The ultrastructure of the apical organ of the Müller's larva of the tiger flatworm Prostheceraeus crozieri

The tiger flatworm Prostheceraeus crozieri (Polycladida) develops via an eight‐lobed, and three‐eyed planktonic Müller's larva. This larva has an apical organ, ultrastructural details of which remain elusive due to a scarcity of studies. The evolution and possible homology of the polyclad larva with other spiralian larvae is still controversial. Here, we provide ultrastructural data and three‐dimensional reconstructions of the apical organ of P. crozieri. The apical organ consists of an apical tuft complex and a dorso‐apical tuft complex. The apical tuft complex features a central tuft of five long cilia, which emerge from four or five individual cells that are themselves encircled by two anchor cells. The necks of six multibranched gland cells are sandwiched between ciliated tuft cell bodies and anchor cells. The proximal parts of the ciliated cell bodies are in contact with the lateral brain neuropil via gap junctions. Located dorsally of the apical tuft complex, the dorso‐apical tuft complex is characterized by several long cilia of sensory neurons, these emerge from an epidermal lumen and are closely associated with several gland cells that form a crescent apically around the dorsal anchor cell, and laterally touch the brain neuropil. Such ciliated sensory neurons emerging from a ciliated lumen are reminiscent of ampullary cells of mollusc and annelid larvae; a similar cell type can be found in the hoplonemertean decidula larva. We hypothesize that the ampullary‐like cells and the tuft‐forming sensory cells in the apical organs of these spiralian larvae could be homologous.

cell types: ciliary tuft cells, ampullary cells, and parampullary cells (Croll & Dickinson, 2004). Similarly, in annelids ciliary tuft cells and ampullary-like cells have been described (Lacalli, 1981;Marlow et al., 2014). One of the central questions of spiralian evolutionary developmental biology is whether some of their larval features like the apical organ, are homologous (Marlow et al., 2014;Nielsen, 2004Nielsen, , 2005Rawlinson, 2014); if so, this would suggest that the larvae themselves are homologous.
While most free-living flatworms are direct developers, some polyclads develop via one of several planktonic larval types known as Müller's, Goette's, and Kato's larvae. These larvae are characterized by different numbers of lobes, eyes, and general body shapes (Martín-Durán & Egger, 2012;Rawlinson, 2014). The most striking peculiarity of polyclad larvae are leg-like lobes that protrude from the body, ciliary bands at the margins of these lobes, a posterior tuft, and an apical organ (also referred to as a "frontal organ") (Lacalli, 1982(Lacalli, , 1983Lapraz et al., 2013;Martín-Durán & Egger, 2012;Rawlinson, 2014). In three transmission electron microscopical studies, the ultrastructure of the apical organ of three Müller's and a Goette's larvae was described as an apical tuft of long cilia with associated gland cells at the anterior pole (Lacalli, 1982(Lacalli, , 1983Ruppert, 1978). In the Müller's larva of the polyclad Prostheceraeus crozieri, a second tuft of long cilia was found just dorsal to the apical tuft in a scanning electron microscopical study (Lapraz et al., 2013). Here, we provide new ultrastructural data concerning the nature of this second dorsoapical ciliary tuft, a detailed description and three-dimensional (3D) reconstruction of the apical organ, and a comparison with other spiralian apical organs.

| Transmission electron microscopy
For transmission electron microscopy, 1-day-old larvae of P. crozieri were anaesthetized in 7.14% aqueous magnesium chloride for 10 min and then fixed in glutaraldehyde and osmium tetroxide in cacodylate buffer following the protocol of Eisenman and Alfert (1982), detailed in Salvenmoser et al. (2010) (for collection of adult animals, see Lapraz et al., 2013). Samples were dehydrated in a graded ethanol series, and embedded in EPON (Sigma-Aldrich) (see Gammoudi et al., 2016;Salvenmoser et al., 2010). Semithin sections (0.35 µm) as well as ultrathin sections (80 nm) were cut using a Leica ultracut UCT microtome. Semithin sections were stained using Richardson's methylene blue azure II disodium tetraborate decahydrate (Richardson et al., 1960). Ultrathin sections from two different individuals were contrasted using lead citrate and examined with a Zeiss Libra 120 energy filter transmission electron microscope using a Tröndle 2 × 2k highspeed camera with ImageSP software (Tröndle). Image processing was performed with ImageSP and Adobe Photoshop 7.

| RESULTS
The apical organ of P. crozieri's Müller's larva consists of two connected parts, which are termed "apical tuft complex" and "dorsoapical tuft complex" which we describe in turn.

| Ultrastructure of the apical tuft complex
The apical tuft complex is located right at the anterior pole ( Figure 1).
It comprises three key components: (1) a cluster of monociliated sensory cells (apical tuft sensory cells, or ATS cells), surrounded by (2) a circle of gland cell necks and the associated gland cells (apical tuft gland cells, or ATG cells), which are anteriorly (above the basal membrane) enclosed by (3)

| The apical tuft complex
In general, there is a broad agreement between our data and the observations of Ruppert (1978) and Lacalli (1982Lacalli ( , 1983. The apical tuft complex of the Müller's larva of P. crozieri includes the two main elements described by Ruppert (1978) and Lacalli (1982Lacalli ( , 1983: apical tuft complex seem to be modified epidermis cells, that is, epidermal cells that lack big vacuoles. A difference between the ATG cells in Goette's larva and in Müller's larva of P. crozieri is the presence of peripheral microtubules in the latter (Ruppert, 1978). We note that the different granule types in ATG cells could not be distinguished in our SBEM images (Figure 2), in contrast to our TEM images (Figures 4 and 6a).

| The number of anchor cells
The anchor cells of the apical tuft complex can, in fact, be recognized in published work. In Ruppert (1978), a slightly oblique transversal TEM section through the very anterior tip of a Goette's larva contains a part of a putative anchor cell (Ruppert, 1978; his figure 4a). A more posterior transverse section shows at least three anchor cells surrounding the circle of gland cell necks (Ruppert, 1978; his figure 4b). The number of anchor cells may apparently vary between species or at least between different larval types: at least three in some Goette's larvae (Ruppert, 1978), and two in some Müller's larvae (this study).
4.1.2 | The number, branching, and arrangement of the ATG cells Lacalli (1983) describes and illustrates the shape of the entire mass of gland cell necks as a "flat-bottomed flask." In his drawing (Lacalli, 1983; his figure 2), the ATG cell necks point straight anterior.
He describes 12 ATG cell necks resulting from the trifurcation of four thick gland cell cords emerging from at least two ATG cells. In the larva of P. crozieri described here, the gland cell necks do not point straight anterior, but rather surround the ATS cells in a cup shape.
We hypothesize that the interleaving of gland cell necks serves a stabilizing function, even more so, in that the two interleaving gland cell neck bundles occupy one-half of each anchor cell. This stresses the importance of the anchor cells as a key component of the apical tuft complex. Regarding the cell bodies of the ATG cells, Ruppert (1978) simply notes that they are located above, below, and behind the brain, while the ATG cell necks run posterior, dorsal, and ventral to the neuropil. It is not clear, however, whether DATG cells are also part of his observations. In P. crozieri's Müller's larva, the ATG cell nuclei are below the brain, but the cell bodies and necks run laterally to both sides (see Figures 7a,b and 8a). Only the DATG cells are   Ruppert (1978) comprises at least six cilia and hence at least six monociliated apical cell processes (Ruppert, 1978; (Ruppert, 1978). In P. crozieri, we think that the missing fifth ATS cell body is not located on top of the brain as are the other four, but rather more ventral. The number of ATS cells is not consistent between the studied species: six or more ATS cells in Ruppert's undetermined Müller's larva and five in P. canadensis and in P. crozieri.

| The dorso-apical tuft complex
In all other ultrastructural studies concerned with polyclad larvae, only a single apically located organ, either termed "frontal organ" (Ruppert, 1978) or "apical organ" (Lacalli, 1982(Lacalli, , 1983, was described. In a light and scanning electron microscopical study, Lapraz et al. (2013) observed for the first time a second tuft of about 15 long apical cilia, shifted slightly dorsal to the first tuft of long apical cilia.
We were able to find this second apical ciliary tuft in our semi and ultrathin sections (Figures 6a and 8c,d).
The distinction of these two anterior cilia tufts is difficult without electron microscopical approaches as they are located quite close (ca. 15 µm) to each other (Figures 4a,b and 6a; see also Lapraz et al., 2013;  Ruppert (1978) are of planktonic origin with unknown age, but the larvae of Lacalli (1983) were fixed within 1 day of hatching like the specimens used in the present study. Curiously, in the specimen used for SBEM, the AmSN cells, and their ciliary tuft could not be detected either, only the associated DATG cells.

| Neuronal differences in the apical and the dorso-apical tufts in polyclad larvae
The function of the apical/frontal organ in polyclad larvae is a muchdiscussed topic (Kato, 1940;Lacalli, 1982Lacalli, , 1983Rawlinson, 2010Rawlinson, , 2014Ruppert, 1978;Younossi-Hartenstein & Hartenstein, 2000). Kato (1940) suggested that the frontal organ is used to break the eggshell at hatching in polyclad larvae. In contrast, Ruppert (1978) hypothesized a sensory and glandular function based on ultrastructural data. Lacalli (1982) observed that the bases of the ciliated apical cells in P. canadensis is located on top of the brain, but not directly connected to the brain and he hence determined that these ciliated apical cells were not sensory neurons (Lacalli, 1982), or that it was not clear if they were sensory neurons or sensory cells (Lacalli, 1983). In P. crozieri, we found neuronal axons in the basal region of the AmSN (Figure 6b (Merkel et al., 2015;Nielsen, 1971;Woollacott & Eakin, 1973). The frontal organ in Loxosomella larvae marks the anterior pole, while the apical organ is located dorsally (see Merkel et al., 2015;their figure 2, Wanninger et al., 2007). The apical organ of the entoproct larva of Loxosomella murmanica shows a high number of serotonin-expressing cells (14-16), but no serotonin expression has been described or shown in the Loxosomella larval frontal organ (see Wanninger et al., 2007). In another Loxosomella larva, a frontal organ ganglion was even described (Woollacott & Eakin, 1973). Comparing the data of the entoproct larva of L. murmanica with our current knowledge of the polyclad Müller's larva of P. crozieri, the following similarities can be found: (1) an anteriorly located, ciliated, glandular organ with a secretory character (P. crozieri: apical tuft complex, L. murmanica: frontal organ) and (2) a second ciliated organ, located dorsal of the frontal organ, with (sensory) neurons (P. crozieri: dorso-apical tuft complex, L. murmanica: apical organ).

| Mollusca
Based on the complexity of the expressed serotonergic pattern in the apical organ, there may be a link between mollusc and entoproct apical organs and even a common ancestral condition (Wanninger et al., 2007).
In molluscs, serotonergic immunoreactivity has been shown in parampullary cells (Croll & Dickinson, 2004;Kempf et al., 1997;Marois & Carew, 1997), and the serotonergic cells in the apical organ of a polyplacophoran larva have been homologised with the serotonergic cells in an entoproct larval apical organ (Wanninger et al., 2007). We suggest that the serotonergic apical organ cells of the polyplacophoran and the entoproct larva are also parampullary cells.

| Nemertea
The apical organ in Müller's larvae of P. crozieri shares a very similar ultrastructural organization with the apical organ in the hoplonemertean decidula larvae of Quasitetrastemma stimpsoni (Magarlamov et al., 2020). Both main components are present: ampullary cells and ATS cells. Additionally, these cells are closely associated with glands in both animal groups. In the decidula, the apical organ consists of a ciliary tuft composed of several multiciliated cells surrounded by a cupshaped structure built by gland cells and braced by the surrounding epidermal cells. The multiciliated tuft cells (called apical plate cells) are arranged in two layers forming an outer and inner concentric ring (Magarlamov et al., 2020). The inner layer forms a ciliated lumen of six to eight cells and is surrounded by an outer layer consisting of four multiciliated cells (Magarlamov et al., 2020). The inner layer forming a ciliated lumen is reminiscent of the AmSN that also form a ciliated lumen in P. crozieri, while the outer multiciliated layer in Q. stimpsoni and the ATS cells in P. crozieri are tuft-building sensory cells, in which the cilia do not emerge from a lumen (Figure 9). In contrast to Müller's larvae, the gland cells of the larva of Q. stimpsoni are not bifurcated.
Interestingly, there are three different gland cell types in the apical organ in early rudiment hoplonemertean larvae (Magarlamov et al., 2020) and in Müller's larvae (Figure 4a According to recent molecular phylogenies, Platyhelminthes and Nemertea are possible sister groups (Marlétaz et al., 2019, Philippe et al., 2019; this clade has been called Parenchymia (Nielsen, 1995). An interesting parallel in polyclad larvae and larvae of hoplonemerteans is that in both groups the multiciliated cells with a lumen, and the ciliated tuft cells of the apical organ are intimately associated with gland cells (Magarlamov et al., 2020).
This is in contrast to other spiralian larvae, making this finding a possible synapomorphy of the hoplonemertean and the polyclad larvae. The overall architecture of the hoplonemertean and the polyclad apical organ is also very similar, with the exception that the tuft-forming, ciliated lumen is in the middle of the apical organ in the hoplonemertean larva, while in the Müller's larva it is slightly dorsally located.

| A revision of the apical organ in spiralians
Based on our data and on an extensive review of the literature, we propose a new definition of the spiralian apical organ, which we consider homologous. In many spiralian larvae, the apical organ consists of two main components: first, multiciliated sensory neurons forming a ciliated lumen (often referred to as ampullary cells  Lacalli, 1982Lacalli, , 1983Ruppert, 1978; this work; nemertean pilidium larva: Lacalli & West, 1985), or multiciliated (annelids, Lacalli, 1981;molluscs, Croll & Dickinson, 2004; nemertean decidula larva, Magarlamov et al., 2020;nemertean pilidium larva, Cantell et al., 1982). These two main components differ in their relative positions. They either form adjacent structures (entoprocts: Nielsen, 1971;Wanninger et al., 2007;molluscs: Page, 2002; annelids : Lacalli, 1981;Marlow et al., 2014; polyclads: this work), or a series of organs (molluscs: Croll & Dickinson, 2004;Kempf et al., 1997;Page & Parries, 2000), or a concentric complex (nemerteans: Magarlamov et al., 2020). Since these two main components are not universal, but are very common in four spiralian phyla (Platyhelminthes, Annelida, Mollusca, Nemertea, Figure 9a-d), we propose them to be an ancestral character for spiralians ( Figure 9e). According to the proposed sister group relationship of Mollusca and Entoprocta, we hypothesize the presence of ampullarylike cells in entoprocts, the apical organ of which has not been studied ultrastructurally to date. In phoronids and other lophophorates, the apical organ may have been substantially modified. One of the components of this putatively homologous apical organ may be absent in some groups such as a ciliary tuft in some molluscs (see Ruthensteiner & Schaefer, 2002) or AmSN in some annelids C Cell ell B Biology iology I International nternational (Lacalli, 1981) and nemertean pilidium larvae (Lacalli & West, 1985;Magarlamov et al., 2020). For polyclads, AmSN have been described for the first time in the present study. The AmSN may either have been overlooked in these polyclad larvae from other species, or they may not be present at all, or they are not present in the examined developmental stages (Lacalli, 1982(Lacalli, , 1983Ruppert, 1978).
According to the current phylogeny of flatworms, polyclad larvae as a plesiomorphic character for the Platyhelminthes would imply the loss of a larva in the Catenulida, the Macrostomorpha, the Prorhynchida, and the Euneoophora (Egger et al., 2015). These losses are necessary to explain the homology of the larval forms in polyclads and the trochophore larva of the Trochozoa (Egger et al., 2015;Martín-Durán & Egger, 2012).
We hypothesize that the two main components of apical organs in many spiralians-the AmSN and the tuft-forming sensory cellsmay comprise a homologous structure (Figure 9e). The alternative is that these components have arisen independently several times, which would suggest that they are required for functions that can best be fulfilled by structures with these forms. No functional studies have been made on the apical organs of polyclad larvae, which may further elucidate these questions. C Cell ell B Biology iology I International nternational Zankel, A., Wagner, J., & Poelt, P. (2014). Serial sectioning methods for 3D investigations in materials science.

SUPPORTING INFORMATION
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