Urban tolerance is phylogenetically constrained and mediated by pre-adaptations in African bats.

With increasing urbanization, particularly in developing countries, it is important to understand how local biota will respond to such landscape changes. Bats comprise one of the most diverse groups of mammals in urban areas, and many species are threatened by habitat destruction and land use change. Yet, in Africa, the response of bats to urban areas is relatively understudied. Therefore, we collated data on urban presence, phylogenetic relationship, and ecological traits of 54 insectivorous bats in Africa from available literature to test if their response to urbanization was phylogenetically and/or ecologically driven. Ancestral state reconstruction of urban tolerance, defined by functional group and presence observed in urban areas, suggests that ancestral African bat species could adapt to urban landscapes, and significant phylogenetic signal for urban tolerance indicates that this ability is evolutionarily conserved and mediated by pre-adaptations. Specifically, traits of high wing loading and aspect ratio, and flexible roosting strategies, enable occupancy of urban areas. Therefore, our results identify the traits that predict which bat species will likely occur in urban areas, and which vulnerable bat clades conservation efforts should focus on to reduce loss of both functional and phylogenetic diversity in Africa. We, additionally, highlight several gaps in research that should be investigated in future studies to provide better monitoring of the impact urbanization will have on African bats.

Rewarming rates of seven insectivorous bat species along an altitudinal gradient in South Africa.

Small endotherms commonly use daily torpor to maintain energetic homeostasis. During daily torpor, rewarming rates have a large impact on overall energy savings because they influence heat loss in this energetically costly phase of torpor. These rates are affected by both intrinsic and extrinsic ecological factors; however, data on rewarming rates along temporal and spatial scales between and within species are scant. We investigated how seven insectivorous bat species differing in body size and thermal stability of preferred roosts vary in rewarming rates along an altitude gradient (0-1400 m.a.s.l.) in South Africa, predicting that rewarming rates would increase with altitude. We kept individuals overnight at 15°C and monitored their surface temperature during induced rewarming with infrared thermal imaging to calculate rewarming rates. In contrast to our prediction, we found no significant variation between species or altitudes despite differences in body mass and life history traits. However, we did find evidence that males rewarmed more rapidly than females at low temperatures despite the higher energetic cost of slow rewarming, possibly due to reluctance of females to initiate active rewarming at low ambient temperatures. We found some support for the prediction that cool temperatures at high altitude result in lower initial surface temperatures during cold-exposure, as an adaptation to mitigate heat loss through thermal conductance to the ambient environment. This is particularly important for small insectivorous bats given their high surface area to volume ratios.

Generation of Human Domain Antibody Fragments as Potential Insecticidal Agents against Helicoverpa armigera by Cadherin-Based Screening.

New insecticidal genes and approaches for pest control are a hot research area. In the present study, we explored a novel strategy for the generation of insecticidal proteins. The midgut cadherin of Helicoverpa armigera (H. armigera) was used as a target to screen materials that have insecticidal activity. After three rounds of panning, the phage-displayed human domain antibody B1F6, which not only binds to the H. armigera cadherin CR9-CR11 but also significantly inhibits Cry1Ac toxins from binding to CR9-CR11, was obtained from a phage-displayed human domain antibody (DAb) library. To better analyze the relevant activity of B1F6, soluble B1F6 protein was expressed by Escherichia coli BL21 (DE3). The cytotoxicity assays demonstrated that soluble B1F6 induced Sf9 cell death when expressing H. armigera cadherin on the cell membrane. The insect bioassay results showed that soluble B1F6 protein (90 µg/cm2) caused 49.5 ± 3.3% H. armigera larvae mortality. The midgut histological results showed that soluble B1F6 caused damage to the midgut epithelium of H. armigera larvae. The present study explored a new strategy and provided a basic material for the generation of new insecticidal materials.

Wastewater treatment works change the intestinal microbiomes of insectivorous bats.

Mammals, born with a near-sterile intestinal tract, are inoculated with their mothers' microbiome during birth. Thereafter, extrinsic and intrinsic factors shape their intestinal microbe assemblage. Wastewater treatment works (WWTW), sites synonymous with pollutants and pathogens, receive influent from domestic, agricultural and industrial sources. The high nutrient content of wastewater supports abundant populations of chironomid midges (Diptera), which transfer these toxicants and potential pathogens to their predators, such as the banana bat Neoromicia nana (Vespertilionidae), thereby influencing their intestinal microbial assemblages. We used next generation sequencing and 16S rRNA gene profiling to identify and compare intestinal bacteria of N. nana at two reference sites and two WWTW sites. We describe the shared intestinal microbiome of the insectivorous bat, N. nana, consisting of seven phyla and eleven classes. Further, multivariate analyses revealed that location was the most significant driver (sex, body size and condition were not significant) of intestinal microbiome diversity. Bats at WWTW sites exhibited greater intestinal microbiota diversity than those at reference sites, likely due to wastewater exposure, stress and/or altered diet. Changes in their intestinal microbiota assemblages may allow these bats to cope with concomitant stressors.

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.

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.

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

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.

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.

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.

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.

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.