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Behaviour, Pollutants and Genomics: exploring their interactions in a fish model

Final Report Summary - BEPOLGEN (Behaviour, Pollutants and Genomics: exploring their interactions in a fish model)

Aquatic habitats are increasingly being exposed to chemicals that, even at low levels, can disturb the endocrinology of organisms. These endocrine disrupting chemicals (EDCs) can have severe impacts on reproductive physiology, yet knowledge of their effects on evolutionary processes and population viability is fundamentally lacking. Using the invasive mosquitofish Gambusia holbrooki, the Fellow will use behavioural and genetic assays to reveal how EDCs impact reproductive success and sexual selection. This study will be among the first to examine the effects of EDCs on both female and male reproductive behaviour and quality of the offspring. Furthermore, cutting-edge next generation sequencing techniques (NGS) will be used to explore EDC-induced changes in gene expression.
The specific aims of the project were to:

• Show that an environmentally relevant concentration of an EDC can influence male and female behaviour and offspring quality.
• To use NGS to determine how gene expression relates to behaviour of exposed individuals.
To achieve these Aims the project adopted the following Objectives:

• to expose mosquitofish to 17-ethynyl estradiol (EE2), a synthetic estrogen used in oral contraceptive pills.
• determine how EE2 exposure affects fish reproductive behaviour and reproductive success.
• establish the transcriptomic profile of the fish brain, ovary or testis with NGS and use digital transcriptomics to analyse the effects of EE2 exposure on gene expression.

The identity and extent of change of the differentially expressed transcripts will be interpreted with respect to the observed behavioural changes. The proposed research set out to allow the Fellow to use existing expertise on behavioural ecotoxicology but importantly to learn new techniques of molecular biology and revolutionary genomics tools of NGS and bioinformatics. In addition, the project plan would provide for the Fellow training in a number of research-related activities that will develop her into an independent scientist able to command her own research funding and supervise a research team.

This report covers only 4-months effort (June-September 2013) after which the Fellowship was terminated consequent on the Fellow receiving longer-term and more generous funding from another European Institution (Academy of Finland, FA). This was to undertake a more ambitious version of the MC proposal. Under the new, continuing arrangements, the Marie Curie (MC) host (John Craft, GCU) and other contributors to the proposal (Kai Lindström, Åbo Akademi, Turku, Finland; Bob Wong Monash University, Melbourne, Australia) still play vital roles in providing training and facilities. Thus many of the benefits to the ERA envisaged in the MC proposal will be realised and the work conducted thus far under the MC award will be useful as a foundation for the continuing FA study.

A number of objectives were reached during the four months:
Objective 1: Source fish for the study. The project took advantage of collaboration with colleagues at Monash University Melbourne, Australia (Dr Bob Wong) to source mosquitofish from the wild at locations believed to be uncontaminated. However, all such sites proved to be contaminated with oestrogens and thus the fish would have been impacted. It would have taken too long to breed unexposed fish (three generations) under uncontaminated laboratory conditions and therefore an alternative was sought. This was found in the guppy, Poecilia reticulata, a Cyprinodontiformes closely related to the mosquitofish in evolution, breeding habits and invasive nature in various continents. Further, a breeding population was available at Monash and known not to be previously exposed to EDCs.
Objective 2: Conduct an initial exposure to EE2, so as to provide material to establish a reference transcriptome for brain. Male and female guppy were exposed to EE2 (nominal concentration 20 ng/L) for three weeks. Matching control groups that were not exposed were also maintained. At the termination of the exposure period animals were sacrificed and brain dissected and stored in RNALater (Ambion). Animal treatments were conducted under appropriate regulatory and ethical approvals for experimentation with animals in Australia.
Objective 3: Organise the preparation of normalised libraries. Tissue samples were transported to Glasgow (with an appropriate import Licence granted by the Scottish Government) where the Fellow developed a suitable protocol for the isolation of high quality RNA. The challenge with respect to guppy brain resulted from the small size of the organ, especially with the males, and the lipid-rich nature of the tissue. A dual approach combining Trizol reagent (Invitrogen) followed by a silica column-based purification (Nucleospin RNA II, Machery and Nagel) proved to yield microgram quantities of total RNA with high quality (RIN ~ 8 measured by Agilent Bioanalyser).
Objective 4: Identify a suitable NGS technology to establish the reference transcriptome and a sequencing centre to conduct the work. Pools of total RNA from both sexes and conditions (EE2-exposed and untreated) have been sent to Evrogen (Moscow) for the preparation of normalised libraries. These are expected to be delivered in the next few weeks and will then be sent for sequencing. It had originally been intended to use pyrosequencing (Roche 454) but it has been decided to use Illumina technology. In particular, the MiSeq instrument can yield 44 million reads of 300 bp paired-end sequence (13.2 Gb) per run. With two runs we compute there will be a 100x depth of coverage and this can be achieved with bioinformatic support by GATC Gmbh (Konstanz, Germany) at economic cost.
Other activities: Development of qPCR assays for guppy gene targets. We have developed qPCR assays, based on a limited sequence database for this species, for 5 genes. These are actin and 28S rRNA (house-keepers), estradiol receptor a (signaling), pyruvate dehydrogenase (intermediary metabolism), heat shock protein 70 (stress response) and NADH dehydrogenase subunit 2 (mitochondrial energy production). These assays will be used for preliminary assessment of gene responses to EE2 in male and female brain in the RNA samples and form the basis for development of further assays once the reference transcriptome is available.
Personal development. The Fellow attended a workshop on Multivariate Data Analysis organised by the Royal Society of Statistics at Northumbria University, Newcastle, on 2 September 2013. She also attended a Postgraduate Supervisor Accreditation Workshop and Women in Science Leadership Workshop.

This study will deepen our knowledge on the genetics of how organisms adapt to human-induced environmental changes and provide evidence on how genome and environment interact. A primary deliverable will be a reference transcriptome of the brain of the guppy and allow identification of genes that differ in expression between male and female and that change expression due to EE2 exposure. Associations between sex-specific gene expression may provide a basis for the understanding of distinctive behavioural characteristics of this species of fish. Changes in gene expression resulting from EE2 will be associated with the effects of this EDC on sexual selection.
Poeciliidae species, such as guppies and mosquitofish are widely used model species in behavioural ecology and ecotoxicology and thus the outcome of our study will benefit and support a wider group of researchers. With the recognition of the potentially serious effects of EDCs on humans (e.g. decreasing sperm counts) and wildlife comes the realization that we need to assess the long-term effects in further detail to ensure a safe and sustainable Europe and knowledge generated by this project will contribute towards that end.