Possible involvement of calcium ions in the hatching of Schistosoma mansoni eggs in water

The possibility of involvement of calcium ions in the hatching of Schistosoma mansoni eggs in water is described. The hatching of S. mansoni eggs under low osmotic pressure was partially inhibited by EGTA (5 mM), lanthanum chloride (1-5 mM), and ruthenium red (0.1-1 mM). The reagents used in these experiments were not toxic to the eggs however, because miracidia hatched normally when the reagents were removed.     

The hatching of the eggs of the liver fluke Fasciola hepatica L. under various CO2-concentrations (author's transl)]

The presupposition for the hatching of the eggs of Fasciola hepatica is the presence of undissociated CO2 with the most effective concentration of 2-10 mMols/l. Higher concentrations show inhibitory effects (Abb. 6). In hatching pH has only importance in dissociation of carbonic acid and so in maintaining free CO2. To provide for sufficient CO2 at higher pH - but below 8.3 - the concentration of carbonic acid must be increased by higher concentrations of metallic ions electrically neutralized by hydrogen carbonate (e.g. in Abb. 1-4). Higher pH is necessary for miracidia surviving. Beside the wellknown hatching factors of exposure to light and cooling, darkening is a new found factor (s. Abb. 7) as well as sufficient increase of CO2 itself. The effect of light stimulation is lasting for at most 3 h with exponential decrease (Abb. 7). At low CO2-pressure the occurrence of hatching is greater without any oxygen (Abb. 5).     

Leucine aminopeptidase and hatching of Schistosoma mansoni eggs

Leucine aminopeptidase (LAP) activity has been measured in extracts of eggs, miracidia, cercariae and adult worms of Schistosoma mansoni. Activity measured at pH 7.2 using L-leu-7-amino-4-trifluoro-methylcoumarin as substrate is 6- to 17-fold greater in eggs than in other life stages. LAP activity is also high in soluble egg antigen preparations and in hatching fluid. The release of LAP from eggs parallels hatching, and inhibitors of LAP also inhibit hatching. The possible role of LAP in the hatching process of S. mansoni eggs is discussed.     

A comparative study of the ultrastructure and mineralogy of calcified land snail eggs (Pulmonata: Stylommatophora)

This study indicates that eggs containing calcium carbonate crystals occur in at least 36 of the 65 known families of the land snails (class Gastropoda: order Stylommatophora). Eggs from 22 of these families were available for examination. The x-ray diffraction data, available for the first time for 21 of these families, shows that these egg shells are all made of calcite only, or of a combination of calcite with smaller amounts of aragonite. All of the snail (body) shells examined were made of aragonite only. This is the first ultrastructural investigation of these egg shells, and it indicates that the eggs exhibit enough structural diversity to allow identification of parental animals to genus, and often to species level solely on the basis of egg shell ultrastructure. All of the calcified eggs may be divided into two groups: (1) partly calcified, with discrete crystals of CaCo₃ dispersed in the jelly layer, and (2) heavily calcified, with a hard, brittle egg shell made of fused crystals of CaCO₃ much like an avian egg. Both types of calcified eggs occur in oviparous as well as in ovoviviparous snails. Because of the wide distribution of calcified eggs in the Stylommatophora, and because of the occurrence of heavily calcified eggs in ancient families such as Partulidae, Endodontidae, and Zonitidae, the calcified egg is viewed as a primitive land snail trait associated with terrestrial adaptation. The function of the calcified egg shell, in addition to mechanical support of egg contents, is to supply the developing embryo with enough calcium to form the embryonic shell by the time of hatching.     

Fertility and hatchability of eggs laid in the pullet-to-breeder transition period and in the initial production period

The initial eggs produced by broiler breeder hens are relatively small compared with later in the production cycle. An evaluation of indices related to hatchability is required when these eggs are to be used for the production of broiler chicks. Two experiments were conducted to evaluate characteristics related to the hatchability of eggs from pullet-to-breeder transition phase, at 25 and 27 weeks of age, and from the peak of production period and five weeks later, at 32 and 37 weeks of age. Eggs from birds 25 weeks had a lesser fertility in Experiment 1. Mortality occurred unevenly in early (1–5 days), middle (6–17 days) and late (18–21 days) incubation, and greater mortality was observed after the internal membrane was ruptured. The younger the hen, the lighter the egg, chick, and shell, and the longer the time required to complete the hatching process. In Experiment 2, greater mortalities were observed at the early period (1–5 days) and after “pipping” of the internal and external membranes. Embryos from heavy eggs of breeder hens 37 weeks of age took less time to complete the hatching process. Results indicated the larger the egg, the heavier the chick and shell, and the lesser the shell percentage. As breeder age advanced, characteristics related to egg fertility and hatchability improved.     

Age of adult worms of Echinostoma caproni does not affect development of miracidia

The effect of the age of adult Echinostoma caproni on egg development was studied. The percentage of fully developed miracidia was determined in eggs derived from adult worms obtained from laboratory mice at 2, 4, 6, and 8 wk postinfection (PI). Regardless of the age of worms from which the eggs were obtained, the percentage of fully developed miracidia was always >90%, and 60-80% of the eggs hatched. Several previous studies have shown that eggs derived from 2- to 4-wk-old E. caproni yielded miracidia that infected Biomphalaria glabrata snails. Results of the present study on E. caproni were in marked contrast to previous results with Echinostoma friedi, for which viable eggs were not obtained at 2 and 3 wk PI and maximal infectivity of miracidia in snails was obtained from eggs derived from worms collected at 8 and 9 wk PI. Further studies are needed to determine if the egg viability of other species in the "revolutum" group follow that of E. caproni or E. friedi.     

Changes in the reproductive biology of Biomphalara glabrata infected with different doses of Echinostoma paraensei miracidia

The egg-laying rate, number of egg masses, number of eggs/mass, number of eggs hatched/snail and egg viability of Biomphalaria glabrata exposed to different doses (5 and 50) of Echinostoma paraensei miracidia were analyzed as indicators of reproductive activity. Polystyrene plates were placed in aquariums containing the snails and every other day for four weeks after infection the plates were removed to count the number of egg masses and eggs laid. After this, the plates were numbered individually and placed in new aquariums free of snails and the egg masses were observed daily to determine the hatching rate. On average there was an increase in the parameters evaluated in the infected snails in relation to the controls (uninfected snails), except for egg viability, which was significantly lower in the groups infected with 50 miracidia. These findings indicate that when infected, this host snail is able to increase its reproductive activity, suggesting an ecological strategy to maintain the species.     

Non-invasive measurement of oxygen partial pressure, lateral diffusion and chorioallantoic blood flow under the avian eggshell.

We measured P(O2) under the shell of avian eggs indirectly, by sealing 0.05 mL glass tubes to the shell, sealing them with mercury and using an oxygen microelectrode to measure the contained gas that equilibrates with the gas in the shell membranes. This technique requires a smaller area of contact with the shell and a shorter equilibration period than established techniques, and allows measurements at several locations simultaneously and over a long period of time without endangering the embryo. P(O2) under the shell of chicken eggs decreased to 14.3 kPa on the day before hatching (day 19). P(O2) was unstable during late development and differences up to 3.1 kPa occurred transiently on opposite sides of the equator. By waxing the shell around sampling tubes, we estimated Krogh's coefficient for lateral oxygen diffusion in the shell membranes at 1.1 mmol cm(-1) d(-1) kPa(-1), a value about a third of a previous estimate. Sampling of gas under sufficiently large regions of waxed shell allowed indirect measurements of chorioallantoic venous P(O2), without affecting embryonic respiration. Venous P(O2) was 3.8 kPa on day 19. Assuming 14.3 kPa represents arterialized blood leaving the chorioallantois, it became possible to calculate the effective chorioallantoic blood flow rate, which was 3.5 mL min(-1) on day 19.     

Pre- and postnatal energetics of the North Island brown kiwi (Apteryx mantelli)

In four eggs and four chicks of the North Island brown kiwi (Apteryx mantelli) we measured pre- and postnatal oxygen consumption rate (VO(2)) daily from day (d)-75 (prior to hatching) until d+25 (after hatching). The increase of embryonic VO(2) reaches a plateau phase between d-22 and d-5 (0.113 ml O(2)/g/h=59.6% of allometrically expected value of a typical 416-g egg). Mean total O(2) uptake per egg (43.01 l O(2)) corresponds to an energy turnover rate of 2.04 kJ/g during embryonic development. This is nearly identical to the expected value for all birds (2.00+/-0.8 kJ/g). Hence, the kiwi neither 'gained nor lost energy' (Calder, 1979.Biosci. 8, 461-467) by its extreme prolongation of incubation time; it is as efficient as other avian embryos. The kiwi embryo expends only approximately 17% (847 kJ) of the energy originally stored in the egg (4942 kJ). Forty-eight percent of the egg's initial yolk mass can be found as spare yolk in the hatchling and can serve as the chick's sole source of energy and substrate for tissue production for up to at least 17 days after hatching.     

Hatching mechanism and delayed hatching of the eggs of three broadcast spawning euphausiid species under laboratory conditions

Three different egg hatching mechanisms were observed underlaboratory conditions in Euphausia pacifica Hansen, Thysanoessaspinifera Holmes and Thysanoessa inspinata Nemoto: backward,forward and flipping. Like all broadcast spawning euphausiids,these species usually hatch as nauplius 1 (N1). Some hours beforehatching the vitelline membrane breaks and the embryo is freelysuspended within the chorion; later the embryo takes on a slightlyoval shape. When ready to hatch, the N1 pushes against the chorionwith the posterior part of the abdomen producing a protuberance.No spine or egg tooth is present to break the chorion. The pressurebreaks the chorion, and the nauplius pushes itself backwardswith the first and second antennae and mandible to slide fromthe chorion. After about three quarters of the body is outside,the nauplius brings all the appendages together to move backwardswithout becoming stuck in the chorion. This is the backwardhatching mechanism. The vitelline membrane remains within theegg after the nauplius leaves the chorion. Hatching takes 5–20s, and most of the eggs in a clutch hatch during <2 h. Severaleggs of E. pacifica hatched as meta-nauplii (MN) (>200 hafter spawning) or as calyptopis 1 (C1) stage (>232 h), ratherthan as N1. Delayed hatching of embryos also was observed inT. spinifera as nauplius 2 (N2) (>120 h) or as MN stage (>180h), and in T. inspinata as N2 (106 h) after spawning. Eggs withlarvae in stages of development beyond N1 have not been observedfrom preserved zooplankton samples. However, eggs spawned inthe field and incubated in the laboratory also had extendeddevelopment and late hatching but with low frequency (<0.06%).It is proposed that, if the backward hatching mechanism fails,alternate hatching mechanisms can be used by the euphausiid.There is high flexibility in their hatching modes. The N2 andMN break the chorion with the first and second antennae, hatchingforwards, and the C1 breaks it with the telson spines and byflipping of the abdomen, resembling the decapod hatching mechanism.Delayed hatching using the forward and flipping mechanisms wereassociated with low hatching success in comparison with thebackward hatching mechanism.     

An ultrastructural study of the egg wall surrounding the miracidium of the digenean Brandesia turgida () (Plagiorchiida: Pleurogenidae), with the description of a unique cocoon-like envelope

The intrauterine eggs of the pleurogenid trematode Brandesia turgida (Brandes, 1888), exhibiting advanced stages of miracidial differentiation and fully formed, ciliated miracidia, were examined by means of transmission electron microscopy (TEM). Each embryonated egg is composed of a mature miracidium surrounded by a four-layered egg wall: (1) an outer, anucleate layer external to the eggshell, which forms a thick cocoon; (2) the operculate egg-shell; (3) a small remnant of the compact, granular cytoplasm of the outer embryonic envelope (sensu stricto); and (4) a relatively distinct cellular remnant of the inner embryonic envelope. Layers enveloping the egg apparently play an important role in the protection, metabolism and storage of nutritive reserves for the developing miracidium. The outer, anucleate layer, or cocoon, situated externally to the eggshell and composed of a transparent, electron-lucent substance with numerous dense, osmiophilic islands attached to its peripheral membrane, has never previously been seen in TEM studies of the eggs of parasitic platyhelminths. The origin, formation, functional ultrastructure and chemical composition of this peculiar layer remain enigmatic, although its function appears to be protective. The thick, electron-dense eggshell resembles that of other trematodes, exhibiting a characteristic fissure zone around the operculum. The very small, indistinct remnants of the outer embryonic envelope appear in the form of a very thin, compact, granular cytoplasm closely attached to the inner surface of the eggshell. Conversely, the inner embryonic envelope is frequently apparent at one or both poles of the developed egg as a syncytial envelope formed by the fusion of mesomeres. This envelope, even in eggs containing a fully formed miracidium, still has the features of a metabolically active layer with an energy storage capability. Lysosome-like structures observed in some eggs may be involved in the autolysis of the embryonic envelopes.     

An ultrastructural study of the egg wall surrounding the miracidium of the digenean Brandesia turgida ( ) (Plagiorchiida: Pleurogenidae), with the description of a unique cocoon-like envelope

The intrauterine eggs of the pleurogenid trematode Brandesia turgida ( Brandes, 1888), exhibiting advanced stages of miracidial differentiation and fully formed, ciliated miracidia, were examined by means of transmission electron microscopy (TEM). Each embryonated egg is composed of a mature miracidium surrounded by a four-layered egg wall: (1) an outer, anucleate layer external to the eggshell, which forms a thick cocoon; (2) the operculate egg-shell; (3) a small remnant of the compact, granular cytoplasm of the outer embryonic envelope (sensu stricto); and (4) a relatively distinct cellular remnant of the inner embryonic envelope. Layers enveloping the egg apparently play an important role in the protection, metabolism and storage of nutritive reserves for the developing miracidium. The outer, anucleate layer, or cocoon, situated externally to the eggshell and composed of a transparent, electron-lucent substance with numerous dense, osmiophilic islands attached to its peripheral membrane, has never previously been seen in TEM studies of the eggs of parasitic platyhelminths. The origin, formation, functional ultrastructure and chemical composition of this peculiar layer remain enigmatic, although its function appears to be protective. The thick, electron-dense eggshell resembles that of other trematodes, exhibiting a characteristic fissure zone around the operculum. The very small, indistinct remnants of the outer embryonic envelope appear in the form of a very thin, compact, granular cytoplasm closely attached to the inner surface of the eggshell. Conversely, the inner embryonic envelope is frequently apparent at one or both poles of the developed egg as a syncytial envelope formed by the fusion of mesomeres. This envelope, even in eggs containing a fully formed miracidium, still has the features of a metabolically active layer with an energy storage capability. Lysosome-like structures observed in some eggs may be involved in the autolysis of the embryonic envelopes.     

Comparison of heart rate in embryonic,young and adult Achatina fulica

We have studied the embryonic heart in the gastropod Achatina fulica using eggs soon after their deposition. We also followed the development of organs, with the main emphasis on the heart and its physiology. The embryos were examined at different stages by carefully removing egg shell until hatching. In this context the heart rate (HR) was analysed in embryonic snails and compared with that of young (from hatching) and adults. The HR of embryos dissected from newly deposited eggs was irregular, with a mean frequency of 0.42?±?0.14?Hz. The heartbeat during the ontogeny was more rhythmic and HR increased to 0.82?±?0.12?Hz. The intersystolic intervals (ISI) in real time showed a Gaussian distribution for all animals. The peak ISI from Gaussian fit closely matched the cumulative probability for corresponding data. Although the HR increased after hatching, comparisons yield a weak correlation (R?=?0.56?±?0.27) with the total body weight of embryonic, young and adult snails.     

The impact of flucycloxuron on eggs weight kinetic and hematological parameters of chicken (Gallus domesticus)

The struggle against the harmful bugs of culture is intensified, and several products are appeared every year without the knowledge how to control their effects on environment and especially on being life. The introduced chemical products in nature are generally, the synthesis products witch are the pesticides. Our study consist the impact mechanism of a pesticides (FCX) on other biological model than harmful bugs, this biological model is a vertebrate model witch is the domestic chicken eggs (Gaollus domesticus). The toxicity of Flucycloxuron reviewed across the eggs weight kinetic accompanied with embryonic hematological parameters, in ovo and after hatching. The tested concentrations of pesticide are 1, 10 and 20 microg/egg injected at first day of incubation. Eggs treatment by three concentrations of pesticides, disturbs the studied parameters, where we observe that the pesticide inhibit the nutriment transformation, translated by eggs decreased weight kinetic according to the control, also the FCX affect the shell weight and cause the alteration of shell integrity. Hematological parameters show a clear impact of the pesticide at the lowest concentration (1 microg/egg). The obtained results confirm that the chosen biological model is good bio-indicator for eventual pollution and they are not far from pesticides toxicity.     

Non-invasive measurement of oxygen partial pressure, lateral diffusion and chorioallantoic blood flow under the avian eggshell

We measured P(O2) under the shell of avian eggs indirectly, by sealing 0.05 mL glass tubes to the shell, sealing them with mercury and using an oxygen microelectrode to measure the contained gas that equilibrates with the gas in the shell membranes. This technique requires a smaller area of contact with the shell and a shorter equilibration period than established techniques, and allows measurements at several locations simultaneously and over a long period of time without endangering the embryo. P(O2) under the shell of chicken eggs decreased to 14.3 kPa on the day before hatching (day 19). P(O2) was unstable during late development and differences up to 3.1 kPa occurred transiently on opposite sides of the equator. By waxing the shell around sampling tubes, we estimated Krogh's coefficient for lateral oxygen diffusion in the shell membranes at 1.1 mmol cm(-1) d(-1) kPa(-1), a value about a third of a previous estimate. Sampling of gas under sufficiently large regions of waxed shell allowed indirect measurements of chorioallantoic venous P(O2), without affecting embryonic respiration. Venous P(O2) was 3.8 kPa on day 19. Assuming 14.3 kPa represents arterialized blood leaving the chorioallantois, it became possible to calculate the effective chorioallantoic blood flow rate, which was 3.5 mL min(-1) on day 19.     

Effects of electromagnetic fields on the reproductive success of American kestrels

Reduced reproductive success of birds nesting near power lines has been documented but never directly attributed to electromagnetic fields (EMFs). Laboratory studies have identified EMF effects on embryonic development, but reproductive success of wild birds is dependent on additional factors, including fertility, egg size, hatching, and fledging success. We tested whether EMFs affect reproductive success of birds. Captive American kestrels (Falco sparverius) were bred for one season per year for 2 yr under either controlled or EMF conditions. EMF exposure was equivalent to that experienced by wild reproducing kestrels and was weakly associated with reduced egg laying in 1 yr only. In both years fertility was higher, but hatching success was lower in EMF pairs than control pairs. Fledging success was higher in EMF pairs than control pairs in 1995 only. Egg composition and embryonic development were examined in 1 yr only, but hatchlings were measured in both years. EMF eggs were larger, with more yolk, albumen, and water, but had thinner egg shells than control eggs. Late-term EMF embryos were larger and longer than control embryos, although hatchlings were similar in body mass and size. EMF exposure affected reproductive success of kestrels, increasing fertility, egg size, embryonic development, and fledging success but reducing hatching success.     

Some effects of suction on the hatching eggs of Meloidogyne javanica

Development and hatch of eggs of Meloidogyne javanica was independent of suction between pF o and 3·6 but decreased rapidly between pF 3·6 and 4·2. Water was lost from the extracellular fluid of eggs containing developing embryos when the suction was above pF 3·6, causing a decrease in the mean volume of the eggs. The vitelline membrane of an egg is probably semipermeable and the osmotic pressure of the contents about 4 atmospheres (pF 3·6). The volume of the cellular contents of an egg did not increase during development and so the hydraulic conductivity of the soil is probably unimportant in hatching. The results suggest that eggs of M. javanica hatch over a wide range of soil moistures. The inhibition of hatch at high suctions, involving the reversible removal of water, is a likely survival mechanism.     

Electroporation Facilitates Introduction of Reporter Transgenes and Virions into Schistosome Eggs

Background

The schistosome egg represents an attractive developmental stage at which to target transgenes because of the high ratio of germ to somatic cells, because the transgene might be propagated and amplified by infecting snails with the miracidia hatched from treated eggs, and because eggs can be readily obtained from experimentally infected rodents.

Methods/Findings

We investigated the utility of square wave electroporation to deliver transgenes and other macromolecules including fluorescent (Cy3) short interference (si) RNA molecules, messenger RNAs, and virions into eggs of Schistosoma mansoni. First, eggs were incubated in Cy3-labeled siRNA with and without square wave electroporation. Cy3-signals were detected by fluorescence microscopy in eggs and miracidia hatched from treated eggs. Second, electroporation was employed to introduce mRNA encoding firefly luciferase into eggs. Luciferase activity was detected three hours later, whereas luciferase was not evident in eggs soaked in the mRNA. Third, schistosome eggs were exposed to Moloney murine leukemia virus virions (MLV) pseudotyped with vesicular stomatitis virus glycoprotein (VSVG). Proviral transgenes were detected by PCR in genomic DNA from miracidia hatched from virion-exposed eggs, indicating the presence of transgenes in larval schistosomes that had been either soaked or electroporated. However, quantitative PCR (qPCR) analysis determined that electroporation of virions resulted in 2–3 times as many copies of provirus in these schistosomes compared to soaking alone. In addition, relative qPCR indicated a copy number for the proviral luciferase transgene of ∼20 copies for 100 copies of a representative single copy endogenous gene (encoding cathepsin D).

Conclusions

Square wave electroporation facilitates introduction of transgenes into the schistosome egg. Electroporation was more effective for the transduction of eggs with pseudotyped MLV than simply soaking the eggs in virions. These findings underscore the potential of targeting the schistosome egg for germ line transgenesis.     

Extremely low frequency magnetic field and the hatching rate of Fasciola hepatica eggs, the fecundity and survival of liver fluke-infected snail, Lymnaea truncatula

Eggs of Fasciola hepatica were exposed for 10 days to extremely low frequency magnetic field (ELFMF) at the frequency of 50 Hz and density of 2 mT (rms). The results show an accelerated hatching of F. hepatica eggs in relation to control (non-exposed) group. The host snails, Lymnaea truncatula, were divided into three groups; those of groups I and II were infected with the miracidia of F. hepatica hatched from control egg culture, whereas those of group III were infected with miracidia hatched from eggs affected by ELFMF. Thereafter, snails of groups II and III were exposed to ELFMF for 53 days, whereas those of group I were not exposed. At day 14 post infection, a significant decrease was observed in the number of cocoons laid by snails of group III, compared with control. Also, significant mortality in group III snails was observed 42 days post infection. The increased mortality and a lower number of cocoons laid by group III snails have probably resulted from enhanced stimulation of metacercarial parthenogenetic reproduction in consequence of infecting the molluscs with miracidia reared under ELFMF.     

Characterisation of proteases involved in egg hatching of the sheep blowfly, Lucilia cuprina

A number of proteases were identified in the egg shell washings (ESW) collected during the egg hatching of Lucilia cuprina (sheep blowfly). Characterization of these proteases indicated a pH optima in a similar pH range that was optimal for L. cuprina egg hatching. Mechanistic characterization of these proteases indicated that they were predominantly of the serine class. Several protease inhibitors were tested for their ability to inhibit L. cuprina egg hatching in vitro. Egg hatching was significantly (P<0.05) inhibited by PMSF (61%), 1,10-Phenanthroline (42%) and Pepstatin (29%). The inhibition of egg hatching by PMSF showed a strong concentration dependence, with its effects ranging from inhibition at high concentrations to enhancement of egg hatching at low concentrations. Addition of ESW to unhatched eggs, significantly (P<0.05) enhanced their rate of hatching above untreated control eggs. This enhancement of egg hatching was significantly (P<0.05) reversed by the protease inhibitors Elastatinal (40%), 1,10-Phenanthroline (40%) and PMSF (38%). These studies indicate a role for serine and/or metallo-proteases in facilitating L. cuprina egg hatch.