Genomics of Secondarily Temperate Adaptation in the Only Non-Antarctic Icefish

White-blooded Antarctic icefishes, a family within the adaptive radiation of Antarctic notothenioid fishes, are an example of extreme biological specialization to both the chronic cold of the Southern Ocean and life without hemoglobin. As a result, icefishes display derived physiology that limits them to the cold and highly oxygenated Antarctic waters. Against these constraints, remarkably one species, the pike icefish Champsocephalus esox, successfully colonized temperate South American waters. To study the genetic mechanisms underlying secondarily temperate adaptation in icefishes, we generated chromosome-level genome assemblies of both C. esox and its Antarctic sister species, Champsocephalus gunnari. The C. esox genome is similar in structure and organization to that of its Antarctic congener; however, we observe evidence of chromosomal rearrangements coinciding with regions of elevated genetic divergence in pike icefish populations. We also find several key biological pathways under selection, including genes related to mitochondria and vision, highlighting candidates behind temperate adaptation in C. esox. Substantial antifreeze glycoprotein (AFGP) pseudogenization has occurred in the pike icefish, likely due to relaxed selection following ancestral escape from Antarctica. The canonical AFGP locus organization is conserved in C. esox and C. gunnari, but both show a translocation of two AFGP copies to a separate locus, previously unobserved in cryonotothenioids. Altogether, the study of this secondarily temperate species provides an insight into the mechanisms underlying adaptation to ecologically disparate environments in this otherwise highly specialized group.     

The unique mitochondrial form and function of Antarctic channichthyid icefishes

Antarctic icefishes of the family Channichthyidae are the only vertebrate animals that as adults do not express the circulating oxygen-binding protein hemoglobin (Hb). Six of the 16 family members also lack the intracellular oxygen-binding protein myoglobin (Mb) in the ventricle of their hearts and all lack Mb in oxidative skeletal muscle. The loss of Hb has led to substantial remodeling in the cardiovascular system of icefishes to facilitate adequate oxygenation of tissues. One of the more curious adaptations to the loss of Hb and Mb is an increase in mitochondrial density in cardiac myocytes and oxidative skeletal muscle fibers. The proliferation of mitochondria in the aerobic musculature of icefishes does not arise through a canonical pathway of mitochondrial biogenesis. Rather, the biosynthesis of mitochondrial phospholipids is up-regulated independently of the synthesis of proteins and mitochondrial DNA, and newly-synthesized phospholipids are targeted primarily to the outer-mitochondrial membrane. Consequently, icefish mitochondria have a higher lipid-to-protein ratio compared to those from red-blooded species. Elevated levels of nitric oxide in the blood plasma of icefishes, compared to red-blooded notothenioids, may mediate alterations in mitochondrial density and architecture. Modifications in mitochondrial structure minimally impact state III respiration rates but may significantly enhance intracellular diffusion of oxygen. The rate of oxygen diffusion is greater within the hydrocarbon core of membrane lipids compared to the aqueous cytosol and impeded only by proteins within the lipid bilayer. Thus, the proliferation of icefish's mitochondrial membranes provides an optimal conduit for the intracellular diffusion of oxygen and compensates for the loss of Hb and Mb. Currently little is known about how mitochondrial phospholipid synthesis is regulated and integrated into mitochondrial biogenesis. The unique architecture of the oxidative muscle cells of icefishes highlights the need for further studies in this area.     

Recent origin of sub-Antarctic notothenioids

Comparison of partial mitochondrial 12S and 16S rDNA sequences from non-Antarctic notothenioid fishes - an icefish Champsocephalus esox and two members of the genus Patagonotothen - and their sister species from the Southern Ocean suggests that their divergence took place 1.7 and 6.6-7 million years ago, respectively, i.e. much later than the formation of the Antarctic Polar Front (20-25 million years ago).     

Chromosomal patterns of major and 5S ribosomal DNA in six icefish species (Perciformes,Notothenioidei, Channichthyidae)

Comparative chromosomal mapping of major and 5S ribosomal genes in six species of the family Channichthyidae, namely Champsocephalus gunnari, Channichthys rhinoceratus, Chionodraco hamatus, Cryodraco atkinsoni, Pagetopsis macropterus and Neopagetopsis ionah, was performed by fluorescence in-situ hybridization, and using 28S and 5S ribosomal gene (rDNA) sequences as probes. Clusters of major and 5S ribosomal genes co-localize and likely compose the entire arm of a single pair of submetacentric chromosomes in all the species. In one species, P. macropterus, a second pair of chromosomes bears an additional common locus for both the two families of ribosomal genes. In all species, except N. ionah, additional copies of 5S rDNA sequences are also present on two other chromosome pairs, including the Y-chromosome in the males of Chionodraco hamatus. The pattern of ribosomal DNAs contributes to species-specific characterization in this fish family, and to our general knowledge and understanding of the chromosomal organization and evolution of the icefish genome.     

Antarctic icefishes (Channichthyidae): a unique family of fishes. A review, Part II

Icefish or white- blooded fish are a family of species unique among vertebrates in that they possess no haemoglobin. With the exception of one species which occurs on the southern Patagonian shelf, icefish live only in the cold-stable and oxygen-rich environment of the Southern Ocean. It is still questionable how old icefish are in evolutionary terms: they may not be older than 6 Ma, i.e. they evolved well after the Southern Ocean started to cool down or they are 15–20 Ma old and started to evolve some time after the formation of the Antarctic Circumpolar Current. Individuals of most icefish species with the exception of species of the genus Champsocephalus have been found down to 700–800 m depth, a few even down to more than 1,500 m. Icefish have been shown to present organ-level adaptations on different levels to compensate for the ‘disadvantages’ of lacking respiratory pigments. These include a low metabolic rate, well perfused gills, increased blood volume, increased cardiac output, cutaneous uptake of oxygen, increased blood flow with low viscosity, enlarged capillaries, large heart, and increased skin vascularity. Biological features, such as reproduction and growth, are not unique and are comparable to other notothenioids living in the same environment. Icefish produce large yolky eggs which have a diameter of more than 4 mm in most species. Consequently, the number of eggs produced is comparatively small and exceeds 10,000–20,000 eggs in only a few cases. With the exception of species of the genus Champsocephalus which mature at an age of 3 to 4 years, icefish do not attain maturity before they are 5–8 years old. Spawning period of most icefish species is autumn–winter. The incubation period spans from 2 to 3 months in the north of the Southern Ocean to more than 6 months close to the continent. Growth in icefish to the extent it is known is fairly rapid. They grow 6–10 cm in length per annum before they reach spawning maturity. Icefish feed primarily on krill and fish. Some icefish species were abundant enough to be exploited by commercial fisheries, primarily in the 1970s and 1980s with Champsocephalus gunnari as the main target species. Most stocks of this species had been overexploited by the beginning of the 1990s, some had further declined due to natural causes. Other species taken as by-catch species in fisheries were Chaenocephalus aceratus, Pseudochaenichthys georgianus, and Chionodraco rastrospinosus. Chaenodraco wilsoni was the only species exploited on a commercial scale in the high-Antarctic. Part I was published in the preceding issue of Polar Biology. DOI 10.1007/s00300-005-0019-z.     

Antarctic icefishes (Channichthyidae): a unique family of fishes. A review, Part I

Icefish or white-blooded fish are a family of species, unique among vertebrates in that they possess no haemoglobin. With the exception of one species which occurs on the southern Patagonian shelf, icefish live only in the cold-stable and oxygen-rich environment of the Southern Ocean. It is still questionable how old icefish are in evolutionary terms: they may not be older than 6 Ma, i.e. they evolved well after the Southern Ocean started to cool down or they are 15–20 Ma old and started to evolve some time after the formation of the Antarctic Circumpolar Current. Individuals of most icefish species with the exception of species of the genus Champsocephalus have been found down to 700–800 m depth, a few even down to more than 1,500 m. Icefish have been shown to present organ-level adaptations on different levels to compensate for the ‘disadvantages’ of lacking respiratory pigments. These include a low metabolic rate, well perfused gills, increased blood volume, increased cardiac output, cutaneous uptake of oxygen, increased blood flow with low viscosity, enlarged capillaries, large heart, and increased skin vascularity. Biological features, such as reproduction and growth, are not unique and are comparable to other notothenioids living in the same environment. Icefish produce large yolky eggs which have a diameter of more than 4 mm in most species. Consequently, the number of eggs produced is comparatively small and exceeds 10,000–20,000 eggs in only a few cases. With the exception of species of the genus Champsocephalus which mature at an age of 3 to 4 years, icefish do not attain maturity before they are 5–8 years old. Spawning period of most icefish species is autumn-winter. The incubation period spans from 2 to 3 months in the north of the Southern Ocean to more than 6 months close to the continent. Growth in icefish to the extent it is known is fairly rapid. They grow 6–10 cm in length per annum before they reach spawning maturity. Icefish feed primarily on krill and fish. Some icefish species were abundant enough to be exploited by commercial fisheries, primarily in the 1970s and 1980s with Champsocephalus gunnari as the main target species. Most stocks of this species had been overexploited by the beginning of the 1990s, some had further declined due to natural causes. Other species taken as by-catch species in fisheries were Chaenocephalus aceratus, Pseudochaenichthys georgianus, and Chionodraco rastrospinosus. Chaenodraco wilsoni was the only species exploited on a commercial scale in the high-Antarctic. Part II will be published in the following issue. DOI 10.1007/s00300-005-0020-6.     

Heart structure and ventricular ultrastructure of hemoglobin- and myoglobin-free icefish Channichthys rhinoceratus

Summary The structural and ultrastructural characteristics of the heart of Channichthys rhinoceratus, an antarctic teleost devoid of respiratory pigments, are described and compared with those obtained from the red-blooded related species Notothenia rossii.The heart of the icefish is characterized by a spongy myocardium supplied with a highly developed arterial coronary system. This vasculature includes a subepicardial system and an extensive intratrabecular capillary network. Arterial hilar network and Thebesian vessels may also be present. The bulbus arteriosus shows unusually large spheroid structures located in the middle layer of the wall.Both white- and red-blooded species display comparable myocardial cell morphology and organelle distribution. However, the mitochondrial cristae of the former are more densely packed and the sarcolemma possesses numerous caveolae. A large proportion of non-contractile cells is also found in the icefish ventricular wall.     

Nitric oxide synthase type I (nNOS), vascular endothelial growth factor (VEGF) and myoglobin-like expression in skeletal muscle of Antarctic icefishes (Notothenioidei: Channichthyidae)

The Antarctic icefishes Channichthyidae lack haemoglobin and are thought to lack myoglobin (Mb) in their skeletal muscle as well. Due to the absence of both respiratory pigments, icefishes may present a variety of physiological adaptations in their skeletal muscles. In mammals, molecular responses to limiting oxygen availability in the skeletal muscle include, among others, the over expression of nitric oxide synthases (NOS), such as type I (neuronal nNOS) and type III (endothelial eNOS), as well as the vascular endothelial growth factor (VEGF). In this paper, we evaluated by western blot analysis whether the skeletal muscle of haemoglobin-less icefishes expresses in a constitutive manner higher levels of the type I and type III NOS isoforms and VEGF. Our results demonstrate that haemoglobin-less icefish of the family Channichthyidae do indeed present higher expression of the type I NOS isoform compared with red-blooded Antarctic fish species of other families of the same suborder Notothenioidei. In contrast, VEGF was not over-expressed. Moreover, we show that some icefish species, thought previously to lack Mb in oxidative muscles, actually present Mb-like immunoreactivity in their skeletal muscle.     

Cardiac myoglobin deficit has evolved repeatedly in teleost fishes

Myoglobin (Mb) is the classic vertebrate oxygen-binding protein present in aerobic striated muscles. It functions principally in oxygen delivery and provides muscle with its characteristic red colour. Members of the Antarctic icefish family (Channichthyidae) are widely thought to be extraordinary for lacking cardiac Mb expression, a fact that has been attributed to their low metabolic rate and unusual evolutionary history. Here, we report that cardiac Mb deficit, associated with pale heart colour, has evolved repeatedly during teleost evolution. This trait affects both gill- and air-breathing species from temperate to tropical habitats across a full range of salinities. Cardiac Mb deficit results from total pseudogenization in three-spined stickleback and is associated with a massive reduction in mRNA level in two species that evidently retain functional Mb. The results suggest that near or complete absence of Mb-assisted oxygen delivery to heart muscle is a common facet of teleost biodiversity, even affecting lineages with notable oxygen demands. We suggest that Mb deficit may affect how different teleost species deal with increased tissue oxygen demands arising under climate change.     

Metabolic pattern of the heart of haemoglobin- and myoglobin-free Antarctic fish Channichthys rhinoceratus

Summary Maximum activities of energy metabolism related enzymes, myofibrillar ATPase and concentrations of carnitine, lipids and myoglobin have been assayed in heart and white muscle of the ice-fish Channichthys rhinoceratus. Results are compared with those of the redblooded Paranotothenia magellanica. Increased activities of glycolytic enzymes and extremely high lactate dehydrogenase activity have been found in icefish heart, suggesting a substantial involvement of anaerobic glycolysis under conditions of oxygen depletion. During normoxia, ATP generation is achieved via oxidative pathways of carbohydrate or fatty acid catabolism. Possible implication of the myocardium in homeostatic regulation by lactate oxidation is also discussed.     

Mitochondrial function in Antarctic notothenioid fishes that differ in the expression of oxygen-binding proteins

State III respiration rates were measured in mitochondria isolated from hearts of Antarctic notothenioid fishes that differ in the expression of hemoglobin (Hb) and myoglobin (Mb). Respiration rates were measured at temperatures between 2 and 40°C in Gobionotothen gibberifrons (+Hb/+Mb), Chaenocephalus aceratus (–Hb/–Mb) and Chionodraco rastrospinosus (–Hb/+Mb). Blood osmolarity was measured in all three species and physiological buffers prepared for isolating mitochondria and measuring respiration rates. Respiration rates were higher in mitochondria from G. gibberifrons compared to those from C. aceratus at 2°C, but were similar among all species at temperatures between 10 and 26°C. Respiration rates were significantly lower in icefishes at 35 and 40°C compared to G. gibberifrons. The respiratory control ratio of isolated mitochondria was lower in C. aceratus compared to G. gibberifrons at all temperatures below 35°C. At 35 and 40°C, mitochondria were uncoupled in all species. The Arrhenius break temperature of state III respiration was similar among all three species (30.5 ± 0.9°C) and higher than values previously reported for Antarctic notothenioids, likely due to the higher osmolarity of buffers used in this study. These results suggest that differences in mitochondrial structure, correlated with the expression of oxygen-binding proteins, minimally impact mitochondrial function.     

The complete mitochondrial genome of the mackerel icefish,Champsocephalus gunnari (Actinopterygii: Channichthyidae), with reference to the evolution of mitochondrial genomes in Antarctic notothenioids

The mackerel icefish (Champsocephalus gunnari Lönnberg, 1905) is a ray‐finned fish living in the Southern Ocean around Antarctica. We sequenced the complete mitochondrial (mt) genome of the mackerel icefish and a segment from cytochrome b to the control region (CR) in 32 individuals. The mt genome of the mackerel icefish was rearranged, containing two nicotinamide adenine dinucleotide (reduced form) dehydrogenase subunit 6 (ND6), two tRNA^Glu, and two CRs. However, variations in numbers of ND6 and tRNA^Glu were observed amongst individuals. These variations included type 1 (containing two ND6 and two tRNA^Glu), type 2 (containing one ND6, one incomplete ND6, and one tRNA^Glu), and type 3 (containing one ND6 and one tRNA^Glu). The gene orders of types 1 and 2, and variations in numbers of ND6 and tRNA^Glu were not previously found in any Antarctic notothenioids, whereas type 3 is the same as that of Racovitzia glacialis. Phylogenetic analyses of CR DNA sequences showed that duplicated CRs of the same species formed a monophyletic group, suggesting that duplication of CRs occurred in each species. The frequent duplication of mt genomes in Antarctic notothenioids is an unusual feature in vertebrates. We propose that interspecific hybridization and impairment of mismatch repair might account for the high frequency of gene duplications and rearrangement of mt genomes in Antarctic notothenioids.     

Microsatellite analysis reveals genetic differentiation between year-classes in the icefish <Emphasis Type="Italic">Chaenocephalus aceratus</Emphasis> at South Shetlands and Elephant Island

Chaenocephalus aceratus is one of the most abundant Antarctic icefish species in the Atlantic sector and has been a by-catch species in the fishery for mackerel icefish, Champsocephalus gunnari, between the mid-1970s and mid-1980s at South Georgia, South Orkney, and South Shetland Islands. The species became the target of the fishery in particular seasons, such as at South Georgia in 1977/78. In our paper, we report results on genetic differentiation for 11 microsatellite loci in C. aceratus samples collected at the South Shetlands and Elephant Island. This study represents the first report on microsatellite variability of an icefish species. Our results support the evidence from previous studies on differences in infestation patterns of parasites that a single panmictic population of C. aceratus exists, spanning the two sampling sites separated by about 100 km. Moreover, our study indicates the presence of a significant genetic differentiation between individual year-classes pointing out the existence of dynamic processes acting at the population genetic level, according to recent results for broadly distributed marine species. Both small effective population size and immigration from unsampled differentiated stocks may be at the base of the differentiation found in C. aceratus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The heart of the Antarctic icefish as paradigm of cold adaptation

The stenothermal Antarctic fishes, particularly the hemoglobinless icefish, have developed biochemical, metabolic and morpho-functional features of cardiac performance that can help to decipher some mechanisms underlying cardiac cold adaptation. Examples taken from different levels of cardiac biology in Antarctic fish as a paradigm of cold adaptation include: the function of myoglobin in the icefish species that either express or do not express this pigment; the metabolic and ultrastructural reshaping of the myocardiocytes; and the intrinsic mechanical characteristics of the icefish heart ventricle as a low rate, low pressure and high volume pump.     

Diet of two icefish species from the South Shetland Islands and Elephant Island,<Emphasis Type="Italic"> Champsocephalus gunnari</Emphasis> and<Emphasis Type="Italic"> Chaenocephalus aceratus</Emphasis>

The summer diet of two species of icefishes (Channichthyidae) from the South Shetland Islands and Elephant Island, Champsocephalus gunnari and Chaenocephalus aceratus, was investigated from 2001 to 2003. Champsocephalus gunnari fed almost exclusively on krill (Euphausia superba) in all years. The importance of other taxa (Themisto gaudichaudii, mysids, myctophids) in the diet was negligible. The average feeding rate of Champsocephalus gunnari inferred from an exponential gastric evacuation model was between 1.0 and 1.5% body weight per day. Most of the stomachs of Chaenocephalus aceratus were empty. Stomachs with food contained mainly krill, mysids and fish. Among the fish taken, locally abundant species formed the bulk of the diet: Gobionotothen gibberifrons in 2001, Lepidonotothen larseni and Champsocephalus gunnari in 2002 and L. larseni in 2003. An ontogenetic shift in feeding preference of Chaenocephalus aceratus was observed: fish smaller than 30 cm fed on krill and mysids, while larger animals relied primarily on fish.     

Preliminary assessment of population structure in the mackerel icefish (Champsocephalus gunnari)

The mackerel icefish (Champsocephalus gunnari Lönnberg E (1905) The Fishes of the Swedish South Polar Expedition. Wiss. Ergebnisse Schwedische Südpol- Exped. 1901–1903, vol 5, p 37 is widely distributed south of the Antarctic convergence and over shelf areas surrounding sub-Antarctic Islands. In order to evaluate global population structure in this species, we examined DNA sequence variation in four mitochondrial regions and four nuclear genes in icefish from four locations in the Atlantic Ocean sector and one location in the Indian Ocean. Despite small sample sizes, mitochondrial and nuclear gene data indicated the existence of at least three genetically distinct stocks: Heard Island, South Shetland Islands, and the remaining Atlantic populations (Shag Rocks, South Georgia, and Bouvet Island). The mitochondrial and nuclear SNP markers developed here will be useful for more extensive analyses of population structure in this species.