Doctoral Degrees (Biological Sciences)

Permanent URI for this collectionhttps://hdl.handle.net/10413/12381

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Influence of human-associated tsetse habitat degradation on tsetse fly (Diptera: Glossinidae) populations and prevalence of infection with trypanosomes in North-Eastern Zambia.
(2021) Chilongo, Kallinga.; Mukaratirwa, Samson.
African trypanosomiasis is among the most important parasitic diseases of livestock and humans caused by several species of trypanosomes, and the disease occurs in 36 countries in sub-Saharan Africa. Human African Trypanosomiasis (HAT) causes a considerable public health burden on rural populations, and Animal African Trypanosomiasis (AAT) is an important constraint to livestock production and full utilization of land for agricultural production, such that if not controlled the disease can induce important losses through limiting crop production and access to land, and diminishing income from meat, milk and other livestock products, consequently resulting in poverty. In Zambia, approximately 40% of the country’s land is tsetse-infested and the infestation in the Luangwa valley is among the most important with respect to occurrence of both human and animal trypanosomiais. In affected areas, occurrence of trypanosomiasis in humans and in livestock normally correlates with the prevalence of trypanosome infection in tsetse flies. Laboratory studies have shown that among the major factors that affect such trypanosome infection in tsetse flies, is occurrence of stress in tsetse flies. Occurrence of stress in wild tsetse fly populations is associated with unfavourable environmental conditions for the flies, and this is usually a consequence of tsetse habitat degradation. In many parts of Zambia’s eastern tsetse belt, human-associated degradation of the tsetse habitat has been on the increase over the last decades. This suggests that research to determine the effects of such human-associated tsetse habitat degradation, on tsetse populations and prevalence of trypanosome infection in the tsetse population in the area, could provide some insights into the epidemiology of trypanosomiasis in the area. In this study undertaken in three sites, Mpika, Lundazi and Rufunsa sites, in north-eastern Zambia (in parts of the eastern tsetse belt), the objectives were, to determine and measure (i) variation in size, age and hunger stages in tsetse flies and (ii) variation in prevalence of trypanosome infection in the tsetse flies, with increase in distance away from the edge into the inner parts of tsetse belt, and in relation to the distribution of human settlements; and (iii) to detect, assess and evaluate the contribution and importance of existing agricultural and other forms of ecosystem utilization, to tsetse-habitat degradation in the three sites. Three study sites were selected based on level and pattern of human settlement, i.e. Mpika and Rufunsa sites with human settlement concentrated at or close to the edge of the tsetse belt, and Lundazi site with human settlement evenly distributed from the edge into the innermost parts of the tsetse belt. Samples of two species of tsetse flies found in the sample sites, i.e. Glossina morsitans morsitans and G. pallidipes, were collected and (i) size, age and hunger stage in the tsetse flies were recorded and assessed with reference to distance away from the edge of the tsetse belt; (ii) variation in prevalence of trypanosome infection in the tsetse population in the study sites, with reference to distance away from the edge of the tsetse belt, and in relation to distribution of human settlements; and (ii) key land-use and socioeconomic factors in the human settlements, with reference to human-associated tsetse habitat degradation in the study sites. Trapping of the tsetse flies was done in defined sample points, identified with use of a Global Position System (GPS) unit, in the transect line, with use of the Black-screen fly round (BFR) and Epsilon traps. From the sampled flies, the following were recorded; species of fly, sex, body size, age and hunger stage (as indicators of levels of occurrence of stress), and screening for trypanosome infection using microscopy and the loop-mediated isothermal amplification (LAMP). A semi-structured questionnaire was administered in each of the settlements within our study location, and national land cover maps for the years 2000 and 2010, produced by the country’s Forest Department, were used to estimate vegetation cover change during the period 2000 to 2010 in each of the sites. Regression models were applied to determine and measure the level of association of the distance from the edge into the inner parts of the tsetse belt with; size, age and hunger stages of tsetse samples, and prevalence of trypanosome infection in the tsetse flies. In each settlement, data were collected on key land-use and socio-economic factors that may be linked to human-associated habitat degradation and changes in the vegetation cover during the period 2000 and 2010, was calculated in QGIS. The results showed that in the Mpika and Rufunsa sites, the number of Glossina morsitans morsitans tsetse flies caught increased along with the increase in distance from the edge into the inner parts of the tsetse belt. This was also associated with increase in the body size (p < 0.0001 in both sites), and reduction in the age (p < 0.001 in each site) and the hunger stages (p < 0.0001 in both sites), and reduction in the prevalence of trypanosome infection (p = 0.024 and p = 0.012 in the case of all sub-species of trypanosomes tested for in the Mpika and Rufunsa sites respectively; and p = 0.013 and p = 0.025 in the case of only nannomonas sub-species in the two sites, respectively). The level of vegetation cover change was insignificant in each of the sites, such that it was unlikely to have had any significant impact on the quality of the tsetse habitat in each of the sites. In the Mpika and Rufunsa sites, human activities associated with access to resources might have had significant effect on the distribution of wild mammals that served as tsetse hosts in the area, such that numbers (of wild mammals) were low in locations that were close to the settlements and high in locations that were furthest from the settlements – giving rise to a gradient of increasing levels of availability of tsetse hosts with increase in distance away from the human settlements. This same trend was observed with regard to the distribution of body size of the flies, age, hunger stages, and prevalence of nannomonas and trypanozoon trypanosome infection, in G. m. morsitans in the Mpika and Rufunsa sites. This was indication that (in the Mpika and Rufunsa sites) increase in the levels of availability of tsetse hosts was associated with increase in levels of tsetse wellbeing – in turn associated with increase in levels of tsetse habitat quality. With regard to the findings in the Lundazi site (where human settlement was evenly distributed in transect line), the absence of any such variation (in each of the respective attributes in G. m. morsitans) with distance from the edge of the belt, could be taken as supportive to the reason indicated above as the likely basis for the existence of a gradient of reducing levels of tsetse habitat degradation in the Mpika and Rufunsa sites. In the case of G. pallidipes, the results showed no variation in the respective features in the tsetse flies, with increase in distance from the edge of the tsetse belt, and factors such as the relatively fewer numbers of the species caught, and a large proportion of the transect length not having registered any catch of the species, in each site in the study, likely contributed to this outcome.
Micromorphology and biological activities of leaves and stem bark of Diospyros villosa (L.) De Winter.
(2021) Adu, Oluwatosin Temilade.; Naidoo, Yougashree.; Lin, Johnson.
Review Article: The genus Diospyros consists of many species which are distributed throughout the world. There is no elaborate information about the holistic importance of the plants. This review aims to delve for more information about the usefulness of the plants for humans. A detailed study of the genus Diospyros with a targeted focus on Diospyros villosa (L.) De Winter plant was carried out information on the plants’ morphology was taken as observed during harvest. Other information with respect to geographical distribution, secretory structures, chemical composition, embedded bioactive constituents and the associated mechanism of action of the bioactive constituents were acquired. The genus Diospyros yields products of medicinal importance throughout the world. This study gives some detailed information about the genus Diospyros as well as the potential use of the species as functional medicinal plants with bioactive compounds. Manuscript 1: This study evaluated the phytochemical constituents, antioxidant and antimicrobial potentials of Diospyros villosa (L.) De Winter leaves and stem bark. The extracts were obtained using different media (methanol, chloroform and hexane). DPPH and FRAP methods were used to investigate the antioxidant potentials of the crude extracts. The antimicrobial potency of Diospyros villosa extracts against five pathogenic bacteria was determined using MIC, MBC and agar well diffusion methods. Flavonoids, alkaloids and phenols were identified in D. villosa extracts. The mean concentrations of methanol extracts Diospyros villosa leaves and stem against DPPH providing 50% inhibition were 9.53 ± 0.25 μg ml-1 and 9.52 ± 0.30 μg ml-1 respectively. The methanolic leaves extracts further showed promising antimicrobial activity against Klebsiella pneumonia, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus and Staphylococcus aureus with inhibition zones which are almost as high as the control. The antimicrobial methods also demonstrated that the leaves and stem bark extracts had wide antimicrobial abilities. The results revealed that D. xxvii villosa leaves and stem bark contain reasonable amount of bioactive compounds. Hence, these compounds may serve as natural antioxidants and antibacterial agents for the treatment of bacterial infections and diseases. Manuscript 2: Nanoparticles are synthesized through wet chemical techniques. Meanwhile, the chemicals used often are toxic and flammable. While considering safe substances, Diospyros villosa leaves and stem bark are both observed to be suitable for the biosynthesis of silver nanoparticles. In this research study, we described a less expensive and environmental-friendly technique for the biosynthesis of silver nanoparticles from silver nitrate (AgNO3) solution and Diospyros villosa extracts. The obtained silver nanoparticles were characterized using the UV-vis absorption spectroscopy, FT-IR, EDX, SEM and TEM, DPPH scavenging ability, ferric reducing antioxidant potential, antimicrobial susceptibility and quorum sensing inhibition tests. The biosynthesized silver nanoparticles showed good antibacterial activity against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermis and Enterococcus faecalis. The D. villosa nanoparticles demonstrated potential antioxidant and quorum sensing inhibition and may thus represent a source of antioxidant as well as quorum sensing inhibition candidate for infection and disease control. Manuscript 3: The structural adaptation, histological compatibility and ecological functions of trichomes in Diospyros villosa, however remain largely unclear. This study aimed to interpret the structure and histochemical analysis of the leaves and stem bark of Diospyros villosa using light and electron microscopy and different histochemical tests as well as to elucidate the composition of the secretory products. Trichome density and length were also determined in three developmental stages of the leaves. The results of histochemical tests also revealed the presence of alkaloids and phenolic compounds which are medicinally important and used to treat multiple ailments. xxviii Manuscript 4: Diospyros villosa is traditionally used for processing anti-bacterial properties. Its cytotoxic effects have not been studied before. Therefore, this study aimed to examine the nutritional properties as well the cytotoxic effects of D. villosa. The leaves and stem barks were subjected to three different extraction methods (methanol, chloroform and hexane) and their nanoparticles were synthesized at two different temperatures (room temperature and at 80 ºC). Thereafter, extracts were assessed using the associated AOCC protocols, for their nutritional content (moisture, fibre, proteins, lipid, ash and hydrolysable carbohydrates). Diospyros villosa extracts and their corresponding nanoparticles were then incubated overnight with cancerous and noncancerous cell lines to evaluate their cytotoxic potential.
The morphological characterization, chemical composition and biological activity of barleria albostellata (Acanthaceae).
(2021) Gangaram, Serisha.; Naidoo, Yashini.; Dewir, Yaser Hassan.
Herbal preparations of plants continue to present mankind with novel remedies as many of these plants contain important secondary metabolites. Medicines manufactured by pharmaceutical companies are largely synthetic. The fear for ineffectiveness, adverse side-effects and toxicity, has brought about further scientific investigations on the potential usage of medicinal plants. Plants of the family Acanthaceae are rich in bioactive phytochemicals. Several plant species are being utilized for their ethnomedicinal properties based on their phytocompounds they acquire, with Barleria (Acanthaceae) being one of such genera. Barleria albostellata C.B. Clarke (Acanthaceae) is a shrub indigenous to South Africa. Herbal use of this plant has not been fully documented, however, several species of Barleria are used in traditional medicine. Little or no investigations have been undertaken to evaluate the micromorphology secretory mechanisms, through which such phytochemicals are synthesized and secreted; floral biology; phytochemical profile; antibacterial and antioxidant activity, cytotoxicity and synthesis of nanoparticles using the leaf and stem extracts of B. albostellata. Therefore, this study aimed at bridging these gaps by first characterizing the morphology, chemical composition and biological activity of leaves and stems of B. albostellata, using various microscopy techniques and biological assays. Floral biology of the plant was conducted using stereo- and scanning electron microscopic (SEM) techniques. ImageJ was used to measure the length and diameter of the different trichome types and pollen structures. Histo-phytochemical, thin-layer chromatography (TLC), fluorescence microscopy and gas-chromatography mass spectrometry (GC-MS) analysis were performed on crude extracts (hexane, chloroform and methanol) to determine the composition of the compounds that may be of medicinal importance. Biological (antibacterial and antioxidant) analyses were also conducted on the crude extracts. Cytotoxicity of the crude extracts were evaluated established using 3-[(4,5- dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay in the human embryonic kidney (HEK293), cervical cancer (HeLa), and breast adenocarcinoma (MCF-7) cell lines. Silver nanoparticles (AgNPs) were synthesized using methanolic, aqueous- powdered and -fresh leaf and stem extracts. These AgNPs were characterized using UV–visible spectroscopy, high resolution transmission electron microscopy (HRTEM), energy-dispersive X-ray (EDX), Fourier transform infrared (FTIR) spectral analysis and nanoparticle tracking analysis (NTA). Antibacterial analysis of the synthesized AgNPs was assessed using the disk diffusion method. Cytotoxicity of the synthesized nanoparticles were investigated using the MTT assay on the three cell lines. Histochemically stained sections, stereo, SEM and TEM micrographs revealed a dense indumentum with the presence of five morphologically distinct glandular capitate trichome types, multangulate-dendritic branched (MDB) non-glandular trichomes and a glandular head attached to a branched non-glandular trichome. Both glandular and non-glandular trichomes in the leaves and stems of B. albostellata are important diagnostic features of this species. The secretory head of glandular typed trichomes varied from 20.72 ± 1.36 µm to 54.23 ± 2.08 µm. Additionally, the length of these stalks varied from 15.11 ± 3.89 µm to 101.92 ± 1.78 µm, and the width ii from 11.62 ± 1.03 µm to 35.32 ± 3.87 µm. Multangulate-dendritic branched trichomes had a stalk length of 554.10 ± 92.27 µm and width of 28.55 ± 2.48 µm. Stained sections and crude extracts indicated the presence of important medicinal compounds such as alkaloids and phenolics. The histochemical tests indicated the presence of various phytocompounds that have medicinal importance and aid in defense against pathogens and herbivores. Pollen grains of B. albostellata are complex, intricate and display reticulate sculpturing, with a diameter of 77.53 ± 5.63 μm and aperture of 14.31 ± 0.59 µm. This will contribute significantly to our growing understanding on the floral and pollen biology of this species. Qualitative phytochemical screening, GC-MS and florescence microscopy of the leaf and stem extracts revealed various biologically active compounds and the presence of different colours in the leaf and stem powder. Different colour intensities obtained from TLC suggested concentrations of the separated compounds were varied. Additionally, the phyto-constituents found in the leaf and stem crude extracts of could inhibit the growth of various pathogenic strains. Results from this study revealed the medicinal potential of B. albostellata in the treatment of various bacterial diseases Ultraviolet spectra, NTA and EDX, revealed varying absorption peaks, size distribution, and elemental Ag in all extracts. Crude extracts and synthesized AgNPs displayed varying degrees of antioxidant, antibacterial and cytotoxic activities. Significance was established at P 63 µg/mL and >9 µg/mL, respectively) may be due to the presence of flavonoids, phenols, and antioxidant activity in the different parts of this species. Synthesized AgNPs showed possible bacteriostatic effects against Gram-positive and -negative human pathogenic bacteria. Their broad spectrum of bioactivity suggested that they may be as promising agents in fighting infections. This study ultimately proved that leaf and stem extracts of B. albostellata contained numerous biologically active compounds such as alkaloids and phenolics. These results are suggestive that the leaves and stems of B. albostellata are rich in bioactive compounds which, could be a possible source of antibacterial agents in treating several diseases .There is a great potential for B. albostellata as this plant displays valuable biological activities. Future studies on this plant are recommended, as this could advance the use of indigenous herbal medicine or product novel drug leads.
The secretory scales and medicinal properties of combretum erythrophyllum.
(2021) Bantho, Sahejna.; Naidoo, Yougasphree.; Yaser, Hassan Dewir.; Singh, Moganavelli.
Medicinal plants are known to contain phytometabolites that could effectively improve an individual’s state of health. Species of Combretum are highly valued in Africa due to the plethora of their traditional medicinal uses. Combretum erythrophyllum. Burch. Sond., commonly known as the river bushwillow, is known to contain medicinally important phytometabolites. Traditionally, the foliage is used to treat venereal diseases and abdominal pain, whilst the bark is used to alleviate sores, infertility, and labour pains. Although C. erythrophyllum has numerous traditional medicinal uses, there is limited scientific knowledge on the micromorphological structures and the associated exudate. Thus, this study aimed to characterize the micromorphological features of leaf and stembark secretory apparatus of C. erythrophyllum, using light and electron microscopy. Furthermore, a histo-phytochemical analysis was conducted to determine the presence and localization of phytometabolites within the trichomes and exudate. The antioxidant, antibacterial, apoptotic and cytotoxic potential of the leaf and stembark extracts were also evaluated. The micromorphological analysis identified the presence of peltate scales and non- glandular trichomes across surfaces. Peltate scales were comprised of a sunken basal cell, bicellular stalk, and a multicellular head. Head cell count appeared to increase upon leaf maturation. The granulocrine pathway was identified as a possible mode of secretion for C. erythrophyllum due to the extensive presence of vesicles, vacuoles, and electron dense material within the peltate scales. Preliminary histo-phytochemical analyses revealed the presence of carbohydrates, sterols, lipids, phenolic compounds, total proteins, alkaloids, and essential oils. Thin-layer chromatography allowed for the visualization of 36 compound classes while gas chromatography-mass spectrometry showed 266 compounds present. Fourier-transform infrared spectroscopy analysis confirmed the presence of phenols, alkenes, amines, alcohols, and esters among many. The antioxidant ability of the generated extracts were evaluated using the 2,2- diphenyl-1-picryl-hydrazyl-hydrate antioxidant assay and Ferric Reducing Antioxidant Power assay. A positive correlation between % inhibition and extract concentrations, was noted in both. In both instances, the methanol stembark extracts performed the best, (Leaf- 5,2866 and Stembark- 4,2866 µg/mL). Furthermore, the results obtained from the total flavonoid assay correlated with the trend observed through the total phenolic assay, whereby methanolic extracts yielded most promising results. Additionally, this study aimed to generate silver nanoparticles using crude extracts. A novel protocol for the synthesis of silver nanoparticles (AgNPs) using the leaf and stembark extracts of C. erythrophyllum was established. The generated AgNPs were characterized and evaluated for its potential antibacterial activity. Methanolic extracts inhibited the growth of Pseudomonas aeruginosa, Bacillus subtilis, Methicillin Resistant Staphylococcus aureus, and Staphylococcus aureus. Lastly, the generated crude extracts displayed promising results when evaluated for their cytotoxic and apoptotic abilities however upon nano- vii encapsulation the cytotoxic and apoptotic capabilities increased significantly. In correlation with the above, the AgNPs appeared to intensify the overall inhibition activity. Based on the findings of the study, Combretum erythrophyllum has a reservoir of unexplored allopathic potential which could revolutionize the medicinal world.

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