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

This paper is aimed at underlining the limited knowledge available about the physiology of triploid fishes compared with diploids. Whereas many aspects (induction, detection, growth, resistance to diseases, etc.) in the production and rearing of triploid fishes have widely been developed and described in the literature, other numerous questions of ecophysiology remain in abeyance, and the study of triploid cells physiology is still in its infancy. Triploid fishes can be considered as models worthwhile for physiological investigations not only for the economical stake in relation to the development of triploid fishes rearing, but also for the cytological and molecular features of their tissues and organs. The functional implications of these features have been poorly studied although they are potential areas of applied and/or fundamental studies.

The physiology of triploid fish: current knowledge and comparisons with diploid fish

This review deals with major areas of triploidy research in fish. It includes not only methods for induction and detection of triploidy but also the impact of triploidy on morphology, anatomy, growth, haematology, energetics, behaviour, endocrinology and gonads in various species of fish, studied so far. The future prospects of research on triploid fish are discussed inviting researchers with diverse areas of interest in fish biology.

The biology of triploid fish

The eyed embryos of raindow trout between diploid female and triploid male were obtained. The chromosome preparations of these embryos were made by the short-term culture method. There were two groups, namely 72 -79 of chromosomal numbers (18 individuals) and 100-108 (3 individuals). Judging from numbers and constitutions of the chromosomes, it is presume that the chromosomes of the triploid during spermatogenesis divide into two by near the common way.

Cytogenetical characteristics of the embryos between diploid female and triploid male in rainbow trout

Triploid Rainbow Trout Induced by High-pH•High-calcium

組織培養した紡錘形細胞肉腫におよぼすマイトマイシンの影響を細胞学的, 細胞化学的に研究した。マイトマイシンは特に静止期にある細胞に第一義的に作用し核のDNAおよび仁のRNAの構成に変化を起こし, 細胞の機能を乱し崩壊に導くようである。核は形態的に著しい変化を受けると同時に, その染色質は染色性を失う。特に核染色質の異常な変形と変質を引き起こす。仁もまたその形態, 染色性に著しい変化を起こし, ついには仁物質は細胞質内に流出する。細胞質には多数の空胞が現われる。分裂期の細胞においては染色体に著しい切断や染色体橋の形成がみられ, 分裂ははなはだしく乱される。仁に著しい変性を起こす点においてマイトマイシンの効果は特異である。

Some cytological and cytochemical studies on effects of “mitomycin-X” on the spindle-cell sarcoma in vitro (A preliminary report)

Gynogenetic haploids were recognized in the abnormal embryos induced by an applica-tion of Pressure shock shortly after fertilization between eggs of masu salmon Oncorhynchus masou and sperms of kokanee O. nerka. In rainbow trout, the haploids were recognized in the abnormal embryos induced by only fertilization and by an application of thermal or pressure shocks shortly after fertilization. In the strain of rainbow trout used here, the XX/XY-sex chro-mosome system were observed. Because all haploids recognized in this study had X chromosomes, their genomes may consist of the maternal chromosomes. The reason why the haploids de-veloped was not clear in this study.

The origin of the genome of haploid Masu salmon and rainbow trout recognized in abnormal embryos.

Silver-banded karyotypes of the rainbow trout Salmo gairdneri, the brook trout Salvelinus fontinalis, and their hybrids were described. Diploid-type and triploid-type hybrids were obtained. Triploid-type hybrids had two maternal genomes of the rainbow trout and one paternal genome of the brook trout. The rainbow trout had one pair of M or SM with Ag-NORs near the satellite. In the brook trout, Ag-NORs were observed in four pairs of chromosomes, but each cell had different number of chromosomes with Ag-NORs. In all observed cells of triploid-type hybrids, Ag-NORs originated from the brook trout were not recognized, whereas those from the rainbow trout were found.

Silver-banded karyotypes of the rainbow trout and the brook trout and their hybrids: disappearance in allotriploids of Ag-NORs originated from the brook trout.

Jindo Kobayashi

Papers

Cytogenetical characteristics of the embryos between diploid female and triploid male in rainbow trout

Triploid Rainbow Trout Induced by High-pH•High-calcium

Some cytological and cytochemical studies on effects of “mitomycin-X” on the spindle-cell sarcoma in vitro (A preliminary report)

The origin of the genome of haploid Masu salmon and rainbow trout recognized in abnormal embryos.

Silver-banded karyotypes of the rainbow trout and the brook trout and their hybrids: disappearance in allotriploids of Ag-NORs originated from the brook trout.