The ultrastructure of the apical organ of Curini‐Galletti's larva, a new polyclad larval type

Polycladida are the only free‐living flatworms with a planktonic larval stage in some species. Currently, it is not clear if a larval stage is ancestral in polyclads, and which type of larva that would be. Known polyclad larvae are Müller's larva, Kato's larva and Goette's larva, differing by body shape and the number of lobes and eyes. A valuable character for the comparison and characterisation of polyclad larval types is the ultrastructural composition of the apical organ. This organ is situated at the anterior pole of the larva and is associated with at least one ciliary tuft. The larval apical organ of Theama mediterranea features two multiciliated apical tuft sensory cells. Six unfurcated apical tuft gland cell necks are sandwiched between the apical tuft sensory cells and two anchor cells and have their cell bodies located lateral to the brain. Another type of apical gland cell necks is embedded in the anchor cells. Ventral to the apical tuft, ciliated sensory neurons are present, which are neighbouring the cell necks of two furcated apical tuft gland cells. Based on the ultrastructural organisation of the apical organ and other morphological features, like a laterally flattened wedge‐shaped body and three very small lobes, we recognise the larva of T. mediterranea as a new larval type, which we name Curini‐Galletti's larva after its first discoverer. The ultrastructural similarities of the apical organ in different polyclad larvae support their possible homology, that is, all polyclad larvae have likely evolved from a common larva.

Are different polyclad larvae homologous to each other, or have they evolved several times within polyclads?This question is difficult to answer, as features like number of lobes and eyes are relatively superficial and do not meet Remane's criteria of homology (Remane, 1952).To make things worse, there is a lack of comparable morphological characteristics and accompanying detailed studies about polyclad larvae (Rawlinson, 2014).
One feature shown to be valuable for the comparison of marine invertebrate larvae in a phylogenetic context is the ultrastructure of the apical organ (Dittmann et al., 2023).The apical organ is a very prominent character present in many spiralian and also in polyclad secretory cell bodies around the brain (Dittmann et al., 2023;Lacalli, 1982Lacalli, , 1983;;Ruppert, 1978).In the apical organ of the Müller's larva of Prostheceraeus crozieri a second ciliary tuft and related gland cells are located dorsally to the apical tuft (AT) (Dittmann et al., 2023).This dorso-apical tuft complex (ATC) consists of sensory neurons (SN) with long cilia emerging from an epidermal lumen and which are closely associated with gland cells (Dittmann et al., 2023).The ciliated-lumen forming cell type resembles the ampullary cells described in the apical organ of mollusc larvae (Haszprunar et al., 2002;Page, 2002;Page & Parries, 2000) and annelid larvae (Marlow et al., 2014), and also resembles SN present in the hoplonemertean decidula larva (Magarlamov et al., 2020).Besides the ampullary cells, the apical organs of these larvae feature further tuft-forming sensory cells, similar to the tuft building sensory cells found in polyclad larvae (Dittmann et al., 2023;Haszprunar et al., 2002;Magarlamov et al., 2020;Marlow et al., 2014;Page, 2002;Page & Parries, 2000).This similarity between the cellular components of the apical organ of the polyclads and other spiralian larvae led to the hypothesis that the apical organ might be homologous in spiralians (Dittmann et al., 2023).
While ultrastructural data about the apical organ in cotylean Müller's and acotylean Goette's larva are available (Dittmann et al., 2023;Lacalli, 1982Lacalli, , 1983;;Ruppert, 1978), nothing is known about the apical organ in other polyclad larval types.This study provides information about the ultrastructure of the apical organ found in a newly described larval type of the polyclad species T. mediterranea, elucidating both the distinction between polyclad larval types, and the bauplan of the apical organ in polyclad larvae.

| Animals
Adult animals of T. mediterranea Curini-Galletti, Campus & Delogu, 2008 were sampled on a sandy beach in March 2016 in Rovinj, Croatia (45.1180406 N, 13.616976 E), as well as in April/May 2019 in Punat, Croatia (45.008536N, 14.622129 E).The upper 5−10 cm of the sand were sampled at the waterline with small plastic containers, extracted with a magnesium chloride solution and kept under laboratory conditions (for more details see Bertemes et al., 2020).
About 1 week after cocoons were laid, juveniles (larvae) emerged from the cocoon into the water column in the petri dishes (also see Curini-Galletti et al., 2008).One-day old larvae of T. mediterranea were sampled from the petri dish, anaesthetised and fixed for further experiments.

| Scanning electron microscopy (SEM)
About 20 specimens of 1-day old larvae were anaesthetised in 7.14%

| Larval shape
The shape of a 1-day old larva of T. mediterranea is a laterally flattened wedge with an apical/anterior tip and a posterior base The mouth opening is recessed in a depression which is surrounded by two lateral lobes (Figures 2a,c,d,f,g,3a,b and 4).
These lobes are rather compact, not far protruding from the body.
The length of the larva along the median line from the anterior to the posterior pole is 89 ± 13 µm (n = 11).At the broadest point (level of the oral hood) the larva measures from ventral to dorsal 56 ± 5 µm (n = 5).Measured from left to right side at the broadest point (level of lateral lobes) the larva is 50 ± 5 µm (n = 6) broad.The whole larva is covered with cilia (Figures 2e and 3).
The similarity between the larvae of Theama, Boninia and Pericelis is especially remarkable due to the close phylogenetic relationship of the three genera (Goodheart et al., 2023) (Figure 1).In the latest phylogeny Boninia and Theama appear as sister groups with Pericelis as their sister clade (Goodheart et al., 2023) (Figure 1).In contrast to the Goette-like larvae of Pericelis or Boninia, the larva of Theama features neither a pair of ventro-lateral lobes (as is the case in Pericelis), nor a dorsal lobe (as is the case in Boninia).Due to the low number and the distinct, very short appearance of lobes, we propose the larva of Theama as new larval type.In honour of its first describer, we name this new larval type Curini-Galletti's larva.Based on their phylogenetic position, together with the number of eyes and the reduced lobes (Table 1), we consider the larvae of Pericelis and Boninia to be Curini-Galletti-like, rather than Goette-like.

| Comparison of the apical organ between different types of polyclad larvae
The particular cell types of the apical organ of Curini-Galletti's larva and their arrangement resemble the apical organ described in other polyclad larvae (Dittmann et al., 2023;Lacalli, 1982Lacalli, , 1983;;Ruppert, 1978).Corresponding to the apical organ of P. crozieri's Müller's larva (Dittmann et al., 2023), also the apical organ of T. mediterranea's larva includes: (1) a group of apical tuft-forming ciliated sensory cells, surrounded by (2) a circle of gland cell necks of two different types, which are (3) sandwiched between apical tuft sensory cells and two anchor cells.The size and electron-density of the apical tuft gland type 1 and type 2 granules of Curini-Galletti's larva are similar to those described in Müller's larva (Dittmann et al., 2023;Ruppert, 1978) (see Table 2).Like P. crozieri's Müller's larva, the Curini-Galletti's larva's apical organ additionally comprises (4) ciliated sensory neurons, as well as (5) a third apical gland cell type (see Table 2).
However, the type of ciliation of sensory neurons, the branching and the presence or absence of peripheral microtubules in the apical gland cells, as well as the number of cells comprising the apical organ differs between different larval types
P. crozieri's Müller's larva and T. mediterranea's Curini-Galletti's larva both have six apical tuft gland type 1 cells (Dittmann et al., 2023).It is unclear if the number of apical tuft gland cells vary between different polyclad species developing via a Müller's or Goette's larva (Dittmann et al., 2023;Lacalli, 1983;Ruppert, 1978).Unlike Müller's larva, which has furcated apical tuft gland type 1 cell necks (Dittmann et al., 2023;Lacalli, 1983), apical tuft gland type 1 cell necks of Curini-Galletti's larva are not furcated, and there are no data from other polyclad larvae.A further difference is the presence of peripheral microtubules in the Müller's larva apical tuft gland cells, while in Goette's larva no microtubules in the apical tuft gland cells were identified (Ruppert, 1978).In Curini-Galletti's larva microtubules in the apical tuft gland type 1 cells are present while in other apical tuft gland cells no microtubules could be found (Table 2).In P. crozieri's Müller's larva as well as in Curini-Galletti's larva the apical tuft gland cells are orientated laterally along both sides of the brain, whereas in Goette's larva they run ventrally and dorsally to the brain (Ruppert, 1978, see also his fig.2) (Table 2).
Similar to P. crozieri's Müller's larva, also in Curini-Galletti's larva sensory neurons closely related to the apical tuft complex can be found.In contrast to the ampullary sensory neurons (AmSN) of P. crozieri's Müller's larva, which are characterised by a central lumen (Dittmann et al., 2023), the sensory neurons in Curini-Galletti's larva show no detectable lumen (Figure 8a).In both larvae, these ciliated sensory neurons are adjacent to a half-circle of gland cell necks, neither belonging to apical tuft gland type 1, nor to apical tuft gland type 2 cells.These gland cells are termed dorso-apical tuft gland (DATG) cells in P. crozieri's Müller's larva (Dittmann et al., 2023) and are referred in Curini-Galletti's larva as apical tuft gland type 3 cells, as they are not related to a tuft-forming structure.In contrast to dorso-apical tuft gland cells, apical tuft gland type 3 cells have smaller granules, and differ in number and position (Table 2).While the ampullary sensory neurons and the dorso-apical tuft gland cells of P. crozieri's Müller's larva are located dorsally of the apical tuft complex, the sensory neurons and the apical tuft gland type 3 cells of Curini-Galletti's larva are located ventrally of the apical tuft.In mollusc larvae the ciliated ampullary cells (sensory neurons) are not always tuft building (Kempf et al., 1997) and their position and c Lacalli (1982Lacalli ( , 1983).
In conclusion, the apical organ of Curini-Galletti's larva combines ultrastructural characters found in both Müller's and Goette's larva, though differing in their number and arrangement (see Table 2).The combination of these ultrastructural characteristics is so far unique, supporting Curini-Galletti's larva as an autonomous larval type.
Currently, no data about the ultrastructure of the apical organ of the larvae of Pericelis or Boninia are available, reducing the comparison between these larval types to morphological characteristics like the number of lobes (see discussion above).Further studies on the ultrastructure of the larval apical organ in Pericelis and Boninia could help to clarify if these larvae are a type of Curini-Galletti's larva, or if they can even be thought to present new larval types.

| Impact on the evolution of polyclad larvae
The finding of a larva in T. mediterranea instead of a direct developing juvenile increases the likelihood of an ancestral polyclad larva, which so far has only received weak support (see Goodheart et al., 2023).
Hypothesising larvae as ancestral for Polycladida, four possible scenarios of the evolution of polyclad larvae are conceivable: (1) Müller's larva represents the ancestral larval type, (2) Goette's larva represents the ancestral larval type, (3) Curini-Galletti's larva represents the ancestral larval type or (4) none of them represents the ancestral larval type and all Müller's, Goette's and Curini-Galletti's larva are derived larval types evolved from a common ancestral larval type, which is yet unknown.
Currently, still too much data about the ultrastructure of the apical organ in other polyclad larvae is missing to make a final statement.
larvae.It is situated at the anterior pole of the animal.The apical organ of Müller's and Goette's larvae comprises a cluster of monociliated (Müller's larvae) or multiciliated (Goette's larvae) sensory cells surrounded by a circle of gland cell necks.The necks of these gland cells pass through the basal membrane extending to F I G U R E 1 Cladogram of the interrelationships of major polyclad taxa on the suborder and superfamily level, except in an unnamed clade comprising of the genera Eucestoplana, Pericelis, Theama and Boninia.After Goodheart et al. (2023).Pictograms indicate known developmental types in the different superfamilies.DITTMANN ET AL.

For
3D reconstructions, three specimens fixed for SEM were embedded in EMBed 812 resin (Sigma-Aldrich).For the 3D model, semithin sections (0.35 μm) were aligned manually and reconstructed using Dragonfly software version 2021.1 for Windows (Object Research Systems) [ORS] Inc., 2020; software available at http:// www.theobjects.com/dragonfly.For further 3D imaging, surface information was acquired from a failed antibody staining, using a Leica TCS SP5 II confocal laser scanning microscope and visualised with Fiji 1.54 f (Schindelin et al., 2012) and Dragonfly software version 2021.1 for Windows (ORS Inc.).

(
Figures 2a,b, 3a,c and 4).In the anterior body half is a pigmented cerebral eye situated on the left side of the brain (Figures2a−d,f and 4).The ventral side is characterised by the oral hood which gives it a convex shape (Figures2a-b,e−g, 3b, and 4).

F
I G U R E 2 One-day old larvae of Theama mediterranea.(a−d) Ventro-lateral views of living animals captured with differential interference contrast.(a) Ventral to the right.Asterisk marks gut.(b) Ventral to the left.(c, d) Ventral to the right.(e) 3D reconstruction based on surface information resulting from antibody staining.(f, g) Schematic drawings.(f) Lateral view.(g) Ventral view.Apical is up for all animals.AT, apical tuft (white arrows); CEy, cerebral eye; G, gut; LL, lateral lobes; M, mouth; OH, oral hood; PT, posterior tuft (black arrow).Scale bars: 25 µm.F I G U R E 3 SEM images of 1-day old larvae of Theama mediterranea from different views.(a) Ventro-lateral view.(b) Ventro-lateral view.(c) Dorsolateral view.(d) Lateral view.Apical is up for all animals.AT, apical tuft; LL, lateral lobe; M, mouth; OH, oral hood; PT, posterior tuft.Coloured arrows show the orientation: a, anterior (orange); p, posterior (orange); l, left (green); r, right (green); v, ventral (blue); d, dorsal (blue).Asterisks mark dirt and probable fixation artefacts.Scale bar: 10 µm.DITTMANN ET AL.Apical organ The apical tuft (AT) at the anterior pole is part of the apical organ (Figure 6a−c).It comprises 14−16 cilia (Figures 5e and 3.2.1 | Ultrastructure of the apical tuft complex (ATC)At the anterior-most tip, the apical organ consists of two multiciliated (together providing 14−16 cilia) apical tuft sensory cells (ATS)(Figures 6b, 8a, and 9).Six necks of apical tuft gland type 1 (ATG1) cells are sandwiched between the apical tuft sensory cells and two anchor (ATAn) cells, while three necks of apical tuft gland type 2 (ATG2) cells are fully enclosed by the anchor cells (Figures7c and 9b).Except for their association with the apical gland cells, the anchor cells closely resemble epidermal cells.Thus, each anchor cell encloses one apical tuft sensory cell, three apical tuft gland type 1 cell necks and one or two apical tuft gland type 2 cell necks (Figures7c and 9b).Together, these four cell types form the apical tuft complex.The anchor cells of the apical tuft complex are situated in the epidermal layer, above the basal membrane (Figures6b,c and 8a,b).The main part of the apical tuft sensory cells, as well as the apical tuft gland cell bodies are located beneath the basal membrane (Figures6b,c, 7d−k, and 8a,b).The nuclei of the apical tuft sensory cells are located just above the anterior tip of the brain (Figure6b,c).The brain is laterally enveloped by a bundle of three unbranching apical tuft gland type 1 cells on each side (Figures7i and 9a,d), which are equipped with peripheral microtubules (Figure8c).The nuclei of the apical tuft gland type 1 cells are situated at the level of the cerebral eye, near to the posterior end of the brain (Figures7i−k and 8a).Only one apical tuft gland type 2 cell, whose nuclei is located at the midlevel of the brain, is present (Figures7h−k and 9c).This cell has no peripheral microtubules.The apical tuft gland type 1 and type 2 cells can be distinguished by their granule size and electron density: apical tuft gland type 1 granules are large (436 ± 86 nm, n = 17), with low electron density; apical tuft gland type 2 granules are small (136 ± 15 nm, n = 17) with high electron density (Figure8a).

F
I G U R E 4 3D reconstruction from serial sections of a 1-day old larva of Theama mediterranea.(a) Ventral view.(b) Ventro-lateral view.(c) Lateral view.Apical is up for all animals.(d, f, g) Position of the cerebral eye in the semitransparent larva seen in (a−c).(e) Position of the cerebral eye in an apical view.CEy, cerebral eye; LL, lateral lobe; M, mouth; OH, oral hood.Coloured arrows show the orientation of the animal: a, anterior (orange); p, posterior (orange); d, dorsal (blue); v, ventral (blue); l, left (green); r, right (green).
3.2.2| Ultrastructure of the ventro-apical complex (VAC)At the side of the apical tuft complex are seven multiciliated sensory neurons (SN)(Figures 7c, 8a,b,d, and 9a,b).The sensory neurons are slender cells located in the epidermis(Figure 8a,b).The only parts of the sensory neurons reaching basally through the basal membrane are the axons (Figure8a).These sensory neurons are neighbouring a third type of apical gland (ATG3) cell (Figures8a and 9a,b).The nuclei of the two apical tuft gland type 3 cells are located ventro-lateral and dorso-lateral to the anterior part of the brain (Figures7e−g and 9c).Compared to the other apical tuft gland cells (ATG1 and ATG2) the granules of the apical tuft gland type 3 cells are medium-sized (228 ± 38 nm, n = 16) and show a high electron density.Together, the sensory neurons and the apical tuft gland type 3 cells form the ventro-apical complex.The sensory neurons and the apical tuft gland type 3 cell necks are arranged in a common half circle around the apical tuft complex (Figures 7c and 9a,b).4| DISCUSSION 4.1 | A new type of polyclad larva Curini-Galletti et al. (2008) described the interstitial polyclad species T. mediterranea from the Mediterranean.They reported flattened, ovoid juveniles with one median eye and a ciliary tuft at the anterior and posterior body ends.The presence of an anterior and posterior F I G U R E 5 SEM images of the apical tuft of 1-day old larvae of Theama mediterranea.(a−e) Apical tuft of different individuals.(c) Detail of the tuft from the same individual shown in (b).(e) Apical tuft with coloured individually recognisable cilia.Coloured arrows showing the orientation of the animal: a, anterior (orange); p, posterior (orange); d, dorsal (blue); v, ventral (blue); l, left (green); r, right (green).Asterisks mark dirt and probable fixation artefacts.Scale bars: (a, b, d, e) 3 µm; (c) 1 µm.AT, apical tuft.at the larval nature of the described juveniles.A larval stage is given, if at least one of the following criteria is applicable: (a) the larval morphology differs from the adult animal by featuring structures which become reduced during the development of the individual (metamorphosis); (b) the larva occupies a different ecological niche; (c) the larva ensures the dispersal of the species (afterHaug, 2020).Due to the presence of apical and posterior tufts, and lobes, its free-swimming behaviour and a lack of dorso-ventral flattening, we determine the juvenile of T. mediterranea to be a larva and not a direct developer.The number of eyes, lobes, as well as the general body shape play an important role in differentiation and classification of polyclad larval types(Martín-Durán & Egger, 2012;Rawlinson, 2014).Named polyclad larval types are Müller's, Goette's and Kato's larvae(Martín- Durán & Egger, 2012).Müller's larvae are eight-lobed, three-eyed and spherical(Lapraz et al., 2013;Martín-Durán & Egger, 2012;
figs.2A, 4B), and Müller's larva at least five multiciliated apical tuft sensory cells (Dittmann et al., 2023; F I G U R E 9 Reconstruction of the cellular arrangement of the apical organ of 1-day old larvae of Theama mediterranea.(a) Ventral view of the apical organ embedded in the larva.(b−d) Cross section of the layers indicated in (a).ATAn, apical tuft anchor cells (red); ATG1, apical tuft gland cells type 1 (light green); ATG2, apical tuft gland cells type 2 (dark green); ATG3, apical tuft gland cells type 3 (purple); ATS, apical tuft sensory cells (yellow); BM, basal membrane (turquoise); Ci, cilia of the ATS (orange); CEy, cerebral eye (black); N, nuclei (blue); Ne, neurons of the brain (light peach); Np, neuropil of the brain (peach); SN, sensory neurons (pink).Coloured arrows showing the orientation: a, anterior; d, dorsal; l, left; p, posterior; r, right; v, ventral.T A B L E 1 Comparison of the lobes and eyes between different polyclad larval types.

ell B Biology iology I International nternational | 691
b Except Hoplopana inquilina with only two cerebral eyes.DITTMANN ET AL.C Cell Comparison of the apical organ between different polyclad larval types.