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.     

Comparison of reproductive development in triploid and diploid female rainbow trout Oncorhynchus mykiss

The diploid rainbow trout Oncorhynchus mykiss reached sexual maturity 3 years after hatching and its oogenesis underwent four stages, which were oogonia, primary oocyte, secondary oocyte and egg. Reproductive development and hormone changes of 4 to 35 month‐old female O. mykiss were investigated using histological and radioimmunoassay methods in order to provide a theoretical and practical basis for the use of triploid female O. mykiss. The oogonium of the triploid female could develop into the oocytes of the prophase with abortion occurring later; the oogonium was surrounded by stroma cells to form the oogonium cluster and the gonads showed a virilescent tendency when the oogonium clusters were gradually replaced by spermatogenic‐like cytocysts. After 13 months, amounts of gonadotropic hormone (GtH‐I, GtH‐II) and oestradiol (17β‐E₂) in triploid females were lower than in diploid fish at corresponding time periods, but the amounts of testosterone (T) increased consistently after 21 months and were more than in diploid fish in the corresponding time periods (P > 0·05). The infertility of triploid females resulted from meiosis failure, which caused developmental abortion of oocytes and oogonium formed cytocysts before the prophase oocytes. The cytocyst formation was due to the lack of the normal interaction of ovum and follicular cells, the development of follicular cells producing steroids were inhibited, the arylate path from T to 17β‐E₂ was interrupted, concentration of 17β‐E₂ decreased and concentration of T increased in the blood, the content of vitellogenin (Vg) decreased in the liver with a low 17β‐E₂ and high T caused to ovaries to show a tendency to be virilescent.     

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.     

Morphometric characteristics and reproductive capacity of nuclear transplants derived from embryonic cells of loach, Misgurnus anguillicaudatus

Nuclear transplants of loach were produced by transplantating blastula cell nuclei into nonenucleated unfertilized eggs, using recipient eggs and donor cells distinguished by different polymorphic microsatellites. Of the total of 2,847 operated eggs, 143 hatched and 119 developed to the feeding larval stage. For 15 nuclear transplants (i.e., 11.1-year-old fish and 4.2-year-old fish) that survived up to the adulthood, DNA analysis and karyotype analysis were performed. Results showed that, of the 15 fish, 14 had only a nucleus derived from the donor; moreover, 12 were diploids, 1 was a triploid, 1 was a tetraploid, and 1 was a diploid-tetraploid mosaic with both donor and recipient nuclei. For the 12 fish with only a 2n donor nucleus, morphometric analysis was performed, and two female fish and two male fish were mated with normal fish. The fish with only a 2n donor nucleus were determined to be morphometrically identical to normal fish: they had normal gametogenesis and were able to reproduce. Currently, nuclear transplantation technology is beginning to be adopted in fisheries. Biological information on nuclear transplants obtained in this study can be used in the development of nuclear transplantation technology.     

Maturation of mosquito spermatozoa during their transit throughout the male and female reproductive systems

The testes, seminal vesicles and spermathecae of 22 species of mosquitoes belonging to the genera Aedes, Anopheles, Culex, Mansonia and Toxorhynchites are investigated under the electron microscope. Modifications of the acrosome and sperm wall occur during the transit of the spermatozoon from the lower region of the testes to the spermathecae throughout the seminal vesicles. The origin and fate of the cell coat and the possible roles of somatic cell layers both in the testes and the seminal vesicles are discussed.     

A cryptic clonal line of the loach Misgurnus anguillicaudatus (Teleostei: Cobitidae) evidenced by induced gynogenesis,interspecific hybridization,microsatellite genotyping and multilocus DNA fingerprinting

In Memanbetsu town, Hokkaido island, Japan, a high frequency of natural triploid loaches Misgurnus anguillicaudatus (7.4% on average) was detected by flow cytometry for relative DNA content. Among sympatric diploid females (n=6) from a single population, we found two unique females that laid unreduced diploid eggs. They gave normal diploid progeny even after induction of gynogenesis with genetically inert UV-irradiated sperm. When fertilized with normal loach sperm, some unreduced eggs developed into triploids, but the rest into diploids. Hybridization using goldfish Carassius auratus sperm gave both normal diploid loaches and inviable allotriploid hybrids possessing the diploid loach genome and the haploid goldfish genome. Microsatellite genotyping and DNA fingerprinting demonstrated that the diploid progeny developing from the unreduced eggs were genetically identical to the mother, while the triploids had some of the paternal DNA. These results indicate that the diploid eggs reproduced unisexually as a diploid clone and in other cases developed into triploids after accidental incorporation of sperm nucleus. The presence of at least one clonal line in this area was shown by the identical DNA fingerprint detected in five out of 17 diploid loaches examined.     

Differences in potential reproductive rates of male and female seahorses related to courtship roles

Dwarf seahorses, Hippocampus zosterae (Syngnathidae), are distinguished by extreme morphological specialization for paternal care, the formation of monogamous pair bonds and mating repeatedly over the course of a breeding season. To determine the potential reproductive rates of male and female dwarf seahorses, we measured (1) the maximum number of offspring produced per breeding cycle when sexually receptive mates were unlimited, and (2) the relative time each sex was unavailable for mating ('time out'). We paired sexually isolated males and females with sexually receptive partners and observed them from the day of introduction through to copulation, to determine the length of time it takes each sex to prepare to mate. We conducted additional experiments to determine the length of gestation, which when added to the time needed to prepare to mate and copulate gives an estimate of total reproductive cycle duration, T. We estimated potential reproductive rate by dividing the mean number of offspring produced per breeding cycle by the duration of the breeding cycle (T). We estimated reproductive 'time out' by identifying the period of time males and females were physiologically capable of mating ('time in', S) and subtracting time S from time T. When provided with sexually receptive partners, females took 2 days longer than males to complete courtship and copulation, but neither males nor females remated during gestation. Therefore, males could potentially produce 17% more offspring than females over the course of one breeding season. Females had reproductive 'times out' 1.2 times longer than did males, as they were only capable of mating during the 4 h directly preceding copulation. Thus, H. zosterae males have higher potential reproductive rates and shorter reproductive 'times out' compared with H. zosterae females. These results and previous work indicating that seahorses display traditional courtship roles support the prediction that the sex having the higher potential reproductive rate, or equivalently, the shorter 'time out', will compete more intensely for access to the opposite sex. Copyright 2000 The Association for the Study of Animal Behaviour.     

Length‐weight relationships of diploid and tetraploid loach Misgurnus anguillicaudatus (Cantor, 1842) from the Yangtze River basin,China

Length‐weight relationships (LWRs) of diploid and tetraploid loach Misgurnus anguillicaudatus from the Yangtze River basin, China were estimated and compared.     

Extended survival and reproductive potential of single-sex male and female Schistosoma japonicum within definitive hosts

Schistosomiasis is caused by dioecious helminths of the genus Schistosoma. Recent work indicated that unpaired female and male schistosomes can survive within their definitive host for at least 1 year, although the viability or fertility of these worms after subsequent pairing remained untested. We performed two experiments on laboratory mice, one with female Schistosoma japonicum exposure first and male schistosomes second and another vice versa. After surviving as single-sex unpaired forms for up to 1 year, 58.5% of male and 70% of female schistosomes were able to mate and produce viable eggs. This highlights an additional biological challenge in achieving elimination of schistosomiasis.     

Movement of cynomolgus and rhesus monkey spermatozoa collected from the lower female reproductive tract

Postcoital (pc) cervical mucus was collected in 73 menstrual cycles of cynomolgus monkeys and in 43 cycles of rhesus monkeys at 2,6,10,30 hr pc. Videomicrography was used to analyze sperm numbers and movement in the mucus. Both cynomolgus and rhesus monkeys had comparable populations of motile sperm in the mucus at 2 hr pc. However, by 6 hr pc, cervical mucus from cynomolgus monkeys contained twice as many total sperm and motile sperm as mucus from rhesus monkeys (P <.05). Mean swimming speeds of the free-swimming cervical sperm were similar for the two species at this time. No motile sperm were recovered in mucus from rhesus monkeys at 30 hr pc. In cynomolgus monkeys, however, 14 of the 26 animals examined at 30 hr pc had motile sperm in their mucus. These sperm exhibited lower percent molility, percent free-swimming sperm, and swimming speed than those sperm observed at 6 hr pc. Uterine sperm were collected by transcervical or transuterine aspiration from cynomolgus monkeys. In the transcervical technique, sperm were successfully obtained in four of nine animals examined at 6 hr and in four of five animals at 30 hr pc. The percentage of motile sperm in the uterine fluid was high, 82% ± 4%, and the swimming speeds (86 ± 2μm/sec) were higher than those observed in cervical mucus. Approximately 5–10% of the uterine sperm exhibited swimming motions similar to the hyperactivated motility seen in most mammals. These findings indicate that the sperm cervical mucus interaction in vivo in cynomolgus monkeys has more similarities to the human situation than does the interaction in rhesus monkeys.     

Double copulation of a female with sterile diploid and polyploid males recovers fertility in Bombyx mori

Silkworm males produce dimorphic sperm, nucleate eupyrene sperm and anucleate apyrene sperm. Apyrene sperm have been speculated to have an assisting role in fertilisation. However, the coexistence of eupyrene and apyrene sperm in the testis and female reproductive organs has made it difficult to define the role of apyrene sperm. Polyploid males are highly sterile. Microscopic observation revealed that the elimination of eupyrene nuclei by peristaltic squeezing caused the sterility of polyploids. Heat-shock applied to pupae of Daizo males (DH) also induced high sterility due to the lack of normal apyrene sperm. When eupyrene sperm of sterile DH males and apyrene sperm of sterile polyploid males were mixed by double copulation, a remarkable increase in fertility of the double-mated females was observed. This finding strongly suggests that the apyrene sperm are indispensable in fertilisation of the silkworm and that polyploid apyrene sperm function as a substitute for diploid sperm. We established an experimental system in which we can separate the two types of sperm for further studies on their functions without chemical and/or mechanical treatments.     

Variations in hepatic estradiol-17beta receptor concentrations during the annual reproductive cycle of diploid and triploid female catfish, Heteropneustes fossilis (Bloch)

Concentrations of hepatic estradiol-17beta (E2) receptors (ER) in cytosolic and nuclear fractions were evaluated in diploid and triploid female catfish Heteropneustes fossilis (Bloch) during four different reproductive periods of a complete reproductive cycle. Basal level of ER concentration was noted in the resting period of both diploids and triploids. Receptor level gradually elevated through the preparatory period and reached a peak in the pre-spawning period in both diploids and triploids. However, ER concentrations were overall reduced in triploid to that of diploid females. In a single point assay, in diploids, ER concentration showed about a 3-fold rise (p<0.001) in the cytosolic and a 4-fold rise (p<0.001) in the nuclear extracts from resting to the pre-spawning period. In triploids, only a 2-fold rise was observed both in cytosolic (p<0.01) and nuclear (p<0.05) ER concentration during the same span. Finally, a sudden fall of receptor level was observed in the spawning period in both the ploidy groups with a lower concentration in the triploids. The K(d) value did not differ between the females of diploids (cytosolic 1.12+/-0.21 nM and nuclear 6.9+/-0.9 nM) and triploids (cytosolic--1.13+/-0.17 nM, nuclear--6.8+/-2 nM). However, B(max) of the diploid showed about double the value than triploid females both in the cytosolic (diploid--367.4+/-33.24 pmol/mg protein, triploid--187.3+/-13.20 pmol/mg protein, p<0.001) and nuclear extracts (diploid--946+/-66 pmol/mg DNA, triploid-558+/-98 pmol/mg DNA, p<0.01) of liver. Lower E2 binding capacity and lower amount of E2 receptors of triploid catfish liver with a stunted vitellogenic status could be one of the major causes for reduced gonadal development and sterility in female triploids.     

Genetic analyses of agronomic and seed quality traits of synthetic oilseedBrassica napus produced from interspecific hybridization ofB. campestris andB. oleracea

The heritability, the number of segregating genes and the type of gene interaction of nine agronomic traits were analysed based on F₂ populations of synthetic oilseedBrassica napus produced from interspecific hybridization ofB. campestris andB. oleracea through ovary culture. The nine traits—plant height, stem width, number of branches, length of main raceme, number of pods per plant, number of seeds per pod, length of pod, seed weight per plant and 1000-seed weight—had heritabilities of 0.927, 0.215, 0.172, 0.381, 0.360, 0.972, 0.952, 0.516 and 0.987 respectively, while the mean numbers of controlling genes for these characters were 7.4, 10.4, 9.9, 12.9, 11.5, 21.7, 20.5, 19.8 and 6.4 respectively. According to estimated coefficients of skewness and kurtosis of the traits tested, no significant gene interaction was found for plant height, stem width, number of branches, length of main raceme, number of seeds per pod and 1000-seed weight. Seed yield per plant is an important target for oilseed production. In partial correlation analysis, number of pods per plant, number of seeds per pod and 1000-seed weight were positively correlated with seed yield per plant. On the other hand, length of pod was negatively correlated (r = -0.69) with seed yield per plant. Other agronomic characters had no significant correlation to seed yield per plant. In this experiment, the linear regressions of seed yield per plant and other agronomic traits were also analysed. The linear regression equation wasy = 0.074x₈ + 1.819x₉ + 6.72x₁₂ -60.78 (R ² = 0.993), wherex ₈, x₉ and x₁₂ represent number of pods per plant, number of seeds per pod and 1000-seed weight respectively. The experiment also showed that erucic acid and oil contents of seeds from F₂ plants were lower than those of their maternal parents. However, glucosinolate content was higher than that of the maternal plants. As for protein content, similar results were found in the F₂ plants and their maternal parents. It was shown that the four quality traits, i.e. erucic acid, glucosinolate, oil content, and protein content, had heritability values of 0.614, 0.405, 0.153 and 0.680 respectively.     

A comparative cytological investigation of the reproductive cycle of an amphimictic diploid and a parthenogenetic triploid form of Lumbricillus lineatus (O.F.M.) (Oligochaeta,Enchytraeidae)

Summary The cytology of a diploid amphimictic (2x=26) and a triploid parthenogenetic (3x=39) cytotype of Lumbricillus lineatus has been described. The triploid type never produces mature sperm, but it must be fertilized by sperm from the diploid cytotype in order to produce viable eggs. However, the sperm does not enter the egg, but eggs which have not been activated by sperm die at an early stage. Oogenesis in the triploid cytotype, which results ultimately in the restoration of the somatic chromosome number, follows a pattern which has not been described in other parthenogenetic organisms. The first meiotic division is asynaptic and no typical metaphase plate is formed; the univalents are distributed to opposite poles without undergoing an equational division. The first division is therefore numerically approximately reductional. The nuclei are at anaphase when the eggs leave the animal, whereas the nuclei of the diploid amphimictic cytotype are in MI when the eggs are laid. Following activation by spermatozoa, the first anaphase spindle is much elongated, and becomes V-shaped. The first anaphase spindle persists and no second anaphase spindle is formed. At the second division the chromosomes divide mitotically. The resulting four chromosome groups fuse two by two in such a way that two nuclei are formed each with the full somatic (triploid) chromosome set (cf. Figs. 14–16).The different steps involved in the evolution of this mechanism are discussed in relation to the evolution of the triploid cytotype.     

Condition dependence of male and female reproductive success: insights from a simultaneous hermaphrodite

Sexually selected traits are predicted to show condition dependence by capturing the genetic quality of its bearer. In separate‐sexed organisms, this will ultimately translate into condition dependence of reproductive success of the sex that experiences sexual selection, which is typically the male. Such condition dependence of reproductive success is predicted to be higher in males than females under conditions promoting intense sexual selection. For simultaneous hermaphrodites, however, sex allocation theory predicts that individuals in poor condition channel relatively more resources into the male sex function at the expense of the female function. Thus, male reproductive success is expected to be less condition dependent than female reproductive success. We subjected individuals of the simultaneously hermaphroditic snail Physa acuta to two feeding treatments to test for condition dependence of male and female reproductive success under varying levels of male–male competition. Condition dependence was found for female, but not for male, reproductive success, meaning that selection on condition is relatively stronger through the female sex function. This effect was consistent over both male–male competition treatments. Decomposition of male and female reproductive performance revealed that individuals in poor condition copulated more in their male role, indicating an increased male allocation to mate acquisition. These findings suggest that sex‐specific condition dependence of reproductive success is at least partially driven by condition‐dependent sex allocation. We discuss the implications of condition‐dependent sex allocation for the evolution of sexually selected traits in simultaneous hermaphrodites.     

Coevolution of female fidelity and male help in populations with alternative reproductive tactics

In socially monogamous species, pair-bonded males often continue to provide care to all offspring in their nests despite some degree of paternity loss due to female extra-pair copulation. Previous theoretical models suggested that females can use their within-pair offspring as ‘hostages'' to blackmail their social mates, so that they continue to provide care to the brood at low levels of cuckoldry. These models, however, rely on the assumption of sufficiently accurate male detection of cuckoldry and the reduction of parental effort in case of suspicion. Therefore, they cannot explain the abundant cases where cuckolded males continue to provide extensive care to the brood. Here we use an analytical population genetics model and an individual-based simulation model to explore the coevolution of female fidelity and male help in populations with two genetically determined alternative reproductive tactics (ARTs): sneakers that achieve paternity solely via extra-pair copulations and bourgeois that form a mating pair and spend some efforts in brood care. We show that when the efficiency of mate guarding is intermediate, the bourgeois males can evolve to ‘specialize'' in providing care by spending more than 90% of time in helping their females while guarding them as much as possible, despite frequent cuckoldry by the sneakers. We also show that when sneakers have tactic-specific adaptations and thus are more competitive than the bourgeois in gaining extra-pair fertilizations, the frequency of sneakers and the degrees of female fidelity and male help can fluctuate in evolutionary cycles. Our theoretical predictions highlight the need for further empirical tests in species with ARTs.