Martin Flajšhans

Genetic studies of tench (Tinca tinca L.): induced triploidy and tetraploidy and first performance data.

Abstract Triploidy was successfully induced with cold shocks of 0–2°C, lasting 35 min and starting 2–5 min after gamete activation and with hydrostatic pressure shocks with 1, 2 and 5 min exposure at 50.01–52.47 MPa starting 2–5 min after gamete activation. Tetraploidy was successfully induced with heat shocks of 40.5°C, lasting 1.5 min and starting 45 min after gamete activation and with hydrostatic pressure shocks starting 50 min after gamete activation without further changes in the remaining parameters. Temperature shocks were considered to be the most suitable treatments, based on observed proportions of viable polyploids and incubation rates. The τ 0 unit for tench was used for standardization of shock initiation. Results of rearing triploid and/or tetraploid tench in experimental earthen ponds were assessed. A sample of 19 4-year-old triploid and their 43 diploid siblings was used for analysis of weight differences, gonadosomatic index and dressing percentage. Triploid females displayed 13.52% higher live weight and 3.94% higher dressing percentage than diploid females ( P P P P P P

Genetic studies in tench (Tinca tinca L.): high incidence of spontaneous triploidy

Abstract A high incidence of spontaneous triploidy was found in tench broodstock hormonally treated to induce spawning but without positive response. Triploidy was also observed in random samples of fingerlings from four tench lines. Triploidy was evaluated by counting Ag-stained NORs in erythrocyte nuclei and was confirmed in 62.5% of the broodstock and in a range from 0.00% to 34.62% in fingerlings. Discrimination of tench diploids and triploids based on differences in shape and length of pelvic fins was tested in the broodstock and also in stocks of earlier artificially induced triploid tench by means of the above-mentioned cytogenetic approach and shown to be a rapid method with a high accuracy (96.43%). Natural disposition for egg overripening is one of the possible causes of highly frequent spontaneous triploidy in the analyzed samples. Genetic predisposition, i.e. occurrence of a recessive allele responsible for the failure of the 2nd polar body extrusion when homozygous, was hypothesized as another possible cause of spontaneous triploidy, based on analysis of the relationships among the tench lines.

The application of image cytometry to viability assessment in dual fluorescence-stained fish spermatozoa

The viability of spermatozoa has been assessed using SYBR 14 staining for DNA of living cells and propidium iodide staining for DNA of degenerate cells. This dual staining was performed on four fish species (Siberian sturgeon, Acipenser baerii; common carp, Cyprinus carpio; tench, Tinca tinca and wels, Silurus glanis) and the proportions of live and dead spermatozoa were assessed by epifluorescence microscopy and image cytometry. Ten phase contrast and epifluorescent images were recorded per sample, corresponding images were overlaid, and the blended images were evaluated for live and dead spermatozoa, represented by green and red fluorescence signals. Live/dead proportions were assessed, after dual threshholding, by imaging software that counted absolute numbers of objects and computed their frequencies. All sperm heads were found to be labelled, emitting either green or red light. Mean numbers of spermatozoa per image were in the ranges 32–113, 61–105, 48–104 and 29–91 for Siberian sturgeon, common carp, tench and wels, respectively. The corresponding proportions of live spermatozoa were in the ranges 83.56–94.59%, 93.92–97.02%, 76.14–97.76% and 79.45–83.76%. Standard deviations did not exceed 5% of the means. The image cytometric system using dual staining with SYBR 14 and propidium iodide was clearly suitable for assessing the viability of freshwater fish spermatozoa.

Sturgeon genetics and cytogenetics: a review related to ploidy levels and interspecific hybridization

Abstract. Sturgeons (Chondrostei: Acipenseriformes) display markedly disjunction distributions with a wide distribution in the northern hemisphere. Their unique benthic specializations and conserved morphology, evolutionary age, the variation in their basic diadromous life history, and the large public interest due to their near extinction or critically endangered status make sturgeons and paddlefishes interesting groups for molecular and cytogenetic studies. From altogether 27 acipenseriform species, seventeen species are supposed to be critically endangered, two species are classified as endangered, four species are vulnerable and other species are near threatened or in low-risk (IUCN Red list 2010). Sturgeons are characteristic by a relatively high number of chromosomes in cell nuclei and differences in ploidy levels. Sturgeons displayed a strong tendency for interspecific and inter-generic hybridization under altered environmental conditions as well as under conditions of artificial propagation. Almost 20 inter-specific sturgeon hybrids were described. The decrease of natural populations and tendencies leading to restocking may result in uncontrolled restocking, production of hybrid specimens (even with non-native species) and decrease of natural genetic diversity of species in their original distribution area. Identification of parental species of natural hybrids by modern methods of molecular biology is still not easy. Here, we attempt to briefly summarize the major aspects of sturgeon genetics and cytogenetics related to ploidy levels and interspecific hybridization.

Are fish immune systems really affected by parasites? an immunoecological study of common carp (Cyprinus carpio)

BackgroundThe basic function of the immune system is to protect an organism against infection in order to minimize the fitness costs of being infected. According to life-history theory, energy resources are in a trade-off between the costly demands of immunity and other physiological demands. Concerning fish, both physiology and immunity are influenced by seasonal changes (i.e. temporal variation) associated to the changes of abiotic factors (such as primarily water temperature) and interactions with pathogens and parasites. In this study, we investigated the potential associations between the physiology and immunocompetence of common carp (Cyprinus carpio) collected during five different periods of a given year. Our sampling included the periods with temporal variability and thus, it presented a different level in exposure to parasites. We analyzed which of two factors, seasonality or parasitism, had the strongest impact on changes in fish physiology and immunity.ResultsWe found that seasonal changes play a key role in affecting the analyzed measurements of physiology, immunity and parasitism. The correlation analysis revealed the relationships between the measures of overall host physiology, immunity and parasite load when temporal variability effect was removed. When analyzing separately parasite groups with different life-strategies, we found that fish with a worse condition status were infected more by monogeneans, representing the most abundant parasite group. The high infection by cestodes seems to activate the phagocytes. A weak relationship was found between spleen size and abundance of trematodes when taking into account seasonal changes.ConclusionsEven if no direct trade-off between the measures of host immunity and physiology was confirmed when taking into account the seasonality, it seems that seasonal variability affects host immunity and physiology through energy allocation in a trade-off between life important functions, especially reproduction and fish condition. Host immunity measures were not found to be in a trade-off with the investigated physiological traits or functions, but we confirmed the immunosuppressive role of 11-ketotestosterone on fish immunity measured by complement activity. We suggest that the different parasite life-strategies influence different aspects of host physiology and activate the different immunity pathways.

Genetic studies with tench, Tinca tinca L.: induced meiotic gynogenesis and sex reversal

Abstract Carp sperm with genome inactivated by gamma irradiation ( 60 Co; 1400 Gy dose) was used to induce gynogenetic development in tench ( Tinca tinca ). The non-participation of paternal genome in tench female X carp male “hybrid” diploidized with cold shock was confirmed morphologically, karyologically and biochemically. Consistent yields of 5–21% viable gynogenetic fry were obtained when eggs were incubated at 20 °C and cold-shocked (30 min at 0 to +2 °C) 2, 5 ( P P τ 0 (duration of one mitotic cycle of synchronous cell division related to water temperature) times after activation were 0.069, 0.173 and 0.346 τ 0 , respectively. In a preliminary study of sex reversal, sex was reversed with tench. The greatest effect of treatment appears to lie in the range of a 0.102 g/kg dose of 17α-methyltestosterone (MT) starting at 40–60 days after activation.

Genetic resources of commercially important fish species in the Czech Republic: present state and future strategy

Abstract Since 1996, the Czech governmental programme on gene resources conservation of commercially important fish species has supported in situ conservation of rare/endangered breeds of common carp ( Cyprinus carpio ; nine breeds) and of rainbow trout ( Oncorhynchus mykiss ; three breeds) by keeping broodstock of each as live gene banks on selected farms. Effective population size ( N e ) is maintained at 120. As fish are not included yet in the Czech animal breeding act, the programme encourages fisheries policy makers and farm managers to protect pure local breeds parallely to a general tendency to import breeds, use crossbreds for production or move breeds among farms. Since most breeds are defined by reproductive and performance traits only, their genetic structure is currently being investigated by enzyme polymorphism analysis to determine their value to the programme. A data records system is being introduced to the breeders that combines fish tagging and a locally-developed computer programme to store information. The experimental cryopreserved sperm bank is being extended to meet the needs of ex situ conservation. Since 1997, the programme has expanded to include eight strains of tench ( Tinca tinca ), two strains of wels ( Silurus glanis ), two coregonid species ( Coregonus lavaretus maraena and Coregonus peled ) and two acipenserid species ( Acipenser ruthenus and Huso huso ).

Leukocyte profiles of diploid and triploid tench, Tinca tinca L.

Abstract Leukocyte profiles were compared in diploid and triploid tench of the same origin. A total of 55 3-year-old (34 diploid and 17 triploid) and 61 4-year-old (28 diploid and 33 triploid) tench were examined in June of 1996 and 1997, respectively. There were no significant differences in total or differential leukocyte counts between diploid and triploid 3-year-old tench, in which lymphocytes dominated (>90%) the total leukocyte count. In the case of 4-year-old tench, there were no significant differences in total leukocyte counts, or in differential lymphocyte, monocyte and myelocyte counts, but triploids had lower metamyelocyte counts and higher neutrophile granulocyte counts ( P

Fertility of a spontaneous hexaploid male Siberian sturgeon, Acipenser baerii

BackgroundEvolution of sturgeons and paddlefishes (order Acipenseriformes) is inherently connected with polyploidization events which resulted in differentiation of ploidy levels and chromosome numbers of present acipenseriform species. Moreover, allopolyploidization as well as autopolyploidization seems to be an ongoing process in these fishes and individuals with abnormal ploidy levels were occasionally observed within sturgeon populations. Here, we reported occurrence of Siberian sturgeon (Acipenser baerii) male with abnormal ploidy level for this species, accessed its ploidy level and chromosome number and investigate its potential sterility or fertility in comparison with normal individuals of sterlet (A. ruthenus), Russian sturgeon (A. gueldenstaedtii) and Siberian sturgeon (A. baerii).ResultsAcipenser ruthenus possessed 120 chromosomes, exhibiting recent diploidy (2n), A. gueldenstaedtii and A. baerii had ~245 chromosomes representing recent tetraploidy (4n), and A. baerii male with abnormal ploidy level had ~ 368 chromosomes, indicating recent hexaploidy (6n). Genealogy assessed from the mtDNA control region did not reveal genome markers of other sturgeon species and this individual was supposed to originate from spontaneous 1.5 fold increment in number of chromosome sets with respect to the number most frequently found in nature for this species. Following hormone stimulation, the spontaneous hexaploid male produced normal sperm with ability for fertilization. Fertilization of A. baerii and A. gueldenstaedtii ova from normal 4n level females with sperm of the hexaploid male produced viable, non-malformed pentaploid (5n) progeny with a ploidy level intermediate to those of the parents.ConclusionThis study firstly described occurrence of hexaploid individual of A. baerii and confirmed its autopolyploid origin. In addition to that, the first detailed evidence about fertility of spontaneous hexaploid sturgeon was provided. If 1.5 fold increment in number of chromosome sets occurring in diploids, resulted triploids possess odd number of chromosome sets causing their sterility or subfertility due to interference of gametogenesis. In contrast, 1.5 fold increment in number of chromosome sets in naturally tetraploid A. baerii resulted in even number of chromosome sets and therefore in fertility of the hexaploid specimen under study.

The First Results on Relationships Among Amphimictic Diploid, Diploid Gynogenic and Triploid Tench, Tinca tinca L. under Communal Testing

Amphimictic diploid (2n), induced meiotic gynogenic (MeiG) and induced triploid (3n) siblings of tench, Tinca tinca L., were separately nursed, marked and kept in long-term communal testing in pond until sex maturation and natural spawning. Survival rates during the initial periods were lesser in 2n and 3n compared to MeiG population (65%). The first overwintering period showed decreased survival of 3n (62.67%) compared to MeiG (90.0%) and 2n populations (91.67%). Survival rates during the next seasons ranged in 70.50–90.97% similarly in both 3n and 2n while in MeiG population it dropped to 38%. Weight and length growth of all populations studied showed significantly higher growth of 3n females and of 3n in general, compared to 2n and MeiG populations (P < 0.001). Health examinations found metacercaria of Diplosthomus spathaceum in eyes of 2-year-old 3n specimens, weak polyparasitic infections of skin and gills by Dactylogyrus, Trichodina, Thrichophrya, Apiosoma and Chilodonella spp. in 3n and 2n specimens but medium strong to strong infections of 100% of MeiG fish which might contribute to their enhanced mortality. Ploidy analysis of progeny obtained from spontaneous spawning of the broodstock showed 100% diploidy, indicating the following possibilities: 2n population might spawn itself, 2n males might also mate to gynogens, or 3n males might also mate to 2n and/or MeiG females causing spontaneous gynogenesis. Further pedigree analyses will be focused to highlight these questions.