Research Article Transcriptome of the Deep-Sea Black...

Research ArticleTranscriptome of the Deep-Sea Black ScabbardfishAphanopus carbo (Perciformes Trichiuridae) Tissue-SpecificExpression Patterns and Candidate Genes Associatedto Depth Adaptation

Sergio Stefanni12 Raul Bettencourt2 Miguel Pinheiro3 Gianluca De Moro4

Lucia Bongiorni5 and Alberto Pallavicini4

1 ISSIA-CNR Via de Marini 6 16149 Genova Italy2 LARSyS Associated Laboratory amp Centre of IMAR of the University of the Azores Department of Oceanography and FisheriesRua Prof Frederico Machado 4 9901-862 Horta Azores Portugal

3 School of Medicine University of St Andrews North Haugh St Andrews KY16 9TF UK4Department of Life Sciences University of Trieste Piazzale Valmaura 9 34148 Trieste Italy5 Institute of Marine Sciences National Research Council (ISMAR-CNR) Castello 2437F 30122 Venezia Italy

Correspondence should be addressed to Sergio Stefanni sergiostefannigeissiacnrit

Received 25 May 2014 Accepted 19 July 2014 Published 17 September 2014

Academic Editor Elena Pasyukova

Copyright copy 2014 Sergio Stefanni et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Deep-sea fishes provide a unique opportunity to study the physiology and evolutionary adaptation to extreme environments Wecarried out a high throughput sequencing analysis on a 454 GS-FLX titanium plate using unnormalized cDNA libraries from sixtissues ofA carbo Assemblage and annotationswere performed byNewbler and InterProPfam analyses respectivelyThe assemblyof 544491 high quality reads provided 8319 contigs 556 of which retrieved blast hits against the NCBI nonredundant database orwere annotated with ESTscan Comparison of functional genes at both the protein sequences and protein stability levels associatedwith adaptations to depth revealed similarities between A carbo and other bathypelagic fishes A selection of putative genes wasstandardized to evaluate the correlation between number of contigs and their normalized expression as determined by qPCRamplificationThe screening of the libraries contributed to the identification of new EST simple-sequence repeats (SSRs) and to thedesign of primer pairs suitable for population genetic studies as well as for tagging and mapping of genes The characterization ofthe deep-sea fish A carbo first transcriptome is expected to provide abundant resources for genetic evolutionary and ecologicalstudies of this species and the basis for further investigation of depth-related adaptation processes in fishes

1 Introduction

The deep-sea (gt1000m depth) covers about 70 of theEarthrsquos surface representing one of the last large unexploredareas on the planet Only within the last few decades thetechnology has advanced sufficiently to reach the deep-sea effectively revealing unexpected high levels of biodi-versity and extremely diverse habitats (canyons cold seepshydrothermal vents deep-water coral reefs mud volcanoesseamounts and trenches) of significant conservation interestand potential high economic values Deep-sea environmentsare characterized by extremely high hydrostatic pressures

(1MPa every 100m) lack of light and low temperatures(down to 1-2∘C)Therefore fish as well as any other organismliving in the deep-sea had to adapt to tolerate conditions ofthis extreme habitat [1]

First studies on adaptation to high pressure and lowtemperatures are dated back in the lsquo70s and they report com-parison of common proteins present in shallow and deep-water fishes [2 3] Key enzymes in muscle tissues that exhibitadaptive differences among species at different depths arethe lactate dehydrogenase (LDH) and malate dehydrogenase(MDH) presenting differences in structural stability (reviewsin [4ndash6]) More recent studies on evolutionary adaptation

Hindawi Publishing CorporationInternational Journal of GenomicsVolume 2014 Article ID 267482 21 pageshttpdxdoiorg1011552014267482

2 International Journal of Genomics

of functional genes to high pressure report unique aminoacid substitutions in 120572-skeletal actin and myosin heavy chain(MyHC) proteins in deep-sea fishes [7ndash10] For deep-seaspecies inhabiting hydrothermal vents and cold seeps envi-ronments characterized by high pressure chronic hypoxiaand high concentrations of toxic compounds molecular andfunctional adaptation of hemoglobins (Hbs) are reviewedin Hourdez and Weber [11] Despite these studies ourknowledge on wide scale gene expression patterns in deep-sea fish remains elusive

The black scabbardfish Aphanopus carbo (Lowe 1839) isa bathypelagic species belonging to the Trichiuridae familyand is distributed in temperate-cold Atlantic waters at depthsbetween 200 and 1800m [12 13] A carbo represents acommercially valuable species for several regions of theIberic peninsula especially in Madeira where catches havereached up to 1000 tons per year [14] amounting to ca55 of the total landings Recently this species has becomeincreasingly targeted by Portuguese French and Irish fishingfleets ([15] and literature therein) and fishery data haveshown a constant decline in population [16]The informationavailable on the biology maturity spawning and growth ofthis species [17 18] is scattered Recent studies are reporting apanmictic distribution of this species in the NE Atlantic withmultiple breeding sites at low latitudes [19] It is also worthmentioning that in southern locations this species lives insympatry with A intermedius a close related species withvery similarmorphology [20] therefore attracting interest forevolutionary studies

High-throughput sequencing approaches applied to tran-scriptomics now provide a global perspective on taxonomicand functional profiling of genes expectedly expressed underthe influence of environmental conditions in which theseorganisms live Also known as next-generation sequencingthese techniques allow for a massive characterization ofexpressed sequence tags (ESTs) providing an overview ofthose genes expressed in a given tissue at any given time[21] In silico analyses of massive gene libraries may serveseveral interests among others For instances from discoveryand identification of new genes characterization of geneexpression to development of novel genetic markers forquantitative trait locus (QTL) and population of genomicanalysesThe breadth of next-generation sequencing applica-tions extends over a variety of biological questions includingthose addressing pertinent questions regarding a speciesrsquoecology life history and evolution [22 23]

Previous studies regarding transcriptome sequencing andgene expression studies in deep-water species were mostlylimited to hydrothermal vents invertebrates [24] microbialcommunities in hydrothermal plumes [25] deep-sediments[26] and in the water column [27] leaving vertebrates speciesvirtually under-represented The present work represents apioneer study for deep-sea fishes providing new insights intothe role of differential gene expression on the environmentaladaptation of deep-sea black scabbardfish

Here we describe the assembly and annotation of thetranscriptome of A carbo obtained by sequencing mRNAlibraries of six tissues (spleen brain heart gonads liverand muscle) and explore functional genes whose sequence

might be associated to depth adaptation Additionally wetested the correlation of selected candidate genes comparingthe number of contigs against the gene expression normalizedto a relative value of 10 as determined by qPCR amplifi-cation Furthermore the screening of the libraries allowedthe identification of newEST-simple-sequence repeats (SSRs)and the design of primer pairs suitable for population geneticstudies as well as tagging and mapping of genes

2 Methods

21 Fish and Tissue Samples Specimens of Aphanopus carbo(two males and two females) were collected in 2009 onboardof the RV ldquoArquipelagordquo A carbo were fished at depth range1100ndash1250m using deep-water long-lines in proximity of theCondor Seamount located approximately at 15 nm SW ofthe island of Fayal (Azores Portugal) The four specimensused in this study were caught on the same longline set andonce onboard the freshly caught animals were dissected andportions (or complete organs) of spleen brain heart gonadsliver and muscle tissues were preserved in formamide solu-tion and kept at minus20∘C until RNA extraction was performed

To validate the correct identification of the species a smallportion ofmuscle tissuewas also preserved in 95 ethanol formolecular screening following Stefanni et al [28] protocols

22 RNA Extraction and Sequencing Total RNA wasextracted from 20 to 40mg of each of the six preservedtissues of a pool of four A carbo individualsusing theRiboPure kit (Ambion Applied Biosystems) Quantity andpurity of the RNAwas determined on a 14 agarose-MOPS-formaldehyde denaturing gels and by assessing the 119860

260280

and 119860260230

ratios using the NanoVue spectrophotometer(GE Healthcare) Poly-A RNA was extracted from 15 120583gof each total RNA sample using the Poly(A)Purist mRNAPurification kit according to manufacturerrsquos instructions(Ambion Applied Biosystems) mRNAs were transcribedinto cDNA utilizing Mint-2 cDNA synthesis kit (Evrogenhttpwwwevrogencom) according to manufacturerrsquosinstructions for NGS platforms Six cDNA libraries wereconstructed from mRNA of individual pools of tissues andsequenced in a single 454 GS FLX Titanium run Each of thecDNA libraries was characterised by unique sequence tags(MIDs) that allowed to trace back the sequences generatedfrom single tissues after assembly

cDNAs were sheared by nebulization to yield randomfragments approximately 500ndash800 bp in length by applying30 psi (21 bar) of nitrogen for 1 minute on 4 120583g of eachlibrary The distribution of fragments was verified on aBioAnalyser DNA 7500 LabChip (Agilent Technologies)The fragmented cDNA sample was end-repaired with T4DNA polymerase and T4 polynucleotide kinase and adaptorsequences ligated according to the manufacturerrsquos instruc-tions [29] The fragments were immobilized onto strepta-vidin beads and nick-repaired with Bst polymerase ThecDNA fragments were denaturated with alkali to yield singlestranded cDNA (sscDNA) library Quality of the library wasassayed on a BioAnalyser RNA 6000 Pico LabChip (AgilentTechnologies) and quantity measured by spectrofluorimetry

International Journal of Genomics 3

with the Quant-iT RiboGreen RNA Assay kit (Invitrogen) Atitrationwas set up at 1 2 4 and 8 copies per bead (cpb) in theclonal amplification by emulsion PCR to optimize yield andsequence quality The percent enrichment of beads carryingthe sscDNA was determined and the amount of library inputcalculated to 18 A large scale emulsion PCR was set-up based on the previous value and sequenced at Biocant(Cantanhede Portugal) using the 454 GS FLX Titaniumpyrosequencing on a full 70X75 PicoTiterPlate according tomanufacturerrsquos instructions (Roche)

23 Bioinformatic Analyses High quality reads were assem-bled using Newbler ver 26 (Roche 454) sequence analysissoftware All reads were identified and grouped by theirunique MIDs to the tissue of origin Trimming and maskingthe polyAs was a common procedure for the assembling tool

The assemblage is characterized by read overlaps andmultiple alignments made in nucleotide space Consensusbase-calling and quality value determination for contigsare performed in flow space The use of flow space indetermining the properties of the consensus sequenceresults in an improved accuracy for the final base-calls Theimplementation of this software was performed using defaultparameters Assembled contigs were annotated throughsequence similarity searches against the National Centrefor Biotechnology Information (NCBI) nonredundant (nr)protein database using the BLASTx [30] with a cut-offcriterion of an expect-value (119890-value) lt 10minus6 The contigsthat did not find a hit were further processed with ESTScan(httpwwwchembnetorgsoftwareESTScan2html) Thetwo assemblages of amino acid sequences resulting from theBLASTx searches at high level of stringency and the ESTScanwere processed by InterProScan for functional annotationof transcripts applying the function for the mapping of geneontology (GO) termsThe GOmethod classifies genes withina hierarchy using a systematic nomenclature of attributesthat can be assigned to all gene products independentlyfrom the organism of origin To reduce the redundancyin the consensus sequences which correspond to the samegene we used BLASTClust to detect similar assemblieswith 95 identity and 90 coverage All the results fromboth assemblage methods were loaded into a SQL databasedeveloped for this purpose

To validate the accuracy of the assembly the result-ing contigs were compared to previously sequenced tran-scriptomes of 6 teleosts including Danio rerio Gasterosteusaculeatus Oreochromis niloticus Oryzias latipes Takifugurubripes and Tetraodon nigroviridis using tBLASTn [30] tofind protein homologs at two levels of stringency (119890-value lt10minus3 and 119890-value lt 10minus10)To identify protein conserved domain specific for each

tissue analysed a new annotation was performed withHmmer against the Pfamdatabase (ver 250) Protein domainrepresentativeness for each tissue was obtained comparingprotein domain abundance in a particular tissue versus all thetissues compiled together using a hypergeometric test

24 cDNA Synthesis and qPCR Validation Tests Fresh cDNAwas synthesised from the six mRNAs that were used for

pyrosequencing cDNA synthesis was performed usingprimers with oligo(dT) and the ThermoScript RT-PCR Sys-tem (Invitrogen) following the manufacturerrsquos instructions

A set of 28 genes were selected including candidates thatwere tissue-specific and genes that were encountered in thetissue expressed at similar as well as at different amountsin all the six libraries with the aim of covering most of thepossible expression scenarios within the dataset Frequenciesof contigs for all candidates genes in themRNA libraries wereobtained by detecting orthologous gene sequences using theBLAST tool included in the A carbo database

For the design of all qPCR primers (Table 1) we usedthe web interfaceNCBI Primer-BLAST (httpblastncbinlmnihgov) Alignments of the sequences provided by theoutput from the internal blast search were used to select allprimer sets

Gene expression calculated as relative expression wasdetermined by means of real-time PCR using the CFX96(Bio-Rad) Primer concentrations and sample dilutions wereoptimized to meet highest efficiency in the PCR reactionin a total reaction volume of 20 120583L Fluorescent signal wasdetermined by the addition of SsoFast EvaGreen Supermix(Bio-Rad) which was included in the cocktail accordinglyto manufacturerrsquos instructions Baseline and threshold cyclewere always set to automatic in the sequence detectionsoftware CFXManager (Bio-Rad) All plates contained a ldquonotemplate controlrdquo (NTC) and each sample was tested in dupli-cate Cycling conditions for gene amplifications were 95∘Cfor 3min followed by 35 cycles of 95∘C for 10 s 56∘C for 10 sand 68∘C for 15 s An additional protocol for melting curvesanalysis included a cycle at 95∘C followed by a progressivereading of fluorescence for every cycle from 65∘C to 95∘Cfor 5 s at intervals of 05∘C Gene expression normalized toa relative value of 10 for all the genes selected and for eachtissue was compared to the contigs frequencies generatedby the assembly platform to determine the significance ofcorrelation between qPCRs values and 454 sequencing readsfrom unnormalized cDNA libraries

25 Characterization of Depth-Related Functional Genes Thepredicted amino acid sequences of functional genes of Acarbo possibly related to depth adaptations were comparedto those deposited in NCBI database searching for homolo-gies We aligned and compared translated sequences oflactate dehydrogenase (LDH-A andLDH-B) cytosolicmalatedehydrogenase (MDHc) hemoglobins (Hb-A and Hb-B)actin (ACTA1) and myosin heavy chain (MyHC) Proteinand nucleotide sequences were aligned using Clustal X [31]while sequence analysis and phylogenetic inferences wereperformed using CLC Main Workbench (v 682 CLC Bio)The neighbor-joining (NJ) algorithm [32] was implementedto construct a phylogenetic tree using HKY substitutionmodel and attributing a gap penalty of 10 The support forinternal branches was assessed using the bootstrap [33] with1000 replicates

Nucleotide alignments and ML trees built implementingthe most appropriate substitution model under the AkaikeInformation Criterion (AIC) were used in the program


Table 1 List of targeted genes using qPCR primer sets specifically designed for this study size for each of the product and NCBI accessionnumber for all the EST sequences

Gene Primer name Primer Sequences (51015840-31015840) Size(bp) NCBI Accession

Elongation factor 1-beta EF-1B L GCTTGGACATGTCGGTCTCGTC 229 bp All gs454 000396EF-1B H GTGGCTGACACCACATCTGGC

Ras-related GTP-binding protein A Rab-1A L AGTAGCCGTTCCACCTTGTCGG 247 bp All gs454 000598Rab-1A H TGCCAAGAAACCGTACGTGGGA

Basic Transcription Factor 3-like 4 BTF3 L CCCAAAGTTCAGGCCTCCCTGT 273 bp All gs454 000873BTF3 H TCATGTGCGTCAGTTCGCTTCG

CuZn Superoxide Dismutase SOD-1 L AAACGTGACTGCAGGAGGGGAT 240 bp All gs454 000925SOD-1 H CAGTGCTCCTGCTCCATGTTCG

2-Cys Peroxiredoxin PRDX1 L CCGATAACCTCGCAGCCGATAC 243 bp All gs454 000558PRDX1 H ACAGTCATTTGCCACCAGCATCA

Heat Shock Protein 90 HSP90 L TGACGATGTCCCCACAGATGAGG 221 bp All gs454 000008HSP90 H GCAACACTGGTCCACCACACAAC

Ferritin heavy subunit Ferr L CCTGCAGCTTGAGAAGAGCGTC 203 bp All gs454 000681Ferr H CAAACAGGTACTCGGCCATGCC

1205722

Globin Hb-A L AAATTGTTGGCCATGCGGAGGA 208 bp All gs454 001919Hb-A H CTGAGGTTCAGCAGACCTGCCT

1205732

Globin Hb-B L TCGTCTACCCCTGGTGTCAGAG 245 bp All gs454 001018Hb-B H AACCACAATGGTCAGGCAGTCC

Ependymin-1 precursor EPD-1 L CAGGTGTGAGGCAGTGCAGT 230 bp All gs454 000469EPD-1 H ACCCCGATCTCCTCCTGGTG

Fatty acid-binding protein brain BLBP L CAACACTTCTTGGCCGGTTTGG 239 bp All gs454 001220BLBP H GAGAGGAGTTCGACGAAGCCAC

CD63-like protein Sm-TSP-2 TSPAN-8 L TCGCTGGCTGCTCTGAGAAAGA 200 bp All gs454 000381TSPAN-8 H GGTCACGCCGAGCTGTATTCTG

Tropomyosin 4 isoform 1 TRPM-1 L GTGGAGGAGGAGTTGGACCGAG 221 bp All gs454 000222TRPM1 H TTGCGAGCCACCTCCTCGTATT

C-Myc-binding protein MYCBP L CGCCAGTTTACCTGCGTTCCAA 182 bp All gs454 001640MYCBP H GGCCGTCAACAACACCACCTTT

Cathepsin S CTSS L AACAGCCTACCCCTACACAGCC 200 bp All gs454 000156CTSS H TGTACACACCGTGGCGGTAGAA

Transferrin STF-1 L AGCTGCACCAGCTTCACAGTTG 215 bp All gs454 000004STF-1 H AAGGATGGCACCAGACAACCCA

Warm Temperature Acclimationrelated-like 65 kDa protein

HPX L TGATACCGGGTGGAACCTGGTG 207 bp All gs454 000060HPX H GCTGCTGTGGAGTGTCCCAAAG

Betaine HomocysteineS-methyltransferase

BHTM L GGGGGTTCGCTGTTACCAAGTG 194 bp All gs454 000088BHTM H TGTGAGACAGCAGCCTCAGGAG

FUCL1 Fucolectin-1 FUCL1 L CGCAAACCCTTTGGCTGGTGTA 196 bp All gs454 000758FUCL1 H GGCTTTTCCTTGGACTGCCAGG

Aldolase B ALDB L GCCATTGGTCTTGGCCCTGATC 220 bp All gs454 000115ALDB H CGCTGTGCCTGGTATCTGCTTC

Type-4 ice-structuring protein LS-12precursor

ISP LS12 L AAGACCTGACAAACCAGGCCCA 198 bp All gs454 001277ISP LS12 H GGAGGATGGCCTCCATCTGCTT


Table 1 Continued


Alcohol Dehydrogenase 8a ADH L GGCAAGAAGGTGCTGCAGTTCA 228 bp All gs454 000105-6ADH H CATGACTGCAGCCAAACCCACA

Glyceraldehyde-3-phosphateDehydrogenase

GAPDH L GTCAACCACTGACACGTTGGGG 229 bp All gs454 000148GAPDH H CGGCATCATTGAGGGCCTGATG

Lactate Dehydrogenase-A LDH-A L TCTTAACCTGGTGCAGCGCAAC 219 bp All gs454 000149LDH-A H TGGAGCTTCTCGCCCATGATGT

Phosphoglycerate Mutase 2-1 (muscle) PglyM L ACACCTCTGTGCTGAAACGTGC 212 bp All gs454 000309PglyM H CATGGGTGGAGGTGGGATGTCA

Heat Shock Protein 70 HSP70 L CGGTGTTGTGTGCTGGGTGAAA 207 bp All gs454 000005HSP70 H CCACATAGCTGGGTGTGGTCCT

Fructose-bisphosphate Aldolase A FBPA L GGAACCAACGGCGAGACAACAA 208 bp All gs454 002732FBPA H CAATGGGGACGATGCCATGCAT

Phosphoglucose Isomerase-2 PGI L CCACACTGGGCCAATTGTCTGG 217 bp All gs454 000011PGI H GGCCTCCTCTGTGGTCTTACCC

Table 2 Global statistics for each of the nonnormalized libraries using Newbler software

Tissue Spleen Brain Heart Gonad Liver Muscle TotalTotal EST 15034 33337 73263 157275 134523 92788 544491Total bases 3426510 8219500 17647600 37123700 31792600 23342900 129412000Contigs 567 651 1260 3875 1274 626 8319Average contig length 470 619 567 465 612 689 555Contigs 119890-value lt 10minus6 220 420 622 951 617 409 2440ESTscan 584 345 977 2838 634 269 2715No similarity 36 70 109 406 211 74 1128GO annotation 202 338 473 623 509 307 1728InterPro annotation 223 417 610 908 649 395 2395

ldquocodemlrdquo in PAML 4 [34] to assess selective pressure onthose genes for which the complete sequences was availablePositive (or negative) selected sites were defined by the ratiobetween nonsynonymous versus synonymous substitutions(dNdS or 120596) Two models were tested comparatively M1which groups codons in two classes (120596 lt 1 and 120596 = 1)clustering sites under negative or neutral selection and M2which groups codons in three classes (120596 lt 1 120596 = 1 and 120596 gt1) adding a cluster for sites under positive selection to theones defined in M1 Probabilistic measures of how well thesemodels fits the evolutionary relationship of individual geneswere calculated from the likelihood values of fitted modelsand the number of ldquofree parametersrdquo for all genes

Protein stability was estimated using a virtual quantifica-tion software [35] calculating Gibbs free energy in terms ofkinetic and thermodynamic quantities taking into accounteach amino acid contribution for the maintenance of thenative structure of the protein For a protein to maintain itsstability there is a need of sufficient hydrophobic residueswhich will utilize free energy to guide proper folding [35]

26 EST-SSR Resources for Population Genetics Among vari-ous molecular markers simple sequence repeats (SSRs) arehighly polymorphic easier to develop and very useful for

researches such as genetic diversity assessment ThereforeA carbo database was further used to detect such regionsin the transcriptome sequencing data and provide a list ofcombination of primer sets on flanking regions that could beused for population genetic studies To identify EST-SSRs allthe contigs were searched using MISA [36] and for primerdesign we used Primer3 [37]The algorithm of the SSR finderidentifies a good quality repeat when one locus is present withadjacent loci at an up or downstream distance higher than100 bp and parameters were set to locate a minimum of 20 bpsequence repeats di-mers (x12) 3-mers (x8) 4-mers (x5) 5-mers (x5) and 6-mers (x5) Primer design was performedsetting parameters of a minimum size of 20 bp and meltingtemperatures of 60∘C

3 Results and Discussion

31 Sequences Assemblage and Functional Annotations Aftersequence trimming a total of 544491 high quality readswere produced with an average length of 237 bp correspond-ing to 1295Mb A total of 8319 contigs were assembledwith Newbler (ver 26) as high quality consensus sequenceswithout the presence of singletons A summary of ESTdata for each of the six tissues is reported in Table 2 Atotal of 2440 assembled contigs were annotated against


the NCBI nonredundant protein database at the cut-off (119890-value) lt 10minus6 Additional 3843 contigs with no proteinmatches were further processed with ESTScan to find 2715homologous proteins All the contigs were then processedby InterProScan for functional annotation of transcriptsand mapping functional information to gene ontology (GO)terms Of the 5155 amino-acid sequences (out of the 6283totally sequenced) 1728 could be annotated within theGO hierarchy (Figure 1) while 2395 could be annotatedaccordingly to InterProScan The complete GO mappingof A carbo for individual tissues can be accessed on thededicated online database (httptranscriptomicsbiocantptAphanopusCarbo) The largest proportions of GO func-tional categories are of similar proportion to the unnormal-ized libraries of Tilapia [38] In the Cellular Components GOgroup (Figure 1(a)) the genes involved in cell and cell partfunctional categories corresponded to the largest percentageof the pie chart (2815 each) In the Molecular FunctionGO group (Figure 1(b)) almost half of the total genes wereinvolved in binding (4740) followed by those related tocatalytic activity (2753) Finally for the Biological ProcessesGO group (Figure 1(c)) the largest portion of the genes wereinvolved in metabolic processes (3345) tightly followed bythe category of genes linked to the Cellular Process (3193)

To further validate the accuracy of the A carbo assemblywe compared our dataset to the transcriptomes of six otherprior sequenced teleosts including Danio rerio Gasterosteusaculeatus Oreochromis niloticus Oryzias latipes Takifugurubripes and Tetraodon nigroviridis Similarity searches wereperformed comparing our assembled contigs against each ofthe available transcriptomes using tBLASTn [30] The resultshighlighted strong similarity between the transcriptomes ofA carbo and other teleosts indicating that about 30 of thetotal contigs matched protein homologs (119890-values lt 10minus3ranging between 2268 and 2457) and that our assemblypresents the highest similarity (by little margin) with thetranscriptome of Gasterosteus aculeatus (119890-value lt 10minus10 =2 223) (Table 3)

A total of 1639 (20) transcripts were annotated by Pfamprotein domainsmatches and the set of protein domains char-acterizing each single tissue was identified by hypergeometrictest (Table 4)

32 qPCR Assays and Validation Tests Quantitative PCRassays were performed using cDNA samples at 1 400 dilution(up to 100 ng approximately in the qPCR master mix)conditions at which the PCR resulted to be more efficientWe tested seven series of dilutions ranging from 1 50 to1 1000 Based on optimized qPCR conditions all targetedcDNAs were tested across all tissues for the complete set ofgenes selection used in this study Normalized expressionto a relative value of 10 for 28 genes is shown in Figure 2(see Figure S1 in Supplementary Material available online athttpdxdoiorg1011552014267482)

Statistical tests were performed to identify the correlationbetween the mean value of normalized expression and arelative value of 10 as determined by qPCR against contigfrequencies using Pearsonrsquos 119903 coefficient and its significance

Table 3 Similarity search for unigenes comparing transcriptomesof other teleosts against Aphanopus carbo using two levels ofstringency

Species Sequencesavailable

A carbo119890-value lt 10minus3


Danio rerio 42787 2457 2199Gasterosteusaculeatus 27576 2445 2223

Oreochromisniloticus 26763 2436 2202

Oryziaslatipes 24674 2412 2173

Takifugurubripes 47841 2268 2062

Tetraodonnigroviridis 23118 2300 2073

(Figure 3) Most of the genes showed highly significantcorrelation however one case indicated a reduced signifi-cance (elongation factor) and a few other cases resulted tobe not significant (eg Ras-related GTP-binding proteinBasic Transcription Factor 3 CuZn Superoxide Dismutaseand 2-Cys Peroxiredoxin) Reduced or lack of significanceappeared to be related to a low number of contigs from the454 sequencing (Table 5) which might be regarded as anindication of failure to reach library saturation Low valuesin contig frequency should correspond to a reduced levelof expression as the libraries are nonnormalized thereforecaution should be used when exploring 454 outputs belowa certain threshold Our data derived from the reading of asingle plate were insufficient for systematical comparison ofall the genes although the majority of the genes show a highcorrelation between the 454 data and the qPCR Howeverit should be emphasized that this exercise was not meant tobe a surrogate to the qPCR approach for the study of geneexpression but more a proxy for a preliminary screening ofdifferentially expressed genes in multiple libraries

33 Candidate Genes Associated to Depth The predictedamino acid sequences of A carbo functional genes puta-tively related to depth adaptations were compared to thosedeposited in NCBI database for homologies searches Com-plete alignments of orthologous protein sequences from theset of functional genes that have been reported as responsiveto depth adaptations included representatives of Teleostsbelonging to several families (Table 6) Exploring the levels ofsimilarities expressed as percentage of amino acid identity ornumber of amino acid that differ among sequences betweenA carbo and other fish species (Table 7) brings evidencesupporting the fact that deep-living aswell as polar species arenot the most similar to the black scabbard fish We attemptedto reconstruct the phylogenies for those species using aminoacid sequences of functional genes (LDH-A LDH-B MDHc-A Hb-A and Hb-B) and corresponding mtCOI nucleotidesequences (Figure 4) The resulting trees showed similartopologies suggesting that the signals embedded in thesefunctional genes reflect evolutionary divergence among taxarather than any enzyme adaptations relationships


Macromolecular complex 1541Organelle part 580Extracellular region 367

Organelle 1616Synapse part 031Synapse 031

Membrane-enclosed lumen 084Extracellular region part 120Cell 2815Cell part 2815

(a) Cellular components

Structural molecule activity 1054Catalytic activity 2753Enzyme regulator activity 294Electron carrier activity 152

Transporter activity 635Transcription factor activity 052Metallochaperone activity 016

Binding 4740Molecular transducer activity 058Antioxidant activity 094

(b) Molecular function

Cellular process 3193Death 010Signaling 127Cellular component biogenesis 215Cell wall organization or biogenesis 010

Metabolic process 3345Biological regulation 446

Viral reproduction 005

Biological adhesion 093

Cellular component organization 289

Signaling process 098Developmental process 024Immune system process 118Establishment of localization 847

Multicellular organismal process 049

(c) Biological processes

Figure 1 Functional categorization of unigenes with gene ontology (GO) term for theAphanopus carboEST collectionThese unigenes resultswere functionally classified from the six tissues pooled together as percentages under three main functional categories with respective GOSlim terms Data refer to assemblage derived by implementation of Newbler Roche 454 sequence analysis software

The lactate dehydrogenase A (LDH-A isotig01401)was encountered abundantly in the muscle tissue (Table 5Figure 2) and its comparison with other orthologoussequences differs for a number of amino acids between 16and 44 One indel at position 75 had to be added to the two

polar species to obtain a complete alignment (Table 7) Thepercentage of identity was higher with the strictly marinespecies ranging between 952 and 937 Unfortunatelyno sequences from neither deep-sea nor abyssal fisheswere available for comparison On the other hand lactate


Table 4 List of Pfam conserved domains that were tissue specifically expressed inAphanopus carbo transcriptome Frequencies and 119875-valuesof tissue specificity analysis are indicated

(a)

Gonads Heart LiverPfam ID Domain Freq 119875 Pfam ID Domain Freq 119875 Pfam ID Domain Freq 119875

PF00100 Zona pellucida 17 315 10minus13 PF00011 HSP20 3 403 10minus4 PF00084 Sushi 14 144 10minus10

PF00125 Histone 8 606 10minus5 PF13405 EF hand 4 5 677 10minus4 PF00089 Trypsin 20 759 10minus9

PF01400 Astacin 3 322 10minus3 PF00412 LIM 3 152 10minus3 PF00079 Serpin 12 563 10minus6

PF00069 Pkinase 3 001 PF05556 Calsarcin 3 360 10minus3 PF07678 A2M comp 4 135 10minus4

PF00653 BIR 2 002 PF01576 Myosin tail 1 3 360 10minus3 PF01042 Ribonuc L-PSP 3 126 10minus3

PF13424 TPR 12 2 002 PF00056 Ldh 1 N 3 360 10minus3 PF00045 Hemopexin 3 126 10minus3

PF13695 zf-3CxxC 2 002 PF05300 DUF737 2 550 10minus3 PF00059 Lectin C 6 169 10minus3

PF09360 zf-CDGSH 2 002 PF00992 Troponin 4 640 10minus3 PF00701 DHDPS 4 464 10minus3

PF01712 dNK 2 002 PF00595 PDZ 3 682 10minus3 PF00386 C1q 8 663 10minus3

PF00538 Linker histone 2 002 PF00022 Actin 3 001 PF00021 UPAR LY6 5 001PF10178 DUF2372 2 002 PF02874 ATP-synt ab N 2 002 PF00754 F5 F8 type C 9 001PF04856 Securin 2 002 PF13895 Ig 2 3 002 PF08702 Fib alpha 2 001PF00250 Fork head 2 002 PF00191 Annexin 2 003 PF03982 DAGAT 2 001PF01498 HTH Tnp Tc3 2 3 003 PF00212 ANP 2 003 PF01048 PNP UDP 1 2 001PF00268 Ribonuc red sm 2 006 PF05347 Complex1 LYR 2 007 PF01014 Uricase 2 001

(b)

Muscle Brain SpleenPfam ID Domain Freq 119875 Pfam ID Domain Freq 119875 Pfam ID Domain Freq 119875

PF01410 Troponin 7 199 10minus5 PF01669 Myelin MBP 4 403 10minus5 PF00042 Globin 5 133 10minus6

PF02807 COLFI 3 967 10minus4 PF01453 B lectin 2 644 10minus3 PF00078 RVT 1 5 133 10minus6

PF00041 ATP-guaPtransN 3 359 10minus3 PF00612 IQ 2 644 10minus3 PF00993 MHC II alpha 4 502 10minus6

PF02453 fn3 3 359 10minus3 PF11414 Suppressor APC 2 644 10minus3 PF07686 V set 5 249 10minus6

PF13895 Reticulon 3 359 10minus3 PF05768 DUF836 2 644 10minus3 PF07654 C1 set 5 471 10minus4

PF00365 Ig 2 4 797 10minus3 PF05196 PTNMK N 2 644 10minus3 PF00089 Trypsin 5 201 10minus3

PF01216 PFK 2 984 10minus3 PF04300 FBA 2 644 10minus3 PF01391 Collagen 2 230 10minus3

PF01267 Calsequestrin 2 984 10minus3 PF00287 Na K-ATPase 2 644 10minus3 PF00240 ubiquitin 3 328 10minus3

PF00261 F-actin cap A 2 984 10minus3 PF11032 ApoM 3 853 10minus3 PF09307 MHC2-interact 2 667 10minus3

PF00856 Tropomyosin 2 984 10minus3 PF00061 Lipocalin 4 001 PF00643 Zf-B box 2 001PF01661 SET 2 984 10minus3 PF00007 Cys knot 2 002 PF13445 Zf-RING LisH 2 001PF01667 Macro 2 003 PF01275 Myelin PLP 2 002 PF01498 HTH Tnp Tc3 2 2 002PF00036 Ribosomal S27e 2 005 PF00300 His Phos 1 2 002 PF02301 HORMA 1 005PF01576 efhand 2 005 PF00230 MIP 2 002 PF14259 RRM 6 1 005PF01410 Myosin tail 1 2 008 PF00091 Tubulin 3 002 PF09004 DUF1891 1 005

dehydrogenase B (LDH-B isotig01423) inA carbowas highlyrepresented in the heart while very scarcely recorded in theother tissues (Table S1) The largest sequence divergence interms of amino acid substitutions ranges between 47 and 55when compared with deep-water macrourids and similarlyto LDH-A an indel had to be added in the sequences ofthe gadiform species at position 76 to obtain a completealignment (Table 7) Previous studies on benthopelagic fishreport a significant decline of LDH activities with increasingwater depth [39] However it is known that variation inenzyme activities of fish at a given depth is influenced byfeeding behaviour and locomotory modes [40 41]

Cytosolic malate dehygrogenase A (MDHc-Aisotig01010) was detected in most of the tissues withthe exception of the muscle (Table S1) An isoform of MDHcwas also detected but its sequence covered only the last 110amino acids (isotig01731) However the type of substitutionsand tissue expression pattern (Table S1) according to Merritand Quattro [42] lead us to believe that this cytosolicisoform is MDHc-B It has been reported that the two teleostMDHc isozymes are the products of a gene duplicationevent after the separation of teleosts and tetrapods (see [42]and references therein) although the exact timing of thisduplication has not been inferred


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

Elongation factor 1-beta

Basic transcription factor 3-like 4

B G H L M S

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

2-Cys peroxiredoxin

B G H L M S

10

08

06

04

02

00Relat

ive f

old

expr

essio

n

Ferritin heavy subunit

B G H L M S

10

08

06

04

02

00Relat

ive f

old

expr

essio

n

1205732-Globin

B G H L M S

10

08

06

04

02

00Relat

ive f

old

expr

essio

n

Ependymin-1 precursor10

08

06

04

02

00Relat

ive f

old

expr

essio

n

B G H L M S

1205722-Globin

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

Heat shock protein 90

12

14

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

CuZn superoxide dismutase

10

08

06

04

02

00Relat

ive f

old

expr

essio

n

B G H L M S

Ras-related GTP-binding protein A12

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H LM S

Figure 2 Continued


Fatty acid-binding protein brain10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B GH LM S

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

CD63-like protein Sm-TSP-2

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H LM S

Tropomyosin 4 isoform 1

10

12

08

06

04

02

00Re

lativ

e fol

d ex

pres

sion

B G H L M S

C-Myc-binding protein

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BG H L M S

Cathepsin S

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

Transferrin

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

GH L M S

Warm temperature acclimation related-like proteinlowast

10

12

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BG H L M S

Betaine homocysteine S-methyltansferase

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BG H L M S

FUCL1 fucolectin-110

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

Aldolase B

Figure 2 Continued


12

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BH L M S

Type-4

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

Alcohol dehydrogenase 8a

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

Glyceraldehyde-3-phosphate dehydrogenase

10

08

06

04

02

00

Relat

ive f

old

expr

essio

nB G H L M S

Lactate dehydrogenase-A

12

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B H L M S

Phosphoglycerate mutase 2-1 (muscle)lowastlowast

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

Fructose-bisphosphate aldolase A

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

Phosphoglucose isomerase-2

ice-structuring protein LS-12 precursorlowastlowast

Figure 2 Gene expression normalized to a relative value of 10 of all genes selected for this study in different tissues from Aphanopus carboS = spleen B = brain H = heart G = gonad L = liver M = muscle lowast= expression in the brain was below detection and lowastlowast= expression in thegonads was below detection

Further comparison analyses were carried out only forMDHc-A with orthologous sequences obtained from NCBIdatabase where only shallow-water fish sequences were avail-able The complete alignment did not include any indel andsequences differed from A carbo between 15 and 35 aminoacid substitutions representing a percentage of identityranging from 955 to 895

Two 120572-skeletal actins were detected from the A carbodatabase the isoform 1 (isotig01459) expressed in shallow-water species and probably not functioning in abyssal fishes

and the isoform 2a (isotig01561) The mutations specific forA carbo in the actin 2a were the substitutions Asp3GluAla155Ser (a mutation also commonwith the isoform 2b [7])Ser234Val Val165Ile Leu261Val and finally Ala278Thr Thistype of isoform was reported in other deep-sea species asCoryphaenoides acrolepis C cinereus C yaquinae and Carmarus [7] The isoform 2b so far reported only in abyssalspecies was not detected in A carbo

Actins 1 and 2a were significantly expressed in muscletissuewith evidence of its presence also in the heart (Table S1)


EF-1Blowast

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SBHSP90

10

08

04

06

02

00

H G L M SB

PRDX1∘10

08

04

06

02

00

H G L M SB

SOD-1∘10

08

04

06

02

00

H G L M SB

Ferr10

08

04

06

02

00

H G L M SB

Hb-A10

08

04

06

02

00

H G L M SB

Hb-B10

08

04

06

02

00

H G L M SB

EPD-110

08

04

06

02

00

H G L M SB

BLBP10

08

04

06

02

00H G L M SB

TSPAN-810

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

BTF3∘

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

MYCBP10

08

04

06

02

00

H G L M SB

CTSS

STF-1

ContigsqPCR

Rab-1A∘

TRPM-1

Figure 3 Comparative plots of relative expression as contig frequencies versusmean qPCR values for all genes selected for this study Codesfor each of the gene are listed in Table 1 S = spleen B = brain H = heart G = gonad L = liver and M = muscle the correlation coefficient(Pearsonrsquos 119903) resulted to be highly significant (119875 lt 00001) for most of the genes except for lowast119875 lt 005 and ∘not significant


Table 5 Summary table of contig frequency for each of the tissues for those genes used to test the correlation with qPCR assays S spleen Bbrain H heart G gonad L liver and M muscle Names of genes based on their coding used in this table are found in table

Gene Contig code Spleen Brain Gonads Heart Liver MuscleEF-1B isotig00991 9 24 33 19 38 54Rab-1A isotig02689 4 0 3 3 0 4BTF3 isotig02213 3 5 7 10 8 2SOD-1 isotig02222 4 16 32 16 25 14PRDX1 isotig01838 4 50 59 17 23 27HSP90 isotig01406 3 64 94 86 86 13Ferr isotig01665 33 121 10 210 264 47Hb-A isotig06973 406 17 2 24 108 1Hb-B isotig00163 2303 81 16 313 707 22EPD-1 isotig01567 0 1190 0 1 0 0BLBP isotig02632 0 171 0 0 0 0TSPAN-8 isotig01397 17 7 3 407 24 9TRPM-1 isotig01595 1 4 0 637 2 2MYCBP isotig01988 0 2 135 1 1 1CTSS isotig01659 0 0 135 0 0 0STF-1 isotig00767 0 1 30 0 3218 0HPX isotig01479 0 0 0 0 1234 0BHTM isotig01489 1 0 0 0 1024 0FUCL1 isotig00473 1 0 0 0 22 0ALDB isotig01524 0 1 30 0 369 0ISP LS12 isotig03004 0 0 0 0 211 1ADH isotig01491-546 1 2 16 7 292 6GAPDH isotig00609 0 8 51 539 134 1281LDH-A isotig01401 2 7 2 5 0 789PglyM isotig01642 0 0 0 36 0 241HSP70 isotig01398 0 0 0 0 2 142FBPA isotig00492 2 2 0 233 0 4471PGI isotig01410 0 0 0 26 0 151

Direct comparison of A carbo actin 1 with orthologoussequences reported for other marine and deep-water speciesdiffered by just 1 amino acid at position 3 (Table 7) Thenumber of substitutions increases to 2 when this sequence iscompared to the isoform 2a of Coryphaenoides species (sub-stitution at position 155) and to 5 amino acids replacement(Table 7) when compared to the isoform 2b two of which areunique (positions 116 and 137) [7] Actin has a function inpolymerization of G-actin to F-actin in neutral salts Whilethe volume is increased following polymerization the reac-tion is strongly affected by high pressure [43] It is reportedthat the substitutions of Val54Ala or Leu67Pro reduced thevolume change associated with actin polymerization [7]

The assemblage of the myosin heavy chain protein(MyHC isotig02568 and isotig1394) obtained in A carbowas incomplete (All gs454 000756 and All gs454 000001)containing a gap of 711 amino acids at the positions from171 to 882 of the 1933 AA of the complete sequence Unfor-tunately within this gap there are the two loop regionswith characteristic structures that are uniquely reported fordeep-sea fish loop-2 region is shorter and the loop-1 regionhas a proresidue [9] MyHC was almost uniquely found in

muscle tissue (Table S1) and for comparison analyses withorthologous genes from other fish we only used the last 305AA (All gs454 000184) of the complete sequenceThe lowestnumber of amino acid substitutions ranged between 53 and69 (corresponding to sequence identity between 9495 to9343) when the sequence from A carbo is compared to itsrelative shallow water marine and freshwater species whilethis value increases up to 92 amino acid substitutions (andcorresponding sequence identity of 9125) when comparedto its relatives from deep water or polar regions (Table 7)

Variation in globin sequences was analysed exploring the1205722- and120573

2-chains (Hb-A isotig00247 andHb-B isotig00163)

whose relative expressions were detected more abundantly inthe spleen followed by liver heart brain andmuscle and vir-tually absent in the gonads (Figure 2 Table 5) Comparisonanalyses with orthologous sequences of deep-sea gadiformsand a notothenoid indicate that the number of amino acidsubstitutions ranged between 39 and 53 (Hb-A) and between31 and 42 (Hb-B) The complete alignments included theadditional single indel for the 120572

2-chain of Notothenia angus-

tata at position 102 Notothenioids acquired a completelydifferent globin genotype with respect to other teleostean


Table6Specieslist

with

inform

ations

ontheirtypeo

fenviro

nment(M

=marineBR

=brackish

andFW

=fre

shwater)climatedepthrange(

inmeters)andthetypeo

fgenes

used

inthe

study

with

relativeG

enbank

accessionnu

mbers

Order

Family

Species

Com

mon

name

Environm

ent

Clim

ate

Depth

range

Gene

NCB

IAccN

r

Percifo

rmes

Trichiuridae

Aphanopu

scarbo

Blackscabbardfish

MDeep-water

200ndash

1700

COI

EU8540

76

Beloniform

esAd

rianichthydae

Oryzias

latip

esJapanese

ricefi

shFW

+BR

Subtropical

shallow

ACTA

1MDHcMyH

CCO

I

NM

00110

4806

NM

00116

3134

XM00

4071618AB4

9806

6

Scorpaenifo

rmes

Hexagrammidae

Pleurogram

mus

azonus

Okh

otsk

atka

mackerel

MTemperate

0ndash240

ACTA

1AB0

73381

Percifo

rmes

Scom

bridae

Scom

berscombrus

Atlanticmackerl

MTemperate

0ndash200

(0ndash100

0)AC

TA1CO

IEF

607093K

C015895

Percifo

rmes

Percihcthyidae

Sinipercachuatsi

Mandarin

fish

FWTemperate

10AC

TA1MyH

CCO

IAY

395872A

Y454304

NC

015822

Percifo

rmes

Sparidae

Sparus

aurata

Gilthead

seabream

MTemperate

1ndash30

(1ndash150)

ACTA

1AF190473

Percifo

rmes

Sphyraenidae

Sphyraenaidiaste

sPelican

barracud

aM

Trop

ical

3ndash24

ACTA

1LD

H-AM

DHc

mMDH

AF503593SIU80001

AF390559AF390561

Tetraodo

ntifo

rmes

Tetraodo

ntidae

Takifugu

rubripes

Japanese

pufferfish

M+FW

+BR

Temperate

0ndash200

(0ndash100

0)Hb-Am

MDHC

OI

XM003964767

XM003965959HM102315

Scorpaenifo

rmes

Ano

plop

omatidae

Anoplopomafim

bria

Sablefish

MDeep-water

0ndash2740

Hb-B

COI

BT082849JQ353978

Percifo

rmes

Serranidae

Epinepheluscoioides

Orange-spottedgrou

per

M+BR

Subtropical

1ndash100

Hb-B

GU982530

Gasteroste

iform

esGasteroste

idae

Gasteroste

usaculeatusTh

ree-spined

stickleb

ack

M+FW

+BR

Temperate

0ndash100

Hb-B

NM

001267638

Percifo

rmes

Nototheniidae

Nototheniacoriiceps

Blackrockcod

MPo

lar

0ndash550

LDH-AM

yHC

COI

AF0

79822AJ

243767

EU326390

Percifo

rmes

Nototheniidae

Nototheniaangusta

taMaorichief

MTemperate

0ndash100

Hb-AH

b-B

P62363P

29628

Gadifo

rmes

Macrouridae

Coryphaenoides

armatus

Abyssalgrenadier

MDeep-water

282ndash5180

LDH-BM

yHC

COI

AJ60

9232A

B330140

FJ1644

97Gadifo

rmes

Gadidae

Gadus

morhu

aAtlanticcod

M+BR

Temperate

0ndash60

0LD

H-BC

OI

AJ60

9233K

C015385

Gadifo

rmes

Gadidae

Arctogadus

glacia

lisArctic

cod

MDeep-water

0ndash1000

Hb-AC

OI

Q1AGS4K

C015200

Percifo

rmes

Latid

aeLa

tescalcarifer

Barram

undi

M+FW

+BR

Trop

ical

10ndash4

0LD

H-BC

OI

FJ439507JQ431879

Gadifo

rmes

Gadidae

Merlangiusm

erlangus

Whitin

gM

Temperate

30ndash100

(10ndash

200)

LDH-BC

OI

AJ60

9234JQ623954

Cyprinod

ontiformes

Poeciliidae

Poeciliareticulata

Gup

pyFW

+BR

Trop

ical

Shallow

LDH-BC

OI

EF40

8825JX9

68696

Gadifo

rmes

Macrouridae

Trachyrin

cusm

urrayi

Roug

hnoseg

renadier

MDeep-water

0ndash1630

LDH-BC

OI

AJ60

9235A

P008990

Percifo

rmes

Channichthyidae

Chionodraco

rastrospinosus

Ocellatedicefish

MPo

lar

0ndash1000

LDH-AC

OI

AF0

79829EU

326337

Percifo

rmes

Pomacentridae

Chromiscaud

alis

Blue-axilchrom

isM

Trop

ical

15ndash55

LDH-A

AY289558

Percifo

rmes

Gob

iidae

Rhinogobiops

nicholsii

Blackeye

goby

MSubtropical

-106

LDH-A

AF0

79534

Cyprinifo

rmes

Cyprinidae

Cyprinus

carpio

Com

mon

carp

FW+BR

Subtropical

shallow

LDH-AM

yHC

COI

AF0

76528D89992

HQ960709


Table6Con

tinued

Order

Family

Species

Com

mon

name

Environm

ent

Clim

ate

Depth

range

Gene

NCB

IAccN

r

Cyprinod

ontiformes

Fund

ulidae

Fund

ulus

heteroclitus

Mum

micho

gM

+FW

+BR

Temperate

shallow

LDH-ALDH-BC

OI

L43525L23792EU

524629

Osm

erifo

rmes

Osm

eridae

Osm

erus

mordax

Rainbo

wsm

eltM

+FW

+BR

Temperate

0ndash425

MDHcmMDH

BT075651B

T07560

0

Salm

onifo

rmes

Salm

onidae

Salm

osalar

Atlanticsalm

onM

+FW

+BR

Temperate

10ndash23

(0ndash210)

MDHcmMDH

BT06

0183B

T048216

Gadifo

rmes

Macrouridae

Coryphaenoides

acrolep

isPacific

grenadier

MDeep-water

900ndash

1300

MyH

CCO

IAB3

30141JQ

3540

60

Gadifo

rmes

Macrouridae

Coryphaenoides

yaquinae

na

MDeep-water

3400ndash5800

MyH

CCO

IAB3

30139GU44

0291

Percifo

rmes

Cirrhitid

aePa

racir

rhitesforste

riBlacksideh

awkfi

shM

Trop

ical

5ndash35

MyH

CCO

IAJ

243770H

Q561521

Percifo

rmes

Carang

idae

Serio

ladu

merili

Greater

amberja

ckM

Subtropical

18ndash72

(1ndash360)

MyH

CCO

IAB0

32020KC

015917

Gadifo

rmes

Gadidae

Boreogadus

saida

Polarc

odM

+BR

Polar

0ndash40

0Hb-AH

b-B

COI

DQ125471Q

1AGS6

KC015250


Table 7 Comparison of protein sequences of depth related genesbetween Aphanopus carbo database (All gs454 xxx) and otherfishes Diff amino acid differences Id percentage of identityGaps number of gaps introduced in the complete alignment andAcc nr NCBI Accession number 1Partial sequence 2only AAsequence available

(a) LDH-A (332 AA)

All gs454 00149 vs Diff Id Gaps Acc nrSphyraena idiastes 16 9518 0 U80001Rhinogobiops nicholsii 17 9488 0 AF079534Chromis caudalis 21 9367 0 AY289558Chionodraco rastrospinosus 26 9217 1 AF079829Notothenia coriiceps 27 9187 1 AF079822Fundulus heteroclitus 27 9187 0 L43525Cyprinus carpio 44 8675 0 AF076528

(b) MDHc-A (333 AA)

All gs454 00146 vs Diff Id Gaps Acc nrOryzias latipes 15 9550 0 NM 1163134Sphyraena idiastes 20 9399 0 AF390559Osmerus mordax 30 9099 0 BT075651Salmo salar 35 8949 0 BT060183

(c) Actin-1 (375 AA)

All gs454 00104 vs Diff Id Gaps Acc nrScomber scombrus 1 0 100 0 EF607093Iniperca chuatsi 1 0 100 0 AY395872Oryzias latipes 1 1 9973 0 NM 1104806Pleurogrammus azonus 1 1 9973 0 AB073381Coryphaenoides acrolepis 1 1 9973 0 AB021649Coryphaenoides cinereus 1 1 9973 0 AB021651Coryphaenoides armatus 2a 2 9947 0 AB086240Coryphaenoides yaquinae 2a 2 9947 0 AB086242Coryphaenoides acrolepis 2a 2 9947 0 AB021650Coryphaenoides cinereus 2a 2 9947 0 AB021652Cyprinus carpio 1 2 9947 0 AY395870Sphyraena idiastes 1 3 9920 0 AF503593Coryphaenoides armatus 2b 5 9867 0 AB086241Coryphaenoides yaquinae 2b 5 9867 0 AB086243Aphanopus carbo 2a 6 9840 0 All gs454 00102Sparus aurata 1 9 9760 0 AF190473

(d) LDH-B (334 AA)

All gs454 00143 vs Diff Id Gaps Acc nrLates calcarifer 14 9581 0 FJ439507Fundulus heteroclitus 21 9371 0 L23792Trachyrincus murrayi 47 8593 0 AJ609235Coryphaenoides armatus 50 8503 0 AJ609232Merlangius merlangus 52 8443 1 AJ609234Gadus morhua 55 8353 1 AJ609233

(e) MyHC1 (305 AA)

All gs454 00001 vs Diff Id Gaps Acc nrParacirrhites forsteri 53 9495 0 AJ243770Seriola dumerilii 54 9486 0 AB032020Siniperca chuatsi 59 9438 0 AY454304Oryzias latipes 62 9410 2 XM 4071618Cyprinus carpio 69 9343 2 D89992Coryphaenoides acrolepis 86 9182 1 AB330141Notothenia coriiceps 86 9182 1 AJ243767Coryphaenoides yaquinae 89 9153 1 AB330139Coryphaenoides armatus 92 9125 1 AB330140

(f) Hb-A1 (143 AA)

All gs454 01074 vs Diff Id Gaps Acc nrNotothenia angustata 39 7273 1 P623632

Boreogadus saida 43 6993 0 DQ125471Arctogadus glacialis 44 6993 0 DQ125475Takifugu rubripes 46 6783 0 XM 3964767Gadus morhua 53 6294 0 O424252

(g) Hb-B1 (146 AA)

All gs454 01018 vs Diff Id Gaps Acc nrEpinephelus coioides 27 8163 0 GU982530Anoplopoma fimbria 31 7891 0 BT082849Gasterosteus aculeatus 31 7891 0 NM 1267638Boreogadus saida 40 7279 0 Q1AGS62

Notothenia angustata 42 7143 0 P296282

groups The Antarctic ichthyofauna (dominated by a singletaxonomically uniform group) lost its globin multiplicity incorrelation with temperature stability On the other handfor the Arctic ichthyofauna it may have been advantageousto maintain a multiple globin system helping to deal withenvironmental changes and metabolic demands [44]

Selective pressure in the site-by-site patterns amongspecies was evaluated for LDH-A -B MDHc and ACTA1(isoform 1) There was no evidence of positive selection atthe nucleotide site level in any of those genes whose global120596 value was very low in all cases (from 0 in ACTA1 and 0032in LDH-B) (Table S2) The proportion of sites supportingpositive selection the model M2 was null (LDH-A and -B)or extremely low (03 in MDHc and 16 in ACTA1) (TableS2) These results indicate that the evolution of these fourgenes in teleosts is constrained by very stringent selectivepressure (model probabilities for M1 versusM2 ranging from87 in MDHc and 88 in all others)

In terms of protein stability calculated as Gibbs freeenergy and taking into consideration kinetic and thermo-dynamic quantities we explored only the functional genesfor which the complete sequences were obtained (Figure 5)In this context protein stability is defined by the ability of


02

01

Ep_coiGa_acuAn_fim

Ap_carBo_sai

No_angNo_ang

Ap_carTa_rub

Bo_saiAr_glaGa_mor

52

5452

66

100

5359

67

100 100

COI

LDH-A

LDH-B

MDHc-A

Hb-B

Hb-A

La_calCy_carAn_fim

Ap_carSi_chu

Se_dumSc_sco

Ta_rubPa_for

Ch_rasNo_cor

Or_latGa_morBo_saiAr_glaMe_mer

Tr_murCo_armCo_yaq

Co_acrPo_ret

Fu_hetLa_cha

62100

73

87

100

100

005

100

99

No_corCh_rasFu_hetCh_cauRh_nicSp_idiAp_car

Cy_carLa_calAp_car

Fu_hetTr_murCo_armGa_morMe_mer

Or_latAp_car

Sp_idiSa_salOs_mor

100

6479

100

100

100

100

100100

9699

7990

8055

70

Figure 4 At the top molecular phylogenetic tree of COI gene estimated by the HKY nucleotide substitution model constructed by neighborjoining and rooted including the sequence of Latimeria chalumnae (acc nr NC 001804) in the middle NJ tree of LDH andMDH genes andat the bottom NJ tree of globin (Hb) gene Numbers in proximity of the nodes denote the bootstrap value (above 50) out of 1000 replicatesThe scale indicates the evolutionary distance of the base substitution per site Taxon coding includes the first two letters for the genus followedby three letters for the species name (see Table 6) A black square helps to locate Aphanopus carbo in the trees


Ac carAc carAc car

Tr murNo corOs mor

Co armCh rasSa sal

Me merSp idiSp idi

Ga mor

Or lat

MDHc-A

LDH-ALDH-B

450

400

350

300

250

200

150

100

50

00

ΔG

(cal

mol

)

(a)

ΔG

(cal

mol

)

Ac car Co acr Co cin Co arm Co yaq460

480

500

520

540

560

580

600

Actin 1

Actin 2bActin 2a

(b)

1000

800

600

400

200

00

minus200

ΔG

(cal

mol

)

Hb-AHb-B

No ang Ac car Bo sai Ar gla Ga morAn fim

(c)

Figure 5 Comparative plots of normalised difference in protein stability (Δ119866) for different functional genes among several representativespecies Taxon coding includes the first two letters for the genus followed by three letters for the species name (see Table 6)

a protein to retain its structural conformation or its activ-ity when subjected to physical or chemical manipulationstherefore the energy consumed by activation to promotedfolding has to be compensated by thermal stability providedby the energy of denaturation [35] Hence protein stability isquantitatively calculated by the standardGibbs energy change(Δ119866) allowing comparison of stabilities for different proteins[45]

The normalised difference in protein stability (Δ119866) oflactate dehydrogenase A (LDH-A) was lower in A carbocompared to fish from polar waters and tropical marine(Figure 5) This gradual variation was primarily dictatedby the lower contributions of leucine and lysine in thehydrophobic trend of kinetic calculation in the A carbosequence (Table S3) The substantial drop in Δ119866 of LDH-B in A carbo compared to the orthologous sequences ofpolar abyssal and shallower marine fishes (Figure 5) wasdue to a larger contribution of glutamic acid (Table S3)promoting the thermal denaturation of the protein Other

studies investigating kinetic physical properties and ability towithstand high pressure of LDH-B of two gadiformes supportour calculations showing that the enzyme from the deep-sea species has a significant increased tolerance to pressureand higher thermostability [6] To provide protection to Acarbo LDH-B from pressure and temperature denaturationosmolytes might play an essential role Experiments addingTrimethylamine-N-oxide (TMAO) to samples resulted insubstantial increment of activity of LDH-B under conditionsat which the enzyme was previously sensitive [6]

In the cytosolicmalate dehygrogenaseA (MDHc-A) therewas a sharp decrease in protein stability of marine fishesgoing from shallower to deeper waters (no sequences ofabyssal specieswere available) Lowest values are encounteredin the sequences of the two euryhaline species included inthe comparison (Figure 5) This progressive decrement instability was associated to the larger contribution of asparticacid (Table S3) also a promoter of thermal denaturation ofthe protein The responses to pressure and temperature of


soluble enzymes like LDH andMDHdiffer adaptively amongspecies found at different depths

The isoforms 1 and 2a of 120572-skeletal actin of A carboshowed very similar numerical values of Δ119866 compared totheir homologues of deep-water (actin 1) and deep andabyssal water (actin 2a) Protein stability drops substantiallywhen these two isoforms were compared with actin 2bonly present in abyssal species (Figure 5) Such reduction instability was due to a higher number of hydrophobic aminoacids contributing to the thermodynamic calculation (TableS3) Morita [7] reported that the substitutions of Gln137Lysand Ala155Ser generate a mechanism for stabilizing enzyme-substrate interactions under high pressure

Comparing the stability of globin 1205722-chain (Hb-A) in

shallow deep-water and polar fish the larger Δ119866 was foundin polar and deep-water cods followed by A carbo beforedropping to negative values for the shallower representativesof Antarctic and temperate environments (Figure 5) Stabilityof protein in A carbo and its deep-water relatives was linkedto larger contributions by leucine in the kinetic calculationspromoting folding and thermal stability (Table S3) Thestability as Δ119866 of globin sequences of the 120573

2-chains (Hb-

B) showed a very similar trend as in Hb-A (although therewere no negative values) and the two deep-living species hadvery similar protein stability value Variation in Δ119866 amongenvironments was linked to the balanced contribution ofhydrophobic amino acids promoting folding versus thoselowering thermal stability (Table S3)

Although for key enzymes it was found that adap-tive differences among species at different depths showedstructural changes as well as structural stability (reviewsin [4ndash6]) several studies remarked on the importance ofregulatory regions of the genome acting on gene regulation(see [46]) Promoter regions often work together with othercys-regulatory elements (eg transcription factors bindingsites enhancers silencers and insulators) to regulate thetranscription and expression of mRNA in a specific tissueat different times and places throughout the genome Inaddition in regards to adaptation to depth it is remarkedthe importance of osmolyte concentrations of methylamines(TMAO is the most relevant) These are protein stabilizersthat counteract inhibition of proteins by hydrostatic pressure[47ndash49]

Finally we should also take into account the stressimposed on this fishes being captured at a depth range of1100ndash1250m and brought to surface in at least 3ndash5 hours Bythe time specimens of black scabbardfish reach the surfacethey are already dead or dying therefore the expressionof some of the transcripts might be different if comparedto a transcriptome of a fish sampled at a thousand metersdepth Notwithstanding several experiments using modelspecies have been targeting specific genes linked to stressfactors [50] and this might prove useful in interpretingtranscriptomes of animals undergoing stressful conditionsbefore being sampled

On the bases of this preliminary but encouraging resultswe are planning to explore further deep water adap-tivity employing different NGS sequence technology andexperimental design for instance using individual RADseq

Table 8 Statistics of EST-SSRs identified in Aphanopus carbotranscriptome

Searching item NumbersTotal number of sequences examined 7920Total size of examined sequences (bp) 4235839Total number of identified SSRs 153Number of SSR containing sequences 142Number of sequences containing more than 1 SSR 9Number of SSRs present in compound formation 8Di-nucleotide 63Tri-nucleotide 52Tetra-nucleotide 35Penta-nucleotide 3

approach on a larger number of specimens caught at deepor shallow waters or by comparing sister species that live atdifferent depth levels

34 EST-SSR Resources for Population Genetics In this studya total of 153 EST-SSRs were detected in 142 contigs (387)with a frequency of one EST-SSR per 2769 kb sequence(Table 8) A further selection was made taking into accountthe type of repeats size of the fragment and quality of theprimer sets reducing the EST-SSR to 98 Some of thosemicrosatellite markers were found in one tissue but notin others (163) while others resolved to be found inmore than one tissue (837) Among the identified EST-SSRs tri-nucleotide repeats represented the largest por-tion (408) followed by di-nucleotide (286) and tetra-nucleotide (257) and only 2 EST-SSRs penta-nucleotidewere identified (Table S4) Primer pairs have sizes rangingbetween 19 and 25 bp and melting temperatures from 571∘Cto 612∘C while expected PCR products are 100 to 280 bp inlength None of these EST-derived primer pairs have beentested for neutrality therefore not allmay be suitable for basicpopulation genetic studies

4 Conclusions

The transcriptome analysis of A carbo revealed a com-prehensive set of genes expressed in six tissues producingover 8000 genes 556 of which annotated by similarity toknown proteins or nucleotide sequences from freely acces-sible database The transcriptome of black scabbardfish setsthe stage for expanding the genetic resources available whileproviding sets of genes that are likely tied with physiologicaladaptations to depth particularly to low temperature andhigh pressure factorsThis study represents the first transcrip-tome analysis for a deep-sea fish providing insights based oncomparison analyses of homologous depth-related functionalgenes from shallow deep-water and abyssal fish highlight-ing similarities for A carbo isozyme patterns and stabilityto other bathypelagic fishes Direct sequence comparisonsuggested that the signals embedded in these functionalgenes reflect evolutionary divergence among taxa rather thanany kind of enzyme adaptations Organisms are adapted to


deep-sea environment and their physiological tolerance mayvary among taxa as well as their enzymatic activities underextreme conditions Osmolyte concentrations as protein sta-bilizers that counteract inhibition of proteins by hydrostaticpressure [6 47ndash49] play a key role in deep-sea adaptationFurthermore contribution to adaptation is also provided bypromoter regions that together with cys- and trans-regulatoryelements work concertedly at the mRNA transcription levelto drive the expression of a gene in a specific tissue or at aspecific time [46]

The strong correlation detected between the values ofstandardized contigs frequency with the expression level ofthe gene by qPCR supports the use of our data to explore geneexpression patterns in A carbo

Finally considering the importance of this species forfishery management a further exploration of this databasehas provided the characterization of new EST-SSR markersas additional resource for basic population genetic studies aswell as tagging and mapping of genes

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contributions

Sergio Stefanni conceived the study and design collectedsamples participated in the data analysis and drafted thepaper Raul Bettencourt contributed to gene validation exper-iments and discussion Miguel Pinheiro and Gianluca DeMoro developed the pipe-line analysis for all functionalannotations and the SQL database Lucia Bongiorni andAlberto Pallavicini participated in the structure discussionand paper drafting

Acknowledgments

All molecular work was supported by the research ProjectsDEECON (European Science Foundation under the EURO-CORES programme proposal no 06-EuroDEEP-FP-008)and ReDEco (MarinERA programme funded by the EU FP6ERA-NET Scheme MARIN-ERAMAR00032008) Speci-mens of A carbo were provided by the CONDOR Project(EEA Grants Financial Mechanism Iceland Liechtensteinand Norway proposal no PT0040) The authors are thank-ful to captain crew and technicians onboard of the RVArquipelago for their excellent contribution while at seaSergio Stefanni is a research fellow supported by the MarieCurie grant cofunded by the EU under the FP7-People-2012-COFUND Cofunding of Regional National and Inter-national Programmes GA no 600407 and the BandieraProject RITMARE IMARDOP is funded through the pluri-annual and programmatic funding scheme as research unitnumber 531 and associate laboratory number 9 The authorsare also grateful to Conceicao Egas for the support providedat Biocant Last but not least the authors thank three

anonymous reviewers for their comments and suggestionsthat greatly improved the paper

References

[1] G N Somero ldquoAdaptations to high hydrostatic pressurerdquoAnnual Review of Physiology vol 54 pp 557ndash577 1992

[2] J J Childress andMHNygaard ldquoThe chemical composition ofmidwater fishes as a function of depth of occurence off southernCaliforniardquo Deep-Sea Research and Oceanographic Abstractsvol 20 no 12 pp 1093ndash1109 1973

[3] J J Childress and G N Somero ldquoDepth-related enzymicactivities in muscle brain and heart of deep-living pelagicmarine teleostsrdquo Marine Biology vol 52 no 3 pp 273ndash2831979

[4] G N Somero ldquoBiochemical ecology of deep-sea animalsrdquoExperientia vol 48 no 6 pp 537ndash543 1992

[5] P Sebert ldquoFish at high pressure a hundred year historyrdquoComparative Biochemistry and Physiology A vol 131 no 3 pp575ndash585 2002

[6] A A Brindley R W Pickersgill J C Partridge D J DunstanD M Hunt and M J Warren ldquoEnzyme sequence and itsrelationship to hyperbaric stability of artificial and natural fishlactate dehydrogenasesrdquo PLoS ONE vol 3 no 4 Article IDe2042 2008

[7] TMorita ldquoStructure-based analysis of high pressure adaptationof 120572-actinrdquo Journal of Biological Chemistry vol 278 no 30 pp28060ndash28066 2003

[8] T Morita ldquoStudies on molecular mechanisms underlying highpressure adaptation of 120572-actin from deep-sea fishrdquo Bulletin ofFisheries Research amp Development Agency vol 13 pp 35ndash772004

[9] T Morita ldquoComparative sequence analysis of myosin heavychain proteins from congeneric shallow- and deep-living rattailfish (genus Coryphaenoides)rdquo The Journal of ExperimentalBiology vol 211 no 9 pp 1362ndash1367 2008

[10] T Morita ldquoHigh-pressure adaptation of muscle proteins fromdeep-sea fishes Coryphaenoides yaquinae and C armatusrdquoAnnals of the New York Academy of Sciences vol 1189 pp 91ndash94 2010

[11] S Hourdez and R E Weber ldquoMolecular and functionaladaptations in deep-sea hemoglobinsrdquo Journal of InorganicBiochemistry vol 99 no 1 pp 130ndash141 2005

[12] D W Tucker ldquoStudies on Trichiuroid fishesmdash3 A preliminaryrevision of the family Trichiuridaerdquo Bulletin of the BritishMuseum (Natural History) Zoology vol 4 pp 73ndash131 1956

[13] M R Martins A M Leite and M L Nunes ldquoPeixe-espada-pretoAlgumas notas acerca da pescaria do peixe-espada-pretordquoInstituto Nacional de Investigacao das Pescas 1987

[14] FAO ldquoFishstatrdquo FAO Fisheries Department Fishery Informa-tion Data and Statistics Unit 2002

[15] M Machete T Morato and G Menezes ldquoExperimental fish-eries for black scabbardfish (Aphanopus carbo) in the AzoresNortheast Atlanticrdquo ICES Journal of Marine Science vol 68 no2 pp 302ndash308 2011

[16] ICES ldquoReport of theWorking Group on the Biology and Assss-ment of Deep-Sea Fisheries Resources (WGDEEP)rdquo Tech RepICES CM 2008ACOM 14 ICEC Headquarters CopenhagenDenmark 2008

[17] I Figueiredo P Bordalo-Machado S Reis et al ldquoObservationson the reproductive cycle of the black scabbardfish (Aphanopuscarbo Lowe 1839) in the NE Atlanticrdquo ICES Journal of MarineScience vol 60 no 4 pp 774ndash779 2003


[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996

[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014

[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007

[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007

[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008

[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008

[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010

[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012

[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011

[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012

[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009

[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005

[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990

[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997

[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985

[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007

[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011

[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003

[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000

[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010

[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007

[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980

[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982

[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003

[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966

[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006

[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993

[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007

[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001

[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002

[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004

[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


of functional genes to high pressure report unique aminoacid substitutions in 120572-skeletal actin and myosin heavy chain(MyHC) proteins in deep-sea fishes [7ndash10] For deep-seaspecies inhabiting hydrothermal vents and cold seeps envi-ronments characterized by high pressure chronic hypoxiaand high concentrations of toxic compounds molecular andfunctional adaptation of hemoglobins (Hbs) are reviewedin Hourdez and Weber [11] Despite these studies ourknowledge on wide scale gene expression patterns in deep-sea fish remains elusive

The black scabbardfish Aphanopus carbo (Lowe 1839) isa bathypelagic species belonging to the Trichiuridae familyand is distributed in temperate-cold Atlantic waters at depthsbetween 200 and 1800m [12 13] A carbo represents acommercially valuable species for several regions of theIberic peninsula especially in Madeira where catches havereached up to 1000 tons per year [14] amounting to ca55 of the total landings Recently this species has becomeincreasingly targeted by Portuguese French and Irish fishingfleets ([15] and literature therein) and fishery data haveshown a constant decline in population [16]The informationavailable on the biology maturity spawning and growth ofthis species [17 18] is scattered Recent studies are reporting apanmictic distribution of this species in the NE Atlantic withmultiple breeding sites at low latitudes [19] It is also worthmentioning that in southern locations this species lives insympatry with A intermedius a close related species withvery similarmorphology [20] therefore attracting interest forevolutionary studies

High-throughput sequencing approaches applied to tran-scriptomics now provide a global perspective on taxonomicand functional profiling of genes expectedly expressed underthe influence of environmental conditions in which theseorganisms live Also known as next-generation sequencingthese techniques allow for a massive characterization ofexpressed sequence tags (ESTs) providing an overview ofthose genes expressed in a given tissue at any given time[21] In silico analyses of massive gene libraries may serveseveral interests among others For instances from discoveryand identification of new genes characterization of geneexpression to development of novel genetic markers forquantitative trait locus (QTL) and population of genomicanalysesThe breadth of next-generation sequencing applica-tions extends over a variety of biological questions includingthose addressing pertinent questions regarding a speciesrsquoecology life history and evolution [22 23]

Previous studies regarding transcriptome sequencing andgene expression studies in deep-water species were mostlylimited to hydrothermal vents invertebrates [24] microbialcommunities in hydrothermal plumes [25] deep-sediments[26] and in the water column [27] leaving vertebrates speciesvirtually under-represented The present work represents apioneer study for deep-sea fishes providing new insights intothe role of differential gene expression on the environmentaladaptation of deep-sea black scabbardfish

Here we describe the assembly and annotation of thetranscriptome of A carbo obtained by sequencing mRNAlibraries of six tissues (spleen brain heart gonads liverand muscle) and explore functional genes whose sequence

might be associated to depth adaptation Additionally wetested the correlation of selected candidate genes comparingthe number of contigs against the gene expression normalizedto a relative value of 10 as determined by qPCR amplifi-cation Furthermore the screening of the libraries allowedthe identification of newEST-simple-sequence repeats (SSRs)and the design of primer pairs suitable for population geneticstudies as well as tagging and mapping of genes

2 Methods

21 Fish and Tissue Samples Specimens of Aphanopus carbo(two males and two females) were collected in 2009 onboardof the RV ldquoArquipelagordquo A carbo were fished at depth range1100ndash1250m using deep-water long-lines in proximity of theCondor Seamount located approximately at 15 nm SW ofthe island of Fayal (Azores Portugal) The four specimensused in this study were caught on the same longline set andonce onboard the freshly caught animals were dissected andportions (or complete organs) of spleen brain heart gonadsliver and muscle tissues were preserved in formamide solu-tion and kept at minus20∘C until RNA extraction was performed

To validate the correct identification of the species a smallportion ofmuscle tissuewas also preserved in 95 ethanol formolecular screening following Stefanni et al [28] protocols

22 RNA Extraction and Sequencing Total RNA wasextracted from 20 to 40mg of each of the six preservedtissues of a pool of four A carbo individualsusing theRiboPure kit (Ambion Applied Biosystems) Quantity andpurity of the RNAwas determined on a 14 agarose-MOPS-formaldehyde denaturing gels and by assessing the 119860

260280

and 119860260230

ratios using the NanoVue spectrophotometer(GE Healthcare) Poly-A RNA was extracted from 15 120583gof each total RNA sample using the Poly(A)Purist mRNAPurification kit according to manufacturerrsquos instructions(Ambion Applied Biosystems) mRNAs were transcribedinto cDNA utilizing Mint-2 cDNA synthesis kit (Evrogenhttpwwwevrogencom) according to manufacturerrsquosinstructions for NGS platforms Six cDNA libraries wereconstructed from mRNA of individual pools of tissues andsequenced in a single 454 GS FLX Titanium run Each of thecDNA libraries was characterised by unique sequence tags(MIDs) that allowed to trace back the sequences generatedfrom single tissues after assembly

cDNAs were sheared by nebulization to yield randomfragments approximately 500ndash800 bp in length by applying30 psi (21 bar) of nitrogen for 1 minute on 4 120583g of eachlibrary The distribution of fragments was verified on aBioAnalyser DNA 7500 LabChip (Agilent Technologies)The fragmented cDNA sample was end-repaired with T4DNA polymerase and T4 polynucleotide kinase and adaptorsequences ligated according to the manufacturerrsquos instruc-tions [29] The fragments were immobilized onto strepta-vidin beads and nick-repaired with Bst polymerase ThecDNA fragments were denaturated with alkali to yield singlestranded cDNA (sscDNA) library Quality of the library wasassayed on a BioAnalyser RNA 6000 Pico LabChip (AgilentTechnologies) and quantity measured by spectrofluorimetry
























1205722


1205732


















Table 1 Continued

























































(a)












(b)















10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S



B G H L M S

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

2-Cys peroxiredoxin

B G H L M S

10

08

06

04

02

00Relat

ive f

old

expr

essio

n


B G H L M S

10

08

06

04

02

00Relat

ive f

old

expr

essio

n

1205732-Globin

B G H L M S

10

08

06

04

02

00Relat

ive f

old

expr

essio

n


08

06

04

02

00Relat

ive f

old

expr

essio

n

B G H L M S

1205722-Globin

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


12

14

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H LM S

Figure 2 Continued



08

06

04

02

00

Relat

ive f

old

expr

essio

n

B GH LM S

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H LM S


10

12

08

06

04

02

00Re

lativ

e fol

d ex

pres

sion

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BG H L M S

Cathepsin S

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

Transferrin

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

GH L M S


10

12

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BG H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BG H L M S


08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

Aldolase B

Figure 2 Continued


12

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BH L M S

Type-4

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

nB G H L M S


12

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S









EF-1Blowast

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SBHSP90

10

08

04

06

02

00

H G L M SB

PRDX1∘10

08

04

06

02

00

H G L M SB

SOD-1∘10

08

04

06

02

00

H G L M SB

Ferr10

08

04

06

02

00

H G L M SB

Hb-A10

08

04

06

02

00

H G L M SB

Hb-B10

08

04

06

02

00

H G L M SB

EPD-110

08

04

06

02

00

H G L M SB

BLBP10

08

04

06

02

00H G L M SB

TSPAN-810

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

BTF3∘

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

MYCBP10

08

04

06

02

00

H G L M SB

CTSS

STF-1

ContigsqPCR

Rab-1A∘

TRPM-1














Table6Specieslist

with

inform

ations

ontheirtypeo

fenviro

nment(M

=marineBR

=brackish

andFW

=fre



fgenes

used

inthe

study

with

relativeG

enbank

accessionnu

mbers

Order

Family

Species

Com

mon

name

Environm

ent

Clim

ate

Depth

range

Gene

NCB

IAccN

r

Percifo

rmes

Trichiuridae

Aphanopu

scarbo

Blackscabbardfish

MDeep-water

200ndash

1700

COI

EU8540

76

Beloniform

esAd

rianichthydae

Oryzias

latip

esJapanese

ricefi

shFW

+BR

Subtropical

shallow

ACTA

1MDHcMyH

CCO

I

NM

00110

4806

NM

00116

3134

XM00

4071618AB4

9806

6

Scorpaenifo

rmes

Hexagrammidae

Pleurogram

mus

azonus

Okh

otsk

atka

mackerel

MTemperate

0ndash240

ACTA

1AB0

73381

Percifo

rmes

Scom

bridae

Scom

berscombrus

Atlanticmackerl

MTemperate

0ndash200

(0ndash100

0)AC

TA1CO

IEF

607093K

C015895

Percifo

rmes

Percihcthyidae

Sinipercachuatsi

Mandarin

fish

FWTemperate

10AC

TA1MyH

CCO

IAY

395872A

Y454304

NC

015822

Percifo

rmes

Sparidae

Sparus

aurata

Gilthead

seabream

MTemperate

1ndash30

(1ndash150)

ACTA

1AF190473

Percifo

rmes

Sphyraenidae

Sphyraenaidiaste

sPelican

barracud

aM

Trop

ical

3ndash24

ACTA

1LD

H-AM

DHc

mMDH

AF503593SIU80001

AF390559AF390561

Tetraodo

ntifo

rmes

Tetraodo

ntidae

Takifugu

rubripes

Japanese

pufferfish

M+FW

+BR

Temperate

0ndash200

(0ndash100

0)Hb-Am

MDHC

OI

XM003964767

XM003965959HM102315

Scorpaenifo

rmes

Ano

plop

omatidae

Anoplopomafim

bria

Sablefish

MDeep-water

0ndash2740

Hb-B

COI

BT082849JQ353978

Percifo

rmes

Serranidae

Epinepheluscoioides

Orange-spottedgrou

per

M+BR

Subtropical

1ndash100

Hb-B

GU982530

Gasteroste

iform

esGasteroste

idae

Gasteroste

usaculeatusTh

ree-spined

stickleb

ack

M+FW

+BR

Temperate

0ndash100

Hb-B

NM

001267638

Percifo

rmes

Nototheniidae

Nototheniacoriiceps

Blackrockcod

MPo

lar

0ndash550

LDH-AM

yHC

COI

AF0

79822AJ

243767

EU326390

Percifo

rmes

Nototheniidae

Nototheniaangusta

taMaorichief

MTemperate

0ndash100

Hb-AH

b-B

P62363P

29628

Gadifo

rmes

Macrouridae

Coryphaenoides

armatus

Abyssalgrenadier

MDeep-water

282ndash5180

LDH-BM

yHC

COI

AJ60

9232A

B330140

FJ1644

97Gadifo

rmes

Gadidae

Gadus

morhu

aAtlanticcod

M+BR

Temperate

0ndash60

0LD

H-BC

OI

AJ60

9233K

C015385

Gadifo

rmes

Gadidae

Arctogadus

glacia

lisArctic

cod

MDeep-water

0ndash1000

Hb-AC

OI

Q1AGS4K

C015200

Percifo

rmes

Latid

aeLa

tescalcarifer

Barram

undi

M+FW

+BR

Trop

ical

10ndash4

0LD

H-BC

OI

FJ439507JQ431879

Gadifo

rmes

Gadidae

Merlangiusm

erlangus

Whitin

gM

Temperate

30ndash100

(10ndash

200)

LDH-BC

OI

AJ60

9234JQ623954

Cyprinod

ontiformes

Poeciliidae

Poeciliareticulata

Gup

pyFW

+BR

Trop

ical

Shallow

LDH-BC

OI

EF40

8825JX9

68696

Gadifo

rmes

Macrouridae

Trachyrin

cusm

urrayi

Roug

hnoseg

renadier

MDeep-water

0ndash1630

LDH-BC

OI

AJ60

9235A

P008990

Percifo

rmes

Channichthyidae

Chionodraco

rastrospinosus

Ocellatedicefish

MPo

lar

0ndash1000

LDH-AC

OI

AF0

79829EU

326337

Percifo

rmes

Pomacentridae

Chromiscaud

alis

Blue-axilchrom

isM

Trop

ical

15ndash55

LDH-A

AY289558

Percifo

rmes

Gob

iidae

Rhinogobiops

nicholsii

Blackeye

goby

MSubtropical

-106

LDH-A

AF0

79534

Cyprinifo

rmes

Cyprinidae

Cyprinus

carpio

Com

mon

carp

FW+BR

Subtropical

shallow

LDH-AM

yHC

COI

AF0

76528D89992

HQ960709


Table6Con

tinued

Order

Family

Species

Com

mon

name

Environm

ent

Clim

ate

Depth

range

Gene

NCB

IAccN

r

Cyprinod

ontiformes

Fund

ulidae

Fund

ulus

heteroclitus

Mum

micho

gM

+FW

+BR

Temperate

shallow

LDH-ALDH-BC

OI

L43525L23792EU

524629

Osm

erifo

rmes

Osm

eridae

Osm

erus

mordax

Rainbo

wsm

eltM

+FW

+BR

Temperate

0ndash425

MDHcmMDH

BT075651B

T07560

0

Salm

onifo

rmes

Salm

onidae

Salm

osalar

Atlanticsalm

onM

+FW

+BR

Temperate

10ndash23

(0ndash210)

MDHcmMDH

BT06

0183B

T048216

Gadifo

rmes

Macrouridae

Coryphaenoides

acrolep

isPacific

grenadier

MDeep-water

900ndash

1300

MyH

CCO

IAB3

30141JQ

3540

60

Gadifo

rmes

Macrouridae

Coryphaenoides

yaquinae

na

MDeep-water

3400ndash5800

MyH

CCO

IAB3

30139GU44

0291

Percifo

rmes

Cirrhitid

aePa

racir

rhitesforste

riBlacksideh

awkfi

shM

Trop

ical

5ndash35

MyH

CCO

IAJ

243770H

Q561521

Percifo

rmes

Carang

idae

Serio

ladu

merili

Greater

amberja

ckM

Subtropical

18ndash72

(1ndash360)

MyH

CCO

IAB0

32020KC

015917

Gadifo

rmes

Gadidae

Boreogadus

saida

Polarc

odM

+BR

Polar

0ndash40

0Hb-AH

b-B

COI

DQ125471Q

1AGS6

KC015250



(a) LDH-A (332 AA)


(b) MDHc-A (333 AA)




(d) LDH-B (334 AA)


(e) MyHC1 (305 AA)


(f) Hb-A1 (143 AA)



(g) Hb-B1 (146 AA)







02

01

Ep_coiGa_acuAn_fim

Ap_carBo_sai

No_angNo_ang

Ap_carTa_rub

Bo_saiAr_glaGa_mor

52

5452

66

100

5359

67

100 100

COI

LDH-A

LDH-B

MDHc-A

Hb-B

Hb-A

La_calCy_carAn_fim

Ap_carSi_chu

Se_dumSc_sco

Ta_rubPa_for

Ch_rasNo_cor


Tr_murCo_armCo_yaq

Co_acrPo_ret

Fu_hetLa_cha

62100

73

87

100

100

005

100

99


Cy_carLa_calAp_car


Or_latAp_car

Sp_idiSa_salOs_mor

100

6479

100

100

100

100

100100

9699

7990

8055

70



Ac carAc carAc car

Tr murNo corOs mor

Co armCh rasSa sal

Me merSp idiSp idi

Ga mor

Or lat

MDHc-A

LDH-ALDH-B

450

400

350

300

250

200

150

100

50

00

ΔG

(cal

mol

)

(a)

ΔG

(cal

mol

)


480

500

520

540

560

580

600

Actin 1

Actin 2bActin 2a

(b)

1000

800

600

400

200

00

minus200

ΔG

(cal

mol

)

Hb-AHb-B


(c)











2-chains (Hb-









4 Conclusions










Acknowledgments



References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
























1205722


1205732


















Table 1 Continued

























































(a)












(b)















10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S



B G H L M S

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

2-Cys peroxiredoxin

B G H L M S

10

08

06

04

02

00Relat

ive f

old

expr

essio

n


B G H L M S

10

08

06

04

02

00Relat

ive f

old

expr

essio

n

1205732-Globin

B G H L M S

10

08

06

04

02

00Relat

ive f

old

expr

essio

n


08

06

04

02

00Relat

ive f

old

expr

essio

n

B G H L M S

1205722-Globin

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


12

14

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H LM S

Figure 2 Continued



08

06

04

02

00

Relat

ive f

old

expr

essio

n

B GH LM S

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H LM S


10

12

08

06

04

02

00Re

lativ

e fol

d ex

pres

sion

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BG H L M S

Cathepsin S

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

Transferrin

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

GH L M S


10

12

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BG H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BG H L M S


08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

Aldolase B

Figure 2 Continued


12

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BH L M S

Type-4

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

nB G H L M S


12

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S









EF-1Blowast

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SBHSP90

10

08

04

06

02

00

H G L M SB

PRDX1∘10

08

04

06

02

00

H G L M SB

SOD-1∘10

08

04

06

02

00

H G L M SB

Ferr10

08

04

06

02

00

H G L M SB

Hb-A10

08

04

06

02

00

H G L M SB

Hb-B10

08

04

06

02

00

H G L M SB

EPD-110

08

04

06

02

00

H G L M SB

BLBP10

08

04

06

02

00H G L M SB

TSPAN-810

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

BTF3∘

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

MYCBP10

08

04

06

02

00

H G L M SB

CTSS

STF-1

ContigsqPCR

Rab-1A∘

TRPM-1














Table6Specieslist

with

inform

ations

ontheirtypeo

fenviro

nment(M

=marineBR

=brackish

andFW

=fre



fgenes

used

inthe

study

with

relativeG

enbank

accessionnu

mbers

Order

Family

Species

Com

mon

name

Environm

ent

Clim

ate

Depth

range

Gene

NCB

IAccN

r

Percifo

rmes

Trichiuridae

Aphanopu

scarbo

Blackscabbardfish

MDeep-water

200ndash

1700

COI

EU8540

76

Beloniform

esAd

rianichthydae

Oryzias

latip

esJapanese

ricefi

shFW

+BR

Subtropical

shallow

ACTA

1MDHcMyH

CCO

I

NM

00110

4806

NM

00116

3134

XM00

4071618AB4

9806

6

Scorpaenifo

rmes

Hexagrammidae

Pleurogram

mus

azonus

Okh

otsk

atka

mackerel

MTemperate

0ndash240

ACTA

1AB0

73381

Percifo

rmes

Scom

bridae

Scom

berscombrus

Atlanticmackerl

MTemperate

0ndash200

(0ndash100

0)AC

TA1CO

IEF

607093K

C015895

Percifo

rmes

Percihcthyidae

Sinipercachuatsi

Mandarin

fish

FWTemperate

10AC

TA1MyH

CCO

IAY

395872A

Y454304

NC

015822

Percifo

rmes

Sparidae

Sparus

aurata

Gilthead

seabream

MTemperate

1ndash30

(1ndash150)

ACTA

1AF190473

Percifo

rmes

Sphyraenidae

Sphyraenaidiaste

sPelican

barracud

aM

Trop

ical

3ndash24

ACTA

1LD

H-AM

DHc

mMDH

AF503593SIU80001

AF390559AF390561

Tetraodo

ntifo

rmes

Tetraodo

ntidae

Takifugu

rubripes

Japanese

pufferfish

M+FW

+BR

Temperate

0ndash200

(0ndash100

0)Hb-Am

MDHC

OI

XM003964767

XM003965959HM102315

Scorpaenifo

rmes

Ano

plop

omatidae

Anoplopomafim

bria

Sablefish

MDeep-water

0ndash2740

Hb-B

COI

BT082849JQ353978

Percifo

rmes

Serranidae

Epinepheluscoioides

Orange-spottedgrou

per

M+BR

Subtropical

1ndash100

Hb-B

GU982530

Gasteroste

iform

esGasteroste

idae

Gasteroste

usaculeatusTh

ree-spined

stickleb

ack

M+FW

+BR

Temperate

0ndash100

Hb-B

NM

001267638

Percifo

rmes

Nototheniidae

Nototheniacoriiceps

Blackrockcod

MPo

lar

0ndash550

LDH-AM

yHC

COI

AF0

79822AJ

243767

EU326390

Percifo

rmes

Nototheniidae

Nototheniaangusta

taMaorichief

MTemperate

0ndash100

Hb-AH

b-B

P62363P

29628

Gadifo

rmes

Macrouridae

Coryphaenoides

armatus

Abyssalgrenadier

MDeep-water

282ndash5180

LDH-BM

yHC

COI

AJ60

9232A

B330140

FJ1644

97Gadifo

rmes

Gadidae

Gadus

morhu

aAtlanticcod

M+BR

Temperate

0ndash60

0LD

H-BC

OI

AJ60

9233K

C015385

Gadifo

rmes

Gadidae

Arctogadus

glacia

lisArctic

cod

MDeep-water

0ndash1000

Hb-AC

OI

Q1AGS4K

C015200

Percifo

rmes

Latid

aeLa

tescalcarifer

Barram

undi

M+FW

+BR

Trop

ical

10ndash4

0LD

H-BC

OI

FJ439507JQ431879

Gadifo

rmes

Gadidae

Merlangiusm

erlangus

Whitin

gM

Temperate

30ndash100

(10ndash

200)

LDH-BC

OI

AJ60

9234JQ623954

Cyprinod

ontiformes

Poeciliidae

Poeciliareticulata

Gup

pyFW

+BR

Trop

ical

Shallow

LDH-BC

OI

EF40

8825JX9

68696

Gadifo

rmes

Macrouridae

Trachyrin

cusm

urrayi

Roug

hnoseg

renadier

MDeep-water

0ndash1630

LDH-BC

OI

AJ60

9235A

P008990

Percifo

rmes

Channichthyidae

Chionodraco

rastrospinosus

Ocellatedicefish

MPo

lar

0ndash1000

LDH-AC

OI

AF0

79829EU

326337

Percifo

rmes

Pomacentridae

Chromiscaud

alis

Blue-axilchrom

isM

Trop

ical

15ndash55

LDH-A

AY289558

Percifo

rmes

Gob

iidae

Rhinogobiops

nicholsii

Blackeye

goby

MSubtropical

-106

LDH-A

AF0

79534

Cyprinifo

rmes

Cyprinidae

Cyprinus

carpio

Com

mon

carp

FW+BR

Subtropical

shallow

LDH-AM

yHC

COI

AF0

76528D89992

HQ960709


Table6Con

tinued

Order

Family

Species

Com

mon

name

Environm

ent

Clim

ate

Depth

range

Gene

NCB

IAccN

r

Cyprinod

ontiformes

Fund

ulidae

Fund

ulus

heteroclitus

Mum

micho

gM

+FW

+BR

Temperate

shallow

LDH-ALDH-BC

OI

L43525L23792EU

524629

Osm

erifo

rmes

Osm

eridae

Osm

erus

mordax

Rainbo

wsm

eltM

+FW

+BR

Temperate

0ndash425

MDHcmMDH

BT075651B

T07560

0

Salm

onifo

rmes

Salm

onidae

Salm

osalar

Atlanticsalm

onM

+FW

+BR

Temperate

10ndash23

(0ndash210)

MDHcmMDH

BT06

0183B

T048216

Gadifo

rmes

Macrouridae

Coryphaenoides

acrolep

isPacific

grenadier

MDeep-water

900ndash

1300

MyH

CCO

IAB3

30141JQ

3540

60

Gadifo

rmes

Macrouridae

Coryphaenoides

yaquinae

na

MDeep-water

3400ndash5800

MyH

CCO

IAB3

30139GU44

0291

Percifo

rmes

Cirrhitid

aePa

racir

rhitesforste

riBlacksideh

awkfi

shM

Trop

ical

5ndash35

MyH

CCO

IAJ

243770H

Q561521

Percifo

rmes

Carang

idae

Serio

ladu

merili

Greater

amberja

ckM

Subtropical

18ndash72

(1ndash360)

MyH

CCO

IAB0

32020KC

015917

Gadifo

rmes

Gadidae

Boreogadus

saida

Polarc

odM

+BR

Polar

0ndash40

0Hb-AH

b-B

COI

DQ125471Q

1AGS6

KC015250



(a) LDH-A (332 AA)


(b) MDHc-A (333 AA)




(d) LDH-B (334 AA)


(e) MyHC1 (305 AA)


(f) Hb-A1 (143 AA)



(g) Hb-B1 (146 AA)







02

01

Ep_coiGa_acuAn_fim

Ap_carBo_sai

No_angNo_ang

Ap_carTa_rub

Bo_saiAr_glaGa_mor

52

5452

66

100

5359

67

100 100

COI

LDH-A

LDH-B

MDHc-A

Hb-B

Hb-A

La_calCy_carAn_fim

Ap_carSi_chu

Se_dumSc_sco

Ta_rubPa_for

Ch_rasNo_cor


Tr_murCo_armCo_yaq

Co_acrPo_ret

Fu_hetLa_cha

62100

73

87

100

100

005

100

99


Cy_carLa_calAp_car


Or_latAp_car

Sp_idiSa_salOs_mor

100

6479

100

100

100

100

100100

9699

7990

8055

70



Ac carAc carAc car

Tr murNo corOs mor

Co armCh rasSa sal

Me merSp idiSp idi

Ga mor

Or lat

MDHc-A

LDH-ALDH-B

450

400

350

300

250

200

150

100

50

00

ΔG

(cal

mol

)

(a)

ΔG

(cal

mol

)


480

500

520

540

560

580

600

Actin 1

Actin 2bActin 2a

(b)

1000

800

600

400

200

00

minus200

ΔG

(cal

mol

)

Hb-AHb-B


(c)











2-chains (Hb-









4 Conclusions










Acknowledgments



References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 1 Continued

























































(a)












(b)















10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S



B G H L M S

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

2-Cys peroxiredoxin

B G H L M S

10

08

06

04

02

00Relat

ive f

old

expr

essio

n


B G H L M S

10

08

06

04

02

00Relat

ive f

old

expr

essio

n

1205732-Globin

B G H L M S

10

08

06

04

02

00Relat

ive f

old

expr

essio

n


08

06

04

02

00Relat

ive f

old

expr

essio

n

B G H L M S

1205722-Globin

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


12

14

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H LM S

Figure 2 Continued



08

06

04

02

00

Relat

ive f

old

expr

essio

n

B GH LM S

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H LM S


10

12

08

06

04

02

00Re

lativ

e fol

d ex

pres

sion

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BG H L M S

Cathepsin S

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

Transferrin

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

GH L M S


10

12

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BG H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BG H L M S


08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

Aldolase B

Figure 2 Continued


12

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BH L M S

Type-4

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

nB G H L M S


12

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S









EF-1Blowast

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SBHSP90

10

08

04

06

02

00

H G L M SB

PRDX1∘10

08

04

06

02

00

H G L M SB

SOD-1∘10

08

04

06

02

00

H G L M SB

Ferr10

08

04

06

02

00

H G L M SB

Hb-A10

08

04

06

02

00

H G L M SB

Hb-B10

08

04

06

02

00

H G L M SB

EPD-110

08

04

06

02

00

H G L M SB

BLBP10

08

04

06

02

00H G L M SB

TSPAN-810

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

BTF3∘

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

MYCBP10

08

04

06

02

00

H G L M SB

CTSS

STF-1

ContigsqPCR

Rab-1A∘

TRPM-1














Table6Specieslist

with

inform

ations

ontheirtypeo

fenviro

nment(M

=marineBR

=brackish

andFW

=fre



fgenes

used

inthe

study

with

relativeG

enbank

accessionnu

mbers

Order

Family

Species

Com

mon

name

Environm

ent

Clim

ate

Depth

range

Gene

NCB

IAccN

r

Percifo

rmes

Trichiuridae

Aphanopu

scarbo

Blackscabbardfish

MDeep-water

200ndash

1700

COI

EU8540

76

Beloniform

esAd

rianichthydae

Oryzias

latip

esJapanese

ricefi

shFW

+BR

Subtropical

shallow

ACTA

1MDHcMyH

CCO

I

NM

00110

4806

NM

00116

3134

XM00

4071618AB4

9806

6

Scorpaenifo

rmes

Hexagrammidae

Pleurogram

mus

azonus

Okh

otsk

atka

mackerel

MTemperate

0ndash240

ACTA

1AB0

73381

Percifo

rmes

Scom

bridae

Scom

berscombrus

Atlanticmackerl

MTemperate

0ndash200

(0ndash100

0)AC

TA1CO

IEF

607093K

C015895

Percifo

rmes

Percihcthyidae

Sinipercachuatsi

Mandarin

fish

FWTemperate

10AC

TA1MyH

CCO

IAY

395872A

Y454304

NC

015822

Percifo

rmes

Sparidae

Sparus

aurata

Gilthead

seabream

MTemperate

1ndash30

(1ndash150)

ACTA

1AF190473

Percifo

rmes

Sphyraenidae

Sphyraenaidiaste

sPelican

barracud

aM

Trop

ical

3ndash24

ACTA

1LD

H-AM

DHc

mMDH

AF503593SIU80001

AF390559AF390561

Tetraodo

ntifo

rmes

Tetraodo

ntidae

Takifugu

rubripes

Japanese

pufferfish

M+FW

+BR

Temperate

0ndash200

(0ndash100

0)Hb-Am

MDHC

OI

XM003964767

XM003965959HM102315

Scorpaenifo

rmes

Ano

plop

omatidae

Anoplopomafim

bria

Sablefish

MDeep-water

0ndash2740

Hb-B

COI

BT082849JQ353978

Percifo

rmes

Serranidae

Epinepheluscoioides

Orange-spottedgrou

per

M+BR

Subtropical

1ndash100

Hb-B

GU982530

Gasteroste

iform

esGasteroste

idae

Gasteroste

usaculeatusTh

ree-spined

stickleb

ack

M+FW

+BR

Temperate

0ndash100

Hb-B

NM

001267638

Percifo

rmes

Nototheniidae

Nototheniacoriiceps

Blackrockcod

MPo

lar

0ndash550

LDH-AM

yHC

COI

AF0

79822AJ

243767

EU326390

Percifo

rmes

Nototheniidae

Nototheniaangusta

taMaorichief

MTemperate

0ndash100

Hb-AH

b-B

P62363P

29628

Gadifo

rmes

Macrouridae

Coryphaenoides

armatus

Abyssalgrenadier

MDeep-water

282ndash5180

LDH-BM

yHC

COI

AJ60

9232A

B330140

FJ1644

97Gadifo

rmes

Gadidae

Gadus

morhu

aAtlanticcod

M+BR

Temperate

0ndash60

0LD

H-BC

OI

AJ60

9233K

C015385

Gadifo

rmes

Gadidae

Arctogadus

glacia

lisArctic

cod

MDeep-water

0ndash1000

Hb-AC

OI

Q1AGS4K

C015200

Percifo

rmes

Latid

aeLa

tescalcarifer

Barram

undi

M+FW

+BR

Trop

ical

10ndash4

0LD

H-BC

OI

FJ439507JQ431879

Gadifo

rmes

Gadidae

Merlangiusm

erlangus

Whitin

gM

Temperate

30ndash100

(10ndash

200)

LDH-BC

OI

AJ60

9234JQ623954

Cyprinod

ontiformes

Poeciliidae

Poeciliareticulata

Gup

pyFW

+BR

Trop

ical

Shallow

LDH-BC

OI

EF40

8825JX9

68696

Gadifo

rmes

Macrouridae

Trachyrin

cusm

urrayi

Roug

hnoseg

renadier

MDeep-water

0ndash1630

LDH-BC

OI

AJ60

9235A

P008990

Percifo

rmes

Channichthyidae

Chionodraco

rastrospinosus

Ocellatedicefish

MPo

lar

0ndash1000

LDH-AC

OI

AF0

79829EU

326337

Percifo

rmes

Pomacentridae

Chromiscaud

alis

Blue-axilchrom

isM

Trop

ical

15ndash55

LDH-A

AY289558

Percifo

rmes

Gob

iidae

Rhinogobiops

nicholsii

Blackeye

goby

MSubtropical

-106

LDH-A

AF0

79534

Cyprinifo

rmes

Cyprinidae

Cyprinus

carpio

Com

mon

carp

FW+BR

Subtropical

shallow

LDH-AM

yHC

COI

AF0

76528D89992

HQ960709


Table6Con

tinued

Order

Family

Species

Com

mon

name

Environm

ent

Clim

ate

Depth

range

Gene

NCB

IAccN

r

Cyprinod

ontiformes

Fund

ulidae

Fund

ulus

heteroclitus

Mum

micho

gM

+FW

+BR

Temperate

shallow

LDH-ALDH-BC

OI

L43525L23792EU

524629

Osm

erifo

rmes

Osm

eridae

Osm

erus

mordax

Rainbo

wsm

eltM

+FW

+BR

Temperate

0ndash425

MDHcmMDH

BT075651B

T07560

0

Salm

onifo

rmes

Salm

onidae

Salm

osalar

Atlanticsalm

onM

+FW

+BR

Temperate

10ndash23

(0ndash210)

MDHcmMDH

BT06

0183B

T048216

Gadifo

rmes

Macrouridae

Coryphaenoides

acrolep

isPacific

grenadier

MDeep-water

900ndash

1300

MyH

CCO

IAB3

30141JQ

3540

60

Gadifo

rmes

Macrouridae

Coryphaenoides

yaquinae

na

MDeep-water

3400ndash5800

MyH

CCO

IAB3

30139GU44

0291

Percifo

rmes

Cirrhitid

aePa

racir

rhitesforste

riBlacksideh

awkfi

shM

Trop

ical

5ndash35

MyH

CCO

IAJ

243770H

Q561521

Percifo

rmes

Carang

idae

Serio

ladu

merili

Greater

amberja

ckM

Subtropical

18ndash72

(1ndash360)

MyH

CCO

IAB0

32020KC

015917

Gadifo

rmes

Gadidae

Boreogadus

saida

Polarc

odM

+BR

Polar

0ndash40

0Hb-AH

b-B

COI

DQ125471Q

1AGS6

KC015250



(a) LDH-A (332 AA)


(b) MDHc-A (333 AA)




(d) LDH-B (334 AA)


(e) MyHC1 (305 AA)


(f) Hb-A1 (143 AA)



(g) Hb-B1 (146 AA)







02

01

Ep_coiGa_acuAn_fim

Ap_carBo_sai

No_angNo_ang

Ap_carTa_rub

Bo_saiAr_glaGa_mor

52

5452

66

100

5359

67

100 100

COI

LDH-A

LDH-B

MDHc-A

Hb-B

Hb-A

La_calCy_carAn_fim

Ap_carSi_chu

Se_dumSc_sco

Ta_rubPa_for

Ch_rasNo_cor


Tr_murCo_armCo_yaq

Co_acrPo_ret

Fu_hetLa_cha

62100

73

87

100

100

005

100

99


Cy_carLa_calAp_car


Or_latAp_car

Sp_idiSa_salOs_mor

100

6479

100

100

100

100

100100

9699

7990

8055

70



Ac carAc carAc car

Tr murNo corOs mor

Co armCh rasSa sal

Me merSp idiSp idi

Ga mor

Or lat

MDHc-A

LDH-ALDH-B

450

400

350

300

250

200

150

100

50

00

ΔG

(cal

mol

)

(a)

ΔG

(cal

mol

)


480

500

520

540

560

580

600

Actin 1

Actin 2bActin 2a

(b)

1000

800

600

400

200

00

minus200

ΔG

(cal

mol

)

Hb-AHb-B


(c)











2-chains (Hb-









4 Conclusions










Acknowledgments



References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








































(a)












(b)















10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S



B G H L M S

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

2-Cys peroxiredoxin

B G H L M S

10

08

06

04

02

00Relat

ive f

old

expr

essio

n


B G H L M S

10

08

06

04

02

00Relat

ive f

old

expr

essio

n

1205732-Globin

B G H L M S

10

08

06

04

02

00Relat

ive f

old

expr

essio

n


08

06

04

02

00Relat

ive f

old

expr

essio

n

B G H L M S

1205722-Globin

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


12

14

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H LM S

Figure 2 Continued



08

06

04

02

00

Relat

ive f

old

expr

essio

n

B GH LM S

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H LM S


10

12

08

06

04

02

00Re

lativ

e fol

d ex

pres

sion

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BG H L M S

Cathepsin S

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

Transferrin

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

GH L M S


10

12

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BG H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BG H L M S


08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

Aldolase B

Figure 2 Continued


12

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BH L M S

Type-4

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

nB G H L M S


12

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S









EF-1Blowast

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SBHSP90

10

08

04

06

02

00

H G L M SB

PRDX1∘10

08

04

06

02

00

H G L M SB

SOD-1∘10

08

04

06

02

00

H G L M SB

Ferr10

08

04

06

02

00

H G L M SB

Hb-A10

08

04

06

02

00

H G L M SB

Hb-B10

08

04

06

02

00

H G L M SB

EPD-110

08

04

06

02

00

H G L M SB

BLBP10

08

04

06

02

00H G L M SB

TSPAN-810

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

BTF3∘

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

MYCBP10

08

04

06

02

00

H G L M SB

CTSS

STF-1

ContigsqPCR

Rab-1A∘

TRPM-1














Table6Specieslist

with

inform

ations

ontheirtypeo

fenviro

nment(M

=marineBR

=brackish

andFW

=fre



fgenes

used

inthe

study

with

relativeG

enbank

accessionnu

mbers

Order

Family

Species

Com

mon

name

Environm

ent

Clim

ate

Depth

range

Gene

NCB

IAccN

r

Percifo

rmes

Trichiuridae

Aphanopu

scarbo

Blackscabbardfish

MDeep-water

200ndash

1700

COI

EU8540

76

Beloniform

esAd

rianichthydae

Oryzias

latip

esJapanese

ricefi

shFW

+BR

Subtropical

shallow

ACTA

1MDHcMyH

CCO

I

NM

00110

4806

NM

00116

3134

XM00

4071618AB4

9806

6

Scorpaenifo

rmes

Hexagrammidae

Pleurogram

mus

azonus

Okh

otsk

atka

mackerel

MTemperate

0ndash240

ACTA

1AB0

73381

Percifo

rmes

Scom

bridae

Scom

berscombrus

Atlanticmackerl

MTemperate

0ndash200

(0ndash100

0)AC

TA1CO

IEF

607093K

C015895

Percifo

rmes

Percihcthyidae

Sinipercachuatsi

Mandarin

fish

FWTemperate

10AC

TA1MyH

CCO

IAY

395872A

Y454304

NC

015822

Percifo

rmes

Sparidae

Sparus

aurata

Gilthead

seabream

MTemperate

1ndash30

(1ndash150)

ACTA

1AF190473

Percifo

rmes

Sphyraenidae

Sphyraenaidiaste

sPelican

barracud

aM

Trop

ical

3ndash24

ACTA

1LD

H-AM

DHc

mMDH

AF503593SIU80001

AF390559AF390561

Tetraodo

ntifo

rmes

Tetraodo

ntidae

Takifugu

rubripes

Japanese

pufferfish

M+FW

+BR

Temperate

0ndash200

(0ndash100

0)Hb-Am

MDHC

OI

XM003964767

XM003965959HM102315

Scorpaenifo

rmes

Ano

plop

omatidae

Anoplopomafim

bria

Sablefish

MDeep-water

0ndash2740

Hb-B

COI

BT082849JQ353978

Percifo

rmes

Serranidae

Epinepheluscoioides

Orange-spottedgrou

per

M+BR

Subtropical

1ndash100

Hb-B

GU982530

Gasteroste

iform

esGasteroste

idae

Gasteroste

usaculeatusTh

ree-spined

stickleb

ack

M+FW

+BR

Temperate

0ndash100

Hb-B

NM

001267638

Percifo

rmes

Nototheniidae

Nototheniacoriiceps

Blackrockcod

MPo

lar

0ndash550

LDH-AM

yHC

COI

AF0

79822AJ

243767

EU326390

Percifo

rmes

Nototheniidae

Nototheniaangusta

taMaorichief

MTemperate

0ndash100

Hb-AH

b-B

P62363P

29628

Gadifo

rmes

Macrouridae

Coryphaenoides

armatus

Abyssalgrenadier

MDeep-water

282ndash5180

LDH-BM

yHC

COI

AJ60

9232A

B330140

FJ1644

97Gadifo

rmes

Gadidae

Gadus

morhu

aAtlanticcod

M+BR

Temperate

0ndash60

0LD

H-BC

OI

AJ60

9233K

C015385

Gadifo

rmes

Gadidae

Arctogadus

glacia

lisArctic

cod

MDeep-water

0ndash1000

Hb-AC

OI

Q1AGS4K

C015200

Percifo

rmes

Latid

aeLa

tescalcarifer

Barram

undi

M+FW

+BR

Trop

ical

10ndash4

0LD

H-BC

OI

FJ439507JQ431879

Gadifo

rmes

Gadidae

Merlangiusm

erlangus

Whitin

gM

Temperate

30ndash100

(10ndash

200)

LDH-BC

OI

AJ60

9234JQ623954

Cyprinod

ontiformes

Poeciliidae

Poeciliareticulata

Gup

pyFW

+BR

Trop

ical

Shallow

LDH-BC

OI

EF40

8825JX9

68696

Gadifo

rmes

Macrouridae

Trachyrin

cusm

urrayi

Roug

hnoseg

renadier

MDeep-water

0ndash1630

LDH-BC

OI

AJ60

9235A

P008990

Percifo

rmes

Channichthyidae

Chionodraco

rastrospinosus

Ocellatedicefish

MPo

lar

0ndash1000

LDH-AC

OI

AF0

79829EU

326337

Percifo

rmes

Pomacentridae

Chromiscaud

alis

Blue-axilchrom

isM

Trop

ical

15ndash55

LDH-A

AY289558

Percifo

rmes

Gob

iidae

Rhinogobiops

nicholsii

Blackeye

goby

MSubtropical

-106

LDH-A

AF0

79534

Cyprinifo

rmes

Cyprinidae

Cyprinus

carpio

Com

mon

carp

FW+BR

Subtropical

shallow

LDH-AM

yHC

COI

AF0

76528D89992

HQ960709


Table6Con

tinued

Order

Family

Species

Com

mon

name

Environm

ent

Clim

ate

Depth

range

Gene

NCB

IAccN

r

Cyprinod

ontiformes

Fund

ulidae

Fund

ulus

heteroclitus

Mum

micho

gM

+FW

+BR

Temperate

shallow

LDH-ALDH-BC

OI

L43525L23792EU

524629

Osm

erifo

rmes

Osm

eridae

Osm

erus

mordax

Rainbo

wsm

eltM

+FW

+BR

Temperate

0ndash425

MDHcmMDH

BT075651B

T07560

0

Salm

onifo

rmes

Salm

onidae

Salm

osalar

Atlanticsalm

onM

+FW

+BR

Temperate

10ndash23

(0ndash210)

MDHcmMDH

BT06

0183B

T048216

Gadifo

rmes

Macrouridae

Coryphaenoides

acrolep

isPacific

grenadier

MDeep-water

900ndash

1300

MyH

CCO

IAB3

30141JQ

3540

60

Gadifo

rmes

Macrouridae

Coryphaenoides

yaquinae

na

MDeep-water

3400ndash5800

MyH

CCO

IAB3

30139GU44

0291

Percifo

rmes

Cirrhitid

aePa

racir

rhitesforste

riBlacksideh

awkfi

shM

Trop

ical

5ndash35

MyH

CCO

IAJ

243770H

Q561521

Percifo

rmes

Carang

idae

Serio

ladu

merili

Greater

amberja

ckM

Subtropical

18ndash72

(1ndash360)

MyH

CCO

IAB0

32020KC

015917

Gadifo

rmes

Gadidae

Boreogadus

saida

Polarc

odM

+BR

Polar

0ndash40

0Hb-AH

b-B

COI

DQ125471Q

1AGS6

KC015250



(a) LDH-A (332 AA)


(b) MDHc-A (333 AA)




(d) LDH-B (334 AA)


(e) MyHC1 (305 AA)


(f) Hb-A1 (143 AA)



(g) Hb-B1 (146 AA)







02

01

Ep_coiGa_acuAn_fim

Ap_carBo_sai

No_angNo_ang

Ap_carTa_rub

Bo_saiAr_glaGa_mor

52

5452

66

100

5359

67

100 100

COI

LDH-A

LDH-B

MDHc-A

Hb-B

Hb-A

La_calCy_carAn_fim

Ap_carSi_chu

Se_dumSc_sco

Ta_rubPa_for

Ch_rasNo_cor


Tr_murCo_armCo_yaq

Co_acrPo_ret

Fu_hetLa_cha

62100

73

87

100

100

005

100

99


Cy_carLa_calAp_car


Or_latAp_car

Sp_idiSa_salOs_mor

100

6479

100

100

100

100

100100

9699

7990

8055

70



Ac carAc carAc car

Tr murNo corOs mor

Co armCh rasSa sal

Me merSp idiSp idi

Ga mor

Or lat

MDHc-A

LDH-ALDH-B

450

400

350

300

250

200

150

100

50

00

ΔG

(cal

mol

)

(a)

ΔG

(cal

mol

)


480

500

520

540

560

580

600

Actin 1

Actin 2bActin 2a

(b)

1000

800

600

400

200

00

minus200

ΔG

(cal

mol

)

Hb-AHb-B


(c)











2-chains (Hb-









4 Conclusions










Acknowledgments



References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S



B G H L M S

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

2-Cys peroxiredoxin

B G H L M S

10

08

06

04

02

00Relat

ive f

old

expr

essio

n


B G H L M S

10

08

06

04

02

00Relat

ive f

old

expr

essio

n

1205732-Globin

B G H L M S

10

08

06

04

02

00Relat

ive f

old

expr

essio

n


08

06

04

02

00Relat

ive f

old

expr

essio

n

B G H L M S

1205722-Globin

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


12

14

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H LM S

Figure 2 Continued



08

06

04

02

00

Relat

ive f

old

expr

essio

n

B GH LM S

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H LM S


10

12

08

06

04

02

00Re

lativ

e fol

d ex

pres

sion

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BG H L M S

Cathepsin S

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

Transferrin

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

GH L M S


10

12

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BG H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BG H L M S


08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

Aldolase B

Figure 2 Continued


12

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BH L M S

Type-4

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

nB G H L M S


12

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S









EF-1Blowast

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SBHSP90

10

08

04

06

02

00

H G L M SB

PRDX1∘10

08

04

06

02

00

H G L M SB

SOD-1∘10

08

04

06

02

00

H G L M SB

Ferr10

08

04

06

02

00

H G L M SB

Hb-A10

08

04

06

02

00

H G L M SB

Hb-B10

08

04

06

02

00

H G L M SB

EPD-110

08

04

06

02

00

H G L M SB

BLBP10

08

04

06

02

00H G L M SB

TSPAN-810

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

BTF3∘

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

MYCBP10

08

04

06

02

00

H G L M SB

CTSS

STF-1

ContigsqPCR

Rab-1A∘

TRPM-1














Table6Specieslist

with

inform

ations

ontheirtypeo

fenviro

nment(M

=marineBR

=brackish

andFW

=fre



fgenes

used

inthe

study

with

relativeG

enbank

accessionnu

mbers

Order

Family

Species

Com

mon

name

Environm

ent

Clim

ate

Depth

range

Gene

NCB

IAccN

r

Percifo

rmes

Trichiuridae

Aphanopu

scarbo

Blackscabbardfish

MDeep-water

200ndash

1700

COI

EU8540

76

Beloniform

esAd

rianichthydae

Oryzias

latip

esJapanese

ricefi

shFW

+BR

Subtropical

shallow

ACTA

1MDHcMyH

CCO

I

NM

00110

4806

NM

00116

3134

XM00

4071618AB4

9806

6

Scorpaenifo

rmes

Hexagrammidae

Pleurogram

mus

azonus

Okh

otsk

atka

mackerel

MTemperate

0ndash240

ACTA

1AB0

73381

Percifo

rmes

Scom

bridae

Scom

berscombrus

Atlanticmackerl

MTemperate

0ndash200

(0ndash100

0)AC

TA1CO

IEF

607093K

C015895

Percifo

rmes

Percihcthyidae

Sinipercachuatsi

Mandarin

fish

FWTemperate

10AC

TA1MyH

CCO

IAY

395872A

Y454304

NC

015822

Percifo

rmes

Sparidae

Sparus

aurata

Gilthead

seabream

MTemperate

1ndash30

(1ndash150)

ACTA

1AF190473

Percifo

rmes

Sphyraenidae

Sphyraenaidiaste

sPelican

barracud

aM

Trop

ical

3ndash24

ACTA

1LD

H-AM

DHc

mMDH

AF503593SIU80001

AF390559AF390561

Tetraodo

ntifo

rmes

Tetraodo

ntidae

Takifugu

rubripes

Japanese

pufferfish

M+FW

+BR

Temperate

0ndash200

(0ndash100

0)Hb-Am

MDHC

OI

XM003964767

XM003965959HM102315

Scorpaenifo

rmes

Ano

plop

omatidae

Anoplopomafim

bria

Sablefish

MDeep-water

0ndash2740

Hb-B

COI

BT082849JQ353978

Percifo

rmes

Serranidae

Epinepheluscoioides

Orange-spottedgrou

per

M+BR

Subtropical

1ndash100

Hb-B

GU982530

Gasteroste

iform

esGasteroste

idae

Gasteroste

usaculeatusTh

ree-spined

stickleb

ack

M+FW

+BR

Temperate

0ndash100

Hb-B

NM

001267638

Percifo

rmes

Nototheniidae

Nototheniacoriiceps

Blackrockcod

MPo

lar

0ndash550

LDH-AM

yHC

COI

AF0

79822AJ

243767

EU326390

Percifo

rmes

Nototheniidae

Nototheniaangusta

taMaorichief

MTemperate

0ndash100

Hb-AH

b-B

P62363P

29628

Gadifo

rmes

Macrouridae

Coryphaenoides

armatus

Abyssalgrenadier

MDeep-water

282ndash5180

LDH-BM

yHC

COI

AJ60

9232A

B330140

FJ1644

97Gadifo

rmes

Gadidae

Gadus

morhu

aAtlanticcod

M+BR

Temperate

0ndash60

0LD

H-BC

OI

AJ60

9233K

C015385

Gadifo

rmes

Gadidae

Arctogadus

glacia

lisArctic

cod

MDeep-water

0ndash1000

Hb-AC

OI

Q1AGS4K

C015200

Percifo

rmes

Latid

aeLa

tescalcarifer

Barram

undi

M+FW

+BR

Trop

ical

10ndash4

0LD

H-BC

OI

FJ439507JQ431879

Gadifo

rmes

Gadidae

Merlangiusm

erlangus

Whitin

gM

Temperate

30ndash100

(10ndash

200)

LDH-BC

OI

AJ60

9234JQ623954

Cyprinod

ontiformes

Poeciliidae

Poeciliareticulata

Gup

pyFW

+BR

Trop

ical

Shallow

LDH-BC

OI

EF40

8825JX9

68696

Gadifo

rmes

Macrouridae

Trachyrin

cusm

urrayi

Roug

hnoseg

renadier

MDeep-water

0ndash1630

LDH-BC

OI

AJ60

9235A

P008990

Percifo

rmes

Channichthyidae

Chionodraco

rastrospinosus

Ocellatedicefish

MPo

lar

0ndash1000

LDH-AC

OI

AF0

79829EU

326337

Percifo

rmes

Pomacentridae

Chromiscaud

alis

Blue-axilchrom

isM

Trop

ical

15ndash55

LDH-A

AY289558

Percifo

rmes

Gob

iidae

Rhinogobiops

nicholsii

Blackeye

goby

MSubtropical

-106

LDH-A

AF0

79534

Cyprinifo

rmes

Cyprinidae

Cyprinus

carpio

Com

mon

carp

FW+BR

Subtropical

shallow

LDH-AM

yHC

COI

AF0

76528D89992

HQ960709


Table6Con

tinued

Order

Family

Species

Com

mon

name

Environm

ent

Clim

ate

Depth

range

Gene

NCB

IAccN

r

Cyprinod

ontiformes

Fund

ulidae

Fund

ulus

heteroclitus

Mum

micho

gM

+FW

+BR

Temperate

shallow

LDH-ALDH-BC

OI

L43525L23792EU

524629

Osm

erifo

rmes

Osm

eridae

Osm

erus

mordax

Rainbo

wsm

eltM

+FW

+BR

Temperate

0ndash425

MDHcmMDH

BT075651B

T07560

0

Salm

onifo

rmes

Salm

onidae

Salm

osalar

Atlanticsalm

onM

+FW

+BR

Temperate

10ndash23

(0ndash210)

MDHcmMDH

BT06

0183B

T048216

Gadifo

rmes

Macrouridae

Coryphaenoides

acrolep

isPacific

grenadier

MDeep-water

900ndash

1300

MyH

CCO

IAB3

30141JQ

3540

60

Gadifo

rmes

Macrouridae

Coryphaenoides

yaquinae

na

MDeep-water

3400ndash5800

MyH

CCO

IAB3

30139GU44

0291

Percifo

rmes

Cirrhitid

aePa

racir

rhitesforste

riBlacksideh

awkfi

shM

Trop

ical

5ndash35

MyH

CCO

IAJ

243770H

Q561521

Percifo

rmes

Carang

idae

Serio

ladu

merili

Greater

amberja

ckM

Subtropical

18ndash72

(1ndash360)

MyH

CCO

IAB0

32020KC

015917

Gadifo

rmes

Gadidae

Boreogadus

saida

Polarc

odM

+BR

Polar

0ndash40

0Hb-AH

b-B

COI

DQ125471Q

1AGS6

KC015250



(a) LDH-A (332 AA)


(b) MDHc-A (333 AA)




(d) LDH-B (334 AA)


(e) MyHC1 (305 AA)


(f) Hb-A1 (143 AA)



(g) Hb-B1 (146 AA)







02

01

Ep_coiGa_acuAn_fim

Ap_carBo_sai

No_angNo_ang

Ap_carTa_rub

Bo_saiAr_glaGa_mor

52

5452

66

100

5359

67

100 100

COI

LDH-A

LDH-B

MDHc-A

Hb-B

Hb-A

La_calCy_carAn_fim

Ap_carSi_chu

Se_dumSc_sco

Ta_rubPa_for

Ch_rasNo_cor


Tr_murCo_armCo_yaq

Co_acrPo_ret

Fu_hetLa_cha

62100

73

87

100

100

005

100

99


Cy_carLa_calAp_car


Or_latAp_car

Sp_idiSa_salOs_mor

100

6479

100

100

100

100

100100

9699

7990

8055

70



Ac carAc carAc car

Tr murNo corOs mor

Co armCh rasSa sal

Me merSp idiSp idi

Ga mor

Or lat

MDHc-A

LDH-ALDH-B

450

400

350

300

250

200

150

100

50

00

ΔG

(cal

mol

)

(a)

ΔG

(cal

mol

)


480

500

520

540

560

580

600

Actin 1

Actin 2bActin 2a

(b)

1000

800

600

400

200

00

minus200

ΔG

(cal

mol

)

Hb-AHb-B


(c)











2-chains (Hb-









4 Conclusions










Acknowledgments



References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



08

06

04

02

00

Relat

ive f

old

expr

essio

n

B GH LM S

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H LM S


10

12

08

06

04

02

00Re

lativ

e fol

d ex

pres

sion

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BG H L M S

Cathepsin S

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

Transferrin

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

GH L M S


10

12

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BG H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BG H L M S


08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S

Aldolase B

Figure 2 Continued


12

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BH L M S

Type-4

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

nB G H L M S


12

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S









EF-1Blowast

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SBHSP90

10

08

04

06

02

00

H G L M SB

PRDX1∘10

08

04

06

02

00

H G L M SB

SOD-1∘10

08

04

06

02

00

H G L M SB

Ferr10

08

04

06

02

00

H G L M SB

Hb-A10

08

04

06

02

00

H G L M SB

Hb-B10

08

04

06

02

00

H G L M SB

EPD-110

08

04

06

02

00

H G L M SB

BLBP10

08

04

06

02

00H G L M SB

TSPAN-810

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

BTF3∘

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

MYCBP10

08

04

06

02

00

H G L M SB

CTSS

STF-1

ContigsqPCR

Rab-1A∘

TRPM-1














Table6Specieslist

with

inform

ations

ontheirtypeo

fenviro

nment(M

=marineBR

=brackish

andFW

=fre



fgenes

used

inthe

study

with

relativeG

enbank

accessionnu

mbers

Order

Family

Species

Com

mon

name

Environm

ent

Clim

ate

Depth

range

Gene

NCB

IAccN

r

Percifo

rmes

Trichiuridae

Aphanopu

scarbo

Blackscabbardfish

MDeep-water

200ndash

1700

COI

EU8540

76

Beloniform

esAd

rianichthydae

Oryzias

latip

esJapanese

ricefi

shFW

+BR

Subtropical

shallow

ACTA

1MDHcMyH

CCO

I

NM

00110

4806

NM

00116

3134

XM00

4071618AB4

9806

6

Scorpaenifo

rmes

Hexagrammidae

Pleurogram

mus

azonus

Okh

otsk

atka

mackerel

MTemperate

0ndash240

ACTA

1AB0

73381

Percifo

rmes

Scom

bridae

Scom

berscombrus

Atlanticmackerl

MTemperate

0ndash200

(0ndash100

0)AC

TA1CO

IEF

607093K

C015895

Percifo

rmes

Percihcthyidae

Sinipercachuatsi

Mandarin

fish

FWTemperate

10AC

TA1MyH

CCO

IAY

395872A

Y454304

NC

015822

Percifo

rmes

Sparidae

Sparus

aurata

Gilthead

seabream

MTemperate

1ndash30

(1ndash150)

ACTA

1AF190473

Percifo

rmes

Sphyraenidae

Sphyraenaidiaste

sPelican

barracud

aM

Trop

ical

3ndash24

ACTA

1LD

H-AM

DHc

mMDH

AF503593SIU80001

AF390559AF390561

Tetraodo

ntifo

rmes

Tetraodo

ntidae

Takifugu

rubripes

Japanese

pufferfish

M+FW

+BR

Temperate

0ndash200

(0ndash100

0)Hb-Am

MDHC

OI

XM003964767

XM003965959HM102315

Scorpaenifo

rmes

Ano

plop

omatidae

Anoplopomafim

bria

Sablefish

MDeep-water

0ndash2740

Hb-B

COI

BT082849JQ353978

Percifo

rmes

Serranidae

Epinepheluscoioides

Orange-spottedgrou

per

M+BR

Subtropical

1ndash100

Hb-B

GU982530

Gasteroste

iform

esGasteroste

idae

Gasteroste

usaculeatusTh

ree-spined

stickleb

ack

M+FW

+BR

Temperate

0ndash100

Hb-B

NM

001267638

Percifo

rmes

Nototheniidae

Nototheniacoriiceps

Blackrockcod

MPo

lar

0ndash550

LDH-AM

yHC

COI

AF0

79822AJ

243767

EU326390

Percifo

rmes

Nototheniidae

Nototheniaangusta

taMaorichief

MTemperate

0ndash100

Hb-AH

b-B

P62363P

29628

Gadifo

rmes

Macrouridae

Coryphaenoides

armatus

Abyssalgrenadier

MDeep-water

282ndash5180

LDH-BM

yHC

COI

AJ60

9232A

B330140

FJ1644

97Gadifo

rmes

Gadidae

Gadus

morhu

aAtlanticcod

M+BR

Temperate

0ndash60

0LD

H-BC

OI

AJ60

9233K

C015385

Gadifo

rmes

Gadidae

Arctogadus

glacia

lisArctic

cod

MDeep-water

0ndash1000

Hb-AC

OI

Q1AGS4K

C015200

Percifo

rmes

Latid

aeLa

tescalcarifer

Barram

undi

M+FW

+BR

Trop

ical

10ndash4

0LD

H-BC

OI

FJ439507JQ431879

Gadifo

rmes

Gadidae

Merlangiusm

erlangus

Whitin

gM

Temperate

30ndash100

(10ndash

200)

LDH-BC

OI

AJ60

9234JQ623954

Cyprinod

ontiformes

Poeciliidae

Poeciliareticulata

Gup

pyFW

+BR

Trop

ical

Shallow

LDH-BC

OI

EF40

8825JX9

68696

Gadifo

rmes

Macrouridae

Trachyrin

cusm

urrayi

Roug

hnoseg

renadier

MDeep-water

0ndash1630

LDH-BC

OI

AJ60

9235A

P008990

Percifo

rmes

Channichthyidae

Chionodraco

rastrospinosus

Ocellatedicefish

MPo

lar

0ndash1000

LDH-AC

OI

AF0

79829EU

326337

Percifo

rmes

Pomacentridae

Chromiscaud

alis

Blue-axilchrom

isM

Trop

ical

15ndash55

LDH-A

AY289558

Percifo

rmes

Gob

iidae

Rhinogobiops

nicholsii

Blackeye

goby

MSubtropical

-106

LDH-A

AF0

79534

Cyprinifo

rmes

Cyprinidae

Cyprinus

carpio

Com

mon

carp

FW+BR

Subtropical

shallow

LDH-AM

yHC

COI

AF0

76528D89992

HQ960709


Table6Con

tinued

Order

Family

Species

Com

mon

name

Environm

ent

Clim

ate

Depth

range

Gene

NCB

IAccN

r

Cyprinod

ontiformes

Fund

ulidae

Fund

ulus

heteroclitus

Mum

micho

gM

+FW

+BR

Temperate

shallow

LDH-ALDH-BC

OI

L43525L23792EU

524629

Osm

erifo

rmes

Osm

eridae

Osm

erus

mordax

Rainbo

wsm

eltM

+FW

+BR

Temperate

0ndash425

MDHcmMDH

BT075651B

T07560

0

Salm

onifo

rmes

Salm

onidae

Salm

osalar

Atlanticsalm

onM

+FW

+BR

Temperate

10ndash23

(0ndash210)

MDHcmMDH

BT06

0183B

T048216

Gadifo

rmes

Macrouridae

Coryphaenoides

acrolep

isPacific

grenadier

MDeep-water

900ndash

1300

MyH

CCO

IAB3

30141JQ

3540

60

Gadifo

rmes

Macrouridae

Coryphaenoides

yaquinae

na

MDeep-water

3400ndash5800

MyH

CCO

IAB3

30139GU44

0291

Percifo

rmes

Cirrhitid

aePa

racir

rhitesforste

riBlacksideh

awkfi

shM

Trop

ical

5ndash35

MyH

CCO

IAJ

243770H

Q561521

Percifo

rmes

Carang

idae

Serio

ladu

merili

Greater

amberja

ckM

Subtropical

18ndash72

(1ndash360)

MyH

CCO

IAB0

32020KC

015917

Gadifo

rmes

Gadidae

Boreogadus

saida

Polarc

odM

+BR

Polar

0ndash40

0Hb-AH

b-B

COI

DQ125471Q

1AGS6

KC015250



(a) LDH-A (332 AA)


(b) MDHc-A (333 AA)




(d) LDH-B (334 AA)


(e) MyHC1 (305 AA)


(f) Hb-A1 (143 AA)



(g) Hb-B1 (146 AA)







02

01

Ep_coiGa_acuAn_fim

Ap_carBo_sai

No_angNo_ang

Ap_carTa_rub

Bo_saiAr_glaGa_mor

52

5452

66

100

5359

67

100 100

COI

LDH-A

LDH-B

MDHc-A

Hb-B

Hb-A

La_calCy_carAn_fim

Ap_carSi_chu

Se_dumSc_sco

Ta_rubPa_for

Ch_rasNo_cor


Tr_murCo_armCo_yaq

Co_acrPo_ret

Fu_hetLa_cha

62100

73

87

100

100

005

100

99


Cy_carLa_calAp_car


Or_latAp_car

Sp_idiSa_salOs_mor

100

6479

100

100

100

100

100100

9699

7990

8055

70



Ac carAc carAc car

Tr murNo corOs mor

Co armCh rasSa sal

Me merSp idiSp idi

Ga mor

Or lat

MDHc-A

LDH-ALDH-B

450

400

350

300

250

200

150

100

50

00

ΔG

(cal

mol

)

(a)

ΔG

(cal

mol

)


480

500

520

540

560

580

600

Actin 1

Actin 2bActin 2a

(b)

1000

800

600

400

200

00

minus200

ΔG

(cal

mol

)

Hb-AHb-B


(c)











2-chains (Hb-









4 Conclusions










Acknowledgments



References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


12

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

BH L M S

Type-4

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

nB G H L M S


12

10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S


10

08

06

04

02

00

Relat

ive f

old

expr

essio

n

B G H L M S









EF-1Blowast

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SBHSP90

10

08

04

06

02

00

H G L M SB

PRDX1∘10

08

04

06

02

00

H G L M SB

SOD-1∘10

08

04

06

02

00

H G L M SB

Ferr10

08

04

06

02

00

H G L M SB

Hb-A10

08

04

06

02

00

H G L M SB

Hb-B10

08

04

06

02

00

H G L M SB

EPD-110

08

04

06

02

00

H G L M SB

BLBP10

08

04

06

02

00H G L M SB

TSPAN-810

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

BTF3∘

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

MYCBP10

08

04

06

02

00

H G L M SB

CTSS

STF-1

ContigsqPCR

Rab-1A∘

TRPM-1














Table6Specieslist

with

inform

ations

ontheirtypeo

fenviro

nment(M

=marineBR

=brackish

andFW

=fre



fgenes

used

inthe

study

with

relativeG

enbank

accessionnu

mbers

Order

Family

Species

Com

mon

name

Environm

ent

Clim

ate

Depth

range

Gene

NCB

IAccN

r

Percifo

rmes

Trichiuridae

Aphanopu

scarbo

Blackscabbardfish

MDeep-water

200ndash

1700

COI

EU8540

76

Beloniform

esAd

rianichthydae

Oryzias

latip

esJapanese

ricefi

shFW

+BR

Subtropical

shallow

ACTA

1MDHcMyH

CCO

I

NM

00110

4806

NM

00116

3134

XM00

4071618AB4

9806

6

Scorpaenifo

rmes

Hexagrammidae

Pleurogram

mus

azonus

Okh

otsk

atka

mackerel

MTemperate

0ndash240

ACTA

1AB0

73381

Percifo

rmes

Scom

bridae

Scom

berscombrus

Atlanticmackerl

MTemperate

0ndash200

(0ndash100

0)AC

TA1CO

IEF

607093K

C015895

Percifo

rmes

Percihcthyidae

Sinipercachuatsi

Mandarin

fish

FWTemperate

10AC

TA1MyH

CCO

IAY

395872A

Y454304

NC

015822

Percifo

rmes

Sparidae

Sparus

aurata

Gilthead

seabream

MTemperate

1ndash30

(1ndash150)

ACTA

1AF190473

Percifo

rmes

Sphyraenidae

Sphyraenaidiaste

sPelican

barracud

aM

Trop

ical

3ndash24

ACTA

1LD

H-AM

DHc

mMDH

AF503593SIU80001

AF390559AF390561

Tetraodo

ntifo

rmes

Tetraodo

ntidae

Takifugu

rubripes

Japanese

pufferfish

M+FW

+BR

Temperate

0ndash200

(0ndash100

0)Hb-Am

MDHC

OI

XM003964767

XM003965959HM102315

Scorpaenifo

rmes

Ano

plop

omatidae

Anoplopomafim

bria

Sablefish

MDeep-water

0ndash2740

Hb-B

COI

BT082849JQ353978

Percifo

rmes

Serranidae

Epinepheluscoioides

Orange-spottedgrou

per

M+BR

Subtropical

1ndash100

Hb-B

GU982530

Gasteroste

iform

esGasteroste

idae

Gasteroste

usaculeatusTh

ree-spined

stickleb

ack

M+FW

+BR

Temperate

0ndash100

Hb-B

NM

001267638

Percifo

rmes

Nototheniidae

Nototheniacoriiceps

Blackrockcod

MPo

lar

0ndash550

LDH-AM

yHC

COI

AF0

79822AJ

243767

EU326390

Percifo

rmes

Nototheniidae

Nototheniaangusta

taMaorichief

MTemperate

0ndash100

Hb-AH

b-B

P62363P

29628

Gadifo

rmes

Macrouridae

Coryphaenoides

armatus

Abyssalgrenadier

MDeep-water

282ndash5180

LDH-BM

yHC

COI

AJ60

9232A

B330140

FJ1644

97Gadifo

rmes

Gadidae

Gadus

morhu

aAtlanticcod

M+BR

Temperate

0ndash60

0LD

H-BC

OI

AJ60

9233K

C015385

Gadifo

rmes

Gadidae

Arctogadus

glacia

lisArctic

cod

MDeep-water

0ndash1000

Hb-AC

OI

Q1AGS4K

C015200

Percifo

rmes

Latid

aeLa

tescalcarifer

Barram

undi

M+FW

+BR

Trop

ical

10ndash4

0LD

H-BC

OI

FJ439507JQ431879

Gadifo

rmes

Gadidae

Merlangiusm

erlangus

Whitin

gM

Temperate

30ndash100

(10ndash

200)

LDH-BC

OI

AJ60

9234JQ623954

Cyprinod

ontiformes

Poeciliidae

Poeciliareticulata

Gup

pyFW

+BR

Trop

ical

Shallow

LDH-BC

OI

EF40

8825JX9

68696

Gadifo

rmes

Macrouridae

Trachyrin

cusm

urrayi

Roug

hnoseg

renadier

MDeep-water

0ndash1630

LDH-BC

OI

AJ60

9235A

P008990

Percifo

rmes

Channichthyidae

Chionodraco

rastrospinosus

Ocellatedicefish

MPo

lar

0ndash1000

LDH-AC

OI

AF0

79829EU

326337

Percifo

rmes

Pomacentridae

Chromiscaud

alis

Blue-axilchrom

isM

Trop

ical

15ndash55

LDH-A

AY289558

Percifo

rmes

Gob

iidae

Rhinogobiops

nicholsii

Blackeye

goby

MSubtropical

-106

LDH-A

AF0

79534

Cyprinifo

rmes

Cyprinidae

Cyprinus

carpio

Com

mon

carp

FW+BR

Subtropical

shallow

LDH-AM

yHC

COI

AF0

76528D89992

HQ960709


Table6Con

tinued

Order

Family

Species

Com

mon

name

Environm

ent

Clim

ate

Depth

range

Gene

NCB

IAccN

r

Cyprinod

ontiformes

Fund

ulidae

Fund

ulus

heteroclitus

Mum

micho

gM

+FW

+BR

Temperate

shallow

LDH-ALDH-BC

OI

L43525L23792EU

524629

Osm

erifo

rmes

Osm

eridae

Osm

erus

mordax

Rainbo

wsm

eltM

+FW

+BR

Temperate

0ndash425

MDHcmMDH

BT075651B

T07560

0

Salm

onifo

rmes

Salm

onidae

Salm

osalar

Atlanticsalm

onM

+FW

+BR

Temperate

10ndash23

(0ndash210)

MDHcmMDH

BT06

0183B

T048216

Gadifo

rmes

Macrouridae

Coryphaenoides

acrolep

isPacific

grenadier

MDeep-water

900ndash

1300

MyH

CCO

IAB3

30141JQ

3540

60

Gadifo

rmes

Macrouridae

Coryphaenoides

yaquinae

na

MDeep-water

3400ndash5800

MyH

CCO

IAB3

30139GU44

0291

Percifo

rmes

Cirrhitid

aePa

racir

rhitesforste

riBlacksideh

awkfi

shM

Trop

ical

5ndash35

MyH

CCO

IAJ

243770H

Q561521

Percifo

rmes

Carang

idae

Serio

ladu

merili

Greater

amberja

ckM

Subtropical

18ndash72

(1ndash360)

MyH

CCO

IAB0

32020KC

015917

Gadifo

rmes

Gadidae

Boreogadus

saida

Polarc

odM

+BR

Polar

0ndash40

0Hb-AH

b-B

COI

DQ125471Q

1AGS6

KC015250



(a) LDH-A (332 AA)


(b) MDHc-A (333 AA)




(d) LDH-B (334 AA)


(e) MyHC1 (305 AA)


(f) Hb-A1 (143 AA)



(g) Hb-B1 (146 AA)







02

01

Ep_coiGa_acuAn_fim

Ap_carBo_sai

No_angNo_ang

Ap_carTa_rub

Bo_saiAr_glaGa_mor

52

5452

66

100

5359

67

100 100

COI

LDH-A

LDH-B

MDHc-A

Hb-B

Hb-A

La_calCy_carAn_fim

Ap_carSi_chu

Se_dumSc_sco

Ta_rubPa_for

Ch_rasNo_cor


Tr_murCo_armCo_yaq

Co_acrPo_ret

Fu_hetLa_cha

62100

73

87

100

100

005

100

99


Cy_carLa_calAp_car


Or_latAp_car

Sp_idiSa_salOs_mor

100

6479

100

100

100

100

100100

9699

7990

8055

70



Ac carAc carAc car

Tr murNo corOs mor

Co armCh rasSa sal

Me merSp idiSp idi

Ga mor

Or lat

MDHc-A

LDH-ALDH-B

450

400

350

300

250

200

150

100

50

00

ΔG

(cal

mol

)

(a)

ΔG

(cal

mol

)


480

500

520

540

560

580

600

Actin 1

Actin 2bActin 2a

(b)

1000

800

600

400

200

00

minus200

ΔG

(cal

mol

)

Hb-AHb-B


(c)











2-chains (Hb-









4 Conclusions










Acknowledgments



References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


EF-1Blowast

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SBHSP90

10

08

04

06

02

00

H G L M SB

PRDX1∘10

08

04

06

02

00

H G L M SB

SOD-1∘10

08

04

06

02

00

H G L M SB

Ferr10

08

04

06

02

00

H G L M SB

Hb-A10

08

04

06

02

00

H G L M SB

Hb-B10

08

04

06

02

00

H G L M SB

EPD-110

08

04

06

02

00

H G L M SB

BLBP10

08

04

06

02

00H G L M SB

TSPAN-810

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

BTF3∘

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

10

08

04

06

02

00

H G L M SB

MYCBP10

08

04

06

02

00

H G L M SB

CTSS

STF-1

ContigsqPCR

Rab-1A∘

TRPM-1














Table6Specieslist

with

inform

ations

ontheirtypeo

fenviro

nment(M

=marineBR

=brackish

andFW

=fre



fgenes

used

inthe

study

with

relativeG

enbank

accessionnu

mbers

Order

Family

Species

Com

mon

name

Environm

ent

Clim

ate

Depth

range

Gene

NCB

IAccN

r

Percifo

rmes

Trichiuridae

Aphanopu

scarbo

Blackscabbardfish

MDeep-water

200ndash

1700

COI

EU8540

76

Beloniform

esAd

rianichthydae

Oryzias

latip

esJapanese

ricefi

shFW

+BR

Subtropical

shallow

ACTA

1MDHcMyH

CCO

I

NM

00110

4806

NM

00116

3134

XM00

4071618AB4

9806

6

Scorpaenifo

rmes

Hexagrammidae

Pleurogram

mus

azonus

Okh

otsk

atka

mackerel

MTemperate

0ndash240

ACTA

1AB0

73381

Percifo

rmes

Scom

bridae

Scom

berscombrus

Atlanticmackerl

MTemperate

0ndash200

(0ndash100

0)AC

TA1CO

IEF

607093K

C015895

Percifo

rmes

Percihcthyidae

Sinipercachuatsi

Mandarin

fish

FWTemperate

10AC

TA1MyH

CCO

IAY

395872A

Y454304

NC

015822

Percifo

rmes

Sparidae

Sparus

aurata

Gilthead

seabream

MTemperate

1ndash30

(1ndash150)

ACTA

1AF190473

Percifo

rmes

Sphyraenidae

Sphyraenaidiaste

sPelican

barracud

aM

Trop

ical

3ndash24

ACTA

1LD

H-AM

DHc

mMDH

AF503593SIU80001

AF390559AF390561

Tetraodo

ntifo

rmes

Tetraodo

ntidae

Takifugu

rubripes

Japanese

pufferfish

M+FW

+BR

Temperate

0ndash200

(0ndash100

0)Hb-Am

MDHC

OI

XM003964767

XM003965959HM102315

Scorpaenifo

rmes

Ano

plop

omatidae

Anoplopomafim

bria

Sablefish

MDeep-water

0ndash2740

Hb-B

COI

BT082849JQ353978

Percifo

rmes

Serranidae

Epinepheluscoioides

Orange-spottedgrou

per

M+BR

Subtropical

1ndash100

Hb-B

GU982530

Gasteroste

iform

esGasteroste

idae

Gasteroste

usaculeatusTh

ree-spined

stickleb

ack

M+FW

+BR

Temperate

0ndash100

Hb-B

NM

001267638

Percifo

rmes

Nototheniidae

Nototheniacoriiceps

Blackrockcod

MPo

lar

0ndash550

LDH-AM

yHC

COI

AF0

79822AJ

243767

EU326390

Percifo

rmes

Nototheniidae

Nototheniaangusta

taMaorichief

MTemperate

0ndash100

Hb-AH

b-B

P62363P

29628

Gadifo

rmes

Macrouridae

Coryphaenoides

armatus

Abyssalgrenadier

MDeep-water

282ndash5180

LDH-BM

yHC

COI

AJ60

9232A

B330140

FJ1644

97Gadifo

rmes

Gadidae

Gadus

morhu

aAtlanticcod

M+BR

Temperate

0ndash60

0LD

H-BC

OI

AJ60

9233K

C015385

Gadifo

rmes

Gadidae

Arctogadus

glacia

lisArctic

cod

MDeep-water

0ndash1000

Hb-AC

OI

Q1AGS4K

C015200

Percifo

rmes

Latid

aeLa

tescalcarifer

Barram

undi

M+FW

+BR

Trop

ical

10ndash4

0LD

H-BC

OI

FJ439507JQ431879

Gadifo

rmes

Gadidae

Merlangiusm

erlangus

Whitin

gM

Temperate

30ndash100

(10ndash

200)

LDH-BC

OI

AJ60

9234JQ623954

Cyprinod

ontiformes

Poeciliidae

Poeciliareticulata

Gup

pyFW

+BR

Trop

ical

Shallow

LDH-BC

OI

EF40

8825JX9

68696

Gadifo

rmes

Macrouridae

Trachyrin

cusm

urrayi

Roug

hnoseg

renadier

MDeep-water

0ndash1630

LDH-BC

OI

AJ60

9235A

P008990

Percifo

rmes

Channichthyidae

Chionodraco

rastrospinosus

Ocellatedicefish

MPo

lar

0ndash1000

LDH-AC

OI

AF0

79829EU

326337

Percifo

rmes

Pomacentridae

Chromiscaud

alis

Blue-axilchrom

isM

Trop

ical

15ndash55

LDH-A

AY289558

Percifo

rmes

Gob

iidae

Rhinogobiops

nicholsii

Blackeye

goby

MSubtropical

-106

LDH-A

AF0

79534

Cyprinifo

rmes

Cyprinidae

Cyprinus

carpio

Com

mon

carp

FW+BR

Subtropical

shallow

LDH-AM

yHC

COI

AF0

76528D89992

HQ960709


Table6Con

tinued

Order

Family

Species

Com

mon

name

Environm

ent

Clim

ate

Depth

range

Gene

NCB

IAccN

r

Cyprinod

ontiformes

Fund

ulidae

Fund

ulus

heteroclitus

Mum

micho

gM

+FW

+BR

Temperate

shallow

LDH-ALDH-BC

OI

L43525L23792EU

524629

Osm

erifo

rmes

Osm

eridae

Osm

erus

mordax

Rainbo

wsm

eltM

+FW

+BR

Temperate

0ndash425

MDHcmMDH

BT075651B

T07560

0

Salm

onifo

rmes

Salm

onidae

Salm

osalar

Atlanticsalm

onM

+FW

+BR

Temperate

10ndash23

(0ndash210)

MDHcmMDH

BT06

0183B

T048216

Gadifo

rmes

Macrouridae

Coryphaenoides

acrolep

isPacific

grenadier

MDeep-water

900ndash

1300

MyH

CCO

IAB3

30141JQ

3540

60

Gadifo

rmes

Macrouridae

Coryphaenoides

yaquinae

na

MDeep-water

3400ndash5800

MyH

CCO

IAB3

30139GU44

0291

Percifo

rmes

Cirrhitid

aePa

racir

rhitesforste

riBlacksideh

awkfi

shM

Trop

ical

5ndash35

MyH

CCO

IAJ

243770H

Q561521

Percifo

rmes

Carang

idae

Serio

ladu

merili

Greater

amberja

ckM

Subtropical

18ndash72

(1ndash360)

MyH

CCO

IAB0

32020KC

015917

Gadifo

rmes

Gadidae

Boreogadus

saida

Polarc

odM

+BR

Polar

0ndash40

0Hb-AH

b-B

COI

DQ125471Q

1AGS6

KC015250



(a) LDH-A (332 AA)


(b) MDHc-A (333 AA)




(d) LDH-B (334 AA)


(e) MyHC1 (305 AA)


(f) Hb-A1 (143 AA)



(g) Hb-B1 (146 AA)







02

01

Ep_coiGa_acuAn_fim

Ap_carBo_sai

No_angNo_ang

Ap_carTa_rub

Bo_saiAr_glaGa_mor

52

5452

66

100

5359

67

100 100

COI

LDH-A

LDH-B

MDHc-A

Hb-B

Hb-A

La_calCy_carAn_fim

Ap_carSi_chu

Se_dumSc_sco

Ta_rubPa_for

Ch_rasNo_cor


Tr_murCo_armCo_yaq

Co_acrPo_ret

Fu_hetLa_cha

62100

73

87

100

100

005

100

99


Cy_carLa_calAp_car


Or_latAp_car

Sp_idiSa_salOs_mor

100

6479

100

100

100

100

100100

9699

7990

8055

70



Ac carAc carAc car

Tr murNo corOs mor

Co armCh rasSa sal

Me merSp idiSp idi

Ga mor

Or lat

MDHc-A

LDH-ALDH-B

450

400

350

300

250

200

150

100

50

00

ΔG

(cal

mol

)

(a)

ΔG

(cal

mol

)


480

500

520

540

560

580

600

Actin 1

Actin 2bActin 2a

(b)

1000

800

600

400

200

00

minus200

ΔG

(cal

mol

)

Hb-AHb-B


(c)











2-chains (Hb-









4 Conclusions










Acknowledgments



References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













Table6Specieslist

with

inform

ations

ontheirtypeo

fenviro

nment(M

=marineBR

=brackish

andFW

=fre



fgenes

used

inthe

study

with

relativeG

enbank

accessionnu

mbers

Order

Family

Species

Com

mon

name

Environm

ent

Clim

ate

Depth

range

Gene

NCB

IAccN

r

Percifo

rmes

Trichiuridae

Aphanopu

scarbo

Blackscabbardfish

MDeep-water

200ndash

1700

COI

EU8540

76

Beloniform

esAd

rianichthydae

Oryzias

latip

esJapanese

ricefi

shFW

+BR

Subtropical

shallow

ACTA

1MDHcMyH

CCO

I

NM

00110

4806

NM

00116

3134

XM00

4071618AB4

9806

6

Scorpaenifo

rmes

Hexagrammidae

Pleurogram

mus

azonus

Okh

otsk

atka

mackerel

MTemperate

0ndash240

ACTA

1AB0

73381

Percifo

rmes

Scom

bridae

Scom

berscombrus

Atlanticmackerl

MTemperate

0ndash200

(0ndash100

0)AC

TA1CO

IEF

607093K

C015895

Percifo

rmes

Percihcthyidae

Sinipercachuatsi

Mandarin

fish

FWTemperate

10AC

TA1MyH

CCO

IAY

395872A

Y454304

NC

015822

Percifo

rmes

Sparidae

Sparus

aurata

Gilthead

seabream

MTemperate

1ndash30

(1ndash150)

ACTA

1AF190473

Percifo

rmes

Sphyraenidae

Sphyraenaidiaste

sPelican

barracud

aM

Trop

ical

3ndash24

ACTA

1LD

H-AM

DHc

mMDH

AF503593SIU80001

AF390559AF390561

Tetraodo

ntifo

rmes

Tetraodo

ntidae

Takifugu

rubripes

Japanese

pufferfish

M+FW

+BR

Temperate

0ndash200

(0ndash100

0)Hb-Am

MDHC

OI

XM003964767

XM003965959HM102315

Scorpaenifo

rmes

Ano

plop

omatidae

Anoplopomafim

bria

Sablefish

MDeep-water

0ndash2740

Hb-B

COI

BT082849JQ353978

Percifo

rmes

Serranidae

Epinepheluscoioides

Orange-spottedgrou

per

M+BR

Subtropical

1ndash100

Hb-B

GU982530

Gasteroste

iform

esGasteroste

idae

Gasteroste

usaculeatusTh

ree-spined

stickleb

ack

M+FW

+BR

Temperate

0ndash100

Hb-B

NM

001267638

Percifo

rmes

Nototheniidae

Nototheniacoriiceps

Blackrockcod

MPo

lar

0ndash550

LDH-AM

yHC

COI

AF0

79822AJ

243767

EU326390

Percifo

rmes

Nototheniidae

Nototheniaangusta

taMaorichief

MTemperate

0ndash100

Hb-AH

b-B

P62363P

29628

Gadifo

rmes

Macrouridae

Coryphaenoides

armatus

Abyssalgrenadier

MDeep-water

282ndash5180

LDH-BM

yHC

COI

AJ60

9232A

B330140

FJ1644

97Gadifo

rmes

Gadidae

Gadus

morhu

aAtlanticcod

M+BR

Temperate

0ndash60

0LD

H-BC

OI

AJ60

9233K

C015385

Gadifo

rmes

Gadidae

Arctogadus

glacia

lisArctic

cod

MDeep-water

0ndash1000

Hb-AC

OI

Q1AGS4K

C015200

Percifo

rmes

Latid

aeLa

tescalcarifer

Barram

undi

M+FW

+BR

Trop

ical

10ndash4

0LD

H-BC

OI

FJ439507JQ431879

Gadifo

rmes

Gadidae

Merlangiusm

erlangus

Whitin

gM

Temperate

30ndash100

(10ndash

200)

LDH-BC

OI

AJ60

9234JQ623954

Cyprinod

ontiformes

Poeciliidae

Poeciliareticulata

Gup

pyFW

+BR

Trop

ical

Shallow

LDH-BC

OI

EF40

8825JX9

68696

Gadifo

rmes

Macrouridae

Trachyrin

cusm

urrayi

Roug

hnoseg

renadier

MDeep-water

0ndash1630

LDH-BC

OI

AJ60

9235A

P008990

Percifo

rmes

Channichthyidae

Chionodraco

rastrospinosus

Ocellatedicefish

MPo

lar

0ndash1000

LDH-AC

OI

AF0

79829EU

326337

Percifo

rmes

Pomacentridae

Chromiscaud

alis

Blue-axilchrom

isM

Trop

ical

15ndash55

LDH-A

AY289558

Percifo

rmes

Gob

iidae

Rhinogobiops

nicholsii

Blackeye

goby

MSubtropical

-106

LDH-A

AF0

79534

Cyprinifo

rmes

Cyprinidae

Cyprinus

carpio

Com

mon

carp

FW+BR

Subtropical

shallow

LDH-AM

yHC

COI

AF0

76528D89992

HQ960709


Table6Con

tinued

Order

Family

Species

Com

mon

name

Environm

ent

Clim

ate

Depth

range

Gene

NCB

IAccN

r

Cyprinod

ontiformes

Fund

ulidae

Fund

ulus

heteroclitus

Mum

micho

gM

+FW

+BR

Temperate

shallow

LDH-ALDH-BC

OI

L43525L23792EU

524629

Osm

erifo

rmes

Osm

eridae

Osm

erus

mordax

Rainbo

wsm

eltM

+FW

+BR

Temperate

0ndash425

MDHcmMDH

BT075651B

T07560

0

Salm

onifo

rmes

Salm

onidae

Salm

osalar

Atlanticsalm

onM

+FW

+BR

Temperate

10ndash23

(0ndash210)

MDHcmMDH

BT06

0183B

T048216

Gadifo

rmes

Macrouridae

Coryphaenoides

acrolep

isPacific

grenadier

MDeep-water

900ndash

1300

MyH

CCO

IAB3

30141JQ

3540

60

Gadifo

rmes

Macrouridae

Coryphaenoides

yaquinae

na

MDeep-water

3400ndash5800

MyH

CCO

IAB3

30139GU44

0291

Percifo

rmes

Cirrhitid

aePa

racir

rhitesforste

riBlacksideh

awkfi

shM

Trop

ical

5ndash35

MyH

CCO

IAJ

243770H

Q561521

Percifo

rmes

Carang

idae

Serio

ladu

merili

Greater

amberja

ckM

Subtropical

18ndash72

(1ndash360)

MyH

CCO

IAB0

32020KC

015917

Gadifo

rmes

Gadidae

Boreogadus

saida

Polarc

odM

+BR

Polar

0ndash40

0Hb-AH

b-B

COI

DQ125471Q

1AGS6

KC015250



(a) LDH-A (332 AA)


(b) MDHc-A (333 AA)




(d) LDH-B (334 AA)


(e) MyHC1 (305 AA)


(f) Hb-A1 (143 AA)



(g) Hb-B1 (146 AA)







02

01

Ep_coiGa_acuAn_fim

Ap_carBo_sai

No_angNo_ang

Ap_carTa_rub

Bo_saiAr_glaGa_mor

52

5452

66

100

5359

67

100 100

COI

LDH-A

LDH-B

MDHc-A

Hb-B

Hb-A

La_calCy_carAn_fim

Ap_carSi_chu

Se_dumSc_sco

Ta_rubPa_for

Ch_rasNo_cor


Tr_murCo_armCo_yaq

Co_acrPo_ret

Fu_hetLa_cha

62100

73

87

100

100

005

100

99


Cy_carLa_calAp_car


Or_latAp_car

Sp_idiSa_salOs_mor

100

6479

100

100

100

100

100100

9699

7990

8055

70



Ac carAc carAc car

Tr murNo corOs mor

Co armCh rasSa sal

Me merSp idiSp idi

Ga mor

Or lat

MDHc-A

LDH-ALDH-B

450

400

350

300

250

200

150

100

50

00

ΔG

(cal

mol

)

(a)

ΔG

(cal

mol

)


480

500

520

540

560

580

600

Actin 1

Actin 2bActin 2a

(b)

1000

800

600

400

200

00

minus200

ΔG

(cal

mol

)

Hb-AHb-B


(c)











2-chains (Hb-









4 Conclusions










Acknowledgments



References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table6Con

tinued

Order

Family

Species

Com

mon

name

Environm

ent

Clim

ate

Depth

range

Gene

NCB

IAccN

r

Cyprinod

ontiformes

Fund

ulidae

Fund

ulus

heteroclitus

Mum

micho

gM

+FW

+BR

Temperate

shallow

LDH-ALDH-BC

OI

L43525L23792EU

524629

Osm

erifo

rmes

Osm

eridae

Osm

erus

mordax

Rainbo

wsm

eltM

+FW

+BR

Temperate

0ndash425

MDHcmMDH

BT075651B

T07560

0

Salm

onifo

rmes

Salm

onidae

Salm

osalar

Atlanticsalm

onM

+FW

+BR

Temperate

10ndash23

(0ndash210)

MDHcmMDH

BT06

0183B

T048216

Gadifo

rmes

Macrouridae

Coryphaenoides

acrolep

isPacific

grenadier

MDeep-water

900ndash

1300

MyH

CCO

IAB3

30141JQ

3540

60

Gadifo

rmes

Macrouridae

Coryphaenoides

yaquinae

na

MDeep-water

3400ndash5800

MyH

CCO

IAB3

30139GU44

0291

Percifo

rmes

Cirrhitid

aePa

racir

rhitesforste

riBlacksideh

awkfi

shM

Trop

ical

5ndash35

MyH

CCO

IAJ

243770H

Q561521

Percifo

rmes

Carang

idae

Serio

ladu

merili

Greater

amberja

ckM

Subtropical

18ndash72

(1ndash360)

MyH

CCO

IAB0

32020KC

015917

Gadifo

rmes

Gadidae

Boreogadus

saida

Polarc

odM

+BR

Polar

0ndash40

0Hb-AH

b-B

COI

DQ125471Q

1AGS6

KC015250



(a) LDH-A (332 AA)


(b) MDHc-A (333 AA)




(d) LDH-B (334 AA)


(e) MyHC1 (305 AA)


(f) Hb-A1 (143 AA)



(g) Hb-B1 (146 AA)







02

01

Ep_coiGa_acuAn_fim

Ap_carBo_sai

No_angNo_ang

Ap_carTa_rub

Bo_saiAr_glaGa_mor

52

5452

66

100

5359

67

100 100

COI

LDH-A

LDH-B

MDHc-A

Hb-B

Hb-A

La_calCy_carAn_fim

Ap_carSi_chu

Se_dumSc_sco

Ta_rubPa_for

Ch_rasNo_cor


Tr_murCo_armCo_yaq

Co_acrPo_ret

Fu_hetLa_cha

62100

73

87

100

100

005

100

99


Cy_carLa_calAp_car


Or_latAp_car

Sp_idiSa_salOs_mor

100

6479

100

100

100

100

100100

9699

7990

8055

70



Ac carAc carAc car

Tr murNo corOs mor

Co armCh rasSa sal

Me merSp idiSp idi

Ga mor

Or lat

MDHc-A

LDH-ALDH-B

450

400

350

300

250

200

150

100

50

00

ΔG

(cal

mol

)

(a)

ΔG

(cal

mol

)


480

500

520

540

560

580

600

Actin 1

Actin 2bActin 2a

(b)

1000

800

600

400

200

00

minus200

ΔG

(cal

mol

)

Hb-AHb-B


(c)











2-chains (Hb-









4 Conclusions










Acknowledgments



References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



(a) LDH-A (332 AA)


(b) MDHc-A (333 AA)




(d) LDH-B (334 AA)


(e) MyHC1 (305 AA)


(f) Hb-A1 (143 AA)



(g) Hb-B1 (146 AA)







02

01

Ep_coiGa_acuAn_fim

Ap_carBo_sai

No_angNo_ang

Ap_carTa_rub

Bo_saiAr_glaGa_mor

52

5452

66

100

5359

67

100 100

COI

LDH-A

LDH-B

MDHc-A

Hb-B

Hb-A

La_calCy_carAn_fim

Ap_carSi_chu

Se_dumSc_sco

Ta_rubPa_for

Ch_rasNo_cor


Tr_murCo_armCo_yaq

Co_acrPo_ret

Fu_hetLa_cha

62100

73

87

100

100

005

100

99


Cy_carLa_calAp_car


Or_latAp_car

Sp_idiSa_salOs_mor

100

6479

100

100

100

100

100100

9699

7990

8055

70



Ac carAc carAc car

Tr murNo corOs mor

Co armCh rasSa sal

Me merSp idiSp idi

Ga mor

Or lat

MDHc-A

LDH-ALDH-B

450

400

350

300

250

200

150

100

50

00

ΔG

(cal

mol

)

(a)

ΔG

(cal

mol

)


480

500

520

540

560

580

600

Actin 1

Actin 2bActin 2a

(b)

1000

800

600

400

200

00

minus200

ΔG

(cal

mol

)

Hb-AHb-B


(c)











2-chains (Hb-









4 Conclusions










Acknowledgments



References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


02

01

Ep_coiGa_acuAn_fim

Ap_carBo_sai

No_angNo_ang

Ap_carTa_rub

Bo_saiAr_glaGa_mor

52

5452

66

100

5359

67

100 100

COI

LDH-A

LDH-B

MDHc-A

Hb-B

Hb-A

La_calCy_carAn_fim

Ap_carSi_chu

Se_dumSc_sco

Ta_rubPa_for

Ch_rasNo_cor


Tr_murCo_armCo_yaq

Co_acrPo_ret

Fu_hetLa_cha

62100

73

87

100

100

005

100

99


Cy_carLa_calAp_car


Or_latAp_car

Sp_idiSa_salOs_mor

100

6479

100

100

100

100

100100

9699

7990

8055

70



Ac carAc carAc car

Tr murNo corOs mor

Co armCh rasSa sal

Me merSp idiSp idi

Ga mor

Or lat

MDHc-A

LDH-ALDH-B

450

400

350

300

250

200

150

100

50

00

ΔG

(cal

mol

)

(a)

ΔG

(cal

mol

)


480

500

520

540

560

580

600

Actin 1

Actin 2bActin 2a

(b)

1000

800

600

400

200

00

minus200

ΔG

(cal

mol

)

Hb-AHb-B


(c)











2-chains (Hb-









4 Conclusions










Acknowledgments



References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Ac carAc carAc car

Tr murNo corOs mor

Co armCh rasSa sal

Me merSp idiSp idi

Ga mor

Or lat

MDHc-A

LDH-ALDH-B

450

400

350

300

250

200

150

100

50

00

ΔG

(cal

mol

)

(a)

ΔG

(cal

mol

)


480

500

520

540

560

580

600

Actin 1

Actin 2bActin 2a

(b)

1000

800

600

400

200

00

minus200

ΔG

(cal

mol

)

Hb-AHb-B


(c)











2-chains (Hb-









4 Conclusions










Acknowledgments



References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






2-chains (Hb-









4 Conclusions










Acknowledgments



References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









Acknowledgments



References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Transcriptome of the Deep-Sea Black...

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Transcript of Research Article Transcriptome of the Deep-Sea Black...