Aspects of the biology of Ergasilus sarsi a gill ectoparasite of Lamprichthys tanganicanus from Lake Tanganyika.
By
ESMARI KILIAN
DISSERTATION
Submitted in fulfilment
of the requirements for the degree
MASTER OF SCIENCE In ZOOLOGY in the FACULTY OF SCIENCE at the UNIVERSITY OF JOHANNESBURG
SUPERVISOR: PROF. A. AVENANT-OLDEWAGE October 2012
i ACKNOWLEDGEMENTS
I would like to acknowledge the following people:
- Professor A. Avenant- Oldewage for her support and guidance during the study. - The National Research Foundation (NRF) and the University of Johannesburg for funding the study. - Tannie Edie Lutch for all her help during the practical part of my study. - Mr. Ebrahim Karim of the Graphic department of the University of Johannesburg for the making of photo plates. - My family and friends for emotional support and encouragement to succeed.
ii ABSTRACT
A literature survey revealed gaps in the knowledge on Ergasilus in Africa. This studied aimed to elucidate on some of these matters.
Ergasilus sarsi was collected from Lamprichthys tanganicanus during an expedition to Lake Tanganyika in March 2010. The gills of the fish were removed and preserved for further studies. Preserved specimens were studied with dissection- and light microscopy. Some specimens were also studied with a scanning electron microscope (SEM). For light microscopy the specimens were sectioned and then stained with AZAN and H&E.
A total of 32 Lamprichthys tanganicanus were collected and studied for ergasilids. The prevalence was 86.40%, the mean intensity 7.56 and the mean abundance 6.38. A total of 204 parasites were collected and only 27 hosts were infected. The highest intensity was 29 parasites. Pearson’s Chi-squared test was used to compare attachment preferences of Ergasilus sarsi. The parasite exhibited site selection but not host specificity. It was noticed that E. sarsi mostly attach to the tip of the gill filament of the second gill arch. The second gill arch receives the largest portion of water flow supporting distribution of newly hatch ergasilid nauplius. There was no significant preference for the dorsal, medial and ventral attachment sites (p-value = 0.000542). However, significant preference between distal, central and proximal regions (p-value = 1.19) was observed.
Fryer (1965) observed that the pathology caused by ergasilids is related to their attachment position on the host. Ergasilids display variation regarding morphology of their second antennae. Some have spines and others elongated antennae that wraps around the entire gill filament. This study shows that Ergasilus sarsi (with no spines on the second antennae) wraps around the gill filament and cause considerable damage to the host. The entire gill filament structure changed due to lamellar fusion and proliferation of mucous – and epithelial cells. Comparison of an infected gill to a healthy gill revealed differences. The compression caused by the second antennae caused some blood vessels to rupture resulting in haemorrhage. The swimming legs of the parasite also cause considerable damage by scraping gill tissue off the host and pushing it towards the mouth parts of the parasite. Mucous cells, gill epithelium and blood cells were observed in the vicinity of the mouth and in the intestine of the parasite. An increase in the number of Rodlet cells and mast cells were also observed on the gills in close proximity to the parasite. Increase in the number of these cells clearly indicate an inflammatory response.
iii This is the first record of Ergasilus sarsi on Lamprichthys tanganicanus. This study also provides the first detailed description of the pathology caused by Ergasilus sarsi as well as the attachment distribution of this parasite.
The shortcomings of the study and future study areas are discussed in the last chapter.
iv OPSOMMING
‘n Literatuur oorsig het gapings in die kennis oor Ergasilus in Afrika bloot gelê. Hierdie studie het probeer om van die gapings te vul.
Ergasilus sarsi is gevind op Lamprichthys tanganicanus tydens ‘n ekspedisie na Tanganjika- meer in Maart 2010. Die kieue van die vis was verwyder en fikseer vir verdere studies. Eksemplare was bestudeer met behulp van disseksie- en ligmikroskopie. Sommige eksemplare was ook bestudeer met ‘n skandeerelektronmikroskoop (SEM). Eksemplare vir ligmikroskopie was in hars ingebed en seriesneë is gemaak. Sneë was gekleur met AZAN en H&E.
‘n Totaal van 32 Lamprichthys tanganicanus is versamel en ondersoek vir verteenwoordigers van die Ergasilidae en daar is gevind dit is besmet met Ergasilus sarsi. Die persentasie besmetting was 86.40%, die gemiddelde besmettingsintensiteit was 7.56 en die besmettingsmoontlikheid was 6.38. ‘n Totaal van 204 parasiete was versamel en 27 gashere was besmet. Die hoogste besmettingsintensiteit was 29 parasiete. Pearson se Chi- kwadraat-toets was gebruik om vashegtings voorkeure van Ergasilus sarsi te vergelyk. Die parasiet toon ‘n voorkeur vir die vashegtings area maar nie vir die gasheer spesie nie. Ergasilus sarsi heg meestal aan die punte van die kieu-filamente van die tweede kieuboog vas. Die tweede kieuboog ontvang die grootste porsie van watervloei wat waarskynlik bydra tot die verspreiding van die nauplii wanneer hul uitbroei. Daar was geen beduidende voorkeur tussen die dorsale, median en ventrale vashegtings areas nie (p-waarde = 0.000542). Beduidende voorkeur tussen die distale, sentrale en proksimale streke (p-waarde = 1.19) was egter waargeneem.
Fryer (1965) het waargeneem dat die patologie wat deur verteenwoordigers van die genus Ergasilus veroorsaak word afhang van die vashegtingposisie op die gasheer. Parasiete verskil ten opsigte van die morfologie van hul tweede antennas. Sommige het hakies en ander is langer wat hul instaat stel om, om die kieufilamente te vou. Die studie wys hoe Ergasilus sarsi (met geen hakies op die tweede antenna) om die kieufilament vou en sodoende aansienlike skade veroorsaak. Die hele kieufilament struktuur word verander as gevolg van lamellêre samesmelting en proliferasie van mukus-en epiteelselle. Vergelyking van 'n besmette kieu met ‘n gesonde kieu vertoon groot verskille. Die kompressie wat veroorsaak word deur die tweede antennas veroorsaak dat sommige bloedvate bars wat lei tot bloeding. Swempote van die parasiet het ook aansienlike skade veroorsaak deur die kieuweefsel van die gasheer af te skraap en dan voorentoe te druk na die mond gedeeltes van die parasiet. Slymselle, kieu-epiteel en bloedselle is waargeneem in die omgewing van
v die parasiet se mond sowel as in die intestinum van die parasiet. ‘n Toename in die aantal Rodletselle en mastselle is waargeneem op die kieue in die omgewing van die parasiet. ‘n Toename in dié selle dui op ‘n inflamatoriese reaksie van die gasheer.
Dit is die eerste rekord van Ergasilus sarsi op Lamprichtys tanganicanus, in Tanganjika- meer. Hierdie studie verskaf ook die eerste gedetallieerde beskrywing van die patologie wat veroorsaak word deur E. sarsi asook die besmettings statistiek van die parasiet.
Tekortkominge van die studie sowel as moontlike toekomstige studies word in die laaste samevattende hoofstuk bespreek.
vi TABLE OF CONTENTS
ABSTRACT ...... III OPSOMMING ...... V LIST OF FIGURES ...... VII LIST OF TABLES...... IX 1 GENERAL INTRODUCTION ...... 2
1.1 MORPHOLOGY AND ECOLOGY ...... 3 1.1.1 Background on Taxonomy ...... 3 1.1.2 Background on Ergasilus ...... 6 1.1.3 Background on the species found in Lake Tanganyika ...... 27 1.2 OBJECTIVES OF THIS STUDY ...... 28 1.2.1 Outline of the dissertation ...... 29 1.2.2 Oral presentations ...... 30 1.2.3 Special awards received during this study ...... 31 2 PATHOLOGY AND INFECTION STATISTICS ...... 33
2.1 INTRODUCTION ...... 33 2.2 MATERIALS AND METHODS: ...... 34 2.3 RESULTS ...... 37 2.3.1 Species identification ...... 37 2.3.2 Infection Statistics ...... 37 2.4 PATHOLOGY ...... 41 2.5 DISCUSSION ...... 48 2.6 CONCLUSION ...... 50 3 SUMMATIVE DISCUSSION AND FUTURE RESEARCH ...... 52 4 GENERAL REFERENCE ...... 57
LIST OF FIGURES
Figure 1: Diagrammatic representation of Ergasilus sp. life cycle. A. Ergasilid nauplii
(Redrawn from Kabata, 1976). B1. Male copepodid/ free- swimming male (Redrawn from
Henderson, 1926). B2. Female copepodid/ free- swimming female (Redrawn from Henderson, 1926). C. Young parasitic female already attached to host (Redrawn from Henderson, 1926). D. Fully developed adult female with egg sacs. (Redrawn from Hoffman, 1977) ...... 8 Figure 2: Diagram illustrating the 4 main regions of a mature female Ergasilus sieboldi von Nordmann, 1832 1) Cephalothorax; 2) Free Thorax; 3) Genital segment and 4) Abdomen. (Redrawn from Kabata, 1979) ...... 10 Figure 3: Diagram showing the large anterior shield (In blue) fused to the bases of the head part of the cephalothorax. (Redrawn from Wilson, 1911) ...... 11 Figure 4: Mouthparts of Ergasilus sp. consist out of the: Labrum (La.), Labium (Lb.), Mandibles (Md.), 1st Maxilla (Mx1.), 2nd Maxilla (Mx2.). (Redrawn from Wilson, 1911) ...... 11
vii Figure 5: Digestive system of Ergasilus sp. A) Median lobe of stomach; B) Diagonal lobe; C) Lateral lobe; D) Excretory tubes; I) Intestine; R) Rectum; S) Stomach. (Redrawn from Wilson, 1911) ...... 12 Figure 6: A) Ergasilus flaccidus. A Ventral view of abdomen (a) and lateral view of second antennae (b). B) Ergasilus kandti. Dorsal view of the cephalo thorax and thoracic segments (a), ventral view of abdomen (b), and ventral view of the second antennae (c). C) Ergasilus sarsi. Dorsal view of the cephalo thorax and the thoracic segments (a), ventral view of the abdomen (b), and lateral view of the second antennae (c). D) Ergasilus megacheir. Dorsal view of the cephalo thorax and thoracic segments (a), ventral view of abdomen (b), and lateral view of second antennae (c). (Redrawn from Fryer 1965) ...... 28 Figure 7: A) A map of Africa to indicate the position of Lake Tanganyika. B) A map of Lake Tanganyika showing the sample sites...... 35 Figure 8: Photomicrograph of a gill filament indicating the six divided regions to determine the exact attachment area of ergasilids. V (ventral), M (medial), Do (dorsal), Di (distal), C (central) and P (proximal)...... 36 Figure 9: A bar chart illustrating the parasite load on the left and the right gills...... 38 Figure 10: A bar chart illustrating the distribution of parasites on the long- axe of the gill filament...... 39 Figure 11: A bar chart illustrating the distribution of parasites on the short- axe attachment sites...... 39 Figure 12: The distribution of the parasites across the four gill arches are illustrated by the bar chart...... 40 Figure 13: In this three dimensional bar chart the overall distribution is illustrated...... 40 Figure 14: A) A micrograph showing the attachment of the parasite, Ergasilus sarsi, to the primary lamella. White arrow indicates the 2nd antennae. The white arrow shows the 2nd antennae that are wrapped around the filament. B) All parasites were observed at the tip of the filament (circles)...... 42 Figure 15: Photomicrographs of gill tissue of Lamprichthys tanganicanus in close proximity of an attached Ergasilus sarsi to show the histology of a normal gill filament stained with AZAN. A) The circle indicates the tissue that becomes eroded when a parasite attaches (see fig B & C). Epithelium lifting can be observed (white arrow). B) Micrograph shows that second antennae (black arrow) embrace the gill filament and proliferation of mucous cells occur (colourless arrow). Compression of the gill filament is also indicated (white arrow). Tissue eroded away in the area indicated by the striped arrow. C) Micrograph to show the pathology caused by attachment. The parasite maxilipede is inserted into the gill tissue (indicated by arrows). D) Micrograph showing secondary lamellar fusion (circle). Parasite egg sacks are indicated by the white arrow...... 43 Figure 16: Specimens stained with AZAN. A) Micrograph indicating a ruptured blood vessel (white arrow) and haemorrhage (black arrow). Tissue eroded in the area of striped arrow. The colourless arrow indicates mucous strand with blood cells. B) Photomicrograph indicating the inflammatory response of the host. All arrows show Rodlet cells and mast cells. C) Micrograph shows second antennae inserted into the filament (black arrows) Compression of the gill filament is also indicated (white arrow). D) This micrograph shows the same type of cell (Red blood cell shown by the circle) that is found in the intestine (Fig 15 A). The white arrow indicates the mouth parts and in the vicinity of the mouth parts around the parasite, Ergasilus sarsi, is lose gill tissue with RBC and mucous (dashed circle)...... 44
viii Figure 17: Specimens stained with AZAN. A) Micrograph with to show red blood cell (small circle) in the intestine (large broken line circle) of Ergasilus sarsi. B) Micrograph of a longitudinal section through the gills to show the difference between unaffected gill lamellae (white arrow) and affected gill lamellae (black arrow). C) The gill lamellae is altered by the insertion of the second antennae (white arrow) and the adjacent gill filament in also affected and mucous cell proliferation is clearly visible (black arrow). Parasite is encircled. The circle indicates a red blood cell within the intestine of the ergasilid. D) The second antennae also push the gill tissue towards the mouth of the parasite (white arrow). Parasite is encircled. The black arrow indicates the intestine of the parasite...... 45 Figure 18: Specimens stained with Hemotoxylin and Eosin is represented in the micrographs above. A) Ergasilus sarsi (encircled) has separated some of the gill tissue (white arrows) using the swimming legs (black arrows). B) The gill tissue in A contains red blood cells (white arrows) covered in a mucous layer (black arrow).The striped arrow indicates the swimming leg of the parasite. C) A micrograph of an infected gill showing numerous amounts of mucous cells (black arrows), mast cells (white arrows) and red blood cells (striped arrows).D) Secondary lamellar fusion (white arrows) and haemorrhage (black arrows) is evident in this micrograph...... 46 Figure 19: A) Scanning electron micrograph of parasites attached to the terminal ends of the filaments (circles). The striped arrow shows that the gill filament adjacent to the parasite is also covered by mucous. The white arrow shows an unaffected gill filament. B) Scanning electron micrograph showing E. sarsi attachment of ventral view, secondary antennae (white arrow); gill filament erosion (striped arrow). C) Scanning electron micrograph showing Ergasilus sarsi attachment dorsal view (circle). Adjacent filament covered by mucous (striped arrow) and the swimming legs (white arrow). D) Swimming legs of E. sarsi covered by mucous (arrows)...... 47
LIST OF TABLES
Table 1: List of all host and distribution records for Ergasilus found in Africa...... 16 Table 2: Table indicating the abundance, mean intensity, and prevalence of Ergasilus sarsi collected from three study sites from Lake Tanganyika. (All answers were rounded to the nearest decimal place) ...... 38
ix GENERAL INTRODUCTION
CHAPTER 1
General Introduction
CHAPTER 1 1 GENERAL INTRODUCTION
1 GENERAL INTRODUCTION
Lake Tanganyika is the oldest Lake in Africa and according to Brichard (1989) it is probably one of the oldest in the world. The lake was formed during the Miocene period approximately 20 million years ago. According to Brichard (1989) it is approximately 700 km long and has a mean width of 50 km. The water depth is +/- 1500 m and the annual runoff is a small 7%. The lake has a remarkable sediment pile up of 7000 m which covers millions of years of history. Lake Tanganyika has a major economical function to provide food for most of the villagers living alongside the lake.
Few surveys of the parasite fauna have been conducted in Lake Tanganyika and those date back to 1909 when Sars described some of the first African Ergasilidae and added a new genus, Ergasiloides to the family. Three species of Ergasiloides from Lake Tanganyika have been described during the Sars survey and they are: Ergasiloides macrodactylus, Ergasiloides brevimanus, and Ergasiloides megacheir. Cunnington conducted a survey in 1914, but it wasn’t before 1920 he described Ergasilus sarsi and Ergasiloides megacheir. However, he omitted the host identification. The next survey was conducted twenty years later by Capart (1944) who studied Ergasilus kandti, Ergasiloides megacheir and Ergasilus sarsi.
Fryer (1965) preformed an intensive study of the material collected during the Capart expedition and thereafter provided a comprehensive review of the biology of ergasilids in Lake Tanganyika. Almost 30 years later Coulter (1991) studied the copepod fauna of Lake Tanganyika and listed 68 species and subspecies which include the free-living and parasitic copepods. In a subsequent study that focused on the shift in life habitat of the parasites, Boxshall et al., (2006) also reported an extremely diverse fauna of both free-living and parasitic copepods.
Although Douëllou and Erlwanger (1994) assert that parasitic crustaceans in Africa have received considerable attention compared to other parasites, still little remains known on some of these parasitic copepods. They are of opinion that it is of great importance that they should be studied as they have implications on the fish fauna but also on an economical level through the trading industry.
CHAPTER 1 2 GENERAL INTRODUCTION
1.1 Morphology and Ecology
1.1.1 Background on Taxonomy
1.1.1.1 Information on the Family Ergasilidae
The family Ergasilidae comprises of over 260 nominal species (Boxshall et al., 2006; The World of Copepods, 2012). von Nordmann (1832) established the family Ergasilidae and it consisted of two genera namely; Ergasilus and Bomolochus. He provided a review of Ergasilus and three Ergasilus species including developmental stages and only a single species of Bomolochus.
Thereafter, Burmeister (1833) established the family Ergasilina which he classified with the Caligina and Lernaeoda and included Nordmann’s genera, Ergasilus and Bomolochus as well as Lamproglena. This arrangement was not long lasting as the family Ergasilina was expanded to include all the above genera (Wilson 1911). Edwards (1840) reclassified the family to Pachycephala and subdivided it into 2 subfamilies: “Dichelestiens and Ergasiliens” (sic). The latter consisted of the genera Ergasilus, Bomolochus and Nicothoë. This grouping forms the basis of the classification of the ergasilid family as it is known today.
Thorell (1859) introduced general copepod systematization that divides it into three different groups: 1) Gnathostoma; containing free mandibles, three pairs of maxillae without a siphon. 2) Poecilostoma; no mandibles, three pairs or no maxillae, no siphon. 3) Siphonostoma; mouth produced into a siphon enclosing the mandibles. Thorell (1859) placed the family Ergasilidae into the second group but this was not entirely factual and Claus (1864) provided factual evidence that the genus Ergasilus possesses mandibles.
Kröyer (1863) added 4 new species to Bomolochus and 4 to Ergasilus. Some of the species placed in the genus Ergasilus did not entirely fit. The species described as Ergasilus gasterostei was previously described as Thersites gasterostei by Pagenstecher (1861) two years before Kröyer. Many of the samples collected by Kröyer were incorrectly described and according to Wilson (1911), Kröyer’s contribution serves chiefly as a source of material for correction.
CHAPTER 1 3 GENERAL INTRODUCTION
Claparède (1870) corroborated the statement by Claus (1864) regarding the mandibles but he added that the mandibles may be greatly reduced and are not used for chewing. He also made attempts at changing the classification but did not add any significant new information.
During the following year Sumpf (1871) established another genus namely Taeniacanthus in the family Ergasilidae. He revised the mouth parts comparing them between the different genera. He also agreed with Claus (1864) that Ergasilus definitely contain mandibles and maxillae.
Gerstaecker (1881) combined the previous classification systems. In this revised classification he placed the family Ergasilidae among tenants and half parasites, between the Corycaeidae and the Ascomyzontidae. It included all genera of the Ergasilidae in a separate family, Lichomolgidae. This family contains only semi parasitic copepods hence including the Ergasilidae. Canu (1892) adopted the general arrangement of Claus and Thorell but expanded the copepods into six divisions instead.
1. Calanoida: Free-living and pelagic. 2. Harpacticoida: Free-living but demersal. 3. Cyclopoida: Partly free-living, commensally & fresh water species. 4. Notodelphyoida: Partly parasitic and partly free. 5. Caligoida: Parasitic upon fishes, moderately degenerate and some freedom of motion. 6. Lernaeoida: Fish parasites, strongly degenerate, fixed in position, and marked sexual dimorphism.
This arrangement of Canu is based on habitat and mode of life as it is sustained by morphological differences. Wilson (1911) concluded the following regarding the classification of copepods:
1) Copepods that are free- swimming (free- living) and have mouth parts which allows them to bite and chew, must be grouped separately. 2) Copepods that are parasitic should be grouped together. These parasites should also have mouth parts that are suited for either piercing or tearing which must be enclosed in a tube or a siphon. 3) Between the above mentioned groups there is a group that includes all the free- swimming, commensals, messmates, complete- and semi parasites. The mouth parts of the organisms in this group are in the process of transforming and contain no tube or siphon.
CHAPTER 1 4 GENERAL INTRODUCTION
Wilson placed the family Ergasilidae in the last group.
Thatcher and Boeger (1984) added a subfamily Abergasilinae to the family with the following characteristics: The female has only 3 pairs of swimming legs and the second antennae bears only 3 segments. Ergasilidae females have 4-5 pairs of swimming legs and 4-5 segments on their second antennae.
1.1.1.2 The development evolution of the genera
Nordmann (1832) classified two genera, Ergasilus and Bomolucus, in the newly established family Ergasilidae but by 1902 two additional genera were included namely Ergasiloides and Thersites Pagenstecher (Sars, 1909). The genus Ergasiloides together with the genus Ergasilus Von Nordmann, 1932, Bomolucus Von Nordmann, 1932, and Thersites Pagenstecher then constituted the family Ergasilidae.
Sars (1909) described the genus Ergasiloides as organisms that contain a reduced number of segments in the urosome of both males and females, whereas, in the equivalent life stage in Ergasilus, the female has four well defined segments and the male has five. Females have bodies that are sub- depressed in the anterior end and are attenuated at the posterior end. The male is more slender than the female. According to Fryer (1965) the difference between Ergasilus and Ergasiloides involved nothing more than the loss of an abdominal somite (urosome) or the fusion thereof. This trait was present in species that were classified as Ergasiloides. In some crustaceans groups this feature is of great significance but was not expected in parasites that are more or less permanently attached to their host.
The first abdominal somite (9th post cephalic segment) bears a reduced pair of segments which are the genital segments. The last abdominal somite (19th post cephalic segment) contains the anus (Stachowitsch, 1992). In adult ergasilids some post cephalic segments are fused. Fryer (1965) states that Ergasilus affinities could best be described and expressed by the union of these two genera. He further held the opinion that a genus should be determined by a phylogenetic unit and not a genetic one and he therefore synonymised the genera (Fryer, 1965). The genus Ergasilus thereafter included the former Ergasilus and Ergasiloides.
CHAPTER 1 5 GENERAL INTRODUCTION
Fryer (1965) then redefined the new genus as organism with the following characteristics: Poecilostomous cyclopoid copepods with parasitic females. In some sense this supports the Canu scheme created in 1892.
1.1.1.3 General Classification
Crustacean parasites include the Ostracoda, Isopoda, Amphipoda and Copepoda. The Ergasilidae is a large family of parasitic copepods which comprises of 17 genera with more than 282 species (The World of Copepods, 2012). About 140 of these species are found in fresh water (Abdelhalim et al., 1993; The World of Copepods, 2012). Ergasilus sp. is ever- present according to Kabata (1984). The current classification for the genus Ergasilus is as follows (The World of Copepods, 2012):
Kingdom: Anamalia Phylum: Arthropoda Subphylum: Crustacea Class: Maxillopoda Subclass: Copepoda Order: Poecilostomatoida Family: Ergasilidae Genus: Ergasilus von Nordmann, 1832
1.1.2 Background on Ergasilus
1.1.2.1 Habitat and Life cycle (Figure 1)
Ergasilids are present in almost all types of water bodies from subterranean waters to large ancient lakes (Boxshall & Defaye, 2008). Parasitism usually occurs on the gills of freshwater fish but has been reported on the gills of some marine fishes. There have also been reports of Ergasilus on the skin of fish (Rogers & Hawke, 1978; Yamashita, 1980).
Only the female Ergasilus is parasitic and becomes more or less fixed upon her host (more or less because she is able to move up or down the gill filament or transfer from one to the other). The male ergasilid remains free living and die off after mating. A female reaches sexual maturity at a very early stage and mating takes place while the female is still free swimming. According to Kabata (1976) the free swimming stages of the females are shorter and less complicated due to their adaptation to parasitism. The free swimming stages are
CHAPTER 1 6 GENERAL INTRODUCTION vitally important in the life cycle, not only because of reproduction, but it also aids in the dispersal of the species and the search for a new host. Without this the life cycle cannot be completed (Kabata, 1976). During the free swimming stages the copepodids feed on nanoplankton (Raibaut, 1985).
These copepods have a single moulting stage from the ergasilid nauplii to the ergasilid copepodid (Kabata, 1976). Development to sexual maturity takes about 10- 70 days depending on the conditions (Hoffman, 1977); fast when the temperature is above 27°C and slows when the temperature is below 14°C (Hoffman, 1977). According to Lahav et al. (1964) temperature affect the rate of development profoundly. When sexual maturity is reached mating will occur and will then be the infective stage.
After mating the female awaits an opportunity to infect a host. She is also capable of swimming around seeking out a host but this tends to be less common. Fish become infected when swimming through a group of copepodids or when fish are feeding (Kabata, 1976). According to Paperna (1996) there is a great variance in host specificity. Some ergasilids are specific to the host genus level where others are less specific and more opportunistic. Ergasilus sarsi shows the least specificity but still displays some predilection (Paperna, 1996).
The attached female enters into the egg-production stage (Henderson, 1926) which hatches in 3-6 days (Lahav & Sarig, 1967; Hoffman, 1977). The number of eggs produced varies between species and is influenced by the age and metabolic health of the female (Paperna, 1996). Females can live up to 1 year on their host (Hoffman, 1977) and produce 2-3 batches of eggs during this period (Lahav & Sarig, 1967).
The gills provide the parasite with the perfect habitat. There is excellent aeration for the eggs and a good position from which she can discharge the nauplii; there is also an abundance of food (Wilson, 1911).
CHAPTER 1 7 GENERAL INTRODUCTION
A. Nauplius ♂ ♀
B1. Male B2. Female Copepodid Copepodid
♀ ♀
C. Young female
D. Adult female
Figure 1: Diagrammatic representation of Ergasilus sp. life cycle. A. Ergasilid nauplii
(Redrawn from Kabata, 1976). B1. Male copepodid/ free- swimming male (Redrawn from
Henderson, 1926). B2. Female copepodid/ free- swimming female (Redrawn from Henderson, 1926). C. Young parasitic female already attached to host (Redrawn from Henderson, 1926). D. Fully developed adult female with egg sacs. (Redrawn from Hoffman, 1977)
CHAPTER 1 8 GENERAL INTRODUCTION
1.1.2.2 General Morphology of Ergasilus
Hoffman (1977) stated that these parasites resemble Cyclops due to the fact that their bodies narrow at the posterior end. The adult female’s cephalothorax is fused with the somite of the first leg. The four succeeding thoracic somites are distinct, sometimes confluent with especially those of legs four and five. The abdomen consists of two or more somites and is usually short with the genital somite being the largest. Segmentation of the abdomen is not always distinct. The Ergasilus body ends in what Wilson (1911) refers to as a “telson” (sic) with simple furcal rami with terminal setae with the inner setae being the longest. The antennae are short with up to 6 segments and the antenna is conspicuous (Wilson, 1911; Kabata, 1979; Abdelhalim et al., 1993).
The mouth parts of the female are minute and located near the centre of the cephalothorax. The mandibles are saw- like and the maxillule is vestigial bearing 2 spines. The tip of the maxilla is armed with minute spinules on an anteriorly- directed lobe. The maxilliped in Ergasilus females is absent. Legs 1 to 4 have broad coxae and bases. Leg 4 has an exopod with two segments while the other exopods and endopods are three segmented. Egg sacs are elongated, paired, and ovoid in shape with the eggs being small and many (Wilson, 1911; Kabata, 1979; Abdelhalim et al., 1993; Pinto da Motta Amado & Falavigna da Rocha, 2001).
The Ergasilus male is not parasitic and is much smaller than the female. The body is cyclopid and the antennae prehensile but still smaller than that of the female. The mouthparts are similar to that of the female but the maxilliped is present with the terminal segment that is long and curved. The rami of all the swimming legs are segmented (three segments). The male abdomen has one more segment than the female (Wilson, 1911; Kabata, 1979).
CHAPTER 1 9 GENERAL INTRODUCTION
1.
2.
3.
♀ 4.
Figure 2: Diagram illustrating the 4 main regions of a mature female Ergasilus sieboldi von Nordmann, 1832 1) Cephalothorax; 2) Free Thorax; 3) Genital segment and 4) Abdomen. (Redrawn from Kabata, 1979)
1.1.2.3 General body form
The female ergasilid body consist of four regions (Figure 2): 1. Cephalothorax 2. Free thorax 3. Genital segments 4. Abdomen
The large anterior shield (Figure 3) is connected to the bases of the second antennae. Eyes are located close to the anterior margin of the carapace and are fused on the mid- line near the ventral surface. The inner margins are heavily pigmented and in contact with one another. The outer margins are clear and transparent (Wilson, 1911). This shield also known as the cephalosome forms a well defined boundary separating it from the first somite (Abdelhalim et al., 1993)
CHAPTER 1 10 GENERAL INTRODUCTION
Figure 3: Diagram showing the large anterior shield (In blue) fused to the bases of the head part of the cephalothorax. (Redrawn from Wilson, 1911)
1.1.2.4 Mouth-parts Consist of the labrum, labium, mandibles, first maxilla, and second maxilla. The labrum is fused with the ventral surface of the carapace and the mandibles are located inside the mouth (Wilson, 1911; Kabata, 1979). According to Abdelhalim et al., (1993) ergasilids lack a true palp and the mandibles consist of a coxa and a gnathobase. The latter comprise of three toothed blades which assist the maxilla in scraping of gill tissue (Abdelhalim et al., 1993) (Figure 4)
Md. Md. 1 Mx1. La. Mx .
2 2 Mx . Mx . Lb.
Figure 4: Mouthparts of Ergasilus sp. consist out of the: Labrum (La.), Labium (Lb.), Mandibles (Md.), 1st Maxilla (Mx1.), 2nd Maxilla (Mx2.). (Redrawn from Wilson, 1911)
1.1.2.5 Digestive system Consist of a stomach, median lobe of the stomach, diagonal lobe, lateral lobe, and excretory tubes (Wilson, 1911). (Figure 5)
CHAPTER 1 11 GENERAL INTRODUCTION
A
B
S D E C
I
R
Figure 5: Digestive system of Ergasilus sp. A) Median lobe of stomach; B) Diagonal lobe; C) Lateral lobe; D) Excretory tubes; I) Intestine; R) Rectum; S) Stomach. (Redrawn from Wilson, 1911)
1.1.2.6 Prehension
The female ergasilid attaches by means of the second antennae and maxillipedes. Both these appendages have powerful muscles and some species bear spines and roughened surfaces. When the female becomes parasitic the basal joint of the second antennae becomes inflated (Henderson, 1926). There are no lunules or sucking disks present in this genus. The body of the parasite always lies parallel with the gill filament, with the head of the parasite towards the base of the filament or gill arch (Fryer, 1965).
CHAPTER 1 12 GENERAL INTRODUCTION
1.1.2.7 Locomotion
The female ergasilid is capable of moving from one filament to the next with the aid of the four well developed pairs of swimming legs (Wilson, 1911; Abdelhalim et al., 1993). However, she loses the swimming setae after attachment (Henderson 1926). Parasites are usually attached towards the ends of the gill arches leaving the medial filaments free, but this might not be true for all species. According to Wilson (1911), the main reason for the parasite to choose the peripheral areas of the filaments is that the water movements are less rapid on the sides than in the medial region. The parasite can move forward towards the branchial arches by creating a vacuum with the cephalothorax, when the vacuum is broken the parasite can push itself forward (Einszporn, 1965). This might be necessary for feeding purposes as will be explained later on.
1.1.2.8 Food
Ergasilid mouth parts are not fit for biting or chewing but are suited for piercing the delicate tissue of the gill and the adult females are able to digest blood, mucous and epithelial tissue (Wilson, 1911). He also states that there is no doubt that ergasilids feed on blood. Henderson (1926) disagreed with Wilson and stated that the parasite only feeds on the excessive mucous that is produced by the host due to its immune response. Smith (1949) agreed with Henderson and stated that the mouth parts of ergasilids are too small to cause any damage to tissue.
The parasite uses oral appendages to cut their food (Wilson, 1911; Kabata, 1979; Abdelhalim et al., 1993); furthermore the swimming appendages play an important role in dislodging the food through rhythmical movements (Wilson, 1911). This movement causes the destruction of the tissue leaving individual cells detached. Blood vessels are exposed by the movement of the cephalothorax (that creates a vacuum on the gill filament) that detaches tissue from gill lamellae (Einszporn, 1965). The first pair of swimming legs bears long bristles extending to the oral pore, this helps to push the food closer to the mouthparts (Einszporn, 1965).
1.1.2.9 General Pathology
Attachment and feeding by ergasilid females can cause an extensive range of pathological alterations on the gills. Once the female ergasilid attaches to the gill filament, compression of
CHAPTER 1 13 GENERAL INTRODUCTION the tissue occur ensuring firm attachment to the host (Oldewage & van As, 1987). This was particularly obvious in Ergasilus mirabilis (Oldewage & van As, 1987). It has been reported on various occasions that ergasilids feed on gill tissue (Einszporn, 1965). The lesions caused by the parasite may become infected by bacteria, fungi and virus growth resulting in secondary infection. An inflammatory response by the host with increased Rodlet cells, mucous cell number (Dezfuli et al., 2003) and cellular proliferation has been reported (Roubal, 1989).
Rodlet cells were first described by Thélohan (1892) as protozoan parasites and Laguesse (1895) classified these protozoan parasites as Rhabdospora thélohani. Plehn (1906) disagreed that these cells are protozoan parasites and claimed that they are part of the fish secretory cells. Rodlet cells were said to be associated with the fish epithelial tissue (Mayberry et al., 1979; Morrison et al., 1978). Catton (1951) and Duthie (1983) claim that Rodlet cells are modified granulocytes that discharge their content at the surface of the epithelium. Leino (1974) stated that these cells have a similar function than mucous cells and are involved in water and electrolyte transport. The cells react to the presence of parasites on the epithelial surfaces and have an antibiotic substance that is secreted to damp the infection (Leino, 1979). Reite (1997) suggested that Rodlet cells are very similar to Mast cells which are involved in the defence mechanisms of the host. Reite (2005) found that Rodlet cells show a form of degranulation in response to acute damage but there is an increase in the number of Rodlet cells during chronic inflammatory reactions.
According to Schmachtenberg (2007) Rodlet cells are part of the non- specific immune response of the fish and they are migratory cells found in every organ that have been investigated. Rodlet cells are cells with a noticeable, enclosed cell cortex and the nature and distribution of these cells has led to an uncertainty on its function (Dezfuli et al., 2000). Dezfuli et al., (2000) reported that Rodlet cells play an important role in fish inflammatory response. In the presence of ectoparasites Rodlet cells react by secreting an antibiotic substance (Leino, 1996). The function is not fully known but it correlates with the fish inflammatory- reactive network (Dezfuli et al., 2000).
In Ergasilus mirabilis the extent of pathological alterations was linked to attachment (Oldewage and van As, 1987). Each species has a unique manner of attachment to the gill filament; some simply embrace the gill filament whereas others insert the entire third segment of the second antennae into the filament (Thatcher & Boeger, 1983). Cellular proliferation is a result of extensive feeding and the parasite must feed regularly to sustain its life style (Oldewage & van As, 1987). According to Paperna (1996) feeding involves the
CHAPTER 1 14 GENERAL INTRODUCTION secretion of proteolytic enzymes that aid in external digestion. The attachment of the parasite can cause the two main blood vessels of the gill filament to become compressed. This may cause hypoxia of the filament and during periods of high infection may result in death (Hoffmann, 1977). The number of gills affected can also be linked to the severity and the damage caused (Abdelhalim, 1990).
1.1.2.10 Hosts in Africa
The Ergasilidae is widely distributed and is not very host specific (Wilson, 1911) table 1 (adapted from Oldewage & Avenant- Oldewage, 1993) below represents a list of all host and distribution records for Ergasilus found in Africa. [*All host scientific names are updated from Fish-Base (2012)]
CHAPTER 1 15 GENERAL INTRODUCTION
Table 1: List of all host and distribution records for Ergasilus found in Africa.
Suborder: Poecilostomatoida
Family: Ergasilidae PARASITE DISTRIBUTION HOSTS * REFERENCE River Galma, small lakes around Ergasilus cunningtoni Capart, 1944 Zaria, Nigeria Brycinus nurse Rüppell, 1832 Shotter, 1977 Raiamas sengalensis (Steindachner, 1870) “ Hydrocynus forskahlii (Cuvier, 1819) “ Barbus macrops Boulenger, 1911 “ Mormyrus macropthalmus Günther, 1866 “ Mormyrops anguilloides (Linnaeus, 1758) “ Ergasilus egyptiacus Abdel-Hady, Bayoumy & Osman, Abdel-Hady, Bayoumy & 2008 Lake Temsah Tilapia zilli (Gervais, 1848) Osman, 2008 Ergasilus flaccidus Fryer, 1965 Lake Tanganyika Oreochromis tanganyicae (Günther, 1894) Fryer 1965 Ergasilus ilani Oldewage & Van As, 1988 Sodwanna Estaury, Sodwanna Mugil cephalus Linnaeus, 1758 Oldewage & van As, 1988 Bay Natal, SA “ Kowie River Estaury, Cape Province, SA “ Berg River, Western Cape Liza richardsonii (Smith, 1846) Oldewage & van As, 1988
CHAPTER 1 16 GENERAL INTRODUCTION
Table 1: List of all host and distribution records for Ergasilus found in Africa.
Suborder: Poecilostomatoida
Family: Ergasilidae PARASITE DISTRIBUTION HOSTS * REFERENCE Province, SA Verlorens Vlei River, Cape Province, SA Liza richardsonii (Smith, 1846) “ Ergasilus inflatupes Cressey, 1971 Volta River, Ghana Strongylura senegalensis (Valenciennes, 1846) Cressey & Collette, 1971 Ebzia Lagoon, Ivory Coast Strongylura senegalensis (Valenciennes, 1846) Cressey & Collette, 1971 Ergasilus kandti van Douwe, 1912 Lake Albert Bagrus bajad (Forsskul, 1775) Thurston, 1970 Lates albertianus (Linnaeus, 1758) “ Lake Albert Lates niloticus (Linnaeus, 1758) Fryer, 1965 Gourao, Sudan Lates niloticus (Linnaeus, 1758) Capart, 1956 Lower Nile Lates niloticus (Linnaeus, 1758) Fryer, 1968a Niger River-system Lates niloticus (Linnaeus, 1758) Fryer, 1968a Lake Tanganyika Pseudosimochromis curviforns (Poll, 1942) Capart, 1944 Lake Tanganyika Limnotilapia dardennii (Boulenger, 1899) Fryer, 1965 Plecodus paradoxus Boulenger, 1898 “ Oreochromis tanganyicae (Günther, 1894) “ Lake Tanganyika Pseudosimochromis curviforns (Poll, 1942) Capart, 1944 Lamprologus lemairii Boulenger, 1899 “
CHAPTER 1 17 GENERAL INTRODUCTION
Table 1: List of all host and distribution records for Ergasilus found in Africa.
Suborder: Poecilostomatoida
Family: Ergasilidae PARASITE DISTRIBUTION HOSTS * REFERENCE Lake Tumba, Lower Zaire Pelmatochromis congicus (Boulenger, 1897) Fryer, 1964a Lake Albert (In Plankton) van Douwe, 1912 Lake Albert - Cunnington, 1920 Black & White Volta confluence Lates niloticus (Linnaeus, 1758) Paperna, 1969 Hemisvnodontis sp. “ Synodontis senegalensis “ Citharinus citharus (Geoffroy Saint- Hilaire, 1809) “ Ergasilus lamellifer Fryer,1961 Lake Victoria Astatoreochromis alluaudi Pellegrin, 1904 Thurston, 1970 Holotilapia retrodens (Hilgendorf, 1888) “ Haplochromis bicolour (Boulenger, 1906 “ Haplochromis degeni (Boulenger, 1906) “ Haplochromis guiarti (Boulenger, 1906) “ Prognathochromis longirostris (Hilgendorf, 1888) “ Haplochromis nuchisquamulatus (Hilgendorf, 1888) “ Haplochromis obesus (Boulenger, 1906) “ Haplochromis obliquidens (Hilgendorf, 1888) “
CHAPTER 1 18 GENERAL INTRODUCTION
Table 1: List of all host and distribution records for Ergasilus found in Africa.
Suborder: Poecilostomatoida
Family: Ergasilidae PARASITE DISTRIBUTION HOSTS * REFERENCE Haplochromis sp. “ Victoria Nile Haplochromis spp. Fryer, 1961 Nile River - Fryer, 1968 Yeji area, Lake Victoria Physalia pellucid Boulenger, 1901 Paperna, 1969 Ergasilus latus Fryer, 1960 Ghana Tilapia spp. Thurston, 1970 Niki River- system Tilapia sp. Fryer, 1968 River Galma, small lakes around Zaria, Nigeria Oreochromis niloticus (Linnaeus, 1758) Shotter, 1977 Tilapia zillii (Gervais, 1848) “ Sarotherodon galilaeus (Linnaeus, 1758) “ Schilbe mystus (Linnaeus, 1758) “ Auchenoglanis occidentalis (Valenciennes, 1840) “ Kitona, near Banana, Zaire mouth Tilapia cabrae Boulenger, 1899 Fryer, 1963 Lake Turkana - Fryer, 1968 Lake Turkana Oreochromis niloticus (Linnaeus, 1758) Fryer, 1960 Sarotherodon galilaeus (Linnaeus, 1758) “ Afram Basin, Volta Lake Tilapia zillii (Gervais, 1848) Paperna, 1969
CHAPTER 1 19 GENERAL INTRODUCTION
Table 1: List of all host and distribution records for Ergasilus found in Africa.
Suborder: Poecilostomatoida
Family: Ergasilidae PARASITE DISTRIBUTION HOSTS * REFERENCE Mawli River, North Ghana Tilapia zillii (Gervais, 1848) “ Peshi Lagoon, east of Accra, Ghana Tilapia guineensis (Günther, 1862) “ Peshi Lagoon, Kete-Krachi area, Sarotherodon melanotheron heudelotii (Duméril, Lake Volta 1861) “ Ergasilus lizae Krøyer, 1863 Lake Ischkeul, Tunisia Liza ramada (Risso, 1827) “ Liza saliens (Risso, 1810) “ Mugil cephalus Linnaeus, 1758 “ Barbus barbus (Linnaeus, 1758) “ Alosa fallax (Lacepéde, 1803) “ Raïbaut, Ben- Hassine & Gulf of Gebès, Tunisia Liza saliens (Risso, 1810) Maamouri, 1971 Lake Ischkeul, Tunisia Solea solea (Linnaeus, 1758) “ Lake Tunis, Tunisia Mugil cephalus Linnaeus, 1758 Ben-Hassine, 1974 Ergasilus macrodactylus (Sars, 1909) Lake Malawi Haplochromis spp. Fryer, 1956 Tilapia spp. “
CHAPTER 1 20 GENERAL INTRODUCTION
Table 1: List of all host and distribution records for Ergasilus found in Africa.
Suborder: Poecilostomatoida
Family: Ergasilidae PARASITE DISTRIBUTION HOSTS * REFERENCE Lethrinops spp. “ Ergasilus megacheir (Sars, 1909) Lake Tanganyika Cyphotilapia frontosa (Boulenger, 1906) Fryer, 1965 Bathybates minor Boulenger, 1906 “ Bathybates fasciatus Boulenger, 1901 “ Haplotaxodon microlepis Boulenger, 1906 “ Plecodus paradoxus Boulenger 1898 “ Limnotilapia dardennii (Boulenger, 1899) “ Synodontis multipunctata Boulenger, 1898 “ Synodontis granulosus Boulenger, 1900 “ Lake Tanganyika Simochromis sp. Capart, 1944 Lake Tumba, Lower Congo Pterochromis congicus (Boulenger, 1877) Fryer, 1964 Lake Tanganyika - Cunnington, 1920 Sumbo, south- western shore of Lake Tanganyika - Sars, 1909 Ergasilus mirabilis Oldewage & Van As, 1987 Pongola flood plain Natal, SA Synodontis leopardina Pellegrin, 1914 Oldewage & van As, 1988 Barbus afrohamiltoni Crass, 1960 “
CHAPTER 1 21 GENERAL INTRODUCTION
Table 1: List of all host and distribution records for Ergasilus found in Africa.
Suborder: Poecilostomatoida
Family: Ergasilidae PARASITE DISTRIBUTION HOSTS * REFERENCE Glossogobius giuris (Hamilton, 1822) “ Labeo rosae Steindachner, 1894 “ Brycinus imberi (Peters, 1852) “ Hydrocynus vittatus Castelnau, 1861 “ Clarias gariepinus (Burchell, 1822) “ Clarias ngamensis Castelnau, 1861 “ Schilbe intermedius Rüppell, 1832 “ Zambezi River, Caprivi Strip Schilbe mystus (Linnaeus, 1758) Oldewage & van As, 1988 Clarias gariepinus (Burchell, 1822) “ “ Clarias ngamensis Castelnau, 1861 “ Synodontis nigromaculata Boulenger, 1911 “ Synodontis leopardina Pellegrin, 1914 “ Synodontis macrostigma Boulenger, 1911 “ Hemichromis elongates (Guichenot, 1861)
CHAPTER 1 22 GENERAL INTRODUCTION
Table 1: List of all host and distribution records for Ergasilus found in Africa.
Suborder: Poecilostomatoida
Family: Ergasilidae PARASITE DISTRIBUTION HOSTS * REFERENCE
Lake Liambezi, Chobe River Hepsetus odoe (Bloch, 1794) Oldewage & van As, 1988 Caprivi Strip Schilbe intermedius Rüppell, 1832 “ Lake Lizekele Marcusenius macrolepidotus (Peters, 1852) Oldewage & van As, 1988 “ Synodontis nigromaculata Boulenger, 1905 “ Synodontis leopardina Pellegrin, 1914
Chobe River, Caprivi Strip Schilbe intermedius Rüppell, 1832 Oldewage & van As, 1988
Limpopo River, SA Synodontis zambezensis Peters, 1852 Oldewage & van As, 1988
Lake Malawi Synodontis zambezensis Peters, 1852 Oldewage & van As, 1988
Lake Kariba, Zimbabwe Hippopotamyrus discorhynchus (Peters, 1852) Douvellou (pers. Comm.)
Ergasilus nodosus Wilson, 1928 White Nile, Omdurman, Sudan Bagrus bajad (Forsskăl, 1775) Wilson, 1928
Sielo Tuni Stream, Ghana Bagrus sp. Fryer, 1964b
Lake Tumba, Zaire Congo Campylomormyrus elephas (Boulenger, 1898) Capart, 1944
Lake Tumba, Lower Congo Distichodus stouentralis Fryer, 1964a
CHAPTER 1 23 GENERAL INTRODUCTION
Table 1: List of all host and distribution records for Ergasilus found in Africa.
Suborder: Poecilostomatoida
Family: Ergasilidae PARASITE DISTRIBUTION HOSTS * REFERENCE “ Tylochromis lateralis (Boulenger, 1898) “ Tylochromis microdon Regan, 1920 “ Pterochromis congicus (Boulenger, 1897) Ikeia, Zaire System Petrophalus grandoculis Boulenger, 1920 Fryer, 1964a “ Hippopotamyrus psittacus (Boulenger, 1897) “ Synodontis nigriventris (cf. E. cunningtoni) Black & White Volta confluence, Lake Volta Brycinus leuciscus (Günther, 1867) Paperna, 1969 “ Pellonula afzeluizi Johnels, 1954 “ Yeji area, Lake Victoria Brycinus nurse (Rüppell, 1832) “ Phago loricatus Günther, 1865 River Galma, small lakes around Ergasilus sarsi Capart, 1944 Zaria, Nigeria Clarias anguillaris (Linnaeus, 1758) Shotter, 1977
CHAPTER 1 24 GENERAL INTRODUCTION
Table 1: List of all host and distribution records for Ergasilus found in Africa.
Suborder: Poecilostomatoida
Family: Ergasilidae PARASITE DISTRIBUTION HOSTS * REFERENCE Heterobranchus bidorsalis Geoffroy Saint- Hibiire, “ 1809 “ Lake Bangwelu Marcusenius macrolepidotus (Peters, 1852) “ Synodontis nigromaculata Boulenger, 1905 Clarias ngamensis Castelnau, 1861 “
Lake Mweru, Katanga Tylochromis mylodon Regan, 1920 Capart, 1944 Lubundiy, Katanga Tylochromis mylodon Regan, 1920 Capart, 1944 Malwi River tributary, Lake Volta Clarias gariepinus (Burchell, 1822) Paperna, 1969 “ Pseudotropheus spp. “ Brycinus imberi (Peters, 1852) Lake Tanganyika - Cunnington, 1920 Lake Tanganyika - Capart, 1944 Sumbo, south- western shore of Lake Tanganyika - Sars, 1909
CHAPTER 1 25 GENERAL INTRODUCTION
Table 1: List of all host and distribution records for Ergasilus found in Africa.
Suborder: Poecilostomatoida
Family: Ergasilidae PARASITE DISTRIBUTION HOSTS * REFERENCE Ergasilus sieboldi Von Nordmann, 1832 Inland brackish waters, Tunisia Mullets Paperna, 1969 “ Mugil cephalus Linnaeus, 1758 “ Liza aurata (Risso, 1810) “ Liza ramada (Risso, 1827) “ Liza saliens (Risso, 1810) Goukamma River mouth, Knysna, SA Mugil cephalus Linnaeus, 1758 Oldewage & van As, 1988
Keurbooms River mouth, SA Mugil cephalus Linnaeus, 1758 Oldewage & van As, 1988
CHAPTER 1 26 GENERAL INTRODUCTION
1.1.3 Background on the species found in Lake Tanganyika
Four species of Ergasilus occur in Lake Tanganyika i.e. Ergasilus flaccidus Fryer, 1965; Ergasilus kandti van Douwe, 1912 (Capart, 1944; Fryer, 1965), Ergasilus megacheir (Sars, 1909) (Cunnington, 1920; Capart, 1944; Fryer, 1965), Ergasilus sarsi Capart, 1944 (Sars, 1909; Cunnington, 1920; Capart, 1944). The four ergasilid species are drawn (Figure 6).
Differences are based on the following characteristics: E. flaccidus is about 900 µm with a longer than wide and bluntly rounded anterior. Inverted T of thickened chitin is present dorsally with no posterior marking. Somites of legs 2 to 5 are distinct with somite 5 being narrow with no distinct tergite (Fryer, 1965).
Ergasilus megacheir anterior is narrow with two distinct circles and an inverted T dorsally. The cephalothorax and thoracic segments are long. Abdomen is very small and inner setae very long. The second antenna has a small terminal hook. Fifth leg is extremely small and narrow.
Ergasilus kandti has an anterior similar to E. flaccidus. Two small circles mark the cephalothorax. Abdomen is long with a thick base. Second antenna has a small hook on the base of the second last segment.
Ergasilus sarsi has a rounded anterior with an indent at the second thoracic segment. The ventral view of the abdomen is short and similar to E. megacheir but inner setae are almost similar to the other species. The lateral view of the second antenna shows that E. sarsi has no small hook present. The antenna is very small and segments can be seen very clearly (Oldewage & van As, 1988). Ergasilus sarsi is the only species out of these four that has a lateral spine on leg 5 (Fryer, 1965).
All ergasilid species do not attach to the same site on the gill filaments and also differ in how they attach to the filament. Some Ergasilus species simply embrace the gill filament (Oldewage & van As, 1987) but others, e.g. E. colomeus Thatcher & Boeger insert the third part of the antennae and the entire claw into the host filament (Thatcher & Boeger, 1983). Fryer (1965) reported that E. kandti and E. megacheir coexist on the same host. Ergasilus kandti is located at the basal third and never at the tip of the gill filament, whereas E. megacheir is located at the tip of the filament. Ergasilus sarsi is also located at the tip of the filament and E. flaccidus near the gill arch.
CHAPTER 1 27 GENERAL INTRODUCTION
A B
c b
b
a a
C D
b b
c a
a c
Figure 6: A) Ergasilus flaccidus. A Ventral view of abdomen (a) and lateral view of second antennae (b). B) Ergasilus kandti. Dorsal view of the cephalo thorax and thoracic segments (a), ventral view of abdomen (b), and ventral view of the second antennae (c). C) Ergasilus sarsi. Dorsal view of the cephalo thorax and the thoracic segments (a), ventral view of the abdomen (b), and lateral view of the second antennae (c). D) Ergasilus megacheir. Dorsal view of the cephalo thorax and thoracic segments (a), ventral view of abdomen (b), and lateral view of second antennae (c). (Redrawn from Fryer 1965)
1.2 Objectives of this study
As evident from the literature review many areas in Ergasilus research especially with regards to the pathology and the quantitative descriptors of infection requires additional attention. Fryer (1965) presented the most recent study on ergasilids from Lake Tanganyika. The most recent work done on African ergasilids was that by Oldewage and van As (1988).
Fryer (1965) described four species of Ergasilus from fishes in Lake Tanganyika. However, little emphasis was placed on the morphology or ecology of these parasites and even less on the pathology caused. The current study aims to address these topics and also to record a new host.
CHAPTER 1 28 GENERAL INTRODUCTION
Therefore, the study to conduct a detailed description of the parasite distribution on the samples collected in Lake Tanganyika during the March 2010 expedition are outlined as follows:
1. Identify the Ergasilus sp. on Lamprichthys tanganicanus: a. Study specimens with scanning electron microscope. b. Study specimens with dissecting microscope. c. Compare findings to existing literature. 2. Describe the infection: a. Study all the gills and record the parasite position. b. Perform statistical analysis to determine preference for attachment site. 3. Determine pathological alterations: a. Study pathology with scanning electron microscope. b. Study parasites and host from histological preparations. c. Describe pathology with light microscopy.
The hypotheses set out for this study based on the objectives were as follows.
1. The Ergasilus sp. found on Lamprichthys tanganicanus in Lake Tanganyika is Ergasilus sarsi.
2. The parasite has an even distribution on all attachment sites.
3. The parasite causes severe pathological alterations to its host.
1.2.1 Outline of the dissertation
Based on the hypotheses and objectives given above the study was planned and executed the best way the author felt possible. Some parts of the dissertation were prepared for the European Association of Fish Pathologist hence all referencing was done in the format required by this journal. Different aspects of the study are discussed in the different sections. The document consists of the following sections:
Chapter 1 provides a general review of ergasilids in general. It provides background knowledge on biological aspects of the parasite and also consists of a review of all African ergasilids. All references are included in the end of the document to minimize repetition.
CHAPTER 1 29 GENERAL INTRODUCTION
Chapter 2 describes the aspects of the biology and infection statistics of Ergasilus sarsi a gill ecto parasite of Lamprichthys tanganicanus from Lake Tanganyika.
Chapter 3 gives a general discussion on this study and also provides ideas for future research to further elucidate on different unknown aspects of ergasilids in general.
Chapter 4 is a combined reference list of all references used during this study.
In addition to the publication of this dissertation the following outputs were delivered during the cause of the study.
1.2.2 Oral presentations
1. Kilian E. Observations on the pathology of ergasilids from Lake Tanganyika on the gills of Lamprichthys tanganicanus. Postgraduate symposium, Department of Zoology, University of Johannesburg. 16 July 2010. 2. Kilian E. en Avenant- Oldewage A. Beskrywing van aspekte van die patologie van Ergasilus (Crustacea: copepoda) indiwidue van Tanganijka-meer op die kieue van Lamprichthys tanganicanus. Die Suid-Afrikaanse Akademie vir Wetenskap en Kuns, Jaarkongres: Afdeling Biologiese Wetenskappe. Die Biosistematiek-afdeling van die Navorsingsinstituut vir Plantbeskerming (NIPB), Universiteit van Pretoria. 1 Oktober 2010. 3. Kilian E. and Avenant Oldewage A. Observations on the pathology of ergasilids from Lake Tanganyika on the gills of Lamprichthys tanganicanus. The 39th Annual Conference of the Parasitological Society of Southern Africa. KwaZulu- Natal. 11- 12 October 2010. 4. Kilian E. Aspects of the Biology of Ergasilus sp. found in Lake Tanganyika. Postgraduate symposium, Department of Zoology, University of Johannesburg. 10 July 2011. 5. Kilian E. en Avenant- Oldewage A. Verspreiding van verteenwoordigers van die genus Ergasilus op die kieue van Lamprichtys tanganicanus in Tanganjikameer. Die Suid-Afrikaanse Akademie vir Wetenskap en Kuns, Jaarkongres: Afdeling Biologiese Wetenskappe. Departement Dierkunde, Universiteit van Johannesburg. 5 September 2011. 6. Kilian E. and Avenant-Oldewage A. Ecological aspects of Ergasilus sp. collected from Lamprichthys tanganicanus from Lake Tanganyika. 8th International Symposium of Fish Parasites, Viňa del mar, Chile. 26- 30 September 2011.
CHAPTER 1 30 GENERAL INTRODUCTION
7. Kilian E. Aspects of the Biology of Ergasilus sp. found in Lake Tanganyika – Results. Postgraduate symposium, Department of Zoology, University of Johannesburg. 4 November 2011.
1.2.3 Special awards received during this study
1. University of Johannesburg merit bursary received on the bases of BSc. Honours mark obtained in 2010. 2. One of 3 honours students chosen to go to Hong Kong on an ecological field trip. 3. Best overall presenter at the Suid-Afrikaanse Akademie vir Wetenskap en Kuns, 2010. For the presentation: Beskrywing van aspekte van die patologie van Ergasilus (Crustacea: copepoda) indiwidue van Tanganijka-meer op die kieue van Lamprichthys tanganicanus. 4. Best junior poster presentation award by the Parasitological Society of Southern Africa. For the presentation: Ecological aspects of Ergasilus sp. collected from Lamprichthys tanganicanus from Lake Tanganyika, at the 39th Annual Conference of the Parasitological Society of Southern Africa. KwaZulu- Natal. 11- 12 October 2010. 5. Best Masters Poster presentation awarded by the Suid-Afrikaanse Akademie vir Wetenskap en Kuns, 2011. For the presentation: Verspreiding van verteenwoordigers van die genus Ergasilus op die kieue van Lamprichtys tanganicanus in Tanganjikameer.
Date of first registration January 2011.
CHAPTER 1 31 PATHOLOGY AND INFECTION STATISTICS
CHAPTER 2
Aspects of the pathology and infection statistics of Ergasilus sarsi a gill ectoparasite of Lamprichthys tanganicanus from Lake Tanganyika.
CHAPTER 2 32 PATHOLOGY AND INFECTION STATISTICS
2 PATHOLOGY AND INFECTION STATISTICS
2.1 Introduction
During a survey in Lake Tanganyika in 2010 ergasilids were collected from Lamprichthys tanganicanus Boulenger, 1989, also known as the Tanganyika Lamp-eye. This is a benthopelagic fish that is non- migratory and endemic to the shore regions of Lake Tanganyika (Huber, 1996).
Ergasilus von Nordmann, 1832 is mainly a gill parasite and is widely distributed throughout Africa. They are not very host specific (Wilson, 1911, Kabata, 1979) inhabiting fresh, marine and brackish water (Boxshall & Defaye, 2008). According to Fryer 4 ergasilid species occur in Lake Tanganyika and they are; Ergasilus flaccidus Fryer, 1965; Ergasilus kandti van Douwe, 1912, Ergasilus megacheir (Sars, 1909), Ergasilus sarsi Capart, 1944.
Only the female ergasilid is parasitic while the male remains free-swimming throughout his lifespan. Ergasilids are specialised parasites with large modified second antennae that are used to attach to the host. Attachment and feeding by ergasilid females can cause extensive pathology to the gills. Some Ergasilus species simply embraces the gill filament (Oldewage & van As, 1987) but others, e.g. E. colomeus Thatcher & Boeger, 1983 insert the third segment of the antennae into the host gill filament (Thatcher & Boeger, 1983).
Furthermore, ergasilids feed on gill tissue consisting of blood, mucous and gill epithelium (Einszporn, 1965). The parasite must feed regularly to sustain the energy levels required for the parasitic lifestyle (Oldewage & van As, 1987). According to Paperna (1996) feeding involves secretion of proteolytic enzymes that aids in external digestion.
Once the female attaches to the gill filament, compression of the tissue occurs ensuring that she becomes attached firmly (Oldewage & van As, 1987). The lesions caused by the parasite may become secondarily infected by bacteria, fungi and virus growth and an inflammatory response by the host is marked by an increase in Rodlet cells, mucous cell number (Dezfuli et al., 2003) and cellular proliferation of these cells (Roubal, 1988). The two main blood vessels of the gill filament become compressed and can cause hypoxia of the filament and during high infections may result in death of the host (Hoffmann, 1977). The number of gills affected is linked to the severity of the damage caused by the parasite
CHAPTER 2 33 PATHOLOGY AND INFECTION STATISTICS
(Abdelhalim, 1990). Impaired respiration may lead to decrease growth rate during an infestation period (Dezfuli et al., 2011).
Upon studying the 4 ergasilid species from Lake Tanganyika, Fryer (1965), noticed that each species attaches to a different location on the gill filament and that the pathology varies according to the attachment site. Ergasilus kandti is known to cause considerable damage to the gill tissue and its antennae become overgrown by the gill tissue. Ergasilus megacheir causes gill compression and makes a visible indent in the gill filament where the antennae embrace it (Fryer, 1965). When Fryer (1965) investigated E. sarsi, he concentrated on the morphology, taxonomy and ecology of the parasite but he did not describe the pathology leaving a gap in the pathological information on this species.
The aim of this study was to determine whether the parasite prefers a specific attachment site on the gills and to describe the pathological alterations caused by the attachment and feeding habits of the parasite.
2.2 Materials and Methods:
A total of 32 Lamprichthys tanganicanus Boulenger, 1898 specimens were collected with hand nets from Lake Tanganyika. Three study sites were selected namely, Kisokwe 4°14'31" S, 29°10'35" E (1), Mufazi 7°05'12" S, 29°54'45" E (2) and Mugayo 6°46'51" S, 29°33'42" E (3) (See Fig. 7).
CHAPTER 2 34 PATHOLOGY AND INFECTION STATISTICS
3
2
1
A B
Figure 7: A) A map of Africa to indicate the position of Lake Tanganyika. B) A map of Lake Tanganyika showing the sample sites.
The fish were killed, immediately after they were caught, by severing the spinal cord and after removal of the gills, parasite specimens were fixed intact on the gills in acetoformaldehyde alcohol (AFA) solution and preserved in 70% ethanol. The gill samples were examined for ectoparasites using a dissection microscope.
In the Parasitology Laboratory of the University of Johannesburg, parasites were identified to species level and the position of attachment on each gill arch was recorded. All gill arches were devided into six regions as indicated in Fig. 8, similar to Gelnar’s (1991) division.
CHAPTER 2 35 PATHOLOGY AND INFECTION STATISTICS
Di M
C Do
V P
Figure 8: Photomicrograph of a gill filament indicating the six divided regions to determine the exact attachment area of ergasilids. V (ventral), M (medial), Do (dorsal), Di (distal), C (central) and P (proximal).
Data regarding the site specificity of ergasilids on the gill filaments was complied and prepared for statistical analysis. A Pearson’s Chi- squared test was used to compare attachment on the left gill and right gill arches and to determine whether a significant difference between the dorsal, ventral, and medial attachment, as well as the distal, proximal, and central regions occur. Descriptive analysis was also undertaken.
Prevalence was determined by dividing the number of infected host individuals by the number of host examined and multiplied by 100 to express it as a percentage.
The Mean intensity was calculated as the total number of parasites in the sample divided by the number of infected hosts in the samples.
Abundance was calculated by dividing the number of parasites in the sample by the number of fish in the sample as suggested by Bush et al., (1997).
Ergasilid samples were prepared for Scanning Electron Microscopy and gill tissue samples for histology in order to study the morphology and pathology respectively. Firstly samples that were placed in 70% ethanol were hydrated to water prior to freeze-drying, gold coated and studied with a JEOL 5600 Scanning Electron Microscope.
CHAPTER 2 36 PATHOLOGY AND INFECTION STATISTICS
Secondly, remainder of the samples were transferred from 70% ethanol to 70% acetone prior to dehydration. Subsequently samples were infiltrated with TAAB Transmit LM resin and 5µm serial sections were made. Sections were stained with a Heidenhains trichrome stain and Hemotoxylin and Eosin (Humason, 1979) and studied and micrographed with an Axio plan 2 Zeiss light microscope.
2.3 Results
2.3.1 Species identification
Ergasilid parasites are identified through various means. The three most important aspects are the shape of the cephalothorax, the shape of the second antennae and the arrangement of the eggs within the egg sacs. Many ergasilids bears spines on their second antennae. Ergasilus sarsi is one of the few in the genus that do not have any spines on the second antennae. The appendages ergasilids use to obtain their food: the second antennae, the swimming legs and the cephalothorax are all important appendages for identification. According to Fryer (1965) the location of the parasite on the gill filament is also very specific for each species. Some ergasilids will attach close to the gill arch and others will attach closer to the tip of the filament. The ergasilid species collected from Lamprichthys tanganicanus was identified as Ergasilus sarsi based on the criteria above (Fig 12 A and B).
2.3.2 Infection Statistics
A total of 32 Lamprichthys tanganicanus were collected and studied for ergasilids. The prevalence was 86.40%, the mean intensity 7.56, and the mean abundance 6.38. The 204 parasites were unevenly distributed among the 27 infected hosts with some of the host being highly infected and some with a low infection. The highest intensity of 29 ergasilids was found on one host. The prevalence, mean intensity, and mean abundance for each site are provided in Table 2.
CHAPTER 2 37 PATHOLOGY AND INFECTION STATISTICS
Table 2: Table indicating the abundance, mean intensity, and prevalence of Ergasilus sarsi collected from three study sites from Lake Tanganyika. (All answers were rounded to the nearest decimal place) Minimum and Mean prevalence maximum intensity Abundance Mean intensity X100 = % for each site. Min Max
Mugayo 6.2 6.9 90% 1 29
Kisokwe 18 18 100% 18 18
Mufazi 5.6 7.75 70% 1 21 (Near Momba)
Pearson’s Chi-squared test was used to compare attachment preference of E. sarsi to either (Fig. 9) left or right gill arches, and to determine whether there are significant differences between attachment to dorsal, ventral, and medial areas as well as between distal, proximal, and central regions.
Results for preference for either gill arch indicated the E. sarsi do not have a preference for either one of the gill archers (p - 0.123485).
120 100
80 60 Observed 40
n of parasitesn Expected 20 0 Left Right Distribution over gills
Figure 9: A bar chart illustrating the parasite load on the left and the right gills.
Related to the distribution of E. sarsi on the gill arch (dorsal, median, ventral, distal, central and proximal), the Pearson Chi-Square indicated that there was no significant difference (p - 0.000542) between the attachment of E. sarsi of the selected regions, dorsal, median and ventral. However there were indeed significant differences (p – 1.19) between regions distal, central and proximal.
CHAPTER 2 38 PATHOLOGY AND INFECTION STATISTICS
Analysis was also done to test whether an equal amount of parasites occur on the 4 gill arches, significant differences (p – 7.88) were noted.
100
80 60 40 Observed
n of parasitesn 20 Expected 0 Dorsal Medial Ventral Long-axe attachment sites. (Horizontal plane)
Figure 10: A bar chart illustrating the distribution of parasites on the long- axe of the gill filament.
200
150
100 Observed
n of parasitesn 50 Expected 0 Distal Central Proximal Short- axe attachment sites (Vertical plane)
Figure 11: A bar chart illustrating the distribution of parasites on the short- axe attachment sites.
CHAPTER 2 39 PATHOLOGY AND INFECTION STATISTICS
100
80 60 40 Observed
n of parasites n 20 Expected 0 Gill1 Gill2 Gill3 Gill4 Distribution over the four gill arches
Figure 12: The distribution of the parasites across the four gill arches are illustrated by the bar chart.
Descriptive statistics to indicate attachment on all the sites are illustrated in the three dimensional bar chart (Fig. 13).
100
80
60 Distal 40 Central 20 Proximal 0
Dorsal Medial Ventral
Figure 13: In this three dimensional bar chart the overall distribution is illustrated.
It can clearly be seen from the results that there is not an equal distribution of parasites throughout all the attachment sites. However this is not true for the distribution between the left and right sides of the host. It should be kept in mind that the sample size was small and that a larger sample may provide different results.
CHAPTER 2 40 PATHOLOGY AND INFECTION STATISTICS
2.4 Pathology
Histological examinations of Lamprichthys tanganicanus gill tissue infected with E. sarsi yielded the following results. Mature female ergasilids attach to the primary gill lamellae with their second antennae that are modified into claw-like structures (Fig. 14A). All Ergasilus sarsi attach to the tip of the filament (Fig. 14B) confirming Fryer’s (1965) observations.
Figure 15A indicates what a healthy, unaffected gill looks like. Superficial tissue erosion of the gill occurred in the area surrounding the second antennae, maxillipedes and swimming legs (Fig.15B & C).The scanning electron micrograph (Fig.19D) shows that the swimming legs are covered by mucous. In Fig. 18B the swimming legs scraped off some gill tissue that consists out of mucous and red blood cells.
Epithelial hyperplasia occurred along the total length of the gill filament adjacent to the parasite and resulted in secondary fusion of gill lamellae (Fig.15D, 18D). Epithelial lifting of the gill filaments occurred in the proximity of the parasite (Fig. 15A).
Haemorrhaging caused by ruptured blood vessels can be observed in Fig. 16A, D and Fig. 18D. The extended production of mucous due to mucous cell proliferation caused blood cells to get caught in the mucous strands (Fig. 16D, 18B & C). Host gill epithelial tissue and blood cells were found to be present in the midgut and the buccal cavity of the parasite (Fig.16D and 17A).
According to the Scanning Electron Micrograph (Fig 19C), the female ergasilid parasite inserts her entire front part of her body into the gill filament. Fig. 19B shows how the second antennae of E. sarsi are wrapped around the gill filament of the host.
CHAPTER 2 41 PATHOLOGY AND INFECTION STATISTICS
Figure 14: A) A micrograph showing the attachment of the parasite, Ergasilus sarsi, to the primary lamella. White arrow indicates the 2nd antennae. The white arrow shows the 2nd antennae that are wrapped around the filament. B) All parasites were observed at the tip of the filament (circles).
CHAPTER 2 42 PATHOLOGY AND INFECTION STATISTICS
Figure 15: Photomicrographs of gill tissue of Lamprichthys tanganicanus in close proximity of an attached Ergasilus sarsi to show the histology of a normal gill filament stained with AZAN. A) The circle indicates the tissue that becomes eroded when a parasite attaches (see fig B & C). Epithelium lifting can be observed (white arrow). B) Micrograph shows that second antennae (black arrow) embrace the gill filament and proliferation of mucous cells occur (colourless arrow). Compression of the gill filament is also indicated (white arrow). Tissue eroded away in the area indicated by the striped arrow. C) Micrograph to show the pathology caused by attachment. The parasite maxilipede is inserted into the gill tissue (indicated by arrows). D) Micrograph showing secondary lamellar fusion (circle). Parasite egg sacks are indicated by the white arrow.
CHAPTER 2 43 PATHOLOGY AND INFECTION STATISTICS
Figure 16: Specimens stained with AZAN. A) Micrograph indicating a ruptured blood vessel (white arrow) and haemorrhage (black arrow). Tissue eroded in the area of striped arrow. The colourless arrow indicates mucous strand with blood cells. B) Photomicrograph indicating the inflammatory response of the host. All arrows show Rodlet cells and mast cells. C) Micrograph shows second antennae inserted into the filament (black arrows) Compression of the gill filament is also indicated (white arrow). D) This micrograph shows the same type of cell (Red blood cell shown by the circle) that is found in the intestine (Fig 15 A). The white arrow indicates the mouth parts and in the vicinity of the mouth parts around the parasite, Ergasilus sarsi, is lose gill tissue with RBC and mucous (dashed circle).
CHAPTER 2 44 PATHOLOGY AND INFECTION STATISTICS
Figure 17: Specimens stained with AZAN. A) Micrograph with to show red blood cell (small circle) in the intestine (large broken line circle) of Ergasilus sarsi. B) Micrograph of a longitudinal section through the gills to show the difference between unaffected gill lamellae (white arrow) and affected gill lamellae (black arrow). C) The gill lamellae is altered by the insertion of the second antennae (white arrow) and the adjacent gill filament in also affected and mucous cell proliferation is clearly visible (black arrow). Parasite is encircled. The circle indicates a red blood cell within the intestine of the ergasilid. D) The second antennae also push the gill tissue towards the mouth of the parasite (white arrow). Parasite is encircled. The black arrow indicates the intestine of the parasite.
CHAPTER 2 45 PATHOLOGY AND INFECTION STATISTICS
Figure 18: Specimens stained with Hemotoxylin and Eosin is represented in the micrographs above. A) Ergasilus sarsi (encircled) has separated some of the gill tissue (white arrows) using the swimming legs (black arrows). B) The gill tissue in A contains red blood cells (white arrows) covered in a mucous layer (black arrow).The striped arrow indicates the swimming leg of the parasite. C) A micrograph of an infected gill showing numerous amounts of mucous cells (black arrows), mast cells (white arrows) and red blood cells (striped arrows).D) Secondary lamellar fusion (white arrows) and haemorrhage (black arrows) is evident in this micrograph.
CHAPTER 2 46 PATHOLOGY AND INFECTION STATISTICS
Figure 19: A) Scanning electron micrograph of parasites attached to the terminal ends of the filaments (circles). The striped arrow shows that the gill filament adjacent to the parasite is also covered by mucous. The white arrow shows an unaffected gill filament. B) Scanning electron micrograph showing E. sarsi attachment of ventral view, secondary antennae (white arrow); gill filament erosion (striped arrow). C) Scanning electron micrograph showing Ergasilus sarsi attachment dorsal view (circle). Adjacent filament covered by mucous (striped arrow) and the swimming legs (white arrow). D) Swimming legs of E. sarsi covered by mucous (arrows).
CHAPTER 2 47 PATHOLOGY AND INFECTION STATISTICS
2.5 Discussion
Ergasilus sarsi was previously reported in Lake Tanganyika (Sars, 1909; Cunnington, 1920; Capart, 1944) but the host species were not reported, however, Ergasilus sarsi have been recorded on individuals of the families Clariidae and Mochokidae (Sars, 1909; Cunnington, 1920; Capart, 1944; Shotter, 1977). Ergasilus sarsi was found for the first time on Lamprichthys tanganicanus during the March 2010 expedition and this represents a new host record.
A correlation between the attachment site and pathology was already noted by Fryer (1965). Few studies on the comparison between the attachment and pathology have been done for ergasilids, especially for those found in African Lakes. Although comparison between the study represented in this paper and other studies are nearly impossible, many similar studies have been conducted around the world and those studies have been used for some comparison.
According to Turgut et al., (2006) the site preference of monogeneans can be related to difference in water current, reproduction and feeding. Austin et al., (2009) found that Lamproglena hoi preferred to attach to the second gill arch. According to that study the increased gill surface and water flow provides an ideal opportunity for attachment. The same can be said for ergasilids if the results obtained are taken into consideration from figure 11 it is clear that the parasite prefers gill arch number 2. This arch has strong water current that flows over it (Turgut et al., 2006) therefore the parasite should be well adapted for this niche environment. To enable ergasilid egg distribution into the water successfully, strong water current is needed. Gill arch 2 will ensure that the newly hatched nauplius stage ergasilids will enter the water column for distribution. By attaching to the tip of the gill filament the egg sacs are closer to the water column and so increase the infection probability.
Rhode and Rhode (2005) identified host range, micro and macro habitats of the host, geographical range, age, and sex of the host, and season as important variables to characterise the multi- dimensional environmental niche. A study conducted by Barse (1998) on Ergasilus manicatus proved that the prevalence was independent of the host sex. The prevalence however was not independent of the host length and the season. Unfortunately this study does not have sufficient data to support Barses’ statement as sampling only took place during March 2010 and the host sex and length was not recorded.
CHAPTER 2 48 PATHOLOGY AND INFECTION STATISTICS
There was no significant difference between the left side and the right side of the gills. Ergasilus sarsi do not have a gill side preference. In a similar study by Austin et al., (2009) on Lamproglena hoi there was also no preference for the left and right side of the gills. Preference for a particular side according to Rhode (1993) is due to the asymmetrical body shape of some parasites; however, Ergasilus sarsi has a bilateral symmetry.
Studies done on Lamproglena (Marx et al., 1996; Tsotetsi et al., 2004; Austin et al., 2009) showed that different species attaches to different sides on the gills. Bush et al., (2001) found that the fitness of a parasite is increased when it attaches to a site where it has adapted to attach. Keeping this statement in mind, Ergasilus sarsi has no spines on the second antennae the antennae are long enabling it to be wrapped around a gill filament. It also predominantly attach to the tip of the gill filament where the filament is narrower. This is clearly indicated by the results obtained. Ergasilus sarsi must ensure attachment otherwise she faces a great risk of being washed off the gills. This manner of attachment leads to severe pathological alteration on the gill filaments, changing the entire shape of the gill filament and ultimately the function of the gill.
Obstructed or ruptured blood vessels are the results of tissue erosion and degradation of gill tissue that extended beyond the epithelium lining (Paperna, 1996). Prolonged infection results in lamellar fusion.
Pathological alterations were observed alongside the entire length of the parasite. The swimming legs also caused damage additional to feeding and attachment of the parasite. The fish exhibit an inflammatory response to the infestation marked by a high number of Rodlet cells and mast cells, especially at the site of the copepods attachment (Dezfuli et al., 2003). In the results obtained in this study a high number of Rodlet cells and mast cells especially at the site of attachment were encountered.
The higher the intensity of parasites on a host the more pathological changes will occur (Oldewage & van As, 1987). Increases in ergasilid number may cause blood-flow restriction to such an extent that the fish might suffer from hypoxia (Oldewage & van As, 1987), if this is enhanced by copious amounts of mucous covering gas exchanging surface of the gills. Lamprichthys tanganicanus was highly infected with E. sarsi but showed no visible signs of hypoxia such as lethargic behaviour.
CHAPTER 2 49 PATHOLOGY AND INFECTION STATISTICS
A correlation between the attachment site and the pathology was observed during the study.
When fish respiration is impaired it causes reduce feeding that lead to weight loss and general deterioration of health. The reduced oxygen binding ability in tropical waters such as that found in Lake Tanganyika can speed up the deterioration of a host that is also infected.
According to Butler (2009) Lamprichthys tanganicanus is a very difficult fish to breed in captivity but still provides great enjoyment in a tropical tank because of its beauty. The pathological effects and the ecological distribution of the parasite are two very important aspects to be fully understood since it may impact the trading industry of these fish. If infected fish are exported the likelihood that the other fish within the tank will get infected is high, as ergasilids are not host specific and due to the confined tank the parasite abundance may increase and lead to great losses.
This study described the pathology of the parasite and highlights the extent to which infection may reach. Further studies must be conducted to determine prevention and treatment to forego the export of Lamprichthys tanganicanus for aquarium trade.
2.6 Conclusion
This study showed that Ergasilus sarsi predominantly attach to gill arch 2. This arch has the biggest surface providing the largest attachment area and it also receives the largest proportion of the water current. This increases the probability of successfully spreading of eggs to decrease interspecies competition.
A new host, Lamprichthys tanganicanus, was recorded and the pathology and infection statistics noted for Ergasilus sarsi.
Pathology that was eminent included cellular proliferation, hyperplasia and lamellar fusion. Compression caused by the attachment of Ergasilus sarsi ruptured the blood vessels causing haemorrhage.
CHAPTER 2 50 SUMMATIVE DISCUSSION AND FUTURE
CHAPTER 3
Summative discussion and future
CHAPTER 3 51 SUMMATIVE DISCUSSION AND FUTURE
3 SUMMATIVE DISCUSSION AND FUTURE RESEARCH
In chapter one a brief literature study of ergasilid biology in Africa was provided. Information pertaining the taxonomy, habitat and lifecycle, morphology, mouth parts, feeding, digestive system and movement were discussed. The objectives and hypotheses for this study were given.
In the literature review it was shown that the parasite distribution is mainly concentrated around the great lakes and rivers of Africa. This may reflect on the distribution of parasitologist rather than the parasites. This study focused on one of these great lakes; Lake Tanganyika where Fryer (1965) previously described 4 ergasilid species and it was shown that since then no studies have been conducted on ergasilids from Lake Tanganyika.
It was hypothesised that the ergasilid species sampled during this study is Ergasilus sarsi. After following a study of the literature this hypothesis was accepted.
Fryer (1965) recorded that each Ergasilus sp. attaches differently to their host. Thatcher and Boeger (1983) elaborated on this and showed that Ergasilus colomeus insert the entire third segment of the second antennae into the gill tissue of the host; other Ergasilus species wraps the antennae around the entire gill filament (Oldewage & van As, 1987) and some species simply just hold onto the filament (Fryer, 1965). Ergasilus sarsi, the parasite relevant to this study, seemed to wrap their antennae around the entire gill filament.
From the ergasilid host record table (table 1) it is clear that ergasilids exhibit little specificity with regards to the host species and the question arises, are such a great number of ergasilid species valid? The four ergasilid species from Lake Tanganyika differs morphologically; some carry spines on the second antennae and specific markings on the cephalothorax. Fryer (1965) recorded two species from the same host and even the same gill filament; Ergasilus kandti attached closer to the gill arch and Ergasilus megacheir attached closer to the tip of the gill filament.
The question therefore, arises whether parasite morphology is influenced by the host or the niche on the host (bony tissue/ soft tissue) in which it is found? Robinson and Avenant- Oldewage (1996) in a study on Lernaea found that the anchor shape depends on the type of host tissue it attaches in. Yet, in Lernaea the species identification is based on the shape of the structure. Similarly, Lamproglena clariae (Tsotetsi et al., 2005) showed a direct correlation between parasite length and gill filament length. Correspondingly Kruger and
CHAPTER 3 52 SUMMATIVE DISCUSSION AND FUTURE
Avenant- Oldewage (1997) showed in Mugilicola smithae that morphoplasticity occurred and was influenced by attachment site.
Future studies addressing the effect of attachment site on the morphology may involve the following; a detailed study of all the ergasilid species found in Lake Tanganyika, morphological identification and re-description of all species collected. Host gill structures should be compared with parasite morphology and genetic studies preformed to validate species.
Different studies showed that pathology depends on how ergasilids attach to the host (Fryer, 1965; Oldewage & van As, 1988). The current study showed Ergasilus sarsi embraces the entire gill filament and cross the tip of the second antennae over each other (see Fig 17 B). Furthermore, it attaches primarily to the tip of the gill filament confirming Fryer’s (1965) observation.
A hypothesis for this study was that E. sarsi exhibit an even distribution on all attachment sites on the gill filaments. It was showed that this was not the case and the hypothesis is therefore rejected. The parasite is site specific and attach primarily to the second gill arch. This arch is the biggest and provides the biggest surface area for attachment. Furthermore, Turgut et al., (2006) found that the second gill arch of most fish species receives the largest water flow. Austin and Avenant-Oldewage (2009) also found that Lamproglena hoi prefers the second gill arch. Attaching to the second gill arch is beneficial not only because it provides a larger surface area but also the largest water current that provides transportation to release the newly hatched nauplia from the egg sacs.
Host- parasite interactions, such as abundance of parasite should be considered in future studies. Specimens should be collected during different seasons to record parasite abundance. Lake Tanganyika is located close to the equator and hence experience short seasons with 2 summers and 2 winters per annum. This may influence the abundance of the parasite or the feeding rituals of the host.
The sensory organs of ergasilids and their influence on infection are not well studied. Fryer (1966) stated that the spatial distribution and infection of new host may simply be an effect of the host behaviour rather than the parasite’s. Ergasilus does not exhibit host specificity which may provide support to Fryer’s theory. However, Bauer (1970) observed that Ergasilus larvae have vertical migratory patterns. This might explain the reason why most ergasilids infect host from most of the trophic levels within an ecosystem. The sensory organs should play a role during this vertical swim pattern or even to locate a male or female for copulation. Chemosensory organs can also play a role in locating a new host.
CHAPTER 3 53 SUMMATIVE DISCUSSION AND FUTURE
The complete lifecycle for ergasilids is not known. Future studies could therefore involve the construction of an artificial ecosystem with different fishes from different trophic levels. A laboratory bread colony of parasites could be used to infect the fish. A good camera apparatus should be used to observe the fish’s swimming and eating patterns. This way the question on exactly how the fishes become infected may be answered. It is plausible that fish gill ventilation may result in parasites being washed over the gills or that parasites are taken up with food. As soon as fish become infected, parasite egg production and development can be monitored. The different stages in the lifecycle can then be collected and described.
Hormone levels could also be measured to determine whether the parasite or the fish excretes chemicals or hormones that attract them to one another or furthermore, to detect the possibility of chemosensory organs in locating a mate for copulation.
One of the objectives of the study was to determine the pathological alterations caused by the parasite. It was hypothesised that the parasite causes severe pathological alterations. This hypothesis infers alia was accepted after the results clearly showed severe alterations resulting in fusion of lamellae and loss of gill tissue. This decreases the surface area for gas exchange. Proliferation of mucous cells occurred. It was also observed that the entire length of the body of the parasite as well as the appendages contributed to the pathology and the cephalothorax forms a vacuum that causes the epithelial layer to lift off the filament, the scraping action of the swimming legs push the loose tissue towards the mouth region of the parasite enabling ingestion. This study is the first to give a detailed description of the pathology caused by Ergasilus sarsi.
Further studies regarding the pathology could focus on digestion. It is unclear whether the parasite digests the food before it is taken up through the release of proteolytic enzymes (Paperna, 1996) or whether digestion is internal. The only information regarding the parasite digestive system was provided by Wilson (1911) who made a drawing of the digestive tract but never discussed it. In order to answer these questions a colony of parasites can be kept and as soon as they feed they can be killed with the fish they are attached to. The presence of proteolytic enzymes can then be tested for. Different staining methods can also be used to track progress of cells along the alimentary canal of the parasites. Einszporn (1965) did a similar study and found that food in the intestine loses its original appearance due to enzyme activity. She used different substances to stain the chromatin and the round nuclei in the lumen to determine the type of cell to tract progress as well as the enzyme activity.
The digestive system study will benefit from a detailed study on the mouth parts of the parasite with the aid of TEM and SEM. A full description of the mouth parts will support the
CHAPTER 3 54 SUMMATIVE DISCUSSION AND FUTURE findings on the type of digestion. This should be done on all four species of Lake Tanganyika to establish whether interspecies variance of mouthparts morphology occurs.
Furthermore, studies can be done on other African ergasilids species. The results thereof can then be compared to that of South- American ergasilid species. These two continents made up Pangaea. Similarities between the ergasilids and their genetic makeup together with morphological comparison may shed further light on the matter.
As already discussed in chapter 1 of this dissertation L. tanganicanus is exported for aquarium use. They are extremely beautiful fish and contribute to a breathtaking exotic aquarium. Ergasilids are not host specific and therefore an infected fish may infect all fishes in a tank. Ramifications thereof may affect the export trade quite significantly. The trade industry should be aware of ergasilid infections and treat fish as part of their export protocols. The treatment for these parasites cannot be done within the lake as it will have devastating effects on the ecosystem and a huge economic impact.
The current study presented infection statistics and it shows that even though these parasites might not be very host specific they are specific to an attachment sites. This enables easy location of the parasites. The exporting company could inspect the fish before exporting. Unfortunately the parasites are not visible to the naked eye and sample fish will have to be sacrificed in order to observe the gills with a dissection microscope. Although many studies have been done on the treatment of copepods in general, limited information is available for treating ergasilids. Paperna (1996) stated that they can be treated with potassium permanganate; reduce oxygen in the water hence it is important to aerate well. It will also be useful to understand the lifecycle of the parasite to be able to intervene. Countries surrounding Lake Tanganyika can also consider setting up fish farms to bread Lake Tanganyika for export. This way it is easier to treat the fish in more predictable environments and provide an income for the local communities.
There are many unanswered questions regarding Ergasilus. Many more interesting studies should be done to fill the gaps in the knowledge especially towards African ergasilids.
CHAPTER 3 55 GENERAL REFERENCES
CHAPTER 4
GENERAL REFERENCES
CHAPTER 4 56 GENERAL REFERENCES
4 GENERAL REFERENCE Abdel-Hady OK, Bayoumy EM and Osman HAM (2008). New copepodal Ergasilid parasitic on Tilapia zilli from Lake Temsah with special reference to its pathological effect. Global Veterinaria 2, 123-129.
Abdelhalim AI (1990). Morphology and epidemiology of some parasitic copepods (Poecilostomatoida: Ergasilidae) from British freshwater fish. PhD thesis, University of London.
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