Genetic and developmental studies of a new Grandchildless mutant of Drosophila melanogaster

Summary A new grandchildless, maternal-effect temperature-sensitive mutant of Drosophila me anogaster, gs (2) M, was isolated in our laboratory. At 28.5° C, homozygous gs (2) M/gs (2) M females lay a normal number of eggs, but about 20% of them fail to hatch and about 40% die just after hatching. The remaining embryos, which pass through this critical stage, complete their development normally, but some of them are devoid of pole cells and thus produce agametic adults. The death of embryos is maternally determined and the hatching probability of an embryo does not depend on its own genotype. The influence of several factors on the phenotypic expression of the new mutant, e.g., age of the females, temperature and number of generations under homozygous condition, is described. Mutants of the type presented here could be useful for further analysis of the establishment of the germ line in Drosophila.     

In situ Development of Perch Eggs,Perca fluviutilis L. (Pisces,Percidae) in a Small Eutrophic Lake,Lake Plussee,Holstein, Germany

Naturally spawned egg strands of perch, Perca fluviatilis L. were sampled from exposed fir trees submerged in Lake Plußsee, Holstein, Northern Germany from April to May 1989. These were incubated in situ and in the laboratory to estimate the viability, development and abnormalities of the embryos. From the in siru incubated eggs 91.6% embryos were viable, the nonviable portion comprising 1.1 % unfertilized eggs, 5.1 % dead and 2.2 % with abnormalities. As compared to the eggs incubated in the laboratory with 84.1 % viable embryos, the nonviable portion consisting 1.1 % unfertilized eggs, 11.3 % dead and 3.5 % with abnormalities. Incubation rate of P. fluviatilis embryos both in situ and laboratory, ranged from 200-210 ATUs (10°C in situ and 8°C in the laboratory) with incubation period of 20-27 days from fertilization to 50 % hatching.     

Organic,inorganic, and caloric composition of eggs,pentaculae, and adults of the brooding sea cucumber Cucumaria curata cowles (Echinodermata: Holothuroidea)

Levels (percentage composition) of water, ash, carbohydrate, lipid, protein, and calories were determined for eggs, pentaculae, and adults of the sea cucumber Cucumaria curata Cowles. Component contents (μg/individual) were calculated for eggs and pentaculae. During the 28 days of development to hatching, the large yolky eggs gain water and ash, the total dry weight increasing from 169 to 190 μg/egg during embryogenesis. There were no statistically significant changes in lipid, protein, and caloric contents during embryogenesis, but carbohydrate decreased by 0.82 μg/egg.The decrease in carbohydrate is sufficient to account for estimated embryonic energy requirements. Based on the utilization of carbohydrate, embryos of C. curata show a nutritional pattern similar to that of the planktonic embryos of sea urchins and different from that of embryos developing from terrestrial eggs, freshwater eggs, and planktonic and demersal marine eggs.Although broods varied widely in egg number and mean egg dry weight, C. curata gives eggs which contain a constant proportion of organic components.Levels of ash, water, and protein in the adults exceeded those in the pentacula, and lipid comprises a much smaller proportion of the adult body than it did of the pentacula.     

Transmission ratio distortion at the growth hormone gene (GH1) in bovine preimplantation embryos: An in vitro culture‐induced phenomenon?

The growth hormone gene (GH1) and its polypeptide product (GH) have a crucial role in reproduction, embryogenesis and general development. A polymorphism present in the fifth exon of the bovine GH1 gene (GH1 p.Leu127Val) has been associated with GH release and milk production in cattle. The objective of the present study was to examine the genotype frequencies of the GH1 p.Leu127Val polymorphism in bovine blastocysts produced in vitro and in vivo to determine if allelic variation of the GH1 gene affects embryo development and survival. A heterozygous (p.Leu127/Val127) sire was used for in vitro fertilization of oocytes of unknown maternal genotype (n = 104) and known maternal genotype (n = 115). PCR amplification and genotyping of the GH1 gene from Day 8 blastocysts derived from these fertilized oocytes demonstrated that there was significant over-representation from the expected Mendelian ratio of GH1 p.Leu127/Leu127 homozygotes from oocytes of known maternal genotype (P = 0.006). Contrary to this, analysis of in vivo-produced bovine blastocysts of known parental GH1 genotype (n = 69) did not reveal an overrepresentation of GH1 p.Leu127/Leu127 homozygotes. These results suggest that developing in vitro-produced embryos are exposed to a selection process, probably due to a less favorable culture environment, that acts to increase the number of GH1 p.Leu127/Leu127 homozygotes, thereby giving rise to the observed transmission ratio distortion (TRD) of GH1 genotypes when compared to in vivo produced embryos.     

Developmental capacity,size and number of nuclei in pig embryos cultured in vitro

Twenty-five surgical embryo recoveries were made from 17 postpuberal gilts 3 to 6 days after mating. A total of 242 eggs was recovered. Recovery rate was 87.5%, fertilization rate was 97.5%, and 98.7% of the fertilized eggs were morphologically intact. The embryos were cultured in vitro in Krebs-Ringer-Bicarbonate (KRB) with 10% heat inactivated lamb serum for 72 or 96 h at +37°C in a humidified 5% CO₂ atmosphere. Of the cultured four-cell embryos 26.6% developed to expanded blastocysts, 16.7% to hatching blastocysts and 5.0% to hatched blastocysts. Of the eight-cell embryos 52.6% developed to hatching blastocysts, 10.5% to hatched blastocysts. When recovered as morulae, the percentage of hatching blastocysts subsequently obtained was 25.8% and 33.9% hatched. A total of 75.0% of the cultured early blastocysts were in the process of hatching (30.6%) or had hatched (44.4%). Significant differences in overall embryo diameter were determined between morulae (156.5 ± 3.94 μm) and early blastocysts (156.9 ± 3.72 μm) versus expanded (197.6 ± 12.57 μm), hatching (207.4 ± 15.86 μm) or hatched (270.0 ± 36.67 μm) blastocysts. The zona pellucida of expanded blastocysts was significantly thinner (5.5 ± 1.59 μm) than that of morulae (12.0 ± 1.01 μm). The number of nuclei was significantly higher for hatching (151 ± 49.8) and hatched (130 ± 17.9) blastocysts cultured as early blastocysts as compared to those cultured from the four-cell stage (88 ± 12.7 and 69 ± 3.6 respectively). Hatching blastocysts that had developed from early blastocysts also had significantly more nuclei than those cultured as eight-cell embryos (99 ± 32.5) or morulae (91 ± 21.2).By the culture method used in this study, a high percentage of pig embryos was capable of developing.     

Methylmercury teratogenesis in the killifish, Fundulus heteroclitus.

Exposure of developing eggs of the killifish, Fundulus heteroclitus, to 0.03 or 0.04 mg/l of methylmercuric chloride resulted in a variety of abnormalities. Percentage of axis formation was reduced somewhat, and many embryos developed cyclopia or intermediate conditions leading to cyclopia, reflecting interference with induction of the forebrain. Defects in the cardiovascular system also appeared in the form of failure of the heart to differentiate properly into chambers. The heart was a thin, feebly beating tube, incapable of causing the blood to circulate. Other tissues, however, continued developing fairly normally, and embryos showed spontaneous movement comparable to controls. Embryos with severe cardiovascular or optic defects did not hatch. Upon hatching, some embryos which had previously appeared normal were found to have skeletal malformations in the form of vertebral bends or the inability to uncurl from the position which they had while still inside the chorion. Exposure to the toxicant for shorter periods of time (6, 12, or 24 hours) reduced the incidence of abnormalities. The second day of development was found to be the most sensitive period.     

Oil droplets and yolk spheres during development of Medaka embryos

The sizes of oil droplets (globules) and the yolk sphere in the Medaka Oryzias latipes egg were measured in the developmental period from fertilization to hatching. Oil droplets coalesced with one another in the process of shifting toward the vegetal pole, and a single large oil droplet was finally located at the vegetal pole region in most eggs 2 days post-fertilization. The volume of the yolk sphere steeply decreased in the period from 2 to 8 days post-fertilization. The volume of oil droplets also declined linearly from 4 to 10 days post-fertilization. Lipid components exhibited no distinct change during embryogenesis. In order to verify whether oil droplets were required for development of Medaka embryos, oil droplets were artificially removed from the early developing embryos without the chorion (egg envelope). Naked embryos without the oil droplet developed normally to fry in the sterilized incubation medium and grew to the same mature fry as those grown from the control embryos.     

Production of chicken progeny (Gallus gallus domesticus) from interspecies germline chimeric duck (Anas domesticus) by primordial germ cell transfer

The present study aimed to investigate the differentiation of chicken (Gallus gallus domesticus) primordial germ cells (PGCs) in duck (Anas domesticus) gonads. Chimeric ducks were produced by transferring chicken PGCs into duck embryos. Transfer of 200 and 400 PGCs resulted in the detection of a total number of 63.0 ± 54.3 and 116.8 ± 47.1 chicken PGCs in the gonads of 7-day-old duck embryos, respectively. The chimeric rate of ducks prior to hatching was 52.9% and 90.9%, respectively. Chicken germ cells were assessed in the gonad of chimeric ducks with chicken-specific DNA probes. Chicken spermatogonia were detected in the seminiferous tubules of duck testis. Chicken oogonia, primitive and primary follicles, and chicken-derived oocytes were also found in the ovaries of chimeric ducks, indicating that chicken PGCs are able to migrate, proliferate, and differentiate in duck ovaries and participate in the progression of duck ovarian folliculogenesis. Chicken DNA was detected using PCR from the semen of chimeric ducks. A total number of 1057 chicken eggs were laid by Barred Rock hens after they were inseminated with chimeric duck semen, of which four chicken offspring hatched and one chicken embryo did not hatch. Female chimeric ducks were inseminated with chicken semen; however, no fertile eggs were obtained. In conclusion, these results demonstrated that chicken PGCs could interact with duck germinal epithelium and complete spermatogenesis and eventually give rise to functional sperm. The PGC-mediated germline chimera technology may provide a novel system for conserving endangered avian species.     

Taxonomic status of the grasshopper Podisma tyatiensis and reproductive isolation between P. tyatiensis and Podisma sapporensis

The grasshopper Podisma tyatiensis, which is distributed only at the summit of Mount Tyatya on Kunashiri Island, the Kuril Islands, is closely related to Podisma sapporensis, which has a broad distribution range on the islands of northern Japan and the Russian Far East (Hokkaido, Sakhalin and Kunashiri). The present study examined the taxonomic status of P. tyatiensis by crossing P. tyatiensis males with P. sapporensis females from Sakhalin. More than 90% of eggs from intrapopulation crosses developed to at least the last embryonic stage, whereas only 64% of eggs from the interpopulation crosses developed into that embryonic stage. Cytogenetic observations of prediapause embryos showed that the interpopulation crosses always led to the production of unfertilized eggs, and that all of the developing embryos had the maternal genome only. A mixture of haploid and diploid cells of maternal origin was found in most of those embryos. This result shows that unfertilized eggs produced by P. sapporensis females from Sakhalin developed parthenogenetically to at least the embryonic stage before hatching. The present crossing experiments revealed a high level of incompatibility between the genomes of the Sakhalin population and the Tyatya population, and confirmed the full species status of P. tyatiensis.     

Effects of feeding high dosages of vitamin E to laying hens on thyroid hormone concentrations of hatching chicks.

Long-term experimental feeding of 20,000 ppm alpha-tocopheryl acetate to laying hens caused a significant (P < 0.05) decrease in hatching rates as compared to the control group, which was fed a diet containing 19 ppm alpha-tocopherol. When the thyroid hormones in the developing chicks were checked on incubation days 16, 19, 21, and 22, the following results were ascertained: During the latter part of incubation, increases in plasma concentrations of thyroxine and triiodothyronine were observed. No significant differences in hormone concentrations (P > 0.05) between the control and the treatment group were observed during incubation days 16, 19, and 22. However, on the day of hatching (day 21 of incubation) significantly lower (P < 0.05) triiodothyronine concentrations in chick embryos of piped eggs were found in the treatment group. Moreover, thyroxine concentrations in non-piped eggs and in hatched chicks were found to be significantly higher as compared to the control group. Given these results, one concludes that extremely high dosages of vitamin E may affect thyroid hormone concentrations of hatching chicks, and therefore, the chicks might be inhibited in pipping the egg shell. Hypothetically, the hepatic enzyme 5'-monodeiodinase is involved in the mechanism of inhibition.     

Gold climates and the evolution of viviparity in reptiles: cold incubation temperatures produce poor-quality offspring in the lizard, Sceloporus virgatus

Evolutionary origins of viviparity among the squamate reptiles are strongly associated with cold climates, and cold environmental temperatures are thought to be an important selective force behind the transition from egg-laying to live-bearing. In particular, the low nest temperatures associated with cold climate habitats are thought to be detrimental to the developing embryos or hatchlings of oviparous squamates, providing a selective advantage for the retention of developing eggs in utero, where the mother can provide warmer incubation temperatures for her eggs (by actively thermoregulating) than they would experience in a nest. However, it is not entirely clear what detrimental effects cold incubation temperatures may have on eggs and hatchlings, and what role these effects may play in favouring the evolution of viviparity. Previous workers have suggested that viviparity may be favoured in cold climates because cold incubation temperatures slow cmbryogenesis and delay hatching of the eggs, or because cold nest temperatures are lethal to developing eggs and reduce hatching success. However, incubation temperature has also been shown to have other, potentially long-term, effects on hatchling phcnotypcs, suggesting that cold climates may favour viviparity because cold incubation temperatures produce offspring of poor quality or low fitness. We experimentally incubated eggs of the oviparous phrynosomatid lizard, Sceloporus virgatus, at temperatures simulating nests in a warm (low elevation) habitat, as is typical for this species, and nests in a colder (high elevation) habitat, to determine the effects of cold incubation temperatures on embryonic development and hatchling phenotypes. Incubation at cold nest temperatures slowed embryonic development and reduced hatching success, but also affected many aspects of the hatchlings' phenotypes. Overall, the directions of these plastic responses indicated that cold-incubated hatchlings did indeed exhibit poorer quality phenotypes; they were smaller at hatching (in body length) and at 20 days of age (in length and mass), grew more slowly (in length and mass), had lower survival rates, and showed greater fluctuating asymmetry than their conspecifics that were incubated at warmer temperatures. Our findings suggest that cold nest temperatures are detrimental to S. virgatus, by delaying hatching of their eggs, reducing their hatching success, and by producing poorer quality offspring. These negative effects would likely provide a selective advantage for any mechanism through which these lizards could maintain warmer incubation temperatures in cold climates, including the evolution of prolonged egg retention and viviparity.     

Vitamin E in early stages of sea bass (Dicentrarchus labrax) development

This study reports titration of vitamin E levels in the sea bass (Dicentrarchus labrax) using high-pressure liquid chromatography. The first part of the work is devoted to vitamin E detection in: (1) plasma of maturing females and males characterized by different body sizes; (2) seminal fluid and eggs; and (3) developing embryos of sea bass fed with vitamin E. In the second part of the study, variations of vitamin E levels during larval development are analyzed. The results show a direct correlation between plasma vitamin E content and body size for both adult male and female sea bass. High vitamin E levels were found in seminal fluid, in eggs before and after fertilization, and in embryos during development and at hatching, whereas vitamin E level was low in dead embryos and in embryos with limited survival. During larval development, the vitamin E content decreased slowly but steadily during the first four days of larval growth; subsequently, it progressively increased from day 9 to day 40. In teratogenic larvae, vitamin E content was significantly higher than in normal larvae. This study provides evidence on how vitamin E exerts an antioxidant defense in sea bass reproduction.     

Temperature effects on embryonic development of Paratlanticus ussuriensis (Orthoptera: Tettigoniidae) in relation to its prolonged diapause

Paratlanticus ussuriensis eggs overwinter by entering diapause, which can be prolonged to more than 1 year depending on environmental conditions. To determine temperature effects on diapause duration of P. ussuriensis eggs, the rates of embryonic development and hatching were compared at various temperatures conditions by measuring embryonic stages and egg weights. Most eggs stayed in a very young stage (blastoderm formation, stage 4) when reared at 15 and 20 °C, 10–30% eggs developed into middle or late stages when reared at 25 °C, and most embryos developed fully (stage 23/24) when reared at 30 °C. Egg weight at 30 °C was 1.5 times higher than those reared at 20 °C. Chilling induced hatching in embryos at stage 23/24. Chilling caused stage 4 embryos to develop into stage 24, but they failed to hatch in response to a second warm period. Thus, P. ussuriensis eggs can overwinter either as young embryos (initial diapause) or as fully-developed embryos (final diapause). Eggs that experience an initial diapause overwinter again the second year in a final stage diapause. The post-diapause period was shorter when embryos overwintered in a final stage diapause. The hatching rate was highest in a temperature range of 7.5–15 °C. Our results suggest that temperature is an important environmental factor for the control of prolonged diapause in P. ussuriensis and initial diapause plays an important role in the control of its life cycle.     

Effects of fetal calf serum, amino acids, vitamins and insulin on blastocoel formation and hatching of in vivo and IVM/IVF-derived porcine embryos developing in vitro

The objective of this study was to determine the effects of fetal calf serum (FCS), non-essential MEM amino acids, MEM vitamins and insulin on blastocoel formation, expansion and hatching in porcine embryos developing in vitro. Addition of 20% FCS to the NCSU 23 medium significantly (P < 0.05) decreased by the compaction and blastocoel formation of 1- to 2-cell embryos developing in vitro. In contrast, more 1- to 2-cell embryos commenced hatching in the media containing amino acids than in control medium (25.7 vs 2.6%, P < 0.01). Amino acids and insulin synergistically enhanced the incidence of blastocoel formation and hatching of porcine embryos developing in vitro (P < 0.05). When early compacted embryos which developed in vitro in NCSU 23 medium were cultured in BSA-free NCSU 23 medium supplemented with 20% FCS, the incidence of hatching was significantly increased compared with that of the control groups (35.7 vs 4.1%, P < 0.01). However, addition of amino acids, vitamins or insulin to the NCSU 23 medium did not enhance the development of early morulae to the hatched embryos (P > 0.1). When either in vivo or IVM/IVF-derived 1- to 2-cell stage embryos were cultured 4 d in the modified NCSU 23 and an additional 4 days in the modified NCSU 23 supplemented in the FCS, the percentages (61.8 and 17.8%, in vivo- and IVM/TVF-derived, respectively) of hatched blastocysts were significantly higher (P < 0.01) than in the control groups (2.9 and 0%, in vivo and IVM/IVF-derived, respectively). These results suggested that dual culture conditions are required to optimize an in vitro culture system for the development of the porcine embryo in vitro.     

Improvement of egg hatch of Ceratitis capitata (Diptera: Tephritidae) for enhanced output

Embryonated eggs of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) genetic sexing strain (GSS), VIENNA 8 were treated with low concentrations of five disinfectants-formaldehyde, iodine, sodium hypochlorite, peracetic acid, and quaternary ammonium--for decontamination and egg hatch improvement. The newly laid eggs were successfully treated with formaldehyde at 100 ppm for 1 min with 74.2% hatching and with quaternary ammonium at 150 ppm for 1 and 2 min with 70.4 and 69% hatching, respectively. Increased formaldehyde concentration may have affected the embryos, because it resulted in a decrease in the hatching percentage. However, egg viability was not impaired and hatch was not affected by quaternary ammonium treatment compared with controls and eggs treated with other disinfectants. Quaternary ammonium shows promise for decontaminating eggs and improving egg hatch.     

Patterns of Embryo Mortality in a Demersally Spawning Coral Reef Fish and the Role of Predatory Fishes

The biological significance of embryo mortality in demersally spawning coral reef fishes is poorly understood. Here we describe patterns of variation in embryo mortality in Pomacentrus amboinensis (Pomacentridae) at Lizard Island (Great Barrier Reef). The aim was to determine whether numbers of embryos hatched substantially differed from egg production, and if so, identify whether predatory fishes were a source of embryo mortality. Spawning success (number of eggs laid), embryo mortality (proportion of embryos that died prior to hatching) and number of embryos hatching were estimated from daily maps of clutches laid on artificial surfaces (PVC tiles) defended by nesting males. Patterns of variation in eggs laid, embryo mortality and numbers of embryos hatched were examined at three spatial scales: (1) among widely-spaced locations around the island; (2) between adjacent reef slope and patch reef habitats occupied by P. amboinensis at a single location; and (3) among different males within these two habitats. The embryo mortality was extremely high, with a mean of 25.9pm ± 6.2% (S.E.) for 4 locations examined in 1994 and a mean of 69.2pm ± 2.9% for two habitats surveyed in 1995. There were no significant differences in embryo mortality among locations or habitats in either year. This meant that spatial patterns in the number of embryos hatching reflected differences in the number of eggs laid on tiles. Embryo mortality was extremely variable on the scale of individual territories, with embryo mortality commonly ranging from 0% to 100%. Much of the mortality could be attributed to diurnal predatory fishes, especially the wrasse Thalassoma lunare. However, variation in predator densities did not explain spatial patterns in embryo mortality rates. Both solitary and group predatory behaviour was observed, with groups often causing 100% embryo mortality. The level of embryo mortality observed suggests that predation prior to hatching may have a substantial effect on the reproductive output of populations of this demersal-nesting fish.     

Survival and ultrastructure of gene-microinjected rabbit embryos after vitrification

Morphological signs of injury and subsequent regeneration following vitrification of either rabbit gene microinjected (Gene-Mi) or intact in vitro cultured embryos derived from in vivo fertilized eggs were evaluated by post-warming recovery in culture and analysed by transmission electron microscopy (TEM). The percentages of vitrified/warmed Gene-Mi embryos that reached the blastocyst stage (69%) and hatched (57%) did not differ significantly from those of intact embryos (78% and 56%, respectively). In contrast, in vitro development of embryos to the blastocyst stage among non-vitrified intact (96%) and Gene-Mi (90%) embryos compared with both the intact vitrified (78%) and Gene-Mi vitrified (69%) groups, as well as hatching rate (94%, 90% vs 56%, 57%, respectively) varied significantly (p < 0.001). Observations by TEM showed that the vitrified/warmed intact or Gene-Mi embryos without post-culture displayed severe degenerative changes among their cells. During 24 h of culture a proportion of the embryos were able to regenerate and complete the compaction process. Nevertheless the signs of previous injury were retained, such as swollen cytoplasmic organelles and remaining cellular debris in the perivitelline space. These observations indicate that the procedure of gene Mi does not significantly compromise embryo tolerance to cryopreservation and post-warming developmental ability. Severe changes in embryo morphology, observed at the ultrastructural level, can be attributed to a direct influence of the vitrification process rather than to the Mi procedure itself.     

Free amino acids in the late embryogenesis and pre-hatching stage of two coregonid fishes

Developing eggs of vendace (Coregonus albula L.) and whitefish (C. lavaretus L.) were experimentally delayed in hatching by incubation at low water temperature (1–2°). Some eggs were taken during this period to a water temperature which was gradually raised up to 8° to provoke mass hatching of embryos. The pattern of free amino acids was followed in eggs incubated at both temperatures. During a 56 days period, the content of several essential amino acids significantly decreased in eggs of both species. For instance, the lysine content dropped from 703 to 270 mg/g dry matter and the arginine content from 257 to 13.3 mg/100 g dry matter in whitefish eggs. A similar pattern of decreasing level of free amino acids in embryonated ova up to hatching was characteristic for essential amino acids and serine. Methionine was exceptional; its level remained approximately the same. On the other hand, non-essential amino acids showed a significant increase in concentration during the experimental period. For instance, the glycine level increased 4.9 and 2.1 times in whitefish and vendace eggs, respectively. Transfer of eggs to 8° accelerated the decrease of nearly all free amino acids before hatching. The consequence of such amino acid metabolism for newly hatched larvae is discussed.     

Development, surface exposure, and embryo behavior affect oxygen levels in eggs of the red-eyed treefrog, Agalychnis callidryas

Oxygen stress can slow development, induce hatching, and kill eggs. Terrestrial anamniote embryos face a potential conflict between oxygen uptake and water loss. We measured oxygen levels within eggs to characterize the respiratory environment for embryos of the red-eyed treefrog, Agalychnis callidryas, a Neotropical frog with arboreal egg masses and plastic hatching timing. Perivitelline oxygen partial pressure (Po2) was extremely variable both within and among eggs. Po2 increased with air-exposed surface of the egg and declined over the developmental period before hatching competence. Through the plastic hatching period, however, average Po2 was stable despite continued rapid development. Development was synchronous across a wide range of perivitelline Po2 (0.5-16.5 kPa), and hatching-competent embryos tolerated Po2 as low as 0.5 kPa without hatching. The variation in Po2 measured over short periods of time within individual eggs was as great as that measured across development or surface exposure, including sharp transients associated with embryo movements. There was also a strong gradient of Po2 across the egg from superficial to deep positions. Ciliary circulation of fluid within the egg is clearly insufficient to keep it mixed. Embryos may maintain development under hypoxic conditions by strategic positioning of respiratory surfaces, particularly external gills, to exploit the patchy distribution of oxygen within their eggs.