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

Improved methods for obtaining, preparing, and staining fish chromosomes are described. Included are procedures for resolving serial or G-type bands. A brief review of various metaphase banding procedures and their use in fishes is also presented.

Improved methods for working with fish chromosomes with a review of metaphase chromosome banding

Mitotic chromosomes, interphase cell nuclei, and male meiosis of 41 species representing all vertebrate classes were analyzed with distamycin A/mithramycin counterstaining. The purpose of the study was to recognize differences and common characteristics in the reverse (R) fluorescent banding patterns in the chromosomes of vertebrate species at various stages of evolution. In contrast to the warm-blooded mammals and birds, the euchromatic segments in the chromosomes of most reptiles, amphibians, and fishes contain no multiple fluorescent R-bands. This is thought to be due to the absence of the long homogeneous regions (isochores) in the DNA of the cold-blooded vertebrates. Distamycin A/mithramycin banding specifically reveals the GC-rich constitutive heterochromatin in all vertebrates. In most of the vertebrate chromosomes examined, the heterochromatic regions have opposite staining properties with mithramycin and quinacrine. Mithramycin labels the nucleolus organizer regions very brightly in the karyotypes of fishes, amphibians, reptiles and birds, but not of mammals. The lack of mithramycin fluorescence at the nucleolus organizer regions of mammals is attributed to the relatively low level of redundancy of the GC-rich ribosomal DNA in their genomes. Studies on the various meiotic stages of the cold-blooded vertebrates show that the mithramycin labeling of the nucleolus organizers is independent of their state of activity. This can be confirmed by mithramycin fluorescence at the nucleoli of actinomycintreated cells.

Evolutionary diversity of reverse (R) fluorescent chromosome bands in vertebrates

A 13-year study of the population dynamics and growth of the European eel Anguilla anguilla in a Norwegian river: evidence for density-dependent mortality, and development of a model for predicting yield

Catadromous eels enter fresh water as sexually undifferentiated glass eels and develop into males and females before migrating back to sea as silver eels. Females develop ovaries directly from the ambiguous primordial gonad whereas males pass through a transitional intersexual stage before developing testes. Eels have sex-specific life-history strategies. Males may grow faster than females initially, but this difference is soon reversed and females attain a greater age- and size-at-metamorphosis than males. Male fitness is maximized by maturing at the smallest size that allows a successful spawning migration (a time-minimizing strategy) whereas females adopt a more flexible size-maximizing strategy that trades off pre-reproductive mortality against fecundity. Although heteromorphic sex chromosomes have been identified in some species, the sex of developing gonads is labile and gender is determined principally by environmental factors. Individuals experiencing rapid growth prior to gonad differentiation tend to develop as males, whereas eels that grow slowly initially are more likely to develop as females. Paradoxically, males tend to predominate under conditions of high density, which may be because a male “grow quickly, mature early” strategy increases an individual’s chances of survival during periods of intraspecific competition. High temperatures and saline conditions have also been proposed to favor development as males but experimental studies have failed to demonstrate a clear effect of either on sex determination. High proportions of female silver eels migrating from some upstream areas, lakes and large rivers may be due to low population density or poor conditions for growth in these habitats. Manipulating sex ratios in favor of females has the potential to increase eel production in aquaculture and to buffer natural populations against fishing pressure. Sex steroids (oestrogens and phytoestrogens) have a strong feminizing effect on undifferentiated individuals and are most effective when targeted at younger eels and administered at high doses for prolonged periods. Modifying local environmental conditions, in particular reducing eel density and limiting interference and social stress, may also promote the development of females. Further research into the timing and mechanisms of sex determination in eels is required to effectively and efficiently manipulate sex for conservation and/or economic benefit.

Sex Determination in Freshwater Eels and Management Options for Manipulation of Sex

Metaphase chromosomes from cultured blood cells of female, male, and hermaphroditic European eels were analyzed. In addition, both gonads from each of the specimens were examined microscopically to ensure correct sexing. The karyological investigation revealed that in some of the specimens a heteromorphic chromosome pair was present. This heteromorphism appeared in both sexes and in the hermaphrodite. C-banding and silver nitrate staining demonstrated that the heteromorphism was due to quantitative differences in constitutive heterochromatin and nucleolar organizing regions in the short arm of chromosome 8. In G-banded preparations it was demonstrated that, except for the heteromorphism mentioned, the karyotypes from both sexes and the hermaphrodite were identical. With the G-band technique it was also easily demonstrated that both the largest metacentric (No. 1) and the smallest metacentric (No. 11) had homologs. Therefore, in contrast to some earlier reports which claimed that these two chromosomes were a heteromorphic pair of sex chromosomes, it is concluded that Anguilla anguilla has no heteromorphic sex chromosomes. The implication of these findings are discussed in relation to the many reports of strongly skewed sex ratios found in commercial eel farms. It is tentatively hypothesized that sex determination in A. anguilla may be metagamic and that sex inversion may occur in this species.

Sex determination in the European eel (Anguilla anguilla, L.). A hypothesis based on cytogenetic results, correlated with the findings of skewed sex ratios in eel culture ponds.

U.H. Wiberg

Papers

Sex determination in the European eel (Anguilla anguilla, L.). A hypothesis based on cytogenetic results, correlated with the findings of skewed sex ratios in eel culture ponds.