Sensitivity of Freshwater Australian Bass (Macquaria novemaculeata) and Silver Perch (Bidyanus bidyanus) to Waterborne Antimony: Exposure-Dose-Response Characteristics and Ion Homeostasis.

We conducted acute toxicity studies using semi-static protocols to examine the lethal responses of Australian bass and silver perch exposed to antimony (Sb) oxidation states in Sb(III) (10.5-30.5 mg L-1) and Sb(V) (95.9-258.7 mg L-1). Bioavailability and the effects of Sb on body ion regulation (Na, Ca, Mg, and K) were also investigated. Antimony species-specific effects were observed with exposure to both Sb oxidation states. Median lethal concentrations (LC50s) for Sb(III) were 13.6 and 18 mg L-1 for Australian bass and silver perch, respectively, and the LC50 for Sb(V) in Australian bass was 165.3 mg L-1. The LC50 could not be calculated for silver perch exposed to Sb(V) as the maximum exposure concentrations produced 40% mortality but a larger-than value of > 258.7 mg L-1 was estimated. Relative median potency values derived from the LC50s were 0.1 Sb(III) and 12.2 and 16.6 Sb(V) for Australian bass and silver perch, respectively, demonstrating greater toxicity of Sb(III) to both fish species. Antimony uptake in fish was observed. Median critical body residue (CBR50) values of 77.7 and 26.6 mg kg-1 for Sb(III) were estimated for Australian bass and silver perch, respectively, and 628.1 mg kg-1 for Sb(V) in Australian bass. Bioconcentration factors (BCFs) for both Sb(III) and Sb(V) did not change with exposure but the greater BCFs for fish exposed to Sb(III) indicate that it is more bioavailable than Sb(V) in acute exposure. No effects on whole-body Na, Ca, Mg, or K ions were observed with fish exposure to either Sb species.

Pathogenicity and infection cycle of Vibrio owensii in larviculture of the ornate spiny lobster (Panulirus ornatus).

The type strain of Vibrio owensii (DY05) was isolated during an epizootic of aquaculture-reared larvae (phyllosomas) of the ornate spiny lobster (Panulirus ornatus). V. owensii DY05 was formally demonstrated to be the etiological agent of a disease causing rapid and reproducible larval mortality with pathologies similar to those seen during disease epizootics. Vectored challenge via the aquaculture live feed organism Artemia (brine shrimp) caused consistent cumulative mortality rates of 84 to 89% after 72 h, in contrast to variable mortality rates seen after immersion challenge. Histopathological examination of vector-challenged phyllosomas revealed bacterial proliferation in the midgut gland (hepatopancreas) concomitant with epithelial cell necrosis. A fluorescent-protein-labeled V. owensii DY05 transconjugant showed dispersal of single cells in the foregut and hepatopancreas 6 h postexposure, leading to colonization of the entire hepatopancreas within 18 h and eventually systemic infection. V. owensii DY05 is a marine enteropathogen highly virulent to P. ornatus phyllosoma that uses vector-mediated transmission and release from host association to a planktonic existence to perpetuate transfer. This understanding of the infection process will improve targeted biocontrol strategies and enhance the prospects of commercially viable larviculture for this valuable spiny lobster species.

Field monitoring of plant-growth-promoting rhizobacteria by colony immunoblotting.

Inoculant plant-growth-promoting bacteria are emerging as an important component of sustainable agriculture. There is a need to develop inexpensive methods for enumerating these organisms after their application in the field, to better understand their survival and impacts on yields. Immunoblotting is one potential method to measure viable cells, but the high cost of the conventionally used nylon membranes makes this method prohibitive. In this study, less expensive alternative materials such as filter papers, glossy photo papers, and transparencies for the purpose of colony immunoblotting were evaluated and the best substance was chosen for further studies. Whatman filter paper No. 541 combined with a 0.01 mol·L(-1) H(2)SO(4) rinsing step gave similar results to nylon membranes but <20% of the overall cost of the original colony immunoblotting assay. The application of the modified immunoblot method was tested on nonsterile clay soil samples that were spiked with high numbers (>10(7) CFU·g(-1)) of the plant-growth-promoting bacteria Pseudomonas fluorescens , Azospirillum brasilense , or Rhizobium leguminosarum . The modified protocol allowed the identification and recovery of over 50% of the inoculated cells of all three strains, amidst a background of the native soil microflora. Subsequently, the survival of P. fluorescens was successfully monitored for several months after application to field-grown rice at Jerilderie, New South Wales, Australia, thus validating the procedure.

Plant-extract-induced changes in the proteome of the soil-borne pathogenic fungus Thielaviopsis basicola.

Thielaviopsis basicola is a hemibiotroph fungus that causes black root rot disease in diverse plants with significant impact on cotton production in Australia. To elucidate how T. basicola growth and proteome are influenced by interactions with natural sources, this fungus was cultured in the presence of root extracts from non-host (wheat, hairy vetch) and susceptible host (cotton, lupin) plants. We found that T. basicola growth was significantly favored in the presence of host extracts, while hierarchical clustering analysis of 2-DE protein profiles of T. basicola showed plant species had a larger effect on the proteome than host/non-host status. Analysis by LC-MS/MS of unique and differentially expressed spots and identification using cross-species similarity searching and de novo sequencing allowed successful identification of 41 spots. These proteins were principally involved in primary metabolism with smaller numbers implicated in other diverse functions. Identification of several "morpho" proteins suggested morphological differences that were further microscopically investigated. Identification of several highly expressed spots suggested that vitamin B(6) is important in the T. basicola response to components present in hairy vetch extract, and finally, three spots, induced in the presence of lupin extract, may correspond to malic enzyme and be involved in lipid accumulation.

The effect of moisture on soil microbial properties and nitrogen cyclers in Mediterranean sweet orange orchards under organic and inorganic fertilization.

Water shortage and soil degradation are common environmental stressors encountered in the Mediterranean area. We evaluated how different soil moisture levels, dependent on distance from drip irrigation points, impact on the biological, chemical and physical properties of citrus soil under organic and inorganic fertilization. We measured soil physicochemical properties, basal soil respiration, soil microbial biomass carbon, soil microbial community structure (phospholipid fatty acid assay), bacterial load (16S rRNA gene abundance), enzymatic activities (urease, dehydrogenase, ß-glucosidase and acid phosphatase) and abundance of microbial nitrogen cyclers (quantitative PCR). A field experiment was established in an orange orchard (Citrus sinensis) in southeast Spain and eighteen soil samples were taken from each plot to compare the impacts of soil moisture: near (wet, w) or away (dry, d) from drip-irrigation points, in plots with inorganic fertilizers under intensive ploughing (PI) or organic fertilization (OA). The results showed that changes in microbial properties and soil microbial indexes were strongly associated with soil moisture content under both organic and inorganic fertilization, and with organic carbon content. Soil moisture influenced soil aggregation, basal soil respiration, phosphatase activity, bacterial and fungal load (PLFAs) and the abundances of bacterial N cycling genes, including nifH (nitrogen fixation) nirS/K and nosZ genes (denitrification) and amoA-B (bacterial nitrification). The potential for N fixation and denitrification, two microbial processes that are crucial for determining the amount of N in the soil, were improved by increased soil moisture in the proximity of the drip irrigation. Soil OC and total N, which are higher under organic fertilization than under inorganic fertilization, were also shown to be highly correlated with the abundance of the N cycling genes. By controlling irrigation doses and applying organic amendments, it may be possible to increase the microbial abundance and function in soil and support greater fertility of soils.

Coring lubricants can increase soil microbial activity in Vertisols.

It is essential that sampling procedures for biological measurements are done in a way that reflects the soil processes, whilst limiting sampling artefacts. In heavy clay Vertisol soils, coring lubricants are often considered necessary in order to extract and recover soil for quality and health assessments. Previous reports into the use of coring lubricants have found soil carbon measurements to be inflated but to date, a study to evaluate the effects of these lubricants on soil microbial activity, has not been forthcoming. We measured soil carbon dioxide (CO2) evolution in response to the addition of common coring lubricants, to determine the effects upon soil microbial activity to the depth of 100 cm. Application of coring lubricants to the surface soil layers of field collected cores did not significantly influence CO2 evolution however, microbial activity increased in deeper soil layers (30-100 cm) with the use of WD-40, mould stripper and silicone oil. When the ratio of coring lubricant to soil was increased to ~5 g coring lubricant to 100 g-1 soil, there was a significant (P = .001) effect on microbial activity, with silicone oil and mould stripper inflating measurements by at least 5%, whilst olive oil and WD-40 were similar to the control. The results imply that when using coring rigs to recover soil for microbial functional analysis in Vertisols, the use of coring lubricants is best avoided, with further research recommended.

Storage and handling of human faecal samples affect the gut microbiome composition: A feasibility study.

Human gut microbiome analysis through faecal sampling typically involves five stages: sample collection, storage, DNA extraction, next generation sequencing and bioinformatics analysis. Of these, the first three are considered irreversible. This feasibility study describes an assessment of methodologies used for faecal DNA extraction and sample handling, using the parameters DNA yield, purity and resultant microbial profile. Six DNA extraction techniques, including commercially available kits and manual protocols were compared on human faecal samples (n = 3). Different extraction techniques produced significant variance in DNA yield (range 2.7-164 ng/mg faeces) and microbial diversity profiles, with considerable variation in phyla dominance (Firmicutes (P < 0.001), Bacteroidetes (P = 0.003), Actinobacteria (P = 0.003), One-way ANOVA). The most effective method, with the highest DNA yield, was a simple and inexpensive extraction technique named MetaHIT. Using this method, DNA was extracted from separate faecal samples (n = 3) and had been aliquoted to seven storage conditions including three stabilizing buffers and three temperature conditions, for a period of 120-h, with storage at -80 °C as a control treatment. DNA yield and purity was not statistically different between the control and remaining treatments. 16S rDNA-based diversity profile was largely comparable across the treatments with only minor differences in genera between samples stored at room temperature in air and - 80 °C control. Overall these results suggest that the choice of DNA extraction method has a greater influence on the resultant microbial diversity profile than the short-term storage method.

Shelter, clothing, and fuel: Often overlooked links between soils, ecosystem services, and human health.

There are clear connections between ecosystem services (ES) and human health, as well as between soils and human health. However, studies to date have not investigated links between soil ES and human health. Viewing the relationship between soils and human health through the ES lens reveals that soil ES such as the provisioning of shelter, clothing, and fuel have been overlooked in the soil and human health literature. Shelter is important to human health because it provides protection against inclement weather, temperature extremes, and other potential threats. Clothing provides a more consistent micro-environment around the skin and also provides protection from ultraviolet radiation and some parasites. Fuel allows us to warm shelters, providing refuge from cold temperatures, and cook food, which reduces disease. The materials supplied by soils in support of these functions are often done so in a more environmentally responsible way than is the case with many modern building and clothing materials or with fossil fuels. However, it is important to realize that sustainable management practices are critical in order to achieve environmentally responsible production of these products. Future studies need to investigate the links between these overlooked soil ES and human health.

The impact of post-fire salvage logging on microbial nitrogen cyclers in Mediterranean forest soil.

Forest fires are a regular occurrence in the Mediterranean basin. High severity fires and post-fire management can affect biological, chemical and physical properties of soil, including the composition and abundance of soil microbial communities. Salvage logging is a post-fire management strategy, which involves the removal of burnt wood from land after a fire. The main objective of this work was to evaluate the impact of post-fire salvage logging and microaggregation on soil microbial communities, specifically on the abundance of nitrogen cyclers and, thus, the potential of the soil for microbial nitrogen cycling. The abundance of nitrogen cyclers was assessed by quantification of microbial nitrogen cycling genes in soil DNA, including nifH (involved in nitrogen fixation), nirS/K and nosZ (involved in denitrification), amoA-B and amoA-Arch (involved in bacterial and archaeal nitrification, respectively). It was demonstrated that salvage logging reduced bacterial load post-fire when compared to tree retention control and resulted in significant changes to the abundance of functional bacteria involved in nitrogen cycling. Microbial gene pools involved in various stages of the nitrogen cycle were larger in control soil than in soil subjected to post-fire salvage logging and were significantly correlated with organic matter, available phosphorous, nitrogen and aggregate stability. The microaggregate fraction of the soil, which has been associated with greater organic carbon, was shown to be a hotspot for nitrogen cyclers particularly under salvage logging. The impact of post-fire management strategies on soil microbial communities needs to be considered in relation to maintaining ecosystem productivity, resilience and potential impact on climate change.

The relative sensitivity of freshwater species to antimony(III): Implications for water quality guidelines and ecological risk assessments.

Antimony (Sb) is a pollutant in many jurisdictions, yet its threat to aquatic biota is unclear. Water quality guidelines (WQGs) for Sb are not well established and large uncertainty factors are commonly applied in derivation. We constructed freshwater species sensitivity distributions (SSDs) for Sb(III) using available acute toxicity data sourced from temperate and tropical regional studies. A tiered ecological risk assessment (ERA) approach using risk quotients (RQs) was applied for characterisation of risks presented by Sb(III) concentrations measured in the freshwater environment. Multiple parametric models were fitted for each SSD, with the optimal model used to derive the 5% hazardous concentration (HC5), defined as protective of 95% of species, and the corresponding predicted no effect concentration (PNEC). The HC5 values for whole and temperate SSDs were estimated at 781 and 976 µg L-1 Sb(III), respectively, while the PNECs for both datasets were 156 and 195 µg L-1 Sb(III), respectively. Due to limited tropical data, a temperate-to-tropic extrapolation factor of 10 was used to estimate an interim PNEC for tropical regions of 20 µg L-1 Sb(III). Based on published freshwater Sb(III) concentration values across a range of locations, potential ecological risks posed by Sb(III) in some freshwater systems studied would be classified as medium to high risk, but the majority of locations sampled would fall into the low ecological risk category. Our results facilitate the understanding of toxic effects of Sb(III) to freshwater species but also demonstrate that data for Sb ERA are extremely limited.

Evaluation of reference genes for gene expression analysis using quantitative RT-PCR in Azospirillum brasilense.

Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data.

Cellular responses during morphological transformation in Azospirillum brasilense and Its flcA knockout mutant.

FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA- strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome.

Probiont niche specialization contributes to additive protection against Vibrio owensii in spiny lobster larvae.

The development of efficient probiotic application protocols for use in marine larviculture relies on comprehensive understanding of pathogen-probiont-host interactions. The probiont combination of Pseudoalteromonas sp. PP107 and Vibrio sp. PP05 provides additive protection against vectored Vibrio owensii DY05 infection in larvae (phyllosomas) of ornate spiny lobster, Panulirus ornatus. Here, fluorescently tagged strains were used to demonstrate niche specialization of these probionts in both the live feed vector organism Artemia and in phyllosomas. The pathogen was vulnerable to direct interaction with PP05 in the bacterioplankton as well as in the Artemia gut and the phyllosoma foregut and midgut gland. In contrast, PP107 was localized on external surfaces of Artemia and phyllosomas, and direct interaction with the pathogen was limited to the bacterioplankton. While PP107 was the overall dominant ectobiont on the phyllosoma cephalothorax and inner leg segments, PP05 was the primary colonizer of outer leg segments, nutrient-rich locales that may promote ingestion during feeding. This study shows that niche specialization can contribute to the additive probiotic effect of a probiotic mixture and highlights that probiotic enrichment of Artemia cultures can intercept the infection cycle of V. owensii DY05 in early-stage P. ornatus phyllosomas.

The role of coral-associated bacterial communities in Australian Subtropical White Syndrome of Turbinaria mesenterina.

Australian Subtropical White Syndrome (ASWS) is an infectious, temperature dependent disease of the subtropical coral Turbinaria mesenterina involving a hitherto unknown transmissible causative agent. This report describes significant changes in the coral associated bacterial community as the disease progresses from the apparently healthy tissue of ASWS affected coral colonies, to areas of the colony affected by ASWS lesions, to the dead coral skeleton exposed by ASWS. In an effort to better understand the potential roles of bacteria in the formation of disease lesions, the effect of antibacterials on the rate of lesion progression was tested, and both culture based and culture independent techniques were used to investigate the bacterial communities associated with colonies of T. mesenterina. Culture-independent analysis was performed using the Oligonucleotide Fingerprinting of Ribosomal Genes (OFRG) technique, which allowed a library of 8094 cloned bacterial 16S ribosomal genes to be analysed. Interestingly, the bacterial communities associated with both healthy and disease affected corals were very diverse and ASWS associated communities were not characterized by a single dominant organism. Treatment with antibacterials had a significant effect on the rate of progress of disease lesions (p = 0.006), suggesting that bacteria may play direct roles as the causative agents of ASWS. A number of potential aetiological agents of ASWS were identified in both the culture-based and culture-independent studies. In the culture-independent study an Alphaproteobacterium closely related to Roseovarius crassostreae, the apparent aetiological agent of juvenile oyster disease, was found to be significantly associated with disease lesions. In the culture-based study Vibrio harveyi was consistently associated with ASWS affected coral colonies and was not isolated from any healthy colonies. The differing results of the culture based and culture-independent studies highlight the importance of using both approaches in the investigation of microbial communities.

Identification of an antagonistic probiotic combination protecting ornate spiny lobster (Panulirus ornatus) larvae against Vibrio owensii infection.

Vibrio owensii DY05 is a serious pathogen causing epizootics in the larviculture of ornate spiny lobster Panulirus ornatus. In the present study a multi-tiered probiotic screening strategy was used to identify a probiotic combination capable of protecting P. ornatus larvae (phyllosomas) from experimental V. owensii DY05 infection. From a pool of more than 500 marine bacterial isolates, 91 showed definitive in vitro antagonistic activity towards the pathogen. Antagonistic candidates were shortlisted based on phylogeny, strength of antagonistic activity, and isolate origin. Miniaturized assays used a green fluorescent protein labelled transconjugant of V. owensii DY05 to assess pathogen growth and biofilm formation in the presence of shortlisted candidates. This approach enabled rapid processing and selection of candidates to be tested in a phyllosoma infection model. When used in combination, strains Vibrio sp. PP05 and Pseudoalteromonas sp. PP107 significantly and reproducibly protected P. ornatus phyllosomas during vectored challenge with V. owensii DY05, with survival not differing significantly from unchallenged controls. The present study has shown the value of multispecies probiotic treatment and demonstrated that natural microbial communities associated with wild phyllosomas and zooplankton prey support antagonistic bacteria capable of in vivo suppression of a pathogen causing epizootics in phyllosoma culture systems.