http://hdl.handle.net/10386/69 2013-06-16T12:53:18Z http://hdl.handle.net/10386/857 Title: Heterologous expression of a Mukwa (pterocarpus angolensis ) seed lectin (Pal) gene in Escherichia coli, Saccharomyces cerevisiae and Yarrowia lipolytica and construction of Pal recombinant vector for expression in Aspergillus niger Authors: Ngoepe, Mafora Gloria Abstract: Pterocarpus angolensis seed lectin (PAL), a 28 kDa non glycosylated protein, was initially successfully cloned and expressed in E. coli for ease of high protein production. It was discovered, however, as in similar studies that the recombinant PAL yield in E. coli is low and localized intracellularly. This makes extraction even more difficult because most of the protein is lost either when the cell undergoes lysing or when there is incomplete extraction. As a result of the low yields in E. coli, expression vectors were constructed for pal expression in S. cerevisiae, Y. lipolytica and A. niger. Colony PCR of S. cerevisiae transformants confirmed the presence of pal gene whilst sequencing revealed a 66% homology to native PAL. Expression of recombinant PAL in S. cerevisiae, which was expected to be intracellular, was doubtfully unsuccessful since no signal was detected following Western blot analysis. A pBARMTE1-pal expression vector was successfully constructed and could be used for expression studies in Aspergillus niger, however, it was not used in this study. A pal gene whose codons were optimized for Y. lipolytica was synthesized and successfully cloned and expressed in Y. lipolytica. Gene sequence alignment of native pal and the codon optimized pal showed 81% homology whilst the amino acid alignment showed 100% homology. A 31 kDa, recombinant PAL was successfully expressed in Y. lipolytica. The recombinant PAL was approximately 3 kDa larger than native PAL. It was established that this is due to glycosylation of the recombinant PAL. This recombinant protein was found to be more thermostable than native PAL since it demonstrated haemagglutination activity after 10 minutes of exposure in a boiling water bath and only lost activity after 2 hours of exposure to boiling. This study succeeded in producing a more stable extracellular recombinant PAL which demonstrated biochemical activity that was largely similar to that of native PAL but only differed in carbohydrate specificity and haemagglutinating strengths. Description: Thesis (M.Sc. (Microbiology)) --University of Limpopo, 2011 2011-01-01T00:00:00Z http://hdl.handle.net/10386/822 Title: Helminth ichthyo-parasitic fauna of a South African Sub-Tropical Lake Authors: Matla, Matsoele Moses Abstract: The diversity of the helminth parasites of fishes in a clear-water, subtropical Lake Tzaneen, in South Africa was investigated. Of the 527 fish specimens sampled approximately 9000 parasites were collected. There are 38 different parasite species discussed comprising 27 Monogenea, 3 Digenea, 4 Cestoda, 3 Nematoda and 1 Acanthocephala. Four new monogenean species are described and these are Dactylogyrus spp. 1 to 4. Three monogenean species are introduced as first records in Africa and these are Actinocleidus fusiformis (Mueller, 1934), Haplocleidus furcatus Mueller, 1937 and Acolpenteron ureteroecetes Fischthal & Allison, 1940. Fourteen monogenean and one acanthocephalan species are discussed as first geographical records for South Africa and these are Gyrodactylus rysavyi Ergens, 1973, Dactylogyrus brevicirrus Paperna, 1973, Dactylogyrus cyclocirrus Paperna, 1973, Dogielius dublicornis Paperna, 1973, Dogielius sp., Schilbetrema quadricornis Paperna & Thurston, 1968, Quadriacanthus aegypticus El Naggar & Serag, 1986, Quadriacanthus clariadis Paperna, 1961, Scutogyrus gravivaginus (Paperna & Thurston, 1969), Cichlidogyrus quaestio Douëllou, 1993, Cichlidogyrus halli Price & Kirk, 1967, Cichlidogyrus sclerosus Paperna & Thurston, 1969, Cichlidogyrus dossoui Douëllou, 1993, Cichlidogyrus tilapiae Paperna, 1960 and Acanthosentis tilapiae Baylis, 1948. Seven species are discussed as first records for their hosts and these are Cichlidogyrus dossoui, Cichlidogyrus halli and Acanthosentis tilapiae on Oreochromis mossambicus; Dactylogyrus sp. 1 on Barbus radiatus and Barbus trimaculatus; Dactylogyrus sp. 2 on Barbus unitaeniatus; Dactylogyrus sp. 3 and Dactylogyrus sp. 4 on Labeo molybdinus. Gyrodactylus rysavyi is the only species with a first site (gills) record. The other monogenean species discussed are Macrogyrodactylus clarii Gussev, 1961, Macrogyrodactylus karibae (Douellou and Chishawa, 1995), Dactylogyrus afrolongicornis afrolongicornis Paperna, 1973, Dactylogyrus allolongionchus Paperna, 1973, Dactylogyrus spinicirrus (Paperna & Thurston, 1968) and Cichlidogyrus philander (Douëllou, 1993). The digeneans discussed are Glossidium pedatum Looss, 1899 and the larvae of Diplostomum van Nordmann, 1832 and Clinostomum Leidy, 1856. The Cestodes discussed are Proteocephalus glanduligerus (Janicki, 1928) Fuhrmann, 1933, Polyonchobothrium clarias Woodland, 1925 and the larvae of Ligula intestinalis Goeze, 1782 and family Gryporhynchidae. The nematodes discussed are Procamallanus laevionchus (Wedl, 1861), Paracamallanus cyathopharynx Baylis, 1923 and larvae of Contracaecum Railliet and Henry, 1912. Monogenea were commonly found on the gills but less on the skin and in the urinary bladder. Digenea were found mainly in the eyes, brain and visceral cavity, with only one species (Glossidium pedatum) present in the intestines of Clarias gariepinus. Cestoda and Nematoda were found in the intestine and body cavity. Only one species of Acanthocephala (Acanthosentis tilapiae) was found in the intestines of Oreochromis mossambicus. No definite seasonal variations of infection and parasite affinities towards the sexes and the sizes of the hosts could be determined. The lake is oligotrophic with the water quality having no influence on the parasite diversity and species richness. Description: Thesis (Ph.D. (Zoology)) --University of Limpopo, 2012 2012-01-01T00:00:00Z http://hdl.handle.net/10386/812 Title: Aspects of the biology of the chestnut-backed sparrow-lark (Eremopterix leucotis) in the Limpopo Province, South Africa Authors: Dikgale, Mahlodi Lucket Abstract: Sparrow-larks form a relatively small genus in the family Alaudidae and comprise only seven species distributed widely throughout Africa and parts of the Eurasian landmass. Sparrow-larks are unique amongst larks in that they are sexually dichromatic and exhibit biparental care. The chestnut-backed sparrow-lark Eremopterix leucotis is endemic to Africa with five subspecies recognized based on differences in plumage colouration. The five subspecies are distributed throughout the arid to semi-arid savannas of Africa with two subspecies (E. l. hoeschi and E. l. smithi) occurring in southern Africa. Despite their widespread occurrence and its interest for research on the evolution of characteristics in the family (e.g. being sexually dichromatic and exhibiting biparental care), very little is known of the biology and ecology of the Eremopterix larks. The chestnut-backed sparrow-lark is no exception and most of what we know of the species is based on incidental observations from a few nests. In an attempt to improve our knowledge of this interesting group of species, it was decided to study various aspects of the breeding biology and ecology, moult, vocalizations and geographical variation in the chestnutbacked sparrow-lark. The breeding biology of the chestnut-backed sparrow-lark was studied at Al3 farm (De Loskop) near Mogwadi in the Limpopo Province of South Africa from January 2008 to December 2010. Data collected during the study included: breeding seasonality, egg and clutch characteristics, duration of the incubation and nestling periods, nest-site characteristics, the roles and relative contribution of the sexes in the breeding cycle, nestling development, diet and nestling provisioning rate, and breeding success. Chestnut-backed sparrow-larks bred mostly during the dry season, which is from April to September in the study area. Nevertheless, the results revealed that breeding is bimodal with a main peak in breeding activity in late summer and autumn (March to April) and a second smaller peak in spring (September to October). The species showed geographical variation in clutch size with a mean of 1.88 eggs recorded in the study area as opposed to 1.00 recorded in the northern parts of its range. Egg dimensions compared well with measurements obtained from the Nest Record Card Scheme of the Animal Demography Unit, University of Cape Town, South Africa. The mean incubation period of 10.33 days recorded in this study compares favourably with that of other Eremopterix species viii (8–10 days), a genus with some of the shortest incubation periods amongst larks. The mean nestling period of 9.2 days (range: 8–10) in the study area was significantly less than the 10–12 days reported for populations in the northern range of the species, but it compares well with those of other sparrow-larks. Nest site characteristics, which were quantified within a 1 m2 quadrant with the nest as the centre, including nest dimensions, were consistent with those reported in the literature. Chestnut-backed sparrow-larks in the study area preferred to nest in areas with a high percentage of bare ground (median = 67.5%) and very little vegetation cover (median = 25%). Most nests faced in a southerly direction compared to nests in the north of the species’ range, which face in a north-easterly or easterly direction. The species’ preference to face the nests away from the midday sun most probably serves a thermoregulatory function to avoid excessive heat during the warmest parts of the day. Most nests (78.2%) had an apron varying in size from small and insignificant to large and well-developed. The functional significance of the apron remains a matter of conjecture and there was no association between breeding success and presence or absence of the apron. In addition, one pair constructed one nest with and another without an apron, suggesting that individual preference or characteristic is not a determinant factor in the construction of an apron. Both sexes took part in nest construction, incubation and feeding and brooding of nestlings. However, the relative contributions were not entirely symmetrical as males incubated a greater proportion (50.1%) of the time compared to females (43.1%), and the mean and median of male incubation shift lengths were longer than that of females, albeit not statistically significant. However, females made statistically significantly (P < 0.05) more nest visits to deliver food compared to males (54.6% vs. 45.5%). The average breeding success, estimated using Mayfield’s method, was 16.1% but there were inter-annual differences with the overall breeding success in 2010 being only 8.1% compared to 20.6% of 2008. Known causes of failure included nest depredation, flooding, starvation, nest abandonment and hatching failure. Statistical analysis of morphometric data of live specimens and museum study skins suggest that, in addition to being sexually dichromatic, chestnut-backed sparrowlarks also exhibit mild sexual size dimorphism. However, there was considerable overlap in these measurements between the sexes and as a result the biological significance of this sexual size dimorphism may be negligible. Nevertheless, the ix results show chestnut-backed sparrow-lark males tend to have longer wings and tails compared to females. This may be adaptive with respect to the extended display flights that males perform during the breeding season. Interestingly, the mean mass of breeding females in the study area was significantly more compared to males, whereas the SAFRING database, representing data recorded throughout the year, showed no significant differences in the mean mass between the sexes. The greater mass of breeding females may relate to physiological changes associated with the acquisition of resources and the development of structures and tissues associated with egg-laying and egg-production. Larks rely heavily on vocalizations to attract mates and advertise territories. Not surprisingly, the study revealed a rich vocal repertoire for the chestnut-backed sparrow-lark. The analysis of the vocalizations shows that chestnut-backed sparrowlarks have a display song performed by males, a sub-song sung by both sexes and various different calls used in different contexts, e.g. flight and alarm calls. The study also presents the first analysis and description of the vocalizations of nestlings. An interesting feature of the vocalizations of the chestnut-backed sparrow-larks was that they performed hetero-specific vocal mimicry, which was incorporated in the subsong. Moult is a relatively unknown aspect in the annual cycle of the majority of larks. Chestnut-backed sparrow-larks undergo post-breeding moult, which is an adaptation to reduce the conflict between moult and breeding as both activities have high energy demands. The moult study also showed that they undergo a partial moult in mid-winter, involving the inner-most secondaries and some of the contour feathers. The results of this study shed valuable light on the natural history of this species and contributed significantly to ornithology and our growing understanding of the biology and ecology of the family. The results can also form a basis for future inter- and intraspecific comparative studies. The study illustrates the importance of undertaking long term studies of species to account for inter-annual differences in various ecological parameters. Description: Thesis (M.Sc. (Zoology)) -- University of Limpopo, 2012 2012-01-01T00:00:00Z http://hdl.handle.net/10386/807 Title: Breeding habitat of Blue Crane (Anthropoides Paradiseus) in Mpumalanga Province, South Africa Authors: Mmonoa, Ernest Mmaphuti Abstract: The aim of this study was to determine the breeding habitat of Blue Crane (Anthropoides paradiseus) by investigating the home range, habitat selection and habitat suitability. Geographic Information System (GIS) was used as the main tool for analysis. Home range sizes of Blue Cranes were studied during the breeding season using direct observation method. A 50% and 95% Adaptive Kernel was used to estimate home range sizes. The home range sizes were 9.0 ha and 43.5 ha for 50% and 95% Adaptive Kernel, respectively. All the nests were located within 50% Adaptive Kernel, often referred to as core area. The nests were located in agricultural land (mainly pasture) and close to water sources. Habitat selection was studied at nest sites (n = 74) and random sites (n = 200) following site attribute design. The Blue Crane showed a preference to breed in agricultural lands, close proximity to water sources, higher elevation areas, within north eastern sandy highveld vegetation, and north facing slope. The Blue Crane also avoided anthropogenic factors such as built-up land, roads and railway line. ModelBuilder extension of ArcGIS software was used to construct a breeding habitat suitability model for Blue Cranes. Nine habitat variables (water source, slope, aspect, elevation, land use, vegetation, built-up land, roads and railway line) were used in the model. The model was constructed using reclassify and weighted overlay command. Highly suitable sites accounted for 601, 448 ha, while moderately suitable sites accounted for 823, 593 ha, and least suitable sites accounted for 3, 000, 153 ha. This study demonstrated the effective use of GIS technology in analysing the breeding ecology of Blue Crane. The GIS technology provided capabilities for capturing and analysing varied and large data. It was also evident that availability of accurate and complete species data remains vital to enable the full utilization of the GIS technology. Description: Thesis (M.Sc. (Zoology)) --University of Limpopo, 2009 2009-01-01T00:00:00Z