Gibbula varia life history
is a dioecious species. The eggs fertilized via copulation are laid in gelatinous egg masses (additional file 1
, Figure S1A, S1B, S1C). Development of the embryos and larvae takes place inside the egg capsule and takes about four days. The main stages of G. varia
development are presented in Table . Epibolic gastrulation occurs by the micromeres rapidly spreading downwards and enclosing the macromeres. The blastopore, being wide at first, gradually becomes constricted at 10 to 12 hours post fertilization (hpf), when the trochoblasts start to become ciliated. At 16 hpf, the prototroch is clearly visible as a circular ciliary band, separating the trochophore larva's episphere from the hyposphere (additional file 1
, Figure S1D). There is no sign of apical cilia (apical tuft) at any stage in the development of the trochophore, although the pretrochal cells were observed to be smaller than those of the posttrochal region (additional file 1
, Figure S1D). By this stage the blastopore gradually moves to a position just below the prototroch, forming the stomodeum. Simultaneously, the shell-gland invagination appears as a thin patch of cells gradually spreading over the dorsal region of the larva (additional file 1
, Figure S1D). At 18 hpf, the late trochophore larva comprises a prototroch, the shell field surrounded by the mantle edge, and a pedal rudiment (additional file 1
, Figure S1E). The late trochophore (24 hpf) turns into an encapsulated pretorsional veliger larva by differentiation of the prototroch to a distinct velum (additional file 1
, Figure S1F). The mantle fold and mantle cavity become visible mid-ventrally on the posterior surface of the pedal rudiment (additional file 1
, Figure S1E, S1G). At 36 hpf, the pretorsional veliger has a velum, an apical organ marked by apical cilia (apical tuft), a mouth opening, and a pedal rudiment with the operculum anlage (additional file 1
, Figure S1H). The first 90° of torsion take place between 36-48 hpf, presumably by contraction of the larval retractor (shell) muscle. This results in a 90° displacement of the mantle cavity to the right side, and, when viewed from the front, the foot and velum are rotated anti-clockwise in relation to the protoconch. The remaining part of torsion is completed within one day while the velum gradually becomes reduced in size and splits ventrally (additional file 1
, Figure S1I). At 60 hpf, the operculum appears in the posttorsional veliger larva (additional file 1
, Figure S1I). The radula and cephalic eyes appear about three days after fertilization. As the eyes form, the cephalic tentacles begin to appear as outgrowths of the prevelar surface. The juvenile hatches on the fourth day of development (about 96 hpf) and after that mineralization of the shell begins. The animals become sexually mature after 11-12 months.
Timing of developmental stages of Gibbula varia (at 22°C); different stages of larval development and metamorphosis of G. varia inside the gelatinous egg capsules before hatching.
Development of gut in G. varia
The development of the digestive tract starts with the development of the stomodeum (future mouth opening) in the trochophore (additional file 1
, Figure S1E). The mouth opens during the pretorsional veliger stage (additional file 1
, Figure S1H, S1J) whereas the anus opens in the late posttorsional stage at the site of a few ciliated cells (anal markers). The development of the digestive tract is very similar to that described in G. cineraria
and Haliotis tuberculata
]. The digestive gland begins to differentiate on the left side of the veliger just before torsion sets in [57
]. The gut develops from differentiated endodermal cells initially scattered within the yolk in the pretorsional veliger. They later migrate to the yolk boundaries to form the definitive midgut in the posttorsional veliger [57
]. Later, the hindgut develops from actively dividing cells of the digestive gland migrating to their final positions in the intestine [57
]. The competent larva's digestive system comprises a mouth opening and a bipartite oesophagus (the anterior part immediately behind the buccal cavity is not effected by torsion, the mid oesophagus includes a portion affected by the torsion), a stomach with the digestive gland, the hindgut leading to the anus that opens into the mantle cavity over the back of the head (additional file 1
, Figure S2A and S2B). The radula anlage is a ventral differentiation of the foregut where mesenchym cells aggregate. The radula teeth become visible in the competent larva at the distal end of the radula sheath (additional file 1
, Figure S2A and S2B).
ParaHox gene sequences
The entire coding sequences for all three G. varia ParaHox
genes were isolated by a combination of 3' and 5' rapid amplification of cDNA ends (RACE, see Methods). 3' and 5' RACE together yielded a complete cDNA of 885 bp with the complete open reading frame (ORF) of 519 bp (172 amino acids) for Gva-Gsx
, a complete cDNA of 1739 bp with complete ORF of 1002 bp (333 amino acids) for Gva-Xlox
, and a complete cDNA of 1466 bp with complete ORF of 976 bp (325 amino acids) for Gva-Cdx
. Alignments of each G. varia ParaHox
amino acid sequence to orthologs of other species are shown in additional file 2
, Figure S3, S4, and S5. Beside the homeobox which is the main region of conservation between ParaHox
genes, further conserved domains are the N-terminal domain in Gsx
, and the hexapeptide motifs just upstream of the homeodomains in both Xlox
(Additional file 2
, Figure S3, S4, and S5). The classification of the G. varia ParaHox
genes into their orthology groups is apparent from phylogenetic analyses (Figure ). The species names and accession number of the genes used in phylogenetic analysis are provided in additional file 2
. Although the phylogenetic analysis clearly assigns the Gibbula paraHox
genes to the Gsx
classes with high support values, the internal grouping remains unclear.
Figure 1 Phylogenetic reconstruction of ParaHox genes. The tree is from Bayesian likelihood analysis using MrBayes: half compatibility consensus from five million replicates, burn-in of 5,000 replicates. The tree is built with the amino-acid sequences of the homeodomain (more ...)
ParaHox gene expression in the trochophore larva
We did not detect Gva-ParaHox transcripts by whole-mount in situ hybridization (WMISH) in developmental stages before the trochophore stage. A scanning electron micrograph (SEM) of a late trochophore larva (18-24 hpf) is shown in Figure .
Figure 2 Expression of Gva-ParaHox during trochophore larval stage. (A) SEM of a late trochophore larvae (18-24 hpf). (B-C) Gva-Gsx expression is first detected in early trochophore larva (12 hpf) in a pair of dorso-medial domains of episphere (red arrow heads). (more ...)
The expression pattern of Gva-Gsx is rather dynamic. The first signs of transcripts of Gva-Gsx are already detected at 12 hpf in early trochophore larvae, when a pair of intensive, bilateral expression domains appears in the dorso-medial episphere (Figure ). When viewed from the anterior, each pair of expression domains appears to be composed of 4-5 Gva-Gsx-positive cells, presumably in the area of future cerebral ganglia (Figure ). This pattern of expression continues in 18 hpf trochophores (Figure and ). Here, the pattern of expression becomes considerably more complex. In addition to the paired expression domains in the dorso-medial episphere, Gva-Gsx transcripts can now be detected in a pair of cells at the tip of the developing apical sensory organ (Figure and ). These two Gva-Gsx-positive cells at the tip of the apical organ do not bear any cilia or apical tuft in the trochophore stage of G. varia (Figure ). The expression of Gva-Gsx in the apical sensory organ is restricted to two groups consisting of three sensory cells (Figure ). Beside the expression in prospective neural or sensory tissues, Gva-Gsx transcripts are also detected around the stomodeum where they appear for the first time in trochophore 18 hpf in two intensely stained bilateral semicircular clusters located anteriorly at the sides of the mouth and a less intensely stained semicircular domain at the posterior part of the mouth (Figure and ). Figures and show the trochophore stomodaeum and Gva-Gsx expression around it at 18 hpf. About 24 hpf, Gva-Gsx is expressed in a complete circle around the stomodeum (Figure ) and in three episphere domains: a pair of adjacent cells at the tip of the apical sensory organ, and two pairs of cell groups dorsolaterally marking presumptive sites of future cephalic neuroectodermal differentiation (Figure ).
Gva-Xlox transcription begins later than Gva-Gsx expression. No expression is detectable until 24 hpf when Gva-Xlox transcripts appear in a group of cells located ventrally in the hyposphere and in a pair of symmetrical expression domains in the medio-ventral episphere of the trochophore larva (Figure and ). These symmetrical expression areas are located ventrally of the more intensely stained Gva-Gsx expression domains in the pretrochal area. Gva-Xlox is also expressed in the hyposphere in 8-9 cells forming a semicircle around the anal marker (Figures and ). These weakly stained Gva-Xlox-positive cells are probably part of ventral neuroectoderm.
Gva-Cdx transcripts are first detected in the early trochophore larva (12 hpf). It is expressed at 12 and 18 hpf in two domains in the ventral vegetal plate: one in an area of presumptive posterior neuroectoderm, the other in a bilateral pair of cells in the interior of the larva (Figure and ). Using Patella vulgata as a reference, the latter expression of Pvu-Cdx probably marks the left and right primary mesentoblasts (green arrows in Figure and ). Gva-Cdx-positive neuroectodermal cells are first observed as a patch of cells expressing this gene in varying intensities (Figure ). Gradually they migrate to the boundary of the expression area (Figure ) so that they from a circle of Gva-Cdx-expressing cells around the anal marker at 24 hpf (Figure and ). The expression of Gva-Cdx around the anal marker at 24 hpf partly overlaps with the expression of Gva-Xlox in the ventral area at this stage, which is visible as a semicircle located ventrally around the anal marker (Figure and ).
ParaHox gene expression in the pretorsional veliger larva
The transcripts of all three Gva-ParaHox genes are detected almost simultaneously in the visceral mass area of the pretorsional veliger larva prior to torsion (36-48 hpf), on the left side of the larva where the digestive gland is forming (Figure ). At this stage, the velum forms a complete circle and a pair of apical tufts is observed in the velar area (Figures , and ). In addition to the apical tufts, there are "sensory cups" in the velar area. These are ciliated pockets embedded within the apical ganglion (Figure ). The expression of Gva-Gsx observed in the area of the mouth opening and of the apical organ of the late trochophore larva (Figure and ) is retained in the pretorsional veliger (Figure ). Gva-Gsx transcripts are also detected in the ventral part of the forming digestive gland in the left side of the visceral mass (Figure and ). Gva-Gsx-positive signals are further detected in the area of the mouth opening (Figure ) and in five cells in the area of the apical organ (Figure ), the two apical tuft cells (Figure ), and the sensory cup cells (compare Figures and ). Similar to Gva-Gsx, Gva-Xlox is expressed in the left side of the pretorsional veliger in the forming digestive gland (Figure ). The expression area of Gva-Xlox is located in the ventral part of the digestive gland, more dorsally but partly overlapping Gva-Gsx expression (Figure and ). The expression pattern of Gva-Xlox detected on the ventral side of the episphere of the late trochophore larva (Figure and red arrow heads) is lost in the pretorsional veliger stage (Figure and ). Additionally, five ectodermally derived Gva-Xlox-positive cells appear on the right side of the larva prior to torsion (Figure ). Similar to the trochophore stage (Figure and ), these ectodermal cells form an incomplete circle and are presumably linked to the ventral nervous system (Figure ). Gva-Cad is expressed weakly in the whole area of the nascent digestive gland of the pretorsional veliger larva (Figure ). The intensity of expression is stronger in a few cells in the dorsal area of the visceral mass in the left side of the larva (Figure and ).
Figure 3 Expression of Gva-ParaHox in the pretorsional larval stage. (A) SEM of pretorsional veliger larva. (B) High magnification of SEM of velar area showing the apical tufts and sensory cups. (C) High magnification of SEM of ciliated cells of the apical organ. (more ...)
Expression of ParaHox genes in veliger and competent larvae
After torsion (60 hpf), the velum reduces in size with a ventral split, and the mantle expands over the back of the head (Figure ). As the digestive tract continues to develop in the posttorsional veliger larva, expression patterns of Gva-ParaHox become more elaborated. At this stage, Gva-Gsx expression in the ventral part of the digestive gland and in the area of the mouth opening persists (Figure and ). Sections reveal Gva-Gsx-positive cells at the ventral border of the area of yolk-filled cells (Figure ). Gva-Gsx transcripts are further apparent as paired domains beneath the apical organ where the formation of the cerebral ganglia commences (Figures and ). At about three days post fertilization, expression of Gva-Gsx fades in the digestive gland. Instead, the gene is now expressed in the foregut around the area of the radula anlage (Figure and ). At metamorphosis, when the apical sensory organ starts to dissociate, Gva-Gsx continues to be expressed in the area of the cerebral ganglia (Figure ). Gva-Xlox expression persists on the left side of the visceral mass from the pretorsional to the posttorsional stages (Figure and ). Sections through the left side of the larva reveal that these Gva-Xlox-positive cells are part of the developing digestive gland (Figure ). Six or seven ectodermally-derived Gva-Xlox-positive cells are located in the ventral part of the visceral mass (Figure , and ). Gva-Cdx is mainly expressed in the newly formed hindgut and rectum, and weakly in the digestive gland (Figures and ).
Figure 4 Expression of Gva-ParaHox in the posttorsional larval stage. (A) SEM of pretorsional veliger larva. (B-D) Gva-Gsx is expressed in the area of the mouth opening (yellow arrow heads), apical ganglion (grey arrow heads), and ventral part of the digestive (more ...)
Post-larval ParaHox gene expression
Serial section in situ hybridizations were used to trace the expression pattern of all three Gva-ParaHox in the hatchling (about four days after fertilization). No positive signals for Gva-Xlox and Gva-Cad transcripts are detected at this stage. Gva-Gsx is the only ParaHox gene that is expressed in the most posterior part of the radula sac during postlarval development (Figure ). The juvenile hatchling has a complete radula with the radula sheath, buccal musculature, and radula bolsters (also called odontoblastic cartilages, Figure ). The posterior end of the radula sac forms the odontoblastic cushion which consists of a single-layered epithelium arranged in a semicircle and protruding into the sac's lumen. The epithelial cells are produced by two separated dorsolateral mitotic centres at the end of the sac (Figure ). Mitotic activity is scattered over the posterior area of odontoblastic cushions where the cells are small and undifferentiated. Towards the anterior of the cushions, the cells gradually elongate and form the tall odontoblastic epithelial cells (Figure ). Gva-Gsx transcripts are mainly detected in the paired odontoblastic cushions at the base of the radula (Figure ; the weak signal observed in the pedal area seems to be unspecific). Gva-Gsx is expressed both in undifferentiated cells located at the back of the cushions and in odontoblastic epithelial cells. No transcripts were detected in the cells separating the two halves of the odontoblastic cushion (Figure and ). The intensity of expression of Gva-Gsx diminishes gradually from posterior to anterior, i.e. from the undifferentiated cells to fully differentiated epithelial odontoblasts (Figure ).
Figure 5 Post-larval expression of Gva-Gsx. (A) Medial longitudinal section through the radula of a hatched juvenile stained with Toluidine Blue. (B) Paired odontoblastic cushions at the base of the radula sac where radula teeth are formed. The staining is Toluidine (more ...)