Egg size and offspring fitness in a bdelloid rotifer

To test if the quality of offspring is affected by egg size and whether it increases with the amount of resources allocated to an individual egg, we compared the offspring hatched from small and large eggs of Macrotrachela quadricornifera(Rotifera, Bdelloidea). Differently sized eggs were obtained by feeding mothers with different food concentrations. Large eggs were expected to provide better offspring in terms of (1) development time of the embryos, (2) age at first reproduction of the juveniles and (3) recovery after desiccation of the newborns. The comparison between offspring hatched from large vs. small eggs of the same bdelloid species revealed that animals hatched from large eggs had shorter embryonic development and earlier age at first reproduction than animals hatched from small eggs. In contrast, the capacity to survive stress, like desiccation, was not affected by egg size. Thus, offspring hatched from larger eggs had shorter generation times, but were not favoured under harsh circumstances.     

Cytological and biochemical analyses of the maternal-effect mutant embryos with abnormal cleavage furrow formation in Xenopus laevis

We describe an embryonic lethal mutation in Xenopus laevis that provokes regression of cleavage furrow formation. The mutant females (designated as af) were obtained by the back-cross of a female with one of her sons. All the fertilized eggs laid by the mutant females, regardless of the wild-type male used in the mating, failed to cleave although each furrow ran at a proper position superficially. Light and electron microscopic observations of the embryos revealed that the cleavage furrows stayed on the surface and cytoplasmic divisions did not take place at all, while nuclear divisions did. Two-dimensional gel-electrophoretic comparisons of af and wild-type embryos demonstrated that two proteins, having estimated molecular masses of about 38 kDa (pI 6.6) and 78 kDa (pI 7.6), were missing in af embryos. Microinjection of clear cytoplasm from a wild-type egg into fertilized af eggs provoked partial surface contraction and cleavage furrow formation in recipient af eggs. The results showed that the af females carry a lethal maternal-effect mutation which causes cleavage furrow regression by being deficient in a few proteins, and that cytoplasm of wild-type eggs can partially rescue the cleavage furrow formation of af eggs by furnishing the corrective material, presumably a product of the normal allele of af.     

Unpredictable offspring survivorship in the damselfly, Megaloprepus coerulatus, shapes parental behavior, constrains sexual selection, and challenges traditional fitness estimates

Evolutionary biologists typically assume that the number of eggs fertilized or developing embryos produced is correlated with an individual's fitness. Using microsatellite markers, we document for the first time estimates of realized fitness quantified as the number of offspring surviving to adulthood in an insect under field conditions. In a territorial damselfly whose males defend tree hole oviposition sites, patterns of offspring survivorship could not be anticipated by adults. Fewer than half of the parents contributing eggs to a larval habitat realized any reproductive success from their investment. The best fitness correlate was the span over which eggs in a clutch hatched. Among parents, female fecundity and male fertilization success were poor predictors of realized fitness. Although body size was correlated with female clutch size and male mating success, larger parents did not realize greater fitness than smaller ones. The uncoupling of traditional fitness surrogates from realized fitness provides strong empirical evidence that selection at the larval stage constrains selection on mated adults.     

Induced gynogenesis in the rainbow trout: Sex and survival of progenies production of all-triploid populations

Summary Gynogenesis could be an efficient way for producing inbred lines in commercial fish species. Gamma-irradiation of sperm gives haploid embryos that all die without hatching; in the present study, we optimized heat treatment of the eggs, in order to produce high rates of diploid gynogenetics. When the eggs are heated to 26 °C for 20 min after 25 min of development, 80% of the embryos hatch, and all the resulting fry are diploid; nevertheless, high mortalities are recorded until feeding start. The monosex female constitution of gynogenetic offspring confirms the female homogamety in the rainbow trout.When the eggs are treated with the same heat shock 25 min after a fertilization with functional sperm, alltriploid populations are obtained; their survival until feeding start is not different from the control.     

Pluripotency of Homozygous-Diploid Mouse Embryos in Chimeras

Pluripotency of mouse uniparental cells (complete homozygous-diploid gynogenetic) produced by embryo manipulation was examined in aggregation chimeras with normally fertilized embryos. A male pronucleus was removed from fertilized eggs by micromanipulation and eggs were diploidized with cytochalasin B. Uniparental cells that developed to 4-cell or more advanced stages were aggregated with normally fertilized 8-cell embryos and transferred to the pseudopregnant female uteri to develop to term. Among the pups, 1 female and 3 males were identified as overt chimeras by their coat color and pigmentation of the retina. Using electophoretic analysis of the isozymes, the contribution of uniparental cells in these chimeras was confirmed by findings in the major organs such as liver, brain, small intestine, kidney, spleen, heart and testis. The female chimera produced offspring derived from oocytes of uniparental origin. Our experiments verified the pluripotency of microsurgically produced mouse uniparental cells.     

Isolation and distribution of elongation factor 2 in eggs and embryos of sea urchins

The subcellular distribution of elongation factor 2 (EF-2) in eggs and early embryos of the sea urchin, Strongylocentrotus purpuratus, was studied by employing the diphtheria toxin dependent ADP-ribosylation of EF-2. When egg and embryo homogenates were fractionated by sedimentation, EF-2 was found associated with a low-speed pellet containing yolk, nuclei, and mitochondria. It also sedimented at 80 S and 5 S. No significant amounts of EF-2 were found on polyribosomes. The 5S form of EF-2 probably represents a monomeric unit of the factor as EF-2 had a molecular weight of 95 000 on sodium dodecyl sulfate-polyacrylamide gels. EF-2 could only be isolated intact if soybean trypsin inhibitor or EGTA was present. The total amount of EF-2 was similar in eggs and embryos. However, the distributions of the factor between the various fractions were substantially different for eggs and embryos. Also, a marked difference in the physical association of EF-2 with material in the low-speed pellet occurred after fertilization. Specifically, in eggs, 23% of the EF-2 was associated with the low-speed pellet; in cleavage-stage embryos, only 11% of the EF-2 was associated with the pellet. In eggs, 65% of the EF-2 sedimented as 80 S; by the 16-cell stage, this amount decreased to 44%. Concomitantly, the amount of EF-2 in the 5S fraction increased from about 8% in eggs to 44% in the 16-cell embryos. In addition, Triton X-100 was required for the extraction of EF-2 from the low-speed pellet of eggs, but not of embryos.(ABSTRACT TRUNCATED AT 250 WORDS)     

Generation of the Y-chromosome linked red fluorescent protein transgenic mouse model and sexing at the preimplantation stage

In mammals, sexual fate is determined by the chromosomes of the male and female gametes during fertilization. Males (XY) or females (XX) are produced when a sperm containing a Y or X-chromosome respectively fertilizes an X-chromosome-containing unfertilized egg. However, sexing of preimplantation stage embryos cannot be conducted visually. To address this, transgenic male mouse models with the ubiquitously expressed green fluorescent protein (GFP) transgene on X- (X-GFP) or Y-chromosomes (Y-GFP) have been established. However, when crossed with wild-type females, sexing of the preimplantation stage embryos by observing the GFP signal is problematic in some cases due to X-inactivation, loss of Y-chromosome (LOY), or loss of transgene fluorescence. In this study, a mouse model with the ubiquitously expressed red fluorescent protein (RFP) transgene on the Y-chromosome was generated since RFP is easily distinguishable from GFP signals. Unfortunately, the ubiquitously expressed tdTomato RFP transgene on the Y-chromosome (Y-RFP) mouse showed the lethal phenotype after birth. No lethal phenotypes were observed when the mitochondrial locating signal N-terminal of tdTomato (mtRFP) was included in the transgene construct. Almost half of the collected fertilized eggs from Y-mtRFP male mice crossed with wild-type females had an RFP signal at the preimplantation stage (E1.5). Therefore, XY eggs were recognized as RFP-positive embryos at the preimplantation stage. Furthermore, 100% sexing was observed at the preimplantation stage using the X-linked GFP/Y-linked RFP male mouse. The established Y-mtRFP mouse models may be used to study sex chromosome related research.     

Yolk androgens and embryo sex: maternal effects or confounding factors?

Maternal effects occur when offspring phenotype is affected by environmental factors experienced by the mother and, in egg-laying species, are often mediated via egg resources. There is currently great interest among behavioural ecologists in maternally allocated yolk androgens, especially their relationship with offspring sex and development. Such studies need embryonic tissue for sexing, however, requiring eggs to be incubated (usually for 3 days). Therefore, there are concerns about whether the androgen concentrations assayed reflect those allocated by the mother. In addition, studies showing sex biases in maternal allocation of androgens could be confounded if male and female embryos uptake or metabolise androgens at different rates. We ran a series of experiments using zebra finch (Taeniopygia guttata) eggs to address these potential confounding factors. First we showed, using eggs naturally incubated for up to 5 days, that eggs containing embryos had lower yolk androgen concentrations than eggs that had failed to form embryos. We then tested various hypotheses for this difference using controlled incubation treatments. Our results suggested that (a) embryo development causes the yolk to become progressively more diluted with albumin; and (b) between 3 and 5 days of incubation embryos start uptaking or metabolising androgens. Crucially, we found no decline in yolk androgen concentration at 3 days incubation, and no evidence for sex-specific rates of uptake or metabolism of androgens. This strongly suggests that yolk androgen levels up to 3 days incubation do reflect those allocated by the mother, and that studies of sex biased maternal allocation of yolk androgens are not confounded by sex differences in embryo development.     

Superovulation of immature hypothyroid rdw rats by thyroxine therapy and the development of eggs after in vitro fertilization.

The aim of this study was to examine the effect of thyroxine on ovulation in immature rdw rats and the fertilization and development of the eggs. Serum thyroxine concentrations at 30 days of age were significantly lower in rdw rats than in normal rats (P < 0.001), and greatly increased after thyroxine replacement therapy (P < 0.001). Although few eggs (1-5 +/- 1-2) were obtained from immature rdw rats treated with gonadotrophins alone, females treated with gonadotrophins and thyroxine ovulated significantly more eggs (85 +/- 5). As a control, normal littermates ovulated 21-45 eggs when treated with gonadotrophins alone, and 68 eggs when administered with gonadotrophins and thyroxine. Of the eggs collected from rdw rats treated with gonadotrophins and thyroxine, and inseminated with spermatozoa from mature F1 males, 98% were penetrated and in almost all (99%) of these eggs, male and female pronuclei formed. Forty-seven per cent of the pronuclear eggs developed to the blastocyst stage in vitro. After transfer to recipients, 21% (14/66) of one-cell and 22% (8/37) of two-cell embryos developed to offspring, and 62% (8/13) of pups were of rdw/rdw genotype. The average body weight (6.9 versus 7.8 g) of offspring derived from one-cell embryos was lower than that for two-cell embryos. The morulae and blastocysts did not develop to term, although 41% implanted in the uterine horns of recipients. In conclusion, in immature rdw rats, superovulation was induced by gonadotrophins combined with thyroxine therapy and the superovulated oocytes were fertilized and developed in vitro and developed to term after embryo transfer.     

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.     

Male‐specific mortality biases secondary sex ratio in Eurasian tree sparrows Passer montanus

Sex allocation theory predicts that parents bias the offspring sex ratio strategically. In avian species, the offspring sex ratio can be biased at multiple growth stages, although the mechanisms are not well known. It is crucial to reveal a cause and timing of biased offspring sex ratio. We investigated (i) offspring sex ratio at multiple growth stages, from laying to fledging; and (ii) the stage at which offspring sex ratio became biased; and (iii) the cause of biased offspring sex ratio in Eurasian tree sparrows Passer montanus. Sex determination of 218 offspring, including hatchlings and unhatched eggs from 41 clutches, suggested that the offspring sex ratio was not biased at the egg‐laying stage but was significantly female‐biased after the laying stage due to higher mortality of male embryos. Half of the unhatched eggs showed no sign of embryo development (37/74, 50.00%), and most undeveloped eggs were male (36/37, 97.30%). Additional experiments using an incubator suggested that the cause of embryo developmental failure was a lack of developmental ability within the egg, rather than a failure of incubation. This study highlights the importance of clarifying offspring sex ratio at multiple stages and suggests that offspring sex ratio is adjusted after fertilization.     

FLPe functions in zebrafish embryos

To assay the efficiency of the FLP/FRT site-specific recombination system in Danio rerio, a construct consisting of a muscle-specific promoter driving EGFP flanked by FRT sites was developed. FLPe capped RNA was microinjected into transgenic single cell stage zebrafish embryos obtained by crossing hemizygous transgenic males with wild-type females. By 48 h post fertilization (hpf), the proportion of embryos displaying green fluorescence following FLPe RNA microinjection was significantly lower (7.7%; P < 0.001) than would be expected from a cross in the absence of the recombinase (50%). Embryos that retained fluorescence displayed marked mosaicism. Inheritance of the excised transgene in non-fluorescent, transgenic embryos was verified by PCR analysis and FLPe-mediated recombination was confirmed by DNA sequencing. Sperm derived from confirmed transgenic males in these experiments was used to fertilize wild-type eggs to determine whether germline excision of the transgene had occurred. Clutches sired by FLPe-microinjected males contained 0–4% fluorescent embryos. Transgenic males that were phenotypically wild-type produced no fluorescent progeny, demonstrating complete excision of the transgene from their germline. FLPe microinjected males that retained some fluorescent muscle expression produced a small proportion of fluorescent offspring, suggesting that in mosaic males not all germline cells had undergone FLPe-mediated transgene excision. Our results show that FLPe, which is derived from Saccharomyces cerevisiae, is an efficient recombinase in zebrafish maintained at 28.5°C.     

Generation of transgenic mice by transfection of pronuclear embryos using lipid-DNA complexes

We had previously developed a methodology to introduce foreign DNA into mouse eggs and embryos using cationic lipids as vectors. In this report we use this technique to produce transgenic animals. Mouse embryos at the pronuclear stage were transfected using a mixture of a plasmid DNA, encoding for a nuclear form of beta-galactosidase, and a commercial lipid transfection reagent. Embryos were washed and incubated overnight. Those that cleaved and develop to the 2-cell stage of normal appearance were transferred to the Fallopian tubes of pseudopregnant foster mothers. We analysed a total of 158 offspring and found two, a female and a male, to be transgenic (1.27% of the total). Integration of the foreign DNA in the female was showed by Southern blot. Both animals expressed the lacz in several organs, but none of them either displayed expression in the germ cells or transmitted the transgene to their offspring. Taken together our results show that lipid transfection can generate transgenic mice, but the efficiency needs to be improved for this method to be widely applied.     

Isolation and characterization ofgrandchildless-like mutants inDrosophila melanogaster

Summary Two temperature-sensitive sex-linkedgrandchildless (gs)-like mutations (gs(1)N26 andgs(1)N441) were induced by ethylmethane sulphonate inDrosophila melanogaster. They complemented each other and mapped at two different loci (1–33.8±0.7 forgs(1)N26 and 1–39.6±1.7 forgs(1)N441), which were not identical to those of any of thegs-like mutants reported in earlier work.Homozygous females of the newly isolated mutants produced eggs that were unable to form pole cells and developed into agametic adults. Competence of the embryos to form pole cells was not restored by wild-type sperm in either mutant; that is, the sterility caused by these mutations is controlled by a maternal effect.Fecundity and fertility ofgs(1)N26 females were low, and their male offspring showed a higher mortality than that of female offspring, causing an abnormal sex ratio. The frequency of agametic progeny was 93.1% and 55.8%, when the female parents were reared at 25° C and 18° C, respectively. In eggs produced by thegs(1)N26 females reared at 25° C, the migration of nuclei to the posterior pole was abnormal, and almost no pole cell formation occurred in these egg. Furthermore, half of these eggs failed to cellularize at the posterior pole. When the females were reared at 18° C, almost all of the eggs underwent complete blastoderm formation, and in half of these blastoderm embryos normal pole cells were formed.In the other mutant,gs(1)N441, the fecundity and fertility of the females were normal. The agametic frequency in the progeny was 70.8% and 18.6% when the female parents were reared at 25° C and 18° C, respectively. In the eggs laid by females reared either at 25° C or at 18° C, the migration of nuclei to the periphery and cellularization proceeded normally; nevertheless, in the majority of the embryos no pole cell formation occured at the stage when nuclei penetrated into the periplasm. When the females were reared at 18° C, some of the embryos from these females formed some round blastoderm cells with cytologically recognizable polar granules and nuclear bodies, which are attributes of pole cells. The temperature sensitive period ofgs(1)N441 was estimated to extend from stage 9 to 13 of King's stages of oogenesis.     

Sexual dimorphism of egg size in the European Blackbird Turdus merula

Recently, a number of studies have shown that female birds are able to control the sex of their progeny at the stage of the gamete. There is also some evidence that females adjust their investment in offspring depending on the sex of the embryo during egg formation. Differential maternal investment to the eggs depending on their sex is usually interpreted as an adaptive strategy, by which females can increase competitive abilities of the smaller sex, or preferentially invest towards the sex with the potentially higher fitness returns. Here, we studied variation in egg size in relation to embryo sex and laying order in the European Blackbird Turdus merula. We found male and female eggs to differ in size, with larger eggs containing male embryos, as well as a significant interaction between embryo sex and laying order. This interaction resulted from the fact that egg size increased with the laying sequence among eggs bearing females but did not change with laying order among eggs bearing males. There was no relationship between offspring sex and the laying sequence within a clutch. We suggest that sexual dimorphism in egg size recorded in the European Blackbird may reflect favouritism of the sex which may give higher fitness returns.     

Generation of fertile and diploid fish, medaka (Oryzias latipes), from nuclear transplantation of blastula and four-somite-stage embryonic cells into nonenucleated unfertilized eggs

In two experimental series of transplantation of embryonic cell nuclei into nonenucleated unfertilized eggs in medaka (Oryzias latipes), fertile and diploid nuclear transplants were successfully generated. In the first experiment, nuclei from blastula cells of a medaka stock with the wild-type body color were transplanted into 1722 eggs from the orange-red variety. Of 26 adult nuclear transplants with the wild-type body color, 22 were, as expected, triploid and sterile, but the other four were fertile. Three of the four were diploid, and the last one was tetraploid. They transmitted the wild-type body color to the F1 and F2 progenies in a Mendelian fashion. In the second experiment, cell nuclei from four-somite-stage embryos of the orangered variety carrying the green fluorescent protein (GFP) transgene were transplanted into 1688 recipients of the same strain. Three adult nuclear transplants expressing GFP were obtained. Two of them were triploid and sterile, but the remaining one was fertile and diploid. The transgene of the donor nuclei was transmitted to the F(1) and F(2) offspring in a Mendelian fashion. These observations that diploid and fertile nuclear transplants could be obtained without enucleation of the recipient eggs may have important implications for future nuclear transplantation in medaka.     

Daughters from daughterless mothers — Rescuing a female-lethal maternal effect by cytoplasmic transplantation inDrosophila embryos

Summary Daughterless is a temperature-sensitive, maternal effect mutation ofDrosophila melanogaster. Homozygousdaughterless females raised at temperatures above 22°C do not produce any female progeny. It was possible to rescue these female embryos by injecting cytoplasm from non-mutant unfertilized eggs into embryos fromdaughterless mothers. Cytoplasm from unfertilized eggs laid by homozygousdaughterless mothers was ineffective. Surprisingly, the cytoplasm from developing embryos with either wild-type ordaughterless mothers could also effect rescue. Based upon this data, we suggest that male and female embryos ofdaughterless mothers differ in their ability to initiate the synthesis of a product during the nuclear multiplication (cleavage) stage of embroniic development in the absence of a putativeda ⁺ maternally-synthesized factor, and that this is the basis for the sex-specific action of theda maternal effect.     

Egg size,postembryonic yolk,and survival ability

Summary Neonates of many species are dependent upon a post-embryonic yolk (PEY), a residual of the energy reserve of the developing embryo. Offspring hatching from large eggs have relatively more PEY than offspring from small eggs. Among daphniid Cladocera, large species produce larger eggs than smaller species. We have found that the proportional amount of energy reserve in eggs of five species of Cladocera is similar, but neonates of the larger Cladocera are born with a greater relative amount of postembryonic yolk, as triacylglycerol, than small species. Apparently, more of the reserve is metabolized by embryos of small species. This is correlated with the higher unitweight metabolic rates of smaller animals. It has been argued that animals should produce relatively larger eggs when exposed to low or unpredictable food conditions to increase the survivorship of their offspring. The physiological constraint of greater relative energy requirements of small embryos may limit PEY and explain why offspring of larger eggs survive better in low or unpredictable food resource environments.     

Biocontrol of Two-spotted Spider Mite Tetranychus urticae on Dwarf Hops by the Phytoseiid Mites Phytoseiulus persimilis and Neoseiulus californicus

Two female Phytoseiulus persimilis and their offspring eliminated two-spotted spider mite Tetranychus urticae from hop leaf discs faster than two female Neoseiulus californicus and their offspring at 25°C. A combination of one female of each species and their offspring eliminated spider mites faster than the N. californicus alone, but slower than P. persimilis alone. Air relative humidities of 55% and 93% had no effect on predation. Both predator species cannibalised eggs and juveniles when spider mite numbers were low. In field experiments in 1996, fewer spider mites were recorded where P. persimilis was released, irrespective of the presence of N. californicus. Pest numbers on cv. 'First Gold' were lower than on cv. 'Herald'. No differences were recorded between the numbers of spider mite eggs in predator release treatments on 'First Gold', but fewer active stages of spider mites were recorded on plots with P. persimilis than controls soon after the time of peak pest population densities. On 'Herald', fewer spider mite active stages and eggs were recorded where predators were released than on untreated controls.     

Mottled coloration in a rainbow trout is associated with mosaicism for albinism

Within a group of rainbow trout at a commercial farm, a single individual was noted for its mottled yellow and dark skin pigmentation. This female fish was reared to sexual maturity and sublots of its eggs were crossed to rainbow trout males from golden, albino, and wild-type (dark-pigmented) strains. Development in other eggs was activated to produce diploid gynogenetic offspring. Coloration within each progeny group was scored at 10 weeks following initiation of feeding. Mottled coloration was observed in none of the progeny at this time. The phenotypes observed (palomino, albino, and/or wild type) and their proportions within progeny groups indicated that the mottled female was originally heterozygous for albinism. The fish apparently became mosaic for this trait following mutation of the wild-type allele at the albinism locus within a cell(s) early in embryonic development. Curiously, at approximately 6 months after initiation of feeding, mottled coloration became apparent in 2 fish from among 25 progeny of the cross to the golden male. No change in phenotype was noted at this time in 9 gynogenetic progeny nor in 68 progeny from the cross to the albino male. Apparently additional mutations and/or other genetic and regulatory processes affecting coloration came into play during juvenile development of these latter two fish.