Øivind Andersen

The genome sequence of Atlantic cod reveals a unique immune system

Atlantic cod (Gadus morhua) is a large, cold-adapted teleost that sustains long-standing commercial fisheries and incipient aquaculture. Here we present the genome sequence of Atlantic cod, showing evidence for complex thermal adaptations in its haemoglobin gene cluster and an unusual immune architecture compared to other sequenced vertebrates. The genome assembly was obtained exclusively by 454 sequencing of shotgun and paired-end libraries, and automated annotation identified 22,154 genes. The major histocompatibility complex (MHC) II is a conserved feature of the adaptive immune system of jawed vertebrates, but we show that Atlantic cod has lost the genes for MHC II, CD4 and invariant chain (Ii) that are essential for the function of this pathway. Nevertheless, Atlantic cod is not exceptionally susceptible to disease under natural conditions. We find a highly expanded number of MHC I genes and a unique composition of its Toll-like receptor (TLR) families. This indicates how the Atlantic cod immune system has evolved compensatory mechanisms in both adaptive and innate immunity in the absence of MHC II. These observations affect fundamental assumptions about the evolution of the adaptive immune system and its components in vertebrates.

Peroxisome proliferator activated receptors in Atlantic salmon (Salmo salar): effects on PPAR transcription and acyl-CoA oxidase activity in hepatocytes by peroxisome proliferators and fatty acids

A cDNA fragment which encodes salmon peroxisome proliferator activated receptor y (sPPARgamma) was amplified by PCR from the liver of Atlantic salmon (Salmo salar L.). The fragment was 627 bp long. The sequence of the amplified PCR product was similar to the PPARgamma of mouse and hamster. 59% of the bases were identical. Northern blot analysis of salmon liver mRNA showed that the amplified sPPARgamma fragment hybridised to three specific transcripts of lengths 1.6, 2.4 and 3.3 kb. Clofibric acid and bezafibrate, administered to salmon hepatocytes in culture, resulted in a 1.7-fold increase of the 1.6 kb sPPARgamma transcript. The activity of acyl-CoA oxidase also increased approx. 1.7-fold after administration of fibrates. These results indicate that PPAR is an important factor in mediating enzymatic response to fibrates in fish.

Haemoglobin polymorphisms affect the oxygen- binding properties in Atlantic cod populations

A major challenge in evolutionary biology is to identify the genes underlying adaptation. The oxygen-transporting haemoglobins directly link external conditions with metabolic needs and therefore represent a unique system for studying environmental effects on molecular evolution. We have discovered two haemoglobin polymorphisms in Atlantic cod populations inhabiting varying temperature and oxygen regimes in the North Atlantic. Three-dimensional modelling of the tetrameric haemoglobin structure demonstrated that the two amino acid replacements Met55β1Val and Lys62β1Ala are located at crucial positions of the α1β1 subunit interface and haem pocket, respectively. The replacements are proposed to affect the oxygen-binding properties by modifying the haemoglobin quaternary structure and electrostatic feature. Intriguingly, the same molecular mechanism for facilitating oxygen binding is found in avian species adapted to high altitudes, illustrating convergent evolution in water- and air-breathing vertebrates to reduction in environmental oxygen availability. Cod populations inhabiting the cold Arctic waters and the low-oxygen Baltic Sea seem well adapted to these conditions by possessing the high oxygen affinity Val55–Ala62 haplotype, while the temperature-insensitive Met55–Lys62 haplotype predominates in the southern populations. The distinct distributions of the functionally different haemoglobin variants indicate that the present biogeography of this ecologically and economically important species might be seriously affected by global warming.

Somite formation and expression of MyoD, myogenin and myosin in Atlantic halibut (Hippoglossus hippoglossus L.) embryos incubated at different temperatures: transient asymmetric expression of MyoD.

SUMMARY Genes encoding the myogenic regulating factors MyoD and myogenin and the structural muscle proteins myosin light chain 2 (MyLC2) and myosin heavy chain (MyHC) were isolated from juvenile Atlantic halibut (Hippoglossus hippoglossus L.). The impact of temperature on their temporal and spatial expression during somitogenesis were examined by incubating halibut embryos at 4, 6 and 8°C, and regularly sampling for whole-mount in situ hybridisation and reverse transcription (RT)–PCR. There were no significant effects of temperature on the onset of somitogenesis or number of somites at hatching. The rate of somite formation increased with increasing temperature, and the expression of MyoD, myogenin and MyHC followed the cranial-to-caudal somite formation. Hence, no significant effect of temperature on the spatial and temporal expression of the genes studied was found in relation to somite stage. MyoD, which has subsequently been shown to encode the MyoD2 isoform, displayed a novel bilaterally asymmetric expression pattern only in white muscle precursor cells during early halibut somitogenesis. The expression of myogenin resembled that previously described for other fish species, and preceded the MyHC expression by approximately five somites. Two MyLC2 cDNA sequences were for the first time described for a flatfish, probably representing embryonic (MyLC2a) and larval/juvenile (MyLC2b) isoforms. Factors regulating muscle determination, differentiation and development have so far mostly been studied in vertebrates with external bilateral symmetry. The findings of the present study suggest that more such investigations of flatfish species could provide valuable information on how muscle-regulating mechanisms work in species with different anatomical, physiological and ecological traits.

Purine-induced expression of urate oxidase and enzyme activity in Atlantic salmon (Salmo salar). Cloning of urate oxidase liver cDNA from three teleost species and the African lungfish Protopterus annectens.

The peroxisomal enzyme urate oxidase plays a pivotal role in the degradation of purines in both prokaryotes and eukaryotes. However, knowledge about the purine‐induced expression of the encoding gene is lacking in vertebrates. These are the first published sequences of fish urate oxidase, which were predicted from PCR amplified liver cDNAs of Atlantic salmon (Salmo salar), Atlantic cod (Gadus morhua), Atlantic halibut (Hippoglossus hippoglossus) and African lungfish (Protopterus annectens). Sequence alignment of different vertebrate urate oxidases revealed amino acid substitutions of putative functional importance in the enzyme of chicken and lungfish. In the adult salmon, expression of urate oxidase mRNA predominated in liver, but was also identified in several nonhepatic organs including brain, but not in skeletal muscle and kidney. Juvenile salmon fed diets containing bacterial protein meal (BPM) rich in nucleic acids showed a significant increase in liver urate oxidase enzyme activity, and urea concentrations in plasma, muscle and liver were elevated. Whereas salmon fed the 18% BPM diet showed a nonsignificant increase in liver mRNA levels of urate oxidase compared with the 0% BPM‐fed fish, no further increase in mRNA levels was found in fish receiving 36% BPM. The discrepancy between urate oxidase mRNA and enzyme activity was explained by rapid mRNA degradation or alternatively, post‐translational control of the activity. Although variable plasma and liver levels of urate were detected, the substrate increased only slightly in 36% BPM‐fed fish, indicating that the uricolytic pathway of Atlantic salmon is intimately regulated to handle high dietary purine levels.

Genomic organization and gene expression of the multiple globins in Atlantic cod: conservation of globin-flanking genes in chordates infers the origin of the vertebrate globin clusters

BackgroundThe vertebrate globin genes encoding the α- and β-subunits of the tetrameric hemoglobins are clustered at two unlinked loci. The highly conserved linear order of the genes flanking the hemoglobins provides a strong anchor for inferring common ancestry of the globin clusters. In fish, the number of α-β-linked globin genes varies considerably between different sublineages and seems to be related to prevailing physico-chemical conditions. Draft sequences of the Atlantic cod genome enabled us to determine the genomic organization of the globin repertoire in this marine species that copes with fluctuating environments of the temperate and Arctic regions.ResultsThe Atlantic cod genome was shown to contain 14 globin genes, including nine hemoglobin genes organized in two unlinked clusters designated β5-α1-β1-α4 and β3-β4-α2-α3-β2. The diverged cod hemoglobin genes displayed different expression levels in adult fish, and tetrameric hemoglobins with or without a Root effect were predicted. The novel finding of maternally inherited hemoglobin mRNAs is consistent with a potential role played by fish hemoglobins in the non-specific immune response. In silico analysis of the six teleost genomes available showed that the two α-β globin clusters are flanked by paralogs of five duplicated genes, in agreement with the proposed teleost-specific duplication of the ancestral vertebrate globin cluster. Screening the genome of extant urochordate and cephalochordate species for conserved globin-flanking genes revealed linkage of RHBDF1, MPG and ARHGAP17 to globin genes in the tunicate Ciona intestinalis, while these genes together with LCMT are closely positioned in amphioxus (Branchiostoma floridae), but seem to be unlinked to the multiple globin genes identified in this species.ConclusionThe plasticity of Atlantic cod to variable environmental conditions probably involves the expression of multiple globins with potentially different properties. The interspecific difference in number of fish hemoglobin genes contrasts with the highly conserved synteny of the flanking genes. The proximity of globin-flanking genes in the tunicate and amphioxus genomes resembles the RHBDF1-MPG-α-globin-ARHGAP17-LCMT linked genes in man and chicken. We hypothesize that the fusion of the three chordate linkage groups 3, 15 and 17 more than 800 MYA led to the ancestral vertebrate globin cluster during a geological period of increased atmospheric oxygen content.

Sex dimorphic expression of five dmrt genes identified in the Atlantic cod genome. The fish-specific dmrt2b diverged from dmrt2a before the fish whole-genome duplication

The Doublesex and Mab-3 related transcription factors (Dmrt) are characterised by the zinc finger-like DM domain binding similar DNA sequences, but show different spatio-temporal expression patterns and functions throughout ontogenesis. Dmrt1 is the master regulator of sex determination in very distant metazoans, while Dmrt2 and Dmrt4 are of crucial importance in vertebrate somitogenesis and neurogenesis, respectively. To elucidate the evolutionary divergence of the fish dmrt genes, we examined the expression patterns and the chromosomal synteny of the five dmrt genes identified in the Atlantic cod genome. Quantitative PCR analyses of cod dmrt1, dmrt2a, dmrt3, dmrt4 and dmrt5 revealed distinct expression patterns in the embryo and larvae, and indicated conserved extragonadal functions during early development. Several dmrt genes seem to be involved in the sexual differentiation of gonads and brain, but the sex-dimorphic expression patterns differed substantially between teleosts, suggesting functional switch between Dmrt members. The dmrt2a-dmrt3-dmrt1 cluster was found to be located in a conserved syntenic region, and the flanking genes have become duplicated in teleosts and are closely linked in a paralogous region lacking the dmrt cluster. Similarly, the region containing the fish-specific dmrt2b gene was found to have a paralogous region without a dmrt2b duplicate in a separate linkage group in the teleost genomes. We propose that the teleost segments paralogous to the dmrt2a- and dmrt2b regions, respectively, were formed through the fish-specific whole genome duplication (3R), while dmrt2a and dmrt2b originated from the second round (2R) of whole genome duplication of the ancestral dmrt2. The dmrt2b paralog seems to have been lost in Atlantic cod as in tetrapods and may be a pseudogene in pufferfish, while dmrt2a and dmrt2b have acquired different functions in zebrafish. Contrasting with the retained duplicates of dmrt flanking genes, the massive losses of dmrt duplicates in the vertebrate tetraploidizations suggest that their functions are exquisitely sensitive to gene dosage.

Divergent and sex-dimorphic expression of the paralogs of the Sox9-Amh-Cyp19a1 regulatory cascade in developing and adult atlantic cod (Gadus morhua L.)

The factors of the Sox9‐Amh‐Cyp19a1 cascade play a crucial role in the complex process of sex differentiation in mammals. The involvement of Sox9 and Cyp19a1 paralogs and the single Amh ortholog in sex differentiation and development of the gonads and the brain in Atlantic cod was examined by analyzing bimodal and sex‐dimorphic gene expression patterns, respectively, during early stages and in maturing males and females. Expression of sox9a and sox9b were initiated at blastulation, and both paralogs were expressed in chondrogenic tissue in the hatched larvae. The male‐specific expression of sox9a in the adult gonads supports a conserved role in testis function, while sox9b was expressed in the maturing testes and ovaries at similar levels. Amh was expressed at low, but variable, levels from late gastrulation prior to the onset of cyp19a1a and cyp19a1b expression. Male‐biased amh expression was found in the maturing gonads, but the increased ovarian levels during maturation suggest a role also in females. The larval expression of cyp19a1a and cyp19a1b increased at the expected time of sex differentiation, but showed large individual variation. The ovarian expression of cyp19a1a and amh increased concomitant with increased plasma estradiol levels during vitellogenesis. The testis‐specific cyp19a1b expression supports the importance of estrogen in the spermatogenesis, while abundant expression in the male and female brain is probably related to the continuous neurogenesis in fish. These divergent and sex‐dimorphic expression patterns of the cod sox9 and cyp19a1 paralogs demonstrate the complexity of the genetic network regulating sexual development in fish. Mol. Reprod. Dev. 80: 358–370, 2013.

Sexually dimorphic expression of dmrt1 in immature and mature Atlantic cod (Gadus morhua L.).

The Doublesex and Mab-3 related transcription factor 1 (Dmrt1) is implicated in testis development in a variety of vertebrates, including teleost fish. Atlantic cod (Gadusmorhua L.) is a promising cold-water aquaculture species, but early sexual maturation of males in particular is a major problem in todays cod farming. Molecular studies of dmrt1 were initiated to gain knowledge about the regulation of gonad development for the first time in a species of the superorder Paracanthopterygii. The predicted cod Dmrt1 of 310 amino acids contains a highly conserved DM domain, including six Cys residues probably involved in the formation of a double zinc-finger motif for DNA binding. The tissue expression analysis revealed that dmrt1 is expressed exclusively in the gonads, and the signal was localized in the germ cells in both genders by in situ hybridization. Sexually dimorphic expression of dmrt1 was documented by quantitative PCR with the highest mRNA levels in immature males corresponding to the start of spermatogenesis. Although significantly less expressed in the ovary, Dmrt1 might also play a role in oogenesis. Southern blot analysis revealed several DM domain-containing genes in the cod genome, but no sex-linked polymorphism was shown.

Muscle fibre number varies with haemoglobin phenotype in Atlantic cod as predicted by the optimal fibre number hypothesis

Atlantic cod (Gadus morhua L.) with the HbI-(2/2) haemoglobin phenotype have a higher blood oxygen affinity at low temperatures and a lower routine metabolic rate than individuals with the HbI-(1/1) phenotype. In the present study, muscle structure was found to be related to haemoglobin phenotype in a coastal population of Atlantic cod from the Saltenfjord region of Northern Norway. The maximum number of fast muscle fibres (FNmax) was reached at approximately 39 cm fork length and was 15% greater in the HbI-(1/1) than in the HbI-(2/2) phenotypes whereas the average fibre diameter for fish of the same fork length was significantly lower. Theoretically, the higher oxygen affinity of the HbI-(2/2) phenotype in the cold water of northern latitudes could have resulted in a relaxation of diffusional constraints at the level of individual muscle fibres, permitting the observed increase in fibre diameter. The results support the optimal fibre number hypothesis which envisages a trade-off between diffusional constraints and the energy cost of maintaining ionic homeostasis with fewer larger diameter muscle fibres in the HbI-(2/2) phenotype contributing to a lower routine metabolic rate.