The regulation of copper stress response genes in the polychaete Nereis diversicolor during prolonged extreme copper contamination.

Polychaetes are frequented in toxicological studies, one reason being that some members occupy shallow burrows in sediments and are maximally exposed to the contaminants that accumulate within them. We have been studying one population of the polychaete Nereis (Hediste) diversicolor exhibiting inheritable tolerance to extreme copper contamination in estuarine sediment. Using transcriptome sequencing data we have identified a suite of genes with putative roles in metal detoxification and tolerance, and measured their regulation. Copper tolerant individuals display significantly different gene expression profiles compared to animals from a nearby population living without remarkable copper levels. Gene transcripts encoding principle copper homeostasis proteins including membrane copper ion transporters, copper ion chaperones and putative metallothionein-like proteins were significantly more abundant in tolerant animals occupying contaminated sediment. In contrast, those encoding antioxidants and cellular repair pathways were unchanged. Nontolerant animals living in contaminated sediment showed no difference in copper homeostasis-related gene expression but did have significantly elevated levels of mRNAs encoding Glutathione Peroxidase enzymes. This study represents the first use of functional genomics to investigate the copper tolerance trait in this species and provides insight into the mechanism used by these individuals to survive and flourish in conditions which are lethal to their conspecifics.

Quantitative Polymerase Chain Reaction for the estimation of toxigenic microalgae abundance in shellfish production waters.

Certain species of marine microalgae produce potent biotoxins that pose a risk to human health if contaminated seafood is consumed, particularly filter feeding bivalve shellfish. In regions where this is likely to occur water and seafood produce are regularly monitored for the presence of harmful algal cells and their associated toxins, but the current approach is flawed by a lengthy delay before results are available to local authorities. Quantitative Polymerase Chain Reaction (qPCR) can be used to measure phytoplankton DNA sequences in a shorter timeframe, however it is not currently used in official testing practices. In this study, samples were collected almost weekly over six months from three sites within a known HAB hotspot, St Austell Bay in Cornwall, England. The abundance of algal cells in water was measured using microscopy and qPCR, and lipophilic toxins were quantified in mussel flesh using LC-MS/MS, focusing on the okadaic acid group. An increase in algal cell abundance occurred alongside an increase in the concentration of okadaic acid group toxins in mussel tissue at all three study sites, during September and October 2021. This event corresponded to an increase in the measured levels of Dinophysis accuminata DNA, measured using qPCR. In the following spring, the qPCR detected an increase in D. accuminata DNA levels in water samples, which was not detected by microscopy. Harmful algal species belonging to Alexandrium spp. and Pseudo-nitzschia spp. were also measured using qPCR, finding a similar increase in abundance in Autumn and Spring. The results are discussed with consideration of the potential merits and limitations of the qPCR technique versus conventional microscopy analysis, and its potential future role in phytoplankton surveillance under the Official Controls Regulations pertaining to shellfish.

'Ready Mixed', improved nucleic acid amplification assays for the detection of Escherichia coli DNA and RNA.

The selective amplification of E. coli nucleic acid sequences could be used for the early warning of faecal contamination in environmental samples. Modified assays for E. coli DNA and RNA markers are presented with improved integrity and performance over existing methods, and demonstrated using 'ready mixed', preserved reagent mixtures.

Molecular-biological sensing in aquatic environments: recent developments and emerging capabilities.

Aquatic microbial communities are central to biogeochemical processes that maintain Earth's habitability. However, there is a significant paucity of data collected from these species in their natural environment. To address this, a suite of ocean-deployable sampling and sensing instrumentation has been developed to retrieve, archive and analyse water samples and their microbial fraction using state of the art genetic assays. Recent deployments have shed new light onto the role microbes play in essential ocean processes and highlight the risks they may pose to coastal populations. Although current designs are generally too large, complex and expensive for widespread use, a host of emerging bio-analytical technologies have the potential to revolutionise this field and open new possibilities in aquatic microbial metrology.

The anti-bacterial effect of an electrochemical anti-fouling method intended for the protection of miniaturised oceanographic sensors.

An electrochemical anti-fouling method, based upon the generation of chlorine from seawater, was applied to a proprietary design of Lab on a Chip conductivity, temperature and dissolved oxygen sensor. The method was evaluated using PCR after a six-week field trial in which it significantly reduced the burden of bacterial biofouling.

Detection and quantification of the toxic microalgae Karenia brevis using lab on a chip mRNA sequence-based amplification.

Now and again, the rapid proliferation of certain species of phytoplankton can give rise to Harmful Algal Blooms, which pose a serious threat to marine life and human health. Current methods of monitoring phytoplankton are limited by poor specificity or by the requirement to return samples to a highly resourced, centralised lab. The Lab Card is a small, microfluidic cassette which, when used in tandem with a portable Lab Card Reader can be used to sensitively and specifically quantify harmful algae in the field, from nucleic acid extracts using RNA amplification; a sensitive and specific method for the enumeration of potentially any species based on their unique genetic signatures. This study reports the culmination of work to develop a Lab Card-based genetic assay to quantify the harmful algae Karenia brevis using mRNA amplification by the Nucleic Acid Sequence Based Amplification (NASBA) method. K. brevis cells were quantified by amplification of the rbcL gene transcript in nucleic acid extracts of K. brevis cell samples. A novel enzyme dehydration and preservation method was combined with a pre-existing reagent Gelification method to prepare fully preserved Lab Cards with a shelf-life of at least six weeks prior to use. Using an internal control (IC), the Lab Card-based rbcL NASBA was demonstrated for the quantification of K. brevis from cell extracts containing between 50 and 5000 cells. This is the first demonstration of quantitation of K. brevis using IC-NASBA on a Lab Card.

Whole-cell Escherichia coli-based bio-sensor assay for dual zinc oxide nanoparticle toxicity mechanisms.

A whole-cell biosensor assay for dual ZnO nanoparticle toxicity mechanisms has been developed based on the transcriptional response of Escherichia coli to: (1) Zn(2+) from ZnO nanoparticle dissolution with genes zntA (Zn(2+) efflux) and znuABC (Zn(2+) uptake); and (2) redox stress from ZnO nanoparticle photo-electron production under ultraviolet light with genes soxS and katG. Both processes occur in a dispersion of ZnO nanoparticles leading to toxicity. ZnO nanoparticle dissolution was measured independently by ICP-MS and photo-radical generation was confirmed by the stochiometric reduction of the redox dye, 2, 6-dichloroindolphenol (DCPIP). The whole-cell biosensor can detect both toxicity mechanisms and is a species-specific assay capable of discriminating between ZnO nanoparticles and the Zn(2+) dissolution product.

Differential gene regulation in the Ag nanoparticle and Ag(+)-induced silver stress response in Escherichia coli: a full transcriptomic profile.

We report the whole-transcriptome response of Escherichia coli bacteria to acute treatment with silver nanoparticles (AgNPs) or silver ions [Ag(I)] as silver nitrate using gene expression microarrays. In total, 188 genes were regulated by both silver treatments, 161 were up-regulated and 27 were down-regulated. Significant regulation was observed for heat shock response genes in line with protein denaturation associated with protein structure vulnerability indicating Ag(I)-labile -SH bonds. Disruption to iron-sulphur clusters led to the positive regulation of iron-sulphur assembly systems and the expression of genes for iron and sulphate homeostasis. Further, Ag ions induced a redox stress response associated with large (>600-fold) up-regulation of the E. coli soxS transcriptional regulator gene. Ag(I) is isoelectronic with Cu(I), and genes associated with copper homeostasis were positively regulated indicating Ag(I)-activation of copper signalling. Differential gene expression was observed for the silver nitrate and AgNP silver delivery. Nanoparticle delivery of Ag(I) induced the differential regulation of 379 genes; 309 genes were uniquely regulated by silver nanoparticles and 70 genes were uniquely regulated by silver nitrate. The differential silver nanoparticle-silver nitrate response indicates that the toxic effect of labile Ag(I) in the system depends upon the mechanism of delivery to the target cell.

Silver nanoparticle enhanced silver ion stress response in Escherichia coli K12.

This study investigated the dissolution-based toxicity mechanism for silver nanoparticles to Escherichia coli K12. The silver nanoparticles, synthesised in the vapour phase, are effective anti-bacterial agents against the Gram-negative bacterium, E. coli K12. The nanoparticles associate with the bacterial cell wall, appearing to interact with the outer and inner membranes, and then dissolve to release Ag(+) into the cell and affect a transcriptional response. The dissolution of these nanoparticles in a modified LB medium was measured by inductively coupled plasma mass spectrometry (ICP-MS) and has been shown to follow a simple first-order dissolution process proportional to the decreasing surface area of the nanoparticles. However, the resulting solution phase concentration of Ag(+), demonstrated by the ICP-MS data, is not sufficient to cause the observed effects, including inhibition of bacterial growth and the differential expression of Cu(+) stress response genes. These data indicate that dissolution at the cell membrane is the primary mechanism of action of silver nanoparticles, and the Ag(+) concentration released into the bulk solution phase has only limited anti-bacterial efficacy.