The use of immobilised metal affinity chromatography (IMAC) to compare expression of copper-binding proteins in control and copper-exposed marine microalgae

Toxicity of metals to aquatic organisms is dependent on both external factors, such as exposure concentration and water quality parameters, and intracellular processes including specific metal-binding sites and detoxification. Current models used to predict copper toxicity in microalgae do not adequately consider these intracellular processes. This study compared the copper-binding proteins from four species of marine microalgae, Dunaliella tertiolecta, Tetraselmis sp., Phaedactylum tricornutum and Ceratoneis closterium, in controls (no added copper) and following a 72-h exposure to copper (sufficient to inhibit growth by approximately 50 %). Cells were lysed by sonication, which was optimised to obtain 54–94 % cell rupture for the different algae. Cell lysates were processed by immobilised metal affinity chromatography (IMAC) using Cu2+ as the bound metal (i.e. Cu-IMAC). Bound proteins were subsequently analysed by SDS-PAGE, comparing proteins recovered from algae that were exposed to copper versus untreated control cells. Individual proteins for which copper exposure resulted in changes to proteins present were excised from gels and further analysed by nano LC ESI-MS/MS; proteins were identified using the Mascot database. Proteins identified in this way included heat-shock proteins, rubisco, α- and β-tubulins and ATP synthase (β subunit). The results established that Cu-IMAC is a useful approach to identify proteins involved in copper binding in algae. This study identified several proteins that may play an active role in responses to copper toxicity in marine microalgae

Time-averaged copper concentrations from continuous exposures predicts pulsed exposure toxicity to the marine diatom, Phaeodactylum tricornutum: importance of uptake and elimination

Intermittent, fluctuating and pulsed contaminant discharges result in organisms receiving highly variable contaminant exposures. Current water quality guidelines are predominantly derived using data from continuous exposure toxicity tests, and most frequently applied by regulators with the assumption that concentrations from a single sampling event will provide a meaningful approach to assessing potential effects. This study investigated the effect of single and multiple (daily) dissolved copper pulses on the marine diatom, Phaeodactylum tricornutum, including measurements of copper uptake and elimination to investigate the toxic mechanism. Copper pulses of between 0.5 and 24 h and continuous exposures with equivalent 72-h time-averaged concentrations (TACs) resulted in similar biomass inhibition of P. tricornutum, with continuous exposures often being marginally more toxic. Rates of cell division generally recovered to control levels within 24 h of the copper pulse removal. Upon resuspension in clean seawater, the extracellular copper per cell decreased rapidly, whereas the intracellular copper per cell decreased slowly. Negligible loss of copper from the total algal biomass indicated that P. tricornutum did not have an effective mechanism for eliminating copper from cells, rather the intracellular copper decreased as a result of dilution by cellular division as the algal growth rate recovered. The measurement of copper uptake after 72-h exposure and kinetics of elimination thereafter suggest that continuous exposures are marginally more toxic to P. tricornutum than pulsed copper exposures with equivalent TACs because slow internalization and saturation of algal membrane transport sites results in less copper uptake into pulse-exposed cells than continuously-exposed cells coupled with dilution of internalized copper via cellular division in the post-exposure period. In the case of P. tricornutum, the results indicate that water quality guidelines for copper based on continuous exposure will be conservative when applied to short-term discharges

Combination of novel and public RNA-seq datasets to generate an mRNA expression atlas for the domestic chicken

Background: The domestic chicken (Gallus gallus) is widely used as a model in developmental biology and is also an important livestock species. We describe a novel approach to data integration to generate an mRNA expression atlas for the chicken spanning major tissue types and developmental stages, using a diverse range of publicly-archived RNA-seq datasets and new data derived from immune cells and tissues. Results: Randomly down-sampling RNA-seq datasets to a common depth and quantifying expression against a reference transcriptome using the mRNA quantitation tool Kallisto ensured that disparate datasets explored comparable transcriptomic space. The network analysis tool Graphia was used to extract clusters of co-expressed genes from the resulting expression atlas, many of which were tissue or cell-type restricted, contained transcription factors that have previously been implicated in their regulation, or were otherwise associated with biological processes, such as the cell cycle. The atlas provides a resource for the functional annotation of genes that currently have only a locus ID. We cross-referenced the RNA-seq atlas to a publicly available embryonic Cap Analysis of Gene Expression (CAGE) dataset to infer the developmental time course of organ systems, and to identify a signature of the expansion of tissue macrophage populations during development. Conclusion: Expression profiles obtained from public RNA-seq datasets - despite being generated by different laboratories using different methodologies - can be made comparable to each other. This meta-analytic approach to RNA-seq can be extended with new datasets from novel tissues, and is applicable to any species

Sensitivity of marine microalgae to copper: The effect of biotic factors on copper adsorption and toxicity

Microalgae are sensitive indicators of environmental change and, as the basis of most freshwater and marine ecosystems, are widely used in the assessment of risk and development of environmental regulations for metals. However, interspecies differences in sensitivity to metals are not well understood. The relationship between metal-algal cell binding and copper sensitivity of marine microalgae was investigated using a series of 72-h growth-rate inhibition bioassays and short-term (1-h) uptake studies. A range of marine algae from different taxonomic groups were screened to determine whether copper adsorption to the cell membrane was influenced by biotic factors, such as the ultrastructure of cell walls and cell size. Minutocellus polymorphus was the most sensitive species to copper and Dunaliella tertiolecta the least sensitive, with 72-h IC50 values (concentration to inhibit growth-rate by 50%) of 0.6 and 530 microg Cu/L, respectively. Copper solution-cell partition coefficients at equilibrium (K(d)) were calculated for six species of algae on a per cell and surface area basis. The largest and smallest cells had the lowest and highest K(d) values, respectively (on a surface area basis), with a general (non-linear) trend of decreasing K(d) with increasing cell surface area (p=0.026), however, no relationship was found between K(d) and copper sensitivity, nor cell size and copper sensitivity. Interspecies differences in copper sensitivity were not related to cell size, cell wall type, taxonomic group or K(d) values. The differences in sensitivity may be due to differences in uptake rates across the plasma membrane, in internal binding mechanisms and/or detoxification mechanisms between the different microalgal species

The importance of slime: does living in a community matrix save algal cells from the toxic effects of copper?

Microscopic algae are often used to assess the toxic effects of chemicals to the environment. They are good indicators of ecosystem health because they form the basis of the aquatic food chain and many algal species are sensitive to metals, like copper, at concentrations which occur naturally in the environment. Most toxicity tests with algae use planktonic species, that is, alga that live in the water as free-living species. To date, little research has been done on the toxicity of metals to attached algal species living in a community matrix known as biofilms, because of the difficulties in quantifying changes in such a complex community

The effects of continuous and fluctuating copper exposures on the marine alga Phaeodactylum tricornutum

Contaminant concentrations in aquatic systems are seldom constant. Erratic inputs such as industrial discharges, rain water flushing, and random spills may cause concentrations to increase rapidly. Environmental processes may contribute through the dispersive actions of tides and currents, adsorptive losses to or release from resuspended sediments, and contaminant losses due to photo-degradation and volatilization. Despite such variability in contaminant concentrations, environmental guidelines are derived from toxicity test data using continuous exposure, where contaminant concentrations at the beginning of the exposure are assumed to remain relatively constant over the test duration. Responses of organisms exposed to fluctuating contaminant concentrations may differ from those exposed continuously to contaminants, even for equal contaminant loads. The current knowledge gap regarding the differing responses of organisms to contaminants from continuous and pulsed exposures is impeding decision making processes of both regulatory bodies and discharging industries. We have investigated the effects of continuous and pulsed copper exposure on the growth of the copper-sensitive microalga Phaeodactylum tricornutum as an indicator of ecosystem health

The effect of field-collected biofilms on the toxicity of copper to a marine microalga (Tetraselmis sp.) in laboratory bioassays

Standard algal growth rate inhibition bioassays can lack environmental realism and may over- or underestimate metal bioavailability in natural systems. In aquatic environments, algal species interact with other biota, including other algae, bacteria and biofilms. In this work, the feasibility of incorporating marine biofilms into 72h algal growth inhibition toxicity tests was explored. The effects of copper on Tetraselmis sp. were tested in the absence and presence of characterised field-collected biofilms. We hypothesised that the addition of biofilm would prevent copper toxicity to the alga primarily through interactions of the metal with other cells and biofilm exudates. The sensitivity of Tetraselmis sp. to copper (based on 72h IC50 values; the copper concentration to inhibit population growth by 50%) in the presence of a blended biofilm inoculum varied 2-fold and was independent of the amount of biofilm added. However, increases in IC10 and IC20 values indicated some amelioration of copper toxicity. When intact biofilms were added to the bioassays, amelioration of toxicity was more consistent, probably due to increased binding of copper to cell surfaces or exudates. Difficulties in characterising biofilms and distinguishing that material from the test alga need to be overcome before biofilms can be routinely incorporated into laboratory bioassays

Copper and zinc tolerance of two tropical microalgae after copper acclimation

Current toxicity tests with microalgae are often criticized as being overly sensitive to metals because algae are cultured in metal-deficient media. If such bioassays overestimate copper toxicity in surface waters, the relevance of water quality guidelines derived from these tests is questionable. In this study, the effect of acclimation to copper at environmentally relevant concentrations, on the sensitivity of the marine diatom Nitzschia closterium and the freshwater green alga Chlorella sp. to copper and zinc was examined. N. closterium was acclimated in culture medium containing 5 or 25 g Cu L-1 for 200 days, while Chlorella sp. was acclimated in medium containing 2 g Cu L-1 for 100 days. Changes in algal growth rates and copper and zinc tolerance were monitored using standard growth inhibition toxicity tests in minimal medium over 72 h. Neither of the two acclimated N. closterium cultures had increased zinc or copper tolerance compared with that of the nonacclimated algae, nor were there any changes in control growth rates. Similarly, no changes in copper tolerance or control growth rates were observed for the acclimated Chlorella sp. culture. This was supported by measurements of intracellular and extracellular copper which confirmed that there were no differences in copper accumulation in either acclimated or nonacclimated algae. These results suggest that these algae grown in standard culture media are generally no more sensitive than algae grown in a metal-enriched medium. This supports the continued use of current laboratory bioassays with microalgae, as part of a suite of tests for assessing metal bioavailability, for use in ecological risk assessments and for providing data for the derivation of water quality guidelines. Copyright 2007 Wiley Periodicals, Inc. Environ Toxicol 22: 234-244, 2007

The effect of bacteria on the sensitivity of microalgae to copper in laboratory bioassays

Although single-species laboratory toxicity tests with microalgae are sensitive and highly reproducible, they lack environmental realism. Interactions between algae and their associated bacteria, either in the plankton or in biofilms, may alter algal sensitivity to contaminants, which are not mimicked in laboratory toxicity tests. This study investigated the effects of simple algal-bacterial relationships on the sensitivity of laboratory-cultured algae to copper using 72-h algal growth-rate inhibition bioassays. Four species of microalgae were used, two isolates of each; a strain of algae with no microscopically visible and no culturable bacteria present (operationally defined as axenic) and a non-axenic strain. The four algae used were the marine diatom Nitzschia closterium, the freshwater green alga Pseudokirchneriella subcapitata and two tropical Chlorella spp. Under control conditions (no copper), N. closterium and P. subcapitata grew better in the presence of the bacterial community. Sensitivity to copper (assessed as the concentration to inhibit the growth rate by 50% after 72-h (IC50)) was not significantly different for the axenic and non-axenic strains of N. closterium, P. subcapitata or for Chlorella sp. (PNG isolate). At pH 5.7, the axenic Chlorella sp. (NT isolate) had a 72-h IC50 of 46 μg Cu L−1, while in the presence of bacteria the IC50 increased (i.e., sensitivity decreased) to 208 μg Cu L−1. However, when the bacterial status of both the operationally defined axenic and non-axenic cultures of N. closterium and Chlorella sp. (NT isolate) was investigated using polymerase chain reaction (PCR) amplification of 16S rRNA followed by DNA fingerprinting using denaturing gradient gel electrophoresis (DGGE), it was found that bacteria were actually present in all the algal cultures, i.e. the axenic cultures were not truly bacteria-free. Based on sequence information, the bacteria present were nearly all identified as alphaproteobacteria, and a number of isolates had high similarity to bacteria previously identified as symbionts or species endophytically associated with marine organisms. The “axenic” cultures contained less bacterial phylotypes than the non-axenic cultures, and based on band-intensity, also contained less bacterial DNA. This supported the findings of few differences in copper sensitivity between strains, and suggests that standard microalgal toxicity tests probably inadvertently use non-axenic cultures in metal assessment

Copper-induced changes in intracellular thiols in two marine diatoms: Phaeodactylum tricornutum and Ceratoneis closterium

Phytochelatins and glutathione (reduced (GSH) and oxidised (GSSG)) are important intracellular ligands involved in metal sequestration and detoxification in algae. Intracellular ratios of GSH:GSSG are sensitive indicators of metal stress in algae, and like phytochelatin production are influenced by metal speciation, concentration, exposure time and the biological species. This study investigated the effect of copper exposure on phytochelatin and glutathione content in two marine diatoms Phaeodactylum tricornutum and Ceratoneis closterium at various time intervals between 0.5 and 72 h. Liberation of cellular glutathione and phytochelatins was optimised using freeze/thaw cycles and chemical extraction, respectively. Extracted phytochelatins were derivatised (by fluorescent tagging of thiol compounds), separated and quantified using HPLC with fluorescence detection. Glutathione ratios were determined using a commercially available kit, which uses the enzyme glutathione reductase to measure total and oxidised glutathione. Despite similarities in size and shape between the two diatoms, differences in internalised copper, phytochelatin production (both chain length and quantity) and reduced glutathione concentrations were observed. P. tricornutum maintained reduced glutathione at between 58 and 80% of total glutathione levels at all time points, which would indicate low cellular stress. In C. closterium reduced glutathione constituted \u3c10% of total glutathione after 48 h. P. tricornutum also produced more phytochelatins and phytochelatins of longer chain length than C. closterium despite the latter species internalising significantly more copper