Interactive effects of sublethal predation and body size on siphon production of the bivalve Nuttallia olivacea

This paper is intended to reveal effects of siphon cropping on siphon production of a tellinacean bivalve Nuttallia olivacea, an important prey for juvenile stone flounder. We carried out a two-way field experiment in which bivalves of three treatments (3-times siphon removal, 1-time removal, no removal) and four size classes (9-50 mm shell length) were caged and placed in the field for 3 mo. Growth of repeatedly-removed bivalves was inhibited, indicating reduced siphon growth (natural increase of siphon size according to somatic growth). However, siphon production of removed bivalves was larger than non-removed bivalves, possibly because of siphon regeneration. Juveniles (N. olivacea < 20 mm shell length) showed high growth performance. Their siphon growth was greater than their siphon regeneration. In all bivalves except juveniles, siphon regeneration was greater than siphon growth and engendered high siphon production. Siphon growth was dependent on bivalve size and was only slightly reduced by siphon loss, but siphon regeneration seemed to be dependent mostly on the extent of siphon loss. Greater siphon removal enhanced larger siphon production. These results indicate that intensive siphon cropping by juvenile stone flounder induces high siphon production without serious impact on N. olivacea.     

Predator-prey interactions from in situ time-lapse observations of a sublittoral mussel bed in the Gulf of Trieste (Northern Adriatic)

Hexaplex trunculus (Linnaeus, 1758) is one of the most abundant and widespread muricid gastropods in the Northern Adriatic Sea, but relatively little is known about the feeding ecology of this predator. We examined the activity of H. trunculus on a sublittoral mussel bed at 24 m depth through in situ time-lapse observations and bulk samples. The camera photographed a 0.25 m² section of the mussel bed at 6-min intervals for ~ 23 h. Photos were examined frame-by-frame for gastropod movement and activities, especially interactions between H. trunculus and Mytilus galloprovincialis (Lamarck, 1819). Our survey indicates high activity-levels of H. trunculus on the sea floor: all gastropods made minor movements, most made major movements, and most left the field of view during the study-interval. On average, individuals remained stationary for only 7.3 h. Two predation attempts on Mytilus involving conspecific competition were documented, and one Hexaplex was consuming a mussel at the onset of the deployment. Additionally, 487 M. galloprovincialis from four diver-taken 0.25 m² quadrates were measured and examined for traces of marginal chipping and drilling predation. Mytilus from surface samples ranged from 11.1 mm to 95.5 mm in length, and one of the four samples had a significantly different average shell length from the others. 114 H. trunculus were collected and measured. Hexaplex ranged from 22.1 mm to 86.1 mm and the mean shell length did not differ among samples, though they were overwhelmingly medium and large. Predation frequency (the ratio of successfully preyed upon bivalves to the total number of bivalves sampled) is high at the studied site (> 55%), and large gastropods preferred a chipping mode of predation to drilling, supporting earlier laboratory studies showing a preference for M. galloprovincialis and this predation strategy. Prey effectiveness (the ratio of failed predatory attacks to total predatory attacks) is also high (63.8%), and no evidence of a size refuge was found. Feeding in H. trunculus is highly facultative, calling for caution when using drill holes to estimate predation intensities; whenever possible, traces of multiple predation modes should be considered.     

The roles of wingless and decapentaplegic in axis and appendage development in the red flour beetle, Tribolium castaneum

Axis patterning and appendage development have been well studied in Drosophila melanogaster, a species in which both limb and segment morphogenesis are derived. In Drosophila, positional information from genes important in anteroposterior and dorsoventral axis formation, including wingless (wg) and decapentaplegic (dpp), is required for allocating and patterning the appendage primordia. We used RNA interference to characterize the functions of wg and dpp in the red flour beetle, Tribolium castaneum, which retains more ancestral modes of limb and segment morphogenesis. We also characterized the expression of potential targets of the WG and DPP signaling pathways in these embryos. Tribolium embryos in which dpp had been downregulated had defects in the dorsalmost body wall, but did not appear to have been globally repatterned and had normal appendages. Downregulation of wg led to the loss of segment boundaries, gnathal and thoracic appendages, and lateral head lobes, and to changes in the expression of dpp, Distal-less, and Engrailed. The functions of wg varied along both the anteroposterior and dorsoventral axes of the embryo. Phylogenetic comparisons indicate that the role of WNT signaling in segment boundary formation is evolutionarily old, but that its role in appendage allocation originated in the common ancestor of holometabolous insects.     

Regeneration of lost siphon tissues in the tellinacean bivalve Nuttallia olivacea

The inhalant siphon of the tellinacean bivalve Nuttallia olivacea is an important prey item for juvenile stone flounder Platichthys bicoloratus in estuaries in Japan. We examined quantitative siphon regeneration of N. olivacea in rearing experiments of siphon-removed bivalves (> 30 mm shell length) both in the laboratory and in their natural habitat. Under laboratory conditions, siphon-removed bivalves regenerated lost tissues quantitatively at 15 and 25 °C 1 mo after siphon removal, although regeneration was incomplete. A 3-mo caging experiment in the field showed that great regeneration occurred in siphon-removed bivalves. However, the siphon weight of removed bivalves was significantly smaller than that of non-amputated bivalves, suggesting the incomplete regeneration. In a 1-mo caging experiment, bivalves that had approximately 15% of their siphons amputated were selected at some intervals to illustrate the quantitative regeneration process. Estimated daily siphon production was remarkably high only a few days after amputation. It decreased greatly thereafter, but regeneration was not completed within 30 d. These results indicate that bivalves regenerate siphons rapidly just after losing siphon tissues and then regeneration is slowed down before it is completed.     

Can bivalve veligers escape feeding currents of adult bivalves?

While the stock of introduced Pacific oysters (Crassostrea gigas) increased in the Oosterschelde estuary (SW Netherlands), so did the filtration pressure of all bivalve species together. In the same period, stocks of native bivalves declined slightly. The expansion of Pacific oysters in Dutch estuaries might be partially due to better abilities of their larvae to avoid or escape filtration, compared to larvae of native bivalves. In this context, escape and swimming abilities of Pacific oyster larvae and the larvae of the native blue mussel (Mytilus edulis) were compared.Swimming behaviour of C. gigas larvae and larvae of M. edulis was recorded in still water and in a suction current mimicking a bivalve feeding current, in a horizontal and in a vertical plane. Larval swimming behaviour in a suction flow field was reconstructed by subtracting local water movement vectors from the total movement of larvae, yielding movement paths due to larval swimming alone.Swimming speeds and the rate of displacement in vertical direction of C. gigas and M. edulis larvae were related to larval shell length, and to the pitch of up- or downward swimming.Larvae of both species did not show escape reactions in a suction flow field. With increasing shell length, larval swimming speeds of both species increased significantly. Swimming speeds of C. gigas larvae were significantly higher than swimming speeds of M. edulis larvae, resulting in a faster vertical displacement. The ability to migrate to more favourable water layers faster may offer C. gigas an advantage over native bivalves with slower swimming larvae.     

Bioaccumulation of copper, lead and zinc by the bivalves Macomona liliana and Austrovenus stutchburyi

Aquatic organisms take up heavy metals from surrounding environments which accumulate in their body tissues. In the region of Auckland, New Zealand, the heavy metals, copper (Cu), lead (Pb) and zinc (Zn) are the primary sediment contaminants of concern. Previous investigations have revealed adverse effects of Cu and Zn, but not of Pb, on estuarine infauna and a higher sensitivity of the deposit-feeding bivalve Macomona liliana than the suspension-feeding bivalve Austrovenus stutchburyi to these metals. In order to further examine the bioavailability of Cu, Pb and/or Zn and their interactive effects, bioaccumulation of Cu, Pb and Zn was measured in M. liliana and A. stutchburyi after 10-day exposure to these metals in the laboratory. Both bivalves accumulated Pb and Zn, while bioaccumulation of Cu only occurred in A. stutchburyi in the presence of Zn. There was some evidence that the presence of Pb could increase bioaccumulation of Zn. Bioaccumulation was generally much higher in M. liliana than in A. stutchburyi, potentially suggesting their higher uptake rates of metals and thus explaining the higher sensitivity of M. liliana to these heavy metals. Bioaccumulation of Pb in the bivalves and its potential influences on the bioavailability of other metals indicated that, despite the lack of any evidence for acute toxicity of Pb in previous studies, it could still pose a potentially important environmental threat. Bioaccumulation of heavy metals found in the present study also highlights the needs for further investigations of potential chronic toxicity of these metals.     

Influence of diet on pre-ingestive particle processing in bivalves: I: Transport velocities on the ctenidium

Suspension-feeding bivalve molluscs can assume a large ecological role by linking benthic and pelagic ecosystems. Therefore, a knowledge of the factors that influence feeding rates and processes at the level of the individual is important in understanding bivalve-dominated environments. We examined the roles of diet quality and concentration on particle processing by the ctenidia of four species of bivalves: the mussels Mytilus edulis L. and Mytilus trossulus Gould, and oysters Crassostrea virginica (Gmelin) and Crassostrea gigas (Thunberg). Bivalves were delivered diets of three different qualities at three different concentrations (1-2×10³, 10⁴, 10⁵ particles ml⁻¹). The high-quality diet consisted of the cryptophyte Rhodomonas lens Pascher et Ruttner; the low-quality diet consisted of particles prepared from ground Spartina sp. detritus; the medium-quality diet consisted of a 50:50 mixture (by particle number) of both particle types. Particle transport velocities on the ventral groove and dorsal tracts were then measured by means of video endoscopy. Ventral-groove transport velocities of M. edulis were the most responsive, demonstrating a significant increase with increasing diet quality, and a significant decrease with increasing diet concentration. Transport velocities in the ventral groove and dorsal tracts of C. virginica were not significantly affected by changes in diet quality, but significantly increased with increasing diet concentration. Transport velocities of M. trossulus and C. gigas demonstrated little change with diet quality or concentration, indicating that responses are species specific. Our data suggest that differential control of particle transport on the bivalve ctenidium is one of the underlying mechanisms that contribute to compensatory feeding responses exhibited by the entire organism.     

A clockwork mollusc: Ultradian rhythms in bivalve activity revealed by digital photography

Time-lapse digital images can be acquired and archived using web-cameras, allowing non-invasive analysis of behavior patterns of bivalve molluscs at ultradian (sub-daily) time-scales over long intervals. These records can be analyzed directly by a human operator or through properly calibrated image analysis software. Preliminary results using species of marine and freshwater bivalves identify several ultradian biological rhythms of similar duration. Wavelet analysis indicates strong periodicity in mantle and siphon activity in the 3 to 7 min range, with longer duration shell contraction periods at 60-90 min. The recurrence of these rhythms among marine and freshwater bivalve species maintained under constant (but differing) conditions suggests the influence of common intrinsic drivers (chemico-physical mechanisms or biological clocks). Sub-daily growth increments preserved in the shells of rapidly growing bivalve species are potentially related to these biological rhythms, with implications for shell growth, biomineralization, and the temporal resolution of paleoclimate proxy data.     

optix functions as a link between the retinal determination network and the dpp pathway to control morphogenetic furrow progression in Drosophila

optix, the Drosophila ortholog of the SIX3/6 gene family in vertebrate, encodes a homeodomain protein with a SIX protein–protein interaction domain. In vertebrates, Six3/6 genes are required for normal eye as well as brain development. However, the normal function of optix in Drosophila remains unknown due to lack of loss-of-function mutation. Previous studies suggest that optix is likely to play an important role as part of the retinal determination (RD) network. To elucidate normal optix function during retinal development, multiple null alleles for optix have been generated. Loss-of-function mutations in optix result in lethality at the pupae stage. Surprisingly, close examination of its function during eye development reveals that, unlike other members of the RD network, optix is required only for morphogenetic furrow (MF) progression, but not initiation. The mechanisms by which optix regulates MF progression is likely through regulation of signaling molecules in the furrow. Specifically, although unaffected during MF initiation, expression of dpp in the MF is dramatically reduced in optix mutant clones. In parallel, we find that optix is regulated by sine oculis and eyes absent, key members of the RD network. Furthermore, positive feedback between optix and sine oculis and eyes absent is observed, which is likely mediated through dpp signaling pathway. Together with the observation that optix expression does not depend on hh or dpp, we propose that optix functions together with hh to regulate dpp in the MF, serving as a link between the RD network and the patterning pathways controlling normal retinal development.     

Expression of the decapentaplegic ortholog in embryos of the onychophoran Euperipatoides rowelli

The gene decapentaplegic (dpp) and its homologs are essential for establishing the dorsoventral body axis in arthropods and vertebrates. However, the expression of dpp is not uniform among different arthropod groups. While this gene is expressed along the dorsal body region in insects, its expression occurs in a mesenchymal group of cells called cumulus in the early spider embryo. A cumulus-like structure has also been reported from centipedes, suggesting that it might be either an ancestral feature of arthropods or a derived feature (=synapomorphy) uniting the chelicerates and myriapods. To decide between these two alternatives, we analysed the expression patterns of a dpp ortholog in a representative of one of the closest arthropod relatives, the onychophoran Euperipatoides rowelli. Our data revealed unique expression patterns in the early mesoderm anlagen of the antennal segment and in the dorsal and ventral extra-embryonic tissue, suggesting a divergent role of dpp in these tissues in Onychophora. In contrast, the expression of dpp in the dorsal limb portions resembles that in arthropods, except that it occurs in the mesoderm rather than in the ectoderm of the onychophoran limbs. A careful inspection of embryos of E. rowelli revealed no cumulus-like accumulation of dpp expressing cells at any developmental stage, suggesting that this feature is either a derived feature of chelicerates or a synapomorphy uniting the chelicerates and myriapods.     

Expression of the decapentaplegic ortholog in embryos of the onychophoran Euperipatoides rowelli

The gene decapentaplegic (dpp) and its homologs are essential for establishing the dorsoventral body axis in arthropods and vertebrates. However, the expression of dpp is not uniform among different arthropod groups. While this gene is expressed along the dorsal body region in insects, its expression occurs in a mesenchymal group of cells called cumulus in the early spider embryo. A cumulus-like structure has also been reported from centipedes, suggesting that it might be either an ancestral feature of arthropods or a derived feature (=synapomorphy) uniting the chelicerates and myriapods. To decide between these two alternatives, we analysed the expression patterns of a dpp ortholog in a representative of one of the closest arthropod relatives, the onychophoran Euperipatoides rowelli. Our data revealed unique expression patterns in the early mesoderm anlagen of the antennal segment and in the dorsal and ventral extra-embryonic tissue, suggesting a divergent role of dpp in these tissues in Onychophora. In contrast, the expression of dpp in the dorsal limb portions resembles that in arthropods, except that it occurs in the mesoderm rather than in the ectoderm of the onychophoran limbs. A careful inspection of embryos of E. rowelli revealed no cumulus-like accumulation of dpp expressing cells at any developmental stage, suggesting that this feature is either a derived feature of chelicerates or a synapomorphy uniting the chelicerates and myriapods.     

Formation of the arthropod labrum by fusion of paired and rotated limb-bud-like primordia

The nature and origin of the arthropod labrum is a matter much under dispute. We show here that in Tribolium castaneum (Herbst, 1797) the labrum develops from two individual primordia, termed labral buds. Expression of the genes decapentaplegic (dpp) and wingless (wg) in these buds is identical to the buds of the metameric appendages (e.g. thoracic legs), except that the patterns are reversed. We propose that this reversal is the result of the rotation of the labral buds through an angle of approximately 180°. We also for the first time study dpp and wg expression in the fully developed labrum of older embryonic stages. Here, gene expression patterns show that the labrum proper is formed by fusion of the labral buds along their dorsal sides, while their ventral sides are facing outward forming the lateral sides of the fused labrum. Furthermore, we show that there are very similar patterns in another arthropod species, the spider Cupiennius salei (Keyserling, 1877), although in this species the labrum develops as a single structure and not from two separate primordia. However, in C. salei the expression of engrailed is also reversed in addition to the reversal of dpp and wg expression: engrailed is expressed in the anterior half of the labrum, and not in the posterior half like in the remaining appendages. Our results suggest that the arthropod labrum is derived evolutionarily from paired limb-bud-like primordia by rotation and fusion, and that this process is recapitulated ontogenetically to a different extent in different arthropod species.     

An environmentally induced tidal periodicity of microgrowth increment formation in subtidal populations of the clam Ruditapes philippinarum

The periodicity of increment formation in the shell of the Manila clam Ruditapes philippinarum was investigated in the subtidal zone of the Auray River estuary (South Brittany, France). Calcein markings were performed at different periods between May and October 2007 using in situ benthic chambers tented by scuba divers. This study shows that shell microgrowth increments were well-defined and deposited with a tidal periodicity in the subtidal zone, providing the calendar base for high-resolution ecological studies and environmental reconstruction from these R. philippinarum shells. Endogenous rhythmicity in shell microgrowth increment formation and oxygen consumption was previously documented in this species from intertidal flats. Our study suggests that, in the subtidal zone, Manila clams' rhythmic activity may be controlled by such an endogenous process, synchronized by tidal cues. As in other bivalves, R. philippinarum is an osmoconformer euryhaline bivalve. The tidal rhythmicity of shell microgrowth increments in subtidal specimens of this species could be explained by a behavioral adaptation of valve closure at low tide to protect the clam from low salinities and/or to synchronize with food availability. Finally, large inter-individual variability in tidally associated growth rates and asynchronous growth breaks were observed, and could be due to genetic variability between individuals, asynchronous partial spawning events or predation.     

Cloning of a<Emphasis Type="Italic"> decapentaplegic</Emphasis> orthologue from the sawfly,<Emphasis Type="Italic"> Athalia rosae</Emphasis> (Hymenoptera), and its expression in the embryonic appendages

The cDNA of a decapentaplegic (dpp) orthologue from the sawfly, Athalia rosae (Hymenoptera), was cloned and characterized. The clone (Ar dpp) was 2,566 bp long and encoded 395 amino acids in a single open reading frame. Genomic Southern blotting showed that Ar dpp is a single copy gene. The deduced amino acid sequence can be aligned along its entire length with known insect DPPs. It shared common characteristics such as a signal sequence, a pro-domain region, and a ligand domain with seven cysteines at conserved locations. Ar dpp was expressed as a single 5.0-kb mRNA in embryos, larvae, pupae and adults. In situ hybridization showed that Ar dpp was expressed in the dorsal region proper in early embryonic stages and in the embryonic appendages of cephalic segments (labrum, antenna, mandible, maxilla, and labium), thoracic segments (thoracic legs), and all abdominal segments except the tenth segment (pleuropodia and proleg primordia). The present results indicate that Ar dpp expression reflects the primary determination of embryonic appendages.Edited by D. TautzThe sequence reported in this paper has been deposited in the DDBJ/EMBL/GenBank database with the accession number AB121072     

Early Shell Formation During Molluscan Embryogenesis, with New Studies on the Surf Clam, Spisula solidissima

Shell formation in molluscs begins early in embryogenesis duringsome stage of archenteron formation. Ultrastructural informationon early formation of external shells is available from onlya few bivalves and gastropods. Secretion of the very first shellmaterial by shell field epithelial cells is preceded by an invaginationof the dorsal ectoderm in the region of the shell field. A centuryago, this invagination was termed the "shell gland." As a secretoryfunction for this invagination has not yet been demonstratedand as the term "shell gland" has taken on various meaningsin the literature, the invagination will be referred to as theshell field invagination. The opening into the shell field invaginationseems to be circular in gastropods and elongate in bivalves.Accordingly initial organic shell material seems to form a ringin gastropods and a saddle in bivalves. As in adult molluscs,shells of pre-metamorphic molluscs are composed of both organicand inorganiccomponents. Ultrastructural data from bivalvesand gastropods indicate that the initial organic shell materialis secreted just outside the shell field invagination (acrossthe pore). Initial inorganic shell materials have not been localizednor their pathway traced into or through any pre-metamorphicmolluscs. New SEM and TEM data show that the invagination inthe bivalve Spisula solidissima is composed of a wide outerregion and very narrow inner region with the first shell materialforming at the junction between the two. This is unlike ultrastructuraldata available for other species. Many sections give the falseimpressions that: 1) the shell field invagination is closedto the outside and, 2) that the first organic shell materiallines the innermost region of the invagination. It is not clearwhether the cells of the outer invagination in this speciesare shell field cells. It is suggested that they are not.     

Trophic transfer of trace metals: Subcellular compartmentalisation in bivalve prey and comparative assimilation efficiencies of two invertebrate predators

We test the hypothesis that accumulated metal in prey that is trophically available to one predator is not necessarily equally trophically available to another predator feeding on the same prey, given the variability between invertebrate digestive systems. We provided two predators, the neogastropod mollusc Hinia reticulata and the palaemonid decapod crustacean Palaemonetes varians, with the digestive glands and adductor muscles of four bivalves radiolabelled with Zn, Cd or Ag. The bivalves (the mussel Mytilus edulis, the clam Ruditapes philippinarum, the scallop Aequipecten opercularis, the oyster Crassostrea gigas) have different metal accumulation patterns with differential dependence on soluble and insoluble detoxification, as confirmed by fractionation of the prey tissues. We found no consistent significant difference between the AE of the two predators for the three trace metals accumulated in the same prey tissues. There were no significant correlations for either predator between percentages of metal in soluble form (or soluble form with organelle-associated metal) and percentage AE for any of the three metals, allowing the conclusion that both predators are assimilating each metal from more than the soluble and organelle-associated metal fractions. For neither predator did an increased percentage of Zn in the form of metal rich granules (MRG) affect its Zn AE, but increases in the percentages of both Cd and Ag bound to MRG decreased the AE of the relevant metal in P. varians but not H. reticulata. Thus the Cd and Ag in some Cd-rich and Ag-rich granules in the bivalve tissues are not as trophically available to P. varians as they are to H. reticulata. This interspecific difference confirms that the neogastropod has the stronger digestive and assimilative powers involving Cd and Ag bound in prey than the palaemonid decapod.     

The pleiohomeotic gene is required for maintaining expression of genes functioning in ventral appendage formation in Drosophila melanogaster

Polycomb group (PcG) proteins are negative regulators that maintain the expression of homeotic genes and affect cell proliferation. Pleiohomeotic (Pho) is a unique PcG member with a DNA-binding zinc finger motif and was proposed to recruit other PcG proteins to form a complex. The pho null mutants exhibited several mutant phenotypes such as the transformation of antennae to mesothoracic legs. We examined the effects of pho on the identification of ventral appendages and proximo-distal axis formation during postembryogenesis. In the antennal disc of the pho mutant, Antennapedia (Antp), which is a selector gene in determining leg identity, was ectopically expressed. The homothorax (hth), dachshund (dac) and Distal-less (Dll) genes involved in proximo-distal axis formation were also abnormally expressed in both the antennal and leg discs of the pho mutant. The engrailed (en) gene, which affects the formation of the anterior-posterior axis, was also misexpressed in the anterior compartment of antennal and leg discs. These mutant phenotypes were enhanced in the mutant background of Posterior sex combs (Psc) and pleiohomeotic-like (phol), which are another PcG genes. These results suggest that pho functions in maintaining expression of genes involved in the formation of ventral appendages and the proximo-distal axis.     

Brown shrimp (Crangon crangon, L.) functional response to density of different sized juvenile bivalves Macoma balthica (L.)

Variability in infaunal bivalve abundance in the Wadden Sea is largely determined by recruitment variability. Post-settlement, but pre-recruitment bivalve mortality is high and related to the occurrence of their most abundant predator, the brown shrimp Crangon crangon. To investigate if the mortality patterns of newly settled bivalves can be explained by the foraging behavior of brown shrimp, we carried out experiments on shrimp functional response to three size classes of juveniles of the Baltic Tellin Macoma balthica. The functional response curves for all three prey sizes (0.62 mm, 0.73 mm, and 0.85 mm) were the hyperbolic Holling's type II. The attack rate was highest for the smallest prey size (a = 0.31, medium and large prey a = 0.22); the handling time was longest for the largest prey size (T_h = 29 s, small and medium prey T_h = 15 s). Thus, a large body size is advantageous for the bivalves over the whole density range. Knowledge of individual foraging behavior is needed to model predation mortality of bivalves. The consumption rates in the experiment were theoretically high enough to account for M. balthica mortality in the field.     

Expression patterns of <Emphasis Type="Italic">engrailed</Emphasis> and <Emphasis Type="Italic">dpp</Emphasis> in the gastropod <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis>

We isolated the full-length cDNAs of engrailed and dpp-BMP2/4 orthologues from the pond snail Lymnaea stagnalis and examined their expression patterns during development by the whole mount in situ hybridization. At the gastrula and trochophore stages, engrailed is expressed in the peripheral ectoderm of the presumptive and invaginating shell gland, corroborating its role in the shell formation that is widely conserved among molluscs. At the same stages, dpp-BMP2/4 is expressed in the right-hand side ectoderm of the shell gland and in the invaginating stomodaeum. Unlike in the gastropod Patella vulgata, our results suggested that dpp-BMP2/4 has a role in the shell formation, rather than in the regional specification and that it could be involved in the specification pathway of the left–right asymmetry of the developing shell in L. stagnalis.