In fish, pre-embryonic events such as insemination, second polar body extrusion and first mitotic cleavage are manipulable and render 37 different types of ploidy induction possible. A classification of physical, chemical, and biological inductors of ploidy is provided. The amazing ability of fish to tolerate genomes from haploid to heptaploid, genomic contributions from the male or female parent alone, and unequal contributions from parents belonging to the same or different species is highlighted; surprisingly, a single species is amenable for 8–12 different types of ploidy induction. Advantages and limitations of different methods and live or preserved tissues for ploidy confirmation are assessed. Live haploids have been induced in Oreochromis mossambicus. With an ability to synthesize rRNAs and metabolic enzymes such as LDH, the haploid embryos are capable of normal translation and transcription, but suffer mass mortality at hatching, perhaps due to expression of lethal mutant genes. Induction of gynogenesis involves egg activation by irradiated homologous or heterologous sperm, and diploidization by retention of the second polar body (meiotic gynogenesis), or suppression of the first mitotic cleavage (mitotic gynogenesis). UV-irradiation inactivates sperm DNA maximally and avoids chromosome fragmentation. Egg activation by UV-irradiated heterologous sperm under dark conditions, and diploidization by pressure shock result in the highest survival of gynogens; meiotic gynogens survive better than mitotic gynogens. The need for confirmation of genetic purity of mitotic gynogens by one or more methods is emphasized. In different species, survival, growth and fertility improve when gynogens are generated successively for two or more generations. Combinations of induction of ploidy and hormonal sex reversal in gynogens renders the scope for generating all-male or all-female populations. In some gynogenetic species genetic homozygosity leads to growth suppression from 3 to 60% however, meiotic gynogens of Clarias macrocephalus and Paralicthys olivaceus display 18 and 35% faster growth. Hypotheses for the unexpected occurrence of males among natural and artificially induced gynogenetic populations are assessed. In a few species, reproductive performance of gynogens is not equivalent to normal females. Maximal elimination of egg genome by UV-radiation, induction of androgenesis using 2n sperm of a tetraploid, facilitation of dispermy using heterologous eggs, and activation by cryopreserved sperm are land-mark events in the history of androgenesis. In male-heterogametic species, androgenesis may produce supermales to establish broodstock for consistent production of all-male populations. In combination with cryopreservation of sperm, androgenesis may prove to be the best method for conservation of fish genomes. As many as 11 different types of triploids are inducible; and most of them require the retention of the second polar body. The yield and survival of triploids are higher than those of gynogens and these values decrease with the kind of inductor used in the following order: pressure < cold < heat shock. Usually triploidy confers sterility, especially in females, and accelerates growth during post-maturation, when reared solitarily in some (Tinca tinca, Oreochromis Mossambicus) or communally (with diploids) in other species (Oncorhynchus mykiss). The presence of two sets of maternal chromosomes does not disturb the manifestation of morphological sexual characters; however, unexpected sex ratios observed in some triploids indicate a preponderance of females in natural populations and a dominance of males in artificially induced populations. In some species, a certain percentage of female triploids are fertile. Possible pathways, through which the natural fertile triploids may form the base for evolution of tetraploids and origin of diploid new species are suggested. Triploids, especially females, suffer delayed maturity, a low gonado-somatic index, and disrupted gametogenesis due to cytogenetic and endocrine incompatibilities; males do not suffer endocrine incompatibility. Three possible pathways, through which oogenesis may be completed in these fertile triploid species, are indicated. Triploid hybridization between salmonids, which can tolerate salinity changes (e.g. chum and pink salmons) and which cannot tolerate early transfer to sea water but are desired for their meat quality (e.g. Atlantic, chinook and coho salmons) is commercially important. Triploid conspecific salmonids grow faster than diploid conspecifics or triploid hybrids. Protocols for the combination of induced ploidy and hormonal sex reversal to generate all-male, all-female or all-sterile triploid populations are described. In triploids, the increase in nuclear DNA per cell is 1.3 to 1.7 times that of diploids, which results in corresponding volumetric increase of a cell. This, in turn, imposes corresponding decreases in total cell surface area and cell number of a tissue or a triploid individual; however, such a decrease in cell surface area does not impair metabolic processes like oxygen uptake and food utilization. Data for effective temperature required to induce cold or heat shock to retain the second polar body suggest that an elevation of 18, 15 and 14 °C successfully retains the polar body in salmonids, cyprinids and cichlids; the corresponding values for the depression of temperature are 11, 19 and 21 °C, respectively. Seven different kinds of tetraploidy are inducible; however, live, feeding tetraploids have been induced in ten species only. Causes for embryonic or post-embryonic mortality of tetraploids are discussed. Increasing maternal genomic contribution and heterologous insemination appear to enhance tetraploid survival. Survival and growth of Oncorhynchus mykiss progressively improve in F1 and F2 tetraploid progenies. In a rare strain of the loach, Misgurnus anguillicaudatus, pentaploids, hexaploids and heptaploids have been successfully induced. An individual polyploid loach (3n) simultaneously spawns small, intermediate and large eggs carrying n, 2n and 3n genomes. It is not clear how during oogenesis the passage of n, 2n and 3n genomes is regulated into small, intermediate and large eggs. However, evidence from other fish species is accumulating for the simultaneous production of at least two kinds of eggs by a single female. Studies on ploidy induction have shown the magnitude of the complicated genetic mechanisms that control sex determination in fish.

Ploidy induction and sex control in fish

Androgenesis, gynogenesis and the production of clones in fishes: A review

Gynogenesis in common carp (Cyprinus carpio L.): II. The production of homozygous gynogenetic clones and F1 hybrids

The physiological response of common carp, Cyprinus carpio (L.) to increased stocking density and an additional acute net confinement stressor was investigated. Stocking densities were increased from 28.4 to 56.8 or 113.6 kg m?3 by the use of crowding screens and fish were sampled from the crowded groups after 15, 39 and 87 hours of crowding (hc). A transient elevation of plasma cortisol was found in the higher density group after 15 hc before values returned to control levels. Increased stocking density also increased plasma levels of glucose, free fatty acids (FFA) and lactate during the experimental period. No effect was found on oxygen radical production in the blood, haemoglobin or haematocrit levels, but leucocrit values were lower after 39 hc in both groups. Net confinement resulted in a significant increase in plasma cortisol levels in all groups. However, after 15 hc, cortisol and FFA levels in both crowded plus confinement groups were higher than in the control and confined groups. Confinement resulted in increased oxygen radical production in the crowded plus confined groups at all times. Results indicate that although carp responded with a mild stress response to increased stocking density and adaptation occurred by 87 hc, the crowded fish were more sensitive to an additional acute stressor

Increased stocking density influences the acute physiological stress response of common carp Cyprinus carpio (L.)

Half-smooth tongue sole (Cynoglossus semilaevis) is an important cultured marine fish as well as a promising model fish for the study of sex determination mechanisms. In the present study, a protocol for artificial gynogenesis of half-smooth tongue sole was developed in order to identify the sex determination mechanism and to generate all-female stock. The optimal UV-irradiation dose for genetically inactivating sea perch spermatozoa was determined to be > or =30 mJ/cm(2). The optimal initiation time for cold shock of gynogenetic embryos was determined to be 5 min after fertilization, while the optimal temperature and treatment duration were determined to be 20-25 min at 5 degrees C. Chromosomes from common diploids, gynogenetic haploids, and diploids were analyzed. WW chromosomes were discovered in some of the gynogenetic diploids. The microsatellite marker was applied to analyze gynogenetic diploid fry. Among the 30 gynogenetic diploid fry, 11 fry contained only one allele, while 19 contained two alleles, which had the same genotype as their mother. The female-specific DNA marker was observed in four individuals out of ten gynogenetic diploid fry. Ploidy analysis of 20 putative gynogenetic fry showed them all to be diploid. Thus, a protocol for the induction of artificial gynogenesis has been developed for the first time in half smooth tongue sole, and the sex determination mechanism in the tongue sole was determined to be female heterogametic with the ZW chromosome.

J. Duynhouwer

Papers

Gynogenesis in common carp (Cyprinus carpio L.): I. Effects of genetic manipulation of sexual products and incubation conditions of eggs