Animals

Crucian carp of mixed sex were caught in a small pond outside of Oslo, Norway, and kept in 750-litre tanks continuously supplied with dechlorinated, aerated Oslo tap water in the aquarium facilities at the Department of Biosciences, University of Oslo, Norway. The photoperiod was kept constant at 12 h/12 h intervals of light/darkness, and water temperature was around 6 °C. The fish were fed commercial carp food (Tetrapond, Tetra, Melle, Germany) on a daily basis. These fish were later subjected to anoxia exposures, as described below. Goldfish and common carp (obtained commercially) were transported to the aquarium facilities at the University of Oslo, where they were sacrificed at the day of arrival.

Anoxia exposures and tissue sampling

Prior to anoxia exposure experiments, randomly selected crucian carp were transferred to experimental tanks with continuous flow-through water supply, and left to acclimate and fast for at least 30 h. Subsequently, these tanks were sealed with tight lids allowing no light to shed through, and the water was continuously bubbled with nitrogen gas (anoxia; AGA) or regular air (normoxia and reoxygenation) through a narrow and 2 m high Plexiglas column to equilibrate the gas with the water. Throughout the experimental period, temperature and oxygen saturation in the tanks were continuously monitored using a galvanometric oxygen electrode (Oxi 340i; WTW, Weilheim, Germany). Water with no detectable oxygen (<0.01 mg O 2 l−1), well below the anaerobic threshold of crucian carp (0.5 mg O 2 l−1)37, was considered anoxic. Upon termination of the exposures, all fish were killed by stunning them with a sharp blow to the head, followed by rapid cutting of the spinal cord. All animal experiments were performed with approval from The National Animal Research Authority of Norway (permit nr 12007), and all methods involving research animals were performed in accordance with relevant guidelines and regulations. No death was observed during the experiment.

Anoxia exposure series

In anoxia exposure series I, randomly selected crucian carp (49 ± 2 g) were divided into four experimental groups (n = 13/group): 7 days of normoxia (N7), 1 day of anoxia (A1), 7 days of anoxia (A7) and reoxygenation (7 days of anoxia followed by 6 days of normoxia; R), and kept in 25 l tanks. Anoxic exposures in this series were conducted at 6.5 ± 0.3 °C during January on fish caught during the previous summer. Tissues were immediately dissected in the following order: (a) brain (b) heart (c) liver, (d) red skeletal muscle and (e) white skeletal muscle, and snap-frozen in liquid nitrogen within 2 min of the initial handling. Tissues were stored at -80 °C until further use in cloning, qPCR or Western blotting experiments.

Anoxia exposure series II: In anoxia exposure series II, randomly selected crucian carp (16 ± 1 g) were divided into three experimental groups (n = 3/group): 7 days of normoxia at 4 °C (N7), 1 day of anoxia at 4 °C (A1) and 7 days of anoxia at 4 °C (A7). Fish acclimated at 4 °C were kept in a 15 l tank with eight compartments of 1.5 l each that were housed in a cold room. Immediately after death, a red skeletal muscle strip (20 mm) from each individual was dissected free from the white muscle and transferred to a chamber containing 0.1% glutaraldehyde and 4% paraformaldehyde in phosphate-buffered saline (PBS). The length of the muscle was maintained by two insect pins during the fixation process. After two hours, a 1 mm3 block was cut (with one side containing the skin) and transferred to a tube containing the same fixatives and stored at 4 °C for later use in electron microscopy studies.

Obtaining sequences for the pyruvate dehydrogenase complex and alcohol dehydrogenase

To our knowledge, none of the genetic components of the pyruvate dehydrogenase complex or alcohol dehydrogenase in the Carassius lineage had been characterized prior to these experiments. Consequently, cloning and sequencing were demanded in order to design species-specific primers for quantitative real-time RT-PCR (qPCR) for Carassius and common carp. Primers were designed from sequences of zebrafish (Danio rerio), belonging to the same family as Carassius (Cyprinidae), using Primer338, and were synthesized by Invitrogen (Invitrogen, Carlsbad, CA, USA). The GSPs are listed in Supplementary Table S4. The primers were designed in regions displaying high degree of conservation between zebrafish and other vertebrates, for which sequences were retrieved from the National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov/) and Ensembl Genome Browser (www.ensembl.org/index.html) databases. Sequences were aligned using GeneDoc (version 2.7; http://www.psc.edu/biomed/genedoc) and ClustalX version 2.0.1239.

For cloning, total RNA was extracted from normoxic tissues of Carassius and common carp using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and an electric homogenizer (Ultra-Turrax T8, IKA, Staufen, Germany). Quality and quantity of the total RNA were assessed using a 2100 BioAnalyzer with RNA 6000 Nano Lab Chip Kit (Agilent Technologies, Palo Alto, CA, USA) and NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Rockland, DE, USA). All samples passed these validity checks. All procedures were carried out according to the manufacturer’s protocols. Total RNA samples were stored at -80 °C.

One µg total RNA of each sample was then treated with DNase I (DNA-free™ Kit, Ambion Applied Biosystems, Foster City, CA, USA), in accordance with the manufacturer’s protocol, and reverse transcribed using SuperScript ™ III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and 500 ng oligo(dT) 18 in reaction volumes of 20 µl, as described in the manufacturer’s protocol. cDNA samples were diluted 1:30 using nuclease-free water (Ambion Applied Biosystems, Foster City, CA, USA), and stored at -20 °C. PCR was carried out using Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA), as described by others40. PCR products were then ligated into pGEM®-T Easy Vector System I (Promega, Madison, WI, USA) and subsequently transformed into CaCl 2 -competent Escherichia coli (E. coli) cells (TOP10 F; Invitrogen, Carlsbad, CA, USA), and cultured on LB plates containing ampicillin and IPTG/X-gal (Promega, Madison, WI, USA). Positive colonies were checked for inserts of correct size using PCR, and at least eight clones of each gene were cleaned with ExoSAP-IT (Affymetrix, Cleveland, OH, USA) and sequenced (ABI-lab, University of Oslo, Norway), using T7 primers (Invitrogen, Carlsbad, CA, USA). Efforts were made to discover all potential sequences for the genes of interest and variants thereof. Consequently, for each gene, a minimum of seven primer pairs resulted in products that were sequenced. All procedures were carried out in accordance with the manufacturer’s protocol.

Expressed sequence tags (ESTs) obtained from cloning were analysed and paralogs identified. Paralogs are designated with a subscript value. In crucian carp, sequencing analyses revealed three paralogs of the E1α enzyme (PDHE1α 1–3 ), two paralogs of the E1β enzyme (PDHE1β 1–2 ), E2 enzyme (PDHE2a-b) and the E3 enzyme (PDHE3a-b) and a singleton of E3BP. Partial sequences of genes encoding PDHE1α, PDHE1β and PDHE2 were retrieved from tissues of goldfish and common carp, using the same abovementioned methods. Resulting genes were denoted in accordance with nomenclature given for crucian carp. Additionally, three paralogs were retrieved for ADH8a (ADH8a1–3) in tissues from crucian carp. Partial and full-length sequences were submitted to Genbank (NCBI; www.ncbi.nlm.nih.gov/). Accession numbers are enlisted in Supplementary Table S4.

Full-length sequences of PDHE1α 1 , PDHE1α 3 , PDHE1β 1 , PDHE1β 2 , PDHE2a, PDHE2b, ADH8a1, ADH8a2 and ADH8a3 from crucian carp were obtained using Rapid Amplification of cDNA Ends (RACE), performed on mRNA purified from total RNA using Dynabeads mRNA Direct Kit (Invitrogen, Carlsbad, CA, USA) and using SMART RACE cDNA Amplification kit (Clontech Laboratories Inc., Mountain View, CA, USA), as described by others41. GSPs for RACE were designed from sequences obtained from cloning using Primer3, and were synthesized by Invitrogen (Invitrogen, Carlsbad, CA, USA). Primers are enlisted in Supplementary Table S4. RACE PCR was carried out on RACE-ready cDNA using Advantage 2 Polymerase (Clontech Laboratories Inc., Mountain View, CA, USA) and the following hot-start PCR program: 1) 94 °C for 30 sec. 2) 72 °C for 3 min, 3) repeat steps 1–2 4x, 4) 94 °C for 30 s, 5) 70 °C for 30 s 6) 72 °C for 3 min, 7) repeat steps 4–6 4x, 8) 94 °C for 30 s, 9) 68 °C for 30 s, 10) 72 °C for 3 min, 11) 24 repeats of steps 8–10. Cloning and sequencing of all RACE products were carried out as previously described.

Three-dimensional structure models of the crucian carp PDHE1 tetramers of different combinations of paralogs were constructed using Swissmodel (www.swissmodel.expasy.org) and the human crystal structure Protein Databank ID 3EXE42; as template. Additionally, a model of the interactions between the E1β dimer and the peripheral subunit binding domain (PSBD) of the E2 monomer was made using the bacterial structure from Bacillus stearothermophilus (PDB ID 1W85)43. The usability of this template was validated by inspecting the superposition of 3EXE and 1W85 (Supplementary Fig. S10). The resulting model of the interaction surfaces in crucian carp were visualized in PyMOL (The PyMOL Molecular Graphics system, version 1.5.0.4, Schrödinger, LCC), and is presented in Fig. 3A.

A three-dimensional model of ADH8a1 and ADH8a3 from crucian carp was constructed with the crystal structure from Baltic cod (Gadus morhua callarias; Protein Databank ID 1CDO)44 as template using Swissmodel (www.swissmodel.expasy.org), aiming at elucidating mutations that could result in altered kinetics. The resulting model was visualized in PyMOL (The PyMOL Molecular Graphics system, version 1.5.0.4, Schrödinger, LLC (Supplementary Fig. S9).

Quantification of gene expression using qPCR

For qPCR, total RNA was extracted from brain, heart, liver, red and white skeletal muscle of crucian carp (anoxia series I), and from normoxic brain and red skeletal muscle of goldfish and common carp, using 15 µl TRIzol/mg, in accordance with the detailed protocol outlined by Ellefsen et al.22. Prior to homogenization, 20 pg external RNA control gene (mw2060) was added per mg tissue. Quality and quantity of the extracted total RNA was assessed using 2100 Bioanalyzer and NanoDrop 2000 UV-Vis Spectrophotometer, as previously described. All samples passed these control tests, and were subsequently DNase I treated and reverse transcribed into cDNA using oligo(dT) 18 and SuperScript III, as previously described. The final cDNA was eluted 1:30 using nuclease-free water (Life Technologies, Carlsbad, CA, USA) and stored at −20 °C. All procedures were carried out according to the manufacturer’s protocols.

qPCR, using LightCycler480 (Roche Diagnostics, Basel, Switzerland) was performed to assess the abundance of the mRNA of PDHc components in the tissues of the abovementioned species, while mRNA abundance of ADH8a1–3 was quantified in skeletal muscles and liver from crucian carp only. qPCR primers were designed based on the sequences obtained by cloning; when possible, primers were designed to span exon-exon transitions. For each target gene, a minimum of three primer pairs for each gene were tested and their products sequenced. The pair that displayed the highest efficiency, lowest crossing point (Cp) value and most distinct melting curve was adopted. Primers were designed as previously described, and were synthesized by Thermo Fisher Scientific (Waltham, MA, USA). Primers are enlisted in Supplementary Table S4.

qPCR was carried out using LightCycler 480 SYBR Green I Master Kit (Roche Diagnostics, Basel, Switzerland) in a reaction volume of 10 µL, using SYBR Green I Master, primers (100 nM; annealing temperature of 60 °C), cDNA (3 µL 1:30 diluted) and sealed LightCycler® 96 multiwell plates (Roche Diagnostics, Basel, Switzerland). The following qPCR program was used: 1) 95 °C for 10 min, 2) 95 °C for 10 s, 3) 60 °C for 10 s, 4) 72 °C for 13 s 5) repeat steps 2–4 42x. All reactions were carried out in duplicate. In the final analysis of gene expression, the mean values of all qPCR reactions were used. All primers were represented in each plate (for PDHc or ADH, respectively), allowing for subsequent analysis of gene-family profiling45, 46. All procedures were carried out according to the manufacturer’s protocol. The relative expression of target genes were calculated from the priming efficiency (E) and the crossing point (Cp) value, and were normalized to the external reference gene (mw2060) 22. Cp values were calculated for each individual sample using the second derivative maximum method, and were obtained using the LightCycler480 Software (Version 1.5; Roche Diagnostics, Basel, Switzerland). Efficiencies were initially calculated for each individual qPCR reaction using the LinRegPCR software47, average priming efficiencies (E mean ) calculated separately for each primer pair in each tissue were utilized in the final calculations.

Phylogenetic analyses of PDHc E1 and E2 subunits

Nucleotide sequences from other selected species were gathered from the GenBank (NCBI; www.ncbi.nlm.nih.gov/), The Gene Indices (TGI (formerly TIGR); http://compbio.dfci.harvard.edu/tgi/) and Ensembl Genome Browser (www.ensembl.org/index.html) databases and aligned with the PDHc sequences from crucian carp, goldfish and common carp found in the present study using ClustalX version 2.0.1239.

The evolutionary histories of E1α, E1β, and E2 sequences were inferred using both nucleotide sequences and translated amino acid sequences by both Maximum Parsimony and Maximum Likelihood methods in MEGA5.248. In the latter case the best sequence evolution model was determined by using the in-built model selection feature in MEGA5.2. For each subunit, the method generating the most highly resolved tree was chosen. Thus, Supplementary Fig. S7 gives the most parsimonious nucleotide tree for E1α and the tree with the highest log likelihood for both E1β and E2. The percentage of replicate trees in which the associated taxa cluster together in the bootstrap test (1000 replicates) are shown next to the branch points49. Accession numbers for sequences included in the analysis are listed in Supplementary Table S11.

Phylogenetic analysis of ADH8a genes

Amino acid sequences from other selected species were gathered from the GenBank (NCBI; www.ncbi.nlm.nih.gov/), The Gene Indices (TGI (formerly TIGR); http://compbio.dfci.harvard.edu/tgi/) and Ensembl Genome Browser (www.ensembl.org/index.html) databases and aligned with the ADH8a sequences from crucian carp described in the present study using ClustalX version 2.0.1239. The evolutionary histories of ADH classes I and III were inferred using the Maximum Likelihood method based on the Jones-Taylor-Thornton (JTT) matrix model50 in MEGA5.248, and the resulting tree is shown in Supplementary Fig. S8. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories ( + G, parameter = 1.5325)). The percentage of replicate trees in which the associated taxa cluster together in the bootstrap test (500 replicates) are shown next to the branch points49. The phylogenetic analysis was based on 85 amino acid sequences with a total of 302 positions in the final dataset. All positions containing gaps and missing data were eliminated. Accession numbers for sequences included in the analysis are enlisted in Supplementary Table S11.

Western blot analysis of phosphorylation status of PDHE1α

Frozen crucian carp brain and red skeletal muscle samples from all groups derived from anoxia series I (n = 7 for all groups) were placed in an ice-cold lysis buffer containing 210 mM sucrose, 40 mM NaCl, 30 mM HEPES, 5 mM EDTA, 100 M sodium orthovanadate and 1% Tween-20. Additionally, one tablet complete EDTA-free protease inhibitor (Roche Diagnostics GmbH, Mannheim, Germany) and 250 µL phosphatase inhibitor cocktail 1 were added to 50 mL of extraction buffer (corresponding to 20 mg tissue/mL extraction buffer). Subsequently, the tissue was homogenized using a Polytron PT 1200 homogenizer. Lysates were further centrifuged at 12 000 * g/4 °C/10 min in order to remove any insoluble material. Next, 1% sodium dodecyl sulphate (SDS) was added to the supernatant, and the samples were vortexed for 15 min at room temperature, and later snap-frozen in liquid N 2 and stored at -80 °C for further analysis.

Protein content in the samples was quantified using Micro BCA protein assay kit (Pierce, Rockford IL). Protein lysates from red muscle (1 µg/lane) and brain (10 µg/lane), respectively, were separated using 10% SDS-PAGE gels and electrophoretically transferred onto a hybond-P membrane (Amersham Biosciences Europe, Freiburg, Germany). To block unspecific binding, membranes were incubated for 2 h in 5% skimmed milk in Tris-buffered saline (20 mM Trizma-base and 140 mM NaCl) with 0.1% Tween-20 (TBST) and subsequently incubated over night with primary antibodies towards either phosphorylated PDHE1α (pSer293 (site 1) (AP1062; Merck, Darmstadt, Germany; 1:10000 for red muscle; 1:1000 for brain51) or PDHE1α (AV48137; Sigma-Aldrich; 1:1000). The antibody against pSer293 (site 1) was chosen as phosphorylation of any of the three phosphorylation sites is sufficient to ablate enzymatic activity of the PDHc52, with site 1 being the most frequent target53, 54.The epitope of the phospho-Ab was found to be intact in crucian carp. Indeed, the sites of phosphorylation have been shown to be invariant in most vertebrates, supporting the suitability of this antibody for a vast selection of species51. After washing with TBST, the membrane was then incubated for 1 h with secondary antibody (goat anti-rabbit; 1:2500, SouthernBiotech, Birmingham, AL, USA), conjugated to horseradish peroxidase. Subsequently, the immunoreactions were visualized by chemiluminescence (ECL + , Amersham Biosciences Europe) and documented using ImageReader LAS-1,000 (Fujifilm Europe). Densitometry of each band was investigated using ImageQuant (Amersham Biosciences Europe). Membranes were stained using Coomassie Brilliant blue (Bio-Rad Laboratories), and scanned (CanonScan Lide 35). Equal loading was investigated using ImageQuant. Membranes displaying uneven blotting were removed from subsequent analyses. The excess E1 transcript level in skeletal muscle was also reflected at the protein level, as Western blot analyses of E1α in lysates from red muscle (Fig. 3A) and brain (Fig. 3B), when diluted to the same degree to avoid saturation, only detected the protein in red muscle (see also Methods and Supplementary Fig. S6 for more details). In contrast, in the anoxia intolerant common carp, overall transcript levels of E1α mRNA were similar between red muscle and brain, with transcription levels being similar to those observed for E1α 1–2 in Carassius brain, and very much below those seen in Carassius skeletal muscle (Supplementary Fig. S3).

Electron microscopy and immunolabeling

Samples for electron microscopy analysis were prepared according to the protocol by Slot and Geuze55. In short, a fixed 1 mm3 tissue block was infiltrated with 2.3 M sucrose over night at 4 °C and subsequently frozen in liquid nitrogen. Ultrathin cryo-sections (60 nm) were cut in a Reichert Ultracut S microtome equipped with a Leica EMFCS cryo-box and using a Diatome Cryo Immuno knife. Sections were picked up with a droplet containing 2.3 M sucrose and 2% methylcellulose (1:1 mixture), placed on copper grids and stored at 4 °C until further use. For immunolabeling, the grids with sections were washed on droplets of PBS (4 × 5 min), quenched with glycine (0.1% in PBS, 2 × 5 min) and blocked with 1% BSA in PBS (1 × 5 min). Sections were incubated with primary antibody (the same antibodies as were used for the western blot analysis; dilution 1/50 in PBS + 1% BSA) before incubation with 10 nm gold particles conjugated to protein A (PAG; diluted in PBS + 1% BSA). Subsequently, sections were washed in PBS + 0.1% BSA and contrasted with Uranyl acetate by placing the grids on ice on droplets containing a Methyl cellulose-Uranyl Acetate mixture. Three randomized pictures were taken with a Philips CM200 transmission electron microscope from each fish from each exposure group in anoxia series II (N14 °C, N4 °C, A1 and A7). Gold particles were quantified per µm2 mitochondria, which in turn had been quantified by a person blinded to the experimental groups. This was performed on three pictures taken randomly from each fish (n = 3 in each treatment). All mitochondria within each picture were quantified (ranging from 3 -11 mitohondria in each).

Chemicals and reagents

Unless otherwise stated, chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Statistics

For statistical evaluation of qPCR data sets, one-way analysis of variance (ANOVA) with Holm-Sidak post hoc test was performed using SigmaPlot (version 12, Systate Software Inc., San Jose, CA, USA) to compare mRNA expression levels of individual target genes between experimental groups. Data sets illustrating gene-family profiling were arcsine-transformed prior to analysis of mRNA expression levels between experimental groups. All statistical tests were performed separately for each tissue. All data are expressed as means ± standard error of the mean (S.E.M.), unless otherwise stated. Graphs were made using SigmaPlot (version 12, Systate Software Inc., San Jose, CA, USA). Data from Western blots were analysed for statistical variations using GraphPad Prism (version 5; GraphPad Software, La Jolla, CA, USA), and ANOVA with Bonferroni multi comparisons test were carried out on all data sets. Results are presented as means ± standard deviations. Electron microscopy data sets were analysed with ANOVA and Dunnet’s multiple comparison test, and the results are presented as means ± standard deviations. For all statistical tests, the confidence level was set at P < 0.05.

Data availability

Sequences derived from cloning have been deposited to GenBank, and their accession numbers are enlisted in Supplementary Tables S4 and S11.