Katrin Deigweiher

Acclimation of ion regulatory capacities in gills of marine fish under environmental hypercapnia

The preservation of ion balance and pH despite environmental fluctuations is essential for the maintenance of vital cellular functions. While several ion transporters contribute to acid-base regulation in fish, the involvement and expression of key transporters under hypercapnia remain to be established. Here, two members of the HCO(3)(-) transporter family (Na(+)/HCO(3)(-) cotransporter NBC1 and Cl(-)/HCO(3)(-) exchanger AE1) were described for the first time in gills of marine fish. Benthic eelpout Zoarces viviparus were acclimated to 10,000 ppm CO(2). Hypercapnia did not affect whole animal oxygen consumption over a period of 4 days. During a time series of 6 wk NBC1 mRNA levels first decreased by about 40% (8 to 24 h) but finally increased about threefold over control. mRNA expression of AE1 decreased transiently by 50% at day 4 but recovered to control levels only. Reduced mRNA levels were also found for two Na(+)/H(+) exchangers (NHE1A, NHE1B) during the first days (by 50-60% at days 1 and 2), followed by restoration of control levels. This pattern was mirrored in a slight decrease of NHE1 protein contents and its subsequent recovery. In contrast, Na(+)-K(+)-ATPase mRNA and protein contents, as well as maximum activity, rose steadily from the onset of hypercapnia, and reached up to twofold control levels at the end. These results indicate shifting acclimation patterns between short- and long-term CO(2) exposures. Overall, ion gradient-dependent transporter mRNA levels were transiently downregulated in the beginning of the disturbance. Upregulation of NBC1 on long timescales stresses the importance of this transporter in the hypercapnia response of marine teleosts. Long-term rearrangements include Na(+)-K(+)-ATPase at higher densities and capacities, indicating a shift to elevated rates of ion and acid-base regulation under environmental hypercapnia.

Differential expression of duplicated LDH-A genes during temperature acclimation of weatherfish Misgurnus fossilis. Functional consequences for the enzyme.

Temperature acclimation in poikilotherms entails metabolic rearrangements provided by variations in enzyme properties. However, in most cases the underlying molecular mechanisms that result in structural changes in the enzymes are obscure. This study reports that acclimation to low (5 °C) and high (18 °C) temperatures leads to differential expression of alternative forms of the LDH‐A gene in white skeletal muscle of weatherfish, Misgurnus fossilis. Two isoforms of LDH‐A mRNA were isolated and characterized: a short isoform (= 1332 bp) and a long isoform ( = 1550 bp), which both have 5′‐UTRs and ORFs of the same length (333 amino acid residues), but differ in the length of the 3′‐UTR. In addition, these two mRNAs have 44 nucleotide point mismatches of an irregular pattern along the complete sequence, resulting in three amino acid mismatches (Gly214Val; Val304Ile and Asp312Glu) between protein products from the short and long mRNA forms, correspondingly LDH‐Aα and LDH‐Aβ subunits. It is expected that the β‐subunit is more aliphatic due to the properties of the mismatched amino acids and therefore sterically more restricted. According to molecular modelling of M. fossilis LDH‐A, the Val304Ile mismatch is located in the subunit contact area of the tetramer, whereas the remaining two mismatches surround the contact area; this is expected to manifest in the kinetic and thermodynamic properties of the assembled tetramer. In warm‐acclimated fish the relative expression between α and β isoforms of the LDH‐A mRNA is around 5 : 1, whereas in cold‐acclimated fish expression of is reduced almost to zero. This indicates that at low temperature the pool of total tetrameric LDH‐A is more homogeneous in terms of α/β‐subunit composition. The temperature acclimation pattern of proportional pooling of subunits with different kinetic and thermodynamic properties of the tetrameric enzyme may result in fine‐tuning of the properties of skeletal LDH‐A, which is in line with previously observed kinetic and thermodynamic differences between ‘cold’ and ‘warm’ LDH‐A purified from weatherfish. Also, an irregular pattern of nucleotide mismatches indicates that these mRNAs are the products of two independently evolving genes, i.e. paralogues. Karyotype analysis has confirmed that the experimental population of M. fossilis is tetraploid (2n = 100), therefore gene duplication, possibly through tetraploidy, may contribute to the adaptability towards temperature variation.