Abstract
BackgroundGlutamate dehydrogenase (GDH) is a key enzyme for the synthesis and catabolism of glutamic acid, proline and alanine, which are important osmolytes in aquatic animals. However, the response of GDH gene expression to salinity alterations has not yet been determined in macro-crustacean species.Methodology/Principal FindingsGDH cDNA was isolated from Eriocheir sinensis. Then, GDH gene expression was analyzed in different tissues from normal crabs and the muscle of crabs following transfer from freshwater (control) directly to water with salinities of 16‰ and 30‰, respectively. Full-length GDH cDNA is 2,349 bp, consisting of a 76 bp 5′- untranslated region, a 1,695 bp open reading frame encoding 564 amino acids and a 578 bp 3′- untranslated region. E. sinensis GDH showed 64–90% identity with protein sequences of mammalian and crustacean species. Muscle was the dominant expression source among all tissues tested. Compared with the control, GDH expression significantly increased at 6 h in crabs transferred to 16‰ and 30‰ salinity, and GDH expression peaked at 48 h and 12 h, respectively, with levels approximately 7.9 and 8.5 fold higher than the control. The free amino acid (FAA) changes in muscle, under acute salinity stress (16‰ and 30‰ salinities), correlated with GDH expression levels. Total FAA content in the muscle, which was based on specific changes in arginine, proline, glycine, alanine, taurine, serine and glutamic acid, tended to increase in crabs following transfer to salt water. Among these, arginine, proline and alanine increased significantly during salinity acclimation and accounted for the highest proportion of total FAA.Conclusions E. sinensis GDH is a conserved protein that serves important functions in controlling osmoregulation. We observed that higher GDH expression after ambient salinity increase led to higher FAA metabolism, especially the synthesis of glutamic acid, which increased the synthesis of proline and alanine to meet the demand of osmoregulation at hyperosmotic conditions.
Highlights
Salinity is one of the most important factors influencing the physiological status of aquatic animals
We observed that higher Glutamate dehydrogenase (GDH) expression after ambient salinity increase led to higher free amino acid (FAA) metabolism, especially the synthesis of glutamic acid, which increased the synthesis of proline and alanine to meet the demand of osmoregulation at hyperosmotic conditions
Characterization of GDH cDNA from E. sinensis The full-length GDH cDNA sequence cloned from E. sinensis was 2,349 bp long, contained a 1,695 bp ORF encoding a 564 amino acid protein, a 76 bp 59UTR and a 578 bp 39UTR (Figure 1), and was deposited in GenBank as JN628041
Summary
Salinity is one of the most important factors influencing the physiological status of aquatic animals. The Chinese mitten crab, Eriocheir sinensis, is a strong osmoregulator and has been used as a model species in a number of physiological investigations [4,5]. Rathmayer and Siebers (2001) reported on the mechanisms that control the balance of sodium and chloride ions in freshwater-acclimated mitten crabs [6]. Their results revealed that the ability of juvenile and adult E. sinensis to cope with salinity variations during their life cycles involves ontogenetic changes in their osmoregulatory capacity [5,9]. The response of GDH gene expression to salinity alterations has not yet been determined in macro-crustacean species
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