Massive production of all-female diploids and triploids in the crucian carp

In many species of aquaculture importance, all-female and sterile populations possess superior productivity due to faster growth and a relatively homogenous size of individuals. However, the production of all-female and sterile fish in a large scale for aquaculture is a challenge in practice, because treatments necessary for gynogenesis induction usually cause massive embryonic and larval mortality, and the number of induced gynogens is too small for their direct use in aquaculture. Here we report the massive production of all-female triploid crucian carp by combining artificial gynogenesis, sex reversal and diploid-tetraploid hybridization. Previously, we have obtained an allotetraploid carp population (4n = 200) by hybridization between red crucian carp (Carassius auratus red var; ♀) and common carp (Cyprinus carpio; ♂). We induced all-female diploid gynogens of the Japanese crucian carp (Carassius cuvieri; 2n = 100). We also generated male diploid gynogens of the same species treated gynogenetic fry with 17-α-methyltestosterone, leading to the production of sex-revered gynogenetic males. Finally, these males were used to cross with the female diploid Japanese crucian carp gynogens and the allotetraploid females, resulting in the production of fertile all-female diploid Japanese crucian carp (2n=100) and sterile all-female triploid hybrids (3n = 150), respectively. Therefore, diploid crucian carp gynogenetic females and sex-reversed male together with an allotetraploid line provide an opportunity to produce all-female triploid populations in a large scale to meet demands in aquaculture industry.     

Induction of meiotic gynogenesis in ship sturgeon Acipenser nudiventris using UV-irradiated heterologous sperm

Diploid gynogenesis was induced in ship sturgeon Acipenser nudiventris using UV-irradiated sperm from Siberian sturgeon Acipenser baerii. The optimal condition for the retention of the second polar body in ship sturgeon was determined to be 10 min after activation/fertilization in experiments. The temperature of cold shock and its duration were 2.5 °C and 30 min, respectively. A total of 30 gynogens of known parentage from experimental treatments were screened using microsatellite DNA analysis, and uniparental transmission in meiogens was confirmed. The results show that heterologous Siberian sturgeon sperm is applicable as UV-irradiated sperm for the induction of gynogenesis in ship sturgeon. This technique may recover the critically endangered sturgeon species that are becoming extinct.     

Production and propagation of fully inbred clonal lines in the Nile tilapia (Oreochromis niloticus L.).

Fully inbred clonal lines of fish are likely to be of great value in research on immunology, sex determination, quantitative genetics, and toxicology. In this study on the Nile tilapia (Oreochromis niloticus), gynogenesis or androgenesis were used to produce a first generation of completely inbred fish, from which clonal lines were established using gynogenesis, androgenesis, hormonal sex reversal and intraline crosses. The clonal nature of these lines was verified by using multilocus DNA fingerprinting and the isozyme locus ADA*. Although these lines might be expected to be monosex in nature (all-female XX or all-male YY depending on the clone), one line did contain both sexes of fish. The presence of males in this gynogenetic clonal line and data from progeny testing of these males suggested that this line was homozygous for an allele or combination of alleles at an autosomal locus or loci which caused female to male sex reversal but with limited penetrance. Outbred clonal lines were also produced by crossing between different inbred clones. J. Exp. Zool. 284:675-685, 1999.     

Induction of gynogenesis in muskellunge with irradiated sperm of yellow perch proves diploid muskellunge male homogamety

Diploid gynogenesis was induced in muskellunge Esox masquinongy using UV-irradiated muskellunge sperm as the first step in producing monosex females. In this approach, we have to rely on negative controls as an indirect reference for sperm genetic material destruction. In the first experiment, equal proportions of gynogenetic females and males were produced. Negative controls, UV-irradiated sperm without heat shock, yielded some normal hatching larvae, described as spontaneous diploids. In the second experiment, muskellunge eggs were activated using sperm from yellow perch. Because hybrids between these species are not viable, we produced unambiguous gynogens. When UV-irradiated yellow perch sperm was used to inseminate muskellunge eggs, haploids resulted (22.5% +/- 2.8% survival to the eyed stage). To produce diploid gynogens, a heat shock of 31 degrees C was applied to inseminated eggs 20 min after activation for a duration of 6 min. This process yielded several hundreds of gynogens for rearing. Several treatments of masculinizing hormone, 17alpha-methyltestosterone (MT), were carried out. Fish were dissected and gonads examined histologically for sex determination. Gynogens produced using yellow-perch sperm confirmed the presence of males in the control group, whereas the MT bath treatment (400 microg/liter) resulted in the production of fish with ovotestis. These results provide evidence for male homogamety in muskellunge and imply that a change of strategy is needed to produce monosex populations.     

Distant hybridization leads to different ploidy fishes

Distant hybridization makes it possible to transfer the genome of one species to another, which results in changes in phenotypes and genotypes of the progenies. This study shows that distant hybridization or the combination of this method with gynogenesis or androgenesis lead to different ploidy fishes with genetic variation, including fertile tetraploid hybrids, sterile triploid hybrids, fertile diploid hybrids, fertile diploid gynogenetic fish, and their derived progenies. The formations of the different ploidy fishes depend on the genetic relationship between the parents. In this study, several types of distant hybridization, including red crucian carp (Carassius auratus red var.) (2n=100, abbreviated as RCC) (♀)×common carp (Cyprinus carpio L.) (2n=100, abbreviated as CC) (♂), and RCC (2n=100) (♀)×blunt snout bream (Megalobrama amblycephala) (2n=48, abbreviated as BSB) (♂) are described. In the distant hybridization of RCC (♀)×CC (♂), bisexual fertile F3–F18 allotetraploid hybrids (4n=200, abbreviated as 4nAT) were formed. The diploid hybrid eggs and diploid sperm generated by the females and males of 4nAT developed into diploid gynogenetic hybrids and diploid androgenetic hybrids, respectively, by gynogenesis and androgenesis, without treatment for doubling the chromosome. Improved tetraploid hybrids and improved diploid fishes with genetic variation were derived from the gynogenetic hybrid line. The improved diploid fishes included the high-body RCC and high-body goldfish. The formation of the tetraploid hybrids was related to the occurrence of unreduced gametes generated from the diploid hybrids, which involved in premeiotic endoreduplication, endomitosis, or fusion of germ cells. The sterile triploid hybrids (3n=150) were produced on a large scale by crossing the males of tetraploid hybrids with females of diploid fish (2n=100). In another distant hybridization of RCC (♀)×BSB (♂), different ploidy fishes were obtained, including diploid bisexual fertile natural gynogenetic fish (2n=100), sterile triploid hybrids (3n=124), and bisexual fertile tetraploid hybrids (4n=148). Furthermore, two kinds of pentaploid hybrids (5n=172 and 5n=198) were formed. The biological characteristics and the mechanisms of formation of the different ploidy fish were compared and discussed at the cellular and molecular level. The results indicated distant hybridization or the combination of this method with gynogenesis or androgenesis affects the formation of different ploidy fish with genetic variation.     

Chromosome pairing and chiasma formation in autopolyploids of different Lathyrus species.

Chromosome pairing and chiasma formation were studied in natural and induced tetraploids (2n = 28) of Lathyrus odoratus (induced), Lathyrus pratensis (natural and induced), Lathyrus sativus (induced), and Lathyrus venosus (natural), as well as in triploids of L. pratensis and diploids of L. odoratus, L. pratensis, and L. sativus. All natural tetraploids appeared to be autotetraploids and their meiotic metaphase I behaviour was very similar to that of the induced autotetraploids, with average numbers of pairing partner switches exceeding 4 or even 5. Multivalent frequencies were high, but the numbers of chiasmata were not much higher than necessary to maintain the configurations. Interstitial chiasmata were common, but not predominant. Fertility was reduced, but sufficient for predominantly vegetatively reproducing species. The triploids of L. pratensis had an even higher multivalent frequency than the tetraploids, but still produced some viable progeny at or close to the tetraploid level, suggesting that in mixed populations of diploids and tetraploids, triploids can contribute to gene flow between the ploidy levels. There was no significant correlation between chiasma frequency and ring bivalent frequency in the diploids and multivalent frequency in the corresponding tetraploids. In the tetraploids, chiasma frequency and multivalent frequency were negatively correlated.     

Development and application of biological technologies in fish genetic breeding

Fish genetic breeding is a process that remolds heritable traits to obtain neotype and improved varieties.For the purpose of genetic improvement,researchers can select for desirable genetic traits,integrate a suite of traits from different donors,or alter the innate genetic traits of a species.These improved varieties have,in many cases,facilitated the development of the aquaculture industry by lowering costs and increasing both quality and yield.In this review,we present the pertinent literatures and summarize the biological bases and application of selection breeding technologies(containing traditional selective breeding,molecular marker-assisted breeding,genome-wide selective breeding and breeding by controlling single-sex groups),integration breeding technologies(containing cross breeding,nuclear transplantation,germline stem cells and germ cells transplantation,artificial gynogenesis,artificial androgenesis and polyploid breeding)and modification breeding technologies(represented by transgenic breeding)in fish genetic breeding.Additionally,we discuss the progress our laboratory has made in the field of chromosomal ploidy breeding of fish,including distant hybridization,gynogenesis,and androgenesis.Finally,we systematically summarize the research status and known problems associated with each technology.     

The role of triploid hybrids in the evolutionary dynamics of mixed-ploidy populations

Theory suggests that the evolution of autotetraploids within diploid populations will be opposed by a minority-cytotype mating disadvantage. The role of triploids in promoting autotetraploid establishment is rarely considered, yet triploids are often found in natural populations and are formed in experimental crosses. Here, I evaluate the effects of triploids on autotetraploid evolution using computer simulations and by synthesizing research on the evolutionary dynamics of mixed-ploidy populations in Chamerion angustifolium (Onagraceae). Simulations show that the fate of a tetraploid in a diploid population varies qualitatively depending on the relative fitness of triploids, the ploidy of their gametes and the fitness of diploids relative to tetraploids. In general, even partially fit triploids can increase the likelihood of diploid–tetraploid coexistence and, in some cases, facilitate tetraploid fixation. Within the diploid–tetraploid contact zone of C. angustifolium , mixed populations are common (43%), and often (39%) contain triploids. Greenhouse and field studies indicate that triploid fitness is low (9% of diploids) but variable. Furthermore, euploid gametes produced by triploids can be x , 2 x or 3 x and contribute the majority (62%) of new polyploids formed in each generation (2.3 × 10⁻³). Although triploid bridge, alone, may not account for the evolution of autotetraploidy in C. angustifolium , it probably contributes to the prevalence of mixed-ploidy populations in this species. Therefore, in contrast to hybrids in homoploid species, triploids may actually facilitate rather than diminish the fixation of tetraploids by enhancing the rate of formation.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 537–546.     

Haematological characterization of loach Misgurnus anguillicaudatus: comparison among diploid, triploid and tetraploid specimens

The purpose of this study was to determine whether diploid, triploid and tetraploid loach (Misgurnus anguillicaudatus) differed in terms of their main haematological and physiological characteristics. Diploid and tetraploid fish were produced by crossing of natural diploids (2n x 2n) and natural tetraploids (4n x 4n), respectively. Triploid fish were produced by hybridization between diploid males and tetraploid females. The blood cells were significantly larger in polyploids, and the volumetric ratios of erythrocytes and leucocytes (thrombocyte and neutrophil) in tetraploids, triploids and diploids were consistent with the ploidy level ratio of 4:3:2. No significant differences were observed in haematocrit among polyploids. The erythrocyte count decreased with increased ploidy level, while total haemoglobin, mean cell volume, mean cellular haemoglobin content, and mean cell haemoglobin concentration all increased with increase in ploidy level. Erythrocyte osmotic brittleness declined in polyploids so that polyploid erythrocytes were more resistant to osmotic stress than diploid ones. Overall, loach with higher ploidy levels showed evidence of some advantages in haematological characteristics.     

Androgenesis,gynogenesis, and parthenogenesis haploids in cucurbit species

Haploids and doubled haploids are critical components of plant breeding. This review is focused on studies on haploids and double haploids inducted in cucurbits through in vitro pollination with irradiated pollen, unfertilized ovule/ovary culture, and anther/microspore culture during the last 30 years, as well as comprehensive analysis of the main factors of each process and comparison between chromosome doubling and ploidy identification methods, with special focus on the application of double haploids in plant breeding and genetics. This review identifies existing problems affecting the efficiency of androgenesis, gynogenesis, and parthenogenesis in cucurbit species. Donor plant genotypes and surrounding environments, developmental stages of explants, culture media, stress factors, and chromosome doubling and ploidy identification are compared at length and discussed as methodologies and protocols for androgenesis, gynogenesis, and parthenogenesis in haploid and double haploid production technologies.     

A Complementary Method for Production of Tetraploid Crassostrea gigas Using Crosses Between Diploids and Tetraploids with Cytochalasin B Treatments

We present a new method to produce tetraploid Crassostrea gigas by cytochalasin B inhibition of polar body 2 expulsion in diploid females crossed with tetraploid males. This offers a means of direct introgression of genetic characters from selected diploid to tetraploid lines, avoiding a triploid step. Offspring larval ploidy shifted over time and depended on size, with tetraploids more frequent among the smaller larvae and triploids among the large. Viable tetraploids were found at 4 and 6 months, indicating the technique was successful. The possibility that gynogenesis occurred was tested by microsatellite analysis to confirm the presence of paternally inherited alleles. These were present in all animals of the 2n × 4n + CB (female first) cross. However, a 4n × 2n + CB cross produced triploids, including some gynogens. Our method illustrates for the first time that diploid C. gigas eggs, if selected for large size, can give viable tetraploid offspring.     

Flow cytometry for assessing the efficacy of interspecific gynogenesis induction in sturgeon

The efficacy of ploidy analysis for separating progeny of Siberian sturgeon Acipenser baerii after induced gynogenesis was demonstrated using sperm of a paternal species differing in ploidy level from the maternal species. Gynogenesis was induced in tetraploid A. baerii with UV‐C irradiated sperm from the diploid sterlet Acipenser ruthenus and vice‐versa. The success of sperm UV irradiation and diploidy restoration by heat‐shock was estimated based on the ploidy level of progeny, confirmed by microsatellite parentage assignment. Hatching rates of interspecific gynogenotes were comparable with rates reported for gynogenesis induction using sperm and eggs of the same species. Juvenile mortality was similar to that observed in the control hybrids. The efficiency and reliability of this method may foster its use for production of gynogenotes in aquaculture, potentially allowing interspecific gynogenesis to replace intraspecific.     

Triploid and Tetraploid Zhikong Scallop, <Emphasis Type="BoldItalic">Chlamys farreri</Emphasis> Jones et Preston,Produced by Inhibiting Polar Body I

: Triploid scallops are valuable for aquaculture because of their enlarged adductor muscle, and tetraploids are important for the commercial production of triploids. We tested tetraploid induction in the zhikong scallop by inhibiting polar body I in newly fertilized eggs. The ploidy of resultant embryos was determined by chromosome counting at 2- to 4-cell stage and by flow cytometry thereafter. Embryos from the control groups were mostly diploids (79%), along with some aneuploids. Embryos from the treated groups were 13% diploids, 18% triploids, 26% tetraploids, 13% pentaploids, and 36% aneuploids. Tetraploids, pentaploids, and most aneuploids suffered heavy mortality during the first week and became undetectable among the larvae at day 14. Five tetraploids (2%) were found among a sample of 267 spat from one of the replicates, and none was detected at day 450. The adductor muscle of triploid scallops was 44% heavier (P < .01) than that of diploids, confirming the value of the triploid technology in this species.     

DNA ploidy-level variation in native and invasive populations of Lythrum salicaria at a large geographical scale

Aim  This study aimed to document precisely the patterns of DNA ploidy variation in the native and secondary ranges of Lythrum salicaria distribution. The hypothesis that species invasiveness had been induced by a switch in ploidy level was addressed.
Location  Europe, Middle East, North America.
Methods  DNA ploidy levels of 1884 progenies of 578+ plants collected at 124 localities were determined by DAPI flow cytometry.
Results  Large cytotype variation (2 x , 3 x , 4 x and 6 x ) was found across the native area of distribution (64 populations covering 12 European and two Middle Eastern countries). DNA hexaploids were detected for the first time, and rare DNA triploids were reliably confirmed. DNA tetraploids largely prevailed across the native range studied, while DNA diploids and DNA hexaploids were recorded only in Israel and Turkey, respectively. DNA triploid progenies occurred in one population from Hungary (together with DNA tetraploids). Sympatric growth of DNA tetraploids and DNA hexaploids was repeatedly encountered in Turkey. In contrast, cytotype uniformity was a typical feature of the invasive North American plants. Sixty populations, covering 13 states of the USA and provinces of Canada, were characterized by the presence of only DNA tetraploids.
Main conclusions  Several L. salicaria cytotypes (2 x , 3 x , 4 x , 6 x ) occur in the native range of distribution, with much variation concentrated in the Middle Eastern countries, whereas only DNA tetraploids appeared to occur in North America. Our data show that the invasive spread of North American populations was not triggered by differences in ploidy level. Alternative explanations should be sought.     

CHROMOSOME RACES IN CLAYTONIA LANCEOLATA (PORTULACACEAE)

Chromosome numbers of n = 8, 12, and 16 were determined for 11 populations of Claytonia lanceolata occurring in the southwestern Rocky Mountains of Utah. No evidence of the wide infra-populational variation of chromosome numbers known in the related eastern species, C. virginica, was observed. The chromosome numbers in C. lanceolata probably evolved from a base number of x = 8. Diploids(n = 8) apparently produced tetraploids (n = 16) of putative autoploid origin. Pairing relationships, including the presence of univalents, bivalents, and trivalents, suggest the chromosome numbers of n = 12 are triploids derived from natural hybridization between diploids and tetraploids. Higher chromosome numbers previously reported in C. lanceolata from Colorado, and presumably based on x = 12, can be explained by subsequent polyploid increases in the triploids. The diploid and tetraploid populations analyzed in this study occupy different ecological habitats. The diploids occur at lower elevations along the foothills, whereas the tetraploids are restricted to higher montane and sub-alpine elevations. The triploids were discovered at intermediate elevations.     

Geographical and environmental range expansion through polyploidy in wild potatoes (Solanum section Petota)

Aim  To assess evidence for geographical and environmental range expansion through polyploidy in wild potatoes ( Solanum sect. Petota ). There are diploids, triploids, tetraploids, pentaploids and hexaploids in this group.
Location  Wild potatoes occur from the south-western USA (Utah and Colorado), throughout the tropical highlands of Mexico, Central America and the Andes, to Argentina, Chile and Uruguay.
Methods  We compiled 5447 reports of ploidy determination, covering 185 of the 187 species, of which 702 determinations are presented here for the first time. We assessed the frequency of cytotypes within species, and analysed the geographical and climatic distribution of ploidy levels.
Results  Thirty-six per cent of the species are entirely or partly polyploid. Multiple cytotypes exist in 21 species, mostly as diploid and triploid, but many more may await discovery. We report the first chromosome count (2 n = 24) for Solanum hintonii . Diploids occupy a larger area than polyploids, but diploid and tetraploid species have similar range sizes, and the two species with by far the largest range sizes are tetraploids. The fraction of the plants that are polyploids is much higher from Mexico to Ecuador than farther south. Compared with diploids, triploids tend to occur in warmer and drier areas, whereas higher-level polyploids tend to occur in relatively cold areas. Diploids are absent from Costa Rica to southern Colombia, the wettest part of the group's range.
Main conclusions  These results suggest that polyploidy played an important role in this group's environmental differentiation and range expansion.     

Cytogenetic markers for X chromosome in a karyotype of rainbow trout from Rutki strain (Poland)

Cytogenetic or molecular identification of sex chromosomes could help in breeding studies in producing monosex fish stocks, estimating success of androgenesis, gynogenesis, etc. Among fish species sex chromosomes are recognizable in only a few cases. Some populations of rainbow trout Oncorhynchus mykiss show morphologically differentiated sex chromosomes. A strain from Rutki, Poland, showed a heteromorphic pair of subtelocentric chromosome: presumably of the XY type in the male and XX in the female. Restriction endonuclease and DAPI banding resulted in a characteristic banding pattern enabling identification of the X chromosome.     

Spontaneous diploidization of maternal chromosome set in ornamental (koi) carp, Cyprinus carpio L.

An unusually high frequency of spontaneous diploidization in a maternal chromosome set (SDM) was discovered in one ornamental carp (koi) female in an experiment on these fish for induced gynogenesis. Spontaneous appearance of diploid embryos in the gynogenetic offspring (intact eggs × irradiated sperm) and appearance of triploids among control fish (intact eggs × intact sperm) obtained from koi female and males of edible carp indicated spontaneous diploidization of maternal chromosomes (SDM). Possible cytological processes causing this phenomenon are discussed.     

Distribution and reproductive capacity of natural triploid individuals and occurrence of unreduced eggs as a cause of polyploidization in the loach,Misgurnus anguillicaudatus

Loaches (Misgurnus anguillicaudatus) were collected from 35 localities in Japan and assayed by flow cytometry to determine ploidy status. No tetraploids were found, with samples from 33 localities having no or few (1.2–3.2%) triploids. Samples collected from Ichinomiya Town, Aichi Prefecture, showed a relatively high rate of triploidy (7.7%). Samples collected from a fish farm in Hirokami Village, Niigata Prefecture, also showed high proportions of triploids (2.0–15.8%), these triploid males being sterile, but the females producing both large-sized triploid and small-sized haploid eggs. Such eggs developed bisexually rather than gynogenetically, giving rise to viable tetraploid and diploid offspring after normal fertilization. Of eight diploid females obtained from the same locality, one produced a high incidence of viable diploid gynogens (55%) after gynogenetic induction by fertilization with UV-irradiated spermatozoa. These observations indicated the presence of diploid fish which produced both diploid and haploid eggs. Thus, triploid and diploid individuals were also produced after fertilization with haploid spermatozoa. These results suggested that the occurrence of such unreduced eggs may be a cause of natural polyploidization in this species.     

Serum-free microcarrier based production of replication deficient Influenza vaccine candidate virus lacking NS1 using Vero cells

Background

Androgenesis (all-male inheritance) is generally induced by means of irradiating the eggs to inactivate the maternal genome, followed by fertilization with normal sperm. In fish, the conventional technique for induced androgenesis has been applied for rapid fixation to traits, recovery of cryopreserved genotypes, sex-control, etc. A new method of androgenesis that eliminates the need to irradiate the egg was proposed using the loach, Misgurnus anguillicaudatus (a teleost fish).

Results

When the eggs of wild-type females were fertilized with sperm of albino or orange phenotype males and cold-shocked at 0 to 3°C for 60 min duration just after fertilization, generally more than 30% (with a peak of 100%) of the hatched progeny were androgenotes. While a few of them were the normal diploid, most of them turned out to be abnormal haploid. All-male inheritance was verified by the expression of the recessive color trait (albino or orange) and microsatellite genotypes comprising only paternally derived alleles. Nuclear behavior after the cold-shock treatment was traced by microscopic observation of DAPI (4'6-diamidino-2-phenylindole)-stained samples and hematoxylin-eosin stained histological sections, and the extrusion of egg (maternal) nucleus was observed in eggs treated in the optimum timing.

Conclusion

In this paper, we demonstrate that cold-shock treatment (at 0 and 3°C) of loach eggs for 60 min just after fertilization successfully induces androgenetic haploid development. The most likely mechanism of cold-shock induced androgenesis is an elimination of the egg nucleus together along with the second polar body and subsequent development of a decondensed sperm nucleus or male pronucleus.