Abstract
Clarias gariepinus (Burchell, 1822) is one of the two most actively cultured freshwater fish in
Africa and therefore, economically important. Specimens of this species were purchased from
a fish farm near Hartbeespoort Dam (North West, South Africa) and introduced into the tanks
of the research aquarium in the Department of Zoology at the University of Johannesburg.
However, the skin of these fish was infected with monogenean ectoparasites that were
preliminarily identified as Macrogyrodactylus Malmberg, 1957. These parasites were observed
to proliferate profusely in the favourable conditions of the aquarium, posing a potential threat
to the fish. The current study was aimed at accurately identifying and describing these
flatworms using morphological and molecular techniques, as well as studying the pathological
effects caused on the skin of the host. The morphological description included light
microscopic examination of whole worms flattened and mounted in glycerine ammonium
picrate (GAP), as well as whole worms stained with Horen’s trichome then cleared and
mounted in lactophenol. Obtained light micrographs of GAP mounted specimens were used to
study and construct line drawings and take measurements of haptoral sclerites, as these
structures are essential for species identification. Generated data was then compared with that
for other species of Macrogyrodactylus parasitising C. gariepinus. Furthermore, a new
technique whereby haptoral sclerites were isolated by removing the encapsulating soft tissue
with a digestion buffer, drying in a desiccator, sputter-coating with gold and studying these
structures with a scanning electron microscope was used for the first time to study this genus.
Molecular identification involved utilising the internal transcribed spacer (ITS) region and a
fragment of 18S rDNA. Generated molecular data were compared to sequences of other
Macrogyrodactylus species from GenBank and topologies were constructed using MEGA 7
software. For the last aim of the study, an examination of the pathology caused by the parasite
was conducted. It involved light microscopy (LM) and scanning electron microscopy (SEM)
of parasites attached to the hosts’ skin. Light microscopy included examination of
haematoxylin and eosin-stained sections (epoxy embedded) of infected skin. For SEM, sections
of infected skin were processed by dehydrating them through a graded series of ethanol
followed by increasing hexamethyldisilazane concentrations, drying in a desiccator, and
sputter-coating with gold. Morphological and molecular analysis of the parasite in question
showed that the parasite was closely related to Macrogyrodactylus congolensis (Prudhoe,
1957). Furthermore, there was intraspecific variation between specimens from the current study
highlighting the need for further molecular investigation with other molecular markers to
elucidate this variability. Pathological examination showed that M. congolensis causes damage
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to the hosts’ skin. SEM of infected skin exhibited excessive mucus production, corroborating
light micrographs showing an increased number of mucous cells alongside proliferated and
abnormally enlarged club cells which resulted in thickening of the epidermal layer as opposed
to uninfected skin. At the site of attachment, the basement membrane was altered, detaching
from the dermis. Moreover, hamulus points and marginal hooks of the parasite were observed
to pierce through the host skin resulting in tearing. Also, epidermal cells and melanin granules
were observed in the intestinal lumen of the parasite. Melanin granules were also absorbed by
the parasite's intestinal epithelium confirming that the parasite feeds on hosts’ skin tissue. This
study was the first to examine isolated haptoral sclerites with SEM, which allowed for a 3-
dimensional study of the respective sclerites for a more accurate morphometric description for
species of the genus as well as a better understanding of the pathology caused by these
structures. Generated morphological, molecular and pathological results presented in this study
contribute to the essential information about M. congolensis. As this parasite infects an
important species of the African aquaculture industry, generated results provide a baseline
study for future research on methods for control of this parasite in aquaria and aquaculture.