Development and juvenile anatomy of the nemertodermatid Meara stichopi (Bock) Westblad 1949 (Acoelomorpha)
© Børve and Hejnol; licensee BioMed Central Ltd. 2014
Received: 17 March 2014
Accepted: 3 July 2014
Published: 7 July 2014
Nemertodermatida is the sister group of the Acoela, which together form the Acoelomorpha, a taxon that comprises bilaterally symmetric, small aquatic worms. While there are several descriptions of the embryology of acoel species, descriptions of nemertodermatid development are scarce. To be able to reconstruct the ground pattern of the Acoelomorpha it is crucial to gain more information about the development of several nemertodermatid species. Here we describe the development of the nemertodermatid Meara stichopi using light and fluorescent microscopic methods.
We have collected Meara stichopi during several seasons and reconstruct the complex annual reproductive cycle dependent on the sea cucumber Parastichopus tremulus. Using common fluorescent markers for musculature (BODIPY FL-phallacidin) and neurons (antibodies against FMRFamide, serotonin, tyrosinated-tubulin) and live imaging techniques, we followed embryogenesis which takes approximately 9–10 weeks. The cleavage pattern is stereotypic up to the 16-cell stage. Ring- and longitudinal musculature start to develop during week 6, followed by the formation of the basiepidermal nervous system. The juvenile is hatching without mouth opening and has a basiepidermal nerve net with two dorsal neurite bundles and an anterior condensation.
The development of Meara stichopi differs from the development of Acoela in that it is less stereotypic and does not follow the typical acoel duet cleavage program. During late development Meara stichopi does not show a temporal anterior to posterior gradient during muscle and nervous system formation.
The clade Nemertodermatida comprises only nine described species of small, completely ciliated, exclusively marine, hermaphroditic worms that live mostly in interstitial habitats [1, 2]. Nemertodermatids possess a medio-ventral mouth that is the sole opening to the epithelial, sack-like gut. The nervous system is located basiepidermally, and all nemertodermatid species possess a characteristic double-statocyst or gravitational sensory organ . Nemertodermatida and Acoela (together forming the Acoelomorpha ) have recently gained attention because of their disputed phylogenetic position, which greatly impacts our understanding of the evolution of animal body plans [5, 6]. These rather simple worms have been placed as sister group to all remaining Bilateria [7–14] – in some studies as separate branches [15, 16] - and thus helpful to understand the evolutionary transition of the cnidarian-bilaterian stem species into the bilaterian stem species . Alternative hypotheses place acoelomorphs either as sister group to all remaining deuterostomes  or as sister group to the Ambulacraria (Echinodermata + Hemichordata) . In both latter cases, the lack of some morphological features in acoelomorphs, such as nephridia and gill slits, would be interpreted as independent losses . Nemertodermatids play a key role for determining the direction of character evolution in the Acoelomorpha . Nemertodermatids share plesiomorphic characters such as a basiepidermal nervous system, monoflagellate sperm, and an epithelial gut [4, 18, 19] and lack acoel novelties, including a subepidermal brain and parenchymal tissues [18, 19]. Nemertodermatids share these characters with members of the Xenoturbellida, a possible sister group of the Acoelomorpha [9, 10, 13]. A thorough comparison of the morphology and development of xenoturbellids, nemertodermatids and acoels is essential to gain a deeper insight into the ancestral character states of this taxon and the changes during cell type and organ system evolution.
Meara stichopi and Nemertoderma westbladi are the two most accessible nemertodermatid species, and both species can be collected relatively easily from the field. Embryos from both species can be obtained for developmental studies (present study and ), but detailed descriptions of the embryology are still missing. Here we describe the development of Meara stichopi and compare it with previous studies of acoel and nemertodermatid embryos.
The annual reproductive cycle of Meara stichopi and presence in the host Parastichopus tremulus (Gunnerus, 1767)
Reproduction and fertilization
Cleavage and gastrulation
Further development and morphogenesis
Anatomy of the hatchling
On the ventral side, no such condensations of axon tracts are observed (Figure 6B). Serotonin-positive sensory cells are located in the epidermis, and are connected to the basiepidermal nerve net and possess extensions through the layer of ciliated cells (Figure 6B inlet, Additional file 1H, I). There are more serotonergic cells detected in the anterior ventral region than in the posterior regions and the dorsal side (Figure 6B). The nervous system of the M. stichopi juvenile appears to have some specialized neurons, as there is a subset of FMRFamide positive neurons within the basiepidermal anterior bundles and commissure (Figure 7, Additional File 1B-F). Additionally, there are serotonin positive sensory cells, including axon tracts in the anterior region (Additional file 1 H, I). Since the statocyst is located internally, below the muscle sheet, axon tracts connect the cells of the double-statocyst to the basiepidermal nerve net (Additional file 1I). The statocyst is also connected to the muscle sheet (Additional file 1G). It is likely that these muscles help to keep the statocyst in place. In addition to the FMRFamide-positive cells of the dorsal neural bundles, we also detect positive cells that are more ventrally and internally located, whose function remains unknown (Figure 7A-C).
A reconstruction of the life cycle of Meara stichopi
Our samplings and observations show that M. stichopi has an annual life cycle that is strongly connected to the host sea cucumber (Figure 2E). Our collections allow us to reconstruct that upon entering the foregut of the sea cucumber, individuals grow inside the host until the reproductive phase in August-October. After depositing the eggs, the adults are then digested by the host. The embryos possess a tough eggshell that probably allows them to exit the gut of the sea cucumber unharmed. Embryogenesis and early postembryonic development takes up to three months and likely happens in the muddy sediment during winter. The hatchlings seem to survive on the remaining yolk until they are taken up by the sea cucumbers in January-March (Figure 2E). We also observed in November and December that the gut of the sea cucumbers is mostly empty of food, and gut parasites, such as the gastropod Enteroxenos, which infest the host. Although first described as a ‘parasite’, Westblad  considered M. stichopi to be commensal because if there is damage to the host, it is only minimal. Our findings that following the reproductive phase, adults even get digested by the host, suggesting that the impact of M. stichopi on the sea cucumber is even less than previously assumed. The possible loss of energy is confined to the homeostasis of the individual worms and to the yolk deposition into the eggs that leave the sea cucumber.
The development and architecture of the nervous system
The nemertodermatid nervous system has previously been investigated using histological [20, 21] and immunocytochemical [24, 25] methods and is described as entirely basiepidermal. Unlike acoels, nemertodermatids have no portions of the nervous system internalized in a way that they are located below the muscle sheath. The exception is the innervation of the statocyst, which is connected via nerve fibers to the outer basiepidermal plexus. There are no brain-like structures described for nemertodermatids – the anterior condensations are exclusively basiepidermal and ring-shaped (Nemertoderma westbladi[24, 25]) or just connected by a commissure composed out of neurite bundles (Meara stichopi). Our results confirm this structure for Meara stichopi and show that the dorsal neurite bundles persist from an anlage in the hatchling to the fully formed structure in the adult. The use of the tyrosinated-tubulin antibody reveals the presence of a larger net of neurons that extend axon tracts also to the internal of the body, while just a subset is stained by the anti-serotonin and anti-FMRFamide antibodies. The dorsal anlage of the two bilateral, longitudinal, thickenings of the nerve plexus are wider than previously described, with a more prominent anterior thickening. Interestingly, such dorsal longitudinal condensations are not found in Nemertoderma westbladi, which instead has a pair of ventral and lateral condensations . A previous study by Raikova et al.  describes the presence of ‘parenchymal fibre bundles’ in M. stichopi. Our results using anti-tyrosinated, anti-FMRFamide and anti-serotonin antibodies, along with BODIPY FL-phallacidin, shows that these ‘fibre bundles’ are basiepidermal, located above the muscle sheet and not internally. Contrary to previous observations [24, 25], we have detected positive immunoreactivity around the statocyst using anti-serotonin and anti-FMRFamide antibodies (Additional file 1B-F). Axon tracts connect the statocyst to anterior epithelial cells and to the dorsal basiepidermal nerve condensations. In accordance with previous reports, we could not detect any stomatogastric nervous system in the juvenile of M. stichopi. The nervous system of M. stichopi, as well as that of other nemertodermatids, is devoid of any prominent internalized structures, such as brains or neurite bundles, which are present in some acoel groups. The nervous system of nemertodermatids is more similar to the nervous system of xenoturbellids, which lacks condensations and only consists of a basiepidermal nerve plexus . Recent phylogenomic analyses [9, 10, 13] suggest that Xenoturbella is closely related to the Acoelomorpha (Xenacoelomorpha). Since Xenoturbella and nemertodermatids both lack subepidermal condensations, this condition has to be considered as plesiomorphic for the whole group and the internalized brain and neurite bundles (‘cords’) found in some acoel taxa have been secondarily evolved from a basiepidermal nerve net. This interpretation hinges on the phylogenetic position of the Xenacoelomorpha as a whole. In the case of Xenacoelomorpha within the Deuterostomia , multiple losses of brain-like and cord-like structures in the Xenacoelomorpha must be considered. However, it is difficult to explain why some lineages display only dorsal condensations (M. stichopi), and some lineages only ventral and lateral condensations (Nemertoderma) , as remnants of an ancestral ventrally condensed nervous system. Further molecular studies are necessary to place the Acoelomorpha in the animal tree of life and to clarify the homology of specific substructures found in this fascinating group of animals.
Comparison of the development with Nemertoderma westbladi and acoels
Although the general pattern of the first divisions of the M. stichopi embryo is similar to the cleavage of N. westbladi, the major differences are the more spherical shape of the N. westbladi embryo versus the oval shape of the M. stichopi embryo and the considerable size differences between the micromeres (Figure 9A-H). The later development of M. stichopi is characterized by an inner cell mass of large, equal-sized blastomeres, which are surrounded by a monolayer of smaller blastomeres. A similar pattern is also present in acoel embryos [6, 29, 30, 33]. The first structure that emerges in acoelomorph embryos is the muscular grid that can be identified by fluorescently labeled phallotoxins  (Figure 5). In the acoel Isodiametra pulchra, the musculature starts to form at the animal pole (=anterior) of the embryo and progresses to the posterior end of the embryo . In contrast, no such gradient is present in M. stichopi, as the musculature appears simultaneously along the entire body axis. Similar to I. pulchra, the ring musculature of M. stichopi is formed before the longitudinal musculature and both are formed before elements of the nervous system are detectable. The formation of the muscular sheath coincides with the differentiation of the outer epidermis and the formation of the cilia (Figure 5). Since the nervous system of M. stichopi is basiepidermal, one should not expect epidermal cells to immigrate internally below the muscle sheet. An exception might be the statocyst sensory complex at the anterior end, but its formation remains unclear. This is different from the nervous system development in acoels where the nervous system is formed by all micromeres  and cells from the outer sheet migrate to form neural structures .
The nemertodermatid Meara stichopi has an annual life cycle with a main reproductive period inside the foregut of the holothurian Parastichopus tremulus. The development of the embryos undergoes an early stereotypic cleavage, and embryogenesis takes 9–10 weeks until the juvenile hatches. The cleavage program of M. stichopi shows significant differences to that of the sister group, Acoela. The musculature is formed before the nervous system, similar to what has been described in acoel embryos. Our study demonstrates the variability of the development in the Acoelomorpha and that further studies are needed to reconstruct the ground pattern of acoelomorph development.
Collection and maintenance of Meara stichopi
Sea cucumbers of the species Parastichopus tremulus (Gunnerus, 1767), the host of Meara stichopi, were collected throughout the year between Winter 2009/2010 – Winter 2013/2014 at collection sites around Bergen, Norway. Between 10 – 50 sea cucumbers were collected from 200 – 350 m depth using the “Schander Sled” (Figure 1) during each collection trip and brought to the lab at the Sars Centre for dissection. Sea cucumbers were opened and the digestive tracts were examined for the presence of M. stichopi. Approximately 3000 M. stichopi have been collected in total and about 800 individuals deposited a total of about 2500 embryos during the reproductive seasons. Juveniles and adults of M. stichopi were transferred to filtered seawater and kept at 5–8 degrees in glass bowls. Seawater was changed every 2–3 days and animals were kept for up to 2–3 months in the lab. Gravid adults deposited oocytes into bottoms of the glass bowls in a jelly-mesh and sperm was surrounding the eggs (Figure 1). Fertilized eggs were cultured in petri dishes containing seawater supplemented with Penicillin (100 Units/ml) and Streptomycin (100 μg/ml) and kept at 5–8 degrees.
Hauglandsosen (60 24.533 N 5 06.566 E).
Lysefjorden (60 12.347 N 05 17.903 E).
Hjeltefjorden (60 24.366 N 05 06.111 E).
Raunefjorden (60 15.896 N 05 08.448 E).
Laboratory work on the species M. stichopi does not raise ethical issues. Therefore approval from a research ethics committee is not required.
Egg shell penetration and fixation of embryos and adults
Egg shells of embryos and pre-hatchlings were penetrated using 1% Thioglycolate/0.05% Pronase (Sigma Aldrichs 5147) in sea water (pH 8.0) for 4 hours at 4°C. During development, the eggshell extends slightly along the long axis and softens, such that late stage embryos are easier to penetrate with a needle than early cleavage stages. Before fixation, embryos, juveniles and adults were relaxed with 7.5% MgCl2 in Millipore water, and fixed using 4% Paraformaldehyde in filtered sea water for 1 hour at 4°C. Fixed specimens were washed four times in PBS containing 0.1% Triton X (PTx) and stored at 4°C before subsequent staining.
Antibody and phallacidin staining
Before the antibody staining, holes were poked into the eggshell of the fixed embryos with insect pins to facilitate the penetration of the antibodies (total n = 500). Antibodies against tyrosinated tubulin (Sigma) and BODIPY®FL labeled phallacidin (Molecular Probes) were used to label the embryos and juveniles following a standard procedure . The phallacidin was used to visualize the F-actin of the cell cortex and the muscle fibers of the embryo, however, it also labeled the centrosomes of some early embryos (e.g. 7 day old, Figure 4D). Anti-serotonin (Sigma) and anti-FMRFamide (Sigma) antibodies were used to label substructures of the nervous system. Specimens were blocked with two 15 min washes in PTx + 0.1% BSA (Bovine Serum Albumin) followed by a 30 min incubation in PTx + 5% normal goat serum. Specimens were incubated with the primary antibody (mouse anti-tyr-tub 1:500, rabbit anti-serotonin 1:200, rabbit anti-FMRFamide 1:200) in PTx + 5% goat serum overnight at 4°C on a shaker. Primary antibody was removed with three 5 min and four 30 min washes in PTx + BSA and an additional blocking step in PTx + normal goat serum for 30 min. Specimens were incubated with the secondary antibody (Cy3 labeled anti-mouse IgM and Cy5 labeled anti-rabbit IgM) diluted 1:200 in PTx + normal goat serum overnight. The secondary antibody was removed with three 5 min and four 30 min washes in PTx + BSA. BODIPY FL phallacidin was added to some samples by first washing the specimens in PBS and incubating in 3–10 Units/ml PTx for 2 hours. Specimens were then washed three times in PBS and prepared for mounting. Propidium Iodide was used to stain the nuclei in some of the samples following a standard protocol in which 0.01 mg/ml propidium iodide was added to the incubation with BODIPY FL-phallacidin.
Specimens were mounted in ‘Murray’s Clear’ (2:1 mixture of benzyl benzoate and benzyl alcohol). Prior to transfer to Murray’s Clear, specimens were subjected to a series of isopropanol washes (70%, 85%, 95%, 100%). Specimens were imaged using a Leica SP5 confocal microscope. Image stacks were rendered using Imaris 7.6 (Bitplane).
Embryos were recorded using a 4D-microscopy system (modified system after Hejnol & Schnabel ). Zygote and 2-cell stages were mounted in seawater, covered with a coverslip and sealed with Vaseline. Recordings (n = 3) were conducted at 10°C and Z-stacks composed out of 50 images were taken every 10 minutes. Cells were traced using the software SIMI°BioCell.
Images of juveniles and adults were taken using a Canon 5D Mark III mounted on a Leica 120 M dissecting scope or with a Zeiss AxioCam HRc mounted on a Zeiss Axio Skope.A1.
We thank the crew of the “Hans Brattström” and Henrik Glenner, Christiane Todt, Glenn Bistrow, Kenneth Meland, Christopher Noever for the continuous help and supply of Parastichopus tremulus. Jonas Bengtsen and Sabrina Schiemann have been helpful with the 4D-microscopy. We thank all S9 team members for helping with the dissection of the sea cucumbers. Kevin Pang edited and improved the manuscript. The study received support by a Marie Curie International Re-Integration grant to AH (FP7-PEOPLE-2009-RG 256450).
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