City-wide wastewater genomic surveillance through the successive emergence of SARS-CoV-2 Alpha and Delta variants.

Genomic surveillance of SARS-CoV-2 has provided a critical evidence base for public health decisions throughout the pandemic. Sequencing data from clinical cases has helped to understand disease transmission and the spread of novel variants. Genomic wastewater surveillance can offer important, complementary information by providing frequency estimates of all variants circulating in a population without sampling biases. Here we show that genomic SARS-CoV-2 wastewater surveillance can detect fine-scale differences within urban centres, specifically within the city of Liverpool, UK, during the emergence of Alpha and Delta variants between November 2020 and June 2021. Furthermore, wastewater and clinical sequencing match well in the estimated timing of new variant rises and the first detection of a new variant in a given area may occur in either clinical or wastewater samples. The study's main limitation was sample quality when infection prevalence was low in spring 2021, resulting in a lower resolution of the rise of the Delta variant compared to the rise of the Alpha variant in the previous winter. The correspondence between wastewater and clinical variant frequencies demonstrates the reliability of wastewater surveillance. However, discrepancies in the first detection of the Alpha variant between the two approaches highlight that wastewater monitoring can also capture missing information, possibly resulting from asymptomatic cases or communities less engaged with testing programmes, as found by a simultaneous surge testing effort across the city.

A seafood risk tool for assessing and mitigating chemical and pathogen hazards in the aquaculture supply chain.

Intricate links between aquatic animals and their environment expose them to chemical and pathogenic hazards, which can disrupt seafood supply. Here we outline a risk schema for assessing potential impacts of chemical and microbial hazards on discrete subsectors of aquaculture-and control measures that may protect supply. As national governments develop strategies to achieve volumetric expansion in seafood production from aquaculture to meet increasing demand, we propose an urgent need for simultaneous focus on controlling those hazards that limit its production, harvesting, processing, trade and safe consumption. Policies aligning national and international water quality control measures for minimizing interaction with, and impact of, hazards on seafood supply will be critical as consumers increasingly rely on the aquaculture sector to supply safe, nutritious and healthy diets.

Sustainable aquaculture through the One Health lens.

Aquaculture is predicted to supply the majority of aquatic dietary protein by 2050. For aquaculture to deliver significantly enhanced volumes of food in a sustainable manner, appropriate account needs to be taken of its impacts on environmental integrity, farmed organism health and welfare, and human health. Here, we explore increased aquaculture production through the One Health lens and define a set of success metrics - underpinned by evidence, policy and legislation - that must be embedded into aquaculture sustainability. We provide a framework for defining, monitoring and averting potential negative impacts of enhanced production - and consider interactions with land-based food systems. These metrics will inform national and international science and policy strategies to support improved aquatic food system design.

Sex-specific transcription and DNA methylation profiles of reproductive and epigenetic associated genes in the gonads and livers of breeding zebrafish.

Reproduction is an essential process for life and is regulated by complex hormone networks and environmental factors. To date, little is known about the contribution of epigenetic mechanisms to the regulation of reproduction, particularly in lower vertebrates. We used the zebrafish (Danio rerio) model to investigate the sex-specific transcription and DNA methylation profiles for genes involved in the regulation of reproduction and in epigenetic signalling in the livers and gonads. We found evidence for associations between DNA promotor methylation and transcription for esr1 (gonads and female livers), amh (gonads) and dnmt1 (livers). In the liver, esr1 was shown to be significantly over-expressed in females compared to males, and its promoter was significantly hypo-methylated in females compared to males. In the gonads, genes involved in epigenetic processes including dnmt1, dnmt3 and hdac1 were over-expressed in the ovary compared to the testis. In addition, dnmt1 and dnmt3 transcription in the testis was found to be strongly correlated with global DNA methylation. These data provide evidence of the sex-specific epigenetic regulation and transcription of genes involved in reproduction and epigenetic signalling in a commonly used vertebrate model.

Advances in the application of high-throughput sequencing in invertebrate virology.

Over the last decade, advances in high-throughput sequencing technologies have revolutionised biological research, making it possible for DNA/RNA sequencing of any organism of interest to be undertaken. Sequencing approaches are now routinely used in the detection and characterisation of (novel) viruses, investigation of host-pathogen interactions, and effective development of disease treatment strategies. For the sequencing and identification of viruses of interest, metagenomics approaches using infected host tissue are frequently used, as it is not always possible to culture and isolate these pathogens. High-throughput sequencing can also be used to investigate host-pathogen interactions by investigating (temporal) transcriptomic responses of both the host and virus, potentially leading to the discovery of novel opportunities for treatment and drug targets. In addition, viruses in environmental samples (e.g. water or soil samples) can be identified using eDNA/metagenomics approaches. The promise that recent developments in sequencing brings to the field of invertebrate virology are not devoid of technical challenges, including the need for better laboratory and bioinformatics strategies to sequence and assemble virus genomes within complex tissue or environmental samples, and the difficulties associated with the annotation of the large number of novel viruses being discovered.

Bisphenol A causes reproductive toxicity, decreases dnmt1 transcription, and reduces global DNA methylation in breeding zebrafish (Danio rerio).

Bisphenol A (BPA) is a commercially important high production chemical widely used in epoxy resins and polycarbonate plastics, and is ubiquitous in the environment. Previous studies demonstrated that BPA activates estrogenic signaling pathways associated with adverse effects on reproduction in vertebrates and that exposure can induce epigenetic changes. We aimed to investigate the reproductive effects of BPA in a fish model and to document its mechanisms of toxicity. We exposed breeding groups of zebrafish (Danio rerio) to 0.01, 0.1, and 1 mg/L BPA for 15 d. We observed a significant increase in egg production, together with a reduced rate of fertilization in fish exposed to 1 mg/L BPA, associated with significant alterations in the transcription of genes involved in reproductive function and epigenetic processes in both liver and gonad tissue at concentrations representing hotspots of environmental contamination (0.1 mg/L) and above. Of note, we observed reduced expression of DNA methyltransferase 1 (dnmt1) at environmentally relevant concentrations of BPA, along with a significant reduction in global DNA methylation, in testes and ovaries following exposure to 1 mg/L BPA. Our findings demonstrate that BPA disrupts reproductive processes in zebrafish, likely via estrogenic mechanisms, and that environmentally relevant concentrations of BPA are associated with altered transcription of key enzymes involved in DNA methylation maintenance. These findings provide evidence of the mechanisms of action of BPA in a model vertebrate and advocate for its reduction in the environment.

Global transcriptome profiling reveals molecular mechanisms of metal tolerance in a chronically exposed wild population of brown trout.

Worldwide, a number of viable populations of fish are found in environments heavily contaminated with metals, including brown trout (Salmo trutta) inhabiting the River Hayle in South-West of England. This population is chronically exposed to a water-borne mixture of metals, including copper and zinc, at concentrations lethal to naïve fish. We aimed to investigate the molecular mechanisms employed by the River Hayle brown trout to tolerate high metal concentrations. To achieve this, we combined tissue metal analysis with whole-transcriptome profiling using RNA-seq on an Illumina platform. Metal concentrations in the Hayle trout, compared to fish from a relatively unimpacted river, were significantly increased in the gills, liver and kidney (63-, 34- and 19-fold respectively), but not the gut. This confirms that these fish can tolerate considerable metal accumulation, highlighting the importance of these tissues in metal uptake (gill), storage and detoxification (liver, kidney). We sequenced, assembled and annotated the brown trout transcriptome using a de novo approach. Subsequent gene expression analysis identified 998 differentially expressed transcripts and functional analysis revealed that metal- and ion-homeostasis pathways are likely to be the most important mechanisms contributing to the metal tolerance exhibited by this population.

Review: Do engineered nanoparticles pose a significant threat to the aquatic environment?

Nanotechnology is a rapidly growing industry of global economic importance, exploiting the novel characteristics of materials manufactured at the nanoscale. The properties of engineered nanoparticles (ENPs) that make them useful in a wide range of industrial applications, however, have led to concerns regarding their potential impact on human and environmental health. The aquatic environment is particularly at risk of exposure to ENPs, as it acts as a sink for most environmental contaminants. This paper critically evaluates what is currently known about sources and discharge of ENPs to the aquatic environment and how the physicochemical characteristics of ENPs affect their fate and behaviour and thus availability for uptake into aquatic organisms, and assesses reported toxicological effects. Having reviewed the ecotoxicological information, the conclusion is that whilst there are data indicating some nanoparticles have the potential to induce harm in exposed aquatic organisms, there is insufficient evidence for harm, for known/modelled environmental concentrations for almost all ENPs considered. This conclusion, however, must be balanced by the fact that there are significant gaps in our understanding on the fate and behaviour of ENPs in the aquatic environment. Greater confidence in the assessments on ENP impacts in aquatic systems to enable effective comparisons across studies urgently requires more standardised approaches for ENP hazard identification, and critically, more thorough characterisations on the exposed particles. There is also an urgent need for the advancement of tools and techniques that can accurately quantify and visualise uptake of nanoparticles into biological tissues.

Evidence for the existence of a functional Kiss1/Kiss1 receptor pathway in fish.

In mammals, the Kiss1 receptor (Kiss1r) and its kisspeptin ligands are key factors regulating the onset of puberty. In fish, however, the mechanisms underlying the initiation of puberty are poorly understood and the role of the Kiss1r/kisspeptin pathway in this process has not been established. In this study, a bioinformatics approach was used to identify the genes for Kiss1 and Kiss1r in five teleost genomes and the information used to clone the corresponding transcripts from zebrafish. Zebrafish kiss1r was expressed predominantly in the brain, with a minor level of expression in the eye, and zebrafish kiss1 was expressed in brain, intestine, adipose tissue and testis. Analysis of the chromosome region containing the kiss1 locus showed high synteny across vertebrate genomes. In contrast to their mammalian homologues, teleost Kiss1 protein sequences were poorly conserved with the exception of the region representing kisspeptin-10. Signal peptide sequences and likely cleavage and amidation sites in the teleost Kiss1 sequences were determined and found to be similar to those in mammalian Kiss1. This is the first report of the existence and characterization of the Kiss1 gene outside the mammalian taxa, suggesting that a functional Kiss1/Kiss1 receptor pathway is conserved across vertebrate species.

Effects of 17alpha-ethinylestradiol in a fathead minnow (Pimephales promelas) gonadal recrudescence assay.

To contribute to the development and evaluation of a practical and cost-effective in vivo testing system for endocrine disruption (specifically environmental estrogens), the effects of 17alpha-ethinylestradiol (EE2) were assessed in a gonadal recrudescence assay with the fathead minnow (Pimephales promelas). Mature male and female fathead minnows were kept first at 15 degrees C on a 8 h light/16 h dark regime and then transferred to 25 degrees C and a 16 h light/8 h dark regime to induce gonadal recrudescence. They were then exposed to various nominal concentrations of the synthetic estrogen EE2 (0, 0.1, 1, 3, 10, 100 ng/L). After 3 weeks of chemical exposure, effects on plasma vitellogenin (VTG), secondary sex characteristics, gonad growth (gonadosomatic index; GSI), and condition factor were assessed. Additionally, the effects on liver and gonad tissue morphology were investigated by means of light (LM) and electron microscopy (EM). Reproductive output (egg production) and fertilization rate were measured during a subsequent 3-week period in breeding adults maintained in clean water. Exposure to EE2 resulted in a significant decrease in GSI, condition factor, and number of batches of eggs and their fertilization rate at EE2 exposure concentrations between 10 and 100 ng/L. A reduction in the extent of parenchymatic areas in ovaries and ultrastructural changes in the livers of females could be detected at EE2 concentrations > or =3 ng/L. The lowest observed effective concentration (LOEC) of EE2 for plasma VTG induction in both sexes and for ultrastructural changes in the testes and livers was 1 ng/L. A significant increase in the mean number of eggs spawned per pair occurred at EE2 exposure doses of 0.1 and 1 ng/L. However, at higher EE2 concentrations, a dose-dependent decrease in the mean number of eggs per pair was apparent. Therefore, the LOEC for a biological effect of EE2 in the fathead minnow using the selected endpoints in the recrudescence assay was 1 ng/L for biomarkers such as plasma VTG and number of tubercles, and 0.1 ng/L for an increased number of eggs spawned per pair.

Development and validation of a homologous zebrafish (Danio rerio Hamilton-Buchanan) vitellogenin enzyme-linked immunosorbent assay (ELISA) and its application for studies on estrogenic chemicals.

Vitellogenin (VTG) was isolated by anion exchange chromatography from plasma of female zebrafish (Danio rerio) induced with 17alpha-ethinylestradiol (EE2). The purity of the VTG isolate was confirmed by polyacrylamide gel electrophoresis (SDS-PAGE). Purified VTG was used to raise polyclonal antibodies in rabbits and the specificity of the antisera for VTG confirmed by Western blot analysis of plasma proteins separated by SDS-PAGE. The antibodies cross-reacted with two proteins in the plasma of female zebrafish, with molecular masses of approximately 142 and 171 kDa. No cross-reactivity was observed with any other plasma proteins. A competitive enzyme-linked immunosorbent assay (ELISA) was developed using the polyclonal zebrafish VTG (z-VTG) antibodies and purified z-VTG as ligand and standard, respectively. The z-VTG ELISA was sensitive with a detection limit of between 2.0 and 3.0 ng purified VTG/ml, and a working range between 3 and 500 ng/ml (30-85% binding). The ELISA demonstrated precision, with inter- and intra-assay variations of 7.5+/-2.7 and 4.9+/-1.4%, respectively. Plasma from adult zebrafish and whole body homogenates from juvenile zebrafish diluted parallel with the z-VTG standard in the ELISA, validating the assay for quantifying z-VTG in both of these tissues. Exposure of adult male zebrafish to EE2 via water induced a concentration-dependent induction of VTG with a lowest observed effect concentration (LOEC) < or =1.67 ng EE2/l (for a 21-day exposure). The homologous z-VTG ELISA provides a valuable tool for the study of environmental estrogens in zebrafish.

Sexual disruption in a second species of wild cyprinid fish (the gudgeon, Gobio gobio) in United Kingdom freshwaters.

To establish whether the intersex condition seen in the roach (Rutilus rutilus) in United Kingdom (UK) rivers was species specific or a more general phenomenon in fish, evidence for sexual disruption was sought in a second cyprinid species, the gudgeon (Gobio gobio). Gudgeon were collected from the Rivers Aire and Lea (at locations that receive high-volume discharges of sewage treatment works [STW] effluent and that contain intersex roach) and from two still waters, and their gonads were examined histologically for evidence of intersexuality (the simultaneous presence of oocytes and testicular tissue). Intersex gonads were found at all sites, with the highest incidences occurring at one of the still waters (Lakeside Fisheries: 15%) and at sites on the River Aire (Thwaite Weir, Silsden Bridge, and Knostrop: 14, 13, and 12%, respectively). In the River Lea and Longton Park Lake, the incidence of intersexuality in gudgeon was 6%. In most cases, intersex gonads were characterized by a few primary oocytes/gonad section in an otherwise normal testis. However, at some sites on the River Aire (Thwaite Weir and Knostrop), the intersex condition was more severe. At Thwaite Weir, for example, more than half of the gonad in 40% of the intersex fish was comprised of ovarian tissue. Elevated concentrations of plasma vitellogenin both in male and in intersex fish indicated that fish had been exposed to estrogen(s). Some of the gudgeon were found at sites several kilometers downstream of any point discharge of STW effluent; therefore, the results likely are representative of this species in wild populations found in typical UK river ecosystems. Together with the findings in the roach, these data on the gudgeon confirm that sexual disruption in fish in UK rivers is not species specific.