General aspects
Due to their specific and highly specialized morphology and biology, Sabellariidae, also known as honeycomb or sandcastle worms, have received special attention since their first description in the 18th century [3]. This interest may be based on the fact that many species are gregarious, building extensive reefs, and thus, they are the major component of specific biocenoses. The division of their body into an operculum, parathorax, abdomen, and cauda is one of the main characteristics of the taxon [2–4]. Sometimes the operculum is further divided into the operculum proper and thorax [6]. Whereas their external morphology is well-known and has been described repeatedly, only a few studies have examined the internal morphology, ultrastructure, and functional morphology [6, 9]. This scarcity of knowledge likewise applies to the various kinds of appendages. In addition to the parapodia, Sabellariids bear four different kinds of appendages, three of which are related to the anterior end or operculum, namely the opercular papillae, the tentacular filaments, and the palps. Only the branchiae are related to the trunk segments. Out of these appendages, merely the ultrastructure and functional morphology of the palps in larval Phragmatopoma californica have been studied thus far [15, 16].
All four types of appendages studied comprise epidermal and mesodermal tissues. Whereas epidermis and cuticle are structurally more uniform, the mesodermal structures differ between the four types of appendages. They may comprise connective tissue, muscle fibers, and coelomic cavities, as well as blood vessels. However, blood vessels and coelomic cavities were not observed in the opercular papillae, blood vessels are absent in the tentacular filaments, and coelomic cavities and blood vessels are only present in the palps and branchiae.
Epidermis and Cuticle. In all appendages investigated, the epidermis comprises just a few and similar cell types. These include unciliated and ciliated supportive cells, gland cells, and several types of receptor cells. However, ciliated supportive cells with motile cilia are only present on the tentacular filaments, palps, and branchiae. Typical for multiciliated cells in annelids, accessory centrioles are lacking, and the basal bodies are equipped with two striated rootlets, a short one parallel to the apical membrane and a long one extending deep into the cell body and anchoring the cilium [25]. The density of cilia has only rarely been determined for annelid epithelia [25–27] but in the appendages of S. alveolata, it is similar to that of pharyngeal epithelia of other annelids with reported densities of up to 6.5–8.5 cilia per µm2 [28, 29]. In these cells, more than 100 cilia per cell may be present [29]. Such densely arranged cilia have been termed compound cilia and the respective cells ciliophores, if these cilia are anchored by long rootlets and more than 100 cilia per cell are present [14, 27, 30]. However, there are no ultrastructural differences from other ciliated cells, and thus justification of this separation and term appears to be questionable. Generally, densely ciliated cells are present on epithelia transporting and collecting materials as well as on epithelia generating water currents for ventilating the branchiae or swimming. The functional significance of the varying density was not addressed.
The general organization of the epidermis is similar to that of polychaetous annelids except for the structure of the cuticle [25, 26]. This organization includes the presence of secretory cells, which are generally abundant in the annelid epidermis [25]. Due to their different functions, gland cells and their secretion exhibit a high structural diversity, as can be observed in their apical apertures. Depending on the fine structure of the cuticle, these often comprise multiple circular microvilli leaving a more or less distinct central pore in the cuticle for the release of secretions [25].
In S. alveolata, the epidermis is covered by a cuticle, similar to a larval cuticle of other annelids in the absence of layers of parallel collagen fibers. Such layers are usually the most characteristic feature of the cuticle in adult annelids of comparable body size [25, 26, 31]. Other sabellariids studied thus far show a similar pattern of cuticular ultrastructure [6, 9–11, 14, 15]. In addition to Sabellariidae, other exceptions to this general pattern are polychaetes having meiofaunal body dimensions and several members of the so-called basal radiation [32, 33] such as Oweniidae, Magelonidae, Chaetopteridae, Psammodrilidae and Apistobranchidae [25, 34–36].
Likewise, there are no differences in the structure of the cuticle between the different body appendages studied here, the operculum or the trunk in S. alveolata [6, 26]. In species possessing a cuticle with collagen fibers, the thickness of the branchial cuticle is considerably less, and collagen fibers are reduced or even absent [21, 22, 37, 38]. Most likely, this is indicative that in Sabellariidae the cuticle does not provide a real barrier for the exchange of molecules with the surrounding environment and there seems to be no functional necessity for a thinner or specialized cuticle in epithelia adapted to gas exchange (see also [6]).
In general, the annelid cuticle is a flexible and soft structure through which many substances may diffuse rather than a tight border as the term cuticle may suggest [25, 31, 39]. Moreover, the cuticle is regularly traversed by more or less densely arranged microvilli, their apices forming a cover above the cuticle proper and in direct contact with the environment. The weak structure of the cuticle might also be an adaptation to the tubicolous lifestyle of all Sabellariidae, which cannot survive outside their tubes after settlement [3]. Thus, their elaborate and firm tubes may have partly taken over the protective role of the cuticle. Absence of collagen fibers and thus similar cuticular features has also been reported for a few species of tubicolous Sabellidae and Serpulidae [40].
Receptor cells. Receptor cells occur everywhere in the epidermis of annelids, but on the various types of appendages, they are often present in higher numbers [25, 41]. Therefore, appendages rich in receptor cells are generally regarded as sensory organs like the antennae, palps, parapodial, and anal cirri [41–43, 45]. On the body and the appendages, the receptor cells may occur scattered between supportive cells or clustered in small groups of mostly just a few cells. These cells are bipolar sensory cells, and their somata are either located in the epidermis or are situated deeper in the body within the nervous system.
The dendritic processes of the receptor cells are generally ciliated, and accordingly, they are often classified by the number of cilia present and whether or not these cilia penetrate the cuticle [43]. The latter, usually called non-penetrative receptor cells, were not encountered on the structures investigated in S. alveolata. Due to their fine structure, receptor cells may be further subdivided into several subtypes [36, 44]. Structural diversity is generally interpreted as indicative of different sensory stimuli or different sensitivity, although the function of these receptor cells is still insufficiently known and mainly speculative [22, 41].
Among the receptor cell types present in S. alveolata, uniciliated sensory cells predominate. Usually, the latter are characterized by a dendritic process diminishing apically in diameter to approximately 1 µm or less, and thus they form tufts of densely arranged sensory cilia that cannot be assigned to a certain number of cells if viewed with SEM. Moreover, such cells intermingle with multiciliated receptor cells possessing a wider cell apex. As a rule, these receptor cells possess microvilli accompanying and surrounding the cilia [41, 43, 45]. One specific receptor cell type of this kind exhibits 8 or 10 strong microvilli forming a regular crown around the single cilium and is usually called a collar receptor [46]. Collar receptors frequently occur in aquatic invertebrates and are the characteristic elements of lateral polychaete organs, representing a typical sensory organ present in many sedentary annelids [36, 41, 43]. Unexpectedly these are obviously absent in the appendages of S. alveolata. Compared to purely sensory appendages of other polychaetes, the receptor cell diversity appears to be lower in S. alveolata. However, it should be noted that only very few species have been investigated for these features [22, 44, 47–51]. These results are consistent with investigations on the median organ in S. alveolata and Idanthyrsus australensis (Haswell, 1883), and the palps of larvae in Phragmatopoma californica (Fewkes, 1889) and Phragmatopoma caudata Krøyer in Mörch, 1863 [as P. lapidosa Kinberg, 1866] [6, 9, 19, 15, 16]. However, in the latter two species, only multiciliated receptor cells have been identified, and probable occurrence of uniciliated cells is not mentioned [10, 15, 16] Thus, the paucity of morphologically distinct ciliated receptor cell types appears to be a general feature of Sabellariidae.
Opercular papillae
A ring of opercular papillae situated immediately beneath the paleae is generally present in members of Sabellariidae [1, 3]. Usually, these papillae are numerous, differing in number, size, and form according to the species and age of the individual considered [2]. The highest numbers recorded are approximately 20 pairs as observed in our material of S. alveolata, and, for instance, up to 18 pairs have been reported in Lygdamis wambiri [1]. The number of papillae is age-dependent, and the development of these papillae starts with only one pair in late larvae [9, 52]. In larger specimens, they are often not easy to count, especially if prepared and mounted for SEM. This challenge might be the main reason why so few authors provide numbers in their descriptions as, for instance, in Capa et al. 2015 [2]. Although these appendages were noted in certain previous studies on the morphology of the anterior end [19, 53], a more precise view of the structure of these appendages was thus far unknown. If viewed with SEM, the opercular papillae with their tufts of sensory cilia distributed all around them (e.g., our Fig. 2B; Fig. 1 in [15]; Fig. 2d, e in [9]) somehow resemble parapodial cirri of other polychaetes [22, 41]. Accordingly, the first pair of these appendages appearing in late pelagic larvae in close proximity to the provisional chaetae has been called cirri or opercular cirri [10, 15, 52, 54]. Somewhat later, after metamorphosis has started, and the provisional chaetae have been replaced, these cirri are situated beneath the first formed paleae and become the first pair of opercular papillae [9, 52].
Generally, the opercular papillae can be classified as outgrowths of the anterior body wall. Due to the presence of connective tissue and musculature, the appendages are moveable structures. In the adult worms, the opercular papillae are situated close to the foremost position of the sabellariid body. Together with the tentacular filaments, the opercular papillae represent those structures which are most likely the first to come in contact with all kinds of potential sensory stimuli when the animals open the operculum and are in the feeding position (see supplementary video in Meyer et al. 2019 [6], [55]). In the absence of coelom, blood vessels and motile cilia indicate that the papillae are neither involved in feeding nor respiration. Instead, the high number of tufts of sensory cilia likely indicates a predominantly sensory function, irrespective of the fact that the receptor cell diversity is lower as in the appendages of the few errant polychaetes studied for this character [22, 47, 56]. Regarding receptor cell diversity and density, sabellariids notably have a completely different life strategy combined with immobility, requiring a different and specific adapted sensory system [5, 6, 9]. Whereas the median organ is most likely involved in triggering of the shadow reflex [2, 5, 6], mechanical or chemical stimuli may play a significant role in the function of the opercular papillae including triggering the withdrawal of the animal into its tube. Unfortunately, our immunostainings did not allow for clarification of the innervation pattern of these appendages, but we assume that the various nerve tracts innervating the operculum as described and imaged by Orrhage [19] include the efferent fibers of the receptor cells. Out of these, the nerves innervating the lateral parts of the operculum or the outer paleae (nmlo1, nmlo2, and nmoop in [19]) appear to be the best candidates for this function. All nerves innervating the corresponding part of the operculum have been shown to represent parapodial nerves emanating outside the brain from the central nervous system by Orrhage [19].
Tentacular filaments
Filamentous appendages inserting on the ventral and inner side of the operculum are present in most sabellariid species except for those of Phalacrostemma [1, 3]. These branched or unbranched appendages are commonly called tentacular or oral filaments and may occur in high numbers, as is the case in S. alveolata [3, 19]. They are involved in the transport of food particles to the mouth and sediment particles for tube construction to the building organ on the ventral side [3, 14, 19, 55, 57, 58]. The particle transport mechanism has been analyzed in high-speed video recordings [14, 55, 58]. In active animals, these appendages are exposed to the exterior and are widely stretched out into the surrounding medium ([55]; see Fig. 1A-D in [5], and supplementary video [6]). As discussed for the opercular papillae, the tentacular filaments are thus the second type of anterior appendage coming in contact with various sensory stimuli and are most likely important sensory structures as well. However, their extensive ciliation, in addition to the sensory cells, speaks in favor of a double function: sensing as well as particle selection and collection.
The external morphology and the histology of the tentacular filaments are comparatively well known [14, 18, 19, 55, 58, 59]. These observations are complemented by preliminary ultrastructural observations of the tentacular filaments by Riisgård and Nielsen [14]. We confirm in our present investigation the general structure of the ciliation pattern of these appendages and clarify some discrepancies. In all studies, frontal cilia have been described as forming a continuous ciliary band, and a more or less distinct groove is formed proximally, which is absent distally [19]. These cilia are supplemented by longer cilia extending laterally from this band. As noted by Riisgård and Nielsen [14], these are made up of three bundles of cilia, each of which belongs to a single cell. So these lateral cilia are formed by three longitudinal rows of densely ciliated cells separated by unciliated cells at regular intervals. These cilia have been called compound cilia, spikes, or grouped frontal and lateral cilia [14, 55, 58]. However, there is no structural evidence that these cilia are somehow structurally interconnected [58].
As in the studies of Dubois et al. [55, 58], we were not able to detect the so-called cirri, somehow separated smaller bundles of long cilia adjacent to the lateral groups of dense cilia, described by Riisgård and Nielsen [14]. It has been argued that they represent artifacts [58]. From live and recorded video observations, it is evident that the lateral grouped cilia often project orthogonally from the surface of the tentacular filaments and appear to be immobile [14, 55, 58]. Nevertheless, according to Riisgård and Nielsen [14], these cilia sporadically bend in the downward longitudinal direction of the tentacular filament. Movability of these cilia has been confirmed, but the direction of ciliary movement has been observed as oblique towards the frontal surface by Dubois et al. [55, 58]. In any case, these previous observations of the activity of these cilia are consistent with our findings on the innervation pattern, strongly suggesting that beating of these long cilia is under nervous control.
Most authors observed that the epidermis is thicker on the frontal side, where the ciliated cells are located [14, 19, 59]. Glandular cells have also only been found on the frontal ciliated side and have been shown to represent only acid mucopolysaccharides belonging to a single type [55]. The conspicuous gutter-like structure situated beneath the epidermis confirms their hyaline cartilaginous nature [14, 19]. However, in contrast to Riisgård and Nielsen [14], it is not part of the basal membrane or ECM, but rather, clearly separated from the ECM and situated beneath it. Likewise, the views that this structure is an artifact and represents an extraordinarily distended basal lamina [58] or represents blood vessels [18] have to be rejected. Since it is entirely cell-free, it should not be termed connective tissue. The two ends of the “U” on the frontal side are connected by the ECM surrounding the entire element. Here it is underlined only by muscle fibers; the presence of a ligament-like structure as discussed by Orrhage [19] cannot be confirmed or may be represented by the ECM.
The movability of the tentacular filaments has been described in detail [14]. Most likely, this movability is due to their cartilaginous structure in combination with the central musculature enclosed by the cartilaginous structure. In this musculature, longitudinal fibers predominate, and these may be responsible for bending the tip, followed by a spiral contraction of the entire tentacle [14]. Stretching might be a more passive process mainly mediated by the cartilaginous structure serving as a kind of flexible skeletal element. The small additional longitudinal fibers situated on the outer side of the cartilaginous structure are described here for the first time. Observations of the presence or absence of food particles in the surrounding medium are correlated with the motion of the tentacles. Furthermore, their ciliation supports our view that the entire system is under nervous control and that at least in part, the different receptor cells present on these filaments are responsible for the necessary sensory input to trigger the uptake of particles.
We can confirm the presence of a coelomic cavity noted by Orrhage [19]. Typical epithelial junctional complexes are present, demonstrating that these spaces represent coelomic cavities sealed by a myoepithelium [60–62]. However, blind-ending blood vessels, as described in the older literature, are definitely absent in the tentacular filaments [17–19] and most likely the cartilaginous structure was mistaken as blood space. Thus a prominent role in gas exchange can be excluded. Whether these appendages are homologous to branchiae or unique for Sabellariidae as discussed by Rouse and Pleijel [4] appears to be answered by the latter hypothesis given their different structure and especially the lack of blood vessels and blood supply.
Palps
The presence of two so-called peristomal palps in larval and adult members of the Sabellariidae has already been observed [2, 7, 19] and their function in chemo- and mechanoreception as well as in feeding behavior assessed [10, 14, 15, 54, 63]. However, ultrastructural analyses of adult palps are rare and mainly based on light microscopic observations [19, 55] or focus on the larval palps [15].
The present ultrastructural analysis of the palps of adult S. alveolata shows strong similarities to the structure of the larval palps of Phragmatopoma californica [15]. In brief, the palp is ciliated and grooved on the frontal side along the longitudinal axis and exhibits two coelomic cavities separated by a single blood vessel and surrounded by mesodermal cells and ECM. Within the epithelium, two prominent nerves are present. This pattern is somewhat similar to earlier descriptions of sabellariid palps [15, 18, 19, 59] and is similar to other grooved palps [4, 64, 65].
The palps of S. alveolata and other sabellariids are multifunctional organs that might have different tasks regarding the developmental stage of the animal. In larval stages, it is assumed that they have a minor part in feeding but mainly function in mechano- and chemoreception. The prominent ciliary band and additional groups of cilia, in combination with the presence of gland cells, make them a highly suitable structure for locomotion [66]. In late larval and juvenile stages, the palps might be responsible for substrate recognition, settlement and the building of the tube, as they sense different chemical signals allowing the animals to recognize others and attach their tube, thus forming the famous large reefs [67, 68].
Branchiae
Annelid branchiae. In annelids, gas exchange may occur through the entire body wall, parapodia, or specialized organs commonly termed branchiae or gills [4, 20, 21, 69]. Polychaete branchiae are generally outgrowths or extensions of the body wall often associated with the parapodia or arising separately from the dorsum [4, 20, 69]. As such, they are composed of epidermis, nerve fibers and receptor cells, blood vessels, musculature, and often coelomic cavities. The occurrence of branchiae is restricted to specific taxa, and especially polychaetes with small body dimensions often don’t possess branchiae. In Clitellata, branchiae are usually absent, and their occurrence is a rare exception [70, 71]. In polychaetes, they are generally found in Amphinomidae, certain Errantia, and many Sedentaria[4, 20, 72]. Probably due to their non-uniform occurrence, they exhibit a certain diversity in structure, form, and position, although several common features have been recognized. The highest complexity thus far has been observed in hydrothermal vent annelids and Siboglinidae [38, 44, 73–75].
Thus far, the ultrastructure of polychaete branchiae has been studied in multiple species, not representing close to the entire range of taxa possessing these organs [4, 20–22, 38, 44, 75–79]. Possibly the entire range of diversity may still not be known. Furthermore, recent investigations indicate that our knowledge of several species investigated previously may still be incomplete [21, 22].
Besides the mentioned diversity, polychaete branchiae show several common features: most polychaete branchiae studied so far are equipped with numerous motile cilia arranged in bands or tufts responsible for generating water currents for an effective, continuous, and powerful exchange of the surrounding water [20]. Thus, the elaboration of concentration gradients above the respiratory surfaces is avoided. In addition, the cuticle is generally thinner above the respiratory epithelia than in other body regions. Polychaete branchiae usually contain a loop of blood vessels giving rise to capillaries basally extending into the epidermal cells [4, 20, 69, 72]. The vessels of this loop are commonly termed afferent and efferent vessels, and within the branchiae, they are differently interconnected. From these vessels, more or less numerous blood spaces or sinuses extend into the epidermis, making the diffusion distances as small as possible. In most cases, the branchiae include mesodermal structures such as muscle fibers and often coelomic spaces. Lastly, branchiae are innervated and bear numerous receptor cells.
Stekolshchikov [80] and Storch and Alberti [82] attempted to assign the diversity of annelid branchiae to three or four different types, respectively. The pros and cons of these classifications were discussed by Belova and Zhadan [21], and it was concluded that this needed revision. Based on the data available, we suggest that the annelid branchiae may be divided into three different types.
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1. Branchiae with coelom and blood vessels: as in S. alveolata, a coelomic cavity following the main blood vessels has been observed in many species [21, 37, 38, 76, 79, 82, 83]. In contrast to the hydrothermal vent Terebellida, the absence of a coelomic cavity has been reported in the intertidal Terebella haplochaeta by Wells et al. [81], but very likely this is due to a misinterpretation of their histological sections (see, e.g., Fig. 12 in [81]). It is unknown whether the coelomic cavities are continuous with the segmental coelom in these species or whether they are separate, as observed in the present study. If the branchiae bear lamellae, filaments, pinnules, and other structures, the coelomic cavity or muscle fibers may reach into these branches [38, 79] or simply comprise epidermal structures with extensions of the blood vessels bulging into the ECM of the epidermis [74]. This latter feature has been observed in the branchial leaflets of Lagis koreni Malmgren, 1866 [as Pectinaria koreni], and most likely, it was erroneously described as a separate type of branchiae by Storch and Alberti [82].
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2. Branchiae with mesodermal tissue and blood vessels: more rarely, the space between the two main vessels is completely occupied by mesodermal cells, mainly musculature [22, 78]. Thus far, this latter pattern has only been found in Amphinomidae and Orbiniidae.
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3. Coelomic branchiae: In certain species with a reduced or absent blood vascular system, the coelom may take over the function of the blood vascular system [77]. Consequently, branchiae, if present, are of different structure and commonly called coelomic branchiae such as those occurring in glycerids and hydrothermal vent polynoids [77]. These species usually appear red due to the presence of hemoglobin in the coelomic fluid or the coelomocytes.
Sabellaria Branchiae. In S. alveolata, the branchiae exhibit the densest ciliation pattern observed, exceeding those of the tentacular filaments and palps. In contrast to the latter appendages, only one pattern of density has been observed. Data on the cellular level of the ciliatation in branchiae are rare; mostly, it is mentioned that a dense ciliation is present [38, 75, 82]. Estimations made based on published micrographs on ciliated cells leads us to assume that the density observed in S. alveolata appears to be among the highest described [22]. As in S. alveolata, each ciliary band is formed by a single row of ciliated cells in most other polychaetes, such as in the amphimonid Eurythoe complanata (Pallas, 1766), the polynoid Branchipolynoe sp. and in the ophelliids Ophelia limacina (Rathke, 1843), Ophelina acuminata Örsted, 1843 and Euzonus arcticus Grube, 1866 [21, 22, 77]. This feature is probably characteristic of alvinellids and trichobranchids, although not explicitly mentioned [38, 79]. In contrast, in an undescribed deep-sea orbiniid, the ciliary band is formed by several rows of cells [78]. This can be confirmed by our unpublished data on the intertidal Scoloplos armiger (Müller, 1776), and thus far, Orbiniidae appears to be the only diverging example. In annelids, only three species have been described thus far possessing branchiae without cilia: Sternaspis scutata (Ranzani, 1817), Travisia forbesii Johnston, 1840 and Arenicola marina (Linnaeus, 1758) [21, 37, 76]. In these species, ventilator currents are solely generated by the peristaltic movements of the body wall [37].
As a rule, the cuticle and epidermis are considerably thinner on branchiae than on other parts of the body [21, 22, 37, 78]. Besides reduction of total thickness, mostly between 1–2 µm, the number of layers of parallel collagen fibers forming a net-like structure is reduced, or even lacking [22, 25, 76]. On initial observation, S. alveolata does not seem to follow this general pattern, since its cuticle is of similar thickness on all body regions. However, as discussed above in S. alveolata and other sabellariids studied thus far, the entire cuticle is highly reduced.
Except for noting the occurrence of receptor cells and neurite bundles [37, 38, 75, 82, 84], innervation of branchiae has usually not been studied in detail. Receptor cells found on branchiae usually have cilia penetrating the cuticle, and the formation of small groups of uniciliated and multiciliated receptor cells has been described [22, 44]. Multiciliated cells are most common in Paralvinella hessleri Desbruyères & Laubier, 1989 (Alvinellidae), whereas in Eurythoe complanata (Amphinomidae) uniciliated receptor cells predominate. Our findings for S. alveolata most closely resemble the former scenario. However, it must be noted that very few species have been studied.
The innervation of the branchiae usually originates from the ventral cord via the segmental nerves [22, 44, 85]. In Eurythoe complanata, the branchial nerve branches off from the main segmental nerve, which innervates the parapodium and also comprises efferent fibers from the dorsal cirrus. This situation appears to be similar in Terebellides cf. stroemii Sars, 1835 (Trichobranchidae), Cossura pygodactyla Jones, 1956 (Cossuridae) and Paralvinella hessleri (Alvinellidae) [44, 85]. Depending on the structure of the respective branchiae, these nerves may split within the branchiae as in S. alveolata, wherein the two nerves mark the extension of the branchial coelomic cavity. In E. complanata, the motile cilia are innervated by separate neurite bundles [22], which is not the case in the other species studied, including S. alveolata.
In annelids possessing a blood vascular system, the branchiae are supplied by efferent and afferent vessels, which in the branchiae are variously connected and often give rise to blind-ending blood spaces (blood sinus). The latter often extend deep into the basal regions of epidermal supportive cells [21–23, 37, 82]. Efferent and afferent vessels unite distally, occasionally forming a long hairpin-like, blind-ending loop as, for example, observed in Eurythoe complanata or Osedax mucofloris Glover, Kallstrom, Smith & Dahlgren, 2005 [22, 75] and in the present study. Thus, blood is driven back into the body by contraction of the branchial musculature, which is present at least along these main vessels. The diffusion distances are often calculated to be 1–3 µm [21, 38, 79]. However, in such measurements, generally, the thickness of the cuticle is included, which might not be accurate since, as discussed above, the cuticle in these areas very likely does not form a diffusion barrier. More reliable might be those values including the smallest thickness of only the epidermal cells where the blind-ending blood spaces most closely approach the apical side. In doing so, values of diffusion distances diminish to approximately 130–350 nm [22, 23, 38], within the same range as that observed in S. alveolata. The micrographs published suggest that this also applies to other species [21, 38]. Outside such lacunae and sinuses, the thickness of the epidermis is reported to be approximately 5–10 µm, twice that of S. alveolata.
The structure of the blood spaces and blood vessels in S. alveolata is typical for coelomate invertebrates in general; they are lined by the ECM of the adjacent epithelia, and an endothelium is always absent [37, 60, 72, 86–88]. The ECM surrounding the afferent and efferent vessels is usually lined by peritoneal and epidermal cells or only by peritoneal cells [21, 22, 37, 38]. The peritoneal cells, or part of them, are usually differentiated as myoepithelial cells, and these cells are responsible for the blood flow inside the branchiae. The same is true for the loops connecting afferent and efferent vessels, whereas the terminal branches (blood spaces or sinus) may only be lined by epidermal cells and, as such are non-contractile. Hemoglobin is usually extracellular in annelids, and only a few cells present in the blood. The blood cells or hemocytes present in S. alveolata exhibit the same structure and high endocytic activity observed in other species [21, 37, 89].