Effects of Cyclic Hypoxia On Gene Expression and Reproduction In a Grass Shrimp, \u3ci\u3ePalaemonetes pugio\u3c/i\u3e

Cyclic changes in dissolved oxygen occur naturally in shallow estuarine systems, yet little is known about the adaptations and responses of estuarine organisms to cyclic hypoxia. Here we examine the responses of Palaemonetes pugio, a species of grass shrimp, to cyclic hypoxia (1.5-8 mg/l dissolved oxygen; 4.20-22.42 kPa) at both the molecular and organismal levels. We measured alterations in gene expression in hepatopancreas tissue of female grass shrimp using custom cDNA macroarrays. After short-term (3-d) exposure to cyclic hypoxia, mitochondrial manganese superoxide dismutase (MnSOD) was upregulated and 70-kd heat shock proteins (HSP70) were downregulated. After 7-d exposure, nuclear genes encoding mitochondrial proteins (ribosomal protein S2, ATP synthase, very-long-chain specific acyl-CoA dehydrogenase [VLCAD]) were downregulated, whereas mitochondrial phosphoenol pyruvate carboxykinase (PEP Cbk) was upregulated. After 14 d, vitellogenin and apolipoprotein A1 were upregulated. Taken together, these changes suggest a shift in metabolism toward gluconeogenesis and lipid export. Long-term (77-d) exposure to hypoxia showed that profiles of gene expression returned to pre-exposure levels. These molecular responses differ markedly from those induced by chronic hypoxia. At the organismal level, cyclic hypoxia reduces the number of broods and eggs a female can produce. Demographic analysis showed a lower estimated rate of population growth in grass shrimp exposed to both continuous and short-term cyclic hypoxia, suggesting population-level impacts on grass shrimp

Applications for next-generation sequencing in fish ecotoxicogenomics

The new technologies for next-generation sequencing (NGS) and global gene expression analyses that are widely used in molecular medicine are increasingly applied to the field of fish biology. This has facilitated new directions to address research areas that could not be previously considered due to the lack of molecular information for ecologically relevant species. Over the past decade, the cost of NGS has decreased significantly, making it possible to use non-model fish species to investigate emerging environmental issues. NGS technologies have permitted researchers to obtain large amounts of raw data in short periods of time. There have also been significant improvements in bioinformatics to assemble the sequences and annotate the genes, thus facilitating the management of these large datasets.The combination of DNA sequencing and bioinformatics has improved our abilities to design custom microarrays and study the genome and transcriptome of a wide variety of organisms. Despite the promising results obtained using these techniques in fish studies, NGS technologies are currently underused in ecotoxicogenomics and few studies have employed these methods. These issues should be addressed in order to exploit the full potential of NGS in ecotoxicological studies and expand our understanding of the biology of non-model organisms

Relationship between reproductive success and male plasma vitellogenin concentrations in cunner, Tautogolabrus adspersus

The gene for vitellogenin, an egg yolk protein precursor, is usually silent in male fish but can be induced by estrogen exposure. For this reason, vitellogenin production in male fish has become a widely used indicator of exposure to exogenous estrogens or estrogen mimics in the aquatic environment. The utility of this indicator to predict impacts on fish reproductive success is unclear because information on the relationship between male plasma vitellogenin and reproductive end points in male and female fish is limited. In the research reported in this article, we investigated whether the presence of male plasma vitellogenin is a reliable indicator of decreased reproductive success in mature fish. Adult and sexually mature male and female cunner (Tautogolabrus adspersus) were exposed to 17β-estradiol, ethynylestradiol, or estrone, three steroidal estrogens that elicit the vitellogenic response. Data were gathered and pooled on egg production, egg viability, egg fertility, sperm motility, and male plasma vitellogenin concentrations. All males, including two with plasma vitellogenin levels exceeding 300 mg/mL, produced motile sperm. Neither percent fertile eggs nor percent viable eggs produced by reproductively active fish demonstrated a significant correlation with male plasma vitellogenin concentrations. Male gonadosomatic index and average daily egg production by females showed significant, but weak, negative correlation with male plasma vitellogenin concentrations. Results suggest that male plasma vitellogenin expression is not a reliable indicator of male reproductive dysfunction in adult cunner exposed to estrogens for 2-8 weeks during their reproductive season, at least in relation to capacity to produce motile sperm or fertilize eggs. Male plasma vitellogenin expression may serve as an indicator of reduced female reproductive function caused by estrogen exposure

Gene expression responses in male fathead minnows exposed to binary mixtures of an estrogen and antiestrogen

<p>Abstract</p> <p>Background</p> <p>Aquatic organisms are continuously exposed to complex mixtures of chemicals, many of which can interfere with their endocrine system, resulting in impaired reproduction, development or survival, among others. In order to analyze the effects and mechanisms of action of estrogen/anti-estrogen mixtures, we exposed male fathead minnows (<it>Pimephales promelas</it>) for 48 hours via the water to 2, 5, 10, and 50 ng 17α-ethinylestradiol (EE₂)/L, 100 ng ZM 189,154/L (a potent antiestrogen known to block activity of estrogen receptors) or mixtures of 5 or 50 ng EE₂/L with 100 ng ZM 189,154/L. We analyzed gene expression changes in the gonad, as well as hormone and vitellogenin plasma levels.</p> <p>Results</p> <p>Steroidogenesis was down-regulated by EE₂as reflected by the reduced plasma levels of testosterone in the exposed fish and down-regulation of genes in the steroidogenic pathway. Microarray analysis of testis of fathead minnows treated with 5 ng EE₂/L or with the mixture of 5 ng EE₂/L and 100 ng ZM 189,154/L indicated that some of the genes whose expression was changed by EE₂were blocked by ZM 189,154, while others were either not blocked or enhanced by the mixture, generating two distinct expression patterns. Gene ontology and pathway analysis programs were used to determine categories of genes for each expression pattern.</p> <p>Conclusion</p> <p>Our results suggest that response to estrogens occurs via multiple mechanisms, including canonical binding to soluble estrogen receptors, membrane estrogen receptors, and other mechanisms that are not blocked by pure antiestrogens.</p

Tissue-Based Mapping of the Fathead Minnow (Pimephales promelas) Transcriptome and Proteome

Omics approaches are broadly used to explore endocrine and toxicity-related pathways and functions. Nevertheless, there is still a significant gap in knowledge in terms of understanding the endocrine system and its numerous connections and intricate feedback loops, especially in non-model organisms. The fathead minnow (Pimephales promelas) is a widely used small fish model for aquatic toxicology and regulatory testing, particularly in North America. A draft genome has been published, but the amount of available genomic or transcriptomic information is still far behind that of other more broadly studied species, such as the zebrafish. Here, we used a proteogenomics approach to survey the tissue-specific proteome and transcriptome profiles in adult male fathead minnow. To do so, we generated a draft transcriptome using short and long sequencing reads from liver, testis, brain, heart, gill, head kidney, trunk kidney, and gastrointestinal tract. We identified 30,378 different putative transcripts overall, with the assembled contigs ranging in size from 264 to over 9,720 nts. Over 17,000 transcripts were &gt;1,000 nts, suggesting a robust transcriptome that can be used to interpret RNA sequencing data in the future. We also performed RNA sequencing and proteomics analysis on four tissues, including the telencephalon, hypothalamus, liver, and gastrointestinal tract of male fish. Transcripts ranged from 0 to 600,000 copies per gene and a large portion were expressed in a tissue-specific manner. Specifically, the telencephalon and hypothalamus shared the most expressed genes, while the gastrointestinal tract and the liver were quite distinct. Using protein profiling techniques, we identified a total of 4,045 proteins in the four tissues investigated, and their tissue-specific expression pattern correlated with the transcripts at the pathway level. Similarly to the findings with the transcriptomic data, the hypothalamus and telencephalon had the highest degree of similarity in the proteins detected. The main purpose of this analysis was to generate tissue-specific omics data in order to support future aquatic ecotoxicogenomic and endocrine-related studies as well as to improve our understanding of the fathead minnow as an ecological model

Towards regulation of Endocrine Disrupting chemicals (EDCs) in water resources using bioassays - A guide to developing a testing strategy

Endocrine disrupting chemicals (EDCs) are found in every environmental medium and are chemically diverse. Their presence in water resources can negatively impact the health of both human and wildlife. Currently, there are no mandatory screening mandates or regulations for EDC levels in complex water samples globally. Bioassays, which allow quantifying in vivo or in vitro biological effects of chemicals are used commonly to assess acute toxicity in water. The existing OECD framework to identify single-compound EDCs offers a set of bioassays that are validated for the Estrogen-, Androgen-, and Thyroid hormones, and for Steroidogenesis pathways (EATS). In this review, we discussed bioassays that could be potentially used to screen EDCs in water resources, including in vivo and in vitro bioassays using invertebrates, fish, amphibians, and/or mammalians species. Strengths and weaknesses of samples preparation for complex water samples are discussed. We also review how to calculate the Effect-Based Trigger values, which could serve as thresholds to determine if a given water sample poses a risk based on existing quality standards. This work aims to assist governments and regulatory agencies in developing a testing strategy towards regulation of EDCs in water resources worldwide. The main recommendations include 1) opting for internationally validated cell reporter in vitro bioassays to reduce animal use & cost; 2) testing for cell viability (a critical parameter) when using in vitro bioassays; and 3) evaluating the recovery of the water sample preparation method selected. This review also highlights future research avenues for the EDC screening revolution (e.g., 3D tissue culture, transgenic animals, OMICs, and Adverse Outcome Pathways (AOPs)).This work was supported by the Fonds de recherche du Québec - Nature et technologies (FRQNT-290501) to JR, Natural Sciences and Engineering Research Council (NSERC) of Canada (NSERC-DG-2020-06475), and Canada Research Chairs to VSL (CRC-950-232235). LNM was supported by a H2020-Marie Skłodowska-Curie Action MSCA-IF-RI- 2017 awarded by the European Commission (ref. 797725-EpiSTOX). The authors are grateful to the Intersectorial Centre for Endocrine Disruptor Analysis (ICEDA)'s researcher network that facilitated this Special Issue. We thank Peta Neale that compiled references from the literature for EBT value that can be found in Table 3.Peer reviewe