Using Illumina sequencing, we investigated transcriptional changes caused by the nematode Anguillicola crassus within yellow and silver eels by comparing swimbladder samples of uninfected yellow with infected yellow eels, and uninfected silver with infected silver eels, respectively. In yellow eel gas gland, the infection caused a modification of steady state mRNA levels of 1675 genes, most of them being upregulated. Functional annotation analysis based on GO terms was used to categorize identified genes with regard to swimbladder metabolism or response to the infection. In yellow eels, the most prominent category was ‘immune response’, including various inflammatory components, complement proteins, and immunoglobulins. The elevated expression of several glucose and monocarboxylate transporters indicated an attempt to maintain the level of glucose metabolism, even in due to the infection thickened swimbladder tissue. In silver eel swimbladder tissue, on the contrary, the mRNA levels of only 291 genes were affected. Genes in the categories ‘glucose metabolism’ and ‘ROS metabolism’ barely responded to the infection and even the reaction of the immune system was much less pronounced compared to infected yellow eels. However, in the category ‘extracellular matrix’, the mRNA levels of several mucin genes were strongly elevated, suggesting increased mucus production as a defense reaction against the parasite. The present study revealed a strong reaction to an Anguillicola crassus infection on mRNA expression levels in swimbladder tissue of yellow eels, whereas in silver eels the changes ware almost negligible. A possible explanation for this difference is that the silvering process requires so much energy that there is not much scope to cope with the additional challenge of a nematode infection. Another possible explanation could be that gas-secreting activity of the silver eel swimbladder was largely reduced, which could coincide with a reduced responsiveness to other challenges, like a nematode infection.

Using Illumina sequencing, transcriptional changes occurring during silvering in swimbladder tissue of the European eel have been analyzed by comparison of yellow and silver eel tissue samples. Functional annotation analysis based on GO terms revealed significant expression changes in a number of genes related to the extracellular matrix, important for the control of gas permeability of the swimbladder, and to reactive oxygen species (ROS) defense, important to cope with ROS generated under hyperbaric oxygen partial pressures. Focusing on swimbladder tissue metabolism, levels of several mRNA species encoding glucose transport proteins were several-fold higher in silver eels, while enzymes of the glycolytic pathway were not affected. The significantly higher steady state level of a transcript encoding for membrane bound carbonic anhydrase, however, suggested that CO2 production in the pentose phosphate shunt and diffusion of CO2 was of particular importance in silver eel swimbladder. In addition, the mRNA level of a large number of genes related to immune response and to sexual maturation was significantly modified in the silver eel swimbladder. The modification of several processes related to protein metabolism and transport, cell cycle, and apoptosis suggested that these changes in swimbladder metabolism and permeability were achieved by increasing cell turn-over. The impact of an infection of the swimbladder with the nematode Anguillicola crassus has been assessed by comparing these expression changes with expression changes observed between uninfected yellow eel swimbladder tissue and infected silver eel swimbladder tissue. In contrast to uninfected silver eel swimbladder tissue, in infected tissue the mRNA level of several glycolytic enzymes was significantly elevated, and with respect to extracellular matrix, several mucin genes were many-fold higher in their mRNA level. Modification of many immune related genes and of the functional categories “response to DNA damage stimulus” and “cellular response to stress” illustrated the damaging effect of the nematode infection. This study has identified a range of cellular processes in the swimbladder of silver eels that appear to be altered by nematode infection. These altered cellular processes could contribute to detrimental changes in swimbladder function that, in turn, may lead to impairment of spawning migration.

In a process called silvering, European eels prepare for their long-distance migration from European freshwater systems to the Sargasso Sea for reproduction. During this journey, eels perform extended diel vertical migrations, and the concomitant changes in hydrostatic pressure significantly affect the swimbladder, functioning as a buoyancy organ. As the swimbladder is primarily filled with oxygen, the tissue has to cope with extreme hyperoxic conditions, which typically are accompanied by the generation of reactive oxygen species (ROS) and oxidative stress. In addition, since the introduction of the parasitic nematode Anguillicola crassus in the early 1980s, swimbladder function of most of the European eels is impaired by the infection with this parasite. However, the exact pathways to detoxify ROS and how these pathways are affected by silvering or the infection are still unknown. In swimbladder and muscle tissue from uninfected and infected yellow, and from uninfected and infected silver eels, we measured the level of lipid peroxidation, which increases with ROS stress. To assess the capacity of the ROS defense systems, we analyzed the activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and glutathione reductase (GR), and determined the concentration of the antioxidant glutathione (GSH + GSSG). In swimbladder tissue, we found increased concentrations of GSH + GSSG as well as higher activities of SOD, GPx and GR, suggesting that SOD and the glutathione cycle are important for ROS detoxification. Comparing swimbladder tissue of uninfected yellow with uninfected silver eels, the concentration of GSH + GSSG and the activity of SOD were higher after silvering, corresponding with lower levels of lipid peroxidation. Whereas in yellow eels the infection with A. crassus had no effect, in silver eels the capacity to cope with ROS was significantly impaired. In muscle tissue, silvering or the infection only affected the activity of SOD but in exactly the same way as in swimbladder tissue.Electronic supplementary materialThe online version of this article (doi:10.1007/s00360-016-0994-0) contains supplementary material, which is available to authorized users.

Background In physoclist fishes filling of the swimbladder requires acid secretion of gas gland cells to switch on the Root effect and subsequent countercurrent concentration of the initial gas partial pressure increase by back-diffusion of gas molecules in the rete mirabile. It is generally assumed that the rete mirabile functions as a passive exchanger, but a detailed analysis of lactate and water movements in the rete mirabile of the eel revealed that lactate is diffusing back in the rete. In the present study we therefore test the hypothesis that expression of transport proteins in rete capillaries allows for back-diffusion of ions and metabolites, which would support the countercurrent concentrating capacity of the rete mirabile. It is also assumed that in silver eels, the migratory stage of the eel, the expression of transport proteins would be enhanced. Results Analysis of the transcriptome and of the proteome of rete mirabile tissue of the European eel revealed the expression of a large number of membrane ion and metabolite transport proteins, including monocarboxylate and glucose transport proteins. In addition, ion channel proteins, Ca2+-ATPase, Na+/K+-ATPase and also F1F0-ATP synthase were detected. In contrast to our expectation in silver eels the expression of these transport proteins was not elevated as compared to yellow eels. A remarkable number of enzymes degrading reactive oxygen species (ROS) was detected in rete capillaries. Conclusions Our results reveal the expression of a large number of transport proteins in rete capillaries, so that the back diffusion of ions and metabolites, in particular lactate, may significantly enhance the countercurrent concentrating ability of the rete. Metabolic pathways allowing for aerobic generation of ATP supporting secondary active transport mechanisms are established. Rete tissue appears to be equipped with a high ROS defense capacity, preventing damage of the tissue due to the high oxygen partial pressures generated in the countercurrent system.

The swim bladder of a fish is a vital organ that with gas gland cells in the swim bladder wall enables key physiological functions including buoyancy regulation in the face of different hydrostatic pressures. Specific gas gland cells produce and secrete acidic metabolites into the blood in order to reduce the physical solubility of gases and blood gas transport capacity for regulating the volume of the swim bladder. Transcriptomic analyses have provided evidence at the RNA level but no specific studies at the protein level have been carried out so far. Herein, it was the aim of the study to show swim bladder proteins of the yellow stage European eel by label-free LCMS (Q-Exactive Plus) that resulted in the identification of 6223 protein groups. Neurotransmitter receptors and transporters were enriched in the membrane fraction and enzymes for acid production were observed. The list of identified proteins may represent a useful tool for further proteomics experiments on this organ. All MS proteomics data are available at the PRIDE repository with the dataset identifier PXD007850.

In spite of many decades of research, the spawning migration of the European eel Anguilla anguilla from the European coast to the Sargasso Sea remains a mystery. In particular, the role of the swimbladder as a buoyancy regulating structure is not yet understood. In this study, we exercised silver eels in a swim tunnel under elevated hydrostatic pressure. The transcriptome of gas gland tissue of these exercised eels was then compared to the known transcriptome of not exercised (control) silver eel gas gland cells. Due to the high infection rate of the eel population with the swimbladder parasite Anguillicola crassus, the comparison also included an exercised group of silver eels with a heavily damaged swimbladder, and we compared the previously published transcriptome of not exercised silver eels with a highly damaged swimbladder with the exercised group of silver eels with a heavily damaged swimbladder. The comparisons of unexercised (control) silver eels with exercised silver eels with functional swimbladder (EF), as well as with exercised silver eels with damaged swimbladder (ED), both showed a significant elevation in transcripts related to glycolytic enzymes. This could also be observed within the comparison of unexercised silver eels with a highly infected swimbladder with exercised eels with a damaged swimbladder (DED). In contrast to EF, in ED a significant elevation in transcript numbers of mitochondrial NADH dehydrogenase was observed. While in EF the transcriptional changes suggested that acid production and secretion was enhanced, in ED these changes appeared to be related to thickened tissue and thus elevated diffusion distances. The remarkable number of differentially expressed transcripts coding for proteins connected to cAMP-dependent signaling pathways indicated that metabolic control in gas gland cells includes cAMP-dependent pathways. In contrast to ED, in EF significant transcriptional changes could be related to the reconstruction of the extracellular matrix, while in ED tissue repair and inflammation was more pronounced. Surprisingly, in exercised eels hypoxia inducible transcription factor expression was elevated. In EF, a large number of genes related to the circadian clock were transcriptionally modified, which may be connected to the circadian vertical migrations observed during the spawning migration.

The rate of glucose metabolism has been shown to be correlated to glucose uptake in swimbladder gas gland cells. Therefore, it is assumed that in the European eel silvering, i.e., the preparation of the eel for the spawning migration to the Sargasso Sea, coincides with an enhanced capacity for glucose uptake. To test this hypothesis expression of all known glucose transport proteins has been assessed at the transcript level in yellow and in silver eels, and we also included Anguillicola crassus infected swimbladders. Glucose uptake by rete mirabile endothelial cells could be crucial for the countercurrent exchange capacity of the rete. Therefore, this tissue was also included in our analysis. The results revealed expression of ten different members of the slc2 family of glucose transporters, of four slc5 family members, and of kiaa1919 in gas gland tissue. Glucose transporters of the slc2 family were expressed at very high level, and slc2a1b made up about 80% of all slc2 family members, irrespective of the developmental state or the infection status of the eel. Overall, the slc5 family contributed to only about 8% of all detected glucose transport transcripts in gas gland tissue, and the slc2 family to more than 85%. In rete capillaries, the contribution of sodium-dependent glucose transporters was significantly higher, leaving only 66% for the slc2 family of glucose transporters. Neither silvering nor the infection status had a significant effect on the expression of glucose transporters in swimbladder gas gland tissue, suggesting that glucose metabolism of eel gas gland cells may not be related to transcriptional changes of glucose transport proteins.

Swimbladder gas gland cells are known to produce lactic acid required for the acidification of swimbladder blood and decreasing the oxygen carrying capacity of swimbladder blood, i.e., the onset of the Root effect. Gas gland cells have also been shown to metabolize glucose via the pentose phosphate shunt, but the role of the pentose phosphate shunt for acid secretion has not yet been evaluated. Similarly, aerobic metabolism of gas gland cells has been largely neglected so far. In the present study, we therefore simultaneously assessed the role of glycolysis and of the pentose phosphate shunt for acid secretion and recorded oxygen consumption of isolated swimbladder gas gland cells of the European eel. Presence of glucose was essential for acid secretion, and at glucose concentrations of about 1.5 mmol l−1 acid secretion of gas gland cells reached a maximum, indicating that glucose concentrations in swimbladder blood should not be limiting acid production and secretion under physiological conditions. The data revealed that most of the acid was produced in the glycolytic pathway, but a significant fraction was also contributed by the pentose phosphate shunt. Addition of glucose to gas gland cells incubated in a glucose-free medium resulted in a reduction of oxygen uptake. Inhibition of mitochondrial respiration significantly reduced oxygen consumption, but a fraction of mitochondria-independent respiration remained in presence of rotenone and antimycin A. In the presence of glucose, application of either iodo-acetate inhibiting glycolysis or 6-AN inhibiting the pentose phosphate shunt did not significantly affect oxygen uptake, indicating an independent regulation of oxidative phosphorylation and of acid production. Inhibition of the muscarinic acetylcholine receptor caused a slight elevation in acid secretion, while forskolin caused a concentration-dependent reduction in acid secretion, indicating muscarinic and c-AMP-dependent control of acid secretion in gas gland cells.