Aerobic mitochondrial capacities in Antarctic and temperate eelpout (Zoarcidae) subjected to warm versus cold acclimation

Capacities and effects of cold or warm acclimation were investigated in two zoarcid species from the North Sea (Zoarces viviparus) and the Antarctic (Pachycara brachycephalum) by investigating temperature dependent mitochondrial respiration and activities of citrate synthase (CS) and NADP⁺ -dependent isocitrate dehydrogenase (IDH) in the liver. Antarctic eelpout were acclimated to 5°C and 0°C (controls) for at least 10 months, whereas boreal eelpout, Z. viviparus (North Sea) were acclimated to 5°C and to 10°C (controls). Liver sizes were found to be increased in both species in the cold, with a concomitant rise in liver mitochondrial protein content. As a result, total liver state III rates were elevated in both cold-versus and warm-exposed P. brachycephalum and Z. viviparus, with the highest rates in boreal eelpout acclimated to 5°C. CS and IDH activities in the total liver were similar in Z. viviparus acclimated to 5°C and 10°C, but decreased in those warm acclimated versus control P. brachycephalum. Enzyme capacities in the total liver were higher in eelpout from Antarctica than those from the North Sea. In conclusion, cold compensation of aerobic capacities in the liver seems to be linked to an increase in organ size with unchanged specific mitochondrial protein content. Despite its life in permanently cold climate, P. brachycephalum was able to reduce liver aerobic capacities in warm climate and thus, displayed a capacity for temperature acclimation.     

Temperature-dependent energy allocation to growth in Antarctic and boreal eelpout (Zoarcidae)

Antarctic fishes display slower annual growth rates than congeneric species from temperate zones. For an analysis of growth in relation to energy turnover, body composition was analysed in two benthic fish species to establish a whole animal energy budget. The Antarctic eelpout, Pachycara brachycephalum, was maintained at 0, 2, 4 and 6°C and the boreal eelpout, Zoarces viviparus at 4, 6, 12 and 18°C. At maximum food supply the weight gain was highest for P. brachycephalum at 4°C. Routine metabolic rate in acclimated Antarctic eelpouts did not differ between temperatures, whereas in Z. viviparus maximized growth benefited from a reduction of metabolic energy demands at 12°C. The lipid content of liver declined with increasing temperature in both species. The thermal window for growth is based on food conversion efficiency and the level of metabolic energy demand and is limited according to the level of aerobic scope available between pejus temperatures.     

Temperature-dependent pH regulation in stenothermal Antarctic and eurythermal temperate eelpout (Zoarcidae): an in-vivo NMR study

Temperature-dependent adjustments of intracellular pH are thought to play a major role in the maintenance of protein function. Comparative studies were carried out in two species from the same fish family (Zoarcidae), the stenothermal Antarctic eelpout (Pachycara brachycephalum) and the eurythermal eelpout (Zoarces viviparus), to find out whether pH regulation is modified by temperature in the closely related species and to what extent the respective pattern differs between eurytherms and stenotherms. Previous invasive studies had compared individual animals sampled at various temperatures and suggested that a decrease in intracellular pH (pHi) values occurs at rising temperatures, as predicted by the alpha-stat hypothesis of acid-base regulation. The present study used non-invasive in vivo ³¹P-NMR spectroscopy in non-anaesthetized, unrestrained fish for long-term online recordings in individual specimens. Control spectra obtained at T=0°C for P. brachycephalum and at 12°C for Z. viviparus indicated low stress conditions, as well as a high stability of energy and acid-base status over time periods longer than 1 week. Temperature changes had no influence on the concentration of high-energy phosphates like phosphocreatine or ATP. Temperature-induced pH changes were monitored continuously in a range between 0 and 6°C for polar, and 12 and 18°C for temperate eelpout. A pHi change of around -0.015 pH units/°C was observed within both species, in accordance with the alpha-stat hypothesis; however, extrapolation to the same temperature revealed different set points of pH regulation in the two species. These findings confirm that an alpha-stat pattern of pH regulation can be found in stenothermal Antarctic animals, at set points deviating from an alpha-stat pattern, however, in a between-species comparison.     

Thermal sensitivity of uncoupling protein expression in polar and temperate fish

Uncoupling proteins (UCP), capable of increasing proton leakage across the inner mitochondrial membrane, may play a role in the temperature-dependent setting of energy turnover in animals (and their mitochondria). Therefore, the genes and expression of fish UCP were investigated in the Antarctic eelpout Pachycara brachycephalum and a temperate confamilial species, the common eelpout Zoarces viviparus. UCP full-length cDNA was amplified from liver and muscle using RT–PCR and rapid amplification of cDNA ends (RACE). The fish UCP mRNA consists of 1906 bp in P. brachycephalum and of 1876 bp in Z. viviparus. Both zoarcid sequences contain open reading frames of 939 bp, encoding 313 amino acids, with 98% and 99% identity, respectively. Protein sequences of zoarcid UCP are closest related to fish and mammalian UCP2. For analysis of temperature-dependent expression common eelpouts were cold-acclimated from 10 °C to 2 °C and Antarctic eelpouts were warm-acclimated from 0 °C to 5 °C. Identical cDNA probes for both species were developed to investigate fish UCP mRNA expression, and protein expression levels were detected by Western Blot in the enriched membrane fraction. During cold-acclimation in Z. viviparus, mRNA levels increased by a factor up to 2.0, protein levels increased up to 1.5, in line with mitochondrial proliferation during cold-acclimation. Despite decreased mitochondrial protein content, in Antarctic eelpout UCP levels rose upon warm acclimation by a factor up to 2.0 (mRNA) and 1.6 (protein), respectively. Besides the ongoing discussion of UCP function in vertebrates, the data are indicative of a significant role of fish UCP in thermal adaptation of fish mitochondria.     

Strong isolation-by-distance in the absence of genetic population structure in the eelpout (Zoarces viviparus,Linnaeus 1758)

The eelpout (Zoarces viviparus) is a benthic marine fish, which has long been assumed to be philopatric. This made it particularly suitable for environmental monitoring programs as it allows matching its content of pollutants to the area of catch. However, a recent small scale genetic study in the Baltic Proper suggested that gene-flow might be stronger than generally believed and may occur frequently up to a distance of at least 90 km. As challenging the assumption of philopatry might have strong implications for environmental monitoring studies, we tested the hypothesis of philopatry at a larger geographical scale using ten microsatellite loci. A total of 220 individuals sampled from eight locations covering almost the entire geographic range of the species was genotyped. Our results show that genetic diversity decreases from the North Sea to the northeastern Baltic Sea. No strong population structuring was found, but a highly significant isolation-by-distance pattern was detected, suggesting a stepwise migration pattern among populations. Thus, the hypothesis of long-distance migration can be falsified. It is more likely that only limited gene flow exists among adjacent populations without any barriers between them. Our results suggest that dispersal in the eelpout is weak enough to retain this species as an important bioindicator. However, we suggest that reference stations should be placed in an appropriate distance to avoid misleading results from migrating individuals. We conclude that a more precise knowledge on migration rates of the eelpout is required in order to get more reliable insights into the potential area over which the concentration of environmental pollutants is integrated.     

Effect of an offshore wind farm on the viviparous eelpout: Biometrics,brood development and population studies in Lillgrund,Sweden

Sufficient, clean and secure energy is the main driver for a worldwide growing welfare and economic development of a society. Environmental concerns on the expansion of offshore renewable energy and its impact on marine organisms need to be scientifically assessed for risks and consequences. In order to observe the effects of an operating wind farm on fish, we studied the rather stationary and benthic-living fish species viviparous eelpout (Zoarces viviparous) as model indicator organisms. We compared local populations of viviparous eelpout in the Lillgrund Offshore Wind Farm (OWF) with natural sites in the Öresund strait in Sweden. Eelpout studies on population dynamics, biometrics, reproductive success and fry development were conducted in 2011 and 2012. Condition index, histosomatic index, gonadosomatic index were measured additionally. Our findings showed that Lillgrund OWF neither had an impact on the condition index (CI), nor on brood development of female viviparous eelpout. Furthermore, populations size estimates in Lillgrund indicated that eelpout neither specifically aggregated in nor avoided the offshore wind farm, and no clear reef effect attracting eelpout to the foundations and scour protections of the OWF was observed. Our conclusion is that the operating wind farm did not have any potentially negative effects, since we did not observe any negative effects neither on the individual health of eelpout nor of the reproductive performance. We suggest that eelpout which may also be used as an indicator species for the environmental status of Lillgrund, as well as for other offshore wind farms and marine renewable energy installations, both in the Baltic and coastal waters in northern Europe.     

Evolution and Regulation of the Downstream Gene of Hypoxia-Inducible Factor-1α in Naked Carp (Gymnocypris przewalskii) from Lake Qinghai, China

The naked carp (Gymnocypris przewalskii) is a native teleost of Lake Qinghai (altitude, 3.2 km) on the Qinghai-Tibet Plateau in China. Hypoxia-inducible factor (HIF)-1α of Gymnocypris przewalskii was cloned and a phylogenetic tree for vertebrate HIF-1α was constructed. By analysis of maximum likelihood models of codon substitutions for HIF-1α, three positive sites in the branch lineages of crucian carp, eelpout, and flounder, and a higher proportion of neutral sites in naked carp, antarctic eelpout, rainbow trout, and grayling, were detected among all teleosts. It seems that low habitat temperatures relax the purifying selection of HIF-1α in these stenothermal coldwater fish, and both cold and hypoxic lake water contributed to the evolution of the HIF-1α gene in the naked carp. Furthermore, Glut 1 mRNA, a gene downstream from HIF-1α, has a time-course- and tissue-specific dependent response to hypoxic challenge.     

Nucleotide sequence variation of the mitochondrial COI gene in several eelpout species of the genus Zoarces (Zoarcidae, Pisces)

The mitochondrial COI gene was for the first time sequenced in eelpout species of the genus Zoarces from the Taui Bay of the Sea of Okhotsk: notched-fin Z. elongatus Kner, 1868 and the new sympatric form Zoarces sp., differing in a large set of morphological traits. The two species were compared with European eelpout Z. viviparus Linnaeus, 1758 from the Finnish Bay of the Baltic Sea. Divergence and phylogenetic analyses demonstrated a greater genetic similarity between Zoarces sp. and Z. viviparus than between sympatric Zoarces sp. and Z. elongatus.     

Features of Transition from Larva to Juvenile in Fishes with Different Types of Early Ontogeny

The transition from the larva state to the juvenile state (i.e. morphological condition characterised by mainly adult characters) was examined in three marine fish species: herring Clupea pallasi marisalbi, wolffish Anarhichas lupus, and eelpout Zoarces viviparus, based on external morphology and skeletal development. In spite of the different reproductive styles (oviparity, facultative viviparity, and obligate viviparity, respectively) and different types of early ontogeny (indirect, transitory, and direct, respectively), the beginning of the juvenile state occurred at similar total lengths (TL), which were approximated as 35 mm TL in herring and eelpout, and 32 mm TL in wolffish. Features of ontogeny were compared, assuming that the beginning of the juvenile state represented an uniform characteristic of morphological development for these species. It was proposed that the beginning of the larva or juvenile periods (sensu Balon) could not coincide with the beginning of the larva and juvenile states in the ontogeny of some species.     

Different stability of posttranslationally modified brain microtubules isolated from cold-temperate fish

Microtubule proteins were isolated by a temperature-dependent assembly-disassembly method from brain tissue of for cold-temperate fish; one fresh water fish (Oncorhynchus mykiss), and three marine fish (Labrus berggylta, Zoarces viviparus andGadus morhua). The -tubulins from all four fish species were acetylated. The -tubulins from the marine fish were composed of a mixture of tyrosinated and detyrosinated tubulin, while the fresh water fish tubulin only reacted with an antibody against detyrosinated tubulin. The isolated microtubules had a similar MAP composition. A 400 kD protein and a MAP2-like protein were found, but MAP1 was missing. All microtubules disassembled upon cooling to 0°C. In spite of these common characteristics, the assembly of microtubules fromLabrus berggylta was inhibited by colchicine and calcium, in contrast to the assembly of microtubules fromOncorhynchus mykiss andZoarces viviparus. For the latter, colchicine was not completely inhibitory even at a concentration as high as 1 mM, and calcium induced the formation of both loosely and densely coiled ribbons. The effects of calcium and colchicine on microtubules fromOncorhynchus mykiss andZoarces viviparus were modulated by either fish or cow MAPs, indicating that the effects are due to intrinsic properties of the fish tubulins and not the MAPs. In view of these findings, our results suggest that there is not correlation between colchicine sensitivity, inability of calcium to inhibit microtubule assembly, and acetylation and detyrosination.     

Panmixia in Zoarces viviparus: implications for environmental monitoring studies

In this study, the genetic population structure of the eelpout Zoarces viviparus was investigated by using microsatellites. Samples were collected at 10 sites in the Baltic Sea, covering a distance of c. 90 km. Ten newly developed microsatellite loci were used to infer the population structure. No global spatial genetic differentiation was found (global F(ST) = 0·0001; D(est) = -0·0003), indicating strong gene flow at this scale, nor any clear pattern of isolation by distance. The results suggest that gene flow among the studied populations of Z. viviparus is stronger than usually thought, which might be caused by environmental homogeneity. This is important for planning and evaluating monitoring activities in this species and for the interpretation of ecotoxicological studies. Strong migration might lead to wrong conclusions concerning the pollution in a given area. Therefore, reference stations should be placed at a larger distance than presently practiced.     

Spatial and length distribution,sex composition,and feeding of the notched-fin eelpout <Emphasis Type="Italic">Zoarces elongatus</Emphasis> (Perciformes: Zoarcidae) in the Sea of Okhotsk

Based on the data from six bottom trawl surveys carried out on the shelf of the Sea of Okhotsk in 1997–2007, the features of the distribution, size and sex structure, and food composition of the notched-fin (east) eelpout Zoarces elongatus were analyzed. It was established that this species regularly occurs in trawl catches only in the eastern part of the sea and that the density of its schools decreases with depth. It was recorded that females of the notched-fin eelpout are larger than males. The sex ratio of the fishes varies from year to year. Females prevail at depths less than 25 m and males prevail at depths greater than 25 m.     

Identification of a two-domain antifreeze protein gene in Antarctic eelpout <Emphasis Type="Italic">Lycodichthys dearborni</Emphasis>

Type III antifreeze proteins (AFP III) in the Antarctic eelpout Lycodichthys dearborni contain at least two size variants—a 7-kDa protein family and a specific 14-kDa isoform composed of two 7-kDa domains linked in tandem. We report the characterization of a two-domain AFP III gene from L. dearborni, and propose that the two-domain AFP III gene arose from a single-domain AFP III gene through duplication and degeneration. AT-rich regions played an important role in the degeneration of the duplicated AFP III gene that resulted in the concatenation of two originally separated 7-kDa AFP-coding exons into a single gene. We also identified a pseudo-AFP III gene interrupted at an AT-rich coding region, supporting AT-rich regions as hotspots for DNA recombination in AFP III gene evolution. Interestingly, study of AFP III genes in the related Antarctic eelpout Pachycara brachycephalum showed absence of two- and multi-domain AFP III genes, indicating that modes of AFP III gene family evolution are specific within species. Nucleotide sequences have been deposited into NCBI Genbank under Accession Numbers: EU627165, EU627166.     

Restriction fragment variation of the mitochondrial gene of cytochrome b in some taxa of the Eelpout family (pisces, perciformes, zoarcidae) from the northern Sea of Okhotsk

The first analysis of restriction fragment length polymorphism (RFLP) of the mitochondrial gene of cytochrome b of fish from the Eelpout Family (Zoarcidae) from the northern Sea of Okhotsk has been performed. The mean genetic distances between the subfamilies Zoarcinae and Lycodinae, subfamilies Lycodinae and Gymnelinae, and subfamilies Zoarcinae and Gymnelinae are 11.14, 13.87, and 14%, respectively. Species of the genus Lycodes (Lycodinae) are characterized by a mtDNA small divergence (on average, 2.04%) and are divided into two groups on the basis of lateral line morphology. Magadania skopetsi, an endemic of Tauiskaya Bay of the Sea of Okhotsk, is genetically close to Hadropareia middendorffii from the same subfamily (Gymnelinae). The mtDNA sequences of species from the genus Lycogrammoides (Lycodinae) differ by 3.83%. The genetic differences between the eelpout Zoarces elongatus Zoarces sp. (d = 4.54%) suggest that the latter is a separate species.     

Molecular evolution of haemoglobins of polar fishes

The Arctic and the Antarctic differ by age and isolation of the respective marine faunas. Antarctic fish are highly stenothermal, in response to stable water temperatures, whereas the Arctic ones are exposed to seasonal and latitudinal temperature variations. The knowledge of the mechanisms of phenotypic response to cold exposure in species of both polar habitats offers fundamental insights into the nature of environmental adaptation. In the process of cold adaptation, the evolutionary trend of Antarctic fish has led to unique specialisations, including modification of haematological characteristics, e.g. decreased amounts and multiplicity of haemoglobins.Unlike Antarctic Notothenioidei, Arctic teleosts have high haemoglobin multiplicity. Although the presence of functionally and structurally distinct haemoglobins is a plesiomorphic condition for many perciform-like fishes, it seems that the oxygen-transport system of teleost fish in the Arctic region has been adjusted to temperature differences and fluctuations of Arctic waters, much larger than in the Antarctic. The amino-acid sequences used to gain insight into the evolution history of α and β globins of polar fish have clearly shown that Antarctic and Arctic globins have different phylogenies, leading to the hypothesis that the selective pressure of environment stability allows the phylogenetic signal to be maintained in the Antarctic sequences, whereas environmental variability would tend to disrupt this signal in Arctic sequences.     

Relationships of eelpouts of the genus <Emphasis Type="Italic">Zoarces</Emphasis> (Zoarcidae,Pisces) inferred from molecular genetic and morphological data

Abstract-Molecular genetic and morphological analysis of eelpouts of the genus Zoarces was carried out. Based on the mitochondrial DNA sequence variation, haplotypes of notched-fin eelpout, Z. elogatus, more closely related Fedorov eelpout, Z. fedorovi, and common eelpout, Z. viviparus, as well as of Andriyashev eelpout, Z. andriashevi, were grouped in one macrocluster. Haplotypes of American eelpout, Z. americanus, and blotched eelpout, Z. gillii, clustered separately from other species. The genetic differences between Z. gillii and the other eelpout species were very high for within-genus comparisons, constituting 7.62%. Species divergence in terms of morphological characters was generally consistent with molecular genetic data and confirmed distinct isolation of American eelpout, and especially of blotched eelpout.     

Development of pigmentation and skeleton in ontogenesis of Fedorov eelpout <Emphasis Type="Italic">Zoarces fedorovi</Emphasis> (Zoarcidae,Perciformes) from the Sea of Okhotsk

The development of pigmentation of the Fedorov eelpout (Zoarces fedorovi) from hatching out of egg integuments to delivery is studied. In Z. fedorovi, the adult coloration develops immediately, without larval coloration, obviously, due to viviparity. Comparative analysis of the sequence of anlage and morphogenesis of elements of the cartilaginous skeleton and the bony skeleton demonstrated that the sequence of anlage of cartilaginous and bony regions of the skeleton of Z. fedorovi principally correspond to the sequence in Z. viviparus and in other perciform fishes. Morphogenesis of the bones of ethmoidal and interorbital regions of the skull, of the axial skeleton, and of the skeleton of the caudal fin is highly similar in eelpouts and other fishes of the suborder Zoarcoidei, differing from that in the generalized perciform fishes. At the same time, the larvae of Z. fedorovi in comparison with Z. vivparus are characterized by a higher rate of development of the skeleton. Advanced structural traits in structure of the skeleton of larvae of Z. viviparus in comparison with Z. fedorovi are pointed out. Differences in size at which anlage of bony elements occurs, as well as in the formation of particular bony elements seem to indicate to a rather prolonged divergence of these species.