Polyploidy has long been recognized to have played an important role in the evolution of plant species (Lewis, 1980). However, polyploidy has only recently been recognized as a potentially important process in the evolution of vertebrates (Ohno, 1974; Fisher et al.,1980; Bogart, 1980; Schultz, 1980). Mammals and birds generally possess more DNA per cell than do fish and other chordates. This observation and the existence of many duplicated gene loci have led Ohno and co-workers (Ohno,1967, 1970a, 1974; Ohno et al., 1968) to propose that genome doubling has taken place at least once in the evolution of vertebrates. One tetraploid event apparently took place about 500 million years (Myr) ago in a common ancestor of all vertebrates. Fisher et al. (1980) describe isozyme studies that are consistent with this idea. Other tetraploid events may have taken place in major lineages of vertebrate evolution, possibly including a genome doubling in a reptilian ancestor of mammals (Ohno, 1967; Comings, 1972).

Tetraploidy and the Evolution of Salmonid Fishes

Polyploid fish and shellfish: Production, biology and applications to aquaculture for performance improvement and genetic containment

Induced triploidy is widely accepted as the most effective method for producing sterile fish for aquaculture and fisheries management. Artificially produced triploids generally differ from conspecific diploids in three fundamental ways: they are more heterozygous, they have larger but fewer cells in most tissues and organs, and their gonadal development is disrupted to some extent. Despite these basic biological differences, triploids are similar in most respects to diploids when examined at the whole animal level. The only clear differences relate to the effects of impaired gametogenesis on the reproductive physiology and behavior of triploids, especially in females. Other apparent differences include reduced aggressiveness, occasional specific morphological abnormalities, and inferior performance when reared under suboptimal conditions. The causes of these latter two problems are poorly understood but must be addressed if triploids are to be used more extensively.

The Physiology and Behavior of Triploid Fishes

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

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.

8 Chromosome Set Manipulation and Sex Control in Fish

Flow cytometry: Assaying experimental polyploid fish and shellfish

Polyploidy induced in the American oyster, Crassostrea virginica , with cytochalasin B.

Erythrocyte nuclear volumes were measured in brook trout (Salvelinus fontinalis) from Phillips Hatchery in Maine to document naturally occurring polyploid sterility. In eight brook trout in which gonads were lacking or undeveloped, the nuclei were large, suggesting polyploidy. The average size of the erythrocyte nuclei in other sterile fish fell within the normal range but all of the eight fish appeared to have some erythrocytes that were polyploid. All polyploids appeared to be mosaics, containing diploid, triploid, tetraploid, or pentaploid cells. The cause of the polyploidy was not determined but may have been caused by the inadvertent exposure of the eggs to low temperatures after fertilization.

Reproductive Sterility in Polyploid Brook Trout, Salvelinus fontinalis

Polyploid mosaicism was induced in landlocked Atlantic salmon by treatment of fertilized eggs with 10 mg/liter solution of cytochalasin B. On the basis of nuclear size in 3,025 erythrocytes and chromosome counts we judged the treated fish to be polyploid mosaics of diploid, triploid, and tetraploid cells. The dominant polyploid cell type in test fish was triploid rather than tetraploid, in contrast to previous reports of Atlantic salmon exposed to cytochalasin B. Control groups had mainly diploid cells but a few triploid cells were measured in most individuals.

Polyploid Mosaics Induced by Cytochalasin B in Landlocked Atlantic Salmon Salmo salar

Flow cytometry was used to identify polyploid fish by means of measuring deoxyribonucleic-acid fluorescence in erythrocytes. One polyploid grass carp Ctenopharyngodon idella and one polyploid bighead carp Hypophthalmichthys (=Aristichthys) nobilis were identified among 38 fish of each species. Ten hybrids identified as diploids on the basis of morphology were confirmed to be diploid. All hybrids identified as polyploid morphologically were indeed polyploid, but two of these were tetraploid rather than triploid. Received May 14, 1982 Accepted February 15, 1983

Standish K. Allen

Papers

Flow cytometry: Assaying experimental polyploid fish and shellfish

Polyploidy induced in the American oyster, Crassostrea virginica , with cytochalasin B.

Reproductive Sterility in Polyploid Brook Trout, Salvelinus fontinalis

Polyploid Mosaics Induced by Cytochalasin B in Landlocked Atlantic Salmon Salmo salar

Ploidy of Hybrid Grass Carp X Bighead Carp Determined by Flow Cytometry