The non-brain anterior nerve center and tentacle crown structure of Owenia borealis (Annelida, Oweniidae): the evolution of the nervous system and tentacles in Bilateria

The Oweniidae are marine annelids with many unusual features of organ system, 22 development, morphology, and ultrastructure. Together with magelionds, oweniids have been 23 placed within the Palaeoannelida, a sister group to all remaining annelids. The study of this 24 group may increase our understanding of the early evolution of annelids (including their 25 radiation and diversification) and of the morphology of the last common bilaterian ancestor. 26 In the current research, scanning electron microscopy revealed that the tentacle apparatus 27 consists of 10 branched arms. The tentacles are covered by monociliary cells that form a 28 ciliar groove that extends along the oral side of the arm base. Light, confocal, and 29 transmission electron microscopy revealed that head region contains two circular 30 intraepidermal nerves (outer and inner) that give rise to the neurites of each tentacle, i.e., 31 intertentacular neurites are absent. Each tentacle contains a coelomic cavity with a network of 32 blood capillaries. Monociliar myoepithelial cells of the tentacle coelomic cavity form both 33 the longitudinal and the circular muscles. The structure of this myoepithelium is intermediate 34 between simple and pseudo-stratified myepithelium. Overall, tentacles lack prominent 35 zonality, i.e., co-localization of ciliary zones, neurite bundles, and muscles. This 36 organization, which indicates a non-specialized tentacle crown in O. borealis and other 37 oweniids with tentacles, is probably ancestral for annelids and for all Bilateria. The outer 38 circular nerve of O. borealis is a dorsal medullary commissure that apparently functions as an 39 anterior nerve center and is organized at the ultrastructural level as a stratified 40 neuroepithelium. Given the hypothesis that the anterior nerve center of the last bilateral 41 ancestor might be a diffuse neural plexus network, these results suggest that the ultra 42 anatomy of that plexus brain might be a stratified neuroepithelium. Alternatively, the results 43 could reflect the simplification of structure of the anterior nerve center in some bilaterian 44 lineages. a cell cortex Our study revealed the absence of a ganglionic organization of the anterior nerve center in O. borealis . In O. borealis , serotonin-lir somata do not form a compact cell cortex, and tubulin-lir neurite bundles do not form a swelling tentacular bilaterian metazoans. These groups have three of tentacle highly specialized tentacles, less specialized tentacles, and non-specialized tentacles. Our anatomical and ultra-anatomical data suggest that O. borealis has the least specialized tentacle apparatus, which can be regarded as an ancestral trait. We that the tentacle apparatuses in the Bilateria evolved from the non-specialized feeders to the highly specialized tentacles of filter-feeders.


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The Annelida is a phylum of bilaterian animals and is the central clade of the 49 Lophotrochozoa superphylum. Annelids exhibit extremely wide patterns of organ system 50 anatomy and ultrastructure (1). According to recent data, the Annelida can be divided into 51 two large clades, Errantia and Sedentaria, and also includes several sister groups, so-called 52 basal branching lineages, including oweniids, chaetopterids, amphinomids, sipunculids, etc.

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(2-7). Members of the family Oweniidae have many unusual morphological, ultrastructural,   (19,20). The organizations of the ganglia and brains have been well annelids, mollusks, hemichordates, echinodermes, and chordates (1,31). The presence of 74 tentacles in many groups suggests that the LCBA may have also had tentacles. If tentacles are 75 inherited from the LCBA, they must have evolved in different directions among bilaterians. 76 Although the directions of tentacle evolution remain uncertain, we know that some organisms 77 have specialized tentacles (32-38). This specialization is expressed in the zonation and co-78 localization of several organ systems: ciliary bands, nerve cords, and muscles (39-47). Such 79 specialized tentacles are present in the lophophorates (48-51). To increase our understanding 80 of how tentacles have evolved among the Bilateria, we require detailed data on the 81 organization and development of tentacles from different groups of recent bilaterians. 82 All oweniids have an intraepidermal non-ganglionic nerve center (11-13,15,26), but its 83 ultrastructural organization remains unclear. The family Oweniidae includes genera that have 84 tentacles (Owenia and Myriowenia) and those that lack tentacles (Galathowenia and 85 Myriochele) (52). Tentacles of Owenia fusiformis were briefly studied in the past, i.e., the 86 cells on the oral side of the tentacles have been described (53,54). Considering that the 87 morphology of oweniids is highly relevant to discussions of the structure of the last common 88 ancestor of the Annelida, in the current report we provide a detailed description of the 89 anatomy and ultra-anatomy of the head and tentacle apparatus of Owenia borealis. We also 90 consider the relevance of the data to the evolution of the structure of the nerve center and 91 tentacles in Bilateria.  Scanning electron microscopy (SEM) 97 The structure of the head was studied by scanning electron microscopy (SEM). The head  Transmission electron microscopy (TEM) 103 The head regions with tentacles were fixed overnight at 4ºC in a 2.5% solution of    119 Adults were fixed in a 4% paraformaldehyde solution in PBS (pH 7.4) (ThermoFisher   150 Our observations indicated that the tentacle crown of O. borealis is not symmetrical and is 151 formed by two lateral groups, which are separated on the dorsal and ventral sides. Each 152 lateral group is represented by five short tentacles, which included (from the ventral to the 153 dorsal side) 2 double tentacles, 1 quadruple tentacle, and 1 double tentacle on the right side or 154 1 triple tentacle and 1 quadruple tentacle on the left side ( Figure 1A). There are two levels of 155 tentacle ramification: for the first level, each tentacle arm splits into two or four branches; for 156 the second level, each tentacle is split into bifid tips. Tentacles and their arms are covered by  This groove is prominent at the base of the tentacle arm (Fig. 2C). The base of the tentacle 160 crown forms a collar that extends along external side of the head (Fig. 1C). The ventral 161 pharyngeal organ, consisting of the dorsal and ventral lips, is very large and is located at the 162 ventral side of the tentacle crown. Two ventrolateral lips are adjacent to the ventral 163 pharyngeal organ and are covered by cilia (Fig. 1C). The mouth resembles a crescent slit 164 (Fig. 1A). The base of the tentacle apparatus is surrounded by a thin collar fold from the 165 outside of the tentacle crown (Fig. 1A).

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Histology and ultrastructure of the tentacles and head 167 Epithelium. Each tentacle is covered by ciliated epithelial cells and contains a coelomic 168 cavity that contains muscles and blood vessels (Fig. 3A, B). The aboral epithelium is formed

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The epithelium lies on the extracellular matrix layer (ECM) (Fig. 3A, B). On the aboral side, 199 the ECM has waves and forms invaginations that contain the bundles of muscles. The aboral 200 ECM is up to 2 µm thick (Fig. 3B). The ECM is 2-3 times thinner on the oral side than on the 201 aboral side of the tentacle (Fig. 3A).

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Coelomic cavity and musculature. The coelomic cavity of the tentacles is connected to the 203 voluminous cavity of first body segment (i.e., the head cavity), which is formed by the  On the dorsal side, the head cavity is occupied by voluminous folds of the pharynx (Fig. 5C).  Blood vessels. Three-dimensional modelling revealed that the ventral and dorsal blood 241 vessels give rise to numerous blood vessels in the head and tentacles (Fig. 5D). At the border 242 between the first trunk segment and the head, the ventral blood vessel splits into two lateral 243 efferent branches, which give rise to two prominent lateral blood plexuses (Fig. 5D). The Peritoneal cells, which cover longitudinal muscles, are shown in dark green. Cell, which includes into 249 wall of blood vessel, is shown in light green. Hemidesmosomes are indicated by double arrowheads. 250 Abbreviations: ajadherence junction; bbbasal body; bvblood vessel; cicilium; ecm -251 extracellular matrix; gcgland cell; nnucleus; npneuropil; oamoral-aboral muscle; pc -252 peritoneal cells; sasecretory area of the gland cell; tcmtentacle circular muscles; tlmtentacle 253 longitudinal muscles. 254 dorsal blood vessel splits into two lateral afferent vessels at the middle of the head (Fig. 5D).

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In each tentacle, there are three longitudinal blood vessels that are connected to each other 256 (Fig. 5E). Together, they form the tentacular blood plexus.   with DAPI: dissepiment between head and trunk coeloms is indicated by arrowheads. (C) Z-300 projection of head and trunk cavity at sagittal optical section after staining with DAPI: dissepiment 301 between head and trunk coeloms is indicated by arrowheads. (D) Three-dimensional reconstruction 302 of blood vessels in head and part of trunk. Trunk and head coeloms are partly transparent. (E) Three-303 dimensional reconstruction of blood capillaries in tentacles. Abbreviations: bcblood capillary; bw -304 body wall; dprdorsal protrusion; dtdigestive tube; dvdorsal blood vessel; hchead coelom; 305 lbdlateral branch of dorsal blood vessel; lbvlateral branch of ventral blood vessel; lbplateral 306 blood plexus; mmouth; pgoparapodial glandular organ; phparynx; ttentacle; sbsetae 307 bundle; trctrunk coelom; vpoventral pharyngeal organ; vprventral protrusion; vvventral 308 blood vessel. 309 310 medullary dorsal commissure to the circumesophageal connectives is diffuse (Fig. 10A), it is 311 impossible to say where one ends and the other begins.

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The circumesophageal connectives give rise to two ventro-lateral medullar nerve cords that 313 fuse together at the border between the head and the first chaetiger (Figs. 8A, 10A). Each 314 ventro-lateral medullar cord gives rise to a root that skirts a tentacle on the oral side and 315 connect the outer and inner nerve rings (Fig. 9C-F). The inner nerve ring gives rise to a few 316 thin neurites that extend along the oral side of the tentacles (Fig. 9A).  Abbreviations: acaccessory centriole; ancanterior nerve center; apjadherence junction; bb -342 basal body; bvblood vessel; cicilium; cfcollar fold; cmcircular muscles; corcircumoral 343 nerve ring; ecmextracellular matrix; Ggolgi apparatus; hchead coelom; lmlongitudinal 344 muscle; mmoth; mfmyofilaments; nnucleus; pcperitoneal cell; srstriated rootlet; vll -345 ventrolateral lip; vpoventral pharyngeal organ. 346 347 As a medullary commissure, the nerve center lies at the tentacle base, in the outer epidermis 348 of the head (Figs. 7C, 11A, B). The epithelium, which includes the nerve center, is up to 349 45µm in height (Figs. 7C, 11A). The neurite bundles, which make up the largest portion of 350 the nerve center, form a layer that is up to 30 µm thick (Fig. 7C). According to TEM, the 351 epithelium, which contains the nerve center, is formed by monociliated cells and has a wide 352 apical part and a narrow basal part that is transformed into a long thin process. The apical 353 surface of the monociliated cells bears thin long microvilli, whose tips are electron dense 354 (Fig. 11A). A thick layer of cuticle is located between the microvilli and cilia. The basal body 355 and accessory centriole are located at the base of the cilium. The cytoplasm of the 356 monociliated cells has many large vesicles and an electron-lucent content (Fig. 11A).  (Fig. 7C). In the nerve center, perikarya are scattered between the somata of 364 the epithelial cells, above the neuropil (Fig. 11A). These perikarya are small (diameter ~ 5 365 µm) (Fig. 12A). The nucleus in these cells has an irregular shape, lacks a nucleolus, has 366 electron-lucent karyoplasm, and contains large aggregations of heterochromatin in the centre 367 (Fig. 12A). The cytoplasm of the perikarya contains many synaptic vesicles that differ in 368 diameter and content. The cytoplasm also contains mitochondria that are small, not abundant, 369 and have an electron-dense matrix (Fig. 12A). The neuropil, which is formed by numerous 370 neurites, is located between the perikarya and the extracellular matrix. In the nerve center, 371 two components of the neuropil can be distinguished at the ultrastructural level. The first 372 component is the upper portion of the neuropil, which is mostly formed by circular neurites 373 that are cut longitudinally in transverse sections of the nerve center (Fig. 11A). The second 374 component is the lower layer, which is mostly formed by longitudinal neurites that are cut 375 transversally in transverse sections of the nerve center (Fig. 11A). In the nerve center, the 376 neuropil consists of neurites that differ in structure. Some of these neurites have small 377 diameters and electron-dense cytoplasm, which regularly form wide varicoses with electron-378 lucent cytoplasm. The cytoplasm of these small neurites contains synaptic vesicles with 379 electron-dense content and with content of intermediate electron density (Fig. 12A, B). Other 380 neurites of the neuropil usually have large diameters and electron-lucent cytoplasm, and 381 contain dense-core synaptic vesicles and vesicles with electron-lucent content (Fig. 12B). In 382 addition to these two types of neurites, the neuropil contains projections of cells that contain 383 ovoid electron-dense granules (Fig. 12A, B).

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In the current research, we used TEM and immunocytochemistry coupled with CLSM to 386 study the anatomy and ultra-anatomy of the anterior nerve center. We report the absence of a 387 brain-like structure in O. borealis. We also used histology, TEM, SEM, and 3D modelling to  (12,13,15,26). We therefore suggest that the anterior nerve center in the entire Oweniidae 428 clade can be termed the "medullary dorsal commissure".

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In a previous study, electron microscopy revealed that O. borealis has a stratified 430 neuroepithelium, similar to that in the brachiopod Coptothyris grayi (57). A stratified 431 neuroepithelium has also been described based on TEM in O. fusiformis and G. oculata (see 432 Fig 8A in (15) and Fig 3D in (13)). We therefore suggest that all oweniids probably have the 433 dorsal medullary commissure, which is organized as a stratified neuroepithelium.  The non-specialized tentacle crown in oweniids 446 Oweniids primarily feed on the surfaces of substrates, and those species with a 447 tentacle crown also use it in suspension feeding (61). The degree of specialization in feeding 448 probably determines the architecture of the tentacle crown, which may differ in number of 449 tentacles, crown length, ramification from the base, rate of branching, and the shape of to the columnar epidermal cells on the oral side. Taking into account all these traits, we 457 consider that O. borealis is not specialized in its mode of feeding, but instead uses its tentacle 458 crown for feeding on suspended particles and on particles on the surface of the substrate. oral side of the tentacles (Fig. 3A). In those annelids that are specialized filter feeders, the 529 outward expansion of the fan of tentacles also occurs due to the contraction of the aboral 530 longitudinal muscles. Those filter feeders, however, also have a cartilaginous skeleton as well 531 as muscles at the base of the tentacular crown that serve as antagonists of the aboral 532 longitudinal muscles, i.e., that enable the organism to withdraw the tentacles and move the 533 captured particles to the mouth (70).

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In the tentacles of O. borealis, the circular muscle layer is very thin. Although this muscle 535 layer may represent only short fragments of individual muscle filaments, we suspect that it 536 represents a complete muscle ring (Fig. 2B, 3А). In the tentacles of the specialized filter   577 It is assumed that various bilaterians, including annelids, cephalopods, onychophorans, 578 echinoderms, and ascidians, have the same genetic program defining the coordinate grid of 579 the various appendages or outgrowths of the body (80-82). At the same time, these appendages of the body are not homologous to each other, have different morphologies and 581 perform completely different functions, for example, sensitive perception, nutrition, 582 movement, etc. Interestingly, in different groups of the bilaterians, including annelids, the 583 anterior outgrowths of the body specialized in parallel in capture of the food particles, the so-584 called tentacular apparatuses. Here, we consider the evolutionary trends of the organization of 585 tentacular apparatus that are used for suspension and filter feeding.

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O. borealis is one of the various annelids that has a tentacle apparatus or anterior appendages.

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A comparative analysis of the organization of tentacles in different groups of Bilateria reveals 588 three main patterns of the tentacle specialization (Fig. 13). The first pattern is represented by 589 highly specialized tentacles with a zonality of the epithelium that is co-localized with nerve tentacles always have at least four zones: one oral, one aboral, and two lateral (Fig. 13A, B).

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Oral and lateral zones are heavily ciliated, whereas cilia are rare or even absent in the aboral 595 zone. The aboral zone can undergo specialization involving the presence of additional 596 skeletal structures and gland cells. These four zones are innervated by different nerve tracts.

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The epidermal zones and nerve tracts are co-localized. The muscle bundles are also co-598 localized with certain zones of tentacle. There are usually two muscular bundles, oral and 599 aboral, which allow each tentacle to bend in two directions. Among all filter feeders, 600 lophophorates have the most specialized tentacles, each of which bears eight zones: one 601 frontal (oral), one abfrontal (aboral), two lateral, two laterofrontal, and two lateroabfrontal.

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Each of these zones is innervated by a specific nerve tract and has a specific function (51).

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The second pattern of tentacle organization is less specialized tentacles, which have at least 604 two zones: heavy ciliated oral and less ciliated aboral. In the second pattern, the innervation 605 of each zone is provided by a specific nerve tract. Less specialized tentacles occur in some 606 annelids that are not specialized filter-feeders, i.e., the Fabriciidae and some Serpulidae 607 (71,83) (Fig. 13C). Although the information is scarce, fabriciids could be deposit and/ or 608 suspension feeders (61,83,85).

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The third pattern of the tentacle organization is the non-specialized tentacles. Such tentacles 610 lack zonality of the epidermis and co-localization of ciliary cells, nerve tracts, and muscular 611 tracts (Fig. 13D, E). In these non-specialized tentacles, all sides are evenly ciliated. If present, 612 ciliary zones are not co-localized with the nerve tracts or neurite bundles, which are evenly 613 scattered in the tentacle. In the tentacles of the third type, muscle cells do not form bundles, 614 and they are evenly distributed in the tentacle. The non-specialized tentacles can be found in 615 oweniids (this study) and in some holothurians (86). Holothurians are deposit or suspension 616 feeders and are able to attach deposited particles to tentacles due to the secretion of a glue by 617 gland cells (87-89). The presence of many gland cells can be regarded as a kind of 618 specialization but cannot be compared with the zonality of the highly specialized tentacles for 619 filter-feeding. 620 If we assume that tentacles have been inherited from the LCBA, we can suggest the evolution 621 of tentacle apparatuses from the non-specialized tentacles of deposit/suspension feeders to the 622 highly specialized tentacles of the filter-feeders. This idea may be supported by data on 623 morphology and diet of the Sabellidae, whose tentacles evolved from less specialized in the 624 Fabriciidae to highly specialized in the Sabellidae (83). In the hypothetical order of tentacle  In this report, we described the anatomy and ultra-anatomy of the tentacular crown of Owenia with the tentacular apparatuses of the other bilaterian metazoans. These groups have three 653 patterns of tentacle organization: highly specialized tentacles, less specialized tentacles, and 654 non-specialized tentacles. Our anatomical and ultra-anatomical data suggest that O. borealis 655 has the least specialized tentacle apparatus, which can be regarded as an ancestral trait. We 656 propose that the tentacle apparatuses in the Bilateria evolved from the non-specialized 657 tentacles of deposit/suspension feeders to the highly specialized tentacles of filter-feeders. The data sets analyzed during this study are available from ET upon request.