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.     

Troponin C of the Antarctic icefish (Champsocephalus gunnari) white muscle

• 1.1. The troponin C (TN-C) from the Antarctic icefish (_{Champsocephalus gunnari}) has been isolated to homogeneity by a procedure involving extraction from acetone powder, DEAE-Sepharose 4B and AcA 54 column chromatography.
• 2.2. The calcium-induced conformational changes of apo TN-C have been studied by absorption difference spectroscopy, circular dichroism and intrinsic fluorescence.
• 3.3. The results indicate that the overall characteristics of icefish TN-C (such as amino acid composition, modifications of helix content and of the microenvironment of aromatic residues as a function of calcium binding) are quite similar to those of rabbit TN-C.
• 4.4. The intrinsic fluorescence properties are close to those reported for pike and carp TN-C.

     

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).     

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.     

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.     

Regulation of splenic contraction persists as a vestigial trait in white-blooded Antarctic fishes

In fishes, the spleen can function as an important reservoir for red blood cells (RBCs), which, following splenic contraction, may be released into the circulation to increase haematocrit during energy-demanding activities. This trait is particularly pronounced in red-blooded Antarctic fishes in which the spleen can sequester a large proportion of RBCs during rest, thereby reducing blood viscosity, which may serve as an adaptation to life in cold environments. In one species, Pagothenia borchgrevinki, it has previously been shown that splenic contraction primarily depends on cholinergic stimulation. The aim of the present study was to investigate the regulation of splenic contraction in five other Antarctic fish species, three red-blooded notothenioids (Dissostichus mawsoni Norman, 1937, Gobionotothen gibberifrons Lönnberg, 1905, Notothenia coriiceps Richardson 1844) and two white-blooded “icefish” (Chaenocephalus aceratus Lönnberg, 1906 and Champsocephalus gunnari Lönnberg, 1905), which lack haemoglobin and RBCs, but nevertheless possess a large spleen. In all species, splenic strips constricted in response to both cholinergic (carbachol) and adrenergic (adrenaline) agonists. Surprisingly, in the two species of icefish, the spleen responded with similar sensitivity to red-blooded species, despite contraction being of little obvious benefit for releasing RBCs into the circulation. Although the icefish lineage lost functional haemoglobin before diversifying over the past 7.8–4.8 millions of years, they retain the capacity to contract the spleen, likely as a vestige inherited from their red-blooded ancestors.     

Geographic isolation and physiological mechanisms underpinning species distributions at the range limit hotspot of South Georgia

In order to allocate quotas for sustainable harvests, that account for climate warming, it is important to incorporate species vulnerabilities that will underlie likely changes in population dynamics. Hotspots, regions with rapidly changing climate, are important locations for rapid advances in mechanistic understanding of the factors driving these changes, particularly if they coincide with regions with a high incidence of range limits, such as the sub-Antarctic Island of South Georgia. This archipelago is at the Northern limit of the Southern Ocean and therefore the northern distribution limit for many Southern Ocean shallow water marine species, which are amongst the most sensitive fauna to increasing temperature. At range limits species may either be living close to their physiological limits, or they may have more resistant phenotypes. In case studies, the northern range limit population of the gastropod limpet, Nacella concinna, has greater physiological plasticity at South Georgia than those from further south, allowing them to cope better with the warmer and more variable seasonal temperatures. Bivalve species, however, alter their depth distributions at South Georgia, to avoid the warmer water masses, indicating that they may not be able to cope with the warmer temperatures. Mackerel icefish, Champsocephalus gunnari, has a unique Antarctic trait, the loss of haemoglobin. A combination of temperature driven change in food web structure, and this extreme physiological cold adaptation, may explain why rapid warming at its northern range limit of South Georgia, has prevented stocks fully recovering from over fishing in the 1980s, despite highly conservative management strategies.     

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.     

Interannual variability in the feeding of ice fish (Notothenioidei, Channichthyidae) in the southern Scotia Arc and the Antarctic Peninsula region (CCAMLR Subareas 48.1 and 48.2)

We have studied the annual variation in food intake of three sub-Antarctic ice fish species (Champsocephalus gunnari, Chaenocephalus aceratus, and Pseudochaenichthys georgianus) and three high-Antarctic ice fish species (Chionodraco rastrospinosus, Cryodraco antarcticus, and Chaenodraco wilsoni). Stomach content analyses were conducted during bottom trawl surveys around the South Shetland Islands in 1998, 2001, 2002, 2003, and 2007, the South Orkney Islands in 1999 and 2009, and off the north-western Antarctic Peninsula in 2002, 2006, and 2006/2007 in order to obtain further insight into the amount of food of Antarctic demersal fish consume during summer. Annual variation in food intake was comparatively low within an area in the krill-feeding species C. gunnari and C. wilsoni. Food intake was much more variable, by a factor of 2 or 3 among years and areas, in larger C. aceratus and C. antarcticus, which rely heavily on fish as their dietary source. Food consumption was intermediate in the two species P. georgianus and C. rastrospinosus, which rely on both krill and fish.     

Population divergences despite long pelagic larval stages: lessons from crocodile icefishes (Channichthyidae)

Dispersal via pelagic larval stages plays a key role in population connectivity of many marine species. The degree of connectivity is often correlated with the time that larvae spend in the water column. The Antarctic notothenioid fishes develop through an unusually long pelagic larval phase often exceeding 1 year. Notothenioids thus represent a prime model system for studying the influence of prolonged larval phases on population structure in otherwise demersal species. Here, we compare the population genetic structure and demographic history of two sub‐Antarctic crocodile icefish species (Chaenocephalus aceratus and Champsocephalus gunnari) from the Scotia Arc and Bouvet Island in the Atlantic sector of the Southern Ocean to delineate the relative importance of species‐specific, oceanographic and paleoclimatic factors to gene flow. Based on 7 (C. aceratus) and 8 (C. gunnari) microsatellites, as well as two mitochondrial DNA markers (cytochrome b, D‐loop), we detect pronounced population genetic structure in both species (amova FSTs range from 0.04 to 0.53). High genetic similarities were found concordantly in the populations sampled at the Southern Scotia Arc between Elephant Island and South Orkney Islands, whereas the populations from Bouvet Island, which is located far to the east of the Scotia Arc, are substantially differentiated from those of the Scotia Arc region. Nonetheless, haplotype genealogies and Bayesian cluster analyses suggest occasional gene flow over thousands of kilometres. Higher divergences between populations of C. gunnari as compared to C. aceratus are probably caused by lower dispersal capabilities and demographic effects. Bayesian skyline plots reveal population size reductions during past glacial events in both species with an estimated onset of population expansions about 25 000 years ago.     

Drinking in Antarctic fishes

Drinking rates have never been measured in Antarctic fish. Drinking rates were measured in four species of notothenioid fish, including a hemoglobinless icefish, found in the near-freezing waters of the Ross Sea of the Southern Ocean. All of the fish, with the exception of the icefish, had low drinking rates and high serum osmolalities relative to temperate seawater fish. The icefish had significantly higher drinking rates and serum osmolalities relative to the Antarctic fishes containing hemoglobin, including Trematomus bernacchii. Warm acclimation of T. bernacchii, from −1.5°C to +4°C for 4 weeks, significantly increased their drinking rates 4.6-fold, significantly decreased their serum and intestinal osmolality by 11% and 12%, respectively, relative to cold-acclimated fish. These results indicate that increased drinking rates in Antarctic fish at elevated temperatures are involved in maintaining a lower serum osmolality.     

Freezing avoidance of the Antarctic icefishes (Channichthyidae) across thermal gradients in the Southern Ocean

Biogeographic studies separate the Antarctic Notothenioid fish fauna into high- and low-latitude species. Past studies indicate that some species found in the high-latitude freezing waters of the High-Antarctic Zone have low-serum hysteresis freezing points, while other species restricted to the low-latitude seasonal pack ice zone have higher serum hysteresis freezing points above the freezing point of seawater (−1.9°C), but the relationship has not been systematically investigated. Freeze avoidance was quantified in 11 species of Antarctic icefishes by determining the hysteresis freezing points of their blood serum, in addition, the freezing point depression from serum osmolytes, the antifreeze activity from serum antifreeze glycoproteins (AFGPs), and the antifreeze activity from serum antifreeze potentiating protein were measured for each species. Serum hysteresis freezing point, a proxy for organismal freeze avoidance, decreased as species were distributed at increasing latitude (linear regression r ² 0.66, slope −0.046°C °latitude⁻¹), which appeared largely independent of phylogenetic influences. Greater freeze avoidance at high latitudes was largely a result of higher levels of antifreeze activity from serum AFGPs relative to those in species inhabiting the low-latitude waters. The icefish fauna could be separated into a circum High-Antarctic Group of eight species that maintained serum hysteresis freezing points below −1.9°C even when sampled from less severe habitats. The remaining three species with low-latitude ranges restricted to the waters of the northern part of the west Antarctic Peninsula and Scotia Arc Islands had serum hysteresis freezing points at or above −1.9°C due to significantly lower combined activity from all of their serum antifreeze proteins than found in the High-Antarctic Zone icefish.     

Fatty acid composition of lipid-rich myctophids and mackerel icefish (<Emphasis Type="Italic">Champsocephalus gunnari</Emphasis>) – Southern Ocean food-web implications

The lipid content, fatty acid composition and calorific value of seven species of mesopelagic deep-sea fish of the family Myctophidae and the mackerel icefish, Champsocephalus gunnari, important in the diet of Southern Ocean marine predators, are presented. Fish were sampled at the Kerguelen Plateau (KP) and Macquarie Ridge (MR) in the Indian and Pacific sectors of the Southern Ocean respectively, to examine geographic variation in lipid compositon. All species of myctophid from KP and Electrona antarctica from MR were high in lipid content (6-18% wet mass), particularly Gymnoscopelus nicholsi (18%) and E. antarctica (15%). The mackerel icefish, and G. fraseri and Protomyctophum tenisoni from MR were generally lower in lipid content (3-5%) and varied significantly in fatty acid composition from KP species. KP myctophids were high in calorific content (9.3 kJ g^-1 wet mass) when compared with icefish (5.4 kJ g^-1 wet mass) and other published values for prey items of marine predators such as squid (1.7-4.5 kJ g^-1). KP myctophids were distinguished from each other and from C. gunnari and MR specimens by cluster and discriminant function analysis using six fatty acids (16:0, 18:1̩, 20:1̩, 22:1Ὃ, 20:5̣, 22:6̣). Findings presented here highlight trophic links between high-latitude fish and their prey and emphasise the importance of myctophids as a significant energy source for marine predators foraging in the Polar Frontal Zone.     

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.     

Age determination of the Antarctic fishes Champsocephalus gunnari and Notothenia rossii marmorata from South Georgia

Summary Investigations were performed on otoliths from Champsocephalus gunnari and Notothenia rossii marmorata as part of an effort to study the population dynamics of Antarctic fishes. Examination of otoliths by scanning electron microscope (SEM) revealed internal microincrements which were similar to daily increments described for other fish species. No easily identifiable annuli could be discerned and fishes were aged by counting microincrements. Multiple regression analyses relating age to otolith morphometrics and fish size, provided a simpler ageing method. It was determined with these techniques that C. gunnari grew to 50 cm in 15 yrs and N. rossii marmorata grew to 50 cm in 6.5 yrs. The lack of larger-sized individuals for N. rossii marmorata may be indicative of recent overfishing with only the younger age groups remaining. It appears that length-at-age data determined on a yearly basis, could provide valuable scientific and management information for these species.     

Fish prey of Antarctic fur seals Arctocephalus gazella at Ile de Croy,Kerguelen

 The composition of Antarctic fur seal prey was assessed through analysis of scats collected in March 1994 on Ile de Croy, Iles Nuageuses. Fish remains predominated in samples, occurring in 95% of droppings. A total of 968 otoliths allowed the identification of 16 fish species. Myctophid fish (12 species) dominated the diet both by number (94% of the otoliths) and by fish reconstituted mass (76%). Three fish species constituted together 87% of the reconstituted mass: the myctophids Gymnoscopelus nicholsi (52%) and G. piabilis (12%), and the channichthyid Champsocephalus gunnari (23%). Prey distribution suggests that during late summer seals forage in upper slope waters in the northeast of the Kerguelen Archipelago. Received:1 March 1996/Accepted: 20 May 1996     

Assembly of the antifreeze glycoprotein/trypsinogen-like protease genomic locus in the Antarctic toothfish Dissostichus mawsoni (Norman)

To investigate the genomic architecture underlying the quintessential adaptive phenotype, antifreeze glycoprotein (AFGP) that enables Antarctic notothenioid survival in the frigid Southern Ocean, we isolated the AFGP genomic locus from a bacterial artificial chromosome library for Dissostichus mawsoni. Through extensive shotgun sequencing of pertinent clones and sequence assembly verifications, we reconstructed the highly repetitive AFGP genomic locus. The locus comprises two haplotypes of different lengths (363.6 kbp and 467.4 kbp) containing tandem AFGP, two TLP (trypsinogen-like protease), and surprisingly three chimeric AFGP/TLP, one of which was previously hypothesized to be a TLP-to-AFGP evolutionary intermediate. The ~ 100 kbp haplotype length variation results from different AFGP copy number, suggesting substantial dynamism existed in the evolutionary history of the AFGP gene family. This study provided the data for fine resolution sequence analyses that would yield insight into the molecular mechanisms of notothenioid AFGP gene family evolution driven by Southern Ocean glaciation.     

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.     

Some like it hot, some like it cold: the heat shock response is found in New Zealand but not Antarctic notothenioid fishes

Previous research on Antarctic notothenioids has demonstrated that cells of cold-adapted Antarctic notothenioids lack a common cellular defense mechanism called the heat shock response (HSR), the induction of a family of heat shock proteins (Hsps) in response to elevated temperatures. The goal of this study was to address how widespread the loss of the HSR is within the Notothenioidei suborder and, specifically, to ask whether cold temperate non-Antarctic notothenioids possess the HSR. In general, Antarctic fish have provided an important opportunity for physiologists to examine responses to selection in the environment and to ask whether traits of the notothenioids represent cold adaptation, or whether the traits are related to history and are characteristics of the notothenioid lineage. Using in vivo metabolic labeling, results indicate that one of the two New Zealand notothenioids possess an HSR. The thornfish, Bovichtus variegatus Richardson, 1846, expressed heat shock proteins (Hsp) in response to heat stress, whereas the black cod, Notothenia angustata Hutton, 1875, did not display robust stress-inducible Hsp synthesis at the protein-level. However, further analysis using Northern blotting clearly demonstrated that mRNA for a common Hsp gene, hsp70, was present in cells of both New Zealand species following exposure to elevated temperatures. Overall, combined evidence on the HSR in notothenioid fishes from temperate New Zealand waters indicate that the loss of the HSR in Antarctic notothenioid fishes occurred after the separation of Bovichtidae from the other Antarctic notothenioid families, and that the HSR was most likely lost during evolution at cold and constant environmental temperatures.