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

Queen Conch (Strombus gigas) Testis Regresses during the Reproductive Season at Nearshore Sites in the Florida Keys

BACKGROUND: Queen conch (Strombus gigas) reproduction is inhibited in nearshore areas of the Florida Keys, relative to the offshore environment where conchs reproduce successfully. Nearshore reproductive failure is possibly a result of exposure to environmental factors, including heavy metals, which are likely to accumulate close to shore. Metals such as Cu and Zn are detrimental to reproduction in many mollusks. METHODOLOGY/PRINCIPAL FINDINGS: Histology shows gonadal atrophy in nearshore conchs as compared to reproductively healthy offshore conchs. In order to determine molecular mechanisms leading to tissue changes and reproductive failure, a microarray was developed. A normalized cDNA library for queen conch was constructed and sequenced using the 454 Life Sciences GS-FLX pyrosequencer, producing 27,723 assembled contigs and 7,740 annotated transcript sequences. The resulting sequences were used to design the microarray. Microarray analysis of conch testis indicated differential regulation of 255 genes (p<0.01) in nearshore conch, relative to offshore. Changes in expression for three of four transcripts of interest were confirmed using real-time reverse transcription polymerase chain reaction. Gene Ontology enrichment analysis indicated changes in biological processes: respiratory chain (GO:0015992), spermatogenesis (GO:0007283), small GTPase-mediated signal transduction (GO:0007264), and others. Inductively coupled plasma-mass spectrometry analysis indicated that Zn and possibly Cu were elevated in some nearshore conch tissues. CONCLUSIONS/SIGNIFICANCE: Congruence between testis histology and microarray data suggests that nearshore conch testes regress during the reproductive season, while offshore conch testes develop normally. Possible mechanisms underlying the testis regression observed in queen conch in the nearshore Florida Keys include a disruption of small GTPase (Ras)-mediated signaling in testis development. Additionally, elevated tissue levels of Cu (34.77 ng/mg in testis) and Zn (831.85 ng/mg in digestive gland, 83.96 ng/mg in testis) nearshore are similar to reported levels resulting in reproductive inhibition in other gastropods, indicating that these metals possibly contribute to NS conch reproductive failure

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