Sex Determination and Polyploid Gigantism in the Dwarf Surfclam (Mulinia Lateralis Say)

Mulinia lateralis, the dwarf surfclam, is a suitable model for bivalve genetics because it is hardy and has a short generation time. In this study, gynogenetic and triploid. M. lateralis were successfully induced. For gynogenesis, eggs were fertilized with sperm irradiated with ultraviolet light and subsequently treated with cytochalasin B to block the release of the second polar body (PB2). Triploidy was induced by blocking PB2 in normally fertilized eggs. The survival of gynogenetic diploids was very low, only 0.7% to 8 days post-fertilization (PF), compared with 15.2% in the triploid groups and 27.5% in the normal diploid control. Larvae in all groups metamorphosed at 8-10 days PF, and there was no significant post-larval mortality. At sexual maturation (2-3 months PF), all gynogenetic diploids were female, and there was no significant difference (P > 0.05) in sex ratio between diploids and triploids. These results suggested that the dwarf surfclam may have an XX-female, XY-male sex determination with Y-domination. Compared with diploids, triploids had a relative fecundity of 59% for females and 80% for males. Eggs produced by triploid females were 53% larger (P < 0.001) in volume than those from diploid females. In both length and weight measurements at three months PF, the gynogenetic diploids were not significantly (P > 0.33) different from normal diploid females, suggesting that inbreeding depression was minimal in meiosis II gynogens. Triploid clams were significantly larger (P < 0.001) than normal diploids. We hypothesize that the increased body-size in triploids was caused by a polyploid gigantism due to the increased cell volume and a lack of cell-number compensation.     

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.     

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.     

Production of second generation triploid and tetraploid rainbow trout by mating tetraploid males and diploid females — Potential of tetraploid fish

Summary First generation tetraploids were produced by hydrostatic pressure treatment before the first cleavage and raised until the adult stage. Their survival and growth were severely depressed when compared to the diploid control: after two years, no ovulated females were found although males produced sperm at 1 and 2 years of age and were mated individually with diploid females. The progenies were consistently normal with high survival rates. They were found to be almost all triploids by karyology, which failed to detect a significant rate of aneuploidies. However, the fertilizing ability of tetraploid males was always low (0 to 97% of the control; average 40%). Several arguments presented here support the hypothesis that diploid spermatozoas, which are wider than haploid ones, would be frequently blocked during their penetration through the micropyle canal. Second generation tetraploids were produced after such matings by heat shocks, causing the retention of the second polar body. Their survival and growth were much more satisfactory than in the first generation, although still lower than in diploid and triploid controls issuing from diploid parents. Performances of second generation triploids were comparable to those of diploids, and slightly better than those of conventional triploids issuing from diploid parents. 94.5% of the second generation tetraploids were male.     

Erratum to Production of diploid and triploid offspring by inbreeding of the triploid planarian <Emphasis Type="Italic">Dugesia ryukyuensis</Emphasis>

Triploidy has generally been considered to be an evolutionary dead end due to problems of chromosomal pairing and segregation during meiosis. Thus, the formation of tetraploids and diploids from triploid types is a rare phenomenon. In the present study, we demonstrated that inbreeding of the triploid planarian Dugesia ryukyuensis resulted in both diploid and triploid offspring in nature. In the triploids of D. ryukyuensis, chiasmata between homologous chromosomes were observed in both female and male germ lines. This result suggests that both diploid and triploid offspring of this species are produced bisexually by zygotic fusion between sperm and eggs. Hence, this phenomenon may be a novel mechanism in planarian for escaping the triploid state.     

Meiotic chromosome configurations in oocytes of <Emphasis Type="Italic">Cobitis taenia</Emphasis> and its polyploid hybrids

Diploid and triploid hybrid females of Cobitis as a rule produce unreduced eggs which mainly develop gynogenetically, but some of the eggs incorporate sperm genome and develop into triploids and tetraploids, respectively. Here, we observed for the first time the meiotic chromosomes in the germinal vesicles (GVs) of mature oocytes of three diploid C. taenia (2n = 48) and 20 allopolyploid females of Cobitis (18 triploid 3n = 74 and 2 tetraploid 4n = 99). The majority of GVs in diploid, triploid and tetraploid females contained 24, 74 and 96 or 99 bivalents, respectively. These results directly indicated premeiotic endomitosis as a mechanism underlying the formation of unreduced eggs in allopolyploid females of Cobitis.     

Production of diploid and triploid offspring by inbreeding of the triploid planarian <Emphasis Type="Italic">Dugesia ryukyuensis</Emphasis>

Triploidy has generally been considered to be an evolutionary dead end due to problems of chromosomal pairing and segregation during meiosis. Thus, the formation of tetraploids and diploids from triploid types is a rare phenomenon. In the present study, we demonstrated that inbreeding of the triploid planarian Dugesia ryukyuensis resulted in both diploid and triploid offspring in nature. In the triploids of D. ryukyuensis, chiasmata between homologous chromosomes were observed in both female and male germ lines. This result suggests that both diploid and triploid offspring of this species are produced bisexually by zygotic fusion between sperm and eggs. Hence, this phenomenon may be a novel mechanism in planarian for escaping the triploid state.     

二倍体鲫鲤F2产生不同倍性卵子的证据

在检测到鲫鲤F2产生3种不同大小(直径分别为0.13 cm,0.17cm和0.2 cm)类型的卵子基础上,进行了F2(♀)×红鲫(♂)及F2(♀)×四倍体鲫鲤(♂)的交配实验.通过染色体计数和流式细胞仪分析,在F2(♀)×红鲫(♂)后代中获得了四倍体、三倍体、二倍体鱼;在F2(♀)×四倍体鲫鲤(♂)后代中获得了四倍体和三倍体鱼.这两个交配组合后代中出现的不同倍性的鱼类为证明鲫鲤F2能产生三倍体、二倍体和单倍体卵子提供了进一步证据.F2(♀)×红鲫(♂)中雄性四倍体鱼的存在说明在四倍体后代中存在基因型为XXXY的个体.对上述两个交配组合后代的四倍体鱼和三倍体鱼的性腺结构观察表明四倍体鱼是可育的,而三倍体鱼是不育的.作者认为鲫鲤F2能够产生二倍体和三倍体卵子与核内复制机制和生殖细胞的融合有关.     

Meiotic hybridogenesis in triploid Misgurnus loach derived from a clonal lineage

Triploid loaches Misgurnus anguillicaudatus are derived from unreduced diploid gametes produced by an asexual clonal lineage that normally undergoes gynogenetic reproduction. Here, we have investigated the reproductive system of two types of triploids: the first type carried maternally inherited clonal diploid genomes and a paternally inherited haploid genome from the same population; the second type had the same clonal diploid genomes but a haploid genome from another, genetically divergent population. The germinal vesicles of oocytes from triploid females (3n=75) contained only 25 bivalents, that is, 50 chromosomes. Flow cytometry revealed that the majority of the progeny resulting from fertilization of eggs from triploid females with normal haploid sperm were diploid. This indicates that triploid females mainly produced haploid eggs. Microsatellite analyses of the diploid progeny of triploid females showed that one allele of the clonal genotype was not transmitted to haploid eggs. Moreover, the identity of the eliminated allele differed between the two types of triploids. Our results demonstrate that there is preferential pairing of homologous chromosomes as well as the elimination of unmatched chromosomes in the course of haploid egg formation, that is, meiotic hybridogenesis. Two distinct genomes in the clone suggest its hybrid origin.     

Triploidy Induction in the Pacific White Shrimp Litopenaeus vannamei: An Assessment of Induction Agents and Parameters, Embryo Viability, and Early Larval Survival

In this study, we trialed 6-dimethylaminopurine (6-DMAP) chemical shocks to induce meiosis I or meiosis II Pacific White shrimp, Litopenaeus vannamei, triploids for the first time, and cold temperature shocks to induce meiosis II L. vannamei triploids as done previously. Inductions were performed on 37 spawnings in total with experiments being progressively designed in a factorial manner to allow optimization of induction parameters. Treatment with a 200-μm 6-DMAP final concentration at 1?min post-spawning detection for a 6 to 8?min duration resulted in the most consistent induction of chemically induced meiosis I triploids while treatment at 7?min 30?s post-spawning detection for a 10-min duration resulted in the most consistent induction of chemically induced meiosis II triploids. A cold temperature shock of 11.7°C to 13.25°C (final treatment temperature; spawning water temperature 28.5°C) applied at 8?min post-spawning detection for a 4 to 10?min duration resulted in the most consistent induction of cold-temperature-induced meiosis II triploids. 6-DMAP shocks resulted in meiosis I induction rates from 29% to 100% in unhatched embryos and 50% in nauplii, and meiosis II induction rates from 65% to 100% in unhatched embryos and 52% to 100% in nauplii. Cold shocks resulted in induction rates from 5% to 100% in unhatched embryos and nauplii. Confocal microscopy analysis of embryos revealed that there are major developmental abnormalities in a large proportion of later stage triploid L. vannamei embryos compared to their diploid sibling controls. Despite this, however, some triploid embryos did appear normal and both shock agents induced small numbers of viable triploid L. vannamei nauplii which were successfully reared to protozoeal stage 3 as confirmed by flow cytometry. Triploids beyond this life-history stage were not observed in the present study as confirmed by flow cytometry at mysis stages. This study adds to our knowledge base of triploid induction in L. vannamei and further highlights the inherent difficulties with triploid embryonic and larval viability in this species.     

Inter-embryo competition in the progeny of autotriploid Aloineae (Liliaceae)

In reciprocal crosses between diploid and triploid Aloineae the progeny are largely diploid or diploid plus one or two chromosomes, but in reciprocal crosses between triploids and tetraploids they are tetraploid or nearly so. Thus the triploids contribute circa haploid gametes to the progeny when crossed with diploids but circa diploid gametes when crossed with tetraploids. These results are compared with those of a number of earlier workers. It is concluded that the bias in the frequency of progeny types towards diploidy or tetraploidy, depending on the ploidy level of the plant which is crossed with the triploid, is caused by inter-embryo competition. Those embryos with an endosperm/embryo factor of 1.5, the value found in normal diploid/diploid crosses having triploid endosperms, are selected in preference to those with factors higher or lower than 1.5.Inter-gamete competition also occurs among the euploid and aneuploid gametes produced by the triploids. This is more pronounced on the male side, because the degree of survival of aneuploid pollen from the triploids into the next generation is much lower than that of aneuploid egg nuclei.Non-reduction in the triploids gives rise to occasional pentaploid progeny in crosses with tetraploids, but it is more probable that in diploid/triploid crosses tetraploid progeny are the products of non-reduction in the diploid.     

Comparison of the gonadal development and plasma levels of sex steroid hormones in diploid and triploid sea bass, Dicentrarchus labrax L

The goal of this study was to compare the reproductive physiology of triploid and diploid European sea bass (Dicentrarchus labrax L.). Gonads of diploid and triploid fish (males and females) were examined both microscopically and macroscopically, together with the plasma levels of the major sex steroids produced (testosterone and estradiol-17beta) when fish were adults. Prior to sexual maturation, the gonadosomatic index (GSI) of triploid males was similar to that of diploids. However, the GSI in 4-year-old adult triploid males was 1.8 times lower than that of diploids (P < 0.05). All diploid males exhibited normal gonadal development. In contrast, in triploid males spermatogenesis was impaired during late meiosis, affecting severely spermiogenesis. This was achieved by an increasing imbalance in the amount of DNA present in daughter cells of the same type as spermatogenesis progressed, as demonstrated by abnormal cell sizes, culminating in inviable spermatids. Thus, no spermiating triploid fish were observed during 4 years, which included three full consecutive maturation cycles. Furthermore, the germ cells from triploids were significantly larger than those from diploids (P < 0.001). Seasonal profiles of plasma levels of testosterone in 4-year-old males were essentially similar in both ploidies. On the other hand, triploid females had rudimentary ovaries containing oogonia and primary oocytes that were arrested during meiotic prophase I, while diploid females exhibited all stages of ovarian development. Diploid females showed levels of testosterone and estradiol-17beta significantly higher than those of triploids (P < 0.05), in which no endocrine signs of maturation were observed at all. Regarding sex ratios, triploids had 10% more females than diploids (P < 0.05) but in both ploidies males predominated, as is usually found in this species under culture conditions. These results show that triploidy blocked the initial phases of meiosis in females and the latter ones in males, resulting in the absence of or reduced gonadal development, respectively. In conclusion, we provide an explanation for the lack of gonadal development in triploid male fish, and, to the best of our knowledge, we report for the first time a case in which induced triploidy completely blocks meiosis in both sexes, thus conferring functional sterility in the sea bass.     

A Tripolar Spindle Formed at Meiosis I Assures the Retention of the Original Ploidy in the Gynogenetic Triploid Crucian Carp, Ginbuna Carassius auratus langsdorfii

A triploid crucian carp, ginbuna ( Carassius auratus langsdorfii ), reproduces by gynogenesis, in which sperm of diploid ginbuna or of other species triggers the development of the triploid eggs, but a male genome makes no contribution to the zygotic genome. Gynogenesis is maintained by two mechanisms: exclusion of male genome during fertilization and retention of somatic ploidy levels during oogenesis. We examined the mechanisms responsible for producing unreduced eggs. Microfluorometry with a DNA staining dye showed that DNA content in the ginbuna oocytes was not reduced in half during meiosis I. Cytological observations revealed that a tripolar spindle was formed at meiosis I and the first polar body was not extruded, whereas an ordinary bipolar spindle was formed and the second polar body was extruded at meiosis II. Activity of histone H1 kinase (as an indicator of maturation-promoting factor) decreased transiently between meiosis I and II, strongly suggesting a "normal" meiotic cycle progression in the ginbuna oocytes. These results have indicated that in the gynogenetic ginbuna the somatic ploidy levels are maintained by inhibiting the first polar body extrusion via the formation of the tripolar spindle at meiosis I.     

Evidence of Different Ploidy Eggs Produced by Diploid F2 Hybrids of Carassius auratus (♀) × Cyprinus carpio (♂)

Based on the presence of three types of eggs with different diameters 0.13, 0.17 and 0.2 cm, we made two crosses: F₂ (♀) × diploid red crucian carp (♂), and F₂ (♀) × F₁₀ tetraploid (♂). The ploidy levels of the progeny of the two crosses were examined by chromosome counting and DNA content measurement by flow cytometer. In the offspring of the former cross, tetraploids, trip-loids, and diploid were obtained. In the progeny of the latter cross, tetraploids and triploids were observed. The production of the different ploidy level fish in the progeny of the two crosses provided a further evidence that F₂ might generate triploid, diploid and haploid eggs. The presence of the male tetraploid found in F₂ (♀) × diploid red crucian carp (♂) suggested that the genotype of XXXY probably existed in the tetraploid progeny. The gonadal structures of the tetraploids and triploids indicated that both female and male tetraploids were fertile and the triploids were sterile. We concluded that the formations of different ploidy level eggs from F₂ were contributed by endoreduplication and fusion of germ cells.     

Effect of triploid fitness on the coexistence of diploids and tetraploids

The conditions for the coexistence of diploids, triploids and tetraploids in a single population were investigated with a deterministic model under the assumptions that diploids might produce 2 n gametes, and that triploids had a lower fitness than other cytotypes and generated equal proportions of haploid and diploid gametes. When diploids produced only haploid gametes, the dynamics of the cytotypes were similar to that of heterozygote disadvantage with two alleles at a single locus, with triploids being equivalent to the heterozygotes. Production of 2 n gametes by diploids increased the pool of diploid gametes and created a stable equilibrium involving a majority of diploids and a minority of polyploids. When the fitness of tetraploids was equal to or higher than that of diploids, increased triploid fitness decreased the threshold of 2 n gametes necessary to deterministically fix tetraploids in the population. Conversely, when tetraploids were less fit than diploids, the rate of 2 n gamete production leading to the exclusion of diploids first decreases and then increased with increasing triploid fitness. Triploids are repeatedly found in diploid-tetraploid hybridizations and are rarely totally sterile. They might play a determinant role in the future of multiple cytotype populations. The effect of triploids depends on the relative fitness of diploids and tetraploids and is also a function of their fitness.     

Sexual maturation in triploid rainbow trout, Salmo gairdneri Richardson

This paper compares some morphological and endocrinological characteristics of diploid and triploid rainbow trout.
Significant differences were found between diploid and triploid females in GSI, condition factor, gut weight, liver weight and percentage dress-out, and between diploid and triploid males in GSI, condition factor and gut weight.
Diploid females had large, well-developed ovaries containing yolk-filled secondary oocytes whereas the triploids had only string-like ovaries containing nests of oogonia. No primary oocytes were present.
All the diploid males produced copious quantities of milt but it was possible to express a thin, watery milt containing motile spermatozoa from only two of the 12 triploid males. Testes weights in triploids were similar to those of diploids but, while the diploid testes were packed with spermatozoa, those of the triploids consisted mainly of spermatocytes and spermatids with few spermatozoa present. Measurements of the heads of spermatozoa revealed that those from triploids were larger and had a wider size range than those from diploids.
Levels of testosterone and 11-ketotestosterone in triploid and diploid males were not significantly different. However, levels of testosterone and 17β-oestradiol in diploid females were considerably higher than those of triploid females.     

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.     

The role of tetraploids in the sexual-asexual cycle in dandelions (Taraxacum)

Apomictic plants often produce pollen that can function in crosses with related sexuals. Moreover, facultative apomicts can produce some sexual offspring. In dandelions, Taraxacum, a sexual-asexual cycle between diploid sexuals and triploid apomicts, has been described, based on experimental crosses and population genetic studies. Little is known about the actual hybridization processes in nature. We therefore studied the sexual-asexual cycle in a mixed dandelion population in the Netherlands. In this population, the frequencies of sexual diploids and triploids were 0.31 and 0.68, respectively. In addition, less than 1% tetraploids were detected. Diploids were strict sexuals, triploids were obligate apomicts, but tetraploids were most often only partly apomictic, lacking certain elements of apomixis. Tetraploid seed fertility in the field was significantly lower than that of apomictic triploids. Field-pollinated sexual diploids produced on average less than 2% polyploid offspring, implying that the effect of hybridization in the 2x-3x cycle in Taraxacum will be low. Until now, 2x-3x crosses were assumed to be the main pathway of new formation of triploid apomicts in the sexual-asexual cycle in Taraxacum. However, tetraploid pollen donors produced 28 times more triploid offspring in experimental crosses with diploid sexuals than triploid pollen donors. Rare tetraploids may therefore act as an important bridge in the formation of new triploid apomicts.     

Morphometrical and acoustical comparison between diploid and tetraploid green toads

In the present paper we compare, on the basis of morphometrical characters and acoustical properties of the advertisement calls, a sample of 158 male green toads (Bufo viridis complex) collected in 12 breeding populations of south Kazakstan and north Kyrgyzstan. The samples of three populations resulted in only diploid toads (2n = 22), those of eight populations in only tetraploid toads (2k=44) whereas in one locality diploid, tetraploid and many triploid toads were collected. Diploid toads show significantly larger body size and proportionally larger head and shorter limbs than both tetraploids and triploids, whereas no evident morphometrical differences were observed between triploids and tetraploids. Diploid advertisement calls have spectral and temporal properties that significantly differ from those of both triploid and tetraploid advertisement calls. In particular, diploids produce significantly longer calls with higher pulse-rates and lower frequencies than those of tetraploids. We address the question of the factors that could be responsible for these differences and we discuss four hypotheses: (1) the direct effect of polyploid mutation, (2) genetic drift, (3) reproductive character displacement and (4) environmental selection.     

四类不同倍性鲫鱼在胚胎发育期间同工酶基因表达的比较分析

Isozyme zymograms of esterase (EST), lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and superoxide dismutase (SOD) were analysed by polyacrylamide gradient gel electrophoresis at different developmental stages of embryogenesis in 4 types of various ploidy crucian carp embryos, including haploids, diploids, natural triploids, and multiple tetraploids, and 2 types of haploid and diploid common carp embryos. Haploid embryos of crucian carp (Carassius auratus) and common carp (Cyprinus carpio) were produced by treating eggs with UV-irradiated milt from blunt snout bream (Megalobrama amblycephala). Natural triploid embryos were obtained from the eggs of gynogenetic silver crucian carp (Carassius auratus gibelio) inseminated with milt from red common carp. Multiple tetraploid embryos were also produced by gynogenesis from eggs of the newly discovered multiple tetraploid females inseminated with milt from red common carp. Gradient gel electrophoresis indicated that the band types and staining intensity of 4 isozymes expressed in haploid embryos of crucian carp and red common carp were similar to that in the correlative diploid embryos. In natural triploid silver crucian carp embryos, the zymograms of MDH and SOD isozymes were identical with that of diploid crucian carp embryos, but the EST and LDH isozymes manifested more new enzyme bands in comparison with diploid embryos. The corresponding expressed products of some bands in the triploid embryos, such as EST5 and EST6, could be observed also in red common carp embryos, which provided evidence for hybrid origin about the gynogenetic fish. The multiple tetraploids incorporated one foreign genome of red common carp, therefore, the effects of genes from the foreign genome could be observed in the multiple tetraploid embryos. Gene expression of the isozymes in the tetraploid embryos was somewhat similar to that in hybrids. Owing to interaction of triploid silver crucian carp genomes and common carp haploid genome, some isozyme bands, such as EST5 and EST6, changed in quantity, and some bands increased, such as s-SOD1, s-SOD2, s-SOD3 and s-SOD4 in the tetraploid embryos. Moreover, the heterogeneity was revealed among embryos developed from gynogenetic eggs of 3 different multiple tetraploid individuals.