reported from a number of different freshwater fish hosts and localities throughout the Ethiopic region. Although 13 species of Chonopeltis have hitherto been described, lack of morphological detail and morphometric data in taxonomic and subsequent descriptions, has led to confusion and much controversy as to, for instance, the phylogenetic origin, identity and function of various structures. Consequently, the validity of the taxonomic identity or status and affinities of the various species are questionable. Lack of important morphological information has also caused a considerable number of gaps and deficiencies in our present understanding of various aspects of ecological and pathogenetic significance.
In the present study, a comprehensive investigation on various aspects of the morphology, anatomy and histology of adult Chonopeltis specimens was conducted, using not only light microscopy, but also scanning electron microscopy (SEM), histological microtomizing and staining techniques and graphic reconstruction. This investigation is based on several specimens of Chonopeltis victori Avenant-Oldewage,1991 collected during surveys of freshwater fish parasites in 1990 and 1991 from four different fish hosts, namely: Labeo rosae S teindachner,1894; L. congoro Peters, 1852; L. ruddi Boulenger,1907 ; and Barbus marequensis A. Smith,1841, sampled at various localities in the Olifants River in the Kruger National Park, as well as several C. australis Boxshal1,1976 specimens found on L. capensis (A. Smith,1841) and L. umbratus (A. Smith,1841), sampled in Boskop Dam near Potchefstroom.
ill By making use of the mentioned methods of investigation a more complete and reliable morphological description of the distinctive external structures, as well as the internal structures of the cephalic shield alae and especially the reproductive system of these parasites could be given. With the aid of semi-thin (2am) serial sections and graphic reconstruction the various components of the reproductive systems could clearly be identified and the mutual relations and histomorphology of these components be described, discussed and compared with those of other members of the Branchiura. In an attempt to determine the true taxonomic position and relationships of the recognised species of Chonopeltis, the original descriptions, morphometric data, geographical distribution, hosts and affinities are compared, summarised and discussed.
The results of the present study reveal a horseshoe-shaped furrow dorsally that separates the alae from the cephalon. Ventrally the frontal ala contains two marginal chitinous grooves and four sclerotized ridges which support the frontal ala. Two pairs of dorso-ventrally orientated muscles account for the movements of the frontal ala, whilst the movements of each lateral ala are controlled by a pair of transverse muscles and some longitudinal muscles. The lateral alae appear to represent the fused pleurae of the cephalic segments.
The male reproductive tract consists of paired testes, vasa efferentia, vasa deferentia, ejaculatory ducts and prostate complex as well as a single median seminal vesicle and a genital atrium. Several prominent secondary sexual structures occur on the four pairs of thoracopods. The mechanism of sperm transfer presumably involves a process during which the two female spermathecal spines, being inserted into the male genital atrium, penetrate the walls of the respective ejaculatory ducts whereafter semen is actively pumped into the spermathecal vesicles.
The female reproductive tract consists of a single large ovary, two anteriorly fused oviducts of which only one is functional, a genital atrium, a single median genital aperture and a crescent-shaped fertilization chamber. The paired spermathecae, each consisting of a spermathecal vesicle, duct and spine, is located separately in the abdomen and are of considerable significance during sperm transfer from the male as well as during oviposition when stored semen is presumably injected into the yet uninseminated ova.
Finally, aspects on the ecology of C. victori concerning the reproductive cycle, life cycle, and epidemiology are investigated, compared and discussed.
iv
ir- G
Branchiuriese Crustacea is redelik algemene parasiete van varswatervisse in Afrika en word huidig verteenwoordig deur nie meer as 200 beskryfde spesies wat aan vier genera behoort, naamlik: Argulus Miiller,1785; Dolops Audouin,1837; Chonopeltis Thiele,1900; en Dipteropeltis Calman,1912. Chonopeltis is egter die enigste een van hierdie genera wat endemies aan Afrika is en spesies van die genus is al aangemeld vanaf heelwat verskillende varswatervisgashere en lokaliteite regdeur die Etiopiese wyk. Alhoewel 13 Chonopeltis spesies tot dusver beskryf is, het gebrek aan morfologiese detail en morfometriese data in taksonomiese en daaropvolgende beskrywings, aanleiding gegee tot verwarring en heelwat kontroversie oor, byvoorbeeld die filogenetiese oorsprong, identiteit en funksie van verskeie strukture. Gevolglik is die geldigheid van die taksonomiese identiteit of status en verwantskappe van die onderskeie spesies betwisbaar. Gebrek aan belangrike morfologiese inligting het ook heelwat leemtes en tekortkominge in ons huidige vertolking van verskeie aspekte van ekologiese en patogenetiese belang veroorsaak.
In die huidige studie is 'n omvattende ondersoek na verskeie aspekte van die morfologie, anatomic en histologic van volwasse Chonopeltis eksemplare uitgevoer, deur gebruik to maak van nie net ligmikroskopie nie, maar ook skandeerelektronmikroskopie (SEM), histologiese mikrotomering- en kleuringstegnieke en grafiese rekonstruksie. Hierdie ondersoeke is gebaseer op verskeie eksemplare van Chonopeltis victori Avenant-Oldewage,1991 wat tydens opnames van varswatervisparasiete in 1990 en 1991 vanaf vier verskillende visgashere gevind is, naamlik: Labeo rosae Steindachner,1894; L. ruddi Boulenger, 1907; L.congoro Peters, 1852 en Barbus marequensis A. Smith,1841 wat versamel is by verskeie lokaliteite in die Olifantsrivier in die Nasionale Kruger Wildtuin, asook verskeie C. australis Boxshal1,1976 eksemplare gevind op L. capensis (A. Smith,1841) en L. umbratus (A. Smith,1841), versamel in Boskopdam naby Potchefstroom. Deur gebruik te maak van die genoemde ondersoekmetodes kon 'n meer volledige en betroubare morfologiese beskrywing van die kenmerkende uitwendige strukture, asook die inwendige strukture van die sefaliese skild-vleuels en veral die geslagstelsels gemaak word. Met behulp van semidun (2/2m) seriesnee en grafiese rekonstruksie kon die verskillende komponente van die geslagstelsels duidelik geidentifiseer word en die onderlinge verhoudings en histomorfologie van hierdie dele beskryf, bespreek en vergelyk word met die van ander verteenwoordigers van die Branchiura. In 'n poging om die ware taksonomiese posisie en verwantskappe van die erkende spesies van Chonopeltis te bepaal, is die oorspronklike beskrywings, morfometriese data, geografiese verspreiding, gashere en verwantskappe vergelyk, opgesom en bespreek.
Die resultate van die huidige studie toon dat 'n perdeskoenvormige groef, die vleuels dorsaal van die sefalon skei. Ventraal bevat die vleuels twee marginale chitienagtige groewe en vier gesklerotiseerde riwwe wat die frontale vleuel ondersteun. Twee paar dorsoventraal georienteerde spiere is verantwoordelik vir die bewegings van die frontale vleuel terwyl die bewegings van elke laterale vleuel deur 'n paar transversale spiere en etlike longitudinale spiere beheer word. Die laterale vleuels verteenwoordig vermoedelik die versmelte pleuras van die sefaliese segmente.
Die manlike geslagstelsel bestaan uit gepaarde testes, vasa efferentia, vasa deferentia, ejakulatoriese buise en prostaatkomplekse asook 'n enkele mediane seminale reservior en 'n genitale atrium. Verskeie prominente sekondere geslagstrukture kom op die vier paar torakale aanhangsels voor. Die meganisme van spermoordrag behels 'n proses waartydens die twee vroulike spermateka-stekels, wat waarskynlik in die mannetjie se genitale artrium ingesteek word, die wande van die onderskeie ejakulatoriese buise penetreer, waarna semen aktief in die spermateka-reservoirs ingepomp word.
Die vroulike geslagstelsel bestaan uit 'n enkele groot ovarium, twee anterior versmelte ovidukte waarvan slegs een funksioneel is, 'n genitale atrium, 'n enkele mediaan gelee genitale opening en 'n halfmaanvormige bevrugtingskamer. Die gepaarde spermatekas, elk bestaande uit 'n spermateka-reservior, -buisie en -stekel, word afsonderlik in die abdomen aangetref en is van aansienlike belang tydens sperm-oordrag vanaf die mannetjie, en ook gedurende eierlegging wanneer gestoorde semen waarskynlik in die, tot dusver, ongeInsemineerde ova ingespuit word.
Laastens is aspekte van die ekologie van C. victori met betrekking tot die reproduktiewe siklus, lewensiklus, en epidemiologie ondersoek, vergelyk en bespreek.
vli " We must not cease from exploration and at the end of our exploring will be to arrive where we began and to know the the place for the first time! "
- 7. S. Eliot
BLE ONTEN
ACKNOWLE 1 GEMENTS U
A ii, STRACT llA
OPSOMEMING
GENERAL INTRODUCTION 1
MATERIAL AND METH S 8
2.1 Introduction 8 2.2 Study areas 10 2.2.1 The Olifants River (KNP) 10 2.2.2 Boskop Dam 13 2.3 Material 14 2.3.1 Sampling of parasites 14 2.4 Methods 14 2.4.1 Sampling of fishes 14 2.4.2 Fixation and preservation 15 2.4.3 Preparation of whole mounts for light microscopy 16 2.4.4 Preparation of histological sections 16 2.4.4.1 Wax-embedding 16 2.4.4.2 Resin-embedding 18 2.4.4.2.1 Embedding in Glycol methacrylate (JB-4) 18 2.4.4.2.2 Embedding in Transmit LM (TAAB) 18 2.4.5 Graphic reconstruction 19 2.4.6 Scanning electron microscopy (SEM) 19
TAXONOMY, GEOGRAPHICAL DISTRIBUTION, 11 ST PRE14ERENCES AND 1411INITIES 23
3.1 Taxonomic position of the Crustacea Pennant,1777 23 3.2 Classification of the Branchiura Thore11,1864 25 3.3 Genera of the Branchiura Thore11,1864 34 3.3.1 Taxonomy and affinities 34 3.3.2 Geographical distribution and host preferences 37 3.3.3 Key to the genera of Branchiura 39 3.4 Species of the genus Chonopeltis Thiele,1900 39 3.4.1 Geographical Distribution, host preferences and affinities 39 3.4.2 Discriminative taxonomic features 44
GENERAL MO 'BIOLOGY OF CH NOPELTIS VICTO AVENANT- OLDEWAGE,1991 54
4.1 Introduction 54 4.2 General morphology of adult Chonopeltis victori 55 4.2.1 Morphometrics 56 4.2.2 Colouration 57 4.2.3 External morphology of the body regions 58 4.2.3.1 Cephalon 58 4.2.3.2 Thorax 62 4.2.3.3 Abdomen 65 4.2.4 Epibionts 65 4.3 Discussion 66
SPECIFIC MORPHOLOGY OF THE CEPHALIC SHIEL @Iy AND ALAE ("CARAPACE") OF CHONOPELTIS THIELE,1900 88
5.1 Introduction 88 5.2 Cephalic shield and alae ("Carapace") 89 5.2.1 Frontal cephalic ala 89 5.2.2 Marginal grooves and alal crests 90 5.2.3 Muscles of the frontal ala 91 5.2.4 Lateral cephalic alae 91 5.2.5 Muscles of the lateral alae 92 5.2.6 Dorsal surface of the cephalic shield 92 5.3 Discussion 93
MALE REPRODUCTIVE SYSTEM AND SPERM TRANSFER CHONOPELTIS THIELE,1900 102
6.1 Introduction 102 6.2 Morphology and other aspects of the male reproductive system 103 6.2.1 Testes 104 6.2.2 Vasa efferentia 105 6.2.3 Seminal vesicle 105 6.2.4 Vasa deferentia 106 6.2.5 Accessory reproductive glands 107 6.2.6 Ejaculatory ducts 108 6.2.7 Genital atrium 108 6.2.8 Secondary sexual structures 109 6.3 Discussion 110 FEMALE REPRS DUCTIVE SYSTEM IN CI ONOPELTIS TH1ELE,1900 121
7.1 Introduction 121 7.2 Morphology and other aspects of the female reproductive system 122 7.2.1 Ovarium 123 7.2.2 Oviducts 126 7.2.3 Genital atrium 127 7.2.4 Fertilization chamber 127 7.2.5 Spermathecae 128 7.2.6 Integumental glands 130 7.2.7 Secondary sexual structures 130 7.3 Discussion 131
ASPECTS OF THE ECOLOGY OF C. VICTOR! CONCERNING THE REP ODUCTIVE CYCLE, LIIIE CYCLE AND EPIDEMIOLOGY 150
8.1 Introduction 150 8.2 Results 151 8.2.1 Population dynamics and reproductive cycle 151 8.2.2 Infestation dynamics and host preferences 154 8.3 Discussion 159
GENERAL CONCLUSIONS AND RECOMMENDATIONS 185
10. REFERENCES 194 " The most beautiful thing we can experience is the mysterious. It is the true source of all art and science. "
- Albert Einstein GENE L INTRODUCTION
The world is not to be put in order, the world is order incarnate. It is for us to put ourselves in unison with this order to be able to discover it in all its grandeur and unmask all its mysteries. Nature has explored virtually all potential avenues of life and has used most sources of energy and nutrition, with a consequent development of an infinite variety of shapes and forms - all in interrelationship with one another. No organism is an entity unto itself, it lives as a member of a community and the life processes both of itself and of its neighbours act and interact on each other. This interaction between different species is never of equal intensity, but the more intimate it is, the more it approaches our concept of the phenomenon we call parasitism - an extremely common phenomenon, and there are probably more parasitic than free-living organisms. Parasitism, however, too often has its real nature as a biological phenomenon clouded by a concept of disease and is regarded subjectively rather than objectively. Like every other branch of biology, parasitology has its roots in morphology and systematics.
Considering their systematics, the delineation of any species is a difficult task, and the more so in parasitic forms which in many cases are even more difficult to recognize than free-living species. Probably the single most important factor contributing to the confusion, is the paucity of known species. Many species of parasites are known from only a few individuals. Obviously nothing, or very little, can be known about infraspecific variation of morphological and physiological characteristics of such animals. Furthermore, even when parasites are common and easily obtained, their uniqueness is often in question due to various contributing
1 factors (cf. Schmidt & Roberts,1989).
Parasitism, in its widest sense, is a specialized type of life, and parasites, in their efforts to pursue this life successfully, have adopted various distinct modifications and compromises - compromises which in some respects parallel those found between free-living sessile animals and particularly among those which have adopted monophagy. These modifications and compromises affect all spheres of their biology, not only the external and internal morphology of the parasite body, but also its metabolic functioning, life-cycle and all other facets of their ecology. While parasitism leads to specialization on the part of the parasite, it also leads to increased hazards in obtaining its goal, and we find many modifications which are directed towards minimizing these obstacles. The most important of these modifications are indubitably concerned with the production of increased offspring to compensate for the obstacles. This implicate optimal reproduction which can only be attained by means of efficiently modified reproductive systems, copulatory structures and mechanisms of sperm transfer.
In general the parasitic way of life is highly successful, since it evolved independently in nearly every phylum of animals as well as in many plant groups. Organisms that are not parasites are usually hosts of other parasites. It is no wonder then that the science of parasitology has developed out of efforts to understand parasites and their relationships with their hosts. Nevertheless, from a human point of view parasites present a continual and unacceptable threat to the well-being of millions of people, to both wild and domesticated animals as well as to various plants (especially those of agricultural importance), in all parts of the world and the cost of parasites in terms of human misery and economic loss is incalculable. Consequently, parasitology has emerged as a science in its own right from zoology, with its emphasis on what parasites are, and tropical and veterinary medicine, with their emphasis on what parasites do (viz. their epidemiology). However, it is sometimes assumed that when a parasite had been identified as a causative agent of a disease or potential lethal stress condition, and its life cycle had been elucidated, its control or eradication would immediately follow with the development of drugs, vaccines, anti-vector measures or any other chemical or biological means of control. Such assumptions, however, seriously underestimate the complete hold a parasite has on its host and the intimacy of the relationship between them.
2 Parasites display combinations of morphological, biochemical, physiological and nutritional adaptations unique in the animal world, and at the same time display a range of methods of evading the immune response of their hosts. Furthermore, their ecology is turning out to be more complex than that of free-living organisms. Parasites, however, are reluctant to yield up their secrets and this thus attracted the attention of not only parasitologists but also biochemists, physiologists, immunologists and epidemiologists. The intellectual rewards of their research are matched by the potential practical benefits to be gained.
Apart from medically orientated parasitology, parasitological research which is considered in southern Africa to be of the utmost importance is that which has a bearing on those parasites which could have a detrimental affect on food production - an aspect which, in the light of the increasing world population, is of cardinal importance to mankind. Besides traditional fishing, subsistence and commercial fishing, fish farming (aquaculture) is a rapid growing section of agriculture (Hecht et a/.,1988), and in South Africa various fish species are cultured at present (Hecht & Britz,1990). Rainbow trout (Oncorhynchus mykiss) and sharptooth catfish (Clarias gariepinus) are the two main species that are cultured presently while others include various "tilapias" such as Oreochromis mossambicus, 0. macrochir, 0. andersonii and Tilapia rendalli, as well as the common carp (Cyprinus carpio), grass carp (Ctenopharyngodon idella), largemouth bass (Micropterus salmoides), goldfish (Carassius auratus) and a wide variety of ornamental species which are cultured for the local and export markets (Skelton,1993). Notwithstanding this, man has changed the landscape of southern Africa in many ways over the past few centuries, and as a result many animal and plant species have declined and been eliminated from much of their natural range. Many of the threats to freshwater fishes are caused directly or indirectly by human actions and within southern Africa the widespread destruction of natural habitat systems is a serious problem. Draining wetlands and vlei's, which often form the source of streams, is a common agriculture practice. Dams, weirs or other obstructions have been built in all our rivers, which restrict the passage of migrating fish and fragment populations into small units that are more susceptible to other threats. Overpopulation, overgrazing, deforestation and the inefficient cultivation of crops cause soil erosion and the introduction of heavy sediment loads into the rivers. Fertilisers and insecticides enter the system from the lands, polluting the water and destroying the living organisms.
3 Pollution from mines, industry and urban centres destroy or degrades long stretches of river and is an everincreasing threat to freshwater environments. Invasive alien plant and animal species, such as invasive predator fish species, are serious problems in freshwater habitats. Beside these threats to freshwater fishes caused directly or indirectly by human actions, fishes are also subject to a wide range of diseases and parasites (Reichenbach - Klinke,1973; Paperna,1980; Schubert,1987), some of which are fairly well known in southern Africa and have had a great impact not only on natural fish populations (Oldewage,1985; Oldewage & Van As,1987), but also on the aquaculture industry, or have caused losses to fish keepers and aquarium traders (Sarig,1971; Paperna & Van As,1984).
In the wild, deteriorating river conditions throughout southern Africa are causing stress to fish populations (Buermann,1994; Nussey,1994) with a consequent increase in parasitic infestations and diseases (Snieszko,1974; Oldewage,1985; Oldewage & Van As,1987). In this sense, the presence or absence of piscine parasites can be indicative of the presence or absence of pollution or other deteriorating conditions in natural freshwater habitats, or any other stress conditions in cultured fish populations.
Documental information on fish diseases and parasites in Africa is derived predominantly from taxonomic studies. Most of such information consists of taxonomic descriptions of individual parasitic species form occasional collections and from museum material. Studies on the biology of the parasites of African fish are scarce or almost non-existing.
In southern Africa freshwater fish parasites are represented by a diverse group of organisms which includes members of the Protozoa, Platyhelminthes, Acanthocephala, Nematoda and Crustacea (Van As & Basson,1984; Skelton,1993). Some of these parasites are ectoparasitic while others are endoparasitic. Among the piscine ectoparasites, members of the Crustacea are commonly found and are mainly represented by species belonging to the Copepoda, Isopoda and Branchiura. The latter, which has frequently been linked throughout their history to the Copepoda, is in contrast to the Copepoda which includes both free-living and parasitic species, an entirely ectoparasitic group. All species of Branchiura are ectoparasites of fishes, although some have been reported from amphibians (Stuhlmann,1891; Bower-Shore,1940), aligators
4 (Ringuelet,1943) and newts (Carvalho,1939) - which most probably represent coincidential infestations rather than genuine hosts.
Presently the Branchiura comprises not more than 200 described species belonging to four genera, namely: Argulus Miiller,1785; Dolops Audouin,1837; Chonopeltis Thiele,1900; and Dipteropeltis Calman,1912. Branchiurans are found worldwide, with the majority of genera and species thus far described from fishes, particularly freshwater fishes. Argulus, in contrast to the other three genera which are known exclusively from freshwater habitats, is ubiquitous and occurs in both marine and freshwater habitats. Dolops, the only spermatophore producing branchiuran genus, is known from South America, Africa and Tasmania. Chonopeltis is endemic to Africa, while Dipteropeltis is described from South America. Morphological adaptations to a piscine ectoparasitic way of life displayed by branchiurans include, amongst others, a dorsoventrally flattened body; a broad, shieldlike carapace (i.e. cephalic shield) with enlarged respiratory areas; two compound eyes and a single trifid ocellus; two pairs of maxillae of which the first maxillae have become modified in adults as large suckers or claws for attachment to the host; four biramous thoracic appendages ("swimming legs") with distinct copulatory structures present on the legs of males; and a short abdomen with fused segments. Ventrally, the parasite body and appendages also possess various denticulated scales and spines which aid in the retention of position of the parasite on the external body surface of the aquatic host, whilst the large combed and serrated setae found on the thoracopods aid in the induction of a water-current to facilitate respiration. The sexes are separate with the females usually larger than the males. The most important adaptations are, however, those concerned with optimal reproduction, thus the anatomy of the reproductive systems, the morphology of the copulatory structures and the mechanisms of sperm transfer. Nevertheless, within the cumulative research done on these parasites, morphological and especially anatomical and histomorphological work has been largely neglected.
Concerning the branchiuran genera found in Africa, Chonopeltis is the only genus endemic to Africa and is presently represented by only 13 species, described from a number of different freshwater fish hosts and localities throughout sub-Saharan Africa (cf. Chapter 3, Table 3.1). The taxonomic descriptions of most of these species are however, typically based on whole
5 mount studies of only a few specimens with no information on the anatomy or histology of any internal structure. Lack of morphological detail and morphometric data in these descriptions as well as in subsequent articles published on Chonopeltis spp., has led to confusion as to, for instance, the phylogenetic origin, identity and function of various structures. Consequently, the validity of the taxonomic identity and affinities of the various species are questionable, and it is most probable that many, or at least some, of the described species do not represent separate species but are either synonyms, different subspecies or even sibling species. More extensive and detailed morphological knowledge of all known species of Chonopeltis is a prerequisite since the distinct morphological features of each species are not only of considerable taxonomic and phylogenetic, but also ecologic and pathogenetic significance since it reflects several important factors such as mode of transmission and adhesion as well as possible mechanical damage inflicted to the host. Furthermore, detailed information on the morphological adaptations concerned with optimal reproduction is a prerequisite for understanding the reproductive adaptations needed for a successful, piscine ectoparasitic existence in an aquatic habitat.
It is against the foregoing background that a comprehensive study on the morphology and anatomy of adult Chonopeltis was planned, using two species, C. victori Avenant-Oldewage, 1991 and C. australis Boxshall,1976, respectively obtained from various freshwater fish species collected at different localities in the Olifants River in the Kruger National Park, and Boskop Dam, a freshwater impoundment in the Mooi River near Potchefstroom, South Africa.
At an early stage of the study, the initial wide extent of the planned investigation was more specific delimited and some aspects were omitted in favour of a more profound investigation of others. Consequently the taxonomy, zoogeographical distribution and affinities of the known species of Chonopeltis are thoroughly investigated, summerized and discussed (Chapter 3). Since it forms the basis of taxonomy and phylogeny, the external morphology and fine structure of adult C.victori are described in detail, using light and scanning electron microscopy, and these results are compared with that of the other described species (Chapter 4). Furthermore, since many of the criteria used in crustacean taxonomy are based on structures subject to relative growth, relative growth relationships are considered to be of
6 more reliable taxonomic significance than absolute growth measurements - thus, the morphological characters of adult parasites are supplemented by a complete set of morphometric data (Chapter 4). The surface morphology of the cephalic shield and alae, as well as the internal structures of the alae in C. australis and C. victori are described from scanning electron micrographs and histological serial sections, whilst the question whether Chonopeltis species do possess a carapace sensu Calman (1909) is also investigated and discussed (Chapter 5). The anatomy and histomorphology of the reproductive system in both males and females, the morphology of the accessory copulatory structures as well as the mechanism of sperm transfer during copulation and oviposition are described and discussed for the first time, using histological serial sections, graphic reconstruction and scanning electron microscopy (Chapters 6 & 7).
Finally, in order to gain a better understanding and insight into the specific reproductive patterns, life cycles, epidemiology and pathobiology of branchiurans in general and of Chonopeltis spp. in particular, aspects of the ecology of C. victori concerning the reproductive cycle, life cycle and epidemiology are investigated, compared and discussed in Chapter 8. Detailed knowledge of all aspects of this unique group of piscine ectoparasites is not only of academic significance, but also of considerable ecological and economical importance since, as mentioned previously (cf. p.4), piscine ectoparasites could not only bear a potential threat to both natural freshwater fish populations and the aquaculture industry (eg. Argulus), but the presence, or absence, of these parasites can be indicative of the presence, or absence, of pollution and/or other deteriorating conditions in natural freshwater habitats (cf. Chapter 8), or any other stress conditions in cultured fish populations. Thus, besides the intellectual rewards of their research and the contribution to be made to our present state of knowledge concerning the biology of these parasites, potential practical benefits are undoubtedly also to be gained. General conclusions and recommendations consequent upon the results of the study are summarized in Chapter 9.
7 " Progress and growth are impossible if you always think and do things the way other's have thought and done! "
- Wayne Dyer MATERIAL AND METHODS
2.]1 ITT DUCTION
All classification is based on the comparison of specimens representing populations and species. We can determine the species-specific characteristics of a species only by comparing it with other similar species, preferably its nearest relatives. An adequite comparative collection is therefore as indispensable to the taxonomist as are light, electron, transmission and scanning electron microscopes, microtomes, Warburg apparatuses, ultracentrifuges, and similar equipment to the morphologist and histologist. Collections of biological organisms can either be borrowed from museums, universities, or other institutions which are repositories of systematic collections, or collected by the researcher himself. However, borrowed material is usually insufficient for certain crucial areas of study and does not allow the biological investigations necessitated in modern taxonomic and consequentional morphologic research. Much of the material preserved by the various institutions are unreplaceable owing to the remoteness of their type-localities or for political reasons, whilst other material is of unique value because it forms the basis of published research and may be needed again at a later period for verification of the original data. Furthermore, collection specimens (especially old material), may not allow renewed study based on modern techniques and methods of investigation. It is therefore legitimate, indeed necessary, that institutions such as museums, universities and other academical and biological research establishments, build up collections of adequate sample sizes - especially since it is known from experience that many crucial biological investigations had to be set aside for lack of adequate material.
8 Considering the great variability of most natural populations, an adequate sample of every population and/or species should be collected and preserved using various techniques of collection, fixation and preservation. Since the study of the diversity of nature requires a far broader approach than was envisioned by taxonomists of bygone generations (Mayr,1969), collections of whole animals must be supplemented by collections that permit histological, cytological (chromosomal), and biochemical research. With more and more species becoming extinct, the problem of "permanent" preservation is raised increasingly often and in this regard, methods such as embedding in plastics and resins which can be preserved in toto or microtomized, mounted on microscope slides, labelled and stored for later study, are being more commonly implemented particularly for the preservation of whole organisms such as invertebrates, or the ultrastructural preservation of organs or other structures of bigger animals.
Almost any attribute of an organism might be useful as a discriminating taxonomic character and can range from superficial external features such as the scale counts of fishes to the highly conservative and phylogenetically significant sutures and sclerites of the arthropod body, whilst the internal anatomy provides an abundant source of taxonomic characters in all groups of animals. New organs and structures are steadily added to those that show taxonomically important differences. Various components of the reproductive organs and especially the spermatozoa of many taxa, for instance, have a highly peculiar and specific morphology and may serve as useful indicators of relationships (cf. Mayr,1969). For reasons that are not yet fully understood (Mayr,1963,p.103), the genitalia of many animals, particularly the arthropods, not only show a great deal of structural detail but are also highly species-specific. In many groups of arthropods, genital structures are more important for species diagnosis than any other character. However, being three dimensional structures they have to be carefully prepared to be strictly comparable (Dreisbach,1952; Humason,1979).
Against the foregoing background, a comprehensive investigation on not only aspects of the taxonomy and external morphology, but also the anatomy and histomorphology of especially the reproductive systems of species of the genus Chonopeltis Thiele,1900 was conducted. Adequate specimens of these piscine ectoparasites were collected from their freshwater fish
9 hosts on a regular basis (every two months) during 1990 and 1991. Various methods of fixation, preservation, light and scanning electron microscopy, as well as embedding in paraffin-wax, glycol methacrylate (JB-4 (Polysciences)) and Transmit LM (TAAB laboratories) resins, were used whilst various histological, microtomizing and staining techniques were also applied. Semi-thin serial sections were utilized for the preparation of three dimensional graphic reconstructions of the different components of both the male and female reproductive systems. All the mentioned methods of investigation are described further on in this chapter (pp.15-20).
Morphological, anatomical and histomorphological investigations are based on adult specimens of Chonopeltis victori Avenant-Oldewage,1991 and C. australis Boxshal1,1976, respectively sampled from various freshwater fish hosts and localities in the Olifants River in the Kruger National Park and Boskop Dam in the Mooi River near Potchefstroom, during surveys of freshwater fish parasites in 1990 and 1991.
2.2 STUDY AREAS
2.2.1 The Olifants River (KNP)
A part of the Olifants River is located in the Kruger National Park (KNP), the largest sanctuary in the Republic of South Africa and one of the largest controlled nature reserves in the world (Fig. 2.1A,p.21). The KNP is situated in the farthest North-Eastern corner of the Republic, between the Crocodile River in the south (25°32'S) and the Limpopo River in the north (22°25'S). The international border with Mozambique (32°2'E) forms the Park's eastern boundary. The western boundary is very irregular. The Park as a whole forms a reversed "L", and covers an area of 1 948 528 ha or nearly 20 000 km2 (Fourie & De Graaff,1992). Average summer day temperatures in the Park vary between 18°C and 30°C, and winter temperatures between 8°C and 23°C. Extreme maximum temperatures can be as high as 47°C in summer and 35°C in winter. Extreme minimum temperatures can fall to 7°C in summer and -4,2°C in winter (Braak,1993).
10 The rainy season starts in September or October and lasts until March or April, and is followed by a period of very little or no rainfall. About 80% of the precipitation occurs in the form of sporadic thunder showers and is very erratic. Rainfall tends to be lowest in the northern parts of the Park and highest in the south. In the south the annual rainfall is about 760mm, in the central area about 540mm per year, while in the north-east it can be as low as 210mm (Fourie & De Graaff,1992; Kleynhans,1992). On average the Olifants River region in the KNP receives 500-600mm of rainfall per annum, however annual evaporation rates are high and often exceed 1800mm per annum. Summer rates are approximately 80% higher than in winter and evaporation exceeds rainfall in each month of the year (PMC Report,1992). Consequently the Olifants River catchment within the KNP is characterised by arid lowveld savannah vegetation types. The banks have a narrow but distinctive strip of riparian vegetation, dominated by larger trees and few shrubs. The vegetation becomes progressively drier with increasing distance from the river banks, large trees become scarcer whilst smaller shrubs and a well developed grass cover predominate in the drier areas. Extensive Phragmites reed-beds and clumps of shrubs and trees occur along sand banks in the river and the edges of the river channel (Venter,1991).
The Olifants River together with several other rivers - the Crocodile, Sabie, Letaba, Shingwedzi, Luvuvhu, and Limpopo - constitute the major perennial rivers of the Park and are spaced more or less evenly over the considerable length of the Park (Fig. 2.1A & B, p.21). These rivers flow from west to east, their catchment areas being largely outside the Park. Consequently these rivers are subjected to pollution even before they enter the Park (Gertenbach,1991) - an aspect of national concern since pollution could not only lead to the deterioration of the water quality, but consequently also influences the freshwater fish fauna and other biota (cf. Chapters 1 & 8). The most important seasonal rivers include the Shisa, Mphongola, Tsende, Timbavati, Nwaswitsonto, Nwatindlopfu, and Mbyamiti. All these rivers drain into the Indian Ocean (Fourie & De Graaff,1992). The Klaserie, Nhlaralumi, Timbavati and Letaba Rivers feed the Olifants River within the Park. The gentle flowing waters of the rivers and streams in the KNP afford sanctuary to 50 species of fish - including four vagrant marine species. Two species of the rare and unusual seasonal killifish, Nothobranchius orthonotus (Peters,1844) and N. rachovii Ah1,1926, are to be found in a few seasonal and
11 1 other pans within the Park. One of the most sought after sporting fish, the tiger fish (Hydrocynus vittatus Castelnau,1861), is also found in the perennial rivers of the Park. Other well-known fish include the large-scaled yellowfish (Barbus marequensis A. Smith,1841), several species of labeo or mudfish (Labeo spp.), a range of tilapia or kurper (Tilapia spp.), and the common sharpthooth catfish (Clarias gariepinus Burche11,1822) (Pienaar,1978).
The catchment area of the Olifants River within the KNP experiences poor water quality, especially during the dry season (DWA & F,1993). The main contributors to the reduced water quality and deterioration of the aquatic environment are mining (particularly that of the Phalaborwa Complex), irrigation and soil erosion from over-grazed areas in the former Lebowa (Buermann,1994). Due to these influences on the ecology, water quality and consequently also the riverine biota of the Olifants River, the study area included three sampling localities inside the KNP along the length of the Olifants River, and one just outside the Park border on the western boundary at a locality a short distance above to the region where the Selati River joins the Olifants River (Fig. 2.1B,p.21). Sample localities were selected in accordance with accessibility and to represent different ecological habitats.
Sample locality one is located just outside the western boundry of the Park, a short distance above the region where the Selati River joins the Olifants River, and close to the copper, asbetos and phosphate mines of the Phalaborwa Complex. The embankments are fringed by dense vegetation, mostly Phragmites species and Cape date palms (Phoenix reclinata) growing in clumps along the river banks.
Sample locality two is situated inside the western boundary of the KNP, near Mamba where the Olifants River forms a single 100-300m wide channel with an open, shallow sandy river- bed. River-banks are steep, with terraces along the way composed of red and brown sand, silt and alluvial deposits of weathered rock. The river-bed consists of sand and gravel alternated by rocky patches where small rapids occur. Species such as Ficus sycamorus, Diospyros mespiliformis, Acacia robusta and a few others are represented in the narrow but dense afforested riparian zone. Phragmites species are limited to patches along the bank. The main soil types are sand and loam.
112 Sample locality three is further downstream, a short distance west to the confluence of the seasonal Mvubu River with the Olifants River. The river-banks, river-bed and vegetation along the river banks are very similar to that of sample locality two. The channel is, however, much wider, the vegetation less dense with characteristic deep pools and short rapids.
Sample locality four lies still further east, close to Balule rest camp and prior to the Letaba River confluence. The 100-250m wide channel branches into a large number of more narrow but deeper channels, forming a range of islets in the braided channel. River banks are composed of alluvial deposits of brown limy silt and sand, while the river-bed is formed from irregular silt and sand deposits on rocks or islands. The river banks are narrow and species such as Ficus sycamorus, Colophospermum mopane, Diospyros mespiliformis and Acacia robusta are found in less afforested riparian zone. Very dense Phragmites spp. patches occur on the islands.
2.202 Boskop Dam
Boskop Dam (27 °08'E,26°33'S) is situated on the Mooi River (a tributary of the Vaal River), about 20km north-east of Potchefstroom, Transvaal, in the heart of a 3 000 ha provincial nature reserve noted for outdoor recreation (Fig. 2.2,p.22). Boskop Dam, the largest dam in the Mooi River, is an oligotrophic irrigation dam and contains, when full, 20 500 000m 3 water, covers an area of approximately 373 ha that extends over a distance of 4,5 km from inflow to overflow, and reaches a dept of about 14 m (Viljoen,1982). Approximately 160 000m3 water is drawn daily for irrigation purposes whilst about the same volume of water enters the dam daily from the Mooi River (Van As & Combrinck,1979; Duggan,1983). The embankments of the dam are fringed by a band of dense reeds and the submerged macrophyte, Potamogeton pectinatus L., which offer excellent breeding places for various fish species as well as water fowl. Ten different fish species have hitherto been identified in Boskop Dam, with Labeo capensis and L. umbratus the dominant fish species in this locality (Koch,1975; Malan,1988).
13 2.3 MATE
2.3.1 Samp arg of parasites
Adult Chonopeltis victori specimens were collected mainly from the body surface and fins of four different host fish species, namely: Labeo congoro Peters,1852; L. rosae Steindachner, 1894; L. ruddi Boulenger,1907; and Barbus marequensis A.Smith,1841, caught at various localities in the Olifants River in the KNP, Republic of South Africa. Since the phenotype of animal populations of the same species often vary according to locality, season, or habitat (Mayr,1969), specimens of this parasite were sampled every two months during a two year period (1990 and 1991) at the same four sampling localities in the Olifants River.
Adult specimens of C. australis were collected from the body surface, fins and gill chambers of two cyprinid hosts, namely: L. capensis (A. Smith,1841) and L. umbratus (A. Smith,1841), caught in Boskop Dam, a freshwater impoundment in the Mooi River. Since these parasites were mainly used for comparison purposes, they were sampled only twice a year during 1990 and 1991, viz. January and July - both periods of established optimal prevalence and abundance (Knight,1991).
The sampling localities where specimens of C. victori and C. australis were collected are respectively illustrated in Figure 2.1B and Figure 2.2B.
2.4 METHODS
2.4.1 Sampling of fishes
Various fish species of different sizes were caught by means of gill-nets, seine-nets and cast- nets in the Olifants River, while only gill-nets and occasionally seine-nets were used in Boskop Dam. Gill-nets of 90,110,120 and 130mm stretched mesh-size, 30m long and 1,5m wide and
14 constructed according to the method of Polling (1979) were used. These nets were placed in the same locations at the different sample localities during each sampling period of the field study. Cast-nets were used for the collecting of smaller fishes, while electro-fishing was only used in fast flowing, stony, shallow streams such as at sample locality one in the Selati River.
All fishes collected during each sampling period (usually between 06:00 and 14:00) were identified and examined for Chonopeltis parasites. The different host species, their total and fork lengths and weight as well as the number of parasites per fish, their position on the fish and the sex of each parasite were recorded. Parasites were removed from the fish using a small brush, and placed into 50m1 specimen bottles filled with either filtered (using filter paper) river water or filtered dam water.
2.4.2 Fixation and preservation
Following removal from the fish host, live specimens were repeatedly washed in filtered river or dam water to remove mucus and other debris prior to fixation. Live parasites were prepared for fixation by positioning each specimen separately on a clean microscope slide containing a drop of water. A small amount of petroleum jelly was placed on either side of the drop of water and the microscope slide then transported to a petri dish for viewing under a dissecting microscope. To ensure correct orientation of the appendages, the specimens were manipulated with a fine camel's hair brush. A small coverslip was next placed onto the petroleum jelly and carefully pressed down to ensure that the specimen remained flat. Using a medicine dropper, the fixative was dropped between the coverslip and microscope slide.
Some specimens were fixed in a 10% solution of steaming hot phosphate-buffered neutral formalin (10% PNF) (Romeis,1968; Humason,1979), whereas others were fixed in either Tellyesniczky's acetic acid-alcohol-formalin solution (AFA) (Lillie & Fullmer,1976; Humason,1979) or in Bouin's fluid (Humason,1979).
Specimens fixed in 10% PNF and Bouin's fluid were removed from the microscope slides after
15 a minimum period of 30 minutes and respectively placed into fresh solutions of 10% PNF and Bouin's fluid for storage in labelled glass bottles, whereas those fixed in AFA were removed from the microscope slides after 10-30 minutes and thereafter preserved in 70% ethanol. Specimens in Bouin's fluid must be fixed for at least 24 hours and may remain in this fixative for several weeks without any damage, but long periods (months) results in poor nuclear staining and large vacuoles often form in tissues (Humason,1979). Contrary to this, specimens fixed in 10% PNF may remain indefinitely in this fixative since its action is progressive.
2.4.3 Preparation of whole mounts for light microscopy
Specimens preserved in 70% ethanol or 10% PNF were used for the preparation of whole mounts for light microscopy. Following fixation in 10% PNF, specimens were rinsed in running tap water for 24 hours and thereafter dehydrated for a minimum of one hour in each concentration of an ascending series of ethanol solutions (30%, 50%, 70%). Parasites in 70% ethanol were then transferred to a concavity microscope slide containing 90% lactic acid solution (Grandjean,1949) which was used as clearing agent. The microscope slide was heated with great caution over a spirits lamp until the desired degree of clearing had been obtained. A coverslip was placed onto the microscope slide and the preparation sealed with a commercial sealant (e.g. Glyceel). The whole mounts were then studied and drawn with the aid of a drawing tube attachment fitted to a Zeiss light microscope.
2.4.4 Preparatio of histological sections
2.4.4.1 Wax-embedding
Following fixation in 10% PNF, specimens were rinsed in running tap water for 24 hours to ensure the removal of all formalin crystals lodged in the tissue. Specimens were thereafter dehydrated through an ascending ethanol series (30%, 50%, 70%, 80%, 90%, 96% and 2 x 100%) for an hour in each concentration. Samples were then placed into a solution of pure
16 benzene for 5 minutes (longer periods tend to harden specimens), whereafter the parasites were transferred to a heated (35°C) solution of benzene-wax (viz. a mixture of equal parts of melted paraffin-wax and benzene) and infiltrated at 35°C in an embedding oven for 30 minutes. The specimens were next placed into a heated solution of pure paraffin-wax and infiltrated with wax at 60°C for an hour. Thereafter the paraffin-wax was changed twice and each time left to infiltrate for one hour at 60°C. After the first 15 minutes the samples were placed under vacuum for 5-10 minutes and again just prior to embedding to ensure the removal of all trapped air bubbles from the specimens. Following embedding and setting at room temperature, wax blocks were placed into ice water to ease sectioning.
Sagittal and transverse serial sections at a thickness of 8,um were cut with a Reichert rotary microtome and sectioned ribbons were placed onto precleaned microscope slides covered with a weak albumen-glycerin solution (Mayer Albumen Fixative (Humason,1979,p.548)). After sections were stretched on a hotplate at 40°C, excessive albumen solution was removed with filter paper and slides were left to dry at room temperature.
Wax-embedded sections were stained with azocarmine and counterstained with Heidenhain's azan solution (Romeis,1968), dehydrated in ethanol (70%, 96%, 2 x 100%), cleared in xylene, and thereafter mounted in Entellan. Drawings of sections were made with the aid of a drawing apparatus fitted to a Zeiss light microscope.
Embedding in wax is a relatively rapid and easy procedure for obtaining histological serial sections. However, serial sections obtained by means of this method were only used for identifying and reconstructing of the relationship of the various parts of the reproductive systems since detailed histological information such as the different types of epithelial cells constituting the walls of ducts and other organs, and the different developmental stages of ova within the ovary, could not be distinguished in wax-embedded sections. Furthermore, fragile, small, hard parts of the exoskeleton of these crustacean parasites were often crumbled or torn during sectioning, consequently distorting the true appearance of the softer internal structures.
17
2.4.4.2 Resin-embedding
2.4.4.2.1 Embedding in Glycol! methacrylate ( J .1Ie -4 (Pollysciences) )
Specimens were fixed in 10% PNF, rinsed in running tap water for 24 hours, and then dehydrated in an ascending series of ethanol - with concentrations ranging from 30% - 70% and 80% - 96% respectively for 40 minutes and 20 minutes in each concentration. Following dehydration, specimens were vacuum infiltrated with Glycol methacrylate resin (JB-4) for 1-2 hours at room temperature and then left overnight (±8 hours) at 4°C for further infiltration. Samples were embedded at 4°C in freshly catalysed resin and polymerised for at least 2 hours at room temperature.
Sagittal, longitudinal and transverse serial sections at a thickness of 2,um and 5Am were obtained by cutting with glass knives fixed to a rotary microtome. Sections were stretched on a waterbath at room temperature using a fine pair of tweezers and a small camel's hair brush. After stretching on the waterbath, sections were transferred onto microscope slides and left to dry. Sections were stained in Mayer's haematoxylin, blued in running tap water, and counterstained with Eosin Y for 10-20 minutes. After quick (1-3 sec.) differentiation in tap water, sections were dehydrated in ethanol (2 x 96%, 2 x 100%), cleared in xylene and mounted in Entellan.
2.4.4.2.2 Embedding in Transmit LM (TAA
Following fixation, specimens were rinsed in running tap water for 24 hours and then dehydrated in an ascending series of ethanol solutions (70%, 80%, 90%, 2 x 100%) for 30 minutes in each concentration followed by another hour in absolute ethanol. Samples were transferred to solutions of Propylene oxide and absolute ethanol with ratios of 1:3, 1:1 and 3:1 respectively for 15 minutes in each and thereafter placed into absolute Propylene oxide for 15 minutes. Following this, specimens were transferred to a solution containing equal parts of Propylene oxide and Transmit LM (TAAB) mixture, infiltrated for an hour and finally placed into absolute Transmit LM (TAAB) mixture and infiltrated overnight (±8 hours) at 4°C. Parasites were individually embedded in fresh Transmit LM (TAAB) mixture and polymerised for 12 - 18 hours at 70°C.
Sagittal, longitudinal, transverse and frontal serial sections at a thickness of 24m as well as 5itm were obtained by cutting with glass knives fixed to a Reichert Jung rotary microtome. Sections were placed directly onto precleaned microscope slides containing a drop of water for each serial section. Sections were stretched on a hotplate at 40°C. After removing the embedding medium with a saturated solution of sodium hydroxide in absolute ethanol, the sections were stained with azocarmine, counterstained with Heidenhain's azan solution (Romeis,1968), or with periodic acid-schiff solution (PAS) (Pearse,1985), followed by heamatoxylin and light green, or PAS, followed by azocarmine, aniline blue and orange-G. Stained sections were cleared in xylene and mounted in Entellan.
Embedding in Transmit LM (TAAB) resin produced the highest quality of detailed histological serial sections and were consequently used for studying and describing the histomorphology of the internal structures of not only the cephalic shield and alae, but also that of both the male and female reproductive systems in C. victori and C. australis.
2.4.5 Graphic reconstruction
Drawings of serial sections, obtained by means of the three above-mentioned methods of embedding, were made with the aid of a drawing tube attached to a Zeiss Standard 18 microscope. Graphic reconstructions were made according to the method devised by Pusey (1939) and measurements were made by means of a stage micrometer.
2.4.6 Scanning electron microscopy (SEM)
Various methods of preparation were followed for scanning electron microscopy and
19 micrography. Specimens fixed in 10% PNF and thereafter rinsed in running tap water, specimens fixed in AFA and preserved in 70% ethanol, as well as specimens directly killed in 70% ethanol, were used for SEM.
Specimens in 70% ethanol were hydrated through a graded ethanol series (70%, 50%, 30%) for ± 15 minutes in each concentration. Thereafter, samples were transferred to ordinary tap water (also applicable to 10% PNF fixed specimens rinsed in tap water), and cleaned several times by using a fine camel's hair brush to remove mucus and other debris. Final cleaning of the body surface was done by placing the parasites for 30 seconds into an ultrasonic-bath containing tap water.
Dehydration for SEM was done either by (1) freeze-drying, using liquid freon as quenching fluid and an Edwards model ETDA 4 tissue dryer; or by (2) prolonged dehydration (1-2 weeks) in an ascending series of ethanol solutions (30 %,50%,70%,80 % ,90 %,96 %,2x100%) and critical point drying in liquid carbon dioxide with amyl acetate as intermediate solvent and utilizing a Polaron critical point drier. Some dehydrated specimens were dissected under a dissecting microscope with the aid of tungsten needles sharpened in NaNO 2 heated to melting point. Dehydrated specimens were attached to aluminium stubs using a chloroform based glue (Cross,1982). Mounted specimens were next sputter coated with carbon and/or gold using Emscope 250 and 500 sputter coaters respectively. Samples were examined and photographed in an ISI SS60 scanning electron microscope at 1-15kV.
20 FIGURE 2.1
A : Map of the Kruger National Park, indicating the position of the Olifants River (B).
B: Enlarged map of the Olifants River as outlined in Figure 2.1A, indicating the position of the four sampling localities (>1 - 4) for Chonopeltis victori Avenant-Oldewage, 1991. ( .• = Rest Camp).
21 f FIGURE 2.2
A : Map of the Transvaal (South Africa), indicating the locality of Boskop Dam.
B : Schematic drawing of Boskop Dam situated in the Mooi River, indicating the sampling localities (*) of Chonopeltis australis Boxshal1,1976.
22 FillOaDOff 2.2
Phalaborwa o
YE3.4100UAAg.,
[80SUCPM
Potchefstroom " Taxonomy can lay claims to being the oldest, the most basic and the most all-embracing of the biological sciences; it is certainly one of the most controversial, misunderstood and maligned. "
- Charles A. Stace TAXON MY, GEOG APHICAL DISTRIBUTION, H ST EFE NC S AND AFFINITIES
3.1 TAX NOMIC POSITION OF T CRUST CEA PENNANT,1777
"Crustacea Pennant,1777 - to be or not to be: Phylum, Subphylum, or Superclass... ?!"
The Crustacea is an arthropodous group of animals whose members exhibit a greater degree of diversity in form than that seen in any other animal group. There may for example be more species of insects and more individual nematodes than any other group in the world, but the inherent capacity of crustaceans to tagmatize and specialize body segments and appendages ensures that there are more basic kinds within the crustaceans than any other animal group in the world (Hickman et a/.,1988).
The arthropods are an enormous assembly of animals - far outnumbering in species all other animals put together. Insects are the most numerous of the arthropods (± 700 000 to 800 000 described species), followed by the arachnids (± 50 000 to 60 000 species) and then the crustaceans (± 30 000 species) (Brusca & Brusca,1990). In the late 1800's the arthropods were viewed as closely related and the merostomes were considered to be Crustacea whilst the arachnids were aligned with the myriapod-hexapods in the Tracheata (cf. Lankester's (1885) diphyletic grouping of the arthropods). Insects and arachnids were supposed to be linked due
23 to the presence of Malpighian tubules in both groups. However, Lankester (1881) effectively demonstrated the affinities of Limulus with scorpions and thus all arachnids. The Chelicerata were established as a group separate and distinct from all others. The arachnids were then realized to be derived from marine merostomes and not myriapods. Later, it was established that the Malpigian tubules were ectodermally derived in the insects whilst endodermally in the arachnids - thus, nullifying the initial linkage between these two arthropodous groups (Schram, 1986).
Concerning the taxonomy, the arthropods have traditionally comprised a single phylum of metameric, coelomate animals - probably descended from a annelid or annelid-like ancestor (Brusca & Brusca,1990). However, in recent years some investigators (e.g. Anderson,1973; Manton,1973,1977) have maintained that arthropodization occurred more than once and that arthropods as a group do not constitute a natural phylum, that is, they are polyphyletic. The controversial contentions concerning arthropod polyphyly of Anderson (1973) - based on his comparative embryological studies, and Manton (1977) - based on her exacting studies of locomotory functional morphology and limb development, provoked a great deal of debate. That is, although the various arthropod groups share some features in common, these features are not in fact unique to arthropods (e.g. compound eyes, chitinous cuticle, segmented body, jointed appendages), and that fundamental aspects of early development and the functional morphology of locomotion force the conclusion that athropod-like organisms form at least four distinct phyla, viz. Uniramia, Cheliceriformes, Trilobitomorpha, and Crustacea. In agreement with Anderson's (1973) and Manton's (1977) scheme, Schram (1978) integrated knowledge about pycnogonid development (cf. Morgan,1891; Dogie1,1913; Sanchez,1959) and locomotion (Schram & Hedgepeth,1978). Schram (1978) concluded that Chelicerata and Pycnogonida were sister groups within the Glade Cheliceriformes and also felt it best to retain a separate phylum status for Trilobitomorpha. Later Sawyer (1984) established the continuum of "arthropodization" from annelids through clitellates to the uniramians. However, a study of the literature revealed that although various authors (e.g. Schram,1986; Barnes,1987; Hickman et a/.,1988) are of the opinion that the different arthropodous groups should have separate phylum status under the superphylum Arthropoda, they submissively follow tradition - thus sticking to the phylum Arthropoda with the Crustacea (among others), either regarded as
24 a subphylum (e.g. Barnes,1980,1987; Bowman & Abele,1982; Marcotte,1982; Hickman et a/.,1988; Schmidt & Roberts,1989; Barnes & Harrison,1992) or as a class under the subphylum Mandibulata (e.g. Martin,1932; Sherman & Sherman,1976; Ryke,1975; Barnes, 1974; Clarke,1979; Holmes,1979). Without taking position on the possible polyphyly of arthropods, Schmidt & Roberts (1989) consider the Arthropoda as a single phylum with three extant subphyla: Crustacea, Uniramia and Chelicerata. Similarly, Adiyodi & Adiyodi (1983a, b,1988,1990,1992) regard the Arthropoda as a single phylum but distinguish only two subphyla: the Chelicerata and Mandibulata, with the Crustacea placed as a class (together with seven others), under the Mandibulata.
In contrast to the foregoing (i.e. arthropod polyphyly), another group of investigators (e.g. Boudreaux,1979; Paulus,1979; Weygoldt,1979) seek to maintain arthropod monophyly. Many of these investigators are, for the most part, involved in entomology, strongly devoted to the cladistic methodology of the Henning school and also strongly committed to maintaining the status of Mandibulata (cf. Snodgrass's (1935,1952,1959) schemes). However, like .Weygoldt (1979), Boudreaux (1979) as well as Paulus (1979) interpret their evidence against arthropod polyphyly towards preconceived ends. Such twisting of facts to fit a preconceived end is all too typical in the monophyletic arguments. In agreement with Schram (1986), it would thus appear that arthropods are represented by at least three or more distinctly separate groups (of which Crustacea are among the most isolated), and are thus polyphyletic with the various lines of arthropods representing separate phyla. However, exactly how many arthropodous phyla there might be still remains an enigma.
3.2 CLASSIFICATION OF T BRANCHIURA THORELL 9 1864
Branchiuran crustaceans are a relatively small group of primarily freshwater fish ectoparasites and occasionally also of amphibians. Presently the Branchiura comprises not more than 200 described species belonging to four different genera: Argulus Miiller,1785; Dolops Audouin, 1837; Chonopeltis Thiele, 1900; and Dipteropeltis Cal man, 1912 (Overstreet et al. ,1992). Throughout their history, branchiurans have been linked to a variety of different groups and
25 consequently also different taxa. They have been repeatedly shifted from Copepoda to Branchiopoda and back, suggesting that for the most part essential morphological detail has been insufficiently comprehended.
Until 1854 the two known species of Argulus (i.e. A. foliaceus Mfiller,1785; A. catostomi Dana & Herrick,1837) were classified as Copepoda under the family Argulidae Maller,1785, suborder Siphonostoma. The latter included all parasitic copepods with the Argulidae regarded as the most primitive. This classification was first challenged by Zenker (1854), claiming that the Argulidae were wrongly placed under the Copepoda since the "sting" in Argulus is not continuous with the mouth-tube, that is not a transformed part of the mouth-tube, as is the case in the copepods. Consequently he separated the Argulidae from the Copepoda and placed them among the Branchiopoda as a group equivalent with the Phyllopoda. In 1860, Gerstaecker initially favoured the inclusion of the Argulidae with the Copepoda, but later supported Zenker's classification (Gerstaecker & Carus,1863).
After discussing various morphological features of the Argulidae and comparing them with the corresponding features found in the Copepoda, Thorell (1864) reached similar conclusions as to the Branchiopoda affinities of the Argulidae and suggested the new name, Branchiura, for the group. Thorell attributed amongst others, the following features to organisms grouped under the Branchiura: body divided into a head/head shield ("scutum cephalicum"), a trunk ("truncus") to which the "swimming-feet" are attached, and a tail ("cauda") with the latter leaf-like, bearing two small appendages (=furcal rami) which, in the newly-hatched larva, are situated at the tip of the tail (as is the case in the Phyllopoda and Copepoda), but in the adult animals, have gradually advanced upwards to the base of the incision which divides the tail into two lobes. Furthermore, the first/foremost two pairs of head appendages are referred to as the first and second pairs of antennae whilst the hindmost pair (i.e. behind the mouth) are referred to as the first and second pairs of "footjaws" (=maxillulae). The first pair of footjaws serve exclusively as fixing-organs which in the adult state take the form of sucking- cups (=suckers/sucking discs/maxillulae) while in the larvae they are armed at the extremity with a hook. The second pair of "footjaws" called "creeping-feet" ("pedes gressorii" of Kroyer,1863) serve as organs of locomotion (=maxillae). The mouth-organs consist of a fused
26 upper and lower lip which forms the mouth-tube which has a recurved, conical, or club-like appearance. The mandibles are situated within the mouth-tube. Thorell stated that the Argulidae are most nearly allied with the phyllopods and copepods not only by the form and position of the corresponding morphological structures, but also by the history of their development. It is Thorell's view that the form of the limbs, the structure of the paired and completely separate eyes, the presence of a carapace, the conspicuous segmentation of the trunk, and the absence of palps and external egg-sacs, are evidence for placing the Branchiura in the Branchiopoda. The small number of appendages and the unsegmented tail are considered to show relationship with the Cladocera. Thorell (1864) thus included the Branchiura as a third suborder together with two other suborders, the Phyllopoda and Cladocera,under the order Branchiopoda.
Thorell's classification was later followed by Leydig (1871) whilst Claus (1975), retaining the name Branchiura, transferred them back among the Copepoda in a separate third group equivalent to the free-living and parasitic forms, or, alternatively as a second group of the Copepoda, the free-living and parasitic forms being put together. Claus (1875) based his classification on the development of the larva and the general morphology of the parasite body of A. foliaceus Mfiller,1785 and A. coregoni NystrOn,1863.
Wilson (1902) supported Claus's (1875) classification as a result of his observations of two new species of Argulus as well as taking the genera Dolops Audouin,1837 and Chonopeltis Thiele,1900 into consideration. Wilson amplified Claus's reasons for grouping the Argulidae with the Copepoda and consequently suggested the following classification for the Argulidae:
Class: Crustacea Order: Copepoda Suborder: Branchiura Family: Argulidae Genera: Argulus Chonopeltis Dolops
27 The following features were used by Wilson (1902) as evidence for his inclusion of the Branchiura with the Copepoda:
The Branchiura have a flattened body which shows the same general form as in the suborder Siphonostoma,the same division into regions and the same segmentation. The head is fused with the first thoracic segment, while the other thoracic appendages are free, and the abdomen is unsegmented - all features not evident in the Phyllopoda. The number and grouping of the appendages are similar in the Argulidae and Eucopepoda but entirely different from that in the Phyllopoda. There are two pairs of antennae which have been modified into fixing organs. The Argulidae possess two pairs of maxillipeds which, in the larval state, are similar to those of the Siphonostoma. The mouth, consisting of a proboscis formed from the lips and jaws, is similar to that of the Siphonostoma with no resemblance to that of the Phyllopoda. The ovary is unpaired and though the oviduct is at first paired, one side afterwards atrophies. The general position, structure, and function of the semen receptacles is the same in the Argulidae and Eucopepoda. The swimming legs are elongated, two-branched appendages with distinctly segmented basipods and long endopods and exopods, furnished with plumose setae.
Thiele (1904) comes to the conclusion that the grouping of the Branchiura with the Copepoda is unsound. He differs from Thorell (1864) and Wilson (1902) with regard to the presence of maxillae in the proboscis, and consequently termed the "footjaws" of Thorell (1864) or "maxillipeds" of Wilson (1902), maxillulae and maxillae. Thiele (1904) suggested that the Branch iura be placed in an entirely separate group equal in status to the Branchiopoda and Copepoda.
Although Grobben (1908, p.225) agrees with Thiele (1904) on the necessity for placing the Branchiura in a separate group, he considers the Branchiura as a primitive group with affinities - not with existing forms - but rather with the early ancestral stock from which both copepods and cirripedes arose, but he formulates no definite classification.
28 According to Martin (1932) the segmentation of the body and the resultant grouping of the appendages as given by Claus (1875) and Wilson (1902) depend on two premises :
The fusion of one thoracic segment with the head; and The presence of one pair of maxillae in the proboscis.
Martin's (1932) detailed work on the morphology of Argulus however, showed that the first thoracic segment is not fused to the head and that the maxillae are not present in the proboscis at all, which thus invalidates Wilson's evidence for grouping the Argulidae with the Copepoda. Martin (1932) concluded as a result of her examination of the evidence put forward by Wilson (1902) in support of Claus's classification of the Argulidae, that the points of resemblance between the Argulidae and Copepoda are mainly due to convergences in structures such as would occur naturally between groups of Crustacea of similar parasitic habits and thus are of negligible value in classification. Furthermore, Wilson's statement that the ovary in the Argulidae is unpaired, is based on inconclusive evidence. Grobben (1908) concludes that the ovary has a double origin with the single ovary derived from paired anlagen which fuse before maturity is reached. This conclusion is based on comparison with the homologous paired testes as well as the fact that there are always two oviducts with probable alternative functioning. The latter is confirmed by Martin (1932) who showed that A. viridis possesses two oviducts of which only one is functional. Martin (1932) also stated that it is probable that these oviducts function either alternately or periodically, particularly since these animals have at least two breeding periods a year. The inclusion of the Branchiura as a suborder under the order Branchiopoda, as suggested by Thorell (1864), is also rejected by Martin (1932) on the basis of insufficient evidence for such a classification as well as the fact that the features in common between the two groups are few compared to the very marked differences. In view of her observations on Argulidae, and in particular A. viridis, Martin concluded that there is enough evidence to necessitate the placing of the Branchiura into an entirely separate group. She suggested that they be raised to an independent subclass of the Crustacea and that the name Branchiura be retained. Martin (1932) listed the following characteristic features which are entirely independent of the parasitic habits of the animals, and as such distinguish them from any other subclass :
29 Carapace forms bilobed dorsal shield; Number of trunk-somites constant and four in number; No trunk-segments completely fused with head; Body behind genital aperture unsegmented, ending in caudal furcae; Mandibles without palp in adult; Trunk-limbs biramous; First two pairs of thoracic limbs with flabellum; Paired compound eyes; Persistent nauplius eyes of one dorsal and two ventral parts; Hatched either in late stage of development or without metamorphosis; Spermatozoa transferred to female spermathecae, without special copulatory organs or formation of spermatophores.
According to Martin (1932), other characteristics which are found in the group as a whole are due to their ectoparasitic habits, and must be regarded to be of little taxonomic value. These characters include :
Modification of antennules and antennae as organs of attachment; and Formation of a suctorial proboscis enclosing the mandibles.
Contrary to Martin's (1932) suggestions, Dahl (1956,1963) proposed that the Mystacocarida, Cirripedia, Copepoda and Branchiura, which had themselves been traditionally considered subclasses, be contained in a new subclass: Maxillopoda.
In 1963 the classification of the Copepoda and Branchiura were reviewed by Yamaguti. He retained the terminology used by Wilson (1902) and suggested a classification in which the subclass Branchiura contains only one order, the Argulidea, with two families: the Argulidae and the Dipteropeltidae. The latter is distinguished on the basis of the presence of the elongated lateral carapace "wings", the absence of a basal plate as well as the absence of furcal rami (Yamaguti,1963).
30 Although Fryer (1969) rejected Yamaguti's (1963) classification of the Branchiura, stating that Yamaguti 's groupings of the known genera of Branchiura are not valid and that his entire treatment of the intergeneric relationships of the Branchiura only causes confusion, Fryer (1969) made no statements as to the taxonomic status of the group Branchiura. He suggested however, that in the present state of ignorance the four known living genera of Branchiura should be resided in only one undivided family, the Argulidae.
McLaughlin (1980) considered the Branchiura as a class under the superclass Crustacea whilst totally ignoring the family Argulidae.
Bowman & Abele (1982) concurred Dahl's (1956,1963) view, but raising Maxillopoda to class status with the other groups as subclasses. In the latest, more authoritative classification of recent Crustacea, Bowman & Abele (1982) recognized and concisely defined six classes of crustaceans with the Branchiura regarded as a subclass under the class Maxillopoda, subphylum Crustacea. This classification, with a few exceptions, was later followed by, amongst others, Barnes & Harrison (1992). Nevertheless, many other workers have strongly disagreed with the Maxillopoda concept (e.g. Boxshall & Lincoln,1983; Schmidt & Roberts, 1989). Various authors retain the Branchiura as a class under the subphylum Crustacea (cf.
Barnes,1980,1987; Hickman et a/.,1988; Schmidt & Roberts,1989; Overstreet et a/.,1992) while others (e.g. Adiyodi & Adiyodi,1983a,b,1988,1990,1992), follow a classification in which the subclass Branchiura is placed under the class Crustacea, subphylum Mandibulata.
Fryer (1982) retained the Branchiura as a subclass, stating that in contrast to the other crustacean subclasses such as the Copepoda, of which there are many free-living as well as parasitic species, the Branchiura is an entirely parasitic group. Its origins are obscure and the more information acquired about branchiurans, the more difficult it becomes to recognize affinities with other crustacean sub-classes. The superficial similarities of branchiurans to certain copepods is the result of convergence (as previously suggested by Martin (1932)), and not surprisingly, similar morphological adaptations to life as ectoparasites have been independently acquired by members of both groups.
31 In a comprehensive study on the phylogeny and higher classification of the Crustacea, Schram (1986) stated that past discussions on crustacean phylogenetic relationships and classification have been, for the most part, of a rather subjective, evolutionary systematic nature. The classic principals of cladistic analysis (Henning,1966) tempered with consideration of structural plans (Schram,1963) and functional morphology (Boxshal1,1983), can prove productive towards a more logical and reasonable understanding of crustacean interrelationships. In this regard, the computerized analysis made by Schram (1986) on the phylogeny and higher classification of the Crustacea, were for the most part performed using Wagner 78 or programs modified there from, which are designed to produce the most parsimonious arrangement of taxa given the total matrix of characters. The crustacean taxonomy thus proposed by Schram (1986) makes both major and minor changes in realigning groups - inevitably leading to the disappearance or "demotion" of traditional "friends". Commenting on a statement made by Gaffney (1979, p.103) that "there is a strong traditional feeling that stability of some sort is important in classification ...", Schram stated that it is unfortunate since formulation of scientific ideas in a context that imposes stability for traditional purposes breeds the illusion that stability of classification demonstrates accuracy and depth of understanding. In fact, temporal stability of classification often reflects ignorance of relationships and lack of work, whilst the maintenance of names (taxa/classification systems) for discarded concepts seems useless and misleading.
Schram's (1986) classification, derived from the cladograms compiled by himself, is as follows:
Phylum : Crustacea Pennant,1777 Class : Maxillopoda Dah1,1956 Subclass : Branchiura Thore11,1864 Order : Arguloida Rafinesque,1815 Family : Argulidae Leach,1819 ?Order : Pentastomida Rafinesque,1815
Concerning the inclusion of the Pentastomida (tongue worms) as an order of the subclass Branchiura as suggested by Schram (1986), Abele et al. (1989) observed that despite more than
32 a century of investigation and speculation, the phylogenetic relationships of many invertebrate phyla remain unknown. This is a particular problem for parasitic groups, which often lack morphological features that suggest relationships. Various authors have consequently allied the pentastomes with tardigrades, mites, onychophorans, annelids, and myriapods (cf. Haugerud,1989); or have treated them, as do most zoology textbooks (e.g. Hickman,1973; Hickman et a/.,1988; Engemann & Hegner,1981), as an independent phylum with arthropod relationships (Self,1969; Noble & Noble,1982; Riley,1983,1986; Cheng,1986); a class of Mandibulata (Beaver et a/.,1984); or an order of the Arachnida (Brown & Neva,1983). In a comprehensive study, Osche's (1963) research on the embriology and anatomy of Pentastomida convincingly establish arthropodan affinities, whilst Wingstrand (1972) proposed that the Pentastomida be regarded as an order of the crustacean class Branchiura. The latter conclusion is based on the results of Wingstrand's study on the development and structure of the spermatozoa that proved to be almost identical in the Branchiura and Pentastomida and that this type of spermatozoon represents a unique type, not encountered in other animals. On the basis of embriogenesis, tegumental structure, and gametogenesis, Riley et al. (1978) concluded similar results. In accordance with the foregoing, Abele et al. (1989) observed that it is not difficult to see a similarity between the life cycle and larval morphology of the branchiuran Chonopeltis brevis (Fryer,1961a) and those of a pentastomid. In addition, the branchiuran genus Dolops has a mouth flanked by hooks not dissimilar in appearance to pentastomid hooks (Abele et a/.,1989). Finally, on the basis of nucleotide sequences of 18 S rRNA, Abele et al. (1989) included the Pentastomida and Branchiura in the subclass Maxillopoda along with the Copepoda, Cirripedia, Mystacocarida, and Tantulocarida.
Since the status of higher taxa in the Crustacea continues to remain in a state of flux and much controversy, even the status of the taxon "Crustacea" itself, it seems prudent not to adopt the Maxillopoda as a class; therefore, for the purpose of this thesis the Branchiura will be considered a class of the subphylum Crustacea (or phylum or superclass, if you prefer).
33 3.3 GENERA OF THE FIE RANCEDITURA THORELL 9 11.064
3.1.1 Taxonomy and affinities
Crustacean branchiurans are presently represented by four recognized genera, i.e. Argulus Miiller,1785; Dolops Audouin,1837; Chonopeltis Thiele,1900; and Dipteropeltis Calman, 1912, consisting of not more than 200 described species. This group has, however, long been represented by a single genus, Argulus, with A. foliaceus Muller,1785 the only described species. Later this species, together with the second species described, A. catostomi Dana & Herrick,1837, were placed in the small crustacean family Argulidae Leach,1819. In 1863 Kroyer gave the number of known species as thirteen, included were the three species which constitute Heller's American genus Gyropeltis Heller,1857.
In 1837 a simple notice was published by Audouin giving a brief and incomplete description of a new genus, Dolops, with D. lacordaire Audouin,1837 as type species, which he claims is analogous to the description of A. foliaceus as given by Jurine (1806). Thorell (1864) pointed out that this "Dolops" is identical with Heller's Gyropeltis, or at least very closely related to it. Although the more detailed description promised by Audouin (1837) was never published, his imperfect description established with sufficient accuracy the characteristics of the genus Dolops. Notwithstanding this, most authors ignored the inclusion of Dolops as a member of the Argulidae, or the now known Branchiura Thore11,1864. In a comprehensive study on the external anatomy, Bouvier (1898) synonymized the genus Dolops and Gyropeltis - retaining the original name Dolops Audouin,1837.
A year later Thiele (1900) erected a third branchiuran genus, Chonopeltis, with C. inermis (based on a single female specimen) as type species. The now three known genera of Branchiura (viz. Argulus, Dolops and Chonopeltis) were recognized by Wilson (1902) and he distinguished them using the following key :
1. First maxillipeds (=maxillulae) modified into sucking discs. 1.1. Two pairs of antennae, the anterior armed with stout hooks; pre-oral
34 sting present Argulus 1.2. Only one (the posterior) pair of antennae; no pre-oral sting Chonopeltis 2. First maxillipeds (=maxillulae) with barbed claws; no sucking discs; no pre-oral sting Dolops
In 1912, Calman described a new branchiuran species, Dipteropeltis hirundo, based on four female specimens collected twenty years ago in southern Brazil, South America. The most striking features of this species include the remarkable form of the carapace with its lateral lobes drawn out into narrow lanceolat wings directed backwards and extending far beyond the tips of the long abdominal lobes; the absence of furcal rami; the absence of the usual radial supports of the discs of the suckers; and the presence of a pre-oral papilla. Calman (1912) consequently proposed the establishment of a new genus, Dipteropeltis, which he defined as follows :
"Argulidae with the so-called maxillae modified as suckers; with the pre-oral papilla present, but without a spine; with antennules and antennae very minute, imperfectly segmented; without large spines or hooks on under side of carapace, body, or appendages; without furcal rami on the abdominal lobes; with the lateral wings of the carapace greatly elongated."
Based on his observations on the larval form of C. inermis Thiele,1900, Fryer (1956) speculated on the relationship between Chonopeltis and Dipteropeltis as well as their relationship to Argulus. Calman (1912) homologized the unarmed pre-oral papilla of
Dipteropeltis with the pre-oral spine or "sting" of Argulus - a suggestion which is according to Fryer (1956), probably correct. Fryer (1956) stated that no trace of this structure exists in the adult or, perhaps more significantly, in the larva of Chonopeltis. This evidence taken alone would tend to suggest that Chonopeltis diverged from the main branchiuran stock before the evolution of a "poison spine" (=pre-oral spine) had occurred rather than that it lost this structure during the course of evolution. Other evidence, however, indicates that this interpretation may be incorrect, that Chonopeltis and Dipteropeltis are fairly closely related, and that both are rather degenerate argulids in which a reduction of structures has taken place and has been continued further in Chonopeltis than in Dipteropeltis (Fryer,1956,1968,1969,
35 1970). In this case, the loss of a "poison spine" would accord well with other evidence and indicates that Chonopeltis is a degenerate and specialized rather than a primitive form. Both it and Dipteropeltis can probably be regarded as degenerate and specialized descendants of an
Argulus - like ancestor from which they have been derived by an elongation of both thorax and abdomen; reduction of the compound eyes; reduction of the antennules, which have disappeared completely in the adult of Chonopeltis but of which a trace remains in the larva (Fryer,1956); reduction of the pre-oral spine, which is represented by an unarmed pre-oral papilla in Dipteropeltis and which has disappeared completely in Chonopeltis; and by a reduction of the thoracic appendages, which again has been carried slightly further in Chonopeltis than in Dipteropeltis (Fryer,1956,1968). Such observations as have been made on the abortive attempts at swimming made by Chonopeltis, support this postulated ancestry. Chonopeltis and Dipteropeltis have diverged in the rather superficial, though striking, characteristic of the form of the cephalic shield ("carapace"), and Dipteropeltis has lost the supporting rods of the suckers.
Concerning the intergeneric relationships of the Branchiura, Yamaguti (1963) presented the following classification :
Subclass : Branchiura Order : Argulidea Family : Argulidae Subfamily : Dolopsinae Genus : Dolops Subfamily : Chonopeltinae Genus : Chonopeltis Subfamily : Argulinae Genus : Argulus Family : Dipteropeltidae Genus : Dipteropeltis Genus : Talaus
36 In this classification, Yamguti (1963) recognizes six branchiuran genera, including Huargulus Yu,1938 and Talaus Moneira,1963 - the latter a synonym of Dipteropeltis as its describer himself acknowledged and as such included in Ringuelet's (1943) complete synonymic list. Concerning Huargulus, in an obscure publication Yu (1935) gave a very brief description of what he called Dolops sinensis and three years later Yu (1938) described the same species as Huargulus chinensis, the alleged genus being new. After examining the type material labelled by Yu, Tokioka (1940) proved that it was undoubtedly juvenile Argulus specimens, most probably related to A. indicus Weber,1892.
Fryer (1969) recognizes only the four known living genera of Branchiura , i.e. Argulus, Dolops, Chonopeltis, and Dipteropeltis. He consequently rejected Yamaguti's (1963) classification, stating that besides erecting a new order - the definition of which includes erroneous statements, his groupings left us, after correcting his errors of synonymy, with a monotypic family in the case of Dipteropeltis and three monotypic subfamilies in the case of Argulus, Dolops and Chonopeltis. These groupings, however, are not valid since the separation of Chonopeltis and Dipteropeltis into different families for instance, is based in part on misinterpretation of the features employed whilst more important similarities and differences between the genera are ignored - consequently obscuring the interrelationships of the various genera. Superficially at least Chonopeltis appears to be more closely related to Dipteropeltis than either is to the other two genera, and it may be that both were derived, as previously suggested (cf. Fryer,1956,1968), from an Argulus-like ancestor. However, whether the similarities point to a derivation from a common ancestor or are the result of convergence, only further information on especially the larval development in Dipteropeltis could confirm (Fryer,1970).
3.3.2 GeograpIli Tcall distribution and host preferences
Concerning the zoogeographical distribution of branchiurans, they are found world-wide in both marine (Argulus) and freshwater (all four genera) habitats. Provided certain hosts are available, the local distribution of these parasites seems to be influenced more by the physical
37 environment than by the properties of the hosts (Fryer,1956,1960a,1961b; Shafir & Van As, 1985,1986). The genus Argulus, represented by more than 100 species (Overstreet et al., 1992), has an essentially ubiquitous distribution both in freshwater and in the sea, whilst the exclusively freshwater genera seem to have a more limited geographical distribution. Dolops, the only spermatophore producing branchiuran genus (Fryer,1958,1960b), is known solely from freshwater and only from the three southern continents with several species recovered from South America, one from Tasmania, and one endemic to Africa. The species of Chonopeltis are endemic to Africa, while Dipteropeltis is known only from South America. According to Fryer (1969,1970,1986), the general conclusion is that the distribution of especially the species known exclusively from freshwater, is best explained on the basis of the continental drift theory.
All species of the Branchiura are ectoparasites of fishes, although some have been reported from amphibians (Stuhlmann,1891; Bower-Shore,1940), aligators (Ringuelet,1943) and newts (Carvalho,1939). However, it is most probable that these exceptions represent coincidential infestations rather than genuine hosts. As a result of their mobility, branchiuran species generally do not exhibit host specificity. Among the Branchiura, the genera Argulus and Dolops include species with wide host tolerance as well as others with more restricted preferences. However, since the swimming abilities of Chonopeltis and Dipteropeltis seem to be more limited than those in Argulus and Dolops, and at least two species of Chonopeltis employ an intermediate host (Fryer,1961a,1986; Van Niekerk,1984), members of Chonopeltis and Dipteropeltis seem to have more restricted host preferences and are usually confined to a single family of fishes - though the family is not necessarily the same in each case (Fryer, 1968,1986; Schram,1986). In some branchiuran species, preference for a particular host is certainly influenced by whether the latter can provide a suitable area of attachment. Attachment site preferences vary and branchiurans have been found on various sites on the host fishes, including the body surface, fins, beneath the operculum, in the gill chamber, on the gills as well as in the mouth or buccal cavity (cf. Table 3.2). By virtue of the fact that they can crawl over the surface of the host, branchiurans tend to be less rigorously restricted to one part of the body, though some species (especially Chonopeltis spp.) exhibit marked preferences for particular sites (Fryer,1968). Attachment in Argulus, Chonopeltis and Dipteropeltis is
38 effected chiefly by means of the suckers, while in Argulus, these are assisted by the hooks borne on the antennules and antennae, and probably by those of the maxillae as well as by minute spinules on the ventral parts of the body. In Dolops attachment is achieved by means of the stout piercing claws (maxillulae) assisted by other anterior appendages as in Argulus.
3.3.3 Key to the ge era of I ram ura (Based on adult morphology)
1. Maxillulae (= first maxillae) modified into large suckers. Two pairs of antennae, anterior (= antennulae) armed with stout hooks, pre-oral spine Argulus Only one pair of antennae (antennulae absent / degenerate); pre-oral spine absent Chonopeltis Two pairs of antennae minute, imperfectly segmented; pre-oral spine absent but with pre-oral papilla; alae (lateral lobes) of cephalic shield (carapace) greatly elongated, extending beyond tips of abdominal lobes; furcal rami absent Dipteropeltis 2. Maxillulae (= first maxillae) modified into robust claws/hooks; no sucker; pre-oral spine absent Dolops
3.4 SPECIES OF THE GENUS CHONO ELVIS THIELE,1900
3.4.1 Geographical distribution, host preferences and affinities
The genus Chonopeltis Thiele,1900 is endemic to Africa and to date comprises 13 described species - all from sub-Saharan Africa, with the most northerly species C. brevis, and the most southerly C. australissimus, while the species in between replace each other geographically.
The species of Chonopeltis, occurring on a number of different freshwater fish species (cf. Table 3.1), include: C. inermis Thiele,1900; C. schoutedeni Brian,1940; C. congicus
39 Fryer,1959; C. flaccifrons Fryer,1960; C. brevis Fryer,1961; C. meridionalis Fryer,1964; C. elongatus Fryer,1974; C. australis Boxshal1,1976; C. australissimus Fryer,1977; C. minutus Fryer,1977; C. fiyeri Van As,1986; C. victori Avenant-Oldewage,1991; and C. koki Van As,1992. The different distribution localities of Chonopeltis species, their fish hosts, attachment sites on hosts as well as the applicable literature references are summarized in Table 3.1. Although the generic names of some of the fish hosts named in Table 3.1 have changed, the host species of the different Chonopeltis species in Table 3.1 are quoted as used by the original authors referred to in each case.
The geographical distribution of Chonopeltis spp. is illustrated in Figure 3.1. Although the species of Chonopeltis show a wide host range (cf. Fryer,1968,1977; Van As & Van As, 1993), each species seems to be in general confined to a single family of host fishes (Table 3.1). From the data presented in Table 3.2 it is apparent that of the 13 species described, seven parasitize cyprinid fishes, three occur on mormyrids, two on clariids, and one on mochokids whilst one species, C. inermis, not only parasitize clariids but also cichlids and mochokids. Although adult C. brevis specimens occur on cyprinids, larvae of this species have been found on schilbeids which, according to Fryer (1961a), most probably represent an intermediate host since it is known with certainty (in at least two cases), that the larval host does not serve as host to the adult and it is presumed with good reason that this applies in the case of the other species of Chonopeltis as well. Undescribed larvae, possibly of the C.
inermis - like species recorded by Monod (1928) and discussed by Fryer (1960a), have been found on Synodontis nigriventris (Mochokidae) in the Mokombe River, Congo System (Fryer, 1964). According to Fryer (1964), this fish species probably also serves as the intermediate host, especially since Barnard (1955) found larval Chonopeltis on the same genus, Synodontis, in the Okavango River, Congo System. As in all previous records, the host is a bottom- dwelling fish (cf. Fryer,1956,1961a,1964). Problems concerning the means whereby the larvae, and subsequently the adults, locate and attach themselves to host fishes are discussed by Fryer (1956,1961a,1968,1986), while Fryer (1968) also gives a comprehensive account on the host preferences, attachment, feeding habits, effect on the host, zoogeographical affinities and distribution of Chonopeltis spp.
40 Concerning the geographical distribution, species of Chonopeltis have hitherto been found in five of the major river systems as well as in five of the major lakes in Africa (Table 3.3.) Concerning the factors affecting the distribution of individual species, Fryer (1968) remarked that presently distribution patterns are the result of many factors which have operated over a long period, which are not the same for all species and which include the biology of the parasites themselves and physiographic changes causing, amongst other, isolation of former connected areas. Species restricted to one river system or lake owe their restricted distribution to various factors of which the most obvious is restriction to a host which is confined, as are many of the fishes of the great lakes, to one lake. The pattern of speciation shown by the genus Chonopeltis illustrates the part played by geographical isolation, host preferences and, perhaps, ecological preferences (Fryer,1968). During isolation, morphological changes have been accompanied by the acquisition of different host preferences - generally within the same host family in the case of Chonopeltis spp. This is illustrated for example by the Congolese species of Chonopeltis where isolated populations of C. flaccifrons and C. schoutedeni presently exist in the Malagarasi region. Similarly, the parasitic crustacean fauna of Lake Victoria is indicative of a period of isolation, but one of insufficient duration to permit much speciation (Fryer,1961b). Based on the non-endemic fishes and parasitic fauna, the Congo, Zambesi and Nile systems must also have been connected in the past (Fryer,1959). Consequently, the reasonable and, indeed almost inevitable, assumption is that the freshwater fauna of the southern part of Africa was largely derived from the north - this is certainly true of various fishes (Skelton,1993) and appears to be so for Chonopeltis spp. as well (Fryer,1959,1968, 1977). The region in which the group of southern species of Chonopeltis originated can not be ascertained with certainty, but according to Fryer (1977), was probably somewhere in East- Central Africa from where it dispersed animals that differentiated in various regions on different hosts to give rise to the present-day southern species.
Except for the three more recent described species (i.e. C. fryeri, C. victori and C. koki), the geographical distribution of the other ten described species of Chonopeltis has been thoroughly summarized and discussed by Fryer (1959,1961b,1968,1977). Based on morphological comparisons, Fryer (1977) concluded that the three more southern species, namely : C. australis, C. australissimus and C. minutus , are closely related and more similar to each other
41 than is any of them to C. meridionalis. However, Van Niekerk (1984) questioned the validity of the discriminative morphological features according to which the said three southern species have been characterised and classified as separate species. Distinct morphological features used to discriminate between these three species include, among others, the presence of a scale- covered prominence on the proximal portion of the second maxilla of C. australissimus and C. minutus (Fryer,1977) and the shape of the pointed tips of the abdomen lobes which are sharply pointed in C. australis but more bluntly pointed in C. australissimus and C. minutus (Fryer,1977) - a feature found, in at least C. australis, to change during larval development from initially rounded to more sharply pointed later (Van Niekerk,1984; Van Niekerk & Kok,1989). Other features used, include the length and mobility of the thoracopods; the relative size of the cephalic shield ("carapace") in relation to the total body length; the size of the spermathecae and the indistinct thoracic segmentation - all features subject to change during development (Van Niekerk,1984). Furthermore, the possibility exists that the material on which the species descriptions of C. australissimus and C. minutus were based, included only immature individuals. In the case of C. australissimus, the said material was old, not so well preserved and it is not certain whether full grown individuals were included (cf. Fryer,1977, p.446). Fryer assumed that the specimens were sexually mature since the legs of the males showed a complete development of the "claspers" (i.e. copulatory structures). However, these structures were found to be already fully developed in the fourth larval stage in C. australis (Van Niekerk,1984). The possibility that C. australissimus and C. minutus are larval forms of C. australis is still further enhanced by the fact that although the adult of C. australis parasitize a Labeo-species, the larval forms are found in the gill chambers of a Barbus host species. The material used for the descriptions of C. australissimus and C. minutus, have also been found on Barbus host species and in the case of C. minutus, assumed adult specimens as well as several small larval stages were collected from the gill chambers of the same Barbus host species (Fryer,1977). Whether C. australissimus and C. minutus represent valid species closely related to C. australis; possible subspecies, since both C. australissimus and C. minutus were collected from geographic isolated localities; or even larval forms of C. australis, only an indepth study of newly collected specimens from the original localities and hosts, could clarify. Nevertheless, another more valid taxonomic feature used by Fryer (1977) to group C. australis, C. australissimus and C. minutus together, is the lack
42 of projections on the posterior face of leg 2 on the males of all three species. Substantiating this, a study of the literature revealed that all other species of which the male is known, some form of sexual characteristic projection does indeed occur on the postero-ventral parts of the protopodite of leg 2 on the male.
Based on the grounds of males possessing projections on the posterior face of leg 2, Fryer (1961b,1968) regards the species C. inermis, C. meridionalis, C. brevis, C. schoutedeni, C. congicus and C. flaccifrons as closely related species, replacing each other from north to south in distribution. Since Fryer (1968) does not mention C. elongatus, C. fryeri and C. victori (all species described after 1968), Avenant-Oldewage (1991) suggests that the latter species, which also possess projections on the said appendages of males, should be included in Fryer's northern section of species. Similarly, C. koki Van As,1992, possessing these projections on leg 2 of males, should thus also be included in this grouping.
Based on her comparison of available relative measurements of all known species of Chonopeltis (except C. koki), Avenant-Oldewage (1991) concluded that the said species groupings of Fryer (1961b,1968,1977), appear to be valid when relative measurements of species within or between sections are compared. Although comparative morphometric evaluation based on relative measurements allows variation in the parameters and thus makes smaller sample sizes possible, meaningful evaluation of interspecies morphometric variation must be based on a statistically significant sample size - which in most cases of Chonopeltis spp. are too inadequate to permit a valid discrimination between species. Furthermore, comparative morphometric evaluation not only necessitate comparative sample sizes, but also that the individuals of such samples should be of similar age or phase of development, especially since age variation is not restricted to differences between larval stages and adults but occurs also between "young" and "old" adults (Mayr,1969). Seasonal variation of individuals must also be taken into consideration as it is possible that relative growth rates during different seasons may vary, especially where a specimen can live as an adult through several breeding periods or seasons. Where several generations are produced in the course of a single year - as in the case of branchiurans (e.g. Martin,1932; Fryer,1964; Shafir & Van As,1985,1986; Avenant & Van As,1986; Avenant et a/.,1989; Shafir & Oldewage,1992), it
43 is not uncommon that individuals which hatch at different times of the year may differ from those produced at other times of the year. Ecological variation, such as variation in the physical aquatic habitat conditions, climatic variation, host-determined variation etc., may also induce differences such as growth rate and colouration of individuals - all possible in Chonopeltis spp. Many of the descriptions of Chonopeltis species are based on only a few specimens - sometimes even a single immature specimen (cf. Thiele,1900), in some cases collected from an unidentified host (cf. Thiele,1900,1904; Brian,1940), whilst in many cases, without any information on the life history, larval stages, biology or ecology of the specific species.
Concluded from the foregoing, it is questionable whether the different species of Chonopeltis really represent separate species. It is most probable that many of the recognised species are synonyms, different subspecies, or even sibling species (cf. Mayr,1969, pp.183-185 on other species) - even, or on the other hand - especially, when the presently available morphometric data of the existing species, which in the case of most these species are of little value due to the small number of individuals examined, are compared (cf. Table 3.4).
3.4.2 Discriminative taxonomic features
Fryer (1960a,1964,1977) compiled identification keys for the then known species of Chonopeltis. The three more recent described species, C. fryeri, C. victori and C. koki, are not included in any of the said keys. Since identification keys become outdated each time a new species is described, Esser et a/.(1976) pointed out some advantages of a diagnostic species compendium over a dichotomous key. Accordingly, the extent of a synoptic species compendium may vary, and the extent and versatility of its contents can be adapted to optimally accommodate the specific group. The more complete such a compendium is, the more effective it can be utilized when information is required on one or more of the concerned species. With regard to Chonopeltis spp., such a diagnostic species compendium should not only include morphometric data and illustrations of both sexes of the specific species, but also a short summary of the most important discriminative taxonomic features of each species.
44 Other appropriate information such as preferred host species, attachment sites on hosts, geographical distribution localities, structure and shape of eggs, etc., may also be of inestimable value, especially since species identification based solely on the comparison of relative measurements can be misleading in the case of Chonopeltis spp. where morphometric data proved to be incomplete and inadequate in most of especially the earlier described species. Such a diagnostic species compendium was recently published by Avenant-Oldewage & Knight (1994) to include all thirteen thus far described species of Chonopeltis.
Although not always used or included in the descriptions by the authors, the distinguishing taxonomic characters used in the literature published on Chonopeltis spp., include:
Relative cephalic shield ("carapace ") length (cephalic shield length vs total length); Presence/absence of alal crests ("chitinous supporting rods") in the frontal ala of the cephalic shield; Presence/absence of median indentation in anterior margin of the frontal cephalic ala; Structure of maxillae: prehensile/not prehensile; Spacing of thoracopods; Structure and relative length of thoracopods; Structure and shape of the natatory lobes of legs 4: bifid/simple, spinous/not spinous; Structure and relative length of abdominal lobes, size and shape of abdomen; In females: shape of spermathecae (i.e. spermathecal vesicles); In males: structure of features on legs 2-4 - especially that of the "peg" on leg 4 (cf. "peg and socket"); and structure of testes - lobated/not lobated, outer or lateral margins crenelated/not crenelated.
Also used by some authors:
Body pigmentation: presence/absence of two longitudinal bands of pigment spots on dorsal surface of thorax; Segmentation of thorax (distinct/indistinct); Segmentation of antennae;
45 Dorsal cephalic shield grooves and sutures (i.e. "dorsal ridge conformations"); Relative body measurements.
In conclusion, to be able to determine the true taxonomic status of each of the described species of Chonopeltis, an indepth examination of the morphology and anatomy of each species, by means of dissection and scanning electron microscopy need to be done. Such an examination will, however, necessitate adequate samples of fresh material (including both males and females) of each species, collected from their original geographic localities at various times of the year and preferably also from their original host fish species. Specimens used need also be proven to be sexually mature adults.
46 FIGURE 3.1
Map of Africa illustrating the zoogeographical distribution of the 13 recognised species of Chonopeltis Thiele,1900. The numbers on the map indicating the different localities of each species correspond with the numbers listed below as well as with the numbers used in all tables.
SPECIES:
C. inermis Thiele, 1900. C. schoutedeni Brian,1940. C. congicus Fryer,1959. C. flaccifrons Fryer,1960. C. brevis Fryer,1961. C. meridionalis Fryer,1964. C. elongatus Fryer,1974. C. australis Boxshal1,1976. C. australissimus Fryer,1977. C. minitus Fryer,1977. C. fryeri Van As,1986. C. victori Avenant-Oldewage,1991. C. koki Van As,1992.
* Unidentified larvae.
47 IFIMUME Z.V T LE 3.1l
Summary of the described species of the genus Chonopeltis, their distribution localities, fish hosts, and attachment sites on hosts
a) East Africa a) Chromic sp. Thiele (1900, 1904) I'hiele; 1900 b) Lake Malawi Cunnington (1913) c) Lake Malawi c) Branchial cavity Monod (1928) Ulele River d) Okavango River d) Synodontis melanostictus Basis of barbes, chin, Barnard (1955) folds-lower lip, axil of pectoral fin Lake Malawi Clarias sp. Mouth, buccal cavity Fryer (1956, 1968) Banga River Clarias mossambicus Lake Malawi Clariid fishes Mouth Fryer (1966) Zambezi River System Fryer (1968) Luphephe River & Dam h) Clarias theodorae h) Gill chamber Van As & Van As (1993)
a) Congo River Brian (1940) b) Tributaries: Congo River Gnathonemus spp. Darteville (1951) c) Lake Bangweula Gnathonemus spp. c) Under operculum Fryer (1959) Stanley Pool G. monteiri Gill chamber Mormyrus longirostris d) Malagarasi Swamps Momiyrus sp. Fryer (1960a) Lake Mweru Marcusenius discorhynchus e) Lake Mweru Gnathonemius moeruensis e) Under operculum Fryer (1965) t) Congo River Mormyrid fishes 0 Fryer (1968) Lake Bangweula Lake Mweru Malagarasi Region Katanga (=Shaba) & Lower Congo
3. C. cpiiic.us. Lake Bangweula Gnathonemus monleiri Flanks Fryer (1959) 12ry0; 1959 Congo River Mormyrid fishes Under operculum Fryer (1968) Lake Bangweula Lake Mweru
Lake Mweru Marcusenius discorhynchus a) Under operculum Fryer (1960a) River Finn M. webverthi Malagarasi Swamps Marcusenius sp. Congo River Mormyrid fishes Fryer (1968) Lake Mweru Malagarasi Region Katanga (=Shaba) & Lower Congo
• 5. C breyis Victoria Nile a) Barbus airmails radchlfi a) Belly & pelvic fins Fryer (1961b) Fryer, 1961 Tana River Mugambuzi River Lake Victoria Lableo victorianus b) Belly & pelvic fins Fryer (1961b) Ragati River (Tana System) Amphilius grandis, c) Innerwall of operculum Fryer (1961a) Garra sp. pectoral & pelvic fins Amphilius sp. Mugambuzi River A. grammatophorus, Konkoure & Mamou Rivers A. rheophihts (Guinea) Tana River d) Labeo cylindrius Fryer (1964) Regati River Nile River e) Lyprinid fishes Fryer (1968) Congo River Eastern Rivers (North of Limpopo) Lake Victoria Tana River Cyprinid Victoria Nile Mugambuzi River
.6 C. mefidit)halif - Nuanetzi (=Mwenezi) River Labeo rosae Fryer (1964) Fryer, •1964::::- Limpopo & Southern Rivers Cyprinid fishes b) Externally Fryer (1968)
48 Table 3A cont.
8. C. (martins a) Vaal River a) Labeo capensis & a) Body surface a) Boxshall (1976) Boxshall, 1976 Boskop Dam Labeo rosae Doomdraai Dam Skin Van As & Basson (1984) Wuras & Tierpoort Dams (Riet Labeo capensis, Ventral body surface, Van Niekerk (1984) River), MockesDam (Modder Barbus holubi, around mouth, pelvic-, River), PMK le Roux Dam L. umbratus, pectoral & anal fins (Orange River) B. kimberlyensis Wuras Dam Barbus holubi Gill chamber & under Van Niekerk (1984) operculum e) Doomdraai Dam Skin Van As & Basson (1984) (Larvae:) f) Wuras Dam Barbus aeneus & t) Gill chamber & body Van Niekerk & Kok L. capensis surface (1989) Boskop Dam, Labeo umbratus & g) Under operculum Knight (1991) Bloemhof Dam L. capensis Orange River System (Namibia) Van As & Van As (1993)
9. C. ausicalt.ssimus a) Groot Berg River a) Barbus burgi a) Skin a) Fryer (1977) Fryer, 1977.
... 10. C. ,ninutus a) Twee & Tra-Tra Rivers a) Barbus a) Gill chamber, a) Fryer (1977) Fryer, 1977 (Olifants River System) B. erubescens on operculum
11. C. frveri a) Mogalakwena River a) Clarias theodorae a) Gill chamber a) Van As (1986) Van As, 1986 Loskop Dam (Olifants River) C. gariepinus
12. C. victori.:: Olifants River Labeo rosae a) Tail & anal fins Avenant-Oldewage (1991) . Oldew age L. rubropundatus L. ruddi Barbus marequensis Olifants River Labeo rosae b) Body Surface & Fins Own unpublished records L. congoro L. ruddi Barbus marequensis
a) Zambezi River at Katimo a) Label, cylindricus a) Tailfm a) Van As (1992) VanAs, 1992 Mulilo, Namibia
• Unidentifiecl , a) Mokombe River (Congo System) a) Synodontis nigriventris a) Fryer (1964) larvae .
(A short line (-) indicates that the information was not available)
49 TA LE 32
Summary of the geographical distribution, fish host families, and attachment sites on hosts of Chonopeltis spp.
SPECIES ...... •:ii*NP CMDISTRJUTON ...... *TOL ...... ...... ...... Lake Malawi (Zambezi System) Cichlidae Lower lip, • Banga River (Zambezi System) Claridae axil pectoral fins, Zambezi River Mochokidae mouth, Okavango River (Zambezi System) buccal cavity, Luphephe River & Dam (Limpopo System) gill chamber
..:C::::schOoNdenV: Congo River & tributaries Mormyridae Gill chamber, Brian, 1940 Lake Bangweula under operulum Stanley Pool (Congo River) Lake Mweru Katanga & Lower Congo Malagarasi Region & Swamps
C. congicus Lake Bangweula Mormyridae Flanks Fryer,.1959 Congo River Lake Mweru
C. flacelfrOns Lake Mweru Mormyridae Under operculum Fryer,.1960 Fimi River (Lower Congo) Malagarasi Swamps & Region Congo River Katanga & Lower Congo
5. C. brePis.. Tana River Cyprinidae Belly, pelvic fins Fryer, 1961 . Regati River (Tana System) Victoria Nile Larvae: Under operculum Lake Victoria Schilbeidae pectoral & pelvic Lake Tanganyika fins Nile River Congo River Mugumbuzi River Konkoure & Mamou Rivers (Guinea)
Cont
50 TABLE 3.2 .... cont.
...•••••••,....".•••,,, ..•,•, ..••••.....•••,.....•••••••••••• 441)1: "04ffainM .--"
6 C. meridiOnalis Nuanetzi River (Limpopo System) Cyprinidae Externally Fryer, 1964 Limpopo & Southern Rivers
7. C. elongatui Lualaba River (Congo System) Mockoridae Fryer, 1974
C. izustra/is.:.. ORANGE RIVER SYSTEM: Cyprinidae Body surface, Boxshall,.: 1976 Vaal River gill chamber Boskop Dam Wuras, Tierpoort & Mockes Dams P.M.K. Le Roux Dam Doorndraai Dam Bloemhof Dam Orange River (Namibia)
C. -qiistra/isSitiiUS: Groot Berg River (adjacent to Olifants Cyprinidae Skin Fryer,. 1977:: System)
Twee & Tra-Tra Rivers Cypranidae Gill chamber, (Olifants River System) on operculum
. Mogalakwena River (Limpopo River Clariidae Gill chamber Van As, 1986 System) Loskop Dam (Olifants River, Limpopo System)
12. C. vEctod Olifants River (Limpopo System) Cyprinidae Fins, Aveniun-Oldewage, body surface 1991
1.3 C. Zambezi River (Namibia) Cyprinidae Tailfin Van As 1992
Unidentified Mokombe River (Congo System) • Mochokidae :::larvat
(A short line (-) indicates that the information was not available)
51 TA LE 3.3
Summary of the host fish families and geographical distribution of Chonopeltis spp. in the major river systems ( & tributaries) and major lakes in Africa.
SPECWS
❑ ❑ X X A schoutedeni 0 Malagarasi Swamps A A
C. congicus ❑ A cctfrons Malagarasi Swamps A brevis 0 Konkoure & Manou Rivers A A (Guinea), Tana River merklionahs 0 X C. elongatus 0 X ausfralis C. australissimus Great Berg River C. MiftiliUS Twee & Tra-Tra Rivers C fiyert 0 X vt.ctort . 0 X .koki X TA LE 3.4
Summary of the relative growth relationships (%) of all the recognised spesies of Chonopeltis Thiele,1900. (Measurements were taken from figures accompanying species descriptions when the information was not available in the text. A short line (-) indicates that the information was not at all available).
ADULT FEMALES:. C. inermis 44 118 24 43 31 31 33 34 61 Thiele (1904) 59 102 28 50 19 28 42 67 Fryer (1956) 47 105 22 55 37 30 42 22 63 Van As & Van As (1993) C. schoittedeni 40 129 26 38 29 40 28 80 Brian (1940) 35 133 19 41 30 42 22 11 85 Fryer (1956) C. congicus 54 104 14 51 26 25 33 21 52 Fryer (1959) C. flaccifrons• 52 104 21 55 36 23 48 33 57 Fryer (1960a) C. hrevis 56 113 26 32 28 27 35 46 54 Fryer (1961b) C meridionalis: 55 107 18 53 29 28 48 40 51 Fryer (1964) -- C. eltingatus --- 40 93 21 43 30 33 28 33 62 Fryer (1974) C. australts 55 113 29 38 25 25 58 36 42 Boxshall (1976) 50 111 45 39 24 60 59 40 Van Niekerk (1984) C. australissitnus 54 114 14 61 44 24 60 40 28 Fryer (1977) C. mthutus 59 105 19 53 40 25 72 28 48 Fryer (1977) C. fryeri 45 114 25 52 30 31 41 23 54 Van As (1986) C. victori 30 103 27 52 26 27 50 30 52 Avenant-Oldewage (1991) 54 110 24 49 20 26 50 35 55 Own unpublished records C. koki 52 112 22 44 21 31 42 49 54 Van As (1992)
ADULT MALES : >' C. inermis 47 106 22 53 31 41 35 64 55 Van As & Van As (1993) C. schoutedeni 43 113 32 24 33 24 73 Brian (1940) C. congicus 26-29 60 Fryer (1959) C..flaccifrons 53 138 19 54 33 26 62 36 57 Fryer (1960a) C. brevts;-:-- 55 113 24 47 29 37 61 66 49 Fryer (1961b) C merdzonahs 51 115 12 50 28 37 40 40 54 Fryer (1964) C elongates:.; 46 100 16 51 43 40 28 66 47 Fryer (1974) C australis 59 108 26 38 33 40 53 61 31 Boxshall (1976) 50 109 47 46 36 51 34 Van Niekerk (1984) C. australissimus 55 31 57 27 Fryer (1977) C. minitus 59 107 13 56 39 30 54 46 32 Fryer (1977) C. fryeri 51 118 14 54 32 41 47 48 38 Van As (1986) C. victori 59 101 25 54 30 34 61 65 51 Avenant-Oldewage (1991) 55 116 24 49 20 34 61 65 51 Own unpublished records C. koki 52 112 26 51 25 38 48 51 54 Van As (1992)
ABBREVIATIONS:
CL/TL = Cephalic shield length / Total length; CW/CL = Cephalic shield width / Cephalic shield length; CcL/CL = Cephalic shield cleft length / Cephalic shield length; FCW/CW = Frontal cephalic ala width / Cephalic shield width; SW/CW = Sucking disc width / Cephalic shield width; AbL/TL = Abdomen length / Total length; AbW/AbL = Abdomen width / Abdomen length; GoL/AbL = Testis or spermatheca length / Abdomen length.; AcL/AbL = Abdomen cleft length / Abdomen length.
53 " Just because I can't see it, or don't fully understand it, does not mean it is i o i -existent. "
- Wayne Dyer APTEL
GENE L MORPHOLOGY OF CH NOPELTIS VICTO AVird21 ANT= OLDEWAGE,19 1
4.1 INT 0 I UCTION
The genus Chonopeltis Thiele,1900 comprises thirteen described species of which Chonopeltis victori, Avenant-Oldewage,1991 is one of the most recent. The taxonomic description of most of these species is, however, like the majority of papers published on Chonopeltis spp. (except for the descriptions by Van As (1986,1992) and Van As & Van As (1993)) who also used scanning electron micrographs (SEM)), typically based on whole mount studies of only a few specimens with little, if any, information given on the surface morphology. Lack of morphological detail in these papers has led to confusion as to, for instance, the phylogenetic origin, identity and function of appendages such as the antennae. Consequently, more extensive and detailed morphological knowledge of all known species of Chonopeltis is a prerequisite, especially since the distinct morphological features of each species are not only of considerable taxonomic and phylogenetic, but also ecologic and pathogenetic significance, since it reflects several important factors such as mode of transmission and adhesion as well as possible mechanical damage inflicted on the host. Furthermore, in order to put the morphometric data of the existing species to use in species identification, it is necessary to have a complete set of relative measurements for each species available. Thus, species identity
54
can eventually be confirmed by comparing not only characteristic morphological features but also relative growth relationships. Presently, this is not possible since relative measurements are either entirely lacking or incompletely presented in the taxonomic descriptions of most Chonopeltis species.
The following description thus aims at systematically describing the fine structure and detailing the surface morphology of a single species, Chonopeltis victori , Avenant-Oldewage,1991, using light and scanning electron microscopy. Although the external morphology of adult C. victori is essentially as originally described by Avenant-Oldewage (1991), a few features were found to differ, whilst several entrenched features and additional structures became apparent when C. victori were examined with SEM. A complete set of relative growth relationships of C. victori (n=30d' + 3M of the present study is also included and compared with those given by Avenant-Oldewage (1991). The terminology used to describe the cephalon and cephalic structures is based on the results of the histomorphological investigation described in Chapter Five.
4.2 GENE AP' MO rrq OIL, GY OF ULT CII PELTIS VICTOR'
The following description is based on 30 male and 30 female specimens used in macroscopical and light microscopical investigations and approximately the same amount used in SEM investigations. The general external morphology of adult C. victori is depicted in Figure 4.3 and Figure 4.4.
Adult C. victori display several distinct morphological adaptations needed for a successful piscine ectoparasitic existence. The parasite body is small, usually almost colourless and transparent, dorsoventrally flattened and differentiated into three distinct tagmata or body regions: cephalon, thorax and abdomen. The segments of the cephalon are dorsally indistinctly fused into a broad, trifoliate cephalic shield ("carapace") bearing two sessile compound eyes and a trifid ocellus. The first maxillae (maxillulae) are transformed into large sucking discs for adhesion and/or attachment to the host (Figs. 4.4B, 4.8A), whilst the second maxillae are
55 prehensile with chitinous terminal claws (Figs. 4.3B, 4.4B & 4.9A,C,D). The proboscis or mouth tube, bearing three to four rows of chitinous denticles on its mandibles, is situated midventrally between the two sucking discs (Figs.4.3B, 4.4B & 4.7A,B). There are four pairs of biramous thoracic appendages ("swimming legs") and a short fused abdomen (i.e. composed of fused segments) (Figs. 4.3,4.4 & 4.5A). Furthermore, the body surface and appendages are distinctly covered by a variety of denticulated scales, spines, setae and circular pits (Figs. 4.5C,D,E & 4.9F).
4.2.1 Morphometrics
The sexes are separate with the females usually larger than the males. The largest female sampled measured 9,952 mm in length and the smallest 2,263 mm whilst the largest male was 6,021 mm long and the smallest 2,113 mm. Measurements of adult C.victori specimens, listed in Table 4.1, were taken as illustrated in Figure 4.1. The relative growth relationships of C. victori recorded during the present study and those given by Avenant-Oldewage (1991) are compared in Table 4.2. The differences evident between these two sets of data can mainly be attributed to the difference in sample size, whilst other factors such as seasonal polymorphism (e.g. female abdomen size) may also be involved.
The relative growth relationships of C. victori of the present study are represented graphically in Figure 4.2 and the regression equations are summarised in Table 4.3. All the relationships show simple isometric growth and only the plot of abdomen length of females in relation to spermatheca length is slightly more dispersed (Fig. 4.2G). This is probably due to a form of seasonal polymorphism where female abdomen size is influenced by the fluctuating volume of content in the spermathecae during breeding and nonbreeding periods (as also reported by Hartnoll (1982) for other crustaceans, and Avenant et a/.(1989) for the branchiuran genus Dolops). These parameters (i.e. abdomen length vs testes length) are constant in males (Fig. 4.2G). There is no significant difference between the relative growth of the total length and cephalic shield ("carapace") length for males and females, with the cephalic shield in both sexes of similar proportions (Fig. 4.2A,C). The relative growth of the cephalic shield cleft
56 length, frontal cephalic ala ("cephalic lobe") width and sucking disc width show no significant differences between males and females (Table 4.2, Fig.4.2D,E). However, in males the abdomen is relatively larger (i.e. greater length and width) than in females (Fig. 4.2B,H), with the abdomen cleft length slightly shorter than in females (Fig. 4.2F).
A comparison of the relative measurements obtained for all known species of Chonopeltis is summarised in Table 4.4.
4.2.2 Collouration
The colouration of live C. victori adults varies from colourless and transparent to white with greyish general flecking. In addition, distinct black, grey or brownish pigment spots (i.e. chromatophores) occur on both ventral and dorsal sides of the abdomen of all male specimens, and in two longitudinal bands overlying the ovaries on the dorsal surface of the thorax and last cephalic segment of all female specimens. The intensity of these pigment spots varies in different individuals. Specimens occurring on light coloured areas on the host such as the lower abdomen and pelvic fins, display pale greyish pigment spots whereas those occurring on the darker upper body surface and fins possess prominent dark pigment spots. This variation in pigment intensity, which are apparently due to specific melanophore response to background colour and other environmental conditions (cf. Discussion, p.67-69), seems not only to protect the gonads against radiation, but also to aid in camouflage. Faint yellow-brown colouration were also observed in specific areas on the body surface and appendages of live as well as preserved specimens. This atypical colouration was more conspicuous in larger specimens and especially in those areas on the parasite body plentifully provided with cuticular openings (i.e. openings of integumental glands) which suggests that this yellow-brown colouration may be indicative of phenolic tanning - at least in those regions subjected to considerable abrasion as were shown in other crustaceans (cf. Patane,1959; Denne11,1960; Stevenson,1961; Stevenson & Schneider,1962).
57 4.2.3 External morphology of the body regions
The body of C. victori is typically differentiated into a cephalon, thorax and abdomen (Figs. 4.3 & 4.4).
4.2.3.1 Cephallon
The segments of the cephalon are indistinguishable and typically fused dorsally into a trifoliate cephalic shield ("carapace") possessing a frontal and two lateral alae ("lobes"), with the latter reaching back to the second pair of legs (thoracopods) in large specimens and third pair of legs in the very small specimens (Figs. 4.3A, 4.4A). The trifid ocellus and two red-brown compound eyes are clearly visible middorsally on the cephalic shield (Figs. 4.3, 4.4). Although greyish general flecking is evident on the dorsal carapace surface of all specimens examined, distinct pigment "tearmarks" running from each compound eye towards the ocellus as described by Avenant-Oldewage (1991), were only observed in immature and very small mature specimens.
Dorsally, the cephalic shield shows little other surface ornamentation except for the presence of minute cuticular openings, a number of sensory pits and setae, as well as a series of grooves, ridges and sutures which are similarly arranged in all specimens examined (Figs. 4.3A, 4.4A & 4.5B). The large number of minute cuticular openings are widely distributed over the entire dorsal surface of the cephalic shield and most probably represent the external openings of the ducts of the integumental glands as were shown in other crustaceans by Dennell (1960) and Felgenhauer (1992). The sensory pits and setae of short spine-like and elongate hair-like structure, circumscribe the dorsal cephalic shield surface while only a few isolated pits occur middorsally (Fig. 4.5C,D,E).
The two dorsal cephalic grooves ("carapace ridges") extend longitudinally backwards from just behind the two compound eyes, on either side of the ocellus, to terminate posteriorly near the fusion of each lateral cephalic ala ("carapace lobe") with the posterodorsal surface of the
58 cephalon (Figs. 4.3A & 4.4A). A horseshoe-shaped furrow, delimiting the cephalic region, extends anterolaterally from the junction between the cephalon and thorax, ending in distinct sinuses at the base of the cephalic shield (Figs. 4.3A, 4.4A & 4.5B). A less conspicuous transverse suture ("anterior cephalic groove" of Martin,1932, in Argulus), bisects the two dorsal grooves immediately behind the ocellus, separating the anterior cephalic area from the rest of the cephalic shield (Figs. 4.3A, 4.4A & 4.5B). A dorsal transverse suture or crease ("posterior cephalic groove" of Martin,1932, in Argulus) clearly marks the posterior limits of the cephalon (Figs. 4.3A & 4.4A).
Ventrally, the cephalic alae ("carapace lobes") are bordered circumferentially by a few small circular pits and irregularly distributed spinules and longer needle-like setae (Fig. 4.5D). Interspersed amongst these structures, minute cuticular openings similar to those on the dorsal cephalic shield surface are found (Fig. 4.5D,E). A few small (5-7Am) posteriorly directed scales with serrated distal edges are concentrated anteromedial to each sucking disc (maxillula), and at wider intervals along the ventrolateral margins on the cephalic shield (Figs. 4.3B, 4.4B & 4.5E). Midventrally on each lateral cephalic ala, a pair of clearly demarcated and unadorned respiratory areas consisting of a small ovoid anterior and a large kidney-shaped posterior area, are found (Figs. 4.3B, 4.4B & 4.5F,G). In live specimens, the respiratory areas appear to be outlined with pale greyish haemolymph (see Discussion).
The anterior margin of the frontal cephalic ala ("anterior/cephalic lobe" of Fryer,1960a) displays a distinct medial indentation from either side of which a ventral marginal groove extends laterally, bordering the anterior and lateral margins of the frontal ala (Fig. 4.6A). Posterior to these two grooves, four radiating alal crests ("Chitienleisten" of Thiele,1904; "cephalic rods" of Fryer,1956; "chitinous supporting rods" of Fryer,1960a,1977; Boxshall, 1976; Van As,1986,1992; Avenant-Oldewage,1991) are found. Distally, the anterior end of each rod is continuous with the anteroventral surface of the frontal ala (viz. the surface of one of the chitinous marginal grooves) while the posterior end terminates in a knob-like structure subproximally (Fig. 4.6A).
59 Cephalic appendages
The cephalic appendages include a pair of antennae, a pair of mandibles situated within the proboscis and paired maxillulae and maxillae (Figs. 4.3B & 4.4B). The pair of uniramous antennae, consisting of four segments each, lies midventrally, to the inside of each lateral alai crest ("outer cephalic rod") (Fig. 4.6A,B). The distal segment of each antenna bears at its apex a slit-like, longitudinal groove on either side of which two terminal setae are situated (Fig. 4.6B,C). Sensory setae and pits are also prominent on all three other segments - implying a primary sensory function of the antennae in adult parasites. A prominent nodular protrusion bearing a group of 14 to 17 setae interspersed by various minute cuticular openings and a few larger sensory pits, are found posterodistally to the basal segment of each antenna (Fig. 4.6B,D). Posteroproximally to each of these protrusions, a previously undescribed, minute biramous structure (see Discussion) with three terminal setae on its distal ramus (Fig. 4.6E), occurs on all specimens examined.
The proboscis or mouth tube is a short dorsoventrally flattened structure occurring midventrally between the two sucking discs, projecting posteriorly from the body wall (Fig. 4.4B). It is composed of an upper and lower lip which is, except for the presence of slits distally, fused for almost the entire length of the proboscis (Fig. 4.7B). The upper lip (labrum) forms the ventral wall and lower lip (metastome) the dorsal wall of the proboscis. Posteriorly, the terminal end of the labrum is bilobed with each lobe bearing a single medial pit containing a papillae-like structure which often projects beyond the upper rim of the pit (Fig. 4.7A). A pair of similar structures are also found medially on the inner lateral wall of each lobe (Fig. 4.7A). These presumed sensory structures resemble the chemoreceptors found in other arthropods - including crustaceans (Barber,1961; Beklemishev,1969; Clarke,1973; Felgenhauer,1992; Govind,1992; Harrison & Humes,1992a,b). Further to the inside, on each inner lateral face of the labrum, a single row of small chitinous denticles is located (Fig. 4.7A).
The metastome is a curved structure with the concavity facing the labrum and the unfused lateral ends (convexities) slightly overlapping the dorsal side of the labrum (Fig. 4.7B).
60 Peripherally, the metastome displays a fringed border of densely packed hair-like setae, followed to the inside by a series of comb-like structures consisting of 21 to 29 spines each (Fig. 4.7A,C). The latter is followed further to the inside by two metastomal spines and a series of minute spinules arranged in comb-like formations consisting of 5 to 11 spinules each (Fig. 4.7C). Beneath the setose marginal fringe, a few irregularly and widely spaced spinous projections are found (Fig. 4.7C). Enclosed within the proboscis, each of the two curved mandibles displays three to four rows of curved chitinous denticles on its convex side (Fig.4.7A) and a double row of spines on its concave side.
The maxillulae are transformed into large sucking discs with the marginal flange of each bearing 57 to 61 radiating rows of chitinous supporting rods (Fig. 4.8A,B,C). Each rod is composed of 8 to 14 interlinked chitinous elements to ensure flexibility and firmness to the sucking disc (Fig. 4.8B,C). Between the supporting rods, small spine-like projections and cuticular pits of presumed mechano- and chemosensorial function respectively, are found (Fig. 4.8B,C). The marginal flange of each sucking disc is bordered peripherally by a series of closely packed papillae-like structures, reminiscent of a fringe (Fig. 4.8D). A large number of these papillae possess a small apical opening surrounded by four terminal setules. These presumed sensory papillae resemble those, also situated peripherally on the marginal sucking disc flange, described as "truncated lobes armed with delicate setae" for the related genus Argulus (Meehean,1940; Sutherland & Wittrock,1986). Directly beneath the medial chitinous ring of each sucking disc, a single row of minute needle-like chitinous spines protruding from shallow cuticular blebs, are followed by a single row of widely spaced circular pits (Fig. 4.8E).
The robust, prehensile maxillae consists of five podomeres each with prominent scabrous areas on the ventral surface of the basal, third and fourth podomeres (Fig. 4.9A,B,E). These scabrous areas contain posteriorly directed ellipsoidal scales with delicate denticulated distal margins, spine-like and hair-like setae, minute cuticular openings and a few sensory pits (Fig. 4.9A,B,E,F). Distally, on the ventral surface of the otherwise smooth second podomere, a single band of large ellipsoidal and less prominent finger-like scales extends medially upward towards the anterodistal edge of the podomere (Fig. 4.9A,E). Two robust setae are apparent
61 beneath this band of scales (Fig. 4.9A,E). The distal podomere of each maxilla terminates in a pulvinar structure bearing two sickle-shaped chitinous claws on its superior half (Fig. 4.9 C,D). Ventrally, a terminal extension of the upper part of the distal podomere forms a slender, dorsoventrally flattened process, bearing at its apex a small chitinous spine (Fig. 4.9A,C,D). This process conceals the basal parts of the claws ventrally whilst the rest of the pulvillus remains visible beneath the process (Fig. 4.9D).
4.2.3.2 Thorax
The thorax is subcylindrical, dorsoventrally flattened and consists of four segments - the first of which has already been referred to as being partly fused along its dorsal surface to the posterior extremity of the cephalon, whilst the posterior end of the fourth thoracic segment articulates with the anterior part of the abdomen (Figs. 4.3, 4.4 & 4.10D). Minute cuticular openings, a few circular pits, slender hair-like setae and four to six cone-shaped spinous projections arising from shallow cuticular blebs, occur middorsally on each of the four thoracic segments (Fig. 4.10B). The four pairs of thoracopods ("swimming legs") are sexually dimorphic, each composed of three podomeres: a precoxa, coxa and basipodite with two rami (the endo- and exopodite) arising from the distal end of the basipodite (Figs. 4.3 & 4.4 ).
Ventrally, the thorax and specific areas on the anterior and ventral sides of the thoracopods are covered by numerous scales, sensory setae and pits, similar to those found on the maxillae (Fig. 4.10F).
Additional finger-like (/hand-shaped) scales (Fig. 4.10E) occur ventrally on the exo- and endopodites as well as between the ellipsoidal scales on the basipodites of all four pairs of legs in females and the first two pairs of legs in males. Dorsally, denticulated scales (Fig.4.10C) which are more closely adhered to the surface, occur on the second and third podomeres of the first three pairs of legs in females and first two pairs of legs in males. The exopodite and second and third segments of the endopodite of the first pair of legs are ventrally unadorned, except for a single setae occurring posterodistally on the second, and two terminally on the
62 third segment of the endopodite. In females, the exopodite of leg four bears two spinous setae terminally and a few small sensory setae posterodorsally, whilst the endopodite possesses a single terminal setae and a few dorsal scales.
The posterior surface of the second and third podomeres of the swimming legs of females possess combed setae (i.e. setae bearing single longitudinal row of setules) on the ventral side (Fig.4.10F) and feathered setae (i.e. setae bearing two opposing longitudinal rows of setules) on the dorsal side. However, combed setae are lacking on the second podomere of leg four whist the third and fourth pairs of legs are devoid of feathered setae.
The natatory lobes of the female are ovoid and bordered ventrally along its posterior and inner posterolateral margins by anteriorly directed denticulated scales interspersed by small spine- like setae and cuticular openings with a border of elongated setae peripherally (Fig. 4.11A,B). A few (4-6) sensory pits are apparent on the otherwise smooth ventral surface of the natatory lobes (Fig. 4.11A). Dorsally, the natatory lobes are unadorned with the outer lateral wall of each lobe extending anterolaterally towards the abdomen. The genital opening situated posteroventrally on the last thoracic segment as well as the two spermathecal spines (Fig. 4.11C,D) present on the anteroventral side of the abdomen, are concealed by the two natatory lobes which meet at the ventral midline (Fig. 4.11A).
In males, the first to fourth pair of thoracopods have accessory structures (Fig. 4.12A) that are presumably used in copulation. These structures include various protrusions covered entirely or partially by some or all of the following superficial structures: denticulated scales, minute spines, small sensory setae and pits as well as various other minute cuticular openings. The first pair of thoracopods is identical to those of females except for the presence of a terminally setigerous (i.e. bearing setae) protrusion occuring posteroventrally on the precoxa in males (Figs. 4.3 & 4.12B). Four terminally adorned finger-like protrusions occur on the posterior side of the second thoracopod of males: one ventrally on the precoxa, two opposing on the ventral and dorsal side of the coxa, and one distally on the ventral side of the basipodite (Fig. 4.12D). Setigerous swollen areas are apparent on the anterior surface of the precoxa and coxa of legs two and three (Fig. 4.12C). On the anterior side of the coxa of leg three there are also
63 two adorned knob-like protrusions and a rounded nodular structure evident (Fig. 4.12E). The latter resembles the so-called "pocket" described by Sutherland & Wittrock (1986) for the related genus Argulus. The "socket", a relatively large slit-like opening, occurs anterodorsally on the coxa of leg three (Fig. 4.12E). Beneath the socket, a posterior marginal flap extends backwards to cover the basal parts of leg four ventrally (Fig. 4.12E). Avenant-Oldewage (1991) described two small protrusions on the ventral side and one on the dorsal side of the flap, as well as a fourth protrusion on the exopodite of leg three. However, during the present study the marginal flap and exopodite were found to be without any protrusions whilst the basipodite bears a large terminally adorned protrusion on the anterior side and a smaller setigerous protrusion on the dorsal side (Fig. 4.12E). Leg four of the male is highly modified with the "peg", an elongate anterolaterally directed process, occuring on the anteroventral side of the basipodite (Fig. 4.12F). A groove which is surrounded and lined by robust papillae-like structures, occurs distally on the ventral side of the peg (Fig. 4.12G). The slender terminal end of the peg which is curved rectangular upwards towards the socket on leg three, is bordered circumferentially by denticulated scales with a short, hollow cylindrical structure at its apex (Fig. 4.12G). An ovoid pad, covered on its flat ventral surface by small serrated scales, occurs proximally on the ventral side and a terminally adorned protrusion on the dorsal side of the basipodite (Fig. 4.12F). Combed setae are found posteroventrally on the basipodite and exopodite, whilst the endopodite bears only two simple setae terminally. The endopodite is sickle-shaped with its convex side extending anteriorly to form a terminally adorned process (Figs. 4.3 & 4.12A,F). The natatory lobes of the male are rounded laterally with the posterior margin of each bearing a central indentation on the one side of which four combed setae and a single simple seta are found (Fig. 4.12H). A few posteriorly directed denticulated scales, interspersed by small spines and setae, occur posteroventrally on each natatory lobe. In contrast to the female, the natatory lobes of the male do not conceal the genital opening which lies at the posterior end on the ventral side of the last thoracic segment (Fig. 4.12H).
64 4.2.3.3 Abdomen
The abdomen of both sexes is essentially as described by Avenant-Oldewage (1991), viz. unsegmented and fused anteriorly with the fourth thoracic segment whilst splitting posteriorly into two lobes with sharply pointed tips directed outwards (Figs. 4.3 & 4.4). However, scanning electron microscopical examination revealed that the entire abdominal surface is covered by numerous cuticular openings, whilst the inner and outer lateral sides also possess a number of small spine-like projections and posteriorly directed scales with comb-like distal edges consisting of 8 to 12 spines each (Fig. 4.13A). Ventrally, a few small, irregularly spaced setae are found medially at the anterior end of the abdomen. The outwards directed tip of each abdomen lobe bears 9 to 14 robust setae terminally (Fig. 4.13C). In females, an additional medial strip of short spines occurs at the anterior end between the two spermathecal spines on the ventral side of the abdomen (Fig. 4.11C). Each ejectable spermathecal spine is borne on a cone-shaped base which is situated in the centre of a nodular protrusion (Fig. 4.11 C,D). This protrusion is bordered terminally by scale-like structures arranged in two or more uneven concentric circles (Fig. 4.11D). In both sexes, the anus is slit-like and situated dorsally in an abdominal sinus which lies anterior to the two minute furcal rami (Fig. 4.13B). The furcal rami, occurring dorsally near the base of the abdominal cleft, are rounded posteriorly with each ramus bearing four to five slender simple setae terminally (Fig. 4.13B).
4.2.4 Epibionts
Occasionally specimens of C. victori were found to be moderately to heavily infested by peritrichous ciliates. Although these infestations occurred mostly on the ventral side of the cephalon (i.e. including the cephalic shield), some of especially the smaller specimens were completely covered by these sessile ciliophorans (Fig. 4.13 D,E,F).
65 403 DISCUSSION
Many of the criteria used in crustacean taxonomy are based on structures subject to relative (isometric or allometric) growth. Consequently, closely related species often show quite dissimilar levels of relative growth for the same structure(s) - a feature also evident when comparing the relative measurements of the thirteen described species of Chonopeltis as summarised in Table 4.4. Some branchiurans like most other crustaceans, not only show discontinuous growth in size, but also reach sexual maturity before attaining maximum size. Therefore, absolute growth measurements are misleading, whilst relative growth relationships are considered to be of more reliable taxonomic significance (Teissier,1960; Haley,1969,1973; Hartnol,1982). With this in mind, as well as the fact that a complete set of relative measurements is lacking in the taxonomic description of most Chonopeltis species, it is suggested that the values of relative growth relationships at a series of body sizes (preferably graphically presented), are included in future taxonomic descriptions of Chonopeltis species. Furthermore, the inclusion of regression equations for these relationships is also recommended since it is clearly the most comprehensive way to present such data so that it may be of maximal descriminative value.
Concerning the colouration of adult Chonopeltis species, an extensive examination of the literature revealed that the ground body colour of adult specimens vary from colourless and transparent to white or greyish - irrespective of the parasite's location on the host. However, the presence of distinct dark pigment spots (melanophores) on the dorsal body surface have been described for six (i.e. C. brevis; C. meridionalis; C. australis; C. australissimus; C. victori; C. koki) of the seven species occurring on the external body surface or fins of their hosts, whilst the absence of pigment spots were reported for five (i.e. C. inermis; C. schoutedeni; C. flaccifrons; C. elongatus; C. fryeri) of the six species found in the gill chambers or mouths of their hosts. The two species found to behave in a contrary manner are C. congicus and C. minutus. The first species, although occurring on the external flanks of its host, apparently lacks pigment spots - a feature attributed by Fryer (1968) to the fact that this species's host often frequents turbid waters where protection from radiation is apparently not needed. Although Fryer (1964,1968) attributed the occurrance of pigment spots in
66 Chonopeltis spp. mainly to the protection of the gonads from some properties of light, C. rninutus which usually occurs in the gill chambers of its host, displays prominent pigment spots on the dorsal body surface with pale olive green flecking on the cephalic shield and dorsal surface of the legs (Fryer,1977). The foregoing, as well as the fact that melanophore intensity, thus the degree of the pigment dispersion or contraction, is influenced by background colour as well as certain physical, chemical and biotic properties of the aquatic habitat (Goodwin,1960; Brown,1961; Kleinholz,1961; Nadaka1,1963; Wetze1,1983), suggest that the characteristic pigment spots displayed by Chonopeltis spp. also aid in camouflage and are most likely present in all species of Chonopeltis. However, these pigment spots may be less conspicuous in some of these species whilst it could become discoloured in museum specimens (as most probably the case in some of the taxonomic descriptions of Chonopeltis spp. where old material was used). Accordingly, Avenant-Oldewage & Knight (1994) suggested that the pigment spots in C. australis are not a permanent species characteristic and that the presence or absence of pigment spots is related to the colouration of the position on the host as well as the sexual maturity of the parasite.
In addition to the characteristic pigment spots overlying the gonads, live specimens of C. victori also display varying degrees of greyish general flecking on both ventral and dorsal sides of the body. Although this flecking may be due to certain other tegumental pigments, in preserved specimens this flecking were found to be darker and mainly concentrated dorsally which suggest that it might be caused by pigment granules present in the haemolymph. Since live C. victori specimens were fixed upside down (sometimes in heated AFA or 10% PNF) between a microscope slide and a coverslip for a prolonged period (20 - 30 minutes), a plausible basis for assuming the latter are provided by the fact that the colour of respiratory pigments intensifies under low oxygen conditions and/or elevated temperature (Goodwin,1960; Nadaka1,1963; Mangum,1982; Avenant-Oldewage et a/. ,1989; Martin & Hose,1992) and that crustacean haemolymph blackens on standing, owing to the action of certain enzymes present in the haemolymph (Busnel & Drilhon,1948; Goodwin,1960). The normal grey or black pigmentation in C. victori which appears brownish in some individuals may be due to the allelic influence of certain genes effecting the synthesis of brown melanoid pigment as were reported in other crustaceans (cf. Hedgecock et a/.,1982).
67 The biochromy of parasitic Crustacea remains almost unexplored with data on their pigmentology extremely meagre. Available evidence indicates that carotenoids (acquired through nutrition) and melanins (formed as a byproduct of protein metabolism) are the principal biochromes involved in the chromatophore system of parasitic crustaceans (Goodwin, 1960; Nadaka1,1963). Examination of the literature revealed that a striking feature of biochromy of crustacean parasites is the obvious sex difference as well as higher frequency of colouration in females than in males. Sexual dichromatism seems to be partly due to the fact that during reproduction, pigments like carotenoids are mobilized from the haemolymph and hepatopancreas into the ovary so that the mature and gravid females take up a vivid colouration, while some crustacean parasites also takes on different hues during its development to sexual maturity (Nadaka1,1963). Qualitative and quantitative aspects of pigmentation in animal species are influenced by enviromental factors such as temperature, pressure, light, oxygen supply, salinity and nutrition. As regards the piscine ectoparasites, the immediate enviromental conditions are provided not only by the host but also the aquatic enviroment in which the host species live. Certain crustacean parasites exhibit adaptive colour changes such as taking on a pale colour when attached to its light coloured normal host, but darken when swimming freely. Attachment to any surface, whether black or white, is said to induce the paling response (Nadaka1,1963). According to Nadakal (1963), carotenoids and melanins may be involved in adaptive colour changes aiding the parasites concealment and escape from various predators, whilst integumentary melanins have generally been assigned a protective function against injurious effects of sunlight.
Although the blood or haemolymph of crustaceans contains several different plasma proteins capable of affecting respiratory capacity, only two respiratory chromoproteins: haemoglobin and haemocyanin, have been identified in several crustacean groups (Mangum,1982). In the Branchiura, haemoglobin has hitherto been detected only in the distinct red blood of Dolops (Fox,1957) whereas no study has been conducted to establish the presence or absence of respiratory pigment in the pale blood of Chonopeltis or brownish blood of Argulus. However, respiratory chromoproteins are often periodically or entirely absent under conditions such as small body size, low capacity for motor performance and/or a normoxic habitat (Mangum, 1982) - all of which are at times applicable in branchiurans.
68 Although no reference is made to the series of grooves, ridges and sutures evident on the dorsal surface of the cephalic shield ("carapace") in any paper previously published on Chonopeltis spp., these features were included in line drawings of C. schoutedeni (Brian, 1940), C. elongatus (Fryer,1974), C. minutus and C. australissimus (Fryer,1977) as well as C. koki (Van As,1992). Dorsal cephalic shield ridges, sutures and grooves have also been described and/or illustrated for various species of all three other branchiuran genera (i.e. Dipteropeltis, Dolops and Argulus). However, in contrast to Chonopeltis spp., the paired dorsal grooves were found to be closer together and extending further anteriorly to pass between the two compound eyes in species of these three genera. Although the basic arrangement of dorsal cephalic shield grooves, ridges and sutures are apparently species specific in branchiurans (Sutherland & Wittrock,1986), inconsequent inclusion of these features in taxonomic descriptions cause it to be of less taxonomical value and should therefore not be readily incorporated in identification keys.
The presence of scales on the ventral surface of the cephalic shield (carapace) as described for C. victori in this study, has hitherto not been described for any other Chonopeltis species. However, it is possible that similar scales might be present in at least some of the other Chonopeltis species, since in other branchiurans (Dolops and Argulus) ventral "carapace" scales or spines are typical for a number of species, while totally absent in others.
Although it is generally accepted that all species of Chonopeltis possess only one pair of antennae, no proof for this assumption could be found in the literature other than the vague description of a single pair of antennae (inconsequently referred to as antennae, first antennae or antennules), in the taxonomic descriptions of nine species. In the taxonomic descriptions of the other four species, a pair of antennae is merely included in line drawings of two of these species (Fryer,1960a,1961b), whilst not mentioned nor illustrated for the other two species (Fryer,1959,1964). During the present study, SEM investigation revealed a previously undescribed pair of minute biramous structures situated posteroproximally to the antennae of C. victori. These structures may merely be superficial adornments or probably represent a reduced or vestigial pair of antennae. However, whether they represent superficial structures or, first or second antennae, only further histological investigations determining whether they
69 are neuro-innervated or not, and if so, whether they are deutocerebrally or tritocerebrally innervated, would confirm (cf. Chapter 5). Since first and second antennae are present in all three other branchiuran genera (i.e. Dipteropeltis, Dolops and Argulus), it is most likely also the case in Chonopeltis, especially since the presumed additional pair of antennae as observed in C. victori is very small, nearly hidden from view by the medial alae crests and thus easily overlooked - even with SEM.
The presence of posteriorly directed denticulated scales on the ventral surface of the parasite body, maxillae and thoracopods, suggests that these scales aid in the retention of position of the parasite on the external body surface of the aquatic host. The simple sensory setae and pits probably have mechano- and chemosensory functions similar to those described for other crustaceans (Ache,1982; Felgenhauer,1992; Harrison & Humes,1992a). The numerous circular cuticular openings present on the body surface and appendages most probably represent the external openings of the integumental glands (Felgenhauer,1992) which, among other functions, are involved in tanning of the integument by the production of phenols (Patane, 1959) and phenol oxidase (Stevenson,1961; Stevenson & Schneider,1962) as well as involved in mucus production (Felgenhauer,1992). Since C. victori, like all other species of this genus, are not active swimmers, we suggest that the larger combed and feathered setae found on the thoracopods are not only mechanosensorial, but also aid in the induction of a water-current to facilitate respiration.
The occurrence of sessile ciliophorans on especially the ventral body surface and appendages of parasitic crustaceans as observed in several specimens of C.victori of the present study,
appears to be a widespread phenomenon. Similar peritrichous ciliates (mostly Epistylis - like organisms) were found on various other parasitic crustaceans (e.g. Amin,1981; Van As & Viljoen,1984; Sutherland & Wittrock,1986; Felgenhauer,1992). In this regard, Bauer (1981, 1989) suggested that some integumental glands in crustaceans may produce an antifouling chemical that would discourage settlement of epibionts and debris.
70 FIGURE 4.1
Schematic drawing of Chonopeltis victori Avenant-Oldewage,1991 to indicate the positions where measurements were taken.
Abbreviations: AbL - Abdomen length. AbW - Abdomen width. AcL - Abdomen cleft length. CL - Cephalic shield length. CcL - Cephalic shield cleft length. CW - Cephalic shield width. FCW - Frontal cephalic ala width. GoL - Testis or spermatheca length. SW - Sucking disc width. TL - Total length.
71
FIGURE 4.2
Graphs showing the relative growth relationships between the different morphometric values (in mm) of Chonopeltis victori Avenant-Oldewage,1991 adults. The values for males (n=30) are indicated in blue (0) and those for females (n=30) in red (*).
Cephalic shield length (CL) . vs Total length (TL). Abdomen length (AbL) vs Total length (TL). Cephalic shield width (CW) vs Cephalic shield length (CL). Cephalic shield cleft length (CcL) vs Cephalic shield length (CL). Frontal cephalic ala width (FCW) vs Cephalic shield width (CW). Abdomen cleft length (AcL) vs Abdomen length (AbL). Spermathecae/Testis length (GoL) vs Abdomen length (AbL). Abdomen width (AbW) vs Abdomen length (AbL).
72 L E3 AbLri
2 3 4 5 6 7 8 9 10 2 3 4 5 6 7 8 9 10 TL TL
CW/CIL. Gala, CcL 1.8
1.4 -
1.2 -
1 -
0.B -
0.0 -
0.4 -
0.2
0 1 2 2.6 3 3.6 4 4.6 6 6.6 0 1 1.6 2 2.6 3 3.6 4 4.6 6 6.6 CL CL
FCW/CW IF Ale_/AbL
Ad.
0.8 1 1.2 1.4 1.8 1.8 2 2.2 24 AbL
GoL/AbIL AbW/AbL
eoL AbW 1.4
1.2 -
1 -
0.8 -
0.8 -
0.4-
0.2 ■ 1 , 1 1 04 0.8 0.8 1 1.2 1.4 1.8 1.8 2 2.2 24 AbL FIGURE 4.3
Chonopeltis victori Avenant-Oldewage,1991. Dorsal view of adult male; Ventral view of adult male.
Abbreviations: ab - Abdomen. by - Basipodite. an - Antenna. at - Anterior transverse suture. cg - Cephalic groove ("dorsal carapace ridges"). co - Coxa. es - Cephalic shield ("carapace"). e - Compound eye. en - Endopodite. ex - Exopodite.
fa - Frontal cephalic ala ("cephalic lobe"). hs - Horeshoe-shaped furrow (i.e. frontal and lateral sulci).
in - Marginal indentation. in - Lateral cephalic ala ("lateral carapace lobe").
lac - Lateral alal crest ("chitinous supporting rod").
mac - Medial alal crest.
mg - Marginal groove of frontal ala. mx - Maxilla/second maxilla. na - Natatory lobe.
o - Trifid ocellus.
pco - Precoxa.
pr - Proboscis.
pt - Posterior transverse suture.
ra(a) - Anterior respiratory area.
ra(p) - Posterior respiratory area. s - Sucking disc (maxillula). sc - Small scales.
tp(1 -4) - Thoracopod (1 to 4). tx - Thorax
73 F11120-d2 6.0 AO] ffirtD FIGURE 4.4
Chonopeltis victori Avenant-Oldewage,1991. Dorsal view of adult female; Ventral view of adult female.
Abbreviations: ab - Abdomen. an - Antenna. by - Basipodite. at - Anterior transverse suture. cg - Cephalic groove. co - Coxa. cs - Cephalic shield. e - Compound eye. en - Endopodite. ex - Exopodite. fa - Frontal cephalic ala. fs - Frontal sulcus (cf. horseshoe-shapped furrow). in - Marginal indentation. la - Lateral cephalic ala. lac - Lateral alal crest. Is - Lateral sulcus (cf. horseshoe-shapped furrow). mac - Medial alal crest. mg - Marginal groove of frontal ala. mx - Maxilla. na - Natatory lobe. o - Trifid ocellus. pco - Precoxa. pr - Proboscis. pt - Posterior transverse suture. ra - Respiratory area. s - Sucking disc (=maxillula). sc - Small scales. tp - Thoracopod. tx - Thorax.
- 74 IP'll@ME 6.6 FIGURE 4.5
Scanning electron micrographs of Chonopeltis victori Avenant-Oldewage,1991.
Dorsal view of parasite body (Scale bar:lmm); Dorsal surface of cephalic shield ("carapace") (Scale bar:200,um); Adornments of the dorsal surface of the cephalic shield (Scale bar:5,um); D,E: Adornments on the ventral surface of the cephalic shield (Scale bar:10,um); Ventral side of lateral ala displaying the posterior respiratory area (Scale bar:200pm); Clearly demarcated and unadorned respiratory areas (Scale bar:104m).
Abbreviations: at - Anterior transverse suture. cg - Cephalic groove. co - Cuticular openings. fa - Frontal cephalic ala. fs - Frontal sulcus (cf. horseshoe-shaped furrow). in - Marginal indentation. la - Lateral cephalic ala. Is - Lateral sulcus (cf. horseshoe-shaped furrow). ns - Needle-like setae. p - Circular pits. ra(a) - Anterior respiratory area. ra(p) - Posterior respiratory area. s - Sucking disc (=maxillula). sc - Small scales. sp - Spinous projections. spi - Spinules. ss - Sensory setae.
75
FIGURE 4.6
Scanning electron micrographs of Chonopeltis victori Avenant-Oldewage,1991.
Ventral surface of the anterior part of the cephalic shield (Scale bar:100,um); Uniramous antenna (Scale bar:10,um); Distal segment of the antenna (Scale bar:10,am); D,E: Setose area and biramous structure at the base of the antenna (Scale bar:10,am).
Abbreviations: an - Antenna. bs - Unidentified biramous structure. in - Medial indentation. lac - Lateral alal crest. mac - Medial alal crest. mg - Marginal groove. np - Setigerous nodular protrusion. ss - Sensory setae.
76 0 Ki N 2
@ RI FIGURE 4.7
Scanning electron micrographs of Chonopeltis victori Avenant-Oldewage,1991.
Opening of proboscis (Scale bar:5um); Proboscis or mouth tube (Scale bar:10Am); Adornments on metastome (Scale bar:2Am).
Abbreviations: cd - Chitinous denticles. cs - Comb-like structures on metastome. f - Setose marginal fringe on metastome. lab - Labrum (bilobed upper lip). ma - Mandible. me - Metastome (lower lip). ms - Metastomal spine. ps - Papillae-like structures. pr - Proboscis. sp - Spinous projections. spf - Spinules in comb-like formations.
77 ND c Ilm M Z1 0 al FIGURE 4.8
Scanning electron micrographs of Chonopeltis victori Avenant-Oldewage,1991.
Sucking discs/suckers (=maxillulae) (Scale bar:20gm); Chitinous supporting rods of the sucking disc flange (Scale bar:5gm); Interlinked chitinous elements of sucking disc (Scale bar: lgm); Papillae-like structures bording each sucking disc (Scale bar:Lum); Adornments beneath medial ring of sucking disc (Scale bar:Lum).
Abbreviations: an - Antenna. cr - Medial chitinous ring.
ie - Interlinked chitinous elements. lac - Lateral alal crest.
mac - Medial alal crest. ns - Needle-like spine. p - Circular pit. s - Sucking disc (maxillula). se - Terminal setules. sp - Spine.
78
FIGURE 4.9
Scanning electron micrographs of Chonopeltis victori Avenant-Oldewage,1991
Prehensile maxilla (Scale bar:20,um); Adorned basipodite of maxilla (Scale bar:10,um); C,D: Distal podomere of maxilla (Scale bar:5p,m). Coxa and precoxa of maxilla (Scale bar:10p,m); Adornments on the ventral body surface (Scale bar:5Arn).
Abbreviations: cl - Chitinous claws. co - Cuticular opening. cx - Coxa.
p - Circular pit.
pu - Pulvinar structure.
pcx - Precoxa. sc - Denticulated scales.
sp - Chitinous spine. ss - Sensory seta.
to - Terminal extention.
79
FIGURE 4.10
Scanning electron micrographs of Chonopeltis victori Avenant-Oldewage,1991.
Dorsal view of the thorax (Scale bar:100gm); Adornments on the dorsal surface of the thorax (Scale bar:10,um); Denticulated scales on the dorsal surface of the thoracopods (Scale bar:10/..tm); Ventral view of the thorax and thoracopods of the female (Scale bar:300/2m); Finger-like scales on the ventral surface of the thoracopods (Scale bar:5,um); Combed setae on the posteroventral side of the thoracopods (Scale bar:20p.m).
Abbreviations: ab - Abdomen.
CO - Cuticular opening. corn - Combed seta. fi - Finger-like scales. hl - Hair-like seta. p - Circular pit. se - Denticulated scales. sp - Spinous projection. ss - Sensory seta. tx - Thorax.
80 , II0(ME 6. W FIGURE 4.11
Scanning electron micrographs of female Chonopeltis victori Avenant-Oldewage,1991.
Ventral view of the natatory lobes of the female (Scale bar:100//m); Adornments on the posteroventral margin of the natatory lobes (Scale bar:10,u,m); Spermathecal spines and triangular medial strip of small spines on the anteroventral side of the abdomen (Scale bar:10,um); Retracted spermathecal spine (Scale bar:4p,m).
Abbreviations: na - Natatory lobe. pr - Nodular protrusion. s - Spine. sc - Denticulated scales. se - Robust setae. sp - Spermathecal spine. ss - Scale-like structures. tri - Triangular medial strip.
81
kg FIGURE 4.12
Scanning electron micrographs of male Chonopeltis victori Avenant-Oldewage,1991.
Dorsal view of legs two through four (Scale bar:100,um); Ventral view of legs one and two (Scale bar:100ktm); Adornments and protrusions on the anterior, ventral and posterior sides of legs two and three (Scale bar:10p,m); Protrusions on the posterior surface of the precoxa and coxa of leg two (Scale bar: lOktm); Dorsal view of leg three displaying the "socket" and "pocket" (Scale bar:10gm); Dorsal view of leg four displaying the "peg" (Scale bar:100,am); Terminal end of the "peg" on leg four (Scale bar:10Am); Genital opening and natatory lobes (Scale bar:10,um).
Abbreviations: ab - Abdomen. p - Papillae-like structures. by - Basipodite. pco - Precoxa. cb - Combed setae. pg - Peg. co - Coxa. po - Pocket. en - Endopodite. pr - Protrusion. ex - Exopodite. sa - Setigerous area. g - Groove. so - Socket. go - Genital opening. tp - Terminally adorned process. mf - Marginal flab. tx - Thorax. na - Natatory lobe.
82 V. 4' 4 Li kr Z FIGURE 4.13
Scanning electron micrographs of Chonopeltis victori Avenant-Oldewage,1991.
Adornments on lateral sides of the abdomen (Scale bar:10,um); Anal sinus and furcal rami (Scale bar:10,um); Tip of abdomen lobe (Scale bar:10,um); Peritrichous ciliates(*) found on the body surface of C. victori specimens (Scale bar:10p.m); E,F: Marks(*) left by sessile ciliophorans on the body surface of C. victori specimens (Scale bar:10p,m);
Abbreviations: as - Anal sinus. fr - Furcal rami. sc - Denticulated scales. sp - Spine-like projections. ss . - Robust setae.
83 1 2, v . V ci ' M a R @ Z
T LE 4.1
Summary of the absolute growth measurements (mean length and width in mm) of adult Chonopeltis victori Avenant-Oldewage,1991.
AVENANT OLDEWAGE -PRESENT STUDY (1991)
1,29 1,28 1,205 1,453 0,64 0,74 0,598 0,889 0,68 0,68 0,661 0,733 2,82 2,25 2,556 2,354 0,76 0,56 0,607 0,572 2,91 2,29 2,816 2,721 1,54 1,25 1,374 1,319 0,39 0,80 0,418 0,939 0,68 0,63 0,554 0,540 4,80 3,10 4,718 4,252
84 TA LE 4.2
Comparison of the relative growth relationships (as %) of Chonopeltis victori Avenant- Oldewage,1991 adults.
AV ENANT ---OL:DEWAGE PRESENT STUDY (1991)
)
30 59 54 55 103 101 110 116 27 25 24 24 52 54 49 49 26 30 20 20 27 34 26 34 50 61 50 61 30 65 35 65 52 51 55 51
Abbreviations: CL/TL=Cephalic shield length / Total length; CW/CL=Cephalic shield width /Cephalic shield length; CcL/CL=Cephalic shield cleft length / Cephalic shield length; FCW/CW= Frontal cephalic ala width / Cephalic shield width; SW/CW =Sucking disc width / Cephalic shield width; AbL/TL=Abdomen length / Total length; AbW/AbL=Abdomen width / Abdomen length; GoL/AbL=Testis or spermatheca length / Abdomen length; AcIL/AbL= abdomen cleft length / Abdomen length.
85 TA LE 4.3
Summary of regression equations for the morphometric data (mm) on adult Chonopeltis victori Avevant-Oldewage,1991.
RELATIVE PARAMETERS SEX n REGRESSION r 2 p y = a + bx
Total length (x) vs Cephalic shield length (y) 9 30 y = 0.44 + 0.45 x 0.98 <0.005 e 30 y = 0.20 + 0.50 x 0.95 <0.005
Total length (x) vs Abdomen length (y) 9 30 y = 0.17 + 0.22 x 0.98 <0.005 a 30 y = -0.02 + 0.34 x 0.95 <0.005
Cephalic shield length (x) vs Cephalic shield width (y) 9 30 y = 0.06 + 1.08 x 1.00 <0.005 a 30 y= 0.11 + 1.11 x 1.00 <0.005
Cephalic shield length (x) vs Cephalic shield cleft length (y) 9 30 y = -0.08 + 0.27 x 0.96 <0.005 e 30 y = -0.12 + 0.29 x 0.92 <0.005
Cephalic shield width (x) vs Cephalic lobe width (y) 9 30 y = 0.29 + 0.39 x 0.99 <0.005 e 30 y = 0.19 + 0.42 x 0.95 <0.005
Cephalic shield width (x) vs Sucking disc width (y) 9 30 y = 0.18 + 0.14 x 0.94 <0.005 d 30 y = 0.11 + 0.16 x 0.96 <0.005
Abdomen length (x) vs Abdomen cleft length (y) 9 30 y = -0.10 + 0.63 x 0.98 <0.005 d 30 y = 0.01 + 0.50 x 0.92 <0.005
Abdomen length (x) vs Spennathecae/testes length (y) 9 30 y = -0.01 + 0.36 x 0.88 <0.005 0^ 30 y = -0.03 + 0.67 x 0.98 <0.005
Abdomen length (x) vs Abdomen width (y) 9 30 y = 0.06 + 0.44 x 0.97 <0.005 0^ 30 y = 0.07 + 0.56 x 0.96 <0.005
86
TA PI LE 4A
Summary of the relative growth relationships (%) of all the recognised spesies of Chonopeltis Thiele,1900. (Measurements were taken from figures accompanying species descriptions when the information was not available in the text. A short line (-) indicates that the information was not at all available).
' Al)ULT C. inennis 44 118 24 43 31 31 33 34 61 Thiele (1904) 59 102 28 50 19 28 42 67 Fryer (1956) 47 105 22 55 37 30 42 22 63 Van As & Van As (1993) C. schoutedeni 4() 129 26 38 29 40 28 80 Brian (1940) 35 133 19 41 30 42 22 11 85 Fryer (1956) C. cougicus : 54 104 14 51 26 25 33 21 52 Fryer (1959) C. tlaccifron..5 52 104 21 55 36 23 48 33 57 Fryer (1960a) C. brevis 56 113 26 32 28 27 35 46 54 Fryer (1961b) C. meridionalis 55 107 18 53 29 28 48 40 51 Fryer (1964) C. elongatus 40 93 21 43 30 33 28 33 62 Fryer (1974) C. australis 55 113 29 38 25 25 58 36 42 Boxshall (1976) 50 111 45 39 24 60 59 40 Van Niekerk (1984) C. auStralisSimus 54 114 14 61 44 24 60 40 28 Fryer (1977) . C. mmutus 59 105 19 53 40 25 72 28 48 Fryer (1977) C. fryeri 45 114 25 52 30 31 41 23 54 Van As (1986) C. victori 3() 103 27 52 26 27 50 30 52 Avenant-Oldewage (1991) 54 110 24 49 20 26 50 35 55 Own unpublished records C. koki 52 112 22 44 21 31 42 49 54 Van As (1992)
ADUL'f:MALES: C. inennis 47 106 22 53 31 41 35 64 55 Van As & Van As (1993) C. schoutedeni 43 113 32 24 33 24 73 Brian (1940) C. cougicus 26-29 60 Fryer (1959) C. flaccifrons 53 138 19 54 33 26 62 36 57 Fryer (1960a) C. brevis 55 113 24 47 29 37 61 66 49 Fryer (1961b) C. meridianalis 51 115 12 50 28 37 40 40 54 Fryer (1964) C. elougatus -- 46 100 16 51 43 40 28 66 47 Fryer (1974) C. australiS 59 108 26 38 33 40 53 61 31 Boxshall (1976) 50 109 47 46 36 51 34 Van Niekerk (1984) C. au.stralissimus 55 31 57 27 Fryer (1977) C. minitus 59 107 13 56 39 30 54 46 32 Fryer (1977) C. fryeri 51 118 14 54 32 41 47 48 38 Van As (1986) C. victori 59 101 25 54 30 34 61 65 51 Avenant-Oldewage (1991) 55 116 24 49 20 34 61 65 51 Own unpublished records 52 112 26 51 25 38 48 51 54 Van As (1992)
ABBREVIATIONS:
CL/TL = Cephalic shield length / Total length; CW/CL = Cephalic shield width / Cephalic shield length; CcL/CL = Cephalic shield cleft length / Cephalic shield length; FCW/CW = Frontal cephalic ala width / Cephalic shield width; SW/CW = Sucking disc width / Cephalic shield width; AbL/TL = Abdomen length / Total length; AbW/AbL = Abdomen width / Abdomen length; GoL/AbL = Testis or spermatheca length / Abdomen length.; AcL/AbL = Abdomen cleft length / Abdomen length.
87 I y not having a ame for it' we lose the power to 'understand it'd "
- Frederick R. Schram HAP E
S IEC C MO T• HOLOGY OF alb CE HAI= S LLD L ("C APACE") 114 CHONOPELTIS THIELE9 1900
TRODUCTION
Since Thiele (1900) described the first Chonopeltis species collected from a chromid fish caught in Lake Nyassa (presently known as Lake Malawi), more than 90 years ago, another 12 species, all from localities in sub-Saharan Africa, were described (cf. Chapter 3, Table 3.1). Except for the descriptions by Van Niekerk (1984), Van As (1986,1992) and Van As & Van As (1993), who used scanning electron micrographs (SEM), all other species descriptions, and in fact most of the information on Chonopeltis species, are based on macroscopical investigations of whole mounts. Consequently, little information is available on the morphology of the internal structures in members of this genus. During the present investigation, studies of transverse, sagittal and frontal serial sections of some Chonopeltis species revealed that many structures of the cephalic shield and its alae, such as ridges, grooves and rods, were incorrectly interpreted and consequently incorrectly named when described from whole mounts.
In this chapter the surface morphology of the cephalic shield and alae, as well as the internal structures of the alae in Chonopeltis australis Boxshal1,1976 and Chonopeltis victori
88 Avenant-Oldewage,1991 are described from SEM micrographs and serial sections. The question whether Chonopeltis species do possess a carapace sensu Calman (1909) is also investigated and discussed.
5.2 CEP s LIC S L AND ALAE ("CA ACE")
In Chonopeltis spp. the parasite body is differentiated into three regions: a cephalon, thorax and an abdomen. The cephalon is composed of five fused segments, the thorax of four definite segments whilst the abdomen appears to be unsegmented (Mclaughlin,1980; Schram,1986) (Fig. 5.1A,B). The segments of the cephalon, bearing the cephalic appendages, are dorsally indistinctly fused into a cephalic shield (Figs. 5.1A & 5.2A). Three distinct wing-shaped structures ("Lappen" of Thiele,1904; "alae" of Brian,1940; "lobes" of Fryer,1956,1959), extend anteriorly and laterally from the cephalon (Fig. 5.1A). Dorsally they are demarcated from the cephalon by furrows or sulci ("sillons" of Brian, 1940). A transverse furrow, which may be referred to as the frontal sulcus, separates the cephalon from the frontal cephalic ala (Fig. 5.1A), and a crescent-shaped furrow, the lateral sulcus, separates the cephalon from each lateral cephalic ala (Figs. 5.1A & 5.2A). These three sulci form a horseshoe-shaped furrow demarcating the cephalon from the alae. The rectangular frontal cephalic ala, covering the antennae dorsally (Fig. 5.1B) is relatively small compared with the large ovoid lateral cephalic alae which extend backward and usually cover the basipodites of the second pair of thoracopods (Fig. 5.1A,B).
5.2.1 Frontal cephalic ala
The frontal ala is rather thin, tapering from a thickness of about 100Am near the frontal sulcus to about 20,um at the distal end (Fig. 5.3A,B). Histologically the frontal ala consists of a dorsal and ventral hypodermis (10/2m) secreting a thin (5iim) cuticle. Between the basal membranes of the dorsal and ventral hypodermis numerous gland cells are present (Fig. 5.3A,B). These ectodermal cells, ranging in size from 20ktm to 50Am in diameter,
89 appear to sink below the hypodermis during development, filling the haemocoelic spaces between the basal membranes of the hypodermis (cf. Felgenhauer,1992). The chitinous efferent ducts of the gland cells open on the cuticle, appearing as minute openings on the surface of the ala (Fig. 5.2D). Some of these gland cells stain intense pink with PAS, indicating the presence of glycoproteins in the apical granules of the cells. The numerous muscle fibres, stretching between the dorsal and ventral integumental walls (Fig. 5.3A,B) could on contraction have a squeezing effect on the gland cells, thus enhancing liberation of secretion.
5.2.2 Marginal grooves and alal crests
Ventrally, along the anterior and lateral margins of the frontal ala are two chitinous grooves (Figs. 5.1B & 5.2B). These grooves, which may be referred to as the marginal grooves of the front alla, are C-shaped in cross section. Each one is secreted by a thickened part of the hypodermis and is sloughed off during moulting (Fig. 5.3B). From each marginal groove two longitudinal sclerotized ridges ("Chitinleisten" of Thiele,1904 "chitinous supporting strips" of Fryer,1956; "chitinous supporting rods" of Fryer,1960a) the me all and Rater alai crests, extend posteromedially across the ventral surface of the frontal ala (Figs. 5.1B, 5.2B), ending in knob-like structures slightly in front of the base of the proboscis (Figs. 5.1B & 5.2B). In cross section an alal crest reveals a distinct eminence of paliform hypodermic cells covered externally by a sclerotized trough-shaped cuticle (Fig. 5.4A,B). The posterior parts of the medial alal crests are more prominent than their anterior parts, with the sclerotized parts U-shaped rather than V-shaped (Fig. 5.4A,B). Furthermore a haemocoelic space is present between the paliform hypodermic cells and a large transversely oriented muscle, each stretching from a point between the two medial alal crests and a sclerotized plate covering the ventrolateral part of the cephalon (Fig. 5.4B). The muscle lies ventrally to the optic nerve, antennary and alal nerves as well as some of the muscles operating the sucker (Fig. 5.4B).
The main function of the four alal crests appears to be that of support to the frontal ala,
90 making it more rigid. Furthermore, contraction of the two transversely oriented muscles would cause a rolling movement of the medial alal crests, bringing their posterior parts in closer apposition. This action could aid in securing a holdfast on the host. The lateral alal crests also cover the greater parts of the compound eyes ventrally (Fig. 5.4A), thus protecting the eyes from objects on the skin of the host when the parasite crawls over the skin. Dorsally the eyes are almost completely covered by the powerful muscles of the suckers (Fig. 5.4A,B), a fact which explains why specimens of Chonopeltis species are so easily caught when swimming freely in for example a Petri dish.
5.2.3 Muscles of the frontal ala
Two pairs of muscles, stretching between the dorsal and ventral walls of the anterior part of the cephalon (Fig. 5.5), accounts for the movements of the frontal ala. One member of a pair originates from the dorsal cephalic wall and inserts on the ventral wall of the proximal part of the frontal ala, slightly in front of the base of the antenna (Fig. 5.5). This muscle, which on contraction would depress the ala, may be referred to as the depressor muscle of the ala usculus depressor ala frontalis). The second member originates from the ventral cephalic wall, slightly behind the medial alal crest, and inserts on the posterodorsal wall of the frontal ala in the region of the frontal sulcus (Fig. 5.5). This muscle, which on contraction would elevate the frontal ala, may be referred to as the levator muscle of the frontal ala (musculus levator ala frontal's).
5.2.4 Lateral cephalic alae
Each of the two lateral alae, like the frontal ala, is separated from the cephalon by a deep furrow, the lateral sulcus (Fig. 5.1A & 5.6A). Distal to the lateral sulcus two secondary furrows, one dorsally and one ventrally are present on the lateral ala (Fig. 5.6A). The ventral secondary sulcus forms the medial boundary of the so-called respiratory area of the lateral ala (Fig. 5.6A). The respiratory area of each lateral ala consists of a small ovoid
91 anterior and large ovoid (almost kidney-shaped) posterior area (Fig. 5.2E). Histologically each respiratory area consists of densely packed columnar epithelial cells with centrally situated nuclei. The basement membrane of the epithelium lines the haemocoel which contains the arborizations of the midgut gland (i.e. enteral diverticules, digestive gland) and some ectodermal mucous glands (Fig. 5.6A). The epithelial cells of the hypodermis in the respiratory area structurally resemble the paliform hypodermic cells of the alal crests (Figs. 5.4A & 5.6A) and they likewise secrete a sclerotized cuticle, though much thinner. The respiratory areas are devoid of surface ornamentation in contrast to the dorsal and remainder of the ventral surface of the alae which contain minute cuticular openings, sensory pits and setae (Fig. 5.2D,E).
5.2.5 Muscles of the lateral alae
The dorso-ventral movements of each lateral ala are controlled by a pair of transverse muscles running close to the dorsal and ventral hypodermic walls of the ala (Fig. 5.6B). The dorsal muscle originates from a median fascia, runs laterally and inserts on the cuticle of the dorsal alal wall in the region opposite the proximal rim of the respiratory area (Fig. 5.6B). This muscle which on contraction would elevate the ala, may be referred to as the levator muscle of the lateral ala (musculus levator ala lateralis). The ventral muscle appears to be a differentiation of the group of muscles operating the maxilla. It inserts on the cuticle of the ventral wall of the lateral ala, close to the medial rim of the respiratory area (Fig. 5.6B). This muscle, which on contraction would depress the lateral ala may be referred to as the depressor muscle of the lateral ala (musculus depressor ala lateralis). The rostro-caudal flexion of the lateral ala is accomplished by some longitudinal muscles situated in the proximal part of the lateral ala (Fig. 5.6 A,B).
5.2.6 Dorsal surface of the cephalic shield
Except for the two compound eyes and trifid ocellus (nauplius eye) the most conspicious
92 structures on the dorsal surface of the cephalic shield are two sclerotized grooves (Fig. 5.1A). Each groove extends backward from the region of the compound eye to the posterior end of the cephalic shield, running parallel to each other and lateral to the trifid ocellus (Fig. 5.1A). Like the alal crests, the sclerotized cephalic grooves are secreted by cells of the hypodermis, having a paliform shape (Fig. 5.4B). During moulting the sloughed sclerotized cuticle appears as a longitudinal rod on the cephalic groove (Fig. 5.4A). Consequently surface studies of the cephalic shield may reveal a ridge instead of a groove in this area (cf. "dorsal ridges" described in other Branchiura - e.g. Meehean,1940; Sutherland & Wittrock,1986). Surface microscopical investigation (i.e. light microscopy and SEM), of the cephalic shield reveals a less conspicious anteriorly situated transverse suture ("anterior cephalic groove" of Martin,1932, in Argulus) which bisects the two dorsal sclerotized grooves just behind the ocellus, whilst a similar more posteriorly located transverse suture ("posterior cephalic groove" of Martin,1932, in Argulus) marks the junction between the cephalon and the first thoracic somite (Fig. 5.1A). Other surface ornamentations of the cephalic shield include minute cuticular openings, sensory pits and setae (Fig. 5.2D).
53 ICISCUSSION
A study of the literature reveals that the terminology applied to the cephalic shield and its extensions in branchiurans in general and in the genus Chonopeltis in particular, is inconsistent and confusing. In the Branchiura (i.e. Argulus Miiller,1785, Dolops Audouin, 1837; Chonopeltis Thiele,1900 and Dipteropeltis Calman,1912), only two body regions were initially identified: a cephalothorax and an abdomen (Jurine,1806). Later three body regions were distinguished but, however, inconsistently termed as: head/head shield, trunk and tail ("scutum cephalicum, truncus, cauda," of Thore11,1864); cephalothorax, thorax and abdomen (e.g. Claus,1875; Wilson,1902; Grobben,1908; Maid1,1912; Fryer,1956,1960a); head, thorax and abdomen (Calman,1912); or cephalon, thorax and abdomen (e.g. Martin, 1932; Meehean,1940; McLaughlin,1980; Schram,1986; Overstreet et al. ,1992).
93 Concerning Chonopeltis in particular, Thiele (1900) distinguished two body regions, viz. a cephalothorax and an abdomen, when he succinctly described the first Chonopeltis species, i.e. Chonopeltis inermis. Brian (1940) later distinguished three body regions, namely a cephalothorax (covered dorsally with a carapace), thorax and abdomen. The carapace, according to Brian (1940) consists of an "area cephalic" (=cephalon), a frontal ala and lateral alae which are separated from the cephalon by distinct "sillons" (=furrows). Fryer (1956), when redescribing C. inermis Thiele,1900, retained the terms cephalothorax, thorax and abdomen but referred to the lateral alae as "posterior lobes of the carapace" as well as "left and right lobes of the carapace". However in Fryer's (1959) redescription of C. schoutedeni Brian,1940 as well as when describing new chonopeltid species, i.e. C. congicus Fryer,1959; C. flaccifrons Fryer,1960; C. brevis Fryer,1961; C. meridionalis Fryer,1964; C. elongatus Fryer,1974; C. minitus Fryer,1977; C. australissimus Fryer, 1977, Fryer referred to a "carapace, thorax and abdomen" and he used the terms "carapace trifoliate". The latter consists of an "anterior (cephalic) lobe" and "right and left lobes" (Fryer,1959,1960a,1961b,1964,1974,1977). Accordingly, Fryer's said left and right lobes corresponds with Brian's (1940) lateral alae and his cephalic lobe with Brian's cephalon plus the frontal ala. Van As & Van As (1993), in their redescription of C. inermis Thiele, 1990, and also Boxshall (1976), Van As (1986,1992) and Avenant-Oldewage (1991) all followed Fryer's (1959) onwards terminology, i.e."carapace trifoliate, cephalic lobe". Since the "carapace" in Chonopeltis spp. consists of a median cephalon or cephalic shield and three wing-like (trifoliate) structures which are clearly separated from the cephalon by a horseshoe-shaped furrow (i.e. frontal and two lateral sulci) it is proposed that the more apt term "ala" (defined as "wing-like projection, structure, extension or outgrowth" by Holmes, 1979), as suggested by Brian (1940), should be used in preference to the term "lobe" (viz. "pendulous part of a structure").
The question whether chonopeltid branchiurans do, in fact, have a carapace as defined by Calman (1909, p.6), i.e. "a structure ... originating as a fold of the integument from the posterior margin of the cephalic region", is still unanswered. Dahl's (1991) thorough investigation of the cephalic and thoracic shield and folds in some crustaceans, which conclusively showed that no carapace fold is formed in the Malacostraca and Branchiopoda
94 investigated by him, did not include the Maxillopoda (i.e. Ostracoda, Copepoda, Branchiura). Since the embryonic development of Chonopeltis species is direct without a nauplius stage (Fryer,1960a; Van Niekerk,1984), we investigated sections of pre-hatched and early post-hatched larvae of C. australis. These sections show that the alae develop as lateral outgrowths of the cephalic tegument, initially consisting of leaf-like structures composed of hypodermic layers separated by haemocoelic spaces. In the frontal ala no cephalic structures invade the haemocoelic spaces, but in the lateral alae the haemocoelic spaces soon become invaded by the arborizations of the midgut glands, parts of the maxillary gland and in males even parts of the prostate gland. The lateral alae in C. australis appear to represent the fused pleurae of the cephalic segments. In accordance with the findings of Dahl (1991) in the Malacostraca and Branchiopoda, a carapace fold does not form in C. australis and possibly also not in the other three branchiuran genera, i.e. Argulus; Dolops and Dipteropeltis. It is therefore, suggested that the term "carapace" in Chonopeltis species be replaced by cephalic shield or cephalon, containing cephalic pleural folds, the alae.
95 FIGURE 5.1
Chonopeltis victori Avenant-Oldewage,1991.
Dorsal view of adult female; Ventral view of adult female.
Abbreviations: ab - Abdomen. an - Antenna. at - Anterior transverse suture. cg - Cephalic groove. co - Compound eye. cs - Cephalic shield.
fa - Frontal cephalic ala. fs - Frontal sulcus.
in - Marginal indentation.
la - Lateral cephalic ala. lac - Lateral alal crest. is - Lateral sulcus.
mac - Medial alal crest.
mg - Marginal groove of frontal ala. pr - Proboscis. pt - Posterior transverse suture.
ra - Respiratory area. s - Sucker. tp - Second thoracopod. tx - Thorax.
96 FilOURZ Z.V FIGURE 5.2
Scanning electron micrographs of Chonopeltis victori Avenant-Oldewage,1991 and Chonopeltis australis Boxshal1,1976.
Dorsal view of the cephalic shield of C. victori (Scale bar:300,um); Ventral view of the anterior part of the cephalic shield of C. victori (Scale bar:200,urn); Ventral view of the anterior part of the cephalic shield of C. australis (Scale bar:2004m); Adornments on the surface of the cephalic shield (Scale bar:10/./m); Clearly demarcated and unadorned respiratory areas on the ventral surface of the cephalic shield (Scale bar:10Am).
Abbreviations: an - Antenna. at - Anterior transverse suture. eg - Cephalic groove. cs - Cephalic shield. cu - Cuticular openings of gland cells. fa - Frontal cephalic ala. fs - Frontal sulcus. in - Marginal indentation. la - Lateral cephalic ala. lac - Lateral alal crest. is - Lateral sulcus. mac - Medial alal crest. mg - Marginal groove of frontal ala. p - Circular pit. pt - Posterior transverse suture. ra(a) - Anterior respiratory area. ra(p) - Posterior respiratory area. s - Sucker. sp - Spine-like projection.
97 am FIGURE 5.3
Chonopeltis australis Boxshal1,1976.
Mid-sagittal section through frontal ala; Parasagittal section through frontal ala showing paliform epithelial cells secreting medial alal crest during ecdysis.
Abbreviations: cu - Cuticle. fs - Frontal sulcus. gs - Gland cells. ha - Haemocoel. by - Hypodermis. im - Integumental muscles. lm - Levator muscle of frontal ala.
mac - Medial alal crest.
mg - Marginal groove of frontal ala.
pc - Paliform cells secreting medial alal crest. sc - Sloughed cuticle.
98 FLIGURE 5.3
,---,-----...... -e .• ilamir flaViirAtargi'N, ao -4"...-4.
IIIIIIIINIIIIIIIiiiiiiill ,;,,,,,agnigirourfle „ FIGURE 5.4
Chonopeltis australis Boxshal1,1976.
Cross section through the region of the eye; Cross section through the posterior parts of the medial alal crest during ecdysis.
Abbreviations: aln - Alal nerve. ann - Antennary nerve. cg - Cephalic groove. cu - Cuticle. dm - Depressor muscle of frontal ala. e - Eye (compound). ha - Haemocoel. lac - Lateral alal crest. lm - Levator muscle of frontal ala. mac - Medial alal crest. ins - Muscles of sucker. on - Optic nerve. pc - Paliform cells of alal crest. sc - Sloughed cuticle. sp - Sclerotized plate. tm - Transverse muscle.
99 MUU 504
,,, AN..,0011A10, 1111"11.7 ", 111010P FIGURE 5.5
Chonopeltis australis Boxshal1,1976. Thick (20//m) parasagittal section to show the muscles operating the frontal ala.
Abbreviations:
ann - Antennary nerve.
ba - Base of antenna. cu - Cuticle.
dm - Depressor muscle of frontal ala. fa - Frontal ala. fs - Frontal sulcus. gs - Gland cells. lac - Lateral alal crest. lm - Levator muscle of frontal ala.
on - Optic nerve. se - Sloughed cuticle.
100 F OUNI, 5.5
VC@ gom FIGURE 5.6
Chonopeltis australis Boxshal1,1976.
Cross section through the maxillary region to show the lateral sulcus and respiratory area on the lateral ala; Thick (20,um) cross section to show the muscles operating the lateral ala.
Abbreviations: bm - Base of maxilla. cu - Cuticle. dm - Depressor muscle of lateral ala. emg - Ectodermal mucous gland. fm - Muscles for rostro-caudal flexion of lateral ala. gm - Gland cells at base of maxilla. la - Haemocoel. lm - Levator muscle of lateral ala. Is - Lateral sulcus. mf - Median fascia. mg - Midgut. mgg - Diverticules of midgut gland. mm - Maxillary muscles. mra - Medial rim of respiratory area. pg - Prostate gland. ra - Epithelium of respiratory area. ss - Secondary sulcus. sv - Seminal vesicle.
101 FEIGURE 5.6
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APTER
PRODUCTIVE SYSTEM ]k SPE 411 SFER IN CHONOPELTIS THIELE,19 0
6.1 INTRODUCTION
Parasitism, in its widest sense, is a specialized type of life, and parasites in their efforts to pursue this life successfully have adopted among others, various distinct morphological modifications. The most important of these adaptations are indubitably concerned with optimal reproduction - thus efficient reproduction systems, copulatory structures and mechanisms of sperm transfer. Extensive and detailed morphological knowledge of these aspects is a prerequisite for understanding the reproductive adaptations needed for a successful parasitic existence. In this sense, branchiuran crustaceans which are primarily ectoparasites of fishes and occasionally also amphibians, are no exception. As previously mentioned, the class Branchiura is presently represented by not more than 200 described species belonging to four genera: Argulus Mtiller,1785; Dolops Audouin,1837; Chonopeltis Thiele,1900; and Dipteropeltis Calman,1912. Within the cumulative research done on these parasites, morphological and especially anatomical and histological work has been largely neglected with the work published by Claus (1875) and Wilson (1902) still regarded as the best sources on the internal anatomy of the group (Schram, 1986).
102 Concerning the morphology of the male reproductive system and the mechanism of sperm transfer during copulation, these aspects have hitherto only been described in Argulus (Jurine, 1806; Leydig , 1850 , 1889 ; Claus, 1875; Grobben, 1908 ; Martin, 1932; Debais ieux , 1953 ; Hirschmann & Partsch,1953; Overstreet et al. ,1992; Avenant-Oldewage & Swanepoe1,1993) and Dolops (Maid!, 1912; Fryer, 1958 , 1960b ; Avenant-Oldewage & Van As , 1990) . In Chonopeltis, the only branchiuran genus endemic to Africa, no studies have to date been published on the said aspects in any of the thirteen described species.
In this chapter the aim is thus to describe for the first time the histomorphology of the male reproduction system, the morphology of the accessory copulatory structures as well as to give an account of the mechanism of sperm transfer in Chonopeltis, using a single species, Chonopeltis victori Avenant-Oldewage,1991. Histological serial sections, graphic reconstructions and scanning electron micrography were used to describe the mentioned features in male C. victori.
6.2 MORPH• LOGY AND OTHER ASPECTS OF THE MALE REPRODUCTIVE SYSTEM
The relative position and different components of the male reproductive system of C. victori are depicted in Figure 6.1 and Figure 6.2. The system consists of two testes from each of which a relatively short vas efferens extends anteriorly to open into the posterior end of the elongated seminal vesicle. The opposite (i.e. anterior) extremity of the latter is joined to paired vasa deferentia of which each one doubles back on itself and proceeds caudally to open together with the efferent prostate duct into the ejaculatory duct. The posterior end of the reproductive tract is formed by the cuticle-lined genital atrium, which opens to the exterior by means of a transverse, slit-like genital aperture. Externally, several distinct secondary sexual structures are present on the first through fourth pair of thoracopods.
103 6.2.1 Testes
As in all branchiurans, the paired testes are located in the abdomen. In C. victori the two ovoid to piriform testes (Fig. 6.2) usually consist of four (sometimes three or five) testicular lobes each and occupy approximately two-thirds (mean: 64%) of the total length of the abdomen. The mean length and width of the testes are respectively 739/2m and 3404m (n=30).
The lumen of each testis, filled by maturing spermatozoa, is lined by a complex stratified epithelium resting on a thin basement membrane (Fig. 6.3H). This seminiferous epithelium which is dorsally usually much thicker than ventrally (with the anterodorsal part probably functioning as the germinal epithelium as in A. foliaceus (Wingstrand,1972)), consists of numerous spermatogenic cells as well as several nurse cells ("Sertoli cells" of Wingstrand, 1972, in A. foliaceus). The structure of the seminiferous epithelium varies with the specific stage of spermatogenesis. The nurse cells is the somatic cells of the testis and are also known as nutritive cells, intercalary cells, Sertoli cells, follicle cells, sustentacular or accessory cells in other crustacean groups (Krol et a/.,1992). In the Crustacea, the nurse cells apparently arise from the lateral and ventral walls of the testes and help sustain germ cells during their development (Pillai,1960; Pochon-Masson,1968a,b).
A thin outer connective tissue wall, reminiscent of a tunica albuginea/perididymis, encapsulates each testis. Although an external muscular coat is lacking, each testis is closely associated with several pairs of variously orientated abdominal muscles (Fig. 6.3G,H) which, on contraction, most probably effectuate constriction of the testis.
Spermatogenesis in one branchiuran genus, Argulus, has been described in detail by Wingstrand (1972), and spermatogenesis and spermatozoa are assumed to be similar among the different genera of the Branchiura, both by Wingstrand (1972) and Mattei (1970). In C. victori only immature spermatozoa were observed within the testes whilst mature spermatozoa occurred in the seminal vesicle, vasa deferentia and genital atrium. This suggests that final maturation occurs after the spermatids leave the testis. Development of spermatozoa may,
104 however, continue as the cells are transported towards the vas deferens.
6.2.2 Vasa efferentia
From the anterior end of each testis a vas efferens (28Am in diameter) extends anteriorly into the thorax where it almost immediately opens together with its partner into the posterior end of the much wider median seminal vesicle (Fig. 6.2). Each inwardly curved vas efferens lies directly beneath the dorsal body wall and dorsolaterally to the hindgut (Fig. 6.3H).
The lumen of the vas efferens is lined by transitional type epithelial cells with indistinct cell bounderies (Fig. 6.3H). The epithelium is enveloped by a thin outer connective tissue layer.
6.2.3 Sembilan vesicle
The seminal vesicle ("terminal ampoule" viz. enlarged end of vas deferens in some crustaceans (Krol et a/.,1992)) is an elongated median structure (21 to 78Am in diameter) which extends anteriorly from the posterior end of the fourth thoracic somite, over the greater part of the length of the thorax (Figs. 6.1 & 6.2). On its longitudinal course within the haemocoel, the seminal vesicle runs dorsally to and in close contact with the muscular wall of the midgut whilst, within the fourth thoracic somite, the triangular heart lies dorsally to the seminal vesicle (Fig. 6.3C,D). At its posterior and anterior ends, the seminal vesicle is joined to the paired vasa efferentia and vasa deferentia respectively, with the latter bounding the outer lateral sides of the seminal vesicle (Figs. 6.2A & 6.3C).
The large lumen of the seminal vesicle is filled by spermatozoa suspended within a clear homogeneous secretion which stains a uniform pale blue with Azocarmine-Azan. The lumen is lined by a cuboidal epithelium (one to two cell layers thick), enclosed by a layer of connective tissue containing striated circular muscle fibers (Fig. 6.3F,G).
105 6.2.4 Vasa deferentia
The vasa deferentia are two muscular ducts through which spermatozoa pass from the seminal vesicle to the genital atrium (Fig. 6.2). The paired vasa deferentia exit from the anterior end of the seminal vesicle, turn at a straight angle backwards to proceed, reflected on either side of the seminal vesicle, caudally (Fig. 6.2A,B). The proximal part of each vas deferens runs dorsolaterally to the anterior half and ventrolaterally to the posterior half of the seminal vesicle, whilst bounded ventrally by the muscular wall of the digestive tract (Fig. 6.3B,D). The distal part of the vas deferens (i.e. the part furthest away from the seminal vesicle) lies dorsolateral to the prostate complex (Figs. 6.2A & 6.3B). In the region of the fourth pair of thoracopods, each vas deferens opens almost simultaneously with the efferent prostate duct into the ejaculatory duct (Fig. 6.3F).
Each vas deferens is lined by a simple cuboidal epithelium followed by a basement membrane and an external muscular coat of variable thickness (Fig. 6.3B-F). The latter consists of individual bundles of circularly orientated muscle fibers displaying a few prominent transverse striations. Interspersed amongst these muscle bundles are a few longitudinally orientated muscle cells. The thickness of the muscular coat increases progressively from anterior to posterior - reaching maximum thickness in the region of the third and fourth thoracic somites where the muscular coats of the vasa deferentia, efferent prostate ducts, ejaculatory ducts and genital atrium seems to form a unity (Figs. 6.2B & 6.3E,F). In this region, the muscular wall on the ventral side of the vas deferens is prominently enlarged, implying a sphincter mechanism guarding this part of the vas deferens (Fig. 6.3E). The specific construction of the muscular coat suggests that, at least in some regions, the vasa deferentia engage in peristalsis. In this regard, McLaughlin (1983) as well as Krol et al. (1992, p.313) observed that the vasa deferentia in crustaceans are divided into various distinct regions with regionally differing functions. Furthermore, Adiyodi & Anikumar (1988) also stated that in crustaceans the length and coiling of each vas deferens serve to increase the effective surface area for secretory, absorptive, and storage functions.
106 6.2.5 Accessory reproductive glands
The accessory reproductive glands, more aptly referred to as the paired prostate complex in A. japonicus (cf. Avenant-Oldewage & Swanepoe1,1993), are two elongated structures situated one on each side of the digestive tract, below and somewhat to the outside of the vas deferens of its side (Fig. 6.2A,B). Both structures proceed further anteriorly than the seminal vesicle and vasa deferentia, usually extending into the basal podomere of the maxillas. Each prostate complex is differentiated from anterior to posterior into three regions: 1) a glandular/ secretory part; 2) a reservoir and 3) an efferent duct (Fig. 6.2A).
Histologically the glandular part is tubulo-alveolar in appearance. It is composed of a system of branched intercellular collecting ducts, each of which is surrounded by 4 to 13 giant secretory cells (16 to 234m in diameter) with large (114m in diameter) spherical nuclei (Fig. 6.3A). The giant secretory cells rest on a thin basement membrane followed by an outer fibrous connective tissue layer. All the intercellular ducts unite to finally discharge via a common collecting duct into the tubular prostate reservoir. The much wider lumen of the latter is lined by large cuboidal epithelial cells with less distinct cell bounderies (Fig. 6.3A,B). The epitelium is separated by a basement membrane from the relatively thin external circular muscular coat. An efferent reservoir duct leads caudally from the prostate reservoir to open, in the region of the fourth thoracic somite, into the ejaculatory duct (Figs. 6.2A & 6.3F). The narrow lumen of the reservoir duct is lined by a layer of cuboidal epithelial cells followed by a thin basement membrane and a thick external muscular coat (Fig. 6.3F). Just before fusion with the ejaculatory duct, the muscular wall of the proximal part of the reservoir duct (i.e. the part closer to the digestive tract) is noticeably thickened, implying that this part of the duct could function as a sphincter (Fig.6.3F). The secretion produced by the glandular part of the prostate complex stains a deep purple-blue with Azocarmine-Azan and is abundantly found in the lumen of all three regions of the prostate complex.
107 6.2.6 Ejaculatory ducts
The two relatively short, dorso-ventrally orientated ejaculatory ducts are situated reflected on either ventrolateral side of the digestive tract in the last thoracic somite (Figs. 6.2B & 6.3E,F). They are lined with tall (18Am) ciliated columnar epithelial cells with prominent basally located nuclei (Fig. 6.3F). The epithelium rests on a thin basement membrane enclosed by an outer, well developed, muscular coat which, on the ventral side of the duct neighbouring the genital atrium, varies from very thin to totally absent (Fig. 6.3E,F). The rest of the muscular coat surrounding the ejaculatory ducts is dorsally continuous with the muscular layers surrounding the vas deferens and prostate duct, whilst ventrally its lateral walls are continuous with the muscular layers covering the lateral surfaces of the genital atrium (Fig. 6.3D,E).
In all (except two) specimens examined, the lumen as well as epithelial lining of the ejaculatory ducts were found to be blind-ending with no direct communication between the lumina of the ejaculatory ducts and the genital atrium (Fig. 6.3D,E). In one large specimen, one of the ejaculatory ducts, and in another specimen both ejaculatory ducts, were found not to be blind-ending but showed a distinct connection between its lumen and that of the genital atrium. The thin epithelial boundary separating the said lumina appeared however to be torn or pierced open.
6.2.7 Genital atrium
The genital atrium is a transversely orientated dorsoventrally flattened chamber (38 to 43,um deep and 80ktm wide) which opens to the exterior by way of a slit-like transverse opening (45,um wide) (Fig. 6.2). Caudally, the body wall forms a anteroventrally directed flap which partially disguises and deforms the slit-like genital opening (Fig. 6.3E). The lumen of the genital atrium, with its two lateral sides anterodorsally curved, is lined with a layer of cuticle continuous with that of the body wall. This cuticular layer is enveloped by an epithelium consisting of cuboidal epithelial cells ventrally and columnar epithelial cells with basally situated nuclei, dorsally (Fig. 6.3D). The epithelium rests on a thin basal layer which is,
108 except for those parts on the dorsal surface which are associated with the ejaculatory ducts, covered by a thin muscular layer (Fig. 6.3D,E).
6.2.8 Secondary sexual structures
In male C. victori the first through fourth pair of thoracopods ("swimming legs") possess accessory structures that are presumably used in copulation (Fig. 6.4). These structures include various epidermal protrusions covered entirely or only partially by some or all of the following superficial structures: denticulated scales, minute spines, small sensory setae and pits as well as various other minute cuticular openings.
The first thoracopod bears a peripherally setigerous protrusion posteroventrally on the precoxa (Fig. 6.4B). Four terminally adorned finger-like protrusions occur on the posterior side of the second thoracopod: one ventrally on the precoxa, two opposing on the ventral and dorsal side of the coxa, and one distally on the ventral side of the basipodite (Fig. 6.4C). Setigerous (i.e. bearing setae) swollen areas are present on the anterior surface of the precoxa and coxa of legs two and three (Fig. 6.4D). On the anterior side of the coxa of leg three there are also two adorned knob-like protrusions and a rounded nodular structure (Fig. 6.4E). The latter resembles the so-called "pocket" described by Sutherland & Wittrock (1986) for the related genus Argulus. The "socket", a cylindrical pouch with a large slit-like opening, occurs anterodorsally on the coxa of leg three (Fig. 6.4E). Beneath the socket, a posterior marginal flap (cf. Fig. 4.12E,p.82) extends backwards to cover the basal parts of leg four ventrally (Fig. 6.4A,E). A large peripherally adorned protrusion occurs on the anterior side and a smaller setigerous protrusion on the dorsal side of the basipodite of leg three (Fig. 6.4E). Leg four is highly modified with the "peg", an elongated anterolaterally directed process, occuring on the anteroventral side of the basipodite (Fig. 6.4F). A groove which is surrounded and lined by robust papilla-like structures with delicately furcated distal ends (cf. Fig. 4.12G, p.82), occur distally on the ventral side of the peg (Fig. 6.4F). The slender distal extremity of the peg, which is curved rectangular upwards towards the socket on leg three, is bordered circumferentially by papillae-like structures similar to those associated with the ventral groove
109 (Fig. 6.4F). The slender cylindrical apex of the peg is dual structured with a single row of small cuticular openings circumscribing the inner rim of the external apical collar, whilst 13 to 14 spindle-shaped, presumed primarily sensory, structures border the apical perimeter of the internal collar (Fig. 6.5A). Cross-sections reveal nervous tissue projecting into the peg, occasionally penetrating the cuticular openings of the peg. The endopodite is sickle-shaped with its convex side extending anteriorly to form a terminally adorned process (Fig. 6.4A). A few setae and posteriorly directed denticulated scales occur on the posteroventral surface of each bilaterally rounded natatory lobe (Fig. 6.5B). In contrast to the female, the natatory lobes of the male do not conceal the genital opening situated posteroventrally on the fourth thoracic somite (Fig. 6.5B).
63 DISCUSSION
When considering the morphological changes and adaptations associated with successful and optimal reproduction coupled with parasitism, we are confronted with two related but opposite phenomena: progressive specialization and regressive elimination of specializations. In this regard, the piscine ectoparasitic branchiurans are no exception, especially when taking the specific morphology of the male reproductive system, the method of sperm transfer and other associated aspects of the reproductive biology into consideration. Substantiating this, branchiurans possess in contrast to the majority of other crustaceans motile flagellate spermatozoa (Fryer,1960b; Wingstrand,1972; Pochon-Masson,1983). However, sperm transfer in one branchiuran genus, Dolops, involves the employment of spermatophores (Fryer,1958,1960b) although spermatophores are generally not produced in those Crustacea with flagellate motile spermatozoa (Pochon-Masson,1983; Mann,1984). Furthermore, the spermatophores formed in Dolops are produced by accessory reproductive glands also present in the non-spermatophore-producing Argulus and Chonopeltis. The following unique features in the Branchiura in general and Chonopeltis in particular thus need to be scrutinised: 1) the nature and function of the accessory reproductive glands and 2) the mechanism of sperm transfer.
110 Concerning the accessory reproductive glands ("Accessorische Geschlechtsdruse" of Leydig, 1850; "Prostata" (=prostate) of Claus,1875, Thiele,1904, Maid1,1912; "accessory blind capsules" of Wilson,1902, Overstreet et a/.,1992; "Beidruse" (=joint glands) of Hirschmann & Partsch,1953; "spermatophore glands" of Fryer,1960b; "prostate complex" of Avenant- Oldewage & Swanepoe1,1993), these structures have hitherto only been described in Argulus and Dolops. In C. victori, the said glands appear to be structurally and functionally very similar to those described in Argulus spp. (Claus,1875; Avenant-Oldewage & Swanepoel, 1993). In both Argulus and Chonopeltis, these structures are histologically differentiated into three distinct regions: a glandular/secretorial part, a reservoir and an efferent duct, thus, more aptly referred to as the paired prostate complex as suggested by Avenant-Oldewage & Swanepoel (1993). In Dolops, Fryer (1960b) describes a pair of accessory glands, or "spermatophore-producing apparatus", each of which is also differentiated into three regions: an arborescent "spermatophoric gland", a "spermatophoric canal" or "storage chamber", and a "spermatophoric vesicle" - inevitable reminicent of the paired prostate complex in the other two genera. Spermatophores are, however, not produced in either Chonopeltis nor Argulus (Martin,1932; Debaisieux,1953; Fryer,1960b,1968), implying a different function of the prostate complex in these two genera. In other spermatophore-producing Crustacea the spermatophore is secreted by cells in the epithelial wall of the vas deferens and/or seminal vesicle or non-germinal cells in the testes (Clarke,1973; Pochon-Masson,1983; Mann,1984; Adiyodi & Anikumar,1988), and not by specialized accessory glands which are independant of the main reproductive tract as described in Dolops (Fryer, 1960b). In Dolops the terminal part of each gland, the partial secretorial spermatophoric vesicle, opens into a "chitin-lined ductus ejaculatorius" which in turn opens to the exterior via the genital aperture (Fryer,1960b, p.412). However, in the Branchiura the ejaculatory ducts are of mesodermal and the genital atrium of ectodermal origin (Avenant-Oldewage & Swanepoe1,1993), as was deduced from the fact that the ejaculatory ducts are not lined by a cuticle whilst the genital atrium is lined with a layer of cuticle continuous with that of the body wall. The structures in Dolops referred to as the spermatophoric vesicle and the ductus ejaculatorius thus appear to actually correspond respectively with the ejaculatory duct and genital atrium in C. victori and Argulus spp., implying that the accessory glands in Dolops are not independant of the main reproductive tract. If it is assumed that the accessory reproductive glands in the branchiurans are homologous structures, the following explanation as to the apparent differences between these structures in the said three genera, is offered. In agreement with Fryer (1960b), homologous accessory glands (presumably secreting some kind of temporary sealant during copulation), were probably present in the primitive branchiuran ancestors. However, on the evolutionary line taken by Argulus and Chonopeltis, leading to the development of more complex copulatory structures and consequently less extensive copious secretion, a negative tendency towards spermatophore formation and thus specialization of the accessory glands probably occurred whilst the opposite process most likely took place in Dolops. With this in mind, the prosate complex in Chonopeltis and Argulus probably still fulfils an important role during sperm transfer, involving not only the possible secretion of a temporary sealant but also functions more similar to those executed by the prostate gland in other animal groups (i.e. enhancing motility and fertility of sperm; counteracts acidity of other fluids; lubricating passageways and providing an additional energy source for sperm (Hickman et a/.,1988)).
Concerning the mechanism of sperm transfer, from the male genital opening to the female spermathecae, Pochon-Masson (1983) observed that in most crustaceans when copulatory structures are not well developed and fertilization joints are not easily approachable, the transmission of the paternal genome carried by immotile spermatozoa is made possible by the formation of spermatophores. In Dolops, especially in D. ranarum, this situation is almost unreservedly applicable except for the presence of motile flagellate spermatozoa (also found in Chonopeltis and Argulus). On the other hand, species of Chonopeltis and Argulus display more extensively developed secondary copulatory structures on the thoracopods and no spermatophore formation. In Dolops, Fryer (1960b) illustrated that sperm transfer in this genus involves the formation of a spermatophore which, during copulation, is pressed against the abdomen of the female, pierced by the perforated spermathecal spines and hence sperm is transferred to the spermathecal vesicles of the female. In Argulus, studies on sperm transfer initially implicated the modified structures on the third and fourth pair of thoracopods as the sperm transferring apparatus in the process. Leydig (1850) was the first to propose that the peg-like structure ("Haken", i.e. hook) on the fourth thoracopod collects the sperm at the male genital aperture and then transfers it to the socket-like structure ("Samenkapsel", i.e. semen capsule) on the third thoracopod from where it then passes through the spermathecal
112 openings to the spermathecal vesicles of the female. In agreement with Leydig (1850), Claus (1875) added that the "hook" also assists in opening the flaps of the semen capsule during sperm transfer. However, neither Leydig nor Claus described exactly how and through which mechanism sperm is transferred from the semen capsule of the male to the spermathecae of the female. According to Martin (1932), the hook and semen capsule, named the "peg-and-socket" by her, are not involved during sperm transfer but are merely used as clasping mechanisms during copulation, whilst sperm transfer is solely accomplished by direct contact of the "male genital opening and ejaculatory duct" with the openings of the spermathecae on the abdomen of the female. Although more recent authors such as Meehean (1940); Debaisieux (1953); Fryer (1968); McLaughlin (1980); Schram (1986) and Sutherland & Wittrock (1986) agree with Martin (1932), none, except Avenant-Oldewage & Swanepoel (1993), could provide an explanation of exactly how sperm is injected into the spermathecae without streaming past the minute opening in each spermathecal spine. Avenant-Oldewage & Swanepoel (1993) suggested that during copulation in Argulus japonicus, contraction of the muscular walls of the vas deferens and prostate duct causes semen to be actively pumped into the ejaculatory duct which is then closed off by sphincters from the vas deferens and prostate duct resulting in a high pressure inside the ejaculatory duct. Once penetrated by a spermathecal spine, semen would then flow from the ejaculatory duct into the spermathecal vesicle due to the higher pressure in the ejaculatory duct. In C. victori, the slit-like transversely orientated genital aperture of the male leads directly into a cuticle-lined chamber, the genital atrium, which in turn neighbours the two opposing ejaculatory ducts. The latter do not open into the genital atrium but are blind-ending tubes with no direct communication between its lumina and that of the genital atrium. However, in one specimen examined, one of the ejaculatory ducts showed a distinct connection between its lumen and that of the genital atrium with the thin epithelial layer separating the said two lumina, torn or pierced open. In another specimen the said epithelial layer between both ejaculatory ducts and the genital atrium were found to be pierced open. These observations in C. victori undoubtedly furnish evidence to a mechanism of sperm transfer very similar to that suggested by Avenant-Oldewage & Swanepoel (1993) - especially when taking the specific morphology of the sperm receiving apparatus (spermathecae) in the female also into consideration. In C. victori females, the two spermathecal vesicles are situated inside the abdomen from where each is connected by means
113 of a coiled spermathecal duct with the sharp external spermathecal spine situated anteriorly on the ventral surface of the abdomen (cf. Chapter 7). Each spermathecal spine is borne on a cone-shaped base which in turn is situated in the centre of a nodular protrusion. The latter is bordered terminally by two to three uneven concentric circles of denticulated scale-like structures which doubtless serve to assist in the firm anchoring of the spermathecal spine during sperm transfer. In additon, both the terminal spine as well as its cone-shaped base is not only fully ejectable but also very flexible, hence permitting the spine to be pointed in various directions. Sub-apically on the outer lateral side of each spine a single slit-like transverse opening is found, proceeding from this opening, a prominent groove extends upwards towards the apex of the spine (cf. Chapter 7). Considering the above described features in C. victori as well as the fact that in some female specimens examined, semen did not occur in both spermathecal vesicles simultaneously, it is suggested that during copulation the two spermathecal spines must be inserted into the genital atrium of the male and then, simultaneously or singly, pushed through the thin epithelial layer separating the lumina of the genital atrium and the two opposing ejaculatory ducts. In accordance with the mechanism of sperm transfer described by Avenant-Oldewage & Swanepoel (1993), sperm and prostate secretion would then, due to the higher pressure in the ejaculatory duct as well as on contraction of the muscular walls of the ejaculatory duct, flow from the ejaculatory duct, down the apical groove and through the opening in the spermathecal spine and then, via the spermathecal duct, into the spermathecal vesicle.
In conclusion, the secretion of the prostate gland in the non-spermatophore-producing Branchiura (i.e. Chonopeltis and Argulus), appears to fulfil more than one important function during sperm transfer - including acting as a temporary sealant which prevents semen from escaping from the ejaculatory duct into the genital atrium. In Chonopeltis, as in Argulus, the mechanism of sperm transfer involves a process during which the respective lumina of the two opposing ejaculatory ducts are, simultaneously or singly, physically penetrated by a spermathecal spine whereafter semen is actively pumped into the female spermathecal vesicle(s) whilst the secondary sexual structures on the thoracopods of the male merely act as clasping mechanisms during copulation.
114 FIGURES 6.1
Ventral view of the adult male of Chonopeltis victori Avenant-Oldewage,1991 to indicate the position of the reproductive organs (----).
Abbreviations : ab - Abdomen. cs - Cephalic shield. fa - Frontal cephalic ala. nix - Maxilla (=second maxilla). na - Natatory lobe. pr - Proboscis. px - Prostate complex. s - Sucker (=first maxilla / maxillula ). to - Testis. tp(1-4) - Thoracopod (1-4). tx - Thorax. vd - Vas deferens. vs - Seminal vesicle.
115
FIGURE 6.2
Chonopeltis victori Avenant-Oldewage,1991. Graphic reconstruction of the male reproductive organs. (A-H represent the different levels illustrated in Figure 6.3A-H).
Ventral view. Lateral view.
Abbreviations: ed - Ejaculatory duct. dp - Efferent duct of prostate reservoir. ga - Genital atrium. go - Genital opening. gp - Glandular part of prostate complex. rp - Reservoir of prostate complex. to - Testis.
vd - Vas deferens. ye - Vas efferens. vs - Seminal vesicle.
116
FIGURE 6.3
Chonopeltis victori Avenant-Oldewage,1991. A-H: Semi-diagrammatic illustrations of consecutive transverse sections of the male reproductive system, corresponding to the different levels (A-H) indicated in Figure 6.2. The top of each figure represents the ventral surface and sections sequenced anterior to posterior.
Abbreviations: am - Anterior midgut. cl - Cuboidal epithelial cells. con - Connective tissue layer containing striated circular muscle fibers. cu - Cuticle. da - Duct of alveolus. dp - Duct of prostate reservoir. dv - Main dorsal blood vessel. ed - Ejaculatory duct. el - Epithelial wall. fl - Ventral flap of body wall. ga - Lumen of genital atrium. gp - Glandular part of prostate complex. ha - Haemocoel. he - Triangular heart. • hg - Hindgut. me - Muscular coat. pm - Posterior midgut. rp - Reservoir of prostate complex. sc - Giant secretory cells. sp - Sphincter. st - Striated muscle. to - Testis. vd - Vas deferens. ye - Vas efferens. vs - Seminal vesicle. Fig. 6.3E-H continue on p.118...
117 gilawcao oao o g) E o cig o g 9 FIGURE 6.3 (continued from p.117)
Chonopeltis victori Avenant-Oldewage,1991. Semi-diagrammatic illustrations of consecutive transverse sections of the male reproductive system, corresponding to the different levels (E-H) indicated in Figure 6.2. The top of each figure represents the ventral surface and sections sequenced anterior to posterior.
Abbreviations: cl - Cuboidal epithelial cells. con - Connective tissue layer containing striated circular muscle fibers. cu - Cuticle. dp - Duct of prostate reservoir. dv - Main dorsal blood vessel. ed - Ejaculatory duct. el - Epithelial wall. fl - Ventral flap of body wall. ha - Haemocoel. hg - Hindgut. me - Muscular coat. Pm - Posterior midgut. sp - Sphincter. st - Striated muscle. to - Testis. vd - Vas deferens. ye - Vas efferens. vs - Seminal vesicle.
118 FllOULT301 0.0
a F go
a®
VG
1 1100 Anm 1 FIGURE 6.4
Scanning electron micrographs of male Chonopeltis victori Avenant-Oldewage,1991.
Dorsal view of thoracopods (Scale bar:100/./m);
Ventral view of legs one and two (Scale bar:100Am);
Ventral view of leg two (Scale bar:10p.m);
Ventral view of legs two and three (Scale bar:10/2m);
Dorsal view of leg three displaying the "socket" and "pocket" (Scale bar:10p.m);
Dorsal view of leg four displaying the "peg" (Scale bar:10,um).
Abbreviations: by - Basipodite. po - Pocket. en - Endopodite. pr - Protrusion. ex - Exopodite. sa - Setigerous area. g - Groove. so - Socket.
mf - Marginal flap. tp - Terminally adorned process.
P - Papillae-like structures. tx - Thorax.
pg - Peg.
119 ozNo z FIGURE 6.5
Scanning electron micrographs of male Chonopeltis victori Avenant-Oldewage,1991.
A : Terminal end of the "peg" on leg four (Scale bar:5,um);
B : Genital opening (go) and natatory lobes (na) (Scale bar:10p,m).
120
" The simplest questions are the most profound. "
- Richard Bach HAPTER
• F MALE REPRODUCTIVE SYST EM IN CHONOPELTIS THIELE9 190
7.11. INTRO RUCTION
Although much work has been done on branchiuran crustaceans, i.e. species belonging to the genera Argulus Maller,1785; Dolops Audouin,1837; Chonopeltis Thiele,1900 and Dipteropeltis Calman,1912, morphological and anatomical information on especially the internal structures of these primarily piscine ectoparasites are scanty. Concerning the morphology and histology of the female reproductive system, information on these aspects has hitherto only been provided on some Argulus spp. (Jurine,1806; Leydig,1850; Claus,1875; Wilson,1902; Grobben,1908; Guberlet,1928; Martin,1932; Debaisieux,1953; Hirshmann & Partsch,1953; Overstreet et a/.,1992) and Dolops spp. (Heller,1857; Stuhlmann,1891; Bouvier,1898; Maidl,1912; Fryer,1960b; Avenant-Oldewage & Van As,1990). No information is available on the histomorphology of the female reproductive system of representatives of both Chonopeltis and Dipteropeltis.
Although thirteen species of the entirely African genus Chonopeltis have to date been described, the majority of papers published on this genus provide little, if any, information on the internal anatomy and a histomorphological description of the female reproductive system does not exist. Extensive and detailed morphological knowledge of all aspects of the female reproductive system of these parasites is, however, a prerequisite for understanding the functional reproductive adaptations needed for their specialized piscine ectoparasitic existence. This is especially applicable to Chonopeltis, as to Argulus, where no spermatophores are formed (Fryer,1960b,1968) and the mechanism of sperm transfer (viz. from the male genital opening to the female spermathecae), still remains an enigma. From the literature it is also evident that no previous histomorphological study on the female reproductive system in branchiurans did show a connection between the ovary and oviducts and it is, therefore not known how the eggs reach the oviducts (Overstreet et a/.,1992). Furthermore, no attention has been given to the maturation of oocytes during the ovarial cycle in branchiurans and it is also not known in which part of the reproductive tract the thick shell surrounding the oocytes are formed.
In order not only to provide a detailed morphological description of the female reproductive system, but also to gain an insight into the functional significance of specific components of the said system during sperm transfer in Chonopeltis, a thorough histological investigation of the female reproductive system in Chonopeltis victori Avenant-Oldewage,1991 as well as Chonopeltis australis Boxshal1,1976 was carried out. Serial sections and graphic reconstructions were used to describe the structure of the said system in both Chonopeltis species. Histological measurements are based on a 7,2 mm long mature C. victori female and a 6,2 mm long mature C. australis female. The histological study was supplemented with a scanning electron microscopical study of the secondary sexual structures apparent externally on the body surface of the female.
7.2 MORPHOLOGY AND OTHER ASPECTS OF THE FE LE PRODUCTIVE SYSTEM
The different components of the female reproductive system in adult Chonopeltis victori Avenant-Oldewage,1991 and Chonopeltis australis Boxshal1,1976 are illustrated in Figures 7.1, 7.2 and 7.3 respectively . The reproductive system consists of an ova-producing and egg
122 storing part or main genital tract and the spermathecae (=seminal receptacles). The main genital tract consists of a single large ovary containing a lumen, the ovarian lumen (=ovarian sac), a rudimentary or vestigial non-functioning oviduct, and a single functioning oviduct opening into the cuticle-lined genital atrium. The latter, in turn communicates with the exterior via a slit-like genital aperture followed by a crescent-shaped fertilization chamber. The paired spermathecal vesicles and spermathecal ducts are situated within the abdomen (Fig. 7.1) whilst the two perforated spermathecal spines are externally visible on the anteroventral surface of the abdomen (Fig. 7.10A). Apart from the two spermathecal spines, the only other distinct secondary sexual structures present on the body surface of the female are the two ovoid natatory lobes which conceal the spermathecal spines as well as the genital opening situated posteroventrally on the last thoracic segment (Fig. 7.10D). The natatory lobes also form the floor of the posterior part of the fertilization chamber.
7.2.1 Ovarium
The ovary in the adult female is a single tubular organ which extends medially from the posterior extremity of the maxillary segment, over the greater part and length of the thorax to the posterior extremity of the fourth thoracic segment, running dorsally to and in close contact with the muscular wall of the digestive tract (anterior midgut to hindgut) and ventrally to the main dorsal blood vessel (Figs. 7.5 & 7.7). This median position of the ovary may deviate asymmetrically to the left or the right in accordance with the relative position of the functioning oviduct as well as the size and number of ova contained within the oviduct. The ovary is closely associated with the two oviducts, partially encapsulating the relatively short non-functioning oviduct whilst usually only the anterior part of the functioning oviduct directly neighbours the ovary (Figs. 7.2 & 7.3). Posteriorly, the latter is separated from the functioning oviduct by means of a dorsoventrally orientated striated muscle bundle or septum (Fig. 7.7D). The ovary and oviducts are enclosed in a fibrous mesodermal capsule (=gonadal sac) which tends to become very thin in the highly distended ovary (Figs. 7.4 & 7.5). The ovary and oviducts lie in a cavity, termed the circumgenital body-cavity by Leydig (1850) and Grobben (1908), that is separated from the rest of the haemocoel. The mesodermal capsule (or
123 gonadal sac) thus isolates the oocytes in the circumgenital body cavity or gonocoel from the haemocoel.
The size of the ovary varies in accordance with the number as well as size of contained oocytes. Sections of the ovary reveal eggs at all stages of development with the more mature eggs usually occupying a more lateral position in the ovary (Figs. 7.4,7.5 & 7.7). The germinal ridge or zone, bordered by immature eggs, lies dorsally (Figs. 7.4 & 7.5) and extends over almost the entire length of the ovary in very young females whilst confined to the posterior region of the ovary in the larger, highly gravid females. The germinal zone, or zone of proliferation (Krol et a/.,1992), may vary in location from species to species or individual to individual because of pressure exerted by growth and differation of the large number of yolk-filled oocytes produced. The developing eggs, present in different stages of meiosis, initially display a finely speckled cytoplasm containing a few prominent vacuoles. The cytoplasm circumscribes a prominent cytocentrum displaying a spherical, translucent nuclear area (Figs. 7.4 & 7.7). In addition to the oolemma, the soft and deformable eggs are covered at first by a delicate primary egg membrane (=vitelline membrane) which progressively thickens as the egg becomes more mature (Fig. 7.5). The cytoplasm becomes increasingly granular and eventually, near the completion of vitellogenesis, it is filled with numerous yolk granules of varying sizes (Fig. 7.5). When reaching the ovarian lumen, the heavily yolked eggs become enveloped by a much thicker tertiary envelope or toughened outer shell displaying a rather coarse texture which stains a bright red with Azocarmine-Azan (Figs. 7.5 & 7.7). The heavily yolked or mature eggs, lacking a micropyle, are ovoid in shaped and vary between 190,um and 264,um in length and 118Am and 164/./m in width (n=30).
Previtellogenic ovary:
In the previtellogenic ovary the germinative zone is represented by a prominent longitudinal ridge of gonadal tissue and oogonia in the dorsal septum, ventral to the heart and dorsal aorta (Fig. 7.4). Ventral and lateral to the germinal ridge, primary oocytes undergoing previtellogenesis make up the bulk of the ovary (Fig. 7.4). The smaller (younger) oocytes
124 (8itm in diameter, n=30) lie close to the germinal ridge whereas the larger (30Am, n=30) previtellogenic primary oocytes occur ventrally along the periphery of the ovary (Fig. 7.4). These larger previtellogenic primary oocytes are separated from the haemocoel by the thin wall of the gonadal sac only (Fig. 7.4). Internally the ovary contains an epithelial lined lumen, the ovarian sac, which is roughly T-shaped in cross section (Fig. 7.4). The ovarian sac, which anteriorly opens into the functional oviduct, subdivides the ovary into medial and lateral divisions (Fig. 7.4). The epithelium lining the ovarian sac contains a very thick basal membrane.
Vitellogenic ovary:
In the vitellogenic ovary the germinative zone is represented by a longitudinal plate of cells occupying a middorsal position (Fig. 7.5). The ovary at this stage is populated with numerous primary oocytes undergoing vitellogenesis as well as eggs surrounded by a thick (15,um) shell (Fig. 7.5). Oocytes undergoing vitellogenesis are usually pear-shapped with their bases facing the haemocoel (Fig. 7.5), occasionally bulging into the haemocoel (Fig. 7.5). Oocytes in advanced stages of vitellogenesis are, like previtellogenic oocytes, surrounded by vitelline membranes (=primary egg membranes) only. No follicle cells (nurse cells) surrounding the oocytes were observed (Fig. 7.5).
In vitellogenic ovaries the tripartite division of the ovary by the ovarian sac is still evident. Encapsulated eggs, i.e. oocytes containing a thick tersiary envelope external to the vitelline membrane, are found in the ovarian sac (Fig. 7.5). Since non-encapsulated eggs are found outside the ovarian lumen, facing the basement membrane of the luminal epithelium, it appears that the egg shell is formed from secretions of the luminal epithelium after ovulation has taken place. Histologically there appears to be no difference between the shells of eggs in the oviduct and those in the ovarian lumen (Fig. 7.5).
125 7.2.2 Oviducts
There is only a single functional oviduct. In the number of specimens examined (n=70), approximately equal numbers have the oviduct on either the left or the right side. In C. victori the functional oviduct extends over the entire length of the thorax with the median, unpaired anterior part of the oviducal system extending into the cephalon where it terminates directly above the digestive tract in the region where the proventriculus enters the anterior end of the anterior midgut (Fig.7.6). The functioning oviduct runs for much of its length in close association with the ovary and the digestive tract. Posteriorly, the proximal part of the functioning oviduct is closely associated with a prominent, dorsoventrally orientated, striated muscle bundle or septum (Fig .7.7C,D), which separates that part of the functioning oviduct from the ovary and the digestive tract (hindgut and posterior midgut).
In both C. australis and C. victori, the ovarian lumen is anteriorly continuous with that of an epithelial sac which shows rostral prolongations (Figs. 7.2 & 7.3). In females with large numbers of encapsulated eggs (i.e. more than 50 eggs) this "egg-containing sac" becomes expanded anteriorly and laterally to invade the haemocoelic spaces at the bases of the maxillae (Fig. 7.1). Posterolaterally this sac opens into the functioning oviduct (Figs. 7.2 & 7.3). The latter, which in a 6,2 mm female contained approximately 20 encapsulated eggs, runs posteriorly, close to the lateral wall of the ovary (Figs. 7.2,7.3 & 7.4). The non-functioning oviduct in both C. australis and C. victori is represented by a short, blind-ending tube which is much shorter than its counterpart (Figs. 7.2 & 7.3). Histologically the structure of the two oviducts (i.e. functioning and non-functioning oviduct) is very similar. Both oviducts are lined with an unpigmented cuboidal epithelium containing a few secretory cells (Fig. 7.6B). The epithelium rests on a very thin basal layer followed by a thin outer circular muscle coat. Although sometimes ill defined and difficult to detect, especially in the smaller specimens, the lumen of the short non-functioning oviduct is not obliterated though, due to its deflated or collapsed nature, it is much smaller than the lumen of the usually highly distended functioning oviduct. The functioning oviduct in previtellogenic ovaries is lined by a cuboidal epithelium covered externally by a layer of circular muscles (Figs. 7.4 & 7.5). In vitellogenic ovaries the functioning oviduct is dilated due to the presence of the large (240Am x 180Am, n=20)
126 eggs and the epithelium appears more squamous than cuboidal. Posteriorly the functioning oviduct opens via a narrow duct into the cavernous genital atrium (Figs. 7.2, 7,3 & 7.7).
7.2.3 Genital atrium
The genital atrium is a cuticle-lined, transversely orientated chamber which opens, via a curved, slit-like genital aperture (± 122/...tm wide) situated posteroventrally on the last thoracic segment, into the fertilization chamber which in turn communicates directly with the exterior (Figs. 7.1, 7.2 & 7.3). The genital atrium measures respectively 120Am by 200itm and 1404m by 360ktm in a 7,2mm long C. victori female and a 6,2mm long C. australis female. The dorsal wall of the genital atrium consists of tall (31/./m) columnar epithelial cells with basally located nuclei whereas the ventral wall consists of cuboidal epithelial cells similar to those of the epidermis (Fig. 7.7A). The epithelium rests on a thin basal layer covered only dorsally by a thin muscle layer (Fig. 7.7). The lumen of the genital atrium is lined by a cuticle continuous with that of the body wall (Fig.7.7).
7.2.4 Fertilization chamber
The fertilization chamber is differentiated into two parts: an anterior and a posterior part. The anterior part is a crescent-shaped, blind-ending pocket situated dorsal to the genital atrium (Figs. 7.8 & 7.9). This part is probably formed by an invagination of the abdominal sternal wall, since its stratified squamous epithelium is lined by a cuticle (Figs. 7.8 & 7.9). The posterior part of the fertilization chamber is also crescent-shaped in cross section and its floor is formed by the two so called "natatory lobes" (Fig. 7.8). At the confluence of the genital atrium and fertilization chamber in C. australis, a cone-shaped papilliform structure projects from the mid-dorsal wall of the fertilization chamber (Fig. 7.8B). In cross section this structure appears to consist of very thick stratified squamous epithelium separated medially by basement membranes. Numerous nerve fibres were observed near the base of the papilliform structure. Muscle fibres originating from the tergal wall, lateral to the hindgut, insert on the
127 sternal wall, lateral to the base of the papilliform structure (Fig. 7.8B). Slightly behind the papilliform structure and lateral to a notch in the abdominal sternal wall, the spermathecal spines are situated (cf. Fig. 7.9). This cone-shaped papilliform structure together with the abdominal notch in C. australis corresponds with the triangular-shaped swollen area, medially situated between the two spermathecal spines in C. victori females (Figs. 7.8A,7.9C & 7.10A) and probably has a mechanosensorial function in both species.
7.2.5 Spermathecae
The spermathecae (=seminal reseptacles) are two separate structures situated within the abdomen, surrounded by giant gland cells, several pairs of variously orientated abdominal muscles, loose parenchymatous cells and a number of blood sinuses (Fig. 7.9). Each spermatheca is differentiated into a spermathecal vesicle, spermathecal duct and a muscular papilla which terminates in a sharp, sclerotized spermathecal spine (Figs. 7.2, 7.3 & 7.9C).
In C. victori the spermathecal vesicles are two ovoid sacs, approximately 689Am in length and 2434m in width, located within the anterior half of the abdomen (Figs. 7.1 & 7.2). The relatively large lumen of each vesicle, usually filled with semen, is lined by a cuticle similar to that covering the body wall (Fig. 7.9). The cuticle-lining is enclosed by a layer of cuboidal epithelial cells bearing prominent spherical nuclei (Fig. 79). A thin but sturdy muscular coat which stains a dark blue with Azocarmyn-Azan capsulates the epithelial wall of each vesicle (Fig. 7.9). Neither of the two spermathecae is connected with the main female genital tract, instead each spermathecal vesicle communicates with the exterior by way of a extremely fine and coiled spermathecal duct which terminates in a minute, singly perforated spine situated anteriorly on the ventral surface of the abdomen (Figs 7.2, 7.9 & 7.10). Movement of semen from the spermathecal vesicle to the exterior is probably accomplished by means of the combined contraction of the thin muscular coat surrounding the spermathecal vesicle and the abdominal muscles closely associated with the vesicle.
The narrow lumen of each spermathecal duct is lined with a relative thick layer of cuticle
12$ surrounded by an epithelium consisting of only a few columnar epithelial cells (15,um in length) with basally situated nuclei (Fig. 7.9). The epithelium rests on a thin basal layer enclosed by a very thin smooth muscle layer (Fig. 7.9). A short, histologically similar but blind-ending accessory duct (Fig. 7.9) is joined to each spermathecal duct at a position anterior to the region where the spermathecal duct opens in the spermathecal spine. Each spermathecal duct terminates in a muscular papilla, bearing at its apex a minute sclerotized spine which possesses a single sub-apical opening (Figs. 7.9C, 7.10A,B,C). Histologically, each cup- shaped spherical papilla is composed of more than one layer of variously orientated striated muscles (Fig. 7.9C). Medially, this multilayered capsule embraces an epithelium consisting of one or two layers of cuboidal epithelial cells with indistinct cell bounderies (Fig. 7.9). A layer of similar cuboidal epithelial cells which is continuous with the epithelial cells medially as well as those of the epidermis, covers the rest of the muscular cup (Fig. 7.9C). Externally each papilla is covered by a cuticle which is much thickened medially around the base of each spermathecal spine (Fig. 7.9C). This cuticle is also continuous with that of the body wall (Figs. 7.8 & 7.9C).
Each muscular papilla presents externally on the ventral surface of the abdomen as a nodular protrusion bearing in its centre the sharp spermathecal spine borne in a cone-shaped base consisting of thickened cuticle (Fig. 7.10A,B). This protrusion is bordered terminally by two to three uneven concentric circles of denticulated scale-like structures (Fig. 7.10B) which doubtless serve to assist in the firm anchoring of the spermathecal spine during sperm transfer. Due to the muscular nature of the papilla, the terminal spermathecal spine together with its cone-shaped base are not only fully ejectable but also very flexible, hence permitting the spine to be pointed in various directions. A single slit-like opening occurs sub-apically on the outer lateral side of each spermathecal spine (Fig. 7.10B,C). Proceeding from this opening a prominent longitudinal groove extends upwards towards the apex of the spine (Fig. 7.10C).
In C. australis, the spermathecal vesicles are two oblong structures, each measuring 530,um by 150,um in a 6,2 mm long female (Fig. 7.3). They occupy the proximal third of the abdominal lobes. The spermathecae in C. australis are histologically very similar to those in C. victor/. Each spermathecal vesicle is lined by a cuboidal epithelium secreting a thin cuticle.
129 Outside the basement membrane of the epithelium, a circular muscle layer is present. Each spermathecal vesicle is connected to the fertilization chamber by means of a short (2504m long), straight duct composed of approximately six epithelial cells which secrete cuticle (Fig. 7.3). A muscular layer surrounds the epithelium of the spermathecal efferent duct. In the region where the spermathecal duct leaves the spermathecal vesicle, a valve occurs which probably regulates the flow of sperm into the spermathecal duct. The latter is joined by an additional coiled duct before the spermathecal duct opens in the spermathecal spine (Fig. 7.3). This accessory duct resembles the spermathecal duct in general structure but is of much larger diameter. Each cone-shaped spermathecal spine (50Am in length), is provided with some 20 muscle bundles arranged in parabolic fashion around the base of the spine (Fig. 7.9C). Contraction of these muscles will cause eversion of the spine. Other muscles, which are responsible for the proximo-distal movements of a spine, originate from the tergal wall, lateral to the hindgut, and insert medially and laterally on the base of the spermathecal spine (Figs. 7.8B & 7.9C). The efferent spermathecal duct opens at the tip of the spermathecal spine.
7.2.6 Integumental glands
Three groups of integumental glands occur in the vicinity of the fertilization chamber. One group of cells lies in the anterior part of the abdomen, occupying the space between the spermathecal vesicle and spermathecal spine (Figs. 7.8 & 7.9). The spermathecal and accessory ducts pass through this glandular mass on their rostral path to the fertilization chamber (Fig. 7.9). The second group of tegumental glands occupies the greater medial part of the natatory lobe (Fig. 7.9A,B). The third group of gland cells lies ventral to the hindgut, occupying the space between the hindgut and ventral abdominal wall (Fig. 7.9A,B).
7.2.7 Secondary sexual structures
Except for the two characteristic spermathecal spines, the only other distinct features apparent externally on the body surface of the female of both Chonopeltis spp. examined, are the two
130 ovoid natatory lobes and the narrow triangular-shaped swollen area bearing a few short cone- shaped spines, medially situated between the two spermathecal spines on the anteroventral surface of the abdomen (Fig. 7.10). The two natatory lobes are bordered ventrally along its posterior and inner postero- lateral margins by anteriorly directed denticulated scales interspersed by small spine-like setae and minute cuticular openings with a border of elongate setae peripherally (Fig. 7.10D). A few (4 to 6) sensory pits and circular openings (probably the external openings of the integumental glands) are apparent on the otherwise smooth ventral surface of the natatory lobes (Fig. 7.10D). The dorsal surface of each natatory lobe, facing the ventral body surface, is unadorned with the outer lateral wall of each lobe extending anterolaterally towards the abdomen (Fig. 7.8), hence making it possible for each natatory lobe to move rectangularly away from or towards the ventral body surface (cf. the fertilization chamber). The two natatory lobes meet, when closed, at the ventral midline, thus, concealing the genital opening situated posteroventrally on the last thoracic segment as well as the two spermathecal spines occurring anteriorly on the ventral surface of the abdomen (Fig. 7.10A,D). The two natatory lobes form the floor of the posterior part of the fertilization chamber (Fig. 7.8) and probably also play a role in manoeuvring and positioning the shelled eggs during oviposition and consequential injection of sperm.
7.3 DISCUSSION
Taking the morphology of the female reproductive system in branchiurans in general and Chonopeltis in particular into consideration, the following questions involuntarily come to mind: why is there only one ovary; how do the eggs pass from the ovary into the functioning oviduct; and how is sperm transferred from the male genital opening to the female spermathecae and consequential insemination of mature eggs accomplished in Chonopeltis, which does not produce spermatophores, and possesses unique spermathecae which are entirely isolated from the main genital tract of the female?
131 Earlier workers who studied the female reproductive system in the Branchiura initially reported the presence of a single ovary and a single short oviduct (Jurine,1806; Leydig,1850; Thore11,1864; Wilson,1902) whilst others (e.g. Heller,1857; Claus,1875; Bouvier,1898; Grobben,1908; Maid1,1912; Martin,1932; Debaisieux,1953; Hirschmann & Partsch,1953; Avenant-Oldewage & Van As,1990; Overstreet et al.,1992) described a single ovary but two oviducts of which only one is functional. Claus (1875, p.232) considers the single ovary in Argulus to be derived from a single anlage in the larva, situated on either the left or the right side of the body. On the other hand, Grobben (1908) - comparing the ovary with the homologous paired testes - postulates that the single ovary in branchiurans is derived from paired anlagen which fuse before sexual maturity is reached. In this regard, Martin (1932) observed that, although not apparent in the actual germinal ridge, the ovary in Argulus viridis displays a bilobed appearance. This is not apparent in C. victori or C. australis. According to Anderson (1982) the primitive condition in crustaceans is represented by paired gonads and gonoducts originating from a pair of germ cells which differentiates in the ventral wall of each pair of segmental coelomic cavities. These cells later proliferate to form segmental groups of cells which link up as paired strands and become connected to coelomoduct gonoducts. Although the precocious development of a single primordial germ cell in the wall of the blastula has been observed in several small-egged crustaceans, Anderson (1982) considers all deviations from the paired primitive condition as the probable result of specializations. Considering the proposed phylogenetic relationship between the Branchiura, Copepoda and Cirripedia (Schram,1986; Barnes,1987; Hickman et a/.,1988), it is noteworthy that in adult females of both latter groups the oviducts are always paired whilst the ovaries are paired (parasitic forms and primitive types) or single (free-living forms) in the Copepoda, while paired and sometimes united anteriorly in the Cirripedia (Schram,1986). In the Pentastomida, which appears to have originated from the Branchiura (Wingstrand,1972; Riley et a/.,1978; Abele et a/.,1989), a single or unpaired genital tract is found in adult females (Riley,1983, 1986). However, according to Self (1990) there is a complete disappearance of the embrionic characters in the transition to the primary larva, implicating a complete metamorphosis between these stages. All considered, the manifestation of an unpaired ovary but paired oviducts in some Branchiura remains an enigma which could only be clarified after a thorough ontogenetic study of the female gonads has been carried out.
132 Concerning the histomorphology of the different components of the female reproductive system in Chonopeltis, the detailed histomorphological investigation of the said system in C. victori and C. australis reveals the single ovary to be an ovoid, tubular organ, enclosed by a peritoneal sheath or "gonadal sac" which separates the circumgenital body cavity from the haemocoel. The ovary contains an epithelial-lined lumen, the ovarian sac, which is anteriorly continous with the functional oviduct. Oogonia apparently leave the germinative zone, (i.e. a longitudinal ridge of mesodermal gonadal tissue and oogonia extending along the entire length of the ovary), to enter the space between the gonadal and ovarian sacs where oocyte maturation and vitellogenesis occur. Contrary to the condition found in most malacostracans (Talbot,1981; Meusy & Payen,1988; Krol et a/.,1992; Hryniewiecka-Szyfter & Tyczewska, 1992), branchiopods (Martin,1992) and copepods (Boxshal1,1992), no follicle cells were observed around previtellogenic oocytes at the onset of vitellogenesis or during vitellogenesis in both Chonopeltis species examined. Follicle cells, variously called "follicullar cells", "nurse cells", "accessory cells", "supporting cells", or "mesodermal stroma" (Adiyodi & Anilkumar, 1988; Krol et a/.,1992), are the only non-germinative cells within the walls of the crustacean ovary and apparently arise from the germinal epithelium (King,1948). According to Adiyodi & Subramoniam (1983) and Krol et a/.(1992), follicle cells may play a role in vitellogenesis (i.e. the synthesis of yolk) in at least some crustaceans, though not in the formation of the egg envelopes. However, Papathanassiou & King (1984) suggested that follicle cells may play a role in the development of the chorion, whilst Talbot (1981) showed that follicle cells do indeed produce components of the chorion in certain crustaceans. Returning to vitellogenesis, Adiyodi & Subramoniam (1983) state that folliculogenesis, the process by which follicle cells come to surround early vitellogenic oocytes, is a prerequisite for the uptake of yolk protein from outside the oocyte. Yano & Chinzei (1987) further found that in some crustaceans, vitellogenin may be synthesized in follicle cells, secreted into the haemolymph, and taken up by the oocytes. Furthermore, a network of intercellular spaces between follicle cells may facilitate passage of extracellular protein to the oocytes (Adiyodi & Subramoniam,1983). In malacostracans the follicular layer plays an important role during vitellogenesis by creating a route for exchange between the haemolymph and vitellogenic oocytes (Jugan & Zerbib,1984; Komm & Hinsch,1987). In C. victori and C. australis vitellogenic oocytes are mostly found along the periphery of the gonadal sac, occasionally bulging into the haemocoel to the extent
133 that more than half of an oocyte's surface is covered by gonadal sac epithelium. It thus seems very likely that the gonadal sac epithelium in both Chonopeltis species functions in facilitating the passage of vitellogenic substances from the haemocoel to the oocyte.
The next step in the ovarian cycle, following vitellogenesis, is ovulation or ovarian spawning (Meusy & Payen,1988). Ovulation in some decapods is preceeded either by a separation of the follicle envelope from the oocyte and a retraction to the periphery of the ovary (Fauve1,1983), or, the follicle cells just disappear from around the oocytes (Yano,1988). In cases where the follicle tissue remains after ovulation it may be used for new folliculogenesis (Meusy & Payen, 1988; Krol et a/.,1992). In C. victori and C. australis however, postvitellogenic oocytes face the basal membrane of the epithelium lining the ovarian lumen, prior to ovulation. Oocytes next appear to move to the ovarian lumen and become completely wrapped in lumenal epithelium. Therefore, after the oocyte has been ovulated and residing in the ovarian lumen, yolk spheres fuse together to form large yolk platelets and a thick (25,um) tertiary envelope (shell) is formed around the oocyte. Shelled oocytes move from the ovarian lumen to the anteriorly situated oviducts. It therefore appears that the functional oviduct in Chonopeltis spp. functions as an ovisac, storing eggs prior to fertilization and oviposition, rather than a true oviduct which usually is responsible for the formation of tertiary membranes around eggs (cf. Hinsch,1990; Krol et al. ,1992; Martin,1992).
Two oviducts have been described in various species of Argulus and Dolops. In all these cases, only one of the two oviducts is functional with its lumen in direct communication with that of the genital atrium, whilst the blind-ending lumen of the non-functioning oviduct is not obliterated, though it has a deflated or collapsed appearance. Since branchiurans, including C. victori and C. australis (own observations), have more than one breeding period a year (Martin,1932; Hirschmann & Partsch,1953; Fryer,1964; Avenant & Van As,1986; Shafir & Van As,1985,1986; Avenant et a/.,1989; Overstreet et a/.,1992; Shafir & Oldewage,1992), it is probable that the two oviducts may function alternately to allow sequential restoration of the cellular material in the epithelial lining of the oviducts - a supposition also proposed by Martin (1932) in Argulus viridis and Avenant-Oldewage & Van As (1990) in Dolops ranarum. However, in contrast to the descriptions given in both latter species the posterior end of the
134 non-functioning oviduct in C. victori and C. australis, as in Dolops longicauda (Heller,1857) (Maid1,1912, p.20), is not connected with the genital atrium - a feature, though contradictorily described, also indicated in the graphic reconstruction of the female reproductive system in D. ranarum (Avenant-Oldewage & Van As,1990, Fig. 1). This suggests that the non- functioning oviduct in Chonopeltis, and probably also Dolops, more likely represents a rudimentary or vestigial structure. In correspondence, Claus (1875, p.232,274) considers the two oviducts in Argulus to be derived from symmetrically situated paired anlagen in the larva whilst regarding the non-functioning oviduct as a "rudimentare" or "verkiimmerten" (i.e. rudimentary or withering) oviduct. In agreement with Claus, Grobben (1908, p.203) stated that the non-functioning oviduct never functions and that its lumen entirely disappears. Concerning the functional significance of the functioning oviduct, Martin (1932, p.790) stated that the second, much thicker egg coat or outer shell is secreted by the walls of the functioning oviduct in A. viridis. In C. victori and C. australis, as in various other crustaceans (Clarke,1973; Hinsch,1990), both egg membranes and outer tertiary envelope (shell) are, however, formed while the eggs are still within the ovary, suggesting that the oocyte itself produces the egg membranes as the egg undergoes growth and maturation whilst the ovarian luminal epithelium is probably responsible for the formation of the tertiary envelope (shell) around the vitelline membrane.
During oviposition in branchiurans, the eggs are coated as they are laid with a gelatinous substance which on hardening cements the eggs firmly to one another and also to the substratum (Claus,1875; Wilson,1902; Grobben,1908; Martin,1932; Tokioka,1936; Bower- Shore,1940; Meehean,1940; Hindle,1949; Fryer,1956,1959,1961a,1968; Shafir & Van As, 1986; Avenant et a/.,1989; Overstreet et a1.,1992). According to Claus (1875), the superficial portion of the egg shell is changed to this gelatinous substance by penetration of the water whilst Grobben (1908) considers it to be a secretion from the functioning oviduct. Although the oviducal secretion in various other crustaceans does not play a role in the adhesion of eggs and may have at best a lubricating effect on eggs during oviposition (Adiyodi & Anilkumar, 1988), the possibility that the functioning oviduct in branchiurans may also be responsible for the secretion of the gelatinous coat surrounding the eggs during oviposition cannot be excluded until evidence to the contrary is given, especially since in various oviparous animal groups
135 secondary protective egg coats such as jelly layers are indeed added as the egg passes through the oviduct (Saunders,1982). However, as early as 1904, Williamson suggested that female crustaceans possess special tegumental glands, the "cement glands", which produce a glue to attach the eggs (cf. Adiyodi & Anilkumar,1988). According to Adiyodi & Anilkumar (1988) female cement glands are known only from macruran decapods and stomatopods, where they have taken on the functions of oviducal secretions in other Malacostraca. The cement glands are in some decapod species situated in the uropods and telson, whereas in other species they are distributed in pleopods, swimmerites and lower regions of the abdomen. The cement glands are said to be homologous with the common tegumental glands, and the tegumental glands present in the swimmerites and lower abdominal regions of macrurans have apparently been suspected for a long time to have a role in cement production (e.g. Herrick,1909). Furthermore, Cheung (1966) suggests the possibility that the secretion of the cement glands in crustaceans may also be involved in the hardening of the egg membrane, formation of the outer egg coat and serves to attach the eggs. Johnson & Talbot (1987) found that the cement glands contain two types of secretory cells, each with its own product, which suggests that these glands have more than one function. Consequently, it seems very likely that one or more of the three groups of tegumental glands found in the vicinity of the fertilization chamber (i.e. anterior part of abdomen, medial part of natatory lobes, between hindgut and ventral abdominal wall) in C. victori and C. australis, are in fact "cement glands" responsible for secreting the adhesive which firmly attach eggs to one another and to the substrate. Nevertheless, only further histochemical investigation would ascertain which group (or groups) of these gland cells are responsible for secreting the adhesive.
Previous investigations on sperm transfer in branchiurans (Jurine,1806; Leydig,1850,1889; Claus ,1875 ; Grobben,1908; Maidl, 1912 ; Martin, 1932; Debaisieux , 1953 ; Fryer, 1958, 1960b ; Avenant-Oldewage & Swanepoe1,1993) mainly concentrated on the transfer of sperm from the male genital opening to the female spermathecae - mostly referring only to the nature and function of the involved male reproductive structures. Concerning the subsequent insemination of mature eggs during oviposition, only a few general assumptions have been made (Fryer, 1960b; Shafir & Van As,1986; Avenant et al. ,1989). In female branchiurans, the essential structures involved in both mentioned cases of sperm transfer are the paired spermathecae.
136 Considering the transfer of sperm from the male genital opening to the female spermathecae, Fryer (1960b) showed that in Dolops this process involves the formation of a spermatophore which during copulation is pressed against the ventral surface of the female's abdomen, pierced by the perforated spermathecal spines and hence sperm is transferred to the spermathecal vesicles of the female (though the actual injection has never been observed). Spermatophores are however not produced in either Chonopeltis nor Argulus (Martin,1932; Debaisieux,1953; Fryer,1960b,1968), implying a different mechanism of sperm transfer in these two genera. In Argulus, earlier studies initially implicated the modified structures on the third and fourth pair of thoracopods of the male as the sperm transferring apparatus (Leydig,1850; Claus,1875; Grobben,1908; Wilson,1902). However, Martin (1932) indicated that these structures, termed the "peg-and-socket" by her, are not involved during sperm transfer but are merely secondary sexual structures used as clasping mechanisms during copulation, whilst sperm transfer is solely accomplished by direct contact of the "male genital opening and ejaculatory duct" with the opening of the spermathecae on the abdomen of the female. Although authors such as Meehean (1940); Debaisieux (1953); Fryer (1968) and Sutherland & Wittrock (1986) agree with Martin (1932), none, except Avenant-Oldewage & Swanepoel (1993), could provide an explanation of exactly how sperm is injected into the spermathecae without streaming past the minute opening in each spermathecal spine. Avenant-Oldewage & Swanepoel (1993) suggested that during copulation in Argulus japonicus Thiele,1900 the two spermathecal spines of the female are inserted into the male genital atrium, penetrating the walls of the respective ejaculatory ducts, whereafter semen is actively pumped into the spermathecal vesicles. In C. victori (own observation), the slit-like transversely orientated genital opening of the male leads directly into the cavernous genital atrium which in turn neighbours the two opposing ejaculatory ducts. The latter do not open into the genital atrium but are blind-ending tubes with no direct communication between its lumina and that of the genital atrium. However, in one male specimen examined, one of the ejaculatory ducts showed a distinct connection between its lumen and that of the genital atrium with the epithelial layer separating the said two lumina, torn or pierced open. In another specimen the said epithelial boundery between both ejaculatory ducts and the genital atrium were found to be pierced open. These observations in male C. victori undoubtedly furnish evidence to a mechanism of sperm transfer very similar to that suggested by Avenant-Oldewage & Swanepoel (1993) - especially when taking the
137 specific morphology of the female spermathecae also into consideration as well as the fact that in some female C. victori specimens, semen was found not to occur in both spermathecal vesicles simultaneously. Furthermore, a marked compatibility has been observed between the distance between the two opposing spermathecal spines of the female and the width of the slit- like genital opening of the male in C. victori specimens examined. It is thus proposed that during copulation in C. victori, the two sharp, ejectable and also very flexible spermathecal spines of the female are inserted into the male genital atrium and then, simultaneously or singly, pushed through the thin epithelial layer separating the lumina of the genital atrium and the two opposing ejaculatory ducts. In accordance with the mechanism described by Avenant- Oldewage & Swanepoel (1993), semen would then, due to the higher pressure inside the ejaculatory duct and on contraction of the muscular walls of the ejaculatory duct, flow from the ejaculatory duct down the apical groove and through the opening in the spermathecal spine and then, via the spermathecal duct, into the spermathecal vesicle.
Although the actual injection of sperm into mature eggs during oviposition has not been directly observed (though deduced from anatomy and observations on egg laying), it is generally assumed that in branchiurans the spermathecal spines penetrate and inseminate each egg on laying by the injection of semen stored in the spermathecal vesicles (Clause,1875; Wilson,1902; Fryer,1960b; Avenant et al. ,1989; Avenant-Oldewage & Van As,1990). There seems to be no reason to doubt this since during oviposition in C. victori (own observation) as well as in Argulus (Clause,1875; Wilson,1902; Shafir & Van As,1986) and Dolops (Fryer, 1960b; Avenant-Oldewage & Van As,1990), each egg must pass that part of the female abdomen bearing the spermathecal spines - thus forcibly comes in contact with the said spines. Furthermore, since the spermathecae in branchiurans are entirely isolated from the main genital tract of the female, internal fertilization as described in other spermathecae-possessing crustaceans (Hinsch,1990),is anatomically not possible in branchiurans. In branchiurans there are also no other means whereby sperm could enter the egg which possesses a very thick shell and lacks a micropyle. Inevitable polyspermy during the process is most probably dealt with as in other animal groups where several sperm may enter a single egg but only one contributes the paternal genome, whilst the others disintegrate when the first sperm reaches the female pronucleus (Saunders,1982, p.114).
138 A structure present in C. victori and C. australis, but not yet described in other branchiurans, is the fertilization chamber. An egg entering the fertilization chamber is probably manoeuvred by a short conical papil projecting from its mid-dorsal wall (cf. triangular swollen area described in C. victori). The so-called natatory lobes, which form the floor of the posterior part of the fertilization chamber, probably also play a role in manoeuvring the egg during oviposition as has been observed in Argulus japonicus Thiele,1900 by Shafir & Van As (1986). This, however, appears not to be the case in Dolops ranarum (Stuhlmann,1891) where the natatory lobes are pulled away when eggs are deposited (Avenant et cd.,1989). The natatory or swimming lobes appear as posteromedial outgrowths of the coxae of the fourth thoracopods (natatory legs) and their shape and size vary among species (Wilson,1902; Brian,1940; Fryer,1960b). Morphologically the natatory lobes agree with the oostergites as described for the amphipod Orchestia gammarella (Pallas,1893) by Charniaux-Cotton (1975) and the Isopod Ligia oceanica Linnaeus,1758 by Besse et al. (1969). In branchiurans, however, there is only one pair of natatory lobes (oostegites) whereas in most amphipods there usually are four pairs contributing to the formation of the brood pouch or marsupium (Meusy & Payen,1988).
In conclusion, the problem concerning the presence of a single median ovary but two oviducts in some Branchiura remains unsolved, but a thorough ontogenetic study may provide an answer. The so-called ovarian sac directly connects the ovary and oviducts in Chonopeltis. Since no spermatophores are produced in Chonopeltis, as in Argulus, the mechanism of sperm transfer involves a process during which the two spermathecal spines of the female are inserted into the male genital atrium, simultaneously or singly penetrating the walls of the respective ejaculatory ducts whereafter semen is actively pumped into the spermathecal vesicles. Although substantiating ultrastructural studies need to be done on various aspects of gametogenesis and fertilization, insemination of mature eggs seems to be accomplished by means of the spermathecal spines which penetrate and inject stored semen into each egg during deposition. During oviposition, shelled eggs entering the fertilization chamber are probably manoeuvred by the natatory lobes and positioned underneath the spermathecal spines so that sperm can be injected into the eggs.
139 FIGURE 7.1
Ventral view of adult female Chonopeltis victori Avenant-Oldewage,1991, indicating the relative position of the components of the reproductive system ( - - - ).
Abbreviations: ab - Abdomen. cs - Cephalic shield.
fa - Frontal cephalic ala. func - Functional oviduct. nix - Maxilla. na - Natatory lobe. ov - Ovarium. pr - Proboscis. s - Sucker (=Maxillula). sv - Spermathecal vesicle. tp(1-4) - Thoracopod (1 to 4). tx - Thorax.
140 g,og zwooll_57 FIGURE 7.2
Chonopeltis victori Avenant-Oldewage,1991. Graphic reconstruction of the female reproductive organs. Ventral view.
Abbreviations: ac - Accessory duct. ao - Anterior part of oviduct. fc - Fertilization chamber. fo - Functioning oviduct. ga - Genital atrium. go - Genital opening. no - Non-functioning oviduct. os - Outline of ovarian lumen (ovarian sac). ov - Ovary. sd - Spermathecal duct. sp - Spermathecal spine. sv - Spermathecal vesicle.
141 F11002Z D.2
MO FIGURE 7.3
Chonopeltis australis Boxshal1,1976. Graphic reconstruction of the female reproductive organs. Ventral view.
Abbreviations:
ac - Accessory duct. ao - Anterior part of oviduct. ee - Encapsulated eggs in ovarian lumen. fc - Fertilization chamber. fo - Functioning oviduct.
ga - Genital atrium.
no - Non-functioning oviduct. os - Outline of ovarian lumen (ovarian sac). ov - Ovary.
rp - Rostral prolongations. sd - Spermathecal duct.
sp - Spermathecal spine. sv - Spermathecal vesicle.
142 FilCOME F.0 FIGURE 7.4
Chonopeltis australis Boxshal1,1976. Semi-diagrammatic drawing of a transverse section through the anterior part of the reproductive system of a young female, showing the ovisac and previtellogenic ovary.
Abbreviations: cb - Circumgenital body cavity containing ovary. cu - Cuticle of body wall. da - Dorsal aorta. el - Epithelial wall of midgut. func - Lumen of functional oviduct. germ - Germinal ridge. go - Wall of gonocoel (=gonadal sac). ha - Haemocoel. mg - Midgut. mu - Circular muscle layer of midgut wall. oc - Young oocyte. ov - Ovarian sac. po - Previtellogenic primary oocytes.
143 FllOWJOZ 27.6
100A.ourn FIGURE 7.5
Chonopeltis australis Boxshal1,1976. Semi-diagrammatic illustration of transverse section through the reproductive system of a mature female showing functional oviduct, ovarian sac and vitellogenic ovary.
Abbreviations: eh - Circumgenital body cavity. ec - Egg shell. ee - Encapsulated egg in lumen of ovarian sac. eo - Egg entering ovarian sac. ev - Early vitellogenic oocyte. func - Lumen of functional oviduct. germ - Germinal ridge. go - Wall of gonadal sac. ha Haemocoel. lv - Late vitellogenic oocyte entering ovarian sac. mg - Midgut. oc - Young oocyte. os - Lumen of ovarian sac. pv - Previtellogenic oocyte. vm - Vitelline membrane. wfunc - Wall of functional oviduct.
144 bsTOME Da
20012m FIGURE 7.6
Chonopeltis victori Avenant-Oldewage,1991. Semi-diagrammatic illustrations of consecutive frontal sections (anterior to posterior) of the female reproductive system showing (A) the ovisac and (B) the anterior part of the ovarium and oviducts.
Abbreviations.
am - Anterior midgut. ao - Anterior part of functional oviduct. dev - Developing ova. func - Lumen of functional oviduct. go - Wall of gonocoel (gonadal sac). nonf - Non-functional oviduct. os - Ovarian sac. ov - Ovarium. pro - Proventriculus in anterior midgut.
145 FiligUJOE YE, 0 FIGURE 7.7
Chonopeltis victori Avenant-Oldewage,1991. A - D : Semi-diagrammatic illustrations of consecutive transverse sections of the . posterior part of the female reproductive system, in the region of the genital atrium. The top of each figure represents the dorsal surface and sections sequenced posterior to anterior.
Abbreviations: cu - Cuticle. dv - Dorsal blood vessel. dev - Developing ovum. el - Epithelial layer. func - Functional oviduct. ga - Genital atrium. germ - Germinal ridge. he - Heart. hg - Hindgut. mat - Mature ovum. os - Ovarian sac. ov - Ovarium. pm - Posterior midgut. sm - Smooth muscle layer.
146 Fil@EVON DoD
cu el
ov
hg
func
el
cu
dev sm
C u
hg mat
func sm
900 um FIGURE 7.8
Semi-diagrammatic drawings of transverse sections through the anterior part of the abdomen of (A) C. victori and (B) C. australis, illustrating the fertilization chamber. The top of each figure represents the dorsal surface.
Abbreviations: con - Cone/triangular shape projection. cu - Cuticle. el - Epithelium. fc - Fertilization chamber. gs - Gland cells. hg - Hindgut. mu - Muscle mus - Muscular papilla of spermathecal spine. na - Natatory lobe. sp - Spermathecal spine.
147 Fil@COOff F.0
0001) FIGURE 7.9
Chonopeltis victori Avenant-Oldewage,1991. Semi-diagrammatic drawings of consecutive frontal sections through the fourth thoracic segment and the anterior part of the abdomen. Sections sequenced dorsal to ventral.
Abbreviations: ac - Accessory duct. con - Cone/triangular shape projection. cu - Cuticle. el - Epithelium. el (fc) - Stratified squamous epitelium of fertilization chamber. fc - Lumen of fertilization chamber. fo - Lumen of functional oviduct. gs - Gland cells. hg - Hindgut. mu - Muscle mus - Muscular papilla of spermathecal spine. na - Natatory lobe. sd - Spermathecal duct. sm - Smooth muscle layer. sp - Spermathecal spine. sv - Spermathecal vesicle. sz - Stored spermatozoa
148 Fil@UOE D. 0
el mus sd mu el ac sv el
sz FIGURE 7.10
Scanning electron micrographs of female Chonopeltis victori Avenant-Oldewage,1991. Ventral view.
Spermathecal spines on anteroventral part of the abdomen (Scale bar:10/um);
Spermathecal spine and slit-like opening (>) (Scale bar:4,um);
Sub-apical opening (>) of spermathecal spine (Scale bar:3Am);
Natatory lobes (Scale,bar:10Am).
Abbreviations: ab - Ventral surface of abdomen.
con - Cone/triangular shape projection. g - Longitudinal groove. mus - Muscular papilla of spermathecal spine.
na - Natatory lobe. sc - Denticulated scale-like structures. sp - Spermathecal spine.
149 FIGURE 7.1 " Sometimes when I consider what tremendous consequences come from little things ... I am tempted to think ... there are no little things. "
- Bruce Barton I-IAPTER
ASPECTS OF THE ECOLOGY OF Co VICTO CONCE G THE PRODUCTIVE CYCL -149 LIFE CYCLE EPIDEMIOLOGY
1
801 INTRODUCTION
In their efforts to pursue the parasitic way of life successfully, parasites - including species of the piscine ectoparasitic genus Chonopeltis Thiele,1900 - have adopted, among others, various distinct morphological adaptations. The most important of these adaptations are indubitably concerned with optimal reproduction. However, in order to gain a better understanding and insight into - not only - the specific mode of reproduction, but also the reproductive cycle, life cycle, epidemiology and pathobiology of this unique group of animals, detailed knowledge of all aspects of their biology, but especialy their morphology, are a prerequisite. In this regard, Schram (1986) stated that " by not having a 'name for it' we lose the power to 'understand it' ". Consequently, as mentioned in the introduction to this thesis (Chapter 1), the aim of the present study was primarily to investigate aspects of the morphology of the branchiuran ectoparasite Chonopeltis Thiele,1900 with special reference to the anatomy and histomorphology of the reproductive systems. However, since the reproductive cycle, sex ratio and growth patterns in branchiurans appear to be influenced by annual physical environmental conditions (cf. Bower-Shore,1940; Paperna,1980; Avenant & van As,1986; Shafir & van As,1986; Oldewage & van As,1987; Poulin & FitzGerald,1989; Shafir & Oldewage,1992) the inclusion of relevant ecological information is inevitable.
150 With the exception of articles published by Fryer (1956,1959,1960b,1961b,1965,1968,1970, 1974,1977,1986) as well as the works published by Van Niekerk (1984) and Knight (1991) on aspects of the ecology of Chonopeltis, little is known about the specific reproductive cycle, annual fluctuation in the sex ratio as well as about specific ecological relations such as prevalence, mean intensity and abundance of infestation in this genus. Some detailed ecological information on the other representatives of the Branchiura in Africa, i.e. Argulus (Brian,1940; Fryer & Talling,1986; Kruger et a/.,1983; Cesare,1986; Shafir & van As,1986; Ogutu- Ohwayo,1989; Shafir & Oldewage,1992) and Dolops ranarum (Fryer,1968; Mbahinzireki, 1980; Avenant & van As,1985,1986; Ogutu-Ohwayo,1989), is however available.
Ecological information on C. victori Avenant-Oldewage,1991 is thus provided in this study for the first time. Ecological information used to determine fluctuation patterns in population structure and distribution as well as the infestation statistics of C. victori on its fish hosts in the Olifants River, Kruger National Park (KNP), and lower Selati River (a tributary of the Olifants River just outside the park's western boundary), were obtained and recorded as described in Chapter 2 (p. 10-15). In analysing these data, the terminology as recommended by Margolis et a/.(1982) for "parasitological ecologists" was used. The data on selected physical and chemical water quality variables of the aquatic habitat in the study area for the period February 1990 to January 1992, were provided by the Reseach Section for Aquatic Toxocology at the Department of Zoology, Rand Afrikaans University. These data were obtained and analysed as described by Seymore et a/.(1994).
8.2 RESULTS
8.2.1 Population dynamics a d reproductive cycle
Since C. victori specimens were never found on any fish collected from the sample locality in the lower Selati River (localitiy 1, Fig. 8.1B), the following ecological information is based on the data obtained from the three sample localities in the Olifants River, KNP (localities 2-4, Fig. 8.1B).
1151 During the present study the total number of C. victori specimens sampled during each bimonthly survey from February 1990 to January 1992, was generally small with the highest number of specimens collected during a single survey 96 and 33, respectively sampled during October 1990 and October 1991 (Table 8.1, Fig. 8.3). Individuals of C. victori showed a progressive increase in total length of both males and females from April/May to January/February during both years (Table 8.2). The smallest male and female specimens were sampled during April/May and measured 2,113mm and 2,263mm in total length respectively, whilst the largest male and female specimens were sampled during January/February and measured 6,021mm and 9,952mm respectively. Gravid females extending from 3,0mm to 9,9mm were present throughout both years. Throughout the two year survey period no newly hatched larvae or early larval stages were observed. However, on one occasion a single, very small immature specimen which could represent the final larval stage in C. victori, was found together with several larger, mature specimens on a fish host. During the extent of the study period (February 1990 to January 1992) females constantly outnumbered males, often by more than 2 : 1 (Table 8.1, Fig. 8.2). The highest ratio's of females to males were recorded during June and October in 1990, and June and August in 1991 (Fig. 8.2), which coincided favourable with the two peaks of gravid females (i.e. % gravid females of the female population sampled during each bimonthly survey), respectively reached during June and October 1990 and June and August 1991 (Table 8.1; Fig. 8.3). During both years, a decrease in the percentage of gravid females coincided with a decrease in the ratio of females to males (Table 8.1; Fig. 8.2).
Although gravid females were present throughout the year during both 1990 and 1991, distinct fluctuations in their proportion of the female population were observed (Table 8.1; Fig. 8.3). The peaks apparent in October 1990 and August 1991 (Fig. 8.3) appear to be directly linked to the marked population increase wittnessed during the late winter and spring months (August to October) in 1990 and the early winter to spring months (June to October) in 1991 (Fig. 8.3). The decline in the percentage gravid (=ovigerous) females began respectively in October during 1990 and August/October during 1991 (corresponding maximum peaks), when the population as a whole apparently reached maximum proportions, and reached its lowest point during February to April (Fig. 8.3). This trend reverses itself thereafter, ascending towards
152 another peak during early winter (Table 8.1; Fig. 8.3). During 1990 the overall population numbers, and consequently also the percentage gravid females, declined during mid-winter, reaching a minimum point during August 1990 (Table 8.1; Fig. 8.3). This trend was not observed during 1991. Furthermore, when comparing the overall fluctuation in population numbers during 1990 and 1991, a more steap and higher population peak was reached during October 1990 in contrast to the much lower, but more progressive maximum peak reached between April and October 1991 (Fig. 8.3). These differences apparent between 1990 and 1991 in the fluctuation in overall population numbers as well as in the proportion of gravid females of the female population, can be attributed to the marked differences in the climatic conditions influencing the physical and chemical properties of the aquatic habitat during the two years respectively.
In contrast to 1990 during which 'normal' climatic conditions prevailed (except for the flood during December when heavy rainfall occurred and the entire length of the Olifants River and its tributaries flowing through the KNP was flooded), 1991/1992 experienced a severe drought and a significant increase in temperatures between April 1991 and February 1992 (cf. Seymore et a1.,1994; Zambatis & Biggs,1995). According to Zambatis & Biggs (1995) the primary reasons for the severity of the drought were a very significant decline in the number of days on which rain occurred (decreasing by almost 50%), and an overall increase in temperatures. Two-thirds of the year experienced maximum atmospheric temperatures of a significantly higher intensity than normal. This, together with slightly increased minimum temperatures and a significant increase in the number of days with maximum temperatures higher than 30 °C, resulted in a hotter than normal year (Zambatis & Biggs,1995). This increase in temperature was also noted when comparing the water temperatures measured in the study area between February 1990 and January 1992 (Table 8.8 & 8.9). Afternoon water temperatures recorded during wintertime were on average 19,2 +1,4 °C for 1990 and 20,7 ±2,2°C for 1991, while afternoon average water temperatures during spring and summer were 26,7 ±2,3 °C for 1990 and 30,6 +1,5 °C for 1991 (Seymore et a/.,1994). This overall higher water temperatures during 1991 in contrast to 1990 can be attributed not only to the increase in average atmospheric temperatures, but also to the lower level and flow of the water in the river during the drought period (cf. Seymore et a1.,1994).
1153 The foregoing results, although not conclusive, provide a basis for speculation on the seasonal cycle and reproductive pattern (cf. Fig. 8.3) of the natural population of C. victori in the Olifants River, KNP (see Discussion).
8.2.2 Infestation dynamics and host preferences
During the present investigation more than 2 800 fishes comprising various fish species were captured during bimonthly surveys between February 1990 and January 1992 at three different localities in the Olifants River (KNP), and at one locality in the lower Selati River (a tributary of the Olifants River just outside the park's western boundary) (Fig. 8.1). Of these fish species collected, only Labeo rosae Steindachner,1894; Labeo congoro Peters,1852; Labeo ruddi Boulenger,1907; and Barbus marequensis A. Smith,1841 were found to be infested with Chonopeltis victori Avenant-Oldewage,1991. However, although a few fish host specimens were occasionally also captured at the sample locality in the lower Selati River (locality 1; Fig. 8.1B), C. victori specimens were never found on any fish host collected from this river.
Infestation statistics of C. victori on the four fish host species collected from the three sample localities in the Olifants River (Fig. 8.1B) are summerized and compared in Tables 8.3-8.7 (Figs. 8.4-8.9). Statistics are based on the data obtained from the examination of 329 specimens of L. rosae, 137 of L. congoro, 28 of L. ruddi, and 232 of B. marequensis. In analysing the data, the following terminology as recommended by Margolis et al. (1982, p.131) for "parasitological ecologists" was used:
Prevalence: Number of individuals of a host species infected with a particular parasite species divided by the number of hosts examined (expressed as a percentage); Mean intensity: Total number of individuals of a particular parasite species in a sample of a host species divided by the number infected individuals of the host species in the sample; Abundance: Total number of individuals of a particular parasite species in a sample of a host species divided by the total number individuals of the host species in the
154 sample; Locality: The geographic place of capture or collection of the host; Site: The tissue, organ, or part of the host in/on which a particular parasite was found.
In addition to the above mentioned terminology provided by Margolis et a/.(1982), the following terms were also used in the present study:
Site prevalence: Number of individuals of a specific host species infected with a particular parasite species on a specific site divided by the total number of infected individuals of the same host species (expressed as a percentage); Mean site intensity: Total number of individuals of a particular parasite species on a specific site on a particular host species divided by the number of individuals of the same host species infected with the parasite on the specific site; Site abundance: Total number of individuals of a particular parasite species on a specific site on a particular host species divided by the total number of infected individuals of the same host species.
In order to avoid any misunderstanding, the term infestation - defined as the "invasion by ectoparasites" (Lawrence,1989) - is used in the present study in terms of prevalence, mean intensity and abundance of the ectoparasite on its host(s).
Infestation statistics of C. victori on the four fish host species collected from the three sample localities in the Olifants River (i.e. localities 2, 3 & 4; Fig. 8.1B) indicate that, prevalence, mean intensity and abundance of C. victori were constantly higher on the host species collected at sample localities 3 and 4 than on those collected from sample locality 2 (Table 8.3; Figs. 8.4A,B,C; 8.5A,B,C & 8.6A,B,C). Maximum infestation (i.e. in terms of prevalence, mean intensity and abundance) were recorded during June and October 1990 and June and August 1991 at both sample localities 3 and 4 with the prevalence, mean intensity and abundance of infestation on the four host species generally higher at locality 4 than at locality 3 (Table 8.3). Prevalence of C. victori on L. congoro sampled at locality 4 (Table 8.3; Fig.
155 8.4C) even reached 100% in June and October 1990 as well as in June and August 1991, while 100% prevalence (recorded on L. congoro) was only reached in October 1990 at sample locality 3 (Table 8.3; Fig. 8.4B). Similarly, during June and October 1990 and June and August 1991 the highest mean intensity and abundance of infestation were recorded at sample locality 4 - also on L. congoro (Table 8.3; Figs. 8.5C & 8.6C).
When comparing the infestation and population dynamics of the natural population of C. victori in the Olifants River (KNP) during the study period (February 1990 - January 1992), an increase in C. victori infestation on the fish hosts species (Table 8.3 & 8.4; Figs. 8.4-8.6) together with an increase in population numbers of C. victori (Table 8.1; Fig. 8.3) were recorded during the winter and spring (June - October/November) in 1990, and late autumn and winter (April/May - October) in 1991. Maximum prevalence, mean intensity and abundance of infestation were reached during October (mid-spring) in 1990, and June to August (early to mid-winter) in 1991 (Figs. 8.4D,8.5D & 8.6D). However, in both years population numbers in C. victori reached a maximum during mid-spring (October) (Table 8.1; Fig. 8.3). Infestation (i.e. prevalence, mean intensity and abundance) on the four fish hosts (Table 8.3 & 8.4; Figs. 8.4-8.6) as well as population numbers in C. victori (Fig. 8.3), declined towards early summer (November/December) during 1990 and early spring (October/November) during 1991. Prevalence, mean intensity and abundance of infestation on the four host species (Figs. 8.4-8.6), maintained basically a similar pattern, but were constantly higher on L. congoro than on any of the other three host species for both 1990 and 1991 (Tables 8.3,8.4 & 8.5; Figs. 8.4-8.7). The prevalence of C. victori on L. congoro even reached 100% in October 1990 and in June 1991 (Table 8.4; Fig. 8.4D). During these two months the highest mean intensity of infestation was also recorded on L. congoro (Tables 8.3 & 8.4; Fig. 8.5D). Similarly, abundance of infestation on L. congoro reached an overall maximum of 4.56 in October 1990 and 2.86 in August 1991 (Table 8.4; Fig. 8.6D). The maximum number of C. victori specimens collected from a single host specimen was 9 (found on a L. congoro specimen), with a maximum mean intensity of 5 recorded in June 1991 - also on L. congoro (Table 8.4).
From the results obtained it thus seems that L. congoro may be the preferred host of C. victori
156 in the Olifants River, KNP. Nevertheless, during summer (December - April) when infestation was relative low, the part of this host in carrying the infestation dropped markedly relative to the other three host species - especially L. rosae (Table 8.3 & 8.4; Figs. 8.4-8.6). The role of L. congoro is however further emphasised when prevalence and abundance of C. victori on all four fish host species are compared (Table 8.5; Fig. 8.7). Despite its smaller numbers in comparison to L. rosae and B. marequensis (Table 8.5), L. congoro was almost the sole carrier of the high-level late autumn/early winter (June) and spring (October) infestations during 1990 as well as during the high-level winter (June - August) infestation during 1991 (Tables 8.3 & 8.4; Figs. 8.4-8.6). Furthermore, the results presented in Table 8.4 and Table 8.5 (Fig. 8.7) reveal that although more individual host specimens were collected during 1991/1992 (higher concentration of fish due to the drought, lower water level and flow) than during 1990, infestation in terms of prevalence, mean intensity and abundance of C. victori on its four fish host species, were higher during 1990 than during 1991/1992 (Table 8.5; Fig. 8.7). During both 1990 and 1991, a decrease in water temperature corresponded with an increase in the population numbers of C. victori (Table 8.9; Fig. 8.3) as well as the infestation on its hosts (Figs. 8.4-8.6), and vice versa. Furthermore, during the second year (1991) when higher average water temperatures prevailed (cf. Seymore et a1.,1994), the maximum peak in population numbers (Fig. 8.3) as well as in infestation of C. victori (Table 8.5; Fig. 8.7) were also noticably lower than during the first year (1990) when the average water temperatures were lower. However, it seems as though not only the temperature, but also the specific time of the year and the physical and chemical properties of the aquatic habitat, have a profound influence on the population numbers in C. victori and, thus, also on the prevalence, mean intensity and abundance of infestation on the four fish host species.
When comparing the selected water quality variables presented in Tables 8.8 & 8.9, a marked seasonal fluctuation that corresponds well with the fluctuation in population numbers of C. victori (Fig. 8.3), its suggested reproductive cycle (Fig. 8.3), and hence also the fluctuation in prevalence, mean intensity and abundance of infestation on the four fish host species (Figs. 8.4-8.6), is witnessed. The data presented in Table 8.9 confirms that a decrease in temperature often coincide with an increase in % 0 2 saturation, conductivity and total dissolved salts (TDS). Furthermore, corresponding annual fluctuation patterns are apparent in the population
157 numbers of C. victori (Fig. 8.3) and the % 02 saturation, conductivity and TDS (Table 8.9), whereas the annual fluctuation in water temperature displays a corresponding but inversed pattern (Table 8.9). When comparing the values of the selected water quality variables obtained for the Olifants River and lower Selati River, higher conductivety and TDS values, accompanied by lower % 02 saturation and pH values, were recorded in the Selati River in comparison to the corresponding values measured in the Olifants River (Table 8.8 & 8.9). This, together with the higher sodium, fluoride, chloride, sulphate, potassium and metal concentrations measured in the Selati River (Seymore et a/.,1994), indicates a poorer water quality for the Selati River than for the Olifants River and might be one of the reasons why just a few host specimens were only occasionally captured there and C. victori specimens never found on any fish host caught in the Selati River.
Since branchiurans can crawl over the surface of the host, they tend to be less rigorously restricted to one part of the hoses body (Fryer,1968). However, since some species - especially Chonopeltis spp. - exhibit marked preferences for particular sites (cf. Chapter 3, Table 3.2), the preferred attachment sites of C. victori on its four fish host species in the Olifants River during the study period (February 1990 to January 1992) were recorded and analysed. In order to analyse the recorded data on site preferences of C. victori, the following sites on the host's body were identified: Site 1 : Ventral body surface; Site 2 : Dorsal body surface; Site 3 : Fins; Site 4 : Head and nose area.
From the results obtained (Table 8.6; Figs. 8.8A-C), it is evident that the ventral body surface (site 1) and fins (site 3) are the preferred sites for parasite attachment on each of the four fish host species. In all four host species the prevalence for site 1 as well as site 3 were more than double the prevalence for both site 2 and site 4 (Table 8.6; Fig. 8.8A). The maximum prevalence for site 1 (80%) was recorded in L. ruddi, whereas the maximum prevalence for site 3 (46.51%) was recorded in L. congoro (Table 8.6; Fig. 8.8A). The preference for parasite attachment on site 1 and site 3 is also confirmed by the site abundance of this parasite
158 on each of the four host species (Table 8.6; Fig. 8.8C). In all four host species, parasite abundance were higher on site 1 and site 3 as compared to parasite abundance on site 2 and site 4 (Table 8.6). However, as far as mean site intensity is concerned, the highest mean parasite intensity in L. rosae was observed on site 2 and site 4; in L. congoro and B. marequensis on site 1 and site 2; and in L. ruddi on site 3 (Table 8.6; Fig. 8.8B). During both 1990 and 1991/1992, C. victori specimens were never found on site 2 and site 4 in L. ruddi (Table 8.6; Figs. 8.8A,B & C).
During the present study fish host specimens of different sizes were caught by means of the various sampling techniques described in Chapter 2 (pp.14-15). However, no host specimen smaller than 10cm in total length or exceeding 50cm in total length were caught. In order to determine a possible relation between C. victori infestation and host size, host specimens were grouped into four different groups based on total fish lengths recorded (cf. Table 8.7; Fig. 8.9) and the prevalence, mean intensity and abundance of C. victori on each of the four host species for the different total fish lengths were calculated (Table 8.7; Figs. 8.9A,B & C). From the results obtained (Figs. 8.9A,B & C), it appears that although an increase in host size coincided with a progressive increase in C. victori infestation, maximum prevalence and abundance of infestation occurred on host specimens with total lengths ranging between 21cm and 40cm (Table 8.7; Figs. 8.9A & C). With the exception of the decline wittnessed in the mean intensity of C. victori on L. ruddi specimens exceeding 30cm in total length (Fig. 8.9B), a progressive increase in the mean intensity of infestation on host specimens coincided with a progressive increase in total fish lengths (Table 8.7; Fig. 8.9B). From the data obtained in the present study it thus appears as though a definite correlation exist between host size and C. victori infestation.
8.3 DISCUSSI N
Natural populations of branciurans are regarded as continuously reproducing populations and accordingly, copulation takes place, eggs are laid and fertilized throughout the year
159 (Martin,1932; Paperna,1980; Shafir & van As,1986; Shafir & Oldewage,1992). Nevertheless, the reproductive cycle, sex ratio and growth pattern appear to be influenced by the annual physical environmental conditions - thus, implying seasonality of a sort within branchiuran populations. In this regard, it has been reported that during winter, prevailing water temperatures can retard, or even prevent, egg development and hatching in branchiurans, which consequently would affect age distribution and structure of the parasite population (Bower-Shore,1940; Paperna,1980; Shafir & van As,1986; Oldewage & van As,1987; Shafir & Oldewage,1992). In one branchiuran genus, Argulus, cases had been cited where some of the eggs laid in autumn, remained dormant throughout winter and hatched in spring, hence extending its reproductive season the year round (Meehean,1940; Hindle,1949; Shafir & Oldewage,1992).
Accordingly, massive hatching of eggs laid during autumn and winter, contribute greatly to the sharp increase in population numbers in Argulus witnessed during the summer (Shafir & Oldewage,1992) - a trend also observed in C. victori during the present study, where maximum population numbers (Fig. 8.3) as well as maximum infestation on fish hosts (Figs. 8.4-8.6) were noted during late spring, early summer (August - October/November). However, increase in population numbers in C. victori was observed during the present study from June and continued to do so - reaching its highest peak at the end of October, beginning November (Fig. 8.3). A possible explanation for this increase in parasite numbers during June (winter) may be found in the well documented fact that despite their retarded development at low temperatures, ectoparasitic crustaceans have a tendency to increase in prevalence during cold seasons (Viljoen,1982,1985; Shafir & van As,1986). It appears that a greater number of individuals succeed in successfully infesting a host during winter which is, according to Oldewage & van As (1987), probably the result of a more lethargic pattern of movement of fish at low temperatures. However, since relative high average water temperatures prevail in the rivers of the KNP throughout the year due to the overall warmer climate of the region (Seymore et a/. ,1994), it seems more likely that this increase in C. victori numbers during winter is the result of their specific reproductive cycle rather than the influence of extreme low temperatures on the behaviour of fish host species. From the results obtained during the present study, the reproductive cycle, hence population numbers, structure and distribution as
160 well as prevalence, mean intensity and abundance of C. victori on its fish hosts, appear to be directly linked to not only temperature changes, but also the annual average water temperatures experienced. Substantiating this, maximum peaks in population numbers (Fig. 8.3) as well as in infestation of C. victori (Table 8.4 & 8.5; Figs. 8.4-8.7) were noticably higher during 1990 when lower average water temperatures than during 1991/1992 prevailed. Furthermore, in the related branchiuran genus, Argulus, Shafir & van As (1986) reported that the mean hatching efficiency lies in the optimal temperature range of 20-30 °C, with hatching considerably lower outside this range.
The natural population of C. victori in the Olifants River displays distinct fluctuation patterns in population numbers, size distribution, proportional distribution of adult males, females and gravid females and, hence, also in the prevalence, mean intensity and abundance of infestation. The differences apparent in these patterns for corresponding periods in the two successive years can, however, be attributed to the differences in the prevailing climatic conditions during each year. Since 1991/1992 experienced 'abnormal' climatic conditions due to the severe drought and elevated temperatures, the fluctuation patterns witnessed during 1990 seem to represent the 'normal' trend in annual fluctuation in population structure and distribution as well as in infestation of C. victori on its fish hosts in the Olifants River. Accordingly, this trend implies seasonality of a sort within the Olifants River population of C. victori. However, it appears to contradict the fact that gravid females were present in the population throughout the year (the latter, suggesting a continuously reproducing population).
AlthotIgh adult C. victori seems to remain associated with their fish hosts throughout the year (Fig. 8.3), it would appear that under 'normal' climatic conditions an increase in parasite numbers and consequently also in infestation occur from early autumn (April) until mid-spring (October), when parasite numbers reach an overall maximum peak (Table 8.1; Fig 8.3). Thereafter, from early to mid-summer (October/November - February), parasite numbers and thus also infestation, drastically decrease (Table 8.1 & 8.4). This coincides with a decrease in the ratio of females to males as well as in the percentage gravid females (Table 8.1; Fig. 8.2), and are probably due to some parasites leaving the hosts during copulation (cf. Fryer,1959; van Niekerk,1984), and females leaving the hosts to deposit eggs on a suitable substrate other
IL61 than the host, since no eggs were ever found on any host specimen. Thereafter the parasites, probably unable to relocate a new host, subsequently die - thus, completed their life span which commenced the previous autumn, early winter (April - June). This is supported by the fact that no host fish were infested by large individuals of C. victori towards late summer and autumn, and by the smaller mean lengths of C. victori specimens sampled during autumn and winter (Table 8.2). The latter most likely represent a new generation of parasites. The slight increase in population numbers wittnessed during mid- to late autumn (April - June) appears to be the result of the hatching of eggs laid during late summer, whereas the massive increase in population numbers towards the end of winter (August - October) coincided with an increase in temperature and, thus probably represent the hatching of eggs laid during autumn and winter (Fig. 8.3). Although drastic declines in parasite numbers occurred, they were never totally absent at any stage throughout the study period which seems to be related to, among others, the overall warmer climate of the region, suitable water quality of the aquatic habitat and the availability of preferred host species.
In agreement with the findings of Van Niekerk (1984) on C. australis, the results obtained during the present study on C. victori indicate that the average water temperature must be at least 20°C or higher (18°C or higher in C. australis according to Van Niekerk (1984)) before females would leave the fish hosts to lay their eggs. Based on the information available on the average water temperatures in the Olifants River during the study period (cf. Seymore et al. ,1994), eggs could thus be laid from October to April during the first year (1990), but already from August during the second year (1991). This corresponds perfectly with the drastic decline in population numbers (especially percentage gravid females), witnessed in C. victori during each respective year (Figs. 8.2 & 8.3). Although the foregoing information thus implicate oviposition in C. victori (as in C. australis) to be seasonal, gravid females were present throughout both years, which suggests ovarium development to be non-seasonal. According to Van Niekerk (1984), it is however possible that the production of eggs (=ovarium development) might be either non-seasonal, or seasonal with the eggs stored in the ovarium until the required optimum temperature for oviposition has been reached. Only further in-depth investigation into the development of the ovarium could, however, clarify this.
162 Concerning the fluctuation in the sex-ratio in C. victori, the ratio of females to males did not alter throughout the study period in favour of males and they consistently remained the minority (Table 8.1). Although the factors determining this are not known, Charniaux-Cotton (1960) suggested two possibilities: 1) a genetic predominance of one sex from birth, or 2) an alteration of the sex-ratio by environmental factors such as emigration and mortality. While mortality undoubtedly contributed towards the shift in the ratio of females to males from 3:1 to 1:1 during both 1990 and 1991 (Table 8.1), it does not alter the fact that females were favoured throughout. Consequently, it is suggested that, although for reasons yet unknown, a genetic predominance of females occurs in the natural population of C. victori from the Olifants River. The opposite were however, noted in the natural population of A. japonicus occuring in Bloemhof Dam where Shafir & Oldewage (1992) suggested that a genetic predominance of males occurs as a result of the differences in energy-input requirements between males and continuously gravid females.
The information presented above, although not conclusive, provides a basis for speculation on the seasonal cycle and reproductive pattern of C. victori. If the conclusions made from the results obtained during the present study are correct, it suggests that the natural population of C. victori in the Olifants River should not be regarded as a continuously reproducing population. Females seem to leave the fish hosts to lay one or more batches of eggs between early to late summer and between late autumn to mid-winter - thus, at the end of their life-span which commenced during the corresponding periods the previous year. This suggested reproductive cycle may, however, be found to vary somewhat if the relevant information is recorded on a monthly instead of a bimonthly basis.
In contrast to the more extended reproductive season suggested for C. victori, it has been reported that various branchiuran species from temperate and cold regions, have only one or two relative short reproductive seasons annually, that is, during the spring and summer months (Wilson,1902; Martin,1932; Meehean, 1940 ; Avenant & van As , 1986) . Accordingly, S header (1977) concluded that the breeding duration of certain invertebrates varies in relation to the location of the population within the geographical range of the species.
163 Within the cumulative research done on branchiurans in general and Chonopeltis in particular, information on reproduction and life cycles of the various species is limited. All information hitherto known indicate that all species of Branchiura are ectoparasitic throughout their lives, mainly parasitizing various species of fish. Although these parasites are considered to be permanent ectoparasites, they leave the host to moult and reproduce and will also change hosts during their lifetime (Fryer,1961a;1968;1986; Paperna,1980; Schram,1986). The life cycle of branchiurans involves several morphologically distinct larval stages, except in Dolops where only one larval form, described as a "juvenile adult" (Fryer,1964), has thusfar been distinguished. In all branchiuran species, parasitism commences from the time of hatching and newly hatched larvae are present in situ on the host. However, according to Fryer (1968) it is possible that the neonatal in some cases has sufficient food reserves to carry it through the first moult.
Copulation in branchiurans can take place either on the fish host, or off the host in a free- swimming mode or attached to a firm substrate (Fryer,1959;1968; van Niekerk,1984; Venter,1988; Shaft' & Oldewage,1992). However, in Chonopeltis where neither the adult nor the larvae are capable of active swimming, copulation has hitherto never been observed in a free-swimming mode (Fryer,1959; van Niekerk,1984; Own Observations). Observations on mating attempts in Chonopeltis spp. made by Fryer (1959) as well as Van Niekerk (1984), suggest that both participants may leave the host during mating, and that copulation may (as does egg-laying) occur away from the host on a suitable hard substrate. This implies that if copulation and egg-laying are to take place more than once, both adults must locate a host more than once. Although copulations between males and females were witnessed repeatedly by various investigators, neither the rate and regularity, nor the stage when first copulation takes place could thusfar be determined for any branchiuran species.
In contrast to Dolops where sperm transfer involves the employment of spermatophores (Fryer,1960b; Avenant-Oldewage & van As,1990), sperm transfer during copulation in Argulus (Avenant-Oldewage & Swanepoe1,1993) and Chonopeltis (cf. Chapters 6 & 7), probably involves a process during which the paired spermathecal spines of the female are inserted into the male genital atrium, penetrating the walls of the respective ejaculatory ducts,
164 whereafter semen is actively pumped into the spermathecal vesicles of the female. In all three genera, eggs are fertilized, while being laid, by the injection of sperm contained in the female spermathecal vesicles (van Niekerk,1984; Shafir & van As,1986; Avenant et a/.,1989; Avenant-Oldewage & Swanepoe1,1993), deposited into them by previous copulation. Information thusfar known also indicates that in all three genera, eggs are laid on any kind of hard substrate (other than the fish host), by females who are gravid from an early stage of their lives (Fryer,1968; Avenant & van As,1986; Shafir & van As,1986; Shafir & Oldewage, 1992).
Among the Crustacea, branchiurans are unique in their manner of egg-laying since, as previously mentioned, females have to leave their fish hosts in order to deposit eggs in clusters on hard substrata. This reproductive pattern leads to a life history which, especially in the case of a few members of the genus Argulus, may contribute to their wide distribution (Fryer,1968; Shafir & van As,1985). Although a great deal of information has accumulated concerning the way in which the eggs of various species of Argulus are laid and develop, (e.g. Wilson,1902; Tokioka,1936; Stammer,1958; Thomas,1961; Shimura,1981; Shafir & van As,1986; Venter, 1988), these aspects have hitherto only been investigated in one species of Dolops (Fryer,1964; Avenant et a/.,1989), and three species of Chonopeltis, viz. C. inermis (Fryer,1956), C. brevis (Fryer,1961a) and C. australis (van Niekerk,1984).
Concerning the development of eggs and larvae, studies on branchiuran development have almost exclusively been restricted to the sequence of events after the first larva hatches from the egg, and only the most general observations have been offered on ontogeny within the egg (e.g. Tokioka,1936). This is regrettable because early ontogenic studies might teach us much about branchiuran relationships and possibly give some insight into the homology of peculiar structural features. This aspect of branchiuran biology thus certainly needs some investigative attention. Nevertheless, information thusfar available indicates that newly hatched branchiuran larvae either resemble modified nauplius larvae (as in certain Argulus spp. - eg. A. japonicus (Tokioka,1936; Lutsch & Avenant-Oldewage,1995)), or more commonly resemble adults in
general form (especially the larval form found in Dolops - eg. D. ranarum (Avenant et a/.,1989)). In Chonopeltis, larval stages differ considerably from adults, their structure being
165 intermediate between the "nauplii type" and "juvenile adult" occuring in Argulus and Dolops respectively (Fryer,1956,1961a,1970; van Niekerk,1984). In Argulus between five and nine morphologically distinct larval stages have hitherto been described (Wilson,1902; Tokioka, 1936; Stammer,1958; Thomas,1961; Shimura,1981; Shafir & van As,1986; Venter,1988), whilst in Chonopeltis, Fryer (1961a) identified eight (possibly nine) different larval stages in C. brevis and Van Niekerk (1984) distinguished seven morphologically distinct larval stages in C. australis.
Since female branchiurans have to leave the host in order to deposit their eggs on firm substrata, an ability to swim well is essential - not only for the female, but also for the young who also need to find a host as quickly as possible. However, in contrast to species of Argulus and Dolops, who are all capable of active swimming, in Chonopeltis spp. neither the adult nor the larvae are capable of active swimming. As if this is not a sufficient handicap, at least two (and most probably all) species of Chonopeltis employ an intermediate host (Fryer,1961a; van Niekerk,1984) - a phenomenon hitherto not reported for any species of Argulus or Dolops. Transmission from one host to another in Chonopeltis spp. thus seems to depend on contact being made with a bottom frequenting fish since the despersal of these parasites must be essentially passive. If the preferred habitat for egg-laying in Chonopeltis coincides with that frequented at times by both the intermediate and definite hosts, chance encounters between them and Chonopeltis larvae as well as adults, are not infrequent. Since the intermediate hosts described for Chonopeltis larvae (i.e. Amphilius sp.; Garra sp., Synodontis sp., and Barbus sp.) are much smaller bottom-dwelling fishes which feed primarily on bentic and planctonic invertebrates, opportunities may be greater at this stage for attachment to them than to the definite host which are usually much larger bottom-dwelling fishes (cf. Chapter 3, Table 3.1). How attachment is initiated is unknown, but according to Fryer (1961a) as well as Van Niekerk (1984), it seems probable that the tiny larvae either enter the host via the host's mouth (during feeding and/or respiration), and hook themselves to the operculum as they leave the oral cavity with the exhalent respiratory current, or hook themselves to the ventrally located fins as the host fishes pass over them. Since the space beneath the operculum and on the fins of the small intermediate hosts is restricted, the increased size of the young adults (i.e. postlarval stage) may be sufficient motivation to cause them to vacate the intermediate host.
166 Since the fishes which serve as definite hosts to all known species of Chonopeltis are, without exception, bottom feeders (cf. Chapter 3, Table 3.1), it is probable that the parasites (both "young" and "old" adults) attach themselves to the bottom, await the arrival of a host and, possibly by the utilization of some, yet unobserved mechanism, contrives to attach themselves to the host when the latter passes closely by.
Branchiuran species in general do not seem to exhibit strict host specificity (Fryer,1968;1986; Schram,1986). In some species preference for a particular host is certainly influenced by whether the host can provide a suitable area of attachment - preferably an external area of smooth skin as in smooth-skinned fishes or in the mouth/buccal cavity if the fish host is scaly (Fryer,1968). Since branchiurans can crawl over the surface of the host, they tend to be less rigorously restricted to one part of the host's body (Fryer,1968), though some species - especially Chonopeltis spp. - exhibit marked preferences for particular sites (cf. Chapter 3, Table 3.2). Furthermore, in all branchiuran genera prevalence, mean intensity and abundance of infestation on the fish hosts seems to be influenced by the physical aquatic environment (Fryer,1956,1959,1960a, 1961b; Paperna,1980; Shen- & van As,1985,1986). Among African branchiurans, species of Argulus as well as D. ranarum exhibit wide host tolerance and occur on fish of diverse families, although some preferences do exist even among the most tolerant of these parasite species (Fryer,1968). In contrast to Argulus and Dolops, species of the genus Chonopeltis have more strict host specificity, each species being primarily confined to one fish family (cf. Chapter 3, Table 3.3). Futhermore, as mentioned previously, host preferences of larval stages in Chonopeltis have also been reported to be different from that of adults. With the exception of a single immature specimen found on one occasion, only adult (i.e. sexually mature) specimens of C. victori were found on the cyprinid host species examined during the present study. This suggests that these cyprinids serve only as the definite host whilst the larvae most probably occur on a much smaller bottom-dwelling intermediate host(s). Similary, Van Niekerk (1984) found larval and adult specimens of C. australis on different bottom- dwelling fish hosts. He collected adults from large cyprinid species (i.e. Labeo capensis, L. umbratus and Barbus kimberleyensis), whereas the larvae were found on the much smaller cyprinid Barbus aeneus, and usually only on specimens smaller than 20cm. Noteworthy of the latter fish host, is the fact that this species spawn over gravel beds with their eggs laid in the
167 gravel (Skelton,1993) - a feature which most definitely enhance chance encounters between them (i.e. fish host) and the chonopeltid larvae.
As previously mentioned, in order to maintain their suggested reproductive pattern, adult C. victori specimens have to leave their fish hosts during reproduction (i.e. copulation and oviposition). This, however, poses the formidable problem of relocating their hosts in the vast volumes of water. In this respect, host species specificity is a distinct disadvantage - especially since C. victori specimens are incapable of active swimming. Accordingly, adult C. victori were found to infest more than one fish species present in the Olifants River (i.e. L. rosae, L. congoro, L. ruddi, and B. marequensis). Nevertheless, the infestation levels in terms of prevalence, mean intensity and abundance of infection, clearly pointed towards L. congoro as the most preferred fish host in the Olifants River (Table 8.3, 8.4 & 8.5). Since L. congoro appears not to be the most common fish in the study area (cf. Table 8.5), the preference for this host species suggests that host specificity in C. victori has more to do with specific factors rather than sheer numbers of fish. In this regard, fish movement, parasite feeding trends and the specific immune response of each fish host species should be considered. Although quantitative experiments are necessary, it seems reasonable to assume that since all four fish host species are bottom-dwelling fish which breed during the spring and summer months (cf. Skelton, 1993), a distinct difference in the immune response of the four fish host species could determine to a considerable extent the nature and magnitude of the infestation pattern of C. victori (cf. Shafir & Oldewage,1992). In a natural population of C. victori, such as the one in the Olifants River, it would also be reasonable to assume that the availability of a range of fish species would create a preference index for infestation based on the nature of the immune response of each such fish species. Furthermore, the accessibility of feeding (i.e. a suitable site for attachment and feeding on the fish hosts), is another factor most definitely contributing towards preference of infestation. During the present study all specimens of C. victori sampled, were found externally on especially the scaleless areas on the host's body surface, i.e. the lower body surface/abdomen, near the basis of fins, on the head and on the operculum (cf. Chapter 3, Table 3.1; Table 8.6). Since it is suspected that all species of Chonopeltis feed on mucus and cellular detritus (Fryer,1968; van Niekerk,1984; Knight,1991), the scaleless Clarias gariepinus (Burche11,1822), commonly present in the Olifants River, would be
168 expected to be the most dominant in determining infestation preferences in C. victori. However, this fish species proved the contrary which further emphasises the importance of immune response in this regard.
The role of L. congoro as a preferred host throughout most of the year during both 1990 and 1991, was conspicuously reversed during the summer months (December - April) when the prevalence of C. victori was low (Table 8.4, Figs. 8.4-8.6) due to the presumed intense reproductive activity of the parasites (Fig. 8.3). As a result of the parasites leaving the fish hosts to reproduce, the problem of host relocation increased, thus attributing more weight to quantitative factors such as number of fish, rather than to qualitative factors such as immune response and feeding accessibility, in choosing hosts. Notwithstanding this, prevalence, mean intensity and abundance of infestation on the other three fish host species remained relative low during this period (cf. Figs. 8.4-8.6), which can be regarded as an indication of their secondary position on the infestation-preference scale applying the qualitative criteria previously mentioned.
Throughout the present study, relatively low infestations of C. victori were recorded on all four fish host species in the Olifants River. Although Knight (1991) recorded a corresponding low mean intensity of C. australis on its fish hosts in Boskop Dam, the prevalence and abundance were more than double that recorded for C. victori. Furthermore, Knight (1991) recorded a maximum number of 16 adult C. australis specimens per host, whereas a maximum number of 9 adult C. victori specimens per host were recorded during the present study. These differences between C. victori and C. australis can most probably be attributed to the particular habitat of each species. Substantiating this, C. australis specimens were sampled in Boskop Dam, a freshwater impoundment in which both the parasites and its hosts are more confined, whereas C. victori specimens occur in a large, natural river where fish hosts and consequently also their parasites are more widely distributed. However, infestation are related to various aspects of the biology and ecology of both the branchiuran parasites and their fish hosts, such as physical environmental conditions, geographical distribution, means of dispersal, host tolerance and specificy, means of attachment and preferred attachment sites of parasites on hosts, mobility of parasites, movement behaviour of parasites on the host's body,
169 gregarious behaviour of both parasites and hosts, as well as the specific immune response of each fish host species. Furthermore, from observations made on other branchiuran species (eg. Fryer,1961b; Mbahinzireki,1980; Otugu-Ohwayo,1989; Knight,1991) as well as from the data obtained on C. victori during the present study, it appears as though a definite correlation exist between host size and prevalence, mean intensity and abundance of infestation (cf. Table 8.7; Figs. 8.9A,B & C).
Observations made on branchiurans, indicate that species of Argulus and Dolops in contrast to Chonopeltis spp., remain capable of active locomotion throughout life, and tend to be more gregarious (Fryer,1966). Furthermore, although heavy infestations of Argulus have been described (cf. Cunnington,1913; Fryer,1968; Paperna,1980; Kruger et a/.,1983; Cesare, 1986; Shafir & Oldewage,1992), heavy infestations of Dolops and Chonopeltis in both natural and cultured fish populations have hitherto not been described.
Among African branchiurans, species of the opportunistic genus Argulus have the highest potential of becoming pathogenic pests of both natural and cultured fish populations, and some Argulus spp. are serious pathogens, causing morbidity and mortalities amoung various freshwater fish species (Reichenbach-Klinke,1973; Paperna,1980; Kruger, van As & Saayman,1983). Nevertheless, the damage done by individual branchiurans is seldom great, though Dolops, which besides drawing blood at times (Fryer,1968), attach themselves to the fish host with piercing claws and sometimes causes a noticeable wound (cf. Avenant- Oldewage,1994). In all branchiuran genera, more heavy infestations are however sometimes encountered, and these, particularly if they involved young fish hosts, may be serious or even fatal (Fryer,1968; Shimura,1981; Shafir & van As,1986; Oldewage & van As,1987).
Pathological damage to the fish host may vary with the specific parasite species as well as with the host fish, and the pathology may be related to both the mode of attachment and of feeding. Although much remains to be learned concerning feeding mechanisms of branchiurans, it is suspected that all species of Chonopeltis and many species of Argulus feed on mucus and cellular detritus, whilst Dolops pierces the skin of its hosts and feeds, though perhaps not always, on blood (cf. Madsen,1964; Fryer,1968; van Niekerk,1984; Baker,1990; Knight,
1170 1991). In fish the skin and mucus act as a natural barrier against infestation (Reichenbach- Klinke,1973), and mechanical damage such as that caused by branchiurans, disrupts this barrier. The latter thus increases susceptibility to secondary bacterial and fungal infections which can contribute to the eventual mortality of the host - especially under unfavourable environmental conditions. Histopathological changes have been investigated by various authors (e.g. Madsen,1964; van Niekerk,1984; Baker,1990; Knight, 1991; Avenant-Oldewage,1994). In all cases, varying degrees of focal lesions (in scaly fish) or extensive lesions (in smooth skinned fish) depending on the site preferences of the parasitic species involved, have been reported. Even when the pathological damage done by these parasites has not been severe, secondary infection of the wound by bacteria and fungi usually followed.
All considered, it seems as though the damage caused to the fish hosts by Chonopeltis spp. is more limited than in Argulus and Dolops, and usually does not directly cause the death of their hosts. However, secondary infections to the wound may result in the death of the fish host (cf. Re ichenbach-K 1 inke, 1973 ; Schubert, 1987) .
171 FIGURE 8.1
A : Map of the Kruger National Park, indicating the position of the Olifants River (B).
B: Map of the Olifants River as outlined in Figure 8.1A, indicating the position of the four sampling localities ()■ 1 - 4) for Chonopeltis victori Avenant-Oldewage,1991. ( 4 = Rest Camp).
172 FileagGlff 63. Y
SOUTH AFRICA ro) ./ FIGURE 8.2
Histogram illustrating the proportional distribution of Chonopeltis victori Avenant- Oldewage,1991 expressed as the percentage of adult males, females and gravid females of the total population sampled bimonthly from February 1990 to January 1992 in the Olifants River, Kruger National Park.
FIGURE 8.3
Histogram illustrating the population distribution (i.e. total parasite numbers) and reproductive cycle (expressed as percentage of gravid females of the female population) of Chonopeltis victori Avenant-Oldewage,1991 based on bimonthly samples from February 1990 to January 1992 in the Olifants River, Kruger National Park.
173
HGUR H 8.2
% Individuals 100
80
60
40
20
F A J A O D F A J AO J Time of year (month)
I 1 % Total males [ 1 % Total females I 1% Gravid females
FIGUR 0.3
Total number/% of parasites 100
80
60
40
20
r— I i 1 I 1 1 F A J ACIDF A J A 0 J Time of year (month)
Total parasites I % Gravid females FIGURE 8.4
Histogram illustrating the prevalence (%) of Chonopeltis victori Avenant-Oldewage,1991 on L. rosae, L. congoro, L. ruddi and B. marequensis at sample locality 2 in the Olifants River during the period February 1990 to January 1992. (The data are presented in Table 8.3).
Histogram illustrating the prevalence (%) of Chonopeltis victori Avenant-Oldewage,1991 on L. rosae, L. congoro, L. ruddi and B. marequensis at sample locality 3 in the Olifants River during the period February 1990 to January 1992. (The data are presented in Table 8.3).
Histogram illustrating the prevalence (%) of Chonopeltis victori Avenant-Oldewage,1991 on L. rosae, L. congoro, L. ruddi and B. marequensis at sample locality 4 in the Olifants River during the period February 1990 to January 1992. (The data are presented in Table 8.3).
Histogram based on the data presented in Table 8.4, illustrating the prevalence (%) of Chonopeltis victori Avenant-Oldewage,1991 on L. rosae, L. congoro, L. ruddi and B. marequensis in the Olifants River during the period February 1990 to January 1992.
174 FIGURE 8.4A FOGURE 8.4 Sample locality 2 Sample locality 3
Prevalence (%) Prevalence (%) 120 120
100 - 100 -
80 - 80 -
60- 80 -
40 - 40 -
20 - 20 1-1 0 n n n F A J A O D F A J A J A O D F A J Time of year (month) Time of year (month)
L. MRS I L. congoro 0 L. rudgl B. marequenola L. 'acme L. congoro L. ruddi 0 B. /norm/ono/a
FOGURE SAC FOGURE SAD Sample locality 4 Olifants River (KNP)
Prevalence (%) Prevalence (%) 120 120
100 100
80 80
80 80
40 40
20 20 1 n F A 0 D F A J F A A O D F A J O J Time of year (month) Time of year (month) L. route I IL. congoro =L. roddl B. marevenslo 1 1 L. room ED L. oongoro L. 'odd! EJ B. moroquenalo FIGURE 8.5
Histogram illustrating the mean intensity of Chonopeltis victori Avenant-Oldewage,1991 on L. rosae, L. congoro, L. ruddi and B. marequensis at sample locality 2 in the Olifants River during the period February 1990 to January 1992. (The data are presented in Table 8.3).
Histogram illustrating the mean intensity of Chonopeltis victori Avenant-Oldewage,1991 on L. rosae, L. congoro, L. ruddi and B. marequensis at sample locality 3 in the Olifants River during the period February 1990 to January 1992. (The data are presented in Table 8.3).
Histogram illustrating the mean intensity of Chonopeltis victori Avenant-Oldewage,1991 on L. rosae, L. congoro, L. ruddi and B. marequensis at sample locality 4 in the Olifants River during the period February 1990 to January 1992. (The data are presented in Table 8.3).
Histogram based on the data presented in Table 8.4, illustrating the mean intensity of Chonopeltis victori Avenant-Oldewage,1991 on L. rosae, L. congoro, L. ruddi and B. marequensis in the Olifants River during the period February 1990 to January 1992..
175
FHGURE 8.5A FDGURE 8.5B Sample locality 2 Sample locality 3 Mean Intensity Mean intensity
II F A J A 0 D F A J A O J F A J A 0 D F A J A0J Time of year (month) Time of year (month) CI L. room = L. congoro CJ L. rigid/ = B. morequenale I I L. rocee = L. congoro = L. ruchll = B. maregoenale
MIRE 8.5C FOG RE 8.5D Sample locality 4 °Wants River (KNP)
Mean Intensity 8 Mean Intensity
1 _F
A J ACM A J A O J F A J A O D F A J A O J Time of year (month) Time of year (month)
( I oongoro = L. ruricil = B. marequenalo 1 f L. roaae L oongoro = L. ruddl = 8. marequenoto FIGURE 8.6
Histogram illustrating the abundance of Chonopeltis victori Avenant-Oldewage,1991 on L. rosae, L. congoro, L. ruddi and B. marequensis at sample locality 2 in the Olifants River during the period February 1990 to January 1992. (The data are presented in Table 8.3).
Histogram illustrating the abundance of Chonopeltis victori Avenant-Oldewage,1991 on L. rosae, L. congoro, L. ruddi and B. marequensis at sample locality 3 in the Olifants River during the period February 1990 to January 1992. (The data are presented in Table 8.3).
Histogram illustrating the abundance of Chonopeltis victori Avenant-Oldewage,1991 on L. rosae, L. congoro, L. ruddi and B. marequensis at sample locality 4 in the Olifants River during the period February 1990 to January 1992. (The data are presented in Table 8.3).
Histogram based on the data presented in Table 8.4, illustrating the abundance of Chonopeltis victori Avenant-Oldewage,1991 on L. rosae, L. congoro, L. ruddi and B. marequensis in the Olifants River during the period February 1990 to January 1992.
176
FlIGURE 806A FOGURE 8.6B Sample locality 2 Sample locality 3
Abundance Abundance 1 3.5 3 0.8 2.5
0.6 2
1.5 0.4 1
0.2 0.5
I r n. 0 ifini [I Finn, F AJAODF A J A O J F A .1 A O D F AJ A 0 J Time of year (month) Time of year (month)
= L. mode I= L. congorg = L. ruddl = B. marequenolo L. roue I I L. oongoro 0 L. ruddl I= B. morequenolo
FHGURE 8.6C FIGURE 8.6D Sample locality 4 Olifants River (KNP)
Abundance Abundance 5 5
5
4
-L, - , n n n 0 n n n rn `1 F A J A O D F A J AO J F A J A O D F A J A 0 Time of year (month) Time of year (month)
L. maw CI L. oongoro O L. ruddl = B. marequen815 I—I L. mese CI L. oongoro = L. ruddl 0 B. marequenola FIGURE 8.7
Histogram illustrating the differences between 1990 and 1991 in the prevalence (P), mean intensity (MI) and abundance (A) of Chonopeltis victori Avenant-Oldewage,1991 on four fish host species (i.e. L. rosae, L. congoro, L. ruddi and B. marequensis) in the Olifants River, KNP.
FIGURE 8.8
Histogram illustrating the prevalence (%) of Chonopeltis victori Avenant-Oldewage,1991 for specific sites on L. rosae, L. congoro, L. ruddi and B. marequensis in the Olifants River during the period February 1990 to January 1992. (Site 1= ventral body surface; Site 2= dorsal body surface; Site 3= fins; Site 4= head and nose area).
Histogram illustrating the mean intensity of Chonopeltis victori Avenant-Oldewage,1991 for specific sites on L. rosae, L. congoro, L. ruddi and B. marequensis in the Olifants River during the period February 1990 to January 1992. (Site 1= ventral body surface; Site 2= dorsal body surface; Site 3= fins; Site 4= head and nose area).
Histogram illustrating the abundance of Chonopeltis victori Avenant-Oldewage,1991 for specific sites on L. rosae, L. congoro, L. ruddi and B. marequensis in the Olifants River during the period February 1990 to January 1992. (Site 1= ventral body surface; Site 2= dorsal body surface; Site 3= fins; Site 4= head and nose area).
177
FDGURE X30 Y FIIGURE 8.8A ®lilan1s River (KNP) OlifaMs River (KNP) Prevalence (%) Mi A Abundance Site prevalence (%) 60 100
40 -4 80 30 3 80
20 2 40
10 20 I n 1 r ~1Ell, 0 I H Site 4 P (1990) P (1991) MI (1990) ka (1991) A (1990) A (1991) Site 1 Site 2 Site 3 Infestation Site on host species
marequenale L roaae I I L. congoro I=1 L. rude, ED B. marequenala = L. roam I I L. congoro O L. red&
FDGURE 8.83 FDGURE 8.8C Olifants Myer (KNP) °Hants River (KNP)
Mean site Intensity Site abundance 6 1.8 1.4 1.2 1 0.8 0.8 0.4 0.2
0 Site 1 Site 2 Site 3 Site 4 Site 1 Slte 2 Site 3 Slte 4 Site on host species Site on host species =IL. roue L oongoro D L. rade!! B. marequenals I 1 L. roaae I I L. congoro O L. rude, O B. marequenala FIGURE 8.9
Histogram illustrating the prevalence (%) of Chonopeltis victori Avenant-Oldewage,1991 on L. rosae, L. congoro, L. ruddi and B. marequensis for specific total fish lengths. The number of fish infested with C. victori for a specific length is indicated above each bar.
Histogram illustrating the mean intensity of Chonopeltis victori Avenant-Oldewage,1991 on L. rosae, L. congoro, L. ruddi and B. marequensis for specific total fish lengths. The number of fish infested with C. victori for a specific length is indicated above each bar.
Histogram illustrating the abundance of Chonopeltis victori Avenant-Oldewage,1991 on L. rosae, L. congoro, L. ruddi and B. marequensis for specific total fish lengths. The number of fish infested with C. victori for a specific length is indicated above each bar.
e.
178
FOGURE 8.9B Olifants River (KNP)
Mean Intenolty 4 nit 21 3 9.5 - 3 3 - 17 2.5 - 2 7 2 RGURE 8.9A 1.6 - 1 00Hants Myer (KNP) 0.6 - 0 41- 60 Prevalence (%) TO - 20 21-30 31 - 40 60 Host length (cm) 26 21 7 L. rooao Ci L. oongoro L. ruddl B. maroccoondo 50 16 2 2
40 1-- 17 30 1 20 3 10 2 FIGURE 8.9C Olifants River (KNP)
10 - 20 21 - 30 31 - 40 41 - 50 Abundance
0-last length (cm) 21
1.6 L. rosae 1 1 L. congoro I L. ruddi B. marequenels TO
1 7
0.6 3 2 1
S0-20 21-30 91 - 40 41- 60 Host length (cm)
L. room L. congoro 173 L. ruslat B. rnaroottondo TA LE 0.1.
Number and proportions of male(d), femaleM and gravid female(G?) Chonopeltis victori in bimonthly samples from February 1990 to January 1992. (Maximum values indicated in bold; F/M = Ratio of females to males).
5 2 3 2 1.50 40.00 60.00 40.00 66.6.7 4 2 2 1 1.00 50.00 50.00 50.00 50:00 22 6 16 12 2.67 27.27 72.73 54.55 75:0Q: 17 5 12 8 2.40 29.41 70.59 47.06 66.67 96 27 69 58 2.56 28.12 71.88 60.42 17 6 11 6 1.83 35.29 64.71 35.29
6 2 4 2 2.00 33.33 66.67 33.33 3 1 2 1 2.00 33.33 66.67 33.33 24 6 18 4 3.00 25.00 75.00 59.33 77.78 29 7 22 18 3.14 24.14 75.86 62.07 81.82 33 10 23 18 2.30 30.30 69.70 54.55 :::::•78.26: • 13 6 7 3 1.17 46.15 53.85 23.08
T LE 8.2
Mean total length (mm) of adult male and female Chonopeltis victori specimens in bimonthly samples from February 1990 to January 1992.
FE M P4.1.•;ES ::(mm
(n= . 3.0}.
6.021 9.782 2.121 2.763 2.297 3.211 2.739 4.702 5.658 7.626 5.075 8.639
5.975 9.952 2.113 2.862 2.275 3.625 4.897 6.175 5.309 8.988 5.813 9.437
179 TABLE 8.3
Prevalence(P), mean intensity(MI) and abundance(A) of Chonopeltis victori on Labeo rosae, Labeo congoro, Labeo ruddi and Barbus marequensis collected at the three sample localities (Loc. 2-4, Fig. 8.1B) in the Olifants River during the period February 1990 to January 1992. (Maximum values in bold).
A (Lot. 3) (toc.-4)
.i'ERROARY:1900 ••• •••• 0 25,00 25,00 0 2,00 2,00 0 0,50 0,50 . • 0 0 0 0 0 0 0 0 0 . : . 0 0 0 0 0 0 0 0 0 0 0 33,33 0 0 1,00 0 0 0,33 APRIL 1990 0 16,67 25,00 0 1,00 1,00 0 0,17 0,25 L. eongoro:: 0 0 0 0 0 0 0 0 0 :..• ...•:. 0 0 0 0 0 0 0 0 0 B. 0 0 43,75 0 0 1,00 0 0 0,29 JUNE:1990)i:. • 12,50 33,33 66,67 1,00 2,00 4,50 0,13 0,67 3,00 L. congoro.: 0 0 100,00 0 0 4,00 0 0 4,00 0 0 0 0 0 0 0 0 0 L3:: marequerisis: 0 50,00 50.00 0 1,00 2,00 0 0,50 1,00
• L.•:rb.siie:.: 12,50 28,57 33,33 1,00 1,00 3,00 0,13 0,29 0,60 L:: congoro 0 0 33,33 0 0 3,50 0 0 1,17 0 0 33,33 0 0 2,00 0 0 0,67 0 20,00 0 0 2,00 0 0 0,40 0 OCTOBER :1990 E: •• • I rosae; 0 0 53,33 0 0 2,50 0 0 1,33 0 100,00 100,00 0 3,00 4,67 0 3,00 4,67 L..:••ruddl.i•j::: 0 50,00 0 0 1,00 0 0 0,50 0 0 0 50,00 0 0 2,00 0 0 1,00
L:.:••redcie:.:.:.:....::::.: 0 33,33 42,86 0 1,00 2,67 0 0,33 1,14 1.;.•:congoro• 0 0 33,33 0 0 3,00 0 0 1,00 • •• • •:•••:::.. ruddi 0 33,33 0 0 1,00 0 0 0,33 0 0 20,00 25,00 0 2,00 2,00 0 0,40 0,50
: FE;BRUARY.IT?.1 •• 10,00 25,00 50,00 1,00 1,00 2,00 0,10 0,25 1,00 L congoro:: 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 73>: mare uensis 0 0 0 0 0 0 0 0 0
L. rosae 0 0 12,50 0 0 1,00 0 0 0,13 L. congoro 0 0 50,00 0 0 2,00 0 0 1,00 L. ruddi 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
7,69 20,00 30,00 1,00 2,50 3,67 0,08 0,70 1,10 14 .01gdro.: 0 0 100,00 0 0 5,00 0 0 5,00 0 0 0 0 0 0 0 0 0 marequensis 0 0 0 0 0 0 0 0 0
0 15,38 71,43 0 1,00 2,00 0 0,15 1,86
L. •congoro • 0 60,00 100,00 0 3,00 5,50 0 1,80 5,50 0 0 0 0 0 0 0 0 0 N. marequensis 0 33,33 0 0 1.00 0 0 0,33 0 OCTOBER 1991: 1,64 40,00 50,00 1,00 1,00 1,33 0.02 0,40 0,67 L.: congoro; 0 69,23 85,71 0 1,22 2,00 0 0,85 1,71 L. too: 0 50,00 0 0 1,00 0 0 0,50 0 B. marequensis. 2,27 0 16,67 1,00 0 1,00 0,02 0 0,17 JANUARY 1992:: L. rosae , 8,33 66,67 66,67 1,00 1,50 2,50 0,08 1,00 1,67 0 0 25,00 0 0 1,00 0 0 0,25 0 100,00 0 0 1,00 0 0 1,00 0 0 11,11 8,33 0 1,00 1,00 0 0,11 0,08
180 T LE OA
Prevalence, mean intensity and abundance of Chonopeltis vietori on Labeo rosae, Labeo congoro, Labeo ruddi and Barbus marequensis in the Olifants River during the period February 1990 to January 1992 (maximum values in bold). The total number(n) of fish hosts as well as the maximum number of parasites per host is recorded in the table.
Ill:41!111111. PRE VALENCE (Fish) parasik,. I (%) • INTENSITY. hosts
L. rosae 11 2 18.18 2.00 0.36 L. congoro 7 0 0 0 0 L. ruddi 1 0 0 0 0 B. marequensis 6 1 16.67 1.00 0.17
L. rosae 24 8.33 1.00 0.08 L. congoro 4 0 0 0 0 L ruddi 3 0 0 0 0 B. marequensis 16 1 12.50 1.00 0.13
L. rosae 27 8 14.82 3.75 0.56 L. congoro 3 4 33.33 4.00 1.33 L. ruddi 2 0 0 0 0 B. marequensis 11 2 18.18 1.50 0.27
L. rosae 20 3 20.00 1.50 0.30 L. congoro 7 4 28.57 3.50 1.00 L. ruddi 3 2 33.33 2.00 0.67 B. marequensis 11 2 9.09 2.00 0.18
L. rosae 32 7 25.00 2.50 0.63 L. congoro 16 9 100.00 4.56 4.56 L. ruddi 2 1 50.00 1.00 0.50 B. marequensis 5 2 20.00 2.00 0.40
L. rosae 18 3 22.22 2.25 0.50 L. congoro 4 3 25.00 3.00 0.75 L ruddi 3 1 33.33 1.00 0.33 B. marequensis 13 2 15.39 2.00 0.31
L rosae 18 2 22.22 1.50 0.33 L. congoro 10 0 0 0 0 L. ruddi 3 0 0 0 0 B. marequensis 6 0 0 0 0
L. rosae 38 1 2.63 1.00 0.03 L. congoro 3 2 33.33 2.00 0.67 L ruddi 8 0 0 0 0 B. marequensis 26 0 0 0 0
L. rosae 33 6 18.18 3.17 0.58 L congoro 1 5 100.00 5.00 1.66 L ruddi 0 0 0 0 0 B. marequensis 16 0 0 0 0
L. rosae 18 2 38.89 1.14 0.44 L. congoro 7 8 71.43 4.00 2.86 L. ruddi 0 0 0 0 0 B. marequensis 19 1 5.26 1.00 0.05
L rosae 72 3 8.33 1.17 0.10 L. congoro 70 6 20.83 1.53 0.33 L. ruddi 3 1 33.33 1.00 0.33 B. marequensis 71 1 2.82 1.00 0.03
L. rosae 18 2 27.78 1.80 0.50 L congoro 5 1 20.00 1.00 0.20 L ruddi 3 1 33.33 1.00 0.33 B. marequensis 32 1 6.25 1.00 0.06
181
T ii ) LE 8.5
Infestation in terms of prevalence, mean intensity and abundance of Chonopeltis victori on Labeo rosae, Labeo congoro, Labeo ruddi and Barbus marequensis in the Olifants River for the period February 1990 to December 1990 and February 1991 to January 1992. The total number (n) of fish hosts, infested fish hosts and parasites collected are recorded in the table. (Maximum values indicated in bold).
19911:: 132 24 56 18.18 2.33 0.42 41 20 87 48.78 4.35 2.12 11 3 4 27.27 1.33 0.36 Barbus marequensis 62 9 14 14.52 1.56 0.23
1991/1992 .Labeo rosae 197 29 50 14.72 1.72 0.25 96 23 51 23.96 2.22 0.53 Labeo congoro Labeo :ruddi 17 2 2 11.77 1.00 0.12 Barbus marequensiS ::;. 170 5 5 2.94 1.00 0.03
TA flJE 8.6
Prevalence, mean intensity and abundance of Chonopeltis victori for specific sites on Labeo rosae, Labeo congoro, Labeo ruddi and Barbus marequensis in the Olifants River for the period February 1990 to January 1992. The number (n) of infested fish hosts as well as the number of parasites collected from specific sites on the hosts are recorded in the table.
SITE MAN5100:!: (C. victori) PREVALENCE INTENSI1 V ABUNDANCE (4)
labeo rosae? :::: 1 22 44 41.51 2.00 0.83 2 6 14 11.32 2.33 0.26 3 22 41 41.51 1.86 0.77 4 3 7 5.66 2.33 0.13
Labeo c()Iigoro 1 18 60 41.86 3.33 1.40 2 3 12 6.98 4.00 0.28 3 20 60 46.51 3.00 1.40 4 2 6 4.65 3.00 0.14
Lezbeo ruddi': 1 4 4 80.00 1.00 0.80 2 0 0 0 0 0 3 1 2 20.00 2.00 0.40 4 0 0 0 0 0
1 5 8 35.71 1.60 0.57 2 2 4 14.29 2.00 0.29 3 6 6 42.86 1.00 0.43 4 1 1 7.14 1.00 0.07
* Site 1 = Ventral boby surface ; Site 2 = Dorsal body surface ; Site 3 = Fins ; Site 4 = Head & nose area.
182 TAmLE 8.7
Prevalence, mean intensity and abundance of Chonopeltis victori for specific total fish lengths on Labeo rosae, Labeo congoro, Labeo ruddi and Barbus marequensis in the Olifants River for the period February 1990 to January 1992. The number (n) of infested fish hosts of specific total lengths(cm) as well as the number of parasites collected on these hosts are recorded in the table.
rota
Labe° rosae 10-20 8 15 15.09 1.88 0.28 21-30 26 55 49.06 2.04 1.00 31-40 17 37 32.08 2.18 0.70 41-50 2 5 3.77 2.50 0.09
Labe° congoro. 10-20 3 8 6.98 2.67 0.19 21-30 16 51 37.20 3.19 1.19 31-40 21 69 48.84 3.29 1.61 41-50 3 10 6.98 3.33 0.23
talieo - rad& : 10-20 1 1 20.00 1.00 0.20 21-30 2 4 40.00 2.00 0.80 31-40 2 1 40.00 0.50 0.20 41-50 0 0 0 0 0
0.07 Barbus marequensis 10-20 1 1 7.14 1.00 21-30 4 6 28.57 1.25 0.43 31-40 7 9 50.00 1.29 0.64 41-50 2 3 14.29 1.50 0.21
TABLE 8.8
Values of selected water quality variables from the sample locality (locality 1, Fig. 8.1B)) in the lower Selati River (a tributary of the Olifants River ), measured during bimonthly surveys between February 1990 to January 1992. (Data were provided by the Reseach Section for Aquatic Toxocology at the Department of Zoology, RAU).
:TeniperAture Conductivgy (mS/tn)
27 7.8 101.00 1837.00 23 7.9 164.00 1267.00 21 7.8 287.00 1990.00 21 7.6 64.00 244.00 2046.00 22 7.9 67.00 225.00 1775.00 27 7.9 64.00 190.00 1472.00
27 7.9 71.00 239.00 1582.00 23 7.8 75.00 244.00 1638.00 19 7.7 54.00 231.00 1694.00 20 8.0 77.00 299.00 1766.00 28 7.8 46.00 255.00 1877.00 27 7.9 112.00 261.00 1824.00
(A short line (-) indicates that the data was not avai able).
183
TA tlYW LE 8.9
Values of selected water quality variables from the three sample localities (localities 2-4, Fig. 8.1B) in the Olifants River measured during bimonthly surveys between February 1990 to January 1992. (Data were provided by the Reseach Section for Aquatic Toxocology at the Department of Zoology, RAU).
• •• •• • ."
FEBRUARY 1990 30 8.8 35.00 265.00 30 8.8 39.00 229.00 28 8.4 37.00 275.00
APRIL 1990 : 24 8.7 55.10 354.00 25 9.0 42.90 335.00 22 9.1 50.30 354.00
JUNE 1990 : 18 8.6 62.50 582.00 19 8.6 64.90 518.00 19 8.5 79.90 572.00
AUGUST 1990 : 22 8.2 150.00 98.80 712.00 17 8.6 99.00 89.70 662.00 19 8.4 114.00 99.00 735.00
OCTOBER 1990 : 24 8.6 121.00 116.70 914.00 26 8.7 134.00 109.10 781.00 27 8.4 148.00 79.30 601.00
liECEMBER 1990 28 8.1 82.00 51.00 356.00 45.20 358.00 24 7.8 76.60 230.00
FEBRUARY 1991 : 28 8.2 109.00 34.20 240.00 28 8.3 124.00 33.90 244.00 29 8.4 107.00 31.40 235.00
24 8.6 92.00 39.00 284.00 26 8.7 108.00 40.00 309.00 25 8.7 100.00 38.00 320.00
JUNE:1991 : 18 8.5 113.00 51.90 420.00 20 8.6 108.00 56.90 395.00 20 8.5 94.00 52.00 412.00
AUGUST 21 9.0 128.00 98.30 722.00 22 8.9 107.00 93.00 675.00 22 8.7 119.00 82.50 601.00
OCTOBEIC1991:: 30 8.8 125.00 157.00 1117.00 33 8.2 141.00 148.00 1068.00 27 8.2 130.00 144.00 1016.00
JANUARY:1992: 31 8.9 117.00 76.00 477.00 31 8.7 112.00 66.00 493.00 32 8.7 114.00 68.00 420.00
(A short line (-) indicates that the data was not available).
184 " Nature will freely offer itself to you to be unmasked, it has no choice, it will roll in ecstacy at your feet. It
- Franz Kafka GENE L CONCLUSIONS AN RECOMM NDATI NS
Parasitology, like every other branch of biology, has its roots in morphology and systematics. Systematic zoology in toto is based on the proper evaluation of taxonomic characters, which are expressions of the biology of their carriers. An understanding of this biology is thus a prerequisite for the proper evaluation of these characters. Furthermore, many aspects of ecology and life history are species specific and the phenotype of animal populations of the same species often varies according to locality, season, or habitat. In this sense, almost any attribute of an organism might be useful as a discriminative taxonomic character. However, morphological characters of adult specimens are still used more frequently than any others and as such represent the most important criteria for systematic differentiation in the majority of animal groups (cf. Mayr,1969). Morphological characters can range from superficial external features to the highly conservative and phylogenetically significant characters, whilst the internal anatomy provides an abundant source of taxonomic characters in all animal groups. Various components of the reproductive organs have a highly peculiar morphology and may serve as useful indicators of relationships.
A study of the literature published on the branchiuran genus Chonopeltis Thiele,1900, revealed that most investigators confined themselves mainly to taxonomic studies of these parasites - concentrating primarily on the description of new species. Presently there are 13 described species of Chonopeltis, of which the taxonomic descriptions and morphological information
185 of most of these species, are solely based on macroscopic investigations of whole mounts of only a few specimens (sometimes even a single immature specimen (cf. Thiele,1900)), with no information on the morphology of any internal structure. Consequently, there are very little known about the internal anatomy or histomorphology, especially of the reproductive systems, of this unique group of piscine ectoparasites. The slight degree of morphological difference between members of Chonopeltis is often used as the only criteria for the discrimination of species.
Lack of morphological detail and morphometric data in the taxonomic descriptions as well as in subsequent literature published on Chonopeltis spp., has inevitably led to confusion and much controversy as to, for instance, the phylogenetic origin, identity and functions of various structures. Furthermore, a meaningful statistic comparison and evaluation of the different species are presently not possible since the necessary comparable relative measurements as well as comparative information regarding anatomical variables, growth rates, seasonal variation, larval development, intermediate and primary hosts and even distribution, are unknown for the majority of species. The validity of the taxonomic identity or status, and the interrelationships of the various recognised species are consequently questionable, and it is most probable that many, or at least some, of the described species do not represent separate species, but are rather synonyms, different subspecies or even sibling species (cf. Chapter 3). Furthermore, lack of important morphological information has also caused a considerable number of gaps and deficiencies in our present understanding of various aspects of ecological and pathogenetic significance. Substantiating this, Schram (1986) stated: "by not having a 'name for it we lose the power to 'understand it' ".
Concluded from the present study, as well as a study of the literature, more detailed information based on adequate samples of all the species of Chonopeltis is needed. Furthermore, morphological and anatomical information should be obtained not only from macroscopical investigations of total preparations, but also by means of scanning electron microscopy (SEM), histological serial sections and graphic reconstruction. Within the cumulative research done on Chonopeltis spp., morphological but especially anatomical and histomorphological work has been largely neglected, with little information available on the
1186 morphology of the internal structures. Except for the study of Van Niekerk (1984), no detailed study has been published on the anatomy and histomorphology of either the male or the female reproductive systems as well as the mechanism of sperm transfer during copulation in any Chonopeltis species. Extensive and detailed morphological knowledge of these aspects is however, a prerequisite for not only understanding the functional reproductive adaptations needed for their specialized piscine ectoparasitic existence, but also to gain an insight into the functional significance of specific components of both the male and female reproductive systems during sperm transfer from the male genital atrium to the female spermathecae and consequential insemination of mature eggs. The latter is of particular interest since Chonopeltis spp. do not produce spermatophores and possess unique spermathecae which are entirely isolated from the main genital tract of the female (cf. Chapter 6 & 7).
Concerning the morphology of Chonopeltis, it is generally accepted that all species of Chonopeltis possess only one pair of antennae. However, no proof for this assumption could be found in the literature other than the vague description of a single pair of antennae (inconsequently referred to as antennae, first antennae or antennules) in the taxonomic descriptions of nine species, whilst a pair of antennae is merely included in line drawings of two of the other four species and not even mentioned nor illustrated for the remaining two species (cf. Chapter 4). During the present study, SEM investigation revealed a previously undescribed pair of minute biramous structures situated posteroproximally to the antennae of C. victori and C. australis. These structures may merely be superficial adornments but probably represent a reduced or vestigial pair of antennae. However, whether they represent superficial structures or, first or second antennae, only further histological investigations determining whether they are neuro-innervated or not, and if so, whether they are deutocerebrally or tritocerebrally innervated, would confirm. Since first and second antennae
are present in all three other branchiuran genera (i.e. Argulus, Dolops and Dipteropeltis) - as in all other Crustacea, it is most likely also the case in Chonopeltis, especially since the presumed additional pair of antennae as observed in C. victori and C. australis are very small, nearly hidden from view by the medial alal crests and thus easily overlooked - even with SEM.
It is evident from the literature that the terminology applied to the cephalic shield and its
187 extentions in branchiurans in general and in the genus Chonopeltis in particular, is inconsistent and confusing. During the present study, studies of histological serial sections of C. victori and C. australis revealed that many structures of the cephalic shield and its alae, such as ridges, grooves and rods, were incorrectly interpreted and consequently incorrectly named when described from total preparations. Since the "carapace" in Chonopeltis spp. consists of a median cephalon or cephalic shield and three wing-like (trifoliate) structures which are clearly separated from the cephalon by a horeshoe-shaped furrow (i.e. frontal and two lateral sulci), it is proposed that the more apt term "ala" should be used in preference to the term "lobe" (cf. Chapter 5). The question whether chonopeltid branchiurans do, in fact, have a carapace as defined by Calman (1909), i.e. "a structure ... originating as a fold of the integument from the posterior margin of the cephalic region", has also been investigated (cf. Chapter 5). Since the embryonic development of Chonopeltis species is direct without a nauplius stage, we investigated sections of pre-hatched and early post-hatched larvae of C. australis. These sections show that the alae develop as lateral outgrowths of the cephalic tegument, initially consisting of leaf-like structures composed of hypodermic layers separated by haemocoelic spaces. In the frontal ala no cephalic structures invade the haemocoelic spaces, but in the lateral alae the haemocoelic spaces soon become invaded by the aborizations of the midgut glands, parts of the maxillary gland and in males, even parts of the prostate gland. The lateral alae in C. australis appear to represent the fused pleurae of the cephalic segments. In accordance with the findings of Dahl (1991) in the Malacostraca and Branchiopoda, a carapace fold does not form in C. australis and possibly also not in the other three branchiuran genera, i.e. Argulus, Dolops and Dipteropeltis. It is therefore, suggested that the term "carapace" in Chonopeltis species be replaced by cephalic shield or cephalon, containing cephalic pleural folds, the alae.
Considering the morphological changes and adaptations associated with successful and optimal reproduction coupled with a parasitic way of life, we are confronted with two related, yet, opposite phenomena in branchiurans, and in particular in members of the genus Chonopeltis. Firstly, branchiurans possess in contrast to the majority of other crustaceans, motile flagellate spermatozoa (Fryer,1960b; Wingstrand,1972; Pochon-Masson,1983) whilst, secondly, sperm transfer in one branchiuran genus, Dolops, involves the employment of spermatophores
188 (Fryer,1958,1960b) although spermatophores are generally not produced in those Crustacea with flagellate motile spermatozoa (Pochon-Masson,1983; Mann,1984). Furthermore, the spermatophores formed in Dolops are produced by accessory reproductive glands (=paired prostate complex), also present in the non-spermatophore-producing Chonopeltis and Argulus, thus, implying a different function of the paired prostate complex in both the latter two genera. If it is assumed that the accessory reproductive glands in branchiurans are homologous structures (cf. Chapter 6), these glands (presumably secreting some kind of temporary sealant during copulation), were probably present in the primitive branchiuran ancestors. However, on the evolutionary line taken by Chonopeltis and Argulus, leading to the development of more complex copulatory structures and consequently less extensive copious secretion, a negative tendency towards spermatophore formation and thus specialization of the accessory glands probably occurred, whilst the opposite process most likely took place in Dolops. Concluded from a comprehensive investigation on the anatomy and histomorphology of the various components of both the male and female reproductive systems in C. victori and C. australis, the secretion of the paired prostate complex in the non-spermatophore-producing branchiurans (i.e. Chonopeltis and Argulus), probably fulfils more than one important function during sperm transfer - including acting as a temporary sealant which prevents semen from escaping from the ejaculatory ducts into the genital atrium (cf. Chapter 6). Other functions involved are probably similar to those executed by the prostate gland in other animal groups (i.e. enhancing motility and fertility of sperm; counteracts acidity of other fluids; lubricating passageways and providing an additional energy source for sperm).
Taking the morphology of the female reproductive system in branchiurans in general and Chonopeltis in particular also into consideration (Chapter 7), the following questions involuntarily come to mind: why is there only one ovary but two oviducts (of which only one is functional) present; how do the eggs pass from the ovary into the functional oviduct; and how is sperm transfer from the male genital opening to the female spermathecae and consequential insemination of mature eggs accomplished in Chonopeltis which does not produce spermatophores and possesses unique spermathecae which are entirely isolated from the main genital tract of the female?
189 Concluded from a thorough examination of the histomorphology of the various components of the female reproductive system in C. victori and C. australis (cf. Chapter 7), as well as considering all the literature published on the female reproductive system in branchiurans, the problem concerning the presence of a single median ovary but two oviducts in some bra. nchiurans remains unsolved. Whether the single ovary in adult branchiuran females is derived from a single or paired anlagen in the larva as suggested by various other authors (e.g. Claus,1875; Grobben,1908; Anderson,1982), only a thorough ontogenetic study of the female gonads could verify.
Concerning the movement of eggs from the ovary into the functional oviduct, the detailed histomorphological investigation of the female reproductive system in C. victori and C. australis (Chapter 7) reveals the single ovary to be an ovoid tubular organ enclosed by a peritoneal sheath or "gonodal sac" which separates the circumgenital body cavity from the haemocoel. The ovary contains an epithelial-lined lumen, the ovarian sac, which is anteriorly continuous with the lumen of the functional oviduct. Oogonia apparently leave the germinal zone (i.e. a longitudinal ridge of mesodermal gonadal tissue and oogonia extending along the entire length of the ovary), to enter the space between the gonadal and ovarian sacs where oocyte maturation and vitellogenesis occur. Ovulation follows vitellogenesis, and in C. victori and C. australis, postvitellogenic oocytes face the basal membrane of the epithelium lining the ovarian lumen, prior to ovulation. Oocytes next move to the ovarian lumen (=ovarian sac) and become completely wrapped in lumenal epithelium. After ovalation, whilst residing in the ovarian lumen, a thick tertiary envelope (shell) is formed around the oocytes. Thereafter, shelled oocytes move from the ovarian lumen (ovarian sac) to the anteriorly situated functional oviduct. The ovarian sac thus directly connects the ovary and functional oviduct in Chonopeltis. It therefore appears that the functional oviduct in Chonopeltis spp. functions as an ovisac, storing eggs prior to fertilization and oviposition, rather than a true oviduct which usually is responsible for the formation of tertiary membranes around eggs.
Two oviducts have been described in various species of Argulus and Dolops (e.g. Claus,1875; Martin,1932; Avenant-Oldewage & van As,1990). In all these cases, only one of the two oviducts is reported functional with its lumen in direct communication with that of the genital
190 atrium, whilst the non-functioning oviduct is blind-ending with its lumen not obliterated though it has a deflated or collapsed appearance. Since branchiurans appear to have more than one breeding period a year, it is probable that the two oviducts may function alternately to allow sequential restoration of the cellular material in the epithelial lining of the oviducts. However, the posterior end of the non-functioning oviduct in C. victori and C. australis, as in various Dolops spp., is not connected with the genital atrium. This suggests that the non-functional oviduct in Chonopeltis, and probably also Dolops, more likely represents a rudimentary or vestigial structure.
Concluded from the results of the present study as well as the fact that no spermatophores are formed in Chonopeltis, it is suggested that during copulation sperm transfer from the male genital atrium to the female spermathecae, involves the following process. During copulation, contraction of the muscular walls of the vasa deferentia and prostate ducts causes sperm and prostate secretion to be pumped into the ejaculatory ducts which are then closed off by sphincters, resulting in the development of a high internal pressure. The two spermathecal spines of the female are presumably inserted into the male genital atrium and then, simultaneously or singly, pushed through the thin epithelial layer separating the lumina of the genital atrium and the two opposing ejaculatory ducts. Thereafter semen would, due to the higher pressure in the ejaculatory ducts as well as on contraction of the muscular walls of the ejaculatory ducts, flow from each ejaculatory duct, down the apical groove and through the opening in each spermathecal spine and then, via the spermathecal duct, into the spermathecal vesicle.
Although substantiating ultrastructural studies need to be done on various aspects of gametogenesis and fertilization, insemination of mature eggs seems to be accomplished by means of the spermathecal spines which penetrate and inject stored semen into each egg during deposition. During oviposition, shelled eggs entering the fertilization chamber are probably maneuvered by the natatory lobes and positioned underneath the spermathecal spines so that sperm can be injected into the eggs.
Concerning the reproductive pattern, life cycle, sex ratio, growth patterns and hence also the
191 prevalence, mean intensity and abundance of branchiurans on fish host species - these aspects appear to be influenced by the fluctuations in annual physical environmental conditions (cf. Chapter 8). This, consequently, implies seasonality of some sort within branchiuran populations. From the results obtained during the present study, the reproductive cycle, hence population numbers, structure and distribution as well as infestation of C. victori, appear to be directly linked to fluctuations in the average water temperatures (cf. Chapter 8). Furthermore, the results from the present study suggest that the natural population of C. victori in the Olifants River should not be regarded as a continuously reproducing population. Females seem to leave the fish hosts to deposit one or more batches of eggs between early to late summer and between late autumn to mid-winter. Although this implicates oviposition in C. victori to be seasonal, gravid females were present throughout the year which suggests ovarium development (=the production of eggs) to be non-seasonal. Notwithstanding this, it is however possible that ovarium development could be seasonal with the eggs stored in the ovarium until the required optimum temperature for oviposition has been reached. To clarify this, further indepth investigation into the development of the ovarium is essential.
When comparing the results obtained from the four different sampling localities in the study area, it appears as though the water quality of the specific aquatic habitat has a profound influence on not only the presence/absence of specific fish species, but also on the presence/absence of specific fish parasites. In this regard, the poorer water quality of the lower Selati River as compared to the Olifants River (cf. Seymore et a/.,1994; Buermann et al., 1995), seems to be one of the reasons why not only fewer fish host specimens - but also no C. victori specimens, were found at the sample locality in the lower Selati River (cf. Chapter 8).
It is also evident from the results of the present study that host specificity in C. victori has to do more with specific factors rather than sheer numbers of fish. In this sense, fish movement, parasite feeding trends and the specific immune response of each fish host species should be considered. Similarly, various aspects of the biology (including the ontogeny), reproductive behaviour, host preferences of both larvae and adults, epidemiology and all other aspects of the ecology of these parasites need to be thoroughly investigated. Chonopeltis infestations
192 should be maintained in the laboratory and monitored daily in order to establish where and how mating and oviposition take place, to study fecundity, egg development as well as the hatching and development of larvae. More detailed knowledge of all aspects of branchiuran crustaceans are not only of academic value, but are also of considerable ecological and economical importance since these piscine ectoparasites bear a potential threat to both natural fish populations and the aquaculture industry.
In conclusion, the extent and nature of the present study have been determined to a great extent by our present state of knowledge regarding the biology of Chonopeltis in general, and C. victori and C. australis in particular. Due to the lack of adequate detailed morphological, but especially anatomical and histomorphological information on this unique group of piscine ectoparasites, the initial wide extent of the present study was at an early stage more specific delimited and some of the planned aspects were omitted in favour of a more profound investigation of others. Consequently a number of new questions arose. By viewing the results of the present study in perspective against our existing state of knowledge regarding the Branchiura in general and Chonopeltis in particular, many deficiencies could clearly be designed and in this way the commission of possible future research, viz. the incisive investigation of specific aspects of the biology of members of the Branchiura, concisely defined.
193 " The earth is the Lord's, and the fulness thereof; the world, and they that dwell therein. For he path founded it upon the seas, and established it upon the floods. "
- Psalm 24 :1-2 CHAPTER 1
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220 This it It and I am It and you are It And so is That aid he is It And she is It and it is It d that is That
- James Broughton