Isolation and evaluation of dextral-specific and dextral-enriched cDNA clones as candidates for the handedness-determining gene in a freshwater gastropod,<Emphasis Type="Italic"> Lymnaea stagnalis</Emphasis>

The handedness of gastropods is genetically determined. The freshwater gastropod Lymnaea stagnalis is a normally dextral species, but contains minor sinistral populations. The gene responsible for handedness determination in this species is predicted to function maternally and specifically in the dextral-ovipositing snail. In this study, we used differential screening and cDNA subtraction to isolate eight dextral genes that are specific to, or enriched in, the dextral-ovipositing strains of L. stagnalis. These genes were promising candidates for the handedness-determining gene. In order to determine whether the true handedness-determining gene was among them, we tested for genetic correlations between the level of expression of each dextral gene and the handedness phenotype, i.e., the chirality of the next generation offspring, by using a collection of backcross F₂ progeny of F₁ offspring from crosses between dextral and sinistral strains. Although the present study could not identify the handedness-determining molecules, this approach appears to be promising for the isolation of such developmentally important genes.Edited by N. Satoh     

The effect of coil phenotypes and genotypes on the fecundity and viability of Partula suturalis and Lymnaea stagnalis: implications for the evolution of sinistral snails

Why are sinistral snails so rare? Two main hypotheses are that selection acts against the establishment of new coiling morphs, because dextral and sinistral snails have trouble mating, or else a developmental constraint prevents the establishment of sinistrals. We therefore used an isolate of the snail Lymnaea stagnalis, in which sinistrals are rare, and populations of Partula suturalis, in which sinistrals are common, as well as a mathematical model, to understand the circumstances by which new morphs evolve. The main finding is that the sinistral genotype is associated with reduced egg viability in L. stagnalis, but in P. suturalis individuals of sinistral and dextral genotype appear equally fecund, implying a lack of a constraint. As positive frequency‐dependent selection against the rare chiral morph in P. suturalis also operates over a narrow range (< 3%), the results suggest a model for chiral evolution in snails in which weak positive frequency‐dependent selection may be overcome by a negative frequency‐dependent selection, such as reproductive character displacement. In snails, there is not always a developmental constraint. As the direction of cleavage, and thus the directional asymmetry of the entire body, does not generally vary in other Spiralia (annelids, echiurans, vestimentiferans, sipunculids and nemerteans), it remains an open question as to whether this is because of a constraint and/or because most taxa do not have a conspicuous external asymmetry (like a shell) upon which selection can act.     

On the origin of the yolk protein ferritin in snails

Summary The iron storage protein, ferritin, is the major yolk protein in freshwater snails. In this report we show by in vitro labelling experiments that yolk ferritin of the snails Lymnaea stagnalis L. and Planorbarius corneus L. is an exogenous protein synthesized in the midgut gland and secreted into the hemolymph. Gonad and mantle tissue are inactive in the synthesis of yolk ferritin, but, together with the midgut gland, they synthesize another ferritin type (soma ferritin) which is not released into the hemolymph and which may be a housekeeping ferritin. Soma ferritin and yolk ferritin are not in a precursor/product relationship since subunits of both ferritins are synthesized as primary translation products in rabbit reticulocyte lysate programmed with poly (A)⁺ RNA from midgut gland and gonad. Results suggest that both ferritins are synthesized on different mRNAs (and possibly on different genes) so they may be regulated in a different way.     

Possible functions of Dpp in gastropod shell formation and shell coiling

We examined dpp expression patterns in the pulmonate snail Lymnaea stagnalis and analyzed the functions of dpp using the Dpp signal inhibitor dorsomorphin in order to understand developmental mechanisms and evolution of shell formation in gastropods. The dpp gene is expressed in the right half of the circular area around the shell gland at the trochophore stage and at the right-hand side of the mantle at the veliger stage in the dextral snails. Two types of shell malformations were observed when the Dpp signals were inhibited by dorsomorphin. When the embryos were treated with dorsomorphin at the 2-cell and blastula stages before the shell gland is formed, the juvenile shells grew imperfectly and were not mineralized. On the other hand, when treated at the trochophore and veliger stage after the shell gland formation, juvenile shells grew to show a cone-like form rather than a normal coiled form. These results indicated that dpp plays important roles in the formation and coiling of the shell in this gastropod species.     

Heterosis and inbreeding depression in bottlenecked populations: a test in the hermaphroditic freshwater snail Lymnaea stagnalis

Small population size is expected to induce heterosis, due to the random fixation and accumulation of mildly deleterious mutations, whereas within‐population inbreeding depression should decrease due to increased homozygosity. Population bottlenecks, although less effective, may have similar consequences. We tested this hypothesis in the self‐fertile freshwater snail Lymnaea stagnalis, by subjecting experimental populations to a single bottleneck of varied magnitude. Although patterns were not strong, heterosis was significant in the most severely bottlenecked populations, under stressful conditions. This was mainly due to hatching rate, suggesting that early acting and highly deleterious alleles were involved. Although L. stagnalis is a preferential outcrosser, inbreeding depression was very low and showed no clear relationship with bottleneck size. In the less reduced populations, inbreeding depression for hatching success increased under high inbreeding. This may be consistent with the occurence of synergistic epistasis between fitness loci, which may contribute to favour outcrossing in L. stagnalis.     

The polarity protein Par6 is coupled to the microtubule network during molluscan early embryogenesis

Cell polarity, which directs the orientation of asymmetric cell division and segregation of fate determinants, is a fundamental feature of development and differentiation. Regulators of polarity have been extensively studied, and the critical importance of the Par (partitioning-defective) complex as the polarity machinery is now recognized in a wide range of eukaryotic systems. The Par polarity module is evolutionarily conserved, but its mechanism and cooperating factors vary among different systems. Here we describe the cloning and characterization of a pond snail Lymnaea stagnalis homologue of partitioning-defective 6 (Lspar6). The protein product LsPar6 shows high affinity for microtubules and localizes to the mitotic apparatus during embryonic cell division. In vitro assays revealed direct binding of LsPar6 to tubulin and microtubules, which is the first evidence of the direct interaction between the two proteins. The interaction is mediated by two distinct regions of LsPar6 both located in the N-terminal half. Atypical PKC, a functional partner of Par6, was also found to localize to the mitotic spindle. These results suggest that the L. stagnalis Par complex employs the microtubule network in cell polarity processes during the early embryogenesis. Identical sequence and localization of LsPar6 for the dextral and the sinistral snails exclude the possibility of the gene being the primary determinant of handedness.     

The rate of oxygen consumption during embryogenesis of Lymnaea stagnalis (Gastropoda)

We studied the rate of oxygen consumption by the Lymnaea stagnalis embryos. The rate of oxygen consumption increased consistently during embryogenesis. The volume specific rate of oxygen consumption increased initially from the early cleavage stages until the gastrula stage and then decreased gradually to the eclosion of snails. There are three periods in embryogenesis of L. stagnalis, which differ in the coefficients of allometric dependence between the rate of oxygen consumption and volume of embryos: (1) early embryogenesis, when the increase in the rate of oxygen consumption is not accompanied by the growth of volume of the embryos; (2) larval period (trochophore and veliger stages; exponential coefficient k = 0.514), and (3) postlarval period (exponential coefficient k = 0.206).     

Embryonic rotational behaviour in the pond snail Lymnaea stagnalis: influences of environmental oxygen and development stage

Responses of freshwater organisms to environmental oxygen tensions (PO₂) have focused on adult (i.e. late developmental) stages, yet responses of embryonic stages to changes in environmental PO₂ must also have implications for organismal biology. Here we assess how the rotational behaviour of the freshwater snail Lymnaea stagnalis changes during development in response to conditions of hypoxia and hyperoxia. As rotation rate is linked to gas mixing in the fluid surrounding the embryo, we predicted that it would increase under hypoxic conditions but decrease under hyperoxia. Contrary to predictions, early, veliger stage embryos showed no change in their rotation rate under hyperoxia, and later, hippo stage embryos showed only a marginally significant increase in rotation under these conditions. Predictions for hypoxia were broadly supported, however, with both veliger and hippo stages showing a marked hypoxia-related increase in their rotation rates. There were also subtle differences between developmental stages, with hippos responding at PO₂s (50% air saturation) greater than those required to elicit a similar response in veligers (20% air saturation). Differences between developmental stages also occurred on return to normoxic conditions following hypoxia: rotation in veligers returned to pre-exposure levels, whereas there was a virtual cessation in embryos at the hippo stage, likely the result of overstimulation of oxygen sensors driving ciliary movement in later, more developed embryos. Together, these findings suggest that the spinning activity of L. stagnalis embryos varies depending on environmental PO₂s and developmental stage, increasing during hypoxia to mix capsular contents and maintain a diffusive gradient for oxygen entry into the capsule from the external environment (“stir-bar” theory of embryonic rotational behaviour).     

Evolution of a dextral lineage by left–right reversal in Cristataria (Gastropoda,Pulmonata, Clausiliidae)

It has long been debated whether mirror image‐like similarity in shell morphology between enantiomorphic pairs of dextral and sinistral taxa represents their sister relationship, or each of them is closer related to other congeners with the same coiling direction. The obligate rock‐dwelling genus Cristataria Vest, 1867 of the eastern Mediterranean region belongs to the Alopiinae subfamily of door snails (Clausiliidae). Cristataria and a few other genera of this subfamily include enantiomorphic pairs that are conchologically very similar to each other. Dextral C. colbeauiana (Pfeiffer, 1861) and its sinistral counterpart of such an enantiomorphic pair occur nearby one another in southern Turkey. However, the latter has been classified either as the sinistral subspecies C. colbeauiana inversa Szekeres, 1998 or as a form of sinistral C. leprevieri (Pallary, 1922). To examine the phylogenetic relationship of this enantiomorphic pair, we carried out molecular phylogenetic analysis of all the Turkish and two other Cristataria taxa based on both mitochondrial and nuclear DNA markers. Our results show that dextral C. colbeauiana and its sinistral counterpart are closest related to one another. This supports the classification of this enantiomorphic pair as dextral C. colbeauiana colbeauiana and sinistral C. colbeauiana inversa. Our results also reveal that these taxa and C. intersita Németh & Szekeres, 1995, sharing a characteristic collar behind the aperture of the shell, represent a monophyletic lineage. By contrast, the Cristataria species of non‐collared shells belong to another clade.     

Transition of Xwnt-11 mRNA from inactive form to polyribosomes in frogs during early embryogenesis

The distribution of Xwnt-11 mRNA between polysomes and informosomes was studied in Xenopus laevis and Rana temporaria during early embryogenesis. The ratio between polysomes and informosomes suggests their involvement in translation of these mRNAs. In eggs and immediately after fertilization the Xwnt-11 mRNAs are mostly positioned in informosomes. During the cleavage stage, these mRNAs have also been recognized in polysomes. Just before the onset of zygote genome functioning (at the stage of mid blastula), Xwnt-11 mRNA rapidly appears in polysomes of Rana embryos. However, in Xenopus, Xwnt-11 mRNA appears in polysomes only at the end of gastrula. Before this stage, the Xwnt-11 mRNA in Xenopus can be found mostly in informosomes.     

Fine Mapping of the Pond Snail Left-Right Asymmetry (Chirality) Locus Using RAD-Seq and Fibre-FISH

The left-right asymmetry of snails, including the direction of shell coiling, is determined by the delayed effect of a maternal gene on the chiral twist that takes place during early embryonic cell divisions. Yet, despite being a well-established classical problem, the identity of the gene and the means by which left-right asymmetry is established in snails remain unknown. We here demonstrate the power of new genomic approaches for identification of the chirality gene, “D”. First, heterozygous (Dd) pond snails Lymnaea stagnalis were self-fertilised or backcrossed, and the genotype of more than six thousand offspring inferred, either dextral (DD/Dd) or sinistral (dd). Then, twenty of the offspring were used for Restriction-site-Associated DNA Sequencing (RAD-Seq) to identify anonymous molecular markers that are linked to the chirality locus. A local genetic map was constructed by genotyping three flanking markers in over three thousand snails. The three markers lie either side of the chirality locus, with one very tightly linked (<0.1 cM). Finally, bacterial artificial chromosomes (BACs) were isolated that contained the three loci. Fluorescent in situ hybridization (FISH) of pachytene cells showed that the three BACs tightly cluster on the same bivalent chromosome. Fibre-FISH identified a region of greater that ∼0.4 Mb between two BAC clone markers that must contain D. This work therefore establishes the resources for molecular identification of the chirality gene and the variation that underpins sinistral and dextral coiling. More generally, the results also show that combining genomic technologies, such as RAD-Seq and high resolution FISH, is a robust approach for mapping key loci in non-model systems.     

Acid-Base Balance Modulates Respiratory and Alimentary Behavior of the Mollusc Lymnaea stagnalis

An active reaction (change of pH) in hemolymph of the freshwater pulmonate mollusc Lymnaea stagnalis is studied in various physiological states: hunger, satiety, and alimentary excitation. In satiated animals the hemolymph pH is shifted to the acid area (7.6 ± 0.003) as compared with hungry animals (7.9 ± 0.05). The satiated individuals are characterized by an increased respiratory activity and decreased food consumption as compared with hungry animals. Acidification of the medium leads to an increase of excitability of the respiratory network interneurons (RPeD1, VD4, VD1/RPD2) and to inhibition of activity of the alimentary network interneurons (R/L CGC), whereas alkalization, to opposite effects. It is suggested that pH is one of factors coordinating activity of functional systems of Lymnaea stagnalis due to effect on neuronal correlates of the respiratory and alimentary behavior.     

Maternal effect of low temperature on handedness determination in C. elegans embryos

C. elegans embryos, larvae, and adults exhibit several left-right asymmetries with an invariant dextral handedness, which first becomes evident in the embryo at the 6-cell stage. Reversed (sinistral) handedness was not observed among > 10,000 N2 adults reared at 16°C or 20°C under standard conditions. However, among the progeny of adults reproducing at 10°C, the frequency of animals with sinistral handedness was increased to ∼0.5%. Cold pulse experiments indicated that the critical period for this increase was in early oogenesis, several hours before the first appearance of left-right asymmetry in the embryo. Hermaphrodites reared at 10°C and mated with males reared at 20°C produced sinistral outcross as well as sinistral self-progeny, indicating that the low temperature effect on oocytes was sufficient to cause reversals. Increased frequency of reversal was also observed among animals developed from embryos lacking the egg shell. Possible mechanisms for the control of embryonic handedness are discussed in the context of these results, including the hypothesis that handedness could be dictated by the chirality of a gametic component. © 1996 Wiley-Liss, Inc.     

Expression of photosynthesis-related genes and their regulation by light during somatic embryogenesis in<Emphasis Type="Italic"> Daucus carota</Emphasis>

To clarify the spatial and temporal pattern of gene expression for photosynthesis-associated proteins during somatic embryogenesis in Daucus carota L., the localization of mRNAs for three genes, rbcL, Lhcb and por, was examined in dark-grown and light-irradiated somatic embryos by in situ hybridization. The three mRNAs were expressed in common in the mesophyll precursor cells of light-irradiated embryos at the late torpedo and plantlet stages, but characteristic expression patterns of each photosynthesis-related gene were also observed. Expression of rbcL mRNA first occurred throughout the embryo but gradually became localized in the mesophyll precursor cells and cortex during early embryogenesis. Localization of Lhcb mRNA in the mesophyll precursor cells and shoot apical meristem became clear in the early torpedo stage. Expression of Lhcb mRNA was not affected by light during early embryogenesis, but could be induced by light in the torpedo stage, suggesting that light-inducible expression of Lhcb mRNA arises within the torpedo stage. At the late torpedo stage, clear localization of por mRNA started in mesophyll precursor cells of the cotyledon in light-irradiated embryos. Greening potency of the embryo also appeared first at this stage. Therefore, greening and initial differentiation of photosynthetic tissues during somatic embryogenesis seem to be associated with coordinated expression of mRNA for rbcL, Lhcb and por in late torpedo-shaped embryos.Abbreviations DIG Digoxigenin - Lhcb3 Gene encoding a type-III light-harvesting chlorophyll a/b-binding protein of photosystem II - LHCII Light-harvesting chlorophyll a/b-binding protein of photosystem II - POR Protochlorophyllide oxidoreductase - rbcL Gene encoding the large subunit of Rubisco - Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase     

Relationship between prevalence of trematode parasite <Emphasis Type="Italic">Diplostomum</Emphasis> sp. and population density of its snail host <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis> in lakes and ponds in Finland

Diplostomum sp. is a trematode parasite that infects aquatic snails, e.g. Lymnaea stagnalis (Gastropoda: Lymnaeidae), fish, and fish-eating birds. Ponds and lakes (n = 28) located in Finland between latitudes 61°45′N and 65°30′N were sampled for L. stagnalis, the first intermediate host for Diplostomum sp. L. stagnalis were found in 22 sites out of 28, and Diplostomum sp. in 10 of the 22 snail populations. Among the L. stagnalis populations that were infected by Diplostomum sp., the mean prevalence was 12.8%. Diplostomum sp. occurred in only one out of the seven L. stagnalis populations in four large lakes, but in 9 out of the 16 L. stagnalis populations in small lakes. In the pooled data, a positive correlation (r _s = 0.427; P = 0.047; n = 22) between L. stagnalis density and Diplostomum sp. prevalence was found. The results suggest that Diplostomum sp. is fairly common in L. stagnalis populations in small lakes but rare in large lakes. Furthermore, although trematode parasites, in general, should have a negative effect on snail population density, the study indicates that the relationship between host density and parasite prevalence may greatly differ for individual trematode species, such as Diplostomum sp.     

Evolution of cell-to-cell communication and the structural brain organization

The review considers key issues of historical development of the nervous system, including evolution of the brain intercellular contacts and neurotransmitter systems. Special attention is given to the structural-functional organization of the central nervous system in a freshwater pulmonate gastropod, Lymnaea stagnalis.     

5′ Untranslated region of the Hsp12 gene contributes to efficient translation in Aspergillus oryzae

We describe a 5′ untranslated region (5′UTR) that dramatically increases the expression level of an exogenous gene in Aspergillus oryzae. Using a series of 5′UTR::GUS (uidA) fusion constructs, we analyzed the translation efficiency of chimeric mRNAs with different 5′UTRs at different temperatures. We found that the 5′UTR of a heat-shock protein gene, Hsp12, greatly enhanced the translation efficiency of the chimeric GUS mRNA at normal temperature (30°C). Moreover, at high temperature (37°C), the translation efficiency of the mRNA containing the Hsp12 5′UTR was far superior to that of mRNAs containing nonheat-shock 5′UTRs, resulting in much more efficient expression of GUS protein (about 20-fold higher GUS activity compared to the control construct). This 5′UTR can be used in combination with various strong promoters to enhance the expression of foreign proteins in A. oryzae.     

In vitro synthesis of lectins in cell-free extracts from dry wheat and rye embryos

Cell-free extracts from dry wheat (Triticum vulgare L.) and rye (Secale cereale L.) embryos do not synthesize their corresponding lectins when incubated under conditions optimalized for translation of either exogenous or endogenous mRNA. Only when the extracts are prepared and incubated in the complete absence of sulphydryl containing compounds lectins are synthesized in vitro. Since immunoprecipitation techniques could not demonstrate unequivocally the presence of lectin among the cell-free translation products a specific affinity purification procedure has been developed in order to proof the presence of stored lectin mRNAs in dry wheat and rye embryos.Abbreviations DTT dithiothreitol - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - TCA trichloracetic acid     

The developmental genetics of dextrality and sinistrality in the gastropodLymnaea peregra

Summary The genetics of body asymmetry inLymnaea peregra follows a maternal mode of inheritance involving a single locus with dextrality being dominant to sinistrality. Maternal inheritance implies that all members of a brood have the same phenotype, however, some broods contain a few individuals of opposite coil. One purpose of this paper is to explain the origin of these anomalous individuals. Genetic analyses of sinistral broods with a few dextral individuals have led to the development of a cross-over model, with the anomalous dextrals originating as a consequence of crossing over either during meiosis or mitosis in the female germ line. The crossover either reconstitutes the dextral gene from previously dissociated parts, or creates a dextral gene by means of a position effect. The probability of a crossover event depends upon the appropriate combination of complementary sinistral chromosomes. Each crossover event has the potential of creating a unique dextral gene. Genetic analyses of dextral broods containing a few sinistral individuals have demonstrated that different dextral genes vary in penetrance.The dextral gene produces a product during oogenesis which influences the pattern of cleavage in the embryo; this cleavage pattern is translated into the appropriate body asymmetry. The other purpose of this paper is to provide an assay for this gene product. Cytoplasm from dextral eggs injected into uncleaved sinistral eggs causes these eggs to cleave in a dextral pattern. Cytoplasm from sinistral eggs has no effect on the cleavage pattern of dextral eggs. While the dextral gene product is made during oogenesis, it does not function in controlling cleavage until just before this process begins.