Can roach, Rutilus rutilus, adapt to the harmful effects of oestrogen exposure from waste water treatment work effluents?

Lead Research Organisation: University of Exeter
Department Name: Biosciences

Abstract

Major worldwide attention has focused on the observations of disruptions of reproduction in both wildlife and human populations that can result from exposure to chemicals that interfere with the body's hormone signalling systems (so-called endocrine disrupting chemicals; EDCs) impacting on reproductive health. We have shown that reproduction in fish (roach, a common member of the carp family of fish) living in many UK Rivers has been damaged by exposure to EDCs contained in the wastewater treatment works (WwTW) effluents and the chemicals responsible for these effects include natural oestrogen hormones and pharmaceutical oestrogens in the contraceptive pill.
Feminised roach have a reduced capability to breed under competitive breeding conditions and we have evidence (as yet, unpublished) for reduced breeding population sizes in wild roach living in rivers with a high effluent content. Populations with low numbers of breeding individuals lose genetic variation over multiple generations with a greater risk of extinction. Nevertheless, we find that in some stretches of these rivers with high oestrogenic exposure roach populations appear to be reproductively self-sustaining. Establishing whether fish (here roach) have adapted to oestrogenic contaminants, how they do this (the mechanisms) and the possible fitness costs of these adaptations are essential in understanding resilience (and thus sustainability) of fish populations living in these polluted environments.
We will examine whether exposure of roach populations to oestrogenic WwTW effluents over multiple generations has resulted in genetic selection and the impacts of selection on the susceptibility in male fish to develop oestrogen-induced effects associated with negative fitness consequences. We will examine for selection by identifying functionally significant changes in their genetic make up. Specifically we will look for differences in genetic poylmorphisms (so called single nucleotide polymorphisms -SNPs) in (1) specific (candidate) genes that we know are important in oestrogen signalling of reproductive (and other life) processes and (2) by using methods that allow us to scan the whole genome for these genetic alterations (using a technique called RAD-tag genotyping). Adopting these methods we expect to find 'footprints' of selection in fish living in rivers with a high oestrogenic effluent content, which should allow identification of novel adaptive processes and suggest important mechanisms of toxicity or survival in effluent rich environments. Uniquely, we are able to do this work because we have in depth knowledge (15 years of study) of the wild populations of roach in the selected UK Rivers that we propose to study.
We will establish whether roach populations exposed to high levels of oestrogenic effluent over many generations have adapted to become less responsive to environmental oestrogens and thus are now less susceptible to their associated adverse effects by comparing responses to a controlled exposure to the contraceptive oestrogen, ethinyloestradiol. We will use roach collected from the same clean and WwTW effluent contaminated sites as for the population genetic analyses described above and fish will be exposed for a period of 1 year and effects quantified on responses that have negative fitness consequences in male fish, including ovotestis.
This work is intended to greatly improve understanding of the ability of fish to adapt to exposure to environmental pollutants (here oestrogen) and how they do so, in turn helping to inform on fish population resilience in UK rivers receiving WwTW effluent discharges. The work will have importance in the regulation of discharges for the better protection of our aquatic resources and biodiversity and is of very wide interest to the government regulatory bodies, environment protection groups, industry and the wider public.

Planned Impact

Endocrine disrupting chemicals (EDCs) potentially have very wide-reaching deleterious environmental impacts and our findings will be of considerable interest (inter)nationally to academia, pharmaceutical and chemical industries, government regulatory bodies, environment protection groups, and the wider public. The major part of our impact activities will be centred on training, engagement with industry partners and (inter)national government regulatory bodies, and informing the wider public.

The approach we propose in this proposal will apply advanced molecular techniques to provide an advanced understanding on the ability of fish to adapt to oestrogenic EDCs, how they do this and the fitness consequences of these adaptations. We expect the work to benefit, both nationally and internationally industry and government regulatory bodies, by supporting evidence-based decision-making on EDCs, a large group of academic researchers working in similar or related fields and by advancing the use of cutting edge molecular and genome methods and understanding adaptive (evolutionary) processes; the water and chemical (including pharmaceutical) industries through better understanding the implications of exposure to EDCs on wildlife health allowing industry to consider remediation strategy options, including additional treatment technologies; fisheries managers, as widespread adaptation would influence management of fish using hatchery stock; NGOs and the public, who will benefit from better evidence of EDC effects on wildlife and that may also affect their own health.

At this time we do not see that this project has any IP with immediate industrial application. If such an opportunity arises however (e.g. in the discovery of molecular biomarkers) we will gain direct support on commercialisation opportunities and be provided with advice on the protection of IP through our University's Research & Knowledge Transfer sections.

The project team has a strong record in public engagement and outreach activities and will make active use of our respective university press offices to maximise publicity opportunities to ensure the results of our work are widely disseminated. The PIs and RCo-I will also participate in a number of public events and scientific discussions on environmental pollution and these will include; the annual National Science and Engineering Week at Exeter, Cafe Scientifique at Exeter, presentations at national and regional levels to the Association of River Trusts, the Environment Agency Fisheries Teams, Natural England and the European Commission. Both PIs participate actively in a series of school-focused events and in higher education programmes at 'Public Open Days' and take on work experience students in their laboratories.

Both RCo-I and RA recruited to this project will be expected to contribute substantially to the communication activities. The RCo-I and RA will attend a NERC training course on science communication. We will ensure that all personnel recruited to this projected are provided with the opportunity to develop their awareness of, and skills in, science communication and knowledge transfer by; a) designating them major roles in the organization and running of the proposed SETAC and Exeter workshops (including as a presenter); b) encouraging the RCo-I and RA to author articles for the popular press and to engage in public discussion related events outlined above; c) working with university publicity team to design project website and briefing leaflets.

Key measures of success will be in the delivery of high quality science with high impact, as evidenced by publication of the work in the best peer reviewed scientific journals, workshop participation and delivery, disseminations at internationally leading events (scientific, government, regulatory), and uptake of this work into these sectors and influence on policy development for EDCs

Publications

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publication icon
Hamilton PB (2017) Adaptive capabilities and fitness consequences associated with pollution exposure in fish. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

 
Description • Key Findings- anything that relates to impact/what impact cases set out

To investigate evidence for adaption in roach we compared responses to oestrogen exposure in the offspring fish derived from rivers which differ in their exposure to oestrogenic effluent. Our completed analysis suggests that the river of origin of the parent fish did not influence whether the fish developed the intersex condition. However, a higher proportion of roach in the exposed group with parents from the Grantham Canal developed ovarian-like cavities when compared to roach from the two Thames rivers (Mole and Gade), which had similar phenotypes. Interestingly the Grantham population substantially differed from the Thames populations in allele frequencies of several oestrogen-responsive genes: AR, FSH receptor, VTG3 and Cyp1A1. EE2 exposure also increased the proportion of females at sexual maturity after two years. This is a novel finding for roach, and a first for species that breed once a year, and could lead to population-level impacts. This was unrelated to the river origin of the parent fish.

Analysis of single nucleotide polymorphisms (SNP) frequencies in wild populations identified several genes with signatures of selection in one or more of the pairwise comparisons between populations from effluent-polluted and clean rivers. Among genes under selection were several oestrogen-responsive candidate genes: the androgen receptor (AR), FSH receptor, cytochrome P4501A (CYP1A), hydroxysteroid (11-beta) dehydrogenase 2 (11ß-HSD), aquaporin 12 (AQP12), oestrogen receptor 1 (ESR1), and vitellogenin 3 (VTG3). However, none of these genes was consistently identified as being under selection in all comparisons between high and low/no effluent populations, and some were also under selection in comparisons of populations at clean rivers. This suggests that factors other than exposure to oestrogenic effluents may be, at least in part, responsible for the pattern.

Strikingly our analysis found the androgen receptor (AR) to be under strong selection in a large number of pairwise comparisons. Notably, this gene was identified as an 'outlier' in all comparisons involving the Lee population, suggesting strong selection at this site. Our collaborative work with CEH has identified that this population has historically been exposed to a range of pesticides and detectable concentrations of the DDT metabolite, p,p '-DDE, an antiandrogen, are still found in the body tissues of roach at this site. SNPs in the AR gene with signatures of selection did not alter the amino acid sequence, so the true SNP under selection may lie in a regulatory region. The xenobiotic metabolizing enzyme, cytochrome P4501A (CYP1A) also showed a strong signature of selection in a number of comparisons, particularly those involving populations from the Lee and the Cuffley with populations from elsewhere in the Thames catchment, and between populations in the Thames and Humber Catchments. This gene plays a key role in the metabolism of a wide range of compounds including polyaromatic hydrocarbons (PAHs) and is under selection in populations of other fish species exposed to PAHs. Another identified as under selection in some comparisons was ALPK2 which is crucial for luminal apoptosis and DNA repair-related gene expression. Overall our results to date indicate that chemical exposure, but not necessarily oestrogens derived from WwTW effluents is an important selective pressure on wild roach populations.

Our analysis identified randomly-selected 'control' genes as having strong signatures of selection in comparisons between populations in the Thames catchment and those in the Humber in northern England. Three of these are involved production of red blood cells: erythroid differentiation related gene (EDRF1) and ADNP homeobox (ADNP2a) that are involved in the expression of globin genes and PIG-T that is involved in essential for attachment of GPI to proteins on the surface of red blood cells. Water oxygen concentration, which is influenced by both temperature and pollution, may therefore have acted as an important selective pressure on roach.

Genome wide SNP genotyping using RAD-seq of roach from the historically polluted River Lee and the low effluent Cuffley Brook provided 11,736 polymorphic RAD loci revealed a large number of genes with a signature of selection. Among the genes providing a strong signature of selection were several that are related to regulation of cell growth and DNA repair. Functional annotation in DAVID identified two overrepresented categories amongst these genes; metal ion binding and exposure to organic substances, which are both consistent with the hypothesis that exposure of roach to chemical pollution has acted as a selective pressure. These analyses are ongoing.

Finally, analyses of the DNA microsatellite dataset demonstrated, for the first time, that populations of roach in rivers in the Humber Catchment are genetically distinct from those from the Thames and other southern rivers. These data also clearly demonstrated that weirs in some of the rivers are effective at restricting movements of roach confining populations to river stretches over multiple generations, consistent with the findings from our previous project. Our microsatellite genotyping also identified a large proportion of the 'roach' from the Hull were, in fact, Ide, an invasive species.
Update for 2017:We have shown the following:

Strong selection at the androgen receptor and Cyp1A suggests local adaptation to chemical pollution in roach (Rulitus rutilus) living in English rivers.

Analysis of SNPs in 465 roach revealed that 3 of the 84 SNPs within 35 candidate genes for estrogen adaptation correlated with estrogen exposure. These were brca2, vasa and ltbp3. However, 4 SNPs in 120 randomly selected genes also correlated with estrogen exposure. Our results therefore did not provide strong evidence for widespread selection at the candidate genes. SNPs in most obvious candidates including the estrogen receptors, the androgen receptor, aromatases and vitellogenin genes showed no correlation with estrogen exposure. Nevertheless, several SNPs showed strong patterns of selection between populations from the Thames and Humber catchments and in individual rivers within each catchment. For example, there was evidence for selection at the androgen receptor in the River Lee, which has had a long history of pollution with estrogenic WwTW effluents and endocrine disrupting pesticides such as DDT and its breakdown products and endosulfan. There was also strong signature selection at Cyp1A within populations from two low-effluent 'rivers', potentially related to adaptation to chemical pollution, but no clear link with estrogen pollution. Our results suggest that selection pressures vary locally and signatures of selection are likely to be population-specific. This adds strong evidence that these genes have a significant role in large-scale adaptation to the abiotic environment.

Ethinyoestradiol in wastewater effluents induce female population skews in roach (Rulitus rutilus) irrespective of their oestrogen exposure history in UK rivers.

The impact of multigenerational exposure of populations of fish to estrogens on the susceptibility of individuals to estrogen is not known. We compared the sexual development of the offspring of roach from a population from a river stretch with a high WwTW content to two with low WwTW inputs. A 2-year controlled exposure to 1.3-1.6 ng ethinyloestradiol (EE2) /L -induced effects in both males and females. Exposure to EE2 resulted in a significantly female-biased genetic sex ratio, which we show include complete sex reversal, and a range of feminising phenotypes in males including vitellogenin induction, feminised reproductive ducts and intersex. EE2 exposure resulted in a significantly higher proportion of females with vitellogenic oocytes compared to the control group. However, no differences in response between the offspring of roach from the three populations were identified except for a significant interaction between exposure and river on vitellogenin concentration in females, but this appears to be related to differences in the vitellogenin levels in control females. This may be attributed to differences between unexposed females, given that exposed females all had high VTG levels. If such effects on females observed in this study occur in wild fish, this has the potential to impact on populations of fish that spawn annually.
Exploitation Route Establishing whether fish (here roach) have adapted to oestrogenic contaminants, how they do this (the mechanisms) and the possible fitness costs of these adaptations are essential in understanding resilience (and thus sustainability) of fish populations living in these polluted environments. This work will inform relevant government agencies, environmental protection groups and industry on adaptive capabilities of fish living in rivers contaminated with wastewater effluents and serve as an important cornerstone for assessing population level impacts of these effluent discharges and regulation needs.
Sectors Chemicals,Communities and Social Services/Policy,Education,Environment,Pharmaceuticals and Medical Biotechnology

 
Description This project set out to investigate whether roach (Rutilus rutilus) exposed to oestrogenic waste water treatment work effluents for multiple generations have adapted to better tolerate the harmful effects of exposure to oestrogens. We investigated evidence of adaptation by conducting an analysis of genetic variants (single nucleotide polymorphisms, SNPs) in wild roach populations from both polluted and clean river stretches, developing significant knowledge on the importance of chemical exposure on selection in roach populations. A literature review identified 65 genes in which variants could potentially alter sensitivity to oestrogen exposure. SNPs were identified in these genes by remapping raw sequence reads to the assembled transcriptome or by Sanger sequencing. Genotyping of roach from nine locations in collaboration with NERC Biomolecular Analysis Facility at Sheffield (NBAF-S) provided genotypes for 217 SNPs, including in 87 SNPs in 36 candidate genes (e.g. oestrogen receptors, VTGs), 9 SNPs in three candidates for being under selection for reasons other than oestrogen exposure and 121 SNPs each in a different random protein coding gene. Genetic outlier analysis identified several of these genes as under selection in roach population including Cyp1A1 and the androgen receptor (detailed in key findings). We also optimised methods for RAD-seq genotyping to conduct genome-wide sequencing of roach in order to investigate adaptive responses to effluent exposure. We have constructed RAD-seq libraries from six populations and analysed two in detail, identifying a suite of genes with strong signatures of having undergone selection in polluted rivers (detailed in key findings). We also conducted population genetic analysis using DNA microsatellite data to provide additional information on population genetic structure and the history of roach in each river. This provided key information on the effectiveness of obstructions such as weirs at restricting movements of roach within rivers and has provided novel knowledge on population genetic structure of roach in UK rivers. Updated for 2017:We have shown the following: Strong selection at the androgen receptor and Cyp1A suggests local adaptation to chemical pollution in roach (Rulitus rutilus) living in English rivers. Analysis of SNPs in 465 roach revealed that 3 of the 84 SNPs within 35 candidate genes for estrogen adaptation correlated with estrogen exposure. These were brca2, vasa and ltbp3. However, 4 SNPs in 120 randomly selected genes also correlated with estrogen exposure. Our results therefore did not provide strong evidence for widespread selection at the candidate genes. SNPs in most obvious candidates including the estrogen receptors, the androgen receptor, aromatases and vitellogenin genes showed no correlation with estrogen exposure. Nevertheless, several SNPs showed strong patterns of selection between populations from the Thames and Humber catchments and in individual rivers within each catchment. For example, there was evidence for selection at the androgen receptor in the River Lee, which has had a long history of pollution with estrogenic WwTW effluents and endocrine disrupting pesticides such as DDT and its breakdown products and endosulfan. There was also strong signature selection at Cyp1A within populations from two low-effluent 'rivers', potentially related to adaptation to chemical pollution, but no clear link with estrogen pollution. Our results suggest that selection pressures vary locally and signatures of selection are likely to be population-specific. This adds strong evidence that these genes have a significant role in large-scale adaptation to the abiotic environment. Ethinyoestradiol in wastewater effluents induce female population skews in roach (Rulitus rutilus) irrespective of their oestrogen exposure history in UK rivers. The impact of multigenerational exposure of populations of fish to estrogens on the susceptibility of individuals to estrogen is not known. We compared the sexual development of the offspring of roach from a population from a river stretch with a high WwTW content to two with low WwTW inputs. A 2-year controlled exposure to 1.3-1.6 ng ethinyloestradiol (EE2) /L -induced effects in both males and females. Exposure to EE2 resulted in a significantly female-biased genetic sex ratio, which we show include complete sex reversal, and a range of feminising phenotypes in males including vitellogenin induction, feminised reproductive ducts and intersex. EE2 exposure resulted in a significantly higher proportion of females with vitellogenic oocytes compared to the control group. However, no differences in response between the offspring of roach from the three populations were identified except for a significant interaction between exposure and river on vitellogenin concentration in females, but this appears to be related to differences in the vitellogenin levels in control females. This may be attributed to differences between unexposed females, given that exposed females all had high VTG levels. If such effects on females observed in this study occur in wild fish, this has the potential to impact on populations of fish that spawn annually.
First Year Of Impact 2017
Sector Chemicals,Communities and Social Services/Policy,Education,Environment,Government, Democracy and Justice
Impact Types Societal,Economic

 
Description Advising UK ministers
Geographic Reach Multiple continents/international 
Policy Influence Type Gave evidence to a government review
Impact We have made considerable efforts to inform people including the UK (e.g. Environment Agency and DEFRA), US and Japanese governments, European Union, environment protection groups, Industry and the wider public (e.g. via the Royal Society, local conservations groups). We are also active in disseminating our research work to Schools, and higher education programmes, principally through University Open Days and International University seminars. Information from this project has been used within a restatement paper with the primary purpose of which is to advising ministers on endocrine disruption in wildlife: Charles J. Godfray, Andrea E. A. Stephens, Paul D. Jepson, Susan Jobling, Andrew C. Johnson, Peter Matthiessen, John P. Sumpter, Charles R. Tyler & Angela R. McLean (2019). A restatement of the natural science evidence base on the effects of endocrine disrupting chemicals on wildlife. Phil Trans Proc Royal Soc. https://doi.org/10.1098/rspb.2018.2416
 
Description Advising ministers across international spectrum of governmental bodies
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a national consultation
Impact Information from this project has been used within a restatement paper with the primary purpose of which is to advising ministers on endocrine disruption in wildlife: Charles J. Godfray, Andrea E. A. Stephens, Paul D. Jepson, Susan Jobling, Andrew C. Johnson, Peter Matthiessen, John P. Sumpter, Charles R. Tyler & Angela R. McLean. A restatement of the natural science evidence base on the effects of endocrine disrupting chemicals on wildlife. Phil Trans Proc Royal Soc. Submitted We have made considerable efforts to inform people including the UK (e.g. Environment Agency and DEFRA), US and Japanese governments, European Union, environment protection groups, Industry and the wider public (e.g. via the Royal Society, local conservations groups). We are also active in disseminating our research work to Schools, and higher education programmes, principally through University Open Days and International University seminars. Data from the project has been presented to DEFRA and the UK Environment Agency, for example at the 19th Annual UK/Japan workshop on endocrine disruption (Yokohama), so the findings are being considered in their deliberations of these governments on the impacts of endocrine disruption on fish populations.
 
Description BBC radio 4 programme
Geographic Reach National 
Policy Influence Type Participation in a national consultation
Impact BBC radio 4 programme 'Costing the Earth' on the subject of fertility. Nationally/internationally accessible
URL https://bbc.in/2O7146g
 
Description DEFRA - impacts of EE2 and endocrine disruption
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a advisory committee
 
Description Data from this project has been presented to DEFRA and the UK Environment Agency in government reports
Geographic Reach National 
Policy Influence Type Gave evidence to a government review
Impact Data from the project has been presented to DEFRA and the UK Environment Agency in the following government reports Charles R. Tyler and Taisen Iguchi (2018) Defra , UK, Min of Evn, Japan Workshop Report. The 20th UK-Japan Annual Scientific Workshop on Research into Environmental Endocrine Disrupting Chemicals 37pp Baynes, A, Lange, A., Jobling, S., Tyler,CR (2019). UK Environment Agency Evidence Report - Revisiting oestrogenic effects in wild fish: assessing and comparing contemporary and historical impacts 68pp
 
Description Environment Agency - DNA microsatellite genotypes
Geographic Reach National 
Policy Influence Type Participation in a national consultation
Impact We have worked with the EA to investigate the ability of the microsatellite dataset generated in the project to determine the effectiveness of the EA's restocking practices. We demonstrated that it is a useful tool to distinguish between roach introduced from the Environment Agency's farm fish from native fish in most rivers using their DNA microsatellite genotypes.
 
Description Press release
Geographic Reach Europe 
Policy Influence Type Influenced training of practitioners or researchers
Impact Wildlife are exposed to a cocktail of hormone-disrupting toxins and our understanding of the risks is limited Oxford Martin restatement of how endocrine disrupting chemicals (EDCs) affect wildlife highlights need for more toxicity data to predict risks before damage to wildlife populations occurs Economic and demographic trends, climate change and increasing human pharmaceutical use all present risks for an acceleration of complex impacts from EDCs
URL https://www.oxfordmartin.ox.ac.uk/policy/restatements/
 
Description Class related developmental toxicity of agrochemicals
Amount £175,500 (GBP)
Organisation Syngenta International AG 
Sector Public
Country Global
Start  
 
Description Contract Research
Amount £80,000 (GBP)
Organisation AstraZeneca 
Sector Private
Country United Kingdom
Start 01/2018 
End 12/2018
 
Description Effects of water chemistry on the exposure risk for pharmaceutical drugs, including antibiotics.
Amount £80,000 (GBP)
Organisation AstraZeneca 
Sector Private
Country United Kingdom
Start 10/2018 
End 09/2022
 
Description Population modelling and risk assessment for EDCs
Amount £105,000 (GBP)
Organisation BASF 
Sector Private
Country Germany
Start  
 
Description SWEEP South West Partnership for Environment and Economic Prosperity
Amount £3,960,000 (GBP)
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 02/2017 
End 01/2021
 
Title A SNP panel for roach 
Description A SNP panel for roach: We used the transcriptome and genome reads to construct a panel of 350 single nucleotide polymorphisms (SNPs) which were then used to genotype 420 wild roach from the United Kingdom. Of these, the majority (217) gave high quality genotypes, thus validating them for other studies. This includes 87 SNPs within 36 key genes involved in responses to endocrine disruption such as hormone receptors (oestrogen/androgen receptors), vitellogenins and aromatases. It also includes 121 SNPs in different protein coding genes, randomly selected from the transcriptome. In addition, a further 6,000 SNPs have been identified in 593 genes but were not genotyped in the project. Of these, ~4000 of these will provide high quality SNPs providing a valuable resource for rapid genotyping of large numbers of roach into other aspects of adaptation, population genetic structure and for practical management of the species (e.g. restocking). Some of these SNPs may be of use in related species such as rudd, ide and bream, but this need to be tested. 
Type Of Material Biological samples 
Provided To Others? No  
Impact as above 
 
Title DNA microsatellite dataset 
Description In order to understand the history of the roach populations, we genotyped 484 wild roach from polluted and clean rivers during this project with 19 DNA microsatellite loci. These data have been integrated with the dataset from the previous NERC-funded research making a combined dataset with genotypes for 2503 fish. The inclusion of roach from rivers in the Humber Catchment (Foss, Aire, Derwent, Hull, Grantham) provided the first information on population-genetic structure in this region and is an important baseline dataset for further studies in population genetic structure and for practical management of the species. 
Type Of Material Biological samples 
Provided To Others? No  
Impact as above 
 
Title Genomic sequences - roach - Bioproject PRJNA295813 
Description Genomic sequences: We sequenced the genome roach at x30 coverage. This is being prepared for submission to GenBank under Bioproject PRJNA295813 
Type Of Material Biological samples 
Provided To Others? No  
Impact as above 
 
Title Improved methods for selecting SNPs for genotyping 
Description Improved methods for selecting SNPs for genotyping: We have written and applied the bioinformatics pipelines to conduct local assemblies around SNPs of interest from raw Illumina genome reads, in order to improve selection of SNPs for genotyping. These contigs have then been used to filter out SNPs close to introns and SNPs in repetitive genomic regions of the genome that are likely to fail in the genotyping assays. 
Type Of Material Biological samples 
Provided To Others? No  
Impact as above 
 
Title RAD-seq (roach) 
Description RAD-seq: We established the methodology for RAD-tag sequencing of roach. We have knowledge of the levels of coverage using the restriction enzyme Sbf1 in the protocol and have optimized the parameters in the program STACKS to generate the genotypes from the raw reads for roach data. The data that we have produced will provide an important baseline of genotypes for further studies on adaptation and population genetic structure using RAD-seq. 
Type Of Material Biological samples 
Provided To Others? No  
Impact AS ABOVE 
 
Title Series of genetic and genomic resources for studies on the roach 
Description A sequenced the transcriptome -annotated and deposited in GenBank under Bioproject PRJNA295813 Sequenced genome roach at x30 coverage deposited in GenBank under Bioproject PRJNA295813 Extensive panel of SNPs (6000) for use in genotyping, investigating adaptation, population genetic structure and for use in the practical management of the species (e.g. restocking). Some of these SNPs may be of use in related species such as rudd, ide and bream. A sex probe for roach (in collaboration with NBAF - Edinburgh) that has been tested on a wide range of populations and has been applied to investigate impacts of long term exposure on sexual development in the species. 
Type Of Material Biological samples 
Year Produced 2017 
Provided To Others? No  
Impact A sex probe for roach (in collaboration with NBAF - Edinburgh) that has been tested on a wide range of populations and has been applied to investigate impacts of long term exposure on sexual development in the species 
 
Title Sex probe for roach 
Description We have also used RAD-seq to develop a sex probe for roach in collaboration with NBAF - Edinburgh. This has been tested on a wide range of populations and has been applied to investigate impacts of long term exposure on sexual development in the species. 
Type Of Material Biological samples 
Provided To Others? No  
Impact as above 
 
Title Transcriptome - Bioproject PRJNA295813 
Description Transcriptome: We sequenced the transcriptome from roach, using RNA extracted from whole fry and adult livers. This has been annotated and deposited in GenBank under Bioproject PRJNA295813 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact as above 
 
Title Illumina sequences and annoted transcriptome have been deposited in GenBank - Bioproject PRJNA295813 
Description The raw Illumina sequences reads from transcriptome sequencing, and the annotated transcriptome have been deposited in GenBank, both under Bioproject PRJNA295813. In addition we have sequence data for the raw genome files and RAD-seq datasets which are being prepared for submission after further analysis under Bioproject PRJNA295813. 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact as above 
 
Title Sequenced the roach genome 
Description We have sequenced the roach genome and together with the RAD-seq datasets They have been deposited in GenBank, both under Bioproject PRJNA295813. 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? Yes  
Impact Some of these SNPs may be of use in related species such as Rudd, Ide and bream.Extensive panel of SNPs (6000) for use in genotyping, investigating adaptation, population genetic structure and for use in the practical management of the species 
 
Description Astra Zeneca BB/L01548X/1 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution Through the FLIP we have strengthened considerably our research partnership with AstraZeneca UK and built a new partnership with Syngenta AG, including via internships (Ross Brown and Jon Green) and regular face-to-face meetings. The partnerships have played a role in securing further major research support to Exeter from AstraZeneca for work on pharmaceuticals, three CASE studentship(s) for advancing the use of transgenic fish/imaging and modeling in chemical hazard identification (Syngenta/AstraZeneca) with further grants (Syngenta) and CASE studentships (AstraZeneca) submitted/being developed for submission to BBSRC.
Collaborator Contribution as above
Impact As above
Start Year 2014
 
Description Brunel/EA/CEH/NBAF - NE/K004263/1 
Organisation Brunel University London
Department Institute of Environment, Health and Societies
Country United Kingdom 
Sector Academic/University 
PI Contribution This project was conducted in collaboration with Professor Jobling's research group at Brunel University. They have played a large part in obtaining information on the river stretches from which roach were sampled including exposure levels, obstructions to movement and history of roach. They co-managed the exposure, undertaking histological analysis, measuring blood vitellogenin concentrations and exposure EE2 concentrations. They also conducted transcriptome assembly and bioinformatics analysis for SNP identification. We have collaborated extensively with the Environment Agency (EA) during the project. They provided us with access to databases and background information on the rivers (history, obstructions to fish movements, restocking) and the roach populations within them. EA fisheries teams also conducted electrofishing of the selected rivers to obtain tissue samples for DNA analysis. The project also has had input from Centre for Ecology & Hydrology (CEH), who provided essential data on the oestrogenic effluent content in the selected UK rivers using catchment modelling. They also provided contaminant levels in roach for several of the locations chosen for the project, providing useful background data. We also collaborated with scientists at NBAF Sheffield, who provided advice on the selection of SNPs for genotyping and also organized genotyping. We have collaborated with scientists at NBAF Edinburgh for development of the sex probe for roach.
Collaborator Contribution as above
Impact n/a
Start Year 2016
 
Description Brunel/EA/CEH/NBAF - NE/K004263/1 
Organisation Environment Agency
Country United Kingdom 
Sector Public 
PI Contribution This project was conducted in collaboration with Professor Jobling's research group at Brunel University. They have played a large part in obtaining information on the river stretches from which roach were sampled including exposure levels, obstructions to movement and history of roach. They co-managed the exposure, undertaking histological analysis, measuring blood vitellogenin concentrations and exposure EE2 concentrations. They also conducted transcriptome assembly and bioinformatics analysis for SNP identification. We have collaborated extensively with the Environment Agency (EA) during the project. They provided us with access to databases and background information on the rivers (history, obstructions to fish movements, restocking) and the roach populations within them. EA fisheries teams also conducted electrofishing of the selected rivers to obtain tissue samples for DNA analysis. The project also has had input from Centre for Ecology & Hydrology (CEH), who provided essential data on the oestrogenic effluent content in the selected UK rivers using catchment modelling. They also provided contaminant levels in roach for several of the locations chosen for the project, providing useful background data. We also collaborated with scientists at NBAF Sheffield, who provided advice on the selection of SNPs for genotyping and also organized genotyping. We have collaborated with scientists at NBAF Edinburgh for development of the sex probe for roach.
Collaborator Contribution as above
Impact n/a
Start Year 2016
 
Description Brunel/EA/CEH/NBAF - NE/K004263/1 
Organisation Natural Environment Research Council
Department Centre for Ecology & Hydrology (CEH)
Country United Kingdom 
Sector Academic/University 
PI Contribution This project was conducted in collaboration with Professor Jobling's research group at Brunel University. They have played a large part in obtaining information on the river stretches from which roach were sampled including exposure levels, obstructions to movement and history of roach. They co-managed the exposure, undertaking histological analysis, measuring blood vitellogenin concentrations and exposure EE2 concentrations. They also conducted transcriptome assembly and bioinformatics analysis for SNP identification. We have collaborated extensively with the Environment Agency (EA) during the project. They provided us with access to databases and background information on the rivers (history, obstructions to fish movements, restocking) and the roach populations within them. EA fisheries teams also conducted electrofishing of the selected rivers to obtain tissue samples for DNA analysis. The project also has had input from Centre for Ecology & Hydrology (CEH), who provided essential data on the oestrogenic effluent content in the selected UK rivers using catchment modelling. They also provided contaminant levels in roach for several of the locations chosen for the project, providing useful background data. We also collaborated with scientists at NBAF Sheffield, who provided advice on the selection of SNPs for genotyping and also organized genotyping. We have collaborated with scientists at NBAF Edinburgh for development of the sex probe for roach.
Collaborator Contribution as above
Impact n/a
Start Year 2016
 
Description Brunel/EA/CEH/NBAF - NE/K004263/1 
Organisation Natural Environment Research Council
Department NERC Biomolecular Analysis Facility (NBAF)
Country United Kingdom 
Sector Public 
PI Contribution This project was conducted in collaboration with Professor Jobling's research group at Brunel University. They have played a large part in obtaining information on the river stretches from which roach were sampled including exposure levels, obstructions to movement and history of roach. They co-managed the exposure, undertaking histological analysis, measuring blood vitellogenin concentrations and exposure EE2 concentrations. They also conducted transcriptome assembly and bioinformatics analysis for SNP identification. We have collaborated extensively with the Environment Agency (EA) during the project. They provided us with access to databases and background information on the rivers (history, obstructions to fish movements, restocking) and the roach populations within them. EA fisheries teams also conducted electrofishing of the selected rivers to obtain tissue samples for DNA analysis. The project also has had input from Centre for Ecology & Hydrology (CEH), who provided essential data on the oestrogenic effluent content in the selected UK rivers using catchment modelling. They also provided contaminant levels in roach for several of the locations chosen for the project, providing useful background data. We also collaborated with scientists at NBAF Sheffield, who provided advice on the selection of SNPs for genotyping and also organized genotyping. We have collaborated with scientists at NBAF Edinburgh for development of the sex probe for roach.
Collaborator Contribution as above
Impact n/a
Start Year 2016
 
Description Collaborations with University college London and obtaining UAS-GCamp6f,m and s lines. 
Organisation University College London
Department Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Sharing of knowledge on transgenic zebrafish systems
Collaborator Contribution provision of UAS-GCamp6f, m and s zebrafish lines.
Impact Joint publication in preparation
Start Year 2014
 
Description Environment Agency and Prof. Jobling 
Organisation Brunel University London
Country United Kingdom 
Sector Academic/University 
PI Contribution Through collaborations with scientists at NBAF Edinburgh we have developed a sex probe for roach which has been important for the new project funding secured from the UK Environment Agency. A research paper on this work will be submitted for publication within the next few months: Anke Lange, Karim Gharbi, Timothée Cézard, Shinichi Miyagawa, Ronny van Aerle, Taisen Iguchi, David J. Studholme, Charles R. Tyler (2018). A newly identified genetic sex marker and its application to population studies on endocrine disruption in roach (Rutilus rutilus). Target journal Molecular Ecology Assessed the status of intersex in roach in UK rivers for an assessment on how the incidence has changed since the original field studies conducted two decades ago
Collaborator Contribution Professor Jobling's research group at Brunel University have completed transcriptome assembly and bioinformatic analysis for SNP identification and a major joint authored research paper is in the final stages of writing for the publication of this work.
Impact Anke Lange, Karim Gharbi, Timothée Cézard, Shinichi Miyagawa, Ronny van Aerle, Taisen Iguchi, David J. Studholme, Charles R. Tyler (2018). A newly identified genetic sex marker and its application to population studies on endocrine disruption in roach (Rutilus rutilus). Target journal Molecular Ecology
Start Year 2018
 
Description Environment Agency and Prof. Jobling 
Organisation Environment Agency
Country United Kingdom 
Sector Public 
PI Contribution Through collaborations with scientists at NBAF Edinburgh we have developed a sex probe for roach which has been important for the new project funding secured from the UK Environment Agency. A research paper on this work will be submitted for publication within the next few months: Anke Lange, Karim Gharbi, Timothée Cézard, Shinichi Miyagawa, Ronny van Aerle, Taisen Iguchi, David J. Studholme, Charles R. Tyler (2018). A newly identified genetic sex marker and its application to population studies on endocrine disruption in roach (Rutilus rutilus). Target journal Molecular Ecology Assessed the status of intersex in roach in UK rivers for an assessment on how the incidence has changed since the original field studies conducted two decades ago
Collaborator Contribution Professor Jobling's research group at Brunel University have completed transcriptome assembly and bioinformatic analysis for SNP identification and a major joint authored research paper is in the final stages of writing for the publication of this work.
Impact Anke Lange, Karim Gharbi, Timothée Cézard, Shinichi Miyagawa, Ronny van Aerle, Taisen Iguchi, David J. Studholme, Charles R. Tyler (2018). A newly identified genetic sex marker and its application to population studies on endocrine disruption in roach (Rutilus rutilus). Target journal Molecular Ecology
Start Year 2018
 
Description Environment Agency and Prof. Jobling 
Organisation Natural Environment Research Council
Department NERC Biomolecular Analysis Facility (NBAF)
Country United Kingdom 
Sector Public 
PI Contribution Through collaborations with scientists at NBAF Edinburgh we have developed a sex probe for roach which has been important for the new project funding secured from the UK Environment Agency. A research paper on this work will be submitted for publication within the next few months: Anke Lange, Karim Gharbi, Timothée Cézard, Shinichi Miyagawa, Ronny van Aerle, Taisen Iguchi, David J. Studholme, Charles R. Tyler (2018). A newly identified genetic sex marker and its application to population studies on endocrine disruption in roach (Rutilus rutilus). Target journal Molecular Ecology Assessed the status of intersex in roach in UK rivers for an assessment on how the incidence has changed since the original field studies conducted two decades ago
Collaborator Contribution Professor Jobling's research group at Brunel University have completed transcriptome assembly and bioinformatic analysis for SNP identification and a major joint authored research paper is in the final stages of writing for the publication of this work.
Impact Anke Lange, Karim Gharbi, Timothée Cézard, Shinichi Miyagawa, Ronny van Aerle, Taisen Iguchi, David J. Studholme, Charles R. Tyler (2018). A newly identified genetic sex marker and its application to population studies on endocrine disruption in roach (Rutilus rutilus). Target journal Molecular Ecology
Start Year 2018
 
Description Syngenta BB/L01548X/1 
Organisation Syngenta International AG
Country Global 
Sector Public 
PI Contribution Tyler, Brown, Hetheridge have continued to work to secure follow-on research collaboration with Richard Currie and Katy Wolton at Syngenta AG UK. They are currently finalising a BBSRC IPA research proposal for submission in April 2018: "Defining critical stages and mechanisms of cleft palate development, which are particularly susceptible to environmental perturbation". The new proposal is a substantial improvement on the previous unsuccessful bid, benefiting from a new collaboration with Dr Steffen Scholpp (formerly from the Karlsruhe Institute of Technology, Germany) concerning imaging of palatal cell signalling events (sonic hedgehog and retinoic acid) and Single Molecule Detection (SMD) for studying protein-protein interactions. The team will also continue to collaborate with Dr Chrissy Hammond from the University of Bristol's Biomedical Science Department, providing expertise in the regulation of cartilage and bone homeostasis, including in the orofacial skeleton. Tyler and Hetheridge have negotiated a further research collaboration with AstraZeneca: Drug Safety Assay Development Research (£252k), which follows-on from a previous consultancy agreement. The majority of this project aims to evaluate the potential for transgenic zebrafish models to aid the prediction of human side effects of pharmaceuticals - including seizure and bone malformation, amongst others. From 2017: Through the FLIP we have strengthened considerably our research partnership with AstraZeneca UK and built a new partnership with Syngenta AG, including via internships (Ross Brown and Jon Green) and regular face-to-face meetings. The partnerships have played a role in securing further major research support to Exeter from AstraZeneca for work on pharmaceuticals, three CASE studentship(s) for advancing the use of transgenic fish/imaging and modeling in chemical hazard identification (Syngenta/AstraZeneca) with further grants (Syngenta) and CASE studentships (AstraZeneca) submitted/being developed for submission to BBSRC.
Collaborator Contribution as above
Impact as above
Start Year 2014
 
Description UEMS Dr John Chilton/Prof Andrew Randall 
Organisation University of Exeter
Department Medical School
Country United Kingdom 
Sector Academic/University 
PI Contribution We have undertaken collaborations with Dr. John Chilton (University of Exeter, Medical School) to test the transient expression of GFP derived from estrogen responsive promotor (ERE) activation in the live chick embryo. The aim of this approach was to examine whether tissue specific estrogen responses seen in our ERE-TG line (e.g. brain, heart, liver and heart) are conserved in a higher vertebrate. Collaboration with Prof. Andrew Randall's lab (Medical School, University of Exeter) for Ca++ imaging of estrogen responsive neuronal cells.
Collaborator Contribution Expertise in neural development and function. Collaborators conducted supporting experiments in neural electrophysiology. Joint research paper in preparation.
Impact Collaborators conducted supporting experiments in neural electrophysiology. Joint research paper in preparation. Joint collaborative research grant application based on this collaborative work submitted to BBSRC.
Start Year 2014
 
Description 19th UK-Japan Annual Scientific Workshop for Research into Environmental Endocrine Disrupting Chemicals 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Anke Lange presented data from this work on roach at the 19th UK-Japan Annual Scientific Workshop for Research into Environmental Endocrine Disrupting Chemicals, Japan December 2017. The conference was attended by UK and Japanese government regulators, and environmental consultants.
Year(s) Of Engagement Activity 2017
 
Description 50th Anniversary of the Fisheries Society of the British Isles 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The Jack Jones Lecture, 50th Anniversary of the Fisheries Society of the British Isles. Feminisation of Nature - An Unnatural History, Exeter, July 2017. C.R.Tyler
Assessing the oestrogenic potency and health impacts of wastewater treatment work effluents using ERE-GFP transgenic zebrafish.
FSBI Annual Symposium, Exeter, UK 3-7 July 2017
Oral presentation
Cooper, R.; Kudoh, T.; Tyler, C.R.; David, A.; Hill, E.
Year(s) Of Engagement Activity 2017
 
Description Annual Scientific Workshop on Research into Environmental Endocrine Disrupting Chemicals 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Progress in our development and applications of transgenic zebrafish. The 20th UK-Japan Annual Scientific Workshop on Research into Environmental Endocrine Disrupting Chemicals, Downing College, Cambridge. International conference to peers, collaborators, industry, policy making
Year(s) Of Engagement Activity 2019
 
Description Axminster and Exeter College visits, Hamilton 2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Hamilton (PDRA and Co-I) has given schools talks in 2015 about his research at Axminster College and Exeter College.
Year(s) Of Engagement Activity 2015
 
Description Chalkstream Headwaters 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Feminisation of Freshwater Nature - Chalk Stream Headwaters Annual Meeting at Sparsholt College, 24th November 2017
Year(s) Of Engagement Activity 2017
 
Description Feminisation of Nature - Linnean Society Keynote 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Linnean Society invited Charles Tyler to speak at an evening lecture which was publicised to members and the general public.
The lecture provided a critical analysis on the effects of environmental estrogens on wildlife and the implications for wildlife populations, with a principal focus on fish, and also illustrated how some molecular technologies are helping in to unravel the complex ways in which oestrogenic chemicals interact within the body.
The event was followed by a Q&A and reception.
Year(s) Of Engagement Activity 2016
URL https://www.linnean.org/meetings-and-events/events/the-feminisation-of-nature
 
Description Government report 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Government report
Year(s) Of Engagement Activity 2018
 
Description International Congress on Comparative Endocrinology, Canada 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Endocrine Disruption in Aquatic Systems. International Congress on Comparative Endocrinology, Canada. June 2017. Keynote speech.
Year(s) Of Engagement Activity 2017
 
Description KEYNOTE PRESENTATION 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact International Symposium on the Reproductive Physiology of Fish, Manaus Brazil.
Year(s) Of Engagement Activity 2018
 
Description Population genetic analysis talk, TGAC course Norwich 2015 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact Hamilton gave an invited talk on the use of population genetic analysis, in particular RAD-seq to investigate the population impacts of pollution in the aquatic environment. TGAC course on RAD-seq Norwich, June 2015.
Year(s) Of Engagement Activity 2015
 
Description SETAC UK Student event at Oxford University 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact A Half hour talk within a workshop event SETAC held at Oxford University, as well as a careers panel activity and a poster event.
Year(s) Of Engagement Activity 2016
 
Description Symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact CRU Symposium attended by international audience, peers and others
Year(s) Of Engagement Activity 2018
 
Description The Feminisation of Nature - Howard Hughes Lecture USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact The Feminisation of Nature. Howard Hughes Lecture - Medical University of South Carolina, USA
Year(s) Of Engagement Activity 2015