Bacteria as biological control agents of freshwater cyanobacteria: is it feasible beyond the laboratory?

Biological control of cyanobacteria is a well-researched area with a central focus on laboratory-scale studies. Numerous reports have been made on algicidal isolates, with bacteria as a major component of the antagonists. The research in this review draws a brief summary of what is currently known in the area of freshwater cyanobacteria being inhibited by bacterial isolates. Proteobacteria, Bacteroidetes and Firmicutes are among the most commonly reported phyla of bacteria associated with or employed in this research area. However, there are limited reports of upscaling these control measures beyond the laboratory scale. Lytic control agents are the most commonly reported in the literature with subsequent cyanotoxin release. From a water quality perspective, this is not feasible. Based on the available literature, temperature, pH and nutrient changes have been explored in this short review as possible contributors to less optimal bacterial performance. Moreover, the investigation into optimising some of these parameters may lead to increased bacterial performance and, therefore, viability for upscaling this biological control. Through the compilation of current research, this review offers insight to live predator-prey cell interactions between cyanobacteria and algicidal bacteria.

Comparative Early Life Stage Toxicity of the African Clawed Frog, Xenopus laevis Following Exposure to Selected Herbicide Formulations Applied to Eradicate Alien Plants in South Africa.

The rise in pesticides application has increased the need for better understanding of their ecological impacts. The global amphibian declines, for example, have been positively correlated with pesticides use. The differential susceptibility in the developmental stages of amphibians to chemical substances are still largely unknown. We examined the 96-h differential toxicity responses of embryos, premetamorphic and transitional larval stage of Xenopus laevis, to six formulated aquatic herbicide products containing the active ingredients of diquat dibromide (Midstream), glufosinate ammonium (Basta), imazapyr (Arsenal), and three glyphosate formulations (Roundup, Kilo Max, and Environ Glyphosate). The results showed the premetamorphic stage as the most sensitive to the herbicides toxicity. This study confirmed that the developmental stage at which amphibian are exposed to contaminants is critical to their survival and that the chemical contamination hypothesis of the global decline of amphibians should continue to be considered. This establishment of the premetamorphic larval as sensitive toxicity representative for all developmental stages of X. laevis means that this stage could be used more extensively in pesticides toxicity assessments.

Evidence for sperm storage in common smoothhound shark Mustelus mustelus and paternity assessment in a single litter from South Africa.

The present histological study of adult female common smoothhound shark Mustelus mustelus demonstrates that the species can store sperm in their oviducal glands. In addition, single paternity of a litter of 22 pups is reported, contributing to further knowledge regarding the genetic and behavioural mating strategy of M. mustelus.

Thyroid function of steatitis-affected Mozambique tilapia Oreochromis mossambicus from a sub-tropical African reservoir.

Thyroid function and nutritional indicators were measured in obese, steatitis-affected Mozambique tilapia Oreochromis mossambicus from Loskop Reservoir (LR), South Africa. Plasma thyroid hormones (especially T3) and thyroid follicle histomorphology revealed high levels of activity in every aspect of the thyroid cascade measured in fish from LR compared to a reference population of steatitis-free fish. Concurrent measurements of nutritional state including plasma lipids, liver lipid content and hepatocyte size showed that fish from LR had significant energy stores indicative of abundant nutritional intake. There were distinct sex and seasonal differences, with the highest plasma lipids and T3 levels observed in steatitis-affected females during spring and summer. Positive correlations were observed between plasma lipids (especially cholesterol) and T3 concentrations in fish from both populations, indicating a link between lipid metabolism and thyroid function. There was no direct evidence of thyroid disruption, but this cannot be ruled out until further research determines the factors that underlie the homeostatic shift leading to elevated plasma and liver lipids and T3 levels in steatitis-affected tilapia.

The tooth, the whole tooth and nothing but the tooth: tooth shape and ontogenetic shift dynamics in the white shark Carcharodon carcharias.

Results from this study of the white shark Carcharodon carcharias include measurements obtained using a novel photographic method that reveal significant differences between the sexes in the relationship between tooth cuspidity and shark total length, and a novel ontogenetic change in male tooth shape. Males exhibit broader upper first teeth and increased distal inclination of upper third teeth with increasing length, while females do not present a consistent morphological change. Substantial individual variation, with implications for pace of life syndrome, was present in males and tooth polymorphism was suggested in females. Sexual differences and individual variation may play major roles in ontogenetic changes in tooth morphology in C. carcharias, with potential implications for their foraging biology. Such individual and sexual differences should be included in studies of ontogenetic shift dynamics in other species and systems.

The expression of selected genes linked to metabolic homeostasis in obese pansteatitis-suffering Mozambique tilapia, Oreochromis mossambicus (Peters).

The Oreochromis mossambicus (Peters) population inhabiting Lake Loskop, South Africa, is characterized by a high incidence of obesity and pansteatitis. We investigated potential links between the impaired health of Lake Loskop O. mossambicus and the endocrine system by assessing the expression of selected genes associated with the thyroid and adrenal endocrine axes as well as peroxisome proliferator-activated receptor gamma (pparg). Moreover, contaminant-induced thyroid and/or metabolic modulation in Lake Loskop water was evaluated using juvenile O. mossambicus in laboratory exposures. The expression of thyroid hormone receptor alpha (thra) and type 2 deiodinase (dio2) was higher in Lake Loskop O. mossambicus than fish from another population, suggesting a degree of thyroid disruption. The altered gene expression may be a consequence, rather than cause of obesity. Expression of dio2 and pparg was higher in juvenile O. mossambicus exposed to unfiltered compared to filtered lake water, and our data suggest fasting as causative factor. Micro-organism abundance can therefore be a confounding factor in studies applying molecular markers to test for thyroid modulation by environmental waters. Pansteatitis was not a significant source of variance in the expression of any of the genes investigated, suggesting that the disease is not associated with disrupted endocrine signalling.

Molasses as a possible cause of an "endocrine disruptive syndrome" in calves.

During the mid 1990s a potentially serious, chronic syndrome was reported in well-managed beef and dairy herds from unrelated parts of South Africa. Farmers reported that it manifested as various combinations of decreased production, decreased weaning masses, apparent immune breakdown in previously immunocompetent animals, increased reproductive disorders, various mineral imbalances in non-deficient areas and goitre, noticeable as enlarged thyroid glands. The farmers associated this syndrome with certain batches of sugar cane molasses and molasses-based products. The syndrome was reminiscent of an "endocrine disruptive syndrome". The objective of this study was to evaluate the suspected endocrine disruptive effect of molasses included in cattle feed. Using existing in vitro assays, four batches of molasses syrup were screened for possible inclusion in a calf feeding trial. Two batches were selected for the trial. Thirty-two, 4- to 6-week-old, weaned Holstein bull calves were included in the single phase, three treatment, parallel design experiment. In two of the groups of calves, two different batches of molasses were included in their rations respectively. The control group was fed a ration to which no molasses was added, but which was balanced for energy and mineral content. The mass gain of the calves was recorded over the 6-month study period. The calves were clinically examined every week and clinical pathology parameters, immune responses and endocrine effects were regularly evaluated. Even though endocrine disrupting effects were detected with the in vitro screening assays, these could not be reproduced in the calves in the experiment. The two batches of molasses utilized in the calf feeding trial did not induce major differences in any of the parameters measured, with the exception of a lower mass gain in one of the molasses-fed groups (Group 1), which tended towards significance. The results of the study indicate that the two batches of molasses had no endocrine disruptive or immunosuppressive effects in calves.

A laboratory based exposure of Microcystis and Oscillatoria cyanobacterial isolates to heterotrophic bacteria.

Biological control of cyanobacteria is a viable means of controlling nuisance bloom occurrences; however the majority of studies done are against Microcystis sp., with a commonly lytic effect caused. Filamentous cyanobacteria such as Oscillatoria are not as extensively studied in this area of biological control and are often part of Microcystis dominated blooms. This study employed heterotrophic bacterial isolates selected from bloom waters that indicated potential predatory behaviour against both filamentous and colonial cyanobacterial isolates. In comparison to a known Bacillus isolate, which is often reported among bacterial control agents, three other bacteria isolates were tested as control agents against non-axenic Oscillatoria and Microcystis cyanobacterial cultures. Assessments of cyanobacterial cell responses to the bacteria were conducted through water chemistry, chlorophyll a, alkaline phosphatase activity, microscopy and cyanotoxin measurements. The changes in these parameters were compared to untreated cyanobacterial cultures where no bacteria were added. The study found that at ratios of bacteria half that of Microcystis, minimal changes in chlorophyll a were observed, whilst Oscillatoria showed a decreased chlorophyll a more in the presence of isolates 1 and 3w. The assessment of alkaline phosphatase activity showed decreased activity in both cyanobacterial isolates exposed to the bacteria, relative to the untreated control sample. Microscopy analysis through fluorescence indicated that the attachment of the bacteria to the surface of the cyanobacteria hampered with the fluorescence and scanning electron microscopy indicated that the cells were damaged by the addition of the bacterial isolates. Cyanotoxin detection through the ELISA kit testing indicated that there was toxin reduction in samples treated with the bacterial isolates, with the highest reduction being close to 60% in the case of Microcystis sp. treated with isolate 3w. Similar reductions were noted in the filamentous cyanobacterium Oscillatoria, in the presence of isolate 1.

Sex, size and isotopes: cryptic trophic ecology of an apex predator, the white shark Carcharodon carcharias.

Demographic differences in resource use are key components of population and species ecology across the animal kingdom. White sharks (Carcharodon carcharias) are migratory, apex predators, which have undergone significant population declines across their range. Understanding their ecology is key to ensuring that management strategies are effective. Here, we carry out the first stable isotope analyses of free-swimming white sharks in South Africa. Biopsies were collected in Gansbaai (34.5805°S, 19.3518°E) between February and July 2015. We used Stable Isotope Bayesian Ellipsis in R and traditional statistical analyses to quantify and compare isotopic niches of male and female sharks of two size classes, and analyse relationships between isotopic values and shark length. Our results reveal cryptic trophic differences between the sexes and life stages. Males, but not females, were inferred to feed in more offshore or westerly habitats as they grow larger, and only males exhibited evidence of an ontogenetic niche shift. Lack of relationship between δ13C, δ15N and female shark length may be caused by females exhibiting multiple migration and foraging strategies, and a greater propensity to travel further north. Sharks < 3 m had much wider, and more diverse niches than sharks > 3 m, drivers of which may include individual dietary specialisation and temporal factors. The differences in migratory and foraging behaviour between sexes, life stages, and individuals will affect their exposure to anthropogenic threats, and should be considered in management strategies.

An overview of cyanobacterial bloom occurrences and research in Africa over the last decade.

Cyanobacterial blooms are a current cause for concern globally, with vital water sources experiencing frequent and increasingly toxic blooms in the past decade. These increases are resultant of both anthropogenic and natural factors, with climate change being the central concern. Of the more affected parts of the world, Africa has been considered particularly vulnerable due to its historical predisposition and lag in social economic development. This review collectively assesses the available information on cyanobacterial blooms in Africa as well as any visible trends associated with reported occurrences over the last decade. Of the 54 countries in Africa, only 21 have notable research information in the area of cyanobacterial blooms within the last decade, although there is substantial reason to attribute these blooms as some of the major water quality threats in Africa collectively. The collected information suggests that civil wars, disease outbreaks and inadequate infrastructure are at the core of Africa's delayed advancement. This is even more so in the area of cyanobacteria related research, with 11 out of 21 countries having recorded toxicity and physicochemical parameters related to cyanobacterial blooms. Compared to the rest of the continent, peripheral countries are at the forefront of research related to cyanobacteria, with countries such as Angola having sufficient rainfall, but poor water quality with limited information on bloom occurrences. An assessment of the reported blooms found nitrogen concentrations to be higher in the water column of more toxic blooms, validating recent global studies and indicating that phosphorous is not the only factor to be monitored in bloom mitigation. Blooms occurred at low TN: TP ratios and at temperatures above 12°C. Nitrogen was linked to toxicity and temperature also had a positive effect on bloom occurrence and toxicity. Microcystis was the most ubiquitous of the cyanobacterial strains reported in Africa and the one most frequently toxic. Cylindrospermopsis was reported more in the dry, north and western parts of the continent countries as opposed to the rest of the continent, whilst Anabaena was more frequent on the south eastern regions. In light of the entire continent, the inadequacy in reported blooms and advances in this area of research require critical intervention and action.

Histological techniques to improve testing of pulpal response of teeth to filling material.

Two fixation fluids, two fixation techniques and two embedding methods were investigated for their effects on the quality of sections of teeth for pulpal response to filling materials to improve evaluation of pulpal responses. Sections from 32 baboon teeth were prepared, half with experimental cavities and half without, using either 10% formaldehyde or 4% glutaraldehyde, longitudinal tooth splitting or removal of the tooth apex, and paraffin or K plast resin embedding; decalcification in a formic acid mixture was a constant throughout. Histometric analysis showed that paraffin embedding produced less shrinkage than the K Plast resin embedding although the difference was not statistically significant. Six parameters of separation at the pulp:dentine interface were studied: embedding, fixative, presence or absence of a cavity, cutting technique and individual animal tooth type. Statistical investigation revealed that fixative, cutting technique, and fixative and cutting technique combined had significant influences on the separation artifact. Of the combinations tested the choice of embedding method depends on which of the two artifacts, shrinkage or separation, is more adverse in the opinion of the investigator. Four percent glutaraldehyde together with the longitudinal split technique of fixation, processed by either K Plast resin embedding or paraffin embedding produced satisfactory pulpal sections.

The use of IL-6 induction as a human biomarker for inflammatory agents in water.

A rapid whole blood culture (WBC) assay system was developed to monitor the inflammatory potential of water samples collected in the Manguang Municipal area, South Africa. Several systems associated with the Modder River catchment area were investigated in this preliminary study to find out whether there were potential problems in this catchment area. Three focus areas were investigated: (a) the impact of the Klein Modder River, which passes through a densely populated residential area, on the inflammatory activity of the Modder River; (b) the efficiency of the treatment plants in eliminating inflammatory activity; and (c) the efficiency of chlorination of treated water in eliminating inflammatory activity that was not eliminated by the treatment system. Water contaminated with inflammatory substances induced the pro-inflammatory hormone interleukin 6 (IL-6). All water samples collected from the Modder River induced IL-6 secretion, and the quantity of IL-6 secreted was dependent on the concentration and origin of the sample. The lowest IL-6 inducing activity for river water was obtained for samples collected near the origin of the river (Rustfontein). Samples at subsequent points downstream from confluence with the Klein Modder River showed a major increase in IL-6 inducing activity. The two treatment plants along the Modder River also received water of different inflammatory activity. Both treatment plants eliminated some of the inflammatory activity during processing, however inflammatory residues remained after processing which might have been potentially harmful for infants and people with suppressed immune systems. Chlorination eliminated most of the inflammatory activity, indicating that chlorination of water was a necessary process in ensuring water of low inflammatory content.

Isolation and characterization of three estrogen receptor transcripts in Oreochromis mossambicus (Peters).

Exposure of aquatic organisms to 17beta-estradiol (E(2)) induces a variety of estrogen-responsive genes, including vitellogenin (vtg)-the precursor protein of egg yolk in oviparous animals and to date the single most used gene product in screening for estrogenic endocrine disruption. Transcription regulation of vtg by E(2) is dependent on binding of the ligand (E(2)) to a specific nuclear receptor (estrogen receptor, ESR) which in turn binds to an estrogen responsive element (ERE) in the promoter of vtg. Since a local tilapiine, Oreochromis mossambicus (Peters), is targeted as a model for estrogenic endocrine disruption in Southern Africa, a platform of knowledge is necessary for the ontogenic and tissue specific behavior of ESR in this species before vtg levels can be interpreted in relation to such endocrine disruption. Therefore, three ESR cDNA sequences (ESR1, ESR2a and ESR2b) in O. mossambicus were isolated and QPCR protocols were developed to ascertain their quantitative transcript levels in adult brain, gonadal and hepatic tissues. ESR1 transcript levels were highest in female liver tissue compared to males and other tissues, whereas the levels for ESR2a and b were not statistically significantly different between male and female tissues. Quantitative gene levels during development demonstrated a sharp increase in ESR1 during the stage of gonad differentiation (50-60 days post-fertilization) in this species. Finally, an induction experiment in adult male liver tissue confirms the upregulation of ESR1 by E(2).

Characterization of vtg-1 mRNA expression during ontogeny in Oreochromis mossambicus (PETERS).

The yolk-precursor lipoprotein, vitellogenin (VTG) has been widely recognized as a biomarker for the detection of estrogenic activity in water-borne chemical pollutants. We characterized the expression status of this important constituent of reproduction in the Mozambique tilapia (Oreochromis mossambicus), a tilapiine freshwater fish species indigenous to Southern Africa, and investigated its utility in detection of exposure to estrogen using a quantitative real-time polymerase chain reaction (QPCR) assay. We initially isolated a 3kb upstream promoter region of the vtg gene and identified putative binding sites for several regulatory factors including estrogen receptor (ESR). Evidence for the expression of several splice-site vtg mRNA variants was found in a number of tissue types. A quantitative real-time polymerase chain reaction (QPCR) assay was subsequently developed based upon a specific primer pair (OMV6/9) that selectively amplified the liver-enriched transcript. The level of this transcript in liver tissue was high in females and lower, but detectable, in males and was significantly increased in male fish following laboratory exposure to 17beta-estradiol (E(2)). This study further established that juvenile whole body homogenates (WBHs) contained extremely low levels of liver-specific vtg mRNA between 5 and 110 days post-fertilization (dpf) compared to adult male liver. Subsequent exposure of 20 dpf juveniles to E(2) showed a substantial increase in this transcript within hours, and when compared to classic male model under same conditions, the juveniles were remarkably more sensitive. We therefore conclude that the quantification, using QPCR methodology, of vtg mRNA expression in 20 dpf O. mossambicus juveniles has promise for assessing estrogenic EDC activity in aquatic sources.

Temporal expression of two cytochrome P450 aromatase isoforms during development in Oreochromis mossambicus, in association with histological development.

Oreochromis mossambicus is targeted as a sentinel species for assay development to detect disruption of estrogen-dependent pathways in Southern Africa. Aromatase--an enzyme encoded by either of two cytochrome P450 19 (cyp19) genes in O. mossambicus is the only enzyme able to catalyze the aromatization of androgens to estrogens. As part of background studies relating to evaluating the use of early life stages of O. mossambicus as an endocrine disruptor screen, we investigated the tissue specificity and temporal expression of cyp19 transcripts, cyp19a (ovarian cyp19) and cyp19b (brain cyp19) during development and estrogen exposure. The cyp19a transcript was detected only in the ovaries of adults, and expression during development reflected this result as induction of cyp19a coincides with histological development of putative ovaries. Using primers that only identify the ovarian transcript, cyp19a transcript levels demonstrated a typical reflection of puberty - after an initial surge in cyp19a, juveniles expressed very low levels, which increased again at the time histologically discernable vitellogenic ovaries were detected. Moreover, we found evidence of putative alternate transcript of cyp19a whose function is currently unknown. cyp19b transcripts were expressed in brain and muscle tissue of both male and female adults, in addition to ovaries in females. During development, cyp19b transcript levels were increased coincidental with cyp19a at 20 days post fertilization but the expression pattern was distinct from that observed for cyp19a. These studies set the foundation for utilizing this native species as a possible indicator of endocrine disruption and accentuates the importance of understanding "normal" basal levels of transcript levels and the nature of amplification of QPCR targets.

Decalcification end point determination by X-rays and flame photometry to obtain superior quality pulpal sections.

Radiography and flame photometry have been compared as means of determining the end point of decalcification in relation to minimizing pulp-dentin separation in histological sections of teeth. Eighteen homologous pairs of vervet monkey incisor teeth were decalcified in a formic-citric acid mixture. At 24 hr intervals decalcification was monitored in half of the teeth by radiography and in the other half by flame photometry. When decalcification was complete as determined by the respective methods, histological specimens were prepared and separation at the pulp-dentin interface evaluated in hematoxylin and eosin stained step serial sections. The median separation was determined for the combined group and the median test applied. There was significantly less separation in the flame photometry group and within each group significantly less separation on the side where the knife cut from dentin to pulp.

Validation of an ex vivo Xenopus liver slice bioassay for environmental estrogens and estrogen mimics.

A sensitive ex vivo bioassay for detecting environmental estrogens and estrogen mimics was developed using Xenopus liver slice culture. Vitellogenin synthesis by these cultures was used as biomarker for estrogenic activity of environmental water samples. Sensitization of the assay for estrogens and mimics was accomplished by employing tissue from animals after in vivo preexposure to estrogen. Effects of various tissue culture factors were investigated in order to obtain optimum conditions for the bioassay. It was found that endogenous vitellogenin and/or estrogen could be "washed out" of cultures and that not only uncontaminated males but also estrogen-pretreated males and females can successfully be used as bioindicators. Estrogenicity was detected, using the ex vivo Xenopus liver slice culture assay, in sewage effluent, lake water, and dam water. This article presents an optimized protocol for effective estrogen detection in environmental water samples.

Inflammatory activity as an indicator of water quality: the use of human whole blood cultures.

A rapid whole blood culture (WBC) assay system was developed to monitor the inflammatory potential of water samples collected in the Western Cape, South Africa. Water contaminated with inflammatory substances induced the pro-inflammatory hormone interleukin 6 (IL-6). All water samples collected from the Eerste River, Stellenbosch, induced IL-6 secretion, and the quantity of IL-6 secreted is dependent on the concentration and origin of the sample. The lowest IL-6 inducing activity for river water was obtained for samples collected near the origin of the river. Samples at subsequent points downstream showed an increase in IL-6 inducing activity. Drinking water samples collected from selected towns in the Western Cape showed that there were major differences between the inflammatory potential of the water. Of the 15 samples assayed, 7 had low inflammatory activity, 4 had an intermediate inflammatory activity and 4 had high inflammatory activity. The water sources that have a high inflammatory activity may pose a health risk to consumers.

The effects of anti-androgenic and estrogenic disrupting contaminants on breeding gland (nuptial pad) morphology, plasma testosterone levels, and plasma vitellogenin levels in male Xenopus laevis (African clawed frog).

Nuptial pads are areas, digits, and forearm characterized by epidermal keratin hooks and dermal breeding glands opening on the epidermis surface in male frogs. The presence of the epidermal hooks and secretory activity of the breeding glands are androgen-dependent and considered functioning during mating. Chemical pollutants released into the aquatic environment by humans are suspected of disrupting the normal hormonal pathways and functioning. Among the best known of these endocrine-disrupting contaminants (EDCs) are compounds that mimic the steroid 17beta-estradiol. However, recently specific anti-androgenic activity by certain EDCs, including DDE and several fungicides, have been shown in mammalian studies. The African clawed frog, Xenopus laevis, has been proposed as an ideal species to be used as a model system for screening of endocrine disruptors. In this study we employed a newly developed ELISA for the measurement of plasma vitellogenin (VTG), normally produced in the liver of females under the influence of estrogens but also in males when exposed to exogenous estrogens. Furthermore, we investigated the potential of breeding (nuptial) gland activity to be employed as a biomarker system for screening for anti-androgenic activity by certain EDCs. Our results indicate that the pharmaceutic anti-androgen flutamide did significantly (p < 0.05) effect the androgen-dependent breeding (nuptial) glands and plasma testosterone concentrations in male X. laevis. Our results further confirm that the dicarboximide fungicide vinclozolin mimics the anti-androgenic action of flutamide. Vinclozolin, however, did not significantly effect the plasma testosterone concentration. Results also confirm the ability of 17beta-estradiol to stimulate production and release of VTG in the liver of male X. laevis, although we could not confirm the in vivo induction of VTG by estrogenic mimics octylphenol and nonylphenol. Although more dose-response research is needed, this preliminary study confirms the potential of VTG production and breeding gland activity as biomarkers to screen chemicals and environmental samples for estrogenic and anti-androgenic activity using X. laevis males.

The development of an ELISA for chicken apolipoprotein II quantitation.

Polyclonal antibodies raised against chicken apoll was characterised for its use in Western blotting and ELISA detection systems of apoll in chicken plasma. The antibody has a high avidity and specificity for apolipoprotein II (apoII). Western blots show that the antibody reacts with a single band at 15 kDa. The antibody was used for setting up both direct and indirect ELISA assays for apoll. The indirect ELISA has a broader detection range (10-1,600U/mL) than the direct ELISA (10-100U/mL). It was found that both ELISA systems discriminate very well between vitellogenic (laying hen) and non-vitellogenic (rooster) plasma. The in- direct ELISA, due to its broad detection range, can potentially be used for monitoring female reproductive cycles, accidental and environmental exposure of males to estrogen, and for apoII secretion by cultured hepatocytes and hepatomas.