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     

Species identification in the trematomid family using nuclear genetic markers

The Trematominae, a subfamily of the Nototheniidae, are typical of the high-Antarctic shelf waters. Within the Trematominae examples of phenotypic plasticity and possible cryptic speciation have been observed. Morphological identification of adult stages can be problematic in cases of high phenotypic plasticity or cryptic speciation. Additionally, postlarval and juvenile stages often have traits still under development and which lack distinction. A microsatellite DNA multiplex of six markers has been developed for Trematomus newnesi (Van Houdt et al. 2006). This multiplex was tested on five additional trematomid taxa: Pagothenia borchgrevinki, Trematomus bernacchii, Trematomus eulepidotus, Trematomus hansoni and Trematomus scotti. We used these six microsatellite loci to assess the genetic differentiation among species and the resolution power of these loci for individual-based assignment methods. The six species could be well discriminated by conventional methods such as principal component analysis and distance-based methods, and individual Bayesian assignment methods. This marker set can be used for a number of purposes, including the identification of eggs and larval and adult stages. It is also useful for the investigation of recent phylogenetic patterns, as well as the detection of cryptic speciation, which has been suggested for T. bernacchii and T. newnesi but never confirmed with high polymorphic genetic markers.     

Cytotaxonomy of antarctic teleosts of the Pagothenia/Trematomus complex (Nototheniidae,Perciformes)

Summary The interspecific relationships and taxonomy of 12 antarctic coastal Nototheniids (the Pagothenia/ Trematomus complex) are still under discussion. Here we present karyological data on eight species in hope that further cytogenetic data can help to clarify phylogenetic problems. Pagothenia bernacchii (2n = 48) has a generalized karyotype which has not developed heterochromosomes. P. hansoni, P. borchgrevinki and Trematomus newnesi share very similar karyotypes with 2n = 46 for females and 2n = 45 for males. The difference between sexes is probably due to the development of a multiple sex-chromosome system with a neo-Y, typical of males, which originated from a centric fusion of an autosome (now X₂) with an undifferentiated Y. The chromosome morphology of the three species seems to be related to that of P. bernacchii. T. nicolai (2n = 58, 57) has a karyotype numerically and morphologically divergent from that of the above species; T. nicolai males may have developed a neo-Y through a tandem translocation. T. pennellii (2n = 32), T. loennbergii (2n = 28; 2n = 30 in a single female) and T. eulepidotus (2n = 24) show karyotypes with progressively fewer chromosomes but with an increasing number of large, bi-armed chromosomes. Such a symmetrization process is generally found in advanced poikilothermic Vertebrates and the three species of Trematomus could therefore be considered as karyologically derived. However, parallelism in the karyotype differentiation producing convergence in the chromosome morphology of distantly related species cannot yet be excluded.     

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.     

Features of gametogenesis and sexual cycles of six notothenioid fishes from East Antarctica

Summary Some features of gametogenesis and sexual cycles of three species of the genus Trematomus (fam. Nototheniidae)-Trematomus centronotus Regan, T. eulepidotus Regan and T. scotti (Boulenger) and three species of icefishes (fam. Channichthyidae)-Chionodraco hamatus (Lönnberg), Ch. myersi Tyler and Neopagetopsis ionah Nybelin-were studied at Davis and Mawson Seas during austral autumn (March–April), 1983. Two peculiarities of the sexual cycles of Trematomus species may be noted: the uniform state of gonads of mature specimens of each species, and differences in time of spawning (austral summer and autumn for T. centronotus and T. eulepidotus, and, evidently, beginning of spring or the end of winter for T. scotti). Among the features of gametogenesis of icefishes are the early accumulation of yolk in oocytes, and the beginning of polarization and homogenization of yolk in oocytes, which are far from definitive sizes. Some differences in spawning time were registered in first maturing and repeatedly maturing specimens of Ch. hamatus. Repeatedly maturing specimens probably begin to spawn in December–January; those maturing for the first time evidently still spawn in March–April. Ch. myersi and N. ionah, judging by singular mature specimens, supposedly spawn during austral autumn.     

Macroscopic and histological analyses of gonads during the spawning season of Chionodraco hamatus (Pisces,Channichthyidae) off Terra Nova Bay,Ross Sea,Southern Ocean

A macroscopic and histological analysis of gonads was carried out during the spawning season of the high-Antarctic channichthyid Chionodraco hamatus in the western Ross Sea. Samples were collected between December and February during several years in the coastal waters of Terra Nova Bay. Gonad maturity stages were described for males and females according to macroscopic and histological scales. Using multi-year data, the estimated length at first spawning of females was about 35 cm TL, very similar to that obtained indirectly for males. Similar to many other high-Antarctic fish, C. hamatus is a summer spawner. The greater part of the stock was indeed in spawning condition between December and February, although a large proportion of females large enough to spawn probably did not spawn in that season. The present data confirm that C. hamatus, as is typical for Antarctic fish, probably spawns a single batch of oocytes once a year. In addition, vitellogenesis is a slow process that extends over at least 1 year. Discrepancies between the macroscopic and histological appearance of gonads were found. These were associated mainly with spent and resting females (maturity stages 5 and 2, respectively). This study demonstrates the importance of histological analysis of gonads in order to confirm the results of the macroscopic analyses routinely carried out in studies of reproductive biology. This is of particular importance in determining size at maturity and spawning stock biomass, for assessment purposes.     

Feeding ecology of two nototheniid fishes,Trematomus hansoni and Trematomus loennbergii,from Terra Nova Bay,Ross Sea

 A study of feeding ecology in Trematomus hansoni Boulenger 1902 and Trematomus loennbergii Regan 1913 was carried out from samples collected in the austral summer 1990–1991 off Terra Nova Bay, Ross Sea. T. hansoni was caught between 35 and 566 m and T loennbergii between 311 and 543 m. Stomach contents analysis shows that T. loennbergii relies on a wider range of prey than T. hansoni. Fish and decapods are the main food resources of T. loennbergii, which feeds also on epifaunal and tube-dwelling polychaetes. T. hansoni mostly relies on fish resources that are made up of juvenile stages of fish and eggs. Despite the common area occupied by the two species, the interspecific competition is mostly mitigated due either to taking different prey or to taking different amounts of the same prey. Received: 31 October 1995/Accepted: 20 February 1996     

Planktivory in benthic nototheniid fish in McMurdo Sound,Antarctica

Synopsis Four species of nototheniid fish were sampled from below the sea ice near Cape Armitage, McMurdo Sound:Pagothenia borchgrevinki from just below the ice 1.5 km offshore,Trematomus bernacchii, Trematomus hansoni andTrematomus centronotus from off the bottom in about 20 m of water near the shore. Scale worms and isopods were conspicuous non-planktonic prey, and present in the three benthic fish species. The planktonic pteropod molluscLimacina helicina was numerically common in all four species of fish. The planktonic hyperiid amphipodHyperiella dilatata was also found in all fish species. WhereasP. borchgrevinki is planktivorous in accord with its pelagic habit, theTrematomus spp. clearly also feed on plankton from the water column.T. hansoni is particularly planktivorous, taking smaller copepods thanP. borchgrevinki.     

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     

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     

Cytomorphology of female and male gonads in Antarctic toothfish <Emphasis Type="Italic">Dissosctichus mawsoni</Emphasis> (Nototheniidae) from the Ross Sea in the summer period

Results of histological analysis of gonads of female and male Antarctic toothfish Dissostichus mawsoni caught in the Ross Sea of the Pacific sector in the period of Antarctic summer (December–February) 2004–2005 are presented. Morphological indices and index of gonad maturity are described and ecological criteria of assessment of maturity stages of ovaries, cytological indices of oocytes, and the type of toothfish oogenesis are determined. It was established that in the period of fishing Antarctic toothfish, females and males with gonads at maturity stage III dominate. Ovaries contain two groups of oocytes of the period of trophoplasmatic growth and large oocytes of the nearest spawning season that comprise the smallest proportion of total sex cells. In the testes of two male toothfish, the primary fusion of renal and generative tissues was revealed. It is suggested that the termination of gonad maturity of toothfish takes place from March to April, and spawning takes place from June to August.     

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     

Age and growth of the nototheniid fishTrematomus bernacchii Boulenger from Terra Nova Bay,Antarctica

Age and growth of the nototheniid fishTrematomus bernacchii Boulenger 1902 were estimated by reading the sagittal otoliths of 457 adult specimens caught off Terra Nova Bay (Ross Sea) in the austral summer 1990–1991. Annuli in ground and polished otoliths were examined using a dissecting microscope under reflected light. The Von Bertalanffy growth equation was L_t=273.5 [1 − e^{−0.109(t+2.10)}] for males (n=122) and L_t=422.2 [1 − e^{−0.055(t+1.92)}] for females (n=211) where L is total length in millimetres. Maximum estimated age was 21 years for females and 16 years for males. This is in agreement with the hypothesis that considers slow growth and old age as a typical feature of Antarctic fishes.     

Spatial and temporal variations in the diet of nototheniid fish in McMurdo Sound,Antarctica

Specimens of 4 species of Antarctic fish were captured at different locations in McMurdo Sound during the early summer, and for one species also during late winter. Stomach contents were analysed to examine resource utilization across species, at different locations, and between late winter and early summer. The results are consistent with earlier findings that there is a gradation in resource utilization across these species.T. pennelli andT. bernacchii tend to take predominantly benthic crawling prey, though they also take prey from the water column.T. hansoni andT. nicolai tend to take more prey from the water column, a tendency which can be related to the visual feeding vector of these species. Substantial differences in diet for the same species captured in different locations indicated significant flexibility in prey selection which would allow utilization of spatial and temporal fluctuations in prey availability. Successful feeding byT. bernacchii in late winter is a further indication that this species can feed non-visually and supports the notion that non-visual feeding mechanisms are likely to be of importance in the biology of the Antarctic fishes.deceased     

Testes development and maturity classification of albacore (Thunnus alalunga ( Bonaterre, 1788)) from the Eastern Mediterranean Sea

The criteria of testicular maturity during the spawning season and for maturity rates are described according to age for 140 male albacore (Thunnus alalunga, Bonnaterre, 1788) sampled between April and August, 2002–2008 in the Eastern Mediterranean Sea. Histology of gonad samples was used to determine maturity status. Spermatogenesis and its relation to reproductive biology are reported. Fish ranged in fork length (FL) from 63 to 90 cm. The reproductive classes were based upon changes in the testicular morphology and stages of the germinative cells, i.e. immature, developing, maturing, spawning, and spent. Mature stages of testes including the smallest sample of a 63 cm male were observed from May to August of each year during the study. The reported evidence indicates that the size and age of first sexual maturity values of males of the Eastern Mediterranean population are nearly the same as in the Western Mediterranean population, but lower than in the Atlantic stock. The results of this histological investigation, conducted for the first time on this species in the Mediterranean Sea, will enable more precise future estimations for sustainability of the male albacore stock in the Eastern Mediterranean.     

Regulatory peptides in the urinary bladder of two genera of Antarctic teleosts

Somatostatin 14, prolactin, atrial natriuretic peptide, galanin and urotensin II were found using immunohistochemistry in the urinary bladders of the Antarctic fishes Trematomus bernacchii (Nototheniidae) and Chionodraco hamatus (Channichthyidae) caught in the Ross Sea. The urinary bladders of the two species showed a different histology in the epithelial layer. In T. bernacchii the epithelium comprises a single type of columnar cells, while in C. hamatus the columnar cells are restricted to the ventral portion of the bladder, and the dorso-lateral region is lined by cuboidal cells. No difference in the intensity of the immunostaining was observed in the two cell types; the only variation was a different distribution of the immunoreactions, which were present in the whole cytoplasm in the cuboidal cells and restricted to the apical and/or basal portion of the columnar cells. Accepted: 1 November 1998     

Gonadal histology and plasma sex steroid concentrations in maturing and mature spring migrants of Caspian lamprey Caspiomyzon wagneri in the Shirud River,southern Caspian Sea,Iran

Maturing degree-days, gonadal histology, and changes in serum sex steroids (progesterone, P; testosterone, T; and 17β-estradiol, E2) were examined in maturing and mature spring migrant Caspian lamprey Caspiomyzon wagneri (Kessler, 1870) in the Shirud River (southern Caspian Sea). Blood and gonad samples were collected from ten fish when they first entered the river (maturing stage) and from ten fish that showed spawning readiness after being held in cages in the river (mature stage). The maturing degree-days of Caspian lamprey from the start of upstream migration to maturation was 208–470°C.day. Serum P and E2 concentrations in maturing females were significantly higher than in maturing males, but in the mature stage, serum P and E2 concentrations of females were lower than males. In both stages, there were no differences in serum concentration of T between females and males. In both males and females, P increased significantly with maturation; T levels likewise appeared to increase, but the difference was not significant. E2 increased significantly with maturation in males, but females showed a significant decrease. Maturing females had similar stage gonads with the germinal vesicle in the polar position. Maturing males had testes that primarily contained secondary spermatocytes with an occasional occurrence of spermatozoa. These results suggest that males mature earlier than females, which is a pattern similar to that found in the sea lamprey.     

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.     

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