A rapid cell counting method utilising acridine orange as a novel discriminating marker for both cultured astrocytes and microglia

Cell culture analyses of growth, morphology and apoptosis commonly require counting of different cell types stained with antibodies to discriminate between them. Previously, we reported the use of l-Leucine methyl ester (l-LME) to prepare purified cultures of type 1 astrocytes with minimal microglia, and staining by GFAP and CD antibodies, respectively. Here, we demonstrate a novel use of acridine orange (AO) for rapid discrimination between these cell types using fluorescence microscopy. AO accumulates in the lysosomes and also binds strongly to nuclear DNA and cytoplasmic/nucleolar RNA. Microglia may contain abundant lysosomes due to known roles in homeostasis and immune response. AO staining of lysosomes was tested at a range of concentrations, and 2.5 μg/mL was most suitable. In agreement with previous reports, microglia treated with AO showed very intense yellow, orange or red granular cytoplasmic staining of lysosomes. Microglia contain a substantially higher number of lysosomes than astrocytes, which have a variable amount. We measured the microglia population at 5.14 ± 0.50% in mixed cultures. Thus, these results show AO is a novel discriminatory marker, as microglia were easily observed and counted in clumps on top of the monolayer of astrocytes, providing a rapid alternative to time-consuming and costly antibody-based assays.

Influence of temperature on evaporative water loss and cutaneous resistance to water vapour diffusion in the orange-thighed frog (Litoria xanthomera)

We evaluated the effect of ambient temperatures between 25 and 43°C on the rate of evaporative water loss (EWL) in eight adult Litoria xanthomera (average body mass = 7.3 ± 0.6 g). Frogs were placed in a cylindrical chamber that permitted them to fully conceal their ventral surfaces using a water-conserving posture. Their EWL was 7.1 ± 0.7 mg g^–1 h^–1 at 25°C and reached 28.0 ± 2.5 mg g^–1 h^–1 at 43°C. Agar replicas of the frogs were used to evaluate boundary-layer resistances associated with the EWL measurements and, thus, to permit evaluation of cutaneous resistance to vapour diffusion (r_c) in live frogs. The r_c of L. xanthomera was stable over the temperature range of 25–35°C, averaging about 28 s cm^–1, and then declined stepwise with ambient temperatures above 37°C. The highest r_c recorded for each individual over the range of temperatures studied averaged 32.0 ± 1.2 s cm^–1. The thermolabile nature of r_c demonstrates a well developed thermoregulatory control of EWL in this species, a trait very similar in pattern and extent to that previously measured in the closely related Litoria chloris.

Optimisation of Bacillus thuringiensis var. israelensis (Vectobac) applications for the blackfly control programme on the Orange River, South Africa

The Orange River, South Africa’s largest river, is a critical water resource for the country. In spite of the clear economic benefits of regulating river flows through a series of impoundments, one of the significant undesirable ecological consequences of this regulation has been the regular outbreaks of the pest blackfly species Simulium chutteri and S. damnosum s.l. (Diptera: Simuliidae). The current control programme, carried out by the South African National Department of Agriculture, uses regular applications, by helicopter, of the target-specific bacterial larvicide Bacillus thuringiensis var. israelensis. While cost-benefit analyses show significant benefits to the control programme, benefits could potentially be further increased through applying smaller volumes of larvicide in an optimised manner, which incorporates upstream residual amounts of pesticide through downstream carry. Using an optimisation technique applied in the West African Onchocerciasis Control Programme, to a 136 km stretch of the Orange River which includes 31 blackfly breeding sites, we demonstrate that 28.5% less larvicide could be used to potentially achieve the same control of blackfly. This translates into potential annual savings of between R540 000 and R1 800 000. A comparison of larvicide volumes estimated using traditional vs. optimised approaches at different discharges, illustrates that the savings on optimisation decline linearly with increasing flow volumes. Larvicide applications at the lowest discharge considered (40 m³·s^-1) showed the greatest benefits from optimisations, with benefits remaining but decreasing to a theoretical 30% up to median flows of 100 m3·s-1. Given that almost 70% of flows in July are less than 100 m³·s^-1, we suggest that an optimised approach is appropriate for the Orange River Blackfly Control Programme, particularly for flow volumes of less than 100 m³·s^-1. We recommend that trials be undertaken over two reaches of the Orange River, one using the traditional approach, and another using the optimised approach, to test the efficacy of using optimised volumes of B.t.i.

Hope for resurrecting a functionally extinct parrot or squandered social capital? Landholder attitudes towards the Orange-bellied Parrot (Neophema chrysogaster) in Victoria, Australia

In early 2010, after 27 years of recovery effort, the orange-bellied parrot (OBP; Neophema chrysogaster) was expected to be extinct in the wild within a few years. Shortly before the imminent wild extinction became evident, we surveyed landholders (114 responses of 783 surveys delivered) in part of the main non-breeding area, according to three classes of modelled habitat suitability ('high', 'medium', and 'low'). Predictions of the habitat models appear to correlate with landholder perceptions of the presence of OBP habitat on private land, thus the models appear a tractable way to identify key stakeholders worthy of priority consultation in relation to habitat works. Landholders were sympathetic to wetlands and birds, including OBPs (89.4% were aware of OBPs). Most indicated that they would be upset if the OBP went extinct and agreed that critical habitat should be protected; 80.7% were prepared to consider changes to the way they managed their land to benefit the species, and sought more information on how they could do so (64.0%). This study suggests that the habitat model usefully identified key stakeholders and the OBP enjoyed high awareness, concern, and engagement among many stakeholders, shortly before the species was considered functionally extinct. The maintenance of landholder support is likely to be critical if future attempts are made to reintroduce the species to the wild.

Microsatellite loci and the complete mitochondrial DNA sequence characterized through next generation sequencing and de novo genome assembly for the critically endangered orange-bellied parrot, Neophema chrysogaster

A suite of polymorphic microsatellite markers and the complete mitochondrial genome sequence was developed by next generation sequencing (NGS) for the critically endangered orange-bellied parrot, Neophema chrysogaster. A total of 14 polymorphic loci were identified and characterized using DNA extractions representing 40 individuals from Melaleuca, Tasmania, sampled in 2002. We observed moderate genetic variation across most loci (mean number of alleles per locus = 2.79; mean expected heterozygosity = 0.53) with no evidence of individual loci deviating significantly from Hardy-Weinberg equilibrium. Marker independence was confirmed with tests for linkage disequilibrium, and analyses indicated no evidence of null alleles across loci. De novo and reference-based genome assemblies performed using MIRA were used to assemble the N. chrysogaster mitochondrial genome sequence with mean coverage of 116-fold (range 89 to 142-fold). The mitochondrial genome consists of 18,034 base pairs, and a typical metazoan mitochondrial gene content consisting of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a single large non-coding region (control region). The arrangement of mitochondrial genes is also typical of Avian taxa. The annotation of the mitochondrial genome and the characterization of 14 microsatellite markers provide a valuable resource for future genetic monitoring of wild and captive N. chrysogaster populations. As found previously, NGS provides a rapid, low cost and reliable method for polymorphic nuclear genetic marker development and determining complete mitochondrial genome sequences when only a fraction of a genome is sequenced.

Weaning of Australian shortfin glass eels (Anguilla Australis): a comparison on the effectiveness of four types of fish roe

Eel culture is solely dependent on wild seed stock, caught in estuaries during the freshwater migratory phase as glass eels. The methods used for weaning glass eels are very variable, and range from the use of live zooplankton to fish roe to fines of commercial fish feeds. The present experiments were conducted on glass eels of the Australian shortfin eel, when the effectiveness of four types of readily available fish roe (European carp, mirror dory, orange roughy and warehou) were evaluated over a 42-day period, in the laboratory.

After 28 days the eels did not show an interest in orange roughy and mirror dory roe, and these two treatments were discontinued to avoid mortality. In all treatments there was a decrease in mean weight during this period, but the survival was over 99%. In the 28th to 42nd day period the mean weight and specific growth rate of glass eels reared on European carp and warehou roe increased, but the differences between these two treatments were not significant.

The physical features of the roe and the oocytes thereof, the proximate composition, amino acid and fatty acid composition indicated major differences amongst the roe types, particularly with regard to the amount of n−6 polyunsaturated fatty acids (PUFA) and the ratio of n−3 to n−6. European carp and warehou roe (and oocytes) had a significantly higher arachidonic acid (AA-20:4n−6; over 60% of PUFA) content and a considerably lower n−3 to n−6 ratio (n−3 to n−6 ratio being 1.32, 5.92, 3.77 and 2.67 for roe types, and 1.25, 4.83, 2.91 and 2.42 for oocytes, of European carp, mirror dory, orange roughy and warehou, respectively), than in the other two roe types. The fatty acid profiles of European carp and warehou roe were similar to that of metamorphosing glass eels.

Sensory adaptability of foods with added Lupin (Lupinus angustifolius) kernel fibre using pre-set criteria

Fiber-enriched white bread, muffin. pasta, orange juice, and breakfast bar were prepared with lupin (Lupin us angusti/olius) kernel fiber. Consumer panelists (n = 44) determined that all these fiber-enriched foods, except orange juice, fulfilled pre-set acceptability criteria. Fiber enrichment did not change overall acceptability (p> 0.05) of the bread and pasta, but reduced overall acceptability (p < 0.05) of the muffin, orange juice, and breakfast bar. In all fiber-enriched products, flavor was the attribute most highly correlated with overall acceptability (p < 0.05). The lupin kernel fiber used in this study therefore appears to have potential as a 'nonintrusive' ingredient in some processed cereal-based foods_ For other applications, fiber modification appears worthy of investigation to accomplish 'nonintrusive' fiber enrichment.

Anaerobic treatment of juice and fruit-processing effluents

Effluents from the juice and fruit processing industries have high organic matter content. Discharge of these effluents without appropriate treatment would therefore have a negative impact on the environment. High organic contents and low contamination levels make such effluents suitable for biological treatment, especially anaerobic digestion. In the latter process, significant amounts of digester gas can be produced, turning a waste stream into a source of renewable energy that can be used for electricity and heat production, leading to financial benefits.This paper investigates the feasibility of anaerobic digestion and the gas generation potential of five different effluents from the carrot-juice, orange-juice and sultana processing industries. Benefits are assessed in terms of digester gas production and organic matter reduction. The results show that the specific gas production ranges between 665 and 860 m3 per tonne of effluent treated (as organic dry matter). Furthermore, nearly 100% of the organic matter is converted into gas in the case of the carrot- and orange-juice processing residues, while a 84.5% reduction of the organic matter was found to be achievable in the case of the sultana wastes. While these results are promising, further testing will be required to validate them in a larger scale.

New light from an old reagent: chemiluminescence from the reaction of potassium permanganate with sodium borohydride

When aqueous sodium borohydride (50 mM) is added to a solution of potassium permanganate (1mM, in sodium hexametaphosphate) at acidic pH, bright red-orange emission is easily visible in a darkened room. This chemiluminescence emission is due to an excited state of manganese (II) that undergoes solution phase phosphorescence and provides an excellent opportunity for students to explore the relationship between the initial oxidation state of the manganese and the likelihood of luminescence. Not surprisingly Mn(VII), Mn(IV) and Mn(III) all give rise to chemiluminescence where as Mn(II) fails to react.

Taxonomy and biology of two new species of gall midge (Diptera: Cecidomyiidae) infesting Sarcocornia quinqueflora (Chenopodiaceae) in Australian salt marshes

Two new species of gall midge associated with two distinct galls on the succulent creeping shrub Sarcocornia quinqueflora are described from salt marshes in south-eastern Australia. The infestations caused by the new species hinder the growth of S. quinqueflora, the seeds of which are the major food of the critically endangered orange-bellied parrot Neophema chrysogaster. Asphondylia floriformis sp. n. Veenstra-Quah & Kolesik transforms leaf segments into flower-like galls, whereas Asphondylia sarcocorniae sp. n. Veenstra-Quah & Kolesik produces simple swellings on branches. Both galls have fungal mycelium growing in the apoplast of the gall tissue and lining the inner surface of the larval chamber where it is presumably grazed by the larva. Descriptions of the larvae, pupae, males, females and the geographical distribution of the two gall midges in south-eastern Australia are given.