Dalene Vosloo

Differential responses of juvenile and adult South African abalone (Haliotis midae Linnaeus) to low and high oxygen levels

Marine invertebrates have evolved multiple responses to naturally variable environmental oxygen, all aimed at either maintaining cellular oxygen homeostasis or limiting cellular damage during or after hypoxic or hyperoxic events. We assessed organismal (rates of oxygen consumption and ammonia excretion) and cellular (heat shock protein expression, anti-oxidant enzymes) responses of juvenile and adult abalone exposed to low (~83% of saturation), intermediate (~95% of saturation) and high (~115% of saturation) oxygen levels for one month. Using the Comet assay, we measured DNA damage to determine whether the observed trends in the protective responses were sufficient to prevent oxidative damage to cells. Juveniles were unaffected by moderately hypoxic and hyperoxic conditions. Elevated basal rates of superoxide dismutase, glutathione peroxidase and catalase were sufficient to prevent DNA fragmentation and protein damage. Adults, with their lower basal rate of anti-oxidant enzymes, had increased DNA damage under hypoxic and hyperoxic conditions, indicating that the antioxidant enzymes were unable to prevent oxidative damage under hypoxic and hyperoxic conditions. The apparent insensitivity of juvenile abalone to decreased and increased oxygen might be related to their life history and development in algal and diatom biofilms where they are exposed to extreme diurnal fluctuations in dissolved oxygen levels.

Seasonal Variation in the Regulation of Redox State and Some Biotransformation Enzyme Activities in the Barn Swallow (Hirundo rustica L.)

Little is known of the normal seasonal variation in redox state and biotransformation activities in birds. In long-distance migratory birds, in particular, seasonal changes could be expected to occur because of the demands of migration and reproduction. In this study, we measured several redox parameters in the barn swallow (Hirundo rustica L.) during the annual cycle. We captured the wintering barn swallows before spring migration in South Africa, and we captured the barn swallows that arrived in spring, bred in summer, and migrated in autumn in Finland. The redox status and biotransformation activities of barn swallows varied seasonally. Wintering birds in South Africa had high biotransformation activities and appeared to experience oxidative stress, whereas in spring and summer, they showed relatively low redox (superoxide dismutase [SOD], catalase [CAT], and glutathione reductase [GR]) and biotransformation enzyme activities. Autumn birds had very low biotransformation enzyme activities and low indication of oxidative stress but high activity of some redox enzymes (GR and glucose 6-phosphate dehydrogenase [G6PDH]). High activities of some redox enzymes (SOD, GR, and G6PDH) seem to be related to migration, whereas low activities of some redox enzymes (SOD, CAT, and GR) may be associated with breeding. Barn swallows in South Africa may experience pollution-related oxidative stress, which may hamper interpretation of normal seasonal variation in redox parameters.

Acute responses of brown mussel (Perna perna) exposed to sub-lethal copper levels: integration of physiological and cellular responses.

This study examined the effect of sub-lethal copper levels on selected physiological and cellular responses of the marine bivalve Perna perna. Animals were exposed to five environmentally relevant concentrations of 12.5, 25.0, 37.5 and 50.0 μg L⁻¹ copper and metal accumulation was found to be significantly increased at the two higher copper concentrations after 24 h of exposure. Physiological responses found to increase during acute copper exposure included mucus secretion rate (at 25 and 50 μg L⁻¹ copper), nitrogen excretion rates and oxygen consumption rates (both at 25 and 50 μg L⁻¹ copper). Perna perna changed its substrate utilisation at 25, 37.5 and 50 μg L⁻¹ copper in favour of protein-based metabolism. A higher degree of ROS induced DNA damage was observed at acute exposure to 37.5 and 50 μg L⁻¹ copper. Filtration rate was unchanged during acute copper exposure. A model is proposed that integrates cellular and physiological responses to copper during short-term acute and long-term chronic exposures.

Haematological and genotoxic responses in an urban adapter, the banana bat, foraging at wastewater treatment works

Wastewater Treatment Works (WWTWs) are a ubiquitous feature of the urban landscape. The Banana Bat, Neoromicia nana specifically exploits the high abundance of chironomid midge prey available at WWTWs but these populations also have higher levels of non-essential metals (Cd, Cr and Ni) in their tissues than bats foraging at unpolluted sites. Pollutant exposure may elicit primary physiological responses such as DNA damage and haematological changes. We investigated whether pollutant exposure from foraging at WWTWs impacts haematological and genotoxic parameters in N. nana. We compared four measures of haematological/genotoxic damage between N. nana foraging at three WWTWs and two unpolluted sites located in KwaZulu-Natal, South Africa: DNA damage measured by the Comet assay, total antioxidant capacity as indicated by the FRAP assay, chromosomal aberration indicated by micronuclei formation and blood oxygen capacity based on haematocrits. There was significantly higher DNA damage in N. nana at WWTWs than in bats from unpolluted sites, suggesting inadequate repair to double stranded DNA breaks. In addition, WWTW bats had a significantly lower antioxidant capacity than bats from unpolluted sites. This suggests that bats at WWTWs may have a diminished capacity to cope with the excess reactive oxidative species (ROS) produced from pollutants such as metals. There was no increase in micronucleus frequency in WWTW bats, indicating that cellular functioning has not yet been disrupted by chemical exposure. Haematocrits, however, were significantly higher in WWTW bats, possibly due to erythrocyte production in response to certain pollutants. Thus, effects of pollutant exposure in bats foraging at WWTWs elicit sub-lethal haematological and genotoxic responses which may pose serious long-term risks. This provides evidence that WWTWs, that are aimed to remove pollutants from the environment, can themselves act as a source of contamination and pose a threat to animals exploiting these habitats.

Pollutant exposure at wastewater treatment works affects the detoxification organs of an urban adapter, the Banana Bat.

The Banana Bat, Neoromicia nana, exploits pollution-tolerant chironomids at wastewater treatment works (WWTWs). We investigated how pollutant exposure impacts the detoxification organs, namely the liver and kidney of N. nana. (i) We performed SEM-EDS to quantify metal content and mineral nutrients, and found significant differences in essential metal (Fe and Zn) content in the liver, and significant differences in Cu and one mineral nutrient (K) in the kidneys. (ii) We performed histological analysis and found more histopathological lesions in detoxification organs of WWTW bats. (iii) We calculated hepatosomatic/renalsomatic indices (HSI/RSI) to investigate whole organ effects, and found significant increases in organ size at WWTWs. (iv) We quantified metallothionein 1E (MT1E), using Western Blot immunodetection. Contrary to predictions, we found no significant upregulation of MT1E in bats at WWTWs. Ultimately, N. nana exploiting WWTWs may suffer chronic health problems from sub-lethal damage to organs responsible for detoxifying pollutants.

Foraging at wastewater treatment works affects brown adipose tissue fatty acid profiles in banana bats

ABSTRACT In this study we tested the hypothesis that the decrease in habitat quality at wastewater treatment works (WWTW), such as limited prey diversity and exposure to the toxic cocktail of pollutants, affect fatty acid profiles of interscapular brown adipose tissue (iBrAT) in bats. Further, the antioxidant capacity of oxidative tissues such as pectoral and cardiac muscle may not be adequate to protect those tissues against reactive molecules resulting from polyunsaturated fatty acid auto-oxidation in the WWTW bats. Bats were sampled at two urban WWTW, and two unpolluted reference sites in KwaZulu-Natal, South Africa. Brown adipose tissue (BrAT) mass was lower in WWTW bats than in reference site bats. We found lower levels of saturated phospholipid fatty acids and higher levels of mono- and polyunsaturated fatty acids in WWTW bats than in reference site bats, while C18 desaturation and n-6 to n-3 ratios were higher in the WWTW bats. This was not associated with high lipid peroxidation levels in pectoral and cardiac muscle. Combined, these results indicate that WWTW bats rely on iBrAT as an energy source, and opportunistic foraging on abundant, pollutant-tolerant prey may change fatty acid profiles in their tissue, with possible effects on mitochondrial functioning, torpor and energy usage. Summary: Brown adipose tissue of banana bats foraging at wastewater treatment works contained lower saturated and higher mono- and polyunsaturated phospholipid fatty acids than that of bats foraging at reference sites.

Corticosterone secretion patterns prior to spring and autumn migration differ in free-living barn swallows (Hirundo rustica L.).

Recent studies of long-distance migratory birds show that behavioural and physiological changes associated with predictable or unpredictable challenges during the annual cycle are distinctively regulated by hormones. Corticosterone is the primary energy regulating hormone in birds. Corticosterone levels are elevated during stresses but they are also modulated seasonally according to environmental conditions and life-history demands. We measured the baseline and stress-induced levels of corticosterone in the barn swallow (Hirundo rustica L.) just before spring and autumn migrations in South Africa and Finland, respectively. Barn swallows completing their pre-breeding moult had low body condition (residual body mass) and high baseline corticosterone levels in the wintering grounds. In contrast, baseline corticosterone levels in Finland were low and not related to residual mass. These data contradict the first prediction of the migration modulation hypothesis (MMH) by showing no association with baseline corticosterone levels and pre-migratory fuelling. Yet, the adrenocortical response to the capture and handling stress was notably blunted in South Africa compared to a strong response in Finland. Further, individuals that had started fuelling in Finland showed a reduced response to the handling stress. Taken together, elevated baseline corticosterone levels and high residual mass may blunt the adrenocortical response in long-distance migrants and aerial feeders such as the barn swallow. This observation lends support to the second prediction of the MMH.

The tobacco carcinogen NNK drives accumulation of DNMT1 at the GR promoter thereby reducing GR expression in untransformed lung fibroblasts

Small cell lung cancer (SCLC) is a highly aggressive, predominantly cigarette smoke-induced tumour with poor prognosis. The glucocorticoid receptor (GR), a SCLC tumour suppressor gene, is typically reduced in SCLC. We now show that SCLC cells express high levels of DNA methyltransferase 1 (DNMT1) which accumulates at the GR promoter. DNMT1 expression is further increased by exposure to the tobacco carcinogen NNK. In the untransformed human lung fibroblast cell line, MRC-5, short term NNK treatment decreases GRα mRNA and protein expression due to accumulation of DNMT1 at the GR promoter. Long term NNK treatment results in persistently augmented DNMT1 levels with lowered GR levels. Long term exposure to NNK slows cell proliferation and induces DNA damage, while the GR antagonist RU486 stimulates proliferation and protects against DNA damage. Although both NNK and RU486 treatment increases methylation at the GR promoter, neither are sufficient to prevent senescence in this context. NNK exposure results in accumulation of DNMT1 at the GR promoter in untransformed lung cells mimicking SCLC cells, directly linking tobacco smoke exposure to silencing of the GR, an important step in SCLC carcinogenesis.

The brains of bats foraging at wastewater treatment works accumulate arsenic, and have low non-enzymatic antioxidant capacities

&NA; Increasing rates of urbanisation cause ubiquitous infrastructures that remove anthropogenic contaminants – particularly Wastewater Treatment Works (WWTWs) – to become stressed, and hence pollute surrounding water systems. Neoromicia nana bats are suitable models to study the effects of pollution in these environments because they exploit abundant pollutant‐tolerant chironomid midges that breed at WWTWs, and consequently accumulate metals such as iron, copper and zinc in their livers and kidneys. If these metals persist in their circulatory systems, and cross the blood brain barrier (BBB) they can have adverse effects on critical functions such as flight and echolocation. The aim of this study was to investigate the potential neurological effects on N. nana foraging at WWTWs versus bats at reference sites in Durban, South Africa. Our objectives were to 1) compare trace metal levels in brain and hair samples (as a proxy for circulating metals) between N. nana foraging at WWTWs and reference sites to determine if excess metals pass through the BBB via the circulatory system; and 2) compare biomarkers of neuron function (acetylcholinesterase activity), protection (antioxidant capacity), DNA integrity (DNA fragmentation), lipid integrity (lipid peroxidation) and cell viability (caspase‐3 activity) between N. nana foraging at WWTW and reference sites. We found a significantly higher concentration of arsenic in hair (p < 0.05) and brain tissue (p < 0.1) of WWTW bats compared to bats at reference sites. By contrast, acetylcholinesterase activity did not differ in bats among sites and there was no evidence of significant differences in lipid peroxidation, compromised DNA integrity or apoptosis in the brains between WWTW bats and reference site bats. However, total antioxidant capacity was significantly lower in brains of WWTW bats than bats at reference sites suggesting that antioxidant protection may be compromised. Long‐term exposure to environmental pollutants at WWTWs may therefore affect cellular processes and protection mechanisms in brains of N. nana bats. It may also affect other mechanisms and functions in the brain such as mitochondrial efficiency and other neurotransmitters but that remains to be tested.

Short Postspawning Recovery Time Affects Dna Integrity and Fertilization Success of South African Abalone (Haliotis midae) Oocytes

ABSTRACT Hydrogen peroxide (H2O2) is used routinely by the abalone farming industry to induce spawning of adult abalone. The aim of the current study was to quantify DNA integrity of adult hemolymph cells as well as germ cells (oocytes and sperm) with different recovery times between spawning events. In addition, fertilization success of exposed germ cells was quantified. The results indicate that spawning events separated by short recovery (2–6 wk) will not provide sufficient recovery from H2O2-induced DNA fragmentation in adult hemocytes. It is recommended that the industry should ideally allow 6–8 wk between spawning events to prevent accumulation of DNA strand breaks in adults over time and to ensure adequate fertilization success to maximize farm production.