Cytogenetic mapping of immunoglobulin heavy chain genes in Antarctic fish

The chromosomal location of the IgH locus has been analyzed in several bony fish of the Antarctic perciform group Notothenioidei. Two IgH probes were prepared from the species Trematomus bernacchii (family Nototheniidae, tribe Trematominae) and mapped onto the chromosomes of ten species belonging to the same genus (Trematomus) and in two outgroups, through one-color and two-color FISH. A single location of the IgH locus was found in the majority of the species examined, including the outgroups, whereas in four of them the IgH genes splited to two chromosomal loci. RT-PCR experiments revealed the presence of three allelic sequences in T. newnesi, a species in which the IgH genes were organized in two chromosomal loci. Possible pathways leading to IgH genes duplication during the diversification of trematomine fishes were inferred from the analysis of the FISH patterns in a phylogenetic context. The present work provides the first comprehensive picture of IgH genes organization at chromosomal level in a bony fish group.     

Molecular and morphological phylogenies of the Antarctic teleostean family Nototheniidae,with emphasis on the Trematominae

Four independent molecular data sets were sequenced in order to solve longstanding phylogenetic problems among Antarctic teleosts of the family Nototheniidae. The anatomical data of Balushkin (2000) were also coded into a matrix of 106 characters in order to test the parsimony of his taxonomic conclusions. Molecular results confirm Balushkin’s Pleuragrammatinae but not his Nototheniinae. Different genes used here found the “clade A” establishing the paraphyly of the Nototheniinae sensu lato; i.e. Lepidonotothen and Patagonotothen are more closely related to the Trematominae than to Notothenia. The genus Notothenia is paraphyletic and Paranotothenia should become Notothenia. Previously no molecular data set could assign a reliable position for the genus Gobionotothen. For the first time robust results are obtained for the phylogeny among the Trematominae. Trematomus scotti is the sister-group of all others, then Trematomus newnesi emerges, then Trematomus eulepidotus. Among the crown group, three clades emerge: 1: Trematomus hansoni + Trematomus bernacchii + Trematomus vicarius; 2: Trematomus pennellii + Trematomus lepidorhinus + Trematomus loennbergii; 3: Trematomus (Pagothenia) borchgrevinki + Trematomus nicolai. Pagothenia should become Trematomus to make the genus Trematomus monophyletic. The Trematomus tree found here did not match the topology obtained with Balushkin’s morphological matrix. The tree shows that the tendencies shown by some trematomines to secondarily colonize the water column are not gained through common ancestry.     

Qualitative and quantitative analysis of serum immunoglobulins of four Antarctic fish species

Immunoglobulins from the Antarctic fish species Trematomus bernacchii, Trematomus hansoni, Trematomus newnesi, and Chionodraco hamatus were analysed in whole serum and after purification by affinity chromatography on protein A-sepharose. Using SDS-PAGE, the apparent masses of the heavy and light chains were, respectively, 83.5 kDa and 27.5 kDa for T. bernacchii, 83.5 kDa and 27 kDa for T. hansoni, 81 kDa and 27.5 kDa for T. newnesi, and 74.5 kDa and 30 kDa for C. hamatus. It was not possible to purify immunoglobulins from T. newnesi due to their low concentration in serum. Heterogeneity in mass of both heavy and light chains was observed in all species. By using a polyclonal antibody raised against sea bass immunoglobulins, cross-reactivity was observed with heavy and light chains of all species. With this antibody, an indirect enzyme-linked immunosorbent assay (ELISA) was developed and results showed the relative immunoglobulin concentration in sera of the Antarctic fish species considered, using as standard sea bass immunoglobulins. Received: 25 November 1996 / Accepted: 3 February 1997     

Fish of Admiralty Bay (King George Island,South Shetland Islands,Antarctica)

Summary The species composition of the ichthyofauna of Admiralty Bay, King George Island was determined from results of sampling using bottom trawls, gill-nets and long-lines. Thirty-five species from 24 genera and 10 families (Table 1) were found. The number of species increased with depth (e.g. 7 species at 100 m, 14 species at 255 m and 21 species at 540 m), a tendency characteristic of Antarctic waters. In the bay, the catch rate obtained with a bottom trawl (greater than 30 kg/h) was roughly ten times lower than the catch rate using the same gear on the shelf around the Island. Notothenia gibberifrons was the dominant species in all trawls. The majority of these fish (about 95%) were immature juveniles (Table 4). Younger fish were found to inhabit shallower waters (Fig. 1). The majority of the fish of species Notothenia coriiceps neglecta, Notothenia rossii marmorata, Notothenia nudifrons, Trematomus newnesi and Trematomus bernacchii preferred waters about 255 m deep. Fourteen specimens of a previously undescribed species of the genus Psilodraco (currently being described by H. DeWitt) were caught in the bay within the 146 to 540 m depth range. The rare zoarcid, Lycenchelys aratrirostris, was also caught in Admirality Bay; previously this species had only been reported from the Elephant Island region. In the case of Trematomus newnesi, the occurrence of scales in the interorbital space was noted (Fig. 2), an observation which verifies this feature as a distinct taxonomical criterion for this species.     

Isolation and multiplex analysis of six polymorphic microsatellites in the Antarctic notothenioid fish,Trematomus newnesi

Six polymorphic microsatellites were isolated from the dusky notothen, Trematomus newnesi (Boulenger 1902), using a microsatellite enrichment protocol and selective hybridization with di‐ tetra‐ and penta‐repeat probes. The loci were screened in 48 individuals captured in the Southern Ocean (coastal zone of Terre‐Adélie), revealing eight to 22 alleles per locus and an observed heterozygosity ranging from 0.70 to 0.92. These microsatellite markers provide a tool to study the relationship between the various morphs observed in this species and can be used for population genetic analysis and biodiversity studies.     

Feeding plasticity of Trematomus newnesi (Pisces, Nototheniidae) in Terra Nova Bay, Ross Sea, in relation to environmental conditions

Forty-three and 49 specimens of the Antarctic fish Trematomus newnesi were collected in the coastal waters of Terra Nova Bay, Ross Sea, during December 1994 and February 1998, respectively. The dietary composition of the two fish samples was determined by means of stomach content analysis and then compared. In general, as reported in most of the previous studies, planktivory appeared to be the main feeding habit of T. newnesi. However, the different environmental conditions occurring in the study area in the two periods investigated, such as the degree of the sea-ice coverage and the related amount of light available below the ice, influenced the food composition of T. newnesi. In December 1994, the whole area investigated was covered by a thick layer of sea ice and the diet of T. newnesi consisted of few prey taxa. Some species that characterize the so-called “cryopelagic habitat”, such as the euphausiid Euphausia crystallorophias and the copepod Metridia gerlachei, were by far the most important prey, followed by amphipods and the pteropod Limacina helicina. Conversely, the ostracod Alacia belgicae and larval stages of fish (Pleuragramma antarcticum) were consumed occasionally. In late summer (February 1998), the lack of sea-ice coverage and changes in the associated fauna influenced the feeding habit of T. newnesi. In this period the food spectrum appeared to be more diverse, revealing an active feeding search in the water column. The bulk of food was composed of several prey groups, such as E. crystallorophias, hyperiid amphipods (Hyperiella dilatata), copepods, L. helicina and several species of fish larvae. Present data provide evidence of a marked feeding plasticity of T. newnesi, in response to diverse environmental conditions that characterize the High-Antarctic Zone. Accepted: 17 July 1999     

Correlation between Hemichrome Stability and the Root Effect in Tetrameric Hemoglobins

Oxidation of Hbs leads to the formation of different forms of Fe(III) that are relevant to a range of biochemical and physiological functions. Here we report a combined EPR/x-ray crystallography study performed at acidic pH on six ferric tetrameric Hbs. Five of the Hbs were isolated from the high-Antarctic notothenioid fishes Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, and one was isolated from the sub-Antarctic notothenioid Cottoperca gobio. Our EPR analysis reveals that 1), in all of these Hbs, at acidic pH the aquomet form and two hemichromes coexist; and 2), only in the three Hbs that exhibit the Root effect is a significant amount of the pentacoordinate (5C) high-spin Fe(III) form found. The crystal structure at acidic pH of the ferric form of the Root-effect Hb from T. bernacchii is also reported at 1.7 Å resolution. This structure reveals a 5C state of the heme iron for both the α- and β-chains within a T quaternary structure. Altogether, the spectroscopic and crystallographic results indicate that the Root effect and hemichrome stability at acidic pH are correlated in tetrameric Hbs. Furthermore, Antarctic fish Hbs exhibit higher peroxidase activity than mammalian and temperate fish Hbs, suggesting that a partial hemichrome state in tetrameric Hbs, unlike in monomeric Hbs, does not remove the need for protection from peroxide attack, in contrast to previous results from monomeric Hbs.     

Gut regulatory peptides in some Antarctic notothenioids

The presence and the comparative distribution of regulatory peptides and serotonin in the gut of four species of Antarctic notothenioid fishes [Cryodraco antarcticus and Chionodraco hamatus (Channichthyidae), and Trematomus bernacchii and Trematomus newnesi (Nototheniidae)], were immunohistochemically studied. In these species, numerous immunoreactive (IR) endocrine cells and nerve elements were detected. In the nototheniids most of the IR nerve fibres were of the intrinsic type, while most of the IR nerve fibres of the channichthyid intestine, besides insulin-like IR fibres, seemed to be of the extrinsic type. The intensity and frequency of immunopositivity are not the same in channnichthyids and nototheniids; the species belonging to the first family show many differences from the teleosts living in temperate water. The finding of insulin-like endocrine cells and nerve fibres in the gut wall of Cryodraco antarcticus is exceptional for vertebrates and deserves special attention.A preliminary account of this work was presented to the VII International Ichthyology Congress, Den Haag, 26–30 August 1991     

The unusual co-assembly of H- and M-chains in the ferritin molecule from the Antarctic teleosts Trematomus bernacchii and Trematomus newnesi

Ferritins from the liver and spleen of the cold-adapted Antarctic teleosts Trematomus bernacchii and Trematomus newnesi have been isolated and characterized. Interestingly, only H- and M-chains are expressed and no L-chains. The H-chains contain the conserved ferroxidase center residues while M-chains harbor both the ferroxidase center and the micelle nucleation site ligands. Ferritins have an organ-specific subunit composition, they are: M homopolymers in spleen and H/M heteropolymers in liver. The M-chain homopolymer mineralizes iron at higher rate with respect to the H/M heteropolymer, which however is endowed with a lower activation energy for the iron incorporation process, indicative of a higher local flexibility. These findings and available literature data on ferritin expression in fish point to the role of tissue-specific expression of different chains in modulating the iron oxidation/mineralization process.     

Isolation and characterization of microsatellite loci from three cryptic species of Ceratosolen emarginatus

From an enriched (AG)_n/(AC)_n library, we developed nine polymorphic microsatellite loci for three cryptic species of the fig‐pollinating wasp Ceratosolen emarginatus. We genotyped one population for each of the three cryptic species across all the nine loci. In total, 204 alleles were detected from the three cryptic species of C. emarginatus. The observed heterozygosity was 0.755 ± 0.034, 0.653 ± 0.030 and 0.603 ± 0.073 in C. emarginatus populations A, B and C, respectively; the expected heterozygosity was 0.850 ± 0.031, 0.724 ± 0.035 and 0.702 ± 0.104, respectively. No linkage disequilibrium was found between any two loci of all three cryptic species. The newly isolated microsatellite markers will be very useful for estimating the genetic variation within and among the cryptic species and for revealing the mechanisms of speciation and inbreeding coexistence hypothesis of the cryptic species.     

Lipids of the notothenioid fishes Trematomus spp. and Pagothenia borchgrevinki from East Antarctica

The notothenioid fishes Trematomus pennellii, T. newnesi, and T. bernacchii had 5–15% skeletal lipid, as percent dry weight, and this comprised 6–8% of the total body lipid. Trematomus hansoni and Pagothenia borchgrevinki had 2–4% skeletal lipid, which comprised 1% of total body lipid. Triacylglycerol was the major lipid class present in all tissues of all fish analyzed (up to 89% of total lipid), with minor components including sterol, phospholipid and wax esters. Monounsaturated fatty acids comprised 38.3–58.0% of the total fatty acids, and included primarily oleic [18 : 1(n-9)] and palmitoleic [16 : 1(n-7)] acids. Polyunsaturated fatty acids comprised 19.1–40.0% of the total fatty acids and included primarily eicosapentaenoic acid [20 : 5(n-3)] and docosahexaenoic acid [22 : 6(n-3)]. These five notothenioid fishes, which include benthic, benthopelagic, and cryopelagic species, are lower in lipid than other important Southern Ocean fishes (such as the Patagonian toothfish) and are estimated to be negatively buoyant. These data will be of use to research groups presently using signature lipid methodology. Accepted: 5 April 1999     

Inducible heat tolerance in Antarctic notothenioid fishes

Significant increases in heat tolerance (time of survival at 14°C) were observed for some, but not all, species of notothenioid fishes collected from McMurdo Sound, Antarctica (77°51′S) following acclimation to 4°C. The increase in thermal tolerance was rapid in Trematomus bernacchii, developing within 1–2 days of acclimation to 4°C. Long-term (6–8 weeks) acclimation to 4°C led to greater heat tolerance in Trematomus pennellii than in T. bernacchii. Unlike its demersal congeners, the cryopelagic notothenioid Pagothenia borchgrevinki did not increase heat tolerance during warm acclimation. A deep-living zoarcid fish, Lycodichthys dearborni, also failed to increase heat tolerance, but survived significantly (> threefold) longer at 14°C than the notothenioids.     

Isolation and characterization of polymorphic microsatellite loci from the Dungeness crab Cancer magister

The Dungeness crab, Cancer magister, is the focus of one of the most intensely harvested fisheries in North America. Given its economic importance, there is considerable interest in assessing the degree and spatial pattern of genetic structure in C. magister. To that end, we developed a series of 17 hypervariable microsatellite loci. Six of these 17 loci could be amplified in a single multiplex PCR reaction. Using dye‐labelled primers all six loci can be coamplified and scored simultaneously on an automated sequencer. The ability to multiplex multiple loci greatly increases the ease and speed of genotyping for this species.     

Feeding habits of juvenile <Emphasis Type="Italic">Trematomus newnesi</Emphasis> (Pisces,Nototheniidae) at Potter Cove,South Shetland Islands,Antarctica

The dietary composition of juvenile Trematomus newnesi, trawled at Potter Cove, South Shetland Islands, in the summers 2004–2005 and 2005–2006 was analysed using frequency of occurrence (F%) and dietary coefficient Q (%) methods. The samples consisted exclusively of immature specimens in the range 4–15.4 cm (total length) and are complementary to those of larger fish from the same site, including adults, which were analysed in previous work. Benthic-demersal organisms such as gammaridean amphipods and harpacticoid copepods were the main (coefficient Q) and most frequent (F%) prey. The importance of the smaller and larger main prey diminished and increased, respectively, during ontogeny. Pelagic krill, being negligible in the diet of the small and medium size fish categories, became secondary food, but only for fish larger than 12 cm. Other taxonomic groups occurred scarcely and constituted occasional food. They were mostly benthic, such as gastropods, bivalves, isopods, cumaceans, and algae, with the exception of an insignificant number of pelagic ostracods and calanoid copepods. Unlike the more pelagic/planktivorous mode of life known for late juvenile-adult stages of T. newnesi, including cryopelagy, present results indicate that early juvenile fish remain sheltered among macroalgae beds preying on the associated community of demersal-benthic organisms.     

Multiplex panels of polymorphic microsatellite loci for two cryptic bat species of the genus Pipistrellus,developed by cross‐species amplification within the family Vespertilionidae

The paper presents multiplex panels of polymorphic microsatellites for two closely related cryptic species Pipistrellus pipistrellus and Pipistrellus pygmaeus. We tested the cross‐species amplification of 34 microsatellite loci, originally developed for five vespertilionid bat species. Ten and nine polymorphic loci in P. pipistrellus (mean number of alleles per locus = 10.5) and P. pygmaeus (8.1), respectively, in three multiplex polymerase chain reactions per species were amplified. All loci can be analysed in a single fragment analysis and can be used as markers to the study of evolution and the ecology of structured populations of socially living bats.     

Ten microsatellite loci for evolutionary research on Nesospiza buntings

We tested 19 loci originally developed for Vidua and Geospiza, of which six Vidua loci were polymorphic and furthermore, developed four microsatellite loci for Nesospiza buntings. Allelic variation ranged from two to 13 alleles, with heterozygosity from 0 to 0.82. Ascertainment bias was evident as increased allelic diversity of loci developed for Nesospiza, versus those developed for Vidua. Two loci showed linkage, and several, deviation from Hardy–Weinberg equilibrium. We suggest such deviations are through introduction of new alleles by introgression, or of the Wahlund effect. These loci will contribute to the understanding of speciation in Nesospiza buntings of the Tristan da Cunha islands.     

Species‐diagnostic microsatellite loci for the fig wasp genus Pegoscapus

To obtain tools for the estimation of inbreeding and assignment of offspring to matrilines, we developed 13 microsatellite loci from the fig wasps that pollinate Ficus obtusifolia. Based on morphological studies, it was thought that a single species (Pegoscapus hoffmeyeri) pollinated this fig. However, our data revealed the presence of two coexisting cryptic species. Several diagnostic microsatellite markers may be used to distinguish these two cryptic species. The new microsatellites can be used across a wide range of fig‐pollinating wasp species for both evolutionary and population genetic studies.     

Relationship between metallothionein and metal contents in red-blooded and white-blooded Antarctic teleosts

Metal bioaccumulation and metallothionein were investigated in different organs of the red-blooded teleost, Trematomus bernacchii and the haemoglobinless Chionodraco hamatus. Specimens of the two Antarctic fish were sampled from Terra Nova Bay (Ross Sea), and their levels of Cd, Cu and Zn in homogenates and in soluble fractions of liver, muscle, gills, heart and plasma were determined. Dosages of metallothioneins (MTs) or MT-like proteins were assayed by the silver saturation method in soluble fractions of the same organs. In both T. bernacchii and C. hamatus the highest MT contents were found in liver. Hepatic MT and Cd, Cu and Zn concentrations correlated positively in T. bernacchii, whereas in C. hamatus hepatic MT showed a positive correlation only with Cd. Positive correlations were also found between Cd and MT in gills of the two species. A metal-binding protein containing a high percentage of cysteine from C. hamatus was purified and compared with the MT from T. bernacchii and mammals. Accepted: 27 November 1999     

Use of the lateral line and tactile senses in feeding in four antarctic nototheniid fishes

Synopsis Fishes of the family Nototheniidae (Pisces: Perciformes) dominate antarctic fish communities and have radiated to fill diverse niches. The most southern species must operate under an extended austral night and under thick sea ice, yet have eyes more typical of shallow coastal fishes. In winter, the eyes are probably useless, except for detecting bioluminescence. I compared the responses of four species to hydromechanical and tactile signals: two benthivores,Trematomus bernacchii andT. pennellii, a benthic planktivore,T. nicolai, andPagothenia borchgrevinki, which feeds near the ice undersurface and within ice cracks. The planktivores have dorsal mouths, with eyes oriented dorsally or laterally (Pagothenia); their lateral line canals and receptor organs are larger dorsally. The benthivores have more ventrally oriented mouths and eyes. All species responded to hydromechanical cues to the head, but only the two benthivores responded to trunk hydromechanical stimuli or tactile stimuli to the ventral trunk or pelvic fins. Possibly responses to plankton along the trunk are of little use if a reorientation washes pelagic prey away. In responding to trunk stimuli,T. bernacchii reorients its head to the target in two stages by slowly pivoting on its pelvic fins. In contrast,T. pennellii reorients in a single quick flip. It is argued that, becauseT. bernacchii has wider canals thanT. pennellii, it must move more slowly to reduce self-generated noise. It is likely that further studies of winter diet and prey behavior may reveal the relative advantages of the two repositioning styles.     

Spermatogenesis in some Antarctic teleosts from the Ross Sea: histological organisation of the testis and localisation of bFGF

Testis structure and spermatogenetic activity were studied in two Antarctic teleostean species, Chionodraco hamatus and Trematomus bernacchii, captured during the austral summer in the Ross Sea. The specimens of C. hamatus showed full reproductive activity but, the spermatogenetic cycle being over, only spermatogonia and Sertoli cells were present in the seminiferous tubules whereas the lumina were full of sperm. By contrast, the specimens of T. bernacchii were in the stage of spermatogenetical recrudescence, having not yet entered the reproductive period. In this species, the seminiferous tubules were devoid of lumen and full of spermatogonial cysts, showing some mitoses. Many tubules contained cysts of meiotic spermatocytes I and, in one case only, small cysts of spermatocytes II. The final stages of spermatogenesis were lacking, presumably occurring later, in autumn/winter. The immunocytochemical tests aimed at identifying bFGF and FGFR1 revealed a positive reaction both in Sertoli cells and spermatogonia in the C. hamatus specimens, indicating that this species was ready to start a new spermatogenetic cycle. The weak reaction in the specimens of T. bernacchii suggests that, in this species, the stage of cell division was over and that of meiosis and differentiation was starting. These data indicate that Antarctic fish have an opportunistic spermatogenetic cycle. Accepted: 24 October 1999