Molecular ecophysiology of Antarctic notothenioid fishes

The notothenioid fishes of the Southern Ocean surrounding Antarctica are remarkable examples of organismal adaptation to extreme cold. Their evolution since the mid-Miocene in geographical isolation and a chronically cold marine environment has resulted in extreme stenothermality of the extant species. Given the unique thermal history of the notothenioids, one may ask what traits have been gained, and conversely, what characters have been lost through change in the information content of their genomes. Two dramatic changes that epitomize such evolutionary transformations are the gain of novel antifreeze proteins, which are obligatory for survival in icy seawater, by most notothenioids and the paradoxical loss of respiratory haemoproteins and red blood cells, normally deemed indispensable for vertebrate life, by the species of a highly derived notothenioid family, the icefishes. Here, we review recent advances in our understanding of these traits and their evolution and suggest future avenues of investigation.The formerly coherent paradigm of notothenioid freeze avoidance, developed from three decades of study of antifreeze glycoprotein (AFGP) based cold adaptation, now faces challenges stemming from the recent discovery of antifreeze-deficient, yet freeze-resistant, early notothenioid life stages and from definitive evidence that the liver is not the physiological source of AFGPs in notothenioid blood. The resolution of these intriguing observations is likely to reveal new physiological traits that are unique to the notothenioids. Similarly, the model of AFGP gene evolution from a notothenioid pancreatic trypsinogen-like gene precursor is being expanded and refined based on genome-level analyses of the linked AFGP loci and their ancestral precursors. Finally, the application of comparative genomics to study evolutionary change in the AFGP genotypes of cool-temperate notothenioids from sub-Antarctic habitats, where these genes are not necessary, will contribute to the mechanistic understanding of the dynamics of AFGP gene gain and loss.In humans and most vertebrates, mutations in the alpha- or beta-globin genes or defects in globin chain synthesis are causes of severe genetic disease. Thus, the 16 species of haemoglobinless, erythrocyte-null icefishes are surprising anomalies -- in fact, they could only have evolved and thrived due to relaxed selection pressure for oxygen-binding proteins in the cold, oxygen-rich waters of the Southern Ocean. Fifteen of the sixteen icefish species have lost most of the adult alphabeta-globin locus and retain only a small 3' fragment of the alpha-globin gene. The only exception to this pattern occurs in Neopagetopsis ionah, which possesses a disrupted alphabeta-globin gene complex that probably represents a non-functional intermediate on the evolutionary pathway to near total globin gene extinction. By contrast, six of the icefish species fail to express myoglobin. The absence of myoglobin expression has occurred by several independent mutations and distinct mechanisms. Haemoprotein loss is correlated with dramatic increases in cellular mitochondrial density, heart size, blood volume and capillary bed volume. Evolution of these compensatory traits was probably facilitated by the homeostatic activity of nitric oxide, a key modulator of angiogenesis and mitochondrial biogenesis. These natural knockouts of the red blood cell lineage are an excellent genomic resource for erythroid gene discovery by comparative genomics, as illustrated for the newly described gene, bloodthirsty.     

Structure and expression of genes involved in transport and storage of iron in red-blooded and hemoglobin-less antarctic notothenioids

Antarctic notothenioids are characterized by a drastic reduction of the hemoglobin content, a condition that reaches its extreme in icefish that, following a gene deletion event, are completely devoid of hemoglobin. To answer the question on what type of adaptive changes occurred in icefish to prevent accumulation of potentially dangerous ferrous iron, we investigated the genes of four proteins known to play a key role in iron metabolism. For this purpose, we cloned and sequenced the cDNAs encoding ceruloplasmin, transferrin, ferritin and divalent metal transporter 1. While the inferred amino acid sequences of transferrin from different Antarctic fish species showed a high level of similarity with the homologous proteins from other species, ceruloplasmin sequence featured amino acid substitutions affecting a copper binding site. Another peculiarity was the presence in subunit H of the icefish ferritin of the two sets of sites involved in iron oxidation and iron mineralization, which in mammals are located on two distinct ferritin subunits. Significant differences in the expression levels of the four genes were found between hemoglobinless and red-blooded notothenioids. An increased expression of ceruloplasmin mRNA in icefish was interpreted as a compensatory mechanism to prevent accumulation of ferrous iron in hemoglobinless fish. In icefish, the amounts of ferritin H-chain mRNA measured in liver, blood and head kidney were lower than in the same organs of the red-blooded fish. In the spleen of both fishes, the expression levels of ferritin H-chain were significantly lower than in the spleen of a "pink-blooded" notothenioid with an intermediate hemoglobin content. Finally, the amount of divalent metal transporter mRNA measured in the head-kidney was lower in the icefish than in the same organ of its red-blooded counterpart. These results indicate that the loss of hemoglobin in icefish is accompanied by remodulation of the iron metabolism.     

Antarctic notothenioid fishes: genomic resources and strategies for analyzing an adaptive radiation

The perciform suborder Notothenoidei provides a compelling opportunity to study the adaptive radiation of a marine species-flock in the cold Southern Ocean that surrounds Antarctica. To facilitate genome-level studies of the diversification of these fishes, we present estimates of the genome sizes of 11 Antarctic species and describe the production of high-quality bacterial artificial chromosome (BAC) libraries for two, the red-blooded notothen Notothenia coriiceps and the white-blooded icefish Chaenocephalus aceratus. Our results indicate that evolution of phylogenetically derived notothenioid families (e.g., the crown group Channichthyidae [icefishes]), was accompanied by genome expansion. Six species from the basal family Nototheniidae had C-values between 0.98 and 1.20?pg, a range that is consistent with the genome sizes of proposed outgroups (e.g., percids) of the notothenioid suborder. In contrast, four icefishes had C-values in the range 1.66-1.83?pg. The BAC libraries VMRC-19 (N. coriiceps) and VMRC-21 (C. aceratus) comprise 12× and 10× coverage of the respective genomes and have average insert sizes of 138 and 168?kb. Paired BAC-end reads representing ～0.1% of each genome showed that the repetitive element landscapes of the two genomes (13.4% of the N. coriiceps genome and 14.5% for C. aceratus) were similar. The availability of these high-quality and well-characterized BAC libraries sets the stage for targeted genomic analyses of the unusual anatomical and physiological adaptations of the notothenioids, some of which mimic human diseases. Here we consider the evolution of secondary pelagicism by various taxa of the group and illustrate the utility of Antarctic icefishes as an evolutionary-mutant model of human osteopenia (low-mineral density of bones).     

Two species of antarctic icefishes (genus <Emphasis Type="Italic">Champsocephalus</Emphasis>) share a common genetic lesion leading to the loss of myoglobin expression

Species of the suborder Notothenioidei dominate the fish fauna of coastal Antarctic waters. Members of one notothenioid family, Channichthyidae (Antarctic icefishes), are unique among all vertebrates in lacking the circulating oxygen-binding protein hemoglobin. Icefish species also do not uniformly express the intracellular oxygen-binding protein myoglobin (Mb) in their oxidative muscles. Our laboratory previously characterized the pattern of cardiac Mb expression in 13 of the 16 known icefish species. In this paper, we complete the survey of cardiac Mb expression among all 16 known species of icefishes. Using PAGE and immunoblot analyses, we demonstrate that both Channichthys rhinoceratus and Cryodraco atkinsoni express Mb in heart ventricle, while Champsocephalus esox does not express the protein. We report Mb gene sequences from Channichthys rhinoceratus and Champsocephalus esox genomic DNA. The Mb gene of C. esox contains the identical 5-bp duplication/insertion to that observed in congeneric Champsocephalus gunnari, a species that also does not produce Mb. This duplication in exon 2 of the Champsocephalus spp. gene causes a shift in reading frame at a position normally encoding for amino acid 91 and also results in a premature stop codon, thus disrupting translation of the normal protein. Thus, 6 of the 16 known icefish species do not express cardiac Mb. These results confirm earlier conclusions that losses of Mb expression have occurred via at least four independent events during the evolution of the icefish family. Extreme similarity of Mb genes in Champsocephalus congeners further suggests recent speciation despite early divergence of this group from the lineage leading to more derived icefishes.     

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.     

Organisation of the Hb 1 genes of the Antarctic skate Bathyraja eatonii: new insights into the evolution of globin genes

An extensive investigation of the organisation of globin genes has greatly contributed to the understanding of universal mechanisms of gene evolution and expression. Cartilaginous fish are the first organisms that have evolved the tetrameric form of hemoglobin (Hb). So far, there has been absolute lack of data about globin genes in chondrichthyans. Bathyraja is the dominant rajid south of 60 degrees S. In the framework of the investigations on globin genes of Antarctic red-blooded and Hb-less fish we obtained the cloning of the alpha- and beta-globin cDNAs of the main Hb (Hb 1) of the skate Bathyraja eatonii. Then, a genomic fragment of 6.2 kb was isolated where the Hb 1 alpha and beta genes are linked in a tail-to-head (3' to 5') orientation. The beta-globin gene promoter region and the chromosomal organisation of the Hb 1 genes of B. eatonii have been compared to their homologues in other vertebrates. The finding of a tail-to-head linkage of the Hb 1 alpha- and beta-globin genes in B. eatonii is the first characterisation of the organisation of globin genes in chondrichthyes; such finding offers a novel contribution to the understanding of the evolution of this class of genes. Moreover, the characterisation of chondrichthyan genes is very important for gaining insight into the ancestral state of vertebrate genomes.     

Thermal tolerance of Antarctic notothenioid fishes correlates with level of circulating hemoglobin

The West Antarctic Peninsula region is experiencing some of the most rapid elevations in temperature of any marine environment. We assessed thermal tolerance of white- and red-blooded Antarctic notothenioid fishes inhabiting these waters, using a modified critical thermal maximum (CT(max)) design. Temperature was elevated acutely from ambient at a constant rate of 3.6°C h(-1), and CT(max) was defined as the temperature where animals lost righting response. CT(max) temperatures of white-blooded icefishes Chionodraco rastrospinosus (13.3° ± 0.2°C) and Chaenocephalus aceratus (13.9° ± 0.4°C) were significantly lower than those of red-blooded fishes Gobionotothen gibberifrons (15.5° ± 0.2°C) and Notothenia coriiceps (17.1° ± 0.2°C). Lepidonotothen squamifrons, a red-blooded species with low hematocrit, exhibited a CT(max) (14.2° ± 0.4°C) that was significantly lower than that of the other red-blooded animals and similar to that of icefishes. A strong relationship between CT(max) and hematocrit (r(2) = 0.76) suggests that the oxygen-carrying capacity of blood may partially dictate acute lethal temperature. Despite a short treatment duration, we detected a rise in the mRNA level of hypoxia response gene HIF-1α in N. coriiceps heart tissue. One-week exposure to 4°C had no effect on the CT(max) of N. coriiceps, indicating an inability to compensate for rising temperature under these experimental conditions. Our results suggest that icefishes are particularly sensitive to temperature elevation because of a lack of hemoglobin and may be a sentinel taxon for climate change.     

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.     

Characterization of embryonic globin genes of the zebrafish

Hemoglobin switching is a complex process by which distinct globin chains are produced during stages of development. In an effort to characterize the process of hemoglobin switching in the zebrafish model system, we have isolated and characterized several embryonic globin genes. The embryonic and adult globin genes are found in clusters in a head-to-head configuration. One cluster of embryonic and adult genes is localized to linkage group 3, whereas another embryonic cluster is localized on linkage group 12. Several embryonic globin genes demonstrate an erythroid-specific pattern of expression early during embryogenesis and later are downregulated as definitive hematopoiesis occurs. We utilized electrospray mass spectroscopy to correlate globin genes and protein expression in developing embryonic red cells. The mutation, zinfandel, has a hypochromic microcytic anemia as an embryo, but later recovers in adulthood. The zinfandel gene maps to linkage group 3 near the major globin gene locus, strongly suggesting that zinfandel represents an embryonic globin defect. Our studies are the first to systematically evaluate the embryonic globins in the zebrafish and will ultimately be useful in evaluating zebrafish mutants with defects in hemoglobin production and switching.     

The primary structure of hemoglobin D from the Aldabra giant tortoise,Geochelone gigantea

The complete primary structures of alpha D-2- and beta-globin of hemoglobin D (Hb D) from the Aldabra giant tortoise, Geochelone gigantea, have been constructed by amino acid sequencing analysis in assistance with nucleotide sequencing analysis of PCR fragments amplified using degenerate oligonucleotide primers. Using computer-assisted sequence comparisons, the alpha D-2-globin shared a 92.0% sequence identity versus alpha D-globin of Geochelone carbonaria, a 75.2% versus alpha D-globin of Aves (Rhea americana) and a 62.4% versus alpha A-globin of Hb A expressed in adult red blood cells of Geochelone gigantea. Additionally, judging from their primary structures, an identical beta-globin was common to the two hemoglobin components, Hb A and Hb D. The alpha D-2- and beta-globin genes contained the three-exon and two-intron configurations and showed the characteristic of all functional vertebrate hemoglobin genes except an abnormal GC dinucleotide instead of the invariant GT at the 5' end of the second intron sequence. The introns of alpha D-2-globin gene were both small (224-bp/first intron, 227-bp/second intron) such that they were quite similar to those of adult alpha-type globins; the beta-globin gene has one small intron (approximately 130-bp) and one large intron (approximately 1590-bp). A phylogenetic tree constructed on primary structures of 7 alpha D-globins from Reptilia (4 species of turtles, 2 species of squamates, and 1 species of sphenodontids) and two embryonic alpha-like globins from Aves (Gullus gullus) and Mammals (Homo sapiens) showed the following results: (1) alpha D-globins except those of squamates were clustered, in which Sphenodon punctatus was a closer species to birds than turtles; (2) separation of the alpha A- and alpha D-globin genes occurred approximately 250 million years ago after the embryonic alpha-type globin-genes (pi' and zeta) first split off from the ancestor of alpha-type globin gene family.     

Correlations of repetitive and AT-rich DNA segments within the chicken globin gene domains

The repetitive DNA segments were mapped within a 30 Kbp genomic domain including (in 5 to 3 order) the chicken embryonic pi and adult alpha D (minor) and alpha A (major) globin genes. Two repeats map 5 and 8 Kbp upstream from the embryonic pi gene and another 3 Kbp downstream of the adult alpha A gene. These repetitive DNA sequences are placed within, or immediately adjacent to the AT-rich DNA segments framing this domain. Similar correlations exist also within the chicken beta globin gene domain. The positions of these AT-rich and repetitive DNA segments framing the alpha globin gene domain also correlate with other already explored features of long range DNA organisation, as clusters of sites of DNAse I hypersensitivity and differential methylation, sites of Matrix-DNA attachment, and with the beginning and end of the transcribed domain.     

Nucleotide sequence of the goat embryonic alpha globin gene (zeta) and linkage and evolutionary analysis of the complete alpha globin cluster

In previous studies we identified and sequenced clones containing two adult alpha globin genes of the goat. Additional studies have revealed the presence of an embryonic alpha globin gene termed zeta. Sequence analysis of the gene shows that it is the largest mammalian or avian globin gene cloned to date. Its unusual size is mainly due to a 14 base-pair tandem repeat sequence in its first intron. A similar sequence is also found in the first intron of the human zeta gene. The goat zeta coding sequence differs greatly from that of the adult alpha, particularly at amino acid position 38, where it codes for the amino acid replacement of Gln for Thr. This change may confer a higher intrinsic O2 affinity on the zeta globin protein, ensuring a sufficient O2 supply for the developing goat embryo. The cloning and sequencing of this gene completes the alpha globin locus of the goat, composed of three genes in the following order 5'-zeta-I alpha-II alpha-3'. Evolutionary comparisons of the goat alpha locus with other amphibian, avian and mammalian loci reveal several interesting features. Statistical analysis confirms the hypothesis that the embryonic alpha gene is much older (400 million years) than the embryonic beta gene (200 million years), and that it is descended from a primordial gene, whose present-day counterpart is the Xenopus larval alpha globin gene. Our results also suggest that after the divergence of the avian line, the alpha A gene converted the alpha D gene during the evolution of the pre-mammalian line. The alpha D globin gene remains unconverted in the avian line, potentially because of insertion/deletion sequences that may prevent any gene conversion event. The divergence rates of specific globin genes have been analyzed and found to form an essentially straight line, in agreement with the neutralist view of evolution.     

Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

Structure and dynamics of Antarctic fish neuroglobin assessed by computer simulations

Neuroglobin (Ngb) is a heme protein, highly conserved along evolution, predominantly found in the nervous system. It is upregulated by hypoxia and ischemia and may have a neuroprotective role under hypoxic stress. Although many other roles have been proposed, the physiological function is still unclear. Antarctic icefishes lack hemoglobin and some species also lack myoglobin, but all have Ngb and thus may help the elucidation of Ngb function. We present the first theoretically derived structure of fish Ngb and describe its behavior using molecular dynamics simulations. Specifically, we sequenced and analyzed Ngbs from a colorless-blooded Antarctic icefish species Chaenocephalus aceratus and a related red-blooded species (Dissostichus mawsoni). Both fish Ngbs are 6-coordinated but have some peculiarities that differentiate them from mammalian counterparts: they have extensions in the N and C termini that can interact with the EF loop, and a gap in the alignment that changes the CD-region structure/dynamics that has been found to play a key role in human neuroglobin. Our results suggest that a single mutation between both fish Ngbs is responsible for significant difference in the behavior of the proteins. The functional role of these characteristics is discussed.     

Genomic organization of zebra finch alpha and beta globin genes and their expression in primitive and definitive blood in comparison with globins in chicken

How alpha and beta globin genes are organized and expressed in amniotes is of interest to researchers in a wide variety of fields. Data regarding this from avian species have been scarce. Using genomic and proteomic approaches, we present here our analysis of alpha and beta globins of zebra finch, a passerine bird. We show that finch alpha globin gene cluster has three genes (alphas 1–3), each orthologous to its chicken counterpart. Finch beta globin gene cluster has three genes (betas 1–3), with an additional pseudogene at the 3′ end. Finch beta3 is orthologous to chicken betaA, but the orthology of beta1 and beta2 to chicken counterparts is less clear. All six finch globins are confirmed to encode functional proteins. Gene expression in both globin gene clusters is regulated developmentally. Adult finch blood has a globin profile similar to that of adult chicken, with high levels of beta3 and alpha3 and moderate levels of alpha2. Finch embryonic primitive blood exhibits a globin profile very different from that of equivalent stage chick embryos, with all six globins expressed at high levels. Overall, our data provide a valuable resource for future studies in avian globin gene evolution and globin switching during erythropoietic development.