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 (Gymnocephalus cernua) and the flounder (Platichthys flesus). 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.

Respiratory proteins are responsible for transport and storage of oxygen. It is well established that specific requirements for oxygen during vertebrate ontogeny cause switches of hemoglobin chain expression. Here, we characterize the developmental profiles of zebrafish (Danio rerio) globins by means of quantitative real-time reverse transcription PCR. The total mRNA levels of the hemoglobin chains, including a newly identified embryonic α-chain, as well as myoglobin, neuroglobin, cytoglobin 1 and 2, and globin X were estimated. mRNAs of all globins were detectable in unfertilized eggs, suggesting maternal storage. Embryonic α- and β-hemoglobin mRNA peaked at hatching and the switch to adult hemoglobin expression occurred 16 dpf. Enhanced myoglobin mRNA levels were detected ~31 h post-fertilization (hpf), coinciding with the heart and the muscle development, while neuroglobin mRNA expression pattern correlates with the formation of the nervous system. Amounts of myoglobin and neuroglobin mRNA were similar within an order of magnitude throughout the ontogeny, tentatively supporting a respiratory role of neuroglobin. Cytoglobin 2 mRNA levels increased gradually, whereas cytoglobin 1 mRNA levels increased strongly after ~31 hpf, which is in agreement with a function in cell proliferation. Globin X mRNA level was highest in oocytes, but low in later stages. Together, these data suggest a specific role for each globin, which are also associated with certain events in fish development.

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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 (Gymnocephalus cernua) and the European flounder (Platichthys flesus). 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.