The Caudal ParaHox gene is required for hindgut development in the mollusc Tritia (a.k.a. Ilyanassa).

Caudal homeobox genes are found across animals, typically linked to two other homeobox genes in what has been called the ParaHox cluster. These genes have been proposed to pattern the anterior-posterior axis of the endoderm ancestrally, but the expression of Caudal in extant groups is varied and often occurs in other germ layers. Here we examine the role of Caudal in the embryo of the mollusc Tritia (Ilyanassa) obsoleta. ToCaudal expression is initially broad, then becomes progressively restricted and is finally only in the developing hindgut (a.k.a. intestine). Knockdown of ToCaudal using morpholino oligonucleotides specifically blocks hindgut development, indicating that despite its initially broad expression, the functional role of ToCaudal is in hindgut patterning. This is the first functional characterization of Caudal in an animal with spiralian development, which is an ancient mode of embryogenesis that arose early in bilaterian animal evolution. These results are consistent with the hypothesis that the ancestral role of the ParaHox genes was anterior-posterior patterning of the endoderm.

A Pilot Evaluation of the Toxicity of EarthTec® QZ on Invasive (Bithynia tentaculata) and Native (Physa gyrina) Snail Species from the Upper Mississippi River.

We used a comparative approach to investigate the effects of a copper-based pesticide (EarthTec® QZ) on embryos of an invasive snail (Bithynia tentaculata) and a native snail (Physa gyrina). Embryos were exposed to one of three treatments: control (0 mg/L Cu2+), low-dose (0.1 mg/L Cu2+), or high-dose (0.6 mg/L Cu2+), which reflect manufacturer-recommended low and medium 4-day molluscicide treatment concentrations. Exposure to 0.6 mg/L Cu2+ over 4 days generated 100% mortality in both invasive and native snail embryos; however, reducing the exposure time from 4 to 1 day resulted in 100% mortality in B. tentaculata but some hatching (7%) in P. gyrina. In contrast, embryos of both species exposed to 0.1 mg/L Cu2+ treatment for 4 days showed almost 100% survivorship. Further manipulations of Cu2+ concentrations and exposure times may yield regimes that maximize mortality in B. tentaculata while minimizing negative impacts on native species.

Early Activation of MAPK and Apoptosis in Nutritive Embryos of Calyptraeid Gastropods.

Investigation of alternative phenotypes, different morphologies produced by a single genome, has contributed novel insights into development and evolution. Yet, the mechanisms underlying developmental switch points between alternative phenotypes remain poorly understood. The calyptraeid snails Crepidula navicella and Calyptraea lichen produce two phenotypes: viable and nutritive embryos, where nutritive embryos arrest their development after gastrulation and are ingested by their viable siblings as a form of intracapsular nutrition. Here, we investigate the activity of mitogen-activated protein kinase (MAPK, ERK1/2) and apoptosis during early cleavage. MAPK and apoptosis, found in a previous transcriptomic study, are known to be involved in organization of other spiralian embryos and nutritive embryo development, respectively. In the model Crepidula fornicata, MAPK activation begins at the 16-cell stage. In contrast, we discovered in C. navicella and C. lichen that many embryos begin MAPK activation at the one-cell stage. A subset of embryos shows a similar pattern of MAPK activation to C. fornicata at later stages. In all stages where MAPK is detected, the activation pattern is highly variable, frequently occurring in all quadrants or in multiple tiers of cells. We also detected apoptosis in cleaving embryos, while C. fornicata and Crepidula lessoni, which do not produce nutritive embryos, show no signs of apoptosis during cleavage. Our results show that MAPK and apoptosis are expressed during early development in species with nutritive embryos, and raises the possibility that these processes may play a role and even interact with one another in producing the nutritive embryo phenotype.

Dpp/BMP2-4 Mediates Signaling from the D-Quadrant Organizer in a Spiralian Embryo.

In some animal groups, the secondary embryonic axis is patterned by a small group of cells, often called an organizer, that signals to other cells to establish the correct pattern of cell fates. The Dpp/BMP2-4 pathway plays a central role in secondary axis patterning in many animals [1-11], but it has not been examined during early axial patterning in spiralian embryogenesis. This is a deeply conserved mode of development found in mollusks, annelids, nemerteans, entoprocts, and some marine platyhelminth groups (reviewed in [12, 13]). In the spiralian embryo of the mollusk Ilyanassa, we find that the Dpp ortholog (IoDpp) is expressed most strongly on the dorsal side, in cells of the embryonic organizer and its neighbors. Phospho-smad staining indicates that the pathway is active in all lineages during organizer signaling, but activation is strongest on the dorsal side. Knockdown of IoDpp by morpholino oligos prevents the development of all structures that require organizer signaling and ventralizes the embryo. Ectopic activation of the pathway can induce eyes and external shell, which require organizer signaling. These results indicate that Dpp/BMP2-4 signaling is a key part of the spiralian organizer and suggest similarity with other metazoan organizers. However, the fact that IoDpp/BMP2-4 is inducing, rather than repressing, the neuroectoderm is a surprising difference that may be conserved among spiralians. These results connect the spiralian organizer to this general aspect of secondary axis patterning but highlight the significant variation across animals in effects of the pathway on particular cell types and tissues.

Comparative Transcriptomics of Alternative Developmental Phenotypes in a Marine Gastropod.

Alternative phenotypes are discrete phenotypic differences that develop in response to both genetic and environmental cues. Nutritive embryos, which arrest their development to serve as nutrition for their viable siblings, are an example of an alternative developmental phenotype found in many animal groups. Females of the marine snail, Crepidula navicella, produce broods that consist mainly of nutritive embryos and a small number of viable embryos. In order to better understand the genetic mechanisms that lead to the development of alternative phenotypes in this species, we compared the transcriptomes of viable and nutritive embryos at the earliest stage that we were able to distinguish visually between the two. Using high-throughput Illumina sequencing, we assembled and annotated a de novo transcriptome and compared transcript levels in viable and nutritive embryos. Viable embryos express high levels of transcripts associated with known developmental events, while nutritive embryos express high levels of apoptosis-related transcripts. Gene Ontology term enrichment with GOSeq found that these are associated with the negative regulation of apoptotic processes. This enrichment, combined with morphological evidence, suggests that apoptosis is important in the formation of gastrula-like nutritive embryos. Apoptosis has been implicated in the development of alternative phenotypes in other animal groups, raising the possibility that this mechanism's role in alternative phenotypes is conserved in gastropod development. We suggest possible alternative mechanisms of nutritive embryo development. Most importantly, we contribute further evidence to the hypothesis that nutritive embryos are an alternative developmental phenotype.

[Individual Growth of the Great Ramshorn Snail Planorbarius corneus (Gastropoda, Planorbidae) Embryos].

Individual growth of the great ramshorn snail Planorbarius corneus has been studied by intravital video imaging. As has been observed, the types of growth change over the embryogenesis. The linear dimensions slightly but in a statistically significant manner decrease during the stages of cleavage to blastula. Starting from the stage of blastula to trochophore, the embryo diameter remains constant to commence increasing at the stage of middle trochophore. During the larval stages (trochophore and veliger), the growth is synchronous (in Dettlaffs, biological time units) for the embryos in both the same clutch and different clutches. The growth at that time is exponential but later desynchronizes in individual clutches. The embryos in eight clutches grew and developed slower and hatched later as compared with the remaining five egg clutches. An accelerated growth follows an asymptomatic pattern according to the von Bertalanffy equation. A retarded growth is describable with a linear equation. The observed differences are likely to be associated with the number of embryos in a clutch. All types of changes in the linear dimensions observed in the great ramshorn snail embryogenesis can be described with the same united equation.

Differential sensitivity of snail embryos to cadmium: relation to age and metallothionein gene expression.

The aim of this study was to determine whether cadmium (Cd) sensitivity of Cantareus aspersus embryos is age-dependent and influenced by metallothionein (MT) gene expression. Hatching success and the expression of three MT isoform genes (Ca-CdMT, Ca-CuMT and Ca-Cd/CuMT) were measured in embryos exposed to increasing Cd concentrations for 24 h starting on the sixth day of development. Isoform gene expression was quantified on days 7 and 12 after exposure. Results were compared to those of embryos exposed to the same conditions as above, but from the beginning of embryogenesis (day 0). Transcription of the Cd-specific MT gene (Ca-CdMT) was observed from the first day of development, whereas the two other genes did not respond to Cd at all. Overall, Cd sensitivity of embryos decreased with increasing age of development, as assessed by age-dependent increase of EC50 values for hatching rate, and increasing Cd threshold concentrations for Ca-CdMT expression.

Metallothionein gene expression in embryos of the terrestrial snail (Cantareus aspersus) exposed to cadmium and copper in the Bordeaux mixture.

The response specificity of three metallothionein (MT) genes (CdMT, CuMT and Cd/CuMT) was assessed after long-term exposure (20 days) of Cantareus aspersus eggs to cadmium (Cd) (2 to 6 mg/L) or to the fungicide Bordeaux mixture (BM) (2.5 and 7.5 g/L). MT gene expression measured by quantitative real-time PCR (qRT-PCR) revealed that in the unexposed embryos, the transcript levels of the three MT genes decreased significantly through embryonic development. However, the CdMT gene was strongly upregulated with increasing Cd exposure concentration, whereas the transcript levels of the other two genes increased less pronouncedly, but significantly above an exposure concentration of 4 mg Cd/L. Upon exposure to BM, all three MT genes were significantly upregulated above a BM concentration of 2.5 g/L. It is concluded that long-term Cd exposure in hatched snails induced patterns of MT gene expression that differed from those obtained after short-term exposure (24 h).

An embryonic transcriptome of the pulmonate snail Radix balthica.

The pond snail, Radix balthica (Linnaeus 1758), is an emerging model species within ecological developmental biology. While its development has been characterised in detail, genomic resources for embryonic stages are lacking. We applied Illumina MiSeq RNA-seq to RNA isolated from pools of embryos at two points during development. Embryos were cultured in either the presence or absence of predator kariomones to increase the diversity of the transcripts assembled. Sequencing produced 47.2M paired-end reads, assembled into 54,360 contigs of which 73% were successfully annotated. This transcriptome provides an invaluable resource to build a mechanistic understanding of developmental plasticity.

Deployment of regulatory genes during gastrulation and germ layer specification in a model spiralian mollusc Crepidula.

BACKGROUND: During gastrulation, endoderm and mesoderm are specified from a bipotential precursor (endomesoderm) that is argued to be homologous across bilaterians. Spiralians also generate mesoderm from ectodermal precursors (ectomesoderm), which arises near the blastopore. While a conserved gene regulatory network controls specification of endomesoderm in deuterostomes and ecdysozoans, little is known about genes controlling specification or behavior of either source of spiralian mesoderm or the digestive tract. RESULTS: Using the mollusc Crepidula, we examined conserved regulatory factors and compared their expression to fate maps to score expression in the germ layers, blastopore lip, and digestive tract. Many genes were expressed in both ecto- and endomesoderm, but only five were expressed in ectomesoderm exclusively. The latter may contribute to epithelial-to-mesenchymal transition seen in ectomesoderm. CONCLUSIONS: We present the first comparison of genes expressed during spiralian gastrulation in the context of high-resolution fate maps. We found variation of genes expressed in the blastopore lip, mouth, and cells that will form the anus. Shared expression of many genes in both mesodermal sources suggests that components of the conserved endomesoderm program were either co-opted for ectomesoderm formation or that ecto- and endomesoderm are derived from a common mesodermal precursor that became subdivided into distinct domains during evolution.

An assessment of the embryotoxicity of cadmium in the terrestrial mollusk Cantareus aspersus: from bioaccumulation to impacts at different levels of biological organization.

This study aims to determine various parameters that allow the evaluation of the toxicity of chemicals to embryos of the ubiquitous land snail Cantareus aspersus. For this purpose, we investigated morphological and physiological endpoints in control embryos and in embryos exposed to a solution of 6mg Cd/L (CdCl2) in a liquid phase bioassay: size at days 3, 6 and 10, heart rate at 7 days, delay in hatching, states of development of non-hatched eggs after 17 days and the fresh mass of newly hatched embryos. The kinetics of Cd accumulation in eggs and DNA fragmentation were also measured. The first detectable sign of adverse effects appeared after 7 days of development, when the heart rate decreased in Cd-exposed embryos compared with the control. After 10 days of exposure, Cd-exposed hatchlings exhibited a lower fresh mass than control individuals. The majority (75 percent) of non-hatched embryos at 17 days was dead and presented signs of disaggregation or malformations. The hatching of Cd-exposed eggs was delayed 4 days, and DNA fragmentation was later detected after 20 days of Cd exposure. The measurement of Cd in the eggs showed that concentrations are relatively stable during the exposure period from 3 days (20-27µg Cd/g DW) to the end of exposure. The present study completes the range of endpoints that can be used to study the effects of contaminants and provides new parameters that are readily measured throughout the embryonic development of a terrestrial mollusk.

Development of blastomere clones in the Ilyanassa embryo: transformation of the spiralian blastula into the larval body plan.

Spiralian embryogenesis is deeply conserved and seems to have been in place in the last common ancestor of the large assemblage of protostome phyla known as the Lophotrochozoa. While the blastula fate maps of several spiralian embryos have been determined, little is known about the events that link the early embryo and the larva. For all cells in the Ilyanassa blastula, we determined the clonal morphology at four time points between the blastula and veliger stages. We found that ectomesoderm comes mostly from 3a and 3b, but also from 2c and 2b. We also observed the ingression and early proliferation of 3a- and 3b-derived ectomesoderm. We found cells in the 2b clone that marked the anterior edge of the blastopore and later the mouth and cells in the 3c/3d clones that marked the posterior edges of these structures. This demonstrates directly that the mouth forms in the same location as the blastopore. In the development of the shell field, we observed dramatic cell migration events that invert the positions of the 2b and 2d clones that contribute to the shell. Using time-lapse imaging, we followed and described the cleavage pattern of the conserved endomesodermal blast cell, 4d, up to 4d + 45 h, when there were 52 cells in the clone. Our results show the growth and movement of clones derived from cells of the spiralian blastula as they transform into the trochophore-like and veliger stages. They have implications for the evolution of the shell in gastropods, the origins of mesoderm in spiralians, and the evolution of mouth formation in metazoans.

Pedal sole immunoreactive axons in terrestrial pulmonates: Limax, Arion, and Helix.

A century ago histological techniques such as formic acid-gold chloride showed the nerve morphology of the pedal sole in Limax and Helix. There have been no similar descriptions since then of the central nervous system relevant to locomotory pedal waves in the foot of slugs and snails. Topical application of 5-HT affects locomotory waves, but the innervation of the pedal sole with 5-HT axons is not known. Three-dimensional morphology of pedal axons in terrestrial pulmonate embryos is shown herein with modern histological techniques using antibodies and the confocal microscope. In Limax maximus, pedal ganglia are shown with Tritonia pedal peptide (TPep) antibodies. Ladder-like cross bridges in the pedal sole are shown with antibodies to both TPep and 5-HT. In Arion ater, pedal ganglia neurons and their axons that form a plexus in the pedal sole are shown with 5-HT antibodies. In Helix aspersa, 5-HT immunoreactive pedal ganglia neurons and a developing pedal sole axon plexus are seen as in A. ater. Axons in this plexus that grow across the pedal sole can be seen growing into pre-existing nerves. No peripheral 5-HT neurons were identified in these three species. This immunoreactive plexus to 5-HT antibodies in A. ater and H. aspersa spreads over the pedal sole epithelium. Axons immunoreactive to 5-HT antibodies in A. ater and H. aspersa extend the length of the foot, primarily in the rim, so that activity in these axons cannot provide local patterned input to produce locomotory waves, but may provide modulatory input to pedal sole muscles.

Spiral cleavages determine the left-right body plan by regulating Nodal pathway in monomorphic gastropods, Physa acuta.

The handedness of gastropods is genetically determined, but the molecular nature of the gene responsible and the associated mechanisms remain unknown. In order to characterize the chiromorphogenesis pathway starting from the gene to the left-right asymmetric body plan, we have closely analyzed the cytoskeletal dynamics of the Physa (P.) acuta embryo, a fresh water non-dimorphic sinistral snail, during the early developmental stage by mechanically altering the handedness of the embryos at the critical spiral third cleavage. A fertile situs inversus was created and the nodal-Pitx gene expression patterns were completely mirror imaged to the wild type at the trochophore stage. Together with our previous work on Lymnaea (L.) stagnalis, we could show that chirality is established at the third cleavage, as dictated by the single handedness-determining gene locus, and then chirality information is transferred via subsequent spiral fourth and fifth cleavages to the later developmental stage, dictating the nodal-Pitx expression pathway. The cytoskeletal dynamics of manipulated and non-manipulated embryos of sinistral P. acuta and dextral dominant L. stagnalis are compared.

Ilyanassa Notch signaling implicated in dynamic signaling between all three germ layers.

Two cells (3D and 4d) in the mud snail Ilyanassa obsoleta function to induce proper cell fate. In this study, we provide support for the hypothesis that Notch signaling in Ilyanassa obsoleta functions in inductive signaling at multiple developmental stages. The expression patterns of Notch, Delta and Suppressor of Hairless (SuH) are consistent with a function for Notch signaling in endoderm formation, the function of 3D/4d and the sublineages of 4d. Veligers treated with DAPT show a range of defects that include a loss of endodermal structures, and varying degrees of loss of targets of 4d inductive signaling. Veligers that result from injection of Ilyanassa Delta siRNAi in general mimic the defects observed in the DAPT treated larvae. The most severe DAPT phenotypes mimic early ablations of 4d. However, the early specification of 4d itself appears normal and MAPK activation in both 3D/4d and the micromeres, which are known to activate MAPK as a result of 3D/4d induction, are normal in DAPT treated larvae. Treating larvae at successively later timepoints with DAPT suggests that Notch/Delta signaling is not only required during early 4d inductive signaling, but during subsequent stages of cell fate determination as well. Based on our results, combined with previous reports implicating the endoderm in maintaining induced fate specification in Ilyanassa, we propose a speculative model that Notch signaling is required to specify endoderm fates and 4d sublineages, as well as to maintain cell fates induced by 4d.

The embryonic and postembryonic developmental toxicity of imidazolium-based ionic liquids on Physa acuta.

The embryonic and postembryonic developmental toxicity of imidazolium-based ionic liquids (ILs) to the snail Physa acuta was evaluated in this study. The results of embryonic toxicity tests showed that lower concentrations of 1-octyl-3-methylimidazolium bromide ([C8 mim]Br) (1.5 and 2.1 mg/L) inhibited the hatching rate of snail embryos, and partial snails hatched normally and died, while all of the treated embryos died when the exposure concentration was higher than 4.16 mg/L, at which IL caused the deformation, death, and decay of snail embryos. Statistical analyses revealed obvious differences in the hatching rates between three developmental stages in the 2.1 and 2.94 mg/L groups, indicating that the veliger stage is more sensitive to [C8 mim]Br exposure than the blastula and gastrula stages. Furthermore, the 96 h LC50 values of [C8 mim]Br on the tested snails at three developmental stages (juvenile, subadult, and adult) were 70.83 ± 2.99, 97.59 ± 4.05, and 109.3 ± 2.22 mg/L, respectively, indicating that young snails were more sensitive to [C8 mim]Br toxicity than adults. In addition, the 96 h LC50 values of ILs with different alkyl chain lengths, that is, [C12 mim], [C10 mim], [C8 mim], and [C6 mim], in adult snails were 1.35 ± 0.24, 8.96 ± 5.66, 109.3 ± 4, and 359.6 ± 11.6 mg/L, respectively, suggesting that longer alkyl chains can increase the toxicity of imidazolium ILs on snails.

Carry-over effects of multiple stressors on benthic embryos are mediated by larval exposure to elevated UVB and temperature.

Damaging effects of UVB in conjunction with other stressors associated with global change are well-established, with many studies focused on vulnerable early life stages and immediate effects (e.g., mortality, developmental abnormalities). However, for organisms with complex life cycles, experiences at one life stage can have carry-over effects on later life stages, such that sublethal effects may mediate later vulnerability to further stress. Here, we exposed embryos in benthic egg masses of the New Zealand intertidal gastropod Siphonaria australis to treatments of either periodic stress (e.g., elevated UVB, salinity, and water temperature mimicking tidepool conditions in which egg masses are commonly found during summer) or control conditions (low UVB, ambient salinity, and water temperatures). Although there was high mortality from stressed egg masses, 24% of larvae hatched successfully. We then exposed the hatching larvae from both egg mass treatments to different combinations of water temperature (15 or 20 °C) and light (high UVB or shade) 12 h per day for 10 days. The most stressful larval conditions of 20 °C/high UVB resulted in low survival and stunted growth. Carry-over effects on survival were apparent for shaded larvae exposed to elevated temperature, where those from stressed egg masses had 1.8× higher mortality than those from control egg masses. Shaded larvae were also larger and had longer velar cilia if they were from control egg masses, independent of larval temperature. These results demonstrate that previous experience of environmental stress can influence vulnerability of later life stages to further stress, and that focus on a single life stage will underestimate cumulative effects of agents of global change.

Parent--offspring similarity in the timing of developmental events: an origin of heterochrony?

Understanding the link between ontogeny (development) and phylogeny (evolution) remains a key aim of biology. Heterochrony, the altered timing of developmental events between ancestors and descendants, could be such a link although the processes responsible for producing heterochrony, widely viewed as an interspecific phenomenon, are still unclear. However, intraspecific variation in developmental event timing, if heritable, could provide the raw material from which heterochronies originate. To date, however, heritable developmental event timing has not been demonstrated, although recent work did suggest a genetic basis for intraspecific differences in event timing in the embryonic development of the pond snail, Radix balthica. Consequently, here we used high-resolution (temporal and spatial) imaging of the entire embryonic development of R. balthica to perform a parent-offspring comparison of the timing of twelve, physiological and morphological developmental events. Between-parent differences in the timing of all events were good predictors of such timing differences between their offspring, and heritability was demonstrated for two of these events (foot attachment and crawling). Such heritable intraspecific variation in developmental event timing could be the raw material for speciation events, providing a fundamental link between ontogeny and phylogeny, via heterochrony.

External and internal shell formation in the ramshorn snail Marisa cornuarietis are extremes in a continuum of gradual variation in development.

BACKGROUND: Toxic substances like heavy metals can inhibit and disrupt the normal embryonic development of organisms. Exposure to platinum during embryogenesis has been shown to lead to a "one fell swoop" internalization of the shell in the ramshorn snail Marisa cornuarietis, an event which has been discussed to be possibly indicative of processes in evolution which may result in dramatic changes in body plans. RESULTS: Whereas at usual cultivation temperature, 26°C, platinum inhibits the growth of both shell gland and mantle edge during embryogenesis leading to an internalization of the mantle and, thus, also of the shell, higher temperatures induce a re-start of the differential growth of the mantle edge and the shell gland after a period of inactivity. Here, developing embryos exhibit a broad spectrum of shell forms: in some individuals only the ventral part of the visceral sac is covered while others develop almost "normal" shells. Histological studies and scanning electron microscopy images revealed platinum to inhibit the differential growth of the shell gland and the mantle edge, and elevated temperature (28 - 30°C) to mitigate this platinum effect with varying efficiency. CONCLUSION: We could show that the formation of internal, external, and intermediate shells is realized within the continuum of a developmental gradient defined by the degree of differential growth of the embryonic mantle edge and shell gland. The artificially induced internal and intermediate shells are first external and then partly internalized, similar to internal shells found in other molluscan groups.

Analysis of ciliary band formation in the mollusc Ilyanassa obsoleta.

Two primary ciliary bands, the prototroch and metatroch, are required for locomotion and in the feeding larvae of many spiralians. The metatroch has been reported to have different cellular origins in the molluscs Crepidula fornicata and Ilyanassa obsoleta, as well as in the annelid Polygordius lacteus, consistent with multiple independent origins of the spiralian metatroch. Here, we describe in further detail the cell lineage of the ciliary bands in the gastropod mollusc I. obsoleta using intracellular lineage tracing and the expression of an acetylated tubulin antigen that serves as a marker for ciliated cells. We find that the I. obsoleta metatroch is formed primarily by third quartet derivatives as well as a small number of second quartet derivatives. These results differ from the described metatrochal lineage in the mollusc C. fornicata that derives solely from the second quartet or the metatrochal lineage in the annelid P. lacteus that derives solely from the third quartet. The present study adds to a growing body of literature concerning the evolution of the metatroch and the plasticity of cell fates in homologous micromeres in spiralian embryos.