Evaluating the thermal stress response of South African abalone, Haliotis midae, to biogeographical temperature variability.

M.Sc. University of KwaZulu-Natal, Durban 2014.A gradient of sea temperatures is created along the South African coastline by the confluence of the cold Benguela Current on the West coast with the warm Agulhas Current on the East coast. This temperature gradient allows for an assortment of species to occupy the variety of microenvironments occurring in this area. Amongst these species is commercially important South African abalone, Haliotis midae, which although being capable of existing across this wide range of temperatures grows larger on the cooler West coast. Abalone reared on the warmer East coast however, experience greater mortalities especially during the more thermally variable summer months. The aim of the study was thus to assess the zone of tolerance for H. midae by exposing abalone to fluctuating temperatures in an attempt to model environmental temperature instability, a scenario which may likely be worsened by global climate change. Animals from the West and East coasts were exposed to two thermal treatments of fluctuating temperatures with the first group being kept at 16°C±2 and the second group kept at 16°C±4. The control group was maintained at a constant 16°C indicating that the mean temperature experienced by all three groups was 16°C. Oxygen consumption, nitrogen excretion and O:N ratio were assessed at the organismal level to give an indication of metabolic rate, amount of protein excreted and type of metabolic substrate utilized respectively. At the biochemical level, D-lactate accumulation was quantified to indicate whether metabolism was proceeding aerobically or anaerobically. Heat shock protein 70 (Hsp70) expression and degree of carbonylation were analyzed at the proteomic level with Hsp70 also being assessed at the transcriptomic level. All biological responses were measured at days 1, 3, 7 and 14 of the two week exposure. Oxygen consumption rates were significantly elevated on day 14 when comparing treatment group animals to control group animals of the same biogeographic region. P < 0.05 for both treatment groups from the West coast, while P < 0.001 for the East coast treatment groups. The ammonia excretion rates of the West coast animals were significantly lower than those of the controls at day 14 with P < 0.001 for both treatment groups, while ammonia excretion rates were elevated in East coast animals at day 14, although not significantly. Trends similar to those seen for ammonia excretion rates were exhibited by O:N ratios. West coast animals showed lower than control O:N ratios at day 14 (P < 0.01 for both treatment groups) while East coast animals displayed higher than control values (P < 0.05 only for the 16°C±2 group) at day 14. D-lactate, having been detected only for the West coast animals, showed no significant differences but large degrees of variation were noted on days 1 and 7. Carbonylation was evident for animals from both biogeographic regions with baseline carbonyl accumulation for East coast animals being greater (non-significantly) than that of the West coast animals. The hsp70 gene expression remained low for both biogeographic groups with West coast animals appearing to show slight elevations in expression at days 1 and 7, days which also displayed high degrees of variability. The West coast animals appeared to be better suited to coping with the thermal fluctuations, as they not only transiently reduced oxygen consumption rate to reduce ROS production, but also utilized the assistance of the D-lactate pathway possibly to maintain metabolism, both of which were not observed in the East coast animals. Although West coast abalone seemed to have slightly elevated hsp70 expression (suggestive of a repair response) when compared to their East counterparts, both groups of abalone were shown to have incurred notable amounts of protein damage (i.e. carbonylation). This suggests impairments in both protective and repair responses for animals from both biogeographic regions. The lack or attenuation of physiological responses noted in East coast abalone may be due to limitations in thermal adaptation but subsequent studies are required to confirm this notion. The information obtained from this study may assist in providing an insight into the mechanisms responsible for thermal limitation in H. midae and how this species is likely to respond to future periods of thermal instability which may be worsened by global climate change. An understanding of the processes leading up to limitations may potentially assist the abalone aquaculture industry in altering culturing practices early on to support optimal performance in abalone

Mucosal-associated invariant T (MAIT) cell heterogeneity in peripheral blood and bronchoalveolar compartment: implications for TB and HIV immunity.

Doctoral Degree. University of KwaZulu-Natal, Durban.Tuberculosis (TB) is the leading cause of infectious mortality globally and the leading cause of death in people living with human immunodeficiency virus (HIV). Understanding the mechanisms leading to impaired anti-TB immunity at mucosal sites and how this is altered during HIV infection, may assist in the development of more effective TB vaccines or immune-based therapies. Mucosalassociated invariant T (MAIT) cells are depleted and dysfunctional in the peripheral blood during HIV infection, but little is known about HIV’s impact on their quantity and quality at the lung’s mucosal surface, the site of TB infection. We aimed to characterise phenotypic, functional and transcriptomic features of MAIT cells in the peripheral blood and lung mucosa of people with latent Mycobacterium tuberculosis (Mtb) infection and HIV co-infection. Matched peripheral blood and bronchoalveolar lavage fluid were collected from consenting participants with confirmed latent TB infection, either with or without HIV. Characterisation of MHC class I-related protein 1 (MR1) 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) tetramer-positive T cells from the lung was performed by cloning, immunophenotyping, functional assays and T cell receptor sequencing. The phenotype, function and transcriptome of MAIT cells from both compartments were characterised by surface marker staining, intracellular cytokine staining, as well as single-cell and bulk RNA-sequencing of MR1 5-OP-RU tetramer-positive T cells from HIVnegative and HIV-positive individuals. Peripheral blood MAIT cells were characterised by the CD161++CD26++ phenotype and produced more interferon (IFN)-γ (P = 0.016) than bronchoalveolar MAIT cells in HIV-negative individuals. Bronchoalveolar MR1 5-OP-RU tetramer-positive cells, included subpopulations with the atypical CD161-CD26++ and CD161-CD26- phenotypes. T cell cloning demonstrated that cells from the typical and atypical CD161/CD26 phenotypic subpopulations all had MR1-restricted function and MAIT cell consistent T cell receptors. In HIV infection, the frequency of both peripheral blood and bronchoalveolar MAIT cells was reduced (P = 0.035 and P = 0.047 respectively), with peripheral blood MAIT cells producing less interleukin (IL)-17 (P = 0.025) and expressing higher levels of the inhibitory co-stimulatory molecule T cell immunoglobulin and mucin domain-containing protein (TIM)-3. Interestingly, the phenotype and function of bronchoalveolar MAIT cells remained relatively unchanged by HIV. Single-cell transcriptional analysis confirmed MAIT cell heterogeneity in the bronchoalveolar compartment which contained two distinct transcriptional subsets, one associated with typical MAIT cell features and effector functions and the other associated with alternative MAIT cell functions including tissue-repair. We report previously undocumented phenotypic and transcriptional heterogeneity in bronchoalveolar MAIT cells, which were also less pro-inflammatory than those in peripheral blood. HIV infection led to depletion of MAIT cells in both compartments, but phenotypic and functional alterations were more pronounced in the peripheral blood compartment. The preservation of function and heterogeneity in bronchoalveolar MAIT cells may represent a potential avenue for therapeutic targeting to restore normal MAIT cell function in people living with HIV. This data suggests that understanding immune responses requires compartment-specific analyses, which may lead to the development of more effective vaccines and immunotherapies targeted at inducing immune responses at the site of infection