Haploid Larvae Research Articles

Haploidy-linked cell proliferation defects limit larval growth in zebrafish.

Haploid larvae in non-mammalian vertebrates are lethal, with characteristic organ growth retardation collectively called 'haploid syndrome'. In contrast to mammals, whose haploid intolerance is attributed to imprinting misregulation, the cellular principle of haploidy-linked defects in non-mammalian vertebrates remains unknown. Here, we investigated cellular defects that disrupt the ontogeny of gynogenetic haploid zebrafish larvae. Unlike diploid control larvae, haploid larvae manifested unscheduled cell death at the organogenesis stage, attributed to haploidy-linked p53 upregulation. Moreover, we found that haploid larvae specifically suffered the gradual aggravation of mitotic spindle monopolarization during 1-3 days post-fertilization, causing spindle assembly checkpoint-mediated mitotic arrest throughout the entire body. High-resolution imaging revealed that this mitotic defect accompanied the haploidy-linked centrosome loss occurring concomitantly with the gradual decrease in larval cell size. Either resolution of mitotic arrest or depletion of p53 partially improved organ growth in haploid larvae. Based on these results, we propose that haploidy-linked mitotic defects and cell death are parts of critical cellular causes shared among vertebrates that limit the larval growth in the haploid state, contributing to an evolutionary constraint on allowable ploidy status in the vertebrate life cycle.

The induction of meiotic gynogenesis in Northern pike (Esox lucius) using the heterologous European perch (Perca fluviatilis) sperm

Abstract Northern pike (Esox lucius L.) is one of the fish species whose production in freshwater aquaculture may increase in the next few years. One method of producing this species is to create monosex stocks of pike, as females grow faster, mature later and gain larger body sizes. They can be obtained in the process of gynogenesis. The aim of this research was to determine and optimize the conditions of UV irradiating European perch (Perca fluviatilis L.) spermatozoa to inactivate them genetically (first experiment). The aim of this study was also to confirm whether perch spermatozoa can be used to induce northern pike gynogenesis using thermal shock (second experiment). During first experiment the highest rate of haploid larvae (29.9 ± 0.85%) was noted in the group inseminated with perch sperm irradiated for 6 min (1548 J m-2). No viable embryos were observed in groups of eggs inseminated with sperm irradiated for more than 10 minutes (2580 J m-2). The heat shock applied 12 or 14 min after gamete activation, for 3 or 5 min at 34.0°C, resulted in obtaining of gynogenetic specimen due to retention of the second polar body in all experimental groups. The most efficient was heat shock applied 14 min after gamete activation and lasting 3 min, and resulted in 18.5 ± 1.3% of gynogenetic larvae for female B. Heat shock applied 12 min after gamete activation, lasting 3 min was also effective in the case of female A, resulting in obtaining of 16.5 ± 2.1% gynogenetic specimen.

Induction of gynogenesis by heterogenous sperm and cold shock treatment in Epinephelus fuscoguttatus

Gynogenesis is a chromosome set manipulation technique to accelerate inbred lines used for genomics analysis and genetic breeding. Groupers are hermaphroditic fish whereby they sexually mature as females and subsequently change sex to male. Production of the homozygous inbred lines of groupers will take a prolonged period with the traditional method. This study presents the meiotic gynogenesis induction of the commercially cultured grouper Epinephelus fuscoguttatus, using a combination of ultraviolet-irradiated heterogenous sperm of E. lanceolatus and recovering second polar body by cold-shock treatment. DNA ploidy verification by flow cytometry showed that the haploid larvae exhibited abnormalities and diploids developed normally. The results indicated that UV irradiation doses at 150 ​mJ/cm2 produced 100% haploid larvae. The high percentage (52.1% ​± ​8.50%) of gynogenetic diploidy was induced by cold shock at 5–7 ​°C for 10 ​min, initiated 6 ​min after fertilization. The gynogenetic offspring presented 48 chromosomes (2n ​= ​48t) and were morphologically identical to maternal chromosomes. Cytological and morphological evidence suggested that gynogenetic individuals were successfully obtained. Histological examination revealed that the gynogens gonads could develop into ovaries. Summarily, the gynogenesis technique was very representative of creating inbred lines in groupers. The successful production of this inbred lines is of great significance in grouper genetic breeding and genomic analysis.

Effective de novo assembly of fish genome using haploid larvae

Recent improvements in next-generation sequencing technology have made it possible to do whole genome sequencing, on even non-model eukaryote species with no available reference genomes. However, de novo assembly of diploid genomes is still a big challenge because of allelic variation. The aim of this study was to determine the feasibility of utilizing the genome of haploid fish larvae for de novo assembly of whole-genome sequences. We compared the efficiency of assembly using the haploid genome of yellowtail (Seriola quinqueradiata) with that using the diploid genome obtained from the dam. De novo assembly from the haploid and the diploid sequence reads (100 million reads per each datasets) generated by the Ion Proton sequencer (200bp) was done under two different assembly algorithms, namely overlap-layout-consensus (OLC) and de Bruijn graph (DBG). This revealed that the assembly of the haploid genome significantly reduced (approximately 22% for OLC, 9% for DBG) the total number of contigs (with longer average and N50 contig lengths) when compared to the diploid genome assembly. The haploid assembly also improved the quality of the scaffolds by reducing the number of regions with unassigned nucleotides (Ns) (total length of Ns; 45,331,916bp for haploids and 67,724,360bp for diploids) in OLC-based assemblies. It appears clear that the haploid genome assembly is better because the allelic variation in the diploid genome disrupts the extension of contigs during the assembly process. Our results indicate that utilizing the genome of haploid larvae leads to a significant improvement in the de novo assembly process, thus providing a novel strategy for the construction of reference genomes from non-model diploid organisms such as fish.

Genetic inactivation of European sea bass (Dicentrarchus labrax L.) eggs using UV-irradiation: observations and perspectives.

Androgenesis is a form of uniparental reproduction leading to progenies inheriting only the paternal set of chromosomes. It has been achieved with variable success in a number of freshwater species and can be attained by artificial fertilization of genetically inactivated eggs following exposure to gamma (γ), X-ray or UV irradiation (haploid androgenesis) and by restoration of diploidy by suppression of mitosis using a pressure or thermal shock. The conditions for the genetic inactivation of the maternal genome in the European sea bass (Dicentrarchus labrax L.) were explored using different combinations of UV irradiation levels and durations. UV treatments significantly affected embryo survival and generated a wide range of developmental abnormalities. Despite the wide range of UV doses tested (from 7.2 to 720 mJ.cm−2), only one dose (60 mJ.cm−2.min−1 with 1 min irradiation) resulted in a small percentage (14%) of haploid larvae at hatching in the initial trials as verified by flow cytometry. Microsatellite marker analyses of three further batches of larvae produced by using this UV treatment showed a majority of larvae with variable levels of paternal and maternal contributions and only one larva displaying pure paternal inheritance. The results are discussed also in the context of an assessment of the UV-absorbance characteristics of egg extracts in this species that revealed the presence of gadusol, a compound structurally related to mycosporine-like amino acids (MAAs) with known UV-screening properties.

Induction of diploid gynogenesis in southern flounder (Paralichthys lethostigma) with heterologous sperm of Pagrosomus major

Female southern flounder(Paralichthys lethostigma) grow faster than males.Therefore,farming all-female progenies could maximize profits from cultured fish.In the present study,diploid gynogenesis was induced by activat-ing flounder egg development with UV-irradiated(72 mJ/cm2) Pagrosomus major sperm for 3 min in sea water,and then subjecting the eggs to a cold-shock(0–2℃) treatment for 45 min.Cold shock was used to prevent extrusion of the second polar body.In the control treatments,one or more of these steps was omitted to separately assess the effective-ness of UV irradiation and cold shock.When the initial treatment was applied 3 min after hatching(treatment duration,45 min),the fertilization rate of eggs activated by UV-irradiated heterologous sperm was(37.2 ± 5.1)%,the malforma-tion rate of newly hatched larvae was(8.3 ± 2.5)%,and the hatching rate was up to(9.4 ± 0.71)%.Flow cytometry and chromosome karyotyping were used to examine the ploidy level of larvae experienced to the cold-shock treatment.All of the gynogenetic larvae were diploid(2n = 48),and no haploid or aneuploid phenomena were detected.The duration of hatching of haploid embryos and gynogenetic embryos was significantly different from that of control normal em-bryos.There were no significant differences in the morphological index between gynogenetic diploids and normal lar-vae(P0.05).All of the haploid larvae(100%) showed abnormal external morphology(P0.05) while normal diploid larvae and gynogenetic diploid larvae showed normal morphologies.In summary,these methods resulted in successful diploid gynogenesis in southern flounder using heterologous sperm.This technique could serve as a useful tool to pro-duce monosex progenies of southern flounder.

Somatic cells derived from haploid larvae are feasible as donors for nuclear transplant in zebrafish. Preliminary results

Summary Somatic cells derived from zebrafish haploid larval (both androgenetic and gynogenetic) cultures were used as donors for nuclear transplant into non-enucleated oocytes. Nuclei were transplanted either before or simultaneously with oocyte activation in the central region and in the incipient animal pole, respectively. Against expected results, 20% of transplanted embryos during oocyte activation using cells of gynogenetic origin reached the 100% epiboly stage, even two survived for up to 5 days, whereas no development was observed when cells from androgenetic origin were used. Results derived from this work open a novel possibility of studying somatic cell reprogramming and imprinting phenomena in zebrafish.

Induction of diploid gynogenesis in southern flounder ( Paralichthys lethostigma) with homologous and heterologous sperm

Effective methods for induction of diploid gynogenesis in North American flounder of the genus Paralichthys are needed to initiate monosex culture, which will allow growers to take advantage of the more rapid growth and larger size attained by females. To test methods for inducing diploid gynogenesis in southern flounder ( Paralichthys lethostigma) using homologous sperm, four treatments, named for their expected outcome, were employed: haploid, diploid, triploid, and gynogenetic diploid. Diploid gynogenesis was induced by activating egg development with UV-irradiated flounder sperm (70 J/cm 2) for 3–4 min in seawater, and then subjecting the eggs to cold shock in 0–2 °C seawater for 45–50 min. Cold shock was used to prevent extrusion of the second polar body. Control treatments omitted one or more of these steps to separately assess the effectiveness of UV irradiation and cold shock. Larvae were observed for physical abnormalities and then histologically processed for ploidy determination. Haploid larvae exhibited abnormal external morphology while diploid, gynogenetic diploid, and triploid larvae showed normal morphologies. Cross-sectional areas of erythrocyte nuclei were measured for larvae in each treatment group and significant differences were found. Nuclear areas for treatment groups corresponded to predicted ploidy (triploid>diploid>haploid) and did not differ between normal diploid controls and gynogenetic diploids. These results suggest that the procedures of sperm irradiation and egg cold shock successfully generated gynogenetic diploids. Due to the low volumes of semen produced by male flounder, and to eliminate any potential genetic contribution by homologous sperm, activation of flounder eggs with heterologous sperm was also investigated. Induction of diploid gynogenesis was successful when flounder eggs were fertilized with irradiated (50 J/cm 2) sperm from striped mullet ( Mugil cephalus), and then cold shocked. This work provides procedures for induction of diploid gynogenesis in southern flounder using homologous and heterologous sperm, and validates a method for verification of ploidy in larval fish.

Testis length distinguishes haploid from diploid drones inMelipona quadrifasciata(Hymenoptera: Meliponinae)

Testes length in diploid and haploid larvae drones of Melipona quadrifasciata were investigated to ascertain whether it is a good character to distinguish haploid from diploid drones. Two groups of haploid and two groups of diploid drones were compared. The results show that diploid males have shorter testes than haploid males. Another interesting observation was that whereas diploid males from different groups had testes of similar sizes, haploid males produced by different queens had testes which differed significantly in size. It is suggested that measurements of the testes length could be used to determine ploidy and contribute to the understanding of the population biology of this and other related Hymenoptera species. Melipona quadrifasciata / stingless bee / diploid drone / testicule

Androgenesis and homozygous gynogenesis in muskellunge (Esox masquinongy): evaluation using flow cytometry.

The purpose of this work was to study the effects of ultraviolet (UV) irradiation on denucleation of eggs and investigate the heat-shock conditions for diploidization for induction of androgenesis in muskellunge, Esox masquinongy. Several egg incubation media, including saline, Ringer's solution, and Ringer's solution supplemented with bovine serum albumin (BSA), were found suitable to maintain the egg fertility as high as in muskellunge ovarian fluid. The optimal doses of UV radiation were 660-1320 J/m2, at which 100% haploid larvae were produced at a hatching rate of 22.5 +/- 2.8%. UV irradiation at low doses (165-330 J/m2) generated abnormal larvae, which were morphologically identical to haploids. Using a flow cytometry method, it was found that cellular DNA content of these larvae was close to that of diploids but significantly lower in value and had a wider distribution (expressed as coefficient of variation) than that of control fish. This suggested that a low dose of UV irradiation might cause gene mutations, alteration of chromosomal conformation and fragmentation, but did not prevent maternal DNA from participating in mitotic division. Interference of maternal DNA residues could be another reason for the poor viability of androgenetic fish. A high dose of UV radiation (1980 J/m2) caused development of severely deformed embryos, indicating that UV radiation also damaged molecules in the eggs other than the denucleation. Our results suggest that classic color and allozyme markers might not be sufficient to prove a complete androgenesis. In order to optimize time and duration of shock for induced diploidization, we investigated the heat-shock conditions for inhibiting the first mitotic cleavage through induction of homozygous gynogenesis. We found that heat-shock treatment at 31 degrees C for 9 min starting at 1.4 tau 0 (a dimensionless factor describing progress in embryo development) after fertilization produced the highest percentage of diploids at hatching.

Facultative Hyperparasitism and Sex Determination of Encarsia smithi (Silvestri) (Hymenoptera: Aphelinidae)

Encarsia smithi (Silvestri) and Encarsia opulenta (Silvestri) are primary parasites of the citrus blackfly, Aleurocanthus woglumi Ashby, but E. smithi is also a hyperparasite. Diploid E. smithi larvae are primary parasites, whereas haploid larvae are parasites of conspecific diploids as well as of diploids of E. opulenta . Virgin females of E. smithi deposit haploid eggs in mature larvae or pupae of E. opulenta or E. smithi to produce male offspring by arrhenotoky. Mated females deposit diploid eggs in A. woglumi nymphs and produce female progeny irrespective of whether the hosts are already parasitized by E. opulenta or unparasitized. Diploid eggs or as many as four newly emerged larvae of E. smithi were found in A. woglumi gastric caeca. Larvae moved into the body cavity of A. woglumi as late second instars. Mated female E. smithi also have the option to produce males by ovipositing haploid eggs in A. woglumi nymphs already parasitized by either E. smithi or E. opulenta .

PARTHENOGENESIS IN CARCINONEMERTES SPP. (NEMERTEA: HOPLONEMERTEA).

When juveniles of Carcinonemertes spp. are removed from male crabs and raised in the laboratory, a 50:50 sex ratio is obtained. Isolated females produce egg strings typical of Carcinonemertes, except that the embryos are haploid, with about 13 chromosomes per cell. Larvae develop at least to hatching, and some do hatch. Nearly all of the 146 females so raised produced egg strings. Larvae of all females raised in isolation were haploid while larvae of females put with males were diploid. Females from both U.S. west coast species, Carcinonemertes epialti and C. errans, and from three hosts, Cancer magister, C. antennarius, and Hemigrapsus oregonensis, produced these haploid larvae by parthenogenesis.

Changes in Tissue Polyploidization During Development of Worker, Queen, Haploid and Diploid Drone Honeybees

SummaryCytophotometric measurements were made of DNA content in nuclei stained by the Feulgen method and direct measurements of length and width of nuclei were also made. There was an enormous increase of polyploidization in larvae 4 days old, from an average of 110 n in workers through 112 n and 200 n in haploid and diploid drones to 320 n in queens. Mean DNA content in nuclei of haploid drone, diploid drone, worker and queen larvae were in the ratios 1·0: 1·7: 1·1: 3·1. Since diploids began with twice as much DNA as haploids, polyploidization was about twice as fast in haploid drone larvae as in worker ones and only a little slower in diploid drone larvae than in haploids. A drastic decrease of polyploidization was found in early pupae from 5 n in haploid drones and workers to 8 n in diploid drones and queens. Some increase of average polyploidization was found in imagines, from 7 n in haploid drones through 8 n in diploid drones and workers to 10 n in queens. Relative polyploidization in imagines as de...

Study on the Comparative Viability of Diploid and Haploid Larval Drone Honeybees

SUMMARYEggs laid by 3 selected inbred queens producing brood of low survival rate were hatched in an incubator. Diploid larvae from worker cells and haploid larvae from drone cells were reared further under identical conditions in an incubator; 60% of 89 haploid larvae and 63% of 128 diploids reached the age of 5 days. About half the diploids were drones. Of the diploid drone larvae that reached 5 days, 43% lived to 9 days, compared with 36% of the haploids. The average weight of drone larvae at transference to pupation dishes was 357·7 mg. for diploids and 296·9 mg. for haploids. (The difference was not statistically significant, but might well be so with a greater number of larvae.) The viability of diploid drone larvae is certainly not lower, and may possibly be higher, than that of the haploids: by using right techniques for rearing, it should be possible to rear larger numbers of diploid drones to the imago.

Unchanged recovery of crossovers after x-irradiation of pupal Habrobracon.

mothers heterozygous for four linked markers showed no significant variation between females, or within age groups from ages 2 to 38 days, or foilowing x-radiation doses of 1000 r, 2000 r, or 3000 r to the white pupal stage. Total fertility was increased among lower dosage females and was drastically reduced in the 3000 r group. The constancy of recombination in the wasp was attributed to the lack of synaptic chromosomes in gonial cells rather than to radioresistance of chromosomes or to complete selection against pseudocrossovers in haploid larvae and pupae. (auth)

STUDIES ON EXPERIMENTAL HAPLOIDY IN SALAMANDER LARVAE

1. Eighty seven per cent of the punctured eggs of Triturus pyrrhogaster began development.2. Advanced haploid larvae were obtained. Nine haploid larvae developed to stages ranging from the limb bud to a swimming larva 47 days of age. One haploid-diploid larva had begun to metamorphose at the time it was fixed.3. Advanced androgenetic larvae can be obtained more readily in Triturus pyrrhogaster than in Triturus viridescens: 27 per cent of the segmenting eggs developed beyond the gastrula stage in Triturus pyrrhogaster as compared to 10 per cent in Triturus viridescens.4. Edema was the most common and serious abnormality associated with haploidy.5. In the majority of cases it was impossible to reduce the edematous condition of the larvae with varying concentrations of salt solutions.6. Two triploid larvae were obtained from punctured eggs. These larvae appeared to be entirely normal and slightly larger than the diploid larvae. This is the eighth species of amphibian in which triploidy has been observed.