Molecular Response of Estuarine Fish to Hypoxia: A Comparative Study with Ruffe and Flounder from Field and Laboratory

<div><p>On a global scale, the frequencies and magnitudes of hypoxic events in coastal and estuarine waters have increased dramatically over the past 20 years. Fish populations are suitable indicators for the assessment of the quality of aquatic ecosystems, as they are omnipresent and often comprise a variety of different lifestyles and adaption strategies. We have investigated on the molecular level the impact of hypoxia on two fish species typical of European estuaries. We monitored the expression of eleven putatively hypoxia-responsive genes by means of quantitative real-time RT-PCR in brains, gills and hearts of the ruffe (<i>Gymnocephalus cernua</i>) and the flounder <i>(Platichthys flesus</i>). We first investigated the effect of naturally occurring hypoxia in the Elbe estuary. In a second approach, expression changes in the response to hypoxia were monitored under controlled laboratory conditions. The genes that showed the strongest effect were two respiratory proteins, myoglobin and neuroglobin, as well as the apoptosis enzyme caspase 3. As previously observed in other fish, myoglobin, which was considered to be muscle-specific, was found in brain and gills as well. Comparison of field and laboratory studies showed that – with the exception of the heart of flounder – that mRNA levels of the selected genes were about the same, suggesting that laboratory conditions reflect natural conditions. Likewise, trends of gene expression changes under hypoxia were the same, although hypoxia response was more pronounced in the Elbe estuary. In general, the flounder displayed a stronger response to hypoxia than the ruffe, suggesting that the flounder is more susceptible to hypoxia. The most pronounced differences were found among tissues within a species, demonstrating that hypoxia response is largely tissue-specific. In summary, our data suggest that laboratory experiments essentially mimic field data, but additional environmental factors enhance hypoxia response in nature.</p></div

Interspecies and experimental site comparison of total mRNA copy numbers of putatively hypoxia responsive genes in ruffe and flounder under normoxic condition analysed in (A) gills, (B) brain and (C) heart.

<p>All expression levels are referred to 1 µg mRNA. qRT-PCR results from the Elbe estuary are represented in black columns for ruffe and grey columns for flounder. Dashed columns indicate qRT-PCR results from laboratory experiments with black background for ruffe and grey background for flounder, respectively. Note that the y-axis has a logarithmic scale. Bars indicate standard deviation (SD). Asterisks indicate significance at p <0.05. N = 3 individuals for results from Elbe estuary for each data point. N = 3 (analysed with a nested experimental design) for results from laboratory for each data point.</p

Bubble charts of relative expression patterns of putatively hypoxia responsive genes in ruffe under different hypoxic conditions in laboratory experiments (A, B and C) and in the Elbe estuary (C, D and E).

<p>Evaluation of qRT-PCR results was performed with ΔΔ CT method by use of RPLP0 as a reference gene. All results of relative expression levels are represented in log base 2. The diameter of bubbles indicates the magnitude of gene expression. Black bubbles represent expression levels >0, pen bubbles represent expression levels <0, and crossing lines without a bubble signify no change in the expression level. Asterisks indicate significance at p<0.1. We analysed the expression pattern in three tissues of ruffe: (A) and (D) gills, (B) and (E) brain and (C) and (F) heart. For more details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090778#pone.0090778.s002" target="_blank">Table S2</a>.</p

Comparison of oxygen concentrations in the Elbe estuary and in laboratory experiments.

<p>Comparison of oxygen concentrations in the Elbe estuary and in laboratory experiments.</p

Venn diagram of expression changes in different tissues of ruffe and flounder.

<p>Black letters indicate genes that are upregulated and white letters indicate genes that are downregulated. Merged Venn diagram shows an overview of genes regulated more than fourfold in an overall comparison of the collected data.</p

Evaluating the Hypoxia Response of Ruffe and Flounder Gills by a Combined Proteome and Transcriptome Approach

<div><p>Hypoxia has gained ecological importance during the last decades, and it is the most dramatically increasing environmental factor in coastal areas and estuaries. The gills of fish are the prime target of hypoxia and other stresses. Here we have studied the impact of the exposure to hypoxia (1.5 mg O₂/l for 48 h) on the protein expression of the gills of two estuarine fish species, the ruffe (<i>Gymnocephalus cernua</i>) and the European flounder (<i>Platichthys flesus</i>). First, we obtained the transcriptomes of mixed tissues (gills, heart and brain) from both species by Illumina next-generation sequencing. Then, the gill proteomes were investigated using two-dimensional gel electrophoresis and mass spectrometry. Quantification of the normalized proteome maps resulted in a total of 148 spots in the ruffe, of which 28 (18.8%) were significantly regulated (> 1.5-fold). In the flounder, 121 spots were found, of which 27 (22.3%) proteins were significantly regulated. The transcriptomes were used for the identification of these proteins, which was successful for 15 proteins of the ruffe and 14 of the flounder. The ruffe transcriptome dataset comprised 87,169,850 reads, resulting in an assembly of 72,108 contigs (N50 = 1,828 bp). 20,860 contigs (26.93%) had blastx hits with E < 1e-5 in the human sequences in the RefSeq database, representing 14,771 unique accession numbers. The flounder transcriptome with 78,943,030 reads assembled into 49,241 contigs (N50 = 2,106 bp). 20,127 contigs (40.87%) had a hit with human proteins, corresponding to 14,455 unique accession numbers. The regulation of selected genes was confirmed by quantitative real-time RT-PCR. Most of the regulated proteins that were identified by this approach function in the energy metabolism, while others are involved in the immune response, cell signalling and the cytoskeleton.</p></div

Changes in the protein level in the gills of the ruffe in response to hypoxia.

<p>Protein spot volumes were normalized according to GAPDH. Protein expression changes were calculated by the comparison of the mean of hypoxia (n = 3) to normoxia (n = 3) ± s.d., and significance levels were determined by a Student's t-test on the log2 transformed values; * = <i>p</i>< 0.1; ** = <i>p</i>< 0.01; *** = <i>p</i>< 0.001. For detailed information see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135911#pone.0135911.s011" target="_blank">S6 Table</a>.</p

Comparison of the changes in protein and mRNA levels in the gills of ruffe (A) and flounder (B).

<p>Changes in protein levels were calculated as stated above (see Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135911#pone.0135911.g004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135911#pone.0135911.g005" target="_blank">5</a>). mRNA copy numbers were measured by qRT-PCR (n = 3). The data are presented as log2 fold changes. The raw qRT-PCR data are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135911#pone.0135911.s013" target="_blank">S8 table</a>, the standard deviations for the proteins are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135911#pone.0135911.s011" target="_blank">S6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135911#pone.0135911.s012" target="_blank">S7</a> tables. A scatter plot that compares the changes of protein and mRNA levels is provided as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135911#pone.0135911.s005" target="_blank">S2 Fig</a>.</p

Gill proteome maps.

<p>The images were composed of six gel images (three from hypoxia and three from normoxia experiment) of the ruffe (A) and the European flounder (B). Protein spots that significantly changed in abundance in response to hypoxia are labelled with numbers. The original gels are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135911#pone.0135911.s001" target="_blank">S1 Appendix</a>. The lists of spots that changed under hypoxia are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135911#pone.0135911.s009" target="_blank">S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135911#pone.0135911.s010" target="_blank">S5</a> Tables.</p

Changes in the protein level in the gills of the European flounder in response to hypoxia.

<p>Protein spot volumes were normalized according to GAPDH. Protein expression changes were calculated by the comparison of the mean of hypoxia (n = 3) to normoxia (n = 3) ± s.d., and significance levels were determined by a Student's t-test on the log2 transformed values; * = <i>p</i>< 0.1; ** = <i>p</i>< 0.01; *** = <i>p</i>< 0.001. For detailed information see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135911#pone.0135911.s012" target="_blank">S7 Table</a>.</p