Successful Induction of Intra-specific Androgenesis in Widow Tetra, Gymnocorymbus ternetzi (Boulenger 1895) Using UV Irradiation
TL;DR: A protocol for elimination of the maternal egg genome in widow tetra (WT), Gymnocorymbus ternetzi (Boulenger 1895) using contrasting black and albino strains was successfully optimised and inactivated the maternal nuclear genome in eggs of the black WT.
Abstract: A protocol for elimination of the maternal egg genome in widow tetra (WT), Gymnocorymbus ternetzi (Boulenger 1895) using contrasting black and albino strains was successfully optimised. Maternal genome elimination is a pre-requisite for inducing androgenesis. In the present study, UV-irradiation for 3 min inactivated the maternal nuclear genome in eggs of the black WT. A single layer of black WT eggs exposed to a UV-G bulb (254 nm, 40 W) at a height of 25 cm and at a final intensity of 4.2 W. m -2 successfully eliminated the WT egg genome. Androgenotes were generated by fusing genome-inactivated eggs with fresh sperm. Fertilisation was achieved by mixing fresh albino tetra sperm with irradiated eggs and adding water to initiate sperm motility. Following fertilisation by fresh albino WT sperm, 22- min-old embryos were shocked at 41 °C for 2 min to restore diploidy. Survival of androgenotes was 11% and 8% at hatching and maturity while that of the controls were 94% and 67%, respectively. Maternal genome inactivation was confirmed by (i) albino body colour in the diploid fry and adult, (ii) haploid karyotype and (iii) embryonic development. Hatched haploid androgenotes showed normal embryonic development but exhibited severe haploid syndrome while diploid androgenotes resembled albino WT and displayed a diploid karyotype.
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TL;DR: Embryos when exposed to samples containing Dibenzothiophene, Phenanthrenes and Pyrenes at varying concentrations exhibited defective embryonic and larval development, but embryonic defects were reversible when transferred to clean water immediately after <24hrs of exposure.
Abstract: Polycyclic aromatic hydrocarbons (PAHs) present in crude oil effluents, when released into aquatic ecosystem pose a major threat to fishes and humans. We investigated the harmful effects of PAHs on early embryonic and larval development of Gymnocorymbus ternetzi. Embryos when exposed to samples containing Dibenzothiophene (3 rings), Phenanthrenes (3 rings) and Pyrenes (4 rings) at varying concentrations (15–90 µg/L) exhibited defective embryonic and larval development. As PAHs targets aryl hydrocarbon receptor (AhR) pathway, cardiac deformities were persistent in embryos exposed to PAHs for >48 hrs; concentration specific defects included arrested embryonic development (36 ± 2.6%), cytoplasmic blubbing (24%), pericardial edema and cardiac looping (43 ± 4.2%) and tail bents and body curvature (19%). However, embryonic defects were reversible (>47%) when transferred to clean water immediately after
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Cites background or methods from "Successful Induction of Intra-speci..."
...For embryos, gametes were collected from brooders for artificial fertilization following established protocols [5-12]....
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...Tetras [10-16] are attractive models for ecotoxicology research....
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...Tetras as Ecotoxicology model: Tetras [10-16] are attractive models for ecotoxicology research....
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...Tetras are excellent vertebrate models for developmental biology, aquatic toxicology and biomedical research owing to genetic homology and visceral organs similarity to humans, optically transparent embryos and larvae that remain amenable to several genome editing technologies, high fecundity (>300 eggs) enabling sibling studies, external and shorter embryonic developmental duration (24 hrs), defined embryonic stages, with fully functional larvae in 3 days post hatching (dph), early (4 months) sexual maturity, and vulnerability to toxic aquatic pollutants attributes to their ability as a humanized vertebrate model [9-11] and International Journal of Fisheries and Aquatic Studies for serving as an in vivo screening tool for identifying novel therapeutic targets in human diseases [5, 7-9]....
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...Tetras also displayed similar phenotypic defects especially pericardial and yolk sac edema which were more pronounced with increasing duration of exposure while lordosis and scoliosis appeared only after >72 hr exposure (Figure....
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Journal Article•
TL;DR: Progeny testing crosses between F0 androgenetic males (Y2Y2) and heterozygous control females (X1X2) generated 96-99% males and 2-4% unexpected females confirming the role of paternal autosomes upon sex determination, and reasons for delayed maturity, reduced reproductive performance and generation of unexpected females by progeny testing are discussed.
Abstract: Production of androgenetic clones using preserved sperm is advantageous in resurrecting a lost population and germplasm conservation. Generation of androgenetic clones to produce 100% males in economically important species is a desirable practice in aquaculture. However, reasons for failure of the clones to generate all male population remains unknown. We investigated reproductive performances of androgenetic widow tetra, Gymnocorymbus ternetzi generated using cryopreserved albino widow tetra (WT) sperm and their ability to generate all male population were assessed. Survival of albino androgenotes at hatching and sexual maturity was 11% and 8%, respectively. Gonado-somatic-index (GSI) and fecundity of F0 androgenetic females (X2X2) was significantly (P
2 citations
TL;DR: The results show a principal possibility of inactivation of ovicells by UV irradiation and use of such cells for producing androgenetic progeny of acipenserids.
Abstract: The results are presented on haploid androgenesis in Siberian sturgeon and sterlet induced by UV irradiation of ovicells. During irradiation, the cells in Ringer solution were rotated around a UV lamp. The efficiency of genetic inactivation of ovicells was estimated by the following parameters: manifestation of Hertwig effect, the fraction of embryos demonstrating haploid syndrome at final developmental stages, by arrest of embryonic development in hybrids Siberian sturgeon × great sturgeon, and by absence of maternal alleles of microsatellite loci in embryos. The dose-effect curve suggests that, during UV irradiation of ovicells of Siberian sturgeon, the complete genetic inactivation is attained at exposition of 120 s, while that in sterlet is 90 or 105 s. The results show a principal possibility of inactivation of ovicells by UV irradiation and use of such cells for producing androgenetic progeny of acipenserids.
1 citations
Cites background from "Successful Induction of Intra-speci..."
...Such a pattern was shown in studies on different fish species (Myers et al., 1995; AbdelHakim et al., 2000; Karayücel and Karayücel, 2001; Kirankumar and Pandian, 2003; Christopher et al., 2012; David and Marimuthu, 2014)....
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..., 2008), black tetra (David and Marimuthu, 2014), and in some other species....
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...…carp (Bongers et al., 1994), Nile tilapia (Myers et al., 1995), clariid catfish and stinging catfish (Bongers et al., 1995; Christopher et al., 2012), Sumatra barb (Kirankumar and Pandian, 2003), dace (Kucharczyk et al., 2008), black tetra (David and Marimuthu, 2014), and in some other species....
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References
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TL;DR: 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.
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.
321 citations
"Successful Induction of Intra-speci..." refers methods in this paper
...For each respective treatment group, samples of freshly hatched fry were transferred into 0.01 % colchicine solution for 6 h. Karyotyping was conducted following the protocol of Kligerman and Bloom (1977)....
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TL;DR: 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, and the need for confirmation of genetic purity of mitotic gynogens by one or more methods is emphasized.
Abstract: 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.
285 citations
"Successful Induction of Intra-speci..." refers methods in this paper
...As both parents in the present study had the same chromosome number, other phenotypic characters including pigmentation and embryonic developmental duration were used as markers of paternal parent (Pandian and Koteeswaran 1998)....
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01 Jan 2000
158 citations
"Successful Induction of Intra-speci..." refers background in this paper
...Hence, this process of enzymatic photo-reactivation can restore the mono pyrimidines (Voet and Voet 1990) to prevent photo-reactivation of the inactivated maternal genome of the egg....
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TL;DR: The survival of androgeneticZebrafish suggests that if paternal imprinting occurs in zebrafish, it does not result in essential genes being inactivated when their expression is required for development, and production of haploid androgenotes can be used to determine the meiotic recombination rate in male zebra fish.
Abstract: To help investigate the evolutionary origin of the imprinting (parent-of-origin mono-allelic expression) of paternal genes observed in mammals, we constructed haploid and diploid androgenetic zebrafish (Danio rerio). Haploid androgenotes were produced by fertilizing eggs that had been X-ray irradiated to eliminate the maternal genome. Subsequent inhibition of the first mitotic division of haploid androgenotes by heat shock produced diploid androgenotes. The lack of inheritance of maternal-specific DNA markers (RAPD and SSR) by putative diploid and haploid androgenotes confirmed the androgenetic origin of their genomes. Marker analysis was performed on 18 putative androgenotes (five diploids and 13 haploids) from six families. None of 157 maternal-specific RAPD markers analyzed, some of which were apparently homozygous, were passed on to any of these putative androgenotes. A mean of 7.7 maternal-specific markers were assessed per family. The survival of androgenetic zebrafish suggests that if paternal imprinting occurs in zebrafish, it does not result in essential genes being inactivated when their expression is required for development. Production of haploid androgenotes can be used to determine the meiotic recombination rate in male zebrafish. Androgenesis may also provide useful information about the mechanism of sex determination in zebrafish.
143 citations
"Successful Induction of Intra-speci..." refers result in this paper
...Colour and appearance of the corresponding pigments have been used in earlier studies to confirm paternity of androgenetic progeny (e.g. Corley-Smith et al. 1996; Bercsenyi et al. 1998; David and Pandian 2006a, b)....
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TL;DR: The genetic characteristics in androgenetic rainbow trout (Oncorhynchus mykiss) and their progeny suggest that both XX and YY androgenetics individuals may develop as males.
Abstract: We analyzed a number of genetic characteristics in androgenetic rainbow trout (Oncorhynchus mykiss) and their progeny. The androgenetic progeny of individual androgenetic males appeared genetically identical to each other based on eight enzyme loci. Their viability was no higher than that of androgenetic progeny of outbred males. Homozygous androgenetic female rainbow trout produced very poor quality eggs. When common eggs and sperm from outbred individuals were used to produce androgenetic and gynogenetic progeny, the yield of gynogenetic progeny was higher but some were heterozygous at protein loci, while no androgenetic progeny were heterozygous. Some androgenetic diploid rainbow trout were successfully produced from cryopreserved sperm. The progeny of some androgenetic males crossed to normal females were virtually all males, while the progeny of other males were virtually all females. This suggests that both XX and YY androgenetic individuals may develop as males. Androgenesis is likely to be useful for generating homozygous clones for research and for recovering strains from cryopreserved sperm.
112 citations
"Successful Induction of Intra-speci..." refers background in this paper
...Androgenesis can also be induced in postmortem preserved (David and Pandian 2006a) or cryopreserved spermatozoa (Scheerer et al. 1991), which may be useful in short-term preservation of valuable breeders for aquarists....
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