Pond Snail Embryos Research Articles

A new set of endogenous control genes for use in quantitative real-time PCR experiments show that formin Ldia2dex transcripts are enriched in the early embryo of the pond snail Lymnaea stagnalis (Panpulmonata)

ABSTRACT Although the pond snail Lymnaea stagnalis is an emerging model organism for molecular studies in a wide variety of fields, there are a limited number of verified endogenous control genes for use in quantitative real-time PCR. As part of a larger study on snail chirality, or left–right asymmetry, we assayed gene expression in pond snail embryos. We evaluated six candidate control genes, by comparing their expression in three tissues (ovotestis, foot and embryo) and used three software programmes (geNorm, Normfinder and Bestkeeper) to do so. The specific utility of these control genes was then tested by investigating the relative expression of six experimental transcripts, including formin Ldia2, a gene that has been associated with chiral variation in L. stagnalis. All six control genes were found to be suitable for use in the three tissues tested. Of the six experimental genes, it was found that all were relatively depleted in the early embryo compared with other tissues, except the formin Ldia2 gene. Instead, transcripts of the wild-type Ldia2dex were enriched in the embryo, whereas a nonfunctional frameshifted version, Ldia2sin, was severely depleted. These differences in Ldia2sin expression were less evident in the ovotestis and were not evident in the foot tissue, possibly because nonsense-mediated decay is obscured in actively transcribing tissues. Our work provides a set of control genes that may be useful to the wider community and illustrates how these genes may be used to assay differences in expression in a variety of tissues.

Radiation-induced cytogenetic and hematologic effects on aquatic biota within the Chernobyl exclusion zone

During 1998–2014 the rate of chromosomal aberrations in embryo tissues of the pond snail (Lymnaea stagnalis) and root meristems of higher aquatic plants, and also hematologic indexes of mantle liquid of the adult snails and peripheral blood of fishes in water bodies within the Chernobyl exclusion zone (EZ) was studied. The absorbed dose rate for hydrobionts from water bodies of the EZ registered in a range from 0.25 to 420 μGy h−1 and in the reference ones – up to 0.09 μGy h−1. The level of chromosomal aberrations in the molluscs from the most contaminated water bodies of the EZ was registered within range of 18–27% and for the molluscs from the reference lakes this index was on the average 1.5% with the maximal values 2.3%. The rate of chromosomal aberrations in root meristematic cells of higher aquatic plants from the contaminated lakes of the EZ was in range of 7–17% and in the plants from reference water bodies was not exceed 2.1%. The positive correlation between chromosomal aberration rate and absorbed dose rate in the pond snail's embryos and root meristems of higher aquatic plants in water bodies of the EZ was registered. Analysis of hemolymph structure of snails from the most contaminated water bodies showed a high rate of dead and phagocytic cells as well as decrease of the young amoebocytes quantity. Hematologic research of fish allows to determine on the one hand an insignificant changes of leukogram structure, and from the other hand a high level of red cells with different abnormalities in the peripheral blood of fishes from the water bodies with high levels of radioactive contamination. It is suppose that qualitative indexes of red cells in peripheral blood of fish are more sensitive to long-term radiation impact in comparison with elements of white blood, which can be used for conducting of the hematologic monitoring of radioactive contaminated water bodies.

Forced Convection Past a Rotating Sphere: Modeling Oxygen Transport to a Pond Snail Embryo

Helisoma trivolvis pond snail embryos are known for their rotation, which is induced by beating of cilia at the embryo's surface. A common hypothesis links this behavior to enhancing oxygen transfer to the embryo's surface. In this paper, this hypothesis is quantified, and the effect of the rotation on the supply of oxygen to an embryo, which is approximately spherical in shape, is studied. To the best of our knowledge, this is the first research presenting a quantitative study on the effect of an embryo's rotation on facilitating gaseous exchange between the embryo and the environment.

Rotational behaviour of encapsulated pond snail embryos in diverse natural environments

Encapsulated freshwater pond snail embryos display a cilia-driven rotation behaviour that is stimulated by artificially induced hypoxia. Previous studies have suggested that the mixing effect of this behaviour causes enhanced oxygen delivery to embryos within their egg capsules. Despite extensive laboratory-based studies describing this behaviour, it is unclear how this behaviour is used to cope with changes in oxygen concentration and other environmental factors in natural water bodies. We made field measurements of embryo rotation rates in laboratory-reared Helisoma trivolvis embryos placed in ponds of different trophic levels that ranged geographically from the southern Alberta prairie to the Rocky Mountains. Abiotic factors including temperature, pH, conductivity and water oxygen concentration were measured to understand how embryonic rotation is influenced by environmental conditions. Results showed that H. trivolvis embryos exhibit differences in rotational behaviour depending on the environmental conditions. Temperature and oxygen concentration were the primary factors significantly affecting rotation rates. The effect of oxygen concentration on rotation rates was not as widespread as observed under laboratory conditions, probably because the measured oxygen concentrations were above the range that influences embryonic rotation in the laboratory. The rotational behaviour of laboratory-reared Lymnaea stagnalis provided confirmation that embryos of other encapsulated pulmonates exhibit a similar rotational response in natural environments. These results suggest that embryo rotation is influenced by a complex interplay of environmental factors.

Serotonin prolongs survival of encapsulated pond snail embryos exposed to long-term anoxia

Embryos of the pond snail, Helisoma trivolvis, develop bilateral serotonergic neurons that innervate ciliary bands and stimulate cilia-driven rotation. This behaviour is postulated to increase oxygen availability during hypoxia by mixing the capsular fluid. We hypothesised that the stimulation of ciliary-driven rotation by serotonin (5-HT) enhances the survival of embryos during prolonged hypoxia. Embryo rotation and survival were monitored in different levels of oxygen for 24-48 h while in the presence or absence of 5-HT (100 micromol l(-1)) or a 5-HT antagonist (50 micromol l(-1)). Long-term hypoxia caused delayed embryonic development that appeared morphologically normal. Hypoxia also induced a transient increase in rotation rate in embryos exposed to artificial pond water (APW) or 5-HT that lasted around 3 h. 5-HT-treated embryos had an elevated rotation rate over embryos in APW throughout the long-term exposure to hypoxia. Long-term anoxia also induced a transient increase in rotation rate in embryos exposed to APW or 5-HT. Rotation ceased in embryos exposed to APW by 13 h but persisted in 5-HT-treated embryos for up to 40 h. Fifty percent mortality was reached at 9 h of anoxia in embryos in APW and at 24 h in 5-HT-treated embryos. The 5-HT antagonist mianserin partially inhibited the 5-HT enhancement of rotation but not the prolongation of survival in anoxia. The ability of 5-HT to prolong survival in anoxia reveals a 5-HT-activated metabolic pathway that liberates an alternative energy source.

Expression of exogenous fluorescent proteins in early freshwater pond snail embryos

We have for the first time succeeded in expressing in vitro-synthesized mRNAs in both the sinistral and the dextral Lymnaea stagnalis early embryos by microinjecting the mRNAs into the eggs before the first polar body stage. Translation of exogenous mRNA in developing embryos was confirmed by expressing various fluorescent proteins; mCherry, DsRed-Express, and enhanced green fluorescent protein. We have found that the protein expression derived from the introduced exogenous mRNA largely depends on the elapsed time after the microinjection and not on the developmental stage of injection, and also on the amount of injected mRNA. Developmental abnormalities were hardly observed. The first notable fluorescent signal was detected within 2-3 h after the injection while the embryos were still in uncleaved stage. Fluorescence gradually increased until 8-9 h and was stable up to 24 h. From these results, it is suggested that there is enough translation machinery necessary for early development and the translation of injected mRNA proceeds immediately and constantly in the early embryos. This is true for both the sinistral and dextral L. stagnalis embryos. Application of the developed method to other freshwater pond snails, dextral Lymnaea peregra, sinistral Physa acuta, and sinistral Indoplanorbis exustus revealed that their early expression mechanisms to be similar to that of L. stagnalis. Thus, in vitro-synthesized mRNA expression is expected to be important for the understanding of evolutional process and the molecular mechanism underlining the handedness determination in these freshwater snail embryos.

Serotonergic sensory-motor neurons mediate a behavioral response to hypoxia in pond snail embryos.

Oxygen (O(2)) is one of the most important environmental factors that affects both physiological processes and development of aerobic animals, yet little is known about the neural mechanism of O(2) sensing and adaptive responses to low O(2) (hypoxia) during development. In the pond snail, Helisoma trivolvis, the first embryonic neurons (ENC1s) to develop are a pair of serotonergic sensory-motor cells that regulate a cilia-driven rotational behavior. Here, we report that the ENC1-ciliary cell circuit mediates an adaptive behavioral response to hypoxia. Exposure of egg masses to hypoxia elicited a dose-dependent and reversible acceleration of embryonic rotation that mixed capsular fluid, thereby facilitating O(2) diffusion to the embryo. The O(2) partial pressures (Po(2)) for threshold, half-maximal, and maximal rotational response were 60, 28, and 13 mm Hg, respectively. During hypoxia, embryos relocated to the periphery of the egg masses where higher Po(2) levels occurred. Furthermore, intermittent hypoxia treatments induced a sensitization of the rotational response. In isolated ciliary cells, ciliary beating was unaffected by hypoxia, suggesting that in the embryo, O(2) sensing occurs upstream of the motile cilia. The rotational response of embryos to hypoxia was attenuated by application of the serotonin receptor antagonist, mianserin, correlated to the development of ENC1-ciliary cell circuit, and abolished by laser-ablation of ENC1s. Together, these data suggest that ENC1s are unique oxygen sensors that may provide a good single cell model for the examination of mechanistic, developmental, and evolutionary aspects of O(2) sensing.