Spectrophotometric parameters of conformational states of hemoglobin and its complexes with ligands in the sturgeons

Parameters of carbo-, carboxy-, oxy-, deoxy-, and methemoglobin of the human and four species of sturgeons (sterlet, Russian sturgeon, starred sturgeon, great sturgeon) were analyzed spectrophotometrically. It has been shown that in the absorption spectra of all forms of hemoglobins there is only one Soret γ₁ band due to the prosthetic group of the pigment; the wavelength of this band is undoubtedly associated with the hemoglobin conformation. This permits the use of the band as an indicator parameter to control an initial state of the fish hemoglobin solutions, the analysis of conformational states of the pigment macromolecules, and the study of hemoglobin derivatives (its complexes with ligands).     

Spectrophotometric parameters of conformational states of hemoglobin and its complexes with ligands in the sturgeons

Parameters of carbo-, carboxy-, oxy-, deoxy-, and methemoglobin of the human and four species of sturgeons (sterlet, Russian sturgeon, starred sturgeon, great sturgeon) were analyzed spectrophotometrically. It has been shown that in the absorption spectra of all forms of hemoglobins there is only one Soret γ₁ band due to the prosthetic group of the pigment; the wavelength of this band is undoubtedly associated with the hemoglobin conformation. This permits the use of the band as an indicator parameter to control an initial state of the fish hemoglobin solutions, the analysis of conformational states of the pigment macromolecules, and the study of hemoglobin derivatives (its complexes with ligands).     

Comparative immunochemical analysis of intraspecies distinctions of serum proteins of starred sturgeon <Emphasis Type="Italic">Acipenser stellatus</Emphasis> (Acipenseriformes,Acipenseridae) from the Caspian basin

By methods of immunochemical analysis, antigenic characteristics of serum proteins of starred sturgeon Acipenser stellatus migrating to the Volga, the Ural, and the Kura are studied. Antigens of serum proteins of starred sturgeon with normal homo- and heteroantisera are differentiated immunoelectochemically into 22–25 components. Cross absorption of antisera revealed the presence of specific antigens. Volga starred sturgeon differs from the Ural starred sturgeon in one antigen which belongs to α₂-globulins and differs from the Kura starred sturgeon in two antigens which manifest themselves in the zone of β₁-globulins. Kura starred sturgeon differs from the Ural starred sturgeon in two antigens which are in the zone of α₂-globilins. It is concluded that presence of specific antigens in the Volga and the Kura population of starred sturgeon and in their absence in the Ural starred sturgeon indicate the reproductive independence of these intraspecies groups.     

Osmotic and ion regulation in different species of acipenserids (acipenseriformes, acipenseridae)

Species specificity of osmotic and ion regulation in five species of acipenserids connected in varying degrees with sea conditions is investigated. The objects of study were freshwater middle-Volga sterlet Acipenser ruthenus; Siberian sturgeon A. baerii, inhabiting the Lena River and migrating to the sea near the river mouth; and the following migratory acipenserids of the Volga-Caspian Basin (regular diadromous migrations): the Russian sturgeon A. gueldenstaedtii, the great sturgeon Huso huso, and the starred sturgeon A. stellatus. The study was carried out on immature specimens (of age 2+). The level of osmolarity and concentrations of cations (Na⁺, K⁺, Ca²⁺, and Mg²⁺) were determined in blood serum, urine, and liquid from the cavity of spiral valve in fish from the fresh water (control) and after their adaptation (period of seven to ten days) to sea water with salinity 12.5–12.7 and 18‰. The increase was found in the functional activity of the mechanism of osmotic and ion homeostasis in sturgeons according to the increase of the environmental salinity of a particular species. The freshwater sterlet adapted to the sea environment as osmoconformer. The other studied species were capable of maintaining a relative stability of osmolarity and ion concentration in blood serum at different levels while adapting to the sea environment; they changed from hyperosmotic (in fresh water) to hypoosmotic type of regulation. The kidney plays a more significant role in the removal of excessive Na⁺ in sturgeons than in bony fish. An increase in the sodium-reabsorption and magnesium-secretion functions of the kidney as well as the sodium-absorption function of the intestines was observed in sturgeons, relative to the increase of environmental salinity.     

Development of mesonephros in prolarvae of acipenserids (acipenseridae)

Development of the excretory system in early ontogenesis of acipenserids—the Russian sturgeon Acipenser gueldenstaedtii, the starred sturgeon A. stellatus, the sterlet A. ruthenus, and the great sturgeon Huso huso—is investigated histologically. In acipenserids, the mesonephros develops after hatching. At the 36th stage of development (after Detlaf et al., 1981), the mesonephros is a consecutively arranged group of spherical rudiments of placodes situated along the Wolffian duct. Morphological differentiation of mesonephros as the first generations of renal corpuscles, vascular glomeruli, and convoluted tubules is completed up to the 45th stage, i.e., to the moment of transition of prolarvae to active feeding.     

Localization of Phosphatase Responsible for Hydrolysis of Inositol 1,4,5-Triphosphate in the Olfactory Lining of True Sturgeons

The paper presents results of a cytochemical study of localization of phosphatase responsible for hydrolysis of inositol 1,4,5-triphosphate (ITP) in the olfactory lining of true sturgeons (the sturgeon, starred sturgeon, and sterlet). Reaction products as a dark discrete granules are localized in the apical parts of epithelium, practically in the same manner in all the species studied. The precipitate is found on the plasma membranes of cilia, microvilli, and clava of the olfactory cells. Occasionally, the precipitate is also found in the cilia, basal bodies, and rootlets of microvillar cells. The ITP-hydrolyzing phosphatase is supposed to restrict development of transduction process by removing excess messengers from the operating system. The data obtained indicate that in the true sturgeons, the phospholipase cascade of olfactory transduction is concentrated predominantly in the cilia and microvilli of olfactory cells.     

Activities of Lactate and Malate Dehydrogenase of the Sterlet and Russian Sturgeon under Conditions of Massive Spread of Muscle Pathology

As a result of the performed study, the basic data were obtained on the level of lactate dehydrogenate (LDH) and malate dehydrogenase (MDH) activity in two species of sturgeons caught in various regions of the Volga basin under conditions of spreading in the population of a pathology, one of its manifestations being stratification of muscle tissue. In the sterlet, a statistically significant increase of muscle LDH was revealed in fish with a pronounced stratification, while in the Russian sturgeon a tendency for such increase was noticed. In the group of sick fish a marked increase of dispersion of individual values of the enzyme activities in tissues was observed, which appears to indicate a disbalance of biochemical processes in the organism. The data obtained indicate that the stratification is not accompanied by a damage of myofibril sheaths and of liver cells. No obvious interspecies differences in the levels of LDH and MDH were observed between the sterlet and Russian sturgeon.     

Cryoprotective activity of phosphorus-containing phenol

The antioxidant and cryoprotective efficiencies of a 3,5-di-tert-butyl-4-hydroxyphenyl)methylenediphosphonic acid (MDPA) differ significantly for sperm cells of various species of sturgeon fish (Russian sturgeon, beluga and Stellate sturgeon). The ability of phosphorus-containing phenol MDPA to decrease the level of lipid peroxidation of sperm, beneficial effect on the activity indicators of the sperm of native sturgeon and of the defrosted one after deep freezing as well as on the fertility of sperm cells was shown.     

New strategies for brood stock management of sturgeon in the Sea of Azov basin in response to changes in patterns of spawning migration

The sturgeon stock in the Sea of Azov is presently maintained by artificial reproduction allowing for the harvest of sturgeons in this basin. Shortened spawning migrations in regulated rivers and long-term artificial selection of the ripest fish at the hatcheries have resulted in high functional maturity of sturgeon spawners in the sea near the river mouth. The traditional technology of artificial sturgeon reproduction is non-effective under modern ecological conditions. The use of ecological and hormonal methods to control seasonal propagation of stellate sturgeon, A.stellatus , Russian sturgeon, A.gueldenstaedti , beluga, Huso huso , and sterlet, A.ruthenus , make it possible to stagger the sexual cycle of breeders from the various biological groups for early (5 months) or later (6 months) spawning. Use of these methods preserves the natural heterogeneity of sturgeon populations maintained through stocking, and also allows for a considerable increase in the quantity of fry reared at the same production base.
The use of the synthetic analogue of mammalian gonadotropin-releasing hormone (Gn-RHa) is better for inducing maturity of the ripest brood fishes, especially for stellate sturgeon, in comparison with the traditional pituitary injections. The schedule of injections was determined for the various species and seasons of spawning.
Short-term rearing of larvae in ponds (no more than 15 days) and subsequent adaptation to sea water in brackish lagoons (1–5 %o) allows for repeated use of nursery ponds in sturgeon hatcheries and a considerable increase in the quantity and survival of sturgeon juveniles in the sea.
The restoration of the sturgeon population structure in the Sea of Azov is not possible without developing a genetic collection of the fish under a controlled culture regime.     

Inheritance of some quantitative morphological characters in reciprocal hybrids of starred sturgeon <Emphasis Type="Italic">Acipenser stellatus</Emphasis>and beluga <Emphasis Type="Italic">A. huso</Emphasis> (Acipenseridae)

Some quantitative characters of three-summer old reciprocal hybrids of beluga Acipenser huso and starred sturgeon A. stellatus,as well as of juveniles of their parental species, are comparatively analyzed. The found maternal and paternal effects in manifestation of some characteristics in hybrids are considered in connection with elucidation of inheritance type of morphological characters in acipenserids. The paternal effect observed by some characters in hybrids beluga × starred sturgeon is explained by prevalence of dominant alleles in the genome of starred sturgeon in comparison with beluga. The maternal effect is elucidated by a few characteristics related to parameters of the head, first of all to the volume of the brain region of skull. Evidently, in this case, the matrocliny depends on the fact that information on the number of segments of embryo and their position is determined by the maternal organism during oogenesis.     

Does body size affect the response to exercise in shortnose sturgeon (Acipenser brevirostrum)?

The effect of body size on various hematological variables was examined in juvenile shortnose sturgeon (Acipenser brevirostrum) ranging in mass from 38 to 730 g. The blood was examined for differences in plasma ionic composition (Na⁺, K⁺, Cl^?), blood oxygen carrying capacities (hemoglobin, hematocrit), and plasma metabolite concentrations (lactate, glucose), before and following a standard 5‐min chasing stress. All measured resting hematological variables were size independent in shortnose sturgeon. After exercise, levels of plasma lactate, potassium, and hemoglobin increased in all fish. Only post‐exercise levels of chloride and hemoglobin changed in a size‐dependent manner; however, the relationships were weak. The general lack of a relationship between body size and hematological variables might reflect the narrow range of fish sizes used in the present study. From a practical perspective, the results suggest that when examining the hematological stress response in juvenile shortnose sturgeon, a range of fish sizes could be used. This is important considering the variability in the growth rates of juvenile shortnose sturgeon under laboratory conditions.     

The surface activity of PVP and other polymers and their antihemolytic capacity

The polymeric cryoprotective agents polyvinylpyrrolidone, dextran, and hydroxyethyl starch do not penetrate the cell membrane and are not present in high osmotic concentrations. Thus, they can exert little of the "antifreeze" behavior generally attributed to glycerol or dimethyl sulfoxide, and must protect cells from freezing injury by some action external to the cell surface. Surface energy measurements of droplets of hemoglobin solution immersed in solutions of cryoprotective polymers indicate that these polymers lower the surface energy of the solution below that of the hemoglobin droplets and form a stable interface. In injured cells, these polymers will therefore hide membrane defects by forming an interface across which hemoglobin cannot easily pass. When freezing is slow, the polymers have little if any true cryoprotective effect but interfere with hemoglobin release as an assay of injury.     

Induced androgenesis in acipenserids may be obtained by ultraviolet radiation

The results are presented on haploid androgenesis in Siberian sturgeon and sterlet induced by UV irradiation of ovicells. During irradiation, the cells in Ringer solution were rotated around a UV lamp. The efficiency of genetic inactivation of ovicells was estimated by the following parameters: manifestation of Hertwig effect, the fraction of embryos demonstrating haploid syndrome at final developmental stages, by arrest of embryonic development in hybrids Siberian sturgeon × great sturgeon, and by absence of maternal alleles of microsatellite loci in embryos. The dose-effect curve suggests that, during UV irradiation of ovicells of Siberian sturgeon, the complete genetic inactivation is attained at exposition of 120 s, while that in sterlet is 90 or 105 s. The results show a principal possibility of inactivation of ovicells by UV irradiation and use of such cells for producing androgenetic progeny of acipenserids.     

Exotic sturgeons in the Vistula Lagoon in 2011, their occurrence,diet and parasites,with notes on the fishery background

The Siberian sturgeon Acipenser baerii and the sterlet Acipenser ruthenus were recorded for the first time in the Vistula Lagoon in 2011. Among 66 sturgeons collected between April and December 2011, the Siberian sturgeon was the most numerous species (77%); however, a significant seasonal variability was observed, with sterlet dominating in the catches in late autumn of the same year. The stomach contents of the two species differed widely: Siberian sturgeon (14.9–42.2 cm standard length, SL) fed on crustaceans (Cercopagis pengoi, Oithona sp., Neomysis integer) (10.7% IRI), larvae and pupas of insects (Chaoborus sp., Chironomus sp., Polypedilum sp., Procladius sp., Culex sp.) (88.9% IRI) and fishes (Neogobius melanostomus, Osmerus eperlanus) (0.5% IRI), whereas sterlet (24.0–34.4 cm SL) consumed crustaceans (N. integer) (64.3% IRI), larvae of insects (Chironomus sp., Polypedilum sp.) (20.3% IRI) and fish (N. melanostomus) (15.4% IRI). Single Siberian sturgeon (4.3%) were found to harbour the parasitic nematode (Raphidascaris acus).     

Lysozyme Content in Organs and Blood Serum in Various Species in the Volga River

The results of study of lysozyme content in kidney, liver, spleen, and blood serum of 12 fish species in the Volga River, which belong to six different orders, are presented. It has been established that the lysozyme level in the organs and blood serum is different in representatives of different taxons. The highest enzyme content was observed in sterlet (order Acipenseriformes), while the lowest content, in burbot (order Gadiformes). The enzyme content and its distribution in visceral organs depended on fish phylogeny, but not on the type of feeding (vegetarian or predatory) and the place of the constant habitation (fresh river or salted sea water). There were seasonal and yearly variations of the lysozyme concentration in fish organs and tissues. The many-year studies have established the regularity in enzyme distribution in sturgeon fish: kidney > spleen > liver > blood serum, this regularity being independent of the sturgeon species and constant during intra- and inter-year variations.     

Dietary protein requirement of fingerling sterlet sturgeon (Acipenser ruthenus)

Although sterlet sturgeon is a valuable candidate for aquaculture development, information regarding the nutrient requirements of this species is limited. Therefore, in this study, we aimed to determine the optimum protein level required by sterlet sturgeon fingerlings. Groups of fingerlings (mean initial body weight ± SD: 7.02 ± 0.02 g; N = 3) were assigned to one of the six dietary protein levels at 29.1%, 39.1%, 46.8%, 49.2%, 53.7%, and 57.6% on a dry matter basis. Isocaloric (18.3 ± 0.2 kJ GE/g diet) semi-purified diets were prepared using white fish meal and casein as the major sources of protein. After the 8-week feeding trial, the fingerlings fed the 49.2% protein diet exhibited the best results in terms of weight gain, feed efficiency, and whole-body nutrient composition (crude protein, crude lipid, gross energy, and amino acid contents), whereas those fed the 53.7% and 57.6% protein diets showed poor performance. Application of the two-slope straight broken-line and quadratic models on the dataset of weight gain revealed the optimum protein level to be 45.9% and 44.1%, respectively. Based on the model selection criteria, including the adjusted coefficient of correlation and corrected Akaike information criterion, the protein level (44.1%) estimated using the quadratic model was more accurate than that obtained using another model (45.9%). Taken together, 44.1% protein level (18.3 kJ GE/g diet on dry matter basis) resulted in the optimal growth of the sterlet sturgeon fingerlings (initial body weight: approximately 7 g).     

Fertility of females of sturgeon hybrids obtained from species with different levels of ploidy (Acipenser ruthenus and A. dauricus) and their cloning

Main purpose of this work is the identification of females of artificial sturgeon hybrids capable to produce unreduced oocytes. The importance of this task is due to the ability to receive clonal all-female lines. Experiments were performed on the previously obtained reciprocal hybrids of sterlet, Acipenser ruthenus (S) with ~120 chromosomes and kaluga, Acipenser dauricus (K) with ~260 chromosomes. Karyotypes of backcross hybrids of (S × K) female (obtained by crossing sterlet female with kaluga male) and sterlet male included 180 – 190 chromosomes. This means that (S × K) female produced eggs with ~125 chromosomes and its karyotype consisted of ~250 chromosomes. This number was confirmed by a comparative analysis of erythrocyte size in this female and species with different ploidy. Karyotype with ~250 chromosomes can occur in (S x K) female only as a result of fertilization of a diploid sterlet egg (120 chromosomes) with kaluga haploid sperm (~130 chromosomes). Eggs of hybrid fertile (S × K) female, inseminated with inactivated sperm of Amur sturgeon and sterlet, developed into viable gynogenetic offspring, confirmed by the analysis of five microsatellite loci in this progeny, (S x K) female, and males used for UV-inactivated sperm. These data allow us to propose a method for obtaining fertile females of sturgeon hybrids from species with different ploidy. For this, experimentally obtained diploidized eggs from diploid 120-chromosome species must be fertilized by 250–270-chromosome male. Karyotypes of backcross hybrids of (K × S) female (obtained by crossing kaluga female with sterlet male) and sterlet male included ~250 chromosomes and hybrids of this female with kaluga male had ~320 chromosomes. These results proved an ability of hybrid (K × S) female to produce unreduced eggs, resulting in triploid backcrosses. The absence of reduction during egg development is well known in clonal forms (species) of vertebrates, which are of hybrid origin, and in artificially created fish hybrids. However, this has not been reported previously for sturgeons. Insemination of eggs of (K × S) female with UV-inactivated sperm of sterlet and Amur sturgeon led to offspring generation for which the genetic identity to their mother was proved using microsatellite analysis. That is, clonal inheritance was observed. These results suggest the possibility of developing a technology to produce all-female offspring. Artificial production of clonal lines in hybrid vertebrates can be also considered as experimental reproduction of the first stages of reticular speciation in nature.     

Analysis of creatine kinase variation in some members of the family Acipenseridae

Creatine kinase (E.C. 2.7.3.2) was examined in stellate sturgeon Acipenser stellatus Pallas, Russian sturgeon A. gueldenstaedtii Brandt, European sterlet A. ruthenus L., Siberian sterlet A. ruthenus marsiglii Brandt, and great sturgeon (beluga) Huso huso L., using polyacrylamide gel electrophoresis. Two loci for creatine kinase were identified: CK-A* in white skeletal muscle and CK-C* in stomach wall muscle. Most species proved to be monomorphic at the CK-A* locus, showing the same phenotype represented by a single band. Heterogeneity and polymorphism in creatine kinase, determined by the CK-A* locus, were found only in Russian sturgeon. Based on the results of densitometric analysis of band staining intensity, we have advanced a hypothesis that synthesis of subunits of the CK-A* product in this species was controlled by eight genes. However, the genotype frequencies in the sample were significantly different from those theoretically expected upon free and independent gene recombination. The results of this study support the hypothesis on the absence of heterodimeric creatine kinase molecules in the skeletal muscle of Russian sturgeon. Locus CK-C* in sterlet was revealed as a single, intensely stained, rapidly migrating fraction, whereas in Russian sturgeon, the enzyme activity in this zone was very weak. No creatine kinase was found in liver, kidneys, spleen, heart, and intestine mucous tunic.     

Ploidy levels and the number of nuclei in cardiomyocytes of the lamprey and fish

It is well known that polyploidization of cardiomyocytes (CMC) is an essential component of heart growth in the warm-blooded vertebrates. Using the Feulgen cytophotometry of alkali-dissociated cells, we determined the ploidy in CMC of the lower vertebrates: lamprey Lampetra fluviatilis (Cyclostomata), skate Bathyraja maculata (Chondrostei), sterlet Acipenser ruthenus, and Russian sturgeon Acipenser güldenst?dti (Ganoids), as well as paradise fish Macropodus opercularis, Amur sleeper Perccottus glehni, and Atlantic salmon Salmo solar (Teleostei). The data obtained have demonstrated a wide variety in CMC ploidy of both cyclostomata and fishes. About 85% of the lamprey CMC contain 2 or more (up to 17) nuclei per cell; with 90 and 10% of the nuclei being, respectively, diploid and tetraploid. Hearts of the skate and sturgeons contain mononucleated diploid CMC. In the perch-like fishes, mononucleated diploid and mononucleated tetraploid CMC make, respectively, 95 and 5%. The salmon heart contains near 50% of mononucleated diploid CMC, 13% of mononucleated tetra- and octaploid CMC, the rest CMC being multinucleated (up to 6 nuclei per cell). In all the examined species, the increased nuclear ploidy is accompanied with a significant increase in the nuclear volume. The number of nucleoli per nucleus does not correlate with the nuclear ploidy level. Evolutionary aspects of CMC polyploidy in chordates are discussed.     

Meiotic gynogenesis in the stellate,Russian sturgeon and sterlet

Diploid gynogenetic progenies were obtained in the stellate sturgeon Acipenser stellatus, Russian sturgeon A. gueldenstaedtii, and sterlet A. ruthenus by means of insemination of the eggs with UV-irradiated spermatozoa and suppression of the second meiotic division by heat shock. The gynogenetic nature of experimental fish was confirmed by RAPD-PCR analysis of DNA. Effective photoreactivation of UV-induced lesions of spermatozoa was shown in the case of illumination of the fertilized eggs with visible light. This phenomenon should be taken into account when determining the doses of irradiation that allow inactivation of the male chromosomes and incubating gynogenetic embryos. Gynogenetic stellate and Russian sturgeons are viable and can be reared in order to study the mechanism of sex determination in sturgeons.