Optimization of UV treatment to induce haploid androgenesis in the stinging catfish, Heteropneustes fossilis

Abstract: Androgenesis is a form of uniparental reproduction leading to progenies inheriting only the paternal set of chromosomes. It has been achieved with variable success in a number of freshwater species and can be attained by artificial fertilization of genetically inactivated eggs following exposure to gamma (γ), X-ray or UV irradiation (haploid androgenesis) and by restoration of diploidy by suppression of mitosis using a pressure or thermal shock. The conditions for the genetic inactivation of the maternal genome in the European sea bass (Dicentrarchus labrax L.) were explored using different combinations of UV irradiation levels and durations. UV treatments significantly affected embryo survival and generated a wide range of developmental abnormalities. Despite the wide range of UV doses tested (from 7.2 to 720 mJ x cm(-2)), only one dose (60 mJ x cm(-2) x min(-1) with 1 min irradiation) resulted in a small percentage (14%) of haploid larvae at hatching in the initial trials as verified by flow cytometry. Microsatellite marker analyses of three further batches of larvae produced by using this UV treatment showed a majority of larvae with variable levels of paternal and maternal contributions and only one larva displaying pure paternal inheritance. The results are discussed also in the context of an assessment of the UV-absorbance characteristics of egg extracts in this species that revealed the presence of gadusol, a compound structurally related to mycosporine-like amino acids (MAAs) with known UV-screening properties.

Abstract: Clonal lines are a powerful scientific tool for improved genetic characterization of organisms used in research. Inbred fish lines can be produced in only two generations using uniparental reproduction techniques. Androgenesis, achieved with variable success in several freshwater species, has been attempted in the European sea bass (Dicentrarchus labrax L), a marine fish of commercial and scientific interest. The low yields of progenies inheriting only the paternal genome after UV-irradiation of eggs led to considerations on the occurrence of UV screening compounds in pelagic eggs. Mycosporine-like amino acids and gadusol were found in many marine and freshwater organisms, but their occurrence in fish eggs was not clearly related to a behavioral pattern and while gadusol appeared in higher proportions in pelagic marine eggs compared to benthic species, this statement did not apply in freshwater, and moreover the kind of compounds was related to phylogeny. Further studies on DNA photorepair could enlighten hypotheses to understand the mechanisms underlying the disparate results obtained in inducing androgenesis in different fish species. Gynogenesis was reported successful to produce clonal founders in the sea bass, but high numbers of meiotic individuals contaminating fully homozygous progenies highlighted the need for efficient DNA markers to distinguish mitotic gynogenetic individuals. Furthermore, gonad development was highly delayed in gynogenetic progenies enhancing the difficulties to produce clonal lines. A high variability between individuals in the success of uniparental reproduction brought out gamete characterization and quality as a prerequisite.

Abstract: The research work was carried out for induction of diploid meyo- and mito- gynogenesis in silver barb (Barbonymus gonionotus) through heat shock treatment in order to produce monosex all-female population. Optimal UV-irradiation achieved when the sperm suspension containing 8 × 108 ml−1 was exposed to a UV dose of 196μWcm−2 for 1.5 min. Diploidization of gynogenetics was done by heat shock at 40 °C for 1 min after 1.5 min of fertilization that produced 40.36 ± 3.46% meyo-gynogens. Similarly, mito-gynogenetic diploidization was achieved using the same heat shock treatment after 27.5 min of fertilization that yielded 33.80 ± 2.84% mito-gynogens. The gynogens were identified by karyotype, gonad identification through sexing and DNA microsatellite analysis. The karyotype analysis showed that the haploids have 23 (N) chromosomes, and the meyo- and mito-gynogenetic diploids and controls have 46 (2 N) chromosomes. Sexing of fish demonstrated that the meyo- and mito-gynogens were nearly all-female, ranging 98.18 to 100% and 96.77 to 100% female sex respectively. The control group had a mean sex ratio of 54.4:52.8, nearly 1:1 female: male sex. DNA microsatellite analysis revealed that the meyo-gynogenetic fry contained the alleles same as their mother and the mito-gynogenetic fry became homozygous by fixing any of the two maternal alleles. None of the gynogens had paternal inheritance. The diploid meyo- and mito-gynogens produced are much valuable as they are nearly all-female and a higher production is expected due to their faster growth rate. In addition, sex inversion of meyo- and mito-gynogenetic larvae using masculinizing hormone will facilitate to produce sex-reversed males (XX) those are capable to generate monosex all-female population upon crossing with genetic females (XX).

Abstract: This study reports the results on induced diploid androgenesis in the Indian catfish, Heteropneustes fossilis. Ultraviolet (UV) irradiation was used to inactivate maternal genome of H. fossilis. Complete inactivation of maternal genome was recorded at 12,500 ergs/mm2. These genome-inactivated eggs of H. fossilis were inseminated with conspecific sperm. The sperm suspension was diluted to 1 × 107 sperm ml/L in Hank's Balanced Salt Solution. Egg viability was assessed for different exposure durations at fertilization, hatching, haploidy, and diploidization. Majority of the larvae derived from irradiated eggs had abnormal appearance. Complete inactivation of maternal genome was detected by haploid syndrome and confirmed by chromosome counting (n = 29). These eggs activated with sperm were subjected to heat shock at 40 and 41 C for different postactivation times and durations. Diploid androgens had a normal appearance as controls and confirmed by chromosome counting (n = 58). A maximum of 21 and 14% of diploidization was recorded at 30 min after activation, at 40 and 41 C, which corresponds to the first cleavage suppression time.

Abstract: The optimum distance and duration of ultraviolet (UV) irradiation for the complete inactivation of African catfish Clarias gariepinus egg nucleus was investigated in this study. The UV light was suspended above the unfertilized eggs at four distances (5, 10, 20 and 30 cm) and for five durations (1, 2, 3, 4 and 5 min). Then, the irradiated eggs were activated with sperm from diploid C. gariepinus and cold shocked at 5°C for 5 min just moments before cell cleavage. Ploidy analysis was performed using karyotype chromosome counting. The results obtained suggested that the further the distance, the better the hatchability rate, however prolonged duration seemed to significantly reduced hatchability. All treatments with surviving progenies at the end of the study showed evidence of successfully diploid gynogen (2n = 56) induction at different percentages. However, the optimal protocol that gave a moderately high hatchability/survival rate and completely induced gynogens was exposure of the eggs to UV irradiation at 20 cm for 1 min. It was concluded that the distance and duration of UV irradiation affects gynogenetic induction in African catfish C. gariepinus.

Abstract: 1 Fisheries Chemotherapy: A Review- 2 Egg Production in the Rainbow Trout- 3 Solar Ultraviolet Radiation: A Potential Environmental Hazard in the Cultivation of Farmed Finfish- 4 Acid Rain: Implications for the Farming of Salmonids- 5 Reproductive Biology and the Hatchery Rearing of Tilapia Eggs and Fry

Abstract: A technique is described for obtaining well-spread metaphases from solid tissues of fishes without the use of methodologies that rely on tissue grinders, centrifuges, digestive enzymes, or tissue culture. This procedure involves the formation of a cell suspension from acetic alcohol fixed tissues using 50% acetic acid. The suspension is applied to a warm (50 °C) slide using a micropipette.Solid tissue preparations may be stained by any of the conventional dyes or treated to reveal Q-bands, C-bands, and nucleolar organizers. Large numbers of slides offish chromosomes can be made easily and rapidly using this procedure.

Abstract: Publisher Summary This chapter describes the techniques used in chromosome set manipulation and reviews the results and prospects in the application of gynogenesis, androgenesis, and induced polyploidy to fish. Chromosome-set manipulation techniques of sperm chromosome inactivation (with radiation or chemicals) and suppression of cell divisions (with heat shock, cold shock, or pressure) can be readily applied to fish to produce gynogenetic and polyploid individuals. Gynogenetic individuals have all their chromosomes from the female parent and should all be females in species with XX females. Polyploids include triploids that are expected to be sterile, and tetraploids that have the potential of being fertile and producing sterile triploids when crossed to normal diploids. Partially inbred gynogenetic diploids and triploids may be produced by treatments causing retention of the second polar body of the egg. Completely homozygous gynogenetic diploids and tetraploids may be produced by treatments blocking the first mitotic division.

Abstract: Manipulation of fish chromosomes dates back to the early part of this century. The earliest experiments involved induction of gynogenesis with sperm inactivated by radiation or chemical treatments. Temperature or pressure shocks applied soon after fertilization resulted in the retention of the second polar body and reconstitution of diploidy; triploidy resulted from shocks to fish ova fertilized with normal sperm. More recently, it has been possible to suppress the first mitotic division offish eggs with high-pressure or -temperature treatments applied at the time of first cleavage to produce mitotic gynogenetic diploids and tetraploids. Androgenesis has been successfully induced in fish by irradiation of ova, fertilization of eggs with normal sperm, and suppression of the first mitosis with high-pressure treatments. Gynogenetic diploids have been used for cytogenetic studies of meiotic phenomena and gene mapping. The general finding to date is that the arrangement of genes on chromosomes is high...

Abstract: CURRENT ideas on fish farming have stimulated interest in fish genetics and highlighted the need for more genetic analysis, particularly of fish of commercial importance. A major difficulty in breeding fish of commercial interest is the long generation time of 3 years or more. The work reported here is an investigation of the possibility of producing homozygous clones of fish rapidly by gynogenesis—a special form of artificial parthenogenesis in which activation of the eggs is achieved by fertilisation with genetically inert spermatozoa. Gynogenesis was first observed in frogs by Hertwig (1911), who showed that a low frequency of apparently normal embryos appeared when eggs were fertilised by spermatozoa which had received radium gamma ray doses much higher than levels normally required to produce 100 per cent, abnormality. It was concluded that, at these very high doses, the genetic material of the spermatozoa was so thoroughly destroyed that it played no part in the subsequent parthenogenetic development of the egg. This \"Hertwig effect\" has been confirmed several times in amphibia (reviewed by Beatty, 1964), and, as in other forms of parthenogenesis in vertebrates, the resulting parthenogenomes are usually haploid, and although developing normally at first are grossly abnormal at hatching. However, sporadic occurrences of more normal hatchlings have been observed following parthenogenesis by pricking and these have been shown to be diploid (Parmenter, 1933; Kawamura, 1939). Several mechanisms have been proposed to explain the process of diploidisation (see Tyler, 1941; Beatty, 1964), but the experimental evidence in amphibia seems to favour diploidisation by doubling of the haploid female genome during cleavage. Thus Parmenter (1933) and Kawamura (1939) observed delayed cleavage in parthenogenetic eggs which subsequently produced diploid organisms, and Subtelny (1958) produced diploid individuals following transplantation of haploid nuclei into enucleated eggs, i.e. diploidisation in the absence of polar bodies. Increased frequencies of both haploid and diploid gynogenomes have been reported by Rostand (1934, 1936) following post-fertilisation cold treatments of amphibian eggs; gynogenesis was produced by irradiated spermatozoa and also by fertilisation of eggs with foreign spermatozoa (\" false hybrids \"). Gynogenesis is a natural form of reproduction in the teleost Mollienesia formosa (Hubbs and Hubbs, 1932), and the existence of distinct clones in laboratory fish has been demonstrated by tissue transplantation tests (Kallman, 1962). Perfectly normal and fully viable broods can easily be reared, and this suggests that diploidy is retained through suppression of meiosis Kaliman, bc. cit.) leading to fixed heterozygosity. Recent work in Russia (Romashov, Belyaeva, Golovinskaia and Pro-